Improvement of dissolution rate of a new antiretroviral drug using an

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um
THÈSE
En vue de l’obtention du
DOCTORAT DE L’UNIVERSITÉ DE TOULOUSE
Délivré par :
Institut National Polytechnique de Toulouse (INP Toulouse)
Présentée et soutenue par :
Suênia de PAIVA LACERDA
Le 01/02/2013
Titre :
Improvement of dissolution rate of a new antiretroviral drug using an
anti-solvent crystallization technology.
Ecole doctorale et discipline ou spécialité :
ED MEGEP : Génie des procédés et de l’Environnement
Unité de recherche :
École des Mines d’Albi Carmaux (RAPSODEE)
Directeurs de Thèse :
M.I RÉ
F. ESPITALIER
P. TCHORELOFF
M. DESCAMPS
Rapporteurs :
Université Paris Sud
Université Lille 1
Rapporteur
Rapporteur
Autres membres du jury :
I. RICO-LATTES
M.GIULIETTI
Directrice de Recherche du CNRS
Universidade Federal de São Carlos
Examinatrice
Examinateur
“Une confrontation permanente entre théorie et expérience est une condition nécessaire
à l'expression de la créativité.”
PIERRE JOLIOT
À mes parents et grands-parents
Remerciements
Cette thèse doit beaucoup aux nombreuses personnes qui m’ont encouragé, soutenu et
conforté au long de toutes ces années. Qu’elles trouvent dans ce travail l’expression de mes
plus sincères remerciements.
Je tiens à remercier très chaleureusement mes directrices de Thèse Maria Inês Ré et
Fabienne Espitalier de m’avoir encadrée et accompagnée tout au long de ces trois années,
pour leur aide, et compétence mais aussi pour leur patience, et leur encouragement à finir ce
travail. Je vous remercie vivement pour vos qualités d’encadrement.
Je remercie les rapporteurs (Pierre Tchoreloff et Marc Descamps) et les membres du
jury pour avoir accepté d’examiner et de participer à mon jury de thèse.
Je tiens ensuite à remercier les Laboratoires Cristalia et RAPSODEE, pour leur soutien
et la confiance qu’ils m’ont témoignés.
Pendant ma thèse j’ai eu le plaisir de travailler avec différentes personens constituant
une formidable équipe. Grâce à leur aide, leur soutien et l’ambiance qu’elles ont fait régner
j’ai pu arriver jusqu’au bout. En particulier, je souhaiterai citer Philippe Accart pour sa
gentillesse et sa bonne humeur ainsi que pour ses conseils avisés, Laurent Devrient pour sa
patience, son amitié et sa disponibilité, Bruno Boyer pour sa gentillesse et toute l’aide qu’il
m’a donné quand j’en ai eu besoin. Je pense aussi « aux filles » Sévérine Patry, Sylvie
Delconfetto, Véronique Nallet et Christine Rolland, pour leur sympathie et leur implication
dans leur ou mon travail.
Je n’oublie pas de remercier tous ceux avec qui j’ai passé de bons moments après avoir
mangé à la salle café et tout particulièrement Rachel Calvet, Laurence Galet et Celine
Boachon. Un grand merci très spécial aussi à Elsa Weiss, Marie Caria et à Manolita Boval.
J’aimerais également remercier Anne-Marie Fontes pour son aide, sa gentillesse,
disponibilité, ainsi que son amabilité.
J’ai une pensé pour les doctorants et stagiaires du laboratoire avec qui j’ai partagé une
tranche de vie et une tranche de vie de laboratoire et auprès de qui j’ai beaucoup appris et
partagé des bons moments. Mon grand merci à Ingrid, Nibal, Sarah, Naomi, Zhoé, Fredj,
William, Anaïs, Graciela, Guillaume, Camille, Fanny, Wellington, Analice, Aline, Antoinette,
Yazmine, Marie, Audrey, Claire, Claire Michel, Fabio, Michelle, Jacqueline, Paulo, Andrea,
Nayane, Tassadite, Moussa, Christophe, Mohamed Raii, Sébastien, Renaud, Haroun, Marta,
Rémis, Vanessa, Maxime et Ludovic.
Je tiens remercier spécialement trois personnes qui ont travaillé directement avec moi
pendant cette thèse Vinciane Magan, Julia Flauder et Bruna Rêgo de Vasconcelos, sans elles
je n’aurai pas pu y arriver. Avec vous trois j’ai beaucoup appris et j’ai essayé de vous passer
un peu du peu que je connais.
J’exprime tous mes remerciements et mon affection à ma famille pour leur précieux
soutien tout au long de ma thèse et surtout tout au long de ma vie, et plus particulièrement à
mes parents. Ils ont toujours cru en moi et j’espère être toujours à la hauteur.
La thèse a parfois été un moment difficile pour mes proches. Elle a été très preneuse de
temps ! Et j’avoue ne pas les avoir consacré le temps qu’ils méritaient. Raffaele D’Elia, je te
remercie pour tes encouragements et ton soutien. Malgré la période difficile qu’on a traversé,
tu as été toujours disponible et compréhensif.
Comme dirait le petit prince : « J’ai des amis à découvrir et beaucoup de choses à
connaître » et ici en France j’ai appris a connaître chacun de vous, vous apprécier et vous
admirer.
Merci à la famille Mayer pour son amitié, sa confiance et pour m’avoir reçu comme un
membre de votre famille dès le début. Merci Audrey Common, la française la plus brésilienne
que je connaisse, pour chaque soirée, les fous rires et tous les bons moments passées
ensembles. Merci Marie Dietemann qui a été une grande amie, toujours là pour me soutenir.
Merci à ma petite famille brésilienne en France : Jacqueline, Vincius, Nayane, Bruna,
Andrea et Paulo. Vous comptez beaucoup dans ma vie, et grâce à vous j’ai pu avoir une
famille en France identique à ma famille au Brésil.Vous tous mes chers amis, vous avez
soulagé le poids de la distance, de la solitude et vous m’avez apporté du bonheur et de la joie
sans rien demander en échange.
Je vous remercie à tous. Merci de m’avoir accompagnée et aidée pendant ces années. En
espérant vous croiser bientôt dans un autre Carnaval !!!
Table of Contents
INTRODUCTION .............................................................................................. 17
CHAPTER 1. ...................................................................................................... 21
1.1. THE BIOAVAILABILITY OF DRUGS .................................................... 27
1.1.1. Absorption and bioavailability ................................................................................................27
1.1.2. Poorly-water soluble drug molecules .....................................................................................30
1.2. ENHANCEMENT OF SOLUBILIZATION AND BIOAVAILABILITY
OF POORLY SOLUBLE DRUGS..................................................................... 31
1.2.1. Concept of dissolution .............................................................................................................31
1.2.2. Dissolution testing for poorly-water soluble drugs ................................................................34
1.2.3. Strategies to increase the amount of dissolved drug at the absorption site...........................38
1.2.3.1. Alteration of pH and solvent composition .......................................................................................... 38
1.2.3.1.1. Salt formation/pH control ........................................................................................................... 38
1.2.3.1.2. Cosolvency ................................................................................................................................. 39
1.2.3.2. Inclusion complexes/complexation ..................................................................................................... 40
1.2.3.2.1. Cyclodextrin (CD) ...................................................................................................................... 40
1.2.3.3. Carrier System..................................................................................................................................... 42
1.2.3.3.1. Micelles....................................................................................................................................... 42
1.2.3.3.2. Micro/Nanoemulsion .................................................................................................................. 43
1.2.3.4. Manipulation of solid state .................................................................................................................. 44
1.2.3.4.1. Co-crystals .................................................................................................................................. 44
1.2.3.4.2. Amorphous Solid dispersion....................................................................................................... 45
1.2.3.5. Physical Modification ......................................................................................................................... 46
1.2.3.5.1. Milling ........................................................................................................................................ 47
1.2.3.5.2. LAS Crystallization .................................................................................................................... 49
1.3. CRYSTALLIZATION ................................................................................. 51
1.3.1. Thermodynamic background ..................................................................................................51
1.3.2. Nucleation................................................................................................................................52
1.3.3. Crystal growth .........................................................................................................................54
1.3.4. Effect of supersaturation on nucleation and growth .............................................................55
1.3.5. Choice of solvent .....................................................................................................................56
1.4.
LAS
CRYSTALLIZATION
TO
IMPROVE
DISSOLUTION
PROPERTIES AND SOLUBILITY OF POORLY SOLUBLE DRUGS ........ 57
1.4.1. Previous works.........................................................................................................................60
1.4.2. Control of process parameters ................................................................................................74
1.5. ADDITIVES IN CRYSTALLIZATION ..................................................... 81
1.5.1. Additives in LAS crystallization ..............................................................................................82
1.6. THESIS OVERVIEW ................................................................................. 89
CHAPTER 2. ...................................................................................................... 91
2.1. INTRODUCTION ....................................................................................... 95
2.1.1. The molecule: CRS 74.............................................................................................................95
2.2. CHARACTERIZATION OF CRS 74 ......................................................... 99
2.2.1. Methods....................................................................................................................................99
2.2.1.1. Particle size measurement ................................................................................................................... 99
2.2.1.2. Density measurement ........................................................................................................................ 100
2.2.1.3. Thermal Analysis .............................................................................................................................. 100
2.2.1.3.1. Thermogravimetric Analysis (TGA) ........................................................................................ 100
2.2.1.3.2. Differential scanning calorimetric analysis (DSC) ................................................................... 101
2.2.1.4. X-Ray Diffraction Analysis (XRD) .................................................................................................. 101
2.2.1.5. Scanning electron microscopy analysis (SEM) ................................................................................ 102
2.2.1.6. Contact angle measurement (sessile drop method) ........................................................................... 102
2.2.1.7. In Vitro Dissolution Testing ............................................................................................................. 103
2.2.1.7.1. Defining the operating conditions for in vitro dissolution........................................................ 103
2.2.1.7.2. High Performance Liquid Chromatography (HPLC) to determine the content of the dissolved
drug ........................................................................................................................................................... 104
2.2.1.7.3. Drug solubility in dissolution media......................................................................................... 108
2.3. RESULTS AND DISCUSSION ................................................................ 109
2.3.1. Particle size, true density and morphology ...........................................................................109
2.3.2. Density ...................................................................................................................................110
2.3.3. XRD analysis .........................................................................................................................110
2.3.4. TGA and DSC analysis .........................................................................................................111
2.3.5. Determination of surface properties .....................................................................................114
2.3.6. Dissolution studies.................................................................................................................116
2.3.6.1. High Performance Liquid Chromatography (HPLC) to determine the content of the dissolved drug
........................................................................................................................................................................ 116
2.3.6.2. In vitro dissolution testing................................................................................................................. 120
2.4. CONCLUSIONS ....................................................................................... 123
CHAPTER 3. ................................................................................................... 125
3.1. INTRODUCTION ..................................................................................... 129
3.2. MATERIALS AND METHODS............................................................... 130
3.2.1. Materials ................................................................................................................................130
3.2.2. Methods for solubilty measurements ....................................................................................130
3.2.3. Methods for Liquid Anti-Solvent (LAS) crystallization .......................................................130
3.2.3.1. Characterization methods for particles in suspension ....................................................................... 133
3.2.3.2. Characterization methods for dried powder ...................................................................................... 135
3.3. RESULTS AND DISCUSSION ON SOLUBILITY STUDY .................. 136
3.3.1. Measurement and correlation of solubility of CRS 74 in water-ethanol mixtures .............136
3.3.1.1. Experimental determination .............................................................................................................. 136
3.3.2. Correlation of solubility data by a UNIQUAC model ..........................................................139
3.3.3. Theoritical yield of solid obtained by LAS crystallization ...................................................144
3.4. RESULTS AND DISCUSSION ON CRYSTALLIZATION STUDY ..... 145
3.4.1. Characterization of particles in suspension .........................................................................146
3.4.1.1. Influence of the type of mixer on mixing ......................................................................................... 146
3.4.1.2. Influence of the type of mixer on particle size.................................................................................. 149
3.4.1.3. Influence of flow rate in the T- mixer on particle size ...................................................................... 154
3.4.1.4. Influence of addition time and steady state ....................................................................................... 159
3.4.2. Characterization of solid particles obtained in experimental design ..................................162
3.4.3. Comparative study of original CRS 74 and LAS recrystallized drug with T-mixer ...........165
3.4.3.1. Particle size and morphology ............................................................................................................ 165
3.4.3.2. XRD analysis .................................................................................................................................... 167
3.4.3.3. DSC analysis ..................................................................................................................................... 167
3.4.3.4. Dissolution studies ............................................................................................................................ 169
3.4.3.5. Determination of surface properties .................................................................................................. 171
3.5. CONCLUSIONS ....................................................................................... 173
CHAPTER 4. .................................................................................................... 175
4.1. INTRODUCTION ..................................................................................... 179
4.2. Materials and methods .............................................................................. 182
4.2.1. Materials ................................................................................................................................182
4.2.2. Methods..................................................................................................................................184
4.2.2.1. Production of particles by LAS crystallization in presence of additives .......................................... 184
4.2.2.2. Determination of solubility in presence of additive .......................................................................... 186
4.2.2.3. Characterization methods .................................................................................................................. 186
4.2.2.3.1. Measurements of particle size during the crystallization process............................................. 186
4.2.2.3.2. Powder physicochemical properties ......................................................................................... 187
4.3. RESULTS AND DISCUSSION ................................................................ 188
4.3.1. CRS 74 solubility in presence of additives............................................................................188
4.3.2. Effect of additives on yields of production of CRS 74 crystals ............................................190
4.3.3. Effect of presence of the additive in the aqueous phase on physical and surface properties
of recrystallized powders .................................................................................................................192
4.3.1. Effect of the presence of the additive in the organic phase on physical and surface
properties of recrystallized powders ................................................................................................200
4.3.2. Effect of the presence of the additive in both, aqueous and organic phases ......................205
4.3.3. Drug concentration in the organic phase.............................................................................210
4.3.4. Reorganized organic phase ...................................................................................................216
4.3.5. Improving process production ..............................................................................................222
4.3.6. Dissolution behaviour of recrystallized powders in presence of additives ..........................224
4.4. CONCLUSION .......................................................................................... 233
CHAPTER 5. ................................................................................................... 235
APPENDIXES ................................................................................................. 247
SYMBOLS AND ABBREVIATIONS ............................................................. 295
LIST OF FIGURES AND TABLES ............................................................... 301
REFERENCES ................................................................................................ 313
Abstract
This study concerns a new antiretroviral drug named CRS 74. This molecule has a
limited bioavailability because of its low aqueous solubility, poor water wettability and low
dissolution rate. In an attempt to improve these properties, CRS 74 was recrystallized by
using a Liquid Anti-Solvent (LAS) crystallization process. The chosen solvent is the ethanol
and the anti-solvent the water. So solid-liquid equilibria in binary mixtures ethanol/water
were measured at 30°C. The obtained solubility data were represented using UNIQUACbased model. The experimental and calculated solubilities permitted to estimate the optimal
ethanol/water mass ratios (25/75 % w/w) in order to maximize the theoretical yield of solid. A
double-jet with premixing (T-mixer) has been used to mix the two solutions. Particles of
recrystallized CRS 74 seemed more agglomerated and the dissolution profile was not
modified compared to the original drug. Furthermore, the study of crystals obtained at the exit
of the mixer showed that the growth and agglomeration rates of crystals are high.In an
attempt to improve its dissolution properties, CRS 74 has been recrystallized using different
additives to optimize process and formulation parameters. Conclusively, produced
microcrystals exhibited significantly faster dissolution rates than the original CRS 74 crystals.
The improved dissolution is attributable to the modification of the particle size of drug
crystals and enhancement of wetting properties due to specific interactions between the drug
and the additives.
Keywords: antiretroviral drug, solubility, bioavailability, dissolution rate, Liquid Anti
Solvent crystallization, additive
Résumé
Cette étude concerne une nouvelle molécule antirétrovirale nommée CRS 74. Cette
molécule présente une biodisponibilité limitée à cause de sa faible solubilité en phase
aqueuse, sa mauvaise mouillabilité et sa faible vitesse de dissolution. Afin d’améliorer sa
biodisponibilité, la molécule CRS 74 a été recristallisée par effet anti-solvant. Le solvant
choisi est l’éthanol et l’anti-solvant l’eau. L'équilibre solide-liquide dans des mélanges
binaires éthanol/eau a été mesuré à 30°C. Les solubilités obtenues ont été représentées en
utilisant le modèle UNIQUAC pour le calcul des coefficients d’activité. Les solubilités
expérimentales et calculées ont permis d’évaluer le ratio éthanol/eau optimum (25/75 % m/m)
pour maximiser le rendement théorique en solide. Un mélange double jet avec pré-mélangeur
type mélangeur en T a été choisi pour réaliser la cristallisation. Le solide cristallisé dans ces
conditions semble plus aggloméré et son profil de dissolution comparé à celui du solide initial
est inchangé. De plus, l’étude des cristaux obtenus en sortie de pré-mélangeur a montré que
les vitesses de croissance et d’agglomération des cristaux sont élevées. Des additifs ont donc
été utilisés en vue de modifier les propriétés de dissolution des cristaux, et d’optimiser les
paramètres de formulation et de cristallisation. Les microcristaux produits en présence
d’additifs présentent des profils de dissolution significativement plus rapides que les cristaux
de la molécule initiale. Cette modification est attribuable à la modification de taille des
cristaux et l'amélioration du mouillage en raison des interactions spécifiques entre la surface
des cristaux et les additifs.
Mots clés : molécule active antirétrovirale, solubilité, vitesse de dissolution,
cristallisation par effet anti-solvant, additifs, biodisponibilité
Introduction
INTRODUCTION
An increasing number of newly developed drugs are poorly soluble in aqueous media.
Poorly water soluble drugs are specially challenging, as they cannot achieve dissolution and
therefore they have a very difficult pass through the dissolving fluid to contact the absorbing
mucosa and to be absorbed. If the dissolution process of the drug molecule is slow, due to the
physicochemical properties of the drug molecules or formulation factors, then dissolution
may be the rate-limiting step in absorption and will influence drug bioavailability. This is the
case of a new compound, (2S, 3S, 5S)-2, -5 bis- [N-[N-[[N- methyl- N-[(2-isopropyl- 4tiazolyl) methyl] amino] carbonyl] vanilyl] amino- 1,6- diphenyl- 3- hydroxyhexane, named
CRS 74.
This promising candidate against HIV infections has high biological activity as
disclosed in PCT documents WO 2005/111006; US 2010/7763733 (BOCKELMANN, M.A.
et al, 2005; BOCKELMANN, M.A. et al, 2010) but its bioavailability is limited because of its
low aqueous solubility and dissolution rate. Such properties pose difficulties not only in the
design of pharmaceutical formulations but may result in biovariability. Moreover, because of
their low solubility, such drugs require high doses to be administered in order to obtain their
pharmacological effect, increasing the side effect incidence.
Drug dissolution is a prerequisite to drug absorption and clinical response for almost all
drugs given orally. Solid forms that have been investigated for drug dissolution enhancement
include salts, polymorphs and amorphous, among others. High energy polymorphs and
amorphous formulations can achieve improved solubility but the system is at serious risk of
crystallizing the thermodynamically stable form, even in the solid state (YU, 2011;
RODRIGUEZ-SPONG et al., 2004). Such transformations can compromise the performance
of the formulation.
To save time and resources in product development, relatively simple approaches
should be tried first like crystallization. The Liquid Anti-Solvent (LAS) crystallization
process is an attractive method. It requires mild conditions (ambient temperature and
atmospheric pressure) with no requirement for expensive equipment. In LAS process,
crystallization of solute is achieved by decreasing the solubility of solid in the system. This is
19
Introduction
done by addition of a non-solvent component for solute called anti-solvent and miscible with
the solvent.
This thesis presents efforts to develop and assemble tools required for improving the
dissolution rate of CRS 74 by LAS crystallization process. It is divided into five chapters.
Chapter 1 is devoted to review literature themes. Many strategies to increase the
dissolution rate of poorly soluble drugs and the key determinants of drug bioavailability are
presented. It also focuses on topics of interest for our work, like properties of drugs
influencing dissolution behavior. Emphasis was given to the LAS crystallization process.
The first elements of the understanding of the drug properties are given in Chapter 2.
The original (as-received) molecule was characterized in terms of its physical properties
(particle shape and size), crystal structure (crystalline, amorphous) and surface properties
(wettability), solubility and dissolution properties in aqueous media. The methodology
utilized and the results obtained are summarized in this Chapter. This characterization study
was crucial to evaluate possible changes on drug properties after recrystallization by LAS
process.
The solvent selection is one of the essential parameters to envisage any crystallization
process. Therefore, the knowledge of the solubility of a target component in different solvents
is required. The solubility of CRS 74 in ethanol and ethanol-water binary mixtures was
measured in the temperature range of 5 -30oC and this study is presented in Chapter 3.
Moreover, Chapter 3 outlines the experimental apparatus and procedures used for LAS
crystallization studies. Two high jet velocity devices were tested to provide adequate mixing
to incorporate the anti-solvent into the bulk solution. Recrystallized solids were compared to
the original drug crystals in terms of particle size, solid state, thermal and dissolution
properties.
Finally, LAS crystallization of CRS 74 in presence of additives is the subject of the
Chapter 4. The study describes the use of different additives, which were introduced in the
drug solution or in the anti-solvent or in both phases, to achieve optimum crystal properties of
CRS 74. The effect of additives on the crystals particle size, dissolution kinetics and drug
wettability could be investigated and are discussed in this Chapter.
Chapter 5 summarizes the main results of this research and suggests future work.
20
Literature Review
Résumé Chapitre 1- Revue Bibliographique
Ce chapitre constitue une revue bibliographique sur l’amélioration de la
biodisponibilité de médicaments peu solubles dans l’eau.
Un nombre croissant des nouveaux médicaments développés par l’industrie
pharmaceutique sont peu solubles dans les milieux aqueux. L’industrialisation de ces
médicaments devient donc un réel défi, du fait de leurs faibles vitesses de dissolution et, par
conséquent, de leurs difficultés à traverser les membranes corporelles afin d'être absorbés. Si
le processus de dissolution de la molécule est lent, en raison de ses propriétés physicochimiques ou des facteurs de formulation, la dissolution peut être le facteur limitant de
l'absorption et aura une influence directe sur la biodisponibilité du médicament.
Le terme médicament faiblement soluble dans l’eau fait généralement référence à un
médicament dont la solubilité aqueuse est inférieure à 1mg/ml. Des exemples de médicaments
couramment commercialisés qui sont peu solubles ou insolubles dans l'eau comprennent les
analgésiques, les cardiovasculaires, les hormones, les antiviraux, les immunosuppresseurs et
les antibiotiques (BERGESE, 2003). De plus un médicament ayant une meilleure solubilité
dans l'eau peut être administré à une plus faible dose réduisant ainsi leurs effets secondaires
systémiques. Ceci est crucial pour les médicaments ayant d'importants effets secondaires, tels
que les antibiotiques, les antifongiques ou les antirétroviraux.
Afin d'améliorer la solubilité dans l'eau et la vitesse de dissolution de ces médicaments
hydrophobes dans le tractus gastro-intestinal et/ou au site d'absorption, plusieurs techniques
peuvent être utilisées, telles que la formation de sels, la complexation, par exemple avec des
cyclodextrines, la vectorisation, des modifications chimiques ou encore des modifications
physiques comme, par exemple, la réduction de taille du solide. En effet, la vitesse de
dissolution est directement proportionnelle à la surface spécifique (loi de Noyes-Whitney). La
dissolution peut donc être effectivement augmentée en réduisant la taille des particules. Par
conséquent, de nombreux efforts ont été consacrés au développement d’opérations faciles,
économiques
et
efficaces
pour
la
fabrication
de
particules
plus
fines
Des micro ou nano-particules peuvent être produites par deux approches différentes.
La première consiste, à partir d’un matériau massif, à le fractionner afin de réduire sa taille,
c’est l’approche « top-down ». Par exemple, le broyage est une méthode « top-down »
traditionnelle de réduction de la taille de matières particulaires (MENG, 2011).
La seconde au contraire, consiste en l’assemblage d’atomes ou de molécules
(cristallisation), c’est l’approche « bottom-up ». La cristallisation par effet anti-solvant est
considérée comme une opération bottom-up. Cette dernière est une approche efficace pour la
précipitation de fines particules en solution, en présence d’un anti-solvant. L'introduction d’un
anti-solvant dans une solution contenant le principe actif, génère une sursaturation élevée qui
induit simultanément la nucléation, la croissance et l’agglomération des particules. Le grand
défi de l’utilisation de cette opération est le contrôle des propriétés du solide cristallisé tels
que la croissance des cristaux et leur agglomération.
Ces dernières années, de nombreuses méthodes de production de particules utilisant la
cristallisation par effet anti-solvant ont été développées. Elles permettent un meilleur contrôle
de la taille, de la cristallinité et de la morphologie des particules obtenues avec des méthodes
« bottom up ». Le procédé de cristallisation est applicable à une large gamme de composées
pharmaceutiques BCS de classes II et IV afin de produire des micro/nanoparticules. La
diminution de taille des particules augmente la surface spécifique, ce qui augmente la vitesse
de dissolution de médicaments peu solubles et, par conséquent, leur biodisponibilité
(LIVERSIDGE et LIVERSIDGE, 2008).
Deux paramètres opératoires influençant les propriétés du solide formé ont été évalués
dans ce chapitre : le mélange et l’utilisation d’additifs. Le premier est une étape essentielle
pour maintenir un niveau constant et homogène de sursaturation dans le cristallisoir, induisant
une nucléation uniforme et le contrôle de la formation de ces petits cristaux (DOUROUMIS
et FAHR, 2006). Les additifs utilisés durant une opération de cristallisation peuvent être
absorbés directement sur les particules formées permettant ainsi de produire des poudres avec
des propriétés physico-chimiques optimales (ZIMMERMANN et coll., 2009). Il a été montré
que la présence d'additifs dans la solution peut affecter les différents mécanismes comme la
nucléation et/ou la croissance et l’agglomération et donc les propriétés du solide, comme sa
solubilité, le faciès des cristaux le composant (KUBOTA et coll, 2000; GARNIER et coll,
2002; VETTER et coll., 2011), et la forme cristalline (CHONG et coll, 2002; SONG et
CÖLFEN, 2011). De plus, l'adsorption des additifs sur la surface des particules peut
provoquer une inhibition de la croissance et de l’agglomération, en occupant des sites
spécifiques et, par conséquence arrêter ou ralentir ces deux processus.
Literature Review
1.1. THE BIOAVAILABILITY OF DRUGS
1.1.1. Absorption and bioavailability
The term bioavailability is usually defined as the rate and extent of absorption of a drug
from its dosage form into the systemic circulation (BLANCHARD and SAWCHUK, 1979).
By definition, when a medication is administered intravenously, its bioavailability is
100%. However, when a medication is administered via other routes (such as orally), its
bioavailability (oral bioavailability) is usually less than 100%, caused by degradation or
metabolism of the drug prior to absorption, incomplete absorption and first-pass metabolism
not seen with intravenous administration (OSCIER et al., 2007).
The therapeutic effect of drugs depends on the drug concentration at the site of action.
The absorption of the drug into the systemic circulation is a prerequisite to reach the site of
action for all drugs, except those drugs that are applied at the site of action, or those that are
intravenously injected.
When a drug is taken orally administration (gastrointestinal route) it passes through the
mouth, esophagus, stomach, duodenum, jejunum (small intestine), colon (large intestine) (see
Figure 1.1) and finally leaves the body if not absorbed. Indeed, it must withstand the effect of
several physiological fluctuations like a large variation in pH along the gastrointestinal tract
(Table 1.1), the presence of bile salts, food, enzymes, bacteria and the motility of the gut.
Esophagus
Stomach
Duodenum
Small intestine Colon
Appendix
Colon
Rectum
Figure 1.1. Anatomy of digestive tract (Modified from: MARTINEZ et al., 2002).
27
CHAPTER 1
Table 1.1. Transit time and pH conditions along the GI tract (MARTINEZ et al., 2002).
GI segment
Transit time
pH
Stomach
2h
2.0 ± 1.9
Duodenum
10 min
6.6 ± 0.5
Jejunum
2h
7.4 ± 0.4
Ileum
1h
7.5 ± 0.4
Colon
36-72 h
7.0 ± 0.7
Absorption of drugs after oral administration of pharmaceutical dosage forms (drug
powder, tablet, capsule…) may occur at the various body sites between the mouth and rectum.
After oral administration, the pharmaceutical product reaches quickly the stomach passing
through esophagus. However, the small intestine has the largest surface for drug absorption in
GI tract (PODCZECK et al. 2007).
After disintegration of the pharmaceutical dosage form in the gastrointestinal tract, the
first requirement for absorption is that the drug dissolves. In fact, only drug that is dissolved
has the ability to permeate the intestinal membrane. The oral bioavailability of a particular
drug is thus determined by the magnitude of the solubility and/or permeability limitations that
exist for it within the GI tract, which is an aqueous environment. These two aspects,
illustrated in Figure 1.2, form the basis of the Biopharmaceutical Classification System
(BCS), which is incorporated in the guidelines of the Food and Drug Administration (FDA)
established by AMIDON et al. (1995) and often cited in the literature. According to the BCS,
four different types of drug absorption regimes are distinguished. They are explained in Table
1.2.
According to Biopharmaceutical Classification System (BCS), Class I drugs dissolve
rapidly in an aqueous environment and are rapidly transported over the absorbing membrane.
For Class II drugs, the dissolution rate in vivo is usually the rate limiting step in drug
absorption. Drugs of this group are poorly water soluble.
Class III drugs dissolve readily, but cannot penetrate biomembranes of GI tract.
28
Literature Review
Class IV drugs are characterized by poor solubility and poor permeability. Oral
administration is not recommended (LINDENBERG et al., 2004) and, subsequently, Class IV
drugs are often administered parentally using formulations containing solubility/permeability
enhancers.
Gastrointestinal tract
Dosage Form
Celular
Membrane
Systematic
Circulation
( Blood)
Drug
Solution
Dissolution
Limited
Permeability
Limited
Figure 1.2. Parameters limiting absorption of drugs taken orally (Adapted from
ENGMAN, 2003).
Table 1.2. Biopharmaceutical Classification System according to AMIDON (1995).
Solubility in aqueous
Class
environment
Permeability over
(intestinal) membrane
I
High
High
II
Low
High
III
High
Low
IV
Low
Low
29
CHAPTER 1
It is clear that, depending on the classification of the drug, different strategies can be
applied to increase or accelerate the absorption of a drug taken orally, either increasing the
amount of dissolved drug or increasing the permeability of the dissolved drug through the
absorbing membrane.
To sum up, Class I drugs do not need a formulation strategy to increase the absorption.
The rate of absorption of Class II drugs can be enhanced by accelerating the dissolution.
This has proven to be effective in many studies (YELLELA, 2010; PATEL et al., 2011;
VIÇOSA et al., 2012).
The strategy for Class III drugs is to increase the permeability of the absorbing
membrane. Numerous studies deal with increasing membrane permeability in the gastrointestinal tract (NEELAM et al., 2012; SHAIKH et al., 2012).
For a Class IV drug, both dissolution as well as permeability must be increased.
Over 90% of the marketed drugs qualify under Class II and Class IV (GRIFFIN. 2012).
The oral bioavailability of these drug compounds is limited due to slow drug dissolution in
the gastrointestinal tract. Therefore, it is desirable to improve the solubility of these drug
compounds by using various pharmaceutical technologies that will be discussed later in this
Chapter.
1.1.2. Poorly-water soluble drug molecules
Poorly-water soluble drugs describe a heterogeneous group of drug compounds that
exhibit poor solubility in water but are typically soluble in various organic solvents.
The degree of water solubility for drug compounds can be defined as slightly soluble (110mg/mL), very slightly soluble (0.1-1 mg/mL), and practically insoluble (<0.1mg/mL)
(MENG, 2011)
The expression poorly-water soluble drug generally refers to a drug whose aqueous
solubility falls into the range of slightly soluble and below. Examples of commonly marketed
drugs that are poorly soluble or insoluble in water include analgesics, cardiovasculars,
hormones, antivirals, immune suppressants and antibiotics (BERGESE, 2003). A drug with
improved water solubility can be administrated in a lower concentrated dose, with a reduction
30
Literature Review
of local and systemic side-effects. This is crucial for drugs with important side-effect profiles,
such as antibiotics, antifungals or antiretrovirals.
The drug we deal with in this thesis belong to the category of insoluble drugs; at the
best of our knowledge, it is not yet classified according to the BCS classification.
1.2. ENHANCEMENT
OF
SOLUBILIZATION
AND
BIOAVAILABILITY OF POORLY SOLUBLE DRUGS
1.2.1. Concept of dissolution
Drug must first be dissolved in the medium at the absorption site. The process that a
drug particle dissolves is called dissolution.
The dissolution of a solid in a solvent is a rather complex process determined by a
multiplicity of physicochemical properties of solute and solvent. However, a more intuitive
approach was interestingly proposed by Bergese (2003) to describe the dissolution of a solid,
regardless of the mechanism by which dissolution occurs, as a consecutive process driven by
energy changes (Figure 1.3).
Energy
ΔEDIFFUSION
ΔESOLVATION
ΔEFUSION
ΔEDISSOLUTION
ΔESURFACE INTERECTION
Reaction Coordinate
Figure 1.3. Energy diagram of the dissolution of a solid phase. Adapted from
BERGESE, 2003.
31
CHAPTER 1
The first step consists in the contact of the solvent with the solid surface (wetting),
which leads to the production of a solid-liquid interface. Following the description given by
Bergese (2003), the break of the molecular bonds of the solid and the molecules passage to
the solid-liquid interface (solvation) are the second and third step.
The final step sees the transfer of the solvated molecules from the interfacial region into
the bulk solution (diffusion). Each stage requires a certain amount of energy to be completed;
the target of drug activation is to lower the overall dissolution energy.
Solvation and diffusion depend on solid and solvent chemical nature and on the system
conditions (temperature, mechanical agitation…), while wetting and fusion also depend on
microstructure of the solid. In the case of a given poorly water-soluble drug that dissolves into
the gastrointestinal tract it is not possible to modify neither the drug molecules nor the
dissolution environment (solvent and system conditions). So the efforts in enhancing drug
dissolution rate are essentially spent (in trying) to tailor the drug microstructure.
To outline the properties of solids that determine their dissolution rate we start from the
simplest model for the dissolution of a solid in a solvent, i.e. the Nernst’s film theory (1904),
considering a single particle dissolving into a large volume of solvent, under agitation (Figure
1.4).
Particle
Surface
Stagnant film
boundary
Ceq
Stagnant Film
Bulk Solution
Concentration
Particle
C
h
Distance from particle surface
Figure 1.4. Dissolution of a single particle in a large volume of solvent (Model of
Nernst). Adapted from BERGESE, 2003.
32
Literature Review
The concentration of the pure molecule at the solid surface, is assumed to be equal to
the saturation concentration, Ceq; the concentration in the bulk solution will be denoted with
C. Despite of agitation, it can be assumed that the solid is surrounded by a stagnant film of
liquid of constant thickness. Solid molecules spontaneously diffuse through this film and
reach the bulk solution. If we assume steady state conditions, diffusion is described by the
Fick’s first law:
(1.1)
where Ji is the diffusion current (defined as the amount of material i passing per unit time
perpendicularly through a unit surface area), Ci the molar concentration of molecule i, Di the
diffusion coefficient of molecule i, and ∂Ci/∂s the concentration gradient through the film.
In this particular case, ∂Ci/∂s is constant, since in the stagnant film laminar condition
holds. To sum up, there is a linear concentration gradient from the solid surface to bulk
solution that can be analytically expressed as ∂Ci/∂s = (Ci,eq –Ci)/h. Using this expression, and
denoting the volume of the solvent V and the surface area of the dissolving solid A, Equation
(1.2) becomes:
(1.2)
The more familiar form of the dissolution equation (also called Noyes-Whitney
equation), which describes the increase of the mass of solute dmi in the time dt due to
dissolution, is obtained rearranging Equation (1.2):
(1.3)
where Mi is the molar weight of solid.
33
CHAPTER 1
To eliminate the saturation effect of the solvent and increase the ability of the solid to
dissolve the expected amount of drug, dissolution methods on “sink condition”, have been
proposed
in
the
literature
(GIBALDI
and
FELDMAN,
1967;
ROHRS,
2001,
GOWTHAMARAJAN and SINGH, 2010). In practice, if the quantity of the medium
dissolution is sufficiently large, between 3 and 10 times, of the volume needed to completely
solubilize the drug, it is said that we are on sink-condition (ABDOU, 1989; ROHRS, 2001;
GOWTHAMARAJAN and SINGH, 2010). When sink condition holds (<< , Equation
simplifies to:
(1.4)
1.2.2. Dissolution testing for poorly-water soluble drugs
For a dosage form to produce its effect after oral administration, drug must be released
and generally should be dissolved in the fluids of the gastrointestinal tract. Drug dissolution
testing plays an important role as a routine quality control test and to characterize the quality
of the product (FDA, 1997).
Dissolution from the dosage form involves mainly two steps: liberation of the drug from
the formulation matrix (disintegration) followed by the dissolution of the drug (solubilization
of the drug particles) in the liquid medium. The overall rate of dissolution depends on the
slower of these two steps. In the first step of dissolution, the cohesive properties of the
formulated drug play a key role.
For solid dosage forms, these properties include disintegration and erosion. If the first
step of dissolution is rate-limiting, then the rate of dissolution is considered disintegration
controlled. In the second step of dissolution (i.e., solubilization of drug particles), the
physicochemical properties of the drug such as its chemical form (e.g., salt, free acid, free
base) and physical form (e.g., amorphous or polymorph and primary particle size) play an
important role. If this latter step is rate-limiting, then the rate of dissolution is dissolution
controlled. This is the case for most poorly soluble compounds in immediate-release
34
Literature Review
formulations whose solubility is less than 1–2 mg/L in the pH range of 2–8
(GOWTHAMARAJAN and SINGH, 2010).
For poorly soluble compounds, dissolution study is particularly important. Different
techniques have been used to improve the maximum dissolvable dose in the dissolution
medium in discriminatory dissolution studies, such as addition of organic solvents to aqueous
medium (DANIEL et al., 2012), increase of the dissolution medium volume, pH changes
(TALARI et al., 2009), and addition of surfactants (JINNO et al., 2000; ROHRS, 2001).
Some dissolution parameters involved in the phase transformation can affect dissolution
method efficiency. The dissolution of most solids is an endothermic phenomenon, thus
increases in temperature tend to increase the speed, which the substance dissolves. Because of
this, the United States Pharmacopeia recommends 37±0.5°C media temperature for
dissolution tests.
The dissolution kinetics depends on the local mass transfer coefficient (which in turn is
a function of suspension state and the local turbulence level). Because of this some authors
have established a relationship between the intensity of agitation and dissolution rate constant
(cm.sec-1). Generally, higher stirring rates result in higher dissolution rates and ideally stirring
conditions must somehow simulate peristalsis physiological. The diffusion rate can decrease
too, with increasing media viscosity, which implies a decrease of the dissolution rate. Another
parameter that can influence the dissolution rate of a solid in a liquid is the solid wettability.
For complete drug dissolution it is necessary that wetting is complete. The ability of wetting
of a solid by a liquid is expressed by the contact angle.
Countless are the variables that can modify the results of dissolution tests. All should be
considered, but some must be strictly monitored to obtain reliable results. Finally,
construction of in vitro-in vivo correlation provides the most valuable data for selection of the
most appropriate dissolution method and testing conditions that can be prognostic of in-vivo
dissolution.
Micro/nanonization during crystallization (RASENACK and MÜLLER, 2002;
RASENACK and MÜLLER, 2004; BADAWI et al., 2011; VIÇOSA et al., 2012), surface
modification (HAN et al., 2011) and crystal structure modification (EERDENBRUGH et al.,
2009) may improve the dissolution rate of poorly water-soluble APIs.
35
CHAPTER 1
Several studies have shown that modification of the crystalline form of the drug , by the
inclusion of specific additives increases the rate of dissolution of the pharmaceutical form,
which can strongly affect the physicochemical properties and kinetic of absorption of the drug
from the final product (SHEKUNOV and YORK, 2000).
It is known that a substance in crystalline form has a lower solubility, while at the same
amorphous form has higher solubility and low thermal stability. Formulations where the drug
is mainly crystalline and amorphous or metastable forms, are much more soluble than the
drug-containing highly crystalline. Drug amorphization can be used to promote a quicker
therapeutic effect by enhancement of drug absorption rate and dissolution rate. However,
formulations containing metastable amorphous forms are less stable than those prepared with
the crystalline forms of the drug. Because there is generally a risk of crystallization during the
production process and shelf life of the product. Such products are generally very reactive and
less stable when exposed to heat and mechanical stress, and very susceptible to moisture
absorption (SINGHAL and CURATOLO, 2004)
The dissolution rate can be enhanced by size reduction that can be explained by the
Noyes-Whitney equation (Equation. 1.3). This is possible due to the special features of drug
size reduction:
1. Increased surface area A;
2. Increased saturation concentration and dissolution pressure ( Ceq; pX );
3. Increased adhesiveness to surfaces/cell membranes (see Figure 1.5).
The increased area increases the dissolution rate. Moreover the transfer of particles from
the macrosize range to the nanodimension changes their physico-chemical properties. This
strategy also modifies the saturation concentration of the drug in solution (Ci,eq), which means
an additional increase with increased speed dissolution of particles in this size range. In
addition, the size reduction can increase the adhesiveness into the skin due to the increase in
the saturation, leading to a larger concentration gradient and the larger gradient promotes
penetration (MÜLLER et al., 2011).
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Literature Review
Factor of surface area (A) increase
1. Increase in surface area (A)
6000
5500
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
Nanonization
Micronization
50 nm
100 nm
500 nm
10 µm 100 µm
0
0,01
0,1
10
1
log particle size (µm)
2. Saturation solubility (Ceq)
= f (size)
=f (curvature)
=f (dissolution pressure-px)
10 µm
100 µm
0.5 µm
Nanonization
Micronization
px
px
px
3. Adhesiveness
=f (size)
=f (contact area)
1 microcrystal
100 µm
18 Nanocrystals
200 nm
Increased contact area
Figure 1.5. Features of size reduction: 1. Increased dissolution velocity due to increased
surface area ; 2. Increased saturation solubility due to increased dissolution pressure of
strongly curved small nanocrystals (upper); 3. Increased adhesiveness of reduced material due
to increased contact area of small versus large particles. Modified from MÜLLER et al., 2011.
