Protoporphyrin IX Fluorescence Research Articles

Review of Neurosurgical Fluorescence Imaging Methodologies

Fluorescence imaging in neurosurgery has a long historical development, with several different biomarkers and biochemical agents being used, and several technological approaches. This review focuses on the different contrast agents, summarizing endogenous fluorescence, exogenously stimulated fluorescence and exogenous contrast agents, and then on tools used for imaging. It ends with a summary of key clinical trials that lead to consensus studies. The practical utility of protoporphyrin IX (PpIX) as stimulated by administration of δ-aminolevulinic acid (ALA) has had substantial pilot clinical studies and basic science research completed. Recently multi-center clinical trials using PpIx fluorescence to guide resection have shown efficacy for improved short term survival. Exogenous agents are being developed and tested pre-clinically, and hopefully hold the potential for long term survival benefit if they provide additional capabilities for resection of micro-invasive disease or certain tumor sub-types that do not produce PpIX or help delineate low grade tumors. The range of technologies used for measurement and imaging ranges widely, with most clinical trials being carried out with either point probes or modified surgical microscopes. At this point in time, optimized probe approaches are showing efficacy in clinical trials, and fully commercialized imaging systems are emerging, which will clearly help lead to adoption into neurosurgical practice.

A comparative study on the enhancement efficacy of specific and non-specific iron chelators for protoporphyrin IX production and photosensitization in HaCat cells

The iron chelators can be utilized in target cells to improve 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT). The purpose of this study is to compare the effect of two kinds of iron chelators, desferrioxamine (DFO) and ethylenediaminetetraacetic acid (EDTA) on the enhancement of ALA-PDT. HaCat cells were cultured in medium containing 2.0 mmol/L of ALA and 0.5 mmol/L of DFO or EDTA. After 3-h incubation in the dark, the concentration of cellular protoporphyrin IX (PpIX) was detected by high performance liquid chromatography (HPLC), and the fluorescence of PpIX was observed at 630 nm emission under confocal laser scanning microscope. For PDT, HaCat cells were irradiated using 632.8 nm laser, and the fractions of apoptotic and necrotic cells were flow cytometrically assayed. Related differences in morphology and ultrastructure of Ha-Cat cells were observed using optical microscope or transmission electron microscope. Compared to incubation with ALA alone, the addition of DFO or EDTA increased the concentration of cellular PpIX and the fluorescent density of PpIX, and also increased cell death ratio after PDT. PDT using ALA plus DFO produced the highest cellular PpIX level, greatest cell death ratio and most severe structural damage to the cells. It was concluded that both DFO and EDTA could enhance ALA-based PpIX production and PDT. Compared to the non-specific iron chelator of EDTA, the specific chelator, DFO, showed more potential for the enhancement.

Regulation of 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation in Human Urothelial Carcinomas

Purpose: The purpose of this study was to clarify the regulatory mechanism of protoporphyrin IX (PpIX) synthesis mediated by 5-aminolevulinic acid (ALA) in human urothelial carcinoma (UC), leading to improved accuracy in photodynamic diagnosis and therapy using ALA. Experimental Design: PpIX accumulation in cultured UC cells after incubation for 1–5 h with 0.5–5 mM ALA was analyzed by fluorescence analysis using fluorescence microscopy and flow cytometry technique. Results: PpIX fluorescence mediated by ALA was increased, and the intensity of PpIX fluorescence was time-dependently increased in UC cells compared to noncancerous cells. The distribution of endogenous PpIX fluorescence primarily coincided with mitochondria, and then increased at a specific perinuclear region in the cells during the time of incubation. The ALA-mediated PpIX synthesis in UC cells was suppressed by β-alanine, an inhibitor of β-transporters of cell membrane, and carbonylcyanide p-trifluoromethoxyphenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, the ALA-mediated PpIX accumulation was increased by deferoxamine, an iron chelator, manganese and nitric oxide, which is contributed to PpIX metabolism by inhibiting ferrochelatase activity, generated by a nitric oxide-generating reagent NOC-18. As observed above, ALA-mediated PpIX synthesis in human UC cells was regulated by the process of ALA uptake, ALA conversion to PpIX and metabolism of accumulated PpIX to heme. Conclusions: This shows that the suppression of ferrochelatase increased PpIX accumulation in UC cells using small amount of ALA, thus leading to an improved clinical practicability of photodynamic diagnosis and therapy.

