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Heather Eng,
Raman Sharma,
Thomas S McDonald,
David J Edmonds,
Jean-Phillippe Fortin,
Xianping Li,
Benjamin D Stevens,
David A Griffith,
Chris Limberakis,
Whitney M Nolte,
David A Price,
Margaret Jackson, Amit S Kalgutkar
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ABSTRACT: 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP) represents a novel small molecule activator of the glucagon-like peptide-1 receptor (GLP-1R), and exhibits glucose-dependent insulin secretion in rats following intravenous (but not oral) administration. In order to explore the quantitative pharmacology associated with GLP-1R agonism in preclinical species, the in vivo pharmacokinetics of BETP were examined in rats after intravenous and oral dosing. Failure to detect BETP in circulation after oral administration of a 10 mg/kg dose in rats was consistent with the lack of an insulinotropic effect of orally administered BETP in this species. Likewise, systemic concentrations of BETP in the rat upon intravenous administration (1 mg/kg) were minimal (and sporadic). In vitro incubations in bovine serum albumin, plasma and liver microsomes from rodents and human indicated a facile degradation of BETP. Failure to detect metabolites in plasma and liver microsomal incubations in the absence of nicotinamide adenine dinucleotide phosphate was suggestive of a covalent interaction between BETP and a protein amino acid residue(s) in these matrices. Incubations of BETP with glutathione (GSH) in buffer revealed a rapid nucleophilic displacement of the ethylsulfoxide functionality by GSH to yield adduct M1, which indicated that BETP was intrinsically electrophilic. The structure of M1 was unambiguously identified by comparison of its chromatographic and mass spectral properties with an authentic standard. The GSH conjugate of BETP was also characterized in NADPH- and GSH-supplemented liver microsomes, and in plasma samples from the pharmacokinetic studies. Unlike BETP, M1 was inactive as an allosteric modulator of the GLP-1R.
Drug metabolism and disposition: the biological fate of chemicals 05/2013; · 3.74 Impact Factor
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Amit S Kalgutkar,
Danny Chen,
Manthena V Varma,
Bo Feng,
Steven G Terra,
Renato J Scialis,
Charles J Rotter,
Kosea S Frederick,
Mark A West,
Theunis C Goosen,
James R Gosset,
Robert L Walsky,
Omar L Francone
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ABSTRACT: Abstract 1. 5-(N-(4-((4-ethylbenzyl)thio)phenyl)sulfamoyl)-2-methyl benzoic acid (CP-778 875), an agonist of the peroxisome proliferator-activated receptor alpha, has been evaluated in the clinic to treat dyslipidemia and type 2 diabetes mellitus. Herein, we investigate the effect of CP-778 875 on the pharmacokinetics of atorvastatin acid and its metabolites in humans. 2. The study incorporated a fixed-sequence design conducted in two groups. Group A was designed to estimate the effects of multiple doses of CP-778 875 on the single dose pharmacokinetics of atorvastatin. Subjects in group A (n = 26) received atorvastatin (40 mg) on days 1 and 9 and CP-778 875 (1.0 mg QD) on days 5-12. Group B was designed to examine the effects of multiple doses of atorvastatin on the single dose pharmacokinetics of CP-778 875. Subjects in group B (n = 29) received CP-778 875 (0.3 mg) on days 1 and 9 and atorvastatin (40 mg QD) on days 5-12. 3. Mean maximum serum concentration (Cmax) and area under the curve of atorvastatin were increased by 45% and 20%, respectively, upon co-administration with CP-778 875. Statistically significant increases in the systemic exposure of ortho- and para-hydroxyatorvastatin were also observed upon concomitant dosing with CP-778 875. CP-778 875 pharmacokinetics, however, were not impacted upon concomitant dosing with atorvastatin. 4. Inhibition of organic anion transporting polypeptide 1B1 by CP-778 875 (IC50 = 2.14 ±0.40 μM) could be the dominant cause of the pharmacokinetic interaction as CP-778 875 did not exhibit significant inhibition of cytochrome P450 3A4/3A5, multidrug resistant protein 1 or breast cancer resistant protein, which are also involved in the hepatobiliary disposition of atorvastatin.
