Glucokinase (GK). It is a small molecule glucokinase activator (GKA). In a glucokinase activity assay, AZD1656 (10 μM) acts as a potent activator. It affects the S₀.₅ (glucose concentration for half-maximal activity) without affecting the Vmax. [1]

In mouse hepatocytes cultured in MEM with 5mM glucose, treatment with 10 μM AZD1656 ex vivo for 4 hours induced the expression of several ChREBP target genes. This includes ChREBP-β, G6pc, Pklr, Acy, Acac, Gpd2, Gckr, Txnip, and FGF21. The magnitude of gene induction by AZD1656 was generally lower compared to the active enantiomer of PF-04991532, consistent with its lower glucokinase activation efficacy in the enzyme assay. Unlike substrate challenges that raise G3P (e.g., xylitol), AZD1656 (at 5mM glucose) primarily raises hexose-phosphates (G6P) rather than triose-phosphates (G3P) and does not lower cellular ATP levels. [1]

C57BL/6 mice treated with AZD1656 (0–9 mg/kg; oral gavage; daily; for 8 weeks) had lower blood glucose and glucose excursions and higher insulin levels. Numerous ChREBP target genes, such as G6pc, Pklr, Acly, Acac, and Gpd2, as well as carbohydrate response element binding protein beta isoform (ChREBP-β), can have their liver mRNA levels increased by AZD1656 [1].

---

In male C57BL/6 mice, acute oral administration of AZD1656 (2-9 mg/kg) 2 hours before an oral glucose tolerance test (GTT) dose-dependently lowered blood glucose and the glucose excursion. It also raised plasma insulin levels. Liver mRNA levels of ChREBP target genes (ChREBP-β, G6pc, Pklr, Acy, Acac, Gpd2) were increased by this acute treatment. [1]
In a 1-week chronic dietary study, AZD1656 (at 0.3, 1, 3, and 9 mg/kg) lowered blood glucose in a dose-dependent manner. Plasma insulin was not significantly increased. Liver mRNA levels of ChREBP target genes were increased at doses of 1 mg/kg and above. Total liver glucokinase activity was increased at doses of 3 mg/kg and above, suggesting liver exposure to a raised insulin/glucagon ratio. [1]
In an 8-week chronic dietary study with AZD1656 (1 and 3 mg/kg), blood glucose lowering was maintained throughout the treatment period. The acute glucose-lowering response to an intragastric load of AZD1656 (drug tolerance test) was also maintained at 4 and 8 weeks. Insulin secretion was stimulated at 4 weeks but not at 8 weeks. Blood and liver triglycerides were unchanged. Liver total glucokinase activity was raised after 4 weeks but was modestly decreased (by 18%) in the 1 mg/kg group at 8 weeks. Liver mRNA levels of most ChREBP target genes were similar to vehicle after 4-8 weeks, except for ChREBP-β, which remained raised. [1]
Hepatocytes isolated from mice treated chronically with AZD1656 (1 and 3 mg/kg) for 4 or 8 weeks showed several adaptations when challenged ex vivo with substrates. They had lower basal glucose-6-phosphate (G6P) levels at 5mM glucose. They demonstrated improved ATP homeostasis, as the fractional lowering of ATP by xylitol or by 25mM glucose + S4048 was significantly attenuated compared to hepatocytes from vehicle-treated mice. The induction of ChREBP-β and other ChREBP target genes (e.g., Pklr, Gckr, Fasn, Acl) by substrate challenge was attenuated in hepatocytes from AZD1656-treated mice. Basal mRNA levels of the AMPK catalytic subunits, Prkaa1 and Prkaa2, were raised in hepatocytes from the 3 mg/kg treatment group. [1]

Glucokinase activity was determined on liver 100,000g supernatant by a kinetic assay. The assay was performed at 100mM and 0.5mM glucose. The efficacy of AZD1656 as a GKA was determined using the same assay at 0.5mM glucose. [1]

Mouse hepatocytes were isolated by collagenase perfusion of the liver. Cells were seeded in 24-well plates and cultured overnight in serum-free Minimum Essential Medium (MEM). For ex vivo experiments, hepatocytes from untreated C57BL/6 mice were incubated in MEM containing 5mM glucose with or without 10 μM AZD1656 or other test substances for either 1 hour (for metabolite analysis of G6P, G3P, and ATP) or 4 hours (for mRNA analysis by qRT-PCR). For studies on chronically treated mice, hepatocytes were isolated after the 4-week or 8-week in vivo treatment period. After overnight culture, they were subjected to similar 1-hour or 4-hour substrate challenges (e.g., 5mM glucose, 25mM glucose ± S4048, 2mM xylitol ± AOA) before metabolite and mRNA analysis. [1]

Animal/Disease Models: C57BL/6 mice[1]
Doses: 0 mg/kg, 2 mg/kg, 4.5 mg/kg, 9 mg/kg
Route of Administration: po (oral gavage); daily; 8 consecutive weeks
Experimental Results: Oral glucose Take it 2 hrs (hrs (hours)) before a tolerance test to reduce blood sugar and glucose excursions and increase insulin levels.

