Comprehensive Insight into Chemical Stability of Important Antidiabetic Drug Vildagliptin Using Chromatography (LC-UV and UHPLC-DAD-MS) and Spectroscopy (Mid-IR and NIR with PCA).

During forced degradation, the intrinsic stability of active pharmaceutical ingredients (APIs) could be determined and possible impurities that would occur during the shelf life of the drug substance or the drug product could be estimated. Vildagliptin belongs to relatively new oral antidiabetic drugs named gliptins, inhibiting dipeptidyl peptidase 4 (DPP-4) and prolonging the activities of the endogenous incretin hormones. At the same time, some gliptins were shown as prone to degradation under specific pH and temperature conditions, as well as in the presence of some reactive excipients. Thus, forced degradation of vildagliptin was performed at high temperature in extreme pH and oxidative conditions. Then, selective LC-UV was used for quantitative determination of non-degraded vildagliptin in the presence of its degradation products and for degradation kinetics. Finally, identification of degradation products of vildagliptin was performed using an UHPLC-DAD-MS with positive ESI. Stability of vildagliptin was also examined in the presence of pharmaceutical excipients, using mid-IR and NIR with principal component analysis (PCA). At 70 °C almost complete disintegration of vildagliptin occurred in acidic, basic, and oxidative media. What is more, high degradation of vildagliptin following the pseudo first-order kinetics was observed at room temperature with calculated k values 4.76 × 10-4 s-1, 3.11 × 10-4 s-1, and 1.73 × 10-4 s-1 for oxidative, basic and acidic conditions, respectively. Next, new degradation products of vildagliptin were detected using UHPLC-DAD-MS and their molecular structures were proposed. Three degradants were formed under basic and acidic conditions, and were identified as [(3-hydroxytricyclo- [3.3.1.13,7]decan-1-yl)amino]acetic acid, 1-{[(3-hydroxytricyclo[3.3.1.13,7]decan-1-yl)amino]acetyl}-pyrrolidine-2-carboxylic acid and its O-methyl ester. The fourth degradant was formed in basic, acidic, and oxidative conditions, and was identified as 1-{[(3-hydroxytricyclo[3.3.1.13,7]-decan-1-yl)amino]acetyl}pyrrolidine-2-carboxamide. When stability of vildagliptin was examined in the presence of four excipients under high temperature and humidity, a visible impact of lactose, mannitol, magnesium stearate, and polyvinylpirrolidone was observed, affecting-NH- and CO groups of the drug. The obtained results (kinetic parameters, interactions with excipients) may serve pharmaceutical industry to prevent chemical changes in final pharmaceutical products containing vildagliptin. Other results (e.g., identification of new degradation products) may serve as a starting point for qualifying new degradants of vildagliptin as it is related to substances in pharmacopoeias.

Gas-Phase Rearrangement of the O-Glucuronide of Vildagliptin Forms Product-Ion Fragments Suggesting Wrongly an N-Glucuronide.

The O-glucuronide of vildagliptin, a dipeptidyl peptidase 4 inhibitor, is a major metabolite in monkeys and a minor metabolite in humans, rats, and dogs. Its product ion spectrum shows fragments that can be explained only by an N-glucuronide. Biotransformation using rat liver yielded milligram amounts of the O-glucuronide, and its structure was assigned unambiguously by nuclear magnetic resonance. The tandem mass spectra (MS/MS) of this compound was investigated in detail using MSn and accurate mass spectrometers and was identical to the animal metabolite. Thus, the MS/MS fragments suggesting an N-glucuronide had to be formed by gas-phase rearrangement. This gas-phase rearrangement can be observed on quadrupole time-of-flight and ion-trap mass instruments. The literature on gas-phase rearrangements is reviewed.

Repurposing of antidiabetics as Serratia marcescens virulence inhibitors.

BACKGROUND: Serratia marcescens becomes an apparent nosocomial pathogen and causes a variety of infections. S. marcescens possess various virulence factors that are regulated by intercellular communication system quorum sensing (QS). Targeting bacterial virulence is a proposed strategy to overcome bacterial resistance. Sitagliptin anti-QS activity has been demonstrated previously and we aimed in this study to investigate the effects of antidiabetic drugs vildagliptin and metformin compared to sitagliptin on S. marcescens pathogenesis. METHODS: We assessed the effects of tested drugs in subinhibitory concentrations phenotypically on the virulence factors and genotypically on the virulence encoding genes' expressions. The protection of tested drugs on S. marcescens pathogenesis was performed in vivo. Molecular docking study has been conducted to evaluate the interference capabilities of tested drugs to the SmaR QS receptor. RESULTS: Vildagliptin reduced the expression of virulence encoding genes but did not show in vitro or in vivo anti-virulence activities. Metformin reduced the expression of virulence encoding genes and inhibited bacterial virulence in vitro but did not show in vivo protection. Sitagliptin significantly inhibited virulence factors in vitro, reduced the expression of virulence factors and protected mice from S. marcescens. Docking study revealed that sitagliptin is more active than metformin and fully binds to SmaR receptor, whereas vildagliptin had single interaction to SmaR. CONCLUSION: The downregulation of virulence genes was not enough to show anti-virulence activities. Hindering of QS receptors may play a crucial role in diminishing bacterial virulence.

