Showing papers in "Drug Metabolism and Disposition in 1991"

Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities.

Abstract: Evidence is presented that human serum contains a single enzyme with both paraoxonase and arylesterase activities. Throughout the steps of purification and after obtaining over 600-fold purification of the enzyme, the arylesterase activity (measured with phenylacetate as the substrate) co-eluted and retained the same ratio of activity to paraoxonase activity as it had in the initial plasma sample. Paraoxon and DFP (diisopropylfluorophosphate) both complete with phenylacetate as substrates; the inhibition is of mixed type with paraoxon and competitive with DFP. Paraoxonase and arylesterase activities require calcium, and both are inhibited to the same degree by EDTA. Purified arylesterase/paraoxonase is a glycoprotein with a minimal molecular weight of about 43,000. It has up to three sugar chains per molecule, and carbohydrate represents about 15.8% of the total weight. The enzyme has an isoelectric point of 5.1. Its amino acid composition shows nothing unusual, except for a relatively high content of leucine. We conclude that human serum arylesterase and paraoxonase activities are catalyzed by a single enzyme, capable of hydrolyzing a broad spectrum of organophosphate substrates and a number of aromatic carboxylic acid esters. Studies on the genetically determined polymorphism responsible for two allozymic forms (A and B) of the esterase are described in the following paper.

Metabolites, pharmacodynamics, and pharmacokinetics of tamoxifen in rats and mice compared to the breast cancer patient

Abstract: The metabolism of tamoxifen was examined in the rat, mouse, and human breast cancer patient. Large oral doses of tamoxifen (200 mg/kg) in the immature ovariectomized rat and mature mouse produced circulating levels of the parent compound, N-desmethyltamoxifen, and 4-hydroxytamoxifen quantifiable by HPLC separation, UV activation, and fluorescence detection. N-Desmethyltamoxifen and 4-hydroxytamoxifen serum levels in the mature ovariectomized mouse paralleled tamoxifen levels throughout a 96-hr time course after a single dose of tamoxifen. On the other hand, N-desmethyltamoxifen was the predominant serum metabolite after an equivalent dose of tamoxifen to the immature rat, but there was little 4-hydroxytamoxifen. Peak levels of tamoxifen occurred 3-6 hr after oral administration of tamoxifen in both species, whereas peak levels of N-desmethyltamoxifen in the immature rat did not occur until 24-48 hr. AUCs for tamoxifen and N-desmethyltamoxifen were approximately 4 times greater in the rat (57.5 and 111 micrograms.hr/ml, respectively) than the mouse (15.9 and 26.3 micrograms.hr/ml, respectively) after equivalent doses of tamoxifen (200 mg/kg). AUC of 4-hydroxytamoxifen for the rat (8.9 micrograms.hr/ml), however, was similar to that for the mouse (13.9 micrograms.hr/ml). The rate of elimination from serum was similar for tamoxifen, N-desmethyltamoxifen, and 4-hydroxytamoxifen in both the rat (t1/2 = 10.3, 12.1, and 17.2 hr, respectively) and the mouse (t1/2 = 11.9, 9.6, and 6 hr, respectively). Administration of large oral doses of tamoxifen (200 mg/kg) every 24 hr to mature ovariectomized mice or immature ovariectomized rats resulted in accumulation for the first 4 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiological disposition of alendronate, a potent anti-osteolytic bisphosphonate, in laboratory animals.

Abstract: Alendronate (4-amino-1-hydroxybutylidene-1,1-bisphosphonate) is currently under investigation as an anti-osteolytic agent in the treatment of a broad range of bone disorders. This study describes the absorption and disposition of the drug in laboratory animals. Following iv administration, alendronate was rapidly cleared from plasma, either taken up and sequestered in the bone or excreted by the kidney. About 30 to 40% of the dose was excreted in the urine in 24 hr, with most of the drug being excreted in the first 3 to 4 hr. There was little or no accumulation of the drug in noncalcified tissues and only a very small fraction of the dose was excreted in the bile. Most of the dose was rapidly taken up by bone tissues: 30% in 5 min, 60% in 1 hr. Absorption of alendronate was very poor. Based on the ratios in bone of the labels from the 14C-labeled oral dose and the 3H-labeled iv dose, absorption was estimated to be about 0.9% for the rat, 1.8% for the dog, and 1.7% for the monkey. Comparison of the concentrations of alendronate in bones of the same rats in fasted (3H-labeled) and fed (14C-labeled) states indicated that food caused a substantial decrease in absorption, by about 6- to 7-fold. The terminal half-life of alendronate in bone was about 200 days for the rat. Based on urinary excretion, the terminal half-life was estimated to be about 1000 days for the dog. The long persistence of alendronate in bone was likely due to its slow dissolution rate from bone tissues.

Immunohistochemical localization of cytochrome P-450IA1 induced by 3,3',4,4'-tetrachlorobiphenyl and by 2,3,7,8-tetrachlorodibenzoafuran in liver and extrahepatic tissues of the teleost Stenotomus chrysops (scup).

