The effect of Pheroid
®
technology on the
bioavailability of artemisone in primates
L Grobler
13065513
BSc, M.Sc. (Biochemistry)
Thesis submitted for the degree Philosophiae Doctor in
Pharmaceutics at the Potchefstroom Campus of the North-West
University
Promoter:
Prof. A.F. Grobler
Co-promoter:
Prof. R.K. Haynes
ACKNOWLEDGEMENTS
Foremost, I would like to express my sincere gratitude to God for giving me this opportunity.
This thesis could not have been written without the encouragement and generous assistance of countless individuals.
Rick Grobler, my husband, thank you for all the love and patience. Thank you for being there through times of tears and smiles and all of the sacrifices that you had to make. Your spiritual support and encouragement throughout this journey really meant the world to me.
J.P and Elmarie Meeding, my parents, thank you for your financial and emotional support. I could not have asked for better parents and am proud to be your daughter. Thank you for giving me this opportunity and never giving up on me.
Hans and Denise Grobler, my parents-in-law, thank you for your love, understanding and encouragement. Thank you for accepting me as your own daughter and supporting me in a way that you do with your own children.
Prof. Anne Grobler, my promoter, thank you for the opportunity to do my PhD in Pharmaceutics. Thank you for all your advice and for your inputs throughout the process of my research. Thank you for the funding and enabling me to visit other laboratories and meet amazing people.
Prof Richard Haynes, my co-promoter, thank you for your enthusiasm for my work. Thank you for always having an open door. I appreciate the way you shared your vast knowledge on malaria and your assistance in the writing of this thesis.
Mike, Marina and Kerryn, my colleagues from the army malaria institute in Australia, thank you for making my visit there a memorable and enjoyable one. I really learned so much from you and was truly blessed by the way I was received in your laboratories. Mike and Maria, thank you for opening your home for me, for all the beautiful places you showed me and the delicious food.
Liezl Gibhard, my PhD buddy, as we walked this road together, I thank God that I could share all those times in the laboratory with you. Whether it was early mornings, late nights or labelling tubes until the break of dawn, we got through it together. I will always remember
and treasure the verse that God gave us for our PhD’s. Rom 5:3-5 “And not only this, but we glory in afflictions also, knowing that afflictions work out patience, and patience works out experience, and experience works out hope.”
I would also like to thank my friends and colleagues for your encouragement, prayers and support.
This thesis is submitted in fulfilment of the requirements of a Doctor of Philosophy in Pharmaceutics. This study forms part of the project T90020: “An investigation into drug resistance reversal of antimalarials using Pheroid™ technology” and was financially supported by the South African Technology Innovation Agency (TIA), the Swiss South African Joint Research Project Initiative and the North-West University. This thesis is submitted in a manuscript format in accordance with the General Academic Rules (A.7.5.7.4) of the North-West University. Each chapter is written in accordance with specific guidelines as stipulated by the journals intended for publication. Manuscript 1 has been submitted to Expert Opinion on Drug Metabolism and Toxicology, manuscript 2 will be submitted to Antimicrobial Agents and Chemotherapy and manuscript 3 will be submitted to The International Journal for Parasitology – Drugs and Drug Resistance
The contribution of each author is as follows:
L. Grobler (Candidate)
- Planning and design of the studies.
- Experimental work.
- Interpretation of results.
- Writing of the thesis and manuscripts. - Corresponding author of manuscripts. Prof. A.F. Grobler (Promotor)
- Supervision of the planning and design of the studies. - Assistance in the interpretation of results.
- Supervision and critical review of the writing of the thesis and manuscripts. - Corresponding author of manuscripts.
Prof. R.K. Haynes (Co-promotor)
- Supervision of the planning and design of the studies. - Assistance in the interpretation of results.
- Supervision and critical review of the writing of the thesis and manuscripts. - Supplied artemisone and metabolite M1.
Prof. P.A. Steenkamp
- Critical review of the writing of the manuscripts 1 and 3.
- Performed the UPLC-QTOF-MS analysis.
- Assistance in the interpretation of metabolism and p-gp results. Prof. C. Masimirembwa
- Critical review of the writing of the manuscript 1.
- Supervision of the planning and design of the metabolism study. - Assistance in the interpretation of the metabolism results. Mrs. R. Thelingwani
- Experimental work and assistance in the interpretation of the metabolism results. Prof. H.S. Steyn
- Performed the statistical analysis.
