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. 1998 Jun;77(12):2112–2119. doi: 10.1038/bjc.1998.355

Evaluation of a novel in vitro assay for assessing drug penetration into avascular regions of tumours.

R M Phillips 1, P M Loadman 1, B P Cronin 1
PMCID: PMC2150429  PMID: 9649122

Abstract

The poor blood supply to solid tumours introduces many factors that affect the outcome of chemotherapy, one of which is the problem of drug delivery to poorly vascularized regions of tumours. Whereas poor drug penetration has been recognized as a contributing factor to the poor response of many solid tumours, the question of drug penetration through multicell layers has not been thoroughly addressed, largely because of restrictions imposed upon these studies by the requirement for either radiolabelled or naturally fluorescent compounds. The aim of this study is to describe modifications made to a recently published assay that broadens the scope for assessing drug penetration during the early stages of drug development and to characterize the ability of various drugs to penetrate multicell layers. DLD-1 human colon carcinoma cells were cultured on Transwell-COL plastic inserts placed into 24-well culture plates so that a top and bottom chamber were established, the two chambers being separated by a microporous membrane. Drugs were added to the top chamber at doses equivalent to peak plasma concentrations in vivo and the rate of appearance of drugs in the bottom chamber determined by high-performance liquid chromatography (HPLC). Both 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine) and 7-[4'-(2-nitroimidazol-1-yl)-butyl]-theophylline (NITP) rapidly penetrated DLD-1 multicell layers (50.9 +/- 12.1 microm thick) with t(1/2) values of 1.36 and 2.38 h respectively, whereas the rate of penetration of 5-aziridino-3-hydroxymethyl-1-methyl-2-[1H-indole-4,7-dione] prop-beta-en-alpha-ol (EO9) and doxorubicin through multicell layers was significantly slower (t(1/2) = 4.62 and 13.1 h respectively). Inclusion of dicoumarol increases the rate of EO9 penetration, whereas reducing the oxygen tension to 5% causes a reduction in tirapazamine penetration through multicell layers, suggesting that the extent of drug metabolism is one factor that determines the rate at which drugs penetrate multicell layers. The fact that EO9 does not readily penetrate a multicell layer, in conjunction with its rapid elimination in vivo (t(1/2) < 10 min), suggests that EO9 is unlikely to penetrate more than a few microm from a blood vessel within its pharmacokinetic lifespan. These results suggest that the failure of EO9 in the clinic is due to a combination of poor drug penetration and rapid elimination in vivo.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chapman J. D., Baer K., Lee J. Characteristics of the metabolism-induced binding of misonidazole to hypoxic mammalian cells. Cancer Res. 1983 Apr;43(4):1523–1528. [PubMed] [Google Scholar]
  2. Chapman J. D., Franko A. J., Sharplin J. A marker for hypoxic cells in tumours with potential clinical applicability. Br J Cancer. 1981 Apr;43(4):546–550. doi: 10.1038/bjc.1981.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Coleman C. N. Hypoxia in tumors: a paradigm for the approach to biochemical and physiologic heterogeneity. J Natl Cancer Inst. 1988 May 4;80(5):310–317. doi: 10.1093/jnci/80.5.310. [DOI] [PubMed] [Google Scholar]
  4. Collard J., Matthew A. M., Double J. A., Bibby M. C. EO9: relationship between DT-diaphorase levels and response in vitro and in vivo. Br J Cancer. 1995 Jun;71(6):1199–1203. doi: 10.1038/bjc.1995.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cowan D. S., Hicks K. O., Wilson W. R. Multicellular membranes as an in vitro model for extravascular diffusion in tumours. Br J Cancer Suppl. 1996 Jul;27:S28–S31. [PMC free article] [PubMed] [Google Scholar]
