Polymeric iron chelators for enhancing 5‐aminolevulinic acid‐induced photodynamic therapy

5‐Aminolevulinic acid (5‐ALA) is an amino acid that can be metabolized into a photosensitizer, protoporphyrin IX (PpIX) selectively in a tumor cell, permitting minimally invasive photodynamic diagnosis/therapy. However, some malignant tumor cells have excess intracellular labile iron and facilitate the conversion of PpIX into heme, which compromises the therapeutic potency of 5‐ALA. Here, we examined the potential of chelation of such unfavorable intratumoral labile iron in photodynamic therapy (PDT) with 5‐ALA hydrochloride, using polymeric iron chelators that we recently developed. The polymeric iron chelator efficiently inactivated the intracellular labile iron in cultured cancer cells and importantly enhanced the accumulation of PpIX, thereby improving the cytotoxicity upon photoirradiation. Even in in vivo study with subcutaneous tumor models, the polymeric iron chelator augmented the intratumoral accumulation of PpIX and the PDT effect. This study suggests that our polymeric iron chelator could be a tool for boosting the effect of 5‐ALA‐induced PDT by modulating tumor microenvironment.     

The monitoring of the accumulation of protoporphyrin IX in Walker tumours by subcutaneous administration of delta-aminolevulinic acid

Photodynamic therapy with protoporphyrin IX induced by delta-aminolevulinic acid (ALA) is mainly applied for the treatment of human superficial skin cancer. In this paper we present our study on photodynamic therapy (PDT) of the implanted Walker tumours using subcutaneous administration of ALA to improve the availability of ALA in the skin. We determined the accumulation and localization of protoporphyrin IX (PpIX) after subcutaneous administration of different concentrations of ALA in a physiological saline solutions, using fluorescence imaging technique. The results obtained indicate that PpIX accumulation depends on the concentration of ALA. The temporal behavior of PpIX fluorescence has shown a clear demarcation of tumoural zone depending on the post-administration time and the administrated concentration of the ALA solution. Further studies are needed to confirm these encouraging results and to define the PDT protocols using subcutaneous administration of ALA solution     

Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters

Aminolevulinic acid (ALA), ALA methylester (ALA-Me) and ALA hexylester (ALA-Hex) were topically applied for 5 and 20 hr, respectively, on normal skin of mice. The distribution of protoporphyrin IX (PpIX) induced in 7 different tissues by these drugs was determined either by spectrofluorometric measurements with an optical fibre probe or by chemical extraction of PpIX from the tissues. The results from these 2 types of measurements were compared. Both methods showed that ALA and the esters induced similar amounts of PpIX at the skin spot where they were applied and that the esters produced much less PpIX at remote skin spots (i.e., spots outside the location where the drugs were applied) than ALA did, notably after 20 hr application. After 20 hr of drug application ALA produced much more PpIX in liver, intestine and lungs than the esters did. In contrast with the direct fluorescence measurements, the extraction method showed detectable amounts of PpIX in liver, intestine and lung after application of the esters, notably of ALA-Me. The discrepancy is probably related to the fact that the pigmented tissues absorb light and, therefore, the direct fluorescence readings are misleading. Notably in the liver, which contains high concentration of light-absorbing pigments, very weak direct fluorescence was seen. In no case there was any accumulation of PpIX in muscle tissue nor in brain. The esters seem to penetrate less into the circulation than ALA, and PpIX formed by them in the skin is faster cleared than PpIX formed from ALA. This is also true after oral and i.p. administration of the drugs.     

Oral versus intravenous administration of 5-aminolaevulinic acid for photodynamic therapy.

