Pharmaceutical Research

, Volume 21, Issue 10, pp 1871–1879

Phototoxicity of 5-Aminolevulinic Acid in the HeLa Cell Line as an Indicative Measure of Photodynamic Effect After Topical Administration to Gynecological Lesions of Intraepithelial Form

  • Paul A. McCarron
  • Ryan F. Donnelly
  • Brendan F. Gilmore
  • A. David Woolfson
  • Raymond McClelland
  • Agnieszka Zawislak
  • John H. Price
Article

Abstract

Purpose. The depth-resolved pattern of aminolevulinic acid (ALA) concentration in excised vaginal tissue was determined after in vitro application of an ALA-loaded bioadhesive patch. From this data, the tissue concentration of ALA achievable at a specified depth from the surface could be related to the concentration needed to elicit a photodynamic effect in a model gynecological tumor cell line (HeLa).

Methods. Excised vaginal tissue was mounted in a modified Franz diffusion cell and exposed to a water-soluble, ALA-loaded, bioadhesive patch. After a period of time, the tissue was cryostatically sectioned and the stratal concentration of radiolabeled ALA determined using scintillation spectroscopy. HeLa cells were cultured in media containing specific concentrations of ALA and exposed to standard photodynamic protocols of light exposure.

Results. An ALA concentration of 65.6 mM was achievable at 2.375 mm from the tissue surface after application of ALA-loaded patch. The photodynamic effectiveness of this concentration was demonstrated in HeLa with exposure to concentrations exceeding 1.0 mM ALA bringing about reductions in viable cell numbers by 90%. An enhancement of PpIX production using adjunctive EDTA over the clinically relevant 4 h application time interval was shown to be minimal in HeLa. Instead, PpIX production was more closely correlated with ALA concentration, with 100 mM ALA producing approximately 3100 ng PpIX mg-1 protein in the same time period.

Conclusions. Given that vaginal intraepithelial neoplasias can extend to 2.0 mm from the lesion surface, the ALA permeability derived from a bioadhesive patch is sufficient to induce photosensitization suitable for light induced destruction at deep sites of this type of lesion.

aminolevulinic acid bioadhesive HeLa intraepithelial photodynamic 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

