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S H Chiu

Publications and source records attributed to S H Chiu.

At least 37 records · Page 2Linked to original sources
PubMed1997

Orally active inhibitors of human leukocyte elastase. II. Disposition of L-694,458 in rats and rhesus monkeys.

The disposition of L-694,458, a potent monocyclic beta-lactam inhibitor of human leukocyte elastase, was studied in male Sprague-Dawley rats and rhesus monkeys. After iv dosing, L-694,458 exhibited similar pharmacokinetic parameters in rats and rhesus monkeys. The mean values for its plasma clearance, terminal half-life, and volume of distribution at steady state were 27 ml/min/kg, 1.8 hr, and 4.0 liters/kg in rats and 34 ml/min/kg, 2.3 hr, and 5 liters/kg in rhesus monkeys. The bioavailability of a 10 mg/kg oral dose was higher in rats (65%) than in rhesus monkeys (39%). In both species, concentrations of L-694,458 in plasma increased more than proportionally when the oral dose was increased from 10 mg/kg to 40 mg/kg. In monkeys a protracted plasma concentration-time profile was observed at 40 mg/kg, characterized by a delayed T(max) (8-24 hr) and a long terminal half-life (6 hr). [3H]L-694,458 was well absorbed after oral dosing to rats at 10 mg/kg, as indicated by the high recovery of radioactivity in bile (83%) and urine (6%) of bile duct-cannulated rats. Only approximately 5% or less of the radioactivity in bile, urine, and feces was a result of intact L-694,458, indicating that the compound was being eliminated by metabolism, followed by excretion of the metabolites in feces, via bile. Demethylenation of the methylenedioxyphenyl group resulting in the catechol was the primary metabolic pathway in human and rhesus monkey liver microsomes. In rat liver microsomes, the major metabolite was the N-oxide of the methyl-substituted piperazine nitrogen. In rats dosed iv and orally with [3H]L-694,458, concentrations of radioactivity were highest in the lung (the primary target tissue), adrenals, and liver. L-694,458 was unstable in rat blood and plasma, degrading via a pathway believed to be catalyzed by B-esterases and to involve cleavage of the beta-lactam ring and loss of the methylpiperazine phenoxy group. In vitro studies indicated that in human liver, L-694,458 was metabolized by CYP3A and 2C isozymes, and in both monkey and human liver microsomes the compound acted as an inhibitor of testosterone 6beta-hydroxylation.

Administration, Oral↗
PubMed1997

Orally active inhibitors of human leukocyte elastase. III. Identification and characterization of metabolites of L-694,458 by liquid chromatography-tandem mass spectrometry.

The in vitro and in vivo metabolism of N-[1(R)-(1,3-benzodioxol-5-yl)butyl]-3,3-diethyl-2(S)-[4-[(4-methy l-1-piperazinyl)carbonyl]phenoxy]-4-oxo-1-azetidinecarboxamide (L-694,458) was studied in male Sprague-Dawley rats and rhesus monkeys. Analysis by LC-MS/MS and NMR revealed that the major metabolite generated in incubations with rat liver microsomes resulted from N-oxidation of the piperazine group, while the major metabolite generated in monkey liver microsomes was the catechol that resulted from O-dealkylation of the methylenedioxyphenyl group. Other metabolites observed in these incubations include the piperazine N-desmethyl, several monohydroxylated derivatives of the parent compound, and three products that resulted from cleavage of the beta-lactam ring. Incubations of parent compound with rat hepatocytes in culture generated two major metabolites that resulted from cleavage of the piperazine ring with the loss of an ethylene group from one side of the ring; one of these metabolites retained the piperazine N-methyl group, while the other did not. The metabolite profiles in vivo were similar to those observed in vitro, but they were much more complex owing to secondary and, in some cases, tertiary biotransformations of many of the primary metabolites. Bile obtained from orally dosed rats contained more than 40 parent-related components, and many of these metabolites had arisen from piperazine ring cleavage.