Xenobiotica 04/2013; · 1.79 Impact Factor
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ABSTRACT: The current study examined the bioactivation potential of ghrelin receptor inverse agonists, 1-(2-(2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(imidazo[2,1-b]thiazol-6-yl)ethanone (1) and 1-(2-(2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)ethanone (2), containing a fused imidazo[2,1-b]thiazole motif in the core structure. Both compounds underwent oxidative metabolism in NADPH- and glutathione-supplemented human liver microsomes to yield glutathione conjugates, which was consistent with their bioactivation to reactive species. Mass spectral fragmentation and NMR analysis indicated that the site of attachment of the glutathionyl moiety in the thiol conjugates was on the thiazole ring within the bicycle. Two glutathione conjugates were discerned with the imidazo[2,1-b]thiazole derivative 1. One adduct was derived from the Michael addition of glutathione to a putative S-oxide metabolite of 1, whereas, the second adduct was formed via the reaction of a second glutathione molecule with the initial glutathione-S-oxide adduct. In the case of the 2-methylimidazo[2,1-b]thiazole analog 2, glutathione conjugation occurred via an oxidative desulfation mechanism, possibly involving thiazole ring epoxidation as the rate-limiting step. Additional insights into the mechanism were obtained via 18O exchange and trapping studies with potassium cyanide. The mechanistic insights into the bioactivation pathways of 1 and 2 allowed the deployment of a rational chemical intervention strategy that involved replacement of the thiazole ring with a 1,2,4-thiadiazole group to yield -(2-(2-chloro-4-(2H-1,2,3-triazol-2-yl)benzyl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1-b][1,3,4]thiadiazol-6-yl)ethanone (3). These structural changes not only abrogated the bioactivation liability but also retained the attractive pharmacological attributes of the prototype agents.
Drug metabolism and disposition: the biological fate of chemicals 04/2013; · 3.74 Impact Factor
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Etzer Darout,
Ralph Pelton Robinson,
Kim Francis McClure,
Matthew Corbett,
Bryan Li,
Andrei Shavnya,
Melissa P Andrews,
Christopher S Jones,
Qifang Li,
Martha L Minich, [......],
Paul D Bonin,
Peter Cornelius,
Ruduan Wang,
Victoria Bagdasarian,
Colleen P Sobota,
Sam Hornby,
Victoria M Masterson,
Reena M Joseph, Amit S Kalgutkar,
Yue Chen
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ABSTRACT: A series of GPR119 agonists based on a 2,6-diazatricyclo[3.3.1.1~3,7~]decane ring system is described. Also provided is a detailed account of the development of a multi-gram scale synthesis of the diazatricyclic ring system, which was achieved using a Hofmann-Löffler-Freytag reaction as the key step. The basis for the use of this complex framework lies in an attempt to constrain one end of the molecule in the "agonist conformation" as was previously described for 3-oxa-7-aza-bicyclo[3.3.1]nonanes. Optimization of carbamate analogs of the diazatricylic compounds led to the identification of 32i as a potent agonist of the GPR119 receptor with low unbound human liver microsomal clearance. The use of an agonist response weighted ligand lipophilic efficiency (LLE) termed AgLLE is discussed along with the issues of applying efficiency measures to agonist programs. Ultimately, solubility limited absorption and poor exposure reduced further interest in these molecules.
Journal of Medicinal Chemistry 12/2012; · 4.80 Impact Factor
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ABSTRACT: The disposition of ertugliflozin (PF-04971729), an orally active selective inhibitor of the sodium-dependent glucose cotransporter 2, was studied after a single 25-mg oral dose of [(14)C]-PF-04971729 to healthy human subjects. Mass balance was achieved with approximately 91% of the administered dose recovered in urine and feces. The total administered radioactivity excreted in feces and urine was 40.9% and 50.2%, respectively. The absorption of PF-04971729 in humans was rapid with a T(max) at ~ 1.0 h. Of the total radioactivity excreted in feces and urine, unchanged PF-04971729 collectively accounted for ~ 35.3% of the dose, suggestive of moderate metabolic elimination in humans. The principal biotransformation pathway involved glucuronidation of the glycoside hydroxyl groups to yield three regioisomeric metabolites M4a, M4b and M4c (~39.3% of the dose in urine) of which M4c was the major regioisomer (~31.7% of the dose). The structure of M4a and M4c were confirmed to be PF-04971729-4-O-β- and -3-O-β-glucuronide, respectively, via comparison of the HPLC retention time and mass spectra with authentic standards. A minor metabolic fate involved oxidation by cytochrome P450 to yield monohydroxylated metabolites M1 and M3 and des-ethyl PF-04971729 (M2), which accounted for ~5.2% of the dose in excreta. In plasma, unchanged PF-04971729 and the corresponding 4-O-β- (M4a) and 3-O-β- (M4c) glucuronides were the principal components, which accounted for 49.9, 12.2 and 24.1% of the circulating radioactivity. Overall, these data suggest that PF-04971729 is well absorbed in humans, and eliminated largely via glucuronidation.
