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.

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.

Mechanistic studies on the reversible metabolism of rofecoxib to 5-hydroxyrofecoxib in the rat: evidence for transient ring opening of a substituted 2-furanone derivative using stable isotope-labeling techniques.

Rofecoxib is a potent and highly selective cyclooxygenase-2 inhibitor used for the treatment of osteoarthritis and pain. Following administration of [4-(14)C]rofecoxib to intact rats, the plasma C(max) (at approximately 1 h) was followed by a secondary C(max) (at approximately 10 h), which was not observed in bile duct-cannulated rats. Following administration of [4-(14)C]5-hydroxyrofecoxib to intact or bile duct-cannulated rats, radiolabeled rofecoxib was detected in plasma, and once again a secondary C(max) for rofecoxib was observed (at approximately 10 h), which occurred only in the intact animals. These results indicate that reversible metabolism of rofecoxib to 5-hydroxyrofecoxib occurs in the rat and that the process is dependent upon an uninterrupted bile flow. Studies on the contents of the gastrointestinal tract of rats showed that conversion of 5-hydroxyrofecoxib to parent compound occurs largely in the lower intestine. Treatment of rats with [5-(18)O]5-hydroxyrofecoxib, followed by liquid chromatography-tandem mass spectrometry analyses of plasma samples, confirmed that 5-hydroxyrofecoxib undergoes metabolism to the parent drug, yielding [1-(18)O]rofecoxib, [2-(18)O]rofecoxib, and unlabeled rofecoxib. Similarly, treatment with [1,2-(18)O(2)]rofecoxib afforded the same three isotopic variants of rofecoxib. These findings are consistent with a metabolic sequence involving 5-hydroxylation of rofecoxib, biliary elimination of the corresponding glucuronide, and deconjugation of the glucuronide in the lower gastrointestinal tract. Reduction of the 5-hydroxyrofecoxib thus liberated yields a hydroxyacid that cyclizes spontaneously to regenerate rofecoxib, which is reabsorbed and enters the systemic circulation. This sequence represents a novel form of enterohepatic recycling and reflects the susceptibility of 5-hydroxyrofecoxib, as well as rofecoxib itself, to reversible 2-furanone ring opening under in vivo conditions.

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.

Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluoromethyl-coumarin bind to different domains within the active site of cytochrome P450 3A4.

Testosterone, 7-benzyloxyquinoline, and 7-benzyloxy-4-trifluoromethyl-coumarin, marker substrates for cytochrome P450 3A4 are commonly used within the pharmaceutical industry to screen new chemical entities as inhibitors of CYP3A4 in a high-throughput manner to predict the potential for drug-drug interactions. However, it has been observed that inhibition data obtained with a given CYP3A4 probe substrate may not correlate well with results from a different probe. As a consequence, the choice of the probe compound becomes an important consideration in such screens. In the present study, kinetic interactions between either two of the above three substrates were evaluated, and three-dimensional nonlinear regression analysis was performed to understand the kinetic mechanisms of drug interaction. Our results demonstrate that the kinetic interaction between each pair of substrates does not appear to be competitive and that the interactions are characterized by an unchanged or a decrease in both apparent K(m) (a = 0.21-0.72, a change of K(m) in the absence of the effector) and V(max) (alpha and beta = 0.09-0.75, changes of V(max) in the absence of the effector). These data suggest that 1) the three substrates bind to different domains; 2) at least two substrates can coexist in the active site of CYP3A4; and 3) the two bound substrates interact kinetically with each other (e.g., through steric hindrance), thereby leading to a change in both apparent kinetic parameters and partial inhibition. Selection of multiple substrates, which are shown not to be competitive, is necessary to accurately predict CYP3A4 inhibition and the potential for drug-drug interaction.

Enzyme kinetics of cytochrome P450-mediated reactions.

The most common drug-drug interactions may be understood in terms of alterations of metabolism, associated primarily with changes in the activity of cytochrome P450 (CYP) enzymes. Kinetic parameters such as Km, Vmax, Ki and Ka, which describe metabolism-based drug interactions, are usually determined by appropriate kinetic models and may be used to predict the pharmacokinetic consequences of exposure to one or multiple drugs. According to classic Michaelis-Menten (M-M) kinetics, one binding site models can be employed to simply interpret inhibition (pure competitive, non-competitive and uncompetitive) or activation of the enzyme. However, some cytochromes P450, in particular CYP3A4, exhibit unusual kinetic characteristics. In this instance, the changes in apparent kinetic constants in the presence of inhibitor or activator or second substrate do not obey the rules of M-M kinetics, and the resulting kinetics are not straightforward and hamper mechanistic interpretation of the interaction in question. These unusual kinetics include substrate activation (autoactivation), substrate inhibition, partial inhibition, activation, differential kinetics and others. To address this problem, several kinetic models can be proposed, based upon the assumption that multiple substrate binding sites exist at the active site of a particular P450, and the resulting kinetic constants are, therefore, solved to adequately describe the observed interaction between multiple drugs. The following is an overview of some cytochrome P450-mediated classic and atypical enzyme kinetics, and the associated kinetic models. Applications of these kinetic models can provide some new insights into the mechanism of P450-mediated drug-drug interactions.

