T-type Ca2+ channel inhibitors hold tremendous therapeutic potential for the treatment of pain, epilepsy, sleep disorders, essential tremor and other neurological disorders; however, a lack of truly selective tools has hindered basic research, and selective tools from the pharmaceutical industry are potentially burdened with intellectual property (IP) constraints. Thus, an MLPCN high-throughput screen (HTS) was conducted to identify novel T-type Ca2+ channel inhibitors free from IP constraints, and freely available through the MLPCN, for use by the biomedical community to study T-type Ca2+ channels. While the HTS provided numerous hits, these compounds could not be optimized to the required level of potency to be appropriate tool compounds. Therefore, a scaffold hopping approach, guided by SurflexSim, ultimately afforded ML218 (CID 45115620) a selective T-Type Ca2+ (Cav3.1, Cav3.2, Cav3.3) inhibitor (Cav3.2, IC50 = 150 nM in Ca2+ flux; Cav3.2 IC50 = 310 nM and Cav3.3 IC50 = 270 nM, respectively in patch clamp electrophysiology) with good DMPK properties, acceptable in vivo rat PK and excellent brain levels. Electrophysiology studies in subthalamic nucleus (STN) neurons demonstrated robust effects of ML218 on the inhibition of T-Type calcium current, inhibition of low threshold spike and rebound burst activity. Based on the basal ganglia circuitry in Parkinson’s disease (PD), the effects of ML218 in STN neurons suggest a therapeutic role for T-type Ca2+ channel inhibitors, and ML218 was found to be orally efficacious in haloperidol-induced catalepsy, a preclinical PD model, with comparable efficacy to an A2A antagonist, a clinically validated PD target. ML218 proves to be a powerful new probe to study T-Type Ca2+ function in vitro and in vivo, and freely available.

ABSTRACT:Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu 5 ) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu 5 . Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (؉16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18 Olabeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (؉18 Da) as well as in a secondary metabolite (M2; ؉36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates.

Inflammation and insulin resistance are characteristics of endotoxemia. While the role of interleukin-6 (IL-6) in insulin resistant states has been characterized, little is known of its role in the metabolic response to inflammation. To study the role of IL-6, conscious chronically catheterized mice were used. Five days prior to being studied, catheters were implanted in the carotid artery and jugular vein. After a 5 h fast, E. coli (250 μg/mouse) LPS was injected in IL6 −/− (KO; n=13), and IL-6 +/+ (WT; n=10) littermates. The IL-6 response to LPS was simulated in an additional group of KO mice (KO+IL6; n=10). IL-6 increased in WT (15±0.7 ng/ml) 4h after LPS and was undetectable in KO. IL-6 replacement in the KO restored circulating IL-6 to levels observed in the WT group (14±0.3ng/ml). Tumor necrosis factor-α (TNF-α) increased more rapidly in WT than in both KO and KO+IL6. KO exhibited a more profound glucose excursion 30 min after LPS injection and no apparent hypoglycemia at 4h (95±5 vs. 70±8 mg/dl; KO vs. WT), despite having a blunted glucagon and epinephrine response. Glucose levels in KO+IL6, while decreased (93±4 mg/dl) at 4h, remained higher than WT. In summary, the absence of IL-6 protected against LPS induced hypoglycemia. Acute restoration of the IL-6 response to LPS did not potentiate hypoglycemia but partially restored the glucagon response. Thus, while IL-6 promotes glucose intolerance in insulin resistant states, IL-6 promotes hypoglycemia during acute inflammation.