DPP-4 (IC50 = 26 nM)

体外活性：沙格列汀对 DPP4 的抑制常数 Ki 为 1.3 nM，比维格列汀或西格列汀（另两种 DPP4 抑制剂）的效力强 10 倍，Ki 分别为 13 和 18 nM。此外，沙格列汀对 DPP4 的特异性比对 DPP8 或 DPP9 酶的特异性更高（分别为 400 倍和 75 倍）。沙格列汀的活性代谢物的效力比母体低两倍。与一系列其他蛋白酶相比，沙格列汀及其代谢物对于预防 DPP4 具有高度选择性（>4000 倍）（与 DPP8 和 DPP9 相比，西格列汀和维格列汀对 DPP4 的选择性分别为 >2600 倍和<250 倍） ）。沙格列汀可减少肠促胰岛素激素胰高血糖素样肽-1 的降解，从而增强其作用，并与改善 β 细胞功能和抑制胰高血糖素分泌有关。激酶测定：Saxagliptin H2O(BMS477118 H2O) 是一种选择性、可逆的 DPP4 抑制剂，IC50 为 26 nM，Ki 为 1.3 nM。细胞测定：沙格列汀对 DPP4 的抑制常数 Ki 为 1.3 nM，比维格列汀或西他列汀（另两种 DPP4 抑制剂）的效力强 10 倍，Ki 分别为 13 和 18 nM。此外，沙格列汀对 DPP4 的特异性比对 DPP8 或 DPP9 酶的特异性更高（分别为 400 倍和 75 倍）。沙格列汀的活性代谢物的效力比母体低两倍。与一系列其他蛋白酶相比，沙格列汀及其代谢物对于预防 DPP4 具有高度选择性（>4000 倍）（与 DPP8 和 DPP9 相比，西格列汀和维格列汀对 DPP4 的选择性分别为 >2600 倍和<250 倍） ）[2]。沙格列汀可减少肠促胰岛素激素胰高血糖素样肽-1 的降解，从而增强其作用，并与改善 β 细胞功能和抑制胰高血糖素分泌有关

在 Zuckerfa/fa 大鼠中，沙格列汀对葡萄糖漂移的最大反应与对照相比约 60% 的血浆 DPP4 抑制相关，并且在较高抑制百分比下没有观察到额外的抗高血糖作用。相对于对照组，沙格列汀在 0.13-1.3 mg/kg 的剂量范围内，可非常有效地在 ob/ob 小鼠中引起明显的剂量依赖性葡萄糖清除率增强。沙格列汀在 oGTT 后 15 分钟以剂量依赖性方式显着升高血浆胰岛素，同时在 oGTT 后 60 分钟改善葡萄糖清除率曲线。

体外DPP-IV抑制试验。[3]
在稳态条件下，通过观察假底物Gly-Pro-pNA裂解后405nm处的吸光度增加来测量对人DPP-IV活性的抑制。使用Thermomax板读数器在96孔板中进行了测定。通常，反应包含100μL ATE缓冲液（100 mM Aces、52 mM Tris、52 mM乙醇胺，pH 7.4）、0.45 nM酶、120或1000μM底物（SKm，Km=180μM）和可变浓度的抑制剂。为了确保慢结合抑制剂的稳态条件，在添加底物之前，酶与化合物预孵育40分钟。所有系列抑制剂稀释液均在DMSO中，最终溶剂浓度不超过1%。通过将抑制数据拟合到结合等温线来评估抑制剂的效力：vi/v=范围/[1+（I/IC50）n]+背景，其中vi是不同浓度抑制剂I下的初始反应速度；v是无抑制剂时的控制速度；范围是未受抑制的速度和背景之间的差异；背景是在没有酶的情况下自发底物水解的速率；n是希尔系数。根据方程式Ki=IC50/[1+（s/Km）]，通过假设竞争性抑制，将每种底物浓度下的计算IC50转化为Ki。通过在高和低底物浓度下从测定中获得的Ki值的密切一致性来判断，所有抑制剂都具有竞争力。在低底物浓度下的IC50接近测定中使用的酶浓度的情况下，数据符合Morrison方程，以解释游离抑制剂的耗竭。30进一步细化IC50值，以确定Ki值，从而使用Ki=IC50/[1+（S/Km）]解释测定中的底物浓度。

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肝微粒体代谢率测定方法。[3]
使用大鼠肝微粒体。孵育物含有50 mM磷酸钾、约1 mg/mL微粒体蛋白、10 mM NADPH和10μM试验化合物。通过加入底物引发反应，并在37°C的振荡水浴中进行。通过加入等体积的乙腈并离心来终止培养。通过LC/MS分析上清液，在0和10分钟时进行母体定量。浓度的百分比变化用于计算母体化合物的代谢速率。

