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Combined antidiabetic benefits of exenatide and dapagliflozin in diabetic mice†

The combined glucose-lowering effect of exenatide and dapagliflozin has not yet been studied. We investigated this combination (single-dose or 4-week dosing) in diabetic ob/ob mice. Vehicle-corrected basal glucose showed greater reduction 1 h following exenatide + dapagliflozin than with exenatide or dapagliflozin alone, and stayed significantly lower for all groups versus vehicle over 3 h. During an oral glucose tolerance test, glucose excursion (30 min post-dose) was significantly lower for exenatide + dapagliflozin versus exenatide or dapagliflozin, or vehicle. Exenatide + dapagliflozin and exenatide, but not dapagliflozin alone, reduced glucose excretion over 24 h versus vehicle. After dosing for 4 weeks, exenatide, dapagliflozin and exenatide + dapagliflozin similarly decreased haemoglobin A1c (HbA1c). Body weight was reduced only with exenatide or exenatide + dapagliflozin. The glomerular filtration rate was similar with exenatide, dapagliflozin and vehicle, and increased with exenatide + dapagliflozin. Optimized combinatorial dosing of these antidiabetic agents may provide additive glucose lowering in type 2 diabetes mellitus.


Introduction
Exenatide is an incretin-based treatment for type 2 diabetes mellitus (T2DM). Exenatide maintains glucose homeostasis through glucagon-like peptide-1 (GLP-1) receptor-mediated actions, including glucose-dependent insulinotropism, reduction of food intake and body weight, suppression of glucagon secretion and slowing of gastric emptying [1]. The majority of T2DM patients receiving exenatide [2] achieve recommended HbA1c levels <7% [3], however, relatively few reach values observed in non-diabetic adults (≤5.6%) [4]. Dapagliflozin belongs to a novel class of antidiabetic therapies that regulate glucose levels through inhibition of renal glucose reabsorption, and increase urinary glucose excretion via selective inhibition of the renal sodium glucose cotransporter-2 (SGLT-2) [5,6]. This study tested whether combining dapagliflozin and exenatide improves glucose homeostasis via additive or synergistic effects on glycaemic control, using diabetic, obese ob/ob mice.
(Jackson Laboratories, Bar Harbor, ME, USA; body weight 43 ± 1 g) were used. Animals were housed individually with a 12-h light/dark cycle and ad libitum access to food/water. Mice subjected to a basal glucose-lowering test or oral glucose tolerance test (OGTT) were fasted for 4 or 15 h, respectively. Urinary glucose excretion was assessed using individual metabolic cages.

Compounds
Exenatide was reconstituted in 10% dimethyl sulfoxide (DMSO) or 30 mM sodium acetate for acute and sub-chronic studies, respectively. Dapagliflozin (AKAAL Organics, Long Beach, CA, USA) was reconstituted in water. Doses for each compound were sub-maximal, allowing for detection of potential additive or synergistic pharmacological effects.

Acute Studies
Single doses of exenatide, dapagliflozin, exenatide + dapagliflozin or vehicle were evaluated for effects on basal glucose lowering, OGTT, or 24-h urinary glucose excretion. Compounds were administered at t = 0 min for basal glucose lowering, and 15 min before oral gavage with 2 g/kg dextrose for OGTT. Urinary glucose excretion was measured to assess dapagliflozininduced changes alone, or combined with exenatide.

Sub-Chronic Studies
Four-week effects on glucose metabolism with exenatide (0.03 mg/kg/day), dapagliflozin (1 mg/kg/day), exenatide + dapagliflozin or combined vehicles were assessed in ob/ob mice. Exenatide was continuously delivered via subcutaneous osmotic minipump. Dapagliflozin was administered once daily via oral gavage.
HbA1c and plasma dapagliflozin levels were measured at weeks 2 and 4. Immediately after final blood sampling at week 4, mice were placed in metabolic cages for 17 h for assessment of glomerular filtration rate [GFR = (urine creatinine × urine volume)/(plasma creatinine × urine time collection)].

Statistical Analyses
Results are presented as mean ± SEM, analysed using Prism 5 ® (GraphPad Software, Inc., La Jolla, CA, USA). Single-point data were analysed using one-way analysis of variance, with Tukey's multiple comparison post hoc test. Significant between-group differences were assumed for p < 0.05.

