Identification of a dithiol intermediate metabolite of malotilate in rats

1. A chemically unstable dithiol intermediate metabolite of malotilate was identified by g.l.c.-mass spectrometry after conversion of the dithiol to a stable derivative by a cyclization reaction with 1,3-dichloroacetone. The dithiol, namely, 2,2-di(isopropoxycarbonyl)ethylene-1,1-dithiol, was present in rat liver at low concentrations. 2. A study of glucuronidation in vitro indicated that the dithiol was converted to the corresponding thio-glucuronide by rat hepatic microsomal enzymes. 3. It was thus confirmed that metabolism of malotilate proceeds via the dithiol intermediate to form the thio-glucuronide, which is a major metabolic pathway.     

Isolation and characterization of a new thio-glucuronide, a biliary metabolite of malotilate in rats

1. The metabolism of malotilate, possessing a 1,3-dithiole ring, was studied in rats. A major biliary metabolite of malotilate was isolated and determined to be a thio-glucuronide of the dithiol formed by ring-opening, namely, 1-mercapto-2,2-di(isopropoxycarbonyl)-ethenyl 1-thio-beta-D-glucosiduronic acid. The structure was elucidated by proton-n.m.r., 13C-n.m.r. and high-resolution mass spectrometry. 2. The thio-glucuronide was completely hydrolysed by beta-glucuronidase. This enzymic reaction was inhibited by saccharo-1,4-lactone.     

Studies on tertiary amine UDP-glucuronosyltransferases from human and rabbit hepatic microsomes.

The conversion of tertiary amines to quaternary ammonium glucuronides was investigated in human liver microsomes, and characteristics of the UDP-glucuronosyltransferase (UGT) catalyzing quaternary ammonium glucuronidation were evaluated. In addition, a rabbit liver microsomal UGT mediating this reaction was studied. The kinetics of quaternary ammonium glucuronidation of cyproheptadine, tripelennamine, amitriptyline, and doxepin in intact human liver microsomes was determined. Tripelennamine was found to have the lowest apparent KM and was used as a representative substrate for further studies. A polyclonal antibody preparation raised in sheep against rabbit liver p-nitrophenol UGT was found to inhibit tripelennamine glucuronidation in solubilized human liver microsomes, but had no effect on p-nitrophenol, 4-methylumbelliferone, 4-aminobiphenyl, estriol, morphine, or naloxone glucuronidation. This antibody also inhibited tripelennamine glucuronidation in solubilized rabbit liver microsomes, but had little or no effect on estrone, testosterone, estradiol, androsterone, and morphine glucuronidation. Chlorpromazine competitively inhibited tripelennamine glucuronidation. This inhibition was markedly enhanced by UV light irradiation. [3H] Chlorpromazine binding to solubilized human liver microsomes was also increased by UV light. The binding was antagonized by substrates for tertiary amine UGT but not by substrates for morphine UGT. These studies suggest that the tertiary amine UGT is photo-affinity-labeled by chlorpromazine. Furthermore, it would appear from immunoinhibition and [3H]chlorpromazine labeling experiments that tertiary ammonium glucuronidation is catalyzed by a unique and distinct UGT in rabbit and human liver microsomes.     

Glucuronidation of the aspirin metabolite salicylic acid by expressed UDP-glucuronosyltransferases and human liver microsomes.

Acetylsalicylic acid (aspirin) is a common nonsteroidal anti-inflammatory drug used for treatment of pain and arthritis. In the body, acetylsalicylic acid is rapidly deacetylated to form salicylic acid. Both compounds have been proposed as anti-inflammatory agents. Major metabolites of salicylic acid are its acyl and phenolic glucuronide conjugates. Formation of these conjugates, catalyzed by UDP-glucuronosyltransferases (UGTs), decreases the amount of pharmacologically active salicylic acid present. We aimed to identify the UGTs catalyzing the glucuronidation of salicylic acid using both heterologously expressed enzymes and pooled human liver microsomes (HLMs) and to develop a liquid chromatography-tandem mass spectrometry method to quantify glucuronidation activity of UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 Supersomes. All UGTs tested, except 1A4, 2B15, and 2B17, catalyzed salicylic acid phenolic and acyl glucuronidation. Ratios of salicylic acid phenolic to acyl glucuronide formation varied more than 12-fold from 0.5 for UGT1A6 to 6.1 for UGT1A1. These results suggest that all UGTs except 1A4, 2B15, and 2B17 might be involved in the glucuronidation of salicylic acid in vivo. From comparisons of apparent Km values determined in pooled HLMs and in expressed UGTs, UGT2B7 was suggested as a likely catalyst of salicylic acid acyl glucuronidation, whereas multiple UGTs were suggested as catalysts of phenolic glucuronidation. The results of this UGT screening may help target future evaluation of the effects of UGT polymorphisms on response to aspirin in clinical and population-based studies.     