Endogenously synthesised protoporphyrin IX (PpIX) following the administration of 5-amino-laevulinic acid (ALA) is an effective photosensitiser for photodynamic therapy (PDT). Following intravenous administration, PpIX accumulates predominantly in mucosa of hollow viscera and on light exposure, mucosal ablation results with relative sparing of the submucosa and muscularis layers. Oral administration is effective with ALA in contrast to conventional exogenous photosensitisers such as haematoporphyrin derivative and phthalocyanines. Oral administration of ALA is also simpler, safer, cheaper and more acceptable to patients. We studied the porphyrin sensitisation kinetics profile in the stomach, colon and bladder in normal rats following enterally and parenterally administered ALA using microscopic fluorescence photometric studies of frozen tissue sections. Mucosal cells in all three organs exhibit higher fluorescence levels as compared with underlying smooth muscle following both intravenous and oral administration. Peak concentration were seen 4 h after sensitisation at the highest doses used (200 mg kg-1 i.v., 400 mg kg-1 oral), and slightly earlier with lower doses. The temporal kinetics of both routes of administration were similar although a higher oral dose was required to achieve the same tissue concentration of PpIX. The highest level of fluorescence was achieved in the gastric mucosa and in decreasing levels, colonic and bladder mucosa. A similar degree of mucosal selectivity was achieved in each organ with each route of administration but an oral dose in excess of 40 mg kg-1 was required to achieve measurable PpIX sensitisation. In a pilot clinical study, two patients with inoperable rectal adenocarcinomas were given 30 mg kg-1 and one patient with sigmoid colon carcinoma was given 60 mg kg-1 ALA orally. Serial biopsies of normal and tumour areas were taken over the subsequent 24 h. Fluorescence microscopy of these specimens showed maximum accumulation of PpIX 4 to 6 h after administration of 30 mg kg-1 ALA. There was greater PpIX accumulation in tumour than adjacent normal mucosa in two patients. Preferential PpIX accumulation in tumour was greater in the patient receiving 60 mg kg-1 ALA.     

Protoporphyrin IX production and its photodynamic effects on glioma cells, neuroblastoma cells and normal cerebellar granule cells in vitro with 5-aminolevulinic acid and its hexylester

5-Aminolevulinic acid (ALA) has shown promising in photodynamic detection and therapy of brain tumor. However, the knowledge on selective accumulation of ALA-induced protoporphyrin IX (PpIX) in brain tumor tissue is still fragment. In the present study, the rat C6 glioma cells, human SK-N-SH neuroblastoma cells, and rat normal cerebellar granule cells (RCG) were used to investigate the PpIX production and photocytotoxicity in vitro. The C6 cells and SK-N-SH cells showed a similar kinetics of PpIX accumulation after exposure to ALA or ALA hexyl ester (ALA-H), with an initial increase up to 6–8 h and then saturated. In the case of RCG cells, the PpIX accumulation slowly increased until 12 h studied. However the cellular PpIX content was more than 10 times higher in the C6 and SK-N-SH cells than that in the normal RCG cells. The intracellular localization of PpIX measured by cofocal laser scanning microscopy was in same pattern in the C6 glioma cells and RCG normal cells with a diffuse cytoplasm distribution. The sensitivity of the C6 cells and SK-N-SH cells to ALA or ALA-H PDT was similar. It appears that ALA-H could achieve similar or slightly better results than ALA with respect to PpIX production and photoinactivation of cells, although a 10 times lower concentration of ALA-H was used.     

Photodetection of early human bladder cancer based on the fluorescence of 5-aminolaevulinic acid hexylester-induced protoporphyrin IX: a pilot study.

Exogenous administration of 5-aminolaevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of protoporphyrin IX (PpIX) in photodynamic therapy (PDT) and fluorescence photodetection (PD). Recently, results have shown that the chemical modification of ALA into its more lipophilic esters circumvents limitations of ALA-induced PpIX like shallow penetration depth into deep tissue layers and inhomogeneous biodistribution and enhances the total PpIX formation. The present clinical pilot study assesses the feasibility and the advantages of a topical ALA ester-based fluorescence photodetection in the human bladder. In this preliminary study 5-aminolaevulinic acid hexylester (h-ALA) solutions, containing concentrations ranging from 4 to 16 mM, were applied intravesically to 25 patients. Effects of time and drug dose on the resulting PpIX fluorescence level were determined in vivo with an optical fibre-based spectrofluorometer. Neither local nor systemic side-effects were observed for the applied conditions. All conditions used yielded a preferential PpIX accumulation in the neoplastic tissue. Our clinical investigations indicate that with h-ALA a twofold increase of PpIX fluorescence intensity can be observed using 20-fold lower concentrations as compared to ALA.     

