Potentiation of flutamide-induced hepatotoxicity in mice by Xian-Ling-Gu-Bao through induction of CYP1A2

Highlights

• Xian-Ling-Gu-Bao (XLGB) is an inducer of CYP1A2 enzyme activity.

• XLGB increases the formation of toxic flutamide metabolite.

• The combination of XLGB and flutamide increases the hepatotoxicity in mice.

• This study suggests potential side effects of the drug-drug interaction between flutamide and XLGB in clinical practice.

Abstract

Ethnopharmacological relevance

Xian-Ling-Gu-Bao (XLGB) Fufang is herbal formula widely used to treat osteoporosis and other bone disorders. Because of its commonality in the clinical use, there is a safety concern over the use of XLGB combined with other androgen deprivation therapy (ADT) drugs such as flutamide (FLU) that is associated with reduced bone density. To date, there have been no evaluations on the side effects of the drug-drug interaction between XLGB and FLU.

Aim of the study

The present study was designed to investigate the hepatotoxicity in the context of the combined treatment of XLGB and FLU in a mouse model, and to determine whether the metabolic activation of FLU through induction of CYP1A2 plays a role in the increased hepatoxicity caused by the combination of XLGB and FLU.

Materials and methods

C57 mice were administered with either XLGB (6,160 mg/kg), FLU (300 mg/kg), or with the combination of the two drugs. Animals were treated with XLGB for 5 days before the combined administration of XLGB and FLU for another 4 days. The serum of mice from single or the combined administration groups was collected for biochemical analysis. The mouse liver was collected to examine liver morphological changes, evaluate liver coefficient, as well as determine the mRNA expression of P450 isozymes (Cyp1a2, Cyp3a11 and Cyp2c37). For metabolism analysis, mice were treated with XLGB, FLU, or the combination of XLGB and FLU for 24 h. The urine samples were collected for the analysis of FLU-NAC conjugate by UPLC-Q-Orbitrap MS. The liver microsomes were prepared from fresh livers to determine the activity of metabolizing enzyme CYP1A2.

Results

The combined treatment of XLGB and FLU caused loss of mice body weight and elicited significant liver toxicity as evidenced by an increased liver coefficient and serum lactate dehydrogenase (LDH) activity as well as pathological changes of fatty lesion of liver tissue. FLU increased hepatic expression of Cyp1a2 mRNA that was further elevated in the liver of mice when administered with both FLU and XLGB. Treatment of FLU resulted in an increase in the expression of Cyp3a11 mRNA that was negated when mice were co-treated with FLU and XLGB. No significant difference in Cyp2c37 mRNA expression was observed among the different treatment groups as compared to the control. Analysis of metabolic activity showed that the combined administration caused a synergic effect in elevating the activity of the CYP1A2 enzyme. Mass spectrometry analysis identified the presence of FLU reactive metabolite derived FLU-NAC conjugate in the urine of mice treated with FLU. Strikingly, about a two-fold increase of the FLU-NAC conjugate was detected when treated with both FLU and XLGB, indicating an elevated amount of toxic metabolite produced from FLU in the present of XLGB.

Conclusion

FLU and XLGB co-treatment potentiated FLU-induced hepatoxicity. This increased hepatoxicity was mediated through the induction of CYP1A2 activity which in turn enhanced bioactivation of FLU leading to over production of FLU-NAC conjugate and oxidative stress. These results offer warnings about serious side effects of the FLU-XLGB interaction in the clinical practice.

Introduction

Prostate cancer is a common malignant tumor in elderly men, and its occurrence and development are closely related to androgen. Anti-androgen deprivation therapy (ADT) has been the most widely used systemic treatment for prostate cancer in clinical practice. Although it is necessary, the long-term use of ADT introduces a variety of adverse events, including osteoporosis with decreased bone mineral density (Chen et al., 2019; Kim et al., 2019). A study of 390 prostate cancer patients followed up for 10 years showed that the incidence rate of osteoporosis in patients who received ADT was 80%, while the rate in patients who did not receive ADT was 35.4% (Morote et al., 2007). Flutamide (FLU) is a first-line drug for ADT, which has been used primarily for the treatment of prostate cancer (Bahnson, 2007). It is also prescribed for other androgen-dependent conditions such as in the treatment of women with polycystic ovary syndrome (PCOS) (Vincenzo et al., 1998). Though highly effective, the treatment of FLU interferes with bone metabolism and causes reduced bone mineral density. Animal studies have shown that FLU-mediated androgen blockade induces osteopenia in rats primarily due to reduced bone formation (Goulding and Gold, 1993). In human studies of patients with PCOS, a 6-month course of therapy with FLU revealed a significant reduction in bone mineral density along with elevated urinary calcium excretion (Moghetti et al., 1999). Considering the serious effects of ADT on bone mass density and the increased risk of fracture, it has been essential to precisely evaluate bone quantity and manage the adverse effects of ADT therapy on bone health. In this regard, ADT therapy has often been used in combination with osteoporosis treatment drugs in clinical practice.

