Preparation Of A Water-soluble DMY-loaded Nanomicelles And Its Role In The Treatment Of Alcoholism

Research background and purpose:Dihydromyricetin（DMY）is a potent anti-inflammatory and antioxidant flavonoid that is widely found in a variety of plants in nature and has good safety and hepatoprotective effects against alcohol.However,its low solubility,poor stability and low permeability lead to low oral bioavailability.In order to improve the oral bioavailability of DMY,we used the polymeric material Solutol^?HS-15 to construct water-soluble DMY-loaded nanomicelles（DMY-Ms）and conducted an in vitro and in vivo evaluation of DMY-Ms to confirm the feasibility of making DMY into DMY-Ms,which is a difficult-to-dissolve drug.The feasibility of making water-soluble DMY-loaded nanomicelles（DMY-Ms）into DMY-Ms was confirmed,which could provide potential clinical application of DMY in the treatment of alcoholism in the future.Materials and Methods:1.Preparation,characterization and quality evaluation of DMY-MsThe DMY-Ms were prepared by a thin film dispersion method and characterised by Malvern laser particle size measurement,transmission electron microscopy（TEM）and differential scanning calorimetry（DSC）.A method was developed for the in vitro and in vivo determination of DMY content,and the quality and physicochemical properties of DMY-Ms were investigated by encapsulation rate,solubility,release and stability.2.Permeability study of DMY-Ms in rat intestineA method for the determination of DMY in rat perfusate was developed and methodologically investigated.Using the rat in vivo gastric study model and the rat in vivo intestinal unidirectional perfusion model,the absorption of DMY-Ms in the stomach and intestine was investigated to elucidate the mechanism by which DMY-Ms enhance the absorption of DMY.3.Pharmacokinetic study of DMY-Ms in ratsA method for the determination of DMY blood concentration in rats by liquid chromatography-mass spectrometry（HPLC-MS/MS）was established and methodologically investigated.The concentrations of DMY in rat plasma were determined by oral gavage,and then the data were analyzed and processed by DAS 3.0software to obtain pharmacokinetic parameters such as Cmax,Tmax and AUC0→24.To verify whether DMY-Ms improves the absorption and bioavailability of DMY.4.Anti-alcoholism effect of DMY-Ms（1）Disappearance of the righting reflex experimentThe mice were randomly divided into 3 groups（n=10）,the control group was gavaged with saline;the other two groups were gavaged with DMY suspension and DMY-Ms（100 mg/kg）respectively.2 h later,all three groups of rats were gavaged with70%（v/v）ethanol（0.16 m L/10 g）,a timer was started,the mice were placed in the supine position,and the disappearance and recovery time of the righting reflex was recorded.To observe whether DMY-Ms could improve the therapeutic effect of anti-alcoholism by prolonging the intoxication tolerance time and shortening the duration of intoxication.（2）Measurement of blood alcohol concentration in ratsRats were randomly divided into 3 groups（n=6）.The rats in the control group were gavaged with saline;the other two groups were gavaged with DMY suspension and DMY-Ms（100 mg/kg）,respectively.2 h later,the rats in all three groups received70%（v/v）ethanol（0.16 m L/10 g）,and blood was collected from the tail vein at 0,5,30,90,180 and 240 min,respectively,to determine the blood ethanol concentration.To verify whether DMY-Ms has the ability to reduce the blood alcohol concentration.（3）Alcoholic gastric mucosal injury experiment in miceC57BL/6 male mice were randomly divided into 4 groups（n=8）.An alcoholic gastric mucosal injury model was established,and the morphological and pathological changes of gastric mucosa were observed in each group of mice,and the gastric mucosal lesions of each group were scored,and the histopathological differences between the groups were compared.To evaluate whether DMY-Ms has a protective effect on gastric mucosal injury in mice with acute alcohol intoxication.To detect the malonaldehyde（MDA）content and superoxide dismutase（SOD）activity in gastric tissues.To evaluate whether DMY-Ms protects the gastric mucosa of mice with acute alcohol intoxication by improving the antioxidant stress capacity of gastric mucosa.