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.

Localization of CGRP receptor components and receptor binding sites in rhesus monkey brainstem: A detailed study using in situ hybridization, immunofluorescence, and autoradiography.

Functional imaging studies have revealed that certain brainstem areas are activated during migraine attacks. The neuropeptide calcitonin gene-related peptide (CGRP) is associated with activation of the trigeminovascular system and transmission of nociceptive information and plays a key role in migraine pathophysiology. Therefore, to elucidate the role of CGRP, it is critical to identify the regions within the brainstem that process CGRP signaling. In situ hybridization and immunofluorescence were performed to detect mRNA expression and define cellular localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), respectively. To define CGRP receptor binding sites, in vitro autoradiography was performed with [(3)H]MK-3207 (a CGRP receptor antagonist). CLR and RAMP1 mRNA and protein expression were detected in the pineal gland, medial mammillary nucleus, median eminence, infundibular stem, periaqueductal gray, area postrema, pontine raphe nucleus, gracile nucleus, spinal trigeminal nucleus, and spinal cord. RAMP1 mRNA expression was also detected in the posterior hypothalamic area, trochlear nucleus, dorsal raphe nucleus, medial lemniscus, pontine nuclei, vagus nerve, inferior olive, abducens nucleus, and motor trigeminal nucleus; protein coexpression of CLR and RAMP1 was observed in these areas via immunofluorescence. [(3)H]MK-3207 showed high binding densities concordant with mRNA and protein expression. The present study suggests that several regions in the brainstem may be involved in CGRP signaling. Interestingly, we found receptor expression and antagonist binding in some areas that are not protected by the blood-brain barrier, which suggests that drugs inhibiting CGRP signaling may not be able to penetrate the central nervous system to antagonize receptors in these brain regions.

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).

Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.

Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated anti-migraine efficacy. One remaining question is where do these blockers act? We hypothesized that the trigeminal ganglion could be one possible site. We examined the binding sites of a CGRP receptor antagonist (MK-3207) and related this to the expression of CGRP and its receptor in rhesus trigeminal ganglion. Pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate were examined and related to the CGRP system. Furthermore, we examined if the trigeminal ganglion is protected by the blood-brain barrier (BBB). Autoradiography was performed with [(3)H]MK-3207 to demonstrate receptor binding sites in rhesus trigeminal ganglion (TG). Immunofluorescence was used to correlate binding and the presence of CGRP and its receptor components, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), and the distribution of PACAP and glutamate in rhesus and rat TG. Evans blue was used to examine large molecule penetration into the rat TG. High receptor binding densities were found in rhesus TG. Immunofluorescence revealed expression of CGRP, CLR and RAMP1 in trigeminal cells. CGRP positive neurons expressed PACAP but not glutamate. Some neurons expressing CLR and RAMP1 co-localized with glutamate. Evans blue revealed that the TG is not protected by BBB. This study demonstrates CGRP receptor binding sites and expression of the CGRP receptor in rhesus and rat TG. The expression pattern of PACAP and glutamate suggests a possible interaction between the glutamatergic and CGRP system. In rat the TG is outside the BBB, suggesting that molecules do not need to be CNS-penetrant to block these receptors.

(E)-Alkenes as replacements of amide bonds: development of novel and potent acyclic CGRP receptor antagonists.

A new class of CGRP receptor antagonists was identified by replacing the central amide of a previously identified anilide lead structure with ethylene, ethane, or ethyne linkers. (E)-Alkenes as well as alkynes were found to preserve the proper bioactive conformation of the amides, necessary for efficient receptor binding. Further exploration resulted in several potent compounds against CGRP-R with low susceptibility to P-gp mediated efflux.

In vivo quantification of calcitonin gene-related peptide receptor occupancy by telcagepant in rhesus monkey and human brain using the positron emission tomography tracer [11C]MK-4232.

