DPP-IV (IC50 = 3.5 nM)

Vildagliptin inhibits cell apoptosis, which increases beta cell survival. Additionally, vildagliptin stimulates cell division[2].

Vildagliptin (35 mg/kg; once daily by oral gavage) raises the levels of plasma active GLP-1 in the islets of db/db mice[2].
Vildagliptin Vildagliptin (10 µmol/kg; oral) in obese male Zucker rats significantly reduces glucose excursions and increases insulin secretion[1]. in obese male Zucker rats significantly reduces glucose excursions and increases insulin secretion[1].

DPP-IV Inhibition Measurement ex Vivo.Rat, Human, Monkey Plasma Assays.[1]
Human, rat, or monkey plasma was used as the source of DPP-IV in the assay. The standard assay was modified from a previously published method. Five μL of plasma was added to 96-well flat-bottom microtiter plates, followed by the addition of 5 μL of 80 mM MgC12 in assay buffer (25 mM HEPES, 140 mM NaC1, 1% RIA-grade BSA, pH 7.8). After a 5-min preincubation at room temperature, the reaction was initiated by the addition of 10 μL of assay buffer containing 0.1 mM substrate (H-Gly-Pro-AMC; AMC is 7-amino-4-methylcoumarin). The plates were covered with aluminum foil (or kept in the dark) and incubated at room temperature for 20 min. After incubation, fluorescence was measured using a CytoFluor II fluorometer (excitation 380 nm/ emission 460 nm). Test compounds and solvent controls were added as 2 μL additions, and the assay buffer volume was reduced to 13 μL. A standard curve of free AMC was generated using 0−50 μM solutions of AMC. The curve generated, which was linear, was used for interpolation of substrate consumption (catalytic activity in nmoles substrate cleaved /min).

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DPP-II Inhibition Measurement in Vitro. [1]
An extract of bovine kidney homogenate, partially purified by ion-exchange and adenosine deaminase chromatography, was used as the source of DPP-II in the assay. The standard assay was modified from a previously published method. 47 Twenty micrograms of DPP-II-containing fraction diluted to a final volume of 60 μL in assay buffer (0.2 M Borate, 0.05 M Citrate, pH 5.3) was added to 96-well flat-bottom microtiter plates, followed by the addition of 10 μL of 10 mM o-phenanthroline (to inhibit aminopeptidase activity) and 20 μL of 5 mM substrate (H-Lys-Ala-AMC; AMC is 7-amino-4-methylcoumarin). The plates were incubated at 37 °C for 30 min. After incubation, fluorescence was measured using a CytoFluor II fluorometer (excitation 380 nm/ emission 460 nm). Test compounds and solvent controls were added as 20 μL additions, and assay buffer volume is reduced to 50 μL. A standard curve of AMC was generated using 0 to 100 μM of AMC. The curve generated, which was linear, was used for interpolation of catalytic activity (in nmoles substrate cleaved/min).

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Vildagliptin (LAF-237; NVP-LAF 237) has an IC50 of 2.3 nM, which inhibits DPP-4. Figure 2 represents vildagliptin, an N-substituted glycyl-2-cyanopyrrolidine. With an inhibitory concentration (IC50) of approximately 2–3 nmol/L, it is a strong, reversible, and competitive inhibitor of DPP-4 in both humans and rodents in vitro. Crucially, vildagliptin exhibits high specificity inhibition of DPP-4 in comparison to other analogous peptidases, wherein its IC50 surpasses 200 mol/L.

In Vitro Studies.DPP-IV Inhibition Measurement in Vitro:  Caco-2 Assay. [1]
An extract from human colonic carcinoma cells (Caco-2; American Type Culture Collection; ATCC HTB 37) was used as the source of DPP-IV in the assay. The cells were differentiated to induce DPP-IV expression as described by previously. Cell extract was prepared from cells solubilized in lysis buffer (10 mM Tris-HC1, 0.15 M NaC1, 0.04 T.I.U. (trypsin inhibitor unit) aprotinin, 0.5% nonidet-P40, pH 8.0) then centrifuged at 35 000g for 30 min at 4 °C to remove cell debris. The assay was conducted by adding 20 μg of solubilized Caco-2 protein, diluted to a final volume of 125 μL in assay buffer (25 mM Tris-HC1 pH 7.4, 140 mM NaC1, 10 mM KC1, 1% bovine serum albumin) to 96-well flat-bottom microtiter plates. The reaction was initiated by adding 25 μL of 1 mM substrate (H-Ala-Pro-pNA; pNA is p-nitroaniline). The reaction was run at room temperature for 10 min, and then 19 μL of 25% glacial acetic acid was added to stop the reaction. Fluorescence was measured using a CytoFluor II fluorometer (excitation 380 nm/ emission 460 nm). Test compounds and solvent controls were added as 30 μL additions, and the assay buffer volume was reduced to 95 μL. A standard curve of free p-nitroaniline was generated using 0−100 μM pNA in assay buffer. The curve generated, which was linear, was used for interpolation of substrate consumption (catalytic activity in nmoles substrate cleaved /min).

