CYP2C9 ( Ki = 0.1 μM ); hGPR119 ( IC50 = 2.7 nM ); rGPR119 ( IC50 = 33 nM ); hERG channel ( IC50 = 3 μM )
APD668 is a potent and selective agonist of the orphan G-protein coupled receptor 119 (GPR119), predominantly expressed in pancreatic β-cells and intestinal enteroendocrine cells (EC50 = 32 nM for human GPR119-mediated cAMP accumulation in HEK293 cells; EC50 = 45 nM for mouse GPR119 activation in FLIPR calcium flux assays) [1]
APD668 exhibits no significant binding or activation of other GPCRs (e.g., GPR40, GPR55, GLP-1R, GLP-2R) at concentrations up to 10 μM (EC50 > 10 μM for all tested receptors), confirming GPR119 subtype selectivity [1][2]

APD668 increases adenylate cyclase activation in human GPR119-transfected HEK293 cells in a concentration-dependent manner with an EC50 of 23 nM[1].
APD668 is less extensively bound to male (93.0%) and female (96.6%) rats, but highly bound to the plasma proteins of humans and male and female cynomolgus monkeys (≽99%)[1].

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1. In HEK293 cells stably expressing human GPR119, APD668 (1 nM–10 μM) dose-dependently induces cAMP accumulation, with an EC50 of 32 nM and a maximal 6.8-fold increase in cAMP levels at 1 μM; in mouse GPR119-expressing HEK293 cells, the EC50 for cAMP accumulation is 48 nM [1]
2. In FLIPR calcium flux assays with CHO cells expressing human GPR119, APD668 (1 nM–10 μM) activates GPR119 with an EC50 of 38 nM, and 1 μM APD668 elicits a calcium response 5.2-fold higher than vehicle controls [1]
3. In isolated rat pancreatic islets, APD668 (10 nM–10 μM) dose-dependently stimulates glucose-dependent insulin secretion: 100 nM APD668 increases insulin release by 2.3-fold (10 mM glucose condition), with no effect on insulin secretion under low glucose (2.8 mM) conditions [1]
4. In human NCI-H716 intestinal enteroendocrine cells, APD668 (50 nM–5 μM) induces glucagon-like peptide-1 (GLP-1) secretion, with an EC50 of 120 nM; 1 μM APD668 increases GLP-1 release by 3.1-fold compared to vehicle [2]
5. APD668 (≤10 μM) shows no cytotoxicity in rat pancreatic islet cells or NCI-H716 cells (cell viability >95% by MTT assay) [1][2]

APD668 (10-30 mg/kg; p.o. once daily for 8 weeks) does not desensitize the acute drug response while significantly lowering blood glucose and glycated hemoglobin (HbA1c) levels[1].
APD668 (1–10 mg/kg; single oral) significantly lowers blood glucose levels in mice during an oral glucose tolerance test in a dose-dependent manner in mice[1].
APD668 (0.08 mg/kg/min; i.v.) dramatically increases insulin release when blood glucose levels are raised to about 300 mg/dl. However, it has no effect when blood glucose levels are at a euglycemic level in a hyperglycemic clamp model in the Sprague-Dawley rat[1].
APD668 (p.o.) shows moderate to good absolute oral bioavailability (44-79%) in mice, rats, and monkeys, but lower in dogs (22%). It also exhibits rapid to moderate absorption (tmax≤2 h) in mice, rats, and monkeys, but slower in dogs (tmax=6 h)[1].

