Biomedical subjects

Adriana Casas

Publications and source records attributed to Adriana Casas.

12 recordsLinked to original sources

PubMed2006

Study of the mechanisms of uptake of 5-aminolevulinic acid derivatives by PEPT1 and PEPT2 transporters as a tool to improve photodynamic therapy of tumours.

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.

Aminolevulinic Acid↗

PubMed2006

Distribution of 5-aminolevulinic acid derivatives and induced porphyrin kinetics in mice tissues.

PURPOSE: Porphyrins synthesised from 5-aminolevulinic acid (ALA) have been successfully used for the photodiagnosis and photodynamic treatment of cancer. To find a more efficient pro-photosensitiser, we synthesised two ALA esters: R,S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester (THP-ALA) and ALA-Undecanoyl ester (Und-ALA). METHODS: In mice bearing a subcutaneous mammary adenocarcinoma, we studied the distribution of the porphyrins formed from these esters in tissues after systemic administration, to establish if these esters are retained in any specific tissue, which could potentially be targeted for photodynamic treatment with ALA derivatives. We also investigated the topical use of these esters. RESULTS: After systemic administration, tumour and skin overlying tumour porphyrin levels were lower from the ALA esters than from ALA. Other tissues such as liver, colon, kidney, skin and spleen also accumulated less porphyrins from the esters, showing that there is no specific retention of the esters in these tissues. However, the brain was the only organ that synthesised more porphyrins from THP-ALA than from ALA. The kinetics of porphyrin synthesis from ALA esters is comparable to those from ALA in almost all tissues, showing that esterases activities are not limiting the availability of the hydrolysed ALA. Both THP-ALA and Und-ALA, applied topically on the skin over the tumour, exhibited higher selectivity than ALA for the site of application, whereas the amount of tumour porphyrin was the same from ALA and THP-ALA but lower from Und-ALA. CONCLUSIONS: THP-ALA may be useful for the treatment of brain tumours after systemic administration, whereas THP-ALA and Und-ALA may be used more suitable for the treatment of superficial tumours due to their higher selectivity.

Aminolevulinic Acid↗

PubMed2006

Photodynamic therapy: regulation of porphyrin synthesis and hydrolysis from ALA esters.

Photodynamic therapy (PDT) is a tool for the treatment of certain cancerous and pre-cancerous conditions. The natural precursor of porphyrins 5-aminolevulinic acid (ALA) has been extensively used as a pro-photosensitiser in PDT. ALA's poor permeability has been enhanced by chemical esterification with aliphatic alcohols. Some of the ALA esters proved to be more efficient than ALA for porphyrin synthesis. In the present work we studied the nature of porphyrin synthesis regulation from the ALA esters Hexyl-ALA (He-ALA) and R,S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester (THP-ALA) in an adenocarcinoma cell line. We found that He-ALA is incorporated into the cells at a higher rate, followed by THP-ALA and ALA, whereas ALA and ALA esters efflux at the same rate mediated by passive diffusion. Although ALA entrance to the cell might be regulatory at low concentrations, ALA derivative uptake is not a limiting factor. At high concentrations, the regulation of ALA conversion into porphyrins is driven by the enzyme porphobilinogenase, whereas ALA esters hydrolysis is regulated by esterases. The key conclusion of this contribution is that the use of ALA esters has to be limited to low concentrations where no regulation on porphyrin synthesis takes place.

Adenocarcinoma↗

PubMed2006

Mechanisms of 5-aminolevulinic acid ester uptake in mammalian cells.

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.

Aminolevulinic Acid↗

PubMed2006

Use of ALA and ALA derivatives for optimizing ALA-based photodynamic therapy: a review of our experience.

5-Aminolevulinic acid (ALA)-based PDT has been gaining increased attention in the last ten years, and become an approved treatment modality for some cancers and other diseases. Different approaches to enhance this therapeutic modality are in progress, including the development of several drug delivery systems and the use of more lipophilic ALA derivatives. This paper focuses on our experience in this field.

Aminolevulinic Acid↗

PubMed2006

Aminolevulinic acid derivatives and liposome delivery as strategies for improving 5-aminolevulinic acid-mediated photodynamic therapy.

