ARNOLD FORKUO DONKOR

266 Pages·2016·3.96 MB·English

1 KWAME NKRUMAH UNIVERSITY OF SCIENCE AND
TECHNOLOGY, KUMASI
2
EFFICACY, PHARMACOKINETICS AND SAFETY EVALUATION
OF CRYPTOLEPINE-ARTEMISININ BASED COMBINATIONS IN
THE MANAGEMENT OF UNCOMPLICATED MALARIA.

By
ARNOLD FORKUO DONKOR (B.PHARM)

A THESIS SUBMITTED IN PARTIAL
FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY
In the

Department of Pharmacology,
Faculty of Pharmacy and Pharmaceutical Sciences
College of Health Sciences

MAY, 2016
DECLARATION
The experimental work described in this thesis was carried out at the Department of Pharmacology,
KNUST; the Department of Biomedical and Forensic Sciences, University of Cape Coast; Noguchi
Institute of Biomedical Research, University of Ghana, Legon, Centre for Scientific and Industrial
Research (CSIR), Pretoria South Africa and the Metabolism and Pharmacokinetics (MAP)/In Vitro
Department, Novartis Pharma AG, Basel, Switzerland. This work has not been submitted for any
other degree.

…………………………..
Arnold Forkuo Donkor (PG 7866612)

……………………………
Rev. Prof. Charles Ansah
(Academic Supervisor)

……………………………
Prof. Dr. David D. Obiri
(Head of Department, Pharmacology)

