Study of the ketohexokinase inhibitor PF-06835919 as a clinical cytochrome P450 3A inducer: Integrated use of oral midazolam and liquid biopsy.

PF-06835919, a ketohexokinase inhibitor, presented as an inducer of cytochrome P450 3A4 (CYP3A4) in vitro (human primary hepatocytes), and static mechanistic modeling exercises predicted significant induction in vivo (oral midazolam area under the plasma concentration-time curve [AUC] ratio [AUCR] = 0.23-0.79). Therefore, a drug-drug interaction study was conducted to evaluate the effect of multiple doses of PF-06835919 (300 mg once daily × 10 days; N = 10 healthy participants) on the pharmacokinetics of a single oral midazolam 7.5 mg dose. The adjusted geometric means for midazolam AUC and its maximal plasma concentration were similar following co-administration with PF-06835919 (vs. midazolam administration alone), with ratios of the adjusted geometric means (90% confidence interval [CI]) of 97.6% (90% CI: 79.9%-119%) and 98.9% (90% CI: 76.4%-128%), respectively, suggesting there was minimal effect of PF-06835919 on midazolam pharmacokinetics. Lack of CYP3A4 induction was confirmed after the preparation of subject plasma-derived small extracellular vesicles (sEVs) and conducting proteomic and activity (midazolam 1'-hydroxylase) analysis. Consistent with the midazolam AUCR observed, the CYP3A4 protein expression fold-induction (geometric mean, 90% CI) was low in liver (0.9, 90% CI: 0.7-1.2) and non-liver (0.9, 90% CI: 0.7-1.2) sEVs (predicted AUCR = 1.0, 90% CI: 0.9-1.2). Likewise, minimal induction of CYP3A4 activity (geometric mean, 90% CI) in both liver (1.1, 90% CI: 0.9-1.3) and non-liver (0.9, 90% CI: 0.5-1.5) sEVs was evident (predicted AUCR = 0.9, 90% CI: 0.6-1.4). The results showcase the integrated use of an oral CYP3A probe (midazolam) and plasma-derived sEVs to assess a drug candidate as inducer.

Discovery of the Potent and Selective MC4R Antagonist PF-07258669 for the Potential Treatment of Appetite Loss.

The melanocortin-4 receptor (MC4R) is a centrally expressed, class A GPCR that plays a key role in the regulation of appetite and food intake. Deficiencies in MC4R signaling result in hyperphagia and increased body mass in humans. Antagonism of MC4R signaling has the potential to mitigate decreased appetite and body weight loss in the setting of anorexia or cachexia due to underlying disease. Herein, we report on the identification of a series of orally bioavailable, small-molecule MC4R antagonists using a focused hit identification effort and the optimization of these antagonists to provide clinical candidate 23. Introduction of a spirocyclic conformational constraint allowed for simultaneous optimization of MC4R potency and ADME attributes while avoiding the production of hERG active metabolites observed in early series leads. Compound 23 is a potent and selective MC4R antagonist with robust efficacy in an aged rat model of cachexia and has progressed into clinical trials.

Industry Perspective on Therapeutic Peptide Drug-Drug Interaction Assessments During Drug Development: A European Federation of Pharmaceutical Industries and Associations White Paper.

Drug-drug interaction (DDI) assessments are well defined in health authority guidelines for small molecule drugs, and US Food and Drug Administration (FDA) draft guidance is now available for therapeutic proteins. However, there are currently no regulatory guidelines outlining DDI assessments for therapeutic peptides, which poses significant uncertainty and challenges during drug development for this heterogenous class of molecules. A cross-industry peptide DDI working group consisting of experts from 10 leading companies was formed under the sponsorship of the European Federation of Pharmaceutical Industries and Associations. We aimed to capture the range of DDI studies undertaken for peptide drugs by (i) anonymously surveying relevant companies involved in peptide drug development to better understand DDI study type/timing currently performed and (ii) compiling a database containing in vitro / clinical DDI data from submission packages for recently approved peptide drugs. Our analyses highlight significant gaps and uncertainty in the field. For example, the reported timing of in vitro peptide DDI studies, if performed, vary substantially across responding companies from early research to phase III. Nearly all in vitro cytochrome P450 / transporter inhibition studies reported in the survey were negative. For the few positive hits reported, no clinical follow-up studies were performed, questioning the clinical relevance of these findings. Furthermore, available submission packages reveal DDI likelihood is low for peptides >2 kDa, making it reasonable to adopt a risk-based approach during drug development for larger peptides. By benchmarking the landscape of peptide DDI activities across the industry, we set the stage for future discussions with health authorities on harmonizing peptide DDI approaches.