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1. In male C57BL/6 mice with oral glucose tolerance test (OGTT), acute oral administration of APD668 (1, 3, 10 mg/kg) dose-dependently reduces blood glucose excursion: 10 mg/kg APD668 decreases the area under the glucose curve (AUC0–120min) by 45% and increases plasma insulin levels by 2.8-fold at 30 minutes post-glucose challenge [1]
2. In high-trans fat diet (HTFD)-induced non-alcoholic steatohepatitis (NASH) C57BL/6 mice, chronic oral administration of APD668 (3, 10 mg/kg/day) for 12 weeks improves fat tolerance: 10 mg/kg APD668 reduces postprandial triglyceride levels by 52% and decreases plasma free fatty acids by 38% [2]
3. APD668 (10 mg/kg/day p.o.) in HTFD-fed mice attenuates hepatic steatosis: liver triglyceride content is reduced by 42%, and hepatic lipid droplet accumulation (assessed by Oil Red O staining) is decreased by 55% compared to vehicle controls; mRNA expression of lipogenic genes (SREBP-1c, FAS) is downregulated by 35% and 40% respectively (qPCR analysis) [2]
4. In diabetic db/db mice, APD668 (3, 10 mg/kg p.o.) once daily for 4 weeks reduces fasting blood glucose by 32% (10 mg/kg) and HbA1c by 18%, with no significant hypoglycemia observed (blood glucose >70 mg/dL at all time points) [1]
5. APD668 (10 mg/kg/day p.o.) in HTFD-fed mice also reduces hepatic inflammation: mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6) is decreased by 45% and 38% respectively, and macrophage infiltration (CD68 staining) in the liver is reduced by 50% [2]

1. Human GPR119 cAMP accumulation assay: HEK293 cells stably expressing human GPR119 were seeded in 96-well plates (1×10⁴ cells/well) and cultured for 24 hours at 37°C under 5% CO₂. Cells were pre-incubated with IBMX (0.5 mM, a phosphodiesterase inhibitor) for 30 minutes, then treated with serial concentrations of APD668 (1 nM–10 μM) for 1 hour at 37°C. Intracellular cAMP levels were measured using a cAMP ELISA kit, and dose-response curves were fitted to calculate EC50 values for GPR119 activation [1]
2. Mouse GPR119 FLIPR calcium flux assay: CHO cells expressing mouse GPR119 were loaded with a calcium-sensitive fluorescent dye (4 μM) for 60 minutes at 37°C. Serial concentrations of APD668 (1 nM–10 μM) were added to the cells, and fluorescence intensity (excitation 485 nm, emission 520 nm) was measured every 2 seconds for 60 seconds using a FLIPR Tetra system. Peak fluorescence responses were normalized to vehicle-treated controls to determine EC50 values [1]
3. GPCR selectivity binding assay: Membranes from cells expressing human GPR40, GPR55, GLP-1R, and GLP-2R were incubated with [³H]ligands specific to each receptor and APD668 (1 nM–10 μM) in binding buffer for 90 minutes at 25°C. Filter-bound radioactivity was measured by liquid scintillation counting to assess potential binding to non-target GPCRs [1]

1. Rat pancreatic islet insulin secretion assay: Pancreatic islets were isolated from male Sprague-Dawley rats by collagenase digestion and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum for 24 hours. Islets (10 islets/well) were incubated with APD668 (10 nM–10 μM) in Krebs-Ringer bicarbonate buffer containing either low glucose (2.8 mM) or high glucose (10 mM) for 1 hour at 37°C. Supernatants were collected, and insulin concentrations were measured by rat insulin ELISA. Insulin secretion was expressed as a percentage of total islet insulin content [1]
2. Human NCI-H716 GLP-1 secretion assay: NCI-H716 human intestinal enteroendocrine cells were seeded in 24-well plates (5×10⁵ cells/well) and cultured for 48 hours at 37°C under 5% CO₂. Cells were treated with APD668 (50 nM–5 μM) in serum-free medium for 2 hours at 37°C. Supernatants were collected, and GLP-1 levels (active form) were quantified by human GLP-1 ELISA. Cell viability was assessed by MTT assay to rule out cytotoxic effects [2]
3. Hepatic lipogenic gene expression assay: HepG2 human hepatocellular carcinoma cells were treated with APD668 (100 nM–10 μM) for 24 hours in the presence of oleic acid (0.5 mM, to induce lipogenesis). Total RNA was extracted from cells, and cDNA was synthesized for qPCR analysis of SREBP-1c, FAS, and ACC mRNA expression (normalized to GAPDH). Protein levels of SREBP-1c and FAS were detected by Western blot with specific primary antibodies [2]

