DPP-4 (IC50 = 26 nM)

Saxagliptin has a DPP4 inhibition constant Ki of 1.3 nM, making it ten times more potent than sitagliptin and vildagliptin, two other DPP4 inhibitors, with Ki values of 13 and 18 nM, respectively. Furthermore, saxagliptin exhibits 400- and 75-fold higher specificity for DPP4 than it does for DPP8 or DPP9. Saxagliptin's active metablite has twice the potency of the parent drug. When compared to various other proteases, saxagliptin and its metabolite are both highly selective (>4000-fold) for preventing DPP4 (selectivity of sitagliptin and vildagliptin for DPP4 is >2600 and <250-fold, respectively, compared with DPP8 and DPP9).[2] Saxagliptin is linked to better β-cell function, suppression of glucagon secretion, and decreased degradation of the incretin hormone glucagon-like peptide-1, which enhances its actions.[3]

Maximum Saxagliptin responses in glucose excursion in Zuckerfa/fa rats are correlated with plasma DPP4 inhibition of about 60% compared to control; at higher percent inhibition, no further antihyperglycemic effects are observed. In the dosage range of 0.13-1.3 mg/kg, saxagliptin significantly increases glucose clearance in ob/ob mice compared to controls in a dose-dependent manner. When saxagliptin is taken in a dose-dependent manner, it significantly raises plasma insulin at 15 minutes after the glucose tolerance test (GTT) and simultaneously improves the glucose clearance curves 60 minutes later.[4]

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.

Stable cell lines were generated by transfecting the expression vector into Chinese hamster ovary (CHO-DG44) cells using electroporation. The CHO-DG44 cell line was grown in PFCHO media supplemented with HT (glycine, hypoxanthine, and thymidine), glutamine, and Recombulin. Then 1 × 107 cells/mL were collected, transfected with 60 μg of DNA using electroporation at 300V, and then transferred to a T75 flask. On the third day following transfection, the HT supplement was removed and selection was initiated with methotrexate (MTX, 10 nM). After a further 10 days, the cells were plated into individual wells of 96-well plates. Every 10 days the concentration of MTX was increased 2−3-fold, up to a maximum of 400 nM. Final stable cell line selection was based on yield and activity of the expressed protein. Protein was further purified using conventional anion exchange, gel filtration (S-200) and high-resolution MonoQ columns. The final protein yielded a single band on SDS−PAGE gels. Amino acid sequence analysis indicated two populations of DPP-IV in the sample. One portion of the protein had 27 amino acids truncated from the N-terminus, while the other was lacking the N-terminal 37 amino acids, suggesting that during isolation the entire transmembrane domain (including the His tag) is removed by proteases present in the CHO cells. Total protein concentration was measured using the Bradford dye method, and the amount of the active DPP-IV was determined by titrating the enzyme with our previously reported inhibitor (compound 29 in ref 18). No biphasic behavior was observed during inhibition or catalysis, suggesting that both protein populations are functionally identical[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, Distribution and Excretion
Following a single oral dose of 5 mg saxagliptin in 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. No accumulation of saxagliptin occurred after repeated dosing. Following a once-daily 5 mg dose, the median time to peak concentration (Tmax) for saxagliptin was 2 hours, and for its active metabolite, it was 4 hours. Bioavailability (2.5–50 mg dose) = 67%. Saxagliptin is primarily excreted via the kidneys and liver. Following a single 50 mg dose of 14C-saxagliptin, 24%, 36%, and 75% of the dose were excreted in the urine as saxagliptin, its active metabolite, and total radioactive material, respectively. 22% of the administered radioactive material was recovered in feces, representing the dose of saxagliptin excreted via bile and/or the drug not absorbed from the gastrointestinal tract.
151 L
Single 50 mg dose renal clearance = 14 L/h
A single-dose, open-label study aimed to evaluate the pharmacokinetics of 10 mg saxagliptin 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 metabolite. 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 metabolites are 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 metabolites to patients with normal renal function, the recommended dose for patients with moderate to severe renal impairment and those with end-stage renal disease requiring hemodialysis is 2.5 mg once daily. Saxagliptin is removed via hemodialysis. Saxagliptin is primarily excreted via the kidneys and liver. Following a single 50 mg (14)-C-saxagliptin dose, 24%, 36%, and 75% of the dose are excreted in the urine as saxagliptin, its active metabolites, and total radioactivity, respectively. The mean renal clearance of saxagliptin (approximately 230 mL/min) is higher than the mean estimated glomerular filtration rate (approximately 120 mL/min), suggesting some active renal excretion. A total of 22% of the administered radioactive material was recovered in feces, representing the portion of the saxagliptin dose excreted via bile and/or not absorbed from the gastrointestinal tract. Following oral administration of saxagliptin on an empty stomach, it is rapidly absorbed, with peak plasma concentrations (Cmax) and peak concentrations of its major metabolite (Tmax) reached within 2 and 4 hours, respectively. The Cmax and AUC values of saxagliptin and its major metabolite increase proportionally with increasing saxagliptin dose, a dose-proportional relationship observed 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. The corresponding plasma Cmax values were 24 ng/mL and 47 ng/mL, respectively. The intra-subject coefficients of variation for both 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 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 metabolites 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 is 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.

