Understanding the clinical pharmacokinetics of a GABAA partial agonist by application of in vitro tools.

1. 4-Oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide (1), developed for general anxiety disorder, was discontinued from clinical development due to unsuitable oral pharmacokinetics. 2. In humans, (1) demonstrated an unacceptable high apparent oral clearance (Cl(p)/F) that also demonstrated a supraproportional dose-exposure relationship. Secondary peaks in the plasma concentration-time profile suggested possible enterohepatic recirculation of (1). A combination of in vitro mechanistic tools was applied to better understand the processes underlying these complex clinical pharmacokinetic profiles of (1). 3. In metabolism experiments, (1) was shown to be a substrate of monoamine oxidase A (MAO-A) as well as being metabolized by cytochrome P450. The former appeared to be a high K(M) process with a high capacity, while the latter showed saturation between 1 and 10 microM, consistent with the supraproportional dose-exposure relationship. 4. In a sandwich-cultured hepatocyte model, (1) was shown to be a substrate for both uptake and efflux into the canicular space, which is consistent with the observation of pharmacokinetics suggestive of enterohepatic recirculation. Finally, in human epithelial colon adenocarcinoma cell line (Caco-2) and Madin-Darby canine kidney cells transwell flux experiments, (1) was shown to have relatively low permeability and a basolateral-to-apical flux ratio consistent with the activity of P-glycoprotein. 5. In combination, a compounding of the contributions of MAO-A, hepatic uptake and efflux transporters, and P-glycoprotein to the disposition of (1) may underlie the low oral exposure, saturable clearance, and aberrant concentration versus time profiles observed for this compound in humans.

Multiple oxidants in cytochrome P450 catalyzed reactions: implications for drug metabolism.

The activation of molecular oxygen by Cytochromes P450 to the ultimate mono-oxygen oxidant species involves three distinct dioxygen species coordinated to the heme iron. These intermediates have different chemical properties, and have recently been proposed to participate in some Cytochrome P450-catalyzed oxidation reactions. This article reviews the extent of our current knowledge on the roles proposed for the heme- peroxo, hydroperoxo, and superoxo complexes in various reactions. The extent to which such species contribute to the breadth of reactions catalyzed by Cytochrome P450 has yet to be defined, and more definitive experiments are needed to establish such species in the reactions they are proposed to effect.

Azole-antifungal binding to a novel cytochrome P450 from Mycobacterium tuberculosis: implications for treatment of tuberculosis.

Although antibiotics against Mycobacterium tuberculosis have decreased the incidence of tuberculosis infections significantly, the emergence of drug-resistant strains of this deadly pathogen renders current treatments ineffective. Therefore, it is imperative to identify biochemical pathways in M. tuberculosis that can serve as targets for new anti-mycobacterial drugs. We recently cloned, expressed, and purified MT CYP51, a soluble protein from M. tuberculosis that is similar in sequence to CYP51 (lanosterol-14alpha-demethylase) isozymes, pharmacological targets for several anti-mycotic compounds. Its striking amino acid sequence similarity to that of mammalian and fungal CYP51s led to the hypothesis that MT CYP51 plays an important role in mycobacterial biology that can be targeted for drug action. In this manuscript, we established through spectral analysis that several azole antifungals bind MT CYP51 with high affinity. The effects of several azole compounds on the growth of M. bovis and M. smegmatis, two mycobacterial species that closely resemble M. tuberculosis were examined. We established a correlation between the affinity of azole compounds to MT CYP51 and their ability to impair the growth of M. bovis and M. smegmatis. These results suggest that the metabolic functions of MT CYP51 may be comparable to those of CYP51 in yeast and fungi and may lead to the development of a new generation of anti-mycobacterial agents.

Assessment of rat liver microsomal epoxide hydrolase as a marker of hepatocarcinogenesis.