Identification of o-hydroxyphenylacetaldehyde as a major metabolite of coumarin in rat hepatic microsomes.

The metabolism of [3-14C]coumarin has been studied in hepatic microsomes from control (corn-oil treated) and Aroclor 1254-treated (100 mg/kg body weight/day, 5 days, ip) rats. [3-14C]Coumarin metabolites in incubate extracts were separated by HPLC and identified by comparison with the retention times of known coumarin metabolites. The major product produced by incubation of 0.25-2.5 mM-[3-14C]coumarin with both control and Aroclor 1254-induced hepatic microsomes was a novel coumarin metabolite. This novel metabolite was extracted from pooled microsomal incubations, purified by semi-preparative HPLC and identified by mass spectrometry as o-hydroxyphenylacetaldehyde (o-HPA). Some possible pathways for the formation of o-HPA from coumarin are proposed.     

Influence of substrate configuration on chlorpheniramine N-demethylation by hepatic microsomes from rats, rabbits, and mice

Measurements of formaldehyde formation in parallel incubations containing either (S)-(+)- or (R)-(-)-chlorpheniramine (CPA) and rat liver microsomes demonstrated that the active antihistamine, (S)-(+)-CPA, is N-demethylated about 35% faster than the inactive (R)-(-)-enantiomer. The KM values for the enantiomers were the same. Phenobarbital pretreatment increased Vmax values without affecting the stereoselectivity. N-Demethylation occurred at a several-fold faster rate with rabbit than with rat liver microsomes, but stereoselectivity was the same. N-Demethylation of CPA enantiomers were studied in microsomes prepared from each of four inbred strains of mice. These experiments demonstrated that stereoselectivity is species-dependent, as no significant differences in metabolism rates of CPA enantiomers could be detected with these microsomes. Pseudoracemic mixtures containing equal quantities of deuterated (S)-(+)-CPA and unlabeled (R)-(-)-CPA were incubated with microsomes from three species. Formation of the enantiomers of N-desmethyl- and N,N-didesmethyl-CPA (DMCPA and DDMCPA, respectively) were measured by GC/MS techniques. With microsomes from rats and mice, the ratio of (S)-DMCPA to (R)-DMCPA was essentially the same as that determined by measuring the formaldehyde formed in separate incubations of (S)-(+)- or (R)-(-)-CPA. Stereoselectivity with rabbit liver microsomes and pseudoracemic CPA was substantially higher than that determined in incubations with the separate enantiomers. The results suggest either that (S)-(+)-CPA inhibits the N-demethylation of (R)-(-)-CPA under these conditions, or that DMCPA undergoes further biotransformation by a route(s) which is stereoselective, favoring the (R)-enantiomer. Formation of DDMCPA could only be detected with rabbit microsomes and was found to occur with approximately the same stereoselectivity as that determined for the formation of DMCPA.     