Abstract: The regulation of different cytochrome P-450 forms and their functions in different organs and cell types could determine the susceptibility of those cells and organs to toxic effects of xenobiotics, including chemical carcinogenesis. Here we describe the cellular localization of cytochrome P-450E (P-450IA1) induced in 10 major organs or organ systems of a marine vertebrate species, the fish, Stenotomus chrysops (scup). Scup were injected ip with 3,3',4,4'-tetrachlorobiphenyl (TCB) at 1 mg/kg, or with 2,3,7,8-tetrachlorodibenzofuran (TCDF) at 3 micrograms/kg. Induction was verified by Western blot analysis of microsomes from selected organs (liver, kidney, and gill) using monoclonal antibody (MAb) 1-12-3 to scup P-450IA1. The localization of P-450IA1 was subsequently determined in sections prepared by standard histological methods (10% buffered formalin fixation, paraffin embedding), and stained with MAb 1-12-3 and peroxidase-labeled second antibody. P-450IA1 was induced in epithelial and endothelial cells in liver (including pancreatic tissue), kidney, gill, gut, spleen, testis, and ovary. Induction also was detected in endothelial cells, but not other types, in heart, brain, and red muscle. In heart, the staining was present in the endocardium as well as in the endothelium of the coronary vasculature and great vessels. Although TCDF and TCB both induced P-450IA1 in various cells of all organs examined, the effect of TCB was in most cases greater than that of TCDF. This may be due to a relatively higher TCB dosage. A wider staining distribution was seen in gut, gill, kidney, and gonad of TCB-treated fish, which might be explained by a greater penetration, or by excretion of parent TCB, as opposed to TCDF. In any case, the results show that these important environmental agents induce P-450IA1 in generally similar patterns in all organs examined. The common finding of a strong induction of P-450IA1 in endothelial cells in all organs examined supports the suggestion that the endothelium may be a primary site of P-450IA1 induction.

Reactions of the nitroso and hydroxylamine metabolites of sulfamethoxazole with reduced glutathione. Implications for idiosyncratic toxicity

Abstract: N4-oxidation of sulfonamides has been implicated in the pathogenesis of idiosyncratic reactions to these antimicrobials. In vitro toxicity assays employing mononuclear leukocytes as target cells have shown that the toxicity of sulfamethoxazole hydroxylamine (SMX-HA) is inhibited by exogenous glutathione, suggesting that conjugation with glutathione is an important detoxification pathway. However, in these experiments, significant depletion of cellular glutathione only occurred at concentrations of SMX-HA greater than or equal to 300 microM. At concentrations of SMX-HA which produce 50% toxicity in mononuclear leukocytes (approximately 100 microM), there was not a significant loss of glutathione. SMX-HA also caused a small but significant increase in oxidized glutathione concentrations. In cell-free experiments, reduced glutathione (GSH) prevented the autooxidation of SMX-HA to nitrososulfamethoxazole (nitroso-SMX). During this process, oxidized glutathione was formed. GSH rapidly reacted with nitroso-SMX to form a labile semimercaptal conjugate. Physiologically relevant concentrations of GSH (i.e. 1 mM) favored thiolytic cleavage of the semimercaptal to form SMX-HA. Isomerization of the semimercaptal to the more stable sulfinamide occurred at low GSH concentrations. Purified glutathione transferases had no effect on the reaction of SMX-HA with GSH. Therefore, glutathione is important in protecting cells from the toxicity of SMX-HA largely by preventing its further oxidation to nitroso-SMX. Stable glutathione conjugates are likely to be formed only in small quantities under physiological conditions. Conjugation with glutathione would not be expected to be a major pathway for clearance of the hydroxylamine and nitroso metabolites of sulfonamides.

Evidence for the involvement of several cytochromes P-450 in the first steps of caffeine metabolism by human liver microsomes.

Abstract: Caffeine biotransformation and four monooxygenase activities involving cytochrome P-450IA2, namely ethoxy- and methoxyresorufin O-dealkylases, phenacetin O-deethylase, and acetanilide 4-hydroxylation were studied in 25 human liver microsomes. All these activities were highly significantly intercorrelated (r greater than 0.72, p less than 0.001) and correlated with the level of immunoreactive P-450IA2 content (r greater than 0.65; p less than 0.001). P-450IA content was measured by immunoblotting with anti-rat P-450 beta-naphthoflavone-B, an antibody that detects only a single band corresponding to P-450IA2. The formation rate of two caffeine metabolites, namely paraxathine and theobromine, was correlated with the four monooxygenase activities measured and P-450IA2-specific content (r greater than 0.75). However, inhibition studies of caffeine metabolism by phenacetin, a specific substrate of P-450IA2, clearly indicated that only the N-3 demethylation of caffeine was supported by this enzyme. These in vitro data demonstrate that P-450IA2 is predominantly responsible for the major metabolic pathway of caffeine and that the formation of other demethylated metabolites is mediated, at least partly, by other P-450 enzymes.

Characteristics of the genetically determined allozymic forms of human serum paraoxonase/arylesterase.

Abstract: Human serum paraoxonase/arylesterase is an esterase with broad substrate specificity. It occurs in two genetically determined allozymic forms, which we have designated types A and B. These allozymes are presumed to be the products of two allelic genes located at the paraoxonase locus on chromosome 7, which is closely linked to the gene for cystic fibrosis. Paraoxonase activity of the B-type isozyme is considerably higher and stimulated more by 1 M NaCl than A-type paraoxonase. The ratio of paraoxonase activity/arylesterase activity of the B-isozyme is about 8, and that of the A-isozyme about 1. Purified isozymes A or B are free of nearly all other serum proteins, and the broad substrate specificity of the serum esterase is preserved after purification. A variety of substrates are hydrolyzed; these include: diisopropylfluorophosphate, soman, sarin, 4-nitro-phenylacetate, 2-nitro-phenylacetate, 2-naphthylacetate, and phenylthioacetate. The isozymic distinctions in kinetic properties and substrate specificity are preserved during purification. It is likely that the allozymes have very similar turnover numbers with phenylacetate (arylesterase activity), but differ considerably in their turnover numbers with paraoxon. Isozymes A and B have about the same minimal molecular weight of 43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Further detailed studies on the individual isozymic proteins (or the DNA coding for their amino acid sequence) will be required to detect the exact structural differences in the isozymes.

