Pharmacokinetic Studies Of Benzoylmesaconine And Ephedrine In Wutou Decoction

BackgroundRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic arthritis, and it has an incidence of0.3%to1%. The etiology of this disease is still unclear. RA is associated with many factors, such as infection, heredity, estrogen levels, fatigue, trauma, and environment. Without timely treatment, the lasting recurrent synovial inflammation can lead to the destruction of joint cartilage and bone, causing severe joint deformity and joint dysfunction, which can seriously reduce the quality of life of patients. The Western medical treatment of RA relieves its symptoms and controls its progression, thereby improving the quality of life of patients. Non-steroidal anti-inflammatory drugs, slow-acting anti-inflammatory drugs, and glucocorticoids are used to treat RA. Although these drugs are fast acting, their long-term use can lead to serious adverse reactions. Numerous clinical studies shown that some Traditional Chinese Medicine (TCM) preparations and TCM combined with chemical drug therapy achieve more satisfactory therapeutic efficacy on RA than chemical drug therapy.Wutou decoction has been used to treat RA, constitutional hypotension, and hemicrania for hundreds of years. Wutou decoction consists of five medicinal herbs, Radix Aconiti(Chuan Wu), Herba Ephedrae (Ma Huang), Radix Paeoniae alba (Bai Shao), Radix Astragali (Huang Qi), and Radix Glycytthizae (Gan Cao). Chuan Wu is the monarch drug and Ma Huang is the ministerial drug of Wutou decoction, and they are the most important effective components. However, Chuan Wu and Ma Huang have serious side effects. Thus, clarifying the material basis and mechanism of the clinical efficacy of Wutou decoction is necessary.Pharmacokinetic study has an important role in the research, development, and clinical application of drugs. Pharmacokinetic parameters can be used to guide clinical application, to assess drug safety, and to promote the development of new drugs. However, the effective components of TCM are much more complicated, and clarifying the pharmacokinetic characteristics of a TCM preparation is more difficult. At present, most of the pharmacokinetic studies on TCM preparations focus on one or several of the active components. Selecting representative compounds is the key to the pharmacokinetic study of Wutou decoction. The representative compounds were selected according to the following conditions. First, the pharmacologic activity of these compounds should be consistent with Wutou decoction. Second, these compounds should have higher content in Wutou decoction.Considering Chuan Wu is the monarch drug of Wutou decoction, it plays the most important role in the decoction. Aconitum alkaloids are the active ingredients of Chuan Wu, including diester diterpenoid alkaloids (DDAs) and monoester diterpenoid alkaloids (MDAs). DDAs are easily hydrolyzed to MDAS during boiling. Most of the DDAs in aconite decoction are hydrolyzed by30min of boiling. Preparing Wutou decoction requires prolonged boiling; therefore, DDA is not an appropriate representative component of Wutou decoction. Benzoylmesaconine (BMA), a mesaconitine hydrolyzate, is one of the active ingredients of Chuan Wu. BMA has significant analgesic effects and a therapeutic effect on RA. In addition, the BMA content of Wutou decoction is significantly higher than other aconitum alkaloids. Thus, BMA was selected as the representative component of Chuan Wu. The pharmacokinetic characteristics of BMA in Wutou decoction were determined in our experiments.Ephedrine exerts the most potent effect of Ma Huang, the ministerial drug of Wutou decoction. The chemical structure of ephedrine is similar to adrenaline, and it interacts with adrenaline receptors in vascular and diastolic blood vessels, thereby increasing blood flow. The ephedrine content of Wutou decoction is much higher than that of other compounds. Therefore, ephedrine was selected as the representative component of Ma Huang. The pharmacokinetic characteristics of ephedrine after oral administration of Wutou decoction and Ma Huang single herb decoction were investigated. A four-site perfusion model was also used to investigate the absorption of ephedrine in Wutou decoction at different intestinal segments.The effective components of Wutou decoction are highly complicated, and the concentrations of target compounds, such as BMA and ephedrine, are quite low in plasma and perfusate samples. Furthermore, many groups and many time points are available for sample acquisition. Thus, a large sample size is needed for analysis. An efficient and sensitive detection method is required for sample analysis. