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The Effect of acetaminophen on isoniazid metabolism Youssefi, Mohammed 1992

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THE EFFECT OF ACETAMINOPHEN ON ISONIAZID METABOLISMbyMOHAMMED YOUSSEFIB.Sc., The University of British Columbia, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESPHARMACOLOGY & THERAPEUTICSWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIA© M. Youssefi, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of Pharmacology & TherapeuticsThe University of British ColumbiaVancouver, CanadaDate  22 December, 1992DE-6 (2/88)iiABSTRACTAcetaminophen (APAP, paracetamol, N-acetyl-p-aminophenol), an analgesic andantipyretic drug, causes liver necrosis in overdose. Isoniazid (INH, isonicotinylhydrazide), an antituberculous drug, also causes liver damage in some patients attherapeutic doses. The two medications are likely to be taken concurrently.Previously we reported that, depending on the condition, INH inhibits or inducesthe toxic pathway of APAP metabolism. In this study the influence of APAP onINH metabolism was investigated in ten healthy volunteers. INH, 300 mg, wasingested daily for 7 days. APAP, 500 mg, was ingested on the day before startingINH (day 0) and on day 7 of INH ingestion. INH and its major metabolites in urine(days 6 and 7) were analyzed by reversed-phase high-performance liquidchromatography. Two metabolites, isonicotinic acid (INA) and isonicotinylglycine(ING), were analyzed directly by using an isocratic mobile phase system;propionylisoniazid was used as an internal standard. INH and its hydrazinemetabolites, hydrazine (Hz) and acetylhydrazine (AcHz), were derivatized with m-anisaldehyde and subsequently analyzed by using a gradient mobile phasesystem; 9-fluorenone was a standard. A different procedure was used to analyzeacetylisoniazid (AcINH) and diacetylhydrazine (DiAcHz). The INH, AcHz, and Hzpresent in the samples were converted to hydrazones by reaction with p-chlorobenzaldehyde. The hydrazones were then extracted with methylenechloride. The AcINH and DiAcHz remaining in the aqueous layer were thenconverted to INH and AcHz, respectively, by partial acid hydrolysis. The partialhydrolysis products were derivatized with m-anisaldehyde and then analyzed asabove. The 2-tailed paired-sample t test suggested that concomitant APAPingestion had no effect on the metabolism of INH, but this conclusion is far fromcertain. A number of explanations are given.iiiTABLE OF CONTENTSCHAPTER^ PAGEAbstract iiTable of contents^ iiiList of Tables viiiList of Figures^ ixList of Abbreviations xAcknowledgements^ xiii1 INTRODUCTION 11.1 GENERAL PRINCIPLES OF ISONIAZID 11.1.1 Chemistry of Isoniazid 11.1.2 Therapeutic Uses and Mechanisms of Action Uses Against Mycobacterium Tuberculosis Antimicrobial Spectrum and Mechanisms of Action Treatment Regimens Clinical Uses 41.1.3 Major Pathways of INH Metabolism Acetylation of IN H Hydrolysis Reactions and Glycine Conjugation Acetylation of Hz and AcHz Condensation With a-Ketoacids Other Pathways of INH Metabolism 91.1.4 Microsomal Metabolism and Hepatotoxicity Isoniazid 10ivTABLE OF CONTENTS (CONT'D)CHAPTER PAGE1.1.4.2 Acetylhydrazine Hydrazine 121.1.5 Factors That Alter INH-induced Liver Toxicity Effects of Age Dose of I N H Co-administration of Other Drugs Cytochrome P450 Isozyme Composition Species Differences Acetylator Status Epidemiological Studies Metabolic Studies 171.1.6 Pharmacokinetics Absorption and Bioavailability Distribution Renal Clearance 201.1.7 Adverse Effects Hepatotoxicity Neurotoxicity Mechanism of CNS Toxicity Treatment of INH Overdose 231.1.8 Future Developments 23VTABLE OF CONTENTS (CONT'D)CHAPTER PAGE1.2 GENERAL PRINCIPLES OF ACETAMINOPHEN 241.2.1 Chemistry of Acetaminophen 241.2.2 Therapeutic Uses 251.2.3 Mechanisms of Action 251.2.4 Biotransformation and Hepatotoxicity Conjugation Reactions Microsomal Metabolism Nontoxic Route Toxic Route 301.2.4.3 Mechanism of NAPO! Formation 311.2.4.4 Mechanisms of NAPO! Hepatotoxicity 311.2.5 Factors that Alter APAP Hepatotoxicity in Man 321.2.5.1 Effects of Age 321.2.5.2 Effects of Pregnancy 331.2.5.3 Interspecies Differences due to Enzyme Variations 331.2.5.4 Variability of P450 Isozyme Composition in Humans 331.2.5.5 Effects of Other Drugs 341.2.5.6 Effect of Diet 351.2.6 Pharmacokinetics 351.2.5.1 Absorption 351.2.5.2 Distribution 351.2.5.3 Excretion 361.2.7 Adverse Effects 361.2.8 Future Developments 37viTABLE OF CONTENTS (CONT'D)CHAPTER^ PAGE1.3 DRUG INTERACTIONS AND OBJECTIVE OF STUDY^381.3.1^The Effect of INH on Drug Metabolism^391.3.1.1^P450 Inhibition and Induction by INH 391.3.1.2 The Effect of INH on APAP Metabolism^411.3.2^The Effect of Other Drugs on INH Metabolism^411.3.3^The Effect of APAP on Drug Metabolism 411.3.4^Objective: The Effect of APAP on INH Metabolism^422 METHODS 432.1 MATERIALS^ 432.1.1^Commercially Obtained Supplies^ 432.1.2^Synthesis of Reference Compounds 432.2 PROTOCOL^ 452.2.1^Subjects 452.2.2^Administration Regimen^ 452.3 HPLC ANALYSIS^ 452.3.1^Column 462.3.2^Assays of INA and ING^ 462.3.2.1^Preparation of Assay 462.3.2.2 Apparatus and Chromatographic Conditions^462.3.3^Assays of Acetylhydrazine, INH, and Hydrazine^472.3.3.1^Preparation of Assay^ 472.3.3.2 Apparatus and Chromatographic Conditions^492.3.4^Assays of Acetylisoniazid and Diacetylhydrazine^502.3.4.1^Preparation of Assay^ 50VIITABLE OF CONTENTS (CONT'D)CHAPTER^ PAGE2.3.4.2^Apparatus and Chromatographic Conditions 522.4 CALCULATIONS 522.5 STATISTICAL ANALYSIS 523 RESULTS 533.1 RESULTS OF INA AND ING 533.2 RESULTS OF INH, AcHz, AND HZ 613.3 RESULTS OF ACETYLISONIAZID AND DIACETYLHYDRAZINE 673.4 SUMMARY OF RESULTS AND STATISTICAL ANALYSIS 703.4.1^Results of Paired Comparison Statistics 703.4.2^Graphical Analysis and Other Methods 773.4.2.1^Box plots 773.4.2.2^Scatter Plots and Variance Statistics 783.4.2.3^Hotelling Multivariate Statistics 793.4.2.4^Power of Test and Sample Size 804 DISCUSSION 915 REFERENCES 101viiiLIST OF TABLESTABLE PAGE1 Summary of acetylator phenotypes, sex, and samplenumbers of the subjects562 Summary of INA and ING as % of total INH dose 603 Summary of INH, AcHz, and Hz as % of total INH dose 664 Summary of AcINH and DiAcHz as % of total INH dose 695 Summary of recovery of total isonicotinyl and totalhydrazide compounds736 The effect of acetaminophen on the hepatic conversionof isoniazid to its metabolites in 5 rapid and 5 slowacetylators747 Summary of the statistical results 768 Minimum Percent Change Detectable 889 Power of Paired-Sample t test 8910 Sample Size Required 90ixLIST OF FIGURESFIGURE PAGE1 The metabolic pathways of INH 62 The metabolic pathways of Hz and AcHz 73 The metabolic pathways of acetaminophen 274 The metabolism of NAPQI and 3-GSH-APAP, 285 Derivatization reactions of AcHz, INH, and Hz 486 HPLC chromatogram of INA and ING 577 The standard plot for INA 588 The standard plot for ING 599 HPLC chromatogram of AcHz, INH, and Hz 6210 The standard plot for INH 6311 The standard plot for AcHz 6412 The standard plot for Hz 6513 The standard plots for AcINH and DiAcHz 6814 Scatter plots of distribution of differences 7515 Box plots of total recoveries of isonicotinyl compoundsin fast ans slow acetylators8216 Box plots of total recoveries of isonicotinyl compoundsin men and women8317 Scatter plots of total recoveries of isonicotinylcompounds in fast and slow acetylators8418 Scatter plots of total recoveries of isonicotinylcompounds in men and women8519 Scatter plots of total recoveries of hydrazidecompounds in fast and slow acetylators8620 Scatter plots of total recoveries of hydrazidecompounds in men and women87LIST OF ABBREVIATIONS3-Cys-APAP^3-cysteinylacetaminophen3-Cys-Gly-APAP^3-cysteinylglycinylacetaminophen3-GSH-APAP 3'-S-glutathionyl-acetaminophen3-0CH3-APAP^3-methoxyacetaminophen3-0H-APAP 3-hydroxyacetaminophen3-SCH3-APAP^3-methylthioacetaminophen9-Fl^ 9-fluorenone, volumetric internal standardAcHz acetylhydrazineAcHz-KA^acetylketoglutaric hydrazoneAcHz-PA acetylpyruvic hydrazoneAcINH^acetylisoniazidACT Acyl-CoA transferaseAPAP^acetaminophen, paracetamolATP adenosine triphosphateA.U.F.S.^detector absorbance units, full scaleBNPP bis-p-nitrophenyl phosphate, amidase inhibitorCNS^ Central Nervous SystemConc ConcentrationCys^ cysteinedayDay 6^INH only ingested by subjectsDay 7 APAP + INH ingested by subjectsDiAcHz^diacetylhydrazineDiINH di-isonicotinylhydrazidexiLIST OF ABBREVIATIONS (CONT'D)F^ female subjects9 gramGABA^gamma-amminobutyric acidGABA-T GABA aminotransferaseGAD^ glutamic acid decarboxylaseGlu glutamic acidGly^ glycineGSH glutathione, reduced formGSSG^glutathione disulfideHPLC high-performance liquid chromatographyhr^ hour(s)Hz hydrazineINA^ isonicotinic acidING isonicotinylglycineINH^ isoniazid, isonicotinyl hydrazineINH-KA isonicotinylketoglutaric hydrazoneINH-PA^isonicotinylpyruvic hydrazonei.v. intravenouskg^ kilogramM male subjectsmg^ milligramMPO myeloperoxidasemin^ minute (s)NAD nicotinamide adenine dinucloetidexiiLIST OF ABBREVIATIONS (CONT'D)NAPO!^N-acetyl-p-benzoquinone imineNAPSQI N-acetyl-p-benzosemiquinone imineNAT^ N-acetyltransferase, N-acetylaseP450 cytochrome P-450P-Azine^pyruvate azinePABA p-aminobenzoic acidPAH^ p-aminohippuric acidPAPS phosphoadenosine phosphosulfatePB^ phenobarbitalPcINH propionylisoniazid, internal standardPHR^ peak height ratioPIH pyridoxal isonicotinyl hydrazonePIP^ pyridoxal phosphate, biologically active form of pyridoxinep.o. oral administrationR^ rapid/fast acetylators of INHRMP rifampin, rifampicins^ slow acetylators of INHt;i apparent half-lifeTB^ tuberculosisTHOPC 1,4,5,6-tetrahydro-6-oxo-3-pyridazine carboxylic acidUDPGA^uridine diphosphate glucuronic aciduv detector ultraviolet wavelengthVd^ volume of distributionWater-I deionized waterACKNOWLEDGEMENTSI am very grateful to Dr. Richard A. Wall and Dr. James M. Wright for theirsupport, encouragement, patience, and continuing advice. Without their supportthis work would not have been possible. Thanks are also due to Dr. M. J. Walkerand Dr. M. Schulzer for consultation regarding statistical analysis.Thanks are also extended to the members of the Department ofPharmacology & Therapeutics for getting me fascinated with the science ofpharmacology and for their assistance in the completion of this degree. Specialthanks are extended to Mrs. Maureen Murphy and Mr. Christian Caritey forproviding technical assistance.The secretarial staff of the department, JaneIle Swetnam, Margaret Wong,and Elaine Jan are thanked for their assistance.The department is thanked for financial sponsorship in the form of teachingassistantships. The financial support of Challenge Awards is also greatlyappreciated.I also express my gratitude to the members of my Supervisory Committee(Dr. Richard A. Wall, Dr. James M. Wright, and Dr. Frank Abbott) for making thecompletion of this thesis possible at a very busy time of the year. All of theirsuggestions and criticisms are greatly appreciated.11 INTRODUCTION1.1 GENERAL PRINCIPLES OF ISONIAZIDTuberculosis (TB) is an infectious disease that has plagued humans sincethe dawn of history. Ancient civilizations made reference to this condition as earlyas 2500 B.C., yet it was not until 1882 that the etiological agent was defined asMycobacterium tuberculosis. Although chemotherapy of TB has been practicedfor 2 millenia, only in 1939 did it became a clinical reality. Dapsone, abreakthrough in the chemotherapy of TB, was made an analog of the antibacterialsulfonamides (Lewis & Shepherd, 1970; Holdiness, 1985). About 1 billion peopleare infected with the tubercle bacillus, and 3 million deaths occur annually.Isoniazid (INH, isonicotinyl hydrazide), the most powerful and besttolerated tuberculostatic drug, was introduced clinically in 1952. Since then it hasbeen used worldwide for the chemoprophylaxis and treatment of TB. Thepharmacology of INH has been extensively studied. Recently the metabolism ofINH has been studied in detail due to the discovery that the drug causes liverinjury in some individuals at therapeutic doses (Mitchell et al., 1975a). In the lastdecade the metabolism of INH has been studied in this laboratory. This thesis isabout a drug interaction: the effect of acetaminophen on INH metabolism.1.1.1 CHEMISTRY OF ISONIAZIDINH (C6H7N30, F.W. 137.15, m.p. 170-174°C), colorless crystals or whitecrystalline powder, a weak base (pKa = 1.8, 3.5, and 10.8 at 20° C), is thehydrazide of isonicotinic acid. It is soluble in water and boiling alcohol, but not inorganic solvents such as ether and benzene. INH forms complexes with divalentcations, and it reduces ammoniacal silver salt solutions to yield elemental silver.Rapid and thermodynamically favorable reactions of INH with aldehydes, ketones,a -ketoacids, and glucose yield hydrazones (Brewer, 1977).2INH was discovered by Meyer & MaIly (1912), but its tuberculostatic actionwas not recognized until its rediscovery in 1951. The quest for antituberculousdrugs found nicotinamide, sulfathiadiazoles, thiosemicarbazones (e.g.thioacetazone; synthetic precursors in the preparation of sulfathiadiazoleanalogs), and cyclic N,N'-phthalylhydrazine to be active (Chorine, 1945; Domagketa!., 1946, Behnisch eta!., 1950).The strong antituberculous activity of INH was reported independently andsimultaneously by 3 groups. Bernstein et al. (1952) made 5000 analogs ofthioacetazone; INH was an intermediate. Following the thiosemicarbazone andnicotinamide leads, Fox (1952) studied many pyridine derivatives, includingisonicotinaldehyde thiosemicarbazone. When methyl isonicotinate was treatedwith hydrazine, INH was obtained as the first of two intermediates. Offe et al.(1952) pursued the thioacetazone and phthalic hydrazide leads, modified thestructure of the latter compounds and made the benzalhydrazone of INH; INHwas an intermediate.The quest for more active and less toxic analogs of INH led to studies ofstructure-activity relationships. Only iproniazid matches INH in activity, but it hasserious liver and CNS toxicity (Lewis & Shepherd, 1970).1.1.2 THERAPEUTIC USES AND MECHANISMS OF ACTION1.1.2.1 Uses Against Mycobacterium Tuberculosis1. Antimicrobial Spectrum and Mechanism of ActionINH is unusually specific for all species of tubercle bacilli of mammals andbirds, especially M. tuberculosis. Low concentrations (0.025-0.05 g/ml) inhibitmycobacteria, but, relatively high concentrations (600 g/m1) are required toinhibit other bacteria (Krishnamurti, 1975). INH does not affect the growth of fungior protozoal parasites.3The precise molecular mechanism of action of INH is still controversial(lwainsky, 1988). INH is taken up by mycobacterial cells by active transport andthen oxidized to isonicotinic acid (INA) by peroxidase (Krishnamurti, 1975). INA isthen converted to 4-hydroxymethylpyridine, with an aldehyde as the intermediate,and then into isonicotinamide. Mycobacterium avium shows an enzyme activitydesignated as "hydrazidase" which converts INH to INA and hydrazine. Theoxidation of INH to INA may generate reactive radicals (Shoeb eta!., 1985).Inhibition of nnycolic acid synthesis, peroxidase metabolism of INH andsubsequent generation of oxygen free radicals, interference with NAD + andpyridoxal phosphate metabolism have all been proposed as possiblemechanisms of action of INH. Zhang eta!. (1992) report that catalase-peroxidasemay chemically convert INH to a biologically active form; catalase-peroxidase mayplay a role in the mode of INH action and nnycobacterial resistance.However, Quemard et al. (1991) claim just the opposite: intact INH isresponsible for the tuberculostatic mechanism and peroxidase plays no importantrole. The loss of acid fastness of INH-treated cells suggested the hypothesis ofinhibition of mycolic acid synthesis. Mycolic acids, unique to mycobacteria, arevery long-chain (C24-C26) a- branched, 13- hydroxy fatty acids that cross-link theouter cell wall to the inner membrane. Apparently the target of INH is eithersynthesis of acetate metabolites used to elongate the fatty chains or some earlysteps of chain elongation (Quemard et al., 1991). Inhibition of cell wall buildingfacilitates the attack on infecting mycobacteria by the host immune system. Treatment RegimensTo combat TB, INH (5 mg/kg or 300 mg/d, p.o., i.m.) is taken for 6-12months. The drug is taken daily, especially in the first 2 months of therapy.Patient compliance, drug toxicity, and the development of microbial resistance4present special therapeutic problems. The use of INH in conjunction with otherdrugs prevents resistance. Concomitant administration of pyridoxine (15-50mg/d) minimizes adverse reactions in malnourished patients and thosepredisposed to neuropathy (Mandell & Sande, 1990). Clinical UsesThe therapeutic value of INH was examined in Huntington's Chorea (Perryet al., 1982), tremors associated with multiple sclerosis (Bozek et al., 1987;Francis eta!., 1986), Parkinson's disease (Gershanik eta!., 1988), and rheumatiodarthritis. Only a small proportion of the multiple sclerosis and Huntington patientsbenefit from INH therapy. A mechanism has been proposed for the beneficialaction of INH. The levels of gamma- aminobutyric acid (GABA), an inhibitoryneurotransmitter, are markedly reduced in many brain regions of Huntingtonpatients. Hydrazine (Hz), a metabolite of INH, inhibits GABA aminotransferase(GABA-T, an enzyme that degrades GABA) and increases GABA levels in thebrain (Perry eta!., 1981, 1982, 1985). The mechanism of action might involve thecarbonyl trapping of the co-factor, pyridoxal phosphate (PIP), by Hz. It has beendocumented, however, that the GABA-elevating effect of INH is not abolished bythe coadministration of pyridoxine. Hz does not inhibit glutamic aciddecarboxylase (GAD), an enzyme that also uses PIP as a co-factor and producesGABA (Perry eta!., 1981). A mechanism other than GABA elevation may underlythe effect of INH, such as inhibition of monoamine oxidase (Francis et al., 1986).Another possibility is that clinical improvement in Huntington patients was maskedor blocked by alterations produced in opposing neurotransmitter systems inwhich synthesis and/or degradation of the neurotransmitter is dependent onpyridoxal phosphate-mediated reactions (Manyam eta!., 1987).51.1.3 MAJOR PATHWAYS OF INH METABOLISMThe biotransformation pathways of INH by the liver are shown in Figure 1.The main pathways involve acetylation, hydrolysis, and condensation reactions.Microsomal and peroxidase pathways are discussed later. Acetylation of INHArylamine N-acetyltransferase (NAT), a cytosolic enzyme, catalyzes theacetylation of a range of drugs and environmental chemicals, such as arylaminesand hydrazines. There are two gene loci in humans encoding different isozymesof NAT. One gene locus (pnat, equivalent to NAT2) is multi-allelic and encodespolymorphic NAT. The other gene locus (mnat, equivalent to NATI), encodesmonomorphic NAT and it does not vary amongst human individuals (Grant et al.,1990). The pnat locus products can confer either the fast (rapid) or the slowacetylator pharmacogenetic phenotype. NAT is inherited by simple autosomalMendelian segregation of two alleles at a single gene locus. Personshomozygous (rr) for mutant alleles are slow acetylators, whereas thosehomozygous (RR) or heterozygous (Rr) for the wild-type allele are fast acetylators(Price-Evans, 1989). Slow acetylation may result from decreased synthesis ofenzyme protein. Polymorphic NAT is expressed in the liver and small intestine,but monomorphic NAT is much more wide-spread in tissue distribution.INH was one of the first substrates to be recognized as being subject topolymorphic acetylation in humans. The acetylation of INH to acetylisoniazid(AcINH), a phase II reaction, is the main pathway in INH elimination in man(Hughes, 1953; Peters et al., 1965b; Boxenbaum & Riegelman, 1976). Slow andfast acetylators differ in their ability to acetylate INH; the rate of acetylation of INHis 4-5 times faster in the latter group. Consequently, there is a bi-modaldistribution in the production of AcINH by fast and slow acetylators.6Figure 1. Metabolic pathways of isoniazid (INH). AcINH is the major metabolite inhumans. Biotransformation enzymes include polymorphic N-acetyltransferase(NAT), amidase, acyl-CoA transferase (ACT), and cytochrome(s) P-450. INH, AcHz,and Hz also react chemically with a -ketoacids, such as pyruvate and a -oxoglutaricacid, to yield hydrazones or azines. The hypothetical toxication pathway(s) (---)generates free radicals that cause hepatotoxicity.COOHCovalent bindingto liver protein2Liver necrosis <--L---CH3^CHsI^IC=N-N=Ci iCOOH COOHPyruvate AzineIP450VIMH2N-NH-OH?