Transporter-Enzyme Interplay in the Pharmacokinetics of PF-06835919, A First-in-class Ketohexokinase Inhibitor for Metabolic Disorders and Non-alcoholic Fatty Liver Disease.

Excess dietary fructose consumption promotes metabolic dysfunction thereby increasing the risk of obesity, type 2 diabetes, non-alcoholic steatohepatitis (NASH), and related comorbidities. PF-06835919, a first-in-class ketohexokinase (KHK) inhibitor, showed reversal of such metabolic disorders in preclinical models and clinical studies, and is under clinical development for the potential treatment of NASH. In this study, we evaluated the transport and metabolic pathways of PF-06835919 disposition and assessed pharmacokinetics in preclinical models. PF-06835919 showed active uptake in cultured primary human hepatocytes, and substrate activity to organic anion transporter (OAT)2 and organic anion transporting-polypeptide (OATP)1B1 in transfected cells. "SLC-phenotyping" studies in human hepatocytes suggested contribution of passive uptake, OAT2- and OATP1B-mediated transport to the overall uptake to be about 15%, 60% and 25%, respectively. PF-06835919 showed low intrinsic metabolic clearance in vitro, and was found to be metabolized via both oxidative pathways (58%) and acyl glucuronidation (42%) by CYP3A, CYP2C8, CYP2C9 and UGT2B7. Following intravenous dosing, PF-06835919 showed low clearance (0.4-1.3 mL/min/kg) and volume of distribution (0.17-0.38 L/kg) in rat, dog and monkey. Human oral pharmacokinetics are predicted within 20% error when considering transporter-enzyme interplay in a PBPK model. Finally, unbound liver-to-plasma ratio (Kpuu) measured in vitro using rat, NHP and human hepatocytes was found to be approximately 4, 25 and 10, respectively. Similarly, liver Kpuu in rat and monkey following intravenous dosing of PF-06835919 was found to be 2.5 and 15, respectively, and notably higher than the muscle and brain Kpuu, consistent with the active uptake mechanisms observed in vitro. Significance Statement This work characterizes the transport/metabolic pathways in the hepatic disposition of PF-06835919, a first-in-class KHK inhibitor for the treatment of metabolic disorders and NASH. Phenotyping studies using transfected systems, human hepatocytes and liver microsomes signifies the role of OAT2 and OATP1B1 in the hepatic uptake and multiple enzymes in the metabolism of PF-06835919. Data presented suggest hepatic transporter-enzyme interplay in determining its systemic concentrations and potential enrichment in liver, a target site for KHK inhibition.

Target occupancy study and whole-body dosimetry with a MAGL PET ligand [11C]PF-06809247 in non-human primates.

BACKGROUND: Monoacylglycerol lipase (MAGL) is a key serine hydrolase which terminates endocannabinoid signaling and regulates arachidonic acid driven inflammatory responses within the central nervous system. To develop [11C]PF-06809247 into a clinically usable MAGL positron emission tomography (PET) radioligand, we assessed the occupancy of MAGL by an inhibitor in the non-human primate (NHP) brain. Additionally, we measured the whole-body distribution of [11C]PF-06809247 in NHP and estimated human effective radiation doses. METHODS: Seven cynomolgus monkeys were enrolled for brain PET measurements. Two PET measurements along with arterial blood sampling were performed in each NHP: one baseline and one pretreatment condition with intravenous administration of PF-06818883, a pro-drug of a selective MAGL inhibitor (total of seven doses between 0.01 and 1.27 mg/kg). Kinetic parameters K1, k2 and k3 were estimated by a two tissue compartment (2TC) model using metabolite corrected plasma radioactivity as the input function. k4 was set as 0 according to the irreversible binding of [11C]PF-06809247. Ki by 2TC and Patlak analysis were calculated as the influx constant. The target occupancy was calculated using Ki at baseline and pretreatment conditions. Two cynomolgus monkeys were enrolled for whole-body PET measurements. Estimates of the absorbed radiation dose in humans were calculated with OLINDA/EXM 1.1 using the adult male reference model. RESULTS: Radioactivity retention was decreased in all brain regions following pretreatment with PF-06818883. Occupancy was measured as 25.4-100.5% in a dose dependent manner. Whole-body PET showed high radioactivity uptake values in the liver, small intestine, kidney, and brain. The effective dose of [11C]PF-06809247 was calculated as 4.3 µSv/MBq. CONCLUSIONS: [11C]PF-06809247 is a promising PET ligand for further studies of MAGL in the human brain.

