DPP-4 (IC50 = 26 nM)

沙格列汀（100 nM；48 小时；INS-1 832/13 细胞）治疗显着增加了 S β 细胞的增殖[1]。用沙格列汀（100 nM；48 小时；INS-1 832/13 细胞）处理可提高 p-AKT 和活性 β-catenin 蛋白的水平，以及 c-myc 和细胞周期蛋白 D1 的产生 [1]。为了增加胰岛素分泌并减少胰高血糖素分泌，沙格列汀抑制胰高血糖素样肽-1的分解[3]。

给高脂饮食并用链脲佐菌素刺激的糖尿病大鼠施用沙格列汀（1 mg/kg）12周。这导致通过高血糖钳测量测量的胰腺胰岛素分泌能力显着增加，以及β细胞和α细胞面积之间的比率增加[1]。在 Han-Wistar 大鼠中，沙格列汀剂量依赖性地抑制血浆 DPP-4 活性； 1mg/kg给药后7小时约发生70%抑制，10mg/kg给药后7小时约90%抑制发生。注射24小时后，抑制效果仍然存在，分别约为20%和70%[2]。

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。

细胞活力测定 [1]
细胞类型： INS-1 832/13 细胞
测试浓度： 100 nM
孵育时间： 48小时
实验结果：显着诱导β细胞增殖。

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蛋白质印迹分析[1]
细胞类型： INS-1 832/13 细胞
测试浓度： 100 nM
孵育时间：48小时
实验结果：p-AKT和活性β-catenin蛋白水平增加，而c-myc和cyclin蛋白水平增加D1 蛋白表达。

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]. Saxagliptin Induces β-Cell Proliferation through Increasing Stromal Cell-Derived Factor-1α In Vivo and In Vitro. Front Endocrinol (Lausanne). 2017 Nov 27;8:326.

[2]. Saxagliptin: A dipeptidyl peptidase-4 inhibitor in the treatment of type 2 diabetes mellitus. J Pharmacol Pharmacother. 2011 Oct;2(4):230-5.

[3]. Discovery and preclinical profile of Saxagliptin (BMS-477118): a highly potent, long-acting, orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. J Med Chem. 2005 Jul 28;48(15):5025-37.

[4]. Saxagliptin: a new dipeptidyl peptidase-4 inhibitor for the treatment of type 2 diabetes. Adv Ther. 2009 May;26(5):488-99.

Saxagliptin Hydrochloride is the hydrochloride salt form of saxagliptin, an orally bioavailable, potent, selective and competitive, cyanopyrrolidine-based inhibitor of dipeptidyl peptidase 4 (DPP-4), with hypoglycemic activity. Saxagliptin is metabolized into an, although less potent, active mono-hydroxy metabolite.
See also: Metformin Hydrochloride; Saxagliptin Hydrochloride (component of); Dapagliflozin; Saxagliptin Hydrochloride (component of) ... View More ...

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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.

C18H26CLN3O2

351.875

351.171

C, 61.44; H, 7.45; Cl, 10.07; N, 11.94; O, 9.09

709031-78-7

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

49800073

White to off-white solid powder

2.598

90.35

3

4

2

24

609

4

C1[C@@H]2C[C@@H]2N([C@@H]1C#N)C(=O)[C@H](C34CC5CC(C3)CC(C5)(C4)O)N.Cl

TUAZNHHHYVBVBR-IGSRIJEQSA-N

InChI=1S/C18H25N3O2.ClH/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,1-7,9,20H21H/t10-,11+,12-,13+,14+,15-,17+,18-/m1./s1

(1S,3S,5S)-2-((2S)-Amino(3-hydroxytricyclo(3.3.1.13,7)dec-1-yl)acetyl)2- azabicyclo(3.1.0)hexane-3-carbonitrile hydrochloride

Saxagliptin hydrochloride; 709031-78-7; Saxagliptin HCl; Onglyza; UNII-Z8J84YIX6L; Z8J84YIX6L; (1S,3S,5S)-2-((2S)-2-Amino-2-(3-hydroxyadamantan-1-yl)acetyl)-2-azabicyclo[3.1.0]hexane-3-carbonitrile hydrochloride; Saxagliptin (hydrochloride);

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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；
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。