Empagliflozin

This study conformed to the National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. It was approved by the Experimental Animal Administration Committee of Tianjin Medical University and Tianjin Municipal Commission for Experimental Animal Control.
HFD and low-dose STZ treatment were used to induce type II diabetes mellitus in rats [8 (link)]. The HFD + STZ model, which similar demonstrates a progression from insulin resistance to hypoinsulinaemia and hyperglycemia, mimics the natural T2DM pathogenesis in humans and is suitable to investigate the pathogenesis of diabetic complications and test the efficiency of anti-diabetic agents. A total of 96 adult male Sprague–Dawley (SD) rats (200 ± 20 g) were purchased from the HuaFuKang Bioscience Co., LTD (Beijing, China). They were kept under a 12 h light/dark cycle at room temperature (20–22 °C) and humidity (50–60%). After 1 week, the rats were divided into two groups: HFD group (n = 72) and control group (n = 24). The rats of the HFD group were fed high-fat chow (H10060, fat energy ratio = 60 kcal%, protein energy ratio = 20 kcal%, carbohydrate energy ratio = 20 kcal%, the Beijing HuaFuKang Bioscience Co., LTD, China) for 4 weeks, then given a single tail vein injection of STZ (30 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) dissolved in citrate buffer at pH 4.5. The rats of control group were fed regular chow and were injected with the same dose of citrate buffer. One week following the STZ injection, blood samples were collected from the tail vein to measure the blood glucose level. The blood glucose level of control group rats was kept within the normal range. HFD group rat with random blood glucose > 16.7 mmol/L was considered successful induction of DM, and was used for further investigation [9 (link)]. The same dose of STZ was injected again in rats with blood glucose level that did not meet the diagnostic criteria. The remaining rats that failed to meet the diagnostic criteria after the injection were excluded from the study. This process was repeated until a sufficient number of DM animals were produced. Blood glucose concentration of the DM models was monitored weekly using the glucometer Optium Xceed (Abbott Laboratories MediSense Products).
The rats were then divided into four groups: control group (CON, n = 24); DM group (DM, 0.5% hydroxyethylcellulose/day, intragastric administration, ig, n = 24); low dose of EMPA (low-EMPA, 10 mg/kg/day, ig, n = 24); and high dose of EMPA (high-EMPA, 30 mg/kg/day, ig, n = 24). The dose of empagliflozin was based on the previous studies [10 (link), 11 (link)]. Empagliflozin was supplied by Boehringer Ingelheim Pharma GmbH & Co. (KG, Germany). Besides from the control group, the remaining three groups were composed of the DM models. The rats were treated for 8 weeks. All rats were anesthetized with sodium pentobarbital by intraperitoneal injection and sacrificed following weeks of treatment. The first 8 rats of each group were used for the first part of the experiments (including echocardiographic, hemodynamic, histological, and serum biochemical and oxidative stress-related markers examination, and western blot analysis). The next eight rats were used for the electrophysiological studies, and the remaining eight rats were used for examinations of mitochondrial function.

A subset of mice from each HFD group was subsequently placed on empagliflozin as described in Section 3. Mice were divided and housed in cages (2–3 cages per group) without a standardised randomisation procedure. Empagliflozin (Advanced ChemBlocks, Hayward, CA, USA) was incorporated into the HFD rodent chow at a concentration of 116.7 mg empagliflozin per 1 kg chow. This incorporation was accomplished by first pulverising the rodent chow with a mortar and pestle and spreading it over a large glass plate. The chow was then evenly sprayed with an empagliflozin solution that consisted of empagliflozin dissolved in 50% ethanol (11.67 mg/mL). The food was allowed to dry on the bench top for 30 min for ethanol evaporation and was then mixed with a 7% starch solution (tapioca starch in water; 2 mL solution per 100 g of chow) to reform the pellets. The control HFD chows used throughout this study were subjected to the same procedure, but empagliflozin was omitted from the 50% ethanol solution. The added starch (1.4 mg/g of chow) did not significantly change the caloric content (kcal/g) or the carbohydrate percentage of either HFD.
Mice were allowed to feed ad libitum. Each mouse consumed approximately 2.5–3 g of food per day delivering an approximate empagliflozin dose of 0.3–0.35 mg/day.

The drug was absorbed by the physio-sorption batch adsorption method. Empagliflozin was dissolved in absolute alcohol to make an exact starting concentration of 1.00 mg per ml which is considered as the stock solution. The concentration of the drug which indicates the maximum elimination percentage was then determined by doing serial dilutions of empagliflozin with various concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg per ml) by using the stock solution. The whole procedure was conducted by using the solution taken in an Erlenmeyer conical flask of 100 ml at room temperature (25 °C). Then the final solution was stirred for 5 h at a fixed stirring rate of 600 RPM using 100 mg of ZnO NPs. The solution was then filtered using a vacuum pump and 0.45 m nylon filter paper after being centrifuged at 10,000 rpm. The previously described HPLC technique was used to assess the concentration of unbound empagliflozin as illustrated in Fig. 2^{31 (link)}.
To evaluate the resilience of the technique repeatability following the standards for quality control and method validation, the adsorption performance for each experiment was repeated three times (as standard without ZnO NPs and evaluated with ZnO NPs. Three experimental replicates were used to get the data, which were then averaged. The drug removal percentage denoted by P (%), is the amount that was loaded and regarded as the “adsorption capacity” at equilibrium, denoted by the qe (mg/g), and was calculated using the following formulas. $$\text{Percentage of drug}\text{in mg/l conc}.=\frac{\text{Areaof drug after loading}}{\text{Conc of drugs without loading}}\times \frac{\text{Area of drug without loading}}{\text{Conc of drug after loading}}\times \text{Percentage of drugs after loading},$$ $$\text{Drug Removed}=100-\text{Percentage of Drug adsorbed}\%,$$ $$\text{Amount of drug}\text{adsorbed}\left(\text{qe}\right)\text{mg/g}=\left(\text{Initial concentrations of drug}-\text{Equilibrium concentration of drug}\right)\times \text{The volume of the drug/Mass of ZnO NPs}=\text{Amount of Drug}.$$

Following the completion of all evaluations, participants will be randomly assigned to three groups. Each group will receive either semaglutide plus placebo, empagliflozin plus placebo or semaglutide plus empagliflozin. The subcutaneous semaglutide injection will be initiated at a dosage of 0.25 mg once weekly for the first 4 weeks and the dose will be doubled every 4 weeks until 1 mg is reached (days 1–28: 0.25 mg semaglutide, days 29–56: 0.5 mg semaglutide and days 57–364: 1.0 mg semaglutide). Patients who cannot tolerate uptitration to 1.0 mg semaglutide will continue with 0.5 mg once weekly. Patients who cannot tolerate 0.5 mg/0.25 mg semaglutide plus placebo/empagliflozin plus placebo/semaglutide plus empagliflozin will be excluded from the study. Patients will receive instruction on the subcutaneous injection technique, which can be self-administered at home. Patients who cannot self-inject will be assisted by a nurse will. Patients will receive 10 mg of empagliflozin daily throughout the study in the empagliflozin plus placebo or semaglutide plus empagliflozin group. Patients will be required to report any development of adverse events (AEs) during the entire treatment period to their provider. Besides taking study medications, patients will be advised on the management of various coexisting illnesses during the trial. Before the commencement of the trial, all participants will receive nutritional and exercise counseling from an experienced dietician. In between follow-up interviews, participants will receive a reminder of scheduled study visits via WeChat.