INVESTIGATION OF THE MECHANISMS OF DRUG-INDUCED AGRANULOCYTOSIS

by

Julia Ring Tin Ip

A thesis submitted in conformity with the requirements for the degree of DOCTOR OF PHILOSOPHY Graduate Department of Pharmaceutical Sciences Faculty of Pharmacy University of Toronto

 Copyright by Julia Ring Tin Ip, 2009 ABSTRACT

Investigation of the Mechanisms of Drug-Induced Agranulocytosis

Julia Ring Tin Ip, Ph.D., 2009

Department of Pharmaceutical Sciences

Faculty of Pharmacy

University of Toronto

Idiosyncratic drug reactions (IDRs) are unpredictable adverse drug reactions.

Their exact mechanisms are unknown but most appear to be immune-mediated.

Mechanistic studies require valid animal models, but there are very few available and none for the study of drug-induced agranulocytosis. Thus, the first part of my thesis has focused on the development of an animal model of agranulocytosis. We pursued many attempts to develop one in rabbits, guinea pigs, and rats by treatment with aminopyrine, amodiaquine, and clozapine and manipulating the factors hypothesized to be involved in the mechanism of IDRs such as reactive metabolite formation/detoxication and immune stimulation. Clozapine-induced agranulocytosis is not associated with immune memory, which suggests that it may not be immune-mediated. Therefore, other factors, specifically selenium and vitamin C deficiencies, were assessed as possible risk factors for clozapine-induced agranulocytosis. Despite many attempts, we were not able to develop an animal model of idiosyncratic drug-induced agranulocytosis.

The second part of this thesis was focused on investigating the effects of clozapine on neutrophils. It is known that the reactive metabolite of clozapine increases neutrophil apoptosis in vitro ; however, it was not clear that the conditions of these experiments reflect in vivo conditions. Therefore, the effect of clozapine on neutrophil

II kinetics in vivo was examined. We found that clozapine treatment decreased the half- life of circulating neutrophils and increased the rate of release of neutrophils in rabbits.

Thus, even though these animals did not develop agranulocytosis clozapine did appear to cause neutrophil damage that was compensated for by an increased production of neutrophils. Failure of the bone marrow to keep up with the increased rate of neutrophil destruction in certain individuals could result in agranulocytosis. Alternatively, damage to neutrophils could lead to an immune response in some patients that results in agranulocytosis.

The failure to develop an animal model of drug-induced agranulocytosis despite many attempts using interventions based on the current mechanistic hypotheses suggests that these hypotheses are wrong. However, it is also possible that we are just unable to overcome the default response of immune tolerance; future studies will examine this possibility and the mechanism of clozapine-induced neutrophil damage.

III ACKNOWLEDGEMENTS

There are many people I need to thank and I do not even know where to begin. But first and foremost, I must thank my supervisor, Dr. Jack Uetrecht. I would not have been able to make it through my Ph.D. without his continuous support. He has always been a role model for me and a guiding light in my scientific research. His wealth of knowledge will always be an inspiration for me to continue to search for answers in life, including but not limited to idiosyncratic drug reactions. I would also like to thank my advisory committee members: Dr. Marciano Reis, Dr. Peter O’Brien and Dr. Douglas

Templeton for their invaluable advice and support throughout the years.

I would like to thank all those I have met and helped me during my Ph.D. study in the Department of Pharmaceutical Sciences. A special thanks to all members of the

Uetrecht lab, especially Suzanne, Marija, Joanne, Jie, Wei, Robert, and Feng. They have greatly enriched my Ph.D. life with their good company and encouragements.

And last but not least I would like to acknowledge my family and the Tse family for their unconditional love and support for everything I do. Joe, thank you for walking it through with me!

IV TABLE OF CONTENTS

ABSTRACT………...... II ACKNOWLEDGEMENTS...... IV TABLE OF CONTENTS ...... V LIST OF THESIS PUBLICATIONS...... XI LIST OF ABBREVIATIONS...... XII LIST OF FIGURES...... XV LIST OF TABLES ...... XVIII

CHAPTER 1 INTRODUCTION...... 1 1.1 Adverse Drug Reactions ...... 2 1.2 Idiosyncratic Drug Reactions...... 6 1.2.1 Proposed Mechanisms of IDRs ...... 7 1.2.2 The Hapten Hypothesis...... 9 1.2.3 The Danger Hypothesis...... 12 1.2.4 The Hapten Hypothesis and the Danger Hypothesis in the context of IDRs...... 15 1.2.5 Nonimmune hypotheses...... 17 1.2.6 Clinical Manifestations of IDRs...... 18 1.2.6.1 Anaphylaxis...... 19 1.2.6.2 Hematotoxicity ...... 19 1.2.6.3 Autoimmunity...... 20 1.2.6.4 Cutaneous Reactions...... 21 1.2.6.5 Hepatotoxicity ...... 23 1.3 Approaches to Study IDRs...... 24 1.3.1 Reactive Metabolites ...... 24 1.3.1.1 Drug Metabolism in the Liver...... 27 1.3.1.2 Drug Metabolism by Leukocytes ...... 29 1.3.1.3 Drug Metabolism in the Skin ...... 36 1.3.1.4 Drugs that Are Intrinsically Chemically Reactive...... 37 1.3.2 Covalent Binding...... 38 1.3.2.1 Identifying Reactive Metabolites and Protein Covalent Binding...... 40 1.3.3 Animal Models...... 42

V 1.3.4 Lymphocyte Transformation Test ...... 44 1.3.5 Genetic Determinants of IDRs ...... 45 1.3.6 mRNA Profiles and Proteomics in the Study of IDRs ...... 46 1.4 Topics in Hematology...... 46 1.4.1 The Hematopoietic Microenvironment...... 46 1.4.2 Blood Cells and Hematopoiesis ...... 47 1.4.3 The Neutrophil ...... 50 1.4.3.1 Neutrophil Kinetics...... 50 1.4.3.2 Neutrophil Function...... 51 1.4.3.3 Neutrophil Apoptosis...... 53 1.4.3.4 Neutrophil Disorders...... 55 1.4.3.5 Effects of Xenobiotics on the Neutrophil Oxidative Burst...... 55 1.5 Drug-induced Blood Dyscrasias...... 56 1.5.1 Predictable Drug-Induced Blood Dyscrasias ...... 56 1.5.2 Idiosyncratic Blood Dyscrasias...... 57 1.5.2.1 Drug-Induced Idiosyncratic Agranulocytosis ...... 57 1.5.2.1.1 Clozapine-Induced Agranulocytosis...... 59 1.5.2.1.2 Aminopyrine-Induced Agranulocytosis...... 64 1.5.2.1.3 Amodiaquine-Induced Agranulocytosis...... 65 1.5.2.2 Drug-Induced Idiosyncratic Aplastic Anemia...... 67 1.5.2.3 Drug-Induced Idiosyncratic Thrombocytopenia ...... 67 1.6 Research Focus and Rationale ...... 68

CHAPTER 2 ATTEMPTS TO DEVELOP AN ANIMAL MODEL OF DRUG-INDUCED AGRANULOCYTOSIS...... 69 2.1 Abstract…...... 70 2.2 Introduction...... 71 2.3 Materials and Methods ...... 77 2.3.1 Chemicals...... 77 2.3.2 Animals...... 77 2.3.3 Drug Treatments...... 78 2.3.4 Preparation of Liposome Entrapped Dipyrone and Poly I:C ...... 80 2.3.5 Blood Sampling...... 81

VI 2.3.6 Blood Cell Counts ...... 81 2.3.7 Serum ALT Level Assessment...... 81 2.3.8 Liver Histology...... 82 2.3.9 Statistical Analysis ...... 82 2.4 Results…...... 83 2.4.1 Amodiaquine-treated Sprague Dawley Rats ...... 83 2.4.2 Aminopyrine-treated Rabbits ...... 88 2.4.3 Aminopyrine-treated Rabbits with Poly I:C Co-treatment ...... 90 2.4.4 Aminopyrine-treated Brown Norway Rats with Poly I:C Co-treatment ...... 93 2.4.5 Aminopyrine-treated Rats with Liposome Entrapped Poly I:C Co- treatment……...... 98 2.4.6 Aminopyrine and Amodiaquine-treated Rats with 1-MT Co-treatment...... 100 2.5 Discussion...... 103

CHAPTER 3 TESTING THE HYPOTHESIS THAT SELENIUM DEFICIENCY IS A RISK FACTOR FOR CLOZAPINE-INDUCED AGRANULOCYTOSIS IN RATS..... 110 3.1 Abstract…...... 111 3.2 Abbreviations ...... 112 3.3 Introduction...... 113 3.4 Materials and Methods ...... 115 3.4.1 Animals...... 115 3.4.2 Chemicals...... 115 2.4.3 Selenium-Deficient Diet and Clozapine Treatment...... 115 3.4.4 Blood Collection and Leukocyte Counts ...... 116 3.4.5 Selenium Status Assessment...... 116 3.4.6 Collection of Bone Marrow and Liver...... 117 3.4.7 SDS-PAGE and Immunoblotting ...... 118 3.4.8 Statistical Analysis ...... 119 3.5 Results…...... 120 3.5.1 Selenium Status ...... 120 3.5.2 Peripheral Leukocyte Counts ...... 121 3.5.3 Covalent Binding of Clozapine to Hepatic and Bone Marrow Proteins...... 122 3.6 Discussion...... 124

VII 3.7 Acknowledgements ...... 126

CHAPTER 4 TESTING THE HYPOTHESIS THAT VITAMIN C DEFICIENCY IS A S RISK FACTOR FOR CLOZAPINE-INDUCED AGRANULOCYTOSIS USING GUINEA PIGS AND ODS RATS...... 127 4.1 Abstract…...... 128 4.2 Abbreviations ...... 129 4.3 Introduction...... 130 4.4 Materials and Methods ...... 132 4.4.1 Animals...... 132 4.4.2 Chemicals...... 133 4.4.3 Vitamin C Deficient Diet and Clozapine Treatments...... 133 4.4.4 Blood Collection and Leukocyte Counts ...... 134 4.4.5 Collection of Bone Marrow and Liver...... 134 4.4.6 SDS-PAGE and Immunoblotting ...... 135 4.4.7 Vitamin C Status Assessment ...... 136 4.4.8 Statistical Analysis ...... 137 4.5 Results…...... 138 4.5.1 Vitamin C Status...... 138 4.5.2 Peripheral Leukocyte Counts ...... 140 4.5.3 Covalent Binding of Clozapine to Hepatic and Bone Marrow Proteins...... 142 4.6 Discussion...... 147 4.6 Acknowledgements ...... 149

CHAPTER 5 INVESTIGATION OF THE MECHANISM OF CLOZAPINE-INDUCED AGRANULOCYTOSIS: A FOCUS ON THE EFFECT OF CLOZAPINE ON NEUTROPHIL KINETICS ...... 150 5.1 Abstract…...... 151 5.2 Introduction...... 152 5.3 Materials and Methods ...... 153 5.3.1 Animals...... 153 5.3.2 Chemicals...... 153

VIII 5.3.3 Measurement of Neutrophil Kinetics...... 153 5.3.4 Measurement of Neutrophil Release from the Bone Marrow...... 155 5.3.5 Blood Cell Counts ...... 156 5.3.6 Measuring Clozapine Blood Levels ...... 157 5.3.7 Statistical Analysis ...... 158 5.4 Results…...... 159 5.4.1 The Effect of Clozapine on Neutrophil Kinetics Measured Using CFSE ...... 159 5.4.2 The Effect of Clozapine on Neutrophil Kinetics Measured Using BrdU...... 164 5.4.3 Clozapine Blood Levels in Rabbits ...... 167 5.5 Discussion...... 171

CHAPTER 6 CONCLUSIONS AND FUTURE WORK...... 177 6.1 Conclusions...... 178 6.2 Future Studies...... 184

REFERENCES...... 188 APPENDICES ...... 225

APPENDIX 1 Investigations of the Effects of Clozapine on Protein Kinase C...... 226 A1.1 Background ...... 226 A1.2 Materials and Methods...... 227 A1.2.1 Animals ...... 227 A1.2.2 Chemicals...... 227 A1.2.3 PKC Activity Assay...... 227 A1.2.4 Covalent Binding Detection ...... 230 A1.2.5 Apoptosis Assessment...... 230 A1.2.6 Statistical Analysis...... 232 A1.3 Results...... 233 A1.3.1 The Effect of Clozapine on PKC Activity ...... 233 A1.3.2 Covalent Binding of Clozapine to PKC...... 238 A1.3.3 The Effect of Clozapine on Apoptosis...... 239 A1.3.4 PMA Down-Regulation of PKC Activity ...... 243

IX A1.4 Discussion ...... 244

APPENDIX 2 Identification of Clozapine Covalently Modified Proteins...... 251 A2.1 Background ...... 251 A2.2 Materials and Methods...... 251 A2.3 Results…...... 254 A2.4 Discussion ...... 256 APPENDICES REFERENCES ...... 257

X

LIST OF THESIS PUBLICATIONS

1. Ip, J. and J.P. Uetrecht, In vitro and animal models of drug-induced blood dyscrasias. Environmental Toxicology and Pharmacology, 2006. 21 : p. 135-140.

2. Ip, J. and J.P. Uetrecht, Testing the hypothesis that selenium deficiency is a risk factor for clozapine-induced agranulocytosis in rats. Chem Res Toxicol, 2008. 21 (4): p. 874-8.

3. Ip, J., J.X. Wilson, and J.P. Uetrecht, Testing the hypothesis that vitamin C deficiency is a risk factor for clozapine-induced agranulocytosis using guinea pigs and ODS rats. Chem Res Toxicol, 2008. 21 (4): p. 869-73.

Elsevier granted permission to include my article in this thesis.

The Amercian Chemical Society granted permission to include my articles in this thesis.

XI LIST OF ABBREVIATIONS

ADR , Adverse drug reactions ALT , Alanine aminotransferase APC , Antigen presenting cell ATP , adenosine triphosphate BrdU , 5-bromo-2-deoxyuridine CFSE , 5-(and-6-)-carboxyfluorescein diacetate succinimidyl ester CFU , Colony-forming unit CFU-G, Granulocyte colony-forming unit CFU-GM , Granulocyte-macrophage colony-forming unit CL , Clozapine co-treatment CSF , Colony-stimulating factor DHBA , 3,4-dihydroxybenzylamine DISC , Death-inducing complex DMSO , Dimethylsulfoxide DTT , DL-Dithiothreitol EDTA , Ethylenediamine-tetraacetic acid disodium salt ELISAs , Enzyme-linked immunosorbant assays FADD , Fas-associated death domain FDA , Food and Drug Administration fMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine G-CSF , Granulocyte colony-stimulating factor GM-CSF , Granulocyte-macrophage colony-stimulating factor HBSS , Hanks’ balanced salt solution HLA , Human lymphocyte antigen HMGB1, High-mobility group box 1

H2O2, Hydrogen peroxide HOCl , Hypochlorous acid HRP , Horseradish peroxidase IDR, Idiosyncratic drug reaction IDO , Indoleamine 2,3-dioxygenase IFN, Interferon

XII IL, Interleukin i.m. , Intramuscular

InsP 3, Inositol 1, 4, 5-trisphosphate i.p. , Intraperitoneal KLH , Keyhole limpet hemocyanin LC/MS , Liquid chromatography/Mass spectrometry LC/MS/MS , Liquid chromatography/Tandem Mass spectrometry LTT , Lymphocyte transformation test MHC, Major histocompatibility complex MOPS, 3-morpholinopropanesulfonic acid MPO , Myeloperoxidase +MRM , Positive ion multiple reaction monitoring 1-MT , 1-methyl-D-tryptophen NAC , N-acetylcysteine NADPH , Nicotinamide diphosphate NaOCl, Sodium hypochlorite NMR , Nuclear magnetic resonance NSAIDs , Non-steroidal anti-inflammatories ODS , Osteogenic Disorder Shionogi P450s , Cytochrome P450s PBMC , Peripheral blood mononuclear cells PBS , Phosphate buffered saline PEG , Polyethylene glycol PKC , Protein kinase C PMA , Phorbol 12-myristate-13-acetate PMSF , Phenylmethanesulfonyl fluoride Poly I:C , Polyinosinic-polycytidylic acid SD , Selenium-deficient SDS-PAGE, Sodium dodecyl sulphate polyacrylamide gel electrophoresis SN , Selenium-adequate SJS , Stevens Johnson syndrome TEN , Toxic epidermal necrolysis TNF , Tumor necrosis factor TNFR , Tumor necrosis factor receptor

XIII TPA , 12-O-tetradecanoylphorbol 13-acetate TRADD , Tumor necrosis factor receptor-associated death domain UTP , Uridine triphosphate VD , Vitamin C-deficient VN , Vitamin C-adequate

XIV LIST OF FIGURES

Figure 1.1. The Hapten Hypothesis...... 11 Figure 1.2. The danger hypothesis ...... 14 Figure 1.3. Induction of IDRs explained by the hapten and danger hypotheses ...... 16 Figure 1.4. Bioactivation of halothane and its proposed pathway for the induction of hepatitis...... 29 Figure 1.5. Neutrophil respiratory burst and the production of hypochlorous acid...... 31 Figure 1.6. The oxidation pathway of neutrophil/monocyte myeloperoxidase ...... 32 Figure 1.7. Drugs and their reactive metabolites formed by activated neutrophils, - MPO/H 2O2/Cl or HOCl...... 34 Figure 1.8. Proposed activation pathway of nevirapine ...... 37 Figure 1.9. Haptenation of protein by penicillin...... 38 Figure 1.10. The formation of thiazolidine ring by D-penicillamine on macrophage ...... 38 Figure 1.11. Hematopoiesis and cytokines involved in its regulation ...... 49 Figure 1.12. Neutrophil kinetics...... 51 Figure 1.13. Chemical structure of clozapine...... 60 Figure 1.14. Chemical structures of aminopyrine and dipyrone...... 64 Figure 1.15. Chemical structure of amodiaquine...... 66 Figure 2.1. Tryptophan metabolism and immune tolerance...... 76 Figure 2.2. Total white blood cell counts in amodiaquine-treated Sprague Dawley rats...84 Figure 2.3. Neutrophil percentages in amodiaquine-treated Sprague Dawley rats...... 85 Figure 2.4. Neutrophil counts in amodiaquine-treated Sprague Dawley rats...... 86 Figure 2.5. Serum ALT levels in amodiaquine-treated Sprague Dawley rats ...... 87 Figure 2.6. Total white blood cell counts in aminopyrine-treated rabbits...... 88 Figure 2.7. Neutrophil percentages in aminopyrine-treated rabbits...... 89 Figure 2.8. Neutrophil counts in aminopyrine-treated rabbits...... 90 Figure 2.9. Total white blood cell counts in aminopyrine-treated rabbits with poly I:C co- treatment ...... 91 Figure 2.10. Neutrophil percentages in aminopyrine-treated rabbits with poly I:C co- treatment ...... 92 Figure 2.11. Neutrophil counts in aminopyrine-treated rabbits with poly I:C co-treatment ...... 93

XV Figure 2.12. Total white blood cell counts in aminopyrine-treated Brown Norway rats with poly I:C co-treatment...... 95 Figure 2.13. Neutrophil percentages in aminopyrine-treated Brown Norway rats with poly I:C co-treatment...... 96 Figure 2.14. Neutrophil counts in aminopyrine-treated Brown Norway rats with poly I:C co-treatment...... 97 Figure 2.15. Neutrophil counts in aminopyrine-treated rats with poly I:C co-treatment...99 Figure 2.16. Total white blood cell counts in aminopyrine and amodiaquine-treated rats with 1-MT co-treatment...... 100 Figure 2.17. Neutrophil percentages in aminopyrine and amodiaquine-treated rats with 1- MT co-treatment...... 101 Figure 2.18. Neutrophil counts in aminopyrine and amodiaquine-treated rats with 1-MT co-treatment...... 102 Figure 3.1. Potential covalent binding of the clozapine nitrenium ion to selenoproteins.114 Figure 3.2. Selenium status assessment...... 120 Figure 3.3. Peripheral leukocyte counts of rats during clozapine treatment ...... 121 Figure 3.4. Peripheral neutrophil counts of rats during clozapine treatment...... 121 Figure 3.5. Covalent binding of clozapine in the liver...... 122 Figure 3.6. Covalent binding of clozapine in the bone marrow...... 123 - Figure 4.1. Clozapine is oxidized by the myeloperoxidase/H 2O2/Cl system of neutrophils forming the reactive nitrenium ion which covalently binds to proteins...... 132 Figure 4.2. Peripheral neutrophil counts of guinea pigs during clozapine treatment ...... 141 Figure 4.3. Peripheral neutrophil counts of ODS rats during clozapine treatment...... 142 Figure 4.4. Covalent binding of clozapine in the guinea pig liver...... 143 Figure 4.5. Covalent binding of clozapine in the guinea pig bone marrow ...... 144 Figure 4.6. Covalent binding of clozapine in the ODS rat liver ...... 145 Figure 4.7. Covalent binding of clozapine in the ODS rat bone marrow...... 146 Figure 5.1. Neutrophil kinetics in rabbits measured by a decrease in CFSE-stained cells ...... 160 Figure 5.2. The effect of 10 days of clozapine treatment on the percentages and half-life of circulating neutrophils ...... 162 Figure 5.3. Neutrophil kinetics of a rabbit after 10 days and 18 days of clozapine treatment ...... 163 Figure 5.4. Kinetics of neutrophils in rabbits measured using BrdU...... 167

XVI Figure 5.5. Product ion mass spectrum of clozapine and the internal standard...... 168 Figure 5.6. LC/MS/MS +MRM extracted ion chromatogram of serum from the rabbit administered clozapine given through the drinking water for 21 days at 40 mg/kg/day ...... 169

Figure A1.1. PKC activity of HL-60 cell lysates incubated with clozapine...... 234 Figure A1.2. PKC activity of rat leukocytes incubated with clozapine ...……………...235 Figure A1.3. Leukocytes and bone marrow cell protein kinase C activity in clozapine- treated rats...... 236 Figure A1.4. Clozapine’s effect on kinase activity in recombinant human PKC...... 237 Figure A1.5. Covalent binding of clozapine to PKC...... 238 Figure A1.6. Western blot analysis of caspase-3 cleavage in clozapine-treated HL-60 cells...... 240 Figure A1.7. DNA fragmentation of HL-60 cells incubated with clozapine for 24 h. ...242 Figure A1.8. PKC activity of HL-60 cells incubated with PMA for 24 h...... 243 Figure A2.1 Isolation of clozapine covalently modified proteins...... 253 Figure A2.2 SDS-PAGE gel of bone marrow cell lysate incubated with and without clozapine.…………………………………………………………………………...254

XVII LIST OF TABLES

Table 1.1. Withdrawn drugs due to adverse reactions ...... 3 Table 1.2. Clinical manifestations of IDRs and the drugs associated...... 18 Table 1.3. Drugs associated with various cutaneous drug reactions...... 22 Table 1.4. Characteristics of hepatocellular and cholestatic liver injury ...... 23 Table 1.5. Examples of electrophiles and nucleophiles ...... 26 Table 1.6. Drugs metabolized by P450s that are associated with idiosyncratic hepatotoxicity...... 28 Table 1.7. Drugs that may be metabolized by activated leukocytes ...... 33 Table 1.8. Xenobiotics associated with agranulocytosis...... 59 Table 4.1. Vitamin C status assessment in the guinea pigs prior to clozapine treatment...... 139 Table 4.2. Vitamin C status assessment in the guinea pigs after clozapine treatment....139 Table 4.3. Vitamin C status assessement in the ODS rats after clozapine treatment...... 140 Table 5.1. Serum concentrations of clozapine in a rabbit given a single dose of clozapine by subcutaneous injection (30 mg/kg) and a rabbit given clozapine through the drinking water for 21 days determined by the LC/MS/MS method...... 170

Table A2.1 Clozapine covalently modified proteins identified by mass spectrometry. …………………………………………………………………………………... 255

XVIII

CHAPTER 1

INTRODUCTION

1 1.1 Adverse Drug Reactions

The definition of an adverse drug reaction (ADR) according to the World Health

Organization is “a response to a drug that is noxious and unintended and occurs at doses normally used in man for the prophylaxis, diagnosis, or therapy of disease, or for modification of physiological function” [1]. A more refined definition was recently proposed by Edwards et al. in which ADRs are defined as: “an appreciably harmful or unpleasant reaction, resulting from an intervention related to the use of a medicinal product, which predicts hazard from future administration and warrants prevention or specific treatment, or alteration of the dosage regimen, or withdrawal of the product” [2].

ADRs represent a major health issue because they may result in diminished quality of life, more frequent physician visits, higher health care costs estimated at over $3.6 billion per year [3], increased hospitalizations, and even death. The incidence of serious ADRs in hospitalized patients in the United States in 1994 was estimated to be 6.7% of which

0.32% were fatal cases [4]. By the year 2000, the number of patient deaths attributed to

ADRs was estimated to be 218,000 annually [3] making it one of the top 10 leading causes of death [5, 6].

ADRs also hamper the drug industry significantly in terms of monetary lost due to drug withdrawals. From 1969 to 2002, within a 33-year span, more than 75 drugs or drug products were removed from the market by the U.S. Food and Drug Administration

(FDA) due to safety problems. Table 1.1 provides a list of drugs that were withdrawn from market and the adverse reactions that they were associated with [7]. In addition, it is estimated that more than 51% of the approved drugs on the market today may have serious adverse effects that were not detected before marketing approval [3].

2 Table 1.1. Withdrawn drugs due to adverse reactions. Adapted from [6-9].

Year Drug Reason 1950s Thalidomide Teratogenicity 1960s Butamben, parenteral Severe tissue slough, transverse myelitis Casein, iodinated Thyrotoxic adverse effects Methopholine Corneal opacities in dogs Sulfadimethoxine Stevens-Johnson syndrome Bithionol Serious skin disorders Cobalt salts Liver/heart damage, claudication Pipamazine Hepatic lesions 1970s Sulfathiazole Renal complications, rash, fever, blood dyscrasias, liver damage Dihydrostreptomysin Ototoxicity sulfate Mepazine Granulocytopenia, granulocytosis, paralytic ileus, seizures, hypotension, jaundice, urinary retention Aminopyrine Bone marrow suppression, agranulocytosis Gonadotropin, chorionic- Allergic reactions animal origin Oxyphenisatin acetate Hepatitis, jaundice Chlormadinone acetate Mammary tumors in dogs Adenosine phosphate Not safe for intended use as vasodilator, anti-inflammatory Methamphetamine Abuse, dependence hydrochloride Oxyphenisatin Hepatitis, jaundice Clioquinol oral Neurotoxicity Vinyl chloride aerosol Acute CNS toxicity Nialamide Liver damage, drug interactions Nitrofurazone nasal and Mammary neoplasia in rats otic drops, vaginal suppository Diethylstibestrol, oral Adenocarcinoma of vagina in daughters after use in early pregnancy and parenteral, with ≥25 mg per unit dose Dibromsalan Serious skin disorders Metabromsalan Serious skin disorders Tribromsalan Serious skin disorders 3,3,4,5- Serious skin disorders Tetrachlorosalicylanilide Chloroform Carcinogenic in animals Azaribine Thromboembolic events Dipyrone Agranulocytosis Diamthazole Neurotoxicity dihydrochloride Reserpine More frequent, more severe adverse effects Trichlorethane aerosol Potential CV toxicity, deaths from misuse, abuse Urethane Carcinogenic Zirconium aerosol Human skin granulomas, toxicity in test animals Potassium chloride Small-bowel lesions (concentrated solid oral dosage forms with ≥100 mg of potassioum per dosage) Gelatin, intravenous Increased blood viscosity, reduced blood clotting, prolonged bleeding time Tetracycline for oral Inhibition of bone growth, permanent staining of teeth, enamel pediatric use (>25mg/ml) hypoplasia Povidone, intravenous Accumulation, storage disease, interferes with blood coagulation, blood-typing, crossmatching

3 Sparteine sulfate Tetanic uterine contractions, obstetrical complications Methapyrilene Potent carcinogen Phenformin Lactic acidosis Buformin Lactic acidosis Diethylstibestrol Teratogenicity 1980s Potassium arsenite Toxicity, potent carcinogen Sweet spirits of nitre Infant methemoglobinemia Camphorated oil Infant, child poisoning Phenacetin Kidney damage, haemolytic anemia, methemoglobinemia from abuse Chlorhexidine gluconate Chemical, thermal burns topical tincture Oxyphenbutazone Blood dyscrasias Neomycin sulphate Toxicity for irrigation of wounds Ticrynafen Hepatotoxicity Benoxyprofen Hepatotoxicity Zomepirac sodium Anaphylaxis Pituitary growth Creutzfeldt-Jakob disease hormone (IND) Nomifensine Hemolytic anemia Suprofen Flank pain syndrome Zimelidine Gullain-Barre syndrome 1990s Glycerol, iodinated Carcinogenic potential Encainide Excess mortality Temafloxacin Hemolytic anemia with renal or hepatic dysfunction and/or coagulopathy Flosequinan Excess mortality Phenolphthalein Carcinogenic Fenfluramine Cardiac valvulopathy Dexfenfluramine Cardiac valvulopathy hydrochloride Terfenadine Drug interactions and ventricular arrhythmias Mibefradil Drug interactions and cardiac events Bromfenac Hepatotoxicity Astemizole Drug interactions and ventricular arrhythmias Grepafloxacin Ventricular arrhythmias hydrochloride Alpidem Hepatotoxicity Tolrestat Hepatotoxicity Etretinate Birth defects, narrow therapeutic index Astemizole Drug interactions and arrhythmias 2000s Troglitazone Hepatotoxicity Cisapride Drug interactions and ventricular arrhythmias Alostron hydrochloride Ischemic colitis and complications of constipation Phenylpropanolamine Hemorrhagic stroke ingredient products Rapacuronium bromide Bronchospasm Cerivastatin sodium Rhabdomyolysis Amineptine Hepatotoxicity, skin disorders, abuse potential Phenylpropanolamine Risk of stroke in women under 50 years of age Trovafloxacin Liver failure Rofecoxib Myocardial infaraction Mixed amphetamine salts Risk of stroke Pemoline Hepatotoxicity Ximelagatran Hepatotoxicity Pergolide Heart valve damage Tegaserod Cardiovascular ischemic events Aprotinin Excess mortality

4 Given such alarming statistics, ADRs are a major public health concern that cannot be overlooked. It is essential that we gain a better mechanistic understanding of these reactions in order to decrease the morbidity and mortality associated with these reactions.

ADRs can be classified into the following five categories according to their mechanistic characteristics [10, 11]:

• Type A: Augmented reactions – These reactions are the most common type

accounting for 80% of all ADRs. They are predictable based on the known

pharmacology of the drug, often representing as an exaggeration of the

pharmacological effect of the drug. This type of reaction is usually non-life

threatening as they are non-host dependent and dose-dependent and can be

alleviated by a reduction in the dose of the drug. Examples include

hypotension which can occur with antihypertensive treatments or

haemorrhage with anticoagulants.

• Type B: Bizarre or idiosyncratic reactions – These are rare reactions that

cannot be predicted based on the known pharmacological properties of the

drug. They are host-dependent and do not show a simple dose-response

relationship. These reactions have a metabolic and/or immunological

component to them. Although the occurrence is less common compared to

Type A reactions, these reactions are often more severe and account for many

drug-induced deaths. Examples include halothane hepatitis and

anticonvulsant hypersensitivity.

• Type C: Chemical reactions – These reactions can be predicted based on the

chemical structure of the drug or its reactive metabolites. Examples include

acetaminophen hepatotoxicity.

5 • Type D: Delayed reactions – These reactions occur from months to years

after drug treatment. They include carcinogenicity and teratogenicity seen in

the children of mothers who took certain drugs during pregnancy. An

example is fetal hydantoin syndrome with phenytoin.

• Type E: End of treatment reactions – These reactions occur due to sudden

withdrawal of a drug. They have a pharmacological basis that usually

involves some form of receptor adaptation during chronic drug exposure.

Examples include withdrawal syndrome with paroxetine and seizures on

stopping phenytoin.

The research in this thesis is focused on Type B reactions, which will be referred from here on as idiosyncratic drug reactions.

1.2 Idiosyncratic Drug Reactions

Idiosyncratic drug reactions (IDRs) are the least understood of all ADRs. They are rare and unpredictable adverse reactions that do not occur in most people at any given dose of the drug; however, in susceptible individuals the incidence may not vary within the therapeutic dose range, although there is always a dose below which no one will have such a reaction [12]. The risk of developing IDRs is relatively low, ranging from

1/1,000 to 1/100,000; however, given the large number of drugs involved and the number of patients taking these drugs the actual number of cases is quite high.

IDRs have been reported to be associated with all classes of drugs, although they are most commonly seen during treatments with anticancer, anticonvulsant, antipsychotic, antidepressant, and non-steroidal anti-inflammatory drugs [11]. These reactions can affect any organ with the most prevalent being the liver, bone marrow or blood, and the

6 skin. The clinical manifestations of these reactions include anaphylaxis, blood dyscrasias, hepatotoxicity, and mild to severe cutaneous reactions.

The idiosyncratic nature of these reactions impedes drug development because they are rarely detected in preclinical and even clinical trials. This is because the low incidence of the reactions requires a large population size in order for them to be detected while the number of patients recruited in a clinical trial averages only about

3000 subjects [13]. Unfortunately, it is only when the drug has been widely marketed that IDRs become apparent. By that time, a drug company would have had invested as much as 1 billion US dollars and well over 10 years of research and development [14].

Furthermore, preclinical studies usually fail to predict these reactions because they are just as idiosyncratic in animals as they are in humans. There are no well-established preclinical or clinical tests that can predict whether a drug candidate will cause IDRs or which patient will development them [15]. Hence, IDRs have become the most common cause for drug withdrawal. Around 10% of the drugs approved between 1975 and 2000 were given a black box warning by the FDA or had to be withdrawn from the market due to such reactions [6]. This resulted in huge monetary losses for the drug industry and it also adds a high degree of uncertainty to drug development. Therefore, it is essential for us to gain a better understanding of the mechanistic basis of these reactions in order to better predict them and ultimately eradicate them if at all possible.

1.2.1 Proposed Mechanisms of IDRs

Despite the extensive effort put forth to try to study these reactions, very little is known regarding the mechanisms of IDRs. This is largely due to the fact that there is a lack of animal models and that prospective and retrospective human studies are difficult to perform. The low incidence of IDRs makes prospective studies almost impossible to

7 perform. Retrospective studies could be used to provide information regarding the genetic disposition of an IDR, but in most cases it appears that more than one gene is involved. It is essential to know the series of events leading up to the onset of a clinically evident adverse reaction, which in turn would provide more useful mechanistic information.

The mechanisms of specific IDRs are not fully understood. From the characteristics of different IDRs it appears that there are significant differences in the mechanisms of IDRs caused by different drugs and even differences in the mechanism of

IDRs caused by the same drug in different individuals. However, there is a large body of circumstantial evidence to suggest that most IDRs are caused by reactive metabolite(s) of the culprit drug [16] and are mediated by the immune system. Although it is still unclear how reactive metabolites can cause IDRs, it is likely that it involves covalently binding to proteins which then triggers an immune response against the parent drug or the reactive metabolite-modified proteins. An example which illustrates the relationship between the amount of reactive metabolite and the incidence of IDRs is in the case of halothane anaesthesia. Halothane is associated with a relatively high incidence of hepatotoxicity compared to other structurally related anaesthetics: isoflurane and desflurane [17]. All three anaesthetics are metabolized in the liver to the same reactive metabolite, trifluoroacetyl chloride which is capable of binding to proteins; however, halothane is metabolized to a much greater extent compared to the other two [16].

Antibodies against the trifluoroacetylated proteins were found in patients diagnosed with halothane-induced hepatotoxicity [18].

Clinical characteristics of IDRs suggest that the immune system is involved in the mechanism. A lot of these reactions are hypersensitivity reactions that are often associated with fever, anaphylaxis, and skin rash. The delay between starting a drug and

8 the onset of an IDR, followed by a decreased time to onset of the reaction on re-exposure indicates the presence of immune memory. Anti-drug antibodies and drug-reactive T cells have been identified in patients diagnosed with IDRs [19]. What further supports the immunological basis of these reactions are the results obtained from immunogenetic studies where specific human lymphocyte antigen (HLA) genotypes [20-23], polymorphisms in cytokines and heat shock genes [22, 24-27] have been found to be associated with certain IDRs.

Although the basic mechanisms of IDRs remain elusive, the aforementioned characteristics led to a series of hypotheses. First was the hapten hypothesis, which was first proposed in the 1930s; then more recently two other hypotheses, the danger hypothesis and the pharmaceutical interaction hypothesis, were added to the working hypotheses for the study of IDRs. For the purpose of this thesis, the hapten and the danger hypothesis will be described in greater detail as these were utilized in my research to investigate idiosyncratic drug-induced agranulocytosis.

