On-line liquid chromatography-accurate radioisotope counting coupled with a radioactivity detector and mass spectrometer for metabolite identification in drug discovery and development.

A novel detection method combining on-line liquid chromatography-accurate radioisotope counting (LC-ARC, advanced stop flow controller) coupled with a radioactivity detector and mass spectrometer has been developed. One of the major benefits of this method is that this system enhances the sensitivity of radioisotope measurement for metabolite identification in drug metabolism studies. Another advantage to this system is the easy interface with the mass spectrometer, which allows acquisition of mass spectrometric data on-line. For purposes of evaluating this system, in vitro microsomal incubations with [3Hlpropranolol were conducted. On-line separation and identification of [3H]propranolol metabolites was achieved without intensive sample preparation, concentration, or fraction collection. Mass spectrometric analysis showed the presence of propranolol major metabolites formed by hydroxylation, correlating with previously published results. Further evaluations of this system also were conducted using two 14C compounds, which are herein labeled X and Y. As our results show, 14C peaks were detected down to 6 cpm, which is approximately 20 times more sensitive than commercially available flow through radioactivity detectors. The overall results suggest that the combination of LC-ARC with radioactivity detection and mass spectrometry has great potential as a powerful tool for improving the sensitivity of radioisotope measurement in metabolite identification studies during drug discovery and development.

Metabolic disposition of the cognition activator tacrine in rats, dogs, and humans. Species comparisons.

The metabolic fate of tacrine [1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate (THA)] was examined in rats, dogs, and humans. After administration of single oral doses of [14C]THA to rats, dogs, and humans, drug-derived material was well absorbed, with urinary excretion being the predominant route of radiolabel elimination. Metabolic profiling of plasma and urine from rats, dogs, and humans showed THA to be extensively metabolized with marked species differences in quantitative amounts of metabolites observed. Plasma profiles were similar to respective urinary profiles in all three species. Present in profiles of urine from rats were 1-hydroxy (OH)-THA (major), 2-OH-THA, and 4-OHA-THA, and unchanged THA. Also observed were trace amounts of more polar metabolites, presumably arising from sequential metabolism. Metabolic profiling of dog urine also showed 1-OH-THA to be the major metabolite, with trace amounts of the 2-OHA-THA and 4-OH-THA regioisomers and THA excreted. In dog urine, more of the radioactivity was associated with polar metabolites, including 1,3-dihydroxy-THA and a dihydrodiol metabolite. Human urinary metabolic profiles were more similar to that in dogs than in rats, with no single metabolite constituting > 10% of urinary radioactivity. Present in human urine were phenol glucuronide metabolites, of which 7-OH-THA was identified as an aglycone. Relevance of the marked quantitative differences in THA metabolism between rats, dogs, and humans to species differences in THA hepatotoxic potential remains to be established.

In vitro and in vivo disposition of 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976). Identification of a novel five-carbon cleavage metabolite in rats.

The metabolism of CI-976, a potent inhibitor of liver and intestinal acyl coenzyme A:cholesterol acyltransferase, was investigated in isolated rat hepatocytes and Wistar rats after oral administration. The major metabolite observed both in vitro and in vivo was identified as the 6-carbon, chain-shortened 5,5-dimethyl-6-oxo-[(2,4,6-trimethoxyphenyl)amino]hexanoic acid (M-4). M-4 was determined to be formed from the omega-carboxylic acid 11,11-dimethyl-12-oxo-12-[(2,4,6-trimethoxyphenyl)amino]dodecanoic acid (M-1) via the 2- and 4-carbon, chain-shortened intermediate metabolites [9,9-dimethyl-10-oxo-10-[(2,4,6-trimethoxyphenyl)amino]decanoic acid (M-2) and 7,7-dimethyl-8-oxo-8-[(2,4,6-trimethoxyphenyl)amino]octanoic acid (M-3)], respectively. M-1 was, in turn, determined to be derived from omega-hydroxy CI-976. A minor metabolite, identified in vitro and in vivo, was a novel 5-carbon, chain-shortened derivative, 6,6-dimethyl-7-oxo-7-[(2,4,6-trimethoxyphenyl)amino]heptanoic acid (M-5). M-5 was shown not to be formed from either M-1 or the omega-hydroxy derivative. Separate incubation of CI-976 (omega-oxidation and beta-oxidation pathways) and M-1 (beta-oxidation only) indicated a potential gender difference in the omega-oxidation of CI-976. Both the omega-oxidation and beta-oxidation pathways were enhanced by clofibrate and phenobarbital induction, and CI-976 metabolism was completely inhibited when coincubated with SKF525A pointing to cytochrome P450-mediated metabolism, presumably CYP4A. Etomoxir and L-carnitine had minor effects on the beta-oxidation of M-1, indicating beta-oxidation occurs predominately within peroxisomes.

