Uncovering the antidiabetic potential of heart-friendly and diuretic bioactive compounds through computer-based drug design.

Avicenna, a pioneer of modern medicine, recommended diuretic therapy to treat diabetes. Like Avicenna's approach, current medicine frequently prescribes oral antidiabetic pills with diuretic and hypoglycemic effects by blocking the absorption of sodium and glucose. To this end, the paper sought natural compounds with potential antidiabetic, cardioprotective, and diuretic properties through computer-based drug design (CADD) techniques, targeting the inhibition of SGLT2 proteins. We identified several bioactive compounds from various sources exhibiting potential multifunctionality through high-throughput virtual screening (HTVS) of vast compound libraries. Subsequent molecular docking and dynamics simulations were employed to assess these compounds' binding efficacy and stability with their respective targets, alongside ADMET prediction, to evaluate their pharmacokinetic and safety profiles. The top hits, phenylalanyltryptophan, tyrosyl-tryptophan, tyrosyl-tyrosine, celecoxib, and DIBOA trihexose, had superior docking scores ranging from -11,4 to -9,8 kcal/mol. The molecular dynamics simulations displayed steady interactions between target proteins and biocompounds throughout 100 ns without significant conformational shifts. These findings lay the groundwork for lead optimization and preclinical testing. This meticulous process ensures the safety and efficacy of potential treatments, marking a meaningful step toward developing innovative treatments for managing diabetes and its associated health complications.

Structures reveal how SGLT inhibitors work.

Sodium glucose cotransporters (SGLTs) transport glucose against its concentration gradient by harnessing the electrochemical potential gradient of sodium ions. SGLT inhibitors are widely prescribed to treat diabetes and other conditions. Recent structural studies have uncovered how chemically diverse SGLT inhibitors bind and inhibit the transporter at the atomic level.

Carbon 14 and Carbon 13 Syntheses of Velagliflozin.

Velagliflozin is the active ingredient of the first oral liquid medication approved by the Food and Drug Administration for the treatment of diabetes in cats. This compound belongs to the known class of sodium-glucose cotransporter 2 inhibitors approved to treat diabetes in human. Here, we report the detailed synthesis of velagliflozin labeled with carbon 14 and carbon 13.

In-silico screening and identification of glycomimetic as potential human sodium-glucose co-transporter 2 inhibitor.

Sodium-glucose co-transporter 2 (SGLT2) is one of the important targets against type II diabetes mellitus. A typical SGLT2 inhibitor acts by inhibiting glucose reabsorption, thus lowering the blood glucose level. Unlike SGLT1, SGLT2 is responsible for almost 90% glucose reabsorption from glomerular filtrate. The current SGLT2 inhibitors include gliflozins, often prescribed as second or third-line agents in diabetes mellitus. The SGLT2 inhibitors also benefit patients with heart and kidney disease. Due to instability issues with the natural O-aryl glycoside analogues C-glycoside analogues were developed and showed improved stability. Despite enhanced bioavailability and selectivity of newer derivatives, some serious side effects are associated with gliflozin analogues. At the present study, we applied in-silico approaches to find new glycomimetic compounds as potent SGLT2 inhibitors that could show improvement in side effects associated with current analogues. This work applied both ligand-based and structure-based drug approaches to find potential compounds. We developed a 3D-QSAR method to screen potential inhibitors from a library of ten thousand compounds and performed docking studies. The compounds were ranked based on predicted pIC50 and docking score. An initial screening of five thousand compounds was conducted, and the subsequently selected top 12 compounds were based on binding free energy calculations. These selected compounds were subjected to molecular dynamics (MD) simulations. Remarkably, our simulations identified nine compounds that exhibited significant and sustained binding affinity compared to the co-crystallized Empagliflozin. Collectively, considering the anticipated pharmacokinetic profiles and toxicity assessments, several of these compounds emerged as promising candidates for further in-depth evaluation.

The potential off-target neuroprotective effect of sister gliflozins suggests their repurposing despite not crossing the blood-brain barrier: From bioanalytical assay in rats into theory genesis.

