Association of respiratory failure with inhibition of NaV1.6 in the phrenic nerve.

As part of a drug discovery effort to identify potent inhibitors of NaV1.7 for the treatment of pain, we observed that inhibitors produced unexpected cardiovascular and respiratory effects in vivo. Specifically, inhibitors administered to rodents produced changes in cardiovascular parameters and respiratory cessation. We sought to determine the mechanism of the in vivo adverse effects by studying the selectivity of the compounds on NaV1.5, NaV1.4, and NaV1.6 in in vitro and ex vivo assays. Inhibitors lacking sufficient NaV1.7 selectivity over NaV1.6 were associated with respiratory cessation after in vivo administration to rodents. Effects on respiratory rate in rats were consistent with effects in an ex vivo hemisected rat diaphragm model and in vitro NaV1.6 potency. Furthermore, direct blockade of the phrenic nerve signaling was observed at exposures known to cause respiratory cessation in rats. Collectively, these results support a significant role for NaV1.6 in phrenic nerve signaling and respiratory function.

Discovery of Arylsulfonamide Nav1.7 Inhibitors: IVIVC, MPO Methods, and Optimization of Selectivity Profile.

The voltage-gated sodium channel Nav1.7 continues to be a high-profile target for the treatment of various pain afflictions due to its strong human genetic validation. While isoform selective molecules have been discovered and advanced into the clinic, to date, this target has yet to bear fruit in the form of marketed therapeutics for the treatment of pain. Lead optimization efforts over the past decade have focused on selectivity over Nav1.5 due to its link to cardiac side effects as well as the translation of preclinical efficacy to man. Inhibition of Nav1.6 was recently reported to yield potential respiratory side effects preclinically, and this finding necessitated a modified target selectivity profile. Herein, we report the continued optimization of a novel series of arylsulfonamide Nav1.7 inhibitors to afford improved selectivity over Nav1.6 while maintaining rodent oral bioavailability through the use of a novel multiparameter optimization (MPO) paradigm. We also report in vitro-in vivo correlations from Nav1.7 electrophysiology protocols to preclinical models of efficacy to assist in projecting clinical doses. These efforts produced inhibitors such as compound 19 with potency against Nav1.7, selectivity over Nav1.5 and Nav1.6, and efficacy in behavioral models of pain in rodents as well as inhibition of rhesus olfactory response indicative of target modulation.

Nav1.7 target modulation and efficacy can be measured in nonhuman primate assays.

Humans with loss-of-function mutations in the Nav1.7 channel gene (SCN9A) show profound insensitivity to pain, whereas those with gain-of-function mutations can have inherited pain syndromes. Therefore, inhibition of the Nav1.7 channel with a small molecule has been considered a promising approach for the treatment of various human pain conditions. To date, clinical studies conducted using selective Nav1.7 inhibitors have not provided analgesic efficacy sufficient to warrant further investment. Clinical studies to date used multiples of in vitro IC50 values derived from electrophysiological studies to calculate anticipated human doses. To increase the chance of clinical success, we developed rhesus macaque models of action potential propagation, nociception, and olfaction, to measure Nav1.7 target modulation in vivo. The potent and selective Nav1.7 inhibitors SSCI-1 and SSCI-2 dose-dependently blocked C-fiber nociceptor conduction in microneurography studies and inhibited withdrawal responses to noxious heat in rhesus monkeys. Pharmacological Nav1.7 inhibition also reduced odor-induced activation of the olfactory bulb (OB), measured by functional magnetic resonance imaging (fMRI) studies consistent with the anosmia reported in Nav1.7 loss-of-function patients. These data demonstrate that it is possible to measure Nav1.7 target modulation in rhesus macaques and determine the plasma concentration required to produce a predetermined level of inhibition. The calculated plasma concentration for preclinical efficacy could be used to guide human efficacious exposure estimates. Given the translatable nature of the assays used, it is anticipated that they can be also used in phase 1 clinical studies to measure target modulation and aid in the interpretation of phase 1 clinical data.

