hSGLT2 ( EC50 = 1.1 nM )

In vitro activity: Dapagliflozin has a 1200-fold IC50 and is therefore not sensitive to hSGLT1.[1]
Dapagliflozin is 32-fold more potent than phlorizin against hSGLT2 but 4-fold less than phlorizin against hSGLT1. Dapagliflozin exhibits a strong selectivity towards GLUT transporters, exhibiting 8–9% inhibition at 20 μM in protein-free buffer and almost no inhibition when 4% bovine serum albumin is present.[2]
Dapagliflozin is a substrate of P-glycoprotein (P-gp) and exhibits good permeability across Caco-2 cell membranes; however, it is not a potent P-gp inhibitor. At 10 μM, dapagliflozin remains stable in serum from rats, dogs, monkeys, and humans. Dapagliflozin does not elicit any induction or inhibitory responses from human P450 enzymes. Dapagliflozin is metabolized in vitro by hydroxylation, O-deethylation, and glucuronidation.[3]

---

In vitro activity.[1]
Dapagliflozin exhibits a mean EC50 against hSGLT2 of 1.12 nmol/l, compared with the EC50 for phlorizin of 35.6 nmol/l (Table 1). Against hSGLT1, Dapagliflozin and phlorizin displayed mean EC50 values of 1,391 and 330 nmol/l, respectively, indicating that dapagliflozin is highly selective (∼1,200-fold) for hSGLT2 vs. hSGLT1. Dapagliflozin is ∼32-fold more potent than phlorizin against hSGLT2 and ∼4-fold less potent than phlorizin against hSGLT1. Dapagliflozin is also a potent selective inhibitor of rSGLT2, displaying a mean EC50 value of 3.0 nmol/l, with ∼200-fold selectivity versus rSGLT1. Dapagliflozin is highly selective versus GLUT transporters as assayed in human adipocytes, displaying 8–9% inhibition in protein-free buffer at 20 μmol/l and virtually no inhibition in the presence of 4% bovine serum albumin (Table 2). Protein was added to this assay to simulate the in vivo condition of plasma protein binding. Phlorizin minimally inhibits adipocyte GLUT activity; however, the aglycone of phlorizin, phloretin, inhibits GLUT activity ∼77% regardless of whether bovine serum albumin is present in the assay.

---

Intracellular Magnesium Concentration [5]
After incubation for 24 h, the intracellular magnesium concentration was measured and calculated. As shown in Table 3 and Figure 3, treatment with Dapagliflozin was associated with a 60% increase in intracellular magnesium concentration compared to the control group at the time of the first measurement (0 min). Treatment with both AG1478 and mesendogen alone significantly decreased the intracellular magnesium concentration. Combined Dapagliflozin with either AG1478 or mesendogen both resulted in lower magnesium concentrations (both p < 0.05). Over 120 min, magnesium concentration was higher in the dapagliflozin group than in the control group, except at 80 min. AG1478 treatment alone was associated with significantly lower concentrations at 20, 60, 100, and 120 min. Mesendogen treatment alone decreased magnesium concentration throughout the 120 min period, except at 80 min. The combined treatment (dapagliflozin with AG1478 and dapagliflozin with mesendogen) significantly decreased the magnesium concentration at most time points.

Dapagliflozin reduces blood glucose levels by 55% in hyperglycemic streptozotocin (STZ) rats following an oral dose of 0.1 mg/kg; this effect is partially attributed to the metabolic stability provided by the C-glucoside linkage. Orally bioavailable dapagliflozin has a favorable absorption, distribution, metabolism, and excretion (ADME) profile.[1]
Dapagliflozin (1 mg/kg) produces a notable 24-hour post-dose rise in urine volume and dose-dependent glucosuria in normal rats. In Zucker diabetic fatty (ZDF) rats, dapagliflozin causes an increase in urine glucose and urine volume excretion six hours after the dose. Even after two weeks of treatment, dapagliflozin lowers the levels of fed and fasting glucose in ZDF rats without causing any signs of liver or renal toxicity.[2]
Dapagliflozin significantly reduces the development of hyperglycaemia, with lowered blood glucose. Dapagliflozin may lessen the mass of β-cells, increase insulin sensitivity, and prevent the onset of pancreatic dysfunction.[4]

