GR/glucagon receptor

With IC50 values of 1020 nM for GIPR, 9200 nM for PAC1, and >10000 nM for GLP-1R, VPAC1, and VPAC2, MK 0893 is selective for the hyperprosin receptor. It is also active against rhesus GCGR, demonstrating an IC50 of 56 nM in a cAMP experiment in CHO cells expressing rhesus GCGR [1]. A potent, selective glucagon receptor antagonist 9m, N-[(4-{(1S)-1-[3-(3,5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-β-alanine, was discovered by optimization of a previously identified lead. Compound 9m is a reversible and competitive antagonist with high binding affinity (IC(50) of 6.6 nM) and functional cAMP activity (IC(50) of 15.7 nM). It is selective for glucagon receptor relative to other family B GPCRs, showing IC(50) values of 1020 nM for GIPR, 9200 nM for PAC1, and >10000 nM for GLP-1R, VPAC1, and VPAC2 [1].

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Compound 9m/MK-0893 is a competitive, reversible GCGR antagonist, as evidenced by Schild Analysis in CHO cells expressing the hGCGR (Figure 1). It dose dependently right-shifted the EC50 of glucagon without changing the maximum effect of glucagon. The binding of 9m was fully reversible within the 30 min of the equilibration period used in the assay. Linear transformation of the data (insert in Figure 1) yielded a straight line with a slope of 1 (Hill coefficient, nh), and a KB of 8.5 nM[1].

Compound 9m/MK-0893 was active against the rhesus monkey GCGR, showing an IC50 of 56 nM in a cAMP assay with CHO cells expressing the rhesus GCGR. The compound was evaluated in vivo in a rhesus glucagon challenge model similar to that utilized in the hGCGR mouse. When compound 9m was administered to chair-restrained rhesus monkeys via a nasogastric tube at 0.3, 1, and 3 mpk four hours prior to an intramuscular glucagon challenge (15 μg/kg), it significantly reduced the glucagon-induced glucose levels at 1 and 3 mpk with a 59 and 55% correction to the vehicle response. A nonstatistically effective dose was observed at 0.3 mpk (29% reduction). The mean plasma levels at the time of glucagon administration were 0.1, 0.3, and 0.7 μM at 0.3, 1.0, and 3.0 mpk, respectively[1].

MK 0893 abolishes glucagon-induced increase of diabetes in hGCGR electrodes and rhesus monkeys. It also reduces ambient electrode levels in acute and chronic electrode models: in hGCGR ob/ob electrodes, supplementation (AUC 0-6 h) was lowered by 32% and 39% at single doses of 3 and 10 mpk, respectively. In hGCGR mice on a high-fat diet, MK 0893 at 3 and 10 mpk po in the diet reduced blood pressure levels by 89% and 94%, respectively, on day 10 relative to the difference between vehicle controls and lean hGCGR mice. Compound 9m/MK-0893 blunted glucagon-induced glucose elevation in hGCGR mice and rhesus monkeys. It also lowered ambient glucose levels in both acute and chronic mouse models: in hGCGR ob/ob mice it reduced glucose (AUC 0-6 h) by 32% and 39% at 3 and 10 mpk single doses, respectively. In hGCGR mice on a high fat diet, compound 9m at 3, and 10 mpk po in feed lowered blood glucose levels by 89% and 94% at day 10, respectively, relative to the difference between the vehicle control and lean hGCGR mice. On the basis of its favorable biological and DMPK properties, compound 9m (MK-0893) was selected for further preclinical and clinical evaluations. [ 1].