The influence of eleven xenobiotics on the activity and amount of hepatic microsomal epoxide hydrolase was determined. Activity was assayed using three different substrates after rats were fed, throughout 3 weeks, diets containing one of six hepatocarcinogens, viz. 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene, 4'-fluoro-4-dimethylaminoazobenzene, thioacetamide, aflatoxin B1 and ethionine. Five hepatocarcinogens induced activity 4- to 10-fold; ethionine was relatively ineffective as an inducer. Two non-carcinogenic analogues of hepatocarcinogens, viz. fluorene and p-aminoazobenzene, caused no appreciable increase in enzyme activity, but phenobarbital, barbital and 1-naphthylisothiocyanate induced activity 2- to 3-fold. All eleven xenobiotics increased the amount of microsomal epoxide hydrolase 2- to 9-fold when examined immunochemically using either a radial diffusion assay or an enzyme-linked immunosorbent assay (ELISA). Serum glutamic oxaloacetic acid transaminase activity was not appreciably elevated by feeding ten of the xenobiotics, suggesting that inductions were not owing to toxicity. Using ELISA, microsomal epoxide hydrolase was detected in post-microsomal (PM) supernatant fractions from control rat liver, thus confirming an earlier report by Gill et al. [Carcinogenesis 3, 1307 (1982)]. The eleven xenobiotics induced the amount of ELISA-detectable antigen in PM supernatant fractions by 3- to 34-fold. Longer centrifugation of PM supernatant fractions yielded a pellet fraction that contained 92 +/- 1.2% of the ELISA-detectable antigen irrespective of the xenobiotic regimen. Relationships between xenobiotic induction of microsomal epoxide hydrolase activity and amount and hepatocarcinogenesis are discussed.

Adrenal proteins bound by a reactive intermediate of mitotane.

PURPOSE: Mitotane (o,p'-DDD), is the only adrenolytic agent available for the treatment of adrenocortical carcinoma. Previous studies have shown that mitotane covalently binds to adrenal proteins following its metabolism in adrenocortical tissue to a reactive acyl chloride intermediate. It was the objective of this study to compare the electrophoresis separation patterns of such adducts following activation of mitotane by various adrenocortical sources. METHODS: With the use of a 125I-labeled analog of mitotane, 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, gel electrophoresis patterns were obtained for homogenates from bovine, canine and human adrenocortical preparations as well as from a human adrenal preparation. Western immunoblotting analysis was used to test the resulting patterns for adducts of cytochrome P-450scc and adrenodoxin. RESULTS: The electrophoresis separations were similar for all preparations, with bands at apparent molecular weights of 49.5 and 11.5 kDa being the most pronounced. Radiolabeling of the proteins of a human adrenal cancer cell line NCI H-295 was weak, but a band at 11.5 kDa was detected. Western immunoblotting analyses indicated that the band at 49.5 kDa corresponded in molecular weight to that of adrenal cytochrome P-450scc, but the band at 11.5 kDa did not correspond to adrenodoxin. CONCLUSIONS: The similarity of the results with canine and bovine adrenal preparations to that of human material offers useful systems for studying mitotane and its analogs. This should aid in understanding the mechanism of action of mitotane and in the design of compounds for the treatment of adrenocortical carcinoma.

Absorption, distribution, metabolism, and excretion of dirlotapide in the dog.

Three once-daily oral doses of 0.2 mg/kg [(14)C]dirlotapide were administered to beagle dogs to study the absorption, distribution, metabolism, and excretion of dirlotapide. Mean (14)C recovered at 2.5 and 4.5 h after the last dose was 90%. Mean (14)C in urine, bile, and feces was <1%, 1.7%, and 56% of the dose, respectively. In tissues, 26% of the (14)C dose was present in the gastrointestinal tract, 6.0% in liver, and <1% each in kidney, gall bladder, heart, and brain. To further characterize drug disposition, a single 2.5-mg/kg oral dose of [(14)C]dirlotapide was administered to beagle dogs. More than 84% of the dose had been eliminated by 72 h in feces, with 21% of the dose present in feces as parent dirlotapide. Less than 1% of the dose was excreted in urine. In bile collected during the first 24-h postdose from three dogs, 32% and 11% of the (14)C dose was present in samples from male and female dogs, respectively. Based upon metabolite profiling of plasma, excreta, and bile samples, dirlotapide was extensively metabolized to more than 20 metabolites. Biliary/fecal excretion and the potential for enterohepatic recycling of metabolites are suggested.

On the mechanism of action of cytochrome P450: evaluation of hydrogen abstraction in oxygen-dependent alcohol oxidation.

