Analgesic potential of PF-06372865, an α2/α3/α5 subtype-selective GABAA partial agonist, in humans.

BACKGROUND: This study investigated the analgesic effects of two doses (15 and 65 mg) of PF-06372865, a novel α2/α3/α5 gamma-aminobutyric acid A (GABAA) subunit selective partial positive allosteric modulator (PAM), compared with placebo and pregabalin (300 mg) as a positive control. METHODS: We performed a randomised placebo-controlled crossover study (NCT02238717) in 20 healthy subjects, using a battery of pain tasks (electrical, pressure, heat, cold and inflammatory pain, including a paradigm of conditioned pain modulation). Pharmacodynamic measurements were performed at baseline and up to 10 h after dose. RESULTS: A dose of 15 mg PF-06372865 increased pain tolerance thresholds (PTTs) for pressure pain at a ratio of 1.11 (90% confidence interval [CI]: 1.02, 1.22) compared with placebo. A dose of 65 mg PF-06372865 led to an increase in PTT for the cold pressor at a ratio of 1.17 (90% CI: 1.03, 1.32), and pressure pain task: 1.11 (90% CI: 1.01, 1.21). Pregabalin showed an increase in PTT for pressure pain at a ratio of 1.15 (95% CI: 1.06, 1.26) and cold pressor task: 1.31 (90% CI: 1.16, 1.48). CONCLUSION: We conclude that PF-06372865 has analgesic potential at doses that do not induce significant sedation or other intolerable adverse events limiting its clinical use. In addition, the present study established the potential role for this battery of pain tasks as a tool in the development of analgesics with a novel mechanism of action, for the treatment of various pain states including neuropathic pain and to establish proof-of-concept. CLINICAL TRIALS REGISTRATION: NCT0223871.

Demonstration of an anti-hyperalgesic effect of a novel pan-Trk inhibitor PF-06273340 in a battery of human evoked pain models.

AIM: Inhibitors of nerve growth factor (NGF) reduce pain in several chronic pain indications. NGF signals through tyrosine kinase receptors of the tropomyosin-related kinase (Trk) family and the unrelated p75 receptor. PF-06273340 is a small molecule inhibitor of Trks A, B and C that reduces pain in nonclinical models, and the present study aimed to investigate the pharmacodynamics of this first-in-class molecule in humans. METHODS: A randomized, double-blind, single-dose, placebo- and active-controlled five-period crossover study was conducted in healthy human subjects (NCT02260947). Subjects received five treatments: PF-06273340 50 mg, PF-06273340 400 mg, pregabalin 300 mg, ibuprofen 600 mg and placebo. The five primary endpoints were the pain detection threshold for the thermal pain tests and the pain tolerance threshold for the cold pressor, electrical stair and pressure pain tests. The trial had predefined decision rules based on 95% confidence that the PF-06273340 effect was better than that of placebo. RESULTS: Twenty subjects entered the study, with 18 completing all five periods. The high dose of PF-06273340 met the decision rules on the ultraviolet (UV) B skin thermal pain endpoint [least squares (LS) mean vs. placebo: 1.13, 95% confidence interval: 0.64-1.61], but not on the other four primary endpoints. The low dose did not meet the decision criteria for any of the five primary endpoints. Pregabalin (cold pressor and electrical stair tests) and ibuprofen (UVB thermal pain) showed significant analgesic effects on expected endpoints. CONCLUSIONS: The study demonstrated, for the first time, the translation of nonclinical effects into man in an inflammatory pain analgesic pharmacodynamic endpoint using a pan-Trk inhibitor.

The use of a battery of pain models to detect analgesic properties of compounds: a two-part four-way crossover study.