Administration, Oral↗
PubMed1997

Pharmacokinetics and disposition of the oxytocin receptor antagonist L-368,899 in rats and dogs.

L-368,899 is a potent, orally-active oxytocin antagonist that completed phase I clinical trials for the prevention of preterm labor. The pharmacokinetics and disposition of L-368,899 were studied in rats (female and male) and dogs (female), the two species used in the toxicology studies. L-368,899 exhibited similar pharmacokinetics in rats and dogs. After iv dosing at 1, 2.5, and 10 mg/kg, the compound had a t1/2 of approximately 2 hr and plasma clearance between 23 and 36 ml/min/kg at all doses and in both species. The exception was female rats at the 10 mg/kg dose where plasma clearance decreased to 18 ml/min/kg. The Vdss was between 2.0 and 2.6 liters/kg for rats and 3.4 to 4.9 liters/kg for dogs. After oral doing, L-368,899 was rapidly absorbed. Mean Cmax values were achieved at <1 hr at the low doses (25 mg/kg in rats and 5 mg/kg in dogs) and between 1 and 4 hr at the higher doses (100 mg/kg in rats and 33 mg/kg in dogs). In bile duct-cannulated female rats, approximately 70% of a radioactive 28 mg/kg dose was recovered in bile and urine within 72 hr post dose. Plasma drug concentrations were higher in female than in male rats especially at the 25 mg/kg dose, where mean AUC values were 4.5-fold higher in the females. In both rats and dogs, plasma drug levels increased more than proportionally with increasing oral dose. In female rats, the mean AUC increased by approximately 8-fold between 25 and 100 mg/kg, while in female dogs, the mean AUC at the 33 mg/kg dose was 12-fold higher than that at 5 mg/kg. Oral bioavailability was estimated at 14% and 18% for the 5 mg/kg dose in female and male rats, respectively, 41% for the 25 mg/kg dose in male rats and 17% and 41%, respectively, for the 5 and 33 mg/kg doses in dogs. Owing to nonlinear kinetics, bioavailability could not be calculated for the other oral doses. L-368,899 was metabolized extensively in both species after iv and oral dosing, with <10% of the dose excreted unchanged. The main route of elimination was via the feces, which contained >70% of the radioactive dose by 48 hr, primarily as metabolites. The gender and dose dependence of the pharmacokinetics of L-389,899 in rats were attributed to gender differences in metabolizing capacity and saturation of hepatic metabolism, respectively. This conclusion was based primarily on results from experiments comparing the rate of in vitro metabolism of L-368,899 in liver microsomes, which showed that the Vmax and KM values for L-368,899 were 4-fold lower in female than in male rat liver microsomes.

Administration, Oral↗
PubMed1996

Pharmacokinetics and disposition of L-692,429. A novel nonpeptidyl growth hormone secretagogue in preclinical species.

L-692,429 is a novel nonpeptidyl growth hormone secretagogue that has been demonstrated to stimulate growth hormone secretion in rats, dogs, and humans after intravenous administration. We have examined the pharmacokinetics and disposition of L-692,429 in male Sprague-Dawley rats, beagle dogs, and chimpanzees. Plasma clearance (CLp) of L-692,429 in dogs after intravenous dosing was approximately 18 ml/min/kg and was constant between the doses of 0.1 and 0.9 mg/kg. In rats, CLp after intravenous dosing increased from 3 to 12 ml/min/kg in a dose-dependent manner between 0.1 and 5 mg/kg. In chimpanzees, CLp after an intravenous dose of L-692,429 at 0.5 mg/kg was 5.7 ml/min/kg. In vitro binding of L-692,429 to plasma proteins of dogs, chimpanzees, and humans was approximately 87%, 94%, and 93.5%, respectively, and was independent of concentration. In contrast, plasma binding of L-692,429 was concentration-dependent in rats and decreased from 98.5% to 90.6% between 0.01 and 10 micrograms/ml. Metabolism of L-692,429 was minimal in rats, but moderate in dogs, with the major metabolite being a derivative monohydroxylated at the benzolactam moiety. Thus, the faster clearance of L-692,429 in dogs likely is caused by less extensive plasma protein binding and higher metabolic clearance. The nonlinear pharmacokinetics in rats probably is the result of concentration-dependence in plasma binding. The results of these studies suggest that plasma protein binding plays a major role in determining the values of CLp of L-692,429 among the species. After an intravenous dose of [3H]L-692,429 to rats, liver, kidney, lung, and heart had the highest levels of radioactivity at the early time points, but the gastrointestinal tract had increasing concentrations at later time points. Most of the radioactivity was cleared from all tissues by 24 hr, indicating that L-692,429 did not accumulate in tissues. After intravenous dosing of [3H]L-692,429 to rats and dogs, recoveries of total radioactivity in urine and feces corresponded to approximately 10% and 90%, respectively. Greater than 70% of radioactivity was recovered in bile of rats within 24 hr after intravenous dosing of [3H]L-692,429, indicating that biliary excretion was the primary route of elimination. Based on the combined recoveries of the radioactive dose in bile and urine after an oral dose of L-692,429, oral absorption in rats was approximately 3%. The poor absorption may be the result of the zwitterionic nature of this compound.