The mechanisms of oxidation of primary and secondary benzylic alcohols to the corresponding carbonyl compounds by purified rabbit liver cytochrome P450 forms 2B4 and 2E1 in a reconstituted enzyme system has been examined by linear free energy relationships, intramolecular and steady-state deuterium isotope effects, and the incorporation of an O2-derived oxygen atom or solvent-derived deuterium. The kcat and Km values were found to be relatively insensitive to the presence of electronic perturbations at the para position. The Hammett reaction constants for the oxidation of benzyl alcohols by P450s 2B4 and 2E1 are -0.46 and -0.37, respectively, and with 1-phenylethyl alcohols the corresponding reaction constants are -1.41 and -1.19, respectively. With [1-2H1]benzyl alcohol, P450s 2B4 and 2E1 show similar intramolecular deuterium isotope effects of 2.6 and 2.8, respectively, whereas with [1-2H2]benzyl alcohol under steady-state conditions, the deuterium isotope effects on the catalytic constants are 2.8 and 1.3, respectively. No significant isotope effect on the catalytic constant was noted for either form of P450 with 1-phenylethyl alcohol. In D2O, acetophenone formed by either form of P450 from 1-phenylethyl alcohol does not contain a deuterium atom at the methyl group, whereas under an atmosphere of 18O2 approximately 30% of the labeled oxygen is incorporated into the carbonyl group with either form of the cytochrome. The results are consistent with a mechanism that involves stepwise oxidation of the alcohol to a carbon radical alpha to the alcohol function, followed by oxygen rebound to yield the gem-diol, dehydration of which gives the carbonyl product.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2,3-Thiadiazole: a novel heterocyclic heme ligand for the design of cytochrome P450 inhibitors.

The 1,2,3-thiadiazole heterocycle has been explored as a heme ligand and mechanism-based inactivator for the design of cytochrome P450 inhibitors. One 4,5-fused bicyclic and three 4,5-disubstituted monocyclic 1,2,3-thiadiazoles have been examined for their spectral interactions, inhibition, mechanism-based inactivation, and oxidation products by the versatile microsomal P450s 2B4, 2E1, and 1A2. The compounds generally show heteroatom coordination to the heme iron; however, the binding mode is influenced by the architecture of the active site. For example, 4,5-diphenyl-1,2,3-thiadiazole shows type I and type II difference spectra with P450s 2B4 and 2E1, respectively, and no spectral perturbation with P450 1A2. Except for the fused bicyclic compound, the spectral dissociation constants are in the 2-50 microM range. The effectiveness as an inhibitor depends on the substituents at the 4- and 5- positions and on the P450 examined. Inhibition of the P450-catalyzed 1-phenylethanol oxidation to acetophenone by the thiadiazoles does not correlate with either the type of binding spectra or the spectral dissociation constants of the compounds. P450s 2E1 and 2B4 are inactivated by the 4,5-fused bicyclic 1,2,3-thiadiazole in a mechanism-based manner. Inactivation of the P450 correlates with loss in absorbance at 450 nm for the ferrous-CO complex. The monocyclic 1,2,3-thiadiazoles do not inactivate any of the P450s examined. The 1,2,3-thiadiazole ring is oxidized by the P450 system. Oxidation of the monocyclic compounds results in extrusion of the three heteroatoms and formation of the corresponding acetylenes, whereas oxidation of the fused bicyclic compound does not yield an acetylenic product.

Hydrocarbon formation in the reductive cleavage of hydroperoxides by cytochrome P-450.

Evidence is presented that cytochrome P-450 catalyzes the reductive cleavage of hydroperoxides. For example, in a reconstituted system containing rabbit liver microsomal P-450 form 2, NADPH-cytochrome P-450 reductase, and NADPH, cumyl hydroperoxide yields acetophenone and methane, but no cumyl alcohol is formed. The stoichiometry of the reaction and similar results with alpha-methylbenzyl, benzyl, and t-butyl hydroperoxides are in accord with the following general equation, in which X represents an alkyl group and R and R' are either alkyl groups or hydrogen atoms in the starting peroxide: XRR'C-OOH + NADPH + H+----XRCO + R'H + H2O + NADP+. Because 13-hydroperoxy-9,11-octadecadienoic acid yields pentane under these conditions, we propose that the known formation of alkanes and aldehydes in membrane lipid peroxidation involves reductive cleavage by P-450 to give the products predicted by the above equation. The cleavage reaction is thought to involve stepwise one-electron transfer, resulting in homolysis of the peroxide oxygen-oxygen bond and generation of an alkoxy radical, with beta-scission of the latter followed by reduction of the secondary radical to the hydrocarbon. In accordance with this scheme, when the cleavage reaction with cumyl hydroperoxide was done in 2H2O, deuteromethane was formed.

