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| Targets |
Gluookinase (GCK). GKA50 quarterhydrate is a potent glucokinase activator that binds to the allosteric site of the glucokinase enzyme. Glucokinase catalyzes the first step of glycolysis, converting glucose to glucose-6-phosphate, and serves as the glucose sensor in pancreatic beta-cells, regulating insulin secretion in a glucose-dependent manner. Activation of glucokinase by GKA50 lowers the glucose threshold for insulin secretion, increases the maximum rate of glucose phosphorylation, and enhances glucose-stimulated insulin secretion (GSIS). By mimicking the effects of elevated glucose, GKA50 enhances beta-cell metabolism, leading to increased ATP/ADP ratio, closure of ATP-sensitive K+ channels, depolarization, Ca2+ influx, and insulin exocytosis.
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| ln Vitro |
GKA50 (0.01-100 μM; 24 hours) increases the proliferation of INS-1 cells, with EC50 values between 1 and 2 μM[2]. In INS-1 cells, treatment with GKA50 (1.2 μM+40 μM glucose; 2-4 days) decreases apoptosis caused by prolonged elevated glucose[2]. With an EC50 of 0.022 μM, GKA50 initiates the enzymatic activity of human glucokinase. With an EC50 of 0.065 μM, GKA50 increases insulin secretion in the pancreatic insulinoma cell line, INS-1. Through the regulation of glucokinase and the apoptotic protein BAD, GKA50 lessens chronically high glucose-induced apoptosis[2].
In vitro, GKA50 quarterhydrate is a potent glucokinase activator with an EC50 value of 33 nM at 5 mM glucose. The compound stimulates insulin release from mouse pancreatic islets of Langerhans and MIN6 insulinoma cells in a concentration-dependent manner. GKA50 acts as a glucose-like activator of beta-cell metabolism, enhancing glucose utilization and ATP production. The compound also increases intracellular Ca2+ levels in a dose-dependent fashion, consistent with its role as a Ca2+-dependent regulator of insulin secretion. In rodent and human islet preparations, GKA50 potentiates glucose-stimulated insulin secretion (GSIS) at sub-threshold glucose concentrations, effectively lowering the glucose set-point for insulin release. It does not induce insulin secretion in the absence of glucose. |
| ln Vivo |
In an oral glucose tolerance test, GKA50 (1-30 mg/kg; po) significantly lowers glucose levels[1].
GKA50 quarterhydrate has been studied in various in vivo models to evaluate its glucose-lowering efficacy. In diabetic rodent models (e.g., db/db mice, high-fat diet-fed mice, streptozotocin-treated rats), acute or chronic administration of GKA50 reduces both fasting and postprandial blood glucose levels. Oral administration of GKA50 (3-30 mg/kg) enhances glucose-stimulated insulin secretion in vivo, leading to improved glucose tolerance in oral glucose tolerance tests (OGTTs). In perfused pancreas preparations, GKA50 enhances first-phase insulin secretion in response to glucose. Chronic treatment with GKA50 may also preserve beta-cell function and mass, as suggested by studies with other glucokinase activators. However, hypoglycemia is a potential risk associated with glucokinase activation, limiting therapeutic development. |
| Enzyme Assay |
For in vitro enzyme activity assays, recombinant human glucokinase (GCK) is incubated in assay buffer (50 mM HEPES, pH 7.1, 100 mM KCl, 2.5 mM MgCl2, 1 mM DTT) with varying concentrations of GKA50 quarterhydrate (0.1-1000 nM), 5 mM glucose, 2.5 mM ATP, and a coupled enzyme system containing glucose-6-phosphate dehydrogenase (G6PDH) and NADP+. The reaction proceeds at 37degC for 30-60 minutes, and NADPH production is monitored by absorbance at 340 nm. Alternatively, glucokinase activity can be measured in a 96-well plate format using a colorimetric assay kit. EC50 values are determined by fitting the dose-response curve with a four-parameter logistic model. The quarterhydrate form does not affect binding kinetics compared to the anhydrous form.
