Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Multiple Rising Doses of Empagliflozin in Patients with Type 2 Diabetes Mellitus

This was a multiple rising dose, randomized, double-blind trial with within-group placebo control. Patients were randomized upon admission to the trial center to one of four doses of empagliflozin (2.5, 10, 25, and 100 mg), which were tested sequentially in ascending order of dose. Within every dose group, patients were randomized 3:1 to receive active drug or placebo. Treatment allocation was carried out according to a randomized list of patient and medication numbers. Within each dose group, patients were randomized to receive placebo or active drug and patients and investigators were double blinded until the study had completed. Patients and investigators were aware of the dosing stage (but not if patients were receiving placebo).

Patients received a single dose on day 1 and once-daily dosing on days 3–9. The dosing on day 2 was skipped to allow estimation of the terminal elimination half-life after a single dose to compare with the steady state half-life. Study drug was administered at the same time every day with 240 mL of water. Patients were fasted overnight for 10 h on days −2, −1, 1, 8, and 9. An end-of-study examination was carried out in the 1-week post-treatment period (days 15–21). An oral glucose tolerance test (OGTT; administration of 75 g glucose solution after overnight fasting) was performed on days −1, 1, and 9.

The trial was conducted at the Profil Institute for Metabolic Research, Neuss, Germany. Eligible subjects were patients with T2DM treated with diet and exercise only or ≤2 oral anti-diabetic agents with at least one agent taken at ≤50% of its maximum dose; HbA_1c ≤8.5% at screening; and body mass index (BMI) 18.5–40 kg/m². Patients on insulin or thiazolidinediones were excluded, as were patients with high fasting [>240 mg/dL (13.3 mmol/L)] or postprandial [>400 mg/dL (>22 mmol/L)] blood glucose and patients with clinically significant concomitant diseases or abnormalities in the screening laboratory.

The primary endpoint was safety and tolerability. Safety parameters, including adverse events (AEs), electrocardiogram (ECG), vital signs, physical examination, and laboratory parameters in blood and urine samples, were measured throughout the study. Meals and fluid intake were standardized during the in-house period of the study (from day −2 to day 9).

Secondary endpoints were pharmacokinetic and pharmacodynamic parameters. Pharmacokinetic endpoints included mean plasma concentration time profiles of empagliflozin, maximum concentration of analyte in plasma (C _max), time to reach peak levels (t _max), terminal elimination half-life (t _1/2), apparent terminal rate constant (λz), area under the plasma concentration–time curve (AUC), renal clearance (CL_R) and the fraction of the dose that was excreted unchanged in urine (fe). Pharmacodynamic measurements included UGE, 8-point weighted mean daily glucose (MDG), and fasting plasma glucose (FPG). Samples for the determination of analyte plasma concentrations were drawn before drug administration on days −2, −1, 1, 2, 3, and 5–9, at time points up to 48 h after dosing on day 1, and up to 72 h after dosing on day 9. On days −2, −1, 1, 8, and 9 all urine voided over a period of 24 h (48 h for day 1 and 72 h for day 9) was collected for UGE measurements. Urine samples collected on days 1 and 9 were also used for pharmacokinetic measurements.

Empagliflozin concentrations in plasma and urine were analyzed using a validated high performance liquid chromatography, tandem mass spectrometry (HPLC–MS/MS) assay with a lower limit of quantification of 1.11 nmol/L (0.5 ng/mL) for plasma, or 4.44 nmol/L (2 ng/mL) for urine. Pharmacokinetic parameters were calculated using WinNonlin™ software (v5.2, Pharsight Corporation, Mountain View, CA, USA). Mean plasma concentration–time profiles of empagliflozin were calculated only if at least 2/3 of the individual patient concentrations at each time point were above the limit of quantification (1.1 nM empagliflozin). C _max and t _max values were directly determined from the plasma concentration–time profiles of each subject. The t _1/2 was calculated as the quotient of the natural log (ln)(2) and λz. The λz was estimated from a regression of ln(C) versus time over the terminal log-linear drug disposition portion of the concentration–time profiles. AUC to the last quantifiable time point was calculated using the linear trapezoidal method for ascending concentrations and the log trapezoidal method for descending concentrations. CL_R was determined as the quotient of the amount of drug excreted unchanged in urine over AUC. The fe was determined by the quotient of the sum of drug excreted over all dosing intervals and the dose administered.

