Eric L. Moore

HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity

BackgroundSafe and effective treatment for chronic inflammatory and neuropathic pain remains a key unmet medical need for many patients. The recent discovery and description of the transient receptor potential family of receptors including TRPV1 and TRPA1 has provided a number of potential new therapeutic targets for treating chronic pain. Recent reports have suggested that TRPA1 may play an important role in acute formalin and CFA induced pain. The current study was designed to further explore the therapeutic potential of pharmacological TRPA1 antagonism to treat inflammatory and neuropathic pain.ResultsThe in vitro potencies of HC-030031 versus cinnamaldehyde or allyl isothiocyanate (AITC or Mustard oil)-induced TRPA1 activation were 4.9 ± 0.1 and 7.5 ± 0.2 μM respectively (IC50). These findings were similar to the previously reported IC50 of 6.2 μM against AITC activation of TRPA1 [1]. In the rat, oral administration of HC-030031 reduced AITC-induced nocifensive behaviors at a dose of 100 mg/kg. Moreover, oral HC-030031 (100 mg/kg) significantly reversed mechanical hypersensitivity in the more chronic models of Complete Freunds Adjuvant (CFA)-induced inflammatory pain and the spinal nerve ligation model of neuropathic pain.ConclusionUsing oral administration of the selective TRPA1 antagonist HC-030031, our results demonstrated that TRPA1 plays an important role in the mechanisms responsible for mechanical hypersensitivity observed in inflammatory and neuropathic pain models. These findings suggested that TRPA1 antagonism may be a suitable new approach for the development of a potent and selective therapeutic agent to treat both inflammatory and neuropathic pain.

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 (Ki = 0.77 nM) and rhesus (Ki = 1.2 nM) CGRP receptors but displays >1500-fold lower affinity for the canine and rat receptors as determined via 125I-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.

Pharmacological Properties of MK-3207, a Potent and Orally Active Calcitonin Gene-Related Peptide Receptor Antagonist

Calcitonin gene-related peptide (CGRP) has long been hypothesized to play a key role in migraine pathophysiology, and the advent of small-molecule antagonists has clearly demonstrated a clinical link between blocking the CGRP receptor and migraine efficacy. 2-[(8R)-8-(3,5-Difluorophenyl)-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(2R)-2′-oxo-1,1′,2′,3-tetrahydrospiro[indene-2,3′-pyrrolo[2,3-b]pyridin]-5-yl]acetamide (MK-3207) represents the third CGRP receptor antagonist to display clinical efficacy in migraine trials. Here, we report the pharmacological characterization of MK-3207, a potent and orally bioavailable CGRP receptor antagonist. In vitro, MK-3207 is a potent antagonist of the human and rhesus monkey CGRP receptors (Ki = 0.024 nM). In common with other CGRP receptor antagonists, MK-3207 displays lower affinity for CGRP receptors from other species, including canine and rodent. As a consequence of species selectivity, the in vivo potency was assessed in a rhesus monkey pharmacodynamic assay measuring capsaicin-induced changes in forearm dermal blood flow via laser Doppler imaging. MK-3207 produced a concentration-dependent inhibition of dermal vasodilation, with plasma concentrations of 0.8 and 7 nM required to block 50 and 90% of the blood flow increase, respectively. The tritiated analog [3H]MK-3207 was used to study the binding characteristics on the human CGRP receptor. [3H]MK-3207 displayed reversible and saturable binding (KD = 0.06 nM), and the off-rate was determined to be 0.012 min−1, with a t1/2 value of 59 min. In vitro autoradiography studies on rhesus monkey brain slices identified the highest level of binding in the cerebellum, brainstem, and meninges. Finally, as an index of central nervous system penetrability, the in vivo cerebrospinal fluid/plasma ratio was determined to be 2 to 3% in cisterna magna-ported rhesus monkeys.

Structural basis for selectivity and diversity in angiotensin II receptors

The angiotensin II receptors AT1R and AT2R serve as key components of the renin–angiotensin–aldosterone system. AT1R has a central role in the regulation of blood pressure, but the function of AT2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT2R bound to an AT2R-selective ligand and to an AT1R/AT2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or β-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure–activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.

The discovery of highly potent CGRP receptor antagonists

Rational modification of a previously identified spirohydantoin lead structure has identified a series of potent spiroazaoxindole CGRP receptor antagonists. The azaoxindole was found to be a general replacement for the hydantoin that consistently improved in vitro potency. The combination of the indanylspiroazaoxindole and optimized benzimidazolinones led to highly potent antagonists (e.g., 25, CGRP K(i)=40pM). The closely related compound 27 demonstrated good oral bioavailability in dog and rhesus.

[(11)C]MK-4232: The First Positron Emission Tomography Tracer for the Calcitonin Gene-Related Peptide Receptor.

Rational modification of the potent calcitonin gene-related peptide (CGRP) receptor antagonist MK-3207 led to a series of analogues with enhanced CNS penetrance and a convenient chemical handle for introduction of a radiolabel. A number of (11)C-tracers were synthesized and evaluated in vivo, leading to the identification of [(11)C]8 ([(11)C]MK-4232), the first positron emission tomography tracer for the CGRP receptor.

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).

Identification of potent, highly constrained CGRP receptor antagonists.

A novel series of potent CGRP receptor antagonists containing a central quinoline ring constraint was identified. The combination of the quinoline constraint with a tricyclic benzimidazolinone left hand fragment produced an analog with picomolar potency (14, CGRP K(i)=23 pM). Further optimization of the tricycle produced a CGRP receptor antagonist that exhibited subnanomolar potency (19, CGRP K(i)=0.52 nM) and displayed a good pharmacokinetic profile in three preclinical species.

Novel CGRP receptor antagonists through a design strategy of target simplification with addition of molecular flexibility.

A novel class of CGRP receptor antagonists was rationally designed by modifying a highly potent, but structurally complex, CGRP receptor antagonist. Initial modifications focused on simplified structures, with increased flexibility. Subsequent to the preparation of a less-potent but more flexible lead, classic medicinal chemistry methods were applied to restore high affinity (compound 22, CGRP Ki=0.035 nM) while maintaining structural diversity relative to the lead. Good selectivity against the closely related adrenomedullin-2 receptor was also achieved.

Novel CGRP receptor antagonists from central amide replacements causing a reversal of preferred chirality

A previously utilized quinoline-for-N-phenylamide replacement strategy was employed against a central amide in a novel class of CGRP receptor antagonists. A unique and unexpected substitution pattern was ultimately required to maintain reasonable affinity for the CGRP receptor, while at the same time predicting acceptable heterocycle positioning for related analogs. Subsequently, specific quinoline and naphthyridine compounds were prepared which supported these structural predictions by displaying CGRP binding affinities in the 0.037-0.15 nM range.