Preparation Of Huang-Gui Solid Dispersion And Its Mechanism Of Antidiabetic Effect

The incidence of type2diabetes increased year by year, which make it the third largestdiseases affecting human health after cancer, cardiovascular disease. Despite continuingintroducting of hypoglycemic drugs, intervention of diabetes and related complication suchas hypoglycemia, lactic acidosis and liver damage, and some even life-threatening stillremain a major global medical problem. Berberine, a traditional drug for treatment ofdiarrhea, was confirmed a significant hypoglycemic effect and also has a significant effecton the complications of diabetes, such as cardiovascular disease, high blood pressure. Thus,berberine has little side effect like hypoglycemia, liver and kidney dysfunction, whichcaused widespread concern in the academic community.Even the hypoglycemic effect of berberine is very perfect, it was also not used in clinicas a hypoglycemic drugs. The major reasons are the poor intestinal absorption and the lowbioavailability. High dose of oral administration usually causes gastrointestinal side effects,which greatly limits its clinical application. So how to improve the bioavailability ofberberine is important. Low oral bioavailability of berberine is due to poor solubility andmembrane permeability. In our previous study, berberine and sodium caprate, a safe andnon-toxic intestinal absorption enhancer, were used in combination, which could achieve agood effect to promote the absorption of berberine. Also the anti-diabetic effects wereincreased significantly. But this method only resolved one problem for the poor intestinalabsorption of berberine, which could not be applied as a formulation in clinical practice.Skeletal muscle is the largest tissue of glucose uptake and consumption in the body,which were mediated by insulin activating the insulin signaling pathway. Impaired insulinsignaling pathway leading to skeletal muscle insulin sensitivity was the most importantreason for insulin resistance, with the performance of impaired glucose transport.Glucocorticoids are important hormonal regulating glucose metabolism in vivo, withsignificant glycemic effects. Glucocorticoids can reduce skeletal muscle tissue response, anddecrease glucose uptake and utilization, leading to occurrance of high blood glucose. 11β-HSD1in skeletal muscle plays a key role in insulin resistance mediated byglucocorticoids.11β-HSD1could alter inactive glucocorticoids to active glucocorticoids,and regulate the combination of glucocorticoids and glucocorticoid receptors, exertingbiological effects of glucocorticoids. When diabetes, obesity and insulin resistance happen,the function11β-HSD1regulating GR was abnormal in insulin-sensitive tissues, causingdysfunction of the insulin signaling pathway, and ultimately inducing insulin resistance. Ourprevious showed that protein levels of11β-HSD1and GR in liver and skeletal muscle type2diabetic rats were extremely high, mediating enhancement of gluconeogenesis and insulinresistance, which were important mechanisms for the occurrence of type2diabetes. Alsoberberine can significantly reduce the protein and mRNA levels of11β-HSD1and GR inliver tissue of type2diabetic rats, reducing endogenous glucose production. But it is not yetclear whether berberine improves insulin resistance by regulating11β-HSD1/GR proteinlevels in skeletal muscle, and restores insulin signaling pathway.Objective: To develop a new dosage form of berberine with dual role of increasingsolubility and intestinal permeability, named Huang-Gui solid dispersion (HGSD), and thento study systematically from the pharmacy, pharmacokinetics, pharmacodynamics andmechanism of hypoglycemic effects of HGSD. Our study could provide theoretical andexperimental basis for berberine as anti-diabetes drug.Method and result:1. Preparation and characterization of HGSDs:①nine different Huang-Gui soliddispersions were prepared by the solvent evaporation method according to the orthogonaldesign. The results showed that the solubility and dissolution of all the nine HGSDs weresignificantly than that of pure berberine and commercial berberine tablet, and higher thanthat of the corresponding physical mixtures. Based on the30min accumulated dissolution ofHGSDs, the best formulation-P9and the optimal prescription were picked up, while the bestpreparation process was determined;②The exist form and the interactions between theinitial materials were studied by differential scanning calorimetry, X-ray diffraction,scanning electron microscopy and infrared spectrum analysis. The results showed thatmajority of berberine in P9existing an amorphous state, and there is no chemical reactionoccuring between berberine, sodium caprate and PEG6000.2. Influence of HGSD on the pharmacokinetics of berberine: The CaCO2cellmonolayer in vitro absorption model, in situ intestinal perfusion experiments and thebioavailability evaluation experiment were applied to study the pharmacokinetics of berberine in P9. The results revealed that on the levels of in vitro, in situ and in vivo, P9could significantly increase the absorption of berberine. The apparent permeabilitycoefficient were increased from smaller than1×10-6cm/s to between1-10×10-6cm/s, makingthe berberine the drug difficult to be absorbed to easyto be adsorbed. Also the intestinalabsorption amount in4h and relative bioavailability were greatly promoted compared withpure berberine and commercial berberine tablet. These results implied that HGSD, as a newdosage form of berberine, could significantly increase the intestinal absorption andbioavailability of berberine.3. Therapy of P9in type2diabetes:①Type2diabetic rat model induced by a high-fatdiet combined with small dose of intraperitoneal injection of STZ and C2C12insulinresistance cell induced by chronic high dose of insulin were used to study the hypoglycemicpharmacodynamics of P9. The results revealed that P9could significantly reduce fastingblood glucose, blood lipid and of increased insulin levels in rat model, improving glucosetolerance. The fasting blood glucose of P9-25mg/Kg group was equal to those of berberine100mg/Kg group and commercial berberine tablet100mg/Kg group. While P9-100mg/Kggroup showed stronger hypoglycemic effect, even reaching blood glucose levels of thecontrol rats. P9with the dose of1/4commercial berberine has remarkable effect on reducingblood glucose equal to commercially available berberine, suggesting P9may havesynergistic effects, low toxicity.