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Quantition of cell-associated doxorubicin by high-performance liquid chromatography after enzymatic desequestration

  • Original Articles
  • Doxorubicin, HPLC, Cellular Concentration
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Abstract

A method for measuring cellular concentrations of the anthracycline doxorubicin was developed. The assay involves cell lysis and protein degradation by detergent and proteinase K treatment followed by DNA hydrolysis using DNase I. Prior to high-performance liquid chromatography, samples are deproteinized by the addition of ZnSO4 and methanol. The assay is linear with respect to both the cellular drug content and the number of cells assayed over the ranges tested, and drug recovery is close to 100%. The method has a limit of detection of 50 fmol injected doxorubicin. Within run and between-day coefficients of variation have consistently been found to be in the 5% and 10% range, respectively, in different cell lines exposed to doxorubicin in vitro. The method has been evaluated in analyses of doxorubicin levels in mononuclear blood cells of patients. The assay offers several advantages over commonly used organic extraction techniques and may improve cellular drug monitoring during anthracycline therapy in patients.

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References

  1. Ackland SP, Ratain MJ, Vogelzang NJ, Choi KE, Ruane M, Sinkule JA (1989) Pharmacokinetics and pharmacodynamics of longterm continuous-infusion doxorubicin. Clin Pharmacol Ther 45: 340

    Google Scholar 

  2. Andersen A, Warren DJ, Slørdal L (1993) A sensitive and simple high-performance liquid chromatographic method for the determination of doxorubicin and its metabolites in plasma. Ther Drug Monit 15: 455

    Google Scholar 

  3. Baurain R, Zenebergh A, Trouet A (1978) Cellular uptake and metabolism of daunorubicin as determined by high-performance liquid chromatography. J Chromatogr 157: 331

    Google Scholar 

  4. Chambers SH, Bleehen NM, Watson JV (1984) Effect of cell density on intracellular adriamycin concentration and cytotoxicity in exponential and plateau phase EMT6 cells. Br J Cancer 49: 301

    Google Scholar 

  5. Chang BK, Brenner DE, Gutman R (1989) Dissociation of the verapamil-induced enhancement of doxorubicins cytotoxicity from changes in cellular accumulation or retention of doxorubicin in pancreatic cancer cell lines. Anticancer Res 9: 347

    Google Scholar 

  6. Cummings J, Smyth JF (1988) Pharmacology of adriamycin: the message to the clinician. Eur J Cancer Clin Oncol 24: 579

    Google Scholar 

  7. Cummings J, Merry S, Willmott N (1986) Disposition kinetics of adriamycin, adriamycinol and their 7-deoxyaglycones in AKR mice bearing a sub-cutaneously growing Ridgway osteogenic sarcoma (ROS). Eur J Cancer Clin Oncol 22: 451

    Google Scholar 

  8. Cummings J, Willmott N, More I, Kerr DJ, Morrison JG, Kaye SB (1987) Comparative cardiotoxicity and antitumour activity of doxorubicin (adriamycin) and 4′-deoxydoxorubicin and the relationship to in vivo disposition and metabolism in the target tissues. Biochem Pharmacol 36: 1521

    Google Scholar 

  9. Cummings J, Willmott N, Smyth JF (1991) The molecular pharmacology of doxorubicin in vivo. Eur J Cancer 27: 532

    Google Scholar 

  10. DeGregorio MW, Holleran WM, Macher BA, Linker CA, Wilbur JR (1984) Kinetics and sensitivity of daunorubicin in patients with acute leukemia. Cancer Chemother Pharmacol 13: 230

    Google Scholar 

  11. Formelli F, Carsana R, Pollini C (1986) comparative pharmacokinetics and metabolism of doxorubicin and 4′-demethoxy-4′-O-methyldoxorubicin in tumor-bearing mice. Cancer Chemother Pharmacol 16: 15

    Google Scholar 

  12. Formelli F, Carsana R, Pollini C (1987) Pharmacokinetics of 4′-deoxy-4′-iodo-doxorubicin in plasma and tissues of tumor-bearing mice compared with doxorubicin. Cancer Res 47: 5401

    Google Scholar 

  13. Gewirtz DA, Yanovich S (1986) Metabolism of the anthracycline antibiotic daunorubicin to daunorubicinol and daunorubicinol aglycone in hepatocytes isolated from the rat and the rabbit. Biochem Pharmacol 35: 4059

    Google Scholar 

  14. Gewirtz DA, Yanovich S (1987) Metabolism of adriamycin in hepatocytes isolated from the rat and the rabbit. Biochem Pharmacol 36: 1793

    Google Scholar 

  15. Israel M, Sweatman TW, Seshadri R, Koseki Y (1989) Comparative uptake and retention of Adriamycin andN-Benzyladriamycin-14-valerate in human CEM leukemic lymphocyte cell cultures. Cancer Chemother Pharmacol 25: 177

    Google Scholar 

  16. Kennedy KA, Siegfried JM, Sartorelli AC, Tritton TR (1983) Effects of anthracyclines on oxygenated and hypoxic tumor cells. Cancer Res 43: 54

