Preclinical in vitro and in vivo pharmacokinetic properties of danicamtiv, a new targeted myosin activator for the treatment of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a disease of the myocardium defined by left ventricular enlargement and systolic dysfunction leading to heart failure. Danicamtiv, a new targeted myosin activator designed for the treatment of DCM, was characterised in in vitro and in vivo preclinical studies. Danicamtiv human hepatic clearance was predicted to be 0.5 mL/min/kg from in vitro metabolic stability studies in human hepatocytes. For human, plasma protein binding was moderate with a fraction unbound of 0.16, whole blood-to-plasma partitioning ratio was 0.8, and danicamtiv showed high permeability and no efflux in a Caco-2 cell line. Danicamtiv metabolism pathways in vitro included CYP-mediated amide-cleavage, N-demethylation, as well as isoxazole- and piperidine-ring-opening. Danicamtiv clearance in vivo was low across species with 15.5, 15.3, 1.6, and 5.7 mL/min/kg in mouse, rat, dog, and monkey, respectively. Volume of distribution ranged from 0.24 L/kg in mouse to 1.7 L/kg in rat. Oral bioavailability ranged from 26% in mouse to 108% in dog. Simple allometric scaling prediction of human plasma clearance, volume of distribution, and half-life was 0.64 mL/min/kg, 0.98 L/kg, and 17.7 h, respectively. Danicamtiv preclinical attributes and predicted human pharmacokinetics supported advancement toward clinical development.

In vitro and in vivo pharmacokinetic characterization of mavacamten, a first-in-class small molecule allosteric modulator of beta cardiac myosin.

Mavacamten is a small molecule modulator of cardiac myosin designed as an orally administered drug for the treatment of patients with hypertrophic cardiomyopathy. The current study objectives were to assess the preclinical pharmacokinetics of mavacamten for the prediction of human dosing and to establish the potential need for clinical pharmacokinetic studies characterizing drug-drug interaction potential. Mavacamten does not inhibit CYP enzymes, but at high concentrations relative to anticipated therapeutic concentrations induces CYP2B6 and CYP3A4 enzymes in vitro. Mavacamten showed high permeability and low efflux transport across Caco-2 cell membranes. In human hepatocytes, mavacamten was not a substrate for drug transporters OATP, OCT and NTCP. Mavacamten was determined to have minimal drug-drug interaction risk. In vitro mavacamten metabolite profiles included phase I- and phase II-mediated metabolism cross-species. Major pathways included aromatic hydroxylation (M1), aliphatic hydroxylation (M2); N-dealkylation (M6), and glucuronidation of the M1-metabolite (M4). Reaction phenotyping revealed CYPs 2C19 and 3A4/3A5 predominating. Mavacamten demonstrated low clearance, high volume of distribution, long terminal elimination half-life and excellent oral bioavailability cross-species. Simple four-species allometric scaling led to predicted plasma clearance, volume of distribution and half-life of 0.51 mL/min/kg, 9.5 L/kg and 9 days, respectively, in human.

Detecting reactive drug metabolites for reducing the potential for drug toxicity.

INTRODUCTION: A number of withdrawn drugs are known to undergo bioactivation by a range of drug metabolizing enzymes to chemically reactive metabolites that bind covalently to protein and DNA resulting in organ toxicity and carcinogenesis, respectively. An important goal in drug discovery is to identify structural sites of bioactivation within discovery molecules for providing strategic modifications that eliminate or minimize reactive metabolite formation, while maintaining target potency, selectivity and desired pharmacokinetic properties leading to the development of efficacious and nontoxic drugs. AREAS COVERED: This review covers experimental techniques currently used to detect reactive drug metabolites and provides recent examples where information from mechanistic in vitro studies was successfully used to redesign candidate drugs leading to blocked or minimized bioactivation. Reviewed techniques include in vitro radiolabeled drug covalent binding to protein and reactive metabolite trapping with reagents such as glutathione, cyanide, semicarbazide and DNA bases. Case studies regarding reactive metabolite detection using a combination of varied techniques, including liquid chromatography-tandem mass spectrometry and NMR analyses and subsequent structural modification are discussed. EXPERT OPINION: Information derived from state-of-art mechanistic drug metabolism studies can be used successfully to direct medicinal chemistry towards the synthesis of candidate drugs devoid of bioactivation liabilities, while maintaining desired pharmacology and pharmacokinetic properties.

