Addressing stability issues of vildagliptin: Method optimization and validation for accurate analysis in human plasma.

This research paper introduces novel strategies to address the stability issues arising with vildagliptin, marking the first attempt to tackle this challenge comprehensively. The study incorporates malic acid into the human plasma, a crucial step in stabilizing vildagliptin and preventing its degradation. Additionally, optimization of the elution process on a C18 Asentis Express column, fine-tuned with a combination of acetonitrile and ammonium trifluoroacetate 5mM, ensures optimal chromatographic conditions. For detection and quantification, electrospray ionization (ESI) is employed, monitoring multiple reactions for vildagliptin (304.2 → 154.2) and vildagliptin D7 (311.1 → 161.2). Meticulous validation of the method demonstrates high accuracy (97.30%-104.15%) and precision [(0.32%-3.09% coefficient of variance (CV)] for vildagliptin calibration curve standards (CC STD), establishing its sensitivity and reliability in measuring vildagliptin levels. This refined methodology offers numerous advantages, including the elimination of stability concerns, reduced human plasma sample volume (100 µL), exceptional reproducibility, shortened run time (~2.2 min), and a wide concentration range (1.00 to 851.81 ng/mL). These attributes make it exceptionally well-suited for diverse research applications, spanning from extensive sampling in therapeutic drug monitoring units to bioequivalence and bioavailability studies, as well as pharmacokinetic investigations of vildagliptin.

Validation of a method for the determination of Aderamastat (FP-025) in K2EDTA human plasma by LC-MS/MS.

Aderamastat (FP-025) is a small molecule, selective matrix metalloproteinase (MMP)-12 inhibitor, under development for respiratory conditions which may include chronic inflammatory airway diseases and pulmonary fibrosis. To support evaluation of the pharmacokinetic parameters of Aderamastat in humans, we developed and validated a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical method for the quantification of Aderamastat in human plasma. This assay was validated in compliance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations (GLP) and European Medicines Agency (EMA) guidelines. K2EDTA human plasma samples were spiked with internal standard, processed by liquid-liquid extraction, and analyzed using reversed-phase HPLC with Turbo Ion Spray® MS/MS detection. Separation was done using a chromatographic gradient on 5 µm C6-Phenyl 110 Å, 50*2 mm analytical column at a temperature of 35 °C. The LC-MS/MS bioanalytical method, developed by QPS Taiwan to determine the concentration of Aderamastat in K2EDTA human plasma, was successfully validated with respect to linearity, sensitivity, accuracy, precision, dilution, selectivity, hemolyzed plasma, lipemic plasma, batch size, recovery, matrix effect, and carry-over. These data indicate that the method for determination of Aderamastat concentrations in human K2EDTA plasma can be used in pharmacokinetics studies and subsequent clinical trials with Aderamastat. Authors declare that, this novel data is not published and not under consideration for publication by another journal than this journal. All data will be made available on request.

Pharmacokinetic/Pharmacodynamic modelling of Saxagliptin and its active metabolite, 5-hydroxy Saxagliptin in rats with Type 2 Diabetes Mellitus.

BACKGROUND AND PURPOSES: It is unclear whether the parent Saxagliptin (SAX) in vivo is the same as that in vitro, which is twice that of 5-hydroxy Saxagliptin (5-OH SAX). This study is to construct a Pharmacokinetic-Pharmacodynamic (PK-PD) link model to evaluate the genuine relationship between the concentration of parent SAX in vivo and the effect. METHODS: First, we established a reliable Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS/MS) method and DPP-4 inhibition ratio determination method. Then, the T2DM rats were randomly divided into four groups, intravenous injection of 5-OH SAX (0.5 mg/kg) and saline group, intragastric administration of SAX (10 mg/kg) and Sodium carboxymethyl cellulose (CMC-Na) group. Plasma samples were collected at different time points for subsequent testing. Finally, we used the measured concentrations and inhibition ratios to construct a PK-PD link model for 5-OH SAX and parent SAX. RESULTS: A two-compartment with additive model showed the pharmacokinetic process of SAX and 5-OH SAX, the concentration-effect relationship was represented by a sigmoidal Emax model and sigmoidal Emax with E0 model for SAX and 5-OH SAX, respectively. Fitting parameters showed SAX was rapidly absorbed after administration (Tmax=0.11 h, t1/2, ka=0.07 h), widely distributed in the body (V ≈ 20 L/kg), plasma exposure reached 3282.06 ng*h/mL, and the elimination half-life was 6.13 h. The maximum plasma dipeptidyl peptidase IV (DPP-4) inhibition ratio of parent SAX was 71.47%. According to the final fitting parameter EC50, EC50, 5-OH SAX=0.46EC50, SAX(parent), it was believed that the inhibitory effect of 5-OH SAX was about half of the parent SAX, which is consistent with the literature. CONCLUSIONS: The PK-PD link model of the parent SAX established in this study can predict its pharmacokinetic process in T2DM rats and the strength of the inhibitory effect of DPP-4 based on non-clinical data.