Protoporphyrin IX occurs naturally in colorectal cancers and their metastases

Colorectal cancers exhibit a red fluorescence. The nature of the responsible fluorophore and its eventual diagnostic potential were investigated. Thirty-three consecutive colorectal resection specimen, 32 of which with histologically confirmed cancer, and a total of 1053 palpable mesenteric nodes were fluorimetrically characterized ex vivo. Furthermore, frozen material from 28 patients was analyzed, selected for the availability of primary tumor material and metastatic tissue, e.g., lymphatic and liver metastases from the same patient. Biochemical characterization was carried out through chemical extraction and reversed phase high-performance liquid chromatography. The fluorescence spectra of tissues, tissue extracts, and standard solutions of porphyrins were determined using a pulsed solid-state laser system for excitation and an imaging polychromator, together with an intensified CCD camera for time-delayed observation. Protoporphyrin IX (PpIX) was identified as the predominant fluorophore in primary tumors and their metastases. The fluorophore occurred in the absence of necrosis and in sterile locations. In untreated cases (n = 24), PpIX fluorescence discriminates metastatically involved lymph nodes from all other palpable nodes with a sensitivity of 62% at a specificity of 78% (P < 0.0001). After neoadjuvant treatment of rectal cancer, the PpIX fluorescence level of the primary tumors was reduced and a discrimination of lymph nodes based on PpIX-fluorescence was impossible. We conclude that colorectal cancer metastases accumulate diagnostic levels of endogenous PpIX as a result of a tumor-specific metabolic alteration.     

Quantitative studies of the kinetics of 5-aminolaevulinic acid-induced fluorescence in bladder transitional cell carcinoma.

Photodynamic therapy is a potential treatment for superficial bladder cancer that utilizes photosensitizer drugs, which are activated by light to cause tissue destruction. However, first-generation photosensitizers cause prolonged phototoxicity, have poor tumour specificity and can accumulate within detrusor muscle, resulting in permanent loss of bladder capacity following treatment. A newer drug, called 5-aminolaevulinic acid (ALA), generates a sensitizer called protoporphyrin IX (PpIX) in situ and has been shown, qualitatively, to be more tumour specific. The fluorescence kinetics of ALA-induced PpIX was investigated in patient biopsies of bladder tumour, normal urothelium and detrusor muscle, both in vitro after incubation of specimens in ALA-rich culture medium for various times and in vivo after instillation of intravesical ALA before endoscopic resection. The fluorescence in tumour tissue was twice that of normal urothelium in vitro and up to tenfold in vivo. There was little ALA-induced fluorescence in detrusor muscle, both in vitro and in vivo. Most importantly, no patients experienced phototoxicity or other adverse events following intravesical instillation of ALA.     

Selective accumulation of ALA-induced PpIX and photodynamic effect in chemically induced hepatocellular carcinoma

The possibility of 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) for liver cancer was investigated using a chemically induced hepatocellular carcinoma (HCC) model. Endogenously synthesised protoporphyrin IX (PpIX) following the administration of ALA is an effective photosensitiser for PDT. We determined the fluorescence intensity of PpIX in HCC and nontumoral tissue in the liver. 5-Aminolaevulinic acid was intravenously injected to male Fisher rats with HCC at a dose of 500 mg x kg(-1), and the fluorescence intensity in each tissue sample excised from liver was measured with a spectrofluorometer at 1, 3 and 6 h after administration. Fluorescence intensity was at a peak of 3 h after administration in both HCC and nontumoral tissue. The accumulation of PpIX in HCC was higher than that in the nontumoral tissue at 1 h (P<0.001) and 3 h (P<0.05) after ALA administration. Based on these results, PDT was performed on HCC at 3 h after 500 mg x kg(-1) ALA administration before laser irradiation of 30 J per tumour. Antitumour effect was more evident in HCC than in the nontumoral tissue surrounding HCC. These findings suggest the possibility to detect HCC by fluorescence and to treat HCC by light.     

