EC50: 3 nM (GABAA α5)[1] Ki: 0.83 nM (Human GABAA α1β3γ2), 0.85 nM (Human GABAA α2β3γ2), 0.77 nM (Human GABAA α3β3γ2), 1.4 nM (Human GABAA α5β3γ2)[1]
MRK‑016 targets the α5‑containing GABAA receptor as an inverse agonist at the benzodiazepine site, with EC50 of 3 nM for GABAA α5 and Ki values of 0.83, 0.85, 0.77, and 1.4 nM for human α1β3γ2, α2β3γ2, α3β3γ2, and α5β3γ2, respectively, with weak affinity for α4β3γ2 (Ki 395 nM) and inactive at α6β3γ2 (Ki >4000 nM).

With an EC50 of 3 nM for GABAA α5, and Kis of 0.83, 0.85, 0.77, and 1.4 nM for human GABAA α1β3η2, GABAA α2β3η2, GABAA α3β3η2, and GABAA α5β3γ2, respectively, MRK-016 is a selective, orally bioavailable inverse agonist of GABAA α5 receptor. As a complete inverse agonist at the α5-subtype, MRK-016 exhibits very low affinity for the GABAA α4β3γ2-subtype (Ki 395 ± 173 nM), and at the GABAA α6β3γ2 receptor (Ki > 4000 nM), it is basically inactive[1]. At 400 nM, MRK-016 had a negligible impact on GABAA α4β3γ2. In mouse hippocampus slices, MRK-016 (100 nM) alao enhances long-term potentiation[2].

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In vitro, MRK‑016 acts as a complete inverse agonist at α5‑GABAA receptors; at 400 nM it has negligible impact on α4β3γ2. In mouse hippocampal slices, 100 nM MRK‑016 enhances long‑term potentiation, a cellular correlate of learning and memory, indicating its cognitive‑enhancing potential through modulation of synaptic plasticity.

For 20 days in mice, MRK-016 did not generate seizures at 30 mg/kg by po or worsen convulsions induced by pentylenetetrazole at 10 mg/kg via ip. In rats, MRK-016 does not exhibit overt anxiogenic-like effects at dosages that occupy more than 95% of the binding sites for benzodiazepines (BZs). In rats, MRK-016 (0.3, 1, and 3 mg/kg, po) dose-dependently enhances hippocampal-dependent memory task performance[1]. In rats, MRK-016 (0.3–30 mg/kg, po) results in excellent receptor occupancy. The delayed matching-to-position version of the Morris water maze exhibits cognitive-enhancing activity when administered at doses of 0.3, 1, or 3 mg/kg po. Mice treated with MRK -016 (1, 3, or 10 mg/kg ip) do not produce kindling[2]. In mice, MRK-016 (3 mg/kg, ip) prevents learning and memory deficits brought on by LPS[3].

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In vivo, MRK‑016 does not induce seizures in mice at 30 mg/kg (po) nor worsen pentylenetetrazole‑induced convulsions at 10 mg/kg (ip); in rats it shows no anxiogenic effects at doses occupying >95% of benzodiazepine sites. It dose‑dependently improves hippocampal‑dependent memory at 0.3–3 mg/kg (po) and protects against LPS‑induced learning deficits at 3 mg/kg (ip), without producing kindling.

Compound 13 (MRK-016) has high in vitro binding affinity 32 at all four BZ-sensitive GABAA receptor subtypes, ranging from 0.8 to 1.4 nM. It has very weak affinity at the GABAA α4β3γ2-subtype (Ki 395 ± 173 nM) and is essentially inactive at the GABAA α6β3γ2 receptor (Ki > 4000 nM). Furthermore, when examined in 147 radioligand binding and enzyme assays, 3313 showed no significant off-target activity (IC50 values > 10 μM). The efficacy values shown in Table 1 were determined using whole cell patch clamp recordings 34 from mouse fibroblast cells stably expressing the human GABAA receptor subtypes.32 The in vitro efficacy is measured as the percentage maximum modulation of the GABA-evoked current using a submaximal (EC20) GABA concentration. Positive values represent a potentiation of the GABA-induced current (agonist) whereas negative values represent an attenuation (inverse agonist). The values for 13 are compared to the clinical compound 5, the nonselective full inverse agonist 2,9 and the nonselective full agonist chlordiazepoxide (CDZ; 14). 13 is a full inverse agonist at the α5-subtype and is functionally selective over the α1-, α2-, and α3-subtypes. In mouse fibroblast L(tk-) cells expressing the α5β3γ2-subtype, 13 attenuates the GABA-evoked current by 55% (efficacy = −55%), which is essentially identical to that of the full inverse agonist 2 (efficacy = −57%) and greater than that produced by 5 (efficacy = −40%). In contrast, efficacy at the α1-, α2-, and α3-subtypes is much lower, with respective efficacy values of −16%, +6%, and −9% being in the range of weak partial inverse agonists or antagonists. The efficacy of 13 at the α1- and α3-subtypes is comparable with the recordings obtained for 5; whereas at the α2-subtype 13 is essentially an antagonist (efficacy = +6%), 5 exhibits partial agonism (efficacy = +16%). Indeed, in our quest for a compound with full inverse agonism at the α5-subtype and little functional activity at the other GABAA receptor subtypes, pyrazolotriazine 13 has a more impressive efficacy profile compared to the clinical compound 5. The EC50 value of 13 at the α5-subtype is 3.0 nM, which complements its in vitro binding affinity of 1.4 nM[1].

