Novel Glutathione-Dependent Thiopurine Prodrugs: Evidence for Enhanced Cytotoxicity in Tumor Cells and for Decreased Bone Marrow Toxicity in Mice

Abstract

Elevated glutathione (GSH) levels have been detected in many tumors compared with the healthy, surrounding tissue. Often, this GSH up-regulation is associated with drug resistance. The prodrugs 6-(2-acetylvinylthio)guanine (AVTG) and 6-(2-acetylvinylthio)purine (AVTP) contain a novel butenone moiety that allows the prodrugs to react selectively with sulfhydryl nucleophiles to release the chemotherapeutic drug 6-thioguanine (6-TG) or 6-mercaptopurine (6-MP), respectively. The cellular uptake and metabolism of trans-AVTG in two human renal carcinoma cell lines that were used as models were rapid and associated with depletion of intracellular GSH. Formation of 6-TG from trans-AVTG correlated positively with intracellular GSH concentrations, and was significantly reduced by diethyl maleate pretreatment. Intracellular concentrations of 6-TG after incubations with trans-AVTG were significantly higher than the 6-TG concentrations obtained after incubations with equimolar concentrations of 6-TG; thus, the prodrug delivered more 6-TG to the cell than did 6-TG itself. Cytotoxicity studies demonstrated that AVTG and AVTP had similar IC 50 values that were comparable with those of 6-TG, but were significantly lower than those of 6-MP. Furthermore, after in vivo treatment of mice with the prodrugs, no reduction was observed in circulating white blood cell counts, whereas white blood cell counts of mice treated with equimolar or 60% lower doses of 6-TG were reduced by 50 to 60%. Collectively, the results show that AVTG and AVTP are novel potential chemotherapeutic agents that may target tumors with up-regulated levels of GSH, and may exhibit less systemic toxicity than the parent thiopurines.The narrow therapeutic index associated with most chemotherapeutic agents often leads to toxicity in nontarget tissues (Hoekman et al., 1999). An approach commonly used to decrease systemic toxicity of chemotherapeutics is prodrug design. Prodrugs are pharmacologically inactive compounds that are metabolized in vivo to yield active drugs. Because quantitative and/or qualitative biochemical differences exist between tumors and the healthy surrounding tissue, it may be possible to selectively deliver drugs to the tumor by designing prodrugs that are metabolized more rapidly or in greater quantities in the tumor than in the surrounding tissue (Dubowchik and Walker, 1999). Thus, prodrugs may decrease adverse drug reactions and systemic toxicity, and increase tissue selectivity and therapeutic efficacy of the parent drug.Glutathione (GSH) plays a major role in the detoxification of numerous chemotherapeutics (Schröder et al., 1996). Changes in GSH content and levels of glutathione S-transferases (GSTs), GSH peroxidase, and GSH reductase (GR) have been detected in tumors (Di Ilio et al., 1991Gajewska et al., 1995;Institoris et al., 1995;Blair et al., 1997). Moreover, increased levels of GSH have been linked with drug resistance (Mickisch et al., 1990;Yang et al., 1992), and up-regulation of the isoform of GST is often associated with dr...

CYTOTOXICITY OF THE NOVEL GLUTATHIONE-ACTIVATED THIOPURINE PRODRUGSCIS-AVTP [CIS-6-(2-ACETYLVINYLTHIO)PURINE] ANDTRANS-AVTG [TRANS-6-(2-ACETYLVINYLTHIO)GUANINE] RESULTS FROM THE NATIONAL CANCER INSTITUTE'S ANTICANCER DRUG SCREEN

“…These results support our previous findings in which cis-AVTP and trans-AVTG exhibited similar cytotoxicities that were greater than or comparable to those of 6-MP and 6-TG, respectively (Gunnarsdottir et al, 2002a).…”

“…Additionally, treatment with cis-AVTP, trans-AVTG, and 6-TG yielded distinct patterns of metabolite composition and quanti-ties in mouse tissues, suggesting differences in tissue distribution and metabolism among the compounds (Gunnarsdottir and Elfarra, 2003). Also, when the in vitro cytotoxicities of cis-AVTP and trans-AVTG were assessed in the renal cell carcinoma cell lines ACHN and A-498, the prodrugs exhibited similar cytotoxicities that were greater than the cytotoxicity of 6-MP but comparable with that of 6-TG after a 72-h drug incubation (Gunnarsdottir et al, 2002a). Likewise, studies comparing the cytotoxicities of cis-AVTP and 6-MP after different incubation times revealed that even after as little as 24 h of drug incubation, extensive cytotoxicity was detected in incubation with cis-AVTP, whereas no cytotoxicity was observed in 6-MP incubations (Gunnarsdottir et al, 2002a).…”

“…The prodrugs cis-AVTP and trans-AVTG were synthesized as previously described (Gunnarsdottir et al, 2002a).…”

“…Also, when the in vitro cytotoxicities of cis-AVTP and trans-AVTG were assessed in the renal cell carcinoma cell lines ACHN and A-498, the prodrugs exhibited similar cytotoxicities that were greater than the cytotoxicity of 6-MP but comparable with that of 6-TG after a 72-h drug incubation (Gunnarsdottir et al, 2002a). Likewise, studies comparing the cytotoxicities of cis-AVTP and 6-MP after different incubation times revealed that even after as little as 24 h of drug incubation, extensive cytotoxicity was detected in incubation with cis-AVTP, whereas no cytotoxicity was observed in 6-MP incubations (Gunnarsdottir et al, 2002a). These findings suggested that the in vitro cytotoxic potency among the thiopurines and their prodrugs was different.…”

“…1) and trans-6-(2-acetylvinylthio)guanine (trans-AVTG), which are novel prodrugs of 6-MP and 6-TG, respectively. cis-AVTP and trans-AVTG are ␣,␤-unsaturated conjugates of the thiopurines that were shown to react rapidly with cellular thiols such as glutathione (GSH) to yield the respective thiopurines as the major metabolites (Gunnarsdottir et al, 2002a). Because overproduction of GSH has been observed in many tumor cells, including colon, breast, lung, ovarian, and renal tumor cells (Mekhail-Ishak et al, 1989;Perry et al, 1993;Oberli-Schrämmli et al, 1994;Joncourt et al, 1998;Lusini et al, 2001), the prodrugs cis-AVTP and trans-AVTG may more selectively deliver the thiopurine moiety to a tumor cell than is achieved using the thiopurine itself.…”