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Effects of 18α-glycyrrhizin on the pharmacodynamics and pharmacokinetics of glibenclamide in alloxan-induced diabetic rats
European Journal of Pharmacology 587 (2008) 330–335
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Effects of 18α-glycyrrhizin on the pharmacodynamics and pharmacokinetics of glibenclamide in alloxan-induced diabetic rats Ying Ao a, Jie Chen b, Jiang Yue a, Ren-Xiu Peng a,⁎ a b
Department of Pharmacology, Medical College of Wuhan University, Wuhan 430071, PR China Biological Engineering Department, Wuhan Institute of Technology, Wuhan 430074, PR China
A R T I C L E
I N F O
Article history: Received 4 September 2007 Received in revised form 28 February 2008 Accepted 13 March 2008 Available online 8 April 2008 Keywords: Cytochrome P-450 3A 18α-glycyrrhizin Glibenclamide Hypoglycemic effect Pharmacokinetic
A B S T R A C T This paper investigated the effects of 18α-glycyrrhizin (18α-GL) on the pharmacodynamics and pharmacokinetics of glibenclamide in experimental diabetic rats. 18α-GL (25 mg/kg) and/or glibenclamide (1 mg/kg) were given to alloxan-induced diabetic rats for consecutive 5 days. When the rats were co-treated with 18α-GL and glibenclamide, fasting plasma glucose concentration was further reduced, plasma insulin content and liver glycogen level were increased markedly as compared with glibenclamide-treated animals. Meanwhile, in co-treated group, elimination rate constant (Ke) of glibenclamide was reduced while peak plasma concentration (Cmax), area under the plasma concentration vs time curve (AUC0–14 h) and elimination half-life (T1/2Ke) were increased significantly vs glibenclamide alone administered rats. The activities of hepatic CYP3A and the markers of liver injury, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were significantly decreased in rats treated with 18α-GL alone and in combination with glibenclamide. Results of immunohistochemistry showed that 18α-GL improved the effects of glibenclamide on the pathological morphology of pancreatic islet β cells and the intensities of positive immunostaining for insulin. Our results revealed that 18α-GL led to the enhancement of the hypoglycemic effect of glibenclamide by inhibiting the activity of CYP3A; on the other hand, 18α-GL protected the pancreatic islet β cells and liver from damage in diabetes which suggested that 18α-GL might be beneficial as an adjuvant drug of glibenclamide in a proper dose, especially to the diabetic patients associated with liver dysfunction. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Glibenclamide is a sulfonylurea oral hypoglycemic agent which is widely used for the treatment of type 2 diabetes mellitus. It produces the hypoglycemic effect primarily by stimulating insulin secretion from β cells of pancreatic islets. It was reported that glibenclamide was metabolized mainly in liver by cytochrome P-450 (CYP) 3A4. In addition, CYP2C9 and 2C19 also took part in its metabolism (van Giersbergen et al., 2002; Naritomi et al., 2004). The hypoglycemic effect of glibenclamide was changed during co-administration with CYP inhibitor ciprofloxacin (Roberge et al., 2000), thus it is necessary for us to study the interaction between glibenclamide and other drugs to avoid adverse effects especially hypoglycemia. 18α-glycyrrhizin (18α-GL) is one of the main active components of traditional Chinese medicine—Licorice (Radix Glycyrrhizae). Due to its efficacy and safety, 18α-GL is often used as a hepatic protective agent in clinic, especially for the treatment of liver dysfunction. Many authors
⁎ Corresponding author. Department of Pharmacology, Medical College of Wuhan University, Donghu Road, Wuhan, 430071, PR China. Tel.: +86 27 68758665; fax: +86 27 87331670. E-mail address: [email protected] (R.-X. Peng). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.03.043
believe that diabetes is accompanied with hepatic impairment (Caldwell et al., 2004; Ratziu et al., 2004), therefore, it is possible that glibenclamide and 18α-GL are administered concomitantly. Our previous studies showed that 18α-GL inhibited liver drug-metabolizing phase enzymes (Yang et al., 2001), but it is unknown whether 18αGL affects the metabolism and hypoglycemic effect of glibenclamide. Thus, the aim of the present study was to investigate the effect and mechanism of 18α-GL on the pharmacodynamics and pharmacokinetics of glibenclamide in experimental diabetic rats to sufficiently recognize the potential combined effect of concomitant treatment with 18α-GL and glibenclamide. 2. Materials and methods 2.1. Chemicals Glibenclamide, butyl 4-hydroxybenzoate, erythromycin, isocitric acid, isocitric acid dehydrogenase, NADP, bovine serum albumin (BSA), alloxan tetrahydrate were purchased from Sigma (U.S.A.). 18αdiammonii glycyrrhizinatis was from Chia-tai Tianqing Pharmaceutical Co (China). Methanol of HPLC-grade was from Fisher (U.S.A.) and all the other chemicals and reagents were of analytical grade.
Y. Ao et al. / European Journal of Pharmacology 587 (2008) 330–335
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2.3. Biochemical analysis Blood sample was withdrawn in a heparinized capillary tube from the retro-orbital venous plexus under light ether anaesthesia. Plasma glucose levels were checked by glucose oxidase method. Plasma insulin content was measured by radioimmunoassay technique using insulin RIA kit (North Institute of Biological Technology, China). Activities of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assessed using test kits (Rongsheng Biotechnology, China). Rats were sacrificed with an overdose of anesthetic and the livers were excised. Hepatic glycogen level was assayed enzymatically using specific kit (Jiancheng Bio-Tek, China). Liver microsomes were prepared by differential ultra-centrifugation. Erythromycin N-demethylase (ERD) activity, as the marker of CYP3A, was assayed as previously described (Peng et al., 1994). Protein was assayed by the method of Lowry et al. (1951). 2.4. Plasma glibenclamide determination
Fig. 1. HPLC chromatograms of glibenclamide in plasma samples of alloxan-induced diabetic rats. (A) blank plasma, (B) blank plasma spiked with glibenclamide and internal standard, (C) a plasma sample spiked with internal standard. Peaks: 1, Internal standard: Butyl 4-phydroxybenzoate; 2, glibenclamide.
