Fosmetpantotenate (RE-024), a phosphopantothenate replacement therapy for pantothenate kinase-associated neurodegeneration: Mechanism of action and efficacy in nonclinical models.

In cells, phosphorylation of pantothenic acid to generate phosphopantothenic acid by the pantothenate kinase enzymes is the first step in coenzyme A synthesis. Pantothenate kinase 2, the isoform localized in neuronal cell mitochondria, is dysfunctional in patients with pantothenate kinase-associated neurodegeneration. Fosmetpantotenate is a phosphopantothenic acid prodrug in clinical development for treatment of pantothenate kinase-associated neurodegeneration, which aims to replenish phosphopantothenic acid in patients. Fosmetpantotenate restored coenzyme A in short-hairpin RNA pantothenate kinase 2 gene-silenced neuroblastoma cells and was permeable in a blood-brain barrier model. The rate of fosmetpantotenate metabolism in blood is species-dependent. Following up to 700 mg/kg orally, blood exposure to fosmetpantotenate was negligible in rat and mouse, but measurable in monkey. Consistent with the difference in whole blood half-life, fosmetpantotenate dosed orally was found in the brains of the monkey (striatal dialysate) but was absent in mice. Following administration of isotopically labeled-fosmetpantotenate to mice, ~40% of liver coenzyme A (after 500 mg/kg orally) and ~50% of brain coenzyme A (after 125 µg intrastriatally) originated from isotopically labeled-fosmetpantotenate. Additionally, 10-day dosing of isotopically labeled-fosmetpantotenate, 12.5 µg, intracerebroventricularly in mice led to ~30% of brain coenzyme A containing the stable isotopic labels. This work supports the hypothesis that fosmetpantotenate acts to replace reduced phosphopantothenic acid in pantothenate kinase 2-deficient tissues.

The discovery of novel 5,6,5- and 5,5,6-tricyclic pyrrolidines as potent and selective DPP-4 inhibitors.

Novel potent and selective 5,6,5- and 5,5,6-tricyclic pyrrolidine dipeptidyl peptidase IV (DPP-4) inhibitors were identified. Structure-activity relationship (SAR) efforts focused on improving the intrinsic DPP-4 inhibition potency, increasing protease selectivity, and demonstrating clean ion channel and cytochrome P450 profiles while trying to achieve a pharmacokinetic profile suitable for once weekly dosing in humans.

Structure-activity-relationship of amide and sulfonamide analogs of omarigliptin.

A series of novel substituted-[(3R)-amino-2-(2,5-difluorophenyl)]tetrahydro-2H-pyran analogs have been prepared and evaluated as potent, selective and orally active DPP-4 inhibitors. These efforts lead to the discovery of a long acting DPP-4 inhibitor, omarigliptin (MK-3102), which recently completed phase III clinical development and has been approved in Japan.

A single-run liquid chromatography mass spectrometry method to quantify neuroactive kynurenine pathway metabolites in rat plasma.

Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are associated with neurodegenerative disorders. Tryptophan is transported across the blood-brain barrier and converted via the kynurenine pathway to N-formyl-L-kynurenine, which is further degraded to L-kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties. The association of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in analytical methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC-MS/MS method to quantify L-kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy-L-kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chemical properties, major efforts were devoted to develop a chromatography suitable for all metabolites that involves plasma protein precipitation with acetonitrile followed by chromatographic separation by C18 RP chromatography, detected by electrospray mass spectrometry. Quantitation range was 0.098-100 ng/ml for 3HK, 9.8-20,000 ng/ml for KYN, 0.49-1000 ng/ml for KYNA and AA. The method was linear (r>0.9963) and validation parameters were within acceptance range (calibration standards and QC accuracy within ±30%).

A novel benzazepinone sodium channel blocker with oral efficacy in a rat model of neuropathic pain.