Photodynamic Therapy employing 5-aminolevulinic acid (ALA) as a precursor of the photosensitizer Protoporphyrin IX has become a promising approach to treat superficial cancers. However, the hydrophilic nature of the ALA molecule somewhat limits the penetration through the skin as well as all cell membranes. Different attempts are currently under investigation to enhance ALA penetration, such as the development of new synthetic and more lipophilic molecules derived from ALA and the incorporation of ALA into lipophilic vehicles such as liposomes. Among the new synthesized molecules, we can find ALA esters, ALA aminoacid derivatives and ALA dendrimers. In general, there is consensus that the promising results obtained in vitro with ALA esters cannot be reproduced in vivo. However, ALA methyl ester (1) has been widely used for treatment of skin malignancies and ALA hexyl ester (15) proved to be more powerful than ALA in bladder imaging. ALA aminoacid derivatives have been designed to use specific cellular aminopeptidases to targeting tumors, and it was shown that they can be metabolized to ALA with some specificity.

Aminolevulinic Acid↗

PubMed2006

Tumor cell lines resistant to ALA-mediated photodynamic therapy and possible tools to target surviving cells.

We isolated and characterized cell lines resistant to aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) derived from a murine adenocarcinoma and studied cross resistance with other injuries. The most resistant clones were numbers 4 and 8, which exhibited 6.7- and 4.2-fold increase in resistance respectively. Several characteristics were altered in these clones. A 2-fold increase in cell volume, higher cell spreading, and a more fibroblastic, dendritic pattern, were the morphology features that led us to think they could have different adhesive, invasive or metastatic phenotypes. The amount of porphyrins synthesized per cell in the resistant clones was similar to the parental line but, when it was expressed per mg protein, there was a 2-fold decrease, with a higher proportion of hydrophilic porphyrins. These cells were not cross-resistant to photosensitization with Benzoporphyrin derivative and Merocyanine 540, but exhibited a slight resistance to exogenous protoporphyrin IX treatment. Both clones displayed higher protein content and increased number of mitochondria, together with a higher oxygen consumption. The distinctive features found in the resistant lines led as to think how to exploit the changes induced by PDT treatment to target surviving cells. Those hypoxic cells can be also a preferential target of bioreductive drugs and hypoxia-directed gene therapy, and would be sensitive to treatment with other photosensitizers.

Aminolevulinic Acid↗

PubMed2005

Sensitivity to ALA-PDT of cell lines with different nitric oxide production and resistance to NO cytotoxicity.

In this work, we studied the in vitro interactions between aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) and nitric oxide (NO), as well as the interactions between ALA, porphyrins and some NO donors and precursors. We employed three murine adenocarcinoma cell lines: LM2, which does not produce NO; LM3, which produces NO, and LM3-SNP, a variant of LM3 resistant to NO producing the same amount of NO as the parental. We did not find cross-resistance between NO-induced cytotoxicity and ALA-PDT. In spite of the lower porphyrin synthesis, LM2 cells show the highest sensitivity to ALA-PDT. However, we hypothesised that this is not related to the lack of endogenous NO production, because modulation of NO levels did not modify the response to PDT in any of the cell lines. Two unexpected results were found: the enhancement of NO production from the donor sodium nitroprusside (SNP) induced by ALA in both cells and medium, and the inhibition by ALA of NO production from arginine. We also found that SNP strongly protected the cells from ALA-PDT by impairing porphyrin biosynthesis as a consequence of an inhibition of the enzyme ALA dehydratase. We were not able to evaluate the action of NO derived from SNP because of the unexpected porphyrin impairment. On the other hand, impairment of NO from Arginine driven by ALA, although not modulating in vitro the ALA-PDT response, by increasing in vivo blood flow, may be contributing to the mechanism of tumour cures.

Adenocarcinoma↗

PubMed2005

No cross-resistance between ALA-mediated photodynamic therapy and nitric oxide.

Photodynamic therapy (PDT) interactions with nitric oxide (NO) are not well understood. In this work, we attempted to elucidate whether NO cytotoxicity and PDT from aminolevulinic acid (ALA) have independent cell damage mechanisms. We employed the murine mammary adenocarcinoma cell line LM3 and its NO-resistant variant LM3-SNP obtained after successive exposures to sodium nitroprusside (SNP). No cross-resistance was found between NO cytotoxicity and ALA-PDT; LM3-SNP cells were not more resistant to ALA-PDT than the parental line, instead they were more sensitive. We also induced resistance to ALA-PDT in LM3-SNP cells after multiple cycles of photodynamic treatment. We isolated two clones, identified as Clon 1 and Clon 3, which were 9.2 and 12.5 times more resistant to ALA-PDT than the parental lines, showing that resistance to NO did not interfere in the development of PDT resistance. In addition, the sensitivity to NO decreased in Clon 1 and increased in Clon 3, but they did not show any modifications in NO production. All the cell lines have similar GSH content and GSH transferases activities. However, GSSG content is markedly lower in LM3-SNP, Clon 1, and Clon 3 compared to parental LM3 line and consequently GSH/GSSG ratios are also higher. Our results suggest that different degrees of NO resistance of tumours would not correlate with resistance to PDT.

