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.

After incubation with human hepatocytes at concentrations of 1 and 10 μM for 48 hours, compound 9m/MK-0893 did not increase CYP3A4 mRNA expression or CYP3A4-mediated testosterone 6β-hydroxylase activity, therefore it is not an inducer of CYP3A4. Furthermore, no treatment-related changes were observed in central nervous system effect assessments of compound 9m in anesthetized dogs (intravenous doses up to 10 mg/kg, drug exposure up to 125 μM) and awake mice (oral dose 100 mg/kg). [1]
Due to the presence of the 6-methoxynaphthyl group, there were concerns that compound 9m might have metabolic instability, but the results of various animal pharmacokinetic studies shown in Table 4 quickly dispelled these concerns. The clearance of compound 9m was low, with Clp values of 7.5, 0.18, and 2.5 mL/min/kg in rats, dogs, and rhesus monkeys, respectively. Elimination half-life varied by species, ranging from 5.9 hours (rat) to 17 hours (dog). The oral bioavailability (F) of compound 9m in rats, dogs, and rhesus monkeys was approximately 43%, 43%, and 57%, respectively. Further studies showed that compound 9m was very metabolically stable, with low levels of metabolism both in vitro and in vivo. In vivo distribution studies using tritium-labeled 9m in cannulated rats and dogs showed that the elimination of compound 9m was almost entirely via bile excretion of the parent compound (see Supplementary Info Figure 1s), and that compound 9m was the only radioactive component in the plasma of rats and dogs at all sampling time points (see Supplementary Info Figures 2s and 3s). In vitro metabolic studies of tritium-labeled 9m in liver microsomes and hepatocytes of rats, dogs, monkeys and humans showed low levels of O-demethylated product (M1), acyl glucuronide (M2), and benzoyl glucuronide (M3) generated by hydrolysis of the amide bond of 9m (Fig. 1s, Supplementary Information). In all cases, the total turnover of these metabolites was less than 2% of that of in vitro incubation. [1]
The plasma protein binding rate of tritium-labeled 9mMK-0893 was also determined in vitro. The binding rate of compound 9m to plasma proteins of rats, dogs, monkeys and humans was extremely high (>99%), making it impossible to accurately determine its free fraction.
In an acute glucagon challenge model in hGCGR mice, compound 9m was found to inhibit glucagon-induced hyperglycemia (Fig. 2). One hour before glucagon challenge (intraperitoneal injection, 15 μg/kg), oral administration of compound 9m at doses of 3, 10, and 30 mpk reduced AUC values by 30%, 56%, and 81% within 0–24 minutes, respectively, compared to the solvent control group (all p < 0.05, compared to the glucagon group). The drug concentrations in the 3, 10, and 30 mpk dose groups were 0.26, 1.15, and 2.88 μM, respectively. In an in vitro study using an hGCGR mouse perfused liver model, glucagon-induced glycogenolysis was determined by tracking the 13C NMR signal of [2–13C]pyruvate-derived glycogen in the perfusion fluid. The results showed that the addition of compound 9m to the perfusion fluid effectively inhibited glycogenolysis (Fig. 5s, Supplementary Information). Compound 9m inhibited glucagon-induced glycogenolysis by 12%/44%/66% at initial concentrations of 0.1/0.3/1.0 μM in the perfusion fluid, and completely blocked glycogenolysis at a concentration of 3 μM. This experiment confirms that compound 9m exerts its effect by inhibiting hepatic glucose production. [1]