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) is sensitive and specific and it has a high throughput. Samples do not need to be separated completely, which simplifies the preparation process and greatly shortens the time for sample analysis. Multiple reaction monitoring (MRM) mode also allows simultaneous determination of several compounds. Using UPLC-MS/MS for pharmacokinetic study facilitates obtaining more accurate pharmacokinetic parameters. ObjectivesThe objective of this study was to investigate the pharmacokinetic characteristic of BMA and ephedrine, the representative compounds of Wutou decoction. We believe that the pharmacokinetic study of the monarch drug (Chuan Wu) and ministerial drug (Ma Huang) of Wutou decoction can provide a material basis and theoretical foundation for the further study and clinical application of Wutou decoction. Our study could be divided into several parts as follows:1. To determine the pharmacokinetic characteristics and bioavailability of BMA after oral administration of Wutou decoction.2. To determine the pharmacokinetic characteristics and bioavailability of ephedrine after the oral administration of Wutou decoction and Mahuang single herb decoction.3. To investigate the absorption of ephedrine in Wutou decoction in different intestinal segments using a four-site perfusion model. Methods and Results1. Pharmacokinetic study of BMA after oral administration of Wutou decoctionA rapid and sensitive UPLC-MS/MS method was developed for quantifying BMA in plasma samples. Wutou decoction was prepared and freeze-dried using a lyophilizer to obtain the lyophilized powder. The BMA content of the lyophilized powder was quantified by UPLC-MS/MS one day before the experiments. Male Sprague-Dawley (SD) rats weighing230g to280g were obtained for the experiments. Ten rats were divided into two groups, namely, BMA and Wutou decoction groups. BMA and Wutou decoction were administered orally at5mg of BMA per kilogram of body weight. Blood samples were collected at5,15,30,45,60,90,120,180,240,420, and600min after dosing. After centrifugation at8000rpm for8min, plasma fractions were transferred into a disposable tube and stored at -80℃until analysis. We mixed80μl of plasma with320μl of methanol containing100nM testosterone. The mixture was centrifuged at13,000rpm for30min. Approximately300μl of the supernatant was transferred into a disposable tube and evaporated to dryness under a stream of nitrogen at room temperature. The residue was reconstituted with100μl of methanol-water (v:v=1:1), and injected into the UPLC-MS/MS system for analysis. The pharmacokinetic parameters were determined using the standard non-compartmental method and calculated using WinNonlin3.3.The Tmax of BMA after oral administration of BMA solution was about45min, and the plasma concentration of BMA was approximately0at600min after dosing. However, the Tmax of BMA after oral administration of Wutou decoction was15min, and BMA was completely eliminated at600min after dosing. The relative bioavailability of BMA after oral administration was19.9%. The Tmax and mean residence time (MRT) after administration were significantly shorter(P<0.05).2. Pharmacokinetic study of ephedrine after oral administration of Wutou decoction and Ma Huang single herb decoctionA rapid and sensitive UPLC-MS/MS method was developed and validated for quantitative determination of ephedrine in plasma samples. Wutou decoction and Ma Huang single herb decoction were prepared and freeze-dried by a lyophilizer to obtain the lyophilized powder. The ephedrine content in lyophilized powders was quantified by HPLC the day before the experiments. Male SD rats weighing from230g to280g were obtained for the experiments. Ephedrine hydrochloride, Wutou decoction, and Ma Huang single herb decoction were administered orally at a dosage of10mg of ephedrine per kilogram of body weight. Ephedrine hydrochloride was also administered via intravenous injection at4mg/kg. Blood