Metabolite Profiling and Characterization of LW6, a Novel HIF-1α Inhibitor, as an Antitumor Drug Candidate in Mice.

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.

Pharmacokinetic Characterization of LW6, a Novel Hypoxia-Inducible Factor-1α (HIF-1α) Inhibitor in Mice.

LW6, an (aryloxyacetylamino)benzoic acid derivative, was recently identified to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α), which is an attractive target for cancer therapeutics. Although LW6 is known to act by inhibiting the accumulation of HIF-1α, pharmacokinetics needs to be evaluated to assess its potential as an anti-tumor agent. Here, we investigated the plasma pharmacokinetics and metabolism of LW6 in mice. LW6 exhibited a small volume of distribution (0.5 ± 0.1 L/kg), and a short terminal half-life (0.6 ± 0.1 h). Following intravenous or oral administration, LW6 was rapidly converted to its active metabolite, (4-adamantan-1-yl-phenoxy)acetic acid (APA). Although LW6 was rapidly absorbed, its oral bioavailability, estimated using AUClast values, was low (1.7 ± 1.8%). It was slowly degraded in mouse liver microsomes (t1/2 > 1 h) and serum (t1/2 > 6 h). About 54% or 44.8% of LW6 was available systemically as APA in the mouse after a single intravenous or oral administration, respectively. Thus, our results indicated the need to simultaneously consider the active metabolite as well as the parent compound for successful evaluation during lead optimization.

Bioequivalence and Food Effect of Dapagliflozin/Saxagliptin/Metformin Extended-release Fixed-combination Drug Products Compared With Coadministration of the Individual Components in Healthy Subjects.

PURPOSE: Fixed-combination drug products (FCDPs) for patients with type 2 diabetes mellitus (T2DM) may show efficacy comparable to their individual components (ICs) while improving adherence to treatment. This study evaluated the bioequivalence and safety of 2 dapagliflozin/saxagliptin/metformin extended-release (XR) FCDPs relative to their ICs: saxagliptin and dapagliflozin/metformin XR. METHODS: This randomized, open-label, single-dose, single-center crossover study was conducted in 84 healthy subjects aged 18-55 years. The primary objective was to evaluate the fed-state bioequivalence of a dapagliflozin 5-mg/saxagliptin 2.5-mg/metformin 1000-mg XR FCDP and a dapagliflozin 10-mg/saxagliptin 5-mg/metformin 1000-mg XR FCDP relative to the ICs. Secondary objectives included the evaluation of the effect of food on the pharmacokinetic (PK) parameters of saxagliptin, dapagliflozin, and metformin in both FCDPs and characterization of the PK parameters of the active metabolite of saxagliptin, 5-hydroxy saxagliptin, in healthy subjects. PK parameters (AUC0-∞, AUC0-t, and Cmax) were used to assess the bioequivalence of the 2 FCDPs with their ICs. The Cmax and AUC0-t of the study drugs were compared between female and male subjects to assess sex differences in exposure. Safety and tolerability of both FCDPs and ICs were also assessed with adverse events, vital signs (systolic and diastolic blood pressures and pulse rate), 12-lead ECG, physical examinations, and laboratory assessments. FINDINGS: Both dapagliflozin/saxagliptin/metformin XR FCDPs were bioequivalent to their ICs. For the dapagliflozin 5-mg/saxagliptin 2.5-mg/metformin 1000-mg XR FCDP, the 90% CI for the geometric mean ratio of dapagliflozin Cmax was slightly above the 80%-125% bioequivalence limit, which is unlikely to be clinically relevant. Food delayed the absorption of the study drugs in both FCDPs, which is unlikely to have a clinically relevant impact on efficacy. In both cohorts, exposure was higher in female subjects compared with male subjects, potentially due to the lower body weight of the female subjects. The safety profile and tolerability of the FCDPs were similar to those of their ICs, and no deaths or serious adverse events were reported. IMPLICATIONS: These data support the use of the dapagliflozin/saxagliptin/metformin XR FCDP in patients with T2DM. ClinicalTrials.gov identifier: NCT03169959.

