Although chemotherapy has been an integral part of cancer treatment for decades, the numerous side effects are a major concern limiting its usage and causing patients’ unwanted clinical consequences in the long-term. As natural products, traditional Chinese medicines have been shown to be an excellent and reliable resource for cancer chemoprevention [1
]. According to recent investigations, the traditional Chinese medicine Schisandra chinensis
) has been demonstrated to possess remarkable protective effects against chemical-induced toxicity [2
] in addition to its widely known beneficial effects on the liver, kidney and nervous system in both experiment animals as well as in vitro human cell incubations [4
]. Therefore, in our previous study the combined therapeutic regime of S. chinensis
extract (SCE) with CTX exhibited desirable ameliorating effects on CTX toxicity, with decreased levels of some biochemical indexes such as serum marker enzymes. Additionally, a significant change in CTX pharmacokinetic parameters was observed along with the toxicity attenuation [8
]. It was then hypothesised and preliminarily proven that the attenuation of toxicity could be at least partially attributed to CYP3A inhibition by SCE as well as a direct protective effect of SCE on tissues, as SCE has been reported to inhibit CYP3A activity in vivo [9
CTX is an alkylating anticancer drug widely employed in chemotherapy and immunosuppressive therapy [10
]. It is mainly activated by CYP2B6 and then metabolized into the effective component phosphoramide mustard [11
] (Scheme 1
Apart from that, a portion of CTX is metabolized by CYP3A into equimolar amounts of an inactive metabolite, 2-dechloroethylcyclophosphamide (DCCTX) and chloroacetaldehyde (CAA) as a by-product [11
]. CAA was reckoned as the toxic product that might result in hepatotoxicity, neurotoxicity and nephrotoxicity [12
]. In our previous study, a large decrease in the blood concentration of DCCTX and CAA was observed in CTX-treated rats with SCE co-administration [8
]. Gomisin A (Gom A, Figure 1
) is one of the most abundant bioactive lignans in S. chinensis
As reported by Iwata and Wan et al., Gom A showed significant CYP3A inhibitory effect in vitro when co-incubated with human/rat liver microsomes (RLMs) and HepG2 cells [15
]. However, the mechanism of CYP3A inhibition by Gom A, or the potential role of Gom A in the DDIs between SCE and CTX along with its detoxification effect of CTX through CYP3A inhibition are poorly understood.
So far, there is no report about the effect of Gom A on CTX metabolism and toxicity. Therefore this study aimed primarily to find out whether and how Gom A participates in the chemopreventive activity of S. chinensis against CTX toxicity, which was tested in in vitro incubation systems by using human liver microsomes (HLMs). Thereafter, the effects of Gom A on the toxic CYP3A-mediated CTX metabolism in rats was discussed based on the pharmacokinetic behaviors of DCCTX in rats with and without Gom A pretreatment.
Previous research about the combination treatment of CTX and traditional Chinese medicines (TCMs) often focused on the changes of pharmacological and/or biochemical indexes, ignoring the potential occurrence of DDIs. This study confirmed that Gom A played an important role in the DDIs between S. chinensis and CTX, and thus might contribute to the observed chemoprotective effects of S. chinensis against CTX toxicity. The above results could lead to the conclusion that Gom A pretreatment might efficiently reduce the production of the toxic CTX metabolite CAA by inactivating CYP3A to some extent. To be exact, Gom A exhibited a multifaceted effect on CYP3A-mediated CTX metabolism with different time intervals between Gom A pretreatment and CTX administration.