Animals↗
PubMed1996

Orally active inhibitors of human leukocyte elastase. I. Disposition of L-683,845 in rats and rhesus monkeys.

L-683,845 is an orally active inhibitor of human leukocyte elastase. Its disposition was studied in rats and rhesus monkeys after dosing with a 3H- or 14C-labeled compound intravenously at 5 mg/kg and orally at 10 mg/kg. L-683,845 exhibited different pharmacokinetics in these two species. In rats, L-683,845 was well-absorbed after oral dosing, with a maximum concentration of 6 microg/ml at 2 hr and bioavailability of approximately 100%. After intravenous dosing, it was cleared slowly at approximately 3 ml/min/kg, with a terminal half-life of approximately 7 hr and a volume of distribution at steady-state of 1 liter/kg. After both intravenous and oral dosing, L-683,845 comprised 50-95% of plasma radioactivity. About 75% of the intravenous and 87% of the oral dose were recovered in the feces as parent and/or conjugates, with the remaining fraction recovered in the urine as polar components. In rhesus monkeys, maximum concentration after oral dosing was only 0.25 microg/ml, and bioavailability was 50%. Plasma clearance was 8-fold higher, at 23 ml/min/kg, and volume of distribution at steady-state larger, at 2 liters/kg, than in rats. The terminal half-life of L-683,845 could not be determined accurately after intravenous dosing, but seemed to be long in orally dosed animals, approximately 13 hr. Intact L-683,845 was a minor component in plasma comprising only approximately 20% of the radioactivity at most time points. Moreover, persistent levels of radioactivity were detected in plasma and urine of rhesus monkeys even at 1-month postdose, and > or = 25% of the radioactivity in plasma was irreversibly bound to proteins at the later time points. Recovery of the radioactivity was incomplete, with only 77% of the intravenous and 43% of the oral dose recovered over a 4-day period. L-683,845-derived radioactivity distributed to all major rat tissues, with highest levels in the liver followed by the small intestine, adrenals, kidneys, and lungs. Radioactivity concentrations in the liver were high even at 24 hr, 22.7 microg eq/g. A large portion of the intravenous dose was recovered in the small intestine, approximately 40% at 2 hr, indicating rapid and extensive biliary excretion. L-683,845 was metabolized primarily to the acyl glucuronide, which was very unstable in rat plasma, and was subject to hydrolysis to L-683,845 and rearrangement. The glucuronide and L-683,845 were degraded in rat plasma by opening the beta-lactam ring and loss of the C4 substituent followed by decarboxylation to give an olefin and/or decomposition to the monosubstituted urea. Based on inhibition by organophosphorus compounds, it is speculated that their degradation is catalyzed by a type B esterase.

Animals↗
PubMed1995

Mechanism of bioactivation and covalent binding of 2,4,6-trinitrotoluene.

