In vitro and in vivo metabolism of a novel cannabinoid-1 receptor inverse agonist, taranabant, in rats and monkeys.

The metabolism and excretion of taranabant (MK-0364, N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2{[5-(trifluoromethyl)pyridine-2-yl]oxy}propanamide), a potent cannabinoid-1 receptor inverse agonist, were evaluated in rats and rhesus monkeys. Following administration of [¹4C]taranabant, the majority of the radioactivity was excreted within 72 h. In both rats and rhesus monkeys, taranabant was eliminated primarily via oxidative metabolism, followed by excretion of metabolites into bile. Major pathways of metabolism that were common to rats and rhesus monkeys included hydroxylation at the benzylic carbon adjacent to the cyanophenyl ring to form a biologically active circulating metabolite M1, and oxidation of one of the two geminal methyl groups of taranabant or M1 to the corresponding diastereomeric carboxylic acids. Oxidation of the cyanophenyl ring, followed by conjugation with glutathione or glucuronic acid, was a major pathway of metabolism only in the rat and was not detected in the rhesus monkey. Metabolism profiles of taranabant in liver microsomes in vitro were qualitatively similar in rats, rhesus monkeys and humans and included formation of M1 and oxidation of taranabant or M1 to the corresponding carboxylic acids via oxidation of a geminal methyl group. In human liver microsomes, metabolism of taranabant was mediated primarily by CYP3A4.

Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: a case study with melanocortin-4 receptor agonists.

1-[(2R)-2-([[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl]amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a lambda(max) at approximately 450 nm. The species responsible for this absorbance required the presence of beta-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug-drug interaction projections based on the k(inact) and K(I) parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug-drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution alpha to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.

Importance of mechanistic drug metabolism studies in support of drug discovery: A case study with an N -sulfonylated dipeptide VLA-4 antagonist in rats.

N-(1-(3,5-dichlorobenzenesulfonyl)-2S-methyl-azetidine-2-carbonyl)-L-4-(2',6'-dimethoxyphenyl)phenylalanine (1) is a potent antagonist of the very late activating (VLA) antigen-4. During initial screening, 1 exhibited an apparent plasma clearance (CL) of 227 ml min(-1) kg(-1) in Sprague-Dawley rats following an intravenous bolus dose formulated in an aqueous solution containing 40% polyethylene glycol. Such a high CL value led to speculation that the elimination of compound 1 involved extra-hepatic tissues. However, the apparent plasma CL was reduced to 97 ml in(-1) kg(-1) when a 2-min time point was added to sample collections, and further decreased to 48 ml min(-1) kg(-1) after the dose was formulated in rat plasma. The lung extraction of 1 in rats was negligible whereas the hepatic extraction was > or =90%, based on comparison of area under the curve (AUC) values derived from intra-artery, intravenous, and portal vein administration. In rats dosed intravenously with [(14)C]-1, approximately 91% of the radioactivity was recovered in bile over 48 h, with 85% accounted for in the first 4-h samples. The biliary radioactivity profile consisted of approximately 30% intact parent compound, 20% 1-glucuronide, and 50% oxidation products resulting from O-demethylation or hydroxylation reactions. When incubated with rat liver microsomes, oxidative metabolism of 1 was inhibited completely by 1-aminobenzotriazole (ABT), whereas the oxidation and glucuronidation reactions were little affected in the presence of cyclosporin A (CsA). In contrast, the hepatic extraction of 1 in rats was unperturbed in animals pre-dosed with ABT, but was reduced approximately 60% following treatment with CsA. In vitro, 1 was a substrate of the rat organic anion transporter Oatp1b2, and its cellular uptake was inhibited by CsA. In addition, the hepatic extraction of 1 was approximately 30% lower in Eisai hyperbilirubinaemic rats which lack functional multidrug resistant protein-2 (MRP2). Collectively, these data suggest that the clearance of 1 in rats likely is a result of the combined processes of hepatic oxidation, glucuronidation and biliary excretion, all of which are facilitated by active hepatic uptake of parent compound and subsequent active efflux of both unchanged parent and its metabolites into bile. It was concluded, therefore, that multiple mechanisms contribute to the clearance of 1 in rats, and suggest that appropriate pharmacokinetic properties might be difficult to achieve for this class of compounds. This case study demonstrates that an integrated strategy, incorporating both rapid screening and mechanistic investigations, is of particular value in supporting drug discovery efforts and decision-making processes.

