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The effect of Ginkgo Biloba extract on valproic acid metabolism by human liver microsomes from donors… Numa, Andres Masato 2005

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THE EFFECT OF GINKGO BILOBA E X T R A C T O N VALPROIC ACID M E T A B O L I S M B Y H U M A N LIVER MICROSOMES F R O M DONORS WITH THE CYP2C9*1/*1 GENOTYPE by A N D R E W M A S A T O N U M A B.Sc , The University of British Columbia, 1999 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH C O L U M B I A August 2005 © Andrew Masato Numa, 2005 ABSTRACT Ginkgo biloba extract (GBE) is a popular herbal preparation used primarily in the treatment of dementia, peripheral vascular diseases, and neurosensory problems. In this study, the effect of G B E on the oxidative metabolism of the anti-epileptic valproic acid (VPA) was investigated. Human liver microsomes (HLM) from donors with the CYP2C9*1/*1 genotype were incubated with V P A and GBE, and the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA were monitored by GC/MS in NICI mode. G B E inhibited the formation of all four metabolites in a dose-dependent manner. GBE from three different sources showed similar inhibition of metabolite formation. Pre-incubation of H L M with a monoclonal antibody against CYP2C9 significantly reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA, demonstrating that CYP2C9 is the major isoform responsible for their formation. Pre-incubation of H L M with monoclonal antibodies against CYP2B6 and CYP2A6 reduced their formation by a smaller amount, suggesting that they are minor isoforms involved in their formation. Pre-incubation of H L M with monoclonal antibodies against CYP2B6 and CYP2A6 followed by incubation with V P A and G B E dramatically reduced their formation. These results show that the inhibition of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation in H L M by G B E is due mostly to inhibition of CYP2C9. These results were confirmed by incubations with recombinant enzymes. GBE inhibited 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by recombinant CYP2C9; 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by recombinant CYP2B6; and 3-OH-VPA formation by recombinant CYP2A6. To elucidate which constituents of GBE are responsible for the inhibition seen with the whole extract, individual terpene trilactones (bilobalide and ginkgolides A, B, C, and J) and u flavonol glycosides (isorhamnetin-3-O-nitinoside, kaempferol-3-O-rutinoside, and quercetin-3-O-rutinoside) were incubated with VP A. However, at the concentrations present in GBE, these constituents failed to inhibit VPA metabolism. The aglycones of isorhamnetin, kaempferol, and quercetin inhibited VPA metabolism. Although the aglycones were not detected in GBE, they may be of importance in vivo, as flavonol glycosides are hydrolyzed to their respective aglycones in the small intestine. In conclusion, GBE inhibited the CYP2C9-, CYP2B6-, and CYP2A6-mediated metabolism of VPA. However, the effect could not be explained by the individual terpene trilactones or flavonol glycosides tested. in TABLE OF CONTENTS Page ABSTRACT ii T A B L E OF CONTENTS i v LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xi A C K N O W L E D G E M E N T S xv 1. INTRODUCTION 1 1.1. The Cytochromes P450 1 1.1.1. B iotransfor mation 1 1.1.2. CYP2C9 2 1.1.3. CYP2B6 3 1.1.4. CYP2A6 • 4 1.2. Ginkgo biloba Extract 4 1.2.1. Background 4 1.2.2. Chemical composition 5 1.2.3. Pharmacological actions 8 1.2.4. Uses 8 1.2.5. Adverse effects 9 1.3. Valproic Acid 10 1.3.1. Therapeutic use 1 0 iv 1.3.2. Mechanism of action 10 1.3.3. Chemistry and biotransformation 11 1.4. Rationale and Hypothesis 14 1.5. Specific Objectives 17 MATERIALS A N D METHODS 19 2.1. Materials 19 2.1.1. Acids, bases, salts, and buffers 19 2.1.2. Organic solvents 20 2.1.3. Gases 20 2.1.4. G B E and its individual constituents 20 2.1.5. V P A 21 2.1.6. N A D P H 21 2.1.7. Derivatization agents 21 2.1.8. Metabolite standards 21 2.1.9. Internal standard 22 2.1.10. Microsomes and antibodies 22 2.2. Standard Curves 22 2.3. Microsomal Incubations 23 2.3.1. Optimization of total C Y P content for incubations with H L M 23 2.3.2. Optimization of incubation time for incubations with H L M 23 2.3.3. Intraday and interday variabilities for incubations with H L M 23 2.3.4. Effect of GBE from different lots and manufacturers on V P A metabolism by H L M 24 2.3.5. Concentration-dependent effect of G B E on V P A metabolism by H L M 25 2.3.6. Effect of G B E on CYP2C9-catalyzed V P A metabolism by H L M 25 2.3.7. Effect of G B E on V P A metabolism by recombinant CYP2C9*1, CYP2B6, and CYP2A6 26 2.3.8. Effect of individual constituents of G B E on V P A metabolism by H L M . . . 2 6 2.4. Sample Preparation for V P A Metabolite Analysis 27 2.4.1. Extraction 30 2.4.2. Derivatization 30 2.5. GC/MS Assay 30 2.6. Data Analysis 33 2.6.1. Calculation of PAR 33 2.6.2. Standard curves 33 2.6.3. Microsomal incubations 34 2.6.4. Limit of quantitation 34 2.6.5. Intraday and interday variabilities..". 35 2.7. Statistical Analysis 35 RESULTS 36 3.1. Validation of VP A GC/MS Assay 36 3.1.1. Chromatograms 36 3.1.2. Standard curves 37 3.1.3. Limit of quantitation 37 3.2. Optimization of V P A Metabolism Assay 44 3.2.1. Optimization of total C Y P content for incubations with H L M 44 vi 3.2.2. Optimization of incubation time for incubations with H L M 44 3.2.3. Intraday and interday variabilities for incubations with H L M 47 3.3. Effect of G B E on V P A Metabolism 47 3.3.1. Overview of experiments 47 3.3.2. Effect of GBE from different lots and manufacturers on V P A metabolism by H L M . : 52 3.3.3. Concentration-dependent effect of G B E on V P A metabolism by H L M 52 3.3.4. Effect of GBE on CYP2C9-catalyzed V P A metabolism by H L M 55 3.3.5. Effect of G B E on V P A metabolism by recombinant CYP2C9*1, CYP2B6, and CYP2A6 ". 57 3.3.6. Effect of individual constituents of GBE on V P A metabolism by H L M . . . 6 1 4. DISCUSSION 66 5. LIMITATIONS A N D FUTURE STUDIES 76 6. S U M M A R Y A N D CONCLUSIONS 79 7. REFERENCES 82 8. APPENDICES 92 8.1. Appendix 1. Amount of Terpene Trilactones and Flavonols in G B E (Indena, lot 1306A) 92 8.2. Appendix 2. H L M Activities 93 8.3. Appendix 3. H L M Donor Profiles 94 8.4. Appendix 4. Information on Recombinant C Y P 95 8.5. Appendix 5. Information on Monoclonal Antibodies 96 vii LIST OF TABLES Page Table 1. GC oven temperature program 32 Table 2. Retention times of V P A and its metabolites on a representative day 41 Table 3. Coefficient of variation and bias of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-V P A standards 43 Table 4. Intraday variabilities of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA formation by H L M 48 Table 5. Interday variabilities of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA formation by H L M 49 -viii LIST OF FIGURES Page Figure 1. Chemical structures of the terpene trilactones 6 Figure 2. Chemical structures of the flavonols and flavonol rutinosides 7 Figure 3. V P A and selected oxidative metabolites 12 Figure 4. Phase I metabolism of V P A 13 Figure 5. Sample preparation scheme 28 Figure 6. Representative total ion chromatogram 38 Figure 7. Representative chromatograms for metabolite standards at mlz of 141 and 148 39 Figure 8. Representative chromatograms for metabolite standards at mlz of 273 and 280 40 Figure 9. Representative standard curves 42 Figure 10. Optimization of total C Y P content for incubations with H L M 45 Figure 11. Optimization of incubation time for incubations with H L M 46 Figure 12. Representative chromatograms for microsomal incubations at mlz of 141 and 148 ...50 Figure 13. Representative chromatograms for microsomal incubations at mlz of 273 and 280 ... 51 Figure 14. Effect of different lots and brands of G B E on V P A metabolism by H L M 53 Figure 15. Effect of GBE on V P A metabolism by H L M 54 Figure 16. Effect of G B E on CYP2C9-mediated V P A metabolism by H L M 56 Figure 17. Effect of G B E on V P A metabolism by recombinant CYP2C9*1 58 Figure 18. Effect of GBE on V P A metabolism by recombinant CYP2B6 59 Figure 19. Effect of GBE on V P A metabolism by recombinant CYP2A6 60 Figure 20. Effect of terpene trilactones on V P A metabolism by H L M 62 ix Figure 21. Effect of flavonol glycosides on VPA metabolism by FILM Figure 22. Effect of flavonol aglycones on VPA metabolism by FfLM LIST OF ABBREVIATIONS 3- OH-VPA 3-hydroxy-valproic acid; 2-«-propyl-3-hydroxypentanoic acid 4- ene-VPA 4-ene-valproic acid; 2-w-propyl-4-pentenoic acid 4- OH-VPA 4-hydroxy-valproic acid; 2-«-propyl-4-hydroxypentanoic acid 5- OH-VPA 5-hydroxy-valproic acid; 2-«-propyl-5-hydroxypentanoic acid pA microampere Mg microgram pL microlitre pm micrometre uM micromolar BROD benzyloxyresorufin O-dealkylation °C degrees Celsius C V coefficient of variation C Y P cytochrome P450 CYP2A6 cytochrome P450 2A6 CYP2B6 cytochrome P450 2B6 CYP2C9 cytochrome P450 2C9 DIPEA /V.V-diisopropylethylamine DMF dimethylformamide DMSO dimethylsulfoxide eV electron volt g gram xi g gravity G A B A Y-aminobutyric acid G B E Ginkgo biloba extract G C gas chromatography GC/MS gas chromatography-mass spectrometry h hour HC1 hydrochloric acid H L M human liver microsomes IC50 inhibitory concentration 50 IgG immunoglobulin G LC/MS liquid chromatography-mass spectrometry L O D limit of detection LOQ limit of quantitation m metre mm millimetre MSI megaohm MAb2A6 monoclonal antibody against CYP2A6 MAb2B6 monoclonal antibody against CYP2B6 MAb2C9 monoclonal antibody against CYP2C9 mg milligram MgCb magnesium chloride min minute mL millilitre Xll mM millimolar MS mass spectrometry M T B S T F A jV-methyl-Ar-(/err-butyldimethylsilyl)-trifluoroacetamide mTorr milliTorr mlz mass-to-charge ratio N A D P H P-nicotinamide adenine dinucleotide phosphate NaOH sodium hydroxide ng nanogram NICI negative ion chemical ionization N M D A JV-methyl-D-aspartate nmol nanomole P A F platelet activating factor P A R peak are ratio pmol ' picomole PROD pentoxyresorufin (9-dealkylase psi pounds per square inch SD standard deviation S E M standard error of the mean SIM selected ion monitoring TBDMSC1 fert-butyldimethylsilyl chloride Tris tris[hydroxymethyl]aminomethane PFBBr pentafluorobenzyl bromide V P A valproic acid; 2-«-propylpentanoic acid; dipropylacetic acid xiii v/v volume/volume w/v weight/volume wlw weight/weight ACKNOWLEDGEMENTS First, I would like to thank my supervisor, Dr. Thomas Chang for his patience and guidance throughout my studies. I would also like to thank my committee members, Dr. Stelvio Bandiera, Dr. Ronald Reid, Dr. Wayne Riggs, and Dr. Zhaoming Xu; and external examiner, Dr. Emma Guns for taking time out of their busy schedules to provide invaluable feedback on this project. I would also like to thank fellow graduate students Vincent Tong, Tony Kiang, and Ted Lakowski; and laboratory members Xiaowei Teng and Jie Chen for their friendship. A special word of gratitude is owed to Dr. Frank Abbott for allowing me to use the GC/MS and for providing the authentic V P A metabolite standards and heptadeuterated metabolites. I would also like to acknowledge Indena S.A. and Pharmaton S.A. for the provision of the Ginkgo biloba extracts. The National Cancer Institute at the National Institutes of Health provided the monoclonal antibodies. I would also like to thank the following sources of financial support: the University of British Columbia for the Graduate Entrance Scholarship, the Rx&D Health Research Foundation and the Canadian Institutes of Health Research for the Graduate Research Scholarship in Pharmacy, and Merck Research Laboratories for the Partial M.Sc. Research Traineeship in Drug Metabolism. An operating grant from the Canadian Institutes of Health funded the research. Finally, I would like to thank my family. Without their love and support, none of this would have been possible. xv 1. INTRODUCTION 1.1. The Cytochromes P450 1.1.1. Biotransformation Humans are exposed to a large number of exogenous compounds each day through the diet, the environment, and pharmaceutical agents. Whereas hydrophilic xenobiotics are usually readily excreted, hydrophobic compounds must usually undergo metabolism into more polar metabolites for excretion. This is achieved via Phase I and Phase II metabolism. Phase I reactions introduce a new functional group into the substrate molecule or modify an existing functional group by oxidation, hydroxylation, reduction, or hydrolysis. The cytochrome P450 (CYP) enzymes are the major mediators of Phase I metabolism. CYP-mediated reactions generally result in the mono-oxygenation of substrates and require molecular oxygen and a supply of reducing equivalents from P-nicotinamide adenine dinucleotide phosphate (NADPH) (Omura, 1999). Phase II metabolism generally involves the conjugation of a hydrophilic moiety such as glucuronic acid or glutathione to the substrate to make it more water-soluble (Meyer, 1996). 1 CYPs comprise a superfamily of heme-thiolate proteins responsible for the biotransformation of a vast array of xenobiotics as well as mostly steroidal endogenous compounds. The CYP enzymes are grouped into families (indicated by Arabic numerals), in which members share at least 40% homology in their amino acid sequences. They are further divided into subfamilies (indicated by letters), whose members share at least 55% homology in their amino acid sequences (Nelson et al., 1996). CYP enzymes have broad but overlapping substrate specificity. Thus, a single CYP may be involved in the metabolism of a large number of different chemicals, and a single chemical may be metabolized by several different CYP isoforms. The majority of xenobiotic metabolizing CYP enzymes are found in the liver. However, certain CYP isoforms are expressed in extrahepatic tissues such as the gastrointestinal tract, lungs, and kidneys (Smith et al., 1998). The major CYP enzymes expressed in the liver are members of the CYP3A subfamily (34% of total hepatic CYP content), members of the CYP2C subfamily (19%), CYP1A2 (13%), and CYP2D6 (3%). CYP3A4 and CYP3A5 metabolize approximately 36% of clinically important drugs. CYP2C8 and CYP2C9 are involved in the metabolism of approximately 17%. CYP2C18 and CYP2C19 metabolize approximately 8%. CYP1A2 is involved in the metabolism of approximately 8%. CYP2D6, though making up only a small proportion of total hepatic CYP, metabolizes 21% of clinically important drugs. Minor isoforms include CYP2E1, CYP2A6, and CYP2B6, which make up 7%, 4%, and 1%, respectively, of total CYP isoforms expressed in the liver (Rendic and Di Carlo, 1997). 