Study On Absorption And Tissue Distribution Of Trametenolic Acid B In Rats

Background Trametenolic acid B（TAB）is a tetracyclic triterpene compound isolated from Poria cocos peel.Previous studies have showed that TAB has good antitumor activity against liver,stomach,and cervical cancers,as well as some neuroprotective effects.In the process of modern drug development,the pharmacokinetic properties of compounds have become an important standard to judge whether they can be developed into drugs.An ideal candidate compound should have high bioavailability and ideal half-life,and be able to reach the target organ to play the drug effect,and should avoid the formation of toxic metabolites in vivo.At present,a large number of literatures have studied the metabolism kinetics and metabolites of other tetracyclic triterpenoids in Poria cocos peel,but there is little research on TAB metabolism,so it is very necessary to study the pharmacokinetics and metabolites of TAB.Objective TAB is insoluble in water so it’s difficult to cross the intestinal barrier when taken orally,resulting in low blood drug concentration,low bioavailability and unable to reach high concentration in target organs.Therefore,it is necessary to increase the solubility of TAB in water first,so as to increase intestinal absorption and improve bioavailability of TAB.Then,the drug concentration in TAB blood was further increased by drug combination so that the drug concentration in the target organ could reach higher concentration.Pharmacokinetics were studied after increasing blood concentration.Methods（1）The solubility of TAB under different cosolvents was determined by vanillic aldehyde-acetic acid-perchloric acid color method,and the best cosolvent was selected.The pharmacokinetics of TAB were studied with the best cosolvent,and the blood concentration and metabolokinetics parameters were obtained.（2）MTT assay was used to detect the effects of TAB combined with curcumin（CUR）on proliferation of SGC9701 cells.Cell uptake method was used to determine the change of intracellular drug concentration under the condition of combination of two drugs.Western blot assay was used to detect the expressions of P-gp and BCRP in SGC9701 cells when TAB and CUR were combined.The concentration curves of the two drugs in rat plasma were determined over time after TAB combined with CUR,and the metabolokinetic parameters were obtained by fitting the curves.（3）TAB and CUR were dissolved by PAP,and orally administered to rats.TAB concentration in plasma,heart,liver,spleen,lung,kidney,brain,stomach,colon and intestine of rats was determined.Results（1）Color reaction showed that the solubilizing effect of PAP on TAB was better than that of sodium carboxymethyl cellulose,Poloxham,Tween-80,glycerol,etc.The solubility of TAB in 10%PAP solution reached 616.0μg·m L^-1.The results of pharmacokinetic study in rats showed that the concentration of TAB in PAP group was significantly higher than that in TAB group at other time points except at 3 h,and the AUC（0-t）in PAP group was 171.28±9.23 mg L^-1·h,which was 2.13 times of that in TAB group.The AUC（0-∞）of PAP group was 337.41±27.12 mg L^-1·h,which was2.47 times that of TAB group.The C_max of PAP group was 18.89±1.55 mg L^-1,which was 1.46 times that of TAB group.（2）The results of MTT showed that the IC₅₀ for single CUR was 37.23μg·m L^-1,the IC₅₀ for single TAB was 119.36μg·m L^-1,and the combined drug index for CUR12μg·m L^-1 and TAB 40μg·m L^-1 was 0.68<1,which showing the synergistic effect.The results of cell uptake experiments showed that the promotion rate of TAB(40μg·m L^-1)in CUR(12μg·m L^-1)was 1.18,and that of TAB(80μg·m L^-1)in CUR(24μg·m L^-1)was 1.63.Western Blot analysis of the expressions of P-gp and BCRP showed that TAB and CUR treatments significantly decreased the expressions of both protein,and the values of TAB and CUR treatments were lower than those of single treatment.In vivo pharmacokinetic experiment of rats showed that the concentration of TAB in TAB+CUR group was higher than that in TAB alone group.The concentration of CUR in the plasma of rats was lower when the CUR was used alone,while the concentration of CUR in the blood increased significantly when the CUR was used with TAB.The C_max of TAB in the combined use group was 2.88 times of that in the single use group.The C_max of CUR in the combined use group was 2.27times of that in the single use group.（3）The pharmacokinetic results of rats showed that the AUC（0-t）of TAB combined with CUR was 277.51±13.08 mg L^-1·h,which was 3.29 times that of TAB alone.The C_max in TAB+CUR+Poria polysaccharide group was 55.49±3.41 mg L^-1,which was 4.46 times of that in TAB+CUR+PAP group.Tissue distribution results showed that TAB concentration in some tissues in the combined treatment group increased significantly at 2.5,6 and 12 h.Conclusion（1）PAP could increase the solubility of TAB in water,thus increasing the absorption of TAB in rats.（2）TAB combined with CUR significantly increases the inhibitory effect on tumor cell proliferation,and promotes mutually in vivo and in vitro absorption by inhibiting efflux transporters.（3）TAB is distributed in the heart,liver,spleen,lung,kidney,stomach,large intestine and small intestine,and can enter the brain through the blood-brain barrier,mainly distributed in the stomach,large intestine and small intestine digestive tract.TAB concentration in rat plasma and tissues under combined action of PAP and CUR.