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.

Changes in glucose-induced plasma active glucagon-like peptide-1 levels by co-administration of sodium-glucose cotransporter inhibitors with dipeptidyl peptidase-4 inhibitors in rodents.

We investigated whether structurally different sodium-glucose cotransporter (SGLT) 2 inhibitors, when co-administered with dipeptidyl peptidase-4 (DPP4) inhibitors, could enhance glucagon-like peptide-1 (GLP-1) secretion during oral glucose tolerance tests (OGTTs) in rodents. Three different SGLT inhibitors-1-(ß-d-Glucopyranosyl)-4-chloro-3-[5-(6-fluoro-2-pyridyl)-2-thienylmethyl]benzene (GTB), TA-1887, and canagliflozin-were examined to assess the effect of chemical structure. Oral treatment with GTB plus a DPP4 inhibitor enhanced glucose-induced plasma active GLP-1 (aGLP-1) elevation and suppressed glucose excursions in both normal and diabetic rodents. In DPP4-deficient rats, GTB enhanced glucose-induced aGLP-1 elevation without affecting the basal level, whereas metformin, previously reported to enhance GLP-1 secretion, increased both the basal level and glucose-induced elevation. Oral treatment with canagliflozin and TA-1887 also enhanced glucose-induced aGLP-1 elevation when co-administered with either teneligliptin or sitagliptin. These data suggest that structurally different SGLT2 inhibitors enhance plasma aGLP-1 elevation and suppress glucose excursions during OGTT when co-administered with DPP4 inhibitors, regardless of the difference in chemical structure. Combination treatment with DPP4 inhibitors and SGLT2 inhibitors having moderate SGLT1 inhibitory activity may be a promising therapeutic option for improving glycemic control in patients with type 2 diabetes mellitus.

Intestinal Sodium Glucose Cotransporter 1 Inhibition Enhances Glucagon-Like Peptide-1 Secretion in Normal and Diabetic Rodents.

The sodium glucose cotransporter (SGLT) 1 plays a major role in glucose absorption and incretin hormone release in the gastrointestinal tract; however, the impact of SGLT1 inhibition on plasma glucagon-like peptide-1 (GLP-1) levels in vivo is controversial. We analyzed the effects of SGLT1 inhibitors on GLP-1 secretion in normoglycemic and hyperglycemic rodents using phloridzin, CGMI [3-(4-cyclopropylphenylmethyl)-1-(ß-d-glucopyranosyl)-4-methylindole], and canagliflozin. These compounds are SGLT2 inhibitors with moderate SGLT1 inhibitory activity, and their IC50 values against rat SGLT1 and mouse SGLT1 were 609 and 760 nM for phloridzin, 39.4 and 41.5 nM for CGMI, and 555 and 613 nM for canagliflozin, respectively. Oral administration of these inhibitors markedly enhanced and prolonged the glucose-induced plasma active GLP-1 (aGLP-1) increase in combination treatment with sitagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, in normoglycemic mice and rats. CGMI, the most potent SGLT1 inhibitor among them, enhanced glucose-induced, but not fat-induced, plasma aGLP-1 increase at a lower dose compared with canagliflozin. Both CGMI and canagliflozin delayed intestinal glucose absorption after oral administration in normoglycemic rats. The combined treatment of canagliflozin and a DPP4 inhibitor increased plasma aGLP-1 levels and improved glucose tolerance compared with single treatment in both 8- and 13-week-old Zucker diabetic fatty rats. These results suggest that transient inhibition of intestinal SGLT1 promotes GLP-1 secretion by delaying glucose absorption and that concomitant inhibition of intestinal SGLT1 and DPP4 is a novel therapeutic option for glycemic control in type 2 diabetes mellitus.

The effect of combined treatment with canagliflozin and teneligliptin on glucose intolerance in Zucker diabetic fatty rats.