- Critical review of the writing of the manuscripts. Dr M. Chavchich
- Critical review of the writing of manuscript 2. - Supplied P. falciparum isolates.
- Supervision of the planning and design of the in vitro efficacy and dormancy study. - Experimental work and assistance in the interpretation of the in vitro efficacy and
dormancy study.
Prof. M. D. Edstein
- Critical review of the writing of the manuscript 2. - Supplied P. falciparum isolates.
- Supervision of the planning and design of the in vitro efficacy and dormancy study. - Assistance in the interpretation of the in vitro efficacy and dormancy study.
Prof. H. J. Viljoen
- Set up of the mathematical model of the caco-2 study and assistance in the
Declarations
I hereby declare that I have approved the manuscripts/thesis and that my role in the study as indicated above is representative of my actual contribution. I give permission as author or co-author for submission of the manuscripts/thesis for degree purposes.
L. Grobler Prof. A.F. Grobler
Prof. R.K. Haynes Prof. P.A. Steenkamp
Prof. C. Masimirembwa Mrs. R. Thelingwani
Prof. F. Steyn Dr M. Chavchich
i
ABSTRACT
Malaria is one the world’s most devastating diseases. Several classes of drugs are used to treat malaria. Artemisinin combination therapy is the first line treatment of uncomplicated malaria. The artemisinin derivative, artemisone in conjunction with the Pheroid® drug delivery system, is the focus of this thesis.
The impact of the Pheroid® on the bioavailability of artemisone was evaluated in vervet monkeys. The resulting artemisone plasma levels were much lower (Cmax of 47 and 114 ng/mL for reference and Pheroid® test formulations respectively) than expected for the dosages administered (60 mg/kg). The Pheroid® improved the pharmacokinetic profile of artemisone in a clinically significant manner. The metabolism of artemisone was assessed
in vitro by using human and monkey liver and intestinal microsomes, and recombinant CYP3A4 enzymes. The Pheroid® inhibits the microsomal metabolism of artemisone. In addition, there is a species difference in artemisone metabolism between man and monkey since the in vitro intrinsic clearance of the reference formulation with monkey liver microsomes is ~8 fold higher in the monkey liver microsomes compared to the human liver microsomes and the estimated in vivo hepatic clearance for the monkey is almost twofold higher than in humans.
Artemisone has potent antimalarial activity. Its in vitro efficacy was approximately twofold higher than that of either artesunate or dihydroartemisinin when evaluated against P.
falciparum W2, D6, 7G8, TM90-C2B, TM91-C235 and TM93-C1088 parasite strains. The Pheroid® drug delivery system did not improve or inhibit the in vitro efficacy of artemisone or DHA. Artemisone (reference and Pheroid® test formulations) and metabolite M1 abruptly arrested the growth of P. falciparum W2 parasites and induced the formation of dormant ring stages in a manner similar to that of DHA.
Interaction of artemisone with the p-glycoprotein (p-gp) efflux transporter was investigated. Artemisone stimulates ATPase activity in a concentration-dependent manner, whereas the Pheroid® inhibited this p-gp ATPase activity. P-gp ATPase activity stimulation was fourfold greater in human than cynomolgus monkey MDR1 expressed insect cell membranes. Artemisone alone and artemisone entrapped in Pheroid® vesicles showed moderate apical to basolateral and high basolateral to apical permeability (Papp) across Caco-2 cells. The Papp efflux ratio of artemisone and artemisone entrapped in Pheroid® vesicles were both >5, and decreased to ~1 when the p-gp inhibitor, verapamil, was added. Therefore, artemisone is a substrate for mammalian p-gp. The cytotoxic properties of Pheroid® on Caco-2 cells were assessed and the pro-Pheroid® seems to be non-toxic at concentrations of ≤1.25%.
ii Vervet monkey plasma caused antibody-mediated growth inhibition of P. falciparum. Heat inactivated or protein A treatment proved useful in the elimination of the growth-inhibitory activity of the drug-free plasma. Plasma samples containing artemisone could not be analysed by the ex-vivo bioassay method. The dual labelling ROS assay did not prove to be useful in the evaluation of ROS production by artemisone and the Pheroid® delivery system.