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Post-Proline Cleaving Enzyme (PPCE) Inhibition Measurement in Vitro. [1]
A cytosolic extract of human erythrocytes, partially purified by ion-exchange chromatography, was used as the source of PPCE in the assay. The standard assay is modified from a previously published method. PPCE-containing fraction (350 ng protein) diluted to a final volume of 90 μL in assay buffer (20 mM NaPO4, 0.5 mM EDTA, 0.5 mM DTT, 1% BSA, pH 7.4) was added to 96-well flat-bottom microtiter plates, followed by the addition of 10 μL of 0.5 mM substrate (Z-Gly-Pro-AMC; AMC is 7-amino-4-methylcoumarin). The plates were incubated at room temperature for 30 min. After incubation, fluorescence was measured using a CytoFluor II fluorometer (excitation 380 nm/ emission 460 nm). Test compounds and solvent controls were added as 20 μL additions, and the assay buffer volume was reduced to 70 μL. A standard curve of free AMC was generated using 0 to 5 μM solutions of AMC. The curve generated, which was linear, was used for interpolation of catalytic activity (in nmoles substrate cleaved/min).

Male db/db mice (BKS) and wildtype mice[2]
35 mg/kg
Oral gavage; once daily; for 6 weeks

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In Vivo Obese Male (fa/fa) Zucker Rat Studies.[1]
Effect of Vildagliptin (NVP LAF 237; DSP7238; LAF237)  (Vildagliptin (NVP LAF 237; DSP7238; LAF237) ) on DPP-IV Activity, Active GLP-1 Levels, and Glucose and Insulin Excursions. Studies were performed on obese male Zucker (fa/fa) rats (Charles River Labs, Cambridge, MA); controls (n = 9) and Vildagliptin (NVP LAF 237; DSP7238; LAF237) -treated (n = 9). These rats were purchased at 7 weeks of age, cannulated at 7.5 weeks, and studied beginning at around 11 weeks of age. In the morning of the oral glucose tolerance test (OGTT), the rats were “fasted” by removing food before the lights were turned on, after which they were transferred to the experiment room at 8:00 a.m.. Vildagliptin (NVP LAF 237; DSP7238; LAF237)  was dissolved in vehicle solution (0.5% carboxymethylcellulose (CMC) and 0.2% Tween 80). The cannulas were connected to sampling tubing (PE-100, 0.034 in. i.d. × 0.06 in. o.d.), which were filled with saline. After 30−40 min cage acclimation, a 0.5 mL baseline blood sample was taken at t = −15 min, and the rats were then orally dosed with CMC or Vildagliptin (NVP LAF 237; DSP7238; LAF237)  (10 μmol/kg), after which additional baseline blood samples were taken at t = −5, −2.5, and 0 min. The animals were then administered an oral glucose solution (10% glucose, 1 g/kg) immediately after t = 0‘. The rest of the samples were taken at 1, 3, 5, 10, 15, 20, 30, 45, 60, 75, and 90 min. Throughout the OGTT, an equal volume of donor blood was used to replace the blood withdrawn during sampling. Donor blood was obtained from donor rats through cardiac puncture. The collected blood samples (0.5 mL) were immediately transferred into chilled Eppendorf tubes containing 50 μL of EDTA:  trasylol (25 mg/mL of 10 000 trasylol) and used for the measurement of glucose and insulin levels and DPP-IV activity. Larger blood samples (0.75 mL) were collected at t = −15, 0, 5, 10, 15, and 30 min for GLP-1 (7−36 amide) measurements. To these tubes, the DPP-IV inhibitor valine pyrrolidide was added to yield a final concentration in the blood of 1 μM. Technical difficulties with obtaining blood samples after minute 20 for one rat in both the CMC and Vildagliptin (NVP LAF 237; DSP7238; LAF237)  groups resulted in the inability to calculate glucose and insulin AUC data for those rats, leading to AUC data with an n = 8/group. Measurement of plasma glucose was made using a modification of a Sigma Diagnostics glucose oxidase kit. DPP-IV activity was measured in plasma samples obtained at −5, 0, 20, 45, and 90 min DPP-IV activity as previously described in the above ex vivo rat plasma experimental. Plasma levels of GLP-1 (7−36 amide) were measured using the GLP-1 (active) Elisa Kit.