Drug metabolism and disposition: the biological fate of chemicals 11/2012; · 3.74 Impact Factor
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Kentaro Futatsugi,
Vincent Mascitti,
Cristiano R W Guimarães,
Nao Morishita,
Cuiman Cai,
Michael P Deninno,
Hua Gao,
Michael D Hamilton,
Richard Hank,
Anthony R Harris, [......],
Michael G Lopaze,
Kim F McClure,
Michael J Munchhof,
Cathy Preville,
Ralph P Robinson,
Stephen W Wright,
Paul D Bonin,
Peter Cornelius,
Yue Chen, Amit S Kalgutkar
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ABSTRACT: A novel GPR119 agonist based on the 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole scaffold was designed through lead optimization starting from pyrazole-based GPR119 agonist 1. The design is centered on the conformational restriction of the core scaffold, while minimizing the change in spatial relationships of two key pharmacophoric elements (piperidine-carbamate and aryl sulfone).
Bioorganic & medicinal chemistry letters 11/2012; · 2.65 Impact Factor
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Samit K Bhattacharya,
Gary E Aspnes,
Scott W Bagley,
Markus Boehm,
Arthur D Brosius,
Leonard Buckbinder,
Jeanne S Chang,
Joseph Dibrino,
Heather Eng,
Kosea S Frederick, [......],
Carmen Garcia-Irizarry,
Jianke Li,
Blaise Lippa,
David A Price,
James A Southers,
Daniel P Walker,
Liuqing Wei,
Jun Xiao,
Michael P Zawistoski,
Xumiao Zhao
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ABSTRACT: Previous drug discovery efforts identified classical PYK2 kinase inhibitors such as 2 and 3 that possess selectivity for PYK2 over its intra-family isoform FAK. Efforts to identify more kinome-selective chemical matter that stabilize a DFG-out conformation of the enzyme are described herein. Two sub-series of PYK2 inhibitors, an indole carboxamide-urea and a pyrazole-urea have been identified and found to have different binding interactions with the hinge region of PYK2. These leads proved to be more selective than the original classical inhibitors.
Bioorganic & medicinal chemistry letters 10/2012; · 2.65 Impact Factor
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Arthur C Rand,
Siegfried S F Leung,
Heather Eng,
Charles J Rotter,
Raman Sharma, Amit S Kalgutkar,
Yizhong Zhang,
Manthena V Varma,
Kathleen A Farley,
Bhagyashree Khunte,
Chris Limberakis,
David A Price,
Spiros Liras,
Alan M Mathiowetz,
Matthew P Jacobson,
R Scott Lokey
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ABSTRACT: A series of cyclic peptides were designed and prepared to investigate the physicochemical properties that affect oral bioavailabilty of this chemotype in rats. In particular, the ionization state of the peptide was examined by the incorporation of naturally occurring amino acid residues that are charged in differing regions of the gut. In addition, data was generated in a variety of in vitro assays and the usefulness of this data in predicting the subsequent oral bioavailability observed in the rat is discussed.