Pharmacokinetic Interaction Study Between Saxagliptin and Omeprazole, Famotidine, or Magnesium and Aluminum Hydroxides Plus Simethicone in Healthy Subjects: An Open-Label Randomized Crossover Study.

Saxagliptin is an orally administered, highly potent, and selective dipeptidyl peptidase-4 inhibitor for the management of type 2 diabetes mellitus. This study was conducted to determine the effect of magnesium and aluminum hydroxides plus simethicone, famotidine, and omeprazole on the pharmacokinetics of saxagliptin and its active metabolite, 5-hydroxy saxagliptin. This was an open-label, randomized, 5-treatment, 5-period, 3-way crossover study in 15 healthy subjects. Mean Cmax of saxagliptin was 26% lower, but AUC was almost unchanged when saxagliptin was coadministered with Maalox Max. Mean Cmax was 14% higher, but AUC was almost unchanged when saxagliptin was coadministered with famotidine. Changes in pharmacokinetics of 5-hydroxy saxagliptin generally paralleled the changes in saxagliptin. These pharmacokinetic changes were unlikely to be clinically meaningful. Coadministration of omeprazole did not affect saxagliptin Cmax or AUC. Saxagliptin in combination with these medicines resulted in no unexpected safety or tolerability findings in these healthy subjects. No dose adjustment of saxagliptin or separation in the time of saxagliptin dosing is necessary with medicines that raise gastric pH when coadministered with saxagliptin.

Quantitative determination of metformin, saxagliptin and 5-hydroxy saxagliptin simultaneously by hydrophilic interaction liquid chromatography - electrospray ionization mass spectrometry and its application to a bioequivalence study with a single-pill combination in human.

A simple, sensitive and specific hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometric (HILIC-MS) method was developed and validated to determine the plasma concentrations of metformin, saxagliptin and 5-hydroxy saxagliptin simultaneously in clinical studies. Plasma samples were first acidified and then protein precipitated with acetonitrile. Chromatographic separation was achieved on a HILIC Chrom Matrix HP amide column (5 µm, 3.0 × 100 mm I.D.). The mobile phase consisted of acetonitrile and 5 mM ammonium formate buffer containing 0.1% formic acid. Multiple reaction monitoring transitions were performed on triple quadrupole mass spectrometric detection in positive-ion mode with an electrospray ionization source. The calibration curves showed good linearity (r ≥ 0.999) over the established concentration range of 1.0-1000 ng/mL for metformin and 0.1-100 ng/mL for saxagliptin and its active metabolite 5-hydroxy saxagliptin. The extraction recovery for all of the analytes was >92% and the matrix effect ranged from 91.0 to 110.0%. After validation, the method was successfully applied to a bioequivalence study with a single-pill combination (SPC) consisting of 5 mg saxagliptin and 500 mg metformin in 10 healthy Chinese subjects.

Analytical confirmation of synthetic cannabinoids in a cohort of 179 presentations with acute recreational drug toxicity to an Emergency Department in London, UK in the first half of 2015.