1.1.2. CYP2C9 In humans, there are at least four members of the CYP2C subfamily: CYP2C8, CYP2C9, CYP2C18, and CYP2C19. Despite sharing over 82% similarity in amino acid identity, they 2 exhibit relatively little overlap in substrate specificity. The CYP2C isoforms are expressed in the liver, the most abundant form being CYP2C9 (Goldstein and de Morais, 1994; Miners and Birkett, 1998). Probes for CYP2C9 activity include (6>warfarin 7-hydroxylation (Rettie et al., 1992), and diclofenac 4'-hydroxylation (Leemann et al., 1993), tolbutamide methylhydroxylation, and phenytoin 4-hydroxylation (Veronese et al., 1991). Other examples of substrates of CYP2C9 are chloramphenicol, flurbiprofen, ibuprofen, and naproxen (Omiecinski' et al., 1999). Specific inhibitors of CYP2C9 include sulphaphenazole (Mancy et al., 1996) and sulfamethoxazole (Wen et al., 2002). Rifampin is an inducer of members of the CYP2C subfamily, but it also induces other isoforms (Rae et al., 2001). Polymorphisms in the CYP2C9 gene result in variant alleles. The wild type CYP2C9*1 protein has relatively high enzyme activity, with the mutant CYP2C9*2 and CYP2C9*3 proteins exhibiting lower enzyme activities (Goldstein, 2001). CYP2C9 polymorphisms has been found to influence the metabolism of warfarin (Furuya et al., 1995), tolbutamide (Sullivan-Klose et a l , 1996), and phenytoin (Hashimoto et al., 1996). Furthermore, the effect of an inhibitor may be genotype-dependent, as is the case for benzbromarone, which is an inhibitor of metabolism by CYP2C9*1 but an activator of metabolism by CYP2C9*3 (Hummel et al., 2005). 1.1.3. CYP2B6 In humans, CYP2B6 is expressed at low levels in the liver (Mimura et al., 1993). CYP2B6 is a polymorphic enzyme (Ariyoshi et al., 2001). CYP2B6 has been considered an enzyme of relatively little importance, but has been receiving more attention due to its role in the activation of pro-drugs such as cyclophosphamide (Chang et a l , 1993). (^-Mephenytoin N-demethylation has been suggested to be a specific marker for CYP2B6 (Ekins et al., 1998). 3 Bupropion hydroxylation also appears to be a fairly selective reaction for CYP2B6 (Faucette et a l , 2000). Pentoxyresorufin O-dealkylation (PROD) and benzyloxyresorufin (9-dealkylation (BROD) are often utilized to measure CYP2B activity in vitro (Lubet et al., 1985; Nerurkar et a l , 1993). Orphenadrine is a mechanism-based inhibitor of CYP2B6, but also inhibits other isoforms (Guo et al., 1997). Phenobarbital and cyclophosphamide are inducers of CYP2B6 (Gervot et al., 1999). 1.1.4. CYP2A6 In humans, CYP2A6 is predominately hepatic, with a limited range of known substrates (Fernandez-Salguero and Gonzalez, 1995). CYP2A6 is a polymorphic enzyme (Fernandez-Salguero et al., 1995). Nicotine is perhaps its most well known substrate (Nakajima et al., 1996). Nitrosamines found in tobacco smoke are activated by CYP2A6 (Yamazaki et al., 1992). Coumarin 7-hydroxylation is commonly used experimentally as a marker for CYP2A6 activity (Pelkonen et al., 1985). Tranylcypromine and diethyldithiocarbamate inhibit CYP2A6, but are not specific for this isoform (Draper et al., 1997). Phenobarbital and pyrazole induce CYP2A6 (Donato et al., 2000). 1.2. Ginkgo biloba Extract /. 2.1. Background Ginkgo biloba, also known as the maidenhair tree, is the sole surviving species of the family Ginkgoaceae. It is the oldest living tree known, and has thus earned the name "living fossil" (Joshi and Kaul, 2001). Fruits and seeds from Ginkgo biloba have been used for millennia in traditional Chinese medicine, mainly in the treatment of asthma (Ernst, 2002). Today, extracts made from the leaves of Ginkgo biloba are used for medicinal purposes. It is now one of the 4 most popular medicinal plants with annual worldwide sales in 1998 estimated to be greater than $ 1 billion US (van Beek, 2002). 1.2.2. Chemical composition Ginkgo biloba extract (GBE) is a complex mixture containing many chemical constituents. Extracts of the leaves are subjected to a multi-step procedure in which unwanted components are removed and desirable compounds are enriched. Commercially available G B E is typically standardized to contain 6% terpene trilactones and 24% flavonol glycosides (Yoshikawa et al., 1999). The terpene trilactones, which have received the most attention due to their chemical uniqueness, include bilobalide and ginkgolides A, B, C, and J (van Beek, 2005). Their structures are shown in Figure 1. The major flavonols are mostly derivatives of kaempferol, quercetin, and isorhamnetin. The flavonols occur mostly as glycosides, with the aglycones occurring at relatively low concentrations (van Beek, 2002). Chemical structures of the aglycones and rutinosides of these flavonols are shown in Figure 2. Other constituents include proanthocyanidins, carboxylic acids, biflavones, and allkylphenols. Proanthocyanidins make up approximately 7% of standardized extracts. Carboxylic acids, which include ascorbic acid, quinic acid, and shikimic acid, comprise approximately 13% of standardized extracts. Biflavones such as amentoflavone, bilobetin, ginkgetin, isoginkgetin, and sciadopitysin are present in leaves, but do not occur in standardized extracts (van Beek, 2002). Allkylphenols include the ginkgolic acids, ginkgols, and bilobols. Standardized extracts now contain little allkylphenols, which can cause contact dermatitis and may be mutagenic (Fuzzati et al., 2003). 5 Ginkgolide A Ginkgolide B Ginkgolide C Ginkgolide J Ri R2 H H OH H O H OH H OH Figure 1. Chemical structures of the terpene trilactones. Structures for (A) the ginkgolides and (B) bilobalide are shown. 6 O H O Ri R 2 Kaempferol Kaempferol-3-O-rutinoside Quercetin Quercetin-3-O-rutinoside Isorhamnetin lsorhamnetin-3-O-rutinoside H H OH OH O C H 3 O C H 3 H 6-O-a-L-rhamnosyl-p-D-glucoside H 6-O-a-L-rhamnosyl-B-D-glucoside H 6-O-a-L-rhamnosyl-p-D-glucoside Figure 2. Chemical structures of the flavonois and flavonol rutinosides. 7 1.2.3. Pharmacological actions G B E possesses anti-oxidant activity. G B E shows the ability to scavenge free radicals such as the peroxyl (Maitra et al., 1995), hydroxyl, and superoxide radicals (Noda et al., 1997). G B E also inhibits lipid peroxidation (Dumont et al., 1995) and suppresses the production of active oxygen (Rong et al., 1996). Ginkgolide B is a potent inhibitor of platelet activating factor (PAF), a potent inflammatory autacoid (Chung et al., 1987). PAF plays an important role in pathologies such as asthma, shock, ischemia, anaphylaxis, graft rejection, renal disease, CNS disorders and numerous inflammatory conditions. (Braquet and Hosford, 1991). However, it is questionable whether after oral consumption of Ginkgo preparations, enough ginkgolides are present in the bloodstream to cause PAF antagonism in humans (Braquet, 1993). 1.2.4. Uses Since GBE possesses many biological actions, it is not surprising that GBE is used in the treatment of a wide range of conditions. In clinical practice, G B E is primarily utilized in the treatment of memory impairment, dementia, peripheral vascular diseases, and neurosensory problems (De Smet, 2002; Ernst, 2002). G B E is marketed for its purported effect on the improvement of memory. A review of 40 clinical trials investigating the efficacy of GBE on memory found 8 trials of good methodological quality. Of these 8 studies, 7 showed positive effects of GBE on cognitive function compared to placebo. However, the authors warned of a possible publication bias (Kleijnen and Knipschild, 1992). Another meta-analysis of 11 clinical studies concluded that G B E is superior to placebo (Hopfenmuller, 1994). 8 Another major use of G B E is in the treatment of dementia. Dementia is defined as an acquired global impairment of cognitive capacities. Approximately 5% of people over 65 years of age are affected by dementia, of which 70% of cases are thought to be due primarily to Alzheimer's disease (Evans et al., 2004). G B E is also used for intermittent claudication, which is characterized by. Symptom relief comparable to pentoxifylline, one of the two drugs for the management of intermittent claudication approved by the US F D A (Jacoby and Mohler, 2004). There are a number of reports in the literature suggesting that G B E may be effective in the management of tinnitus, a condition characterized by the perception of sound in the absence of external acoustic stimulation. However, there also appears to be a strong placebo effect in tinnitus management. The limited evidence does not demonstrate that G B E is effective for tinnitus (Hilton and Stuart, 2004). G B E may be useful in the treatment of glaucoma, which can be caused by either increased intraocular pressure or poor circulation resulting in damage to the optic nerve. GBE may be useful in glaucoma caused by the latter, as it derives its effect not through the lowering of intraocular pressure, but by improving blood flow to the optic nerve (Head, 2001; Rhee et al., 2001; Evans etal., 2004). 1.2.5. Adverse effects Common adverse effects of Ginkgo are mild, transient, and reversible, such as gastrointestinal symptoms, headache, nausea, and vomiting. Serious effects include bleeding and seizures (Ernst, 2002). 9 1.3. Valproic Acid 1.3. J. Therapeutic use Valproic acid (VPA), otherwise known as 2-w-propylpentanoic acid or dipropylacetic acid, is used in the treatment of epilepsy (Pinder et al., 1977). Several clinical trials have demonstrated its safety and efficacy in adults and children for the treatment of generalized seizures (absence, tonic-clonic, and myoclonic), partial seizures (simple, complex, and secondarily generalized), and compound or combination seizures (Davis et al., 1994). More recently, V P A has found its way into the treatment of bipolar disorder and migraines. A meta-analysis of clinical trials showed that for bipolar disorder, V P A was more effective than placebo, equally effective as lithium and carbamazepine, but less effective than olanzapine (Macritchie et al., 2003). According to a recent review of clinical trials, V P A was an efficacious and well-tolerated agent for the preventive treatment of migraine, chronic daily headache, and cluster headache, as well as for the treatment of acute migraine attacks (Freitag, 2003). 1.3.2. Mechanism of action The mechanism of the anti-epileptic action of V P A is not well understood. Several mechanisms of action have been proposed. V P A potentiates the effect of y-aminobutyric acid (GAB A), but does not directly interact with postsynaptic G A B A receptors (Owens and Nemeroff, 2003). Instead, V P A increases regional neuronal concentrations of G A B A by inhibiting its degradation and increasing its synthesis (Loscher, 2002). V P A also inhibits iV-methyl-D-aspartate (NMDA) receptor-mediated excitation (Zeise et al., 1991). Another possible mechanism is a non-specific membrane stabilizing effect by blockade of voltage-dependent sodium currents (McLean and Macdonald, 1986). 10 1.3.3. Chemistry and biotransformation V P A is a C-8 branched fatty acid, as shown in Figure 3. The pure acid, which is a colourless liquid with a molecular weight of 144.2 g/mol, is highly soluble in organic solvents. The sodium salt is very soluble in water (Kuo et al., 2004). In contrast with its simple chemical structure, the biotransformation of V P A is highly complex. Phase I metabolism of VPA, shown in Figure 4, utilizes pathways such as P-oxidation, co-hydroxylation, and (co-l)-hydroxylation, which are normally reserved for the biotransformation of endogenous fatty acids (Abbott and Anari, 1999). Mitochondrial P -oxidation is involved in the formation of (£)-2-ene-VPA, 3-keto-VPA, and 3-OH-VPA (Schafer and Luhrs, 1978). Another pathway of 3-OH-VPA formation is CYP-dependent (co-2)-hydroxylation. The (w-l)-hydroxylation of V P A to 4-OH-VPA and OD-hydroxylation to 5-OH-V P A are also CYP-dependent (Prickett and Baillie, 1984). The 3-ene-VPA metabolite is formed by CYP-dependent desaturation of V P A (Bjorge and Baillie, 1991). Further P-oxidation of 3-ene-VPA leads to (£,£)-2,3'-diene-VPA (Rettenmeier et al., 1987). Of particular interest is the CYP-dependent desaturation of V P A to 4-ene-VPA (Rettie et al., 1992). The 4-ene-VPA metabolite has been shown to be the most toxic metabolite of V P A in isolated hepatocytes (Kingsley et al., 1983), and is teratogenic in mice (Nau and Loscher, 1986) Subsequent P-oxidation of 4-ene-VPA leads to the production of another hepatotoxic metabolite, (£)-2,4-diene-VPA (Kassahun et al., 1991). 11 (C) 4-OH-VPA (A) V P A COOH OH COOH HQ. (D) 5-OH-VPA COOH (B) 4-ene-VPA COOH (E) 3-OH-VPA COOH OH Figure 3. V P A and selected oxidative metabolites. Structures for (A) VPA, (B) 4-ene-VPA, (C) 4-OH-VPA, (D) 5-OH-VPA, and (E) 3-OH-VPA are shown, indicates a chiral centre. 12 C O O H C O O H H O . 3 - e n e - V P A 2 , 3 ' - d i e n e - V P A A i i i C O O H 5 - O H - V P A V P A ( A ) 2 - e n e - V P A J/B) C O O H ( A ) (B) f C O O H 4 - e n e - V P A 2 , 4 - d i e n e - V P A 4 - O H - V P A C O O H C O O H O 3 - k e t o - V P A Figure 4. Phase I metabolism of VPA. Enzymatic pathways include (A) P-oxidation, (B) C Y P -dependent desaturation, (C) CYP-dependent co-hydroxylation, (D) CYP-dependent (co-1)-hydroxylation, and (E) CYP-dependent (oo-2)-hydroxylation. Adapted from Abbott and Anari (1999). 13 V P A also undergoes phase II conjugation. The major V P A metabolite in humans is V P A glucuronide (Dickinson et al., 1989). The conjugation of V P A with D-glucuronic acid is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes UGT1A3, UGT1A6, UGT1A9, and UGT2B7 (Ethell et al., 2003; Sakaguchi et al., 2004). Phase I metabolites of V P A have also been found to undergo glucuronidation (Granneman et al., 1984; Tatsuhara et al., 1987). 