In conclusion, entrapment of artemisone in the Pheroid® delivery system improves the pharmacokinetic properties of artemisone, but does not improve or inhibit its antimalarial efficacy in vitro. The Pheroid® inhibited both the microsomal metabolism of artemisone and P-gp ATPase activity and was shown to be non-toxic at clinically usable concentrations.
Keywords: artemisone, bioavailability, clearance, drug delivery, efficacy, malaria,
iii
UITTREKSEL
Malaria is een van die wêreld se mees verwoestende siektes. Verskeie geneesmiddelklasse word gebruik om malaria te behandel. Artemisinin gebaseerde kombinasie terapie is die eerste linie van behandeling van ongekompliseerde malaria. Die artemisinin derivaat, artemisoon, in kombinasie met die Pheroid® geneesmiddel afleweringsisteem, is die fokus van hierdie proefskrif.
Die impak van die Pheroid® op die biobeskikbaarheid van artemisoon is geëvalueer in blouape. Die artemisoon plasma vlakke was laer (Cmax van 47 en 114 ng/mL vir verwysing en Pheroid® toets formulerings onderskeidelik) as wat verwag is vir die dosis wat toegedien is (60 mg/kg). Inkorporering van artemisoon in die Pheroid® afleweringsisteem het die farmakokinetiese profiel van artemisoon op 'n klinies beduidende wyse verbeter. Die metabolisme van artemisoon is beoordeel in vitro deur die gebruik van 5 toetsisteme: menslike lewer, aaplewer, menslike en aap intestinale mikrosome, en die rekombinante ensiem CYP3A4. Die Pheroid® inhibeer die mikrosomale metabolisme van artemisoon. Daarbenewens is daar is 'n spesie verskil in artemisoon metabolisme tussen mens en aap aangesien die in vitro intrinsieke opruiming van die verwysing formulering gemeet in aap lewer mikrosome ~8 keer hoër is in die aap lewer mikrosome in vergelyking met die menslike lewer mikrosome. Die beraamde in vivo hepatiese opruiming vir die aap is byna twee keer hoër as in die mens.
Artemisoon toon goeie anti-malaria effektiwiteit in vitro. Die effektiwiteit was ongeveer twee keer hoër as dié van óf artesunaat óf dihydroartemisinin teen P. falciparum W2 , D6 , 7G8 , TM90 - C2B , TM91 - C235 en TM93 - C1088 parasiet isolate in in vitro effektiewiteits toetse. Die Pheroid® het nie die in vitro effektiwiteit van artemisoon of DHA verbeter of geinhibeer nie. Artemisoon (verwysing- en Pheroid® formulerings) en metaboliet M1 het ‘n vinnige staking in die groei van P. falciparum W2 parasiete veroorsaak en het ook gelei tot die vorming van dormant ring stadia op 'n wyse soortgelyk aan dié van DHA .
Interaksie van artemisoon met p-glikoproteïen (p-gp) effluks sisteem se ATPase is ondersoek. Artemisoon stimuleer ATPase aktiwiteit op 'n konsentrasie-afhanklike wyse, terwyl die Pheroid® hierdie aktiwiteit inhibeer. Stimulasie van p-gp ATPase aktiwiteit was vier keer hoër in menslike MDR1 uitgedrukte insek selmembrane as in dié van die cynomolgus aap. Artemisoon alleen en artemisoon in die Pheroid® vesikels het matige apikale tot basolaterale en hoë basolaterale tot apikale deurlaatbaarheid (Papp ) deur Caco-2 selle getoon. Die Papp uitvloei verhouding van artemisoon en artemisoon vasgevang in Pheroid® vesikels was albei >5, en het tot ~1 gedaal wanneer die p-gp inhibitor, verapamil,
iv bygevoeg is. Artemisoon is dus 'n substraat vir soogdier p-gp. Die sitotoksiese eienskappe van Pheroid® op Caco-2 selle is bepaal en die pro-Pheroid® blyk nie-toksies te wees by konsentrasies van ≤1,25% nie.
Blouaap plasma veroorsaak teenliggaam-bemiddelde inhibisie van die vermeerdering van P.
falciparum parasiete. Inaktivering van plasma deur verhitting of protein A behandeling is gebruik vir die vernietiging van die groei- inhiberende effek van die geneesmiddel-vrye plasma. Artemisoon bevattende plasma monsters kon nie ontleed word deur die ex-vivo bioassay metode nie. Die dubbele merking ROS metode was nie bruikbaar vir die evaluering van ROS produksie deur artemisoon en die Pheroid® nie.