1.2.2 The Hapten Hypothesis

The hapten hypothesis is based on the observation reported by Landsteiner in

1935 in which he had found that small molecules (< 1000 Da) were unable to induce an immune response unless they were bound to proteins [28]. These low molecular weight compounds, known as haptens, can only become immunogenic upon covalent binding to high molecular weight proteins or other macromolecules [29]. Landsteiner’s experiments showed that chemicals become potent sensitizing agents only when they bind to a protein of molecular weight greater than 50,000 Da, forming a hapten-protein complex [28]. This classical theory was then adopted as a working hypothesis to explain the role of reactive metabolites in the induction of an immune response. When

9 applied to the mechanisms of IDRs, the hapten hypothesis proposes that a chemically reactive drug or its reactive metabolite must first covalently bind to host endogenous proteins, forming a hapten-protein complex, in order to be able to induce an immune response [30]. The drug-modified proteins are seen as foreign by the host immune system leading to an immune response according to the widely accepted theory in immunology where the immune system differentiates “self” from “nonself” and responds with tolerance to self while mounting an immune response to anything considered nonself or foreign [12, 31].

Some drugs such as penicillin are chemically reactive and covalently bind to proteins, but the majority require bioactivation to form reactive metabolites in order for binding to occur. The electrophilic group on haptens form a stable bond with nucleophilic groups on tissue proteins which include the thiol group of cysteinyl, the amino group of lysyl, and the carbon or nitrogen of the imidazole group of histidine [29].

The covalent bond between the hapten and the protein must be relatively stable in order for this complex to survive antigen processing which follows once it is taken up by antigen-presenting cells (APCs) [29]. However, the bond between hapten and protein does not have to be completely irreversible; for example, the reaction of acrolein with protein thiols is slowly reversible. These drug-modified peptides are presented on major histocompatibility complex (MHC) class II molecules on the APCs to helper (CD4 +) T cells in order to elicit an immune response [12, 29]. The hapten hypothesis is illustrated in Figure 1.1.

10

Reactive drug Antigen presenting cell

or +

Protein Modified protein MHC Drug Reactive metabolite

T cell receptor

Helper T cell

CD8 + B T cell cell

Immune response

Figure 1.1. The hapten hypothesis.

11 An IDR in which the mechanism is consistent with the hapten hypothesis is penicillin-induced anaphylaxis. Penicillin is chemically reactive because of the ring strain involved in its β-lactam ring structure; hence, it does not require bioactivation in order to covalently bind to proteins [12]. These are IgE-mediated reactions in which most of the antibodies are directed against the penicillin-modified proteins [32]. These

IgE antibodies are pathogenic since they lead to degranulation of mast cells with the release of histamine and leukotrienes [12]. However, many drugs that are able to generate reactive metabolites are not associated with a significant incidence of IDRs.

Therefore, covalent binding alone may be insufficient to induce an IDRs [33]. A newly proposed hypothesis in immunology: the danger hypothesis, provides a possible explanation for this observation and further insights into the mechanisms of IDRs.

1.2.3 The Danger Hypothesis

Insufficiencies in the basic self-nonself concept, such as why organisms fail to generate significant immune responses to vaccines composed of foreign proteins in the absence of an adjuvant or why proteins that are expressed for the first time during puberty and pregnancy do not invoke an immune response triggered an immunologist,

Polly Matzinger, to challenge this concept [34, 35]. In 1994 she proposed the danger hypothesis, which states that it is not foreignness but rather the ability to cause cell stress or damage that induces an immune response [34]. She argued that differentiating between self and nonself is inefficient and can even be potentially harmful for the host if the immune system mounts a response to something that is not causing any danger [36].

The presence of a danger signal activates local APCs, which then results in up-regulation of co-stimulatory molecules, i.e. the B7 receptor on the APC, needed to activate T cells and initiate an immune response [37]. This up-regulation of co-stimulatory molecules on

12 APCs, and interaction with co-stimulatory molecules, i.e. CD28 on T cells, is known as signal 2. Signal 1 refers to the interaction between MHC-restricted antigen and the T cell receptor as described in the hapten hypothesis [37]. A full blown immune response only occurs in the presence of both signal 1 and 2, hence only when danger is present. In the absence of signal 2, the result is immune tolerance [12, 37] (refer to Figure 1.2).

According to Matzinger, danger signals are endogenous and are released by stressed cells or those undergoing necrotic cell death. However, stimulation of cytotoxic

T cell responses has been observed with endogenous signals released by cells that were dying from either apoptosis and necrosis as shown in an in vivo study by Shi et al . [38].

We still do not have a very complete understanding of the nature of danger signals at present, but several things have been proposed to act as danger signals including hydrophobic biological molecules [39], stress proteins such as heat shock proteins and high-mobility group box 1 (HMGB1) [40], intracellular molecules such as DNA/RNA or nucleotides (ATP), reactive oxygen intermediates, extracellular-matrix breakdown products, neuromediators, and cytokines such as TNF-α and interferons [41].

13 Stressed or necrotic cell Homeostatic cell

Danger No danger Signal signal

Antigen presenting cell Antigen presenting cell

Signal 1 Signal 2 Signal 1

MHC B7 MHC

CD 28 T cell receptor T cell receptor

Helper T cell Helper T cell

CD8 + B T cell cell Immune Tolerance

Immune response

Figure 1.2. The danger hypothesis.

14 1.2.4 The Hapten Hypothesis and the Danger Hypothesis in the context of IDRs

Together, the hapten hypothesis and the danger hypothesis form the framework of the working hypothesis for the study of the mechanisms of IDRs. They predict the following sequence of events: reactive metabolite(s) of the culprit drug (or a reactive parent drug) bind to tissue proteins in patients taking the drug. The hapten-protein complex after processing by APCs is presented to helper T cells through MHC II - signal

1 is represented by this MHC II-T cell receptor interaction. It is postulated that in the presence of signal 1 only, an immune response is not elicited, thus resulting in immune tolerance. However, with the addition of signal 2, which is represented by the interaction between co-stimulatory molecules on the APC and corresponding receptors on T cells, an immune response occurs. This signal 2 is thought to be stimulated by danger signals released from cells undergoing stress. Reactive metabolites of drugs associated with

IDRs may cause cell damage which generates a danger signal. Alternatively, reactive metabolites may also directly induce stress in APCs. An immune response leads to destruction of target tissues, which is then manifested as an IDR. Figure 1.3 provides a summary of the two hypotheses and the proposed mechanism of IDR.

15 Stressed or necrotic cell

Danger Signal

Reactive drug

or + Antigen presenting cell Protein Modified protein Drug Reactive MHC B7 metabolite CD 28 T cell receptor Helper T cell

+ CD8 B cell T cell Tolerance

Immune response

Destruction of target tissues

IDR

Figure 1.3. Induction of IDRs explained by the hapten and danger hypotheses.

16 If the hapten and the danger hypotheses are correct - they are not mutually exclusive - they may explain why many drugs that form reactive metabolites are not associated with IDRs. This could be because a determinant of whether a drug, which forms reactive metabolites, will be associated with a significant incidence of IDRs is the ability of its reactive metabolites to cause cell damage. Likewise factors leading to cell damage such as underlying bacterial or viral infections, tissue trauma from surgery, or stress in the host can increase the risk of an IDR [42]. Examples in which a higher incidence of IDRs are observed include HIV patients taking sulfonamides and patients who were treated with procainamide after surgery [36]. However, the danger hypothesis does not explain why some drugs or their reactive metabolites that induce cell stress or even cell death do not result in an immune response. An example is acetaminophen; it causes hepatic necrosis when taken in overdose, but it does not appear to cause idiosyncratic liver toxicity [43]. Furthermore, it is not clear to what extent nondrug sources of danger contribute to the induction of IDRs.

1.2.5 Nonimmune hypotheses

Even though most IDRs appear to be immune-mediated, some do not have characteristics typical of an immune-mediated reaction. Nonimmune-mediated IDRs, especially those hepatotoxic reactions that are not accompanied by fever, rash, eosinophilia, anti-drug antibodies, and do not occur rapidly on rechallenge are thought to involve metabolic idiosyncrasy. Genetic polymorphisms in metabolic enzymes, oxidative stress, mitochondrial damage, inhibition of bile salt transportation, and drug- induced apoptosis were proposed to be mediators of these types of IDRs. However, there are no convincing examples in which such polymorphisms can explain the idiosyncratic nature of these IDRs [12, 42].

17 1.2.6 Clinical Manifestations of IDRs

IDRs affect just about any organ, but the common targets are the skin, liver, and the haematological system. It is likely that these reactions usually occur where the reactive metabolites of the drug are formed, presumably because they are too reactive to escape in significant concentration from the site of formation. IDRs are most often clinically represented as cutaneous reactions, hepatotoxicity, hematotoxicity, anaphylaxis, and autoimmunity. They can range from mild reactions that may go undiagnosed to severe reactions that can lead to death. Most drugs associated with IDRs are not limited to one type of IDR. In fact a lot of the drugs can cause different reactions in different people. For example, some patients on nevirapine can develop a skin rash, while others may experience hepatotoxicity, and yet there are some whom will develop both reactions

[44, 45]. Table 1.2 provides examples of drugs and their associated forms of IDRs.

Table 1.2. Clinical manifestations of IDRs and the drugs associated [11, 46-52].

Anaphylaxis Blood Autoimmunity Cutaneous Hepatotoxicity dyscrasias reactions

Cyclosporine Clozapine Procainamide Sulfonamides Felbamate Penicillin Minocycline Nydralazine Phenytoin Valproate NSAIDs Mianserin Chloropromazine Carbamazapine Amodiaquine Suxamethonium Felbamate Isoniazid Phenobarbital Isoniazid Sulfonamides Aminopyrine Methyldopa Lamotrigine Nimesulide Vesnarinone Penicillamine Penicillins Tienillic acid Dipyrone Minocycline Allopurinol Carbamazepine Amodiaquine Hydralazine Thalidomide Phenytoin Dapsone Sulfonamides Nevirapine Phenobarbital Methyldopa Dapsone Troglitazone Ketoconazole Halothane Diclofenac

18 1.2.6.1 Anaphylaxis

Anaphylaxis is an acute, systemic, hypersensitivity reaction to an allergen that is

IgE-mediated. This type of IDR typically involves multiple organs and immediate medical attention is required in order to prevent death of the patient. Of the various types of IDRs, anaphylactic reaction is one which we have a reasonable understanding of the mechanism. In these reactions, cross-linking of mast cell Fc receptors by drug- specific IgE antibodies occurs and results in degranulation of mast cells , causing a release of histamine and leukotrienes, and activation of cytokine synthesis [53].

Systemic effects including bronchospasm, mucosal edema, and inflammation are observed. Clinical manifestations are primarily observed in the skin, gastrointestinal tract, and cardiovascular system. Symptoms include urticaria and angioedema, wheezing, upper airway edema, hypotension, and shock [53]. The classes of drugs which are most often associated with anaphylactic reactions are antibiotics and sulfonamides [54, 55].

IgE antibodies against penicillin-derived hapten-carrier conjugates were detected in patients who experienced anaphylaxis with penicillin treatment. Although we have a reasonably good understanding of the mechanism of anaphylactic reactions at present, we still cannot predict which patients will develop this type of IDR because little is known in terms of individual susceptibility.

1.2.6.2 Hematotoxicity

Idiosyncratic drug-induced blood toxicity can affect any formed element of the blood, which includes erythrocytes, leukocytes, and platelets. These reactions can affect peripheral blood cells in the circulation and/or progenitors cells in the bone marrow.

Destruction of peripheral red blood cells results in haemolytic anemia. Drugs known to

19 cause haemolytic anemia include methyldopa, cephalosporins, chlorpromazine, indinavir, penicillins, and aminopyrine [10]. A decrease in neutrophils results in neutropenia or agranulocytosis. Drugs associated with agranulocytosis that were studied in our lab include clozapine, aminopyrine, and amodiaquine. Thrombocytopenia results when platelets are affected. Drugs known to be associated with thrombocytopenia include heparin, gold salts, sulfa drugs, rifampin, carbamazepine, and valproate [10, 56].

Aplastic anemia occurs when all blood cell types in the bone marrow are affected.

Chloramphenicol, carbamazepine, felbamate, phenytoin, quinine, and phenylbutazone are examples of drug that can cause aplastic anemia [57-61] . Cell destruction has been shown to be mediated by anti-drug antibodies in many cases [62-64]. However, nonimmune-mediated mechanisms such as direct cytotoxicity have also been proposed to be involved in other cases where obvious characteristics suggestive of the involvement of immune system were not observed. Drug-induced blood dyscrasias, especially agranulocytosis, will be discussed in greater detail in the following sections of this introduction.

1.2.6.3 Autoimmunity

Drug-induced autoimmunity is a type of immune-mediated IDR against native antigens. A classic example of this type of IDR is drug-induced lupus. These reactions are characterised by autoantibodies (anti-nuclear antibodies) and are similar in character to lupus [65]. However, drug-induced lupus is generally milder compared to the idiopathic form and usually resolves quickly when treatment with the culprit drug is discontinued. Vital organs, such as the kidney and brain, are usually not affected in the drug-induced form while patients do suffer from arthralgia, myalgia, pleuropulmonary symptoms, and fever as in the idiopathic form of the disease [66]. The mechanism of

20 drug-induce lupus is still not clear, but there are several hypotheses. One hypothesis is that activation of T cells is brought about by the inhibition of DNA methylation.

Another is that reactive metabolites of drugs can break tolerance leading to the reaction.

Other hypotheses focus on activation of APCs. It appears that one co-stimulatory signal between APCs and T cells involves a reversible imine bond formed between an amine on

T cells and an aldehyde on the APCs. Some drugs such as penicillamine and hydralazine form virtually irreversible bonds with aldehydes and lead to macrophage activation.

Such generalized activation of macrophages/APCs could lead to an autoimmune reaction

[65]. Drugs associated with lupus include procainamide, hydralazine, isoniazid, sulfonamides, and penicillamine. It is likely that several mechanisms are involved in drug-induced autoimmunity and they may be different for different drugs [65].

1.2.6.4 Cutaneous Reactions

Cutaneous reactions induced by drugs are fairly common. They can range from mild skin reactions such as exanthematous rashes or urticaria to very severe cases such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). The majority of skin reactions are thought to be immune-mediated [11]. The skin is capable of metabolizing drugs because it contains both phase I and phase II drug-metabolizing enzymes; however, the level of most drug-metabolizing enzymes are quite low in the skin. It is also a highly developed and active immunological defence system. Dendritic cells, Langerhans cells and other cells capable of antigen presentation can be found in the skin [11]. Drug-induced vasculitis and, in some cases, urticaria are thought to be antibody-mediated, while exanthematous reactions, SJS, TEN, and fixed drug eruptions are believed to be T cell-mediated [67]. Table 1.3 provides a list of drugs associated with various cutaneous drug reactions.

21

Table 1.3. Drugs associated with various cutaneous drug reactions. Adapted from [68].

Morbilliform Urticaria Fixed Drug SJS/TEN Drug Eruption Eruption

Allopurinol ACE inhibitors Allopurinol Allopurinol Amphotericin B Aminoglycosides Barbiturates Amoxicillin Barbiturates Azole antifungals Dapsone Ampicillin Benzodiazepines Cephalosporins NSAIDs Barbiturates Captopril Hydralazine Oral contraceptives Carbamazepine Carbamazepine Narcotic analgesics Metronidazole Diclofenac Gold NSAIDs Pseudoephedrine Nevirapine Lithium Penicillin Sulfonamides Nitrofurantoin NSAIDs Phenytoin Tetracyclines Phenobarbital Penicillin Quinidine Phenytoin Phenothiazines Protamine Piroxicam Phenytoin Salicylates Sulfonamides Quinidine Sulfonamides Sulfonamides Tetracyclines Thiazides

22 1.2.6.5 Hepatotoxicity

Hepatotoxicity is the major cause of drug withdrawal from the market because it is the most common life-threatening IDR [69, 70]. Drug-induced hepatotoxicity can affect any cells in the liver and can manifest as several forms of either acute or chronic hepatic lesions [71]. The liver injury can be hepatocellular (overt damage to hepatocytes) or cholestatic (arrested bile flow) or a mixture of both with clinical presentations resembling acute hepatitis or cholestatic liver disease [72] (Table 1.4).

Table 1.4. Characteristics of hepatocellular and cholestatic liver injury. Adapted from [71, 73].

Hepatocellular Injury Cholestatic Injury Clinical • Jaundice • Jaundice Manifestations • Pruritus Histology • Hepatocyte necrosis/apoptosis • Minimal hepatocyte injury and/or steatosis • Some or no portal inflammation Clinical • Increased ALT (> 8-20 fold) • Alk phos levels elevated < 3 Biochemistry • Alk phos levels elevated < 3 fold fold • Increased ALT (< 8 x ULN) *mixed hepatocellular/cholestatic injury → ALT/alk phos >2 to <5 x ULN Prognosis • ≥10% mortality rate • <1% mortality rate

Alk phos, alkaline phosphatase; ALT, alanine aminotransferase; ULN, upper limit of normal

Approximately 13-17% of all acute liver failure cases are attributed to IDRs [74].

The mechanisms of idiosyncratic drug-induced liver injuries can be divided into two categories as described by Zimmerman [71]. Those reactions that are characterized by fever, rash, eosinophilia, and eosinophilic or granulomatous inflammation in the liver as well as a decreased time to onset of the reaction on re-exposure are classified as immune- mediated. For those that lack clear evidence of an immune-mediated mechanism

23 Zimmerman proposes that the reaction is attributed to metabolic idiosyncracy, i.e. due to aberrant metabolism of the drug in susceptible individuals. However, it is likely that both metabolism and the immune system are involved in the mechanism of drug-induced liver injuries. Many drugs are associated with drug-induced liver injuries. Being the site of most drug metabolism, the largest amount of reactive metabolite is formed in the liver, and this is presumably why they are a common site of IDRs. Drugs with aromatic amines, certain types of halogenated groups, acyl glucuronide, and reactive sulfate conjugates are often associated with idiosyncratic liver toxicity [75, 76].

1.3 Approaches to Study IDRs

There can be no doubt that the study of IDRs is a difficult endeavour, which in part is due to the fact that very little is known regarding the basic mechanisms of these reactions. Nevertheless, progress has been made as our lab and others working in this field have taken various approaches to the study of IDRs, both in terms of their basic mechanisms and clinical characteristics.

1.3.1 Reactive Metabolites

As indicated above, there is a large amount of circumstantial evidence for a role of reactive metabolites in the mechanism of IDRs. Although some drugs are excreted unchanged, most drugs are cleared by a combination of several phase I and phase II metabolizing pathways. This modification of drug molecules usually leads to increased water solubility and/or decreased pharmacological activity; however, many drugs are also metabolized to chemically reactive metabolites that can react with DNA, proteins, or other molecules leading to a variety of toxicities such as mutations, cancer, birth defects,

24 and idiosyncratic reactions as described earlier in the hapten hypothesis. Some of metabolites can also undergo redox cycling and induce oxidative stress, which could lead to a danger signal [77].

There are several types of reactive metabolites, and their reactivity depends on their chemical structures. Most reactive metabolites are electrophiles, which are electron-deficient molecules, or free radicals, which have an unpaired electron. In the case of electrophiles, soft electrophiles have a more diffuse area of electron-deficiency

[78] and prefer to react with soft nucleophiles that possess diffuse electron-rich moieties.

In some cases, the covalent bond formed between soft electrophiles and soft nucleophiles is reversible. Examples of soft electrophiles include α,β-unsaturated carbonyls, quinones (and the related quinone imines and quinone methides) and Michael acceptors.

Soft nucleophiles include sulfhydryl groups such as those found on glutathione and cysteineyl residues of proteins (Table 1.6). Hard electrophiles have either a highly localized electron deficiency or a formal positive charge [79]. They tend to be more reactive than soft electrophiles and preferentially react with hard nucleophiles to form a strong covalent bond. Examples of hard nucleophiles include the ε-amino group of lysine and the oxygen atoms of purines and pyrimidines (Table 1.5). Almost all drugs that contain a primary aromatic amine or aromatic nitro group are associated with a significant incidence of IDRs [77]. This is likely because they are oxidized, in the case of primary aromatic amines, or reduced, in the case of aromatic nitro groups, to similar reactive intermediates [80].

Free radical reactive metabolites do not usually form covalent bonds, but they can abstract hydrogen atoms from other molecules such as an endogeneous lipid. This results in lipid peroxidation and can cause cell stress [78]. However, free radicals can be electrophilic as in the case of the oxidation of aminopyrine by hypochlorous acid where a

25 cation radial is formed [81]. Free radicals can also add to double bonds, most commonly in lipids. This can lead to chain reactions that are analogous to hydrogen abstraction reactions. Such oxidations can also lead to the oxidation of protein sulfhydryl groups, which can cause changes in protein structure [80].

The site of drug reactive metabolite formation leading to an IDR is usually in the target tissue of the IDR. This is because these bioactivated drug molecules are usually very reactive and would have been chemically reduced by endogenous detoxifying entities such as glutathione as they travel through the body. In general, the main target sites of tissue destruction in IDRs are the liver, leukocytes, particularly neutrophils, and the skin. Likewise, these sites have active drug-metabolizing systems, or in the case of the skin, is immunologically quite reactive because it is a major barrier to various potential pathogenic substances.

Table 1.5. Examples of electrophiles and nucleophiles. Adapted from [79].

Electrophiles Nucleophiles

Hard • Alkyl carbonium ions • Oxygen atoms of • Benzylic carbonium ions purine/pyrimidine bases in DNA • Endocyclic nitrogens of purine bases in DNA • Oxygen atoms of protein serine and threonine residues Soft • Michael acceptors • Protein thiol groups (acrylamide, acrolein, • Sulfhydryl groups of acrylonitrile) glutathione • Quinones • Primary/secondary amino groups of protein lyseine and histidine residues

26 1.3.1.1 Drug Metabolism in the Liver

As much as drug metabolism in the liver is required for the elimination of xenobiotics from the body, metabolism of drugs can paradoxically lead to formation of chemically reactive metabolites. Cytochrome P450s (P450s) are the main oxidative phase I metabolizing enzymes responsible for the generation of reactive metabolites in the liver. They account for approximately 70% of total drug metabolism [82]. P450 is a heme-containing monooxygenase, which is found in highest concentration in the liver, but is also present to a lesser extend in other organs including the kidney, lung, intestine, and to a lesser extent in the brain, leukocytes (macrophage, lymphocytes and dendritic cells), and skin [83-86]. All mammalian P450s are generally membrane bound enzymes.

Microsomal P450s reside in the endoplasmic reticulum while the mitochondrial P450s reside in the inner mitochondrial membrane[84, 85]. The enzyme is anchored to the surface of the endoplasmic reticulum by its lipophilic NH 2-terminal region. Most of the enzyme is exposed to the cytoplasmic surface which provides access of the substrates to the catalytic region of the enzyme. Catalysis of P450s requires NADPH-cytochrome

P450 reductase as a co-enzyme. This is a flavoprotein that delivers electrons to P450 to initiate the catalytic cycle. P450 can also utilize peroxide as a co-substrate that oxidizes

Fe III via the peroxide shunt [78].

Humans have 57 P450 genes. Various oxidative reactions are catalyzed by the different isozymes of P450. These include carbon hydroxylation, heteroatom release (S-,

O-, or N-dealkylation), heteroatom oxygenation, epoxidation, and olefininic suicide destruction (heme destruction) [87]. These reactions can lead to the formation of reactive metabolites which are likely responsible for much of the idiosyncratic liver toxicity. Examples of some of the drugs implicated in causing hepatic IDRs and their reactive metabolites are provided in Table 1.6.

27 Table 1.6. Drugs metabolized by P450s that are associated with idiosyncratic hepatotoxicity. Compiled from [78, 88]. Immunological Drug Reactive Metabolite Perturbation (Antibodies formed) Halothane Acyl halide Anti-hapten Auto-antibodies Anti-CYP2E1 Tienillic acid Epoxide Anti-CYP2C9 Carbamazepine Arene oxide Anti-CYP3A Iminoquinone Autoantibodies Amodiaquine Iminoquinone Anti-hapten Sulfamethoxazole Nitroso Nitroso-specific IgG (in rat)

A well studied example of idiosyncractic liver toxicity caused by a reactive metabolite is halothane. This anesthetic is associated with a low but significant risk of liver failure and a relatively high incidence of idiosyncratic hepatitis: approximately

1/35,000 on primary exposure and 1/3,500 on re-exposure [17, 89]. As much as 20% of halothane is metabolized in the liver by P450 2E1 to a highly reactive trifluoroacetyl chloride [90, 91]. Trifluoroacetyl chloride can covalently modify proteins, including the

P450 isozyme that metabolizes it and microsomal epoxide hydrolase. This is a hard electrophile and it binds to the ε-amino groups of lysine residues on the proteins, mainly those in the endoplasmic reticulum (Figure 1.4) [89, 92-94]. Antibodies against trifluroacetylated proteins were found in the sera of patients with halothane-induced hepatitis indicating that the covalent binding elicited an immune response [18].

Autoantibodies against the native hepatic proteins were also identified in these patients

[95]. Analogues of halothane: enflurane, isoflurane, and desflurane, were found to be associated with lower incidences of idiosyncratic hepatitis [90]. Enflurane, isoflurane, and desflurane are also metabolized by P450s to form the same or a similar reactive

28 metabolite as halothane, but to a much lesser degree, approximately 3% and <1% for the

latter two, respectively [10, 90]. This strongly suggests that the amount of reactive

metabolite formed is a factor in the relative incidence of idiosyncratic drug-induced

hepatitis. However, the involvement of the trifluroacetyl chloride reactive metabolite in

this IDR is circumstantial and it is not clear whether antibodies against

trifluoroacetylated proteins are pathogenic. Trifluoroacetylated protein adducts were

found in the liver tissues of all patients treated with halothane and yet most patients do

not develop hepatitis [16, 96].

F Cl F O F O CYP2E1 Covalent binding Sensitization F C CH F C C F C C Hepatitis F Br F Cl F NH Halothane Trifluoroacetylchloride Protein Trifloroacetylated protein adduct

Figure 1.4. Bioactivation of halothane and its proposed pathway for the induction of hepatitis.

1.3.1.2 Drug Metabolism by Leukocytes

Drug metabolism has also been observed to occur in peripheral leukocytes (white

blood cells) [97]. Leukocytes and their bone marrow precursors contain drug

metabolizing enzymes such as P450s. Aryl hydrocarbon hydroxylase, which is a P450

isozyme, has been found to be capable of metabolizing benzo[a]pyrine to potentially

reactive species in lymphocytes and monocytes [98, 99]. However the amount of P450s

29 in leukocytes is quite low [100]; thus, they are not considered to be the main pathway for drug metabolism in these cells.

Neutrophils and macrophages are known to contain nitric oxide synthase which converts arginine to nitric oxide. Nitric oxide is released by these cells upon activation

- and can react with superoxide anion (O 2 ) to form peroxynitrite [101]. Peroxynitrite has been shown to oxidize thiols, and to decompose to other oxidizing agents such as the hydroxylradical and nitrogen dioxide [102, 103]. All of these oxidizing agents can potentially oxidize drugs.

Prostaglandin endoperoxide synthetase is another potential drug metabolizing enzyme in leukocytes. It is present in relatively high concentrations in lymphocytes, monocytes, neutrophils, and platelets [78]. In the presence of arachidonic acid, this enzyme has been shown to co-oxidize various drugs such as acetaminophen [104] and phenytoin [105]. Some drugs have been demonstrated to oxidize to free radical intermediates [106-108]. However, the significance of drug metabolism via this pathway in vivo is not known [109].

In neutrophils, oxidation via myeloperoxidase (MPO) is probably the most significant pathway for drug bioactivation. MPO is abundant and constitutively present in phagocytotic cells which include neutrophils and macrophages. During the respiratory burst (oxidative burst), MPO is released from the primary granules of neutrophils into the phagosome. In this process, stimulated neutrophils also convert extracellular oxygen

- - to O 2 by cell surface NADPH oxidase activity. The O 2 then undergoes spontaneous and enzymatic conversion (by superoxide dismutase) to hydrogen peroxide (H 2O2), which then acts as a co-substrate along with chloride ion for the production of hypochlorous acid (HOCl) by MPO (Figure 1.5). HOCl is a strong oxidant produced by neutrophils to kill bacteria as part of the innate immune system.

30

Neutrophil MPO

MPO H2O2 HOCl Cl - NADPH oxidase Superoxide dismutase O2 - O2

Figure 1.5. Neutrophil respiratory burst and the production of hypochlorous acid.

MPO is a heme-containing enzyme. It is a peroxidase/oxidase, which catalyzes oxidations using hydrogen peroxide or another hydroperoxide [109]. MPO exists as the ferric enzyme (Fe 3+ ) in its native state as shown in Figure 1.6. Like horseradish peroxidase, MPO undergoes oxidation in the presence of hydrogen peroxide to form compound I. Compound I is a strong oxidant and is presumed to be the major oxidant in the absence of chloride ion. In the presence of chloride, Compound I reacts with chloride to produce HOCl and regenerate MPO. In the absence of chloride, compound I undergoes a one-electron reduction to form compound II, which can then be further reduced back to the native MPO by electron donors such as ascorbate or certain drugs.

- Compound III can be produced by the reaction of O 2 with MPO or through the oxidation of compound II by H 2O2. Compound III then spontaneously decays back to MPO [109].

31 3+ MP + H2O2 Compound I + H2O

- O2 O2 O2 - O2 - O2 - O2 O2

Compound III Compound II

Abbreviation: MP3+, ferric myeloperoxidase

Figure 1.6. The oxidation pathway of myeloperoxidase. Adapted from [110]. Aside from being a bactericide, HOCl produced by MPO can oxidize many drugs, especially those which contain easily oxidizable nitrogen-containing functional groups

[111]. Drugs that possess primary aromatic amines, other aromatic nitrogens, and even aliaphatic amines are susceptible to oxidation by activated neutrophils, the

- MPO/H 2O2/Cl system or HOCl. The mechanism is thought to involve N-chlorination followed by the loss of HCl. Oxidation of sulphur compounds and even oxidation at carbon centres by MPO or HOCl can lead to the formation of reactive intermediates.

Our lab has demonstrated that various compounds can be oxidized to reactive intermediates by MPO that are capable of binding to leukocytes [109]. Table 1.7 and

Figure 1.7 provide examples of drugs that are metabolized by activated neutrophils, the

- MPO/H 2O2/Cl system, HOCl or MPO/H 2O2. in the absence of chloride.

32 Table 1.7. Drugs that may be metabolized by activated leukocytes. Adapted from [109].

Drug Chemical species oxidized

5-Aminosalicylic acid Primary aromatic amines Chloramphenicol (bacterial metabolite) Daspone Procainamide Sulfadiazene Sulfamethoxazole

Acetaminophen Other nitrogen-containing functional Aminopyrine groups Amodiaquine Chlorpromazine Clozapine Diclofenac Hydralazine Isoniazid Phenylhydrazine Phenytoin Vesnarinone

Methimazole Sulphur compounds Propylthiouracil Tenoxicam

Benzoic Acid Carbon centres Carbamazepine Diethylstiebestrol Phenol Phenylbutazone Salicylic acid

Gold compounds Others

33 CH 3 CH3 CH3 N +N N CH N N 3 -Cl- CH3 - N CH Myeloperoxidase/H2O2/Cl 3 Activated neutrophils Cl O N O CH O + N 3 N + CH3 H3C CH3 H3C H3C Aminopyrine dication intermediate

N-chloro

Cl + CHO

- Myeloperoxidase/H2O2/Cl N N Activated neutrophils N C C O C O NH NH2 2 O NH2 Carbamazepine carbocation acridan derivative

CH3 CH3 CH3 N N N

N N -Cl- N Myeloperoxidase/H O /Cl- N 2 2 N N Cl Activated neutrophils Cl Cl N N N + H Cl Clozapine Nitrenium ion

OCH3 OCH3 H

H3CO N NH2 H3CO N N - Cl Myeloperoxidase/H2O2/Cl N N Activated neutrophils H3CO H3CO H H NH2 NH2 Trimethoprim

OCH3

H3CO N NH

N H3CO

NH2

iminoquinone methide

Figure 1.7. Drugs and their reactive metabolites formed by activated neutrophils,

- MPO/H 2O2/Cl or HOCl.

34

HO O O O NH2 NH N N

- O2 or H O - Myeloperoxidase/H2O2/Cl 2 2 Myeloperoxidase/H2O2/Cl Activated neutrophils Activated neutrophils Ascorbic acid O C O C O C O C NHCH2CH2N(C2H5)2 R R R Procainamide procainamide nitroso- nitro- hydroxylamine procainamide procainamide

C3H7 N SH C H N S S N C H - C3H7 N SCl 3 7 3 7 Myeloperoxidase/H2O2/Cl N N N N Activated neutrophils OH OH OH OH Propylthiouracil sulfenyl chloride

C H N SO H C3H7 N SO3H 3 7 2

N N

OH OH reactive intermediate

O OH C2H5 C2H5 CH2N CH2N C2H5 C2H5 - Myeloperoxidase/H2O2/Cl NH Activated neutrophils N

Cl N Cl N

Amodiaquine quinoneimine O O H CO H3CO 3 C C N Cl H CO Myeloperoxidase/H O /Cl- H3CO N 3 N 2 2 N + Activated neutrophils

HN N Vesnarinone -HCl H O O O H3CO C H CO N N 3 +

N O

imidoiminium ion

Figure 1.7 Continued.

35 1.3.1.3 Drug Metabolism in the Skin

Although metabolism by the liver is generally considered the primary pathway for most drugs, P450s and other drug metabolizing enzymes are also found in the skin.

Keratinocytes express several P450 isozymes [112, 113] and the messenger RNAs of

P450s have been identified in fibroblasts and melanocytes [114]. Bioactivation of drugs in the skin can also lead to hapten-protein adduct formation in this organ; hence, IDRs can occur. However, there are still no convincing data to show that there are sufficient

P450s in the skin to lead to an IDR.

Besides drug metabolizing enzymes, the skin is also rich in APCs, such as dendritic cells, macrophages, and Langerhan’s cells. Monocytes, like neutrophils, contain myeloperoxidases and can undergo an oxidative burst to produce reactive oxygen species that are capable of oxidizing drugs. An example of a drug that can cause idiosyncratic cutaneous reactions is sulfamethoxazole. It is thought that the severe skin eruptions that occur is a result of reactions induced by either the hydroxylamine or the nitroso reactive metabolite of this drug [115]. It was inferred that metabolism of sulfamethoxazole first occurs in the liver to a hydroxylamine which then circulates to the skin where it is oxidized to the nitroso reactive metabolite by reactive oxygen species.

Another example of a drug which can cause IDR in the skin is nevirapine. This is a non- nucleoside reverse transcriptase inhibitor associated with a relatively high incidence of life-threatening skin rash and liver toxicity. Using a rat model, Chen et al. in our lab proposed that the 12-hydroxylation pathway of nevirapine is responsible for the IDRs

[44]. It is thought that hepatotoxicity of nevirapine in humans is due to the quinone methide formed by P450 in the liver, while the skin rash may be due to the quinone methide formed in the skin by sulfation of the 12-OH metabolite followed by loss of sulfate [44] (Figure 1.8).

36 N N N N N N N N P450 2D6, 3A OH N

NH CH2 NH H C OH NH CH3 2 O O O Nevirapine 12 OH-nevirapine

-H N N N O

NH H2C O S O N O O N N

N CH2 O quinone methide

Figure 1.8. Proposed activation pathway of nevirapine.

1.3.1.4 Drugs that Are Intrinsically Chemically Reactive

Not all drugs require bioactivation in order to become chemically reactive. These include compounds that are reactive because of ring strain. A carbon in a three- membered ring in which the bond angle is forced to be 60 o is under considerable strain since normal bond angle of an sp 3-hybridized carbon is 109 o. Reactions that open the ring are facilitated. Examples of drugs that possess ring strain and are associated with

IDRs are penicillins and cephalosporins. This class of drug contains a β-lactam ring and are reactive with amino and sulfhydryl groups because of ring strain (Figure 1.9), although their reactions with proteins are quite slow and only a small fraction of administered drug becomes covalently bound to protein [116]. As mentioned earlier, allergic reactions to penicillin are mediated by IgE antibodies against penicillin-modified proteins [32], Another example of drugs which do not require metabolism to become

37 chemically reactive is D-penicillamine. This drug, used in the treatment of rheumatoid arthritis, is associated with a variety of autoimmune syndromes. It is known that D- penicillamine can bind to aldehyde groups to form a thiazolidine ring [117]. The formation of a thiazolidine ring by penicillamine is irreversible and could lead to activation of antigen-presenting cells [65] (Figure 1.10).

O O H S H S CH CH2 C N CH3 CH2 C N 3 N HN CH3 CH3 O O NH Penicillin COOH COOH Protein Protein - NH2

Figure 1.9. Haptenation of protein by penicillin.

H3C CH3 COOH HS H3C NH2 CH3 HOOC D-penicillamine S Activation of antigen- N presenting cells O HC Macrophage

Macrophage

Figure 1.10. The reaction of penicillamine with macrophage aldehydes to form a thiazolidine ring.

