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Simple Summary While photodynamic therapy (PDT) has emerged as an attractive treatment for certain cancer types, it still lacks cancer specificity, which limits therapeutic efficacy and damages normal tissues. The present study aimed to evaluate the targeting potential of a novel minibody that recognizes a cell surface receptor expressed on various cancer cell lines. The engineered minibody-photosensitizer conjugate (MS5-IR700) killed target cells in both cell monolayer and tumor spheroid cultures. Additionally, the conjugate induced immunogenic cell death. Hence, the developed minibody could be used as a photosensitizer carrier for -cancer cell-targeted PDT. Abstract Photodynamic therapy (PDT) is a treatment strategy that utilizes photosensitizers (PSs) and light of a specific wavelength to kill cancer cells. However, limited tumor specificity is still a drawback for the clinical application of PDT. To increase the therapeutic efficacy and specificity of PDT, a novel human minibody (MS5) that recognizes a cell surface receptor expressed on various cancer cells was labeled with the hydrophilic phthalocyanine PS IR700 to generate an MS5-IR700 conjugate that is activated by near-infrared (NIR) light. The phototoxicity of the conjugate was mainly tested against the PC3 prostate cancer cell line. The MS5-IR700 conjugate killed PC3 cells after NIR light irradiation as compared to untreated cells or cells treated with IR700 alone. Time-course analysis of cell viability revealed a high percentage of cell death during the first hour in PC3 cells exposed to the MS5-IR700 conjugate and NIR light irradiation. After irradiation, the MS5-IR700 conjugate-treated PC3 cells displayed cellular swelling, round shape, and rupture of the cell and nuclear membranes. In a co-culture model, the MS5-IR700 conjugate killed MS5-positive Ramos lymphoma cells specifically, while leaving MS5-negative cells unaffected. In line with the data obtained with the monolayer cultures, the MS5-IR700 conjugate also killed PC3 cancer cell spheroids. The treatment induced relocation of heat shock protein 70 and calreticulin to the cell surface, implying the induction of immunogenic cell death. Overall, the data suggest that the developed MS5-IR700 conjugate is a promising therapeutic agent that warrants further preclinical studies.

Extracorporeal photopheresis (ECP), a modality that exposes isolated leukocytes to the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light, is used to treat conditions such as cutaneous T-cell lymphoma and graft-versus-host disease. However, the current procedure of ECP has limited selectivity and efficiency, and produces only partial response in the majority of treated patients. Additionally, the treatment is expensive and time-consuming, so the improvement for this modality is needed. In this study, we used the concept of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA), a precursor of an endogenously synthesized photosensitizer protoporphyrin IX (PpIX) in combination with blue light to explore the possibility of targeting activated human blood T cells ex vivo. With various T-cell activation protocols, a high ALA-induced PpIX production took place in activated CD3+, CD4+CD25+, and CD8+ T cell populations with their subsequent killing after blue light exposure. By contrast, resting T cells were much less damaged by the treatment. The selective and effective killing effect on the activated cells was also seen after co-cultivating activated and resting T cells. Under our clinically relevant experimental conditions, ALA-PDT killed activated T cells more selectively and efficiently than 8-MOP/UV-A. Monocyte-derived dendritic cells (DCs) were not affected by the treatment. Incubation of ALA-PDT damaged T cells with autologous DCs induced a downregulation of the co-stimulatory molecules CD80/CD86 and also upregulation of interleukin 10 (IL-10) and indoleamine 2,3-dioxygenase expression, two immunosuppressive factors that may account for the generation of tolerogenic DCs. Overall, the data support the potential use of ALA-PDT strategy for improving ECP by selective and effective killing of activated T cells and induction of immune tolerance.

Extracorporeal photopheresis (ECP), an immunomodulatory therapy for the treatment of chronic graft-versus-host disease (cGvHD), exposes isolated white blood cells to photoactivatable 8-methoxypsoralen (8-MOP) and UVA light to induce the apoptosis of T-cells and, hence, to modulate immune responses. However, 8-MOP-ECP kills diseased and healthy cells with no selectivity and has limited efficacy in many cases. The use of 5-aminolevulinic acid (ALA) and light (ALA-based photodynamic therapy) may be an alternative, as ex vivo investigations show that ALA-ECP kills T-cells from cGvHD patients more selectively and efficiently than those treated with 8-MOP-ECP. The purpose of this phase I-(II) study was to evaluate the safety and tolerability of ALA-ECP in cGvHD patients. The study included 82 treatments in five patients. One patient was discharged due to the progression of the haematological disease. No significant persistent changes in vital signs or laboratory values were detected. In total, 62 adverse events were reported. Two events were severe, 17 were moderate, and 43 were mild symptoms. None of the adverse events evaluated by the internal safety review committee were considered to be likely related to the study medication. The results indicate that ALA-ECP is safe and is mainly tolerated well by cGvHD patients.

Radioligand therapy targeting the prostate-specific membrane antigen (PSMA) is rapidly evolving as a promising treatment for metastatic castration-resistant prostate cancer. The PSMA-targeting ligand p-SCN-Bn-TCMC-PSMA (NG001) labelled with 212Pb efficiently targets PSMA-positive cells in vitro and in vivo. The aim of this preclinical study was to evaluate the therapeutic potential of 212Pb-NG001 in multicellular tumour spheroid and mouse models of prostate cancer. The cytotoxic effect of 212Pb-NG001 was tested in human prostate C4-2 spheroids. Biodistribution at various time points and therapeutic effects of different activities of the radioligand were investigated in male athymic nude mice bearing C4-2 tumours, while long-term toxicity was studied in immunocompetent BALB/c mice. The radioligand induced a selective cytotoxic effect in spheroids at activity concentrations of 3–10 kBq/mL. In mice, the radioligand accumulated rapidly in tumours and was retained over 24 h, while it rapidly cleared from nontargeted tissues. Treatment with 0.25, 0.30 or 0.40 MBq of 212Pb-NG001 significantly inhibited tumour growth and improved median survival with therapeutic indexes of 1.5, 2.3 and 2.7, respectively. In BALB/c mice, no signs of long-term radiation toxicity were observed at activities of 0.05 and 0.33 MBq. The obtained results warrant clinical studies to evaluate the biodistribution, therapeutic efficacy and toxicity of 212Pb-NG001.

