Sodium-glucose co-transporter 2 inhibitor canagliflozin modulates myocardial metabolism and inflammation in a swine model for chronic myocardial ischemia.

BACKGROUND: Inflammation and disruption of cardiac metabolism are prevalent in the setting of myocardial ischemia. Canagliflozin, a sodium-glucose costransporter-2 inhibitor, has beneficial effects on the heart, though the precise mechanisms are unknown. This study investigated the effects of canagliflozin therapy on metabolic pathways and inflammation in ischemic myocardial tissue using a swine model of chronic myocardial ischemia. METHODS: Sixteen Yorkshire swine underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic ischemia. Two weeks later, pigs received either no drug (n = 8) or 300 mg canagliflozin (n = 8) daily. Five weeks later, pigs underwent terminal harvest and tissue collection. RESULTS: Canagliflozin treatment was associated with a trend toward decreased expression of fatty acid oxidation inhibitor acetyl-CoA carboxylase and decreased phosphorylated/inactivated acetyl-CoA carboxylase, a promotor of fatty acid oxidation, compared with control ischemic myocardium (P = .08, P = .03). There was also a significant modulation in insulin resistance markers p-IRS1, p-PKCα, and phosphoinositide 3-kinase in ischemic myocardium of the canagliflozin group compared with the control group (all P < .05). Canagliflozin treatment was associated with a significant increase in inflammatory markers interleukin 6, interleukin 17, interferon-gamma, and inducible nitric oxide synthase (all P < .05). There was a trend toward decreased expression of the anti-inflammatory cytokines interleukin 10 (P = .16) and interleukin 4 (P = .31) with canagliflozin treatment. CONCLUSION: The beneficial effects of canagliflozin therapy appear to be associated with inhibition of fatty acid oxidation and enhancement of insulin signaling in ischemic myocardium. Interestingly, canagliflozin appears to increase the levels of several inflammatory markers, but further studies are required to better understand how canagliflozin modulates inflammatory signaling pathways.

Canagliflozin improves fatty acid oxidation and ferroptosis of renal tubular epithelial cells via FOXA1-CPT1A axis in diabetic kidney disease.

Impaired fatty acid oxidation (FAO) is a metabolic hallmark of renal tubular epithelial cells (RTECs) under diabetic conditions. Disturbed FAO may promote cellular oxidative stress and insufficient energy production, leading to ferroptosis subsequently. Canagliflozin, an effective anti-hyperglycemic drug, may exert potential reno-protective effects by upregulating FAO and inhibiting ferroptosis in RTECs. However, the mechanisms involved remain unclear. The present study is aimed to characterize the detailed mechanisms underlying the impact of canagliflozin on FAO and ferroptosis. Type 2 diabetic db/db mice were administrated daily by gavage with canagliflozin (20 mg/kg/day, 40 mg/kg/day) or positive control drug pioglitazone (10 mg/kg/day) for 12 weeks. The results showed canagliflozin effectively improved renal function and structure, reduced lipid droplet accumulation, enhanced FAO with increased ATP contents and CPT1A expression, a rate-limiting enzyme of FAO, and relieved ferroptosis in diabetic mice. Moreover, overexpression of FOXA1, a transcription factor related with lipid metabolism, was observed to upregulate the level of CPT1A, and further alleviated ferroptosis in high glucose cultured HK-2 cells. Whereas FOXA1 knockdown had the opposite effect. Mechanistically, chromatin immunoprecipitation assay and dual-luciferase reporter gene assay results demonstrated that FOXA1 transcriptionally promoted the expression of CPT1A through a sis-inducible element located in the promoter region of the protein. In conclusion, these data suggest that canagliflozin improves FAO and attenuates ferroptosis of RTECs via FOXA1-CPT1A axis in diabetic kidney disease.

Role of canagliflozin in ameliorating isoprenaline induced cardiomyocyte oxidative stress via the heme oxygenase-1 mediated pathway.