AIM: The aim was to investigate the ability of a battery of pain models to detect analgesic properties of commonly used analgesics in healthy subjects. METHODS: The battery consisted of tests eliciting electrical, mechanical and thermal (contact heat and cold pressor)-pain and included a UVB model, the thermal grill illusion and a paradigm of conditioned pain modulation. Subjects were administered fentanyl 3 µg kg-1 , phenytoin 300 mg, (S)-ketamine 10 mg and placebo (part I), or imipramine 100 mg, pregabalin 300 mg, ibuprofen 600 mg and placebo (part II). Pain measurements were performed at baseline and up to 10 h post-dose. Endpoints were analysed using a mixed model analysis of variance. RESULTS: Sixteen subjects (8 female) completed each part. The pain tolerance threshold (PTT) for electrical stimulation was increased (all P < 0.05) compared to placebo for (S)-ketamine (+10.1%), phenytoin (+8.5%) and pregabalin (+10.8%). The PTT for mechanical pain was increased by pregabalin (+14.1%). The cold pressor PTT was increased by fentanyl (+17.1%) and pregabalin (+46.4%). Normal skin heat pain detection threshold was increased by (S)-ketamine (+3.3%), fentanyl (+2.8%) and pregabalin (+4.1%). UVB treated skin pain detection threshold was increased by fentanyl (+2.6%) and ibuprofen (+4.0%). No differences in conditioned pain modulation were observed. CONCLUSION: This study shows that these pain models are able to detect changes in pain thresholds after administration of different classes of analgesics in healthy subjects. The analgesic compounds all showed a unique profile in their effects on the pain tasks administered.

Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels.

Voltage-gated sodium (Nav) channels play a fundamental role in the generation and propagation of electrical impulses in excitable cells. Here we describe two unique structurally related nanomolar potent small molecule Nav channel inhibitors that exhibit up to 1,000-fold selectivity for human Nav1.3/Nav1.1 (ICA-121431, IC50, 19 nM) or Nav1.7 (PF-04856264, IC50, 28 nM) vs. other TTX-sensitive or resistant (i.e., Nav1.5) sodium channels. Using both chimeras and single point mutations, we demonstrate that this unique class of sodium channel inhibitor interacts with the S1-S4 voltage sensor segment of homologous Domain 4. Amino acid residues in the "extracellular" facing regions of the S2 and S3 transmembrane segments of Nav1.3 and Nav1.7 seem to be major determinants of Nav subtype selectivity and to confer differences in species sensitivity to these inhibitors. The unique interaction region on the Domain 4 voltage sensor segment is distinct from the structural domains forming the channel pore, as well as previously characterized interaction sites for other small molecule inhibitors, including local anesthetics and TTX. However, this interaction region does include at least one amino acid residue [E1559 (Nav1.3)/D1586 (Nav1.7)] that is important for Site 3 α-scorpion and anemone polypeptide toxin modulators of Nav channel inactivation. The present study provides a potential framework for identifying subtype selective small molecule sodium channel inhibitors targeting interaction sites away from the pore region.

Reproducibility of a battery of human evoked pain models to detect pharmacological effects of analgesic drugs.

BACKGROUND: Although reproducibility is considered essential for any method used in scientific research, it is investigated only rarely; thus, strikingly little has been published regarding the reproducibility of evoked pain models involving human subjects. Here, we studied the reproducibility of a battery of evoked pain models for demonstrating the analgesic effects of two analgesic compounds. METHODS: A total of 81 healthy subjects participated in four studies involving a battery of evoked pain tests in which mechanical, thermal and electrical stimuli were used to measure pain detection and tolerance thresholds. Pharmacodynamic outcome variables were analysed using a mixed model analysis of variance, and a coefficient of variation was calculated by dividing the standard deviation by the least squares means. RESULTS: A total of 76 subjects completed the studies. After being administered pregabalin, the subjects' pain tolerance thresholds in the cold pressor and pressure stimulation tests were significantly increased compared to the placebo group. Moreover, the heat pain detection threshold in UVB-irradiated skin was significantly increased in subjects who were administered ibuprofen compared to the placebo group. Variation among all evoked pain tests ranged from 2.2% to 30.6%. CONCLUSIONS: Four studies using a similar design showed reproducibility with respect to the included evoked pain models. The relatively high consistency and reproducibility of two analgesics at doses known to be effective in treating clinically relevant pain supports the validity of using this pain test battery to investigate the analgesic activity and determine the active dosage of putative analgesic compounds in early clinical development. SIGNIFICANCE: The consistency and reproducibility of measuring the profile of an analgesic at clinically relevant doses illustrates that this pain test battery is a valid tool for demonstrating the analgesic activity of a test compound and for determining the optimal active dose in early clinical drug development.

