Development of sustained-release pellets to modulate the in vivo processes of the main active components of Danshen: A pharmacokinetic and pharmacodynamic evaluation
Graphical abstract
Introduction
Danshen is a typical traditional Chinese medicine (TCM) derived from the root of Salvia miltiorrhiza Bunge, which has been widely applied in clinics for cardiovascular disease, particularly for variant angina (VA). (Cai et al., 2018). At present, over 100 chemical constituents have been isolated and identified from this plant. (Chen et al., 2014). Among them, tanshinone IIA (TA), tanshinol (TS), protocatechuic aldehyde (PD), and salvianolic acid B (Sal B) are the four major active components (Fig. 1A) (Chang et al., 2008) that exert diverse pharmacological effects on cardiovascular diseases. Specifically, TA can reduce myocardial infarct size by increasing the scavenging of oxygen free radicals, diminishing lipid peroxidation and upregulating the Bcl-2/Bax ratio (Pan et al., 2011). TS exerts the effects of dilating coronary arteries, reducing platelet aggregation, and improving microcirculation (Zhou et al., 2005). Additionally, PD directly attenuates the proliferation and migration of vascular smooth muscle cells and inhibits thrombosis (Moon et al., 2012). Sal B is capable of reducing ischemia-reperfusion injury owing to the production of NO and inhibition of stress-activated protein kinase activity (Ouyang et al., 2001). Taken together, all of the active components might generate greatly amplified pharmacological effect by hitting multiple targets synergistically when used in combination (Cai et al., 2018, Zhang et al., 2016a).
The combined effect of the multiple active components is related to the pharmacokinetic performance and the bioavailability of each ingredient, which are heavily dependent on their physicochemical properties (Yang et al., 2014, Zhang et al., 2016b). However, TA, TS, PD, and Sal B show different physiochemical properties, For example, TA is a major hydrophobic constituent with poor water solubility (2.8 ng/ml) and exhibits extensive first-pass metabolism (Yan et al., 2015). Although TS, Sal B, and PD are hydrophilic bioactive constituents, TS and Sal B show limited permeability (Zhou et al., 2009), while PD undergoes substantial first-pass metabolism (Zhang et al., 2016c). The aforementioned physicochemical properties result in their extremely low bioavailability. In our previous studies, Sal B phospholipids complex (Li et al., 2013), PD proliposome (Zhang et al., 2016c), TA solid dispersion pellets (Yan et al., 2015), and TS pellets with absorption enhancer (Yu et al., 2017) have been developed to modify their physicochemical properties for superior oral bioavailability, respectively.
However, the different physiochemical properties of the four active components in Danshen also lead to their dissimilar pharmacokinetic performances. Consequently, TS, PD, and Sal B are usually absorbed and eliminated rapidly after oral administration as they all reach peak plasma concentrations within 1–2 h and their mean residence time (MRT) is approximately 3.5 h, 3 h, and 1.5 h, respectively (Li et al., 2013, Yu et al., 2017, Zhang et al., 2016a). Nevertheless, the peak plasma concentration of TA is reached at 5.5 h with an MRT of approximately 10 h (Yan et al., 2015). Theoretically, the multiple components might generate better effect if they could be absorbed and eliminated synchronously and exert their pharmacological actions simultaneously, (Song et al., 2002), which is in accordance with the organic whole concept of compatibility of TCM. Unfortunately, the divergent pharmacokinetic characteristics of the four active components in Danshen might restrict their co-existence and co-action in vivo, thus compromising their overall effect. To mitigate this problem, sustained- or controlled-release formulations might be a promising approach for modulating the pharmacokinetic behaviours of the multiple components.
Well-designed sustained- or controlled-release formulations could alter the drug release or pharmacokinetic profiles via different rate-controlling mechanisms such as diffusion, swelling and erosion (Chavanpatil et al., 2006). These novel preparations were recently applied to synchronise the release of multiple components. Cheng et al. developed an erodible glyceryl monostearate matrix system from which the active components with extremely different physicochemical properties in silymarin can dissolve and release synchronously as the drug release is mostly related to the erosion rate of the glyceryl monostearate matrix (Lu et al., 2007). Song et al. reported pH-dependent gradient heart-protecting musk pellets that were capable of releasing the active components simultaneously at pH 5.5, 6.2 and 6.8, which can also be regarded as “synchronous” (Song et al., 2002). However, the in vivo performances of the multiple components in the sustained- or controlled-release system did not depend only on their release behaviors from the formulations, but also might be influenced by complex and varied physiological conditions (Zahirul and Khan, 1996). Therefore, to regulate the absorbed and eliminated processes of the four active components in Danshen for the prolongation of their coexistence and achieve the desired therapeutic efficiency, a new method should be established by considering the physicochemical properties of the drugs, the physiological environment, and the clinical requirements.
