Endocrine Journal 2015, 62 (1), 13-20

Original

Teneligliptin improves glycemic control with the reduction of postprandial insulin requirement in Japanese diabetic patients Wakaba Tsuchimochi1), Hiroaki Ueno1), Eiichiro Yamashita1), Chikako Tsubouchi1), Hideyuki Sakoda1), 2), Shuji Nakamura3) and Masamitsu Nakazato1) 1)

Division of Neurology, Respirology, Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan 2) Department of Metabolic Disease, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan 3) Heiwadai Hospital, Miyazaki 880-0034, Japan

Abstract. Teneligliptin is a novel peptidomimetic-chemotype prolylthiazolidine-based inhibitor of dipeptidyl peptidase-4 (DPP-4). The aim of this study was to evaluate the effects of teneligliptin on 24 h blood glucose control and gastrointestinal hormone responses to a meal tolerance test, and to investigate the glucose-lowering mechanisms of teneligliptin. Ten patients with type 2 diabetes mellitus (T2DM) were treated for 3 days with teneligliptin (20 mg/day). Postprandial profiles for glucose, insulin, glucagon, active glucagon-like peptide-1 (GLP-1), active glucose-dependent insulinotropic polypeptide (GIP), ghrelin, des-acyl ghrelin, and 24 h glycemic fluctuations were measured via continuous glucose monitoring for 4 days. Once daily teneligliptin administration for 3 days significantly lowered postprandial and fasting glucose levels. Significant elevations of fasting and postprandial active GLP-1 and postprandial active GIP levels were observed. Teneligliptin lowered postprandial glucose elevations, 24 h mean blood glucose levels, standard deviation of 24 h glucose levels and mean amplitude of glycemic excursions (MAGE) without hypoglycemia. Serum insulin levels in the fasting state and 30 min after a meal were similar before and after teneligliptin treatment; however significant reductions at 60 to 180 min after treatment were observed. A significant elevation in early-phase insulin secretion estimated by insulinogenic and oral disposition indices, and a significant reduction in postprandial glucagon AUC were observed. Both plasma ghrelin and des-acyl ghrelin levels were unaltered following teneligliptin treatment. Teneligliptin improved 24 h blood glucose levels by increasing active incretin levels and early-phase insulin secretion, reducing the postprandial insulin requirement, and reducing glucagon secretion. Even short-term teneligliptin treatment may offer benefits for patients with T2DM. Key words: DPP-4 inhibitor, Continuous glucose monitoring, Incretin, Type 2 diabetes mellitus

THE GASTROINTESTINAL tract produces more than 20 regulatory peptides, including ghrelin, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP), which function in various physiological responses including the regulation of appetite and glucose homeostasis. Ghrelin, which was initially isolated from gastric extracts, exists in two major forms: n-octanoyl–modified ghrelin and des-acyl ghrelin (a non-acylated form of ghrelin) [1]. Submitted Jun. 5, 2014 as EJ14-0269; Accepted Sep. 3, 2014 as EJ14-0393 Released online in J-STAGE as advance publication Sep. 25, 2014

Correspondence to: Masamitsu Nakazato, M.D., Ph.D., Division of Neurology, Respirology, Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. E-mail: [email protected] ©The Japan Endocrine Society

Ghrelin stimulates feeding, gastric motility, and secretion of growth hormone [2, 3]. The two major ghrelin isoforms appear to have distinct actions and regulate glucose metabolism. The effects of them are still a matter of debate [4]. GLP-1 stimulates postprandial insulin secretion and suppresses glucagon secretion in a glucose-dependent manner, and also slows gastric emptying [5]. GIP stimulates postprandial insulin secretion and fat biosynthesis [5]. In people with type 2 diabetes mellitus (T2DM), the GLP-1 response to meal test is reduced, while the GIP response to glucose tolerance is elevated [6]. The lower plasma levels of active GLP-1 in people with diabetes are thought to be due to both impairment of GLP-1 secretion and an increase in its degradation by dipeptidyl peptidase-4 (DPP-4) expressed by endothelial cells and circulat-

Tsuchimochi et al.

