Patrick Trapa

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.

Discovery and preclinical characterization of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-[3,4-b]pyrazine (PF470): a highly potent, selective, and efficacious metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulator.

A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (1), a systematic structure-activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of 14 to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-mediated type IV hypersensitivity in a 90-day NHP regulatory toxicology study.

Strategies to minimize CNS toxicity: in vitro high-throughput assays and computational modeling

Introduction: Healthy functioning of the brain is dependent on the ability of the blood–brain barrier (BBB) and other central nervous system (CNS) barriers to protect the neurocompartments from potential disruptive and damaging xenobiotic agents. In vitro high-throughput (HT) screens and computational models that assess a compounds ability to pass through or disrupt the BBB have become important tools in the identification of new well-tolerated peripheral drugs and safer chemical products such as pesticides. Leveraging these HT in vitro assays and computational BBB tools together with the current understanding of brain penetration may enable the drug discovery community to minimize access of drug candidates into the CNS compartment. Areas covered: This article reviews aspects of the most recent in vitro and computational approaches designed to provide an early assessment of a compounds ability to access the neurocompartment. This article also provides insight into using these tools to identify compounds that have restricted access to the neurocompartment. Expert opinion: The development of safer peripheral-acting medicines and chemical products can be achieved through prospective design and early assessment with HT assays of the BBB in conjunction with computational models. Exclusion or significantly reduced access of a compound to the neurocompartment will increase the odds of identifying a compound with reduced CNS-related adverse drug reactions. A holistic approach to compound design and evaluation that incorporates prospective design principles (e.g., optimization of physicochemical properties), leverages HT in vitro assays and integrates the use of BBB computational models may yield the ‘best-in-class’ peripherally acting product.

Enhancing ketamine translational pharmacology via receptor occupancy normalization

Ketamine is used preclinically and clinically to study schizophrenia and depression. Accordingly, it is imperative to understand the temporal relationship between the central concentrations and N-methyl-d-aspartate receptor (NMDAR) interactions of both ketamine and norketamine, its primary active metabolite, across species to assess the translatability of animal models to humans and the back-translation of clinical observations to the preclinical realm. However, such an interspecies normalization of ketamine and norketamine exposures at different clinical and preclinical doses (and their different routes and regimens) is lacking. This work defines the NMDAR occupancy (RO) time course following single doses of ketamine in rats, nonhuman primates (nhp) and humans to allow direct interspecies comparisons of specific ketamine-mediated pharmacodynamics via RO normalization. Total plasma concentration (Cp)-time profiles of ketamine and norketamine were generated from rats and nhp following a single, memory-impairing dose of ketamine; neuropharmacokinetics were determined in rats. [(3)H]MK-801-displacement studies in rats determined estimated mean (95% confidence interval) unbound plasma concentrations (Cp,u) for ketamine and norketamine producing 50% RO (IC50) of 1420 (990, 2140) nM and 9110 (5870, 13700) nM, respectively. Together, these datasets transformed Cp,u-time data to predicted RO (ROpred)-time profiles for rats, nhp and humans at behaviorally relevant ketamine doses. Subsequently, this approach helped determine an infusion paradigm in rats producing a ROpred-time profile mirroring that for a clinically antidepressant infusion. The described indication-independent methodology allows normalization to RO at any time following any ketamine dose (regardless of route or regimen) in any species by simply quantifying the Cp of ketamine and norketamine. Matching temporal RO relationships in animals and humans should allow direct comparisons of specific ketamine-dependent NMDAR-based pharmacodynamics.

Impact of recovery on fraction unbound using equilibrium dialysis

Historically, recovery had been used to evaluate the data quality of plasma protein binding or tissue binding obtained from equilibrium dialysis assays. Low recovery was often indicative of high nonspecific binding, instability, or low solubility. This study showed that, when equilibrium was fully established in the dialysis assay, low recovery due to nonspecific binding had no impact on the determination of fraction unbound. The conclusion was supported by the principles of the equilibrium dialysis assay, experimental data, and mathematic simulations. The results suggested that the use of recovery as an acceptance criterion for the equilibrium dialysis assay in drug discovery was too restrictive, and introduced the additional burden of repeating studies unnecessarily.

Discovery of a Highly Selective Glycogen Synthase Kinase-3 Inhibitor (PF-04802367) That Modulates Tau Phosphorylation in the Brain: Translation for PET Neuroimaging.

Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology, and neurology. N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A (11) C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding.

Insights From an Integrated Physiologically Based Pharmacokinetic Model for Brain Penetration

Central-nervous-system, physiologically based pharmacokinetic (PBPK) models predict exposure profiles in the brain, that is, the rate and extent of distribution. The current work develops one such model and presents improved methods for determining key input parameters. A simple linear regression statistical model estimates the passive permeability at the blood-brain barrier from brain uptake index data and descriptors, and a novel analysis extracts the relative active transport parameter from in vitro assays taking into consideration both paracellular transport and unstirred water layers. The integrated PBPK model captures the concentration profiles of both rate-restricted and effluxed compounds with high passive permeability. In many cases, compounds distribute rapidly into the brain and are, therefore, not rate limited. The PBPK model is then simplified to a straightforward equation to describe brain-to-plasma ratios at steady state. The equation can estimate brain penetration either from in vitro efflux data or from in vivo results from another species and, therefore, is a valuable tool in the discovery setting.

Discovery and Characterization of (R)-6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.

Translational pharmacokinetic-pharmacodynamic analysis in the pharmaceutical industry: an IQ Consortium PK-PD Discussion Group perspective

With inadequate efficacy being the primary cause for the attrition of drug candidates in clinical development, the need to better predict clinical efficacy earlier in the drug development process has increased in importance in the pharmaceutical industry. Here, we review current applications of translational pharmacokinetic-pharmacodynamic (PK-PD) modeling of preclinical data in the pharmaceutical industry, including best practices. Preclinical translational PK-PD modeling has been used in many therapeutic areas and has been impactful to drug development. The role of preclinical translational PK-PD modeling in drug discovery and development will continue to evolve and broaden, given that its broad implementation in the pharmaceutical industry is relatively recent and many opportunities still exist for its further application.

Understanding the target and evaluating the consequences of gamma-secretase modulation from in vitro models to higher-order species

P4-298 UNDERSTANDING THE TARGETAND EVALUATING THE CONSEQUENCES OF GAMMASECRETASE MODULATION FROM IN VITRO MODELS TO HIGHER-ORDER SPECIES Kathleen Wood, Nikolay Pozdnyakov, Emily Sylvain, Michael Marconi, Ashley Robshaw, Leslie Pustilnik, Stefanus Steyn, Patrick Trapa, Yasong Lu, Douglas Johnson, Martin Pettersson, Kelly Bales, Pfizer Inc., Cambridge, Massachusetts, United States; Pfizer, Cambridge, Massachusetts, United States; Pfizer, Groton, Connecticut, United States; Pfizer Inc., Cambridge, Massachusetts, United States; Pfizer Inc., Groton, Connecticut, United States. Contact e-mail: kathleen.m.wood@pfizer.com