Structure-assisted discovery of the first non-retinoid ligands for Retinol-Binding Protein 4.

Retinol-Binding Protein 4 (RBP4) is a plasma protein that transports retinol (vitamin A) from the liver to peripheral tissues. This Letter highlights our efforts in discovering the first, to our knowledge, non-retinoid small molecules that bind to RBP4 at the retinol site and reduce serum RBP4 levels in mice, by disrupting the interaction between RBP4 and transthyretin (TTR), a plasma protein that binds RBP4 and protects it from renal excretion. Potent compounds were discovered and optimized quickly from high-throughput screen (HTS) hits utilizing a structure-based approach. Inhibitor co-crystal X-ray structures revealed unique disruptions of RBP4-TTR interactions by our compounds through induced loop conformational changes instead of steric hindrance exemplified by fenretinide. When administered to mice, A1120, a representative compound in the series, showed concentration-dependent retinol and RBP4 lowering.

Discovery and optimization of 5-(2-((1-(phenylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)oxy)pyridin-4-yl)-1,2,4-oxadiazoles as novel gpr119 agonists.

We describe the discovery and optimization of 5-(2-((1-(phenylsulfonyl)-1,2,3,4-tetrahydroquinolin-7-yl)oxy)pyridin-4-yl)-1,2,4-oxadiazoles as novel agonists of GPR119. Previously described aniline 2 had suboptimal efficacy in signaling assays using cynomolgus monkey (cyno) GPR119 making evaluation of the target in preclinical models difficult. Replacement of the aniline ring with a tetrahydroquinoline ring constrained the rotation of the aniline C-N bond and gave compounds with increased efficacy on human and cyno receptors. Additional optimization led to the discovery of 10, which possesses higher free fraction in plasma and improved pharmacokinetic properties in rat and cyno compared to 2.

Discovery and optimization of N-(3-(1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-4-yloxy)phenyl)benzenesulfonamides as novel GPR119 agonists.

The discovery and optimization of novel N-(3-(1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-4-yloxy)phenyl)benzenesulfonamide GPR119 agonists is described. Modification of the pyridylphthalimide motif of the molecule with R(1)=-Me and R(2)=-(i)Pr substituents, incorporated with a 6-fluoro substitution on the central phenyl ring offered a potent and metabolically stable tool compound 22.

Metabolism-guided discovery of a potent and orally bioavailable urea-based calcimimetic for the treatment of secondary hyperparathyroidism.

A series of urea based calcimimetics was optimized for potency and oral bioavailability. Crucial to this process was overcoming the poor pharmacokinetic properties of lead thiazole 1. Metabolism-guided modifications, characterized by the use of metabolite identification (ID) and measurement of time dependent inhibition (TDI) of CYP3A4, were essential to finding a compound suitable for oral dosing. Calcimimetic 18 exhibited excellent in vivo potency in a 5/6 nephrectomized rat model and cross-species pharmacokinetics.

Discovery and optimization of arylsulfonyl 3-(pyridin-2-yloxy)anilines as novel GPR119 agonists.

We describe the discovery of a series of arylsulfonyl 3-(pyridin-2-yloxy)anilines as GPR119 agonists derived from compound 1. Replacement of the three methyl groups in 1 with metabolically stable moieties led to the identification of compound 34, a potent and efficacious GPR119 agonist with improved pharmacokinetic (PK) properties.

