Susan M. Lotarski

Behavioral Characterization of A53T Mice Reveals Early and Late Stage Deficits Related to Parkinson’s Disease

Parkinsons disease (PD) pathology is characterized by the formation of intra-neuronal inclusions called Lewy bodies, which are comprised of alpha-synuclein (α-syn). Duplication, triplication or genetic mutations in α-syn (A53T, A30P and E46K) are linked to autosomal dominant PD; thus implicating its role in the pathogenesis of PD. In both PD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of protein aggregates (i.e., α-syn) and neurodegeneration. Characterization of the timing and nature of symptomatic dysfunction is important for understanding the impact of α-syn on disease progression. Furthermore, this knowledge is essential for identifying pathways and molecular targets for therapeutic intervention. To this end, we examined various functional and morphological endpoints in the transgenic mouse model expressing the human A53T α-syn variant directed by the mouse prion promoter at specific ages relating to disease progression (2, 6 and 12 months of age). Our findings indicate A53T mice develop fine, sensorimotor, and synaptic deficits before the onset of age-related gross motor and cognitive dysfunction. Results from open field and rotarod tests show A53T mice develop age-dependent changes in locomotor activity and reduced anxiety-like behavior. Additionally, digigait analysis shows these mice develop an abnormal gait by 12 months of age. A53T mice also exhibit spatial memory deficits at 6 and 12 months, as demonstrated by Y-maze performance. In contrast to gross motor and cognitive changes, A53T mice display significant impairments in fine- and sensorimotor tasks such as grooming, nest building and acoustic startle as early as 1–2 months of age. These mice also show significant abnormalities in basal synaptic transmission, paired-pulse facilitation and long-term depression (LTD). Combined, these data indicate the A53T model exhibits early- and late-onset behavioral and synaptic impairments similar to PD patients and may provide useful endpoints for assessing novel therapeutic interventions for PD.

Anxiolytic-Like Activity of Pregabalin in the Vogel Conflict Test in α2δ-1 (R217A) and α2δ-2 (R279A) Mouse Mutants

The α2δ auxiliary subunits (α2δ-1 and α2δ-2) of voltage-sensitive calcium channels are thought to be the site of action of pregabalin (Lyrica), a drug that has been shown to be anxiolytic in clinical trials for generalized anxiety disorder. Pregabalin and the chemically related drug gabapentin have similar binding and pharmacology profiles, demonstrating high-affinity, in vitro binding to both α2δ-1 and α2δ-2 subunits. Two independent point mutant mouse strains were generated in which either the α2δ-1 subunit (arginine-to-alanine mutation at amino acid 217; R217A) or the α2δ-2 subunit (arginine-to-alanine mutation at amino acid 279; R279A) were rendered insensitive to gabapentin or pregabalin binding. These strains were used to characterize the activity of pregabalin in the Vogel conflict test, a measure of anxiolytic-like activity. Pregabalin showed robust anticonflict activity in wild-type littermates from each strain at a dose of 10 mg/kg but was inactive in the α2δ-1 (R217A) mutants up to a dose of 320 mg/kg. In contrast, pregabalin was active in the α2δ-2 (R279A) point mutants at 10 and 32 mg/kg. The positive control phenobarbital was active in mice carrying either mutation. These data suggest that the anxiolytic-like effects of pregabalin are mediated by binding of the drug to the α2δ-1 subunit.

Discovery of the Potent and Selective M1 PAM-Agonist N-[(3R,4S)-3-Hydroxytetrahydro-2H-pyran-4-yl]-5-methyl-4-[4-(1,3-thiazol-4-yl)benzyl]pyridine-2-carboxamide (PF-06767832): Evaluation of Efficacy and Cholinergic Side Effects

It is hypothesized that selective muscarinic M1 subtype activation could be a strategy to provide cognitive benefits to schizophrenia and Alzheimers disease patients while minimizing the cholinergic side effects observed with nonselective muscarinic orthosteric agonists. Selective activation of M1 with a positive allosteric modulator (PAM) has emerged as a new approach to achieve selective M1 activation. This manuscript describes the development of a series of M1-selective pyridone and pyridine amides and their key pharmacophores. Compound 38 (PF-06767832) is a high quality M1 selective PAM that has well-aligned physicochemical properties, good brain penetration and pharmacokinetic properties. Extensive safety profiling suggested that despite being devoid of mAChR M2/M3 subtype activity, compound 38 still carries gastrointestinal and cardiovascular side effects. These data provide strong evidence that M1 activation contributes to the cholinergic liabilities that were previously attributed to activation of the M2 and M3 receptors.