Studies were undertaken to investigate the mechanism of bioactivation and covalent binding of TNT. Incubation of [14C]TNT with rat liver microsomes in the presence of an NADPH generating system resulted in metabolism and covalent binding to microsomal proteins. Time-dependence studies showed that TNT was rapidly reduced to yield 4-hydroxylamino-2,6-dinitrotoluene (4HA), 4-amino-2,6-dinitrotoluene (4A) and 2-amino-4,6-dinitrotoluene (2A) as intermediates which were further metabolized to form 2,4-diamino-6-nitrotoluene (2,4DA) and 2,6-diamino-4-nitrotoluene (2,6DA). In contrast to the rapid disappearance of TNT, formation of covalent protein adducts increased with time, suggesting that the reactive intermediate was likely to be formed not directly from TNT but from proximal intermediates such as 4HA. The hypothesis that 4HA was more readily converted to the reactive intermediate than TNT was further supported by the increased levels of covalent adduct formation when [14C]4HA was incubated directly with liver microsomes. Covalent binding of TNT and 4HA was dependent on oxygen concentration. Higher levels of covalent adducts were formed when TNT was incubated aerobically (up to 50% oxygen concentration) than under anaerobic conditions. Covalent binding of [14C]4HA also increased with increasing oxygen concentrations. These results suggest that the reactive intermediate is likely to be an oxidized metabolite of 4HA, e.g. 4-nitroso-2,6-dinitrotoluene. Compounds containing a free sulfhydryl group (cysteine, N-acetylcysteine, GSH or 3,4-dichlorobenzenethiol) decreased the amount of covalent binding to various degrees, suggesting the involvement of the sulfhydryl group in adduct formation with TNT following bioactivation. Metabolic activation of TNT by liver microsomes required NADPH but not NADH as the cofactor. Incubation of [14C]TNT with purified rat liver NADPH cytochrome P450 reductase under either aerobic or anaerobic conditions yielded exclusively 4HA. In contrast, 2A and 4A were formed following incubation of TNT with the reconstituted system containing cytochrome P450, NADPH cytochrome P450, reductase and dilauroyl phosphatidylcholine. These observations suggest that the initial reduction of the nitro group can be catalyzed by NADPH cytochrome P450 reductase alone but cytochrome P450 is needed in the reduction of the hydroxylamine to the amine.

Animals↗
PubMed1995

Biotransformation of losartan to its active carboxylic acid metabolite in human liver microsomes. Role of cytochrome P4502C and 3A subfamily members.

Losartan is a 4-chloro-5-hydroxymethylimidazole derivative that is a potent and highly selective angiotensin II receptor antagonist. Losartan is metabolized in vivo in rats, monkeys, and humans to a carboxylic acid derivative E3174 that is pharmacologically more active than the parent compound. We have investigated the mechanism of this biotransformation in human liver preparations. The oxidation of both losartan and the putative aldehyde intermediate E3179 was catalyzed by the microsomal fraction, required both NADPH and molecular oxygen, and was inhibited by SKF 525-A, implicating cytochrome P450 (CYP). When incubations with each substrate were performed under an atmosphere of 18O2, the extent of 18O incorporation into the carboxylic acid product was consistent with a mechanism for losartan oxidation involving an aldehyde intermediate. To substantiate the involvement of CYP in these reactions, incubations with losartan and the aldehyde E3179 were performed in the presence of isoform-selective inhibitors. Inhibitors of CYP3A4/5 (gestodene and ketoconazole) and CYP2C9/10 (sulfaphenazole) attenuated the oxidation of both substrates. It was then demonstrated that microsomes containing either recombinant human liver CYP2C9 or CYP3A4 were capable of oxidizing both losartan and the aldehyde E3179 to the carboxylic acid E3174. Subsequently, it was shown that rabbit anti-CYP2C9 and anti-CYP3A3/4 inhibited the oxidation of losartan to E3174 in incubations with human liver microsomes. These studies support the hypothesis that the aldehyde E3179 is an intermediate in the oxidation of losartan and that this two-step reaction is catalyzed in human liver microsomes by members of the CYP3A and CYP2C subfamilies.