Medicinal Chemistry Communication 10/2012; 3(10):1282-1289. · 2.80 Impact Factor
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ABSTRACT: Inhibition of intestinal and hepatic microsomal triglyceride transfer protein (MTP) is a potential strategy for the treatment of dyslipidemia and related metabolic disorders. Inhibition of hepatic MTP, however, results in elevated liver transaminases and increased hepatic fat deposition consistent with hepatic steatosis. Diethyl 2-((2-(3-(dimethylcarbamoyl)-4-(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-ylcarboxamido)phenyl)acetoxy)methyl)-2-phenylmalonate (JTT-130) is an intestine-specific inhibitor of MTP and does not cause increases in transaminases in short-term clinical trials in patients with dyslipidemia. Selective inhibition of intestinal MTP is achieved via rapid hydrolysis of its ester linkage by liver-specific carboxylesterase(s), resulting in the formation of an inactive carboxylic acid metabolite 1. In the course of discovery efforts around tissue-specific inhibitors of MTP, the mechanism of JTT-130 hydrolysis was examined in detail. Lack of (18)O incorporation in 1 following the incubation of JTT-130 in human liver microsomes in the presence of H(2)(18)O suggested that hydrolysis did not occur via a simple cleavage of the ester linkage. The characterization of atropic acid (2-phenylacrylic acid) as a metabolite was consistent with a hydrolytic pathway involving initial hydrolysis of one of the pendant malonate ethyl ester groups followed by decarboxylative fragmentation to 1 and the concomitant liberation of the potentially electrophilic acrylate species. Glutathione conjugates of atropic acid and its ethyl ester were also observed in microsomal incubations of JTT-130 that were supplemented with the thiol nucleophile. Additional support for the hydrolysis mechanism was obtained from analogous studies on diethyl 2-(2-(2-(3-(dimethylcarbamoyl)-4-(4'-trifluoromethyl)-[1,1'-biphenyl]-2-ylcarboxamido)phenyl)acetoxy)ethyl)-2-phenylmalonate (3), which cannot participate in hydrolysis via the fragmentation pathway because of the additional methylene group. Unlike the case with JTT-130, (18)O was readily incorporated into 1 during the enzymatic hydrolysis of 3, suggestive of a mechanism involving direct hydrolytic cleavage of the ester group in 3. Finally, 3-(ethylamino)-2-(ethylcarbamoyl)-3-oxo-2-phenylpropyl 2-(3-(dimethylcarbamoyl)-4-(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-ylcarboxamido)phenyl)acetate (4), which possessed an N,N-diethyl-2-phenylmalonamide substituent (in lieu of the diethyl-2-phenylmalonate motif in JTT-130) proved to be resistant to the hydrolytic cleavage/decarboxylative fragmentation pathway that yielded 1, a phenomenon that further confirmed our hypothesis. From a toxicological standpoint, it is noteworthy to point out that the liberation of the electrophilic acrylic acid species as a byproduct of JTT-130 hydrolysis is similar to the bioactivation mechanism established for felbamate, an anticonvulsant agent associated with idiosyncratic aplastic anemia and hepatotoxicity.
Chemical Research in Toxicology 09/2012; 25(10):2138-52. · 3.78 Impact Factor
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ABSTRACT: INTRODUCTION: The design of target-specific covalent inhibitors is conceptually attractive because of increased biochemical efficiency through covalency and increased duration of action that outlasts the pharmacokinetics of the agent. Although many covalent inhibitors have been approved or are in advanced clinical trials to treat indications such as cancer and hepatitis C, there is a general tendency to avoid them as drug candidates because of concerns regarding immune-mediated toxicity that can arise from indiscriminate reactivity with off-target proteins. AREAS COVERED: The review examines potential reason(s) for the excellent safety record of marketed covalent agents and advanced clinical candidates for emerging therapeutic targets. A significant emphasis is placed on proteomic techniques and chemical/biochemical reactivity assays that aim to provide a systematic rank ordering of pharmacologic selectivity relative to off-target protein reactivity of covalent inhibitors. EXPERT OPINION: While tactics to examine selective covalent modification of the pharmacologic target are broadly applicable in drug discovery, it is unclear whether the output from such studies can prospectively predict idiosyncratic immune-mediated drug toxicity. Opinions regarding an acceptable threshold of protein reactivity/body burden for a toxic electrophile and a non-toxic electrophilic covalent drug have not been defined. Increasing confidence in proteomic and chemical/biochemical reactivity screens will require a retrospective side-by-side profiling of marketed covalent drugs and electrophiles known to cause deleterious toxic effects via non-selective covalent binding.
