DPP-4 (IC50 = 4 nM)

体外活性：曲格列汀（也称为 SYR-472）是武田正在开发的一种有效、高选择性、长效的 DPP-4（二肽基肽酶-4）抑制剂，用于治疗 2 型糖尿病 (T2D)。 -每周曲格列汀治疗对 2 型糖尿病患者的血糖控制产生了临床和统计学上的显着改善。它具有良好的耐受性，可能成为这种疾病患者的一种新的治疗选择。曲格列汀在日本被批准用于治疗 2 型糖尿病 (T2DM)。

Trelagliptin/曲格列汀（口服灌胃；7 mg/kg；单次剂量）在狗体内抑制DPP-4活性的时间超过80%，即使在24小时后也是如此，这表明了持续的帕金森病效应[1]。
与赋形剂组相比，Trelagliptin（口服灌胃；3 mg/kg；单次剂量；口服葡萄糖前60分钟）使ob/ob小鼠的AUC_0-120min降低了19.3%，大大提高了葡萄糖耐量[3]。
Trelagliptin（口服灌胃；10mg/kg；每周一次；8周）显著降低了空腹血糖（FBG）水平；在治疗期间，平均下降幅度比对照组低16.8%。此外，它提高了胰岛素水平，在ob/ob小鼠中，AUC_0-120min[3]的胰岛素水平提高了1.7倍。

酶抑制试验[2]
这些研究中使用的人DPP-4酶来自几个来源。如前所述，使用从ATCC（ATCC编号HTB-37；www.ATCC.org）购买的Caco-2细胞部分纯化的人DPP-4来确认trelagliptin抑制剂的效力。为了比较DPP-4抑制剂，trelagliptin、阿格列汀和西格列汀，使用市售重组人DPP-4（台湾Abnova）。为了进行详细的动力学研究，如前所述克隆、表达和纯化重组人DPP-4。此外，使用人、狗和大鼠的血浆样本测定了血浆DPP-4活性的抑制作用。根据之前报道的方法，DPP-4相关蛋白酶，二肽基肽酶-2（DPP-2）和脯氨酰内肽酶（PEP）分别从大鼠肾脏和脑中制备。通过亲和层析从表达每种FLAG标记蛋白的293-F细胞中纯化人二肽基肽酶-8（DPP-8）、二肽基多肽酶-9（DPP-9）和成纤维细胞活化蛋白α（FAPα）。[2]
为了进行详细的动力学研究，使用GP pNA作为底物，并在室温下在pH 7.4的缓冲液中进行测定，该缓冲液含有20 mmol/L HEPES、20 mmol/L MgCl2、0.1 mg/ml牛血清白蛋白和1%（v/v）DMSO。在大多数情况下，最后加入DPP-4酶（终浓度为1 nmol/L）以启动酶反应，但在测量预先形成的DPP-4抑制剂复合物中DPP-4酶活性的恢复时除外，在这种情况下，酶首先与trelagliptin预孵育70分钟，然后通过稀释50倍到含有大量过量（2 mmol/L，约17xKm）GP pNA底物的反应缓冲液中来启动反应。所有测定均以96孔格式重复进行，总测定体积为200uL，每10秒测量405nm处的吸光度，以确定反应时间过程。在大多数情况下，整个反应过程曲线如下所述进行分析。然而，对于通过trelagliptin建立GP pNA底物竞争性抑制的初始速率研究，仅使用了前40秒的吸光度测量值。

---

SD大鼠的体外生物测定、晶体结构测定和药代动力学测定[3]
体外DPP-4抑制研究（至少三个独立实验）、使用表面等离子体共振的结合动力学研究、DPP-4与化合物5的共结晶以及结构测定，以及SD大鼠的药代动力学测定，都是使用我们之前工作中报告的相同操作方法进行的。

使用发色底物Gly-Pro-p-硝基苯胺（GP pNA）（终浓度为0.5 mmol/L）测定Caco-2细胞或血浆中的DPP-4活性，并在pH 7.5的缓冲液中进行，该缓冲液含有100 mmol/L Tris-HCl、1 mg/mL牛血清白蛋白和0.5 mg/mL CHAPS（3-[（3-甲酰氨基丙基）二甲基铵]-1-丙磺酸），在37°C（Caco-2电池的DPP-4组分）或30°C（血浆）下进行60分钟。测量405nm处的吸光度变化以确定反应速率。使用荧光底物Gly-Pro-7-氨基-4-甲基香豆素（GP-AMC）（终浓度90μmol/L）测定重组人DPP-4活性，并在含有25 mmol/L HEPES、140 mmol/L NaCl、1 mg/mL牛血清白蛋白的pH 7.8缓冲液中在37°C下进行15分钟。通过加入100μL 25%（v/v）乙酸停止反应，并使用Envision 2103 Multilabel Reader测量荧光（380 nm激发/460 nm发射）。表1中描述了测量DPP-2、DPP-8、DPP-9、PEP和FAPα活性的反应条件。测量405nm处的吸光度变化以确定反应速率[2]。

ICR ob/ob mice[3]
10 mg/kg
Oral gavage; 10 mg/kg; once a week; 8 weeks

---

Effect on DPP-4 Activity in ob/ob Mice[3]
Eight-week-old ob/ob mice (n = 10 in each group, 5 male and 5 female) were randomly assigned to treatment groups. After 2 h of fasting, baseline blood was collected into a tube containing EDTA. Mice were then treated orally with vehicle (0.5% sodium carboxymethyl cellulose, 10 mL/kg), compound 5 (0.3, 1, 3, 1, and 10 mg/kg), omarigliptin (3 mg/kg), or trelagliptin (3 mg/kg). Subsequently, blood per animal was collected at 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 h. All samples were centrifuged at 10 000 rpm for 2 min, and the plasma was harvested. Aliquots of plasma samples were stored at −80 °C until analysis. The measurement of in vivo DPP-4 activity was the same as the method with ICR mice.