Effect of a Ketohexokinase Inhibitor (PF-06835919) on In Vivo OATP1B Activity: Integrative Risk Assessment Using Endogenous Biomarker and a Probe Drug.

PF-06835919 is a first-in-class ketohexokinase inhibitor (KHKi), recently under development for the treatment of metabolic and fatty liver diseases, which inhibited organic anion transporting polypeptide (OATP)1B1 in vitro and presented drug-drug interaction (DDI) risk. This study aims to investigate the dose-dependent effect of KHKi on OATP1B in vivo activity. We performed an open-label study comparing pharmacokinetics of atorvastatin (OATP1B probe) dosed alone (20 mg single dose) and coadministered with two dose strengths of KHKi (50 and 280 mg once daily) in 12 healthy participants. Additionally, changes in exposure of coproporphyrin-I (CP-I), an endogenous biomarker for OATP1B, were assessed in the atorvastatin study (1.12-fold and 1.49-fold increase in area under the plasma concentration-time profile (AUC) with once-daily 50 and 280 mg, respectively), and a separate single oral dose study of KHKi alone (100-600 mg, n = 6 healthy participants; up to a 1.80-fold increase in AUC). Geometric mean ratios (90% confidence interval) of atorvastatin AUC following 50 and 280 mg KHKi were 1.14 (1.00-1.30) and 1.54 (1.37-1.74), respectively. Physiologically-based pharmacokinetic modeling of CP-I plasma exposure following a single dose of KHKi predicted in vivo OATP1B inhibition from about 13% to 70% over the 100 to 600 mg dose range, while using the in vitro inhibition potency (1.9 µM). Model-based analysis correctly predicted "no-effect" (AUC ratio < 1.25) at the low dose range and "weak" effect (AUC ratio < 2) on atorvastatin pharmacokinetics at the high dose range of KHKi. This study exemplified the utility of biomarker-informed model-based approach in discerning even small effects on OATP1B activity in vivo, and to project DDI risk at the clinically relevant doses.

The Effects of a Novel Non-catechol Dopamine Partial Agonist on Working Memory in the Aged Rhesus Monkey.

Aged-related declines in cognition, especially working memory and executive function, begin in middle-age and these abilities are known to be mediated by the prefrontal cortex (PFC) and more specifically the dopamine (DA) system within the PFC. In both humans and monkeys, there is significant evidence that the PFC is the first cortical region to change with age and the PFC appears to be particularly vulnerable to age-related loss of dopamine (DA). Therefore, the DA system is a strong candidate for therapeutic intervention to slow or reverse age related declines in cognition. In the present study, we administered a novel selective, potent, non-catechol DA D1 R agonist PF-6294 (Pfizer, Inc.) to aged female rhesus monkeys and assessed their performance on two benchmark tasks of working memory - the Delayed Non-match to Sample Task (DNMS) and Delayed Recognition Span Task (DRST). The DNMS task was administered first with the standard 10 s delay and then with 5 min delays, with and without distractors. The DRST was administered each day with four trials with unique sequences and one trial of a repeated sequence to assess evidence learning and retention. Overall, there was no significant effect of drug on performance on any aspect of the DNMS task. In contrast, we demonstrated that a middle range dose of PF-6294 significantly increased memory span on the DRST on the first and last days of testing and by the last day of testing the increased memory span was driven by the performance on the repeated trials.

D1 Agonist Improved Movement of Parkinsonian Nonhuman Primates with Limited Dyskinesia Side Effects.

Parkinson's disease is a progressive neurodegenerative disease characterized by striatal dopaminergic loss. L-DOPA treatment replaces lost dopamine and enables motor function; however, eventually, fluctuating efficacy and side effects associated with its use become challenging for many patients. Here, we demonstrate, in a clinically translatable nonhuman primate model of parkinsonian motor symptoms, that treatment with the partial D1 receptor agonist CVL-751, formerly known as PF-06649751, is just as effective as L-DOPA in enabling movement and reducing disability. Importantly, CVL-751 efficacy is observed with less of the concomitant dyskinesia side effect associated with L-DOPA treatment. Data presented suggest that partial D1 agonists may be an effective and important treatment strategy for the management of Parkinson's patients.