Pharmacokinetic characteristics and hepatic distribution of IH-901, a novel intestinal metabolite of ginseng saponin, in rats

We investigated the pharmacokinetic characteristics of 20- O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH-901), a metabolite that is formed by intestinal bacteria, after its intravenous (i.v.) or oral administration in rats. We developed an LC/MS/MS-based method to analyze IH-901 levels in plasma, bile, urine and tissue homogenates and validated its use in a pharmacokinetic study. After i.v. administration of 3 - 30 mg/kg IH-901, it disappeared rapidly from the plasma at alpha phase followed by slow disappearance at beta phase (t(1/2,)(alpha) of 0.042 - 0.055 h and t (1/2,)(beta) of 6.98 - 10.6 h, respectively). The oral route slightly prolongs IH-901 plasma levels (terminal phase t(1/2) of 26.1 h) yet leads to a bioavailability of only 4.54 %. Of the various organs tested, the liver contained the majority of the i.v. bolus or orally administered IH-901, and liver IH-901 levels shortly after i.v. administration were 6-fold higher than the initial plasma concentration. The R(h) (hepatic recovery ratio) was calculated to be 0.417, and the uptake clearance (CL(uptake)) for i.v. administered IH-901 was 0.401 mL.min(-1).g liver(-1). Additionally, IH-901 is mostly excreted into the bile, since 40.5 % of the i.v.-administered dose (30 mg/kg) was recovered in the bile within 6 h, and only 15 % was found in the urine. Moreover, at steady state after i. v. infusion of IH-901, C(ss,liver) was about 11.3-fold higher than C(ss,plasma), whereas C(ss,bile) was about (1/2)-fold lower than C(ss,liver). These results indicated that the liver is largely responsible for removing IH-901 from the circulation. Oral administration of IH-901 leads to a low bioavailability; thus, the parenteral route may be the suitable way to deliver IH-901 for clinical applications.     

Inhibition of UDP-glucuronosyltransferases in rat liver microsomes by natural mutagens and carcinogens

Eight toxic compounds of natural origin present in the human diet or used as drugs were tested as inhibitors of UDP-glucuronosyltransferase (UGT) activity in rat liver microsomes with 1-naphthol (1-NA), phenolphthalein (PPh) and 4-nitrophenol (4-NP) as substrates. Strong inhibitory effects were observed with tannic acid (tannin) and the antifungal drug griseofulvin (GF): at a concentration of 1 mM, the two compounds completely suppressed the glucuronidation of 1-NA and PPh, respectively. A concentration of 0.1 mM still proved to be highly inhibitory, and even at a concentration as low as 50 microM, tannin produced nearly 50% inhibition of 1-NA conjugation. The UGT isoforms converting 4-NP were less sensitive to the tested compounds (with the exception of GF). Kinetic studies with tannin revealed an uncompetitive type of inhibition toward 1-NA, with an apparent Ki value of 20 microM. The inhibition by GF was non-competitive with respect to PPh and was of a mixed type toward UDP-glucuronic acid, with apparent Ki values of 40 microM and 30 microM, respectively. Tannin and GF did not act as substrates for rat microsomal UGT.     

Glucuronidation of olanzapine by cDNA-expressed human UDP-glucuronosyltransferases and human liver microsomes

Olanzapine is a widely used, newer antipsychotic agent, which is metabolized by various pathways: hydroxylation and N-demethylation by cytochrome P450, N-oxidation by flavin monooxygenase and direct glucuronidation. In vivo studies have pointed towards the latter pathway as being of major importance. Accordingly, the glucuronidation reaction was studied in vitro using cDNA-expressed human UDP-glucuronosyltransferase (UGT) enzymes and a pooled human liver microsomal preparation (HLM). Glucuronidated olanzapine was determined by HPLC after acid or enzymatic hydrolysis. The following UGT-isoenzymes were screened for their ability to glucuronidate olanzapine: 1A1, 1A3, 1A4, 1A6, 1A9, 2B7 and 2B15. Only UGT1A4 was able to glucuronidate olanzapine obeying saturation kinetics. The K(m) value was 227 micromol/l (SE 43), i.e. of the same order of magnitude as for other psychotropic drugs, and the V(max) value was 2370 pmol/(min mg) (SE 170). Glucuronidation was also mediated by the HLM preparation, but a saturation level was not reached. The olanzapine glucuronidation reaction was inhibited by several drugs known as substrates for UGT1A4, e.g. amitriptyline, trifluoperazine and lamotrigine. Thus, competition for glucuronidation by UGT1A4 represents a possibility for drug-drug interactions in subjects receiving several of these psychotropic drugs at the same time. Whether such possible interactions are of any clinical importance may await further studies in patients.     