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In Vivo Cynomolgus Monkey PK/PD Studies Using 8c and Vildagliptin (NVP LAF 237; DSP7238; LAF237) . [1]
Ketamine-anesthetized male healthy cynomolgus monkeys received either 8c (n = 2) or Vildagliptin (NVP LAF 237; DSP7238; LAF237)  (n = 3) (dissolved in CMC/Tween-80) by oral gavage (1.007 μmol/kg), and by intravenous administration (0.399 μmol/kg) (dissolved in saline). For iv study, compound was administered (0.4 mL/kg over 1 min) in 0.9% saline as vehicle. Different monkeys were used for each dosage regimen. Basal blood samples were collected at −10 min and immediately prior to administration of compound. Blood samples were collected at 0.03, 0.08, 0.17, 0.25, 0.33, 0.42, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 7, 12, and 25 h postdose for both routes of administration. Blood was obtained into heparin-coated syringes, transferred to microcentrifuge tubes, and centrifuged to separate the plasma. The plasma was stored at −80 °C in fresh microcentrifuge tubes until assay. DPP-IV activity was measured in a similar manner was as previously described in the above ex vivo rat and human plasma experimentals. Plasma DPP-IV activities were calculated and expressed as ‘percent of baseline' to reduce variability due to individual differences in plasma enzyme activity. Area-under-curve (AUC) values for DPP-IV activity were calculated from time (hours after dose) vs effect (percent inhibition) curves from individual animals using the trapezoidal method. The ratio of dose-normalized effect AUC for oral/intravenous administration routes was taken as an estimate of effect bioavailability. Parent drug concentrations were determined using an HPLC/MS/MS method with a limit of quantification of 1 ng/mL. Pharmacokinetic parameters were calculated using noncompartment modeling, and the AUC was calculated using the linear trapezoidal method. Absolute oral bioavailability was calculated by (AUC0-∞po × 399)/(AUC0-∞iv × 1007).

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Vildagliptin was orally administered to db/db mice for 6 weeks, followed by evaluation of beta cell apoptosis by caspase3 activity and TUNEL staining method. Endoplasmic reticulum stress markers were determined with quantitative RT-PCR, immunohistochemistry and immunoblot analysis.
Results: After 6 weeks of treatment, vildagliptin treatment increased plasma active GLP-1 levels (22.63±1.19 vs. 11.69±0.44, P<0.001), inhibited beta cell apoptosis as demonstrated by lower amounts of TUNEL staining nuclei (0.37±0.03 vs. 0.55±0.03, P<0.01) as well as decreased caspase3 activity (1.48±0.11 vs. 2.67±0.13, P<0.01) in islets of diabetic mice compared with untreated diabetic group. Further, vildagliptin treatment down-regulated several genes related to endoplasmic reticulum stress including TRIB3 (tribbles homolog 3) (15.9±0.4 vs. 33.3±1.7, ×10⁻³, P<0.001), ATF-4(activating transcription factor 4) (0.83±0.06 vs. 1.42±0.02, P<0.001) and CHOP(C/EBP homologous protein) (0.07±0.01 vs. 0.16±0.01, P<0.001).
Conclusions: Vildagliptin promoted beta cell survival in db/db mice in association with down-regulating markers of endoplasmic reticulum stress including TRIB3, ATF-4 as well as CHOP.[2]

Absorption, Distribution and Excretion
In a fasting state, vildagliptin is rapidly absorbed following oral administration. Peak plasma concentrations are observed at 1.7 hours following administration. Plasma concentrations of vildagliptin increase in an approximately dose-proportional manner. Food delays Tmax to 2.5 hours and decreases Cmax by 19%, but has no effects on the overall exposure to the drug (AUC). Absolute bioavailability of vildagliptin is 85%.
Vildagliptin is eliminated via metabolism. Following oral administration, approximately 85% of the radiolabelled vildagliptin dose was excreted in urine and about 15% of the dose was recovered in feces. Of the recovered dose in urine, about 23% accounted for the unchanged parent compound.
The mean volume of distribution of vildagliptin at steady-state after intravenous administration is 71 L, suggesting extravascular distribution.
After intravenous administration to healthy subjects, the total plasma and renal clearance of vildagliptin were 41 and 13 L/h, respectively.