1.4. Rationale and Hypothesis One case report has suggested a possible interaction between G B E and warfarin. A 78-year-old woman stabilized on warfarin for 5 years experienced an intracerebral hemorrhage after 2 months of chronic Ginkgo biloba use (Matthews, 1998). It is possible that this interaction may be either pharmacodynamic or pharmacokinetic in nature. The pharmacodynamic explanation rests on the fact that both Ginkgo biloba and warfarin possess anti-coagulant properties. In vitro tests have shown that ginkgolide B is a potent inhibitor of PAF (Chung et al., 1987). Warfarin derives its anticoagulant effect through antagonism of Vitamin K, a cofactor in the bioactivation of coagulation factors II, VII, IX, and X (Hirsh et al., 2001). Thus, G B E may have had an additive effect on the anticoagulant effect of warfarin. This theory is supported by another case report in which after one week of ingesting 80 mg of Ginkgo biloba daily, a 70-year-old man taking 325 mg of aspirin daily developed hyphema, a spontaneous bleeding from the iris into the anterior chamber of the eye. Aspirin is an inhibitor of platelet aggregation. The patient continued to take aspirin, but the bleeding stopped upon discontinuation of Ginkgo biloba. (Rosenblatt and Mindel, 1997). There have also been a number of case reports in which patients taking Ginkgo biloba have experienced bleeding without the concomitant use of anticoagulant drugs. A 61-year-old man developed subarachnoid hemorrhage after consuming 40 mg tablets of Ginkgo biloba 3 or 4 14 times daily for more than six months. Bleeding time was increased, but returned to normal after discontinuation of Ginkgo biloba (Vale, 1998). Right parietal hematoma was detected in a man taking 120 mg of G B E daily for the previous 18 months (Benjamin et al., 2001). In another case report, an otherwise-healthy 33-year-old woman taking 120 mg of Ginkgo biloba daily for 2 years presented with headaches, nausea, and vomiting. An MRI of the brain revealed bilateral subdural hematomas. Bleeding times were prolonged but retuned to normal after cessation of Ginkgo biloba ingestion (Rowin and Lewis, 1996). However, some doubt was raised as to whether the hematomas can be attributed to GBE (Odawara et al., 1997). In another case report, a 72-year-old woman who had been taking 50 mg of Ginkgo biloba daily for 6 to 7 months complained of memory impairment and dizziness. A brain CT showed left frontal subdural hematoma (Gilbert, 1997). However, caution was advised in interpreting whether this was due to coincidence or if there was a causal relationship between the use of Ginkgo and the patient's hematoma (Lewis and Rowin, 1997). Indeed, there is debate over whether enough ginkgolides are present in GBE and whether enough ginkgolides are absorbed after oral ingestion to cause clinically relevant P A F antagonism in humans (Chung et al., 1987; Braquet, 1993). In one clinical trial of G B E for dementia involving 309 patients, there was one case of subdural hematoma, and that case occurred in the placebo group. (Le Bars et al., 1997) Since there are some doubts as to whether the interaction between G B E and warfarin is a pharmacodynamic one, an alternative explanation was explored. It is also possible that the interaction of GBE with warfarin is a pharmacokinetic one involving the CYP enzymes. It has been shown that CYP2C9 is the major isoform responsible for the metabolism of (5)-warfarin (Rettie et al., 1992). In addition, several constituents of G B E have been shown to inhibit CYP2C9 (Zou et al., 2002). This led to the research hypothesis: 15 GBE reduces CYP2C9-mediated drug clearance in humans. The effect by GBE may be due to one or more of its constituents such as kaempferol and its glycosides, quercetin and its glycosides, isorhamnetin and its glycosides, the ginkgolides, or bilobalide. To probe the effect of G B E on CYP2C9, a substrate probe was needed. For this project, VPA, which is metabolized by CYP2C9 (Sadeque et al., 1997, Anari et al., 2000; Ho et al., 2003), was chosen. Thus, the experimental hypothesis was: GBE and some of its constituents such as kaempferol and its glycosides, quercetin and its glycosides, isorhamnetin and its glycosides, the ginkgolides, and bilobalide inhibit CYP-mediated oxidative metabolism of VPA in vitro. In addition to CYP2C9, CYP2B6 and CYP2A6 also appear to be involved in the oxidative metabolism of V P A (Sadeque et al., 1997; Anari et al., 2000). In the present study, the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA was monitored. The formation of the four metabolites were measured using a slightly modified version of the GC/MS assay utilized by Ho et al. (2003). The in vitro system used to study V P A metabolism was human liver microsomes (HLM). Al l H L M were from donors with the CYP2C9*1/*1 genotype. This one genotype was chosen since it is the most prevalent, and in order to reduce variability in the results, since CYP2C9 allelic variants have been shown to exhibit different enzyme kinetics towards V P A (Ho et al., 2003). 16 1.5. Specific Objectives To validate the VPA GC/MS assay by: • ensuring that V P A metabolites and internal standards are adequately resolved in chromatograms; • determining the dynamic range of the standard curves of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA; and • determining accuracy and precision for limits of quantitation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA. To optimize the VPA enzyme assay by: • optimizing total CYP content of H L M for incubations with VPA; • optimizing incubation time of H L M for incubations with VPA; and • determining intraday and interday variabilities for incubations of H L M with V P A using optimized conditions. To investigate the effect of GBE on VPA metabolism by: • comparing the effect of G B E from different lots and manufacturers on V P A metabolism by H L M ; • determining the effect of GBE concetration on V P A metabolism by H L M ; • determining the effect of GBE on CYP2C9-catalyzed V P A metabolism by H L M ; • determining the effect of GBE on VPA metabolism by recombinant CYP2C9*1, CYP2B6, and CYP2A6; and 17 determining the effect of some of the individual constituents of G B E on V P A metabolism by H L M . 18 2. MATERIALS AND METHODS 2.1. Materials 2.1.1. Acids, bases, salts, and buffers Tris[hydroxymethyl]aminomethane (Tris) and magnesium chloride (MgCk) were purchased from Sigma (St. Louis, MO). Anhydrous sodium sulfate and solid sodium hydroxide (NaOH) were acquired from E M Science (Darmstadt, Germany). Monobasic potassium phosphate, phosphoric acid and hydrochloric acid (HC1) were purchased from Fisher (Fair Lawn, NJ). A 1 M solution of NaOH was made by dissolving solid NaOH in water. Phosphoric acid was diluted to 0.1 M with water. Potassium phosphate buffer of 1 M concentration was prepared by adding monobasic potassium phosphate to water, and adjusting the pH to 3.0 with phosphoric acid. Tris buffer of 0.2 M concentration containing 6 mM MgCb was made by adding Tris and MgCl2 to water, and adjusting the pH to 7.4 by drop-wise addition of HC1. These stock buffers were stored at 4°C until use. Al l water used was of 18 M Q quality. 19 2.1.2. Organic solvents Ethyl acetate and hexane were purchased from Fisher. Acetone was acquired from Galdeon Laboratories (Georgetown, ON). Dimethylsulfoxide (DMSO) was from Sigma. Ethyl acetate and DMSO were of H P L C grade. Hexane and acetone were of GC grade. 2.1.3. Gases Nitrogen, helium, and methane were purchased from Praxair (Mississauga, ON). Helium and methane were of Ultra High Purity 5.0 quality. 2.1.4. GBE and its individual constituents Two different lots of G B E (1306A and 302831) were obtained from Indena S.A. (Milan, Italy). Another lot (63964, GK501™) was provided by Pharmaton S.A. (Bioggio, Switzerland). The chemical composition of G B E of lot 1306A is shown in Appendix 1. GBE was prepared fresh daily in 0.1 M Tris containing 3 mM MgCb. For experiments with individual G B E constituents, GBE was prepared in 0.1 M Tris buffer containing 3 mM MgCb and 0.05% DMSO (v/v). Bilobalide and ginkgolides A, B, and C were purchased from L K T (St. Paul, MN). Ginkgolide J was acquired from ChromaDex (Santa Ana, CA). Quercetin was obtained from Sigma. Kaempferol, isorhamnetin, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, and quercetin-3-O-rutinoside were purchased from Indofine Chemical Company (Hillsborough, NJ). Individual constituents were prepared fresh each day in 0.1 M Tris buffer containing 3 m M M g C l 2 and 0.05% DMSO (v/v). 20 2.1.5. VPA The free base form of V P A was purchased from Acros Organics (New Jersey, NJ). The free base was hydrolyzed by the addition of an equimolar amount of 1 M NaOH. After one day at 4°C, the solution was diluted to 10 mM by the addition of water. The stock solution was aliquoted and stored at -20°C until use. 2.1.6. NADPH N A D P H was purchased from Sigma. It was prepared fresh each day in water and placed on ice, shielded from exposure to light, until use. 2.1.7. Derivatization agents V.iV-Diisopropylethylamine (DIPEA) and ferr-butyldimethylsilyl chloride (TBDMSC1) were purchased from Sigma. Pentafluorobenzyl bromide (PFBBr), dimethylformamide (DMF), and Af-methyl-V-(fer/-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) were acquired from Pierce (Rockford, EL). MTBSTFA containing 2% TBDMSC1 (w/v) was prepared by adding 0.1 g of T B D M S C l to a 5 g bottle of MTBSTFA. 2.1.8. Metabolite standards The stock solution of metabolite standards used in the preparation of standard curves contained 0.5 pg/mL each of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA. These V P A metabolites were synthesized in the laboratory of Dr. Frank Abbott (Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC). This solution was aliquoted and stored at -20°C. 21 2.I.9. Internal standard The internal standard stock solution contained 2 pg/mL each of heptadeuterated V P A metabolites 4-ene-[ 2H 7]-VPA, 2-ene-[2H7]-VPA, 4-OH-[ 2H 7]-VPA, 5-OH-[ 2H 7]-VPA, and 3-OH-[ 2 H 7 ]-VPA. These heptadeuterated metabolites were synthesized in the laboratory of Dr. Frank Abbott (Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC). This solution was aliquoted and stored at -20°C. 2. /. 10. Microsomes and antibodies H L M from individuals with the CYP2C9*1/*1 genotype (donors HH18, HG30, HH47, HH91, and HG95), control insect cell microsomes, and recombinant CYP2C9*1, CYP2B6, and CYP2A6 were purchased from Gentest (Woburn, MA) . H L M activities are shown in Appendix 2. Donor history is provided in Appendix 3. Information in recombinant CYP is provided in Appendix 4. Monoclonal antibodies against CYP2C9 (MAb2C9) (Krausz et al., 2001), CYP2B6 (MAb2B6) (Yang et al., 1998), and CYP2A6 (MAb2A6) (Sai et a l , 1999), and control IgG were provided by the National Cancer Institute at the National Institutes of Health (Bethesda, MD). Information about the antibodies is shown in Appendix 5. 2.2. Standard Curves Standard curves were prepared on each experimental day. Metabolite standards (0-200 uL) were added to borosilicate glass screw-top vials. The volume of each tube was topped up to 1 mL with water. To each tube, 75 uL of 0.1 M phosphoric acid and a 200 uL mixture containing 1 m M V P A in 0.1 M Tris buffer 3 mM M g C l 2 (pH 7.4) were added. To each tube, 50 uL of internal standard was added. 22 2.3. Microsomal Incubations 2.3.1. Optimization of total CYP content for incubations with HLM In a polypropylene eppendorf tube, a mixture containing 1 mM VPA, 1 m M NADPH, and 0.1 M Tris buffer containing 3 mM MgCb (pH 7.4) was pre-warmed at 37°C for 2 min in a shaking water bath. The reaction was initiated with the addition of H L M (donor HH47, 3 determinations) containing 20, 40, 60, 80, or 100 pmol of total CYP. The final incubation volume was 200 pL. The blank did not contain any microsomes. After 20 min, the incubation was terminated with the addition of 75 pL of ice-cold 0.1 M phosphoric acid. To each tube, 50 uL of internal standard was added. The contents were then transferred to a borosilicate glass screw-top vial containing 1 mL of water. 2.3.2. Optimization of incubation time for incubations with HLM In a polypropylene eppendorf tube, a mixture containing 1 mM VPA, 1 mM NADPH, and 0.1 M Tris buffer containing 3 mM MgCb (pH 7.4) was pre-warmed at 37°C for 2 min in a shaking water bath. The reaction was initiated with the addition of H L M (donor HH47, 3 determinations) containing 60 pmol of total CYP. The final incubation volume was 200 uL. After 10, 20, 30, 40, or 50 min, the incubation was terminated with the addition of 75 uL of ice-cold 0.1 M phosphoric acid. For the blank, the reaction was terminated immediately after the addition of microsomes. To each tube, 50 pL of internal standard was added. The contents were then transferred to a borosilicate glass screw-top vial containing 1 mL of water. 2.3.3. Intraday and interday variabilities for incubations with HLM In a polypropylene eppendorf tube, a mixture containing 1 mM VPA, 1 mM NADPH, and 0.1 M Tris buffer containing 3 mM MgCb (pH 7.4) was pre-warmed at 37°C for 2 min in a 23 shaking water bath. The reaction was initiated with the addition of H L M (donor HG30) containing 60 pmol of total CYP. An equivalent amount of inactivated microsomes, which were prepared by heating H L M at 65°C for 20 min, was used for the blank. The final incubation volume was 200 uL. After 20 min, the incubation was terminated with the addition of 75 uL of ice-cold 0.1 M phosphoric acid. To each tube, 50 uL of internal standard was added. The contents were then transferred to a borosilicate glass screw-top vial containing 1 mL of water. This procedure was repeated on four separate days. On each day, four determinations were performed. 2.3.4. Effect of GBE from different lots and manufacturers on VPA metabolism by HLM In a polypropylene eppendorf tube, a mixture containing 1 mM VPA, 1 mM N A D P H , and 0.3 mg/mL of G B E (Indena, lot 1306A), G B E from a different lot (Indena, lot 302831), or G B E from a different manufacturer (Pharmaton, lot 63964) in 0.1 M Tris buffer containing 3 m M M g C l 2 (pH 7.4) was pre-warmed at 37°C for 2 min in a shaking water bath. For the control, 0.1 M Tris buffer containing no GBE was used. The reaction was initiated with the addition of H L M (donors HH18, HH47, HH91, and HG95; two determinations each) containing 60 pmol of total CYP. An equivalent amount of inactivated microsomes was used for the blank. The final incubation volume was 200 uL. After 20 min, the incubation was terminated with the addition of 75 uL of ice-cold 0.1 M phosphoric acid. To each tube, 50 pL of internal standard was added. The contents were then transferred to a borosilicate glass screw-top vial containing 1 mL of water. 