This single center study was conducted at Profil Institute for Clinical Research (Chula Vista, CA, USA) and was conducted in accordance with Good Clinical Practice and the principles of the Declaration of Helsinki. The study protocol and subject information were reviewed and approved by Biomedical Research Institute of America investigational reviewer board (San Diego, CA, USA) and all subjects provided written, informed consent prior to start of study-related procedures.
Ten healthy male and female subjects followed by a separate group of 6 subjects with T2DM were enrolled in this study. Enrollment of women was restricted to those who were postmenopausal or surgically sterile. All subjects gave written informed consent prior to participation in any study-related procedures. Healthy subjects were required to be 18–55 years of age, and have a body mass index (BMI) of 19.0 to 30.0 kg/m2 inclusive. Subjects with T2DM were required to be 30–60 years of age, have a BMI of 22–35 kg/m2, to be healthy other than having been diagnosed with T2DM at least 6 months prior to entry in the study, and to have been maintained on a stable treatment regimen for at least 3 months. Diabetic subjects were required to have hemoglobin A1c (HbA1c) ≤10% and fasting plasma glucose <280 mg/dL at screening. Participants with diabetes were required to be on a stable treatment regimen using a single oral antidiabetic agent (either sulfonylureas, rosiglitazone, metformin or acarbose) or management by diet and exercise. All T2DM subjects also had to be willing and medically able to discontinue their diabetes medications for up to 72 h during each treatment period. Subjects were excluded if they had been taking diuretics, corticosteroids or other medications that might result in electrolyte depletion; had required insulin during the last 3 months; had significant renal disease; or if their participation would have resulted in donation of blood in excess of 550 mL within an 8-week period.
This double-blind, randomized, placebo-controlled, single escalating-dose crossover study was conducted in two parts: Part A consisted of a randomized, dose-escalation in healthy subjects; Part B was of similar design, but conducted in subjects with T2DM and included an evaluation of pharmacodynamics using a 50 g oral glucose tolerance test (OGTT).
Ten healthy subjects were evaluated in 5 study sessions, each separated by approximately 2 weeks. At each study session, subjects received either an oral dose of remogliflozin etabonate or placebo after an overnight fast. Remogliflozin etabonate doses were 20 mg, 50 mg, 150 mg, 500 mg and 1000 mg. Over the course of participation in the study, each subject received 4 of the 5 active remogliflozin etabonate doses and 1 dose of placebo (4:1 active to placebo ratio per treatment period). The available safety and PK results from each dosing period were evaluated before proceeding to the next dose level.
Six subjects with T2DM received two doses of remogliflozin etabonate and a placebo dose, in a randomized, dose escalating, crossover design, along with an oral glucose load on three study sessions separated by 7–14 days. Full PK and safety profiles were measured on Day 1 of each dosing period. The doses selected for this portion of the study, 50 mg and 500 mg, were based on data obtained in Part A. In each dosing period, subjects were assigned to active vs placebo in a 2:1 ratio.
All subjects were admitted to the unit two nights prior to receiving study drug to establish baseline safety parameters and fluid intake levels over a 36 h period. Subjects remained in the unit for at least 24 h after doses were administered for monitoring of clinical laboratory parameters exploratory biomarkers, vital signs, ECGs and adverse events. While confined to the clinical research unit, all subjects received meals standardized with respect to calories, fat, protein, carbohydrate, and sodium content; however, detailed dietary information was not captured in this study. In Part A, subjects were dosed following an overnight fast; lunch and dinner were provided at 4 and 10 h after dosing, respectively. Fifteen minutes after dosing in each treatment period in Part B, a fasting OGTT was performed using 50 g glucose (administered as 50 g Glucola™). A 50 g glucose load was chosen since the OGTT was being performed in subjects already known to have diabetes. The glucose drink was consumed by subjects within approximately 5 minutes. Blood samples for the measurement of glucose, insulin, and intact glucagon-like peptide 1 (GLP-1) were collected for 24 hours following dose administration. Provided that there were no safety or tolerability concerns, subjects were released from the clinic on day 2 of each treatment period until their return for the next treatment or follow-up period. Each subject was involved in the study for approximately 8 weeks (from screening to follow-up).