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Compound 9m/MK-0893 demonstrated efficacy in glucose lowering in a hGCGR ob/ob mice in an acute model, Figure 4s, Supporting Information. Compared to vehicle control group, compound 9m at 10 and 3 mpk oral doses lowered blood glucose level (AUC 0–6 h post dose) by 39%, 32% respectively. At 1 mpk, compound 9m lowered glucose at 1 and 3 h but not at 6 h post dose. At 0.3 mpk, there was no effect at 1, 3, and 6 h time points. Compound 9m was also efficacious in lowering ambient glucose level in a chronic setting using diet-induced obese hGCGR mice. This group of hGCGR mice on a high fat diet developed a mild degree of nonfasting hyperglycemia, hyperinsulinemia, and hyperglucagonemia and offered a good opportunity to evaluate the GCGR antagonists’ efficacy in ambient glucose reduction. When dosed in feed (high fat diet S3282 from Bio-Serv) at 3 and 10 mpk per day, compound 9m demonstrated a glucose lowering effect by day 3 and maintained lower glucose levels in the treatment groups throughout the duration of the study, Figure 4. At day 3, the glucose levels relative to the difference between the vehicle control group and lean group were reduced by 70% and 105% for the 3 and 10 mpk groups, respectively. At day 10, the corresponding glucose reductions were 89% and 94% for the 3 and 10 mpk groups, respectively. In this 10-day chronic treatment, the glucagon and GLP-1 levels were also increased relative to vehicle control group, a result of feedback upregulation of proglucagon expression. Glucagon levels were elevated by 1.5- and 2.6-fold in the 3 and 10 mpk groups, respectively. Total GLP-1 also increased by 2.1- and 4.0-fold in the 3 and 10 mpk groups, respectively. Note that these increases in glucagon were far less than the reported increase observed in the GCGR knockout mice (>100×). In keeping with previously reported data, no gross morphological changes in pancreatic tissues were observed.

Glucagon Binding Assay [1]
A CHO cell line expressing the human glucagon receptor (CHO hGCGR) was maintained and membranes prepared as described in Chicchi et al. Membranes (2–5 μg) were incubated in buffer containing 50 mM Tris, pH 7.5, 5 mM MgCl2, 2 mM EDTA, 1% bovine serum albumin, 12% glycerol, 0.2 mg of wheat germ agglutinin-coated polyvinyltoluene scintillation proximity assay beads, increasing concentration of compound (diluted in 100% DMSO and added to the assay at a final concentration of 2.5%), and 50 pM 125I-glucagon. The assay was incubated for 3 h at room temperature, and the total bound radioactivity was measured with a Wallac-Microbeta counter. Nonspecific counts were determined using 1 μM unlabeled glucagon. Data were analyzed using the nonlinear regression analysis software GraphPad Prism, v4.

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cAMP Assay [1]
CHO hGCGR cells were grown in Iscove’s Modified Dulbecco’s Medium (IMDM), 10% FBS, 1 mM l-glutamine, penicillin–streptomycin (100 u/mL), and 500 ug G418/mL for 3–4 days before harvesting using Enzyme-Free Dissociation Media. The cells were centrifuged at low speed and resuspended in stimulation buffer. Compounds were diluted from DMSO stocks and added to the assay at a final concentration of 5% DMSO. Cells were preincubated with compound or DMSO controls for 30 min. Glucagon (250 pM) was added, and the samples were incubated at room temperature for an additional 30 min. The assay was terminated with the addition of the FlashPlate kit detection buffer. The assay was then incubated for an additional 3 h at room temperature, and bound radioactivity was measured using a liquid scintillation counter. cAMP levels were determined as per manufacturer’s instructions. For Schild Plot analysis, aliquots of cells were preincubated with 56, 100, 178, 300, 560, and 1000 nM MK-0893/9m for 30 min at room temperature prior to the addition of 0.001–1000 nM glucagon to initiate the assay. Data were analyzed using the linear and nonlinear regression analysis software GraphPad Prism, v4.

hGCGR Mice were anesthetized (Nembutal IP, 50 mg/kg) at approximately the middle of the dark cycle. The portal vein was then cannulated and tied off and the liver was excised and perfused with a pre-oxygenated Krebs-Bicarbonate buffered solution for 5-10 minutes which initially was not recirculated to wash out any endogenous substrates. The liver was then excised and put into an NMR tube and the initial Krebs solution was exchanged for a BSA-Krebs perfusate (approx. 72ml) which was recirculated. 31P-NMR spectroscopy was performed initially to examine the ATP and inorganic phosphate (Pi) levels in the liver which can be used to assess the viability of the liver. A 13C-NMRvisible pool of glycogen was then created by the addition of the gluconeogenic substrate [2-13C]Pyruvate + NH4Cl. The amount of glycogen contained in the liver was monitored via the C1 resonance of the glucosyl units in the glycogen chain in real time until a suitable level was reached. At this time a novel glucagon receptor (GCGR) antagonist or DMSO was infused followed 20 minutes later by a glucagon challenge. The response of glycogen levels to the glucagon challenge was used to assess the efficacy of the GCGR antagonist.