Analytical Method Development and Validation of UV-visible Spectrophotometric Method for the Estimation of Vildagliptin in Gastric Medium.

BACKGROUND: Vildagliptin is an antidiabetic agent, belongs to the dipeptidyl peptidase IV (DPP-4) inhibitors. OBJECTIVE: The aim of investigation was to develop a simple UV-visible Spectrophotometric method for the determination of vildagliptin in its pure form and pharmaceutical formulations, further to validate the developed method. MATERIAL AND METHODS: Vildagliptin was estimated using UV-Visible double beam spectrophotometer at the wavelength of maximum absorption (210 nm) in acidic medium containing 0.1N HCl. The drug was characterized by melting point, Differential Scanning Calorimetry (DSC), and Fourier Transform Infra-Red (FTIR) techniques. The analysis of the drug was carried out by novel UV-Visible method which was validated analytical parameters like linearity, precision, and accuracy as per guidelines laid down by International Conference on Harmonization (ICH). RESULT: Melting point of drug was found 154°C which is corresponds to its actual melting range. Similarly by the interpretation of spectra the drug was confirmed. The linear response for concentration range of 5-60 µg/ml of vildagliptin was recorded with regression coefficient 0.999. The accuracy was found between 98-101%. Precision for intraday and interday was found to be 1.263 and 1.162 respectively, which are within the limits. To establish the sensitivity of the method, limit of detection (LOD) and limit of quantification (LOQ) were determined which were found to be 0.951 µg/ml and 2.513 µg/ml respectively. CONCLUSION: The UV method developed and validated for vildagliptin drug was found to be linear, accurate, precise and economical which can be used for the testing of its pharmaceutical formulations.

UPLC-ESI/Q-TOF MS/MS Method for Determination of Vildagliptin and its Organic Impurities.

Vildagliptin (VLG) corresponds to a drug used for the treatment of diabetes mellitus. This disease requires continuous treatment, and so the control of impurities present in it is important to assure the quality of this drug. Thus, it is necessary to use sensitive and selective detection techniques and the ultra-performance liquid chromatography is a better option compared with high-performance liquid chromatography because it enhances the separation efficiency with a shorter analysis time and an increased resolution. This research analysis was accomplished by using liquid chromatography/tandem mass spectrometry, and the quantification was performed by using an extracted ion from the VLG drug and its main organic impurities of synthesis. During the validation process, following international standards, the method proved to be linear for the tree substances (R2 = 0.997-0.998) and the analysis of variance showed a non-significant linearity deviation (P > 0.05). Three critical factors were selected to evaluate method robustness with a full factorial experimental design, and the changes in the parameters were found to be not significant for the quantification of VLG and its impurities. The ultra-performance liquid chromatography-tandem mass spectrometry for the determination of impurities in VLG was precise, accurate and robust proving to be effective for analysis in the pharmaceutical industry and to improve the quality, safety and effectiveness of the new drug developed.

In vitro toxic evaluation of two gliptins and their main impurities of synthesis.

BACKGROUND: The presence of impurities in some drugs may compromise the safety and efficacy of the patient's treatment. Therefore, establishing of the biological safety of the impurities is essential. Diabetic patients are predisposed to tissue damage due to an increased oxidative stress process; and drug impurities may contribute to these toxic effects. In this context, the aim of this work was to study the toxicity, in 3 T3 cells, of the antidiabetic agents sitagliptin, vildagliptin, and their two main impurities of synthesis (S1 and S2; V1 and V2, respectively). METHODS: MTT reduction and neutral red uptake assays were performed in cytotoxicity tests. In addition, DNA damage (measured by comet assay), intracellular free radicals (by DCF), NO production, and mitochondrial membrane potential (ΔψM) were evaluated. RESULTS: Cytotoxicity was observed for impurity V2. Free radicals generation was found at 1000 µM of sitagliptin and 10 µM of both vildagliptin impurities (V1 and V2). A decrease in NO production was observed for all vildagliptin concentrations. No alterations were observed in ΔψM or DNA damage at the tested concentrations. CONCLUSIONS: This study demonstrated that the presence of impurities might increase the cytotoxicity and oxidative stress of the pharmaceutical formulations at the concentrations studied.