Quantitative Translational Analysis of Brain Kynurenic Acid Modulation via Irreversible Kynurenine Aminotransferase II Inhibition.

Kynurenic acid (KYNA) plays a significant role in maintaining normal brain function, and abnormalities in KYNA levels have been associated with various central nervous system disorders. Confirmation of its causality in human diseases requires safe and effective modulation of central KYNA levels in the clinic. The kynurenine aminotransferases (KAT) II enzyme represents an attractive target for pharmacologic modulation of central KYNA levels; however, KAT II and KYNA turnover kinetics, which could contribute to the duration of pharmacologic effect, have not been reported. In this study, the kinetics of central KYNA-lowering effect in rats and nonhuman primates (NHPs, Cynomolgus macaques) was investigated using multiple KAT II irreversible inhibitors as pharmacologic probes. Mechanistic pharmacokinetic-pharmacodynamic analysis of in vivo responses to irreversible inhibition quantitatively revealed that 1) KAT II turnover is relatively slow [16-76 hours' half-life (t1/2)], whereas KYNA is cleared more rapidly from the brain (<1 hour t1/2) in both rats and NHPs, 2) KAT II turnover is slower in NHPs than in rats (76 hours vs. 16 hours t1/2, respectively), and 3) the percent contribution of KAT II to KYNA formation is constant (∼80%) across rats and NHPs. Additionally, modeling results enabled establishment of in vitro-in vivo correlation for both enzyme turnover rates and drug potencies. In summary, quantitative translational analysis confirmed the feasibility of central KYNA modulation in humans. Model-based analysis, where system-specific properties and drug-specific properties are mechanistically separated from in vivo responses, enabled quantitative understanding of the KAT II-KYNA pathway, as well as assisted development of promising candidates to test KYNA hypothesis in humans.

Discovery of Trifluoromethyl Glycol Carbamates as Potent and Selective Covalent Monoacylglycerol Lipase (MAGL) Inhibitors for Treatment of Neuroinflammation.

Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log  D, improving solubility, and minimizing chemical lability. Compound 15 achieves excellent CNS exposure, extended 2-AG elevation effect in vivo, and decreased brain inflammatory markers in response to an inflammatory challenge.

Azetidine and Piperidine Carbamates as Efficient, Covalent Inhibitors of Monoacylglycerol Lipase.

Monoacylglycerol lipase (MAGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the CNS. MAGL catalyzes the conversion of 2-AG to arachidonic acid (AA), a precursor to the proinflammatory eicosannoids such as prostaglandins. Herein we describe highly efficient MAGL inhibitors, identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of azetidine and piperidine-derived carbamates. The discovery and optimization of 3-substituted azetidine carbamate irreversible inhibitors of MAGL were aided by the generation of inhibitor-bound MAGL crystal structures. Compound 6, a highly efficient and selective MAGL inhibitor against recombinant enzyme and in a cellular context, was tested in vivo and shown to elevate central 2-AG levels at a 10 mg/kg dose.

Systematic N-methylation of oxytocin: Impact on pharmacology and intramolecular hydrogen bonding network.

Oxytocin (OT) is a peptide hormone agonist of the OT receptor (OTR) that plays an important role in social behaviors such as pair bonding, maternal bonding and trust. The pharmaceutical development of OT as an oral peptide therapeutic has been hindered historically by its unfavorable physicochemical properties, including molecular weight, polarity and number of hydrogen bond donors, which determines poor cell permeability. Here we describe the first systematic study of single and multiple N-methylations of OT and their effect on physicochemical properties as well as potency at the OT receptor. The agonist EC50 and percent effect for OTR are reported and show that most N-methylations are tolerated but with some loss in potency compared to OT. The effect of N-methylation on exposed polarity is assessed through the EPSA chromatographic method and the results validated against NMR temperature coefficient experiments and the determination of NMR solution structures. We found that backbone methylation of residues not involved in IMHB and removal of the N-terminal amine can significantly reduce the exposed polarity of peptides, and yet retain a significant OTR agonist activity. The results of this study also expose the potential challenge of using the N-methylation strategy for the OT system; while exposed polarity is reduced, in some cases backbone methylation produces a significant conformational change that compromises agonist activity. The data presented provides useful insights on the SAR of OT and suggests future design strategies that can be used to develop more permeable OTR agonists based on the OT framework.

Peripheral Administration of a Long-Acting Peptide Oxytocin Receptor Agonist Inhibits Fear-Induced Freezing.

