DPP-4 (IC50 = 19 nM)

体外活性：作为口服活性剂，西他列汀磷酸盐对 DPP-4 表现出有效的抑制作用，Caco-2 细胞提取物的 IC50 为 19 nM。 MK0431 通过涉及 cAMP/PKA/Rac1 激活的途径减少分离的脾 CD4 T 细胞的体外迁移。最近的一项研究表明，西他列汀发挥一种新颖的直接作用，通过不依赖 DPP-4、依赖蛋白激酶 A 和 MEK-ERK1/2 的途径刺激肠道 L 细胞分泌 GLP-1。因此，它减少了自身免疫对移植物存活的影响。激酶测定：DPP-4 是从汇合的 Caco-2 细胞中提取的。使用裂解缓冲液（10 mM Tris-HCl、150 mM NaCl、0.04 U/mL 抑肽酶、0.5% Nonidet P40、pH 8.0）在室温下孵育 5 分钟后，将细胞在 4 °C 下以 35,000 g 离心 30 分钟，上清液保存于-80°C。通过将 20 μL 适当的化合物稀释液与 50 μL DPP-4 酶的底物 H-Ala-Pro-7-amido-4-三氟甲基香豆素（测定中的最终浓度，100 μM）和 30 μL 混合来进行测定Caco-2 细胞提取物（用 100 mM Tris-HCl、100 mM NaCl、pH 7.8 稀释 1000 倍）。将板在室温下孵育 1 小时，并使用 SpectraMax GeminiXS 在 405/535 nm 的激发/发射波长下测量荧光。将 Caco-2 细胞提取物与高抑制剂浓度（BI 1356 为 30 nM，维格列汀为 3 μM）预孵育 1 小时后，测定抑制剂与 DPP-4 酶的解离动力学。用测定缓冲液将预孵育混合物稀释 3000 倍后，添加底物 H-Ala-Pro-7-amido-4-triflumethylcoumarin，开始酶促反应。在这些条件下，在存在或不存在抑制剂的情况下，在某个时间点DPP-4活性的差异反映了仍然与DPP-4酶结合的该抑制剂的量。使用 SpectraMax 的 SoftMax 软件计算 10 分钟间隔的最大反应速率（荧光单位/秒 × 1000），并针对未抑制反应的速率进行校正 [(vcontrol-vinhibitor)/vcontrol]。细胞测定：将 CD4T 细胞铺在无血清 RPMI 1640 的膜插入物上，并在存在或不存在纯化猪肾 DPP-4（32.1 单位/mg；100 mU）的情况下使用 Transwell 小室（Corning）测定细胞迁移/mL 最终浓度）和 DPP-4 抑制剂（100 μM）。 1小时后，机械除去上表面的细胞，对迁移到下室的细胞进行计数。迁移程度是相对于对照样品来表示的。

在体内，在自由喂养的 Han-Wistar 大鼠中，磷酸西格列汀抑制血浆 DPP-4 活性的 ED50 值计算为给药后 7 小时 2.3 mg/kg 和给药后 24 小时 30 mg/kg。链脲佐菌素诱导的 1 型糖尿病小鼠模型表现出血浆中 DPP-4 水平升高，而在服用磷酸西他列汀饮食的小鼠中，DPP-4 水平可得到显着抑制。这是通过对高血糖调节的积极作用来实现的，可能是通过延长胰岛移植物的存活时间来实现的。大鼠中磷酸西格列汀的血浆清除率和分布容积（40-48 mL/min/kg，7-9 L/kg）高于狗（9 mL/min/kg，3 L/kg）；其半衰期在大鼠中较短，为2小时，而在狗中为4小时。

汇合的 Caco-2 细胞用于提取 DPP-4。用裂解缓冲液（10 mM Tris-HCl、150 mM NaCl、0.04 U/mL 抑肽酶、0.5% Nonidet P40、pH 8.0）在室温下孵育 5 分钟后，将细胞在 35,000 g、4 ℃下离心 30 分钟。 °C，然后将上清液保存在-80°C。将二十微升合适的化合物稀释液与五十微升作为 DPP-4 酶底物的 H-Ala-Pro-7-酰胺基-4-三氟甲基香豆素（测定中的最终浓度：100 微升）和三十微升Caco-2 细胞提取物（用 100 mM Tris-HCl、100 mM NaCl、pH 7.8 稀释 1000 倍）。将板在室温下孵育一小时后，使用 SpectraMax GeminiXS 在 405/535 nm 的激发/发射波长下测量荧光。将 Caco-2 细胞提取物暴露于高浓度抑制剂（BI 1356 为 30 nM，维格列汀为 3 μM）一小时后，确定抑制剂与 DPP-4 酶的解离动力学。一旦用测定缓冲液将预孵育混合物稀释 3000 倍，就通过添加底物 H-Ala-Pro-7-amido-4-triflumethylcoumarini 来启动酶促反应。仍然与DPP-4酶结合的抑制剂的量通过在存在或不存在抑制剂的情况下给定时间的DPP-4活性的差异来指示。使用 SpectraMax 的 SoftMax 软件，以 10 分钟的间隔计算最大反应速率（荧光单位/秒 × 1000），并针对未抑制反应的速率进行校正 [(vcontrol-vinhibitor)/vcontrol]。