37
CHAPTER 1
1.2.3. Strategies to increase the amount of dissolved drug at the absorption site
To enhance the aqueous solubility and the dissolution rate of hydrophobic drugs at the
absorption site several strategies can be used. These methods (alteration of solvent
composition, complexation, carrier system, chemical modification and physical modification)
are shown in Figure 1.6. The following section is a brief review on the state of the art of this
topic.
Poorly Water Soluble
Chemical Entities (BCS
Class II and IV)
Alteration of
pH/Solvent
Composition
Complexation
Carrier
System
Cyclodextrin
Salt formation/
pHcontrol
Co-solvency
Chemical
Modification
Manipulation of
solid state
Physical
Modification
Size Reduction
Micelles
Microemulsion
Co-crystals
Solid Dispersions
High Pressure
Homogenization
Milling
LAS*crystallization
Figure 1.6. Strategies to increase the amount of dissolved drug at the absorption site
(Modified from LAKSHMI et al., 2012).
1.2.3.1. Alteration of pH and solvent composition
1.2.3.1.1. Salt formation/pH control
It is well documented that the bioavailability of pharmaceuticals products is influenced
by the changes in pH within the GI tract. The absorption of drug is largely dependent on
38
Literature Review
diffusion, which varies with pH of the individual regions within the GI tract, pKa of the drug,
and permeability, that depends of regional pH effects upon drug ionization.
Poorly water soluble drugs can be protonated (base) or deprotonated (acid) and may
potentially be dissolved in water by applying a pH change. The presence of salts can act as
alkalising or acidifying agents, and may increase the solubility of weakly basic or acid drugs.
After pH adjustment, ionisable compounds are stable and soluble at selected pH.
Salts of acidic and basic drug have, in general higher solubility values than their
corresponding acid or basic forms (GRAHAM et al., 1986).
Generally a salt of a drug is frequently less stable chemically compared to crystalline
solid. At pH extremes drug is dissolved, which can increase the chance of hydrolysis, catalyze
or other degradation mechanisms (VENKATESH et al., 1996). Another disadvantage is the
risk for precipitation upon dilution with aqueous media having a pH at which the compound is
less soluble. Intravenously this may lead to emboli, orally it may cause variability and toxicity
(local and systemic) related to the use of non physiological and extreme pH (GIACONA et
al., 1982).
1.2.3.1.2. Cosolvency
Co-solvent system works by reducing the interfacial tension between the aqueous
solutions and hydrophobic solute by solvent blending or cosolvency. This method is based on
the mixing of solvents of different polarities to form a solvent system of optimum polarity to
dissolve the solute. Co-solvent formulations of poorly soluble drugs can be administered
orally and parentally (THELLY et al., 2000; BOYLAN, 1987). This type of formulation can
increase the solubility of poorly soluble compounds several thousand times compared to the
aqueous solubility of the drug alone. However, these modifications can cause serious side
effects due to solvents use.
Yeh (2009) realized a formulation study of tenoxicam, a poorly water-soluble drug, by
use of a ternary cosolvent system, DMSO/polyethoxylated castor oil/ethanol system, that had
significantly enhanced the solubility. Additionally, the relative bioavailability was improved.
This study provided a novel strategy for improving tenoxicam solubility, but also helps
further scientific knowledge for the development of parenteral formulations.
39
CHAPTER 1
Seedher and Bhatia (2003) investigated that the aqueous solubility of celecoxib,
rofecoxib and nimesulide could be enhanced significantly by using ethanol as the second
solvent and PEG-400-ethanol had highest solubilization potentiality among the mixed solvent
systems.
Large quantities of organic solvent used in cosolvent formulations may result in the loss
of solvent capacity of the formulation upon dilution in aqueous media in vivo. For drugs Class
II and IV, administered orally, the use of cosolvents technique may not increase the
bioavailability dramatically because the poorly soluble drug can typically uncontrollably
precipitate into a crystalline or amorphous precipitate. In this case, dissolution of this
precipitate is required for oral absorption. A major concern for the use of this technique is the
biocompatibility, tolerability and toxicity of used solvents formulations and the loss of solvent
capacity in aqueous media in vivo.
1.2.3.2. Inclusion complexes/complexation
1.2.3.2.1. Cyclodextrin (CD)
Another approach used to improve drug solubilization is based on the formation of
inclusion complexes between molecular assemblies and drug molecules, like cyclodextrins
(CD). CDs are cyclic oligosaccharides composed of glucopyranose units and adopt a
truncated cone structure with hydrophobic cavity (SAENGER, 1980).
Cyclodextrins and their derivatives have been employed as complexing agents to
increase water solubility, dissolution rate and bioavailability of lipophilic drugs for parenteral
or oral delivery (SRIDEVI et al., 2003; MORIWAKI et al., 2008).
An inclusion complex is produced by inclusion of a non polar molecule or the nonpolar
region of a molecule (known as the guest) into the nonpolar cavity of another molecule or
group of molecules (known as the host), as shown in Figure 1.7. When the guest molecule
enters the host molecule, they are temporarily locked or caged within the host cavity. Giving
rise to beneficial modifications of guest molecules, as solubility improvement for example.
Furthermore these complexes have a broad range of utilizations in different applications; they
are used in the food and cosmetics industries and the pharmaceutical sector (DEL VALLE,
2004).
40
Literature Review
Hydrophobic cavity
Drug-CD complex
Hydrophilic exterior
Figure 1.7. Schematic illustration of the association of free cyclodextrin (CD) and drug
to form drug–CD complexes and a truncated cone structure of CD (Modified from: DEL
VALLE, 2004).
The three cyclodextrins α-, β-, and γ- (CD) are composed of six, seven, and eight
glucopyranose units, and are produced in industrial scales (MORIN-CRINI and CRINI,
2012). These agents have a torus structure with primary and secondary hydroxyl groups
orientated outwards, as shown in Figure 1.8.
Figure 1.8. Schematic representations of Cds (a) α- CD, (b) β-CD, (c) γ-CD.
Containing 6,7 and 8 glucopyranoside units, respectively (MORIN-CRINI and CRINI, 2012).
For Class IV drugs, the cyclodextrin complexation may not improve their oral
bioavailability. However, cyclodextrins are able to improve aqueous solubility of some large
lipophilic molecules leading to increase drug availability at the mucosal surface. This will
frequently lead to increased oral bioavailability (LOFTSSON et al., 2005).
Even though CD seems to be ideal carriers, they possess some limitations, related to
toxicological considerations, formulation and production cost. In general, the complexation
41
CHAPTER 1
efficiency of cyclodextrins is low and thus relatively large amounts of cyclodextrins are
needed to complex small amounts of drug (LOFTSSON and O’FEE, 2003).
1.2.3.3. Carrier System
1.2.3.3.1. Micelles
Micelle solution is another formulation strategy to increase drug solubility. According
to IUPAC (Compendium of Chemical Terminology) (1972) micelle solubilization can be
defined as colloidal association in a solvent system for solubilization of a component into or
on micelles.
Of the above mentioned, for colloidal association, the use of surfactants into the media
is the most popular method for micelle formation. Various synthetic surfactants can simulate
the surfactants present in the gastrointestinal fluid, e.g., bile salts, lecithin, cholesterol and its
esters (YALKOWSKY, 1999).
Surfactants are molecules with distinct polar and non polar regions. In water, as the
concentration of surfactant increases above a critical value, its molecules self-associate into
soluble structures called micelles. The concentration at which they begin to form is called the
critical micelle concentration (CMC) (FLORENCE and ATTWOOD, 1981 ; MYRDAL and
YALKOWSKY, 2002 ). These micelles are normally spherical with the nonpolar regions of
surfactant molecules gathered in the center (core) and surrounded by a shell of the polar
region which are in contact with the water.
Micelle is a nanoparticle structured by one hydrophilic shell and one hydrophobic core
(Figure 1.9), which are capable of encapsulating drug molecules, resulting in reduction in the
interfacial tension and improved solubility of the drug in the medium (SUBHASHI et al.,
2009).
The stability of drug carrier micelles is the biggest challenge for the use of this
technique. When in aqueous media, in vivo micelles become much diluted by blood (below
CMC) and may be gradually disintegrated into unimers. In addition, the low drug loading
efficiency and the difficulty in transporting through cell membranes, has also retarded the
development of effective micellar drug (KIM et al., 2010).
42
Literature Review
Figure 1.9. Schematic representation of a hydrophobically assembled polymer micelle.
The hydrophobic core loading lipophilic drugs is protected from the environment by the
hydrophilic shell (KIM et al., 2010).
1.2.3.3.2. Micro/Nanoemulsion
Microemulsion consists of dispersions of two immiscible or partially miscible liquids
(water in oil or oil in water) that present droplets ranging from 5-100 nm. They are
thermodynamically stable and do not require power supply for their formation (ROSSI et al.,
2007). The main difference between microemulsions and nanoemulsions is that
microemulsions are self-assembling nano-scale emulsions, whereas nanoemulsions are nanoscale emulsions formed under intense mechanical shear (WHITESIDE and GRZYBOWSKI,
2002).
All types of emulsions should be prepared with a certain amount of surfactant.
Surfactants can promote the formation of emulsion as they reduce the interfacial tension
between oil and water by attaching to the liquid-liquid interface (SHINODA, 1967). The main
difference between surfactant micelles and emulsion is the liquid phase. Typically, micelles
are formed by adding surfactant to a single liquid phase, either oil (reversed micelles) or
water, whereas emulsions are prepared by adding surfactant to a double liquid phase (oil and
water)
Microemulsions are suitable carriers for poorly water soluble drugs, because they can
be dispersed easily in gastrointestinal juice in microemulsion form. Furthermore,
microemulsions can enhance the drug absorption due to their small particle size. Also the
drugs can be stored longer because of the stability of microemulsions (MALMSTEN, 1999).
43
CHAPTER 1
The disadvantage of microemulsions as drug carriers is that the toxicity of the drugs tends to
increase due to a large amount of the surfactant utilized in microemulsion formulation.
1.2.3.4. Manipulation of solid state
1.2.3.4.1. Co-crystals
Co-crystals are solids that are crystalline materials composed of two or more molecules
in the same crystal lattice (FDA, 2011). The formation of pharmaceutical co-crystals has
gained attention as attractive alternate solid forms for drug development. Their formation
involves incorporation of a given API with one or more pharmaceutically acceptable molecule
(Coformers) in the crystal lattice (Figure 1.10), that is a solid under ambient conditions. The
co-crystals do not affect pharmacological activity of API but can improve physical properties,
such as solubility, hygroscopicity, compaction behavior (RODRIGUEZ-HORNEDO et al.,
2007 ; ZAWOROTKO, 2008). Of these properties, solubility is the most widely appreciated
in the literature.
API
Coformers
Figure 1.10. Possible solid forms of a drug cocrystal . Modified from ALHALAWEH,
2012).
Poor aqueous solubility can compromise drug performance, and co-crystals are an
emerging strategy to design materials with desirable properties. Co-crystal solution phase
behavior was first investigated by Higuchi and Connors (HIGUCHI and CONNORS, 1965).
Co-crystals are usually prepared by evaporation from a solution containing
stoichiometric amounts of components (co-crystal formers). However sublimation, blending
of powders, sonication, growth from melt, slurries and grinding of the components together
are suitable methodologies (KRISHNAIAH, 2010).
The major disadvantage of co-crystals technique is the notoriously difficult situation of
these systems related to their preparation— it has been known to take 6 months to prepare a
single co-crystal of suitable quality for single X-ray diffraction analysis (PORTALONE and
44
Literature Review
COLAPIETRO, 2004). In addition, for solution co-crystallization, the two components must
have similar solubility; otherwise the least soluble component will precipitate out exclusively.
On the other hand, similar solubility of two components alone will not promise success. It has
been recommended that it possibly useful to consider polymorphic compounds which exist in
more than one crystalline form as co-crystallising components (BLAGDEN, 2007).
1.2.3.4.2. Amorphous Solid dispersion
In amorphous solid dispersion, drug molecules are dispersed molecularly but irregularly
within amorphous excipient (biologically inert matrix) (Figure 1.11). Chiou and Riegelman
(1971) defined the term solid dispersion as ‘‘a dispersion of one or more active ingredient in
an inert carrier or matrix at solid state prepared by the melting (fusion), solvent or meltingsolvent method’’. This method, involves the formation of eutectic mixtures of drugs with
water-soluble carriers by the melting of their physical mixtures.
Figure 1.11. Amorphous solid dispersion (From GHASTE et al., 2009).
There are various reasons for the improvement of solubility of poorly water-soluble
drug using solid dispersion technology. The reasons for solid dispersion or advantages of
solid dispersions are as follows: particle size reduction, improved wettability, higher degree
of porosity, drugs in amorphous state (KUMAR et al., 2011).
The major disadvantages of solid dispersions are related to their instability. During
processing (mechanical stress) or storage (temperature and humidity stress) the amorphous
state may undergo crystallization and dissolution rate decrease with ageing. The effect of
moisture on the storage stability of amorphous pharmaceuticals is also a significant concern.
By absorbing moisture, phase separation, crystal growth or a change from metastable
crystalline form to stable form can take place which leads to the reduction of drug solubility,
dissolution rate, and consequently a bad in vivo drug performance (WANG et al., 2005).
45
CHAPTER 1
1.2.3.5. Physical Modification
It has been proven, as expressed by Noyes-Whitney (equation 1.3, section 1.2.1), that
the dissolution rate is directly proportional to the specific surface area, which can be
effectively increased by reducing the particle size. Therefore, considerable efforts have gone
into developing reliable and efficient methods for the manufacture of fine particles.
Micro- or nano-particles can be produces by two different technology approaches as
illustrated in Figure 1.12, called “top-down” and “bottom-up” technologies. Top-down
technologies start with coarse materials and apply forces to break down into micro or nanoparticles, while bottom-up technologies start with the molecules in solution and the molecules
are aggregated to form the solid particles (RABINOW, 2004).
Top Down
Bulk
Powder
MICRO/NANOPARTICLES
Clusters
Atoms
Bottom Up
Figure 1.12 Schematic representation of ‘bottom up’ and top down’ technology
approaches for ultra-fine particles production (micro- or nanometric scale of particle size).
Modified from FLORENCE and KONSTANTIN, 2010.
46
Literature Review
1.2.3.5.1.Milling
Milling and homogenization are the traditional top-down methods for size reduction of
large quantities of particulate materials (MENG, 2011). In the milling operation, the applied
stress is applied on the material, which causes the breakage of the particle.
Drug particles can be broken between moving pearls by shear forces and forces of
impaction generated by a movement of the milling media (MÜLLER and AKKAR, 2004), as
schematized in Figure 1.13.
Moving Pearls
Drug particle
ticle
Figure 1.13. Particle size reduction by milling of drug particles between moving pearls
schema.
This process can reduce drug particle size, depending on the drug hardness and drug
quantities to be milled. Milling period and speed had critical impact on particle size
distribution (CHE et al., 2012). However, this method has limited opportunity to control the
final particle size, shape, morphology, surface properties and electrostatic charge and it is
difficult to reduce the particle size below 1 μm because of the cohesiveness of the particle
(BHAKAY et al., 2011).
There are different milling materials available, traditionally steel, glass, and zircon
oxide and more recently, special polymers (hard polystyrene) are used. A problem associated
with the pearl milling technology is the erosion from the milling material during the milling
process. In general, very few data have been published on contamination of pharmaceutical
drug suspensions by erosion from the milling material. Of course it should be noted that the
extent of erosion depends on the solid concentration of the macrosuspension to be processed,
47
CHAPTER 1
the hardness of the drug, and based on this, the required milling time and milling material.
Apart from the milling material, the erosion from the container also needs to be considered.
Moreover, disadvantages that have also been reported are potential growth of germs in
the water phase when milling for a long time, and time and costs associated with the
separation procedure of the milling material from the drug suspensions (CHE et al., 2012).
High-pressure homogenization (HPH) has been utilized for many years for production
of emulsion and suspensions (TIPPETTS and MARTINI, 2009; LOVELYN and ATTAMA,
2011 ; LACERDA et al., 2011). Piston-gap principle and jet-stream technology are the two
basic technologies for most homogenizers, illustrated in Figure 1.14. For instance, in the
piston-gap homogenizer, particles to be milled suspended in a liquid medium coming from
the sample container are forced to pass through a tiny gap (e.g., 10 mm), and the particle
diminution is affected by shear force, cavitation and impaction. In jet-stream homogenizers,
the collision of two high-velocity streams leads to particle diminution mainly by impact
forces.
Particle suspension with reduced particle granulometry
Dissipation volume
Original dispersion
Figure 1.14. Basic homogenization principles: piston-gap (left) and jet-stream
arrangement (right). Adapted from SIVASANKAR and KUMAR, 2010.
High pressure-homogenization (HPH) has been used to generate nanosuspensions of
many poorly water soluble drugs such as azithromycin, quercetin and nitrendipine (ZHANG
et al., 2007; KAKRAN etl., 2012; QUAN et al., 2012).
48
Literature Review
Generally, high-energy input resulting in enormous impact forces produced effective
size reduction of a suspension (median diameters less than 500 nm have been reported by
Muller and co-workers (MÜLLER et al., 2001; MÜLLER et al., 2004). However, only fragile
drug candidates can be broken up into nanoparticulates using this technique (RADZUAN,
2010).
In general, the particle size decreases with an increasing number of cycles and
increasing homogenization pressure and they are mainly influenced by the hardness of the
drug, the finesse of the starting material (GRAU et al., 2000; SALAZAR et al., 2011). The
optimum number of homogenization cycles is determined by considering the particulate size
and polydispersity index of the drug after each cycle.
During milling, additives such as surfactants and stabilizers have been used for the
physical stability of the produced drug with reduced particle size (BHAKAY et al., 2011).
1.2.3.5.2. LAS Crystallization
Particle size reduction technologies such as milling or high-pressure homogenization
have been used over the years. However, controlling of size distribution, morphology, and
surface properties can be challenging. As many hydrophobic drugs are soluble in various
water miscible organic solvents, an effective approach is the precipitation of fine particles
from solution phase while mixing with an anti-solvent.
LAS crystallization is considered as a bottom-up process, which means that one starts
from the molecular level, and goes via molecular association to the formation of a solid
particle (GASSMANN et al., 1994; RASENACK and MÜLLER, 2003).
The basic advantage of anti-solvent technique is the use of simple and low cost
equipment, compared with milling and high-pressure homogenization technique. Typically,
the drug is first dissolved in an organic solvent and the drug solution is mixed with an antisolvent (Figure 1.15). The solvent and the anti-solvent must be miscible at the operating
conditions. One of the advantages of the method is to avoid the use of high energy like in
disintegration technique as used for milling, which prevents denaturation of drug due to high
energy input (ZHONG et al., 2005).
49
CHAPTER 1
Drug+ solvent
Anti- solvent
Crystallization
Solid-liquid separation
(filtration,drying)
Micro-particles
Figure 1.15. Approach for drug particle formation by LAS crystallization.
The introduction of the drug solution to the anti-solvent generates high supersaturation
that subsequently induces nucleation and simultaneous growth by condensation and
coagulation. The whole process is schematized in Figure 1.16. One formidable challenge
remains to control the properties of the crystallized solid such as crystal growth and
agglomeration to ensure good further dispersion for dissolution.
+
Nucleus
Anti-solvent
i-solvvent
Nucleation
Supersaturation
on
Homogeneous
Solution
Coagulation
Condensation
Precipitation
Particle agglomeration
Figure 1.16. Schematic representation of the different steps involved in drug particle
formation by an LAS crystallization process. Adapted from MENG, 2011.
50
Literature Review
The next section reviews in more details some concepts in the crystal engineering field,
with emphasis on the driving force for crystallization, control of the two key steps (nucleation
and growth) and of solid properties.
1.3. CRYSTALLIZATION
Crystallization is a key process in the manufacturing of most pharmaceutical
compounds. Over 90% of all pharmaceutical products, such as tablets, aerosols, capsules,
suspensions and suppositories contain drug in particulate, generally crystalline, form
(VALDER and MERRIFIELD, 1996). The most common type of crystallization is
crystallization from solution, in which a material that is a crystalline solid at a temperature of
interest is dissolved in a solvent while crystallization is induced by changing the state of the
system in some way that reduces the solubility of the solute. That results in the formation of a
crystalline or amorphous solid (MYERSON, 1999).
Crystallization process is considered a two-stage process. Nucleation is the first step in
which the “birth” of nuclei (a new solid phase) from the supersaturated solution occurs. The
stable crystal grows in size, going from dissolved drug in solution to solid in suspension.
The crystallization process is governed mainly by the kinetics of nucleation and crystal
growth, and these processes depend on the driving force called supersaturation (i.e. the
concentration of the solute exceeds its equilibrium concentration). This can be achieved in
several ways − for example by cooling a solution, or by solvent evaporation, or by the
addition of an anti-solvent, or by changing the solution pH.
1.3.1. Thermodynamic background
For crystallization to occur, the system must be brought into a non-equilibrium state
where the concentration of the solute exceeds its equilibrium concentration (i.e. the solution is
supersaturated). This phenomenon is better represented in the phase diagram (Figure 1.17),
represented by a curve of the equilibrium concentration of solute as a function of temperature
(solid line). At concentrations below solid line, the solid will dissolve until equilibrium is
reached. At solid concentrations between the dashed and solid lines, crystals will grow by
seeding but fresh crystals will not nucleate. This is the "metastable zone", which defines the
compositions at which spontaneous crystallization occurs and the region bounded by the
51
CHAPTER 1
solubility curve and the metastable limit. Above the dashed line, the solution produces
crystals spontaneous; in this region the supersaturation is achieved.
Metastable zone widthh
UNSTABLE
Solubility
Solubili
ity curve
Supersaturartion
Solute Concentration
Heterogeneous area (solid+liquid)
Metastable
Zone
Undersaturation
Homogeneous area (one liquidd phase)
STABLE
Temperature
Figure 1.17. The solubility / supersolubility phase diagram. Modified from DAVEY
and GARSIDE, 2000.
1.3.2. Nucleation
The nucleation is the stage of solid formation from a supersaturated mother phase
(DAVEY and GARSIDE, 2000). Supersaturation generated leads to nucleation and
precipitation. According to equation 1.5, nucleation rate J depends to the supersaturation
ratio. It is defined as:
(1.5)
52
Literature Review
where is the supersaturation ratio, T the temperature, C the actual concentration of
API in the solution (mol/L) and Ceq the solubility (mol/L) of API in a mixture of organic
solvent and water. The ratio of activity coefficient
is assumed to tend towards 1.
Unfortunately, the mechanisms of nucleation are very poorly understood which leads to
significant problems in the design, operation and control of crystallization processes.
Nucleation is therefore an important issue. Crystal nucleation denotes the formation and
physical characteristics (i.e. size distribution, habit morphology) of new crystalline solid. It
involves the aggregation of dissolved molecules in solution, leading to the formation of a
nucleus.
In general, nucleation mechanisms can be divided into three main categories:
homogeneous, heterogeneous, and secondary nucleation (Figure 1.18).
If a solution contains neither solid foreign particles nor crystals of its own type, nucleus
can be formed only by homogeneous nucleation. This type of nucleation rarely occurs in
volumes larger than 100 μl, since “real” solutions tend to contain random impurities which
may induce nucleation (PEREPEZKO, 1997). If foreign particles are present, nucleation is
facilitated and the process is know by heterogeneous nucleation.
On the other hand, secondary nucleation can only happen when crystals of the solute are
already present or are deliberately added to the solution, as seeds. This nucleation mechanism
generally occurs at much lower supersaturations than homogeneous or heterogeneous
nucleation, and it is caused mostly by collisions of crystals with crystallizer and mixer.
Nucleation processes are of practical importance in production of pharmaceutical
compounds, for example: A great interest in pharmaceutical research are the concepts of
heterogeneous nucleation to be applied in the directed nucleation of specific polymorphs
(WEISSBUCH et al., 1987). These studies provide us with the attractive possibility that “a
library” of organic seeds can be used to control polymorphism, or to search for unknown
polymorphs (WARD, 1997).
53
CHAPTER 1
Homogeneous
Heterogeneous
Contact
Shear
Fracture
Attritition
Needle
Figure 1.18. Various kinds of nucleation. Modified from MARSMANN et al., 2001.
1.3.3. Crystal growth
Once formed, nucleus begins to grow larger through the addition of solute molecules to
the crystal lattice, and this stage of crystallization process is known as crystal growth. The
nucleation and growth processes compete for solute molecules in function of their dependence
on supersaturation, and their relative rates will determine the crystal size distribution
(RODRIGUEZ-HORNEDO and MURPHY, 1999). The sites to capture arriving growth units
can be differentiated in kink, ledge and flat faces (Figure 1.19). Growth process occurs by
diffusion of solute molecules (atoms, molecules, ions) from the bulk solution to the interface
by diffusion and convection and then integration of the solute molecules into the crystal
lattice, under supersaturation conditions. The first step is the diffusion of growth units from
the growth medium to an impingement site (step I) of the crystal to adsorb on the surface. In
most cases (except rough growth), the growth can migrate to the surface of crystal to a step or
a growth site (step II). Finally, either the growth unit back in the solution (phase II*), either it
becomes part of the growth site and incorporate into the crystal lattice. Desolvation of the
growth unit may take place anywhere in step II or the solvent may be adsorbed with the
growth unit. The relative importance of each step depends on the surface structure of the
crystals and the properties of the solution (MEENAN et al., 2002).
54
Literature Review
Kink
Step
Ledge
II*
I
II
Growth unit
Figure 1.19. A three-dimensional crystal surface showing three type of growth sites and
different steps ( I, II and II*) involved in the process of growth to unit cubic. Modified from
MERSMANN, 2001.
In pharmaceutical field, particles of uniform size in a product are desirable for the
convenience of selection of filter, washing, uniform time of dissolution and good appearance
of product. Besides this, caking tendency of crystals during pharmaceutical form storage
period can be prevented since number of points of contact between crystalline particles is
significantly less in uniform crystals size.
1.3.4. Effect of supersaturation on nucleation and growth
Supersaturation (see section 1.3.2) is defined as a measure of the deviation of a
dissolved molecule from its equilibrium value (MYERSON, 1993).
Supersaturation can have a profound effect on crystal growth, resulting in
morphological changes in the crystallizing compound over a range of supersaturations
(YOSHIZAKI et al., 2001; RISTIC, 2002). The variation of the ratio C/Ceq (section 1.2.3)
enables control of particle size. In general, the number of crystals produced increases with the
supersaturation, while the size of crystals decreases (GARNIER and COQUEREL, 2002). For
a very high supersaturated solution, crystals nuclei are formed rapidly and may produce a vast
quantity of small, elongated crystals over a very short timescale. In the extreme case, when
the material is completely and fastly precipitated (larger number of nuclei produced per unit
of time) in the form of primary particles, lacking time sufficient for crystal growth, so that
55
CHAPTER 1
smaller crystals are obtained. If the degree of supersaturation is low, relatively few nuclei are
formed and the growth rate is low due to the dissolution of the material. In these conditions, a
few crystals are formed, but perfect. In this case is likely to produce larger, granular-shaped
crystals over a longer period (HALEBLIAN, 1975). The size and habit of many crystalline
products are particularly sensitive to degree of supersaturation (RISTIC et al., 2001).
1.3.5. Choice of solvent
The manufacture of pharmaceuticals often involves crystallization from organic
solvents or mixtures of solvents. Generally, solvents are selected based on the resulting
solubility, the mode of crystallization and the type of crystals, but the role of solvents in
different parts of pharmaceutical processes and some of the limitations and difficulties are
related to their toxicity.
Solvent can strongly affects the nucleation rate and habit of crystalline materials in
enhancing or inhibiting crystal growth of each crystal face by its properties in solution (e.g.
density, viscosity, diffusivity), solubility of the crystallizing species and surface-solvent
interactions. For example, changing the solvent polarity during crystallization of ibuprofen
provided crystals with a polyhedral crystal habit for ethanol and methanol and needlelike for
hexane. Furthermore the results showed that crystal habit modification had a great influence
on the mechanical properties (compressibility, flow rate, and bulk density) (GAREKANI et
al., 2001).
The solvent composition may also influence the aggregation properties, as well as the
solvent incorporation of the crystals (KLUG, 1993).
To date, although the role played by the solvent at the molecular level is still not
completely understood (WEISSBUCH et al., 1995), two theories have been proposed
regarding the influence of the solvent on nucleation and growth.
In one theory, favourable interactions between solute and solvent on specific faces may
lead to a reduction in solid-liquid interface energy. Hence, the activation energy for
nucleation on the crystal is reduced and the crystal becomes rougher, leading to enhanced
faster surface growth. In the second one, it has been proposed for solvent molecule interaction
that can be strongly bound to the crystallizing compound; the preferential adsorption of
solvent molecules at specific faces may inhibit their growth as removal of bound solvent
56
Literature Review
poses an additional barrier for continued growth. In the latter case, solute−solvent interactions
at the crystal interfaces could be similar to stereospecific interaction of tailor-made impurities
(LAHAV and LEISEROWITZ, 2001).
1.4. LAS CRYSTALLIZATION TO IMPROVE DISSOLUTION
PROPERTIES
AND
SOLUBILITY
OF
POORLY
SOLUBLE
DRUGS
Considerable research efforts have been made to optimize crystals properties for
pharmaceutical products. The complexity and variety of crystallization pharmaceutical
processes can provoke significant changes in the crystal properties, such as their
granulometry.
Particle size is one of the physicochemical properties influencing the performance of the
drug product and its manufacturability. In addition, a very strict control is needed to achieve
particle size requirements, which is defined depending on the pharmaceutical dosage form
prepared with the drug crystals and of its administration route (see Table 1.3).
Table 1.3. Particle size distribution of pharmaceuticals with respect to dosage form and
route of administration (SHEKUNOV et al., 2007).
Dosage form or route of administration
Particle size (µm)
Min
Max
Oral granules
200
1000
Oral depot
50
200
Intraperitoneal
10
50
Nasal
5
20
Aerosols
1
5
Ocular
0.1
2.5
Intravenous/intramuscular
0.2
2
Gene delivery
0.2
0.9
Transdermal
0.06
0.6
Long-circulating (brain, tumor)
0.06
0.2
Lymphatic
0.01
0.06
57
CHAPTER 1
In practice, crystal morphology is usually described in terms of length, width and
thickness. The classifications of crystal shapes adopted by either British Standard (BS
2955:1993) or the US Pharmacopoeia (monograph 776) (Table 1.4) are commonly used for
routine microscopically examination of solid pharmaceutical materials.
Table 1.4. Classification of common crystal morphologies for pharmaceutical solids
accepted by the US Pharmacopoeia
Morphology
Description
Equant
Crystal with similar
length, width, and
thickness
Flakes
Thin, flat crystals of
similar width and length
Plates
Flat, tabular crystals
with similar width and
length but thicker than
flakes
Laths
Elongated, thin and
blade-like crystals
Needles
Columnar
Acicular, thin and
highly elongated crystals
having similar width and
breadth
Elongated, prismatic
crystals with greater width
and thickness than needles
Diagram
Plate
(tabular)
Columnar
(prismatic)
Flake
Needle
(acicular)
Lath
(blade)
Equant
Particle size control during crystallization is a challenging area, mainly for the synthesis
of nano/microparticles (CHOW et al., 2007). As already said, the growth rates of the different
crystal faces are determined by intermolecular interactions between molecules in the crystal
as well as by a number of external parameters such as solvent, supersaturation, temperature,
and impurities (MYERSON, 1999). Changes in any of these parameters may lead to
significant modifications in crystal morphology. This gives rise to crystal habit diversity of a
chemical entity grown under various crystallization conditions and provides the basis for
morphological crystal engineering.
58
Literature Review
Besides particle size, other crystal properties can be strongly modified by
crystallization as summarized in Table 1.5.
Table 1.5. The most important solid-state characteristics, which are affected by
crystallization, and the influence of these properties on the stability and downstream
processing of pharmaceutical materials. Modified from SHEKUNOV and YORK, 2000.
Crystal properties
Effect on drug substance and/or drug product
•
•
Physical and chemical stability
Polymorphism
Polymorphs
Solvates (hydrates)
Salts
Crystal defects
•
•
Solubility profile and dissolution rate
All aspects of processing
Dimensional
•
Structural
Crystallinity (existence of amorphous
and semi-crystalline forms)
Particle size distribution
Particle surface structure
Processing behavior: bulk density,
agglomeration, flow, compaction
• Particle permeability (i.e. particle adsorption)
• Bioavailability (drug absorption)
• Consistency and uniformity of the dosage form
Chemical
Presence of impurities, residual solvent,
and decomposition products
Chiral forms and chiral separation
•
•
Toxicity
Chemical, physical, and enantiomeric stability
Brittle/ductile transitions, fracture stress,
indentation hardness, stress/strain
relaxation, Young’s modulus
•
Milling and tableting behavior
Electrical
•
Agglomeration and flow properties
Mechanical
Electrostatic charge distribution
59
CHAPTER 1
The next sections introduce LAS crystallization in the context of process design and
what role additives currently play in LAS crystallization research.
1.4.1.Previous works
In recent years, many methods of particle production using LAS crystallization have
been developed, as shown in Table 1.6. They allow a better control of the size, crystallinity,
and morphology of particles that not achieved with top-down methods. The crystallization
method is applicable for a wide range of pharmaceutical drugs BCS Class II and IV to prepare
micro/nanoparticles. Decrease in particle size increases the surface area, which increases the
solubility and dissolution rate of poorly water soluble drugs and hence bioavailability
(LIVERSIDGE and LIVERSIDGE, 2008), as discussed befeore in the section 1.2.2.
Table 1.6 reveals that several poorly soluble drugs are crystallized by LAS
crystallization using different apparatus in laboratory scale like batch reactors, magnetic
stirring, injection method and sonoreactors. The drug powders obtained in these studies
presented particle size in a range between 0.010- 20 µm.
It could be noted from this literature review, that the major crystallization assays were
realized in presence of additives like surfactants (ionic or non ionic) and polymers or both.
Since the molecules have a different orientation at the different crystal faces, the additives
will have different effect on the growth rate of different faces. Each additive can act like a
stabilization agent through a different mechanism; these mechanisms are a relatively less
explored area but are important for morphologic, polymorphic and technologic screening of a
compound during its developmental stage. Any relationship between the chemical structure of
the stabilization agent and the chemical structure of the drug to be stabilized against growth is
related in the literature. However the attempt of the use of stabilizers agents is actually turned
on the changes of drug compound characteristics.
The majority of the recrystallized drugs investigated is asymmetric drugs and do not
posses a very large molecular structure, which facilitated the achievement of crystals with a
reduced size, generally in presence of additives. The different aspects to controlled LAS
crystallization will be discussed in more detail in the following sections.
60
Sonoreactor
Magnetic/Mechanic
stirring - Injection
method
Batch reactor
Batch reactor
Batch reactor
Magnetic/Mechanic
stirring
Apparatus
X
HPMC
HPMC
HPMC
HPMC
X
Stabilizer
X
Steric
Steric and
removal of
solvent
Acetone
NaOH solution
Acetone
Acetone
Isopropyl alcohol
Steric and
removal of
solvent
Steric and
removal of
solvent
Isopropyl acetate
Solvent
X
Stabilization
mechanism
Table 1.6. Obtention of drug crystals by LAS precipitation method.
Literature Review
Water
HCl solution
Water
Water
Water
Hexane
Anti-solvent
Roxithromycin
5-(3-ethoxy-4pentyloxyphenyl)2,4-thiazolidinedione
Ketoconazole
Itraconazole
Ibuprofen
Abecarnil
Drug
X
10–15
1.200
0.600
2
20
ParticleSize
(µm)
61
GUO et al.,2005
TERAYAMA et al.,
2004
RASENACK and
MÜLLER, 2002
RASENACK and
MÜLLER, 2002
RASENACK and
MÜLLER, 2002
BECKMANN, 1999
Author
X
Magnetic/Mechanic
stirring
Magnetic/Mechanic
stirring
Sonoreactor
HPMC
SDS
and Miglyol®
PVP
P407
X
Rotating packed bed
Magnetic/Mechanic
StirringInjection
method
Stabilizer
Apparatus
Table 1.6. Continued
CHAPTER 1
Steric
Electrostatic,
steric and
inhibition of
Ostwald
ripening
Steric
X
X
Stabilization
mechanism
N-Methyl-2pyrrolidinone
N,Ndimethylacetamide
Tetrahydrofuran
Acetone
HCl solution
Solvent
Water
Water
Water
Isopropyl ether
Acetone and
Triethylamine
Anti-solvent
Prednisolone
AZ68
Itraconazole
Cefuroxime axetil
Cephradine
Drug
1.460
0.145-0.162
0.300
0.300
0.200-0.400
Particle Size
(µm)
62
LI et al., 2007
SIGFRIDSSON et
al., 2007
MATTEUCCI et al,
2006
ZHANG et al, 2006
ZHONG et al. 2005
Author
Magnetic/Mechanic
stirring
Magnetic/Mechanic
stirring
HPMC 606
Magnetic/Mechanic
stirring
PEG-400
Tween 80
PVP PF17
Stabilizer
Apparatus
Table 1.6. (continued)
Literature Review
Steric
Steric
Steric
Stabilization
mechanism
PEG-400
DMSO
PEG-300
Solvent
Water
Water
Water
Anti-solvent
Salmeterol xinafoate
2-2-devinyl
pyropheophorbide-a
Carbamezepine
Drug
2.540
0.100-0.200
0.010-0.020
Particle Size
(µm)
63
MURNANE et al.,
2008
BABA et al., 2007
DOUROUMIS and
FAHR, 2007
Author
Sonoreactor
Magnetic/Mechanic
stirring
Batch reactor
Magnetic/Mechanic
stirring
Apparatus
X
PVP
P407
HPMC
Tyloxapol
HPC
Stabilizer
Table 1.6. (continued)
CHAPTER 1
X
Steric
Steric
Steric
Stabilization
mechanism
Acetone
N,Ndimethylacetamide
Tetrahydrofuran
Acetone and MeOH
Solvent
Isopropyl ether
Water
Water
Water
Anti-solvent
Cefuroxime axetil
Bicalutamide
Itraconazole
Budesonide
Drug
0.800
0.115
0.300
5
Particle Size
(µm)
64
DHUMAL et al.,
2008
LINDFORS et al.
2008
MATTEUCCI et al.,
2008
HU et al.,2008
Author
PS 1k-b -PEO3k
Sonoreactor
Sonoreactor
P407
P407
PS 1k-b -PEO3k
Sonoreactor
Sonoreactor
X
HPMC
Stabilizer
Magnetic/Mechanic
stirring
Magnetic/Mechanic
stirring
Apparatus
Table 1.6. (continued)
Literature Review
Steric
Steric
Steric
Steric
X
Steric
Stabilization
mechanism
Tetrahydrofuran
Tetrahydrofuran
Tetrahydrofuran
Tetrahydrofuran
EtOH and DMSO
Acetone
Solvent
Water
Water
Water
Water
Water
Water
Anti-solvent
Odanacatib
Itraconazole
Odanacatib
Itraconazole
Bicalutamide
Gliclazide
Drug
0.350
0.158
0.352
0.145
0.450
6.200
Particle Size
(µm)
65
KUMAR et al, 2009
KUMAR et al., 2009
KUMAR et al, 2009
KUMAR et al., 2009
Y.LE et al., 2009
VARSHOSAZ et al.,
2008
Author
X
X
Rotating packed bed
PVP K90
Magnetic/Mechanic
stirring
Sonoreactor
HPMC K3
X
Magnetic/Mechanic
stirring
Sonoreactor
Stabilizer
Apparatus
Table 1.6. (continued)
CHAPTER 1
X
X
Steric
Steric
X
Stabilization
mechanism
EtOH
X
Acetone
Acetone
DMSO
Solvent
Water
Water
Water
Water
Buffer pH 7.4
Anti-solvent
Danazol
Atropine sulfate
Paclitaxel
Ibuprofen
Hypocrellin A
Drug
0.190
0.100-0.600
0.299-0.359
0.702
0.350-0.800
Particle Size
(µm)
66
ZHAO et al., 2009
ALI et al., 2009
EL-GENDY and
CBERKLAND, 2009
VERMA et al., 2009
L. ZHOU et al., 2009
Author
Multi-stage liquid
impingers
Magnetic/Mechanic
stirring
Magnetic/Mechanic
stirring
X
HPMC
X
HPMC
X
Magnetic/Mechanic
stirring
Magnetic/Mechanic
stirring
Stabilizer
Apparatus
Table 1.6. (continued)
Literature Review
MeOH
Steric and
removal of
solvent
X
Steric
HCl sol. and NaOH
sol.
