Mark P. Grillo

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.

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.

NMR spectroscopy and MRI investigation of a potential bioartificial liver

NMR feasibility was established for a coaxial hydrophobic‐membrane bioreactor containing isolated rat hepatocytes with features designed to mimic the human liver. A novel triple‐tuned NMR probe and a perfusion system controlling temperature, gas concentrations, flow‐rate, and pH were used. We determined the optimum coaxial interfiber distance (i.e. diffusion distance) for maintaining hepatocyte viability in two bioreactor prototypes. Prototype no. 1 and no. 2 had diffusion distances of 500 μm and 200 μm, respectively. Cell viability was established by 31P NMR and trypan blue exclusion. Only prototype no. 2 maintained cell viability for more than 6 h, indicating the importance of diffusion distance. 31P spectra obtained over this 6 h time period were similar to in vivo spectra of rat liver. The 31P spectra were found to be more sensitive to subacute cell viability than trypan blue exclusion. In the 1H and 31P spectra, 1H2O and inorganic phosphate signals were split in two at all flow‐rates, probably due to bulk magnetic susceptibility effects originating from the three bioreactor compartments. MRI was useful for quality control and determining flow dynamics, fiber integrity, and cell inoculate distribution. MRI revealed that the inner fibers were not centered in either prototype. Although an increased flow‐rate did not influence spectral resolution or chemical shifts, significant degradation of MRI quality occurred above 50 mL/min. NMR spectroscopy and imaging provide valuable, real‐time information on cell biochemistry and flow dynamics which can be used in development and monitoring of bioreactors designed as artificial livers.

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.

In vitro studies on the chemical reactivity of 2,4-dichlorophenoxyacetyl-S-acyl-CoA thioester.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a widely used broadleaf herbicide that has been associated with acute liver toxicity in exposed humans or animals. Chemically reactive metabolites of 2,4-D are proposed as mediators of 2,4-D-induced hepatotoxicity. The aim of the present study was to investigate a novel reactive metabolite of 2,4-D, namely 2,4-dichlorophenoxyacetyl-S-acyl-CoA (2,4-D-CoA), and to determine its involvement in 2,4-D covalent adduct formation. Thus, incubations of synthetic 2,4-D-CoA (106 microM) with GSH (1 mM) in phosphate buffer (pH 7.4) showed 2,4-D-CoA to be able to transacylate the cysteine sulfhydryl of GSH, resulting in the formation of 2,4-D-S-acyl-glutathione (2,4-D-SG) thioester and reaching a concentration of 65 microM after 1 h of incubation. Under similar conditions, 2,4-D-CoA was shown to covalently bind to nucleophilic groups on human serum albumin (HSA, 30 mg/ml), resulting in time-dependent 2,4-D-HSA covalent adduct formation that reached a maximum of 440 pmol/mg HSA after 1 h of incubation. In addition to these studies, incubations of [1-(14)C]2,4-D (1 mM) with rat hepatocytes showed a time-dependent covalent binding of 2,4-D to hepatocyte protein. Inhibition of acyl-CoA formation by trimethylacetic acid (2 mM) decreased the amount of covalent binding to protein in rat hepatocytes by 50%. These results indicate that 2,4-D-CoA thioester is a reactive metabolite of 2,4-D that may contribute to 2,4-D-protein adduct formation in vivo and therefore the associated hepatotoxicity.

Discovery of AMG 853, a CRTH2 and DP Dual Antagonist.

Prostaglandin D2 (PGD2) plays a key role in mediating allergic reactions seen in asthma, allergic rhinitis, and atopic dermatitis. PGD2 exerts its activity through two G protein-coupled receptors (GPCRs), prostanoid D receptor (DP or DP1), and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2 or DP2). We report the optimization of a series of phenylacetic acid derivatives in an effort to improve the dual activity of AMG 009 against DP and CRTH2. These efforts led to the discovery of AMG 853 (2-(4-(4-(tert-butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenyl sulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid), which is being evaluated in human clinical trials for asthma.

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 μM) 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 μM) with ketoconazole (1 μM), 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 μM) or the P450 2D6 inhibitor quinidine (40 μM) 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.

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.

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.

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.