Cancer Chemotherapy and Pharmacology

, Volume 67, Issue 4, pp 775–781 | Cite as

Biodistribution of PVP-hypericin and hexaminolevulinate-induced PpIX in normal and orthotopic tumor-bearing rat urinary bladder

  • Joachim Vandepitte
  • Ben Van Cleynenbreugel
  • Klaudia Hettinger
  • Hendrik Van Poppel
  • Peter A. M. de WitteEmail author
Original Article



In this preclinical study, we examined the biodistribution of hypericin formulated as its water-soluble PVP-hypericin complex in the different layers (urothelium, submucosa, muscle) of a normal rat bladder and a rat bladder bearing a malignant urothelium composed of syngeneic AY-27 tumor cells. The results were compared with the biodistribution of hexaminolevulinate (HAL)-induced protoporphyrin IX (PpIX).


Freshly prepared PVP-hypericin and HAL solutions were instilled in both normal as well as tumor-bearing rat bladders. Following instillation, bladders were removed and snap frozen in liquid nitrogen. Fluorescence of PVP-hypericin or PpIX-induced HAL was measured in the bladder layers and quantified using image analysis software.


The results of these experiments show that PVP-hypericin (30 μM) accumulated about 3.5-fold more in malignant urothelial tissue when compared to normal urothelium, whereas PpIX accumulated to the same extent in malignant and normal urothelium, both after intrabladder instillation of 8 or 16 mM HAL. Besides, PVP-hypericin and PpIX accumulated selectively in the urothelium with a tumor-to-muscle ratio of 30.6 for PVP-hypericin and 3.7–8.3 for 16 and 8 mM HAL, respectively.


This study shows that PVP-hypericin appears to have great potential as a photodynamic agent against non-muscle-invasive bladder cancers after intravesical administration, with a limited risk of affecting the deeper layers of the bladder.


Bladder cancer Orthotopic animal model PVP-hypericin HAL 







Protoporphyrin IX


3,3′-Dioctadecyloxacarbocyanine perchlorate


Hematoxylin and eosin


Non-muscle-invasive bladder cancer



This work was supported by grants awarded by Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (F.W.O-Vlaanderen), by the K.U.Leuven (onderzoekstoelage) and by Sanochemia Pharmazeutika AG (Austria).