Biotransformation of pravastatin sodium in humans

Abstract: Pravastatin sodium (PV) is a potent cholesterol-lowering agent that acts by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase Biotransformation profiles of PV in pooled human urine, plasma, and feces from healthy male volunteers given single 192-mg oral or 99-mg iv doses of [14C]PV were determined by HPLC The predominant drug-related component in urine, plasma, and feces corresponded to intact PV; in the pooled urine samples, PV constituted 29 and 69% of the radioactivity after the po and iv doses, respectively The delta 45-3 alpha-hydroxy isomer of PV constituted 10% (po) and 2% (iv), and 6-epi-PV constituted 3% (po) and 1% (iv) of the urinary radioactivity Negligible amounts of the lactones of PV or its isomers were detected in urine, plasma, or feces At least 15 other metabolites were also present; none of these accounted for more than 6% of the total urinary radioactivity For metabolite isolation, an aliquot of pooled urine samples, obtained after administration of the radioactive dose, was added as a tracer to urine samples obtained from healthy subjects after administration of single nonradiolabeled 40-mg oral doses of PV Urinary metabolites were concentrated on an XAD-2 column, extracted with ethyl acetate, and purified by extensive preparative HPLC In addition to isolation and identification of unchanged drug and the two isomeric metabolites described above, eight other metabolites were isolated and structural assignments were made based on HPLC, UV spectra, mass spectral analysis, and proton NMR(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of the glutathione and N-acetylcysteine conjugates of (E)-2-propyl-2,4-pentadienoic acid, a toxic metabolite of valproic acid, in rats and humans.

Abstract: The severe hepatotoxicity of valproic acid (VPA) is believed to be mediated through reactive metabolites. The formation of glutathione (GSH) and N-acetylcysteine (NAC) adducts of reactive intermediates derived from VPA and two of its metabolites, 2-propyl-4-pentenoic acid (4-ene-) and 2-propyl-2,4-pentadienoic acid [(E)-2,4-diene VPA], was investigated in the rat. Rats were dosed ip with 100 mg/kg of VPA, 4-ene-, or 2,4-diene-VPA, and methylated bile and urine extracts were analyzed by LC/MS/MS and GC/MS, respectively. The GSH conjugate of (E)-2,4-diene VPA was detected in the bile of rats treated with 4-ene- and (E)-2,4-diene VPA. The NAC conjugate was a major urinary metabolite of rats given (E)-2,4-diene VPA and was a prominent urinary metabolite of those animals given 4-ene VPA. The NAC conjugate was also found to be a metabolite of VPA in patients. Both the GSH and NAC adducts were chemically synthesized and their structures established to be 5-(glutathion-S-yl)3-ene VPA and 5-(N-acetylcystein-S-yl)3-ene VPA by NMR and mass spectrometry. In contrast to the very slow reaction of the free acid of (E)-2,4-diene VPA with GSH, the methyl ester reacted rapidly with GSH to yield the adduct. In vivo it appears the diene forms an intermediate with enhanced electrophilic reactivity to GSH as indicated by the facile reaction of the diene with GSH in vivo [about 40% of the (E)-2,4-diene VPA administered to rats was excreted as the NAC conjugate in 24 hr]. The characterization of the GSH and NAC (in humans and rats) conjugates of (E)-2,4-diene VPA suggests that VPA is metabolized to a chemically reactive intermediate that may contribute to the hepatotoxicity of the drug.

Nordihydroguaiaretic acid, an inhibitor of lipoxygenase, also inhibits cytochrome P-450-mediated monooxygenase activity in rat epidermal and hepatic microsomes.

Abstract: Nordihydroguaiaretic acid (NDGA), a plant lignan and phenolic antioxidant, is a known lipoxygenase inhibitor. In this study, we investigated the effect of NDGA on rat epidermal and hepatic monooxygenase activity and its interaction with rat hepatic microsomal cytochrome P-450. The addition of NDGA to epidermal microsomes prepared from control and 3-methylcholanthrene (3-MC)-pretreated rats and hepatic microsomal preparations from control, 3-MC-pretreated, and phenobarbital (PB)-pretreated rats resulted in a concentration-dependent inhibition of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase (ERD) activities. The 50% inhibitory dose for NDGA ranged from 4.1 x 10(-5) to 13.1 x 10(-5) M for AHH and ERD activities in these microsomal preparations. The addition of NDGA to hepatic microsomes prepared from PB-pretreated rats resulted in spectral changes characterized by absorbance maxima at 380 nm and minima at 414 nm, typical of type I binding difference spectra. It also showed time- and concentration-dependent inhibition of the binding of carbon monoxide to dithionite or NADPH-reduced cytochrome P-450. We speculate that perhaps hydroxyl groups present in NDGA play an important role in inhibiting the monooxygenase activity and suggest that NDGA may have potential as an antimutagen and/or anticarcinogen. Furthermore, caution must be exercised in elucidating the role of lipoxygenase in metabolic pathways based solely on the criterion of inhibition by NDGA.

Oxidation of propylthiouracil to reactive metabolites by activated neutrophils. Implications for agranulocytosis.

