Renal Glucose Reabsorption in Response to Dapagliflozin
Renal Glucose Reabsorption in Response to Dapagliflozin
Twelve healthy subjects (41 years of age, BMI 27.0 kg/m, HbA1c 5.5%, FPG 4.7 mmol/L [85 mg/dL], 7 males and 5 females, 11 white and 1 black) and 12 subjects with type 2 diabetes (53 years of age, BMI 29.8 kg/m, HbA1c 6.5%, FPG 6.0 mmol/L [108 mg/dL], 7 males and 5 females, and 9 white, 2 black, and 1 Asian) participated in the study (Table 1). Other than diabetes, subjects were in good health as assessed by screening laboratory measurements, electrocardiogram, medical history, and physical examination. All subjects had an estimated GFR ≥60 mL/min by Modification of Diet in Renal Disease equation. No subject had microalbuminuria. Body weight was stable for ≥3 months before the study, and no subject participated in an excessively heavy exercise program. Subjects receiving antidiabetic therapy (Table 1) continued their usual regimen throughout the study except for metformin, which was withheld for 48 h before each stepped hyperglycemic clamp (SHC) procedure (because of iohexol administration). Sulfonylurea was not taken on the day of study.
Eligible subjects underwent combined pancreatic/SHC to determine 1) renal TmG, 2) splay in the renal glucose titration curve, 3) threshold for glucose excretion, and 4) percentage of filtered glucose load excreted at any given plasma glucose concentration. Subjects fasted overnight at the research center (10–12 h). Subjects were connected to a Biostator (Life Science Instruments, Elkhart, IN) by an intravenous line in the lower arm or dorsal hand, and arterialized blood was continuously sampled to determine blood glucose level in minute intervals. Another intravenous line for sampling of pharmacokinetic/pharmacodynamic parameters was inserted in same arm. An antecubital vein in the contralateral arm was cannulated for infusion of test substances.
A pancreatic clamp approach was used to suppress endogenous insulin secretion, reduce the volume of glucose required to achieve the maximal target glucose in healthy subjects during SHC, reduce the time required to stabilize blood glucose at each target level, and obviate potential effects of hyperinsulinemia on TmG. Type 2 diabetic subjects received an overnight insulin infusion (Novolin R 100 IU/mL; Novo Nordisk, Bagsvaerd, Denmark) before SHC to achieve and maintain an FPG of ~100 mg/dL (target plasma glucose for the initial step of SHC [5.6 mmol/L]) without administration of glucose. Before the SHC procedure (~2.25 h), octreotide (30 ng/kg min) was infused to suppress endogenous pancreatic hormone secretion, and infusions of glucagon (1 ng/kg min) and growth hormone (3 ng/kg min) were started to restore basal plasma levels of these hormones (pancreatic clamp). An insulin infusion was started at 0.1 mU/kg min to replace basal insulin levels in healthy subjects. Subjects with type 2 diabetes received individualized insulin infusion rates based on the insulin infusion rate that controlled the plasma glucose level to 5.6 mmol/L (100 mg/dL) without the need for exogenous glucose administration for 60 min before the start of the pancreatic clamp. Basal insulin infusion rates were constantly maintained throughout the procedure.
A bolus of iohexol providing 45 mg organic iodine followed by a continuous infusion delivering 15 mg/h organic iodine was initiated ~1 h before the SHC to assess GFR. Plasma samples for iohexol measurement were obtained at –60, –30, and 0 min and every 40 min during SHC. Plasma samples for insulin and additional glucose measurements were collected at –30, –15, and 0 min and every 40 min during SHC. The baseline plasma glucose concentration at the start of SHC was 5.6 mmol/L (100 mg/dL). An automated SHC technique was performed with the Biostator and standard infusion pumps to infuse glucose (20% volume for volume), acutely raise plasma glucose to consecutive target levels (50 mg/dL [2.8 mmol/L] increments), and constantly maintain these levels for 40 min up to a maximum target glucose concentration of 550 mg/dL (30.5 mmol/L).
Subjects received a water load (25 mL/kg ideal body weight) ~2 h before the SHC, and the volume of spontaneously voided urine was replaced to initiate and sustain a water diuresis and facilitate urine collections during the procedure. Subjects voided at –30 min, and urine was collected from –30 to 0 min and every 40 min thereafter. Each urine sample volume was recorded, and concentrations of glucose and iohexol were measured.
On days 1–7 after completion of the baseline SHC, subjects ingested 10 mg of dapagliflozin each morning. On day 7, the SHC was repeated. Plasma samples for determination of dapagliflozin pharmacokinetics were obtained at 0, 40, 80, 120, 160, 200, 240, 320, 360, and 400 min and at 24 h postdosing.