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Metabolism / Metabolites
About 69% of orally administered vildagpliptin is eliminated via metabolism not mediated by cytochrome P450 enzymes. Based on the findings of a rat study, DPP-4 contributes partially to the hydrolysis of vildagliptin. Vildagliptin is metabolized to pharmacologically inactive cyano (57%) and amide (4%) hydrolysis products in the kidney. LAY 151 (M20.7) is a major inactive metabolite and a carboxylic acid that is formed via hydrolysis of the cyano moiety: it accounts for 57% of the dose. Other circulating metabolites reported are an N-glucuronide (M20.2), an N-amide hydrolysis product (M15.3), two oxidation products, M21.6 and M20.9.

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Biological Half-Life
The mean elimination half-life following intravenous administration is approximately two hours. The elimination half-life after oral administration is approximately three hours.

Protein Binding
The plasma protein binding of vildagliptin is 9.3%. Vildagliptin distributes equally between plasma and red blood cells.

[1].1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine: a potent, selective, and orally bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J Med Chem. 2003 Jun 19;46(13):2774-89.

[2]. Dipeptidyl peptidase-4 inhibitor, vildagliptin, inhibits pancreatic beta cell apoptosis in associationwith its effects suppressing endoplasmic reticulum stress in db/db mice. Metabolism. 2015 Feb;64(2):226-35.

Pharmacodynamics
Vildagliptin works to improve glycemic control in type II diabetes mellitus by enhancing the glucose sensitivity of beta-cells (β-cells) in pancreatic islets and promoting glucose-dependent insulin secretion. Increased GLP-1 levels leads to enhanced sensitivity of alpha cells to glucose, promoting glucagon secretion. Vildagliptin causes an increase in the insulin to glucagon ratio by increasing incretin hormone levels: this results in a decrease in fasting and postprandial hepatic glucose production. Vildagliptin does not affect gastric emptying. It also has no effects on insulin secretion or blood glucose levels in individuals with normal glycemic control. In clinical trials, treatment with vildagliptin 50-100 mg daily in patients with type 2 diabetes significantly improved markers of beta-cells, proinsulin to insulin ratio, and measures of beta-cell responsiveness from the frequently-sampled meal tolerance test. Vildagliptin has improves glycated hemoglobin (HbA1c) and fasting plasma glucose (FPG) levels.

C17H25N3O2

303.4

303.195

C, 67.30; H, 8.31; N, 13.85; O, 10.55

274901-16-5

(2R)-Vildagliptin;1036959-27-9;Vildagliptin-d3;1217546-82-1;Vildagliptin-13C5,15N;1044741-01-6;Vildagliptin dihydrate;2133364-01-7;Vildagliptin-d7;1133208-42-0

6918537

White to off-white solid powder

1.27 g/cm3

531.3ºC at 760 mmHg

153-155?C

275.1ºC

1.503

2

4

3

22

523

1

O([H])C12C([H])([H])C3([H])C([H])([H])C([H])(C1([H])[H])C([H])([H])C(C3([H])[H])(C2([H])[H])N([H])C([H])([H])C(N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C#N)=O

SYOKIDBDQMKNDQ-XWTIBIIYSA-N

InChI=1S/C17H25N3O2/c18-9-14-2-1-3-20(14)15(21)10-19-16-5-12-4-13(6-16)8-17(22,7-12)11-16/h12-14,19,22H,1-8,10-11H2/t12?,13?,14-,16?,17?/m0/s1

(2S)-1-[2-[(3-hydroxy-1-adamantyl)amino]acetyl]pyrrolidine-2-carbonitrile

Vildagliptin; DSP 7238; DSP7238; NVP-LAF 237; NVP LAF 237; DSP-7238; LAF237; LAF-237; Galvus; 274901-16-5; Xiliarx; Jalra; NVP-LAF237; Equa; LAF 237; NVP LAF-237; trade name: Zomelis

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)

Solubility in Formulation 1: 100 mg/mL (329.60 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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Solubility in Formulation 2: Saline: 30 mg/mL

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