Photosensitive epilepsy: Robust clinical efficacy of a selective GABA potentiator.

OBJECTIVE: The objective of this phase 2a study was to assess the activity of PF-06372865, a positive allosteric modulator (PAM) of α2/3/5 subunit-containing GABAA receptors with minimal activity at α1-containing receptors, which are believed to mediate many of the adverse events associated with benzodiazepines, in the epilepsy photosensitivity model as a proof-of-principle of efficacy. METHODS: Seven participants with a photoparoxysmal response to intermittent photic stimulation (IPS) at baseline were randomized in a double-blind, 4-period cross-over study examining single doses of 17.5 and 52.5 mg PF-06372865, 2 mg lorazepam (active control), and placebo. Standardized photosensitivity ranges (SPRs) to IPS were recorded at screening, predose, and 1, 2, 4, and 6 hours postdose. The primary endpoint was the average least squares mean change in the SPR in the participant's most sensitive eye condition, across all time points. RESULTS: Both doses of PF-06372865 produced a marked and statistically significant mean reduction in SPR compared to placebo, which was similar in degree to lorazepam. There was complete suppression of SPR in 6/7 participants following PF-06372865 or lorazepam administration. PF-06372865 was safe and well-tolerated. CONCLUSION: PF-06372865 demonstrated highly robust efficacy. This demonstrates anticonvulsant activity of a novel α2/3/5-subtype selective GABAA PAM in humans. Further study of the antiepileptic properties of PF-06372865 is warranted. CLINICALTRIALSGOV IDENTIFIER: NCT02564029. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that for people with a stable photoparoxysmal response to intermittent photic stimulation, PF-06372865 reduces the SPR.

A randomised, placebo-controlled clinical trial with the α2/3/5 subunit selective GABAA positive allosteric modulator PF-06372865 in patients with chronic low back pain.

The effect of PF-06372865, a subtype-selective positive allosteric modulator of the γ-aminobutyric acid type A (GABAA) receptor, on chronic low back pain was investigated in a randomised, placebo- and active-controlled phase 2 clinical trial. The parallel treatment group trial consisted of a 1-week single-blind placebo run in the phase, followed by 4-week double-blind treatment. Patients were randomised to receive either PF-06372865, naproxen, or placebo twice a day for 4 weeks. The primary end point was the numerical rating score of low back pain intensity after 4 weeks of active treatment. Secondary end points included the Roland Morris Disability Questionnaire and the Hopkins Verbal Learning Test-Revised. The trial had predefined decision rules based on the probability that PF-06372865 was better than placebo. The study was stopped at the interim analysis for futility. At this time, a total of 222 patients were randomised and the mean PF-06372865 4-week response on the low back pain intensity was 0.16 units higher (worse) than placebo (90% confidence interval -0.28 to 0.60). There were small, statistically significant reductions in the delayed recall test score with PF-06372865, as measured by Hopkins Verbal Learning Test-Revised. The effects of naproxen were in line with expectations. PF-06372865 was well tolerated. The most common treatment-related adverse events in the PF-06372865 arm were somnolence (5 mild and 4 moderate), dizziness (2 mild and 3 moderate), and nausea (2 mild). Although the reason for the lack of analgesic effect is not completely clear, it may be a result of not achieving sufficient receptor occupancy to drive efficacy.

Efficacy of the Nav1.7 blocker PF-05089771 in a randomised, placebo-controlled, double-blind clinical study in subjects with painful diabetic peripheral neuropathy.

