Whiteness and greenness assessments of a sensitive HPLC method with fluorimetric detection for dapagliflozin quantitation in human plasma: Application to a healthy human volunteer.

The evident ecological impact of human actions, like air pollution, global warming, and ozone depletion, underscores the need for environmentally friendly approaches across various domains, including analytical chemistry. This study aimed to establish a validated, eco-friendly, and sustainable approach utilizing a fluorescence detector coupled with high-performance liquid chromatography for quantifying the antihyperglycemic agent dapagliflozin (DAPA), in human plasma. This method employed a C18 Microsorb MV (4.5 × 250 mm, 5 µm [particle size]) column at 40°C, with 40:60% v/v isocratic elution of acetonitrile and (0.1%) orthophosphoric acid as the mobile phase at 1 mL/min flow rate. DAPA and the internal standard demonstrated their greatest response by performing excitation at 225 nm (λex) and recording chromatograms at an emission wavelength (λem) equal to 305 nm. The presented approach demonstrated high linearity between 50 and 2000 ng/mL and full adherence to the guidelines of the US Food and Drug Administration regarding the validation of bioanalytical methods. The described technique was effectively used for quantification of DAPA in human plasma samples from a healthy male participant who received a tablet of 10 mg DAPA. Analytical Eco-Scale, Analytical GREEnness metric, and the recently created ChlorTox Scale were utilized for greenness assessment. Additionally, the "Red, Green, and Blue 12" model was used in whiteness evaluation.

A new HPLC-MS/MS method for the simultaneous quantification of SGLT2 inhibitors and metformin in plasma and its application to a pharmacokinetic study in healthy volunteers.

Monitoring the plasma concentrations of metformin and sodium-glucose cotransporter-2 inhibitors (canagliflozin, dapagliflozin and empagliflozin) is essential for pharmacokinetic and bioequivalence studies and therapeutic monitoring. The present work therefore aimed to develop and validate a high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous quantification of these drugs in human plasma. The analyses were performed using an Agilent 1200 HPLC system coupled to an Applied Biosystems API 3200 triple quadrupole MS/MS with electrospray ionization in positive ion mode. After one-step protein precipitation of plasma with acetonitrile containing 0.1% formic acid, chromatographic separation was achieved on an Xbridge C18 column, with a mobile phase consisting of a gradient of water and acetonitrile, both containing 1 mm ammonium formate and 0.1% formic acid. Quantification was performed in multiple reaction monitoring mode using m/z 130.1 → 71.1 for metformin, m/z 462.0 → 191.2 for canagliflozin, m/z 426.1 → 167.1 for dapagliflozin and m/z 468.0 → 354.9 for empagliflozin. The proposed method was validated and demonstrated to be adequate for the quantification of metformin, canagliflozin, dapagliflozin and empagliflozin for clinical monitoring, pharmacokinetics and bioequivalence studies.

Pathogenesis of Renal Injury and Gene Expression Changes in the Male CD-1 Mouse Associated with Exposure to Empagliflozin.

An increased incidence of renal tubular adenomas and carcinomas was identified in the 2-year CD-1 mouse carcinogenicity study with empagliflozin (sodium-glucose transporter 2 inhibitor) in high dose (1,000 mg/kg/day) male mice. A 13-week mouse renal investigative pathogenesis study was conducted with empagliflozin to evaluate dose dependency and temporal onset of nonneoplastic degenerative/regenerative renal tubular and molecular (genes, pathways) changes which precede neoplasia. Male and female CD-1 mice were given daily oral doses of 0, 100, 300, or 1,000 mg/kg/day (corresponding carcinogenicity study dose levels) for 1, 2, 4, 8, or 13 weeks. The maximum expected pharmacology with secondary osmotic diuresis was observed by week 1 at ≥100 mg/kg/day in both genders. Histopathologic kidney changes were first detected after 4 weeks of dosing in the male 1,000 mg/kg/day dose group, with progressive increases in the incidence and/or number of findings in this dose group so that they were more readily detected during weeks 8 and 13. Changes detected starting on week 4 consisted of minimal single-cell necrosis and minimal increases in mitotic figures. These changes persisted at an increased incidence at weeks 8 and 13 and were accompanied by minimal to mild tubular epithelial karyomegaly, minimal proximal convoluted tubular epithelial cell hyperplasia, and a corresponding increase in Ki-67-positive nuclei in epithelial cells of the proximal convoluted tubules. There were no corresponding changes in serum chemistry or urinalysis parameters indicative of any physiologically meaningful effect on renal function and thus these findings were not considered to be adverse. Similar changes were not identified in lower-dose groups in males nor were they present in females of any dose group. RNA-sequencing analysis revealed male mouse-specific changes in kidney over 13 weeks of dosing at 1,000 mg/kg/day. Treatment-related changes included genes and pathways related to p53-regulated cell cycle and proliferation, transforming growth factor ß, oxidative stress, and renal injury and the number of genes with significant expression change dramatically increased at week 13. These treatment-related changes in genes and pathways were predominant in high-dose males and complemented the observed temporal renal tubular changes. Overall, these mouse investigative study results support the role of early empagliflozin-related degenerative/regenerative changes only observed in high-dose male CD-1 mice as a key contributing feature to a nongenotoxic mode of renal tumor pathogenesis.