For the pharmacokinetic study, blood samples were collected in heparinized tubes before and 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 14 h after final administration via the right external jugular vein. Plasma glibenclamide concentration was determined by reverse phase high performance liquid chromatography (HPLC) (Srinivas and Nayanabhirama, 2005). The solvent delivery system was a Shimadzu pump model LC-9A (Shimadzu, Japan). The analytical column was Zorbax SB-C18 (4.6 mm ID × 25 cm, 5 μm particle size) purchased from Yilite Scientific Instrument Co., China. Column effluent was monitored with SPD-6AV ultraviolet spectrophotometric detector (Shimadzu, Japan) at 228 nm. The HPLC system was equilibrated with the mobile phase consisting of ammonium dihydrogen phosphate 50 mmol/l (pH 3.0): methanol (3:5, by volume), at a flow-rate of 1.0 ml/min. Plasma samples were denatured by trichloracetic acid, and then centrifuged at 15,000 ×g for 10 min. The supernatant was transferred and a 20 μl volume was injected into the HPLC system for quantitation. The calibration curve of glibenclamide concentration (C) vs the ratio for area (Y) of glibenclamide to butyl 4-hydroxybenzoate resulted in a correlation coefficient (r) of 0.999. And the linear range for glibenclamide in plasma was from 10 to 250 μg/l. The regression equation was Y = 0.009C + 0.0069. The average recovery rate was 100.1%, and the relative standard deviations (RSD) of intra-day and inter-day were all less than 5% (Fig. 1). 2.5. Immunohistochemical assay
2.2. Animals and treatment Male Wistar rats weighing 190–200 g (Certificate No 19-088), SPF grade, were supplied by Experimental Animals Center, Hubei Province, China. Throughout the study, rats were housed under the same adequate environmental conditions, with a 12/12 h light/dark cycle. They were fed with standard laboratory chow and water ad libitum. All the animals were cared for according to the rules and regulations of the Institutional Animal Ethics Committee (IAEC) guidelines of the Wuhan University, China. For the induction of diabetes, rats were kept on fasting for 24 h prior to alloxan injection. On the day of administration, alloxan tetrahydrate was freshly prepared in normal saline and intraperitoneal injection (i.p.) was given at the dosage of 150 mg/kg. Fasting plasma glucose concentration was checked by glucose oxidase method (Trinder, 1969) 3 days after alloxan treatment. The animals with glucose level exceeding 13.89 mmol/l were considered as hyperglycemic. All the rats were divided into 5 groups, each containing 6 animals. ➀ Control group. ➁ Diabetic group. ➂ Diabetic rats treated with glibenclamide (1 mg/kg, i.g.). ➃ Diabetic rats treated with 18α-GL (25 mg/kg, i.p.). ➄ Diabetic animals treated with glibenclamide (1 mg/ kg, i.g.) and 18α-GL (25 mg/kg, i.p.). The treatment was continued for 5 days. Before the final administration, animals were fasted overnight.
Cauda pancreatis was preserved in neutral buffered 10% formalin and then embedded in paraffin, sectioned at 5 μm. Immunohistochemistry for insulin was performed using a combination streptavidin–biotin–peroxidase method. The negative control was performed by omitting the primary antibody. The quantitative expression of
Table 1 Plasma glucose concentration in alloxan-induced diabetic rats administered with glibenclamide and/or 18α-GL Groups
Control Diabetic Glibenclamide 18α-GL Glibenclamide +18α-GL
Plasma glucose concentration (mmol/l) 0h
2h
4h
6h
4.27 ± 0.49 21.81 ± 5.27a 21.72 ± 5.14a 19.91 ± 3.90a 21.52 ± 7.00a
4.14 ± 0.43 22.70 ± 4.16a 17.64 ± 4.76a, b 20.29 ± 5.10a 16.95 ± 2.98a, b
3.92 ± 0.22 22.76 ± 3.29a 17.29 ± 5.90a, b 19.90 ± 6.78a 12.84 ± 3.47a, c
4.01 ± 0.93 22.65 ± 2.66a 16.28 ± 7.20a, b 19.20 ± 5.88a 7.08 ± 3.39c, d
Glibenclamide (1 mg/kg, i.g.) and/or 18α-GL (25 mg/kg, i.p.) were given once daily for 5 days to the alloxan (150 mg/kg, i.p.)-induced diabetic rats. Plasma glucose concentration was measured at 0, 2, 4 and 6 h after final administration. Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin. aP b 0.01 vs control group; bP b 0.05, cP b 0.01 vs diabetic group; dP b 0.01 vs glibenclamide group.
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Table 2 The fasting plasma insulin, Insulin Sensitivity Index (ISI) and liver glycogen in alloxaninduced diabetic rats administered with glibenclamide and/or 18α-GL
Table 3 The activities of plasma transaminases and liver index in alloxan-induced diabetic rats administered with glibenclamide and/or 18α-GL
Groups
Groups
Control Diabetic Glibenclamide 18α-GL Glibenclamide + 18α-GL
Insulin
ISI
Liver glycogen
(mU/l)
(×10 3)
(mg/g)
24.8 ± 9.6 7.1 ± 1.7b 11.7 ± 5.8a, c 8.6 ± 3.4b 19.3 ± 8.2d, e
11.3 ± 2.9 6.7 ± 1.9b 7.4 ± 4.2 7.3 ± 2.9a 8.9 ± 1.4c
27.9 ± 22.5 14.4 ± 7.8 22.5 ± 7.4c 28.3 ± 11.6c 39.3 ± 15.2d, e
See Table 1 for treatment. ISI = 1 / fasting plasma glucose × insulin. The indices were measured at 6 h after final administration. Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin. aP b 0.05, bP b 0.01 vs control group; c P b 0.05, dP b 0.01 vs diabetic group; eP b 0.05 vs glibenclamide group.
Control Diabetic Glibenclamide 18α-GL Glibenclamide +18α-GL
Plasma ALT
Plasma AST
Liver index
(µmol/min/l)
(µmol/min/l)
(× 103)
38 ± 12 109 ± 19b 92 ± 18b 50 ± 14d, f 54 ± 20d, f
90 ± 23 186 ± 31b 165 ± 21b 86 ± 29d, f 94 ± 20d, f
29 ± 4 45 ± 8a 40 ± 4a 35 ± 9 27 ± 5c, e
See Table 1 for treatment. Liver index = liver weight / body weight. The indices were measured at 6 h after final administration. Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin; ALT = alanine aminotransferase; AST = aspartate aminotransferase. a P b 0.05, b P b 0.01 vs control group; c P b 0.05, d P b 0.01 vs diabetic group; eP b 0.05, fP b 0.01 vs glibenclamide group.
insulin was detected by HPIAS-1000 pathological image analysis system (Qianping Image Technology, China) via calculating the average optical density in each field.
57% (P b 0.01) vs glibenclamide-treated rats, which indicated that 18αGL potentiated the hypoglycemic effect of glibenclamide.