‘H2N-N=0 -H20HN=NHX. or X+ ....NI,Cavalen/ binding ?to liver protein_0^iv,47 DiAcHzIICH3 C-NH-NH2AcHzIP450_0IICH3-C-NH-N-00IICH3-C-NH-NH-OH7^-H200IICH3-C-N=NHIIMINHN — N0IICH3-C-OHAcetic acid/CO2?^?----> N2 •---Figure 2. The metabolic pathways of Hz and AcHz. Both metabolites get acetylatedby NAT. Condensation reactions of Hz and AcHz with pyruvate yield pyruvate azineand AcHz-PA, respectively; reactions with a -oxoglutarate yield THOPC and AcHz-KA,respectively. Postulated microsomal pathways create hepatotoxins.7o^AcHz-KAUCH3-C-NH-N=C-COOH1CH2-CH2-COON0 AcHz-PAICH3-C-NH-N=C-COOHICH3_0^0II IICH3- C+ , CH3-C• . CH2 = C =0_THOPCUrea....NH3Z NATNH2 NH2 <^Hz81.1.3.2 Hydrolysis Reactions and Glycine ConjugationIn a phase I reaction in the cytosol, amidase catalyzes the hydrolysis ofINH to isonicotinic acid (INA) and hydrazine (Hz). Amidase also catalyzes thehydrolysis of AcINH to INA and acetylhydrazine (AcHz); this pathway is the mainroute of formation of INA (Ellard eta!., 1972). Hz and AcHz are minor metabolites,however they are toxic. Via a phase II reaction, INA is conjugated with glycine bythe nnitochondrial enzyme, acyl-CoA glycine transferase (ACT), to yieldisonicotinylglycine (ING, isonicotinuric acid). Individuals differ in their ability toconjugate INA with glycine, but these differences are unrelated to acetylatorstatus. INA was identified as a metabolite of INH as soon as INH came intoclinical use (Kelley et a/., 1952). The first published clinical report of the metabolicfate of INH identified intact INH, INA, and ING in the urine (Cuthbertson et al.,1953). Acetylation of Hz and AcHzIn addition to acetylating INH, polymorphic NAT also acetylates Hz to AcHz(Fig. 2); AcHz is acetylated further to N,N'-diacetylhydrazine (DiAcHz)(Ellard &Gammon, 1976; Hein & Weber, 1982; Lauterburg et aL, 1985a). About 5% of Hzis acetylated to AcHz and about 10% is excreted unchanged (Wright & Timbrell,1978). As expected, fast acetylators have a greater capacity to convert AcHz toDiAcHz than slow acetylators. A small amount of AcHz may be hydrolyzed to Hzand acetate by amidase (Timbrell eta!., 1980; Wright & Timbrell, 1978). Hz, AcHz,and DiAcHz were first identified as INH metabolites by McKennis eta!. (1959). Condensation with a-KetoacidsINH undergoes nonenzymatic reactions with endogenous a -ketoacidssuch as pyruvic acid and a- oxoglutaric acid, to yield acid-labile hydrazones:9pyruvic acid isonicotinylhydrazone (INH-PA) and a- ketoglutaric acidisonicotinylhydrazone (INH-KA)(Zamboni & Defranceschi, 1954). The formationof INH-PA exceeds that of INH-KA by 2 or 3 times (Colvin, 1969). The extent offormation of hydrazones of INH depends on the amount of INH available forcondensation (Peters et al., 1965b).In a similar fashion (Fig. 2), AcHz also reacts with the endogenous a-ketoacids to form pyruvic acid acetylhydrazone (AcHz-PA) and a -oxoglutaric acidacetylhydrazone (AcHz-KA)(Ellard & Gammon, 1976; Wright & Timbrell, 1978). Amolecule of Hz reacts chemically with two molecules of pyruvic acid to yield anontoxic metabolite, pyruvate azine (P-Azine). In vivo 15N-NMR studies in rathepatocytes have revealed that Hz reacts with 2-oxoglutarate to form 2-oxoglutaric acid hydrazone which immediately undergoes intramolecularcyclization to yield 1,4,5,6-tetrahydro-6-oxo-3-pyridazine carboxylic acid(THOPC). Hz is also metabolized to ammonia which is then converted to urea bythe hepatic urea cycle (Nelson & Gordon, 1982; Preece eta!., 1991). Other Pathways of INH MetabolismDi-isonicotinylhydrazide (DiINH) is apparently formed from thecondensation of INH with INA. In mice and guinea pigs DiINH accounts for about1-4% of the total urinary excretion of INH. Isonicotinamide and the glucosehydrazone of INH also appear to be INH metabolites (Peters et al., 1965a; Colvin,1969; Brewer, 1977). INA could be converted to 4-hydroxymethylpyridine, with 4-pyridyl aldehyde as the intermediate, and then into isonicotinamide. Thealdehyde intermediate is difficult to detect because it is metabolized rapidly bycells. There is also the possibility for the direct conversion of INH to 4-hydroxymethylpyridine (wall, personal communication).101.1.4 MICROSOMAL METABOLISM AND HEPATOTOXICITYINH, AcINH (Snodgrass eta!., 1974), AcHz (Nelson eta!., 1976; Timbrell etal., 1980; Wright & Timbrell, 1978), and Hz (Noda et al., 1983; Blair et al., 1985)are believed to be responsible for causing liver injury. For a number of reasons, itis unlikely that AcINH itself is toxic: i) pretreatment of rats with a competitiveamidase inhibitor bis-para-nitrophenyl phosphate (BNPP), a compound thatinhibits the hydrolysis of AcINH to AcHz, prevents necrosis caused by AcINH (andINH), ii) but AcHz-induced necrosis can not be prevented by BNPPpretreatment, and iii) AcHz is a more potent hepatotoxin than AcINH (Mitchell etal., 1976; Timbrell et al., 1980). It is not known which hydrazine metabolite isresponsible for INH-induced liver injury in humans.In man, the metabolism, disposition, and toxicity of hydrazines are not wellknown (Preece et al., 1992). The oxidation of hydrazines apparently producesfree radical intermediates (Noda et al., 1986) which may induce cellular toxicity bycovalent binding to tissue macromolecules (Timbrell eta!., 1980).Free radicals from hydrazines can occur in a variety of oxidizing systemsincluding cytochrome P-450/NADPH, FAD-dependent monooxygenases,peroxidases (Prough et al., 1981), prostaglandin synthase/arachidonic acid,cmhemoglobin, and neutrophil derived oxidants. The possibility exists that thesechemicals cause liver injury by a mechanism independent of microsomaloxidation. Hydrazines are very good nucleophiles, and they may also covalentlybind to cell components without metabolic activation. IsoniazidIn the last two decades liver injury and fatal intoxication cases caused bytherapeutic doses of INH have been reported (Black et al., 1975). It is widelybelieved that 2 metabolites of INH, Hz and AcHz, are heptotoxins but not INH11itself. INH may be directly oxidized to INA and free radicals by cytochrome P4502E1 (Wall, personal communication). In fact INH was oxidized directly to INA bymyeloperoxidase present in activated leukocytes (Hofstra et al., 1992). Theperoxidase-catalyzed direct oxidation of INH to INA may generate isoniazidyl andpyridinyl free radicals that may cause lipid peroxidation (Fig. 1)(Sinha, 1983;Shoeb et al., 1985). These reactive radicals could be responsible forhepatotoxicity. AcetylhydrazineAs shown in Figure 2, AcHz is eliminated by 3 processes (Timbre!' et al.,1977a): i) it is excreted intact in the urine and as hydrazones, ii) it is acetylated toDiAcHz, and iii) it is converted to reactive electophilic intermediates by the hepaticmicrosomal enzyme system. These radicals alkylate liver proteins and interactwith thiols. The adducts produced with cysteine and glutathione have beencharacterized, revealing that the acetyl group is the alkylating species (Nelson etal., 1976a-b). The P450 oxidation also yields acetate, which is further metabolizedto CO2 (TimbreII eta!., 1980; Lauterburg eta!., 1985a).Basic studies led to the postulation that AcHz is responsible for INH-induced hepatotoxicity (Mitchell et al., 1975b; Mitchell et al., 1976; Nelson et al.,1976). The administration of AcHz (or AcINH) causes dose-related hepaticnecrosis in rats pre-treated with PB. PB is an inducer of microsomal enzymes.Pretreatment of rat hepatocytes with inhibitors of cytochrome P450 such asCoCl2, carbon monoxide, SKF 525A, metyrapone, and 1-naphthyl-isothiocyanateinhibits the microsomal metabolism of AcHz (Albano & Tomasi, 1985).A number of workers have been unable to reproduce the rat model ofAcHz liver injury (Wright et al., 1986). AcHz-induced hepatitis occurs only whenrats are pretreated with PB (Mitchell eta!., 1976; Bahri eta!., 1982). HydrazineHz was identified and isolated in the 1870s, and synthesized in 1907 (citedby Juchau & Horita, 1972). In nature Hz occurs in tobacco and mushrooms. Hzis a metabolite of INH and other drugs (Noda et al., 1978; Blair et al., 1985).Several drugs are derivatives of Hz. Hz is widely used in the industry in thepreparation of blowing agents (used in the manufacture of plastics), boiler feedwater (Hz is an oxygen scavenger in high temperature boilers), plant growthretardants, and spacecraft propellants (the combustion of Hz is very exothermic)(Preece et al., 1992).Basic studies indicate that Hz is a toxic compound that causes fatty liver,liver necrosis, disturbances of the CNS and red blood cell, and tumours in variousorgans (Moloney & Plough, 1983; Juchau & Horita, 1972; Colvin, 1969).Hz undergoes a variety of biotransformations including microsonnaloxidation, acetylation, and nonenzymatic Schiff's base formation with carbonylcompounds (Fig. 2). Studies in rabbits have shown that microsomal metabolismof Hz generates radical intermediates and nitrogen, probably from the diimideintermediate (HN =NH), hydroxyhydrazine ((H2NNHOH), and other hepatotoxicspecies (Noda eta!., 1983, 1985). Expired nitrogen is a major metabolite of Hz inmice in vivo (Nelson & Gordon, 1982). In mice 25-35% of the dose of Hz isexpired as N2. Both oxyhemoglobin and liver P450 are capable of oxidizing Hz toN2, and diimide, a powerful diazene reducing agent (Nelson & Gordon, 1982;Springer et al., 1981). These reactive intermediates, like those of AcHz, arebelieved to bind covalently to hepatic macromolecules and cause hepatic injury.Despite the large number of studies conducted on the toxicity of Hz, the invivo fate of Hz and its mechanism of hepatotoxicity are not well known. Recentpharmacokinetic studies suggest some correlation between levels of Hz andhepatotoxicity (Gent et al., 1992; Woo et al., 1992). Although the liver toxicity of13Hz may be due to its microsomal metabolism, the toxicity may be caused by intactHz (TimbreII et al., 1982). Both in vivo and in vitro studies in rat hepatocytessuggest that ATP depletion may underlie the hepatotoxicity of Hz. Hz coulddeplete hepatic ATP by a number of possible mechanisms (Preece et al., 1990;Sannins et al., 1992).1.1.5 FACTORS THAT ALTER INH-INDUCED LIVER TOXICITYThe risk of INH liver damage increases with increasing age, with increaseddose, and with some co-administered drugs. Other factors that may influenceINH-induced liver toxicity include genetic factors such as species differences,cytochrome P450 isozyme composition, and acetylator phenotype. Effects of AgeAge appears to be the most important factor in determining the risk of INH-induced hepatotoxicity. Age has no significant influence on acetylator phenotypeor on the half-life of INH, but it is associated with a decrease in the apparentvolume of distribution (Kergueris et al., 1986). Increasing age leads to a strikingincrease in the incidence of hepatic injury from INH (Black et al., 1975; Dickinsonet al., 1981; Mitchell eta!., 1976): the incidence is rare in those patients less than20 years old, 0.3% in those 20 to 34 years old, 1.2% in individuals 35 to 49, and2.3% in individuals older than 50 years of age. Dose of INHWright & Wall (1985) provide clinical evidence that as the dose of INHincreases, hepatotoxicity also increases. This is supported by a report that theincidence of hepatitis was appreciably lower with a 12 mg/kg dose as comparedwith 20 mg/kg in children with TB (Parthasarathy et al., 1986). Co-administration of Other DrugsUpon co-administration with INH, carbamazepine induces hepaticmicrosomal enzymes and, subsequently, increases production of hepatotoxicINH metabolites (Wright et aL, 1982, 1984; Barbare et al., 1986). According toepidemiological studies, chronic alcohol consumption increases the risk of liverdamage with INH (Dickinson et al., 1981). There are at least 2 possiblemechanisms: i) induction of microsomal metabolism of INH by alcohol, and ii)possible additive hepatotoxicity. PB is a well-known inducer of the microsomalmetabolism of INH. The effect of RMP on the microsomal metabolism of INH inhumans is still unclear. RMP does not influence the metabolism of INH and itsmetabolites in man (TimbreII et al., 1985; Jenner & Ellard, 1989), however theseauthors did not measure the effect of AMP on Hz metabolism. In rabbits, RMPpretreatment increases the microsomal metabolism of Hz but not AcHz (Noda etal., 1984). Based on indirect measurements, Sarma et al. (1986) suggested thatRMP influences the metabolism of Hz. Pharmacokinetic studies of Hz in TBpatients on multiple drug therapy suggest that AMP has no major effect on Hzformation (Gent et al., 1992). Concomitant use of INH and RMP increases livertoxicity, but the effect may be additive since RMP itself is known to cause liverdamage (Steele eta!., 1991).Some compounds such as Cu2+ , zn2+, mn2+, Ni2+, sulfhydrylinhibitors, N-ethylmaleimide, and p-chloromercuribenzoate act as blocking agentsof polymorphic NAT, but they are not used as drugs (Weber & King, 1981). Drugssharing the acetylation pathway, such as procainamide, are able to interfere withthe acetylation of INH (Ladero eta!., 1989). Prednisolone enhances acetylation ofINH in slow acetylators (Sarma et al., 1980). However, such interactions are notclinically significant. Cytochrome P450 Isozyme CompositionAmongst humans, differences in the complement of P450 forms present invarious tissues, especially in the liver, is a major factor responsible forinterindividual variation in the metabolism and effects of drugs and toxic chemicals(Kalow, 1987). Individuals who have higher levels of the specific P450 isozymesthat metabolize INH and/or its metabolites to toxic intermediates, should have ahigher risk of INH-induced liver damage. Species DifferencesDespite the fact that many papers appeared on INH hepatotoxicity, only asmall number of researchers actually demonstrated liver necrosis in rats resultingfrom the actions of INH or one of its metabolites (Snodgrass et al., 1974; Erill etal., 1977; Bahri et al., 1981), and so far none of these groups have been able toreproduce their original experiments. Covalent binding of liver macromolecules inrats has been demonstrated, but it was also shown that the pathological damageof the liver is not necessarily correlated to covalent acetyl binding to hepaticmicrosomal macromolecules (Wodward & TimbreII, 1984).Since the rat was not a good model for the study of INH-inducedhepatotoxicity, rabbits were studied by Whitehouse et al. (1983) and ourlaboratory. Interestingly, NAT is polymorphically inherited in rabbits, like humans,but not in rats, mice, or guinea pigs. It appears that the levels of amidase inrabbits is higher than that in rat, and this allows liver damage to occur by INHalone ((Whitehouse et al., 1983). However, as Noda et al. (1983) have shown,and also demonstrated in our laboratory, Hz may be the cause of hepatotoxicity inrabbits not AcHz. Acetylator StatusAcetylator phenotype has been associated with differing susceptibility todisease. Slow acetylators are at a greater risk of predisposition to bladdercancer. Colonic cancer, in contrast, may be associated with the fast acetylatorphenotype. Despite many epidemiological and metabolic studies and debates,the precise relationship, if any, between acetylator status and the incidence ofINH-induced hepatotoxicity is unclear. Epidemiological StudiesThe results of epidemiological studies are conflicting. Evidence has beenobtained that i) fast acetylators are at a greater risk of IN H-induced liver damage(Mitchell et al., 1975b, 1976); ii) slow acetylators are at a greater risk accordingto Dickinson eta!., 1981, Musch et a!., 1982, and Parthasarathy eta!., 1986; andiii) the risk is about the same in both groups (Gurumurthy eta!., 1984; Ellard eta!.,1978).The experimental conditions were not identical in the above studies.Mitchell did not have proper controls for age differences of his patients. It seemsthat the significance of the acetylator status as a predictor of susceptibility to liverdamage