Protein kinase B (Akt), similar to many other protein kinases, is at the crossroads of cell death and survival, playing a pivotal role in multiple interconnected cell signaling mechanisms implicated in cell metabolism, growth and division, apoptosis suppression and angiogenesis. Akt protein kinase displays important metabolic effects, among which are glucose uptake in muscle and fat cells or the suppression of neuronal cell death. Disruptions in the Akt-regulated pathways are associated with cancer, diabetes, cardiovascular and neurological diseases. The regulation of the Akt signaling pathway renders Akt a valuable therapeutic target. The discovery process of Akt inhibitors using various strategies has led to the identification of inhibitors with great selectivity, low side-effects and toxicity. The usefulness of Akt emerges beyond cancer therapy and extends to other major diseases, such as diabetes, heart diseases, or neurodegeneration. This review presents key features of Akt structure and functions, and presents the progress of Akt inhibitors in regards to drug development, and their preclinical and clinical activity in regards to therapeutic efficacy and safety for patients.

Nanocarriers based on polymers, metals and lipids have been extensively developed for cancer therapy and diagnosis due to their ability to enhance drug accumulation in cancer cells and decrease undesired drug toxicity in healthy tissues. Overcoming multidrug resistance by designing proper drug nanocarriers will improve outcome of existing oncologic treatments such as chemotherapy and radiotherapy. In this article the relation between physicochemical properties and capacity of a nanosystem to deliver therapeutic agents into pathological sites is discussed. Most promising examples of drug delivery systems are reviewed, and, in particular, the design of a carbohydrate based matrix with entrapped gold nanoparticles is highlighted.

Background: Proteasome inhibition is a current therapeutic strategy used in the treatment of multiple myeloma. Drugs controlling proteasome activity are ideally suited for unidirectional manipulation of cellular pathways such as apoptosis. The first proteasome inhibitor approved in clinics was bortezomib. This drug is currently used in combination with other anticancer agents. Objectives: In this study, the enhancement of bortezomib activity was evaluated using gold nanoparticles coated with poly(ethylene glycol). The uptake mechanism of the gold nanoparticles in pancreatic cell lines, S2-013 and hTERT-HPNE, was assessed by laser scanning confocal microscopy (LSCM). Results: Pancreatic cancer cells internalized the nanoparticles together with the drug in few minutes through the formation of endocytic vesicles. This rapid uptake leads to an increase in the concentration and diffusion of bortezomib in the cytoplasm yielding an increased toxicity on the cells when compared to the drug alone. Conclusion: Gold nanoparticles can be used as effective delivery systems to increasing the permeation and retention of drugs in cancer cells.

Simona Steponkiene1-3, Simona Kavaliauskiene1, Rasa Purviniene4, Ricardas Rotomskis3,5, Petras Juzenas11Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Radiumhospital, Oslo, Norway; 2Faculty of Natural Sciences, Vilnius University, Vilnius, Lithuania; 3Biomedical Physics Laboratory of Oncology Institute, Vilnius University, Vilnius, Lithuania; 4Immunology Laboratory of Oncology Institute, Vilnius University, Vilnius, Lithuania; 5Biophotonics Laboratory, Laser Research Center, Vilnius University, Vilnius, LithuaniaBackground: In novel treatment approaches, therapeutics should be designed to target cancer stem cells (CSCs). Quantum dots (QDs) are a promising new tool in fighting against cancer. However, little is known about accumulation and cytotoxicity of QDs in CSCs.Methods: Accumulation and cytotoxicity of CdTe-MPA (mercaptopropionic acid) QDs in CSCs were assessed using flow cytometry and fluorescence-activated cell sorting techniques as well as a colorimetric cell viability assay.Results: We investigated the expression of two cell surface-associated glycoproteins, CD44 and CD133, in four different cancer cell lines (glioblastoma, melanoma, pancreatic, and prostate adenocarcinoma). Only the melanoma cells were positive to both markers of CD44 and CD133, whereas the other cells were only CD44-positive. The QDs accumulated to a similar extent in all subpopulations of the melanoma cells. The phenotypical response after QD treatment was compared with the response after ionizing radiation treatment. The percentage of the CD44high-CD133high subpopulation decreased from 72% to 55%–58% for both treatments. The stem-like subpopulation CD44highCD133low/- increased from 26%–28% in the untreated melanoma cells to 36%–40% for both treatments.Conclusion: Treatment of melanoma cells with QDs results in an increase of stem-like cell subpopulations. The changes in phenotype distribution of the melanoma cells after the treatment with QDs are comparable with the changes after ionizing radiation.Keywords: prominin-1, CD44, glycoproteins, flow cytometry, FACS, nanoparticles