Precise detection of lymph node metastases in mouse rectal cancer by using 5‐aminolevulinic acid

Accurate diagnosis of metastatic lymph nodes (LNs) is essential in choosing appropriate treatment for gastrointestinal carcinoma. Our aim was to evaluate the diagnostic power of 5-aminolevulinic acid (5-ALA) for LN metastasis in mouse rectal cancer. Colorectal cancer cell lines, isolated cells from normal LNs, and orthotopic mouse model incorporating enhanced green fluorescent protein-tagged and untagged human rectal cancer cells were studied after 5-ALA administration by using confocal microscopy, fluorescence stereomicroscopy, fluorescence lifetime imaging microscopy (FLIM), multichannel spectrophotometry and macroconfocal imaging system to precisely detect LN metastases. In vitro confocal microscopic analyses showed that all colorectal cancer cell lines tested were positive for 5-ALA-induced fluorescence, whereas isolated normal LN cells were negative. 5-ALA-induced protoporphyrin IX (PPIX) fluorescence, verified by FLIM and multichannel spectrophotometry, revealed LN metastases in mice-bearing human rectal cancer cells. Occult LN metastases, unrecognized on white-light imaging and simplified hematoxylin-eosin analyses, were readily detectable on 5-ALA-induced PPIX fluorescence imaging. In vivo macroconfocal images clearly revealed PPIX-fluorescence-positive cancer cells in draining lymph vessels and nodes. Together with specific speckled patterns of PPIX-fluorescence in metastatic lesions, the PPIX-fluorescence intensity ratio of metastatic and nonmetastatic lesions discriminated metastasis with 100% sensitivity and 100% specificity in excised whole LN samples. These results show that fluorescence diagnosis with 5-ALA is very accurate in the detection of LN micrometastases of mouse rectal cancer, suggesting that this feasible diagnostic approach is applicable to target sectioning of metastases of resected fresh whole node samples in pathology laboratories. (c) 2009 UICC.

Desferrioxamine shows different potentials for enhancing 5-aminolaevulinic acid-based photodynamic therapy in several cutaneous cell lines

Protoporphyrin IX (PpIX) is the photosensitizer in 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT). Its further bioconversion to heme requires iron and can be suppressed by iron chelators such as desferrioxamine (DFO). To investigate the effectiveness of DFO in enhancing PpIX-based PDT in skin tissue, we selected fibroblasts, HaCat cells and Hep-2 cells as targets co-cultured with ALA, that have different biological characteristics for PpIX conversion. Evaluated interventions included: (1) blank control (no ALA, no DFO); (2) DFO alone; (3) ALA alone; and (4) DFO in combination with ALA. Before photodynamic irradiation, cellular PpIX level and fluorescence were measured. After irradiation, cell death ratio was calculated and morphological changes in the cells were observed. The results showed that the content and photodynamic effects of cellular PpIX presented in the order Hep-2 cells > HaCat cells > fibroblasts, either co-cultured with ALA alone or with ALA plus DFO. DFO was found to have increasing effects on both PpIX level and cell death ratio in the same order. It was found that DFO had different potentials for augmenting ALA-PDT in these cutaneous cell lines. The cells proliferating more rapidly might be more susceptible to enhancement of DFO.

Topical photodynamic therapy with 5-aminolevulinic acid for acne vulgaris.