    Google Scholar 

  17. Kokenberg E, Sonneveld P, Nooter K, Steuyt K van der, Lövenberg B (1986) Quantitative evaluation of intracellular uptake of daunorubicin in acute myeloid leukemia: a method analysis. Cancer Chemother Pharmacol 17: 63

    Google Scholar 

  18. Lee FYF, Sciandra J, Siemann DW (1989) A study of the mechanism of resistance to adriamycin in vivo. Glutathione metabolism, p-glycoprotein expression, and drug transport. Biochem Pharmacol 38:3697

    Google Scholar 

  19. Legha SS, Benjamin RS, Mackay B, et al (1982) Reduction of doxorubicin cardiotoxicity by prolonged continuous intravenous infusion. Ann Intern Med 96: 133

    Google Scholar 

  20. Mahdadi R, Pommery N, Pommery J, Lhermitte M (1987) Quantitative determination of adriamycin in rat hepatocytes using a volatile extraction buffer, HPLC and fluorescence detection. Biomed Chromatogr 2: 91

    Google Scholar 

  21. Paul C, Baurain R, Gahrton G, Peterson C (1980) Determination of daunorubicin and its main metabolites in plasma, urine and leukaemic cells in patients with acute myeloblastic leukaemia. Cancer Lett 9: 263

    Google Scholar 

  22. Reed E, Ozols R, Tarone R, Yuspa SH, Poirier MC (1987) Platinum-DNA adducts in leukocyte DNA correlate with disease response in ovarian cancer patients receiving platinum-based chemotherapy. Proc Natl Acad Sci USA 84: 5024

    Google Scholar 

  23. Rose LM, Tillery KF, Dareer SME, Hill DL (1988) High-performance liquid chromatographic determination of doxorubicin and its metabolites in plasma and tissue. J Chromatogr 425: 419

    Google Scholar 

  24. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual (2nd edn) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  25. Schwartz HS (1973) A fluorometric assay for daunomycin and adriamycin in animal tissues. Biochem Med 7: 396

    Google Scholar 

  26. Speth PAJ, Linssen PCM, Boezeman JBM, Wessels HMC, Haanen C (1985) Quantitation of anthracyclines in human hematopoietic cell subpopulations by flow cytometry correlated with high pressure liquid chromatography. Cytometry 6: 143

    Google Scholar 

  27. Speth PAJ, Linssen PCM, Boezeman JBM, Wessels JCM, Haanen C (1986) Rapid quantitative determination of four anthracyclines and their main metabolites in human nucleated haematopoietic cells. J Chromatogr 377: 415

    Google Scholar 

  28. Speth PAJ, Raijmakers RAP, Boezeman JBM, et al (1988) In vivio cellular adriamycin concentrations related to growth inhibition of normal and leukemic human bone marrow cells. Eur J Cancer Clin Oncol 24: 667

    Google Scholar 

  29. Speth PAJ, Hoesel QGC van, Haanen C (1988) Clinical pharmacokinetics of doxorubicin. Clin Pharmacokinet 15: 15

    Google Scholar 

  30. Strauss JF, Kitchens RL, Patrizi VW, Frenkel EP (1980) Extraction and quantification of daunomycin and doxorubicin in tissues. J Chromatogr 221: 139

    Google Scholar 

  31. Sundman-Engberg B, Tidefelt U, Liliemark J, Paul C (1990) Intracellular concentrations of anticancer drugs in leukemic cells in vitro versus in vivo. Cancer Chemother Pharmacol 25: 252

    Google Scholar 

  32. Takemura Y, Kobayashi H, Miyachi H, Hayashi K, Sekiguchi S, Ohnuma T (1991) The influence of tumor cell density on cellular accumulation of doxorubicin or cisplatin in vitro. Cancer Chemother Pharmacol 27: 417

    Google Scholar 

  33. Tidefelt U, Sundman-Engberg B, Paul C (1989) Comparison of the intracellular pharmacokinetics of doxorubicin and 4′-epi-doxorubicin in patients with acute leukemia. Cancer Chemother Pharmacol 24: 225

    Google Scholar 

  34. Zhang Y, Sweet KM, Sognier MA, Belli JA (1992) An enhanced ability for transforming adriamycin into a noncytotoxic form in a multidrug-resistant cell line (LZ-8). Biochem Pharmacol 44: 1869

    Google Scholar 

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Authors and Affiliations

  1. Department of Clinical Pharmacology, The Norwegian Radium Hospital, Montebello, N-0310, Oslo, Norway

    Anders Andersen, David J. Warren & Lars Slørdal

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  1. Anders Andersen
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  2. David J. Warren
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  3. Lars Slørdal
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Andersen, A., Warren, D.J. & Slørdal, L. Quantition of cell-associated doxorubicin by high-performance liquid chromatography after enzymatic desequestration. Cancer Chemother. Pharmacol. 34, 197–202 (1994). https://doi.org/10.1007/BF00685077

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  • DOI: https://doi.org/10.1007/BF00685077

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