Abstract

Cyclophosphamide (Cy) is commonly used in conditioning therapy for bone marrow transplantation (BMT). The conversion of Cy to its first active metabolite 4-hydroxy cyclophosphamide (4-HCy) is mediated by several enzymes of the cytochrome P450 (CYP450) family [CYP2B6, 2C9, 2C19 and 3A4]. Since the pharmacokinetics (PK) of 4-HCy is formation rate limited, the AUC ratio of 4-HCy/Cy reflects better the degree of systemic exposure to 4-HCy. The PK of Cy and HCy exhibits up to 20 fold inter-individual variation in patients during conditioning. To understand the mechanisms underlying the variation in Cy metabolism, we analyzed the PK of Cy and 4-HCy in 40 consecutive patients with beta thalassemia major undergoing BMT and correlated them with the common polymorphisms of CYP450 2B6, 2C9 and 2C19 genes and the pre transplant hepatic status (Lucarelli class) of the patient. All patients received Cy at a dose of 50mg/kg as one-hour intravenous infusion for 4 days (day -5 to -2) after 4 days of busulfan (day-9 to -6). Levels of Cy and 4-HCy were measured using high performance liquid chromatography. Genotyping for CYP2B6 G516T, CYP2C9 *2, *3 and CYP2C19*2 and *3 were done using PCR-RFLP methods. There was a 4–20 fold inter-patient variation in the PK of Cy and 4-HCy. Mean AUC of Cy was 2288±1169 mg* h/ml, (range: 674–5126), while that of AUC of 4-HCy was 5.67±2.52 mg* h/ml (range: 0.817–11.17). Patients with wild type CYP2B6 G/G genotype had significantly higher Cy 4-hydroxylation than those with the homozygous mutant genotype (T/T).

CYP4502B6, 2C9, 2C19 genotypes and ratio of AUC 4HCy/AUC Cy

CYP2B6 G/G (n=15) G/T (n=8) T/T (n=16)   t-test p value 
 0.0033±0.0014 0.003±0.0013 0.0023±0.0011   0.03 (G/G vs. T/T) 
CYP2C9 *1/*1 (n=25) *1/*2 (n=3) *1/*3 (n=9) *2/*3 (n=2) *3/*3 (n=1)  
 0.003±0.0016 0.0037±0.0009 0.0023±0.0011 0.0014, 0.0015 0.0073 0.076 (*1/*2 vs. *1/*3) 
CYP2C19 *1/*1 (n=26) *1/*2 (n=10) *2/*2 (n=3)    
 0.0026±0.001 0.0036±0.0012 0.0029±0.0013   0.016 (1/1 vs. 1/2) 
CYP2B6 G/G (n=15) G/T (n=8) T/T (n=16)   t-test p value 
 0.0033±0.0014 0.003±0.0013 0.0023±0.0011   0.03 (G/G vs. T/T) 
CYP2C9 *1/*1 (n=25) *1/*2 (n=3) *1/*3 (n=9) *2/*3 (n=2) *3/*3 (n=1)  
 0.003±0.0016 0.0037±0.0009 0.0023±0.0011 0.0014, 0.0015 0.0073 0.076 (*1/*2 vs. *1/*3) 
CYP2C19 *1/*1 (n=26) *1/*2 (n=10) *2/*2 (n=3)    
 0.0026±0.001 0.0036±0.0012 0.0029±0.0013   0.016 (1/1 vs. 1/2) 

One patient with homozygous mutant genotype CYP 2C9 3/3 showed 2.6 fold higher Cy 4-hydroxylation (0.0073 vs. 0.0026), while two patients with CYP2C9 2/3 genotype had 2 fold lower than the mean of the ratio for all 2C9 genotypes. Patients with CYP2C19 1/2 genotype showed significantly higher ratio than those with CYP2C19 1/1 genotype. No association was found between patients’ pre- transplant liver status (Lucarelli class) and Cy 4-hydroxylation. We conclude that 4-hydroxylation of Cy is significantly influenced by CYP2B6, 2C9 and 2C19 genotypes. This could explain the wide inter individual variation in Cy PK which should be correlated with toxicity and outcome of BMT.

Author notes

Corresponding author