On the pharmacokinetics of topically applied 5-aminolevulinic acid and two of its esters

The kinetics of protoporphyrin IX (PpIX) production in normal tissues and WiDr tumors of mice were studied after topical application of 5-aminolevulinic acid (ALA) and its methyl ester and hexyl ester. ALA and ALA esters were applied on a spot of 1.0 cm diameter on normal skin and on skin overlaying tumors. PpIX production was studied by fluorescence measurements. ALA induced PpIX not only on the spot of application but also on remote skin areas. This was not found for the ALA esters. They produced PpIX only on the spot of application. Thus, ALA, but neither its esters nor PpIX, is passing into the circulation. The time needed for ALA to enter the circulation through normal skin was about 5 hr. Even when looking normal, the skin overlaying tumors was more permeable to ALA than normal skin. Thus, when applied on the tumor, ALA induced PpIX on remote skin areas without any lag phase. Mainly, PpIX was found in all tissues although small amounts of a porphyrin with an excitation peak at about 400 nm, supposedly uroporphyrin and/or coproporphyrin, were found, notably in remote skin areas. An altered stratum corneum of the skin overlaying tumors probably contributes to the tumor-selectivity, although in the present tumor system less PpIX was found in tumors than in muscles. This is probably related to biochemical and physiological conditions in this particular tumor, since i.p. injection of ALA also leads to less PpIX formation in the tumor than in skin/muscle tissue. Nevertheless, it seems evident that ALA can diffuse more easily from the skin surface and down to the vasculature in the tumor than in the normal tissue and that this leads to a higher concentration of PpIX in the tumor than would have been found if the physiological factors relevant for drug diffusion were the same for tumors as for skin/muscles.     

Photodynamic therapy involves an antiangiogenic mechanism and is enhanced by ferrochelatase inhibitor in urothelial carcinoma

The purpose of the present study was to investigate the mechanism of photodynamic therapy (PDT) supplemented with exogenously added 5‐aminolevulinic acid (ALA) on human urothelial cancer (UC). Moreover, we aimed to determine whether the therapeutic effects of ALA‐based PDT (ALA‐PDT) for UC could be enhanced by deferoxamine (DFX), an inhibitor of ferrochelatase. The efficiency of ALA‐PDT on these cells was analyzed using flow cytometry and the type of cell death was also assessed. The ALA‐PDT promoting effect of DFX was examined on both UC cells and human umbilical vein endothelial cells (HUVEC). The ALA‐PDT decreased levels of mitochondrial membrane potential and induced cell death mainly via apoptosis in these cells. Moreover, inhibition of ferrochelatase by DFX led to an increase of protoporphyrin IX (PpIX) accumulation and enhanced the effect of ALA‐PDT on UC cells. We further investigated the effect of DFX on in vivo PDT with a tumor‐bearing animal model and found that DFX efficiently enhanced tumor cell apoptosis. ALA‐PDT induced death of neovascular endothelial cells in tumors but did not affect small vessel endothelial cells in normal tissues surrounding the tumor. Furthermore, DFX enhanced inhibition of neovascularization. These results demonstrated ALA‐PDT dominantly induced apoptosis over necrosis by direct action on UC as well as via antiangiogenic action on neovacular endothelial cells, suggesting that the therapeutic damage by ALA‐PDT could be kept to a minimum in the surrounding normal tissues. In addition, increased accumulation of PpIX by DFX could enhance this effectiveness of ALA‐PDT.     

Comparative effect of ALA derivatives on protoporphyrin IX production in human and rat skin organ cultures.