2.6. Statistical analysis
3.2. Effects of glibenclamide and/or 18α-GL administration on the levels of plasma insulin, Insulin Sensitivity Index (ISI) and liver glycogen
Data were presented as mean ± standard deviation (S.E.M.) and repeated measures analysis of variance (ANOVA) was used for comparing the time-course of plasma glucose concentration. Other results were analyzed by Student's t test or one-way ANOVA. The pharmacokinetic analysis was conducted using the DAS ver 1.0 software. Differences were considered to be significant when P b 0.05. 3. Results 3.1. Effect of 18α-GL on the hypoglycemic action of glibenclamide All the animals which received alloxan injection developed diabetes. The rats that were made diabetes had higher glucose concentration than control non-diabetic animals (Table 1). It was reported that elimination half-life of glibenclamide was 6.4 h (Srinivas and Nayanabhirama, 2005). Therefore, in this study, we monitored the plasma glucose levels at 0, 2, 4 and 6 h after final administration to observe the hypoglycemic effect of glibenclamide. As shown in Table 1, glibenclamide significantly decreased plasma glucose level at 2 h and maintained the hypoglycemic effect during 2–6 h after final treatment, plasma glucose concentration at 6 h was decreased by 28% vs the diabetic rats (P b 0.05). 18α-GL itself did not show significant effect on plasma glucose content vs alloxan treatment, however, plasma glucose level in rats co-treated with 18α-GL and glibenclamide was decreased by 25% (P b 0.05), 44% (P b 0.01) and 69% (P b 0.01) at 2, 4 and 6 h after final administration, respectively, as compared with the diabetic animals; plasma glucose content at 6 h was significantly reduced by
Fig. 2. Liver CYP3A (erythromycin N-demethylase) activity in alloxan-induced diabetic rats administered with glibenclamide and/or 18α-GL. The activity of CYP3A was measured at 6 h after final administration. Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin. ⁎P b 0.05 vs control group.
As shown in Table 2, alloxan-induced diabetic rats showed marked decrease in plasma insulin concentration and Insulin Sensitivity Index (ISI) when compared to the control non-diabetic animals. Administration of glibenclamide caused a significant increase in plasma insulin (P b 0.05), which indicated that glibenclamide provoked insulin secretion, but ISI was not changed by this drug. 18α-GL showed poor effect on insulin content or ISI, whereas it increased liver glycogen level vs alloxan treatment (P b 0.05). When 18α-GL was administrated concurrently with glibenclamide, both of plasma insulin content and liver glycogen level were further increased (P b 0.05) as compared to the animals treated with glibenclamide alone, ISI was also markedly elevated (P b 0.05) vs the diabetic rats. 3.3. Effects of glibenclamide and/or 18α-GL administration on the activities of liver CYP3A, plasma ALT and AST It was reported that glibenclamide was metabolized by CYP3A in liver (van Giersbergen et al., 2002; Naritomi et al., 2004). To investigate the mechanism of 18α-GL enhancing the hypoglycemic effect of glibenclamide, the activity of liver CYP3A was determined. As demonstrated in Fig. 2, hepatic CYP3A activity in diabetic or glibenclamide-treated rats had no change compared with the control. For rats treated with 18α-GL only or co-treated with 18α-GL and glibenclamide, CYP3A level was markedly (P b 0.05) decreased by 54% and 51%, respectively, vs that in diabetic status. The result suggested
Fig. 3. Mean plasma concentration–time curve of glibenclamide in alloxan-induced diabetic rats administrated with glibenclamide alone (empty symbols) and coadministrated with 18α-GL (solid symbols). Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin.
Y. Ao et al. / European Journal of Pharmacology 587 (2008) 330–335 Table 4 Main pharmacokinetic parameters of glibenclamide in alloxan-induced diabetic rats administrated with glibenclamide alone and co-administrated with 18α-GL Parameters
Glibenclamide
Glibenclamide+18α-GL
Ka (1/h) Ke (1/h) T1/2Ka (h) T1/2Ke (h) AUC0–14 h (µg h/l) Tmax (h) Cmax (µg/l)
0.46 ± 0.08 0.34 ± 0.05 1.53 ± 0.23 2.11 ± 0.40 828.56 ± 78.36 3.00 ± 0.55 127.62 ± 8.02
0.39 ± 0.08 0.21 ± 0.04a 1.86 ± 0.40 3.43 ± 0.62a 1315.00 ± 153.05a 3.50 ± 0.55 150.70 ± 13.59a
Ka = absorption rate constant; Ke = elimination rate constant; T1/2Ka = absorption halflife; T1/2Ke = elimination half-life; AUC = area under the plasma concentration vs time curve; Tmax = time to reach Cmax; Cmax = peak plasma concentration. Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin. aP b 0.05 vs glibenclamide group.
that 18α-GL potentiated the glucose-lowering effect of glibenclamide probably by inhibiting the activity of hepatic CYP3A and in turn reducing the metabolism of glibenclamide. It has been pointed out that 18α-GL possesses the effect to diminish liver injury (Zheng and Lou, 2003). To observe whether 18α-GL may protect against liver impairment accompanied with diabetes, we investigate the influence of 18α-GL to the levels of the hepatic damage
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makers. As shown in Table 3, in alloxan-diabetic rats, the activities of plasma ALT and AST were significantly (P b 0.01) increased by 187% and 107%, respectively, relative to their normal levels. When the diabetic rats were treated with glibenclamide, plasma ALT, AST and liver index were not significantly different from that in diabetic status. The results indicated that glibenclamide exerted the antihyperglycemic effect (Table 2), but it could not improve the impaired liver function. However, after the treatment with 18α-GL, the activities of plasma ALT and AST were markedly (P b 0.01) decreased by 54.1% and 53.7%, respectively, compared to the alloxan-diabetic group. When the diabetic rats were co-administered with 18α-GL and glibenclamide, plasma ALT, AST and liver index were significantly reduced by 41% (P b 0.01), 43% (P b 0.01) and 33% (P b 0.05), respectively, compared to the glibenclamide-treated animals. The results demonstrated that 18α-GL could protect the damaged liver in alloxan-induced diabetic rats. 3.4. Pharmacokinetic analysis Mean plasma concentration vs time curve for glibenclamide was illustrated in Fig. 3, and mean plasma pharmacokinetic parameters were summarized in Table 4. For the animals co-treated with 18α-GL and glibenclamide, elimination rate constant (Ke) of glibenclamide
Fig. 4. Immunohistochemical staining of insulin in pancreatic islets of rats. Pancreatic β cells in positive immunostaining were distributed over the center of pancreatic islets, aligned regularly and the endochylema was full of gross insulin particles in deep brown color in control group (A). The immunostaining particles for insulin were decreased significantly in the diabetic group (B). When treated with glibenclamide or 18α-GL, the staining particles were increased (C, D) and the effect further strengthened in rats co-treated with 18α-GL and glibenclamide (E). ×400. 18α-GL = 18α-glycyrrhizin.