Objective To investigate the optimal concentration and incubation time of 5-aminole-vulinic acid (ALA) in photodynamic therapy of acne vulgaris. Methods Thirty patients with facial acne vulgaris were equally divided into two groups. In group 1, ALA (10%)cream was applied to acne lesions and protoporphyrin Ⅸ (PpⅨ) fluorescence was examined visually and spectroscopically at 1, 2, 3, 4 and 5 hours. In group 2, ALA cream of 3%, 5% and 10% was applied to lesions in the right cheek, left cheek, and forehead, respectively, of the same patient and incubated for 3 hours followed by photodynamie diagnosis and quantification of fluorescence intensity; clinical outcome and side effects were analyzed. Results Strong brick red PpⅨ fluorescence was observed in inflammatory papules, pustules, and cysts alter applica-tion of 10% ALA cream and irradiation with excitation light. The relative intensity of Ppix fluorescence was 1.3, 4.3, 5.1 and 5.8 in comedones, inflammatory papules, pustules and cysts, respectively, and it increased with the severity of lesions. A higher intensity of PpⅨ fluorescence was noted in patients with longer incu-bation period (3, 4 or 5 hours) compared with those with shorter incubation period (1 or 2 hours), and the difference was significant (P 0.05). The overall clearance rate was 73% (11/15) after two courses of ALA-photodynamic therapy (PDT) in group 2. Side effects mainly included mild to moderate erythema, swelling and little exudation (occasionally). A transient pigmentation was observed in 2 patients. Neither ulceration nor scarring was noted. Conclusions ALA-PDT is suitable for the manage-ment of acne vulgaris mainly characterized by inflammatory papules, pustules and cysts. The results strongly suggest that 3% and 3 hours are the optimal concentration and incubation time of ALA in PDT of acne vulgaris. Key words: Acue vulgaris; Aminolevulinic acid; Photochemotherapy

A time course investigation of the fluorescence induced by topical application of 5‐aminolevulinic acid and methyl aminolevulinate on normal human skin

Treatment of non-melanoma skin cancers (NMSC) with topical photodynamic therapy (PDT) is a treatment of choice for many clinicians. The two most commonly used PDT photosensitizer precursors are 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL). Current PDT treatment regimes advise longer (4-6 h) application times for ALA and shorter times (3 h) for MAL. To establish the time course characteristics of protoporphyrin IX (PpIX) fluorescence following the application of ALA and MAL in normal skin. A total of 17 healthy volunteers were recruited, and both ALA and MAL were applied to the inner forearm for varying times (1-6 h). PpIX fluorescence was detected using a non-invasive spectroscopy system. PpIX fluorescence (following the application of either ALA or MAL) is dependent on duration of application. Following the application of ALA for 1-3 h peak fluorescence was noted at 7 h. Longer duration times (4-6 h) resulted in sustained fluorescence, which peaked at 24 h. MAL-induced fluorescence peaked at 7 h and was significantly decreased by 24 h for all application times. ALA induced fluorescence was shown to be significantly greater than MAL. The findings from this study have shown that potentially it would be more beneficial to apply ALA for shorter periods of time and MAL for longer than current practice.

Improved Peptide Prodrugs of 5-ALA for PDT: Rationalization of Cellular Accumulation and Protoporphyrin IX Production by Direct Determination of Cellular Prodrug Uptake and Prodrug Metabolization

Twenty-seven dipeptide derivatives of general structure Ac-Xaa-ALA-OR were synthesized as potential prodrugs for 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT). Xaa is an alpha-amino acid, chosen to provide a prodrug with appropriately tailored lipophilicity and water solubility. Although no simple correlation is observed between downstream production of protoporphyrin IX (PpIX) in PAM212 keratinocytes and HPLC-derived descriptors of compound lipophilicity, quantification of prodrug uptake reveals that most of the dipeptides are actually more efficiently accumulated than ALA in PAM212 and also A549 and Caco-2 cell lines. Subsequent ALA release is the limiting factor, which emphasizes the importance of decoupling prodrug uptake and intracellular metabolization when assessing the efficacy of ALA derivatives for PDT. In agreement with PpIX fluorescence studies, at a concentration of 0.1 mM, l-Phe derivatives 4m and 4o, and l-Leu, l-Met, and l-Glu derivatives 4f, 4k, and 4u, exhibit significantly enhanced photoxicity in PAM212 cells compared to ALA.

