DPP-4 (IC50 = 26 nM)

Treatment with saxagliptin (100 nM; 48 hours; INS-1 832/13 cells) markedly increased the proliferation of S β cells [1]. Treatment with saxagliptin (100 nM; 48 hours; INS-1 832/13 cells) raises the levels of p-AKT and active β-catenin proteins, as well as the production of c-myc and cyclin D1 [1]. In order to increase insulin secretion and decrease glucagon secretion, saxagliptin inhibits the breakdown of glucagon-like peptide-1 [3].

Saxagliptin (1 mg/kg) was administered for 12 weeks to diabetic rats fed a high-fat diet and stimulated with streptozotocin. This resulted in a notable increase in the pancreatic insulin secretory capacity measured by hyperglycemic clamp, as well as an increase in the ratio between the β-cell and α-cell area [1]. In Han-Wistar rats, saxagliptin dose-dependently suppresses plasma DPP-4 activity; approximately 70% inhibition occurs 7 hours after 1 mg/kg administration, and approximately 90% inhibition occurs 7 hours after 10 mg/kg administration. Twenty-four hours after injection, the inhibitory effect was still present at about 20% and 70%, respectively [2].

In Vitro DPP-IV Inhibition Assays. [3]
Inhibition of human DPP-IV activity was measured under steady-state conditions by following the absorbance increase at 405 nm upon the cleavage of the pseudosubstrate, Gly-Pro-pNA. Assays were performed in 96-well plates using a Thermomax plate reader. Typically reactions contained 100 μL of ATE buffer (100 mM Aces, 52 mM Tris, 52 mM ethanolamine, pH 7.4), 0.45 nM enzyme, either 120 or 1000 μM of substrate (S < Km and S > Km, Km = 180 μM) and variable concentration of the inhibitor. To ensure steady-state conditions for slow-binding inhibitors, enzyme was preincubated with the compound for 40 min prior to substrate addition. All serial inhibitor dilutions were in DMSO and final solvent concentration did not exceed 1%. Inhibitor potency was evaluated by fitting inhibition data to the binding isotherm:  vi/v = range/[1 + (I/IC50)n] + background, where vi is the initial reaction velocity at different concentrations of inhibitor, I; v is the control velocity in the absence of inhibitor; range is the difference between the uninhibited velocity and background; background is the rate of spontaneous substrate hydrolysis in the absent of enzyme; n is the Hill coefficient. Calculated IC50's at each substrate concentration were converted to Ki's by assuming competitive inhibition according to the equation Ki = IC50/[1 + (S/Km)]. All inhibitors were competitive as judged by close agreement of Ki values obtained from assays at high and low substrate concentrations. In cases where IC50 at the low substrate concentration was close to the enzyme concentration used in the assay, the data were fit to the Morrison equation to account for the depletion of the free inhibitor.30 IC50 values were further refined to determine Ki values to account for the substrate concentration in the assay using Ki = IC50/[1 + (S/Km)].

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Liver Microsomal Metabolic Rate Determination Methods. [3]
Rat liver microsomes were used. Incubations contained 50 mM potassium phosphate, ca. 1 mg/mL microsomal protein, 10 mM NADPH, and 10 μM test compound. Reactions were initiated by the addition of substrate and were carried out in a shaking water bath at 37 °C. Incubations were terminated by the addition of an equal volume of acetonitrile and centrifugation. The supernatants were analyzed by LC/MS with parent quantitation at 0 and 10 min. The percent change in concentration was used to calculate a rate of metabolism of parent compound.

Cell viability assay [1]
Cell Types: INS-1 832/13 Cell
Tested Concentrations: 100 nM
Incubation Duration: 48 hrs (hours)
Experimental Results: Dramatically induced β-cell proliferation.

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Western Blot Analysis[1]
Cell Types: INS-1 832/13 Cell
Tested Concentrations: 100 nM
Incubation Duration: 48 hrs (hours)
Experimental Results: p-AKT and active β-catenin protein levels increased, while c-myc and cyclin increased D1 protein Express.

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
In healthy subjects, after a single oral dose of 5 mg saxagliptin, the mean plasma AUC values of 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. No accumulation of saxagliptin occurred after repeated dosing. After a once-daily dose of 5 mg, the median time to peak concentration (Tmax) of saxagliptin was 2 hours, and the median time to peak concentration of its active metabolite was 4 hours. Bioavailability (2.5–50 mg dose) = 67%
Elimination Pathway
Saxagliptin is primarily eliminated via the renal and hepatic routes. After a single dose of 50 mg 14C-saxagliptin, 24%, 36%, and 75% of the dose were excreted in the urine as saxagliptin, its active metabolite, and total radioactivity, respectively. 22% of the administered radioactivity was recovered in feces, representing the dose of saxagliptin excreted in bile and/or the drug not absorbed 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 aimed to evaluate the pharmacokinetics of saxagliptin (10 mg dose) in subjects with varying degrees of chronic renal impairment (8 patients per group) and subjects with normal renal function. The 10 mg dose is not the approved dose. The study included patients with renal impairment categorized by 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 undergoing hemodialysis. …The degree of renal impairment did not affect the Cmax of saxagliptin and its active metabolites. In patients with mild renal impairment, the AUC values of saxagliptin and its active metabolite were 20% and 70% higher, respectively, than in patients with normal renal function. Since this increase is not clinically significant, dose adjustment is not recommended for patients with mild renal impairment. In patients with moderate or severe renal impairment, the AUC values of saxagliptin and its active metabolite were 2.1 times and 4.5 times higher, respectively, than in patients with normal renal function. To ensure similar plasma exposure of saxagliptin and its active metabolite to patients with normal renal function, the recommended dose for patients with moderate to severe renal impairment and end-stage renal disease requiring hemodialysis is 2.5 mg once daily. Saxagliptin is removed by hemodialysis.