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Fig. 5. Expression of insulin in pancreatic islets of alloxan-induced diabetic rats administered with glibenclamide and/or 18α-GL. The index was measured at 6 h after final administration. Data are expressed as means ± S.E.M. for six rats in each group. ⁎ P b 0.05, ⁎⁎ P b 0.01 vs control group; # P b 0.05, ## P b 0.01 vs diabetic group; Δ P b 0.05 vs glibenclamide group. 18α-GL = 18α-glycyrrhizin.
was decreased by 38% (P b 0.05) while peak plasma concentration (Cmax), area under the plasma concentration vs time curve (AUC0–14h) and elimination half-life (T1/2Ke) were markedly (P b 0.05) increased by 18%, 59% and 63%, respectively, as compared with glibenclamidetreated group. The results indicated that 18α-GL exerted the effect to inhibit the elimination of glibenclamide and led to the elevation of Cmax and AUC. 3.5. Immunohistochemical assay for insulin To investigate if 18α-GL has protective effect on damaged pancreas in experimental diabetic rats, we observed the effects of 18α-GL on the pathological morphology of pancreatic islet β cells and the secretion of insulin. The result of image analysis was demonstrated in Fig. 5. In control group, islet β cells in positive immunostaining were distributed over the center of pancreatic islets, aligned regularly and the endochylema was full of gross insulin particles in deep brown color (Fig. 4A). In alloxan-diabetic rats, β cells were aligned loosely and distributed irregularly, the immunostaining particles for insulin were decreased significantly as evidenced by much lower staining density and intensity (P b 0.01) (Fig. 4B and 5). When diabetic rats were treated with glibenclamide or 18α-GL, the staining particles were increased (Fig. 4C and D) and the staining intensities for both groups were reinforced markedly (P b 0.01 or P b 0.05) vs diabetic rats (Fig. 5). The immunostaining particles for insulin were further increased (Fig. 4E) and intensities further strengthened (P b 0.05) in rats co-treated with 18α-GL and glibenclamide as compared with the diabetic animals treated with glibenclamide or 18α-GL only (Fig. 5). The results showed that 18α-GL and glibenclamide could improve damaged pancreatic islets and β cells, especially when these two drugs were co-administrated. 4. Discussion Glibenclamide produces the hypoglycemic effect by stimulating insulin secretion from β cells of pancreatic islets. The results in our study showed that the pharmacokinetic process and hypoglycemic effect of glibenclamide altered when co-administrated with 18α-GL. In this study, peak plasma concentration of glibenclamide was markedly (P b 0.05) increased 18% by the co-treatment with 18α-GL and glibenclamide in alloxan-diabetic rats. AUC and T1/2Ke of glibenclamide were significantly (P b 0.05) increased 59% and 63%, respectively, by those two drugs co-administration. This result indicated that 18α-GL could inhibit the metabolism of glibenclamide. It is known that glibenclamide is metabolized in the liver by CYP 3A4, 2C9 and 2C19 (van Giersbergen et al., 2002; Naritomi et al., 2004). Studies have revealed that 96.4% of glibenclamide was metabolized by CYP3A4 (Naritomi et al., 2004). The results in our study demonstrated
that CYP3A activity was significantly inhibited (P b 0.05) in alloxandiabetic rats when treated with 18α-GL alone or in combination with glibenclamide. The mechanism underlying the interactions between 18α-GL and glibenclamide probably is the inhibition of CYP3Amediated metabolism of glibenclamide by 18α-GL during elimination. Although the role of CYP2C9 and CYP2C19 in the metabolism of glibenclamide in the present study is unclear, only the inhibition to the activity of CYP3A leading to the changes of the pharmacokinetics and glucose-lowing effect of glibenclamide is obviously. The immunohistochemistry study showed the morphology and structure of pancreatic islets and β cells were improved with the effect of 18α-GL. The role of promoting the repair and regeneration of damaged islet β cells might be one of other mechanisms which 18α-GL took part in affecting the hypoglycemic effect of glibenclamide. The increase in the activities of plasma ALT and AST (Table 3) indicated that alloxan-induced diabetes might cause hepatic dysfunction. Supporting our finding, it has been found by El-Demerdash et al. (2005) in alloxan-diabetic rats and by Deng et al. (2006) in patients that the liver was necrotic accompanied with diabetes. The elevation of the activities of ALT and AST in plasma may be mainly due to the leakage of these enzymes from liver cytosol into blood stream, which gives an indication on the hepatotoxic effect of alloxan. Glibenclamide showed no improvement on the change of ALT and AST. On the other hand, treatment of diabetic rats with 18α-GL caused reduction of the activities of these enzymes in plasma (Table 3) compared to the mean values of diabetic group. 18α-GL has been widely used in clinic due to its effects of protecting liver and lowering serum transaminase (Wang et al., 2004). It was reported that 18α-GL could prevent hepatocyte apoptosis, protect liver cell membranes (Guo et al., 2004), inhibit hepatotoxicant activation (Yang et al., 2001), depress hepatocyte steatosis and necrosis (Lu et al., 2005), which may contribute to the protective effect of 18α-GL on the damaged liver in alloxaninduced diabetic rats in the present study. Our results suggested that 18α-GL might be beneficial to the diabetic patients accompanied with liver impairment. Drug interactions are usually seen in clinical practice and the mechanisms of interactions are evaluated usually in animal models (Satyanarayana and Kilari, 2006). The diabetic rat model served to validate the occurrence of the drug interaction in the actually used condition of hypoglycemic drugs. From our results, we can conclude two aspects: on the one hand, 18α-GL enhanced the hypoglycemic effects of glibenclamide. Hence care should be taken and dosage adjustment is needed when the combination of these two drugs is prescribed. On the other hand, 18α-GL protected the damaged pancreas and liver, which predicted that co-administration of the two drugs might produce synergism action and the curative effect of glibenclamide might be extended. According to the guidance of rational administration principle, whether the combination of glibenclamide and 18α-GL can be used for the treatment of diabetes mellitus is worthy of further study. In conclusion, the effects of 18α-GL on the pharmacodynamics and pharmacokinetics of glibenclamide suggest that 18α-GL affects the metabolism of glibenclamide in alloxan-induced diabetic rats, possibly by the inhibition of CYP3A. Concomitant use of 18α-GL with glibenclamide considerably increases the glucose-lowering effect of glibenclamide.