Three complementary liquid chromatographic methods for determination of the peptoid cholecystokinin-B antagonist, CI-988, in rat plasma.

Three different liquid chromatographic methods (two quantitative methods which employ fluorescence detection and one qualitative method which employs selected ion-monitoring detection) were developed and validated to provide complementary specificity for determination of CI-988, a cholecystokinin-B antagonist, in rat plasma. The first quantitative method involves isocratic separation of "non-ionized" CI-988 and internal standard on a C-18 column, whereas the alternative quantitative method involves isocratic separation of the "anionic" analytes. These two quantitative HPLC methods rely on the intrinsic fluorescence of CI-988 and internal standard for detection, and both methods are equally sensitive (linear range of 2.0-1000 ng ml-1), accurate (+/- 15% relative error), and precise (< or = 15% relative standard deviation). Plasma CI-988 concentrations for samples (N = 69) assayed with the "non-ionized" separation are linearly correlated with concentrations for the same samples assayed with the "anionic" separation (y = 1.08 chi - 0.57, R = 0.999). In addition, a third qualitative method, HPLC-thermospray mass spectrometry, was developed to provide complementary evaluation of assay specificity through the use of selected CI-988 fragment ion monitoring. When investigating an anomalous chromatographic result that calls into question the specificity of a method, the availability and use of alternative validated chromatographic separations and orthogonal detection schemes are beneficial.

Bioactivation and irreversible binding of the cognition activator tacrine using human and rat liver microsomal preparations. Species difference.

Tacrine's [1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate, (THA)] metabolic fate was examined using human and rat liver microsomal preparations. Following 1-hr incubations with human microsomes, [14C]THA (0.4 microM) was extensively metabolized to 1-hydroxyTHA with trace amounts of 2-, 4-, and 7-hydroxyTHA also produced. Poor recovery of radioactivity in the postreaction incubates suggested association of THA-derived radioactivity with precipitated microsomal protein. After exhaustive extraction, 0.034, 0.145, 0.126, and 0.012 nmol eq bound/mg protein/60 min of THA-derived radioactivity was bound to human liver preparations H109, H111, H116, and H118, respectively. Preparations H109 and H118 were lower in P4501A2 content and catalytic activity as compared with preparations H111 and H116. Incubations of equimolar [14C]1-hydroxyTHA with human liver microsomes also resulted in binding to protein, although to a lesser extent than observed with THA. [14C]THA (0.4 microM) was incubated for 1 hr with rat liver microsomes (1 microM P-450) prepared from noninduced (N), phenobarbital (PB), isoniazid (I), and 3-methylcholanthrene (3-MC)-pretreated animals. In all incubations, 1-hydroxyTHA was the major biotransformation product detected. After exhaustive extraction, 0.048, 0.054, 0.049, and 0.153 nmol eq/mg protein/60 min of THA-derived radioactivity was bound to microsomal protein from N, PB, I, and 3-MC pretreated rats. Increased binding with 3-MC induced rat liver preparations suggests the involvement of the P-450 1A subfamily in THA bioactivation. Glutathione (5 mM) coincubation inhibited the irreversible binding of THA-derived radioactivity in both human and 3-MC-induced rat liver preparations, whereas human epoxide hydrase (100 micrograms/incubate) had a relative minor effect. A mechanism is proposed involving a putative quinone methide(s) intermediate in the bioactivation and irreversible binding of THA. A species difference in THA-derived irreversible binding exists between human and noninduced rat liver microsomes, suggesting that the rat is a poor model for studying the underlying mechanism(s) of THA-induced elevations in liver marker enzymes found in clinical investigations.

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

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 of the acyl-CoA:cholesterol acyltransferase inhibitor 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide in rat and monkey. Omega-/beta-oxidation pathway.