The effect of PF-05089771, a selective, peripherally restricted Nav1.7 sodium channel blocker on pain due to diabetic peripheral neuropathy was investigated in a randomised, placebo and active-controlled parallel group clinical trial (NCT02215252). A 1-week placebo-run in the period was followed by a 4-week treatment period and a 1-week placebo run-out/taper-down period. Single-blind placebo was administered throughout run-in and run-out periods. Subjects were randomised to receive either PF-05089771 150 mg twice daily, pregabalin 150 mg twice daily, or placebo during the 4-week treatment period. One hundred thirty-five subjects were randomised. The primary endpoint was the average pain score derived from subjects' Numerical Rating Scale scores over the past 7 days of week 4 of the double-blind treatment period. Predefined efficacy criteria for the trial were the effect of PF-05089771 being >0.5 units better than placebo at interim analysis after completion of the first part of the study. Although a trend for a reduction in the weekly average pain score in the PF-05089771 treatment group was observed, this was not statistically significant when compared with placebo at week 4, with a mean posterior difference of -0.41 (90% credible interval: -1.00 to 0.17). The effect of PF-05089771 was smaller than that seen with pregabalin, which was statistically significant when compared with placebo at week 4, with a mean posterior difference of -0.53 (90% credible interval: -0.91 to -0.20). As predefined efficacy criteria were not met, the study did not proceed to the second part. PF-05089771 was well tolerated. Possible reasons for the modest efficacy observed with PF-05089771 are discussed.

Pharmacology in translation: the preclinical and early clinical profile of the novel α2/3 functionally selective GABAA receptor positive allosteric modulator PF-06372865.

BACKGROUND AND PURPOSE: Benzodiazepines, non-selective positive allosteric modulators (PAMs) of GABAA receptors, have significant side effects that limit their clinical utility. As many of these side effects are mediated by the α1 subunit, there has been a concerted effort to develop α2/3 subtype-selective PAMs. EXPERIMENTAL APPROACH: In vitro screening assays were used to identify molecules with functional selectivity for receptors containing α2/3 subunits over those containing α1 subunits. In vivo receptor occupancy (RO) was conducted, prior to confirmation of in vivo α2/3 and α1 pharmacology through quantitative EEG (qEEG) beta frequency and zolpidem drug discrimination in rats respectively. PF-06372865 was then progressed to Phase 1 clinical trials. KEY RESULTS: PF-06372865 exhibited functional selectivity for those receptors containing α2/3/5 subunits, with significant positive allosteric modulation (90-140%) but negligible activity (≤20%) at GABAA receptors containing α1 subunits. PF-06372865 exhibited concentration-dependent occupancy of GABAA receptors in preclinical species. There was an occupancy-dependent increase in qEEG beta frequency and no generalization to a GABAA α1 cue in the drug-discrimination assay, clearly demonstrating the lack of modulation at the GABAA receptors containing an α1 subtype. In a Phase 1 single ascending dose study in healthy volunteers, evaluation of the pharmacodynamics of PF-06372865 demonstrated a robust increase in saccadic peak velocity (a marker of α2/3 pharmacology), increases in beta frequency qEEG and a slight saturating increase in body sway. CONCLUSIONS AND IMPLICATIONS: PF-06372865 has a unique clinical pharmacology profile and a highly predictive translational data package from preclinical species to the clinical setting.

Discovery of Clinical Candidate 4-[2-(5-Amino-1H-pyrazol-4-yl)-4-chlorophenoxy]-5-chloro-2-fluoro-N-1,3-thiazol-4-ylbenzenesulfonamide (PF-05089771): Design and Optimization of Diaryl Ether Aryl Sulfonamides as Selective Inhibitors of NaV1.7.

A series of acidic diaryl ether heterocyclic sulfonamides that are potent and subtype selective NaV1.7 inhibitors is described. Optimization of early lead matter focused on removal of structural alerts, improving metabolic stability and reducing cytochrome P450 inhibition driven drug-drug interaction concerns to deliver the desired balance of preclinical in vitro properties. Concerns over nonmetabolic routes of clearance, variable clearance in preclinical species, and subsequent low confidence human pharmacokinetic predictions led to the decision to conduct a human microdose study to determine clinical pharmacokinetics. The design strategies and results from preclinical PK and clinical human microdose PK data are described leading to the discovery of the first subtype selective NaV1.7 inhibitor clinical candidate PF-05089771 (34) which binds to a site in the voltage sensing domain.