Abstract: Propylthiouracil (PTU) is associated with idiosyncratic agranulocytosis that may be due to reactive metabolites generated from oxidative metabolism by neutrophils. Therefore, the metabolism of PTU was investigated in activated neutrophils. Three oxidized metabolites were observed on HPLC: PTU-disulfide, propyluracil-2-sulfinate, and propyluracil-2-sulfonate (PTU-SO3-). No metabolism was detected in cells that had not been activated. Metabolism was inhibited by sodium azide and by catalase. The same products were produced by myeloperoxidase (MPO) in an MPO/H2O2/Cl- system. PTU inhibited its own metabolism; however, complete conversion to PTU-SO3- could be achieved with optimal PTU concentrations. MPO/H2O2 without Cl- produced only slight metabolism. The PTU-sulfenyl chloride is a postulated intermediate. In the absence of chloride, oxidation might proceed through propyluracil-2-sulfenic acid. The sulfenyl chloride and PTU-SO3- are both chemically reactive with sulfhydryl compounds such as N-acetylcysteine. Such reactive metabolites, generated by activated neutrophils, may be involved in hypersensitivity reactions associated with PTU, such as agranulocytosis.

Glutathione conjugation with phosphoramide mustard and cyclophosphamide. A mechanistic study using tandem mass spectrometry.

Abstract: The conjugations of cyclophosphamide and of phosphoramide mustard with glutathione are shown to be catalyzed by hepatic cytosolic glutathione-S-transferases. Cyclophosphamide conjugation is also catalyzed by microsomal glutathione-S-transferases, both in intact microsomes and after solubilization and immobilization. Deuterium isotope labels are used to test whether chloride is directly displaced by glutathione in the enzyme-catalyzed conjugations, or whether conjugation takes place via symmetrical cyclic aziridinium ions. Tandem mass spectrometry with high energy collisional activation is shown to provide reliable analysis of the isotope-labeling patterns in the conjugated products. This experiment leads to the conclusion that the aziridinium ion is opened in the conjugation of phosphoramide mustard in both the enzyme-catalyzed and the chemical reactions. Cyclophosphamide, on the other hand, is shown to be conjugated through direct displacement of chloride.

Stereoselective degradation of the fenoprofen acyl glucuronide enantiomers and irreversible binding to plasma protein

Abstract: Stereoselective degradation of fenoprofen (FEN) glucuronides and irreversible binding of FEN enantiomers to human serum albumin via their glucuronides were studied. At different pH values, 37 degrees C, and in the absence of albumin, degradation half-lives were diastereomeric, resulting mainly from a combination of hydrolysis and acyl migration. Lower pH enhanced FEN glucuronide stability and reduced the extent of irreversible binding. The degradation rate of R-FEN glucuronide was greater than that of the S-glucuronide (S-FEN). When human serum albumin was added to the medium, stability was decreased as compared to protein-free buffer. FEN glucuronides were readily hydrolyzed to parent drug, indicating an esterase-like activity of the albumin molecule. In vitro irreversible binding was higher for R-FEN (1.22% +/- 0.36) than for S-FEN glucuronide (0.76% +/- 0.12), when a 0.1 mM concentration of each conjugate enantiomer was incubated under physiological conditions (pH 7.4, 37 degrees C). Incubation with unconjugated FEN did not lead to measurable irreversible binding. Analysis of plasma samples from a clinical study showed that enantioselective irreversible binding of FEN to plasma proteins also occurs in vivo. After administration of a single 600-mg dose of racemic FEN to six healthy volunteers, covalent binding of R- and S-FEN to plasma proteins was measured in all subjects. The percentage of S-FEN protein adduct was greater than that of its R-enantiomer adduct. Total amounts of FEN irreversibly bound to plasma protein in vivo were also very low (1.02 +/- 0.32 and 3.23 +/- 0.85 mol/mol protein x 10(-4) for R- and S-FEN, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma pharmacokinetics and metabolism of 13-cis- and all-trans-retinoic acid in the cynomolgus monkey and the identification of 13-cis- and all-trans-retinoyl-beta-glucuronides. A comparison to one human case study with isotretinoin.

Abstract: In order to compare the disposition and metabolism of 13-cis-retinoic acid (13-cis-RA) and all-trans-retinoic acid (all-trans-RA) in the nonpregnant female cynomolgus monkey, the plasma concentrations of the parent compound, the oxidized metabolites 4-oxo-13-cis-retinoic acid and 4-oxo-all-trans-retinoic acid, and the conjugate metabolites 13-cis-retinoyl-beta-glucuronide (13-cis-RAG) and all trans-retinoyl-beta-glucuronide (all-trans-RAG), were determined on day 1 and day 10 after oral dosing of 2 and 10 mg 13-cis- and all-trans-RA/kg/day. Both 13-cis-RAG and all-trans-RAG have been identified as major plasma metabolites in these studies using thermospray/HPLC/mass-spectrometry of the intact conjugates. AUC comparisons from 0-24 hr after administration indicated that 13-cis-RA treatment resulted in primarily cis metabolites and all-trans-RA treatment resulted in primarily trans metabolites, although low levels of isomerization products were observed. Comparison of the two doses (2 and 10 mg/kg, po) revealed that the AUCs were proportional to the dose administered. Although qualitatively similar, elimination of 13-cis-RA in the monkey was more rapid than in the human, and approximately a 10-fold greater dose of 13-cis-RA was required in the monkey to produce the AUC values comparable to the human. The elimination of all-trans-RA in monkey was faster than that of 13-cis-RA and tended to increase with repeated dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the beta-oxidation of valproic acid in rat liver mitochondrial preparations.