24 2.3.5. Concentration-dependent effect of GBE on VPA metabolism by HLM In a polypropylene eppendorf tube, a mixture containing 1 m M VPA, 1 m M N A D P H , and 0.01, 0.03, 0.1, 0.3, 0.6, or 1 mg/mL of G B E (Indena, lot 1306A) in 0.1 M Tris buffer containing 3 m M MgCl2 (pH 7.4) was pre-warmed at 37°C for 2 min in a shaking water bath. For the control, 0.1 M Tris buffer containing no G B E was used. The reaction was initiated with the addition of H L M (donors HH18, HH47, HH91, and HG95; two determinations each) containing 60 pmol of total CYP. An equivalent amount of inactivated microsomes was used for the blank. The final incubation volume was 200 pL. After 20 min, the incubation was terminated with the addition of 75 pL of ice-cold 0.1 M phosphoric acid. To each tube, 50 uL of internal standard was added. The contents were then transferred to a borosilicate glass screw-top vial containing 1 mL of water. 2.3.6. Effect of GBE on CYP2C9-catalyzed VPA metabolism by HLM For incubations involving antibodies, the protocol was changed slightly. H L M (donors HH18, HG30, and HH47; two determinations each) containing 60 pmol of total CYP were pre-incubated at 37°C with 3 pL of MAb2C9 or 3 pL of MAb2B6 and 3 pL of MAb2A6 in 0.1 M Tris buffer containing 3 mM MgCh (100 pL incubation volume) for 5 min in a shaking water bath. A previous study determined that near maximal inhibition of V P A metabolism by MAb2C9, MAb2B6, or MAb2A6 alone was seen in H L M using 3 pL of antibody (Kiang et al., 2005). Product formation for incubations with antibodies was normalized to product formation for pre-incubations with an equivalent volume of control IgG (3 or 6 pL). Inactivated H L M containing 60 pmol of total CYP were used for the blank. The reaction was initiated with the addition of 100 uL of a pre-warmed mixture containing 2 mM VPA, 2 mM NADPH, and 0.1 M Tris buffer or 2 mg/mL G B E (Indena, lot 1306A) in 0.1 M Tris buffer. The final concentrations were the same as 25 in other experiments (1 m M V P A , 1 m M N A D P H , and 1 mg/mL G B E in 200 uL of 0.1 M Tris buffer). After 20 min, the incubation was terminated with the addition of 75 uL of ice-cold 0.1 M phosphoric acid. To each tube, 50 uL of internal standard was added. The contents were then transferred to a borosilicate glass screw-top vial containing 1 mL of water. 2.3.7. Effect of GBE on VPA metabolism by recombinant CYP2C9*1, CYP2B6, and CYP2A6 In a polypropylene eppendorf tube, a mixture containing 1 mM VPA, 1 m M NADPH, and 0.3 or 1 mg/mL G B E (Indena, lot 1306A) in 0.1 M Tris buffer containing 3 m M M g C l 2 (pH 7.4) was pre-warmed at 37°C for 2 min in a shaking water bath. For the control, 0.1 M Tris buffer containing no G B E was used. The reaction was initiated with the addition of microsomes containing 40 pmol of cDNA-expressed CYP2C9*1, CYP2B6, or CYP2A6 (3 determinations each). Control insect cell microsomes were used for the blank. The final incubation volume was 200 uL. After 30 min, the incubation was terminated with the addition of 75 uL of ice-cold 0.1 M phosphoric acid. To each tube, 50 uL of internal standard was added. The contents were then transferred to a borosilicate glass screw-top vial containing 1 mL of water. 2.3.8. Effect of individual constituents of GBE on VPA metabolism by HLM In a polypropylene eppendorf tube, a mixture containing 1 mM VPA, 1 mM NADPH, and 500 pg/mL of G B E (Indena, lot 1306A) or its individual constituents in 0.1 M Tris buffer containing 3 mM M g C l 2 (pH 7.4) and 0.05% DMSO (v/v) was pre-warmed at 37°C for 2 min in a shaking water bath. For the vehicle control, 0.1 M Tris buffer containing 3 mM M g C l 2 (pH 7.4) and 0.05% DMSO (v/v) was used. The reaction was initiated with the addition of H L M (donors HH18, HG30, HH47, and HH91; two determinations each) containing 60 pmol of total CYP. Inactivated H L M containing 60 pmol of total CYP were used for the blank. The final incubation 26 volume was 200 uL. After 20 min, the incubation was terminated with the addition of 75 pL of ice-cold 0.1 M phosphoric acid. To each tube, 50 uL of internal standard was added. The contents were then transferred to a borosilicate glass screw-top vial containing 1 mL of water. The concentrations of the terpene trilactones and isorhamnetin-3-O-rutinoside were the concentrations present in 500 pg/mL of G B E (bilobalide; ginkgolides A, B, C, and J; and isorhamnetin-3-O-rutinoside in Appendix 1). The concentrations of kaempferol-3-O-rutinoside and quercetin-3-O-rutinoside were the concentrations of their more abundant diglycoside present in 500 pg/mL of GBE (kaempferol diglycoside 1 and quercetin diglycoside 2 in Appendix 1). The concentrations of the flavonol aglycones were the concentrations that would be present in 500 pg/mL of GBE if all the flavonol glycosides were converted to their respective aglycones (sum under isorhamnetin, kaempferol, and quercetin in Appendix 1). The individual constituents tested were 14 pg/mL (43 uM) of bilobalide, 5.5 pg/mL (13 uM) of ginkgolide A, 1.5 pg/mL (4 pM) of ginkgolide B, 7 pg/mL (16 uM) of ginkgolide C, 3 pg/mL (7 uM) of ginkgolide J, 3 pg/mL (5 pM) of isorhamnetin-3-O-rutinoside, 5 pg/mL (8 uM) of kaempferol-3-O-rutinoside, 12 pg/mL (20 uM) of quercetin-3-O-rutinoside, 20.5 pg/mL (65 pM) of isorhamnetin aglycone, 31.5 pg/mL (110 pM) of kaempferol aglycone, and 53 pg/mL (175 uM) of quercetin aglycone). 2.4. Sample Preparation for VPA Metabolite Analysis Samples from microsomal incubations and standards were processed according to the scheme shown in Figure 5. Extraction and derivatization are described in detail below. 27 Incubations Standards 100 uL of 0.2 M Tris (pH 7.4) + (1000 - y) uL of water + 20 uL of 10 mM V P A + y uL of metabolite standards 20 uLo f 10mM NADPH + (60 - x) uL of water j i Pre-warmed for 2 min A D D E D 1 0 0 U L O F 0 2 M Tris (pH 7.4) + I 20 uL of 10 mM V P A + Initiated reaction with 30 u|_ Q f water x ul_ of microsomes I i -Incubated for 30 min T . . . ^ .. ... Added 75 uL of 0.1 M phosphoric acid Terminated reaction with M K K i Added 50 pL of internal standard 75 uL of 0.1 M phosphoric acid I Added 1000 uL of water 1 Added 50 pL of internal standard 1 i Extraction Added 1 mL of 1 M potassium phosphate buffer (pH 3.0) + 8 mL of ethyl acetate i Capped then gently rotated for 30 min 1 Centrifuged at 1600 x g for 10 min I Transferred organic layer to new vial 1 Added 1 g of anhydrous sodium sulfate i Capped then gently rotated for 15 min 1 Centrifuged at 1600 x g for 10 min i Decanted into new vial i Dried under stream of 2.5 psi nitrogen gas until volume reduced to 100-200 pL 1 (continued on next page) »ure 5. Sample preparation scheme. 28 Derivatization Added 30 uL of DIPEA + 10 uL of PFBBr I Capped then vortexed 1 Incubated for 1 h at 45°C 1 Added 20 pL of DMF + 40 pL of MTBSTFA containing 2% TBDMSCI i Capped then vortexed 1 Incubated for 2 h at 65°C 1 Dried under stream of 2.5 psi nitrogen gas I Added 200 uL of hexane 1 Capped then vortex for 1 min i Centrifuged at 1600 x g for 10 min i Transferred hexane layer to glass insert Figure 5 (continued). 29 2.4.1. Extraction The pH was adjusted to 3.0 with 1 mL of 1 M potassium phosphate buffer. Extraction was performed by adding 8 mL of ethyl acetate to tubes containing incubation samples or standards which were then gently rotated for 30 min. Following centrifugation at 1600 xg for 10 min in a Beckman GP centrifuge (Fullerton, CA), the organic layer was transferred to a new vial. The organic phase was dried over approximately 1 g of anhydrous sodium sulfate for 15 min. Following centrifugation at 1600 x g for 10 min, the organic phase was decanted into another new vial. The organic phase was evaporated under a stream of 2.5 psi nitrogen gas for 2-3 h until the volume was reduced to 100-200 pL in a Zymark TurboVap L V evaporator (Hopkinton, MA) , with the bath temperature set at 25°C. 2.4.2. Derivatization Samples were incubated for 1 h at 45°C with 30 pL of DIPEA and 10 uL of PFBBr. Samples were then incubated for 2 h at 65°C with 20 pL of D M F and 40 pL of M T B S T F A containing 2% TBDMSC1. The samples were dried under a stream of 2.5 psi nitrogen for 30 min. Following the addition of 200 pL of hexane, each tube was vortexed for 1 min, then centrifuged at 1600 x g for 10 min. The hexane layer was transferred to a 250 pL glass insert, placed in an autosampler vial, and capped with a Teflon-silicone septum (all from Chromatographic Specialties, Brockville, ON). 2.5. G C / M S Assay The GC/MS system consisted of an HP 7683 Series autosampler, an HP 7683 Series injector, and an HP 6890 Series GC system interfaced with an HP 5973 Mass Selective detector (Avondale, PA). The front inlet, GC/MS interface, and ion source temperatures were set at 30 250°C, 270°C, and 200°C, respectively. The septum purge flow rate of the carrier gas, helium, was 20 mL/min for 2 min. The nominal initial flow was set at 19.18 psi and 0.5 mL/min. A l l instruments were controlled by HP Enhanced ChemStation (version B.01.00). Autosampler vials were loaded onto the autosampler, and 1 pL was injected in splitless mode. The oven was held steady at 40°C for 0.5 min. Next, the temperature was rapidly increased to 140°C at a rate of 75°C/min. The temperature was then raised at a rate of l°C/min to 160°C. Finally, the temperature was increased to 270°C at a rate of 10°C/min. Al l temperatures were raised using a linear gradient. The oven temperature program is shown in Table 1. Separation was achieved using a SolGel-lms GC capillary column (60 m x 0.25 mm id, 0.25 pm film thickness) from SGE (Austin, TX), which was connected via a fused silica union to a Z guard column (5 m x 0.25 mm id) from Phenomenex (Torrance, CA). The MS was operated in negative ion chemical ionization mode (NICI) with selected ion monitoring (SIM). The fixed element emission current was 50 pA and the electron energy was 150 eV. The reagent gas was methane, and its pressure was set at 0.18 mTorr. Ions at mlz of 141 (4-ene-VPA), 148 (2-ene-[ 2 H 7 ]-VPA), 273 (4-OH-VPA, 5-OH-VPA, and 3-OH-VPA), and 280 (4-OH-[ 2H 7]-VPA, 5-OH-[ 2 H 7 ]-VPA, and 3,-OH-[ 2H 7]-VPA) were monitored. 31 Table 1. GC oven temperature program. Start Time (min) End Time (min) Start Temp (°C) Final Temp (°C) Ramp (°C/min) 0 0.5 40 40 0 0.5 1.83 40 140 75 1.83 21.83 140 160 1 21.83 37.83 160 270 10 32 2.6. Data Analysis 2.6. J. Calculation of PAR Chromatograms were integrated using ChemStation Integrator. Peaks were manually identified and peak areas were entered into Microsoft Excel (Redmond, WA). Peak area ratio (PAR) was calculated by dividing the peak area of a metabolite by the peak area of its heptadeuterated analog. For 4-ene-VPA, 2-ene-[ 2H 7]-VPA was used as the internal standard due to poor resolution of 4-ene-[ 2H 7]-VPA. To calculate the P A R for 4-OH-VPA, the sum of the peak areas of the two diastereomers of 4-OH-VPA was divided by the sum of the peak areas of the two diastereomers of 4-OH-[ 2 H 7 ]-VPA. r , » * * \ us A Peak area of 4-ene-VPA PAR of 4-ene-VPA = P e a k area of 2-ene-f H 7 ] -VPA r ^ r . r . ^ . w ™ Peak areas of 4-OH-VPA (1) + 4-OH-VPA (2) PAR of 4 -OH-VPA = 0 „ , , „ ^ u r A — Peak areas of 4-OH-f H 7 ] -VPA (1) + 4 -OH-fH 7 ] -VPA (2) r m u wr, A Peak area of 5-OH-VPA PAR of 5-OH-VPA = P e a k a r e a of 5-OH-[^H7]-VPA PAR of 3-OH-VPA = Peak area of 3-OH-VPA Peak area of 3-OH-f H 7 ] -VPA 2.6.2. Standard curves PAR values for the metabolite standards were calculated. Standard curves for 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA were constructed by plotting the PAR values against the amount of standard in grams. A best fit line was drawn using the least squares method. A relationship in the form y = mx + b, where y is the PAR, m is the slope, x is the amount of standard in grams, and b is the ^ -intercept, was determined for each of the four metabolites. 33 2.6.3. Microsomal incubations P A R values for enzyme incubations and blanks were calculated. The PAR values for enzyme incubations were corrected by subtracting from them the P A R value for the blank. The amount of metabolite in a particular sample was determined by calculating the values of x from the standard curve equation, based on the corrected P A R values of that particular sample. For incubations with FELM, product formation is expressed as pmol/min/nmol total CYP, and for incubations with recombinant CYP, as pmol/min/nmol CYP. 2.6.4. Limit of quantitation The limit of quantitation (LOQ) was determined for 4-ene-VPA, 4 -OH-VPA 5-OH-VPA, and 3-OH-VPA. LOQ is defined as the minimum amount of metabolite that can be detected with acceptable precision and accuracy. According to convention, coefficient of variation (CV), a measure of precision, of less than 20% is considered acceptable. Bias, a measure of accuracy, of less than 20% is acceptable (Shah et al., 1991). Standard curves were constructed and 0.5, 1- 1.5 and 2 ng of metabolite standards were spiked into the same matrix as that of the standard curve. Six replicates were performed for each amount of standard. The measured amounts of standard were calculated from the standard curves, and C V and bias were determined. C V = Standard Deviation of Measured Amount x 100% Mean of Measured Amount Bias = Mean of Measured Amount - Nominal Amount x 100% Nominal Amount 34 2.6.5. Intraday and inter day variabilities The intraday and interday variabilities of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA formation were determined. Standard curves were constructed. Microsomal incubations were performed on four different days. Four determinations were carried out on each day. Intraday variability, as measured by the C V of product formation of four replicates on one day, of less than 20% was considered acceptable. Interday variability, as measured by the C V of the average product formation over four days, of less than 20% was considered acceptable (Shah et al., 1991). 