---

Male C57BL/6J0laHSD mice were used. For acute studies, food was withdrawn 4h before glucose gavage. Mice were gavaged with AZD1656 at doses of 2-9 mg/kg. Blood was sampled after 2h, followed by glucose gavage (2g/kg body wt) and further blood sampling. For chronic studies, mice received a powdered diet without or with AZD1656 added at doses to achieve 1 or 3 mg/kg body wt/day (based on a consumption of 4g diet/day). Treatment lasted for 4 or 8 weeks. Free-feeding blood samples were collected from the tail vein. For drug tolerance tests (DTT), mice were fasted for 2h, a blood sample was collected, followed by gavage with AZD1656 (dose equivalent to their daily dietary intake), and blood was sampled after 2h. At the end of the study, mice were culled for whole liver analysis or hepatocyte isolation. [1]

---

Male C57BL/6J0laHSD mice were used. For acute studies, food was withdrawn 4h before glucose gavage. Mice were gavaged with AZD1656 at doses of 2-9 mg/kg. Blood was sampled after 2h, followed by glucose gavage (2g/kg body wt) and further blood sampling. For chronic studies, mice received a powdered diet without or with AZD1656 added at doses to achieve 1 or 3 mg/kg body wt/day (based on a consumption of 4g diet/day). Treatment lasted for 4 or 8 weeks. Free-feeding blood samples were collected from the tail vein. For drug tolerance tests (DTT), mice were fasted for 2h, a blood sample was collected, followed by gavage with AZD1656 (dose equivalent to their daily dietary intake), and blood was sampled after 2h. At the end of the study, mice were culled for whole liver analysis or hepatocyte isolation. [1]

AZD1656 is described as a glucokinase activator developed as a potential blood glucose-lowering drug for type 2 diabetes. It was administered orally (by gavage or in diet) in these studies. No specific PK parameters (half-life, bioavailability, etc.) are provided. [1]

The study reports that during 8-week treatment with AZD1656 (1 and 3 mg/kg) in C57BL/6 mice, body weight gain was unchanged. Blood triglycerides and liver triglycerides were also unchanged, indicating no significant drug-induced dyslipidemia or hepatic steatosis at these doses under the conditions of a standard rodent diet. The study notes that AZD1656 has a good safety record based on prior clinical experience. No other toxicity data (e.g., half-lethal dose, organ toxicity) are provided in this document. [1]

[1]. Chronic glucokinase activator treatment activates liver Carbohydrate response element binding protein and improves hepatocyte ATP homeostasis during substrate challenge. Diabetes Obes Metab. 2020 Jun 10.

[2]. Design and Development of the Glucokinase Activator AZD1656. Complete Accounts of Integrated Drug Discovery and Development: Recent Examples from the Pharmaceutical Industry Volume 1, 185-220.

[3]. The novel use of a heterozygous knockout mouse for embryofetal development assessment of a glucokinase activator. Birth Defects Res B Dev Reprod Toxicol. 2014 Apr;101(2):152-61.

AZD-1656 is being studied in the clinical trial NCT00747175 (a study evaluating the safety, tolerability, and P-Glucose of AZD1656 in patients with type 2 diabetes after multiple escalations of oral dose).

---

AZD1656 is a small molecule glucokinase activator (GKA) that was developed as a potential therapy for type 2 diabetes. GKAs work by activating the enzyme glucokinase, which plays a key role in glucose sensing and metabolism in the liver and pancreatic islets. This study investigates the hepatic adaptations that occur during chronic treatment with AZD1656 in mice, seeking to understand the mechanism behind the decline in glycaemic efficacy observed in some clinical trials. The research shows that chronic treatment activates the transcription factor ChREBP in the liver, leading to raised basal levels of ChREBP-β mRNA and improved resilience of hepatocytes to ATP depletion under high-substrate challenge. These changes are interpreted as an adaptive, protective response to maintain metabolite homeostasis, rather than a cytotoxic effect. The study concludes that ChREBP activation has a more prominent role than glucokinase repression in the chronic hepatic adaptations to AZD1656. [1]

478.196

919783-22-5

16039797

White to off-white solid powder

1.3±0.1 g/cm3

635.0±55.0 °C at 760 mmHg

337.8±31.5 °C

0.0±1.9 mmHg at 25°C

1.631

-0.21

1

9

9

35

710

1

O=C(C1C=NC(=CN=1)OC1=CC(C(NC2C=NC(C)=CN=2)=O)=CC(=C1)O[C@@H](C)COC)N1CCC1

FJEJHJINOKKDCW-INIZCTEOSA-N

InChI=1S/C24H26N6O5/c1-15-10-27-21(12-25-15)29-23(31)17-7-18(34-16(2)14-33-3)9-19(8-17)35-22-13-26-20(11-28-22)24(32)30-5-4-6-30/h7-13,16H,4-6,14H2,1-3H3,(H,27,29,31)/t16-/m0/s1

3-[5-(azetidine-1-carbonyl)pyrazin-2-yl]oxy-5-[(2S)-1-methoxypropan-2-yl]oxy-N-(5-methylpyrazin-2-yl)benzamide

AZD-1656 AZD 1656 AZD1656

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)

DMSO : ~250 mg/mL (~522.47 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.35 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (4.35 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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: ≥ 2.08 mg/mL (4.35 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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