Interaction of γ-glutamyltranspeptidase with ibuprofen-S-acyl-glutathione in vitro and in vivo in human.

Ibuprofen is metabolized to chemically reactive acyl glucuronide and S-acyl-CoA metabolites that are proposed to transacylate glutathione (GSH) forming ibuprofen-S-acyl-GSH (I-SG) in vivo. Herein, we report the detection of novel metabolites of ibuprofen, namely ibuprofen-N-acyl-cysteinylglycine (I-N-CG), ibuprofen-N-acyl-cysteine (I-N-C), and the mercapturic acid conjugate, ibuprofen-S-acyl-N-acetylcysteine (I-S-NAC), in urine from an ibuprofen-dosed volunteer. Thus, analysis of ibuprofen-dosed (Advil, 800 mg, Pfizer, Madison, NJ) human urine extracts by sensitive liquid chromatography tandem mass spectrometric detection resulted in the identification of I-N-CG, I-N-C, and I-S-NAC derivatives as minor metabolites (6.0, 1.7, and 0.2 µg excreted 10-hours postadministration, respectively). I-N-CG is proposed to be formed from the degradation of I-SG by γ-glutamyltranspeptidase (γ-GT)-mediated cleavage of the γ-glutamyl group, leading to an unstable ibuprofen-S-acyl-cysteinylglycine (I-S-CG) intermediate that undergoes spontaneous S to N intramolecular rearrangement. Then, dipeptidase-mediated cleavage of glycine from I-N-CG leads to the formation of I-N-C. Treatment of racemic I-SG (100 µM) in vitro with commercially available bovine kidney γ-GT (0.1 units/ml) in buffer at pH 7.4 and 37°C resulted in its complete degradation, yielding (R)- and (S)-I-N-CG after 15 minutes of incubation. In vitro enzyme kinetic studies with bovine kidney γ-GT incubated separately with (R)- and (S)-I-SG isomers revealed no enantioselective degradation. Results from these studies provided evidence that ibuprofen is metabolized in human to reactive transacylating-type intermediates that react with GSH, forming I-SG thioester that, following degradation by γ-GT and dipeptidase enzymes and following S to N intramolecular rearrangement, leads to the urinary excretion of the I-N-CG and I-N-C amide-linked conjugates, respectively.

Metabolic activation of mefenamic acid leading to mefenamyl-S-acyl-glutathione adduct formation in vitro and in vivo in rat.

Carboxylic acid-containing nonsteroidal anti-inflammatory drugs (NSAIDs) can be metabolized to chemically reactive acyl glucuronide and/or S-acyl-CoA thioester metabolites capable of transacylating GSH. We investigated the metabolism of the NSAID mefenamic acid (MFA) to metabolites that transacylate GSH, leading to MFA-S-acyl-GSH thioester (MFA-SG) formation in incubations with rat and human hepatocytes and in vivo in rat bile. Thus, incubation of MFA (1-500 µM) with rat hepatocytes led to the detection of MFA-1-ß-O-acyl glucuronide (MFA-1-ß-O-G), MFA-S-acyl-CoA (MFA-SCoA), and MFA-SG by liquid chromatography-tandem mass spectrometric analysis. The C(max) of MFA-SG (330 nM; 10-min incubation with 100 µM MFA) was 120- to 1400-fold higher than the C(max) of drug S-acyl-GSH adducts detected from studies with other carboxylic acid drugs to date. MFA-SG was also detected in incubations with human hepatocytes, but at much lower concentrations. Inhibition of MFA acyl glucuronidation in rat hepatocytes had no effect on MFA-SG formation, whereas a 58 ± 1.7% inhibition of MFA-SCoA formation led to a corresponding 66 ± 3.5% inhibition of MFA-SG production. Reactivity comparisons with GSH in buffer showed MFA-SCoA to be 80-fold more reactive than MFA-1-ß-O-G forming MFA-SG. MFA-SG was detected in MFA-dosed (100 mg/kg) rat bile, where 17.4 µg was excreted after administration. In summary, MFA exhibited bioactivation in rat and human hepatocytes and in vivo in rat, leading to reactive acylating derivatives that transacylate GSH. The formation of MFA-SG in hepatocytes was shown not to be mediated by reaction with MFA-1-ß-O-G, and not solely by MFA-SCoA, but perhaps also by intermediary MFA-acyl-adenylate formation, which is currently under investigation.