1-Methyl-2pyrrolidone
Acetonitrile
Water
Removal of
solvent
Removal of
solvent
Solvent
Stabilization
mechanism
Isopropyl
alcohol
Water
Water
Water
MeOH
Anti-solvent
Ciprofloxacin
Spironolactone
Salbutamol sulphate
Atorvastatin calcium
Insulin
Drug
1-2
0.300
0.060
0.240-0.410
2
Particle Size
(µm)
67
ZHOA et al.,2009
DONG et al., 2009
BHAVNA et al.,
2009
ZHANG et al., 2009
KLINGLER et al.,
2009
Author
Sonoreactor
Sonoreactor
Sonoreactor
Sonoreactor
Apparatus
HPMC
HPMC
HPMC
SDS
HPMC
SDS
Stabilizer
Table 1.6.(continued)
CHAPTER 1
Steric
Steric
Electrostatic and
steric
Electrostatic and
steric
Stabilization
mechanism
Acetone
Tetrahydrofuran
Acetone
Acetone
Solvent
Water
Water
Water
Water
Anti-solvent
Griseofulvin
Itraconazole
Griseofulvin
Fenofibrate
Drug
2.400
0.830
1.900
3.400
Particle Size
(µm)
68
DALVI and DAVE,
2010
DALVI and DAVE,
2010
MENG et al., 2009
MENG et al., 2009
Author
Magnetic/Mechanic
stirring - Injection
method
Magnetic/Mechanic
stirring
Sonoreactor
Sonoreactor
Apparatus
X
Kollidon VA 6
HPMC
HPMC
Stabilizer
Table 1.6. (continued)
Literature Review
X
Steric
Steric
Steric
Stabilization
mechanism
EtOH
Acetone
Acetone
Acetone
Solvent
Water
Water
Water
Water
Anti-solvent
Isoxyl
Megestrol acetate
Sulfamethoxazole
Ibuprofen
Drug
0.220
1.654
30
8.400
Particle Size
(µm)
69
WANG and
HICKEY, 2010
CHO et al., 2010
DALVI and DAVE,
2010
DALVI and DAVE,
2010
Author
Batch reactor
Sonoreactor
Injection
method
under
sonication
Sonoreactor
Apparatus
HPMC
SDS
X
Stearic acid
PEG 200
PVA
Stabilizer
Table 1.6. (continued)
CHAPTER 1
Electrost
atic, steric and
removal of
solvent
X
Steric
Steric
Stabilization
mechanism
EtOH
EtOH
EtOH
PEG 200 and
Acetone
Solvent
Water
Water
Water
Water
Antisolvent
Fenofibrate
Diatrizoic
acid
Progesterone
Nitrendipine
Drug
0.318
2.400
0.267
0.209
Particl
e Size (µm)
70
HU et al.,
2011
EL-GENDY
et al., 2010
SALEM, 2010
XIA et al.,
2010
Author
Sonoreactor
Magnetic/Mechanic
stirring
Magnetic/Mechanic
stirring
Injection method
under sonication
Apparatus
X
PVA
P188
Lecithin
Stearic acid
Stabilizer
Table 1.6. (continued)
Literature Review
X
DMSO
Water
Tert-butyl alcohol
Vesicles and
freeze drying
Steric and
removal of
solvent
Water
Dimethylformamide
and EtOH
Steric
Water pH4
Acetonitrile
Water
Anti-solvent
Solvent
Stabilization
mechanism
Camptothecin
Alpha ketoglutarate
Lysozyme
Theophylline
Drug
0.234
0.110
0.200
0.290
Particle Size
(µm)
71
ZHANG et al., 2011
SULTANA et al.,
2011
TAN et al., 2011
SALEM et al., 2011
Author
Magnetic/Mechanic
stirring
Sonoreactor
Magnetic/Mechanic
stirring
Apparatus
HPMC
Tween 80
HPMC
Inutec SP1®
P407
TEA
SDS
Stabilizer
Table 1.6. (continued)
CHAPTER 1
Steric
Steric
Electrostatic and
steric
Stabilization
mechanism
1-Ethyl 3- Methyl
imidazolium methylphosphonate
Methylene chloride
Isopropyl alcohol
Solvent
Phosphate
Buffer pH6,8
Ethyl alcohol
Water
Anti-solvent
Rifampicin
Itraconazole
Ibuprofen
Drug
0.283-0. 353
0.170-0.380
0.300-0.400
Particle Size
(µm)
72
VIÇOSA et al., 2012
BADAWI et al., 2011
MANSOURI et al.,
2011
Author
HPMC
P188
Stabilizer
Steric and freeze
drying
Stabilization
mechanism
EtOH
Solvent
Water
Anti-solvent
Riccardin D
Drug
1k-b
LIU et al., 2012
Author
-PEO3k – 1,000 Mw polystyrene-block-
0.184
Particle Size
(µm)
407 / P188- Ploxamer 188 / TEA- Triethanolamine/ Tween 80 - Polysorbate 80/ PVA- Polyvinyl alcohol/ NaOH- Sodium Hydroxide / HCl- Hydrochloric acid.
73
3,000 Mw polyethylene oxide) / PVP- Polyvinylpyrrolidone / MeOH- Methanol / EtOH- Ethanol / PEG- Polyethylene glycol / DMSO- Dimethylùsulfoxide/ P407-Poloxamer
PVP K30
Legend: SDS- Sodium dodecyl sulfate / HPC-Hydroxypropyl Cellulose / HPMC- hydroxypropyl methyl cellulose / PS
Magnetic/Mechanic
stirring
Apparatus
Table 1.6. (continued)
Literature Review
CHAPTER 1
1.4.2. Control of process parameters
Mixing devices
Mixing between drug solution and anti-solvent is a critical step to maintain a constant
level of supersaturation throughout the crystallizer, uniform nucleation and control of small
embryo particles formed in the process (DOUROUMIS and FAHR, 2006).
The initial contacting between the drug solution and the anti-solvent is an important
parameter, which will influence the nucleation rate. Two different manners to bring them into
contact are illustrated in Figure 1.20.
Single-jet configuration is the simplest manner by which one solution is pumped into
the stirred vessel containing the other liquid (Fig. 1.20a). Alternatively, two solutions can be
premixed before entering the tank as a single jet (Fig. 1.20b). The two feeding configurations
have different temporal and spatial supersaturation profiles.
(a) Single-jet
(b) Double-jet with premixing
Figure 1.20. Feeding configuration for anti-solvent crystallization
(a) single-jet, (b) double-jet with premixing.
Typically, mixing is divided into three main groups: macromixing, mesomixing and
micromixing. Macromixing is the mixing occurring on a crystallizer scale, which represents
the uniformity of the local average of the concentrations of all the species within the entire
crystallizer. Mesomixing is the mass transfer of a solution, also known as turbulent mixing
and micromixing comprises the molecular diffusion and engulfment of different solvent
composition region, which represents molecular scale mixing (BALDYGA and BOURNE,
1989).
74
Literature Review
Actually, to achieve the rapid mixing the high jet velocity mixing devices have been
described in the literature. These mixing devices are reported in the literature as static mixer,
high gravity jet, confined impining jet, multi-inlet vortex mixer (MIVM), Y-shaped
microchannel reactor, Y and T-mixer, Roughton mixer, as illustrate in Figure 1.21. They
show advantages when compared to the traditional CSTR (Continuous Stirring Tank
Reactor), such as small space requirement, low equipment cost, no power required except
pumps, short residence times and good mixing at low shear rates (THAKUR et al., 2003).
Because of this they have been extensively related in the literature for LAS crystallization of
drugs. The details of such mixing devices are reported in Table 1.7.
A- Roughton mixer
B- Tee-mixer
C- Multi-inlet vortex mixer
D- Static mixer
E- Y-mixer
F- Confined impining jet
G- High gravity jet
Figure 1.21. Schematics of mixing devices used in chemical and pharmaceutical field.
Modified from THORAT and DALVI, 2012.
75
High-gravity
reactive
precipitation
(HGRP)
0.97 ± 0.11
0.5
0. 328 ± 0.0022
0.3
Oxcarbazepine
Spironolactone
Fenofibrate
Cephradine
x
x
x
x
Antisolvent flow rate: 450 mL/min
Solvent flow rate: 0.1-0.3 L/min,
antisolvent flow rate: 0.9-2.7 L/min
Solvent flow rate: 50 mL/min,
antisolvent flow rate: 500 mL/min
Continuous addition of solvent and
antisolvent
Nozzle diameters: 0.5 and
1.5mm
x
4.09 ± 0.47
Antisolvent flow rate: 500 mL/min
Solvent flow rate: 150 mL/min
Carbamazepine
x
Solvent flow rate: 83.33 mL/min
0.25±0.03
x
Solvent flow rate: 18.75 mL/min
Antisolvent flow rate: 300 mL/min
ß-metahsone
valerate- 17
1.45 ± 0.17
Progesterone
x
Solvent flow rate: 12.5 mL/min
Antisolvent flow rate: 400 mL/min
SMX DN3 static mixer
Static mixer
Mixing elements Diameter:
3.2 mm, length: 3.2 mm.
welded together with offset
of 90° with each other in
stainless steel tube of inner
diameter: 3.3 mm 6element of a size 25 mm
welded together with
offset of 90° with each
other in glass tube-SMV
DN25 static mixer
Particle size
(µm)
Drug
Mixing time and
energy dissipation
Flow rates/velocity
Dimensions of mixing
device
Mixing device
76
ZHONG et al.,
2005.
HU etal., 2011
DONG et al.,
2010.
DOUROUMIS
and FAHR,
2006.
Author
Table 1.7. Summary of mixing intensification reports using mixing devices for LAS drug crystallization. Modified from THORAT and
DALVI, 2012
CHAPTER 1
Mixing time~16.7ms
Cyclosporine A
0.18-0.7
77
CHIOU et al.,
2008
ZHU et al.,
2007
0.1
ß-Carotene
x
Solvent and antisolvent
flow rate: 72 mL/min
At the maximum flow of
120mL/min
ZHAO et al.,
2009.
0.19
Danazol
Micromixing time:
100µs
Solvent flow rate: 70ml/min
Antisolvent flow rate: 1500 mL/min
Rotor
Inner diameter: 25 mm
Outer diameter: 90.Axial
height: 28 mm mm
Packing: Material: wire
mesh. Voidage: 95%
Production capacity:
116.1 g/h
Jet inlet diameter 0.50 mm,
outlet diameter 1 mm,
chamber diameter: 2.38 mm
CHEN et al.,
2006.
0.305
Cefuroxime axetil
Micromixing time:
100µs
Solvent: 10 L/h
Antisolvent: 200 L/h
Rotating packed bed (RPB)
Inner diametrs : 50mmOuter
diameters: 150 mm, Axial
width of rotating bed: 50
mm, Distributor: two pipes
(10 mm in outer diameter
and 1.5 mm in wall
thickness), each having a
slot (1 mm in width and 48
mm in length)
High-gravity
reaction antisolvent
precipitation
(HGAP)
Confined liquid
impinging jets
(CLIJ)
Author
Particle size
(µm)
Drug
Mixing time and
energy dissipation
Flow rates/velocity
Dimensions of mixing
device
Mixing device
Table 1.7. (continued)
Literature Review
78
PANAGIOTOU
et al., 2009.
Norfloxacin
Micromixing time: 4ms,
mesomixing time :
20s, energy dissipation
rates ~10 7 W/kg
Inlet fluid velocity 500m/s
Channels depth: 50-150
µm, channels width: 50150 µm, scalable to tens of
L/min
0.17
ZHU et al.,
2011
0.1
ß-Carotene
x
Microfluidic
reaction
technology
(MRT)
D’ADDIO et
al., 2010.
0.14
ß-Carotene
x
In let velocity from 0.12 to 0.37
m/s. Antisolvent flow rate:120
mL/min, solvent flow rate: 13.3
mL/min.
x
Multi-inlet
vortex mixer
DONG et al.,
2011.
0.302-0.360
Spironolactone
x
Solvent flow rate : 25-100 mL/min,
antisolvent flow rate: 22-900
mL/min, rate ratio of solvent to
antisolvent (1:9)
Solvent inlet diameter: 0.5,
1, 1.5mm, antisolvent inlet
diameter 1.5, 3, 4.5mm,
outlet diameter 0.5mm
Impinging
device
MAHAJAN and
KIRWAN, 1996
2-10
Lovastatin
(2) CIJR-d2(0.05-0.1 s)
Micromixing time: 65
and 145ms
Inlet fluid velocity ~40m/s
Jet nozzle diameter: 0.5, 1
and 2mm. Mixing chamber
diameter: 2.54 cm
Twoimpinging-jets
(TIJ)
Author
Particle size
(µm)
Drug
Mixing time and
energy dissipation
Flow rates/velocity
Dimensions of mixing
device
Mixing device
Table 1.7. (continued)
CHAPTER 1
Cefuroxime axetil
0.35
79
WANG et al.,
2010.
ZHANG et al.,
2010
0.48
Atorvastatin
calcium
0.04-0.4ms
Antisolvent flow rate:30 mL/min,
solvent flow rate: 3 mL/min
Mixing chamber: 60
(width) × 10 (depth) × 105
length) mm
0.04-0.4ms
ALI et al.,
2009 ; ALI et
al., 2011
0.08-0.45 and
0.295±0.0032
Hydrocortisone
x
Antisolvent flow rate:2.5mL/min,
solvent flow rate: 1mL/min
Channel diameters: 0.1, 0.5
and 1mm, inlet angles 25
and 50.
Antisolvent flow rate:50-60
mL/min, solvent flow rate: 2
mL/min, overall flow rate : 63
mL/min
ZHAO et al.,
2007.
0 .364
Danazol
Micromixing time : 0.250.35ms, energy
dissipation rates :
2000-4000 W/kg
Antisolvent flow rate:80 mL/min,
solvent
flow rate: 4 mL/min
Inlet diameter: 300µm,
inner length: 300 µm,
outlet diameter: 300 µm,
outlet length: 600 µm
Y-shaped
microfluidic
reactor
(YMCR)
Unlet channels: 400 × 500
× (width × depth m) × 20
length mm), cross section
of the mixing channel: 800
× 500 (width × depth) µm,
×40 (length, mm)
Author
Particle size
(µm)
Drug
Mixing time and
energy dissipation
Flow rates/velocity
Dimensions of mixing
device
Mixing device
Table 1.7. (continued)
Literature Review
ZHU et al.,
2010.
LIU et al.,
2010.
HE et al.,
2010 ; HE et
al., 2011.
0.4-1.4
0.19
0.03-0.05
Cefuroxime axetil
Curcumin
Curcumin
Micromixing time: 2ms
Mixing time: 0.18
x
Total volumetric flow rate :6L/min
Antisolvent flow rate: 1.2 mL/min,
solvent flow rate: 1.2 mL/min
Antisolvent flow rate: 10 µL/min,
solvent flow rate: 10 µL/min
Micropore size: 5 µm
length of micropore zone:
1 mm porosity: 46%, outer
tube diameter: 15.5 mm,
inner tube diameter: 15 mm
Inlet and outlet: 250 µm
(depth × width of channel)
Inlet diameter: 1 mm, outlet
diameter: 1 mm, crosssection: width 200 µm ×
height 200 µm
Microporous
tube-in-tube
microchannnel
reactor
T-shaped
micro-channel
Micromixer
80
Author
Particle size
(µm)
Drug
Mixing time and
energy dissipation
Flow rates/velocity
Dimensions of mixing
device
Mixing device
Table 1.7. (continued)
CHAPTER 1
Literature Review
1.5. ADDITIVES IN CRYSTALLIZATION
Additives in crystallization processes have gained attention in recent years,
because they can be adsorbed directly onto drug particles to produce powders with
optimized physicochemical properties (ZIMMERMANN et al., 2009).
It has been shown that the presence of additives in solution can affect all
parameters of crystallization, either the solubility, the nucleation or growth, and hence
the resulting morphology (KUBOTA et al., 2000; GARNIER et al., 2002; VETTER et
al., 2011) and polymorphic form (CHONG et al., 2002; SONG and CÖLFEN, 2011)
Some theories are presented in literature to describe the mechanisms of additive
influence on crystallization process, which comprises:
•
Inhibition of nucleation by formation of hydrogen bonds between the additive
and the molecule. This interaction inhibits or retards the formation of nucleus by
collision and therefore the induction time period is different from that of pure
systems (TAYLOR and ZOGRAFI, 1997);
•
Adsorbed additives on crystal surface form a mechanical barrier that prevents
the diffusion of solute molecules from the bulk solution to the crystal surface
(ZILLER and RUPPRECHT, 1988);
•
Additives do not inhibit nucleation, but just reduce supersaturation of the
solution by modifying the saturation of solute. Consequently the width of the
metastable zone is also influenced (RODRIGUEZ-HORNEDO and MURPHY,
1999; MERSMANN, 2001);
•
Non-adsorbed additive are rejected by the crystal surface and accumulate in the
boundary region. This creates a greater resistance to drug molecules limiting
their diffusion through the barrier and leads to growth inhibition, as shown in
Figure 1.22 (RAGHAVAN et al., 2001).
The adsorption of additives at different sites can cause growth inhibitions, even
block the growing surface and in consequence stop the growth process. However, the
adsorbed impurities may simultaneously lead to a reduction in the edge free energy,
81
CHAPTER 1
which results in an increase in crystal growth rate (RAK et al., 2005). For
pharmaceutical technology, understanding of these mechanisms may help to control the
quality and purity of raw crystalline substances and, consequently, to improve the
manufacture and performance of the final dosage forms.
Drug molecule
Additive (polymer)
Crystal
Liquid
Diffusional boundary layer
Figure 1.22. Schematic diagram showing the mechanism of growth inhibition
and habit modification of crystals by polymers. Modified from RAGHAVAN et al.,
2001.
1.5.1. Additives in LAS crystallization
The stabilization and controlled crystal growth mechanism during LAS
crystallization depend on the strength of adsorption of stabilizer molecules on the drug
surface. Two main mechanisms have been investigated in additive screening studies on
drug LAS recrystallization: steric stabilization and electrostatic repulsion (WU et al.,
2011; THORAT and DALVI, 2012), as illustrated in Figure 1.23.
Each mechanism has its benefits for particulate systems. The stabilization
mechanisms used in LAS crystallization, described in the literature, are summarized in
Table 1.6.
In the case of steric stabilization, non ionic surfactants, polymers and amphiphilic
block copolymers are usually used (MATTEUCCI et al., 2008; KUMAR et al., 2009; ).
82
Literature Review
Block copolymers are particularly well suited for steric stabilization, as they can
provide a high degree of coverage with strong adsorption while still having extended
tails which interact favorably with the medium (BUDIJONO et al., 2010).
Steric stabilization
Electrostatic stabilization
Figure 1.23. Types of colloidal stabilization. Modified from WU et al., 2011.
Stabilization by steric mechanism is achieved by attaching of hydrophobic group
of the drug and polymer. The resulting polymer layer masks the attractive force and also
provides a repulsive force. There are several mechanisms proposed in the literature for
steric stabilization as discussed below:
•
Drug-polymer interaction: The interaction between polymers and particle
surface has been extensively studied for decades due to its wide application. The
adsorption properties of stabilizers can be affected by the nature of stabilizer and drug
surface, for example molecular weight is an important factor for polymeric
stabilizers. The chain length should be high enough, so that polymers chain have an
optimum length to overcome the Van der Waals forces of attraction (PELTONEN and
HIRVONEN, 2010). Furthermore, another important factor is the size of the polymer.
The polymer’s chain was divided into three sub-types: trains, loops and tails. (as shown
in Figure 1.24). Trains are all contacted (adsorbed) with the particle surface. Loops are
83
CHAPTER 1
not in contact with the particle surface, but connect with two trains. Tails are nonadsorbing chain ends. The portion of polymer chain in solution provides the steric
protection depending on molecular weight of polymer (NAPPER, 1983).
Figure 1.24. Schematic illustration of adsorbed polymer layer. From NYLANDE
et al., 2006.
Apart the size and the molecular weight the effect of polymer concentration is an
important aspect to be considered. For polymer concentration, there is a maximum
value
full
coverage
of
surface,
or
saturated
adsorption
(SCHOTT,1980;
OTSUBO,2003) concentration with free polymer in the dispersion medium, above a
critical volume fraction of polymer in solution. This phenomenon is called depletion
flocculation. This one occurs when the distance separating two colloidal particles is too
small to admit the presence of polymer coils in solution. They are forced out of the
space in between, resulting in an osmotic pressure difference between the area in
between the particles and the rest of the bulk solution. As a result, an attractive force is
generated which pushes the particles together, leading to agglomeration. (OTSUBO,
2003).
Another such mechanism is the formation of hydrogen bond between the
stabilizer molecule and the particle surface. Some functional groups in polymers, such
as carboxyl, hydroxyl, amine, and ester group play an important role in the steric
stabilization. These functional groups can interact with the particle surface and act as
good anchors (SHI, 2002)
The adsorption of polymer on crystal surface can occur as a result of forces as
those arising from hydrophobic interactions. It has been observed that these
84
Literature Review
hydrophobic interactions can enhance the stability by occupying the adsorption sites and
inhibiting the incorporation of drug molecules in solution in crystal lattice
(RAGHAVAN et al., 2001).
Actually in the literature there are a lot of reports that describe the use of block
co-polymers for stabilization of crystals obtained by LAS crystallization. Hydrophobic
part of block copolymers gets anchored on the hydrophobic drug surface and
hydrophilic chains extend in solution. Depending on the solvent quality, affinity of the
chain monomers to the surface and crowding conditions on the surface, the
conformation of polymer chains on a surface can be rather different. Chain exists as
isolated free coils in solution, also called the “mushroom” regime (Figure1.25 A). In
this case, chain will lay almost flat on the surface. When more block copolymer is
attached the chains will extend perpendicularly to the hydrophobic surface, called “semi
brush”(Figure 1.25B). When the surface gets more crowded, the chains will start to
mutually interact, eventually resulting in a stretching of the individual chains away from
the surface leading to a so-called “brush” formation (Figure 1.25C). This state is the
result of a balance, where at full equilibrium the free energy of the stretched chain
balances the interfacial energy of the solvent–particle surface (S.J. BUDIJONO et
al.,2010).
(a)
(b)
(c)
Figure 1.25. Different conformations of polymers at surfaces: (a) mushrooms
conformation of a single adsorbed, where chains are non-interacting on the surface and
achieve a size given by the polymer (b) semi-brush where crowding among chains
causes extension of the chain from the surface, and (c) brush conformation for high
grafting densities, leading to extension of the chains away from the surface. Modified
from J. BUDIJONO et al., 2010.
85
CHAPTER 1
The use of block co-polymers play a key role in both halting particle growth and
stabilizing particle suspensions during rapid LAS crystallization. Its use as stabilization
agent has been reported for LAS crystallization of Itraconazole, Odanacatib (KUMAR
et al., 2009), ß-carotene (JOHNSON and PRUD'HOMME, 2003 ; J. BUDIJONO et
al.,2010 ; SHEN et al., 2011 ; CAPRETTO et al., 2012). According to these reports,
with a rapid change in solvent conditions, the drug and polymer precipitate, the polymer
assembling around the drug. The particle size is controlled by the polymer concentration
and the mixing time.
•
Solvent-polymer interactions: Solvent-polymer affinity is a very important factor
in steric stabilization. Choi et al. (2002) have shown that solvent–polymer interaction is
very important for the effective formation of nanoparticles. Choi et al. (2002) obtained
PLGA using different solvents (ethyl acetate, methyl ethyl ketone, propylene carbonate,
and benzyl alcohol). Ethyl acetate solvent formed the smallest nanospheres (approx. 120
nm in size). In this case, the authors suggest that solvents with low exchange ratio
between diffusion from solvent to water and vice versa, form small nanoparticles due to
small supersaturation region produced.
•
Drug-polymer-solvent interaction: The polarity of the solvent can affect the
relative diffusion rates of polymer and drug molecules towards the solid–liquid
interface. It has been also reported that slower diffusion of API molecules and relatively
quick adsorption of stabilizer molecules on particle surface leads to inhibition of growth
and hence, smaller particle size (THORAT and DALVI, 2012).
In electrostatic stabilization ionic surfactants and polymers are usually used to
prevent aggregation and have stable crystalline particles. The ions in solution adsorb
onto the surface of a particle and the substances acquire surface electrical charges when
brought in contact with a polar medium. The surface charge influences the spatial
distribution of ions or molecules in the surrounding solution, attracting ions of opposite
charge but repelling ions of similar charge from the surface.
The electrostatic stabilization depends on DLVO theory (DERJAGUIN and
LANDAU, 1941 ; VERWEY and OVERBEEK in 1948) i.e. repulsive electrostatic
forces and attractive Van der Waals forces. Therefore, electrostatic stabilization of
dispersion occurs when the electrostatic repulsive force overcomes the attractive Van
86
Literature Review
der Waals forces between the particles (WU et al., 2011). Due to the charge on particle
surface, there exists a double layer surrounding the particle surface. This double layer is
referred as electric double layer (GRAHAME, 1947). There exists a potential difference
between bulk of solution and the outer layer of double layer which is called zeta
potential. The magnitude of the zeta potential gives an indication of the potential
stability of the colloidal system. The zeta potential is a measure of the stabilization
provided by electrostatic stabilization. If all the particles in suspension have a large
negative or positive zeta potential (>±
± 30 mV) then they will tend to repel each other,
i.e. good suspension stability.
Surfactants can provide stabilization at concentrations below critical micelle
concentration (CMC). When concentration is increased above the CMC, the number of
micelles increases leaving the particles unprotected (WU et al., 2011 ; THORAT and
DALVI, 2012). In the order hand, some literature reports have focused on beneficial
effects of additives, which have the capacity to form micelles, above the CMC. For
example, Santander-Ortega et al., (2006) observed a variety of stabilization mechanisms
for the Pluronic-coated PLGA nanoparticles. These complexes were completely stable
by adding poloxamer at concentrations above the CMC, The explanation was the
formation of surface aggregates that gives a highly enriched polymer layer
concentration.
The synergistic effects of a neutral polymer and an ionic surfactant together can
enhance the stabilization effect. This synergistic behavior is typically brought about by
the interaction between different types of stabilizing molecules, which provides a
driving force for the mixtures to form mixed aggregates or structures. However, the
ability of synergistically stabilizing depends on the pair of stabilizers used. Hu et al.
(2011) observed the synergistic effect of Sodium dodecyl sulfate (SDS) and
hydroxylpropyl methyl cellulose (HPMC) E3, such as an effective system to retain the
particles of fenofibrate within the nanosized range by minimizing particle aggregation.
Actually both steric and electrostatic stabilizing agent (polyelectrolytes) have been
studied in the literature (ZHU et al., 2010 ; PATTEKARI et al., 2011 . They form a
87
CHAPTER 1
strong double layer around hydrophobic drug particle and, adsorbs loosely, the
extending polymer loops provide the steric stabilization, if the polyelectrolyte adsorbs
on particle surface. Chitosan can be cited as an example. The chitosan is a cationic
polyelectrolyte of natural origin. Furthermore, chitosan is physiologically safe. It can be
biodegraded by several human enzymes. It is inexpensive and approved as a safe
dietary. The properties of chitosan include the ability to adhere to mucosal layers, due to
the electrostatic interaction with negatively charged mucus. This makes chitosan
especially useful for poorly water soluble drug delivery and targeting (ZHU et al.,
2010 ).
Finally, it has been shown that additives can increase their dissolution rate by
increasing aqueous wettability of LAS recrystallized drugs of (SRITAPUNYA et al.,
2012). This ability of additives will permit the gastrointestinal fluids to wet more
effectively and to come into more intimate contact with the solid dosage forms, which
will tend to increase the dissolution and absorption rates of the drugs (BUCH et al.,
2011 ; SAHARAN and CHOUDHURY, 2012).
It has been reported in the literature, that poloxamer 407 (P-407) has improved
significantly the dissolution of poorly water soluble drugs (DUMORTIER et al., 2006 ;
VIKRANT et al., 2009 ; CHOWDARY and ANNAMMA, 2012). P-407 is characterized
by its highest hydrophilicity and surfactant property, that results in greater wetting and
increases the surface available to dissolution by reducing interfacial tension between the
hydrophobic drug and dissolution medium (VIKRANT et al., 2009).
88
Literature Review
1.6. THESIS OVERVIEW
This Chapter reviewed the literature about the improvement of bioavailability of
poorly water soluble drugs. It focuses on topics of interest for our work, like properties
of drugs influencing dissolution behaviour, technologies to enhance dissolution rate and
solubility of this category of drugs. Emphasis was given to the LAS crystallization
process.
An overview of the literature on LAS crystallization summarized the main results
of previous works. The main points to be highlighted are:
•
Till now, several poorly water soluble drugs have been micronized by
various particle formation processes for particle size and morphology but the
main challenges are the control of particle size distribution and particle
growth.
•
The continuous production of recrystallized drug particles can be achieved
by using rapid mixing devices such as impinging jets and static mixers,
among others. Moreover, polymers and surfactants are used in most cases for
prevent crystal growth and/or agglomeration. However, the selection of
correct stabilizers for the LAS crystallization of a given drug is of crucial
importance.
This chapter concludes with a statement of research objectives:
LAS crystallization is used for particle formation of a new antiretroviral drug
named CRS 74. The objective is to enhance dissolution properties by reducing particle
size of an original (as-received) sample from industry.
To generate high mixing rates and help in generating rapid and uniform
supersaturation, rapid mixers are used (T-mixer, Roughton mixer).
The best conditions for LAS crystallization of this new molecule are defined
through solubility studies, dissolution method development and screening of LAS
89
CHAPTER 1
crystallization process parameters such as ratio between drug solution and anti-solvent
and additive screening study.
90
Characterization of CRS 74
Résumé Chapitre 2- Caractérisation du CRS 74
Cette étude porte sur un nouveau composé, l'acide (2S, 3S, 5S) -2, -5 bis-[N-[N[[N-méthyl-N-[(2-isopropyl-4 - tiazolyl) méthyl] amino] carbonyl ] vanilyl] amino-1,6 diphényl-3 - hydroxyhexane, nommée par la suite CRS 74. Ce principe actif possède
une activité de l’inhibition du VIH (virus de l'immunodéficience humaine) protéase, une
enzyme essentielle impliquée dans le processus de réplication du VIH.
Dans la première partie de notre étude, cette molécule a été caractérisée sous
forme solide en terme de faciès et de taille de particules, de structure cristalline, de
mouillabilité, de solubilité en milieu aqueux à 37 ° C. Un test de dissolution a été
développé en milieu aqueux à 37 °C afin de déterminer les profils de libération de la
poudre initiale et des poudres synthétisées, car ce test n’existait pas dans le littérature.
Cette molécule sous forme solide a une solubilité aqueuse limitée (<0.5µg/mL),
qui peut être expliqué par sa cristallinité, son point de fusion et son enthalpie de fusion
élevés (188,6 °C et 86,6 J/g). Elle présente aussi un faible taux de dissolution dans l’eau
pouvant être liée à sa large distribution granulométrique, sa faible solubilité et sa
mouillabilité très faible (θ = 136,4 ± 0,8 °).
Afin d’améliorer son taux de dissolution une opération de cristallisation par effet
anti-solvant a été proposée afin d’augmenter sa surface spécifique et améliorer sa
mouillabilité.
Characterization of CRS 74
2.1. INTRODUCTION
In the first part of our study, the selected molecule (as-received) was characterized
in terms of their physical properties (particle shape and size), crystal structure, surface
properties (wettability), solubility and dissolution properties in aqueous media at 37°C.
The methodology used and the results obtained are summarized in this Chapter.
2.1.1. The molecule: CRS 74
This study concerns a new compound, (2S, 3S, 5S)-2, -5 bis- [N-[N-[[N- methylN-[(2-isopropyl- 4- tiazolyl) methyl] amino] carbonyl] vanilyl] amino- 1,6- diphenyl- 3hydroxyhexane, named CRS 74. Its chemical formula is C46H66N8O5S2 and its
molecular weight 875.2 g/mol. Its molecular structure is shown in Figure 2.1. It is
obtained as courtesy from Cristália Ltda (Itapira, SP, Brazil). The drug samples have a
purity of 99 %.
Ph
S
H3C
CH3
CH3
O
H
N
N
H
N
N
H3C
H 3C
O
N
H
O
OH
H3 C
N
O
CH3
Ph
CH3
CH3
N
N
H
CH3
S
(a) CRS 74 - (2S, 3S, 5S)-2, -5 bis- [N-[N-[[N- methyl- N-[(2-isopropyl- 4- tiazolyl)
methyl] amino] carbonyl] vanilyl] amino- 1,6- diphenyl- 3- hydroxyhexane, is disclosed at
PCT document number WO 20005/111006; US 2010/7763733 (BOCKELMANN et al.,
2005; BOCKELMANN et al., 2010).
Ph
H3
C
S
CH3
O
N
OH
H
N
N
O
N
S
N
H
H3C
O
O
H 3C
CH3
Ph
(b) RITONAVIR - (2S, 3S, 5S)-5-[N-[N-[[N-methyl-N-[(2- isopropyl-4-thiazolyl)
methyl] amino] carbonyl] vanilyl] amino- 2- [N [(5-thiazolyl) methoxycarbonyl] amino-1,6diphenyl-3-hydroxyhexane, is disclosed at PCT document number WO 94/14436 (KEMPF
et al., 1994).
Figure 2.1. Chemical structures of (a) CRS 74 and (b) Ritonavir.
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CHAPTER 2
This compound can be prepared by methods disclosed in PCT document WO
111006 and US 7763733 (BOCKELMANN et al., 2005; BOCKELMANN et al., 2010)
following the scheme shown on Figure 2.2. In a general way, the compound (4) can be
obtained by coupling the compound (1) and (2), wherein Y can be OH or an activate
ester group and P is a N-protective group, to the formation of (3) following the Ndeprotection. Finally the compound (4) is coupled to the compound (5) wherein Z can
be OH or an activate ester group to provide the analogous compound (6).
Ph
O
NHP
+
A
H 2N
Y
(1)
(2)
OH
Ph
Ph
O
NHP
A
HN
(3)
OH
Ph
Ph
O
O
NH2
A
HN
+
(4)
B
Z
(5)
OH
Ph
Ph
O
B
NH
A
NH
(6)
OH
O
Ph
Figure 2.2. Schematic synthesis of CRS 74 (BOCKELMANN et al., 2005;
BOCKELMANN et al., 2010).
96
Characterization of CRS 74
CRS 74 has activity for inhibiting HIV (Human immunodeficiency virus)
protease, an essential enzyme involved in HIV replication process (see Appendix I).
Consequently, this new compound can be used for the treatment of HIV infections,
itself or in combination with other anti-HIV medicines.
CRS 74 is an analogous compound of Ritonavir, an important antiretroviral drug
developed and patented by Abbot Laboratories (WO 94/14436; KEMPF et al., 1994.).
The chemical structure of Ritonavir can be compared to that of CRS 74 in Figure 2.1
and their physicochemical properties in Table 2.1.
Table 2.1. Physicochemical properties of Ritonavir and its analogous compound
CRS 74 .
Drug identification
Name of the drug
Ritonavir
CRS 74
Molecular formula
C37H48N6O5S21
C46H66N8O5S23
Molecular weight (g/mol)
7211
875.23
Innovator
Abbot Laboratories (USA)
Cristalia Laboratories (BR)
Therapeutic category
HIV protease inhibitor
Melting point (°C)
HIV protease inhibitor
Physicochemical properties
122-124 2
Polymorphism
Form I and II4
*
Description
white or almost white
powder1
white powder3
Solubility
Soluble in ethanol,
methanol, sparingly soluble
in acetone R and very
slightly soluble in
acetonitrile and insoluble in
water 1
*
180-1853
*Data not determined
1-The International Pharmacopoeia. Fourth edition, 2011.
2-ALENCAR et al., 2006.
3- Patent WO 2005/111006 and US 7763733 (BOCKELMANN et al, 2005 ; BOCKELMANN et al., 2010)
4-BAUER et al., 2001
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CHAPTER 2
To date, there are some ambiguities regarding the BCS of Ritonavir. This
molecule has been classified as a molecule belonging to Class II (WU and BENET,
2005; SINHA et al., 2010) or Class IV (HERMAN et al., 1989; WILLIAMS and
SINKO, 1999;
LINDENBERG et al., 2004; GOYAL and VAVIA, 2012) of the
Biopharmaceutical Classification System (AMIDON et al., 1995).
Published data have shown that Ritonavir presents crystalline polymorphism
(BAUER et al., 2001). Crystalline polymorphism, or the ability of a compound to exist
in multiple solid-state structures, has significant impact on the physical properties,
performance, and safety of an active pharmaceutical ingredient (API) and its formulated
product(s). From the discovery of Ritonavir until the new drug application (NDA)
filing, only one crystalline form was known to exist (Form I). Attempts to identify other
possible crystal forms were unsuccessful. Ritonavir is marketed as Norvir. Two years
after the launch of Norvir to the market, some lots of Norvir capsules failed a
dissolution specification. Investigation of this phenomenum revealed the existence of a
crystal form of Ritonavir other than the one already known (Form I). This new crystal
form was designated as Form II. The two crystal forms are polymorphs and differ
substantially in their physical properties such as solubility, as given in Table 2.2.
Table 2.2. Solubility of Ritonavir polymorphs at 5°C in hydroalcoholic solvent
systems (CHEMBURKAR et al., 2000).
Ethanol/water (w/w)
100/0 75/25
Form I (mg/mL)
90
170
Form II (mg/mL)
19
30
The key challenge of this antiviral drug present on the Market is still drug
reformulation to modify its bioavailability and pharmacokinetics. Improving
bioavailability of Ritonavir (in most case, solubility) is a subject of current interest as
confirmed by numerous research works in the last decade, among them:
•
Nanonization by wet milling, homogenization or sonication (BALKUNDI et al,
98
Characterization of CRS 74
2011);
•
Prodrug synthesis (HAMADA et al., 2002). A prodrug is a drug that is
administered in an inactive (or significantly less active) form. Once
administered, the prodrug is metabolised in vivo into an active metabolite;
•
Inclusion complexes with cyclodextrins and surfactants (GOYAL and VAVIA,
2012; CHOWDARY et al., 2012);
•
Solid Dispersions (LAW et al., 2004 ; SINHA et al., 2010; MUSLE, 2012);
•
Micro/nano-encapsulation in polymeric micelles (BORGMANN et al., 2011);
•
Self-emulsifying drug delivery systems (SEEDS) (LEI et al., 2010).
One of the requirements for a drug to be considered suitable for a therapeutic
usage is its therapeutic efficacy, so, to achieve such requirement, the drug should
present adequate characteristics of bio-absorption and bioavailability. CRS 74 has high
biological activity as disclosed at PCT document WO 111006 and US 7763733
(BOCKELMANN et al, 2005; BOCKELMANN et al., 2010, respectively) but is
bioavailability is limited because of its low aqueous solubility and dissolution rate. Such
properties pose difficulties not only in the design of pharmaceutical formulations but
may result in bio-variability.
2.2. CHARACTERIZATION OF CRS 74
2.2.1. Methods
Samples of CRS 74 were characterized as received by using different instrument
and measurement techniques that will be described in the following.
2.2.1.1. Particle size measurement
Mean particle size and particle-size distribution are the most important parameters
in designing a novel formulation and are routinely the first to be measured. Particle size
distribution of the powder samples was determined by laser diffractometry. The
technique of laser diffraction is based on the principle that particles passing through a
laser beam will scatter light at an angle that is directly related to their size: large
99
CHAPTER 2
particles scatter at low angles, whereas small particles scatter at high angles. The laser
diffraction is accurately described by the Fraunhofer approximation and the Mie theory,
with the assumption of spherical particle morphology.
The equipment used for the measurements was a MasterSizer 3000 laser
granulometer (Malvern Instruments, United Kingdom), which has a reading range of
0.1-3500 μm. All the dry samples were firstly mixed with Tween 20 and then dispersed
in water until achieve the good obscuration. The laser diffraction data obtained were
evaluated using the volume distribution diameters dv10%, dv50% and dv90%. The diameter
values 10% to 90% indicate the percentage of particles possessing a diameter equal or
lower than the given size value.
2.2.1.2. Density measurement
In this work, the true densities of CRS 74 powder sample (0.9282 g) was
determined using a helium pycnometer (Accupyc 1330, Micromeritics, UK) that was
operated according to the manufacturer’s recommended procedures. Calibration was
performed using standard stainless steel spheres of known mass and volume. The
sample was used as they were received from the supplier. Mean values and standard
deviations were determined from 25 successive measurements.
2.2.1.3. Thermal Analysis
Thermal analysis of the CRS 74 powder was performed to characterize the
properties of this material as they change with temperature. Two different thermal
techniques were employed here distinguished by the property which is measured: mass
loss by thermogravimetric analysis (TGA) and enthalpy of transition (melting,
crystallization) by differential scanning calorimetry (DSC).
2.2.1.3.1. Thermogravimetric Analysis (TGA)
Thermogravimetric Analysis (TGA) measures the amount and rate of change in
the weight of a material as a function of temperature or time in a controlled atmosphere.
The technique can characterize materials that exhibit weight loss or gain due to
decomposition, oxidation, or desolvatation (dehydration). Measurements were
conducted here to predict the thermal stability of CRS 74 at temperatures up to 300°C.
100
Characterization of CRS 74
Thermogravimetric analysis (TGA) of original CRS 74 crystals was performed by a
thermogravimetric analyser TG-DSC 111 (SETARAM, France). The dynamic
thermogravimetric curve was recorded with a mass of sample of around 5 mg packed in
aluminium cell under a dynamic nitrogen atmosphere (50 mL.min-1). The experiments
were run from 20 to 300˚C at a heating rate of 10˚C /min.
2.2.1.3.2. Differential scanning calorimetric analysis (DSC)
Differential scanning calorimetric (DSC) is based upon the detection of changes
in the heat content (enthalpy) or the specific heat of a sample at a certain temperature.
As thermal energy is supplied to the sample, its enthalpy increases and its temperature
rises by an amount determined for a given energy input by the specific heat of the
sample. The specific heat of a material changes slowly with temperature in a particular
physical state, but alters discontinuously at a change of state. As well as increasing the
sample temperature, the supply of thermal energy may induce physical or chemical
processes in the sample, e.g. melting or decomposition, accompanied by a change in
enthalpy, the latent heat of fusion, heat of reaction etc. Such enthalpy changes may be
detected by thermal analysis and related to the processes occurring in the sample (GILL,
1984).
DSC measurements were carried out using a DSC-Q200 thermal analyzer (TA
Instruments, France) in a temperature range of 20 to 210°C at a heating rate of
10°C/min under nitrogen atmosphere (50 mL.min-1). The samples (about 3 mg) were
placed in a hermetically closed aluminium pan. The transition temperature and the
enthalpy of fusion (ΔHt ) were calculated using the DSC software.
2.2.1.4. X-Ray Diffraction Analysis (XRD)
X-ray diffraction is based on constructive interference of monochromatic X-rays
and a crystalline sample. These X-rays are generated by a cathode ray tube, filtered to
produce monochromatic radiation, collimated to concentrate, and directed toward the
sample. The interaction of the incident rays with the sample produces constructive
interference (and a diffracted ray) when conditions satisfy Bragg's Law (nλ=2d sin θ).
101
CHAPTER 2
These diffracted X-rays are then detected, processed and counted. Conversion of the
diffraction peaks to d-spacing allows identification of the material.