Translational Pharmacokinetic-Pharmacodynamic Modeling of NaV1.7 Inhibitor MK-2075 to Inform Human Efficacious Dose.

MK-2075 is a small-molecule selective inhibitor of the NaV1.7 channel investigated for the treatment of postoperative pain. A translational strategy was developed for MK-2075 to quantitatively interrelate drug exposure, target modulation, and the desired pharmacological response in preclinical animal models for the purpose of human translation. Analgesics used as a standard of care in postoperative pain were evaluated in preclinical animal models of nociceptive behavior (mouse tail flick latency and rhesus thermode heat withdrawal) to determine the magnitude of pharmacodynamic (PD) response at plasma concentrations associated with efficacy in the clinic. MK-2075 was evaluated in those same animal models to determine the concentration of MK-2075 required to achieve the desired level of response. Translation of MK-2075 efficacious concentrations in preclinical animal models to a clinical PKPD target in humans was achieved by accounting for species differences in plasma protein binding and in vitro potency against the NaV1.7 channel. Estimates of human pharmacokinetic (PK) parameters were obtained from allometric scaling of a PK model from preclinical species and used to predict the dose required to achieve the clinical exposure. MK-2075 exposure-response in a preclinical target modulation assay (rhesus olfaction) was characterized using a computational PKPD model which included a biophase compartment to account for the observed hysteresis. Translation of this model to humans was accomplished by correcting for species differences in PK NaV1.7 potency, and plasma protein binding while assuming that the kinetics of distribution to the target site is the same between humans and rhesus monkeys. This enabled prediction of the level of target modulation anticipated to be achieved over the dosing interval at the projected clinical efficacious human dose. Integration of these efforts into the early development plan informed clinical study design and decision criteria.

fMRI study of olfactory processing in mice under three anesthesia protocols: Insight into the effect of ketamine on olfactory processing.

Functional magnetic resonance imaging (fMRI) is a valuable tool for studying neural activations in the central nervous system of animals due to its wide spatial coverage and non-invasive nature. However, the advantages of fMRI have not been fully realized in functional studies in mice, especially in the olfactory system, possibly due to the lack of suitable anesthesia protocols with spontaneous breathing. Since mice are widely used in biomedical research, it is desirable to evaluate different anesthesia protocols for olfactory fMRI studies in mice. Dexmedetomidine (DEX) as a sedative/anesthetic has been introduced to fMRI studies in mice, but it has a limited anesthesia duration. To extend the anesthesia duration, DEX has been combined with a low dose of isoflurane (ISO) or ketamine (KET) in previous functional studies in mice. In this report, olfactory fMRI studies were performed under three anesthesia protocols (DEX alone, DEX/ISO, and DEX/KET) in three different groups of mice. Isoamyl-acetate was used as an odorant, and the odorant-induced neural activations were measured by blood oxygenation-level dependent (BOLD) fMRI. BOLD fMRI responses were observed in the olfactory bulb (OB), anterior olfactory nuclei (AON), and piriform cortex (Pir). Interestingly, BOLD fMRI activations were also observed in the prefrontal cortical region (PFC), which are most likely caused by the draining vein effect. The response in the OB showed no adaptation to either repeated odor stimulations or continuous odor exposure, but the response in the Pir showed adaptation during the continuous odor exposure. The data also shows that ISO suppresses the olfactory response in the OB and AON, while KET enhances the olfactory response in the Pir. Thus, DEX/KET should be an attractive anesthesia for olfactory fMRI in mice.

fMRI study of the role of glutamate NMDA receptor in the olfactory processing in monkeys.