Abstract: Incubation of valproic acid (VPA) with freshly isolated rat liver mitochondria led to the identification of the beta-keto acid, 3-oxo-VPA, together with three unsaturated metabolites, viz. delta 2(E)-VPA, delta 3-VPA, and delta 2(E),3'(E)-VPA. The corresponding beta-hydroxy acid, 3-hydroxy-VPA, was not detected as a metabolite of VPA in this in vitro system. Studies with subcellular fractions of rat liver and with inhibitors of fatty acid metabolism demonstrated that these metabolites are products of the beta-oxidation system in mitochondria, and are not formed to any significant extent by the related peroxisomal beta-oxidation complex. When incubations with mitochondria were carried out in a medium enriched in H218O, metabolites were labeled in a manner consistent with the involvement of coenzyme A thioester intermediates. Incubation of the authentic metabolites with mitochondrial preparations showed that delta 2(E)-VPA, delta 3-VPA, and delta 2(E),3'(E)-VPA are interconverted by isomerization and reduction processes, and that all three serve as precursors of 3-oxo-VPA. It is concluded that VPA is a substrate for beta-oxidation in liver mitochondria, and that competition by VPA for enzymes and/or cofactors of the fatty acid beta-oxidation complex may contribute to VPA-mediated inhibition of fatty acid metabolism and associated hepatic steatosis.

Metabolism and penetration through blood-brain barrier of parkinsonism-related compounds. 1,2,3,4-Tetrahydroisoquinoline and 1-methyl-1,2,3,4-tetrahydroisoquinoline.

Abstract: 14C-Labeled 1,2,3,4-tetrahydroisoquinoline (TIQ) and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) were synthesized, and their metabolism and tissue distribution were studied. Both compounds showed similar metabolic patterns. In 24 hr after po administration (50 mg/kg) to rats, 76% of TIQ and 72% of 1MeTIQ were excreted unchanged, and 2.7 and 8.7% were excreted as the 4-hydroxyl derivatives, 4-hydroxy-TIQ and 4-hydroxy-1MeTIQ, respectively. Small amounts of N-methylated metabolites, 2-methyl-TIQ (0.4%) and 2-methyl-1MeTIQ (0.7%) were detected. Isoquinoline (2.5%) also was found as a metabolite of TIQ and 1-methyl-3,4-dihydroisoquinoline (1.0%) was found as a metabolite of 1MeTIQ. The concentration of labeled compounds in the brain was about 4.5-fold higher than the blood concentration at 4 hr after dosing, and over 90% was unchanged TIQ or 1MeTIQ. These data indicated that TIQ and 1MeTIQ easily passed through the blood-brain barrier and were concentrated in the brain. Thus, it appears that TIQ and 1MeTIQ as endogenous or exogenous amines may accumulate in the brain and may be related to the onset of Parkinson's disease.

The disposition and metabolism of [14C]fluconazole in humans.

Abstract: The disposition and metabolism of 14C-labeled fluconazole (100 microCi) was determined in three healthy male subjects after administration of a single oral capsule containing 50 mg of drug. Blood samples, total voided urine, and feces were collected at intervals after dosing for up to 12 days post-dose. Pharmacokinetic analysis of fluconazole concentrations showed a mean plasma half-life of 24.5 hr. Mean apparent plasma clearance and apparent volume of distribution were 0.23 ml/min/kg and 0.5 liter/kg, respectively. There was no evidence of any significant concentrations of metabolites circulating either in plasma or blood cells. Mean total radioactivity excreted in urine and feces represented 91.0 and 2.3%, respectively, of the administered dose. Mean excretion of unchanged drug in urine represented 80% of the administered dose; thus, only 11% was excreted in urine as metabolites. Only two metabolites were present in detectable quantities, a glucuronide conjugate of unchanged fluconazole and a fluconazole N-oxide, which accounted for 6.5 and 2.0% of urinary radioactivity, respectively. No metabolic cleavage products of fluconazole were detected.

Metabolism of nicotine by rat liver cytochromes P-450. Assessment utilizing monoclonal antibodies to nicotine and cotinine.

Abstract: Because of the prevalence of cigarette smoking in the general population and because studies suggest that a large percentage of nicotine is metabolized to cotinine in humans, it is important to study the enzymes responsible for nicotine metabolism. The cytochromes P-450 have long been implicated in the first step in the conversion of nicotine to nicotine delta 1'(5')-iminium ion. We demonstrate here that rat liver P-450IIB1 is able to convert nicotine to cotinine in the presence of cytosol with a Km of 5-7 microM. A constitutive form of P-450 is also implicated in nicotine metabolism, while purified P-450IA1 and P-450IIC6 show no detectable activity. The lack of P-450IA1 activity substantiates work by others who also failed to observe an increase in the efficiency of nicotine metabolism to cotinine by microsomes from rats that had been pretreated with benzanthracene. This result is in contrast to work with purified rabbit liver enzymes, in which P-450IA1 exhibited low but measurable activity. Our results support the notion that nicotine metabolism to cotinine by P-450 enzymes is highly species dependent. Thus, it is unwise in some cases to extrapolate results obtained by animal model study to the possible role of specific forms of the P-450 enzymes in nicotine metabolism in humans.