  6. Dexter D. L., Barbosa J. A., Calabresi P. N,N-dimethylformamide-induced alteration of cell culture characteristics and loss of tumorigenicity in cultured human colon carcinoma cells. Cancer Res. 1979 Mar;39(3):1020–1025. [PubMed] [Google Scholar]
  7. Dirix L. Y., Tonnesen F., Cassidy J., Epelbaum R., ten Bokkel Huinink W. W., Pavlidis N., Sorio R., Gamucci T., Wolff I., Te Velde A. EO9 phase II study in advanced breast, gastric, pancreatic and colorectal carcinoma by the EORTC Early Clinical Studies Group. Eur J Cancer. 1996 Oct;32A(11):2019–2022. doi: 10.1016/0959-8049(96)00226-2. [DOI] [PubMed] [Google Scholar]
  8. Durand R. E. Distribution and activity of antineoplastic drugs in a tumor model. J Natl Cancer Inst. 1989 Jan 18;81(2):146–152. doi: 10.1093/jnci/81.2.146. [DOI] [PubMed] [Google Scholar]
  9. Erlanson M., Daniel-Szolgay E., Carlsson J. Relations between the penetration, binding and average concentration of cytostatic drugs in human tumour spheroids. Cancer Chemother Pharmacol. 1992;29(5):343–353. doi: 10.1007/BF00686002. [DOI] [PubMed] [Google Scholar]
  10. Fisher R. I. Report of workshop 5: early high-dose chemotherapy. Ann Oncol. 1996;7 (Suppl 4):131–133. doi: 10.1093/annonc/7.suppl_4.s131. [DOI] [PubMed] [Google Scholar]
  11. Franko A. J. Hypoxic fraction and binding of misonidazole in EMT6/Ed multicellular tumor spheroids. Radiat Res. 1985 Jul;103(1):89–97. [PubMed] [Google Scholar]
  12. GOLDACRE R. J., SYLVEN B. On the access of blood-borne dyes to various tumour regions. Br J Cancer. 1962 Jun;16:306–322. doi: 10.1038/bjc.1962.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garrecht B. M., Chapman J. D. The labelling of EMT-6 tumours in BALB/C mice with 14C-misonidazole. Br J Radiol. 1983 Oct;56(670):745–753. doi: 10.1259/0007-1285-56-670-745. [DOI] [PubMed] [Google Scholar]
  14. Graham M. A., Senan S., Robin H., Jr, Eckhardt N., Lendrem D., Hincks J., Greenslade D., Rampling R., Kaye S. B., von Roemeling R. Pharmacokinetics of the hypoxic cell cytotoxic agent tirapazamine and its major bioreductive metabolites in mice and humans: retrospective analysis of a pharmacokinetically guided dose-escalation strategy in a phase I trial. Cancer Chemother Pharmacol. 1997;40(1):1–10. doi: 10.1007/s002800050617. [DOI] [PubMed] [Google Scholar]
  15. Helmlinger G., Yuan F., Dellian M., Jain R. K. Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat Med. 1997 Feb;3(2):177–182. doi: 10.1038/nm0297-177. [DOI] [PubMed] [Google Scholar]
  16. Hendriks H. R., Pizao P. E., Berger D. P., Kooistra K. L., Bibby M. C., Boven E., Dreef-van der Meulen H. C., Henrar R. E., Fiebig H. H., Double J. A. EO9: a novel bioreductive alkylating indoloquinone with preferential solid tumour activity and lack of bone marrow toxicity in preclinical models. Eur J Cancer. 1993;29A(6):897–906. doi: 10.1016/s0959-8049(05)80434-4. [DOI] [PubMed] [Google Scholar]
  17. Hodgkiss R. J., Stratford M. R., Dennis M. F., Hill S. A. Pharmacokinetics and binding of the bioreductive probe for hypoxia, NITP: effect of route of administration. Br J Cancer. 1995 Dec;72(6):1462–1468. doi: 10.1038/bjc.1995.530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kerr D. J., Kaye S. B. Aspects of cytotoxic drug penetration, with particular reference to anthracyclines. Cancer Chemother Pharmacol. 1987;19(1):1–5. doi: 10.1007/BF00296245. [DOI] [PubMed] [Google Scholar]
  19. Keyes S. R., Heimbrook D. C., Fracasso P. M., Rockwell S., Sligar S. G., Sartorelli A. C. Chemotherapeutic attack of hypoxic tumor cells by the bioreductive alkylating agent mitomycin C. Adv Enzyme Regul. 1985;23:291–307. doi: 10.1016/0065-2571(85)90053-6. [DOI] [PubMed] [Google Scholar]