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ABSTRACT
The emergence of Plasmodium falciparum strains (multidrug resistant) resistant to commonly used
antimalarial agents has motivated the research for novel drugs and drug combinations as new
strategies for the prophylaxis and treatment of malaria. Cryptolepis sanguinolenta, a popular West
African antimalarial plant has been used for several decades for the treatment of malaria and other
infections. Cryptolepine is the major alkaloid isolated from the plant with remarkable antimalarial
activity and by far the most studied. This study was conducted to characterize some drug-likeness
properties of cryptolepine and determine whether a safe and novel antimalarial combination could
be developed in combination with the artemisinin derivatives. The in vitro efficacy of cryptolepine
and the aqueous root extract of Cryptolepis sanguinolenta against the late stage gametocytes of P.
falciparum (NF54) using the PrestoBlue® assay was also ascertained in this study. In vivo
pharmacokinetics in rat and the in vitro absorption, distribution and metabolism of cryptolepine were
also characterised in rat and human models. Cryptolepine hydrochloride was isolated from the root of
C. sanguinolenta and identified by Ultra Violet (UV) spectra, Thin Layer chromatography (TLC), High
Performance Liquid chromatography (HPLC) and melting point determination studies. A SYBR Green
I fluorescent-based in vitro drug sensitivity assay using a fixed ratio method was performed on the
chloroquine-sensitive plasmodial strain 3D7 to build isobolograms from cryptolepine-based
combinations with standard antimalarial drugs. Cryptolepine exhibited promising synergistic
interactions in vitro with artesunate, artemether, dihydroartemisinin and amodiaquine. The
combination of cryptolepine with chloroquine and lumefantrine showed an additive effect whereas
antagonism was observed with mefloquine in the isobologram analysis. In vivo, the Rane’s test in
ICR mice infected with Plasmodium berghei NK-65 strains was used to build an isobologram from
cryptolepine-artesunate fixed ratio combination (1:1) and fractions of their ED s. Cryptolepine
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combination with artesunate again showed synergy as the Zexp was 1.02±0.02 mg/kg which was
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significantly lesser than the Z of 8.3±0.31 mg/kg. The aqueous root extract of C. sanguinolenta
add
and its major alkaloid, cryptolepine had minimal inhibitory effects on the late stage gametocytes
from Plasmodium falciparum strain NF54. In the in vitro pharmacokinetic assays, cryptolepine
showed a high passive permeability, a low human P-gp efflux potential, a good metabolic stability
and a moderate protein binding in rat and human plasma. The preliminary incubation in human
and rat hepatocyte showed a low to moderate hepatic extraction. Nine metabolites were identified
in human and rat hepatocytes, resulting from metabolic pathways involving oxidation (M2-M9)
and glucuronidation (M1, M2, M4, M8 and M9). Some of the metabolites were also identified in
the urine (M2, M6 and M9) and plasma of rats (M6). The enzyme phenotyping assay and the
metabolites identified in the hepatocytes suggests that both cytosolic and microsomal liver
enzymes may be involved in the metabolism of cryptolepine in rat and human hepatocytes and
among them may be aldehyde oxidase, UDP-glucuronyltransferase and the cytochrome P450
enzyme system. The in vivo rat Pharmacokinetic profile of cryptolepine showed very high
clearance and volume of distribution (Vss), a moderate half-life, low oral exposure, early Tmax,
and a low Cmax. Elimination was faster and systemic exposure (AUC) to cryptolepine was low to
moderate in rats with unchanged excretion of cryptolepine in the urine less than 0.1% of the
administered dose. This indicates metabolism, unchanged drug and/or biliary excretion as the main
clearance pathway(s). The haematological, biochemical, organ/body weight ratio and
histopathology indices in the rats treated with cryptolepine at all doses (25, 50, 100 mg/kg p.o) and
in combination with artesunate (4 mg/kg) or artemether (50 mg/kg) showed no significant acute
toxicity compared to the control group. All treatments presented no morphological changes in the
kidney, spleen, stomach, and liver tissues used in the histopathology study. These findings provide
a strong basis for the selection of cryptolepine as a potential lead compound in the development
of combination therapy against malaria.
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ACKNOWLEDGEMENT
I am thankful to the Almighty God, Giver of life, knowledge and wisdom for seeing me to this point
of my postgraduate studies. My profound gratitude also goes to my lead supervisor, Rev. Prof.
Charles Ansah of the Department of Pharmacology, KNUST for his immense contribution, fatherly
advice, thorough supervision and mentorship role during the period of the programme.
I am also grateful to Caroline Rynn of the Metabolism and Pharmacokinetic (MAP/IN VITRO)
Department, Novartis Institute of Biomedical Research (NIBR), Basel, Switzerland for her guidance
and supervision during my stay in her laboratory at Novartis. Not forgetting David Pearson, Werner
Gertsch, Markus Trunzer and Laurent Hoffman for their immense help and training during my stay
in Novartis AG, Basel, Switzerland. I will also want to thank Professor Dr. David Obiri, the Head of
the Pharmacology Department and all my Laboratory Scientific associates Sabinne Arnold, Yannick
Friche, Gaelle Chenal and Thomas Ansah for their technical contribution to my research. My sincere
appreciation goes to the Education, Diversity and Inclusion group of Novartis AG for funding my
stay and research in Basel, Switzerland for 3 months. I appreciate greatly the help offered me by
Prof. Boampong and Dr. Ameyaw of the Department of Human Biology during the in vivo
antimalarial assay in their laboratory. Not forgetting Profrssor Ben Gyan and Dr. Michael Ofori of
NMIMR, University of Ghana, Legon for their assistance and support in the in vitro antimalarial
assays and Dr. Dalu Mancama of the Centre for Scientific and Industrial Research in Pretoria, South
Africa for his assistance with the gametocyte assay. Finally, my heartfelt gratitude goes to my family,
postgraduate colleagues and staff of the Department of Pharmacology for their encouragement. Not
forgetting Jennifer, Aaron, Paapa, Nana Ama, Robert, Charles, Prince, Patience, Eyram and Soudzi
for their friendship.
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TABLE OF CONTENTS
DECLARATION ................................................................................................................................. II
ABSTRACT ........................................................................................................................................ III
ACKNOWLEDGEMENT ................................................................................................................... V
TABLE OF CONTENTS....................................................................................................................VI
LIST OF FIGURES ......................................................................................................................... XIV
LIST OF TABLES ......................................................................................................................... XVII
