The physico-chemical properties of
spiramycin and clarithromycin
Rodé van Eeden
20573901
B. Pharm
Dissertation submitted in partial
fulfilment of the requirements
for the degree Magister Scientiae
in Pharmaceutics at the
Potchefstroom Campus of the North-West University
Supervisor/Promoter:
Dr. M. Aucamp
Co-supervisor/Co-promoter:
Prof. W. Liebenberg
I
Table of Contents
Abstract VI
Uittreksel VII
Aims and objectives X
Chapter 1: General principles of the pharmaceutical solid state
1.1 Introduction 1
1.2 Phases 2
1.2.1 Energy landscapes 3
1.3 The crystalline state 6
1.3.1 Crystal structure 7
1.3.2 Nucleation and crystal growth 8
1.4 Polymorphism 10
1.4.1 Packing and conformational polymorphism 11
1.4.2 Generating different crystal forms 12
1.4.3 Polymorphic nature and thermodynamic stability 13
1.4.3.1 Enantiotropy and monotropy 14
1.4.3.2 The metastable state 16
1.4.4 Significance of polymorphism 18
1.4.4.1 Bioavailability and bioequivalence 18
1.4.4.2 Processability 20
1.5 Solvatomorphism 21
1.5.1 Hydrates 22
1.5.2 Solvates 23
1.6 The amorphous or glassy state 24
1.6.1 Generation of amorphous materials by cooling of a melt 25 1.6.2 The measurement of Tg by differential scanning calorimetry (DSC) 26 1.6.3 The effect of plasticisers on the value of Tg 29 1.6.4 Structural heterogeneity and relaxation of amorphous materials 30
II
Chapter 2: Characterisation methods for clarithromycin and spiramycin
2.1 Introduction 32
2.2 Thermal analysis 32
2.2.1 Differential scanning calorimetry (DSC) 33
2.2.1.1 Heating rate 34
2.2.2 Thermal gravimetric analysis (TGA) 37
2.3 Crystallography 37
2.3.1 X-ray powder diffraction (XRPD) 38
2.4 Microscopy 39
2.4.1 Thermal microscopy (TM) 39
2.4.2 Scanning electron microscopy (SEM) 40
2.5 Molecular motion 42
2.5.1 Infrared microscopy 42
2.6 Karl Fischer analysis (KF) 43
2.7 Stability studies 44
2.8 Solubility studies 45
2.8.1 Sample preparation 45
2.8.2 Equilibration 46
2.8.3 Separation of phases 46
2.8.4 Analysis of the supersaturated solution and residual solid 46
2.8.5 Data analysis and interpretation 47
2.8.6 HPLC method validation 47 2.9 Conclusion 49 Chapter 3: Spiramycin 3.1 Introduction 50 3.2 Description 51 3.2.1 Chemical name 51 3.2.2 Non-proprietary name 51 3.2.3 Proprietary names 51 3.3 Formulae 52 3.3.1 Structural formula 52 3.4 Physical properties 52
3.4.1 Appearance and colour 52
III 3.5 Pharmacology 53 3.5.1 Mode of action 53 3.5.2 Therapeutic activity 53 3.5.3 Dosage 54 3.5.3.1 Antibacterial 54
3.5.3.1.1 Usual adult and adolescent dose 54 3.5.3.1.2 Usual paediatric dose 54
3.5.4 Adverse reactions 54 3.6 Pharmacokinetics 55 3.6.1 Absorption 55 3.6.2 Distribution 55 3.6.3 Metabolism 55 3.6.4 Excretion 56 3.7 Pharmacodynamics 56 3.7.1 Tissue penetration 56 3.7.2 Intracellular penetration 59 3.7.3 Post-antibiotic effect 60 3.7.4 Inhibitory quotient 60 3.7.5 Residential time 60
3.8 The physico-chemical properties of spiramycin 61
3.9 Recrystallisation method 61
3.10 Results 62
3.10.1 Spiramycin raw material 62
3.10.2 Recrystallisation from alcohols 67
3.10.2.1 n-butanol 67 3.10.2.2 2-butanol 71 3.10.2.3 Ethanol (EtOH) 75 3.10.2.4 Methanol (MeOH) 80 3.10.2.5 1-propanol 83 3.10.2.6 Iso-propanol 87
3.10.2.7 Discussion of the data generated from alcohol solvents 91 3.10.3 Recrystallisation from diverse solvents 94 3.10.3.1 Acetone 94
3.10.3.2 Acetonitrile (ACN) 99
3.10.3.3 Chloroform 103
3.10.3.4 Dichlorometane (DCM) 107
IV
3.10.3.6 Ethyl Acetate (EA) 114
3.10.3.7 Tetrahydrofuran (THF) 119
3.10.3.8 Toluene 123
3.10.3.9 Discussion of the results generated from diverse solvents 126
3.11 Solubility studies 129
3.11.1 Discussion of the solubility data 130
3.12 Conclusion 132
Chapter 4: Clarithromycin
4.1 Introduction 134
4.2 Description and nomenclature 134
4.2.1 Chemical name 134 4.2.2 Non-proprietary name 134 4.2.3 Proprietary names 134 4.3 Formulae 134 4.3.1 Empirical formula 134 4.3.2 Structural formula 135 4.4 Physical properties 135 4.4.1 Molecular weight 135
4.4.2 Appearance and colour 135
4.4.3 Solubility 135 4.5 Pharmacology 135 4.5.1 Mode of action 135 4.5.2 Therapeutic activity 136 4.5.3 Dosage 136 4.5.3.1 Adult dose 136 4.5.3.2 Paediatric dose 137 4.5.4 Adverse effects 137 4.6 Pharmacokinetics 138 4.6.1 Absorption 138 4.6.2 Bioavailability 138 4.6.3 Distribution 138
