Angela Wolford

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.

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.

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.

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.

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.

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.

Optimization of triazole-based TGR5 agonists towards orally available agents

Reported herein is a medicinal chemistry effort towards the identification of orally available TGR5 agonist 12, which served as a dog tool compound for studies to increase confidence in this mechanism. With the challenge of striking the balance of TGR5 potency and desired clearance profile, the screening strategy as well as medicinal chemistry strategy are discussed in this article.

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 Parkinsons 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.

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.