Quantification of busulfan in plasma by liquid chromatography-ion spray mass spectrometry. Application to pharmacokinetic studies in children.

Optimisation of busulfan dosage in patients undergoing bone marrow transplantation is recommended in order to reduce toxic effects associated with high drug exposure. A new method was developed coupling liquid chromatography with mass spectrometry (LC-MS) and was validated for the determination of busulfan concentrations in plasma. Recovery was 86.7%, the limit of detection was 2.5 ng/ml and linearity ranged from 5 to 2500 ng/ml. The correlation between the busulfan concentrations measured by our previously published HPLC-UV method and the new HPLC-MS method was highly significant (P<0.0001). Sample volume was reduced and the method was rapid, sensitive and less expensive than the methods previously used in our laboratory. This method was used to determine the pharmacokinetic parameters of busulfan after the first administration of 1 mg/kg orally, in 13 children receiving the drug as part of the preparative regimen for bone marrow transplantation. Our results were similar to previously reported data. They showed that the apparent oral clearance of busulfan was 0.299+/-0.08 l/h/kg, and that it was significantly higher (P=0.02) in patients below the age of 5 years than in older children.

Pharmacokinetics of oral busulphan in children with beta thalassaemia major undergoing allogeneic bone marrow transplantation.

The pharmacokinetics of busulphan were studied in 23 thalassaemic children undergoing BMT. Patients received busulphan at a dose of either 16 mg/kg with cyclophosphamide and ATG (Group A) or 600 mg/m2 (with cyclophosphamide alone) (Group B) in 16 divided doses every 6 h over 4 days. Busulphan levels were analyzed by a modified GC-MS method. The dose of busulphan/kg for patients in group B was 64% (range 56-71%) higher than that for patients in group A. The mean AUC, Css, Cmax and MRV were significantly higher in group B as compared with group A for both doses 1 and 13. There was no significant difference in Vd/F, T1/2 and Kel between the two groups. A significant decrease in AUC and Css was found between 1st and 13th doses in group B, but not in group A. The Cl/F values in group A were significantly higher than those in group B after dose 1, but not after dose 13. No increase in toxicity due to the higher dose of busulphan was noted. We conclude that busulphan at 600 mg/m2 results in much higher systemic exposure to the drug as compared to 16 mg/kg, without increase in toxicity in children with beta thalassaemia major.

In-vitro cytochrome P450 dependent metabolism of oxybutynin to N-deethyloxybutynin in humans.

Oxybutynin (Ditropan), a direct antagonist of acetylcholine on muscarinic receptors, is administered in children for the treatment of vesical immaturity and is responsible for atropinic adverse effects, which are more frequent in paediatric than in adult patients. Oxybutynin is metabolized by oxidation to N-desethyloxybutynin, a stable and toxic metabolite, and previous results in vivo have suggested that cytochrome P450 2D6 (CYP2D6) may be involved in this pathway of metabolism. We used human liver microsomes genotyped as extensive metabolizers for CYP2D6 to determine the kinetic parameters of N-desethyloxybutynin formation: Km was 16.5+/-5.2 microM and Vmax was 76.8+/-3.7 mmol/mg/h. Quinidine and anti-liver kidney microsome antibody type I had no inhibitory effects on N-desethyloxybutynin formation, demonstrating that CYP2D6 has no role in oxybutynin metabolism. The effects of specific inhibitors of other cytochromes P450 were also investigated. Recombinant human CYP 3A4 but not 2B6, 2D6, 2C8 and 2E1, displayed significant N-desethylation activity. Our results demonstrate that the CYP3A subfamily, and not CYP2D6, is involved in N-desethyloxybutynin formation.