Correlation of intrinsic in vitro and in vivo clearance for drugs metabolized by hepatic UDP-glucuronosyltransferases in rats

A method for quantitatively predicting the hepatic clearance of drugs by UDP-glucuronosyltransferases (UGTs) from in vitro data has not yet been established. We examined the relationship between in vitro and in vivo intrinsic clearance by rat hepatic UGTs using 10 drugs. For these 10 drugs, the in vitro intrinsic clearance by UGTs (CL(int, in vitro)) measured using alamethicin-activated rat liver microsomes was in the range 0.10-4500 ml/min/kg. Microsomal binding (f(u, mic)) was determined to be in the range 0.29-0.95 and the unbound intrinsic clearance (CL(uint, in vitro)) to be in the range 0.11-9600 ml/min/kg. The contribution of rat hepatic glucuronidation to drug elimination was 12.0%-76.6% and in vivo intrinsic clearance by UGTs was 5.7-9000 ml/min/kg. To evaluate the discrepancy between the in vitro and in vivo values, a scaling factor was calculated (CL(int, in vivo)/CL(int, in vitro)); the values were found to be in the range 0.89-110. The average fold error of the scaling factor values incorporating f(u, mic) was closer to unity than that without f(u, mic). The scaling factor values incorporating f(u, mic) were <10 in 8/10 drugs and <2 in 6/10 drugs, indicating a small discrepancy between in vitro and in vivo values. Thus, using alamethicin-activated liver microsomes, incorporating f(u, mic) into CL(int, in vitro), and considering the contribution of glucuronidation may enable us to quantitatively predict in vivo hepatic glucuronidation from in vitro data.     

A new pyrrolizidine alkaloid metabolite, 19-hydroxysenecionine isolated from mouse hepatic microsomes in vitro

The in vitro mouse hepatic microsomal metabolism of the macrocyclic pyrrolizidine alkaloid senecionine was studied by high-performance liquid chromatography. A muBondapak-C18 reverse-phase system was developed to study the senecionine metabolites over a wide range of polarities. Methods were further developed for the isolation of each individual metabolite. Senecic acid, senecionine N-oxide, and 19-hydroxysenecionine, a new metabolite, were isolated from the microsomal enzyme system of BALB/c mice. The metabolite, dehydroretronecine, which had previously been isolated from rat hepatic microsomes, was not detected, and minor metabolites were not identified.     

Glucuronidation of dihydroartemisinin in vivo and by human liver microsomes and expressed UDP-glucuronosyltransferases.

The aim of this study was to elucidate the metabolic pathways for dihydroartemisinin (DHA), the active metabolite of the artemisinin derivative artesunate (ARTS). Urine was collected from 17 Vietnamese adults with falciparum malaria who had received 120 mg of ARTS i.v., and metabolites were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Human liver microsomes were incubated with [12-(3)H]DHA and cofactors for either glucuronidation or cytochrome P450-catalyzed oxidation. Human liver cytosol was incubated with cofactor for sulfation. Metabolites were detected by HPLC-MS and/or HPLC with radiochemical detection. Metabolism of DHA by recombinant human UDP-glucuronosyltransferases (UGTs) was studied. HPLC-MS analysis of urine identified alpha-DHA-beta-glucuronide (alpha-DHA-G) and a product characterized as the tetrahydrofuran isomer of alpha-DHA-G. DHA was present only in very small amounts. The ratio of the tetrahydrofuran isomer, alpha-DHA-G, was highly variable (median 0.75; range 0.09-64). Nevertheless, alpha-DHA-G was generally the major urinary product of DHA glucuronidation in patients. The tetrahydrofuran isomer appeared to be at least partly a product of nonenzymic reactions occurring in urine and was readily formed from alpha-DHA-G by iron-mediated isomerization. In human liver microsomal incubations, DHA-G (diastereomer unspecified) was the only metabolite found (V(max) 177 +/- 47 pmol min(-1) mg(-1), K(m) 90 +/- 16 microM). Alpha-DHA-G was formed in incubations of DHA with expressed UGT1A9 (K(m) 32 microM, V(max) 8.9 pmol min(-1) mg(-1)) or UGT2B7 (K(m) 438 microM, V(max) 10.9 pmol mg(-1) min(-1)) but not with UGT1A1 or UGT1A6. There was no significant metabolism of DHA by cytochrome-P450 oxidation or by cytosolic sulfotransferases. We conclude that alpha-DHA-G is an important metabolite of DHA in humans and that its formation is catalyzed by UGT1A9 and UGT2B7.     