1.3.2 Covalent Binding

The concept that small organic molecules can undergo bioactivation to electrophiles and free radicals and elicit toxicity by chemical modification of cellular

38 macromolecules began with the Millers’ studies on chemical carcinogenicity in the

1940s [118, 119]. Covalent binding was later applied to the study of drug-induced toxicity which began with the work of Brodie, Gillette, and Mitchell on the hepatotoxicity of acetaminophen induced by covalent modification of tissue macromolecules by the drug [120, 121]. The formation of reactive metabolites through bioactivation of drugs is now established as the primary step in many chemical toxicities, including carcinogenesis, necrosis, and apoptosis. Nevertheless, the precise role of reactive metabolite covalent binding to proteins in these processes is still unclear. In the context of IDRs, covalent binding of reactive metabolites is thought to be crucial in the pathogenesis of these reactions as described in the proposed mechanism of IDRs where a response directed against the drug or a drug-peptide epitope may be induced.

The major mechanisms for the covalent binding of reactive species to protein molecules involve nucleophilic substitutions or Michael additions but Schiff’s base mechanism can also be involved [122]. Nucleophilic substitution is the formation of a new covalent bond from an unshared pair of electrons in the nucleophiles (the reactive metabolite). The reaction of a nucleophile with a C=C that is conjugated with a carbonyl group is called a Michael addition. Alternatively, aldehydes on reactive metabolites can react reversibly with nucleophiles, such as amines, to generate Schiff’s bases via formation of carbinolamines.

Many drugs which have been shown to extensively bind to proteins in a covalent manner and yet are not associated with a high incidence of IDR. Therefore, covalent binding alone is not enough to cause IDRs and it is likely that the type of protein and the level of protein adduct formation is also crucial to the induction of IDRs. However, at the present we still do not know what constitutes significant covalent binding. Lance

Pohl, an expert in this field, has proposed a safe upper limit of 50 ρmol/mg microsomal

39 protein which is currently used as a rough guide at Merck for drug development [15], but there is no evidence that this limit has produced safer drugs. There are also a few examples where the identities of the proteins to which reactive metabolites bind have been identified; however, in most cases this information is still lacking. These proteins include P450s and microsomal proteins in the liver, myeloperoxidase in neutrophils and monocytes and their bone marrow precursors. Databases of protein targets of reactive metabolites have been established. As the data expands, correlation between specific protein adducts and specific types of IDRs may be found, thereby, facilitating our ability to predict the risk of IDRs.

1.3.2.1 Identifying Reactive Metabolites and Protein Covalent Binding

Many methods have been proposed to screen for reactive metabolites of drugs.

These include covalent binding studies, screening for suicide inhibitors and screening for glutathione conjugates [123]. The half-lives of most reactive metabolites are quite short; thus, they cannot be easily detected in biological systems. However, their existence can be inferred from the detection of the protein adducts that they form. The screening procedure for reactive metabolites begins with the utilization of in vitro bioactivation systems such as hepatic microsomes or hepatocytes. Reactive electrophilic metabolites form adducts with nucleophilic trapping agents, either with soft nucleophiles such as thiols from glutathione, or with hard nucleophiles such as amines form N-acetyllysine.

These adducts can then be analyzed and characterized by liquid chromatography coupled to mass spectrometry (LC/MS) and/or NMR spectroscopy. The identity of the reactive metabolite can be deduced from the structure of the adduct. However such methods may not detect all reactive metabolites responsible for protein covalent binding, either

40 because the glutathione adduct is not stable or because the reactive metabolites preferentially form adducts with other nucleophiles [15].

Covalent binding of drugs to proteins can be detected using radiolabelled compounds. A radiolabelled compound, or a drug in this case, can be incubated with an enzymatic system such as rat liver microsomes and NADPH. The protein is then precipitated and washed to remove all unbound radiolabelled drug. The remaining radioactivity is considered to be covalently bound, and the amount of covalent binding is expressed in terms of ρmol of bound drug/mg protein. In vitro detection of protein covalent binding using radiolabelled drugs provides a quantitative method to compare the amount of protein binding between different drug candidates. A generalized cut-off point for the drug industry is 50 ρmol of bound drug/mg protein as mentioned earlier.

However, there is no evidence that this results in safer drugs.

Detection of protein covalent binding using radiolabelled compounds is limited by the availability of the radiolabelled drug. In vitro studies of this kind may overestimate the amount of covalent binding because of a lack of detoxication systems which would be present in vivo . On the other hand, they can also underestimate the amount of binding because the activation system is not present in vitro . It is also difficult to perform these studies in vivo because of the amount of radiolabelled drug required.

Another method used to detect drug-protein covalent binding is to use immunochemical techniques with Western blot analysis using antibodies against the drug or its reactive metabolite. This is a qualitative technique, which has been used extensively in our lab to detect and compare the amount and pattern of protein covalent binding in in vitro systems as well as in tissues of animals given drug treatments [124].

The method consists of first synthesizing the reactive metabolite of the drug of interest

41 followed by its conjugation with N-acetylcysteine (NAC). Then the carboxylate of the cysteine is activated with a carbodiimide and conjugated with Keyhole limpet hemocyanin (KLH). KLH is a large protein molecule from the limpet mollusk which facilities the induction of antibody against the drug-NAC adduct. The drug-NAC-KLH is then injected into rabbits. Blood is collected from the animal and serum which contains the polyclonal anti-drug-NAC-KLH antibodies is isolated. All of the studies to detect covalent binding in this thesis used this technique, which will be further discussed in the chapters to follow.

1.3.3 Animal Models

The term ‘animal model’ in this thesis is used to indicate a reaction occurring in animals in response to a drug treatment that mimics the reaction that occurs in some humans. One of the major reasons why the study of IDRs is difficult is due to the lack of good animal models. In vitro studies are very unlikely to mimic the mechanism of an

IDR. Studies with patients, especially prospective studies, are virtually impossible because of low incidence of these reactions. There are obvious ethical issues in exposing patients to a drug to which they have had an IDR. There are also practical and technical limitations to obtaining human tissue samples for analysis. Retrospective studies with patients are more feasible, but they do not permit the study of events leading up to the

IDR. Therefore, it is difficult for us to gain a better understanding of the mechanistic basis of these reactions without the use of animal models. Unfortunately, these reactions are just as idiosyncratic in humans as they are in animals. To date our lab has only found two rodent models of IDRs that appear to involve the same mechanism as the IDR that occurs in humans: the penicillamine-induced autoimmunity in the Brown Norway rats

42 and the nevirapine-induced skin rash in rats. The second model was developed in our lab by Shenton et al .

D-penicillamine is associated with idiosyncratic drug-induced lupus in humans.

When treated with a daily dose of 20 mg D-penicillamine, approximately 60-80% of male Brown Norway rats develop an autoimmune reaction [125]. The autoimmune reaction is not observed in the Lewis or Sprague Dawley rats. Work with this animal model has shown that the incidence of penicillamine-induced autoimmunity can be modified with various treatments, such as polyinosinic-polycytidylic acid (Poly I:C), misoprostol, and aminoguanidine. Tolerance can be induced by treatment with a T cell immunosuppressant [126]. In addition, a correlation between the incidence of penicillamine-induced autoimmunity and the expression of co-stimulatory molecules by macrophages in the spleen was observed with the various treatments which modified the incidence [125, 126]).

In the nevirapine model, female Brown Norway rats treated with the drug at a daily dose of 150 mg/kg all developed skin rash, while only 20% of Sprague Dawley rats developed a rash [45]. Studies with this model demonstrated that the nevirapine-induced skin rash is immune-mediated. Rechallenge led to a more rapid onset of the rash and this sensitivity could be transferred to naïve animals with spleen cells. CD4 + T cells appear to be essential because since partial depletion of CD4 + T cells was partially protective while depletion of CD8 + T cells appeared to make the reaction worse [127]. In addition, studies with this model have allowed us to deduce that the 12-hydroxy metabolic pathway is responsible for this IDR [44].

Although there are very few good animal models of IDRs (a review by Shenton et al. describes some of them [128]), and most of the currently available ones are far from ideal; nevertheless, they have the potential to be an invaluable tool for the study of

43 the mechanisms of IDRs. In fact, it is rather hard, if not impossible, to test some of our mechanistic hypotheses of IDRs without the use of animal models. For example, some of the studies in our lab had begun to look at the immunological basis of certain IDRs by studying changes in cytokine levels in animals given drugs that cause IDRs in humans.

1.3.4 Lymphocyte Transformation Test

The symptoms of an IDR are often indistinguishable from other diseases not caused by drugs. In order to determine whether a patient is experiencing an IDR and also to determine what drug is causing the IDR if the patient is taking more than one drug, the lymphocyte transformation test (LTT) can be performed. The LTT also offers insights into the type of immune reaction involved, specifically the type of cells that are activated and the cytokines that are released by the drug.

The LTT is an in vitro assay based on the fact that lymphocytes that have been sensitized by a certain immunogen may proliferate when they are re-exposed to this immunogen. The procedure involves incubation of peripheral blood mononuclear cells

(PBMC) from a patient with the suspected drug at non-toxic concentrations for approximately 3-7 days. Proliferation of the lymphocytes is then detected by measuring the incorporation of 3H-thymidine into replicating DNA. An enhanced proliferation of lymphocytes leading to an increase in thymidine uptake of greater than two fold over that of a solvent control is interpreted as a sign of drug-specific T cell sensitization [129].

Alternatively, T-lymphocyte activation can be detected by an increase in the release of cytokines or other immune-related proteins to measure antigen-dependent proteins [130,

131]. Specifically, activated T cells can produce cytokines such as IFN-γ, IL-5, and IL-

10, which can be assayed in the supernatant of stimulated cell cultures by enzyme-linked immunosorbant assays (ELISAs) or intracellularly by flow cytometry of permeablized

44 cells [130]. It is important to point out that the usefulness and accuracy of this test for drug hypersensitivity diagnosis is still controversial and it can be difficult to achieve sufficient sensitivity and reproducibility. Nevertheless, the LTT is an invaluable tool to gain a better understanding of the mechanisms of IDRs. Tharmanathan et al. in our lab attempted to use the LTT to determine if it is the parent drug or its metabolites that is responsible for initiating the immune response in nevirapine-induced IDRs [132].

1.3.5 Genetic Determinants of IDRs

It is likely that genetic factors are major determinants of the idiosyncratic nature of IDRs. Studies were done to try to associate genetic polymorphisms of drug metabolism as a risk factor of IDR. However, in most cases no link was found and any observations to date are too weak to explain the idiosyncratic nature of IDRs [42]. On the other hand, several strong associations with specific HLA genotypes have been found.

HLA is the human version of the MHC protein, which is involved in antigen presentation.

An example is the studies on abacavir-induced hypersensitivity reactions which was found to be strongly associated with the HLA-B*5701 genotype; the haplotype Hsp 70-

Hom variant was also associated but to a lesser degree [22]. Other examples of genetic determinants include studies on carbamazepine-induced Stevens-Johnson syndrome which was found to be strongly associated with HLA-B*1502 in Han Chinese [133], and allopurinol-induced toxic epidermal necrolysis was strongly associated with HlA-

B*5801 [134].

45 1.3.6 mRNA Profiles and Proteomics in the Study of IDRs

It is likely that drugs that are associated with IDRs cause some biochemical changes in all those who take the drug even though they do not cause clinically apparent toxicity in most people possibly because these people develop immune tolerance. These biochemical changes are probably the precursors to the IDR and we could, in theory, monitor such changes using mRNA microarrays and proteomics to identify patterns that may predict a drug’s propensity to cause IDRs. Research groups and industry working in this field have begun to utilize these techniques to gain a better understanding of IDRs.

Studies are now underway in our lab to determine the patterns in the expression levels of certain genes that encode for proteins that can act as danger signals. In a microarray study with hepatic tissues from Brown Norway rats treated with D-penicillamine, some of the animals exhibited substantial expression changes in genes involved in cell stress, energy metabolism, acute phase response, and inflammation [135]. In a more recent microarray study performed by Pacitto et al. , tienillic acid was found to induce changes in gene expression involving oxidative stress, inflammation, cytotoxicity, and liver regeneration in rats [136]. These data support the hypothesis that danger signals in the form of cell stress may be involved in initiating the immune response observed in tienillic acid- and penicillamine-induced IDRs.

1.4 Topics in Hematology

1.4.1 The Hematopoietic Microenvironment

The main components of the hematologic system are blood and the organ that forms it. The bone marrow is one of the largest organs in the body constituting approximately 4% of total body weight. Bone marrow is the tissue found in the hollow

46 interior of bones. The main function of this organ is to produce blood cells both under steady-state conditions as well as during periods of increased demands. Daily production of blood cells in a normal adult is around 6 billion cells/kg of body weight [137]. There are two types of bone marrow: the red marrow, found mainly in the flat bones and epiphyseal ends of the long bones, consisting mainly of myeloid tissue which is where red blood cells, platelets, and most white blood cells are formed, and the yellow marrow, found in the middle portion of the long bones, consisting of fat cells. Constituents of the bone marrow include proliferating and mature blood cells, and an extracellular matrix made up of stromal cells (fibroblastoid cells, endothelial cells, adipocytes, reticular cells

(osteoblasts and osteoclasts) and macrophages), collagen, glycoproteins (fibronectin, tenascin, laminin, thrombospondin and hemonectin) and proteoglycans (heparin sulphate, dermatan sulphate and chondroitin sulphate) [138]. The proliferative activity of the various cell pools in the bone marrow involves humoral feedback from peripheral target tissues and cell-cell and/or cell-matrix interactions within the microenvironment of the marrow [137]. There is substantive evidence for specialized niches within the bone marrow that promote linage-restricted cell maturation [138-140].

1.4.2 Blood Cells and Hematopoiesis

Blood cells consist of red blood cells (erythrocytes), white blood cells

(leukocytes) and platelets (thrombocytes). White blood cells are classified into cells that possess granules, which are the granulocytes (neutrophils, basophils and eosinophils), and mononuclear cells that lack granules (monocytes and lymphocytes). All of the cellular elements of blood are derived from the same progenitor or precursor cells – the hematopoietic bone marrow stem cells. These are known as pluripotent hematopoietic stem cells because they can give rise to all of the different types of blood cells [141].

47 The hematopoietic stem cells are capable of both self-renewal and differentiating into all hematopoietic lineages. When they divide, some of their daughter cells remain as hematopoietic stem cells while others become either myeloid cells, which give rise to the myeloid, megakaryocytic or erythroid multipoteintial stem cells, called the “colony forming unit” (CFU), and the lymphoid progenitor cells which give rise to the lymphocytes. Figure 1.11 provides the sequence of development of each of the cell types.

Factors that stimulate the production of committed stem cells are called colony- stimulating factors (CSFs). Cytokines such as interleukin 3 (IL-3) and granulocyte- macrophage-CSF (GM-CSF) stimulate myeloid stem cell differentiation into granulocyte-macrophage CFU (CFU-GM). GM-CSF stimulates the differentiation and proliferation of neutrophils, monocytes, and eosinophils, whereas granulocyte-CSF (G-

CSF) specifically stimulates the production of neutrophils in the later stage of the maturation process. In neutrophil production, CFU-GM gives rise to CFU-G, which then differentiates into the morphologically recognizable myeloid precursors of the mitotic pool of cells: myeloblasts, promyelocytes, and myelocytes. The myelocytes then differentiate into the metamylocytes, which can no longer divide. These form the post- mitotic pool of cells. Band cells, which are immature neutrophils that have nuclei which have not yet fully segmented, are also found in this pool of cells. Such cells can sometimes be found in the peripheral blood, composing less than 8% of circulating neutrophils, but their numbers can increase upon hematopoietic stress such as during an infection.

48

Figure 1.11. Hematopoiesis and cytokines involved in its regulation.

49 1.4.3 The Neutrophil

Neutrophils are polymorphonuclear cells, which account for 50-70% of the total white blood cells in humans. The absolute count in the peripheral circulation ranges from 2.5-7.5 x 10 9 cells/L. Neutrophils stain a neutral pink on hematoxylin and eosin histological preparations. Their cytoplasm contains granules and a nucleus divided into

2-5 lobes.

1.4.3.1 Neutrophil Kinetics

Except in disease states, only functionally mature neutrophils are released from the bone marrow, although low numbers of immature band neutrophils are found in the peripheral circulation. However, during stress conditions their maturation time in the bone marrow may be shortened. Corticosteroids, fragments or components of the complement system, and catecholamines released during stress can accelerate the release of mature and band neutrophils into the circulation [142]. Upon release from the bone marrow, the life of a neutrophil is relatively short, having a half-life of approximately 6-

8 hours in the circulation [143]. A similar half-life was measured in rats [144, 145], dogs

[146], and rabbits [147, 148]. Neutrophils can adhere to the endothelium of blood vessels by adhesion receptor-ligand interactions [149]. These cells form the marginated pool which constitutes about half of the total blood neutrophils. When activated, neutrophils can undergo selectin-dependent capture and roll along the vascular endothelium, followed by integrin-dependent adhesion and migration into tissues where they can survive for 1-2 days [149]. Under homeostatic conditions, senescent neutrophils are cleared from the circulation by the spleen, liver, and bone marrow. The bone marrow also has a reserve of mature neutrophils. Following infection or inflammation, neutrophil production and release from the bone marrow, mediated by

50 cytokines and chemokines such as TNF-α, IL-1, IL-6, and IL-12, is elevated. Figure

1.12 provides a schematic diagram of neutrophil kinetics.

Mitotic Pool Post -Mitotic Pool

Myeloblasts Metamylocytes Marginated Promyelocytes Bands Pool Tissues Myelocytes Segmented Neutrophils Stem 3-6 days Cell 5-7 days Circulating Pool Pool

6-8 hours

Bone Marrow Peripheral Blood

Figure 1.12. Neutrophil kinetics.

1.4.3.2 Neutrophil Function

As an integral part of the innate immune system, neutrophils are essential for the destruction of invading microorganisms and to initiate and maintain conditions of inflammation. Neutrophils undergo chemotaxis, which allows them to migrate towards sites of infection or inflammation. This is mediated by chemotatic substances such as

C5a (a complement fragment), interleukin-8 (IL-8), interferon gamma (IFN-γ), leukotrienes, and bacterial peptides [150]. Neutrophils in the marginated pool will undergo transendothelial migration to sites of infection outside of the vasculature. This process is mediated by β2-integrin CD11b/CD18 present on the surface of mature neutrophils [149, 151].

Neutrophils are capable of ingesting microorganisms or particles by phagocytosis.

This is mediated by complement receptors and Fc receptors that recognize opsonic

51 proteins, such as immunoglobulins, complement fragment C3b, and lectin [150]. Upon phagocytosis of foreign material, neutrophils undergo an oxidative (respiratory) burst. In this process, reactive oxygen species are quickly released to facilitate bacteria killing.

The NADPH oxidase enzyme is assembled and activated on the neutrophil membrane.

NADPH oxidase is a multicomponent enzyme containing cytosolic proteins, which include four soluble factors: p67 phox , p47 phox , p40 phox , and a small G-protein, Rac, as well as membrane-bound proteins, which includes a stable, heterodimeric flavocytochrome

phox phox b558 composed of two subunits: gp91 and p22 [152, 153]. In resting cells, the

NADPH oxidase is unassembled and inactive. Upon cell surface receptor activation by inflammatory mediators, cytosolic components of NADPH oxidase translocate to the plasma or phagosomal membrane where this enzyme complex is assembled [153].

The interaction of inflammatory mediators such as N-formyl-L-methionyl-L- leucyl-L-phenylalanine (fMLP) with G-protein-coupled membrane receptors results in the generation of inositol 1,4,5-triphosphate (InsP3) by phospholipase C β, which activates the release of Ca 2+ from intracellular stores through its channel receptor. As stored Ca 2+ decreases, stromal-interacting molecule 1 interacts with store-operated calcium channels, and extracellular Ca 2+ entry occurs. The resulting elevation in cytosolic Ca 2+ is required in regulating the assembly of NADPH oxidase components at the plasma or granular membrane. Store-operated calcium entry mediates the translocation of Rac, activates myeloid-related proteins, which enhance the organization and recruitment of cytosolic factors to the membrane-bound flavocytochrome b558 , and stimulates protein kinase Cs (PKCs) involved in the phosphorylation of cytosolic phox proteins [152]. NADPH oxidase reduces extracellular oxygen to form superoxide anion.

The superoxide anion is then catalyzed by superoxide dismutase to form H 2O2. In the presence of chloride ions, MPO, which is released from the azurophilic granules,

52 catalyses the production of hypochlorous acid from H 2O2. Hypochlorous acid is a strong oxidant with potent bactericidal effect. Besides MPO, azurophilic granules also contain bactericidal/permeability increasing protein, defensins, serine proteases, neutrophil elastase, and cathepesin G [154]. These granules are formed early in granulopoiesis during the mitotic phase while specific and tertiary granules, which lack MPO, are formed near the end of the mitotic phase and in the post-mitotic phase. Changes in the serum concentration of cytokines such as TNF-α, G-CSF, and GM-CSF are thought to have an influence on the level of neutrophil oxidative burst [155].

1.4.3.3 Neutrophil Apoptosis

The maintenance of homeostasis in the blood cell lineages is carried out by the apoptotic process. Apoptosis is responsible for the physiological death of virtually all cells in every organ including neutrophils. Apoptosis is programmed cell death, which causes a variety of morphological changes in the cells, including blebbing of the plasma membrane, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. This process minimizes the leakage of cellular contents from dying cells, in contrast to necrosis which usually results from trauma that causes injured cells to swell and lyse resulting in the release of cell contents that can damage surrounding tissues. Cell apoptosis can be induced by either extrinsic signals or intrinsic inducers. The initiation of apoptotic mechanisms involves members of the tumor necrosis factor receptor (TNFR) family and is mediated through either the TNF- induced pathway or the Fas-Fas ligand-mediated pathway. The binding of TNF to the

TNF-R1 has been shown to initiate the pathway that leads to caspase activation via the

TNF receptor-associated death domain (TRADD) and the Fas-associated death domain

(FADD) protein [156]. In the pathway involving the Fas receptor, its interaction with the

53 Fas ligand results in the formation of the death-inducing complex (DISC), which contains the FADD, caspase-8, and caspase-10 [157].

Apoptosis is crucial in eliminating neutrophils from inflamed tissues and to maintain appropriate neutrophil numbers under physiological conditions. Apoptotic neutrophils are quickly phagocytosed by macrophages. Induction of the apoptotic program in the older neutrophils in the circulation and during the resolution of inflammation has been proposed to involve the Fas/CD95/Apo-1 signalling pathway

[158]. Although neutrophils express both Fas receptors and ligands, results from in vitro studies do not seem to support the idea that spontaneous apoptosis of these cells is mediated by autocrine or paracrine Fas ligand/Fas receptor interactions [159]. Studies on the role of TNF-α in the induction of neutrophil apoptosis also yielded inconsistent results. In vitro studies show that low serum concentrations of TNF-α are protective while high concentrations are pro-apoptotic [160]. TNF-α induces a dose-dependent increase in the expression of the adhesion molecule CD11b/CD18, and it also stimulates neutrophils to undergo oxidative bursts [160]. Various caspases such as caspase-1, 3, 8, and 9 have been shown to be involved in the spontaneous and Fas receptor-mediated neutrophil apoptosis [159]. Members of the Bcl-2 family also seem to play a role in modulating this process, where Bax, Bak, Bid, Bad, and Bim are thought to be pro- apoptotic and Bcl-XL, A1, and Mcl-1 are anti-apoptotic [159]. In terms of delaying apoptosis, many pro-inflammatory mediators including G-CSF, GM-CSF, LP, C5a, fMLP, ATP, leukotriene B4, IL-1β, IL-2, IL-3, IL6, IL-15, and INF-γ can have an effect

[159]. Recent studies show that PKC too can be involved in regulating neutrophil apoptosis [161]. Some isozymes of the PKC family are pro-apoptotic ( δ, θ, and βI), while others are anti-apoptotic ( α and βII ) in neutrophils [161, 162].

54 1.4.3.4 Neutrophil Disorders

Neutrophil disorders can result from a reduced number of cells or defective cell function. Low neutrophil counts are known as neutropenia. When the neutrophil count drops to 0.5 x 10 9/L cells or lower, it is termed agranulocytosis. Low neutrophil counts can be a result of congenital disorders such as severe congenital neutropenia, cyclic neutropenia, myelokathexis, and the Shwachman-Diamond syndrome. In these cases, neutrophil progenitor cells fail to complete the process of proliferation, differentiation, and maturation in the bone marrow and fail to enter the circulation [163]. Some of these disorders may be associated with accelerated apoptotic cell death of the myeloid precursors. Alternatively, low neutrophil counts can also be induced by xenobiotics.

This will be discussed in greater detail in the sections to follow.

Functional disorders of neutrophils include disorders of chemotaxis, adhesion, phagocytosis, and the oxidative burst. These are often heredity as in the case of chronic granulomatous disease, which involves mutations in the CYBB gene located on the X chromosome. This produces defects in the NADPH oxidase and hence an absence or a reduction in oxidative burst in those with the disease.

Both low neutrophil counts and compromised function may result in increased susceptibility of infections in an individual. Clinical recognition and proper management are warranted.

1.4.3.5 Effects of Xenobiotics on the Neutrophil Oxidative Burst

One of the most important functions of neutrophils is the oxidative burst which can be stimulated, potentiated, or inhibited by various exogenous or endogenous agents.

Receptor-mediated stimulation of oxidative burst can be induced by agents such as the

55 chemotactic peptide fMLP and opsonized zymosan [164, 165], whereas stimulation by phorbol 12-myristate-13-acetate (PMA) or calcium ionophores are not receptor-mediated but are more powerful oxidative burst stimulants [152, 166]. Drugs such as indomethacin and procaine have been shown to act synergistically with fMLP to enhance the oxidative burst in neutrophils, while nonsteroidal antiinflammatory drugs (NSAIDS), phenothiazine compounds, and drugs that bind to β-adrenergic receptors on neutrophils have inhibitory effects [109, 164, 167-170].

1.5 Drug-induced Blood Dyscrasias

Drug-induced blood dyscrasias can affect any type of blood cell. Common examples include agranulocytosis, which is a loss of granulocytes leading to an increased risk of infection; thrombocytopenia, which is a decrease in the number of platelets leading to an increased risk of bleeding; and aplastic anemia, in which bone marrow- derived blood cells are decreased and cells in the bone marrow are replaced by fat.

Drug-induced blood dyscrasias can either be acute and predictable, as in the case of myelotoxicity induced by most anticancer agents during chemotherapy, or delayed and idiosyncratic such as clozapine-induced agranulocytosis.

1.5.1 Predictable Drug-Induced Blood Dyscrasias

While the goal of anticancer drugs is to kill cancer cells, most of these agents are not very selective and tend to cause acute toxicity to bone marrow cells as well because of the rapid turnover rate of this type of cell. Bone marrow toxicity associated with

56 anticancer drugs is relatively easy to predict based on the pharmacological effects of these drugs. It is usually detected in in vitro assays using CFU-GM and in in vivo animal toxicity studies. Such studies can refine safety margins and provide a rational basis for calculating clinical dosages and for setting human exposure limits.

1.5.2 Idiosyncratic Blood Dyscrasias

The haematological system is one of the most common organ systems affected by

IDRs. As with other forms of IDRs, the mechanisms of drug-induced blood dyscrasias are still unknown. They are likely to involve the bioactivation of dugs by blood and bone marrow cells. The generated reactive metabolites can either be directly cytotoxic or they can generate immune responses leading to this IDR as described in the proposed mechanisms of IDRs. There are also no in vitro tests that can predict which drug will cause idiosyncratic blood dyscrasias.

1.5.2.1 Drug-Induced Idiosyncratic Agranulocytosis

Acute agranulocytosis is a potentially life-threatening condition with an incidence estimated at 2-9/million inhabitants/year, and 70 to >90% of the cases are attributable to drugs [171]. A wide spectrum of drugs can cause agranulocytosis, some of which are listed in Table 1.9.

Drug-induced agranulocytosis is characterized by a neutrophil count of less than

0.5 x 10 9/L with no relevant decrease in haemoglobin and platelet counts in the peripheral blood. Examination of the bone marrow may show one or more of the following conditions: complete lack of granulopoiesis, myeloid hypocellularity, neutrophilic maturation arrest, or hypercellularity with increased myeloid precursors and

57 decreased maturation in the case of peripheral destruction of neutrophils [171].

Neutrophil counts usually return to normal with cessation of the culprit drug. The prognosis has greatly improved over recent years with better intensive care treatment and increased alertness by health care professionals, which has brought the fatality rate owing to drug-induced agranulocytosis down to approximately 5% [171].

The pathogenesis of drug-induced agranulocytosis is not completely understood, especially those which are idiosyncratic in nature. In some cases, there is rapid onset of the reaction upon re-challenge which is suggestive of the involvement of an adaptive immune mechanism. In fact, drug-specific antibodies were also found to be involved in the mechanism of certain drugs associated with agranulocytosis. These include aminopyrine, amodiaquine, penicillin, propylthiouracil, sulfamethoxazole, sulfasalazine, and trimethoprim. Autoantibodies were also found in the case of propylthiouracil.

However, for drugs such as clozapine, mianserin, captopril, gold and phenothiazines, there were no evidence of antibodies. Thus, we cannot rule out other mechanisms, such as cytotoxic effects. Such is the focus of this thesis research, particularly in the case of clozapine, as we are trying to shed light on how this drug causes agranulocytosis.

58 Table 1.8. Xenobiotics associated with agranulocytosis [172-174].

Analgesics and NSAIDs Aminopyrine, Dipyrone, Phenylbutazone, Ibuprofen, Indomethacin, Diflunisal, Benoxaprofen, Sulindac, Fenoprofen, Tolmetin, Cinchophen, Acetylsalicylic acid, Aminophenazone, Diclofenac, Piroxicam, Temoxicam Antipsychotics, Sedatives, Antidepressants Phenothiazines, Clozapine, Imipramine, Chlordiazepoxide, Amoxapine, Meprobamate, Diazepam, Haloperidol, Indalpin, Meprobamate, Mianserin, Risperidone, Tiapride Anticonvulsants Phenytoin, Ethosuximide, Carbamazepine, Trimethadione, Valporic acid Antithyroid drugs Thiouracils, Methimazole, Potassium perchlorate, Thiocyanate, Carbinazole Cardiovascular drugs Procainamides, Captropril, Aprindine, Propafenone, Nifedipine, Quinidine, Flurbiprofen, Furosemide, Hydralazine, Lisinopril, Methyldopa, Phenindione, Propafenone, Propranolol, Spironolactone, Thiazide diuretics, Ticlopidine Heavy metals Gold, Arsenic compounds, Mercurial diuretics Sulfa drugs Sulfonamides, Methazolamide, Acetazolamide, Oral hypoglycemics, Sulfasalazine, Dapsone, Sulfa antibiotics Antibiotics Penicillins, Vancomycin, Cephalosporins, Clindamycin, Nitrofurantoin, Novobiocin, Isoniazid, Rifampin, Streptomycin, Thiocetazone, Flucytosin, Pyrimethamine, Mebendazole, Levamisole, Quinine, Chloroquine, Hydroxychloroquine, Quinacrine, Zidovudine, Acyclovir, Chloramphenicol, Ciprofloxacin, Cotrimoxazole, Ethambutol, Lincomycin, Terbinafine, Tetracylcines, Tinidazole, Vancomycin Antihistamines Cimetindine, Ranitidine, Tripelennamine, Thenalidine, Chlorpheniramine, Brompheniramine, Methaphenilene Miscellaneous drugs Retinoic acid, Phenindione, Cochicine, Allopurinol, Aminoglutethimide, Metoclopramide, Ticlopidine, Tamoxifen, Penicillamine, Acetazolamide, Benzafibrate, Tripelemamine, 1,2-dimethyl-3, hydroxypyrid-4-one Miscellaneous chemicals Dinitrophenol, DDT, Mustard gas, certain Herbal medicines and Hair dyes

1.5.2.1.1 Clozapine-Induced Agranulocytosis

Clozapine is a tricyclic dibenzodiazepine (Figure 1.13) atypical antipsychotic agent, which is effective in the treatment of refractory schizophrenia. Its therapeutic

59 effects are probably mediated by dopaminergic and seretonergic activities [175, 176]. It displays strong affinity for several dopaminergic receptors, especially the D 4 receptor, but shows only weak antagonism for the D 2 receptor. It is also a serotonin antagonist, which displays a strong binding to the 5-HT 2A/2C receptor subtype and a partial agonist for the 5-HT1A receptor. Clozapine is also a strong antagonist at different subtypes of adrenergic, cholinergic, and histaminergic receptors. In spite of its effectiveness and lack of extrapryramidal side effects, its use has been limited because of its propensity to cause agranulocytosis [177]. Clozapine can also cause other hematological toxicities such as neutropenia, anemia, eosinophilia, leukocytosis, lymphopenia, thrombocytopenia, and thrombocytosis [178] as well as liver and cardiac toxicities [179, 180].

CH3 N

N N Cl

N H 8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo [b,e] [1,4] diazepine

C18H19ClN4 Mol. Wt. 326.82 Figure 1.13. Chemical structure of clozapine.

Developed by Sandoz in 1961, Clozapine was first introduced in Europe in 1971, but it was withdrawn from market in 1975 after it was shown to cause agranulocytosis that lead to death in some patients [181]. Because of its effectiveness, the FDA approved

60 its clinical use in 1990, but only for treatment-resistant schizophrenia [182]. The FDA requires that clozapine carries a black box warning for agranulocytosis, as well as one for seizures, myocarditis, “other adverse cardiovascular and respiratory effects”, and for

“increased mortality in elderly patients with dementia-related psychosis”. Patients on clozapine are required to have weekly blood tests to monitor blood cell counts for the first six months of therapy and less frequently thereafter. This is because the risk of developing neutropenia and agranulocytosis with clozapine is approximately 3% and

0.8%, respectively [177, 183-185]. The highest risk of developing this IDR is within the first 6-18 weeks of treatment with most cases occurring within the first 6 months, but the risk then drops to 0.08% after 1 year of treatment [177]. Initial symptoms include fever, sore throat, and malaise. Eosinophilia may also occur in some patients, but is considered transient and asymptomatic [186]. Hypocellularity of the granulocytic series in the bone marrow have been described in some cases [187]. Clozapine-treated patients have also been shown to have a significant increase in band neutrophils in the peripheral circulation; although this is not associated with the ability of the drug to cause agranulocytosis [188]. Clozapine-induced neutropenia and agranulocytosis are normally fully reversible upon discontinuation of the drug with leukocyte counts returning to pretreatment values within 2-3 weeks [189]. In addition to cessation of clozapine, treatment may involve administration G-CSF to stimulate granulopoiesis. Aside from full-fledge agranulocytosis, around 22% of clozapine-treated patients have been shown to develop a transient neutropenia (neutrophil count between 0.5 - 1.5 x 10 9/L) [189-

191]. There are also some patients who developed mild leukocytosis on starting clozapine which spontaneously resolves 3-4 weeks after onset [178, 189]. Transient and asymptomatic eosinophilia was observed in some patients [189, 190, 192, 193]. It has been reported that changes in cytokine levels were observed during clozapine treatment.

61 These include an increase in plasma levels of TNF-α, IL-6, G-CSF, and leptin [194] which may be related to the incidence of transient fever, weight gain, myocarditis, and pancreatitis observed in some patients treated with clozapine [194, 195].

Clozapine is metabolized extensively in the liver and the two major metabolites formed by P450s are desmethylchlozapine and clozapine N-oxide. In chronically treated patients, the serum level of clozapine ranges from 60-600 ng/ml (0.18-1.8 µM) at approximately 12 hours after oral daily doses of 100-500 mg [196], while another clinical study have shown that serum level can reach as high as 3 µM [197]. The elimination half-life is 6-17 hours with a volume of distribution of 2-7 L/kg and a clearance of 2-13 ml/min/kg in humans [196]. In vivo and in vitro animal studies have shown the presence of glutathione adducts which implies the formation of reactive metabolites [198]. Furthermore, incubations of clozapine with PMA-stimulated neutrophils and bone marrow cells yielded a reactive intermediate -- the nitrenium ion, which is presumably formed from an N-chloro intermediate that spontaneously looses

HCl [198, 199] (Figure 1.7).

The mechanism of clozapine-induced agranulocytosis is elusive. However, it is likely to involve the nitrenium reactive metabolite. Using an antibody that recognizes clozapine bound to protein, clozapine has been shown to covalently bind to neutrophils in vitro and also to neutrophils of patients who take clozapine [124]. Interestingly, olanzapine, which has a very similar structure to clozapine, covalently binds to neutrophils in vitro to the same as extend as clozapine, but in vivo binding to human neutrophils was not detected [124]. Olanzapine is also not associated with a significant incidence of agranulocytosis. This may probably be due in part to its much lower therapeutic dose compared to clozapine and the lack of covalent binding in vivo [200] .

Clozapine-induced agranulocytosis has also been proposed to be immune-mediated.

62 Studies by Pisciotta et al. detected antibodies that inhibited neutrophils [201], but this appears to be an artifact due to platelet aggregation because studies from our lab had found inhibitory activity in serum and not in plasma [202]. Our lymphocyte transformation studies were also unable to find evidence of T cells specific for clozapine- modified leukocytes (unpublished data). A characteristic of clozapine-induced agranulocytosis is that its time to onset is the same on re-exposure as on initial exposure to the drug [202]. It was initially thought that this suggests that the reaction is not likely to be immune-mediated because most immune-mediated reactions have immune memory

(there are “memory” T lymphocytes that can respond quickly) and occur rapidly on re- exposure. However, there are examples of immune-mediated reactions that do not have immune memory. A good example is heparin-induced thrombocytopenia [203]. This

IDR is mediated by antibodies against the heparin-platelet factor 4 complex [204].

