The Preparation And Pharmacokinetic Studies Of The New Traditional Chinese Medicine Jiawei Foshou San Capsule

BackgroundThe Jiawei Foshou San (JWFSS), which consists of A, B and C component, is a drug developed from Chinese Angelica sinensis, Ligusticum chuanxiong in the Foshou San formula, a popular traditional Chinese medicine for irregular menses. Now JWFSS is used for treating endometriosis. A series of detections in vivo have proved that the combination of the three ingredients is safety to use. For clinic, we have encapsulated A, B and C for oral administration. This study was supported by National Key New Drug Development Project of China (Number:2014ZX09304-306-04)ObjectiveThis study will investigate JWFSS capsule’s quality standard, stability, pharmacokinetics and the effect on rat hepatic CYP450 enzyme activity. These experiments will be foundation for developing " JWFSS capsule" in future. Methods1. The preparation methods of JWFSS capsuleThe medicine granules were made via wet granulation, and then loaded in capsule. The formation technology was optimized by the difficulty level of the granulating, the hardness, the uniformity, the rate of powder, the percentage of the moisture absorption, the liquidity, and the bulk density of the granule. A suitable filler were selected from soluble starch, lactose and microcrystalline cellulose, and adhesive from PVP K30, HPMC, CMC-Na and model number of HPMC from HPMC-K100M, HPMC-K15M, HPMC-K4M. Then, all of the doses of them in JWFSS capsule were determined.2. Quality control of JWFSS capsuleThe quality standard of JWFSS capsule was researched from the following four aspects:(1) Characteristics:the appearance and color were described. (2) Identification: HPLC was used to identify the uniformity of retention time between the test sample solution and the control sample solution. (3) Inspection:basing on the appendix IE in 2010th edition of Chinese Pharmacopeia, JWFSS capsules were inspected on the content uniformity, the particle size, the moisture content, the dissolution rate, etc. (4) Determination:HPLC was used to make sure the stability of A, B and C component in JWFSS capsule. The quality standard draft of the JWFSS capsule was established.3. Stability of JWFSS capsuleThe stability of JWFSS capsule was detected by influential factor test, accelerated test and long-term test. (1) Influential factor test:to investigate the stability of JWFSS capsule under severe environment, JWFSS capsules were respectively placed under situations of high temperature (60 ℃±2 ℃), high humidity (relative humidity is 90%±5% and 75%±5%), and highlight (4500 Lx±500 Lx). (2) Accelerated test:JWFSS capsules were placed under conditions of 40℃±2 ℃ and humidity of 75% for 6 months. (3) Long-time test:JWFSS capsules were placed under environment of 25℃±2℃ and humidity of 65%±5% for 9 months.4. Pharmacokinetics of JWFSS capsule in rats(1) The method of detecting the A, B and C in JWFSS formula in rat plasma was established by HPLC. (2) 20 SD female rats were divided into 2 groups at random:ig and iv administration group at the dose of 180 mg-kg"1 bulk drugs of JWFSS,6 in each group. The whole blood was collected at 0,5,10,20,30,60,120,240,480,720 min after administration. The plasma was tested by HPLC to detect the concentration of the drug at different time after administration. The pharmacokinetic parameters Cmax、Tmax、 t1/2、AUC(0-t)、AUC(0-∞) and MRT(0-∞) were analyzed by DAS 3.2.1 software. Moreover, the absolute bioavilability was calculated. (3) 20 SD female rats were divided into 2 groups at random:normal and endometriosis model group at the dose of 180 mg·kg-1 JWFSS capsule’s content,10 in each group. The whole blood was collected at 0,5,10, 20,30,60,120,240,480,720 min after administration. The plasma was tested by HPLC to detect the concentration of the drug at different time after administration. The pharmacokinetic parameters Cmax、Tmax、t1/2、AU(0-t)、AUC(0-∞) and MRT(0-∞) were analyzed by DAS 3.2.1 software.5. The effect of the JWFSS capsule on liver microsome CYP450 enzyme activity(1) The test of inhibitory effect of JWFSS capsule on human liver microsomes CYP450 enzyme activity in vitro:The in vitro test groups included negative control group, inhibitor positive control group, A group, B group, C group, and JWFSS capsule group. After incubating liver microsomes with