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| Cell Assay |
Cell Proliferation Assay[2]
Cell Types: INS-1 cells(starved overnight with 3 μM glucose) Tested Concentrations: 0.01-100 μM Incubation Duration: 24 hrs (hours) Experimental Results: Stimulated cell proliferation in a dose-dependent manner, with EC50 values ranging from 1 to 2 μM. For cellular function assays, MIN6 mouse insulinoma cells or isolated primary mouse islets are seeded in 96-well plates in RPMI-1640 medium containing 10% FBS and 11 mM glucose. For insulin secretion studies, cells or islets are pre-incubated for 2 hours in low-glucose (2.8 mM) KRB buffer. After pre-incubation, cells are treated with GKA50 quarterhydrate (0.1-1000 nM) in KRB buffer containing 2.8 or 5-20 mM glucose for 1 hour at 37degC. The supernatant is collected, and insulin concentration is measured by ELISA or HTRF assay. For intracellular Ca2+ measurement, MIN6 cells loaded with Fluo-4 AM (2-5 uM for 30-40 minutes) are treated with GKA50, and Ca2+ flux is monitored using a fluorescence plate reader (excitation 494 nm, emission 516 nm). For glucose utilization studies, cells are incubated with 3H-2-deoxyglucose, and uptake is quantified by liquid scintillation counting. |
| Animal Protocol |
Animal/Disease Models: High-fat-fed obese female Zucker (fa/fa) rats[1]
Doses: 1, 3 , 10, 30 mg/kg Route of Administration: Oral administration Experimental Results: Significant percentage glucose lowering. For acute in vivo efficacy studies, male C57BL/6J mice (8-10 weeks old) or db/db diabetic mice are fasted overnight (14-16 hours). GKA50 quarterhydrate is formulated in 0.5% methylcellulose or 10% DMSO/0.5% hydroxypropyl methylcellulose (HPMC) and administered orally by gavage at doses ranging from 1-30 mg/kg. For oral glucose tolerance tests (OGTT), mice receive compound or vehicle 30 minutes prior to an oral glucose challenge (1-2 g/kg glucose solution). Blood glucose levels are measured from tail vein using a glucometer at -30, 0, 15, 30, 60, 90, and 120 minutes post-glucose. For insulin secretion assessment, blood samples are collected into heparinized capillary tubes, and plasma is separated for insulin ELISA. For chronic studies (7-28 days), mice are dosed once or twice daily, and body weight, food intake, HbA1c, and glucose levels are monitored throughout the study. Pharmacodynamics are assessed via OGTT at the end of the treatment period. |
| ADME/Pharmacokinetics |
No specific pharmacokinetic data are available for GKA50 quarterhydrate. As a small-molecule glucokinase activator, GKA50 is expected to be orally bioavailable based on its in vivo efficacy after oral administration. The quarterhydrate form (0.25 H2O per molecule) does not significantly alter solubility or dissolution rate compared to the anhydrous form. For a typical glucokinase activator in this chemical class, the plasma half-life in rodents is 2-6 hours, Cmax occurs at 1-2 hours post-dose, and oral bioavailability is 30-70%. The compound likely undergoes hepatic metabolism via cytochrome P450 enzymes (CYP3A4, CYP2C9). Plasma protein binding may be moderate to high (>80%). The quarterhydrate is a pharmaceutically acceptable crystalline hydrate form, which is a solid-state form commonly encountered in drug development to improve stability and handling properties.
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| Toxicity/Toxicokinetics |
No specific toxicity data are available for GKA50 quarterhydrate. As a glucokinase activator, the primary dose-limiting toxicity risk is hypoglycemia due to enhanced insulin secretion independent of glucose levels. In animal studies at high doses (e.g., >30 mg/kg), GKA50 may cause blood glucose levels to drop below normal range (hypoglycemia), leading to symptoms including lethargy, seizures, or mortality. No genotoxicity or organ toxicity has been reported for this compound. In chronic toxicity studies with other glucokinase activators, hepatic steatosis (fatty liver) has been observed in some cases, attributed to increased hepatic glucose utilization and lipid synthesis. The quarterhydrate form is not associated with additional toxicities. Standard precautions for handling research chemicals should be followed.
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| References |
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| Additional Infomation |
GKA50 is a prototypical glucokinase activator from a chemical class that includes the clinical candidate AZD1656. The quarterhydrate designation indicates a stoichiometric hydrate containing 0.25 molecules of water per molecule of the parent compound (C26H28N2O6·0.25H2O). This water content is consistent with a crystalline solid form that may have improved pharmaceutical properties (e.g., solubility, stability) compared to the anhydrous form. Glucokinase (GK) is a key enzyme in glucose homeostasis, and GK activators have been extensively pursued for the treatment of type 2 diabetes (T2DM). GKA50 quarterhydrate is a research tool used to study the role of glucokinase in glucose sensing and insulin secretion. No glucokinase activator has received FDA approval, though several have entered clinical trials; development has been limited by the risk of hypoglycemia and long-term safety concerns (e.g., hepatic steatosis). This product is for research use only.
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| Molecular Formula |
C26H28N2O6.1/4H2O
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| Molecular Weight |
469.01
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| Related CAS # |
GKA50;851884-87-2
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| Appearance |
White to off-white solid powder
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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 |
| 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 :~46 mg/mL (~98.08 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1322 mL | 10.6608 mL | 21.3215 mL | |
| 5 mM | 0.4264 mL | 2.1322 mL | 4.2643 mL | |
| 10 mM | 0.2132 mL | 1.0661 mL | 2.1322 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.
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