2.7. Statistical Analysis In experiments involving recombinant CYP or H L M from only one donor, data are expressed as mean ± standard deviation (SD). In experiments where H L M from more than one donor were used, data are expressed as mean ± standard error of the mean (SEM). In cases where product formation was less than LOQ, a value midway between limit of detection (LOD) and LOQ is used in the calculation of the mean. Since L O D was not determined in this study, in such cases the midpoint of nought and LOQ (i.e. half of LOQ) was used. Statistical significance for differences between treatment groups was determined by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test using SigmaStat for Windows (version 1.0) from Jandel (San Rafael, CA). Statistical significance for differences between treatment groups and control was determined using one-way A N O V A followed by Dunnett's test. Differences were considered statistically significant if/? < 0.05. 35 3. RESULTS 3.1. Validation of VPA GC/MS Assay 3.1.1. Chromatograms The M S recorded the total ion current. A representative total ion chromatogram is shown in Figure 6. The M S also performed S E M . The molecular ion of 4-ene-VPA, visible at mlz =141, was clearly resolved as shown in Figure 7A. Due to the relatively large amount of V P A {mlz = 143), some carry-over was seen at mlz = 141. As shown in Figure 7B, at mlz = 148, 4-ene-[2F£7]-VPA was not adequately resolved. Therefore, 2-ene- [ 2 H 7 ] -VPA was used as the internal standard for 4-ene-VPA. On a representative day, the retention time for 4-ene-VPA was 21.16 min. Its heptadeuterated analog, 4-ene-[2H7]-VPA, eluted 0.25 min earlier, at 20.91 min. Elution of 2-ene-[ 2H 7]-VPA occurred at 24.34 min. As shown in Figure 8A, at mlz = 273, the molecular ions of 3-OH-VPA, 5-OH-VPA, and the two diastereomers of 4-OH-VPA were resolved. At mlz = 280, 3-OH-[ 2H 7]-VPA, 5-OH-[ 2 H 7 ] -VPA and the two diastereomers of 4-OH-[ 2 H 7 ]-VPA were resolved, as shown in Figure 8B. 36 The two diastereomers of 3-OH-[ H7]-VPA were not separated and appeared as one peak. On a representative day, the retention times for the diastereomers of 4-OH-VPA were 32.13 and 32.39 min. Elution of 3-OH-VPA occurred at 32.24 min. The retention time of 5-OH-VPA was 33.45 min. Their heptadeuterated analogs eluted approximately 0.05 min earlier, at 32.08, 32.34, 32.19, and 33.40 min, respectively. Retention times are listed in Table 2. 3.1.2. Standard curves Standard curves were constructed for 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA. For the 4-ene-VPA standard curve, 0.5-10 ng of 4-ene-VPA standard was used. For 4-OH-VPA, 2-100 ng of 4-OH-VPA standard was used to construct the standard curve. For the 5-OH-V P A standard curve, 2-100 ng of 5-OH-VPA standard was used. For 3-OH-VPA, 0.5-5 ng of 3-O H - V P A standard was used to construct the standard curve. The standard curves were linear in their dynamic ranges. Representative standard curves from one experiment are shown in Figure 9. 3.1.3. Limit of quantitation The LOQs of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA were determined. Their values were 0.5 ng (3.5 pmol), 2 ng (12.5 pmol), 2 ng (12.5 pmol), and 0.5 ng (3.1 pmol), respectively, as shown in Error! Reference source not found.. At the LOQ, accuracy (bias) and precision (CV) were both less than 20% for all four metabolites. 37 IV Abundance 360000 340000; 320000: 300000^  280000 260000 240000: 220000: 200000 180000 160000 140000 120000 100000 80000 60000 40000 20000 ffime-> II 4--Slob' 'iQ.'ob' '2'o!ob' 21.00 22!ob' '23!00 24!ob' •2S.'oV -26:06 27:00• •28.'0O' 29:00' •aolOb' '31:00 32:00 33.00 34.00 35.00 36.00 37.00 Figure 6. Representative total ion chromatogram. This sample contained 100 ng of each metabolite and internal standard. Peaks for (I) 4-ene-VPA and 4-ene-[2H7]-VPA; (II) 2-ene-[2H7]-VPA; (IH) 3-OH-VPA, 3-OH-[ 2H 7]-VPA, 4-OH-VPA, and 4-OH-[ 2 H 7 ]-VPA; and (IV) 5-OH-VPA and 5-OH-[ 2H 7]-VPA can be seen. Abundance {k)mlz= 141 II Jlloo™ 2l!so 22.'00 22|50 23J00 23!60 (B) m/r= 148 frh»-> 20.00 20.50 Abundance 24.00 24.50. Ill IV U n e - ^ 20!00 20150 21100 21 to 22.'00 ' 22.50 23100 ZJX 24100 24.50 Figure 7 Representative chromatograms for metabolite standards at mlz of 141 and 148. In this sample, the metabolite standard contained 100 ng each of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA and 3-OH-VPA. The internal standard contained 100 each of 2-ene-[2H7]-VPA, 4-ene-[2H7]-VPA, 4-OH-[ 2H 7]-VPA, 5-OH-[ 2H 7]-VPA, and 3-OH-[ 2H 7]-VPA. At (A) mlz- 141, (I) 4-ene-VPA and (II) V P A are visible. At (B) mlz = 148, (III) 4-ene-[ 2H 7]-VPA and (TV) 2-ene-[ H 7 ] -VPA are visible. Ill 260000 240000 220000 200000 180000 160000 140000 120000 100000 aoooo 60000 40000 20000 (A) mlz = 273 II |nme-» ' ailBO 32.0032)10 32,'20 32,'3Q 32>0 32150 32!60 yjjj 32^ 80 33)00 33)10 33120 33.30 33.40 33.50 33.60 33.70 (B) mlz = 280 (•Kbundwioe 260000! 240000 220000 200000 190000 160000 140000 120000 100000 80000 60000 40000 20000 IV 31190 32:0O 32!10 32:20 32:30 32:40 32:50 32:60 32>0 32.'60 32)90 33.00 33.10 33.20 33.30 33.40 33.50 33.60 33.70 Figure 8. Representative chromatograms for metabolite standards at mlz of 273 and 280. In this sample, the metabolite standard contained 100 ng each of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA. The internal standard contained 100 ng each of 2-ene-[2H7]-VPA, 4-ene-[2H7]-VPA, 4-OH-[ 2H 7]-VPA, 5-OH-[ 2H 7]-VPA, and 3-OH-[ 2H 7]-VPA. At (A) mlz = 273, (I) 4-OH-VPA, (IT) 5-OH-VPA, and (UI) 3-OH-VPA are visible. At (B) mlz = 280, (IV) 4-OH-[ 2 H 7 ]-VPA, (V) 5-OH-[ 2H 7]-VPA, and (VI) 3-OH-[ 2H 7]-VPA are visible. Table 2. Retention times of V P A and its metabolites on a representative day. Metabolite mlz Retention Time (min) 4-ene-VPA 141 21.16 V P A 141 b 21.51 4-ene-[ 2H 7]-VPA a 148 20.91 2-ene-[ 2H 7]-VPA 148 24.34 4-OHA/PA 273 32.13 3-OH-VPA 273 32.24 4-OH-VPA 273 32.39 5-OH-VPA 273 33.45 4-OH-[ 2H 7 ] -VPA 280 32.08 3-OH-[ 2H 7 ]-VPA 280 32.19 4-OH-[ 2H 7 ] -VPA 280 32.34 5-OH-[ 2H 7 ]-VPA 280 33.40 "The peak for 4-ene-[ 2H 7]-VPA was not adequately resolved. ftVPA has mlz =143, but due to its relatively high concentration, some carry-over is seen at mlz =141. 41 (A) 4-ene-VPA (C) 5-OH-VPA 0 4 6 4-ene-VPA (ng) 8 10 1.2 o CD CO CD < CD CD CL 0.4 0 0 20 40 60 5-OH-VPA (ng) 80 100 (B) 4-OH-VPA (D) 3-OH-VPA 0 20 40 60 80 100 0 1 ^ o 4-OH-VPA (ng) 3-OH-VPA (ng) Figure 9. Representative standard curves. For (A) 4-ene-VPA 0.5-10 ng of standard 4-ene-VPA was used. For (B) 4-OH-VPA, 2-100 ng of 4-OH-VPA was used. For (C) 5-OH-VPA, 2-100 ng of 5-OH-VPA was used. For (D) 3-OH-VPA, 0.5-5 ng of 5-OH-VPA was used. The internal standard contained 100 ng each of 2-ene-[2H7]-VPA, 4-OH-[ 2 H 7 ]-VPA, 5-OH-[ 2H 7]-VPA, and 3-OH-[ 2H 7]-VPA. P A R values were calculated by dividing the peak areas of the metabolite standards by the peak areas of their respective internal standards. Table 3. Coefficient of variation and bias of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA standards. Metabolite CV (%) Bias(%) 3.5 pmol 4-ene-VPA 15 -7 12.5 pmol 4-OH-VPA 11 -20 12.5 pmol 5-OH-VPA 14 -3 3.1 pmol 3-OH-VPA 7 4 Samples containing nominal amounts of standards were assayed. Amounts of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA were calculated from standard curves. The C V of 6 replicates was calculated for measured amounts of standard. Bias comparing measured versus nominal amounts of spiked standard were determined. 43 3.2. Optimization of VPA Metabolism Assay 3.2.1. Optimization of total CYP content for incubations with HLM H L M containing 20, 40, 60, 80, or 100 pmol of total C Y P were incubated for 20 min with 1 mM V P A . The formation of 4-ene-VPA and was linear up to 60 pmol of total CYP (Figure 10A). The formation of 4-OH-VPA was linear up to at least 100 pmol of total C Y P (Figure 10B). The formation of 5-OH-VPA was linear up to at least 100 pmol of total C Y P (Figure 10C). The formation of 3-OH-VPA was linear up to 60 pmol of total C Y P (Figure 10D). Thus, 60 pmol of total C Y P was chosen as the amount to use in all further experiments with H L M . 3.2.2. Optimization of incubation time for incubations with HLM H L M containing 60 pmol of total C Y P were incubated for 10, 20, 30, 40, or 50 min with 1 mM VPA. The formation of 4-ene-VPA was linear up to 20 min (Figure 11 A). The formation of 4-OH-VPA was linear up to 30 min (Figure 1 IB). The formation of 5-OH-VPA was linear up to 30 min (Figure 11C). The formation of 3-OH-VPA was linear up to 20 min (Figure 11D). Thus, an incubation time of 20 min was used in all further experiments with H L M . 44 (A) 4-ene-VPA 0 20 40 60 80 100 Total CYP Content (pmol) (B) 4-OH-VPA 600 i Total CYP Content (pmol) (C) 5-OH-VPA 400 n 0 20 40 60 80 100 Total CYP Content (pmol) (D) 3-OH-VPA 15 ! Total CYP Content (pmol) Figure 10. Optimization of total CYP content for incubations with H L M . H L M containing 0, 20, 40, 60, 80, or 100 pmol of total C Y P were incubated with 1 m M V P A for 20 min. The formation of (A) 4-ene-VPA, (B) 4-OH-VPA, (C) 5-OH-VPA, and (D) 3-OH-VPA was monitored. Error bars represent SD (3 determinations). (A) 4-ene-VPA (C) 5-OH-VPA Incubation Time (min) 750 i 0 10 20 30 40 50 Incubation Time (min) (B) 4-OH-VPA (D) 3-OH-VPA Incubation Time (min) Incubation Time (min) Figure 11. Optimization of incubation time for incubations with H L M . H L M containing 60 pmol of total C Y P were incubated with 1 m M V P A for 0, 10, 20, 30, 40 or 50 min. The formation of (A) 4-ene-VPA, (B) 4-OH-VPA, (C) 5-OH-VPA, and (D) 3-OH-VPA was monitored. Error bars represent SD (3 determinations). 3.2.3. Intraday and interday variabilities for incubations with HLM H L M containing 60 pmol of total C Y P were incubated for 20 min with 1 m M V P A . Mean formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA over the four days were 30±4, 436±49, 303±20, and 11±3 pmol/min/nmol total CYP, respectively. As shown in Table 4, the intraday variabilities of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA were less than 20%. As shown in Table 5, although the interday variabilities of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA were less than 20%, the interday variability was not less than 20% for 3-OH-VPA. Therefore, for samples from the same donor, all treatments in one experiment were performed on the same day to avoid any potential issues with interday variability. 3.3. Effect of GBE on VPA Metabolism 3.3.1. Overview of experiments The effect of GBE on V P A metabolism was investigated in a series of experiments. First, the effect of different brands and lots of G B E on V P A metabolism by H L M was examined (Section 3.3.2). Then, using one lot of one brand of GBE, the concentration of GBE was varied to observe the dose-dependence of inhibition and to determine the IC50, the concentration at which 50% inhibition is observed (Section 3.3.3). Next, immunoinhibition studies were employed to ascertain which C Y P isoform in H L M G B E inhibits (Section 3.3.5). Recombinant CYPs were then used to confirm which CYP isoforms are inhibited by G B E (Section 3.3.4). Finally, individual constituents of GBE were tested to determine if they are responsible for the inhibitory effect of G B E (Section 3.3.5). 47 Table 4. Intraday variabilities of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA formation by H L M . Metabolite CV (%) 4-ene-VPA 7 4- OH-VPA 5 5- OH-VPA 6 3-OH-VPA 16 H L M from donor HG30 containing 60 pmol of total CYP were incubated with 1 mM V P A for 20 min. The formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA and 3-OH-VPA was monitored. C V is that of 4 determinations performed on one day. 48 Table 5. Interday variabilities of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA formation by H L M . Metabolite C V (%) 4-ene-VPA 14 4-OH-VPA 11 5-OH-VPA 7 3-OH-VPA 30 H L M from donor HG30 containing 60 pmol of total C Y P were incubated with 1 mM V P A for 20 min. The formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA was monitored. C V is that of 4 determinations performed on 4 separate days. 49 (A)m/z= 141 (A)m/z= 141 lOO. 23150 2<00 24.50 Fissure 12. Representative chromatograms for microsomal incubations at mlz of 141 and 148 HLM were mcubated w,th l mM VPA At(A}m/z=141 the formation of (I) 4-ene-VPA can be seen, and at (B) mlz = 148, the internal standard „ W r t . W A vi ib ;. HIM were incubid with 1 mM VPA and 0.3 mg/mL GBE (Indena, lot 1306AX At (C) mlz = 141, a decrease in L formation of 4-ene-VPA can be seen, and a. (D) mlz = 148, the internal standard 2-ene-[JH,]-VPA . . v1S1ble. jfVbundanca I6OO00 160000 140000| 1200001 lOOOOOi 800001 60000 400001 20000 (A) mlz = 273 II (Ttma-> 31.90 32.00 III 32)10 u^h^iWo iilso iiloo 32)70 32)ao 3i:«o 33:00 S'i'o 33:20 33:30' iWri 33:50 33)60 33:70 Abundance 180000 160000: 140000J 120000: 100000] 80000 60000! 40000J 20000 IV IV (B) mlz = 280 V 1 200000 180000 180000 I4OOOOI 120000 100000 soooo 60000 40000 20000 (C) mlz = 273 I I III II ,<>| n i | . |^7>. yunjndanca 200000 160000 160000 140000 120000 100000 80000 eoooo 40000 20000! 31J90 ttloo"5ll0 32'20' Via 3iS 32.60 3^ 70 tt!»0 32I9O 33100 33)10 33:20 335) 33.40 33.50 33.60 3370 31)90 silod n l io 32.'20 32!30 32l40 32)&0 32)60 32>0 tt)80 32)90 SJIoo'.UllO 33:20 33)30 33)40 33)50 33)60 33.70 I V I V | (D) mlz = 280 V VI i i lw 32l00 32.'l0 32l20 32!30 32l40 32)50 32)60 32)70 32)60 32)60 33)00 33)10 33)20 33)30 33)40 33.50 33.60 33.70 Figure 13. Representative chromatograms for microsomal incubations at mlz of 273 and 280. H L M were incubated with 1 m M VPA. At (A) mlz = 273, the formation of (I) 4-OH-VPA, (II) 5-OH-VPA, and (UI) 3-OH-VPA can be seen, and at (B) mlz = 280, the internal standards (IV) 4-OH-[ 2H 7]-VPA, (V) 5-OH-[ 2H 7]-VPA, and (VI) 3-OH-[ 2 H 7 ]-VPA are visible. H L M were incubated with 1 mM V P A and 0.3 mg/mL GBE (Indena, lot 1306A). At (C) mlz = 273, the formation of 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA can be seen, and at (B) an mlz = 280, the internal standards 4-OH-[ 2 H 7 ]-VPA, 5-OH-[ 2H 7]-VPA, And 3-OH-[ 2 H 7 ]-VPA are visible. The formation of 4-ene-VPA (Figure 12A) and of 4-OH-VPA, 5-OH-VPA, and 3-OH-V P A (Figure 13 A) can be seen in chromatograms. The inhibition of the formation of 4-ene-VPA (Figure 12C) and of 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA (Figure 13C) by G B E can also be seen visually in chromatograms. 