Design, synthesis and evaluation of DNA nano-cubes as a core material protected by the alginate coating for oral administration of anti-diabetic drug.

Poor control towards glycemic levels among diabetic patients may lead to severe micro/macro-vascular and neuropathic complexities. Proper functioning of alpha-beta cells of pancreases is required to attain long term glycemic control among type 2 diabetics. The recent developments to manage diabetes are focused on controlling the insulin-glucagon secretions from the pancreases. DPP-4 inhibitors class of drugs after elevating GLP-1/GIP (incretins) levels in the blood, not only raise the insulin levels but also suppress the glucagon level. Vildagliptin (VI) is a potent DPP-4 inhibitor with least adverse events compared to other DPP-4 inhibitors. We encapsulated VI into 3D nanocube that gets bind to the DNA due to secondary amine in its chemical structure. DNA-nanocube being negatively charged was incubated with the PLL to attain positive surface. Ultimately VI loaded nanocubes were coated with the negatively charged Na-alginate via electrostatic attraction method to get stable spherical nanospheres for oral delivery of VI. Nanospheres were evaluated physically through native PAGE analysis, DSC, TGA, dissolution testing, XRD and FTIR. We attained uniformed and spherical nanospheres with stable topology, nanoscale size precision (40-150 nm in diameter), Entrapment efficiency (up to 90%), prolonged drug release (13 ± 4 h) at basic pH, and superior oral antidiabetic effects with improved GLP1 and glycemic levels. The formulated nanospheres attained size uniformity and better therapeutic outcomes in terms of reduced adverse events and better control of glycemic levels than previously reported methods with decreased dosage frequency tested in Db/Db mice.

DNA scaffold nanoparticles coated with HPMC/EC for oral delivery.

The control of the glycemic level among diabetes/T2 patients is very important for their long term survival and avoiding further complexities including micro/macrovascular diseases as well as diabetic neuropathy. Vildagliptin (VD) is a drug that has addressed these issues successfully with the desired safety portfolio. We used DNA-nanocubes for initial nano-encapsulation of VD followed by HPMC/EC coating. The results revealed the stable, smooth, spherical and nano-sized nanoparticles with improved size uniformity (from 100 to 400 nm in diameter) and encapsulation-efficiency (E.E.%) than previously reported (500-2000 nm) with the chemical compatibility evident in ATR/FTIR and DSC results. Animal experiments results revealed the improvement of incretin level in the serums due to potent DPP-4 inhibition compared to the free-VD/solution with better maintenance of glycemic levels after feeding. The safety of these HPMC/EC-DNA-VD nanoparticles was assessed through the histological-examination after completion of the treatment turn. The solvent evaporation technique provided the better coating of HPMC around DNA-core with gastro-resistant and effervescent property due to presence of NaHCO3 (0.01%) in the formulations that caused delayed delivery of VD as well as nanoparticles to the intestine, increasing the availability time of the drug and nanospheres at the target sites (intestine and blood) where DPP-4 enzyme is most abundant (to degrade the GLP-1 and GIP causing loss of control of the postprandial glycemic levels. So the availability of sustained release nanospheres near the target sites and prolonged DPP-4 inhibition improved the outcomes of the therapy.

Antidiabetic Drugs and Their Nanoconjugates Repurposed as Novel Antimicrobial Agents against Acanthamoeba castellanii.

Acanthamoeba castellanii belonging to the T4 genotype may cause a fatal brain infection known as granulomatous amoebic encephalitis, and the vision-threatening eye infection Acanthamoeba keratitis. The aim of this study was to evaluate the antiamoebic effects of three clinically available antidiabetic drugs, Glimepiride, Vildagliptin and Repaglinide, against A. castellanii belonging to the T4 genotype. Furthermore, we attempted to conjugate these drugs with silver nanoparticles (AgNPs) to enhance their antiamoebic effects. Amoebicidal, encystation, excystation, and host cell cytotoxicity assays were performed to unravel any antiacanthamoebic effects. Vildagliptin conjugated silver nanoparticles (Vgt-AgNPs) characterized by spectroscopic techniques and atomic force microscopy were synthesized. All three drugs showed antiamoebic effects against A. castellanii and significantly blocked the encystation. These drugs also showed significant cysticidal effects and reduced host cell cytotoxicity caused by A. castellanii. Moreover, Vildagliptin-coated silver nanoparticles were successfully synthesized and are shown to enhance its antiacanthamoebic potency at significantly reduced concentration. The repurposed application of the tested antidiabetic drugs and their nanoparticles against free-living amoeba such as Acanthamoeba castellanii described here is a novel outcome that holds tremendous potential for future applications against devastating infection.

