Evaluation of In Vitro Bone Marrow Culture as a Tool for Assessing Mechanisms of Haematotoxicity

Abstract

Dose limiting haematotoxicity has been associated with a range of therapeutic agents used for the treatment of a number of different conditions. Haematotoxicity is usually assessed as part of the preclinical safety studies in experimental animals, where changes in peripheral blood cell numbers and bone marrow cellularity are determined at the end of the study. Often no information on the mechanism of the haematotoxicity is revealed. This thesis demonstrates how in vitro bone marrow cultures can be utilized to assist in the assessment of haematotoxicity by two different approaches; firstly, in vitro bone marrow cultures can be used to assess the haematopoietic lineage specificity of vincristine sulphate, vinblastine sulphate, hydroxyurea and anagrelide hydrochloride using clonogenic cultures, enabling ranking of these compounds according to their haematotoxicity. Secondly, using in vitro assays only, elucidate the mechanism(s) of the megakaryocytic lineage specific inhibition of anagrelide hydrochloride. To this end both clonogenic cultures and LTBMC offer the ability to elucidate mechanisms of action on multipotent stem cells, lineage specific cells and effects on the bone marrow microenvironment following single and repeated administration. In addition, the combination of cell identification techniques flow cytometry and light microscopy was shown to provide a more detailed understanding of the different cell populations within the non-adherent cell layer. In vivo AN reduces platelet counts only, however, the mechanism of the megakaryocyte specific toxicity by AN is not understood. In these studies, the mechanism (s) of the megakaryocytic lineage haematotoxicity of AN was examined using the established human clonogenic and LTBMC. The action of AN was shown to be focused at a late stage in megakaryocyte (Mk) colony development. Ranking the potential mechanisms of action of AN by concentration at which they were noted, the inability to organize the microtubules appears to be secondary to 1) alteration in cell cycling, 2) surface receptor expression and 3) inhibition in achieving high (greater than 8N) ploidy number. However, identification of the primary mechanism based solely on concentration seems to be very crude and most probably reflects a limitation of in vitro systems. The inhibition of platelet production by AN is most likely a result from a combination of mechanisms; inhibition of cell cycling, disruption in the expression of cell surface receptors, inhibition of the ability of the cells to increase ploidy number and an associated inability to organize microtubules leading to a reduction in platelet release. This work also demonstrated the importance of the selection of the source of bone marrow used in the cultures. The concentration at which 50 percent of Mk colony growth was inhibited (IC50) by AN for murine cells was markedly (46 fold) different (88.6μM) compared to the IC50 with human cord blood (hCB) (1.92μM). This disparity is indicative of differences in species sensitivity possibly related to AN having a greater affinity towards the human c-mpl chrombopoietin (TPO) receptor than the equivalent murine receptor as suggested by McCarty et al (2006). This work highlights the utility of in vitro bone marrow cultures as a tool for investigating the lineage specific haematotoxicity by evaluating compounds used in the treatment of ET. In addition in vitro haematopoietic cultures can successfully be used as a tool to investigate potential mechanism(s) of haematotoxicity as demonstrated herein by providing an insight to mechanism of platelet count reduction by AN.