Role of cytochrome P-450 in hydrocarbon formation from xenobiotic and lipid hydroperoxides.

Cytochrome P-450 has recently been found to catalyze the reductive cleavage of both xenobiotic hydroperoxides and biologically occurring lipid hydroperoxides. In a reconstituted enzyme system containing phenobarbital-inducible P-450 form 2, NADPH-cytochrome P-450 reductase, and NADPH, the corresponding alcohols are not produced, but instead carbonyl compounds and hydrocarbons. In the following equation for the reaction, X represents any of a variety of alkyl groups and R and R' are either hydrogen or alkyl groups, of which only methyl has been studied so far: XRR'C-OOH + NADPH + H+----XRCO + R'H + H2O + NADP+ The products derived from cumyl, alpha-methylbenzyl, benzyl, and t-butyl hydroperoxides as well as 13-hydroperoxy-9,11-octadecadienoic acid indicate that the reaction involves stepwise one-electron transfer, resulting in homolysis of the peroxide oxygen-oxygen bond. The alkoxy radical thus generated undergoes beta-scission to yield the carbonyl compound and an alkyl radical that is reduced to the alkane. As predicted by this radical mechanism, when the reductive cleavage of cumyl hydroperoxide was carried out in D2O, deuteromethane was formed.

Affinity labeling of bovine opsin by trans-retinoyl chloromethane.

All-trans-retinoyl chloromethane is a potent irreversible inactivator of bovine opsin in retinal rod outer segments. The inactivation appears to be due to specific modification of the apoprotein at the 11-cis-retinaldehyde binding site. The reaction follows pseudo first order kinetics at 37 degrees C. A moderate dissociation constant for the initial reversible complex of 6.1 mM could be derived with a first order rate constant of 1.8x10(-1) min-1 for the conversion to the irreversible inactivated form. Native rhodopsin or rhodopsin regenerated from opsin by addition of 11-cis-retinaldehyde is completely protected against inactivation by trans-retinoyl chloromethane. All-trans-retinaldehyde does not provide this protection for the irreversible inactivation.

Radical intermediates in the catalytic cycles of cytochrome P-450.

Schemes are presented summarizing current knowledge of the mechanism of action of cytochrome P-450 when it functions either as a monooxygenase with molecular oxygen as the oxygen donor or as a peroxygenase with peroxy compounds as the oxygen donor. In the process, a large variety of physiologically occurring and foreign compounds undergo hydroxylation and oxy and peroxy radicals are generated. In addition, cytochrome P-450 catalyzes reductive reactions, including a recently discovered reaction in which organic hydroperoxides are cleaved to yield hydrocarbons and aldehydes or ketones. The reaction is believed to involve homolysis of the oxygen-oxygen bond and generation of an alkoxy radical, with beta-scission of the latter followed by reduction of the secondary radical to the hydrocarbon. Evidence has been obtained that lipid hydroperoxides are physiological substrates for this reductive cleavage reaction catalyzed by cytochrome P-450.

Role of isozymes of rabbit microsomal cytochrome P-450 in the metabolism of retinoic acid, retinol, and retinal.