Dipeptidyl peptidase IV Inhibitory activity of Terminalia arjuna attributes to its cardioprotective effects in experimental diabetes: In silico, in vitro and in vivo analyses.

BACKGROUND: The marketed synthetic (Dipeptidyl peptidase-IV) DPP-IV Inhibitors are expensive antidiabetic drugs and have been reported to cause unacceptable adverse effects such as pancreatitis, angioedema, thyroid and pancreatic cancers. In this scenario research to develop novel DPP-IV Inhibitors from alternative sources is the need of the hour. HYPOTHESIS/PURPOSE: Terminalia arjuna, a medicinal herb with antidiabetic and cardioprotective activities may represent a natural DPP-IV Inhibitor, the DPP-IV Inhibitory activity of which may translate into demonstrable therapeutic benefits in setting of diabetes with cardiovascular co-morbidities. STUDY DESIGN: The study type used for the present study was an experimental (In vitro, In vivo and In silico) design. METHOD: The DPP-IV Inhibitory, antidiabetic and cardioprotective effects of Terminalia arjuna was evaluated in the experimental model of myocardial infarction co-existing with diabetes. To determine the active principle of Terminalia arjuna responsible for DPP-IV Inhibitory activity, the crystal structure of DPP-IV was considered as receptor which was docked against Arjunetin, Arjungenin, Arjunic acid, Arjunone, Ellagic acid, Gallic acid, Sitagliptin and Vildagliptin. The binding sites as well as affinity of various active ingredients of Terminalia arjuna for DPP- IV enzyme was elucidated using in silico studies and compared to Vildagliptin. RESULTS: Terminalia arjuna demonstrated significant DPP-IV Inhibitory, antidiabetic (significant reduction in HbA1C) and cardioprotective effects (restoration of myocardial CPK-MB) in the experimental model of myocardial infarction co-existing with diabetes. The cardioprotective efficacy correlated to its DPP-IV Inhibitory activity. The active ingredients of Terminalia arjuna (Arjunetin, Arjungenin, Arjunic Acid Arjunone, Ellagic acid and Gallic acid) demonstrated significant inhibition of DPP-IV enzyme. Arjunic acid and Arjunone prefers the active site pocket of DPP-IV enzyme. Compounds like Arjunetin and Vildagliptin prefers to bind near the interface region of the DPP-IV as their biological active forms are homodimer. Sitagliptin binds near the α/ß hydrolase domain. CONCLUSION: The DPP-IV Inhibitory activity of Terminalia arjuna was found to be comparable to Vildagliptin. The DPP-IV Inhibitory activity translated into significant cardioprotective effects in the setting of diabetes. The active ingredient of Terminalia arjuna; Arjunetin, Arjungenin, Ellagic acid and Arjunic acid showed superior DPP-IV Inhibitory activity as compared to synthetic DPP-IV inhibitors (Sitagliptin and Vildagliptin) based on results of docking studies.

Vildagliptin protects endothelial cells against high glucose-induced damage.

Hyperglycemia or high blood sugar is one of the major pathological characteristics of diabetes. The endothelium is the inner layer of the vascular wall and is directly exposed to various stimuli in blood vessels. As a characteristic of diabetes, chronic high glucose is known to be harmful to endothelial cells. In this study, we found that vildagliptin, an available type 2 diabetes agent, protects primary human aortic endothelial cells (HAECs) against damage induced by high glucose. Our data indicate that vildagliptin improves the decrease in endothelial viability and reduces the release of lactate dehydrogenase (LDH) induced by high glucose. Vildagliptin potently suppresses high glucose-induced generation of reactive oxygen species (ROS) and production of vascular inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), intercellular cell adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein 1 (MCP-1). Moreover, vildagliptin suppresses adhesion of monocytes to endothelial cells and induction of toll-like receptor 4 (TLR-4) in HAECs caused by high glucose. Mechanistically, we found that the cellular protective effects mediated by vildagliptin involve suppression of nuclear factor-kappa B (NF-κB) nuclear signals. Collectively, our data indicate that vildagliptin possesses a protective function in vascular cells.