To assess the impact of concomitant inhibition of sodium-glucose cotransporter (SGLT) 2 and dipeptidyl peptidase IV (DPP4) for the treatment of type 2 diabetes mellitus (T2DM), the effect of combined treatment with canagliflozin, a novel SGLT2 inhibitor, and teneligliptin, a DPP4 inhibitor, on glucose intolerance was investigated in Zucker diabetic fatty (ZDF) rats. Canagliflozin potently inhibited human and rat SGLT2 and moderately inhibited human and rat SGLT1 activities but did not affect DPP4 activity. In contrast, teneligliptin inhibited human and rat DPP4 activities but not SGLT activities. A single oral treatment of canagliflozin and teneligliptin suppressed plasma glucose elevation in an oral glucose tolerance test in 13 week-old ZDF rats. This combination of agents elevated plasma active GLP-1 levels in a synergistic manner, probably mediated by intestinal SGLT1 inhibition, and further improved glucose intolerance. In the combination-treated animals, there was no pharmacokinetic interaction of the drugs and no further inhibition of plasma DPP4 activity compared with that in the teneligliptin-treated animals. These results suggest that the inhibition of SGLT2 and DPP4 improves glucose intolerance and that combined treatment with canagliflozin and teneligliptin is a novel therapeutic option for glycemic control in T2DM.

Analysis of the effect of canagliflozin on renal glucose reabsorption and progression of hyperglycemia in zucker diabetic Fatty rats.

Sodium-glucose cotransporter 2 (SGLT2) plays a major role in renal glucose reabsorption. To analyze the potential of insulin-independent blood glucose control, the effects of the novel SGLT2 inhibitor canagliflozin on renal glucose reabsorption and the progression of hyperglycemia were analyzed in Zucker diabetic fatty (ZDF) rats. The transporter activity of recombinant human and rat SGLT2 was inhibited by canagliflozin, with 150- to 12,000-fold selectivity over other glucose transporters. Moreover, in vivo treatment with canagliflozin induced glucosuria in mice, rats, and dogs in a dose-dependent manner. It inhibited apparent glucose reabsorption by 55% in normoglycemic rats and by 94% in hyperglycemic rats. The inhibition of glucose reabsorption markedly reduced hyperglycemia in ZDF rats but did not induce hypoglycemia in normoglycemic animals. The change in urinary glucose excretion should not be used as a marker to predict the glycemic effects of this SGLT2 inhibitor. In ZDF rats, plasma glucose and HbA1c levels progressively increased with age, and pancreatic ß-cell failure developed at 13 weeks of age. Treatment with canagliflozin for 8 weeks from the prediabetic stage suppressed the progression of hyperglycemia, prevented the decrease in plasma insulin levels, increased pancreatic insulin contents, and minimized the deterioration of islet structure. These results indicate that selective inhibition of SGLT2 with canagliflozin controls the progression of hyperglycemia by inhibiting renal glucose reabsorption in ZDF rats. In addition, the preservation of ß-cell function suggests that canagliflozin treatment reduces glucose toxicity via an insulin-independent mechanism.

N-Glucosides as human sodium-dependent glucose cotransporter 2 (hSGLT2) inhibitors.

Inhibition of renal sodium-dependent glucose cotransporter 2 (SGLT2) increases urinary glucose excretion (UGE), and thus reduces blood glucose levels in hyperglycemia. A series of N-glucosides was synthesized for biological evaluation as human SGLT2 (hSGLT2) inhibitors. Among these compounds, N-glucoside 9d possessing an indole core structure showed good in vitro activity (IC50=7.1 nM against hSGLT2). Furthermore, 9d exhibited favorable in vivo potency with regard to UGE in rats based on good pharmacokinetic profiles.

C-Glucosides with heteroaryl thiophene as novel sodium-dependent glucose cotransporter 2 inhibitors.

Canagliflozin (1), a novel inhibitor for sodium-dependent glucose cotransporter 2 (SGLT2), has been developed for the treatment of type 2 diabetes. To investigate the effect of replacement of the phenyl ring in 1 with heteroaromatics, C-glucosides 2 were designed, synthesized, and evaluated for their inhibitory activities against SGLT2. Of these, 3-pyridyl, 2-pyrimidyl or 5-membered heteroaryl substituted derivatives showed highly potent inhibitory activity against SGLT2, while 5-pyrimidyl substitution was associated with slightly reduced activity. In particular, 2g (TA-3404) had remarkable anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice.

Synthesis and biological evaluation of thiophene-C-glucosides as sodium-dependent glucose cotransporter 2 inhibitors.

The synthesis and structure-activity relationship (SAR) of thiophene-C-glucosides have been explored, and the human sodium-dependent glucose cotransporter 2 (hSGLT2) inhibitory activities and rat urinary glucose excretion (UGE) effects of 3a-f were evaluated. As a result, they showed good hSGLT2 inhibitory activities and rat UGE effects. In particular, the chlorothiophene derivative 3f showed remarkable inhibitory activity against hSGLT2.