Canagliflozin (CZ) is commonly prescribed for management of type-2 diabetes mellitus (T2DM); it also can reduce the risk of myocardial infarction. We used 80 albino Wistar rats to investigate the cardioprotective potential of CZ against oxidative stress caused by administration of isoprenaline (ISO). We found that ISO stimulates production of reactive oxygen species and that CZ administration caused up-regulation of antioxidants and down-regulation of oxidants due to nuclear factor erythroid-2 related factor-2, as well as by enhancement of the heme oxygenase-1 mediated cascade. CZ monotherapy may play a cardioprotective role in diabetic patients. CZ possesses strong antioxidant potential that ameliorates cardiac damage induced by ISO administration.

Canagliflozin improves coronary microvascular vasodilation and increases absolute blood flow to the myocardium independent of angiogenesis.

OBJECTIVES: Sodium-glucose cotransporter-2 inhibitor, canagliflozin, improves myocardial perfusion to ischemic territory without accompanying changes in vascular density. We aimed to (1) characterize effects on angiogenic pathways, (2) use multiomics to identify gene expression and metabolite profiles relevant to regulation of myocardial blood flow, and (3) investigate drug effect on coronary microvascular reactivity. METHODS: A nondiabetic swine model of chronic myocardial ischemia and nondiabetic rat model were used to study functional and molecular effects of canagliflozin on myocardium and in vitro microvascular reactivity. RESULTS: Canagliflozin resulted in increased coronary microvascular vasodilation and decreased vasoconstriction (P < .05) without changes in microvascular density (P > .3). Expression of the angiogenic modulator, endostatin, increased (P = .008), along with its precursor, collagen 18 (P < .001), and factors that increase its production, including cathepsin L (P = .004). Endostatin and collagen 18 levels trended toward an inverse correlation with blood flow to ischemic territory at rest. Proangiogenic fibroblast growth factor receptor was increased (P = .03) and matrix metalloproteinase-9 was decreased (P < .001) with canagliflozin treatment. Proangiogenic vascular endothelial growth factor A (P = .13), Tie-2 (P = .10), and Ras (P = .18) were not significantly altered. Gene expression related to the cardiac renin-angiotensin system was significantly decreased. CONCLUSIONS: In chronic myocardial ischemia, canagliflozin increased absolute blood flow to the myocardium without robustly increasing vascular density or proangiogenic signaling. Canagliflozin resulted in altered coronary microvascular reactivity to favor vasodilation, likely through direct effect on vascular smooth muscle. Downregulation of cardiac renin-angiotensin system demonstrated local regulation of perfusion. VIDEO ABSTRACT.

The SGLT2 Inhibitor Canagliflozin Reduces Atherosclerosis by Enhancing Macrophage Autophagy.

It has been shown that SGLT2 suppresses atherosclerosis (AS). Recent studies indicate that autophagy widely participates in atherogenesis. This study aimed to assess the effect of canagliflozin (CAN) on atherogenesis via autophagy. Macrophages and ApoE - / - mice were used in this study. In macrophages, the results showed that CAN promoted LC3II expression and autophagosome formation. Furthermore, the cholesterol efflux assay demonstrated that CAN enhanced cholesterol efflux from macrophages via autophagy, resulting in lower lipid droplet concentrations in macrophages. The western blot revealed that CAN regulated autophagy via the AMPK/ULK1/Beclin1 signaling pathway. CAN resulted in increased macrophage autophagy in atherosclerotic plaques of ApoE - / - mice, confirming that CAN could inhibit the progression of AS via promoting macrophage autophagy. The current study found that CAN reduced the production of atherosclerotic lesions, which adds to our understanding of how SGLT2 inhibitors function to delay the progression of AS.

Canagliflozin attenuates chronic unpredictable mild stress induced neuroinflammation via modulating AMPK/mTOR autophagic signaling.