  20. Koch C. J. Unusual oxygen concentration dependence of toxicity of SR-4233, a hypoxic cell toxin. Cancer Res. 1993 Sep 1;53(17):3992–3997. [PubMed] [Google Scholar]
  21. Kotake A. N., Vogelzang N. J., Larson R. A., Choporis N. New high-performance liquid chromatographic assay for plasma doxorubicin. J Chromatogr. 1985 Jan 11;337(1):194–200. doi: 10.1016/0378-4347(85)80030-x. [DOI] [PubMed] [Google Scholar]
  22. Malkinson A. M., Siegel D., Forrest G. L., Gazdar A. F., Oie H. K., Chan D. C., Bunn P. A., Mabry M., Dykes D. J., Harrison S. D. Elevated DT-diaphorase activity and messenger RNA content in human non-small cell lung carcinoma: relationship to the response of lung tumor xenografts to mitomycin Cł. Cancer Res. 1992 Sep 1;52(17):4752–4757. [PubMed] [Google Scholar]
  23. Nederman T., Carlsson J. Penetration and binding of vinblastine and 5-fluorouracil in cellular spheroids. Cancer Chemother Pharmacol. 1984;13(2):131–135. doi: 10.1007/BF00257130. [DOI] [PubMed] [Google Scholar]
  24. Olive P. L. Detection of hypoxia by measurement of DNA damage in individual cells from spheroids and murine tumours exposed to bioreductive drugs. I. Tirapazamine. Br J Cancer. 1995 Mar;71(3):529–536. doi: 10.1038/bjc.1995.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pavlidis N., Hanauske A. R., Gamucci T., Smyth J., Lehnert M., te Velde A., Lan J., Verweij J. A randomized phase II study with two schedules of the novel indoloquinone EO9 in non-small-cell lung cancer: a study of the EORTC Early Clinical Studies Group (ECSG). Ann Oncol. 1996 Jul;7(5):529–531. doi: 10.1093/oxfordjournals.annonc.a010645. [DOI] [PubMed] [Google Scholar]
  26. Phillips R. M. Bioreductive activation of a series of analogues of 5-aziridinyl-3-hydroxymethyl-1-methyl-2-[1H-indole-4, 7-dione] prop-beta-en-alpha-ol (EO9) by human DT-diaphorase. Biochem Pharmacol. 1996 Dec 13;52(11):1711–1718. doi: 10.1016/s0006-2952(96)00521-7. [DOI] [PubMed] [Google Scholar]
  27. Phillips R. M., Hulbert P. B., Bibby M. C., Sleigh N. R., Double J. A. In vitro activity of the novel indoloquinone EO-9 and the influence of pH on cytotoxicity. Br J Cancer. 1992 Mar;65(3):359–364. doi: 10.1038/bjc.1992.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rasey J. S., Grunbaum Z., Krohn K., Nelson N., Chin L. Comparison of binding of [3H]misonidazole and [14C]misonidazole in multicell spheroids. Radiat Res. 1985 Mar;101(3):473–479. [PubMed] [Google Scholar]
  29. Robertson N., Haigh A., Adams G. E., Stratford I. J. Factors affecting sensitivity to EO9 in rodent and human tumour cells in vitro: DT-diaphorase activity and hypoxia. Eur J Cancer. 1994;30A(7):1013–1019. doi: 10.1016/0959-8049(94)90134-1. [DOI] [PubMed] [Google Scholar]
  30. Robin H., Jr, Senan S., Workman P., Graham M. A. Development and validation of a sensitive solid-phase-extraction and high-performance liquid chromatography assay for the bioreductive agent tirapazamine and its major metabolites in mouse and human plasma for pharmacokinetically guided dose escalation. Cancer Chemother Pharmacol. 1995;36(3):266–270. doi: 10.1007/BF00685859. [DOI] [PubMed] [Google Scholar]
  31. Sartorelli A. C. Therapeutic attack of hypoxic cells of solid tumors: presidential address. Cancer Res. 1988 Feb 15;48(4):775–778. [PubMed] [Google Scholar]
  32. Schellens J. H., Planting A. S., van Acker B. A., Loos W. J., de Boer-Dennert M., van der Burg M. E., Koier I., Krediet R. T., Stoter G., Verweij J. Phase I and pharmacologic study of the novel indoloquinone bioreductive alkylating cytotoxic drug E09. J Natl Cancer Inst. 1994 Jun 15;86(12):906–912. doi: 10.1093/jnci/86.12.906. [DOI] [PubMed] [Google Scholar]