将含有 CD4T 细胞的膜插入物铺板于无血清 RPMI 1640 中。使用 Corning Transwell 小室测量细胞迁移，使用或不使用 DPP-4 抑制剂 (100 μM) 和纯化的猪肾 DPP-4（32.1 单位/毫克；最终浓度为 100 mU/mL)。一小时后，对移入下室的细胞进行计数，并机械去除上表面的细胞。迁移量的表达式与对照样品相关。

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胰高血糖素样肽-1（GLP-1）是由肠L细胞分泌到循环中的肠促胰岛素激素。二肽基肽酶IV（DPP-IV）抑制剂西格列汀可防止GLP-1降解，并在临床上用于治疗2型糖尿病患者，从而改善糖化血红蛋白水平。当在2型糖尿病模型新生链脲佐菌素大鼠中检查西格列汀对GLP-1水平的影响时，观察到活性GLP-1的基础血浆水平增加4.9±0.9倍，口服葡萄糖刺激的血浆水平增加3.6±0.4倍（P<0.001），肠道L细胞总数增加1.5±0.1倍（P<0.01）。因此，在体外小鼠GLUTag（mGLUTag）和人hNCI-H716肠L细胞中研究了西格列汀对GLP-1分泌和L细胞信号传导的直接影响。西格列汀（0.1-2μM）增加了mGLUTag和hNCI-H716细胞的GLP-1总分泌量（P<0.01-0.001）。然而，MK0626（1-50μM）是一种结构上无关的DPP-IV抑制剂，在两种模型中均不影响GLP-1的分泌。用GLP-1受体激动剂毒蜥外泌肽-4处理mGLUTag细胞没有调节GLP-1的释放，表明GLP-1对L细胞没有反馈作用。西格列汀增加了mGLUTag和hNCI-H716细胞中的cAMP水平（P<0.01）和ERK1/2磷酸化（P<0.05），但没有改变细胞内钙或磷酸化Akt水平。用蛋白激酶A（H89和蛋白激酶抑制剂）或MAPK激酶-ERK1/2（PD98059和U0126）抑制剂预处理mGLUTag细胞可防止西格列汀诱导的GLP-1分泌（P<0.05-0.01）。这些研究首次证明，西格列汀对肠道L细胞具有直接的、不依赖DPP IV的作用，激活cAMP和ERK1/2信号传导，刺激GLP-1的总分泌[3]。

Mice: C57BL/6J mice that have been fasted overnight are challenged with an oral glucose load (2 g/kg) 45 minutes after the compound is administered. Tail bleed predose and successive time points following the glucose load are used to draw blood samples for glucose measurement. DPP-4 inhibitors or a vehicle are given 16 hours prior to the glucose challenge in order to assess how long the effect lasts on glucose tolerance.

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Effects of MK0431 on islet graft survival in diabetic NOD mice were determined with metabolic studies and micropositron emission tomography imaging, and its underlying molecular mechanisms were assessed.
Results: Treatment of NOD mice with MK0431 before and after islet transplantation resulted in prolongation of islet graft survival, whereas treatment after transplantation alone resulted in small beneficial effects compared with nontreated controls. Subsequent studies demonstrated that MK0431 pretreatment resulted in decreased insulitis in diabetic NOD mice and reduced in vitro migration of isolated splenic CD4+ T-cells. Furthermore, in vitro treatment of splenic CD4+ T-cells with DPP-IV resulted in increased migration and activation of protein kinase A (PKA) and Rac1.
Conclusions: Treatment with MK0431 therefore reduced the effect of autoimmunity on graft survival partially by decreasing the homing of CD4+ T-cells into pancreatic beta-cells through a pathway involving cAMP/PKA/Rac1 activation.[2]