Effect of culture time on the basal expression levels of drug transporters in sandwich-cultured primary rat hepatocytes.

Sandwich-cultured rat hepatocytes are used in drug discovery for pharmacological and toxicological assessment of drug candidates, yet their utility as a functional model for drug transporters has not been fully characterized. To evaluate the system as an in vitro model for drug transport, expression changes of hepatic transporters relative to whole liver and freshly isolated hepatocytes (day 0) were examined by real-time quantitative reverse transcription-polymerase chain reaction for 4 consecutive days of culture. No significant differences in transporter expression levels were observed between freshly isolated hepatocytes and whole liver. Two distinct mRNA profiles were detected over time showing 1) a more than 5-fold decline in levels of uptake transporters such as Na(+)-taurocholate cotransporting polypeptide (Ntcp), organic anion transporter (Oat) 2, organic anion-transporting polypeptide (Oatp) 1a1, Oatp1a4, and Oatp1b2 and 2) a greater than 5-fold increase of efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-related proteins (Mrp) 1, 2, 3, and 4. In addition, protein levels and functional activities for selected transporters were also determined. Protein levels for Mrp2, Bcrp, P-gp, Ntcp, and Oatp1a4 corresponded to changes in mRNA. Functional activities of Oatps and Oct1 exhibited a 3- and 4-fold decrease on day 2 and day 4, respectively, relative to that on day 0, whereas a more than 10-fold reduction in Oat2 activity was observed. These results indicate that the cell culture conditions used herein did not provide an optimal environment for expression of all hepatic transporters. Significant time-dependent alterations in basal gene expression patterns of transporters were detected compared with those in liver or freshly isolated hepatocytes. Further work and new strategies are required to improve the validity of this model as an in vitro tool for in vivo drug transport or biliary clearance prediction.

Constituents in kava extracts potentially involved in hepatotoxicity: a review.

Aqueous kava root preparations have been consumed in the South Pacific as an apparently safe ceremonial and cultural drink for centuries. However, several reports of hepatotoxicity have been linked to the consumption of kava extracts in Western countries, where mainly ethanolic or acetonic extracts are used. The mechanism of toxicity has not been established, although several theories have been put forward. The composition of the major constituents, the kava lactones, varies according to preparation method and species of kava plant, and thus, the toxicity of the individual lactones has been tested in order to establish whether a single lactone or a certain composition of lactones may be responsible for the increased prevalence of kava-induced hepatotoxicity in Western countries. However, no such conclusion has been made on the basis of current data. Inhibition or induction of the major metabolizing enzymes, which might result in drug interactions, has also gained attention, but ambiguous results have been reported. On the basis of the chemical structures of kava constituents, the formation of reactive metabolites has also been suggested as an explanation of toxicity. Furthermore, skin rash is a side effect in kava consumers, which may be indicative of the formation of reactive metabolites and covalent binding to skin proteins leading to immune-mediated responses. Reactive metabolites of kava lactones have been identified in vitro as glutathione (GSH) conjugates and in vivo as mercapturates excreted in urine. Addition of GSH to kava extracts has been shown to reduce cytotoxicity in vitro, which suggests the presence of inherently reactive constituents. Only a few studies have investigated the toxicity of the minor constituents present in kava extract, such as pipermethystine and the flavokavains, where some have been shown to display higher in vitro cytotoxicity than the lactones. To date, there remains no indisputable reason for the increased prevalence of kava-induced hepatotoxicity in Western countries.