X-rays diffraction patterns (diffractograms) can be used to confirm the crystalline
nature of a sample. Therefore, this information is used to verify whether the substances
are amorphous, partially amorphous crystalline or fully crystalline (MAULUDIN, 2008)
as well as the polymorphic form being present. A powder X-ray diffractometer
(XPERT,Philips) was used here for diffraction studies. X-ray diffraction analysis was
conducted with CuKα radiation at a scanning rate of 1.228°/min from 5 to 30°, applying
40 kV at 30 mA.
2.2.1.5. Scanning electron microscopy analysis (SEM)
The surface morphology of powder samples was viewed under a scanning
electron microscope (ESEM, FEG, Philips) operated at an excitation voltage of 20 kV.
The powder samples were fixed on an SEM stub using double-sided adhesive tape and
sputter coated with platinum at 50 mA for 6 min using an ion sputter (SC7640,
Polaron), before analysis.
The principle of this technique consists in the emission of primary electrons. Once
they reach the sample surface, they interact with the atoms of the material, giving rise to
secondary electrons, backscattered electrons and photons. The number of electrons
emitted varies according to the geometry and other properties of the sample. These
electrons are collected by a detector, producing image.
2.2.1.6. Contact angle measurement (sessile drop method)
Contact angles (θ) of water on drug substrates were measured by the sessile drop
method using a Contact Angle Measuring Instrument DSA30E (Kruss Instruments,
France). This method of contact angle measurement uses optics to measure the angle of
a drop sitting level on a surface. The drop shape is recorded with a high speed framing
camera, images are then processed by a computer and stored. The camera determines a
baseline, forms a line around the drop, and calculates the contact angle. The powder
sample was placed on the sample holder. A 5 µL droplet of the liquid probe (deionized
water) was placed on the sample surface and the image of the drop was captured by a
102
Characterization of CRS 74
CCD digital video camera. All measurements were performed in air under ambient
conditions and the reported values are an average of at least three measurements for
each experiment.
2.2.1.7. In Vitro Dissolution Testing
2.2.1.7.1. Defining the operating conditions for in vitro dissolution
To date, there is no published dissolution test for the evaluation of in vitro release
profiles of CRS 74 from immediate-release solid oral dosage forms (powders for
example). To study the dissolution properties of the CRS 74 powder sample, this part of
the work was planned with the following objectives:
1.
To develop and validate a dissolution methodology for this newer
antiretroviral drug;
2.
To estimate antiretroviral drugs by HPLC method.
A dissolution tester DT 60 (ERWEKA, Germany) was used in this study. A
schematic diagram of the type II dissolution apparatus is shown in Figure 2.3, where
paddle was used as the source of agitation (the paddle method).
Three different dissolution media (pH 6.8 phosphate buffer, 0.1 M hydrochloric
acid and deionized water) without additives were tested to find the best conditions to
evaluate the drug dissolution rate. From preliminary experiments whose results will be
shown in the section 2.3.6, the selected medium was 0.1 M HCl.
Three different rotation speeds were tested before setting a rotation speed of 75
rpm for the CRS 74 dissolution study (no enough powder dispersion in the medium at
50 rpm; no additional effect on powder dispersion at 100 rpm). In the following,
experiments were carried out using 30 mg of the reference product (as received CRS 74)
in 900 g of 0.1 M HCl at 75 rpm and 37.0 ± 0.5°C. Two milliliters samples were
withdrawn at specific intervals. The samples were filtered through a 0.22 µm filter
before the injection (20 µL) into the HPLC system (Agilent 1100 Series) for evaluate
103
CHAPTER 2
the amount of CRS 74 dissolved. The sample volume taken was not replaced by fresh
dissolution medium to prevent any possible interference with the chemical equilibrium.
D
H
h
0.1M HCl
C
Thermostated bath- 37°C
D= 10.5 cm
c= 2.85 cm
h= 6.5 cm
H= 11.7 cm
HPLC
Figure 2.3. Schematic diagram of Apparatus II (Paddle Apparatus):D: vessel diameter;
h: paddle width, c: distance between the paddle and the vessel bottom, H: liquid height
into the vessel.
2.2.1.7.2. High Performance Liquid Chromatography (HPLC) to determine the
content of the dissolved drug
Dissolved drug in dissolution media was determined by high performance liquid
chromatography (HPLC). This technique utilizes different types of stationary phases
contained in columns, a pump that moves the mobile phase and sample components
through the column and a detector capable of providing characteristic retention times for
the sample components and area counts reflecting the amount of each analyte passing
through a detector (Figure 2.4).
The HPLC system consists of an Agilent chromatograph (Model 1100 series)
equipped with a UV-vis detector. There are several parameters that quantitatively
measure how well a HPLC column separates the components of interest. These
104
Characterization of CRS 74
parameters will vary based on the dimension of the column, type of column, type of
mobile phase or stationary phase used, and HPLC instrument. The different HPLC
conditions tested during our method development were shown in Table 2.3. The mobile
phase was chosen after several trials with acetonitrile, methanol and phosphate buffer
pH 4.0 in different volume proportions. Finally, a mobile phase consisting of
acetonitrile/water (50:50) in an isocratic mode was selected to achieve maximum
separation.
HPMC Column
Packing Material
Chromatograms
Injector
Auto Sampler
Sample Manager
Computer Data Station
Solvent
(Mobile Phase)
Reservoir
Sample
Pump Solvent Manager
Solvent Delivery Ssytem
Detector
Waste
Figure 2.4. Schematic representation of a High Performance Liquid
Chromatography system.
Flow rates were arranged between 0.8 and 1.3 mL/min (Table 2.3). A flow rate
of 1.0 mL/min gave an optimum signal/noise ratio with a reasonable separation time (10
min) between the analyte peak and dissolution media peak. After comparison between
the different columns shown in Table 2.3 such as C8 Prontosil 100 mm x 4.6 mm, 5µm,
C8 Prontosil 250 mm x 4.6 mm, 5 µm and C18 Prontosil 250 mm x 4.6 mm, 5 µm, the
105
CHAPTER 2
best separation efficiency was obtained using ProntoSIL 120-5 C8 SH, 150 x 4.0 mm
column.
The retention time, i.e., the time taken for CRS 74 molecule to reach the detector
once it has been injected into the system, was observed to be 12 min. UV spectrum – of
CRS 74 showed maximum absorption at 210 nm; therefore, the compounds were
monitored at this wavelength. In summary the best conditions were set to the HPLC
analyses: the elution was done using a mobile phase consisting of acetonitrile/water in
the ratio of 50:50 on HPLC column ProntoSIL 120-5 C8 SH, 150 x 4.0 mm ID, at a
flow rate of 1.0 mLmin-1 with UV detection at 210 nm. Each experiment was carried out
in quadruplicate.
There is no monograph of this drug in any pharmacopoeia. After setting the HPLC
parameters for analysis, the HPLC quantification method was developed for the
quantification of CRS 74 concentrations and two validation parameters were tested
through specificity and linearity.
Specificity of the method was determined by analyzing the dissolution media with
and without the standard substance to verify the interference of the eluent in the CRS 74
concentration measurements. To assess the linearity of the method, seven calibration
standard solutions of CRS 74 dissolved in 0.1 M HCl were prepared over the
concentration range of 13- 289 µg/gsolution. The calculation of regression line was carried
out by plotting the peak area against standard concentration.
106
Characterization of CRS 74
Table 2.3. Different HPLC parameters tested during the HPLC method
development.
Column
Mobile phase (volume %)
Flow rate
(mL/min)
C8 PRONTOSIL
100mmx4.6mm,
5µm
ACN: 50% ; PB : 40% ; MeOH :
10%
1.3
ACN : 50% ; PB : 40% ; MeOH :
10%
C8 PRONTOSIL
250mmx4.6mm,
5µm
10
10
0.8
1.0
ACN : 50% ; PB : 50%
20
0.8
ACN : 30% ; PB: 70%
ACN: 50%; PB: 40% ; MeOH: 10%
C18 PRONTOSIL
250mmx4.6mm,
5µm
1.0
Volume
injection
(µL)
ACN : 50% ; PB: 30% ; MeOH :
20%
ACN : 60% ; PB: 30% ; MeOH :
10%
0.8
1.0
1.0
20
10
10
1.3
10
1.0
Prontosil 300-5ODSH, 5µm
ACN : 70 % ; Pure water : 30%
1.0
ProntoSIL 120-5 C8
SH, 150 x 4.0 mm,
5µm
ACN : 50 % ; Pure water : 50%
1.0
20
20
ACN: Acetonitrile; PB: Phosphate buffer ; MeOH: Methanol.
107
CHAPTER 2
The obtained results in HPLC analysis were used to calculate the percentage
dissolved at each time of dissolution profile. The cumulative percentage of dissolved
drug was plotted against time, in order to obtain the dissolution profile and to calculate
the in vitro dissolution data, using the equation (2.1).
Dissolved Drug (%) =
Abssample
StdPurity(%)
Absstd
(2.1)
where Absstd is the absorbance of the standard solution containing the original CRS 74
100% dissolved, Abssample the absorbance of the sample during the dissolution essay as a
function of time and StdPurity (%) the purity of the raw material, provided by the
supplier.
2.2.1.7.3.Drug solubility in dissolution media
The solubility of CRS 74 was determined by equilibrating an excess of CRS 74 in
5g of 0.1 M HCl at 37°C ± 0.5°C in a temperature-controlled bath for 24 h (Figure 2.5).
Figure 2.5. Experimental setup for solubility measurements.
The flasks were sealed for the duration of the tests and the concentration was
determined by removing the solid phase by filtration (0.22 µm pore size) and injection
of the filtered solution into the HPLC system to be analyzed at wavelength of 210 nm.
The HPLC parameters were already presented in the methodology.
108
Characterization of CRS 74
2.3. RESULTS AND DISCUSSION
2.3.1. Particle size, true density and morphology
The particle size distribution is shown in Figure 2.6. Laser diffractometry yields
the volume-weighted diameters. Particle size analysis values of dv90% dv50% and dv10%
were 515 µm, 101 µm and 4.3 µm, respectively. The broad particle size distribution
exhibited by the original drug powder is confirmed by the SEM micrographs (Figure
2.7).
5
4.5
4
Volume (%)
3.5
3
2.5
2
1.5
1
0.5
0
0.1
1
10
100
1000
10000
Size (µm)
Figure 2.6. Particle size distribution for as-received CRS 74 powder sample.
In addition, the SEM images at a higher magnification showed that the original
CRS 74 sample consisted of columnar crystals that were several micrometers long
(Figure 2.7 (b)). SEM images show the presence of agglomerates of small columnar
crystals (Fig 2.7 (a))
109
CHAPTER 2
(a)
(b)
Figure 2.7. SEM micrographs of the original CRS 74.
2.3.2. Density
The powder had a true density of 1.2295 ± 0.0205 g/cm3 when analyzed using
helium pycnometry.
2.3.3. XRD analysis
XRD analysis was performed to detect the changes in the physical state and
crystalline phases of the drug, before and after LAS crystallization. Figure 2.8 shows
the XRD patterns for the as-received powder sample. CRS 74 shows peaks at
approximately 8.5, 14, 16.9, 18.7, 19.4 and 21.3°, indicating that the drug was a
crystalline powder.
110
Characterization of CRS 74
25000
Intensité [I]
20000
15000
10000
5000
0
5
10
15
20
25
30
Position [2Theta]
Figure 2.8. X-Ray diffractograms of the as-received CRS 74 sample.
2.3.4. TGA and DSC analysis
Thermogravimetric analysis (TGA) was used in this study to determine the
thermal stability of CRS 74 by monitoring the weight change that occurred as the
sample was heated. From TGA data (Figure 2.9) it was observed that the drug becomes
thermally unstable from 215°C. Mass loss and a large peak were observed respectively
on the mass loss and on the heat flow at 242oC. This endothermic peak was observed in
an only one step. It probably characterizes the degradation of the drug. For this reason,
in the following, DSC analyses were carried out between 20 and 210°C to avoid the
drug thermal degradation.
111
CHAPTER 2
0,2
2
0
0
-0,2
Heat flow (W/g)
-0,6
EXO
-4
-0,8
-1
-6
-1,2
-8
-1,4
-1,6
-10
-1,8
-12
0
50
100
150
200
250
-2
300
Temperature (°C)
Figure 2.9. TGA-DSC curve of CRS 74 run in a nitrogen atmosphere and heating
rate of 10°C/min.
The DSC thermograms of CRS 74 powder, is presented in Figure 2.10. During the
heating-cooling cycle it was observed a broad endotherm peak at 188.6°C, which may
correspond to the melting of crystalline product.
Crystal energy is known to correlate with Tm(Onset) (onset melting point) and ΔHm
(enthalpy of melting), which refers to the energy a compound must overcome to
dissolve the drug (VIPPAGUNTA et al., 2007). The onset melting point and fusion
enthalpy obtained from the DSC study are summarized in Table 2.4. The molecule was
then characterized by a high melting point (188.6oC) and a high enthalpy of fusion (86.6
J/g).
112
Mass loss (%)
-0,4
-2
Characterization of CRS 74
0
Heat Flow (mW )
-5
E
EXO
-10
-15
-20
-25
140
150
160
170
180
190
200
Temperature (°C)
Figure 2.10. DSC thermograms of as-received CRS 74 sample (first heating),
which consists of a melting endotherm (peak onset temperature) Tm(Onset) = 188.6oC.
Table 2.4. Melting Temperature (Tm(Onset)), Enthalpy of fusion (ΔHm) for asreceived CRS74 sample.
Thermal
Cycle 1
parameters
(heating-cooling)
Tm(Onset)(°C)
188.6
Δ Hm (J/g)
86.6
113
CHAPTER 2
2.3.5. Determination of surface properties
The surface properties of drug samples were investigated to find a possible
relation between surface properties and dissolution. Sessile drop contact is most
commonly measured on compacted powder disc surface. However, compaction of the
material can alter the particle morphology and surface energy (BUCKTON, 1955).
Alternatively, some of the authors (AHFAT et al, 2000; HE et al, 2008) have reported
the use of powder layer adhered to an inert support. The powder layer was adopted for
the present work as it allows the study of “as is” powder properties. This method gave
reproducible values. It has been seen that contact angle measurements can vary with
experimental methodology, however the technique was used for evaluation of the
surface properties of original drug powder.
The contact angle of drop deposited on powder surface was plotted as a function
of time from 0 to 10 s, beyond which there was no significant change in the contact
angle. With water as the wetting liquid, original drug crystals exhibited a contact angle
almost unchanged with time from 136.4 ± 0.8° (0 s) to 136.6 ± 0.6°(10 s), as shown in
Figure 2.11.
150
145
Contact Angle (°)
140
135
130
125
θ= 136.6°±0.57 θ=136.4°±0.85 120
115
110
105
100
0
1
2
3
4
5
6
7
8
9
10
Time (seconds)
Figure 2.11. Contact angle (o) of water as a function of time for original CRS 74.
114
Characterization of CRS 74
The wetting process with water was quantified for the work of adhesion (Wa),
calculated using a combination of the Young and Dupré Equations (SCHRADER,
1995), cohesion (WCL) and the spreading coefficient (λ
λ LS), are calculated by the
following equations, derived from the Young’s equation (IVESON et al., 2001):
WCL = 2γ LV
(2.2)
WA = γ SL − (γ SV + γ LV ) = γ LV (cosθ +1)
(2.3)
λLS = W A − WCL
(2.4)
where, γ is the surface free energy and L, S and V refer to the state as being liquid, solid
and vapor respectively. Equation 2.3 is only valid for θ higher than 0, which is a
common case for hydrophobic powders. The contact angle made at 0 s was considered
as the initial contact angle and the surface tension of water was taken from the literature
(72.8 mN/m; PURI et al., 2010). These values were used to determine Wa, WCL and λLS
from equations (2.1), (2.2) and (2.3) for the drug powder (Table 2.5).
Table 2.5. Work of adhesion (WA), work of cohesion (WCL) and spreading
coefficient (λ LS) for CRS 74.
Sample
WA (mN/m)
WCL (mN/m)
λ LS (mN/m)
CRS 74
20.08
145.6
-125.6
The thermodynamic driving force for each process is indicated by the work value,
where a negative value denotes the spontaneity of the process (YOUNG and
BUCKTON, 1990). The positive Wa and WCL obtained for this new antiretroviral drug
115
CHAPTER 2
indicated that the adhesion and the cohesion processes are not spontaneous over the
original powder sample. Further, a negative spreading coefficient (λLS < 0) means that
this powder displayed an unfavorable spreading of water (NGUYEN and HAPGOOD et
al., 2010).
2.3.6. Dissolution studies
2.3.6.1. High Performance Liquid Chromatography (HPLC) to determine the
content of the dissolved drug
Specificity of the HPLC method
The specificity of a method gives us the guarantee that the result of the method
only comes from the analyte. Specificity of our method was determined by analysing
the dissolution media with and without the reference product to verify the interference
of the eluent in the CRS 74 concentration measurements.
The specificity of the HPLC quantification method was demonstrated in Figure
2.12. No interferences from the dissolution medium with the peak of interest were
observed in the HPLC chromatograms, confirming the selectivity of the method.
116
Characterization of CRS 74
HCl peak
(a)
CRS 74 peak
HCl peak
(b)
Figure 2.12. Specific test for CRS 74 in dissolution medium (0.1M HCl); (a)
HPLC chromatograms of placebo (dissolution medium); (b) HPLC chromatograms of
the component of interest dissolved in the dissolution medium. Flow rate of 1.0
mL/min; mobile phase consisted of acetonitrile:water (50:50).
117
CHAPTER 2
Linearity of the HPLC method
A linear analytical method indicates that it has the ability to demonstrate
experimentally that the results obtained are directly proportional to the concentration of
analyte in the sample within a specified range (FDA, 1997). To assess the linearity of
the method, seven solutions of CRS 74 dissolved in 0.1M HCl were prepared: 13
µg/gsolution, 26 µg/gsolution, 34 µg/gsolution, 51 µg/gsolution, 99 µg/gsolution, 213 µg/gsolution and
289 µg/gsolution.
The standard curve (Figure 2.13) showed an excellent correlation in the
concentration range of 13- 289 µg/gsolution (y = 36.2x, r2 = 0.99917, exceeding 0.99,
which is the minimum recommended by the regulating agencies ICH, 1996 and FDA,
1997). The limit of quantification (LOQ) of the assay was 0.5 μg/mL. The calculation
method used is based on the standard deviation (SD) of the y-intercepts of regression
lines and the slope of the calibration curve (S), according to the formula: LOQ =
10(SD/S) (ICH, 1996).
12 000
Absorbance (mAU)
10 000
8 000
6 000
4 000
2 000
0
0
50
100
150
200
250
300
350
Conc. (µg/gsolution)
Figure 2.13. Standard curve used for the determination of CRS 74 in samples
produced during dissolution experiments.
118
Characterization of CRS 74
CRS 74 solubility in dissolution medium and sink conditions
Sink conditions describe a dissolution system that is sufficiently diluted so that
dissolution process is not impeded by approach of saturation of compound of interest
(ROHRS, 2001). The amount of reference product (30 mg) set for our experiments
assured the presence of sink conditions in the dissolution medium because it
corresponds to a maximum drug concentration in the acidic medium 33 µg/gsolution, three
times less the equilibrium concentration of the drug in this medium, also determined
experimentally, 102 ± 8 µg/gsolution at 37°C.
Any optimal separations and symmetrical chromatographic peaks were observed
in HPLC chromatograms for CRS 74 in water or phosphate buffer 6.8 (Figures 2.14-a
and 2.14-b, respectively), reason for which the dissolution method were tested only in
acidic medium.
Most drugs are weak acids or weak bases that are present in solution as both the
ionized and unionized species. The solubility of a weak acid or a weak base is related to
pH. Acids ionize in alkaline medium, while bases ionize in acidic medium. The pKa is
the pH at which concentrations of ionized and un-ionized forms are equal (NEAU,
2008).
CRS 74 is a new ritonavir analogous compound (BOCKELMANN et al., 2005;
BOCKELMANN et al., 2010). Ritonavir is a weak base with two ionisable sites that
dissociate below pH 3 (BERTZ et al, 2004). The pKa of 2.8 for ritonavir refers to loss of
a hydrogen from a protonated thiazole group because thiazole itself is a weak base
(pKBH+, 2.4) (LIDE, 1988). Analogous to Ritonavir, CRS 74 is probably a weak base
with a pKa close to 3 with similar pH-dependent solubility behaviour.
According to the literature, Ritonavir is poorly water soluble with a solubility of
400μg/mL in 0.1M HCl at 37oC, which is reduced to 1 μg/mL at phosphate buffer pH
6.8 (LAW et al, 2001). In comparison, the solubility of CRS 74 experimentally
measured in this work was 102 μg/mL in 0.1M HCl and not detected (<0.5 μg/mL) by
HPLC in phosphate buffer pH 6.8 or water at the same temperature.
119
CHAPTER 2
Water pH 7.0
(a)
Phosphate buffer pH 6.8
(b)
Figure 2.14. HPLC chromatograms of the component of interest dissolved in (a)
water and (b) phosphate buffer pH 6.8.
2.3.6.2. In vitro dissolution testing
Figure 2.15 shows the dissolution profiles of original CRS 74 drug in 0.1 M HCl
(pH 1.2). It can be seen that the as-received drug did not even reach 20% dissolution
within 3h, confirming its poor tendency to dissolve in aqueous media. The poor
dissolution rate measured in this study can be related to the poor surface properties of
the powder presented in section 2.3.5, such as unfavourable spreading of water (λLS <0)
120
Characterization of CRS 74
and not spontaneous adhesion and the cohesion processes (positives values of Wa and
WCL).
100
90
Dissolveld Drug (%)
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (min)
Figure 2.15. Dissolution profile of CRS 74 in 0.1 M HCl at 37°C (n=4; SD =±2).
The dissolution step seems to be the limiting step of this process of CRS 74
release in the aqueous medium. The model of Hixson–Crowell (HIXSON and
CROWELL, 1931) describes the release from dosage forms, which show dissolution
rate limitation and which do not dramatically change during the release process. The
equation of Hixson–Crowell cube-root kinetic model is given by Equation 2.5.
(2.5)
where k is the kinetic constant (min -1), Mt is the mass of the drug dissolved in time t
and M0 is the initial drug mass in the dissolution medium.
121
CHAPTER 2
Our dissolution data were then plotted in accordance with Hixson–Crowell cube
root law (Figure 2.16) (correlation coefficient r2 = 0.984) to determine the dissolution
rate constant k, as given in Table 2.6.
0.2
0.18
0.16
1-[ ( 1-%D/100)1/3 ]
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
5
10
15
20
25
30
Time (min)
Figure 2.16. Application of Hixson-Crowell mathematical model on CRS 74
release profile.
Table 2.6. Values of k and regression equations for the mathematical models of
Hixson-Crowell applied to the CRS 74 dissolution data.
Sample
CRS 74
Hixson-Crowell
k (min -1)
Linear equation
0.0008
y = 0.0008 x - 0.0017**
k = dissolution rate constant.
** x = t (min) and y = 1-[(1-%Dissolved/100) 1/3 ]
122
Characterization of CRS 74
2.4.CONCLUSIONS
As-received CRS 74 was characterized by laser diffractometry, powder X-ray
diffraction (XRD), thermal gravimetric analysis (TG), differential scanning calorimetry
(DSC), scan electronic microscopy (SEM), surface properties and dissolution testing.
The results were presented and discussed in this chapter.
The saturation concentration of this molecule in water and phosphate buffer 6.8
was not detected by HPLC analysis. The molecule is slightly soluble in acid medium
(0.1M HCl pH 1.2) : 102 ± 8 µg/gsolution. Its low aqueous solubility can be explained by
its high crystallinity, its high melting point (188.6oC) and high enthalpy of fusion
(86.6 J/g).
The dissolution in vitro test is an important tool in quality control of drugs and it
becomes more important for drugs with low aqueous solubility such as CRS 74. Some
characteristics of this drug are not well defined, for instance, its classification in the
biopharmaceutical classification system. The aim of the dissolution study carried out
here was to contribute to define CRS 74 dissolution conditions, what can be the focus
for further studies. To date, there is no published dissolution test for the evaluation of in
vitro release profiles of CRS 74 from immediate-release solid oral dosage forms.
Therefore, we developed a dissolution method for CRS 74 to determine its release
profiles from powder samples. The low dissolution rate presented by this molecule can
be related to its large particle size (micrometric range, with a broad particle size
dispersion) and very poor water wettability (θ =136.4 ± 0.8°).
Physical factors important to drug dissolution include particle size, molecular size,
hydrophobicity, and crystalline structure. Physical modifications often aim to increase
the surface area, solubility, and wettability of the powder particles and, therefore,
typically focus on particle size reduction or generation of amorphous states. This work
planned to improve the dissolution rate of as-received CRS 74 molecule using a Liquid
Anti-Solvent crystallization process to increase the surface area and improve its
wettability.
123
CRS 74 solubility studies in hydroethanolic solutions
and design of experimental apparatus for crystallization
Résumé Chapitre 3- Étude de la solubilité et développement du
protocole expérimental
Le choix du solvant est un des paramètres essentiels pour pouvoir envisager une
opération de cristallisation. La connaissance de la solubilité d'un composant dans
différents solvants est requise. L’éthanol et l’eau ont été retenus respectivement comme
solvant et anti-solvant.
Dans ce chapitre, la solubilité du CRS 74 en mélanges binaires éthanol et éthanoleau a été mesurée dans la plage de température de 5 – 30 °C, afin de déterminer le cadre
expérimental pour permettre la cristallisation par effet anti-solvant.
Dans un premier temps, les équilibres solide-liquide dans l’éthanol pur et dans des
mélanges binaires (éthanol/eau) ont été étudiés pour une gamme de température
comprise entre 5 et 30 °C. Le solide étudié est très soluble dans l'éthanol à 30 °C : 92,6
mg/g de solution. A 30°C, la solubilité de la molécule diminue quand la quantité d’eau
dans le mélange augmente. Les solubilités obtenues ont été représentées en utilisant le
modèle UNIQUAC pour le calcul des coefficients d’activité. Les solubilités
expérimentales et calculées présentent un bon accord. La solubilité calculée dans des
mélanges eau-éthanol présente un maximum de 130,20 mg/g de solution pour un ratio
massique éthanol/eau de 0,83/0,17 (w/w). Les résultats de la modélisation indiquent que
ce modèle est l'outil approprié pour représenter le comportement de solubilité de CRS
74 dans des mélanges de solvants (éthanol-eau). Les solubilités expérimentales et
calculées ont permis d’évaluer le ratio éthanol/eau optimum (25/75 % m/m) pour
maximiser le rendement théorique en solide.
Dans un second temps, un mélange double jet avec pré-mélangeur type mélangeur
en T ou mélangeur Roughton a été choisi pour réaliser la cristallisation. Dans les deux
cas, des nanoparticules sont créées en sortie du pré-mélangeur. Des expériences
préliminaires ont montré que le solide présente des vitesses de croissance et
d’agglomération élevées. Les particules obtenues avec le mélangeur en T semblent
moins agglomérées.
Ce type de mélangeur a donc été retenu pour la suite de l’étude. Deux principaux
résultats ont été obtenus.
Le solide cristallisé est toujours aggloméré. Son profil de dissolution dans une
solution acide (0,1M HCl) reste inchangé. À partir de ces résultats, il a été conclu que
les débits d’entrée des solutions (paramètre opératoire) n'ont pas d'influence sur les
particules recristallisées. Après avoir défini, les paramètres du processus de
cristallisation, le produit synthétisé ne présente pas de changements de cristallinité ni de
changements liés aux propriétés de surface (mouillabilité). Toutefois, une diminution de
la taille des particules a été observée. Aucun réel impact sur la vitesse de dissolution de
la poudre synthétisée par effet anti-solvant n’a été observé.
Le mélangeur se colmate après quelques minutes d’utilisation. Il peut s’agir d’un
problème d’affinité du solide avec la paroi inox du pré-mélangeur et/ou d’un
phénomène d’agglomération très rapide. Afin d’améliorer la cinétique de dissolution de
la poudre recristallisée, il est nécessaire de rendre leur surface plus hydrophile en
utilisant par exemple des additifs différents afin d'optimiser les paramètres du procédé
et de formulation.
Solubility study and experimental design
3.1. INTRODUCTION
Chapter 2 summarizes the crystal properties, which are experimentally
determined in this work. The poor water solubility, bad wettability and the low
dissolution rate in acidic medium could be confirmed. In order to modify these
properties, this work proposed a recrystallization using an Anti-Solvent-Liquid process.
The solvent selection is one of the essential parameters to envisage any
crystallization process. Therefore, the knowledge of the solubility of a target component
in different solvents is required. In this work, the solubility of CRS 74 in ethanol and
ethanol-water binary mixtures was measured in the temperature range of 5 -30oC.
Although experimental data on solubility are essential to provide information
about a system and help to understand its behavior, correlations and prediction models
are also required for the correct design of crystallization processes. Solid-liquid
equilibria of ternary mixtures containing ethanol (solvent), water (anti-solvent) and the
new antiretroviral drug were studied. The solubility data were estimated using
UNIQUAC-based model.
After determining the solubility of the active ingredient in ethanol (solvent)water (anti-solvent), an experimental system was developed to study CRS 74
recrystallization. As mentioned in Chapter 1, crystal properties are influenced by a
number of operating variables in anti-solvent crystallization, some of which are more
influential than others.
The present Chapter is composed of two sections. The first one concerns the
solubility study and the second one the crystallization process design. The methodology
and the results obtained are presented.
129
CHAPTER 3
3.2. MATERIALS AND METHODS
3.2.1. Materials
The active pharmaceutical ingredient (CRS 74) with 99% purity was provided as
courtesy from Cristalia Ltda (Itapira, SP, Brazil), ethanol (EtOH) from Fluka Analytical
(Sigma–Aldrich,
France)
and
acetonitrile
(ACN)
high
performance
liquid
chromatography (HPLC) grade from Scharlau Chemie (Barcelona, Spain). Ethanol and
Acetonitrile had purity higher than 99%. All products were used as supplied.
3.2.2. Methods for solubilty measurements
The solubility of CRS 74 was determined by equilibrating an excess of CRS 74
in 5 g of water, ethanol and different ethanol/water combinations at 30 ± 0.5°C in a
temperature-controlled bath for 72 h (Figure 2.5). The flasks were sealed for the
duration of the tests and the concentration was determined by removing the solid phase
by filtration (0.22 µm pore size) and injection of the filtered solution into the HPLC
system to be analyzed at wavelength of 210 nm. The HPLC system consisted of an
Agilent Chromatograph (Model1100 series) equipped with a UV-vis detector, and of an
HPLC column ProntoSIL 300-5-ODSH 5µm, 250x4 mm ID. The flow rate of mobile
phase (acetonitrile/water in the ratio of 70:30) is 1.0 mLmin-1.
3.2.3. Methods for Liquid Anti-Solvent (LAS) crystallization
After drug solubility characterization and definition of the best crystallization
conditions, some process parameters were evaluated. In this part of the study, different
essays at different operational conditions were realized in order to evaluate the influence
of the process parameters on the final size and agglomeration state of particles at the
end of the crystallization process.
The original CRS 74 crystals were recrystallized by a LAS crystallization
method. As already discussed in Chapter 1, this method is based on saturation changes,
when the drug is dissolved into a solvent and then this solution is mixed with an antisolvent of solute.
130
Solubility study and experimental design
Anti-solvent and saturated solutions can be brought into contact in several
manners: single-jet, double-jet or double-jet with premixing (Chapter 1).
In the crystallization proceeding for CRS 74 (expensive pharmaceutical drug)
the use of single jet was not viable due to use of a large amount of drug for each
experiment. So, a double-jet with premixing has been used. So rapid mixers combined
with a stirred vessel were used as process configuration. Briefly, the drug was
recrystallized via the concurrent introduction of the CRS 74 ethanol solution and an
anti-solvent stream of water in stainless steel mixers specially manufactured for this
study, on the basis of previous works published in the literature (LINDENBERG et al.,
2008; LINDENBERG, 2009). The experimental setup is shown in Figure 3.1, in which
the mixing device can be easily changed to test different mixers, which are presented in
more details hereafter.
Briefly, a certain amount of original CRS 74 samples was completely dissolved
in ethanol at 30 ± 0.5°C at definite concentration (90 mgCRS 74/gsolution). The solution was
filtrated through 0.22 µm pore size membranes to remove the possible particulate
impurities. To each experiment the solutions were fed into the mixing device by gear
pumps (mzr-7255-hs-f S, mzr-7205-hs-f S; HNP Mycrosysteme). The freshly formed
crystals were collected in a vessel under magnetic stirring and then they were filtered
and dried under vacuum at 50 ± 1°C for 24h. The dried samples produced by this LAS
crystallization process were characterized by laser diffractometry, differential scanning
calorimetric analysis, X-ray diffraction analysis, scanning electron microscopy analysis,
wetting properties and dissolution testing.
The flow rates of CRS 74 ethanol solution and water were fixed at flow rates of
11.29 g/min and 33.22 g/min respectively, such after mixing a definite supersaturation
ratio (S) was calculated from the conditions at the entries of the T-mixer
(S = C/Ceq = 894). Gear pumps and a digital mass flow meter/controller (M1X,
Bronkhorst) were used in the experimental process to ensure minimal flow rate
fluctuations and good mixing. In order to validate the flow of gear pumps the pumps
were previously calibrated (data shown in Appendix II).
131
CHAPTER 3
Gear pump
L
Gear pump
Drug solution at 30°C*
Solvent=Ethanol
Final drug suspension in
the water-ethanol
solution
Water at 30°C*
°C*
nt)
(anti-solvent)
L
* Temperature-controlled bath
Figure 3.1. Schematic of the experimental apparatus used for the LAS
crystallization experiments.
Mixers
Two different types of mixers were tested: a T-mixer with two radial entries and
a two jets vortex mixer also called Roughton mixer. The T-mixer had two radial entries
with a diameter of 1 mm and its outlet tube had a diameter of 2 mm and a length of
17.5 mm. The Roughton mixer has a mixing chamber diameter of 3 mm and its outlet
tube has a diameter of 1.75 mm and a length of 15 mm. A schematic of both mixers is
showed in Figures 3.2a and 3.2b.
132
Solubility study and experimental design
2mm
2mm
3mm
1mm
3mm
2mm
15mm
17.5mm
1.75mm
(a)
(b)
Figure 3.2. Sketch of the mixers used: (a) Roughton mixer, (b) T-mixer.
3.2.3.1.Characterization methods for particles in suspension
Particle size analysis
The mean size and particle size distribution of the drug particles generated by
LAS recrystallization were analysed in different phases of the process.
Firstly, freshly particles were analysed by Photon correlation spectroscopy (PCS)
using a Zetasizer Nano Zs (Malvern Instruments, United Kingdom) to measure the
mean size at the exit of the mixer right after the mixing proceeding (from the outlet of
the mixer), as shown in Figure 3.3. Two measurements are made at 0 and 150 seconds
(measure time). Before analysis, the suspension was diluted 5 times using a saturated at
30°C. This saturated solution was composed by water, ethanol and drug, to achieve
appropriate measurement concentration. It was filtrated through 0.22 µm pore size
membranes to remove the possible particles. The new supersaturation of solution S’ can
be calculated by this expression:
133
CHAPTER 3
S’= 5/6 + S/6
(3.1)
In a second time the drug suspension was analysed by laser granulometry, using
a MasterSizer 3000 (Malvern Instruments, United Kingdom). The samples were taken
from the outlet of the mixer (right after the mixing) and immediately diluted with the
same saturated solution in order to monitor the evolution of crystal size as a function of
the time in the cell of the granulometer, as shown in Figure 3.3.
Particle size evolution
Laser Granulometry
Particle size evolution
PCS
Particle size range 0.1-3500µm
Particle size range 0.15nm-10µm
Sampling
Sampling
Dilutionn
Dilution
Measure time (s)
()
t0
Measure time (min)
t150
t0
t10
Final drug suspensionn
in the water-ethanoll
solution
Figure 3.3. Sketch of particle size analysis for particles in suspension by PCS and
laser granulometry.
Optical microscopy analysis
Samples were taken from the final suspension into the vessel and immediately
analysed by optical microscopy.
134
Solubility study and experimental design
3.2.3.2. Characterization methods for dried powder
The dried samples were analysed by laser diffractometry, powder X-ray
diffraction (XRD), thermal gravimetric analysis (TG), differential scanning calorimetry
(DSC), scan electronic microscopy (SEM), contact angle measurement and dissolution
testing, using the same procedure already described in Chapter 2. Furthermore, purity
and solvent content were determined too.
Purity
In order to evaluate the purity of drug solid, approximately 1.5 mg of CRS 74
was solubilized in approximately 40 g of ethanol. This solution was then assayed by
HPLC analysis at wavelength of 210 nm to evaluate the amounts of dissolved drug. The
HPLC system consisted of an Agilent Chromatograph (Model1100 series), equipped
with a UV-vis detector, column Prontosil 300-5-ODSH 5µm (5 µm, 250x4 mm). The
flow rate of mobile phase (acetonitrile/water in the ratio of 70:30) is 1.0 mL min-1. All
experiments were carried out in triplicate. The purity is the ratio between the measured
concentration of the synthetized powder and the initial concentration of the raw
material. The purity of the synthesized powders was calculated as shown below.
Purity(%) =
Abssample
× Purity CRS 74 (%)
AbsCRS 74
(3.2)
where AbsCRS 74 is the absorbance of the standard solution containing the raw material
100% dissolved, Abssample is the absorbance of the sample containing the synthesized
material 100% dissolved. And PurityCRS 74 is the purity of the raw material, provided by
supplier. For all purity results, the standard deviation was not shown, because it was not
significant.
135
CHAPTER 3
Residual solvent content
The residual solvent (water+ethanol) content was determined using Infrared
balance (LJ16, Mettler) at 100°C until constant weight was achieved.
3.3. RESULTS AND DISCUSSION ON SOLUBILITY STUDY
3.3.1. Measurement and correlation of solubility of CRS 74 in water-ethanol
mixtures
3.3.1.1. Experimental determination
Solubility data were experimentally measured in the temperature range of 5 to
30oC for pure ethanol and for a mixed solvent system containing 95%(w/w) water and
5% (w/w) ethanol. For analytical purposes, the solubility in pure water could not be
measured. Indeed, the low concentration of a solid in solution, that should lead to
analytical results unreliable, that can be not quantified due to sensitivity of the
quantification method.
Concentration measurements have made as function of time and are shown in
Tables 3.1 and 3.2.
The standard deviation obtained (± 5 %), is mainly due to evaporation losses of
solvent after sampling, but also closely related to systematic error (analytical method),
like dilution and calibration curve.
For the two mass ratios, the concentrations measured at 24, 48, 72 h are identical
(Tables 3.1 and 3.2). Subsequently we have considered, for all solutions, the liquid-solid
system has reached equilibrium after 24 h of stirring at a controlled temperature ±
0.5°C. Thereafter, all the experimental results are the average of the three
concentrations measured respectively at 24, 48 and 72 h (Table 3.1 and Table 3.2).
136
Solubility study and experimental design
As shown in Table 3.1, the solubility of CRS 74 in pure ethanol was enhanced
by temperature rise above 15oC, whereas the solubility in the mixed aqueous system
containing 95% (w/w) water - 5% (w/w) ethanol was not affected by the temperature in
all studied temperature range. With this last ratio, the solubility of CRS 74 is equal to
0.0040 ± 0.0001 mg/g solution.
The solubility of in different water-ethanol mixtures at 30oC was listed in
Table 3.3.
Table 3.1. Solubility of CRS 74 in ethanol as function of sample time in
temperature range (5-30°C).
C (mg/g) C (mg/g) C (mg/g)
C
SD
T (°C)
24h
48h
72h
(mg/g)
(mg/g)
5
68.1
65.3
73.4
68.9
4.1
10
67. 5
67.8
68.9
68.1
0.8
15
66.4
69.7
61.4
65.8
4.1
20
72.0
74.8
77.7
74.8
2.8
25
78.3
82.7
81.6
80.9
2.3
30
90.6
93.0
94.3
92.6
1.9
137
CHAPTER 3
Table 3.2. Solubility of CRS 74 in 95% (w/w) water - 5% (w/w) ethanol mixture
as function of sample time in temperature range (5-30°C).
C (mg/g) C (mg/g) C (mg/g)
C
SD
T (°C)
24h
48h
72h
(mg/g)
(mg/g)
5
0.0042
0.0048
0.0036
0.0042
0.0006
10
0.0042
0.0041
0.0038
0.0040
0.0002
15
0.0042
0.0041
0.0038
0.0040
0.0002
20
0.0036
0.0036
0.0036
0.0036
0.0000
25
0.0039
0.0041
0.0039
0.0040
0.0001
30
0.0041
0.0041
0.0040
0.0041
0.0001
The results revealed that this molecule exhibited a very poor solubility in water
(not detected by HPLC analysis) and a solubility of 92.6 mg/gsolution in pure ethanol. On
the one hand, Table 3.3 shows that the addition of ethanol to water at 30°C changed the
solubility of and that ethanol could be used as an organic co-solvent to change
its solubility in aqueous media. It is well-known that the addition of an organic cosolvent to water can dramatically change the solubility of drugs (YALKOWSKY and
ROSEMAN, 1981) as observed here for more than 40(w/w) . It is reported
that a solvent is sufficient for pharmaceutical processing when the solubility exceeds 1
mg/mL (LEE et al., 2006; SMITH et al., 2011). On the other hand, combinations
containing more than 60% (w/w) water as an anti-solvent are favourable for further
crystallization studies.
138
Solubility study and experimental design
Table 3.3. Solubility of CRS 74 in Ethanol/water mixtures at 30 °C.
Solvent
Mean Solubility (mg/gsolution) ± SD
Water
not detected by HPLC analysis
95%(w/w) water - 5%(w/w) ethanol
0.004±0.0001
85%(w/w) water - 15%(w/w) ethanol
0.010±0.0004
80%(w/w) water - 20%(w/w) ethanol
0.020±0.0020
75%(w/w) water - 25%(w/w) ethanol
0.030±0.0020
70%(w/w) water - 30%(w/w) ethanol
0.070±0.0030
60%(w/w) water - 40%(w/w) ethanol
2.290±0.0900
50%(w/w) water - 50%(w/w) ethanol
12.300±1.4100
40%(w/w) water - 60%(w/w) ethanol
60.960±5.2400
30%(w/w) water - 70%(w/w) ethanol
87.890±4.0000
Ethanol
92.600±1.9000
3.3.2. Correlation of solubility data by a UNIQUAC model
At the liquid-solid equilibrium, the chemical potential of the constituent CRS 74
in solid phase is equal to the chemical potential of the constituent CRS 74 in liquidphase at constant temperature and pressure. From this equality of chemical potential, we
can
show
that
the
activity
of
the
solute
CRS
74
in
liquid-phase
(
) is a function of melting enthalpy of CRS 74,
∆Hm, melting temperature Tm and solution temperature T (WALAS, 1985):
(3.3)
139
CHAPTER 3
xCRS 74 solute being the molar fraction of the solute at saturation, γCRS 74 solute the
activity coefficient of solute.
