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

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.

Homology Modeling of Human γ-Butyric Acid Transporters and the Binding of Pro-Drugs 5-Aminolevulinic Acid and Methyl Aminolevulinic Acid Used in Photodynamic Therapy

“…Whereas studies have suggested that the PDT pro-drug ALA is a GAT substrate [16], [17], studies regarding MAL are more ambiguous as this compound seemingly is transported via GAT in some cell types but not in others [15], [17]. Molecular insight into the binding interactions of GABA, ALA and MAL in the central substrate binding site of the four GAT subtypes may help shed light on this question.…”

“…In adenocarcinoma WiDr and LM3 cells, studies have indicated that MAL is transported via non-polar amino acid transporters rather than GAT [15], [18]. MAL uptake was also recently suggested to be via GATs and other amino acid transporters in rat peripheral DRG sensory neurons [16] and in human A431 and CCD skin cells [17].…”

A review of BF-200 ALA for the photodynamic treatment of mild-to-moderate actinic keratosis

“…The drawback of this approach lies in the biological need to have the ester cleaved by cellular enzymes before ALA can enter the heme biosynthetic pathway. Thus, some ALA esters, especially MAL, have been shown to induce less PpIX compared with ALA after the same incubation time [57][58][59]. Additionally, the concentration of short-chained ALA-esters (C1-C3), which is required to induce half-maximal PpIX accumulation, was clearly higher in several cell lines than the corresponding concentration of ALA [56].…”

“…Pain during the illumination has received much attention in research and pain mechanisms were discussed in some detail elsewhere [57,86,87].…”

Assessing daylight & low-dose rate photodynamic therapy efficacy, using biomarkers of photophysical, biochemical and biological damage metrics in situ

“…Despite ALA-PDT being highly effective to treat AKs and non-melanoma skin cancers, patients often report moderate to severe pain associated to the treatment [7–10], and this has been viewed as one of the more problematic issues in acceptance of the treatment. The source of pain in ALA-PDT is believed to be from protoporphyrin IX (PpIX) production or accumulation in nerve endings [11] which leads to damage during illumination. This pain has been related to the PpIX concentration in AK lesions [12].…”