Heather Eng

Optimizing PK properties of cyclic peptides: the effect of side chain substitutions on permeability and clearance

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.

Utility of the carboxylesterase inhibitor bis-para-nitrophenylphosphate (BNPP) in the plasma unbound fraction determination for a hydrolytically unstable amide derivative and agonist of the TGR5 receptor

The potent, functional agonist of the bile acid Takeda G-protein-coupled receptor 5 (TGR5), (S)-1-(6-fluoro-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-(isoquinolin-5-yloxy)ethanone (3), represents a useful tool to probe in vivo TGR5 pharmacology. Rapid degradation of 3 in both rat and mouse plasma, however, hindered the conduct of in vivo pharmacokinetic/pharmacodynamic investigations (including plasma-free fraction (fu plasma) determination) in rodent models of pharmacology. Studies were therefore initiated to understand the biochemical basis for plasma instability so that appropriate methodology could be implemented in in vivo pharmacology studies to prevent the breakdown of 3. Compound 3 underwent amide bond cleavage in both rat and mouse plasma with half-lives (T1/2) of 39 ± 7 and 9.9 ± 0.1 min. bis(p-nitrophenyl) phosphate (BNPP), a specific inhibitor of carboxylesterases, was found to inhibit hydrolytic cleavage in a time- and concentration-dependent manner, which suggested the involvement of carboxylesterases in the metabolism of 3. In contrast with the findings in rodents, 3 was resistant to hydrolytic cleavage in both dog and human plasma. The instability of 3 was also observed in rat and mouse liver microsomes. β-Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-dependent metabolism of 3 occurred more rapidly (T1/2 approximately 2.22–6.4 min) compared with the metabolic component observed in the absence of the co-factor (T1/2 approximately 89–130 min). Oxidative metabolism dominated the NADPH-dependent decline of 3, whereas NADPH-independent metabolism of 3 proceeded via simple amide bond hydrolysis. Compound 3 was highly bound (approximately 95%) to both dog and human plasmas. Rat and mouse plasma, pre-treated with BNPP to inhibit carboxylesterases activity, were used to determine the fu plasma of 3. A BNPP concentration of 500 μM was determined to be optimal for these studies. Higher BNPP concentrations (1000 μM) appeared to displace 3 from its plasma protein-binding sites in preclinical species and human. Under the conditions of carboxylesterases-inhibited rat and mouse plasma, the level of protein binding displayed by 3 was similar to those observed in dog and human. In conclusion, a novel system has been devised to measure fu plasma for a plasma-labile compound. The BNPP methodology can be potentially applied to stabilize hydrolytic cleavage of structurally diverse carboxylesterase substrates in the plasma (and other tissue), thereby allowing the characterization of pharmacology studies on plasma-labile compounds if and when they emerge as hits in exploratory drug-discovery programmes.

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.

Peptide to Peptoid Substitutions Increase Cell Permeability in Cyclic Hexapeptides

The effect of peptide-to-peptoid substitutions on the passive membrane permeability of an N-methylated cyclic hexapeptide is examined. In general, substitutions maintained permeability but increased conformational heterogeneity. Diversification with nonproteinogenic side chains increased permeability up to 3-fold. Additionally, the conformational impact of peptoid substitutions within a β-turn are explored. Based on these results, the strategic incorporation of peptoid residues into cyclic peptides can maintain or improve cell permeability, while increasing access to diverse side-chain functionality.

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 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.

In Vitro Metabolism of the Glucagon-Like Peptide-1 (GLP-1)-Derived Metabolites GLP-1(9-36)amide and GLP-1(28-36)amide in Mouse and Human Hepatocytes

Previous studies have revealed that the glucoincretin hormone glucagon-like peptide-1 (GLP-1)(7-36)amide is metabolized by dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase 24.11 (NEP) to yield GLP-1(9-36)amide and GLP-1(28-36)amide, respectively, as the principal metabolites. Contrary to the previous notion that GLP-1(7-36)amide metabolites are pharmacologically inactive, recent studies have demonstrated cardioprotective and insulinomimetic effects with both GLP-1(9-36)amide and GLP-1(28-36)amide in animals and humans. In the present work, we examined the metabolic stability of the two GLP-1(7-36)amide metabolites in cryopreserved hepatocytes, which have been used to demonstrate the in vitro insulin-like effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis. To examine the metabolic stability of the GLP-1(7-36)amide metabolites, a liquid chromatography–tandem mass spectrometry assay was developed for the quantitation of the intact peptides in hepatocyte incubations. GLP-1(9-36)amide and GLP-1(28-36)amide were rapidly metabolized in mouse [GLP-1(9-36)amide: t1/2 = 52 minutes; GLP-1(28-36)amide: t1/2 = 13 minutes] and human hepatocytes [GLP-1(9-36)amide: t1/2 = 180 minutes; GLP-1(28-36)amide: t1/2 = 24 minutes), yielding a variety of N-terminal cleavage products that were characterized using mass spectrometry. Metabolism at the C terminus was not observed for either peptides. The DPP-IV and NEP inhibitors diprotin A and phosphoramidon, respectively, did not induce resistance in the two peptides toward proteolytic cleavage. Overall, our in vitro findings raise the intriguing possibility that the insulinomimetic effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis and oxidative stress might be due, at least in part, to the actions of additional downstream metabolites, which are obtained from the enzymatic cleavage of the peptide backbone in the parent compounds.

Demonstration of the Innate Electrophilicity of 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP), a Small-Molecule Positive Allosteric Modulator of the Glucagon-Like Peptide-1 Receptor

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 i.v. (but not oral) administration. To explore the quantitative pharmacology associated with GLP-1R agonism in preclinical species, the in vivo pharmacokinetics of BETP were examined in rats after i.v. 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 i.v. administration (1 mg/kg) were minimal (and sporadic). In vitro incubations in bovine serum albumin, plasma, and liver microsomes from rodents and humans indicated a facile degradation of BETP. Failure to detect metabolites in plasma and liver microsomal incubations in the absence of NADP 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.

Statistical Methods for Analysis of Time-Dependent Inhibition of Cytochrome P450 Enzymes

Time-dependent inhibition (TDI) of cytochrome P450 (P450) enzymes, especially CYP3A4, is an important attribute of drugs in evaluating the potential for pharmacokinetic drug-drug interactions. The analysis of TDI data for P450 enzymes can be challenging, yet it is important to be able to reliably evaluate whether a drug is a TDI or not, and if so, how best to derive the inactivation kinetic parameters KI and kinact. In the present investigation a two-step statistical evaluation was developed to evaluate CYP3A4 TDI data. In the first step, a two-sided two-sample z-test is used to compare the kobs values measured in the absence and presence of the test compound to answer the question of whether the test compound is a TDI or not. In the second step, kobs values are plotted versus both [I] and ln[I] to determine whether a significant correlation exists, which can then inform the investigator of whether the inactivation kinetic parameters, KI and kinact, can be reliably estimated. Use of this two-step statistical evaluation is illustrated with the examination of five drugs of varying capabilities to inactivate CYP3A4: ketoconazole, erythromycin, raloxifene, rosiglitazone, and pioglitazone. The use of a set statistical algorithm offers a more robust and objective approach to the analysis of P450 TDI data than frequently employed empirically derived or heuristic approaches.