Cytochrome P-450 and monooxygenase activity in hepatic microsomes from N-phenylimidazole-treated rats

Repeated administration of N-phenylimidazole (PI) to rats (3 daily doses of 200 mumol/kg/day) enhanced hepatic microsomal cytochrome P-450 levels (approx. 130%) and aminopyrine N-demethylase (APDM) and aniline p-hydroxylase (APH) activities (approx. 140%); aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) activities were not enhanced over control values under similar conditions. Spectral studies with PI-induced microsomes indicated that although type II PI-binding characteristics were similar to those observed in controls, the 427 nm/455 nm absorbance ratio of the type III dihydrosafrole metabolite-cytochrome P-450 complex was lower than that in control microsomes. The results suggest that the inducing characteristics of PI bear some resemblance to those of phenobarbital (PB).     

Comparative in vitro metabolism of T-2 toxin by hepatic microsomes prepared from phenobarbital-induced or control rats, mice, rabbits and chickens

Hepatic microsomes were prepared from phenobarbital (PB)-treated and control rats, mice, rabbits and chickens and were incubated with T-2 toxin (100 micrograms/mg microsomal protein). Additional microsomes from PB-induced animals were incubated with T-2 toxin and the esterase inhibitor paraoxon (PA) at 2.5 nmol/mg microsomal protein. The major metabolite in microsomal preparations from both control and PB-induced rats, rabbits and mice was HT-2. In microsomes isolated from PB-treated chickens, 3'-hydroxy T-2 was the major metabolite, but 30 and 79% of the added T-2 toxin remained unmetabolized at 60 min in incubations from PB-induced and control birds, respectively. The percentage of hydroxylated metabolites formed in the microsomal preparations of the four species studied was significantly increased following PB treatment compared with the non-treated controls. The addition of PA to the incubation system effectively inhibited the hydrolysis of the ester groups in T-2 toxin, resulting in 1.4- and 1.25-fold increases in the percentage of 3'-hydroxy T-2 in the mouse and rat microsomal samples, respectively. In the rabbit microsomal preparations, 3'-hydroxy T-2, which was not detected in the absence of PA, represented 11% of the added substrate in the PB/PA incubation samples. Addition of PA did not cause a significant change in the amount of 3'-hydroxy T-2 formed in chicken microsomal samples, since competition between hydrolysis and hydroxylation pathways for the T-2 toxin substrate was not an important factor in this species. Two new metabolites, designated RLM-2 and RLM-3 were detected in chicken, rat and mouse microsomal preparations. On the basis of gas chromatography/mass spectrometry data, the compounds were tentatively identified as isomers of 3'-hydroxy T-2.     

Acceleration of liver regeneration by malotilate in partially hepatectomized rats

The effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) on liver regeneration was studied by using partially hepatectomized rats. Malotilate administration (100 mg/kg/day, p.o.) facilitated the weight gain of the liver after partial hepatectomy. Protein, RNA and DNA contents of the regenerating liver correlated well with the weight gain. The weight gain, RNA and DNA contents, and mitotic index were significantly suppressed in the alloxan-diabetic rats 24 hr after partial hepatectomy. However, malotilate administration significantly improved the delayed recovery of RNA content. Other parameters were not significantly improved by malotilate, but tended to increase to a level comparable to those of partially hepatectomized control rats. These results show that malotilate accelerates cell proliferation, resulting in facilitated liver regeneration in rats (as well as in alloxan-diabetic rats).     