Studies in rodents show that olfactory processing in the principal neurons of olfactory bulb (OB) and piriform cortex (PC) is controlled by local inhibitory interneurons, and glutamate NMDA receptor plays a role in this inhibitory control. It is not clear if findings from studies in rodents translate to olfactory processing in nonhuman primates (NHPs). In this study, the effect of the glutamate NMDA receptor antagonist MK801 on odorant-induced olfactory responses in the OB and PC of anesthetized NHPs (rhesus monkeys) was investigated by cerebral blood volume (CBV) fMRI. Isoamyl-acetate was used as the odor stimulant. For each NHP, sixty fMRI measurements were made during a 4-h period, with each 4-min measurement consisting of a 1-min baseline period, a 1-min odor stimulation period, and a 2-min recovery period. MK801 (0.3 mg/kg) was intravenously delivered 1 hour after starting fMRI. Before MK801 injection, olfactory fMRI activations were observed only in the OB, not in the PC. After MK801 injection, olfactory fMRI activations in the OB increased, and robust olfactory fMRI activations were observed in the PC. The data indicate that MK801 enhances the olfactory responses in both the OB and PC. The enhancement effects of MK801 are most likely from its blockage of NMDA receptors on local inhibitory interneurons and the attenuation of the inhibition onto principal neurons. This study suggests that the mechanism of local inhibitory control of principal neurons in the OB and PC derived from studies in rodents translates to NHPs.

Pharmacological validation of a novel nonhuman primate measure of thermal responsivity with utility for predicting analgesic effects.

INTRODUCTION: The development of novel analgesics to treat acute or chronic pain has been a challenge due to a lack of translatable measurements. Preclinical end points with improved translatability are necessary to more accurately inform clinical testing paradigms, which may help guide selection of viable drug candidates. METHODS: In this study, a nonhuman primate biomarker which is sensitive to standard analgesics at clinically relevant plasma concentrations, can differentiate analgesia from sedation and utilizes a protocol very similar to that which can be employed in human clinical studies is described. Specifically, acute heat stimuli were delivered to the volar forearm using a contact heat thermode in the same manner as the clinical setting. RESULTS: Clinically efficacious exposures of morphine, fentanyl, and tramadol produced robust analgesic effects, whereas doses of diazepam that produce sedation had no effect. CONCLUSION: We propose that this assay has predictive utility that can help improve the probability of success for developing novel analgesics.

Benzoxazolinone aryl sulfonamides as potent, selective Nav1.7 inhibitors with in vivo efficacy in a preclinical pain model.

Studies on human genetics have suggested that inhibitors of the Nav1.7 voltage-gated sodium channel hold considerable promise as therapies for the treatment of chronic pain syndromes. Herein, we report novel, peripherally-restricted benzoxazolinone aryl sulfonamides as potent Nav1.7 inhibitors with excellent selectivity against the Nav1.5 isoform, which is expressed in the heart muscle. Elaboration of initial lead compound 3d afforded exemplar 13, which featured attractive physicochemical properties, outstanding lipophilic ligand efficiency and pharmacological selectivity against Nav1.5 exceeding 1000-fold. Key structure-activity relationships associated with oral bioavailability were leveraged to discover compound 17, which exhibited a comparable potency/selectivity profile as well as full efficacy following oral administration in a preclinical model indicative of antinociceptive behavior.

Discovery of selective, orally bioavailable, N-linked arylsulfonamide Nav1.7 inhibitors with pain efficacy in mice.

The voltage-gated sodium channel Nav1.7 is a genetically validated target for the treatment of pain with gain-of-function mutations in man eliciting a variety of painful disorders and loss-of-function mutations affording insensitivity to pain. Unfortunately, drugs thought to garner efficacy via Nav1 inhibition have undesirable side effect profiles due to their lack of selectivity over channel isoforms. Herein we report the discovery of a novel series of orally bioavailable arylsulfonamide Nav1.7 inhibitors with high levels of selectivity over Nav1.5, the Nav isoform responsible for cardiovascular side effects, through judicious use of parallel medicinal chemistry and physicochemical property optimization. This effort produced inhibitors such as compound 5 with excellent potency, selectivity, behavioral efficacy in a rodent pain model, and efficacy in a mouse itch model suggestive of target modulation.

fMRI study of the role of glutamate NMDA receptor in the olfactory adaptation in rats: Insights into cellular and molecular mechanisms of olfactory adaptation.

