Improved Peptide Prodrugs of 5-ALA for PDT: Rationalization of Cellular Accumulation and Protoporphyrin IX Production by Direct Determination of Cellular Prodrug Uptake and Prodrug Metabolization

Abstract

Twenty-seven dipeptide derivatives of general structure Ac-Xaa-ALA-OR were synthesized as potential prodrugs for 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT). Xaa is an alpha-amino acid, chosen to provide a prodrug with appropriately tailored lipophilicity and water solubility. Although no simple correlation is observed between downstream production of protoporphyrin IX (PpIX) in PAM212 keratinocytes and HPLC-derived descriptors of compound lipophilicity, quantification of prodrug uptake reveals that most of the dipeptides are actually more efficiently accumulated than ALA in PAM212 and also A549 and Caco-2 cell lines. Subsequent ALA release is the limiting factor, which emphasizes the importance of decoupling prodrug uptake and intracellular metabolization when assessing the efficacy of ALA derivatives for PDT. In agreement with PpIX fluorescence studies, at a concentration of 0.1 mM, l-Phe derivatives 4m and 4o, and l-Leu, l-Met, and l-Glu derivatives 4f, 4k, and 4u, exhibit significantly enhanced photoxicity in PAM212 cells compared to ALA.

Activity of phosphatase-sensitive 5-aminolevulinic acid prodrugs in cancer cell lines

“…29,30 Although different strategies have been employed in order to protect the 5-amino group of 5-ALA while keeping some pharmacological activity, they did not really provide any promising candidates for further development. 31,32 Our approach to protect the amino group in 5-ALA from degradation by integration of phosphatase-sensitive moieties aims at providing the stability needed for the systemic administration of 5-ALA whilst keeping the toxicity profile low. As it was previously reported, both protease-sensitive prodrugs are better tolerated in chick embryos than ALA-Hex.…”

The Use of Dipeptide Derivatives of 5-Aminolaevulinic Acid Promotes Their Entry to Tumor Cells and Improves Tumor Selectivity of Photodynamic Therapy

“…In previous work, we have shown that both Ac-Phe-ALA-Me and Ac-Leu-ALA-Me penetrate PAM212 cells more easily than ALA itself (38,40) and that ALA dipeptides entry to the cells is partly driven by passive transport, whereas He-ALA, which is more lipophilic, penetrates mainly through passive diffusion (40).…”

“…It has been previously shown that Ac-Phe-ALA-Me and Ac-Leu-ALA-Me are efficient at inducing PpIX in PAM212 cells and in pig skin explants (38,40). N-acetyl termination appears to play an important role in metabolic processes leading to the production of PpIX from such compounds, whereas masking the C-terminus as a methyl ester does not exert a major effect (40).…”

“…A relatively recent development in ALA-PDT concerns the preparation of peptide-based ALA prodrugs, and we (37,38,47) and others (35,36) have described the synthesis and evaluation of short ALA peptide derivatives in which either the amino or the carboxyl functions of the latter are masked, thereby providing improved physical properties and the potential for cell line-specific ALA release, according to which peptidases are expressed.…”

“…ALA-HCl and MTT were from Sigma Chem Co. Ac-Leu-ALA-Me and Ac-Phe-ALA-Me, were synthesized according to the method described by Rogers and colleagues (37) and Giuntini and colleagues (38). Stock solutions were prepared by dissolving at 10 mmol/L in 0.01 mol/L HCl and buffered with 0.01 mol/L NaOH before use.…”