血清饥饿一晚后，将亚汇合细胞与 1.5 或 15 μM 沙格列汀、FBS (1%)、胰岛素 (5 ng/mL) 或 IGF1 (10-8 M) 一起孵育一小时（影响信号）转导机制）或二十四小时（影响细胞增殖）。

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通过电穿孔将表达载体转染到中国仓鼠卵巢（CHO-DG44）细胞中，产生稳定的细胞系。CHO-DG44细胞系在添加了HT（甘氨酸、次黄嘌呤和胸苷）、谷氨酰胺和重组蛋白的PFCHO培养基中生长。然后收集1×107个细胞/mL，在300V下用60μg DNA进行电穿孔转染，然后转移到T75烧瓶中。转染后第三天，移除HT补充剂，用甲氨蝶呤（MTX，10 nM）开始选择。再过10天后，将细胞接种到96孔板的单个孔中。每10天，MTX的浓度增加2-3倍，最高可达400 nM。最终稳定的细胞系选择是基于表达蛋白的产量和活性。使用常规阴离子交换、凝胶过滤（S-200）和高分辨率MonoQ柱进一步纯化蛋白质。最终的蛋白质在SDS-PAGE凝胶上产生了一条带。氨基酸序列分析表明样品中有两个DPP-IV群体。该蛋白质的一部分从N端截短了27个氨基酸，而另一部分缺少N端37个氨基酸，这表明在分离过程中，整个跨膜结构域（包括His标签）被CHO细胞中存在的蛋白酶去除。使用Bradford染料法测量总蛋白浓度，并通过用我们之前报道的抑制剂（参考文献18中的化合物29）滴定酶来确定活性DPP-IV的量。在抑制或催化过程中没有观察到两相行为，这表明两种蛋白质群体在功能上是相同的[3]。

Male 13−14 week-old ob/ob mice
10 μmol/kg
Orally

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Pharmacokinetic and BioavailabilityStudies in Rats. [3]
Rats were housed under standard conditions and had free access to water and standard rodent laboratory diet. Adult male Sprague Dawley rats were surgically prepared with indwelling jugular vein cannulae 1 day prior to drug administration. Rats were fasted overnight prior to dosing and were fed 8 h after dosing. The animals had free access to water and were conscious and unrestrained throughout the study. Each rat was given either a single intravenous (iv) or oral dose (10 mg/kg, n = 2, both routes). The iv doses were administered as a bolus through the jugular vein cannula and the oral doses were by gavage. The compounds were administered as a solution in water. Blood samples (250 μL) were collected at serial time points for 12 h after dose into heparin-containing tubes. Plasma was prepared immediately, frozen, and stored at −20 °C prior to analysis.

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Rat ex Vivo Plasma DPP-IV Inhibition. [3]
DPP-IV activity in rat plasma was assayed ex vivo using Ala-Pro-AFC·TFA, a fluorescence-generating substrate from Enzyme Systems Products. Plasma samples were collected from normal male Sprague−Dawley rats at various timepoints following an oral dose of test compound as previously described.18 A 20 μL plasma sample was mixed with 200 μL of reaction buffer, 50 mM Hepes, and 140 mM NaCl. The buffer contained 0.1 mM Ala-Pro-AFC·TFA. Fluorescence was then read for 20 min on a Perseptive Biosystem Cytofluor-II at 360 nm excitation wavelength, and 530 nm emission wavelength. The initial rate of DPP-IV enzyme activity was calculated over the first 20 min of the reaction, with units/mL defined as the rate of increase of fluorescence intensity (arbitrary units) per mL plasma. All in vivo data presented are mean ± SE (n = 6). Data analysis was performed using ANOVA followed by Fisher Post-hoc.

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Oral Glucose Tolerance Test in Zucker Rats. [3]
Male Zuckerfa/fa rats (Harlan) weighing between 400 and 450 g were housed in a room that was maintained on a 12 h light/dark cycle and were allowed free access to normal rodent chow and tap water. The day before the experiment, the rats were weighed and divided into control and treated groups of six. Rats were fasted 17 h prior to the start of the study. On the day of the experiment, animals were dosed orally with vehicle (water) or DPP-IV inhibitors (0.3, 1, or 3 μmol/kg) at −240 min. Two blood samples were collected at −240 and 0 min by tail bleed. Glucose (2 g/kg) was administered orally at 0 min. Additional blood samples were collected at 15, 30, 60, and 120 min. Blood samples were collected into EDTA-containing tubes from Starstedt. Plasma glucose was determined by Cobas Mira by the glucose oxidation method.