Summary Background Measurements of tissue oxygenation and blood supply are of vital importance during photodynamic therapy. The aim of this study was to measure photosensitization responses in healthy mouse skin using non-invasive reflectance spectroscopy and fluorescein angiography. Methods Healthy mouse skin was treated topically with hexylaminolevulinate (HAL) for 3 h and then exposed to red light (632 nm). Results Non-invasive reflectance spectroscopy showed that the fraction of oxygenized haemoglobin decreased considerably after treatment with HAL and red light. This indicates reduced tissue oxygenation. Fluorescein angiography showed no vasoconstriction in the mouse skin after short (3 h) treatment with HAL and light, indicating that the oxygen concentration was high enough to give a photodynamic effect consistent with the skin damage (scar formation) observed for a few days after the treatment. However, longer treatment with HAL (17 h) resulted in complete vasoconstriction with oxygen depletion and no skin damage in the centre, while a circular (rim) wound was seen around the light exposed area. Skin damage was larger for the lowest fluence rate (20 mW/cm2) than for the highest one (100 mW/cm2) applied. Conclusion Skin treatment with topical HAL and red light in fluencies and fluence rates as applied here does not result in complete vasoconstriction. Haemoglobin oxygenation monitored by reflectance spectroscopy can be used as a rough measure of the resulting photodynamic response. However, haemoglobin oxygenation alone is not a complete predictive factor since reflectance spectroscopy did not reveal any differences in the status of haemoglobin oxygenation for different fluence rates.

Summary Background 5-Aminolevulinic acid (ALA) or its methyl ester mediated photodynamic therapy (PDT) is the most widely practiced form of PDT in dermatology. One of the advantages of topical PDT is that undesirable photosensitization lasts only for 24–48 h. However, patients are still sensitive in the areas where the cream has been applied, and are advised to avoid exposure to the sun and other light sources for at least 40 h after ALA-PDT. Therefore, the main aim of this work is to improve post-treatment procedures for increasing patient's comfort. The present study was carried out to investigate clearance of protoporphyrin IX (PpIX) in the presence of metal ions in the cream applied on healthy skin of mice in vivo. Methods The photosensitizer PpIX was induced in normal mouse skin by topical application of ALA. Fluorescence spectroscopy was used to study porphyrin kinetics. Results Topical application of ferrous, cobalt or zinc sulphate significantly diminished the fluorescence of PpIX in mouse skin. These results show that the clearance kinetics of PpIX observed after exogenous application of ALA are determined by the conversion of PpIX into haem, and not by the clearance of PpIX from the body. Conclusions Application of a vehicle containing ferrous or cobalt sulphate after PDT might be an approach in clinical practice for the reduction of cutaneous photosensitivity and elimination of undesirable photoreactions in skin and lesions.

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 12h applications. Tumours not treated with photodynamic therapy continued to grow over the 17 days of the anti-tumour study. However, those treated with 12h 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. Copyright r 2009 John Wiley & Sons, Ltd.

Semiconductor quantum dots and nanoparticles composed of metals, lipids or polymers have emerged with promising applications for early detection and therapy of cancer. Quantum dots with unique optical properties are commonly composed of cadmium contained semiconductors. Cadmium is potentially hazardous, and toxicity of such quantum dots to living cells, and humans, is not yet systematically investigated. Therefore, search for less toxic materials with similar targeting and optical properties is of further interest. Whereas, the investigation of luminescence nanoparticles as light sources for cancer therapy is very interesting. Despite advances in neurosurgery and radiotherapy the prognosis for patients with malignant gliomas has changed little for the last decades. Cancer treatment requires high accuracy in delivering ionizing radiation to reduce toxicity to surrounding tissues. Recently some research has been focused in developing photosensitizing quantum dots for production of radicals upon absorption of visible light. In spite of the fact that visible light is safe, this approach is suitable to treat only superficial tumours. Ionizing radiation (X-rays and gamma rays) penetrate much deeper thus offering a big advantage in treating patients with tumours in internal organs. Such concept of using quantum dots and nanoparticles to yield electrons and radicals in photodynamic and radiation therapies as well their combination is reviewed in this article.

5-Aminolevulinic acid (ALA) is a natural precursor of protoporphyrin IX (PpIX) and heme in cells. Photodynamic therapy (PDT) utilizes a metabolic imbalance in cancer cells, leading to increased PpIX generation from exogenous ALA. Due to chemical instability of ALA in therapeutic concentrations at pH values larger than 5.0 and at high temperatures, it looses its activity by spontaneous dimerization to 2,5-dicarboxyethyl-3,6-dihydropyrazine (DHPY). ALA esters are now supplementing ALA in PDT, but little is known about their stability. We have studied the stability of ALA and its methyl ester (MAL) stored under different conditions (temperatures, pH values) by measuring their ability to generate PpIX. 100mM solutions of both compounds were found to be stable at pH 4 and at 4 degrees C. However, at pH 5.5 they lost almost 10% of the initial activity during 5days of storage at 4 degrees C. The fastest decay of ALA and MAL was seen at pH 7.4 and at 37 degrees C, and followed first order kinetics. At pH 7.4 and at 4 degrees C MAL lost its PpIX producing ability more slowly than at 37 degrees C. Our work shows that solutions should be prepared immediately before use and stored at low temperatures. The pH of stock solutions should not exceed 5.

Significant amounts of protoporphyrin IX (PpIX) are formed after 6 min of topical application of 5-aminolevulinic acid (ALA) and its hexylester derivative, whereas PpIX is formed after 10 min of topical application of ALA-methylester derivative in normal mouse skin at 37 degrees C. Lowering the skin temperature to 28-32 degrees C by the administration of the anesthetic Hypnorm-Dormicum reduces the PpIX fluorescence by a factor of 2-3. Practically no PpIX was formed as long as the skin temperature was kept at 12-18 degrees C. At around 30 degrees C PpIX fluorescence appears later after application of ALA-ester derivatives (14-20 min) than after application of ALA (8 min), indicating differences in their bioavailability (delayed penetration through the stratum corneum, cellular uptake, conversion to ALA, PpIX production) in mouse skin in vivo. The difference in lag time in the PpIX formation after application of ALA and ALA-esters may be partly related to deesterification of the ALA-ester molecules. The temperature dependence of PpIX production may be used for improvement of photodynamic therapy with ALA and ALA-ester derivatives, where accumulation of PpIX can be selectively enhanced by increasing the temperature of the target tissue.