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ing in the blood [7]. An understanding of the secretion profile of incretin hormones helps predict the efficacy of anti-diabetic drugs that target incretin-related pathways. When cleaved by DPP-4, GLP-1 and GIP no longer act as incretin hormones. Therefore, active GLP-1(7-36)NH2 and active GIP(1-42) should be measured using the plasma extraction method, because factors that interfere with immunoassays for these compounds are present in the blood [8, 9]. Seven DPP-4 inhibitors are available in Japan. DPP-4 inhibitors are classified into peptidomimetic (i.e., vildagliptin, saxagliptin, anagliptin, and teneligliptin) or non-peptidomimetic (i.e., sitagliptin, alogliptin, and linagliptin) subtypes. Teneligliptin, a novel peptidomimetic-chemotype prolylthiazolidine-based DPP-4 inhibitor, was approved in 2013 in Japan. The drug, with a unique structure characterized by five consecutive rings, produces a potent and long-lasting glucose-lowering effect [10]. An X-ray structural analysis of the co-crystal of teneligliptin with DPP-4 demonstrated that an interaction occurs between the phenyl ring on the pyrazole of teneligliptin and the S2 extensive subsite of DPP-4. This interaction increased not only the potency of the drug, but also its selectivity [10]. The effects of teneligliptin on 24-h glycemic fluctuations and gastrointestinal peptides in people with T2DM have yet to be investigated. We studied the effect of teneligliptin on Japanese people with T2DM by analyzing 24-h glycemic fluctuations via continuous glucose monitoring (CGM), as well as changes in glucose and insulin during a meal tolerance test. We also studied the effects of teneligliptin on gastrointestinal hormones including glucagon, active GLP-1, active GIP, ghrelin, and des-acyl ghrelin.

Subjects and Methods Subjects Japanese people with T2DM aged 30–79 years, who had inappropriate control of blood glucose levels (HbA1c ≥ 7.0%) in spite of diet and exercise therapies, were recruited from our outpatient clinic. Exclusion criteria were as follows: 1) serious infection, pre- or postoperative condition, or severe trauma; 2) pregnancy, possible pregnancy, or breast-feeding; 3) moderate or severe renal dysfunction (estimated glomerular filtrating ratio [mL/min/1.73 m2]) < 50 mL/min, serum creatinine level > 1.5 mg/dL in men or > 1.3 mg/dL in women); 4) severe liver dysfunction; 5) insulin treat-

ment; and 6) treatment with anti-diabetic agents other than sulfonylurea or thiazolidine alone. This study was approved by the ethics committee of the Faculty of Medicine of the University of Miyazaki and conducted in accordance with the Helsinki Declaration. Written informed consent was obtained from all participants before enrolment. Study protocol All participants were admitted to our hospital for 10 days. The study started 1 week after admission, after stable glycemic control had been obtained by dietary therapy (25–30 kcal/kg ideal body weight). Daily plasma glucose fluctuations were monitored using CGM (iPro2, Medtronic Incorporated, Northridge, CA) starting on the seventh day. Administration of teneligliptin (20 mg/day, once daily before breakfast) started on the eighth day and continued for 3 consecutive days. In CGM, mean and standard deviation (SD) of 24-h blood glucose level, mean amplitude of glycemic excursions (MAGE) [11], and proportion of time in hyperglycemia (> 180 mg/dL) or hypoglycemia (< 70 mg/dL) were calculated. Meal tolerance tests (592 kcal, carbohydrate 75 g, protein 8.0 g, fat 28.5 g, Saraya Corp, Osaka, Japan) [12] were conducted on the seventh day (before the initiation of teneligliptin treatment) and the tenth day (3 days after starting teneligliptin). The blood concentrations of glucose, immunoreactive insulin (IRI), glucagon, active GLP-1, active GIP, ghrelin, and des-acyl ghrelin were measured before and 30, 60, 90, 120, and 180 min after the subject consumed the test meal. Serum DPP-4 activity was measured before the meal test. Laboratory measurements Plasma glucose was measured by the hexokinase method. Serum insulin and plasma glucagon were measured by chemiluminescent enzyme immunoassay and double antibody radioimmunoassay method, respectively, using kits obtained from SRL Inc (Tokyo, Japan). Serum DPP-4 activity was determined by measuring the concentration of 7-amino-4-methylcoumarin (AMC) generated from a synthetic DPP-4-specific substrate (H-Gly-Pro-AMC) (BioVision Inc, Milpitas, CA) incubated for 1 h in a 5-fold–diluted serum sample [13]. The percent inhibition of DPP-4 was calculated by comparison with the value before teneligliptin administration. To measure plasma concentrations of active GLP-1 and active GIP, blood was collected directly into chilled tubes containing DPP-4 inhibitor, protease inhibitor, and ester-