Drug-S-acyl-glutathione thioesters: synthesis, bioanalytical properties, chemical reactivity, biological formation and degradation.

Carboxylic acid-containing drugs can be metabolized to chemically-reactive acyl glucuronide, S-acyl-CoA thioester, and/or intermediate acyl-adenylate metabolites that are capable of transacylating the cysteinyl-thiol of glutathione (GSH) resulting in the formation of drug-S-acyl-GSH thioesters detected in-vivo in bile and in-vitro in hepatocytes. Authentic S-acyl-GSH thioesters of carboxylic acids can be readily synthesized by modifying the cysteinyl-thiol group of GSH with an applicable acylating reagent. Bionanalytical characterization of S-acyl-GSH derivatives has demonstrated enhanced extraction efficiency from biological samples when formic acid is included in appropriate extraction solvents, and that tandem mass spectrometry of S-acyl-GSH conjugates results in fragmentation producing a common MH+-147 Da product ion. Chemical reactivity comparisons have shown that S-acyl-CoA thioester and acyl-adenylate conjugates are more reactive than their corresponding 1-ß-O-acyl glucuronides toward the transacylation of GSH forming S-acyl-GSH thioesters. S-Acyl-GSH thioester derivatives are also chemically-reactive electrophiles capable of transacylating biological nucleophiles. Glutathione S-transferases (GSTs) weakly catalyze S-acyl-GSH conjugate formation from S-acyl-CoA, acyl-adenylate, and 1-ß-O-acyl glucuronide substrates; however purified-GSTs have also been shown to hydrolyze S-acyl-GSH thioesters. Mechanistic in vitro studies in hepatocytes have revealed the primary importance of the S-acyl-CoA formation pathway leading to S-acyl-GSH-adduct formation. In addition to being hydrolytically-unstable in hepatocytes and plasma, S-acyl-GSH thioesters undergo γ-glutamyltranspeptidase-mediated cleavage of the γ-glutamyl-group leading to N-acyl-cysteinylglycine amide-linked products. In summary, S-acyl GSH thioesters are indicators of reactive transacylating metabolite formation produced from the biotransformation of carboxylic acids, but since they are also chemically-reactive, perhaps these derivatives can contribute to covalent binding to tissue proteins and potential toxicity.

Stereoselective flunoxaprofen-S-acyl-glutathione thioester formation mediated by acyl-CoA formation in rat hepatocytes.

Flunoxaprofen (FLX) is a chiral nonsteroidal anti-inflammatory drug that was withdrawn from clinical use because of concerns of potential hepatotoxicity. FLX undergoes highly stereoselective chiral inversion mediated through the FLX-S-acyl-CoA thioester (FLX-CoA) in favor of the (R)-(-)-isomer. Acyl-CoA thioester derivatives of acidic drugs are chemically reactive species that are known to transacylate protein nucleophiles and glutathione (GSH). In this study, we investigated the relationship between the stereoselective metabolism of (R)-(-)- and (S)-(+)-FLX to FLX-CoA and the subsequent transacylation of GSH forming FLX-S-acyl-glutathione (FLX-SG) in incubations with rat hepatocytes in suspension. Thus, when hepatocytes (2 million cells/ml) were treated with (R)-(-)- or (S)-(+)-FLX (100 microM), both FLX-CoA and FLX-SG were detected by sensitive liquid chromatography-tandem mass spectrometry techniques. However, these derivatives were observed primarily from (R)-(-)-FLX incubation extracts, for which the formation rates of FLX-CoA and FLX-SG were rapid, reaching maximum concentrations of 42 and 2.8 nM, respectively, after 6 min of incubation. Incubations with (S)-(+)-FLX over 60 min displayed 8.1 and 2.7% as much FLX-CoA and FLX-SG area under the concentration versus time curves, respectively, compared with corresponding incubations with (R)-(-)-FLX. Coincubation of lauric acid (1000 microM) with (R)-(-)-FLX (10 microM) led to the complete inhibition of FLX-CoA formation and a 98% inhibition of FLX-SG formation. Reaction of authentic (R,S)-FLX-CoA (2 microM) with GSH (10 mM) in buffer (pH 7.4, 37 degrees C) showed the quantitative formation of FLX-SG after 3 h of incubation. Together, these results demonstrate the stereoselective transacylation of GSH in hepatocyte incubations containing (R)-(-)-FLX, which is consistent with bioactivation by stereoselective (R)-FLX-CoA formation.