Although vast progress has been made to understand the pathogenesis of depression, existing antidepressant remedies, with several adverse effects, are not fully adequate. Interestingly, new emerging theories implicating an altered HPA-axis, tryptophan metabolism, neuroinflammation and altered gut integrity were proposed to further identify novel therapeutic targets. Along these lines, canagliflozin (CAN), a novel antidiabetic medication with anti-inflammatory and neuroprotective activity may present an effective treatment for depression; nevertheless, no studies have explored its effect on depressive disorder yet. To this end, this study aimed to investigate the possible antidepressant activity of CAN in CUMS and the mechanisms underlying its action on the gut-brain inflammation axis as well as the alteration in the TRY/KYN pathway in addition to its role in modulating the autophagic signaling cascade. Interestingly, CAN successfully attenuated the CUMS-induced elevations in despair and anhedonic behaviors as well as the elevated serum CORT. Furthermore, it enhanced gut integrity via hampering the CUMS-induced colonic inflammation and amending colonic tight junction proteins. The enhanced gut integrity was further corroborated by a notable anti-inflammatory and neuroprotective activity manifested via the observed mitigation of immune cell activation in addition to IDO hippocampal protein content and promotion of the autophagy cascade. Our findings postulate the possible anti-inflammatory and neuroprotective effects of CAN and the implication of TRY/KYN and AMPK/mTOR signaling pathways in the CUMS-induced MDD. Hence, this study shed light to the promising role of CAN in the augmentation of the current antidepressant treatments.

Canagliflozin reverses Th1/Th2 imbalance and promotes podocyte autophagy in rats with membranous nephropathy.

Idiopathic membranous nephropathy is the main cause of chronic kidney disease (CKD). Studies have shown sodium-glucose co-transporter 2 (SGLT2) inhibitors significantly delay renal outcomes in patients with CKD, but the exact mechanism remains unknown. In this study, we investigated the mechanism by which the SGLT2 inhibitor canagliflozin attenuates podocyte injury by reversing the imbalance in Helper T cell 1 (Th1)/Helper T cell 2 (Th2) in peripheral blood of rats with membranous nephropathy (MN). MN rats were gavaged with canagliflozin (10 mg/kg/d) and losartan (10 mg/kg/d), respectively, for eight weeks. Compared with the MN group, the urinary ratio of total protein and the creatinine levels of the canagliflozin group decreased significantly. Canagliflozin improved the glomerulus pathological damage, increased the expression levels of podocyte marker proteins. The protective effect of canagliflozin on kidneys was more obvious than that of losartan. Treatment with canagliflozin increased the proportion of Th1 cells by 2.3 times, decreased the proportion of Th2 cells by 68.5%, and significantly restrained the synthesis of immunoglobulin G1 in B-cells and glomerulus subepithelial immune complex deposition. Co-culture of B-cells derived from MN rats with podocytes triggered the activation of phosphorylation of mTOR and ULK1 of podocytes, inhibited podocyte autophagy and resulted in podocyte injury. B-cells derived from canagliflozin treatment rats reversed these effects above. In conclusion, canagliflozin exerts a protective effect on kidneys by reversing the imbalance in Th1/Th2 cells in MN rats and restoring the autophagy of podocytes inhibited by the abnormal immunoglobulin G secretion from B-cells.

Canagliflozin Ameliorates Nonalcoholic Fatty Liver Disease by Regulating Lipid Metabolism and Inhibiting Inflammation through Induction of Autophagy.

PURPOSE: Nonalcoholic fatty liver disease (NAFLD) is closely associated with metabolic diseases, including obesity and diabetes, and has gradually become the most common cause of chronic liver disease. We investigated the effects of sodium glucose cotransporter 2 (SGLT2) inhibitor canagliflozin on NAFLD in high-fat diet (HFD)-induced obese mice and possible underlying mechanisms. MATERIALS AND METHODS: Male C57BL/6 mice were fed a normal-diet, HFD, or HFD with canagliflozin for 14 weeks. AML-12 hepatocytes were treated with canagliflozin. Expression of related pathways was assessed. RESULTS: Canagliflozin administration reduced body weight and fat mass, compared with HFD alone. Canagliflozin improved glucose and lipid metabolic disorders. Compared with HFD-fed mice, liver weight, serum alanine transaminase (ALT) levels, and hepatic lipid accumulation were decreased after canagliflozin administration. Additionally, canagliflozin upregulated lipolysis markers (CPT1a, ACOX1, and ACADM), downregulated lipogenesis markers (SREBP-1c and FASN), and suppressed the production of inflammatory cytokines (TNFα, MCP1, IL-1ß, and IL-6), consistent with significantly increased LC3 II/I and Atg7 levels in the liver following canagliflozin treatment. In vitro, canagliflozin increased CPT1a, ACOX1, and ACADM expression, decreased SREBP-1c and FASN protein expression, and reduced TNFα, MCP1, IL-1ß, and IL-6 mRNA levels in lipid mixture (LM)-induced hepatocytes in a dose-dependent manner. These changes were reversed by 3-MA, an autophagy inhibitor. CONCLUSION: Our findings suggest that canagliflozin ameliorates the pathogenesis of NAFLD by regulating lipid metabolism and inhibiting inflammation, which may be associated with its promotion of autophagy.