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Effects of the DPP-IV inhibitor MK0431 (sitagliptin) on glycemic control and functional islet mass in a streptozotocin (STZ)-induced type 1 diabetes mouse model were determined with metabolic studies and microPET imaging.
Results: The type 1 diabetes mouse model exhibited elevated plasma DPP-IV levels that were substantially inhibited in mice on an MK0431 diet. Residual beta-cell mass was extremely low in STZ-induced diabetic mice, and although active GLP-1 levels were increased by the MK0431 diet, there were no significant effects on glycemic control. After islet transplantation, mice fed normal diet rapidly lost their ability to regulate blood glucose, reflecting the suboptimal islet transplant. By contrast, the MK0431 group fully regulated blood glucose throughout the study, and PET imaging demonstrated a profound protective effect of MK0431 on islet graft size.
Conclusions: Treatment with a DPP-IV inhibitor can prolong islet graft retention in an animal model of type 1 diabetes.[4]

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The pharmacokinetics, metabolism, and excretion of sitagliptin [MK-0431; (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine], a potent dipeptidyl peptidase 4 inhibitor, were evaluated in male Sprague-Dawley rats and beagle dogs. The plasma clearance and volume of distribution of sitagliptin were higher in rats (40-48 ml/min/kg, 7-9 l/kg) than in dogs ( approximately 9 ml/min/kg, approximately 3 l/kg), and its half-life was shorter in rats, approximately 2 h compared with approximately 4 h in dogs. Sitagliptin was absorbed rapidly after oral administration of a solution of the phosphate salt. The absolute oral bioavailability was high, and the pharmacokinetics were fairly dose-proportional. After administration of [(14)C]sitagliptin, parent drug was the major radioactive component in rat and dog plasma, urine, bile, and feces. Sitagliptin was eliminated primarily by renal excretion of parent drug; biliary excretion was an important pathway in rats, whereas metabolism was minimal in both species in vitro and in vivo. Approximately 10 to 16% of the radiolabeled dose was recovered in the rat and dog excreta as phase I and II metabolites, which were formed by N-sulfation, N-carbamoyl glucuronidation, hydroxylation of the triazolopiperazine ring, and oxidative desaturation of the piperazine ring followed by cyclization via the primary amine. The renal clearance of unbound drug in rats, 32 to 39 ml/min/kg, far exceeded the glomerular filtration rate, indicative of active renal elimination of parent drug.[5]

Absorption
Sitagliptin is 87% orally bioavailable and taking it with or without food does not affect its pharmacokinetics. Sitagliptin reaches maximum plasma concentration in 2 hours.

Route of Elimination
Approximately 79% of sitagliptin is excreted in the urine as the unchanged parent compound. 87% of the dose is eliminated in the urine and 13% in the feces.

Volume of Distribution
198L.

Clearance
350mL/min.

Sitagliptin is secreted in the milk of lactating rats at a milk to plasma ratio of 4:1. It is not known whether sitagliptin is excreted in human milk.

Placental transfer of sitagliptin administered to pregnant rats was approximately 45% at 2 hours and 80% at 24 hours postdose. Placental transfer of sitagliptin administered to pregnant rabbits was approximately 66% at 2 hours and 30% at 24 hours.

Approximately 79% of sitagliptin is excreted unchanged in the urine with metabolism being a minor pathway of elimination.

Elimination of sitagliptin occurs primarily via renal excretion and involves active tubular secretion. Sitagliptin is a substrate for human organic anion transporter-3 (hOAT-3), which may be involved in the renal elimination of sitagliptin. The clinical relevance of hOAT-3 in sitagliptin transport has not been established. Sitagliptin is also a substrate of p-glycoprotein, which may also be involved in mediating the renal elimination of sitagliptin. However, cyclosporine, a p-glycoprotein inhibitor, did not reduce the renal clearance of sitagliptin.

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Metabolism / Metabolites
Sitagliptin is mostly not metabolised, with 79% of the dose excreted in the urine as the unchanged parent compound. Minor metabolic pathways are mediated mainly by cytochrome p450(CYP)3A4 and to a lesser extent by CYP2C8. After 18 hours, 81% of the dose has remained unchanged, while 2% has been N-sulfated to the M1 metabolite, 6% has been oxidatively desaturated and cyclized to the M2 metabolite, <1% glucuronidated at an unknown site to the M3 metabolite, <1% has been carbamoylated and glucuronidated to the M4 metabolite, 6% has been oxidatively saturated and cyclized to the M5 metabolite, and 2% has been hydroxylated at an unknown site to the M6 metabolite. The M2 metabolite is the cis isomer while the M5 metabolite is the trans isomer of the same metabolite.