End-to-end application of model-informed drug development for ertugliflozin, a novel sodium-glucose cotransporter 2 inhibitor.

Model-informed drug development (MIDD) is critical in all stages of the drug-development process and almost all regulatory submissions for new agents incorporate some form of modeling and simulation. This review describes the MIDD approaches used in the end-to-end development of ertugliflozin, a sodium-glucose cotransporter 2 inhibitor approved for the treatment of adults with type 2 diabetes mellitus. Approaches included (1) quantitative systems pharmacology modeling to predict dose-response relationships, (2) dose-response modeling and model-based meta-analysis for dose selection and efficacy comparisons, (3) population pharmacokinetics (PKs) modeling to characterize PKs and quantify population variability in PK parameters, (4) regression modeling to evaluate ertugliflozin dose-proportionality and the impact of uridine 5'-diphospho-glucuronosyltransferase (UGT) 1A9 genotype on ertugliflozin PKs, and (5) physiologically-based PK modeling to assess the risk of UGT-mediated drug-drug interactions. These end-to-end MIDD approaches for ertugliflozin facilitated decision making, resulted in time/cost savings, and supported registration and labeling.

Ultrasound-assisted dispersive liquid-liquid microextraction for determination of three gliflozins in human plasma by HPLC/DAD.

A novel, highly sensitive ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) high performance liquid chromatography with diode-array detection (HPLC/DAD) method was developed for the determination of empagliflozin, dapagliflozin and canagliflozin in human plasma using methanol as protein precipitating agent/disperser and 1-dodecanol as extracting solvent. The analytes were eluted with an isocratic mobile phase consisting of acetonitrile:aqueous 0.1% trifluoroacetic acid pH 2.5, (40:60, v/v), at a flow rate of 1 mL/min and UV detection at 210 nm. The microextraction conditions were optimized regarding type and volume of extractant, type of disperser, sample pH, extraction time and centrifugation time. Under the optimal conditions, the enrichment factors were 19 for empagliflozin, 27 for dapagliflozin and 50 for canagliflozin. Linearity ranges were 2-2500 ng/mL, 3.5-2500 ng/mL and 1.1-2500 ng/mL for empagliflozin, dapagliflozin and canagliflozin, respectively. The developed method employs very small volumes of organic solvents in sample extraction and allows determination of small concetrations of gliflozins in human plasma.

Pharmacokinetic Properties of Single and Multiple Doses of Ertugliflozin, a Selective Inhibitor of SGLT2, in Healthy Chinese Subjects.

Ertugliflozin, a sodium-glucose cotransporter 2 inhibitor for the treatment of type 2 diabetes mellitus, prevents renal glucose reabsorption resulting in urinary glucose excretion. This open-label, parallel cohort, randomized study conducted in healthy Chinese adults residing in China assessed the pharmacokinetics, tolerability, and safety of 5 mg and 15 mg of ertugliflozin following single (fasted condition) and multiple-dose (fed condition) administration. Sixteen subjects were randomized and completed the study. Ertugliflozin absorption was rapid, with maximum plasma concentrations observed 1 hour after dosing under fasted conditions and 2 to 4 hours after dosing under fed conditions. Following single- and multiple-dose administration, ertugliflozin exhibited dose-proportional exposures with an apparent mean terminal half-life of approximately 9.5 to 11.9 hours. Steady state was reached after 4 once-daily doses. The accumulation ratio based on the area under the plasma concentration-time curve after multiple-dose administration was approximately 1.3 and 1.2 for ertugliflozin 5 mg and 15 mg, respectively. Ertugliflozin was generally well tolerated following administration of single and multiple oral doses of 5 mg and 15 mg in healthy Chinese subjects. Pharmacokinetic comparison with non-Asian subjects indicated that there are no clinically meaningful racial differences and no dose modification of ertugliflozin is required based on race or body weight.