The metabolism of retinoic acid, retinol, and retinal has been investigated with eight purified rabbit cytochrome P-450 (P-450) isozymes, including the major forms in nasal and liver microsomes. Retinoids hydroxylated at the 4-position were found to be major metabolites with each of the isozymes examined. Only two of the isozymes, polycyclic aromatic hydrocarbon-inducible P-450 1A2 and antibiotic-inducible P-450 3A6, also catalyze the oxidation of retinal to retinoic acid, a reaction not previously attributed to P-450. P-450 1A2 showed high activities in both the 4-hydroxylation and aldehyde oxidation reactions. Phenobarbital-inducible P-450 2B4 also had high activity in the 4-hydroxylation reaction of retinoids, and cytochrome b5 was found to increase the activity of P-450 2B4 with each substrate but to increase the activity of P-450 1A2 only with retinoic acid. In microsomes, retinoic acid is converted in an NADPH-dependent manner to both 4-hydroxyretinoic acid and 4-oxoretinoic acid, but none of the isozymes investigated was found to convert the 4-hydroxy derivative to the 4-oxo derivative. Microsomes from animals treated with phenobarbital were more active than those from untreated animals in the 4-hydroxylation reaction and, consequently, showed an increase in the ratio of 4-hydroxy to 4-oxo derivatives produced. These results show that the individual forms of P-450 metabolize retinoic acid, retinol, and retinal to multiple products, and they indicate that the amounts formed may be dependent on the exposure of animals to various inducers of P-450.

Metabolic activation of trans-4-hydroxy-2-nonenal, a toxic product of membrane lipid peroxidation and inhibitor of P450 cytochromes.

Lipid peroxidation in biological membranes is known to yield reactive aldehydes, of which trans-4-hydroxy-2-nonenal (HNE) is particularly cytotoxic. This laboratory previously reported that purified liver microsomal P450 cytochromes are directly inactivated to varying extents by HNE. We have now found a mechanism-based reaction in which P450s are inactivated by HNE in the presence of molecular oxygen, NADPH, and NADPH-cytochrome P450 reductase. The sensitivity of the various isozymes in the two pathways is different as follows: P450 2B4 and the orthologous 2B1 are inactivated to the greatest extent and 2C3, 1A2, 2E1, and 1A1 to a somewhat lesser extent by the pathway in which HNE undergoes metabolic activation. In contrast, 2B4 and 2B1 are insensitive to direct inactivation, and the reductase is unaffected by HNE by either route. Recent studies on the catalytic activities of the T302A mutant of P450 2B4 have shown that the rate of oxidation of a variety of xenobiotic aldehydes to carboxylic acids is decreased, but the rates of aldehyde deformylation and mechanism-based inactivation of the cytochrome are stimulated over those of the wild-type enzyme (Raner, G. M., Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902). Inactivation by those aldehydes apparently occurs by homolytic cleavage of a peroxyhemiacetal intermediate to yield formate and an alkyl radical that reacts with the heme. In sharp contrast, the rate of mechanism-based inactivation by HNE is decreased with the T302A mutant relative to that of the wild-type P450 2B4, and mass spectral analysis of the heme adduct formed shows that deformylation does not occur. We therefore propose that the metabolic activation of HNE involves formation of an acyl carbon radical that leads to the carboxylic acid or alternatively reacts with the heme.

Aromatization of a bicyclic steroid analog, 3-oxodecalin-4-ene-10-carboxaldehyde, by liver microsomal cytochrome P450 2B4.

Several purified isoforms of microsomal cytochrome P450 were previously shown in this laboratory to catalyze the oxidative deformylation of a variety of alpha- or beta-branched aldehydes with the production of olefins and formic acid. In the present study, 3-oxodecalin-4-ene-10-carboxaldehyde (ODEC, numbered according to the convention for steroids) was synthesized as a bicyclic analog of the aldehyde that is known to be the terminal intermediate in the enzymatic conversion of androgens to estrogens. ODEC undergoes aromatization in a reconstituted enzyme system containing liver microsomal cytochrome P450 2B4 and NADPH-cytochrome P450 reductase, along with NADPH and phosphatidylcholine, under aerobic conditions. The products, 3-hydroxy-6,7,8,9-tetrahydronaphthalene (HTN) and formic acid, were identified by mass spectrometry. The corresponding 10-carbinol does not undergo oxidative aromatization with P450 2B4, and with ODEC as substrate, other microsomal P450 cytochromes are either weakly active (isoforms 2C3 and 3A6) or inactive (isoforms 2E1, 1A2, and 2G1). Cytochrome b5 stimulates the P450 2B4-catalyzed reaction with ODEC about 2.6-fold but has no effect with the other P450s. In two respects the conversion of the bicyclic model compound to HTN with P450 2B4 was shown to be similar to that of the steroid aromatase reaction. Deuterium in the formyl group of ODEC was retained in the formic acid that was produced and isolated as the 4-nitrobenzyl derivative, and with preparations of ODEC containing deuterium in the 1 alpha position or the 1 alpha and 2 alpha positions, it was shown that the desaturation reaction is specific for removal of the 1 beta-hydrogen, thus involving a stereospecific cis elimination of formate. Cytochrome b5 has no effect on the stereospecificity of the reaction.