Clinical Micro-Dose Studies to Explore the Human Pharmacokinetics of Four Selective Inhibitors of Human Nav1.7 Voltage-Dependent Sodium Channels.

BACKGROUND: The emergence of genetic data linking Nav1.7 sodium channel over- and under- expression to human pain signalling has led to an interest in the treatment of chronic pain through inhibition of Nav1.7 channels. OBJECTIVE: We describe the pharmacokinetic (PK) results of a clinical microdose study performed with four potent and selective Nav1.7 inhibitors and the subsequent modelling resulting in the selection of a single compound to explore Nav1.7 pharmacology at higher doses. METHODS: A clinical microdose study to investigate the intravenous and oral PK of four compounds (PF-05089771, PF-05150122, PF-05186462 and PF-05241328) was performed in healthy volunteers. PK parameters were derived via noncompartmental analysis. A physiologically-based PK (PBPK) model was used to predict exposure and multiples of Nav1.7 50 % inhibitory concentration (IC50) for each compound at higher doses. RESULTS: Plasma clearance, volume of distribution and bioavailability ranged from 45 to 392 mL/min/kg, 13 to 36 L/kg and 38 to 110 %, respectively. The PBPK model for PF-05089771 predicted a 1 g oral dose would be required to achieve exposures of approximately 12× Nav1.7 IC50 at maximum concentration (C max), and approximately 3× IC50 after 12 h (minimum concentration [C min] for a twice-daily regimen). Lower multiples of Nav1.7 IC50 were predicted with the same oral doses of PF-05150122, PF-05186462, and PF-05241328. In a subsequent single ascending oral dose clinical study, the predictions for PF-05089771 compared well with observed data. CONCLUSION: Based on the human PK data obtained from the microdose study and subsequent modelling, PF-05089771 provided the best opportunity to explore Nav1.7 blockade for the treatment of acute or chronic pain conditions.

Subtype-Selective Small Molecule Inhibitors Reveal a Fundamental Role for Nav1.7 in Nociceptor Electrogenesis, Axonal Conduction and Presynaptic Release.

Human genetic studies show that the voltage gated sodium channel 1.7 (Nav1.7) is a key molecular determinant of pain sensation. However, defining the Nav1.7 contribution to nociceptive signalling has been hampered by a lack of selective inhibitors. Here we report two potent and selective arylsulfonamide Nav1.7 inhibitors; PF-05198007 and PF-05089771, which we have used to directly interrogate Nav1.7's role in nociceptor physiology. We report that Nav1.7 is the predominant functional TTX-sensitive Nav in mouse and human nociceptors and contributes to the initiation and the upstroke phase of the nociceptor action potential. Moreover, we confirm a role for Nav1.7 in influencing synaptic transmission in the dorsal horn of the spinal cord as well as peripheral neuropeptide release in the skin. These findings demonstrate multiple contributions of Nav1.7 to nociceptor signalling and shed new light on the relative functional contribution of this channel to peripheral and central noxious signal transmission.

Oral administration of PF-01247324, a subtype-selective Nav1.8 blocker, reverses cerebellar deficits in a mouse model of multiple sclerosis.

Cerebellar symptoms significantly diminish quality of life in patients with multiple sclerosis (MS). We previously showed that sodium channel Nav1.8, although normally restricted to peripheral somatosensory neurons, is upregulated in the cerebellum in MS, and that Nav1.8 expression is linked to ataxia and MS-like symptoms in mice. Furthermore, intracerebroventricular administration of the Nav1.8 blocker A-803467 temporarily reversed electrophysiological and behavioral manifestations of disease in a mouse MS model; unfortunately A-803467 is not orally bioavailable, diminishing the potential for translation to human patients. In the present study, we assessed the effect of per os (p.o.) dosing of a new orally bioavailable Nav1.8-selective blocker, PF-01247324, in transgenic mice expressing Nav1.8 in Purkinje neurons, and in wildtype mice in the experimental autoimmune encephalomyelitis (EAE) model. PF-01247324 was administered by oral gavage at 1000 mg/kg; control groups received an equal volume of vehicle. Behavioral assays of motor coordination, grip strength, and ataxia were performed. We observed significant improvements in motor coordination and cerebellar-like symptoms in mice that received PF-01247324 compared to control littermates that received vehicle. These preclinical proof-of-concept data suggest that PF-01247324, its derivatives, or other Nav1.8-selective blockers merit further study for providing symptomatic therapy for cerebellar dysfunction in MS and related disorders.