Plasma glucose was measured with the YSI 2300 Stat analyzer (YSI Life Sciences, Yellow Springs, OH) and Biostator glucose analyzer module.
With the use of a one-sided test (significance of 0.05), it was determined that 10 subjects/group would provide 99% power, assuming a 40% TmG reduction with 7 days of dapagliflozin treatment. With two one-sided tests (significance of 0.05 for each test), 10 subjects/group provided 87% power to conclude that the effect of dapagliflozin on TmG would be equivalent using a 20% equivalence limit (set a priori as a reasonable estimate), assuming that TmG posttreatment-to-pretreatment ratios were log-normally distributed and an SD of the log was <0.15. To ensure that 10 subjects completed the study, 12 subjects per group were recruited.
To characterize the reduction in TmG after dapagliflozin administration and to identify potential differences in the renal glucosuric effect of dapagliflozin between groups, absolute values were expressed as percent change in TmG, and TmG values were log-transformed. Percent changes in TmG from baseline to day 7 were analyzed by ANCOVA of logarithms of posttreatment versus pretreatment ratios in TmG, with group as the main effect and logarithm of the baseline TmG value as a covariate. From that statistical model, point estimates and 90% CIs for geometric mean percent change from baseline TmG within each group were constructed, P values were determined, and the point estimate and its 90% CI were calculated for the diabetic group-to-control group ratio of geometric means of posttreatment-to-pretreatment baseline ratios in TmG. Equivalence in dapagliflozin effect on TmG between the two groups can be concluded if the 90% CI for diabetic-to-control group ratio of geometric means for TmG at baseline is entirely contained within 80–125%. Similar analyses were performed for splay and threshold.
To identify potential differences in baseline TmG between type 2 diabetic and control subjects, the point estimate and its 90% CI were calculated for the diabetic-to-control group ratio of geometric means for TmG at baseline. These were estimated from a fitted ANOVA model for log-transformed data, with treatment group as a fixed effect. The point estimate of the difference and its 90% CI in the log scale were exponentiated to obtain the estimate and CI for the ratio of geometric means in the original scale. Similar analyses were performed for splay.
Equations used to calculate parameters reported herein are included as Supplementary Data. Data are mean ± SD, with the exception of data presented for TmG and splay, which are adjusted geometric means.
Research Design and Methods
Subjects
Twelve healthy subjects (41 years of age, BMI 27.0 kg/m, HbA1c 5.5%, FPG 4.7 mmol/L [85 mg/dL], 7 males and 5 females, 11 white and 1 black) and 12 subjects with type 2 diabetes (53 years of age, BMI 29.8 kg/m, HbA1c 6.5%, FPG 6.0 mmol/L [108 mg/dL], 7 males and 5 females, and 9 white, 2 black, and 1 Asian) participated in the study (Table 1). Other than diabetes, subjects were in good health as assessed by screening laboratory measurements, electrocardiogram, medical history, and physical examination. All subjects had an estimated GFR ≥60 mL/min by Modification of Diet in Renal Disease equation. No subject had microalbuminuria. Body weight was stable for ≥3 months before the study, and no subject participated in an excessively heavy exercise program. Subjects receiving antidiabetic therapy (Table 1) continued their usual regimen throughout the study except for metformin, which was withheld for 48 h before each stepped hyperglycemic clamp (SHC) procedure (because of iohexol administration). Sulfonylurea was not taken on the day of study.
Experimental Design
Eligible subjects underwent combined pancreatic/SHC to determine 1) renal TmG, 2) splay in the renal glucose titration curve, 3) threshold for glucose excretion, and 4) percentage of filtered glucose load excreted at any given plasma glucose concentration. Subjects fasted overnight at the research center (10–12 h). Subjects were connected to a Biostator (Life Science Instruments, Elkhart, IN) by an intravenous line in the lower arm or dorsal hand, and arterialized blood was continuously sampled to determine blood glucose level in minute intervals. Another intravenous line for sampling of pharmacokinetic/pharmacodynamic parameters was inserted in same arm. An antecubital vein in the contralateral arm was cannulated for infusion of test substances.