Expert Opinion on Drug Discovery 05/2012; 7(7):561-81. · 2.12 Impact Factor
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ABSTRACT: The importance of mitigating drug-drug interaction (DDI) risks, which arise from inhibition of major human cytochrome P450 enzymes is a well-established component of the lead optimization process in drug discovery. More recently, there has been much interest in clinical DDIs potentially arising via time- and concentration-dependent cytochrome P450 inhibition, a phenomenon consistent with mechanism-based inactivation. Inactivated P450 is catalytically incompetent and must be replenished by newly synthesized protein. Consequently, time-dependent inhibition of P450s presents a greater safety concern compared to reversible inhibition because of the increased propensity for pharmacokinetic interactions upon multiple dosing and the sustained duration of these interactions after discontinuation of the mechanism-based inactivator. Mechanism-based or time-dependent P450 inhibitors pose an additional risk of idiosyncratic drug toxicity since the mechanism of time-dependency often involves the formation of reactive metabolites, which can react with proteins other than the P450 isozyme responsible for catalysis. in vitro time-dependent inhibition (TDI) of P450 enzymes is now routinely assessed as part of lead optimization efforts in preclinical drug discovery. However, identification of an in vitro TDI liability can raise several questions such as: What is the mechanism of TDI? Does it involve the formation of reactive metabolites? Is there a 1:1 correlation between P450 TDI and RM formation (as measured from reactive metabolite trapping studies)? What is the likelihood that a P450 time-dependent inhibitor will also cause toxicity? What are the DDI risk mitigation options when dealing with P450 inactivators in drug discovery - compound progression or termination? Several drugs exhibit in vitro TDI of P450 enzymes, but only a fraction thereof causes clinical DDIs. Hence, when do we initiate labor-intensive medicinal chemistry efforts to design compounds devoid of P450 TDI liability? What are the best methods to precisely predict the likelihood of occurrence of clinical DDIs with drug candidates that inactivate P450 enzymes? What are (if any) the qualifying considerations for clinical progression of a P450 time-dependent inactivator with projected clinical DDI risks? In an effort to address these questions and hopefully provide answers to some of them, we embarked on the present venture wherein we highlight the current state-of-the-art knowledge in this field with a special emphasis on (a) available biochemical and mechanistic approaches in drug discovery to examine TDI of P450 isozymes with new chemical entities, (b) structure-activity relationship studies with marketed drugs associated with DDIs via P450 inactivation, (c) case studies of medicinal chemistry tactics to abrogate P450 inactivation liability, (d) strategies for progression of P450 TDI-positive drug candidates, and (e) the utility of in silico methodology, including the use of physiologically-based pharmacokinetic simulators, in drug discovery to predict the magnitude of clinical DDIs risks anticipated with new clinical candidates.
Journal of Medicinal Chemistry 03/2012; 55(11):4896-933. · 4.80 Impact Factor
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ABSTRACT: The United States Public Health Service Administration is alerting medical professionals that a substantial percentage of cocaine imported into the United States is adulterated with levamisole, a veterinary pharmaceutical that can cause blood cell disorders such as severe neutropenia and agranulocytosis. Levamisole was previously approved in combination with fluorouracil for the treatment of colon cancer; however, the drug was withdrawn from the U.S. market in 2000 because of the frequent occurrence of agranulocytosis. The detection of autoantibodies such as antithrombin (lupus anticoagulant) and an increased risk of agranulocytosis in patients carrying the human leukocyte antigen B27 genotype suggest that toxicity is immune-mediated. In this perspective, we provide an historical account of the levamisole/cocaine story as it first surfaced in 2008, including a succinct review of levamisole pharmacology, pharmacokinetics, and preclinical/clinical evidence for levamisole-induced agranulocytosis. Based on the available information on levamisole metabolism in humans, we propose that reactive metabolite formation is the rate-limiting step in the etiology of agranulocytosis associated with levamisole, in a manner similar to other drugs (e.g., propylthiouracil, methimazole, captopril, etc.) associated with blood dyscrasias. Finally, considering the toxicity associated with levamisole, we propose that the 2,3,5,6-tetrahydroimidazo[2,1-b]thiazole scaffold found in levamisole be categorized as a new structural alert, which is to be avoided in drug design.
Drug metabolism and disposition: the biological fate of chemicals 03/2012; 40(6):1067-75. · 3.74 Impact Factor
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Tina R White,
Chad M Renzelman,
Arthur C Rand,
Taha Rezai,
Cayla M McEwen,
Vladimir M Gelev,
Rushia A Turner,
Roger G Linington,
Siegfried S F Leung, Amit S Kalgutkar,
Jonathan N Bauman,
Yizhong Zhang,
Spiros Liras,
David A Price,
Alan M Mathiowetz,
Matthew P Jacobson,
R Scott Lokey
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ABSTRACT: Backbone N-methylation is common among peptide natural products and has a substantial impact on both the physical properties and the conformational states of cyclic peptides. However, the specific impact of N-methylation on passive membrane diffusion in cyclic peptides has not been investigated systematically. Here we report a method for the selective, on-resin N-methylation of cyclic peptides to generate compounds with drug-like membrane permeability and oral bioavailability. The selectivity and degree of N-methylation of the cyclic peptide was dependent on backbone stereochemistry, suggesting that conformation dictates the regiochemistry of the N-methylation reaction. The permeabilities of the N-methyl variants were corroborated by computational studies on a 1,024-member virtual library of N-methyl cyclic peptides. One of the most permeable compounds, a cyclic hexapeptide (molecular mass = 755 Da) with three N-methyl groups, showed an oral bioavailability of 28% in rat.