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.

Studies on the metabolism of troglitazone to reactive intermediates in vitro and in vivo. Evidence for novel biotransformation pathways involving quinone methide formation and thiazolidinedione ring scission.

Therapy with the oral antidiabetic agent troglitazone (Rezulin) has been associated with cases of severe hepatotoxicity and drug-induced liver failure, which led to the recent withdrawal of the product from the U.S. market. While the mechanism of this toxicity remains unknown, it is possible that chemically reactive metabolites of the drug play a causative role. In an effort to address this possibility, this study was undertaken to determine whether troglitazone undergoes metabolism in human liver microsomal preparations to electrophilic intermediates. Following incubation of troglitazone with human liver microsomes and with cDNA-expressed cytochrome P450 isoforms in the presence of glutathione (GSH), a total of five GSH conjugates (M1-M5) were detected and identified tentatively by LC-MS/MS analysis. In two cases (M1 and M5), the structures of the adducts were confirmed by NMR spectroscopy and/or by comparison with an authentic standard prepared by synthesis. The formation of GSH conjugates M1-M5 revealed the operation of two distinct metabolic activation pathways for troglitazone, one of which involves oxidation of the substituted chromane ring system to a reactive o-quinone methide derivative, while the second involves a novel oxidative cleavage of the thiazolidinedione (TZD) ring, potentially generating highly electrophilic alpha-ketoisocyanate and sulfenic acid intermediates. When troglitazone was administered orally to a rat, samples of bile were found to contain GSH conjugates which reflected the operation of these same metabolic pathways in vivo. The finding that metabolism of the TZD ring of troglitazone was catalyzed selectively by P450 3A enzymes is significant in light of the recent report that troglitazone is an inducer of this isoform in human hepatocytes. The implications of these results are discussed in the context of the potential for troglitazone to covalently modify hepatic proteins and to cause oxidative stress through redox cycling processes, either of which may play a role in drug-induced liver injury.

A kinetic model for the metabolic interaction of two substrates at the active site of cytochrome P450 3A4.

In many cases, CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrates that coexist at the active site. In the present study, we observed that alpha-naphthoflavone (alpha-NF) exhibited a differential effect on CYP3A4-mediated product formation as shown by an increase and decrease, respectively, of the carboxylic acid (P(2)) and omega-3-hydroxylated (P(1)) metabolites of losartan, while losartan was found to be an inhibitor of the formation of the 5,6-epoxide of alpha-NF. Thus, to address this problem, a kinetic model was developed on the assumption that CYP3A4 can accommodate two distinct and independent binding domains for the substrates within the active site, and the resulting velocity equations were employed to predict the kinetic parameters for all possible enzyme-substrate species. Our results indicate that the predicted values had a good fit with the experimental observations. Therefore, the kinetic constants can be used to adequately describe the nature of the metabolic interaction between the two substrates. Applications of the model provide some new insights into the mechanism of drug-drug interactions at the level of CYP3A4.

Metabolites of caspofungin acetate, a potent antifungal agent, in human plasma and urine.

Caspofungin acetate (MK-0991) is a semisynthetic pneumocandin derivative being developed as a parenteral antifungal agent with broad-spectrum activity against systemic infections such as those caused by Candida and Aspergillus species. Following a 1-h i.v. infusion of 70 mg of [(3)H]MK-0991 to healthy subjects, excretion of drug-related material was very slow, such that 41 and 35% of the dosed radioactivity was recovered in urine and feces, respectively, over 27 days. Plasma and urine samples collected around 24 h postdose contained predominantly unchanged MK-0991, together with trace amounts of a peptide hydrolysis product, M0, a linear peptide. However, at later sampling times, M0 proved to be the major circulating component, whereas corresponding urine specimens contained mainly the hydrolytic metabolites M1 and M2, together with M0 and unchanged MK-0991, whose cumulative urinary excretion over the first 16 days postdose represented 13, 71, 1, and 9%, respectively, of the urinary radioactivity. The major metabolite, M2, was highly polar and extremely unstable under acidic conditions when it was converted to a less polar product identified as N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine gamma-lactone. Derivatization of M2 in aqueous media led to its identification as the corresponding gamma-hydroxy acid, N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine. Metabolite M1, which was extremely polar, eluting from HPLC column just after the void volume, was identified by chemical derivatization as des-acetyl-M2. Thus, the major urinary and plasma metabolites of MK-0991 resulted from peptide hydrolysis and/or N-acetylation.