Gliflozins are successfully marketed antidiabetic agents with a reported neuroprotective effect, and this study tests their blood-brain barrier crossing ability. Henceforward, a computational hypothesis interpreting their effects was reasonable after failure to cross into the brain. A chromatographic bioassay for canagliflozin, dapagliflozin, and empagliflozin was developed, validated, and applied to the rat's and rat's plasma and brain. HPLC method robustness was tested over two levels using Design of Experiment on MINITAB. It is the first method for gliflozins' detection in rats' brain tissue. The method was applied on 18 rats and six for each drug. Concentrations in plasma were determined but neither of them was detected in brain at the described chromatographic conditions. A computational study for the three drugs was endorsing two techniques. First, ligand-based target fishing reveals possible targets for gliflozins. They showed an ability to bind with human equilibrative nucleoside transporter 1, a regulator of adenosine extracellularly. Second, a docking study was carried out on this protein receptor. Results showed perfect alignment with a minimum of one hydrogen bond. Dapagliflozin achieved the lowest energy score with two hocking hydrogen bonds. This is proposing gliflozins ability to regulate equilibrative nucleoside transporter 1 receptors in peripheries, elevating the centrally acting neuroprotective adenosine.

The dynamic interplay between AMPK/NFκB signaling and NLRP3 is a new therapeutic target in inflammation: Emerging role of dapagliflozin in overcoming lipopolysaccharide-mediated lung injury.

Acute lung injury (ALI) is one the most common causes of morbidity and mortality in critically ill patients. In this study, we examined for first time the role of dapagliflozin (DPGZ) in lipopolysaccharide (LPS)-induced ALI in rats and determined the underlying molecular mechanisms by evaluating the effects of DPGZ on adenosine monophosphate kinase (AMPK), nuclear transcription factor kappa B, nucleotide-binding and oligomerization domain-like receptor 3 inflammasome activation. Treatment of acute lung injured rats with either low dose (5 mg/kg) or high dose (10 mg/kg) DPGZ significantly decreased oxidative stress by decreasing malondialdehyde and nitric oxide tissue levels with a significant increase in spectrophotometric measurements of superoxide dismutase, catalase, and reduced glutathione levels. DPGZ treatment resulted in a significant anti-inflammatory effect as indicated by suppression in myeloperoxidase activity, MCP-1, IL-1ß, IL-18, and TNF-α levels. DPGZ treatment also increased p-AMPK/t-AMPK with a significant reduction in NF-kB P65 binding activity and NFĸB p65 (pSer536) levels. These effects of DPGZ were accompanied by a significant reduction in NLRP3 levels and NLRP3 gene expression and a significant decrease in caspase-1 activity, which were also confirmed by histopathological examinations. We conclude that DPGZ antioxidant and anti-inflammatory activity may occur through regulation of AMPK/NFĸB pathway and inhibition of NLRP3 activation. These results suggest that DPGZ represents a promising intervention for the treatment of ALI, particularly in patients with type 2 diabetes.

In-silico screening and identification of phytochemicals from Centella asiatica as potential inhibitors of sodium-glucose co-transporter 2 for treating diabetes.

Sodium-glucose co-transporter 2 (SGLT-2) is a major transport protein responsible for reabsorption of glucose from the kidney back to the bloodstream. Inhibiting this protein effectively lowers the glucose level of diabetic patients; however, the use of synthetic SGLT-2 inhibitors has been linked to some serious adverse effects. There is a need to identify safer alternatives that are equally or more effective as the current inhibitor drugs. Phytochemicals are known for their efficacy as herbal remedies, but these molecules remain underexplored as source of therapeutic agents. In this study, we performed in silico screening to identify potential SGLT-2 inhibitors from the 21 phytochemicals from Centella asiatica. Docking results identified eleven compounds with estimated binding energies comparable to that of known inhibitors drugs. The stability of the complexes was then elucidated using 100 ns MD simulations. From our dynamic binding free energy calculations using MM/PBSA, asiaticoside, betulinic acid, centellasapogenol, methyl brahmate, and rutin exceeded at least one of the binding energies of the reference compounds, which highlights their strong affinity towards SGLT-2. Among the five, betulinic acid, centellasapogenol, and methyl brahmate maintained their structural stability to the same extent as the references and exhibited better oral bioavailability and excellent drug-like properties. Because of these results, it is recommended to prioritize betulinic acid, centellasapogenol, and methyl brahmate in future in vitro and in vivo studies to verify their potential as inhibitor drugs for diabetes therapies. Communicated by Ramaswamy H. Sarma.