Acute Studies
Ob/ob mice were severely diabetic: 4 h fasting blood glucose was 34.6 ± 2.2 mmol/l. At 60 min post-dose, exenatide + dapagliflozin produced significantly greater reductions in glucose (−27.9 ± 2.3 mmol/l, p < 0.05) than exenatide (−17.7 ± 0.8 mmol/l) or dapagliflozin (−12.2 ± 4.5 mmol/l; Figure 1A) alone. Blood glucose plateaued in the combination group after 1 h; the exenatide and dapagliflozin groups had similar profiles with maximum decreases at 3 h. Exenatide + dapagliflozin significantly suppressed glucose excursion during a 2-h OGTT and was more effective than either agent alone ( Figure 1B). Blood glucose was significantly lower with exenatide versus dapagliflozin (29.1 ± 1.8 vs. Dapagliflozin had no significant effect on 24-h total excreted urinary glucose or total urine volume, while exenatide significantly reduced total urine glucose and volume versus vehicle ( Figure 1C, D). Exenatide + dapagliflozin also lowered total urine glucose and volume versus vehicle or dapagliflozin alone.

Sub-Chronic Studies
In vehicle-treated mice, HbA1c increased from 7.34 ± 0.01% to 9.09 ± 0.40% during the 4-week study. Administration of exenatide, dapagliflozin or the combination significantly reduced HbA1c versus vehicle at study end ( Figure 2A); all treatment groups had similar declines, with the combination showing a trend towards being most effective. Exenatide + dapagliflozin and exenatide alone decreased body weight ( Figure 2B). Dapagliflozin alone increased food intake ( Figure 2C).
Exenatide + dapagliflozin significantly increased GFR ( Figure 2D) and decreased plasma creatinine ( Figure 2E), without significant effects on urine volume ( Figure 2F) or total urine creatinine. Exenatide or dapagliflozin alone did not modify GFR, urine volume or urine and plasma creatinine levels after 4-week treatment.

Discussion
This study in diabetic mice reports the novel observation that GLP-1 receptor agonism with exenatide plus SGLT-2 inhibition with dapagliflozin has the potential for additive effects in T2DM. Assessment of basal glucose lowering and oral glucose tolerance showed that exenatide + dapagliflozin controlled blood glucose significantly better than each monotherapy. Longer-term combination therapy significantly reduced HbA1c versus vehicle, with a trend towards greater efficacy than either agent alone.
The apparent additive glucose-lowering effect seen with exenatide + dapagliflozin in the acute studies did not significantly translate to additive HbA1c lowering or weight loss in the sub-chronic study. Several factors may contribute to this discrepancy. First, HbA1c did not reach a plateau in any group by study end; therefore, the sub-chronic study could have been too short to provide definitive results. Second, the chronic dosing regimen for dapagliflozin might not have been optimal. Since ob/ob mice are hyperphagic, chronic administration of dapagliflozin in food could provide improved exposure/effectiveness. Detailed, multiple dose-interaction studies may be required (e.g. response surface methodology) to elucidate optimal long-term dose combinations that may reveal potential additivity or synergy.
In this study, dapagliflozin acutely lowered blood glucose but did not increase 24-h urinary glucose excretion. Diabetic ob/ob mice are already polyuric/glucosuric; therefore, further enhancing glucosuria with dapagliflozin may not have been feasible. Dapagliflozin-induced increases in excreted glucose might have been measurable after overnight fasting or earlier than 24 h post-dose. Previous data in Zucker diabetic fatty (ZDF) rats support this, as dapagliflozin-induced effects on urine glucose were more pronounced at 6 versus 24 h [7]. Han et al. reported that oral dapagliflozin produced a 400-fold increase in urine glucose in normal Sprague-Dawley rats 24 h post-dose, whereas urine glucose was increased only twofold in diabetic ZDF rats [7]. Elvert et al. reported glucose excretion in ZDF rats to be dramatically higher than in lean animals, and the SGLT-2 inhibitor AVE2268 dose-dependently lowered blood glucose, with no effect on glucose excretion [8].
Type 2 diabetes is often associated with chronic kidney disease resulting in gradual renal impairment, which ultimately leads to renal failure. Renal function may affect exenatide pharmacokinetics, due to renal clearance [9], and influence dapagliflozin exposure, metabolism and efficacy [10]. Diabetic ob/ob mice have dysfunctional kidneys and it has been reported that exenatide exerts renoprotective effects in rodent models [11]; hence, we evaluated renal function at the end of the subchronic study. Exenatide + dapagliflozin improved GFR and reduced plasma creatinine, indicating combinatorial benefits on kidney function.
In conclusion, exenatide and dapagliflozin monotherapies significantly improved glycaemia in ob/ob mice. Exenatide + dapagliflozin regulated basal and postprandial glucose better than either monotherapy, suggesting additive effects. The additive glucoregulatory effects of longer-term combination therapy were less pronounced in this mouse model; however, renal function significantly improved. Thus, combining GLP-1 receptor agonism and SGLT-2 inhibition may be a promising approach in diabetes. Further experimentation to establish optimal doses of exenatide + dapagliflozin to maximize long-term benefits is warranted.