Translational prediction of first-in-human pharmacokinetics and pharmacodynamics of janagliflozin, a selective SGLT2 inhibitor, using allometric scaling, dedrick and PK/PD modeling methods.

AIM: Janagliflozin is an orally selective SGLT2 inhibitor. To predict human pharmacokinetics/pharmacodynamics (PK/PD) characteristics of janagliflozin. To design optimal starting dose and effective dose for janagliflozin first-in-human (FIH) study. METHODS: Animal PK/PD properties of janagliflozin were obtained from preclinical in vivo and in vitro study. Pharmacologically effective level of same class SGLT2 inhibitors were assessed through preclinical and clinical efficacy data of dapagliflozin, empagliflozin and canagliflozin. Human PK parameters and profiles of janagliflozin were predicted by various methods such as allometric scaling (AS), dedrick and PK/PD modeling analysis. Mechanistic PK/PD model was developed to describe janagliflozin-mediated impact on urinary glucose excretion (UGE). Human IC50 was scaled from rat model-estimated IC50 by correcting interspecies difference of in vitro IC50 and plasma fu of rat and human. The quantitative PK/PD prediction of janagliflozin was evaluated via observed PK/PD profiles of healthy subjects. Predicted PK/PD characteristics of janagliflozin were applied in FIH dose design. Optimal starting dose was suggested by considering preclinical PD and toxicity data of janagliflozin. Effective dose was suggested by considering pharmacologically effective level of same class drugs. RESULTS: PK/PD characteristics of janagliflozin in preclinical species were summarized. Pharmacologically effective level for SGLT2 inhibitors was defined as 25~30% ΔUGE (ΔUGE=--(PG*GFR)within24h) based on efficacy data of three same class drugs. Human predicted CL, Vss and F were 1.04 L/h, 77.5 L and 0.80. Predicted AUC and Cmax of janagliflozin of 10 and 50 mg were within 0.47~2.08 fold of observed values. Predicted human UGE0-24 h and UGE0-144 h of 10 and 50 mg dose range were within 0.66~1.41 fold of observed values. Optimal starting dose and pharmacologically active dose (PAD) were suggested as 10 mg and 50 mg. Dose range for FIH study was designed as 10-450 mg. CONCLUSIONS: This study predicted human PK/PD characteristics of janagliflozin based on preclinical data and provide optimal dose design for janagliflozin FIH study based on pharmacologically effective level of same class drugs.

Simple, fast and robust LC-MS/MS method for the simultaneous quantification of canagliflozin, dapagliflozin and empagliflozin in human plasma and urine.

Sodium-glucose cotransporter 2 -inhibitors (SGLT2i) are oral glucose-lowering drugs that have also demonstrated cardioprotective and renoprotective effects. SGLT2i play an increasingly important role in the treatment of type 2 diabetes. Here we report a simple and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of three SGLT2i (canagliflozin, dapagliflozin and empagliflozin) in human plasma, serum and urine with a runtime of 1 min. Methanol was used as protein precipitating agent. Chromatographic separation was accomplished using a Waters ACQUITY UPLC HSS T3 1.8 µm; 2.1 × 50 mm column with a Waters ACQUITY UPLC HSS T3 1.8 µm VanGuard Pre-column; 2.1 × 5 mm, using gradient elution with ammonium acetate 20 mM (pH 5) and acetonitrile as mobile phase at a flow rate of 0.8 ml/min. Mass spectrometric analysis of the acetate adduct ions was carried out using electrospray with negative ionization and SRM mode. The assay was validated according to FDA and EMA guidelines, including selectivity, linearity, accuracy and precision, dilution integrity, stability and recovery. With a sample volume of 200 µl, linear ranges of 10-5000 µg/L, 1-500 µg/L and 2-1000 µg/L for canagliflozin, dapagliflozin and empagliflozin respectively, were achieved. The assay was successfully applied in two pharmacokinetic studies with dapagliflozin and empagliflozin. In conclusion, we developed and validated a simple, fast and robust LC-MS/MS method for the simultaneous quantification of canagliflozin, dapagliflozin and empagliflozin, that allows rapid analysis of large numbers of samples and can be used for both pharmacokinetic studies and biomedical analysis of canagliflozin, dapagliflozin and empagliflozin.

An Open-Label, Single-Period, Two-Stage, Single Oral Dose Pharmacokinetic Study of Remogliflozin Etabonate Tablet 100 and 250 mg in Healthy Asian Indian Male Subjects Under Fasting and Fed Conditions.