is highly dependent on the index of liver damage chosen. If one choosesclinical jaundice as Gurumurthy et al. have done, "liver damage" is unrelated toacetylator status. On the other hand, if a biochemical index such as serumtransaminase elevations are chosen (Dickinson et al., 1981; Musch et al., 1982),"liver damage" depends on acetylator status. Clearly, failure to define such termsadequately may lead to loss of valuable information if not invalidate anyconclusions. In some of the studies multiple drugs were used for the treatment ofTB; this may invalidate the conclusions. In other cases the therapeutic regimenwas not the same as those of others. Metabolic StudiesClinical studies with healthy subjects show that upon administration of INH,the levels of INH, AcHz, and Hz are higher in the plasma of slow acetylators thanfast acetylators (Blair et al., 1985). Most metabolic studies suggest that slowacetylators are at a greater risk of INH-induced hepatotoxicity if AcHz is thecausative agent. An initial report suggested that fast acetylators have a higherrisk of INH-induced liver damage, presumably because they convert more INH toAcINH and therefore AcHz. It was confirmed that fast acetylator humans formhigher amounts of AcHz, but they also detoxify more AcHz (by conversion toDiAcHz) than slow acetylators. After ingestion, about 23-28% of INH is excretedas DiAcHz by fast acetylators vs 5-8% by slow acetylators (Ellard & Gammon,1976; TimbreII et aL, 1977a). Based on these measurements, TimbreII et al.estimated that the amount of AcHz that should undergo metabolic activationthrough the cytochrome P-450 pathway to hepatotoxicity in the fast acetylators(16.3%) should not be significantly different from the slow acetylators (18.2%).Based on results from the study of patients on triple therapy (INH, RMP,and ethambutol) for TB (Musch et al. (1982; Eichelbaum et al. (1982), fastacetylators (n=26) formed almost twice as much AcHz (40.1% of INH dose) ascompared to slow acetylators (n=37; 25.8% of INH dose). In fast and slowacetylators about 70% (25.9% of INH dose) and 27% (4.9% of INH dose),respectively, of the AcHz formed is acetylated to DiAcHz. Renal excretion of AcHzand its hydrazones in fast and slow acetylators amounted to 2.3% and 3.5% ofINH dose, respectively. In slow acetylators 68% of AcHz (17.5% of INH dose) isoxidatively metabolized, whereas in fast acetylators only 30% (12% of INH dose)is metabolized along this pathway. If AcHz is the compound responsible forhepatotoxicity, then this study suggests that the risk of hepatitis is higher in slowacetylators. Hz levels were not measured.18It was found that the formation of 14CO2 after administration of [14C)-AcHz or [14q-AcINH in rats reflects the fraction of the dose that passes throughthe toxic pathway (TimbreII eta!., 1980). Building on these observations, a similarstudy was done in humans (Lauterburg et a!., 1985a). The cumulative exhalationof 14co2 increases as the rate of acetylation of INH decreases, such that slowacetylators generate more 14CO2 than fast acetylators. Thomas et a/. (1987)found that in rabbits, covalent binding of 14C to hepatic protein was inverselyproportional to the rate of acetylation. This is an important finding if we asssumethat covalent binding is a prerequisite for hepatotoxicity. These observationssuggest that slow acetylators metabolize more INH via the toxic pathway andhence would probably be more susceptible to INH hepatitis, particularly at highdoses of the drug.The acetylation of AcHz to DiAcHz is the most important detoxifying step(Wright & Timbrell, 1978). The balance between microsomal metabolism andacetylation of AcHz may be a key determinant of the toxicity of INH (Lauterburg etal., 1985b). INH has been reported to alter the metabolism of AcHz. In vivo, INHinhibits the acetylation of AcHz to DiAcHz (Wright & Timbrell, 1978; Timbrell,1979). The authors predicted correctly that normal therapeutic doses of INH inhumans would cause inhibition of AcHz acetylation (Peretti et a!., 1987). Becausethe levels of INH (and AcHz and Hz) are higher in slow acetylators (Boxenbaum &Riegelman, 1976; Weber & Hein, 1979), acetylation of AcHz is probably moreinhibited in slow as compared to fast acetylators. Lauterburg et a/. (1985b) did apharmacokinetic study of INH metabolism in man. Since the half-life of eliminationof AcHz is fivefold slower than that of INH, repeated doses of INH are expected tocause accumulation of AcHz. One might expect that, due to higher blood levelsof INH, slow acetylators would have a greater risk of developing hepatotoxicity.When a large dose of INH such as 10 mg/kg is administered, saturation of19acetylation may occur, particularly in patients who are slow acetylators. Thissaturation of acetylation may be especially contributory to an accumulation ofAcHz in slow acetylators by decreasing its clearance to DiAcHz (Lauterburg et al.,1985b).However, Bahri et al. (1981) showed histologically that INH, whencoadministered with AcHz, decreased the liver injury of AcHz when the rats werepretreated with PB. This could be due to the inhibition of cytochrome P-450mediated activation of AcHz by INH (Wright & TimbreII, 1978; TimbreII & Wright,1979; TimbreII, 1979). INH inhibits liver microsomal enzymes in man (Kutt et al.,1970; Wright eta!., 1982). This may partly explain why several small doses of INHcause mild liver toxicity in PB-pretreated animals, but large single doses of INH donot (Mitchell et al., 1976). Timbrell & Wright (1979) found that INH inhibits thecovalent binding of 14C-AcHz to rat microsomal proteins in vitro, but theconcentration required is unlikely to be attained in vivo after therapeutic dose ofthe drug. Therefore, it is not clear if high levels of INH, as compared to low levels,enhance or inhibit the hepatotoxicity of AcHz in man.1.1.6 PHARMACOKINETICS1.1.6.1 Absorption and BioavailabilitySince INH is a weak base that is effectively unionized at body pH and isboth water and fat soluble and not significantly protein bound, it readily diffusesacross lipid membranes. As a consequence, it is rapidly and completelyabsorbed after oral or parenteral dosage (Ellard & Gammon, 1976; Mitchell et a!.,1975b; Weber & Hein, 1979). Good estimates of possible loss due to first-passmetabolism are not available; therefore, the bioavailability of INH is not known(Wright, personal communication; Mandell & Sande, 1990).2 DistributionINH and its 2 major metabolites, AcINH and INA, are distributed in totalbody water (Vd= 0.8 L/kg). The peak levels of INH attained in slow acetylatorsexceed those in rapid acetylators by about 25% (Ellard, 1984). Therapeutic dosesof INH (5 ring/kg) cause peak plasma concentrations (Cm) of 3-5 A g/ml within1-2 hours of oral ingestion (Weber & Hein, 1979). The therapeutic concentrationsof INH in the plasma can range from 3 to 10 A g/ml. In the whole population, thehalf-life of INH generally averages 1 hr (fast acetylators) and 3 hrs (slowacetylators). Hepatic insufficiency prolongs the half-life of INH. Renal ClearanceIntact INH, its hydrazones, AcINH, INA, ING, intact AcHz, its hydrazones,DiAcHz, intact Hz and its derivatives, get excreted in the urine. About 85-98% ofthe total dose of INH is excreted in the urine. About 0.5-10% of the total dose isexcreted in the bile (lwainsky, 1988). From 75 to 95% of a dose of INH is excretedin the urine within 24 hours, mostly as metabolites.INH undergoes glomerular filtration and tubular reabsorption. Eliminationof INH depends mainly on its metabolic biotransformation. The clearance of INHis dependent to only a small degree on the status of renal function, howeverpatients who are slow acetylators may accumulate toxic concentrations if theirrenal function is impaired (Mandell & Sande, 1990). AcINH also undergoesglomerular filtration. INA and ING are actively secreted. The apparent first-orderrate constants for the excretion of some of the metabolites of INH have beendetermined (Ellard & Gammon, 1976).211.1.7 ADVERSE EFFECTSHepatitis and peripheral neuritis are the most serious and the mostcommon side effects, respectively, of INH use. Other adverse effects includehypersensitivity/allergic reactions, idiosyncratic reactions, lupus erythrematosus,etc. (Mandell & Sande, 1990). HepatotoxicityLiver toxicity secondary to INH therapy was first reported one year after thedrug's remarkable effectiveness against TB was recognized. Most INH-inducedhepatic damage occurs 4 to 8 weeks after the start of therapy. Chronicadministration of therapeutic doses of INH causes subclinical hepatic dysfunction(which is mild and reversible) in 10 to 20% of patients (Mitchell et a!., 1975a,1976). The overall incidence of overt INH-induced hepatitis is ca. 1-2% (Black etal., 1975; Mitchell et al., 1976; Dickinson et a!., 1981; Gurumurthy et a!., 1984); upto 10% of these patients may die from hepatic failure. NeurotoxicityIf pyridoxine (vitamin B6) is not given concurrently, peripheral neuritis(especially in slow acetylators) is the most common reaction to INH and occurs inabout 2% of patients receiving 5 mg/kg of the drug daily. Higher doses mayresult in peripheral and central nervous system (CNS) toxicity in 10-20% ofpatients. These toxic reactions include peripheral neuritis, insomnia,restlessness, urinary retention, lactic acidosis, muscle twitching, convulsions(when the dose is over 17 mg/kg), psychotic episodes, seizures and coma(Mandell & Sande, 1990; Ellard, 1984). Acute neurological toxicity, associatedwith overdose, may be fatal if not recognized and treated promptly.2 Mechanism of CNS ToxicityThe adverse effects produced by INH intoxication may be due to thelowering of the effective tissue and serum levels of pyridoxine through theformation of two types of pyridoxal isonicotinyl hydrazones (PIH's) which arerapidly excreted through the kidney. The PIH's also cause competitive inhibitionof pyridoxine kinase, an activating enzyme, which converts pyridoxine to thephysiologically active pyridoxal phosphate (PIP).As mentioned before, therapeutic doses of INH inhibit GABA-T andconsequently raise GABA levels in the brain (Perry et al., 1982). GABA is aninhibitory neurotransmitter in the CNS. GAD and GABA-T are involved in thesynthesis and degradation of GABA, respectively. PIP is an important cofactor inthe metabolism of multiple neurotransmitters. Many enzymes require PIP as acofactor, e.g., apotryptophanase (which degrades tryptophan). PIP is importantin the metabolism of GABA, because it is a cofactor for both GAD and GABA-T. Ithas been speculated that overdoses of INH causes inhibition of GAD.INH-induced seizures may be produced by an overwhelming stimulation ofthe CNS, apparently caused by i) depletion of GABA with subsequentheightened CNS excitability, and ii) inhibition of nnonoamine oxidase which leadsto increased sympathetic activity (Cash & Zawada, 1991). Most of theseproblems can be effectively prevented by daily administration of supplementalpyridoxine.Another adverse effect, lactic acidosis, caused by INH overdose ispostulated to be induced by seizure activity, which results in an anerobic state.Alternatively, INH may also interfere with nicotinamide-adenine dinucleotide (NAD)that is necessary for the conversion of lactate to pyruvate in the Kreb's cycle.2 Treatment of INH OverdoseLethality from INH overdose has occurred from an oral dose of as little as 5grams, although patients have ingested as much as 30 grams and survived(Leibowitz et al., 1989). Toxic effects have been generally associated with plasmaconcentrations greater than 20 A g/ml. The standard procedure is used for thetreatment of INH overdose: lavage, emesis (but not in conscious patients),followed by activated charcoal. Vasopressors are occasionally required. Forceddiuresis is not recommended. Intravenous pyridoxine in a dose equivalent to thatof INH ingested aborts and prevents seizures. Hemodialysis is used in severecases (Cash & Zawada, 1991). Charcoal hemoperfusion was shown to be muchmore effective than standard hemodialysis (Leibowitz et aL, 1989). Severehepatotoxicity does not appear to be a consequence of acute overdose of INH(Cash & Zawada, 1991).1.1.8 FUTURE DEVELOPMENTSAt present INH is usually administered for 6-12 months for preventivetherapy. However, this approach has many deficiencies. These include theexpense of treating and monitoring patients for such a long time, noncompliancewith preventive therapy of long duration, and the occurrence of drug toxicity.Because hepatotoxicity is a major limitation to the use of INH preventive therapy,research efforts should be encouraged that i) contribute to the understanding ofthe mechanisms of INH-induced hepatitis, ii) contribute to the understanding ofthe mechanism by which INH acts, which will in turn facilitate the development ofcongeners that retain antimicrobial potency while eliminating sites of hepatotoxicpotential, iii) investigate the cytoprotective effects of drugs that may block orinterfere with the the toxic effects of INH, and iv) identify more specific riskfactors, other than age, for the development of INH-hepatitis (CDC, 1989).2 41.2 GENERAL PRINCIPLES OF ACETAMINOPHENAcetaminophen^(paracetamol,^4'-hydroxyacetanilide,^N-acetyl-p-aminophenol, APAP), a widely used analgesic and antipyretic medication that hasweak anti-inflammatory action, has been available in most countries since the1950s. It has become a common household analgesic because it lacks theoccasional side effects of aspirin, is well tolerated, and is available withoutprescription (Penna & Buchanan, 1991). The drug is remarkably safe when usedin therapeutic doses, but when taken in overdose, it causes acute centrilobularhepatic necrosis in man (Black, 1984; Hinson, 1980; Mitchell & Jollow, 1975;Prescott et al., 1983) and animals (Mitchell et al., 1973a). The pharmacologicalproperties of APAP have been reviewed by Clissold (1986).1.2.1 CHEMISTRY OF ACETAMINOPHENAPAP (C8H9NO2, F.W. = 151.16, m.p. 169-172°C, white crystals orcrystalline powder), a moderately water- and lipid-soluble weak organic acid(pKa =9.5), is largely un-ionized over the physiological range of pH. APAP issynthesized by the electrolytic reduction of nitrobenzene to p-aminophenol,followed by acetylation with acetic anhydride. In industry APAP is used as aphotographic chemical and in the manufacture of azo dyes (Merck Index, 1989;Forrest et a!., 1982; Fairbrother, 1974).Acetanilide, accidentally discovered to have antipyretic action about 1886,was too toxic. In the search for less toxic analogs, p-aminophenol, phenacetin,and APAP were tested. APAP was first synthesized by Morse in 1878 byreduction of p-nitrophenol with tin in glacial acetic acid (cited by Fairbrother,1974). The p-aminophenol produced by the reducing action of the tin wasacetylated by acetic acid. APAP was first tested clinically as an antipyretic-analgesic drug by von Mering in 1893 (cited by Prescott, 1983). APAP became2 5popular in 1949, when it was found that i) it is the active metabolite of acetanilideand phenacetin, and ii) it is less toxic than the other 2 drugs (Insel, 1990).The antipyretic activity of APAP and other 4-aminophenol derivativesresides in the aminobenzene structure. Some APAP analogs, such as N-methyl-APAP and 2,6-dimethyl-APAP, do not cause liver injury but unfortunately they lackanalgesic activity. Methylation of APAP at position 2 or 3 of the benzene ringretains the analgesic activity but has hepatotoxicity as well. Positional isomers ofAPAP, 2- and 3-hydroxyacetanilide, retain some pharmacological activity and theyare not as hepatotoxic as APAP. Some 3,5-dialkyl analogs of APAP are morepotent than APAP itself but their hepatotoxic effects are controversial. None is aseffective as APAP in terms of analgesia (Vermeulen et a!., 1992).1.2.2 THERAPEUTIC USESAPAP is useful in mild to moderate pain, especially when patients areallergic to aspirin or when salicylates are poorly tolerated. Effective analgesic-antipyretic levels of APAP are 10-20 1.4g/m1 (Insel, 1990). The conventional oraldose of APAP is 325-1000 mg; the total daily dose should not exceed 4 g.1.2.3 MECHANISMS OF ACTIONThe rapid, reversible, and non-competitive inhibition of cyclooxygenase byAPAP involves antioxidant or free radical trapping properties (Lands, 1985). Thiseffect is significant since it reduces the hydroperoxides, which are believed tohave an essential role in the generation of prostaglandins by cyclooxygenase. Inareas of inflammation, hydroperoxides generated by activated leukocytes negatethe effect of APAP; this may explain the weak anti-inflammatory action of APAP.However, in conditions where leukocye infiltration is not a major factor, such aspain and fever, APAP is an effective medication.2 61.2.4 BIOTRANSFORMATION AND HEPATOTOXICITYThe biotransformation pathways of APAP by the liver are shown in Figs. 3and 4. The major pathways of APAP metabolism are conjugation reactions andP450 oxidative pathways that lead to both catechol and glutathione metabolites(Andrews et al., 1976; Mitchell et al., 1974; Slattery et a/., 1987). Large doses ofAPAP saturate the conjugation pathways and a higher amount is cleared throughthe microsomal oxidative pathways (Davis et al., 1976; Mitchell et al., 1974;Slattery eta!., 1987).Small amounts of deacetylated (4-aminophenol) and hydroxylatedmetabolites have also been found. The P450 metabolism of 4-aminophenol isapparently responsible for the nephrotoxicity of APAP in rats (Klos et al., 1992).The metabolites of APAP are therapeutically inert. Conjugation ReactionsThe conjugations of APAP and some of its metabolites with UDP-glucuronic acid (UDPGA) and phosphoadenosine phosphosulfate (PAPS) lead toglucuronide and sulfate conjugates, respectively (Figs. 3 & 4). APAP glucuronideand APAP sulfate together account for about 70-80% of therapeutic doses ofAPAP in humans (Prescott, 1983). The proportion of APAP eliminated as thesulfate vs. the glucuronide conjugate depends on the dose. The ratio ofglucuronide to sulfate conjugates in human urine is usually between 1.8 and 2.3.Sulfation is a high-affinity, low capacity pathway whereas glucuronidation is a low-affinity, high-capacity pathway (Hinson et al., 1981). At high doses of APAP theconjugation pathways become saturated and greater