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Saxagliptin is cleared via the kidneys and liver. Following a single oral dose of 50 mg (14)-C-saxagliptin, 24%, 36%, and 75% of the dose were excreted in the urine as saxagliptin, its active metabolites, and total radioactivity, respectively. The mean renal clearance of saxagliptin (approximately 230 mL/min) was greater than the mean estimated glomerular filtration rate (approximately 120 mL/min), suggesting partial active renal excretion. The total radioactivity recovered in the feces was 22% of the administered dose, representing the dose of saxagliptin excreted via bile and/or not absorbed from the gastrointestinal tract.

Saxagliptin is rapidly absorbed after oral administration on an empty stomach, with peak plasma concentrations (Cmax) of saxagliptin and its major metabolite reached within 2 and 4 hours, respectively. The Cmax and AUC values of saxagliptin and its major metabolite increase proportionally with increasing saxagliptin dose, and this dose-proportionality is still observed at doses up to 400 mg. In healthy subjects, after a single oral dose of 5 mg saxagliptin, the mean plasma AUC values of saxagliptin and its major metabolite were 78 nghr/mL and 214 nghr/mL, respectively, with corresponding plasma Cmax values of 24 ng/mL and 47 ng/mL. The intra-subject coefficients of variation for both saxagliptin Cmax and AUC were less than 12%.
Metabolism/Metabolites
The metabolism of saxagliptin is primarily mediated by cytochrome P450 3A4/5 (CYP3A4/5). 50% of the absorbed dose is metabolized by the liver. Saxagliptin's major metabolite, 5-hydroxysaxagliptin, is also a DPP4 inhibitor, with approximately half the potency of saxagliptin. The metabolism of saxagliptin is primarily mediated by CYP3A4/5. In vitro studies have shown that saxagliptin and its active metabolite do not inhibit CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, or 3A4, nor do they induce CYP1A2, 2B6, 2C9, or 3A4. Therefore, saxagliptin is not expected to alter the metabolic clearance of drugs co-metabolized with these enzymes. Saxagliptin is a substrate of P-glycoprotein (P-gp), but not a significant inhibitor or inducer of P-gp. The major metabolite of saxagliptin is also a DPP4 inhibitor, with approximately half the potency of saxagliptin.

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Biological Half-Life
Saxagliptin = 2.5 hours; 5-Hydroxysaxagliptin = 3.1 hours;
Following a single oral dose of 5 mg Onglyza in healthy subjects, the mean plasma terminal half-lives of saxagliptin and its active metabolite were 2.5 hours and 3.1 hours, respectively.

Effects During Pregnancy and Lactation
◉ Overview of use during lactation There is currently no information on the clinical use of saxagliptin during lactation. Saxagliptin has a shorter half-life than other dipeptidyl peptidase IV inhibitors, so it may be a better option among these drugs for lactating women. It is recommended to monitor the blood glucose levels of breastfed infants while the mother is taking saxagliptin. [1] However, other drugs may be preferred, especially when breastfeeding newborns or preterm infants. ◉ Effects on breastfed infants No relevant published information was found as of the revision date. ◉ Effects on lactation and breast milk No relevant published information was 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 form of saxagliptin, a highly bioavailable, potent, selective, and competitive cyanopyrrolidine dipeptidyl peptidase-4 (DPP-4) inhibitor with hypoglycemic activity. Saxagliptin is metabolized into a less active monohydroxy metabolite. See also: Metformin Hydrochloride; Saxagliptin Hydrochloride (ingredient); Dapagliflozin; Saxagliptin Hydrochloride (ingredient)... View more...

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Drug Indications
Combination therapy with saxagliptin is indicated for individuals aged 18 years and older. For adult patients with type 2 diabetes, metformin is used to improve glycemic control in the following ways: Monotherapy: For patients whose blood glucose is poorly controlled by diet and exercise alone, and who are unsuitable for metformin due to contraindications or intolerance; Dual oral therapy: When metformin alone, combined with diet and exercise, fails to adequately control blood glucose; When sulfonylureas alone, combined with diet and exercise, fail to adequately control blood glucose, and are unsuitable for metformin; When thiazolidinediones alone, combined with diet and exercise, fail to adequately control blood glucose, and are suitable for thiazolidinediones; Triple oral therapy: When metformin alone, combined with sulfonylureas, combined with diet and exercise, fails to adequately control blood glucose; Insulin combination therapy (with or without metformin): When insulin alone, combined with diet and exercise, fails to adequately control blood glucose.

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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)