However, when the drug is stopped and when pathogenic antibodies are no longer detectable (after about 100 days), rechallenge with the drug does not usually result in thrombocytopenia, and if it does, the time course is not accelerated [205]. Thus, the hypothesis of an immune-mediated mechanism cannot be ruled out for clozapine- induced agranulocytosis. Alternatively, other research groups have looked at the cytotoxic effect of clozapine and its metabolites. They were found to inhibit the growth of CFU-GM in a dose-dependent manner in in vitro cell cultures [206, 207]. Other studies have found the nitrenium ion to induce cytotoxicity and apoptosis in neutrophils incubations [200, 208, 209]. While such in vitro studies have demonstrated the toxic potential of clozapine and its reactive metabolite, it is not clear whether this represents the situation in vivo and it would not explain the idiosyncratic nature of the reaction because covalent binding appears to occur in most patients and yet most of them do not develop the IDR. In addition, genetic or host-dependent components to clozapine-

63 induced agranulocytosis have also been explored as a risk factor of this IDR. An increased frequency of the HLA-B38, DR4 and DQ3 haplotype among Ashkenazi Jewish patients has been identified to be a possible risk factor [210].

1.5.2.1.2 Aminopyrine-Induced Agranulocytosis

Aminopyrine or dipyrone, its sodium sulphonate derivative (Figure 1.14), is a pyrazole formulated in the latter 19 th century and was widely used as an analgesic, anti- inflammatory, and antipyretic agent in the early 1900s [211, 212]. It is no longer licensed for use in most countries because of its associated risk of agranulocytosis. In fact, aminopyrine was one of the first drugs known to be associated with drug-induced agranulocytosis in the 1930s [213, 214]. However, it is still available in over-the- counter medications or present in herbal preparations in certain countries [215] and can be taken at doses of as much as 4 g per day [216]. The incidence of aminopyrine- induced agranulocytosis varies broadly from country to country, estimated at 1:2,000 to

1:1,000,000 [217, 218]. The basis for this variation is unknown.

CH CH3 3 N N N N CH CH3 3

O O N N CH CH3 H C 3 H3C 2 NaO3S Aminopyrine

4-(dimethylamino)-1,5-dimethyl-2-phenylpyrazol-3-one Dipyrone

C13H17N3O Mol. Wt. 231.29 g/mol

Figure 1.14. Chemical structures of aminopyrine and dipyrone.

64 Aminopyrine is metabolized by liver P450s to four main metabolites in humans:

4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine, and 4- formylaminoantipyrine [219]. It is also known to be oxidized by peroxidases to a cation radical. It is also oxidized by hypochlorous acid to a reactive species, likely a dication, that coproportionates to two radical cations (Figure 1.7) [81]. This reactive dication was demonstrated to form by activated neutrophils and could be responsible for aminopyrine- induced agranulocytosis [81].

The mechanism of aminopyrine-induced agranulocytosis is still not yet fully understood, but several studies found strong evidence to suggest that it is immune- mediated. Human studies done in the 1950s found a drug-dependent antineutrophil antibody [220]. It has been shown that the in vitro proliferation of myeloid progenitor cells (CFU-GM) is inhibited by the serum from sensitized patients when aminopyrine is also present in the marrow cell cultures [221]. CFU-GM growth was not inhibited by either the drug alone, nor the drug combined with normal serum, or with the sensitized patient’s serum alone [212]. It is likely that the reactive dication metabolite of aminopyrine is involved in the induction of this drug-dependent antibody-mediated IDR.

Unfortunately, there is presently no animal model or in vitro system that can be used to further explore the mechanism.

1.5.2.1.3 Amodiaquine-Induced Agranulocytosis

Amodiaquine is a 4-aminoquinoline compound (Figure 1.15) effective for the treatment and prevention of malaria [222]. It was synthesized in the late 1940s [223] and was found in the 1980s to be particularly effective against some species of chloroquine- resistant Plasmodium flaciparum malaria infections [224]. However, its association with idiosyncratic agranulocytosis and hepatotoxicity have severely restricted its use. The

65 incidence of amodiaquine-induced agranulocytosis was 1/2000 patients during prophylactic treatment, and this resulted in the drug’s withdrawal for use as prophylaxis

[225, 226], but is still widely used in countries in Africa.

OH C2H5

CH2N

C2H5

NH

Cl N

4-[(7-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)phenol

C20H22ClN3O Mol. Wt. 355.86 g/mol

Figure 1.15. Chemical structure of amodiaquine.

The mechanism of amodiaquine-induced agranulocytosis remains unclear. This drug undergoes extensive bioactivation to a reactive electrophilic quinoneimine metabolite in vivo in rats [227] and in vitro by both hepatic microsomes [228]and phorbol ester-stimulated neutrophils [229] (Figure 1.7). Subsequent oxidative stress or covalent binding of this reactive metabolite to proteins is likely involved in the proposed cytotoxicity or immune-mediated mechanisms of amodiaquine-induced agranulocytosis

[230, 231]. This IDR appears to be immune-mediated because anti-amodiaquine antibodies have been detected in sera from patients who had experienced serious adverse reactions to the drug [232] and also in rats treated with the drug [231]. However, the

66 evidence for toxicity of amodiaquine to neutrophils or their precursors is conflicting

[233-235].

1.5.2.2 Drug-Induced Idiosyncratic Aplastic Anemia

Aplastic anemia is manifested as a deficiency of red blood cells, monocytes, neutrophils, and platelets in the blood. There is an absence of hematopoietic precursors in the bone marrow which are replaced by fat cells. This marked diminution of blood cell production can occur as a result of: direct toxicity to hematopoietic stem cells, a defect in the stromal microenvironment of the marrow required for hematopoietic cell development, impaired production or release of essential hematopoietic growth factors, and cellular or humoral immune suppression of marrow progenitor cells [236]. The occurrence of aplastic anemia appears to be idiosyncratic with many drugs. An example of a drug that causes this IDR is felbamate. Its use as an anticonvulsant has been limited because of its associated risk of aplastic anemia or hepatotoxicity in about 1/10,000 patients [237]. Felbamate is metabolized to a reactive Michael acceptor, namely atropaldehyde, which was found to be toxic to bone marrow cells [238]. No in vitro assay would predict the bone marrow toxicity of this drug because the initial step in the bioactivation does not seem to occur in the liver [237].

1.5.2.3 Drug-Induced Idiosyncratic Thrombocytopenia

Thrombocytopenia is defined as a platelet count of less than 150 x 10 9/L in the blood. A low platelet count could result from peripheral destruction by an immune or nonimmune-mediated mechanism, decreased production resulting from an inherited or acquired bone marrow disease, or splenic pooling [239]. An example of a drug that is

67 known to cause this IDR is heparin. This drug binds to platelet factor 4, and in some patients, this leads to the production of an antibody against this complex that causes aggregation and depletion of platelets [240].

1.6 Research Focus and Rationale

Despite their significance, our mechanistic understanding of IDRs is minimal.

The goal of this research is to develop and use animal models to study the mechanism of one type of IDR, namely agranulocytosis.

Chapters 2, 3 and 4 of this thesis are focused on attempts to develop an animal model of idiosyncratic drug-induced agranulocytosis with aminopyrine, amodiaquine, and clozapine by manipulating various aspects of the proposed mechanisms of IDRs.

Because the normal neutrophil count varies significantly with species we could not use the definition of agranulocytosis used for humans. Therefore, we used a decrease in the neutrophil count to less than 20% of the count prior to drug treatment. Chapter 5 describes various mechanistic studies to elucidate the mechanism of clozapine-induced agranulocytosis.

68

CHAPTER 2

ATTEMPTS TO DEVELOP AN ANIMAL MODEL OF DRUG-INDUCED AGRANULOCYTOSIS

69 2.1 Abstract

Animal models are an essential tool in understanding the mechanisms of idiosyncratic drug reactions (IDRs). They are indispensable in the study of IDRs because many hypotheses can only be tested in an in vivo system. Unfortunately, there are very few animal models available for IDRs. In fact, there are no practical animal models for the study of drug-induced agranulocytosis. Therefore, the first part of this thesis research was devoted to the development of an animal model of drug-induced agranulocytosis. Many attempts were pursued in order to develop a rabbit or rodent model of aminopyrine and amodiaquine-induced agranulocytosis by manipulating certain aspects of the proposed mechanisms of IDRs. We began by stimulating neutrophils in rats with the administration of a phorbol ester, PMA, in order to induce the production of more drug reactive metabolites. We then “tweaked” the immune system with an immunostimulant and even tried to break immune tolerance by inhibition of the tryptophan metabolism pathway. However, these attempts did not result in an animal model of this IDR.

70 2.2 Introduction

Animal models have always been an essential tool in biomedical research.

They allow investigation of disease states in ways which would be impossible in humans.

The criteria for a viable animal model depends on how useful it is, which in turn depends on how well it resembles the disease or condition in terms of the clinical, morphological, biochemical, and functional features of the specific disease. But more importantly, especially for the use of animal models in the study of IDRs, the basic mechanism involved in the adverse reaction seen in the animal should be very similar to that of the adverse reaction that occurs in humans [128]. Drugs often cause biochemical changes without causing clinical disease, and it is important to understand what causes some patients to develop a clinical adverse reaction. Therefore, it is also important that a useful animal model should present some biological endpoint that is analogous to the adverse reaction in humans [128]. Often times in vitro studies do not reflect the conditions in vivo . Studies with humans, especially prospective studies, are virtually impossible because of the low incidence of these reactions as well as the ethically issues of exposing susceptible patients to the drug. Obtaining human tissue samples for research analyses often also presents practical and technical limitations. Retrospective studies with human patients are more feasible, but they do not permit the study of events leading up to the IDR. More importantly, in vitro systems are inadequate for the study of the involvement of the immune system in the mechanisms of IDRs. Assays with isolated tissues and cells simply cannot mimic the complexicity of the whole immune system in an animal. Therefore, it is difficult to gain a better understanding of the mechanistic basis of these reactions without the use of animal models.

Animal models of IDRs are rare because these reactions are just as idiosyncratic in animals as they are in humans. Thus most animals models used in the

71 study of IDRs have been found by serendipity. Although there have been case reports from veterinarians of domestic animals such as cats and dogs suffering from IDRs similar to humans, practical issues prevent the routine use of these models, especially when the incidence of the IDR is low [128]. So far, our lab has worked extensively with only two rodent models of IDRs, the penicillamine-induced autoimmunity in the Brown

Norway rats and the nevirapine-induced skin rash in rats, which were described earlier in

Chapter 1. Unfortunately, there are still no practical animal models of drug-induced idiosyncratic blood dyscrasias, particularly agranulocytosis.

This chapter of the thesis will describe all the attempts I have made in trying to develop an animal model of drug-induced idiosyncratic agranulocytosis. The main objective of these studies was to use leukocyte count, especially neutrophil count, as an end-point to assess whether aminopyrine or amodiaquine, which are associated with agranulocytosis in humans, can induce the same pathological condition in the rats or rabbits. It should be mentioned that the neutrophils in rabbits and guinea pigs are known as heterophils, which are the lapine and cavian equivalent of neutrophils. Heterophils have the same function as other mammalian neutrophils, but they have acidophilic or eosinophilic granules in their cytoplasm. From here on the heterophiles will also be referred to as neutrophils for simplicity.

The mechanism of IDRs remains obscure, much like a complicated jigsaw puzzle, where the full picture is not revealed until most or all the puzzle pieces have been put together. So far, we have just begun to discover some of these puzzle pieces, those being reactive metabolites, the immune system, and host factors. The picture which is revealed as they are put together seems to provide us with a generalized proposed mechanism of IDRs as described in the first chapter. We have tried various interventions

72 along this proposed mechanism in order to develop an animal of drug-induced agranulocytosis.

We began with a study in which the production of reactive metabolites was manipulated. Reactive metabolites, as mentioned earlier in the introduction, is likely to be involved in the induction of IDRs either by acting as haptens or causing cell damage.

More specifically, we tested the hypothesis that an increase in reactive metabolite production by stimulated neutrophils would lead to agranulocytosis in amodiaquine- treated rats. As mentioned earlier, it is likely that the reactive metabolites involved in drug-induced agranulocytosis are produced at the target site, in this case is neutrophils or their precursors. By stimulating an oxidative burst in the neutrophils, we can increase the formation of reactive metabolites of drugs which are metabolized by neutrophils.

Amodiaquine undergoes bioactivation to a reactive electrophilic quinoneimine metabolite by phorbol ester-stimulated neutrophils [229]. Subsequent oxidative stress or covalent binding of this reactive metabolite to proteins is likely involved in the proposed cytotoxicity or immune-mediated mechanisms of amodiaquine-induced agranulocytosis

[230, 231]. In the induction of an oxidative burst, PKCs directly activate NADPH oxidase assembly by phosphorylating cytosolic phox proteins [153, 241]. PKCs can be activated by phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (PMA or TPA)

[242, 243]. PMA is a powerful activator of neutrophils and has been used extensively to stimulate an oxidative burst [152, 153, 241, 244-250]. We chose to use rats as the model species for amodiaquine studies because it was reported by Clarke et al. that administration of the drug to rats induced IgG anti-amodiaquine antibodies [231]. In addition, their study showed a significant decrease in the peripheral white blood cell count after the fourth dose of amodiaquine treatment. This seemed like a good starting point for further studies.

73 In addition to reactive metabolites, the immune system also seems to have a significant role in the induction of IDRs. According to the proposed mechanism of IDRs, the induction of an immune response requires both signal 1, which is the interaction of

MHC complex on the antigen-presenting cells with the T cell receptor, and signal 2, which is the interaction of co-stimulatory receptors on these cells. Therefore, agents that increase signal 2 should increase the risk of an immune response, and in this case idiosyncratic drug-induced agranulocytosis. Poly I:C is a synthetic double-stranded ribonucleic acid consisting of a pair of strands of poly-inosinic and poly-cytidylic acids structurally similar to viral RNA found in some viruses but not in mammalian cells. It is a potent inducer of type I interferon in vitro and in vivo by interacting with Toll-like receptor 3 expressed on B-cells and dendritic cells [251-253]. Low dose Poly I:C can also activate T cells, natural killer cells, and dendritic cells, and induce the release of cytokines such as interleukins (IL2, IL6), corticosteroids, and tumor necrosis factor [253].

A single dose of poly I:C injection been shown to be capable of significantly increasing the occurrence and accelerated the onset of D-penicillamine-induced lupus in the Brown

Norway rat in our lab [254]. In this study, rabbits were treated with aminopyrine, another drug known to cause immune-mediated idiosyncratic agranulocytosis in humans along with Poly I:C. Rabbits were chosen as the model animal for these studies because of an earlier study that found that aminopyrine caused a marked decrease in white blood cell count in some rabbits treated with the drug [213]. We have also performed this experiment using the Brown Norway rats because successful models of drug-induced autoimmunity and IDRs have been obtained using this strain of rats [128]. To further test this hypothesis, we have also conducted an experiment where we had packaged the

Poly I:C along with dipyrone, the sodium sulfonate derivative of aminopyrine, into liposomes and injected them into the rats. Packaging both the immunostimulant and the

74 offending drug into lipsomes would facility uptake by phagocytic cells such as macrophages which are the antigen-presenting cells required in initiating an immune response. This also allows the immunostimulation to be physically linked with the offending drug such that both of these may be presented to the antigen presenting cells

(APCs) simultaneously.

One possible reason that it is difficult to develop animal models of IDRs is because IDRs are quite injurious and therefore in most individuals the immune response is tolerance. Therefore, overcoming immune tolerance could be the key to developing an animal model. There is strong evidence that dendritic cells, the major APC, have a crucial role in peripheral T cell tolerance [255]. Recently, certain APCs have been found to express the enzyme indoleamine 2,3-dioxygenase (IDO). IDO catalyzes the initial and rate-limiting step of tryptophan degradation [256]. The local degradation of tryptophan has been shown to be capable of modulating T cell activity, specifically by depleting tryptophan which is required for T cell proliferation (Figure 2.1) [255]. Recent studies have shown that regulatory T cells can induce activation of IDO in dendritic cells [257].

However, it is not certain at this point whether the hindrance of T cell proliferation is due to the depletion of tryptophan or the increase in its degradation products [258].

Regulation of T cell survival through tryptophan depletion seems to play a part in many physiologic and pathophysiologic conditions [259]. IDO + dendritic cells have been detected preferentially and in advance of tumor cells in metastic lymph nodes, which suggest that IDO may be involved in tumor immune escape. The depletion of tryptophan by IDO had also been shown to be involved in antibacterial defense mechanisms. IDO was also described as crucial in establishing tolerance (inhibit maternal T cell immunity to fetal tissues) during pregnancy [256, 260]. Several recent mechanistic studies from a range of mouse models demonstrate that IDO regulates adaptive T cell immunity [261]. A

75 pharmacological inhibitor of IDO, 1-methyl-D-tryptophan (1-MT), was used in many of these mice studies. Injection of 1-MT to pregnant mice resulted in rejection of the allogenic fetuses [262]. 1-MT treatment also exacerbated symptoms of certain autoimmune disease models [261]. We performed two experiments, one with aminopyrine and the other with amodiaquine in which the animals were co-treated with 1-MT.

Dendrtic Cell

Treg Tryptophan IDO Kynurenine

Tryptophan

Teff proliferation

apoptosis

Immune Tolerance

Figure 2.1. Tryptophan metabolism and immune tolerance.

76 2.3 Materials and Methods

2.3.1 Chemicals

Aminopyrine, dipyrone, and amodiaquine were purchased from Sigma-Aldrich

(Oakville, ON). Polyethylene glycol (PEG) 200, phorbol 12-myristate-13-acetate (PMA), dimethyl sulfoxide (DMSO), ethylenediamine-tetraacetic acid disodium salt (EDTA), polyinosinic:polydytidylic acid potassium salt (Poly I:C), and 1-methyl-DL-tryptophan were purchased from Sigma-Aldrich (Oakville, ON). Phosphate buffered saline (PBS, 10 mM, pH 7.4) and RPMI 1640 culture medium were prepared by University of Toronto

Media Services.

2.3.2 Animals

Female rats (150-175 g) of the Sprague Dawley and Brown Norway strains and female rabbits (1.25-2.5 kg) of the New Zealand White strain were purchased from

Charles River (Montreal, QC). The rats were housed in pairs in plastic cages with corncob bedding in a 12:12 h light:dark cycle at 22oC. Rabbits were housed singly in metal cages under similar conditions. Animals were given either the regular lab rodent chow diet (powdered or pellets) or rabbit diet. Daily food and water intake were monitored. All of the animals were given drinking water which was deionized water filtered through a reverse osmosis system. All animals were acclimatized for 1 week prior to the commencement of each experiment which was approved by University of

Toronto’s animal care committee.

77 2.3.3 Drug Treatments

In each study animals in the different treatment groups were given drug treatments as follows:

• Amodiaquine-treated Sprague Dawley rats

Group: A: amodiaquine only (n=3)

AP: amodiaquine with PMA (n=3)

PC: PEG vehicle only (n=3)

P: PEG vehicle with PMA (n=1)

In the groups that received amodiaquine, the drug was suspended in PEG (0.25

g/ml) and injected subcutaneously daily (0.25 g/kg) for 10 days. PMA was

dissolved in DMSO (0.25 µg/0.2 ml) and injected intraperitoneally (0.2 ml) every

week for a total of 2 doses. In the control group, only PEG was injected

subcutaneously at 0.25 ml for 10 days.

• Aminopyrine-treated rabbits

Group: Aminopyrine (n= 4)

Untreated Control (n = 1)

Aminopyrine was given at an approximate dose of 200 mg/kg/day orally by

dissolving it in the drinking water.

• Aminopyrine-treated rabbits with poly I:C co-treatment

Group: Aminopyrine with Poly I:C (n=4, Rabbits 1,2,3)

Aminopyrine Alone (n =1, Rabbit 4)

Untreated Control (n=1, Rabbit 5)

In the groups that received aminopyrine, the drug was administered at 200

mg/kg/day orally by dissolving into drinking water. Poly I:C was administered at 5

mg/kg/three times per week by subcutaneous injections to four rabbits, initially in

78 the Aminopyrine with Poly I:C group. However, due to toxicity in the poly I:C

rabbits, in which the decrease in food and water consumption became significant,

administration of poly I:C was discontinued after 1 week and administered again to

only one of the rabbits (i.e. Rabbit 2) at a much lower dose of 1 mg/kg/three times

per week.

• Aminopyrine-treated Brown Norway rats with poly I:C co-treatment

Group: Aminopyrine Alone (n = 4)

Aminopyrine with Poly I:C (n=3)

Aminopyrine was administered at 200 mg/kg/day orally by mixing into rodent

chow. Poly I:C was dissolved in PBS (4.8 mg/ml) and injected i.p. three times a

week at a dose of 5 mg/kg.

• Aminopyrine-treated rats with poly I:C co-treatment using liposomes

Group: Dipyrone with Poly I:C Liposomes (Brown Norway n=2, Sprague

Dawley n=2)

Empty Liposomes (PBS filled) (Brown Norway, n=2; Sprague

Dawley, n=2)

Dipyrone was given to the treated group both orally through the diet at 800

mg/kg/day and in liposomes at approximately 250 mg/kg/day along with poly I:C

at 10 mg/kg/day. Liposomes containing dipyrone and poly I:C or empty ones

(containing PBS) were given to each rat as a suspension in a volume of 1 ml by

intraperitoneal injections 3 times a week for 3 weeks. The first injection was given

at day 1 of the dipyrone-dosed diet. After 3 weeks of liposome injections the rats

remained on the dipyrone diet for 3 more weeks.

• Aminopyrine and amodiaquine-treated rats with 1-MT co-treatment

Group: AP: 1-MT with Aminopyrine (n=2)

79 AM: 1-MT with Amodiaquine (n=2)

CON: 1-MT Alone (n=2)

Aminopyrine at a dose of 800 mg/kg/day or amodiaquine at dose of 200 mg/kg/day

was given orally to the rats in the treated groups by mixing it in the rodent chow. 1-

MT (20 mg/day) was injected intraperitoneally daily for 3 weeks, the first dose was

given on the first day the drug was administered. A suspension was made with 4%

starch solution because 1-MT was insoluble in aqueous solution at neutral pH.

2.3.4 Preparation of Liposome Entrapped Dipyrone and Poly I:C

Multilamellar liposomes were prepared using the method described by Van

Rooijen and Claassen [263]. Briefly, phosphatidylcholine (86 mg) and cholesterol (8 mg) at a molar ratio of 6:1 were dissolved in 10 ml of chloroform in a 500 ml round bottom flask. A thin film was formed along the interior of the flask by low vacuum rotary evaporation at 37 °C. 10 ml of dipyrone and poly I:C solution (200 mg dipyrone and 8 mg poly I:C dissolved in 10 ml of sterile 10 mM PBS) was gently swirled in the flask for 10 min, dispersing the thin film. Control liposomes were made by dispersing the thin film with 10 ml of PBS. Liposomes were then incubated under nitrogen at room temperature for 2 h to allow swelling, followed by sonication for 5 min for separation.

They were stored under nitrogen at 4 °C overnight. The next day, free dipyrone and poly

I:C were removed by centrifuging the liposome mixture at 10,000 g for 30 min.

Liposomes formed a white band at the top of the suspension, and the solution underneath was removed using a Pasteur pipette. The liposomes were then washed three times using

30 ml of sterile PBS and centrifuged at 10,000 g for 15 min. They were resuspended in 4 ml of sterile PBS for injection.

80 2.3.5 Blood Sampling

Peripheral blood samples were collected from each animal at various time points.

They were obtained from the tail vein in rats and from the marginal ear vein in rabbits.

Blood samples of no more than 200 µl per week from each animal were collected with a

25G needle and Microvettes® CB 300 Kalium-EDTA capillary tubes (Sarstedt, Montreal,

QC) from the rats or with a 25G Vacutainer® blood collection set (butterfly needle, BD

Biosciences, Mississauga, ON) connected to a EDTA-coated syringe (0.05 µl at 1.5%) or

EDTA Vacutainer® (BD Biosciences, Mississauga, ON).

2.3.6 Blood Cell Counts

Peripheral blood cell counts were obtained immediately after blood samples were collected. Total white blood cell counts were obtained by manual counting using an inverted light microscope and Turk’s solution to lyse the red blood cells and to dilute the samples (1:10). Cell differential counts were obtained by manually counting white blood cell percentages on blood smear slides stained with Giemsa-Wright stain. Cell counts were performed in triplicate to obtain standard deviations.

2.3.7 Serum ALT Level Assessment

Serum (100 µl) was obtained from blood drawn from the tail vein into untreated microvettes. Serum alanine aminotransferase (ALT) levels were assayed with the

Infinity TM ALT (GPT) reagent from Thermo Electron Corp. (Melbourne, Australia) using the UV method of Frankel and Reitman.

81 2.3.8 Liver Histology

All rats were sacrificed on day 14 and their livers were harvested. These were then stained with haematoxylin and eosin to investigate liver pathology.

2.3.9 Statistical Analysis

Statistical analyses were performed using GraphPad Prism (GraphPad, San Diego,

CA). Unpaired t tests (two tailed, 95% confidence interval) were used to compare the treatment groups. Data were expressed as the mean ± s.d. and results were considered statistically significant if P< 0.05.

82 2.4 Results

2.4.1 Amodiaquine-treated Sprague Dawley Rats

We were unable to administer amodiaquine through daily subcutaneous injections in the rats for longer than ten days. In fact, one of the rats in the PMA drug co-treatment group was sacrificed after the fifth dose due to apparent toxicity. We had found that daily subcutaneous injections of amodiaquine at 0.25 g/kg (538 µmol/kg) resulted in general malaise and weight loss in the rats. The rats did not tolerate the drug very well at this dose after the fifth day. In the autopsies of some of the rats given amodiaquine we had found that most of the drug was accumulating underneath the skin. Thus, we were not certain if most of the administered drug administered was absorbed into the systemic circulation. We were not able to extend the treatment for longer than 10 days.

We found that amodiaquine treatment after the second dose produced an increase in the absolute and percent neutrophil counts, but decreased after the fourth dose although cell counts were still higher than pre-treatment levels (Figure 2.3 and 2.4).

Then a gradual increase in leukocyte and neutrophil counts were observed in some of the treated rats after the eighth dose. We did not observe any significant decrease in leukocyte counts as described in Clarke’s study (Figure 2.2).

Amodiaquine appears to cause liver injury in rats as noted by liver histology which showed degenerative fatty infiltration and by the serum ALT levels, which tended to be high in the treatment group (Figure 2.5). Drug-induced degenerative changes varied in severity in the treatment groups. In Group A, mild to moderate steatosis was noted in 2/3 rats. In Group AP, mild to severe steatosis was noted in 2/3 rats. In Group

PC, liver histology was unremarkable in 3/3 rats. In Group P, liver histology was unremarkable in 1/1 rat. There was a rough correlation between the pathology seen and

83 the ALT levels measured. While drug-induced steatosis was observed, hepatic necrosis was not evident.

Total White Blood Cell Count

30 A1 A2 A3 25 AP1 AP2 AP3 20 PC1 PC2 PC3 P1 ) / ml blood

6 6 15

10 Cellsx ( 10

5

0 0 2 4 6 8 10 12 14 16 Days

Figure 2.2. Total white blood cell counts of amodiaquine-treated Sprague Dawley rats. Treatment groups were A (amodiaquine only, n=3), AP (amodiaquine with PMA, n=3), PC (PEG only n=3) and P (PMA only, n=1). Amodiaquine (0.25 g/ml suspended in PEG 200) was injected subcutaneously daily (0.25 g/kg) for 10 days. PMA (0.25 µg/0.2 ml in DMSO) was injected intraperitoneally (0.2 ml) every week for a total of 2 doses. PEG 200 (the vehicle) was injected in subcutaneously daily (0.25 ml) for 10 days. Values represent the total white blood cell counts from each rat from blood samples were taken from the tail vein on day 0, 1, 3, 7, 10 and 14 of the amodiaquine treatment.

84

Neutrophil Percentage

90

80

70 A1 A2 60 A3 AP1 50 AP2 AP3 PC1

Percent 40 PC2 PC3 30 P1

20

10

0 0 2 4 6 8 10 12 14 16 Days

Figure 2.3. Neutrophil percentages of amodiaquine-treated Sprague Dawley rats. Treatment groups were A (amodiaquine only, n=3), AP (amodiaquine + PMA, n=3), PC (PEG only n=3) and P (PMA only, n=1). Amodiaquine (0.25 g/ml suspended in PEG 200) was injected subcutaneously daily (0.25 g/kg) for 10 days. PMA (0.25 µg/0.2 ml in DMSO) was injected intraperitoneally (0.2 ml) every week for a total of 2 doses. PEG 200 (the vehicle) was injected in subcutaneously daily (0.25 ml) for 10 days. Values represent the neutrophil percentages from each rat from blood samples taken on days 0, 1, 3, 7, 10 and 14 of the amodiaquine treatment.

85 Absolute Neutrophil Count

25 A1 A2 A3 AP1 20 AP2 AP3 PC1 PC2 15 PC3 P1 ) / ml blood 6

10 Cellsx 10 (

5

0 0 2 4 6 8 10 12 14 16 Days

Figure 2.4. Neutrophil counts of amodiaquine-treated Sprague Dawley rats. Treatment groups were A (amodiaquine only, n=3), AP (amodiaquine + PMA, n=3), PC (PEG only n=3) and P (PMA only, n=1). Amodiaquine (0.25 g/ml suspended in PEG 200) was injected subcutaneously daily (0.25 g/kg) for 10 days. PMA (0.25 µg/0.2 ml in DMSO) was injected intraperitoneally (0.2 ml) every week for a total of 2 doses. PEG 200 (the vehicle) was injected in subcutaneously daily (0.25 ml) for 10 days. Values represent the peripheral absolute neutrophil counts from each rat on days 0, 1, 3, 7, 10 and 14 of the amodiaquine treatment.

86 Serum ALT Levels

350 AP1 AP2 300 AP3 A1 A2 250 A3 P1

200 PC1 PC2 PC3 150 ALT(Units/L)

100

50

0 0 2 4 6 8 10 12 14 16 Day

Figure 2.5. Serum ALT levels of amodiaquine-treated Sprague Dawley rats. Treatment groups were A (amodiaquine only, n=3), AP (amodiaquine + PMA, n=3), PC (PEG only n=3) and P (PMA only, n=1). Amodiaquine (0.25 g/ml suspended in PEG 200) was injected subcutaneously daily (0.25 g/kg) for 10 days. PMA (0.25 µg/0.2 ml in DMSO) was injected intraperitoneally (0.2 ml) every week for a total of 2 doses. PEG 200 (the vehicle) was injected in subcutaneously daily (0.25 ml) for 10 days. Values represent the serum ALT levels in each rat from blood samples taken on days 0, 1, 3, 7,10 and 14 of the amodiaquine treatment.

87 2.4.2 Aminopyrine-treated Rabbits

Leukocytosis was observed in all three aminopyrine-treated rabbits after 20 days, with one of the rabbits (Aminopyrine 3) presenting with an unusual spike in the total white cell and absolute neutrophil count on the 26 th day (Figure 2.6). However, a depression in the white cell and neutrophil counts was not observed as described by

Hoffman et al. following the leukocytosis (Figure 2.6, 2.7 and 2.8). The weights and feed intake were similar in all groups, but water consumption was slightly lower in the aminopyrine-treated rabbit presumably due to bitterness of aminopyrine. No visual difference or general malaise was observed in any of the rabbits.

Total White Blood Cell Count

14

12

10

8 Aminopyrine 1

) ml / blood Aminopyrine 2

6 Aminopyrine 3 6 Control

Cells (10 Cells x 4

2

0 0 5 10 15 20 25 30 35 40 45 50 Days

Figure 2.6. Total white blood cell counts of aminopyrine-treated rabbits. Treatment groups were Aminopyrine (n=3) and Control (n=1). Aminopyrine was administered at 200 mg/kg/day orally by dissolving it in the drinking water. Values represent the total white blood cell counts during aminopyrine treatment.

88

Neutrophil Percentage

40

35

30

25 Aminopyrine 1 Aminopyrine 2 20 Aminopyrine 3 Percent Control 15

10

5

0 0 5 10 15 20 25 30 35 40 45 50 Days

Figure 2.7. Neutrophil percentages of aminopyrine-treated rabbits. Treatment groups were Aminopyrine (n=3) and Control (n=1). Aminopyrine was administered at 200 mg/kg/day orally by dissolving it in the drinking water. Values represent the neutrophil percentages during aminopyrine treatment.

89 Absolute Neutrophil Count

80

70

60

50 Aminopyrine 1

) / ml ml ) / blood Aminopyrine 2

6 40 Aminopyrine 3 Control 30 Cells ( x 10 (Cells x 20

10

0 0 5 10 15 20 25 30 35 40 45 50 Days

Figure 2.8. Neutrophil counts of aminopyrine-treated rabbits. Treatment groups were Aminopyrine (n=3) and Control (n=1). Aminopyrine was administered at 200 mg/kg/day by dissolving it in the drinking water. Values represent the absolute peripheral neutrophil counts during aminopyrine treatment.

2.4.3 Aminopyrine-treated Rabbits with Poly I:C Co-treatment

A dramatic increase in leukocyte counts, especially in neutrophils, was observed in all of the rabbits given poly I:C in the first ten days (Figure 2.9 and 2.11). No decreasing or increasing trends in leukocyte and neutrophil counts or percentages were observed other than those presumably brought about by poly I:C (Figure 2.9, 2.10 and

2.11). Leukocyte and neutrophil counts in all treated rabbits were similar to and never significantly lower than the control rabbit. The weights, food intake, and water consumption were much lower than the control animal when they were given poly I:C.

90 Rabbits appeared ill. However, subsequent to the cessation of poly I:C or a decrease in

dose to 1 mg/kg, rabbits regained normal food intake and reasonable water consumption

(the volume was still lower than the control rabbit). Growth (in terms of increase in

body weight) was stunted in rabbits treated with poly I:C. No visual difference was

otherwise observed in any of the rabbits.

Total White Blood Cell Count

14

12

10

Rabbit 1 8 Rabbit 2 ) / ml ) blood ml /

9 Rabbit 3 6 Rabbit 4 Rabbit 5

4 Cells 10 ( Cells x

2

0 0 10 20 30 40 50 60 70 Days Poly I:C Poly I:C given to Rabbit 2 only

Figure 2.9. Total white blood cell counts of aminopyrine-treated rabbits with poly I:C co-treatment. Treatment groups were: Aminopyrine with Poly I:C (n=4, Rabbit 1, 2, 3); Aminopyrine Alone (n =1, Rabbit 4) and Untreated Control (n=1, Rabbit 5). Aminopyrine was administered at 200 mg/kg/day by dissolving it in the drinking water. Poly I:C was initially administered at 5 mg/kg/three times per week by subcutaneous injections to four rabbits. However, due to decreased weight in the poly I:C rabbits, administration of poly I:C was discontinued after 1 week and administered again to only one of the rabbits (i.e. Rabbit 2) at a much lower dose of 1 mg/kg/three times per week. Values represent the total white blood cell counts from each rabbit from blood samples taken twice a week from the marginal ear vein during the aminopyrine treatment.

91

Neutrophil Percentage

90

80

70

60

Rabbit 1 50 Rabbit 2 Rabbit 4

Percent 40 Rabbit 3 Rabbit 5 30

20

10

0 0 10 20 30 40 50 60 70 Day Poly I:C Poly I:C given to Rabbit 2 only

Figure 2.10. Neutrophil percentages of aminopyrine-treated rabbits with poly I:C co-treatment. Treatment groups were: Aminopyrine with Poly I:C (n=4, Rabbit 1, 2, 3); Aminopyrine Alone (n =1, Rabbit 4) and Untreated Control (n=1, Rabbit 5). Aminopyrine was administered at 200 mg/kg/day by dissolving it in the drinking water. Poly I:C was initially administered at 5 mg/kg/three times per week by subcutaneous injections to four rabbits. However, weight loss in the poly I:C rabbits, administration of poly I:C was discontinued after 1 week and administered again to only one of the rabbits (i.e. Rabbit 2) at a much lower dose of 1 mg/kg/three times per week. Values represent the neutrophil percentages from each rabbit from blood samples taken during the aminopyrine treatment.