Structural basis of inhibition of the human SGLT2-MAP17 glucose transporter.

Human sodium-glucose cotransporter 2 (hSGLT2) mediates the reabsorption of the majority of filtrated glucose in the kidney1. Pharmacological inhibition of hSGLT2 by oral small-molecule inhibitors, such as empagliflozin, leads to enhanced excretion of glucose and is widely used in the clinic to manage blood glucose levels for the treatment of type 2 diabetes1. Here we determined the cryogenic electron microscopy structure of the hSGLT2-MAP17 complex in the empagliflozin-bound state to an overall resolution of 2.95 Å. Our structure shows eukaryotic SGLT-specific structural features. MAP17 interacts with transmembrane helix 13 of hSGLT2. Empagliflozin occupies both the sugar-substrate-binding site and the external vestibule to lock hSGLT2 in an outward-open conformation, thus inhibiting the transport cycle. Our work provides a framework for understanding the mechanism of SLC5A family glucose transporters and also develops a foundation for the future rational design and optimization of new inhibitors targeting these transporters.

Discovery of GCC5694A: A potent and selective sodium glucose co-transporter 2 inhibitor for the treatment of type 2 diabetes.

Sodium-dependent glucose co-transporter 2 (SGLT2) has emerged as a promising drug target for the treatment of type 2 diabetes, and recently, several SGLT2 inhibitors have been approved for clinical use. A series of molecules with a C-aryl glucoside scaffold was designed and synthesized for biological evaluation. Among the molecules tested, a dihydrobenzofuran-containing analog, 14g (GCC5694A), exhibited excellentin vitro activity against SGLT2 (IC50 = 0.460 nM), good selectivity for SGLT1, and good metabolic stability. Data from further evaluation of the compound in animal models showed that this molecule is a promising candidate for development as an anti-diabetic agent.

Optimization of empagliflozin immediate release tablets (10 mg) using central composite rotatable design with response surface methodology.

Empagliflozin is a selective inhibitor of sodium glucose co-transporter II, given as mono therapy or an add-on treatment to reduce the glycated hemoglobin levels in type 2 diabetes. This work deals with designing, formulating and optimizing empagliflozin (10mg) immediate release (IR) tablets by direct compression technique using different excipients. Through central composite rotatable design (CCRD), total nine formulations (EF1-EF9) were generated by changing the composition of binder avicel PH 102® (X1) and superdisintegrant acdisol⌖ (X2). Formulation runs with in suitable weight range and powder properties were subjected to compression. The influence of interaction of excipients on friability (Y1), hardness (Y2) and disintegration (Y3) were analyzed by fitting the polynomial quadratic model with response surface methodology (RSM). Trials EF2, EF7, EF8 and EF9 exhibited acceptable tablet attributes upon physico-chemical testing. Different dissolution models were applied to observe the in vitro drug release pattern in phosphate buffer of pH 6.8. The cumulative drug release of IR tablet batches followed the Weibull kinetics with regression coefficient (r2) values of 0.983-0.992. Empagliflozin trials were exposed to accelerated storage conditions (40±2°C/ 75±5% RH) for stability testing. Shelf life period of exposed formulations were computed in range of 22 to 25 months. Keeping in view of the results, it is concluded that the employed technique of preparation and optimization are observed to be excellent for developing immediate release empagliflozin (10mg) tablets.

QSAR analysis of sodium glucose co-transporter 2 (SGLT2) inhibitors for anti-hyperglycaemic lead development.

