Pilot Study of Miglitol and Sitagliptin for T2DM
Pilot Study of Miglitol and Sitagliptin for T2DM
The baseline characteristics of the 3 Japanese patients with type 2 diabetes who were treated with diet therapy alone or with oral hypoglycemic agents other than α-GIs or DPP-4 inhibitors, and 1 Japanese female control subject are summarized in Table 1 The value for haemoglobin A1c (HbA1c) (%) was converted to National Glycohemoglobin Standardization Program (NGSP) levels by using the formula: HbA1c (%)(NGSP) = HbA1c [Japan Diabetic Society (JDS)] (%) + 0.4%, considering the relational expression of HbA1c (JDS) (%) measured by the previous Japanese standard substance and measurement methods. All anti-diabetic drugs were washed out for 5 days before each experimental day. The study protocol was approved by the Ethics Committee of the National Center for Global Health and Medicine (NCGM), and written informed consent was obtained from each study subject. The study was conducted in accordance with the ethical principles stated in the Declaration of Helsinki.
CGMS The patients were admitted to a diabetic ward at the NCGM and equipped with a CGMS device (CGMS-GOLD; Medtronic MiniMed, Northridge, CA, USA). The subjects were monitored for 3 days before the study and until the end of the 5-day study period. The CGMS soft sensor was changed at the proper times according to the manufacturer's instructions. Glucose levels measured with a self-monitoring blood glucose (SMBG) device (Nipro Stat Strip XP; Nipro, Japan) were checked at least 4 times per day for calibration of the CGMS. The recorded data were analyzed with CGMS Solutions software.
Breakfast The calories of each breakfast through the study period were determined, considering age and body mass index (BMI) of each patient and the control subject. For example, the mean calories for patients in case 1, 2, 3, and the control subject were 310, 413, 400, and 470 kcal, respectively.
On the first day of the study, after an overnight fast for 14 h, an intravenous line was inserted into 1 forearm vein and flushed with sterile 0.9% NaCl solution for repeated blood sampling. Blood was drawn at 0, 30, 60, 120, 180, and 240 min after breakfast (the meal was ingested within 10–15 min) for measurements of serum insulin, 1,5-AG, plasma glucagon, GLP-1, and GIP. Blood samples were immediately cooled and centrifuged at 4°C, and plasma was stored at ™20°C until analysis. Blood samples for determination of active GLP-1 were collected into chilled BD P700 tubes containing spray-dried K2EDTA anticoagulant and proprietary DPP-4 protease inhibitor (Becton Dickinson, Franklin Lakes, NJ, USA), and the GLP-1 concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Millipore Corporation, Billerica, MA, USA). Plasma concentrations of amidated GLP-1 (7–36) and (7–37) were measured using an antibody that is highly specific for the N-terminus of GLP-1 and does not react with GLP-1 (9–36), GLP-2, or glucagon. The detection limit of the ELISA was 2 pmol/l, with an intra-assay coefficient of variation (CV) of 2.6%-6.0% and interassay CV of 7.1%-9.8%. Total GIP was measured using a human GIP ELISA kit (Millipore Corporation). This kit has 100% cross reactivity to human GIP (1–42) and GIP (3–42), with a detection limit of 1.8 pmol/l, an intraassay CV of 4.8%-6.3%, and an interassay CV of 2.2%-5.0%. Serum insulin was measured using a chemiluminescent enzyme immunoassay (CLEIA). Blood samples for glucagon measurements were collected in tubes containing EDTA-2Na plus aprotinin and analyzed with a double-antibody radioimmunoassay. Serum 1,5 AG was measured using an enzymatic method (Nippon Kayaku, Tokyo, Japan). All sample measurements were performed at SRL, Inc. (Tokyo, Japan). Areas under the curve (AUC) values for these hormones after meal ingestion were calculated using the trapezoidal rule.
From the second day to the end of the study period, the subjects were prescribed miglitol (50 mg), 3 tablets per day just before every meal. From day 4 through day 5, the subjects were also prescribed sitagliptin (50 mg), 1 tablet per day before breakfast. Blood sampling was also performed on the third day and the fifth day of the study. The sampling methods and timing were identical for each blood sampling period.