92 Absolute Neutrophil Count

90

80

70

60 Rabbit 1 50 Rabbit 2 ) blood / ml 8 Rabbit 3 40 Rabbit 4 Rabbit 5 30 Cells ( x 10 ( Cells x

20

10

0 0 10 20 30 40 50 60 70 Days Poly I:C Poly I:C given to Rabbit 2 only

Figure 2.11. Neutrophil counts of aminopyrine-treated rabbits with poly I:C co- treatment. Treatment groups were: Aminopyrine with Poly I:C (n=4, Rabbit 1, 2, 3); Aminopyrine Alone (n =1, Rabbit 4) and Untreated Control (n=1, Rabbit 5). Aminopyrine was administered at 200 mg/kg/day by dissolving it in the drinking water. Poly I:C was initially administered at 5 mg/kg/three times per week by subcutaneous injections to four rabbits. However, due to weight loss in the poly I:C rabbits, poly I:C was discontinued after 1 week and administered again to only one of the rabbits (i.e. Rabbit 2) at a much lower dose of 1 mg/kg/three times per week. Values represent the peripheral absolute neutrophil counts from each rabbit from blood samples taken twice a week from the marginal ear vein during the aminopyrine treatment.

2.4.4 Aminopyrine-treated Brown Norway Rats with Poly I:C Co-treatment

There was a slight downward trend in neutrophil percentages and absolute

counts in the treatment group given aminopyrine alone for the first 20 days, whereas in

93 the aminopyrine with Poly I:C co-treatment group, the downward trend was only observed starting at day 15 of the treatment (Figure 2.13 and 2.14). However, this downward trend did not persist in both of the treatment groups and seemed to follow by a slight upward trend. No significant changes were observed in the total white blood cell counts (Figure 2.12).

The weights and food intake were similar in all groups. No visual difference was observed in any of the rats. However, after 28 days of treatment one of the rats in the poly I:C with aminopyrine treatment group died. The cause of death was not due to aminopyrine-induced agranulocytosis since the rat did not show a decreasing trend in the absolute white cell counts nor in the neutrophil counts prior to its death.

94

Mean Total White Blood Cell Count

25

20 PolyIC + aminopyrinopyrine

Aminopyrine Alone 15 ) / ml blood 6 10

Cells ( x 10 5

0 0 10 20 30 40 50 Days

Figure 2.12. Total white blood cell counts of aminopyrine-treated Brown Norway rats with poly I:C co-treatment. Treatment groups were Aminopyrine Alone (n=4) and Aminopyrine with Poly I:C (n=3). Aminopyrine was administered at 200 mg/kg/day by mixing it in the rodent chow. Poly I:C was dissolved in PBS (4.8 mg/ml) and injected i.p. three times a week at a dose of 5 mg/kg. Values represent the mean total white blood cell counts with standard deviations of each treatment group from blood samples taken during the aminopyrine treatment.

95

Mean Neutrophil Percentage

50

40 Poly IC + Aminopyrine Aminopyrine Alone

30

Percent 20

10

0 0 10 20 30 40 50 Days

Figure 2.13. Neutrophil percentages of aminopyrine-treated Brown Norway rats with poly I:C co-treatment. Treatment groups were Aminopyrine Alone (n=4) and Aminopyrine with Poly I:C (n=3). Aminopyrine was administered at 200 mg/kg/day by mixing it in the rodent chow. Poly I:C was dissolved in PBS (4.8 mg/ml) and injected i.p. three times a week at a dose of 5 mg/kg. Values represent the mean neutrophil percentages with standard deviations of each treatment group from blood samples taken during the aminopyrine treatment.

96

Mean Absolute Neutrophil Count

50 PolyIC + aminopyrine

Aminopyrine Alone 40

30 ) / ml blood 5

20

Cells ( 10 x 10

0 0 10 20 30 40 50 Days

Figure 2.14. Neutrophil counts of aminopyrine-treated Brown Norway rats with poly I:C co-treatment. Treatment groups were Aminopyrine Alone (n=4) and Aminopyrine with Poly I:C (n=3). Aminopyrine was administered at 200 mg/kg/day by mixing it in the rodent chow. Poly I:C was dissolved in PBS (4.8 mg/ml) and injected i.p. three times a week at a dose of 5 mg/kg. Values represent the mean peripheral absolute neutrophil counts with standard deviations of each treatment group from blood samples taken during the aminopyrine treatment.

97 2.4.5 Aminopyrine-treated Rats with Liposome Entrapped Poly I:C Co-

treatment

Injection of liposome entrapped dipyrone and poly I:C did not result in any significant changes in the peripheral neutrophil counts (Figure 2.15). Decreasing trends in the neutrophil counts were not observed and values were within the normal range. To ensure that the liposomes did contain enough dipyrone and poly I:C, liposomes were solublized in methanol and the UV absorbance measured. The liposomes contained dipyrone and poly I:C at approximately the intended concentrations.

98

Absolute Neutrophil Count

25

20

) /) blood ml 15 5

DPLipoBN 10 ConLipBN DPLipoSD

Cells x 10 ( ConLipoSD 5

0 0 7 14 21 28 35 42 Days

Liposome Injection Dipyrone Diet

Figure 2.15. Neutrophil counts of dipyrone-treated rats with poly I:C co-treatment using liposomes. Treatment groups were Dipyrone with Poly I:C (DPLipo) (Sprague Dawley rats, SD, n=2; Brown Norway rats, BN, n=2) and Empty Liposomes containing PBS vehicle (ConLipo) (SD n=2; BN n=2). Dipyrone was administered in the diet at 800 mg/kg per day for those rats given the dipyrone with poly I:C liposomes injections. The dose of dipyrone administered by liposomal injections was approximately 250 mg/kg/day. Poly I:C given by liposomal injections was approximately 10 mg/kg/day. Each rat was injected i.p. with either 1 ml of dipyrone and poly I:C liposomes or empty liposomes 3 times a week for 3 weeks. The first injection was given on day 1 of the dipyrone-dosed diet. After 3 weeks of liposome injections the rats remained on the dipyrone diet for 3 more weeks. Values represent the mean peripheral absolute neutrophil counts of each treatment group from blood samples taken during the aminopyrine treatment.

99 2.4.6 Aminopyrine and Amodiaquine-treated Rats with 1-MT Co-treatment

After 70 days of amodiaquine or 80 days of aminopyrine treatment, agranulocytosis was not induced in any of the rats. Both total white blood cell and neutrophil counts were within the normal range (Figure 2.16 and 2.18). However, in the amodiaquine group both rats showed an increase in neutrophil percentage and absolute counts in the first 3 weeks of drug treatment (Figure 2.17).

Total White Blood Cell Count

25

20 ) blood ml/ ) 6 15 CON AP AM

Cells ( x 10 x Cells ( 10

5

0 0 10 20 30 40 50 60 70 80 90 Days

Figure 2.16. Total white blood cell counts of aminopyrine and amodiaquine-treated rats with 1-MT co-treatment. Treatment groups were AP (aminopyrine with 1-MT, n=2), AM (amodiaquine with 1-MT, n=2) and CON (1-MT alone, n=2). Aminopyrine was given orally at 800 mg/kg/day and amodiaquine at 200 mg/kg/day by mixing it in the powdered rodent diet. 1-MT was administered in a suspension of 4% starch solution at 20 mg/day by i.p. injections for 3 weeks; the first dose was given on the first day of aminopyrine or amodiaquine treatment. Values represent the mean total white blood cell counts of each treatment group from blood samples taken during the aminopyrine or amodiaquine treatment.

100

Neutrophil Percentage

45

40

35

30 CON AP 25 AM Percent 20

15

10

5

0 0 10 20 30 40 50 60 70 80 90 Days

Figure 2.17. Neutrophil percentages of aminopyrine and amodiaquine-treated rats with 1-MT co-treatment. Treatment groups were AP (aminopyrine with 1-MT, n=2), AM (amodiaquine with 1-MT, n=2) and CON (1-MT alone, n=2). Aminopyrine was given orally at 800 mg/kg/day and amodiaquine at 200 mg/kg/day by mixing it in the powdered rodent diet. 1-MT was administered in a suspension of 4% starch solution at 20 mg/day by i.p. injections for 3 weeks; the first dose was given on the first day of aminopyrine or amodiaquine treatment. Values represent the mean neutrophil percentages of each treatment group from blood samples taken during the aminopyrine or amodiaquine treatment.

101 Absolute Neutrophil Count

60

50

40 CON AP AM 30 ) / ml blood 5

20 Cells ( x 10

10

0 0 10 20 30 40 50 60 70 80 90

Days

Figure 2.18. Neutrophil counts of aminopyrine and amodiaquine-treated rats with 1-MT co-treatment. Treatment groups were AP (aminopyrine with 1-MT, n=2), AM (amodiaquine with 1-MT, n=2) and CON (1-MT alone, n=2). Aminopyrine was given orally at 800 mg/kg/day and amodiaquine at 200 mg/kg/day by mixing it in the powdered rodent diet. 1-MT was administered in a suspension of 4% starch solution at 20 mg/day by i.p. injections for 3 weeks; the first dose was given on the first day of aminopyrine or amodiaquine treatment. Values represent the mean peripheral absolute neutrophil counts of each treatment group from blood samples taken during the aminopyrine or amodiaquine treatment.

102 2.5 Discussion

In all the studies done in an attempt to develop an animal model of idiosyncratic drug-induced agranulocytosis, neutrophil counts were within the normal range.

Although spikes and fluctuations in neutrophil counts were observed in the animals throughout the drug treatment, persistent increasing or decreasing trends in the neutrophil counts or percentages were not observed in any of the animals with any of the treatments. Such spikes and fluctuations in the neutrophil counts were considered normal because the release of neutrophils from storage pools, particularly demargination, is known to occur during exercise and “fight” sympathetic responses. The handling of the animals could have been enough to induce a stress response.

Although the mechanisms of amodiaquine-induced agranulocytosis and hepatotoxicity remain unclear, both direct cytotoxicity and immune-mediated mechanisms have been implicated [231, 264, 265]. Amodiaquine is metabolized to an electrophilic quinoneimine metabolite in vivo in rats and by hepatic microsomes and phorbol ester-stimulated neutrophils in vitro [227-229]. Oxidative stress induced by this metabolite and/or its conjugation to cysteinyl sulfydryl groups on proteins are thought to be involved in the induction of these idiosyncratic adverse drug reactions [230, 231].

Unfortunately, there are no animal models to demonstrate such IDRs in vivo , but a study conducted by Clarke et. al had found a decrease in leukocyte counts and an increase in serum ALT levels in amodiaquine treated rats [231]. In this study, specific IgG anti- amodiaquine antibodies were also detected after administration of the drug to rats (269

µmol/kg for 4 days). The magnitude of the humoral immune response in terms of antibody titer was in the order intraperitoneal administration>intramuscular administration>oral administration. Anti-amodiaquine antibodies were produced at doses that did not produce direct hepatotoxicity nor leukopenia in the rat. A significant

103 decrease in the peripheral leukocyte count was observed in all rats only after the fourth dose of the highest dose of amodiaquine given i.m. (538 µmol/kg). Upon cessation of the drug, leukocyte counts recovered and remained higher than pre-drug treatment until day 14. Serum ALT levels were increased only after the fourth dose of the highest dose of the drug given i.m. This could potentially be a very useful animal model to study the mechanisms of immune-mediated drug induced agranulocytosis. We conducted a pilot study in which amodiaquine was administered to rats for a longer period of time to determine whether a persistent decrease in leukocytes, particularly neutrophils, could be produced and to observe its effect on the liver. In addition to administering amodiaquine alone, we have also treated some rats with both amodiaquine and PMA. We expected that co-treatment with PMA would activate neutrophils or more specifically trigger them to go through an oxidative burst because exposure to nanomolar concentrations of PMA have been shown to be capable of activating PKC both in vitro and in vivo [266, 267] and activated PKC is part of the signal transduction pathway in the initiation of a neutrophil oxidative burst.

We had initially wanted to conduct a study where we would treat rats with amodiaquine for a longer period of time to determine whether a persistent decrease in neutrophil counts could be obtained. However we were unable to administer amodiaquine through daily subcutaneous injections in the rats for longer than ten days.

We had found that daily subcutaneous injections of amodiaquine at 0.25 g/kg (538

µmol/kg) resulted in general malaise and weight loss in the rats. The rats did not tolerate the drug very well at this dose after the fifth dose. In the autopsies of some of the rats given amodiaquine, we had found that most of the drug had accumulated underneath the skin. Thus, we were not certain if most of the drug administered was absorbed. In addition, in subsequent studies, it was found that amodiaquine has a very long half-life

104 and accumulates with chronic dosing. We found that amodiaquine treatment after the second dose produced an increase in neutrophil count and percentage, but returned to pre-treatment levels after the fourth dose. Then a gradual increase in leukocyte and neutrophil counts were observed in some of the treated rats after the eighth dose. In fact, an increase in neutrophil percentages and absolute counts was also observed in a subsequent study conducted by Dr. Ping Cai in our lab with treatments of amodiaquine at

62.5 mg/kg/day in the Wistar rats by oral gavage (unpublished data). We did not observe any significant decrease in leukocyte counts as described in Clarke’s study.

Amodiaquine appears to cause liver injury in rats as noted by liver histology which showed degenerative fatty infiltration and by the serum ALT levels, which tended to be higher in the drug-treated groups. While we produced drug-induced steatosis, we failed to demonstrate any clinically evident hepatic necrosis in this study.

In both the liver pathology and leukocyte counts, we did not observe any significant difference in rats that were given PMA along with the amodiaquine treatment compared to rats that were given amodiaquine alone. We are not certain whether there was an increase in the amount of amodiaquine reactive metabolites produced in these rats co-treated with PMA. In fact, we are not even certain whether an increase in neutrophil oxidative burst has been produced in these animals. We could assay for the level of oxidative burst in isolated rat neutrophils incubated with PMA by using conventional methods based on the reduction or oxidation of probes to fluorescent, luminescent, or coloured species [268-272]. This would be more reflective of the ability of the drug to induce oxidative burst in vitro but not the situation in vivo which is what is more of interest to us. In a sense one may just collect blood and isolate neutrophils from PMA treated rats to assay for the level of oxidative burst in these cells. However, it is likely that the neutrophils would have gone through oxidative burst already by the time the

105 blood samples have been collected and the cells isolated and prepared from the assay.

Thus we need to devise a method to measure oxidative burst in vivo and with such an assay we can also proceed to optimize the dose of PMA administered in these rats to obtain an increase in oxidative burst which would increase the amount of drug reactive metabolites produced. We also could not determine the amount of amodiaquine produced in the rats because we did not have an antibody against the reactive metabolite of amodiaquine produced at the time of this study. With such an antibody, we would be able to detect amodiaquine covalent binding to proteins in treated animals. Thus, our data on PMA co-treatment in amodiaquine-treated rats remains inconclusive without confirming the level of oxidative burst and the level of reactive metabolites produced in these animals.

In an earlier paper published in the Journal of American Medical Association in

1934, Hoffman et al. dosed rabbits with aminopyrine, varying from 0.2 to 0.9g/kg.

Leukocytosis was observed, followed a few weeks later by a depression of the total white blood cell count. In the differentials of some of the rabbits the proportion of polymorphonuclear leukocytes was reduced to as low as 8%. In all of the rabbits the granulocytes were less than 20% (the normal neutrophil percentage in healthy adult New

Zealand White rabbit is 33.5% - 10.8%), while controls remained unchanged. However, with similar treatments we did not observe a depression in the white cell and neutrophil counts. It may be worthwhile to look at higher doses of aminopyrine since 200 mg/kg was the lowest dose used in the Hoffman’s study in which their highest dose was 900 mg/Kg. However, achieving such as high doses would not be feasible without gavaging the animal. We have not attempted to gavage rabbits in our lab because it is not a normal procedure done at the medical sciences animal facility in the University of Toronto and we anticipate it to be a very difficult task with rabbits.

106 A dramatic increase in leukocyte counts, especially in neutrophils, was observed in all the rabbits given poly I:C in the first ten days. This was the opposite of what was observed in the studies conducted by Climenko et al . in the 1930’s in which they did not find an increase in leukocytes in aminopyrine-treated rabbits co-treated with nucleic acid, while leukocytosis was observed in those given only the nucleic acid treatment [273].

The reason why leukocytosis was observed in poly I:C and aminopyrine-treated rabbits in this experiment was probably because the dose of aminopyrine was not high enough to have an effect on the neutrophils. Aminopyrine has a short half-life, thus a reasonably high plasma concentration could be hard to obtain when the drug was ingested through the drinking water. No decreasing or increasing trend in leukocyte or neutrophil counts was observed other than those presumably brought about by poly I:C. Leukocyte and neutrophil counts in all treated rabbits were similar to and never significantly lower than the control rabbit. In the study with Brown Norway rats treated with aminopyrine neutropenia was not observed and studies conducted by Ferguson et al ., also failed to observe neutropenia in dipyrone (a sodium sulphonate derivative of aminopyrine)- treated white rats [274]

The characteristics of IDRs, such as the delay for onset of the reaction, strongly suggest that these reactions are immune-mediated. In one of the animal models studied in our lab, Brown Norway rats given penicillamine develop an autoimmune syndrome similar to that observed in humans. The incidence is 50-80% at a dose of 20 mg/day, but can be increased to 100% with the administration of a polymer of inosine and cytosine

(poly I:C) that stimulates macrophages through toll like receptor 3. We treated Brown

Norway rats and rabbits with a combination of aminopyrine and poly I:C, but agranulocytosis was not observed. We then hypothesized that the location and the timing of the immune stimulation may affect the induction of an immune response.

107 Therefore, we packaged the poly I:C with dipyrone in liposomes that should be taken up by macrophages and neutrophils where the poly I:C could stimulate an immune response and the aminopyrine could form a reactive metabolite, both in the target organ of the toxicity. However, this strategy also failed to induce agranulocytosis. There may be several reasons why agranulocytosis could not be induced in these animals. First, the dose of poly I:C we used in this experiment may not be right dose needed to induce drug- induced agranulocytosis. A dose lower or higher than 10 mg/kg/day or injection of more or less than three times a week may be required. However, poly I:C is a potent immunostimulant. High doses and prolonged administration have been shown to cause weight loss and depression in animals. The dose of dipyrone is probably also important for the induction of agranulocytosis. Again, an appropriate blood concentration may have not been achieved. Another obvious reason may be the number of liposomes taken up by the macrophages. We assume that most of the liposomes are taken up the macrophages as this is how clodronate filled liposomes deplete macrophages. In order to determine whether enough of the dipyrone and poly I:C liposomes had been taken up by macrophages we can measure whether there is upregulation of pro-inflammatory cytokines such as IFN-α and β and IL-6, IL-12, and TNF-α as they are known to be induced in poly I:C treated animals. Most importantly, we are not sure if poly I:C induces the optimal spectrum of cytokines to lead to agranulocytosis because different types of immune responses are associated with different cytokine/chemokine profiles. In addition, it now appears that repeated treatments with Poly I:C can lead to a loss of response to this immunostimulant (unpublished data).

After 70 days of amodiaquine or 80 days of aminopyrine treatment, agranulocytosis was not induced in any of the rats. However, in the amodiaquine group, both rats showed an increase in neutrophil counts in the first three weeks of drug

108 treatment. This suggests that the drug is having an effect on bone marrow but it still fails to induce agranulocytosis.

The attempt to induce agranulocytosis with a combination of amodiaquine or aminopyrine and 1-MT were also unsuccessful. It may be that the dose of 1-MT was insufficient to adequately inhibit IDO because most of the published studies have been done in mice rather than rats. In a more recent published study, it was demonstrated that immune tolerance in Lewis rats was inhibited by IDO inhibition using 1-MT administered at 50 mg/rat by oral gavage twice a day [275].

In summary, several different strategies were used to try to develop an animal model of idiosyncratic drug-induced agranulocytosis using two different drugs that are associated with a relatively high incidence of agranulocytosis in humans. In both cases the IDR in humans appears to be immune-mediated. Some IDRs appear to require a specific MHC and it is possible that the animals do not have the appropriate MHC to mount an immune response against the drug-modified proteins associated with these drugs. However, although the data are far from complete, it does not appear that most

IDRs require a specific MHC. The problem may be analogous to the problem of trying to get the immune system to attack cancer cells that display different proteins from other cells in the body. The problem appears to be one of overcoming immune tolerance, and despite the large amount of work that has been done in this area, progress has been limited.

109

CHAPTER 3

TESTING THE HYPOTHESIS THAT SELENIUM DEFICIENCY IS A RISK FACTOR FOR CLOZAPINE- INDUCED AGRANULOCYTOSIS IN RATS

Reproduced with permission from: Julia Ip and Jack P. Uetrecht, Chemical Research in Toxicology (2008), 21 (4), 874-878. Copyright 2008 American Chemical Society. (The American Chemical Society granted permission to include my own articles in this thesis).

110 3.1 Abstract

Clozapine is an effective atypical antipsychotic associated with a relatively high incidence of drug-induced agranulocytosis. It forms a reactive nitrenium ion metabolite upon oxidation by peripheral neutrophils and their precursors in the bone marrow.

Although the mechanism of this idiosyncratic drug reaction is still unknown, the observation that it does not occur rapidly on rechallenge of patients with a history of clozapine-induced agranulocytosis suggests that it is not immune-mediated. Previous studies by other research groups had found that patients on clozapine had lower plasma and red blood cell levels of selenium. The reactive metabolite of clozapine reacts with glutathione and therefore it is likely that it also binds to selenocysteine-containing proteins, such as glutathione peroxidase, thioredoxin reductase, and protein disulfide isomerase. We set out to test the hypothesis that clozapine-induced agranulocytosis is associated with selenium deficiency with rats on a selenium-deficient diet. We studied the effects of clozapine on selenium levels and the effect of selenium deficiency on leukocyte and neutrophil counts and clozapine covalent binding. We did not observe any significant difference between clozapine-treated rats given a selenium-adequate or deficient diet. Therefore, it is unlikely that selenium deficiency is a major risk factor for clozapine-induced agranulocytosis.

111 3.2 Abbreviations

SD , Selenium deficient diet

SN , Diet containing a normal amount of selenium

CL , Clozapine co-treatment

112 3.3 Introduction

Clozapine is an atypical antipsychotic used in the treatment of refractory schizophrenia. Although highly effective, its use has been limited due to its propensity to cause agranulocytosis in 0.8% of patients [177]. Clozapine has also been found to be associated with other adverse reactions including myocarditis, cardiomyopathy, hepatotoxicity, and nephritis [276-281]. The mechanisms of these reactions, including agranulocytosis, are still not understood; however, it is believed to be due to a reactive nitrenium ion generated by neutrophils and their precursors [199].

In a small study conducted by Linday et al . that measured the free radical scavenging enzyme activity and levels of related trace metals, clozapine-treated patients had lower plasma selenium concentrations than normal healthy controls [282]. However, a significant difference was not observed between those that developed agranulocytosis while on clozapine and those that did not. Another study carried out by Vaddadi et al . to assess the difference in selenium levels between schizophrenic patients that were or were not on clozapine also found lower plasma and red cell selenium concentrations in those treated with this drug [283]. It is plausible to hypothesize that the observed lower levels of selenium in clozapine-treated patients could be a result of the binding of the nitrenium ion to the selenocysteine of those selenium-containing proteins (selenoproteins). Known as the 21 st amino acid, selenocysteine is an analogue of cysteine with a selenium atom replacing the sulfur. We have shown in our previous studies that clozapine covalently binds to nucleophiles such as glutathione upon bioactivation by activated neutrophils or hypochlorous acid [199]. Selenium displays many similarities with sulfur, yet it is different enough that a selenocyteine residue is usually more reactive than the cysteine analogue, by two orders of magnitude, because it has a much lower pKa and is ionized at

113 physiological pH [284, 285]. Therefore, it is very likely that clozapine will covalently bind to selenocyteine in a similar fashion to the thiol group in cysteine (Figure 3.1).

Such covalent binding could alter the structure and/or function of selenoproteins leading to their destruction and depletion of selenium, which can potentially be a risk factor for clozapine-induced agranulocytosis. The importance of selenoproteins to human health and its antioxidant properties had been well described in recent reviews [286-291]. They are essential to many enzymatic functions in thyroid hormone metabolism, antioxidant defence systems, and the immune system [286, 292, 293]. Selenoproteins such as glutathione peroxidases and thioredoxin reductases are among the key antioxidant enzymes needed to prevent excessive oxidative stress [286]. Therefore, alterations in the function of such selenoproteins and their removal would induce oxidative stress, which may be a risk factor of idiosyncratic drug reactions. The aim of this study is to determine whether selenium deficiency can increase the risk of clozapine-induced agranulocytosis in rats and also to determine its effect on the amount of clozapine- protein covalent binding and the effect of clozapine treatment on selenium levels.

CH3 CH3 CH3 N N N

N myeloperoxidase N selenoproteins N - N H2O2/Cl N N Cl Cl Cl N N N H H Se clozapine nitrenium ion protein covalent binding with selenoproteins

Figure 3.1. Potential covalent binding of the clozapine nitrenium ion to selenoproteins.

114 3.4 Materials and Methods

3.4.1 Animals

Sixteen female Sprague Dawley rats weighing approximately 50 g were purchased from Charles River (Montreal, QC). All rats were housed in pairs in plastic cages with corncob chip bedding in a 12:12 h light:dark cycle at 22 oC. They were acclimatized and given access to regular rodent powder diet (Harlen Teklad, Madison,

WI) and tap water ad libitum for 1 week prior to the beginning of the experiment. The experimental protocol was approved by University of Toronto’s animal care committee.

3.4.2 Chemicals

Clozapine was provided by Novartis Pharmaceuticals Inc. (Dorval, QC). DTT and Ponceau S solution were purchased from Sigma-Aldrich (Oakville, ON). Stock acrylamide solution (30%) and nitrocellulose were purchased from Bio-Rad

(Mississuaga, ON). Horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L chains) was purchased from Cedarlane (Burlington, ON). SuperSignal West Pico

Chemiluminescent Substrate was purchased from Pierce (Rockford, IL).

2.4.3 Selenium-Deficient Diet and Clozapine Treatment

Half of the rats were placed on a selenium-deficient (SD) 1 purified powder rodent diet (TD. 92163) containing 0.03 ppm of background selenium from Harlan Teklad

(Madison, WI). The rest were given a selenium-adequate (SN) diet supplemented with

0.1 ppm of selenium. All rats were given deionized water filtered through a reverse osmosis system. Fifty-four days after the commencement of the selenium-deficient diet,

115 four of the eight rats were subjected to clozapine treatment at 50 mg/kg body weight/day by addition of the drug to the diet (SDCL). This dose is approximately 5 times the therapeutic dose and higher doses are not well tolerated by the animals. Four of the eight rats on the selenium supplemented diet were given the same dose of clozapine in their diet (SNCL). The rats were treated with clozapine for 62 days.

3.4.4 Blood Collection and Leukocyte Counts

Blood samples were collected from each rat once a week. A sample of blood

(200 µL) was obtained with a 25 G needle from the tail vein and collected into

Microvettes® CB 300 Kalium-EDTA capillary tubes (Sarstedt, Montreal, QC). Total leukocyte counts were performed by mixing 10 µL of blood with Turk Blood Diluting

Fluid (Ricca Chemical Co., Arlington, TX) at 1:9 and using a hemacytometer.

Leukocyte differential counts were obtained from blood smears on slides stained with

Wright-Giemsa stain (Fisher Scientific Co., Middletwon, VA). Peripheral neutrophil counts were calculated by multiplying the total leukocyte counts by the percentage of neutrophils in each blood sample.

3.4.5 Selenium Status Assessment

The selenium status of each rat was obtained using the Ransel kit by Randox

Laboratories (Crumlin, UK) which is based on the principle of NADPH oxidation.

Glutathione peroxidase is a selenoprotein in which selenium is present in the form of selenocysteine [289]. Glutathione peroxidase activity decreases in response to selenium deficiency in most animals [289, 294-296]. Therefore, whole blood glutathione

116 peroxidase activities in the rats provided an index of their selenium status and were measured in this experiment.

3.4.6 Collection of Bone Marrow and Liver

At the end of the study, rats were sacrificed with an overdose of anesthetic

(ketamine, 50 mg/rat)/xylazine, 5 mg/rat). The femurs and tibia were removed and bone marrows were collected by flushing them with 20 ml of RPMI 1640 culture medium

(University of Toronto, Tissue Culture). The bone marrow cells were resuspended by a five times passage through a 1 ml serological pipette tip. The cell suspension was centrifuged at 125 x g for 6 min. Red blood cells were then removed by resuspension of the cell pellet in red cell lysis buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA) for 6 min and centrifugation at 125 x g for 6 min. Tissue debris was removed by passing the cell suspension through a 70 µm nylon cell strainer

(BD Biosciences, Bedford, MA) upon resuspension in PBS (University of Toronto,

Tissue Culture). The bone marrow cells were washed again in PBS and resuspended in

500 µL of cell lysis buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.2% Triton X-100, protease inhibitor cocktail). Livers were excised from the rats and stored at -80 oC.

Liver tissue homogenate was prepared by homogenizing a small aliquot of the frozen liver in cell lysis buffer using a tissue homogenizer (9500 rpm, 3 bursts of 10 s). Bone marrow cell lysate and liver tissue homogenate samples were analyzed for protein concentration using a BCA protein assay kit from Pierce (Rockford, IL).

117 3.4.7 SDS-PAGE and Immunoblotting

Bone marrow cell lysate and liver tissue homogenate samples were diluted to a final protein concentration of 1 µg/ µL with cell lysis buffer. One part of a 6X SDS-

PAGE sample buffer (0.35 M Tris-Cl, 10% SDS, 4% glycerol, 0.02% bromophenol blue,

18 mg/ml DTT) was added to 5 parts of sample and then heated at 90 oC for 10 min.

SDS-PAGE was performed using a mini-gel system (Mini-PROTEAN II, Bio-Rad).

Stacking and resolving gels were 4% and 10% acrylamide, respectively. Prestained broad range molecular mass makers were used (Bio-Rad). A sample (20 µL) was loaded into each well. Gels were run at 120 V for 90 min until the dye front reached the bottom of the resolving gel. Electrophoretic transfer to nitrocellulose membrane was carried out at 100 V for 60 min using a mini Trans-Blot transfer cell (Bio-Rad) in a transfer buffer

(25 mM Tris-HCl, 0.19 M glycine, 20% methanol, 0.1% SDS). The nitrocellulose membrane was stained with Ponceau S solution for 5 min to assess the efficiency of the transfer. Lane densitometry was performed on the Ponceau S-stained blots. Only blots with all lanes having a net arbitrary lane density differing no more than 10% of the mean density of all lanes were used. The membrane was destained with wash buffer (100 mM

Tris-Cl, 0.9% NaCl, 0.1% Tween 20).

The subsequent steps were conducted at room temperature with gentle shaking on a rocker. The membrane was blocked with 5% (w/v) skimmed milk powder in 100 mM

Tris-HCl buffer (pH 7.5) containing 0.9% NaCl and 0.1% Tween 20 for 1 h. The blocked membrane was then incubated for 15 h with an anti-clozapine antibody diluted

(1:3000 for liver tissue homogenate blots, 1:2000 for bone marrow cell lysate blots) in the Tris-HCl buffer; production of the anti-clozapine antibody has been described previously [124]. The membrane was washed with wash buffer for 10 min 5 times to remove any unbound antibodies. It was then incubated for 2 h with horseradish

118 peroxidase-conjugated goat anti-rabbit IgG (H + L chain) antiserum diluted 1: 20 000 with wash buffer. After washing 5 times with wash buffer to remove any unbound antibodies, the membrane was incubated in SuperSignal West Pico Chemiluminescent

Substrate for 10 min. Chemiluminescence on the blot was immediately captured using the FluorChem TM 8800 imaging system by Alpha Innotech (San Leandro, CA) by exposing it for 10 min to visualize the bound antibodies.

3.4.8 Statistical Analysis

Statistical analyses were performed using the GraphPad Prism 4 software

(GraphPad Software Inc.). Unpaired t tests (two tailed, 95% confidence interval) were used to compare between treatment groups.

119 3.5 Results

3.5.1 Selenium Status

Glutathione peroxidase activities of whole blood in the rats given a selenium deficient diet for 116 days were significantly lower than the rats given an adequate level

(p < 0.05) (Figure 3.2). However, clozapine treatment did not result in any significant changes in the level of glutathione peroxidase activities either in the rats given a selenium-deficient or a selenium-adequate diet.

9

8

7

6 5

4 /Litre of whole blood) 4 3

2

GlutathioneActivity Peroxidase

(Units x 10 * 1 *

0 SN SNCL SD SDCL Treatment Groups

Figure 3.2. Selenium status assessment. Glutathione peroxidase activity after 116 days of selenium-deficient (SD) or adequate diet (SN) in the presence (CL) and absence of clozapine co-treatment. Values are expressed as the mean ± SD from 4 animals. * p < 0.05 compared to the selenium-adequate group with or without clozapine treatment.

120 3.5.2 Peripheral Leukocyte Counts

Total leukocytes and peripheral neutrophil counts were within the normal ranges of Sprague Dawley rats in this study [297]. Changes in total leukocyte and peripheral neutrophil counts were not observed during the course of clozapine treatment in either the group of rats given a selenium-deficient or those given a selenium-adequate diet

(Figures 3.3 and 3.4) .

25000 SN SNCL 20000 SD L blood) µ µ µ µ SDCL

15000

10000

5000 Leukocyte Count (cells/ 0 0 7 14 21 28 35 62 Days of Clozapine Treatment

Figure 3.3. Peripheral leukocyte counts of rats during clozapine treatment. Values are expressed as the mean ± SD of 4 animals for each treatment group.

6000 SN SNCL 5000 SD

L blood) SDCL µ µ µ µ 4000

3000

2000

1000

Neutrophil Count (cells/ 0 0 7 14 21 28 35 62 Days of Clozapine Treatment

Figure 3.4. Peripheral neutrophil counts of rats during clozapine treatment. Values are expressed in mean ± SD of 4 animals for each treatment group.

121 3.5.3 Covalent Binding of Clozapine to Hepatic and Bone Marrow Proteins

In both the liver homogenates and bone marrow cell lysates from drug-treated rats covalent binding of clozapine was detected. Immunoblots of the liver homogenate showed bands with molecular masses ranging from 30 to 240 kDa, whereas immunoblot of the bone marrow cell lysate blot showed only one prominent band at ~75 kDa.

Differences in terms of the amount of clozapine covalent binding in the rats given an adequate selenium diet and those given a deficient diet were not observed in either the liver or bone marrow (Figures 3.5 and 3.6).

kDa SN SNCL SDCL

204 95

54

29

Figure 3.5. Covalent binding of clozapine in the liver. Western blot detection of clozapine-modified hepatic proteins from selenium-deficient (SDCL) and adequate (SNCL) rats given a daily dose of 50 mg/kg clozapine for 62 days. Lane SN represent sample from a rat on the selenium-adequate diet without clozapine treatment. Thirty micrograms of total protein from liver tissue homogenate were loaded per lane, and the primary antiserum was used at a dilution of 1:3000. Each lane represents sample from an individual animal.

122

SN SNCL SDCL kDa

75

Figure 3.6. Covalent binding of clozapine in the bone marrow. Western blot detection of clozapine-modified bone marrow proteins from selenium-deficient (SDCL) and adequate (SNCL) rats given a dose of 50 mg/kg clozapine for 62 days. Lane SN represent sample from a rat on the selenium-adequate diet without clozapine treatment. Thirty micrograms of total protein from bone marrow cell lysate were loaded per lane, and the primary antiserum was used at a dilution of 1:2000. Each lane represents sample from an individual animal.

123 3.6 Discussion

Plasma and red blood cell selenium levels have been found to be lower in clozapine-treated schizophrenic patients [282, 283]. It is not clear whether this atypical antipsychotic depletes this essential trace element in these patients or whether the disease itself is associated with decreased selenium levels. Selenium has an important role in reducing oxidative stress; for example, glutathione peroxidase can reduce hydrogen peroxide and modulate the oxidative burst [286]. Other selenium-containing proteins are also thought to be involved in a defense mechanism against arylating agents because they are the common target of the electrophilic metabolites of many chemicals [298]. In mice, one of the proteins to which the reactive metabolite of acetaminophen covalently binds is a 56 kDa selenium-binding protein [298-300].

Glutathione peroxidase activity was significantly lower in the selenium-deficient rats, dropping to 12% of the adequate control groups after 116 days of selenium-deficient diet. These results confirm that this commercial selenium-deficient diet induced selenium-deficiency in these rats. However, clozapine treatment did not result in a significant decrease in whole blood selenium level in either selenium-deficient or adequate rats as measured in the glutathione peroxidase activity. Although there is a suggestion of a decrease in selenium levels associated with clozapine treatment in the selenium deficient animals but even with a one-tailed T test it did not quite reach statistical significance (p = 0.059); it is difficult to know if this represents a biologically significant difference. However, this is only with a relatively extreme selenium deficiency that is unlikely to occur in humans. In Linday’s study, plasma glutathione peroxidase levels in the post-clozapine agranulocytosis group were lower than healthy controls as well as those that did not develop agranulocytosis while on clozapine [282].

124 From our study, it did not appear that clozapine had a direct effect on selenium status.

Recent findings showed a possible association between the pathogenesis of schizophrenia and increased oxidative stress and cellular injuries [301-303]. It is possible that schizophrenic patients, especially those that require clozapine treatment, are predisposed to lower selenium levels because of poor diet.

Despite prolonged treatment with a combination of a selenium-deficient diet and clozapine, trends suggesting a decrease in white blood cell or neutrophil counts were not observed. Agranulocytosis clearly was not induced. It appeared that there may be slightly more covalent binding of clozapine to bone marrow proteins in the deficient animals; however, using densitometry these differences were not statistically significant.