QSAR (Quantitative Structure Activity Relationship) modelling was performed on a dataset of 90 sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors. The quantitative and explicative evaluations revealed some of the subtle and distinguished structural features that are responsible for the inhibitory potency of these compounds against SGLT2, such as less possible number of ring carbons at 8 Å from the lipophilic atoms in the molecule (fringClipo8A) and more possible value for the sum of the partial charges of the lipophilic atoms present within seven bonds from the donor atoms (lipo_don_7Bc). Multivariate GA-MLR (genetic algorithm-multi linear regression) and thorough validation methodology out-turned a statistically robust QSAR model with a very high predictability shown from various statistical parameters. A QSAR model with r2 = 0.83, F = 51.54, Q2LOO = 0.79, Q2LMO = 0.79, CCCcv = 0.88, Q2Fn = 0.76-0.81, r2ext = 0.77, CCCext = 0.85, and with RMSEtr < RMSEcv was proposed. This QSAR model will assist synthetic chemists in the development of the SGLT2 inhibitors as the antidiabetic leads.

Sympatholytic Mechanisms for the Beneficial Cardiovascular Effects of SGLT2 Inhibitors: A Research Hypothesis for Dapagliflozin's Effects in the Adrenal Gland.

Heart failure (HF) remains the leading cause of morbidity and death in the western world, and new therapeutic modalities are urgently needed to improve the lifespan and quality of life of HF patients. The sodium-glucose co-transporter-2 (SGLT2) inhibitors, originally developed and mainly indicated for diabetes mellitus treatment, have been increasingly shown to ameliorate heart disease, and specifically HF, in humans, regardless of diabetes co-existence. Indeed, dapagliflozin has been reported to reduce cardiovascular mortality and hospitalizations in patients with HF and reduced ejection fraction (HFrEF). This SGLT2 inhibitor demonstrates these benefits also in non-diabetic subjects, indicating that dapagliflozin's efficacy in HF is independent of blood glucose control. Evidence for the effectiveness of various SGLT2 inhibitors in providing cardiovascular benefits irrespective of their effects on blood glucose regulation have spurred the use of these agents in HFrEF treatment and resulted in FDA approvals for cardiovascular indications. The obvious question arising from all these studies is, of course, which molecular/pharmacological mechanisms underlie these cardiovascular benefits of the drugs in diabetics and non-diabetics alike. The fact that SGLT2 is not significantly expressed in cardiac myocytes (SGLT1 appears to be the dominant isoform) adds even greater perplexity to this answer. A variety of mechanisms have been proposed over the past few years and tested in cell and animal models and prominent among those is the potential for sympatholysis, i.e., reduction in sympathetic nervous system activity. The latter is known to be high in HF patients, contributing significantly to the morbidity and mortality of the disease. The present minireview first summarizes the current evidence in the literature supporting the notion that SGLT2 inhibitors, such as dapagliflozin and empagliflozin, exert sympatholysis, and also outlines the main putative underlying mechanisms for these sympatholytic effects. Then, we propose a novel hypothesis, centered on the adrenal medulla, for the sympatholytic effects specifically of dapagliflozin. Adrenal medulla is responsible for the production and secretion of almost the entire amount of circulating epinephrine and of a significant percentage of circulating norepinephrine in the human body. If proven true experimentally, this hypothesis, along with other emerging experimental evidence for sympatholytic effects in neurons, will shed new light on the pharmacological effects that mediate the cardiovascular benefits of SGLT2 inhibitor drugs, independently of their blood glucose-lowering effects.

Application of experimental design in HPLC method optimization and robustness for the simultaneous determination of canagliflozin, empagliflozin, linagliptin, and metformin in tablet.