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.

Metabolism of A dopamine D(4)-selective antagonist in rat, monkey, and humans: formation of A novel mercapturic acid adduct.

3-([4-(4-Chlorophenyl)piperazin-1-yl]-methyl)-1H-pyrrolo-2, 3-beta-pyridine (L-745,870) is a dopamine D(4) selective antagonist that has been studied as a potential treatment for schizophrenia, with the expectation that it would not exhibit the extrapyramidal side effects often observed with the use of classical antipsychotic agents. The metabolism of L-745,870 in vivo was investigated in the rat, rhesus monkey, and human using liquid chromatography-tandem mass spectrometry and/or NMR techniques in conjunction with radiochemical detection. In all three species, two major metabolic pathways were identified, namely N-dealkylation at the substituted piperazine moiety and the formation of a novel mercapturic acid adduct. It is proposed that the latter biotransformation process involves the formation of an electrophilic imine methide intermediate, analogous to that produced from 3-methyl indole. This report appears to represent the first example of metabolic activation of a 3-alkyl-7-azaindole nucleus.

Identification of S-(n-butylcarbamoyl)glutathione, a reactive carbamoylating metabolite of tolbutamide in the rat, and evaluation of its inhibitory effects on glutathione reductase in vitro.

Tolbutamide (TOLB), a widely used hypoglycemic agent in the therapy of non-insulin-dependent diabetes mellitus, has been reported to be teratogenic and/or embryotoxic in several animal species and humans. It has been proposed that the teratogenic effects of TOLB are linked to drug-mediated depletion of glutathione (GSH) through inhibition of the enzyme glutathione reductase (GR), although the mechanism by which this inhibition occurs remains unknown. In the study presented here, rats were injected with TOLB (200 mg/kg ip), and bile was collected for analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This led to the identification of S-(n-butylcarbamoyl)glutathione (SBuG), a reactive GSH conjugate derived from n-butyl isocyanate, as a minor metabolite of TOLB in bile. Upon incubation of SBuG (0.25-1.0 mM) with GR from either yeast or bovine intestinal mucosa in the presence of NADPH (0.20 mM), enzyme activity was lost in a time- and concentration-dependent manner. No inhibition was observed when NADPH was omitted from incubations, or when the natural substrate for the enzyme, glutathione disulfide (GSSG, 0.05 mM), was added. TOLB itself did not inhibit GR over the concentration range of 0.8-2.0 mM. It is concluded that metabolic activation of TOLB in vivo leads to the generation of reactive intermediates (n-butyl isocyanate and SBuG) which carbamoylate and thereby inhibit GR. At critical periods of organogenesis, the resulting perturbation of GSH homeostasis in exposed tissues may play a key role in the teratogenic and/or embryotoxic effects of TOLB.

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.

Role of a potent inhibitory monoclonal antibody to cytochrome P-450 3A4 in assessment of human drug metabolism.

Cytochrome P-450 (CYP) 3A4 is an inordinately important CYP enzyme that catalyzes the metabolism of a vast array of clinically used drugs. Microsomal proteins of Spodoptera frugiperda (Sf21) insect cells infected with recombinant baculoviruses encoding CYP3A4 cDNA were used to immunize mice and to develop a monoclonal antibody (mAb(3A4a)) specific to CYP3A4 through the use of hybridoma technology. The mAb is both a potent inhibitor and a strong binder of CYP3A4. One and 5 microl (0.5 and 2.5 microM IgG(2a)) of the mAb mouse ascites in 1-ml incubation containing 20 pmol of CYP3A4 strongly inhibited the testosterone 6beta-hydroxylation by 95 and 99%, respectively, and, to a lesser extent, cross-inhibited CYP3A5 and CYP3A7 activity. mAb(3A4a) exhibited no cross-reactivity with any of the other recombinant human CYP isoforms (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) in the course of CYP reaction phenotyping and Western immunoblot analyses. The potency of mAb-induced inhibition is insensitive to substrate concentration in human liver microsomes. Therefore, mAb(3A4a) was used to assess the quantitative role of CYP3A4/5 to the metabolism of testosterone and diazepam in five human liver microsomes. The results showed that CYP3A4 and CYP3A5 contribute >95% to both testosterone 6beta-hydroxylation and diazepam 3-hydroxylation and 52 to 73% to diazepam N-demethylation, respectively. In addition, mAb(3A4a) significantly inhibited testosterone 6beta-hydroxylase activity in rhesus monkey liver microsomes to a degree equal to that observed with CYP3A4 in human liver microsomes. By comparison, no inhibition of testosterone 6beta-hydroxylase activity was observed in the presence of dog, rat, and mouse liver microsomes. The selectivity of ketoconazole, a chemical inhibitor of CYP3A4, was probed with mAb(3A4a) and was shown to be highly concentration dependent in the diazepam N-demethylation by human liver microsomes. The results demonstrate that inhibitory and immunoblotting mAb(3A4a) can offer a precise and useful tool for quantitative identification of CYP3A4/5 in the metabolism of drugs in clinical use and drugs in development.