②Serum pharmacological method was used to study theinfluence of berberine on glucose uptake and consumption in C2C12cell insulin resistancemodel. The results showed that P9could increase glucose consumption and uptake, implyingberberine was the major ingredient of P9for hypoglycemic effects, which may be related tothe promotion of skeletal muscle glucose uptake.The anti-diabetic mechanism of HGSD:①Effects of P9on insulin signal pathway relatedproteins in skeletal muscle of type2diabetic rats and insulin resistance cell model wereevaluated. The in vivo results show IRS and AKT phosphorylation levels and GLUT4translocation in skeletal muscle of control rats were significantly increased with insulinstimulation. Compared to the control group, IRS and AKT phosphorylation levels andGLUT4translocation of diabetic rats had no change. No. P9Huang-Gui solid dispersioncould dose-dependently increase AKT and IRS1phosphorylation level and GLUT4membrane translocation, while reduce the protein level of11β-HSD1and GR in skeletalmuscle of diabetic rats. On the cellular level, the insulin resistance model of skeletal musclecell line C2C12was established by the sustained high concentrations of insulin stimulation. Then the cells of model group were giving serum with P9for treatment. The results showedthat glucose uptake and consumption the model group were significantly lower than that ofcontrol group, while AKT and IRS1phosphorylation levels and GLUT4translocation weresignificantly decreased after insulin stimulation compared to the control group. In contrast,P9administration group could increase AKT and IRS1phosphorylation levels together withGLUT4translocation, while reducing the expression11β-HSD1and GR of the model group,and the concentration of corticosterone.②Then we apply11β-HSD1siRNA and GRinhibitor RU486in the same cell model. The results showed that found glucose uptake andconsumption could be increased by11β-HSD1siRNA and RU486compared to the modelgroup, which implied insulin resistance could be improved while reducing11β-HSD1andGR protein levels. These results show Huang-Gui solid dispersion could significantlyimprove insulin resistance in the skeletal muscle of type2diabetes, increase insulinsensitivity. And this improvement effect of Huang-Gui solid dispersion may relate todecreasing of11β-HSD1and GR protein levels.③To study how Huang-Gui improvesinsulin resistance according to11β-HSD1and GR protein levels, the mice model of insulinresistance were established by intraperitoneal injection of dexamethasone, meanwhile P9(150mg/Kg) and berberine (150mg/Kg) were given for treatment. The results showed asignificant increase fasting blood glucose and blood insulin levels in model mice comparedto the control group, together with impaired glucose tolerance. P9and berberine can reducemodel mice fasting blood glucose and serum insulin levels and improve insulin resistance.Then, we established C2C12cell line model of insulin resistance induced by dexamethasone.The glucose uptake and consuption were significantly reduced in the model group, whileberberine and RU486is able to increase the cellular glucose uptake and consumption.Further on the in vivo and cellular level, we studied the varition of proteins level of insulinsignaling pathway11β-HSD and GRα, which subtype completed the vast major biologicalfunction of GR. It is showed that in the model group IRS1and AKT phosphorylation levelsand GLUT4membrane translocation were significantly lower than the control group afterinsulin stimulation. P9and berberine could increase IRS1and AKT activity, increasedGLUT4membrane translocation, while the effect of P9was stronger than berberine. On thecellular level berberine and RU486can increase AKT and IRS1phosphorylation level andGLUT4translocation after insulin stimulation. This result is consistent with the previousresults of this experiment.④GR could competitively bind with IRS1, reducing IRS bindingwith PI3K and leading to insulin resistance. Co-immunoprecipitation method was imployed to vary the combination and situation of PI3K and GRα in the model group and the influnceof berberine on them. The results showed that compared to the control group, GRα combinedwith PI3K increased in cytoplasm of model cell, while PI3K binding with IRS1decreased.Berberine and RU486is able to reverse the increased combination of GRα and PI3K,recoverying PI3K combined with IRS1, thereby promoting the phosphorylation of AKT,increasing membrane GLUT4translocation, which in the end promoted glucose uptake andimprove insulin resistance, this results verified in immunofluorescence.In summary, the P9Huang-Gui solid dispersion we prepared could significantlyincreased intestinal absorption of berberine and improved its oral bioavailability. Huang-Guisolid dispersion can significantly reduce fasting blood glucose and lipids in type2diabeticrats, improve insulin glucose tolerance and insulin resistance. Skeletal muscle insulinresistance is the main mechanism in type2diabetes. Berberine, the main medicinalingredient of Huang-Gui solid dispersion improved insulin resistance by reducing11β-HSD1and GR protein levels in skeletal muscle, inhibiting the binding of GRα with PI3K,increasing AKT activity levels, promoting membrane translocation of GLUT4, andincreasing glucose uptake of skeletal muscle, which is an important mechanism for thetreatment of type2diabetes.