Targeted cancer therapies are used to inhibit the growth, progression, and metastasis of the tumor by interfering with specific molecular targets and are currently the focus of anticancer drug development. Protein kinase B, also known as Akt, plays a central role in many types of cancer and has been validated as a therapeutic target nearly two decades ago. This review summarizes the intracellular functions of Akt as a pivotal point of converging signaling pathways involved in cell growth, proliferation, apoptotis and neo-angiogenesis, and focuses on the drug design strategies to develop potent anticancer agents targeting Akt. The discovery process of Akt inhibitors has evolved from adenosine triphosphate (ATP)-competitive agents to alternative approaches employing allosteric sites in order to overcome the high degree of structural similarity between Akt isoforms in the catalytic domain, and considerable structural analogy to the AGC kinase family. This process has led to the discovery of inhibitors with greater specificity, reduced side-effects and lower toxicity. A second generation of Akt has inhibitors emerged by incorporating a chemically reactive Michael acceptor template to target the nucleophile cysteines in the catalytic activation loop. The review outlines the development of several promising drug candidates emphasizing the importance of each chemical scaffold. We explore the pipeline of Akt inhibitors and their preclinical and clinical examination status, presenting the potential clinical application of these agents as a monotherapy or in combination with ionizing radiation, other targeted therapies, or chemotherapy.

Summary Photodynamic therapy (PDT) is a medical treatment by which a combination of a photosensitising drug and visible light cause the destruction of selected cells. Thick lesions, such as nodular basal cell carcinomas (BCCs), or lesions with overlying keratinous debris, are reported as being difficult to eradicate using 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT). Such treatment failures have been attributed to the shallow penetration of water-soluble drugs like ALA. In addition, the current scarcity of sophisticated drug delivery research centered on PDT applications has meant that accurate comparison of similar clinical studies is difficult. This paper investigates, for the first time, novel drug delivery systems for controlled drug delivery of methyl-ALA (M-ALA). Pressure sensitive adhesive (PSA) and bioadhesive patches containing defined M-ALA loadings and a standard cream containing equivalent amounts of drug were applied to the skin of mice for defined periods of time and the fluorescence of the protoporphyrin IX (PpIX) induced measured over 24 h. Of major importance, the PSA patches containing low drug loadings induced high PpIX levels, which were limited to the site of application, after only 1 h applications. Such systems have the potential to improve selectivity of PpIX accumulation, increase simplicity of treatment and, due to the low drug loadings required, reduce costs of clinical PDT. PSA patches would be most suitable for application to areas of dry skin, while bioadhesive patches would be suitable for moist areas, such as the mouth or lower female reproductive tract and have been shown here to induce significant PpIX production at the site of application after 4 h applications of patches containing high drug loadings.

Summary Background 5-Aminolaevulinic acid (ALA) and its ester derivatives are used in photodynamic therapy. Despite extensive investigations, the differences in biodistribution and pharmacokinetics of protoporphyrin IX (PpIX) induced by ALA and its derivatives are still not well understood, notably for humans. Objectives To study porphyrin accumulation after topical application of ALA and two of its ester derivatives in normal human skin. Methods Creams containing 0·2%, 2% and 20% (w/w) of ALA, methyl 5-aminolaevulinate (MAL) and hexyl 5-aminolaevulinate (HAL) were applied on normal human skin of six volunteers. The amount and distribution of porphyrins formed in the skin was investigated noninvasively by means of fluorescence spectroscopy. Results Fluorescence emission and excitation spectra exhibited similar spectral shapes for the all drugs, indicating that mainly PpIX was formed. Low concentrations (0·2% and 2%) of MAL induced considerably less PpIX in normal human skin than similar concentrations of ALA and HAL. A high concentration (20%) of ALA gave higher PpIX fluorescence in normal human skin than was found for MAL and HAL. Conclusions The concentrations inducing half of the maximal PpIX fluorescence are around 2% for ALA, 8% for MAL and 1% for HAL.

Cell survival was investigated after exposing cells in vitro to different temperatures before or after photodynamic therapy with 5-aminolevulinic acid. The photodynamic process was found to be temperature dependent. Cells exposed for 1h to 41 degrees C before light exposure or to 7 degrees C after light exposure showed decreased survival. Furthermore, the photobleaching rate of protoporphyrin IX in the cells was found to increase with increasing temperature during the light exposure. Thus, the photodynamic effect with 5-aminolevulinic acid may be enhanced by heating the tumour area before, and by cooling it immediately after the treatment.

Formation of protoporphyrin IX (PpIX) after topical application of 5-aminolevulinic acid (ALA) and its hexylester derivative (ALA-Hex) was studied on healthy human skin. Temperature, density of hai ...

In this work, the effect of 10% of dimethylsulfoxide (DMSO), 1-[2-(decylthio)ethyl]azacyclopentan-2-one (HPE-101) and Labrafac(®)CC (a mixture of caprylic and capric acid triglycerides) on porphyrin formation in mouse skin during topical application of methyl 5-aminolevulinate (MAL) was studied. The porphyrin level in mouse skin was determined by measuring directly fluorescence and by extraction method. The porphyrin fluorescence kinetics during continuous application of MAL in creams in concentrations 2, 10 and 20% (wt./wt.) for up to 7h showed that in this concentration range the kinetics of porphyrin production in the site of application does not depend significantly on the MAL concentration. After 24h of application of all studied creams the porphyrin fluorescence in the area of treatment was dramatically reduced to be about two-fold higher than the skin autofluorescence, suggesting a significant decrease of the porphyrin concentration in these sites, although in all cases traces of porphyrins were found. It was found by extraction method that a 10% MAL cream with 10% DMSO for 4h increased the concentration of porphyrin about four-fold compared with 10% MAL cream alone. The presence of 10% HPE-101 or 10% Labrafac(®)CC in the 10% MAL cream increased the porphyrin concentration in the area of application about 2.5- and 2-fold, respectively, as compared with MAL cream without enhancers. No statistically significant difference was found between the effects of the creams containing 10% MAL with 10% HPE-101 or 10% Labrafac(®)CC. The results obtained after 24h of mouse skin treatment with the same creams showed a large decrease of porphyrin formation in comparison with results found after 4h. All porphyrin concentrations measured after this time of MAL creams application were similar. Skin erythema was observed using MAL cream with DMSO and HPE-101, but not with Labrafac(®)CC. Our work suggests that the new penetration enhancer Labrafac(®)CC in creams with MAL may be used to increase the penetration of MAL through the mouse skin and at the same time increase production of porphyrin in the skin. This may reduce skin erythema, which is induced by DMSO and HPE-101.