Impacts of Sodium/Glucose Cotransporter-2 Inhibitors on Circulating Uric Acid Concentrations: A Systematic Review and Meta-Analysis.

BACKGROUND: Several trials have assessed the antihyperglycemic effects of sodium/glucose cotransporter-2 inhibitors (SGLT2i) in patients with type 2 diabetes mellitus (T2DM). We conducted a quantitative analysis to assess the impact of SGLT2is on serum uric acid (SUA) in patients with T2DM. METHODS: Placebo-controlled trials published before 13 August 2021 were identified by searching PubMed, Embase, Web of Science, and Scopus. The intervention group received SGLT2i as monotherapy or add-on treatment, and the control group received a placebo that was replaced with SGLT2i. Clinical trials providing changes in SUA were included. The mean change of SUA, glycated hemoglobin (HbA1c), fasting plasma glucose (FPG), and body weight were calculated (PROSPERO CRD42021287019). RESULTS: After screening of 1172 papers, 59 papers were included in the systematic review. A total of 55 trials (122 groups) of 7 types of SGLT2i on patients with T2DM were eligible for meta-analysis. All SGLT2is significantly decreased SUA levels compared with the placebo groups: empagliflozin mean difference (MD) = -40.98 µmol/L, 95% CI [-47.63, -34.32], dapagliflozin MD = -35.17 µmol/L, 95% CI [-39.68, -30.66], canagliflozin MD = -36.27 µmol/L, 95% CI [-41.62, -30.93], luseogliflozin MD = -24.269 µmol/L, 95% CI [-33.31, -15.22], tofogliflozin MD = -19.47 µmol/L, 95% CI [-27.40, -11.55], and ipragliflozin MD = -18.85 µmol/L, 95% CI [-27.20, -10.49]. SGLT2i also decreased FPG, body weight, and HbA1c levels. SUA reduction persisted during long-term treatment with SGLT2i (except for empagliflozin), while the SUA reduction was affected by the duration of diabetes. CONCLUSIONS: SGLT2i can be a valid therapeutic strategy for patients with T2DM and comorbid hyperuricemia. Besides reducing FPG, body weight, and HbA1c, SGLT2i can significantly decrease SUA levels compared to placebo (Total MD = -34.07 µmol/L, 95% CI [-37.00, -31.14]).

Anti-diabetic drug canagliflozin hinders skeletal muscle regeneration in mice.

Canagliflozin is an antidiabetic medicine that inhibits sodium-glucose cotransporter 2 (SGLT2) in proximal tubules. Recently, it was reported to have several noncanonical effects other than SGLT2 inhibiting. However, the effects of canagliflozin on skeletal muscle regeneration remain largely unexplored. Thus, in vivo muscle contractile properties recovery in mice ischemic lower limbs following gliflozins treatment was evaluated. The C2C12 myoblast differentiation after gliflozins treatment was also assessed in vitro. As a result, both in vivo and in vitro data indicate that canagliflozin impairs intrinsic myogenic regeneration, thus hindering ischemic limb muscle contractile properties, fatigue resistance recovery, and tissue regeneration. Mitochondrial structure and activity are both disrupted by canagliflozin in myoblasts. Single-cell RNA sequencing of ischemic tibialis anterior reveals a decrease in leucyl-tRNA synthetase 2 (LARS2) in muscle stem cells attributable to canagliflozin. Further investigation explicates the noncanonical function of LARS2, which plays pivotal roles in regulating myoblast differentiation and muscle regeneration by affecting mitochondrial structure and activity. Enhanced expression of LARS2 restores the differentiation of canagliflozin-treated myoblasts, and accelerates ischemic skeletal muscle regeneration in canagliflozin-treated mice. Our data suggest that canagliflozin directly impairs ischemic skeletal muscle recovery in mice by downregulating LARS2 expression in muscle stem cells, and that LARS2 may be a promising therapeutic target for injured skeletal muscle regeneration.