a cocktail of probe drugs, the metabolites were quantitated using liquid chromatography tandem mass spectrometry (LC-MS/MS) to evaluate the effect of JWFSS capsule on human microsomes CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 enzyme activity in vitro. (2) The test of inhibitory effect of JWFSS capsule on rat liver microsomes CYP450 enzyme activity in vitro:The grouping and method were the same as above. The effect of JWFSS capsule on rat liver microsomes the activity of the above five enzymes were tested. (3) The test of inducing effect of JWFSS capsule on rat liver microsome CYP450 enzyme activity in vivo:in vivo tests, female rats were randomly divided into 9 groups: negative control, a phenobarbital-induced positive control, A, B, C, A+B+C and three different doses of JWFSS capsule. After 28 days of treatment, liver microsomes were obtained from the rats and assayed for CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 activity.6. The test of influence of JWFSS capsule on rat hepatocyte morphology:female rats were randomly divided into 8 groups:negative control, A, B, C, A+B+C and three different doses of JWFSS capsule. After 28 days of treatment, the livers from all animals were prepared for pathological observation using hematoxylin and eosin (HE) staining.Results1. The established preparation methods of JWFSS capsule is stable and feasibleThe three kinds of main medicine A, B and C were mixed based on the previous pharmacodynamics research. Every 1.8g mixed powder,0.9g filler lactose and glue 0.5% HPMC-K15M 0.25 mL were mixed, and sieved; medicine granules were made via wet granulation, and then loaded in capsules; prepared capsules were sealed and stored in aluminum plastic foamed mask packages. Three batches of JWFSS capsules based on thees conditions show consistent colour and lustre, uniform granules, high molding rate, good liquidity and low moisture absorption rate. Comprehensive evaluation shows that this process condition of preparation is stable and feasible. And it is the best molding process for the capsule.2. several sides are used for controlling the quality of JWFSS capsule(1) Characteristics:The JWFSS capsules were sealed and stored in aluminum plastic foamed mask packages. Its content presented as faint yellow.(2) Identification:By HPLC, JWFSS capsule were identified to be the same retention time with controls.(3) Inspection:All of the content uniformity, the particle size, the moisture content, and the dissolution rate of JWFSS capsule were in accordance with the Chinese Pharmacopeia.(4) Determination:The content of A, B and C component in these batches of JWFSS capsules varied in 98%-105%, which were in accordance with the Chinese Pharmacopeia.3. JWFSS capsule has good stability.The influential factor test, the accelerated test and the long-time test showed the satisfactory stability of JWFSS capsule.4. The in vivo pharmacokinetics of JWFSS capsule in rats(1) The bioavailability of investigation of JWFSS formula:the AUC(0-∞) of A, B, and C in rat plasma after intravenous injection administration of JWFSS were 44.35±17.08 mg·h·L-1,139.65±22.28 mg-h-L-1,61.43±7.76 mg-h-L-1, respectively; the AUC(o～∞) of A, B, and C in rat plasma after intragastric administration of JWFSS were 31.77±8.64 mg·h·L-1,97.70±15.26 mg-h-L-1,44.22±10.85mg·h·L-1, respectively. These results showed the absolute bioavailability (F) of A, B, and C were 71.63%,69.96% and 71.98% respectively. Therefore, JWFSS was suitable for oral because of the high bioavailability.(2) The pharmacokinetic comparison of JWFSS capsule in the normal rats and endometriosis rats:there was no significant difference on the absorption of A between the normal and the EMs model rats (P>0.05); compared with the normal rats, the Tmax was shortened in the model rats (P<0.05), which showed the absorption speed of B in the model rats was much faster; compared with the normal rats, the Cmax, AUC(0-t) and AUC(0-∞) were higher in the models rats (P<0.05), which showed the uptake was better than the normal rats. These results indicated that the clinical application of JWFSS capsule was rational in the treatment of EMs disease.5. The effect of JWFSS capsule on rat hepatic cytochrome P450(1) The in vitro inhibition test of JWFSS capsule on the activity of CYP450 enzyme in human liver microsomes:the ICsos of the gradient A and