Oxygen is a ubiquitous element and a vitally important substance for life on the Earth, and especially for human life. Living organisms need oxygen for most, if not all, of their cellular functions. On the other hand, oxygen can produce metabolites that are toxic and potentially lethal to the same cells. Being reactive and chemically unstable reactive oxygen species (ROS) are the most important metabolites that initiate reduction and oxidation (redox) reactions under physiological conditions. Oxygen in its excited singlet state (1O2) is probably the most important intermediate in such reactions. Since the discovery of oxygen by Joseph Priestley in 1775 it has been recognized that oxygen can be both beneficial and harmful to life.

Although 5-aminolevulinic acid, ALA, and its derivatives, have been widely studied and applied in clinical photodynamic therapy (PDT), there is still a lack of reliable and non-invasive methods and technologies to evaluate physiological parameters of relevance for the therapy, such as erythema, melanogenesis, and oxygen level. We have investigated the kinetics of these parameters in human skin in vivo during and after PDT with the hexyl ester of ALA, ALA-Hex. Furthermore, the depth of photosensitizer (protoporphyrin IX, PpIX) production after different application times was investigated. It was found that the depth increased with increasing application time of ALA-Hex. We also investigated the depth of PpIX before and after light exposure causing 50% photobleaching at 407 nm. The PpIX localized in superficial layers of the normal tissue was removed during the bleaching. Thus, after bleaching, the remaining PpIX was localized mainly in the deeper layers of normal tissue. We have applied fluorescence emission spectroscopy, fluorescence excitation spectroscopy, and reflectance spectroscopy in the study of the above-mentioned parameters. In conclusion, fluorescence excitation spectroscopy and reflectance spectroscopy are simple, useful, reliable, and noninvasive techniques in the evaluation of the processes taking place in human skin in vivo during and after PDT. Using these methods we were able to quantify melanogenesis, O 2 level, erythema, vasoconstriction, and vasodilatation.

Effect of the iron chelator deferoxamine (DF) on the production of endogenous porphyrins was studied in adenocarcinoma WiDr cells and erythroid K562 cells in vitro. Porphyrin fluorescence was observed in the cells in vitro incubated with DF. The fluorescence spectra recorded in the cells were similar to that of protoporphyrin IX (PpIX). The amount of PpIX generated by DF was around 5% of the ALA effect. Around 90% of the WiDr cells incubated in vitro with DF (0.5 mM, 24 h) and then exposed to light (400-460 nm, 20 min) were photodynamically inactivated. In conclusion, the present study describes a novel approach of using iron chelating agents without 5-aminolevulinic acid (ALA) to photosensitize cancer cells.

Summary Background: Stability of ALA is an important factor for photodynamic therapy (PDT). The dimerization of ALA to pyrazines takes place via the amine group. It is, therefore, to be expected that blocking this group by addition of a formyl group should result in a more stable compound. Methods: The ability of a new N -formyl derivative of ALA ( N -f-ALA) to form protoporphyrin IX (PPIX) was compared with that of ALA and three of its ester (methyl, butyl and hexyl) derivatives. Dark toxicity of the compounds was measured using MTT assay. Formation of PPIX was measured by fluorescence spectroscopy. Results and conclusions: N -f-ALA showed an outstanding stability in water solutions even at pH 7. However, it induced no PPIX neither in WiDr cells in vitro, nor in mouse skin in vivo. A probable reason is lack of an enzyme that can cleave the bond between the formyl group and ALA. Thus, steric hindrance may prevent processing of the compound into porphobilinogen. N -f-ALA did not inhibit PpIX formation from ALA and is unable to enter the heme cycle. Generation of ALA from its derivatives, therefore, seems to be an essential step in PPIX synthesis.

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. © 2002 Wiley-Liss, Inc.

No fluorescence of protoporphyrin IX (PpIX) was measured using a fiber optic probe in pigmented B16F10 melanoma in mice after topical application of 5-aminolevulinic acid methylester (ALA-Me). However, chemical extraction of tissues excised from mice after intratumoral administration of ALA-Me or its parent compound ALA revealed that this tumor had the capability to produce PpIX. Small amounts of endogenous porphyrins, mainly PpIX, were found in the melanoma not treated with these drugs. Topical application of ALA-Me followed by exposure with laser light (633nm) delayed the growth of the tumors slightly. Light alone also had a significant effect on the tumor growth.