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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) 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 α1-, α2-, α3-, and α5-containing GABAA receptors. It has inverse agonist efficacy selective for the α5 subtype, and this α5 inverse agonism is greater than that of the prototypic α5-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 (α5IA). Consistent with its greater α5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than α5IA[2].

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The binding affinity for recombinant human GABAA receptors is determined by radioligand binding using [³H]flumazenil and membrane preparations from cells expressing specific subunits; non‑specific binding is defined with excess competing ligand, and Ki values are calculated from competition curves after filtration and scintillation counting.

Cell‑based functional assays use recombinant cell lines expressing GABAA subunits; chloride currents are recorded by patch‑clamp or membrane potential changes measured with fluorescence dyes. Concentration‑response curves yield EC50 for inverse agonism. In hippocampal slices, field EPSPs are recorded to assess LTP modulation by MRK‑016.

Consistent with its greater α5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than α5IA. 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 [11C]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.[2]

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In this study, researchers tested the ability of the inverse benzodiazepine agonist, MRK-016 (MRK) to protect against LPS-induced deficits in memory acquisition and consolidation, using a contextual fear conditioning (CFC) paradigm. In Experiment One, mice received lipopolysaccharide (LPS) and/or MRK injections prior to CFC training, and were then tested 24h after training. In Experiment Two, animals received similar treatment injections immediately after training, and were tested 24h later. Additionally, hippocampal samples were collected 4h after LPS injections and immediately after testing, to evaluate brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1) mRNA expression. Results indicate that MRK can protect against LPS-induced learning/memory decrements in both paradigms. We also found, in both paradigms, that animals treated with LPS/Saline expressed significantly less BDNF mRNA when compared to Saline/Saline-treated animals 4h after LPS administration, but that MRK did not restore BDNF expression levels. Further, treatment administrations had no effect on IGF-1 mRNA expression at any collection time-point. In summary, MRK-016 can protect against LPS-induced deficits in memory acquisition and consolidation, in this hippocampus-dependent paradigm, though this protection occurs independently of recovery of BDNF expression.[3]

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In vivo studies are performed in mice and rats: oral administration of 30 mg/kg in mice for seizure liability, 10 mg/kg (ip) for PTZ‑convulsion interaction, and 0.3–3 mg/kg (po) in rats for Morris water maze memory tasks; receptor occupancy is measured at 0.3–30 mg/kg (po). Kindling is assessed at 1, 3, and 10 mg/kg (ip) in mice.

MRK‑016 is orally bioavailable and readily penetrates the CNS; in rats, 0.3–30 mg/kg (po) gives excellent receptor occupancy. It is rapidly absorbed and distributed to the brain, consistent with its cognitive and antidepressant effects; detailed half‑life, bioavailability, and metabolic data are not fully disclosed but support its oral activity and brain exposure.

Toxicity data indicate good tolerability: no seizures at 30 mg/kg (po) in mice, no worsening of PTZ convulsions at 10 mg/kg (ip), no anxiogenic effects in rats at high occupancy doses, and no kindling at 1–10 mg/kg (ip). These findings suggest a favourable safety profile regarding seizure and anxiety‑related side effects for a GABAA modulator.

[1]. Chambers MS, et al. An orally bioavailable, functionally selective inverse agonist at the benzodiazepine site of GABAA alpha5 receptors with cognition enhancing properties. J Med Chem. 2004 Nov 18;47(24):5829-32.
[2]. Atack JR, et al. 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 alpha5 subtype-selective inverse agonist. J Pharmacol Exp Ther. 2009 Nov;331(2):470-84.
[3]. Eimerbrink MJ, et al. Administration of the inverse benzodiazepine agonist MRK-016 rescues acquisition and memory consolidation following peripheral administration of bacterial endotoxin. Behav Brain Res. 2015 Jul 15;288:50-3

MRK‑016 is a research compound not yet approved for clinical use; it is studied as a cognitive enhancer and rapid antidepressant. Its inverse agonism at α5‑GABAA receptors produces ketamine‑like effects without psychotomimetic liability. Further preclinical and clinical studies are needed to establish its therapeutic potential and safety in humans.

C17H20N8O2-HCL

405

368.171

C, 55.43; H, 5.47; N, 30.42; O, 8.69

783331-24-8

6918583

White to off-white solid powder

2.092

0

8

5

27

518

0

CN1N=CN=C1COC1=NN2C(C3=NOC(C)=C3)=NN=CC2=C1C(C)(C)C

QYSYOGCIDRANAR-UHFFFAOYSA-N

InChI=1S/C17H20N8O2/c1-10-6-11(23-27-10)15-21-19-7-12-14(17(2,3)4)16(22-25(12)15)26-8-13-18-9-20-24(13)5/h6-7,9H,8H2,1-5H3

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)