Toxicity Summary
Identification and Use: Saxagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor used to treat type 2 diabetes. It has been shown in various clinical settings as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes. Human Exposure and Toxicity: Saxagliptin treatment significantly improves glycated hemoglobin (A1C) levels compared to placebo. Cases of overdose have been reported, but most were accidental. Most adults and children/adolescents taking gliptins can be safely managed at home without hospitalization upon assessment at a healthcare facility. Cases of adults intentionally self-harming from taking gliptins have been treated at healthcare facilities, but even at doses up to 18 times the adult treatment dose, hospitalization or serious complications are rare. In healthy subjects, daily administration of up to 400 mg of saxagliptin for 2 weeks, equivalent to 80 times the maximum recommended human dose (MRHD), did not result in any dose-related adverse clinical events or clinically significant effects on corrected QT interval (QTc) or heart rate. Animal studies: Saxagliptin caused adverse skin reactions on the extremities of cynomolgus monkeys (scabs and/or ulcers on the tail, toes, scrotum, and/or nose). At doses up to 20 times the MRHD, skin damage was reversible, but at higher doses, irreversible necrosis occurred in some cases. In developmental studies, higher doses of saxagliptin that caused maternal toxicity also increased fetal uptake (approximately 2069 times and 6138 times the MRHD). Additional effects on estrous cycles, fertility, ovulation, and implantation were observed at doses approximately 6138 times the MRHD. In in vitro Ames bacterial assays, in vitro primary human lymphocyte cytogenetics assays, in vivo rat oral micronucleus assays, in vivo rat oral DNA repair studies, and rat peripheral blood lymphocyte oral in vivo/in vitro cytogenetics studies, saxagliptin, regardless of metabolic activation, did not exhibit mutagenicity or chromosome breakage. The active metabolite did not show mutagenicity in the in vitro Ames bacterial assay.

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Hepatotoxicity
In large clinical trials, the rate of serum enzyme elevation in the saxagliptin treatment group was similar to that of other drugs (
Probability score: E (unproven but suspected rare cause of clinically significant liver injury)).

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Pregnancy and Lactation Effects
◉ Overview of Use During Lactation
Currently, there is no information on the clinical use of saxagliptin during lactation. Saxagliptin has a shorter half-life than other dipeptidyl peptidase IV inhibitors, therefore it may be a better option among drugs in this class for breastfeeding women. Monitoring of blood glucose levels in breastfed infants is recommended during maternal treatment with saxagliptin. [1] However, especially in breastfed newborns or preterm infants, alternative medications may be preferred.
◉ Effects of breastfeeding on infants
No published information found as of the revision date.
◉ Effects on breastfeeding and breast milk
No published information found as of the revision date.

