Effects of normothermic hepatic ischemia-reperfusion injury on the in vivo, isolated perfused liver, and microsomal disposition of chlorzoxazone, a cytochrome P450 2E1 probe, in rats

In vitro studies have shown that the activities of cytochrome P450 (P450) enzymes may be altered after hepatic ischemia-reperfusion (IR) injury. Here, we investigated the effects of 1 h of partial ischemia, followed by 3 (IR3) or 24 (IR24) h of in vivo reperfusion, on the in vivo, isolated perfused rat liver (IPRL), and microsomal disposition of chlorzoxazone (CZX) and its cytochrome P450 2E1 (CYP2E1)-mediated metabolite, 6-hydroxychlorzoxazone (HCZX), in rats. Although IR3 caused a 30% reduction in the in vivo clearance of CZX, the area under the plasma concentration-time curve of HCZX was not affected. IPRL experiments showed that IR3, in addition to a 30% reduction in the clearance of CZX, causes a 70% decrease in the biliary clearance of HCZX. Microsomal data revealed a 50% decline in the intrinsic clearance of HCZX formation due to an IR3-induced significant decline in maximum velocity. Although IR3 did not affect the microsomal CYP2E1 protein, it caused approximately 30% reduction in the cytochrome P450 reductase activity. IR24 did not have any effect on the disposition of CZX or HCZX. In conclusion, metabolism of xenobiotics and endogenous compounds that are substrates for CYP2E1, and possibly other P450 isoenzymes, may be reduced shortly after surgical procedures that require transient interruption of the hepatic blood flow.     

Chlorzoxazone: a probe drug the metabolism of which can be used to monitor one-point blood sampling in the carbon tetrachloride-intoxicated rat.

In this study, we have carried out an investigation to determine if chlorzoxazone (CZX) is a suitable probe drug for predicting hepatic injury in carbon tetrachloride (CCl4)-intoxicated rats. The animals received oral doses of CCl4 (0.25, 0.5 and 1 ml/kg) 24 h prior to intraperitoneal administration of CZX. The total CYP and CYP2E1 content, as well as the aniline and CZX hydroxylase activity (Vmax and CLint), was reduced depending on the dose of CCl4 administered. At the highest concentration (128 mM) of diethyldithiocarbamate, a specific inhibitor of CYP2E1, the production of 6-hydroxychlorzoxazone (HCZX) in microsomes from CCl4-treated rats was reduced by about 85%. The IC50 value in microsomes from CCl4-treated rats was between 3 and 5 microM. The production of HCZX and the activity of aniline hydroxylase in CCl4-treated rats correlated with the amount of rat CYP2E1 protein (r=0.881, P<0.001 and r=0.822, P<0.001, respectively). The elimination of CZX by CCl4-treated rats was reduced and the HCXZ production in the CCl4-treated group was less than that in the olive oil-treated control group. The correlations between the intrinsic clearance [CLint: Vmax/Km) in vitro and the total body clearance (CLtot) of CZX hydroxylation and the elimination half-life (t1/2) of CZX in vivo in CCl4-treated rats were high (r=0.839, P<0.001 and r= -0.828, P<0.001, respectively). In addition, the metabolic plasma HCZX/CZX ratio did not require multiple blood sampling and, 2 h after CZX administration in vivo, there was also a high correlation with the CLint (Vmax/Km) in vitro (r= -0.909, P<0.01). In conclusion, these results from this study demonstrate that CZX is a good probe for monitoring the inhibition of metabolism in rats due to CCl4 treatment.     

Metabolism and excretion of 3-hydroxyphenyltrimethylammonium and neostigmine

1. Carbon-14 labelled 3-hydroxyphenyltrimethylammonium (3-OH PTMA), an active metabolite of neostigmine, has been given to rats by intramuscular injection and its excretion, distribution and metabolism have been studied.2. A method is described for the separation and estimation of free and conjugated 3-OH PTMA in urine and liver.3. In the first hour, about 20% of a dose is excreted in the urine as free 3-OH PTMA and thereafter the rate of excretion of glucuronide conjugate exceeds that of free 3-OH PTMA. In 24 hr 76.8% of the dose is excreted in urine mainly as the conjugate.4. The peak concentration of radioactivity in blood occurs within 30 min and in liver within 1 hr after administration. More than 90% of the radioactivity in liver occurs as the glucuronide conjugate. Relatively high concentrations of radioactivity were found in liver and heart.5. In the hen 3-OH PTMA is rapidly excreted by renal tubular secretion.6. Experiments with carbon-14 labelled neostigmine show that up to 1 hr mainly unchanged neostigmine is excreted in urine; thereafter increasing amounts of free 3-OH PTMA and its glucuronide conjugate are excreted.7. It is concluded that the duration of action of neostigmine is determined by its rapid renal excretion and by its metabolism to the glucuronide conjugate of 3-OH PTMA.     