Depth Profile of Protoporphyrin IX Fluorescence in an Amelanotic Mouse Melanoma Model

Protoporphyrin IX (PpIX) fluorescence was measured at different depths in a subcutaneous amelanotic melanoma model (LOX) in mice. PpIX was induced by topical application of 5-aminolevulinic acid (ALA) and two of its derivatives, the methylester (MAL) and hexylester (HAL) onto the normal skin covering the tumor. The PpIX fluorescence intensity on the surface of the tumors was the highest for HAL, followed by ALA and MAL. Using equimolar concentrations (0.5 mmol g(-1)), HAL induced nearly twice as much fluorescence as ALA did. The depth profile of PpIX fluorescence was measured at different layers of the tumor, which was carefully sliced and controlled in situ ex vivo. The PpIX fluorescence was mainly localized within the upper 2 mm of the tissue for ALA and within 1 mm for MAL and HAL. There were no significant differences in the shape of the fluorescence excitation spectra, but the long wavelength excitation peak (633 nm) was so weak that these results are unreliable for depth estimation. When considering the low fluorescence intensity (around 5% of the intensity at the tumor surface), the actual penetration depth of HAL was comparable to that of ALA. The fluorescence after topical application of ALA and HAL was significantly above the background level down to a depth of around 6 mm, and there were traces of PpIX fluorescence even at the tumor base (10 mm). The fluorescence after topical application of MAL was detectable down to 1 mm. In the depth of 2-6 mm, the fluorescence was slightly higher for HAL than for ALA. Using the estimated diffusion coefficients for topically applied ALA (0.16 +/- 0.03 mm(2) h(-1)), MAL (0.045 +/- 0.005 mm(2) h(-1)) and HAL (0.037 +/- 0.003 mm(2) h(-1)), the behavior of the drugs after different application times could be estimated in this tumor model.

Influence of formulation factors on PpIX production and photodynamic action of novel ALA‐loaded microparticles

A novel 5-aminolevulinic acid (ALA)-containing microparticulate system was produced recently, based on incorporation of ALA into particles prepared from a suppository base that maintains drug stability during storage and melts at skin temperature to release its drug payload. The novel particulate system was applied to the skin of living animals, followed by study of protoporphyrin IX (PpIX) production. The effect of formulating the microparticles in different vehicles was investigated and also the phototoxicity of the PpIX produced using a model tumour.Particles formulated in propylene glycol gels (10% w/w ALA loading) generated the highest peak PpIX fluorescence levels in normal mouse skin. Peak PpIX levels induced in skin overlying subcutaneously implanted WiDr tumours were significantly lower than in normal skin for both the 10% w/w ALA microparticles alone and the 10% w/w ALA microparticles in propylene glycol gels during continuous 12 h applications. Tumours not treated with photodynamic therapy continued to grow over the 17 days of the anti-tumour study. However, those treated with 12 h applications of either the 10% w/w ALA microparticles alone or the 10% w/w ALA microparticles in propylene glycol gel followed by a single laser irradiation showed no growth. The gel formulation performed slightly better once again, reducing the tumour growth rate by approximately 105%, compared with the 89% reduction achieved using particles alone. Following the promising results obtained in this study, work is now going on to prepare particle-loaded gels under GMP conditions with the aim of initiating an exploratory clinical trial.

Noninvasive measurement of aminolevulinic acid-induced protoporphyrin IX fluorescence allowing detection of murine glioma <italic>in vivo</italic>

Aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence is studied as a contrast agent for noninvasive detection of murine glioma, using the fluorescence-to-transmission ratio measured through the cranium. Signals measured prior to administration of ALA are very similar between control animals, 9L-GFP, and U251 tumor-bearing animals. However, 2 h after ALA administration, the PpIX signal from both tumor-bearing groups is significantly higher than the control group (9L-GFP group p-value=0.016, and U251 group p-value=0.004, relative to the control group). The variance in signal from the 9L-GFP group is much larger than either the control group or the U251 group, which is consistent with higher intrinsic PpIX fluorescence heterogeneity as seen in situ at ex vivo analysis. Decreasing the skin PpIX fluorescence via intentional photobleaching using red light (635 nm) is examined as a tool for increasing PpIX contrast between the tumor-bearing and control groups. The red light bleaching is found to increase the ability to accurately quantify PpIX fluorescence in vivo, but decreases the specificity of detection between tumor-bearing and nontumor-bearing groups.