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Protein binding
The in vitro protein binding of saxagliptin and its active metabolites in human serum is negligible (<10%).

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Interactions
Concomitant administration of a single dose of saxagliptin (10 mg) and glibenclamide (5 mg) increased the peak plasma concentrations of glibenclamide and saxagliptin by 16% and 8%, respectively; the AUC of glibenclamide increased by 6%, and the AUC of saxagliptin decreased by 2%. The manufacturer states that no dose adjustment is required when saxagliptin and glibenclamide are administered concurrently due to changes in systemic exposure. However, in treated patients, when saxagliptin is used in combination with sulfonylureas, the dose of the sulfonylureas may need to be reduced to decrease the risk of hypoglycemia. A single dose of saxagliptin (100 mg) and metformin hydrochloride (1 g) decreased peak plasma concentration of saxagliptin by 21% and AUC by 2%; metformin increased AUC and peak plasma concentration by 20% and 9%, respectively. Concomitant administration of saxagliptin (5 mg once daily for 21 days) and an estrogen-progestin combined oral contraceptive (ethinylestradiol 35 mcg and norgestrel 0.25 mg fixed combination, once daily for 21 days) did not significantly alter the steady-state pharmacokinetics of ethinylestradiol or the main active progestin component. Concomitant administration of a single dose of saxagliptin (10 mg) and a single dose of famotidine (40 mg) increased peak plasma concentration of saxagliptin by 14% and AUC by 3%. For more complete (8 items) data on drug interactions of saxagliptin, please visit the HSDB record page.

[1]. Vasc Health Risk Manag. 2008;4(4):753-68.

[2]. Adv Ther. 2009 May;26(5):488-99.

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

[4]. Adv Ther. 2009 Mar;26(3):249-62.

Therapeutic Uses
Onglyza is indicated for use in a variety of clinical situations as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes. /US Product Label Includes/ Onglyza should not be used to treat type 1 diabetes or diabetic ketoacidosis, as it is ineffective in these conditions.