---

Acute in vivo activity. [2]
In normal rats, Dapagliflozin administration caused significant dose-dependent glucosuria (Fig. 1) and increase in urine volume, with 1 mg/kg producing a 400-fold increase in urine glucose and a threefold increase in urine volume versus vehicle over 24 h post-dose. During an oral glucose tolerance test in normal rats, dapagliflozin administration was associated with a reduction in glucose area under the curve over 1 h post-dose at 1 and 10 mg/kg doses (Fig. 2), demonstrating that this glucosuric agent was able to reduce glucose excursions after an acute glucose challenge in normal rats. In ZDF rats administered single oral doses of dapagliflozin, a dose-dependent increase in urine glucose and urine volume excretion was apparent at 6 h post-dose (Fig. 3A) simultaneous with plasma glucose lowering in the same rats (Fig. 4) at doses of 0.01–1.0 mg/kg. The clear dose dependency of the urine glucose excretion effect declined when examined over a 24-h period (Fig. 3B), with treated rats in all dose groups demonstrating a twofold enhancement of urine glucose levels compared with vehicle-treated rats. Efficacy in lowering plasma glucose was still observed at 24 h post-dose after a period of refeeding in these rats at a dose of 1 mg/kg. No evidence of hypoglycemia was observed during the course of these studies. At 1 mg/kg, plasma exposure to dapagliflozin at 1 h post-dose was 1.2 μmol/l and estimated to be 5.2 μmol · l−1 · h−1 over the first 6 h of the experiment.

---

Chronic in vivo efficacy. [2]
In the first of two chronic studies, Dapagliflozin dose-dependently lowered fasting glucose levels in 18-h–fasted ZDF rats by day 8 of treatment, measured 24 h after the previous dose (Fig. 5). This effect was also evident on day 15 of treatment, where rats were fasted for a 24-h period, and in fed animals, measured on day 14 of the study. These data demonstrate that efficacy in lowering FPG was maintained over a 2-week once-daily treatment regimen. No body weight changes compared with vehicle-treated rats were noted, and no abnormal behavior was observed; the rats appeared to be well. No marker of renal or liver toxicity was measured. [2]
In the second chronic study, when measured 24 h after the final dose on day 15, ZDF rats treated with 0.5 mg/kg dDapagliflozin displayed a 53% decrease in 18-h FPG level compared with vehicle-treated rats (Table 3). On the third day after the final dose, a hyperinsulinemic-euglycemic clamp study was initiated to assess the metabolic effects of dapagliflozin versus vehicle treatment. In the basal stage, urine glucose loss rate was significantly higher in the vehicle-treated rats compared with the dapagliflozin-treated rats (Table 3), perhaps reflective of a trend for higher plasma glucose levels in vehicle-treated rats. The fact that the clamp procedure was initiated 48 h after the last dose of a compound that exhibits a 4- to 5-h half-life in rats (W. Humphreys, W.N.W., unpublished data) suggests that plasma drug levels were negligible during the clamp procedure, although they were not measured. Thus, we expect that urine glucose excretion acutely induced by dapagliflozin was not a significant contributor to the metabolic effects observed during the clamp. Urine volumes were also significantly reduced in dapagliflozin-treated rats during this procedure (S.H., L.X., J.M.W., W.G. Humphreys, W.N.W., J.R.T., unpublished data). Although there appeared to be reduced urinary glucose loss during the insulin infusion stage of the clamp in dapagliflozin-treated rats compared with vehicle-treated rats, this difference was not statistically significant. [2]
Glucose infusion rate in Dapagliflozin-treated rats was increased significantly during the insulin infusion stage of the clamp compared with that observed in vehicle-treated rats, suggesting an improvement in whole-body glucose utilization (Table 3). Glucose production during the insulin infusion stage was also reduced significantly in dapagliflozin-treated rats compared with vehicle-treated rats (Table 3). In addition, radio-labeled glucose uptake into liver during the clamp was significantly increased in dapagliflozin-treated rats, whereas glucose uptake into skeletal muscle or white adipose tissue was not significantly changed. These data suggest that 2-week treatment with dapagliflozin ameliorated elevated glucose production in ZDF rats and enhanced liver insulin sensitivity.

---

Dapagliflozin, administered from initiation of high-fat feeding, reduced the development of hyperglycaemia; after 24 days, blood glucose was 8.6 ± 0.5 vs. 13.3 ± 1.3 mmol/l (p < 0.005 vs. vehicle) and glycated haemoglobin 3.6 ± 0.1 vs. 4.8 ± 0.26% (p < 0.003 vs. vehicle). Dapagliflozin improved insulin sensitivity index: 0.08 ± 0.01 vs. 0.02 ± 0.01 in obese controls (p < 0.03). DI was improved to the level of lean control rats (dapagliflozin 0.29 ± 0.04; obese control 0.15 ± 0.01; lean 0.28 ± 0.01). In dapagliflozin-treated rats, β-cell mass was less variable and significant improvement in islet morphology was observed compared to vehicle-treated rats, although there was no change in mean β-cell mass with dapagliflozin. Results were similar when dapagliflozin treatment was initiated when animals were already moderately hyperglycaemic. Conclusion: Sustained glucose lowering with dapagliflozin in this model of type 2 diabetes prevented the continued decline in functional adaptation of pancreatic β-cells. [4]