Upon incubation with human hepatocytes at 1 and 10 μM for 48 h, compound 9m/MK-0893 did not increase CYP3A4 mRNA expression or CYP3A4-mediated testosterone 6β-hydroxylase activity, and thus it was not an inducer of CYP3A4. Furthermore, compound 9m produced no treatment-related changes in assays evaluating cardiovascular effect in anesthetized dogs (up to 10 mpk IV, reaching drug exposure of 125 μM) and central nervous system effect in conscious mice (100 mpk PO).[1]

Concern over potential metabolic instability of compound 9mMK-0893/ due to the 6-methoxynaphthalene group was quickly dispelled by the results of pharmacokinetic studies in several species, as shown in Table 4. Compound 9m was characterized by low clearance, with a Clp of 7.5, 0.18, and 2.5 mL/min/kg, in rat, dog, and rhesus monkey, respectively. The elimination half-life was species-dependent, varying from 5.9 h (rat) to 17 h (dog). The oral bioavailability (F) of 9m was ∼43% in rat, 43% in dog, and 57% in rhesus. Further studies indicated that compound 9m was metabolically very stable, undergoing only minor metabolism in vitro and in vivo. In vivo disposition studies in bile duct cannulated rats and dogs using tritium-labeled 9m indicated that elimination occurred almost exclusively via biliary excretion of the parent compound (Figure 1s, Supporting Information) and that compound 9m was the only radioactive plasma component in both rat and dog at all the time points sampled (Figures 2s, 3s, Supporting Information). In vitro metabolism studies in liver microsomes and hepatocytes from rat, dog, monkey, and human using tritium-labeled 9m revealed minor amounts of O-demethylation (M1), acyl glucuronide (M2) of 9m, and acyl glucuronide of the benzoic acid (M3) derived from hydrolysis of the amide bond of 9m (Figure 1s, Supporting Information). In all cases, the total metabolites represented <2% turnover in the in vitro incubations. [1]

Plasma protein binding was also determined in vitro with the tritium-labeled 9mMK-0893. Compound 9m was highly bound (>99%) to rat, dog, monkey, and human plasma proteins, and the unbound fraction could not be accurately determined.
In an acute glucagon challenge model in hGCGR mice, compound 9m was found to be active in blunting glucagon-induced glucose excursion, Figure 2. When dosed orally at 3, 10, and 30 mpk one hour prior to a glucagon challenge (IP, 15 ug/kg), compound 9m reduced glucose elevation relative to vehicle control by 30%, 56%, and 81%, respectively, as determined by AUC over a 0–24 min period (all with p < 0.05 vs the glucagon group). The drug levels were found to be 0.26, 1.15, and 2.88 μM for 3, 10, and 30 mpk dose groups, respectively. In an ex vivo study using an hGCGR mouse perfused liver model, the glucagon-induced glycogenolysis (determined by following the 13C NMR signal of glycogen derived from [2-13C]pyruvate in perfusate) was effectively inhibited by 9m added to the perfusate (Figure 5s, Supporting Information). Compound 9m inhibited glucagon-induced glycogenolysis by 12/44/66% at 0.1/0.3/1.0 μM initial concentration in perfusate and completely blocked glucagon-induced glycogenolysis at 3 μM. This experiment confirmed that compound 9m acted in the liver by inhibiting hepatic glucose production.[1]

Compound 9m/MK-0893 was well-tolerated in a five-week safety study in rat, with no treatment-related antemortem or postmortem findings at doses ≤100 mpk daily (Cmax reached 21.3 ± 1.4 μM). On the basis of the efficacy and good selectivity profile, compound 9m was selected for further evaluation in preclinical safety species and entered into clinical studies for type II diabetes treatment. In a phase IIa study in diabetic patients, compound 9m, at 200 and 1000 mg single doses, achieved ∼59% and near maximal blockade of glucagon-induced glucose excursions, respectively. In a 12-week phase IIb clinical study in type II diabetic patients, compound 9m demonstrated robust reduction from baseline level in fasting plasma glucose (−53 and −63 mg/dL) and HbA1c (−1.1 and −1.5%) at 60 and 80 mg qd doses, respectively (p < 0.001), surpassing metformin at 1000 mg bid. [1]

[1]. Discovery of a novel glucagon receptor antagonist N-[(4-{(1S)-1-[3-(3, 5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-β-alanine (MK-0893) for the treatment of type II diabetes. J Med Chem. 2012 Jul 12;55(13):6137-48.

MK0893 has been used in trials studying the treatment of Type 2 Diabetes Mellitus and Diabetes Mellitus, Type 2. MK-0893 is a small molecule drug with a maximum clinical trial phase of II and has 1 investigational indication.