LIST OF ABBREVIATIONS ....................................................................................................... XXII
1.0 CHAPTER ONE (INTRODUCTION) .......................................................................................... 1
1.1 GENERAL INTRODUCTION ................................................................................................ 1
1.2 CRYPTOLEPIS SANGUINOLENTA. ....................................................................................... 3
1.3 TRADITIONAL USES OF CRYPTOLEPIS SANGUINOLENTA .......................................... 4
1.4 CRYPTOLEPINE ..................................................................................................................... 5
1.4.1 Sources ................................................................................................................................. 5
1.4.2 Biological activities of Cryptolepine ................................................................................... 7
1.5 MALARIA .............................................................................................................................. 12
1.5.1 Disease definition, incidence and trends. ......................................................................... 12
1.5.2 Geographical Distribution and Populations at Risk ........................................................ 13
1.5.3 Transmission and Developmental stages of the Malaria Parasite ................................. 14
1.5.4 Malaria Symptoms and Diagnosis ................................................................................... 17
1.5.5 Malaria Prevention and Treatment ................................................................................... 18
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1.5.6 Antimalarial Agents-A Historical Perspective ................................................................. 19
1.5.7 Artemisinin Derivatives .................................................................................................... 21
1.5.8 Antimalarial Combination Therapy .................................................................................. 25
1.5.8.1 Artemisinin Based Combinations. ............................................................................. 26
1.5.9 Herbal Antimalarial Remedies. ......................................................................................... 27
1.6 ASSESSMENT OF ANTIMALARIAL ACTIVITY ............................................................ 29
1.6.1 In Vitro Assessment of Antimalarial Activity .................................................................. 29
1.6.1.2 SYBR® Green I Assay .............................................................................................. 30
1.6.2 In vivo assessment of antimalarial activity ....................................................................... 31
1.6.2.1 Parasite Species .......................................................................................................... 33
1.6.2.2 Suppressive Test (Peters’ 4 Day test) ........................................................................ 33
1.6.2.3 Curative or Rane Test ................................................................................................. 34
1.6.2.4 Prophylactic Activity (Repository Test) .................................................................... 34
1.6.3 Gametocyte Assay .............................................................................................................. 35
1.7 DRUG METABOLISM AND PHARMACOKINETICS ..................................................... 36
1.7.1 Phase I and Phase II reactions .......................................................................................... 37
1.7.3 Drug Metabolizing Enzymes (DME) ................................................................................. 38
1.7.4 Drug-drug interaction ....................................................................................................... 40
1.7.5 Herb-drug interaction ........................................................................................................ 41
1.8 TOXICOLOGICAL STUDIES .............................................................................................. 42
1.8.1 Target organ Toxicity ........................................................................................................ 44
1.8.1.1 Toxicity to the liver .................................................................................................... 44
1.8.1.2 Toxicity to the kidneys ............................................................................................... 46
1.8.1.3 Toxicity to the blood .................................................................................................. 47
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1.9 JUSTIFICATION, AIMS AND OBJECTIVES .................................................................... 48
1.9.1 JUSTIFICATION OF STUDY ........................................................................................... 48
1.9.2 AIMS AND OBJECTIVES ................................................................................................. 51
2.0 CHAPTER TWO ........................................................................................................................ 52
PLANT COLLECTION, ISOLATION AND CHARACTERIZATION ................................. 52
2.1 PLANT COLLECTION AND EXTRACTION .................................................................... 53
2.1.1 Plant Collection ................................................................................................................. 53
2.1.2 Preparation of the aqueous extract from the roots. .......................................................... 53
2.2 ISOLATION AND CHARACTERIZATION OF CRYPTOLEPINE HYDROCHLORIDE
54
2.2.1 Chemicals/Reagent ............................................................................................................ 54
2.2.2 Methodology ...................................................................................................................... 55
2.2.3 Determination of Ultraviolet (UV) absorption spectrum of isolated Cryptolepine. ........ 56
2.2.4 High Performance Liquid Chromatography (HPLC) spectra of reference and isolated
cryptolepine. ................................................................................................................................ 57
2.3 RESULTS ............................................................................................................................... 58
2.3.1 Melting Point Determination ............................................................................................. 58
2.3.2 Thin Layer Chromatography (TLC) .................................................................................. 58
2.3.4 Ultraviolet/Visible absorption spectrum ........................................................................... 60
2.3.5 High Performance Liquid Chromatography (HPLC) spectra ......................................... 62
2.4 DISCUSSION ......................................................................................................................... 65
2.5 CONCLUSION ......................................................................................................................... 66

