RESUMEN
The delta-amino acid 5-aminolevulinic acid (ALA), is the precursor of the endogenous photosensitiser Protoporphyrin IX (PpIX), and is currently approved for Photodynamic Therapy (PDT) of certain superficial cancers. However, ALA-PDT is not very effective in diseases in which T-cells play a significant role. Cutaneous T-cell lymphomas (CTCL) is a group of non-Hodgkin malignant diseases, which includes mycosis fungoides (MF) and Sézary syndrome (SS). In previous work, we have designed new ALA esters synthesised by three-component Passerini reactions, and some of them showed higher performance as compared to ALA. This work aimed to determine the efficacy as pro-photosensitisers of five new ALA esters of 2-hydroxy-N-arylacetamides (1f, 1 g, 1 h, 1i and 1 k) of higher lipophilicity than ALA in Myla cells of MF and HuT-78 cells of SS. We have also tested its effectiveness against ALA and the already marketed ALA methyl ester (Me-ALA) and ALA hexyl ester (He-ALA). Both cell Myla and SS cells were effectively and equally photoinactivated by ALA-PDT. Besides, the concentration of ALA required to induce half the maximal porphyrin synthesis was 209 µM for Myla and 169 µM for HuT-78 cells. As a criterion of efficacy, we calculated the concentration of the ALA derivatives necessary to induce half the plateau porphyrin values obtained from ALA. These values were achieved at concentrations 4 and 12 times lower compared to ALA, according to the derivative used. For He-ALA, concentrations were 24 to 25 times lower than required for ALA for inducing comparable porphyrin synthesis in both CTCL cells. The light doses for inducing 50% of cell death (LD50) for He-ALA, 1f, 1 g, 1 h and 1i were around 18 and 25 J/cm2 for Myla and HuT-78 cells respectively, after exposure to 0.05 mM concentrations of the compounds. On the other hand, the LD50s for the compound 1 k were 40 and 57 J/cm2 for Myla and HuT-78, respectively. In contrast, 0.05 mM of ALA and Me-ALA did not provoke photokilling since the concentration employed was far below the porphyrin saturation point for these compounds. Our results suggest the potential use of ALA derivatives for topical application in PDT treatment of MF and extracorporeal PDT for the depletion of activated T-cells in SS.
Asunto(s)
Ácido Aminolevulínico/análogos & derivados , Fármacos Fotosensibilizantes/farmacología , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Humanos , Luz , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Linfocitos/fisiología , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
Photodynamic therapy (PDT) is an effective procedure for the treatment of lesions diseases based on the selectivity of a photosensitising compound with the ability to accumulate in the target cell. Atherosclerotic plaque is a suitable target for PDT because of the preferential accumulation of photosensitisers in atherosclerotic plaques. Dendrimers are hyperbranched polymers conjugated to drugs. The dendrimers of ALA hold ester bonds that inside the cells are cleaved and release ALA, yielding PpIX production. The dendrimer 6m-ALA was chosen to perform this study since in previous studies it induced the highest porphyrin macrophage: endothelial cell ratio (Rodriguez et al. in Photochem Photobiol Sci 14:1617-1627, 2015). We transformed Raw 264.7 macrophages to foam cells by exposure to oxidised LDLs, and we employed a co-culture model of HMEC-1 endothelial cells and foam cells to study the affinity of ALA dendrimers for the foam cells. In this work it was proposed an in vitro model of atheromatous plaque, the aim was to study the selectivity of an ALA dendrimer for the foam cells as compared to the endothelial cells in a co-culture system and the type of cell death triggered by the photodynamic treatment. The ALA dendrimer 6m-ALA showed selectivity PDT response for foam cells against endothelial cells. A light dose of 1 J/cm2 eliminate foam cells, whereas less than 50% of HMEC-1 is killed, and apoptosis cell death is involved in this process, and no necrosis is present. We propose the use of ALA dendrimers as pro-photosensitisers to be employed in photoangioplasty to aid in the treatment of obstructive cardiovascular diseases, and these molecules can also be employed as a theranostic agent.