Conjugation kinetics of acetaminophen by the perfused rat liver preparation

Sulfate conjugation of acetaminophen was studied in the perfused rat liver preparation. Varying input concentrations of unlabeled acetaminophen (0.22 to 6.0 μg/ml or 1.5 to 40 μM) were added in a stepwise fashion once-through the liver preparation at a flow rate of 10 ml/min. For the concentration range tested, acetaminophen sulfate conjugate remained the only detectable metabolite in perfusate plasma. Other metabolites such as the glucuronide and the glutathione conjugates, however, were detected only in bile and, together with the sulfate conjugate and unconjugated acetaminophen, constituted 5 per cent of the total administered dose. Hepatic elimination of acetaminophen in the formation of sulfate conjugate was apparently maximal at input concentrations ? 1.0 μg/ml (6.7 μM) and could be viewed as mediated via a uni-enzyme system. Sulfate conjugation also decreased with preloading of the liver, especially with high concentrations of acetaminophen. A plot of the ratio of the steady-state output concentrations of drug to metabolite versus the reciprocal of drug extraction ratio, which might prove useful in the prediction of metabolite concentrations in the liver, was introduced.     

Disposition and effect of the new thromboxane synthetase inhibitor 6-(1-imidazolylmethyl)-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid in man

The disposition of a new thromboxane synthetase inhibitor, 6-(1-imidazolylmethyl)-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid (DP-1904) upon administration of a single 200-mg oral dose to normal Japanese volunteers was studied. DP-1904 proved to be rapidly absorbed from the gastrointestinal tract and converted to its ester glucuronide, which appeared in plasma within 30 min after dosing. The AUCs of DP-1904 and its ester glucuronide were 7.23 +/- 0.54 and 7.93 +/- 0.86 micrograms.h/ml (mean +/- S.E., n = 5), respectively. Both compounds were also eliminated very rapidly from the body (half-lives greater than 60 min). The primary route of elimination was renal, with 52.1 +/- 2.2 and 37.6 +/- 1.6% of the dose being excreted in the urine as the unchanged form and the glucuronide conjugate within 48 h, respectively. The cumulative fecal excretion rates of DP-1904 up to 48 h after dosing were approximately 0.5%. The main metabolite of DP-1904 in humans was DP-1904 glucuronide. Serum thromboxane (TX) B2 levels were reduced more than 98% within 1 h after dosing. There was still more than 75% suppression of serum TXB2 levels at 12 h after dosing. At 72 h TXB2 concentrations returned to control levels. These data indicate that DP-1904 is a potent and long-acting thromboxane synthetase inhibitor.     

Effect of 1-aminobenzotriazole on the in vitro metabolism and single-dose pharmacokinetics of chlorzoxazone, a selective CYP2E1 substrate in Wistar rats

The aim of this study was to study the effect of 1-aminobenzotriazole (ABT) on in vitro metabolism, oral, and intravenous (IV) pharmacokinetics of chlorzoxazone (CZX) in rats. Enzyme kinetics of CZX was performed with rat and human liver microsomes and pure isozyme (CYP2E1) with and without ABT. The enzyme kinetics (V(max) and K(m)) of the formation of 6-hydroxychlorzoxazone (OH-CZX) was found to be similar among rat liver microsomes (3486 pmol mg protein(-1) min(-1) and 345 microM), human liver microsomes (3194 pmol mg protein(-1) min(-1) and 335 microM) and pure isozyme (3423 pmol mg protein(-1) min(-1) and 403 microM), but K(I) and K(inact) values for ABT towards the ability to inhibit the formation of OH-CZX from CZX varied between liver microsomes (rat: 32.09 microM and 0.12 min(-1); human: 27.19 microM and 0.14 min(-1)) and pure isozyme (3.18 microM and 0.29 min(-1)). The novel robust analytical method was capable of quantifying CZX, OH-CZX, and ABT simultaneously in a single run, and the method was used for both in vitro and in vivo studies. Pre-treatment of rats with ABT prior to oral and IV administration of CZX significantly decreased the clearance (threefold) and consequently increased the AUC of CZX (approx. three- to fourfold). When rats were pre-treated with ABT, the formation of OH-CZX was completely blocked after oral and IV administration; however, we were able to measure OH-CZX in rats administered with CZX by oral and IV routes without pre-treatment of ABT. The oral bioavailability of CZX was approximately 71% when dosed alone and reached 100% under pre-treatment with ABT. The t(1/2) values of CZX was significantly prolonged for oral dosing compared with IV dosing under pre-treated conditions with ABT, suggesting an involvement of pre-systemic component in the disposition of CZX. The pharmacokinetic parameters of ABT did not change when it was dosed along with CZX (oral and IV), indicating that either CZX or OH-CZX had no effect on disposition of ABT. The plasma concentrations of ABT were above and beyond the required levels to inhibit CYP2E1 enzyme for at least 36 h post-treatment.     