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Teneligliptin improves hyperglycemia

ase inhibitor (P800, Becton, Dickinson and Company, NJ), and then immediately centrifuged at 1,468 g for 15 min at 4°C. The plasma samples were subjected to solid-phase extraction as described elsewhere [9]. Oasis HLB Extraction Cartridges (Waters, Milford, MA) were equilibrated with 1 mL of methanol, and then with 1 mL of distilled water. Plasma samples were applied to the wells of the equilibrated plates. Samples were then washed twice with 1 mL of 10% methanol in distilled water, and then peptides were eluted with 0.5 mL of 0.5% NH3/75% ethanol in distilled water. Eluates were freeze-dried and reconstituted in solutions supplemented with commercial immunoassay kits. Active GLP-1[736]NH2 and active GIP[1-42] were measured using the Glucagon Like Peptide-1 (Active) ELISA kit (Millipore, St. Charles, MO) and the GIP Active Form Assay kit (Immuno-Biological Laboratories, Gunma, Japan), respectively. To measure plasma ghrelin and des-acyl ghrelin, blood was withdrawn directly into chilled P800 tubes and centrifuged at 1,468 g for 15 min at 4°C. The plasma was treated with 10% volume of 1 N hydrochloric acid. The acylated and des-acylated forms of ghrelin were measured using fluorescence enzyme immunoassays (Tosoh Corp, Tokyo, Japan) [14]. Indices of glucose metabolism Insulin sensitivity was estimated by homeostasis model assessment of insulin resistance (HOMA-R) [15]. Test meal–stimulated insulin secretion was evaluated based on the insulinogenic index [16], oral disposition index = ΔIRI0,30 (μU.mL-1) / Δglucose 0,30 (mg.dL-1) × 1 / fasting insulin (μU.mL-1) [17], AUC insulin (μU.h.mL-1) / AUC glucose (mg.h.dL-1) ratio [18], and homeostasis model assessment of beta cell function (HOMA-β) [15]. Statistical analysis Data are shown as means ± SD. Statistical analyses of data were performed with JMP 8 (SAS Institute, Cary, NC) using non-parametrical statistical analyses. Repeated measures analyses of variance (ANOVA) were performed to determine overall differences between before and after teneligliptin administration. The Wilcoxon signed-ranks test was applied to paired comparisons if a significant overall difference was detected. Paired comparisons between measurements taken before and after teneligliptin treatment were made using the Wilcoxon signed-ranks test for paired samples. A P value less than 0.05 was considered to

represent a statistically significant difference.