Methods
Subjects
The baseline characteristics of the 3 Japanese patients with type 2 diabetes who were treated with diet therapy alone or with oral hypoglycemic agents other than α-GIs or DPP-4 inhibitors, and 1 Japanese female control subject are summarized in Table 1 The value for haemoglobin A1c (HbA1c) (%) was converted to National Glycohemoglobin Standardization Program (NGSP) levels by using the formula: HbA1c (%)(NGSP) = HbA1c [Japan Diabetic Society (JDS)] (%) + 0.4%, considering the relational expression of HbA1c (JDS) (%) measured by the previous Japanese standard substance and measurement methods. All anti-diabetic drugs were washed out for 5 days before each experimental day. The study protocol was approved by the Ethics Committee of the National Center for Global Health and Medicine (NCGM), and written informed consent was obtained from each study subject. The study was conducted in accordance with the ethical principles stated in the Declaration of Helsinki.
Procedure
CGMS The patients were admitted to a diabetic ward at the NCGM and equipped with a CGMS device (CGMS-GOLD; Medtronic MiniMed, Northridge, CA, USA). The subjects were monitored for 3 days before the study and until the end of the 5-day study period. The CGMS soft sensor was changed at the proper times according to the manufacturer's instructions. Glucose levels measured with a self-monitoring blood glucose (SMBG) device (Nipro Stat Strip XP; Nipro, Japan) were checked at least 4 times per day for calibration of the CGMS. The recorded data were analyzed with CGMS Solutions software.
Breakfast The calories of each breakfast through the study period were determined, considering age and body mass index (BMI) of each patient and the control subject. For example, the mean calories for patients in case 1, 2, 3, and the control subject were 310, 413, 400, and 470 kcal, respectively.
Sample Collection and Analysis
On the first day of the study, after an overnight fast for 14 h, an intravenous line was inserted into 1 forearm vein and flushed with sterile 0.9% NaCl solution for repeated blood sampling. Blood was drawn at 0, 30, 60, 120, 180, and 240 min after breakfast (the meal was ingested within 10–15 min) for measurements of serum insulin, 1,5-AG, plasma glucagon, GLP-1, and GIP. Blood samples were immediately cooled and centrifuged at 4°C, and plasma was stored at ™20°C until analysis. Blood samples for determination of active GLP-1 were collected into chilled BD P700 tubes containing spray-dried K2EDTA anticoagulant and proprietary DPP-4 protease inhibitor (Becton Dickinson, Franklin Lakes, NJ, USA), and the GLP-1 concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Millipore Corporation, Billerica, MA, USA). Plasma concentrations of amidated GLP-1 (7–36) and (7–37) were measured using an antibody that is highly specific for the N-terminus of GLP-1 and does not react with GLP-1 (9–36), GLP-2, or glucagon. The detection limit of the ELISA was 2 pmol/l, with an intra-assay coefficient of variation (CV) of 2.6%-6.0% and interassay CV of 7.1%-9.8%. Total GIP was measured using a human GIP ELISA kit (Millipore Corporation). This kit has 100% cross reactivity to human GIP (1–42) and GIP (3–42), with a detection limit of 1.8 pmol/l, an intraassay CV of 4.8%-6.3%, and an interassay CV of 2.2%-5.0%. Serum insulin was measured using a chemiluminescent enzyme immunoassay (CLEIA). Blood samples for glucagon measurements were collected in tubes containing EDTA-2Na plus aprotinin and analyzed with a double-antibody radioimmunoassay. Serum 1,5 AG was measured using an enzymatic method (Nippon Kayaku, Tokyo, Japan). All sample measurements were performed at SRL, Inc. (Tokyo, Japan). Areas under the curve (AUC) values for these hormones after meal ingestion were calculated using the trapezoidal rule.
Medications
From the second day to the end of the study period, the subjects were prescribed miglitol (50 mg), 3 tablets per day just before every meal. From day 4 through day 5, the subjects were also prescribed sitagliptin (50 mg), 1 tablet per day before breakfast. Blood sampling was also performed on the third day and the fifth day of the study. The sampling methods and timing were identical for each blood sampling period.
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