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Effects of 18α-glycyrrhizin on the pharmacodynamics and pharmacokinetics of glibenclamide in alloxan-induced diabetic rats Ying Ao a, Jie Chen b, Jiang Yue a, Ren-Xiu Peng a,⁎ a b
Department of Pharmacology, Medical College of Wuhan University, Wuhan 430071, PR China Biological Engineering Department, Wuhan Institute of Technology, Wuhan 430074, PR China
A R T I C L E
I N F O
Article history: Received 4 September 2007 Received in revised form 28 February 2008 Accepted 13 March 2008 Available online 8 April 2008 Keywords: Cytochrome P-450 3A 18α-glycyrrhizin Glibenclamide Hypoglycemic effect Pharmacokinetic
A B S T R A C T This paper investigated the effects of 18α-glycyrrhizin (18α-GL) on the pharmacodynamics and pharmacokinetics of glibenclamide in experimental diabetic rats. 18α-GL (25 mg/kg) and/or glibenclamide (1 mg/kg) were given to alloxan-induced diabetic rats for consecutive 5 days. When the rats were co-treated with 18α-GL and glibenclamide, fasting plasma glucose concentration was further reduced, plasma insulin content and liver glycogen level were increased markedly as compared with glibenclamide-treated animals. Meanwhile, in co-treated group, elimination rate constant (Ke) of glibenclamide was reduced while peak plasma concentration (Cmax), area under the plasma concentration vs time curve (AUC0–14 h) and elimination half-life (T1/2Ke) were increased significantly vs glibenclamide alone administered rats. The activities of hepatic CYP3A and the markers of liver injury, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were significantly decreased in rats treated with 18α-GL alone and in combination with glibenclamide. Results of immunohistochemistry showed that 18α-GL improved the effects of glibenclamide on the pathological morphology of pancreatic islet β cells and the intensities of positive immunostaining for insulin. Our results revealed that 18α-GL led to the enhancement of the hypoglycemic effect of glibenclamide by inhibiting the activity of CYP3A; on the other hand, 18α-GL protected the pancreatic islet β cells and liver from damage in diabetes which suggested that 18α-GL might be beneficial as an adjuvant drug of glibenclamide in a proper dose, especially to the diabetic patients associated with liver dysfunction. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Glibenclamide is a sulfonylurea oral hypoglycemic agent which is widely used for the treatment of type 2 diabetes mellitus. It produces the hypoglycemic effect primarily by stimulating insulin secretion from β cells of pancreatic islets. It was reported that glibenclamide was metabolized mainly in liver by cytochrome P-450 (CYP) 3A4. In addition, CYP2C9 and 2C19 also took part in its metabolism (van Giersbergen et al., 2002; Naritomi et al., 2004). The hypoglycemic effect of glibenclamide was changed during co-administration with CYP inhibitor ciprofloxacin (Roberge et al., 2000), thus it is necessary for us to study the interaction between glibenclamide and other drugs to avoid adverse effects especially hypoglycemia. 18α-glycyrrhizin (18α-GL) is one of the main active components of traditional Chinese medicine—Licorice (Radix Glycyrrhizae). Due to its efficacy and safety, 18α-GL is often used as a hepatic protective agent in clinic, especially for the treatment of liver dysfunction. Many authors
⁎ Corresponding author. Department of Pharmacology, Medical College of Wuhan University, Donghu Road, Wuhan, 430071, PR China. Tel.: +86 27 68758665; fax: +86 27 87331670. E-mail address: [email protected] (R.-X. Peng). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.03.043
believe that diabetes is accompanied with hepatic impairment (Caldwell et al., 2004; Ratziu et al., 2004), therefore, it is possible that glibenclamide and 18α-GL are administered concomitantly. Our previous studies showed that 18α-GL inhibited liver drug-metabolizing phase enzymes (Yang et al., 2001), but it is unknown whether 18αGL affects the metabolism and hypoglycemic effect of glibenclamide. Thus, the aim of the present study was to investigate the effect and mechanism of 18α-GL on the pharmacodynamics and pharmacokinetics of glibenclamide in experimental diabetic rats to sufficiently recognize the potential combined effect of concomitant treatment with 18α-GL and glibenclamide. 2. Materials and methods 2.1. Chemicals Glibenclamide, butyl 4-hydroxybenzoate, erythromycin, isocitric acid, isocitric acid dehydrogenase, NADP, bovine serum albumin (BSA), alloxan tetrahydrate were purchased from Sigma (U.S.A.). 18αdiammonii glycyrrhizinatis was from Chia-tai Tianqing Pharmaceutical Co (China). Methanol of HPLC-grade was from Fisher (U.S.A.) and all the other chemicals and reagents were of analytical grade.
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2.3. Biochemical analysis Blood sample was withdrawn in a heparinized capillary tube from the retro-orbital venous plexus under light ether anaesthesia. Plasma glucose levels were checked by glucose oxidase method. Plasma insulin content was measured by radioimmunoassay technique using insulin RIA kit (North Institute of Biological Technology, China). Activities of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assessed using test kits (Rongsheng Biotechnology, China). Rats were sacrificed with an overdose of anesthetic and the livers were excised. Hepatic glycogen level was assayed enzymatically using specific kit (Jiancheng Bio-Tek, China). Liver microsomes were prepared by differential ultra-centrifugation. Erythromycin N-demethylase (ERD) activity, as the marker of CYP3A, was assayed as previously described (Peng et al., 1994). Protein was assayed by the method of Lowry et al. (1951). 2.4. Plasma glibenclamide determination
Fig. 1. HPLC chromatograms of glibenclamide in plasma samples of alloxan-induced diabetic rats. (A) blank plasma, (B) blank plasma spiked with glibenclamide and internal standard, (C) a plasma sample spiked with internal standard. Peaks: 1, Internal standard: Butyl 4-phydroxybenzoate; 2, glibenclamide.