A HCl/alcohol formulation increased 5‐aminolevulinic acid skin distribution using an ex vivo full thickness porcine skin model

Topical photodynamic therapy with 5-aminolevulinic acid (5-ALA) is a newly established treatment modality for epithelial skin cancer. Skin distribution of different 20% 5-ALA formulations was investigated by detection of protoporphyrin IX (PpIX) using an ex vivo porcine skin model. Fluorescence of PpIX was first detectable at 2 h after application of a 5-ALA/HCl-alcohol solution, followed by a 5-ALA gel +/- 40% DMSO > 5-ALA lipophilic ointment > 5-ALA hydrophilic ointment at 4 h after application. Intensity of PpIX was 10-fold higher after 5-ALA/HCl-alcohol application in contrast to 5-ALA hydrophilic ointment. Maxima of PpIX fluorescence were measured between 18 and 24 h after application. PpIX accumulation induced by 5-ALA within the lower parts of the epidermis was increased in order of 5-ALA hydrophilic ointment < 5-ALA lipophilic ointment < 5-ALA gel w/o DMSO < 5-ALA gel with DMSO < 5-ALA/HCl-alcohol. Illumination of skin incubated with different 5-ALA formulations led to a marked increase of apoptotic cells in the epidermis depending on the penetration depth of the 5-ALA formulations. A formulation containing short chain alcohols have been observed to increase the permeation and distribution of 5-ALA. PpIX fluorescence was detected earlier yielding to photodynamic effective amounts of PpIX in the epidermis as compared with all other 5-ALA formulations used. These data emphasizes the potential of investigating properties of new formulations using such a full thickness porcine skin model.

Fluorescence Kinetics of Protoporphyrin‐IX Induced from 5‐ALA Compounds in Rabbit Postballoon Injury Model for ALA‐Photoangioplasty

Protoporphyrin IX (PpIX) is one of the photodynamically active substances that are endogenously synthesized in the metabolic pathway for heme as a precursor. Aminolevulinic acid-esters are more lipophilic than conventional 5-aminolevulinic acid (ALA) and some of them are currently being approved as new drugs for photodynamic diagnosis (PDD) and photodynamic therapy (PDT). In order to investigate the pharmacokinetics of ALA and ALA-ethyl ester (ALA-ethyl) in the atheromatous plaque and normal aortic wall of rabbit postballoon injured artery, each 60 mg kg(-1) of ALA or ALA-ethyl was injected intravenously followed by serial detection of PpIX fluorescence of harvested arteries at 0-48 h post-injection. Maximum PpIX build-up in the atheromatous plaque was seen at 2 h after injecting ALA. In contrast, it occurred at 9 h after injecting ALA-ethyl. In addition, the selective build-up of ALA in the atheromatous plaque compared to normal vessel wall was much higher (10 times) than that of ALA-ethyl. The time of maximum fluorescence intensity of PpIX was employed as drug-light-interval for subsequent PDT treatment of the atheromatous plaque with 50-150 J cm(-1) of light dose. Significant reduction in plaque was observed without damage of the medial wall at both groups, but smooth muscle cell (SMC) was still present in the media region below the PDT-treated atheromatous plaque. In conclusion, ALA may be a more effective compound for endovascular PDT treatment of the atheromatous plaque compared with ALA-ethyl based on their pharmacokinetics, but further optimization of PDT methodology remains to remove completely residual SMC in the media for preventing potential restenosis.