A pilot study verifying the effectiveness of high-frequency repetitive transcranial magnetic stimulation in combination with intensive speech-language-hearing therapy in patients with chronic aphasia.

BACKGROUND: A lot of research on the effectiveness of repetitive transcranial magnetic stimulation (rTMS) in patients with aphasia in the chronic stage deals with low-frequency stimulation, and reports on high-frequency stimulation (HF-rTMS) are scarce. OBJECTIVE: The purpose of this study was to evaluate retrospectively the effectiveness of high-frequency rTMS in combination with intensive speech-language-hearing therapy (ST) following the identification of the stimulation site using functional magnetic resonance imaging (fMRI) before, after the procedure and at 3 months in patients with aphasia in the chronic stage. METHODS: 20 patients with aphasia in the chronic stage who met the eligibility criteria for rTMS therapy and who underwent HF-rTMS for the first time were included in the present study. All patients received fMRI before hospitalization, and the stimulation site was decided accordingly. RESULTS: There was a significant improvement in all patients, rTMS to the left hemisphere, fluent and non-fluent groups by the evaluation of pre- and post- treatment by hospitalization. All groups had a significant improvement in total Standard Language Test of Aphasia score at 3 months when compared with the time of hospitalization. CONCLUSIONS: The current study indicates that HF-rTMS in combination with intensive ST is an effective therapeutic approach for patients with aphasia in the chronic stage.

Dose-response of rPMS for upper Limb hemiparesis after stroke.

INTRODUCTION: Repetitive peripheral magnetic stimulation (rPMS) therapy is an innovative and minimally invasive neurorehabilitative technique and has been shown to facilitate neural plasticity. However, there is at present no research that clarifies the dose-response of rPMS therapy on the recovery of upper limb hemiparesis after stroke. This trial aims to clarify the dose-response of rPMS therapy combined with intensive occupational therapy (OT) for chronic stroke patients with moderate to severe upper limb hemiparesis. METHODS AND ANALYSIS: This multicenter, prospective, assessor-blinded, randomized controlled study with 3 parallel groups will be conducted from January 20, 2020 to September 30, 2022. Fifty patients will be randomly assigned in a ratio of 1:2:2 to the control group, the group receiving daily 2400 pulses of rPMS, or the group receiving daily 4800 pulses of rPMS, respectively. From the day after admission (Day 1), rPMS therapy and intensive OT will be initiated. The primary outcome is the change in the motor function of the affected upper extremity (Fugl-Meyer Assessment) between the time of admission (Day 0) and the day after 2 weeks of treatment (Day 14). Secondary outcomes will include the changes in spasticity, active range of motion, motor evoked potential, and activity of daily living. ETHICS AND DISSEMINATION: The study was approved by the Jikei University Certified Review Board for all institutions (reference number: JKI19-020). Results of the primary and secondary outcomes will be published in a peer-reviewed journal and presented at international congresses. The results will also be disseminated to patients. TRIAL REGISTRATION NUMBER: jRCTs032190191.

Beneficial effects of canagliflozin in combination with pioglitazone on insulin sensitivity in rodent models of obese type 2 diabetes.

BACKGROUND: Despite its insulin sensitizing effects, pioglitazone may induce weight gain leading to an increased risk of development of insulin resistance. A novel sodium glucose co-transporter 2 (SGLT2) inhibitor, canagliflozin, provides not only glycemic control but also body weight reduction through an insulin-independent mechanism. The aim of this study was to investigate the combined effects of these agents on body weight control and insulin sensitivity. METHODS: Effects of combination therapy with canagliflozin and pioglitazone were evaluated in established diabetic KK-Ay mice and prediabetic Zucker diabetic fatty (ZDF) rats. RESULTS: In the KK-Ay mice, the combination therapy further improved glycemic control compared with canagliflozin or pioglitazone monotherapy. Furthermore, the combination significantly attenuated body weight and fat gain induced by pioglitazone and improved hyperinsulinemia. In the ZDF rats, early intervention with pioglitazone monotherapy almost completely prevented the progressive development of hyperglycemia, and no further improvement was observed by add-on treatment with canagliflozin. However, the combination significantly reduced pioglitazone-induced weight gain and adiposity and improved the Matsuda index, suggesting improved whole-body insulin sensitivity. CONCLUSIONS: Our study indicates that combination therapy with canagliflozin and pioglitazone improves insulin sensitivity partly by preventing glucotoxicity and, at least partly, by attenuating pioglitazone-induced body weight gain in two different obese diabetic animal models. This combination therapy may prove to be a valuable option for the treatment and prevention of obese type 2 diabetes.