For the pharmacokinetic study, blood samples were collected in heparinized tubes before and 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 14 h after final administration via the right external jugular vein. Plasma glibenclamide concentration was determined by reverse phase high performance liquid chromatography (HPLC) (Srinivas and Nayanabhirama, 2005). The solvent delivery system was a Shimadzu pump model LC-9A (Shimadzu, Japan). The analytical column was Zorbax SB-C18 (4.6 mm ID × 25 cm, 5 μm particle size) purchased from Yilite Scientific Instrument Co., China. Column effluent was monitored with SPD-6AV ultraviolet spectrophotometric detector (Shimadzu, Japan) at 228 nm. The HPLC system was equilibrated with the mobile phase consisting of ammonium dihydrogen phosphate 50 mmol/l (pH 3.0): methanol (3:5, by volume), at a flow-rate of 1.0 ml/min. Plasma samples were denatured by trichloracetic acid, and then centrifuged at 15,000 ×g for 10 min. The supernatant was transferred and a 20 μl volume was injected into the HPLC system for quantitation. The calibration curve of glibenclamide concentration (C) vs the ratio for area (Y) of glibenclamide to butyl 4-hydroxybenzoate resulted in a correlation coefficient (r) of 0.999. And the linear range for glibenclamide in plasma was from 10 to 250 μg/l. The regression equation was Y = 0.009C + 0.0069. The average recovery rate was 100.1%, and the relative standard deviations (RSD) of intra-day and inter-day were all less than 5% (Fig. 1). 2.5. Immunohistochemical assay
2.2. Animals and treatment Male Wistar rats weighing 190–200 g (Certificate No 19-088), SPF grade, were supplied by Experimental Animals Center, Hubei Province, China. Throughout the study, rats were housed under the same adequate environmental conditions, with a 12/12 h light/dark cycle. They were fed with standard laboratory chow and water ad libitum. All the animals were cared for according to the rules and regulations of the Institutional Animal Ethics Committee (IAEC) guidelines of the Wuhan University, China. For the induction of diabetes, rats were kept on fasting for 24 h prior to alloxan injection. On the day of administration, alloxan tetrahydrate was freshly prepared in normal saline and intraperitoneal injection (i.p.) was given at the dosage of 150 mg/kg. Fasting plasma glucose concentration was checked by glucose oxidase method (Trinder, 1969) 3 days after alloxan treatment. The animals with glucose level exceeding 13.89 mmol/l were considered as hyperglycemic. All the rats were divided into 5 groups, each containing 6 animals. ➀ Control group. ➁ Diabetic group. ➂ Diabetic rats treated with glibenclamide (1 mg/kg, i.g.). ➃ Diabetic rats treated with 18α-GL (25 mg/kg, i.p.). ➄ Diabetic animals treated with glibenclamide (1 mg/ kg, i.g.) and 18α-GL (25 mg/kg, i.p.). The treatment was continued for 5 days. Before the final administration, animals were fasted overnight.
Cauda pancreatis was preserved in neutral buffered 10% formalin and then embedded in paraffin, sectioned at 5 μm. Immunohistochemistry for insulin was performed using a combination streptavidin–biotin–peroxidase method. The negative control was performed by omitting the primary antibody. The quantitative expression of
Table 1 Plasma glucose concentration in alloxan-induced diabetic rats administered with glibenclamide and/or 18α-GL Groups
Control Diabetic Glibenclamide 18α-GL Glibenclamide +18α-GL
Plasma glucose concentration (mmol/l) 0h
2h
4h
6h
4.27 ± 0.49 21.81 ± 5.27a 21.72 ± 5.14a 19.91 ± 3.90a 21.52 ± 7.00a
4.14 ± 0.43 22.70 ± 4.16a 17.64 ± 4.76a, b 20.29 ± 5.10a 16.95 ± 2.98a, b
3.92 ± 0.22 22.76 ± 3.29a 17.29 ± 5.90a, b 19.90 ± 6.78a 12.84 ± 3.47a, c
4.01 ± 0.93 22.65 ± 2.66a 16.28 ± 7.20a, b 19.20 ± 5.88a 7.08 ± 3.39c, d
Glibenclamide (1 mg/kg, i.g.) and/or 18α-GL (25 mg/kg, i.p.) were given once daily for 5 days to the alloxan (150 mg/kg, i.p.)-induced diabetic rats. Plasma glucose concentration was measured at 0, 2, 4 and 6 h after final administration. Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin. aP b 0.01 vs control group; bP b 0.05, cP b 0.01 vs diabetic group; dP b 0.01 vs glibenclamide group.
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Table 2 The fasting plasma insulin, Insulin Sensitivity Index (ISI) and liver glycogen in alloxaninduced diabetic rats administered with glibenclamide and/or 18α-GL
Table 3 The activities of plasma transaminases and liver index in alloxan-induced diabetic rats administered with glibenclamide and/or 18α-GL
Groups
Groups
Control Diabetic Glibenclamide 18α-GL Glibenclamide + 18α-GL
Insulin
ISI
Liver glycogen
(mU/l)
(×10 3)
(mg/g)
24.8 ± 9.6 7.1 ± 1.7b 11.7 ± 5.8a, c 8.6 ± 3.4b 19.3 ± 8.2d, e
11.3 ± 2.9 6.7 ± 1.9b 7.4 ± 4.2 7.3 ± 2.9a 8.9 ± 1.4c
27.9 ± 22.5 14.4 ± 7.8 22.5 ± 7.4c 28.3 ± 11.6c 39.3 ± 15.2d, e
See Table 1 for treatment. ISI = 1 / fasting plasma glucose × insulin. The indices were measured at 6 h after final administration. Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin. aP b 0.05, bP b 0.01 vs control group; c P b 0.05, dP b 0.01 vs diabetic group; eP b 0.05 vs glibenclamide group.
Control Diabetic Glibenclamide 18α-GL Glibenclamide +18α-GL
Plasma ALT
Plasma AST
Liver index
(µmol/min/l)
(µmol/min/l)
(× 103)
38 ± 12 109 ± 19b 92 ± 18b 50 ± 14d, f 54 ± 20d, f
90 ± 23 186 ± 31b 165 ± 21b 86 ± 29d, f 94 ± 20d, f
29 ± 4 45 ± 8a 40 ± 4a 35 ± 9 27 ± 5c, e
See Table 1 for treatment. Liver index = liver weight / body weight. The indices were measured at 6 h after final administration. Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin; ALT = alanine aminotransferase; AST = aspartate aminotransferase. a P b 0.05, b P b 0.01 vs control group; c P b 0.05, d P b 0.01 vs diabetic group; eP b 0.05, fP b 0.01 vs glibenclamide group.
insulin was detected by HPIAS-1000 pathological image analysis system (Qianping Image Technology, China) via calculating the average optical density in each field.
57% (P b 0.01) vs glibenclamide-treated rats, which indicated that 18αGL potentiated the hypoglycemic effect of glibenclamide.