Covalent binding of phenylacetic acid to protein in incubations with freshly isolated rat hepatocytes.

Phenylacetic acid (PAA) represents a substructure of a class of nonsteroidal anti-inflammatory carboxylic acid-containing drugs capable of undergoing metabolic activation in the liver to acylcoenzyme A (CoA)- and/or acyl glucuronide-linked metabolites that are proposed to be associated with the formation of immunogenic, and hence potentially hepatotoxic, drug-protein adducts. Herein, we investigated the ability of PAA to undergo phenylacetyl-S-acyl-CoA thioester (PA-CoA)-mediated covalent binding to protein in incubations with freshly isolated rat hepatocytes in suspension. Thus, when hepatocytes were incubated with phenylacetic acid carboxy-(14)C (100 microM) and analyzed for PA-CoA formation and covalent binding of PAA to protein and over a 3-h time period, both PA-CoA formation and covalent binding to protein increased rapidly, reaching 1.3 microM and 291 pmol equivalents/mg protein after 4 and 6 min of incubation, respectively. However, the covalent binding of PAA to protein was reversible and decreased by 72% at the 3-h time point. After 3 h of incubation, PAA was shown to be metabolized primarily to phenylacetyl-glycine amide (84%). No PAA-acyl glucuronide was detected in the incubation extracts. PA-CoA reacted readily with glutathione in buffer, forming PA-S-acyl-glutathione; however, this glutathione conjugate was not detected in hepatocyte incubation extracts. Coincubation of hepatocytes with lauric acid led to a marked inhibition of PA-CoA formation and a corresponding inhibition of covalent binding to protein. SDS-polyacrylamide gel electrophoresis analysis showed the formation of two protein adducts having molecular masses of approximately 29 and approximately 33 kDa. In summary, PA-CoA formation in rat hepatocytes leads to the highly selective, but reversible, covalent binding to hepatocyte proteins, but not to the transacylation of glutathione.

Gamma-glutamyltranspeptidase-mediated degradation of diclofenac-S-acyl-glutathione in vitro and in vivo in rat.

Diclofenac, a nonsteroidal antiinflammatory drug, is known to be metabolized to chemically reactive intermediates that transacylate GSH forming diclofenac-S-acyl-glutathione (D-SG) in vivo in rat and in vitro in rat and human hepatocytes. Recently, it was reported that the treatment of rats with diclofenac led to a substantial decrease in the activity of hepatic gamma-glutamyltranspeptidase (gamma-GT), an extracellular canalicular membrane enzyme. Because studies have indicated that D-SG is a chemically reactive transacylating species that is excreted into rat bile, we propose that D-SG formed in the liver may be a substrate for, and potential inhibitor of, hepatic gamma-GT. The present experiments were performed to investigate the ability of D-SG to be a substrate for gamma-GT in vivo in rat and in vitro with commercially available gamma-GT enzyme. We also examined the ability of D-SG to inhibit gamma-GT in vitro. Thus, LC-MS/MS analysis of bile extracts from diclofenac-dosed rats (200 mg/kg, iv) showed the presence of the gamma-GT-mediated D-SG degradation product diclofenac-N-acyl-cysteinylglycine (D- N-CG), where a total of approximately 8 microg was excreted 6 h postadministration. When D-SG (100 microM) was incubated with gamma-GT (1 unit/mL), the GSH adduct was degraded in a linear time-dependent fashion where approximately 94 microM D- N-CG was formed after 20 min of incubation. Dialysis studies showed that inhibition of gamma-GT by D-SG was completely reversible. Further inhibition studies showed that D-SG is a competitive inhibitor of the gamma-GT enzyme. Results from theses studies indicate that D-SG is a substrate for gamma-GT; however, the conjugate may not contribute significantly to the decrease in gamma-GT activity reported to occur in vivo in rat.