In vitro hepatic conversion of the anticancer agent nemorubicin to its active metabolite PNU-159682 in mice, rats and dogs: a comparison with human liver microsomes

We recently demonstrated that nemorubicin (MMDX), an investigational antitumor drug, is converted to an active metabolite, PNU-159682, by human liver cytochrome P450 (CYP) 3A4. The objectives of this study were: (1) to investigate MMDX metabolism by liver microsomes from laboratory animals (mice, rats, and dogs of both sexes) to ascertain whether PNU-159682 is also produced in these species, and to identify the CYP form(s) responsible for its formation; (2) to compare the animal metabolism of MMDX with that by human liver microsomes (HLMs), in order to determine which animal species is closest to human beings; (3) to explore whether differences in PNU-159682 formation are responsible for previously reported species- and sex-related differences in MMDX host toxicity. The animal metabolism of MMDX proved to be qualitatively similar to that observed with HLMs since, in all tested species, MMDX was mainly converted to PNU-159682 by a single CYP3A form. However, there were marked quantitative inter- and intra-species differences in kinetic parameters. The mouse and the male rat exhibited V(max) and intrinsic metabolic clearance (CL(int)) values closest to those of human beings, suggesting that these species are the most suitable animal models to investigate MMDX biotransformation. A close inverse correlation was found between MMDX CL(int) and previously reported values of MMDX LD(50) for animals of the species, sex and strain tested here, indicating that differences in the in vivo toxicity of MMDX are most probably due to sex- and species-related differences in the extent of PNU-159682 formation.     

Kinetic characteristics of norcocaine N-hydroxylation in mouse and human liver microsomes: involvement of CYP enzymes

The first step in the oxidative metabolism of cocaine is N-demethylation to norcocaine, which is further N-hydroxylated to more toxic N-hydroxynorcocaine. In this study we examined the kinetics of norcocaine N-hydroxylation mediated by cytochrome P450 (CYP) in mouse and human liver microsomes. N-hydroxynorcocaine was identified by analytical HPLC-MS after incubation of norcocaine with mouse liver microsomes in the presence of NADPH. In mouse liver microsomes, there was no apparent difference in Km values for norcocaine N-hydroxylation between male and female microsomes, while the Vmax rate was approximately two times higher in female than in male microsomes (34+/-10 v. 16+/-4 pmol/min per mg protein). The Km value for norcocaine N-hydroxylation in human liver microsomes was approximately three times higher than that observed in comparable incubations using mouse liver microsomes, whereas the Vmax rate was ten times lower. Both cocaine and norcocaine induced type I difference spectra upon interaction with CYP in mouse liver microsomes. In contrast, in human microsomes both type I and type II spectra were recorded. In the 0.01 to 1 mM concentration range, cocaine and norcocaine inhibited mouse microsomal testosterone 6alpha-, 7alpha- and 16alpha-hydroxylation reactions by 20% to 30%. Testosterone 6beta- and 15alpha-hydroxylations were blocked by 60% and 50%, respectively, by 1 mM norcocaine, while only 40% inhibition was obtained with 1 mM cocaine. Coumarin 7-hydroxylation and pentoxyresorufin O-deethylation were inhibited by 50% by 1 and 0.4 mM norcocaine, respectively. In contrast, 10 and 2 mM cocaine, respectively, were needed to obtain the same degrees of inhibition. In human liver microsomes, 1 mM norcocaine and cocaine blocked testosterone 6beta-hydroxylase by 60% and 40%, respectively. Coumarin 7-hydroxylation was inhibited by only 30% by norcocaine (5.4 mM) and cocaine (10 mM). Norcocaine N-hydroxylation in mouse and human liver microsomes was blocked by 30% and 60%, respectively, by alpha-naphthoflavone (0.1 mM). The reaction was inhibited by 30-40% by metyrapone, cimetidine and gestodene at a concentration of 1 mM in mouse microsomes, while in human microsomes, 70% inhibition was obtained with 1 mM metyrapone and cimetidine. Taken together, these results indicate that (1) norcocaine N-hydroxylation is at least partly a CYP-mediated reaction, (2) the rate of reaction is considerably lower in human liver microsomes than in mouse liver microsomes and (3) several CYP subfamilies including 1A, 2A, 3A and possibly 2B may contribute to the formation of N-hydroxynorcocaine.     

N-glucuronidation of nicotine and cotinine by human liver microsomes and heterologously expressed UDP-glucuronosyltransferases.