Canagliflozin is a potential cardioprotective drug but exerts no significant effects on pirarubicin­induced cardiotoxicity in rats.

Pirarubicin (THP), one of the anthracycline anticancer drugs, is widely used in the treatment of various types of cancer, but its cardiotoxicity cannot be ignored. Canagliflozin, the first sodium­glucose co­transporter­2 inhibitor approved by the USA FDA, has been shown to have a significant effect on cardiovascular damage caused by diabetes. However, it has not been reported whether it can resist THP­induced cardiotoxicity. The aim of the present study was to investigate the effect of canagliflozin on THP­induced cardiotoxicity and its mechanism. A rat model of cardiotoxicity induced by THP was established and canagliflozin treatment was performed at the same time. The changes of electrocardiography, cardiac coefficient and echocardiogram were observed. The levels of lactate dehydrogenase, brain natriuretic peptide, creatine kinase MB, cardiac troponin T, superoxide dismutase (SOD) and malondialdehyde were detected. The expression of SOD2, NADPH oxidase 2, pro/cleaved­caspase­ and Bcl­2/Bax were evaluated by western blotting. The primary culture of cardiomyocytes was prepared to explore the effect in vitro. After eight weeks, a series of cardiotoxicity manifestations were observed in THP rats. However, canagliflozin treatment had no significant effect on the above adverse reactions. Similarly, further studies showed that canagliflozin had no significant effect on THP­induced cardiomyocyte injury in vitro. The present study showed that there was no significant protective effect of canagliflozin on THP­induced cardiotoxicity and cardiomyocyte injury.

Effects of canagliflozin on human myocardial redox signalling: clinical implications.

AIMS: Recent clinical trials indicate that sodium-glucose cotransporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in heart failure patients, but the underlying mechanisms remain unknown. We explored the direct effects of canagliflozin, an SGLT2 inhibitor with mild SGLT1 inhibitory effects, on myocardial redox signalling in humans. METHODS AND RESULTS: Study 1 included 364 patients undergoing cardiac surgery. Right atrial appendage biopsies were harvested to quantify superoxide (O2.-) sources and the expression of inflammation, fibrosis, and myocardial stretch genes. In Study 2, atrial tissue from 51 patients was used ex vivo to study the direct effects of canagliflozin on NADPH oxidase activity and nitric oxide synthase (NOS) uncoupling. Differentiated H9C2 and primary human cardiomyocytes (hCM) were used to further characterize the underlying mechanisms (Study 3). SGLT1 was abundantly expressed in human atrial tissue and hCM, contrary to SGLT2. Myocardial SGLT1 expression was positively associated with O2.- production and pro-fibrotic, pro-inflammatory, and wall stretch gene expression. Canagliflozin reduced NADPH oxidase activity via AMP kinase (AMPK)/Rac1signalling and improved NOS coupling via increased tetrahydrobiopterin bioavailability ex vivo and in vitro. These were attenuated by knocking down SGLT1 in hCM. Canagliflozin had striking ex vivo transcriptomic effects on myocardial redox signalling, suppressing apoptotic and inflammatory pathways in hCM. CONCLUSIONS: We demonstrate for the first time that canagliflozin suppresses myocardial NADPH oxidase activity and improves NOS coupling via SGLT1/AMPK/Rac1 signalling, leading to global anti-inflammatory and anti-apoptotic effects in the human myocardium. These findings reveal a novel mechanism contributing to the beneficial cardiac effects of canagliflozin.

Canagliflozin Facilitates Reverse Cholesterol Transport Through Activation of AMPK/ABC Transporter Pathway.