Metabolic activation of a pentafluorophenylethylamine derivative: formation of glutathione conjugates in vitro in the rat.

The aim was to investigate the metabolic activation potential of a pentafluorophenylethylamine derivative (compound I) in vitro in the rat and to identify the cytochrome P450 (CYP) enzymes that catalyse these metabolic activation processes. Reduced glutathione (GSH) was fortified in rat hepatocytes and liver microsomes to trap possible reactive intermediates. Four glutathione conjugates (M1-4) were identified by LC-MS(n) following incubation of compound I in GSH-enriched rat hepatocytes and liver microsomes. Three of these conjugates (M2-4) have not been reported previously for pentafluorophenyl derivatives. Elemental composition analysis of these conjugates was obtained using high-resolution quadrupole time-of-flight mass spectrometry. The formation of GSH conjugate M1 was rationalized as a direct nucleophilic replacement of fluoride by glutathione, whereas the formation of the GSH conjugates M2-4 was proposed to occur by NADPH-dependent metabolic activation of the pentafluorophenyl ring via arene oxide, quinone and/or quinoneimine reactive intermediates. Formation of these conjugates was enhanced three- to five-fold in liver microsomes obtained from phenobarbital- and dexamethasone-treated rats. In incubations with pooled rat liver microsomes and recombinant rat CYP3A1 and CYP3A2, troleandomycin (TAO) reduced the formation of GSH conjugates M2-4 by 80-90%, but it had no effect on the formation of M1. Incubation of compound I with rat supersomes indicated that only CYP3A1 and CYP3A2 were capable of mediating these metabolic activation processes.

Influence of different recombinant systems on the cooperativity exhibited by cytochrome P4503A4.

1. The in vitro cooperativity exhibited by cytochrome P450 (CYP) 3A4 is influenced by the nature of the recombinant system in which the phenomenon is studied. Diclofenac, piroxicam and R-warfarin were used as model substrates, and quinidine was the effector. 2. The 5-, 5'- and 10-hydroxylation of diclofenac, piroxicam and R-warfarin, respectively, were enhanced five- to sevenfold by quinidine in human liver microsomal incubations. Whereas these cooperative drug interactions were apparent in incubations with CYP3A4 expressed in human lymphoblast cells, similar phenomena were not observed with the enzyme expressed in insect cells. 3. Insect cell microsomes were treated with a detergent and CYP3A4 was solubilized into a buffer medium. In incubations with CYP3A4 'freed' from its host membrane, the 5-hydroxylation of diclofenac increased with increasing quinidine concentrations, reaching a maximal eightfold elevation relative to controls. The metabolism of piroxicam and warfarin was similarly enhanced by quinidine. 4. Kinetically, enhancement by quinidine of the 5-hydroxylation of diclofenac in incubations with solubilized CYP3A4 was characterized by increases in the rate of metabolism with little change in the substrate-binding affinity. Conversely, the 3-hydroxylation of quinidine was not affected by diclofenac. 5. The data suggest that certain properties of CYP3A4 are masked by expression of the protein in insect cells and reinforce the concept that the enzyme possesses multiple binding domains. The absence of cooperative drug interactions with quinidine when CYP3A4 was expressed in insect cells might be due to an absence of enzyme conformation changes on quinidine binding, or the inability of quinidine to gain access to a putative effector-binding domain. 6. Caution should be exercised when comparing models for CYP3A4 cooperativity derived from different recombinant preparations of the enzyme.