Comparison of ALA- and ALA hexyl-ester-induced PpIX depth distribution in human skin carcinoma

Photodynamic therapy (PDT) based on the use of photoactivable porphyrins, such as protoporphyrin IX (PpIX), induced by the topical application of amino-levulinic acid (ALA) or its derivatives, ALA methyl-ester (m-ALA), is a treatment for superficial basal cell carcinoma (BCC), with complete response rates of over 80%. However, in the case of deep, nodular-ulcerative lesions, the complete response rates are lower, possibly related to a lower bioavailability of PpIX. Previous in vitro skin permeation studies demonstrated an increased penetration of amino-levulinic acid hexyl-ester (h-ALA) over ALA. In this study, we tested the validity of this approach in vivo on human BCCs. An emulsion containing 20% ALA (w/w) and preparations of h-ALA at different concentrations were applied topically to the normal skin of Caucasian volunteers to compare the PpIX fluorescence intensities with an optical fiber-based spectrofluorometer. In addition, the PpIX depth distribution and fluorescence intensity in 26 BCCs were investigated by fluorescence microscopy following topical application of 20% ALA and 1% h-ALA. We found that, for application times up to 24 h, h-ALA is identical to ALA as a PpIX precursor with respect to PpIX fluorescence intensity, depth of penetration, and distribution in basal cell carcinoma, but has the added advantage that much smaller h-ALA concentrations can be used (up to a factor 13). We observed a non-homogenous distribution in BCCs with both precursors, independent of the histological type and depth of invasion in the dermis.

Effect of (R)L-sulforaphane on 5-aminolevulinic acid-mediated photodynamic therapy

Topical photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA), or so-called ALA-PDT, is a standard procedure in the clinical practice. For optimal treatment of nonmelanoma skin cancer, actinic keratoses and other dermatoses improvements are required because of adverse side effects, which include pruritus, erythema, edema, and pain. (R)L-sulforaphane (SF) is a compound that protects against erythema, but it can also induce DNA fragmentation that leads to cell death by apoptosis. The aim of our study was to investigate whether SF has any impact on protoporphyrin IX (PpIX) production and on PDT effectiveness. We have investigated some relevant properties of SF: its photostability in dimethyl sulfoxide (DMSO), its effect on ALA-induced production of PpIX in A431 human squamous carcinoma cells and in human skin, its effect on the photoinactivation of PpIX sensitized cells, and its effect on the rate of photobleaching of PpIX. SF had no influence on PpIX photodegradation, neither in solution nor in A431 cells. The synthesis of PpIX was increased by SF in human skin, but not in A431 cells. The average increase in PpIX fluorescence in human skin was 18% +/- 6% and 43% +/- 10% for ALA combined with 80 nmol/L SF and 120 nmol/L SF, respectively. Pretreatment with (R)L-sulforaphane before topical ALA-PDT may improve penetration of ALA through the stratum corneum, and, subsequently, increase PpIX synthesis.

Irradiance-Dependent Photobleaching and Pain in δ-Aminolevulinic Acid-Photodynamic Therapy of Superficial Basal Cell Carcinomas

In superficial basal cell carcinomas treated with photodynamic therapy with topical delta-aminolevulinic acid, we examined effects of light irradiance on photodynamic efficiency and pain. The rate of singlet-oxygen production depends on the product of irradiance and photosensitizer and oxygen concentrations. High irradiance and/or photosensitizer levels cause inefficient treatment from oxygen depletion in preclinical models. Self-sensitized photobleaching of protoporphyrin IX (PpIX) fluorescence was used as a surrogate metric for photodynamic dose. We developed instrumentation measuring fluorescence and reflectance from lesions and margins during treatment at 633 nm with various irradiances. When PpIX was 90% bleached, irradiance was increased to 150 mW/cm(2) until 200 J/cm(2) were delivered. Pain was monitored. In 33 superficial basal cell carcinomas in 26 patients, photobleaching efficiency decreased with increasing irradiance above 20 mW/cm(2), consistent with oxygen depletion. Fluences bleaching PpIX fluorescence 80% (D80) were 5.7 +/- 1.6, 4.5 +/- 0.3, 7.5 +/- 0.8, 7.4 +/- 0.3, 12.4 +/- 0.3, and 28.7 +/- 7.1 J/cm(2), respectively, at 10, 20, 40, 50, 60 and 150 mW/cm(2). At 20-150 mW/cm(2), D80 doses required 2.5-3.5 min; times for the total 200 J/cm(2) were 22.2-25.3 min. No significant pain occurred up to 50 mW/cm(2); pain was not significant when irradiance then increased. Clinical responses were comparable to continuous 150 mW/cm(2) treatment. Photodynamic therapy with topical delta-aminolevulinic acid using approximately 40 mW/cm(2) at 633 nm is photodynamically efficient with minimum pain. Once PpIX is largely photobleached, higher irradiances allow efficient, rapid delivery of additional light. Optimal fluence at a single low irradiance is yet to be determined.