Asunto(s)
Ácido Aminolevulínico/farmacología , Apoptosis/efectos de los fármacos , Células Espumosas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Ácido Aminolevulínico/química , Animales , Línea Celular , Técnicas de Cocultivo , Células Espumosas/fisiología , Humanos , Macrófagos/fisiología , Ratones , Fármacos Fotosensibilizantes/químicaRESUMEN
The use of endogenous protoporphyrin IX generated after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). However, the bioavailability of ALA is limited by its hydrophilic properties and limited cell uptake. A promising approach to optimize the efficacy of ALA-PDT is to deliver ALA in the form of prodrugs to mask its hydrophilic nature. The aim of this work was to evaluate the potential of two ALA dipeptide derivatives, N-acetyl terminated leucinyl-ALA methyl ester (Ac-Leu-ALA-Me) and phenylalanyl-ALA methyl ester (Ac-Phe-ALA-Me), for their use in PDT of cancer, by investigating the generation of protoporphyrin IX in an oncogenic cell line (PAM212-Ras), and in a subcutaneous tumor model. In our in vitro studies, both derivatives were more effective than ALA in PDT treatment, at inducing the same protoporphyrin IX levels but at 50- to 100-fold lower concentrations, with the phenylalanyl derivative being the most effective. The efficient release of ALA from Ac-Phe-ALA-Me appears to be consistent with the reported substrate and inhibitor preferences of acylpeptide hydrolase. In vivo studies revealed that topical application of the peptide prodrug Ac-Phe-ALA-Me gave greater selectivity than with ALA itself, and induced tumor photodamage, whereas systemic administration improved ALA-induced porphyrin generation in terms of equivalent doses administered, without induction of toxic effects. Our data support the possibility of using particularly Ac-Phe-ALA-Me both for topical treatment of basal cell carcinomas and for systemic administration. Further chemical fine-tuning of this prodrug template should yield additional compounds for enhanced ALA-PDT with potential for translation to the clinic.
Asunto(s)
Ácido Aminolevulínico/uso terapéutico , Dipéptidos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Fotoquimioterapia , Ácido Aminolevulínico/administración & dosificación , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dipéptidos/administración & dosificación , Dipéptidos/química , Dipéptidos/farmacología , Humanos , Cinética , Masculino , Ratones Endogámicos BALB C , Microscopía Fluorescente , Neoplasias/patología , Porfirinas/biosíntesis , Proteínas ras/metabolismoRESUMEN
The use of endogenous protoporphyrin IX (PpIX) after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). However the efficacy of ALA-PDT is sub-optimal for thicker tumours and improved ALA delivery and therapeutic response are required. We have investigated the conjugation of ALA to a second-generation dxcendrimer for enhancing porphyrin synthesis in vitro and in vivo in a murine tumour model using systemic i.p. administration. In vitro, the dendrimer was more efficient than ALA for porphyrin synthesis at low concentrations in good correlation with higher cellular ALA dendrimer accumulation. In vivo, the porphyrin kinetics from ALA exhibited an early peak between 3 and 4 h in most tissues, whereas the dendrimer induced sustained porphyrin production for over 24 h and basal values were not reached until 48 h after administration. Integrated porphyrin accumulation from the dendrimer and ALA, at equivalent molar ratios, was comparable showing that the majority of ALA residues were liberated from the dendrimer. The porphyrin kinetics appear to be governed by the rate of enzymatic cleavage of ALA from the dendrimer, which is consistent with in vitro results. ALA dendrimers may be useful for metronomic PDT, and multiple low-dose ALA-PDT treatments.
Asunto(s)
Ácido Aminolevulínico/farmacología , Dendrímeros/química , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Porfirinas/metabolismo , Adenocarcinoma/tratamiento farmacológico , Ácido Aminolevulínico/administración & dosificación , Ácido Aminolevulínico/química , Ácido Aminolevulínico/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Colorantes/metabolismo , Dendrímeros/síntesis química , Relación Dosis-Respuesta a Droga , Inyecciones Subcutáneas , Masculino , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Peso Molecular , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Protoporfirinas/biosíntesis , Relación Estructura-Actividad , Sales de Tetrazolio/metabolismo , Tiazoles/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Endogenous porphyrin accumulation after administration of 5-aminolevulinic acid is employed in photodynamic therapy of tumours. Due to its low membrane permeability, esterified 5-aminolevulinic acid derivatives less hydrophilic than the parental compound are under investigation. Knowledge of the mechanisms of 5-aminolevulinic acid derivatives uptake into target cells is essential to understand and improve photodynamic therapy and useful in the design of new derivatives with better affinity and with higher selectivity for tumour cells in specific tissues. The aim of this work was to assess the interaction of 5-aminolevulinic acid derivatives with the intestinal PEPT1 and renal transporter PEPT2 expressed in Pichia pastoris yeasts. We found that Undecanoyl, Hexyl, Methyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters and the dendron 3m-ALA inhibited (14)C-5-aminolevulinic acid uptake by PEPT2. However, only the Undecanoyl ester inhibited 5-aminolevulinic acid uptake by PEPT1. We have also found through a new developed colorimetric method, that Hexyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters display more affinity than 5-aminolevulinic acid for PEPT2 whereas none of the compounds surpass 5-aminolevulinic acid affinity for PEPT1. In addition, the Undecanoyl ester binds with high affinity to the membranes of PEPT2 and PEPT1-expressing yeasts and to the control yeasts. The main finding of this work was that some derivatives have the potential to improve 5-aminolevulinic acid-based photodynamic therapy by increased efficiency of transport into cells expressing PEPT2 such as kidney, mammary gland, brain or lung whereas in tissues expressing exclusively PEPT1 the parent 5-aminolevulinic acid remains the compound of choice.