（4）Alcoholic liver injury experiment in miceC57BL/6 male mice were randomly divided into 4 groups（n=8）.The mice were administered by gavage once/day for 6 days.The mice in the normal control and model groups were gavaged with saline（2 m L/100 g）;the other two groups were gavaged with DMY suspension and DMY-Ms（100 mg/kg）,respectively.On day 6,1 h after the last gavage,mice in the normal control group were gavaged with saline,and mice in all other groups were orally gavaged with 50%（v/v）ethanol（0.1 m L/10 g）once every 12 h,for a total of 3 times.After the last gavage of ethanol for 4 h,the mice were executed.The liver and serum of mice were collected,and the levels of Aspartic acid aminotransferase（AST）and Alanine aminotransferase（ALT）were measured in each group of mice.To evaluate the protective effect of DMY-Ms on liver injury in mice with acute alcohol intoxication.To detect the levels of malondialdehyde（MDA）and superoxide dismutase（SOD）activity in liver tissues of mice in each group.To evaluate whether DMY-Ms improves the antioxidant stress capacity of liver tissues to protect the liver of acute alcohol intoxicated mice.Results:1.Preparation,characterization and quality evaluation of DMY-MsThe optimal prescription ratio of Solutol（?）HS-15 to DMY was 20:1 after prescription screening;the prepared DMY-Ms were spherical in shape;the average particle size was 13.97±0.82 nm;the DSC analysis showed that DMY was dispersed in DMY-Ms in an amorphous state;the encapsulation rate of DMY-Ms was 87.26%±1.93%;the drug loading was 4.69%±0.05%;and the laboratory-made DMY-Ms could significantly improve the solubility of DMY,slow down the release of DMY and improve the stability of DMY in different buffers and gastrointestinal liver homogenates.The solubility of DMY-Ms produced in the laboratory（0.20±0.02 mg/m L）was 25times higher than that of DMY（5.07±0.15 mg/m L）,which significantly improved the solubility,dissolution rate and stability of DMY in different buffers and gastrointestinal liver homogenates.2.Permeability study of DMY-Ms in rat intestineThe total absorption of DMY-Ms in the stomach（18.3%±4.6%）was 8 times higher than that of DMY（2.3%±0.9%）.In the rat in vivo intestinal unidirectional perfusion experiment,the absorption rate constant（K_a）and effective permeability coefficient(P_eff)of DMY in jejunum were 5.45 and 2.82 times higher than those of DMY,respectively.The experiments showed that DMY-Ms had higher membrane permeability.3.Pharmacokinetic study of DMY-Ms in ratsThe pharmacokinetic results showed that the Cmax of DMY-Ms in plasma（2143.38μg/L）was 3.36 times higher than that of DMY（637.57μg/L）;the value of AUC_0→24（8981.66μg/L*h）was 2.03 times higher than that of DMY（4414.95μg/L*h）.The DMY-Ms prepared in this experiment had higher bioavailability.4.Anti-alcoholism effect of DMY-MsIn the experiment on the disappearance of the reflex,the duration of drunkenness was increased by 2.35 times and 5.73 times（P<0.05）in the DMY suspension group and DMY-Ms group respectively,and the duration of drunkenness was decreased by1.34 times and 2.10 times（P<0.05）in the mice compared with the model group.In the blood alcohol concentration assay in rats,DMY-Ms reduced the alcohol concentration by 73.6%at 30 min and 45.1%at 90 min compared to the model group.In the alcoholic gastric mucosal injury,DMY-Ms reduced the GUI value of mice to 21.09±1.60,which was 66.10%lower than that of mice in the model group;DMY-Ms made the gastric mucosa intact and transparent with only mild inflammatory infiltration.The difference was statistically significant（P<0.05）compared with the DMY-Ms group.In alcoholic liver injury,DMY-Ms significantly reduced serum ALT and AST levels,compared to the model group（P<0.01）;DMY suspension and DMY-Ms group increased SOD activity and reduced MDA levels in liver tissue（P<0.01）,and there was a statistically significant difference between DMY-Ms and DMY suspension（P<0.05）.Conclusion:DMY-Ms can significantly improve the solubility of DMY,slow down the release of DMY,improve the stability of DMY and increase the permeability of DMY in the intestine,thus increasing the absorption and the oral bioavailability of DMY,and the anti-alcoholism ability of DMY-Ms is also significantly improved.DMY-Ms provides an experimental basis for the development of oral DMY formulations with good absorption and high bioavailability in the future,and provides a clinical application for DMY against alcoholism.DMY-Ms provides an experimental basis for the future development of oral DMY formulations with good absorption and high bioavailability,as well as clinical application of DMY against alcoholism.