Novel Indole-N-glucoside, TA-1887 As a Sodium Glucose Cotransporter 2 Inhibitor for Treatment of Type 2 Diabetes.

Inhibition of the renal sodium glucose cotransporter (SGLT) increases urinary glucose excretion (UGE) and thus reduces blood glucose levels during hyperglycemia. To explore the potential of new antihyperglycemic agents, we synthesized and determined the human SGLT2 (hSGLT2) inhibitory potential of novel substituted 3-benzylindole-N-glucosides 6. Optimization of 6 resulted in the discovery of 3-(4-cyclopropylbenzyl)-4-fluoroindole-N-glucoside 6a-4 (TA-1887), a highly potent and selective hSGLT2 inhibitor, with pronounced antihyperglycemic effects in high-fat diet-fed KK (HF-KK) mice. Our results suggest the potential of indole-N-glucosides as novel antihyperglycemic agents through inhibition of renal SGLT2.

Effect of canagliflozin on renal threshold for glucose, glycemia, and body weight in normal and diabetic animal models.

BACKGROUND: Canagliflozin is a sodium glucose co-transporter (SGLT) 2 inhibitor in clinical development for the treatment of type 2 diabetes mellitus (T2DM). METHODS: (14)C-alpha-methylglucoside uptake in Chinese hamster ovary-K cells expressing human, rat, or mouse SGLT2 or SGLT1; (3)H-2-deoxy-d-glucose uptake in L6 myoblasts; and 2-electrode voltage clamp recording of oocytes expressing human SGLT3 were analyzed. Graded glucose infusions were performed to determine rate of urinary glucose excretion (UGE) at different blood glucose (BG) concentrations and the renal threshold for glucose excretion (RT(G)) in vehicle or canagliflozin-treated Zucker diabetic fatty (ZDF) rats. This study aimed to characterize the pharmacodynamic effects of canagliflozin in vitro and in preclinical models of T2DM and obesity. RESULTS: Treatment with canagliflozin 1 mg/kg lowered RT(G) from 415±12 mg/dl to 94±10 mg/dl in ZDF rats while maintaining a threshold relationship between BG and UGE with virtually no UGE observed when BG was below RT(G). Canagliflozin dose-dependently decreased BG concentrations in db/db mice treated acutely. In ZDF rats treated for 4 weeks, canagliflozin decreased glycated hemoglobin (HbA1c) and improved measures of insulin secretion. In obese animal models, canagliflozin increased UGE and decreased BG, body weight gain, epididymal fat, liver weight, and the respiratory exchange ratio. CONCLUSIONS: Canagliflozin lowered RT(G) and increased UGE, improved glycemic control and beta-cell function in rodent models of T2DM, and reduced body weight gain in rodent models of obesity.

Gene organization of the liverwort Y chromosome reveals distinct sex chromosome evolution in a haploid system.

Y chromosomes are different from other chromosomes because of a lack of recombination. Until now, complete sequence information of Y chromosomes has been available only for some primates, although considerable information is available for other organisms, e.g., several species of Drosophila. Here, we report the gene organization of the Y chromosome in the dioecious liverwort Marchantia polymorpha and provide a detailed view of a Y chromosome in a haploid organism. On the 10-Mb Y chromosome, 64 genes are identified, 14 of which are detected only in the male genome and are expressed in reproductive organs but not in vegetative thalli, suggesting their participation in male reproductive functions. Another 40 genes on the Y chromosome are expressed in thalli and male sexual organs. At least six of these genes have diverged X-linked counterparts that are in turn expressed in thalli and sexual organs in female plants, suggesting that these X- and Y-linked genes have essential cellular functions. These findings indicate that the Y and X chromosomes share the same ancestral autosome and support the prediction that in a haploid organism essential genes on sex chromosomes are more likely to persist than in a diploid organism.