Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes.

N1-Substituted-6-arylthiouracils, represented by compound 1 [6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one], are a novel class of selective irreversible inhibitors of human myeloperoxidase. The present account is a summary of our in vitro studies on the facile oxidative desulfurization in compound 1 to a cyclic ether metabolite M1 [5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one] in NADPH-supplemented rats (t1/2 [half-life = mean ± S.D.] = 8.6 ± 0.4 minutes) and dog liver microsomes (t1/2 = 11.2 ± 0.4 minutes), but not in human liver microsomes (t1/2 > 120 minutes). The in vitro metabolic instability also manifested in moderate-to-high plasma clearances of the parent compound in rats and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of liver microsomes or coincubation with the flavin-containing monooxygenase (FMO) inhibitor imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of compound 1 to M1 was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally catalyzed the conversion of compound 1 to M1. FMO1 is not expressed in adult human liver, which rationalizes the species difference in oxidative desulfurization. Oxidation by FMO1 followed Michaelis-Menten kinetics with Michaelis-Menten constant, maximum rate of oxidative desulfurization, and intrinsic clearance values of 209 µM, 20.4 nmol/min/mg protein, and 82.7 µl/min/mg protein, respectively. Addition of excess glutathione essentially eliminated the conversion of compound 1 to M1 in NADPH-supplemented rat and dog liver microsomes, which suggests that the initial FMO1-mediated S-oxygenation of compound 1 yields a sulfenic acid intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion or undergoing further oxidation to a more electrophilic sulfinic acid species that is trapped intramolecularly by the pendant alcohol motif in compound 1.

Pharmacokinetics and Disposition of the Thiouracil Derivative PF-06282999, an Orally Bioavailable, Irreversible Inactivator of Myeloperoxidase Enzyme, Across Animals and Humans.

The thiouracil derivative PF-06282999 [2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide] is an irreversible inactivator of myeloperoxidase and is currently in clinical trials for the potential treatment of cardiovascular diseases. Concerns over idiosyncratic toxicity arising from bioactivation of the thiouracil motif to reactive species in the liver have been largely mitigated through the physicochemical (molecular weight, lipophilicity, and topological polar surface area) characteristics of PF-06282999, which generally favor elimination via nonmetabolic routes. To test this hypothesis, pharmacokinetics and disposition studies were initiated with PF-06282999 using animals and in vitro assays, with the ultimate goal of predicting human pharmacokinetics and elimination mechanisms. Consistent with its physicochemical properties, PF-06282999 was resistant to metabolic turnover from liver microsomes and hepatocytes from animals and humans and was devoid of cytochrome P450 inhibition. In vitro transport studies suggested moderate intestinal permeability and minimal transporter-mediated hepatobiliary disposition. PF-06282999 demonstrated moderate plasma protein binding across all of the species. Pharmacokinetics in preclinical species characterized by low to moderate plasma clearances, good oral bioavailability at 3- to 5-mg/kg doses, and renal clearance as the projected major clearance mechanism in humans. Human pharmacokinetic predictions using single-species scaling of dog and/or monkey pharmacokinetics were consistent with the parameters observed in the first-in-human study, conducted in healthy volunteers at a dose range of 20-200 mg PF-06282999. In summary, disposition characteristics of PF-06282999 were relatively similar across preclinical species and humans, with renal excretion of the unchanged parent emerging as the principal clearance mechanism in humans, which was anticipated based on its physicochemical properties and supported by preclinical studies.

Toxicities associated with 1-month treatment with propylthiouracil (PTU) and methimazole (MMI) in male rats.

Thionamides such as propylthiouracil (PTU) and methimazole (MMI) have been used for more than 50 years to treat the more common causes of thyrotoxicosis/hyperthyroidism such as Graves' disease. Serious adverse effects associated with thionamides in humans include idiosyncratic liver damage, agranulocytosis, aplastic anemia, and vasculitis. Both prospective and retrospective clinical studies with these drugs have failed to identify predictive biomarker for these adverse effects. To assess whether rat is a good model for predicting drug-related adverse events in the liver and in the bone marrow, we conducted a comprehensive study in male rats with multiple doses of PTU and MMI. As expected, euthyroid animals became hypothyroid along with several secondary changes associated with hypothyroidism. There were slight reductions in red blood cell parameters along with some marginal effects on the bone marrow elements. However, there was no evidence of significant neutropenia and liver injury in both PTU-treated and MMI-treated cohorts. MMI-related effects were noted in the seminiferous tubules of the testes. Overall, 1-month daily treatment of euthyroid rats with PTU or MMI resulted in hypothyroidism, minor bone marrow effects, and several secondary effects associated with hypothyroidism, but without any evidence of adverse effects reported in humans including liver injury and agranulocytosis.