A series of benzazepinones were synthesized and evaluated for block of Nav1.7 sodium channels. Compound 30 from this series displayed potent channel block, good selectivity versus other targets, and dose-dependent oral efficacy in a rat model of neuropathic pain.

Improved Cav2.2 Channel Inhibitors through a gem-Dimethylsulfone Bioisostere Replacement of a Labile Sulfonamide.

We report the investigation of sulfonamide-derived Cav2.2 inhibitors to address drug-metabolism liabilities with this lead class of analgesics. Modification of the benzamide substituent provided improvements in both potency and selectivity. However, we discovered that formation of the persistent 3-(trifluoromethyl)benzenesulfonamide metabolite was an endemic problem in the sulfonamide series and that the replacement of the center aminopiperidine scaffold failed to prevent this metabolic pathway. This issue was eventually addressed by application of a bioisostere strategy. The new gem-dimethyl sulfone series retained Cav2.2 potency without the liability of the circulating sulfonamide metabolite.

Aminopiperidine sulfonamide Cav2.2 channel inhibitors for the treatment of chronic pain.

The voltage-gated calcium channel Ca(v)2.2 (N-type calcium channel) is a critical regulator of synaptic transmission and has emerged as an attractive target for the treatment of chronic pain. We report here the discovery of sulfonamide-derived, state-dependent inhibitors of Ca(v)2.2. In particular, 19 is an inhibitor of Ca(v)2.2 that is selective over cardiac ion channels, with a good preclinical PK and biodistribution profile. This compound exhibits dose-dependent efficacy in preclinical models of inflammatory hyperalgesia and neuropathic allodynia and is devoid of ancillary cardiovascular or CNS pharmacology at the doses tested. Importantly, 19 exhibited no efficacy in Ca(v)2.2 gene-deleted mice. The discovery of metabolite 26 confounds further development of members of this aminopiperidine sulfonamide series. This discovery also suggests specific structural liabilities of this class of compounds that must be addressed.

Metabolism and pharmacokinetics of JM6 in mice: JM6 is not a prodrug for Ro-61-8048.

Understanding whether regulation of tryptophan metabolites can ameliorate neurodegeneration is of high interest to investigators. A recent publication describes 3,4-dimethoxy-N-(4-(3-nitrophenyl)-5-(piperidin-1-ylmethyl)thiazol-2-yl)benzenesulfonamide (JM6) as a novel prodrug for the kynurenine 3-monooxygenase (KMO) inhibitor 3,4-dimethoxy-N-(4-(3-nitrophenyl)thiazol-2-yl)benzenesulfonamide (Ro-61-8048) that elicits therapeutic effects in mouse models of Huntington's and Alzheimer's diseases (Cell 145:863-874, 2011). Our evaluation of the metabolism and pharmacokinetics of JM6 and Ro-61-8048 indicate instead that Ro-61-8048 concentrations in mouse plasma after JM6 administration originate from a Ro-61-8048 impurity (<0.1%) in JM6. After a 0.05 mg/kg Ro-61-8048 oral dose alone or coadministered with 10 mg/kg JM6 to mice, the Ro-61-8048 areas under the concentration-time curves (AUCs) from 0 to infinity were similar (4300 and 4900 nM × h, respectively), indicating no detectable contributions of JM6 metabolism to the Ro-61-8048 AUCs. JM6 was stable in incubations under acidic conditions and Ro-61-8048 was not a product of JM6 metabolism in vitro (plasma, blood, or hepatic models). Species differences in the quantitative rate of oxidative metabolism indicate that major circulating JM6 metabolite(s) in mice are unlikely to be major in humans: JM6 is rapidly metabolized via the piperidyl moiety in mouse (forming an iminium ion reactive intermediate) but is slowly metabolized in human (in vitro), primarily via O-dealkylation at the phenyl ring. Our data indicate that JM6 is not a prodrug for Ro-61-8048 and is not a potent KMO inhibitor.

