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Substantial Effects of Luseogliflozin Revealed by Analyzing Responses to Postprandial Hyperglycemia: Post Hoc Subanalyses of a Randomized Controlled Study

Introduction In our previous study investigating effects of luseogliflozin, a sodium–glucose cotransporter 2 inhibitor, on 24-h glycemic variability by continuous glucose monitoring (CGM), luseogliflozin elicited parallel downward shifts in fasting and postprandial glucose levels. However, further review of individual patients’ data revealed that postprandial hyperglycemia was not reduced in some patients, while preprandial glucose was ameliorated in most patients. Therefore, we divided patients into two groups according to their postprandial glucose responses and conducted a post hoc subanalyses to elucidate which factors contributed to the differential effects of luseogliflozin. Methods Thirty-four Japanese type 2 diabetic patients in our previous randomized, double-blind, placebo-controlled, crossover study with 7-day luseogliflozin administration were divided into postprandial glucose responders (PGR, n = 23, ameliorated peak glucose) and postprandial glucose non-responders (PGNR; n = 11, non-ameliorated peak glucose). Baseline characteristics, variations in CGM-measured 24-h glucose levels, and other pharmacodynamic variabilities were compared. Results Baseline characteristics did not differ significantly between groups. Placebo-subtracted peak glucose was significantly lowered in PGR and significantly increased in PGNR (−43.8 and 17.9 mg/dL; both p < 0.05). Luseogliflozin significantly lowered “lowest glucose” (defined as the lowest level measured throughout a 24-h period) similarly in PGR and PGNR (−19.2 and −24.0 mg/dL; both p < 0.05), significantly reduced the mean amplitude of glucose excursions in PGR (−15.50 mg/dL; p < 0.05), and increased the area under the curve for plasma glucagon over 24 h in PGNR (median difference vs. placebo: 240 pg/mL h; p < 0.05). Luseogliflozin increased urinary glucose excretion (UGE) and decreased serum insulin by similar magnitudes in both groups. Conclusions Luseogliflozin diminished glucose fluctuations in most patients by lowering peak glucose to a greater extent than lowest glucose. Luseogliflozin may also lower lowest glucose in patients whose peak glucose was not ameliorated despite increasing UGE. The glucagon increase in PGNR might explain its hypoglycemic effect on postprandial glucose. Funding Taisho Pharmaceutical Co., Ltd, Tokyo, Japan. Trial Registration JapicCTI-142548. Electronic supplementary material The online version of this article (doi:10.1007/s12325-016-0350-5) contains supplementary material, which is available to authorized users.

parallel downward shifts in fasting and postprandial glucose levels. However, further review of individual patients' data revealed that postprandial hyperglycemia was not reduced in some patients, while preprandial glucose was ameliorated in most patients. Therefore, we divided patients into two groups according to their postprandial glucose responses and conducted a post hoc subanalyses to elucidate which factors contributed to the differential effects of luseogliflozin.
Results: Baseline characteristics did not differ significantly between groups.
Placebo-subtracted peak glucose was significantly lowered in PGR and significantly increased in PGNR (-43. 8

INTRODUCTION
The updated American Diabetes Association (ADA)/European Association for the Study of Diabetes (EASD) position statement [1] evaluated recent data and developed recommendations, including the advantages and disadvantages, for anti-hyperglycemic agents for the treatment of type 2 diabetes mellitus (T2DM). The position statement places emphasis on a patient-centered approach, in which patient preference, cost, potential side effects, effects on body weight, and risk of hypoglycemia associated with each drug class should be considered when selecting a treatment approach.
Sodium-glucose cotransporter 2 (SGLT2) inhibitors are newly approved drugs that correct hyperglycemia without increasing the risk of hypoglycemia or body weight gain [2][3][4][5][6], and have been included as a treatment option for combination therapy of T2DM in the ADA/ EASD position statement [1]. To date, however, few studies have examined the effect of SGLT2 inhibitors on 24-h glycemic variability including nocturnal glycemia and the risk of asymptomatic hypoglycemia. Moreover, no studies have described the effects of SGLT2 inhibitors on the changes in serum insulin or plasma glucagon concentrations throughout the day; these data are important in terms of understanding the effects of these drugs on pancreatic a and b cells [7,8].
Luseogliflozin is a highly selective SGLT2 inhibitor that was recently approved and launched in Japan for the treatment of T2DM [5].  [10,11]. Similar glycemic-lowering effects on 24-h glucose levels were also reported in a clinical study of empagliflozin, another SGLT2 inhibitor [12]. However, when we reviewed in detail the changes in 24-h glucose levels in the individual patients' data from our study [10], we found that postprandial hyperglycemia was not reduced in some patients, even though fasting glucose was ameliorated after luseogliflozin administration in almost all patients.
Therefore, we divided the patients into two groups according to their responses to peak glucose, and performed post hoc subanalyses to elucidate the factors associated with the differential effects of luseogliflozin in these patients.

