| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 101 === Backgrounds and Aims: Tuberculosis (TB) is the second leading cause of death from an infectious disease worldwide, after the human immunodeficiency virus (HIV). Drug-induced adverse effects: isoniazid (INH), rifampin (RIF) and pyrazinamide (PZA) may induce hepatic injury, incidences various 1-36%. Cytochrome P450 2E1 (CYP2E1) and amidase are thought to contribute to the anti-tuberculosis drugs-induced hepatotoxicity (ATDH). In addition, the severity of PZA-induced hepatotoxicity is no less than that associated with INH or RIF. However, relatively little is known about the hepatotoxicity of PZA. The objectives of this study were to: (1) investigate the mechanism of ATDH and improve the ATDH through metabolic enzymes regulation; (2) to evaluate whether the genetic polymorphisms of xanthine oxidase (XO) influence susceptibility to ATDH.
Methods: We screened 111 known compounds from known pharmaceutical excipients and natural flavonoids compounds as inhibitors of CYP2E1. The hepatotoxic fixed-dose of INH/RIF/PZA were 50/100/250 mg/kg/day, respectively. Hepatotoxicity was assessed by the galactose single point (GSP) method (a US Food and Drug Administration (FDA) recommended quantitative liver function test), liver histopathology, malondialdehyde (MDA) assay, and measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity. We chose the CYP2E1-specific substrate chlorzoxazone to assess CYP2E1 activity in mice and human. In addition, PZA metabolites were identified and cytotoxicity in HepG2 cells was assessed. Potential PZA and PA hepatotoxicity was then tested in rats. Urine specimens were collected from 191 TB patients and the results were evaluated to confirm whether a correlation existed between PZA metabolite concentrations and hepatotoxicity. This led to the hypothesis that co-administration of amidase inhibitor decreases or prevents PZA (and its metabolites)-induced hepatotoxicity in rats and human. Finally, we included 411 TB patients to evaluate whether the genetic polymorphisms of XO influence susceptibility to ATDH.
Results: Selected CYP2E1 inhibitor, HUEXC030 (mannitol) inhibited CYP2E1 activity by 60% to 88% in HLMs and RLMs in a dose-dependent manner. HUEXC030 diminished or even eradicated INH/RIF-induced hepatotoxicity in mice. Serum AST, ALT and GSP levels were significantly increased 3.8- to 7.8-fold in these mice (p < 0.005), and these levels could be lowered by selected CYP2E1 inhibitors. Moreover, HUEXC030 treatment prevented INH/RIF-mediated lipid peroxidation, as indicated by the reduction in hepatic GSH depletion and MDA formation (p < 0.005). Pharmacokinetic studies with INH/RIF showed that co-treatment with HUEXC030 inhibited CYP2E1 activity but did not affect the absorption of INH and RIF in mice. We also demonstrated the pharmacodynamically effective CYP2E1 inhibitor HUEXC030 had the same inhibitory effect on CYP2E1 in healthy volunteers as well as in mice, with inhibition of up to 58% (p < 0.005). These results implied that above selected CYP2E1 inhibitors have the high potential for use in developing a novel INH/RIF pharmaceutical formulation that does not cause hepatotoxicity and consequently improving the compliance of TB patients taking anti-TB drugs. To determine whether PZA metabolites are responsible for PZA-induced hepatotoxicity, PA and 5-OH-PA are more toxic than PZA. PZA and PA treatment of rats significantly increases AST and ALT activity and GSP levels (p < 0.005). In analysis of PZA metabolites in 191 TB patients’ urine with hepatotoxicity, provides addition evidence support for 5-OH-PA significantly increased the phenomenon (p < 0.005). PA and 5-OH-PA, the metabolites of PZA through amidase catalysis, were more toxic effect than PZA in HepG2 cell. In pharmacokinetic study of PZA without or with HUCHE033 (quercetin) combinations in healthy volunteers, HUCHE033 could decreased amidase activity in human, pharmacokinetic parameters of 5-OH-PA with AUC and metabolic ratio (AUC5-OH-PA/ AUCPZA and AUCPA/ AUCPZA) were significantly inhibited 25% and 39%, respectively (p < 0.01). HUCHE033 did decrease plasma level of PZA toxic metabolites, such as 5-OH-PA and PA, but did not affect PZA in human. Furthermore, an anti-TB efficacy assay revealed that above selected CYP2E1 or amidase inhibitor combinations did not affect the anti-TB effects of anti-TB drugs. In addition, we found that XO rs1884725 or rs2295475 mutant type could be a risk factor of ATDH in 411 TB patients (mutant vs. wildtype: Odds ratio [OR], 1.71, p = 0.043; mutant vs. wildtype: [OR], 4.32, p = 0.006, respectively).
Conclusions: (1) To our knowledge, this is the first report of kaempferol’s utility as an adjuvant for preventing CYP2E1-mediated hepatotoxicity induced by drugs such as INH and RIF; (2) Mannitol was an FDA-approved excipient, was identified as a CYP2E1 inhibitor that could be a useful component to eradicate the anti-TB drugs-induced hepatotoxicity; (3) This is the first report of a cell-line, animal, and clinical trial confirming that the metabolite 5-OH-PA is responsible for PZA-induced hepatotoxicity; (4) Xanthine oxidase SNP rs2295475 polymorphism could be a risk factor associated with susceptibility to ATDH.
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