Results Baseline characteristics of subjects Ten subjects (five men and five women) were enrolled in this study. Their characteristics are shown in Table 1. Glucose metabolism Plasma glucose levels in the meal tolerance test performed 3 days after teneligliptin treatment were significantly reduced at all time points relative to the levels before administration (Fig. 1A). Glucose AUC0-180 was significantly reduced after teneligliptin treatment (Fig. 1B). Serum insulin levels from 0 to 30 min were similar between before and after the teneligliptin treatment, but those from 60 to 180 min were significantly lower after teneligliptin administration than before treatment (Fig. 1C). Both insulin AUC0-180 (Fig. 1D) glucose and the AUCinsulin 60-180 /AUC 60-180 ratio (Table 2) were significantly reduced following teneligliptin treatment, by 29.6% and 13.3%, respectively. HOMA-R and HOMA-β were unaltered; however, the insulinogenic and oral disposition indices were significantly elevated, by 1.7- and 2.5-fold, respectively, after teneligliptin treatment (Table 2). CGM revealed that teneligliptin administration significantly reduced the mean daytime (6:00–21:00) and 24-hour blood glucose levels, SD, and MAGE. The proportion of time in hyperglycemia reduced to one-third after teneligliptin treatment (Table 3). Table 1 Baseline characteristics of participants Characteristics Age (years) Sex (men/women) Body mass index (kg/m2) HbA1c (%) Fasting blood glucose (mg/dL) Creatinine (mg/dL) estimated glomerular filtrating ratio (mL/min/1.73 m2) Total cholesterol (mg/dL) Low-density lipoprotein cholesterol (mg/dL) High-density lipoprotein cholesterol (mg/dL) Triglyceride (mg/dL) Duration of T2DM (years) Treatment None Sulfonylurea Thiazoridine

Value 68.6 ± 6.4 5 /5 24.4 ± 3.1 8.3 ± 1.4 123.3 ± 23.7 0.77 ± 0.20 69.7 ± 17.0 204 ± 39.8 129.5 ± 33.4 43.2 ± 6.7 138.4 ± 36.9 5.6 ± 4.7 5 3 2

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Fig. 1 Effects of teneligliptin on glucose and insulin in the meal tolerance test. The changes in (A) plasma glucose (before vs. after, P < 0.0001), (B) glucose AUC0-180, (C) insulin (before vs. after, P = 0.047), and (D) insulin AUC0-180. Open and closed circles indicate before and after teneligliptin administration, respectively. *, P < 0.05 vs. before teneligliptin administration.

Table 2 Parameters of beta-cell function and insulin resistance before and 3 days after the start of teneligliptin treatment Before 3 days after P value Insulinogenic index 0.17 ± 0.06 0.29 ± 0.15 0.002 Oral disposition index

0.031 ± 0.008

0.076 ± 0.04

0.002

AUC 60-180 /AUC 60-180 ratio

0.15 ± 0.12

0.13 ± 0.11

0.0078

HOMA-R

1.78 ± 0.84

1.35 ± 1.19

0.11

HOMA-β

35.5 ± 17.9

33.2 ± 27.0

0.56

insulin

glucose

Values are means ± SD.

Table 3 Indices of daily blood glucose fluctuations measured by CGM in patients before or 1–3 days after the start of teneligliptin treatment Before 1–3 days after P value 24-h mean glucose level (mg/dL) 162.6 ± 16.7 144.7 ± 13.9 0.014 Day time (6:00–21:00) mean glucose level (mg/dL) 177.3 ± 14.4 154.8 ± 18.0 0.016 Night time (21:00–6:00) mean glucose level (mg/dL) 136.8 ± 25.2 127.6 ± 14.3 0.20 SD of 24-h glucose levels (mg/dL) 38.9 ± 12.1 27.6 ± 12.8 0.0078 MAGE (mg/dL) 83.1 ± 31.5 64.5 ± 29.1 0.047 Proportion (%) of time of Hyperglycemia (> 180 mg/dL) 33.5 ± 11.1 13.4 ± 14.3 0.0078 Hypoglycemia (< 70 mg/dL) 0 0 Values are means ± SD.

