Optimization of cyclophosphamide therapy based on pharmacogenetics

Cyclophosphamide (CPA) is one of the most broadly used anticancer agents. CPA is also a potent immunosuppressive agent that is used for the treatment of autoimmune diseases. CPA is a prodrug that is activated in liver by a 4-hydroxylation reaction catalyzed by cytochrome P450 (CYP) enzymes. The main enzyme in its activation is CYP2B6 that has a polymorphic gene. Several studies have reported high degree of inter- and intraindividual variation in CPA pharmacokinetics.

The aim of this thesis was to increase the knowledge about the role of specific CYPS in CPA bioactivation, to determine the effect of dose and administration schedule on the CYP induction by CPA and to study drug-drug interaction between CPA and ciprofloxacin, in order to individualize and optimize CPA treatment.

In rats treated with single dose CPA (i.v.), the CYP2B1 and CYP2B2 mRNAs and the proteins CYP2B1/2 were significantly induced. Microsomal activity of CYP2B was significantly increased as well. The maximum levels of mRNA, proteins and microsomal activity were reached at 4-8 hours after the dose. The induction in mRNA, protien and the enzyme activity were dose dependent. These results demonstrate that CPA has a high inducing effect on CYP2B1 and 2B2 in rat.

In multiple dose schedule of CPA when the animals were treated with four low doses (75 mg/kg) in short intervals (6 h), we observed high induction in CYP2B1 mRNA and protein over the 24 hours. Using the same administration schedule but high doses of CPA (150 mg/kg), a decrease in CYP2B1 mRNA and protein levels were observed after the second injection which might be due to toxicity and/or down-regulation caused by CPA and/or CPA metabolites.

The role of the polymorphic human CYP2B6 in CPA bioactivation using cDNAs expressed wild type (allele*l) and 3 different mutations of enzyme (alleles *4 and *6, and mutation G516T) was investigated. All three mutations have less activity for the hydroxylation of CPA compared to CYP2116* 1 (wild type).

Ciprofloxacin altered CPA pharmacokinetics in patients with NHI, that were treated with standard CHOP. A decrease in the exposure to drug as expressed as AUC of 4-hydroxyCPA/AUC of CPA was observed in all patients, which might influence CPA therapeutic efficacy.

In conclusion, the present results show the importance of dosing regimes, administration intervals, specific CYP alleles and drug-drug interaction in CPA bioactivation. Genotyping of the patients prior to the treatment with CPA and following the effect of the therapy on gene expression can be of a high clinical importance. Inherited combinations of CYPS can determine the clinical outcome, i.e. resistance to the drug, risk of toxicity or inefficient therapy. In cases where repeated treatment with cyclophosphamide can modify the gene expression, other treatment strategy should be introduced.

List of scientific papers

I. Xie H, Afsharian P, Terelius Y, Mirghani RA, Yasar U, Hagbjork AL, Lundgren S, Hu Y, Rane A, Hassan M (2005). Cyclophosphamide induces mRNA, protein and enzyme activity of cytochrome P450 in rat. Xenobiotica. 35(3): 239-51.
https://pubmed.ncbi.nlm.nih.gov/16019949

II. Afsharian P, Terelius Y, Nilsson C, Hassan Z, Lundgren S, Hassan M (2006). The effect of repeated administration of cyclophosphamide, on CYP2B in rat. [Submitted]

III. Afsharian P, Terelius Y, Hidestrand M, Ingelman-Sundberg M, Hassan M, Lundgren S (2006). The role of human CYP2B6 polymorphism in the bioactivation of cyclophosphamide using cDNA expressed enzymes. [Manuscript]

IV. Afsharian P, Mollgard L, Hassan Z, Xie H, Kimby E, Hassan M (2005). The effect of ciprofloxacin on cyclophosphamide pharmacokinetics in patients with non-Hodgkin lymphoma. Eur J Haematol. 75(3): 206-11.
https://pubmed.ncbi.nlm.nih.gov/16104876