Protoporphyrin IX fluorescence photobleaching is a useful tool to predict the response of rat ovarian cancer following hexaminolevulinate photodynamic therapy

Accurate dosimetry was shown to be critical to achieve effective photodynamic therapy (PDT). This study aimed to assess the reliability of in vivo protoporphyrin IX (PpIX) fluorescence photobleaching as a predictive tool of the hexaminolevulinate PDT (HAL-PDT) response in a rat model of advanced ovarian cancer. Intraperitoneal 10(6) NuTu 19 cells were injected in 26 female rats Fisher 344. Peritoneal carcinomatosis was obtained 26 days post-tumor induction. Four hours post-intraperitoneal HAL (Photocure ASA, Oslo, Norway) injection, a laparoscopic procedure (D-light AutoFluorescence system, Karl Storz endoscope, Tuttlingen, Germany) and a fluorescence examination were made for 22 rats. The first group (LASER group, n=26) was illuminated with laser light using a 532 nm KTP laser (Laser Quantum, Stockport, UK) on 1 cm(2) surface at 45 J/cm(2). The second group (NO LASER group, n=26) served as controls. Biopsies were taken 24 hours after PDT. Semi-quantitative histology was performed and necrosis value was determined: 0--no necrosis to 4--full necrosis. Fluorescence was monitored before and after illumination on complete responders (NV=3-4; n=20) and non-responders (NV=0-2; n=6). High PpIX photobleaching corresponded with complete responders whereas low photobleaching corresponded with non-responders (P<0.05). A direct linear correlation was shown between photobleaching and necrosis (R(2)=0.89). In vivo PpIX fluorescence photobleaching is useful to predict the tissue response to HAL-PDT.

5-Aminolaevulinic acid peptide prodrugs enhance photosensitization for photodynamic therapy

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (ALA) has been widely used in photodynamic therapy for a range of malignant and nonmalignant lesions. However, ALA is relatively hydrophilic and lacks stability at physiologic pH, limiting its bioavailability. We have investigated more lipophilic, uncharged ALA-peptide prodrugs based on phenylalanyl-ALA conjugates, which are water soluble and chemically stable for improving ALA delivery. Pharmacokinetics of the induced protoporphyrin IX (PpIX) were studied in transformed PAM212 keratinocyte cells and pig skin explants. The intracellular porphyrin production was substantially increased with Ac-L-Phe-ALA-Me (compound 1) and Ac-L-Phe-ALA (compound 3) compared with equimolar ALA: after 6-h incubation, the PpIX fluorescence measured using 0.01 mmol/L of compound 1 was enhanced by a factor of 5 compared with ALA. Phototoxicity results showed good correlation with PpIX levels, giving a LD(50) (2.5 J/cm(2)) of 25 micromol/L for ALA, 6 micromol/L for 5-aminolaevulinic hexyl ester, and 2.6 micromol/L for compound 1, which exhibited the highest phototoxicity. However, these results were stereospecific because the corresponding D-enantiomer, Ac-D-Phe-ALA-Me (compound 2), induced neither porphyrin synthesis nor phototoxicity. PpIX levels were considerably reduced when cells were incubated with compound 1 at low temperatures, consistent with active transport. In pig skin explants, compound 1 induced higher porphyrin fluorescence than ALA by a factor of 3. These results show that water-soluble peptide prodrugs of ALA can greatly increase its cellular uptake, generating more intracellular PpIX and improved tumor cell photosensitization. The derivatives are comparable in efficacy with 5-aminolaevulinic hexyl ester but less toxic and more stable at physiologic pH.