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In summary, the development of a modular synthesis of 1,3,5-trisubstituted pyrazoles allowed thorough investigation of the SAR around the pyrazole 3- and 5- positions. Two key findings of the SAR studies in this pyrazole GCGR antagonist series were the potency enhancing effect of 6-substituted naphth-2-yl groups at the pyrazole 5-position, and the incorporation of a chiral methyl group at the benzylic position off the pyrazole-N-1. Consequently a large number of potent GCGR antagonists were synthesized; among them, compound 9m was further profiled due to its balanced potency and selectivity profile. Compared with the initial lead compound 2 in this pyrazole series, compound 9m showed increased potency on the GCGR (13-fold in binding, 7-fold in functional assay) and improved off-target selectivity profile, particularly in hERG channel binding activity (IC50 >10 μM for 9m vs 5.1 μM for 2). It is interesting to note that incorporation of CF3 group in the para (or pseudo para) position in pharmacophore Z was associated with hERG channel activity: IC50 in hERG channel binding was <1.2 μM for compound 9p and 9r. Compound 9m showed an improved selectivity in reversible binding to CYP3A4 but had moderate reversible inhibition of CYP2C8. Compound 9m is a reversible, competitive human GCGR antagonist with KB of 8.5 nM. It is moderately active against the mouse, dog, and rhesus GCGRs but significantly less active at the rat GCGR. Compound 9m is selective for the glucagon receptor relative to other family B GPCRs tested, GIPR (IC50, 1020 nM), PAC1 (IC50, 9200 nM), and GLP-1R/VPAC1/VPAC2 (IC50 >10000 nM). Compound 9m demonstrated acute efficacy in blunting glucagon-induced glucose elevation in hGCGR mouse and rhesus monkey models. In a hGCGR mice PD study, it reduced glucose levels (AUC 0–1 h post dose) by 56%, and 81% at 10 and 30 mpk oral doses, respectively. In a rhesus monkey PD study, it demonstrated 59% and 55% reduction in glucose level relative to vehicle group at 1 and 3 mpk oral doses, respectively. Compound 9m was also efficacious in lowering ambient glucose levels in hGCGR mice models. In a hGCGR ob/ob mice acute model, it reduced glucose level (AUC 0–6 h post dosing) by 32% and 39% at 3 and 10 mpk oral doses, respectively. In a subchronic study in hGCGR mice on a high fat diet, it reduced ambient glucose levels by 89% and 94% at 3 and 10 mpk doses, respectively, in feed at day 10. In spite of having a 6-methoxynaphthalene group on the periphery, compound 9m is actually very stable metabolically in vitro and in vivo. It has low clearance in preclinical species tested (Clp of 7.5, 0.2, and 2.5 mL/min/kg, in rats, dogs, and rhesus monkeys, respectively), and the major clearance route of compound 9m is via biliary excretion as the parent compound. Except for moderate inhibition of CYP2C8 (IC50 2700 nM), compound 9m does not inhibit major CYPs nor is it a CYP3A4 inducer. [1]

C33H27F4N3O4

605.578802347183

605.19

C, 65.45; H, 4.49; F, 12.55; N, 6.94; O, 10.57

870823-19-1

870823-12-4

11621378

Solid powder

6.5

2

9

9

44

977

1

C[C@@H](C1=CC=C(C=C1)C(=O)NCCC(=O)O)N2C(=CC(=N2)C3=C(C=CC(=C3)C(F)(F)F)F)C4=CC5=C(C=C4)C=C(C=C5)OC

UXPYCHMVTKGDJP-IBGZPJMESA-N

InChI=1S/C33H27F4N3O4/c1-19(20-3-5-21(6-4-20)32(43)38-14-13-31(41)42)40-30(24-8-7-23-16-26(44-2)11-9-22(23)15-24)18-29(39-40)27-17-25(33(35,36)37)10-12-28(27)34/h3-12,15-19H,13-14H2,1-2H3,(H,38,43)(H,41,42)/t19-/m0/s1

3-[[4-[(1S)-1-[3-[2-fluoro-5-(trifluoromethyl)phenyl]-5-(6-methoxynaphthalen-2-yl)pyrazol-1-yl]ethyl]benzoyl]amino]propanoic acid

MK 0893 analog; MK 0893; MK-0893; GRA-1; GRA1; GRA 1

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)