Flavin-containing monooxygenase 3 (FMO3) role in busulphan metabolic pathway

Abstract

Busulphan (Bu) is an alkylating agent used in the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Bu is extensively metabolized in the liver via conjugations with glutathione to form the intermediate metabolite (sulfonium ion) which subsequently is degraded to tetrahydrothiophene (THT). THT was reported to be oxidized forming THT-1-oxide that is further oxidized to sulfolane and finally 3-hydroxysulfolane. However, the underlying mechanisms for the formation of these metabolites remain poorly understood. In the present study, we performed in vitro and in vivo investigations to elucidate the involvement of flavin-containing monooxygenase-3 (FMO3) and cytochrome P450 enzymes (CYPs) in Bu metabolic pathway. Rapid clearance of THT was observed when incubated with human liver microsomes. Furthermore, among different recombinant microsomal enzymes, the highest intrinsic clearance for THT was obtained via FMO3 followed by several CYPs including 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. In Bu- or THT-treated mice, inhibition of FMO3 by phenylthiourea significantly suppressed the clearance of both Bu and THT. Moreover, the simultaneous administration of a high dose of THT (200μmol/kg) to Bu-treated mice reduced the clearance of Bu. Consistently, in patients undergoing HSCT, repeated administration of Bu resulted in a significant up-regulation of FMO3 and glutathione-S-transfrase -1 (GSTA1) genes. Finally, in a Bu-treated patient, additional treatment with voriconazole (an antimycotic drug known as an FMO3-substrate) significantly altered the Bu clearance. In conclusion, we demonstrate for the first time that FMO3 along with CYPs contribute a major part in busulphan metabolic pathway and certainly can affect its kinetics. The present results have high clinical impact. Furthermore, these findings might be important for reducing the treatment-related toxicity of Bu, through avoiding interaction with other concomitant used drugs during conditioning and hence improving the clinical outcomes of HSCT.

Fig 1. Metabolic pathway of busulphan.

Fig 2. Concentration-time curves for tetrahydrothiophene and its three metabolites after incubation with recombinant FMO3.

Microsomes with cDNA-expressed human FMO3 were incubated with 25μM tetrahydrothiophene (THT). A time curve (0, 5, 15, 30 and 60min) was performed with triplicate incubations and a protein concentration of 0.35mg/mL. 96% of THT was metabolized by recombinant FMO3 in 15 min. The disappearance was accompanied by the formation of metabolites, mainly THT 1-oxide. The other two metabolites (sulfolane and 3-OH-sulfolane) started to appear at later time points, in line with sequential metabolism. (□) THT, solid line; (Δ) THT 1-oxide, dash/dotted; (○) sulfolane, dotted; (◊) 3-OH-sulfolane, solid line.

Fig 3. The effect of FMO3 inhibition on concentration-time curve of busulphan (Bu) and tetrahydrothiophene (THT) in mice.

To study the effect of FMO3 inhibition on Bu kinetics, animals were divided into three groups. All experiments were run with 3 mice per time point in each group. Animals were euthanized and blood samples collected at different time points. Group1. A single dose of Bu (25mg/kg) was administered i.p. to mice Group2. A single dose of Bu (25mg/kg) was administered i.p. 24h after pretreatment with the FMO3-inhibitor phenylthiourea (PTU, 3mg/kg; i.p.) for 3 days. Group3. Bu (25mg/kg) was administered concomitantly with a high dose of THT (17.6mg/kg). A. Pretreatment with PTU significantly (P<0.05) increased plasma levels of Bu (Δ, dashed line) compared to that observed when Bu was administered alone (□, dotted line). THT, when given together with Bu, also affected Bu metabolism and increased in Bu plasma concentrations (○; solid line) compared to Bu alone. B. Pretreatment with PTU significantly (P<0.05) increased plasma concentrations of Bu first stable metabolite (THT) in mice injected with Bu (Δ, dashed line) compared to that observed when Bu was administered alone (□, dotted line). THT concentrations in mice injected with Bu and PTU (Δ, dashed line) were almost the same as in mice injected with Bu and concomitant high doses of THT without pretreatment with PTU (○; solid line).

Fig 4. The effect of FMO3 inhibition on concentration-time curve of tetrahydrothiophene (THT) in mice.

To study the effect of FMO3 inhibition on THT kinetics, animals were divided into two groups. All experiments were run with 3 mice per time point in each group. Animals were euthanized and blood samples collected at different time points. Group 1. A single dose of THT (8.8mg/kg) was administered i.p. to mice. Group 2. THT (8.8mg/kg) was administered 24h after pretreatment with the FMO3-inhibitor phenylthiourea (PTU, 3mg/kg; i.p.) for 3 days. Pretreatment with PTU significantly (P<0.05) increased plasma levels of THT (Δ, dashed line) compared to that observed when THT was administered alone (□, solid line).

Fig 5

The effect of busulphan treatment on mRNA of FMO3 (A) and GSTA1 (B) in patients during conditioning prior to HSCT. The gene array analysis showed that FMO3 gene expression was significantly (P<0.05) increased after busulphan (Bu) conditioning (2mg/kg b.i.d. for 4 days) compared to before the start of Bu conditioning (confirmed by qRT-PCR). The expression was normalized to that of GAPDH. The inter-individual variation in the relative expression increased from 2.8-fold before Bu conditioning, to 5.6-fold by the end of the treatment. GSTA1 was also significantly up-regulated (P<0.05). Inter-individual variation in the relative expression of GSTA1 increased from 1.6-fold before Bu conditioning to 2.3-fold by the end of the treatment.

Fig 6. The effect of voriconazole treatment on busulphan and tetrahydrothiophene kinetics in patients conditioned prior to HSCT.

Busulphan (Bu) and tetrahydrothiophene (THT) were determined in two patients undergoing stem cell transplantation and conditioned with high doses of busulphan (2mg/kg b.i.d.) for four days. One patient received only Bu during conditioning, while the other patient was concomitantly administered voriconazole for Candida infection (3mg/kg i.v. b.i.d). Samples were collected at several time points for therapeutic drug monitoring. (A) Significantly (P<0.05) higher levels of Bu were observed in the patient receiving concomitant voriconazole (Δ, dashed line), compared to the levels observed after the same dose of Bu when administered alone (□, solid line). (B) THT levels were significantly (P<0.05) higher already after the first Bu dose with high accumulation and slower metabolism (Δ, dashed line) compared to Bu alone (□, solid line). Voriconazole was withdrawn after Bu second dose and Bu dose was reduced to 1.2mg/kg b.i.d for two days.