Disposition of the selective cholesterol absorption inhibitor ezetimibe in healthy male subjects.

Ezetimibe [SCH 58235; 1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone], a selective cholesterol absorption inhibitor, is being developed for the treatment of primary hypercholesterolemia. The absorption, metabolism, and excretion of ezetimibe were characterized in eight healthy male volunteers in this single-center, single-dose, open-label study. Subjects received a single oral 20-mg dose of [14C]ezetimibe (approximately 100 microCi) with 200 ml of noncarbonated water after a 10-h fast. Concentrations of radioactivity and/or ezetimibe (conjugated and unconjugated) were determined in plasma, urine, and fecal samples. Ezetimibe was rapidly absorbed and extensively conjugated following oral administration. The main circulating metabolite in plasma was SCH 60663 [1-O-[4-[trans-(2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-azetidinyl]phenyl]-beta-D-glucuronic acid], the glucuronide conjugate of ezetimibe. Plasma concentration-time profiles of unconjugated and conjugated drug exhibited multiple peaks, indicating enterohepatic recycling. Approximately 78 and 11% of the administered [14C]ezetimibe dose were excreted in feces and urine, respectively, by 240 h after drug administration. Total recovery of radioactivity averaged 89% of the administered dose. The main excreted metabolite was the glucuronide conjugate of ezetimibe. The primary metabolite in urine (0- to72-h composite) was also the glucuronide conjugate (about 9% of the administered dose). Significant amounts (69% of the dose) of ezetimibe were present in the feces, presumably as a result of SCH 60663 hydrolysis and/or unabsorbed drug. No adverse events were reported in this study. A single 20-mg capsule of [(14)C]ezetimibe was safe and well tolerated after oral administration. The pharmacokinetics of ezetimibe are consistent with extensive glucuronidation and enterohepatic recirculation. The primary metabolic pathway for ezetimibe is by glucuronidation of the 4-hydroxyphenyl group.     

Fasting increases the sensitivity of hepatic harmol glucuronidation to hypoxia.

Livers from fasted (N = 16) and fed (N = 22) rats were perfused with harmol (50 microM) for an initial 30 min with normal oxygen delivery (6-10 mumol/min/g liver), then for 45 min with perfusate equilibrated with O2/N2 mixtures, which reduced hepatic oxygen delivery to 0.9-6 mumol/min/g liver, and finally for a further 30 min period of normal oxygenation. Seventy per cent of the harmol eliminated was accounted for as the glucuronide conjugate and approximately 5% as the sulphate conjugate. During the hypoxia phase with fed preparations, decreasing oxygenation did not reduce harmol clearance or harmol glucuronide formation clearance until oxygen delivery was less than 2.5 mumol/min/g liver, whereas with fasted preparations this hypoxic threshold was much higher (5 mumol/min/g liver). Below the hypoxic threshold, harmol clearance was linearly related to oxygen delivery in both groups. Hepatic tissue concentrations of unchanged harmol at the end of the hypoxia phase were double those after the same period of normal oxygenation, whereas tissue harmol glucuronide concentrations were similar. By establishing a hypoxic threshold for reduced oxygen availability this study shows that harmol glucuronidation is relatively insensitive to hypoxia, but sensitivity increases markedly in fasted animals.     

