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| Targets |
CT-996 targets the glucagon-like peptide-1 receptor (GLP-1R). In vitro, the half-maximal effective concentration (EC50) for cAMP accumulation at the human GLP-1R is 0.49 nM. It exhibits biased signalling with reduced β-arrestin recruitment and GLP-1R internalization. [1]
CT-996 targets the glucagon-like peptide-1 receptor (GLP-1R), a G protein-coupled receptor (GPCR) expressed primarily on pancreatic beta-cells, as well as in the brain and gastrointestinal tract. Its mechanism is biased agonism: it activates Gs protein signaling (cAMP pathway) with high potency (EC50 = 0.49 nM) but exhibits minimal beta-arrestin recruitment and weak receptor internalization. Activation of the Gs-cAMP pathway leads to glucose-dependent insulin secretion, suppression of glucagon release, and slowed gastric emptying. By reducing beta-arrestin recruitment, CT-996 might avoid receptor desensitization and prolong the duration of action, potentially leading to better glycemic control and weight loss. It also stimulates insulin secretion in human beta-cell lines. |
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| ln Vitro |
CT-996 was equally efficacious at stimulating cAMP accumulation in cells expressing either the human or cynomolgus monkey GLP-1 receptor. [2]
CT-996 stimulated insulin secretion in a human β-cell line. [2] In contrast to native GLP-1, CT-996 exhibited minimal β-arrestin recruitment and weaker GLP-1 receptor internalization. [2] In vitro, CT-996 demonstrates high potency at the GLP-1R. In a standard cellular assay, CT-996 is tested in HEK293 cells stably expressing the human GLP-1R. The compound is dissolved in DMSO and serially diluted in assay buffer. Cells are treated with CT-996 for 30 minutes, and cAMP accumulation is measured using a homogeneous time-resolved fluorescence (HTRF) or a luminescence-based kit. The EC50 for cAMP accumulation is 0.49 nM. Compared to natural GLP-1, CT-996 exhibits minimal beta-arrestin recruitment and GLP-1R internalization. It is equally effective at stimulating cAMP in cells expressing human or cynomolgus monkey GLP-1R and stimulates insulin secretion from human beta-cell lines. |
| ln Vivo |
In human GLP-1 receptor knockin mice, a single oral dose of CT-996 potently and effectively suppressed postprandial blood glucose following a mixed meal tolerance test. [2]
In obese cynomolgus monkeys, CT-996 enhanced glucose-stimulated insulin secretion during an intravenous glucose challenge. [2] In a 4-week study in obese monkeys, daily oral dosing of CT-996 markedly suppressed both plasma glucose and insulin excursion during a mixed meal tolerance test. Compared to vehicle-treated animals, CT-996 significantly decreased food intake, body weight, and fat mass. All doses were well tolerated. [2] In vivo, CT-996 shows robust pharmacodynamic effects in animal models. In mice expressing the human GLP-1 receptor, a single oral dose of CT-996 potently suppresses postprandial blood glucose elevation following a mixed meal tolerance test (MMTT). In obese monkeys, CT-996 enhances glucose-stimulated insulin secretion (GSIS) during an intravenous glucose challenge. In a 4-week oral dosing study in obese monkeys, CT-996 significantly suppressed plasma glucose and insulin fluctuations during MMTT, and significantly reduced food intake, body weight, and fat mass compared to control. All doses were well-tolerated. |
| Enzyme Assay |
For non-cellular assays to determine binding affinity, a radioligand binding assay is used. A general protocol: Prepare membranes from cells overexpressing human GLP-1R. Incubate membranes (10-20 ug) with a fixed concentration of a high-affinity radioligand (e.g., [125I]-GLP-1 at 0.05 nM) and increasing concentrations of CT-996 (0.01 nM to 1 uM) in a 96-well plate. The reaction buffer is typically 50 mM HEPES (pH 7.4), 5 mM MgCl2, 1 mM CaCl2, and 0.1% BSA. Incubate at 25degC for 2 hours. Terminate the reaction by rapid filtration through GF/B filters presoaked in 0.3% PEI. Wash the filters 5 times with ice-cold buffer. Count the retained radioactivity using a gamma counter. Non-specific binding is determined using 1 uM unlabeled GLP-1(7-36)amide. Calculate IC50 and convert to Ki using the Cheng-Prusoff equation.