In a five-week safety study in rats, compound 9m/MK-0893 was well tolerated, with no treatment-related antemortem or postmortem adverse events observed at daily doses ≤100 mg/kg (Cmax reaching 21.3 ± 1.4 μM). Based on its efficacy and good selectivity, compound 9m was selected for further safety evaluation in preclinical animal models and entered into clinical studies for the treatment of type 2 diabetes. In a phase IIa study in diabetic patients, single doses of 200 mg and 1000 mg of compound 9m achieved approximately 59% and near-maximal blockade of glucagon-induced glycemic variability, respectively. In a 12-week phase IIb clinical trial in patients with type II diabetes, compound 9m significantly reduced baseline levels of fasting blood glucose (-53 and -63 mg/dL) and HbA1c (-1.1 and -1.5%) at doses of 60 mg and 80 mg qd, respectively (p < 0.001), which was superior to metformin at 1000 mg twice daily. [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 clinical trials investigating type 2 diabetes and diabetic anemia. MK-0893 is a small molecule drug currently in Phase II clinical trials with one investigational indication. In summary, the development of modular synthetic methods for 1,3,5-trisubstituted pyrazoles has enabled in-depth studies of the structure-activity relationship (SAR) around the 3 and 5 positions of pyrazole. Two key findings from the SAR study of this series of pyrazole GCGR antagonists are that the 6-substituted naphth-2-yl group at the 5-position of pyrazole enhances its potency, and the introduction of a chiral methyl group at the benzylic position of the N-1 position of pyrazole enhances its potency. Therefore, a large number of highly effective GCGR antagonists were synthesized; among them, compound 9m was further investigated due to its balanced potency and selectivity. Compared to the initial lead compound 2 in this pyrazole series, compound 9m exhibits significantly enhanced potency against GCGR (13-fold increase in binding activity and 7-fold increase in assay activity) and better off-target selectivity, particularly in hERG channel binding activity (IC50 of 9m > 10 μM, compared to 5.1 μM for 2). Notably, the introduction of a CF3 group at the para (or pseudo-para) position of pharmacophore Z is associated with hERG channel activity: compounds 9p and 9r show IC50 < 1.2 μM for hERG channel binding. Compound 9m shows improved reversible binding selectivity to CYP3A4, but moderate reversible inhibition of CYP2C8. Compound 9m is a reversible competitive human GCGR antagonist with a KB value of 8.5 nM. This compound exhibits moderate activity against GCGR in mice, dogs, and rhesus monkeys, but significantly reduced activity against GCGR in rats. Compared to other tested group B GPCRs, compound 9m exhibits selectivity for glucagon receptors, such as GIPR (IC50 1020 nM), PAC1 (IC50 9200 nM), and GLP-1R/VPAC1/VPAC2 (IC50 >10000 nM). Compound 9m demonstrated acute efficacy in inhibiting glucagon-induced hyperglycemia in both hGCGR mouse and rhesus monkey models. In pharmacodynamic studies in hGCGR mice, oral doses of 10 mg/kg and 30 mg/kg reduced blood glucose levels (AUC 0–1 hour post-administration) by 56% and 81%, respectively. In PD studies in rhesus monkeys, oral doses of 1 mg/kcal (mpk) and 3 mg/kcal (mpk) of compound 9m reduced blood glucose levels by 59% and 55%, respectively, compared to the solvent group. Compound 9m also effectively reduced blood glucose levels in the hGCGR mouse model. In an acute model of hGCGR ob/ob mice, oral administration of 3 and 10 mg/kcal (mpk) reduced blood glucose levels (AUC 0–6 hours post-administration) by 32% and 39%, respectively. In a subchronic study of hGCGR mice fed a high-fat diet, supplementation with 3 and 10 mg/kcal (mpk) of compound 9m on day 10 reduced blood glucose levels by 89% and 94%, respectively. Despite the 6-methoxynaphthalene group at the molecule's end, compound 9m is highly stable in both in vitro and in vivo metabolism. In preclinical animal models, compound 9m exhibited low clearance (7.5 mL/min/kg in rats, 0.2 in dogs, and 2.5 in rhesus monkeys), primarily through bile excretion, similar to the parent compound. Apart from moderate inhibition of CYP2C8 (IC50 2700 nM), compound 9m does not inhibit major CYP enzymes or induce CYP3A4. [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.)