3.3.2. Effect of GBEfrom different lots and manufacturers on VPA metabolism by HLM H L M containing 60 pmol of total C Y P were incubated for 20 min with 1 mM V P A and 0.3 mg/mL of G B E (Indena, lot 1306A), GBE from a different lot (Indena, lot 302831), or G B E from a different manufacturer (Pharmaton, lot 63964). Control values for the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA were 19±3, 311±39, 206±28, and 9±2 pmol/min/nmol total CYP, respectively. The three G B E preparations inhibited the formation of all four metabolites. Formation of all four metabolites was significantly different from control. As shown in Figure 14, all three extracts inhibited 4 -OH-VPA 5-OH-VPA, and 3-OH-VPA formation to a similar extent. Inhibition of 4-ene-VPA formation, however, varied among the extracts. For all further experiments, the extract which was the most characterized chemically (Indena, lot 1306A, Appendix 1) was used. 3.3.3. Concentration-dependent effect of GBE on VPA metabolism by HLM H L M containing 60 pmol of total CYP were incubated for 20 min with 1 mM V P A and 0.01, 0.03, 0.1, 0.3, 0.6, or 1 mg/mL of G B E (Indena, lot 1306A). Control values for the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA were 18±5, 256±40, 173±22, and 9±2 pmol/min/nmol total C Y P , respectively. As shown in Figure 15, G B E inhibited the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA in a dose-dependent manner with IC 5 0 values of 0.34±0.04, 0.18±0.03, 0.21±0.02, and 0.37±0.10 mg/mL, respectively. 52 eg U J o ON > > A * O CL 3 O 5i K § 3 CL < S 3 2 o 3, 3. Ef> O o r> 3 a 13 C L G> 3 P wo •-I cr P Q 3 o oo n> SS i—> ^ ^ 00 A W V II * era' 3 o cr C L O « o ,3 *• - 3 S O M 3 } <^  S. 5. ON hi ~> A » a r? 2 3 "t 2 C L 0) C O O QJ _ r+ J " Q I—• <-K O O <*> 3 rt » 3 • - ' 3 B. w a. 3". o O ON a* 3 > 3 i". w s, ™ s w o 3 t*J 8 p ° 3 & 1 3 ~ 35 3 ^ 3 P 3 H » © 3* 9 - ET. « § 2 t+> p 3 § ~ e r 3 o vj 3% »*> W o to CX) bo g CL •-i 3 >-h • V O 3 CL <D 3 P S o P > 3 S ffi & & I > Product Formation (Percentage of Control) to x± en co CD o o o o o o CD 6 TJ > Product Formation (Percentage of Control) N> O) CO o o o o o o o CO i O TJ > Product Formation (Percentage of Control) cb => CD k TJ > Product Formation (Percentage of Control) o en • O TJ > (A) 4-ene-VPA (C) 5-OH-VPA 0.01 0.1 G B E (mg/mL) 0.01 0.1 G B E (mg/mL) (B) 4-OH-VPA 125 0.01 0.1 G B E (mg/mL) (D) 3-OH-VPA 125 0.01 0.1 G B E (mg/mL) Figure 15. Effect of GBE on V P A metabolism by H L M . H L M were incubated with 1 m M V P A and 0.01, 0.03, 0.1, 0.3, 0.6, or 1 mg/mL of G B E (Indena, lot 1306A). Product formation is relative to control (water). The formation of (A) 4-ene-VPA, (B) 4-OH-VPA, (C) 5-OH-VPA, and (D) 3-OH-VPA was monitored. Error bars represent S E M (» = 4). 3.3.4. Effect of GBE on CYP2C9-catalyzed VPA metabolism by ELM H L M containing 60 pmol of total CYP were pre-incubated with 3 pL of MAb2C9 then incubated for 20 min with 1 m M VPA, pre-incubated with 3 pL of MAb2B6 and 3 pL of MAb2A6 then incubated for 20 min with 1 mM VPA, or pre-incubated with 3 pL of MAb2B6 and 3 pL of MAb2A6 then incubated for 20 min with 1 m M V P A and 1 mg/mL of G B E (Indena, lot 1306A). Mean values for the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by microsomes in the absence of antibodies and control IgG were 22±2, 333±42, and 230±26 pmol/min/nmol total CYP, respectively. A l l inhibition by treatment groups were compared to control incubations, in which an equivalent volume of control IgG was pre-incubated with H L M . The formation of 4-ene-VPA, 4 -OH-VPA and 5-OH-VPA by H L M was reduced by 73±1%, 75±0%, and 77±2%, respectively, when microsomes were pre-incubated with MAb2C9. The formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA was reduced slightly after pre-incubation with MAb2B6 and MAb2A6. Inhibition was 19±2%, 20±10%, and 17±5%, respectively. The formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by H L M pre-incubated with MAb2B6 and MAb2A6 was strongly inhibited by GBE. GBE reduced the formation of 4-ene-VPA to levels below LOQ. The formation of 4-OH-VPA and 5-OH-VPA was reduced by 89±1% and 86±1%, respectively. Al l inhibition seen was statistically different from control. 55 c o E c o o o o CD O) O CO 3 c CD 2 o D_ CD CL 100 75 50 25 0 (A) 4-ene-VPA <L0Q MAb2C9 MAb2B6 + MAb2B6 + MAb2A6 MAb2A6 + G B E _ (D) 4-ene-VPA CL >-ca 2 o o o u- E o -5 D C O t CL O E Q. 30 i 20 10 0 0 F Control IgG (pL) c o CD E o LL C o o o CD O CD Z3 "a o CD 2 CD CL 100 75 50 25 0 (B) 4-OH-VPA MAb2C9 MAb2B6 + MAb2B6 + MAb2A6 MAb2A6 + G B E CL >-EH '+Z CD t o o o ^ E o -5 "a c o .E CL O E CL (E) 4-OH-VPA 600 400 i 200 0 i i 0 3 6 Control IgG (pL) (C) 5-OH-VPA o o c £ o b ° LL. <D O CD •a § 2 o CL CD CL 100 75 50 25 0 MAb2C9 MAb2B6 + MAb2B6 + MAb2A6 MAb2A6 + G B E CL >-EH CD * i O O E o -5 D C o fc CL O E a. (F) 5-OH-VPA 300 200 100 0 0 3 6 Control IgG (pL) Figure 16. Effect of GBE on CYP2C9-mediated VPA metabolism by H L M . H L M were pre-incubated with MAb2C9 then incubated with 1 mM VPA, pre-incubated with MAb2B6 and MAb2A6 then incubated with 1 mM V P A , or pre-incubated with MAb2B6 and MAb2A6 then incubated with 1 mM V P A and 1 mg/mL of GBE (Indena, lot 1306A). The formation of (A) 4-ene-VPA, (B) 4-OH-VPA, and (C) 5-OH-VPA is shown. Values are relative to control activities (equivalent volume of control IgG), shown in panels (D), (E), and (F). Error bars represent S E M (n = 3). * Significantly different from control (p < 0.05). 56 3.3.5. Effect of GBE on VPA metabolism by recombinant CYP2C9*!, CYP2B6, and CYP2A6 Recombinant CYP2C9*1, CYP2B6, and CYP2A6 containing 40 pmol of C Y P were incubated for 30 min with 1 m M V P A and 0.3 or 1 mg/mL G B E (Indena, lot 1306A). Control values for the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by cDNA-expressed CYP2C9*1 were 18±4, 252±66, and 143±46 pmol/min/nmol CYP, respectively. No 3-OH-VPA was detected. As shown in Figure 17, G B E at both concentrations significantly inhibited the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by cDNA-expressed CYP2C9*1. Control values for the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by cDNA-expressed CYP2B6 were 12±1, 224±17, and 229±40 pmol/min/nmol C Y P , respectively. No 3-OH-VPA was detected. G B E at both concentrations significantly inhibited the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by cDNA-expressed CYP2B6, as shown in Figure 18. The inhibitory effect of G B E was not as strong as for recombinant CYP2C9* 1. Control values for the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA by cDNA-expressed CYP2A6 were 6±1, 82±2, 31±1, and 16±1 pmol/min/nmol C Y P , respectively. As shown in Figure 19, 1 mg/mL of G B E significantly inhibited the formation of 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA by cDNA-expressed CYP2A6. At 1 mg/mL of GBE, the formation of 4-ene-VPA was below LOQ. The inhibitory effect of G B E on recombinant CYP2A6 was weak compared to the effect on recombinant CYP2C9* 1 and CYP2B6. 57 (A) 4-ene-VPA <LOQ <LOQ Control 0.3 G B E (mg/mL) (B) 4-OH-VPA <LOQ Control 0.3 G B E (mg/mL) (C) 5-OH-VPA 200 Control 0.3 G B E (mg/mL) Figure 17. Effect of GBE on VPA metabolism by recombinant CYP2C9*1. Recombinant CYP2C9*1 microsomes were incubated with 1 mM V P A and 0.3 or 1 mg/mL of G B E (Indena, lot 1306A). The formation of (A) 4-ene-VPA, (B) 4-OH-VPA, and (C) 5-OH-VPA is shown. Error bars represent SD (3 determinations). * Significantly different from control (p < 0.05). 58 (A) 4-ene-VPA <LOQ Control 0.3 1 G B E (mg/mL) (B) 4-OH-VPA Control 0.3 G B E (mg/mL) (C) 5-OH-VPA Control 0.3 G B E (mg/mL) Figure 18. Effect of GBE on VPA metabolism by recombinant CYP2B6. Recombinant CYP2B6 microsomes were incubated with 1 mM V P A and 0.3 or 1 mg/mL of GBE (Indena, lot 1306A). The formation of (A) 4-ene-VPA, (B) 4 -OH-VPA and (C) 5-OH-VPA is shown. Error bars represent SD (3 determinations). * Significantly different from control (p < 0.05). 59 (A) 4-ene-VPA (C) 5-OH-VPA G B E (mg/mL) G B E (mg/mL) (B) 4-OH-VPA (D) 3-OH-VPA G B E (mg/mL) G B E (mg/mL) Figure 19. Effect of GBE on V P A metabolism by recombinant CYP2A6. Recombinant CYP2A6 microsomes were incubated with 1 m M V P A and 0.3 or 1 mg/mL of GBE (Indena, lot 1306A). The formation of 3-OH-VPA is shown. Error bars represent SD (3 determinations). * Significantly different from control (p < 0.05). 3.3.6. Effect of individual constituents of GBE on VPA metabolism by HLM H L M were incubated for 20 min with 1 mM V P A and 500 ug/mL of G B E (Indena, lot 1306A, Appendix 1), 14 pg/mL of bilobalide, 5.5 pg/mL of ginkgolide A, 1.5 pg/mL of ginkgolide B, 7 pg/mL of ginkgolide C, 3 pg/mL of ginkgolide J, 3 pg/mL of isorhamnetin-3-O-rutinoside, 5 pg/mL of kaempferol-3-O-rutinoside, 12 pg/mL of quercetin-3-O-rutinoside, 20.5 pg/mL of isorhamnetin aglycone, 31.5 pg/mL of kaempferol aglycone, or 53 pg/mL of quercetin aglycone. The concentrations of the terpene trilactones and isorhamnetin-3-O-rutinoside were the concentrations present in 500 pg/mL of GBE. The concentrations of kaempferol-3-O-rutinoside and quercetin-3-O-rutinoside were the concentrations of their more abundant unidentified diglycoside (kaempferol diglycoside 1 and quercetin diglycoside 2 in Appendix 1). The concentrations of the flavonol aglycones were the concentrations that would be present in 500 pg/mL of G B E if all the flavonol glycosides were converted to their respective aglycones (sum under isorhamnetin, kaempferol, and quercetin in Appendix 1). Control values (no DMSO) for the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA by H L M were 23±5, 325±31, 213±17, and 12±1 pmol/min/nmol total CYP, respectively. For vehicle control (0.05% DMSO, v/v), formation was 21-27% lower. As shown in Figure 20, bilobalide and ginkgolides A, B, C, and J did not inhibit V P A metabolism. G B E appeared to inhibit the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and perhaps 3-OH-VPA; however, due to the large variability seen on this day, G B E was not significantly different from control by one-way A N O V A . 61 Z9 3 CD c r CD 0 2 3 rt <: 00 1 w 3 ^ o H 3" CD CD $ CD O f- t-o 3 P 3 U II P M t3 m <^ _ 3 3-* 3 <? 9-Cu <T> 2". ^ °* 5 CD © CD CD 3 O 3 o o 3 o td n "i T3 CD 3 CD 2*. > CD D-3* CD o1 3 > o a "1 o jsa CD 5' 3 3 w o 4^ t CD 3 CD 3-CD o o 3 o <T> 3 p o x o 3 3 as O" o O 3 w o X k i> 1 o x P 3 Cu o X 3* 1 CD < O ^ ffi 3 ^ 3 Cu O fa O > P >-» CD w> 3* O 3 W o -t a* CO 3 CD O CD » 3 o c cr R CD Cl. < CD 2*. S" £ § ^ S 3 p o o o 00 o 3 > p 3 Cu Q to ffl Product Formation (pmol/min/nmol total CYP) Control G B E O Bilobalide Ginkgolide A Ginkgolide B Ginkgolide C Ginkgolide J CD 1 o k TJ > Product Formation (pmol/min/nmol total CYP) Control G B E Bilobalide Ginkgolide A Ginkgolide^ B Ginkgolide C Ginkgolide co • O <: TJ > Product Formation (pmol/min/nmol total CYP) Control GBE Bilobalide Ginkgolide A Ginkgolide B Ginkgolide C Ginkgolide J cb ID CD k TJ > Product Formation (pmol/min/nmol total CYP) cn o ai o 0 0 0 0 0 Control GBE Bilobalide Ginkgolide A Ginkgolide B Ginkgolide C Ginkgolide O cn • O X <: TJ > £9 o g o 3 W o o* 3 " - i <T> CO fl> 1 3 re-W as II 4^ o O p H a* bo G?5 I—> W o >-1 3 * p 3 a ? o 12. ' a. O n> o r + o a re-s-ell o5 i f i p C/> TO 3. o p 3 a* a> o o 3 o o 3 r + •-I &. o 3 P O s fJQ 2 a. 3 O CO O O P O 3 O >-+> CO >-t CD O o o 3 2. •V 3 o <-% s-c 3 - Q , > P ' 3 3 a o s > 3 » 3 a* a> o o 3 O 3 r + -\ &. o 3 c 3 » o 5 & 3 TO a O o X w i-n A o o o CO oi CD o5 c 3 5' o 7t P 3 s t 3 ^ °-"3 P a a. < T> a H P o 3 O cn _ 51 CD Product Formation (pmol/min/nmol total CYP) en o en o o o o Control GBE Isorhamnetin -3-0-rutinoside Kaempferol -3-0-rutinoside Quercetin -3-0-rutinoside CO • O k TJ > Product Formation (pmol/min/nmol total CYP) O M CO CO O Control GBE Isorhamnetin | -3-0-rutinoside Kaempferol -3-0-rutinoside Quercetin -3-0-rutinoside co 6 k TJ > Product Formation (pmol/min/nmol total CYP) o cn o - i M M cn o cn Control GBE Isorhamnetin -3-0-rutinoside Kaempferol -3-0-rutinoside Quercetin -3-0-rutinoside <b u CD k TJ > Product Formation (pmol/min/nmol total CYP) cn o cn o o o o o o Control GBE Isorhamnetin -3-0-rutinoside Kaempferol -3-0-rutinoside Quercetin -3-0-rutinoside O • O X k TJ > v9 TO' • O P 3 « c f 5 ' '§•8 3 ft) to CD CD CD — a 3 Cu CO cd *<* w o 4^  O • CO A o o W CD o 3 o o 3 i CD O T3 K g > p p. <& °- 8 * — ' CO 1 <-O » X BT. > 3 CD o O ~) ° s. 3 5? 3 O CD 3 O 3 — CD <<3 3 o 3 P 3 Cu 42 C CD "i o CD 3' & r-f 3* CD O o 3 O CD < > 3 CD S3 O " o_ La" 3 o* w is CO 3* O 3 P 3" P CO Cu o' X 3 r* ffl o p co "i CD T3 >-l CD co CD 3 !-+• on 3 CD "-I CD 5' 8 3 3 H Cu 3," 3- 3* CD 3 _ CD >-i O ffl c? 3 3 II 4^  O 3 3* CD 3 ffl S P w 3-. ^ O O P 1 " ! 3 & Product Formation (pmol/min/nmol total CYP) Product Formation (pmol/min/nmol total CYP) — * I O C O o o o o o o o cn o IO ro cn o cn Control GBE Isorhamnetin Kaempferol DO 4^ i O X k T J > Control GBE O D Isorhamnetin Kaempferol 4^  cb CD <: TJ > Quercetin Quercetin Product Formation (pmol/min/nmol total CYP) o cn o cn Product Formation (pmol/min/nmol total CYP) cn o O O O Control GBE Isorhamnetin Kaempferol Quercetin D co 6 <: TJ > Control GBE Isorhamnetin Kaempferol O cn i O I k TJ > Quercetin As shown in Figure 21, GBE significantly reduced the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA. Formation of all four metabolites in incubations with the rutinosides of isorhamnetin, kaempferol, and quercetin was not significantly different from vehicle control. GBE and the aglycones of kaempferol and quercetin significantly inhibited the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA, as shown in Figure 22. The aglycone of isorhamnetin inhibited 4-OH-VPA, and 5-OH-VPA formation. Quercetin exhibited the strongest effect, followed by kaempferol and isorhamnetin. No significant reduction of 3-OH-VPA formation was observed in incubations with any of the flavonol aglycones or GBE. 65 4. DISCUSSION The V P A assay has been shown to be a sensitive assay for quantitating the V P A metabolites 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA (Abbott et al., 1986; Kassahun et al., 1989; Kassahun et al., 1990; Anari et al., 2000; Ho et al., 2003). In this study, the assay was used to measure the inhibition of their formation by GBE. This assay proved especially suitable for the measurement of the inhibition of 4-OH-VPA and 5-OH-VPA formation. The amounts of 4-OH-VPA and 5-OH-VPA formation by H L M in the absence of G B E were quite high, averaging at about 64 and 42 ng (333 and 219 pmol/min/nmol total CYP), respectively. With the LOQ for both 4-OH-VPA and 5-OH-VPA being 2 ng, it was possible to quantify inhibition of up to 97 and 95 % on average, respectively. The assay presented more challenges in the measurement of the inhibition of 4-ene-VPA and 3-OH-VPA formation. The amounts of 4-ene-VPA and 3-OH-VPA formation by H L M in the absence of GBE were only slightly greater than their LOQ of 0.5 ng, with production averaging about 3.7 and 2.2 ng (22 and 11 pmol/min/nmol total CYP), respectively. Thus, for 4-ene-VPA and 3-OH-VPA, it was possible to measure inhibition of up to 86 and 77 % on average, respectively. 