BACKGROUND AND OBJECTIVE: Remogliflozin etabonate is an orally available prodrug of remogliflozin, an inhibitor of renal sodium glucose co-transporter-2 (SGLT2) with antihyperglycemic activity. The present study was conducted to characterize the pharmacokinetic and safety profile of remogliflozin etabonate under fasting and fed conditions at single oral doses of 100 and 250 mg in healthy Asian Indian adults. METHODS: Sixty-five healthy, adult Asian Indian male subjects were enrolled in an open-label, two-stage, single-period pharmacokinetic study. Remogliflozin was given under fasting and/or fed conditions as a single oral dose of 100 or 250 mg. The plasma concentrations of remogliflozin etabonate, remogliflozin, and the metabolite GSK279782 were quantified by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Pharmacokinetic parameters were determined from the plasma concentration-time profile by non-compartmental analysis. Safety was assessed through monitoring of adverse events. Descriptive statistics were calculated and reported for all parameters. RESULTS: The plasma concentration profiles showed rapid absorption of the prodrug remogliflozin etabonate and rapid conversion to the active moiety, remogliflozin, which is then further metabolized to another active metabolite, GSK279782. The geometric mean maximum concentration (Cmax) and area under the plasma concentration-time curve (AUC) were comparable for all three analytes between the fasted and fed state. The fed/fasted ratio for Cmax ranged from 0.77 to 1.44 at the 100 mg dose, and from 0.80 to 1.12 at the 250 mg dose. The fed/fasted ratio for AUC was 1.22 and 1.35 at 100 and 250 mg, respectively. An early time to Cmax (tmax) was observed for all three analytes while being administered in the fasted state. Both the Cmax and AUClast of all the three analytes increased in a dose-proportional manner under the fasted and fed states. The terminal half-life for remogliflozin ranged from 1.53 to 2.07 h. All three analytes had comparable terminal half-lives irrespective of dose levels or dietary conditions. CONCLUSIONS: Following single oral administration at 100 and 250 mg, remogliflozin etabonate showed favorable, linear pharmacokinetics. There were no clinically relevant food effects on the pharmacokinetics at both the 100 and 250 mg dose levels. Remogliflozin etabonate was well-tolerated without any safety concerns or hypoglycemic events. CLINICAL TRIAL REGISTRATION: Clinical Trial Registry-India identifier number CTRI/2017/10/010043.

The Role of Glucagon in the Acute Therapeutic Effects of SGLT2 Inhibition.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) effectively lower plasma glucose (PG) concentration in patients with type 2 diabetes, but studies have suggested that circulating glucagon concentrations and endogenous glucose production (EGP) are increased by SGLT2i, possibly compromising their glucose-lowering ability. To tease out whether and how glucagon may influence the glucose-lowering effect of SGLT2 inhibition, we subjected 12 patients with type 2 diabetes to a randomized, placebo-controlled, double-blinded, crossover, double-dummy study comprising, on 4 separate days, a liquid mixed-meal test preceded by single-dose administration of either 1) placebo, 2) the SGLT2i empagliflozin (25 mg), 3) the glucagon receptor antagonist LY2409021 (300 mg), or 4) the combination empagliflozin + LY2409021. Empagliflozin and LY2409021 individually lowered fasting PG compared with placebo, and the combination further decreased fasting PG. Previous findings of increased glucagon concentrations and EGP during acute administration of SGLT2i were not replicated in this study. Empagliflozin reduced postprandial PG through increased urinary glucose excretion. LY2409021 reduced EGP significantly but gave rise to a paradoxical increase in postprandial PG excursion, which was annulled by empagliflozin during their combination (empagliflozin + LY2409021). In conclusion, our findings do not support that an SGLT2i-induced glucagonotropic effect is of importance for the glucose-lowering property of SGLT2 inhibition.

Effect of Rifampin on the Pharmacokinetics of Ertugliflozin in Healthy Subjects.