Anticonvulsant activity of pregabalin in the maximal electroshock-induced seizure assay in α2δ1 (R217A) and α2δ2 (R279A) mouse mutants

Pregabalin has been shown to have anticonvulsant, analgesic, and anxiolytic activity in animal models. Pregabalin binds with high affinity to the α2δ1 and α2δ2 subunits of voltage-gated calcium channels. In order to better understand the relative contribution that binding to either the α2δ1 or α2δ2 subunits confers on the anticonvulsant activity of pregabalin, we characterized the anticonvulsant activity of pregabalin in different wild-type (WT) and mutant mouse strains. Two targeted mouse mutants have been made in which either the α2δ1 subunit was mutated (arginine-to-alanine mutation at amino acid 217; R217A) or the α2δ2 subunit was mutated (arginine-to-alanine mutation at amino acid 279; R279A). These mutations in α2δ1 or α2δ2 render the subunits relatively insensitive to pregabalin binding. The anticonvulsant activity of pregabalin was assessed in these different mouse lines using the maximal electroshock-induced seizure (MES) model. Pregabalin reduced the percentage of seizures and increased the latency to seizure in the MES model in two parental mouse strains used to construct the mutants. Pregabalin also reduced the percentage of seizures and increased latency to seizure similarly in the α2δ2 (R279A) and WT littermate control mice. In contrast, pregabalins anticonvulsant efficacy was significantly reduced in α2δ1 (R217A) mutants compared with WT littermate control mice. Phenytoin showed anticonvulsant activity across all WT and mutant mice. These data show that the anticonvulsant activity of pregabalin in the MES model requires binding to the α2δ1 subunit.

Positive Allosteric Modulation of AMPA Receptors from Efficacy to Toxicity: The Interspecies Exposure-Response Continuum of the Novel Potentiator PF-4778574

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) positive allosteric modulation (i.e., “potentiation”) has been proposed to overcome cognitive impairments in schizophrenia, but AMPAR overstimulation can be excitotoxic. Thus, it is critical to define carefully a potentiator’s mechanism-based therapeutic index (TI) and to determine confidently its translatability from rodents to higher-order species. Accordingly, the novel AMPAR potentiator N-{(3R,4S)-3-[4-(5-cyano-2-thienyl)phenyl]tetrahydro-2H-pyran-4-yl}propane-2-sulfonamide (PF-4778574) was characterized in a series of in vitro assays and single-dose animal studies evaluating AMPAR-mediated activities related to cognition and safety to afford an unbound brain compound concentration (Cb,u)–normalized interspecies exposure-response relationship. Because it is unknown which AMPAR subtype(s) may be selectively potentiated for an optimal TI, PF-4778574 binding affinity and functional potency were determined in rodent tissues expected to express a native mixture of AMPAR subunits and their associated proteins to afford composite pharmacological values. Functional activity was also quantified in recombinant cell lines stably expressing human GluA2 flip or flop homotetramers. Procognitive effects of PF-4778574 were evaluated in both rat electrophysiological and nonhuman primate (nhp) behavioral models of pharmacologically induced N-methyl-d-aspartate receptor hypofunction. Safety studies assessed cerebellum-based AMPAR activation (mouse) and motor coordination disruptions (mouse, dog, and nhp), as well as convulsion (mouse, rat, and dog). The resulting empirically derived exposure-response continuum for PF-4778574 defines a single-dose-based TI of 8- to 16-fold for self-limiting tremor, a readily monitorable clinical adverse event. Importantly, the Cb,u mediating each physiological effect were highly consistent across species, with efficacy and convulsion occurring at just fractions of the in vitro–derived pharmacological values.

