Restless running behavior was first described
‘Restless running behavior’ was first described in mice after administration of 30mg/kg i.p. ALX-5407 and was thought to be due to an augmentation of NMDA-induced dopamine activity . The motor impairments caused by ALX-5407 were confirmed by Perry et al.  and extended to another sarcosine-derived compound, LY2365109. The hallmark of the behavior was a compulsive walking even when confronted with an obstacle, such as the corner of the cage, where the animals continued to walk in place while pushing their heads into the corner. The mice were capable of walking in any direction in the circular arena, and conducted no other exploratory or grooming behavior. This behavior is reminiscent of the syndrome of obstinate progression originally reported in cats with lesions of the nucleus interpeduncularis  and in 6-hydroxydopamine lesioned rats after atropine treatment . Similar effects were observed in the present study after dosing with ALX-5407 and (S)-13h. The OP produced by ALX-5407 was striking for the apparent time- and dose-dependence of the effect, which may be due to the pseudo-irreversible nature of ALX-5407 binding to GlyT1 , , . Lower doses of ALX-5407 presumably occupied a lower percentage of the available GlyT1, and therefore the rise in glycine concentration required to produce OP was delayed. Consistent with the time delay for onset of OP, 6mg/kg ALX-5407 was ineffective in PPI when tested 2h post dosing, but produced efficacy comparable to 20mg/kg ALX-5407 when tested 4 and 6h post dosing. The delay in OP of up to 3h at 10mg/kg is of interest, as all reports of efficacy in PPI at 10mg/kg have been reported 2h post dosing , . For ALX-5407, the time delay between the generation of efficacy in PPI and the adverse effect of OP provides a plausible explanation as to how such a robust effect as OP did not confound the PPI results in earlier studies , . For (S)-13h, there was a much shorter time delay between efficacy in PPI, tested at 0–30min after dosing, and the adverse effect of OP, observed at 30–90min post dosing. These observations suggest that Ivabradine HCl glycine concentrations achieved efficacious levels at the time of testing in the PPI assay, but continued to increase, ultimately reaching concentrations that induced OP for both ALX-5407 and (S)-13h. It is also important to note that OP and increases in PPI were produced by similar doses (6–20mg/kg ALX-5407 and 10–20mg/kg (S)-13h), thus indicating no separation between efficacious doses and those causing adverse effect. The apparent recovery from OP observed over time with 10 and 30mg/kg ALX-5407 must be interpreted with caution since these mice developed the more advanced symptoms of lateral recumbency and respiratory distress , , . Therefore, the return to baseline levels of locomotor activity was not recovery, but a worsening of adverse effect. The adverse effects of respiratory distress and hypotonia were also observed in GlyT1 homozygous knockout mice leading to death on postnatal day 1 . The symptoms in the GlyT1 knockout mice resembled the human condition of nonketotic hyperglycinemia, in which a defect in glycine metabolism leads to high levels of glycine in the central nervous system, respiratory insufficiency and death . Roche-7, sarcosine, and d-serine, while they did not produce OP, did produce hypoactivity at doses similar to or higher than doses that increased percent PPI. Hypoactivity may be seen as a potential side effect. However, sarcosine and d-serine were well-tolerated clinically, and there were no significant adverse effects as compared to placebo, particularly with regard to sedation and fatigue , . Thus, the hypoactivity observed herein may represent a tolerable effect. Obstinate progression appeared to be mediated by the action of the elevated glycine concentration acting at the glycinergic receptor, because strychnine, a glycinergic receptor antagonist, inhibited OP induced by (S)-13h. However, direct activation of the NMDA receptor with d-serine did not induce OP, suggesting that activation of glutamatergic networks is not involved with OP. These data are in agreement with results reported by Perry et al. , where ALX-5407 blocked strychnine-induced elevations in cGMP levels in mouse cerebellum.