Studies On The Metabolism Of Novel Anticancer Candidate BFBTS

BFBTS is a novel antitumor candidate obtained by systematic screen of derivatives from dibenzyl trisulfide which is the pharmacologically important constituents from Petiveria alliacea. The antitumor tests revealed that BFBTS exhibited the highest activity against several human cancer cell lines and even multidrug resistant cancer cells showed vunerable to BFBTS. Thus BFBTS is a very promising drug candidate for cancer therapy. There were few reports about the metabolism or excretion study of trisulfide and so does those about the mechanism of trisulfide metabolism and their metabolic interactions. In this study, in vitro and in vivo studies were applied to investigate the excretion and metabolism of BFBTS. We also investigated the metabolic interactions between BFBTS and biosystems. The present study will provide support for the clinical development of BFBTS and intrigue the development of trisulfide.1. Mechanism of BFBTS metabolism in bloodOur preliminary investigation and PK study had indicated that BFBTS was extremely unstable in blood while it was not metabolized in hepatic micosomes. Therefore RBCs were used as in vitro model for BFBTS metabolism. We investigated main metabolic site of BFBTS. HPLC-UV, GC-MS, LC-MS was used to identify the metabolites in blood. BFBDS, p-FBM, and p-FBA were the three metabolites identified in rat blood or whole blood. The reduction of BFBTS was fast in RBCs, and the half life was less than lmin. The formation of p-FBM and p-FBA was compared which showed no significant diference. Thus reduction of BFBTS and formation of metabolites were mainly occurred in RBCs.To investigate the factors in BFBTS metabolism, we compared the ability of endogrnous thiols like GSH, Cys, N-acetyl-Cys and CoA to reduce BFBTS. It was found that Cys and GSH showed the highest ability of reducing BFBTS. Iodoacetamide was used to evaluate the involvement of thiols in the BFBTS metabolism, which confirmed the critical importance of thiols in BFBTS metabolism. The involvement of proteins in BFBTS metabolism was investigated by superfiltration with different MW cutoff. It was found that proteins enhance the BFBTS metabolism in RBCs. After comparing the BFBTS metabolism in rat and human RBCs, we found that rat RBCs had the highest activity toward BFBTS and the lower activity of human RBCs in BFBTS metabolism may indicated a prolonged half-life of BFBTS in clinical.2. Investigation of BFBTS derived hemoglobin adductRBCs were used as in vitro models, and HPLC-UV, MALDI-TOF, ESI-TOF, LC-TRQ were applied for BFBTS derived hemoglobin adduct. The results showed that Hb adduct was detected only when concentration of BFBTS was as high as 250μmol/L in vitro. Trypsinized peptides were ananlysed, and Cys125βand Cys1O4/111αwere identified as site of modification. A moiety ofρ-FBM was adducted with a mass shift of 140 Da. When concentration of BFBTS reached 500μmol/L, all the Cys residues were modified except Cys13α. There was no Hb adduct detected in rat after a single i.v. high dosing while continuous administration of BFBTS will induce Hb adduct. Formation of human hemoglobin adduct was investigated in vitro, and no Hb addut was found. The result indicated that the risk of forming human Hb adduct in vivo was low, and Hb contributed limitedly and indirectly to BFBTS metabolism.3. Excretion study of BFBTS in ratAn HPLC-UV method was established for the analysis of BFBTS and BFBDS in rat bile, urine and feces, but there were no BFBTS and BFBDS detected in rat urine and feces after dosing and only trace BFBTS and BFBDS were detected in rat bile. We applied HPLC-UV, GC-MS, LC-MS to investigate the metavolites in rat urine and two metabolites were detected and identified as p-FHA and p-FBA. We developed a simple HPLC method for the analysis of p-FHA and p-FBA in rat urine. Investigation of BFBTS excretion in rat was conducted and excretion rate of p-FHA and p-FBA was calculated. As final metabolite, the cumulative excretion of p-FHA and p-FBA reached 67%in urine and about 4%in bile.4. Investigation of BFBTS's influence on efflux transportersWe compared the antitumor potency between BFBTS, BFBDS andρ-FBM aginst HepG2, H46, K562, K562/adr, MCF-7, MCF-7/adr by SRB method. The highest potency of BFBTS was also observed and multidrug resistant cancer cells were equally vunerable to BFBTS and it metabolite. For the investigation of BFBTS, BFBDS and p-FBM influence on P-gp and BCRP, we use MDCK-MDR1 and LLC-PK1-BCRP as in vitro models. Rhodaminel23 and mitoxantrone were used as classic substrate of P-gp and BCRP. The uptake of R123 in MDCK-MDR1 cell showed little induction of BFBTS and its metabolites on P-gp and transport experiment showed no inhibition on the efflux of R123. BFBTS, BFBDS and p-FBM also showed little influence on BCRP in the uptake experiment of mitoxantrone on LLC-PK1-BCRP. Non-reudced SDS-PAG coupled with western blot showed influence on BCRP monomers and dimers by BFBTS and BFBDS while function of BCRP was not affected. Collectively, there were no influence between BFBTS, BFBDS, p-FBM and efflux transport like P-gp and BCRP.