Ten slotte, toevoeging van artemisoon tot die Pheroid® verbeter die farmakokinetiese eienskappe van artemisoon, maar daar is nie ‘n verbetering of inhibisie van die in vitro anti-malaria effektiwiteit nie. Die Pheroid® inhibeer beide die mikrosomale metabolisme van artemisoon en P-gp ATPase aktiwiteit en bleik nie-toksies te wees by klinies toepaslike konsentrasies.
Sleutelwoorde: aap, artemisoon, biobeskikbaarheid, effektiwiteit geneesmiddel aflewering, ,
v
TABLE OF CONTENTS
PAGE
ABSTRACT i
UITTREKSEL iii
CHAPTER 1: PROBLEM STATEMENT AND AIM OF THE STUDY
Problem statement and aim of the study 1
References 7
CHAPTER 2: LITERATURE REVIEW
1. MALARIA 15
1.1 Introduction 15
1.2. The malaria parasite 16
1.3. Symptoms 18
1.3.1. Uncomplicated malaria 18
1.3.2. Complicated (severe) malaria 18
1.4. Diagnosis 19
1.5. Control strategies 21
1.5.1. Malaria control policies and strategies 21 1.5.2. Malaria prevention through vector control 21 1.5.3. Antimalarial chemotherapy 21
1.5.3.1. Quinolines 22
1.5.3.2. Antifolates 23
1.5.3.3. Antibiotics 24
1.5.3.4. Peroxides 25
1.5.4. Antimalarial chemotherapy resistance 30
1.5.5. Preventive chemotherapy 33
1.5.6. Case management: Treatment 35
1.5.6.1. Uncomplicated malaria 35
1.5.6.2. Severe/complicated malaria 36
2. ARTEMISONE, A NOVEL ARTEMISININ DERIVATIVE 37
2.1. Introduction 37
2.2. Synthesis and physico-chemical properties 39 2.3. The mechanism of action of artemisinins 41
vi
2.4. Pharmacokinetics 45
2.5. Metabolism 46
2.6. Antimalarial Activities 48
2.7. Toxicity 53
3. DRUG DELIVERY OF ARTEMISININS 54 3.1. Pheroid® vesicles as a drug delivery system 55 3.2. Application of Pheroid technology as an anti-malarial (specifically artemisone) drug
delivery system 58
4. REFERENCES 61
CHAPTER 3: MANUSCRIPT 1
Guide for Authors: Expert Opinion on Drug Metabolism and Toxicology 82
Proof of submission 88 Title page 89 Abstract 90 1. Introduction 91 2. Methods 94 2.1. Materials 94
2.2. In vivo pharmacokinetic studies 94 2.2.1. Preparation of artemisone reference and test formulations 94 2.2.2. Drug administration and sample analysis 95
2.2.3. Statistical analysis 96
2.3. In vitro metabolism studies 97 2.3.1. Preparation of artemisone reference and test formulations 97 2.3.2. Metabolic stability and inhibitor phenotype assays 97 2.3.3. Determination on intrinsic clearance 98
2.3.4. Statistical analysis 99
2.4. Pro-Pheroid characterization 100
3. Results and discussion 100
3.1. Pro-Pheroid characterization 100 3.2. In vivo pharmacokinetics 101
3.2.1. LC-MS/MS assay 101
3.3. In vitro metabolic stability and inhibition 104
vii
5. Conclusion 114
6. Acknowledgements 115
7. Reference 115
CHAPTER 4: MANUSCRIPT 2
Guide for Authors: Antimicrobial Agents and Chemotherapy 123
Title page 145
Abstract 146
1. Introduction 146
2. Materials and methods 150
2.1. Drug preparation 150
2.2. In vitro cultivation of P. falciparum 151 2.3. In vitro antimalarial activity assay 151
2.4. In vitro drug dormancy 152
3. Results 153
3.1. In vitro antimalarial drug activity 153 3.2. In vitro drug induced dormancy 156
4. Discussion 157
5. Acknowledgements 160
6. References 160
CHAPTER 5: MANUSCRIPT 3
Guide for Authors: The International Journal for Parasitology 169
Title page 181
Abstract. 182
1. Introduction 183
2. Materials and methods 185
2.1. Materials 185
2.2. Preparation of solutions 186
2.2.1. ATPase assay 186
2.3. Pheroid characterization 187 2.4. Cultivation of Caco-2 cells 188
2.5.P-gp ATPase Assay 188
2.6. Cytotoxicicity of the Pheroid drug delivery system (Live/Dead assay) 189 2.7. Caco-2 transport studies 190