  1. 1.
    Kirkali Z, Chan T, Manoharan M, Algaba F, Busch C, Cheng L, Kiemeney L, Kriegmair M, Montironi R, Murphy WM (2005) Bladder cancer: epidemiology, staging and grading, and diagnosis. Urol Int Consult Bladder Tumors 66:4–34Google Scholar
  2. 2.
    Kausch I, Doehn C, Jocham D (2006) Recent improvements in the detection and treatment of nonmuscle-invasive bladder cancer. Expert Rev Anticancer Ther 6:1301–1311PubMedCrossRefGoogle Scholar
  3. 3.
    Hendricksen K, Witjes JA (2007) Current strategies for first and second line intravesical therapy for nonmuscle invasive bladder cancer. Curr Opin Urol 17:352–357PubMedCrossRefGoogle Scholar
  4. 4.
    Hungerhuber E, Stepp H, Kriegmair M, Stief C, Hofstetter A, Hartmann A, Knuechel R, Karl A, Tritschler S, Zaak D (2007) Seven years’ experience with 5-aminolevulinic acid in detection of transitional cell carcinoma of the bladder. Urology 69:260–264PubMedCrossRefGoogle Scholar
  5. 5.
    Jocham D, Stepp H, Waidelich R (2008) Photodynamic diagnosis in urology: state-of-the-art. Eur Urol 53:1138–1148PubMedCrossRefGoogle Scholar
  6. 6.
    Kubin A, Wierrani F, Burner U, Alth G, Grunberger W (2005) Hypericin–the facts about a controversial agent. Curr Pharm Des 11:233–253PubMedCrossRefGoogle Scholar
  7. 7.
    Kiesslich T, Krammer B, Plaetzer K (2006) Cellular mechanisms and prospective applications of hypericin in photodynamic therapy. Curr Med Chem 13:2189–2204PubMedCrossRefGoogle Scholar
  8. 8.
    D’Hallewin MA, De Witte PA, Waelkens E, Merlevede W, Baert L (2000) Fluorescence detection of flat bladder carcinoma in situ after intravesical instillation of hypericin. J Urol 164:349–351PubMedCrossRefGoogle Scholar
  9. 9.
    D’Hallewin MA, Kamuhabwa AR, Roskams T, De Witte PA, Baert L (2002) Hypericin-based fluorescence diagnosis of bladder carcinoma. BJU Int 89:760–763PubMedCrossRefGoogle Scholar
  10. 10.
    Sim HG, Lau WK, Olivo M, Tan PH, Cheng CW (2005) Is photodynamic diagnosis using hypericin better than white-light cystoscopy for detecting superficial bladder carcinoma? BJU Int 95:1215–1218PubMedCrossRefGoogle Scholar
  11. 11.
    Pytel A, Schmeller N (2002) New aspect of photodynamic diagnosis of bladder tumors: fluorescence cytology. Urology 59:216–219PubMedCrossRefGoogle Scholar
  12. 12.
    Fu CY, Ng BK, Razul SG, Chin WW, Tan PH, Lau WK, Olivo M (2007) Fluorescence detection of bladder cancer using urine cytology. Int J Oncol 31:525–530PubMedGoogle Scholar
  13. 13.
    Van De Putte M, Roskams T, Bormans G, Verbruggen A, De Witte PA (2006) The impact of aggregation on the biodistribution of hypericin. Int J Oncol 28:655–660Google Scholar
  14. 14.
    Kubin A, Loew HG, Burner U, Jessner G, Kolbabek H, Wierrani F (2008) How to make hypericin water-soluble. Pharmazie 63:263–269PubMedGoogle Scholar
  15. 15.
    Liu J, Saw CL, Olivo M, Sudhaharan T, Ahmed S, Heng PW, Wohland T (2009) Study of interaction of hypericin and its pharmaceutical preparation by fluorescence techniques. J Biomed Opt 14:014003PubMedCrossRefGoogle Scholar
  16. 16.
    Leveckis J, Burn JL, Brown NJ, Reed MW (1994) Kinetics of endogenous protoporphyrin IX induction by aminolevulinic acid: preliminary studies in the bladder. J Urol 152:550–553PubMedGoogle Scholar
  17. 17.
    Van Hillegersberg R, Van den Berg JW, Kort WJ, Terpstra OT, Wilson JH (1992) Selective accumulation of endogenously produced porphyrins in a liver metastasis model in rats. Gastroenterology 103:647–651PubMedGoogle Scholar
  18. 18.
    Rodriguez L, Batlle A, Di Venosa G, Battah S, Dobbin P, Macrobert AJ, Casas A (2006) Mechanisms of 5-aminolevulinic acid ester uptake in mammalian cells. Br J Pharmacol 147:825–833PubMedCrossRefGoogle Scholar
  19. 19.
    Schmidbauer J, Witjes F, Schmeller N, Donat R, Susani M, Marberger M (2004) Improved detection of urothelial carcinoma in situ with hexaminolevulinate fluorescence cystoscopy. J Urol 171:135–138PubMedCrossRefGoogle Scholar
  20. 20.
    Fotinos N, Campo MA, Popowycz F, Gurny R, Lange N (2006) 5-Aminolevulinic acid derivatives in photomedicine: Characteristics, application and perspectives. Photochem Photobiol 82:994–1015PubMedCrossRefGoogle Scholar
  21. 21.
    Kamuhabwa A, Agostinis P, Ahmed B, Landuyt W, van Cleynenbreugel B, van Poppel H, de Witte P (2004) Hypericin as a potential phototherapeutic agent in superficial transitional cell carcinoma of the bladder. Photochem Photobiol Sci 3:772–780PubMedCrossRefGoogle Scholar
  22. 22.
    Berger AP, Steiner H, Stenzl A, Akkad T, Bartsch G, Holtl L (2003) Photodynamic therapy with intravesical instillation of 5-aminolevulinic acid for patients with recurrent superficial bladder cancer: a single-center study. Urology 61:338–341PubMedCrossRefGoogle Scholar