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide whose agonist interaction with the CGRP receptor (CGRP-R) in the periphery promotes vasodilation, neurogenic inflammation and trigeminovascular sensory activation. This process is implicated in the cause of migraine headaches, and CGRP-R antagonists in clinical development have proven effective in treating migraine-related pain in humans. CGRP-R is expressed on blood vessel smooth muscle and sensory trigeminal neurons and fibers in the periphery as well as in the central nervous system. However, it is not clear what role the inhibition of central CGRP-R plays in migraine pain relief. To this end, the CGRP-R positron emission tomography (PET) tracer [(11)C]MK-4232 (2-[(8R)-8-(3,5-difluorophenyl)-6,8-[6-(11)C]dimethyl-10-oxo-6,9-diazaspiro[4.5]decan-9-yl]-N-[(2R)-2'-oxospiro[1,3-dihydroindene-2,3'-1H-pyrrolo[2,3-b]pyridine]-5-yl]acetamide) was discovered and developed for use in clinical PET studies. In rhesus monkeys and humans, [(11)C]MK-4232 displayed rapid brain uptake and a regional brain distribution consistent with the known distribution of CGRP-R. Monkey PET studies with [(11)C]MK-4232 after intravenous dosing with CGRP-R antagonists validated the ability of [(11)C]MK-4232 to detect changes in CGRP-R occupancy in proportion to drug plasma concentration. Application of [(11)C]MK-4232 in human PET studies revealed that telcagepant achieved only low receptor occupancy at an efficacious dose (140 mg PO). Therefore, it is unlikely that antagonism of central CGRP-R is required for migraine efficacy. However, it is not known whether high central CGRP-R antagonism may provide additional therapeutic benefit.

Localization of CGRP receptor components, CGRP, and receptor binding sites in human and rhesus cerebellar cortex.

The cerebellum is classically considered to be mainly involved in motor processing, but studies have suggested several other functions, including pain processing. Calcitonin-gene-related peptide (CGRP) is a neuropeptide involved in migraine pathology, where there is elevated release of CGRP during migraine attacks and CGRP receptor antagonists have antimigraine efficacy. In the present study, we examined CGRP and CGRP receptor binding sites and protein expression in primate cerebellar cortex. Additionally, mRNA expression of the CGRP receptor components, calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1), was examined. In addition, expression of procalcitonin was studied. We observed high [(3)H]MK-3207 (CGRP receptor antagonist) binding densities in the molecular layer of rhesus cerebellar cortex; however, due to the limit of resolution of the autoradiographic image the exact cellular localization could not be determined. Similarly, [(125)I]CGRP binding was observed in the molecular layer and Purkinje cell layer of human cerebellum. CLR and RAMP1 mRNA was expressed within the Purkinje cell layer and some expression was found in the molecular layer. Immunofluorescence revealed expression of CGRP, CLR, and RAMP1 in the Purkinje cells and in cells in the molecular layer. Procalcitonin was found in the same localization. Recent research in the biology of cerebellum indicates that it may have a role in nociception. For the first time we have identified CGRP and CGRP receptor binding sites together with CGRP receptor expression through protein and mRNA localization in primate cerebellar cortex. These results point toward a functional role of CGRP in cerebellum. Further efforts are needed to evaluate this.

[(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.

MK-8825: a potent and selective CGRP receptor antagonist with good oral activity in rats.

Rational modification of the clinically tested CGRP receptor antagonist MK-3207 (3) afforded an analogue with increased unbound fraction in rat plasma and enhanced aqueous solubility, 2-[(8R)-8-(3,5-difluorophenyl)-8-methyl-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(6S)-2'-oxo-1',2',5,7-tetrahydrospiro[cyclopenta[b]pyridine-6,3'-pyrrolo[2,3-b]pyridin]-3-yl]acetamide (MK-8825) (6). Compound 6 maintained similar affinity to 3 at the human and rat CGRP receptors but possessed significantly improved in vivo potency in a rat pharmacodynamic model. The overall profile of 6 indicates it should find utility as a rat tool to investigate effects of CGRP receptor blockade in vivo.

Targeting a family B GPCR/RAMP receptor complex: CGRP receptor antagonists and migraine.