Nature Chemical Biology 09/2011; 7(11):810-7. · 14.69 Impact Factor
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ABSTRACT: It is generally believed that metabolic bioactivation of drug molecules to form reactive metabolites, followed by their covalent binding to endogenous macromolecules, is one of the mechanisms that can lead to hepatotoxicity or idiosyncratic adverse drug reactions (IADRs). Although the role of bioactivation in drug-induced liver injury has been reasonably well established and accepted, and methodologies (e.g., structural alerts, reactive metabolite trapping, and covalent binding) continue to emerge in an attempt to detect the occurrence of bioactivation, the challenge remains to accurately predict the likelihood for idiosyncratic liver toxicity. Recent advances in risk-assessment methodologies, such as by the estimate of total body burden of covalent binding or by zone classification, taking the clinical dose into consideration, are positive steps toward improving risk assessment. The ability to better predict the potential of a drug candidate to cause IADRs will further be dependent upon a better understanding of the pathophysiological mechanisms of such reactions. Until a thorough understanding of the relationship between liver toxicity and the formation of reactive metabolites is achieved, it appears, at present, that the most practical strategy in drug discovery and development to reduce the likelihood of idiosyncratic liver toxicity via metabolic activation is to minimize or eliminate the occurrence of bioactivation and, at the same time, to maximize the pharmacological potency (to minimze the clinical dose) of the drug of interest.
Drug Metabolism Reviews 09/2011; 44(1):18-33. · 6.40 Impact Factor
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ABSTRACT: As part of efforts directed at the G protein-coupled receptor 119 agonist program for type 2 diabetes, a series of cyanopyridine derivatives exemplified by isopropyl-4-(3-cyano-5-(quinoxalin-6-yl)pyridine-2-yl)piperazine-1-carboxylate (1) were identified as novel chemotypes worthy of further hit-to-lead optimization. Compound 1, however, was found to be unstable in plasma (37 °C, pH 7.4) from rat (T(1/2) = 16 min), mouse (T(1/2) = 61 min), and guinea pig (T(1/2) = 4 min). Lowering the temperature of plasma incubations (4-25 °C) attenuated the degradation of 1, implicating the involvement of an enzyme-mediated process. Failure to detect any appreciable amount of 1 in plasma samples from protein binding and pharmacokinetic studies in rats was consistent with its labile nature in plasma. Instability noted in rodent plasma was not observed in plasma from dogs, monkeys, and humans (T(1/2) > 370 min at 37 °C, pH 7.4). Metabolite identification studies in rodent plasma revealed the formation of a single metabolite (M1), which was 16 Da higher than the molecular weight of 1 (compound 1, MH(+) = 403; M1, MH(+) = 419). Pretreatment of rat plasma with allopurinol, but not raloxifene, abolished the conversion of 1 to M1, suggesting that xanthine oxidase (XO) was responsible for the oxidative instability. Consistent with the known catalytic mechanism of XO, the source of oxygen incorporated in M1 was derived from water rather than molecular oxygen. The formation of M1 was also demonstrated in incubations of 1 with purified bovine XO. The structure of M1 was determined by NMR analysis to be isopropyl-4-(3-cyano-5-(3-oxo-3,4-dihydroquinoxalin-6-yl)pyridine-2-yl)piperazine-1-carboxylate. The regiochemistry of quinoxaline ring oxidation in 1 was consistent with ab initio calculations and molecular docking studies using a published crystal structure of bovine XO. A close-in analogue of 1, which lacked the quinoxaline motif (e.g., 5-(4-cyano-3-methylphenyl)-2-(4-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)nicotinitrile (2)) was stable in rat plasma and possessed substantially improved GPR119 agonist properties. To the best of our knowledge, our studies constitute the first report on the involvement of rodent XO in oxidative drug metabolism in plasma.
