DPP-4 (IC50 = 18 nM)

Sitagliptin phosphate potently inhibits DPP-4 in Caco-2 cell extracts with an IC50 of 19 nM [1]. Sitagliptin inhibits the migration of isolated splenic CD4 T cells in vitro via activating the cAMP/PKA/Rac1 pathway [2]. According to a recent study, sitagliptin stimulates intestinal L cells' production of GLP-1 by a unique direct effect that is dependent on MEK-ERK1/2 and protein kinase A but not DPP-4. As a result, it lessens autoimmunity's negative effects on graft survival [3].

The ED50 of sitagliptin phosphate for suppressing plasma DPP-4 activity was determined to be 2.3 mg/kg 7 hours post-dose and 30 mg/kg 24 hours post-dose in vivo in Han-Wistar rats fed at ad libitum [1]. Elevated levels of plasma DPP-4 are shown in a mouse model of type 1 diabetes caused by streptozotocin; mice given a diet high in sitagliptin phosphate had much lower levels of this protein. This is accomplished by improving the management of hyperglycemia, maybe by extending islet transplant survival [4]. Rats have a greater volume of distribution and plasma clearance of sitagliptin phosphate (40-48 mL/min/kg, 7-9 L/kg) than dogs (9 mL/min/kg, 3 L/kg); the half-life of the drug is lower in rats (2 hours) than in dogs (4 hours) [5].

Confluent Caco-2 cells are used to extract DPP-4. Following a 5-minute room temperature incubation period with lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 0.04 U/mL aprotinin, 0.5% Nonidet P40, pH 8.0), the cells are centrifuged at 35,000 g for 30 minutes at 4 °C, and the supernatant is kept at -80°C afterwards. Twenty microliters of suitable compound dilutions are combined with fifty microliters of H-Ala-Pro-7-amido-4-trifluoromethylcoumarin (final concentration in the assay: 100 microliters) as the substrate for the DPP-4 enzyme, and thirty microliters of the Caco-2 cell extract (diluted 1000 times with 100 mM Tris-HCl, 100 mM NaCl, pH 7.8). Fluorescence is measured using a SpectraMax GeminiXS at excitation/emission wavelengths of 405/535 nm after plates are incubated for one hour at room temperature. After exposing Caco-2 cell extracts to high inhibitor concentrations (30 nM for BI 1356 and 3 μM for vildagliptin) for one hour, the dissociation kinetics of the inhibitors from the DPP-4 enzyme are ascertained. Once the preincubation mixture has been diluted 3000-fold with assay buffer, the enzymatic reaction is initiated by adding the substrate, H-Ala-Pro-7-amido-4-trifluoromethylcoumarini. The amount of an inhibitor that is still bound to the DPP-4 enzyme is indicated by the difference in DPP-4 activity at a given time in the presence or absence of the inhibitor. Using the SoftMax software of the SpectraMax, maximum reaction rates (fluorescence units/seconds × 1000) are calculated at 10-minute intervals and corrected for the rate of an uninhibited reaction [(vcontrol-vinhibitor)/vcontrol].

Membrane inserts containing CD4T-cells are plated in serum-free RPMI 1640. Cell migration is measured using Corning Transwell chambers, either with or without DPP-4 inhibitor (100 μM) and purified porcine kidney DPP-4 (32.1 units/mg; final concentration of 100 mU/mL). Following an hour, cells that have moved into the lower compartment are counted and those on the upper surface are mechanically removed. The expression for the amount of migration is in relation to the control sample.

---

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted into the circulation by the intestinal L cell. The dipeptidylpeptidase-IV (DPP-IV) inhibitor, sitagliptin, prevents GLP-1 degradation and is used in the clinic to treat patients with type 2 diabetes mellitus, leading to improved glycated hemoglobin levels. When the effect of sitagliptin on GLP-1 levels was examined in neonatal streptozotocin rats, a model of type 2 diabetes mellitus, a 4.9 ± 0.9-fold increase in basal and 3.6 ± 0.4-fold increase in oral glucose-stimulated plasma levels of active GLP-1 was observed (P < 0.001), in association with a 1.5 ± 0.1-fold increase in the total number of intestinal L cells (P < 0.01). The direct effects of sitagliptin on GLP-1 secretion and L cell signaling were therefore examined in murine GLUTag (mGLUTag) and human hNCI-H716 intestinal L cells in vitro. Sitagliptin (0.1-2 μM) increased total GLP-1 secretion by mGLUTag and hNCI-H716 cells (P < 0.01-0.001). However, MK0626 (1-50 μM), a structurally unrelated inhibitor of DPP-IV, did not affect GLP-1 secretion in either model. Treatment of mGLUTag cells with the GLP-1 receptor agonist, exendin-4, did not modulate GLP-1 release, indicating the absence of feedback effects of GLP-1 on the L cell. Sitagliptin increased cAMP levels (P < 0.01) and ERK1/2 phosphorylation (P < 0.05) in both mGLUTag and hNCI-H716 cells but did not alter either intracellular calcium or phospho-Akt levels. Pretreatment of mGLUTag cells with protein kinase A (H89 and protein kinase inhibitor) or MAPK kinase-ERK1/2 (PD98059 and U0126) inhibitors prevented sitagliptin-induced GLP-1 secretion (P < 0.05-0.01). These studies demonstrate, for the first time, that sitagliptin exerts direct, DPP-IV-independent effects on intestinal L cells, activating cAMP and ERK1/2 signaling and stimulating total GLP-1 secretion[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.