The model UNIQUAC (UNIversal QUASI Chemical) is chosen to calculate the
activity coefficient of solute.
For a ternary system, twelve parameters are necessary: six parameters
concerning the geometry of molecules and six binary interaction parameters. The first
parameters, Rk and Qk, are estimated from the molecular formula of component. The
second parameters, binary interaction parameter water/CRS 74 and ethanol/CRS 74,
will be identified from solubilities measurement. Binary interaction parameters
water/ethanol are available in DIPPR table.
The volume and surface area parameters Rk and Qk of three components are
given in Table 3.4.
Table 3.4. Parameter Rk and Qk.
Molecule
Water (DIPPR)
Ethanol (DIPPR)
CRS 74
Rk (cm3/mol)
0.9200
2.1055
37.4015
Qk (cm3/mol)
1.4000
1.9720
29.4640
The binary interaction parameters of the UNIQUAC model have been identified
in two steps. Initially, solubility data on the binary ethanol/CRS 74 as function of
temperature were considered to estimate the two binary interaction parameters
ethanol/CRS 74 from the activity coefficient of the solute calculated by the equation
(3.3) (Table 3.5).
In a second stage, the binary interaction parameters of water/CRS 74 were
calculated from experimental data over the ternary water/ethanol/CRS 74 with a mass
ratio ethanol/(ethanol-water) of 5% and variable temperature (Table 3.5). A ternary
mixture has been chosen because the solubility data in water as a function of
temperature are not available.
140
Solubility study and experimental design
In both cases, the minimized function fmin used for parameter estimation is
calculated the following relation:
(3.4)
Table 3.5. Binary interaction parameters.
Binary Systems
i-j
uij (cal/mol)
Water-CRS 74
1-3
-400.55
3-1
22 132.13
2-3
-381.47
3-2
23 701.66
Water-Ethanol
1-2
-96.4730 + 0.6843 * T(K)
(database Simulis)
2-1
31.6290 + 0.4759 * T(K)
Ethanol-CRS 74
The experimental and calculated activity coefficients ( and
) for the two systems are reported in Table 3.6. The means of standard
deviation (equation 3.5) for the binary system ethanol/CRS 74 and for the ternary
system water/ethanol/CRS 74 are respectively 0.123 and 0.196 on the ternary system.
(3.5)
141
CHAPTER 3
Table 3.7 gives the solubilities calculated in pure ethanol as a function of
temperature. The calculated concentrations are in good agreement with experimental
values for temperatures above 15°C. Below this temperature, the calculated
concentrations are significantly underestimated.
Figure 3.4 shows the calculated solubility at 30°C as a function of the ethanol
mass proportion in the mixture ethanol-water with the identified parameters. The
calculated data showed good agreement with experimental results and revealed a
maximum solubility of CRS 74 of 130.20 mg/gsolution for a mass ratio of 17%(w/w)
water - 83%(w/w) ethanol. The maximum solubility for a solute in a mixed solvent
system has been observed experimentally for other systems (water/acetone/ketoprofenESPITALIER et al., 1995; water /ethanol /paracetamol and water/dioxane/phenacetinRUCKENSTEIN and SHULGIN, 2003; water/ethanol /hydrocortisone- ALI et al., 2009
and n-heptane/ethanol/eflucimide - TEYCHENE and BISCANS, 2011). Different
studies on Hidelbrand solubility approach have shown that the location and the height of
the peaks could be linked with the polariry of the solute (JOUYBAN-GHARAMALEKI
et al., 2000, PEÑA et al. 2006). Hydroxyl and amine groups of solute give a polar
character to this molecule that could explain the maximum of solubility calculated for a
high ratio of ethanol in the mixture.
Figure 3.4. Experimental (characters) and calculated CRS 74 solubilities (__) in
different ethanol-water mixtures at 30 ± 0.5oC.
142
Solubility study and experimental design
Table 3.6. Experimental and calculated activity coefficients for ethanol/CRS 74
and ethanol/water/CRS 74.
System ethanol/CRS 74
System water/ethanol/CRS 74
SD = 0.123
Ethanol/(Ethanol+water) = 0.05
SD = 0.196
T (°C)
10-4
10-4
5
0.0957
0.1842
0.4165
1.0533
10
0.1765
0.2562
0.7947
1.5338
15
0.3261
0.3475
1.4161
2.2045
20
0.4965
0.5200
2.7487
3.1296
25
0.7829
0.7449
4.2884
4.3908
30
1.1373
1.1081
6.9886
6.0919
Table 3.7. Solubility of CRS 74 in pure ethanol at different temperatures.
C
SD
Ccalc
(mg/g)
(mg/g)
(mg/g)
5
68.9
4.1
46.4
10
68.1
0.8
54.6
15
65.8
4.1
63.4
20
74.8
2.8
72.9
25
80.9
2.3
83.1
30
92.6
1.9
93.9
T (°C)
143
CHAPTER 3
3.3.3.Theoritical yield of solid obtained by LAS crystallization
Measurements and correlation of solubility of CRS 74 in different ethanol/water
mixtures provided useful data for a better understanding of the solubility phenomenon
in these media and to estimate the theoretical efficiency of the LAS recrystallization
process as a function of ethanol/water mass ratios. Theoretical solid efficiency is the
theoretical solid yield of crystallization based on the drug solubility in the liquid media,
calculated from the ratio of weight particles obtained by assuming solid-liquid
equilibrium attained and the initial weight of solute. Figure 3.5 depicts the theoretical
solid efficiency of the LAS crystallization for the drug. It can also be seen that an
ethanol concentration up to 50%(w/w) in ethanol/water mixtures is still favorable for
the crystallization process, resulting in recovery of 82.6% (50 %Ethanol) of CRS 74
based on the drug solubility in the liquid media.
In an attempt to improve its dissolution properties, CRS 74 can be recrystallized
by using a Liquid Anti-solvent (LAS) crystallization process and the data generated
here can represent a useful tool to define the mass proportion between solvent (ethanol)
and anti-solvent (water) for LAS crystallization studies.
100%
90%
Theoretical yield (%)
80%
70%
60%
50%
40%
30%
20%
10%
0%
0
5
15
20
25
30
40
50
60
70
100
Ethanol/(Ethanol-water) (w/w)
Figure 3.5. Theoretical yield of CRS 74 in different ethanol/(ethanol-water) mass
ratios.
144
Solubility study and experimental design
3.4. RESULTS AND DISCUSSION ON CRYSTALLIZATION
STUDY
After determining the solubility of the active ingredient in ethanol (solvent)water (anti-solvent), it was developped an experimental system to study CRS 74
recrystallization solvent. The crystals of CRS 74 were prepared by LAS crystallization
from an ethanolic saturated solution at 30 °C (90 mg/gsolution) and water, used like antisolvent. The crystals suspension obtained after the crystallization process had a milky
aspect that changed into a clear solution, after some seconds under magnetic stirring.
This phenomenon can indicate the growth and the subsequent agglomeration of the
crystals in solution. That can be observed in the form of long and big macroscopically
visible agglomerates.
Two different rapid mixing devices for controlling the properties of CRS 74
particles produced by LAS crystallization were used. Two process parameters were
tested: type of mixer and flow rate, which will be discussed here. For all essays at least
two replicates were realized to evaluate the repeatability of the process and particle size
measure method. The process conditions are given in the Table 3.8.
The solid has been characterized after crystallization (still in suspension) and after
crystallization, filtration and drying. The characterization step concerns the particle size
measurement at the outlet tube of the mixer and the characterization of dried powder by
particle size measurement, morphology, crystalline structure and dissolution properties.
145
CHAPTER 3
Table 3.8 Experimental LAS crystallization process conditions
Experiment
Solvent flow
rate (g/min)
Antisolvent
flow rate
(g/min)
Total flow
(g/min)
Type of
mixer
1
11.29
33.22
44.51
Roughton
2
11.29
33.22
44.51
T
3
6.77
20.32
27.09
T
4
17.46
52.1
69.56
T
3.4.1. Characterization of particles in suspension
3.4.1.1. Influence of the type of mixer on mixing
In order to compare the efficiency of both mixers parameters such as residence
time (τ), Reynolds number (Re) and energy dissipation rate (ED) were calculated for
each mixer and for each flow rate tested.
The residence time τ in the mixer is calculated from V the volume of the mixer
(mL) and Q the volume flow rate (mL/s):
(3.6)
In our system, the Reynolds number can be calculated as follows (FOX et al.,
2004).
(3.7)
with Re = Reynolds number, = density of the mixture (kg/m3), u = velocity of the
mixture (m/s),= flow rate of the mixture (m3/s), D = diameter of the outlet tube (m),
µ = viscosity of the mixture (N.s/m2), A = cross sectional area of the outlet tube (m2).
146
Solubility study and experimental design
It has been described that for Reynolds number above 1600, the mixing process
is completed in the main mixing chamber. On the other hand, for low Reynolds number,
this process continues at the outlet tube. Therefore, the larger the Reynolds number, the
faster the mixing process and, consequently, a uniform composition in the mixer will be
faster achieved.
The energy dissipation is a factor that strongly influences the quality of mixing.
The purpose of the mechanical energy dissipated by the moving fluid is to homogenize
fields of concentrations by displacement of fluid portions. The perfect mixing at the
molecular level is sometimes achieved so late that the solute molecules are already
chemically transformed or in phase change. The energy dissipated can be calculated as
follow.
(3.8)
where ED = energy dissipated (W/kg), f = friction factor, V = velocity of the fluid at the
outlet tube (m/s), D = diameter of the outlet tube (m).
By generating homogenized fields of concentration in the solution, the energy
dissipation influences the mean velocity convection, the turbulent diffusion and the
viscous-convective deformation (LINDENBERG and al, 2008). The improvement of
these factors means an improved mixing.
Properties of solvent mixture used, like density (ρ) and viscosity (µ) were
calculated as shown in Table 3.9. These values were determined for the solvents
mixture composed by ethanol (25%, w/w) and water (75%, w/w) at 30°C, without
taking into account the influence of the solid (KHATTAB et al., 2012).
147
CHAPTER 3
Table 3.9 Density (ρ) and viscosity (µ) of ethanol/water mixtures at 30°C
(KHATTAB et al., 2012).
ρmixture (kg/m3)
µmixture (N.s/m2)
955.4
0.0017
According to Table 3.10, Reynolds numbers were lower than 1600, so the
mixing process continues at the outlet tube. Moreover, Roughton mixer shows better
mixing efficiency when compared to the T-mixer. For the same flow rate, the Roughton
mixer presents lower mixing time, which is required in order to have a uniform
concentration distribution in the mixer, wide causes particle size distribution (ZHAO et
al., 2011). It also shows higher Reynolds numbers and energy dissipation rates. It means
a greater turbulence in the system that favours a closer contact between the fluid for
total flow rate higher than 7 g/min and, consequently, results in improved mixing.
Table 3.10. Calculated mixers parameters for: Roughton mixer (a) and T-mixer
(b).
QEtOH (g/min)
QH2O (g/min)
Qtotal (g/min)
τ (s)
Re
ED (W/Kg)
6.77
20.32
27 09
0.08
195
0.7
11.29
33.22
44 .51
0.05 321
17.46
52.1
69.56
0.03
503
2
4.9
(a)
QEtOH (g/min)
QH2O (g/min) Qtotal (g/min) τ (s) Re
ED (W/Kg)
6.77
20.32
27.09
0.11 171
0.3
11.29
33.22
44.51
0.07 281
0.9
17.46
52.1
9.56
0.04 440
2.2
(b)
148
Solubility study and experimental design
3.4.1.2. Influence of the type of mixer on particle size
For CRS 74 LAS crystallization the proportion ethanol:water equal to 25%:75%
(w/w) and total flow rate of 44.51 g/min, were used, as given in Table 3.8. Results
obtained by PCS and laser granulometry are presented.
PCS results
In a first time the influence of the type of mixer on particle size (PSD) by PCS
was evaluated. Different essays using Roughton and T mixers at the same process
conditions were realized. In order to follow the evolution of particle size and understand
the influence of each mixer on the size of the final product.
It was clear observed, that the particle size at 0 s for both mixers is small
(nanoscale), but they evolved very fast inside the equipment during the measurement
time (150 s), as shown in Table 3.11. This fast evolution of size could be explained by
two phenomena: growth or aggregation/agglomeration. A certain variation of size value
for the same essay was noticed as well. This difference of the measures values can be
acceptable due to the high growth or agglomeration rates of the particles produced,
which complicates the measurement accuracy. This variation among the measures was
noted at 0 and 150 s for both mixers.
It was noted, that the mean size of particle obtained with Roughton mixer seems
to be smaller than that obtained with T-mixer at t0 = 0 s. After tf =150 s, none difference
is observed. This behaviour can be explained by the fact that it provokes a better mixing
when compared to the T-mixer, as showed before in Table 3.10 of this chapter.
149
CHAPTER 3
Table 3.11. Evolution of crystallized particle size by PCS in different mixers,
Roughton mixer (a) and T-mixer (b) at total flow rate of 44.51 g/min and ethanol-water
ratio of 25-75 % (w/w).
Experiment
Size (nm) at t0 = 0 s
Size (nm) at tf = 150 s
1A
224
778
1B
153
926
1C
120
655
Average
166± 54
787± 136
(a)
Experiment
Size (nm) at t0 = 0 s
Size (nm) at tf = 150s
2A
269
845
2B
393
815
Average
331± 88
830± 21
(b)
Laser granulometry results
The evolution of the particle size was still followed by laser granulometry, from
the outlet of the mixer, until size stabilization of crystals during 10 minutes, in which
each measure lasts around one minute. The first and the last measure of this period of 10
minutes were shown in Figures 3.6 and 3.7.
Analysing the results for each mixer separately, it was noted the fast crystal
growth or agglomeration. During the 10 minutes analysis the particle size increased 30
times of its initial size of 2 µm, i.e. almost a mean growth rate of 7 µm/min. This
behaviour is due to tendency of the molecule to grow quickly and/or to form larger
agglomerates.
150
Solubility study and experimental design
In figure 3.7 it was noted that T-mixer presents particles with smaller sizes at the
end of the measure (10 min) when compared with the size particle distribution of the
particles crystallized in the Roughton mixer (see Figure 3.6).
Optical microscopy photos can confirm the agglomeration phenomenon in the
cell of laser granulometer. However, as shown in Table 3.12, the particles obtained
using the Roughton mixer presented an agglomeration state of particles more
significant, when compared to the particles obtained using the T-mixer. The presence of
these agglomerates was observed too in Figure 3.6, where a family of particles was
noticed at 500 µm.
Table 3.12. Crystals at tf =10 min in suspension observed by optical microscopy
Experiment
Type of mixer
Total Flow rate
500 µm
1A
Roughton
44.51 g/min
2A
T
44.51 g/min
151
CHAPTER 3
12
10
1A
Volume (%)
8
1B
1C
6
4
2
0
0.01
0.1
1
10
100
1000
10000
Size (µm)
Experiment
D[4;3] (µm)
Span
1A
3
1.8
1B
1
1.2
1C
2
1.4
Average
2±1.0
1.5±0.3
t0 = 0 min
14
12
1A
10
Volume (%)
1B
8
1C
6
4
2
0
0.01
0.1
1
10
100
1000
10000
Size (µm)
Experiment
D[4;3] (µm)
Span
1A
85
6.3
1B
72
2.6
1C
51
1.1
Average
69±17.0
3.3±2.7
tf = 10 min
Figure 3.6. Particle size distributions by laser granulometry for Roughton mixer
at t0 = 0 min and tf = 10 min at total flow rate of 44.51 g/min and ethanol-water ratio of
25-75 % (w/w).
152
Solubility study and experimental design
14
12
10
2A
Volume (%)
8
2B
6
4
2
0
0.01
0.1
1
10
100
1000
10000
Size (µm)
Experiment
D[4;3] (µm)
Span
2A
2
1.3
2B
2
2.6
Average
2±0.0
1.9±0.9
t0 = 0 s
12
10
Volume (%)
8
2A
2B
6
4
2
0
0.01
0.1
1
10
100
1000
10000
Size (µm)
Experiment
D[4;3] (µm)
Span
2A
56
1.8
2B
62
1.4
Average
59±4.0
1.6±0.3
tf = 10 min
Figure 3.7. Particle size distribution by laser granulometry for T-mixer at t0 = 0
min and tf = 10 min at total flow rate of 44.51 g/min and ethanol-water ratio of 25-75 %
(w/w).
153
CHAPTER 3
3.4.1.3. Influence of flow rate in the T- mixer on particle size
In this section the influence of the flow rate on particle size was analyzed using
the T-mixer. Three different experiments varying the flow rate were performed (Table
3.8) and the particle size was measured with the same experimental procedure.
PCS results
Analyzing the results for initial size and final size in different flow rates by PCS
in Table 3.13. It was noted that there is no a great influence of flow rate on the particle
size.
Laser granulometry results
The evolution of the particle size was still followed by laser granulometry during
10 minutes. Like it was made in previous section.
For all experiments the quickly grow or agglomeration rates of crystals was
observed as illustrate in Figures 3.8, 3.9 and 3.10. During the 10 minutes analysis the
particle size increased between 20 and 30 times of its initial size of 2 and 4 µm, i.e.
almost a mean growth rate between 9 and 7 µm/min.
According these results, the particles still continue to grow fast and/or to form
larger agglomerates, which means that the flow rate does not influence the growth of the
particle size after the mixing. The average flow (44.51 g/min) was chosen in the
following essays.
154
Solubility study and experimental design
Table 3.13. Evolution of crystallized particle growth by PCS in different total
flow rate: a) 27.09 g/min; b) 44.51 g/min; c) 69.56 g/min and ethanol-water ratio of 2575 % (w/w).
Experiment
Total flow (g/min)
Size (nm) at t0 = 0 s
Size (nm) at tf = 150 s
3A
27.09
354
942
3B
27.09
350
1195
352 ± 3
1068 ± 179
Average
(a)
Experiment
Total flow (g/min)
Size (nm) at t0=0s
Size (nm) at tf=150s
2A
44.51
269
845
2B
44.51
393
815
331 ± 88
830 ± 22
Average
(b)
Experiment
Total flow (g/min)
Size (nm) at t0 = 0 s
Size (nm) at tf = 150 s
4A
69.56
385
1241
4B
69.56
257
963
321 ± 63
1102 ± 197
Average
(c)
155
CHAPTER 3
9
8
7
6
Volume (%)
3A
5
3B
4
3
2
1
0
0.01
0.1
1
10
100
1000
10000
Size (µm)
Experiment
3A
3B
Average
D[4;3] (µm)
4
4
4±0.0
Span
1.7
2.1
1.9±0.3
t0=0s
12
10
Volume (%)
8
3A
3B
6
4
2
0
0.01
0.1
1
10
100
1000
10000
Size (µm)
Experiment
3A
3B
Average
D[4;3] (µm)
92
97
94±3.0
Span
1.2
1.3
1.2±0.1
tf=10min
Figure 3.8. Particle size distribution by laser granulometry for T-mixer at total
flow rate of 27.09 g/min and ethanol-water ratio of 25-75 % (w/w)., at t0 = 0 min and tf
= 10 min.
156
Solubility study and experimental design
14
12
10
2A
Volume (%)
8
2B
6
4
2
0
0.01
0.1
1
10
100
1000
10000
Size (µm)
Experiment
D[4;3] (µm)
Span
2A
2
1.3
2B
2
2.6
Average
2±0
1.9±0
t0 = 0 s
12
10
Volume (%)
8
2A
2B
6
4
2
0
0.01
0.1
1
10
100
1000
10000
Size (µm)
Experiment
D[4;3] (µm)
Span
2A
56
1.8
2B
62
1.4
Average
59±4
1.6±0
tf = 10 min
Figure 3.9. Particle size distribution by laser granulometry for T-mixer at total flow rate
of 44.51 g/min and ethanol-water ratio of 25-75 % (w/w), at t0 = 0 min and tf = 10 min.
157
CHAPTER 3
10
9
8
Volume (%)
7
6
4A
5
4B
4
3
2
1
0
0.01
0.1
1
10
100
1000
10000
Size (µm)
Experiment
4A
4B
Average
D[4;3] (µm)
4
2
3±1.0
Span
1.7
1.5
1.6±0.1
t0 = 0 s
10
9
8
Volume (%)
7
6
4A
5
4B
4
3
2
1
0
0.01
0.1
1
10
100
1000
10000
Size (µm)
Experiment
4A
4B
Average
D[4;3] (µm)
93
85
89±6.0
Span
2.1
1.8
1.9±0.2
tf = 10 min
Figure 3.10. Particle size distribution by laser granulometry for T-mixer at total
flow rate of 69.56 g/min and ethanol-water ratio of 25-75 % (w/w), at t0 = 0 min and tf =
10 min.
158
Solubility study and experimental design
3.4.1.4. Influence of addition time and steady state
From the previous section, for all experiments the particle size average (PCS)
presented a nanometric range right after crystallization. But the crystals size has evolved
quickly until stabilization, i.e. micrometric range.
In the case of uncontrolled crystallization, the size evolution of crystals can be
due to growth or agglomeration mechanism. To know which crystallization
phenomenon control the increase of size crystal, the population balance was proposed.
For this simple calculation the steady state must be achieved, i.e. particle of size
constant.
An experiment over the time under the best crystallization condition was
realized. The ethanol-water ratio equal to 25-75 % (w/w) and total flow rate of 44.51
g/min, were chosen, based on previous results.
The experiment does not last very long, only 6 min. This is due to problems of
blockage, caused by affixation of solid on the surface of the mixer, as shown in Figure
3.11. This problem can be caused by the affinity of the molecule with the material,
which constitutes the surface of the mixer. It precludes the system from reaching the
steady state and consequently, it prevents to calculate the growth and nucleation rates
from population balance.
Figure 3.11. Blockage and affixation of CRS 74 crystals on surface of the Tmixer.
159
CHAPTER 3
The blockage problem can be related to high saturation (S) of the alcoholic
solution. In order to prevent the blockage problem, a second experiment with a low flow
rate and a low supersaturation was performed, as shown in Table 3.14. In this
experiment, different samplings from the outlet of the mixer as a function of time were
realized.
Table 3.14. Experimental LAS crystallization process conditions
Experiment
Total flow
(g/min)
ethanol-water ratio
(w/w)
S*
5
44.51
25-75 %
894.0
6
27.09
25-75 %
15.4
*S= C /Ceq, - C is concentration after dilution with anti-solvent by assuming no crystallization
(mg/g) and Ceq is the equilibrium concentration of the pharmaceutical active in the solution water+ethanol
(mg/g)
Comparing the results of both supersaturations at the same time, it was perceived
that there is no big difference between the size particles values, as a function of time, as
given in Table 3.15. It means that the supersaturation does not have a great influence on
the particle size. It can be noted too, that the particle size for both experiments increases
as a function of time. It means that, the steady state was not achieved for population
balance calculus.
160
Solubility study and experimental design
Table 3.15. Particle size distribution by PCS: S = 894.04 and S = 15.4.
S= 894.0
S= 15.4
t (min)
Size (nm) at t0 = 0s
Size (nm) at t0 = 0s
0
438
421
2
455
491
4
503
579
6
688
x
For both experiments, the particle size measurement as a function of time does
not last very long (t < 6 min) due to problems of blockage.
The essay with the lower supersaturation and flow rate lasted less than the other
one, only 4 minutes. In this process condition the residence time of the crystals is
bigger, as shown in Table 3.10 (0.11 s for 27.09 g/min and 0.07 for 44.51 g/min). It
means, that the crystals have more time to be in contact with the surface of mixer,
causing problems of blockage. Furthermore, the particles in nanometric range have a
high-energy surface; this property can potentiate the high affinity of CRS 74 on the
surface material.
Because of this drug behaviour, which precluded the realization of essays over
time, it was not possible to simply calculate the nucleation and agglomeration rates. In
addition this drug behaviour prevent the use of this crystallization proceeding in
industrial applications.
161
CHAPTER 3
3.4.2. Characterization of solid particles obtained in experimental design
By comparing the SEM photos of raw material and the synthetized powders, a
decrease of the crystal size (elementary particles and agglomerates) and no shape
changes can be observed in Table 3.16. This size reduction can indicate an influence of
the crystallization process on the size particle. On the other hand, the presence of
powder compact agglomerates was noted. This agglomeration phenomenon may be due
to the crystallization process (particle surface state) and to the powder recuperation
process (filtration and drying).
The powder obtained were characterized by purity ranging from 97.00% ± 2.76
%. This value can prove that the experimental proceeding does not induce the product
contamination by germs in tubing elements of pump or in mixer device.
162
44.51
44.51
Roughton
T
1A
2A
X
Total flow
rate (g/min)
X
Type of
Mixer
Original
CRS
Exp.
25-75 %
25-75 %
X
Ethanol-water ratio
(w/w)
200 µm
SEM Image
Table 3.16. Identification and characterization of dried powder regarding solid state properties.
Solubility study and experimental design
20 µm
SEM Image
0.56
1.00
0.37
163
Residual
solvent
Content
(%)
Type of
Mixer
T
T
Exp.
3A
4A
Total flow
rate
(g/min)
69.56
27.09
Table 3.16. Continued
CHAPTER 3
25-75 %
25-75 %
Ethanol-water ratio
(w/w)
200 µm
SEM Image
20 µm
SEM Image
0.54
0.56
164
Residual
solvent
Content
(%)
Solubility study and experimental design
3.4.3. Comparative study of original CRS 74 and LAS recrystallized drug with Tmixer
According to experimental result (see section 3.4.1.2) the T-mixer was chosen for the
following steps. The original CRS 74 (raw material) and recrystallized powder using T-mixer
using the best experimental conditions was characterized in order to verify physicochemical
changes, among the products after recrystallization process.
3.4.3.1. Particle size and morphology
Laser diffractometry yields the volume-weighted diameters. The dv10%,dv50% and
dv90% represent the sizes in which 10%, 50% and 90% of the particles are below the given
sizes, respectively. From Figure 3.12, it can be seen that the particle size distribution changed
as follows: the original CRS 74 with 90%, 50% and 10% of the particles smaller than 515 µm,
101 µm and 4.3 µm were reduced respectively to 138 µm (dv90%), 34 µm (dv50%) and 4.4 µm
(dv10%) after LAS recrystallization.
600
515
Particle size (µm)
500
400
300
200
101
138
100
4.29
4.37
33.8
0
d10%
d50%
d90%
Original CRS 74
LAS recrystallized drug
Figure 3.12. Particle size (laser diffraction data) of CRS 74, before and after the LAS
recrystallization process.
165
CHAPTER 3
These results are supported by the powder morphology observed by SEM (Figure 3.13).
The particles of the original powder were found to be larger and exhibiting a broad particle
size distribution compared to the LAS recrystallized drug.
(a)
(b)
(c)
(d)
Figure 3.13. SEM micrographs of the original (a, b) and LAS recrystallized CRS 74 (c,
d).
166
Solubility study and experimental design
3.4.3.2. XRD analysis
XRD analysis was performed to detect the changes in the physical state and crystalline
phases of the drug, before and after LAS recrystallization. Figure 3.14 shows the XRD
patterns for the two studied samples. CRS 74 shows peaks at approximately 8.5, 14, 16.9,
18.7, 19.4 and 21.3°, indicating that the drug was a crystalline powder. Comparing the DRX
diffractograms of LAS recrystallized drug to the original CRS 74 it can be seen that the
crystalline phases were preserved.
35000
30000
LAS recrystallized drug
Intensity [I]
25000
20000
Original CRS 74
15000
10000
5000
0
5
10
15
20
25
30
Position [2Theta]
Figure 3.14.
X-Ray diffractograms of the original and LAS recrystallized drug
powders.
3.4.3.3. DSC analysis
The DSC thermograms of original and LAS recrystallized drug are presented in Figure
3.15. The onset melting point and fusion enthalpy obtained from the DSC study are
summarized in Table 3.17. Relative enthalpy is calculated by taking the fusion enthalpy of
original CRS 74 powder as 100%.
167
CHAPTER 3
From DSC data, it was concluded that LAS recrystallization changed marginally the
0
0
-2
-5
-4
-10
-6
-8
Original CRS 74
-15
-10
LAS recrystallized drug
Heat Flow (mW ) - LAS recrystallized drug
Heat Flow (mW ) - Original CRS 74
thermal properties of the drug.
-20
-12
-25
-14
140
150
160
170
180
190
200
Temperature (°C)
4,5
1,8
4
1,6
3,5
1,4
3
1,2
Original CRS 74
2,5
1
2
0,8
1,5
0,6
1
Heat Flow (mW ) - LAS recrystallized drug
Heat Flow (mW )
- Original CRS 74
(a)
0,4
LAS recrystallized drug
0,5
0,2
0
0
70
90
110
130
150
170
190
210
Temperature (°C)
(b)
Figure 3.15. a) DSC thermograms of original CRS 74 (first heating), which consists of
a melting endotherm (peak onset temperature Tm(Onset) = 188.64oC) and LAS recrystallized
drug, which consists of a melting endotherm (peak onset temperature Tm(Onset) = 187.79oC); b)
DSC thermograms of original CRS 74 (cooling after first heating), which consists of a
crystallization exotherm (peak onset temperature Tc(Onset) = 132.12oC) and LAS recrystallized
drug, which consists of a crystallization exotherm (peak onset temperature Tc(Onset) =
138.43oC).
168
Solubility study and experimental design
The observed reduction of the fusion enthalpy (Table 3.17) may be caused by the
higher surface/volume ratio exhibited by the LAS recrystallized powder, needing lower
energy for melting. Crystal energy is known to correlate with Tm(Onset) (onset melting point)
and ∆hf (enthalpy of fusion), which refers to the energy a compound must overcome to
dissolve (VIPPAGUNTA et al., 2007).
Table 3.17. Melting Temperature (Tm(Onset)), Heat of Fusion (Δhm) for the original and
LAS recrystallized drug.
Thermal and dissolution
parameters
Original
CRS 74
LAS recrystallized
CRS 74
Tm(Onset)(°C)
188.6
187.8
Δ hm (J/g)
86.6
79.2
Relative enthalpy(%)
100
91.5
3.4.3.4. Dissolution studies
Figure 3.16 shows the dissolution profiles of original and LAS recrystallized CRS 74
in 0.1 M HCl (pH 1.2). It can be seen from Figure 3.13 that both drug samples did not even
reach 50% dissolution within 3 h.
The dissolution profiles were compared through the model-independent simple
method. The model-independent simple method includes the difference factor (f1) and the
similarity factor (f2), calculated by using the following equations (3.7) and (3.8) (MOORE
and FLANNER, 1996; COSTA and LOBO, 2001).
(3.9)
(3.10)
169
CHAPTER 3
where Rt and Tt are the percentage of drug dissolved at each time point for the reference and
test products, respectively; n is the number of dissolution sample times and t is the time points
for collecting dissolution samples.
The f1 factor measures the percentage of the error between two curves over all time
points. This percentage is zero when the test and drug reference profiles are identical and
increases proportionally with the dissimilarity between the two dissolution profiles. The f2
factor is a logarithmic transformation of the sum-squared error of differences between the test
and the reference products over all time points. When the two profiles are identical, f2=100.
An average difference of 10% at all measured time point results in a f2 value of 50. FDA has
set a public standard of f2 value in the range 50-100 to indicate similarity between two
dissolution profiles.
Since original CRS 74 is the reference, the factors f1 and f2 are calculated between this
product and the LAS recrystallized powder. The results of f1 and f2, 24.8 and 98.8,
respectively, showed that the profiles of original CRS 74 and LAS recrystallized drug are
almost similar.
80
70
Dissolveld Drug (%)
60
LAS recrystallized drug
50
Original CRS 74
40
30
20
10
0
0
20
40
60
80
100
120
140
160
180
200
Time (min)
Figure 3.16. Dissolution profiles of CRS 74, before and after LAS recrystallization (in
0.1 M HCl at 37 °C, n = 4).
170
Solubility study and experimental design
3.4.3.5. Determination of surface properties
The surface properties of drug samples were investigated to find a possible relation
between surface properties and dissolution. Sessile drop contact is most commonly measured
on compacted powder disc surface. However, compaction of the material can alter the particle
morphology and surface energy (BUCKTON, 1955). Alternatively, some of the authors
(AHFAT et al, 2000; HE et al, 2008) have reported the use of powder layer adhered to an
inert support. The powder layer was adopted for the present work as it allows the study of “as
is” powder properties. This method gave reproducible values. It has been seen that contact
angle measurements can vary with experimental methodology, however the technique was
used for comparative evaluation between LAS recrystallized samples and the original drug
powder.
The contact angle of drop deposited on powder surface was plotted as a function of
time from 0 to 10 s, beyond which there was no significant change in the contact angle. With
water as the wetting liquid, original drug crystals exhibited a contact angle almost unchanged
with time from 136.4 ± 0.8° (0 s) to 136.6 ± 0.6°(10 s), as shown in Figure 3.17. In turn, LAS
recrystallized drug exhibited an initial contact angle of 133.5 ± 1.8° which changed
marginally to 132.9 ± 1.7° over the 10 s period.
The wetting process with water was quantified for the work of adhesion (Wa),
cohesion (WCL) and the spreading coefficient (λ
λ LS), as already calculated in Chapter 2,
section 2.3.5.
The contact angle made at 0 s was considered as the initial contact angle and the surface
tension of water was taken from the literature (72.8 mN/m; PURI et al., 2010). These values
were used to determine Wa, WCL and λLS from equations (2.2), (2.3) and (2.4) for the drug
powders (Table 3.18).
The thermodynamic driving force for each process is indicated by the work value,
where a negative value denotes the spontaneity of the process (Young and Buckton, 1990).
The positive Wa and WCL obtained for both drug powders indicated that the adhesion and the
cohesion processes are not spontaneous in both cases. Further, a negative spreading
coefficient (λLS < 0) means that the original CRS 74 displayed an unfavorable spreading of
171
CHAPTER 3
water (NGUYEN and HAPGOOD et al., 2010), characteristic which remained unchangeable
after LAS recrystallization of the drug. This poor wettability can be related to the poor
dissolution rates measured.
150
145
Contact Angle (°)
140
135
130
125
θ= 136.6°±0.57 θ=136.4°±0.85 120
115
110
105
100
0
1
2
3
4
5
6
7
8
9
10
Time (seconds)
(a)
(b)
Figure 3.17. Contact angle (o) of water as a function of time for: a) original CRS 74; b)
LAS recrystallized drug.
172
Solubility study and experimental design
Table 3.18. Work of adhesion (WA), cohesion (WCL) and spreading coefficient (λ
λ LS)
for CRS 74.
Sample
WA (mN/m) WCL (mN/m)
λ LS (mN/m)
CRS 74
20.08
145.6
-125.6
LAS recrystallized powder
22.69
145.6
-122.9
3.5. CONCLUSIONS
The present chapter had the purpose to determine the solubility of CRS 74 in binary
solvent mixture (ethanol-water) and to study the influence of operational parameters on the
synthesis of a pharmaceutical drug.
The CRS 74 is soluble in ethanol (92.6 mg/gsolution) and seems to present a maximum
in solubility in ethanol-water mixtures as determined in this study using UNIQUAC-based
model at 30°C. To the best of our knowledge, there are no published data of the solubility of
such given solute in ethanol-water mixtures to compare with.
The synthesis process should result in a powder with small crystal size with the view
to improve the bioavailability of the drug.
According to two main parameters tested, type of mixer and flow rate, uncontrolled
crystal growth and no change on the final product was observed, i.e. the operational
parameters do not have influence on recrystallized particles. These experiments proved to be
quite laborious due to the rapid growth and agglomeration of the produced particles and
problems of blockage of the mixer. Because of this drug behaviour, which precluded the
realization of essays over time, it was not possible to simply calculate the nucleation and
agglomeration rates. In addition this drug behaviour prevent the use of this crystallization
proceeding in industrial applications.
173
CHAPTER 3
After setting process conditions, a solid crystallized by LAS crystallization (operating
conditions) was compared (or has been compared) to the raw material. After crystallization
process the synthesized product do not present any crystallinity changes. However, a decrease
of particle size was observed and consequently a considerable agglomeration of crystals.
Furthermore, in the case of CRS 74 LAS crystallization does not have a real impact on the
dissolution rate of synthesized powder.
In an attempt to improve its dissolution properties, CRS 74 can be recrystallized by
using a Liquid Anti-solvent (LAS) crystallization process and the data generated here can
represent a useful tool to define the mass proportion between solvent (ethanol) and antisolvent (water) for LAS crystallization studies. However, to improve its dissolution kinetics,
smaller particles with a more hydrophilic surface need to be produced. This research is going
on and our specific aims are to consider the feasibility of the LAS crystallization using
different excipients to optimize process and formulation parameters.
174
Effect of additives in LAS crystallization
Résumé Chapitre 4- Etude de la recristallisation par effet anti-solvant en
présence d’additifs
En vue d’améliorer sa dissolution en milieux aqueux, le CRS 74 a été recristallisé en
utilisant une opération de cristallisation par effet anti-solvant en présence d’additifs.
Des additifs ont donc été utilisés en vue de modifier les propriétés de dissolution des
cristaux, et les vitesses de cristallisation (nucléation, agglomération, croissance).
L'effet des additifs sur la taille des cristaux, la cinétique de dissolution et la
mouillabilité du solide synthétisé a été étudié.
Les additifs ont été choisis en accord avec des applications pharmaceutiques futures de
cette molécule. Ils sont de quatre types
-des tensio-actifs non ioniques : Tween 20 (ester de sorbitan polyoxyéthylénique) et
HPMC (Hydroxypropylméthylcellulose) pour une stabilisation stérique,
- un copolymère à bloc, le poloxamer 407 (P-407) pour une stabilisation stérique,
-un tensio-actif anionique, le dodécylsulfate de sodium (SDS) pour une répulsion
électrostatique,
-un copolymère composé de D-glucosamine et de N-acétyl-D-glucosamine (chitosane)
pour un effet combiné de stabilisation stérique et répulsion électrostatique.
Ils ont été introduits dans le solvant (éthanol) ou dans l'anti-solvant (eau), ou dans les
deux phases.
Par rapport aux additifs testés, des changements des propriétés de surface ont été
constatés. Une diminution remarquable de l'angle de contact a été observée pour deux
formulations, une avec le copolymère à bloc P-407 (concentration de 0,02 %, > CMC dans la
phase organique) et l'autre avec ce dernier combiné avec du chitosane (P-407 concentration de
0.02 %, > CMC dans la phase organique et le chitosane avec une concentration de 0,5 % dans
la phase aqueuse). De plus, la dissolution a été améliorée de façon très notable : à 20 min de
dissolution, le pourcentage dissous du principe actif est de 4% avec la poudre initiale et de
l’ordre
40%
pour
la
poudre
recristallisée
avec
ces
deux
formulations.
Dans l'ensemble, les résultats ont montré de façon concluante que la technique de
cristallisation par effet anti-solvant en présence d'additifs a permis de produire des
microcristaux présentant des profils de dissolution nettement plus rapide que la poudre
originale. L’amélioration de la dissolution peut-être due à la réduction de la taille des
particules des cristaux du principe actif mais aussi à l'amélioration des propriétés de
mouillage due aux interactions spécifiques entre le principe actif, les additifs et le milieu
aqueux de dissolution. Il est important de noter qu’aucun colmatage n’a été observé avec la
formulation combinant les additifs P-407 et chitosane.
Effect of Additives in LAS crystallization
4.1.INTRODUCTION
Up to now, we could demonstrate that LAS recrystallization process reduced the mean
particle size and particle size distribution of the CRS 74 crystals. However, this particle size
reduction did not change the dissolution rate as it could be expected with the increased
surface area of the powder. In fact, the powder is characterized by a very high hydrophobicity
as confirmed experimentally, and the particles once formed in a nanometric size range grew
up very fast and finally agglomerated, reducing the powder surface area in contact with the
dissolution medium. In addition, a very strong attraction between the drug particles and the
metallic surface of the rapid mixers used in the process led to a retention of the solid inside
the mixers and to the interruption of the continuous process.
In this last part of the work, we investigated the effect of organic additives in the CRS
74 recrystallization using the LAS process. Changes in drug-crystal properties were evaluated
for particle size and shape, crystal structure, surface characteristics such as wettability and
dissolution rate. The methodology and the results obtained are presented in this Chapter.
Considerations for selection of stabilizers
In LAS crystallization, the mixing process and the control of supersaturation are crucial
factors to control the production of uniform and small solid drug particles. However, as
already discussed in Chapter 1, the addition of stabilizers in the process in order to inhibit
excess crystal growth or particle aggregation has been related by a great number of scientific
publications (ALI et al., 2011, GHOSH et al., 2011, SU et al., 2011).
Stabilizers can be added either in solvent or anti-solvent, acting as surface modifiers.
Polymers are often used for steric stabilization, while surfactants are often used for
electrostatic stabilization. The suitable stabilization depends on properties of the substance
that should crystallize. Charge and hydrophilicity are some important properties to consider.
The stabilizer must of course have sufficient affinity for the particles surface, and it must also
have a rather high diffusion rate in order to rapidly cover the generated surface.
Unfortunately, there exists no systematic technique to determine the effectiveness of additives
a priori. Stabilizing agents are chosen from the list of pharmaceutically acceptable substances,
with surface active properties capable to provide steric and/or ionic stabilization against
179
CHAPTER 4
growth and agglomeration of drug particles, during LAS crystallization. In this study,
stabilizers were screened and introduced in the solvent, or in the anti-solvent, or in both
phases.
Two main mechanisms of stabilization were tested: steric stabilization and electrostatic
repulsion.