Chemical Research in Toxicology 09/2011; 24(12):2207-16. · 3.78 Impact Factor
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Amit S Kalgutkar
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ABSTRACT: Because of the inability to predict and quantify the risk of idiosyncratic adverse drug reactions (IADRs) and because reactive metabolites (RMs) as opposed to the parent molecules from which they are derived are thought to be responsible for the pathogenesis of some IADRs, procedures (RM trapping/covalent binding) are being incorporated into the discovery screening funnel early-on to assess the risk of RM formation. Utility of the methodology in structure-toxicity relationships and scope in abrogating RM formation at the lead optimization stage are discussed in this article. Interpretation of the output from RM assessment assays, however, is confounded by the fact that many successfully marketed drugs are false positives. Therefore, caution must be exercised in deprioritizing a compound based on a positive result, so that the development of a useful and potentially profitable compound won't be unnecessarily halted. Risk mitigation strategies (e.g., competing detoxication pathways, low daily dose, etc.) when selecting RM positives for clinical development are also reviewed.
Chemico-biological interactions 06/2011; 192(1-2):46-55. · 2.46 Impact Factor
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ABSTRACT: Because of a preconceived notion that eliminating reactive metabolite (RM) formation with new drug candidates could mitigate the risk of idiosyncratic drug toxicity, the potential for RM formation is routinely examined as part of lead optimization efforts in drug discovery. Likewise, avoidance of "structural alerts" is almost a norm in drug design. However, there is a growing concern that the perceived safety hazards associated with structural alerts and/or RM screening tools as standalone predictors of toxicity risks may be over exaggerated. In addition, the multifactorial nature of idiosyncratic toxicity is now well recognized based upon observations that mechanisms other than RM formation (e.g., mitochondrial toxicity and inhibition of bile salt export pump (BSEP)) also can account for certain target organ toxicities. Hence, fundamental questions arise such as: When is a molecule that contains a structural alert (RM positive or negative) a cause for concern? Could the molecule in its parent form exert toxicity? Can a low dose drug candidate truly mitigate metabolism-dependent and -independent idiosyncratic toxicity risks? In an effort to address these questions, we have retrospectively examined 68 drugs (recalled or associated with a black box warning due to idiosyncratic toxicity) and the top 200 drugs (prescription and sales) in the United States in 2009 for trends in physiochemical characteristics, daily doses, presence of structural alerts, evidence for RM formation as well as toxicity mechanism(s) potentially mediated by parent drugs. Collectively, our analysis revealed that a significant proportion (∼78-86%) of drugs associated with toxicity contained structural alerts and evidence indicating that RM formation as a causative factor for toxicity has been presented in 62-69% of these molecules. In several cases, mitochondrial toxicity and BSEP inhibition mediated by parent drugs were also noted as potential causative factors. Most drugs were administered at daily doses exceeding several hundred milligrams. There was no obvious link between idiosyncratic toxicity and physicochemical properties such as molecular weight, lipophilicity, etc. Approximately half of the top 200 drugs for 2009 (prescription and sales) also contained one or more alerts in their chemical architecture, and many were found to be RM-positive. Several instances of BSEP and mitochondrial liabilities were also noted with agents in the top 200 category. However, with relatively few exceptions, the vast majority of these drugs are rarely associated with idiosyncratic toxicity, despite years of patient use. The major differentiating factor appeared to be the daily dose; most of the drugs in the top 200 list are administered at low daily doses. In addition, competing detoxication pathways and/or alternate nonmetabolic clearance routes provided suitable justifications for the safety records of RM-positive drugs in the top 200 category. Thus, while RM elimination may be a useful and pragmatic starting point in mitigating idiosyncratic toxicity risks, our analysis suggests a need for a more integrated screening paradigm for chemical hazard identification in drug discovery. Thus, in addition to a detailed assessment of RM formation potential (in relationship to the overall elimination mechanisms of the compound(s)) for lead compounds, effects on cellular health (e.g., cytotoxicity assays), BSEP inhibition, and mitochondrial toxicity are the recommended suite of assays to characterize compound liabilities. However, the prospective use of such data in compound selection will require further validation of the cellular assays using marketed agents. Until we gain a better understanding of the pathophysiological mechanisms associated with idiosyncratic toxicities, improving pharmacokinetics and intrinsic potency as means of decreasing the dose size and the associated "body burden" of the parent drug and its metabolites will remain an overarching goal in drug discovery.