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Oral Glucose Tolerance Test in ob/ob Mice. [3]
Male 13−14 week-old ob/ob mice were maintained under constant temperature and humidity conditions, a 12:12 light-dark cycle, and had free access to a 10% fat rodent diet and tap water. After an overnight fasting period of 16 h, animals were dosed orally with vehicle (water) or DPP-IV inhibitor (1, 3, 10 μmol/kg) at −60 min. Two blood samples were collected at −60 and 0 min by tail bleed for glucose and insulin determinations. Glucose (2 g/kg) was administered orally at 0 min. Additional blood samples were collected at 15, 30, 60, 90, and 120 min for glucose and insulin determinations. Blood samples were collected into EDTA-containing tubes. Plasma glucose was determined with a Accu-Chek Advantage glucometer. Plasma insulin was assayed using a mouse insulin ELISA kit. Data represent the mean of 12−24 mice/group. Data analysis was performed using one way ANOVA followed by Dunnett's test.

Absorption
Following a 5 mg single oral dose of saxagliptin to healthy subjects, the mean plasma AUC values for saxagliptin and its active metabolite were 78 ng•h/mL and 214 ng•h/mL, respectively. The corresponding plasma Cmax values were 24 ng/mL and 47 ng/mL, respectively. Saxagliptin did not accumulate following repeated doses. The median time to maximum concentration (Tmax) following the 5 mg once daily dose was 2 hours for saxagliptin and 4 hours for its active metabolite. Bioavailability, 2.5 - 50 mg dose = 67%

Route of Elimination
Saxagliptin is eliminated by both renal and hepatic pathways. Following a single 50 mg dose of 14C-saxagliptin, 24%, 36%, and 75% of the dose was excreted in the urine as saxagliptin, its active metabolite, and total radioactivity, respectively. A total of 22% of the administered radioactivity was recovered in feces representing the fraction of the saxagliptin dose excreted in bile and/or unabsorbed drug from the gastrointestinal tract.

Volume of Distribution
151 L

Clearance
Renal clearance, single 50 mg dose = 14 L/h

A single-dose, open-label study was conducted to evaluate the pharmacokinetics of saxagliptin (10 mg dose) in subjects with varying degrees of chronic renal impairment (N=8 per group) compared to subjects with normal renal function. The 10 mg dosage is not an approved dosage. The study included patients with renal impairment classified on the basis of creatinine clearance as mild (>50 to =80 mL/min), moderate (30 to =50 mL/min), and severe (<30 mL/min), as well as patients with end-stage renal disease on hemodialysis. ... The degree of renal impairment did not affect the Cmax of saxagliptin or its active metabolite. In subjects with mild renal impairment, the AUC values of saxagliptin and its active metabolite were 20% and 70% higher, respectively, than AUC values in subjects with normal renal function. Because increases of this magnitude are not considered to be clinically relevant, dosage adjustment in patients with mild renal impairment is not recommended. In subjects with moderate or severe renal impairment, the AUC values of saxagliptin and its active metabolite were up to 2.1- and 4.5-fold higher, respectively, than AUC values in subjects with normal renal function. To achieve plasma exposures of saxagliptin and its active metabolite similar to those in patients with normal renal function, the recommended dose is 2.5 mg once daily in patients with moderate and severe renal impairment, as well as in patients with end-stage renal disease requiring hemodialysis. Saxagliptin is removed by hemodialysis.

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Saxagliptin is eliminated by both renal and hepatic pathways. Following a single 50 mg dose of (14)-C-saxagliptin, 24%, 36%, and 75% of the dose was excreted in the urine as saxagliptin, its active metabolite, and total radioactivity, respectively. The average renal clearance of saxagliptin (~230 mL/min) was greater than the average estimated glomerular filtration rate (approximately 120 mL/min), suggesting some active renal excretion. A total of 22% of the administered radioactivity was recovered in feces representing the fraction of the saxagliptin dose excreted in bile and/or unabsorbed drug from the gastrointestinal tract.

Saxagliptin was rapidly absorbed after oral administration in the fasted state, with maximum plasma concentrations (Cmax) of saxagliptin and its major metabolite attained within 2 and 4 hours (Tmax), respectively. The Cmax and AUC values of saxagliptin and its major metabolite increased proportionally with the increment in the saxagliptin dose, and this dose-proportionality was observed in doses up to 400 mg. Following a 5 mg single oral dose of saxagliptin to healthy subjects, the mean plasma AUC values for saxagliptin and its major metabolite were 78 ng*hr/mL and 214 ng*hr/mL, respectively. The corresponding plasma Cmax values were 24 ng/mL and 47 ng/mL, respectively. The intra-subject coefficients of variation for saxagliptin Cmax and AUC were less than 12%.

