Daunorubicin kinetics and drug resistance in leukaemia
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The aims of this thesis were to examine: (1) plasma and cellular pharmacokinetics of daunorubicin and its major metabolite daunorubicinol in patients with acute leukaemia, and the relationships between pharmacokinetics, patient response and the presence of P glycoprotein; (2) actions of the multidrug resistance reversing agents cyclosporin A and trifluoperazine, at clinically achievable concentrations, on daunorubicin accumulation and retention in human leukaemia cell lines and patients with acute leukaemia; and (3) effect of daunorubicin on the cell membrane of both sensitive and resistant cell lines, with and without the multidrug resistance reversing agents. Twenty-seven patients with acute leukaemia received daunorubicin as part of induction therapy. The plasma and cellular levels of daunorubicin and its metabolite daunorubicinol were determined using HPLC. There were no significant differences between patients who went into complete remission (12 out of 23) compared to those who did not respond for any of the plasma pharmacokinetic parameters. There was a significant difference in the cellular daunorubicin and daunorubicinol area under the concentration-time curve between responders and non responders (p less than 0.02), as well as in cellular Cmax, cellular clearance and cellular volume of distribution. Eleven patients were P glycoprotein positive and 10 P glycoprotein negative (no sample available for 2 patients). There was no correlation between patient response and the presence of P glycoprotein; nor a correlation between the cellular concentration of daunorubicin or daunorubicinol and P glycoprotein. Patients responding to chemotherapy had higher cellular daunorubicin and daunorubicinol compared to non responders. In contrast to in vitro studies, overexpression of P glycoprotein was not the reason for the lower cellular daunorubicin levels. Cyclosporin A was capable of increasing both cellular accumulation and retention in the drug resistant CEM/VLB and HL 60/ADR cell lines, but not in the drug sensitive CEM and HL 60 cell lines. Trifluoperazine had no effect in any of the four cell lines. In contrast to the cell line findings, only the combination of cyclosporin A and trifluoperazine were able to increase both accumulation and retention in the blast cells of patients at initial presentation. The multidrug resistant reversing agents alone had no effect in increasing accumulation or retention in the blast cells of P glycoprotein positive patients, nor patients in relapse. The cell line studies show that at clinically relevant concentrations only cyclosporin A is capable of increasing daunorubicin accumulation in both the drug resistant P glycoprotein positive (VLB) and P glycoprotein negative (ADR) cell lines. Thus, cyclosporin A does not work only by inhibiting the actions of P glycoprotein. Trifluoperazine was unable to reverse drug resistance at clinically relevant concentrations in either cell lines or patient blast cells. However, the combination of cyclosporin A and trifluoperazine increased accumulation in patient blast cells at initial presentation, suggesting that these agents may be more useful in patients at initial presentation than relapse. Daunorubicin was immobilised by linking it to poly vinyl alcohol and the effect of immobilised-daunorubicin was studied on the four cell lines above. The immobilised-daunorubicin was able to decrease cell growth in the drug sensitive HL 60 cell line but not in the drug resistant VLB or ADR cell lines. Poly vinyl alcohol itself was cytotoxic to the CEM cell line. The multidrug resistance reversing agents cyclosporin A and trifluoperazine were only capable of increasing cytotoxicity in the HL 60 cell line, with no effect in the drug resistant VLB or ADR cell lines.
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