Asunto(s)
Ácido Aminolevulínico/metabolismo , Fotoquimioterapia/métodos , Simportadores/metabolismo , Ácido Aminolevulínico/análogos & derivados , Organismos Modificados Genéticamente/metabolismo , Transportador de Péptidos 1 , Pichia/metabolismoRESUMEN
The porphyrin precursor 5-aminolevulinic acid (ALA) is being widely used in photodynamic therapy of cancer. Improvement in ALA delivery has been sought through the use of ALA derivatives, in particular the esterification of ALA with aliphatic alcohols, which in certain cases can improve cellular penetration and selectivity. ALA uptake systems appear to be distinctive for each cell type. The LM3 mammary adenocarcinoma cell line takes ALA up by BETA transporters. In this work, we investigated ALA derivative transport systems through the inhibition of radiolabelled ALA uptake in the LM3 cells. We also performed inhibition studies of gamma-aminobutyric acid (GABA) uptake. The more lipohilic ALA derivatives hexyl-ALA and undecanoyl-ALA inhibit ALA uptake, whereas methyl-ALA, R, S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester and the dendron aminomethane tris methyl 5-ALA does not inhibit ALA uptake. A similar pattern was found for GABA, except that the dendron inhibited GABA uptake. However, hexyl-ALA and undecanoyl-ALA are not taken up by BETA transporters, but by simple diffusion, although they still inhibit ALA uptake by binding to the cell membrane. These results show that different modifications to the ALA molecule lead to different uptake mechanisms. Whereas ALA is taken up by BETA transporters, none of the ALA derivatives shares the same mechanism. Knowledge of the mechanisms of ALA derivatives entry into the cells is essential to understand and improve ALA-mediated PDT and to the design of new ALA derivatives that may be taken up at a higher rate than ALA.
Asunto(s)
Ácido Aminolevulínico/metabolismo , Ácido Aminolevulínico/análogos & derivados , Ácido Aminolevulínico/antagonistas & inhibidores , Animales , Transporte Biológico Activo/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Difusión , Femenino , Antagonistas del GABA/farmacología , Ratones , Porfirinas/biosíntesis , Ácido Succínico/metabolismo , Temperatura , Sales de Tetrazolio , Tiazoles , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Photodynamic therapy is a treatment for malignant and certain non-malignant lesions that involves administration of a photosensitising drug. The use of 5-aminolaevulinic acid-induced porphyrins has become one of the most active fields of photodynamic therapy research. Since the efficacy of the treatment is somewhat limited by the hydrophilic nature of 5-aminolaevulinic acid, chemical modifications such as esterification with aliphatic alcohols have been made to induce higher porphyrin production. In an attempt to improve delivery of 5-aminolaevulinic acid to tissue, we have investigated the use of dendritic derivatives capable of bearing several drug molecules. The aim of this work was to evaluate in vivo and in vitro the efficacy of the first generation dendron, aminomethane tris-methyl 5-aminolaevulinic acid (containing three 5-aminolaevulinic acid residues) in terms of porphyrin synthesis. In LM3 cells, the dendron induced similar porphyrin levels compared to equimolar concentrations of 5-aminolaevulinic acid. Although the dendron is taken up with comparable efficiency to 5-aminolaevulinic acid, we found that there is only partial intracellular liberation of 5-aminolaevulinic acid residues. Both systemic and topical administration of the dendron to tumour-bearing mice induced higher porphyrin levels than the widely investigated hexyl ester derivative in most tissues studied, although it was not possible to surpass the levels induced by 5-aminolaevulinic acid. In conclusion, aminomethane tris-methyl 5-aminolaevulinic acid is capable of being taken up by cells efficiently, and liberating the active residues, although in vivo it was not possible to improve upon the efficacy of 5-aminolevulinic acid. Studies of accessibility and regulation of the esterases are needed to improve the design of these dendritic derivatives.