The metabolism and excretion of (14)C sitagliptin ... were investigated in humans after a single oral dose of 83 mg/193 muCi. Urine, feces, and plasma were collected at regular intervals for up to 7 days. The primary route of excretion of radioactivity was via the kidneys, with a mean value of 87% of the administered dose recovered in urine. Mean fecal excretion was 13% of the administered dose. Parent drug was the major radioactive component in plasma, urine, and feces, with only 16% of the dose excreted as metabolites (13% in urine and 3% in feces), indicating that sitagliptin was eliminated primarily by renal excretion. Approximately 74% of plasma AUC of total radioactivity was accounted for by parent drug. Six metabolites were detected at trace levels, each representing <1 to 7% of the radioactivity in plasma. These metabolites were the N-sulfate and N-carbamoyl glucuronic acid conjugates of parent drug, a mixture of hydroxylated derivatives, an ether glucuronide of a hydroxylated metabolite, and two metabolites formed by oxidative desaturation of the piperazine ring followed by cyclization. These metabolites were detected also in urine, at low levels. Metabolite profiles in feces were similar to those in urine and plasma, except that the glucuronides were not detected in feces. CYP3A4 was the major cytochrome P450 isozyme responsible for the limited oxidative metabolism of sitagliptin, with some minor contribution from CYP2C8. PMID:17220239

Following a (14)C sitagliptin oral dose, approximately 16% of the radioactivity was excreted as metabolites of sitagliptin. Six metabolites were detected at trace levels and are not expected to contribute to the plasma DPP-4 inhibitory activity of sitagliptin. In vitro studies indicated that the primary enzyme responsible for the limited metabolism of sitagliptin was CYP3A4, with contribution from CYP2C8.

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Biological Half-Life
Approximately 12.4 hours. Other studies have reported a half life of approximately 11 hours.

Two double-blind, randomized, placebo-controlled, alternating-panel studies evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of single oral doses of sitagliptin (1.5-600 mg) in healthy male volunteers. Sitagliptin was well absorbed (approximately 80% excreted unchanged in the urine) with an apparent terminal half-life ranging from 8 to 14 hours. ... PMID:16338283

The apparent terminal half life following a 100 mg oral dose of sitagliptin was approximately 12.4 hours

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of sitagliptin during breastfeeding. Sitagliptin has a shorter half-life than most other dipeptidyl-peptidase IV inhibitors, so it might be a better choice among drugs in this class for nursing mothers. Monitoring of the breastfed infant's blood glucose is advisable during maternal therapy with sitagliptin. However, an alternate drug may be preferred, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[1]. (R)-8-(3-amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione (BI 1356), a novel xanthine-based dipeptidyl peptidase 4 inhibitor, has a superior potency and longer duration of action compared with other dipeptidyl peptidase-4 inhibitors. J Pharmacol Exp Ther. 2008 Apr;325(1):175-82.

[2]. Dipeptidyl peptidase IV inhibition with MK0431 improves islet graft survival in diabetic NOD mice partially via T-cell modulation. Diabetes, 2009. 58(3): p. 641-51.

[3]. Novel biological action of the dipeptidylpeptidase-IV inhibitor, sitagliptin, as a GLP-1 secretagogue. Endocrinology, 2012. 153(2): p. 564-73.

[4]. Inhibition of dipeptidyl peptidase IV with sitagliptin (MK0431) prolongs islet graft survival in streptozotocin-induced diabetic mice. Diabetes, 2008. 57(5): p. 1331-9.

[5]. Disposition of the dipeptidyl peptidase 4 inhibitor sitagliptin in rats and dogs. Drug Metab Dispos, 2007. 35(4): p. 525-32.

Sitagliptin Phosphate is the phosphate salt form of sitagliptin, an orally available, competitive, beta-amino acid-derived inhibitor of dipeptidyl peptidase 4 (DDP-4) with hypoglycemic activity. Sitagliptin may cause an increased risk in the development of pancreatitis.
A pyrazine-derived DIPEPTIDYL-PEPTIDASE IV INHIBITOR and HYPOGLYCEMIC AGENT that increases the levels of the INCRETIN hormones GLUCAGON-LIKE PEPTIDE-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). It is used in the treatment of TYPE 2 DIABETES.
See also: Sitagliptin Phosphate (annotation moved to).

