Effects of Gastric pH on Mitiglinide Absorption（Cmax and Tmax）
Objectives: Mitiglinide（MGN）, a rapid-acting secretagogue （glinides）, is an oral hypoglycemic medication which can be taken by type-2 diabetic patients before meal. We examined the possibility of gastric absorption of MGN, and the relationship between this absorption and the gastric pH in rats.
Methods: We investigated the absorption of orally administered MGN（4.2 mg/kg）with and without an antacid, in both untreated rats（the control and antacid groups）and in pylorus-ligated（PL）rats（the PL-control and PL-antacid groups）. Gastric pH was higher than 5 in the rats with an antacid.
Results & Discussion: In untreated rats, Cmax significantly decreased and Tmax significantly prolonged（P < 0.05）in the antacid group as compared to the control group, while there was no significant difference in AUC between the control group and the antacid group. Additionally, this change in absorption caused the loss of the rapid- and short-acting properties of antihyperglycemic action by MGN. In pylorus-ligated rats, there was no significant difference in Cmax, Tmax, and AUC between the PL-control group and the control group, and Cmax and AUC significantly decreased（P < 0.05）and Tmax significantly prolonged（P < 0.05）in the PL-antacid group when compared to the PL-control group. It appears that MGN, a mildlyacidic drug（pKa = 4.43）, was absorbed via the stomach and the gastric absorption was delayed when the gastric pH is higher than 5.
Conclusion : This study has shown that the delay of absorption of MGN could induce prolonged antihyperglycemic action（as do sulfonylurea hypoglycemic agents）where the gastric pH was higher than 5.
Mitiglinide（MGN）is an oral hypoglycemic medication sold in Japan and China for type-2 diabetic patients. MGN stimulates insulin secretion in a short period of time and induces hypoglycemic action by blocking the ATP-dependent K+ channels in pancreatic beta-cells（islets of Langerhans）1）. Maximum drug concentration time（Tmax）and half-life（t1/2）of MGN in healthy volunteers are 0.25 and 1.2 hr, which suggests that MGN is quickly absorbed and metabolized2）. It is these properties that make MGN an agent designed to reduce postprandial hyperglycemia when taken orally 5 min before meal.
It is reported that rapid-absorption drugs are absorbed via the stomach when administered orally3-5）. Drugs in molecular form can pass through the gastric membrane by passive diffusion because transporters do not exist in the stomach. Therefore, the rate of absorption via the stomach of the molecular forms of mildly-acidic substances increases when the gastric pH value is low（e.g., on an empty stomach）, according to the Henderson-Hasselbalch equation. However, when the gastric pH value is high, the absorption of mildlyacidic substances can be suppressed6）.
Consequently, we hypothesized that as a mildlyacidic drug（pKa = 4.43）MGN would demonstrate immediate efficacy because of its absorption via the stomach, and that this absorption would be affected by the gastric pH, however there was no report about absorption of MGN. In this study, we examined the possibility of gastric absorption of MGN, and relationship between this absorption and the gastric pH in rats.
Materials and Methods
1. Reagents and Preparation of MGN solution
MGN calcium hydrate（99.7% pure MGN）was generously provided by Kissei Pharmaceutical Co., Ltd. Other reagents and solvents were of HPLC or Guaranteed Reagent grade. As a solvent of MGN, distilled water and 1.3% or 7.8% of sodium bicarbonate （NaHCO3）solution were used to adjust MGN concentration to be 1.4 mg/mL（hereafter MGN/H2O, MGN/1.3%NaHCO3 and MGN/7.8%NaHCO3）.
Sprague-Dawley male rats（6 to 7 weeks old, Japan SLC Inc., Shizuoka, Japan）were used as experimental animals. The rats were acclimated for at least one week, during which they were allowed to ingest feed and water ad libitum, with a 12-hr dark/light cycle. The experiment was conducted after the animals had fasted for 18 hr. All experiments were conducted in accordance with the Osaka University of Pharmaceutical Sciences Animal Facility Guidelines.