Chemical Research in Toxicology 06/2011; 24(9):1345-410. · 3.78 Impact Factor
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Amit S Kalgutkar,
Meera Tugnait,
Tong Zhu,
Emi Kimoto,
Zhuang Miao,
Vincent Mascitti,
Xin Yang,
Beijing Tan,
Robert L Walsky,
Jonathan Chupka,
Bo Feng,
Ralph P Robinson
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ABSTRACT: (1S,2S,3S,4R,5S)-5-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-1-hydroxymethyl-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol (PF-04971729), a potent and selective inhibitor of the sodium-dependent glucose cotransporter 2, is currently in phase 2 trials for the treatment of diabetes mellitus. This article describes the preclinical species and in vitro human disposition characteristics of PF-04971729 that were used in experiments performed to support the first-in-human study. Plasma clearance was low in rats (4.04 ml · min(-1) · kg(-1)) and dogs (1.64 ml · min(-1) · kg(-1)), resulting in half-lives of 4.10 and 7.63 h, respectively. Moderate to good bioavailability in rats (69%) and dogs (94%) was observed after oral dosing. The in vitro biotransformation profile of PF-04971729 in liver microsomes and cryopreserved hepatocytes from rat, dog, and human was qualitatively similar; prominent metabolic pathways included monohydroxylation, O-deethylation, and glucuronidation. No human-specific metabolites of PF-04971729 were detected in in vitro studies. Reaction phenotyping studies using recombinant enzymes indicated a role of CYP3A4/3A5, CYP2D6, and UGT1A9/2B7 in the metabolism of PF-04971729. No competitive or time-dependent inhibition of the major human cytochrome P450 enzymes was discerned with PF-04971729. Inhibitory effects against the organic cation transporter 2-mediated uptake of [(14)C]metformin by PF-04971729 also were very weak (IC(50) = ∼900 μM). Single-species allometric scaling of rat pharmacokinetics of PF-04971729 was used to predict human clearance, distribution volume, and oral bioavailability. Human pharmacokinetic predictions were consistent with the potential for a low daily dose. First-in-human studies after oral administration indicated that the human pharmacokinetics/dose predictions for PF-04971729 were in the range that is likely to yield a favorable pharmacodynamic response.
Drug metabolism and disposition: the biological fate of chemicals 06/2011; 39(9):1609-19. · 3.74 Impact Factor
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Allen J Duplantier,
Mark A Dombroski,
Chakrapani Subramanyam,
Aimee M Beaulieu,
Shang-Poa Chang,
Christopher A Gabel,
Crystal Jordan, Amit S Kalgutkar,
Kenneth G Kraus,
Jeff M Labasi,
Christopher Mussari,
David G Perregaux,
Rick Shepard,
Timothy J Taylor,
Kristen A Trevena,
Carrie Whitney-Pickett,
Kwansik Yoon
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ABSTRACT: High throughput screening (HTS) of our compound file provided an attractive lead compound with modest P2X(7) receptor antagonist potency and high selectivity against a panel of receptors and channels, but also with high human plasma protein binding and a predicted short half-life in humans. Multi-parameter optimization was used to address the potency, physicochemical and pharmacokinetic properties which led to potent P2X(7)R antagonists with good disposition properties. Compound 33 (CE-224,535) was advanced to clinical studies for the treatment of rheumatoid arthritis.
Bioorganic & medicinal chemistry letters 06/2011; 21(12):3708-11. · 2.65 Impact Factor
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Vincent Mascitti,
Benjamin D Stevens,
Chulho Choi,
Kim F McClure,
Cristiano R W Guimarães,
Kathleen A Farley,
Michael J Munchhof,
Ralph P Robinson,
Kentaro Futatsugi,
Sophie Y Lavergne,
Bruce A Lefker,
Peter Cornelius,
Paul D Bonin, Amit S Kalgutkar,
Raman Sharma,
Yue Chen
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ABSTRACT: The design and synthesis of a GPR119 agonist bearing a 2-(2,3,6-trifluorophenyl)acetamide group is described. The design capitalized on the conformational restriction found in N-β-fluoroethylamide derivatives to help maintain good levels of potency while driving down both lipophilicity and oxidative metabolism in human liver microsomes. The chemical stability and bioactivation potential are discussed.
Bioorganic & medicinal chemistry letters 03/2011; 21(5):1306-9. · 2.65 Impact Factor