Samples of human and rat skin in short-term organ culture exposed to ALA or a range of hydrophobic derivatives were examined for their effect on the accumulation of protoporphyrin IX (PpIX) measured using fluorescence spectroscopy. With the exception of carbobenzoyloxy-D-phenylalanyl-5-ALA-ethyl ester the data presented indicate that, in normal tissues, ALA derivatives generate protoporphyrin IX more slowly than ALA, suggesting that they are less rapidly taken up and/or converted to free ALA. However, the resultant depot effect may lead to the enhanced accumulation of porphyrin over long exposure periods, particularly in the case of ALA-methyl ester or ALA-hexyl ester, depending on the applied concentration and the exposed tissue. Addition of the iron chelator, CP94, greatly increased PpIX accumulation in human skin exposed to ALA, ALA-methyl ester and ALA-hexyl ester. The effect in rat skin was less marked.     

Differentiation-dependent photodynamic therapy regulated by porphobilinogen deaminase in B16 melanoma

Protoporphyrin IX (PpIX) synthesis by malignant cells is clinically exploited for photodiagnosis and photodynamic therapy following administration of 5-aminolevulinic acid (ALA). The expression and activity of the housekeeping porphobilinogen deaminase (PBGD) was correlated to PpIX synthesis in differentiating B16 melanoma cells. Differentiation was stimulated by two inducers, butyrate and hexamethylene bisacetamide (HMBA), both of which promote the formation of typical melanosomes and melanin, as well as morphological changeover. A marked decrease in total PBGD activity and PpIX synthesis was observed following stimulation by butyrate, while HMBA induced an opposite effect. In contrast, ferrochelatase levels remained unchanged. Photodynamic inactivation of the cells undergoing differentiation was largely dependent on the PpIX accumulation, which was modulated by the two inducers butyrate and HMBA. Fluorescence immunostaining with anti-PBGD antibodies revealed a major PBGD fraction in the nucleus and a minor fraction in the cytosol. This nuclear localisation pattern was confirmed by expression of PBGD fused to green fluorescence protein. We suggest that efficient photodynamic therapy of cancer facilitated by ALA administration can be enhanced using combined therapeutic modalities.     

Production of protoporphyrin IX induced by 5-aminolevulinic acid in transplanted human colon adenocarcinoma of nude mice can be increased by ultrasound

BALB/c nude mice bearing WiDr human colon adenocarcinoma were used to determine the effect of ultrasound on the production of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) both in the tumors and in skin overlying the tumors. Ultrasound (1 MHz) with pulsed irradiation at an average intensity of 3 W/cm2 was given 10 min to the tumor area 10 min after administration of ALA (20% in an oil-in-water emulsion applied topically on the surface of the tumor for 30 min to 3 hr). An approximately 45% increase in the amount of PpIX produced by ALA in the tumors was obtained within 1 to 2 hr following ultrasound treatment. In particular, 1 hr after ultrasound treatment, the amount of PpIX in the tumors was at the same level as that 3 hr after ALA application alone. However, pulsed ultrasound irradiation for 5 min or continuous irradiation for 5 or 10 min had no significant effect on the production of PpIX by the tumor 1 hr after topical ALA application. Furthermore, in most cases, the amount of PpIX in the tumors was significantly decreased when ultrasound was given immediately before ALA application. There was no significant change in the ratio of the amount of PpIX in tumor to that in skin after ultrasound treatment. Most likely, the distribution of PpIX fluorescence in the tumors treated with ultrasound was more homogeneous than that in the tumors given ALA only. Our results provide a theoretical basis for possible clinical use of ultrasound-combined ALA or ALA based photodynamic therapy. Int. J. Cancer 78:464–469, 1998. © 1998 Wiley-Liss, Inc.     

Cell cycle phase influences tumour cell sensitivity to aminolaevulinic acid-induced photodynamic therapy in vitro.

