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δ-Aminolevulinic acid and its methyl ester induce the formation of Protoporphyrin IX in cultured sensory neurones

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Abstract

Application of δ-aminolevulinic acid (ALA) or its methyl ester (MAL) onto cutaneous tumours increases intracellular Protoporphyrin IX (PpIX), serving as photosensitizer in photodynamic therapy (PDT). While PDT is highly effective as treatment of neoplastic skin lesions, it may induce severe pain in some patients. Here, we investigated ALA and MAL uptake and PpIX formation in sensory neurones as potential contributor to the pain. PpIX formation was induced in cultured sensory neurones from rat dorsal root ganglion by incubation with ALA or MAL. Using inhibitors of GABA transporters (GAT), a pharmacological profile of ALA and MAL uptake was assessed. GAT mRNA expression in the cultures was determined by RT-PCR. Cultured sensory neurones synthesised Protoporphyrin IX (PpIX) from extracellularly administered ALA and MAL. PpIX formation was dose- and time-dependent with considerably different kinetics for both compounds. While partial inhibition occurred using l-arginine, PpIX formation from both ALA and MAL could be fully blocked by the GABA-Transporter (GAT)-2/3 inhibitor (S)-SNAP 5114 with similar K i (ALA: 195 ± 6 μM; MAL: 129 ± 13 μM). GAT-1 and GAT-3 could be detected in sensory neurons using RT-PCR on mRNA level and using [³H]-GABA uptake on protein level. Cultured sensory neurones take up ALA and MAL and synthesize PpIX from both, enabling a direct impact of photodynamic therapy on cutaneous free nerve endings. The pharmacological profile of ALA and MAL uptake in our test system was very similar and suggests uptake via GABA and amino acid transporters.

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Abbreviations

ALA:

δ-Aminolevulinic acid

BGT-1:

Betaine-GABA transporter 1

BNPP:

Bis(p-nitrophenyl) phosphate sodium salt

DMEM:

Dulbecco’s modified Eagle’s medium

DRG:

Dorsal root ganglion

F12:

Nutrient mixture F12

GABA:

γ-Aminobutyric acid

GAT:

GABA transporter

l-Arg:

l-Arginine

MAL:

ALA-methyl ester

PpIX:

Protoporphyrin IX

RFU:

Relative fluorescence units

(S)-SNAP 5114:

1-[2-[Tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3-piperidinecarboxylic acid

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Acknowledgements

The authors are indebted to the excellent technical support by Silvia Schweer and would also like to thank Xinran Zhu, Frank Paris, Beate Schmitz and Mirella Gwarek for many helpful discussions and inspirations. Special thanks to Anna Suedkamp and Mirella Gwarek for their kind help with the PpIX assay system and the radiolabelled GABA uptake experiments.

This work was supported with grants by the DFG (German research council) Graduiertenkolleg 736 and the Ruhr-University Bochum research school.

Conflict of interest

One of the authors (HL) is closely linked as general manager to Biofrontera Bioscience GmbH which has a PDT drug containing ALA in the European approval process.

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  1. Department of Animal Physiology, Faculty of Biology and Biotechnology, Ruhr-University Bochum, Building ND 5/122, Universitätsstr. 150, 44780, Bochum, Germany

    B. Novak, R. Schulten & H. Lübbert

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  1. B. Novak
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Correspondence to H. Lübbert.

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Novak, B., Schulten, R. & Lübbert, H. δ-Aminolevulinic acid and its methyl ester induce the formation of Protoporphyrin IX in cultured sensory neurones. Naunyn-Schmiedeberg's Arch Pharmacol 384, 583–602 (2011). https://doi.org/10.1007/s00210-011-0683-1

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