Bioactivation of diclofenac via benzoquinone imine intermediates-identification of urinary mercapturic acid derivatives in rats and humans.

The metabolism of diclofenac has been reported to produce reactive benzoquinone imine intermediates. We describe the identification of mercapturic acid derivatives of diclofenac in rats and humans. Three male Sprague-Dawley rats were administered diclofenac in aqueous solution (pH 7) at 50 mg/kg by intraperitoneal injection, and urine was collected for 24 h. Human urine specimens were obtained, and samples were pooled from 50 individuals. Urine samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Two metabolites with MH(+) ions at m/z 473 were detected in rat urine and identified tentatively as N-acetylcysteine conjugates of monohydroxydiclofenac. Based upon collision-induced fragmentation of the MH(+) ions, accurate mass measurements of product ions, and comparison of LC/MS/MS properties of the metabolites with those of synthetic reference compounds, one metabolite was assigned as 5-hydroxy-4-(N-acetylcystein-S-yl)diclofenac and the other as 4'-hydroxy-3'-(N-acetylcystein-S-yl)diclofenac. The former conjugate also was detected in the pooled human urine sample by multiple reaction-monitoring LC/MS/MS analysis. It is likely that these mercapturic acid derivatives represent degradation products of the corresponding glutathione adducts derived from diclofenac-2,5-quinone imine and 1',4'-quinone imine, respectively. Our data are consistent with previous findings, which suggest that oxidative bioactivation of diclofenac in humans proceeds via benzoquinone imine intermediates.

Heterotropic cooperativity of cytochrome P450 3A4 and potential drug-drug interactions.

Cytochromes P450 (CYP) 3A4 is the most abundant human hepatic CYP isoform catalyzing the metabolism of approximately 50% of therapeutic agents. In addition to inhibition or induction, CYP3A4 is subject to stimulation, termed homotropic (substrate stimulation) and heterotropic (stimulation by effectors) cooperativity. The heterotropic cooperativity of CYP3A4 may result from an increase in Vmax, a decrease in Km or a combination of the two and sometimes exhibits regio-selectivity when the enzyme is involved in two or more metabolic pathways for a single substrate. An effector of CYP3A4 can also be a substrate; its metabolism may or may not be inhibited by another substrate. These characteristics of heterotropic cooperativity of CYP3A4 have been interpreted in the context of two binding domains in the active site of the enzyme, two substrate binding plus a distinct allosteric binding site, multiple enzyme conformations or multiple binding sites accompanied by conformational changes. Examples of in vivo CYP cooperativity are rare; representative cases include flavone-dependent stimulation of zoxazolamine metabolism in rats and enhancement of CYP3A-mediated hepatic clearance of diclofenac by quinidine in monkeys. Effector-induced increases in CYP3A4 activity were observed during the 1'-hydroxylation of midazolam and 4'- and 10-hydroxylation of warfarin in human hepatocyte systems. These data imply that CYP cooperativity has the potential to cause in vivo drug-drug interactions. Because cooperative and inhibitory responses from CYP3A4 are known to be substrate-dependent, projection of the pharmacokinetics of an investigational drug and CYP-associated risks of drug-drug interactions in humans can be very complex. Further investigation of CYP cooperativity is warranted.

In vitro stimulation of warfarin metabolism by quinidine: increases in the formation of 4'- and 10-hydroxywarfarin.