In vivo metabolites of S-(2-benzothiazolyl)-L-cysteine as markers of in vivo cysteine conjugate beta-lyase and thiol glucuronosyl transferase activities

Cysteine conjugate beta-lyases (beta-lyase), enzymes that are present in mammalian liver, kidneys, and intestinal microflora, were exploited recently for site-selective delivery of 6-mercaptopurine to the kidneys. In this study, in vivo beta-lyase activity was assessed using S-(2-benzothiazolyl)-L-cysteine (BTC). 2-Mercaptobenzothiazole and 2-mercaptobenzothiazole S-glucuronic acid were major metabolites of BTC in rat liver, kidney, plasma, and urine. Total metabolite concentrations in liver, kidney, or plasma at 30 min were similar and were higher than that detected at 3 hr; metabolites were mostly in the glucuronide form. The portions of metabolites excreted in urine at 8 and 24 hr were nearly 93 and 99% of that excreted at 40 hr, respectively. Pretreatment of rats with aminooxyacetic acid did not alter kidney, liver, plasma, or urinary metabolite concentrations. The portion of the BTC dose excreted as metabolites at 24 hr was independent of the BTC dose (100-400 mumol/kg), age (5-12 weeks), or sex of the rats. The rates of in vitro BTC metabolism by guinea pig hepatic and renal beta-lyases were slower than those of rats, but the portion of the BTC dose recovered as metabolites in guinea pig urine at 24 hr was nearly 60%, which was nearly 2-fold higher than that recovered in urine of rats, mice, or hamsters. The amounts of total metabolites excreted into urine by mice or hamsters were similar, but the portion of metabolites that was in the glucuronide form in hamster urine was higher than that in mouse urine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effect of Valproate on the Metabolism of Phenobarbital in the Rat

Valproate has been shown to interact with all major antiepileptic drugs. The interaction with phenobarbital is the most clinically significant. The mechanism of the interaction was evaluated in the in vivo rat and in vitro liver perfusion system. Phenobarbital and parahydroxyphenobarbital (PbOH) were administered with and without valproate treatment. In vivo, after administration of PbOH, valproate caused a significant inhibition of both the renal clearance of unchanged PbOH (40%) and the formation clearance (ClF) of its glucuronide conjugate (44%). When coadministered with phenobarbital, valproate caused a significant decrease in the total plasma clearance of phenobarbital (95.4 ± 29.0 to 65.8 ± 20.2 ml/hr/kg), with no apparent effect on the phenobarbital renal clearance or the ClF of PbOH. Valproate did cause a significant inhibition (50%) of formation of a minor metabolite, metahydroxyphenobarbital. The largest effect of valproate appears to be on unknown pathways of phenobarbital elimination. In the isolated perfused rat liver, the ClF of PbOH and its glucuronide conjugate were determined. Valproate caused a small (10%) but significant decrease in the ClF of PbOH. As seen in vivo, the most significant effect of valproate was on the ClF of the PbOH glucuronide (66% decrease). In conclusion, inhibition of PbOH formation by valproate cannot account entirely for the increased plasma concentrations of phenobarbital that occur when valproate is added to therapy. A complete understanding of the mechanism will require a complete accounting of the phenobarbital dose in rat or in humans.     

Selective effects of nitric oxide on the disposition of chlorzoxazone and dextromethorphan in isolated perfused rat livers.

The rapid and direct effects of nitric oxide (NO) donors sodium nitroprusside (SNP) and isosorbide dinitrate (ISDN) on the hepatic and biliary disposition of chlorzoxazone (CZX), a marker of CYP2E1, and dextromethorphan (DEM), a marker of CYP2D1, were studied in a single-pass isolated perfused rat liver model. Livers (n = 30) were perfused with constant concentrations of NO donors (0-120 min) in addition to infusion of CZX or DEM (60-120 min), and periodical outlet and bile samples were collected. Both ISDN and SNP significantly reduced (30 and 60%, respectively) the hepatic extraction ratio of CZX and decreased (50 and 70%, respectively) the recovery of the CYP2E1-mediated metabolite, 6-hydroxychlorzoxazone, in the outlet perfusate and bile. As for DEM, both NO donors increased (up to 3.5-fold) the recovery of the CYP2D1-mediated metabolite dextrorphan (DOR) in the outlet perfusate. However, this was associated with a simultaneous decrease (50-75%) in the excretion of the metabolite into the bile, thus resulting in no change in the overall recovery of DOR as a result of NO donor treatment. The decrease in the biliary excretion of DOR was caused by NO-induced simultaneous reductions in both the conjugation of DOR and biliary clearance of DOR conjugate. Additionally, both SNP and ISDN significantly reduced the metabolism of DEM to 3-hydroxymorphinan, which is mostly regulated by CYP3A2. These studies in an intact liver model confirm the selectivity of the inhibitory effects of NO donors on cytochrome P450 enzymes, which was recently reported in microsomal studies, and expand these inhibitory effects to conjugation pathways.     