Pyridinesulfonylureas and pyridinesulfonamides as selective bombesin receptor subtype-3 (BRS-3) agonists.

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein coupled receptor belonging to the subfamily of bombesin-like receptors. BRS-3 is implicated in the development of obesity and diabetes. We report here small-molecule agonists that are based on a 4-(alkylamino)pyridine-3-sulfonamide core. We describe the discovery of 2a, which has mid-nanomolar potency, selectivity for human BRS-3 versus the other bombesin-like receptors, and good bioavailability.

Synthesis and evaluation of [(1R)-1-amino-2-(2,5-difluorophenyl)ethyl]cyclohexanes and 4-[(1R)-1-amino-2-(2,5-difluorophenyl)ethyl]piperidines as DPP-4 inhibitors.

A series of 4-amino cyclohexanes and 4-substituted piperidines were prepared and evaluated for inhibition of DPP-4. Analog 20q displayed both good DPP-4 potency and selectivity against other proteases, while derivative 20k displayed long half life and modest oral bioavailability in rat. The most potent analog, 3-(5-aminocarbonylpyridyl piperidine 53j, displayed excellent DPP-4 activity with good selectivity versus other proline enzymes.

A potent and selective indole N-type calcium channel (Ca(v)2.2) blocker for the treatment of pain.

N-type calcium channels (Ca(v)2.2) have been shown to play a critical role in pain. A series of low molecular weight 2-aryl indoles were identified as potent Ca(v)2.2 blockers with good in vitro and in vivo potency.

Discovery of MK-5046, a Potent, Selective Bombesin Receptor Subtype-3 Agonist for the Treatment of Obesity.

We report the development and characterization of compound 22 (MK-5046), a potent, selective small molecule agonist of BRS-3 (bombesin receptor subtype-3). In pharmacological testing using diet-induced obese mice, compound 22 caused mechanism-based, dose-dependent reductions in food intake and body weight.

Pharmacokinetics of memantine in rats and mice.

To evaluate the potential of memantine as a therapeutic agent for Huntington's disease (HD) we have undertaken a series of in vitro, ex vivo and whole animal studies to characterize its pharmacokinetics (PK) and pharmacodynamics (PD) in rats and mice. Results from these studies will enable determination of memantine exposures needed to engage the related functional PD marker and help predict the dose regimen for clinical trials to test its proposed mechanism of action; the selective blockade of extrasynaptic, but not synaptic, NMDA receptors. The studies reported here describe the PK of memantine in rats and mice at low (1 mg/kg) and high (10 mg/kg) doses. Our studies indicate that the clearance mechanisms of memantine in rats and mice are different from those in human, and that clearance needs to be taken into account when extrapolating to the human. In rats only, there is a significant metabolic contribution to memantine clearance at lower dose levels. While memantine is primarily cleared renally in all three species, the proportion of total systemic clearance above the glomerular filtration rate (GFR) is much higher in rats and mice (~13, 4.5, and 1.4 times higher than GFR in rats, mice, and humans, respectively), suggesting that the contribution of active transport to memantine elimination in rats and mice is more significant than in the human. In rats and mice, memantine had a short half-life (<4 h) and steep Cmax/Cmin ratios (>100). In the human, the half-life of memantine was reported to be very long (60-80 h) with a Cmax/Cmin ratio at steady state concentrations of ~1.5. A small change in the clearance of memantine - for example due to renal impairment or competition for the elimination pathway with a co-administered drug - will likely affect exposure and, therefore, the selectivity of memantine on NMDA receptors . The PK differences observed between these species demonstrate that the PK in mice and rats cannot be directly extrapolated to the human. Further, the relationship between the plasma concentration (and therefore dose) needed to elicit a mechanism-related in vivo functional effect (PD readout) while maintaining the selectivity of the extrasynaptic blockade of the NMDA receptors needs to be established before clinical trials can be appropriately planned.

Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain.

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.