Clinical research has demonstrated that VA predominantly occurs at rest from midnight to early morning and peaks at approximately 4:00 am (Lemmer, 2006), showing obvious circadian rhythm. These temporal patterns of VA are important for pharmacotherapy from a chronotherapeutic perspective. Herein, we attempted to regulate the pharmacokinetic performances of TS, TA, PD, and Sal B based on the circadian rhythm of VA. In this study, TA, TS, PD and Sal B sustained-release pellets (SRPs) were respectively prepared based on the optimised formulations reported in our previous researches, in which the desired drug-release curves of the four kinds of SRPs were calculated via deconvolution method based on the circadian rhythm of VA (Li et al., 2013, Xia, 2016, Yan et al., 2015, Zhang et al., 2016c). They were then filled into capsules according to the original weight ratio of the four active components in pure Salvia miltiorrhiza extract. Release tests of the combined Danshen capsules in different media were conducted to investigate the release stability of the four kinds of SRPs in the combined Danshen capsules. The pharmacokinetic behaviors of the combined Danshen capsules were also determined to verify whether the coexistent time of the four active components were prolonged in vivo. Finally, pharmacodynamic studies were conducted to compare the cardiovascular protective effect of the combined Danshen capsules and commercial Danshen capsules on an angina model of New Zealand White (NZW) rabbits by taking electrocardiogram, heart histopathological and the level of nitric oxide (NO), endothelin (ET), creatine kinase (CK-MB), cardiac troponin-I (cTn-I), Superoxide Dismutase (SOD) and malondialdehyde (MDA) as the evaluation indicators.
Section snippets
Materials
TA (98.63%) and Sal B (98%) were purchased from Xi'an Honson Biotechnology Co., Ltd. (Xi'an, Shanxi, China). PD (99%) and TS (>99%) were purchased from Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China). Gelatin capsules were from Suzhou Capsugel Ltd. (Suzhou, China). High-fat fodder was provided by Jiangsu Xietong Biological Engineering Co., Ltd. (Nanjing, China). Cholesterol was obtained from Sigma-Aldrich Co., Ltd. NO reagent box, SOD reagent box, MDA reagent box, cTn-I reagent
Quantitative analysis of the drug content in the combined Danshen capsules
The proposed HPLC method was used for a quantitative analysis of the contents of the four active compounds of Danshen. The drug loading of TA, TS, PD, and Sal B was 2.5%, 3.7%, 3.7%, and 11.4%, respectively.
In vitro release studies of the combined Danshen capsules
Due to the pH variations in the gastrointestinal tract, the release stability of the combined Danshen capsules in three release conditions with different pH was evaluated. As shown in Fig. 2, each kind of SRPs in the combined Danshen capsules maintained sustained release for 24 h. The
Discussion
VA is a kind of clinical presentation of atherosclerotic that is caused by a variety of pathophysiological mechanisms, including inflammatory reaction, reduction of antioxidant defenses, vasoconstriction, plaque associated with thrombosis and so on (Michelle et al., 2008, Misra et al., 2009, Nakashima et al., 2007). Accordingly, rather than focusing on a single target, combination treatment with multiple effect targets tend to be a more effective way (Yang et al., 2014). Danshen, whose
Conclusion
In this paper, the TA, TS, PD and Sal B sustained-release pellets were prepared according to their individual optimised formulations developed in our previous studies, and filled into the capsules based on their original weight ratios in pure Salvia miltiorrhiza extract. The dissolution performances of the four kinds of SRPs contained in the combined Danshen capsules were stable in different pH media. Furthermore, the pharmacokinetic results confirmed that the combined Danshen capsules could
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
This study was financially supported by National Natural Science Foundation of China (no. 81473151) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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