Sigmoidal kinetic model for two co-operative substrate-binding sites in a cytochrome P450 3A4 active site: an example of the metabolism of diazepam and its derivatives.

Cytochrome P450 3A4 (CYP3A4) plays a prominent role in the metabolism of a vast array of drugs and xenobiotics and exhibits broad substrate specificities. Most cytochrome P450-mediated reactions follow simple Michaelis-Menten kinetics. These parameters are widely accepted to predict pharmacokinetic and pharmacodynamic consequences in vivo caused by exposure to one or multiple drugs. However, CYP3A4 in many cases exhibits allosteric (sigmoidal) characteristics that make the Michaelis constants difficult to estimate. In the present study, diazepam, temazepam and nordiazepam were employed as substrates of CYP3A4 to propose a kinetic model. The model hypothesized that CYP3A4 contains two substrate-binding sites in a single active site that are both distinct and co-operative, and the resulting velocity equation had a good fit with the sigmoidal kinetic observations. Therefore, four pairs of the kinetic estimates (KS1, kalpha, KS2, kbeta, KS3, kdelta, KS4 and kgamma) were resolved to interpret the features of binding affinity and catalytic ability of CYP3A4. Dissociation constants KS1 and KS2 for two single-substrate-bound enzyme molecules (SE and ES) were 3-50-fold greater than KS3 and KS4 for a two-substrate-bound enzyme (SES), while respective rate constants kdelta and kgamma were 3-218-fold greater than kalpha and kbeta, implying that access and binding of the first molecule to either site in an active pocket of CYP3A4 can enhance the binding affinity and reaction rate of the vacant site for the second substrate. Thus our results provide some new insights into the co-operative binding of two substrates in the inner portions of an allosteric CYP3A4 active site.

Metabolic interactions between mibefradil and HMG-CoA reductase inhibitors: an in vitro investigation with human liver preparations.

AIMS: To determine the effects of mibefradil on the nletabolism in human liver microsomal preparations of the HMG-CoA reductase inhibitors simvastatin, lovastatin, atorvastatin, cerivastatin and fluvastatin. METHODS: Metabolism of the above five statins (0.5, 5 or 10 microM), as well as of specific CYP3A4/5 and CYP2C8/9 marker substrates, was examined in human liver microsomal preparations in the presence and absence of mibefradil (0.1-50 microM). RESULTS: Mibefradil inhibited, in a concentration-dependent fashion, the metabolism of the four statins (simvastatin, lovastatin, atorvastatin and cerivastatin) known to be substrates for CYP3A. The potency of inhibition was such that the IC50 values (<1 microM) for inhibition of all of the CYP3A substrates fell within the therapeutic plasma concentrations of mibefradil, and was comparable with that of ketoconazole. However, the inhibition by mibefradil, unlike that of ketoconazole, was at least in part mechanism-based. Based on the kinetics of its inhibition of hepatic testosterone 6beta-hydroxylase activity, mibefradil was judged to be a powerful mechanism-based inhibitor of CYP3A4/5, with values for Kinactivation, Ki and partition ratio (moles of mibefradil metabolized per moles of enzyme inactivated) of 0.4 min(-1), 2.3 microM and 1.7, respectively. In contrast to the results with substrates of CYP3A, metabolism of fluvastatin, a substrate of CYP2C8/9, and the hydroxylation of tolbutamide, a functional probe for CYP2C8/9, were not inhibited by mibefradil. CONCLUSION: Mibefradil, at therapeutically relevant concentrations, strongly suppressed the metabolism in human liver microsomes of simvastatin, lovastatin, atorvastatin and cerivastatin through its inhibitory effects on CYP3A4/5, while the effects of mibefradil on fluvastatin, a substrate for CYP2C8/9, were minimal in this system. Since mibefradil is a potent mechanism-based inhibitor of CYP3A4/5, it is anticipated that clinically significant drug-drug interactions will likely ensue when mibefradil is coadministered with agents which are cleared primarily by CYP3A-mediated pathways.