Olfactory adaptation, characterized by attenuation of response to repeated odor stimulations or continuous odor exposure, is an intrinsic feature of olfactory processing. Adaptation can be induced by either "synaptic depression" due to depletion of neurotransmitters, or "enhanced inhibition" onto principle neurons by local inhibitory interneurons in olfactory structures. It is not clear which mechanism plays a major role in olfactory adaptation. More importantly, molecular sources of enhanced inhibition have not been identified. In this study, olfactory responses to either repeated 40-s stimulations with interstimulus intervals (ISI) of 140-s or 30-min, or a single prolonged 200-s stimulus were measured by fMRI in different naïve rats. Olfactory adaptations in the olfactory bulb (OB), anterior olfactory nucleus (AON), and piriform cortex (PC) were observed only with repeated 40-s odor stimulations, and no olfactory adaptations were detected during the prolonged 200-s stimulation. Interestingly, in responses to repeated 40-s odor stimulations in the PC, the first odor stimulation induced positive activations, and odor stimulations under adapted condition induced negative activations. The negative activations suggest that "sparse coding" and "global inhibition" are the characteristics of olfactory processing in PC, and the global inhibition manifests only under an adapted condition, not a naïve condition. Further, we found that these adaptations were NMDA receptor dependent; an NMDA receptor antagonist (MK801) blocked the adaptations. Based on the mechanism that glutamate NMDA receptor plays a role in the inhibition onto principle neurons by interneurons, our data suggest that the olfactory adaptations are caused by enhanced inhibition from interneurons. Combined with the necessity of the interruption of odor stimulation to observe the adaptations, the molecular source for the enhanced inhibition is most likely an increased glutamate release from presynaptic terminals due to glutamate over-replenishment during the interruption of odor stimulation. Furthermore, with blockage of the adaptations, the data reveal that orbital, medial & prefrontal, and cingulate cortices (OmPFC) are involved in the olfactory processing.

fMRI study of olfaction in the olfactory bulb and high olfactory structures of rats: Insight into their roles in habituation.

Cerebral blood volume (CBV) fMRI with ultrasmall superparamagnetic iron oxide particles (USPIO) as a contrast agent was used to investigate olfactory processing in rats. fMRI data were acquired in sixteen 0.75-mm coronal slices covering the olfactory bulb (OB) and higher olfactory regions (HOR), including the anterior olfactory nucleus and piriform cortex. For each animal, multiple consecutive fMRI measurements were made during a 3-h experiment session, with each measurement consisting of a baseline period, an odorant stimulation period, and a recovery period. Two different stimulation paradigms with a stimulation period of 40s or 80s, respectively, were used to study olfactory processing. Odorant-induced CBV increases were robustly observed in the OB and HOR of each individual animal. Olfactory adaptation, which is characterized by an attenuation of responses to continuous exposure or repeated stimulations, has different characteristics in the OB and HOR. For adaptation to repeated stimuli, while it was observed in both the OB and HOR, CBV responses in the HOR were attenuated more significantly than responses in the OB. In contrast, within each continuous 40-s or 80-s odor exposure, CBV responses in the OB were stable and did not show adaptation, but the CBV responses in the HOR were state dependent, with no adaptation during initial exposures, but significant adaptation during following exposures. These results support previous reports that HOR plays a more significant role than OB in olfactory habituation. The technical approach presented in this study should enable more extensive fMRI studies of olfactory processing in rats.

Functional imaging of olfaction by CBV fMRI in monkeys: insight into the role of olfactory bulb in habituation.

