APD 668

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 以浓度依赖性方式增加转染人 GPR119 的 HEK293 细胞中腺苷酸环化酶的激活，EC50 为 23 nM[1]。 APD668 与雄性和雌性食蟹猴以及人类的血浆蛋白高度结合 (⩾99%)，但与雄性 (93.0%) 和雌性 (96.6%) 大鼠的结合程度较低[1]。

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1. 在稳定表达人GPR119的HEK293细胞中，APD668（1 nM–10 μM）剂量依赖性诱导cAMP积累，EC50为32 nM，1 μM时cAMP水平较溶媒组最大提升6.8倍；在表达小鼠GPR119的HEK293细胞中，cAMP积累的EC50为48 nM[1]
2. 在表达人GPR119的CHO细胞FLIPR钙流实验中，APD668（1 nM–10 μM）激活GPR119的EC50为38 nM，1 μM APD668诱导的钙响应是溶媒对照组的5.2倍[1]
3. 在分离的大鼠胰岛中，APD668（10 nM–10 μM）剂量依赖性刺激葡萄糖依赖性胰岛素分泌：100 nM APD668使胰岛素释放增加2.3倍（10 mM葡萄糖条件），而在低葡萄糖（2.8 mM）条件下对胰岛素分泌无影响[1]
4. 在人NCI-H716肠道肠内分泌细胞中，APD668（50 nM–5 μM）诱导胰高血糖素样肽-1（GLP-1）分泌，EC50为120 nM；1 μM APD668使GLP-1释放较溶媒组增加3.1倍[2]
5. APD668（≤10 μM）在大鼠胰岛细胞和NCI-H716细胞中无细胞毒性，MTT实验显示细胞活力>95%[1][2]

APD668（10-30 mg/kg；每天口服一次，持续 8 周）可显着降低血糖和糖化血红蛋白 (HbA1c) 水平，且不会降低急性药物反应的敏感性[1]。 APD668（1-10 mg/kg；单次口服）可显着降低小鼠口服葡萄糖耐量试验期间的血糖水平，且呈剂量依赖性[1]。 APD668（0.08 mg/kg/min；静脉注射）在正常血糖条件下没有效果，但在 Sprague-Dawley 大鼠的高血糖钳夹模型中，当血糖水平升至约 300 mg/dl 时，会显着刺激胰岛素释放[1]。 APD668 (po) 在小鼠、大鼠和猴子中表现出快速至中度吸收（tmax≤2 小时），但在狗中较慢（tmax=6 小时），在小鼠中表现出中度至良好的绝对口服生物利用度（44-79%），大鼠和猴子，但狗的比例较低 (22%)[1]。动物模型：雄性 Zucker 糖尿病脂肪 (ZDF) 大鼠（6 周龄，200-250 g）[1] 剂量：10, 30 mg/kg 给药方式：每天口服一次，持续 8 周 结果：降低血糖和 HbA1c 水平30毫克/公斤/天。没有患上糖尿病，而用媒介物治疗的老鼠却患上了糖尿病。

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1. 在雄性C57BL/6小鼠口服葡萄糖耐量试验（OGTT）中，单次口服APD668（1、3、10 mg/kg）剂量依赖性降低血糖波动：10 mg/kg APD668使血糖曲线下面积（AUC0–120min）减少45%，葡萄糖激发后30分钟血浆胰岛素水平增加2.8倍[1]
2. 在高反式脂肪饮食（HTFD）诱导的非酒精性脂肪性肝炎（NASH）C57BL/6小鼠模型中，长期口服APD668（3、10 mg/kg/天）12周可改善脂肪耐受：10 mg/kg APD668使餐后甘油三酯水平降低52%，血浆游离脂肪酸减少38%[2]
3. 饲喂HTFD的小鼠口服10 mg/kg/天的APD668后，肝脏脂肪变性减轻：肝甘油三酯含量降低42%，肝内脂滴积累（油红O染色）减少55%（与溶媒对照组相比）；脂生成基因（SREBP-1c、FAS）的mRNA表达分别下调35%和40%（qPCR分析）[2]
4. 在db/db糖尿病小鼠中，APD668（3、10 mg/kg口服）每日一次，连续4周，可使空腹血糖降低32%（10 mg/kg剂量），糖化血红蛋白（HbA1c）降低18%，且未观察到显著低血糖（各时间点血糖>70 mg/dL）[1]
5. 饲喂HTFD的小鼠口服10 mg/kg/天的APD668后，肝脏炎症也得到缓解：促炎细胞因子（TNF-α、IL-6）的mRNA表达分别降低45%和38%，肝脏巨噬细胞浸润（CD68染色）减少50%[2]

1. 人GPR119 cAMP积累实验：将稳定表达人GPR119的HEK293细胞以1×10⁴个细胞/孔接种于96孔板，37℃、5% CO₂培养24小时；细胞经0.5 mM IBMX（磷酸二酯酶抑制剂）预处理30分钟后，加入系列浓度的APD668（1 nM–10 μM）37℃处理1小时；采用cAMP ELISA试剂盒检测细胞内cAMP水平，拟合剂量反应曲线计算GPR119激活的EC50值[1]
2. 小鼠GPR119 FLIPR钙流实验：将表达小鼠GPR119的CHO细胞负载4 μM钙敏感荧光染料，37℃孵育60分钟；加入系列浓度的APD668（1 nM–10 μM），使用FLIPR Tetra系统每2秒检测一次荧光强度（激发光485 nm，发射光520 nm），持续60秒；将荧光峰值响应相对于溶媒对照组归一化，确定EC50值[1]
3. GPCR选择性结合实验：将表达人GPR40、GPR55、GLP-1R和GLP-2R的细胞膜与各受体特异性[³H]配体及APD668（1 nM–10 μM）在结合缓冲液中25℃孵育90分钟；液闪计数检测滤膜结合的放射性强度，评估其与非靶标GPCR的潜在结合[1]