Study Design and Participants
The present subanalyses are based on a previous

Statistical Analysis
We investigated and compared the baseline values, the effect on 24-h glucose variability, and other pharmacodynamic variability changes in the PGR and PGNR groups.
Baseline characteristics are presented as the mean ± standard deviation (SD). Regarding pharmacodynamic and glucose variables, normally distributed variables are presented as the least-squares mean (LSM) and two-sided 95% confidence interval. The differences between the two treatments were analyzed using a mixed-effects model, which included treatment, sequence, and period as fixed effects and patients as a random effect. Non-normally distributed variables are presented as the median and interquartile range, and differences between the two treatments were determined using paired Wilcoxon tests. To evaluate the difference between PGR and PGNR groups in terms of the difference between luseogliflozin and placebo in each group, a two-sample t test was used to analyze normally distributed variables. The Wilcoxon rank sum test was used to analyze non-normally distributed variables. All statistical analyses were performed using SAS software version 9.2 (SAS Institute Japan, Tokyo, Japan).

Patient Baseline Characteristics
Baseline demographic and clinical characteristics of the study patients are shown in Table 1

Pharmacodynamics
The variations in 24-h glucose levels measured by CGM after 7 days of treatment with luseogliflozin and placebo in each group are shown in Fig. 1, and the indices derived from the CGM data are presented in Table 2.
Because patients were divided according to the responses in postprandial glucose concentrations, the placebo-subtracted peak glucose level throughout the day was significantly decreased in the PGR (p\0.001, Table 2) and was significantly increased in the PGNR (p = 0.004, Table 2). Consistent with this, the placebo-subtracted peak glucose levels after each meal were also significantly decreased in the PGR (all p\0.001, Table 2). Such changes were not observed in the PGNR.
Luseogliflozin significantly lowered the lowest glucose throughout the day, as well preprandial (before each meal) and midnight (3 h after sleep) glucose compared with placebo in the PGR and PGNR (all p\0.05, Table 2). Furthermore, daily glucose fluctuations for the mean amplitudes of glucose excursions (MAGE) and for SD over 24 h were significantly decreased in the PGR (p = 0.009 and p\0.001, respectively, Table 2). By contrast, MAGE and SD over 24 h increased significantly in the PGNR (p = 0.002 and p\0.001, respectively).
The urinary glucose excretion (UGE) rate on day 7 is shown in Fig. 2a, and cumulative UGE Data are the mean ± standard deviation or n (%) Glucose: 1 mg/dL = 0.0556 mmol/L Insulin: 1 lU/mL = 6.945 pmol/L Glucagon: 1 pg/mL = 1 ng/L BMI body mass index, eGFR estimated glomerular filtration rate, FPG fasting plasma glucose, HbA1c glycated hemoglobin, PGR postprandial glucose responders, PGNR postprandial glucose non-responders a Two-sample t test   Values are derived from 24-h continuous glucose monitoring. Normally distributed variables are presented as the least-squares mean and 95% confidence interval, and the differences between the two treatments were analyzed using a mixed-effects model that included treatment, sequence and period as fixed effects and patients as a random effect. Non-normally distributed variables are presented as the median and interquartile range, and differences between the two treatments were determined using paired Wilcoxon tests. Data are shown for the pharmacodynamic analysis set Glucose: 1 mg/dL =  Fig. 2b (0-24 h only) and Table 3. Luseogliflozin significantly increased the UGE rate and cumulative UGE by similar magnitudes in both groups (all p\0.001, Fig. 2; Table 3). The magnitude of the difference between luseogliflozin and placebo for the cumulative UGE was not significantly different between PGR and PGNR at each measurement period ( Table 3).
The concentrations of serum insulin and plasma glucagon on day 7 with luseogliflozin and placebo in each group are shown in Fig. 3a and b, respectively, and the area under the curve (AUC) and the maximum concentration of serum insulin and plasma glucagon at 0-24 h, 0-5 h, 5-11 h, 11-15 h, and 15-24 h on day 7 are shown in Table 3. Luseogliflozin decreased serum insulin throughout the 24-h measurement period compared with placebo in both PGR and PGNR, and the AUC for serum insulin over 24 h was significantly decreased compared with placebo in both PGR and PGNR (both p\0.05, Table 3).
Luseogliflozin increased plasma glucagon throughout the 24-h measurement period compared with placebo in both PGR and PGNR (Fig. 3b), as well as the AUC for plasma glucagon over 24 h in the PGNR (p\0.05, Table 3).
The concentrations of serum acetoacetic acid and serum b-hydroxybutanoic acid on day 7 with luseogliflozin and placebo in the PGR and PGNR are shown in Fig. 4, and the AUC and the    (Fig. 4a, b), and numerically increased the AUC for serum acetoacetic acid and serum b-hydroxybutanoic acid over 24 h in PGNR but not PGR (Table 3).