proportions of the drug aremetabolized by P450.27Figure 3. The biotransformation pathways of acetaminophen (APAP) are catalyzedby microsomal (*) and cytosolic (**) enzymes. Glucuronidation and sulfation are themajor pathways at therapeutic doses of APAP; these pathways get saturated atoverdoses of APAP, and more toxic metabolite, NAPO', gets formed. When most ofthe glutathione (GSH) gets depleted, NAPQI may cause hepatotoxicity by covalentattachment to macromolecules or by other mechanisms. +6 indicates that the metaposition of NAPQI has a partial positive charge; this part of the molecule is attachedto GSH or to macromolecules.2 8Figure 4. The fate of NAPQI, the toxic metabolite of APAP. NAPQI generated fromtherapeutic doses of APAP is detoxified easily by glutathione (GSH); the GSH-derivedconjugates account for 5-10% of the dose of APAP. Overdoses of APAP lead todepletion of GSH; the excess NAPQI then causes liver damage by a number ofpostulated mechanisms. Glutamyl transpeptidase and cysteinyl-glycinase are foundon the extracellular face of the plasma membrane. +s indicates that the metaposition of NAPQI has a partial positive charge; this part of the molecule is attachedto GSH or to macromolecules.2 Microsomal MetabolismLiver microsomes contain several P450 isoforms that are involved with theoxidation of APAP, including the ethanol-inducible form from rabbits, rats, andhumans (P450 2E1; Morgan eta!., 1983; Ryan et al., 1986), the 13 -naphtoflavone-inducible form in rats and humans (P450 1A2; Harvison eta!., 1988a; Raucy eta!.,1989), the pregnenolone-16a-carbonitrile (PCN)-inducible form in rats (P450 3A1),and other isoforms such as 2C11 and 1A1 in rats (Lee et al., 1991). P450 1A2metabolizes APAP mainly to a relatively nontoxic catechol metabolite, but it canalso oxidize APAP to NAPQI. P450 2E1, P450 3A1, and myeloperoxidase(MPO/H202) oxidize APAP to NAPQI. Purified human liver P450 2E1 and itsortholog in rabbits (P450 3A) are relatively good catalysts of APAP oxidation toNAPQI, but only at high APAP concentrations (Nelson, 1990). Nontoxic RouteThe oxidation of APAP by cytochrome P450 1A2 generates the catecholmetabolite, 3-hydroxyacetaminophen (3-0H-APAP), which is then converted to 3-methoxyacetaminophen (3-0CH3-APAP). After therapeutic doses in man, thesearomatic oxidation products account for approximately 5 to 10% of the dose(Slattery et al., 1987; Raucy et al., 1989). The catechols are interestingmetabolites because, like the thioether metabolites, their formation clearancesdecrease with increasing dose (Slattery et al., 1987), and they are found in largeamounts in urines of overdosed patients (Andrews eta!., 1976).Some reactive quinones (benzoquinone, the quinone of the APAP catecholmetabolite, and the quinone imine of the methylated catechol metabolite) areformed to a lesser extent, but their role in hepatotoxicity is not clear (Nelson,1990).3 Toxic RouteAs mentioned above, APAP is oxidized to NAPQI by a number of P450isoforms. APAP causes hepatic necrosis in both animals and man when ingestedin high enough doses. The exact sequence of events that ultimately leads tohepatic necrosis has not been elucidated. In man the oxidation of APAP byhepatic cytochrome P450 2E1 and 3A4 (and perhaps others) generates N-acetyl-p-benzoquinone imine (NAPQI), an electrophilic hepatotoxic metabolite (Raucy etal., 1989; Lee et al., 1991). The detoxification of NAPQI occurs by at least 2mechanisms; NAPO! oxidizes reduced glutathione (GSH) to glutathione disulfide(GSSG), with the concomitant regeneration of APAP, and NAPQI reacts with GSHto form 3'-S-glutathionyl-acetaminophen (3-GSH-APAP). Events leading to celldeath are initiated by the depletion of GSH and protein-bound thiols or by thebinding of NAPO! to other constituents of the liver cell once GSH stores aredepleted (Albano et al., 1985a; Dahlin eta!., 1984).The degradation of 3-GSH-APAP in the liver, gut and the kidney leads tothe formation of other metabolites. About 5 to 10% of the dose of APAP isexcreted as glutathione-derived conjugates (Fig. 4): 3-cysteinyl (3-Cys-APAP), 3-mercapturate (APAP-3-mercapturate) and 3-methylthio (3-SCH3-APAP)conjugates. These thioethers are detoxication products of the reactive, putativetoxic metabolite of APAP, NAPQI. Therefore they are indicators of flux throughthe toxic pathways.Cytochrome P450 can function as a peroxidase as well as an oxygenase(Nelson et al., 1981). There is a direct two-electron oxidation of APAP to NAPO!by cytochrome P450 and MPO or, alternatively, a one-electron oxidation to N-acetyl-p-benzosemiquinone imine (NAPSQI) by peroxidase, prostaglandin Hsynthase (PGS) or cytochrome P450 (Dahlin et al., 1984; Harvison et al., 1988b;van de Straat et al., 1988). The relationship of NAPSQI to APAP-induced31hepatotoxicity is unclear. NAPSQI can also react with molecular oxygen to formsuperoxide or other active oxygen species responsible for lipid peroxidation. Mechanism of NAPQI FormationThe mechanism of NAPQI formation from APAP is still unclear. Becausedifferent isozymes of cytochromes P450 form NAPQI and a catechol metabolite(3-0H-APAP) at different rates (Harvison et al., 1988a), two different schemeswere proposed. Initial reactions are abstraction of hydrogen radicals by aferryloxy form of P450 from either the phenolic oxygen (in the case of 3-0H-APAP)or amide nitrogen (in the case of NAPQI). Radical recombination reactions wouldthen occur so rapidly that the radicals do not diffuse from the enzyme active site.Such a mechanism is in accord with what is known about the formation of radicalsby cytochromes P450 and their subsequent fate. It is proposed that therecombination products essentially undergo a dehydration reaction to yield theobserved two electron oxidation products, 3-0H-APAP and NAPQI. Mechanism of NAPQI HepatotoxicityWhen the GSH levels fall to 20-30% of normal, the excess metabolite(NAPQI) is free to produce cell damage (Mitchell et al., 1973a, b, 1974) Hepaticglutathione plays a vital role in protecting against APAP hepatotoxicity. Upondepletion of GSH, NAPQI causes liver injury by covalent binding and/or oxidativestress (thiol oxidation or lipid peroxidation). There is not enough evidence for therole of lipid peroxidation. Covalent binding appears to be the most damagingevent. The resulting hepatotoxicity has often been correlated with the extent ofthe covalent binding of APAP to proteins (Mitchell eta!., 1973a; Jollow eta!., 1973;Potter et al., 1973; Hinson, 1980; Albano et al., 1985). The protein-bound3 2residues in vitro and in vivo were identified as 3-cysteinyl thioether conjugates(Nelson, 1990).Studies with positional isomers of APAP show that covalent binding maynot be necessarily related to liver damage. For example, some positional isomersbind to the same extent to hepatic proteins, and also form GSH conjugates, butdo not cause liver damage. APAP, however, damages the mitochondria muchmore than its isomers; this is consistent with the known oxidative properties ofAPAP (Dahlin eta!., 1984; Albano eta!., 1985). Thiols in the mitochondria may bean important target in the pathogenesis of APAP hepatotoxicity (Nelson et al.,1991). The mitochondrial proteins that are arylated have not been characterizedyet.APAP, primarily through its reactive metabolite NAPQI, depletes cellularthiols, which in turn disrupts Ca2+ and ATP homeostasis. These changes canlead to increases in cell Ca2+ levels, mitochondrial Ca2+ cycling, and activationof proteases and endonucleases, which may be involved in the propagation ofliver damage (Nelson, 1990).One can not rule out the possibility that both covalent binding and oxidantstress caused by APAP are important factors in liver cell damage, and theirrelative importance may be dependent on the redox state of the cell. It is to beexpected that the mechanism of APAP liver injury is quite complex andmultifactorial.1.2.5 FACTORS THAT ALTER APAP HEPATOTOXICITY IN MAN1.2.5.1 Effects of AgeAPAP overdose in young children has generally been associated with amuch lower incidence of hepatotoxicity than the incidence in adults (Penna &Buchanan (1991). Limited studies in humans show an increased clearance of3 3APAP in young subjects, and this may be the result of increased clearance viasulfation. Effects of PregnancyClearance of APAP in pregnant women through pathways ofglucuronidation and oxidation (as measured by thioether conjugates) is increasedsignificantly (about 2 times) in the third trimester (Miners eta!., 1986). While theseeffects were not seen in pregnant mice, GSH was depleted at a faster rate and theanimals were more susceptible to APAP-induced liver injury (Larrey et al., 1986).However, an increased incidence of liver injury caused by APAP has not beendocumented in pregnant women. lnterspecies Differences Due to Enzyme VariationsGenetic effects on the oxidation of APAP to its hepatotoxic metabolite ormetabolites have been observed in laboratory animals and man. Speciesdifferences in hepatotoxicity and metabolism are quite dramatic (Gregus et al.,1988). Hamsters are the species most susceptible to toxicity, followed by mice.Rats, rabbits, and guinea pigs are relatively resistant to APAP-induced liver injury.Mice and hamsters have high levels of the alcohol-inducible P450 2E in the liver,but rats have low levels. The more susceptible species clear a larger fraction ofthe dose through NAPQI. The susceptibility of humans is somewhere in the rangebetween the mouse and the rat. Variability of P450 Isozyme Composition in HumansThe oxidation of APAP to NAPOI by human liver samples varied over atenfold range, which suggests that P450 isozyme composition may play animportant role in man when large doses are taken; not all subjects taking an3 4overdose of APAP are equally susceptible to its hepatotoxic effects (Prescott,1983; Seddon et al., 1987; Parkinson & Hurwitz, 1991). In fact it was recentlyshown that the interindividual variation in the amount of hepatic P450 2E1 isconsiderable (Ingelman-Sundberg et al., 1990). The data of Ingelman-Sundberget al. might indicate the presence of polymorphically distributed variant P450 2E1genes which may be linked to the incidence of alcohol-induced liver damage. Effects of Other DrugsThe risk of APAP-induced hepatotoxicity is increased in alcoholics,presumably because of the induction of the toxic pathway of APAP metabolism(Seeff et al., 1986). Anticonvulsants such as phenytoin, PB, carbamazepine, andprimidone may increase the risk of APAP-induced hepatotoxicity (Minton et al.,1988; Smith et al., 1986). There is evidence to support the hypothesis that P4502E1 may contribute to the enhanced susceptibility of chronic alcoholics to APAPhepatotoxicity (Lieber, 1988). However, when given acutely, ethanol decreasesthe metabolism of APAP to toxic metabolites and prevents APAP hepatotoxicity inanimals (Sato & Lieber, 1981) and possibly in man (Critchley eta!., 1983).The activity of liver microsomal enzymes is also important since this maydetermine the rate of NAPO' formation. The hepatotoxicity of APAP is increasedin most species by pretreatment with PB, pregnenalone 16 -carbonitrile, 3-methylcholanthrene, and reduced by inhibitors such as SKF 525A, piperonylbutoxide, ketoconazole, cimetidine, and cobaltous chloride (Mitchell et al., 1973a;Jollow et al., 1973). Cimetidine has been shown to protect mice and man fromAPAP toxicity (Rolband et al., 1991). Aniline, a P450 2E1 substrate, competitivelyinhibits the activation of APAP by INH in mice (Seddon eta!., 1987).3 Effect of DietProlonged fasting, ingestion of protein-deficient diets, or diethyl maleateadministration, have all been shown to reduce the liver levels of GSH and increasethe hepatotoxicity of APAP. Dietary supplementation of fish-oil protects miceagainst APAP hepatotoxicity in vivo (Speck & Lauterburg, 1991).1.2.6 PHARMACOKINETICS1.2.5.1 AbsorptionAlthough APAP is rapidly and almost completely absorbed from the gut, itis incompletely available to the systemic circulation after oral administration, avariable proportion being lost through first-pass metabolism (Perucca & Richens,1979). The bioavailability appears to depend upon the amount administered,decreasing from 90% after 1-2 g to 68% after 0.5 g (Forrest eta!., 1982).APAP absorption depends on the rate of gastric emptying. The mean half-time for absorption from the small intestine is about 7 minutes, whereas gastricemptying half-times are about 3 times longer. In man, APAP absorption appearsto be negligible from the stomach but very rapid from the small intestine.Absorption is by passive transport with first-order kinetics (Forrest et a/., 1982). DistributionThe concentration in plasma reaches a peak in 30 to 60 minutes, and thet1/2 in plasma is about 2-4 hours after therapeutic doses. Therapeutic levels inplasma range from 10 to 20 II g/ml. With toxic levels of APAP or liver disease, thehalf-life may be increased. APAP is relatively uniformly distributed throughoutmost body fluids. Binding of APAP to plasma proteins is variable; about 20 to50% is bound at plasma concentrations associated with overdosage. The volumeof distribution is about 0.95 I/kg.3 6APAP distributes throughout most tissues and fluids. After i.v. doses,plasma APAP concentration-time curves are multiexponential and the relativelyshort half-times for the initial disposition phase (t½ce equal to 3 to 19 minutes)indicate rapid tissue distribution (Forrest et a!., 1982). ExcretionAfter therapeutic doses, 90 to 100% of the drug may be recovered in theurine within the first day (Insel, 1990), with about 4, 55, 30, 4 and 4% appearing asunchanged APAP, and its glucuronide, sulfate, cysteine and mercapturic acidconjugates, respectively. The minor metabolites are also detected in the urine.The total body clearance of APAP is 5.0 ± 1.4 ml/min/kg (Insel, 1990). SinceAPAP is a moderately lipid-soluble weak organic acid (pKa =9.5) which is notextensively bound to plasma proteins, it is likely to undergo considerableglomerular filtration with subsequent passive tubular reabsorption. In healthyadults the renal clearance of APAP at normal urine flow rates is about 12 ml/min(range 5 to 20 ml/min).The highly polar glucuronide and sulfate conjugates appear to be bothfiltered at the glomerulus and actively secreted by the tubules since their renalclearances are approximately 130 and 170 ml/min, respectively. The clearance ofthese metabolites is independent of changes in urine flow and pH (Forrest et al.,1982). These two metabolites accumulate in subjects with impaired renalfunction.1.2.7 ADVERSE EFFECTSThe side effects of APAP may include hepatotoxicity, nephrotoxicity, skinrash, other allergic reactions, neutropenia, thrombocytopenia, and hypoglycemiccoma in overdose. In adults, hepatotoxicity may occur after ingestion of a single3 7dose of 10 to 15 g (150 to 250 mg/kg) of APAP; doses of 20 to 25 g or more arepotentially fatal. Clinical signs of liver damage become apparent in 2-4 days.Perhaps 10% of poisoned patients who do not receive specific treatment developsevere liver damage; of these, 10 to 20% eventually die of liver failure. Severe liverdamage occurs in 90% of patients with plasma concentrations of APAP greaterthan 300 A g/ml at 4 hours or 45 A g/ml at 15 hours after the ingestion of the drug.Minimal hepatic damage can be anticipated when the drug concentration is lessthan 120 A g/mL at 4 hours or 30 A g/ml at 12 hours after ingestion (Prescott,1983; Insel, 1990).Early diagnosis is vital in the treatment of APAP overdoses.Decontamination in overdose cases include gastric lavage and emesis.Supportive therapy may be necessary. N-acetylcysteine, cysteamine, ormethionine given within 10 hours can prevent impeding liver toxicity. N-acetylcysteine is now the preferred therapy. These antidotes are believed to actprimarily by stimulating GSH synthesis and hence facilitate GSH conjugation ofNAPQI.1.2.8 FUTURE DEVELOPMENTSFuture studies need to focus on the time course of the hepatotoxic eventscaused by APAP to determine what kind of intervention may be appropriate atparticular time periods in the treatment of APAP poisoning. Ideally, anonhepatotoxic APAP analogue could be developed. It is unlikely that APAP,which is a relatively safe and inexpensive analgesic and antipyretic, will bereplaced unless an analog can be developed with greater anti-inflammatoryactivity that retains the non-gastrointestinal irritant properties of APAP (Nelson,1990).3 81.3 DRUG INTERACTIONS AND OBJECT OF STUDYA significant change in the magnitude and/or duration of action of onedrug (index drug) by concomitant or prior use of a second drug is defined as adrug interaction. The interaction can be desirable, adverse, or insignificant.Adverse drug interactions account for about 0.2% of all in-hospital adverse drugreactions (Wright, 1992).Drug interactions involve either pharmacokinetic or pharmacodynamicmechanisms. Pharmacokinetic interactions include many distinct processes suchas drug absorption, distribution (including protein binding), hepatic metabolism,and renal excretion. Among these, modifications of hepatic metabolism appear toconstitute the major source of drug interactions. The consequences of metabolicdrug interactions are often difficult to predict, especially if a number of differentpathways are involved.Hepatic drug interactions are usually the result of inhibition or induction ofliver enzymes. Most inhibitory interactions that are of any clinical significanceinvolve the microsomal oxidative enzymes. These interactions are not alwayspredictable because there are many different mechanisms of inhibition and alsobecause some inhibitors can also cause induction under certain conditions (e.g.chronic ethanol, isoniazid, etc.). Some drugs that inhibit P450s includecimetidine, disulfiram, acute ethanol, erythromycin, INH, ketoconazole, oralcontraceptives, etc. Also, there are important interindividual variations in bothP450-dependent catalytic activities and amounts of enzymes in human livermicrosomes (Guengerich, 1989).The selective induction of a subset of the reactions catalyzed by the P450monooxygenases suggested that distinct forms of P450 are induced by specificinducers. Studies carried out with laboratory animals have revealed the existenceof a multigenic superfamily (Nebert & Gonzalez, 1987) and made possible the3 9classification of the inducers into 5 distinct groups, according to the form(s) ofP450 specifically induced: i) polycyclic aromatic hydrocarbons (1A), ii) PB (2B),iii) chronic ethanol, INH, acetone, benzene, ether, imidazole, pyridine (2E), iv)glucocorticoids such as PCN and macrolide antibiotics, and AMP (3A), and v)clofibrate (4A).Induction is sometimes associated with de novo synthesis of the P450(s).Other possible mechanisms, such as protein stabilization, are discussed by Zandet al. (1992). P450 enzyme induction by xenobiotics can be species-specific aswell as tissue-specific (Alvares & Pratt, 1990). Some drugs can induce their ownmetabolism (autoinduction). Some enzyme inductions make it necessary to forthe clinician to increase the dose of the index drug appropriately. Some enzymeinductions are harmful. For example, carbamazepine induces the microsomalmetabolism of INH and increases formation of hepatotoxic INH metabolites(Wright, 1992; Wright eta!., 1982, 1984; Barbare eta!., 1986).1.3.1 THE EFFECT OF INH ON DRUG METABOLISMINH is involved in a number of complex drug interactions. INH can inhibitor induce the metabolism of many other drugs. P450 Inhibition and Induction by INHINH inhibits the microsomal metabolism of carbamazepine (Wright et al.,1982), phenytoin (Kutt eta!., 1970), APAP (Epstein et al., 1991; Zand et al., 1992),theophylline (Samigun et al., 1990), benzodiazepines (Ochs et al., 1981, 1983),and valproic acid (Dockweiler eta!., 1987; Jonville eta!., 1991). Higher than usualdoses of INH have been reported to inhibit the metabolism of warfarin (Baciewicz& Self, 1985). The interactions of INH with carbamazepine and phenytoin areclinically important, the former is actually a double-drug interaction. The4 0interactions involving carbamazepine, phenytoin, theophylline, valproic acid, andwarfarin are more important in slow acetylators of INH.The microsomal enzyme inhibitory action of INH may be due to a metabolicintermediate of INH that forms a complex with the enzyme. This action of INHaffects many P450 enzymes and appears to be nonspecific (Muakkassah et al.,1981; Moloney eta!., 1984; Zand eta!., 1992). The major routes of elimination ofphenytoin and carbamazepine are 4-hydroxylation (P450 209; Veronese et al.,1991) and 10,11-epoxidation (P450 3A3/4; Kerr eta!., 1991), respectively. P4502C9 also 7-hydroxylates S-warfarin; this is the major route of inactivation of thisanticoagulant in humans (Gonzalez, 1992). Theophylline is oxidized by P4501A1/2 (Sarkar et al., 1992). INH seems to inhibit both microsomal oxidativepathways of APAP, namely P450 1A2 and 2E1 (Zand et al., 1992). In humans themicrosomal metabolism of APAP by P450 3A4 is also likely to be inhibited by INH.Triazolam and diazepam are metabolized by P450 3A4 (Kronbach et al., 1989)and the P450 2B/3A family (Inaba et al., 1988), respectively, in humans. Thesestudies suggest that INH does not selectively inhibit one specific P450 form; itinhibits many isoforms.Studies in experimental rats have shown that INH induces hepaticmicrosomal P450 2E1 (Ryan et al., 1986) and potentiates the hepatotoxicity ofAPAP (Burk eta!., 1990) and halothane (Rice eta!., 1987) in rats and halothane inguinea pigs (Lind et al., 1991). INH also induces the same P450 isozyme inrabbits and it induces enflurane defluorination in humans (Mazze et al., 1982;Hoffman et al., 1989). In these studies, INH administration was stopped beforethe administration of the other drugs.411.3.1.2 The effect of INH on APAP MetabolismWe previously reported that INH inhibits the oxidative metabolism of APAP(Epstein et al., 1991). The effect of INH on the metabolism of APAP is actuallyquite complex (Zand et al., 1992). Chlorozoxazone, a drug metabolized primarilyby P450 2E1 (Peter et al., 1991), was used to test the inductive effect of INH onthis isozyme. Apparently when INH is present in the body it stabilizes P450 2E1and inhibits its catalytic activity, but when INH is eliminated from the body there isinduction of this P450 isozyme. Consequently, depending on the condition, INHinhibits or induces the toxic pathway of APAP metabolism.In fact, cases of APAP hepatotoxicity have been reported following theingestion of long-term INH and high doses of APAP (Moulding et al., 1991;Murphy et al., 1990). The levels of INH and its metabolites were not measured inany of these cases. Murphy et al. (1990) report an individual who had takenabout 11.5 g of APAP 12 hr after a dose of INH. The hepatotoxicity was mostlikely due to induction of microsomal P450 2E1 metabolism of APAP by isoniazid.It is unlikely that the high dose of APAP per se could have caused the liverdamage; in one of the cases the level of APAP in blood was reported, and thatlevel is generally not associated wih hepatotoxicity.1.3.2 THE EFFECT OF OTHER DRUGS ON INH METABOLISMThe effects of carbamazepine, ethanol, and procainamide on themetabolism of INH are discussed in section THE EFFECT OF APAP ON DRUG METABOLISMAt doses of 1.6 g/d or more, APAP may inhibit the microsomal oxidativepathway of warfarin, but this has not been substantiated (Boeijinga et al., 1982;Bartle & Blakely, 1991). APAP competitively inhibits the sulfation of ethinyl4 2estradiol, an oral contraceptive (Rogers et al., 1987). APAP might alsocompetitively inhibit the glucuronidation of chloramphenicol (Spika & Aranda,1987; Stein eta!., 1989).1.3.4 OBJECTIVE: THE EFFECT OF APAP ON INH METABOLISMThe interaction between APAP and INH is of clinical interest for a numberof reasons: i) each medication can cause liver injury under conditions which canbe altered when other drugs are taken concomitantly (Mitchell et al., 1976; Black,1984; Nelson, 1990), ii) the 2 drugs are likely to be taken concurrently becauseTB patients ingest INH chronically for many months and APAP is a majornonprescription analgesic-antipyretic medication, and iii) after many years ofdecline, TB cases have been rising since 1985. The excess cases were mostlikely caused by the increased risk of TB in persons infected with the humanimmunodificiency virus (HIV). Widespread undernutrition and the spread of HIVare likely to worsen TB morbidity in the future. The treatment of mycobacterialinfections will become an even more important and challenging problem (CDC,1989; Starke, 1989). If these trends continue, combinations of INH/APAP couldbecome even more common in the near future.The effect of APAP on the risk of INH-induced hepatotoxicity and INHmetabolism is currently unknown (Epstein eta!., 1991). The aim of this study wasto examine the effect of a single therapeutic oral dose of APAP on INHmetabolism.4 32 METHODS2.1 MATERIALS2.1.1 COMMERICALLY OBTAINED SUPPLIESINA was obtained from Eastman Organic Chemicals (Rochester, NY, USA),AcHz (95% pure), m-anisaldehyde (3-methoxybenzaldehyde), and p-chlorobenzaldehyde were purchased from Aldrich Chemical Co. (Milwaukee, WI,USA). Hydrazinium sulfate (Hz-sulfate) and 9-fluorenone were obtained fromFisher Chemical Co. (Fair Lawn, NJ, USA). N,N'-diacetylhydrazine waspurchased from ICN Pharmaceuticals (Plainview, NY, USA).All the hygroscopic compounds were dried in the dessicator before making upstandard solutions. INH, dichloromethane, HPLC grade acetonitrile andmethanol, propan-/-ol, formic acid, acetic acid anhydride, diethyl ether and allacids and bases were obtained from British Drug House (BDH) ChemicalsCanada Ltd. The mobile phases were filtered through a 0.2A m cellulose acetatefilter (Micron Separations, Inc., Westboro, MA, USA) prior to use. The urinesamples were filtered with 0.45A m filters prior to use (Millipore Corp., Bedford,MA, USA). Sulfamethazine required for the determination of acetylator phenotypewas obtained from Mattheison, Coleman and Bell Co.2.1.2 SYNTHESIS OF REFERENCE COMPOUNDSAcINH was semi-synthesized by the method of von Sassen et al. (1985),wherein INH was reacted with a four-fold excess of acetic acid anhydride at roomtemperature for 1.5 hr with continuous stirring. After recrystallization frommethanol-diethyl ether (1:4), colorless and needle-like crystals were obtained witha melting point of 164° C (uncorrected). The literature value of the melting point is162-163° C; von Sassen et al. obtained 157-160° C. HPLC analysis of the AcINH4 4showed that contamination by INH, AcHz, or Hz was below the level of detection(ca. 0.2-0.4% at the detector sensitivity used).PropionylINH (Pcl NH, 1-isonicotiny1-2-propionyl-hydrazine, C9Fl1 1 N302)was synthesized by a modification of the method of von Sassen et al. (1985) forpropionylhydrazine. PcINH was synthesized by reacting a four-fold excess of themethyl ester of propionic acid with INH. The reaction was allowed to proceed for12 hr at room temperature with continuous stirring. The excess methylpropionate was removed by vacuum distillation. PcINH was recrystallized indichloromethane/n-hexane (2:1, v/v). The melting point of the final product was133-138°C, which is close to the literature values of 130.5-131.5°C (von Sasseneta!., 1985).ING was prepared by a slight modification (Gardner et a/. (1954) of theoriginal method (Rohrlich, 1951) for the preparation of nicotinylglycine. Fox &Field (1943) had previously reported a similar method for the preparation ofnicotinylglycine. These methods used the acid azide method. One normal NaOHwas used in place of 0.1N NaOH, and 1M HCI was subsequently used to achieveneutralization. The product was recrystallized a few times from water/ethanol(8:2, v/v) mixture, melting point 220° C (uncorrected) with decomposition. Variousmelting points have been reported in the literature: 230-232° C (Gardner et al.1954), 224-225° C (corrected) with decomposition (Boxenbaum et al., 1974), and256-258° C (El-Naggar et al., 1985). HPLC analysis showed that no INA wasdetected (or very little less than 1%). We were not able to synthesize ING by theacid chloride method of El-Naggar et al. (1985). However, Kubo et al. (1990)reported that they synthesized ING from isonicotinyl chloride and glycine.4 52.2 PROTOCOL2.2.1 SUBJECTSThe subjects, acetylator phenotyping, 24 hr urinary creatinine excretion,and clinical laboratory measurements are described in the first part of this study(Epstein eta!., 1991).2.2.2 ADMINISTRATION REGIMENIsoniazid (300 mg p.o.) daily was administered for 7 days. Acetaminophen(500 mg p.o.) was given on the day prior to starting isoniazid (day 0) and on day 7of isoniazid administration. Twenty-four hour urine samples were collected over 3g of ascorbic acid and maintained at 4° C, on days 0, 6, and 7. Sample volumeswere measured and small amounts were stored at -70° C until analysis.2.3 HPLC ANALYSISVarious analytical procedures have been used for quantitative analysis ofINH and its metabolites in blood and urine. These include colorimetric,fluorometric, microbiological, radioimmunological, mass fragmentography andmore recently gas chromatographic and liquid chromatographic assays.INH exhibits a number of color reactions with many colorimetric reagents.Colorimetric techniques are relatively insensitive and cumbersome. Fluorometrictechniques are more sensitive than colorimetric methods; both methods havebeen used to measure INH only. Microbiologic procedures, although highlysensitive, are too time consuming to perform, and this method does notdifferentiate INH from its hydrazones.Gas chromatographic (Timbrell et al., 1977b; Noda et al., 1978) or high-performance liquid chromatographic (Lauterburg et al., 1981; Holdiness, 1982;Blair et al., 1985; von Sassen et al., 1985; Svensson eta!., 1985; Jenner & Ellard,4 61987) procedures alone, or in combination with mass spectrophotometry, enableINH and its metabolites to be measured simultaneously. The advantages anddisadvantages of various methods have been reviewed (Holdiness, 1985).2.3.1 COLUMNINH and all of its metabolites were separated by HPLC using a reversed-phase column (125 X 4.6mm, i.d.) packed with Spherisorb 5/./ m ODS 2 (PhaseSeparations Ltd., Norwalk, CT, USA).2.3.2 ASSAYS OF INA AND ING2.3.2.1 Preparation of AssayPcINH was the internal standard. The urine samples were filtered withMillipore filters, portions were aliquoted, PcINH was added, and the samples werediluted 40-fold with deionized water (water-l). Apparatus and Chromatographic ConditionsSample volumes of 40 Al were injected per analysis. Ion-pair HPLC wasused to analyze the two metabolites. The prepared samples were elutedisocratically at ambient temperature (approximately 22° C) with a mobile phasethat consisted of 22% v/v acetonitrile, 3% v/v glacial acetic acid, 1 mM each SDS,triethylamine, and H3PO4 made up with deionized water (water-I). The flow ratewas 0.8 ml/min (ca. 1200 psi). The mobile phase was filtered and then degassedin situ by helium. The two metabolites were analysed using a Waters Model M-6000A chromatography pump (Waters Associates, Milford, MA, USA) equippedwith a CE 212 variable wavelength UV detector (Cecil Instruments, Cambridge,UK), connected to an Apple Ile microcomputer; a chromatography program was4 7used (Interactive Microware, Inc., State College, PA, USA) to measure peakheights and/or areas. Quantification was based on uv absorbance at 267 nm.2.3.3 ASSAYS OF ACETYLHYDRAZINE, INH, AND HYDRAZINE2.3.3.1 Preparation of AssayThe derivatization technique was previously developed in our laboratory(Wall, personal communication). The derivatizing reagent was made up asfollows: 250 ilL of 3-methoxybenzaldehyde, 5 ml of 10mM 9-fluorenone solution(made up in propan-l-ol), and 3.75 ml of formic acid (minimum assay 88%) wereput in a volumetric flask and made up to 50 ml with propan-/-ol. The final solutionconsisted of 41.1mM m-anisaldehyde (i.e. excess concentration), 1mM 9-fluorenone, and 7.5% formic acid (v/v). The solution was transfered to a darkbottle and kept in the dark when not in use.The urine samples containing INH and its metabolites were dilutedappropriately and then filtered. The urine solution and the derivatizing reagentwere mixed in the ratio of 4:1 (e.g., 200 p I of INH solution was put in a plastic vial,and 50 th I of the reagent was added, and the vial was inverted a few times). Thereaction was allowed to occur for 2 hr at room temperature in the dark. The acidpresent in the derivatizing reagent set the urine pH to 3 so that the a -ketoacidhydrazones of AcHz and INH, and the a -ketoacid azines of Hz could be readilyhydrolyzed. The AcHz, Hz, and INH released in these reactions then reacted with3-methoxpenzaldehyde to form N-acetyl-3-methoxybenzalhydrazone, 3-methoxybenzaldiazine,^and^isonicotiny1-3-methoxybenzaldhydrazone,respectively (Fig. 5).4 8Figure 5. The derivatization of AcHz, INH, and Hz with m-anisaldehyde (3-methoxybenzaldehyde). The reactions were allowed to occur at pH 3 for 2 hours atroom temperature. Because m-anisaldehyde and 9-fluorenone are bothphotosensitive, the reaction vessels were kept in the dark.49Incubation of samples at pH 3 for 2 hr led to the hydrolysis of virtually allthe a -ketoacids, and probably most of the azines. The azines of Hz are a littlemore stable than the hydrazones of INH and AcHz. A vast excess quantity of m-anisaldehyde made it very unlikely for the free a -ketoacids to react with INH andAcHz again (Wall, personal communication).The unreactive compound, 9-fluorenone, served as an internal check onvolumetric accuracy. The derivatized products were analyzed with the HPLCsystem described below. Sample volumes of 80 g I were injected per analysis. Apparatus and Chromatographic ConditionsAcHz, INH, and Hz were separated by using a 125mm-length column. Thederivatized products were analysed using a Spectra-Physics SP8000B liquidchromatograph (Santa Clara, CA, USA) fitted with a Spectra-Physics SP8400uv/vis detector set at 300 nm. A gradient system was set up. The oventemperature was 48° C, and the flow rate was 1 ml/min.Mobile phase A consisted of 20% MeCN/ 10% Me0H solution (unbuffered,for cleaning column). Mobile phase B consisted of 20% MeCN/ 10% Me0H, and5mM Sodium acetate. The stock solution of sodium acetate was 1M (pH =5).Mobile phase C consisted of 40% MeCN/ 40% Me0H, and 5mM sodium acetate.The solutions were made up with water-I, and the buffered solutions were filteredbefore use and degassed in situ. The HPLC gradient for the 125mm-column was:Time (min.) %B %C0 100 05 60 4010 0 10016.66 0 10017.5 100 05 02.3.4 ASSAYS OF ACETYLISONIAZID AND DIACETYLHYDRAZINE2.3.4.1 Preparation of AssaySome of the urine samples were diluted with blank urine. The sampleswere then filtered as usual. The procedure used to prepare samples involved 3steps.First, the AcHz and INH that were present in the urine samples werederivatized with p-chlorobenzaldehyde to form hydrazones which were thenextracted with CH2Cl2. The procedure is now discussed in more detail. Thederivatizing reagent was a propan- /-ol solution containing 41.09mM p-chlorobenzaldehyde and 7.5% (v/v) formic acid. One ml of this derivatizingreagent and 1 ml of sample urine were added to a 15mL eppendorf glass testtube (reaction vessel), which was capped and inverted a few times. The reactionwas allowed to occur at room temperature in the dark for 2 hr.The derivatization reaction allowed AcHz and INH (present in the reactionvessel) to react with p-chlorobenzaldehyde to yield hydrazones. Thesehydrazone products were then eliminated by extraction with CH2Cl2. CH2Cl2 (4ml) was added to the solution 3 times and each time most of the organic portion(bottom layer) was carefully removed with Pasteur capillary pipets. The residualCH2Cl2 was blown off with a stream of nitrogen gas. AcINH and DiAcHzremained in the aqueous layer.Second, the AcINH and DiAcHz remaining in the aqueous layer were thenconverted to INH and AcHz, respectively, by partial acid hydrolysis. In partial acidhydrolysis a fraction of the total amount of AcINH and DiAcHz gets hydrolyzed. Incomplete acid hydrolysis, all of the AcINH and DiAcHz molecules would gethydrolyzed. Partial acid hydrolysis was done by adding 100 A I of concentratedHCI (12.1M) to the reaction vessel (1.1M HCI in the reaction mixture), the reaction5 1vessels were capped, and then placed in a hot water bath. The reaction vesselswere heated at 80°C for 10 min. The reactions are shown below:Heating for a longer period of time (or a higher HCI concentration, or ahigher water bath temperature) would hydrolyze all of the AcINH and DiAcHz.However, it was not necessary to do this since proper standard solutions werealso prepared at the same time.At the end of the 10 minutes, the hydrolysis reaction was stopped byputting all the reaction vessels in cold water for 1 min. Then 500 p.I of 1.04Msodium citrate was added to each reaction vessel to partly neutralize the solutionand bring the pH up to about 2.9. The solutions were filtered once more, and 200p. 1 portions were aliquoted into 4 or 5 plastic vials and frozen at -70° C for analysisthe next day.Third, each plastic vial was taken out of the freezer and the contents wereallowed to thaw. Then 50 41 of the derivatizing reagent (41.1 mM m-anisaldehyde52and 9-fluorenone in propan-/-ol) was added and the reaction was allowed toproceed for 2 hr. These prepared samples were subsequently analyzed as above(i.e., same as the procedure for INH and AcHz from here on). Apparatus and Chromatographic ConditionsThe same SP8000B system was used to analyze hydrolysis products ofAcINH and DiAcHz.2.4 CALCULATIONSPeak height ratios were calculated by dividing the peak heights of INA andING by that of the internal standard (PcINH), and the peak heights of AcHz, INH,Hz, AcINH, and DiAcHz by that of 9-fluorenone (unreactive internal standard).Calibration curves were constructed by plotting peak height ratios versus theconcentration of the respective compound (II M). The concentrations of INH andits metabolites were calculated from the standard calibration plot using the slope(y =mx) obtained by a linear regression analysis.Quantification of all the compounds was achieved by comparison of theirpeak heights with that of their respective internal standards.2.5 STATISTICAL ANALYSISThe parametric two-tailed paired-sample t test was used (Zar, 1984). Foreach drug or metabolite analysed, the differences were calculated within each pairof measurement (days 6 and 7) for each subject. P < 0.05 was accepted as anindication of significance. Single-sample Hotelling T2 statistic was also used (Zar,1984). In addition, boxplots and scatter plots were prepared (Chambers et al.,1983).533 RESULTSThe ten healthy volunteers were between the ages of 23 and 56 years.Five subjects were slow acetylators (2 women and 3 men) and the other five wererapid acetylators (3 women and 2 men). All subjects completed the study withoutadverse effects. The data from all subjects were analyzed and comparison of INHmetabolism was made on days 6 (INH) and 7 (INH + APAP). Table 1 shows thesubject acetylator phenotypes, sex, and urine sample numbers.3.1 RESULTS OF INA AND INGFig. 