Cerebral blood volume (CBV) fMRI with superparamagnetic iron oxide nanoparticles (USPIO) as contrast agent was used to investigate the odorant-induced olfaction in anesthetized rhesus monkeys. fMRI data were acquired in 24 axial slices covering the entire brain, with isoamyl-acetate as the odor stimulant. For each experiment, multiple fMRI measurements were made during a 1- or 2-h period, with each measurement consisting of a baseline period, a stimulation period, and a recovery period. Three different stimulation paradigms with a stimulation period of 1 min, 2 min, or 8 min, respectively, were used to study the olfactory responses in the olfactory bulb (OB). Odorant-induced CBV increases were observed in the OB of each individual monkey. The spatial and temporal activation patterns were reproducible within and between animals. The sensitivity of CBV fMRI in OB was comparable with the sensitivities reported in previous animal fMRI studies. The CBV responses during the 1-min, 2-min, or 8-min odor stimulation period were relatively stable, and did not show attenuation. The amplitudes of CBV response to the repeated stimuli during the 1- or 2-h period were also stable. The stable CBV response in the OB to both continuous and repeated odor stimuli suggests that the OB may not play a major role in olfactory habituation. The technical approach described in this report can enable more extensive fMRI studies of olfactory processing in OB of both humans and non-human primates.

Design, synthesis, and structure-activity relationship study of bicyclic piperazine analogs of indole-3-carboxamides as novel cannabinoid CB1 receptor agonists.

Bicyclic piperazine derivatives were synthesized as conformationally constrained analogs of N-alkyl piperazines and were found to be potent CB1 receptor agonists. The CB1 receptor agonist activity was dependent upon the absolute configuration of the chiral center of the bicyclic ring system. Although the conformational constraint did not protect the compounds from metabolism by N-dealkylation, several bicyclic analogs were found to be more potent than the unconstrained lead compound. Compound 8b demonstrated potent antinociceptive activity in vivo.

Design, synthesis, and structure-activity relationship study of conformationally constrained analogs of indole-3-carboxamides as novel CB1 cannabinoid receptor agonists.

Novel tricyclic indole-3-carboxamides were synthesized as structurally restricted analogs of bicyclic indoles, and found to be potent CB1 cannabinoid receptor agonists. The CB1 agonist activity depended on the absolute configuration of the chiral center of the tricyclic ring. The preferred enantiomer was more potent than the structurally unconstrained lead compound. Structure-activity relationships in the amide side chain of the indole C-3 position were also investigated.

Rapid access towards follow-up NOP receptor agonists using a knowledge based approach.

A knowledge based approach has been adopted to identify novel NOP receptor agonists with simplified hydrophobes. Substitution of the benzimidazol-2-one piperidine motif with a range of hydrophobic groups and pharmacophore guided bio-isosteric replacement of the benzimidazol-2-one moiety was explored. Compound 51 was found to be a high affinity, potent NOP receptor agonist with reduced affinity for the hERG channel.

Structure-activity relationships and CoMFA of N-3 substituted phenoxypropyl piperidine benzimidazol-2-one analogues as NOP receptor agonists with analgesic properties.

The N-3 position of a series of 3-phenoxypropyl piperidine benzimidazol-2-one analogues was optimised using the predictive power of a CoMFA model. The model was used to prioritise compounds for synthesis culminating in the triazole (+)-24. (+)-24 was found to be a high affinity, potent NOP agonist and demonstrated both antinociceptive and antiallodynic effects when administered iv to rodents.

The hypnotic, electroencephalographic, and antinociceptive properties of nonpeptide ORL1 receptor agonists after intravenous injection in rodents.