A "middle-out" approach to human pharmacokinetic predictions for OATP substrates using physiologically-based pharmacokinetic modeling.

Physiologically based pharmacokinetic (PBPK) models provide a framework useful for generating credible human pharmacokinetic predictions from data available at the earliest, preclinical stages of pharmaceutical research. With this approach, the pharmacokinetic implications of in vitro data are contextualized via scaling according to independent physiological information. However, in many cases these models also require model-based estimation of additional empirical scaling factors (SFs) in order to accurately recapitulate known human pharmacokinetic behavior. While this practice clearly improves data characterization, the introduction of empirically derived SFs may belie the extrapolative power commonly attributed to PBPK. This is particularly true when such SFs are compound dependent and/or when there are issues with regard to identifiability. As such, when empirically-derived SFs are necessary, a critical evaluation of parameter estimation and model structure are prudent. In this study, we applied a global optimization method to support model-based estimation of a single set of empirical SFs from intravenous clinical data on seven OATP substrates within the context of a previously published PBPK model as well as a revised PBPK model. The revised model with experimentally measured unbound fraction in liver, permeability between liver compartments, and permeability limited distribution to selected tissues improved data characterization. We utilized large-sample approximation and resampling approaches to estimate confidence intervals for the revised model in support of forward predictions that reflect the derived uncertainty. This work illustrates an objective approach to estimating empirically-derived SFs, systematically refining PBPK model performance and conveying the associated confidence in subsequent forward predictions.

Immune-mediated agranulocytosis caused by the cocaine adulterant levamisole: a case for reactive metabolite(s) involvement.

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.

A novel relay method for determining low-clearance values.

A novel relay method has been developed using cryopreserved human hepatocytes to measure intrinsic clearance of low-clearance compounds. The relay method involved transferring the supernatant from hepatocyte incubations to freshly thawed hepatocytes at the end of the 4-h incubation to prolong the exposure time to active enzymes in hepatocytes. An accumulative incubation time of 20 h or longer in hepatoctyes can be achieved using the method. The relay method was validated using seven commercial drugs (diazepam, disopyramide, theophylline, timolol, tolbutamide, S-warfarin, and zolmitriptan) that were metabolized by various cytochrome P450s with low human in vivo intrinsic clearance at approximately 2 to 15 ml · min⁻¹ · kg⁻¹. The results showed that the relay method produced excellent predictions of human in vivo clearance. The difference between in vitro and in vivo intrinsic clearance was within 2-fold for most compounds, which is similar to the standard prediction accuracy for moderate to high clearance compounds using hepatocytes. The relay method is a straightforward, relatively low cost, and easy-to-use new tool to address the challenges of low clearance in drug discovery and development.

Oxidative metabolism of a quinoxaline derivative by xanthine oxidase in rodent plasma.

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.

Discovery of microsomal triglyceride transfer protein (MTP) inhibitors with potential for decreased active metabolite load compared to dirlotapide.

Analogues related to dirlotapide (1), a gut-selective inhibitor of microsomal triglyceride transfer protein (MTP) were prepared with the goal of further reducing the potential for unwanted liver MTP inhibition and associated side-effects. Compounds were designed to decrease active metabolite load: reducing MTP activity of likely human metabolites and increasing metabolite clearance to reduce exposure. Introduction of 4'-alkyl and 4'-alkoxy substituents afforded compounds exhibiting improved therapeutic index in rats with respect to liver triglyceride accumulation and enzyme elevation. Likely human metabolites of select compounds were prepared and characterized for their potential to inhibit MTP in vivo. Based on preclinical efficacy and safety data and its potential for producing short-lived, weakly active metabolites, compound 13 (PF-02575799) advanced into phase 1 clinical studies.

Deuterium isotope effects in drug pharmacokinetics II: Substrate-dependence of the reaction mechanism influences outcome for cytochrome P450 cleared drugs.