66 In this study, the ratio of formation rates of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by H L M was 1:15:10. This is comparable to the ratio of 1:18:9 observed in H L M by Sadeque et al. (1997). The ratio of formation rates of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by recombinant CYP2C9*1 was 1:14:8. This is roughly comparable to the 1:10:11 ratio obtained by Ho et al. (2003). The relatively lower proportion of 4-OH-VPA obtained by Ho et al. may be due to slight differences in methodology. Ho et al. froze their incubation mixtures at -20°C until analysis. However, 4-OH-VPA has been shown to readily form a y-lactone (Schafer and Luhrs, 1978), and the lower proportion seen may have been due to a loss of 4-OH-VPA between the time of incubation and derivatization. In this study, incubation mixtures immediately underwent extraction and derivatization, with 4-OH-VPA undergoing ferr-butyldimethylsilylation to form a more stable molecule. The ratio of formation rates of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by recombinant CYP2B6 was 1:18:19. For recombinant CYP2A6, the ratio of formation rates of 4-ene-VPA, 4-OH-VPA and 5-OH-VPA was 1:15:6. Since natural products are complex mixtures of many different constituents and may differ from lot to lot and between manufacturers, to ensure that the inhibition seen with the G B E used in this project is representative of commercially available G B E products, it was compared to G B E from another lot from the same company and G B E from another manufacturer. The three preparations yielded similar inhibition of 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA formation. This is not surprising since they had been standardized to contain 24% flavonols and 6% terpene trilactones. There was, however, statistically different difference for 4-ene-VPA inhibition by the three G B E preparations, but this may be due to chance. GBE inhibited the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA in a dose-dependent manner. In order to determine which isoform G B E inhibits to cause a reduction 67 in the formation of these metabolites, immunoinhibition studies were carried out. Key to determining which specific CYP isoforms are inhibited by G B E is an understanding of which isoforms are involved in the formation of these four V P A metabolites. In the present study, MAb2C9 decreased the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by H L M from all three donors tested by approximately 75%. Similar inhibition was seen in another study, where MAb2C9 inhibited the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by H L M by at least 75%, with inhibition of at least 80% for recombinant CYP2C9*1 (Kiang et al., 2005). MAb2C9 has been shown to be highly specific against recombinant human CYP2C9*1, CYP2C9*2, and CYP2C9*3, while showing no cross-reactivity towards recombinant human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, as measured by ELISA. Tolbutamide methylhydroxylation by recombinant CYP2C9*1 was inhibited by greater than 90% with MAb2C9. MAb2C9 inhibited diclofenac 4'-hydroxylation by 80-90% in recombinant CYP2C9*1, CYP2C9*2, and CYP2C9*3. In H L M , MAb2C9 inhibited diclofenac activity by 85-90% (Krausz et al., 2001). These results suggest that MAb2C9 inhibits CYP2C9*1 under these experimental conditions, and that CYP2C9 is the major isoform in H L M responsible for the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA. The residual activity is likely due to CYP2B6 and CYP2A6. The present study showed that in H L M from all three donors tested, pre-incubation with MAb2B6 and MAb2A6 decreased the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by approximately 20%. Another study showed that pre-incubation of H L M with MAb2B6 or MAb2A6 alone had little or no effect on the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA. Pre-incubation with MAb2B6 alone resulted in at least 90% inhibition of the formation of 68 these three metabolites by CYP2B6. Pre-incubation with MAb2A6 alone resulted in at least 90% inhibition of the formation of these three metabolites by CYP2A6 (Kiang et a l , 2005). MAb2B6 has been shown to exhibit strong binding to recombinant human CYP2B6, but without significant cross-reactivity to recombinant human CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, as tested by ELISA. MAb2B6 inhibited the metabolism of phenanthrene, diazepam, 7-ethoxycoumarin, and testosterone by cDNA-expressed CYP2B6 by 90-91% (Yang et al., 2003). ELISA and immunoblot analyses showed strong binding of MAb2A6 to recombinant human CYP2A6, while showing no significant cross-reactivity to CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5. MAb2A6 inhibited CYP2A6-catalyzed metabolism of coumarin by at least 94% in both cDNA-expressed CYP2A6 and H L M (Sai et al., 1999). Therefore, MAb2B6 and MAb2A6 inhibit CYP2B6 and CYP2A6 under these experimental conditions. With CYP2B6 and CYP2A6 inhibited, the approximately 80% of remaining activity is likely due to CYP2C9. In H L M pre-incubated with MAb2B6 and MAb2A6, G B E decreased the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA to approximately 10% of control IgG activity. With the CYP2B6 and CYP2A6 being blocked by antibodies, the inhibition seen was due to the inhibition of CYP2C9 by GBE. In other words, the inhibition by G B E of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by H L M is due mostly to the inhibition of CYP2C9 by GBE. In another study performed using H L M , an extract of Ginkgo biloba Special herbal tea inhibited CYP2C9 (Foster et al., 2003). However, this blended tea contained other herbal ingredients such as lemongrass, licorice root, spearmint leaf, peppermint leaf, cinnamon bark, ginger root, and sage leaf in addition to standardized GBE. Since some of these other components are known to inhibit 69 C Y P (Paolini et al., 1998; Unger and Frank, 2004), the inhibition seen cannot be attributed solely to Ginkgo biloba. To confirm the results seen in immunoinhibition studies with FILM, the effect of G B E on V P A metabolism by recombinant C Y P was examined. G B E inhibited the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by cDNA-expressed CYP2C9*1 in a dose-dependent manner. G B E strongly inhibited the formation of these three metabolites. Gaudineau et al. (2004) also observed inhibition of cDNA-expressed CYP2C9 by GBE, with a K{ of 14±4 pg/mL. The formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by cDNA-expressed CYP2B6 was also inhibited by G B E in a dose-dependent manner, although the inhibition seen was not as strong as for CYP2C9*1. G B E inhibited the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA and 3-OH-VPA by cDNA-expressed CYP2A6 in a dose-dependent manner. Inhibition of CYP2A6 was relatively weak. Experiments with recombinant C Y P confirmed the results observed with the immunoinhibition studies. Namely, that the major C Y P isoforms involved in the oxidative metabolism of V P A to 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA are CYP2C9, CYP2B6, and CYP2A6; and that G B E inhibits all three isoforms. CYP2A6 is the major isoform responsible for 3-OH-VPA formation. These results are in agreement with those of Kiang et al. (2005), who used a panel of human cDNA-expressed microsomes to investigate the metabolism of V P A to 4-ene-VPA 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA. The desaturation of V P A to 4-ene-VPA was catalyzed by CYP2C9*1, CYP2B6, and CYP2A6, with CYP2C9*1 exhibiting the highest activity. Formation of 4-OH-VPA by (co-l)-hydroxylation was catalyzed by CYP2C9*1, CYP2B6, and CYP2A6 with CYP2C9*1 being the most active. CYP2C9*1, CYP2B6, and CYP2A6 catalyzed oo-hydroxylation of V P A to 5-OH-VPA. CYP2A6 was the most active in the 70 (ffi-2)-hydroxylation of V P A to 3-OH-VPA, with CYP1A1, CYP2B6 CYP4F3B, and CYP4F2 exhibiting lower activity. CYP1A2, C Y P IB 1, CYP2B8, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4A11, and CYP4F3A did not display any significant activity in the formation of these four metabolites. G B E is a complex mixture of many constituents. In an effort to discover which constituent of G B E is responsible for its inhibitory effect seen on V P A metabolism, the effect of individually quantified and commercially available constituents on V P A metabolism by H L M was investigated. None of the individual terpene trilactones at the concentrations found in the extract, namely 14 pg/mL (43 uM) of bilobalide, 5.5 pg/mL (13 pM) of ginkgolide A, 1.5 pg/mL (4 pM) of ginkgolide B , 7 pg/mL (16 pM) of ginkgolide C, and 3 pg/mL (7 pM) of ginkgolide J, inhibited V P A metabolism. This is in agreement with another study which showed that 153 p M bilobalide, 200 p M ginkgolide A, 200 u M ginkgolide B, and 200 uM ginkgolide C did not inhibit cDNA-expressed CYP2C9 (Zou et a l , 2002). Similarly, bilobalide and ginkgolides A, B, C, and J had either weak or negligible inhibitory capacity (defined as less than 50% inhibition at a concentration of 100 pg/mL) in recombinant CYP2C9 (von Moltke et a l , 2004). In contrast, He and Edeki (2004) reported that ginkgolides A and B inhibited CYP2C9 in H L M with I C 5 0 values of 113 and 168 pM, respectively. However, the concentrations at which they saw inhibition were approximately 10-fold greater than the concentrations used in this study, and thus would not explain the inhibition by G B E of CYP2C9-mediated V P A metabolism. Gaudineau et al. (2004) observed that the terpenoid fraction of GBE strongly inhibited recombinant CYP2C9, with a K{ of 15±6 pg/mL. However, such this crude terpenoid fraction likely contained compounds other than the terpene trilactones tested in this project. Thus, individually, the 71 terpene trilactones were not responsible for the inhibitory effect of G B E on CYP2C9. No studies pertaining to the effect of terpene trilactones on CYP2B6 or CYP2A6 have been published to date. The flavonol glycosides tested did not inhibit V P A metabolism. Isorhamnetin-3-O-rutinoside was identified in the extract. The identities of the two diglycosides of kaempferol and the two diglycosides quercetin were not determined. It was assumed that one of kaempferol diglycosides was kaempferol-3-O-rutinoside and one of quercetin diglycosides was quercetin-3-O-rutinoside. This was most likely a safe assumption, since it is known that the major diglycosides of kaempferol and quercetin contain biloside or rutinose as sugar moieties (Bedir et al., 2002). Therefore, individually, these three flavonol diglycosides do not explain the inhibitory effect of GBE. This is in agreement with a study by von Moltke et al. (2004), in which four kaempferol glycosides and five quercetin glycosides extracted from Ginkgo biloba leaves showed either weak or negligible (less than 50%) inhibition of CYP2C9 at a much higher concentration of 100 pg/mL. The concentrations of kaempferol-3-O-rutinoside and quercetin-3-O-rutinoside used for this project were 5 pg/mL and 12 pg/mL, respectively. However, there were other flavonol glycosides present in G B E that were not identified and thus not tested. A flavonoid fraction isolated from GBE by Gaudineau et al. (2004) inhibited CYP2C9. However, it is possible that chemical constituents other than that flavonol glycosides eluted in this fraction. Thus, it cannot be concluded that flavonol glycosides are responsible for the inhibition of V P A metabolism by GBE. Although the terpene trilactones and flavonol glycosides tested did not inhibit V P A metabolism, there are many other constituents in GBE which could explain the inhibition seen with the extract. Other constituents known to be inhibitory towards CYP2C9 in H L M include 72 amentoflavone, sesamin, (Z,Z)-4,4'-(l,4-pentadiene-l,5-diyl)diphenol and 3-nonadec-8-enyl-benzene-l,2-diol. In particular, amentoflavone exhibited strong inhibition of CYP2C9, with an IC50 of 0.035 p M (von Moltke et al., 2004). Another candidate is tamarixetin, which is metabolized to quercetin by H L M and cDNA-expressed CYP2C9 (Breinholt et al., 2002). However, whether these constituents were present in the extract used in this study is not known. Ginkgolic acids I and II significantly inhibited cDNA-expressed CYP2C9, with IC50 values of 2.41 and 1.94 pM, respectively (Zou et al., 2002). However, the ginkgolic acids are usually removed from standardized extracts due to their toxicity (van Beek, 2002). The identities of the constituents responsible for the inhibitory effect of GBE in vitro were not ascertained in this project. Although the flavonol glycosides did not inhibit V P A metabolism in vitro, flavonols may be of importance in vivo since flavonol glycosides are deglycosylated to their respective aglycones in human small intestine mucosa (Rasmussen and Breinholt, 2003). Lactase-phlorizin hydrolase, which is localized to the apical membrane of small intestinal epithelial cells, and cytosolic P-glucosidase have exhibited activity towards flavonoid glycosides (Nemeth et al., 2003; Jiang et al., 2005). In humans, both flavonol glycosides and aglycones are absorbed (Hollman and Katan, 1997). The aglycones of kaempferol and quercetin inhibited the in vitro formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA at the concentrations that would be present in 500 pg/mL of GBE if all the flavonol glycosides were converted to their respective aglycones. The over 50% inhibition of formation of the three CYP2C9-catalyzed V P A metabolites by 175 p M quercetin is consistent with other studies. Zou et al. Values of IC50 for inhibition of CYP2G9 in H L M range from 25.8 p M (von Moltke et al., 2004) to 35 p M (He and Edeki, 2004). Zou et al. (2002) reported an IC50 for recombinant CYP2C9 of 3.14 pM, but warned that this may be an underestimate due to the 73 fluorescence of quercetin which may have interfered with their assay. In the present study, 65 p M isorhamnetin inhibited the formation of 4-OH-VPA and 5-OH-VPA. However, Zou et al. (2002) reported that isorhamnetin at 100 p M did not inhibit cDNA-expressed CYP2C9. Overall, these results point to inhibition of CYP2C9, the major isoform responsible for the formation of these metabolites, and perhaps CYP2B6 by the aglycones of isorhamnetin, kaempferol and quercetin: Since the formation of 3-OH-VPA was not inhibited by any of the flavonol aglycones, they are unlikely to inhibit CYP2A6 in vivo. One possible reason that the aglycones inhibited V P A metabolism whereas the rutinosides did not is that rutinoside is a large sugar moiety. In rat hepatic microsomes, the aglycones of isorhamnetin, kaempferol, and quercetin inhibited EROD activity in rat hepatic microsomes, with inhibition decreasing as the size of the sugar moiety increased. Monoglycosides (isorhamnetin-3-O-glucoside, kaempferol-3-O-glucoside, and quercetin-3-O-glucoside) exhibited less inhibition than aglycones, and diglycosides (isorhamnetin-3-O-rutinoside, kaempferol-3-O-rutinoside, and quercetin-3-O-rutinoside) exhibited less inhibition than monoglycosides (Kuo et al., 2004). Thus, it is possible that the presence of bulky sugar moieties may decrease the inhibition by flavonols of human CYP2C9, CYP2B6, and CYP2A6 as they did i n r a t C Y P l A . While it is difficult to predict what will happen in vivo based on the results from in vitro studies, the inhibitory effect of GBE on CYP2C9, CYP2B6, and CYP2A6 may have several consequences. The inhibition of CYP2C9 may lead to higher plasma levels of drugs metabolized by CYP2C9 such as tolbutamide (Leemann et al., 1993), as well as drugs with a narrow therapeutic window, such as phenytoin (Veronese et al., 1991) and warfarin (Rettie et al., 1992). G B E may be involved in a pharmacokinetic interaction with warfarin through inhibition of 74 CYP2C9. However, it would be difficult to discern whether an interaction between G B E and warfarin is pharmacokinetic or pharmacodynamic, since the ginkgolides possess anti-platelet activity (Chung et al., 1987). Inhibition of CYP2B6 by G B E could lead to prodrugs such as cyclophosphamide not being activated (Chang et al., 1993). Inhibition of CYP2A6 by G B E could have a protective effect, since many nitrosamines in tobacco smoke are activated by CYP2A6 (Yamazaki et al., 1992). 75 5. LIMITATIONS AND FUTURE STUDIES One advantage of the V P A assay is that it allows the study of multiple C Y P isoforms by using only one substrate, VPA. However, since each V P A metabolite is formed by more than one C Y P isoform, it is difficult to determine which isoform is being affected by a particular inhibitor. Also, since in this study V P A was used as the substrate, with the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA being monitored, the effect of G B E on only those isoforms involved in their production (CYP2C9, CYP2B6, and CYP2A6) could be ascertained. Other studies have shown the effect of G B E on other isoforms of CYP. For example, in addition to CYP2C9, G B E inhibits cDNA-expressed human CYP1A2, CYP2E1, and CYP3A4 (Budzinski et a l , 2000; Gaudineau et a l , 2004). In rat hepatic microsomes, G B E inhibits CYP1A (Kuo et a l , 2004). In future studies, to gain a more complete profile of the effect of G B E on different CYP isoforms, specific probe substrates could be used to measure the activity of each isoform in H L M as well as in cDNA-expressed CYP. This study demonstrated that G B E inhibits CYP2C9, CYP2B6, and CYP2A6 in H L M and cDNA-expressed microsomes. However, the results of experiments performed in vitro cannot always be extrapolated to the in vivo situation. It may be worthwhile to perform a study 76 comparing in vitro and in vivo inhibition of CYP2C in an animal model. For example, Ohnishi et al. (2003) found that G B E inhibited rat CYP3A in vitro in small intestine and liver microsomes, ex vivo in small intestine and liver microsomes after a single oral administration of GBE, and in vivo after a single oral administration of G B E using diltiazem as a probe for CYP3A. A similar design could be utilized, except using substrate probes for CYP2C, CYP2B, and CYP2A. Likewise, in humans, clearance of CYP2C9, CYP2B6, and CYP2A6 substrates after a single oral dose of G B E versus placebo could be investigated. Another possible experiment would be to investigate induction by GBE. When G B E and tolbutamide were co-administered in rats, the hypoglycemic activity of tolbutamide was potentiated, pointing to inhibition of CYP2C by GBE. However, the hypoglycemic action of tolbutamide in rats pre-treated with G B E for 5 days was significantly attenuated compared to rats receiving no GBE, suggesting induction of CYP2C. In rat liver microsomes, GBE competitively inhibited (5)-warfarin 7-hydroxylase (Sugiyama et al., 2004a), although it is not known which enzyme in rats is responsible for the metabolism of (^-warfarin. Likewise, liver microsomes from rats treated with G B E for 5 days showed an increase in (^-warfarin 7-hydroxylase and PROD, pointing to the possible induction of CYP2C and CYP2B (Umegaki et al., 2002; Yang et al., 2003). After discontinuation of GBE treatment, activities recovered to normal levels (Sugiyama et al., 2004b). Treatment of rats with G B E for 4 weeks increased the levels of CYP2B mRNA (Shinozuka et al., 2002). Another possible experiment would be to look at the effect of G B E on induction of CYP2C9, CYP2B6, and CYP2A6 in humans by measuring the clearance of CYP2C9, CYP2B6, and CYP2A6 substrates after chronic administration of GBE versus placebo. In this study, individual terpene trilactones and flavonol glycosides did not inhibit the formation of V P A metabolites. It is possible that other chemicals not tested in this study were 77 responsible for the inhibitory effect of GBE. Further studies could focus on identifying and quantifying such compounds, and investigating their effect on VPA metabolism using microsomes. Another possibility is that while individually, the compounds tested did not inhibit CYP2C9, in combination they may exhibit synergy, as often occurs with phytomedicines (Williamson, 2001). If so, then in future experiments, combinations of different constituents could be tested to see if they have inhibitory potential. 78 6. SUMMARY AND CONCLUSIONS The VPA assay is a versatile assay, allowing the simultaneous measurement of CYP2C9, CYP2B6, and CYP2A6 activity. The desaturation of V P A to 4-ene-VPA is catalyzed by CYP2C9, CYP2B6, and CYP2A6. The formation of 4-OH-VPA by (co-1)-hydroxylation is mainly catalyzed by CYP2C9, with CYP2B6 and CYP2A6 playing minor roles. The cn-hydroxylation of V P A to 5-OH-VPA is catalyzed mainly by CYP2C9, with CYP2B6 and CYP2A6 playing minor roles. CYP2A6 is the major isoform responsible for the (co-2)-hydroxylation of V P A to 3-OH-VPA. GBE inhibits VPA metabolism in vitro. G B E decreased the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA in H L M . G B E may interact with V P A in vivo. GBE inhibits CYP2C9 in vitro. G B E decreased the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA in H L M by cDNA-expressed CYP2C9*1. G B E may interact with drugs metabolized by CYP2C9. The interaction seen with warfarin in vivo is more difficult to explain as the ginkgolides have been shown to possess anti-platelet activity (Chung et al., 1987). Thus, it could be a pharmacodynamic interaction between the two 79 blood thinning agents, a pharmacokinetic interaction in which G B E inhibits CYP2C9-mediated metabolism of (5)-warfarin, or a combination of both. GBE inhibits CYP2B6 in vitro. GBE decreased the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by H L M and cDNA-expressed CYP2B6. GBE may be contraindicated for patients on drugs metabolized by CYP2B6. GBE inhibits CYP2A6 in vitro. GBE decreased the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA by H L M and cDNA-expressed CYP2A6. G B E may be contraindicated for patients on drugs metabolized by CYP2A6. The inhibitory effect of GBE seen in H L M is due mostly to inhibition of CYP2C9. CYP2C9 is responsible for approximately 75% of 4-ene-VPA, 4-OH-VPA, and 5-OH-V P A formation in H L M . In H L M pre-incubated with MAb2B6 and MAb2A6, G B E dramatically reduced the formation of these three largely CYP2C9-mediated metabolites. The terpene trilactones at the levels present in the extract are not responsible for inhibition of VPA metabolism by GBE in vitro. Bilobalide and ginkgolides A, B, C, and J did not inhibit V P A metabolism at the concentrations found in GBE. The flavonol rutinosides at the levels present in the extract are not responsible for inhibition of VPA metabolism by GBE in vitro. 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Amount of Terpene Trilactones and Flavonols in GBE (Indena, lot 1306A) Constituent Amount (% wlw) Constituent Amount (% w/w) Kaempferol Quercetin Aglycone not detected Aglycone not detected Diglycoside 1 2.4 Diglycoside 1 0.9 Diglycoside 2 1.6 Diglycoside 2 1.0 Other glycosides 6.6 Other Glycosides 4.4 Sum 10.6 Sum 6.3 Isorhamnetin Terpene trilactones Aglycone not detected Bilobalide 2.8 3-O-Rutinoside 0.6 Ginkgolide A 1.1 Other Glycosides 3.5 Ginkgolide B 0.3 Sum 4.1 Ginkgolide C 1.4 Ginkgolide J 0.6 GBE (Indena, lot 1306A) was standardized to contain 6% terpene trilactones and 24% flavonol glycosides. The terpene trilactones were quantified using LC/MS by ChomaDex (Santa Ana, CA). The flavonols were quantified using GC by Indena (Milan, Italy). 92 00 Donor Lot Protein Content (mg/mL) Total C Y P Content (pmol total C Y P / mg-protein) a Rate of Product Formation (pmol/min/mg protein) Rate of Product Formation (pmol/min/pmol total CYP) C Y P 2 C 9 b CYP2B6 c CYP2A6 d C Y P 2 C 9 CYP2B6 CYP2A6 HH18 1 20 370 3900 32 1100 10500 86 3000 HG30 2 20 535 6190 48 940 11600 90 1800 HH47 1 20 260 2900 6.5 510 11200 25 2000 HH91 1 20 340 3400 28 580 10000 82 1700 HG95 1 20 230 2100 12 200 9100 52 570 Total C Y P content and rate of product formation (pmol/min/mg protein) were drawn from information sheets provided by Gentest (Woburn, M A ) . "Total C Y P content was measured by the method of Omura and Sato. 6 CYP2C9 activity was measured by the diclofenac 4'-hydoxylase assay. C CYP2B6 activity, was measured by the (5)-mephenytoin /V-demethylase assay. rfCYP2A6 activity was measured by the coumarin 7-hydroxylase assay. A l l assays were conducted using 0.8 mg/mL of protein incubated with an N A D P H generating system (1.3 m M N A D P + , 3.3 m M glucose 6-phosphate, and 0.4 U/mL of glucose 6-phosphate dehydrogenase), and 3.3 m M MgCb. The incubation time was 20 min for CYP2B6 and CYP2A6, and 10 min for CYP2C9. For CYP2B6, 0.05 M potassium phosphate buffer (pH 7.4) was used, and for CYP2C9 and CYP2A6, 0.1 M Tris (pH 7.4) was used. The rate of product formation (pmol/min/pmol total CYP) was calculated by dividing the rate of product formation (pmol/min/mg protein) by the total CYP content. > T3 T3 n s a w s > n Vi Donor HH18 HG30 HH47 HH91 HG95 00 C Y P 2 C 9 Genotype CYP2C19 Genotype Gender Age *1/*1 *1/*1 Female 78 *1/*1 *1/*1 Female 28 *1/*1 *1/*1 Female 53 Cause of Death C V A * Cardio-pulmonary arrest CVA No tobacco use Social History Tobacco use No tobacco use Alcohol use Medical History Medication Given During Hospitalization Hypertension NIDDM h Asthma Arthritis Insulin Zantac Rocephin Zantac Decadron Dopamine Atropine Lidocaine Not available Hypertension Diabetes Dopamine Fortaz Insulin *1/*2 Female 55 Race African American Caucasian African American Caucasian Cerebro-vascualar Healthy *1/*1 *1/*1 Female 47 Hispanic Closed head trauma Not available Tobacco use Not available Not available Ancef Chloramphenicol Synthroid n s a-w w W O o s o o S n" Donor history was provided by Gentest (Woburn, M A ) . a C V A = cerebrovascular accident. ' 'NTDDM = non-insulin dependent diabetes mellitus. Protein C Y P R a t g Q f P r o d u c t Formation Product Name Lot Content Contort (p m 0 | /min/pmol CYP) (mg/mL) (pmol/mL) ^ Human CYP2C9*1 (Arg i 4 4) + P450 Reductase 20 3.6 2000 21 a Supersomes™ Human CYP2B6 +.P450 Reductase + Cytochrome b 5 13 11 2000 Supersomes™ Human C Y P 2 A 6 + P450 Reductase + Cytochrome b 5 5 13.8 2000 21 C Supersomes™ Insect Cell Control 3 1 5 Q N / A d N / A Supersomes™ A l l information was drawn from information sheets provided by Gentest (Woburn, M A ) . Insect cells (BTI-TN-5B1-4) infected With wild type baculovirus (Autographa californica) were used to prepare all microsomes. a CYP2C9 activity was measured by the diclofenac 4'-hydoxylase assay. X Y P 2 B 6 activity was measured by the 7-ethoxy-4-trifluoromethylcoumarin assay. C CYP2A6 activity was measured by the coumarin 7-hydroxylase assay. ^Not applicable. Al l assays were conducted using 0.8 mg/mL of protein incubated with an N A D P H generating system (1.3 m M N A D P + , 3.3 m M glucose 6-phosphate, and 0.4 U/mL of glucose 6-phosphate dehydrogenase), and 3.3 m M MgCb. For CYP2B6, 0.05 M potassium phosphate buffer (pH 7.4) was used, and for CYP2C9 and CYP2A6, 0.1 M Tris (pH 7.4) was used. The incubation time was 15 min for CYP2B6, and 10 min for CYP2C9 and CYP2A6. For CYP2C9, 20 pmol of C Y P was used. For CYP2B6 and CYP2A6, 10 pmol was used. 8.5. Appendix 5 . Information on Monoclonal Antibodies Antibody Clone Isotype MAb2C9 763-15-5 lgG1 MAb2B6 49-10-20 lgG2b MAb2A6 151-45-4 lgG1 Control (Egg Lysozyme) Hy-Hel-9 I g d Information on antibodies was provided by the National Cancer Institute at the National Institutes of Health (Bethesda, MD). 96 

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