---

Adult rats were fed a fructose-rich diet to induce MetS in the first 3 months and were then treated with either Dapagliflozin or magnesium sulfate-containing drinking water for another 3 months. Fructose-fed animals had increased insulin resistance, hypomagnesemia, and decreased urinary magnesium excretion. Dapagliflozin treatment improved insulin resistance by decreasing glucose and insulin levels, increased serum magnesium levels, and reduced urinary magnesium excretion. Serum vitamin D and parathyroid hormone levels were decreased in fructose-fed animals, and the levels remained low despite dapagliflozin and magnesium supplementation. In the kidney, claudin-16, TRPM6/7, and FXDY expression was increased in fructose-fed animals. Dapagliflozin increased intracellular magnesium concentration, and this effect was inhibited by TRPM6 blockade and the EGFR antagonist. We concluded that high fructose intake combined with a low-magnesium diet induced MetS and hypomagnesemia. Both dapagliflozin and magnesium sulfate supplementation improved the features of MetS and increased serum magnesium levels. Expression levels of magnesium transporters such as claudin-16, TRPM6/7, and FXYD2 were increased in fructose-fed animals and in those administered dapagliflozin and magnesium sulfate. Dapagliflozin enhances TRPM6-mediated trans-epithelial magnesium transport in renal tubule cells [5].

The selective SGLT substrate α-methyl-D-glucopyranoside (AMG) is used in the development of transport assays using Chinese hamster ovary (CHO) cells that stably express human SGLT2 (hSGLT2) and human SGLT1 (\hSGLT1). The ability of dapagliflozin to inhibit [14C]AMG uptake is measured over the course of two hours of incubation in a protein-free buffer. The inhibitor concentration at half maximal response, or EC50, is found by fitting the response curve to an empirical four-parameter model. The low-protein environment of the glomerular filtrate, which covers the SGLT targets on the kidney's proximal tubule lumenal surface, is replicated using protein-free buffer.

---

SGLT1 and SGLT2 assays. [2]
Cells expressing hSGLT1, hSGLT2, rSGLT1, or rSGLT2 were maintained using standard cell culture techniques. Assays for sodium-dependent glucose transport in 96-well plates were initiated by adding 100 μl/well of protein-free assay buffer containing sodium (HEPES/Tris pH 7.4, 137 mmol/l NaCl, 5.4 mmol/l KCl, 2.8 mmol/l CaCl2, 1.2 mmol/l MgSO4), 10 μmol/l 14C-α-methyl-d-glucopyranoside and inhibitor, or DMSO vehicle, and plates were incubated for 2 h at 37°C. Sodium-dependent 14C-α-methyl-d-glucopyranoside uptake was calculated by subtracting the counts per minute observed under sodium-free uptake conditions from the counts observed under sodium-containing conditions. Inhibitors were assayed at eight concentrations in triplicate in the presence of sodium, and the percent inhibition was calculated by comparing counts per minute in inhibitor-containing wells with counts per minute in wells containing only DMSO vehicle. Phlorizin was evaluated in parallel in every assay. A dose-response curve was fitted to an empirical four-parameter model using XL Fit to determine the inhibitor concentration at half-maximal response (EC50).

---

Adipocyte glucose uptake assays. [2]
Before the assay, predifferentiated human adipocytes were washed once in Dulbecco's modified Eagle's medium, low glucose, without fetal bovine serum, and incubated for 2 h at 37°C. The cells were then washed twice in Krebs-Ringer bicarbonate HEPES buffer, without glucose. The assay buffer (100 μl/well) consisted of Krebs-Ringer bicarbonate HEPES buffer containing either no insulin or 100 nmol/l insulin, 10 μmol/l 2-14C-deoxy-d-glucose, inhibitor or cytochalasin B, and a DMSO control (n = 6 per set). Cells were incubated at 37°C for 20 min, washed three times in PBS, and lysed in 50 μl/well of 0.1 N NaOH. MicroScint-40 was added, and cells were counted in a TopCount scintillation counter. Percent inhibition was calculated by comparing counts per minute in inhibitor-containing wells to counts per minute in wells without inhibitor.

Cells are collected for the cell survival assay following a 24-hour incubation period with either vehicle or dapagliflozin pretreatment in a 30-minute ischemia period. Any remaining cells are then counted using Trypan blue staining. The relative viable number of treated cells divided by the viable number of untreated cells is quantized to get the percentage survival.

---

Caco-2 Permeability and P-gp Interactions. [3]
The permeability coefficient of Dapagliflozin in the apical (A) to basolateral (B) direction was 60 nm/s at an initial concentration of 50 μM at pH 7.4. This value is comparable with compounds that exhibit moderate absorption in humans (Marino et al., 2005). Under the same conditions, the average B-to-A permeability coefficient value of dapagliflozin was 227 nm/s (BA/AB ratio of 3.8). In the presence of a P-gp inhibitor, the A-to-B permeability of dapagliflozin was 159 nm/s, and the B-to-A...