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Drug Indication
For patients with type-2 diabetes mellitus, Tesavel is indicated to improve glycaemic control: as monotherapy: in patients inadequately controlled by diet and exercise alone and for whom metformin is inappropriate due to contraindications or intolerance; as dual oral therapy in combination with: metformin when diet and exercise plus metformin alone do not provide adequate glycaemic control; a sulphonylurea when diet and exercise plus maximal tolerated dose of a sulphonylurea alone do not provide adequate glycaemic control and when metformin is inappropriate due to contraindications or intolerance; a PPARγ agonist (i. e. a thiazolidinedione) when use of a PPARγ agonist is appropriate and when diet and exercise plus the PPARγ agonist alone do not provide adequate glycaemic control; as triple oral therapy in combination witha sulphonylurea and metformin when diet and exercise plus dual therapy with these agents do not provide adequate glycaemic control; a peroxisome-proliferator-activated-receptor-gamma (PPARγ) agonist and metformin when use of a PPARγ agonist is appropriate and when diet and exercise plus dual therapy with these agents do not provide adequate glycaemic control. Tesavel is also indicated as add on to insulin (with or without metformin) when diet and exercise plus stable dosage of insulin do not provide adequate glycaemic control.
For adult patients with type-2 diabetes mellitus, Januvia is indicated to improve glycaemic control: as monotherapy: in patients inadequately controlled by diet and exercise alone and for whom metformin is inappropriate due to contraindications or intolerance; as dual oral therapy in combination with: metformin when diet and exercise plus metformin alone do not provide adequate glycaemic control; a sulphonylurea when diet and exercise plus maximal tolerated dose of a sulphonylurea alone do not provide adequate glycaemic control and when metformin is inappropriate due to contraindications or intolerance; a peroxisome-proliferator-activated-receptor-gamma (PPARγ) agonist (i. e. a thiazolidinedione) when use of a PPARγ agonist is appropriate and when diet and exercise plus the PPARγ agonist alone do not provide adequate glycaemic control; a PPARγ agonist (i. e. a thiazolidinedione) when use of a PPARγ agonist is appropriate and when diet and exercise plus the PPARγ agonist alone do not provide adequate glycaemic control; as triple oral therapy in combination with: a sulphonylurea and metformin when diet and exercise plus dual therapy with these medicinal products do not provide adequate glycaemic control; a PPARγ agonist and metformin when use of a PPARγ agonist is appropriate and when diet and exercise plus dual therapy with these medicinal products do not provide adequate glycaemic control. Januvia is also indicated as add-on to insulin (with or without metformin) when diet and exercise plus stable dose of insulin do not provide adequate glycaemic control.
Treatment of type II diabetes mellitus
Treatment of type II diabetes mellitus

C16H20F6N5O6P

523.32

523.105

C, 36.72; H, 3.85; F, 21.78; N, 13.38; O, 18.34; P, 5.92

654671-77-9

Sitagliptin;486460-32-6;Sitagliptin phosphate;654671-78-0;(S)-Sitagliptin phosphate;823817-58-9;(Rac)-Sitagliptin;823817-56-7

11591741

White to off-white solid powder

529.9ºC at 760 mmHg

274.3ºC

1.661

173.84

5

15

4

34

616

1

[H]O[H].O=C(N1CC2=NN=C(C(F)(F)F)N2CC1)C[C@H](N)CC3=CC(F)=C(F)C=C3F.O=P(O)(O)O

GQPYTJVDPQTBQC-KLQYNRQASA-N

InChI=1S/C16H15F6N5O.H3O4P.H2O/c17-10-6-12(19)11(18)4-8(10)3-9(23)5-14(28)26-1-2-27-13(7-26)24-25-15(27)16(20,21)22;1-5(2,3)4;/h4,6,9H,1-3,5,7,23H2;(H3,1,2,3,4);1H2/t9-;;/m1../s1

(3R)-3-amino-1-[3-(trifluoromethyl)-6,8-dihydro-5H-[1,2,4]triazolo[4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one;phosphoric acid;hydrate

MK 431; Sitagliptin Phosphate; MK-0431; MK0431; MK 0431; Sitagliptin Phosphate Monohydrate; Sitagliptin phosphate monohydrate; 654671-77-9; Januvia; Sitagliptin phosphate hydrate; Glactiv; (R)-3-Amino-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one phosphate hydrate; sitagliptin monophosphate monohydrate; MK-431; MK431; trade name: Januvia Xelevia Janumet

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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: 50 mg/mL (95.54 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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配方 2 中的溶解度: Saline: 30 mg/mL

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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));
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；
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。