CONTEXT: Synthetic cannabinoid receptor agonists are the largest group of new psychoactive substances reported in the last decade; in this study we investigated how commonly these drugs are found in patients presenting to the Emergency Department with acute recreational drug toxicity. METHODS: We conducted an observational cohort study enrolling consecutive adult patients presenting to an Emergency Department (ED) in London (UK) January-July 2015 (6 months) with acute recreational drug toxicity. Residual serum obtained from a serum sample taken as part of routine clinical care was analyzed using high-resolution accurate mass-spectrometry with liquid-chromatography (HRAM-LCMSMS). Minimum clinical data were obtained from ED medical records. RESULTS: 18 (10%) of the 179 patient samples were positive for synthetic cannabinoid receptor agonists. The most common was 5F AKB-48 (13 samples, concentration 50-7600 pg/ml), followed by 5F PB-22 (7, 30-400 pg/mL), MDMB-CHMICA (7, 80-8000 pg/mL), AB-CHMINACA (3, 50-1800 pg/mL), Cumyl 5F-PINACA (1, 800 pg/mL) and BB-22 (1, 60 pg/mL). Only 9/18 (50%) in whom synthetic cannabinoid receptor agonists were detected self-reported synthetic cannabinoid receptor agonist use. The most common clinical features were seizures and agitation, both recorded in four (22%) individuals. Fourteen patients (78%) were discharged from the ED, one of the four admitted to hospital was admitted to critical care. CONCLUSIONS: Synthetic cannabinoid receptor agonists were found in 10% of this cohort with acute recreational drug toxicity but self-reported in only half of these. This suggests that presentations to the ED with acute synthetic cannabinoid receptor agonist toxicity may be more common than reported.

Structure-Activity Relationship of the Antimalarial Ozonide Artefenomel (OZ439).

Building on insights gained from the discovery of the antimalarial ozonide arterolane (OZ277), we now describe the structure-activity relationship (SAR) of the antimalarial ozonide artefenomel (OZ439). Primary and secondary amino ozonides had higher metabolic stabilities than tertiary amino ozonides, consistent with their higher pKa and lower log D7.4 values. For primary amino ozonides, addition of polar functional groups decreased in vivo antimalarial efficacy. For secondary amino ozonides, additional functional groups had variable effects on metabolic stability and efficacy, but the most effective members of this series also had the highest log D7.4 values. For tertiary amino ozonides, addition of polar functional groups with H-bond donors increased metabolic stability but decreased in vivo antimalarial efficacy. Primary and tertiary amino ozonides with cycloalkyl and heterocycle substructures were superior to their acyclic counterparts. The high curative efficacy of these ozonides was most often associated with high and prolonged plasma exposure, but exposure on its own did not explain the presence or absence of either curative efficacy or in vivo toxicity.

Hepatic Dipeptidyl Peptidase-4 Controls Pharmacokinetics of Vildagliptin In Vivo.

The main route of elimination of vildagliptin, which is an inhibitor of dipeptidyl peptidase-4 (DPP-4), in humans is cyano group hydrolysis to produce a carboxylic acid metabolite M20.7. Our in vitro study previously demonstrated that DPP-4 itself greatly contributed to the hydrolysis of vildagliptin in mouse, rat, and human livers. To investigate whether hepatic DPP-4 contributes to the hydrolysis of vildagliptin in vivo, in the present study, we conducted in vivo pharmacokinetics studies of vildagliptin in mice coadministered with vildagliptin and sitagliptin, which is another DPP-4 inhibitor, and also in streptozotocin (STZ)-induced diabetic mice. The area under the plasma concentration-time curve (AUC) value of M20.7 in mice coadministered with vildagliptin and sitagliptin was significantly lower than that in mice administered vildagliptin alone (P < 0.01). Although plasma DPP-4 expression level was increased 1.9-fold, hepatic DPP-4 activity was decreased in STZ-induced diabetic mice. The AUC values of M20.7 in STZ-induced diabetic mice were lower than those in control mice (P < 0.01). Additionally, the AUC values of M20.7 significantly positively correlated with hepatic DPP-4 activities in the individual mice (Rs = 0.943, P < 0.05). These findings indicated that DPP-4 greatly contributed to the hydrolysis of vildagliptin in vivo and that not plasma, but hepatic DPP-4 controlled pharmacokinetics of vildagliptin. Furthermore, enzyme assays of 23 individual human liver samples showed that there was a 3.6-fold interindividual variability in vildagliptin-hydrolyzing activities. Predetermination of the interindividual variability of hepatic vildagliptin-hydrolyzing activity might be useful for the prediction of blood vildagliptin concentrations in vivo.

LC-MS/MS analysis of metformin, saxagliptin and 5-hydroxy saxagliptin in human plasma and its pharmacokinetic study with a fixed-dose formulation in healthy Indian subjects.

