Peter J. Bungay

A comprehensive non-clinical evaluation of the CNS penetration potential of antimuscarinic agents for the treatment of overactive bladder

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT This study provides antimuscarinic agents for overactive bladder (OAB) display variable association with side effects mediated by the central nervous system (CNS), which may be of particular concern in the elderly. Adverse effects on CNS functioning are related to muscarinic receptor subtype selectivity and the ability of the agent to cross the blood-brain barrier, where P-gp plays a role in limiting permeability. WHAT THIS STUDY ADDS This study provides a parallel investigation of CNS penetration of antimuscarinic OAB agents in vivo and assessment of physical properties and permeability in cell monolayers in vitro. It adds further understanding of the roles of passive transcellular permeability and P-gp in determining CNS penetration of antimuscarinic OAB agents. It also enables a comparison of CNS side-effect profiles of OAB agents with preclinical CNS penetration data. AIMS To assess and compare the mechanisms of central nervous system (CNS) penetration of antimuscarinic overactive bladder (OAB) agents. METHODS Physical properties were computed or compiled from the literature. Rats were administered 5-hydroxymethyl tolterodine (HMT), darifenacin, oxybutynin, solifenacin, tolterodine or trospium subcutaneously. At 1 h postdose, plasma, brain and cerebrospinal fluid (CSF) concentrations were determined using LC-MS/MS assays. Brain and plasma protein binding were determined in vitro. Permeability in the presence and absence of the efflux transporter P-glycoprotein (P-gp) was assessed in RRCK and MDCK-MDR1 transwell assays. RESULTS Oxybutynin displayed extensive CNS penetration, with brain:plasma ratios (B:P), unbound brain:unbound plasma ratios (Kp,free) and CSF:free plasma ratios each >1. Tolterodine (B:P = 2.95, Kp,free = 0.23 and CSF:free plasma = 0.16) and solifenacin (B:P = 3.04, Kp,free = 0.28 and CSF:free plasma = 1.41) showed significant CNS penetration but with some restriction from CNS as indicated by Kp,free values significantly <1. 5-HMT, darifenacin and trospium displayed much lower B:P (0.03-0.16), Kp,free (0.01-0.04) and CSF:free plasma (0.004-0.06), consistent with poor CNS penetration. Permeability in RRCK cells was low for trospium (0.63 × 10(-6) cm s(-1) ), moderate for 5-HMT (11.7 × 10(-6) cm s(-1) ) and high for darifenacin, solifenacin, tolterodine and oxybutynin (21.5-38.2 × 10(-6) cm s(-1) ). In MDCK-MDR1 cells 5-HMT, darifenacin and trospium, were P-gp substrates, whereas oxybutynin, solifenacin and tolterodine were not P-gp substrates. CONCLUSIONS Brain penetration was low for antimuscarinics that are P-gp substrates (5-HMT, darifenacin and trospium), and significant for those that are not P-gp substrates (oxybutynin, solifenacin and tolterodine). CNS adverse events reported in randomized controlled clinical trials show general alignment with the preclinical data described in this study.

Functional coupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive luciferase reporter gene

Abstract: A cyclic AMP‐responsive reporter cell line has been established through the stable expression of a luciferase reporter plasmid in Chinese hamster ovary (CHO) cells. Reporter cells showed a dose‐dependent expression of luciferase in response to incubation with forskolin. These CHO cells were screened for endogenous G protein‐coupled receptors capable of stimulating or inhibiting adenylyl cyclase, by monitoring changes in luciferase expression. Serotonin (5‐HT) receptor agonist ligands caused an inhibition of forskolin‐stimulated luciferase expression in the rank order 5‐carboxamidotryptamine > 5‐HT > sumatriptan > 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin. The response to 5‐HT was reversed by the 5‐HT1 receptor antagonists cyanopindolol and pindolol, but not the 5‐HT2 receptor antagonist ketanserin. Calcitonin was more potent than calcitonin gene‐related peptide (CGRP) at stimulating luciferase expression in this cell line, and these responses were insensitive to the CGRP receptor antagonist, CGRP (8–37). These results were consistent with the presence of 5‐HT1B‐like and calcitonin (C1a‐like) receptors in CHO cells, with the responses to 5‐HT and CGRP being pertussis and cholera toxin‐sensitive, respectively. This reporter gene assay gave the expected pharmacological profile for these receptors when compared with cyclic AMP accumulation assays, confirming its value as a functional assay for G protein‐coupled receptors linked to adenylyl cyclase.