6 shows sample chromatograms of INA and ING. The retention times(tR) and absorbance units full-scale (AUFS) are also shown. AcINH was alsoanalyzed by this method. However something present in the urine (most likelyascorbic acid, as explained below) interfered with the separation of AcINH.Therefore, AcINH was analyzed by another method.All urine samples (blanks, etc.) were diluted 40-fold with deionized waterbefore analysis. Each diluted sample contained 10 pg/m1 (51 /.1 M) PcINH (internalstandard). The standards (for calibration plots) were made up in deionized water.Figure 6 (A) shows the chromatogram of a blank urine that does notcontain any INH, APAP, or ascorbic acid (sample 148M); PcINH (peak 4) has aretention time (tR) of 12.1 min.Figure 6 (B) displays a chromatogram of a urine sample from day 0 (APAPwas taken, urine was collected over ascorbic acid). The arrow will be discussedlater. Peak 4 is that of PcINH. Peak 5 (tR =9.84 min) belongs to either ascorbicacid, or a metabolite of APAP; this peak does not appear in chromatogram A(where the subject took no APAP, and the blank urine contained no ascorbicacid).5 4Figure 6 (C) shows the chromatogram of a standard solution containing20 AM each of INA (peak 1, tR =3.89 min), AcINH (peak 2, tR =9.40 min), and ING(peak 3, tR = 10.8 min). The solution also contained 10 A g/ml of PcINH (peak 4,tR =12.1 min). All the four peaks were resolved well.Figure 6 (D) displays the chromatogram of a urine sample from day 6(219M). The subject had taken INH only, and the urine was collected overascorbic acid. INA has a low retention time because its peak shows up just afterthe peaks of unretained compounds. The Arrow (chromatogram B) points to theposition where the peak of INA would appear. Therefore, the peaks of unretainedcompounds do not interfere too much with the peak of INA. Peak 5 ( please seechromatogram B) appears again; the reason is because the AcINH peak has ashoulder. Also, peak 5 starts at about 9.70 min and ends about 9.98 min. Thepeak of AcINH normally ends at about 9.93 min. Therefore the 2 peaks interferewith each other. For some reason, whenever there is such an interference, the tRof AcINH goes down. The the tR of AcINH is 9.40 min in chromatogram C(standard solution) and its about 8.9 min in chromatogram D (INH ingested,sample collected over ascorbic acid). Therefore, it seems likely that peak 5belongs to ascorbic acid. The tR's of INA, ING, and PcINH are very similar tothose in chromatogram C.Figure 6 (E) shows the chromatogram of a urine sample from day 7(246M). The subject had taken INH + APAP, and the urine was collected overascorbic acid. As expected, there is interference between peaks 5 (ascorbicacid?) and 2 (AcINH). The tR's of INA, ING, and Pc1NH are very similar to those inchromatograms C and D.For the above reasons, the results of AcINH were considered unreliable.Furthermore, the standard plot of AcINH was not linear above 20 A M; above this55concentration, the plot leveled off. Therefore, AcINH was analyzed by the acidhydrolysis method.Figures 7 and 8 show the standard calibration plots for INA and ING. Thepeak height ratio (PHR) is the ratio of the chromatogram peak height of themetabolite to that of an internal standard, PcINH. The points represent mean ±s.e.Table 2 displays summaries of the urinary recoveries of INA and ING (as %of total INH dose). The recovery of each metabolite (as % of total INH dose) wascalculated as follows:% dose = Moles metabolite ÷ MOleS INH ingested X 100%.Moles IIVH ingested =0.3 g ÷ 137.15 (F.W. INH).Moles metabolite =Peak Height Ratio ÷ Standard Slope X Total volume of 24-hr urine(I) X 40 (dilution factor) X 10-6 (to convert from p, moles to moles).For example, % of dose for INA of subject ME (day 6) is calculated asfollows: 0.352 (Peak Height Ratio) ÷ 0.0418 (slope, Fig. 7) X 1.136 I (total urinevolume, Table 1) X 40 X i064- (0.3 g ÷ 137.15 Daltons) X i00%= 17.5% (Table2).56Table 1. Summary of acetylator phenotype (R =rapid, s=slow), sex, and samplenumbers of the 10 subjects from days 0, 6, and 7. Blank urine samples, 003E and 148M,are not included in the table. The volumes shown are for 24-hr urine collections.Subject Sex AcetylationDay 0(APAP )Day 6(INH )Day 7(INH+ APAP)Sample Volume (I)No.Sample Volume (I)No.Sample Volume (I)No.ME F s 024E 1.640 063E 1.136 077E 0.550JW M s 023W 2.258 064W 2.746 081W 2.815JL F R 074L 1.540 112L 2.060 135L 1.527DW M R 091DW 0.715 138DW 0.847 158DW 1.715AC M s 139AC 3.265 186AC 3.955 251AC 3.920MM F R 160M 0.840 219M 1.675 246M 1.060DC M s 235DC 1.580 277DC 2.595 295DC 1.785TZ F R 241Z 1.275 289Z 0.905 313Z 1.118MC F s 252MC 1.673 290MC 0.465 330MC 1.210SC M R 2695C 0.845 3125C 0.748 3355C 0.79557Figure 6. Chromatograms of urine samples analyzed at 0.5 A.U.F.S. A) 148M(blank), B) 160M (day 0: APAP), C) standard solution, D) 219M (day 6: INH),and E) 246M (day 7: APAP + INH). The compounds and their correspondingretention times (tR) are INA (peak 1, tR =3.89 min), AcINH (peak 2, tR discussedin text), ING (peak 3, tR =10.8 min), PcINH (peak 4, tR= 12.1 min) and interferingcompound (peak 5, tR =9.84 min). Please see text for details.58Figure 7. Standard calibration plot for isonicotinic acid (INA). Each pointsrepresent the mean (n =3) ± s.e.5 9Figure 8. Standard calibration plot for isonicotinylglycine (ING). Each pointsrepresent the mean (n =3) ± s.e.60Table 2. Urinary recoveries (% of total dose of INH) of INA and ING on days 6 (INH) and7 (INH + APAP). Values are the means (± S.E.M.) of a number of measurements (n).Subject Sex Acetylation Day n INA INGMean + S.E. Mean + S.E.ME F s 6 4 18 ±0.33 9.0 ±0.837 3 21 + 0.21 9.6 + 0.21JW M s 6 3 18 + 0.18 9.5 + 0.287 3 18.+ 0.18 11 + 0.11JL F R 6 3 24 + 0.13 16 + 1.17 4 22 + 0.48 14 +0.56DW M R 6 3 19 + 0.35 11 + 0.207 4 24 + 0.42 14 + 1.3AC M s 6 4 19 + 0.52 15 + 0.917 3 18 +0.57 16 +0.64MM F R 6 3 25 + 0.36 16 ±0.837 3 32 ±0.37 18 ±0.23DC M s 6 3 20 + 0.79 13 + 0.777 3 16 + 0.40 12 +0.42TZ F R 6 4 24 ±0.52 8.5 + 0.297 3 23 +0.18 7.3 +0.43MC F s 6 3 12 + 0.17 7.6 + 0.237 3 18 ±0.42 12 + 0.55SC M R 6 4 15 + 0.32 7.8 + 0.237 4 23 + 0.25 12 + 0.35613.2 RESULTS OF INH, ACHZ, AND HZFig. 9 shows sample chromatograms of the derivatized hydrazones ofAcHz and INH, and the derivatized azine of Hz. The retention times (tR) andabsorbance units full-scale (AUFS) are also shown.Figures 10, 11, and 12 show the standard calibration plots of thehydrazones of INH and AcHz, and the azine of Hz, respectively. The peak heightratio is the ratio of the chromatogram peak height of the derivatized metabolite tothat of an internal standard, 9-fluorenone (9-Fl). The points represent mean ± s.e.Table 3 displays summaries of the urinary recoveries of INH, AcHz, and Hz(as % of total INH dose). The recovery of INH and each metabolite (as % of totaldose of INH) was calculated as above. The dilution factors ranged from 1.33 to10.6 2Figure 9. Chromatograms of standard solutions and retention times (tR) of thederivatized hydrazones of AcHz (1, tR =6.92 min) and INH (2, tR =7.76 min), andthe azine of Hz (4, tR=40.4 min). 9-fluorenone (3, tR =16.5 min) is a volumetricinternal standard. The large peak (located between peaks 2 and 3) belongs tom-anisaldehyde, the derivatizing compound. A sample chromatogram (0.32A.U.F.S.) is shown on the left (sample 077E). The chromatogram (0.08 A.U.F.S.)of a solution containing 20 M N-acetyl-3-methoxybenzaldhydrazone (a measureof AcHz), 20 j M isonicotiny1-3-methoxybenzaldhydrazone (a measure of IN H),and 10 u M 3-methoxybenzaldiazine (a measure of Hz) is displayed on the right.The chromatogram of AcINH and DiAcHz is the same as that of the hydrazones ofINH and AcHz, respectively.y .0315xr=0.99980^50^100^150^200Conc. INH, pM63250Figure 10. Standard calibration plot for isonicotiny1-3-methoxybenzaldhydrazone(a measure of INH). Each point represents the mean (n=3) ± s.e.6 4Figure 11. Standard calibration plot for N-acetyl-3-methmrybenzaldhydrazone (ameasure of AcHz). Each point represents the mean (n=3) ± s.e.0.00a 2 4 6 80.300.200.10y=.0337x + 0.0211r=0.9990Conc. Hz, pMFigure 12. Standard calibration plot for 3-methoxybenzaldiazine (a measure ofHz). Each point represents the mean (n=3) ± s.e. The intercept is most likelydue to contamination; a standard sample contained AcHz, INH, and Hz. TheAcHz was contaminated with a small amount of Hz.6566Table 3. Urinary recoveries (% of total INH dose) of INH, AcHz, and Hz on days 6 (INH)and 7 (INH + APAP). Values are the means (± S.E.M.) of a number of measurements(n).Subject Sex Acetylation Day n INH AcHz HzMean + S.E. Mean + S.E. Mean + S.E.ME F s 6 3 20 + 0.30 3.7 + 0.053 0.70 + 0.0307 3 31 + 0.42 5.0 + 0.044 0.74 + 0.039JW M s 6 3 26 + 0.15 3.8 + 0.10 0.70 + 0.0127 3 28 + 0.53 4.1 + 0.082 1.1 + 0.027JL F R 6 3 7.8 + 0.076 2.4 + 0.046 0.22 + 0.0827 3 6.5 +0.080 2.0 + 0.067 0.26 + 0.014DW M R 6 3 6.2 + 0.098 1.5 + 0.064 0.30 + 0.00607 3 9.8 + 0.15 1.8 + 0.065 0.64 + 0.0037AC M s 6 3 29 + 0.49 4.7 + 0.082 1.5 + 0.0347 3 26 + 0.68 4.4 + 0.17 0.52 + 0.0085MM F R 6 3 8.6 + 0.23 2.4 + 0.071 0.56 + 0.0127 3 11 +0.23 2.6 + 0.020 0.41 + 0.012DC M s 6 3 23 + 0.57 4.1 + 0.086 0.50 + 0.0187 3 20 + 0.73 2.8 + 0.12 0.34 + 0.017TZ F R 6 3 14 + 0.86 2.7 + 0.21 0.31 + 0.0157 4 13 + 0.19 3.1 + 0.094 0.099 + 0.0064MC F s 6 3 28 + 0.21 3.2 + 0.061 0.38 + 0.00657 3 37 + 2.0 5.7 + 0.35 0.85 + 0.046SC M R 6 3 7.4 -I- 0.042 1.6 + 0.020 0.11 + 0.00407 3 12 + 0.37 2.4 + 0.092 0.42 + 0.0081673.3 RESULTS OF ACETYLISONIAZID AND DIACETYLHYDRAZINEThe chromatogram of AcINH and DiAcHz is the same as that of INH andAcHz, respectively (Fig. 9). As mentioned already, the original INH and AcHz inthe urine samples were eliminated. Then AcINH and DiAcHz were converted to'INH' and 'AcHz', respectively. 'INK and 'AcHz' were then derivatized with m-anisaldehyde and analyzed (the same procedure as that of the orininal INH andAcHz).Because of large inter-day variation in peak heights (due to variations of afew seconds in the length of partial acid hydrolysis) standard plots were set upeach day as the samples of a subject were run. Fig. 13 shows the standardcalibration plots for subject JL. The peak height ratio is the ratio of thechromatogram peak height of the metabolite to that of an internal volumetricstandard, 9-fluorenone (9-Fl). The points represent mean ± s.e. The slopes ofthe calibration plots for AcINH ranged from 7.78X10-3 to 1.00X10-2 (r= 0.9935 to0.9995). The slopes of the calibration plots for DiAcHz ranged from 2.43X10-3 to2.98X10-3 (r= 0.9531 to 0.9992). The dilution factors ranged from 1 to 5. Thefinal results are shown in Table 4. The recovery of each metabolite (as % of totaldose of INH) was calculated as above.Extraction with dichloromethane eliminated essentially all of the originalINH. After extraction, the residual INH peak was not larger than that of thebaseline noise.The hydrolysis of DiAcHz generated AcHz. The concurrent hydrolysis ofAcINH also generated a small amount of AcHz as a by-product. However, thepeak height of this by-product was not larger than that of the baseline noise.• AcINH A DiAcHz3.002.502.00y= .00895xr-0.99951.501.00y=.00281xr=0.99220.500.000^50^100 150 200 250 300 35068Concentration of AcINH or DiAcHz, pMFigure 13.^Standard calibration plots for acetylisoniazid (AcINH) anddiacetylhydrazine (DiAcHz). Each points represent the mean (n=3) ± s.e.69Table 4. Urinary recoveries (% of total dose of INH) of AcINH and DiAcHz on days 6(INH) and 7 (INH + APAP). Values are the means (± S.E.M.) of a number ofmeasurements (n).Subject Sex Acetylation Day n AcINH McHzMean + S.E. Mean + S.E.ME F s 6 4 11 + 0.18 3.6 + 0.0527 3 14.3 + 0.719 5.5 + 0.25JW M s 6 3 24 + 0.32 4.8 + 0.0337 3 24 + 0.26 4.9 + 0.059JL F R 6 3 63 + 1.2 38 + 0.647 4 49 + 0.43 25 + 0.092DW M R 6 3 50 + 1.1 22 + 0.447 4 57 + 1.2 27 + 0.12AC M s 6 4 28 + 0.70 8.1 + 0.117 3 26 + 0.47 6.5 + 0.094MM F R 6 3 32 + 0.83 26 + 0.297 3 38 + 1.4 34 + 1.1DC M s 6 3 38 + 0.53 9.3 + 0.227 3 37 + 0.27 9.5 + 0.033TZ F R 6 4 68 + 0.36 25 + 0.0207 3 60 + 0.84 20 + 0.13MC F s 6 3 24 + 0.38 3.0 + 0.127 3 16 + 0.25 2.8 + 0.11SC M R 6 4 38 + 0.91 13.0 + 0.317 4 62 + 0.72 29 + 0.557 03.4 SUMMARY OF RESULTS AND STATISTICS3.4.1 RESULTS OF PAIRED COMPARISON STATISTICSTable 5 shows a summary of recovery of the sum of isonicotiny (INH,AcINH, INA, and ING) and hydrazide (INH, AcINH, Hz, AcHz, and DiAcHz)compounds. Some recoveries were apparently greater than 100%. This isprobably due to the fact that the standard errors are additive as the % recovery ofvarious compounds were pooled together.The recoveries of the 7 compounds in rapid and slow acetylators aredisplayed in Table 6. The recoveries of all the metabolites for slow and rapidacetylator on day 6 (INH only) are similar to those reported in the literature. Thisstudy confirmed previous findings that the recovery of Hz and AcHz, suspectedhepatotoxins, are greater in slow acetylators than rapid acetylators. The recoveryof all the hydrazide compounds is greater in rapid acetylators (86% recovered)than slow acetylators (61% recovered). This suggests that more of INHundergoes microsomal metabolism in slow acetylators as compared to fastacetylators.The parametric two-tailed paired-sample t test was used to analyze thedata. The differences were calculated within each pair of measurements (day 6 -day 7). The paired-sample t test does not have the normality and equality ofvariances assumptions, but assumes only that the differences, di, come from anormally distributed population of differences. A histogram or stem-and-leafdisplay of the d's can provide a rough check for approximate normality. Oursample number (n =10) is too small for a stem-and-leaf plot. Histograms wereprepared, but it was difficult to tell if the differences were normally distributed.Scatter plots were prepared that displayed distribution of differences. Figure 14displays a log plot for the differences of the sum of isonicotinyl and hydrazidecompounds. In order to do a log plot, the differences were all transformed linearly71by adding 100. Such coding does not change the measures of dispersion, exceptfor the coefficient of variation. Figure 14 shows that it is difficult to tell if thedifferences are normally distributed or not. Alternatively, a nonparametricanalogue to the paired-sample t test (the Wilcoxon paired-sample rank test) couldbe used. This test does not have the assumption of normality of differences, but ithas an underlying assumption that the sampled population is symmetrical aboutthe median (Zar, 1984). Two boxplots (Figs. 15 and 16; discussed later) showthat the data are not symmetrical about the median. Another nonparametric testfor paired samples is the sign test, one of ther oldest statistical procedures.However, the rank test is less powerful than the Wilcoxon test (Zar, 1984). TheWilcoxon test could not be applied because subgroup analysis (slow or rapidacetylators, n = 5) could not be done due to the small sample number. Since, itcould not be assumed that the differences come from a normal distribution, wedecided to do a log transformation of the original data before doing theparametric paired-sample t test. It was decided that if anything was significant bythe parametric test, then it would be backed-up by the Wilcoxon test for n =10 (allsubjects, slow and rapid acetylators).The null hypothesis was that concomitant ingestion of APAP would notalter the recoveries of INH and its metabolites. P < 0.05 was accepted as anindication of significance. There was no need to do Bonferroni corrections sincenothing was statistically significant. Also, the 24-hr urinary creatinine recoveriesdid