Rakesh Nagilla

AMTB, a TRPM8 channel blocker: evidence in rats for activity in overactive bladder and painful bladder syndrome

The activation of the TRPM8 channel, a member of the large class of TRP ion channels, has been reported to be involved in overactive bladder and painful bladder syndrome, although an endogenous activator has not been identified. In this study, N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt (AMTB) was evaluated as a TRPM8 channel blocker and used as a tool to evaluate the effects of this class of ion channel blocker on volume-induced bladder contraction and nociceptive reflex responses to noxious bladder distension in the rat. AMTB inhibits icilin-induced TRPM8 channel activation as measured in a Ca(2+) influx assay, with a pIC(50) of 6.23. In the anesthetized rat, intravenous administration of AMTB (3 mg/kg) decreased the frequency of volume-induced bladder contractions, without reducing the amplitude of contraction. The nociceptive response was measured by analyzing both visceromotor reflex (VMR) and cardiovascular (pressor) responses to urinary bladder distension (UBD) under 1% isoflurane. AMTB (10 mg/kg) significantly attenuated reflex responses to noxious UBD to 5.42 and 56.51% of the maximal VMR response and pressor response, respectively. The ID50 value on VMR response was 2.42 +/- 0.46 mg/kg. These results demonstrate that TRPM8 channel blocker can act on the bladder afferent pathway to attenuate the bladder micturition reflex and nociceptive reflex responses in the rat. Targeting TRPM8 channel may provide a new therapeutic opportunity for overactive bladder and painful bladder syndrome.

Discovery of Small Molecule RIP1 Kinase Inhibitors for the Treatment of Pathologies Associated with Necroptosis.

Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.

Discovery of a First-in-Class Receptor Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate (GSK2982772) for the Treatment of Inflammatory Diseases

RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.

Comparative evaluation of oral systemic exposure of 56 xenobiotics in rat, dog, monkey and human.

The prediction of human pharmacokinetics is often based on in vivo preclinical pharmacokinetic data. However, to date, no clear guidance has been available about the relative ability of the major preclinical species to estimate human oral exposure. The study was conducted to survey the literature on oral pharmacokinetic parameters in rat, dog, monkey and human, and to compare various methods for prediction of oral exposure in humans. Fifty–six non–peptide xenobiotics were identified with oral pharmacokinetic data in rat, dog, monkey and human, and comparison of the data from each species to humans was conducted along with an evaluation of the molecular features of these compounds. Monkey liver blood flow–based oral exposure was qualitatively and quantitatively more predictive of human oral exposure than rat or dog. Furthermore, generation of data in three versus two preclinical species did not always improve human predictivity. The use of molecular properties did not substantially improve the prediction of human oral exposure compared with the prediction from monkey alone. These observations confirm the continued importance of non–human primates in preclinical pharmacokinetics, and also have implications for pharmacokinetic lead optimization and for prediction of human pharmacokinetic parameters from in vivo preclinical data.

Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors

The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.

Extrapolation of Preclinical Pharmacokinetics and Molecular Feature Analysis of 'Discovery-Like' Molecules to Predict Human Pharmacokinetics

The prediction of human pharmacokinetics from preclinical species is an integral component of drug discovery. Recent studies with a 103-compound dataset suggested that scaling from monkey pharmacokinetic data tended to be the most accurate method for predicting human clearance. Additionally, interrogation of the two-dimensional molecular properties of these molecules produced a set of associations which predict the likely extrapolative outcome (success or failure) of preclinical data to project human pharmacokinetics. However, a limitation of the previous analyses was the relative paucity of data for typical “discovery-like” molecules (molecular weight >300 and/or clogP >3). The objective of this investigation was to generate preclinical data required for extension of this dataset for additional discovery-like molecules and determine whether the aforementioned findings continue to apply for these molecules. In vivo nonrodent intravenous pharmacokinetic data were generated for 13 molecules, and data for 8 additional molecules were obtained from the literature. Additionally, the various scaling methodologies and molecular features analysis were applied to this new dataset to predict human pharmacokinetics. Whereas the predictive accuracies demonstrated across all of the various methodologies were lower for this higher clearance compound dataset, scaling from monkey liver blood flow continued to be an accurate methodology, and human volume of distribution was similarly well predicted regardless of scaling methodology. Lastly, application of the molecular feature associations, particularly data-dependent associations, afforded an improved predictivity compared with the liver blood flow scaling approaches, and provides insight into the extrapolation of high clearance compounds in the preclinical species to human.

Discovery of novel aminothiadiazole amides as selective EP3 receptor antagonists

This Letter discloses a series of 2-aminothiadiazole amides as selective EP(3) receptor antagonists. SAR optimization resulted in compounds with excellent functional activity in vitro. In addition, efforts to optimize DMPK properties in the rat are discussed. These efforts have resulted in the identification of potent, selective EP(3) receptor antagonists with excellent DMPK properties suitable for in vivo studies.

Cassette dosing for pharmacokinetic screening in drug discovery: Comparison of clearance, volume of distribution, half-life, mean residence time, and oral bioavailability obtained by cassette and discrete dosing in rats†

The purpose of this investigation was to compare selected pharmacokinetic (PK) parameters obtained by cassette and discrete dosing of compounds in rats. The concordance of PK properties obtained by the two dosing strategies was evaluated for 116 compounds representing various therapeutic programs and diverse chemical structures. The correspondence between cassette- and discrete-dosing-derived PK properties was examined semiquantitatively and qualitatively. For semiquantitative comparison, compounds with cassette-to-discrete PK parameter ratios between 0.5 and 2 (inclusive) were considered to be in agreement. For qualitative comparison, compounds were divided into three categories (low, moderate, and high) based on the value of the PK parameter; compounds that fell into the same category following cassette and discrete dosing were considered to be in agreement. Of the 116 compounds evaluated, 89%, 91%, 80%, and 91% of the compounds were semiquantitatively equivalent for the intravenous PK parameters of clearance (CL), volume of distribution (Vdss), terminal elimination plasma half-life (HL), and mean residence time (MRT), respectively, whereas 79%, 80%, 79%, and 72% were qualitatively similar for CL, Vdss, MRT, and terminal elimination plasma HL, respectively. Following oral administration, bioavailability concordance was 72% when assessed qualitatively and 78% when determined semiquantitatively. Results from these analyses indicate that a cassette dosing strategy is a viable approach to screen compounds for PK properties within a drug discovery setting.

The Identification and Pharmacological Characterization of 6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine (GSK583), a Highly Potent and Selective Inhibitor of RIP2 Kinase

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.

Discovery of orally active, pyrrolidinone-based progesterone receptor partial agonists.

We have designed and synthesized a novel series of pyrrolidinones as progesterone receptor partial agonists. Compounds from this series had improved AR selectivity, rat pharmacokinetic properties, and in vivo potency compared to the lead compound. In addition, these compounds had improved selectivity against hERG channel inhibition.