Chlorambucil and fludarabine as a new pre-transplant conditioning for patients with chronic lymphocytic leukemia: results of in vivo experiments

Chronic lymphatic leukemia (CLL), incurable by standard treatments, may be potentially cured by allogeneic hematopoietic stem cell transplantation. Since CLL affects predominantly older people, there is a need for some low-toxicity conditioning with, on the other hand, strong antileukemic activity. Since there are very encouraging results with busulfan + fludarabine conditionings in myeloid malignancies and since the clinical study with the combination treatment with chlorambucil and fludarabine was stopped prematurely for myelotoxicity, we hypothesized that this chlorambucil + fludarabine combination would have the potential as a good conditioning for high-risk lymphoid malignancies. The aim of this study was to test the chlorambucil + fludarabine combination in vivo (in rats) for toxicity. Male Wistar rats were used in all experiments. First, the maximum tolerated dose (MTD) of each drug was tested. For fludarabine, doses of 0.75-60 mg/kg/day, and for chlorambucil, doses of 0.15-50 mg/kg/day were used, all administered for five days. Then, the combination treatment was tested: (1) fludarabine and chlorambucil simultaneously (F+CH), (2) fludarabine followed by chlorambucil (F-CH), (3) chlorambucil followed by fludarabine (CH-F); all drugs were administered for five days. For fludarabine alone, the MTD was not reached. Clinically, the rats tolerated well even the highest doses. Moreover, no myelotoxicity was seen. However, pneumotoxicity, hepatotoxicity, nephrotoxicity, and gastrointestinal toxicity were found by a histological examination. For chlorambucil alone, the MTD is about 40-50 mg/kg/day. Pneumotoxicity, nephrotoxicity, gastrointestinal toxicity, and myelotoxicity were observed. The combination treatment tested a fixed dose of fludarabine (3 mg/kg per day) and three doses of chlorambucil (1, 2, and 4 mg/kg/day). Clinically, the best tolerated combination was fludarabine followed by chlorambucil (F-CH). Haematological toxicity was observed, usually affecting predominantly lymphocytes, and interestingly, it was most pronounced in the clinically best tolerated regimen. Rats can tolerate extremely high doses of fludarabine and chlorambucil. Based on these experiments, for further development, hopefully into the clinical usage, we could recommend the administration of fludarabine, followed by chlorambucil. This combination will further be tested together with monoclonal antibodies and total lymphoid irradiation as a conditioning regimen for allogeneic bone marrow transplantation.