Two chemotypes were examined in vitro with CYPs 3A4 and 2C19 by molecular docking, metabolic profiles, and intrinsic clearance deuterium isotope effects with specifically deuterated form to assess the potential for enhancement of pharmacokinetic parameters. The results show the complexity of deuteration as an approach for pharmacokinetic enhancement when CYP enzymes are involved in metabolic clearance. With CYP3A4 the rate limiting step was chemotype-dependent. With one chemotype no intrinsic clearance deuterium isotope effect was observed with any deuterated form, whereas with the other chemotype the rate limiting step was isotopically sensitive, and the magnitude of the intrinsic clearance isotope effect was dependent on the position(s) and extent of deuteration. Molecular docking and metabolic profiles aided in identifying sites for deuteration and predicted the possibility for metabolic switching. However, the potential for an isotope effect on the intrinsic clearance cannot be predicted and must be established by examining select deuterated versions of the chemotypes. The results show how in a deuteration strategy molecular docking, in-vitro metabolic profiles, and intrinsic clearance assessments with select deuterated versions of new chemical entities can be applied to determine the potential for pharmacokinetic enhancement in a discovery setting. They also help explain the substantial failures reported in the literature of deuterated versions of drugs to elicit a systemic enhancement on pharmacokinetic parameters.

Acyl Glucuronide Metabolites of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1 H-indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphate-Activated Protein Kinase (AMPK).

Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1ß1γ1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of ß1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK α1ß1γ1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.

Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy.

Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

Discovery and characterization of novel inhibitors of the sodium-coupled citrate transporter (NaCT or SLC13A5).

Citrate is a key regulatory metabolic intermediate as it facilitates the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake, by blocking the sodium-coupled citrate transporter (NaCT or SLC13A5), has been suggested as a potential therapeutic approach to treat metabolic disorders. NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions. The studies herein report the identification and characterization of a novel small dicarboxylate molecule (compound 2) capable of selectively and potently inhibiting citrate transport through NaCT, both in vitro and in vivo. Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT. The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints.

Discovery of 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999): A Highly Selective Mechanism-Based Myeloperoxidase Inhibitor for the Treatment of Cardiovascular Diseases.

Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Clinical evidence suggests a causal role for MPO in various autoimmune and inflammatory disorders including vasculitis and cardiovascular and Parkinson's diseases, implying that MPO inhibitors may represent a therapeutic treatment option. Herein, we present the design, synthesis, and preclinical evaluation of N1-substituted-6-arylthiouracils as potent and selective inhibitors of MPO. Inhibition proceeded in a time-dependent manner by a covalent, irreversible mechanism, which was dependent upon MPO catalysis, consistent with mechanism-based inactivation. N1-Substituted-6-arylthiouracils exhibited low partition ratios and high selectivity for MPO over thyroid peroxidase and cytochrome P450 isoforms. N1-Substituted-6-arylthiouracils also demonstrated inhibition of MPO activity in lipopolysaccharide-stimulated human whole blood. Robust inhibition of plasma MPO activity was demonstrated with the lead compound 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999, 8) upon oral administration to lipopolysaccharide-treated cynomolgus monkeys. On the basis of its pharmacological and pharmacokinetic profile, PF-06282999 has been advanced to first-in-human pharmacokinetic and safety studies.

Identification of Tetrahydropyrido[4,3-d]pyrimidine Amides as a New Class of Orally Bioavailable TGR5 Agonists.

Takeda G-protein-coupled receptor 5 (TGR5) represents an exciting biological target for the potential treatment of diabetes and metabolic syndrome. A new class of high-throughput screening (HTS)-derived tetrahydropyrido[4,3-d]pyrimidine amide TGR5 agonists is disclosed. We describe our effort to identify an orally available agonist suitable for assessment of systemic TGR5 agonism. This effort resulted in identification of 16, which had acceptable potency and pharmacokinetic properties to allow for in vivo assessment in dog. A key aspect of this work was the calibration of human and dog in vitro assay systems that could be linked with data from a human ex vivo peripheral blood monocyte assay that expresses receptor at endogenous levels. Potency from the human in vitro assay was also found to correlate with data from an ex vivo human whole blood assay. This calibration exercise provided confidence that 16 could be used to drive plasma exposures sufficient to test the effects of systemic activation of TGR5.