Adult↗
PubMed1995

Identification of human cytochrome P450 isozymes responsible for the in vitro oxidative metabolism of finasteride.

Finasteride, a prescription drug for the treatment of benign prostatic hypertrophy and alleviation of symptoms associated with benign prostatic hypertrophy and alleviation of symptoms associated with benign prostatic hypertrophy, has been shown to be metabolized in rat hepatic microsomes by hydroxylation at the t-butyl group (omega-OH finasteride), followed by further oxidation to the corresponding acid (omega-oic acid finasteride), with omega-aldehyde finasteride as an intermediate. In this study, we identified specific human cytochrome P450 (CYP) isozyme(s) involved in the in vitro metabolism of [14C]finasteride using CYP isozyme-selective inhibitors and microsomes containing specific recombinant human CYP isozymes (expressed in human AHH-1 TK+/-cells). Each of the three steps of the oxidative pathway was examined separately by using [14C]finasteride and its consecutive metabolites (omega-OH finasteride and omega-aldehyde finasteride) as substrates, and human liver microsomes or expressed recombinant CYP isozymes as the enzyme source. Gestodene, a mechanism-based inhibitor of CYP3A isozymes, showed a concentration-dependent inhibition of the oxidative metabolism of [14C]finasteride. In addition, the respective omega-OH finasteride and omega-oic acid finasteride metabolites were generated only by microsomes containing recombinant CYP3A4, but not the other isozymes (CYP1A1, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP2E1). Similar results were obtained for the oxidation of omega-OH finasteride to omega-aldehyde finasteride, suggesting that human CYP3A isozymes were involved in the oxidation of omega-OH finasteride. When omega-aldehyde finasteride was incubated with human liver microsomes in the presence of an NADPH regenerating system, both the omega-oic acid finasteride and the omega-OH finasteride were detected, suggesting that oxidative and reductive reactions were occurring simultaneously and that they were NADPH- or NADP-dependent. Inhibitors of CYP3A isozymes inhibited the oxidation of omega-aldehyde finasteride in a concentration-dependent manner; an increase in the reduction was also observed, presumably caused by inhibition of the competitive oxidative reaction. Other selective CYP inhibitors for CYP1A1/2 (alpha-naphthoflavone), CYP2C8-10 (sulfaphenazole), CYP2D6 (quinidine), and CYP2E1 (diallylsulfone) showed minor or no effects on both reactions. Consistent with these results, only microsomes containing human recombinant CYP3A4 catalyzed the oxidation of omega-aldehyde finasteride to omega-oic acid finasteride. These results indicate that the oxidation of omega-aldehyde finasteride was NADPH-dependent and was mediated at least in part by CYP3A4. In addition, NAD-dependent enzymes in cytosolic, microsomal, and mitochondrial fractions were capable of oxidizing omega-aldehyde finasteride to omega-oic acid finasteride. Other cellular fractions, particularly mitochondria, were shown to convert finasteride to omega-oic acid finasteride in a similar fashion.

5-alpha Reductase Inhibitors↗
PubMed1994

1-((7,7-Dimethyl-2(S)-(2(S)-amino-4-(methylsulfonyl)butyramido)bicyclo [2.2.1]-heptan-1(S)-yl)methyl)sulfonyl)-4-(2-methylphenyl)piperaz ine (L-368,899): an orally bioavailable, non-peptide oxytocin antagonist with potential utility for managing preterm labor.