A specific and rapid liquid chromatography-tandem mass spectrometry method is proposed for the simultaneous determination of metformin (MET), saxagliptin (SAXA) and its active metabolite, 5-hydroxy saxagliptin (5-OH SAXA) in human plasma. Sample preparation was accomplished from 50 µL plasma sample by solid-phase extraction using sodium dodecyl sulfate as an ion-pair reagent. Reversed-phase chromatographic resolution of analytes was possible within 3.5 min on ACE 5CN (150 × 4.6 mm, 5 µm) column using acetonitrile and10.0 mm ammonium formate buffer, pH 5.0 (80:20, v/v) as the mobile phase. Triple quadrupole mass spectrometric detection was performed using electrospray ionization in the positive ionization mode. The calibration curves showed good linearity (r2 ≥ 0.9992) over the established concentration range with limit of quantification of 1.50, 0.10 and 0.20 ng/mL for MET, SAXA and 5-OH SAXA respectively. The extraction recoveries obtained from spiked plasma samples were highly consistent for MET (75.12-77.84%), SAXA (85.90-87.84%) and 5-OH SAXA (80.32-82.69%) across quality controls. The validated method was successfully applied to a bioequivalence study with a fixed-dose formulation consisting of 5 mg SAXA and 500 mg MET in 18 healthy subjects. The reproducibility of the assay was demonstrated by reanalysis of 87 incurred samples.

Acute Toxicity Associated with Use of 5F-Derivations of Synthetic Cannabinoid Receptor Agonists with Analytical Confirmation.

INTRODUCTION: Synthetic Cannabinoid Receptor Agonists (SCRAs) are the largest group of new psychoactive substances reported to the European Warning System and the United Nations Office on Drugs and Crime to date. The heterogeneous nature and speed of diversification of these compounds make it challenging to accurately characterise and predict harms of these compounds in pre-clinical studies, ahead of their appearance. CASE REPORT: We report the case of a 19-year-old female who purchased three products from a headshop: two new psychoactive substances (sachets of "cannabis tea" and "mushroom tea") as well as two LSD blotters. After the "cannabis tea" was smoked and the two LSD blotters and "mushroom tea" were ingested, the patient became tachycardic (HR 128), developed seizures, agitation, visual hallucinations as well as suspected serotonergic toxicity (sustained ankle clonus 20-30 beats) 1-2 hours after use. She was treated with 1 mg of intravenous midazolam. Symptoms/signs resolved within 13 hours. No further supportive care was required. Plasma, blood, and urine samples confirmed the presence of two SCRAs: 5FAKB-48 and 5F-PB-22. The patient also reported therapeutic use of both fluoxetine and citalopram for depression. DISCUSSION: To the best of our knowledge, this is the first case report of non-fatal intoxication with 5F-AKB-48 with analytical confirmation and exposure times. It also highlights the difficulties in understanding the pattern of toxicity of certain SCRAs in the context of psychotropic medications/co-morbid mental illness.

Behavioural and pharmacological characterization of a novel cannabinomimetic adamantane-derived indole, APICA, and considerations on the possible misuse as a psychotropic spice abuse, in C57bl/6J mice.

The novel adamantane derivative APICA (N-(adamantan-1-yl)-1-pentyl-1H-indole-3-carboxamide) was recently identified as a cannabinomimetic indole of abuse. Despite its novel structure, APICA recalls cannabinomimetic indoles, such as representative member JWH-018. In present study, the effects of APICA (1-3mg/kg, i.p.) were tested in C57BL/6J mice, in the Tetrad task which includes the assessment of: body temperature; locomotor activity and behavioural reactivity; nociception; motor coordination; declarative memory. Furthermore, pre-treatment with the CB1 antagonist AM251 (3mg/kg, i.p.) or the CB2 antagonist AM630 (3mg/kg, i.p.) was carried out to characterize APICA activity. Our results show that APICA was able to dose-dependently decrease locomotor activity and behavioural reactivity in the open field, whereas only the highest dose was able to induce hypothermia, analgesia, motor incoordination and recognition memory impairment, with respect to vehicle (p<0.01; p<0.001). The pretreatment with the CB1 antagonist AM251 elicited an increase in body temperature, total distance travelled in the open field, latency to fall down in the Rotarod, and a decrease in tail flick latency (p<0.05; p<0.01). On the other hand, pretreatment with AM630 did not induced significant differences on APICA effects. This study supports preliminary reports on APICA cannabinomimetic properties, extending its detrimental effects on cognitive function. Moreover, these properties can be attributed to the CB1 receptor activity, indicating APICA as a selective CB1 receptor agonist.