---

Cell Culture and Intracellular Magnesium Concentration Measurement. [5]
NRK52E cells were seeded on 96-well fluorescent plates, and cells were treated under the following conditions for 24 h: 1. regular medium; 2. 10 μM mesendogen; 3. 10 μM AG1478; 4. 0.2 μM Dapagliflozin ; 5. 10 μM mesendogen and 0.2 μM dapagliflozin; 6. 10 μM AG1478 (0.2 μM) and dapagliflozin. The cells were then incubated with 5 μM Mg-Fura-2 AM at 37 °C for 60 min and then washed with the required final incubation medium three times. The cells were then incubated for a further 60 min to allow complete de-esterification of intracellular AM esters before fluorescence measurements. All experiments were repeated 4–6 times, and results were then averaged.

Dissolved in 5% mpyrol, 20% PEG400, and 20 mM sodium diphosphate; 0.01-10 mg/kg (1 mL/kg) followed by a 50% glucose solution (2 g/kg); oral administration. Normal Sprague Dawley rats or streptozotocin induced male Sprague Dawley rats.

---

Diagrammatic representation of the protocols used in these experiments are available in an online appendix at http://dx.doi.org/10.2337/db07-1472. The animals were allowed ad libitum access to food and water unless otherwise stated, and rooms were maintained at 22°C and 50% humidity on a 12-h light/dark cycle. Male Sprague-Dawley rats were maintained on Harlan Teklad 2018 diet and weighed 250 g at the time of the experiment. Male Zucker diabetic fatty (ZDF) rats were maintained on Purina 5008 chow, and for acute studies, rats were 19 weeks of age and had a mean weight of 422 g. For the first chronic study, male ZDF rats were 17 weeks of age with a mean weight of 399 g; in the second chronic study, male ZDF rats were 15 weeks old with a mean body weight of 397 g. For all ZDF rat studies, rats were randomized into groups where body weight and plasma glucose levels were not statistically different between groups. Blood samples were collected and centrifuged (Eppendorf) at 4°C, 2,500 rpm, for 10 min. Plasma (5 μ1) was removed and mixed with 25 μl saline for glucose analysis using the Cobas Mira Analyzer. All urine volumes were measured and recorded. For urine glucose analysis, 5 μl urine was removed and mixed with 250 μl saline and analyzed on the Cobas Mira Analyzer.

---

Acute normal and diabetic rat studies.[2]
For all animal studies, the vehicle used for drug administration was 5% 1-methyl-2-pyrrolidinone, 20% polyethylene glycol, and 20 mmol/l sodium diphosphate. For glucose tolerance testing, 15 Sprague-Dawley rats were fasted overnight (18 h), weighed, bled via tail tip (30–40 μl), and randomized into five groups (n = 3). Rats were dosed orally with single doses of vehicle or drug (1 ml/kg; 0.01–10 mg/kg drug) and subsequently dosed orally with 50% aqueous glucose solution (2 g/kg). Rats were then bled at 15, 30, and 60 min and 24 h post-dose. Insulin was not measured in these studies.
For glucosuria assessment, overnight-fasted Sprague-Dawley rats were placed into metabolism cages for baseline urine collection over 24 h. Rats were weighed, randomized into five groups (n = 3), dosed orally with single doses of vehicle or drug (1 ml/kg; 0.01–10 mg/kg drug), and subsequently dosed orally with 50% aqueous glucose solution (2 g/kg). Immediately after dosing, rats were returned to metabolism cages for 24-h urine collection and re-fed at 1 h after the glucose challenge. The delta area under the curve for plasma glucose from baseline glucose value was calculated using GraphPad Prism. The urine glucose and urine volume data were normalized per 200 g body weight.
For assessment of acute glucosuria and plasma glucose effects in ZDF rats, the animals were weighed, bled via the tail tip (40–50 μl) in the fed state, and randomized into four groups (n = 6). Rats were dosed with vehicle or drug (1 ml/kg; 0.01–1.0 mg/kg drug) and placed into metabolism cages (without prior acclimation). Blood samples were collected immediately before dosing and at 2, 4, 6, and 24 h post-dose. Urine collections were obtained at 2, 4, 6, and 24 h post-dose. The animals were allowed to re-feed after the 6-h time point. Plasma samples at each time point were analyzed for the presence of glucose and Dapagliflozin. Urine glucose and urine volume data were normalized per 400 g body weight. Insulin was not measured in these studies.