Metabolism of bisphenol a in primary cultured hepatocytes from mice, rats, and humans.

Studies have shown that in the rat, bisphenol A (BPA) is metabolized and eliminated primarily as a monoglucuronide, a metabolite without estrogenic activity. The purpose of this study was to determine the extent of monoglucuronide formation in monolayers of hepatocytes from rats, mice, and humans. Noncytotoxic concentrations of BPA (10, 20, and 35 microM; 1.0 microCi), as assessed by lactate dehydrogenase leakage, were incubated with isolated hepatocytes for 0-6 h. Media were collected and analyzed for metabolites by radiochemical high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. The metabolites identified include a monoglucuronide (major metabolite), a sulfate conjugate, and a glucuronide/sulfate diconjugate (minor metabolites). In hepatocytes of male Fischer-344 rats, the predominate metabolite was the diconjugate (glucuronide/sulfate). Under these conditions, the extent of metabolism by 3 h was similar in all species tested because all BPA was converted to conjugates by 3 h. Initial rates of metabolism in hepatocytes followed the order of mice > rats > humans. However, when extrapolated to the whole liver (i.e., cells per liver), the hepatic capacity for BPA glucuronidation is predicted to be humans > rats > mice. This research was supported in part by The Society of Plastics Industry Inc., and Southwest Environmental Health Science Center (ES 06694).     

The pharmacokinetics of naproxen, its metabolite O-desmethylnaproxen, and their acyl glucuronides in humans. Effect of cimetidine.

1. The pharmacokinetics of 500 mg naproxen given orally were described in 10 subjects using a direct h.p.l.c. analysis of the acyl glucuronide conjugates of naproxen and its metabolite O-desmethylnaproxen. 2. The mean elimination half-life of naproxen was 24.7 +/- 6.4 h (range 7 to 36 h). 3. Naproxen acyl glucuronide accounted for 50.8 +/- 7.3% of the dose recovered in the urine, its isomerised conjugate isoglucuronide for 6.5 +/- 2.0%, O-desmethylnaproxen acyl glucuronide for 14.3 +/- 3.4%, and its isoglucuronide for 5.5 +/- 1.3%. Naproxen and O-desmethylnaproxen were excreted in negligible amounts (< 1%). 4. Even though the urine pH of the subjects was kept acid in order to stabilize the acyl glucuronides, isomerisation took place in blood. 5. The extents of plasma binding of the unconjugated compounds were 98% (naproxen) and 100% (O-desmethylnaproxen), while naproxen acyl glucuronide binding was 92%; that of its isomer isoglucuronide 66%. O-desmethylnaproxen acyl glucuronide was 72% bound and its isoglucuronide was 42% bound. 6. Cimetidine (400 mg twice daily) decreased the t1/2 of naproxen by 39-60% (mean 47.3 +/- 11.5%; P = 0.0014) from 24.7 +/- 6.4 h to 13.2 +/- 1.0 h. It increased (10%) the urinary recovery of naproxen acyl glucuronide (P = 0.0492). The urinary recoveries of naproxen isoglucuronide and O-desmethylnaproxen acyl glucuronide remained unchanged.     

First-pass metabolism of salicylamide. Studies in the once-through vascularly perfused rat intestine-liver preparation

