Douglas K. Spracklin

Discovery of 4-(5-Methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane (CP-810,123), a Novel α7 Nicotinic Acetylcholine Receptor Agonist for the Treatment of Cognitive Disorders in Schizophrenia: Synthesis, SAR Development, and in Vivo Efficacy in Cognition Models

A novel alpha 7 nAChR agonist, 4-(5-methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane (24, CP-810,123), has been identified as a potential treatment for cognitive deficits associated with psychiatric or neurological conditions including schizophrenia and Alzheimers disease. Compound 24 is a potent and selective compound with excellent pharmaceutical properties. In rodent, the compound displays high oral bioavailability and excellent brain penetration affording high levels of receptor occupancy and in vivo efficacy in auditory sensory gating and novel object recognition. The structural diversity of this compound and its preclinical in vitro and in vivo package support the hypothesis that alpha 7 nAChR agonists may have potential as a pharmacotherapy for the treatment of cognitive deficits in schizophrenia.

Differential Effects of Various Typical and Atypical Antipsychotics on Plasma Glucose and Insulin Levels in the Mouse: Evidence for the Involvement of Sympathetic Regulation

Atypical antipsychotic treatment has been associated with serious metabolic adverse events, such as glucose dysregulation and development of type 2 diabetes. As part of our studies on possible underlying mechanisms, we investigated the acute effects of various typical and atypical antipsychotics on plasma glucose and insulin in FVB/N mice, a strain that showed a more pronounced hyperglycemic response to clozapine than C57BL/6 and CD-1 mice. Acute administration of high doses of clozapine, olanzapine, quetiapine, perphenazine, or chlorpromazine significantly increased plasma glucose by 100%-140% above basal levels without significant effects on insulin levels. In contrast, risperidone reduced plasma glucose (-30%) and markedly enhanced plasma insulin levels. Doses of ziprasidone that gave 50-fold higher free plasma concentrations than therapeutic plasma levels, as well as high doses of aripiprazole and haloperidol, did not significantly alter either glucose or insulin levels. Clozapine- and olanzapine-induced hyperglycemia occurred at free plasma concentrations that were within, or one order of magnitude above, the range of therapeutic plasma levels. Pretreatment with either the ganglionic blocker hexamethonium, or the alpha(2) adrenergic receptor antagonist yohimbine, blocked the clozapine- and chlorpromazine-induced increase in glucose levels. Taken together, these results suggest that typical and atypical antipsychotics with known metabolic liability produce acute hyperglycemia in mice and that this effect is likely driven by activation of the sympathetic autonomic nervous system via a central mechanism.

Deuterium Isotope Effects on Drug Pharmacokinetics. I. System- Dependent Effects of Specific Deuteration with Aldehyde Oxidase Cleared Drugs

The pharmacokinetic properties of drugs may be altered by kinetic deuterium isotope effects. With specifically deuterated model substrates and drugs metabolized by aldehyde oxidase, we demonstrate how knowledge of the enzymes reaction mechanism, species differences in the role played by other enzymes in a drugs metabolic clearance, and differences in systemic clearance mechanisms are critically important for the pharmacokinetic application of deuterium isotope effects. Ex vivo methods to project the in vivo outcome using deuterated carbazeran and zoniporide with hepatic systems demonstrate the importance of establishing the extent to which other metabolic enzymes contribute to the metabolic clearance mechanism. Differences in pharmacokinetic outcomes in guinea pig and rat, with the same metabolic clearance mechanism, show how species differences in the systemic clearance mechanism can affect the in vivo outcome. Overall, to gain from the application of deuteration as a strategy to alter drug pharmacokinetics, these studies demonstrate the importance of understanding the systemic clearance mechanism and knowing the identity of the metabolic enzymes involved, the extent to which they contribute to metabolic clearance, and the extent to which metabolism contributes to the systemic clearance.

Discovery of Two Clinical Histamine H3 Receptor Antagonists: trans-N-Ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidinylmethyl)phenyl]cyclobutanecarboxamide (PF-03654746) and trans-3-Fluoro-3-[3-fluoro-4-(pyrrolidin-1-ylmethyl)phenyl]-N-(2-methylpropyl)cyclobutanecarboxamide (PF-03654764)

The discovery of two histamine H(3) antagonist clinical candidates is disclosed. The pathway to identification of the two clinical candidates, 6 (PF-03654746) and 7 (PF-03654764) required five hypothesis driven design cycles. The key to success in identifying these clinical candidates was the development of a compound design strategy that leveraged medicinal chemistry knowledge and traditional assays in conjunction with computational and in vitro safety tools. Overall, clinical compounds 6 and 7 exceeded conservative safety margins and possessed optimal pharmacological and pharmacokinetic profiles, thus achieving our initial goal of identifying compounds with fully aligned oral drug attributes, best-in-class molecules.

3-Benzyl-1,3-oxazolidin-2-ones as mGluR2 positive allosteric modulators: Hit-to lead and lead optimization.

The discovery, synthesis and SAR of a novel series of 3-benzyl-1,3-oxazolidin-2-ones as positive allosteric modulators (PAMs) of mGluR2 is described. Expedient hit-to-lead work on a single HTS hit led to the identification of a ligand-efficient and structurally attractive series of mGluR2 PAMs. Human microsomal clearance and suboptimal physicochemical properties of the initial lead were improved to give potent, metabolically stable and orally available mGluR2 PAMs.

