?ALA dipeptides derivatives for their use in Photodynamic Therapy

VALLECORSA P.; DI VENOSA G; MAMONE L; GIUNTINI F.; GÁNDARA L; BATLLE A; ROSSETTI MV; MACROBERT A; EGGLESTON I.; CASAS, A.

Congreso; International Congress of Porphyrins and Porphyrias; 2013

ALA dipeptides derivatives for their use in Photodynamic Therapy. Pablo Vallecorsa, G. Di Venosaa, L. Mamonea, Francesca Giuntinib, L. Gándaraa, A. Batllea, A. Rossetti MV, A.J. MacRobertd, Ian Egglestonb & A. Casasa   Photodynamic Therapy (PDT) is a non-thermal technique for inducing tissue damage with light following administration of a light-activated photosensitising drug which can be selectively retained in malignant or diseased lesions relative to normal adjacent tissue In last years, 5-aminolaevulinic acid (ALA)-mediated PDT has become one of the most promising fields in PDT research. ALA is the pro-drug of the photosensitiser Protoporphyrin IX (PpIX).  The efficacy of ALA-PDT is somewhat limited by the hydrophilic nature of the molecule, leading to poor penetration through certain malignant tissues. An attractive way to obtain ALA prodrugs that have both improved physicochemical properties and can selectively release ALA in specific cell lines is to incorporate ALA into a short peptide derivative. The aim of this work was test two novel ALA dipeptides derivatives, AcLeuALAMe and AcPheALAMe in different cell lines, for their use in PDT.  The cell lines employed were F3II and LM3 mammary carcinomas with different invasion phenotypes, PAM212 normal keratinocytes, and B16 melanoma, and PpIX was evaluated fluorimetrically after chemical extraction and was expressed as fluorescence units (FU). PpIX synthesised from ALA was as follows: PAM212>LM3>F3II,>B16. And porphyrins from ALA reached plateau values employing 0.4-0.6 mM concentrations in all the cell lines, (showing) PAM212: 30 FU/105 cells, LM3; 15  FU/105 cells, F3II: 10 FU/105 cells and B16: 5 FU/105 cells. On the other hand, porphyrins synthesized from ALA dipeptides were reached employing 0.001 mM and 0.05 mM from Phe-ALA and Leu-ALA respectively, in the four cell lines, whereas maximal PpIX synthesis reached was similar to ALA. The results demonstrate that 2 orders of magnitude lower concentrations are needed as compared to ALA to synthesise plateau porphyrin values in tumour and non-tumour cell lines.  Application of PDT treatment employing 0.05 mM of derivatives not always led to lethal doses 50 (LD50) proportional to porphyrin synthesis: PAM212: 34,2 mJ/cm2 (Phe-ALA and Leu-ALA), F3II: 24.7  mJ/cm2 (Phe-ALA and Leu-ALA), LM3: 20 mJ/cm2 (Phe-ALA and Leu-ALA), B16: 13.3 and 17.1 mJ/cm2 for Phe-ALA and Leu-ALA respectively. Employing equimolar doses, ALA did not induce any PDT damage in any of the cell lines.  It is worth to note that PAM212, which is the best PpIX producer, is the most resistant to ALA dipetides treatment, showing that the cell type is a keypoint in resistance to photodamage. Uptake mechanism studies suggest that ALA dipeptides are partly taken up by cells by passive transport. Mice skin explants exposed to either 0.1 and 1 mM ALA and ALA-peptides, induced equal porphyrin synthesis (around 15 ug porphyrins/g tissue), thus showing that in vitro penetration of the ALA derivatives through the skin is different in vivo as compared to in vitro models.