Evaluation of the QT effect of a combination of piperaquine and a novel anti-malarial drug candidate OZ439, for the treatment of uncomplicated malaria.

AIMS: The aim was to investigate the QT effect of a single dose combination regimen of piperaquine phosphate (PQP) and a novel aromatic trioxolane, OZ439, for malaria treatment. METHODS: Exposure-response (ER) analysis was performed on data from a placebo-controlled, single dose, study with OZ439 and PQP. Fifty-nine healthy subjects aged 18 to 55 years received OZ439 alone or placebo in a first period, followed by OZ439 plus PQP or matching placebos in period 2. OZ439 and PQP doses ranged from 100-800 mg and 160-1440 mg, respectively. Twelve-lead ECG tracings and PK samples were collected serially pre- and post-dosing. RESULTS: A significant relation between plasma concentrations and placebo-corrected change from baseline QTc F (ΔΔQTc F) was demonstrated for piperaquine, but not for OZ439, with a mean slope of 0.047 ms per ng ml(-1) (90% CI 0.038, 0.057). Using an ER model that accounts for plasma concentrations of both piperaquine and OZ439, a largest mean QTc F effect of 14 ms (90% CI 10, 18 ms) and 18 ms (90% CI 14, 22 ms) was predicted at expected plasma concentrations of a single dose 800 mg OZ439 combined with PQP 960 mg (188 ng ml(-1) ) and 1440 mg (281 ng ml(-1) ), respectively, administered in the fasted state. CONCLUSIONS: Piperaquine prolongs the QTc interval in a concentration-dependent way. A single dose regimen combining 800 mg OZ439 with 960 mg or 1440 mg PQP is expected to result in lower peak piperaquine plasma concentrations compared with available 3 day PQP-artemisinin combinations and can therefore be predicted to cause less QTc prolongation.

Development of miniaturized sorbent membrane funnel-based spray platform for biological analysis.

In this work, a miniaturized solid-phase extraction (SPE) platform, called sorbent membrane funnel, which permits in situ cleanup prior to membrane funnel-based spray analysis was developed. The fabrication of funnel and the mounting of SPE sorbent were simple and straightforward by a homemade punching system. Using different sorbents, the SPE sorbent funnel has been successfully applied in spray analysis of drug molecules spiked in human plasma, trypsin digested solution of bovine serum albumin in the presence of high concentration of chaotropic reagents, and phosphopeptides in the tryptic digested solution of casein. The results demonstrated that SPE sorbent attached membrane funnels can be a useful tool in common metabolomic and proteomic applications.

A rapid and sensitive LC-MS/MS assay for the determination of saxagliptin and its active metabolite 5-hydroxy saxagliptin in human plasma and its application to a pharmacokinetic study.

The authors proposed a simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method for the simultaneous determination of saxagliptin and its active metabolite 5-hydroxy saxagliptin in human plasma. The developed method was fully validated as per the US FDA guidelines. The method utilized stable labeled isotopes saxagliptin-15 N d2 (IS1) and 5-hydroxy saxagliptin-15 N-d2 (IS2) as internal standards for the quantification of saxagliptin and 5-hydroxy saxagliptin, respectively. Analytes and the internal standards were extracted from human plasma by a single step solid-phase extraction technique without drying, evaporation and reconstitution steps. The optimized mobile phase was composed of 0.1% acetic acid in 5 mM ammonium acetate and acetonitrile (30:70, v/v) and delivered at a flow rate of 0.85 mL/min. The method exhibits the linear calibration range of 0.05-100 ng/mL for both the analytes. The precision and accuracy results for both the analytes were well within the acceptance limits. The different stability experiments conducted in aqueous samples and in matrix samples are meeting the acceptance criteria. The chromatographic run time was set at 1.8 min; hence more than 400 samples can be analyzed in a single day.

Simultaneous determination of a novel oral Janus kinase inhibitor ASP015K and its sulfated metabolite in rat plasma using LC-MS/MS.