2.6. Statistical analysis
3.2. Effects of glibenclamide and/or 18α-GL administration on the levels of plasma insulin, Insulin Sensitivity Index (ISI) and liver glycogen
Data were presented as mean ± standard deviation (S.E.M.) and repeated measures analysis of variance (ANOVA) was used for comparing the time-course of plasma glucose concentration. Other results were analyzed by Student's t test or one-way ANOVA. The pharmacokinetic analysis was conducted using the DAS ver 1.0 software. Differences were considered to be significant when P b 0.05. 3. Results 3.1. Effect of 18α-GL on the hypoglycemic action of glibenclamide All the animals which received alloxan injection developed diabetes. The rats that were made diabetes had higher glucose concentration than control non-diabetic animals (Table 1). It was reported that elimination half-life of glibenclamide was 6.4 h (Srinivas and Nayanabhirama, 2005). Therefore, in this study, we monitored the plasma glucose levels at 0, 2, 4 and 6 h after final administration to observe the hypoglycemic effect of glibenclamide. As shown in Table 1, glibenclamide significantly decreased plasma glucose level at 2 h and maintained the hypoglycemic effect during 2–6 h after final treatment, plasma glucose concentration at 6 h was decreased by 28% vs the diabetic rats (P b 0.05). 18α-GL itself did not show significant effect on plasma glucose content vs alloxan treatment, however, plasma glucose level in rats co-treated with 18α-GL and glibenclamide was decreased by 25% (P b 0.05), 44% (P b 0.01) and 69% (P b 0.01) at 2, 4 and 6 h after final administration, respectively, as compared with the diabetic animals; plasma glucose content at 6 h was significantly reduced by
Fig. 2. Liver CYP3A (erythromycin N-demethylase) activity in alloxan-induced diabetic rats administered with glibenclamide and/or 18α-GL. The activity of CYP3A was measured at 6 h after final administration. Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin. ⁎P b 0.05 vs control group.
As shown in Table 2, alloxan-induced diabetic rats showed marked decrease in plasma insulin concentration and Insulin Sensitivity Index (ISI) when compared to the control non-diabetic animals. Administration of glibenclamide caused a significant increase in plasma insulin (P b 0.05), which indicated that glibenclamide provoked insulin secretion, but ISI was not changed by this drug. 18α-GL showed poor effect on insulin content or ISI, whereas it increased liver glycogen level vs alloxan treatment (P b 0.05). When 18α-GL was administrated concurrently with glibenclamide, both of plasma insulin content and liver glycogen level were further increased (P b 0.05) as compared to the animals treated with glibenclamide alone, ISI was also markedly elevated (P b 0.05) vs the diabetic rats. 3.3. Effects of glibenclamide and/or 18α-GL administration on the activities of liver CYP3A, plasma ALT and AST It was reported that glibenclamide was metabolized by CYP3A in liver (van Giersbergen et al., 2002; Naritomi et al., 2004). To investigate the mechanism of 18α-GL enhancing the hypoglycemic effect of glibenclamide, the activity of liver CYP3A was determined. As demonstrated in Fig. 2, hepatic CYP3A activity in diabetic or glibenclamide-treated rats had no change compared with the control. For rats treated with 18α-GL only or co-treated with 18α-GL and glibenclamide, CYP3A level was markedly (P b 0.05) decreased by 54% and 51%, respectively, vs that in diabetic status. The result suggested
Fig. 3. Mean plasma concentration–time curve of glibenclamide in alloxan-induced diabetic rats administrated with glibenclamide alone (empty symbols) and coadministrated with 18α-GL (solid symbols). Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin.
Y. Ao et al. / European Journal of Pharmacology 587 (2008) 330–335 Table 4 Main pharmacokinetic parameters of glibenclamide in alloxan-induced diabetic rats administrated with glibenclamide alone and co-administrated with 18α-GL Parameters
Glibenclamide
Glibenclamide+18α-GL
Ka (1/h) Ke (1/h) T1/2Ka (h) T1/2Ke (h) AUC0–14 h (µg h/l) Tmax (h) Cmax (µg/l)
0.46 ± 0.08 0.34 ± 0.05 1.53 ± 0.23 2.11 ± 0.40 828.56 ± 78.36 3.00 ± 0.55 127.62 ± 8.02
0.39 ± 0.08 0.21 ± 0.04a 1.86 ± 0.40 3.43 ± 0.62a 1315.00 ± 153.05a 3.50 ± 0.55 150.70 ± 13.59a
Ka = absorption rate constant; Ke = elimination rate constant; T1/2Ka = absorption halflife; T1/2Ke = elimination half-life; AUC = area under the plasma concentration vs time curve; Tmax = time to reach Cmax; Cmax = peak plasma concentration. Data are expressed as means ± S.E.M. for six rats in each group. 18α-GL = 18α-glycyrrhizin. aP b 0.05 vs glibenclamide group.
that 18α-GL potentiated the glucose-lowering effect of glibenclamide probably by inhibiting the activity of hepatic CYP3A and in turn reducing the metabolism of glibenclamide. It has been pointed out that 18α-GL possesses the effect to diminish liver injury (Zheng and Lou, 2003). To observe whether 18α-GL may protect against liver impairment accompanied with diabetes, we investigate the influence of 18α-GL to the levels of the hepatic damage
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makers. As shown in Table 3, in alloxan-diabetic rats, the activities of plasma ALT and AST were significantly (P b 0.01) increased by 187% and 107%, respectively, relative to their normal levels. When the diabetic rats were treated with glibenclamide, plasma ALT, AST and liver index were not significantly different from that in diabetic status. The results indicated that glibenclamide exerted the antihyperglycemic effect (Table 2), but it could not improve the impaired liver function. However, after the treatment with 18α-GL, the activities of plasma ALT and AST were markedly (P b 0.01) decreased by 54.1% and 53.7%, respectively, compared to the alloxan-diabetic group. When the diabetic rats were co-administered with 18α-GL and glibenclamide, plasma ALT, AST and liver index were significantly reduced by 41% (P b 0.01), 43% (P b 0.01) and 33% (P b 0.05), respectively, compared to the glibenclamide-treated animals. The results demonstrated that 18α-GL could protect the damaged liver in alloxan-induced diabetic rats. 3.4. Pharmacokinetic analysis Mean plasma concentration vs time curve for glibenclamide was illustrated in Fig. 3, and mean plasma pharmacokinetic parameters were summarized in Table 4. For the animals co-treated with 18α-GL and glibenclamide, elimination rate constant (Ke) of glibenclamide
Fig. 4. Immunohistochemical staining of insulin in pancreatic islets of rats. Pancreatic β cells in positive immunostaining were distributed over the center of pancreatic islets, aligned regularly and the endochylema was full of gross insulin particles in deep brown color in control group (A). The immunostaining particles for insulin were decreased significantly in the diabetic group (B). When treated with glibenclamide or 18α-GL, the staining particles were increased (C, D) and the effect further strengthened in rats co-treated with 18α-GL and glibenclamide (E). ×400. 18α-GL = 18α-glycyrrhizin.