REFERENCES

  1. 1.
    T. R. Moore, R. C. Reiter, R. W. Rebar, and V. V. Baker. Gynecology and Obstetrics: A Longitudinal Approach,W.B. Saunders, New York, 1993.Google Scholar
  2. 2.
    H. A. Kurwa, R. J. Barlow, and S. Neill. Single-episode photodynamic therapy and vulval intraepithelial neoplasia type III resistant to conventional therapy. Br. J. Dermatol. 143:10401042(2000).Google Scholar
  3. 3.
    E. A. Joura. Epidemiology, diagnosis and treatment of vulvar intraepithelial neoplasia. Curr. Opin. Obstet. Gyn. 14:3943(2002).Google Scholar
  4. 4.
    P. Hillemanns, M. Untch, C. Dannecker, R. Baumgartner, H. Stepp, J. Diebold, H. Weingandt, F. Prove, and M. Korell. Photodynamic therapy of vulvar intraepithelial neoplasia using 5-aminolevulinic acid. Int. J. Cancer 85:649653(2000).Google Scholar
  5. 5.
    A. Zawislak and J. H. Price. Vulval intraepithelial neoplasia a rare but distressing condition. Northern Ireland Medical Review 4:3839 (2002).Google Scholar
  6. 6.
    G. Damasus-Awatai and T. Freeman-Wang. Human papilloma virus and cervical screening. Curr. Opin. Obstet. Gyn. 15:473477(2003).Google Scholar
  7. 7.
    N. J. Nwabineli and J. M. Monaghan. Vaginal epithelial abnormalities in patients with CIN: clinical and pathological features and management. Brit. J. Obstet. Gynaec. 98:2529 (1991).Google Scholar
  8. 8.
    A. B. MacLean. Benign Diseases of the Vagina, Cervix and Ovary. In K. D. Edmonds (ed.), Dewhurst's Textbook of Obstetrics and Gynecology for Postgraduates Sixth Edition.Blackwell Science, Malden, MA, 1999, pp. 582601.Google Scholar
  9. 9.
    P. A. McCarron, A. D. Woolfson, D. F. McCafferty, J. H. Price, H. Sidhu, and G. I. Hickey. Cytotoxicity of 5-fluorouracil re-leased from a bioadhesive patch into uterine cervical tissue. Int. J. Pharm. 151:6974 (1997).Google Scholar
  10. 10.
    T. E. Carson, W. J. Hoskins, and J. F. Wurzel. Topical 5-fluorouracil in the treatment of carcinoma in situof the vulva. Obstet. Gynecol. 47:59S62S (1976).Google Scholar
  11. 11.
    J. G. Moore and N. F. Hacker. Essentials of Obstetrics and Gynecology.Churchill Livingstone, Philadelphia, pp. 3681 (1998).Google Scholar
  12. 12.
    Q. Peng, K. Berg, J. Moan, M. Kongshaug, and J. M. Nesland. 5-Aminolevulinic acid-based photodynamic therapy: principles and experimental research. Photochem. Photobiol. 65:235251 (1997).Google Scholar
  13. 13.
    F. S. De Rosa and M. V. L. B. Bentley. Photodynamic therapy of skin cancers: sensitizers, clinical studies and future directives. Pharm. Res. 17:14471455 (2000).Google Scholar
  14. 14.
    J. C. Kennedy and R. H. Pottier. Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy. J. Photoch. Photobio. B. 14:275292(1992).Google Scholar
  15. 15.
    E. R. Gallegos, I. D. Rodriguez, L. A. M. Guzman, and A. J. P. Zapata. In vitro study of biosynthesis of protoporphyrin IX in-duced by delta-aminolevulinic acid in normal and cancerous cells of the human cervix. Arch. Med. Res. 30:163170(1999).Google Scholar
  16. 16.
    Y. Ninomiya, Y. Itoh, S. Tajima, and A. Ishibashi. In vitro and in vivo expression of protoporphyrin IX induced by lipophilic 5-aminolevulinic acid derivatives. J. Dermatol. Sci. 27:114120 (2001).Google Scholar
  17. 17.
    A. Frenga, P. Stentella, F. DiRenzi. L. DelleChiaie, L. Cipriano, and A. Pachi. Assessment of self application of four topical agents on genital warts. J. Eur. Acad. Dermatol. Venereol. 8:112 115 (1997).Google Scholar
  18. 18.
    B. J. Monk, C. Brewer, K. Van Nostrand, M. W. Berns, J. L. McCullough, Y. Tadir, and A. Manetta. Photodynamic therapy using topically applied dihematoporphyrin ether in the treatment of cervical intraepithelial neoplasia. Gynecol. Oncol. 64:7075 (1997).Google Scholar
  19. 19.
    P. A. McCarron, A. D. Woolfson, R. F. Donnelly, G. P. Andrews, A. Zawislak, and J. H. Price. Influence of plasticiser type and storage conditions on properties of poly(methylvinylether-co-maleic anhydride) bioadhesive films. J. Appl. Polym. Sci. 91: 15761589 (2004).Google Scholar
