The association of cytochrome P450 genetic polymorphisms with sulfolane formation and the efficacy of a busulfan-based conditioning regimen in pediatric patients undergoing hematopoietic stem cell transplantation

Abstract

Cytochrome P450 enzymes (CYPs) and flavin-containing monooxygenases (FMOs) likely have a role in the oxidation of intermediate metabolites of busulfan (Bu). In vitro studies to investigate the involvement of these enzymes are cumbersome because of the volatile nature of the intermediate metabolite tetrahydrothiophene (THT) and the lack of sensitive quantitation methods. This study explored the association between the CYP2C9, CYP2C19, CYP2B6 and FMO3 genotypes and sulfolane (Su, a water soluble metabolite of Bu) plasma levels in children undergoing hematopoietic stem cell transplantation (HSCT). The relationship between these genotypes and the effectiveness of myeloablative conditioning was also analyzed. Sixty-six children receiving an intravenous Bu-based myeloablative conditioning regimen were genotyped for common functional variant alleles in CYP2C9 (*2 and *3), CYP2C19 (*2 and *17), FMO3 (rs2266780, rs2266782 and rs1736557) and CYP2B6 (*5 and *9). The plasma levels of Bu and its metabolite Su were measured after the ninth Bu dose in a subset of 44 patients for whom plasma samples were available. The ratio of Bu to Su was considered the metabolic ratio (MR) and was compared across the genotype groups. Higher MRs were observed in CYP2C9*2 and *3 allele carriers (mean±s.d.: 7.8±3.6 in carriers vs 4.4±2.2 in non-carriers; P=0.003). An increased incidence of graft failure was observed among patients with an MR>5 compared with those with MR values <5 (20% vs 0%; P=0.02). In contrast, a significantly higher incidence of relapse and graft failure (evaluated as event-free survival) was observed in patients with malignant disease who carried CYP2B6 alleles with reduced function on both chromosomes compared with carriers of at least one normal allele (100% vs 40%; P=0.0001). These results suggest that CYP2C9 has a role in the oxidation reactions of THT and indicate that it may be possible to predict the efficacy of Bu-based myeloablative conditioning before HSCT on the basis of CYP genotypes and Bu MRs.

The Association of Combined GSTM1 and CYP2C9 Genotype Status with the Occurrence of Hemorrhagic Cystitis in Pediatric Patients Receiving Myeloablative Conditioning Regimen Prior to Allogeneic Hematopoietic Stem Cell Transplantation

“…The cumulative BU doses did not differ between the patients with and without HC ( Table 2 ) indicating that the variability could be at the level of Su formation from the parent drug by the CYP2C9 enzyme. We previously demonstrated the role of CYP2C9 in Su formation from BU (Uppugunduri et al, 2014). In this analysis we did observe a trend of higher Su levels in children carrying both normal CYP2C9 and GSTM1 genotypes, but was not statistically significant (Supplementary Figure S3).…”

“…Su did not display any influence on CYP2C9, 19, 3A4, and 2B6 activities at the level of phenotyping or at the level of mRNA expression in vitro , supporting the hypothesis that depletion of GSH and BU action on cysteine rich proteins might be the cause for increased toxicity of CY metabolites when CY is given after BU. The formation of Su indicates the simultaneous formation of GSH analog γ-glutamyl-dehydroalanyl -glycine (EdAG) (Uppugunduri et al, 2014), another metabolite which inhibits GSH irreversibly, and also participates in increased formation of protein adducts of glutaredoxins and thus increasing oxidative stress and cellular apoptosis together with BU (Scian and Atkins, 2015). Thus Su levels might reflect these sequential events predisposing to HC.…”

“…With the availability of an analytical method for quantifying Su levels in plasma (Versace et al, 2012), it is now possible to investigate its relation to HC incidence in a clinical setting. Several enzymes contribute to the metabolism of Su and CY, for example, CYP2C9 is involved in both the processes of activation of CY and formation of Su from BU (Uppugunduri et al, 2014). Flavin monooxygenase (FMO3) is involved in Su formation, while CYP2B6, CYP2C19, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH3A1) are involved in either activation or inactivation of CY (Timm et al, 2005; Xie et al, 2006).…”

Genotypes Affecting the Pharmacokinetics of Anticancer Drugs

“…However, other genetic and clinical factors may also influence the metabolism of busulfan [19]. The CYP2C19 enzyme plays a role in the metabolism of cyclophosphamide, ifosfamide, tamoxifen, and thalidomide.…”

The busulfan metabolite EdAG irreversibly glutathionylates glutaredoxins

“…Metabolites of busulfan have been considered as potential sources of toxicity, and the enzymes responsible for the metabolism have been considered as genetic factors in toxicity, but no definitive mechanisms have been demonstrated (14–16). The metabolite pool of busulfan is derived from its initial Glutathione Transferase (GST)-catalyzed reaction with GSH and includes tetrahydrothiophene (THT + ) and oxidized THT + variants (17,18).…”