Studies on mechanisms of busulphan cytotoxicity and pharmacokinetics : with special reference to liposomal busulphan

Busulphan is an alkylating agent currently used in conditioning regimen prior to stem cell transplantation (SCT). High dose therapy with busulphan has been shown to contribute to transplantation-related toxicities, such as veno-occlusive disease (VOID) and interstitial pneumonia, Previous pharmacodynamic studies have attempted to define a target systemic exposure to busulphan, and thresholds for different side effects. Pharmacokinetic studies have shown a wide inter-patient variation in the systemic exposure to busulphan after a fixed dose. The wide variation may be due to bioavailability, age, underlying disease, drug-drug interaction and circadian rhythm.

The aim of the first part of this thesis was to develop and evaluate an intravenous formulation of busulphan encapsulated in liposomes. Small, uncharged liposomes containing cholesterol suspended in 5% glucose were selected for the preparation of liposomal busulphan (LBu). The distribution of LBu was assessed with 14C labeled busulphan in a rat model, and compared with a solution of free drug. The distribution of LBu was higher to bone marrow and spleen, and lower to brain, lung and heart, compared with free drug, while the distribution to liver was similar. The myelosuppressive effect of LBu was stable and reproducible in a mouse model. Pharmacokinetics of liposomal busulphan was studied in man after either low dose or high dose of LBu. A linear relation between the systemic exposure and the dose was found. The pharmacokinetic parameters were in agreement with those previously reported for orally administered drug. It was feasible to use liposomal busulphan for high dose therapy. The advantageous distribution and good myelosuppressive effect in animals, and the more predictable pharmacokinetics in man have motivated the launching of a phase 1/11 clinical trial.

The aim of the second part of the thesis was to investigate mechanisms of busulphan-induced cytotoxicity, and the role of glutathione (GSH) in this process. The myeloid P39 cell line was used in vitro as a model for induction of differentiation and/or apoptosis with the differentiating agent all-trans retinoic acid and the cytostatic agent etoposide. Both agents induced apoptosis that was mediated through caspase activation, but different pathways were involved. These pathways diverged in kinetics, preceding maturation, cleavage of BcI-2 and actin, and rescue from apoptosis by granulocyte colonystimulating factor (G-CSF). Busulphan- and etoposide-induced apoptosis shared common features, but differed in kinetics. The P39 cells were arrested in G2 phase of the cell cycle before apoptotic morphology developed. Proliferation and clonogenic capacity of the P39 cells showed an inverse linear relation to the exposure to busulphan expressed as AUC. Also in busulphan-treated human CD34+ hematopoietic progenitors, clonogenic capacity was inversely and linearly related to the exposure to busulphan expressed as AUC. Myeloid progenitors were more sensitive than erythroid progenitors (AUCs completely inhibiting colony formation were 69 ± 7.5 µg.hr/ml and 140 ± 36 µg.hr/ml for CFUGM and erythroid colonies, respectively). Neither an increase (induced by N-acetylcysteine), nor a decrease (induced by buthionine sulfoximine) of the cellular content of GSH affected busulphan-induced cytotoxicity in human CD34+ hernatopoietic progenitors in vitro, or in murine bone marrow cells in vivo.

In conclusion, liposomal busulphan is a suitable formulation for high dose treatment in conditioning regimen prior to stem cell transplantation. N-acetylcysteine does not decrease busulphan-induced cytotoxicity in hernatopoietic progenitor cells, and may thus serve as a potential prophylactic agent against transplantation-related hepatotoxicity.

List of scientific papers

I. Pharmacokinetics and distribution of liposomal busulfan in the rat: a new formulation for intravenous administration. (1998). ": Hassan M, Hassan Z, Nilsson C, Rehim MA, Kumlien S, Elfsson B, Kallberg N " Cancer Chemother Pharmacol 42(6): 471-8
https://pubmed.ncbi.nlm.nih.gov/9788573

II. Hassan Z, Nilsson C, Hassan M (1998). "Liposomal busulphan: bioavailability and effect on bone marrow in mice. " Bone Marrow Transplant 22(9): 913-8
https://pubmed.ncbi.nlm.nih.gov/9827821

III. Hassan Z, Ljungman P, Ringden O, Winiarski J, Nilsson C, Aschan J, Whitley HR, Hassan M (2001). "Pharmacokinetics of liposomal busulphan in man. " Bone Marrow Transplant 27(5): 479-85
https://pubmed.ncbi.nlm.nih.gov/11313681

IV. Hassan Z, Fadeel B, Zhivotovsky B, Hellstrom-Lindberg E (1999). "Two pathways of apoptosis induced with all-trans retinoic acid and etoposide in the myeloid cell line P39. " Exp Hematol 27(8): 1322-9
https://pubmed.ncbi.nlm.nih.gov/10428509

V. Hassan Z, Hassan M, Hellstrom-Lindberg E (2001). "The pharmacodynamic effect of busulfan in the P39 myeloid cell line in vitro. " Leukemia 15(8): 1240-7
https://pubmed.ncbi.nlm.nih.gov/11480566

VI. Hassan Z, Hellstrom-Lindberg E, Alsadi S, Edgren MR, Hagglund H, Hassan M (2001). "The role of glutathione in busulphan-induced cytotoxicity in hemaopoietic cells in vitro and in vivo." (Manuscript)