Thromboembolic disease, comprising VTE and ATE [5], is caused by vascular endothelial damage, stasis of blood flow, and hyper-coagulability of blood [6]. It has become a major global health problem and poses a substantial threat to patient life safety [1, 30]. Therefore, it is of great significance to prevent and treat thromboembolic disease with scientific and systematic methods.
Anticoagulant drugs, including heparin, coumarins, antiplatelet drugs and new oral anticoagulants (NOACs), are used to prevent and treat thromboembolic disease in clinical [31–33]. At present, coumarins (warfarin) and NOACs are the commonly used drugs, which have become the primary choice for prevention and treatment of thromboembolic disease due to their significant anticoagulant effect [34, 35]. Moreover, there is conclusive evidence that haemorrhagic strokes, intracranial bleedings and the risk of thromboembolic complications of NOACs are fewer comparing with warfarin. It indicated NOACs might be more safe than warfarin and become a substitute for warfarin [36, 37]. NOACs mainly include direct thrombin inhibitor (dabigatran) and FXa inhibitors (rivaroxaban, apixaban, edoxaban) [38, 39]. Coagulation FXa is located at the intersection of endogenous and exogenous coagulation pathways [40, 41]. Rivaroxaban can interrupt the endogenous and exogenous pathways of the clotting waterfall by inhibiting FXa, thereby inhibiting thrombin production and thrombosis [42, 43]. Although NOACs have the advantages of fast action and well anticoagulant effect [44], severe bleeding adverse reactions (intracranial hemorrhage [45], gastric hemorrhage [46]) of NOACs have been reported with the wide application in clinical, especially when combined with the strong inhibitors of CYP3A4 or P-glycoprotein (P-gp) [47].
Rivaroxaban is often used in combination with some TCMs in clinical in China, such as Salvia miltiorrhiza [48], Hypericum perforatum [49] and Saffron [50]. However, there are more and more reports that HDIs might be present between rivaroxaban and TCMs due to the complex active components of herbs [27]. The interactions generally result from the effect on CYP3A4 enzyme activity [51, 52]. It has been reported that rivaroxaban is a substrate for CYP3A4 [53]. Cmax and AUC of rivaroxaban would increase significantly when combined with strong CYP3A4 inhibitor (fluconazole [54], ritonavir [55], verapamil [56]). On the contrary, Cmax and AUC of rivaroxaban decreased significantly when combined with potent CYP3A4 inducers (rifampicin [57], phenytoin [58], carbamazepine [59]). DHI could inhibit the activity of CYP3A4 enzyme in vitro [26], but the effect on CYP3A4 enzyme activity in vivo is still unclear. In order to determine whether it is safe when rivaroxaban combined with DHI, we investigated the effects of DHI on the pharmacokinetics and pharmacodynamics of rivaroxaban in rats, including the effect on activity of CYP3A2.
In this study, the effects of DHI on the pharmacokinetics of rivaroxaban in rats were evaluated. In addition, the effect on pharmacodynamics of DHI on rivaroxaban was evaluated based on APTT, PT, TT, FIB, INR, vWF, t-PA, PAI-1, IL-1β, TNF-α and histopathology. Furthermore, the effect of DHI on CYP3A2 enzyme activity in rats was investigated by dapsone (probe substrate of CYP3A2).
The pharmacokinetics study showed that Cmax and AUC of rivaroxaban increased significantly in combination group. It suggested that rivaroxaban combined with DHI might increase the risk of bleeding and the dose of should be adjusted to avoid adverse reactions. Furthermore, DHI could inhibit the activity of CYP3A2 enzyme in rats, which might slow down elimination and increase the blood concentration of rivaroxaban. The result of effect on CYP3A2 enzyme from DHI in vivo is consistent with the conclusion of in vitro.
The pharmacodynamics study indicated that the anticoagulant and antithrombotic effects of combination group were more effective than rivaroxaban or DHI alone. APTT, PT, INR, TT and t-PA values were significantly increased, FIB, black tail length, vWF, PAI-1, IL-1β and TNF-α were significantly decreased in combination group. The tail thrombus was significantly improved. The enhancement of pharmacodynamics effects might be related to the increase of rivaroxaban plasma exposure and the synergistic effect of DHI.