A sensitive and selective liquid chromatography with tandem mass spectrometry (LC-MS/MS) was developed for determining the concentrations of novel Janus kinase inhibitor ASP015K and its sulfated metabolite M2 in rat plasma. This method involves solid-phase extraction (SPE) from 25 µL of rat plasma. LC separation was performed on an Inertsil PH-3 column (100 mm L ×4.6 mm I.D., 5 µm) with a mobile phase consisting of 10 mM ammonium acetate and methanol under linear gradient conditions. Analytes were introduced to the LC-MS/MS through an electrospray ionization source and detected in positive-ion mode using selected reaction monitoring. Standard curves were linear from 0.25 to 500 ng/mL (r ≥0.9964). This assay enabled quantification of ASP015K and M2 at a concentration as low as 0.25 ng/mL in rat plasma. Validation data demonstrated that the method is selective, sensitive and accurate. Further, we also successfully applied this method to a preclinical pharmacokinetic study in rats.

Bioequivalence of saxagliptin/metformin extended-release (XR) fixed-dose combination tablets and single-component saxagliptin and metformin XR tablets in healthy adult Chinese subjects.

BACKGROUND AND OBJECTIVES: As compared with individual tablets, saxagliptin/metformin extended-release (XR) fixed-dose combination (FDC) tablets offer the potential for increased patient compliance with the convenience of once daily dosing. The aim of the present study was to show that the FDC of saxagliptin and metformin XR is bioequivalent to co-administration of the individual components when given to Chinese subjects residing in China. METHODS: This was a randomized, open-label, single-dose, two-period, cross-over pharmacokinetic study in two cohorts of healthy adult Chinese male subjects (n = 32 in each cohort) under fed conditions. In cohort 1, the pharmacokinetic properties of a saxagliptin/metformin XR 5/500 mg FDC tablet were compared with those of co-administration of a 5 mg saxagliptin tablet and a 500 mg metformin XR tablet. In cohort 2, the pharmacokinetic properties of a saxagliptin/metformin XR 5/1,000 mg FDC tablet were compared with those of co-administration of a 5 mg saxagliptin tablet and 2 × 500 mg metformin XR tablets. The two cohorts were independent of each other with respect to treatment and results. The pharmacokinetic properties of the active metabolite of saxagliptin (5-hydroxy-saxagliptin), as well as the safety and tolerability of each treatment, were also evaluated. RESULTS: For both cohorts, saxagliptin and metformin in the FDCs were bioequivalent to the individual components, as the limits of the 90 % confidence intervals of the geometric least squares mean ratios were contained within the 80-125 % bioequivalence limits for the area under the plasma concentration-time curve parameters and within the 70-143 % bioequivalence limits for the maximum plasma concentration. Similar exposures of 5-hydroxy-saxagliptin were observed with the two treatment regimens within each cohort. Co-administration of saxagliptin and metformin XR was generally safe and well tolerated as the FDCs or as individual tablets. CONCLUSION: Saxagliptin/metformin XR 5/500 mg and saxagliptin/metformin XR 5/1,000 mg FDCs were bioequivalent to individual tablets of saxagliptin and metformin XR of the same strengths and were generally well tolerated. These results in healthy Chinese subjects are consistent with those of previous assessments of saxagliptin/metformin XR FDC in the saxagliptin clinical development programme.

Simultaneous determination of metformin and vildagliptin in human plasma by a HILIC-MS/MS method.

The objective of this work was to develop and validate a HILIC-MS/MS method for the simultaneous determination of metformin and vildagliptin in human plasma. Chromatographic separation was achieved using an Atlantis HILIC Silica 150-mm × 2.1-mm, 3-µm particle size column maintained at 40°C. The isocratic mobile phase consisted of 20% water and 80% acetonitrile/water solution 95:5 (v/v), containing both 0.1% formic acid and 3mM ammonium formate. The flow rate was maintained at 400 µL min(-1). Data from validation studies demonstrated that the new method is highly selective, sensitive (limits of detection <1.5 ng mL(-1)) and free of matrix and residual effects. The new method was also precise (RSD<9.0%), accurate (RE<11.2%) and linear (r ≥ 0.99) over the ranges of 5-500 ng mL(-1) for each compound. The developed method was successfully applied to determine metformin and vildagliptin in plasma volunteers who orally received a single dose of metformin (850 mg), vildagliptin (50mg) or drug association (metformin 850 mg+vildagliptin 50mg). The new method can thus also be used as a tool for the clinical monitoring of metformin and vildagliptin.