It has been demonstrated that the activity of cytochrome P450 (CYP)3A4 in certain cases is stimulated by quinidine (positive heterotropic cooperativity). We report herein that the 4'- and 10-hydroxylation of S- and R-warfarin are enhanced in human liver microsomal incubations containing quinidine. These reactions were catalyzed by CYP3A4, based on data derived from immunoinhibitory studies, with 4'-hydroxylation being preferentially associated with S-warfarin and 10-hydroxylation with R-warfarin. The 4'-hydroxylation of S-warfarin and 10-hydroxylation of R-warfarin increased with increasing quinidine concentrations and maximized at ~3- and 5-fold the values of controls, respectively. Stimulatory effects of quinidine also were observed with recombinant CYP3A4, suggesting that increases in warfarin metabolism were due to quinidine-mediated enhancement of CYP3A4 activity. This positive cooperativity of CYP3A4 was characterized by a 2.5-fold increase in V(max) for the 4'-hydroxylation of S-warfarin and a 5-fold increase in V(max) for the 10-hydroxylation of R-warfarin, with little change in K(m) values. Conversely, V(max) for the 3-hydroxylation of quinidine was not influenced by the presence of warfarin. These results are consistent with previous findings suggesting the existence of more than one binding site in CYP3A4 through which interactions may occur between substrate and effector at the active site of the enzyme. Such interactions were subsequently illustrated by a kinetic model containing two binding domains, and a good regression fit was obtained for the experimental data. Finally, stimulation of warfarin metabolism by quinidine was investigated in suspensions of human hepatocytes, and increases in the formation of 4'- and 10-hydroxywarfarin again were observed in the presence of quinidine, indicating that this type of drug-drug interaction occurs in intact cells.

Discovery of a potent, orally bioavailable beta(3) adrenergic receptor agonist, (R)-N-[4-[2-[[2-hydroxy-2-(3-pyridinyl)ethyl]amino]ethyl]phenyl]-4-[4 -[4-(trifluoromethyl)phenyl]thiazol-2-yl]benzenesulfonamide.

As part of our investigation into the development of orally bioavailable beta(3) adrenergic receptor agonists, we have identified a series of pyridylethanolamine analogues possessing a substituted thiazole benzenesulfonamide pharmacophore that are potent human beta(3) agonists with excellent selectivity against other human beta receptor subtypes. Several of these compounds also exhibited an improved pharmacokinetic profile in dogs. For example, thiazole sulfonamide 2e (R = 4-F(3)C-C(6)H(4)) is a potent full beta(3) agonist (EC(50) = 3.6 nM, 94% activation) with >600-fold selectivity over the human beta(1) and beta(2) receptors, which also displays good oral bioavailability in several mammalian species, as well as an extended duration of action.

Determination of brain and plasma drug concentrations by liquid chromatography/tandem mass spectrometry.

A method is described for the evaluation of drug concentrations in plasma and brain from treated rats. The analyte is recovered from plasma or brain homogenate by liquid-liquid extraction and subsequently analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A simple experimental protocol renders the procedure valuable for obtaining information rapidly on brain penetration and plasma exposure of specific classes of compounds. This methodology has been applied to evaluate brain penetration with 30 different compounds from the same discovery program. In an attempt to increase throughput in our screening efforts, mixture dosing was evaluated. Results from single compound administration were compared with results following administration of a mixture of four compounds. Preliminary results, with specific classes of compounds, show no major differences (ranking order) in brain or plasma concentrations between mixture dosing and single compound administration, suggesting that mixture dosing could be applicable to brain penetration studies in the drug discovery phase.

Cytochrome P450 3A4-mediated interaction of diclofenac and quinidine.