Xian-Ling-Gu-Bao (XLGB) is a well-known patent herbal medicine formula, which is composed of six herbal medicines as follows: foliage of Epimedium brevicornu Maxim (Berberidaceae, Yin Yang Huo in Chinese, 70% g/g), stem of Dipsacus asper Wall. ex C.B. Clarke (Caprifoliaceae, Xu Duan in Chinese, 10% g/g), fructus of Cullen corylifolium (L.) Medik (Fabaceae, Bu Gu Zhi in Chinese, 5% g/g), root and rhizome of Salvia miltiorrhiza Bunge (Lamiaceae, Dan Shen in Chinese, 5% g/g), rhizome of Anemarrhena asphodeloides Bunge (Asparagaceae, Zhi Mu in Chinese, 5% g/g), and root and rhizome of Rehmannia glutinosa (Gaertn.) DC. (Orobanchaceae, Di Huang in Chinese, 5% g/g) (Wu et al., 2019). XLGB is herbal formulation approved by the China Food and Drug Administration (CFDA; China, Z20025337). It has been widely used in the clinic to treat osteoporosis, osteoarthritis, fractures and other diseases. The efficacy and quality control of XLGB have been widely described previously (Cheng et al., 2013; Guan et al., 2011; Yao et al., 2017; Zhu et al., 2012). Chinese herbal medicine has a long history of medical applications both in prescribed and self-prescribed medications for various acute and chronic conditions. Patients generally have a high acceptance of Chinese patent medicines in the belief that Chinese herbal medicines are associated with lower side effects. Combinations of Chinese patent medicines with other drugs are therefore quite common. However, herbal medicine presents a greater risk of adverse effects compared to other complementary therapies, especially in the context of potential drug interactions (Tachjian et al., 2010; Vickers et al., 2001). XLGB as a preventive drug for osteoporosis is often chosen to be used in combination with FLU in the treatment of prostate cancer patients, yet the safety of this combination has not been evaluated.

The combined use of drugs can increase the possibility of pharmacokinetic or pharmacodynamic interactions. Clinical studies have demonstrated that the combination of Chinese patent medicines and other drugs can enhance or reduce the efficacy and toxicity of drugs (Liu et al., 2011; Xu et al., 2019). Hepatotoxicity of FLU or XLGB has been reported in a number of clinical applications (Giorgetti et al., 2017; Manso et al., 2006; Tavakkoli et al., 2011; Yang and Peng, 2013; Yang and Zhou, 2007; Zheng et al., 2014). Will the combination of two hepatotoxic drugs further increase the risk? At present, the safety risks associated with the combination of FLU and XLGB in clinical practice remain unknown. Drug interactions are complex in clinical practice and often difficult to identify and predict. Given the commonality of the clinical usage of FLU and XLGB, it is necessary to evaluate the toxicity of the interaction of the combination of FLU and XLGB in an animal model.

The toxicity of FLU is mainly caused by its metabolites, and the key metabolic enzymes of FLU are cytochrome P450 1A2 (CYP1A2) and cytochrome P450 3A4 (CYP3A4) (Bahnson, 2007; Ohbuchi et al., 2009). In our previous studies, XLGB was found to induce the expression of Cyp1a2 mRNA in mouse liver (Ding et al., 2019). On the basis of these findings, it is likely that there may exist a high possibility of drug interaction between XLGB and FLU when used in combination. Therefore, in the present study, we evaluated the hepatotoxicity of XLGB combined with FLU in mice. Using mass spectrometry analysis, we further explored the underlying mechanism by dissecting the reactive metabolites of FLU in combination with XLGB.