Oxytocin (OT) modulates the expression of social and emotional behaviors and consequently has been proposed as a pharmacologic treatment of psychiatric diseases, including autism spectrum disorders and schizophrenia; however, endogenous OT has a short half-life in plasma and poor permeability across the blood-brain barrier. Recent efforts have focused on the development of novel drug delivery methods to enhance brain penetration, but few efforts have aimed at improving its half-life. To explore the behavioral efficacy of an OT analog with enhanced plasma stability, we developed PF-06655075 (PF1), a novel non-brain-penetrant OT receptor agonist with increased selectivity for the OT receptor and significantly increased pharmacokinetic stability. PF-06478939 was generated with only increased stability to disambiguate changes to selectivity versus stability. The efficacy of these compounds in evoking behavioral effects was tested in a conditioned fear paradigm. Both central and peripheral administration of PF1 inhibited freezing in response to a conditioned fear stimulus. Peripheral administration of PF1 resulted in a sustained level of plasma concentrations for greater than 20 hours but no detectable accumulation in brain tissue, suggesting that plasma or cerebrospinal fluid exposure was sufficient to evoke behavioral effects. Behavioral efficacy of peripherally administered OT receptor agonists on conditioned fear response opens the door to potential peripheral mechanisms in other behavioral paradigms, whether they are mediated by direct peripheral activation or feed-forward responses. Compound PF1 is freely available as a tool compound to further explore the role of peripheral OT in behavioral response.

Reduction of brain kynurenic acid improves cognitive function.

The elevation of kynurenic acid (KYNA) observed in schizophrenic patients may contribute to core symptoms arising from glutamate hypofunction, including cognitive impairments. Although increased KYNA levels reduce excitatory neurotransmission, KYNA has been proposed to act as an endogenous antagonist at the glycine site of the glutamate NMDA receptor (NMDAR) and as a negative allosteric modulator at the α7 nicotinic acetylcholine receptor. Levels of KYNA are elevated in CSF and the postmortem brain of schizophrenia patients, and these elevated levels of KYNA could contribute to NMDAR hypofunction and the cognitive deficits and negative symptoms associated with this disease. However, the impact of endogenously produced KYNA on brain function and behavior is less well understood due to a paucity of pharmacological tools. To address this issue, we identified PF-04859989, a brain-penetrable inhibitor of kynurenine aminotransferase II (KAT II), the enzyme responsible for most brain KYNA synthesis. In rats, systemic administration of PF-04859989 dose-dependently reduced brain KYNA to as little as 28% of basal levels, and prevented amphetamine- and ketamine-induced disruption of auditory gating and improved performance in a sustained attention task. It also prevented ketamine-induced disruption of performance in a working memory task and a spatial memory task in rodents and nonhuman primates, respectively. Together, these findings support the hypotheses that endogenous KYNA impacts cognitive function and that inhibition of KAT II, and consequent lowering of endogenous brain KYNA levels, improves cognitive performance under conditions considered relevant for schizophrenia.

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.

Structure-Based Design of Irreversible Human KAT II Inhibitors: Discovery of New Potency-Enhancing Interactions.

A series of aryl hydroxamates recently have been disclosed as irreversible inhibitors of kynurenine amino transferase II (KAT II), an enzyme that may play a role in schizophrenia and other psychiatric and neurological disorders. The utilization of structure-activity relationships (SAR) in conjunction with X-ray crystallography led to the discovery of hydroxamate 4, a disubstituted analogue that has a significant potency enhancement due to a novel interaction with KAT II. The use of k inact/K i to assess potency was critical for understanding the SAR in this series and for identifying compounds with improved pharmacodynamic profiles.

Application of structure-based drug design and parallel chemistry to identify selective, brain penetrant, in vivo active phosphodiesterase 9A inhibitors.

Phosphodiesterase 9A inhibitors have shown activity in preclinical models of cognition with potential application as novel therapies for treating Alzheimer's disease. Our clinical candidate, PF-04447943 (2), demonstrated acceptable CNS permeability in rats with modest asymmetry between central and peripheral compartments (free brain/free plasma = 0.32; CSF/free plasma = 0.19) yet had physicochemical properties outside the range associated with traditional CNS drugs. To address the potential risk of restricted CNS penetration with 2 in human clinical trials, we sought to identify a preclinical candidate with no asymmetry in rat brain penetration and that could advance into development. Merging the medicinal chemistry strategies of structure-based design with parallel chemistry, a novel series of PDE9A inhibitors was identified that showed improved selectivity over PDE1C. Optimization afforded preclinical candidate 19 that demonstrated free brain/free plasma ≥ 1 in rat and reduced microsomal clearance along with the ability to increase cyclic guanosine monophosphosphate levels in rat CSF.