Adenocarcinoma↗

PubMed2005

Aminolevulinic acid: from its unique biological function to its star role in photodynamic therapy.

Porphyrins are molecules essential for life. They are involved in the key processes of photosynthesis and respiration. The biosynthesis of tetrapyrroles in all living cells occurs through several steps where the formation of aminolevulinic acid (ALA) is the first committed intermediate. Two alternative routes for the formation of ALA have been proposed: one involves the condensation of Succinyl CoA and glycine catalyzed by ALA synthetase taking place in the mitochondria, and the second one is the so called 5-carbon route, occurring in the stroma of plastids. Eight molecules of ALA are used in the formation of protoporphyrin IX. Specific deficiencies in one of the enzymes of the heme pathway produce the porphyrias. In the acute porphyrias, the pathogenesis of the neurological dysfunction is attributed to the accumulation of ALA. Fluorescent and photosensitizing properties of protoporphyrin accumulated after the exogenous administration of ALA, can be used to visualize and destroy malignant cells in the so-called photodynamic diagnosis (PDD) and photodynamic therapy (PDT) of cancer. Many clinical ALA-PDT applications to malignant and non-malignant pathologies are currently in use. Different approaches to enhance ALA penetration in cells are under investigation, including the use of more lipophilic ALA derivatives and studies of the transport mechanisms of ALA. ALA has also been proposed to be used as a biodegradable herbicide, as an insecticide and as a plant growth regulator.

Aminolevulinic Acid↗

PubMed2004

A method for separating ALA from ALA derivatives using ionic exchange extraction.

Photodynamic therapy using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX is a recent approach to detect and treat some malignancies. The use of lipophilic derivatives of ALA has been exploited in the last years to enhance ALA penetration. In this paper, we describe the application of the Mauzerall and Granick's method [J. Biol. Chem. 219 (1956) 435] to the quantification of ALA derivatives. We also describe the employment of reusable ion-exchange chromatographic columns for separating mixtures of ALA and ALA derivatives present in biological samples. The relation between 555 nm absorbance and ALA or ALA derivative concentration was linear up to 100 nmol/ml and the limit of detection of ALA and ALA derivatives was 1 nmol per ml. We employed a Dowex 50 X8 hydrogen form resin to separate ALA from the derivatives. Whereas 90+/-4% of the total ALA was eluted using sodium acetate, only 3-9% of the ALA derivatives was recovered. Only upon exposure of the resin to a high HCl concentration, the ALA derivatives were completely released. We employed this new method for the separation of ALA from ALA derivatives in cells exposed to different ALA compounds.

Aminolevulinic Acid↗

PubMed2002

Rational design of 5-aminolevulinic acid derivatives aimed at improving photodynamic therapy.

5-aminolevulinic acid (ALA) is the first intermediate in heme biosynthesis and is therefore a precursor of protoporphyrin IX (PpIX). PpIX is used as an endogenous photosensitizer in photodynamic therapy (PDT). Several chemical modifications have been made, both on the amino and carboxyl groups of ALA to induce higher PpIX production and photosensitisation. Esterification of ALA with aliphatic lineal and cyclic alcohols was found to reduce the amount of ALA required for photosensitization. Esterification by aliphatic alcohols with carbohydrate chains equal or lower than C4 leads to porphyrin accumulation lower than ALA, whereas equal or longer than C6 chains leads to greater synthesis of porphyrin. A branch point in the alcohol located next to the site of ester cleavage limits access of the esters to the esterase active site, resulting in lower PpIX production. ALA esters of the polyethylenglycol family can induce high levels of PpIX, with some selectivity for endothelial cells toward tumor cells. On the basis of the differential expression of some aminopeptidases in tumor vasculature when compared to normal vasculature, some ALA-pseudopeptides were synthesized. In a rational design of ALA derivatives, the transport mechanism of these aminoacids into the cell is central. Due to the similar characteristics between ALA and GABA transport, a novel approach for designing new ALA derivatives which could penetrate more easily into tumoral cells, would be to take into account the structures of the inhibitors of GABA transport.

Aminolevulinic Acid↗