3. Blood sampling
Rats were cannulated via the right jugular vein 90 min before MGN（4.2 mg/kg）was orally administered. First, untreated rats were divided into two groups: the control group and the antacid group. MGN/H2O was administered to the control group through a gastric tube, and MGN/1.3%NaHCO3 was administered to the antacid group. Next, pylorus-ligated（PL）rats were divided into two groups: the PL control group and the PL-antacid group. MGN/H2O was administered to the PL-control group, and MGN/7.8%NaHCO3 was administered to the PLantacid group. The concentration level of the NaHCO3 solution was determined in a preliminary experiment to raise the gastric pH up to or over 5.0 for both untreated and pylorus-ligated rats（data not shown）. Blood samples（0.3 mL）were collected through the jugular vein cannula at 5, 10, 20, 30, 60, 120, 180, 240, and 300 min after administration of MGN. The blood samples were placed in 200 IU heparinized micro tubes and centrifuged（2100 ×g, 4℃, 10 min）, and the resultant supernatant was used as the plasma sample.
4. Measurement of MGN
One hundred μL of phosphate buffer （pH = 2.0） and 2.0 mL of dichloromethane were added to 100 μL of the plasma sample, and the mixture was stirred for 2 min and then centrifuged（2100 ×g, 10 min, room temperature）. The dichloromethane layer（1.5 mL） was separated, evaporated to dryness（30 min）, and then reconstituted in 50 μL of the mobile phase. An aliquot of the resulting specimen（40 μL）was injected into a high performance liquid chromatography （HPLC）to determine the amount of MGN.
The following HPLC system was used. Pump: LC-10ADvp（Shimadzu Co., Kyoto, Japan）; detector: SPD-10Avp（Shimadzu Co.）; automated sampler: SIL-10AD（Shimadzu Co.）; and column: COSMOSIL 5C18-MS-II 250 L × 4.6 nm I.D.（Nacalai Tesque, Inc. Kyoto, Japan）. A mixture of acetonitrile : phosphoric acid（pH = 1.9）= 43 : 57 was used as the mobile phase for separation. A flow rate of 1.2 mL/min was used to determine the absorbance（at 210 nm）of samples pretreated at a column temperature of 40 ℃. The measurable range was from 100 to 1000 ng/mL. All samples were measured in such dilute quantities that the concentration of MGN fell within 100 to 1000 ng/mL.
5. Measurement of blood-glucose level
Blood-glucose level was measured by the glucose dehydrogenase method using 1.0 μL of a whole blood sample.
6. Statistical analysis
Both Cmax and Tmax were obtained directly from the plasma MGN concentration-time course data. The area under the plasma concentration time curve（AUC）of MGN was calculated by the trapezoidal approximation method. The Student’s t-test, the Dunnett’s or the Tukey’s multiple comparison test was used for comparison of plasma concentration of MGN, pharmacokinetic parameters, and blood-glucose level. The numerical values were expressed as mean ± SD, and P <0.05 were considered to indicate a significant difference. The statistical software add-in for Microsoft Excel called“Statcel”（ver.2, OSM publishing Inc.） was used for statistical analysis.
Plasma concentration and pharmacokinetic parameters of MGN in the control and antacid groups were compared（Fig. 1, Table 1）. Cmax and Tmax in the control group were 6961 ± 1316 ng/mL and 5 min; those in the antacid group were 3925 ± 1230 ng/mL and 15 ± 5.8 min. Cmax significantly decreased and Tmax significantly prolonged in the antacid group as compared to the control group（P < 0.01）. While plasma concentrations of MGN in the control group at 240 and 300 min after administration were 48 ± 20 ng/mL and 38 ± 11 ng/mL, those in the antacid group were 139 ± 61 ng/mL and 97 ± 31 ng/mL. Therefore, in the antacid group, plasma concentration of MGN was significantly higher than that in the control group（P < 0.05）. In contrast, there was no significant difference in AUC between both groups（the control group: 2471 ± 196 ng・hr/mL, the antacid group: 2413 ± 840 ng・hr/mL）. There was no significant difference in Cmax, Tmax, and AUC of MGN between the PL-control group and the control group（Table 1）.
Plasma concentration and pharmacokinetic parameters of MGN in the PL-control and PL-antacid groups were compared （Fig. 2, Table 1）. Cmax, Tmax, and AUC of MGN in the PL-control group were 6034 ± 1853 ng/mL, 5 min, and 2776 ± 647 ng・hr/mL; those in thePL-antacid group were 354 ± 188 ng/mL, 44 ± 25 min and 977 ± 434 ng・hr/mL. A significant decrease of Cmax, significant prolongation of Tmax, and significant decrease of AUC（P < 0.01）were shown in the PLantacid group as compared to the PL-control group.