not change significantly from day 6 (1443 ± 140 mg) to day 7 (1510 ± 110mg).Although treatment with APAP was associated with increased recoveries ofmost metabolites in fast acetylators, the effect was not statistically significant(Table 7). In slow acetylators, treatment with APAP was associated with7 2increased recoveries of some metabolites, but not all. However, the effect wasnot statistically significant.We are allowed to pool the results of the 5 slow and 5 fast acetylators(n=10) and then do a paired comparison. Although the paired comparison t-testis the traditional method of solving this type of problem, Sokal & Rohlf (1987)recommend the two-way anova (paired comparison, randomized completeblocks design with only two treatments (a = 2)). The second test has theadvantage of providing a measure of the variance component among blocks(b = 10). This is useful knowledge because if there is no significant addedvariance component among blocks one might simplify the analysis and design offuture, similar studies by employing a completely randomized anova. Failure toallow for differences among individuals can lead to erroneous results about thedifferences in INH metabolism on days 6 and 7.The two-way anova indicates that the individuals (b = 10) are different fromeach other (i.e., as expected, slow and fast acetylators are different from eachother), but APAP has no effect on the metabolism of INH. However, concomitantingestion of APAP was apparently not associated with changes in INHmetabolism. Table 7 reports the calculated t-values (ts) from the pairedcomparison t-test. It is known that ts2 = Fs (Fs = calculated F-value of two-wayanova paired comparison, i.e., randomized complete blocks design). Therecoveries of INA (19% and 6% increase in fast and slow acetylators, respectively)and ING (8% and 9% increase in fast and slow acetylators, respectively) wereincreased upon concomitant ingestion of APAP, but these effects were notstatistically significant. INA and ING both belong to the same metabolic pathway.When the results of INA and ING are pooled together (n=10), the t-test for pairedcomparisons indicates that APAP administration was not associated with asignificant change in the conversion of AcINH and INH to INA/ING.73Table 5. Summary of recovery of total isonicotinyl (sum of INH, AcINH, INA, and ING)and total hydrazide (sum of INH, AcINH, Hz, AcHz, and DiAcHz) compounds. Thevalues over 100% are most likely due to the additive effects of coefficients of variations.Subject Sex Acetylation Day TotalIsonicotinylsTotal Hydrazides(% of dose) (% of dose)ME F s 6 58 397 76 57JW M s 6 78 597 80 62JL F R 6 110 1117 92 82DW M R 6 86 807 104 96AC M s 6 91 727 85 63MM F R 6 81 707 100 86DC M s 6 94 757 84 69TZ F R 6 115 1107 104 96MC F s 6 71 587 83 62SC M R 6 69 607 108 10574Table 6. Effect of APAP on the hepatic conversion of INH to its metabolites in 5 rapidacetylators and 5 slow acetylators. Values (mean + S.E.) show urinary recoveries (% ofdose) on days 6 (IN H) and 7 (INH + APAP).Compound Rapid Acetylators Slow AcetylatorsDay 6 Day 7 Day 6 Day 7Isoniazid 8.8 + 1.4 10 + 1.1 25 + 1.7 28 + 2.7(INH)Acetylisoniazid 50 + 6.9 53 + 4.4 25 + 4.4 23 + 4.0(AcINH)Isonicotinic acid 21 + 1.8 25 + 1.8 17 + 1.4 18 + 0.84(INA)Isonicotinylglycine 12 + 1.8 13 + 1.8 11 + 1.3 12 + 1.0(ING)Diacetylhydrazine 25 + 4.0 27 + 2.3 5.7 + 1.3 5.8 + 1.1(DiAcHz)Acetylhydrazine 2.1 + 0.25 2.4 + 0.24 3.9 + 0.25 4.4 + 0.48(AcHz)Hydrazine 0.30 + 0.075 0.36 + 0.090 0.75 + 0.19 0.72 + 0.14(Hz)Total isonicotinyls1 92 ±8.8 102 + 2.7 78 + 6.6 82 + 1.7Total hydrazides2 86 + 10 93 + 4.0 61 + 6.3 63 + 2.01 Sum of INH, AcINH, INA, and ING.2Sum of INH, AcINH, Hz, AcHz, and DiAcHz.A+0^ 0••A+7510030Fast (I) Slow (I) Fast (H) Slow (H)Phenotype (compound)Figure 14. Log plot of the distribution of differences (between days 6 and 7) ofthe sum of isonicotinyl (I) or hydrazide (H) compounds in slow or fast acetylators.The raw data were coded by adding 100. The 10 different symbols represent the10 subjects. Each subject is represented by the same symbol in Figure 14, andFigures 17-20.76Table 7. Statistical results. The parametric paired-sample two-tailed t test was used toexamine the effect of APAP on the conversion of INH to its metabolites in slow acetylators(n=5), rapid acetylators (n=5), or all the subjects (5 rapid and 5 slow acetylators, n=10).The values show the calculated t-values. The critical t-values are 2.78 (n=5), and 2.26(n =10). Bonferroni corrections were not necessary since none of the calculated t-valueswere significant.Compound AcetylationAll 10 SubjectsRapid SlowIsoniazid -1.37 -0.96 -1.74(INH)Acetylisoniazid -0.627 0.470 -0.184(Acl NH)Isonicotinic acid (INA) -1.73 -0.448 -1.47Isonicotinylglycine (ING) -0.947 -1.22 -1.22INA/ING -1.48 -0.770 -1.64Diacetylhydrazine -0.604 -0.302 -0.709(DiAcHz)Acetylhydrazine -1.41 -0.590 -1.32(AcHz)Hydrazine -0.368 0.0494 -0.277(Hz)Total Isonicotinyls1 -0.995 -0.780 -1.32Total Hydrazides2 -0.713 -0.621 -0.9791Sum of INH, AcINH, INA, and ING.2Sum of INH, AcINH, Hz, AcHz, and DiAcHz.773.4.2 GRAPHICAL ANALYSIS AND OTHER METHODSSince the results of the paired-sample tests were not significant, attemptswere made to obtain some information from i) boxplots, ii) scatterplots, iii)Hotelling T 2 statistics, and iv) calculations of the power of the tests and samplesizes required. Box PlotsAt this stage of analysis it would be useful to have summary displays of thedistribution. One method of summarization is by preparing box plots. A box plotallows a partial assessment of symmetry. If the distribution is symmetric then thebox plot is symmetric about the median: the median cuts the box in half and theupper and lower vertical lines are about the same length. The upper and lowerquartiles of the data are portrayed by the top and bottom, respectively, of the box.The median is displayed by a horizontal line segment within the box; it shows thecentre of the distribution. The spread of the bulk of the data (the central 50%) isseen as the length of the box. The lengths of the vertical lines relative to the boxshow how stretched the tails of the distribution are (Chambers et al., 1983;Wilkinson, 1990a).Fig. 15 displays box plots of total recoveries of isonicotinyl compounds ondays 6 (INH) and 7 (INH + APAP) in 5 fast and 5 slow acetylators. The data arenot symmetrical about the median. The upper components of the box plot for day6 data (fast and slow acetylators) are stretched relative to their counterpartsbelow the median, revealing that the distribution is skewed to the right.Conversely, the distributions from day 7 are skewed to the left. None of the tails issymmetric. It is also apparent that the distribution of data of fast and slowacetylators are quite different from each other: this is due partly to the fact thatrecoveries of isonicotinyl compounds is greater in fast than slow acetylators78(Table 6). The recoveries of isonicotinyl compounds increase on day 7 comparedto day 6; the increase is more drastic in fast acetylators. The variabilities of theday 7 distributions are drastically small compared to the respective day 6distributions. Thus concomitant APAP ingestion is associated with reduction inthe variation of the distributions. It should be noted that the sample size (n = 5)was small and that we would quite likely see different behavior in larger samplesizes.Fig. 16 displays box plots of total recoveries of isonicotinyl compounds ondays 6 (IN H) and 7 (INH + APAP) in 5 men and 5 women on days 6 and 7. Again,the distributions are not symmetric. The variation of the distribution of the data ofwomen is reduced on day 7 compared to day 6. Interestingly, the correspondingvariation of the data of men is not reduced on day 7. These could be due tochance effects. These could also reflect the fact that the proportions of fast/slowacetylators in men and women were not equal. It is obvious the sex is not a majordeterminant of INH metabolism but acetylation phenotype is (this was also shownby variance statistics, discussed in section box plots of total recoveries of hydrazide compounds (not shown) arevery similar to those of total isonicotinyl compounds. The box plots of individualmetabolites were similar to those of the sums.  Scatter Plots and Variance StatisticsFig. 17 shows scatterplots (log) of total recoveries of isonicotinylcompounds in slow and fast acetylators on days 6 and 7. It is difficult to tell if thedata are normally distributed or not. However, it is obvious that the variations ofthe distributions decrease on day 7 compared to day 6 (as shown also by the boxplots). Fig. 18 confirms Fig. 16: on day 7 the variations of the distributionsdecrease in women but not in men.7 9Fig. 19 shows scatterplots (log) of total recoveries of hydrazidecompounds in slow and fast acetylators on days 6 and 7. Again the variationsdecrease on day 7 compared to day 6. Fig. 20 (like Fig. 18) shows that thevariability of distributions of female data decreases on day 7 but not those ofmales.Two-sample hypotheses were used to test the differences betweenvariances (Zar, 1984). The results of day 6 or 7 were compared: 5 male versus 5female subjects, and 5 slow versus 5 fast acetylators. The test assumes that thedata are normally distributed. As discussed above, we are not able to show thatthe data are normally distributed. As mentioned above, the variation of thedistribution of the data of women looks different on day 7 compared to thecorresponding variation of the data of men. Subgroup analysis showed maleversus female comparisons were not statistically significant (2-tailed test). Butbased on the small sample size, it is difficult to reach a definite conclusion.Previous studies have shown that the recoveries of INH, AcINH, Hz, AcHz,and DiAcHz are different between slow and fast acetylators but not between menand women. It was also shown before that the recoveries of INA and ING aresimilar in fast and slow acetylators, and men and women. Our study confirmsthese previous findings (Table 6). We could test for difference between twomeans but this is only done when the data is normally distributed. Therefore, itmakes no sense to test these since our data do not look normally distributed.  Hotelling Multivariate StatisticsWhen we test simultaneously the hypothesis that several population means donot differ from a specified set of constants, a statistic that considers all variablestogether is required. The multivariate Hotelling's T2 statistic (one-sample test) isusually used for this purpose (Wilkinson, 1990b; Zar, 1984). The test assumes8 0that the several variables are dependent on each other (the recovery of INH andits metabolites are dependent on each other). The differences of the original datawere taken for days 6 and 7. The hypothesis was tested that the differences forall isonicotinyl compounds (or all hydrazide compounds) do not differ from zero(i.e., were equal). The test was also performed for INA and ING since they bothbelong to the same metabolic pathway. In all cases there was no statisticalsignificance.  Power of Test and Sample SizeThe 95% confidence intervals (original data) of the differences (betweendays 6 and 7) were computed for INH and each of its metabolites in slowacetylators, fast acetylators, or slow and fast acetylators combined (n=10). In allcases zero was within the computed interval. Therefore, it is not surprising thatnothing was statistically significant by the paired-sample t test.By considering the paired-sample t test to be a one-sample t test for asample of differences, cli, we can address the questions of minimum detectabledifference, power, and required sample size (Zar, 1984).Table 8 displays the minimum detectable differences based on a samplesize of 10 (5 Slow + 5 fast acetylators), or 5 (5 slow or 5 fast acetylators). Anestimate was made of the smallest difference (i.e., difference between recoveriesof each metabolite on days 6 and 7) that is detectable 80% of the time using asample size of 10 or 5 data and a significance level of 0.05. With a sample size of10, almost all the minimum detectable values are unacceptable. The test coulddetect about a 20% change in the recovery of INH and sum of isonicotinylcompounds with a reasonable degree of accuracy. In the case of Hz, an 80%change in recovery must occur so that the test can detect the change. When onelooks at slow or fast acetylators separately, one expects homogeneity to increase81and variation to decrease. Therefore we expect the minimum detectabledifferences to decrease. However the estimations do not generally support this(probably due to the smaller sample sizes of 5).Table 9 shows calculations of the power of the test. A power of 0.80 isconsidered reasonably good. In agreement with Table 8, the power was 0.80 ormore only in the case of ING and sum of isonicotinyl compounds. In the case ofHz, AcHz, and DiAcHz, there was no power at all.The required sample sizes were also estimated (Table 10). Only ING andsum of the isonicotinyl compounds required a sample size close to 10 (oursituation). If we had equal numbers of slow and fast acetylators, we would needabout 25 subjects to reject the null hypothesis. However proper detection of therecoveries of DiAcHz and Hz would require about 52 and 130 subjects,respectively. Alternatively, we would expect to reduce variability by studying onlyslow or fast acetylators. The required sample size of only slow or fast acetylatorsshould be less than the situation where we have a mixture. However theestimates (Table 10) are not in full agreement; this is probably due to the fact thatthe estimates were based on data of only 5 subjects. It appears that if we had 25slow acetylators, we could make definite conclusions about the study. Theexception is the case of Hz: about 116 subjects may be required to draw a definiteconclusion.----1T-----^I^1^ITT-■•••■■^182120110100co00 90-iz,_ 800cx).706050Fast (6) Fast (7) Slow (6) Slow (7)CLASSFigure 15. Box plots of total recoveries of isonicotinyl compounds in fast andslow acetylators on days 6 (INH) and 7 (INH + APAP). The top and the bottomborders of each box show the 75% quartile and the 25% quartile, respectively, ofthe distribution. The median is shown by the horizontal line segment within eachbox. The data are not symmetrical about the median. In addition, the tails are notsymmetric since the two vertical lines of each box are not of equal length. Thevariations of distributions decrease on day 7 compared to day 6.1^I^I--.■•■--1^1^ITTTI------^83120110100EDCl)o 90-oT7,_ 80ocx)706050F (6)^F (7)^V (6)^V (7)SEXFigure 16. Box plots of total recoveries of isonicotinyl compounds in women (F)and men (M) on days 6 (INH) and 7 (INH + APAP). The top and the bottomborders of each box show the 75% quartile and the 25% quartile, respectively, ofthe distribution. The median is shown by the horizontal line segment within eachbox. The distributions are not symmetric. On day 7 the variation of thedistribution of the data of women is reduced, but not those of men.COoe4084++A_V0•i+Fast (6) Fast (7) Slow (6) Slow (7)Phenotype (day)Figure 17. Scatter plots (log) of total recoveries of isonicotinyl compounds in fastand slow acetylators on days 6 and 7. The variations of distributions decrease onday 7 compared to day 6. The 10 different symbols represent the 10 subjects.Each subject is represented by the same symbol in Figure 14, and Figures 17-20.+a)o•■•■••O 1 00co0-0=z....."6e40+A^ •+^v- oA^0V tl•. 0^ ••+85F (6)^F (7)^M (6)^M(7)Sex (day)Figure 18. Scatter plots (log) of total recoveries of isonicotinyl compounds infemales (F) and males (M) on days 6 and 7. The variations of distributions appearto decrease in women but not men. The 10 different symbols represent the 10subjects. Each subject is represented by the same symbol in Figure 14, andFigures 17-20.g,^100-.7_--0co0-0=Z—0e8630_4•_+0Aao^0iIt^++Fast (6) Fast (7) Slow (6) Slow (7)Phenotype (day)Figure 19. Scatter plots (log) of total recoveries of hydrazide compounds in fastand slow acetylators on days 6 and 7. The variations of distributions decrease onday 7 compared to day 6. The 10 different symbols represent the 10 subjects.Each subject is represented by the same symbol in Figure 14, and Figures 17-20.30oV0o+0A•V087-4+F (6)^F (7)^M (6)^M (7)Sex (day)Figure 20. Scatter plots (log) of total recoveries of hydrazide compounds infemales (F) and males (M) on days 6 and 7. The variations of distributions appearto decrease in women but not men. The 10 different symbols represent the 10subjects. Each subject is represented by the same symbol in Figure 14, andFigures 17-20.88Table 8. Minimum percent change detectable. The minimum value of 8 (the differencebetween the mean values of recoveries on days 6 and 7 for each metabolite), that isdetectable 80% of the time by the paired-sample t test at the a (0.05) level ofsignificance, using a sample of specified size n. A 8 was calculated by the formula below(Zar, 1984). A 5 was then divided by the mean value of each metabolite (day 6) to getthe minimum percent change detectable. The mean % change observed is the % ofincrease (or decrease) of the recovery of each metabolite on day 7 compared to day 6.All Subjects^Rapid Acetylators^Slow AcetylatorsCompound(n=10) (n=5) (n=5)Mean^Min. %% Change^ChangeObserved^able toDetectMean% ChangeObservedMin. %Changeable toDetectMean^Min. %% Change^ChangeObserved^able toDetectINH 14 27 19 51 12 42AcINH 1.6 27 5.7 48 -6.8 27INA 11 22 16 34 4.0 39ING 9.7 20 9.8 36 10 33DiAcHz 7.2 50 8.9 76 1.1 37AcHz 13 34 14 36 12 62Hz 2.9 80 21 42 -4.6 128Total isonicotinyls 7.4 20 10 42 4.2 25(I INH, AcINH, INA,and ING)Total hydrazides 5.9 28 8.1 55 3.1 28(E INH, AcINH, Hz,AcHz, and DiAcHz)s5 =--v71-(tay + towy )s =standard deviation of the differences (day 6 - day 7) of the original datatocy:t 0.05(2),9= 2.26, t 0.05(44=2.78t 0 toy : t o.20(1),9 =0.880, t 0.2o(11,4= 0.94189Table 9. Power of the paired-sample t test. The probability of detecting a truedifference, i.e., a difference between mean recoveries on day 6 and those on day 7 of atleast 20%. The value of 8 (the difference between the mean values of recoveries ondays 6 and 7 for each metabolite), is arbitrarily set to 20%; i.e., 8 is equal to 20% of therecoveries on day 6. a =0.05, n = 10, and t 0(11,9 was calculated as