Impact of valproic acid on busulfan pharmacokinetics: In vitro assessment of potential drug-drug interaction

Abstract

Busulfan (Bu) is an alkylating agent commonly used at high doses in the preparative regimens of hematopoietic stem cell transplantation (HSCT). It has been shown that such high doses of Bu are associated with generalized seizures which are usually managed by prophylactic antiepileptic drugs (AEDs) such as valproic acid (VPA). Being a strong enzyme inhibitor, VPA may inhibit Bu metabolism and thus increase its potential toxicity. Despite its clinical relevance, the potential interaction between Bu and VPA has not yet been evaluated. The aim of the present study was to assess and evaluate the potential drug-drug interaction (DDI) between Bu and VPA. This study was carried out by incubating Bu in laboratory-prepared rat liver-subcellular fractions including S9, microsomes, and cytosol, alone or in combination with VPA. The liver fractions were prepared by differential centrifugation of the liver homogenate. Analysis of Bu was employed using a fully validated LC-MS/MS method. The validation parameters were within the proposed limits of the international standards guidelines. Bu metabolic stability was assessed by incubating Bu at a concentration of 8 μg/ml in liver fractions at 37°C. There were significant reductions in Bu levels in S9 and cytosolic fractions, whereas these levels were not significantly (P ˃ 0.05) changed in microsomes. However, in presence of VPA, Bu levels in S9 fraction remained unchanged. These results indicated, for the first time, the potential metabolic interaction of Bu and VPA being in S9 only. This could be explained by inhibiting Bu cytosolic metabolism by the interaction with VPA either by sharing the same metabolic enzyme or the required co-factor. In conclusion, the present findings suggest, for the first time, a potential DDI between Bu and VPA in vitro using rat liver fractions. Further investigations are warranted in human-derived liver fractions to confirm such an interaction.

Fig 1. Busulfan metabolic pathway (El-Serafi et al., 2017).

Fig 2. Oxidative-o-deethylation of 7-ER by active CYP1A1/2 to resorufin (Short and Springman, 2016).

Fig 3. Conjugation of CDNB with GSH via active GST enzyme (Gonzalez, 2006).

Fig 4. Concentration of resorufin in microsomal fraction versus incubation time.

The 7-ER was incubated with 2 mg/ml PRLM containing NADPH, UDPGA and MgCl₂ at 37°C for 120 min. Each point represents the mean value (n = 2) of two duplicated assays. 7-ER; 7-Ethoxyresorufin, PRLM; Pooled Rat Liver Microsomes, NADPH; Nicotinamide Adenine Dinucleotide Phosphate, UDPGA; Uridine-5`-Diphosphoglucuronic Acid.

Fig 5. Absorbance of CDNB-GS in cytosolic fraction versus incubation time.

The CDNB was incubated with 0.075 mg/ml PRLC containing glutathione at 37°C for 120 min. Each point represents the mean value of two readings from duplicated reactions. CDNB-GS; 1-Chloro-2,4-DinitroBenzen-Glutathione conjugate, PRLC; Pooled Rat Liver Cytosol.

Fig 6. Stability profiles of Bu in the presence and absence of VPA.

Bu is incubated alone or in presence of VPA at 37°C for 180 min in (A) mobile phase, (B) ACN, (C) incubation buffer, or (D) rat plasma. Each point represents the average value of duplicated incubations. Bu; Busulfan, VPA; Valproic Acid, ACN; Acetonitrile.

Fig 7. Concentration of Bu incubated in microsomal fraction in presence and absence of VPA.

Bu was incubated in PRLM containing NADPH, UDPGA and MgCl₂ at 37°C for 60 min. The left panel represents analyzed samples for Bu at 2 and 60 min (A and B, respectively) alone and in presence of VPA, while the right panel represents the same analyzed Bu samples alone and in presence of VPA (C and D, respectively) at 2 and 60 min. Each value represents mean ± SD; calculated from 6 assays. Bu; Busulfan, VPA; Valproic Acid, PRLM; Pooled Rat Liver Microsomes, NADPH; Nicotinamide Adenine Dinucleotide Phosphate, UDPGA; Uridine-5`-Diphosphoglucuronic Acid.

Fig 8. Concentration of Bu incubated in cytosolic fraction in presence and absence of VPA.

Bu was incubated in PRLC containing glutathione at 37°C for 60 min. The left panel represents analyzed samples for Bu at 2 and 60 min (A and B, respectively) alone and in presence of VPA, while the right panel represents the same analyzed Bu samples alone and in presence of VPA (C and D, respectively) at 2 and 60 min. Each value represents mean ± SD; calculated from 6 assays. Bu; Busulfan, VPA; Valproic Acid, PRLC; Pooled Rat Liver Cytosol.

Fig 9. Concentration of Bu incubated in S9 fraction in presence and absence of VPA.

Bu was incubated in PRLS9 containing NADPH, UDPGA, MgCl₂ and glutathione at 37°C for 60 min. The left panel represents analyzed samples for Bu at 2 and 60 min (A and B, respectively) alone and in presence of VPA, while the right panel represents the same analyzed Bu samples alone and in presence of VPA (C and D, respectively) at 2 and 60 min. Each value represents mean ± SD; calculated from 6 assays. Bu; Busulfan, VPA; Valproic Acid, PRLS9; Pooled Rat Liver S9, NADPH; Nicotinamide Adenine Dinucleotide Phosphate, UDPGA; Uridine-5`-Diphosphoglucuronic Acid.