Nicotine is considered the major addictive agent in tobacco. Tobacco users extensively metabolize nicotine to cotinine. Both nicotine and cotinine undergo N-glucuronidation. Human liver microsomes have been shown to catalyze the formation of these N-glucuronides. However, which UDP-glucuronosyltransferases contribute to this catalysis has not been identified. To identify these enzymes, we initially measured the rates of glucuronidation by 15 human liver microsome samples. Fourteen of the samples glucuronidated both nicotine and cotinine at rates ranging from 146 to 673 pmol/min/mg protein and 140 to 908 pmol/min/mg protein, respectively. The rates of nicotine glucuronidation and cotinine glucuronidation by these 14 samples were correlated, r = 0.97 (p < 0.0001). The glucuronidation of nicotine and cotinine by heterologously expressed UGT1A3, UGT1A4, and UGT1A9 was also determined. All three enzymes catalyzed the glucuronidation of nicotine. However, the rate of catalysis by UGT1A4 Supersomes was more than 30-fold greater than that by either UGT1A3 Supersomes or UGT1A9 Supersomes. Interestingly, when expressed per UGT1A protein, measured by a UGT1A specific antibody, cell lysate from V79-expressed UGT1A9 catalyzed nicotine glucuronidation at a rate 17-fold greater than did UGT1A9 Supersomes. UGT1A4 Supersomes also catalyzed cotinine N-glucuronidation, but at one-tenth the rate of nicotine glucuronidation. Cotinine glucuronidation by either UGT1A3 or UGT1A9 was not detected. Both propofol, a UGT1A9 substrate, and imipramine, a UGT1A4 substrate, inhibited the glucuronidation of nicotine and cotinine by human liver microsomes. Taken together, these data support a role for both UGT1A9 and UGT1A4 in the catalysis of nicotine and cotinine N-glucuronidation.     

Effect of pregnancy on hepatic microsomal drug metabolism in rabbits and rats

In Dutch-belted rabbits, pregnancy caused several-fold decrease of in vitro hepatic microsomal aminopyrine, benzphetamine, and hexobarbital biotransformations. In pregnant Sprague-Dawley rats, various kinds of expressing the in vitro rates of hexobarbital biotransformation (per mg of microsomal protein, g of liver, 100 g of body weight) indicated unchanged or slightly elevated microsomal enzyme activity. In vivo, the course of hexobarbital blood levels after i. p. hexobarbital sodium, 100 mg/kg, indicated that the fate of hexobarbital was not primarily determined by the small changes of microsomal enzyme activity but, rather, by changed hexobarbital distribution. Different ways of expressing in vitro rates of aniline biotransformation showed decreased or unchanged enzyme activity during pregnancy and in vivo experiments indicated that these changes did not affect aniline metabolism in living rats. The results pointed out marked species differences in the effect of pregnancy on drug metabolism. Interpretation of in vitro biotransformation data for living animals suggested that with different substrates, microsomal enzyme activity and distribution, respectively, may exert different effects playing either significant or apparently minor role in drug disposition.Supported in part by U.S. Public Health Grant NIGMS 12,675.     

The role of hepatic and extrahepatic UDP-glucuronosyltransferases in human drug metabolism.

At present, the methods and enzymology of the UDP-glucuronosyltransferases (UGTs) lag behind that of the cytochromes P450 (CYPs). About 15 human UGTs have been identified, and knowledge about their regulation, substrate selectivity, and tissue distribution has progressed recently. Alamethicin has been characterized as a treatment to remove the latency of microsomal glucuronidations. Most UGT isoforms appear to have a distinct hepatic and/or extrahepatic expression, resulting in significant expression in kidney, intestine, and steroid target tissues. The gastrointestinal tract possesses a complex expression pattern largely containing members of the UGT1A subfamily. Thus, these forms are poised to participate in the first pass metabolism of oral drugs. The authors and others have identified a significant expression of UGT1A1 in human small intestine, an enzyme possessing considerable allelic variability and a polymorphic expression pattern in intestine. Intestinal glucuronidation therefore plays a major role not only in first pass metabolism, but also in the degree of interindividual variation in overall oral bioavailability. Due to issues such as significant genetic variability and tissue localization in first-pass organs, clearance due to UGT1A1 should be minimized for new drugs.