---

Chronic diabetic rat studies.[2]
Two studies were performed in ZDF rats to evaluate the effects of multi-dose treatment with Dapagliflozin on prandial and fasting plasma glucose (FPG) and the metabolic profile of the rats after 2 weeks of treatment. In study 1, rats were fasted overnight, weighed, bled via the tail tip (40–50 μl), and randomized into four groups (n = 6 per group). Rats were dosed orally with vehicle or drug (1 ml/kg; 0.01–1.0 mg/kg drug) once daily for 14 days. Fasting body weight and plasma samples were obtained on days 8 (18-h fast) and 15 (24-h fast), and fed plasma samples were taken on day 14, 24 h after the previous dose. In study 2, ZDF rats were randomized into two groups (n = 6 per group) and dosed orally with vehicle or drug (1 ml/kg; 0.5 mg/kg drug) once daily for 15 days. Blood samples (40 μl) were obtained from 18-h–fasted rats from all groups by tail bleed on days 1, 8, and 15 of the study to determine plasma glucose levels. Neither fasting nor fed insulin was measured in these studies. A hyperinsulinemic-euglycemic clamp study was conducted with vehicle- and dapagliflozin-treated rats on day 17 and 48 h after the last dose of vehicle or dapagliflozin. Neither food nor water intake were monitored in these studies.

---

In a preliminary study, four obese female ZDF rats were placed on C13004 high-fat diet for 12–15 days, while four matched obese and four lean animals remained on RM1 chow diet. A hyperglycaemic clamp study was then carried out as described in subsequent text. For the evaluation of Dapagliflozin, two separate studies were performed in parallel in two separate batches of rats. In the first study (‘prevention’), Dapagliflozin (n = 14) was administered from the initiation of high-fat feeding. In the second study (‘intervention’), dapagliflozin treatment (n = 14) was initiated 10 days after the start of high-fat diet when animals had become moderately hyperglycaemic. Dapagliflozin (1 mg/kg, p.o. in water) or vehicle was administered once daily at 08:00 h for 33 days. In both studies, 48 h after the final dose, following an overnight fast, all groups were subdivided into two matched subgroups based on day 24 glucose and glycated haemoglobin (gHb) levels. One subgroup (5–6 animals) was used for evaluation of pancreatic function by hyperglycaemic clamp. The remaining animals were rendered insentient by inhalation of a 5 : 1 mixture of CO2: O2 to minimize changes of glucose and insulin levels in blood samples taken by cardiac puncture into ethylenediaminetetraacetic acid (EDTA) blood syringes. The pancreas was removed and fixed in 10% neutral buffered formalin for 24–48 h, followed by conventional processing and embedding in paraffin wax.[4]

---

Blood Sampling and Plasma Assays [4]
Disease progression was monitored prestudy, and on days 14 and 24, by measurement of gHb and blood glucose from small samples taken from the tail vein in conscious animals. Samples were taken at 08:00 h prior to the dose of Dapagliflozin. Plasma insulin was measured by enzyme-linked immunosorbent assay (ELISA). Plasma C-peptide was measured by radioimmunoassay. The plasma triglyceride assay was carried out using a Roche modular system by the Glycero-3-phosphate oxidase - para aminophenazone method.

Adult male Sprague Dawley rats weighing 200–250 g were used in this experiment. The animals were maintained under a constant 12 h photoperiod at temperatures between 21 °C and 23 °C. The animals were allowed free access to water and food. The animals were allocated to control and fructose-diet groups. Fructose diet groups were fed a fructose-rich diet (60% fructose, 0.05% magnesium wt/wt), and control animals (n = 10) received standard rat chow for 6 months. In animals receiving a high-fructose diet, drug treatments were started after 3 months of feeding. The animals were divided into three groups as follows: 1. continued fructose feeding for six months (FR, n = 10); 2. continued fructose diet with Dapagliflozin treatment (FR+Dapa, 1 mg·kg−1day−1 via oral gavage; n = 10) for 3 months; 3. continued fructose diet with magnesium sulfate supplementation (FR+Mg, 296 mg/L of magnesium in drinking water, Taiwan Biotech Co., Ltd., Taoyuan, Taiwan, n = 10) for 3 months. Blood pressure of each animal was measured by indirect tail cuff method twice a week. At least 5 readings were obtained and averaged. Body weight was measured weekly. At the end of the study, 24 h urine samples were collected in an individualized metabolic cage. The rats were then sacrificed, and blood samples were withdrawn from the inferior vena cava for biochemical analysis. [5]

Absorption, Distribution and Excretion
Oral dapagliflozin reaches a maximum concentration within 1 hour of administration when patients have been fasting. Following oral administration of dapagliflozin, the maximum plasma concentration (Cmax) is usually attained within 2 hours under fasting state. The Cmax and AUC values increase dose proportionally with an increase in dapagliflozin dose in the therapeutic dose range. The absolute oral bioavailability of dapagliflozin following the administration of a 10 mg dose is 78%. Administration of dapagliflozin with a high-fat meal decreases its Cmax by up to 50% and prolongs Tmax by approximately 1 hour but does not alter AUC as compared with the fasted state. These changes are not considered to be clinically meaningful and dapagliflozin can be administered with or without food.
Dapagliflozin and related metabolites are primarily eliminated via the renal pathway. Following a single 50 mg dose of [¹⁴C]-dapagliflozin, 75% and 21% of total radioactivity is excreted in urine and feces, respectively. In urine, less than 2% of the dose is excreted as the parent drug. In feces, approximately 15% of the dose is excreted as the parent drug.
The volume of distribution was estimated to be 118L.
Oral plasma clearance was 4.9 mL/min/kg, and renal clearance was 5.6 mL/min.