2,2-Dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976) is a newly developed hypocholesterolemic agent. In pharmacokinetic studies, CI-976 was found to have a much shorter elimination half-life in monkey (0.6 hr) compared to rat (8 hr). Radioactivity analysis of biological samples from rats and monkeys administered [14C]CI-976 either iv or orally showed CI-976 to be extensively metabolized to a single major common metabolite. This metabolite was isolated and its structure determined by GC/MS, LC/MS, and NMR spectroscopy as a 6-carbon chain-shortened carboxylic acid derivative, 5,5-dimethyl-6-oxo-6-[(2,4,6-trimethoxyphenyl)amino]-hexanoic acid. A potential pathway leading to this carboxylic acid derivative may involve initial omega-oxidation followed by beta-oxidation. A potential species difference in omega-/beta-oxidative biotransformation capabilities appears to exist.

Comparison of glycolysis and glutaminolysis in Onchocerca volvulus and Brugia pahangi by 13C nuclear magnetic resonance spectroscopy.

Comparison of glycolysis in Brugia pahangi and Onchocerca volvulus by 13C nuclear magnetic resonance (NMR) spectroscopy showed that the former organism is predominantly a lactate fermenter and the latter resembles more closely the metabolism of a mixed acid fermenter producing lactate, succinate, acetate, ethanol, formate and carbon dioxide. Both organisms synthesize glycogen as a storage carbohydrate. Glutaminolysis in both organisms proceeds by the delta-amino-butyrate shunt to produce succinate which is then further metabolized to acetate and carbon dioxide as end-products.

Inhibition of medium-chain fatty acid beta-oxidation in vitro by valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid.

Valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid, were found to inhibit strongly the metabolism of decanoic acid in homogenates of rat liver. Reductions in decanoate consumption in response to inhibitors were paralleled by decreases in the formation of octanoic and hexanoic acids, two products of decanoate beta-oxidation. In contrast, 4-pentenoic acid, an established inhibitor of long-chain fatty acid beta-oxidation, had little effect on the metabolism of decanoate. It is concluded that the title compounds are potent, broad-spectrum inhibitors of fatty acid beta-oxidation, a property which may be of key toxicological importance in the pathology of valproate-induced liver injury.

Studies on the biotransformation in the perfused rat liver of 2-n-propyl-4-pentenoic acid, a metabolite of the antiepileptic drug valproic acid. Evidence for the formation of chemically reactive intermediates.

The metabolism of 2-n-propyl-4-pentenoic acid (delta 4-VPA), a putative toxic biotransformation product of valproic acid (VPA), was examined in the isolated perfused rat liver. Metabolites excreted into perfusion medium and bile were characterized by GLC and GC/MS techniques and their identities were verified by synthesis. A total of eight metabolites was detected, the structures of which could be best accounted for by initial oxidation reactions catalyzed by either cytochrome P-450 or the fatty acid beta-oxidation complex. Evidence was obtained which indicates that metabolism of delta 4-VPA by each of these enzyme systems can lead to the generation of chemically reactive intermediates which may contribute to the hepatotoxic properties of VPA.

The microsomal metabolism and site of covalent binding to protein of 3'-hydroxyacetanilide, a nonhepatotoxic positional isomer of acetaminophen.

Incubations of 3'-hydroxyacetanilide (3HAA) with hepatic microsomal preparations from phenobarbital-pretreated mice led to the formation of three products of aromatic hydroxylation, viz. 2',5'-, 3',4'-, and 2',3'-dihydroxyacetanilide, which were identified by GC/MS techniques and quantified by GLC analysis. NADPH-dependent covalent binding of radioactivity from [14C]3HAA to microsomal protein took place at almost four times the rate at which [14C]acetaminophen became irreversibly bound to protein under the same experimental conditions. This binding was inhibited by the addition to incubation media of ascorbate, glutathione, and the soluble proteins bovine serum albumin and bovine alpha s1-casein, but not by superoxide dismutase. Radioactivity from [14C]3HAA also became covalently bound to the added soluble proteins, the extent of which was greatest when the proteins contained a high content of free -SH groups. From an analysis of the effect of ascorbate and glutathione on both the covalent binding of 14C to protein and the production of the noncovalently bound products of 3HAA metabolism, it is concluded that reactive intermediates most likely derive from further oxidation of the primary 3HAA metabolites to electrophilic semiquinone and/or quinone species. Sulfhydryl groups appear to be the principal sites on protein at which covalent binding of these reactive metabolites of 3HAA takes place, a feature shared by the reactive species generated during acetaminophen metabolism.