Pharmacologic characterization of atogepant: A potent and selective calcitonin gene-related peptide receptor antagonist.

BACKGROUND: The trigeminal sensory neuropeptide calcitonin gene-related peptide (CGRP) is identified as an essential element in migraine pathogenesis. METHODS: In vitro and in vivo studies evaluated pharmacologic properties of the CGRP receptor antagonist atogepant. Radioligand binding using 125I-CGRP and cyclic adenosine monophosphate (cAMP) accumulation assays were conducted in human embryonic kidney 293 cells to assess affinity, functional potency and selectivity. Atogepant in vivo potency was assessed in the rat nitroglycerine model of facial allodynia and primate capsaicin-induced dermal vasodilation (CIDV) pharmacodynamic model. Cerebrospinal fluid/brain penetration and behavioral effects of chronic dosing and upon withdrawal were evaluated in rats. RESULTS: Atogepant exhibited high human CGRP receptor-binding affinity and potently inhibited human α-CGRP-stimulated cAMP responses. Atogepant exhibited significant affinity for the amylin1 receptor but lacked appreciable affinities for adrenomedullin, calcitonin and other known neurotransmitter receptor targets. Atogepant dose-dependently inhibited facial allodynia in the rat nitroglycerine model and produced significant CIDV inhibition in primates. Brain penetration and behavioral/physical signs during chronic dosing and abrupt withdrawal were minimal in rats. CONCLUSIONS: Atogepant is a competitive antagonist with high affinity, potency and selectivity for the human CGRP receptor. Atogepant demonstrated a potent, concentration-dependent exposure/efficacy relationship between atogepant plasma concentrations and inhibition of CGRP-dependent effects.

Lead optimization of cathepsin K inhibitors for the treatment of Osteoarthritis.

Cathepsin K (Cat K) is a cysteine protease involved in bone remodeling. In addition to its role in bone biology, Cat K is upregulated in osteoclasts, chondrocytes and synoviocytes in osteoarthritic (OA) disease states making it a potential therapeutic target for disease-modifying OA. Starting from a prior preclinical compound, MK-1256, lead optimization efforts were carried out in the search for potent Cat K inhibitors with improved selectivity profiles with an emphasis on cathepsin F. Herein, we report the SAR studies which led to the discovery of the highly selective oxazole compound 23, which was subsequently shown to inhibit cathepsin K in vivo as measured by reduced levels of urinary C-telopeptide of collagen type I in dog.

Development of ProTx-II Analogues as Highly Selective Peptide Blockers of Nav1.7 for the Treatment of Pain.

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.

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.

Application of Pharmacokinetic-Pharmacodynamic Modeling to Inform Translation of In Vitro NaV1.7 Inhibition to In Vivo Pharmacological Response in Non-human Primate.

PURPOSE: This work describes a staged approach to the application of pharmacokinetic-pharmacodynamic (PK-PD) modeling in the voltage-gated sodium ion channel (NaV1.7) inhibitor drug discovery effort to address strategic questions regarding in vitro to in vivo translation of target modulation. METHODS: PK-PD analysis was applied to data from a functional magnetic resonance imaging (fMRI) technique to non-invasively measure treatment mediated inhibition of olfaction signaling in non-human primates (NHPs). Initial exposure-response was evaluated using single time point data pooled across 27 compounds to inform on in vitro to in vivo correlation (IVIVC). More robust effect compartment PK-PD modeling was conducted for a subset of 10 compounds with additional PD and PK data to characterize hysteresis. RESULTS: The pooled compound exposure-response facilitated an early exploration of IVIVC with a limited dataset for each individual compound, and it suggested a 2.4-fold in vitro to in vivo scaling factor for the NaV1.7 target. Accounting for hysteresis with an effect compartment PK-PD model as compounds advanced towards preclinical development provided a more robust determination of in vivo potency values, which resulted in a statistically significant positive IVIVC with a slope of 1.057 ± 0.210, R-squared of 0.7831, and p value of 0.006. Subsequent simulations with the PK-PD model informed the design of anti-nociception efficacy studies in NHPs. CONCLUSIONS: A staged approach to PK-PD modeling and simulation enabled integration of in vitro NaV1.7 potency, plasma protein binding, and pharmacokinetics to describe the exposure-response profile and inform future study design as the NaV1.7 inhibitor effort progressed through drug discovery.

Characterization of Ubrogepant: A Potent and Selective Antagonist of the Human Calcitonin Gene‒Related Peptide Receptor.