Blood-glucose level in the antacid group reached its lowest level at 60 min after administration（64.5 mg/dL）and was significantly higher（P < 0.05）than that in the control group（53.3 mg/dL, Fig. 3）. The blood-glucose level at 5 min after MGN administration of each group was defined as the initial glucose level, and the initial glucose level was compared with the level at 10, 20, 30, 60, 120, 180, 240, 300 min after administration. In the control group, the blood-glucose level significantly decreased from the initial glucose level（124 mg/dL）from 20 to 120 and 240 min（P <0.05）, while there was no significant difference at 180 and 300 min. In the antacid group, on the other hand, the blood-glucose level significantly decreased from the initial glucose level（124 mg/dL）from 30 to 300 min after MGN administration（P < 0.05）.
MGN, a rapid-acting secretagogue（glinides）, hasrapid- and short-acting activity in stimulating insulin secretion, which effectively prevents postprandial hyperglycemia1）. When drugs are administered orally, they are usually absorbed by the small intestine. Thus, the effect of these drugs usually manifests itself one hour or later after administration. We hypothesized that MGN（Tmax is 0.25 hr）would be absorbed via the stomach by passive diffusion and thereby demonstrated immediate efficacy.
In the antacid group, Cmax significantly decreased （P < 0.05）and Tmax significantly prolonged（P < 0.05） as compared to the control group, but there was no significant difference in AUC between the control group and the antacid group（Table 1）. Additionally, plasma concentrations of MGN at 240 and 300 min after MGN administration were significantly higher in the antacid group than that in the control group（Fig.1）. These suggested that the absorption of MGN was delayed when the gastric pH is higher than 5.
Blood-glucose level in the antacid group reached its lowest level at 60 min after administration and it was significantly higher（P < 0.05）than that in the control group（Fig. 3）. In the control group, the bloodglucose level significantly decreased from its initial level from 20 to 120 and at 240 min after administration（P < 0.05）, but there was no significant difference at 180 and 300 min. MGN appears to remain effective from 20 to 120 min after administration in the control group. In the antacid group, the blood-glucose level significantly decreased from its initial level from 30 to 300 min after MGN administration（P < 0.05）, indicating that MGN began to exert the effect at 30 min after administration and remained effective from 30 to 300 min after MGN administration in the antacid group. These results indicate the fact that decreased Cmax and prolonged Tmax affected the antihyperglycemic action of MGN, the loss of rapid- and short-acting property.
Then, pylorus-ligation was performed to limit absorption of MGN to only the stomach. There were no significant differences in Cmax, Tmax, and AUC between the PL-control group and the control group （Table 1）. This is consistent with the fact that MGN is absorbed almost exclusively from the stomach. As compared to the PL-control group, Cmax and AUC significantly decreased（P < 0.05）and Tmax significantly prolonged（P < 0.05）in the PL-antacid group, suggesting that the absorption value via the stomach decreased when the gastric pH was higher than 5（due to passive diffusion）. The AUC ratio of the PL-antacid group to the PL-control group was 39.5%, whereas in fact the ratio of MGN in molecular form, which can be absorbed by passive diffusion, is less than 21% when the gastric pH is higher than 5. The difference between theoretical value and the actual absorption value is considered to be attributable to a gradual increase of MGN in molecular form. MGN in molecular and ionic form maintains a balanced ratio, unless MGN in molecular form in the stomach increases with its absorption into the blood.
In this study we showed that MGN, a mildly-acidic drug（pKa = 4.43）, was absorbed via the stomach and began to exert efficacy from 20 min after administration under fasting conditions, although this efficacy disappeared within 120 min. When the gastric pH was higher than 5, this gastric absorption of MGN delayed （decrease of Cmax, prolongation of Tmax）and remained effective from 30 to 300 min after administration, as sulfonylurea hypoglycemic agents would. Therefore it is preferable to take MGN 5-15 min before meal. In clinical practice, there were reports about delayed absorption of MGN taken after meal and other mildlyacidic drugs with antacids7,8）. This study indicated that increased the gastric pH at 5 and over could induce this delay in absorption. Additionally, the gastric pH of patients taking acid reducers may be higher than 5 as in the antacid group in this study9,10, so further investigation is required about the relationship between the delayed action of MGN and hypoglycemia in the patients taking acid reducers.
This study has shown that the delay of absorption of MGN could induce prolonged antihyperglycaemic action（as do sulfonylurea hypoglycemic agents） when the gastric pH was higher than 5.