Teneligliptin improves hyperglycemia

Hypoglycemia occurred neither before nor after teneligliptin treatment. Gastrointestinal hormones Although plasma glucagon levels did not differ significantly between before and after teneligliptin treatment at any time points during the meal tolerance test (Fig. 2A), the glucagon AUC0–180 3 days after teneligliptin treatment was significantly lower than the value before administration (Fig. 2B). Plasma active GLP-1 levels at all time points, as well as active GLP-1 AUC0– 180, significantly increased after teneligliptin treatment (Fig. 2C, D). Basal active GIP level tended to increase after teneligliptin treatment (P = 0.055), and plasma active GIP levels from 30 to 180 min and active GIP AUC0–180 were significantly elevated after teneligliptin treatment (Fig. 2E, F). The percentage inhibition of plasma DPP-4 activity 3 days after teneligliptin administration was 68.8 ± 23.8 %. Plasma ghrelin and des-acyl ghrelin levels at 30 to 180 min after the meal fell significantly from the baseline as described previously [19] in both before and after the teneligliptin treatment. Both plasma ghrelin and des-acyl ghrelin levels, as well as the AUCs for both compounds, were unaltered by teneligliptin treatment (Fig. 2G–J).

Discussion Here we showed that 3-day teneligliptin treatment improved early-phase glucose-stimulated insulin secretion and decreased inappropriate glucagon secretion, resulting in reduction of the postprandial insulin requirement and blood glucose levels throughout the day. Furthermore, using incretin immunoassays in combination with the solid-phase extraction method, we showed that teneligliptin increased pre- and postprandial active incretin levels. In previous studies of people with T2DM, both insulin and C-peptide responses to mixed meals were unaltered both after 1–12-week sitagliptin treatments and after 5–10-day vildagliptin treatments [20-22]. In this study, early-phase mixed meal initiated insulin secretion was improved, and both postprandial insulin levels and glucose levels were significantly reduced, after 3-day teneligliptin treatment. This is the first demonstration of a reduction in postprandial insulin requirement concomitant with a glucose-lowering effect in response to treatment with a DPP-4 inhibitor. Because teneligliptin treatment was started 1 week after hos-

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pitalization, appropriate diet therapy might have augmented the drug’s effects. In addition, the test meal, which contained 43.3% fat, may have influenced insulin secretion and nutrient absorption. Fasting blood glucose levels were reduced after teneligliptin treatment, whereas serum insulin levels from 0 to 30 min were similar before and after the treatment. The early-phase insulin secretion parameters, estimated from the insulinogenic and oral disposition indices, were significantly higher after teneligliptin treatment. Impaired early-phase insulin secretion causes postprandial hyperglycemia and late-phase excessive insulin secretion, whereas elevated early-phase insulin secretion results in reductions in postprandial total insulin requirement and blood glucose concentrations. Hyperinsulinemia accelerates obesity and atherosclerosis [23], and is associated with cancer risk [24]. Thus, long-term teneligliptin treatment may offer aid in preventing complications of diabetes by reducing the insulin requirement. Future studies should evaluate the effect of long-term efficacy of teneligliptin. The use of DPP-4 inhibitors for the treatment of T2DM has been firmly established. Teneligliptin binds to three sites on DPP-4: the S1, S2, and S2 extensive subsites [25]. This may increase DPP-4 inhibition beyond the level afforded by the fundamental interactions with the S1 and S2 subsites that occur between DPP-4 and other types of inhibitors [25]. The mean half-life of teneligliptin (administered once daily, 20 mg/day) was 24.2 h [26]. The percentage inhibition of plasma DPP-4 activity 24 h after administration in this study was 68.8%, consistent with a previously published result (61.8%) [26]. The results of CGM and the meal tolerance test indicated that teneligliptin improved not only postprandial blood glucose levels but also overall management of blood glucose fluctuations. The long-lasting blood glucose-lowering effect of teneligliptin could result from its long half-life and high inhibitory potency. The elevation of early-phase insulin secretion and the reduction in fasting blood glucose level and glucagon levels after teneligliptin treatment may be due to the drug’s long half-life. Glucagon secretion is suppressed after meals in healthy participants, but elevated in people with diabetes [27]. In one study, 4-week vildagliptin treatment of people with diabetes significantly suppressed glucagon AUC0–120 by 9% (from 281.3 ± 10.8 to 254.1 ± 11.9 ng∙h /L) [28]. In another study, performed on Japanese people with diabetes, 12-week sitagliptin treat-

