The Study Of Preclinical Pharmacokinetics Of A Novel Anti-inflammatory Drug M10-Na Derived From Myricetin

Object:Myricetin has a variety of biological activities,such as anti-inflammatory,bactericidal,but because of its poor water solubility,difficult to absorb in the body and other factors are difficult to make medicine,so its structure was modified.As myricetin derivatives,M10-H and M10-Na inhibit the intestinal tumorigenesis via strong inhibition of the NF-B/IL-6/STAT3 pathway in a mice model with ulcerative colitis induced by azoxymethane（AOM）/dextran sulfate sodium（DSS）.It’s pharmacological activity was significantly better than that of myricetin and mesalazine.The aim of this research was to evaluate the preclinical pharmacokinetic feature of M10-Na in animals,so as to support the data for the study of druggability,preparation and route of administration,and to provide reference for the pharmacokinetic research and clinical application of human body.Methods:In this study,we developed a sensitive UPLC-MS/MS method for the measurement of M10-H and M10-Na in rat plasma.This method was applied to study the pharmacokinetics of M10-H and M10-Na after oral administration in Wistar rats.This work describes the details of method establishment,validations.This method was applied to study the pharmacokinetics and tissue distribution of M10-H and M10-Na in Wistar rats.The absorption characteristics of M10-H and M10-Na in the intestine were studied by the everted intestine method.The rectal thromboembolus of M10-Na rats was prepared,and the rat model of ulcerative colitis was successfully replicated（symptoms of hematochezia and diarrhea were found by enema of tnitrobenzene sulfonic acid/50%ethanol solution）,and the pharmacokinetic differences of oral and rectal administration of M10-Na were compared between healthy rats and model rats.Results:1.A selective,sensitive,accurate and reliable UPLC-MS/MS method was established and validated for determination of M10-H,M10-Na and myricetin in preclinical pharmacokinetic study under the guiding principles for biological sample analysis method of 2015 edition of Chinese pharmacopoeia.2.After intragastric administration with 100 mg·kg^-1M10-H,M10-Na and myricetin,the results of the pharmacokinetic study showed that the area under the plasma concentration-time curve(AUC_last)of M10-H,M10-Na and myricetin were93±30 h·ng·m L^-1,156±147 h·ng·m L^-1and 132±91 h·ng·m L^-1,respectively.The area under the plasma concentration-time curve(AUC_last)of three different dose of M10-Na were 91±79 h·ng·m L^-1,156±147 h·ng·m L^-1and 752±709 h·ng·m L^-1,respectively,speculation in the dose range M10-Na in the changing process of rats can be linear pharmacokinetic,prompt animal individual differences between larger at the same time.3.Following a single intravenous infusion administration with 0.5 mg·kg^-1M10-H,M10-Na and myricetin to Wistar rats,the blood samples were collected at different time points after dosing.The results of the pharmacokinetic study showed that the area under the plasma concentration-time curve(AUC_last)of M10-H,M10-Na and myricetin were 394±63 h·ng·m L^-1,276±52 h·ng·m L^-1and 375±133 h·ng·m L^-1,respectively.The bioavailability of M10-H,M10-Na and myricetin were 0.12%±0.04%,0.28%±0.27%and 0.18%±0.12%.The absolute bioavailability ratios of M10-H and myricetin to M10-Na were 43%±14%and 64%±43%,respectively.Among them,M10-Na has the highest bioavailability,which is about 2 times of M10-H and myricetin.4.The intestinal absorption function of rats in the M10-H group and M10-Na group were detected in vitro by using the everted intestine method.The cumulative absorption and absorption rate constants of the intestine were calculated and compared.There was no significant difference in the absorption rate of M10-Nabetween the duodenum,jejunum and ileum.Both M10-H and M10-Na were linearly absorbed in the range of parenteral fluid concentration at 0.1 mg·ml^-1.The relative cumulative absorption of M10-H and M10-Na was 43%±18%,which was consistent with the absolute bioavailability ratio（43%±14%）of M10-H and M10-Na in the whole animal experiment.5.Wistar rats were serially enthanized by spondylopathy luxation at 0.25 h,1 h and 2 h after oral administration of M10-Na(100 mg·kg^-1).Tissues were taken out and made into homogenate preparing with 3 times volume normal saline,which followed