Salicylamide (SAM) metabolism was studied in a once-through in situ perfused rat intestine-liver preparation in a manner which mimicked the first-pass effect. SAM (40 or 200 microM) was delivered into the intestine via the superior mesenteric artery at a flow rate of 7.5 ml/min. The intestine venous outflow into the portal vein and the hepatic arterial flow (2.5 ml/min; without drug) served as dual inflows into the liver. The steady state intestinal and hepatic extraction ratios were 0.262 +/- 0.055 and 0.992 +/- 0.014, respectively, at 40 microM, and 0.206 +/- 0.035 and 0.638 +/- 0.117, respectively, at 200 microM. SAM glucuronide was found to be the only metabolite formed by the intestine at both doses. Less than 3% of the dose was secreted into the intestinal lumen, with SAM glucuronide and SAM as the major and minor components, respectively. Hepatic metabolism of SAM, however, revealed SAM sulfation as the predominant pathway, while glucuronidation and hydroxylation were minor metabolic pathways. About 6% of the dose was excreted into bile, mostly as SAM and gentisamide glucuronides. The interrelationship between the intestine and liver clearances was also examined by mass balance considerations and simulation of data. The total rate of elimination of substrate across the two organs is the sum of the rates of metabolism by each organ. However, the overall effective extraction ratio and, hence, the clearance, are less than the sum of the individual extraction ratios and organ clearances, respectively. Our results showed that intestinal metabolism regulated the available substrate for hepatic elimination, and hence modified the contribution of hepatic metabolism in the overall first-pass effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism and disposition of sulfinalol in laboratory animals

Peak levels of radioactivity in blood occurred 1.0 hr after oral administration of 3H-sulfinalol hydrochloride to rats, dogs, and monkeys. The plasma decay curve for intact sulfinalol in the dog was biphasic, with apparent first-order half-lives of 0.55 and 6.2 hr. Rats excreted 42.5% of the dose in the urine and 31.8% in the feces after 24 hr. Urinary and fecal recovery were 53.8% and 41.2%, respectively, after 10 days for dogs and 57.8% and 38.0%, respectively, after 9 days for monkeys. Free sulfinalol (11.8% of the dose) was the major component in dog feces with lesser amounts of the sulfide and sulfone metabolites, also in the unconjugated form. All metabolites in dog urine were conjugated with glucuronic acid, with sulfinalol (28.5%) and desmethylsulfinalol (8.5%) representing the major constituents, whereas the sulfone and sulfide metabolites were minor ones. Monkey feces contained primarily unconjugated forms of the desmethyl sulfide metabolite (17.0%) and sulfinalol (7.5%); lesser amounts of desmethylsulfinalol and the sulfone metabolite were present. Desmethylsulfinalol (8.7%) and its sulfate (7.0%) and glucuronide (4.0%) conjugates were the major urinary metabolites in the monkey; sulfinalol (1.4%), its glucuronide conjugate (5.1%), the desmethyl sulfide metabolite (and its sulfate conjugate), and the sulfone metabolite were also present.     

Extent of urinary excretion of p-hydroxyphenytoin in healthy subjects given phenytoin

Urinary excretion of p-hydroxyphenytoin and its glucuronide conjugate was measured in eight healthy young adults in a comparative bioavailability study of oral sodium phenytoin (approximately 5 mg/kg/dose). Among these subjects the percentage of the phenytoin dose converted to p-hydroxyphenytoin and appearing in urine was relatively similar (mean 79%, range 67-88%). The great majority of the p-hydroxyphenytoin appeared in urine as conjugates; only 1.4-3.4% of the excreted p-hydroxyphenytoin was in the form of unconjugated metabolite. The proportion of a single phenytoin dose excreted in urine as p-hydroxyphenytoin or its conjugate increased from the first dose (mean +/- SD) 74.9 +/- 4.6% to the second dose, given 2 weeks later 79.3 +/- 4.6% (p less than 0.05). This finding suggests that autoinduction of phenytoin metabolism may occur after relatively brief exposure to the drug.     

Pharmacokinetics of ceftizoxime suppository in experimental animals

Ceftizoxime suppository (CZX-S) was administered rectally to mice, rats and dogs, and the pharmacokinetics were studied in comparison with those after intravenous, intramuscular and subcutaneous administration of ceftizoxime (CZX). Absorption of CZX given rectally was rapid in all animals, similar to intramuscular or subcutaneous administration. The peak serum levels of CZX in mice, rats and dogs when administered rectally at a dose of 25 mg/kg were 23.1 micrograms/ml at 7.5 minutes, 23.5 micrograms/ml at 15 minutes and 25.2 micrograms/ml at 15 minutes, respectively. These values were about 76%, 68% and 42% of the values for subcutaneous or intramuscular administration in mice, rats and dogs at the same respective doses. Urinary recoveries of CZX after rectal administration of 25 mg/kg were 44.2% (0-12 12 hours) in rats and 27.7% (0-6 hours) in dogs, and 2.7% (0-6 hours) of the dose was excreted into bile fluid in rats. Organ distribution of CZX when administered rectally to rats was similar in distribution pattern to that of muscular administration, although its concentrations in various organs were slightly lower than those for intramuscular administration, as was the case for serum concentration. Serum concentrations of CZX were proportionately elevated with dose when dogs were rectally administered CZX-S in doses of 12.5, 25 and 50 mg/kg. In the case of multiple administrations (t.i.d. for 10 days) of CZX-S to dogs, no remarkable difference was found in serum concentrations of CZX in comparison with single doses, and no accumulation of CZX was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chlorzoxazone: a probe drug whose metabolism can be used to monitor toluene exposure in rats