shown below. Thecorresponding p values were found from a statistical table, and the power (1- 0) wascalculated.CompoundMeanRecoveryDay 6 (%INH dose)Calculated8StandardDeviation,sCalculatedt p (1),90 Values(StatisticalTable)Power ofTest(1-0)INH 17 3.4 4.7 0.050 0.48 0.52AcINH 38 7.5 10 0.040 0.48 0.52INA 19 3.9 4.2 0.65 0.26 0.74ING 11 2.26 2.2 0.94 0.17 0.83DiAcHz 15 3.1 7.7 -1.0 NoneAcHz 3.0 0.60 1.02 -0.40 - NoneHz 0.53 0.11 0.42 -1.48 NoneTotal isonicotinyls 85 17 17 0.85 0.20 0.80(E INH, AcINH, INA,and ING)Total hydrazides 74 15 20 0.01 0.50 0.50( I INH, AcINH, Hz,AcHz, and DiAcHz)Jet 4t (30),A, =8 x - - taxSS=standard deviation of the differences (day 6 - day 7) of the original datatax: t 0.05(2),9 = 2.26,90Table 10. Sample size required. We want to be able to detect a difference betweendays 6 and 7 as small as 5 (20%). We wish to test at the 0.05 level of significance with a80% chance of detecting means (day 7) significantly different from day 6, means by aslittle as 20%. The required sample size was calculated by rearranging the equation(Table 9) for n. For all subjects, the estimated minimum sample size was based onn=10 set of values; for slow or rapid acetylators, the estimated minimum sample sizewas based on n =5 set of values.All Subjects Rapid acetylators^Slow AcetylatorsCompound Calculated Minimum^Calculated Minimum^Calculated MinimumSample Size Required Sample Size Required Sample Size RequiredINH 17 21 15AcINH 17 19 8INA 12 11 13ING 10 12 10DiAcHz - 52 - 43 12AcHz 25 12 21Hz -130 -142 -116Total isonicotinyls 11 15 7(E INH, AcINH, INA,and ING)Total hydrazides 18 24 8(E INH, AcINH, Hz,AcHz, and DiAcHz)914 DISCUSSIONWe previously reported that INH inhibits the oxidative metabolism (minorpathway) of APAP (Epstein et al., 1991). A more thorough study suggests thatthe effect of INH on the metabolism of APAP is actually quite complex (Zand eta!.,1992). Depending on the condition, INH inhibits or induces P450 2E1, the majorenzyme that catalyzes the toxic pathway of APAP metabolism.Cases of APAP hepatotoxicity have been reported following the ingestionof long-term INH and high doses of APAP (Moulding eta!., 1991; Murphy eta!.,1990). The hepatotoxicity was most likely due to induction of P450 2E1metabolism of APAP by isoniazid (Zand eta!., 1992).At doses of 1.6 g/day or more, APAP may inhibit the microsomal oxidativepathway of warfarin, but this has not been substantiated (Boeijinga et al., 1982;Bartle & Blakely, 1991). APAP competitively inhibits the sulfation of ethinylestradiol, an oral contraceptive (Rogers et al., 1987). APAP has been reported toincrease, decrease, and have no effect on the clearance of chloramphenicol.Intravenous, but not oral, doses of APAP might competitively inhibit theglucuronidation of chloramphenicol (Spika & Aranda, 1987; Stein et al., 1989).The reasons for the disparate results in the chloramphenicol studies probablyresult from differences in study design, number of subjects, assay techniques, orroute of APAP administration. In addition, the clearance of chloramphenicol hasbeen reported to increase with continuous dosing. The clinical significance of thisinteraction is still speculative.The present study suggests that a low dose of APAP (500 mg) has verylittle effect on INH metabolism. However, due to the small sample size and great9 2variability in the metabolism of INH we can not draw a definite conclusion. APAPadministration was associated with increases in the conversion of INH to some ofits metabolites, but the effects were not statistically significant. This will not resultin an adverse drug interaction. For example, the recoveries of INA and INGincreased, but these two metabolites are relatively nontoxic; the effect of APAP, ifany, represents a clinically inconsequential drug interaction. If APAP does in facthave an effect on the metabolism of INH, we were not able to detect it. A highdose of APAP may alter the metabolism of INH significantly, but it is unethical toconduct such a study because of the higher risk of liver toxicity. Just like the caseof APAP/warfarin, a low dose of APAP may have no effect, whereas a higher dosemight have. There are some possible explanations for the results of this study.Due to our sample size and great variability in the metabolism of INH inhumans, the power of our paired-sample t-test was generally not satisfactory. Inorder to be able to do proper statistics, either the sample size would have to beincreased, or variability decreased; e.g., all slow acetylator or all fast acetylators.Perhaps with a larger sample size (e.g., 21 slow acetylators) the data will benormally distributed. The recoveries of INH and its metabolites in slow acetylatorsare more homogeneous than those in fast acetylators. To increase homogeneity,fast acetylators could be classified into 2 groups with acetylator genotyping.However, as Table 8 (p. 88) shows, this study still provided some usefulinformation. For example, we can be reasonably confident that APAP does notcause a change of 42% or more in the metabolism of INH. Similarly, the urinaryrecovery of AcINH would not be changed by more than 27% (Table 8). We canbe fairly confident that APAP does not cause a change of 50% or more in therecoveries of INH and most of its metabolites in fast or slow acetylators (Table 8).9 3The mean recovery of isonicotinyl compounds is higher by 15-20% in fastacetylators than slow acetylators (Table 6, p. 74). Ideally, the recoveries shouldbe identical in slow and fast acetylators. There are many possible explanations.First, it is possible that more of INH is converted to other isonicotinyl compounds(e.g., DiINH, 4-hydroxymethylpyridine, etc.) in slow acetylators than fastacetylators. This appears to be a reasonable explanation, considering the factthat the levels of INH are higher in the body of slow acetylators compared to fastacetylators. In this study, DiINH and 4-hydroxymethylpyridine were not analyzed.Literature reports suggest almost equal recoveries of isonicotinyl compounds inslow and fast acetylators (Ellard & Gammon, 1976).Second, it is possible that the determinations of AcINH by the acidhydrolysis method were not very accurate. For example, five times out of six themethod could accurately determine the concentration of AcINH present in spikedurine. But one time out of six the determination was off by 14%. It is quitepossible that overestimation of AcINH levels in some urine samples andunderestimation of others have led to different estimates in slow and fastacetylators. If so, then the recoveries of hydrazide compounds are very differentdue to the same reason. This artefact of methodology obviously would havemajor effects on the distributions of the box plots and scatter plots. The secondreason is more likely; this is supported by the fact that the the estimatedrecoveries of isonicotinyl compounds were over 100% for some subjects (Table 5,p. 73).One positive finding of this experiment is that APAP causes a largerreduction in the variability of recoveries (of isonicotinyl compounds) in fastacetylators as compared to slow acetylators (Figure 15, p. 82). If this finding is9 4not caused by any artefact of methodology, then it would suggest an interactionbetween APAP and the enzyme N-acetyltransferase (NAT). High levels of AcINHinhibit NAT. Since APAP has an acetyl group like AcINH, it would be expected toinhibit NAT. Furthermore, the inhibition should be most dramatic in the fastestfast acetylator subject (i.e., the faster the acetylation rate, the more inhibition).However, the results of AcINH recoveries in Table 4 (p. 69) are conflicting. Therecovery AcINH in subject TZ, the fastest of fast acetylators, decreases by 13%when INH and APAP are taken together. The recovery of AcINH in subject JL, thesecond fastest of fast acetylators, decreases by 22%. The correspondingrecoveries in subject DW, the third fastest of fast acetylators, increases by 14%.Therefore, there does not appear to be a trend.Although calculation of intrinsic metabolic clearance is the preferred meansof assessing drug metabolizing enzyme activity in vivo, simpler but indirectmeasures of metabolic capacity are often utilized (Miners et al., 1992). Oneexample is the urinary metabolic ratio. For example, in the study ofcarbamazepine-induced INH hepatotoxicity, it was shown that carbamazepinedecreased the ratios, DiAcHz/AcHz, AcINH/INH, and ING/INA, by 37%, 31% and63%, respectively (Wright et al., 1984). In our present study, the correspondingratios were decreased by 5.4%, 6.7%, and 8.8% in fast acetylators; none of theratios changed that drastically. If APAP had drastic effects, e.g. 50% change inrecoveries of any of INH, AcINH, INA, ING, AcHz, or Hz in fast acetylators, thepaired-sample t-test would have been powerful enough to detect the changes(Table 8, p. 88).In a follow-up clinical study, the effect of different doses of APAP (e.g., 325mg, 650 mg, and maybe 1000 mg) could be tested on the conversion of INH to95AcINH in 21 fast acetylators. The effect of APAP on the pharmacokineticparameters of INH could also be investigated. Alternatively, basic studies couldbe done with rabbits. In addition, in vitro enzyme studies could be conducted.If APAP had shown significantly important effects on the metabolism ofINH, there could be many possible explanations. First, the significance couldhave been due to a chance effect. Second, INH could have altered its ownmetabolism: i) there could have been accumulation of some of the metabolites inthe body due to long half-lives and subsequent increased clearance by thekidneys. For example, Blair et al. (1985) administered INH (300 mg) daily for 14days to 4 slow and 4 rapid acetylators. They reported that accumulation of Hzoccurred in slow acetylators by the end of the study. The authors did not suggestany possible mechanisms. INH might have inhibited the P-450 metabolism of Hz,and subsequently caused the levels of Hz to rise in the blood. As the subjectscontinued to ingest more INH every day, accumulation of Hz occurred. If thelevels of Hz continue to rise in the blood, then the clearance of Hz by the kidneyswould be expected to increase. Beyond all this, it is known that Hz (and AcHz)have longer half-lives than INH. From our study we would not be able to tell if INHaltered its own metabolism.INH could have altered its own metabolism by another mechanism: ii) INHinduced its own metabolism through P450 2E1 (Wall, personal communication).Microsonnal metabolism of both INH and AcINH could occur. Generally, a drugthat induces a cytochrome P450 enzyme is also usually a substrate for it. N-hydroxylation of many compounds is carried out by P450 2E1. We believe(although not proven yet) that P450 2E1 hydroxylates the hydrazine nitrogen ofINH adjacent to the isonicotinyl ring, and converts INH to INA and other by-9 6products (Wall, personal communication). In fact, it was just shown thatmyeloperoxidases (MPO)/H202 in activated neutrophils and monocytes convertINH to INA by oxidation, and it is likely that a radical intermediate is involved in thismetabolic pathway (Hofstra eta!., 1992).P450 2E1 is responsible for the metabolic activation of many chemicalsand carcinogens (Hyland et al., 1992; Gonzalez, 1992). P450 2E1 is known toefficiently catalyzes the oxidation or reduction of more than 75 low-molecular-weight substrates (Koop, 1992; Ingelman-Sundberg et al., 1992). INH was notlisted as an oxidative substrate of P450 2E1, because no one has studied this.Third, the renal clearance of some INH metabolites might have beenincreased by APAP administration. APAP, INH, and AcINH undergo glomerularfiltration. INA, ING, APAP glucuronide, and APAP sulfate are all actively secretedby the kidneys. APAP (4.0 g/day for 3 days) had no significant effect on theglomerular filtration rate in healthy volunteers (Prescott et al., 1990). The renalclearance of INA and ING might have been increased by APAP administration, butwe have no reason to believe so. APAP glucuronide and APAP sulfate mightinhibit the clearance of INA and ING, not increase it.Fourth, APAP might have interacted allosterically with the enzyme amidaseand increased its activity. If this happened, we would expect increased recoveriesof INA/ING, AcHz, and Hz. However, AcHz and especially Hz are very reactive,and they are rapidly converted to other products; we would not be able to tell ifthe activity of amidase increased. The only way to find out would be to do a directenzyme study with amidase.97Fifth, APAP could potentially affect the metabolism of INH by interactingwith amidase, NAT, or cytochrome P450(s). The enzyme amidase is probablyinvolved in the hydrolysis of INH and AcINH. Studies in rats have shown thatannidase catalyzes the hydrolysis of APAP to yield p-aminophenol, a minorpathway of APAP metabolism (Klos et al., 1992). Therefore APAP and INH areboth substrates for amidase, but the latter is a much better substrate. A numberof amidases are present in the mitochondrial, microsomal, and soluble fractions ofcells. In vitro studies, using rat liver subcellular fractions, revealed that INHamidase activity was widespread in all liver subfractions; however, relatively highconcentrations of the enzyme were localized in the liver microsomal andlysosomal fractions (Sendo et al., 1984). The INH amidase activity was inhibitedextensively by BNPP, moderately by AcINH, and slightly by acetanilide. BecauseAcINH is also a substrate for amidase, the inhibition by AcINH was probably acompetitive one. It is possible that the slight inhibition by acetanilide, a p-aminophenol derivative that gets converted to aniline by amidase, was acompetitive one. Assuming that amidase also catalyzes the hydrolysis of APAP(another p-aminophenol derivative) in humans, there is a potential for druginteraction. But this would be unlikely because, at therapeutic doses, thehydrolysis of APAP is a minor pathway. It remains to be seen how low doses andhigh doses of APAP affect INH amidase in humans.The cytosolic enzyme, polymorphic N-acetyltransferase (NAT; E.C., catalyzes the acetylation of INH and its metabolites, Hz and AcHz. Amicrosomal NAT (cysteine S-conjugate NAT; E.C. acetylates the APAPmetabolite, 3-Cys-APAP, to APAP 3-mercapturate. This enzyme has beenpartially purified from rat kidney microsomes and appears to favor morehydrophobic thioethers (Duffel & Jakoby, 1982). Both liver and kidney contain the9 8enzyme with the specific activity in the kidney being about twice that in liver(Green & Elce, 1975). This microsomal NAT is distinct from the soluble NAT thatcatalyzes the N-acetylation of many endogenous and exogenus amines and ispresent in these and other tissues (Stevens & Jones, 1989). Microsomal NAT isinibited by the same agents that inhibit cytosolic NAT. Rat kidney microsomalNAT is unaffected by 1 mM GNI" , Mg2+, Ca2+, and Co2+ while Zn2+ and Mn2+are moderately inhibitory and Cu2+ is totally inhibitory at that concentration(Weber et al., 1980). EDTA is slightly stimulatory but does not reverse Cu2 4.inhibition. N-ethylmaleimide and p-chloromercuribenzoate are both inhibitory at 1mM concentration.Studies on the metabolism of the glutathione S-conjugate of APAP byisolated rat kidney cells showed that these cells can take up the cysteine S-conjugate and N-acetylate it (Stevens & Jones, 1989). Liver and kidney probablyrepresent most of the cysteine S-conjugate removal from the plasma. It is notknown if uptake and N-acetylation of cysteine S-conjugates occurs in all othertissues. Apparently rat brain, spleen, stomach, heart and small intestine lackactivity (Weber et al., 1980). This is an important concern given the recognitionthat cysteine S-conjugates can be toxic (Stevens & Jones, 1989).Substrates effective with cytosolic NAT, e.g., aniline and p-aminobenzoicacid (PABA), were not acetylated by rat microsomal NAT (Duffel & Jakoby, 1982).In humans, PABA and aniline are preferentially acetylated by the monomorphicand polymorphic NAT, respectively. However, studies of genetic variation in N-acetylation in a rat model are few. In the rat liver, apparently a common NATcatalyzes the acetylation of PABA and INH. Other rat tissues such as lung,kidney, gut, and spleen also acetylate PABA (Weber, 1987). It appears that rat9 9NAT acetylates drugs monomorphically. However, there is a high variation in liverPABA NAT activity across inbred rat strains (Weber, 1987). Since there are noreports in the literature of studies on human microsomal NAT, it is not not known ifINH is a substrate for this enzyme. Even if human microsomal NAT couldacetylate INH, a drug interaction would not be expected since the acetylation of 3-Cys-APAP is a minor pathway (at therapeutic doses of APAP).APAP (4.0 g/d for 3 days) apparently inhibits the acetylation of p-amminohippurate (PAH) in humans since it reduced the urinary excretion of themetabolite while the recovery of the total PAH was unchanged (Prescott et al.,1990). Apparently the mouse kidney can rapidly acetylate PAH (Carpenter &Mudge, 1981); if the human kidney is also the primary organ where PAH isacetylated, then it would suggest the involvement of a monomorphic NAT.Conversely, acetanilide, a compound structurally related to APAP, is preferentiallyacetylated by polymorphic NAT in humans (Weber, 1987). Therefore, theinterference of APAP with the acetylation of PAH suggests that the latter could bea substrate for polymorphic NAT, i.e., APAP interferes with polymorphic NAT. Ifthis is the case, then high doses of APAP would be expected to affect theacetylation of INH.Because APAP is metabolized by many types of P450 enzymes (P450 2E1,3A4, 1A2, and perhaps other types in humans) it would have the potential tointerfer with the P450(s) that metabolize INH, AcHz, and Hz. As mentionedalready, APAP (doses of 1.6 g/day or more) may inhibit the microsomal oxidativepathway of warfarin; but this has not been substantiated. APAP, so far, has notbeen reported to affect the microsomal metabolism of any other drug.100Based on the results of this study, we can conclude that APAP does nothave a major effect (e.g., 50% change) on the metabolism of INH. However,APAP might have small effects on the metabolism of INH. For example, APAPmight interfere with the enzyme NAT. 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