Modifications to the previously reported spiroindenylpiperidine camphor-sulfonamide oxytocin (OT) antagonist L-366,509 have produced a new series of o-tolylpiperazine (TP) camphor-sulfonamides. A number of analogues in the TP series that incorporate a modified or unmodified L-methionine sulfone amide at the C2 endo position on the camphor ring exhibit high affinity for OT receptors (IC50 = 1.3-15 nM) and good selectivity for binding to OT versus arginine vasopressin V1a and V2 receptors. Several of these analogues were additionally characterized as potent antagonists of OT-stimulated contractions of the isolated and/or in situ rat uterus. Compound 7 (L-368,899) exhibited the best overall profile of OT receptor affinity (IC50 = 8.9 nM, rat uterus; 26 nM, human uterus), potency for inhibition of OT-stimulated contractions of the isolated rat uterus (pA2 = 8.9) and in situ rat uterus (AD50 = 0.35 mg/kg after intravenous (i.v.) administration and 7.0 mg/kg after intraduodenal administration), aqueous solubility (3.7 mg/mL at pH 5.0), and oral bioavailability in several species (35% (rat), 25% (dog), and 21% (chimpanzee) as estimated from radioreceptor determination of drug levels in plasma after oral and i.v. dosing). On the basis of these favorable properties, 7 has begun clinical testing for use as an oral and i.v. tocolytic agent. Molecular modeling alignment studies have provided support for the hypothesis that the TP camphor-sulfonamide portion of the non-peptide structures may serve as a mimetic of the important D-AA2-Ile3 dipeptide (AA = aromatic amino acid) found in many potent OT antagonists from the cyclic hexapeptide and OT analogue structural classes.

Animals↗
PubMed1993

The use of in vitro metabolism studies in the understanding of new drugs.

In vitro models derived from various animal species are routinely used for the evaluation of pharmacological potency and toxicological potential of new drug candidates. It is well known that metabolism of the drug molecule often contributes to the efficacy and toxicity observed in vivo. In vitro metabolism studies conducted in biological matrixes ranging from intact organ, tissue slice to subcellular fraction offer the advantage of reduced complexity of the study system, and allow the evaluation of intrinsic metabolic potential or mechanism with respect to a specific reaction. In addition, in vitro systems derived from various animal species offer the possibility of comparing metabolic pathways among species, including humans, before a compound can be tested clinically. Two areas of particular interest for the use of in vitro studies in the understanding of new drugs are 1) the relationship of metabolic pathway and pharmacodynamics and 2) the prediction of drug interaction potential in humans. To illustrate these aspects, I review metabolism studies on losartan, an angiotensin-II receptor antagonist currently in phase-III clinical development for hypertension, and cyclosporin A, a widely prescribed immunosuppressant.

Angiotensin Receptor Antagonists↗
PubMed1992

Determination of enantiomeric concentrations of a 2,5-diaryltetrahydrofuran (L-668,750), a platelet-activating factor receptor antagonist, in rat plasma using a chiral alpha 1-acid glycoprotein high-performance liquid chromatographic column.

Racemic sulfonylated 2,5-diaryltetrahydrofuran [L-668,750, (+-)-trans-2-[3-methoxy-5-(2-hydroxy)ethylsulfonyl-4-n-propoxy]-p henyl-5-(3,4,5-trimethoxyphenyl)-tetrahydrofuran, I] is a potent, specific and orally active platelet-activating factor (PAF) receptor antagonist. Its (-)-(2S,5S) enantiomer [L-680,573, (S)-I] exhibited higher PAF antagonistic potency than the (+)-(2R,5R) enantiomer [L-680,574, (R)-I] in vitro and in animal models. For assay of drug concentrations in plasma of rats dosed intravenously or orally with tritium-labeled I, we have developed a high-performance liquid chromatographic (HPLC) method which directly resolved the two enantiomers. The column contained alpha 1-acid glycoprotein as the chiral stationary phase and was eluted with phosphate buffer, methanol and ethanol at neutral pH. The concentration of each enantiomer in the plasma was then determined by reverse isotope dilution assay. Results showed that the plasma clearance rate of the more potent (S)-I enantiomer was more than ten-fold faster than that of the (R)-I enantiomer; the enantioselective clearance resulted in nearly ten-fold higher concentrations of the latter in plasma at all time points regardless of the dosing route. This paper describes the HPLC chiral resolution method and its application in plasma analysis.