Microscopic localisation of protoporphyrin IX in normal mouse skin after topical application of 5-aminolevulinic acid or methyl 5-aminolevulinate

Light fractionation does not enhance the response to photodynamic therapy (PDT) after topical methyl-aminolevulinate (MAL) application, whereas it is after topical 5-aminolevulinic acid (ALA). The differences in biophysical and biochemical characteristics between MAL and ALA may result in differences in localisation that cause the differences in response to PDT. We therefore investigated the spatial distribution of protoporphyrin IX (PpIX) fluorescence in normal mouse skin using fluorescence microscopy and correlated that with the PDT response histologically observed at 2.5, 24 and 48 h after PDT. As expected high fluorescence intensities were observed in the epidermis and pilosebaceous units and no fluorescence in the cutaneous musculature after both MAL and ALA application. The dermis showed localised fluorescence that corresponds to the cytoplasma of dermal cells like fibroblast and mast cells. Spectral analysis showed a typical PpIX fluorescence spectrum confirming that it is PpIX fluorescence. There was no clear difference in the depth and spatial distribution of PpIX fluorescence between the two precursors in these normal mouse skin samples. This result combined with the conclusion of Moan et al. that ALA but not MAL is systemically distributed after topical application on mouse skin [Moan et al., Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters, Int. J. Cancer 103 (2003) 132–135] suggests that endothelial cells are involved in increased response of tissues to ALA-PDT using light fractionation. Histological analysis 2.5 h after PDT showed more edema formation after ALA-PDT compared to MAL-PDT that was not accompanied by a difference in the inflammatory response. This suggests that endothelial cells respond differently to ALA and MAL-PDT. Further investigation is needed to determine the role of endothelial cells in ALA-PDT and the underlying mechanism behind the increased effectiveness of light fractionation using a dark interval of 2 h found after ALA but not after MAL-PDT.

Photodynamic Diagnosis of Prostate Cancer Using 5-Aminolevulinic Acid—First Clinical Experiences

To study the feasibility of 5-aminolevulinic-acid (5-ALA)-induced photodynamic diagnosis (PDD) for the evaluation of the surgical margins (SMs) during radical prostatectomy (RP) in patients with prostate cancer (PCa). A total of 18 patients with histologically confirmed PCa (Gleason score 4 to 8, prostate-specific antigen 1 to 20 ng/mL) underwent RP. Of the 18 patients, 16 received 5-ALA (20 mg/kg) orally 2 hours before RP, and 2 served as controls without any application of 5-ALA. To study the protoporphyrin IX (PPIX) accumulation after application of 5-ALA, all harvested specimens were investigated by fluorescence microscopy and spectroscopy. In 10 of 16 patients, PDD of the SMs and the prostate was performed during RP using an incoherent light source filtered for efficient fluorescence excitation. In all 16 patients, who had received 5-ALA fluorescence microscopy showed a selective accumulation of PPIX in the PCa cells, and only weak PPIX fluorescence could be detected in benign epithelial cells and none in the adjacent stroma. The 2 patients, who had not received 5-ALA had no PPIX fluorescence in the prostate. Of 10 patients, 8 demonstrated fluorescence-negative and histologically confirmed negative margins during PDD, and 1 each demonstrated a fluorescence-positive SM that was also confirmed by histologic examination and a positive SM that was not confirmed by PPD. This is the first report of PDD for PCa using 5-ALA. These initial results have demonstrated that PPIX is selectively enhanced in malignant tissue, an essential prerequisite of PDD. Additional studies are warranted to validate these preliminary data and the efficacy of PDD for PCa during RP.