Wan et al. and Iwata et al. pointed out that Gom A could cause moderate to strong CYP3A inhibition when co-incubated with HLMs or human HepG2 hepatoma cells [15
]. Results from our study confirmed and extended previous findings, and characterized for the first time the time- and NADPH-dependent CYP3A inhibition of Gom A. In detail, Figure 2
A (Lineweaver-Burk plot) suggests that Gom A was a competitive inhibitor in reversible inhibition assay, as little change of Vm
was observed. In CYP3A TDI assessment, the IC50
value of Gom A decreased by 1.8-fold with a preincubation step, meeting the 1.5-fold shift criteria [17
]. Since TDI had been closely related to mechanism-based inactivation (MBI) [18
], in vitro data suggested that Gom A was likely to cause a long-term inhibition of CYP3A activity in rats, as the inactivated P450 enzyme had to be replaced to restore activity [19
In HLMs, Gom A significantly inhibited CYP3A-mediated CTX metabolism. A better understanding was obtained that Gom A exhibited multifaceted effects on DCCTX production in vivo from subsequent pharmacokinetic data, which could be explained by the short- and long-term effect of Gom A administration on CYP3A-mediated metabolism of CTX. Therefore, Gom A and CTX were co-administered to rats with different time intervals. When Gom A was pretreated 0.5 h and 6 h before CTX injection, the production of DCCTX was significantly decreased. However, an increase in DCCTX blood concentration was observed when Gom A was pretreated 24 h and 72 h before CTX administration (Figure 7
A, Table 1
When CTX was administered within 6 h after Gom A pretreatment, the blood exposure of DCCTX was strongly decreased (Figure 7
A, Table 1
). As shown in Figure 7
C, the blood concentration of Gom A reached a peak at or before the first blood sample collection and then decreased rapidly in experimental rats. Therefore, CTX was administered to rats with a much lower exposure of Gom A in group 3 (6 h time interval) compared with group 2 (0.5 h time interval). However, the pharmacokinetic parameters of DCCTX in groups 2 and 3 were similar. Moreover, according to Figure 5
, the hepatic CYP3A in rat did not regain activity within 12 h after Gom A administration. The above observations indicated CYP3A inactivation, which agreed well with the in vitro study. Nevertheless, Qin et al. reported that Gom A co-administration had little effect on the in vivo metabolism of intravenously administered tacrolimus, which was also metabolized by CYP3A [20
]. One possible explanation for the different outcomes could be a difference in plasma protein binding rates of the two chemicals. Tacrolimus is strongly bound to plasma protein, whereas the plasma protein binding rate of CTX is only approximately 9% [21
]. With relatively low hepatic extraction ratios, only unbounded tacrolimus and CTX could be eliminated by hepatic CYPs [23
]. With a much lower protein binding rate, it is possible that the hepatic CYP3A metabolism of CTX is more prone to be affected by CYP3A inhibitors compared with that of tacrolimus.
When CTX was administered 24 h and 72 h after Gom A pretreatment, a substantial increase in DCCTX production was observed. The major contributor to the increase could be CYP3A induction, as Gom A had been found as a potent inducer of CYP3A [26
]. Figure 6
shows that the hepatic CYP3A mRNA expression in rats was substantially increased after single-dose Gom A pretreatment. The obtained data suggests that the CYP3A inductive effect by Gom A in rats lasted for more than 3 days, as the mRNA expression was still increased at 72 h after Gom A pretreatment compared with the control group. In general, the effect of Gom A on DCCTX production was a composite of CYP3A inhibition and induction. It is possible that though CYP3A mRNA expression kept accumulating within 24 h after Gom A administration, the CYP3A induction effect was covered up by CYP3A inactivation. 24 h after Gom A pretreatment, though a portion of CYP3A was still inactivated, the CYP3A inactivation was not of sufficient magnitude to have an effect in vivo with the substantially augmented hepatic CYP3A content.
In our previous study, the time interval between S. chinensis
and CTX administration was 0.5 h, which meant than Gom A exhibited a strong inhibitory effect on rat hepatic CYP3A activity when CTX was injected. CAA is the CTX metabolite produced by CYP3A, which could result in broad toxic effects on the liver, kidney and nervous system [12
]. It was demonstrated that Gom A acted as an important component in S. chinensis
during the detoxification of CTX by inhibiting CYP3A activity and consequently reducing CAA blood concentrations. Furthermore, it was possible that this detoxification effect was not limited to the pharmacokinetic interference by Gom A. CAA could cause GSH depletion and lipid peroxidation, ultimately resulting in cytotoxicity and cell death [30
]. Gom A treatment has been reported to greatly increase the total liver and mitochondrial GSH levels in mice [32
]. Thus, Gom A may enhance the resistance against CTX toxicity by increasing GSH levels in experiment rats. As the major bioactive component of S. chinensis
, Gom A has also been indicated to attenuate chemical-induced damages in liver and kidney by modulating NRF2/ARE and MAPK signal pathways [4
]. In summary, it could be concluded that Gom A was an essential component in S. chinensis
for the chemoprevention against CTX toxicity.
The current study was aimed primarily to evaluate the in vivo effects of Gom A pretreatment on CYP3A-mediated CTX metabolism and investigate the pharmacokinetic behaviors of Gom A in rats. Gom A contains the methylenedioxyphenyl group in common with another Schisandrae
lignan gomisin C, which had been found to potently inactivate CYP3A [15
]. Methylenedioxyphenyl-containing compounds could be transformed by CYPs into a reactive carbene metabolite, and the carbene metabolite is apt to react with CYPs to form a catalytically inactive metabolic-inhibitor (MI) complex, which had been demonstrated to play an important role in CYPs inactivation [19
Several limitations in this study need to be addressed. Our in vitro study only provided indirect evidence for the Gom A-induced MBI on CYP3A. Future investigations need to be performed to investigate the specific mechanisms of the CYP3A inactivation induced by Gom A. In addition, only HLMs was used in CYP3A inhibition investigations. In further mechanism studies, both rat and human liver microsomes should be involved to provide a more comprehensive mechanism description. Also, to provide visual evidence of the protective effects of Gom A against CTX toxicity, changes in related biochemical indexes of CTX-treated rats with and without Gom A pretreatment should be further investigated.