---

Effect on OGTT in db/db Mice[3]
To examine the effect of compound 5 on blood glucose after an oral glucose challenge in 6 week old db/db mice (n = 10 in each group, 5 male and 5 female), compound 5 (3 and 10 mg/kg), omarigliptin (10 mg/kg), trelagliptin (10 mg/kg), or vehicle (0.5% sodium carboxymethyl cellulose) was orally administered to 6 h-fasted db/db mice 60 min prior to the oral glucose challenge (1.5 g/kg). Blood glucose was estimated using a glucometer at 60 min before the glucose load and 0, 15, 30, 60, 90, and 120 min post-glucose challenge. The AUC for the glucose tolerance test was calculated using the trapezoidal method.

---

Long-Term Antidiabetic Effects in db/db Mice[3]
Six-week-old db/db mice were divided into 5 groups (n = 10 in each group, 5 male and 5 female) based on nonfasting blood glucose and 6 h FBG, serum insulin levels, PBW (non-FBW), and 6 h FBW. Lean littermates were used as the lean control. Compound 5 (3 and 10 mg/kg), omarigliptin (10 mg/kg), trelagliptin (10 mg/kg), or vehicle (0.5% sodium carboxymethyl cellulose) was orally administered once weekly for 8 weeks. Nonfasting glucose and FBG, PBW, and 6 h FBW were determined at 7 d intervals. After 7 weeks of treatment, the 6 h-fasted animal was challenged by 1.5 g/kg glucose. Blood glucose was estimated using a glucometer at 0, 15, 30, 60, 90, and 120 min post-glucose challenge. After 8 weeks of treatment, the 6 h-fasted animal was challenged by 1.5 g/kg glucose. Blood samples were collected at 0, 15, 30, and 60 min post-glucose challenge to test plasma insulin levels. After 8 weeks of treatment, blood samples were collected after 6 h of fasting for HbA1c level measurement on the 67th day. The detailed dosing regimen is provided in the Supporting Information (Figure S11).

[1]. Recent approaches to medicinal chemistry and therapeutic potential of dipeptidyl peptidase-4 (DPP-4) inhibitors. Eur J Med Chem. 2014 Mar 3;74:574-605.

[2]. Trelagliptin (SYR-472, Zafatek), Novel Once-Weekly Treatment for Type 2 Diabetes, Inhibits Dipeptidyl Peptidase-4 (DPP-4) via a Non-Covalent Mechanism. PLoS One. 2016 Jun 21;11(6):e0157509.

[3]. Discovery of a Natural-Product-Derived Preclinical Candidate for Once-Weekly Treatment of Type 2 Diabetes. J Med Chem. 2019 Mar 14;62(5):2348-2361.

Trelagliptin is a member of benzenes and a nitrile.
Trelagliptin is under investigation in clinical trial NCT03555591 (Specified Drug-Use Survey of Trelagliptin Tablets "Survey on Long-term Use in Patients With Type 2 Diabetes Mellitus").

C18H20FN5O2

357.38

357.16

C, 60.49; H, 5.64; F, 5.32; N, 19.60; O, 8.95

865759-25-7

Trelagliptin succinate;1029877-94-8

15983988

White to off-white solid powder

1.4±0.1 g/cm3

519.0±60.0 °C at 760 mmHg

267.7±32.9 °C

0.0±1.4 mmHg at 25°C

1.646

1.88

97.05

1

6

3

26

657

1

C(C1C=C(F)C=CC=1C#N)N1C(=O)N(C)C(=O)C=C1N1CCC[C@@H](N)C1

IWYJYHUNXVAVAA-OAHLLOKOSA-N

InChI=1S/C18H20FN5O2/c1-22-17(25)8-16(23-6-2-3-15(21)11-23)24(18(22)26)10-13-7-14(19)5-4-12(13)9-20/h4-5,7-8,15H,2-3,6,10-11,21H2,1H3/t15-/m1/s1

2-[[6-[(3R)-3-aminopiperidin-1-yl]-3-methyl-2,4-dioxopyrimidin-1-yl]methyl]-4-fluorobenzonitrile

SYR 472; Trelagliptin; SYR-472; TRELAGLIPTIN; 865759-25-7; Trelagliptin [USAN]; Trelagliptin [USAN:INN]; UNII-Q836OWG55H; Q836OWG55H; Trelagliptin (USAN); SYR472; trade name: Zafatek

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 (7.00 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (7.00 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 25.0 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。

---

View More

配方 3 中的溶解度: ≥ 2.5 mg/mL (7.00 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 储备液加入到 900 μL 玉米油中并混合均匀。

---

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));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。