For steric stabilization, we used non ionic polymers and amphiphilic block
copolymers:
• Polyoxyethylene sorbitan monooleate (Tween 20), a nonionic surfactant.
If this additive is adsorbed on the surface of a hydrophobic drug like CRS 74, we could
expect that a mechanical barrier would be formed against crystal growth and agglomeration.
The additive would occupy the adsorption sites on the surface of freshly formed CRS 74
crystals during LAS crystallization, and inhibits subsequent growth by inhibiting the
incorporation of drug molecules from solution into crystal lattices, as already observed for
other drugs such as Fenofibrate and Acid Folic (WU et al. 2011; PARDEIKE et al., 2011); In
addition, Tween 20 was selected because it is also very well tolerated in pharmaceutical
formulations, being accepted even for intravenous injection (FDA, 2007).
•
Hydroxypropylmethylcellulose (HPMC), a neutral polymer.
It could be expected that this polymer adsorbs on the hydrophobic CRS 74 surface and
the portions of polymer chain extending in solution provides steric protection. HPMC is a
stabilizer frequently used in LAS crystallization studies due to its characteristic of good
solubility in water and not being toxic (DONG et al., 2009).
• Poloxamer 407 (P-407) copolymer (ethylene oxide and propylene oxide blocks), a
hydrophilic non-ionic surfactant of the more general class of copolymers known as
poloxamers.
Poloxamer-407 can induce the steric stabilization and its use in pharmaceutical
formulations is approved (oral solutions, ophthalmic solutions, periodontal gels and
topical emulsions (REHMAN, 2011).
180
Effect of Additives in LAS crystallization
For electrostatic repulsion, the anionic surfactant Sodium dodecyl sulfate (SDS) was
chosen. This surfactant has been used as an electrostatic stabilizer with high affinity to absorb
onto particle surfaces leading to high zeta potentials. It is a regulatory accepted stabilizer for
oral dosage forms (e.g. tablets and capsules), and is therefore suitable to be used in
nanosuspensions (HU et al., 2011).
For a combined effect of steric stabilization and electrostatic repulsion, we tested
Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, a polycationic,
biocompatible and biodegradable polymer.
Chitosan is a compound which combines the electrostatic stabilization due to its
positive charge in acidic medium, and the steric stabilization because of its polymeric nature.
Theoretically, that means that if the polyelectrolyte adsorbs onto the surface of small embryo
drug particles surface, it should be the ideal stabilizer because it combines electrostatic and
steric stabilization, it forms a strong double layer around hydrophobic drug particle. In
addition, the chitosan can increase the drug bioavailability due to mucoadhesion, that is able
to increase cellular permeability (BOWMAN and LEONG, 2006).
Concerning surfactants, in aqueous solution, dilute concentrations of surfactant act
much as normal electrolytes, but at higher concentrations a very different behavior is
observed. This behavior is explained in terms of formation of organized aggregates of large
numbers of molecules called micelles, in which the lipophilic parts of the surfactants associate
in the interior of the aggregate leaving hydrophilic parts to face the aqueous medium.
The physico-chemical properties of surfactants vary markedly above and below the
CMC value. As an example, below the CMC value, the physico-chemical properties of ionic
surfactants like sodium dodecylsulfate, SDS, (e.g., conductivities, electromotive force
measurements) resemble those of a strong electrolyte. Above the CMC value, these properties
change dramatically, indicating a highly cooperative association process is taking place, This
is illustrated in Figure 4.1. ( LAURIER et al., 2003).
Based on this discussion, the CMC values can be important in our additives study, and
the additive concentration for the surfactants Tween 20, SDS and Poloxamer 407 were varied
from below to above their CMC. The CMC is also of interest because at concentrations above
181
CHAPTER 4
this value, the adsorption of surfactant at interfaces usually increases very little. This means
that the CMC frequently represents the solution concentration of surfactant from which nearly
maximum adsorption occurs (LAURIER et al., 2003).
Figure 4.1. Variation in physical properties of (or SDS)
surfactant solutions below and above the CMC value (from Laurier et al, 2003).
To sum up, Tween 20, SDS, Poloxamer 407, HPMC and Chitosan are the chosen
additives expected to improve particle size control during the LAS crystallization process of
CRS 74, and they were introduced in the solvent (Poloxamer 407) or in the anti-solvent (all
the other ones) in our CRS 74-ethanol-water system.
4.2. Materials and methods
4.2.1. Materials
The additives used are shown in Table 4.1.
CMCs of Tween 20, SDS and Poloxamer 407 are given in Table 4.2. The CMC values
for Tween 20 and SDS were found in the literature (Silva and Volpato, 2002).The CMC for P-
182
Effect of Additives in LAS crystallization
407 was measured in the laboratory, using a tensiometer 3S ILMS (GBX Instruments,
France).
Table 4.1. Different additives used in LAS crystallization of CRS 74.
Additives
Sodium dodecyl sulfate (SDS)
M.W. : 288 g/mol
Chemical structure
H 3C
O
Supplier
SO3Na
Fluka, France.
Polyoxyethylene sorbitan
monolaurate
(Tween 20)
M.W. : 1228 g/mol
Sigma–Aldrich, France.
Poloxamer 407 or Pluronic
F127 M.W. : 8400 g/mol
Basf, France.
Hydroxypropylmethylcellulose
(HPMC)
M.W. : 22000 g/mol
Sigma–Aldrich, France.
Chitosan
Low molecular weight
M.W . : 227000 g/mol
Sigma–Aldrich, France.
M.W.: Molecular weight
A maximum concentration was fixed for all additives to give a mass ratio of 1:4
(additive:drug). This corresponds to 0.5%w/w for HPMC, Chitosan, Tween 20 and SDS in
water and 0.02% (w/w) for P-407 in ethanol.
183
CHAPTER 4
Table 4.2. Critical micelle concentration of additives used in LAS crystallization at 25°C.
Addtive
CMC value (w/w)
Reference
Tween 20
0.05 %
SILVA and VOLPATO, 2002
SDS
0.1 %
SILVA and VOLPATO, 2002
Poloxamer P-407
0.0008 %
Measured experimentally
4.2.2. Methods
4.2.2.1. Production of particles by LAS crystallization in presence of additives
The original CRS 74 crystals were recrystallized by the LAS crystallization method
already described in Chapter 3, using the same experimental set-up. Briefly, a certain amount
of original CRS 74 samples was completely dissolved in ethanol at 30 ± 0.5°C at definite
concentration (90 mgCRS 74/gsolution). The solution was filtrated through 0.22 µm pore size
membranes to remove the possible particulate impurities. The drug was then recrystallized via
the concurrent introduction of the CRS 74 ethanol solution and an anti-solvent stream of
water in the T-mixer. The freshly formed crystals were collected in a vessel under magnetic
stirring and then filtered and dried under vacuum at 50 ± 1°C for 24 h. The dried samples
produced in the process were characterized by laser diffractometry, differential scanning
calorimetric analysis, X-ray diffraction analysis, scanning electron microscopy analysis,
sessile drop method and dissolution testing.
The experimental conditions are summarized in Table 4.3. The studied parameters were:
1. Phase for incorporation of the additive (organic solution, aqueous solution or both
solutions);
2. Additive concentration (above and below CMC);
3. Drug concentration in the organic phase;
4. Re-structured organic phase containing P-407: Poloxamer 407 was introduced in a
dilute ethanol solution (concentration below its CMC), before the drug. The resulting
solution was then heated under controlled conditions (rotoevaporator) to promote a
partial evaporation of the solvent and concentrate the solution to a surfactant
concentration above its CMC.
184
Effect of Additives in LAS crystallization
Table 4.3. Experimental conditions for LAS crystallization in presence of additives.
Experiment
AdditiveOrganic phase
1
X
Additive in the aqueous phase
2
X
Ratioaddit.org.:drug
AdditiveAqueous phase
Ratioaddit.aqu.:drug
X
X
X
S
(S=C/Ceq)
894.04
X
HPMC 0.5% w/w
SDS < CMC
(0.008% w/w)
SDS 0.5% w/w
Tween 20 < CMC
(0.008% w/w)
Tween 20
0.5% w/w
Chitosan
0.5% w/w
1:6
445.63
1:4000
1:6
391.85
150.51
1:4000
541.13
1:6
398.72
1:6
*
X
X
901.97
X
X
673.40
3
4
X
X
X
X
5
X
X
X
6
X
X
7
X
Additive in the organic phase
P-407 < CMC
8
(0.0003%w/w)
1:8000
P-407> CMC
9
(0.02% w/w)
1:4
Additive in both, organic and aqueous phases
P-407 < CMC
1 :10000
10
(0.0003%w/w)
P-407> CMC
1:4
11
(0.02% w/w)
P-407 < CMC
1 :10000
12
(0.0003% w/w)
1:4
P-407 > CMC
(0.02% w/w)
13
Drug concentration in the organic phase
P-407 > CMC
1:4
14
(0.02% w/w)
P-407 > CMC
1:4
15
(0.02% w/w)
P-407 > CMC
1:4
16
(0.02% w/w)
P-407 > CMC
1:4
17
(0.02% w/w)
P-407 > CMC
1:4
18
(0.02% w/w)
P-407 > CMC
1:4
19
(0.02% w/w)
Reorganized organic phase containing P-407
P-407 < CMC
1:4
20
(0.0003% w/w)
P-407 < CMC
1:4
21
(0.0003% w/w)
*Data not determined
SDS
0.5% w/w
SDS
0.5% w/w
Chitosan
0.5% w/w
Chitosan
0.5% w/w
X
X
SDS 0.5% w/w
SDS 0.5% w/w
Chitosan 0.5% w/w
Chitosan 0.5% w/w
X
Chitosan 0.5% w/w
1:6
1:6
217.11
194.93
1:6
*
1:6
*
X
X
1:3
1:1.5
374.51
156.41
104.50
43.25
1:3
*
1:1.5
*
X
77.59
*
X
185
CHAPTER 4
4.2.2.2. Determination of solubility in presence of additive
The equilibrium concentration (Ceq) for the CRS 74-ethanol-water system was
previously measured and presented in Chapter 3. The presence of additives can modify the
drug saturation concentration in this system. The solubility of CRS 74 was then determined in
presence of additives, for a mass proportion of 1 ( 25% solvent) to 3 ( 75% anti-solvent) using
the same procedure as described previously. The new solubility data were used to estimate the
theoretical efficiency of the process as a function of the additive used.
4.2.2.3. Characterization methods
4.2.2.3.1. Measurements of particle size during the crystallization process
Particle size was measured during the crystallization process and at the end of the
process, after product drying.
In a first step, at the exit of the T-mixer, the mixed phases were poured into a vessel
under agitation and a sample was immediately submitted to a particle size analysis in a
Zetasizer Nano Zs (Malvern Instruments, United Kingdom) to follow the growth of small
embryo particles in the crystallization medium during 150 seconds. To achieve appropriate
measurement concentration for analysis, the sample was diluted 5 times using a saturated
solution composed of water, ethanol, drug and the additive.
At the end of the process (2 min), the final suspension was filtered and the solids were
dried under vacuum at 50 ± 1°C for 24h. The dry powder was then analyzed by laser
granulometry, using a MasterSizer 3000 (Malvern Instruments, United Kingdom). All
samples were firstly mixed with Tween 20 to increase the powder dispersion in dispersive
media, and then dispersed in water until achievement of the good obscuration. The sampling
procedure is schematized in Figure 4.2.
Zeta potential
The zeta potential was also determined in the Zetasizer Nano Zs (Malvern
Instruments, United Kingdom) by measuring the electrophoretic mobility of the particles. The
186
Effect of Additives in LAS crystallization
measurements were performed on the same sample taken at the exit of the T-mixer
immediately diluted with the saturated solution.
Particle size evolution
Sampling
Particle size distribution
Dilution
Measure time (s)
()
Tween 20
0
150
Final drug suspension
in the water-ethanol
solution
Figure 4.2. Particle size analysis using Zetasizer Nano Zs (particles in suspension) and
MasterSizer 3000 (dried powders).
4.2.2.3.2.Powder physicochemical properties
Size distribution, surface properties, morphology, drug purity and dissolution kinetics
of recrystallized powders were determined as previously described in Chapter 3.
187
CHAPTER 4
4.3. RESULTS AND DISCUSSION
4.3.1. CRS 74 solubility in presence of additives
The results obtained for the solubility of CRS 74 in presence of additives are presented
in Table 4.4. Figure 4.3 displays the same data graphically. It can be seen that the effect of
additives on the drug solubility in the ethanol-water mixture depended on the type and the
concentration of the additive used.
Surfactants in solution below their critical micelle concentration (CMC) improve drug
solubility by providing regions for hydrophobic drug interactions in solution (NARANG et al,
2007). It can be seen that the presence of the three additives used here in a concentration
below their CMC (Tween 20, SDS and P-407) improved only modestly the drug solubility in
the ethanol-water mixture (0.025-0.05 mg/gsolution ).
Above the CMC, surfactants self-aggregate in defined orientation to form micelles with
a hydrophobic core and a hydrophilic surface. The hydrophobic core enhances the entrapment
of drug, thus increasing its solubility (RANGEL-YAGUI et al., 2005), which probably
happened to CRS 74 but the effect is still marginal with Tween 20 and P-407 (0.025-0.057
mg/gsolution).
The extent of CRS 74 solubility in the micelle cores formed with the different additives
is probably dependent on the compatibility between the drug and the micelle core. In contrast,
SDS in concentration above its CMC increased the Ceq from 0.025 to 0.151 mg/gsolution. The
highest solubilization capacity of SDS can be attributed in part to its anionic charge.
According to the literature, the solubilizing powers of the surfactants is in the order of anionic
< cationic < nonionic (TOKIWA, 1968).
Finally, the presence of HPMC did not have a significant influence on the Ceq.
The solubility of CRS 74 in presence of chitosan could not be measured because any
drug peak was detected by HPLC analysis.
188
Effect of Additives in LAS crystallization
Table 4.4. CRS 74 solubility in ethanol-water mixture with a ratio of 25-75 % (ww) at
30°C.
Additive
Solubility (mg/gsolution) ± SD
No additive (original CRS 74)
0.025±0.000
No additive (recrystallized CRS 74)
0.025±0.000
P407< CMC (0.0003%w/w)
0.025±0.000
P407> CMC (0.02%w/w)
0.033±0.002
HPMC 0.5%w/w
0.051±0.002
Tween 20 < CMC (0.008%w/w)
0.042±0.000
Tween 20 0.5%w/w
0.057±0.001
SDS<CMC (0.008%w/w)
0.058±0.003
SDS 0.5%w/w
0.151±0.003
SDS 0.5% +P407< CMC (0.0003%w/w)
0.106±0.001
SDS 05%+P407> CMC (0.02%w/w)
0.114±0.003
0.200
0.180
0.160
SDS 0.5%
Concentration(mg/gsolution)
0.140
SDS 0.5%+
P407> CMC
0.120
SDS 0.5% +
P407 < CMC
0.100
0.080
Tween 0.5% SDS<CMC
0.060
HPMC 0.5%
Tween<CMC
0.040
P407>CMC
CRS 74 No additive P407<CMC
0.020
0.000
Systems
Figure 4.3. CRS 74 solubility in ethanol-water mixture with a ratio of 25-75 % (w/w) at
30oC, in presence of different additives.
189
CHAPTER 4
4.3.2. Effect of additives on yields of production of CRS 74 crystals
The crystals suspension obtained immediately after the crystallization process, in
absence of additives, had a milky aspect, which changed into a clear solution after several
minutes. In contrast, for the particles crystallized in presence of additives, this phenomenon
was not observed. The crystal suspension maintained a milky appearance until the filtration
step, which could represent a first indication on the additive effect for covering the CRS drug
crystal surfaces and inducing steric or electrostatic hindrance to prevent crystal growth or/and
agglomeration.
Theoretical yield is the maximum amount of recrystallized dry crystals that can be
created by the given amount of initial mass of raw crystals. The yields of production were
calculated as the weight percentage of the crystal powder after drying with respect to the
initial total amount of drug used for recrystallization. Table 4.5 shows that the percent yield of
production that was obtained from the LAS crystallization ranged from approximate 50 to
87%. Tween 20 0.5% was proven to be the most effective stabilizer to improve the percent
yield of crystals.
There are some unavoidable errors that prevented the experiments from achieving
100% yield. Probably the most common error was mass losses in all experiments due to
operation of solid and liquid transfer. The filtration process may allow some of the product to
pass through. Also during recovery there was some product lost. For instance, some of the
product was stuck inside the T-mixer and could not be completely removed.
The next sections will present and discuss experimental data related to the solid
properties when additives were placed respectively in the aqueous phase, in the organic phase
and in both phases.
190
Effect of Additives in LAS crystallization
Table 4.5. Theoretical and Practical Yield (%) of CRS 74 crystals obtained by LAS
crystallization process.
Yield of
Theoretical
Experiment
Additive
production
Yield (%)
(%)
X
99.67
79.44
2
HPMC 0.5% w/w
99.84
50.23
3
SDS<CMC (0.008%w/w)
99.86
76.61
4
SDS 0.5% w/w
99.95
75.56
5
Tween 20<CMC (0.008%w/w)
99.80
47.15
6
Tween 20 0.5% w/w
99.86
86.77
7
Chitosan 0.5% w/w
*
64.78
8
P-407<CMC (0.0003%w/w)
99.67
65.63
9
P-407>CMC (0.02%w/w)
99.75
70.96
1
Additive in aqueous phase
Additive in organic phase
Additive in both, organic and aqueous phase
10
P-407<CMC (0.0003%w/w)+ SDS 0.5% w/w
99.92
72.74
11
P-407>CMC(0.02%w/w)+ SDS 0.5% w/w
99.91
87.83
12
P-407<CMC(0.0003%w/w)+Chitosan 0.5% w/w
*
76.43
13
P-407>CMC(0.02%w/w)+Chitosan 0.5% w/w
*
83.74
Drug concentration in the organic phase
14
P-407>CMC (0.0003%w/w)
99.75
52.81
15
P-407>CMC(0.02%w/w)
99.75
74.55
16
P-407>CMC(0.02%w/w)+ SDS 0.5% w/w
99.91
72.48
17
P-407>CMC(0.02%w/w)+ SDS 0.5% w/w
99.91
65.43
18
P-407>CMC(0.02%w/w)+Chitosan 0.5% w/w
*
65.00
19
P-407>CMC(0.02%w/w)+Chitosan 0.5% w/w
*
60.19
99.75
57.56
*
57.92
Reorganized organic phase
20
P-407<CMC(0.0003%w/w)
21
P-407<CMC(0.0003%w/w)+Chitosan 0.5% w/w
*Data not determined
191
CHAPTER 4
4.3.3.Effect of presence of the additive in the aqueous phase on physical and
surface properties of recrystallized powders
Table 4.6 summarizes the results related to the particle size evolution of the small
embryo particles formed at the exit of the T-mixer, when the additive was added in the
aqueous phase (HPMC, SDS, Tween 20 and Chitosan). As it can be seen, in all studied
systems, the drug particles presented almost the same size (140 - 240 nm), regardless of the
type of additive used. However, these particles grew fast reaching in 150 s (t150) almost 2 times
their initial particle size (t0).
Table 4.6. Initial average particle size (APS) and characteristics of CRS 74 crystals in
suspension in presence of different additives in the aqueous phase
Zeta potential
Experiment
Additive
Time (s)
APS(nm)
pH suspension
(mV)
1
2
t0
248
*
*
t150
417
-2.11± 0.28
3.28
t0
245
*
*
t150
459
-2.44±0.27
4.88
t0
174
*
*
t150
325
-18.57±0.31
4.48
t0
161
*
*
t150
358
-38.90±2.12
4.44
Tween 20<CMC
t0
138
*
*
(0.008%w/w)
t150
411
-8.67±1.27
4.53
t0
154
*
*
t150
308
-6.77±1.12
4.45
t0
204
*
*
t150
653
+35.20±1.40
4.44
X
HPMC 0.5% w/w
3
SDS<CMC(0.008%w/w)
4
SDS 0.5% w/w
5
6
7
Tween 20 0.5% w/w
Chitosan 0.5% w/w
The particle charge was quantified as the so-called zeta potential, which was measured
via the electrophoretic mobility of the particles in an electrical field. The zeta potential theory
is described in very detail in the literature (HUNTER, 1981), here only a brief explanation is
192
Effect of Additives in LAS crystallization
given. In general, particles possess a surface charge which occurs due to the dissociation of
surface functional groups, the so-called Nernst potential. Of course, the degree of dissociation
of the functional groups depends on the pH of the suspension; therefore the zeta potential is
pH dependent.
Table 4.6 shows the changes on the zeta potential of the particles in presence of
additives. When the crystals were synthesized without additives, they possessed a low
negative surface charge (-2.1mV). The zeta potential remained almost unchanged when
nonionic stabilizers, i.e. HPMC (-2.4mV) and Tween 20 (-8.7/-6.8mV) were used. However,
charged additives like SDS (negatively charged) and Chitosan (positively charged) adsorbed
with the charged parts of the respective molecules onto the drug particle surface. The surface
coverage increased with the increase in SDS concentration, leading subsequently to an
increase of the zeta potential (-18.6/-38.9mV). The higher particles are equally charged, the
higher is the electrostatic repulsion between particles.
As a rule of thumb, suspensions with zeta potential above |30| mV are physically stable.
Suspensions with a potential above |60| mV show excellent stability. Suspensions below
|20|mV are of limited stability; below |5| mV they undergo pronounced aggregation (LEE et
al., 2008; PATEL and AGRAWAL, 2011). Chitosan and SDS 0.5% were proven to be the
best additives to ensure an electrostatic repulsion between freshly CRS 74 crystal formed in
the LAS process.
Table 4.7 summarizes some properties of the dried powder obtained at the end of the
LAS crystallization process e.g., particle size, SEM images, contact angle and composition
(drug content and residual solvent). For easier comparaison, the dv10%, dv50% and dv90% for the
dried powders produced in presence of all additives tested in this study are gathered in Figure
4.4. The corresponding particle size distributions are graphically presented in Appendix III.
193
CHAPTER 4
800
767
600
515
500
400
300
200
138
33.8
4.37
81.4
3.27
12
132
77.9
52.7
5.76
2.15 7.55
C
20.2
C
/w
/w
(w
5%
0.
sa
n
ito
Tw
ee
n
20
Tw
0.
ee
n
5%
20
(w
)
/w
)
<C
M
(w
5%
0.
S
SD
dv90%
23.6
4.26
)
CM
(w
5%
0.
C
PM
H
re
cr
y
S
LA
dv50%
/w
dr
sta
lli
ze
d
in
al
C
rig
O
dv10%
16.6
3.55
)
ug
RS
74
2.51
S<
4.29
0
226
134
Ch
101
100
SD
Particle size (µm)
700
Figure 4.4. Particle size of the drug powders synthesized in presence of additives in the
aqueous phase (laser diffraction data).
The primary role of stabilizers is to inhibit excessive crystal growth. In a general way,
Figure 4.4 shows that most CRS 74 crystals produced in presence of additives had a smaller
particle size compared to original or recrystallized powder without additives. Additives such
as HPMC and Tween 20 (concentration below CMC) were less effectives to inhibit
agglomeration, while a higher concentration of Tween 20 (above CMC) had a very positive
effect on particle size control.
Concerning particle morphology (see Table 4.7), the SEM images revealed that in the
case of SDS, Tween 20 and Chitosan there was not particle shape changes on the columnar
crystals or very little; Contrarily, clusters of thinner (predominantly needle-shaped) primary
particles forming agglomerates or aggregates were obtained in presence of HPMC. This effect
can be also confirmed by light microscopy (Appendix V) revealing agglomerates or particle
assemblies composed of a large number of individual needle-shaped crystals). In this case, a
possible explanation could be an inappropriate diffusion (too slow) of the solvent toward the
anti-solvent caused by a high viscosity of the anti-solvent containing 0.5%w/w HPMC. A
high viscosity could prevent diffusion between solution and anti-solvent and resulted in
nonuniform supersaturation and agglomeration.
194
Effect of Additives in LAS crystallization
XRD analysis was performed to detect the changes in the physical state and crystalline
phases of the drug due to the presence of additives in the aqueous phase. Figure 4.5 shows the
XRD patterns for all powder samples. Comparing the DRX diffractograms of recrystallized
drug in presence of HPMC, Tween 20 (below and above CMC), SDS (below and above
CMC) and Chitosan, it can be seen that the crystalline phases were preserved (the peaks at
8.5, 14, 16.9, 18.7, 19.4 and 21.3° detected for the original drug remained unchanged after
recrystallization in presence of these additives).
HPMC 0.5% (w/w)
LAS recrystallized drug
LAS recrystallized drug
Original CRS 74
Tween 20 0.5% (w/w)
Original CRS 74
Tween 20<CMC
(a)
(b)
LAS recrystallized drug
SDS<CMC
SDS 0.5% (w/w)
Chitosan 0.5% (w/w)
LAS recrystallized drug
Original CRS 74
Original CRS 74
(c)
(d)
Figure 4.5. X-Ray diffractograms of the drug powders synthesized with additives in the
aqueous phase: a) HPMC 0.5% (w/w); b) Tween 20 <CMC and 0.5% (w/w); c) SDS<CMC
and 0.5% (w/w); d) Chitosan 0.5% (w/w).
195
CHAPTER 4
The contact angle measurements were employed to describe the effect of the presence
of additives in the aqueous phase on the wettability of the recrystallized powders compared to
original CRS 74. The contact angle of drop deposited on all powders surface was plotted as a
function of time from 0 to 10 s, and the results are given in Appendix IV. Table 4.6 presents
the initial contact angle made at 0 s for all powders. The original powder and the powder
recrystallized without additives were strongly hydrophobic (θ >132°) as already discussed in
Chapter 3. This surface characteristic remains practically unchanged after recrystallization in
presence of most of additives tested up to here. It can be observed that the powder produced
in presence of Chitosan possessed a contact angle higher than the contact angle for the
original drug (θ = 140.9°± 0.4). In fact, the measure is not totally effective as it became
difficult to measure experimentally liquid contact angles on high charged surfaces. To sum
up, the unique additive that was able to reduce consistently the contact angle of this drug with
water to approximately 100° was Tween 20 0.5 % (w/w).
Finally, Table 4.7 also gives the powder composition in terms of drug content and
residual solvent. The drug contents of the CRS 74 microcrystals were above 98% for all CRS
74 recrystallized samples. These results show that almost all of the additives were removed by
washing.
196
Contact Angle
(°)
136.4±0.85
133.5±1.77
124.5±0.14
Additive
X
X
HPMC 0.5% w/w
Exp.
Original CRS 74
1
2
images and contact angle.
D[10] : 2.51
D[50] : 134
D[90] : 767
D[4.3] : 287
Span : 5.68
D[10] : 4.37
D[50] : 33.8
D[90] : 138
D[4.3] :77.7
Span : 3.94
D[10] : 4.29
D[50] : 101
D[90] : 515
D[4.3] : 191
Span : 5.06
Size distribution
(µm)
200µm
SEM images
20µm
SEM images
197
98.64
97.00
98.90
Purity
(%w/w)
Table 4. 7. Summarizes some properties of the dried powder obtained at the end of the LAS crystallization process e.g., particle size, SEM
Effect of Additives in LAS crystallization
0.89
0.60
0.37
Residual
solvent
(%)
Contact Angle
(°)
135.7±0.78
136.2±1.77
136.8±2.33
Additive
SDS<CMC
(0.008%w/w)
SDS 0.5% w/w
Tween 20<CMC
(0.008%w/w)
Exp.
3
4
5
Table 4.7. Continued
CHAPTER 4
D[10] : 5.76
D[50] : 77.9
D[90] : 226
D[4.3] : 103
Span : 2.82
D[10] : 3.27
D[50] : 12.0
D[90] : 52.7
D[4.3] :21.0
Span : 4.11
D[10] : 3.55
D[50] : 16.6
D[90] : 81.4
D[4.3] :32.8
Span : 4.67
Size distribution
(µm)
200µm
SEM images
20µm
SEM images
198
101.50
100.45
99.14
Purity
(%w/w)
0.57
0.74
0.59
Residual
solvent
(%)
Contact Angle
(°)
100.5±0.21
140.9±0.42
Additive
Tween 20
0.5% w/w
Chitosan 0.5% w/w
Exp.
6
7
Table 4.7 Continued
Effect of Additives in LAS crystallization
D[10] : 4.26
D[50] : 23.6
D[90] : 132
D[4.3] :58.2
Span : 5.40
D[10] : 2.15
D[50] : 7.55
D[90] : 20.2
D[4.3] :9.56
Span : 2.39
Size distribution
(µm)
200µm
SEM images
20µm
SEM images
199
100.17
98.16
Purity
(%w/w)
0.56
0.59
Residual
solvent
(%)
CHAPTER 4
4.3.1. Effect of the presence of the additive in the organic phase on physical and
surface properties of recrystallized powders
In the second part of the additive screening study, P-407 was incorporated in the
organic phase (ethanol + drug + P-407).
Block co-polymeric surfactants such as P-407, consist of ethyleneglycol and propylene
glycol, and are very efficient non-ionic stabilizers owing to multiple attachments of
hydrophobic domains at the drug particle surface (KIPP, 2004; CHO et al, 2010). Because P407 has hydrophobic moieties that adsorb onto hydrophobic drug particle surface and two
hydrophilic blocks, the adsorption of P-407 onto CRS 74 drug particle surface could provide
an effective steric barrier against crystal growth. From the polymer screening, P-407 was
selected as an additive for this part of the work. The effect of the polymer concentration was
investigated by incorporating the additive in ethanol in two different concentrations (Table
4.4): 0.0003%w/w (<CMC) and 0.02%w/w (> CMC), before the incorporation of the drug.
Following the same experimental procedure already described, Table 4.8 shows the
particle size evolution of the small embryo particles formed in presence of P-407 when the
mixed phases exiting the T-mixer were poured into a vessel and immediately sampling for
analysis. From the results, it can be observed that nanometric particles were obtained, which
continued to grow to reach an approximate particle size average of 350 nm at the end of the
measure time (150 s), independently of the additive concentration. These particles were
slightly more negative charged (-5.4/-7.5 mV) compared to those of drug suspension without
additives (-2.1 mV).
200
Effect of Additives in LAS crystallization
Table 4.8. Initial particle size average (APS) and characteristics of CRS 74 crystals in
suspension in presence of P-407 in the organic phase
Zeta potential
Experiment
Additive
Time (s)
APS (nm)
pH suspension
(mV)
X
1
P-407<CMC(0.0003%w/w)
8
P-407>CMC(0.02%w/w)
9
t0
248
*
*
t150
417
-2.11± 0.28
3.28
t0
199
*
*
t150
351
-5.43±0.51
4.41
t0
133
*
*
t150
341
-7.52±0.37
5.68
Particle size, SEM micrographs, contact angle, drug content and residual solvent of
recrystallized particles are given in Table 4.9. Dv10%, dv50% and dv90% for the dried powders
produced are also presented in Figure 4.6. The corresponding particle size distributions are
graphically presented in Appendix III. When compared to the original or the recrystallized
powder without additives (Figure 4.6), a remarkable effect of reduction of particle size (dv50%
and dv90%) was observed with P-407 at the higher concentration (> CMC). SEM images
confirmed that the CRS 74 microcrystals crystals were clearly homogeneously distributed.
Hardly any agglomerated or aggregated particles were found. This result confirmed that the
microcrystals, which normally aggregate in order to lower the surface energy, could be
stabilized sterically against crystal growth by a layer of protective polymer.
The higher concentration of P-407 also modified the surface properties of the
recrystallized powder (see Table 4.9). Contact angle of the powder with water was reduced
from 133.5 ± 1.8° (without additive) to 85.3 ± 0.8° (P-407 0.5 %w/w).
Table 4.9 also shows a drug content of 96 % for the recrystallized sample with the
higher concentration of P-407. This results shows that all additive was probably not removed
by washing. The remarkable effect on the contact angle can confirm this finding the additive
probably remained attached to the hydrophobic drug particle surface lowering the interfacial
tension.
201
CHAPTER 4
600
Particle size (µm)
515
500
400
300
200
220
138
101
100
83.7
33.8
4.29
4.37
0
6.4
CM
C
40
40
7>
P-
ys
cr
P-
LA
S
re
7<
ta
lli
ze
d
CM
C
dr
lC
in
a
rig
O
dv10%
dv50%
6.47 23.5
ug
RS
74
2.20
dv90%
Figure 4.6. Particle size of the drug powders synthesized in presence of P-407 in the
organic phase.
Figure 4.7 shows the XRD patterns for the powder samples obtained in presence of P407. It can be noted that the crystalline phases were preserved after recrystallization in
presence of this additive.
P-407>CMC
LAS recrystallized drug
Original CRS 74
P-407<CMC
Figure 4.7. X-Ray diffractograms of the drug powders synthesized in presence of P-407
in the organic phase.
202
Contact Angle
(°)
136.4±0.85
133.5±1.77
133.1±3.04
Additive
X
X
P-407<CMC
(0.0003% w/w)
Exp.
Original CRS 74
1
8
D[10] : 6.40
D[50] : 83.7
D[90] : 220
D[4.3] : 101
Span :2.55
D[10] : 4.37
D[50] : 33.8
D[90] : 138
D[4.3] :77.7
Span : 3.94
D[10] : 4.29
D[50] : 101
D[90] : 515
D[4.3] : 191
Span : 5.06
Size distribution
(µm)
200µm
SEM images
Table 4.9. Identification and characterization of dried powder regarding solid state properties
Effect of Additives in LAS crystallization
20µm
SEM images
100.45
97.00
98.90
Purity
(%w/w)
203
0.75
0.60
0.37
Residual
solvent
(%)
Contact Angle
(°)
85.3±0.77
Additive
P-407>CMC
(0.02% w/w)
Exp.
9
Table 4.9. Continued
CHAPTER 4
D[10] : 2.20
D[50] : 6.47
D[90] : 23.5
D[4.3] : 11.2
Span : 3.29
Size distribution
(µm)
200µm
SEM images
20µm
SEM images
96.13
Purity
(%w/w)
204
0.58
Residual
solvent
(%)
Effect of Additives in LAS crystallization
4.3.2. Effect of the presence of the additive in both, aqueous and organic phases
Additives can be placed either in solvent or anti-solvent. The strength of adsorption of
additive molecules on the drug surface depends on the nature of the additive and drug surface
(THORAT and DALVI 2012). Additives are being introduced in the LAS crystallization of
CRS 74 molecule, firstly, to inhibit particle growth and, subsequently, particle agglomeration.
Factors controlling the amount of additive adsorbed on the CRS 74 will probably be related to
the solubility of the additive in liquid phase, to the strength of additive-liquid phase
interactions and to the strength of additive-drug particle interaction. The later can be complex
function of parameters such as functional groups and surface energy as suggested by CHOI et
al (2005).
Stabilizing agents such as polymer and surfactant can be combined together to enhance
the stabilization through synergistic effect (THORAT and DALVI 2012). In such cases the
overall stabilization depends on the pair of the stabilizers used. For instance, synergistic effect
have been reported in previous works combining ionic and non-ionic stabilizer (WU et al.,
2011) or neutral polymers (HPMC) with anionic surfactant (SDS) (DALVI and DAVE,
2009).
Effect of additives on the CRS 74 particles size were analysed by measuring the drug
particles immediately after LAS crystallization. The same experimental sampling already
described in previous sections was followed. It was found that the particles at 0 s were
discrete with a size of approximate two hundred nanometers with SDS, however two times
bigger with Chitosan (Table 4.10). These particles were also examined 150 s after LAS
crystallization and found to be 2 times bigger but always in nanometric range of size. At the
end of the process, after drying, they were examined and found to be several microns as given
in Table 4.10 and graphically represented in Figure 4.8. Surprisingly, the dry particles
produced in presence of Chitosan were not bigger than the other ones. Maybe, Chitosan was
less effective than SDS in arresting the particle growth, but not less effective to prevent
aggregation (high absolute zeta potential given in Table 4.10).
Table 4.11 also shows that the association of additives in both aqueous and organic
phases had a positive effect, reducing the contact angle of the drug powder with water to
94.1°±4.3 with P-407/Chitosan and to 74.7°± 5 with P-407/SDS, in comparison to the original
205
CHAPTER 4
drug (136.4°± 0.8). The powders were characterized by purity higher than 98.8% and residual
solvent content ranged from 0.6% to 1.5% (see Table 4.11).
Table 4.10. Initial particle size average (APS) of CRS 74 crystals in suspension in
presence of additives in both, aqueous and organic phases.
Zeta potential
Experiment
Additive
Time (s)
pH suspension
APS (nm)
(mV)
t0
248
*
*
t150
417
-2.11± 0.28
3.28
t0
150
*
*
t150
368
-17.53±4.77
4.73
P407> CMC (0.02%w/w)+
t0
234
*
*
SDS 0.5% w/w
t150
440
-3.96±0.17
4.27
P407< CMC
t0
457
*
*
t150
990
+39.40±3.24
4.60
P407> CMC (0.02%w/w +
t0
596
*
*
Chitosan 0.5% w/w
t150
918
+16.33±2.73
4.64
X
1
P407< CMC
(0.0003%w/w)+
10
SDS 0.5% w/w
11
(0.0003%w/w)+
12
Chitosan 0.5% w/w
13
600
400
300
200
138
101
100
4.37
4.7
n
sa
sa
to
C+
Ch
i
CM
CM
7<
7>
7>
40
P-
40
n
to
C+
Ch
i
C+
CM
7<
40
P-
P-
dv90%
4.08 13.8
5%
0.
SD
C+
CM
cr
re
S
LA
dv50%
76.5
45
3.27 11.9
S
SD
ta
ys
O
dv10%
29.5
5%
S
lli
rig
ze
0.
d
in
al
dr
ug
CR
S
74
3.55
16.6
40
0
104
81.4
33.8
4.29
P-
Particle size (µm)
515
500
Figure 4.8. Particle size (laser diffraction data) of the drug powders synthesized in
presence of additives in both, organic and aqueous phases.
206
Effect of Additives in LAS crystallization
X-ray powder diffraction patterns shown in Figure 4.9 identical to original CRS 74 for
crystals generated using P-407/Chitosan and P-407/SDS suggested that these additive are not
within the crystal lattice but only adsorbed on the surface.
P-407>CMC+
SDS 0.5% (w/w)
LAS recrystallized drug
Original CRS 74
P-407<CMC+
SDS 0.5% (w/w)
(a)
P-407>CMC+
SDS 0.5% (w/w)
LAS recrystallized drug
Original CRS 74
P-407<CMC+
SDS 0.5% (w/w)
(b)
Figure 4.9. X-Ray diffractograms of drug powders produced with additives in organic
phase: a) P-407/SDS; b) P-407/Chitosan.
207
Contact Angle (°)
136.4±0.85
133.5±1.77
107.9±1.20
74.7±5.52
Addtive
X
X
P-407<CMC
(0.0003% w/w)+
SDS 0.5% w/w
P-407>CMC
(0.02%w/w) +
SDS 0.5% w/w
Exp.
Original CRS 74
1
10
11
D[10] : 4.70
D[50] : 29.5
D[90] : 104
D[4.3] : 43.5
Span : 3.35
D[10] : 3.55
D[50] : 16.6
D[90] : 81.4
D[4.3] :32.8
Span : 4.67
D[10] : 4.37
D[50] : 33.8
D[90] : 138
D[4.3] :77.7
Span : 3.94
D[10] : 4.29
D[50] : 101
D[90] : 515
D[4.3] : 191
Span : 5.06
Size distribution
(µm)
200µm
SEM images
Table 4.11. Identification and characterization of dried powder regarding solid state properties
CHAPTER 4
20µm
SEM images
99.56
99.87
97.00
98.90
Purity
(%w/w)
208
0.94
0.60
0.60
0.37
Residual
solvent
(%)
Contact Angle (°)
141.8±2.97
94.1±4.35
Addtive
P-407<CMC
(0.0003% w/w)+
Chitosan 0.5%
w/w
P-407>CMC
(0.02%w/w) +
Chitosan 0.5%
w/w
Exp.
12
13
Table 4.11. Continued
Effect of Additives in LAS crystallization
D[10] : 4.08
D[50] : 13.8
D[90] : 76.5
D[4.3] : 29.2
Span : 5.23
D[10] : 3.27
D[50] : 11.9
D[90] : 45.0
D[4.3] : 18.5
Span : 3.51
Size distribution
(µm)
200µm
SEMimages
20µm
SEMimages
98.77
98.90
Purity
(%w/w)
209
0.97
1.52
Residual
solvent
(%)
CHAPTER 4
4.3.3. Drug concentration in the organic phase
At we known, an uniform and extremely high supersaturation is required to produce
ultra-fine particles by LAS recrystallization. With the aim to improve control on particle size
of the recrystallized powder, some additional experiments were made to investigate the effect
of supersaturation on the properties of the drug recrystallized in presence of the pairs of
stabilizers, P-407/Chitosan and P-407/SDS. Experiments were conducted with reduced initial
drug concentration in the organic phase. The corresponding supersaturation values are given
in Table 4.12. No remarkable effect of the supersaturation level was observed on the
reduction of the drug particle size, as confirmed by Figure 4.10. The ability to achieve high
particle concentrations without compromising the particle size significantly is of great interest
for scaling up this process. Furthermore, the utilization of relatively concentrated organic
solutions to reduce the amount of organic solvent is also beneficial.
Table 4.12 also shows that drug particles recrystallized in presence of Chitosan were
always positively charged. Powders presented high purity and low residual solvent content
(Table 4.13).
Table 4.12. Initial average particle size (APS) of the CRS 74 crystals in suspension in
presence of different stabilizing systems into internal phase and external phase in different
supersaturations.
Experiment
Additive
S
1
X
894.04
14
P-407>CMC(0.02%w/w)
374.51
15
P-407>CMC(0.02%w/w)
156.41
16
17
18
19
P-407>CMC(0.02%w/w)
+ SDS 0.5% w/w
P-407>CMC(0.02%w/w)
+ SDS0.5% w/w
P-407>CMC+
Chitosan 0.5% w/w
P-407>CMC(0.02%w/w)
+Chitosan 0.5% w/w
104.50
43.25
*
*
Time (s)
APS (nm)
t0
t150
t0
t150
t0
t150
t0
t150
t0
t150
t0
t150
t0
t150
248
417
199
290
140
301
186
404
125
348
489
971
453
1649
Zeta
potential
(mV)
*
-2.11± 0.28
*
-9.70±0.85
*
*
*
-6.39±0.04
*
*
*
+23.87±1.07
*
+23.03±4.11
pH
suspension
*
3.28
*
4.97
*
4.96
*
5.27
*
5.46
*
4.64
*
4.76
*Data not determined
210
Effect of Additives in LAS crystallization
600
400
300
S=894.04
200
138
101
100
S=374.51
S=156.41
44.4
11.4
3.46
4.39 13.7
4.6
)
/w
(w
/w
(w
0.
