Mechanism based neurotoxicity of mGlu5 positive allosteric modulators--development challenges for a promising novel antipsychotic target.

Previous work has suggested that activation of mGlu5 receptor augments NMDA receptor function and thereby may constitute a rational approach addressing glutamate hypofunction in schizophrenia and a target for novel antipsychotic drug development. Here, we report the in vitro activity, in vivo efficacy and safety profile of 5PAM523 (4-Fluorophenyl){(2R,5S)-5-[5-(5-fluoropyridin-2-yl)-1,2,4-oxadiazol-3-yl]-2-methylpiperidin-1-yl}methanone), a structurally novel positive allosteric modulator selective of mGlu5. In cells expressing human mGlu5 receptor, 5PAM523 potentiated threshold responses to glutamate in fluorometric calcium assays, but does not have any intrinsic agonist activity. 5PAM523 acts as an allosteric modulator as suggested by the binding studies showing that 5PAM523 did not displace the binding of the orthosteric ligand quisqualic acid, but did partially compete with the negative allosteric modulator, MPyEP. In vivo, 5PAM523 reversed amphetamine-induced locomotor activity in rats. Therefore, both the in vitro and in vivo data demonstrate that 5PAM523 acts as a selective mGlu5 PAM and exhibits anti-psychotic like activity. To study the potential for adverse effects and particularly neurotoxicity, brain histopathological exams were performed in rats treated for 4 days with 5PAM523 or vehicle. The brain exam revealed moderate to severe neuronal necrosis in the rats treated with the doses of 30 and 50 mg/kg, particularly in the auditory cortex and hippocampus. To investigate whether this neurotoxicity is mechanism specific to 5PAM523, similar safety studies were carried out with three other structurally distinct selective mGlu5 PAMs. Results revealed a comparable pattern of neuronal cell death. Finally, 5PAM523 was tested in mGlu5 knock-out (KO) and wild type (WT) mice. mGlu5 WT mice treated with 5PAM523 for 4 days at 100 mg/kg presented significant neuronal death in the auditory cortex and hippocampus. Conversely, mGlu5 KO mice did not show any neuronal loss by histopathology, suggesting that enhancement of mGlu5 function is responsible for the toxicity of 5PAM523. This study reveals for the first time that augmentation of mGlu5 function with selective allosteric modulators results in neurotoxicity.

[Macrophage migration-inhibitory factors expression and its effects on proliferation in human dental pulps].

OBJECTIVE: To investigate the expression of macrophage migration-inhibitory factors (MIF) in clinically healthy and inflamed human pulp tissues and the effects of rhMIF on the proliferation of human dental pulp cells (HDPC). METHODS: Immunohistochemistry was used to detect the localization of MIF expression in clinically healthy pulp and inflamed pulp tissues. Quantitative real-time polymerase chain reaction (PCR) was performed to evaluate the mRNA levels of MIF in pulp specimens. In addition, the culture supernatants of HDPC were collected after HDPC was stimulated by lipopolysaccharide (LPS) for 24 h, and then the MIF levels were assayed by quantitative sandwich enzyme-linked immunosorbent assay. Meanwhile, the effects of rhMIF on the proliferation of HDPC at different concentrations for 24 and 48 h were observed by cell counting kit-8 (CCK-8). RESULTS: MIF was mainly distributed in odontoblasts of healthy pulp tissue, however, in inflamed pulp tissue, it was widely detected in fibroblasts, inflammatory infiltrates and endothelial cells as well as odontoblasts. Quantitative real-time PCR showed that there was no significant difference in MIF mRNA levels between inflamed pulps and healthy pulps (P > 0.05). Additionally, the secretion of MIF was significantly increased by stimulation with LPS at the concentration of 0.1 and 1.0 mg/L [(1772.58 ± 495.05), (1692.58 ± 337.45) ng/L] (P < 0.05), and the concentration was (1048.53 ± 161.81) ng/L in control group. rhMIF stimulated the HDPC's proliferation at the concentration of 10, 30, 60 µg/L for 24 and 48 h. CONCLUSIONS: MIF was expressed in pulp tissue and its expression was increased after stimulation by LPS. rhMIF increased the proliferation of HDPC. These results suggest that MIF may be involved in the process of pulpal inflammation.

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.

Potent benzimidazolone-based CGRP receptor antagonists.

The previously disclosed spirohydantoin-based CGRP receptor antagonists were optimized for potency through modification of the benzimidazolone substituents. Compounds were identified which had minimal shift in the cAMP functional assay containing 50% human serum. Blockade of CGRP-mediated vasodilation was observed with these compounds in a rhesus pharmacodynamic assay and the in vivo potency correlated with the in vitro activity in the serum-shifted functional assay.

Identification of novel, orally bioavailable spirohydantoin CGRP receptor antagonists.

A rapid analogue approach to identification of spirohydantoin-based CGRP antagonists provided novel, low molecular weight leads. Modification of these leads afforded a series of nanomolar benzimidazolinone-based CGRP receptor antagonists. The oral bioavailability of these antagonists was inversely correlated with polar surface area, suggesting that membrane permeability was a key limitation to absorption. Optimization provided compound 12, a potent CGRP receptor antagonist (K(i)=21nM) with good oral bioavailability in three species.