---

Metabolism / Metabolites
Dapagliflozin is primarily glucuronidated to become the inactive 3-O-glucuronide metabolite(60.7%). Dapagliflozin also produces another minor glucuronidated metabolite(5.4%), a de-ethylated metabolite(<5%), and a hydroxylated metabolite(<5%). Metabolism of dapagliflozin is mediated by cytochrome p-450(CYP)1A1, CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP3A4, uridine diphosphate glucuronyltransferase(UGT)1A9, UGT2B4, and UGT2B7. Glucuronidation to the major metabolite is mediated by UGT1A9.

---

Biological Half-Life
The mean plasma terminal half-life (t_1/2) for dapagliflozin is approximately 12.9 hours following a single oral dose of 10 mg. In healthy subjects given a single oral dose of 50 mg of dapagliflozin, the mean terminal half-life was 13.8 hours.

---

(2S,3R,4R,5S,6R)-2-(3-(4-Ethoxybenzyl)-4-chlorophenyl)-6-hydroxymethyl-tetrahydro-2H-pyran-3,4,5-triol (dapagliflozin; BMS-512148) is a potent sodium-glucose cotransporter type II inhibitor in animals and humans and is currently under development for the treatment of type 2 diabetes. The preclinical characterization of dapagliflozin, to allow compound selection and prediction of pharmacological and dispositional behavior in the clinic, involved Caco-2 cell permeability studies, cytochrome P450 (P450) inhibition and induction studies, P450 reaction phenotyping, metabolite identification in hepatocytes, and pharmacokinetics in rats, dogs, and monkeys. Dapagliflozin was found to have good permeability across Caco-2 cell membranes. It was found to be a substrate for P-glycoprotein (P-gp) but not a significant P-gp inhibitor. Dapagliflozin was not found to be an inhibitor or an inducer of human P450 enzymes. The in vitro metabolic profiles of dapagliflozin after incubation with hepatocytes from mice, rats, dogs, monkeys, and humans were qualitatively similar. Rat hepatocyte incubations showed the highest turnover, and dapagliflozin was most stable in human hepatocytes. Prominent in vitro metabolic pathways observed were glucuronidation, hydroxylation, and O-deethylation. Pharmacokinetic parameters for dapagliflozin in preclinical species revealed a compound with adequate oral exposure, clearance, and elimination half-life, consistent with the potential for single daily dosing in humans. The pharmacokinetics in humans after a single dose of 50 mg of [(14)C]dapagliflozin showed good exposure, low clearance, adequate half-life, and no metabolites with significant pharmacological activity or toxicological concern. [3]

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of dapagliflozin during breastfeeding. Dapagliflozin is an uncharged molecule that is 91% protein bound in plasma, so it is unlikely to pass into breastmilk in clinically important amounts. The manufacturer does not recommend dapagliflozin during breastfeeding because of a theoretical risk to the infant's developing kidney. An alternate drug may be preferred, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

---

Protein Binding
Dapagliflozin is approximately 91% protein bound. Protein binding is not altered in patients with renal or hepatic impairment.

[1]. Discovery of dapagliflozin: a potent, selective renal sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor for the treatment of type 2 diabetes. J Med Chem. 2008 Mar 13;51(5):1145-9.

[2]. Dapagliflozin, a selective SGLT2 inhibitor, improves glucose homeostasis in normal and diabetic rats. Diabetes. 2008 Jun;57(6):1723-9.

[3]. In vitro characterization and pharmacokinetics of dapagliflozin (BMS-512148), a potent sodium-glucose cotransporter type II inhibitor, in animals and humans. Drug Metab Dispos. 2010 Mar;38(3):405-14.

[4]. The novel sodium glucose transporter 2 inhibitor dapagliflozin sustains pancreatic function and preserves islet morphology in obese, diabetic rats. Diabetes Obes Metab. 2010 Nov;12(11):1004-12.

[5]. Effect of Dapagliflozin and Magnesium Supplementation on Renal Magnesium Handling and Magnesium Homeostasis in Metabolic Syndrome. Nutrients. 2021 Nov 15;13(11):4088.