Biosynthesis of Drug Metabolites and Quantitation Using NMR Spectroscopy for Use in Pharmacologic and Drug Metabolism Studies

The contribution of drug metabolites to the pharmacologic and toxicologic activity of a drug can be important; however, for a variety of reasons metabolites can frequently be difficult to synthesize. To meet the need of having samples of drug metabolites for further study, we have developed biosynthetic methods coupled with quantitative NMR spectroscopy (qNMR) to generate solutions of metabolites of known structure and concentration. These quantitative samples can be used in a variety of ways when a synthetic sample is unavailable, including pharmacologic assays, standards for in vitro work to help establish clearance pathways, and/or as analytical standards for bioanalytical work to ascertain exposure, among others. We illustrate five examples of metabolite biosynthesis and qNMR. The types of metabolites include one glucuronide and four oxidative products. Concentrations of the isolated metabolite stock solutions ranged from 0.048 to 8.3 mM, with volumes from approximately 0.04 to 0.150 ml in hexadeutarated dimethylsulfoxide. These specific quantified isolates were used as standards in the drug discovery setting as substrates in pharmacology assays, for bioanalytical assays to establish exposure, and in variety of routine absorption, distribution, metabolism, and excretion assays, such as protein binding and determining blood-to-plasma ratios. The methods used to generate these materials are described in detail with the objective that these methods can be generally used for metabolite biosynthesis and isolation.

Synthesis and SAR studies of 1,4-diazabicyclo[3.2.2]nonane phenyl carbamates – subtype selective, high affinity α7 nicotinic acetylcholine receptor agonists

The synthesis and SAR studies about the bicyclic amine, carbamate linker and aromatic ring of a 1,4-diazabicyclo[3.2.2]nonane phenyl carbamate series of alpha7 nAChR agonists is described. The development of the medicinal chemistry strategy and SAR which led to the identification of 5 and 7aa as subtype selective, high affinity alpha7 agonists as excellent leads for further evaluation is discussed, along with key physicochemical and pharmacokinetic data highlighting their lead potential.

Concordance between trifluoroacetic acid and hepatic protein trifluoroacetylation after disulfiram inhibition of halothane metabolism in rats

Background:u2002 Cytochrome P4502E1(CYP2E1)‐mediated oxidation of halothane to a reactive intermediate (trifluoroacyl chloride) that covalently binds to hepatic proteins forming trifluoroacetylated neoantigens is believed to be the initiating event in a complex immunologic cascade culminating in antibody formation and severe hepatic necrosis (‘halothane hepatitis’) in susceptible patients. Trifluoroacyl chloride may also hydrolyze to the stable metabolite trifluoroacetic acid (TFA). CYP2E1 inactivation by disulfiram or its primary metabolite, diethyldithiocarbamate, inhibits human halothane oxidation to TFA in vitro and in vivo. Nevertheless, disulfiram effects on hepatic protein trifluoroacetylation by halothane in vivo are unknown. This investigation tested the hypotheses that disulfiram prevents halothane‐dependent protein trifluoroacetylation in vivo, and that TFA represents a biomarker for hepatic protein trifluoroacetylation.

Quantitative projection of human brain penetration of the H3 antagonist PF-03654746 by integrating rat-derived brain partitioning and PET receptor occupancy.

Abstract 1.u2002Unbound brain drug concentration (Cb,u), a valid surrogate of interstitial fluid drug concentration (CISF), cannot be directly determined in humans, which limits accurately defining the human Cb,u:Cp,u of investigational molecules. 2.u2002For the H3R antagonist (1R,3R)-N-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-lmethyl)phenyl]cyclobutane-1-carboxamide (PF-03654746), we interrogated Cb,u:Cp,u in humans and nonhuman primate (NHP). 3.u2002In rat, PF-03654746 achieved net blood–brain barrier (BBB) equilibrium (Cb,u:Cp,u of 2.11). 4.u2002In NHP and humans, the PET receptor occupancy-based Cp,u IC50 of PF-03654746 was 0.99u2009nM and 0.31u2009nM, respectively, which were 2.1- and 7.4-fold lower than its in vitro human H3 Ki (2.3u2009nM). 5.u2002In an attempt to understand this higher-than-expected potency in humans and NHP, rat-derived Cb,u:Cp,u of PF-03654746 was integrated with Cp,u IC50 to identify unbound (neuro) potency of PF-03654746, nIC50. 6.u2002The nIC50 of PF-03654746 was 2.1u2009nM in NHP and 0.66u2009nM in human which better correlated (1.1- and 3.49-fold lower) with in vitro human H3 Ki (2.3u2009nM). 7.u2002This correlation of the nIC50 and in vitro hH3 Ki suggested the translation of net BBB equilibrium of PF-03654746 from rat to NHP and humans, and confirmed the use of Cp,u as a reliable surrogate of Cb,u. 8.u2002Thus, nIC50 quantitatively informed the human Cb,u:Cp,u of PF-03654746.

The Role of Biotransformation Studies in Reducing Drug Attrition

Biotransformation has evolved beyond simple structural elucidation of metabolites to provide data that is highly impactful and influential to various aspects of drug discovery. Based on our experience, we have grouped areas where biotransformation-related understanding can make an impact on drug design as follows: (1) defining clearance mechanisms, particularly for drug metabolizing enzymes other than P450s, (2) identifying metabolic hot spots, (3) identifying reactive metabolites, (4) characterizing active metabolites, and (5) assessing metabolite safety. This review will describe how these studies may be used to guide the development of structure–activity relationships to identify and mitigate potential safety liabilities and to interpret pharmacokinetic/pharmacodynamic (PKPD) relationships. Ultimately, a better understanding in all these aspects of drug disposition will aid in reducing candidate attrition.