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Fig. 5. Expression of insulin in pancreatic islets of alloxan-induced diabetic rats administered with glibenclamide and/or 18α-GL. The index was measured at 6 h after final administration. Data are expressed as means ± S.E.M. for six rats in each group. ⁎ P b 0.05, ⁎⁎ P b 0.01 vs control group; # P b 0.05, ## P b 0.01 vs diabetic group; Δ P b 0.05 vs glibenclamide group. 18α-GL = 18α-glycyrrhizin.
was decreased by 38% (P b 0.05) while peak plasma concentration (Cmax), area under the plasma concentration vs time curve (AUC0–14h) and elimination half-life (T1/2Ke) were markedly (P b 0.05) increased by 18%, 59% and 63%, respectively, as compared with glibenclamidetreated group. The results indicated that 18α-GL exerted the effect to inhibit the elimination of glibenclamide and led to the elevation of Cmax and AUC. 3.5. Immunohistochemical assay for insulin To investigate if 18α-GL has protective effect on damaged pancreas in experimental diabetic rats, we observed the effects of 18α-GL on the pathological morphology of pancreatic islet β cells and the secretion of insulin. The result of image analysis was demonstrated in Fig. 5. In control group, islet β cells in positive immunostaining were distributed over the center of pancreatic islets, aligned regularly and the endochylema was full of gross insulin particles in deep brown color (Fig. 4A). In alloxan-diabetic rats, β cells were aligned loosely and distributed irregularly, the immunostaining particles for insulin were decreased significantly as evidenced by much lower staining density and intensity (P b 0.01) (Fig. 4B and 5). When diabetic rats were treated with glibenclamide or 18α-GL, the staining particles were increased (Fig. 4C and D) and the staining intensities for both groups were reinforced markedly (P b 0.01 or P b 0.05) vs diabetic rats (Fig. 5). The immunostaining particles for insulin were further increased (Fig. 4E) and intensities further strengthened (P b 0.05) in rats co-treated with 18α-GL and glibenclamide as compared with the diabetic animals treated with glibenclamide or 18α-GL only (Fig. 5). The results showed that 18α-GL and glibenclamide could improve damaged pancreatic islets and β cells, especially when these two drugs were co-administrated. 4. Discussion Glibenclamide produces the hypoglycemic effect by stimulating insulin secretion from β cells of pancreatic islets. The results in our study showed that the pharmacokinetic process and hypoglycemic effect of glibenclamide altered when co-administrated with 18α-GL. In this study, peak plasma concentration of glibenclamide was markedly (P b 0.05) increased 18% by the co-treatment with 18α-GL and glibenclamide in alloxan-diabetic rats. AUC and T1/2Ke of glibenclamide were significantly (P b 0.05) increased 59% and 63%, respectively, by those two drugs co-administration. This result indicated that 18α-GL could inhibit the metabolism of glibenclamide. It is known that glibenclamide is metabolized in the liver by CYP 3A4, 2C9 and 2C19 (van Giersbergen et al., 2002; Naritomi et al., 2004). Studies have revealed that 96.4% of glibenclamide was metabolized by CYP3A4 (Naritomi et al., 2004). The results in our study demonstrated
that CYP3A activity was significantly inhibited (P b 0.05) in alloxandiabetic rats when treated with 18α-GL alone or in combination with glibenclamide. The mechanism underlying the interactions between 18α-GL and glibenclamide probably is the inhibition of CYP3Amediated metabolism of glibenclamide by 18α-GL during elimination. Although the role of CYP2C9 and CYP2C19 in the metabolism of glibenclamide in the present study is unclear, only the inhibition to the activity of CYP3A leading to the changes of the pharmacokinetics and glucose-lowing effect of glibenclamide is obviously. The immunohistochemistry study showed the morphology and structure of pancreatic islets and β cells were improved with the effect of 18α-GL. The role of promoting the repair and regeneration of damaged islet β cells might be one of other mechanisms which 18α-GL took part in affecting the hypoglycemic effect of glibenclamide. The increase in the activities of plasma ALT and AST (Table 3) indicated that alloxan-induced diabetes might cause hepatic dysfunction. Supporting our finding, it has been found by El-Demerdash et al. (2005) in alloxan-diabetic rats and by Deng et al. (2006) in patients that the liver was necrotic accompanied with diabetes. The elevation of the activities of ALT and AST in plasma may be mainly due to the leakage of these enzymes from liver cytosol into blood stream, which gives an indication on the hepatotoxic effect of alloxan. Glibenclamide showed no improvement on the change of ALT and AST. On the other hand, treatment of diabetic rats with 18α-GL caused reduction of the activities of these enzymes in plasma (Table 3) compared to the mean values of diabetic group. 18α-GL has been widely used in clinic due to its effects of protecting liver and lowering serum transaminase (Wang et al., 2004). It was reported that 18α-GL could prevent hepatocyte apoptosis, protect liver cell membranes (Guo et al., 2004), inhibit hepatotoxicant activation (Yang et al., 2001), depress hepatocyte steatosis and necrosis (Lu et al., 2005), which may contribute to the protective effect of 18α-GL on the damaged liver in alloxaninduced diabetic rats in the present study. Our results suggested that 18α-GL might be beneficial to the diabetic patients accompanied with liver impairment. Drug interactions are usually seen in clinical practice and the mechanisms of interactions are evaluated usually in animal models (Satyanarayana and Kilari, 2006). The diabetic rat model served to validate the occurrence of the drug interaction in the actually used condition of hypoglycemic drugs. From our results, we can conclude two aspects: on the one hand, 18α-GL enhanced the hypoglycemic effects of glibenclamide. Hence care should be taken and dosage adjustment is needed when the combination of these two drugs is prescribed. On the other hand, 18α-GL protected the damaged pancreas and liver, which predicted that co-administration of the two drugs might produce synergism action and the curative effect of glibenclamide might be extended. According to the guidance of rational administration principle, whether the combination of glibenclamide and 18α-GL can be used for the treatment of diabetes mellitus is worthy of further study. In conclusion, the effects of 18α-GL on the pharmacodynamics and pharmacokinetics of glibenclamide suggest that 18α-GL affects the metabolism of glibenclamide in alloxan-induced diabetic rats, possibly by the inhibition of CYP3A. Concomitant use of 18α-GL with glibenclamide considerably increases the glucose-lowering effect of glibenclamide.
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