  23. 23.
    Kriegmair M, Waidelich R, Lumper W, Ehsan A, Baumgartner R, Hofstetter A (1995) Integral photodynamic treatment of refractory superficial bladder cancer. J Urol 154:1339–1341PubMedCrossRefGoogle Scholar
  24. 24.
    Pervaiz S, Olivo M (2006) Art and science of photodynamic therapy. Clin Exp Pharmacol Physiol 33:551–556PubMedCrossRefGoogle Scholar
  25. 25.
    Xiao Z, McCallum TJ, Brown KM, Miller GG, Halls SB, Parney I, Moore RB (1999) Characterization of a novel transplantable orthotopic rat bladder transitional cell tumour model. Br J Cancer 81:638–646PubMedCrossRefGoogle Scholar
  26. 26.
    Kamuhabwa AA, Cosserat-Gerardin I, Didelon J, Notter D, Guillemin F, Roskams T, D’Hallewin MA, Baert L, de Witte PA (2002) Biodistribution of hypericin in orthotopic transitional cell carcinoma bladder tumors: implication for whole bladder wall photodynamic therapy. Int J Cancer 97:253–260PubMedCrossRefGoogle Scholar
  27. 27.
    El Khatib S, Didelon J, Leroux A, Bezdetnaya L, Notter D, D’Hallewin M (2004) Kinetics, biodistribution and therapeutic efficacy of hexylester 5-aminolevulinate induced photodynamic therapy in an orthotopic rat bladder tumor model. J Urol 172:2013–2017PubMedCrossRefGoogle Scholar
  28. 28.
    Kubin A, Meissner P, Wierrani F, Burner U, Bodenteich A, Pytel A, Schmeller N (2008) Fluorescence diagnosis of bladder cancer with new water soluble hypericin bound to polyvinylpyrrolidone: PVP-hypericin. Photochem Photobiol 84:1560–1563PubMedCrossRefGoogle Scholar
  29. 29.
    Jichlinski P, Guillou L, Karlsen SJ, Malmstrom PU, Jocham D, Brennhovd B, Johansson E, Gartner T, Lange N, van den Bergh H, Leisinger HJ (2003) Hexyl aminolevulinate fluorescence cystoscopy: new diagnostic tool for photodiagnosis of superficial bladder cancer–a multicenter study. J Urol 170:226–229PubMedCrossRefGoogle Scholar
  30. 30.
    Malmstrom PU, Hedelin H, Thomas YK, Thompson GJ, Durrant H, Furniss J (2009) Fluorescence-guided transurethral resection of bladder cancer using hexaminolevulinate: analysis of health economic impact in Sweden. Scand J Urol Nephrol 43:192–198PubMedCrossRefGoogle Scholar
  31. 31.
    Gronlund-Pakkanen S, Wahlfors J, Makinen K, Pakkanen TM, Talja M, Ala-Opas M, Alhava E, Moore RB (2002) The fluorescence biodistribution and kinetics of aminolevulinic acid induced protoporphyrin IX in the bladder of a rat model with orthotopic urothelial carcinoma. J Urol 167:1848–1853PubMedCrossRefGoogle Scholar
  32. 32.
    Berrahmoune S, Fotinos N, Bezdetnaya L, Lange N, Guedenet JC, Guillemin F, D’Hallewin MA (2008) Analysis of differential PDT effect in rat bladder tumor models according to concentrations of intravesical hexyl-aminolevulinate. Photochem Photobiol Sci 7:1018–1024PubMedCrossRefGoogle Scholar
  33. 33.
    Manyak MJ (1991) Photodynamic therapy: principles and urologic applications. Semin Urol 9:192–202PubMedGoogle Scholar
  34. 34.
    Harty JI, Amin M, Wieman TJ, Tseng MT, Ackerman D, Broghamer W (1989) Complications of whole bladder dihematoporphyrin ether photodynamic therapy. J Urol 141:1341–1346PubMedGoogle Scholar
  35. 35.
    Kamuhabwa AA, Roskams T, Baert L, De Witte PA (2003) Microscopic quantification of hypercin fluorescence in an orthotopic rat bladder tumor model after intravesical instillation. Int J Oncol 22:933–937PubMedGoogle Scholar
  36. 36.
    Kamuhabwa AA, Roskams T, D’Hallewin MA, Baert L, Van Poppel H, de Witte PA (2003) Whole bladder wall photodynamic therapy of transitional cell carcinoma rat bladder tumors using intravesically administered hypericin. Int J Cancer 107:460–467PubMedCrossRefGoogle Scholar
  37. 37.
    Pope AJ, Bown SG (1991) The morphological and functional changes in rat bladder following photodynamic therapy with phthalocyanine photosensitization. J Urol 145:1064–1070PubMedGoogle Scholar
  38. 38.
    Chang SC, MacRobert AJ, Bown SG (1996) Photodynamic therapy on rat urinary bladder with intravesical instillation of 5-aminolevulinic acid: light diffusion and histological changes. J Urol 155:1749–1753PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Joachim Vandepitte
    • 1
  • Ben Van Cleynenbreugel
    • 2
  • Klaudia Hettinger
    • 3
  • Hendrik Van Poppel
    • 2
  • Peter A. M. de Witte
    • 1
    Email author
  1. 1.Faculty of Pharmaceutical Sciences, Laboratory for Pharmaceutical BiologyKatholieke Universiteit LeuvenLeuvenBelgium
  2. 2.Department of UrologyUniversity Hospital LeuvenLeuvenBelgium
  3. 3.Sanochemia Pharmazeutika AGViennaAustria

Personalised recommendations