Gliflozins and gliptins represent two different pharmacological drug classes that exert different and potentially complementary glucose-lowering effect in patients with type II diabetes mellitus. A novel, selective, and sensitive HPLC method was developed for the determination of canagliflozin, empaglifozin, linagliptin, and metformin in pure form, in laboratory prepared mixtures, and in pharmaceutical dosage form. Experimental design optimization was applied by using Plackett-Burman and face-centered composite designs to achieve the best resolution with minimum experimental trials. Three significant variables affecting optimization, namely buffer pH, percentage of methanol, and percentage of acetonitrile, were studied. Chromatographic separation was achieved using an Agilent Eclipse C8 column, and column temperature was kept at 45°C. The mobile phase was composed of dipotassium hydrogen phosphate buffer (0.05 M, adjusted to pH 6 using o-phosphoric acid):acetonitrile:methanol (50:25:25, v/v/v) at a flow rate of 1.5 mL/min. Sharp and well-resolved peaks of the cited drugs were obtained. The method was fully validated in terms of linearity, accuracy, precision, selectivity and robustness in agreement with the International Council of Harmonization (ICH) guidelines Q2 (R1). Satisfactory results were obtained by the analysis of tablets through applying the developed method. Therefore, it could be performed for the analysis of the cited drugs in quality control laboratories.

Discovery of remogliflozin etabonate: A potent and highly selective SGLT2 inhibitor.

We optimized the structure of an active metabolite (1) of WAY-123783, which was obtained from mouse urine after oral administration, to improve selectivity for SGLT2 and oral bioavailability. O-glucoside derivative 24 (remogliflozin etabonate) was subsequently identified as a potent, highly selective, and orally available SGLT2 inhibitor.

Biocomputational Prediction Approach Targeting FimH by Natural SGLT2 Inhibitors: A Possible Way to Overcome the Uropathogenic Effect of SGLT2 Inhibitor Drugs.

The Food and Drug Administration (FDA) approved a new class of anti-diabetic medication (a sodium-glucose co-transporter 2 (SGLT2) inhibitor) in 2013. However, SGLT2 inhibitor drugs are under evaluation due to their associative side effects, such as urinary tract and genital infection, urinary discomfort, diabetic ketosis, and kidney problems. Even clinicians have difficulty in recommending it to diabetic patients due to the increased probability of urinary tract infection. In our study, we selected natural SGLT2 inhibitors, namely acerogenin B, formononetin, (-)-kurarinone, (+)-pteryxin, and quinidine, to explore their potential against an emerging uropathogenic bacterial therapeutic target, i.e., FimH. FimH plays a critical role in the colonization of uropathogenic bacteria on the urinary tract surface. Thus, FimH antagonists show promising effects against uropathogenic bacterial strains via their targeting of FimH's adherence mechanism with less chance of resistance. The molecular docking results showed that, among natural SGLT2 inhibitors, formononetin, (+)-pteryxin, and quinidine have a strong interaction with FimH proteins, with binding energy (∆G) and inhibition constant (ki) values of -5.65 kcal/mol and 71.95 µM, -5.50 kcal/mol and 92.97 µM, and -5.70 kcal/mol and 66.40 µM, respectively. These interactions were better than those of the positive control heptyl α-d-mannopyranoside and far better than those of the SGLT2 inhibitor drug canagliflozin. Furthermore, a 50 ns molecular dynamics simulation was conducted to optimize the interaction, and the resulting complexes were found to be stable. Physicochemical property assessments predicted little toxicity and good drug-likeness properties for these three compounds. Therefore, formononetin, (+)-pteryxin, and quinidine can be proposed as promising SGLT2 inhibitors drugs, with add-on FimH inhibition potential that might reduce the probability of uropathogenic side effects.

Development of an HPLC-UV Method to Assay Empagliflozin Tablets and Identification of the Major Photoproduct by Quadrupole Time-of-Flight Mass Spectrometry.

Diabetes is a set of metabolic disorders that affect >400 million individuals worldwide. Empagliflozin belongs to the gliflozin class and is used orally to treat type 2 diabetes. In this study, a simple stability-indicating HPLC-UV method was developed to assay empagliflozin tablets and its main photoproduct was identified by high-resolution mass spectrometry. The mobile phase, which was optimized by Central Composite Design, was composed of methanol, acetonitrile and purified water (60:5:35 v/v), at a flow rate of 1 mL min-1. The calibration curve was linear in the range of 5-150 µg mL-1. All the validation parameters were met and the method was specific, even in the presence of degradation products. In the forced degradation study, empagliflozin standard and empagliflozin tablets were submitted to several conditions (acidic, alkaline, neutral and oxidant media, thermal, photolytic and humidity), and empagliflozin showed instability under all these conditions. A degradation product generated after drug exposure to ultraviolet C radiation was isolated and analyzed by quadrupole time-of-flight mass spectrometry, and the results suggested that empagliflozin undergoes decomposition by a dechlorination pathway. In silico toxicity was predicted for the degradation product, which showed a high risk of genotoxicity and hepatotoxicity.