Photodynamic therapy (PDT) is a form of cancer treatment based on the destruction of cells by the interaction of light, oxygen and a photosensitizer. Aminolaevulinic acid (ALA) is the prodrug of the photosensitizer protoporphyrin IX (PpIX). ALA-induced PDT depends on the rate of cellular synthesis of PpIX, which may vary with cell cycle phase. This study has investigated the relationship between cell cycle phase, PpIX generation and phototoxicity in synchronized and unsynchronized bladder cancer cells (HT1197). In unsynchronized cells, relative PpIX fluorescence values (arbitrary units) were significantly different between cell cycle phases after a 1-h ALA incubation (G1 24.8 +/- 0.7; S-phase, 32.7 +/- 0.8, P < 0.05; G2 35.4 +/- 0.8, P < 0.05). In synchronized cells after a 1-h ALA incubation, cells in G1 produced less PpIX than those in S-phase or G2 [6.65 +/- 1.1 ng per 10(5) cells compared with 15.5 +/- 2.1 (P < 0.05), and 8.1 +/- 1.8 ng per 10(5) cells (not significant) respectively] and were significantly less sensitive to ALA-induced PDT (% survival, G1 76.2 +/- 8.3; S-phase 49.7 +/- 4.6, P < 0.05; G2 44.2 +/- 2.4, P < 0.05). This differential response in tumour cells may have implications for clinical PDT, resulting in treatment resistance and possible failure in complete tumour response.     

Kinetics and subcellular localization of 5-ALA-induced PpIX in DHL cells via two-photon excitation fluorescence microscopy

Two-photon excitation fluorescence (TPEF) microscopy was used to measure the 5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence in follicular lymphoma DHL cells. Kinetics of 5-ALA-induced PpIX accumulation in DHL cells under various 5-ALA concentrations was studied. We found that during the course of continuous incubation with 5-ALA, the relationship between the DHL cell fluorescence signal and the incubation time showed a biphasic variation. Initially the PpIX signal increased with the incubation time and reached the maximal value at about 3 h, and then it decreased with time during the subsequent incubation period. By labeling the 5-ALA incubated DHL cells with different organelle-specific fluorescence probes: Rhodamine 123 (for mitochondria), DioC6(3) (for endoplasmic reticulum) and LysoTracker Green (for lysosomes) respectively, we found that 5-ALA-induced PpIX was primarily localized in endoplasmic reticulum and mitochondria; its concentration in the lysosome was much lower. The results suggested that 5-ALA could potentially be an effective photosensitizer in photodynamic purging of DHL cells. Two-photon excitation fluorescence microscope is a useful tool for studying 5-ALA-induced PpIX subcellular localization.     

Photobleaching during and re-appearance after photodynamic therapy of topical ALA-induced fluorescence in UVB-treated mouse skin

Photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA) seems a promising alternative for the treatment of superficial non-melanoma skin cancer and actinic keratosis. In this study, the kinetics of new PpIX fluorescence arising after a PDT treatment that had photobleached the original fluorescence were determined. Our purpose was to examine the feasibility of multiple irradiations, following a single topical ALA application, to increase PDT efficacy. In addition, photobleaching during PDT and the fluorescence spectra during and after PDT were studied. As a model we used hairless mice with and without UVB-induced skin lesions. ALA was applied to the skin for 4 hr. An illumination was delivered either immediately after application or 6 hr after the end of the application (at interval of maximum fluorescence). During PDT, the fluorescence of normal skin decreased at a faster rate than the fluorescence of the skin lesions. In the fluorescence study after PDT, the areas treated immediately post-application showed a fluorescence increase over time similar to that in non-treated areas on the same mice. A remarkable result was that the fluorescence of areas treated at maximum fluorescence increased, whereas the fluorescence of non-treated areas did not increase over time. With both treatment intervals the new fluorescence showed a characteristic PpIX spectrum. Our results demonstrate that a second illumination, when new PpIX fluorescence has been formed, may increase PDT efficacy after topical ALA application. This finding has been demonstrated previously for systemic ALA administration. Int. J. Cancer 72:110–118, 1997. © 1997 Wiley-Liss Inc.     