A growing body of evidence has implicated the calcitonin gene-related peptide (CGRP) receptors in migraine pathophysiology. With the recent approval of monoclonal antibodies targeting CGRP or the CGRP receptor, the inhibition of CGRP-mediated signaling has emerged as a promising approach for preventive treatments of migraine in adults. However, there are no small-molecule anti-CGRP treatments available for treating migraine. The current studies aimed to characterize the pharmacologic properties of ubrogepant, an orally bioavailable, CGRP receptor antagonist for the acute treatment of migraine. In a series of ligand binding assays, ubrogepant exhibited a high binding affinity for native (K i=0.067 nM) and cloned human (K i=0.070 nM) and rhesus CGRP receptors (K i=0.079 nM), with relatively lower affinities for CGRP receptors from rat, mouse, rabbit and dog. In functional assays, ubrogepant potently blocked human α-CGRP stimulated cAMP response (IC50 of 0.08 nM) and exhibited highly selective antagonist activity for the CGRP receptor compared with other members of the human calcitonin receptor family. Furthermore, the in vivo CGRP receptor antagonist activity of ubrogepant was evaluated in a pharmacodynamic model of capsaicin-induced dermal vasodilation (CIDV) in rhesus monkeys and humans. Results demonstrated that ubrogepant produced concentration-dependent inhibition of CIDV with a mean EC50 of 3.2 and 2.6 nM in rhesus monkeys and humans, respectively. Brain penetration studies with ubrogepant in monkeys showed a CSF/plasma ratio of 0.03 and low CGRP receptor occupancy. In summary, ubrogepant is a competitive antagonist with high affinity, potency, and selectivity for the human CGRP receptor. SIGNIFICANCE STATEMENT: Ubrogepant is a potent, selective, orally delivered, small-molecule competitive antagonist of the human calcitonin generelated peptide receptor. In vivo studies using a pharmacodynamic model of capsaicin-induced dermal vasodilation (CIDV) in rhesus monkeys and humans demonstrated that ubrogepant produced concentration-dependent inhibition of CIDV, indicating a predictable pharmacokinetic-pharmacodynamic relationship.

Identification of second-generation P2X3 antagonists for treatment of pain.

A second-generation small molecule P2X3 receptor antagonist has been developed. The lead optimization strategy to address shortcomings of the first-generation preclinical lead compound is described herein. These studies were directed towards the identification and amelioration of preclinical hepatobiliary findings, reducing potential for drug-drug interactions, and decreasing the projected human dose of the first-generation lead.

Novel oxazolidinone calcitonin gene-related peptide (CGRP) receptor antagonists for the acute treatment of migraine.

In our efforts to develop CGRP receptor antagonists as backups to MK-3207, 2, we employed a scaffold hopping approach to identify a series of novel oxazolidinone-based compounds. The development of a structurally diverse, potent (20, cAMP+HS IC50=0.67 nM), and selective compound (hERG IC50=19 µM) with favorable rodent pharmacokinetics (F=100%, t1/2=7h) is described. Key to this development was identification of a 3-substituted spirotetrahydropyran ring that afforded a substantial gain in potency (10 to 35-fold).

Decahydroquinoline amides as highly selective CB2 agonists: role of selectivity on in vivo efficacy in a rodent model of analgesia.

A novel series of decahydroquinoline CB2 agonists is described. Optimization of the amide substituent led to improvements in CB2/CB1 selectivity as well as physical properties. Two key compounds were examined in the rat CFA model of acute inflammatory pain. A moderately selective CB2 agonist was active in this model. A CB2 agonist lacking functional CB1 activity was inactive in this model despite high in vivo exposure both peripherally and centrally.

Imidazopyridine CB2 agonists: optimization of CB2/CB1 selectivity and implications for in vivo analgesic efficacy.

A new series of imidazopyridine CB2 agonists is described. Structural optimization improved CB2/CB1 selectivity in this series and conferred physical properties that facilitated high in vivo exposure, both centrally and peripherally. Administration of a highly selective CB2 agonist in a rat model of analgesia was ineffective despite substantial CNS exposure, while administration of a moderately selective CB2/CB1 agonist exhibited significant analgesic effects.

Orally bioavailable imidazoazepanes as calcitonin gene-related peptide (CGRP) receptor antagonists: discovery of MK-2918.

In our ongoing efforts to develop CGRP receptor antagonists for the treatment of migraine, we aimed to improve upon telecagepant by targeting a compound with a lower projected clinical dose. Imidazoazepanes were identified as potent caprolactam replacements and SAR of the imidazole yielded the tertiary methyl ether as an optimal substituent for potency and hERG selectivity. Combination with the azabenzoxazinone spiropiperidine ultimately led to preclinical candidate 30 (MK-2918).

Optimization of azepanone calcitonin gene-related peptide (CGRP) receptor antagonists: development of novel spiropiperidines.

Several novel spiropiperidine-based CGRP receptor antagonists have been developed that maintain good potency and have reduced potential for metabolism.

Copper-facilitated Suzuki reactions: application to 2-heterocyclic boronates.