[14C]monocrotaline kinetics and metabolism in the rat

Abstract: The pyrrolizidine alkaloid monocrotaline (MCT) has been shown to cause hepatic necrosis and pulmonary hypertension in the rat. To better understand the mechanism of action, tissue distribution and covalent binding studies were conducted at 4 and 24 hr following administration of [14C]MCT (60 mg/kg, 200 microCi/kg, sc). For the 4 hr study, the levels of MCT equivalents were 85, 74, 67, 36, and 8 nmol/g of tissue for red blood cells (RBC), liver, kidney, lung, and plasma, respectively, while the covalent binding levels were 125, 132, 39, 64, 44 pmol/mg of protein for tissues as listed above. The 24-hr tissue distribution levels were 49, 25, 9, 10, 2 nmol/g of tissue for RBC, liver, kidney, lung, and plasma, respectively, while covalent binding was 74, 28, and 55 pmol/mg of protein for liver, kidney, and lung, respectively. We also studied the kinetics of [14C]MCT (60 mg/kg, 10 microCi/kg, iv), which demonstrated rapid elimination of radioactivity with approximately 90% recovery of the injected radioactivity in the urine and bile by 7 hr. The plasma levels of radioactivity dropped from 113 nmol/g of MCT equivalents to 11 nmol/g at 7 hr while RBC levels decreased from 144 to only 81 nmol/g at the same time point. The apparent retention of MCT equivalents in the RBC suggests that this organ may act as the carrier of metabolites from the liver to other organs including the lung and may play a role in the pulmonary toxicity.

Potential error in the measurement of tissue to blood distribution coefficients in physiological pharmacokinetic modeling. Residual tissue blood. II. Distribution of phencyclidine in the rat.

Abstract: A model for predicting the magnitude of error (% Err) in measuring tissue concentrations of a compound that have not been corrected for residual blood in the tissue was previously developed. The model was tested using data for phencyclidine tissue distribution in the rat. It is shown that % Err may be expressed as a function of volume fraction of blood in tissue (VF)B and tissue-to-blood distribution coefficient. Correction for residual blood is important when the volume fraction of the blood in the tissue is large and when the compound is not taken up substantially by the tissue. On the other hand, a correction may not be necessary when (VF)B is small and uptake of the compound into the tissue is substantial.

Renal tubular transport of morphine, morphine-6-glucuronide, and morphine-3-glucuronide in the isolated perfused rat kidney.

Abstract: The isolated perfused rat kidney was used to examine the renal handling of morphine and its inactive metabolite morphine-3-glucuronide (M3G), and active metabolite morphine-6-glucuronide (M6G). The kidneys were perfused with Krebs-Henseleit buffer (pH 7.4) containing albumin, glucose, and amino acids, and drug concentrations were measured by high performance liquid chromatography. There was no conversion of morphine to the glucuronides or deconjugation of M3G or M6G. At an initial morphine concentration of 100 ng/ml, the unbound renal clearance to glomerular filtration rate ratio (CLur/GFR) was 5.5 +/- 3.2 (mean +/- SD), indicating that net tubular secretion of morphine occurred. In the presence of M3G (2000 ng/ml) and M6G (500 ng/ml) this Clur/GFR ratio was elevated to 17.3 +/- 4.8 (p less than .001), which implicates an interaction between these compounds at an active reabsorption transport system. The CLur/GFR ratio for M3G at 2000 ng/ml was 0.90 +/- 0.04, indicating the possibility of a small component of tubular reabsorption, and this ratio was not significantly altered in the presence of morphine and M6G. M6G was reabsorbed, probably actively, to a greater extent than M3G, with an initial CLur/GFR ratio of 0.67 +/- 0.04, which was not affected when morphine and M3G were coadministered. These data demonstrate an unusual phenomenon in that the glucuronide metabolites, which are larger and less lipophilic than the parent drug morphine, undergo net tubular reabsorption. The renal handling of morphine is a complex combination of glomerular filtration, active tubular secretion, and possibly active reabsorption.

Disposition of the cephalosporin cefepime in normal and renally impaired subjects.

Abstract: The metabolism and disposition of an iv-administered, 1000 mg (100 microCi) single dose of the 14C-labeled cephalosporin cefepime was studied in healthy and renally impaired male volunteers. The 14C-label was located in the methyl group of the N-methyl pyrrolidine (NMP) moiety at the 3'-position of cefepime. Concentrations of cefepime and its metabolites were determined in plasma and urine as a function of time after drug administration. Cefepime comprised 95 and 76% of the total plasma radioactivity in subjects with normal and impaired renal functions, respectively. The elimination half-life of cefepime was 2 hr in normal volunteers and increased to 4 and 12 hr in subjects with moderate and severe renal impairment, respectively. Steady-state volume of distribution was about 18 liters and was independent of the degree of renal impairment. Cefepime was primarily excreted unchanged in the urine of normal subjects, as 87.9% of the total recovered radioactivity. The major cefepime metabolites, NMP N-oxide, the 7-epimer of cefepime and NMP, constituted 6.8, 2.5, and less than 1% of the total radioactivity excreted in urine, respectively. As the severity of renal impairment increased, the proportion of radioactivity recovered in urine as cefepime decreased and that of NMP-N-oxide increased. Our results indicate that cefepime undergoes minimal metabolism and is excreted primarily as unchanged drug in urine in humans with normal kidney functions.

Oxidation of butylated hydroxytoluene to toxic metabolites. Factors influencing hydroxylation and quinone methide formation by hepatic and pulmonary microsomes.