samples were collected at5,15,30,45,60,90,120,180,240,420,600, and1440min after oral administration; and at1,5,12,18,30,45,72,100,150,240,480, and600min after intravenous injection. After centrifugation at8000rpm for8min, plasma fractions were transferred into a disposable tube and stored at-80℃until analysis. We mixed80μl of plasma with520μl of ethyl acetate containing20μM testosterone. The mixture was centrifuged at13000rpm for30min. Approximately400μl of the supernatant was transferred to a disposable tube and evaporated to dryness under a stream of nitrogen at room temperature. The residue was reconstituted with100μl of methanol-water (v:v=1:1), and injected into the UPLC-MS/MS system for analysis. The pharmacokinetic parameters were determined using the standard non-compartmental method and calculated using WinNonlin3.3.Ephedrine was completely eliminated at600min after oral administration of ephedrine hydrochloride, Wutou decoction, and Ma Huang single herb decoction. The absolute bioavailabilities of ephedrine in the rats after oral administration of ephedrine hydrochloride, Ma Huang single herb decoction, and Wutou decoction were83.4%,71.4%, and29.9%, respectively. The relative bioavailabilities of ephedrine in the rats after oral administration of Ma Huang single herb and Wutou decoctions were85.6%and35.9%, respectively. The Tma of ephedrine after oral administration of Wutou decoction and Mahuang single herb decoction were significantly shortened (P<0.05).3. Absorption study of ephedrine in Wutou decoction at different intestinal segmentsWutou decoction was prepared and freeze-dried using a lyophilizer to obtain the lyophilized powder. The ephedrine content of the lyophilized powder was quantified by high-performance liquid chromatography (HPLC) the day before the experiments. Ephedrine hydrochloride and lyophilized powder were dissolved in Hank’s balanced salt solution to obtain perfusate solutions containing40μg/ml ephedrine. Male SD rats weighing from230g to280g were obtained for the experiments. A four-site perfusion model was used to investigate the absorption of ephedrine at different intestinal segments. Four segments of the intestine (duodenum, upper jejunum, terminal ileum, and colon) were perfused simultaneously with the prepared perfusate. The perfusate samples were collected every30min. Acetonitrile was added into the perfusate (v:v=1:2) and centrifuged at13,000rpm for30min. We injected10μl of supernatant into the UPLC system for analysis.After perfusion with ephedrine hydrochloride solution, the permeability coefficients (Peff) of ephedrine in the duodenum, jejunum, ileum, and colon were2.20,1.81,2.33, and1.68, respectively. Approximately24.9%,19.5%,25.2%, and17.0%of ephedrine was absorbed in the duodenum, jejunum, ileum, and colon, respectively. After perfusion with Wutou decoction, the Peff of ephedrine at duodenum, jejunum, ileum, and colon were2.33,2.31,3.35, and2.68, respectively. Approximately27.7%,26.4%,37.4%, and30.3%of ephedrine was absorbed at duodenum, jejunum, ileum, and colon, respectively. The absorption of ephedrine at jejunum, ileum, and colon following Wutou decoction perfusion was significantly higher than those with ephedrine hydrochloride (P<0.05).Conclusions1. The relative bioavailability of BMA after oral administration of Wutou decoction was19.9%. The Tmax. of BMA after oral administration was significantly shortened, which indicates that some compounds in the decoction accelerate BMA absorption. MRT was also significantly shortened, indicating that some compounds in the decoction promote BMA clearance.2. The absolute bioavailabilities of ephedrine in the rats after oral administration of Wutou decoction was29.9%, which was much lower than the other groups. The Tmax of ephedrine after oral administration of Wutou decoction and Mahuang single herb decoction were significantly shortened, which indicates some compounds in the decoctions accelerate ephedrine absorption.3. Ephedrine was well absorbed at different intestinal regions in rats perfused with ephedrine hydrochloride solution and Wutou decoction. The absorption of ephedrine in the jejunum, ileum, and colon after Wutou decoction perfusion was significantly higher than those with ephedrine hydrochloride, which suggests that some compounds in the decoction promote ephedrine absorption.