Rivaroxaban, an direct FXa inhibitors, that inhibits not only free FXa but also the prothrombinase complex and clot bound FXa, thus effectively blocking thrombin generation [8]. FXa is situated at the juncture of intrinsic and extrinsic pathways in the coagulation cascade and catalyzes the conversion of prothrombin to thrombin, and plays prominent roles in various thromboembolic complications [60]. DHI has multiple pharmacological effects for its multi-component and multi-target therapeutic characteristics. DHI inhibited multiple G protein-coupled receptors agonists-induced platelet adhesion, aggregation and Ca2+ signaling pathways [61]. The salvianolic acids(SAs) is a core anti-thrombotic component of DHI. It was reported that Salvianolic acid A (SAA), the most active compound in SAs, exert an anti-thrombotic activity by inhibiting PI3K pathway of platelet [62]. Similar to SAA, salvianolic acid B (SAB) could inhibit platelets as a P2Y12 antagonist [63]. These results proved that DHI had effect of anti-platelet aggregation through multi-target, which might have synergistic effects with other anticoagulants. APTT, PT, INR, TT, and FIB are the main indicators of coagulation [64, 65]. PT and APTT mainly reflect the status of exogenous and endogenous coagulation systems [66]. FIB and TT mainly reflect the content of fibrinogen and the time of conversion to fibrin [67]. Fibrinogen is an important component of coagulation factors and can promote platelet aggregation [68]. These parameters are important clinical biomarkers that are frequently used in clinical to assess efficacy and safety of anticoagulants [69]. The results indicated that rivaroxaban could play an anticoagulant role by affecting the coagulation factors in endogenous and exogenous coagulation pathways. DHI plays an anticoagulant role by inhibiting platelet aggregation with multiple components and targets. The enhanced efficacy of combination group might be related to the synergistic effect of rivaroxaban and DHI.
Evidence has indicated that inflammation is closely related to thrombosis [70]. IL-1β and TNF-α were pro-inflammatory cytokines. They can damage endothelial function and promote blood clots forming by inducing inflammation [71]. Therefore, the increasing of IL-1β and TNF-α indicates the risk of thrombosis. NF-κB is an essential multi-channel nuclear transcription factor involved in the inflammatory process, cell proliferation and differentiation [72]. It has been suggested that FXa could activate nuclear factor κB (NF-κB) of endothelial cells, and result in the release of inflammatory factors [73, 74]. Therefore, as an Fxa inhibitor, rivaroxaban might significantly inhibit inflammation by inhibiting the NF-κB signaling pathway. A previous study demonstrated that the main active components of DHI were salvianolic acids (from Salvia miltiorrhiza), hydroxysafflower yellow A (from Carthamus tinctorius) [19]. SAA could inhibit NF-κB, phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) signaling pathway [75, 76], which could depress the expression of downstream pro-inflammatory factors. SAB could activate SIRT1-mediated autophagy [77]. Safflower yellow could regulate inflammation by directly acting on BV2 microglia [78]. Therefore, the efficacy of DHI is not a simple superposition of the efficacy of the two herbs. Compared with chemical drugs, DHI could exert a holistic therapeutic effect. The content of IL-1β and TNF-α decreased significantly in combination group compared with model group and rivaroxaban group (P < 0.01). This might be due to the dual inhibition of rivaroxaban and DHI on NF-κB signaling pathway, which significantly inhibits inflammation and plays an antithrombotic role. The pathological section results showed that the vascular wall necrosis and inflammatory cell infiltration in the tail tissue of Danhong injection group and rivaroxaban group were less severe to compare with model group. It indicated that both rivaroxaban and DHI could protect vascular endothelial cells, inhibit the release of inflammatory factors, reduce inflammatory response, and improve thrombus.
The formation of thromboembolic disease is also related to pathological factors such as vascular endothelial injury and fibrinolytic system disorder [79, 80]. Vascular inflammation might affect vascular endothelial function and also increase fibrinogen synthesis, resulting in elevated fibrinogen levels [81, 82]. vWF is a marker of vascular endothelial injury and a bridge for platelet adhesion to collagen fibers, which can promote platelet adhesion to vascular endothelial to form thrombosis [83]. The increase of the level of this factor in plasma may indicate the occurrence of thrombosis [84]. t-PA and PAI-1 maintain blood fluidity and vascular patency [85]. t-PA is a primary rational activator of plasminase, which plays a key role in the process of thrombolysis [86]. The destruction of dynamic balance between t-PA and PAI-1 can lead to the reduction of the local fibrin decomposition rate in the blood vessels and result in fibrin deposition and intravascular thrombosis [87]. In this study, vWF and PAI-1 in Danhong injection group, rivaroxaban group and combination group significantly decreased, while t-PA significantly increased compared with model group (P < 0.01). Among them, the changes in the combination group were more significant. It indicated that the combination of rivaroxaban and DHI might inhibit the formation of thrombus by repairing damaged vascular endothelial cells, enhancing the activity of the fibrinolytic system and improving inflammation.
The antithrombotic effects of the combination might be exerted by affecting coagulation pathways and platelet aggregation, inhibiting the release of inflammatory factors, repairing the damaged vascular endothelial cells and enhancing fibrinolytic system activity. We evaluated the changes of biomarkers in rats through kit tests to clarify the effect of DHI on the pharmacodynamics of rivaroxaban. To sum up, it follows that the detection of coagulation indexes, vascular endothelial cells, fibrin, inflammatory factors and other molecular markers has important clinical significance for the formation, development and treatment of thrombus.
In addition, rivaroxaban is also a substrate for P-gp [88]. However, the effect of DHI on P-gp is unknown, whether the change of rivaroxaban pharmacokinetics is related to the downregulation of P-gp expression needs further study. Because the study was conducted in rats, further research should be done to investigate the consistency of pharmacokinetics and pharmacodynamics between rats and humans.