Metabolism of all-trans, 9-cis, and 13-cis isomers of retinal by purified isozymes of microsomal cytochrome P450 and mechanism-based inhibition of retinoid oxidation by citral.

The involvement of a series of microsomal cytochrome P450 (P450) isozymes in all-trans-retinoid metabolism, including the conversion of all-trans-retinal to all-trans-retinoic acid, was previously described. In the current study, we examined the role of seven liver microsomal P450 isozymes in the oxidation of three isomers of retinal. P450 1A1, which was not tested previously, is by far the most active in the conversion of all-trans-, 9-cis-, and 13-cis-retinal to the corresponding acids, as well as in the 4-hydroxylation of all-trans- and 13-cis retinal. In contrast, P450s 2B4 and 2C3 are the most active in the 4-hydroxylation of 9-cis-retinal, with turnover numbers approximately 7 times as great as that of P450 1A1. The inclusion of cytochrome b5 in the reconstituted enzyme system is without effect or inhibitory in most cases but stimulates the 4-hydroxylation of 9-cis-retinal by P450 2B4, giving a turnover of 3.7 nmol of product/min/nmol of this isozyme, the highest for any of the retinoid conversions we have studied. Evidence was obtained for two additional catalytic reactions not previously attributed to P450 oxygenases: the oxidation of all-trans- and 9-cis-retinal to the corresponding 4-oxo derivatives by isoform 1A2, and the oxidative cleavage of the acetyl ester of vitamin A (retinyl acetate) to all-trans-retinal, also by isoform 1A2. The physiological significance of the latter reaction, with a Km for the ester of 32 microM and a Vmax of 18 pmol/min/nmol of P450, remains to be established. We also examined the effect on P450 of citral, a terpenoid alpha, beta-unsaturated aldehyde and a known inhibitor of cytosolic retinoid dehydrogenases. Evidence was obtained that citral is an effective mechanism-based inactivator of isozyme 2B4, with a KI of 44 microM as determined by the oxidation of 1-phenylethanol to acetophenone, and by isozyme 1A2 in the oxidation of all-trans-retinal to the corresponding acid and by isozyme 2B4 in the 4-hydroxylation of all-trans-retinol and retinoic acid. Thus, citral is not suitable for use in attempts to distinguish between retinoid conversions catalyzed by dehydrogenases in the cytoplasm and by P450 cytochromes in the endoplasmic reticulum.

Cytochrome P450 2: peroxidative reactions of diversozymes.

Cytochrome P450, the most versatile biological catalyst known, was originally named as a pigment having a carbon monoxide difference spectrum at about 450 nm and no known function. Recent progress in many laboratories has revealed that the P450 superfamily has immense diversity in its functions, with hundreds of isoforms in many species catalyzing many types of chemical reactions. We believe it is safe to predict that each mammalian species may be found to have up to a hundred P450 isoforms that respond in toto to a thousand or more inducers and that, along with P450s from other sources, metabolize a million or more potential substrates. Accordingly, the name DIVERSOZYMES is proposed for this remarkable family of hemoproteins. This paper reviews the peroxidative reactions of Diversozymes, including peroxides as oxygen donors in hydroxylation reactions, as substrates for reductive beta-scission, and as peroxyhemiacetal intermediates in the cleavage of aldehydes to formate and alkenes. Lipid hydroperoxides undergo reductive beta-cleavage to give hydrocarbons and aldehydic acids. One of these products, trans-4-hydroxynonenal, inactivates P450, particularly alcohol-inducible 2E1, in what may be a negative regulatory process. Although a P450 iron-oxene species is believed to be the oxygen donor in most hydroxylation reactions, an iron-peroxy species is apparently involved in the deformylation of many aldehydes with desaturation of the remaining structure, as in aromatization reactions.