Analgesic effects of a substituted N-triazole oxindole (TROX-1), a state-dependent, voltage-gated calcium channel 2 blocker.

Voltage-gated calcium channel (Ca(v))2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca(v)2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1), as a small-molecule, state-dependent blocker of Ca(v)2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca(v)2.2 channels under depolarized conditions (IC(50) = 0.27 microM) compared with hyperpolarized conditions (IC(50) > 20 microM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited omega-conotoxin GVIA-sensitive calcium currents (Ca(v)2.2 channel currents), with greater potency under depolarized conditions (IC(50) = 0.4 microM) than under hyperpolarized conditions (IC(50) = 2.6 microM), indicating state-dependent Ca(v)2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca(v)2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca(v)2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca(v)2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20- to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca(v)2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.

Synthesis of 7-benzyl-5-(piperidin-1-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c][2,7]naphthyridin-1-ylamine and its analogs as bombesin receptor subtype-3 agonists.

The original structure of a high-throughput screening hit obtained from an external vendor was revised based on multiple NMR studies. The active compound was re-synthesized via a novel route and its structure and biological activity as a BRS-3 agonist were unambiguously confirmed. Multi-gram quantities of the hit were prepared for pharmacokinetic and efficacy studies. The synthetic strategy allowed for the preparation of multiple analogs for SAR exploration.

A peripherally acting Na(v)1.7 sodium channel blocker reverses hyperalgesia and allodynia on rat models of inflammatory and neuropathic pain.

BACKGROUND: Voltage-gated sodium channels (Na(v)1) are expressed in primary sensory neurons where they influence excitability via their role in the generation and propagation of action potentials. Recently, human genetic data have shown that one sodium channel subtype, Na(v)1.7, plays a major role in pain. We performed these studies to characterize the antinociceptive effects of N-[(R)-1-((R)-7-chloro-1-isopropyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-ylcarbamoyl)-2-(2-fluorophenyl)-ethyl]-4-fluoro-2-trifluoromethyl-benzamide (BZP), a non-central nervous system (CNS) penetrant small molecule with high affinity and preferential selectivity for Na(v)1.7 over Na(v)1.8 and Na(v)1.5. METHODS: BZP was evaluated in rat preclinical models of inflammatory and neuropathic pain and compared with standard analgesics. Two models were used: the complete Freund's adjuvant model of inflammatory pain and the spinal nerve ligation model of neuropathic pain. BZP was also evaluated in a motor coordination assay to assess its propensity for CNS side effects. RESULTS: In preclinical models of chronic pain, BZP displayed efficacy comparable with that of leading analgesics. In the complete Freund's adjuvant model, BZP produced reversal of hyperalgesia comparable with nonsteroidal antiinflammatory drugs, and in the spinal nerve ligation model, BZP produced reversal of allodynia comparable with gabapentin and mexiletine. Unlike the CNS penetrant compounds gabapentin and mexiletine, BZP did not induce any impairment of motor coordination. CONCLUSIONS: These data suggest that a peripherally acting sodium channel blocker, preferentially acting through Na(v)1.7, could provide clinical relief of chronic pain without the CNS side effects typical of many existing pain treatments.

Discovery of isoxazole voltage gated sodium channel blockers for treatment of chronic pain.

A series of novel isoxazole voltage gated sodium channel blockers have been synthesized and evaluated. Substitutions on the benzylic position of benzamide were investigated to determine their effect on Na(v)1.7 inhibitory potency. The spirocyclobutyl substitution had the most significant enhancement on Na(v)1.7 inhibitory activity.

Discovery of a novel class of isoxazoline voltage gated sodium channel blockers.

Analogs of the previously reported voltage gated sodium channel blocker CDA54 were prepared in which one of the amide functions was replaced with aromatic and non-aromatic heterocycles. Replacement of the amide with an aromatic heterocycle resulted in significant loss of sodium channel blocking activity, while non-aromatic heterocycle replacements were well tolerated.