In Vitro and in Vivo Properties of 3-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), a GABAA Receptor 5 Subtype- Selective Inverse Agonist

3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4triazol-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-triazol4-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 [C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance 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 corresponding 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 pharmacokinetics, precluded its further development. This work was conducted while all authors were employees of Merck and Co., Inc. 1 Current affiliation: Johnson and Johnson Pharmaceutical Research and Development, Beerse, Belgium. 2 Current affiliation: BTG International, London, United Kingdom. 3 Current affiliation: Eli-Lilly, Windlesham, Surrey, United Kingdom. 4 Current affiliation: Novartis, Horsham, Surrey, United Kingdom. 5 Current affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey. 6 Current affiliation: Pfizer Global Research and Development, Sandwich, Kent, United Kingdom. 7 Current affiliation: BioFocus, Saffron Walden, Essex, United Kingdom. 8 Current affiliation: P1Vital, University of Oxford, Oxford, United Kingdom. 9 Current affiliation: Centocor R&D USA, Malvern, Pennsylvania. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.157636. ABBREVIATIONS: DMCM, methyl-6,7-dimethoxy-4-ethyl-carboline-3-carboxylate; L-655,708, ethyl[S]-11,12,13,13a-tetrahydro-7-methoxy-9-oxo9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxlate; 5IA, 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine; RO4938581, 3-bromo-10-difluoromethyl-9H-imidazo(1,5-a)(1,2,4)triazolo(1,5-d)(1,4)benzodiazepine; MRK-016, 3-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; FG-7142, N-methyl-carboline-3-carboxamide; Ro 15-1788, [flumazenil, 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylic acid, ethyl ester]; Ro 15-4513, ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate; PPF, paired pulse facilitation; LTP, long-term potentiation; fEPSP, field excitatory postsynaptic potential; ANOVA, analysis of variance; Occ50, dose required to produce 50% occupancy; PET, positron emission tomography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; ROI, region of interest; PTZ, pentylenetetrazole; AUC, area under the curve; AE, adverse event. 0022-3565/09/3312-470–484$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 2 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 157636/3527847 JPET 331:470–484, 2009 Printed in U.S.A. 470 at A PE T Jornals on M ay 3, 2016 jpet.asjournals.org D ow nladed from Classical benzodiazepines, such as diazepam (Valium), exert their effects via GABAA receptors that are GABA-gated chloride ion channels. These receptors are pentameric assemblies of members of the GABAA gene family ( 1–6, 1–3, 1–3, , ε, , and ), the most abundant of which contain , , and subunits in a 2:2:1 stoichiometry (Barnard et al., 1998). Benzodiazepines interact with GABAA receptors containing either an 1, 2, 3, or 5 subunit along with and 2 subunits, and their specific recognition site occurs at the interface of the subunit and 2 subunit. Clinically used benzodiazepines have agonist activity at the benzodiazepine binding site in that they enhance the inhibitory effects of GABA (positive allosteric modulation), thereby reducing neuronal excitability (Sieghart, 1995, 2006). In contrast, benzodiazepine site inverse agonists (negative allosteric modulators), the prototypic example of which is the -carboline DMCM, attenuate the inhibitory effects of GABA, with a consequent increase in neuronal excitability. The opposing effects of benzodiazepine site agonists and inverse agonists at the molecular level are reflected in vivo. Hence, benzodiazepine site agonists possess a variety of pharmacological actions in human, including anxiolytic, sedative, myorelaxant, anticonvulsant, and cognition-impairing activities (Argyropoulos and Nutt, 1999; Buffett-Jerrott and Stewart, 2002), whereas inverse agonists are anxiogenic and convulsant or proconvulsant and also enhance vigilance and improve cognitive performance in preclinical species (Sieghart, 2006). Unfortunately, the anxiogenic and proconvulsant properties of nonselective inverse agonists have precluded their clinical use as cognition and/or vigilance enhancers (Dorow et al., 1983; Horowski and Dorow, 2002). Clearly, however, a compound that retained the cognition-enhancing properties yet was devoid of the anxiogenic and proconvulsant liabilities would offer the potential for clinical utility. The use of molecular genetic (knockout and point-mutated mice) and pharmacological (subtype-selective compound) approaches has begun to define which of the pharmacological features of benzodiazepine site ligands are associated with particular GABAA receptor subtypes (Rudolph et al., 1999; McKernan et al., 2000; Rudolph and Möhler, 2004). As a consequence, molecular targets for the development of compounds that selectively interact with specific GABAA receptor subtypes and that therefore have unique pharmacological profiles relative to the nonselective benzodiazepines have been identified (McKernan et al., 2000; Dias et al., 2005; Atack et al., 2006b). With respect to cognition, the 5 subtype is preferentially localized within the hippocampus (Fritschy and Mohler, 1995; Sur et al., 1999) and has been shown to play a role in aspects of cognitive performance (Collinson et al., 2002; Crestani et al., 2002; Maubach, 2003). More specifically, compounds that possess either 5-selective affinity, 5-selective efficacy, or a mixture of both these features (e.g., L-655,708, 5IA, and RO4938581, respectively) have been shown to enhance cognition in rodents (Atack et al., 2006a; Collinson et al., 2006; Dawson et al., 2006; Ballard et al., 2009) as well as primates (Ballard et al., 2009). Regarding human data, 5IA did not improve performance in the paired-associate learning task in normal elderly (mean age, 72 years) volunteers (indeed, if anything, performance got worse; Atack, 2009), but it did attenuate the ethanol-induced impairment of word recall in healthy young (25-year-old) volunteers (Nutt et al., 2007). However, due to preclinical hepatotoxicity issues, development of this compound was stopped (Atack, 2009). Here, we provide a more detailed description of the in vitro and in vivo properties of the back-up compound MRK-016, an initial characterization of which has been presented previously (compound 13 in Chambers et al., 2004). Materials and Methods