4.6.4 Metabolism and elimination 139
4.7 The physico-chemical properties of clarithromycin 139
V 4.7.2 Acetonitrile 145 4.7.3 Chloroform 152 4.7.4 Ethyl acetate 164 4.8 Solubility studies 169 4.8.1 Results 170
4.8.2 Discussion of the solubility results 171 4.9 Conclusion 172
Chapter 5: Summary and conclusion 174 Acknowledgements 178
VI
Abstract
The physico-chemical properties of spiramycin and clarithromycin
In most cases, organic materials exist in the solid phase as polymorphs, solvatomorphs or amorphous forms. Physico-chemical properties in the solid-state are all affected primarily in terms of dissolution, solubility, bioavailability, stability and processability. Therefore investigation into the polymorphic behaviour of APIs has become a mandatory part of drug characterisation studies by pharmaceutical companies (Giron, 2001).
The influence polymorphism has on bioavailability and the need for the development of drugs in the amorphous form have instigated regulatory bodies such as the FDA to require solid-state characterisation of pharmaceuticals (Strachan et al., 2005). Subsequently a study was conducted to determine the physico-chemical properties of two poorly water-soluble macrolides; clarithromycin and spiramycin. Characterisation methods included: XRPD, IR, TGA, DSC, SEM, Karl Fischer titration, solubility and stability studies.
Recrystallisations of spiramycin from various solvents indicated that this API mainly exists in the amorphous form. The DSC proved to be of little value in the characterisation of this particular macromolecular antibiotic, since wide inter-sample variations were mostly obtained. TGA results showed higher solvent uptake than expected. This was ascribed to the amorphous, sponge-like character of this drug.
For the sake of reproducibility and quality of the results, characterisation of spiramycin was more reliant on spectroscopic and crystallographic methods. Samples generated from 2-butanol, chloroform, ethyl acetate, 1.4-dioxane, methanol, n-propanol, iso-propanol and tetrahydrofuran showed characteristic peaks in the range of 2000-2400 cm-1 that were not present in the IR spectrum of the raw material. Conversely, the XRPD patterns were all
VII identical, exhibiting a characteristic “halo” pattern with no detectable Bragg diffraction peaks. A solubility assessment showed no significant differences between the raw material and the recrystallisation products. In fact the raw material seemed to be the form with the highest solubility, albeit it only by a small margin.
According to the literature, clarithromycin exists in five forms. Form 0 exists as a solvate, form I is a metastable form, form II is the stable form (Liu & Riley 1998; Deshpande et al., 2006), form III is a solvate of acetonitrile (Liu et al., 2003; Liang & Yao, 2008) and form IV is a hydrate (Avrutov et al., 2003). The stable form II is used in formulations currently on the market.
A follow-up study was done relating to a study performed by De Jager (2005). The raw material (form II) was recrystallised from acetonitrile, chloroform and ethyl acetate.
Two new crystal forms were prepared from chloroform and acetonitrile. With the necessary driving force, both of these crystals forms are able to convert to the thermodynamically stable form II. In addition, a solvate recrystallised from chloroform together with its corresponding desolvate, showed a 4 and 1.5 fold respective increase in solubility when compared to the raw material.
The recrystallisations from ethyl acetate delivered crystals with an XRPD pattern similar to form II. This proved that clarithromycin can be recrystallised directly from this solvent without the need of an additional conversion step, as was the case in the study done by De Jager (2005).
VIII
Uittreksel
Die fisies-chemiese eienskappe van spiramisien en klaritromisien
Organiese stowwe bestaan in die algemeen in die soliede vorm as polimorfe, solvate of amorfe. Die fisies-chemiese eienskappe word primêr beïnvloed in terme van dissolusie, oplosbaarheid, biobeskikbaarheid, stabiliteit en vervaardigbaarheid. Die bestudering van die polimorfe gedrag van aktiewe bestanddele maak daarom deeluit van die roetine-ondersoek deur farmaseutiese maatskappye gedurende karakteriseringstudies (Giron, 2001).