2.8. Statistical Analysis 192
viii 3.1. Pheroid characterization 192 3.2. Effects of artemisone and Pheroid on p-gp-ATPase and p-gp drug interaction 193 3.3. Cytotoxic effects of the Pheroid particles on Caco-2 cells 197
3.4. Transport study 198
3.5. Mathematical model of the caco-2 study 202
3.5.1. The mathematical model 202
3.5.2. Parameters of the model 203
3.5.3. Results 204
4. Discussion 205
5. Conclusion 208
6. References 208
CHAPTER 6: MISCELLANEOUS RESULTS
1. Introduction 213
2. Materials and Methods 214
2.1. Materials 214
2.2. Methods 215
2.2.1. Preparation of drug solutions and plasma samples 215 2.2.2. In vitro cultivation of P. falciparum 217 2.2.3. In vitro and ex vivo antimalarial activities of artemisone against P. falciparum 217 2.2.4. Determination of oxidative stress formation by artemisone reference and pro-Pheroid®
formulation 218
2.2.5. Statistical analysis 219
3. Results 219
3.1. Pro-Pheroid® characterization 219 3.2. In vitro and ex vivo antimalarial activities of artemisone against P. falciparum 220 3.3. Determination of ROS formation in P.falciparum parasites. 224
4. Discussion 229
5. Conclusion 232
6. References 232
CHAPTER 7: SUMMARY AND FUTURE PROSPECTS
1. Introduction 239
2. Summary and future prospects 239
3. References 245
ix Annexure A 250 Annexure B 252 Annexure C 254 Annexure D 255 Annexure E 256
LIST OF FIGURES (SHORT LIST)
CHAPTER 2:
Figure 1.1: Countries and areas at risk of malaria transmission in 2009. 16 Figure 1.2: Illustration of the life cycle of the malaria parasite. 17 Figure 1.3: Illustrations of the different stages of Plasmodium. 20 Figure 1.4: Structure of several quinoline antimalarial drugs. 22 Figure 1.5: Structures of several resistance reversal agents and modified chloroquine
molecules. 23
Figure 1.6: Structures of several antifolate antimalarial drugs. 24 Figure 1.7: Structures of some antibiotic antimalarial drugs. 25 Figure 1.8: Structures of artemisinin, its derivatives and Yingzhaosu A. 26
Figure 1.9: Peroxides. 26
Figure 1.10: Structures of several cyclic endoperoxides. 27 Figure 1.12: Structures of the 1,2,4-trioxolanes OZ277 and OZ439. 28 Figure 1.13: Structures of several of the 1,2,4,5-tetraoxanes. 29 Figure 1.14: Structures of examples of trioxaquines, trioxolaquines and tetraoxaquines. 30 Figure 1.15: Sites where suspected or confirmed artemisinin resistance has been detected in therapeutic efficacy studies conducted from 2007 to 2012 32 Figure 2.1: The global portfolio of anti malarial medicines under development 39 Figure 2.2: The synthesis of artemisone from dihydroartemisinin 40 Figure 2.3: Artemisone and metabolites M1–M5 47 Figure 3.1: Size distribution of a typical Pheroid formulation measured by CLSM 57 Figure 3.2: Confocal micrographs 58
CHAPTER 3:
Figure 1: Artemisone and the metabolites M1-5 formed during metabolism. 92 Figure 2: Effect of the Pheroid delivery system on artemisone and metabolite M1 plasma
concentration. 103
Figure 3: Metabolic stability of artemisone reference standard and pro-Pheroid formulation in a) HLM, b) MLM, c) HIM and (d) MIM. 105
x Figure 4: Selected ion chromatograms of artemisone and its metabolites. 108 Figure 5: Percentage representation of the artemisone parent compound depletion and
metabolite formation. 109
Figure 6: Effect of the Pheroid delivery system on artemisone clearance in the presence of the CYP3A4 inhibitor azamulin. 110 Figure 7: a) The effect of Pheroid technology on artemisone metabolism in HLM vs.