The palladium-catalyzed Suzuki-Miyaura reaction has been utilized as one of the most powerful methods for C-C bond formation. However, Suzuki reactions of electron-deficient 2-heterocyclic boronates generally give low conversions and remain challenging. The successful copper(I) facilitated Suzuki coupling of 2-heterocyclic boronates that is broad in scope is reported. Use of this methodology affords greatly enhanced yields of these notoriously difficult couplings. Furthermore, mechanistic investigations suggest a possible role of copper in the catalytic cycle.

Examining the binding properties of MK-0974: a CGRP receptor antagonist for the acute treatment of migraine.

Calcitonin gene-related peptide (CGRP) is a neuropeptide that plays a key role in the pathophysiology of migraine headache. MK-0974 (telcagepant) is a potent and selective antagonist of the human and rhesus CGRP receptors and is currently in Phase III clinical studies for the acute treatment of migraine. The pharmacology of MK-0974 has been studied extensively, but there has not been a thorough characterization of its binding properties. Here, we characterize the binding of a tritiated analog of MK-0974 on human neuroblastoma (SK-N-MC) membranes and rhesus cerebellum. [(3)H]MK-0974 displayed reversible and saturable binding to both SK-N-MC membranes and rhesus cerebellum with a K(D) of 1.9 nM and 1.3 nM, respectively. Agonists and antagonists of the CGRP receptor displaced [(3)H]MK-0974 in a concentration-dependent manner in competition binding experiments. Both CGRP and adrenomedullin demonstrated biphasic competition while MK-0974 and the peptide antagonist CGRP(8-37) displaced [(3)H]MK-0974 in a monophasic fashion. In competitive binding studies with [(3)H]MK-0974 and CGRP, the fraction of high-affinity binding was reduced significantly by incubating the membranes with GTPgammaS. In kinetic binding experiments, the off-rate of [(3)H]MK-0974 was determined to be 0.51 min(-1) with a half-life of 1.3 min. In conclusion, the radioligand [(3)H]MK-0974 has proven to be a useful tool for studying the binding characteristics of MK-0974 and has broadened our understanding of this promising molecule.

Synthesis of the (3R,6S)-3-amino-6-(2,3-difluorophenyl)azepan-2-one of telcagepant (MK-0974), a calcitonin gene-related peptide receptor antagonist for the treatment of migraine headache.

Two novel routes have been developed to the (3 R,6 S)-3-amino-6-(2,3-difluorophenyl)-1-(2,2,2-trifluoroethyl)azepan-2-one 2 of the CGRP receptor antagonist clinical candidate telcagepant (MK-0974, 1). The first employs a ring-closing metathesis of the styrene 7 as the key reaction, while the second makes use of a highly diastereoselective Hayashi-Miyaura Rh-catalyzed arylboronic acid addition to nitroalkene 16. The latter route has been implemented to produce multigram quantities of telcagepant for extensive preclinical evaluation.

Calcitonin gene-related peptide (CGRP) receptor antagonists: investigations of a pyridinone template.

In our effort to find potent, orally bioavailable CGRP receptor antagonists for the treatment of migraine, a novel series based on a pyridinone template was investigated. After optimizing the privileged structure and the placement of the attached phenyl ring, systematic SAR was carried out on both the N-alkyl and C-5 aryl substituents. Several analogs with good potency and pharmacokinetic profiles were identified.

Pharmacological characterization of MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide], a potent and orally active calcitonin gene-related peptide receptor antagonist for the treatment of migraine.

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide that plays a key role in the pathophysiology of migraine headache. CGRP levels in the cranial circulation are increased during a migraine attack, and CGRP itself has been shown to trigger migraine-like headache. The correlation between CGRP release and migraine headache points to the potential utility of CGRP receptor antagonists as novel therapeutics in the treatment of migraine. Indeed, clinical proof-of-concept in the acute treatment of migraine was demonstrated with an intravenous formulation of the CGRP receptor antagonist BIBN4096BS (olcegepant). Here we report on the pharmacological characterization of the first orally bioavailable CGRP receptor antagonist in clinical development, MK-0974 [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxamide]. In vitro, MK-0974 is a potent antagonist of the human (K(i) = 0.77 nM) and rhesus (K(i) = 1.2 nM) CGRP receptors but displays >1500-fold lower affinity for the canine and rat receptors as determined via (125)I-human CGRP competition binding assays. A rhesus pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging was utilized to determine the in vivo activity of CGRP receptor antagonism. MK-0974 produced a concentration-dependent inhibition of dermal vasodilation, generated by capsaicin-induced release of endogenous CGRP, with plasma concentrations of 127 and 994 nM required to block 50 and 90% of the blood flow increase, respectively. In conclusion, MK-0974 is a highly potent, selective, and orally bioavailable CGRP receptor antagonist, which may be valuable in the acute treatment of migraine.