  33. Schipper H., Turley E. A., Baum M. A new biological framework for cancer research. Lancet. 1996 Oct 26;348(9035):1149–1151. doi: 10.1016/S0140-6736(96)06184-3. [DOI] [PubMed] [Google Scholar]
  34. Scourides P. A., Brownlee R. T., Phillips D. R., Reiss J. A. Application of analytical and semi-preparative high-performance liquid chromatography to anthracyclines and bis-anthracycline derivatives. J Chromatogr. 1984 Apr 20;288(1):127–136. doi: 10.1016/s0021-9673(01)93687-8. [DOI] [PubMed] [Google Scholar]
  35. Simpson-Herren L., Noker P. E. Diversity of penetration of anti-cancer agents into solid tumours. Cell Prolif. 1991 Jul;24(4):355–365. doi: 10.1111/j.1365-2184.1991.tb01164.x. [DOI] [PubMed] [Google Scholar]
  36. Smitskamp-Wilms E., Hendriks H. R., Peters G. J. Development, pharmacology, role of DT-diaphorase and prospects of the indoloquinone EO9. Gen Pharmacol. 1996 Apr;27(3):421–429. doi: 10.1016/0306-3623(95)00118-2. [DOI] [PubMed] [Google Scholar]
  37. Sutherland R. M. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science. 1988 Apr 8;240(4849):177–184. doi: 10.1126/science.2451290. [DOI] [PubMed] [Google Scholar]
  38. Sutherland R. M., Durand R. E. Radiation response of multicell spheroids--an in vitro tumour model. Curr Top Radiat Res Q. 1976 Jan;11(1):87–139. [PubMed] [Google Scholar]
  39. Sutherland R. M., Eddy H. A., Bareham B., Reich K., Vanantwerp D. Resistance to adriamycin in multicellular spheroids. Int J Radiat Oncol Biol Phys. 1979 Aug;5(8):1225–1230. doi: 10.1016/0360-3016(79)90643-6. [DOI] [PubMed] [Google Scholar]
  40. Sutherland R. M., Sordat B., Bamat J., Gabbert H., Bourrat B., Mueller-Klieser W. Oxygenation and differentiation in multicellular spheroids of human colon carcinoma. Cancer Res. 1986 Oct;46(10):5320–5329. [PubMed] [Google Scholar]
  41. THOMLINSON R. H., GRAY L. H. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer. 1955 Dec;9(4):539–549. doi: 10.1038/bjc.1955.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vaupel P., Kallinowski F., Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 1989 Dec 1;49(23):6449–6465. [PubMed] [Google Scholar]
  43. Walton M. I., Workman P. Pharmacokinetics and bioreductive metabolism of the novel benzotriazine di-N-oxide hypoxic cell cytotoxin tirapazamine (WIN 59075; SR 4233; NSC 130181) in mice. J Pharmacol Exp Ther. 1993 May;265(2):938–947. [PubMed] [Google Scholar]
  44. West G. W., Weichselbaum R., Little J. B. Limited penetration of methotrexate into human osteosarcoma spheroids as a proposed model for solid tumor resistance to adjuvant chemotherapy. Cancer Res. 1980 Oct;40(10):3665–3668. [PubMed] [Google Scholar]
  45. Wilson W. R., Whitmore G. F., Hill R. P. Activity of 4'-(9-acridinylamino)methanesulfon-m-anisidide against Chinese hamster cells in multicellular spheroids. Cancer Res. 1981 Jul;41(7):2817–2822. [PubMed] [Google Scholar]
  46. Workman P., Binger M., Kooistra K. L. Pharmacokinetics, distribution, and metabolism of the novel bioreductive alkylating indoloquinone EO9 in rodents. Int J Radiat Oncol Biol Phys. 1992;22(4):713–716. doi: 10.1016/0360-3016(92)90509-g. [DOI] [PubMed] [Google Scholar]
  47. Workman P., Stratford I. J. The experimental development of bioreductive drugs and their role in cancer therapy. Cancer Metastasis Rev. 1993 Jun;12(2):73–82. doi: 10.1007/BF00689802. [DOI] [PubMed] [Google Scholar]
  48. van der Vijgh W. J., Maessen P. A., Pinedo H. M. Comparative metabolism and pharmacokinetics of doxorubicin and 4'-epidoxorubicin in plasma, heart and tumor of tumor-bearing mice. Cancer Chemother Pharmacol. 1990;26(1):9–12. doi: 10.1007/BF02940286. [DOI] [PubMed] [Google Scholar]

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