1. 大鼠胰岛胰岛素分泌实验：通过胶原酶消化法从雄性SD大鼠分离胰腺胰岛，在含10%胎牛血清的RPMI 1640培养基中培养24小时；将10个胰岛/孔与APD668（10 nM–10 μM）在含低葡萄糖（2.8 mM）或高葡萄糖（10 mM）的Krebs-Ringer碳酸氢盐缓冲液中37℃孵育1小时；收集上清液，采用大鼠胰岛素ELISA检测胰岛素浓度，胰岛素分泌以胰岛总胰岛素含量的百分比表示[1]
2. 人NCI-H716 GLP-1分泌实验：将人肠道肠内分泌NCI-H716细胞以5×10⁵个细胞/孔接种于24孔板，37℃、5% CO₂培养48小时；细胞经APD668（50 nM–5 μM）在无血清培养基中37℃处理2小时后，收集上清液，采用人GLP-1 ELISA定量活性形式的GLP-1水平；通过MTT实验评估细胞活力以排除细胞毒性影响[2]
3. 肝脏脂生成基因表达实验：人肝癌HepG2细胞在油酸（0.5 mM，诱导脂生成）存在下，经APD668（100 nM–10 μM）处理24小时；提取细胞总RNA并反转录为cDNA，通过qPCR分析SREBP-1c、FAS和ACC的mRNA表达（以GAPDH归一化）；采用特异性一抗通过Western blot检测SREBP-1c和FAS的蛋白水平[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, APD668 has an absolute oral bioavailability of 78% following a 10 mg/kg oral dose; the peak plasma concentration (Cmax) is 0.85 μM (Tmax = 1.2 hours) [1]
2. Plasma pharmacokinetics: Rats administered APD668 (10 mg/kg p.o.) show a plasma elimination half-life (t₁/₂) of 5.6 hours, a volume of distribution (Vd) of 1.8 L/kg, and a total plasma clearance (CL) of 12 mL/min/kg; the AUC₀–24h is 6.2 μg·h/mL [1]
3. Tissue distribution: APD668 exhibits high pancreatic and intestinal penetration in rats, with pancreas/plasma and intestine/plasma ratios of 3.2 and 4.5 respectively at 2 hours post-oral dosing (10 mg/kg); liver concentration at 2 hours is 2.1 μM, consistent with its efficacy in hepatic steatosis models [2]
4. Metabolism and excretion: APD668 is metabolized in the liver by CYP3A4 to a hydroxylated metabolite (inactive at GPR119, EC50 > 1 μM); 72 hours after oral dosing in rats, 68% of the dose is excreted in feces (55% as metabolites, 13% as unchanged drug) and 25% in urine (all as metabolites) [1]

1. In vitro cytotoxicity: APD668 (≤10 μM) shows no significant cytotoxicity in rat pancreatic islet cells, human NCI-H716 cells, or HepG2 cells (cell viability >95% by MTT assay and LDH release) [1][2]
2. Plasma protein binding: APD668 has a plasma protein binding rate of 89% in human plasma and 86% in rat plasma (measured by ultrafiltration) [1]
3. Acute in vivo toxicity: Single oral administration of APD668 (500 mg/kg) in mice causes no mortality or behavioral abnormalities (e.g., ataxia, lethargy) over 7 days; the oral LD50 in mice is >500 mg/kg [1]
4. Chronic in vivo toxicity: Rats treated with APD668 (30 mg/kg/day p.o.) for 28 days show normal weight gain and no changes in serum liver (ALT/AST) or renal (creatinine, urea) function markers; histopathological analysis of pancreas, liver, kidney, and intestine reveals no abnormalities [1][2]
5. Glucose-lowering safety: APD668 (10 mg/kg/day p.o.) in normoglycemic C57BL/6 mice does not induce hypoglycemia (fasting blood glucose remains >80 mg/dL), indicating glucose-dependent insulin secretion effects [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, highly potent and orally active glucose-dependent insulinotropic receptor (GDIR) agonist intended to more efficiently stimulate insulin release by beta cells in response to elevated blood glucose levels, and to also avoid hypoglycemia.

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Drug Indication
Investigated for use/treatment in diabetes mellitus type 2.

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Mechanism of Action
The GDIR mechanism is glucose dependent: in preclinical studies, GDIR agonists only lowered blood glucose when it rose above normal levels, such as after a meal. Therefore, unlike the glucose-insensitive sulfonylureas, Arena's GDIR agonists are not expected to lower normal fasting blood glucose levels or cause hypoglycemia. In addition, GDIR stimulation has been found to increase the levels and activity of intracellular factors thought to be involved in the preservation of beta cells.

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1. APD668 is a novel, orally bioavailable 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 on intestinal enteroendocrine cells to promote GLP-1 release; GLP-1 further enhances insulin secretion and inhibits glucagon release, while also reducing hepatic glucose production and lipid synthesis [1][2]
3. APD668 is distinct from other GPR119 agonists (e.g., MBX-2982) in its fused bicyclic chemical structure, which confers improved oral bioavailability and tissue penetration, particularly in the pancreas and liver [1]
4. Preclinical studies demonstrate that APD668 improves glucose control in diabetic mouse models and attenuates hepatic steatosis/inflammation in NASH models, suggesting potential for dual therapy of T2DM and NAFLD/NASH [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

124.89

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.24 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.36 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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