Busulphan Metabolism Via Flavin-Containing Monooxygenase 3 (FMO3) Can Explain Several Interactions with Other Drugs
Authors: El Serafi Ibrahim , Abedi-Valugerdi Manuchehr , Naughton Seán , Saghafian Maryam , Mattsson Jonas , Moshfegh Ali , Terelius Ylva , Potacova Zuzana , Hassan Moustapha 
Published: October 2019

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for several malignant and non-malignant diseases. Busulphan (Bu) is an alkylating agent that is used in high doses as a part of the conditioning regime prior to HSCT. Busulphan is metabolized mainly in the liver by conjugation with glutathione (GSH) by glutathione transferases (GST), in particular, GSTA1. Busulphan interactions with drugs metabolized by hepatic enzymes other than GSH/GST have been reported. The aim of the present study was to investigate the role of other enzymes than GSH/GST in the busulphan metabolism including FMO3 and cytochrome P450 enzymes (CYPs).

In our in vitro studies, human liver microsomes were incubated with busulphan first core metabolite, tetrahydrothiophene (THT). The incubation results showed a rapid disappearance of THT and gradual formation of the other metabolites. THT incubation with recombinant enzymes showed FMO3 to give the highest Initial THT disappearance rate (v0, 6.87 µmol/min/mL) and the highest intrinsic clearance(CLint, 0.26 µL/min/mg protein) followed by other CYPs. Comparing CYPs based on the initial THT disappearance rate (v0) per POR/CYP ratio, CYP2C8 had the highest rate for THT metabolism (5.03 nmol/min/(POR/CYP ratio) followed by CYPs 2C9, 2C19, 2E1 and 3A4. These results showed that THT is metabolized by microsomal enzymes and FMO3 is the main enzyme.

For in vivo studies, we determined the kinetics of Bu in mice in the presence or absence of phenylthiourea (PTU), an inhibitor of FMO3. Moreover, the effect of THT accumulation on Bu metabolism was also determined in the same species. Treatment with PTU resulted in increase of Bu plasma concentrations as expressed as AUC. Furthermore, synchronized treatment of mice with THT and Bu also enhanced the plasma levels of Bu as compared to animals receiving Bu alone. After THT dosing, THT concentrations and AUC significantly (P < 0.05) increased after concurrent PTU injection. THT concentrations and AUC for mice treated with Bu and PTU also significantly (P < 0.05) increased compared to mice injected with Bu alone.

To evaluate the role of FMO3 in the kinetics of Bu in the clinical settings, we investigated twelve patients undergoing HSCT and conditioned with busulphan / cyclophosphamide. A significant up-regulation (P < 0.05) of mRNA was found for FMO3 after Bu conditioning as confirmed using qRT-PCR. The up-regulation observed for FMO3 was similar to what was observed for GSTA1 (P < 0.05).

To further confirm the involvement of FMO3 on in vivo Bu kinetics in humans, blood samples were drawn routinely from a patient treated with high dose busulphan and concomitant administration of voriconazole. Voriconazole is an antimycotic drug is wieldy used in hematology and HSCT and is known to be metabolized via FMO3. High busulphan levels that were not relevant to the administered dose were detected in this patient. Measuring THT in the samples showed that THT levels detectable with high accumulation and slow oxidation rate.

In conclusion, our results showed that FMO3 and, to a lesser extent, CYPs are involved in busulphan metabolism through metabolism of its first core metabolite, THT. FMO3 is up regulated during Bu conditioning. FMO3 inhibition affects Bu kinetics. Our findings may offer valuable explanations into several drug interactions involving busulphan during HSCT conditioning therapy and thus, lower treatment related toxicity reported in HSCT.

Disclosures

No relevant conflicts of interest to declare.