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Fig. 2 Effects of teneligliptin on gut hormones in the meal tolerance test. The changes in (A) plasma glucagon (before vs. after, P = 0.13), (B) glucagon AUC0-180, (C) active GLP-1 (before vs. after, P < 0.0001), (D) active GLP-1 AUC0-180, (E) active GIP (before vs. after, P < 0.0001), (F) active GIP AUC0-180, (G) ghrelin (before vs. after, P = 0.13), (H) ghrelin AUC0-180, (I) desacyl ghrelin (before vs. after, P = 0.13), and (J) des-acyl ghrelin AUC0-180. Open and closed circles indicate before and after teneligliptin administration, respectively. *, P < 0.05 vs. before teneligliptin administration.

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ment significantly reduced glucagon AUC0–120 from 238.9 ± 75.6 to 213.1 ± 76.9 pg∙h/mL after a meal tolerance test. In this study, glucagon AUC0–180 exhibited a significant decrease, from 226.2 ± 74.9 to 201.7 ± 60.5 pg∙h/mL, following teneligliptin administration. Thus, teneligliptin-induced suppression of glucagon secretion could also be responsible for improved glucose metabolism. Because GLP-1 and GIP are rapidly degraded by DPP-4 [29], blood samples must be treated carefully between collection and measurements. The blood samples used in this study were directly withdrawn into pre-chilled tubes containing DPP-4 inhibitor, and then immediately centrifuged. Peptides were extracted from the plasma by the solid-phase extraction method in order to prevent substances present in the plasma from interfering with the assay [9]. Using this method, we showed that teneligliptin significantly increased both fasting and postprandial plasma concentrations of active GLP-1 and postprandial active GIP. Eto et al. reported that teneligliptin increased the concentration of postprandial plasma active GLP-1, relative to placebo [26]. Teneligliptin’s long half-life and high inhibitory activity might be responsible for these effects. Teneligliptin did not alter pre- or postprandial plasma ghrelin, des-acyl ghrelin, or the ghrelin/desacyl ghrelin ratio (data not shown). Ghrelin, an orexigenic gastric peptide, is not a substrate of DPP-4. In earlier studies, the meal-induced decrease in ghrelin did not differ in the presence or absence of single dose of sitagliptin [30] or after 10-day vildagliptin treatment [31]. In this study, we confirmed these findings using

highly sensitive fluorescence-based enzyme immunoassays specific for the individual acylated and des-acylated forms of ghrelin [14]. This study has some limitations. Specifically, the number of participants studied was small, and the duration of teneligliptin administration was short. Furthermore, circulating DPP-4 does not necessarily reflect the activity of the enzyme expressed on the vascular endothelium, which could play a major role in GLP-1 inactivation. Further studies are therefore necessary in order to more completely characterize the efficacy of teneligliptin. In summary, our results show that short-term teneligliptin treatment improves glycemic control in Japanese diabetic patients, associated with an increase in early-phase insulin secretion and reductions in both postprandial insulin requirement and inappropriate glucagon secretion.

Acknowledgments The authors are very thankful to H. Shibata and M. Oshikawa in the Division of Neurology, Respirology, Endocrinology and Metabolism, Department of Internal Medicine, University of Miyazaki. This study was funded in part by the Japan Vascular Disease Research Foundation, whose support is deeply appreciated.

Disclosure M. Nakazato has received research grants and honoraria from the Mitsubishi Tanabe Corporation.

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