BACKGROUND AND PURPOSE: Cholesterol is an essential lipid and its homeostasis is a major factor for many diseases, such as hyperlipidemia, atherosclerosis, diabetes, and obesity. Sodium-glucose cotransporter 2 (SGLT2) inhibitor canagliflozin (Cana) is a new kind of hypoglycemic agent, which decreases urinary glucose reabsorption and reduces hyperglycemia. Cana has been shown to regulate serum lipid, decrease serum triglyceride and increase serum high-density lipoprotein-cholesterol (HDL-C), and improve cardiovascular outcomes. But evidence of how Cana impacted the cholesterol metabolism remains elusive. METHODS: We treated Cana on mice with chow diet or western diet and then detected cholesterol metabolism in the liver and intestine. To explore the mechanism, we also treated hepG2 cells and Caco2 cells with different concentrations of Cana. RESULTS: In this study, we showed that Cana facilitated hepatic and intestinal cholesterol efflux. Mechanically, Cana via activating adenosine monophosphate-activated protein kinase (AMPK) increased the expression of ATP-binding cassette (ABC) transporters ABCG5 and ABCG8 in liver and intestine, increased biliary and fecal cholesterol excretion. CONCLUSION: This research confirms that Cana regulates cholesterol efflux and improves blood and hepatic lipid; this may be a partial reason for improving cardiovascular disease.

Lower limb events in individuals with type 2 diabetes: evidence for an increased risk associated with diuretic use.

AIMS/HYPOTHESIS: Recently, safety data signalled an increased risk of amputations in people taking canagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor. If this side effect is due to drug-induced hypovolaemia, diuretics should also increase that risk. The aim of this study was to analyse the association between diuretic use and the risk of lower limb events (LLEs) in people with type 2 diabetes. METHODS: SURDIAGENE (SUivi Rénal, DIAbète de type 2 et GENEtique) is a prospective observational cohort that includes people with type 2 diabetes enrolled from 2002 to 2012 and followed-up until onset of LLE, death or 31 December 2015, whichever came first. Primary outcome was the first occurrence of LLE, a composite of lower limb amputation (LLA) and lower limb revascularisation (LLR). The rates of primary outcome were compared between participants taking and not taking diuretics at baseline in a Cox-adjusted model. RESULTS: At baseline, of the 1459 participants included, 670 were taking diuretics. In participants with and without diuretics, the mean ages were 67.1 and 62.9 years and 55.8% and 59.8% were men, respectively. During a median follow-up of 7.1 years, the incidence of LLE was 1.80 per 100 patient-years in diuretic users vs 1.00 in non-users (p < 0.001). The HR for LLE in users vs non-users was 2.08 (95% CI 1.49, 2.93), p < 0.001. This association remained significant in a multivariable-adjusted model (1.49 [1.01, 2.19]; p = 0.04) and similar after considering death as a competing risk (subhazard ratio 1.89 [1.35, 2.64]; p < 0.001). When separated, LLA but not LLR, was associated with the use of diuretics: 2.01 (1.14, 3.54), p = 0.02 and 1.05 (0.67, 1.64), p = 0.84, respectively, in the multivariable-adjusted model. CONCLUSIONS/INTERPRETATION: Among people with type 2 diabetes treated with diuretics, there was a significant increase in the risk of LLE, predominantly in the risk of LLA.

In Vitro and In Vivo Correlation of Hepatic Fraction of Metabolism by P450 in Dogs.

1-Aminobenzotriazole (ABT) has been widely used as a nonspecific mechanism-based inhibitor of cytochrome P450 (P450) enzymes. It is extensively used in preclinical studies to determine the relative contribution of oxidative metabolism mediated by P450 in vitro and in vivo. The aim of present study was to understand the translation of fraction metabolized by P450 in dog hepatocytes to in vivo using ABT, for canagliflozin, known to be cleared by P450-mediated oxidation and UDP-glucuronosyltransferases-mediated glucuronidation, and 3 drug discovery project compounds mainly cleared by hepatic metabolism. In a dog hepatocyte, intrinsic clearance assay with and without preincubation of ABT, 3 Lilly compounds exhibited a wide range of fraction metabolized by P450. Subsequent metabolite profiling in dog hepatocytes demonstrated a combination of metabolism by P450 and UDP-glucuronosyltransferases. In vivo, dogs were pretreated with 50 mg/kg ABT or vehicle at 2 h before intravenous administration of canagliflozin and Lilly compounds. The areas under the concentration-time curve (AUC) were compared for the ABT-pretreated and vehicle-pretreated groups. The measured AUCABT/AUCveh ratios were correlated to fraction of metabolism by P450 in dog hepatocytes, suggesting that in vitro ABT inhibition in hepatocytes is useful to rank order compounds for in vivo fraction of metabolism assessment.