Differences in the amount of covalent binding in hepatic tissue between selenium- deficient and adequate rats were not observed.

In summary, although there may have been subtle changes in selenium levels caused by clozapine treatment in animals already very selenium deficient as determined by glutathione peroxidase activity, there were no effects on neutrophil numbers. These results do not support the hypothesis that clozapine treatment causes selenium deficiency nor that selenium deficiency is the major risk factor for clozapine-induced agranulocytosis.

125 3.7 Acknowledgements

Dr. Jack P.Uetrecht is the recipient of the Canada Research Chair in Adverse Drug

Reactions. The work was supported by grants from the Canadian Institutes of Health

Research.

126

CHAPTER 4

TESTING THE HYPOTHESIS THAT VITAMIN C DEFICIENCY IS A S RISK FACTOR FOR CLOZAPINE- INDUCED AGRANULOCYTOSIS USING GUINEA PIGS AND ODS RATS

Reproduced with permission from: Julia Ip, John X. Wilson, and Jack P. Uetrecht, Chemical Research in Toxicology (2008), 21 (4), 869-873. Copyright 2008 American Chemical Society. (The American Chemical Society granted permission to include my own articles in this thesis).

127 4.1 Abstract

The use of clozapine is limited by a relatively high incidence of drug-induced agranulocytosis. Clozapine is oxidized by bone marrow cells to a reactive nitrenium ion.

Although many idiosyncratic drug reactions are immune-mediated, the fact that patients with a history of clozapine-induced agranulocytosis do not immediately develop agranulocytosis on rechallenge suggests that some other factor may be responsible for the idiosyncratic nature of this reaction. The reactive nitrenium ion is very rapidly reduced back to clozapine by vitamin C and many schizophrenic patients are vitamin C deficient. We set out to test the hypothesis that vitamin C deficiency is a major risk factor for clozapine-induced agranulocytosis using a vitamin C deficient guinea pig model. Although the vitamin C deficient guinea pigs did not develop agranulocytosis, the amount of clozapine covalent binding in these animals was less than we had previously observed in samples from rats and humans. Therefore we studied ODS rats which also cannot synthesize vitamin C. Vitamin C deficient ODS rats also did not develop agranulocytosis, and furthermore, although covalent binding in the bone marrow was greater than in the guinea pig, it was not increased in the vitamin C deficient ODS rats relative to ODS rats that had adequate vitamin C in their diet. Therefore, it is very unlikely that vitamin C deficiency is a major risk factor for clozapine-induced agranulocytosis.

128 4.2 Abbreviations

ODS , Osteogenic Disorder Shionogi

VN , diet containing an adequate amount of vitamin C

VD , vitamin C-deficient diet

CL , clozapine co-treatment

DHBA , 3,4-dihydroxybenzylamine

129 4.3 Introduction

Clozapine is an atypical dibenzodiazepine antipsychotic lacking extrapyramidal side effects. Despite its unique effectiveness in treating refractory schizophrenia, its use is limited because of the propensity to cause agranulocytosis in ~0.8% of patients [304,

305]. Agranulocytosis is a potentially fatal blood dyscrasia characterized by a dramatic decrease in neutrophil count. Therefore, patients on clozapine are required to monitor their neutrophil count weekly for the first 6-12 months after which the frequency of monitoring is often decreased [304]. Little is known about the mechanism of drug- induced agranulocytosis and how clozapine causes this idiosyncratic drug reaction.

Clozapine is bioactivated (oxidized) by activated human neutrophils and bone marrow cells to a reactive nitrenium ion through the myeloperoxidase-hydrogen peroxide system generating hypochlorous acid during the respiratory burst [306, 307]. The idiosyncratic nature of this reaction suggests that it might be immune-mediated; however, the observation that it does not recur rapidly on rechallenge of patients with a past history of clozapine-induced agranulocytosis argues against, but does not completely exclude, this possibility [202]. Studies have shown that the reactive metabolite of clozapine is capable of inducing apoptosis and cytotoxicity in neutrophils and bone marrow cells [200, 209,

308-310]. It has also been shown that the nitrenium ion is capable of covalently binding to proteins in neutrophils and bone marrow tissue [124]. Induction of apoptosis, cytotoxicity, and the formation of protein adducts by this reactive metabolite have been postulated to be associated with clozapine-induced agranulocytosis. Notably, the addition of ascorbate prevented detection of clozapine oxidation by the myeloperoxidase-hydrogen peroxide system [311]. We have demonstrated that the nitrenium ion is reduced back to clozapine by vitamin C (ascorbic acid) extremely rapidly such that we were unable to measure the rate (unpublished data). Neutrophils

130 normally have high levels of vitamin C (in the millimolar range compared to the plasma level which is only about 50 µM) [312]. This is presumably because vitamin C is an antioxidant much needed to protect neutrophils from the oxidative stress generated by the large amounts of oxidants produced in these cells [313]. There is even evidence linking the pathogenesis of schizophrenia with increased oxidative damage [301-303].

Studies have shown that as many as 2% of institutionalized psychiatric patients might have vitamin C levels less than 0.1 mg/dL; detrimental effects on immunity are thought to occur at levels three times higher [314]. Therefore, it is conceivable that vitamin C deficiency may be a major risk factor for clozapine-induced agranulocytosis because we know that vitamin C can detoxify the nitrenium ion (Figure 4.1). Indeed, ascorbic acid had been shown to attenuate clozapine-mediated cytotoxicity toward HAS303 stromal cells and neutrophils presumably because of its ability to reduce the reactive metabolite and thus, resulting in a decrease in product adduct formation [208, 308]. The present study evaluated the hypothesis that vitamin C deficiency is a risk factor for clozapine- induced agranulocytosis using guinea pigs and Osteogenic Disorder Shionogi (ODS) rats as models. Both of these animals are unable to produce vitamin C because, similar to humans, they are both genetically deficient in l-gulonolactone oxidase required in the synthesis of ascorbic acid from glucose via the glucuronic pathway [315, 316]. We also tested the hypothesis that depletion of intracellular vitamin C would result in an increase in clozapine protein binding.

131

CH3 CH3 CH3 N N N

N myeloperoxidase N protein -S-H N - N H2O2/Cl N N Cl Cl Cl N N N H H S clozapine nitrenium ion protein

vitamin C

- Figure 4.1. Clozapine is oxidized by the myeloperoxidase/H 2O2/Cl system of neutrophils forming the reactive nitrenium ion which covalently binds to proteins. In the presence of vitamin C, the nitrenium ion is very rapidly reduced back to clozapine.

4.4 Materials and Methods

4.4.1 Animals

Sixteen female Harley guinea pigs weighing approximately 700 g were purchased from Charles River (Montreal, QC) and 15 female ODS rats at 200 g from CLEA Japan

(Tokyo, Japan). The guinea pigs were housed in pairs in metal cages with paper bedding and the rats were in pairs in plastic cages with corncob bedding in a 12:12 h light:dark cycle at 22 oC. They were acclimatized and given access to either standard guinea pig diet or rodent diet supplemented with vitamin C (Harlen Teklad, Madison, WI) and tap water ad libitum for 1 week prior to the beginning of the experiment. The experiment performed on these laboratory animals was approved by University of Toronto’s animal care committee.

132 4.4.2 Chemicals

Clozapine was provided by Novartis Pharmaceuticals Inc. (Dorval, QC). DL-

Dithiothreitol (DTT), Ponceau S solution, 3,4-dihydroxybenzylamine (DHBA) 1, and L- ascorbic acid were purchased from Sigma-Aldrich (Oakville, ON). Stock acrylamide solution (30%) and nitrocellulose were purchased from Bio-Rad (Mississuaga, ON).

Horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L chains) was purchased from Cedarlane (Burlington, ON). SuperSignal West Pico Chemiluminescent Substrate was purchased from Pierce (Rockford, IL).

4.4.3 Vitamin C Deficient Diet and Clozapine Treatments

After acclimatization, all guinea pigs and rats were placed on the vitamin C-free purified rodent diet AIN-93G purchased from Harlen Teklad (Madison, WI). All animals were given drinking water which was deionized water filtered through a reverse osmosis system. Eight guinea pigs were placed in the vitamin C-adequate group (VN) and their diets were supplemented with ascorbic acid at 75 mg/kg/day given in the drinking water. The other eight guinea pigs were placed in the vitamin C-deficient group

(VD) without supplementation of ascorbic acid in their diet. In both the vitamin C- deficient and adequate groups, four animals (VNCL and VDCL) were dosed with clozapine at 50 mg/kg/day in the drinking water starting on Day 28 of the study and treated for 55 days. The rats were divided into 3 treatment groups: vitamin C-adequate

(VN); vitamin C-deficient A (VD-A); vitamin C-deficient B (VD-B) each receiving a different level of vitamin C supplementation in their drinking water (1.67 g/L, 0.33 g/L,

0.2 g/L). In each of these groups, 3 of the 5 rats (VD-ACL and VD-BCL) were dosed with clozapine at 50 mg/kg/day in their diet starting on Day 28 of the study and treated

133 for 42 days. This dose is approximately 5 times the therapeutic dose and higher doses are not well tolerated by the animals.

4.4.4 Blood Collection and Leukocyte Counts

Blood samples were collected from each animal once a week. A sample of blood

(200 µL) was obtained with a 25 G needle from the saphenous vein on the leg of the guinea pig or the tail vein of the rat and collected into Microvettes® CB 300 Kalium-

EDTA capillary tubes (Sarstedt, Montreal, QC). Total leukocyte counts were performed by mixing 10 µL of the blood sample with Turk Blood Diluting Fluid (Ricca Chemical

Co., Arlington, TX) at 1:9 and using a hemocytometer. Leukocyte differentials were obtained by preparing blood smears on slides and stained with Wright-Giemsa stain

(Fisher Scientific Co., Middletown, VA). Peripheral neutrophil counts were calculated by multiplying the total leukocyte counts by the percentage of neutrophils in each blood sample.

4.4.5 Collection of Bone Marrow and Liver

After 55 days and 42 days of clozapine treatment in the guinea pigs and ODS rats, respectively, all animals were sacrificed with an overdose of anesthetic (ketamine

/xylazine). The femurs and tibia were removed and bone marrows were collected by flushing with 20 ml of RPMI 1640 culture medium (University of Toronto, Tissue

Culture). The bone marrows were resuspended by a five times passage through a 1 ml serological pipette tip. The cell suspension was centrifuged at 125 x g for 6 min. The red blood cells were then removed by resuspension of the cell pellet in red cell lysis buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA) for

134 6 min and centrifuged at 125 x g for 6 min. Tissue debris was removed by passing the cell suspension through a 70 µm nylon cell strainer (BD Biosciences, Bedford, MA) upon resuspension in phosphate-buffered saline (PBS; University of Toronto, Tissue

Culture). The bone marrow cells were washed again in PBS and resuspended in 500 µL of cell lysis buffer (10 mM Tris-Cl pH 7.4, 1 mM EDTA, 0.2% Triton X-100, protease inhibitor cocktail). Livers were excised from the guinea pigs and stored at -80 oC. Liver tissue homogenate was prepared by taking a small aliquot of the frozen liver and homogenizing it in cell lysis buffer using a tissue homogenizer (9500 rpm, 3 bursts of 10 s). Bone marrow cell lysate and liver tissue homogenate samples were analyzed for protein concentration using BCA protein assay kit from Pierce (Rockford, IL).

4.4.6 SDS-PAGE and Immunoblotting

Bone marrow cell lysate and liver tissue homogenate samples were diluted to a final protein concentration of 1 µg/ µL with cell lysis buffer. One part of a 6X SDS-

PAGE sample buffer (0.35 M Tris-Cl, 10% SDS, 4% glycerol, 0.02% bromophenol blue,

18 mg/ml DTT) was add to 5 parts of samples. They were then heated at 90 oC for 10 min. SDS-PAGE was performed using a mini-gel system (Mini-PROTEAN II, Bio-Rad).

Stacking and resolving gels were 4% and 10% acrylamide, respectively. Prestained broad range molecular mass makers were used (Bio-Rad). A sample (20 µL) was loaded into each well. Gels were run at 120 V for 90 min until the dye front reached the bottom of the resolving gel. Electrophoretic transfer to nitrocellulose membrane was carried out at 100 V for 60 min using a mini Trans-Blot transfer cell (Bio-Rad) in a transfer buffer

(25 mM Tris-Cl, 0.19 M glycine, 20% methanol, 0.1% SDS). The nitrocellulose membrane was stained with Ponceau S solution for 5 min to assess the efficiency of the

135 transfer. Lane densitometry was performed on the Ponceau S-stained blots. Only blots with all lanes having a net arbitrary lane density differing no more than 10% of the mean density of all lanes were used. The membrane was destained with wash buffer (100 mM

Tris-Cl, 0.9% NaCl, 0.1% Tween 20).

The subsequent steps were conducted at room temperature with gentle shaking on a rocker. The membrane was blocked with 5% (w/v) skimmed milk powder in 100 mM

Tris-HCl buffer (pH 7.5) containing 0.9% NaCl and 0.1% Tween 20 for 1 h. The blocked membrane was then incubated for 15 h with an anti-clozapine antibody diluted

(1:3000 for liver tissue homogenate blots and 1:2000 for bone marrow cell lysate blots) in the Tris-HCl buffer, production of the anti-clozapine antibody has been described previously [200]. The membrane was washed with wash buffer for 10 min 5 times to remove any unbound antibodies. It was then incubated for 2 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L chain) antiserum diluted 1: 20 000 with wash buffer. After washing 5 times with wash buffer to remove any unbound antibodies, the membrane was incubated in SuperSignal West Pico Chemiluminescent

Substrate for 10 min. The chemiluminescence on the blot was immediately captured using the FluorChem TM 8800 imaging system by Alpha Innotech (San Leandro, CA) by exposing it for 10 min to visualize bound antibodies.

4.4.7 Vitamin C Status Assessment

Upon sacrifice of each guinea pig, blood was obtained by cardiac puncture for serum, plasma, and peripheral leukocytes isolation; liver, heart, and skeletal muscle were excised; and bone marrow cells were isolated for ascorbate level assessment in each of these organs. Ascorbate was assayed by acidic extraction and HPLC with electrochemical detection, according to a previously described procedure [317]. Organ

136 samples were homogenized (Kontes Pestle Motor Mixer) at 4 °C in a metaphosphoric acid solution (8.5 g/L) that contained 3,4-dihydroxybenzylamine (DHBA) as an internal standard. Plasma and serum samples were extracted with equal volumes of methanol with 200 µM DHBA. The homogenates or extracts were then centrifuged at 4 °C, passed through a 45 um Millex filter and then injected into the HPLC system. A Resolve C18 90

A silica 3.9 x 150 mm column was used with a flow rate of 0.5 ml/min. The mobile phase contained 80 mM sodium acetate, 0.015% metaphosphoric acid, 1 mM n- octylamine, and 15% methanol (pH 4.6). Ascorbate and DHBA in samples and standards were quantified with a Waters M460 amperometric detector. Assay sensitivity was 2 pmol. The ascorbate concentrations of samples were determined by interpolation on an external standard curve and corrected for DHBA recovery. Blood and organ tissues were taken from two guinea pigs, one on the vitamin C-deficient diet and one from the vitamin C-adequate diet, after 28 days on the diet and from the rest of the guinea pigs at the end of the study. Blood was taken from each rat at day 28 of the study for serum ascorbate level assay prior to clozapine treatment.

4.4.8 Statistical Analysis

Statistical analyses were performed using the GraphPad Prism 4 software

(GraphPad Software Inc.). Unpaired t tests (two tailed, 95% confidence interval) were used to compare between treatment groups; values of p ≤ 0.05 were considered statistically significant.

137 4.5 Results

4.5.1 Vitamin C Status

In a preliminary study, the effect of 28 days of the vitamin C-deficient diet was determined. Ascorbate concentrations in the guinea pig given the deficient diet were an order of magnitude less than a control given a normal vitamin C diet, and the plasma, serum, heart, and skeletal muscle ascorbate concentrations were actually below the detection limit (data not shown). Ascorbate levels reminded very low in the guinea pigs on the vitamin C-deficient diet both with and without 55 days of clozapine treatment compared to those on the vitamin C-adequate diet ( p < 0.05; Table 4.1). Although there was a trend to lower ascorbate levels in clozapine-treated animals in all tissues and with both vitamin C deficient and adequate vitamin C diets, the difference was only significant in the heart. In the ODS rats, after 28 days the serum ascorbate levels of animals on the lowest vitamin C diet were only one fifth of that of animals receiving the vitamin C-adequate diet (Table 4.2).

138 Table 4.1. Vitamin C status assessment in the guinea pigs prior to clozapine treatment. Ascorbate concentrations in the plasma, serum, peripheral leukocytes, bone marrow cells, heart, skeletal muscle, and liver tissue in guinea pigs after 28 days of vitamin C-deficient or vitamin C-adequate diet. a

Plasma Serum Leukocytes Bone Marrow Heart Skeletal Liver (µM) (µM) (nmol/10 6 Cells (nmol/mg) Muscle (nmol/mg) cells) (nmol/10 6 cells) (nmol/mg) Vitamin C-Deficient ND ND 0.52 0.17 ND ND 24.2 Diet Vitamin C-Adequate 41.1 123.9 2.09 1.59 32.2 31.6 463.3 Diet

a This was a preliminary experiment with only two animals to determine the effect of this degree of vitamin C deficiency. Assay sensitivity was 2 pmol. ND denotes nondetectable levels.

Table 4.2. Vitamin C status assessment in the guinea pigs after clozapine treatment. Plasma, serum, peripheral leukocytes, bone marrow cells, and heart tissue ascorbate level of guinea pigs in each treatment groups after 55 days of clozapine treatment. a

Plasma Serum Leukocytes Bone Marrow Heart (µM) (µM) (nmol/10 6 cells) Cells (nmol/mg) (nmol/10 6 cells) Vitamin C-Deficient Diet VD ND ND 0.09 ± 0.03 b 0.04 ± 0.01 b ND With clozapine treatment VDCL ND ND 0.06 ± 0.01 c 0.03 ± 0.01 c 0.72 ± 0.12 c Vitamin C-Adequate Diet VN 150.8 ± 46.6 141.3 ± 44.6 0.69 ± 0.16 0.74 ± 0.10 74.1 ± 12.7 With clozapine treatment VNCL 132.3 ± 81.5 130.7 ± 63.3 0.67 ± 0.26 0.60 ± 0.07 44.0 ± 21.2 b

a Ascorbate was assayed by acidic extraction and HPLC with electrochemical detection. Assay sensitivity was 2 pmol. ND denotes nondetectable levels. Values are expressed in mean ± SD in each treatment group ( n = 4). b p < 0.05 compared to treatment group VN c p < 0.05 compared to treatment group VNCL

139 Table 4.3. Vitamin C status assessement in the ODS rats after clozapine treatment. Serum ascorbate levels of ODS rats after 28 days of vitamin C supplemented diet prior to clozapine treatment. a

Serum Ascorbate Concentration (µM) VD-A 10.82 ± 8.42 b VD-B 5.83 ± 2.96 b VN 29.24 ± 15.62 a Ascorbate was assayed by acidic extraction and HPLC with electrochemical detection. Assay sensitivity was 2 pmol. Values are expressed in mean ± SD in each treatment group ( n = 5). The VN group received vitamin C through the drinking water at 1.67 g/L, VD-A at 0.33 g/L and VD-B at 0.2 g/L. b p < 0.05 compared to treatment group VN.

4.5.2 Peripheral Leukocyte Counts

Total leukocytes and peripheral neutrophil counts were within the normal ranges of guinea pigs and rats in these studies [318, 319]. Significant changes in total leukocyte and peripheral neutrophil counts were not observed during the course of clozapine treatment in either the group of guinea pigs or rats given an adequate or deficient vitamin

C diet (Figure 4.2 and 4.3).

140 3500 VN VNCL 3000 VD VDCL 2500

2000

1500

1000

500 Neutrophil Count (cells/uL blood) (cells/uL CountNeutrophil

0 0 14 28 42 52 Days of Clozapine Treatment

Figure 4.2. Peripheral neutrophil counts of guinea pigs during clozapine treatment. Values are expressed as the mean ± SD of each treatment group, n = 4.

141

3500 VN VNCL 3000 VD-A VD-ACL 2500 VD-B VD-BCL 2000

1500

1000

500 NeutrophilCount (cells/uLblood) 0 0 14 28 42 Days of Clozapine Treatment

Figure 4.3. Peripheral neutrophil counts of ODS rats during clozapine treatment. Values are expressed as the mean ± SD of each treatment group, n = 4.

4.5.3 Covalent Binding of Clozapine to Hepatic and Bone Marrow Proteins

Covalent binding of clozapine to hepatic proteins in the liver homogenate and bone marrow proteins in the bone marrow cell lysate were detected in all guinea pig and rats dosed with the drug. However, the binding in samples from the guinea pigs produced only faint bands ranging from 7.2 to 203 kDa (Figures 4.4 and 4.5). In contrast, immunoblots of the rat liver homogenate showed bands with molecular masses ranging from 30 to 240 kDa (Figure 4.6), whereas immunoblot of the bone marrow lysate blot showed only one prominent band at 49 kDa (Figure 4.7). From previous studies we believe that this band represents myeloperoxidase, the enzyme responsible for reactive

142 metabolite formation. There was no significant difference in the amount of clozapine covalent binding in samples from guinea pigs or rats between animals given an adequate vitamin C diet and those on the deficient diet.

VN VNCL VDCL

kDa

94 72

54

Figure 4.4. Covalent binding of clozapine in the guinea pig liver. Immunochemical detection of covalent binding of clozapine to hepatic proteins of vitamin C-deficient guinea pigs (VDCL) and those which received adequate vitamin C (VNCL) given a daily dose of 50 mg/kg clozapine for 55 days. Lane VN represent sample from a guinea pig on the vitamin C-adequate diet without clozapine treatment. Protein loading was 10 µg/lane, and the primary antiserum was used at a dilution of 1:3000. Each lane represents sample from an individual animal.

143 VDCL kDa VN VNCL 203

49.1

20.6

Figure 4.5. Covalent binding of clozapine in the guinea pig bone marrow. Immunochemical detection of covalent binding of clozapine to bone marrow proteins of vitamin C-deficient guinea pigs (VDCL) and those which received adequate vitamin C (VNCL) given a daily dose of 50 mg/kg clozapine for 55 days. Lane VN represent sample from a guinea pig on the vitamin C-adequate diet without clozapine treatment. Protein loading was 60 µg/lane, and the primary antiserum was used at a dilution of 1:2000. Each lane represents sample from an individual animal.

144 kDa VNCL VD-ACL VD-BCL 203

49.1

20.6

Figure 4.6. Covalent binding of clozapine in the ODS rat liver. Immunochemical detection of clozapine-modified hepatic proteins from clozapine- treated vitamin C- deficient ODS rats given drinking water supplemented with L-ascorbic acid at 1.67 g/L (VNCL), 0.33 g/L (VD-ACL) and 0.2 g/L (VD-BCL). Rats were administered a daily dose of 50 mg/kg clozapine for 42 days. Protein loading was 30 µg/lane, and the primary antiserum was used at a dilution of 1:3000. Each lane represents sample from an individual animal. An untreated control sample (VN) was analyzed on a separate blot and did not show any clozapine protein covalent binding.

145 kDa VNCL VD-ACL VD-BCL

49

Figure 4.7. Covalent binding of clozapine in the ODS rat bone marrow. Immunochemical detection of clozapine-modified bone marrow proteins from clozapine- treated vitamin C-deficient ODS rats given drinking water supplemented with L-ascorbic acid at 1.67 g/L (VNCL), 0.33 g/L (VD-ACL) and 0.2 g/L (VD-BCL). Rats were administered a daily dose of 50 mg/kg clozapine for 42 days. Protein loading was 30 µg/lane, and the primary antiserum was used at a dilution of 1:2000. Each lane represents sample from an individual animal. An untreated control sample (VN) was analyzed on a separate blot and did not show any clozapine protein covalent binding.

146 4.6 Discussion

In an in vitro study conducted by Pereira et al. the addition of ascorbic acid significantly decreased bone marrow stromal cell death caused by clozapine [308]. This is likely due to the ability of ascorbic acid to inhibit clozapine oxidation or reduce the reactive nitrenium ion back to clozapine [311]. Although scurvy is not common in present day society, schizophrenic patients have been reported to have low to scorbutic levels of vitamin C for reasons such as poor diets that resulted in nutritional deficiency

[314, 320]. It has been suggested that co-administration of vitamin C with clozapine may reduce risk of agranulocytosis by its cytoprotective properties [321]. The aim of this study is to evaluate the hypothesis that vitamin C deficiency is a risk factor for clozapine-induced agranulocytosis in the guinea pigs. Beside humans, and other primates, the guinea pig is one of the few species that cannot produce vitamin C. As expected, ascorbate levels in plasma, serum, peripheral leukocytes, bone marrow cells, and heart were much lower or even below the detection limit in guinea pigs given a vitamin C-deficient diet (Table 4.1). Despite prolonged treatment of vitamin C-deficient guinea pigs with clozapine, decreasing trends in white blood cell or neutrophil counts were not observed (Figure 4.2). However, the degree of covalent binding observed in guinea pig samples (Figures 4.4 and 4.5) was much less than we had observed previously in samples from rats treated with clozapine or neutrophils from humans treated with clozapine, suggesting the degree of bioactivation in guinea pigs is less than that in rats and humans. Therefore, we judged that the guinea pig is not a good animal in which to test the hypothesis.

Normal rats cannot be made vitamin C-deficient because they are capable of producing ascorbic acid from glucose via the glucuronic pathway. However, a mutant strain, the ODS rat, bears an inborn deficiency of 1-gulonolactone oxidase and thus it

147 lacks the ability to produce ascorbic acid. These animals can be given a marginally scorbutic diet without showing any overt signs of scurvy for a few months. Therefore this animal offered a good alternate to the guinea pig for these studies because we had already studied covalent binding of clozapine in rats and covalent binding of clozapine in rat neutrophils is comparable to that in human neutrophils. As with guinea pigs, the serum levels of ascorbate in ODS rats on a vitamin C-deficient diet were significantly lower compared to those measured in vitamin C-adequate group or in Wistar rats (Table

4.2). However, as with guinea pigs, there was no effect of the vitamin C deficient diet on neutrophil counts (Figure 4.3). Furthermore, vitamin C deficiency did not appear to have any significant effect on the amount of clozapine covalent binding in either the hepatic or bone marrow tissues from guinea pigs or ODS rats (Figure 4.4-4.7). It is interesting that clozapine treatment appeared to decrease ascorbate levels in the heart and it is possible that this is related to the myocarditis sometimes observed in patients treated with clozapine [279, 322]. Given the very rapid inactivation of the nitrenium ion by vitamin

C, it is somewhat surprising that vitamin C deficiency did not lead to an increase in covalent binding. This suggests that other detoxification pathways for the nitrenium ion exist. It has been shown that a decrease in glutathione in rats can induce a compensatory increase in ascorbic acid level [323]. If the reverse is also true it may explain why covalent binding was not affected by vitamin C deficiency. If vitamin C deficiency had failed to lead to agranulocytosis in this model but did significantly increase covalent binding, it would suggest that vitamin C deficiency might be necessary but not sufficient to lead to clozapine-induced agranulocytosis. However, the observation that vitamin C deficiency did not even significantly increase covalent binding of clozapine makes it very unlikely that vitamin C deficiency is a major risk factor for clozapine-induced agranulocytosis.

148 4.6 Acknowledgements

Dr. Jack P. Uetrecht is the recipient of the Canada Research Chair in Adverse Drug

Reactions. The work was supported by grants from the Canadian Institutes of Health

Research

149

CHAPTER 5

INVESTIGATION OF THE MECHANISM OF CLOZAPINE-INDUCED AGRANULOCYTOSIS: A FOCUS ON THE EFFECT OF CLOZAPINE ON NEUTROPHIL KINETICS

150 5.1 Abstract

Clozapine is an atypical antipsychotic effective in the treatment of refractory schizophrenia; however, its use is limited due to its propensity to cause agranulocytosis in some patients. Little is known about the mechanisms of idiosyncratic drug-induced agranulocytosis, in part because of the lack of a valid animal model. Clozapine is oxidized by activated human neutrophils and bone marrow cells to a reactive nitrenium ion by the myeloperoxidase-hydrogen peroxide system of neutrophils. This reactive metabolite has been shown to induce apoptosis and cytotoxicity in neutrophils and bone marrow cells. While in vitro studies demonstrated the toxic potential of clozapine, the in vivo conditions could be quite different. Therefore, we conducted this study in rabbits to study the effect of clozapine treatment on neutrophil kinetics in vivo using the fluorescein dye, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and the thymidine analogue, 5-bromo-2-deoxyuridine (BrdU) to label neutrophils. Clozapine, indeed, increased the rate of both the release of neutrophils from the bone marrow and their subsequent disappearance from the circulation. Failure of the bone marrow to compensate for this apparent shortened neutrophil half-life could lead to agranulocytosis.

Alternatively, it is also possible that the damage to neutrophils caused by clozapine can lead to an idiosyncratic immune response resulting in agranulocytosis.

151 5.2 Introduction

Clozapine belongs to the class of atypical dibenzodiazepine antipsychotics drugs that lacks extrapyramidal side effects. It is the drug of choice for the treatment of refractory schizophrenia; however, it’s use is limited due to its propensity to cause agranulocytosis in ~0.8% of patients [177, 305]. Agranulocytosis is a potentially fatal blood dyscrasia characterized by a dramatic decrease in the neutrophil count. Patients who are on clozapine are required to monitor their neutrophil counts closely [177]. Little is known about the mechanism of idiosyncratic drug-induced agranulocytosis, including that caused by clozapine. We had previously reported that clozapine is oxidized to a reactive nitrenium ion by activated human neutrophils and bone marrow cells through the myeloperoxidase-hydrogen peroxide system of neutrophils [198, 199]. This nitrenium ion was found to covalently bind to the neutrophils of patients who take the drug [124, 322]. Studies have also shown that clozapine is capable of inducing apoptosis and cytotoxicity in neutrophils and bone marrow cells in vitro in the presence of a bioactivation system [200, 202, 209, 308-310]. It is difficult to determine if the in vitro conditions mimicked in vivo conditions, and treatment of most patients and animals does not lead to a decrease in their neutrophil counts. However, the bone marrow has a large capacity to generate neutrophils; therefore, it is possible that although clozapine causes neutrophil damage and cell death, the bone marrow is able to keep up with the production and release of neutrophils to maintain normal neutrophil counts. If clozapine does cause significant neutrophil damage in vivo , according to the danger hypothesis, it is possible that this could lead to an immune response resulting in an IDR, in this case agranulocytosis. Therefore we had set out to determine if clozapine can cause a change in neutrophil kinetics in vivo that would reflect neutrophil damage.

152 5.3 Materials and Methods

5.3.1 Animals

Female New Zealand White rabbits (2.5-4 kg, specific pathogen-free) were purchased from Charles River Laboratories (St. Constant, QC). All animals were housed singly with a 12:12 h light:dark cycle at 22 oC. They were acclimatized and given access to regular rabbit diet (Harlen Teklad, Madison, WI) and tap water ad libitum for 1 week prior to the beginning of the experiment. The experimental protocols were approved by the University of Toronto’s Animal Care Committee.

5.3.2 Chemicals

Clozapine was kindly provided by Novartis (Dorval, QC) and DMP 406 by

Dupont-Pharma (Wilmington, DW). Dimethyl sulfoxide (DMSO), 5-bromo-2’- deoxyuridine (BrdU), paraformaldehyde, polyethylene glycol 400 (PEG), acetic acid, ammonium acetate, and dextran were purchased from Sigma-Aldrich (St. Louis, MO).

The source of 5-(and-6-)-carboxyfluorescein diacetate succinimidyl ester (CFSE) was

Molecular Probes (Eugene, Oregon). Sterile phosphate buffered saline (PBS), Hanks’ balanced salt solution (HBSS), RPMI 1640, and Iscove's Modified Dulbecco's medium

(4 mM L-glutamine, 4500 mg/L glucose and 1500 mg/L sodium bicarbonate) were prepared by University of Toronto Tissue Culture Centre.

5.3.3 Measurement of Neutrophil Kinetics

Measurements of neutrophil kinetics using the fluorescent dye, CFSE, were performed according to the method described by Iverson [78] with slight modifications.

153 Female New Zealand White rabbits were dosed with clozapine (33.3 mg/ml of the hydrochloride salt in sterile saline by subcutaneous injection). Control rabbits were given similar injections of sterile saline. The rabbits were given clozapine for 10 or 18 days at 30 mg/kg/day.

A stock solution of the CFSE dye was made by dissolving it in DMSO at 16.13 mg/ml. Aliquots of 100 µL were prepared and stored at -20 oC until used. Prior to injection of the dye, the stock CFSE solution aliquots were thawed and combined.

Dilution of the dye was made at 1 part stock to 5 parts PEG 400 prior to injection.

Previous studies conducted by Hay et al. [324, 325] had found that 2 µg of CFSE per ml of blood is needed for adequate staining of the neutrophils. Therefore, CFSE was administered at 0.14 mg/kg body weight with the assumption that rabbits have 70 ml blood/kg. One hour after the last dose of clozapine or vehicle, local anesthetic cream

(Emla, 2.5% lidocaine, 2.5% prilocaine; Astra Pharma, Inc.) was applied to the ear and then the CFSE dye was injected into the marginal ear vein over a period of 2 minutes.

Blood samples (300 µL) were drawn from the ear vein using a heparinized syringe at various time points up to 10 hours post dye injection. The samples were prepared for flow cytometry analysis no longer than three hours after collection. Blood was transferred to 12 x 75 mm polystyrene tubes suitable for flow cytometry analysis and 4 ml of red cell lysis buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate,

0.1 mM EDTA) was added. After a 5-10 min incubation at room temperature, the samples were centrifuged at 800 g for 6 min and washed in 4 ml PBS to isolate the white blood cells. The cells were then resuspended in 250 µL PBS and fixed by adding 250 µL of 2% (w/v) paraformaldehyde in PBS. Samples were stored at 4 oC in the dark overnight until flow cytometry analysis.

154 Fluorescence of stained white blood cells was measured on a dual laser Becton-

Dickenson FACSCalibur using FL1 (530 ± 15 nm) for detection. Data were acquired and analyzed using the Cellquest software (Becton-Dickenson, Mountainview, CA). The neutrophil population was selected within the white blood cells for analysis based on the forward and side angle light scatter of neutrophils. The population with the highest side scatter was selected since neutrophils are the most granular white blood cells and this gate was labeled R1. Within R1, a second gate, R2, was set on the brightest population such that 79-85% of these cells were inside the gate. R2 was set using the sample collected from the first time point and was kept constant for the analyses of the remaining samples from the same animal. The percentage of cells in R2 was determined for each time point for each rabbit.

5.3.4 Measurement of Neutrophil Release from the Bone Marrow

In the acute study, 3 female New Zealand White rabbits were treated with 30 mg/kg clozapine per day for 12 days by subcutaneous injection as previous described.

Three rabbits in the control group were given the saline vehicle. In the chronic study, 3 rabbits were treated with clozapine in the drinking water. Clozapine was added to the drinking water at concentrations required for a daily dose of 40 mg/kg for 29 days.

Three rabbits were in the corresponding untreated control group.

BrdU was dissolved at 10 mg/ml sterile PBS (100 mg/kg) and injected slowly over 10 min into the left marginal ear vein 1 h after the 4 th dose of clozapine in the acute study or after the 21 st dose in the chronic study. Clozapine treatments were continued for 8 days after the BrdU injection.

Blood samples (1 ml) were collected from the marginal ear vein using EDTA- coated syringes at various time points after BrdU administration. A 100 µL aliquot was

155 taken for leukocyte counts and blood smear slide preparation to obtain an absolute neutrophil count. Leukocytes were isolated from the rest of the blood sample with removal of the red blood cells by incubation in 9 ml red cell lysis buffer at room temperature for 5-10 min followed by centrifugation at 800 g for 6 min. The remaining leukocytes were washed in PBS and transferred into 12 x 75 mm polystyrene tubes. The leukocyte samples were then fixed and permeabilized using the BD Pharmingen TM FITC

BrdU Flow kit (BD Biosciences, San Diego, CA). They were then washed in staining buffer and stored in freezing media at -80 oC until flow cytometry analysis. Upon thawing of the leukocyte samples, the cells were washed with staining buffer and the fixation and permeabilization steps were repeated. They were then treated with DNase to expose the BrdU epitopes and stained with the FITC-conjugated anti-BrdU antibody, washed with staining buffer, and subjected to flow cytometry analysis similar to that described in the CFSE analysis to determine the percentage of cells within gate R2 (i.e. neutrophils labelled with BrdU).

5.3.5 Blood Cell Counts

Peripheral blood cell counts were obtained immediately after blood samples were collected. Total white blood cell counts were obtained by manual counting using an inverted light microscope and Turk’s solution to lyse the red blood cells and dilute the samples (1:10). Differential counts were obtained by manually counting white blood cell percentages on blood smear slides stained with Giemsa-Wright stain. Absolute cell counts of each type of white blood cells were calculated by multiplying the percentage from the differential count with the total white blood cell count. Cell counts were performed in triplicate to obtain standard deviations.