Investigation of Solubility Behavior of Canagliflozin Hydrate Crystals Combining Crystallographic and Hirshfeld Surface Calculations.

Canagliflozin (CG) was a highly effective, selective and reversible inhibitor of sodium-dependent glucose co-transporter 2 developed for the treatment of type 2 diabetes mellitus. The crystal structure of CG monohydrate (CG-H2O) was reported for the first time while CG hemihydrate (CG-Hemi) had been reported in our previous research. Solubility and dissolution rate results showed that the solubility of CG-Hemi was 1.4 times higher than that of CG-H2O in water and hydrochloric acid solution, and the dissolution rates of CG-Hemi were more than 3 folds than CG-H2O in both solutions. Hirshfeld surface analysis showed that CG-H2O had stronger intermolecular forces than CG-Hemi, and water molecules in CG-H2O participated three hydrogen bonds, forming hydrogen bond networks. These crystal structure features might make it more difficult for solvent molecules to dissolve CG-H2O than CG-Hemi. All these analyses might explain why the dissolution performance of CG-Hemi was better than CG-H2O. This work provided an approach to predict the dissolution performance of the drug based on its crystal structure.

SGLT2 Inhibitors: A Novel Player in the Treatment and Prevention of Diabetic Cardiomyopathy.

Diabetic cardiomyopathy (DCM) characterized by diastolic and systolic dysfunction independently of hypertension and coronary heart disease, eventually develops into heart failure, which is strongly linked to a high prevalence of mortality in people with diabetes mellitus (DM). Sodium-glucose cotransporter type2 inhibitors (SGLT2Is) are a novel type of hypoglycemic agent in increasing urinary glucose and sodium excretion. Excitingly, the EMPA-REG clinical trial proved that empagliflozin significantly reduced the relative risk of cardiovascular (CV) death and hospitalization for heart failure (HHF) in patients with type 2 DM (T2DM) plus CV disease (CVD). The EMPRISE trial showed that empagliflozin decreased the risk of HHF in T2DM patients with and without a CVD history in routine care. These beneficial effects of SGLT2Is could not be entirely attributed to glucose-lowering or natriuretic action. There could be potential direct mechanisms of SGLT2Is in cardioprotection. Recent studies have shown the effects of SGLT2Is on cardiac iron homeostasis, mitochondrial function, anti-inflammation, anti-fibrosis, antioxidative stress, and renin-angiotensin-aldosterone system activity, as well as GlcNAcylation in the heart. This article reviews the current literature on the effects of SGLT2Is on DCM in preclinical studies. Possible molecular mechanisms regarding potential benefits of SGLT2Is for DCM are highlighted, with the purpose of providing a novel strategy for preventing DCM.

SGLT2 inhibitors, an accomplished development in field of medicinal chemistry: an extensive review.

Diabetes is a chronic progressive metabolic disease caused by insulin deficiency or insulin resistance. In spite of the availability of several antihyperglycaemics, there is a need for the development of safer antidiabetic drugs due to their undesirable effects. Sodium-glucose cotransporter-2 inhibitors are a class of antidiabetics, which hinder the reabsorption of glucose in the kidneys, causing excretion of glucose via urine. Sodium-glucose cotransporter-2 inhibitors are a well-tolerated class with no significant adverse effects and are found to be favorable in certain conditions, which may be rudimentary to cardiovascular and renal diseases. The current advancements in their design and development, their mechanism of action, structure-activity relationship, synthesis and in silico development along with their auxiliary roles have been extensively reviewed.

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.