Inhibitory effects of sulfonylureas and non-steroidal anti-inflammatory drugs on in vitro metabolism of canagliflozin in human liver microsomes.

Canagliflozin, used to treat type 2 diabetes mellitus (T2DM), is commonly co-administered with sulfonylureas. The objective of the present study was to evaluate the possible inhibitory effect of sulfonylureas and non-steroidal anti-inflammatory drugs (NSAIDs) on canagliflozin metabolism in vitro. Three sulfonylurea derivatives were evaluated as inhibitors: chlorpropamide, glimepiride and gliclazide. Two other NSAIDs were used as positive control inhibitors: niflumic acid and diclofenac. The rate of formation of canagliflozin metabolites was determined by HPLC analysis of in vitro incubations of canagliflozin as a substrate with and without inhibitors, using human liver microsomes (HLMs). Among sulfonylureas, glimepiride showed the most potent inhibitory effect against canagliflozin M7 metabolite formation, with an IC50 value of 88 µm, compared to chlorpropamide and gliclazide with IC50 values of more than 500 µm. Diclofenac inhibited M5 metabolite formation more than M7, with IC50 values of 32 µm for M5 and 80 µm for M7. Niflumic acid showed no inhibition activity against M5 formation, but had relatively selective inhibitory potency against M7 formation, which is catalysed by UGT1A9, with an IC50 value of 1.9 µm and an inhibition constant value of 0.8 µm. A clinical pharmacokinetic interaction between canagliflozin and sulfonylureas is unlikely. However, a possible clinically important drug interaction between niflumic acid and canagliflozin has been identified.

Canagliflozin stability study and ecofriendly chromatographic determination of its degradation product: A comparative study.

Canagliflozin is a newly approved drug for type II diabetes mellitus. A full stability study of canagliflozin was performed following international conference on harmonization strategies. The drug was stable against all conditions except oxidation where only one degradation product was separated and structurally elucidated using mass spectrometry and infrared spectroscopy. A green high-performance thin-layer chromatographic densitometric determination was developed and validated for the accurate quantification of canagliflozin and its main oxidative degradation product. Separation was performed on high-performance aluminum plates precoated with silica gel using acetone/ethanol (80:20, v/v) as a developing system and scanning at 290 nm. Retardation factor values were 0.64 and 0.81 and linearity ranges were 0.4-3.6 and 0.2-3.2 µg/band for the drug and the degradation product, respectively. It was a matter of interest to use green solvents with no harmful effects on the environment. The comparison between the proposed and the reported high-performance liquid chromatography method regarding greenness profile showed that the proposed method was greener and so could be used as an alternative method to the reported one with no environmental harm. Method validity was tested as per international conference on harmonization and method utility was verified by application to Invokana® tablets.

Characterization of forced degradation products of canagliflozine by liquid chromatography/quadrupole time-of-flight tandem mass spectrometry and in silico toxicity predictions.

RATIONALE: Forced degradation studies are useful for better understanding of the stability of active pharmaceutical ingredients and drugs and to generate information about drug degradation pathways and formation of degradation products (DPs). Identification of DPs plays a vital role in establishing the safety and therapeutic benefit of a drug. METHODS: Canagliflozin (CAN) was subjected to different stress conditions as per International Conference on Harmonization guidelines (Q1A R2). All the DPs and the drug were well separated on an Aquity CSH C18 (100 × 2.1 mm, 1.7 µm) column using acetonitrile-methanol (70:30, v/v) and formic acid in gradient mode. The same UPLC method was employed for LC/HRMS for the characterization of DPs. In addition, in silico toxicity was predicted for all the DPs by using TOPKAT and DEREK software tools. RESULTS: CAN was found to degrade under oxidative stress condition and formed DP1 and DP2. This is a typical case of degradation where co-solvents acetonitrile-water (50:50, v/v) and methanol-water (50:50, v/v) react with CAN under acid hydrolytic conditions leading to the formation of pseudo-DPs, DP3 and DP4, respectively. Among these, DP2 and DP3 showed ocular irritancy whereas DP1 showed skin sensitization. CONCLUSIONS: The drug was labile under oxidative stress condition. CAN reacted with co-solvent under acid hydrolytic conditions and gave pseudo-DPs. All the DPs were separated using UPLC and characterized by LC/QTOF/MS/MS. Toxicity of DPs was evaluated using TOPKAT and DEREK software tools.