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Drug Warnings /Black Box Warning/ Warning: Lactic acidosis. Lactic acidosis is a rare but serious complication that can be caused by metformin accumulation. Risk is increased in conditions such as sepsis, dehydration, excessive alcohol consumption, liver impairment, kidney impairment, and acute congestive heart failure. The onset of lactic acidosis is often insidious, with only nonspecific symptoms such as malaise, myalgia, dyspnea, increased drowsiness, and nonspecific abdominal discomfort. Laboratory abnormalities include low pH, increased anion gap, and elevated blood lactate levels. If acidosis is suspected, Kombiglyze XR should be discontinued immediately and the patient taken to a hospital. /Saxagliptin and Metformin Hydrochloride Combination/
The FDA is evaluating new, unpublished findings from a group of academic researchers that suggest an increased risk of pancreatitis and a precancerous cellular lesion called pancreatic duct metaplasia in patients with type 2 diabetes treated with a class of drugs called incretin analogs. These findings are based on examination of pancreatic tissue samples from a small number of post-mortem patients whose cause of death is unknown. The FDA has requested that the researchers provide methods for collecting and studying these samples, as well as tissue samples, so that the FDA can further investigate potential pancreatic toxicities associated with incretin analogs. Incretin analogs include exenatide (Byetta, Baidu Ruian), liraglutide (Vituzar), sitagliptin (Jenova, Genomex, Genomex Extended-Release, Uvitin), saxagliptin (Amrita, Combigliza Extended-Release), alogliptin (Nessina, Kazaro, Oseni), and linagliptin (Trajeta, Gentaduto). These drugs work by mimicking the body's naturally produced incretin hormones, stimulating the release of insulin after meals. They are used in conjunction with diet and exercise to lower blood sugar in adults with type 2 diabetes. The FDA has not yet reached any new conclusions regarding the safety risks of incretin analogues. This preliminary notification is intended only to inform the public and healthcare professionals that the FDA plans to obtain and evaluate this new information. …The FDA will release its final conclusions and recommendations after completing its review or obtaining more information. The “Warnings and Precautions” section of the drug label and patient guide for incretin analogues contains warnings about the risk of acute pancreatitis. The FDA has not previously issued any announcements regarding the risk that incretin analogues may cause precancerous lesions of the pancreas. The FDA has not concluded that these drugs may cause or promote the development of pancreatic cancer. Currently, patients should continue to take the medication as prescribed until they consult a healthcare professional; healthcare professionals should also continue to follow the prescribing recommendations on the drug label. …
Post-marketing surveillance data shows that patients receiving saxagliptin have reported experiencing acute pancreatitis. The U.S. Food and Drug Administration (FDA) is evaluating unpublished study results suggesting a potentially increased risk of pancreatitis and precancerous cellularity (pancreatic metaplasia) in patients with type 2 diabetes treated with incretin analogues (exenatide, liraglutide, sitagliptin, saxagliptin, alogliptin, or linagliptin). These findings are based on examination of small samples of pancreatic tissue from patients who died of unknown causes while receiving incretin analogue treatment. The FDA has not yet drawn any new conclusions regarding the safety risks of incretin analogues. The FDA will notify healthcare professionals of its conclusions and recommendations upon completion of its review or when more information becomes available. The FDA states that clinicians should continue to follow the recommendations in the prescribing information for incretin analogues. The manufacturer states that patients receiving treatment with saxagliptin should be monitored for symptoms of pancreatitis. If pancreatitis is suspected, saxagliptin should be discontinued immediately and appropriate treatment should be initiated. Saxagliptin has not been studied in patients with a history of pancreatitis, therefore it is unclear whether treatment with saxagliptin in such patients increases the risk of pancreatitis. Post-marketing reports indicate that saxagliptin can cause serious anaphylactic and hypersensitivity reactions (e.g., anaphylactic shock, angioedema, exfoliative dermatitis). These reactions typically occur within the first 3 months of treatment; some reactions occur immediately after the first dose. For more complete data on drug warnings for saxagliptin (19 in total), please visit the HSDB records page. Pharmacodynamics: GLP-1 and GIP levels can increase 2 to 3 times after administration of saxagliptin. Systemic side effects are less common due to its high selectivity for DPP-4 inhibitors. Saxagliptin inhibits DPP-4 enzyme activity for up to 24 hours. It also reduces glucagon concentrations and increases glucose-dependent insulin secretion from pancreatic β-cells. The half-maximal inhibitory concentration (IC50) is 0.5 nmol/L. Saxagliptin does not cause clinically significant QTc interval prolongation.

C18H30CLN3O4

387.901504039764

387.192

1073057-20-1

945667-22-1 (hydrate); 709031-78-7 (HCl); 1073057-20-1 (HCl hydrate); 1073057-33-6 (benzoate hydrate)

11243969

Typically exists as solid at room temperature

2.469

2

4

2

23

609

4

Cl.OC12CC3CC(C1)CC([C@@H](C(N1[C@H](C#N)C[C@@H]4C[C@H]14)=O)N)(C3)C2.O.O

QGJUIPDUBHWZPV-SGTAVMJGSA-N

InChI=1S/C18H25N3O2/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/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

Saxagliptin hydrochloride dihydrate; UNII-4N19ON48ZN; 4N19ON48ZN; 1073057-20-1; (1S,3S,5S)-2-((2S)-2-Amino-2-(3-hydroxyadamantan-1-yl)acetyl)-2-azabicyclo[3.1.0]hexane-3-carbonitrile hydrochloride dihydrate; DTXSID30861540; Q27260182

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.)