  20. 20.
    P. A. McCarron, R. F. Donnelly, A. D. Woolfson, and A. Zawislak. Photodynamic therapy of vulval intraepithelial neoplasia using bioadhesive patch-based delivery of aminolevulinic acid. Drug Delivery Systems and Sciences 3:5964 (2003).Google Scholar
  21. 21.
    O. B. Gadmar, J. Moan, E. Scheie, L. W. Ma, and Q. Peng. The Stability of 5-aminolevulinic acid in Solution. J. Photochem. Photobiol. B 67:187193 (2002).Google Scholar
  22. 22.
    J. Hanania and Z. Malik. The effect of EDTA and serum on endogenous porphyrin accumulation and photodynamic sensiti-zation of human K562 leukemic cells. Cancer Lett. 65:127131(1992).Google Scholar
  23. 23.
    C. Fuchs, R. Riesenberg, J. Siegert, and R. Baumgartner. pH-Dependent formation of 5-aminolevulinic acid-induced protopor-phyrin IX in fibrosarcoma cells. J. Photoch. Photobio. B. 40:4954(1997).Google Scholar
  24. 24.
    C. J. Gomer, J. V. Jester, N. J. Razum, B. C. Szirth, and L. Murphree. Photodynamic therapy of intraocular tumours. Examination of hematoporphyrin derivative distribution and long-term damage in rabbit ocular tissue. Cancer Res. 45:37183725 (1985).Google Scholar
  25. 25.
    J. Leveckis, J. L. Burn, N. J. Brown, and M. W. R. Reed. Kinetics of endogenous protoporphyrin IX induction by aminolevulinic acid: preliminary studies in the bladder. J. Urol. 152:550553 (1994).Google Scholar
  26. 26.
    P. Shatz, C. Bergeron, E. J. Wilkinson, J. Arseneau, and A. Ferenczy. Vulvar intraepithelial neoplasia and skin appendage in-volvement. Obstet. Gynecol. 74:769774 (1989).Google Scholar
  27. 27.
    J. Moan, G. Streckyte, S. Bagdonas, O. Bech, and K. Berg. Photobleaching of protoporphyrin IX in cells incubated with 5-ami-nolevulinic acid. Int. J. Cancer 70:9097 (1997).Google Scholar
  28. 28.
    W. Xiang, H. Weingandt, F. Liebmann, S. Klein, H. Stepp, R. Baumgartner, and P. Hillemanns. Photodynamic effects induced by aminolevulinic acid esters on human cervical carcinoma cells in culture. Photochem. Photobiol. 74:617623 (2001).Google Scholar
  29. 29.
    L. Wyld, M. W. R. Reed, and N. J. Brown. The influence of hypoxia and pH on aminolevulinic acid-induced photodynamic therapy in bladder cancer cells in vitro. Br. J. Cancer 77:1621 1627 (1998).Google Scholar
  30. 30.
    B. M. Moretti, S. C. Garci, C. Stella, E. Ramos, A.M. Del, and C. Batlle. 5-aminolevulinic acid transport in Saccharomyces Cerevisiae. Int. J. Biochem. 25:19171924 (1993).Google Scholar
  31. 31.
    E. Musgrove, M. Seaman, and D. Hedley. Relationship between cytoplasmic pH and proliferation during exponential growth and cellular quiescence. Exp. Cell Res. 172:6575 (1987).Google Scholar
  32. 32.
    E. Schick, R. Kaufmann, A. Ruck, A. Hainzl, and W.-H. Boehncke. On the effectiveness of photodynamic therapy. Acta Derm. Venerol. 75:276279 (1995).Google Scholar
  33. 33.
    O. Bech, K. Berg, and J. Moan. The pH dependancy of protoporphyrin IX formation in cells incubated with 5-aminolevulinic acid. Cancer Lett. 113:2529 (1997).Google Scholar
  34. 34.
    J. Moan, K. Berg, O. Gadmar, V. Iani, L. W. Ma, and P. Juzenas. The temperature dependence of protoporphyrin IX production in cells and tissues. Photochem. Photobiol. 70:669673 (1999).Google Scholar
  35. 35.
    K. Berg, H. Anholt, O. Bech, and J. Moan. The influence of iron chelators on the accumulation of protoporphyrin IX in 5-amino-levulinic acid-treated cells. Br. J. Cancer 74:688697 (1996).Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2004

Authors and Affiliations

  • Paul A. McCarron
    • 1
  • Ryan F. Donnelly
    • 1
  • Brendan F. Gilmore
    • 1
  • A. David Woolfson
    • 1
  • Raymond McClelland
    • 2
  • Agnieszka Zawislak
    • 2
  • John H. Price
    • 2
  1. 1.School of PharmacyQueens University Belfast, Medical Biology CentreBelfastUK. (e-mail
  2. 2.Department of Obstetrics and GynaecologyBelfast City HospitalBelfastUK

Personalised recommendations