DISCUSSION
When we examined the 24-h glucose variations in the individual patient's data obtained in our previous study [10], we found that patients could be divided into two groups according to whether postprandial hyperglycemia was ameliorated. Therefore, we divided the patients into PGR and PGNR, and investigated the differential effects of luseogliflozin in each group.
No significant differences in baseline characteristics were observed between the PGR and PGNR. Previous studies indicated the effects of SGLT2 inhibitors diminish depending on baseline plasma glucose concentrations [13].
Therefore, we expected that the baseline glycemic parameters (HbA1c, FPG, and GA) would differ between the PGR and PGNR because the patients were divided according to their postprandial glucose responses. However, we observed no between-group differences in the baseline glycemic parameters. Of note, insulin concentrations tended to be higher and glucagon concentrations tended to be lower in the PGR compared with the PGNR at baseline, although these differences were not significant. Furthermore, because the UGE increasing effect of SGLT2 inhibitors is diminished in patients with a low eGFR, we also expected there would be a difference in the baseline eGFR, but no between-group differences were observed. From these Table 3 continued Laboratory tests were performed using blood samples obtained over 24 h. Normally distributed variables are presented as the least-squares mean and 95% confidence interval, and differences between the two treatments were analyzed using a mixed-effects model which included treatment, sequence, and period as fixed effects and patients as a random effect. Non-normally distributed variables are presented as the median and interquartile range, and differences between the two treatments were determined using paired Wilcoxon tests. Data are shown for the pharmacodynamic analysis set Glucose: 1 g = 0.00556 mol Insulin: 1 lU/mL To evaluate the difference between PGR and PGNR in terms of the difference between luseogliflozin and placebo in each group, a two-sample t test was used to analyze normally distributed variables, and the Wilcoxon rank sum test was used to analyze non-normally distributed variables subanalyses, we could not identify which baseline characteristic determined the difference between the PGR and PGNR.
We previously investigated the hypoglycemic effect of an SGLT2 inhibitor using CGM [10,11] and revealed a parallel Interestingly, the magnitude of the increase in UGE, which is a direct pharmacological effect of luseogliflozin, was similar in the PGR and PGNR. Furthermore, the decrease in FPG in the PGNR was similar to that in the PGR. Because there was an apparent difference in the response to postprandial glucose levels between the PGR and PGNR, a difference in the increase in UGE and decrease in FPG was expected between these groups of patients. However, the increase in UGE and the decrease in FPG were similar in the PGR and PGNR. Therefore, we investigated other factors which might affect glucose concentrations.
The decrease of serum insulin secretion throughout the day by luseogliflozin was similar in each group and was consistent with previous studies [8,[14][15][16]. We expected the Values are presented as the mean ± standard deviation. PGR postprandial glucose responders, PGNR postprandial glucose non-responders was not ameliorated in the PGNR, even though the increase in UGE and the decrease in FPG were similar in both the PGNR and PGR. SGLT2 inhibitors have been reported to increase plasma glucagon secretion and enhance endogenous glucose production [17]. In this subanalysis, glucagon secretion was increased in both the PGR and PGNR. Compared with the PGR, the increase in glucagon secretion in the PGNR was especially high during the postprandial periods, although this difference did not reach statistical significance (Fig. 3b).
We consider that luseogliflozin increased glucagon secretion, enhanced gluconeogenesis, In this subanalysis, ketone bodies increased in both the PGR and PGNR; the increase was larger in the PGNR compared with the PGR although it did not reach statistical significance.
It is thought that euglycemic diabetic ketoacidosis is induced by use of SGLT2 inhibitors and glucagon may also be involved [18]. Therefore, production of ketone bodies may be enhanced, particularly in the PGNR relative to the PGR. Meanwhile, no AEs related to ketone body increase (other than slight increases in laboratory parameters) occurred in either PGR or PGNR groups. Further investigation is needed to clarify the difference between the groups in the effects of luseogliflozin with respect to ketone body increase.
The present study had some limitations. It involved a subanalysis of our previous study [10], which was not powered for the analyses reported here. Although other factors might have affected the responses to postprandial hyperglycemia in patients treated with luseogliflozin, the interpretations of the present study are limited because of the inclusion/exclusion criteria of the original study. Additionally, luseogliflozin was only administered for 7 days, so the results might differ if the treatment period were longer. In the future, we plan to investigate the effects of long-term luseogliflozin treatment on 24-h glycemic variability.

CONCLUSION
The results of these post hoc subanalyses suggest that luseogliflozin diminishes glucose fluctuations in most patients by lowering postprandial glucose to a greater extent than its effects on fasting glucose. Luseogliflozin may also lower fasting glucose in patients whose postprandial glucose was not ameliorated despite an increase in UGE. Furthermore, the increase in glucagon secretion in patients whose postprandial glucose was not ameliorated might contribute to the differential hypoglycemic effects of luseogliflozin on postprandial glucose.