Topical methyl aminolevulinate (MAL) is currently under study for the treatment of actinic keratoses. This approach involves topical application of MAL to the lesion to be exposed to light as well as a few millimetres of adjacent normal skin. We studied the effects of whole-body white light exposure following topical application of MAL to the entire back of hairless mice chronically exposed to UV radiation. Groups of mice were exposed to UV from FS20 lamps. One group of mice was treated weekly with 8% topical MAL followed 2 h later by suberythematous exposure to 1.2 J/cm(2) of light from a slide projector. MAL followed by light induced a significant delay in the time of appearance of the first tumour as compared to mice exposed only to UV ( P0.0001). After 26 weeks of UV exposure large tumours (/=4 mm) were present in 14 mice in the UV group as compared to only one mouse in the UV-MAL group. In mice treated on one side with MAL and the other side with vehicle, the delay in the appearance of tumour was only observed on the side treated with MAL, suggesting that a local rather than systemic effect was responsible for this phenomenon. In vivo fluorescence spectroscopy and quantitative fluorescence microscopy showed that there was a preferential accumulation of protoporphyrin IX in tumours as compared to adjacent UV-exposed skin and normal skin at the time of light exposure. In conclusion, topical MAL followed by light under suberythematous conditions delayed the appearance of UV-induced skin tumours without increasing mortality or morbidity, and thus acted in a prophylactic manner.

Aim: In this study, Ce3+-doped lanthanum(III) fluoride (LaF3:Ce3+) nanoparticles were synthesized by a wet-chemistry method in dimethyl sulfoxide (DMSO) and their application as an intracellular light source for photodynamic activation was demonstrated. Materials & methods: The LaF3:Ce3+/DMSO nanoparticles have a strong green emission with a peak at approximately 520 nm, which is effectively overlapped with the absorption of protoporphyrin IX (PPIX). The nanoparticles were encapsulated into poly(D,L-lactide-co-glycolide (PLGA) microspheres along with PPIX. Upon irradiation with x-rays (90 kV), energy transfer from the LaF3:Ce3+/DMSO nanoparticles to PPIX occurs and singlet oxygen is generated for cancer cell damage. Results: The LaF3:Ce3+/DMSO/PLGA or LaF3:Ce3+/DMSO/PPIX/PLGA microspheres alone caused only sublethal cytotoxicity to the cancer cells. Upon x-ray irradiation, the LaF3:Ce3+/DMSO/PPIX/PLGA microspheres induced oxidative stress, mitochondrial damage and DNA fragmentation on prostate cancer cells (PC3). Discussion: The results indicate that x-rays can activate LaF3:Ce3+ and PPIX nanocomposites, which can be a novel method for cancer destruction. Original submitted 7 June 2013; Revised submitted 25 September 2013

Colloidal gold nanoparticles intensify the anticancer response of the drug bortezomib, a proteasome inhibitor. Polyethylene glycol-coated gold nanoparticles and the drug show a synergistic effect in reducing the cell viability of prostate cancer cell line Du145. It was observed a significant cell viability reduction with bortezomib concentrations as low as 4 nM. The proteasome inhibitor alone had to be present at concentrations in the ranger of 120 nM to induce identical cytotoxicity response. These findings demonstrate that gold nanoparticles enhancing the permeation and retention (EPR) effect in Du145 cells and open the possibility to decrease multi-drug resistance (MDR). The in vitro results of functionalized gold nanoparticles, internalized by cancer cells, pave the way for a more efficient proteasome inhibitor delivery and release in adenocarcinoma cells. Copyright

Gold nanoparticles are versatile vectors for biological applications including in cancer therapy and diagnosis. Combining gold nanoparticles/anticancer drugs with X-rays radiation may contribute to tumor eradication and treatment efficacy. The conjugation of a proteasome inhibitor (BTZ) with functionalized gold nanoparticles were tested. The drug activity and the anticancer effects in human prostate cancer cells (Du-145) were evaluated. It was observed that gold nanoparticles, with small diameters (24 nm), at concentrations of 0.1 nM are not toxic for Du-145 cells. Finally, nanoparticle parameters were discussed and new ideas are proposed to optimize the anticancer properties of conjugated gold nanoparticles leading to a better delivery of the drug, minimizing the side effects.

Objectives The inclusion 01 chemical penetration enhancers in a novel patch-based system for the delivery of 5-aminolevulinic acid (ALA) was examined in vitro and in vivo. Poor penetration of ALA has been implicated as the primary factor for low response rates achieved with topical ALA-based photodynamic therapy of thicker neoplastic lesions. such as nodular basal cell carcinomas. Methods Several chemical permeation enhancers (dimethylsulfoxide, Labrafac CC. Labrafac PG and Labrafil M1944CS) were incorporated into bioadhesive patches tailored to deliver 19 mg ALA/cm(2). Key findings In-vitro depth penetration studies into excised porcine skin showed that high concentrations of ALA (>9 mu mol/cm(3)) could be delivered to a depth of 1.875 mm. However, inclusion of permeation enhancers did not significantly increase ALA delivery, relative to the control (P > 0.05). In-vivo studies were in strong agreement with in-vitro results, with formulations containing chemical enhancers showing no improvement in delivery compared with the control. Conclusions The patches designed in this work are suited to defineable ALA delivery without the need to immobilise patients for up to 6 h, as IS common with the cream-under-occlusion approach. Overall, permeation enhancers were not found to markedly enhance the topical delivery of ALA. 11 chemical penetration enhancers may have a greater effect on the delivery of more lipophilic ALA prodrugs, which are thought to primarily permeate the stratum corneum via the intercellular pathway.