156 5.3.6 Measuring Clozapine Blood Levels

Clozapine blood levels in the serum of rabbits treated with the drug by a single subcutaneous injection at 30 mg/kg or chronic administration of the drug at 40 mg/kg/day for 21 days through the drinking water were determined by a LC/MS/MS assay method. Serum was isolated from 1 ml of blood samples collected from a rabbit given a single dose of clozapine at 0.5, 1, 4, 6, and 24 h after the injection and from a rabbit given clozapine chronically by administration through drinking water at 9 am, 12 pm, and 3 pm on the 19 th , 20 th, and the 21 st day of drug treatment. Whole blood samples were allowed to coagulate after collection from the marginal ear vein of the rabbits and centrifuged at 500 g for 10 min. Serum samples were stored at -80 oC until further processing. Serum (50 µl) was mixed with 150 µl of the internal standard (DMP406, 0.1

µg/ml in methanol) and 5 µl of 1:1 methanol:water. The mixture was vortexed and protein was allowed to precipitate at -20 oC for 1 h.

The supernatant was then collected and analyzed by LC/MS/MS using a PE

Sciex API 3000 quadrupole system with an electrospray ionization source (Sciex, ON) interfaced with an HPLC system (Shimadzu). The HPLC column was a Luna 3 µ C18 50 mm x 2 mm 3 µm column (Phenomenex). The mobile phase consisted of 40% methanol,

1% acetic acid and 2 mM ammonium acetate. The compounds were eluted isocratically with a flow rate of 0.2 ml/min. Mass spectrometry was operated in the positive ion mode at 500 oC. The detection and quantification of compounds were performed by positive ion multiple reaction monitoring (+MRM) mode. The ion transitions monitored were m/z 327.06/270.1 (Q1/Q3) for clozapine and 322.12/279.2 for the internal standard. The standard curve prepared for clozapine was 0.03-3 µg/ml with a R2 value >0.99. The mass spectrometer, LC system, mass calibration, data acquisition, data representation,

157 and post acquisition analyses were carried out using Analyst 1.4.2 Software (Applied

Biosystems/MDS SCIEX Instruments).

5.3.7 Statistical Analysis

Statistical analyses were performed using the Student’s t test or one-way

ANOVA with the GraphPad Prism program (GraphPad, San Diego, CA). Individual comparisons were performed using Newman-Keuls post hoc tests. Data were expressed as the mean ± s.d. Results were considered statistically significant if P<0.05.

158 5.4 Results

5.4.1 The Effect of Clozapine on Neutrophil Kinetics Measured Using CFSE

The effects of clozapine treatment on the half-life of circulating neutrophils and on their release profile from the bone marrow were studied in a rabbit model using CFSE to stain the cells. Flow cytometry showing the neutrophil population is shown in Figure

5.1A. Figure 5.1B provides flow cytometry data from two animals that are representative of the results obtained in the treated and control groups. A decrease in the percentage of CFSE-stained neutrophils was observed over time and the decrease was much higher in the rabbits that had been treated with clozapine for 10 days compared to the control group. Plots of the mean percentage of stained neutrophils in each treatment group or animal were prepared. The rate of disappearance of neutrophils in the

-kt circulation (t 1/2 ) was calculated by the following equation: N t = N 0e , where k is the rate of disappearance of CFSE-labeled neutrophils from the circulation, t is the time after the

CFSE injection, N t is the percent CFSE-labeled neutrophils in the circulation at time t, and N o is the percent CFSE-labeled neutrophils in the circulation at time 0. Since t 1/2 can be estimated as the time at which N t is equal to half of N o], the rate decay equation for t 1/2 becomes:

t1/2 = ln2/k

The constant, k, was calculated with the Prism version 2.0c graphing and statistics software (San Diego, CA) using a first-order non-linear regression model. The half-life can also be calculated by plotting the logged values against time, where t1/2 = [log e (ln

2)] / slope.

159 A)

B)

Figure 5.1. Neutrophil kinetics in rabbits measured by a decrease in CFSE-stained cells. One hour after the last dose of clozapine (30 mg/kg), each rabbit was injected with CFSE. A) neutrophil population as determined by forward and side scatter. B) flow cytometry data showing the disappearance of CFSE-labelled neutrophils over time in a representative animal from each of the treated and control group.

160 In the experiment where three rabbits were treated with clozapine at a dose of

33.3 mg/kg for 10 days, the mean circulating half-life of neutrophils in treated group was

1.8 ± 0.1 h while the control group had a significantly longer half-life of 5.5 ± 1.9 h (p

=0.028, Figure 5.2 A and B). The half-life was calculated using the values obtained in the first two hours because the decay curve seems to be bi-phasic. The decay constant appeared to be different in the initial portion of the curve compared to the latter portion.

It is not clear why the kinetics appear to be bi-phasic. It is possible that it is consistent with a 2-compartment model. However, we have no intention of applying a complicated mathematical analysis to the data until we have found out the reason for such bi-phasic kinetics. Therefore, only the initial portion of the curve (first 2 hours) would be more representative of the disappearance of neutrophils and were compared between the treated and control group.

A)

161 B)

Treated t1/2 (hours) Control t1/2 (hours) t-test T1 1.9 C1 9.2 T2 1.6 C2 3.3 T3 1.9 C3 4.0 Mean t 1/2 1.8 ± 0.1 5.5 ± 1.9 p= 0.028

Figure 5.2. The effect of 10 days of clozapine treatment on the percentages and half-life of circulating neutrophils. A) Three rabbits (T1, T2, and T3) were dosed with clozapine by subcutaneous injections for 10 days at 30 mg/kg/day. Three control rabbits (C1, C2, and C3) were injected similarly with the vehicle. The fluorescent dye CFSE (2 µg/ml blood) was injected into each rabbit one hour after the last dose of clozapine and blood was collected at various time points up to 7 h. B) To obtain the circulating half-life of stained neutrophils by plotting Log (% stained neutrophils) vs. time and calculating the slope by linear regression.

162 In the second experiment, where neutrophil kinetics were measured in a rabbit after 10 days and 18 days of clozapine treatment, the circulating half-lives were similar on both occasions: 1.3 h and 1.2 h, respectively (Figure 5.3). These values were also similar to the ones obtained for the treated group in the second experiment where the rabbits had been treated with clozapine for 10 days.

t1/2 after 10-days t1/2 after 18-days Treatment Treatment CL-2-1 1.3 hours 1.2 hours

Figure 5.3. Neutrophil kinetics of a rabbit after 10 days and 18 days of clozapine treatment. The circulating half-life of neutrophils was measured at two different time points (10 and 18 days) of the clozapine treatment in one rabbit (CL-2-1).

163 5.4.2 The Effect of Clozapine on Neutrophil Kinetics Measured Using BrdU

Measurement of neutrophil kinetics in terms of their release from the bone marrow was performed using BrdU. A significant increase in the number of circulating

BrdU-stained neutrophils was observed in the acute clozapine-treated group compared to control (Figure 5.4A). The peak level of labelled neutrophils, which increased to a count of 1500 x 10 6 labelled cells/ml blood, occurred at 48 hours post-BrdU injection while the control group attainted a peak level of only approximately 750 x 10 6 labelled cells/ml blood and only after 72 hours. Figure 5.5B shows the stained neutrophil profile from the longer clozapine treatment. Both the control and treated group achieved a peak level at

78 hours and the number of stained neutrophils was similar and significantly less than those rabbits with a shorter treatment.

164 A)

2500 Treated Control / L 6 2000 48 h

1500 * 72 h

1000

500 * BrdU-labeled Neutrophils x 10

0 0 24 30 48 54 72 78 96 102 120 144 168 192 Time Post BrdU-Injection (h)

B)

1200 Treated

1000

/ L Control 6

800

600

400

200 BrdU-Labeled Neutrophils Neutrophils x 10 BrdU-Labeled

0 0 6 24 42 48 54 72 78 96 102 120 144 168 192 Time Post BrdU-Injection (h)

165 C)

8.0×10 6 Treated Control 7.0×10 6

6.0×10 6

5.0×10 6

4.0×10 6

3.0×10 6

2.0×10 6

1.0×10 6

Total neutrophil (cells/mlcount blood) 0 0 25 50 75 100 125 150 175 200 225 Time post-BrdU injection (h)

D)

3.5×10 6 Treated 3.0×10 6 Control

2.5×10 6

2.0×10 6

1.5×10 6

1.0×10 6

Total neutrophil count (cells/ml blood) 5.0×10 5

0 0 25 50 75 100 125 150 175 200 225 Time post-BrdU injection (h)

166 Figure 5.4. Kinetics of neutrophils in rabbits measured using BrdU. A) In the acute study, rabbits were treated with clozapine (30 mg/kg/day) for 12 days. B) In the chronic study, rabbits were treated with clozapine in the drinking water at a daily dose of 40 mg/kg for 29 days. BrdU was injected 1 h after the fourth dose of clozapine treatment in the first study or after the 21 st dose in the second study. Clozapine treatments were continued for 8 days after BrdU injection. The number of BrdU-labelled neutrophils in circulation was obtained by multiplying the absolute neutrophil counts by the percentage labelled. The total neutrophil counts (BrdU-stained and unstained) in the acute study C) and chronic study D) were also obtained. Results are presented as the mean neutrophil count ± s.d.; n= 3. *Significant increase from control (P < 0.05).

5.4.3 Clozapine Blood Levels in Rabbits

To compare the blood levels of clozapine in rabbits given the drug by two different routes of administrations, serum concentrations were quantified by a

LC/MS/MS method. Figure 5.5 shows the product ion mass spectrum of clozapine and the internal standard. A representative LC/MS/MS +MRM extracted ion chromatogram of the serum samples is shown in Figure 5.6. The highest serum concentration of clozapine in a rabbit given clozapine through the drinking water for 21 days at 40 mg/kg/day was less than one tenth of the highest concentration quantified in a rabbit given a single dose of the drug at 30 mg/kg by subcutaneous injection (Table 5.1).

167

Figure 5.5. Product ion mass spectrum of clozapine and the internal standard.

168

Figure 5.6. LC/MS/MS +MRM extracted ion chromatogram of serum from the rabbit administered clozapine given through the drinking water for 21 days at 40 mg/kg/day.

169 Table 5.1. Serum concentrations of clozapine in a rabbit given a single dose of clozapine by subcutaneous injection (30 mg/kg) and a rabbit given clozapine through the drinking water for 21 days determined by the LC/MS/MS method.

Subcutaneous injection of a single dose (30 mg/kg)

Blood samples collected at: Serum Concentration of Clozapine ( µg/ml) 0.5 h 1.71 1 h 1.69 4 h 1.22 6 h 1.01 24 h 0.04

In drinking water (40 mg/kg/day) for 21 days

Blood samples collected at: Serum Concentration of Clozapine ( µg/ml) 9 am 0.07 12 pm 0.13 3 pm 0.07

170 5.5 Discussion

Many studies have been conducted over the last two decades to investigate how clozapine causes idiosyncratic agranulocytosis, but none so far has led to a clear mechanistic understanding. I began my study on clozapine in this chapter with some neutrophil kinetic experiments in which we sought to determine the half-lives of these cells in the peripheral circulation as well as their release profiles from the bone marrow.

Pharmacological alterations of neutorphil kinetics are rarely measured in humans because of ethical issues and the lack of a simple and feasible cell tracking technique that is not toxic to the cells. To our knowledge, this is one of the first few studies that looks at the effect of clozapine treatment on neutorphil kinetics in animals. The New Zealand

White rabbit is a suitable model for this type of cell kinetics study because it has a larger blood volume than rats, which enables the collection of serial blood samples. Although the rabbits’ neutrophils, known as heterophils, are morphologically different from human neutrophils, they are functionally similar in that they possess the same receptors and oxidative burst machinery such as the Fc receptors, NADPH oxidases, and myeloperoxidase (MPO), which are similar to human neutrophils [326-329]. The CFSE staining technique used in this study to measure neutrophil kinetics was first optimized by Dr. Suzanne Iverson in our lab [78]. It involved labelling peripheral blood cells with a fluorescent dye, CFSE, which is a diacetate succimidyl ester. It is able to diffuse across cell membranes and becomes fluorescent and non-membrane permeable when the acetate groups are cleaved by intracellular esterases. The fluorescent dye is trapped in the cells with the formation of protein conjugates when the succinmidyl ester moiety reacts with amines forming covalent bonds with proteins inside the cells. CFSE has been found to be relatively non-toxic to animals and cells at low concentrations [330]. It has been used in many studies to label cells in vivo via intravenous injections of the dye [324,

171 331]. Another labelling technique used in this study was with BrdU. BrdU is a thymidine analogue that becomes incorporated into the DNA of proliferating cells. It has been widely used to measure cell proliferation and cell kinetics in animals [332, 333]. In this case, the cells are labelled in the bone marrow and then can be tracked as they enter the peripheral circulation and ultimately are removed as they undergo apoptosis.

Results from studies conducted by Iverson found that the circulating half-life of neutrophils in rabbits after 10 days of clozapine treatment was significantly shortened,

1.7 h (2.9-1.2), r 2=0.94 compared to control at 4.7 h (6.7-3.6), r 2=0.99 [78]. I have repeated this study with the same conditions and the results obtained from this experiment, in which all three rabbits had a significantly shorter circulating half-lives with a mean of 1.8 ± 0.1 h vs. 5.5 ± 1.9 h in the control group, was similar to what

Iverson had obtained in her study. In addition, extending clozapine treatment to 18 days did not result in any significant difference in the neutrophil half-life compared to 10 days.

The in vivo labelling of cells with the fluorescent dye, CFSE, allowed us to show that circulating half-lives of neutrophils in clozpaine-treated rabbits were shorter compared to control untreated rabbits. In Iverson’s studies, an acute response neutrophilia was observed in rats and rabbits given one dose of clozapine [78]. Demargination of neutrophils into the circulation, release of neutrophils from the bone marrow, decreased apoptosis of circulating neutrophils, or a combination of two or all of the above can lead to clozapine-induced neutrophilia. Neutrophilia is common in patients, particuraly during the first few weeks of clozapine treatment [183]. Therefore, we wanted to measure the neutrophil kinetics in clozapine-treated rabbits when their hematological parameters were more stable. Moreover, we were interested in the effect of a more chronic clozapine treatment on circulating neutrophils. As the white blood cell counts in treated animals return to similar levels as those before and after clozapine injections, a

172 difference in neutrophil kinetics was also observed after ten days of drug treatment.

Neutrophils in the clozapine-treated group had a significantly shorter half-life compared to untreated controls. The mechanism involved with this change in neutrophil kinetics upon clozapine treatment is unclear at this point.

Clozapine could have an effect on circulating neutrophils and/or the bone marrow.

Bone marrow being the site of initial toxicity in clozapine-induced agranulocytosis has long been suspected and studied [201, 202]. Indeed, the BrdU assay showed neutrophils in rabbits treated with clozapine for 4 days had a much shorter transit time throught the bone marrow and significantly more new cells were released compared to controls. Most of the new neutrophils were produced and released within 48 h in the clozapine-treated group as represented by the length of time required to attain the highest stained neutrophil count of around 1500 x 10 6 cells/L post-BrdU injection. In contrast, the control group had a mean bone marrow transit time of 72 h and the highest stained neutrophil count was ~750 x 10 6 cells/L indicating that a lesser number of new cells were released. These differences in terms of the bone marrow transit time and number of new cells released between the treated and control group were not observed in the longer treatment of 21 days with the drug given in the drinking water. However, in a separate experiment, we found that a rabbit given 40 mg/kg of clozapine in the drinking water for more than 2 weeks had serum levels of clozapine between 0.071 to 0.13 µg/ml while the highest serum concentration measured in a rabbit given a single dose of clozapine by subcutaneous injection was 1.71 µg/ml. Therefore, clozapine administered by drinking water achieved less than one tenth of the serum level as when the drug was given subcutaneously, and it was also significantly lower than the usual therapeutic concentration (0.35-0.6 µg/ml). This is consistent with the oral bioavailability observed in humans of about 50% which is likely due to a first pass effect [334].

173 The shortened bone marrow transit time along with the decrease in circulating half-life combined with the increase in peripheral neutrophil counts indicates that the bone marrow is able to keep up with the increased rate of destruction in neutrophils by releasing more new cells at a faster rate. Indeed, clozapine-treated patients have a significant increase in band neutrophils in the peripheral circulation when compared to the control group as described in a study by Delieu et al. , even though most of these patients do not develop clozapine-induced agranulocytosis [188]. If the ability of the bone marrow to compensate for the increased turnover of neutrophils were compromised it could be one possible cause of clozapine-induced agranulocytosis.

There is increasing evidence from various studies to show that clozapine-induced agranulocytosis may be accompanied by apoptosis of neutrophils or its precursors. In vitro studies by Williams et al . found that therapeutic concentrations of clozapine with the addition of an oxidizing system can induce apoptosis in human neutrophils [209]. In another study, Husain et al. found a decrease in surface expression of CD16 along with an increase in Fas ligand gene expression in clozapine-treated human polymorphronuclear cells which were also found to be apoptotic [310]. CD16 is the receptor on neutrophil plasma membrane for the Fc portion of IgG. Loss of CD16 expression in granulocytes has been found to be closely correlated with apoptosis of neutrophils in culutre [335]. It is generally accepted that the fas-fas ligand interaction is one of the mechanisms which triggers granulocyte apoptosis [336]. In addition to in vitro incubations, neutrophils isolated from a patient on clozapine also displayed elevated expression levels of pro-apoptotic genes which included p53 , bax α, and bik

[337]. Given that we saw a decrease in neutrophil half-life, it is resonable to hypothsize that their disapparrance from the circulation is a result of destruction of these cells possibly due to accelerated apoptosis induced by clozapine.

174 In a preliminary study, we have shown using Western blot analysis of caspase 3 cleavage and electrophoresis to detect DNA fragmentation that clozapine was capapble of inducing apoptosis in the HL-60 cell line (refer to section A1.4.3 of Appendix 1). In addition to in vitro incubations, studies in our lab have also attempted to quantify apoptosis in animals treated with clozapine. Apoptotic cells in the circulation are hard to detect because their numbers are diluted by new cells entering the circulation dilute and also because they are swiftly removed by the reticuloendothelial system. Therefore

Iverson had devised a method in which CFSE dye was injected into animals and the level of apoptosis in neutrophils stained with the dye was determined [78]. In her studies, both

CFSE-stained and total neutrophils in rabbit treated with four doses of clozapine showed only a slight increase in apoptosis as measured by the Annexin V assay. It is likely that most of the apoptotic cells have already been removed from circulation by phagocytic cells as this is the fate of these dead cells. I have also attempted to study the effect of clozapine on neutrophil apoptosis in vivo using a different staining technique. FLIVO TM , from Immunochemistry Technologies LLC, detects activation of caspases in situ . A fluorescent probe was injected i.v. into the animal. The probe permeates cells and covalently binds to activated caspases. Therefore, only cells undergoing apoptosis will fluoresce, which can then be detected and quantified by flow cytometry upon sacrifice of the animal and isolation of their leukocytes. Two attempts were made using this kit, but no cells, especially neutrophils, with sufficient fluorescent intensity were detected with flow cytometry. This kit was made for the purpose of studying apoptosis and cytotoxicity in mice. We have made an attempt to use this method in rats, which are much bigger in size and body weight. Therefore, more of the probe may have to be injected in order to obtain the same effect, but toxicity and efficacy of this probe have not been fully assessed in rats yet. Despite all these attempts to try to detect and quantify

175 the level of apoptosis in animals during clozapine treatment, it is possible that we may not see a significant difference between treated and control animals because, if the rate of apoptosis is increased, it is likely that the rate of cell removal by the reticuloendothelial system is also increased; thus the steady state concentration of apoptotic cells may not change significantly.

In summary, based on the results obtained from this study, one possible mechanism of clozapine-induced agranulocytosis is the following. The fate of neutrophils, after being produced in the bone marrow, begins with their release from the bone marrow into the circulation, followed shortly by apoptotic death. Clozapine may accelerate this process by inducing apoptosis in circulating neutrophils. The bone marrow could compensate for this by releasing more cells into the circulation in order to maintain normal neutrophil counts. However, failure of the bone marrow to compensate may lead to a decrease in the number of circulating neutrophils. In fact, studies by

Pereira et al. found a dose-dependent inhibition of cell viability when bone marrow stromal cells were incubated with clozapine in an in vitro system [308]. This may very well be the case in a small percentage of the population who are susceptible to clozapine- induced agranulocytosis. Alternatively, damage done to neutrophils by clozapine could induce an immune response in some patients that results in agranulocytosis. Further studies and more importantly animal models are needed to validate this proposed mechanism and to understand the pathogenesis of this idiosyncratic reaction.

176

CHAPTER 6

CONCLUSIONS AND FUTURE WORK

177 6.1 Conclusions

The aim of these studies was to investigate the mechanisms of idiosyncratic drug- induced agranulocytosis. It is rather hard to study IDRs without valid animal models.

Thus, the first part of this thesis focused on developing an animal model of this IDR.

Animal models of IDRs are rare because these reactions are just as idiosyncratic in animals as they are in humans. We did not anticipate developing an animal model of drug-induced agranulocytosis to be trivial because many past attempts have been unsuccessful. Thus, it was not surprising that the attempts described in this work were unsuccessful. Nevertheless, animal models are an essential tool for studying IDRs.

They allow us to test certain hypotheses that are very difficult, if not impossible, to test in any other way.

In our attempts to develop an animal model of idiosyncratic drug-induced agranulocytosis, we began by taking the generalized hypothesis of the mechanism of

IDRs and intervening at various points along the proposed pathway to try to induce the

IDR in different animal species. Reactive metabolites are thought to play an important role in the induction of IDRs because they can potentially form hapten-protein complexes, which can be immunogenic, or be involved in some other non-immune mechanisms such as apoptosis or cytotoxicity. Attempts were made to develop an animal model of amodiaquine-induced agranulocytosis by treating Sprague-Dawley rats with subcutaneous injections of the drug along with PMA co-treatment to induce the production of reactive metabolites. PMA has been found to induce an oxidative burst in neutrophils. Activated neutrophils that go through an oxidative burst can metabolize many drugs, including amodiaquine, which forms a reactive iminoquinone metabolite.

We did not observe a decrease in neutrophil counts in the amodiaquine-treated animals either with or without PMA co-treatment. However, we did observe an increasing trend

178 in the total white blood cell counts and neutrophil counts after eight doses of the drug. It is possible that neutropenia or even agranulocytosis could occur after a longer period of amodiaquine treatment. However, we were unable to carry out extended experiments because chronic treatment of the rats with amodiaquine led to weight loss and general malaise. It was later found that amodiaquine has a long half-live and so it was accumulating in the animals with chronic treatment. Studies are now underway to develop an animal model of amodiaquine-induced hepatotoxicity in our lab. But so far, agranulocytosis has not been observed in these studies.

Most IDRs are thought to be immune-mediated. Although we do not have direct proof of this, the presence of drug-specific antibodies in many IDRs is strong evidence that the immune system is involved. Two such drugs that are known to cause agranulocytosis and drug-specific antibodies are aminopyrine and amodiaquine. Since the immune system is likely to be involved in the induction of agranulocytosis for these two drugs, we decided to “tweak” the immune system of the animals given these drugs.

Poly I:C is a potent immunostimulant; treatment with this synthetic RNA mimics a viral infection in the host and subsequent endogenous events that occur can induce “danger signals”. We placed New Zealand White rabbits and Brown Norway rats on a chronic treatment with aminopyrine along with Poly I:C co-treatment. However, none of these animals showed any decreasing trend in neutrophil counts that might be a precursor to agranulocytosis either in the presence or absence of poly I:C co-treatment. We even treated some rats with a combination of poly I:C with dipyrone (the sulfonated derivative of aminopyrine) which had been encapsulated in liposomes. The rationale was that putting the culprit drug in close proximity to a danger signal so that they are presented simultaneously to APCs would increase the chance of an immune response. Yet none of these animals developed agranulocytosis. One likely reason that it is so hard to develop

179 animal models of IDRs is that the usual response is immune tolerance. In another study in an attempt to break immune tolerance we added a co-treatment of 1-methyl-D- tryptophen (1-MT) to chronic treatment with aminopyrine or amodiaquine. 1-MT is an inhibitor of indoleamine 2,3-dioxygenase (IDO) which is the enzyme involved in the degradation of tryptophan. IDO has been found to mediate immune tolerance by inducing tryptophan deprivation, which leads to a decrease in effector T cell proliferation and an increase in apoptosis. However, these treatments did not lead to agranulocytosis.

In our next set of attempts to develop an animal model of drug-induced agranulocytosis, we focused on host factors. The reason we focused on host factors is that there was circumstantial evidence that points to the fact that the IDR induced by clozapine may not be immune-mediated. This includes the absence of a shortened time to onset of the reaction upon re-exposure to the drug characteristic of immune memory

[30, 338]. Taken together, such circumstantial evidence led us (at least at the beginning of my research) to think that the mechanism of clozapine-induced agranulocytosis could potentially be independent of an adaptive (T cell- and/or antibody-mediated) immune response. It appears that certain host factors such as sex, age, environmental factors such as infections and genetic disposition put certain people at a higher risk of developing

IDRs. However, none of the studies have looked at the nutritional status as a risk factor for IDRs. Based on studies which found lower levels of plasma and red blood cell selenium levels in patients on clozapine, we conducted a series of experiments to determine whether selenium deficiency could be a risk factor for this IDR. Selenium deficiency, which takes time to develop in a patient while on clozapine, could explain why there is a delay in onset of the IDR and this length of time to onset would be the same on re-exposure to the drug since it will take just as long for selenium deficiency to occur on subsequent exposures. Rats subjected to a selenium-deficient diet were then

180 given clozapine; however, prolonged treatment of the drug did not result in agranulocytosis. There was also no increase in the amount of clozapine-protein covalent binding in the liver and bone marrow of rats that were deficient in selenium. Thus, it seems unlikely that selenium-deficiency is a risk factor for clozapine-induced agranulocytosis. In another set of studies we also looked at vitamin C-deficiency as a risk factor for this IDR. Vitamin C has been found in previous in vitro studies to very rapidly reduce the nitrenium ion reactive metabolite of clozapine back to the parent drug.

Therefore, it has been suggested that patients on clozapine could reduce the risk of developing agranulocytosis by vitamin C supplementation. We placed guinea pigs, which turned out not to produce much clozapine reactive metabolite, and ODS rats, which have a genetic deficiency in the ability to synthesize vitamin C, on vitamin C- deficient diets and treated them with clozapine for more than a month. None of these animals developed agranulocytosis. Moreover, surprisingly, clozapine covalent binding to proteins in the liver and bone marrow was not increased in vitamin C deficient animals.

Thus, it also seems unlikely that vitamin C-deficiency is a significant risk factor for clozapine-induced agranulocytosis. Despite the many attempts in this thesis and the attempts of others in the lab, none produced a valid animal model of an IDR.

The second part of this thesis was focused on using various in vitro and in vivo assays to study the effects of clozapine on neutrophils. Although clozapine does not cause neutropenia in most patients or animals, its reactive metabolite has been shown to induce apoptosis in vitro . The bone marrow has a large capacity to produce neutrophils and so it might easily be able to compensate for an increase rate of neutrophil apoptosis.

On the other hand, the in vitro experiments may not reproduce in vivo conditions. I have continued the studies initiated by Dr. Susanne Iverson using CFSE to determine the effects of clozapine on neutrophil kinetics in vivo . She had found that the circulating

181 half-life of neutrophils appeared to be significantly longer in rabbits treated with clozapine for two days, but the half-life was significantly shortened after 10 days of clozapine treatment. I repeated this study and found similar results for the ten day treatment but did not observe a lengthened half-life in the two day treatment study.

CFSE studies are complicated by the fact that some of the decrease in fluorescence could be due to leakage of the dye from labelled cells. Therefore we used another method that labels the DNA of dividing cells with BrdU. This label does not leak out of cells and also provides a measure of the production and release of neutrophils from bone marrow.

The method clearly demonstrated that clozapine treatment led to an earlier release of neutrophils from the bone marrow and there was also a significant increase in the number of labelled cells that were released. This indicates that clozapine leads to an increase in the production of neutrophils and implies a decrease in neutrophil half-life, presumably due to an increase in the rate of apoptosis. This increase in neutrophil turnover was not observed when the rabbits were dosed in their drinking water rather than by subcutaneous injection, but the blood levels of clozapine administered in the drinking water were quite low; about 1/10 of that achieved by injection and they were also lower than the therapeutic concentration in humans.

The next step was to determine the mechanism by which clozapine increased the rate of neutrophil turnover. As indicated above, this is likely to be due to an increase in the rate of apoptosis, but we were unable to detect an increased number of apoptotic neutrophils in vivo in clozapine-treated animals. This is not surprising because, at steady state, the increase is likely to be quite small. One interesting result from Iverson’s studies was that clozapine decreased the respiratory burst in neutrophils stimulated with

PMA. PMA is a direct activator of PKC and activated PKC is required in the signalling pathway leading to a respiratory burst. This led us to test the hypothesis that clozapine is

182 PKC inhibitor. PKC is involved in the process of apoptosis, and the dominant effect of

PKC inhibition would be expected to be inhibition of apoptosis. However, the role of

PKC in the mechanism of apoptosis is complex, so it is possible that PKC inhibition could lead to either an increased or a decreased rate of apoptosis depending on the isozymes involved and the cell type. In our preliminary study on the effect of clozapine on PKC, we first looked at its effect on PKC activity in HL-60 cell lysate and found that clozapine at 3 to 4 µM, in the presence of the HRP/H 2O2 metabolizing system, appeared to have signifcantly inhibited PKC activity. In a second experiment with purified whole rat leukocytes, decreases in PKC activity upon clozapine incubation in the presence of the HRP/H 2O2 were also noted. In the in vivo experiments to determine clozapine’s ability to decrease PKC activity in rats, we treated these animals with clozapine for 6 weeks. Clozapine treatment did not lead to significant inhibition of PKC in the peripheral leukocytes, but a slight decrease was observed in the bone marrow cells. In the set of experiments with purified active human recombinant PKC, we found inconsistency in the inhibitory profiles among the three isozymes tested. And whether the reactive metabolite of clozapine was required for the inhibition was also inconsistent.

In addition to PKC inhibition we have also investigated the ability of clozapine to covalently bind with PKC. Results from our in vitro study show that clozapine is capable of covalently binding to the α, βII, and δ isozymes of PKC. However, we were unable to detect any clozapine covlant binding to PKC in rats treated with the drug on our first attempt using Protein A beads and mass spectrometry to identify the proteins that were covalently modified by clozapine (refer to Appendix 2). It is possible that covalent binding may lead to inhibiton of kinase activity in PKC. All in all, these preliminary results seem to show that clozapine may have an effect on PKC but further

183 studies are required to verify these results and to investigate how it may be involved in apoptosis.

In summary, the first part of this thesis focused on developing an animal model of idiosyncratic drug-induced agranulocytosis. Despite the large number of experiments based on several different hypotheses, none were sucessful in producing agranulocytosis or even significant neutropenia. However, we were sucessful at showing that clozapine causes a significant increase in the rate of neutrophil turnover. The results obtained from these studies, allowed us to propose a very basic mechanism of clozapine-induced agranulocytosis. Clozapine, or more specifically its nitrenium reactive metabolite, may accelerate apoptosis in circulating neutrophils. This may explain why some patients on clozapine experience neutropenia, as the drug may have an apoptotic effect on peripheral blood neutrophils. The bone marrow would compensate for this by releasing more cells into the circulation. However, failure of the bone marrow to compensate in certain individuals may lead to a more drastic decrease in the number of circulating neutrophils; hence, the clinical manifestation of agranulocytosis. In this case, clozapine may have an effect both on the bone marrow as well as peripheral blood neutrophils. We cannot rule out at this point that this is an immune-mediated reaction. Further studies are required to validate this hypothesis, but without an animal model of this IDR, testing this hypothesis may be impossible.

6.2 Future Studies

Much work still needs to be done to study the effect of clozapine on neutrophil kinetics. But from the data which we were able to gather so far, it appears that clozapine leads to an increase in the rate of neutrophil turnover. One could question whether this occurs in humans because we saw an effect in the CFSE and BrdU studies the blood

184 levels of clozapine were likely to be above therapeutic levels and at subtherapeutic levels there was no effect. What would happen at therapeutic levels? We should conduct future studies with doses of clozapine that would produce blood levels closer to the therapeutic levels in humans to see whether there are any appreciable changes in neutrophil kinetics and apoptosis. Besides blood levels, the level of clozapine reactive metabolite should also be considered. Future studies should be carried out to determine whether rabbit neutrophils, known as heterophils, also produce similar levels of the clozapine nitrenium ion as in rats and humans. These would involve quantification of the amount of covalent binding of clozapine to rabbit neutrophils.

The faster disappearance of neutrophils during chronic clozapine treatment is presumably due to an increase in apoptosis of these cells as this is what leads to their clearance. Although verification is required, the question then is by what mechanism does clozapine induce apoptosis? In an attempt to answer this question, we begun by conducting a series of preliminary studies to look at the effect of clozapine on PKC activity. Experiments conducted so far seem to produce inconsistent results in terms of the inhibitory profile of clozapine on three specific PKC isozymes, namely the α, δ, βII isozymes. More experiments are required in order to verify the results that we have observed. As mentioned earlier, perhaps the next step in determining whether PKC is involved in clozapine-induced agranulocytosis is to pre-incubate the HL-60 cell lysate with either a known activator or an inhibitor of PKC. By doing so, if clozapine is a PKC inhibitor and its inhibitory effect on PKC is what induces apoptosis then addition of a

PKC activator should block the apoptosis while apoptosis should still be observed or may even be potentiated with addition of a PKC inhibitor. In addition, other methods of down-regulating PKC activity should be sought to substantiate the role of this enyzme in clozapine-induced agranulocytosis. All in all, these studies may imply that future

185 investigations of the PKC signaling pathway involved in clozapine-mediated apoptotic activities should focus on specific PKC isoforms, which may help to better understand the mechanism. The use of antisense oligonucleotides, RNAi and selective pharmacological inhibitors, to specifically knock down the levels or kinase activity of individual PKCs in relevant cell lines may help to address these potential caveats.

If there is an increased release of neutrophils from the bone marrow during clozapine treatment to compensate for the accelerated apoptosis in the peripheral circulation, does clozapine have a direct effect on the bone marrow which causes it to release more cells or is it an epiphenomenon of the apoptosis induced by the drug? Thus we need to establish whether clozapine has an effect on the bone marrow, both on changes in the microenvironment and on the expression of cell surface receptors.

We also need to investigate why only a very small percentage of the patients on clozapine develop agranulocytosis. Are these patients for some reason seeing a higher level of the drug? In fact, a study had found that a patient who developed leukocytosis while on clozapine had a leukocyte level of the drug eight times higher than a control of patients who did not develop the IDR [339]. The results may suggest that patients at risk of clozapine-induced agranulocytosis show increased clozapine concentration in their leukocytes even though the clozapine plasma concentration is in the therapeutic range. It would be interesting and probably necessary to look at why and how clozapine is accumulated in the neutrophils in these individuals. Clozapine uptake in cultured HL-

60 cells has been shown to be mediated by a mechanism suggestive of an active unidirectional transport [340]. It is possible that there is a genetic polymorphism in the transport protein responsible for clozapine influx which may play a role in the development of clozapine-induced agranulocytosis. Further studies, particularly on the

186 molecular and biochemical characterization of this transporter, would be required to determine its involvement in the pathogenesis of this IDR.

Besides clozapine, other agranulocytosis-associated drugs should also be considered when carrying out the aforementioned studies and those in this thesis. A particular drug of interest would be olanzapine. It is an atypical antipsychotic structurally similar to clozapine and is also metabolized to a reactive nitrenium ion by neutrophils [200]. However, olanzapine is not associated with agranulocytosis to the extend of clozapine and no covalent binding of the drug was detected in neutrophils isolated from patients on the drug while incubation of the drug with neutrophils in vitro showed less protein covalent binding compared to clozapine [124]. This is presumably due to lower blood levels of olanzapine, but a lower affinity of the antibody for olanzapine could also play a significant role. A much higher concentration of olanzapine was required to induce cytotoxicity [124], but it did have similar effects on neutrophil oxidative burst in vitro [78].

Finally, although the development of an animal model of idiosyncratic drug- induced agranulocytosis seems far-fetched after failure of the countless attempts, they are still an important, if not the only way, to test certain hypothesis of IDRs in an in vivo system.

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224

APPENDICES

(PLEASE NOTE: references for the appendices are found at the end of this section)

225 APPENDIX 1

Investigation of the Effects of Clozapine on Protein Kinase C

A1.1 Background

Results from previous studies in our lab had shown that clozapine inhibited the

PMA-induced neutrophil respiratory burst [1]. This lead us to further hypothesize that clozapine may be a PKC inhibitor because PKC activation is required for a respiratory burst, and PMA is an activator of this kinase. We began to test this hypothesis with a series of in vitro and in vivo experiments. In the in vitro studies, lysates from the human promyelocytic leukemia (HL-60) cell line (which is closely related to neutrophil progenitors) and lysates from rat leukocytes were incubated with various concentrations of clozapine in the presence of the HRP/H 2O2 metabolizing system and PKC activity was assayed. In the in vivo experiments PKC activity were assayed in leukocytes and bone marrow cell lysates of clozapine-treated rats. We then further assayed the activity of specific isozymes of purified active recombinant PKCs after incubation with clozapine.