Mean cumulative UGE over 24 h (Ae_0–24) was measured after single doses of empagliflozin with OGTT (day 1) and after multiple doses after fasting (day 8) or with OGTT (day 9). Weighted MDG was estimated by dividing the area under the 24-h glucose curve (AUEC_0–24) by 24 h. Inhibition of glucose reabsorption was calculated by comparing renal tubular glucose reabsorption rate (TG) on day 8 with day −2 as baseline. TG was calculated as the difference between filtered load (FL) and glucose excretion rate (ER). The FL was calculated as the product of estimated glomerular filtration rate (eGFR) and plasma MDG. ER was estimated by dividing the cumulative amount of glucose excreted in urine (UGE) over a dosing interval by 24 h. MDG measurements were performed during the OGTT on days −1, 1, and 9, and under ‘normal’ trial conditions (standardized meals without OGTT) on days −2 and 8. The impact of empagliflozin on postprandial glucose (PPG) levels was analyzed from the area under the glucose concentration–time curve AUEC 1–5 h (AUEC_1–5) after OGTT.

Safety variables, pharmacokinetic and pharmacodynamic parameters were evaluated by descriptive statistical methods. Safety and pharmacodynamic analyses were based on the randomized set, which was identical to the treated set and included all 48 patients treated with active drug or placebo. Pharmacokinetic analyses were based on the pharmacokinetic set, which included all 36 patients treated with active drug. Attainment of steady state using trough concentrations between days 5 and 9 was analyzed using a repeated measures linear model on the logarithmic scale including ‘patient’ as a random effect and ‘time’ as a repeated effect. Dose proportionality was explored using a regression model applied to log-transformed data. UGE, weighted MDG, and FPG were analyzed daily for change from baseline using an analysis of covariance (ANCOVA) model. The effect ‘patient’ was considered random while the effect ‘treatment’ was considered fixed. The baseline value was included as a continuous covariate. Statistical analysis utilized SAS^® statistics software version 8.2 (SAS Institute Inc. Cary, NC, USA).

Seventy-four patients were screened and 48 (39 white males, 8 white females, 1 black female) were randomized (12 to each dose group, with 9 patients randomized to receive empagliflozin treatment and 3 to placebo). All randomized patients received at least 1 dose of study drug; 46 patients received 8 doses of empagliflozin or placebo over 8 days, 1 patient received 6 doses of placebo over 6 days, and 1 patient received 2 doses of empagliflozin over 2 days. Forty-six patients completed the study, with 2 patients discontinuing treatment prematurely due to adverse events. Baseline characteristics are summarized in Table 1.

In total, 24 (50.0%) patients experienced AEs during the treatment period. No AEs were reported in the 1-week post-treatment period. The frequency of AEs was 52.8% in patients receiving empagliflozin treatment and 41.7% with placebo. The most frequently reported AE was headache, reported by 4 patients (8.3%) during the treatment period, occurring in 0–2 patients per group (Table 2). There were no fatalities or serious AEs. A total of 16 patients (33.3%) experienced AEs that the investigator considered related to study medication, but there were no treatment-related differences in their frequency. The most common investigator-defined treatment-related AEs were headache, diarrhea, and hypoglycemia. Three patients in the empagliflozin treatment group experienced hypoglycemic events, defined as blood glucose levels <3.5 mmol/L (<63 mg/dL), but all of them occurred within 5 h of OGTT administration. These events were considered by the investigator to be moderate in intensity, but all patients recovered following therapy. Two patients experienced AEs that led to discontinuation of study treatment [1 patient in the placebo group had an increase in hepatic enzymes (maximum alanine transaminase level of 335 U/L) considered to be of severe intensity and 1 patient treated with empagliflozin experienced cellulitis (phlegmon of the lower left arm) considered to be of moderate intensity, which the investigator did not believe to be related to the study medication]. No urinary tract or genital tract infections were reported. There were no findings of clinical significance in the laboratory clinical evaluations (including electrolytes and lipid parameters; Table 3), 12-lead ECG, vital signs, weight, or waist circumference. No major differences in total urine volume were observed between the empagliflozin and placebo groups. On day 8, the mean volume of urine collected ranged from 2.36 to 4.08 L following empagliflozin dosing, compared with a mean of 3.23 L for the placebo group. Similarly, there were no changes to micturition frequency after empagliflozin administration compared with placebo under the controlled fluid intake conditions of the trial (Table 4). Creatinine clearance was similar in all dose groups except for the 2.5 mg empagliflozin dose group at 166 h (Table 5). In this group, two patients had high derived creatinine clearance values in urine fractions collected over a 2-h period pre-dose on day 8 (1,392 mL/min × 1.73 m² and 1,124 mL/min × 1.73 m² change from baseline), based on urinary creatinine concentrations of 251 mg/dL in a volume of 670 mL and 84.1 mg/dL in a volume of 2,115 mL of urine, respectively, and plasma creatinine concentrations of 0.68 mg/dL and 0.94 mg/dL, respectively. This resulted in a mean (SD) change from baseline in derived creatinine clearance of 360 (620) mL/min × 1.73 m² in the 2.5 mg empagliflozin group at 166 h, compared with −12.1 (54.6) mL/min × 1.73 m² for the placebo group (Table 5).