with the test of M10-Na concentrations using UPLC-MS/MS method.After oral administration of M10-Na,the concentration of M10-Na was very high in gastrointestinal tract.The significant difference between the contents of the small intestine and large intestine suggest that the M10-Na was absorbed faster and may be metabolized in the intestine.In addition,the concentration of M10-Na in liver was higher than the majority of tissues at 2 h,which pointed out that the drug may be metabolized in liver.6.In order to evaluate the rat by rectal drug absorption properties,and to compare the differences of pharmacokinetic of M10-Na in the two kinds of different conditions rats（physiological and pathological conditions）,ulcerative colitis rats model was established,and the evaluation of rats in the two kinds of state（health）and those with colitis oral and rectal pharmacokinetic characteristics of the drug.Following a single intragastric and rectal administration with 100 mg·kg^-1M10-Na to health and ulcerative colitis in Wistar rats,the blood samples were collected at different time points after dosing.Compare the group of intragastric administration with M10-Na in health rats,the group of rectal administration with M10-Na in health rats,the group of rectal administration with M10-Na in ulcerative colitis rats and the group of intragastric administration with M10-Na in ulcerative colitis rats.The results of the pharmacokinetic study showed that the area under the plasma concentration-time curve(AUC_last)were 156±147 h·ng·m L^-1,4174±2357 h·ng·m L^-1,2167±806 h·ng·m L^-1and 150±119 h·ng·m L^-1,respectively.The bioavailability of a single intragastric and rectal administration with 100 mg·kg^-1M10-Na were 0.28%±0.27%and 7.56%±4.27%.In the healthy rat-rectum group,which was about 27 times higher than that in the gavage group.There was almost no difference in bioavailability between the model rat-gavage group and the healthy rat-gavage group.It was speculated that the gavage of M10-Na was mainly absorbed by the stomach and small intestine,while the main pathological site of colitis rats was the distal colon,so there was no significant effect on the absorption of the tested compound after gavage.Model rats rectum to medicine group and healthy rats-rectal dose group the relative bioavailability of 52%±19%,presumably because of model group rats colon ulceration erosion,so the rectum to medicine,model group rats caused by the absorption of rats decreased significantly than health M10-Na in model rats-rectal administration group exposed to decrease nearly half in the body.Conclusion:This method was validated for the carryover effect,selectivity,accuracy,precision,matrix effect,stability,and recovery.A linear correlation was established between concentration and response by the calibration curves of M10-H and M10-Na over 10～2000 ng·m L^-1.Myricetin is linear in the range of 5～1000ng·ml^-1.Compared with myricetin and M10-H,the bioavailability of oral administration of M10-Na was significantly improved.Comparison of the absorption characteristics of M10-H and M10-Na in the small intestine by evagination intestinal capsule experiment further verified that the absorption of M10-Na in the small intestine was significantly higher than that of M10-H.After oral administration of M10-Na,the concentration of M10-Na was very high in gastrointestinal tract.The conclusion suggest that the M10-Na may be was metabolized in the liver and intestine,and was excreted in the kidney and bladder.Rectum administration can greatly improve the bioavailability of M10-Na in vivo.It is speculated that the rectum administration can effectively avoid the first-pass effect,leading to a significant increase in bioavailability.Since ulcerative colitis is most common in the rectum and the distal colon near the rectum,the results of this study suggest that rectum administration maybe a more effective route than oral administration.The results provide important theoretical reference and research ideas for the improvement of M10-Na administration pathway and the research of clinical application.According to the results of pharmacokinetic experiments in vivo,the rectal route of administration is significantly better than the oral route.Whether the pharmacodynamic results are consistent with the results still needs to be further explored by pharmacodynamic experiments.