Blood collections and analysis
On each dosing day, a series of 2.0 mL blood samples were collected at pre-dose and 10, 20, 30 and 45 min, and 1, 1.25, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, and 24 h post-dose for the determination of remogliflozin etabonate, remogliflozin and GSK279782 in plasma by using high-performance liquid chromatography with tandem mass spectrometry (MS/MS) as described .
Non-compartmental PK analysis of plasma concentration–time data was performed using WinNonlin Version 4.1 (Pharsight Corporation, Mountainview, CA, USA). The Cmax and Tmax were obtained directly from the data. Areas under the plasma concentration–time curves from time zero to the last quantifiable time point (AUC[0–last]) and extrapolated to infinity (AUC[0–∞]) were calculated using the log-linear trapezoidal method. The terminal plasma elimination rate-constant (λz) was estimated from log-linear regression analysis of the terminal phase of the plasma concentration–time curve, and the T½ was calculated as T½ = ln2/λz. Ratios of AUC(0–∞) remogliflozin to AUC(0–last) remogliflozin etabonate were calculated including molecular weight corrections.
Blood samples for glucose were taken at 0 (pre-dose), and 1, 2, 4, 8 and 12 h after dosing in each treatment period (Part A). For Part B, blood samples for glucose, insulin and GLP-1 were taken at check-in on day -2, and at 0 h (pre-dose), and 0.5, 1, 1.5, 2, 4 h (prior to lunch), 4.5, 5, 6, 8, 10, 12 and 24 h after dosing on day 1 of each treatment period.
Glucose and insulin sample handling
For glucose, plasma was analyzed using a YSI 2300 Glucose Analyzer (Yellow Springs International Life Sciences, Yellow Springs, OH, USA). For insulin, plasma was rapidly prepared and frozen at -70°C until analyzed by LabCorp (San Diego, CA, USA) using a chemiluminescent immunometric assay method (Siemen’s Immulite 2000 analyzer with Immulite Insulin Kit L2KIN2).
GLP-1 sample handling
For assay of intact GLP-1, blood was collected into a chilled EDTA tube and protease inhibitors (DPP4 inhibitor obtained from EMD Millipore, St. Charles, MO) were immediately added. Samples were then spun down and plasma split into two separate tubes and frozen at -70°C until analyzed by Pathway Diagnostics (Malibu, CA, USA) by ELISA (kit # EGLP-35 K, EMD Millipore). The lowest level of intact GLP-1 this assay can detect is 2 pM (with a minimum plasma sample size of 0.4 mL).
Urine samples were collected at pre-dose, and over a series of intervals (0–2, 2–4, 4–6, 6–8, 8–12 and 12–24 h post-dose) for the analysis of creatinine, glucose and electrolytes (Na, K and Cl). All fluid intake was recorded, as well as urine volume, over the 24 hours before and after dosing in each treatment period. Urine was tested for protein on the first morning void of day -1 and 24 h after dosing.
Creatinine clearance (CLCR) was calculated as the amount of creatinine excreted in 24 h (Ae0–24 h) divided by the mean of the pre-dose and 24-h post-dose plasma creatinine levels. The percentage of filtered glucose excreted in the urine for each individual time period was calculated as the amount of glucose excreted during that time period divided by (CLCR × PG × time interval length), where CLCR is the creatinine clearance for the time interval, PG is the plasma glucose concentration closest to the midpoint of the time interval, and the time interval is the period (min) of urine collection (CLCR × PG represents the glucose filtered load). For the 24-h period, the percentage of filtered glucose excreted was calculated as the amount of glucose excreted over 24 h divided by the sum over the individual time intervals of (CLCR × PG × time interval length).
Safety and pharmacodynamic data were summarized using descriptive statistics. This was a small exploratory study and no formal hypothesis-testing was conducted. Dose proportionality with respect to Cmax, and AUC was assessed using the power model y = α doseβ, where y = Cmax or AUC, and α denotes a random subject effect. The exponent β in the power model will be estimated by regressing the loge-transformed PK parameters on loge dose, i.e. ln(PK parameter) = ln(α) + β * ln(dose). Dose proportionality implied that β = 1 and was assessed by estimating β and its corresponding 90% confidence interval. The power model was fitted by restricted maximum likelihood using SAS Proc Mixed.