BACKGROUND: Agonists at the opioid receptor-like receptor 1 (ORL1) induce motor impairment, sedation, and loss of righting reflex (LRR) in rodents. This receptor may provide a novel target in the field of anesthesia. METHODS: We examined the hypnotic, electroencephalographic (EEG), and antinociceptive effects of two IV administered nonpeptide ORL1 agonists, (Ro 65-6570 and Org 26383), using LRR in mice and rats, percent EEG burst suppression in rats, and formalin paw test in mice. RESULTS: In mice, Ro 65-6570 and Org 26383 produced LRR (hypnotic dose 0.6 and 3.7 micromol/kg for Ro 65-6570 and Org 26383, respectively). Naloxone had no significant effect on sleep times produced by both compounds. In rats, Ro 65-6570 (0.6-2.4 micromol/kg) and Org 26383 (4-8 micromol/kg) produced LRR and burst suppression activity in the EEG. Both sleep times and burst suppression activity were significantly reduced with a selective ORL1 antagonist. In mice, dose-dependent inhibition of formalin-induced nociceptive behaviors occurred (Phase 1 ED50 0.4 and 1.8 micromol/kg and Phase 2 ED50 0.4 and 4.2 micromol/kg for Ro 65-6570 and Org 26383, respectively). CONCLUSIONS: These results show that Ro 65-6570 and Org 26383 (probably via the ORL1 receptor) behave as IV hypnotics and analgesics in mice and rats, and that the hypnotic and antinociceptive doses are similar.

Synthesis and evaluation of N-3 substituted phenoxypropyl piperidine benzimidazol-2-one analogues as NOP receptor agonists with analgesic and sedative properties.

A series of 3-phenoxypropyl piperidine benzimidazol-2-one analogues have been discovered as novel NOP receptor agonists. Structure-activity relationships have been explored via N-3 substitution of the benzimidazol-2-one with a range of functionality. The N-methyl acetamide derivative (+)-7f was found to be a high-affinity, potent NOP agonist with greater than 100-fold selectivity over the MOP receptor. Furthermore (+)-7f was shown to be both antinociceptive and sedative when administered iv to rodents.

Synthesis and SAR studies of 3-phenoxypropyl piperidine analogues as ORL1 (NOP) receptor agonists.

A series of 3-phenoxypropyl piperidine analogues have been discovered as novel ORL1 receptor agonists. Structure-activity relationships have been explored around the 3-phenoxypropyl region with several potent and selective analogues identified.

Mrz 2/579, a fast kinetic NMDA channel blocker, reduces the development of morphine tolerance in awake rats.

The purpose of the present study was to investigate whether uncompetitive NMDA antagonists with fast channel blocking kinetics, which show fewer side effects in man than compounds such as ketamine, affect the development of tolerance to continuous exposure to morphine. Rats were trained on the Randall--Selitto apparatus before being implanted, under halothane anaesthesia, with primed mini-osmotic pumps (240 microl/day). Six rats were implanted with a vehicle filled pump, seven with a morphine filled pump (28.8 mg/kg/day), and eight with a pair of pumps, one containing morphine and the other Mrz 2/579, a new NMDA antagonist (40 mg/kg/day). A fourth group was implanted with a morphine filled pump followed 25 h later by a Mrz 2/579 filled pump. Paw withdrawal tests were undertaken immediately before, and at 2, 4, 6, 8, 10, 12, 24, 48 and 72 h after the first pump was implanted. Before pump implantation, withdrawal thresholds were 120+/-7 g (mean+/-SEM, n=30). Vehicle infusion had no effect on withdrawal thresholds, whereas morphine infusion increased them significantly at 2 and 4 h after pump implantation (+2 h: 208+/-14 g; P<0.001 vs. control). From 6 h the antinociception elicited by morphine declined progressively; at 10 h withdrawal thresholds were significantly lower than the 2 h post-treatment value (P<0.001). In rats treated with morphine plus Mrz 2/579, thresholds remained significantly higher between 10--72 h post-implantation than with morphine alone (P<0.05). In contrast, infusion of the same level of Mrz 2/579 once tolerance had developed did not reverse tolerance. These results indicate that fast NMDA channel blockers such as Mrz 2/579 may prove to be useful in enhancing analgesia to continuous morphine administration.