Application of PBPK modelling in drug discovery and development at Pfizer

Early prediction of human pharmacokinetics (PK) and drug–drug interactions (DDI) in drug discovery and development allows for more informed decision making. Physiologically based pharmacokinetic (PBPK) modelling can be used to answer a number of questions throughout the process of drug discovery and development and is thus becoming a very popular tool. PBPK models provide the opportunity to integrate key input parameters from different sources to not only estimate PK parameters and plasma concentration-time profiles, but also to gain mechanistic insight into compound properties. Using examples from the literature and our own company, we have shown how PBPK techniques can be utilized through the stages of drug discovery and development to increase efficiency, reduce the need for animal studies, replace clinical trials and to increase PK understanding. Given the mechanistic nature of these models, the future use of PBPK modelling in drug discovery and development is promising, however, some limitations need to be addressed to realize its application and utility more broadly.

Evaluation of a CRE-Directed Luciferase Reporter Gene Assay as an Alternative to Measuring cAMP Accumulation

A CHO reporter cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was used to investigate the relationship between cAMP accumulation and cAMP dependent reporter gene expression and therefore, to assess the reporter gene system as an alternative functional assay. Timecourse experiments showed that cAMP accumulation preceded luciferase expression and that longer incubations (>2 h) were required to gain results with the reporter gene system. However, forskolin concentration dose-response studies revealed a 100-fold amplification of the signal measured by luciferase expression compared with direct cAMP accumulation, indicating that the reporter gene system afforded greater sensitivity. EC50 values determined for agonist activation of an inhibitory (5-HTlB-like) G-protein-coupled receptor (GPCR) were the same, and for a stimulatory GPCR (calcitonin Cla-like) were 10-fold lower, using the reporter gene system compared to cAMP accumulation assays, indicating the suitability of the reporter system for measuring the activity of receptors differentially coupled to the cAMP pathway. The phorbol ester, PMA, and the Ca2+ ionophore, A23187, were able to potentiate forskolin-stimulated luciferase expression but not cAMP accumulation, suggesting that the former could also be used to monitor cross-talk between different signal transduction pathways at the level of gene transcription.

The combinatorial synthesis of a 30,752-compound library: discovery of SAR around the endothelin antagonist, FR-139,317

Abstract A combinatorial library of 30,752 compounds has been synthesised from a set of 32 natural and unnatural amino acids. The library was designed to include the known endothelin antagonist, FR-139,317, as a positive control, and this and a number of close analogues were shown to be the most potent compounds. Thus, combinatorial libraries may be used both to discover leads and rapidly explore SAR. A combinatorial library of 30,752 compounds has been synthesised from a set of 32 natural and unnatural amino acids. The library was designed to include the known endothelin antagonist, FR-139,317, as a positive control, and this and a number of close analogues were shown to be the most potent compounds. Thus, combinatorial libraries may be used both to discover leads and rapidly explore SAR.

A novel selective and orally bioavailable Nav1.8 channel blocker, PF‐01247324, attenuates nociception and sensory neuron excitability

NaV1.8 ion channels have been highlighted as important molecular targets for the design of low MW blockers for the treatment of chronic pain. Here, we describe the effects of PF‐01247324, a new generation, selective, orally bioavailable Nav1.8 channel blocker of novel chemotype.

Functional analysis of the D2L dopamine receptor expressed in a cAMP-responsive luciferase reporter cell line.