A novel selective and orally bioavailable Nav 1.8 channel blocker, PF-01247324, attenuates nociception and sensory neuron excitability.

BACKGROUND AND PURPOSE: NaV 1.8 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 Nav 1.8 channel blocker of novel chemotype. EXPERIMENTAL APPROACH: The inhibition of Nav 1.8 channels by PF-01247324 was studied using in vitro patch-clamp electrophysiology and the oral bioavailability and antinociceptive effects demonstrated using in vivo rodent models of inflammatory and neuropathic pain. KEY RESULTS: PF-01247324 inhibited native tetrodotoxin-resistant (TTX-R) currents in human dorsal root ganglion (DRG) neurons (IC50 : 331 nM) and in recombinantly expressed h Nav 1.8 channels (IC50 : 196 nM), with 50-fold selectivity over recombinantly expressed TTX-R hNav 1.5 channels (IC50 : ∼10 µM) and 65-100-fold selectivity over TTX-sensitive (TTX-S) channels (IC50 : ∼10-18 µM). Native TTX-R currents in small-diameter rodent DRG neurons were inhibited with an IC50 448 nM, and the block of both human recombinant Nav 1.8 channels and TTX-R from rat DRG neurons was both frequency and state dependent. In vitro current clamp showed that PF-01247324 reduced excitability in both rat and human DRG neurons and also altered the waveform of the action potential. In vivo experiments n rodents demonstrated efficacy in both inflammatory and neuropathic pain models. CONCLUSIONS AND IMPLICATIONS: Using PF-01247324, we have confirmed a role for Nav 1.8 channels in both inflammatory and neuropathic pain. We have also demonstrated a key role for Nav 1.8 channels in action potential upstroke and repetitive firing of rat and human DRG neurons.

Estimation of binding rate constants using a simultaneous mixed-effects method: application to monoamine transporter reuptake inhibitor reboxetine.

BACKGROUND AND PURPOSE: Reboxetine is a clinically used antidepressant and is a racemic mixture of two enantiomers, SS- and RR-reboxetine. The aim of the work described in this manuscript was to determine the kinetics of binding of the RR- and SS-reboxetine to the human noradrenaline transporter (hNET). EXPERIMENTAL APPROACH: We have applied a simultaneous mixed-effects method to the analysis of the transient kinetics of binding of SS-, RR- and racemic reboxetine to hNET. This method allowed simultaneous modelling of multiple datasets, taking into account inter-experiment variability, thereby facilitating robust parameter estimation and minimizing the assumptions made. KEY RESULTS: The mixed-effects method proved simple and robust. SS-reboxetine bound to hNET according to a one-step binding model with the SS-enantiomer having 130-fold higher steady state affinity than the RR-enantiomer (K(d)= 0.076 +/- 0.009 nM vs. 9.7 +/- 0.8 nM respectively). The k(on) for SS-reboxetine was c. 1.4 x 10(5) M(-1).s(-1) and k(off) 1.05 x 10(-5) s(-1) (t(1/2) approximately 18 h). The k(on) for RR-reboxetine was c. 4.3 x 10(5) M(-1).s(-1) and k(off) 4.2 x 10(-3) s(-1) (t(1/2) approximately 3 min). The racemate behaved as expected for an equimolar mixture of RR- and SS-reboxetine, assuming mutually exclusive binding. CONCLUSIONS AND IMPLICATIONS: These data will be useful for the interpretation of the behaviour of reboxetine and its enantiomers in man and the method used could be applied to other candidate drugs.