---

\n\nEffects 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.\n
\nResults: 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.\n
\nConclusions: 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]

---

\n\n\nEffects 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.\n
\nResults: 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.\n
\nConclusions: Treatment with a DPP-IV inhibitor can prolong islet graft retention in an animal model of type 1 diabetes.[4]

---

\n \nThe 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
The oral bioavailability of sitagliptin is 87%, and its pharmacokinetics are not affected by administration on an empty stomach or with food. Sitagliptin reaches peak plasma concentration within 2 hours.
Elimination pathway
Approximately 79% of sitagliptin is excreted unchanged in the urine. 87% of the dose is excreted in the urine and 13% in the feces.
Volume of distribution
198 liters.
Clearance
350 mL/min.
Sitagliptin is secreted in the milk of lactating rats at a milk-to-plasma ratio of 4:1. It is unknown whether sitagliptin is secreted into human milk.
In pregnant rats, placental translocation is approximately 45% at 2 hours and approximately 80% at 24 hours after administration. In pregnant rabbits, placental translocation is approximately 66% at 2 hours and approximately 30% at 24 hours after administration of sitagliptin. Approximately 79% of sitagliptin is excreted unchanged in the urine, with a very small percentage metabolized. Sitagliptin is primarily excreted through the kidneys, involving active tubular secretion. Sitagliptin is a substrate of human organic anion transporter-3 (hOAT-3), which may be involved in its renal excretion. The clinical significance of hOAT-3 in sitagliptin transport remains unclear. Sitagliptin is also a substrate of P-glycoprotein, which may also be involved in its renal excretion. However, cyclosporine (a P-glycoprotein inhibitor) has not reduced the renal clearance of sitagliptin.

---

View More

Metabolism/Metabolites
Sitagliptin is not primarily metabolized; 79% of the dose is excreted unchanged in the urine. Secondary metabolic pathways are primarily mediated by cytochrome P450 (CYP)3A4, with less involvement of CYP2C8. After 18 hours, 81% of the dose remained unchanged; 2% was N-sulfated to the M1 metabolite; 6% was oxidized, desaturated, and cyclized to the M2 metabolite; <1% was glucuronized at an unknown site to the M3 metabolite; <1% was carbamylated and glucuronized to the M4 metabolite; 6% was oxidized, saturated, and cyclized to the M5 metabolite; and 2% was hydroxylated at an unknown site to the M6 metabolite. The M2 metabolite is the cis isomer of the M2 metabolite, while the M5 metabolite is the trans isomer.
Metabolism and excretion were investigated after a single oral administration of 83 mg/193 μCi sitagliptin in humans. Urine, fecal, and plasma samples were collected periodically over a period of up to 7 days. The primary route of radioactive excretion was the kidneys, with an average of 87% of the administered dose recovered in urine. The average amount excreted in feces was 13% of the administered dose. The parent drug is 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 is primarily excreted via the kidneys. The parent drug accounts for approximately 74% of the total radioactive AUC in plasma. Six trace metabolites were detected, each with a radioactivity level in plasma ranging from less than 1% to 7%. These metabolites include N-sulfate and N-carbamoyl glucuronide conjugates of the parent drug, a mixture of hydroxylated derivatives, an ether glucuronide of a hydroxylated metabolite, and two metabolites formed by the cyclization of piperazine after epoxidation and desaturation. These metabolites were also detected in urine, but at low levels. The metabolite profile in feces was similar to that in urine and plasma, except that glucuronide was not detected in feces. CYP3A4 is the major cytochrome P450 isoenzyme for the limited oxidative metabolism of sitagliptin, with CYP2C8 also contributing slightly. PMID:17220239
After oral administration of (14)C-labeled sitagliptin, approximately 16% of the radioactivity is excreted as sitagliptin metabolites. Six trace metabolites were detected, which are not expected to affect the plasma DPP-4 inhibitory activity of sitagliptin. In vitro studies have shown that the major enzyme in the limited metabolism of sitagliptin is CYP3A4, with CYP2C8 also contributing.

---

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 group studies evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of a single oral dose of sitagliptin (1.5–600 mg) in healthy male volunteers. Sitagliptin is well absorbed (approximately 80% is excreted unchanged in the urine), with an apparent terminal half-life of 8 to 14 hours. ...PMID: 16338283
The apparent terminal half-life after oral administration of 100 mg sitagliptin is approximately 12.4 hours.