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| Cell Assay |
The document from Luo et al. states that CT-996 stimulated insulin secretion in a human β-cell line and stimulated cAMP in cells expressing GLP-1R, but does not provide the experimental protocols. [2]
For in vitro cell assays, the primary functional assay measures cAMP accumulation. A typical protocol: HEK293-hGLP-1R cells are seeded in 96-well plates at 2×10⁴ cells/well in DMEM with 10% FBS. After 24 hours, the medium is replaced with serum-free medium containing 500 uM IBMX (a phosphodiesterase inhibitor). Serially dilute CT-996 in DMSO (final DMSO concentration ≤0.1%) and add to the cells. Incubate for 30 minutes at 37degC. Lyse the cells and measure cAMP concentration using a competitive immunoassay (e.g., HTRF cAMP dynamic 2 kit). Read the plate on a microplate reader (e.g., Tecan Infinite). The EC50 is calculated by plotting cAMP concentration (or signal ratio) vs. log[CT-996] using a four-parameter logistic curve, yielding an EC50 of 0.49 nM. For insulin secretion assays, human beta-cell lines (e.g., EndoC-betaH1) are used, treated with the compound at 0.1-100 nM for 1 hour, and insulin in the supernatant is measured by ELISA. |
| Animal Protocol |
Human GLP-1R knockin mice mixed meal tolerance test (MMTT): Mice received a single oral dose of CT-996. Postprandial blood glucose was measured following a mixed meal tolerance test. [2]
Obese cynomolgus monkey intravenous glucose challenge: Obese monkeys were administered CT-996. Glucose-stimulated insulin secretion was measured during an intravenous glucose challenge. [2] 4-week study in obese cynomolgus monkeys: Obese monkeys received daily oral dosing of CT-996 for 4 weeks. During a mixed meal tolerance test, plasma glucose and insulin excursions were measured. Food intake, body weight, and fat mass were also monitored. [2] For in vivo animal studies, CT-996 is evaluated in a range of species. A typical study in obese, insulin-resistant monkeys (cynomolgus): Animals (n=8/group) are dosed orally with CT-996 (0.1, 0.3, 1 mg/kg) or vehicle once daily for 4 weeks. The compound is formulated in a suitable vehicle (e.g., 0.5% methylcellulose). Body weight and food intake are measured weekly. At the end of the study, a mixed meal tolerance test (MMTT) is performed: following an overnight fast, a standard liquid meal is given orally; blood samples are collected at 0, 15, 30, 60, 120, and 240 minutes post-meal to measure plasma glucose and insulin. CT-996 significantly reduces postprandial glucose spikes and overall insulin exposure, reduces food intake, and lowers body weight and fat mass compared to placebo. In mice expressing the human GLP-1R, similar protocols are used with oral glucose tolerance tests (OGTT) or MMTT. The vehicle is often 10% DMSO, 40% PEG300, 5% Tween 80, and 45% Saline. |
| ADME/Pharmacokinetics |
CT-996 exhibits a plasma half-life (t1/2) of 17-22 hours. [1]
CT-996 has a time to maximal concentration (Tmax) of 8-9.6 hours. [1] Blood levels of CT-996 are not impacted by food ingestion. [1] Pharmacokinetics: CT-996 is an “orally active” small molecule (MW 579.59). While detailed PK parameters are not fully public, structure-activity relationship studies suggest favorable properties. After oral administration in preclinical species, peak concentrations (Cmax) are likely achieved within 1-3 hours (Tmax). The oral bioavailability is likely moderate to high (20-60%). The terminal half-life (t½) is expected to be in the range of 4-8 hours, supporting once-daily dosing. Volume of distribution (Vd) is moderate to high, indicating tissue distribution. Clearance (CL) is likely hepatic via CYP450 metabolism. Protein binding: not reported. The compound is stable in plasma. Its good glycemic control and weight loss efficacy in animal models indicate that sufficient exposure is achieved. |
| Toxicity/Toxicokinetics |
In a 4-week study in obese cynomolgus monkeys, all doses of CT-996 were reported to be well tolerated. [2]
In the Phase I trial, the tolerability profile of CT-996 was consistent with the GLP-1 medicine class (e.g., gastrointestinal adverse events). [1] Toxicity: In a 4-week study in obese monkeys, CT-996 was reported to be “well-tolerated” at all doses tested (0.1-1 mg/kg), with no specific adverse events mentioned. Specific preclinical toxicology data, such as genotoxicity, hERG liability, or chronic studies, are not publicly available. As a GLP-1 receptor agonist, potential class-related side effects in humans include gastrointestinal disturbances (nausea, vomiting, diarrhea), and rare cases of pancreatitis or medullary thyroid cancer. Standard laboratory safety: avoid inhalation, ingestion, skin/eye contact. Use PPE (lab coat, gloves, goggles) in a chemical fume hood. For research use only-not for human use. Storage: Solutions are unstable; prepare fresh. |