The metabolism of diclofenac to its 5-hydroxylated derivative in humans is catalyzed by cytochrome P450 (CYP)3A4. We report herein that in vitro this biotransformation pathway is stimulated by quinidine. When diclofenac was incubated with human liver microsomes in the presence of quinidine, the formation of 5-hydroxydiclofenac increased approximately 6-fold relative to controls. Similar phenomena were observed with diastereoisomers of quinidine, including quinine and the threo epimers, which produced an enhancement in the formation of 5-hydroxydiclofenac in the order of 6- to 9-fold. This stimulation of diclofenac metabolism was diminished when human liver microsomes were pretreated with a monoclonal inhibitory antibody against CYP3A4. In contrast, neither cytochrome b(5) nor CYP oxidoreductase appeared to mediate the stimulation of diclofenac metabolism by quinidine, suggesting that the effect of quinidine is mediated through CYP3A4 protein. Further kinetic analyses indicated that V(max) values for the conversion of diclofenac to its 5-hydroxy derivative increased 4.5-fold from 13.2 to 57.6 nmol/min/nmol of CYP with little change in K(m) (71-56 microM) over a quinidine concentration range of 0 to 30 microM. Conversely, the metabolism of quinidine was not affected by the presence of diclofenac; the K(m) value estimated for the formation of 3-hydroxyquinidine was approximately 1.5 microM, similar to the quinidine concentration required to produce 50% of the maximum stimulatory effect on diclofenac metabolism. It appears that the enhancement of diclofenac metabolism does not interfere with quinidine's access to the ferriheme-oxygen complex, implicating the presence of both compounds in the active site of CYP3A4 at the same time. Finally, a approximately 4-fold increase in 5-hydroxydiclofenac formation was observed in human hepatocyte suspensions containing diclofenac and quinidine, demonstrating that this type of drug-drug interaction occurs in intact cells.

Discovery of an orally bioavailable alkyl oxadiazole beta3 adrenergic receptor agonist.

5-n-Pentyl oxadiazole substituted benzenesulfonamide 8 is a potent and selective beta3 adrenergic receptor agonist (beta3 EC50 = 23 nM, beta1 IC50 = 3000 nM, beta2 IC50 = 3000 nM). The compound has high oral bioavailability in dogs (62%) and rats (36%) and is among the most orally bioavailable beta3 adrenergic receptor agonists reported to date.

Synthesis and SAR of benzyl and phenoxymethylene oxadiazole benzenesulfonamides as selective beta3 adrenergic receptor agonist antiobesity agents.

Benzyl and phenoxymethylene substituted oxadiazoles are potent and orally bioavailable beta3 adrenergic receptor (AR) agonists. The 4-trifluormethoxy substituted 5-benzyl oxadiazole 5f has an EC50 of 8 nM in the beta3 AR agonist assay with 100-fold selectivity over beta1 and beta2 AR binding inhibition activity. Its oral bioavailability in dogs is 30 +/- 4%, with a half-life of 3.8 +/- 0.4 h. In the anesthetized rhesus, 5f evoked a dose-dependent glycerolemia (ED50Gly = 0.15 mg/kg). Under these conditions a heart rate increase of 15% was observed at a dose level of 10 mg/kg.

Interaction of diclofenac and quinidine in monkeys: stimulation of diclofenac metabolism.

The cytochrome P-450 (CYP)3A4-mediated metabolism of diclofenac is stimulated in vitro by quinidine. A similar effect is observed in incubations with monkey liver microsomes. We describe an in vivo interaction of diclofenac and quinidine that leads to enhanced clearance of diclofenac in monkeys. After a dose of diclofenac via portal vein infusion at 0.055 mg/kg/h, steady-state systemic plasma drug concentrations in three male rhesus monkeys were 87, 104, and 32 ng/ml, respectively (control). When diclofenac was coadministered with quinidine (0.25 mg/kg/h) via the same route, the corresponding plasma diclofenac concentrations were 50, 59, and 18 ng/ml, representing 57, 56, and 56% of control values, respectively. In contrast, steady-state systemic diclofenac concentrations in the same three monkeys were elevated 1.4 to 2.5 times when the monkeys were pretreated with L-754,394 (10 mg/kg i.v.), an inhibitor of CYP3A. Further investigation indicated that the plasma protein binding (>99%) and blood/plasma ratio (0.7) of diclofenac remained unchanged in the presence of quinidine. Therefore, the decreases in plasma concentrations of diclofenac after a combined dose of diclofenac and quinidine are taken to reflect increased hepatic clearance of the drug, presumably resulting from the stimulation of CYP3A-catalyzed oxidative metabolism. Consistent with this proposed mechanism, a 2-fold increase in the formation of 5-hydroxydiclofenac derivatives was observed in monkey hepatocyte suspensions containing diclofenac and quinidine. Stimulation of diclofenac metabolism by quinidine was diminished when monkey liver microsomes were pretreated with antibodies against CYP3A. Subsequent kinetic studies indicated that the K(m) value for the CYP-mediated conversion of diclofenac to its 5-hydroxy derivatives was little changed (75 versus 59 microM), whereas V(max) increased 2.5-fold in the presence of quinidine. These data suggest that the catalytic capacity of monkey hepatic CYP3A toward diclofenac metabolism is enhanced by quinidine.