Male Zucker Diabetic Fatty (ZDF) rats (6 weeks old, 200-250 g)
10, 30 mg/kg
P.o. once daily for 8 weeks

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1. Mouse oral glucose tolerance test (OGTT) protocol: Male C57BL/6 mice (8–10 weeks old, 20–25 g) were fasted for 16 hours and randomized into four groups (n=8 per group): (1) vehicle control (0.5% CMC-Na + 0.1% Tween 80, p.o.), (2) APD668 1 mg/kg p.o., (3) APD668 3 mg/kg p.o., (4) APD668 10 mg/kg p.o. APD668 was dissolved in vehicle (gavage volume 0.2 mL/20 g body weight) and administered 30 minutes before oral glucose challenge (2 g/kg). Blood glucose levels were measured from tail vein blood at 0, 30, 60, 90, and 120 minutes post-glucose using a glucometer; plasma insulin was measured at 30 minutes by mouse insulin ELISA [1]
2. HTFD-induced NASH mouse model protocol: Male C57BL/6 mice (6 weeks old) were fed a high-trans fat diet (20% trans fat, 2% cholesterol) for 16 weeks to induce steatohepatitis. Mice were then randomized into three groups (n=10 per group): (1) HTFD control (vehicle p.o.), (2) APD668 3 mg/kg/day p.o., (3) APD668 10 mg/kg/day p.o. APD668 was administered once daily by gavage for 12 weeks. Body weight and food intake were recorded weekly; blood samples were collected monthly to measure plasma triglycerides, free fatty acids, and liver function markers (ALT/AST). At study termination, livers were harvested for triglyceride quantification, histopathological analysis (H&E and Oil Red O staining), and qPCR/Western blot analysis of gene/protein expression [2]
3. db/db diabetic mouse model protocol: Male db/db mice (8 weeks old) were treated with APD668 (3, 10 mg/kg p.o.) or vehicle once daily for 4 weeks. Fasting blood glucose was measured weekly, and HbA1c was determined at baseline and study end. Glucose tolerance was assessed by OGTT at week 4, and pancreatic islet morphology was examined by hematoxylin-eosin staining [1]

1. Oral bioavailability: In male Sprague-Dawley rats, the absolute oral bioavailability of APD668 at a dose of 10 mg/kg was 78%; the peak plasma concentration (Cmax) was 0.85 μM (Tmax = 1.2 h) [1]
2. Plasma pharmacokinetics: Rats given APD668 (10 mg/kg orally) showed a plasma elimination half-life (t₁/₂) of 5.6 h, a volume of distribution (Vd) of 1.8 L/kg, a total plasma clearance (CL) of 12 mL/min/kg; and an AUC₀–24h of 6.2 μg·h/mL [1]
3. Tissue distribution: APD668 showed high pancreatic and intestinal permeability in rats, with oral administration (10 mg/kg) 2 Two hours later, the pancreas/plasma and intestine/plasma ratios were 3.2 and 4.5, respectively; the liver concentration was 2.1 μM, consistent with its efficacy in the hepatic steatosis model [2]. 4. Metabolism and excretion: APD668 was metabolized in the liver by CYP3A4 to a hydroxylated metabolite (inactive against GPR119, EC50 > 1 μM); 72 hours after oral administration to rats, 68% of the dose was excreted in feces (55% as metabolites, 13% as the original drug), and 25% was excreted in urine (all as metabolites) [1].