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Metabolism / Metabolites
The metabolism of saxagliptin is primarily mediated by cytochrome P450 3A4/5 (CYP3A4/5). 50% of the absorbed dose will undergo hepatic metabolism. The major metabolite of saxagliptin, 5-hydroxy saxagliptin, is also a DPP4 inhibitor, which is one-half as potent as saxagliptin.

The metabolism of saxagliptin is primarily mediated by CYP3A4/5. In in vitro studies, saxagliptin and its active metabolite did not inhibit CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, or 3A4, or induce CYP1A2, 2B6, 2C9, or 3A4. Therefore, saxagliptin is not expected to alter the metabolic clearance of coadministered drugs that are metabolized by these enzymes. Saxagliptin is a P-glycoprotein (P-gp) substrate but is not a significant inhibitor or inducer of P-gp. ... The major metabolite of saxagliptin is also a DPP4 inhibitor, which is one-half as potent as saxagliptin.

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Biological Half-Life
Saxagliptin = 2.5 hours; 5-hydroxy saxagliptin = 3.1 hours;

Following a single oral dose of Onglyza 5 mg to healthy subjects, the mean plasma terminal half-life for saxagliptin and its active metabolite was 2.5 and 3.1 hours, respectively.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of saxagliptin during breastfeeding. Saxagliptin has a shorter half-life than the 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 saxagliptin.[1] 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]. Eur J Pharmacol. 2014 Mar 15:727:8-14.

[2]. Diabetes Obes Metab. 2011 Sep;13(9):850-8.

[3]. J Med Chem. 2005 Jul 28;48(15):5025-37.

[4]. Vascul Pharmacol. 2016 Jan:76:62-71.

[5]. Am J Health Syst Pharm. 2010 Sep 15;67(18):1515-25.

Saxagliptin hydrate is a hydrate that is the monohydrate form of anhydrous saxagliptin. Used for the treatment of Type II diabetes. It has a role as a hypoglycemic agent and an EC 3.4.14.5 (dipeptidyl-peptidase IV) inhibitor. It contains a saxagliptin.
Saxagliptin is a potent, selective and competitive, cyanopyrrolidine-based, orally bioavailable inhibitor of dipeptidyl peptidase 4 (DPP-4), with hypoglycemic activity. Saxagliptin is metabolized into an, although less potent, active mono-hydroxy metabolite.
See also: Saxagliptin Hydrochloride (annotation moved to).

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Drug Indication
Add-on combination therapyOnglyza is indicated in adult patients aged 18 years and older with type-2 diabetes mellitus 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 metformin alone, with diet and exercise, does not provide adequate glycaemic control; in combination with a sulphonylurea, when the sulphonylurea alone, with diet and exercise, does not provide adequate glycaemic control in patients for whom use of metformin is considered inappropriate; in combination with a thiazolidinedione, when the thiazolidinedione alone with diet and exercise, does not provide adequate glycaemic control in patients for whom use of a thiazolidinedione is considered appropriate; as triple oral therapy: in combination with metformin plus a sulphonylurea when this regimen alone, with diet and exercise, does not provide adequate glycaemic control; as combination therapy with insulin (with or without metformin), when this regimen alone, with diet and exercise, does not provide adequate glycaemic control.

C18H25N3O2.H2O

333.43

333.205

C, 64.84; H, 8.16; N, 12.60; O, 14.39

945667-22-1

Saxagliptin;361442-04-8; Saxagliptin hydrochloride; 709031-78-7; Saxagliptin hydrate;945667-22-1; 361442-04-8; 1073057-20-1 (HCl hydrate); 1073057-33-6 (benzoate hydrate)

53297473

Off-white to light yellow solid powder

1.731

99.58

3

5

2

24

609

4

[C@@H](C12CC3CC(CC(C3)C1)(O)C2)(N)C(N1[C@H](C#N)C[C@@H]2C[C@H]12)=O.O

AFNTWHMDBNQQPX-NHKADLRUSA-N

InChI=1S/C18H25N3O2.H2O/c19-8-13-2-12-3-14(12)21(13)16(22)15(20)17-4-10-1-11(5-17)7-18(23,6-10)9-17;/h10-15,23H,1-7,9,20H2;1H2/t10?,11?,12-,13+,14+,15-,17?,18?;/m1./s1

(1S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxy-1-adamantyl)acetyl]-2-azabicyclo[3.1.0]hexane-3-carbonitrile;hydrate

BMS-477118 hydrate; Onglyza hydrate; Saxagliptin hydrate; 945667-22-1; saxagliptin monohydrate; Onglyza; Saxagliptin (hydrate); 9GB927LAJW; BMS-477118-11; (1S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxy-1-adamantyl)acetyl]-2-azabicyclo[3.1.0]hexane-3-carbonitrile;hydrate; BMS 477118 hydrate; BMS477118 hydrate; brand name: Onglyza

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)