rCYP3A4, b) Percentage representation of the artemisone parent compound depletion and metabolite formation in HLM and rCYP3A4. 111
CHAPTER 4:
Figure 1: Structure diagrams of artemisone and its metabolite M1. 148 Figure 2: In vitro antimalarial activities against six P. falciparum strains. 155 Figure 3: Comparison of control parasites with parasites exposed to drugs. 156 Figure 4: The effect of DHA, AMS), artemisone entrapped into the Pheroid, metabolite M1 and MQ against the P. falciparum W2 strain. 157
CHAPTER 5:
Figure 1: CLSM micrograph of the Pheroid vesicle formulations. 193 Figure 2: The effect of artemisone on the p-gp ATPase activity. 194 Figure 3: The effect of verapamil, artemisone and artemisone-pro-Pheroid on the p-gp ATPase activity in human MDR1 and cynomolgus monkey Mdr1 expressed insect cell
membranes. 195
Figure 5: The toxicity of the Pheroid on Caco-2 cells was analysed by Caco-2 cell
attachment . 198
Figure 6: Artemisone (diamonds) and artemisone-Pheroid (squares) transport kinetics. 199 Figure 7: a) The apparent permeabilities of artemisone and artemisone-Pheroid 201
CHAPTER 6:
Figure 1: Confocal laser scanning electron microscopy micrograph 220 Figure 2: Effect of the Pheroid® formulation on the antimalarial activity of artemisone. 221 Figure 3: The effect of plasma dilutions from plasma collected from three time intervals on
parasite proliferation. 221
Figure 4: The effect of drug free plasma concentration on the parasite proliferation. 222 Figure 5: The effect of heat inactivated artemisone spiked plasma concentration on parasite
proliferation. 223
Figure 6: The effect of (a) heat inactivated and (b) protein A inactivated artemisone spiked plasma concentration on the parasite proliferation. 223
xi Figure 7: Two-dimensional plots in uninfected, ring and trophozoite stage infected red blood cells analyzed by flow cytometry. 225 Figure 8: DCF fluorescent signals obtained with drug treatment of trophozoite stage
parasites. 225
Figure 9: DCF fluorescent signals obtained with drug treatment of trophozoite stage
parasites. 226
Figure 10: DCF fluorescent signals obtained with drug treatment of trophozoite stage
parasites. 227
Figure 11: DCF fluorescent signals obtained with drug treatment of trophozoite stage
parasites. 228
LIST OF TABLES (SHORT LIST)
CHAPTER 2:
Table 1.1: Dates of the first reports of antimalarial drug resistance. 31 Table 2.1: Aqueous solubility and octanol–water partition coefficients. 41 Table 2.2: In vitro and ex vivo activities artemisone and other artemisinins, synthetic
peroxides and standard antimalarial drugs. 49 Table 2.3: In vitro activities of artemisone and other artemisinins. 50 Table 2.4: In vivo activity of artemisone and artesunate. 50 Table 2.5: In vivo activity of artemisone and artesunate. 51 Table 2.6: Responses of malaria-infected monkeys to oral treatment with artemisone alone or with artemisone combined with mefloquine, amodiaquine or clindamycin. 52 Table 3.1: The in vitro efficacy results of artemisone reference and artemisone entrapped into the Pheroid® drug delivery system. 60 Table 3.2: The in vivo bioavailability results of artemisone reference and artemisone
entrapped into the Pheroid® drug delivery system 60
CHAPTER 3:
Table 1: The particle size of the different Pheroid formulations 101 Table 2: The plasma pharmacokinetics of artemisone 102 Table 3: The in vitro intrinsic clearance of the artemisone reference and artemisone
pro-Pheroid formulation . 106
CHAPTER 4:
xii
CHAPTER 5:
Table 1: The particle size, span and zeta potential of the different Pheroid formulations 193 Table 2: The effect of pro-Pheroid on the p-gp ATPase activity of the substrates 197 Table 3: The apparent permeabilities 201 Table 4: The parameter values that reproduced the experimental values 204
CHAPTER 6:
Table 1: The composition of the Pheroid® formulations used in the drug susceptibility and
ROS studies. 216
Table 2: The particle size of the different Pheroid® formulations 220
CHAPTER 7:
Table 1: The composition of the starting oil phase of the two formulations used in the in vitro