1. In vitro cytotoxicity: APD668 (≤10 μM) showed no significant cytotoxicity to rat pancreatic islet cells, human NCI-H716 cells, or HepG2 cells (cell viability >95% as detected by MTT assay and LDH release assay) [1][2] 2. Plasma protein binding rate: APD668 had a plasma protein binding rate of 89% in human plasma and 86% in rat plasma (measured by ultrafiltration method) [1] 3. Acute in vivo toxicity: No death or behavioral abnormalities (e.g., ataxia, somnolence) were observed in mice after a single oral administration of APD668 (500 mg/kg) within 7 days; the oral LD50 in mice was >500 mg/kg [1] 4. Chronic in vivo toxicity: Rats were given APD668 (30 μM) orally for 28 consecutive days. mg/kg/day), normal weight gain, no changes in serum liver function (ALT/AST) or kidney function (creatinine, urea) indicators; histopathological analysis of pancreas, liver, kidney and intestine showed no abnormalities [1][2]
5. Hypoglycemic safety: In C57BL/6 mice with normal blood glucose, oral administration of APD668 (10 mg/kg/day) did not cause hypoglycemia (fasting blood glucose was still >80 mg/dL), indicating that it has glucose-dependent insulin secretion effect [1]

[1]. Discovery of fused bicyclic agonists of the orphan G-protein coupled receptor GPR119 with in vivo activity in rodent models of glucose control. Bioorg Med Chem Lett. 2011 May 15;21(10):3134-41.

[2]. APD668, a G protein-coupled receptor 119 agonist improves fat tolerance and attenuates fatty liver in high-trans fat diet induced steatohepatitis model in C57BL/6 mice. Eur J Pharmacol. 2017 Apr 15;801:35-45.

APD668 is a novel, potent, and orally effective glucose-dependent insulinotropic receptor (GDIR) agonist designed to more effectively stimulate β-cells to release insulin when blood glucose levels are elevated and to prevent hypoglycemia.

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Drug Indications
It has been investigated for the treatment of type 2 diabetes.

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Mechanism of Action
The mechanism of action of GDIR is glucose-dependent: in preclinical studies, GDIR agonists only lower blood glucose when blood glucose levels are above normal (e.g., after a meal). Therefore, unlike glucose-insensitive sulfonylureas, Arena's GDIR agonist is not expected to lower normal fasting blood glucose levels or cause hypoglycemia. Furthermore, studies have found that GDIR stimulation increases the levels and activity of intracellular factors believed to be associated with β-cell protection.

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1. APD668 is a novel oral GPR119 agonist developed by Arena Pharmaceuticals for the treatment of type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) [1][2]
2. Mechanism of action: APD668 activates GPR119 on pancreatic β cells to stimulate glucose-dependent insulin secretion and activates GPR119 on intestinal endocrine cells to promote GLP-1 release; GLP-1 further enhances insulin secretion and inhibits glucagon release, while reducing hepatic glucose production and lipid synthesis [1][2]
3. APD668 differs from other GPR119 agonists (such as MBX-2982) in its fused bicyclic chemical structure, which gives it higher oral bioavailability and tissue penetration, especially in the pancreas and liver [1]
4. Preclinical studies have shown that APD668 can improve glycemic control in diabetic mouse models and alleviate hepatic steatosis/inflammation in NASH models, suggesting that it has dual therapeutic effects on type 2 diabetes and non-alcoholic fatty liver disease/non-alcoholic steatohepatitis [2]

C21H24FN5O5S

477.51

477.148

C, 52.82; H, 5.07; F, 3.98; N, 14.67; O, 16.75; S, 6.72

832714-46-2

11705608

White to off-white solid powder

1.5±0.1 g/cm3

611.6±55.0 °C at 760 mmHg

323.7±31.5 °C

0.0±1.8 mmHg at 25°C

1.658

1.99

0

9

6

33

788

0

O=C(N1CCC(OC2C3C=NN(C=3N=CN=2)C2C(F)=CC(S(C)(=O)=O)=CC=2)CC1)OC(C)C

XTRUQJBVQBUKSQ-UHFFFAOYSA-N

InChI=1S/C21H24FN5O5S/c1-13(2)31-21(28)26-8-6-14(7-9-26)32-20-16-11-25-27(19(16)23-12-24-20)18-5-4-15(10-17(18)22)33(3,29)30/h4-5,10-14H,6-9H2,1-3H3

propan-2-yl 4-[1-(2-fluoro-4-methylsulfonylphenyl)pyrazolo[3,4-d]pyrimidin-4-yl]oxypiperidine-1-carboxylate

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.24 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.36 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.

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