5%
5%
0.
S
7>
P-
P-
40
7>
CM
C+
CM
Ch
C+
ito
Ch
sa
ito
n
sa
0.
n
SD
CM
7>
40
P-
13.1
)
/w
(w
5%
0.
S
SD
C+
CM
7>
S=*
61.8
)
/w
(w
5%
40
P40
P-
dv90%
79.9
40.1
4.6313.9
)
CM
7>
7>
40
P-
LA
dv50%
S=*
S=43.25
C
CM
dr
ed
liz
tal
ys
cr
S
re
66.3
14.9
3.73
C
ug
S
CR
al
in
ig
Or
dv10%
S=104.50
51.7
C+
0
11.2
3.36
40
33.8
4.37
4.29
74
Particle size (µm)
515
500
S= Supersaturation value
*= Not determined
Figure 4.10. Particle size (laser diffraction data) of the drug powders synthesized in
presence of additives in organic phase and in both (organic and aqueous) at different
supersaturation degrees.
Moreover, no changes on the powder crystallinity were observed as proved by the Xray diffractograms presented in Figure 4.11.
211
CHAPTER 4
P-407>CMC
S=156.41
LAS recrystallized drug
S=894.04
LAS recrystallized drug
S=894.04
P-407>CMC
S=374.51
P-407>CMC+
SDS 0.5% (w/w)
S=104.50
Original CRS 74
Original CRS 74
P-407>CMC+
SDS 0.5% (w/w)
S=43.25
S= Supersaturation value
S= Supersaturation value
(a)
(b)
LAS recrystallized drug
S=894.04
P-407>CMC+
Chitosan 0.5% (w/w)
S=*
(20%)
Original CRS 74
P-407>CMC+
Chitosan 0.5% (w/w)
S=*
(50%)
S= Supersaturation value
*= Not determined
(c)
Figure 4.11. X-Ray diffractograms of the drug powders synthesized in presence of
additives in organic phase and in both (organic and aqueous) at different supersaturation
degrees: a) P-407; b) P-407/SDS 07; c) P-407/Chitosan.
212
S
*
894.04
374.51
156.41
Additive
X
X
P407>CMC(0.02%w/w)
P407>CMC(0.02%w/w)
Exp.
Original CRS 74
1
14
15
D[10] : 3.46
D[50] : 11.4
D[90] : 51.7
D[4.3] : 20.8
Span : 4.23
D[10] : 3.36
D[50] : 11.2
D[90] : 44.4
D[4.3] : 19.5
Span : 3.66
D[10] : 4.37
D[50] : 33.8
D[90] : 138
D[4.3] :77.7
Span : 3.94
D[10] : 4.29
D[50] : 101
D[90] : 515
D[4.3] : 191
Span : 5.06
Size distribution
(µm)
200µm
SEM images
Table 4.13. Identification and characterization of dried powder regarding solid state properties
Effect of Additives in LAS crystallization
20µm
SEM images
213
100.82
102.07
97.00
98.90
Purity
(%w/w)
*
1.00
0.60
0.37
Residual
solvent
(%)
S
104.50
43.25
*
Additive
P407>CMC(0.02%w/w)+
SDS 0.5% w/w
P407>CMC(0.02%w/w)+
SDS0.5% w/w
P407>CMC(0.02%w/w)+
Chitosan 0.5% w/w
Exp.
16
17
18
Table 4.13. Continued
CHAPTER 4
D[10] : 4.39
D[50] : 13.7
D[90] : 79.9
D[4.3] : 30.3
Span : 5.50
D[10] : 4.63
D[50] : 13.9
D[90] : 40.1
D[4.3] : 18.5
Span : 2.55
D[10] : 3.73
D[50] : 14.9
D[90] : 66.3
D[4.3] : 26.0
Span : 4.20
Size distribution
(µm)
200µm
SEM images
20µm
SEM images
214
100.07
102.17
101.54
Purity
(%w/w)
0.78
*
0 .96
Residual
solvent
(%)
*
P407>CMC(0.02%w/w)+
Chitosan 0.5% w/w
19
*Data not determined
S
Additive
Exp.
Table 4.13. Continued
Effect of Additives in LAS crystallization
D[10] : 4.60
D[50] : 13.1
D[90] : 61.8
D[4.3] : 23.9
Span : 4.36
Size distribution
(µm)
200µm
SEM images
20µm
SEM images
215
101.89
Purity
(%w/w)
*
Residual
solvent
(%)
CHAPTER 4
4.3.4. Reorganized organic phase
In the second part of the additive screening study, P-407 was incorporated into the
organic phase (ethanol + drug + P-407). Two concentrations were studied: 0.0003%w/w
(<CMC) and 0.02%w/w (> CMC), before the incorporation of the drug.
The results obtained already discussed in the previous section, showed that the higher
concentration (> CMC) could prevent crystal growth and agglomeration, and modify the
surface properties of the recrystallized powder. Contact angle of the powder with water was
reduced from 133.5 ± 1.8° (without additive) to 85.3 ± 0.8° (P-407 0.5%w/w). These results
were attributed to the probable attachment of the additive to the hydrophobic drug particle
surface lowering the interfacial tension. However, CRS 74 crystals obtained are always
micron-sized.
Due to the molecular characteristics of CRS 74, we hypothesized the possibility of a
molecular pre-organization of this drug in organic solution favoring uncontrolled growth of
crystals after nuclei formation. This hypothesis is investigated in this section.
In fact, a large number of drug molecules are amphiphilic, and self-associate in aqueous
solution to form small aggregates. The self-assembly and self-organization are natural and
spontaneous processes, occurring mainly through non-covalent interactions such as Van der
Waals, hydrogen-bonding, hydrophilic/hydrophobic, electrostatic, donor and acceptor, and
metal-ligand coordination networks (WHITESIDES and GRZYBOWSKI, 2002).
Many molecules are amphiphilic, such as phenothiazines, tranquilizers, analgesics,
peptides, antibiotics, tricyclic antidepressants, and self-associate in surfactant like-manner in
aqueous environment, above a critical concentration value (ATTWOOD and FLORENCE,
1983). It is known that the amphiphilic drug molecules in water form aggregates (MOHD et
al., 2010), but it is described in the literature that the amphiphilic aggregates can be formed on
nano- or micron-sized in selective solvents, like ethanol (LEE et al., 2008).
Some amphiphilic drugs are well studied in the literature, among them Promethazine
hydrochloride (PMT) and Imprimine chloride (IMP), both being used in clinics as
antidepressant and antipsychotic drugs. These amphiphilic compounds possess a hydrophobic
216
Effect of Additives in LAS crystallization
nitrogen-containing heterocyclic bound to a short chain carrying a charged amino group
(ALAM et al., 2008), as shown in Figure 4.12.
S
N
CH3
H3C
N
.
HCl
CH3
PMT
IMP
Figure 4.12 Molecular Structure of Promethazine hydrochloride (PMT) and Imprimine
chloride (IMP). (ALAM et al., 2008).
Regarding the studied molecule CRS 74 (see Figure 4.13), it has the same chemical
function responsible for the drug self-organization in the refered molecules. It possesses a
hydrophobic nitrogen-containing heterocyclic bound to a short chain carrying a charged
amino group, as discussed before for the PMT and IMP. It means that this molecule could
have the same capacity to self-associate in solution above a critical concentration value.
Figure 4.13. Chemical structure of CRS 74.
217
CHAPTER 4
Some experiments were then carried out in our study to verify the effect of drug
concentration in ethanol solution, by dynamic light scattering (PCS) measurements. This
methodology has proved to be a useful tool in characterizing colloidal systems (MOHD et al.,
2010). The study was realized in order to verify the appearance of micelles depending on the
concentration of drug in solution. Figure 4.14 confirms the appearance of drug micelles (1.6 –
1.8 nm) in ethanol at drug concentrations above 0.001 mol/L.
A micellar organization in solution could be the starting point of all LAS crystallization
studies conducted up to here, as schematically represented on Figure 4.15. From a preaggregated molecular organization, it could be difficult to stop growth after nucleation, which
could maybe explain the strong difficulty to stop drug nuclei growth within a nanosize range,
even in presence of different additives.
2
1.8
1.6
Size (nm)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.01
Concentration (mol/L)
Figure 4.14. Diameter of drug micelles in ethanol as a function of drug concentration
(PCS measurements).
218
Effect of Additives in LAS crystallization
Drug Self assembled
Drug-molecular structure
EtOH
CRS 74 concentration
Figure 4.15. Illustration of the effect of increasing concentration of drug in ethanolic
media leading to a micellar organization.
Keeping in mind the above hypothesis, complexes molecular aggregates of CRS 74 in
organic solution can hinder the crystal size control during LAS crystallization. In order to
perturb this molecular organization, some new experiments were carried out. The aim was to
introduce the additive P-407 in the organic phase in a dilute system, in order to promote
interaction between the additive and the CRS 74 molecule in concentrations below the drug
critical micelle organization (0.001 mol/L), as represented on Figure 4.16.
Drug Self assembled
Drug-molecular structure
Amphiphilic Polymer
CRS 74 concentration
EtOH
Micelle Amphiphilic Polymer
C
Concentrated
solution by rotoevaporation
Concentrated solution by rotoevaporation
EtOH
Figure 4.16. Possible schematic representation of drug self-assembled at molecular level in
ethanolic solution.
219
CHAPTER 4
The additive was added to ethanol in a concentration below its CMC (0.0002% w/w). In
the following, the drug was added in a concentration below its critical micellar concentration
(0.00036 mol/L) to prevent drug self-assembly. The organic phase was then concentrated by
rotary evaporation under reduced pressure to a final more concentrated drug solution. Table
4.14 summarizes the results of experiments conducted under the described conditions. A
combined effect of Chitosan was also investigated as shown in the same table. Table 4.15
shows SEM images drug and water contents of the powders obtained. Due to low
supersaturation, the yield of production was too low and the amount of drug powder obtained
was insufficient for further analysis (dry particle size, surface properties, XRD and DSC
analysis).
Compared to the previous experiments for which the drug was in contact with the
additive in concentrations above its critical micelle concentration, no positive effect in particle
size control within the first 150s during LAS crystallization was noted.
Table 4.14. Initial average particle size (APS) of CRS 74 nanocrystals during LAS
crystallization, in presence of P-407 in organic phase, which was concentrated by rotary
evaporation.
Experiment
1
19
APS
(s)
(nm)
894.0
t0
248
*
*
4
t150
417
-2.11± 0.28
3.28
P-407<CMC
77.59
t0
203
*
*
(0.0003%w/w)
*
t150
319
*
4.87
t0
687
t150
1237
Additive
X
S
P407<CMC(0.0003%
20
Zeta
Time
w/w)+Chitosan 0.5%
(w/w)
*
potential
(mV)
pH
suspension
*
+19.93±0.25
4.64
*Data not determined; * Supersaturation calculated after ethanol evaporation
220
*
894.04
77.59
*
X
X
P407<CMC(0.0003%w/w)
P407<CMC(0.0003%w/w)+
Chitosan 0.5% w/w
Original CRS 74
1
19
20
*Data not determined
S
Additive
Exp.
*
*
D[10] : 4.37
D[50] : 33.8
D[90] : 138
D[4.3] :77.7
Span : 3.94
D[10] : 4.29
D[50] : 101
D[90] : 515
D[4.3] : 191
Span : 5.06
Size distribution
(µm)
200µm
SEM images
Table 4.15. Identification and characterization of dried powder regarding solid state properties
Effect of Additives in LAS crystallization
20µm
SEM images
221
101.27
100.34
97.00
98.90
Purity
(%w/w)
*
*
0.60
0.37
Residual
solvent
(%)
CHAPTER 4
4.3.5. Improving process production
Most of LAS recrystallizations realized up to here in order to investigate the influence
of several processes or formulation parameters on the properties of the recrystallized drug
powder were conducted during a short period of time of approximately 2- 6 min. Experiments
in absence of additives, as described in Chapter 3, proved to be quite laborious due to the
rapid growth of the produced particles and problems of blockage of the mixer, which
precluded the realization of essays over time. This is not interesting for the industrial
proceeding.
It seemed that these problems were caused by the affinity of the drug molecule with the
material which constitutes the surface of the rapid mixer. Furthermore, the nano-sized drug
crystals, freshly formed, have a high-energy surface; this property can increase the high
affinity for CRS 74 surface material adsorption reported in Chapter 3.
To solve this blockage problem and to make the LAS crystallization process industrially
viable, some studied systems were selected (Table 4.16). The criteria of choice was the
surface properties of recrystallized drugs: low contact angle values (θ ≈ 90°), that means a
decrease in surface hydrophobicity, and probably, a decrease in affinity of the molecule with
the material which constitutes the hydrophobic surface of the mixer (stainless steel).
Table 4.16. Stabilizing agents studied to solve the T-mixer blockage problem
Experiment
Additive
θ (°)
9
P-407>CMC
85.3
11
P-407>CMC+SDS 0.5% (w/w)
74.7
13
P-407>CMC+Chitosan 0.5% (w/w)
94.1
As shown in Table 4.16, all the stabilizing systems presented a significant reduction of
contact angle value compared to recrystallized drug in absence of additives (136o). However,
the better drug surface characteristics were not enough to prevent the affinity of the molecule
with the surface material for Experiments 9 and 11 (see Table 4.17). On the contrary, drug
particles highly negatively charged by SDS 0.5% showed a strong affinity to T-mixer material
walls (blockage of the mixer operation after 2 min). On the other hand, the combined effect of
222
Effect of Additives in LAS crystallization
P-407 placed in the organic phase and Chitosan in the aqueous phase could solve this
problem, ensuring a continuous process of production through the T-mixer.
Table 4.17. Initial particle size average (APS) of the nanosuspensions and
characteristics of CRS 74 nanocrystals in suspension.
Zeta potential (mV)
pH suspension
Blockage
time (min)
Experiment
Additive
APS (nm)
9
P-407>CMC
169.5
-3,94±0.37
4.51
6
11
P-407>CMC+SDS 0.5%
(w/w)
225.3
-5,26±0.35
4.78
2
13
P-407>CMC+Chitosan
0.5% (w/w)
706.6
+23,80±
± 2.12
4.65
No blockage
To explain this finding, it was necessary to consider the characteristics of the T-mixer
material, i.e. stainless steel. Some materials can be ionized on specific pH; among them, as
reported in the literature, stainless steels can present different surface charge properties as a
function of pH. The material surface is positively charged over the pH range of 2.5 to 5.0
(TAKEHARA and FUKUZAKI, 2002). Because of this, the surface charge of stainless steel
is believed to act as adsorption sites for negatively charged CRS 74, as schematically
represented in Figure 4.17.
pH: 4-5
(a)
pH: 4-5
(b)
Figure 4.17. The charge interaction between the negatively (a) and positively (b)
charged CRS 74 nanocrystals and the negatively surface charged stainless steel (pH in the
range 3-5).
223
CHAPTER 4
In conclusion, the combination of P-407 (organic phase) and Chitosan (aqueous phase)
represented a key solution to a continuous production of recrystallized CRS 74 through
stainless steel rapid mixers.
4.3.6. Dissolution behaviour of recrystallized powders in presence of additives
In previous sections, it was observed changes on some powders properties, like:
wettability, powder agglomeration state and size reduction. These properties will probably
have a direct impact on dissolution profile, which will be verified in this section.
In order to evaluate the impact of powders on dissolution kinetics, the powders for this
section were selected according the lower agglomeration state and/or low contact angle value.
The dissolution of each sample was determined under sink conditions, as discussed in Chapter
2.
Additives can change the drug solubility In the dissolution medium. To ensure sink
conditions (maxium drug concentration << Ceq), the solubility of the recrystallized samples in
presence of the different additives was firstly measured in 0.1M HCl at 37oC. The same
experimental procedure described in Chapter 2, was used. The results are given in Table 4.18
and represented graphically in Figure 4.18. It can be concluded that the probably association
of additives to the drug in some cases did not change the drug solubility in the dissolution
medium, or very little (Ceq < 0.2 mg/gsolution in all cases). To sum up, although additives can
increase the solubility of poorly soluble compounds, the amounts adsorbed onto the CRS 74
particles crystallized in the presence of additives in our study are probably too small to affect
the solubility of the drug in bulk solution.
To assess the differences between original drug and the generated samples, 0.1M HCl
was chosen as a discriminatory dissolution medium (The dissolution method was developed
and presented in Chapter 2).
224
Effect of Additives in LAS crystallization
Table 4.18. CRS 74 solubility and synthesized powders solubility in HCl 0.1M at
37°C.
Stabilizing system
Solubility
(mg/gsolution) ± SD
0.102±0.008
0.134±0.005
0.117±0.006
0.144±0.008
0.091±0.003
0.094±0.003
Original CRS 74
No additive (recrystallized CRS 74)
P407> CMC (0.02%w/w)
HPMC 0.5% w/w
Tween>CMC 0.5%w/w
SDS 0.5%/P-407< CMC (0.0003%w/w)
SDS 0.5% /P-407 > CMC (0.02%w/w)
0.110±0.004
0.088±0.004
0.161±0.009
0.121±0.006
Chitosan 0.5%/ P-407> CMC (0.02%w/w)
P-407<CMC (0.0003%w/w) Reorganized
Chitosan 0.5% / P-407(0.0003%w/w) Reorganized
0.500
0.450
0.400
Concentration(mg/gsolution)
0.350
0.300
0.250
0.200
P407<CMC
0.150
No additive
HPMC 0.5%
P407>CMC
CRS 74
0.100
SDS 0.5%+
P-407> CMC
SDS 0.5% +
P-407 < CMC
Tween>CMC
Chitosan 0.5% +
P407<CMC
Chitosan 0.5%
+ P407> CMC
0.050
0.000
Systems
Figure 4.18. Solubility of CRS 74 samples in HCl 0.1M at 37°C
(samples
recrystallized in presence of different additives; original drug also presented for comparison)
225
CHAPTER 4
Figure 4.19 shows the dissolution profiles of CRS 74 crystallized in presence of the
additives studied in this section. The dissolution profiles of original CRS 74 and the sample
crystallized in the absence of additive are also included in this Figure.
90
80
Dissolved drug (%)
70
60
Original CRS 74
LAS recrystallized drug-No additive
50
P-407>CMC
HPMC 0.5%
Tween>CMC
40
P-407 > CMC+SDS 0.5%
P-407< CMC+SDS 0.5% /
30
P-407> CMC+Chitosan 0.5%
P-407<CMC Reorganized
20
P-407<CMCReorganized+Chitosan 0.5%
10
0
0
50
100
150
200
Time (min)
Figure 4.19. Dissolution profiles using 0.1M HCl as discriminatory dissolution medium
at 37oC and paddle at 75 rpm. LAS recrystallized drug in presence of additive. Original CRS
74 also included.
The dissolution profiles of recrystallized samples in presence of additives and original
CRS 74 were compared by calculating difference factor (f1) and similarity factor (f2) for each
dissolution profile given in Figure 4.19. Table 4.19 shows the calculated f1 and f2 values. f1
(>0) and f2 (50<f2 <85) values confirmed significant difference in dissolution profiles of
recrystallized samples in presence of additives compared to the original drug.
The percentage of drug dissolved within the first 20 min. was used to compare
dissolution rate of various samples. The values are given in Table 4.20. The data indicates that
226
Effect of Additives in LAS crystallization
there is a marked increase in the dissolution rate of CRS 74 from the recrystallized samples in
presence of additive compared to the original drug. The faster dissolution profile led to 80%
of drug dissolved in 3 h of against approximate 20% for original drug or even the drug
recrystallized whitout additives.
Table 4.19. Comparison of dissolution profiles through the difference factor (f1) and the
similarity factor (f2). Original CRS 74 is the reference product for test.
Additive
f1
f2
Original CRS 74
X
*
*
1
No additive
24.8
98.8
2
HPMC 0.5% w/w
389.92
64.18
6
Tween 20 0.5% w/w
518.62
58.89
9
P-407>CMC
749.52
50.50
10
P-407<CMC+SDS 0.5% w/w
239.86
72.65
11
P-407>CMC+ SDS 0.5% w/w
296.38
69.89
13
P-407>CMC+Chitosan 0.5% w/w
659.33
53.54
19
P-407<CMC Reorganized
130.4
84.49
20
P-407<CMCReorganized+Chitosan 0.5% w/w
309.13
69.49
Experiment
Table 4.20. Percentage of drug dissolved within the first 20 min.
Experiment
Additive
Original CRS 74
1
19
10
11
2
6
20
13
9
X
No additive
P-407<CMC Reorganized
P-407<CMC+SDS 0.5% w/w
P P-407>CMC+ SDS 0.5% w/w
HPMC 0.5% w/w
Tween 20 0.5% w/w
P-407<CMCReorganized+Chitosan 0.5% w/w
P-407>CMC+Chitosan 0.5% w/w
P-407>CMC
Percentage of drug
dissolved in 20 min
4.36 ± 0.75
3.37 ± 1.86
11.45 ± 2.71
12.53 ± 2.88
16.40 ± 2.01
22.92 ± 3.79
26.66 ± 1.04
27.74 ± 0.64
34.46±1.59
39.96 ± 1.98
227
CHAPTER 4
Drug release kinetics
In order to describe the kinetics of the release process of drug from the different
samples, two equations were used:
•
The first-order equation, which describes the release from systems where dissolution
rate is dependent on the concentration of the dissolving species (ISHI, 1996; SERRA,
2007; RATNA et al., 2012):
•
(4.1)
The Hixson-Crowell cube root law, which describes the release the release from
dosage forms which show dissolution rate limitation and which do not dramatically
change during the release process. (HIXSON, 1931):
(4.2)
The applicability of these two equations was tested for selected dissolution data giving
the faster drug release rates. The dissolution data were plotted in accordance with the firstorder equation, i.e., the logarithm of the percent remained as a function of time between 10
and 25 min (Figure 4.20) and were also plotted in accordance with the Hixson-Crowell cube
root law, i.e., the cube root of the initial concentration minus the cube root of percent
remained, as a function of time between 0 and 25 min (Figure 4.21). It is evident from Figures
4.20 and 4.21 that a linear relationship was obtained with r2 values close to unity as shown in
Table 4.21.
228
Effect of Additives in LAS crystallization
4.9
4.7
ln % no dissolved
4.5
CRS 74
4.3
No additive
P407>CMC
4.1
P407> CMC+Chitosan 0,5%
3.9
3.7
3.5
5
10
15
20
25
30
Time (min)
Figure 4.20. A linear plot for the dissolution data of CRS 74 powder samples in
accordance with the first-order model equation.
0.2
0.18
0.16
1-[ ( 1-%D/100)1/3 ]
0.14
0.12
CRS 74
0.1
No addtive
0.08
P407>CMC
0.06
P407> CMC+Chitosan 0,5%
0.04
0.02
0
5
10
15
20
25
30
Time (min)
Figure 4.21. A linear plot for the dissolution data of CRS 74 powder samples in
accordance with the Hixson-Crowell cube root law.
229
CHAPTER 4
The first order model describes the release systems for which the dissolution rate is
dependent on the concentration of the drug dissolved in the media, whereas the drug surface
area remains constant. The Hixson-Crowell model has been used to describe a decrease of the
drug particle surface as dissolution occurs. Table 4.21 shows that both mathematical models
fitted well the experimental dissolution data probably because during 25 min the decrease of
particle surface is very low.
Table 4.21. Release parameters of CRS 74 powder samples recrystallized in presence of
additives in comparison to original drug sample.
First order
Exp.
r2
k (min-1)
CRS 74
0.9846
-0.0024
1
0.9973
9
13
Hixson-Crowell
r2
k (min-1)
Linear equation
y = -0.0024x + 4.6104
0.9847
0.0008
y = 0.0008x - 0.0017
-0.0014
y = -0.0014x + 4.598
0.9974
0.0005
y = 0.0005x + 0.0024
0.9964
-0.0246
y = -0.0246x + 4.5899
0.9939
0.0071
y = 0.0071x + 0.0131
0.9961
-0.0157
y = -0.0157x + 4.502
0.9945
0.0046
y = 0.0046x + 0.0371
Linear equation
k = dissolution rate constant
x = t- e y = ln % No dissolved
** x = t e y = 1-[(1-%Dissolved/100) 1/3 ]
DSC was performed on the recrystallized drug in presence of additive in the organic
phase (P-407) and in both organic (P-407) and aqueous phase (Chitosan) in comparison to
DSC data for original and recrystallized CRS 74 without additives. The DSC curves of
samples are shown in Figure 4.22. From DSC data summarized in Table 4.22, it was
concluded that stabilizers did not change the physical state of CRS 74.
Interestingly, CRS 74 recrystallized in presence of P-407 and Chitosan needs less
energy for melting when compared to the other samples (ΔHm=74.8 J/g), which could be
related to a less energy it must overcome to dissolve in according to its faster dissolution rate.
230
Effect of Additives in LAS crystallization
Table 4.22. Melting Temperature (Tm(Onset)), Heat of Fusion (ΔHf) for the original and
LAS recrystallized drug in presence and absence of additives.
Thermal and
LAS
Original
dissolution
LAS recrystallized
LAS recrystallized
recrystallized
CRS 74
parameters
CRS 74-PCRS 74-P-407
CRS 74
407+Chitosan
Tm(Onset)(°C)
188.6
187.8
189.0
186.6
ΔHm(J/g)
86.6
79.2
87.9
74.8
Relative enthalpy(%)
100
91.5
101.5
86.37
231
4,5
1,8
4
1,6
3,5
1,4
3
1,2
Original CRS 74
2,5
1
2
0,8
1,5
0,6
1
Heat Flow (mW ) - LAS recrystallized drug
Heat Flow (mW ) - Original CRS 74
CHAPTER 4
0,4
LAS recrystallized drug
0,5
0,2
0
0
70
90
110
130
150
170
190
210
Temperature (°C)
(a)
(b)
4.5
0
2.5
-5
-8
-15
Original CRS 74
-20
LAS recrystallized drug
P-407>CMC
-10
Heat Flow (mW )
Heat Flow (mW )
-6
Heat Flow (mW )
3.5
-4
-10
3
1.5
160
170
180
LAS recrystallized drug
P-407>CMC
2
1.5
-12
1
-14
0.5
1
0
-16
150
2
Original CRS 74
2.5
0.5
-25
140
3
4
-2
Heat Flow (mW )
0
190
0
70
200
90
110
130
150
170
190
210
Temperature (°C)
Temperature (°C)
(c)
(d)
0
4.5
0
2.5
4
-2
-6
Original CRS 74
-15
-8
LAS recrystallized drug
P-407>CMC+Chitosan 0.5%(w/w)
-20
-12
150
160
170
Temperature (°C)
(e)
180
Original CRS 74
1.5
2.5
LAS recrystallized drug
P-407>CMC+Chitosan 0.5%(w/w)
2
1
1.5
1
-10
-25
140
Heat Flow (mW )
-4
-10
3
190
200
Heat Flow (mW )
2
3.5
Heat Flow (mW )
Heat Flow (mW )
-5
0.5
0.5
0
0
70
90
110
130
150
170
190
210
Temperature (°C)
(f)
Figure 4.22. a) DSC thermograms of original CRS 74 (first heating), which consists of a melting
endotherm (peak onset temperature Tm(Onset) = 188.64oC) and LAS recrystallized drug, which consists of a
melting endotherm (peak onset temperature Tm(Onset) = 187.79oC); b) DSC thermograms of original CRS 74
(cooling after first heating), which consists of a crystallization exotherm (peak onset temperature Tc(Onset) =
132.12oC) and LAS recrystallized drug, which consists of a crystallization exotherm (peak onset temperature
Tc(Onset) = 138.43oC). c) DSC thermograms of original CRS 74 (first heating), and LAS recrystallized drug in
presence of P-407>CMC, which consists of a melting endotherm (peak onset temperature Tm(Onset) = 189.01oC);
d) DSC thermograms of original CRS 74 (cooling after first heating), and LAS recrystallized drug in presence
of P-407>CMC, which consists of a crystallization exotherm (peak onset temperature Tc(Onset) = 137.61oC) ; e)
DSC thermograms of original CRS 74 (first heating) and LAS recrystallized drug in presence of P407>CMC+Chitosan 0.5% (w/w), which consists of a melting endotherm (peak onset temperature Tm(Onset) =
186.58oC); f) DSC thermograms of original CRS 74 (cooling after first heating) and LAS recrystallized drug in
presence of P-407>CMC+Chitosan 0.5% (w/w), which consists of a crystallization exotherm (peak onset
temperature Tc(Onset) = 136.21oC).
232
Effect of Additives in LAS crystallization
4.4. CONCLUSION
To improve particle size control during the LAS crystallization process of CRS 74, and
they were introduced in the solvent (P-407) or in the anti-solvent (all the other ones) in our
CRS 74-ethanol-water system. A maximum concentration was fixed for all additives to give a
mass ratio of 1:4 (additive:drug). This corresponds to 0.5%w/w for HPMC, Chitosan, Tween
20 and SDS and 0.02% (w/w) for P-407.
The first results showed that additives enhanced the drug solubility in the ethanol-water
mixture, markedly or very little, depending on the additive used. Among them, SDS 0.5%
presented the higher capacity to solubilize CRS 74 in the binary solvent mixture, which is
attributed to the better solubilization power of anionic surfactants.
Chitosan and SDS 0.5% were proven to be the best additives to ensure an electrostatic
repulsion between freshly CRS 74 crystals formed in the LAS process. The higher particles
are equally charged, the higher is the electrostatic repulsion between particles. The
electrostatic repulsion given by Chitosan (crystals positively charged) played a decisive role
in controlling aggregation of freshly formed crystal drug to the mixer walls, also charged
positively at pH of the liquid mixture. Contrarily, drug particles highly negatively charged by
SDS 0.5% showed a strong affinity to T-mixer material walls (blockage of the mixer
operation in a shorter time).
The primary role of stabilizers is to inhibit excessive crystal growth. In a general way,
most CRS 74 crystals produced in presence of additives had a smaller particle size compared
to original or recrystallized powder without additives. Additives such as HPMC and Tween
20 (concentration below CMC) modestly inhibited agglomeration, while a higher
concentration of Tween 20 (above CMC) had a very positive effect on particle size control.
Agglomerates or aggregates were obtained in presence of HPMC. A possible
explanation could be an inappropriate diffusion (too slow) of the solvent toward the antisolvent caused by a high viscosity of the anti-solvent containing 0.5%w/w HPMC. A high
viscosity could prevent diffusion between solution and anti-solvent and result in nonuniform
supersaturation and agglomeration.
233
CHAPTER 4
Concerning surface properties, the unique additive placed in the aqueous phase, which
was able to reduce consistently the contact angle of this drug with water to approximately
100°, was Tween 20 0.5%.
In the second part of the additive screening study, P-407 was incorporated in the organic
phase (ethanol + drug + P-407). The effect of the polymer concentration was investigated by
incorporating the additive in ethanol in two different concentrations: 0.0003%w/w (<CMC)
and 0.02%w/w (> CMC), before the incorporation of the drug.
These particles were slightly more negative charged (-5.4/-7.5mV) compared to those of
drug suspension without additives (-2.1mV).
This result confirmed that the microcrystals, which normally aggregate in order to lower
the surface energy, could be stabilized sterically against crystal growth by a layer of
protective polymer. The higher concentration of P-407 also modified the surface properties of
the recrystallized powder. Contact angle of the powder with water was reduced from
133.5o±1.8 (without additive) to 85.3 o ±0.8 (P-407 0.5%w/w). The remarkable effect on the
contact angle can confirm this finding: the additive probably remained attached to the
hydrophobic drug particle surface lowering the interfacial tension.
The dry particles produced in presence of Chitosan were not bigger than the other ones.
Maybe, Chitosan was less effective than SDS in stopping the particle growth, but more
effective to prevent aggregation (higher absolute zeta potential)
The association of additives in both aqueous and organic phases had a positive effect,
reducing the contact angle of the drug powder with water to 94.1°± 4.3 with P-407/Chitosan
and to 74.7°± 5 with P-407/SDS, in comparison to the original drug (136.4°± 0.8).
Finally, X-ray powder diffraction patterns confirmed that these additives are not within
the crystal lattice, but only adsorbed on the surface.
Dissolution study confirmed the enhancement of the dissolution rate of CRS 74 crystals
in presence of additives because of an increase of surface area and of a modification of
surface properties of crystal.
234
General Conclusions and Perspectives
Résumé Chapitre 5- Conclusion
Dans cette étude, la cristallisation par effet anti-solvant a été utilisée pour recristalliser
un nouveau médicament antirétroviral. L'objectif était d'améliorer ses propriétés de
dissolution. Pour générer un mélange efficace et une sursaturation rapide et uniforme, des
mélangeurs spécifiques ont été utilisés (T-mélangeur, mélangeur Roughton). Les meilleures
conditions de cristallisation de cette nouvelle molécule ont été définies par une étude de
solubilité, un développement de méthodes de dissolution et un screening des paramètres
opératoire de la cristallisation et d’additifs.
Il a été démontré la poudre présente une faible solubilité en phase aqueuse et une vitesse
de dissolution lente en milieu acide. Ce travail a permis de construire à travers différentes
caractérisations une monographie préliminaire de ce solide.
La molécule étudiée est soluble dans l'éthanol (92,6 mg/g de solution) à 30°C. Les
résultats de la modélisation des équilibres liquide-solide par un modèle à coefficient d’activité
(UNIQUAC) indiquent que ce modèle est un outil approprié pour représenter le
comportement de solubilité de ce solide dans des mélanges de solvants (éthanol-eau). Les
solubilités expérimentales et calculées présentent un bon accord. La modélisation a permis
d’évaluer un ratio éthanol/eau optimum (25/75 % m/m) pour maximiser le rendement
théorique en solide.
Le développement du protocole expérimental de recristallisation a montré que les
paramètres opératoires (débits d’entrée des solutions, type de pré-mélangeur, concentration)
n'ont pas d'influence sur les particules recristallisées. De plus, préparer de fines particules de
ce solide est un véritable défi, en raison de la croissance et l’agglomération très rapides des
particules produites. Des problèmes de colmatage des canaux du pré-mélangeur ont aussi été
constatés.
Il a été montré que la cristallisation de ce solide donne naissance à des particules de
tailles nanométriques en sortie de pré-mélangeur. Cependant ces nanoparticules ont une forte
tendance à croître et à s’agglomérer. Afin de limiter la croissance et l’agglomération des
cristaux, des additifs ont été sélectionnés et introduits dans les solutions initiales. Chaque
additif a montré un comportement différent en empêchant la croissance des cristaux. La
combinaison de deux additifs (P-407 et chitosane) a été la plus efficace : diminution de la
croissance des cristaux, amélioration de la mouillabilité des particules formées,
modification de charge de surface de particules (elle devient positive). Cette modification de
charge de surface a empêché le colmatage des canaux au cours de la cristallisation (répulsion
électrostatique avec les parois de mélangeur). Dans l’ensemble, les microcristaux synthétisés
en présence d'additifs ont montré une vitesse de dissolution nettement plus élevée que les
cristaux de la poudre initiale.
L’amélioration de la vitesse de dissolution est principalement due à l'amélioration des
propriétés de mouillage grâce à des interactions spécifiques entre la poudre et les additifs. Par
conséquent, les microcristaux synthétisés ont de meilleures propriétés physico-chimiques.
L’utilisation de cette technique serait donc une alternative pour l’obtention d’une poudre avec
de meilleures caractéristiques de dissolution et par conséquent une meilleure biodisponibilité.
Pour continuer ses travaux sur l'amélioration de la biodisponibilité du CRS 74,
différentes voies peuvent être suggérées en terme de procédé, de compréhension du
mécanisme de stabilisation, et du potentiel du principe actif.
En termes de procédé, il serait intéressant d'étudier l'influence des paramètres
opératoires de la cristallisation (débit, concentration en additifs, température …) pour la
formulation comprenant les deux additifs (P-407 et chitosane) afin d'optimiser le processus de
production des cristaux. De plus, la cristallisation par effet anti-solvant pourrait être comparée
à d'autres technologies « bottom-up » prometteuses pour la préparation de nanocristaux de
CRS 74, comme par exemple : la préparation de dispersions solides par atomisation ou par
extrusion à chaud.
Au cours de cette étude, il était clair que la cristallisation en présence d'additifs peut
avoir un effet positif sur la vitesse de dissolution. Il est donc nécessaire d'approfondir la
compréhension du mécanisme d'adsorption de l'additif et de déterminer quel est le niveau
d'adsorption nécessaire pour obtenir un effet prononcé sur les propriétés de surface des
particules. Pour comprendre cette propriété, une détermination quantitative de l’adsorption de
l'excipient devra être effectuée. Il sera intéressant d'étudier différentes hypothèses
d’attachement à l'échelle moléculaire. L’interaction de l'additif sur la surface cristalline
pourrait être étudiée, par exemple, par spectroscopie Raman.
Enfin, la méthodologie de dissolution développée dans cette étude devra être validée
afin de pouvoir l’utiliser en tant que technique sensible, fiable et reproductible pour les tests
de bioéquivalence. De plus l'effet biologique du solide recristallisé par rapport au solide
d'origine devra être identifié afin d'évaluer les changements de la poudre synthétisée et
déterminer le réel effet sur l’amélioration de la biodisponibilité.
Conclusions and Perspectives
This study concerned a novel antiretroviral promising drug candidate, named CRS 74.
It could be proven in this study that the original drug powder (as currently produced
industrially) exhibits poor aqueous solubility and slow dissolution rate.
The limited low aqueous solubility at 37
o
C (< 0.004 mg/g) can be explained by its
high crystallinity, its high melting point (188.6oC) and high enthalpy of fusion (86.6 J/g). The
low dissolution rate is related to its large particle size (micrometric range, with a broad
particle size dispersion) and very poor water wettability (Θ =136.4 ± 0.8°).
This molecule is soluble in ethanol (92.6mg/gsolution) and presented a maximum in
solubility in ethanol-water mixtures as determined in this study using UNIQUAC-based
model. To the best of our knowledge, there are no published data of the solubility of such
given solute in ethanol-water mixtures to compare with.
There is no monograph of this drug in any pharmacopoeia and, from the
characterization study carried out in this thesis, a monograph proposition is suggested:
CRS 74 monograph
Synonym:
(2S, 3S, 5S)-2, -5 bis- [N-[N-[[N- methylN-[(2-isopropyl- 4- tiazolyl) methyl]
amino] carbonyl] vanilyl] amino- 1,6diphenyl- 3- hydroxyhexane
Therapeutic category
HIV protease inhibitor
Formula
C46H66N8O5S2
Description
white powder
Molecular weight (g/mol)
875.2
Melting point (°C)
187-189
Melting enthalpy (J/g)
78-87
Solubility
0.102 mg/gsolution in 0.1M HCl pH 1.2,
37oC - Very slightly soluble
lower than 0.004 mg/g in water at 37 o C –
Insoluble
92.6 mg/gsolution in ethanol (solvent) at
30°C -Very soluble
241
CHAPTER 5
To predict in vitro CRS 74 dissolution behavior, a conventional acid medium was
chosen. The development of the analytical methodology of quantification and dissolution
assays is very important. The inexistence of specific method for CRS 74 in official summaries
highlights the importance of above-cited tests for the definition of specific method for such
purpose appropriates method to quality control of the drug. The quantification method was
developed in this work to quantify the drug in acid media (0.1M HCl) or in water-ethanol
solutions. Both developed methods were linear and specific for the drug.
Several original data were experimentally obtained concerning the solubility of CRS 74
in liquid media. They are:
•
Acidic aqueous medium (0.1 M HCl) at 37°C;
•
Acidic aqueous medium in presence of five different additives at 37°C;
•
Hydroalcoholic solutions in different water-ethanol mass proportions at 30°C;
•
Pure ethanol at different temperatures (5, 10, 15, 20, 25 and 30°C)
•
Water (LAS recrystallized drug) at 37°C.
In hydro-alcoholic solutions, the drug solubility increases as the ethanol ratio in ethanolwater increases, presenting a maximum as determined in this study using UNIQUAC-based
model. The calculated data showed good agreement with experimental results and revealed a
maximum solubility of CRS 74 of 130.20 mg/gsolution for a mass ratio of ethanol/water of
0.83/0.17 (w/w).
The results of the modeling indicate that this model is the appropriate tool for
representing the solubility behavior of CRS 74 in ethanol-water solvent mixtures. The
measurements and correlation of CRS 74 solubility in binary solvent mixtures provided useful
data to estimate the theoretical efficiency of the LAS recrystallization process as a function of
ethanol/water mass ratios. An ethanol-water mixture containing 25% (w/w) ethanol was found
favorable for the crystallization process and was fixed for the LAS crystallization.
For experimental design to CRS 74 LAS crystallization, two different types of mixers
were tested: a T-mixer with two radial entries and a two jets vortex mixer also called
Roughton mixer. It was clearly observed, that nano-sized particles were obtained at the outlet
of both mixers. However, no remarkable effect of the supersaturation level was observed on
the reduction of the drug particle size.
242
Conclusions and Perspectives
The T-mixer was chosen for further experiments because it generated less
agglomerated particles. However to prepare fine particles of CRS 74 without polymer became
quite a challenge, due to the rapid growth and agglomeration of the produced particles and
problems of blockage of the mixer.
Recrystallized solids were compared to the original drug crystals in terms of particle
size, solid state, physical and dissolution properties. The particles of the original powder were
found to be larger and exhibited a broad particle size distribution compared to the LAS
recrystallized drug. In turn, recrystallized solids seemed more agglomerated than the original
ones. In addition, recrystallization did not modify the dissolution profile compared to the
original drug. It could be concluded that to improve dissolution kinetics, smaller particles
with a more hydrophilic surface need to be produced. So the LAS crystallization was then
carried out in presence of different additives.