Animals↗
PubMed1992

In vitro metabolism of FK-506 in rat, rabbit, and human liver microsomes: identification of a major metabolite and of cytochrome P450 3A as the major enzymes responsible for its metabolism.

The metabolism of the immunosuppressant FK-506 was shown to be catalyzed primarily by cytochrome P450 isozymes of the P450 3A subfamily. Antibodies against rat P450 3A inhibited FK-506 metabolism by 82% in rat liver microsomes and by 35-56% in liver microsomes from humans, dexamethasone-induced rats, and erythromycin-induced rabbits. Poor species cross-reactivity of the antibodies, metabolic switching, and/or some metabolism by P450 isozymes other than P450 3A may be responsible for the incomplete inhibition observed. Besides anti-rat P450 3A, antibodies against rat P450 1A also appeared to have some inhibitory effect implicating these particular cytochrome P450 isozymes as having a minor role in FK-506 metabolism. The formation of 13-desmethyl FK-506, identified here as a major metabolite of FK-506 in all types of microsomes examined, was inhibited completely by anti-P450 3A in liver microsomes from dexamethasone-induced rats and erythromycin-induced rabbits but only partially in human and control rat liver microsomes.

Adult↗
PubMed1992

In vivo covalent binding of [14C]trinitrotoluene to proteins in the rat.

When a single dose of [14C]trinitrotoluene was administered intraperitoneally (i.p.) to rats at 1, 10 or 50 mg/kg of body weight, covalently bound radioactivity was detected in globin, plasma proteins and proteins in the liver and kidney. The extent of covalent binding was dose dependent and was highest in plasma and renal proteins at all times up to 4 h after dosing. Covalent adduct levels in globin, however, decline slower than others. At a dose of 50 mg/kg of body weight, globin covalent adduct levels peaked at 1 h after dosing at 182 pmol/mg protein and subsequently decreased to approximately 50 pmol/mg protein between days 1 and 8. Of the covalent adduct levels in liver and kidney, those in the 10,000 x g and microsomal fractions were found to be higher than that in the cytosolic fraction. Radioactivity covalently bound to globin and the hepatic proteins was susceptible to dilute acid hydrolysis from which 2-amino-4,6-dinitrotoluene (2A) and 4-amino 2,6-dinitrotoluene (4A) were the major products recovered by solvent extraction. Upon acetylation, the hydrolysate gave rise to derivatives identified as the acetates of 2A and 4A on the basis of mass spectrometry and HPLC cochromatography with authentic samples. Four hours after an i.p. dose of [14C]TNT at 50 mg/kg of body weight about 0.4% of the dose was found as bound adducts to hemoglobin, of which approximately 48% was recovered as solvent extractable radioactivity after acid hydrolysis. About 2% of the radioactive dose was in the liver, of which approximately 30% was covalently bound to hepatic proteins, and approximately 49% of that was convertible to solvent extractable radioactivity upon acid hydrolysis. In vitro incubation of [14C]TNT with blood showed that there was a linear increase of covalent adducts in globin during the first 2 h of incubation; the concentration of covalent adducts was slightly higher than that with plasma proteins. The major compounds recovered from the hydrolysate of the globin adducts were also 2A and 4A as obtained from globin in the in vivo studies. On the basis of the in vitro and in vivo study results, we have confirmed the formation of protein adducts following a single i.p. administration of [14C]TNT at 1, 10 or 50 mg/kg of body weight to the rat or by in vitro incubation with blood.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Effects of the immunosuppressant FK-506 and its analog FK-520 on hepatic and renal cytochrome P450 mixed-function oxidase.