Empagliflozin was rapidly absorbed after oral administration, reaching peak levels between 1.5 and 3 h after a single dose, thereafter, plasma levels declined in a biphasic fashion with a rapid distribution phase and slower elimination phase (Fig. 1). Both AUC and C _max increased approximately proportionally with dose over the dose range from 2.5 to 100 mg (Table 6). After repeated dosing, trough concentrations were similar on days 5–8, indicating that steady state was reached by day 5. At steady state, empagliflozin concentration–time profiles showed a trend similar to single dose profiles on day 1 (Fig. 1) [note, 2 patients (1 each from the 10 mg and 100 mg dose groups on day 9) were excluded due to incomplete data or low exposure]. The mean t _1/2 after a single dose ranged from 10.8 (25 mg dose) to 13.6 h (100 mg dose) and was similar at steady state (day 9; range 10.3–18.8 h) (Table 6). Consistent with the drug’s half-life, up to 23% accumulation of empagliflozin was observed at steady state. The fe ranged from 12.2% to 18.7% at steady state. On day 1, renal clearance of empagliflozin over 48 h (CL_{R, 0–48h}) was 15.0–29.1 mL/min and at steady state on day 9 (CL_R,τ,ss) was 23.5–34.4 mL/min (Table 6). Overall, steady state parameters were similar to single dose parameters, suggesting linear pharmacokinetics with respect to time (Table 6).

At all doses, oral administration of empagliflozin increased UGE above the level of placebo, with both the amount and rate of glucose excretion increasing with dose. After a single dose under OGTT conditions (day 1; Fig. 2), the amount of glucose excreted in the urine over 24 h (Ae_0–24) was 46.3 g with 2.5 mg empagliflozin compared with 5.84 g with placebo. There was more than a twofold increase in UGE with the fourfold increase in dose from 2.5 to 10 mg, in contrast with about a 1.2-fold increase in UGE with the fourfold dose increase in dose from 25 to 100 mg. Glucose excretion seemed to plateau after the 10 mg dose, with total cumulative amounts excreted ranging from 77.9 mg to 89.8 g with 10 to 100 mg empagliflozin after a single dose. Similar results were observed after multiple doses with or without OGTT (days 9 and 8, respectively; Fig. 2). Empagliflozin inhibited reabsorption of 39%, 46%, 58%, and 64% of filtered glucose with 2.5, 10, 25, and 100 mg once-daily doses, respectively (Fig. 3). The mean decrease in FPG from baseline (day −1) to day 9 (based on least squares means) ranged from 17.2% to 25.8% with empagliflozin, compared with 12.7% with placebo. The overall decline in FPG was 4.6–13.2% greater with empagliflozin than with placebo (Fig. 4). The decrease in FPG from baseline was significantly different compared to placebo with the 10 mg dose (Fig. 4). A respective decrease in plasma glucose levels was also observed following OGTT immediately after the first dose. The mean decrease in MDG from baseline (based on least squares means) was 24.1–37.0% with empagliflozin, compared with 13.5% for placebo [comparison of day 8 (without OGTT) with baseline (day −2)] (Table 5). The decrease in MDG from baseline was significantly different compared to placebo with the 2.5 and 10 mg doses (Table 7). After OGTT, reductions in plasma glucose levels in empagliflozin groups were greater than in the placebo group. Mean AUEC_1–5 values decreased approximately 22.9–27.5% from baseline with empagliflozin, compared with 14.2% with placebo (Table 8). Serum insulin levels did not change to a relevant degree (data not shown).