A Chinese hamster ovary (CHO) cell line expressing the firefly luciferase gene under the control of six cAMP response elements (CREs) was stably transfected with the long form of the rat D2 dopamine receptor. Saturation binding analysis using [3H]spiperone showed that the receptor was expressed at low levels (Bmax = 96.5+/-15.8 fmol/mg), but with an affinity characteristic of the D2 receptor (Kd = 21.5+/-3.7 pM). Luciferase expression in this cell line was modified in a dose dependent manner with dopamine receptor agonists (N-propylapomorphine > apomorphine > quinpirole > dopamine) and antagonists (spiperone > (+)-butaclamol > D0710 > (-)-sulpiride > tiapride > remoxipride), according to their rank order of potency in binding and cAMP accumulation studies. Dopamine-mediated inhibition of forskolin-stimulated luciferase expression was pertussis toxin sensitive. This demonstrated the efficiency of the luciferase reporter gene assay for the functional testing of D2 dopamine receptors, which are negatively coupled to the adenylyl cyclase signaling pathway, when heterogously expressed at low levels in CHO cells.

Preclinical and Clinical Pharmacokinetics of PF-02413873, a Nonsteroidal Progesterone Receptor Antagonist

The recently discovered selective nonsteroidal progesterone receptor (PR) antagonist 4-[3-cyclopropyl-1-(methylsulfonylmethyl)-5-methyl-1H-pyrazol-4-yl]oxy-2,6-dimethylbenzonitrile (PF-02413873) was characterized in metabolism studies in vitro, in preclinical pharmacokinetics in rat and dog, and in an initial pharmacokinetic study in human volunteers. Clearance (CL) of PF-02413873 was found to be high in rat (84 ml · min−1 · kg−1) and low in dog (3.8 ml · min−1 · kg−1), consistent with metabolic stability determined in liver microsomes and hepatocytes in these species. In human, CL was low in relation to hepatic blood flow, consistent with metabolic stability in human in vitro systems, where identified metabolites suggested predominant cytochrome P450 (P450)-catalyzed oxidative metabolism. Prediction of CL using intrinsic CL determined in human liver microsomes (HLM), recombinant human P450 enzymes, and single species scaling (SSS) from pharmacokinetic studies showed that dog SSS and HLM scaling provided the closest estimates of CL of PF-02413873 in human. These CL estimates were combined with a physiologically based pharmacokinetic (PBPK) model to predict pharmacokinetic profiles after oral suspension administration of PF-02413873 in fasted and fed states in human. Predicted plasma concentration versus time profiles were found to be similar to those observed in human over the PF-02413873 dose range 50 to 500 mg and captured the enhanced exposure in fed subjects. This case study of a novel nonsteroidal PR antagonist underlines the utility of PBPK modeling techniques in guiding prediction confidence and design of early clinical trials of novel chemical agents.

Multiparameter Optimization in CNS Drug Discovery: Design of Pyrimido[4,5-d]azepines as Potent 5-Hydroxytryptamine 2C (5-HT2C) Receptor Agonists with Exquisite Functional Selectivity over 5-HT2A and 5-HT2B Receptors

A series of 4-substituted pyrimido[4,5-d]azepines that are potent, selective 5-HT2C receptor partial agonists is described. A rational medicinal chemistry design strategy to deliver CNS penetration coupled with SAR-based optimization of selectivity and agonist potency provided compounds with the desired balance of preclinical properties. Lead compounds 17 (PF-4479745) and 18 (PF-4522654) displayed robust pharmacology in a preclinical canine model of stress urinary incontinence (SUI) and no measurable functional agonism at the key selectivity targets 5-HT2A and 5-HT2B in relevant tissue-based assay systems. Utilizing recent advances in the structural biology of GPCRs, homology modeling has been carried out to rationalize binding and agonist efficacy of these compounds.

Discovery and Optimization of Selective Nav1.8 Modulator Series That Demonstrate Efficacy in Preclinical Models of Pain

Voltage-gated sodium channels, in particular Nav1.8, can be targeted for the treatment of neuropathic and inflammatory pain. Herein, we described the optimization of Nav1.8 modulator series to deliver subtype selective, state, and use-dependent chemical matter that is efficacious in preclinical models of neuropathic and inflammatory pain.