Tunable phosphatase-sensitive stable prodrugs of 5-aminolevulinic acid for tumor fluorescence photodetection

Abstract

5-aminolevulinic acid (5-ALA) has been at the forefront of small molecule based fluorescence-guided tumor resection and photodynamic therapy. 5-ALA and two of its esters received marketing authorization but suffer from several major limitations, namely low stability and poor pharmacokinetic profile. Here, we present a new class of 5-ALA derivatives aiming at the stabilization of 5-ALA by incorporating a phosphatase sensitive group, with or without self-cleavable linker. Compared to 5-ALA hexyl ester (5-ALA-Hex), these compounds display an excellent stability under acidic, basic and physiological conditions. The activation and conversion into the 5-ALA is controlled and can be structure-tailored. The prodrugs display reduced acute toxicity compared to 5-ALA-Hex with superior dose response profiles of protoporphyrin IX synthesis and fluorescence intensity in human glioblastoma cells in vitro. Clinically relevant fluorescence kinetics in vivo shown in U87Mg glioblastoma spheroid tumor model in chick embryos provide a solid basis for their further development and translation to clinical fluorescence guided tumor resection and photodynamic therapy.

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

“…Moreover, the acute in vivo toxicity studies showed a 5-fold increase in the tolerated dose of P-ALA-Hex compared to ALA-Hex. 21 In this study we compared PSI-ALA-Hex and P-ALA-Hex to ALA-Hex by assessing the efficacy of the novel prodrugs to induce the formation of PpIX in five different cancer cell lines.…”

“…On the other hand, a higher dose than 1.00 mM of P-ALA-Hex is needed to induce the optimal cell response. This is most likely a consequence of different activation rates between the two phosphatase-sensitive prodrug classes needed for the conversion to 5-ALA. 21 The phenyl-phospho group of P-ALA-Hex is not an optimal substrate for alkaline phosphatases and has a very narrow concentration range for optimal activity hence the higher concentrations needed for activity. 33 Furthermore, this study revealed that T24 and A549 cells produce significantly less PpIX in comparison to PC3 and MCF7 when exposed to 5-ALA prodrugs.…”

“…20 In order to circumvent the problem of poor drug-like properties of 5-ALA and its esters, we recently reported new 5-ALA prodrugs where the amino group of ALA-Hex was modified with a phosphatase-sensitive linker. 21 The presence of phosphatase activity in various tissues and their upregulated expression in certain types of cancer might provide a solution for a broader clinical application of 5-ALA PDT and PD. [22][23][24][25] Phospho-self-immolative 5-ALA (PSI-ALA-Hex) and phospho-5-ALA (P-ALA-Hex) keep the lipophilicity of ALA-Hex which facilitates their uptake into cells compared to 5-ALA while having reduced acute toxicity and improved stability over a wide range of pH values.…”

Advanced Photosensitizer Activation Strategies for Smarter Photodynamic Therapy Beacons

“…The molecule's zwitterionic character at physiological conditions contributes to its poor bioavailability with only 0.1 % of administered doses reaching their target tissue in the brain . Phosphate‐conjugated 5‐ALA derivatives were developed to enhance the stability of 5‐ALA, establishing an alkaline phosphatase (AP)‐activated beacon system for downstream protoporphyrin IX (PpIX) fluorescence and PDT . As AP is overexpressed in certain types of tumours, cleavage activation of the stabilized 5‐ALA derivative beacons by AP yielded preferential accumulation of 5‐ALA in tumour cells, biasing PpIX synthesis and accumulation to this target tissue.…”

Advanced Photosensitizer Activation Strategies for Smarter Photodynamic Therapy Beacons

“…The molecule's zwitterionic character at physiological conditions contributes to its poor bioavailability with only 0.1 % of administered doses reaching their target tissue in the brain . Phosphate‐conjugated 5‐ALA derivatives were developed to enhance the stability of 5‐ALA, establishing an alkaline phosphatase (AP)‐activated beacon system for downstream protoporphyrin IX (PpIX) fluorescence and PDT . As AP is overexpressed in certain types of tumours, cleavage activation of the stabilized 5‐ALA derivative beacons by AP yielded preferential accumulation of 5‐ALA in tumour cells, biasing PpIX synthesis and accumulation to this target tissue.…”