In vitro and in vivo preclinical pharmacokinetic characterization of aficamten, a small molecule cardiac myosin inhibitor.

Aficamten, a small molecule selective inhibitor of cardiac myosin, was characterised in preclinical and studies.Protein binding in human plasma was 10.4% unbound and ranged from 1.

Pharmacokinetics, mass balance, tissue distribution, metabolism, and excretion of [C]aficamten following single oral dose administration to rats.

The pharmacokinetics, metabolism, excretion, mass balance, and tissue distribution of [C]aficamten were evaluated following oral administration of an 8 mg/kg dose in Sprague Dawley rats and in a quantitative whole-body autoradiography study in Long Evans rats.[C]Aficamten accounted for ∼80% and a hydroxylated metabolite (M1) accounted for ∼12% of total radioactivity in plasma over 48-h (AUC). Plasma was 4-h and the of total plasma radioactivity was 5.

Preclinical and 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 and preclinical studies. Danicamtiv human hepatic clearance was predicted to be 0.

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.

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Introduction: A number of withdrawn drugs are known to undergo bioactivation by a range of drug metabolizing enzymes to chemically reactive metabolites that bind covalently to protein and DNA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.

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.

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.

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.

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.

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-β-O-acyl glucuronide (MFA-1-β-O-G), MFA-S-acyl-CoA (MFA-SCoA), and MFA-SG by liquid chromatography-tandem mass spectrometric analysis.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

Mechanistic studies on the bioactivation of diclofenac: identification of diclofenac-S-acyl-glutathione in vitro in incubations with rat and human hepatocytes.

Diclofenac, a nonsteroidal anti-inflammatory drug, is metabolized to diclofenac-1-O-acyl glucuronide (D-1-O-G), a chemically reactive conjugate that has been implicated as playing a role in the idiosyncratic hepatoxicity associated with its use. The present studies investigated the ability of diclofenac to be metabolized to diclofenac-S-acyl-glutathione thioester (D-SG) in vitro in incubations with rat and human hepatocytes and whether its formation is dependent on a transacylation-type reaction between D-1-O-G and glutathione. When diclofenac (100 microM) was incubated with hepatocytes, D-SG was detected in both rat and human incubation extracts by a sensitive LC-MS/MS technique.

Identification of zomepirac-S-acyl-glutathione in vitro in incubations with rat hepatocytes and in vivo in rat bile.

Zomepirac (ZP), a nonsteroidal anti-inflammatory drug that was withdrawn from use, is metabolized to zomepirac-1-O-acyl-glucuronide (ZP-1-O-G), a chemically reactive conjugate that has been implicated in the toxicity of the drug. In the present studies, we investigated the ability of ZP to become bioactivated to reactive metabolites that transacylate glutathione (GSH) forming ZP-S-acyl-glutathione thioester (ZP-SG) in vitro and in vivo in rat. When ZP (100 microM) was incubated with rat hepatocytes, ZP-SG was detected in incubation extracts by a sensitive selected reaction monitoring liquid chromatography/tandem mass spectrometry (LC/MS-MS) technique.

Studies on the chemical reactivity of diclofenac acyl glucuronide with glutathione: identification of diclofenac-S-acyl-glutathione in rat bile.

Diclofenac, a nonsteroidal anti-inflammatory drug, is metabolized to a reactive acyl glucuronide that has been proposed to mediate toxic adverse drug reactions associated with its use. In the present study, we examined the ability of diclofenac acyl glucuronide (D-1-O-G) to transacylate glutathione (GSH) in vitro in buffer and in vivo in rats. Thus, in vitro reactions of D-1-O-G (100 microM) with GSH (10 mM) at pH 7.