Aminolaevulinic acid (ALA) is known to poorly penetrate into thick lesions, such as nodular basal cell carcinomas Short chain ALA esters, possessing increased lipophilicity relative to their hydrophilic parent, have previously been shown to be highly efficient at inducing protoporphyrin IX (PpIX) production in cell culture, at equimolar concentrations. In contrast, in vitro skin permeation and in vivo animal studies, which up to now have compared prodrugs on a % w/vv basis, have failed to demonstrate such benefits For the first time, equimolar concentrations of ALA, methyl-ALA (m-ALA) and hexyl-ALA (h-ALA) have been incorporated into an o/w cream preparation. In vitro penetration studies into excised porcine skin revealed that increased levels of h-ALA, compared to ALA and m-ALA were found in the upper skin layers, at all drug loadings studied. Topical application of the formulations to nude murine skin in vivo, revealed that creams containing h-ALA induced significantly higher levels of peak PpIX fluorescence (F-max = 289.0) at low concentrations compared to m-ALA (F-max = 159.2) and ALA (F-max = 191 9). Importantly, this study indicates that when compared on an equimolar basis, h-ALA has improved skin penetration, leading to enhanced PpIX production compared to the parent drug and m-ALA (C) 2010 Wiley-Liss, Inc and the American Pharmacists Association J Pharm Sci 99 3486-3498, 2010

Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) is the most widely used form of PDT in clinical practice. Topical application of ALA leads to overproduction of the endogenous photosensitizer protoporphyrin IX (PpIX). ALA-PDT is efficient treatment of superficial skin lesions, but not for thicker lesions. The main reason for this is suboptimal penetration of ALA molecules through cellular membranes and through stratum corneum of intact skin. Different approaches (formulations, mechanical and physical penetration enhancers, ALA derivatives) are currently used to increase the penetration. The content and distribution of ALA intracellularly and in tissues is difficult to measure, but PpIX content, on a relative scale, can be easily measured by fluorimetric assays.

In photodynamic therapy (PDT) a combination of visible light and a sensitising drug causes the destruction of selected cells. Aminolaevulinic acid (ALA) has been widely used in topical PDT for over 15 years. However, ALA does not possess favourable physicochemical properties for skin penetration. Consequently, the clearance rates for difficult to treat lesions, such as nodular basal cell carcinomas are relatively low. For the first time, equimolar concentrations of ALA, methyl-ALA (m-ALA) and hexyl-ALA (h-ALA) have been incorporated into a bioadhesive patch-based system. In vitro penetration studies into excised porcine skin revealed that ALA patches containing relatively high loadings (226.7 μmol cm −2 ) were associated with significantly greater tissue concentrations (70.7 μmol cm −3 ) than patches containing m-ALA (16.3 μmol cm −3 ) or h-ALA (17.4 μmol cm −3 ). ALA was also found to be the most efficient inducer of protoporphyrin (PpIX) fluorescence in mice, in vivo (maximum mean fluorescence: ALA = 236.2 a.u., m-ALA = 175.1 a.u., h-ALA = 193.5 a.u.). However, when the lipophilic hexylester was formulated in a pressure sensitive adhesive (PSA) patch, significantly higher PpIX levels were achieved compared to all bioadhesive systems tested. Of major importance, PSA patches containing relatively low h-ALA loadings induced high PpIX levels, which were localised to the application area. This study has highlighted the importance of rational selection of both the active agent and the delivery system. Bioadhesive preparations containing ALA are ideal for delivery to moist environments; whereas h-ALA-loaded PSA systems may facilitate enhanced delivery to dry areas of skin. In addition, owing to the relatively low loadings of h-ALA required in PSA patches, the costs of clinical PDT may potentially be reduced.

Silicon microneedle (MN) arrays were used to puncture excised murine and porcine skin in vitro and transdermal and intradermal delivery of meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP) investigated using topical application of a bioadhesive patch containing 19 mg TMP cm(-2). Animal studies, using nude mice, were then conducted to investigate the in vivo performance of the bioadhesive patch following MN puncture of skin. MN puncture significantly enhanced both intradermal and transdermal delivery of TMP in vitro, though the total amounts of drug delivered (25.22% into porcine skin and 0.07% across murine skin) were still quite small in each case. Notwithstanding this, in vivo experiments showed that MN puncture was capable of permitting a prolonged increase in TMP fluorescence at the site of application. Importantly, fluorescence was negligible at distant sites, meaning systemic delivery of the drug was not sufficient to induce TMP accumulation other than at the application site. In this study we have conclusively demonstrated proof of principle; MN puncture allows true intradermal delivery of a preformed photosensitizer in animal skin models in vitro and in vivo. Importantly, transdermal delivery was much reduced in each case. Increasing MN density would allow increased amounts of photosensitizer to be delivered. However, as MNs create aqueous pores in the stratum corneum, a preformed photosensitizer must possess at least some degree of water solubility in order to permit enhanced intradermal delivery in this way. We believe that use of MN array technology in this way has the potential to significantly improve topical photodynamic therapy of skin tumors.

Summary The fluorescence kinetics of a new photosensitizer for photodynamic therapy, di- l -arginine protoporphyrinate (PP(Arg)2), was studied in the skin of healthy mice. Furthermore, induction of necrosis in WiDr human colon adenocarcinoma xenografts in athymic nude mice was studied after photodynamic therapy (PDT) with PP(Arg)2. After intravenous administration of PP(Arg)2 maximal fluorescence was reached after 72 h in normal mouse skin. Complete elimination of the drug from the mouse skin was not found even after 32 days. Exposure of WiDr tumours in mice to red light (λ = 632 nm, fluence 150 J/cm2, fluence rate 250 mW/cm2) 24 and 72 h after intravenous administration of 10 mg/kg of PP(Arg)2 caused extensive tumour necrosis. Epidermal damage and infiltration of inflammatory cells was seen 24 h after light exposure but not after 72 h.

5-Aminolevulinic acid (ALA) is now being increasingly used in photodynamic therapy (PDT) and photodiagnosis (PDD) of lesions of the oral cavity, esophagus, and female genital tract. Clearly, topically applied creams and solutions are not the most suitable drug delivery systems for such moist and irregularly shaped areas, where shear forces may be high. As a result, such areas of the body are currently relatively inaccessible to efficient topical delivery of ALA. The use of a bioadhesive patch for topical delivery of ALA to the lower female genital tract and now, for the first time, to the human lip, is described. Use of the patch, containing 38 mg ALA cm-2, allowed successful PDT of vulval intraepithelial neoplasia lesions and induced protoporphyrin IX production in Herpes simplex lesions of the lip.