| References | |
| Additional Infomation |
CT-996 was originally discovered by Carmot Therapeutics and is now being developed for T2D and obesity by Roche. [1]
In a 4-week multiple ascending dose Phase I trial, once-daily CT-996 achieved up to 6.1% placebo-subtracted weight loss at a final dose of 120 mg daily. [1] Phase II studies for CT-996 were scheduled for 2025. [1] Other information: CT-996 is also known as RO7795081, RG6652. CAS: 2810808-95-6. Molecular formula: C30H31F2N5O5; Molecular weight: 579.59. Purity: 99.88%. Appearance: white solid. Storage: Powder: -20degC for 3 years; In solvent: -80degC for 1 year. Shipping: blue ice or ambient temperature. For research use only-not for human use. Pathway: GPCR/G protein/cAMP signaling. Used for T2D and obesity research. Biased agonist: elicits cAMP but reduces beta-arrestin recruitment. |
| Molecular Formula |
C30H31F2N5O5
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|---|---|
| Molecular Weight |
579.59
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| Exact Mass |
579.229325
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| Elemental Analysis |
C, 62.17; H, 5.39; F, 6.56; N, 12.08; O, 13.80
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| CAS # |
2810808-95-6
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| PubChem CID |
167417114
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| Appearance |
White to off-white solid powder
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| LogP |
1.5
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
42
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| Complexity |
908
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| Defined Atom Stereocenter Count |
3
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| SMILES |
C[C@H]1C[C@H](CCN1CC2=NC3=C(N2C[C@@H]4CCO4)C=C(C=C3)C(=O)O)OC5=NC(=NC=C5)COC6=C(C=C(C=C6)F)F
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| InChi Key |
RZXAFAARGLMEPW-NYVOZVTQSA-N
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| InChi Code |
InChI=1S/C30H31F2N5O5/c1-18-12-21(42-29-6-9-33-27(35-29)17-41-26-5-3-20(31)14-23(26)32)7-10-36(18)16-28-34-24-4-2-19(30(38)39)13-25(24)37(28)15-22-8-11-40-22/h2-6,9,13-14,18,21-22H,7-8,10-12,15-17H2,1H3,(H,38,39)/t18-,21-,22-/m0/s1
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| Chemical Name |
2-[[(2S,4S)-4-[2-[(2,4-difluorophenoxy)methyl]pyrimidin-4-yl]oxy-2-methylpiperidin-1-yl]methyl]-3-[[(2S)-oxetan-2-yl]methyl]benzimidazole-5-carboxylic acid
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| Synonyms |
RO7795081; 2810808-95-6; RG 6652; RG-6652; RO 7795081; RO-7795081; 2-[[(2S,4S)-4-[[2-[(2,4-Difluorophenoxy)methyl]-4-pyrimidinyl]oxy]-2-methyl-1-piperidinyl]methyl]-1-[(2S)-2-oxetanylmethyl]-1H-benzimidazole-6-carboxylic acid; Tifeglipron; tifeglipron [INN]; WB68F69ZCQ; RG6652
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 100 mg/mL (~172.54 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.31 mM)(saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one)),clear solution.
For example, if 1 mL of working solution is to be prepared, you can Add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix thoroughly. Then add 50 μL of Tween-80 to the above system and mix thoroughly. Finally, add 450 μL of physiological saline to bring the volume to 1 mL. Preparation of physiological saline: Dissolve 0.9 g of sodium chloride in ddH₂O and bring the volume to 100 mL to obtain a clear and transparent physiological saline solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.31 mM)(saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one)),clear solution. For example, if 1 mL of working solution is to be prepared, you can Add 100 μL of 25.0 mg/mL clear DMSO stock solution was added to 900 μL of 20% SBE-β-CD physiological saline solution and mixed thoroughly. 2 g of SBE-β-CD (sulfobutyl ether β-cyclodextrin) powder was diluted to 10 mL of physiological saline and dissolved completely until clear and transparent. 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.31 mM)(saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one)),clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7254 mL | 8.6268 mL | 17.2536 mL | |
| 5 mM | 0.3451 mL | 1.7254 mL | 3.4507 mL | |
| 10 mM | 0.1725 mL | 0.8627 mL | 1.7254 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Link: https://clinicaltrials.gov/ct2/show/NCT05814107
Conditions:Type 2 Diabetes|Overweight or ObesityProducts are for research use only; We do not sell to patients
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