Dapagliflozin is a C-glycosyl comprising beta-D-glucose in which the anomeric hydroxy group is replaced by a 4-chloro-3-(4-ethoxybenzyl)phenyl group. Used (in the form of its propanediol monohydrate) to improve glycemic control, along with diet and exercise, in adults with type 2 diabetes. It has a role as a hypoglycemic agent and a sodium-glucose transport protein subtype 2 inhibitor. It is a C-glycosyl compound, an aromatic ether and a member of monochlorobenzenes.
Dapagliflozin is a sodium-glucose cotransporter 2 (SGLT2) inhibitor, and it was the first SLGT2 inhibitor to be approved. indicated for managing diabetes mellitus type 2. When combined with diet and exercise in adults, dapagliflozin helps to improve glycemic control by inhibiting glucose reabsorption in the proximal tubule of the nephron and causing glycosuria. Dapagliflozin has been investigated either as monotherapy or as an adjunct treatment with insulin or other oral hypoglycemic agents. Dapagliflozin was originally approved by the FDA on Jan 08, 2014, to improve glycemic control in adults with type 2 diabetes in conjunction with diet and exercise. It was later approved to reduce the risk of kidney function decline, kidney failure, cardiovascular death, and hospitalization for heart failure in adults with chronic kidney disease in April 2021.

Dapagliflozin is a Sodium-Glucose Cotransporter 2 Inhibitor. The mechanism of action of dapagliflozin is as a Sodium-Glucose Transporter 2 Inhibitor.
Dapagliflozin is a selective sodium-glucose co-transporter subtype 2 (SGLT2) inhibitor with antihyperglycemic activity. Dapagliflozin selectively and potently inhibits SGLT2 compared to SGLT1, which is the cotransporter of glucose in the gut.
DAPAGLIFLOZIN is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2012 and has 3 approved and 37 investigational indications.

---

Pharmacodynamics
Dapagliflozin also reduces sodium reabsorption and increases the delivery of sodium to the distal tubule. This may influence several physiological functions including, but not restricted to, lowering both pre- and afterload of the heart and downregulation of sympathetic activity, and decreased intraglomerular pressure which is believed to be mediated by increased tubuloglomerular feedback. Increases in the amount of glucose excreted in the urine were observed in healthy subjects and in patients with type 2 diabetes mellitus following the administration of dapagliflozin. Dapagliflozin doses of 5 or 10 mg per day in patients with type 2 diabetes mellitus for 12 weeks resulted in excretion of approximately 70 grams of glucose in the urine per day at Week 12. A near-maximum glucose excretion was observed at the dapagliflozin daily dose of 20 mg. This urinary glucose excretion with dapagliflozin also results in increases in urinary volume. After discontinuation of dapagliflozin, on average, the elevation in urinary glucose excretion approaches baseline by about 3 days for the 10 mg dose. Dapagliflozin was not associated with clinically meaningful prolongation of QTc interval at daily doses up to 150 mg (15 times the recommended maximum dose) in a study of healthy subjects. In addition, no clinically meaningful effect on QTc interval was observed following single doses of up to 500 mg (50 times the recommended maximum dose) of dapagliflozin in healthy subjects.

---

The C-aryl glucoside 6 (dapagliflozin) was identified as a potent and selective hSGLT2 inhibitor which reduced blood glucose levels in a dose-dependent manner by as much as 55% in hyperglycemic streptozotocin (STZ) rats. These findings, combined with a favorable ADME profile, have prompted clinical evaluation of dapagliflozin for the treatment of type 2 diabetes.[1]

---

Objective: The inhibition of gut and renal sodium-glucose cotransporters (SGLTs) has been proposed as a novel therapeutic approach to the treatment of diabetes. We have identified dapagliflozin as a potent and selective inhibitor of the renal sodium-glucose cotransporter SGLT2 in vitro and characterized its in vitro and in vivo pharmacology. Research design and methods: Cell-based assays measuring glucose analog uptake were used to assess dapagliflozin's ability to inhibit sodium-dependent and facilitative glucose transport activity. Acute and multi-dose studies in normal and diabetic rats were performed to assess the ability of dapagliflozin to improve fed and fasting plasma glucose levels. A hyperinsulinemic-euglycemic clamp study was performed to assess the ability of dapagliflozin to improve glucose utilization after multi-dose treatment. Results: Dapagliflozin potently and selectively inhibited human SGLT2 versus human SGLT1, the major cotransporter of glucose in the gut, and did not significantly inhibit facilitative glucose transport in human adipocytes. In vivo, dapagliflozin acutely induced renal glucose excretion in normal and diabetic rats, improved glucose tolerance in normal rats, and reduced hyperglycemia in Zucker diabetic fatty (ZDF) rats after single oral doses ranging from 0.1 to 1.0 mg/kg. Once-daily dapagliflozin treatment over 2 weeks significantly lowered fasting and fed glucose levels at doses ranging from 0.1 to 1.0 mg/kg and resulted in a significant increase in glucose utilization rate accompanied by a significant reduction in glucose production. Conclusions: These data suggest that dapagliflozin has the potential to be an efficacious treatment for type 2 diabetes. [2]