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3.0 CHAPTER THREE ...................................................................................................................... 67
ANTIPLASMODIAL ASSAYS ........................................................................................................ 67
3.1 INTRODUCTION............................................................................................................. 68
3.2 MATERIALS AND METHODS ............................................................................................ 70
3.3 IN VITRO ANTIMALARIAL ASSAYS .................................................................................. 72
3.3.1 In vitro cultivation of malaria parasite ............................................................................. 72
3.3.2 In vitro Drug Sensitivity Assay .......................................................................................... 74
3.4 IN VIVO ANTIMALARIAL ASSAY .................................................................................................. 76
3.4.1 Animals ............................................................................................................................... 76
3.4.2 Antimalarial assay of Cryptolepine hydrochloride. ......................................................... 76
3.4.3 In vivo antimalarial interaction assay. ............................................................................. 77
3.5 TRANSMISSION BLOCKING PROPERTIES OF CRYPTOLEPINE AND CRYPTOLEPIS
SANGUINOLENTA. ........................................................................................................................ 78
3.5.1 In vitro cultivation of asexual stage Plasmodium falciparum .......................................... 78
3.5.2 Induction of Gametocytogenesis and maintenance of gametocyte cultures. ................... 78
3.5.3 Gametocytocidal activity assays ....................................................................................... 79
3.6 STATISTICAL ANALYSIS OF DATA .................................................................................. 80
3.7 RESULTS .................................................................................................................................. 82
3.7.1 In vitro drug interaction assay .......................................................................................... 82
3.7.2 In vivo antimalarial assay. ................................................................................................ 87
3.7.2.1 Determination of ED of cryptolepine in P.berghei infected mice. ........................ 87
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3.7.2.2 Combination antimalarial assay of cryptolepine and artesunate ............................... 88
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3.7.2.3 In vivo synergistic interaction of cryptolepine and artesunate (Isobologram
analysis)................................................................................................................................... 89
3.7.3 In vitro Gametocyte assay ................................................................................................. 91
3.7.4 Haematological analysis of P. berghei infected mice treated with various drug
combinations ............................................................................................................................... 92
3.7.5 Histopathology ................................................................................................................... 94
3.8 DISCUSSION ........................................................................................................................... 96
3.9 CONCLUSION ....................................................................................................................... 100
4.0 CHAPTER FOUR ....................................................................................................................... 101
IN VITRO PHARMACOKINETIC STUDIES ON CRYPTOLEPINE .......................................... 101
4.1 INTRODUCTION ............................................................................................................... 102
4.2 DRUGS AND CHEMICALS ................................................................................................. 104
4.3 METHODOLOGY ............................................................................................................... 106
4.3.1 Permeability Assays of cryptolepine ............................................................................... 106
4.3.1.1 MDR1-MDCK II Assay (Cryptolepine assay in MDR1-MDCK cells) ................. 106
4.3.1.2 Parallel Artificial Membrane Permeability (PAMPA) Assay ................................. 110
4.3.2 Distribution Assays ........................................................................................................ 112
4.3.2.1 Plasma Protein binding of cryptolepine using the Pierce Rapid Equilibrium Dialysis
(RED) .................................................................................................................................... 112
4.3.2.2 In vitro stability of cryptolepine in human and rat plasma ...................................... 113
4.3.3 In vitro metabolism of cryptolepine in the liver .............................................................. 114
4.3.3.1 Preliminary metabolic stability of cryptolepine in human and rat hepatocytes ...... 114
4.3.3.2 In vitro stability of cryptolepine in human, rat and dog liver S9 and cytosol ........ 116
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