Methotrexate used in combination with aminolaevulinic acid for photodynamic killing of prostate cancer cells

Photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA) to drive production of an intracellular photosensitiser, protoporphyrin IX (PpIX), is a promising cancer treatment. However, ALA-PDT is still suboptimal for thick or refractory tumours. Searching for new approaches, we tested a known inducer of cellular differentiation, methotrexate (MTX), in combination with ALA-PDT in LNCaP cells. Methotrexate alone promoted growth arrest, differentiation, and apoptosis. Methotrexate pretreatment (1 mg l(-1), 72 h) followed by ALA (0.3 mM, 4 h) resulted in a three-fold increase in intracellular PpIX, by biochemical and confocal analyses. After exposure to 512 nm light, killing was significantly enhanced in MTX-preconditioned cells. The reverse order of treatments, ALA-PDT followed by MTX, yielded no enhancement. Methotrexate caused a similar relative increase in PpIX, whether cells were incubated with ALA, methyl-ALA, or hexyl-ALA, arguing against a major effect upon ALA transport. Searching for an effect among porphyrin synthetic enzymes, we found that coproporphyrinogen oxidase (CPO) was increased three-fold by MTX at the mRNA and protein levels. Transfection of LNCaP cells with a CPO-expressing vector stimulated the accumulation of PpIX. Our data suggest that MTX, when used to modulate intracellular production of endogenous PpIX, may provide a new combination PDT approach for certain cancers.     

Polymeric iron chelators for enhancing 5-aminolevulinic acid-induced photodynamic therapy

5-Aminolevulinic acid (5-ALA) is an amino acid that can be metabolized into a photosensitizer, protoporphyrin IX (PpIX) selectively in a tumor cell, permitting minimally invasive photodynamic diagnosis/therapy. However, some malignant tumor cells have excess intracellular labile iron and facilitate the conversion of PpIX into heme, which compromises the therapeutic potency of 5-ALA. Here, we examined the potential of chelation of such unfavorable intratumoral labile iron in photodynamic therapy (PDT) with 5-ALA hydrochloride, using polymeric iron chelators that we recently developed. The polymeric iron chelator efficiently inactivated the intracellular labile iron in cultured cancer cells and importantly enhanced the accumulation of PpIX, thereby improving the cytotoxicity upon photoirradiation. Even in in vivo study with subcutaneous tumor models, the polymeric iron chelator augmented the intratumoral accumulation of PpIX and the PDT effect. This study suggests that our polymeric iron chelator could be a tool for boosting the effect of 5-ALA-induced PDT by modulating tumor microenvironment.     

Photodynamic therapy using intravenous delta-aminolaevulinic acid-induced protoporphyrin IX sensitisation in experimental hepatic tumours in rats.

The efficacy of photodynamic therapy (PDT) using delta-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) sensitisation and laser light at 635 nm was investigated in the treatment of experimental hepatic tumours. The model of liver tumours was induced either by local inoculation or by administration of tumour cells through the portal vein in rats. ALA at a dose of 60 mg kg(-1) b.w. was intravenously administered 60 min before PDT. PpIX accumulation in tumour, normal liver and abdominal wall muscle was detected by means of laser-induced fluorescence (LIF). Laser Doppler imaging (LDI) was used to determine changes in the superficial blood flow in connection with PDT. Histopathological examinations were performed to evaluate the PDT effects on the tumour and the surrounding liver tissue, including pathological features in the microvascular system. The accumulation of PpIX, as monitored by LIF, showed high fluorescence intensities at about 635 nm in both the hepatic tumour tissue and normal liver and low values in the abdominal wall. LDI demonstrated that the blood flow in the treated tumour and its surrounding normal liver tissue decreased immediately after the PDT, indicating an effect on the vascular system. A large number of thrombi in the irradiated tumour were found microscopically 3 h after the PDT. The tumour growth rate showed a marked decrease when evaluated 3 and 6 days after the treatment. These results show that the ALA-PDT is effective in the inhibition of growth of experimental hepatic tumours.