Enantioselective formation of ibuprofen-S-acyl-glutathione in vitro in incubations of ibuprofen with rat hepatocytes.

Ibuprofen is metabolized to chemically reactive ibuprofen-1- O-acyl-glucuronide (I-1- O-G) and ibuprofen- S-acyl-CoA (I-CoA) derivatives, which are proposed to mediate the formation of drug-protein adducts via the transacylation of protein nucleophiles. We examined the ability of ibuprofen to undergo enantioselective metabolism to ibuprofen- S-acyl-glutathione thioester (I-SG) in incubations with rat hepatocytes, where I-CoA formation is known to be highly enantioselective in favor of the (R)-(-)-ibuprofen isomer. We proposed that potential enantioselective transacylation of glutathione forming I-SG in favor of the (R)-(-)-isomer would reveal the importance of acyl-CoA formation, versus acyl glucuronidation, in the generation of reactive transacylating-type intermediates of the drug. Thus, when (R)-(-)- and (S)-(+)-ibuprofen (100 microM) were incubated with hepatocytes, the presence of I-CoA and I-SG was detected in incubation extracts by LC-MS/MS techniques. The formation of I-CoA and I-SG in hepatocyte incubations with (R)-(-)-ibuprofen was rapid and reached maximum concentrations of 2.6 microM and 1.3 nM, respectively, after 8-10 min of incubation. By contrast, incubations with (S)-(+)-ibuprofen resulted in 8% and 3.9% as much I-CoA and I-SG formation, respectively, compared to that in corresponding incubations with the (R)-(-)-isomer. Experiments with a pseudoracemic mixture of (R)-(-)-[3,3,3-(2)H3]- and (S)-(+)-ibuprofen showed that >99% of the I-SG detected in hepatocyte incubations contained deuterium and therefore was derived primarily from (R)-(-)-ibuprofen bioactivation. Inhibition of (R)-(-)-ibuprofen (10 microM) glucuronidation with (-)-borneol (100 microM) led to a 98% decrease in I-1-O-G formation; however, no decrease in I-SG production was observed. Coincubation with pivalic, valproic, or lauric acid (500 microM each) was shown to lead to a significant inhibition of I-CoA formation and a corresponding decrease in I-SG production. Results from these studies demonstrate that the reactive I-CoA derivative, and not the I-1-O-G metabolite, plays a central role in the transacylation of GSH in incubations with rat hepatocytes.

A novel bioactivation pathway for 2-[2-(2,6-dichlorophenyl)aminophenyl]ethanoic acid (diclofenac) initiated by cytochrome P450-mediated oxidative decarboxylation.

