Doubly protected ester prodrug of 5-aminolevulinic acid for enhanced cell uptake and photoinactivation

Photodynamic therapy (PDT) employs a molecular photosensitizer combined with light to induce localized cellular toxicity. The natural product, 5-aminolevulinic acid (5-ALA), is used in clinical PDT as a precursor drug molecule that is converted inside cancer cells to the photosensitizer, protoporphyrin IX (PpIX). However, the therapeutic efficacy of 5-ALA is limited by its relatively short chemical lifetime under physiological conditions and its poor bioavailability. To obviate these limitations, we have developed 5-eMAL as a doubly protected ester prodrug of 5-ALA with greater chemical stability and improved capacity to enter cells. Intracellular esterase activity converts 5-eMAL into free 5-ALA for subsequent biosynthesis into PpIX. Cell culture experiments with three different cancer cell lines (HepG2, 4T1, A549) showed greatly enhanced production of intracellular PpIX when the cells were treated with 5-eMAL compared to cells treated with 5-ALA. Inhibition of the membrane peptide transporters in HepG2 cells did not lower PpIX formation when the cells were treated with 5-eMAL suggesting that cellular uptake of 5-eMAL is independent of the membrane transporters. Cell photoinactivation experiments using blue light irradiation produced much greater cell death when the cells were pre-treated with 5-eMAL compared to cells that were pretreated with 5-ALA. Cell microscopy showed that the photoinactivated cells exhibited blebbing and were co-stained by the fluorescent indicators Annexin V FITC and Propidium Iodide suggesting the formation of necrotic cells. Cell studies using different nanoparticle formulations of 5-eMAL also produced high levels of intracellular PpIX highlighting the potential for in-vivo application.