Design and Synthesis of γ- and δ-Lactam M1 Positive Allosteric Modulators (PAMs): Convulsion and Cholinergic Toxicity of an M1-Selective PAM with Weak Agonist Activity

Recent data demonstrated that activation of the muscarinic M1 receptor by a subtype-selective positive allosteric modulator (PAM) contributes to the gastrointestinal (GI) and cardiovascular (CV) cholinergic adverse events (AEs) previously attributed to M2 and M3 activation. These studies were conducted using PAMs that also exhibited allosteric agonist activity, leaving open the possibility that direct activation by allosteric agonism, rather than allosteric modulation, could be responsible for the adverse effects. This article describes the design and synthesis of lactam-derived M1 PAMs that address this hypothesis. The lead molecule from this series, compound 1 (PF-06827443), is a potent, low-clearance, orally bioavailable, and CNS-penetrant M1-selective PAM with minimal agonist activity. Compound 1 was tested in dose escalation studies in rats and dogs and was found to induce cholinergic AEs and convulsion at therapeutic indices similar to previous compounds with more agonist activity. These findings provide preliminary evidence that positive allosteric modulation of M1 is sufficient to elicit cholinergic AEs.

Design and optimization of selective azaindole amide M1 positive allosteric modulators

Selective activation of the M1 receptor via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimers disease. A novel series of azaindole amides and their key pharmacophore elements are described. The nitrogen of the azaindole core is a key design element as it forms an intramolecular hydrogen bond with the amide N-H thus reinforcing the bioactive conformation predicted by published SAR and our homology model. Representative compound 25 is a potent and selective M1 PAM that has well aligned physicochemical properties, adequate brain penetration and pharmacokinetic (PK) properties, and is active in vivo. These favorable properties indicate that this series possesses suitable qualities for further development and studies.

Oxadiazolone bioisosteres of pregabalin and gabapentin.

A series of oxadiazolone bioisosteres of pregabalin 1 and gabapentin 2 were prepared, and several were found to exhibit similar potency for the alpha(2)-delta subunit of voltage-gated calcium channels. Oxadiazolone 9 derived from 2 achieved low brain uptake but was nevertheless active in models of osteoarthritis. The high clearance associated with compound 9 was postulated to be a consequence of efflux by OAT and/or OCT, and was attenuated on co-administration with cimetidine or probenecid.

The Discovery and Characterization of the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Potentiator N-{(3S,4S)-4-[4-(5-Cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242)

A unique tetrahydrofuran ether class of highly potent α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiators has been identified using rational and structure-based drug design. An acyclic lead compound, containing an ether-linked isopropylsulfonamide and biphenyl group, was pharmacologically augmented by converting it to a conformationally constrained tetrahydrofuran to improve key interactions with the human GluA2 ligand-binding domain. Subsequent replacement of the distal phenyl motif with 2-cyanothiophene to enhance its potency, selectivity, and metabolic stability afforded N-{(3S,4S)-4-[4-(5-cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242, 3), whose preclinical characterization suggests an adequate therapeutic index, aided by low projected human oral pharmacokinetic variability, for clinical studies exploring its ability to attenuate cognitive deficits in patients with schizophrenia.

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M1 Receptor Activation

The rationale for using M1 selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well-established in the literature. Here, we investigate measurement of inositol phosphate accumulation, an end point immediately downstream of the M1 muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical readout for M1 muscarinic acetylcholine receptor activation. Five brain penetrant M1-subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine-stimulated locomotor activity behavioral assay and the inositol phosphate accumulation biochemical assay demonstrated that the latter has utility for assessing functional potency of M1 activators. Efficacy measured by inositol phosphate accumulation in mouse striatum compared favorably to efficacy in reversing amphetamine-induced locomotor activity, suggesting that the inositol phosphate accumulation assay has utility for the evaluation of M1 muscarinic acetylcholine receptor activators in vivo. The benefits of this in vivo biochemical approach include a wide response window, interrogation of specific brain circuit activation, an ability to model responses in the context of brain exposure, an ability to rank order compounds based on in vivo efficacy, and minimization of animal use.