In this study we investigated cytochrome P450 (CYP) 2E1 expression using a probe drug, chlorzoxazone (CZX), whose metabolism can be used to monitor toluene exposure in rats. The animals received an i.p. injection of toluene (0.25, 0.5 and 1 ml/kg) once a day for 3 days. The total CYP and CYP2E1 content and the aniline and CZX hydroxylase activity (V _max and CL_int) increased depending on the dose of toluene administered. At the highest concentration (128 mM) of diethyldithiocarbamate, a specific inhibitor of CYP2E1, the production of 6-hydroxychlorzoxazone (HCZX) in microsomes from toluene-treated rats was reduced by about 80%. The IC₅₀ values in microsomes from toluene-treated rats were between 3 and 5 μM. The production of HCZX and the activity of aniline hydroxylase in toluene-treated rats were correlated with the amount of rat CYP2E1 protein (r=0.88 and r=0.88, respectively). The elimination of CZX by toluene-treated rats was increased and the HCXZ production in the toluene-treated group was greater than that in the olive oil control group. The correlations between intrinsic clearance (CL_int: V _max/K _m) in vitro and total body clearance (CL_tot) of CZX hydroxylation and the elimination half-life (t _1/2) of CZX in vivo in toluene-treated rats were high (r=0.784, P < 0.001; r=−0.678, P < 0.001, respectively). In addition, the metabolic plasma HCZX/CZX ratio did not require multiple blood sampling and 2 h after CZX administration in vivo there was also a high correlation with CL_int (V _max/K _m) in vitro (r=−0.729, P < 0.001). In conclusion, these results demonstrate that CZX is a very good probe for monitoring induction in toluene-treated rats. Received: 28 September 1999 / Accepted: 10 January 2000     

Metabolism and enterohepatic circulation of benzo(a)pyrene-4,5-epoxide in the rat

After i.v. administration of 3H-benzo(a)pyrene-4,5-epoxide (32.5 mumol/kg) to rats, 76% of the 3H appeared in bile within 3 h. The glutathione conjugate of benzo(a)pyrene-4,5-epoxide was the major biliary metabolite (33% of dose), together with a glucuronic acid conjugate of benzo(a)pyrene-4,5-diol (18%) and an unidentified metabolite (10%). The glutathione and glucuronic acid conjugates both undergo extensive enterohepatic circulation. Thus, following the intraduodenal administration of the 3H-labelled conjugates, 26% of the radioactivity was excreted in the bile after 24 h in the case of the glutathione derivative, and 40% in the case of the glucuronide. The benzo(a)pyrene-4,5-diol glucuronide, on enterohepatic circulation, appears in the bile in the same form as the conjugate administered with no evidence of further metabolism of the polycyclic hydrocarbon moiety. The glutathione conjugate of benzo(a)pyrene-4,5-epoxide, on recirculation, is reexcreted in bile as one unidentified metabolite, which is susceptible to the action of arylsulphatase.     

Pathways of disposition of acetaminophen conjugates in the mouse

After a single dose of [¹⁴C] acetaminophen (150 mg/kg) was administered orally to bile duct cannulated mice, 13.9% of the radioactivity was recovered in the bile while 41.2% was found in the urine in the first 3 h after drug administration. Analyses of biles revealed that the major biliary metabolite was acetaminophen glutathione (AG) conjugate which was derived from the hepatotoxic acetaminophen intermediate. Examination of urines showed that they contained mostly glucuronide and sulfate conjugates with no AG or its degradation products (cysteine and mercapturate). Analysis of urines collected from non-cannulated animals at 4 h showed that they contained glucuronide, sulfate, cysteine and mereapturate metabolites. Our results suggest that after formation in the liver, the majority of the glucuronide and sulfate conjugates were directly eliminated by the kidney. On the other hand, the pathway for the disposition of the glutathione conjugate was first into the bile, then reabsorption, and finally disposition into the urine as cysteine and mercapturate metabolites.