Stabilizers were screened and introduced in the solvent, or in the anti-solvent, or in both
phases. For steric stabilization, we used nonionic surfactant (Polyoxyethylene sorbitan
monooleate –Tween 20) polymers (Hydroxypropylmethylcellulose-HPMC) and amphiphilic
block copolymers (Poloxamer 407-P-407). For electrostatic repulsion, the anionic surfactant
Sodium Dodecyl Sulfate (SDS) was chosen. For a combined effect of steric stabilization and
electrostatic repulsion, we tested Chitosan, a copolymer of glucosamine and N-acetyl
glucosamine.
The primary role of stabilizers is to inhibit excessive crystal growth. In a general way,
most CRS 74 crystals produced in presence of additives had a smaller particle size compared
to the original or recrystallized powder without additives.
As a result of introduction of additives in the LAS crystallization study, the crystal size,
agglomeration state, dissolution kinetics and drug wettability varied over a wide range
depending on the additive involved and its concentration. According to the results, the zeta
potential remained almost unchanged when nonionic stabilizers were used. In other hand,
charged additives like SDS (negatively charged) and Chitosan (positively charged) adsorbed
with the charged parts of the respective molecules onto the drug particle surface.
Over the additives screened, changes on surface properties were observed. A
remarkable decrease of contact angle (θ<100°) was observed for two formulations, one with
P-407 (concentration of 0.02 % in organic phase) and another with P-407 combined with
243
CHAPTER 5
chitosan (P-407 concentration of 0.02 % in organic phase and chitosan concentration equal to
0.5 % in aqueous phase) at the recrystallized powder in the higher concentration of P- 407, P407 and Chitosan. The enhancement of wetting properties can be attributed to multiple
attachments of hydrophobic domains of P-407 on the drug particle surface.
Each additive showed different behavior in preventing crystal growth, but the
association of P-407 in the organic phase and chitosan in the aqueous phase was most
effective in preventing crystal growth, improving surface wettability, and providing enough
electrostatic repulsion due to highly positive charged drug surface.
When placed in the organic phase, P-407 chains are more available to precipitate drug
particles upon mixing, since they do not need to diffuse across the interface through the
volume of the aqueous phase, which exceeded the organic phase volume. This reduction in
time for diffusion of polymer to the drug surface leads to more rapid stabilization against
coagulation and condensation. The presence of surfactant at the surface of the clusters may be
expected to lower the interfacial tension between the solid surface and solvent mixture and
raise the nucleation rate.
Drug microcrystals recrystallized in presence of additives showed significantly
improved dissolution velocity in comparison to microcrystals (raw material and without
additive). Among the additives tested, P-407 and P-407 + Chitosan gave the best results. They
enhanced dramatically the drug dissolution rate from 4% to almost 40% drug dissolved at 20
min. In addition, the presence of chitosan in the crystallization process solved the problem
related to the process, the blockage, due to charge repulsion between the positively charged
crystals and surface charge of stainless steel.
Overall, the results of this research showed conclusively that the liquid anti-solvent
crystallization technique in presence of additives used in this research produced microcrystals
that exhibited significantly faster dissolution rates than the original (as-received) CRS 74
crystals. The improved dissolution is attributable to the modification of the particle size of
drug crystals and enhancement of wetting properties due to specific interactions between the
drug and the additives. Therefore, CRS 74 microcrystals yielded better physicochemical
properties and would be one alternative for better CRS 74 dissolution characteristics and
thereby bioavailability for commercial purposes.
To continue the work concerning the improvement of the bioavailability of CRS 74,
244
Conclusions and Perspectives
some strategies are suggested in different fields (process, stabilization mechanism and drug
potential).
In term of process, it will be interesting to study the influence of crystallization
operating conditions with the association of two additives (P-407, Chitosan) on solid
properties in order to optimize and eventually scale up the process. This process could be
compared to others promising technologies based on bottom-up preparation of CRS 74 drug
nanocrystals, like the preparation of solid dispersions by spray drying and hot melt extrusion.
Still in terms of process, there is a need for further understanding excipient adsorption, e.g.,
what is the level of adsorption needed to provide a pronounced effect on particle properties?
In order to understand this point, a quantitative determination of excipient adsorption should
be carried out.
During this study it was clear that crystallization in the presence of additives can have a
positive effect on dissolution rate. For instance, some hypotheses about the attachment on
molecular level of additive on crystal surface were explored. However, a better understanding
of these interactions should be carried out. The interaction additive crystal-surface could be
explored by Confocal Raman Spectroscopy.
The last research field is related to the drug. The dissolution methodology developed in
this study could be validated and used as a sensitive, reliable, and reproducible for
bioequivalence testing. Finally, the biological effect of recrystallized solid compared to
original solid should to investigate in order to assess the changes of the synthesized powder
and its improved bioavailability.
245
HIV and its life cycle
Human Immunodeficiency Virus (HIV) is a retrovirus (constituted by RNA)
belonging to the Lentivirinae subfamily, capable to sponge human immunologic system
causing the infectious disease known by the acronym AIDS (Acquired Immuno Deficiency
Syndrome).
HIV comprises an outer envelope consisting of a lipid bilayer with spikes of
glycoproteins (gp), gp41 and gp120. These glycoproteins are linked in such a way that gp 120
protrudes from the surface of the virus. Inside this envelope is a nucleocapsid (p 17), which
surrounds a central core of protein, p24. Within this core, are two copies of single-stranded
RNA (the virus genome). Proteins, p7 and p9, are bound to the RNA and are believed to be
involved in regulation of gene expression. Multiple molecules of the enzyme, reverse
transcriptase (R T), are also found in the core. This enzyme is responsible for converting the
viral RNA into proviral DNA (ABBAS, 2000), as shown in the Figure I.1.
Figure I.1. Schematic drawing of the lentivirus HIV (ENKIRCH, 2011)
HIV may only infects certain types of cells. In general, these are cells which carry
CD4 receptors on their surface. Some cells in the immune system have these receptors, in
particular, T4-lymphocytes or T-helper cells. Cellular infection occurs when HIV virus binds
to a cellular receptor, generally the T CD4+, by means of the gp120 protein; then, virus
merges to the cell membrane and the capsid content is released into the cell cytoplasm. The
HIV enzyme, reverse transcriptase, catalyses the DNA copy production starting from HIV
virus RNA. The double helix DNA copy is then transported to the cellular nucleus where a
second HIV enzyme, the integrase, catalyses the incorporation of viral DNA to the host
genetic material. Subsequent viral genes expression results in RNA transcription starting from
251
HIV DNA and in translation of viral proteins. However, newly formed viral proteins are
produced in the form of polyproteins precursors that are long unities consisting of viral
enzymes and structural proteins added to each other. Polyproteins and viral RNA move to the
cell surface where they are incorporated to the new viruses that spring from cell membrane
taking part of it with them to form the external layer of the viruses (figure I.2). Newly formed
viruses, however, could not be infectious without the action of a third essential HIV enzyme,
the protease, that turns viral polyproteins in functional and structural proteins and enzymes.
Proteases are enzymes that cleave others proteins at highly specific sites. HIV protease, an
aspartyl protease, cleaves viral polyproteins in essential functional proteins during the process
of maturation of the "virion" (complete viral particle). This process occurs when each new
"virion" springs to outside of the infected cell membrane and it continues after the release of
immature virus by the cell. If polyproteins are not cleft, virus formation does not finish and it
becomes unable to infect a new cell. Protease inhibitors, as this name implicate, are
substances able to inhibit protease enzyme function. They perform their inhibitory effect
disabling the enzyme before it cleaves gag/pol polyprotein to form its essential products. It
means that, blockage of HIV protease leads to the formation of immature non-infectious- PCT
document number 111006 (BOCKELMANN et al., 2005; BOCKELMANN et al., 2010 ).
Figure
I.2.
Life
cycle
of
HIV
(HOGGARD
and
OWEN,
2003).
252
Calibration pumps
In order to validate the flow of gear pumps (mzr-7255-hs-f S, mzr-7205-hs-f S; HNP
Mycrosysteme) for the solvent and the anti-solvent system and digital mass flow
meter/controller (MIX, Bronhorst), the pumps calibration was realized.
For this purpose, a beaker with the fluid to be pumped was weighed and the software
was activated, starting the pump. At the end, after all the fluid has being pumped, the beaker
was weighed and the theoretical mass flow of fluid was calculated with the mass used and the
time spent in the experiment. Finally, the value of the calculated flow was compared with the
flow indicated by the software.
The calibration test was realized for both pumps with water used as the fluid for the
anti-solvent’s pumps and ethanol to the solvent’s pump. According to the table II.1, the value
of the calculated flow is in agreement with the flow value indicated by the software.
Table II.1. Data obtained in the pumps calibration
Pumped liquid
Mass (g)
Time
(min)
Flow rate calculated
(g/min)
Flow rate software
(g/min)
Error
(%)
Ethanol
109.3
15
7.29
7
4.0
Water
749.5
15
49.97
50.17
0.4
In a second time, to ensure that the pumps were pumping fluids in the correct ratio a
simple test was performed. Mixtures between ethanol and water in different proportions were
made without the aid of the pumps and measured up their refractive indices. Later, the same
mixtures were performed using the pumps and they also had their refractive indices measured.
Each result is an average of at least 3 replicates.
No changes of refractive indices were observed, for all different ratio ethanol/water
using different manners of preparation, as seen in the table II.2. It can be supposed that, the
pumps can respond correctly to request flow values and they pumped the fluids in the correct
proportions. It means that pumps are working as it should.
255
Table II.2. Values of the refractive indices measured ± standard deviation .
Proportion
Physical mixture
Mixture with pumps
ethanol/water (w:w)
1:1
1.3582 ± 0.0001
1.3592 ± 0.0002
2:1
1.3523 ± 0.0004
1.3548 ± 0.0001
3:1
1.3476 ± 0.0004
1.3478 ± 0.0002
256
Paticle size distribution
LAS crystallization process conditions
•
T-Mixer
•
Ratio solvent (ethanol):anti-solvent (water) - 1:3 (25%:75%) ;
•
Presence of additive
•
Temperature of organic and aqueous phases at 30°C
•
Crystals collection: vessel under magnetic stirring
259
Additive in aqueous phase
Figure III.1. Particle size distribution pattern for as HPMC 0.5 % (w/w)
Figure III.2. Particle size distribution pattern for as SDS 0.5 % (w/w) and SDS <
CMC.
260
Figure III.3.
Tween20 < CMC.
Particle size distribution pattern for as Tween20 0.5 % (w/w) and
Figure III.4. Particle size distribution pattern for as Chitosan 0.5 % (w/w).
261
Addtive in organic phase
Figure III.5. Particle size distribution pattern for as P-407 0.5 % (w/w) and P-407 <
CMC.
262
Additive in both, organic and aqueous phase
Figure III.6. Particle size distribution pattern for as P-407 < CMC + SDS 0.5 %
(w/w) and P-407 > CMC + SDS 0.5 %(w/w).
Figure III.7. Particle size distribution pattern for as P-407 < CMC + Chitosan 0.5 %
(w/w) and P-407 > CMC + Chitosan 0.5 %(w/w).
263
Supersaturation degree
Figure III.8. Particle size distribution pattern for as P-407 0.5 % (w/w) (S =156.41)
and P-407 0.5 % (S =374.51)
Figure III.9. Particle size distribution pattern for as P-407 > CMC + SDS 0.5 % (w/w)
(S = 43.25) and P-407 > CMC + SDS 0.5 % (w/w) (S = 104.5).
264
Figure III.10. Particle size distribution pattern for as P-407 > CMC + Chitosan 0.5 %
(w/w) (20 %) and P-407 > CMC + Chitosan 0.5 % (w/w) (50 % ).
265
Contact angle (θ °) as a function of time
LAS crystallization process conditions
•
T-Mixer
•
Ratio solvent (ethanol):anti-solvent (water) - 1:3 (25%:75%) ;
•
Presence of additive
•
Temperature of organic and aqueous phases at 30°C
•
Crystals collection: vessel under magnetic stirring
269
Original CRS 074
Figure IV.1 Contact angle (°) as a function of time for as CRS 74.
LAS recrystalized drug
Figure IV.2. Contact angle (°) as a function of time for as LAS recrystalized drug .
270
Additive in aqueos phase
Figure IV.3. Contact angle (°) as a function of time for as HPMC 0.5 % (w/w).
Figure IV.4. Contact angle (°) as a function of time for as SDS < CMC.
271
Figure IV.5. Contact angle (°) as a function of time for as SDS 0.5 % (w/w).
Figure IV.6. Contact angle (°) as a function of time for as Tween 20 < CMC.
272
Figure IV.7. Contact angle (°) as a function of time for as Tween 20 0.5 % (w/w).
Figure IV.8. Contact angle (°) as a function of time for as Chitosan 0.5 % (w/w).
273
Additif in the organic phase
Figure IV.9. Contact angle (°) as a function of time for as P-407 < CMC.
Figure IV.10. Contact angle (°) as a function of time for as P-407 0.5 % (w/w).
274
Additif in both, organic and aqueos phases
Figure IV.11. Contact angle (°) as a function of time for as P-407< CMC + SDS
0.5%(w/w).
Figure IV.12. Contact angle (°) as a function of time for as P-407 >CMC + SDS
0.5%(w/w).
275
Figure IV.13. Contact angle (°) as a function of time for as P-407< CMC + Chitosan
0.5 % (w/w).
Figure IV.14. Contact angle (°) as a function of time for as P-407 > CMC + Chitosan
0.5 % (w/w).
276
Morphology of crystals in suspension
HPMC 0.5% (w/w)
X
1
2
Additive
Exp
Additive in aqueous phase
S = 445.63
Ratio
S:AS*1:3
S = 894.04
Ratio
S:AS*1:3
Experimental
Conditions
500 µm
500 µm
20x
Microscopic image
500 µm
500 µm
40x
Microscopic image
279
4
3
Exp
SDS 0.5%
(w/w)
(0.008 % (w/w))
SDS<CMC
Additive
S = 150.51
Ratio
S:AS*1:3
S = 391.85
Ratio
S:AS*1:3
Experimental
conditions
500 µm
500 µm
20x
Microscopic image
500 µm
500 µm
40x
Microscopic image
280
6
5
Exp
S = 398,72
Ratio
S:AS*1:3
S = 541.13
(0.008 % (w/w))
Tween 20 0.5%
(w/w)
Ratio
S:AS*1:3
Experimental
conditions
Tween
20<CMC
Additive
500 µm
500 µm
20x
Microscopic image
500 µm
500 µm
40x
Microscopic image
281
AS: anti-solvent
*S: solvent
7
Exp
Chitosan 0.5%
(w/w)
Additive
x: data not dertemined
S=x
Ratio
S:AS*1:3
Experimental
conditions
500 µm
20x
Microscopic image
500 µm
40x
Microscopic image
282
8
1
Exp
P-407<CMC
(0.0003%(w/w))
X
Additive
Additive in organic phase
S = 901.97
Ratio
S:AS*1:3
S = 894.04
Ratio
S:AS*1:3
Experimental
conditions
500 µm
500 µm
20x
Microscopic image
500 µm
500 µm
40x
Microscopic image
283
Additive
P407>CMC
(0.02% (w/w))
AS: anti-solvent
*S: solvent
9
Exp
d
S = 673.40
Ratio
S:AS*1:3
Experimental
conditions
500 µm
20x
Microscopic image
500 µm
40x
Microscopic image
284
10
1
Exp
P-407<CMC
(0.0003%(w/w))
+SDS 0.5%
(w/w)
X
Additive
S = 217.11
Ratio
S:AS*1:3
S = 894.04
Ratio
S:AS*1:3
Experimental
conditions
Additif in both, organic and aqueos phases
500 µm
500 µm
20x
Microscopic image
500 µm
500 µm
40x
Microscopic image
285
12
11
Exp
P-407<CMC
(0.0003%(w/w))
+Chitosan 0.5%
(w/w)
P-407>CMC
(0.02% (w/w))+
SDS 0.5%
(w/w)
Additive
S=x
Ratio
S:AS*1:3
S = 194.93
Ratio
S:AS*1:3
Experimental
conditions
500 µm
500 µm
20x
Microscopic image
500 µm
500 µm
40x
Microscopic image
286
Additive
P-407>CMC
(0.02% (w/w))
+Chitosan 0.5%
(w/w)
AS: anti-solvent
*S: solvent
13
Exp
500 µm
x: data not dertemined
S=x
Ratio
S:AS*1:3
Experimental
conditions
20x
Microscopic image
500 µm
40x
Microscopic image
287
14
Exp
1
P-407>CMC
(0.02%(w/w))
X
Additive
Supersaturation degree
S = 374.51
Ratio
S:AS*1:3
S = 894.04
Ratio
S:AS*1:3
Experimental
conditions
500 µm
500 µm
20x
Microscopic image
500 µm
500 µm
40x
Microscopic image
288
P-407>CMC
(0.02%(w/w))
+ SDS 0.5%
(w/w)
15
16
Additive
P-407>CMC
(0.02%(w/w)
Exp
S = 104.50
Ratio
S:AS*1:3
S = 156.41
Ratio
S:AS*1:3
Experimental
condition
500 µm
500 µm
20x
Microscopic image
500 µm
40x
Microscopic image
289
P-407>CMC
(0.02%(w/w))
+Chitosan
0.5% (w/w)
17
18
Additive
P-407>CMC
(0.02%(w/w))
+SDS0.5%
(w/w)
Exp
S = 50 % CI
Ratio
S:AS*1:3
S = 43.25
Ratio
S:AS*1:3
Experimental
conditions
500 µm
500 µm
20x
Microscopic image
500 µm
500 µm
40x
Microscopic image
290
P-407>CMC
(0.02%(w/w))
+Chitosan
0.5% (w/w)
19
AS: anti-solvent
*S: solvent
Additive
Exp
500 µm
CI: initial concentration
S = 20% CI
Ratio
S:AS*1:3
Experimental
conditions
20x
Microscopic image
500 µm
40x
Microscopic image
291
20
1
Exp
P-407<CMC
(0.0003%(w/w))
X
Additive
S = 77.59
Ratio
S:AS*1:3
S = 894.04
Ratio
S:AS*1:3
Experimental
conditions
Re-structured organic phase containing P-407
500 µm
500 µm
20x
Microscopic image
500 µm
500 µm
40x
Microscopic image
292
Additive
P-407<CMC
(0.0003%(w/w))
+Chitosan 0.5%
(w/w)
AS: anti-solvent
*S: solvent
21
Exp
500 µm
x: data not dertemined
S=x
Ratio
S:AS*1:3
Experimental
conditions
20x
Microscopic image
500 µm
40x
Microscopic image
293
List of Abbreviations
LAS
Liquid Anti-Solvent
API
Active Pharmaceutical Ingredient
GI
Gastrointestinal
BCS
Biopharmaceutical
System
Classification
FDA
Food and Drug Administration
CD
Cyclodextrin
CMC
Critical Miscellar Concentration
HPH
High Pressure Homogenization
CSTR
Continuous Stirring Tank Reactor
SDS
Sodium dodecyl sulfate
HPC
Hydroxypropyl Cellulose
HPMC
Hydroxypropyl methyl cellulose
PS 1k-b -PEO3k
1000 Mw polystyrene-block-3000 Mw
polyethylene oxide
PVP
Polyvinylpyrrolidone
MeOH
Methanol
EtOH
Ethanol
ACN
Acetonitrile
PEG
Polyethylene glycol
DMSO
Dimethylsulfoxide
P-407
Poloxamer 407
P-188
Poloxamer 188
297
TEA
Triethanolamine
Tween
Polyoxyethylene sorbitan monooleate
PVA
Polyvinyl alcohol
NaOH
Sodium Hydroxide
HCl
Hydrochloric acid
PLGA
poly(lactic-co-glycolic acid)
Creal
Concentration after dilution
PCS
photon correlation spectroscopy
STD
Standard deviation
J
Nucleation rate
S
Supersaturation ratio
T
Temperature
C
Concentration
Ceq
Solubility
S’
supersaturation of solution
Abs
Absorbance
PMT
Promethazine hydrochloride
IMP
Imprimine chloride
APS
Average Particle Size
298
List of Symbols
γ
Activity coeficient
Rk
Volume parameter
Qk
Area parameter
aCRS 74 solute
Activity of the solute CRS 74 I liquid
phase
xCRS 74 solute
Molar fraction of the solute at saturation
γCRS 74 solute
Activity coefficient of the solute
fmin
Minimized function
τ
Residence time
Re
Reynolds number
ED
Energy dissipation
Q
Volume flow rate
ρ
Density of the mixture
u
Velocity of the flow of the mixture
Flow rate of the mixture
D
Diameter of the outlet tube
μ
Viscosity of the mixture
A
Cross sectional area of the outlet tube
f
Friction factor
C
Average concentration
V
Velocity of the fluid at the tube
Rt
Percentage of drug dissolved at each
time for Reference product
299
Tt
Percentage of drug dissolved at each
time for test products
Wa
Work of adhesion
WCL
Work of cohesion
λLS
Spreading coefficient
γ
Surface free energy
w
Watt
f1
Difference factor
f2
Similarity factor
Tm(onset)
Peak onset melting temperature (thermal
analysis)
Tc(onset)
Peak onset crystalization temperature
(thermal analysis)
ΔHf, ΔHm
Heat of fusion, melting
300
List of Figures
Figure 1.1. Anatomy of digestive tract (Modified from MARTINEZ et al., 2002) ................ 27
Figure 1.2. Parameters limiting absorption of drugs taken orally (Adapted from ENGMAN,
2003)......................................................................................................................................... 29
Figure 1.3. Energy diagram of the dissolution of a solid phase (Adapted from BERGESE,
2003)......................................................................................................................................... 31
Figure 1.4. Dissolution of a single particle in a large volume of solvent - Model of Nernst
(Adapted from BERGESE, 2003) ............................................................................................ 32
Figure 1.5. Features of size reduction: 1. Increased dissolution velocity due to increased
surface are; 2. Increased saturation solubility due to increased dissolution pressure of strongly
curved small nanocrystals (upper); 3. Increased adhesiveness of reduced material due to
increased contact area of small versus large particles (Modified from MÜLLER et al., 2011)
.................................................................................................................................................. 37
Figure 1.6. Strategies to increase the amount of dissolved drug at the absorption site
(Modified from LAKSHMI et al., 2012).................................................................................. 38
Figure 1.7. Schematic illustration of the association of free cyclodextrin (CD) and drug to
form drug–CD complexes and a truncated cone structure of CD (Modified from : DEL
VALLE, 2004) ......................................................................................................................... 41
Figure 1.8. Schematic representations of Cds (a) α- CD, (b) β-CD, (c) γ-CD. Containing 6,7
and 8 glucopyranoside units, respectively (MORIN-CRINI and CRINI, 2012) ..................... 41
Figure 1.9. Schematic representation of a hydrophobically assembled polymer micelle. The
hydrophobic core loading lipophilic drugs is protected from the environment by the
hydrophilic shell (KIM et al., 2010) ......................................................................................... 43
Figure 1.10. Possible solid forms of a drug cocrystal (Modified from ALHALAWEH, 2012)
.................................................................................................................................................. 44
Figure 1.11. Amorphous solid dispersion (From GHASTE et al., 2009). ............................... 45
Figure 1.12. Schematic representation of ‘bottom up’ and top down’ technology approaches
for ultra-fine particles production (micro- or nanometric scale of particle size), (Modified
from FLORENCE and KONSTANTIN, 2010)........................................................................ 46
Figure 1.13. Particle size reduction by milling of drug particles between moving pearls ...... 47
303
Figure 1.14. Basic homogenization principles: piston-gap (left) and jet-stream arrangement
(right) (Adapted from SIVASANKAR and KUMAR, 2010) .................................................. 48
Figure 1.15. Approach for drug particle formation by LAS crystallization ............................. 50
Figure 1.16. Schematic representation of the different steps involved in drug particle
formation by an LAS crystallization process (Adapted from MENG, 2011) ......................... 50
Figure 1.17. The solubility/ supersolubility phase diagram (Modified from DAVEY and
GARSIDE, 2000) ..................................................................................................................... 52
Figure 1.18. Various kinds of nucleation (Modified from MARSMANN et al., 2001) .......... 54
Figure 1.19. A three-dimensional crystal surface showing three type of growth sites and
different steps ( I, II and II*) involved in the process of growth to unit cubic (Modified from
MERSMANN, 2001) ............................................................................................................... 55
Figure 1.20. Feeding configuration for anti-solvent crystallization (a) single-jet, (c) double-jet
with premixing ......................................................................................................................... 74
Figure 1.21. Schematics of mixing devices used in chemical and pharmaceutical field
(Modified from THORAT and DALVI, 2012) ........................................................................ 75
Figure 1.22. Schematic diagram showing the mechanism of growth inhibition and habit
modification of crystals by polymers (Modified from RAGHAVAN et al., 2001) ................. 82
Figure 1.23. Types of colloidal stabilization (Modified from WU et al., 2011) ...................... 83
Figure 1.24. Schematic illustration of adsorbed polymer layer (From NYLANDE et al., 2006)
.................................................................................................................................................. 84
Figure 1.25. Different conformations of polymers at surfaces: (a) mushrooms conformation of
a single adsorbed, where chains are non-interacting on the surface and achieve a size given by
the polymer (b) semi-brush where crowding among chains causes extension of the chain from
the surface, and (c) brush conformation for high grafting densities, leading to extension of the
chains away from the surface (Modified from .J. BUDIJONO et al., 2010) ........................... 85
Figure 2.1. Chemical structures of (a) CRS 74 and (b) Ritonavir ........................................... 95
Figure 2.2. Schematic synthesis of CRS 74 (BOCKELMANN et al., 2005; BOCKELMANN
et al., 2010) ............................................................................................................................... 96
Figure 2.3. Schematic diagram of Apparatus II (Paddle Apparatus):D: vessel diameter; h:
paddle width, c: distance between the paddle and the vessel bottom, H: liquid height into the
vessel ..................................................................................................................................... 104
304
Figure 2.4. Schematic representation of a High Performance Liquid Chromatography system
................................................................................................................................................ 105
Figure 2.5. Experimental setup for solubility measurements ................................................ 108
Figure 2.6. Particle size distribution for as-received CRS 74 powder sample ...................... 109
Figure 2.7. SEM micrographs of the original CRS 74 ........................................................... 110
Figure 2.8. X-Ray diffractograms of the as-received CRS 74 sample ................................. 111
Figure 2.9. TGA-DSC curve of CRS 74 run in a nitrogen atmosphere and heating rate of
10°C/min ............................................................................................................................... 112
Figure 2.10. DSC thermograms of as-received CRS 74 sample (first heating), which consists
of a melting endotherm (peak onset temperature) Tm(Onset) = 188.6oC ................................... 113
Figure 2.11. Contact angle (o) of water as a function of time for original CRS 74................ 114
Figure 2.12. Specific test for CRS 74 in dissolution medium (0.1M HCl); (a) HPLC
chromatograms of placebo (dissolution medium); (b) HPLC chromatograms of the component
of interest dissolved in the dissolution medium. Flow rate of 1.0 ml/min; mobile phase
consisted of acetonitrile:water (50:50) ................................................................................... 117
Figure 2.13. Standard curve used for the determination of CRS 74 in samples produced during
dissolution experiments .......................................................................................................... 118
Figure 2.14. HPLC chromatograms of the component of interest dissolved in (a) water and (b)
phosphate buffer pH 6.8 ......................................................................................................... 120
Figure 2.15. Dissolution profile of CRS 74 in 0.1 M HCl at 37°C (n=4; SD =±2) ............... 121
Figure 2.16. Application of Hixson-Crowell mathematical model on CRS 74 release profile
................................................................................................................................................ 122
Figure 3.1. Schematic of the experimental apparatus used for the LAS crystallization
experiments ........................................................................................................................... 132
Figure 3.2. Sketch of the mixers used:(a)Roughton mixer, (b) T-mixer ................................ 133
Figure 3.3. Sketch of particle size analysis for particles in suspension by PCS and laser
granulometry ......................................................................................................................... 134
Figure 3.4. Experimental (characters) and calculated CRS 74 solubilities (__) in different
ethanol-water mixtures at 30 ± 0.5oC .................................................................................... 142
Figure 3.5. Theoretical yield of CRIS074 in different ethanol/(ethanol-water) mass ratios . 144
305
Figure 3.6. Particle size distributions by laser granulometry for Roughton mixer at t0 = 0 min
and tf = 10 min at total flow rate of 44.51 g/min and ethanol-water ratio of 25-75 % (w/w) 152
Figure 3.7. Particle size distribution by laser granulometry for T-mixer at t0 = 0 min and tf =
10 min at total flow rate of 44.51 g/min and ethanol-water ratio of 25-75 % (w/w) ............. 153
Figure 3.8. Particle size distribution by laser granulometry for T-mixer at total flow rate of
27.09 g/min and ethanol-water ratio of 25-75 % (w/w)., at t0 = 0 min and tf = 10 min ......... 156
Figure 3.9. Particle size distribution by laser granulometry for T-mixer at total flow rate of
44.51 g/min and ethanol-water ratio of 25-75 % (w/w)., at t0 = 0 min and tf = 10 min ......... 157
Figure 3.10. Particle size distribution by laser granulometry for T-mixer at total flow rate of
69.56 g/min and ethanol-water ratio of 25-75 % (w/w), at t0 = 0 min and tf = 10 min........... 158
Figure 3.11. Blockage and affixation of CRS 74 crystals on surface of the T-mixer ........... 159
Figure 3.12. Particle size (laser diffraction data) of CRS 74, before and after the LAS
recrystallization process ........................................................................................................ 165
Figure 3.13. SEM micrographs of the original (a,b) and LAS recrystallized CRS 74 (c,d) .. 166
Figure 3.14. X-Ray diffractograms of the original and LAS recrystallized drug powders .. 167
Figure 3.15. a) DSC thermograms of original CRS 74 (first heating), which consists of a
melting endotherm (peak onset temperature Tm(Onset) = 188.64oC) and LAS recrystallized drug,
which consists of a melting endotherm (peak onset temperature Tm(Onset) = 187.79oC); b) DSC
thermograms of original CRS 74 (cooling after first heating), which consists of a
crystallization exotherm (peak onset temperature Tc(Onset) = 132.12oC) and LAS recrystallized
drug, which consists of a crystallization exotherm (peak onset temperature Tc(Onset) =
138.43oC)................................................................................................................................ 168
Figure 3.16. Dissolution profiles of CRS 74, before and after LAS recrystallization (in 0.1 M
HCl at 37 °C, n = 4) ............................................................................................................... 170
Figure 3.17.Contact angle (o) of water as a function of time for: a) original CRS 74; b) LAS
recrystallized drug ................................................................................................................. 172
Figure 4.1. Variation in physical properties of surfactant solutions below and above the CMC
value (from Laurier et al, 2003) ............................................................................................. 182
Figure 4.2. Particle size analysis using Zetasizer Nano Zs (particles in suspension) and
MasterSizer 3000 (dried powders) ......................................................................................... 187
Figure 4.3. CRS 74 solubility in ethanol-water mixture (1:3) at 30oC, in presence of different
additives. ................................................................................................................................ 189
306
Figure 4.4. Particle size of the drug powders synthesized in presence of additives in the
aqueous phase (laser diffraction data) .................................................................................... 194
Figure 4.5. X-Ray diffractograms of the drug powders synthesized with additives in the
aqueous phase: a) HPMC 0.5% (w/w); b) Tween 20 <CMC and 0.5% (w/w); c) SDS<CMC
and 0.5% (w/w); d) Chitosan 0.5% (w/w) .............................................................................. 195
Figure 4.6. Particle size of the drug powders synthesized in presence of P-407 in the organic
phase ....................................................................................................................................... 202
Figure 4.7. X-Ray diffractograms of the drug powders synthesized in presence of P-407 in the
organic phase .......................................................................................................................... 202
Figure 4.8. Particle size (laser diffraction data) of the drug powders synthesized in presence of
additives in both, organic and aqueous phases. ...................................................................... 206
Figure 4.9. X-Ray diffractograms of drug powders produced with additives in organic phase:
a) P-407/SDS; b) P-407/Chitosan. ......................................................................................... 207
Figure 4.10. Particle size (laser diffraction data) of the drug powders synthesized in presence
of additives in organic phase and in both (organic and aqueous) at different supersaturation
degrees .................................................................................................................................... 211
Figure 4.11. X-Ray diffractograms of the drug powders synthesized in presence of additives
in organic phase and in both (organic and aqueous) at different supersaturation degrees: a) P407; b) P-407/SDS 07; c) P-407/Chitosan ............................................................................. 212
Figure 4.12 Molecular Structure of Promethazine hydrochloride (PMT) and Imprimine
chloride (IMP) ........................................................................................................................ 217
Figure 4.13. Chemical structure of CRS 74 ........................................................................... 217
Figure 4.14. Diameter of drug micelles in ethanol as a function of drug concentration (PCS
measurements). ...................................................................................................................... 218
Figure 4.15. Illustration of the effect of increasing concentration of drug in ethanolic media
leading to a micellar organisation .......................................................................................... 219
Figure 4.16. Possible schematic representation of drug self-assembled at molecular level in
ethanolic solution ................................................................................................................... 219
Figure 4.17. The charge interaction between the negatively (a) and positively (b) charged
CRS 74 nanocrystals and the negatively surface charged stainless steel (pH in the range 3-5)
................................................................................................................................................ 223
Figure 4.18. Solubility of CRS 74 samples in HCl 0.1M at 37°C (samples recrystallized in
presence of different additives; original drug also presented for comparison) ...................... 225
307
Figure 4.19. Dissolution profiles using 0.1M HCl as discriminatory dissolution medium at
37oC and paddle at 75 rpm. LAS recrystallized drug in presence of additive. Original CRS 74
also included .......................................................................................................................... 226
Figure 4.20. A linear plot for the dissolution data of CRS 74 powder samples in accordance
with the first-order model equation ........................................................................................ 229
Figure 4.21. A linear plot for the dissolution data of CRS 74 powder samples in accordance
with the Hixson-Crowell cube root law ................................................................................. 229
Figure 4.22. a) DSC thermograms of original CRS 74 (first heating), which consists of a
melting endotherm (peak onset temperature Tm(Onset) = 188.64oC) and LAS recrystallized drug,
which consists of a melting endotherm (peak onset temperature Tm(Onset) = 187.79oC); b) DSC
thermograms of original CRS 74 (cooling after first heating), which consists of a
crystallization exotherm (peak onset temperature Tc(Onset) = 132.12oC) and LAS recrystallized
drug, which consists of a crystallization exotherm (peak onset temperature Tc(Onset) =
138.43oC). c) DSC thermograms of original CRS 74 (first heating), and LAS recrystallized
drug in presence of P-407>CMC, which consists of a melting endotherm (peak onset
temperature Tm(Onset) = 189.01oC); d) DSC thermograms of original CRS 74 (cooling after first
heating), and LAS recrystallized drug in presence of P-407>CMC, which consists of a
crystallization exotherm (peak onset temperature Tc(Onset) = 137.61oC) ; e) DSC thermograms
of original CRS 74 (first heating) and LAS recrystallized drug in presence of P407>CMC+Chitosan 0.5% (w/w), which consists of a melting endotherm (peak onset
temperature Tm(Onset) = 186.58oC); f) DSC thermograms of original CRS 74 (cooling after first
heating) and LAS recrystallized drug in presence of P-407>CMC+Chitosan 0.5% (w/w),
which consists of a crystallization exotherm (peak onset temperature Tc(onset)=136.21 °C
………………………………………………………………………………………………232
308
List of Tables
Table 1.1. Transit time and pH conditions along the GI tract (MARTINEZ et al., 2002) ...... 28
Table 1.2. Biopharmaceutical Classification System according to AMIDON (1995) ............. 29
Table 1.3. Particle size distribution of pharmaceuticals with respect to dosage form and route
of administration (SHEKUNOV et al., 2007) .......................................................................... 57
Table 1.4. Classification of common crystal morphologies for pharmaceutical solids accepted
by the US Pharmacopoeia ........................................................................................................ 58
Table 1.5. The most important solid-state characteristics, which are affected by
crystallization, and the influence of these properties on the stability and downstream
processing of pharmaceutical materials. (Modified from SHEKUNOV and YORK, 2000) ... 59
Table 1.6. Obtention of drug crystals by LAS precipitation method. ...................................... 61
Table 1.7. Summary of mixing intensification reports using mixing devices for LAS drug
crystallization. (Modified from THORAT and DALVI, 2012) ............................................... 76
Table 2.1. Physicochemical properties of Ritonavir and its analogous compound CRS 74 ... 97
Table 2.2. Solubility of Ritonavir polymorphs at 5°C in hydroalcoholic solvent systems
(CHEMBURKAR et al., 2000) ................................................................................................ 98
Table 2.3. Different HPLC parameters tested during the HPLC method development ......... 107
Table 2.4. Melting Temperature (Tm(Onset)), Enthalpy of fusion (ΔHm) for as-received CRS74
sample..................................................................................................................................... 113
Table 2.5. Work of adhesion (WA), work of cohesion (WCL) and spreading coefficient (λLS)
for CRS 74 .............................................................................................................................. 115
Table 2.6. Values of k and regression equations for the mathematical models of HixsonCrowell applied to the CRS 74 dissolution data. ................................................................... 122
Table 3.1. Solubility of CRS 74 in ethanol as function of sample time in temperature range (530°C) ...................................................................................................................................... 137
Table 3.2. Solubility of CRS 74 in 95% (w/w) water - 5% (w/w) ethanol mixture as function
of sample time in temperature range (5-30°C) ....................................................................... 138
Table 3.3. Solubility of CRS 74 in Ethanol/water mixtures at 30 °C .................................... 139
309
Table 3.4. Parameter Rk and Qk.............................................................................................. 140
Table 3.5. Binary interaction parameters ............................................................................... 141
Table 3.6. Experimental and calculated activity coefficients for ethanol/CRS 74 and
ethanol/water/CRS 74 ............................................................................................................ 143
Table 3.7. Solubility of CRS 74 in pure ethanol at different temperatures ............................ 143
Table 3.8. Experimental LAS crystallization process conditions .......................................... 146
Table 3.9. Density (ρ) and viscosity (µ) of ethanol/water mixtures at 30°C (KHATTAB et al.,
2012)....................................................................................................................................... 148
Table 3.10. Calculated mixers parameters for: Roughton mixer (a) and T-mixer (b) .......... 148
Tabela 3.11. Evolution of crystallized particle size by PCS in different mixers, Roughton
mixer (a) and T-mixer (b) at total flow rate of 44.51 g/min and ratio ethanol:water 1:3....... 150
Table 3.12. Crystals at tf =10 min in suspension observed by optical microscopy ................ 151
Table 3.13. Evolution of crystallized particle growth by PCS in different total flow rate: a)
27.09 g/min; b) 44.51 g/min; c) 69.56 g/min and ethanol-water ratio of 25-75 % (w/w)...... 155
Table 3.14. Experimental LAS crystallization process conditions ........................................ 160
Table 3.15. Particle size distribution by PCS: S = 894.04 and S = 15.4 ................................ 161
Table 3.16. Identification and characterization of dried powder regarding solid state properties
................................................................................................................................................ 163
Table 3.17. Melting Temperature (Tm(Onset)), Heat of Fusion (Δhm) for the original and LAS
recrystallized drug .................................................................................................................. 169
Table 3.18. Work of adhesion (WA), cohesion (WCL) and spreading coefficient (λLS) for CRS
74 ............................................................................................................................................ 173
Table 4.1. Different additives used in LAS crystallization of CRS 74 .................................. 183
Table 4.2. Critical micelle concentration in water of additives used in LAS crystallization at
25°C. ...................................................................................................................................... 184
Table 4.3. Experimental conditions for LAS crystallization in presence of additive .......... 185
Table 4.4. CRS 74 solubility in ethanol-water mixture (ratio 1:3) at 30°C ........................... 189
Table 4.5. Theoretical and Practical Yield (%) of CRS 74 crystals obtained by LAS
crystallization process ............................................................................................................ 191
310
Table 4.6. Initial average particle size (APS) and characteristics of CRS 74 crystals in
suspension in presence of different additives in the aqueous phase ...................................... 192
Table 4.7. Summary of some properties of the dried powder obtained at the end of the LAS
crystallization process e.g., particle size, SEM images and contact angle ............................. 197
Table 4.8. Initial average particle size (APS) and characteristics of CRS 74 crystals in
suspension in presence of P-407 in the organic phase ........................................................... 201
Table 4.9. Identification and characterization of dried powder regarding solid state properties
................................................................................................................................................ 203
Table 4.10. Initial average particle size (APS) of CRS 74 crystals in suspension in presence of
additives in both, aqueous and organic phases. .................................................................... 206
Table 4.11. Identification and characterization of dried powder regarding solid state properties
................................................................................................................................................ 208
Table 4.12. Initial particle size average (APS) of the CRS 74 crystals in suspension in
presence of different stabilizing systems into internal phase and external phase in different
supersaturations. .................................................................................................................... 210
Table 4.13. Identification and characterization of dried powder regarding solid state properties
................................................................................................................................................ 213
Table 4.14. Initial average particle diameter (APD) of CRS 74 nanocrystals during LAS
crystallization, in presence of P-407 in organic phase, which was concentrated by rotary
evaporation ............................................................................................................................ 220
Table 4.15. Identification and characterization of dried powder regarding solid state properties
................................................................................................................................................ 221
Table 4.16. Stabilizing agents studied to solve the T-mixer blockage problem .................... 222
Table 4.17. Initial average particle size (APS) of the nanosuspensions and characteristics of
CRS 74 nanocrystals in suspension ........................................................................................ 223
Table 4.18. CRS 74 solubility and synthesized powders solubility in HCl 0.1M at 37°C..... 225
Table 4.19. Comparison of dissolution profiles through the difference factor (f1) and the
similarity factor (f2). Original CRS 74 is the reference product for test ............................... 227
Table 4.20. Percentage of drug dissolved within the first 20 min .......................................... 227
Table 4.21. Release parameters of CRS 74 powder samples recrystallized in presence of
additives in comparison to original drug sample. .................................................................. 230
311
Table 4.22. Melting Temperature (Tm(Onset)), Heat of Fusion (ΔHf) for the original and LAS
recrystallized drug in presence and absence of additives ....................................................... 231
312
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