It has been shown by other investigators that clozapine is able to induce neutrophil apoptosis in vitro [2, 3] and possibly in patients treated with the drug [4, 5]. It is possible that clozapine’s inhibitory effect on PKC is responsible for the induction of neutrophil apoptosis. In this study, we incubated HL-60 cells with clozapine and demonstrated its ability to induce apoptosis by looking at the level of caspase 3 cleavage and DNA fragmentation. Using these assays, we then tried to determine whether clozapine-induced apoptosis is associated with its effect on PKC by down-regulating

PKC activities in these cells prior to exposing them to clozapine.

226 A1.2 Materials and Methods

A1.2.1 Animals

Female Sprague-Dawley rats (200-250 g) were purchased from Charles River

Laboratories (St. Constant, QC). All rats were housed in pairs in plastic cages with corncob chip bedding in a 12:12 h light:dark cycle at 22 oC. They were acclimatized and given access to regular rodent powder diet (Harlen Teklad, Madison, WI) and tap water ad libitum for 1 week prior to the beginning of the experiment. The experimental protocols were approved by the University of Toronto’s Animal Care Committee.

A1.2.2 Chemicals

Clozapine was kindly provided by Novartis (Dorval, QC). Sodium hypochlorite solution (NaOCl), ethylenediaminetetraacetic acid disodium salt (EDTA), phorbol 12- myristate-13-acetate (PMA), horseradish peroxidase (Type VI, HRP), hydrogen peroxide

(H 2O2), dimethyl sulfoxide (DMSO), and dextran were purchased from Sigma-Aldrich

(St. Louis, MO). Sterile phosphate buffered saline (PBS), Hanks’ balanced salt solution

(HBSS), RPMI 1640, and Iscove's Modified Dulbecco's medium (4 mM L-glutamine,

4500 mg/L glucose, and 1500 mg/L sodium bicarbonate) were prepared by University of

Toronto Tissue Culture Centre.

A1.2.3 PKC Activity Assay

PKC activity was quantified using the Omnia  Ser/Thr Recombinant kit, a commercial PKC kinase activity assay kit from Invitrogen (Camarillo, CA) for the experiments using purified recombinant PKC, and the StressXpress  PKC Kinase

227 Activity Assay Kit by Stressgene Bioreagents from Assay Designs (Ann Arbor, MI) for the experiments with cell lysates. Both are non-radioactive assays in which the former kit uses a fluorophore attached to a PKC substrate which fluoresces upon phosphorlyation, and the later kit is based on a solid phase enzyme-linked immuno- absorbent assay that utilizes a specific synthetic peptide as a substrate for PKC and an antibody that recognizes the phosphorylated form of the substrate. PKC activity was quantified in each sample in triplicate following the protocol provided in the kit.

In vitro experiments with purified active recombinant human PKC ( α, δ, and βII isozymes) obtained from Invitrogen and Stressgene Bioreagents were conducted by incubating 30 ng of PKC with various concentrations of clozapine (0-20 µM in DMSO at

0.5% of the incubation volume) for 20 min on ice prior to the kinase activity assay. In experiments where NaOCl was added, 0-20 µM was added to the incubation on ice 20 min before the kinase activity assay with clozapine at 18 times higher than the NaOCl concentration to ensure most of the NaOCl is consumed. NaOCl was diluted in a Tris buffer containing 20 mM Tris (pH 7.4) and 0.1 M NaCl. PKC activity for each sample was assayed.

In vitro experiments with rat leukocytes were conducted by isolating the cells from 8-9 ml of blood obtained from 1 rat by cardiac puncture with the addition of EDTA as an anticoagulant. Leukocytes were obtained by incubating the whole blood with equal volume of 3% dextran in saline for 30 min at 37 oC to settle the red blood cells. The upper fraction excluding the red blood cell layer was collected and centrifuged at 200g for 8 min to obtain the leukocyte pellet. Residual red blood cells were lysed using the isotonic red blood cell lysis buffer. The cells were then centrifuged and resuspended in

HBSS. Leukocytes (3.5 x 10 6 cells) were incubated with various concentrations of clozapine (0- 20 µM) at 37 oC for 1 h in the presence and absence of 20 units of HRP and

228 20 µM H 2O2. Clozapine was dissolved in DMSO and added at 0.5% of the incubation volume. HRP and H 2O2 were dissolved in PBS. Cells were washed and lysed with a lysis buffer consisting of 20 mM 3-morpholinopropanesulfonic acid (MOPS), 5 mM

EGTA, 1% Nonidet P-40, 1 mM DL-dithiothreitol (DTT), protease inhibitor cocktail with EDTA, phosphatase inhibitor cocktail, and 1 mM phenylmethanesulfonyl fluoride

(PMSF). Cell lysis was carried out for 10 min on ice with 3 X 20 sec bursts of sonication. The lysate was then centrifuged at 13,000 rpm for 15 min at 4 oC. The supernatant of the cell lysate was collected and kept frozen at -80 oC for protein quantification and PKC activity assays. Protein concentration was assayed in each sample with the Bio-Rad Bradford Reagent using the standard microplate method. PKC activity was quantified in 30 µg of proteins.

In vitro experiments with HL-60 cell lysates were done by isolating the cells from the culture medium and washing them with PBS. Cells were then lysed with lysis buffer on ice for 10 min. Protein concentrations were determined. Lysates containing 30 µg of protein were incubated in various concentrations of clozapine dissolved in DMSO (0.5% of the incubation volume) at 0-40 µM with 20 units of HRP and 20 µM H2O2 for 1 h at 4 oC prior to PKC activity quantification.

In vivo experiments in rats were conducted by treating four female Sprague-

Dawley rats (200 g) with clozapine at a daily oral dose of 50 mg/kg added in their diet for 6 weeks. Four untreated rats were kept as controls. Leukocytes and bone marrow cells were collected from each rat after the 6 week treatment. They were sacrificed with an overdose of anesthetic (ketamine, 50 mg/rat)/xylazine, 5 mg/rat). Blood was obtained by cardiac puncture with EDTA as the anticoagulant. Isolation of the leukocytes was as previously described. The femurs and tibia were then removed and bone marrows were collected by flushing them with 20 ml of RPMI 1640 culture medium (University of

229 Toronto, Tissue Culture). The bone marrow cells were resuspended by a five times passage through a 1 ml serological pipette tip. The cell suspension was centrifuged at

125g for 6 min. Red blood cells were then removed by resuspension of the cell pellet in red cell lysis buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA) for 6 min and centrifugation at 125g for 6 min. After resuspension in PBS

(University of Toronto, Tissue Culture), tissue debris were removed by passing the cell suspension through a 70 µm nylon cell strainer (BD Biosciences, Bedford, MA). The bone marrow cells were washed again in PBS and resuspended in 500 µL of cell lysis buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.2% Triton X-100, protease inhibitor cocktail). Protein quantification was carried out for both the leukocyte and bone marrow samples. PKC activity was assessed in 20 µg of protein in triplicate for each animal.

A1.2.4 Covalent Binding Detection

Clozapine covalent binding to PKC was detected by incubating 1 µg of active recombinant PKC, either isozyme α, δ, or βII (Invitrogen, CA) with 20 µM clozapine, 5 units of horseradish peroxidase (HRP), and 100 µM hydrogen peroxide (H 2O2) in 1 ml

HBSS at 37 oC for 30 min. Control samples with only PKC or no PKC were made.

Samples were boiled in a water bath upon addition of a 6X loading buffer containing

DTT. SDA-PAGE and immunoblots were prepared as described in a previous publication using an anti-serum against clozapine produced in rabbits [6].

230 A1.2.5 Apoptosis Assessment

The ability of clozapine to induce apoptosis in vitro was assessed using HL-60 cells. The effect of protein kinase C down-regulation was also studied to elucidate its role in clozapine-induced apoptosis. HL-60 cells were pretreated with PMA at 200

o ng/ml or its vehicle control (0.05 % DMSO) in culture medium at 37 C and 5% CO 2 for

24 h. The cells (2 x 10 6) were then suspended by scraping and washed in PBS prior to incubation with various concentrations of clozapine from 0-40 µM dissolved in DMSO.

The final concentration of DMSO in the incubation was 0.5% v/v. Twenty units of HRP and H 2O2 (at a final concentration of 20 µM) were added to the incubation to generate of the nitrenium ion metabolite. Cells were incubated with clozapine for 2 h (for the caspase 3 Western blot analysis) and 24 h (for the DNA fragmentation analysis) at 37 oC and 5% CO 2. Cells were then suspended by scraping and washed in PBS to be assayed for apoptosis using western blot to determine caspase 3 cleavage and agarose gel electrophoresis for DNA fragmentation.

HL-60 cells were lysed with lysis buffer containing 20 mM Tris-HCl (pH 7.5),

150 mM NaCl, 1 mM Na 2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na 3VO 4, 1 µg/ml leupeptin, 1mM

PMSF (Cell Signaling Technology, MA) on ice for 10 min. Protein in the cell lysate was quantified with Bio-Rad Bradford reagent. Samples containing 50 µg of protein were boiled in a water bath for 5 min with the addition of a 6X loading buffer containing DTT

(Cell Signaling Technology, MA). Samples were loaded onto a 4-20% SDS polyacrylamide gel for electrophoresis at 120 V for 1 h. Proteins were transferred onto a

PVDF membrane for 1.5 h at 40 V. The membrane was blocked for 1.5 h with 5% skimmed milk in tris-buffered saline Tween-20 (TBST) followed by an overnight incubation in rabbit anti-caspase 3 antibody (Cell Signaling Technology, MA) at 1:1000

231 dilution in TBST with 2% skimmed milk at 4 oC. Blots were thoroughly washed for 45 min with TBST before incubation with goat anti-rabbit IgG HRP conjugated antibody

(Sigma Inc., MO) at 1:10000 in TBST with 2% skimmed milk for 1.5 h. Blots were washed and visualized with ECL Plus western blotting detection reagents (GE

Healthcare, UK) and the FluorChem TM 8800 imaging system (Alpha Innotech, San

Leandro, CA).

DNA fragmentation was analyzed by agarose gel electrophoresis. HL-60 cells (2 x 10 6) were lysed in 0.5 ml TTE buffer which consisted of 10 mM Tris-Cl (pH 7.5),

0.2% TritonX-1001 and mM EDTA; vortexed vigorously to release fragmented chromatin from the nuclei. Proteinase K (10 µL, Roche, Germany) was added to each sample, incubated overnight at 55 oC and then vortexed vigorously with the addition of

0.1 ml ice-cold 5 M NaCl to remove histones from the DNA. DNA was precipitated by adding 0.7 µL of ice-cold isopropanol and allowed to stand overnight at 20 oC. DNA was recovered by centrifugation at 20,000g, 4 oC for 10 min. The DNA pellets were rinsed with 0.5 ml ice-cold 70% ethanol, centrifuged again, and the pellets were allowed to air dry for 3 h. The DNA was then resuspended by adding 20 µL TE buffer which consisted of 10 mM Tris-Cl (pH 7.5) and 1 mM EDTA and incubated at 37 oC for 3 days.

Samples were prepared for loading onto a 1.8% agarose gel by adding 4 µL of 6X loading dye (Fermentas Life Sciences, ON) and electrophoresed for 1 h at 100 V. DNA on the gel were visualized with SYBR TM Safe DNA Gel Stain (Invitrogen, CA) and the

FluorChem TM 8800 imaging system (Alpha Innotech, San Leandro, CA).

A1.2.6 Statistical Analysis

Statistical analyses were performed using the Student’s t test or one-way

ANOVA with the GraphPad Prism program (GraphPad, San Diego, CA). Individual

232 comparisons were performed using Newman-Keuls post hoc tests. Data were expressed as the mean ± s.d. Results were considered statistically significant if P<0.05.

A1.3 Results

A1.3.1 The Effect of Clozapine on PKC Activity

Incubations of clozapine with the HL-60 cell lysate in the presence of HRP and

H2O2 for 1 h resulted in a dose-dependent decrease in PKC activity from 1-10 µM with significant decreases at 3, 10, and 40 µM compared to the control (Figure A1.1), although a higer level of PKC activity was measured in the 40 µM incubation compared to 10 µM.

Treatment of purified rat leukocytes with clozapine for 1 h resulted in a similar profile of PKC activity with significant decreases at concentrations of 0.2 and 2 µM clozapine (Figure A1.2A). Addition of the HRP/H 2O2 oxididizing system also resulted in a decrease in PKC activity with increasing clozapine concentrations, but the decrease was only significant at 20 µM, which was not observed at the same concentration without the oxidizing system (Figure A1.2B).

In the in vivo system, a slight increase in PKC activity was measured in leukocytes of rats treated with 50 mg/kg clozapine for six weeks; whereas in the bone marrow cells a decrease in PKC activity was observed compared to the control group

(Figure A1.3A and B). However, these differences were not statistically significant.

Incubation of clozapine with active human recombinant PKC resulted in various degrees of kinase activity inhibition for the three different isozymes (Figure A1.4A, C, and E). Significant decreases in PKC activity were observed particularly at higher clozapine concentrations and also in the incubtions with the addition of the oxidant,

233 NaOCl, for the δ and βII isozymes (Figure A1.4D, and E). A decrease in PKC activity was observed in the incubations with all concentrations of clozapine compared to 0 µM with the α isozyme in the absence of HOCl (Figure A1.4A) while a decrease was observed only at 320 µM in the presence of HOCl (Figure A1.4B). A dose-dependent decrease in kinase activity was more obvious with the δ isozyme in the presence of

HOCl (Figure A1.4D).

Figure A1.1. PKC activity of HL-60 cell lysates incubated with clozapine. HL-60 cell lysates containing 30 µg were incubated with various concentrations of clozapine and 20 units of HRP and 20 µM H2O2 for 1 h prior to PKC activity quantification assay. Results are presented as the mean relative PKC activity ± s.d. from 3 assay wells. *Significant decrease from control (P < 0.05).

234 A)

B)

Figure A1.2. PKC activity of rat leukocytes incubated with clozapine. Leukocytes (3.5 x 10 6 whole cells) were incubated with clozapine for 1 hr in the absence A) or presence B) of a HRP/H 2O2 activating system. PKC activity was assayed in 30 µg of proteins for each sample. Results are presented as mean relative PKC activity ± s.d. from 3 wells. *Significant decrease from control (P < 0.05).

235 A)

B)

Figure A1.3. Leukocytes and bone marrow cell protein kinase C activity in clozapine-treated rats. Protein kinase C activity in leukocyte lysate A) and bone marrow cell lysate B) from four rats treated with 50 mg/kg clozapine for 6 weeks and four control animals. The kinase activity was assayed using 20 µg of proteins in triplicate and the results are presented as mean relative PKC activity ± s.d.

236 -NaOCl +NaOCl

A) B)

PKC ααα

C) D)

PKC δδδ

E) F)

PKC βββII

Figure A1.4. Clozapine’s effect on kinase activity in recombinant human PKC.

Recombinant human PKC ( α, δ and βII isozymes) were incubated with clozapine for 20 min in the absence of NaOCl (Figure A1.4A, C, and E) or in the presence of NaOCl (Figure A1.4B, D and F) prior to the kinase activity assay. Results are presented as mean relative PKC activity of 30 ng of the enzyme ± s.d. from 3 wells. *Significant decrease from control (P < 0.05).

237 A1.3.2 Covalent Binding of Clozapine to PKC

The immunoblot in Figure A1.5 shows clozapine covalent binding to proteins with molecular masses of 75 and 44 kDa which correspond to PKC and HRP, respectively. Clozapine covalent binding was detected in all of the PKC isozymes tested, namely α, δ, and βII . Control samples with the addition of PKC and HRP but without clozapine showed a lack of the 75 and 44 kDa bands indicating that the rabbit anti-serum was specific for clozapine.

Figure A1.5. Covalent binding of clozapine to PKC. Clozapine covalent binding to PKC was analyzed by incubating 1 µg of active recombinant PKC of the isozyme α, δ, and βII, represented in lanes 1,3, and 5, respectively, with 20 µM clozapine, 5 units of horseradish peroxidase (HRP) and 100 µM hydrogen peroxide (H 2O2). Incubations with

PKC only, are in lanes 2 (PKC α), lane 4 (PKC δ), and lane 6 (PKC βII ) or no PKC but clozapine and HRP with H 2O2 (lane 7) were also performed.

238 A1.3.3 The Effect of Clozapine on Apoptosis

The ability of clozapine to induce apoptosis was assessed in vitro with HL-60 cells incubated with clozapine. The immunoblot shown in Figure A1.6A and the relative band densities in Figure A1.6B show an increase in cleaved caspase 3 with increasing clozapine concentrations except for 10 µM of clozapine. Only a very low level of detectable cleaved enzyme was observed in the absence of clozapine. In the experiment where the cells were pre-treated with PMA for 24 h prior to clozapine treatment, the amount of cleaved caspase 3 were less compared to those without PMA pre-treatment.

The level of cleaved caspase 3 was also similar for all concentrations of clozapine

(Figure A1.6 C and D).

239

- 24 h PMA + 24 h PMA C A Treatment Treatment ) )

0.3 B 0.6 D ) 0.5 ) 0.4 0.2

0.3

0.2 0.1 Relative density 0.1 Relative density

0.0 0 1 3 10 40 0.0 0 1 3 10 40 Clozapine concentration ( µµµM) Clozapine concentration ( µµµM)

Figure A1.6. Western blot analysis of caspase-3 cleavage in clozapine-treated HL- 60 cells. Cells were pre-treated with PMA at 200 ng/ml C) or its vehicle control of 0.05% DMSO A) for 24 h. Cells (2 x 10 6) were then incubated with various o concentrations of clozapine from 0-40 µM for 2 h at 37 C and 5% CO 2. Twenty units of horseradish peroxidase Type VI (HRP) and 20 µM hydrogen peroxide (H 2O2) were added to the incubation to allow the generation of the nitrenium ion metabolite. Cell lysates were then assayed for cleaved caspase 3 using Western blotting. An extract from cytochrome c-treated Jurkat cells served as a positive control while the negative control was from untreated Jurkat cells. Figure B (without 24 h PMA pre-treatment) and D (with 24 h PMA pre-treatment) show the relative band density of the cleaved caspase-3 in each lane compared to the β-actin loading control for 50 µg of proteins.

240

In the agarose gel showing qualitative analaysis of DNA fragmenation, cells incubated with clozapine showed increasing levels of small DNA fragments at around

100-200 bp in a dose-dependent manner (Figure A1.7A). DNA fragmentation was not observed at 0 µM clozapine. Samples from 24 h PMA pre-treated cells showed much lower levels of DNA fragmenation for all clozapine concentrations (Figure A1.7B).

A)

241

B)

Figure A1.7. DNA fragmentation of HL-60 cells incubated with clozapine for 24 h. HL-60 cells were pre-treated for 24 h with PMA at 200 ng/ml B) or its vehicle control of 0.05% DMSO A) . Cells (2x10 6) were then incubated with various concentrations of clozapine from 0-40 µM for 24 h. Twenty units of horseradish peroxidase Type VI

(HRP) and 20 µM H 2O2 were added to the incubation to generate the nitrenium ion metabolite. Cell lysates were then assayed for DNA fragmentation by electrophoresis.

242

A1.3.4 PMA Down-Regulation of PKC Activity

Incubation of HL-60 cells with PMA at 200 ng/ml for 24 h resulted in a significant decrease in PKC activity (Figure A1.8). PKC activity was reduced by approximately 75% compared to cells exposed to the corresponding DMSO vehicle.

Figure A1.8. PKC activity of HL-60 cells incubated with PMA for 24 h. Cells (2x10 6) were treated with PMA at 200 ng/ml or its vehicle control of 0.05% DMSO for 24 h. Cell lysates containing 30 µg of proteins were then assayed for PKC activity. Results are presented as mean relative PKC activity ± s.d.

243 A1.4 Discussion

Studies conducted by Iverson on the effect of clozapine on neutrophil function found that it is able to inhibit the oxidative burst of cells stimulated with PMA [1]. She had began by testing the hypothesis that clozapine can stimulate an oxidative burst in neutorphils because previous studies from our lab had found a high level of covalent binding of clozapine to neutrophils in patients taking the drug [7], which suggested that was an activation neutrophils leading to the production of H 2O2 to form the reactive metabolite for covalent binding and that neutrophils which have undergone respiratory burst are likely to die quickly. This may explain why some patients suffer from a drastic decrease in neutrophil counts (agranulocytosis) during clozapine treatment. Surprisingly, she found that clozapine actually inhibited the neutrophil respiratory burst [1]. The results of those neutrophil function assays are in agreement with the results from studies conducted by Joffe et al. who demonstrated that in drug-free neuroleptic-resistant schizophrenic patients, clozapine-induced a decrease in the production of reactive oxygen species by unstimulated mononuclear cells [8]. It is thought that neutrophils live longer in the absence of an oxidative burst, but this is contrary to our finding of a decreased half-life [9-11]. In addition, Iverson’s studies also showed that clozapine inhibited the respiratory burst in neutrophils that were stimulated with PMA [1]. PMA is a direct stimulator of PKC, and the activation of this kinase is required for the assembly

- of NADPH oxidase to generate O 2 production in neutrophils during an oxidative burst.

Since an increase in the respiratory burst was not observed when clozapine was added in the presence of PMA, it is possible that clozapine was acting as a PKC inhibitor. This led us to the hypothesis that clozapine is a PKC inhibitor. It is not clear how the inhibition of oxidative burst might be involved in the pathogenesis of neutropenia or agranulocytosis, if at all. Perphaps this has to do with clozapine acting as a PKC

244 inhibitor, and its effect on kinase inhibition might cause neutrophil apoptosis since various PKC inhibitors have been found to induce apoptosis. Indeed, an in vivo study had shown that chronic treatment with clozpaine decreased PKC activity in discrete regions of the rat brain [12-14]. In a separate study, clozapine was found to decrease

PKC activity in the Neuro-2A mouse neuroblastoma cell line when they were incubated with the drug at a therapeutic concentration of 3 µM for 5 days [15]. Clozapine has also been found to markedly suppress concanavalin A and lipopolysaccharide-induced splenocyte proliferation and it is thought that the inhibitory effect of clozapine maybe associated with PKC inhibition because the addition of a PKC activator in the study attenuated this inhibitory effect and clozapine also strongly inhibited PKC activity in splenocytes [15]. Moreover, it has been suggested that modification of the PKC isozymes by antipsychotic drugs is linked to their pharmacological effect [12].

Therefore, it is reasonable to infer that clozapine is a PKC inhibitor. In order to test this hypothesis, we had conducted a series of experiments to look at PKC activity in in vitro and in vivo systems.

We first looked at its effect on PKC activity in HL-60 cell lysate. Incubation of the cell lysate with increasing concentrations of clozapine in the presence of the

HRP/H 2O2 metabolizing system resulted in signifcant inhibition of PKC activity in a dose-dependent manner. A further experiment with purified whole rat leukocytes also found similar results. However the decrease in PKC was more prominent only at the higher concentration of clozapine (20 µM) in the presence of HRP/H 2O2 when compared to the decrease in PKC activity observed in the experiment using HL-60 cell lysate.

Perhaps this can be explained by the fact that the PKCs are not as readily available to interact with clozapine or its nitrenium metabolite when they are inside cells. In the in vivo experiments to determine clozapine’s ability to decrease PKC activity in rats, we

245 treated these animals with clozapine for six weeks. The treated group may have a decrease in PKC activity, although statistically insignificant, which was only observed in bone marrow cells but not in the peripheral leukocytes. It is possible that the effect on neutrophils and their bone marrow precursor PKCs would have been diluted with the various cell types present in these samples. Isolation of the neutrophils would have been necessary in order to observe the effect in this specific population of cells. In the set of experiments with purified active human recombinant PKC, we found that incubation with clozapine resulted in various degrees of kinase activity inhibition for the three different isozymes. The inhibitory profiles were inconsistent between the different isozymes and inconsistency was also observed in the need for HOCl to induce the inhibitory effect. The inhibitory effect of clozapine seem to be more prominent in the incubations with higher concentrations of clozapine and also in those with the addition of

NaOCl for the δ and βII isozymes, whereas a decrease in PKC activity was observed in the incubations with all concentrations of clozapine compared to 0 µM clozapine with the α isozyme in the absence of HOCl. A dose-dependent decrease in kinase activity appeared to be more obvious in the δ isozyme in the presence of HOCl even at the lower concentrations of clozapine but only at 20 µM or higher for the βII isozyme. This suggests that the ability of clozapine to inhibit protein kinase C activity is likely to be different between the parent compound and the reactive nitrenium ion metabolite.

Studies have been carried out by other investigators to look at PKC’s role in mediating cell apoptosis. The α, β, ε, λ, and ζ isozymes have been found to be associated with cell survival and anti-apoptotic effects, while δ,θ and µ were found to act mostly as pro- apoptotic kinases [16, 17]. However, most PKC isozymes classified as primarily anti- apoptotic can also function to promote apoptosis and vis a versa depending on the stimulus and cell type. Specific PKC inhibitors such as RO-31-8220 and chelerythrine

246 with inhibitory activity against the α,β,and γ isozymes were found to induce apoptotsis in gastric cancer cells. These inhibitors act at the catalytic domain (C3 and C4) of PKC

[18]. In studies with HL-60 cells, specific and nonspecific PKC inhibitors which act on the catalytic or regulatory domains have also been shown to induce apoptosis leading to cell death [19]. Elevated levels of apoptosis were noted in reponse to both highly selective inhibitors, such as calphosin C and chelerythrine that act at the diglyceride binding site within the PKC regulatory domain, as well as in relatively nonspecific inhibitors, such as H7, staurosporine, and gossypol that act at the ATP-binding site within the catalytic domain of the enzyme [19]. Certain oxidants, alkylating agents, and cancer-preventive agents have been found to be capable of modifying cysteine residues present within the catalytic domain, leading to the inactivation of PKC [20-22]. Since various PKC inhibitors have been found to induce apoptosis it is possible that clozapine or its reactive nitrenium ion, given that they may have an inhibitory effect on PKC, could induce cell apoptosis in a similar way. It is possible that the nitrenium ion, a reactive intermediate of clozapine upon oxidation, may covalently bind to the C1 regulatory domain of PKC since this region of the enzyme is known to be rich in cysteine [10, 23,

24] or to the cysteine residues also present in the catalytic domain. This nitrenium ion binds readily to neucleophiles such as the thiol group on cysteine. In this study, we incubated clozapine with PKC α, δ, and βII and found covalent binding of the drug to all three isozymes. Such binding may affect the function of these kinases.

In a preliminary study, we have shown using Western blot analysis of caspase 3 cleavage and electrophoresis to detect DNA fragmentation that clozapine was capapble of inducing apoptosis in the HL-60 cell line. Caspases, cysteine-aspartic acid proteases, are essential effctor molecules of apoptosis. Activation of caspases requires proteolytic processing of its inactive zymogen into active fragments. Assaying for cleaved caspases

247 allows one to detect early apoptosis. We have employed a commerical antibody against caspase 3 to detect its level of activation in HL-60 cells. Caspase 3 is a downstream execution caspase in the cascade which is activated both by the intrinsic and extrinsic pathways of apoptosis [25]. Incubation of HL-60 cells with clozapine, along with an oxidizing system of HRP and H 2O2 to allow for the formation of the reactive metabolite, resulted in cleavage of caspase 3 in these cells which was dose-dependent up to 3 µM of clozapine. At 10 µM of clozapine the amount of cleaved caspase 3 was much less suggesting a decrease in cell apoptosis at this concentration; however, it increased again at 40 µM. Concentrations between 0 to 3 µM correspond to therapeutic concentrations in vivo [26]. As shown earlier by Williams et al., concentrations of clozapine above 3

µM in the presence of an activating system produced necrotic cell death which could explain why there was a decrease in the amount of cleaved caspase 3 in the 10 µM incubation. However, it is not known why there appeared to be a much higher level of cleaved caspase 3 in the incubation with 40 µM of clozapine. The effect of clozapine on

HL-60 cell apoptosis is in accordance with the results obtained in the in vitro studies conducted by Williams et al . with assessment of neutrophil apoptosis using Annexin V

[2]. To further show that these cells did undergo apoptosis we also looked for DNA fragmentation, which is a morphrologic change that occurs later on in the apoptosis process. Results were in line with what we had found with the immunoblots. There was a dose-dependent increase in DNA laddering, as shown by the increase in the smaller fragments of 200 bp or less, in HL-60 cells incubated with clozapine along with an oxidizing system.

In order to determine whether clozapine’s induction of apoptosis is associated with its effect on PKC, we needed to somehow remove PKC from the picture in order to test this. That is to say if we have removed PKC activity from HL-60 cells and were still

248 able to observe a similar level of apoptosis with clozapine incubation, then we can rule out the involvement of PKC on clozapine-induced apoptosis. In an in vitro system, one way to do so was to utilize PMA to decrease PKC activity in these cells prior to clozapine incubation. Prolonged activation of PKC by PMA has long been known to lead to PKC down-regulation [27, 28]. Some studies have shown that chronic exposure of cells to phorbol esters leads to loss or down-regulation of phrobol-ester-binding sites

[29-31]. Other studies have shown that the decrease in enzymatic activity is due to an increased rate of PKC degradation with a consequent decrease in the steady-state amounts of the intact polypeptide [27]. In this study, PMA treatment for 24 h decreased

PKC activity by ~75% compared to that measured in cells without the PMA treatment.

Indeed, PMA pre-treatment inhibited apoptosis in clozapine-treated HL-60 cells as shown by the lower levels of caspase-3 cleavage and DNA fragmenation. The level of apoptosis in these cells was much less after incubation with clozapine and a dose- dependent effect was not observed compared to those without PMA pre-treatment. Thus this suggests that PKC could be involved in clozapine-induced agranulcytosis; however, these results cannot definitively show that PKC is involved because the unobserved accelerated apoptosis can also be due to PMA’s ability to inhibit apoptosis possibly by causing changes in the cell machineries necessary for apoptosis to occur since PMA has been shown to decrease apoptosis in certain cell types. In an in vitro study conducted by

Solary et al. where they found no apoptotic cells among the adherent PMA-differentiated

HL-60 cells for up to 5 days after PMA treatment, while apoptosis was observed in the population of cells that failed to attach early to the culture dish.[32]. They also found that the phenotypic changes in HL-60 cells induced by PMA treatment include inactivation of a cytoplasmic activity, where this cytoplasmic activity can induce DNA fragmentation associated with apoptosis. Perhaps the next step in determining whether

249 PKC is involved in clozapine-induced agranulocytosis is to pre-incubate the HL-60 cell lysate with either a known activator or an inhibitor of PKC. By doing so, if clozapine is a PKC inhibitor and its inhibitory effect on PKC is what induces apoptosis then addition of a PKC activator should block the apoptosis while apoptosis should still be observed or may even be potentiated with the addition of a PKC inhibitor. In addition, other methods of down-regulating PKC activity, other than prolonged PMA treatment, should be explored to substantiate the role of this enyzme in clozapine-induced agranulocytosis.

All in all, these studies suggest that future investigation of the PKC signaling pathway involved in clozapine-mediated apoptotic activities should be focused on specific PKC isoforms, which may help to better understand the mechanism. Perhaps the use of antisense oligonucleotides, RNAi and selective pharmacological inhibitors to specifically knock down the levels or kinase activity of individual PKC in relevant cell lines may help address these potential caveats.

250 APPENDIX 2

Identification of Clozapine Covalently Modified Proteins

A2.1 Background

Previous studies from our lab and those of others have found that clozapine can covalently bind to proteins in human neutrophils, rat bone marrow cells, liver, and heart tissues [7, 33]. In the liver and heart immunoblots, many bands ranging from 30-120 kDa were observed, while in the neutrophil and bone marrow blots, only one or two bands were obvious. These bands were around 77, 49, or 58 kDa, where 58 kDa corresponds to the molecular weight of the half dimer of the human myeloperoxidase. In order to obtain a better idea of what these proteins are and whether myeloperoxidase is one of them, previous students in our lab had attempted to purify the polyclonal anti- clozapine antibody from the rabbit antiserum for further study. However, the purification was unsuccessful because the conditions required for the elution of antibodies from the column were too harsh and the antibodies were then no longer capable of binding to clozapine. Therefore, this study was performed in an attempt to try to identify these covalently modified proteins using Protein A beads and mass spectrometry.

A2.2 Materials and Methods

A female Sprague Dawley rat was treated for 6 weeks with 50 mg/kg/day clozapine given in the diet. The animal was sacrificed at the end of the drug treatment and bone marrow cells were obtained and cell lysate prepared as previously described in

251 Chapter 3 section 3.4.6 of this thesis. Protein A beads purchased from New England

BioLabs, Inc. (Ipswich, MA) were prepared and washed according to the manufacturer’s directions. These Protein A beads, which are capable of binding to IgG, were then incubated with rabbit antiserum containing the polyclonal anti-clozapine antibodies for

30 min at 4 oC (Figure A2.1). The bone marrow cell lysates were subsequently added to the incubation and allowed to incubate at 4 oC for 1.5 h. Covalently modified proteins should bind to the antibodies on the Protein A beads. The Protein A beads were then subjected to SDS and reducing conditions in a sample buffer containing 0.35 M Tris-Cl,

10% SDS, 4% glycerol, 0.02% bromophenol blue, 18 mg/ml DTT to allow cleavage of the antibodies and the covalently-modified proteins from the beads. Samples were then subjected to SDS-PAGE on a 12 % gel at 120 Volts for 1.5 h. The protein bands from the gel were excised and stained with Coomassie blue for 2 min and destained overnight.

In gel tryptic digestion was then preformed on the excised gel pieces using the Pierce In-

Gel Tryptic Digestion kit (Rockford, IL). The processed samples were subjected to liquid chromatographic separation and electrospray ionization mass spectrometry (LC-

ESI MS) for protein identification.

252

Clozapine - Protein A bead

+ rat bone + rabbit marrow cell Clozapine - clozapine lysate antiseru

+SDS And reducing

Clozapine -

Run

Mass Protein(s) (49 kDa) Spec to Cut band identify out pro tein(s And tryptic

Figure A2.1 Isolation of clozapine covalently modified proteins.

253 A2.3 Results

In the SDS-PAGE gel of the bone marrow cell lysate after Coomassie staining, the second lane contains cell lysate sample incubated with protein A beads and the fourth lane represents the negative control where no cell lysate was added to the protein A beads incubation. The single most obvious band (~49 kDa) from lane 2 and 4 was cut out and subjected to tryptic digestion (Figure A2.2). After matching the fragmentation pattern to the NCBInr database of rat proteins, the various proteins were identified

(Table A2).

Figure A2.2 SDS-PAGE gel of bone marrow cell lysate incubated with and without clozapine. Lane 1 contains the protein molecular weight marker, lane 2 contains cell lysate sample incubated with protein A beads and lane 4 represents the negative control where no cell lysate was added to the protein A beads incubation. The single most obvious band (~49 kDa) from lane 2 and 4 was cut out and subjected to tryptic digestion.

254

Table A2.1 Clozapine covalently modified proteins identified by mass spectrometry.

Clozapine treated Lysate buffer control Anionic trypsin-1 precursor Anionic trypsin-1 precursor

Tubulin alpha-1A chain Keratin, type I cytoskeletal 42 Keratin, type I cytoskeletal 42

Ig gamma-2A chain C region Anionic trypsin-2 precursor Keratin, type II cytoskeletal 1

255 A2.4 Discussion

Previous experiments which identified clozapine binding of clozapine in human neutrophils and rat bone marrow cells have shown protein bands around 49 kDa that were thought to be myeloperoxidase. However, we still do not have experimental data that can prove the identities of the proteins modified by clozapine. In order to do so, we need to work with purified clozapine antibodies rather than the crude anti-serum from the rabbit. Previous experiments conducted in our lab have attempted to purify the clozapine antibody but were unsuccessful. Thus, in this study we devised a method to isolate the anti-clozapine antibody by allowing it to bind to Protein A beads. These Protein A beads are capable of binding to IgG. By using this method it is expected that most of the IgG in the rabbit anti-serum are antibodies against clozapine. Subsequent incubation with bone marrow cell lysate obtained from a clozapine-treated rat would allow the covalently modified proteins to bind to the anti-clozapine antibody which is in turn bound to the

Protein A beads. Then cleaving off the beads and the antibody from the covalently modified protein would produce a purified sample of the proteins which were modified by clozapine. These proteins would then by identify by mass spectrometry.

Although this protein purification method seemed feasible and straightforward, the study failed to identify the 49 kDa proteins as myeloperoxidase. In fact, all of the proteins identified were either contaminants such as keratin or left over trypsin from the digestion step. It is likely that the contaminants were in such high concentrations in the protein samples that the clozapine covalently modified protein(s) could not be identified.

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261