Toxicity Overview
Identification and Use: Sitagliptin is a viscous liquid. It is a dipeptidyl peptidase-4 inhibitor used to improve glycemic control in patients with type 2 diabetes. Human Exposure and Toxicity: Sitagliptin improves glycemic control and is generally well tolerated in patients with type 2 diabetes. Use of sitagliptin is associated with an increased risk of heart failure-related hospitalization in patients with type 2 diabetes who have a history of heart failure. A recent study indicated that sitagliptin may be useful for treating certain neurodegenerative diseases of the peripheral nervous system. Sitagliptin does not appear to cause adverse reactions such as weight gain and hypoglycemia as some other treatments have. Animal Studies: In rodents, renal and hepatic toxicity was observed at systemic exposure to sitagliptin doses up to 58 times the human exposure level. In dogs, several treatment-related transient signs were observed at exposure doses approximately 23 times the clinical exposure dose, some of which suggested neurotoxicity, such as open-mouth breathing, salivation, white frothy vomiting, ataxia, tremor, decreased activity, and/or arched posture. Mouse carcinogenicity studies showed no increase in tumor incidence in any organ at doses up to 500 mg/kg; however, in rats, at 500 mg/kg, the incidence of mixed hepatic adenomas/carcinomas increased in both male and female rats, and the incidence of hepatocellular carcinoma also increased in female rats. Reproductive toxicity in rats and rabbits was only observed at doses above 250 mg/kg. Abnormalities in rat incisors were observed at exposure doses 67 times the clinical exposure dose. Sitagliptin did not exhibit mutagenicity or chromosomal breakage in the Ames bacterial mutagenicity test, Chinese hamster ovary (CHO) chromosome aberration test, CHO cell in vitro cytogenetics test, rat hepatocyte DNA in vitro alkaline elution test, and in vivo micronucleus test, regardless of metabolic activation.

---

Hepatotoxicity
Liver injury caused by sitagliptin is rare. In large clinical trials, the incidence of elevated serum enzymes in the sitagliptin treatment group (0.5%) was not significantly different from that in the placebo group (0.4%), and no clinically significant cases of liver injury were reported. Since its market launch, both the FDA and the sponsor have received reports of cases of elevated serum enzymes associated with sitagliptin. One case report of clinically significant liver injury has been published, but the patient also had hepatitis C. The serum enzyme elevation pattern was hepatocellular, with a peak serum bilirubin level of 9.4 mg/dL, which rapidly returned to normal upon discontinuation of sitagliptin. No signs of immune hypersensitivity or autoantibodies were observed. Probability score: D (likely a rare cause of clinically significant liver injury).

---

View More

Use during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the clinical use of sitagliptin during lactation. Sitagliptin has a shorter half-life than most other dipeptidyl peptidase IV inhibitors, therefore it may be a better option among such drugs for breastfeeding women. It is recommended to monitor the blood glucose levels of breastfed infants while the mother is taking sitagliptin. However, especially when breastfeeding newborns or premature infants, other medications may be preferred.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

---

Non-human toxicity values
Oral LD50 in mice: 4000 mg/kg
Oral LD50 in rats: >3000 mg/kg

---

Protein binding: 38%.

[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 form of sitagliptin, an orally potent competitive β-amino acid derivative that inhibits dipeptidyl peptidase-4 (DDP-4) and has hypoglycemic activity. Sitagliptin may increase the risk of pancreatitis. It is a pyrazine derivative dipeptidyl peptidase IV inhibitor and hypoglycemic agent that increases the levels of the incretin hormone glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). It is used to treat type 2 diabetes. See also: sitagliptin (containing the active ingredient); etagliflozin; sitagliptin phosphate (ingredient); metformin hydrochloride; sitagliptin phosphate (ingredient)... See more...

C₁₆H₁₈F₆N₅O₅P

505.31

505.094

C, 38.03; H, 3.59; F, 22.56; N, 13.86; O, 15.83; P, 6.13

654671-78-0

Sitagliptin;486460-32-6;Sitagliptin-d4 phosphate;1432063-88-1;Sitagliptin phosphate monohydrate;654671-77-9;(S)-Sitagliptin phosphate;823817-58-9;(Rac)-Sitagliptin;823817-56-7

6451150

White to off-white solid powder

529.9ºC at 760 mmHg

202-204ºC

274.3ºC

1.726

4

14

4

33

616

1

C1CN2C(=NN=C2C(F)(F)F)CN1C(=O)C[C@@H](CC3=CC(=C(C=C3F)F)F)N.OP(=O)(O)O

IQFYVLUXQXSJJN-SBSPUUFOSA-N

InChI=1S/C16H15F6N5O.H3O4P/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)/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

Januvia; MK0431 phosphate; Sitagliptin phosphate; 654671-78-0; Sitagliptin (phosphate); MK-0431; sitagliptin phosphate anhydrous; UNII-494P4635I6; Sitagliptin monophosphate; MK 431; MK 0431 phosphate; MK-0431 phosphate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~197.90 mM)
H2O : ~50 mg/mL (~98.95 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (4.95 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 50 mg/mL (98.95 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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