Die invloed wat polimorfisme het op biobeskikbaarheid en die toenemende aanvraag na amorfe geneesmiddels, het regulatoriese instansies soos die FDA genoodsaak om karakterisering van die soliede vorm van farmaseutiese middels te vereis (Strachan et al., 2005). Gevolglik is ʼn studie uitgevoer om die fisies-chemiese eienskappe van twee swak wateroplosbare makroliede te bepaal. Die karakteriseringsmetodes het ingesluit: XRPD (x-straalpoeierdiffraksie), IR (infrarooispektroskopie), TGA (termogravimetriese analises), DSC (differensiële skanderingskalorimetrie), SEM (skanderingelektronmikroskopie), Karl Fischer-titrasie, oplosbaarheid- en stabiliteitstudies.
Rekristallisasies van spiramisien vanuit verskeie oplosmiddels het aangedui dat hierdie middel hoofsaaklik in die amorfe vorm bestaan. Die feit dat groot variasies verkry is tussen monsters, het bewys dat die DSC nie ʼn geskikte karakeriseringsmetodeis vir hierdie spesifieke makro-molekulêre antibiotika nie. Die TGA-resultate het gedui op ʼn boverwagte hoeveelheid vasgevangde oplosmiddel. Dit kan toegeskryf word aan die amorfe, sponsagtige karakter van hierdie geneesmiddel.
Ter wille van herhaalbaarheid en die kwaliteit van die resultate het karakteriseringstudies meer staat gemaak op spektroskopiese en kristallografiese metodes. Monsters verkry vanuit 2-butanol, chloroform, etielasetaat, 1.4-di-oksaan, metanol, n-propanol, isopropanol
IX en tetrahidrofuraan het beduidende verskille getoon met die grondstof in die area tussen 2000-2400 cm-1 tydens die IR-bepalings. In teenstelling hiermee was daar geen verskille wat die XRPD-patrone betref nie en ʼn karakteristieke “halo” patroon met geen Bragg-diffraksiepieke is verkry. ʼn Oplosbaarheidsbepaling het op geen noemenswaardige verskille gedui tussen die rekristallisasieprodukte en die grondstof nie. Inteendeel, uit die resultate lyk dit of die laasgenoemde effens meer oplosbaar is as die ander vorms.
Volgens die literatuur bestaan klaritromisien uit vyf vorms. Vorm 0 is ʼn solvaat, vorm I is ʼn metastabiele vorm, vorm II is die stabiele vorm (Liu & Riley, 1998; Deshpande et al., 2006), vorm III is ʼn solvaat van asetonitriel (Liu et al., 2003; Liang & Yao, 2008) en vorm IV is ʼn hidraat (Avrutov et al., 2003). Die stabiele vorm, vorm II, is die vorm wat tans in formulerings op die mark verskyn.
ʼn Opvolgstudie is gedoen na aanleiding van ʼn studie gedoen deur De Jager (2005). Die grondstof (vorm II) was gerekristalliseer vanuit asetonitriel, chloroform en etielasetaat.
Twee nuwe kristalvorms is berei vanaf chloroform en asetonitriel. Met die nodige dryfkrag, kan beide hierdie kristalvorms oorskakel na die termodinamiese stabiele vorm. Voorts het die solvaat gerekristalliseer vanuit chloroform tesame met die ooreenstemmende desolvaat ʼn 4- en 1.5-maal dienooreenkomstige verhoogde oplosbaarheid getoon in vergelyking met die grondstof. Herkristallisasie vanuit etielasetaat het kristalle gelewer met XRPD-patrone soortgelyk aan vorm II. Dus kan die stabiele vorm direk vanaf etielasetaat gerekristalliseer word sonder die addisionele tussenstap wat benodig was in die studie deur De Jager (2005).
X
Aims and Objectives
The physico-chemical properties of spiramycin and clarithromycin
Most pharmaceuticals in the solid phase are able to crystallise into more than one crystal form. While many others exist in either a partially or entirely disordered or amorphous state. It is therefore crucial to have knowledge of the physico-chemical concepts underpinning the behaviour of these systems (Craig et al., 1999).
Clarithromycin is a semi-synthetic 6-O-methyl derivative of erythromycin with a better pharmacokinetic profile, fewer gastro-intestinal side-effects, increased acid stability and bioavailability (Rodvold, 1999; Amini & Ahmadiani, 2005). Its approval in the treatment of Mycobacterium avium infection in patients with acquired immunodeficiency syndrome (AIDS) is another major advantage over the parent compound (Salem, 1996). Spiramycin, a 16-membered macrolide, has been effectively implemented in the treatment of toxoplasmosis in pregnant women (Rubinstein & Keller, 1998).
A drawback for both these macrolides however, is their poor water solubility profiles. The solubility of the API has a direct influence on its bioavailability and can often be the rate limiting factor in dissolution and oral absorption. An investigation into the physico-chemical properties of the macrolide group would offer insight into the most appropriate form to be incorporated into the solid dosage form.
Subsequently the following objectives were set:
Through the recrystallisation of the raw materials in various organic solvents, prepare different crystal forms and possibly find a novel crystal form with improved solubility characteristics.
XI Solid-state characterisation of all the obtained forms of each API in terms of its
physico-chemical properties with special emphasis on the solubility and thermodynamic stability.
Implementation of different analytical techniques to identify the obtained forms as being either polymorphs, solvates or amorphous.
