Study design
This was a randomized, double-blinded, repeat dose parallel group study. Subjects currently taking metformin for the treatment of T2DM and fulfilling the inclusion criteria were recruited. The study protocol was approved by the institutional review board (IRB) and the study was conducted in accordance with the major ethical principles specified in the Declaration of Helsinki and good clinical practice (GCP) guidelines. Written informed consent was obtained from each subject before any study related procedure was performed. This was a multicenter study conducted at a total of four centers: two centers in the USA, one center in Germany, and one center in Argentina. The study protocol, any amendments, the informed consent, and other information were reviewed and approved by relevant ethics committee or review board at each study center.
The study was comprised of three phases; run-in, randomization, and treatment. In the run-in phase, subjects who were taking < 2000 mg of metformin were titrated up over a 14-day period to a minimum daily dose of 2000 mg immediate release metformin. If subjects were taking metformin once daily (QD) or three times daily (TID), then they were titrated to BID for the duration of the study. Following the run-in phase, treatment phase of 13 days included in-house assessment in clinical research unit from day − 2 to day 4 and day 12 to day 14 and outpatient basis assessment from Day 5 to Day 11 with follow-up visit from Day 21–24. In treatment phase the patients were considered in two cohorts. Cohort 1 subject were randomly allocated to receive either RE 500 mg BID or placebo BID (2:1) in addition to metformin. Cohort 2 subjects received either RE 750 mg BID or placebo BID (2:1) in addition to metformin. Cohort 2 dosing commenced after at least 18 subjects in Cohort 1 had completed dosing and preliminary safety information had been reviewed (Fig. 1). The decision to begin Cohort 2 was based upon blinded review of AE reports, vital signs, ECG findings and clinical laboratory results by the site physicians and medical monitor.
Treatment assignment
Treatment assignments were in accordance with the randomization schedule stratified by cohort with fixed block size. Upon successful completion of the metformin titration period, each subject is randomly assigned to one of the two cohorts with either one of the two RE dose regimens or placebo in a ratio of 2:1. A central randomization was applicable for all sites, and performed using a GSK proprietary IWRS randomization system.
Sample size
The sample size planned for this study arose primarily from practical feasibility. There was no formal calculation of power or sample size. However, considering mean difference of plasma lactate level between treatment and placebo as one of evaluable end-points, a sample size estimation using the plasma lactate levels had been determined. A sample size of 12 subjects or 16 subjects with evaluable data completed for each active arm and placebo arm was estimated. This was based on larger inter-subject standard deviation of lactate level of 0.45 mmol/L in metformin alone group observed in previous report of 3-day metformin-RE drug-drug interaction study [15].
Inclusion and exclusion criteria
Men and women (with adequate birth control, if of childbearing potential) aged 30–64 years (both inclusive) having a body mass index (BMI) of 22–40 kg/m2 with a documented T2DM diagnosis (diagnosed not less than 3 months; HbA1c range from 7 to< 10% and fasting plasma glucose (FPG) < 280 mg/dL at screening, fasting C-peptide lower limit of the reference range for an approved local laboratory assay) who were not adequately controlled by monotherapy with metformin were included in the study. Only those subjects were included who were willing and medically able to titrate their metformin dose to at least 2000 mg/day for 2 weeks, if on a lesser dose and have FPG > 126 mg/dL near the end of the run-in period.
Subjects with a prior history of autoimmune type 1 diabetes mellitus, diabetic ketoacidosis or lactic acidosis, gastrointestinal, hepatic or renal diseases were excluded from the study. Subjects requiring insulin therapy and any other anti-diabetic drug were also excluded from the study.
Study objectives
The primary objective of the study was to access the safety and tolerability of RE (500 mg and 750 mg twice daily) along with metformin at doses 2000 mg daily in subjects with T2DM in terms of incident adverse events including hypoglycemia, plasma lactic acid levels and lactate levels and clinically relevant changes in vital signs and laboratory assessments.
The secondary objectives included the assessment of the effect of RE on steady state plasma concentrations of metformin, pharmacokinetics of RE and key metabolites while on metformin treatment and the response to an oral glucose tolerance test (OGTT) when RE was added to metformin versus metformin alone. The study assessment therefore included safety assessments (adverse events, hypoglycemia, plasma lactic acid level and lactic acidosis events, vital signs, ambulatory blood pressure monitoring, ECG, clinical laboratory values, fluid balance and body weight) as well as pharmacokinetic sampling, and plasma glucose, plasma insulin levels after OGTT.