PURPOSE: Ertugliflozin is a selective sodium glucose cotransporter 2 inhibitor being developed for the treatment of type 2 diabetes mellitus. The primary enzyme involved in the metabolism of ertugliflozin is uridine diphosphate-glucuronosyltransferase (UGT) 1A9, with minor contributions from UGT2B7 and cytochrome P450 (CYP) isoenzymes 3A4, 3A5, and 2C8. Rifampin induces UGT1A9, UGT2B7, CYP3A4, and CYP3A5. Because concurrent induction of these enzymes could affect ertugliflozin exposure, this study assessed the effect of multiple doses of rifampin on the pharmacokinetic properties of single-dose ertugliflozin. METHODS: Twelve healthy adult subjects were enrolled in this open-label, 2-period, fixed-sequence study and received ertugliflozin 15mg on day 1 of period 1, followed by rifampin 600mg once daily on days 1 to 10 in period 2. On day 8 of period 2, ertugliflozin 15mg was coadministered with rifampin 600mg. Plasma samples for ertugliflozin pharmacokinetic analysis were collected during 72hours after dosing on day 1 of period 1 and day 8 of period 2 and analyzed using a validated HPLC-MS/MS method. Pharmacokinetic parameters were calculated using noncompartmental analysis of concentration-time data. Natural log transformed AUC0-∞ and Cmax of ertugliflozin were analyzed using a mixed-effects model with treatment as a fixed effect and subject as a random effect. FINDINGS: After administration of ertugliflozin 15mg alone or with rifampin, the Tmax was 1hour. The mean t½ was 12.3hours for ertugliflozin alone and 9.2hours with steady-state rifampin. Geometric mean ratios for AUC0-∞ and Cmax were 61.2% (90% CI, 57.2%-65.4%) and 84.6% (90% CI, 74.2%-96.5%), respectively. Ertugliflozin was well tolerated when administered alone or with rifampin. IMPLICATIONS: Coadministration of ertugliflozin with rifampin decreased ertugliflozin AUC0-∞ and Cmax by 39% and 15%, respectively. The effect of the reduced exposure was evaluated using the ertugliflozin dose-response model. The model predicted that a 5-mg ertugliflozin dose after coadministration with rifampin is expected to maintain clinically meaningful glycemic efficacy. Therefore, no dose adjustment of ertugliflozin is recommended when ertugliflozin is coadministered with a UGT and CYP inducer, such as rifampin.

Weight loss associated with sodium-glucose cotransporter-2 inhibition: a review of evidence and underlying mechanisms.

With their novel, insulin-independent mechanism, sodium-glucose cotransporter-2 (SGLT2) inhibitors are a major turning point in the management of type 2 diabetes mellitus. At present, there are several SGLT2 inhibitors available or in development, and these oral anti-hyperglycaemic agents lower plasma glucose through the inhibition of SGLT2-mediated reuptake of filtered glucose in the kidney. This unique mechanism of action is also expected to result in other beneficial effects, such as weight loss and blood pressure reduction. In various studies, including randomized controlled trials and real-world studies, patients treated with SGLT2 inhibitors have reported weight loss of around 1 to 3 kg. This review describes the characteristics of weight loss associated with SGLT2 inhibitor therapy, the clinical factors affecting SGLT2 inhibitor-associated weight loss and the possible underlying mechanisms of SGLT2 inhibitor-associated weight loss, including changes in metabolism and body composition, and the role of a reduction in insulin dose and compensatory hyperphagia. Understanding the weight loss effect of SGLT2 inhibitors, its related factors and underlying mechanisms can aid clinicians in optimal treatment decision-making, provide valuable insight on both obesity and diabetes management and reveal areas of future research and new therapeutic options.

Pharmacokinetic Evaluation of Empagliflozin in Healthy Egyptian Volunteers Using LC-MS/MS and Comparison with Other Ethnic Populations.

The present study considered the pharmacokinetic evaluation of empagliflozin after administration to Egyptian volunteers, and the results were compared with other ethnic populations. The FDA recognizes that standard methods of defining racial subgroups are necessary to compare results across pharmacokinetic studies and to assess potential subgroup differences. The design of the study was as an open labeled, randomized, one treatment, one period, single dose pharmacokinetic study. The main pharmacokinetic parameters estimated were Cmax, Tmax, t1/2, elimination rate constant, AUC0-t and AUC0-inf. The insignificant difference in pharmacokinetic parameters between Egyptians and white German subjects suggests that no dose adjustment should be considered with administration of 25 mg empagliflozin to Egyptian population. A new LC-MS/MS method was developed and validated, allowing sensitive estimation of empagliflozin (25-600 ng mL-1) in human plasma using dapagliflozin as an internal standard (IS). The method was applied successfully on the underlying pharmacokinetic study with enhanced sample preparation that involved liquid-liquid extraction. Multiple Reaction Monitoring (MRM) of the transition pairs of m/z 449.01 to 371.21 for empagliflozin and m/z 407.00 to 328.81 for dapagliflozin (IS) was employed utilizing negative mode Electro Spray Ionization (ESI). The validated LC-MS/MS method is suitable for further toxicodynamic and bioequivalence studies.