Design and discovery of 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943), a selective brain penetrant PDE9A inhibitor for the treatment of cognitive disorders.

6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943) is a novel PDE9A inhibitor identified using parallel synthetic chemistry and structure-based drug design (SBDD) and has advanced into clinical trials. Selectivity for PDE9A over other PDE family members was achieved by targeting key residue differences between the PDE9A and PDE1C catalytic site. The physicochemical properties of the series were optimized to provide excellent in vitro and in vivo pharmacokinetics properties in multiple species including humans. It has been reported to elevate central cGMP levels in the brain and CSF of rodents. In addition, it exhibits procognitive activity in several rodent models and synaptic stabilization in an amyloid precursor protein (APP) transgenic mouse model. Recent disclosures from clinical trials confirm that it is well tolerated in humans and elevates cGMP in cerebral spinal fluid of healthy volunteers, confirming that it is a quality pharmacological tool for testing clinical hypotheses in disease states associated with impairment of cGMP signaling or cognition.

Negative allosteric modulation of the mGluR5 receptor reduces repetitive behaviors and rescues social deficits in mouse models of autism.

Neurodevelopmental disorders such as autism and fragile X syndrome were long thought to be medically untreatable, on the assumption that brain dysfunctions were immutably hardwired before diagnosis. Recent revelations that many cases of autism are caused by mutations in genes that control the ongoing formation and maturation of synapses have challenged this dogma. Antagonists of metabotropic glutamate receptor subtype 5 (mGluR5), which modulate excitatory neurotransmission, are in clinical trials for fragile X syndrome, a major genetic cause of intellectual disabilities. About 30% of patients with fragile X syndrome meet the diagnostic criteria for autism. Reasoning by analogy, we considered the mGluR5 receptor as a potential target for intervention in autism. We used BTBR T+tf/J (BTBR) mice, an established model with robust behavioral phenotypes relevant to the three diagnostic behavioral symptoms of autism--unusual social interactions, impaired communication, and repetitive behaviors--to probe the efficacy of a selective negative allosteric modulator of the mGluR5 receptor, GRN-529. GRN-529 reduced repetitive behaviors in three cohorts of BTBR mice at doses that did not induce sedation in control assays of open field locomotion. In addition, the same nonsedating doses reduced the spontaneous stereotyped jumping that characterizes a second inbred strain of mice, C58/J. Further, GRN-529 partially reversed the striking lack of sociability in BTBR mice on some parameters of social approach and reciprocal social interactions. These findings raise the possibility that a single targeted pharmacological intervention may alleviate multiple diagnostic behavioral symptoms of autism.

Phosphodiesterase 9A regulates central cGMP and modulates responses to cholinergic and monoaminergic perturbation in vivo.

Cyclic nucleotides are critical regulators of synaptic plasticity and participate in requisite signaling cascades implicated across multiple neurotransmitter systems. Phosphodiesterase 9A (PDE9A) is a high-affinity, cGMP-specific enzyme widely expressed in the rodent central nervous system. In the current study, we observed neuronal staining with antibodies raised against PDE9A protein in human cortex, cerebellum, and subiculum. We have also developed several potent, selective, and brain-penetrant PDE9A inhibitors and used them to probe the function of PDE9A in vivo. Administration of these compounds to animals led to dose-dependent accumulation of cGMP in brain tissue and cerebrospinal fluid, producing a range of biological effects that implied functional significance for PDE9A-regulated cGMP in dopaminergic, cholinergic, and serotonergic neurotransmission and were consistent with the widespread distribution of PDE9A. In vivo effects of PDE9A inhibition included reversal of the respective disruptions of working memory by ketamine, episodic and spatial memory by scopolamine, and auditory gating by amphetamine, as well as potentiation of risperidone-induced improvements in sensorimotor gating and reversal of the stereotypic scratching response to the hallucinogenic 5-hydroxytryptamine 2A agonist mescaline. The results suggested a role for PDE9A in the regulation of monoaminergic circuitry associated with sensory processing and memory. Thus, PDE9A activity regulates neuronal cGMP signaling downstream of multiple neurotransmitter systems, and inhibition of PDE9A may provide therapeutic benefits in psychiatric and neurodegenerative diseases promoted by the dysfunction of these diverse neurotransmitter systems.