Diclofenac (2-[2-(2,6-dichlorophenyl)aminophenyl]ethanoic acid), a nonsteroidal antiinflammatory drug, undergoes bioactivation by cytochrome P450 oxidation to chemically reactive metabolites that are capable of reacting with endogenous nucleophiles such as glutathione (GSH) and proteins and that may play a role in the idiosyncratic hepatotoxicity associated with the drug. Here, we investigated the ability of diclofenac to be metabolized to 2-(2,6-dichloro-phenylamino)benzyl-S-thioether glutathione (DPAB-SG) in incubations with rat liver microsomes (RLMs) and human liver microsomes (HLMs) fortified with NADPH and GSH. Thus, after incubation of diclofenac (50 microM) with liver microsomes (1 mg protein/ml), the presence of DPAB-SG was detected in both RLM and HLM incubation extracts by liquid chromatography-tandem mass spectrometry techniques. The formation of DPAB-SG was NADPH-, concentration-, and time-dependent. Coincubation of diclofenac (10 microM) with ketoconazole (1 microM), an inhibitor of cytochrome P450 (P450) 3A4, with HLMs led to a 75% decrease in DPAB-SG formation. However, in contrast, coincubation with the P450 2C9 inhibitor sulfaphenazole (10 microM) or the P450 2D6 inhibitor quinidine (40 microM) led to a 1.9- and 1.6-fold increase in DPAB-SG production, respectively. From these data, we propose that P450 3A4 mediates the oxidative decarboxylation of diclofenac, resulting in the formation of a transient benzylic carbon-centered free radical intermediate that partitions between elimination (o-imine methide production) and recombination (alcohol formation) pathways. The benzyl alcohol intermediate, which was not analyzed for in the present studies, if formed could undergo dehydration to provide a reactive o-imine methide species. The o-imine methide intermediate then is proposed to react covalently with GSH, forming DPAB-SG.

Metabolic activation of carboxylic acids.

BACKGROUND: Carboxylic acids constitute a large and heterogeneous class of both endogenous and xenobiotic compounds. A number of carboxylic acid drugs have been associated with adverse reactions, linked to the metabolic activation of the carboxylic acid moiety of the compounds, i.e., formation of acyl-glucuronides and acyl-CoA thioesters. OBJECTIVE: The objective is to give an overview of the current knowledge on metabolic activation of carboxylic acids and how such metabolites may play a role in adverse reactions and toxicity. METHODS: Literature concerning the formation and disposition of acyl glucuronides and acyl-CoA thioesters was searched. Also included were papers on the chemical reactivity of acyl glutathione-thioesters, and literature concerning possible links between metabolic activation of carboxylic acids and reported cellular and clinical effects. RESULTS/CONCLUSION: This review demonstrates that metabolites of carboxylic acid drugs must be considered chemically reactive, and that the current knowledge about metabolic activation of this compound class can be a good starting-point for further studies on the consequences of chemically reactive metabolites.

Differential effects of fibrates on the metabolic activation of 2-phenylpropionic acid in rats.

A series of studies were conducted to explore the inductive potential of different fibric acid derivatives on the two alternative metabolic activation pathways of 2-phenylpropionic acid (2-PPA) (a model substrate for profen drugs), namely acyl-CoA formation and acyl glucuronidation, in vivo in rats, and to evaluate whether such treatment could potentially modulate the covalent binding of profens to hepatic protein. After administration of a single dose of 2-PPA (130 mg/kg) to rats pretreated with equimolar doses of clofibric acid (160 mg/kg/day), fenofibrate (260 mg/kg/day), or gemfibrozil (180 mg/kg/day) for 7 days, rat livers were collected and analyzed for covalent binding and hepatic levels of the two reactive metabolites over a 2-h period. Results showed that the three fibrates exhibited very different effects on the hepatic levels of 2-PPA-S-acyl CoA (2-PPA-CoA) in vivo, even though all three significantly increased acyl-CoA synthetase activity in vitro in liver homogenate. Treatment with clofibric acid markedly increased the hepatic exposure of 2-PPA-CoA by 2.9-fold and led to a 25% increase (p < 0.05) in covalent binding of 2-PPA to liver protein. In contrast, significant decreases of the hepatic levels of 2-PPA acyl glucuronide and/or 2-PPA-CoA by fenofibrate and gemfibrozil significantly lowered the covalent binding of 2-PPA to hepatic protein. Together, these results suggest that fibrates exhibit markedly different abilities to alter the extent of covalent binding of 2-PPA to hepatic protein by differentially modulating the hepatic exposure of the two reactive metabolites of 2-PPA, namely 2-PPA-CoA thioester and acyl glucuronide.