A pancreatic clamp approach was used to suppress endogenous insulin secretion, reduce the volume of glucose required to achieve the maximal target glucose in healthy subjects during SHC, reduce the time required to stabilize blood glucose at each target level, and obviate potential effects of hyperinsulinemia on TmG. Type 2 diabetic subjects received an overnight insulin infusion (Novolin R 100 IU/mL; Novo Nordisk, Bagsvaerd, Denmark) before SHC to achieve and maintain an FPG of ~100 mg/dL (target plasma glucose for the initial step of SHC [5.6 mmol/L]) without administration of glucose. Before the SHC procedure (~2.25 h), octreotide (30 ng/kg min) was infused to suppress endogenous pancreatic hormone secretion, and infusions of glucagon (1 ng/kg min) and growth hormone (3 ng/kg min) were started to restore basal plasma levels of these hormones (pancreatic clamp). An insulin infusion was started at 0.1 mU/kg min to replace basal insulin levels in healthy subjects. Subjects with type 2 diabetes received individualized insulin infusion rates based on the insulin infusion rate that controlled the plasma glucose level to 5.6 mmol/L (100 mg/dL) without the need for exogenous glucose administration for 60 min before the start of the pancreatic clamp. Basal insulin infusion rates were constantly maintained throughout the procedure.
A bolus of iohexol providing 45 mg organic iodine followed by a continuous infusion delivering 15 mg/h organic iodine was initiated ~1 h before the SHC to assess GFR. Plasma samples for iohexol measurement were obtained at –60, –30, and 0 min and every 40 min during SHC. Plasma samples for insulin and additional glucose measurements were collected at –30, –15, and 0 min and every 40 min during SHC. The baseline plasma glucose concentration at the start of SHC was 5.6 mmol/L (100 mg/dL). An automated SHC technique was performed with the Biostator and standard infusion pumps to infuse glucose (20% volume for volume), acutely raise plasma glucose to consecutive target levels (50 mg/dL [2.8 mmol/L] increments), and constantly maintain these levels for 40 min up to a maximum target glucose concentration of 550 mg/dL (30.5 mmol/L).
Subjects received a water load (25 mL/kg ideal body weight) ~2 h before the SHC, and the volume of spontaneously voided urine was replaced to initiate and sustain a water diuresis and facilitate urine collections during the procedure. Subjects voided at –30 min, and urine was collected from –30 to 0 min and every 40 min thereafter. Each urine sample volume was recorded, and concentrations of glucose and iohexol were measured.
On days 1–7 after completion of the baseline SHC, subjects ingested 10 mg of dapagliflozin each morning. On day 7, the SHC was repeated. Plasma samples for determination of dapagliflozin pharmacokinetics were obtained at 0, 40, 80, 120, 160, 200, 240, 320, 360, and 400 min and at 24 h postdosing.
Measurements
Plasma glucose was measured with the YSI 2300 Stat analyzer (YSI Life Sciences, Yellow Springs, OH) and Biostator glucose analyzer module.
Sample Size Determination
With the use of a one-sided test (significance of 0.05), it was determined that 10 subjects/group would provide 99% power, assuming a 40% TmG reduction with 7 days of dapagliflozin treatment. With two one-sided tests (significance of 0.05 for each test), 10 subjects/group provided 87% power to conclude that the effect of dapagliflozin on TmG would be equivalent using a 20% equivalence limit (set a priori as a reasonable estimate), assuming that TmG posttreatment-to-pretreatment ratios were log-normally distributed and an SD of the log was <0.15. To ensure that 10 subjects completed the study, 12 subjects per group were recruited.
Statistical Comparisons
To characterize the reduction in TmG after dapagliflozin administration and to identify potential differences in the renal glucosuric effect of dapagliflozin between groups, absolute values were expressed as percent change in TmG, and TmG values were log-transformed. Percent changes in TmG from baseline to day 7 were analyzed by ANCOVA of logarithms of posttreatment versus pretreatment ratios in TmG, with group as the main effect and logarithm of the baseline TmG value as a covariate. From that statistical model, point estimates and 90% CIs for geometric mean percent change from baseline TmG within each group were constructed, P values were determined, and the point estimate and its 90% CI were calculated for the diabetic group-to-control group ratio of geometric means of posttreatment-to-pretreatment baseline ratios in TmG. Equivalence in dapagliflozin effect on TmG between the two groups can be concluded if the 90% CI for diabetic-to-control group ratio of geometric means for TmG at baseline is entirely contained within 80–125%. Similar analyses were performed for splay and threshold.
To identify potential differences in baseline TmG between type 2 diabetic and control subjects, the point estimate and its 90% CI were calculated for the diabetic-to-control group ratio of geometric means for TmG at baseline. These were estimated from a fitted ANOVA model for log-transformed data, with treatment group as a fixed effect. The point estimate of the difference and its 90% CI in the log scale were exponentiated to obtain the estimate and CI for the ratio of geometric means in the original scale. Similar analyses were performed for splay.
Calculations
Equations used to calculate parameters reported herein are included as Supplementary Data. Data are mean ± SD, with the exception of data presented for TmG and splay, which are adjusted geometric means.