Abstract: The effects of inducing agents and inhibitors on the cytochrome P-450-catalyzed oxidations of butylated hydroxytoluene (BHT) to form three metabolites were investigated with liver and lung microsomes from rats and mice. These compounds, the quinone methide (QM) formed by two-electron oxidation of BHT, the hydroxy-tert-butyl analog of BHT (BHT-OH) resulting from aliphatic hydroxylation, and the hydroxy-quinone methide (QM-OH) derived from BHT-OH, have been implicated previously as intermediates or products involved in BHT bioactivation and toxicity. Although there was little or no increase in BHT metabolism in pulmonary microsomes from either species following phenobarbital (PB) administration, 6- to 37-fold enhancements occurred in the transformation of BHT to QM, and the conversion of BHT-OH to QM-OH with hepatic microsomes from both species. The first step in QM-OH formation, hydroxylation of BHT to BHT-OH, is a minor pathway with hepatic microsomes from treated or untreated rats, thereby explaining the lack of QM-OH formation from BHT by that species. The two-step oxidation of BHT to QM-OH, however, is a relatively important metabolic pathway with hepatic microsomes from PB-treated mice, due to an unusually large (111-fold) PB-induced increase in the tert-butyl hydroxylation step. These results demonstrate that pulmonary microsomes from mice, but not rats, have relatively high constitutive P-450 activity for the formation of QM-OH from BHT, supporting the proposal that this metabolite is involved in BHT-induced pneumotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of a novel quaternary ammonium-linked glucuronide of lamotrigine.

Abstract: Lamotrigine (LTG) is a novel triazine anticonvulsant currently undergoing clinical trials. LTG N-glucuronide, the major human metabolite of LTG, was isolated from human urine by means of XAD-2 column chromatography and semi-preparative HPLC. The structure of the suspected lamotrigine 2-N-glucuronide was proven by mass spectroscopy and NMR spectroscopy, along with chemical and enzymatic hydrolysis studies. High resolution fast atom bombardment mass spectrometry and Electrospray tandem mass spectrometry of the glucuronide gave an M+ ion at 432.0 amu and a fragment ion at 256.0 (M - 176)+ amu. The proton NMR of the glucuronide indicated the presence of a glucuronic acid moiety. A downfield anomeric proton (5.35-5.60 ppm) implied direct attachment to the aromatic triazine ring. Carbon-13 NMR of the glucuronide revealed an upfield shift (delta = -7.0 ppm) of the C-3 carbon of the triazine ring compared to LTG, indicating attachment of the glucuronide to the N-2 position. Chemical degradation or rearrangement of the glucuronide occurs at neutral pH to produce an unknown product (RP-1), while at basic pH a different unknown product (RP-2) is formed. The glucuronide is unusually stable at acidic pH. Treatment of the glucuronide with beta-glucuronidase resulted in hydrolysis to LTG, and enzymatic hydrolysis was inhibited by saccharo-1,4-lactone.

Gastrointestinal absorption of xenobiotics in physiologically based pharmacokinetic models. A two-compartment description.

Abstract: Gastrointestinal (GI) absorption in physiologically based pharmacokinetic models is typically described as first-order transfer from one compartment directly into the liver. This approach appears to be adequate for water but not for oily vehicles. In this study, a two-compartment description of GI absorption was developed in which the chemical moved from the first compartment to the second (first-order rate constant KT) and was absorbed from both compartments (first-order rate constants KAS and KAD, respectively) into the liver. Rat blood time-course data sets for methylene chloride, chloroform, dichloroethane, and trichloroethylene after oral gavage in water or corn oil obtained from the literature were used for model validation. Optimization of the KAS, KAD, and KT values for each dosing solution allowed accurate simulation of each data set. In general, the KAS values were 3-4 times greater when water rather than corn oil vehicle was used. The KAD and KT values were similar for the two vehicles. By comparison, a one-compartment description resulted in a poor simulation of the oil gavage data. The two-compartment model rate constant values obtained by optimization of the rat blood time-course data set for trichloroethylene after oral gavage in water were used in the model to predict rat exhaled breath concentrations after oral gavage of trichloroethylene in water (data generated in this laboratory). Exhaled breath trichloroethylene concentrations compared favorably with model predictions.

Metabolism and disposition of diethylene glycol in rat and dog.

Abstract: The disposition of carbon-14-labeled diethylene glycol (DEG) was determined in rats after oral, iv, and dermal administration, and in dogs after oral administration. Oral administration of DEG to rats was by gavage of 50 or 5000 mg/kg doses, or by provision of 0.3 1.0, and 3.0% in drinking water. Oral doses were well absorbed and excreted primarily (approximately 80%) in urine within 24 hr of administration. Greater than half of the dose was excreted unchanged, with 10-30% of the dose appearing as a single metabolite. The metabolite was isolated and characterized by 13C-NMR to be 2-(hydroxy) ethoxyacetic acid (HEAA). Confirmation of identity was provided by synthesis of HEAA and comparison of its NMR spectra and chromatographic behavior with those of the metabolite. Intravenous doses (50 mg/kg) were eliminated by the same routes and at the same rates as those administered orally and exhibited the same metabolic profile. The fate of oral doses of DEG administered to dogs (500 mg/kg) was similar to that of DEG in rats, with about 30% of the administered dose being excreted in urine as HEAA. DEG slowly penetrated the skin of rats after application of 50 mg to a 12-cm2 area. Only about 10% of the dose was absorbed in 72 hr of exposure, and the absorbed dose appeared to have the same fate as doses administered iv or orally. In all studies with rats, excretion of radiolabel in feces and persistence in tissues were low. The highest percentage of conversion to 14CO2 was 7%, found for doses of 0.3% DEG in drinking water.

Felbamate pharmacokinetics in the rat, rabbit, and dog.

