In Vitro and in Vivo Properties of 3-tert-Butyl-7-(5-methylisoxazol-

tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4- triazol-5-ylmethoxy)-pyrazolo(1,5-d)(1,2,4)triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human 1-, 2-, 3-, and 5-containing GABAA receptors. It has inverse agonist efficacy selective for the 5 subtype, and this 5 inverse agonism is greater than that of the prototypic 5-selective compound 3-(5-methylisoxazol-3-yl)-6-((1-methyl-1,2,3-triazol- 4-hdyl)methyloxy)-1,2,4-triazolo(3,4-a)phthalazine (5IA). Consistent with its greater 5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than 5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC50 value of 15 ng/ml that was similar to the rhesus monkey plasma EC50 value of 21 ng/ml obtained using ( 11 C)flumazenil positron emission tomogra- phy. In normal rats, MRK-016 enhanced cognitive perfor- mance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3-0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species (3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corre- sponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human phar- macokinetics, precluded its further development.