L-770,644: a potent and selective human beta3 adrenergic receptor agonist with improved oral bioavailability.

L-770,644 (9c) is a potent and selective agonist of the human beta3 adrenergic receptor (EC50 = 13 nM). It shows good oral bioavailability in both dogs and rats (%F = 27), and is a full agonist for glycerolemia in the rhesus monkey (ED50 = 0.21 mg/kg). Based on its desirable in vitro and in vivo properties, L-770,644 was chosen for further preclinical evaluation.

Human beta3 adrenergic receptor agonists containing cyclic ureidobenzenesulfonamides.

Human beta3 adrenergic receptor agonists containing 5-membered ring ureas were shown to be potent partial agonists with excellent selectivity over beta1 and beta2 binding. L-760,087 (4a) and L-764,646 (5a) (beta3 EC50 = 18 and 14 nM, respectively) stimulate lipolysis in rhesus monkeys (ED50 = 0.2 and 0.1 mg/kg, respectively) with minimal effects on heart rate. Oral absorption in dogs is improved over other urea analogs.

Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites.

The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury.

Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac.

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.

Disposition of L-732,531, a potent immunosuppressant, in rats and baboons.

L-732,531 is a semi-synthetic analog of the macrolide tacrolimus (Prograf(R)). Like tacrolimus, L-732,531 is a potent immunosuppressant. In this study, its absorption, distribution, metabolism, and excretion were studied in rats and baboons. In rats, its blood and plasma levels were similar, whereas in baboons, its blood levels were, on average, twice as high as those in plasma. This was consistent with the in vitro blood-to-plasma ratio of L-732, 531, which in these two species, as well as in humans, was much lower than that of tacrolimus and showed a minimal concentration dependence. After iv administration to rats, the blood and plasma clearance of L-732,531 decreased from approximately 60 ml/min/kg at 0.2 mg/kg to 30 ml/min/kg when dosed at 1 and 3 mg/kg. After oral administration, plasma area under the concentration vs. time curve (AUC) and maximal plasma concentration (Cmax) increased more than proportionally to the dose. At 1, 5, and 15 mg/kg, plasma AUC was 29, 466, and 2832 ng.hr/ml, respectively, and Cmax was 10, 129, and 304 ng/ml, respectively. Bioavailability, although compromised by nonlinear kinetics, was estimated to be between 8% and 18%. In baboons, the clearance of L-732,531 was lower than that in rats, especially when calculated from blood concentrations (12 ml/min/kg at 0.2 mg/kg and 8 ml/min/kg at 1 mg/kg). After oral dosing, baboon plasma AUC and Cmax were much lower than those in rats, but as in rats, they increased more than proportionally with increasing doses. The bioavailability of L-732,531 in baboons was estimated at 3%, 9%, and 24% when animals were dosed at 5, 15, and 26 mg/kg po, respectively. After oral administration of [3H]L-732,531 at 5 mg/kg, approximately 32% of the radioactivity was recovered in bile and urine of rats, compared with 9% in baboons. High-performance liquid chromatography profiles of rat and baboon plasma, bile, urine, and feces indicated that L-732,531 was metabolized extensively to a complex mixture of products. Some intact parent drug was observed in feces of orally dosed animals, indicating incomplete absorption. In vitro, L-732,531 was metabolized more extensively by baboon liver microsomes than rat or human microsomes. Its metabolism in human liver microsomes was shown to be catalyzed primarily by cytochrome P450 3A isozymes.

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys.

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.