Abstract: Rats, rabbits, and dogs were given single iv or single and multiple oral doses of felbamate ranging from 1.6-1000 mg/kg. Absorption of oral drug was complete in all species. The mean Cmax increased with dose from 13.9 to 185.9 micrograms/ml in rats, from 19.1 to 161.9 micrograms/ml in rabbits, and from 12.6 to 168.4 micrograms/ml in dogs. The tmax also increased with dose from 1-8 hr in rats, 8-24 hr in rabbits, and 3-7 hr in dogs. The plasma elimination half-life for the drug increased with dose from 2-16.7 hr in rats, 7.2-17.8 hr in rabbits, and 4.1-4.5 hr in dogs. A proportional increase in Cmax with dose was observed in all species up to 300-400 mg/kg doses. A biexponential equation fitted the drug plasma concentration vs. time data well. For multiple oral doses of 50 mg/kg or less, projected and observed steady-state concentrations agreed well. Animals dosed with [14C]felbamate eliminated most of the radioactivity in urine (58-87.7%), less in feces (7-23.7%), with considerable amounts in the bile. In rats, radioactivity was readily distributed into tissues and crossed the placenta and blood-brain barrier, but no accumulation in any tissue was observed. The volume of distribution was 131, 54, and 72% of body weight for rats, rabbits, and dogs, respectively. Binding of drug to rat, rabbit, and dog plasma proteins ranged from 22.4-35.9%. The overall plasma clearance of the drug for rats, rabbits, and dogs was 327, 52, and 108 ml.h-1.kg-1, respectively. Renal clearance of unchanged drug accounted for an estimated 20-35% and hepatic clearance due to metabolism for 65-80% of the overall clearance.

Potential error in the measurement of tissue to blood distribution coefficients in physiological pharmacokinetic modeling. Residual tissue blood. I. Theoretical considerations.

Abstract: Physiological pharmacokinetic models require the determination of tissue to blood distribution coefficients. A theoretical model has been developed and the resulting equations indicate that under certain conditions it is necessary to correct for the presence of drug in the residual blood remaining in the tissue. The potential error in ignoring this residual blood is expressed mathematically in terms of several important factors that include the anatomical features of the tissue (volume fractions of the blood, interstitial fluid, and cellular space) as well as the physicochemical properties of the drug (extent of binding in the blood and tissues). These theoretical considerations and resulting simulations have been applied to experimental literature data for several compounds (methotrexate, digoxin, and biperiden). We conclude that correction for the residual blood is necessary when the values of tissue to blood distribution coefficients are very small or large (relative to one) and when the volume fraction of the blood in tissue is substantial.

Evidence for oxidative activation of mitoxantrone in human, pig, and rat

Abstract: A new metabolite of mitoxantrone in human, rat, and pig urine has been discovered by means of HPLC. The metabolite has been isolated by preparative HPLC from patient urine and is characterized by tandem mass spectrometry and UV-visible spectroscopy as 8,11-dihydroxy-4-(2-hydroxyethyl)-6-[[2-[(2-hydroxyethyl)amino]ethyl] amino]-1,2,3,4,7,12-hexahydronaphtho-[2,3-f]-chinoxaline-7,1 2-dione. Final structural proof has been obtained by independent synthesis. The new metabolite is a product of the enzymatic oxidation of the phenylenediamine substructure of mitoxantrone. An important biological consequence of the oxidative biotransformation is the possibility of covalent binding to intracellular targets via a highly electrophilic intermediate. Thus, alkylation may be an important mode of action of mitoxantrone. Incubation of mitoxantrone with horseradish peroxidase/hydrogen peroxide in the presence of glutathione led to the formation of two glutathione conjugates of mitoxantrone. Their structures have been elucidated by combination of IonSpray (Sciex, Canada) ionization and tandem mass spectrometry. Radioactive mitoxantrone, synthesized from sodium [14C]cyanide, was used to determine interspecies variations between human and rat. The collected rat urine was analyzed by HPLC using a radioactivity monitoring detector and revealed significant differences in the biotransformation of mitoxantrone in rat compared to human. The main metabolites thus far described in human urine are not observed in rat urine.

Mechanistic studies on the metabolic chiral inversion of R-ibuprofen in the rat

Abstract: Deuterium labeling techniques and stereoselective GC/MS methodology have been employed to investigate the mechanism by which R-ibuprofen undergoes metabolic chiral inversion in the rat in vivo. Following oral administration of a mixture of R-ibuprofen (7.5 mg kg-1) and R-[ring-2H4; 2-2H]ibuprofen (R-[2H5]ibuprofen) (7.5 mg kg-1) to male Sprague-Dawley rats, the enantiomeric composition and deuterium excess of the drug were determined in serial plasma samples and in pooled urine collected over 10 hr. The results demonstrate that: (i) R-ibuprofen undergoes extensive inversion of configuration to its S antipode in the rat; (ii) chiral inversion of R-[2H5]ibuprofen yields S-[2H4]ibuprofen in a process that involves quantitative loss of the deuterium atom present originally at C-2; (iii) labeling of R-ibuprofen with deuterium at C-2 does not introduce a measurable kinetic deuterium isotope effect on the chiral inversion reaction; and (iv) metabolism of R-[2H5]ibuprofen leads to the appearance in plasma and urine of molecules of R-ibuprofen labeled with 4 atoms of deuterium. On the basis of these findings, a mechanism is proposed for the chiral inversion reaction that invokes the stereoselective formation of the coenzyme A thioester of R-ibuprofen as a key metabolite; conversion of this species to the corresponding enolate tautomer affords a symmetrical intermediate through which racemization of ibuprofen occurs in vivo.