Adverse event monitoring
The adverse events monitoring included recording of any adverse event (AE) and serious adverse event (SAE) reported or observed during the entire study and follow-up period. All the subjects were counseled to inform study staff of any AEs that occurred during the study. Any abnormal laboratory findings (e.g., clinical chemistry, hematology, and urinalysis) or other abnormal assessments (e.g., ECGs, vital signs, physical examination) that were judged by the investigator as clinically significant were recorded as AEs or SAE.
Clinical laboratory evaluations
For all measures, blood sample was obtained prior to having breakfast and were collected on baseline (Day-1), mornings (prior to first dose and breakfast) of Day 1, 2, 3, and 4 (inpatient) and Days 6, 8, and 10 (outpatient) and Days 13 and 14 (inpatient). Free fatty acids (FFAs) were also measured on Day-1 and 13. Samples were kept at 4 °C and were analyzed immediately.
Surveillance for lactic acidosis
The samples for acidosis analysis were collected at screening (following an overnight fast and prior to morning dose), once during the run-in phase, at baseline (Day- 1), mornings (prior to first dose and breakfast) of Days 1, 2, 3, and 4 (inpatient) and Days 6, 8, and 10 (outpatient) and Days 13 and 14 (inpatient). Subjects were not allowed to do rigorous exercise or physical labor during the treatment phase of the study and instructed to limit exercise 12 h before sampling.
Electrocardiogram
A 12-lead ECG was performed at the Screening Visit, Day- 1, pre-dose Days 4, 6, 8, 10, and 13; and at the follow-up Visit.
Fluid balance assessments
The amount of all liquids consumed and urine volume were collected beginning the morning of Day 1 (prior to metformin dose) and the morning of Day 13 (prior to metformin and RE dosing), and continued until the subject was discharged from the clinical research facility after completing the inpatient portions of the study.
Body weight
Body weight measurement was performed at screening and during inpatient Days- 1, 2, 4, 8, 13, and 14.
Ambulatory blood pressure monitoring
The ambulatory blood pressure was monitored for 24 during inpatient days (beginning Day 1 at 08.00, Day 2 at 08.00 and Day 13 at 08.00). The unit was programmed to take blood pressure readings every 10 min from 08.00 to 10.00, every 20 min from 10.00 to 22.00, and every 30 min from 22.00 to 08.00 the following morning.
Pharmacokinetic analysis
Serial blood samples (2 ml) were collected on Day 1 and Day 13 at different time-points for the determination of remogliflozin etabonate, remogliflozin (GSK189074), GSK279782 and metformin concentrations in plasma; pre-dose (within 10 min prior to next dose), 15, 30, 45 min and 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10 and 12 h (before second dose of the day). PK analysis was conducted by INDAPharma, LLC (Chapel Hill, NC, USA), under the direction of Clinical Pharmacokinetics/Modelling and Simulation, GSK using the non-compartmental analysis Model 200 (for extra vascular administration) of WinNonlin Professional Edition version 4.1 (Pharsight Corporation, Mountain View, CA). The drug assay techniques utilized for each of the drug and metabolites has been reported prior [14].
Pharmacodynamic analysis
Oral glucose tolerance testing was performed on Day 1 and Day 13 using a 75 g oral glucose solution. Blood samples for the measurement of insulin and glucose were taken immediately before drug dosing (within 5 min of drug dosing), and at 30 min, 1, 1.5, 2, 3, 4, and 6 h following drug dosing.
Statistical analysis
Descriptive statistics were used to summarize baseline demographics, safety data, and pharmacokinetic parameters. Geometric mean and % CVb (between-subject coefficient of variation) were calculated for all pharmacokinetic parameters except tmax. Comparisons of placebo with each active dose of RE were assessed in terms of change from baseline in fasting plasma lactate acid using repeated measures analysis of covariance model. The model included terms for baseline measurement, visit, treatment and visit-by-treatment interaction. The same model was applied to change from baseline in weighted means of day-time, night-time and 0–24 h of blood pressures. Within group treatment comparison was performed using mixed model on log transformed Cmax and AUC (0-last).