the B for the five CYP450s were too high to determine, so A and B did not show any significant inhibitory effects on these five CYP450s in human microsomes in vitro; The IC50 of C for CYP2D6 was 9.24 μM, which is lower than 10 μM and higher than 1 μM, showing that C had a moderate-intensity inhibitory effect on the activity of CYP2D6 in human microsomes in vitro. The IC50S of JWFSS capsule for CYP2D6, CYP2E1 and CYP3A4 were 123.9 mg·L-1,189.9 mg·L-1and 171.3 mg·L-1 respectively, all of which are much lower than the half of the highest concentration 1600 mg·L-1, showing that JWFSS capsule had a inhibitory effect on the activity of CYP2D6, CYP2E1 and CYP3A4 in human microsomes in vitro.(2) The in vitro inhibition test of JWFSS capsule for the activity of CYP450 enzyme in rat liver microsomes:the IC50S of A and B for five CYP450s were too high to determine, suggesting that A and B do not show any significant inhibitory effects on the activities of these five CYP450 in rat microsomes in vitro; The IC50 of the C for CYP3A4 was 7.46 μM, which is lower than 10 μM and higher than 1 uM, showing that C had a moderate-intensity inhibitory effect on the activity of CYP3A4 in rat microsomes in vitro. The IC50S of the JWFSS capsule for CYP2D6, CYP2E1 and CYP3A4 were 241.3 mg·L-1,369.8 mg·L-1 and 293.0 mg·L-1 respectively, which is much less than the half of the highest concentration 1600 mg·L-1, showing that JWFSS capsule had a inhibitory effect on the activity of CYP2D6, CYP2E1 and CYP3A4 in rat microsomes in vitro.(3) The in vivo induction test of JWFSS capsule on the activity of CYP450 enzyme in rat liver microsomes:Compared with the negative control group, the five CYP450 activities in the A, B and C groups were not significantly different (P>0.05), but the enzyme activities of CYP1A2, CYP2C9 and CYP3A4 showed a significant increase in the JWFSS capsule 180 mg·L-1·d-1 group and the A+B+C group (P<0.05), which suggested that A, B and C did not significantly induce the five CYPs in rat microsomes in vivo, but JWFSS capsule and A+B+C showed a significant induction on CYP1A2, CYP2C9 and CYP3A4.6. The effect of the JWFSS capsule on the hepatocyte morphologyAfter administration, the cells morphology were normal, and the liver portions in A-treated and B-treated rats did not show any obvious morphological changes compared to the control. The pathological damages were observed in C-treated rats, the boundaries of the hepatic lobules were unclear, and the hepatic cord was disorderly and characterized by a blurred edge. Additionally, the infiltration of inflammatory cells was observed. Both the (A+B+C)-treated and JWFSS capsule-treated rats showed pathological changes, such as the infiltration of inflammatory cells, but the extent of the lesions was generally less than the C-treated rats. These results show the combination of A, B and C could significantly reduce the liver injury of C component.Conclusions1. We obtained the production craft of JWFSS capsule, a novel candidate of TCM to treat endometriosis. The production craft was simple, and the quality control standards could control the quality of JWFSS capsule, the stability was good.2. The bioavailability’s studies show that the absorption of the drug was good after intragastric administration of JWFSS formula. The bioavailability of JWFSS was high. Therefore, it was suitable for oral. The in vivo absorption rate of B component and the absorption’s degree of C component in the EMs modle rats after administration of JWFSS capsule were better than in the normal rats, which provided the rationality of the pharmacokinetic experiment basis of JWFSS capsule to treat endometriosis.3. JWFSS capsule significantly induced the CYP1A2, CYP2C9 and CYP3A4 in rat liver microsomes in vivo; it significantly inhibited the CYP2D6, CYP2E1 and CYP3A4 in rat and human liver microsomes in vitro, our results suggested a strengthened efficacy and a prolonged effective time when the drugs metabolized by CYP2D6 and CYP2E1 are used in combination with JWFSS capsule. On the converse, when the drugs metabolized by CYP1A2 and CYP2C9 are used in combination with JWFSS capsule, the effect of these drugs is reduced.4. C component could induce liver injury alone, but while it used in combination with A and B, its adverse effect on liver were reduced, suggesting the reasonable formula of JWFSS capsule.