The novel immunosuppressant FK-506 and its analog FK-520 were found to inhibit the hepatic microsomal mixed-function oxidase system in male Sprague-Dawley rats. At 5 and 10 mg/kg/day, s.c., for 6 days they caused 30-80% decreases in cytochrome P450 levels, NADPH-cytochrome P450 reductase, and benzphetamine N-demethylase activities. The metabolism of FK-506 itself was inhibited by 50%. FK-506 and FK-520 had a minimal effect on the renal cytochrome P450 levels unlike cyclosporin A which produced a 67% increase after six daily 25 mg/kg doses. A single dose of FK-506 (25 mg/kg, s.c.) had a minimal effect on the hepatic or renal metabolizing enzyme system. In vitro, addition of FK-506 and FK-520 to human and control rat liver microsomes resulted in a concentration-dependent inhibition of benzphetamine N-demethylation (10-20% at 50 microM, 60-75% at 250 microM). We suggest that in view of its potential to inhibit hepatic cytochrome P450-dependent mixed-function oxidase, resulting in the inhibition of its own metabolism, FK-506 should be administered with caution to transplant patients.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1988

Application of two-dimensional high-performance liquid chromatography in metabolism studies of ivermectin.

Two-dimensional high-performance liquid chromatography (HPLC) of alternate reversed-phase and normal-phase columns was used in the purification, quantification and identification of submicrogram quantities of drug residue and metabolites in tissues from animals dosed with ivermectin. In reverse isotope dilution assay of the parent drug, two-dimensional HPLC assured constant specific activity of the drug isolates. For identification of metabolites from liver tissue, HPLC in two dimensions not only facilitated the purification, but also provided information (capacity factor k') in both systems for the elimination of possible metabolite structures in the course of compound identification. These approaches exemplified by the ivermectin studies should be generally applicable in analyses of complex biological samples in which quantity is frequently a limiting factor.

Animals↗
PubMed1988

A step-wise logistic regression analysis of hepatocellular carcinoma. An aspiration biopsy study.

Fine needle aspiration biopsy (FNAB) has become a popular method to diagnose mass lesions of the liver. Although several reports have listed FNAB criteria to be used to diagnose both primary and metastatic tumors of the liver, none have separated key cytologic criteria from secondary criteria. We reviewed the FNAB smears from 35 patients with proven hepatocellular carcinoma and 74 patients with proven metastatic tumors in the liver. All specimens were coded as to the presence or absence of the following variables: polygonal cells with centrally placed nuclei; well-defined, granular cytoplasm; large nucleoli; small cytoplasmic vacuoles; large cytoplasmic vacuoles; bile; polymorphonuclear leukocytes; malignant cells separated by sinusoidal vessels; endothelial cells surrounding tumor cell clusters; multinucleated tumor giant cells; basophilic intracytoplasmic inclusions; eosinophilic intracytoplasmic inclusions; and intranuclear cytoplasmic inclusions. A step-wise logistic regression analysis was performed on the data to determine the variables predictive of hepatocellular carcinoma. The statistical analysis selected polygonal cells with centrally placed nuclei, malignant cells separated by sinusoidal capillaries, and bile as the key cytologic criteria for hepatocellular carcinoma. Endothelial cells surrounding tumor cell clusters and intranuclear cytoplasmic inclusions were selected as secondary criteria by this analysis.

Biopsy, Needle↗
PubMed1988

Cytologic criteria for fibroadenoma. A step-wise logistic regression analysis.

The authors reviewed the fine-needle aspiration biopsy smears from 62 subjects with proven fibroadenoma, 60 subjects with proven ductal carcinoma, and 42 subjects with proven fibrocystic disease. All smears were coded as to the presence or absence of the following variables: epithelial cells, stroma, honeycomb sheets, antler horn clusters, naked nuclei, nucleoli, marked cellularity, foam cells, apocrine cells, anisokaryosis, atypical nuclear hyperchromasia, single cells with cytoplasm, mitotic figures, and nuclei greater than two red blood cell diameters. Step-wise logistic regression analyses were performed to determine the variables predictive of fibroadenoma. The statistical analyses selected stroma, antler horn clusters, and marked cellularity as the key cytologic criteria to differentiate fibroadenomas from fibrocystic disease. The statistical analyses selected stroma, antler horn clusters, and honeycomb sheets as the key cytologic criteria to differentiate fibroadenoma from ductal carcinoma.

Adenofibroma↗