The clinical effectiveness of antagonizing the calcitonin gene-related peptide (CGRP) receptor for relief of migraine pain has been clearly demonstrated, but the road to the development of these small molecule antagonists has been daunting. The key hurdle that needed to be overcome was the CGRP receptor itself. The vast majority of the current antagonists recognize similar epitopes on the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). RAMP1 is a relatively small, single, transmembrane-spanning protein and along with the G-protein-coupled receptor CLR comprise a functional CGRP receptor. The tri-helical extracellular domain of RAMP1 plays a key role in the high affinity binding of CGRP receptor antagonists and drives their species-selective pharmacology. Over the years, a significant amount of mutagenesis data has been generated to identify specific amino acids or regions within CLR and RAMP1 that are critical to antagonist binding and has directed attention to the CLR/RAMP1 extracellular domain (ECD) complex. Recently, the crystal structure of the CGRP receptor ECD has been elucidated and not only reinforces the early mutagenesis data, but provides critical insight into the molecular mechanism of CGRP receptor antagonism. This review will highlight the drug design hurdles that must be overcome to meet the desired potency, selectivity and pharmacokinetic profile while retaining drug-like properties. Although the development of these antagonists has proved challenging, blocking the CGRP receptor may one day represent a new way to manage migraine and offer hope to migraine sufferers.

Identification of a novel RAMP-independent CGRP receptor antagonist.

Identification of an HIV integrase inhibitor with micromolar affinity for the CGRP receptor led to the discovery of a series of structurally novel CGRP receptor antagonists. Optimization of this series produced compound 16, a low-molecular weight CGRP receptor antagonist with good pharmacokinetic properties in both rat and dog. In contrast to other nonpeptide antagonists, the activity of 16 was affected by the presence of divalent cations and showed evidence of an alternative, RAMP-independent CGRP receptor binding site.

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).

Cerebellar distribution of calcitonin gene-related peptide (CGRP) and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) in rat.

Clinical and experimental results have revealed a fundamental role of calcitonin gene-related peptide (CGRP) in primary headaches. CGRP is widely expressed in neurons both in the central nervous system (CNS) and in peripheral sensory nerves. In the CNS there is a wide distribution of CGRP-containing neurons with the highest levels seen in striatum, amygdale and cerebellum. Moreover, in acute attacks of migraine there is evidence of cerebellar activation. To understand the role of CGRP, antibodies towards the CGRP receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein type 1 (RAMP1) have been developed. In the present study we therefore examined immunohistochemically the distribution of CGRP and its receptor components in the cerebellum. CGRP immunoreactivity was only found intracellularly in the cerebellar Purkinje cell bodies, whereas CLR and RAMP1 were detected on the surface of the Purkinje cell bodies and in their processes. The elaborate dendritic tree of Purkinje cell fibers was distinctly visualized with the RAMP1 antibody. In addition, profoundly stained fibers spanning from the molecular layer into the medulla was observed with the RAMP1 antibody. Judged from the high density of immunoreactive cells expressing CGRP, RAMP1 or CLR, and from the double staining of CGRP and RAMP1 it is likely that most, if not all, Purkinje cells express both the peptide and the receptor components. Double staining with RAMP1 and the glial cell markers glial fibrillary acidic protein (GFAP) and S-100 revealed an almost identical staining pattern of the antibodies in the area of the cell body surfaces. However, as judged by confocal microscopy, no double staining was present. Instead, it was discovered that the glial cells tightly surrounded the Purkinje cells which easily could be interpreted as co-localization in the epifluorescence microscope. Our observations demonstrate that there is a rich expression of CGRP and CGRP receptor elements in the cerebellum which points towards a functional role of CGRP in cerebellar Purkinje cells. Recent advances in the biology of the cerebellum indicate that there may be a role in nociception; hence a target of the recently discovered CGRP receptor antagonists that have demonstrated improvement in migraine pain and associated symptoms could be cerebellar CGRP receptors.

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.

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.

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 (K(i) = 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 (K(D) = 0.06 nM), and the off-rate was determined to be 0.012 min(-1), with a t(1/2) 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.

Discovery of MK-3207: A Highly Potent, Orally Bioavailable CGRP Receptor Antagonist.

Incorporation of polar functionality into a series of highly potent calcitonin gene-related peptide (CGRP) receptor antagonists was explored in an effort to improve pharmacokinetics. This strategy identified piperazinone analogues that possessed improved solubility at acidic pH and increased oral bioavailability in monkeys. Further optimization led to the discovery of the clinical candidate 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) (4), the most potent orally active CGRP receptor antagonist described to date.

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.

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.

The identification of potent, orally bioavailable tricyclic CGRP receptor antagonists.

A series of tricyclic CGRP receptor antagonists was optimized in order to improve oral bioavailability. Attenuation of polar surface area and incorporation of a weakly basic indoline nitrogen led to compound 5, a potent antagonist with good oral bioavailability in three species.