Interaction of the Sodium/Glucose Cotransporter (SGLT) 2 inhibitor Canagliflozin with SGLT1 and SGLT2.

Canagliflozin, a selective sodium/glucose cotransporter (SGLT) 2 inhibitor, suppresses the renal reabsorption of glucose and decreases blood glucose level in patients with type 2 diabetes. A characteristic of canagliflozin is its modest SGLT1 inhibitory action in the intestine at clinical dosage. To reveal its mechanism of action, we investigated the interaction of canagliflozin with SGLT1 and SGLT2. Inhibition kinetics and transporter-mediated uptake were examined in human SGLT1- or SGLT2-expressing cells. Whole-cell patch-clamp recording was conducted to examine the sidedness of drug action. Canagliflozin competitively inhibited SGLT1 and SGLT2, with high potency and selectivity for SGLT2. Inhibition constant (Ki) values for SGLT1 and SGLT2 were 770.5 and 4.0 nM, respectively. (14)C-canagliflozin was suggested to be transported by SGLT2; however, the transport rate was less than that of α-methyl-d-glucopyranoside. Canagliflozin inhibited α-methyl-d-glucopyranoside-induced SGLT1- and SGLT2-mediated inward currents preferentially from the extracellular side and not from the intracellular side. Based on the Ki value, canagliflozin is estimated to sufficiently inhibit SGLT2 from the urinary side in renal proximal tubules. The Ki value for SGLT1 suggests that canagliflozin suppresses SGLT1 in the small intestine from the luminal side, whereas it does not affect SGLT1 in the heart and skeletal muscle, considering the maximal concentration of plasma-unbound canagliflozin. Similarly, SGLT1 in the kidney would not be inhibited, thereby aiding in the prevention of hypoglycemia. After binding to SGLT2, canagliflozin may be reabsorbed by SGLT2, which leads to the low urinary excretion and prolonged drug action of canagliflozin.

Inhibition of Human UDP-Glucuronosyltransferase Enzymes by Canagliflozin and Dapagliflozin: Implications for Drug-Drug Interactions.

Canagliflozin (CNF) and dapagliflozin (DPF) are the first sodium-glucose cotransporter 2 inhibitors to be approved for clinical use. Although available evidence excludes clinically significant inhibition of cytochromes P450, the effects of CNF and DPF on human UDP-glucuronosyltransferase (UGT) enzymes are unknown. Here, we report the inhibition of human recombinant UGTs by CNF and DPF, along with the Ki values for selected recombinant and human liver microsomal UGTs. CNF inhibited all UGT1A subfamily enzymes, but the greatest inhibition was observed with UGT1A1, UGT1A9, and UGT1A10 (IC50 values ≤ 10 µM). DPF similarly inhibited UGT1A1, UGT1A9, and UGT1A10, with IC50 values ranging from 39 to 66 µM. In subsequent kinetic studies, CNF inhibited recombinant and human liver microsomal UGT1A9; Ki values ranged from 1.4 to 3.0 µM, depending on the substrate (propofol/4-methylumbelliferone) enzyme combination. Ki values for CNF inhibition of UGT1A1 were approximately 3-fold higher. Consistent with the activity screening data, DPF was a less potent inhibitor of UGT1A1 and UGT1A9. The Ki for DPF inhibition of UGT1A1 was 81 µM, whereas the Ki values for inhibition of UGT1A9 ranged from 12 to 15 µM. Based on the in vitro Ki values and plasma concentrations reported in the literature, DPF may be excluded as a perpetrator of DDIs arising from inhibition of UGT enzymes, but CNF inhibition of UGT1A1 and UGT1A9 in vivo cannot be discounted. Since the sodium-glucose cotransporter 2 inhibitors share common structural features, notably a glycoside moiety, investigation of drugs in this class for effects on UGT to identify (or exclude) potential drug-drug interactions is warranted.