---

Aims: To investigate whether glucose lowering with the selective sodium glucose transporter 2 (SGLT2) inhibitor dapagliflozin would prevent or reduce the decline of pancreatic function and disruption of normal islet morphology. Methods: Female Zucker diabetic fatty (ZDF) rats, 7-8 weeks old, were placed on high-fat diet. Dapagliflozin (1 mg/kg/day, p.o.) was administered for ∼33 days either from initiation of high-fat diet or when rats were moderately hyperglycaemic. Insulin sensitivity and pancreatic function were evaluated using a hyperglycaemic clamp in anaesthetized animals (n = 5-6); β-cell function was quantified using the disposition index (DI) to account for insulin resistance compensation. Pancreata from a matched subgroup (n = 7-8) were fixed and β-cell mass and islet morphology investigated using immunohistochemical methods.[4]

---

The prevalence of metabolic syndrome (MetS) is increasing, and patients with MetS are at an increased risk of cardiovascular disease and diabetes. There is a close link between hypomagnesemia and MetS. Administration of sodium-glucose transporter 2 (SGLT2) inhibitors has been reported to increase serum magnesium levels in patients with diabetes. We investigated the alterations in renal magnesium handling in an animal model of MetS and analyzed the effects of SGLT2 inhibitors. Adult rats were fed a fructose-rich diet to induce MetS in the first 3 months and were then treated with either dapagliflozin or magnesium sulfate-containing drinking water for another 3 months. Fructose-fed animals had increased insulin resistance, hypomagnesemia, and decreased urinary magnesium excretion. Dapagliflozin treatment improved insulin resistance by decreasing glucose and insulin levels, increased serum magnesium levels, and reduced urinary magnesium excretion. Serum vitamin D and parathyroid hormone levels were decreased in fructose-fed animals, and the levels remained low despite dapagliflozin and magnesium supplementation. In the kidney, claudin-16, TRPM6/7, and FXDY expression was increased in fructose-fed animals. Dapagliflozin increased intracellular magnesium concentration, and this effect was inhibited by TRPM6 blockade and the EGFR antagonist. We concluded that high fructose intake combined with a low-magnesium diet induced MetS and hypomagnesemia. Both dapagliflozin and magnesium sulfate supplementation improved the features of MetS and increased serum magnesium levels. Expression levels of magnesium transporters such as claudin-16, TRPM6/7, and FXYD2 were increased in fructose-fed animals and in those administered dapagliflozin and magnesium sulfate. Dapagliflozin enhances TRPM6-mediated trans-epithelial magnesium transport in renal tubule cells.[5]

C21H25CLO6

408.87

408.133

C, 61.69; H, 6.16; Cl, 8.67; O, 23.48

461432-26-8

Dapagliflozin ((2S)-1,2-propanediol, hydrate); 960404-48-2; Dapagliflozin-d5; 1204219-80-6

9887712

White solid powder

1.3±0.1 g/cm3

609.0±55.0 °C at 760 mmHg

322.1±31.5 °C

0.0±1.8 mmHg at 25°C

1.614

4.42

4

6

6

28

472

5

ClC1C([H])=C([H])C(=C([H])C=1C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])C([H])([H])[H])[C@@]1([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O1)O[H])O[H])O[H]

JVHXJTBJCFBINQ-ADAARDCZSA-N

InChI=1S/C21H25ClO6/c1-2-27-15-6-3-12(4-7-15)9-14-10-13(5-8-16(14)22)21-20(26)19(25)18(24)17(11-23)28-21/h3-8,10,17-21,23-26H,2,9,11H2,1H3/t17-,18-,19+,20-,21+/m1/s1

(2S,3R,4R,5S,6R)-2-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-6-(hydroxymethyl)oxane-3,4,5-triol

Dapagliflozin; BMS 512148; BMS512148; Dapagliflozin; 461432-26-8; BMS-512,148; Forxiga; Farxiga; BMS 512,148; (2S,3R,4R,5S,6R)-2-(4-chloro-3-(4-ethoxybenzyl)phenyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol; dapagliflozine; BMS-512148; trade name Farxiga in the US and Forxiga in the EU

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.11 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (6.11 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (5.09 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 4: ≥ 2.08 mg/mL (5.09 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

Solubility in Formulation 5: ≥ 2.08 mg/mL (5.09 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 6: ≥ 0.5 mg/mL (1.22 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 7: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30 mg/mL

---

Solubility in Formulation 8: 33.33 mg/mL (81.52 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

---

 (Please use freshly prepared in vivo formulations for optimal results.)