Our previous data indicate, that hematoporphyrin dimethyl ether (HPde) can totally inhibit the growth of aggressive Ehrlich ascite tumour, when combined with low doses (2Gy) of ionizing radiation. Taking into account these findings, it appears of particular interest to evaluate the dependence of radiosensitizing efficiency of porphyrins on tumour aggressiveness. For this purpose two experimental tumour models (aggressive murine Ehrlich ascite carcinoma, (EAT), and not-aggressive hepatoma MH-22A) were used. Moreover, radiosensitizing properties of three porphyrin-type compounds of different chemical heterogeneity were evaluated (hematoporphyrin dimethyl ether (HPde), photofrin II (PII) and hematoporphyrin derivative (HPD)). Data obtained indicate, that HPde is the most effective one in this context (HPde>PII>HPD). It is important to note, that only the aggressive EAT tumours were radiosensitized by these dyes. No signs of radiosensitization (inhibition of tumour growth, injury of tumour tissue, evaluated by histological analysis) were observed in not-aggressive MH-22A hepatoma. Moreover, it was shown, that ligands of peripheral benzodiazepine receptors (PBR) might diminish the cell growth in aggressive EAT, but not in not-aggressive MH-22A hepatoma. The mechanism of radiosensitization by porphyrins, proposed in our previous studies, was strongly confirmed by these data. Actually, dicarboxylic porphyrins, being ligands of PBR, which are highly expressed in just aggressive tumours, can inhibit tumour cell proliferation and act in concert with ionizing radiation. Thus, combination of porphyrin and ionising radiation reflects the action of two antiproliferative factors, what eventually increases the response of aggressive tumours to the low doses of ionising radiation.

5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy (PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370-450 nm blue light, 0.6 mW/cm(2) after 2-h incubation with 1 mM ALA) has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.

Aqueous solutions of aluminium phthalocyanine tetrasulphonate (AlPcS(4)) were investigated by means of absorption and fluorescence spectroscopy. The absorption spectrum of AlPcS(4) is independent of concentration in a wide range (from 10(-8) to 10(-4) M). The fluorescence spectrum measured with a standard setup is strongly dependent on AlPcS(4) concentration, and the fluorescence maximum is gradually red-shifted with increasing concentration. Calculations that take into account reabsorption of fluorescence (inner-filter effect) fit the experimental observations at low concentrations (up to 10(-6) M). Disagreement between the calculations and spectra recorded at higher concentrations (above 10(-5) M) shows that the reabsorbed light may be reemitted as fluorescence. The influence of inner-filter effects on the spectral shape was demonstrated by the experiments where a fibre-optic front-face fluorescence setup was applied: Under such conditions the shape of the fluorescence spectra for a high concentration (10(-3) M) coincided with that of a low concentration (10(-8) M). In conclusion, the present spectroscopic results show that AlPcS(4) does not form aggregates and is a very stable compound in aqueous solutions.

Many medical applications, including photodynamic therapy for cancer (PDT), involve the use of lasers. However, the coherence of laser light is not necessary for PDT, and attempts have been made to construct non-coherent light sources for PDT, which are relatively inexpensive, stable and easy to operate, require simple maintenance but differ fundamentally from the lasers in their output characteristics. In the present work we compared two clinically used lamps, CureLight1, which is a broadband source (560–740 nm) based on a filtered halogen lamp, and CureLight2, which is a narrowband source based on light-emitting diodes (LEDs), with respect to several parameters of crucial significance for PDT efficiency in vivo: (a) depth of action in tissues, (b) heating effects, (c) pain generation, (d) photodegradation of PpIX in solution, in cells and in mouse skin and (e) photo-inactivation of cells in vitro. We conclude that CureLight2 (LED), relative to CureLight1 (halogen) has deeper PDT action in tissue, similar efficiency for bleaching PpIX in mouse skin, better efficiency for bleaching PpIX in cells and solutions and good efficiency for inactivating cells in vitro. CureLight2 gives less heating of the tissue and less pain in unsensitised human skin. All these differences are related to difference in the spectra of the lamps. Thus, PDT light sources with emissions that are visually similar have significantly different photobiological properties.

Ester derivatives of 5-aminolevulinic acid (ALA-esters) have been proposed as alternative drugs for ALA in photodynamic therapy. After topical application of creams containing ALA, ALA methylester (ALA-Me), ALA hexylester (ALA-Hex) and ALA octylester (ALA-Oct) on mouse skin, typical fluorescence excitation and emission spectra of protoporphyrin IX (PpIX) were recorded, exhibiting a similar spectral shape for all the drugs in the range of concentrations (0.5-20%) studied. The accumulation kinetics of PpIX followed nearly a similar profile for all the drug formulations. The fluorescence of PpIX peaked at around 6-12 h of continuous cream application. Nevertheless, some differences in pharmacokinetics were noticed. For ALA cream, the highest PpIX fluorescence was achieved using 20% of ALA in an ointment. Conversely, 10% of ALA-Me and ALA-Hex, but not of ALA-Oct, in the cream was more efficient (P < 0.05) than was 20%. The cream becomes rather fluid when 20% of any of these ALA-esters is used in ointment, whereas 10% and lower concentrations of ALA-esters do not significantly increase fluidity of the cream. The dependence of PpIX accumulation on the concentration of ALA and ALA-ester in the applied cream followed (P < 0.002) kinetics as described by a mathematical model based on the Michaelis-Menten equation for enzymatic processes. Under the present conditions, the PpIX amount in the skin increased by around 50% by the application of ALA-Me, ALA-Hex or ALA-Oct for 4-12 h as compared with ALA for the same period. Observations of the mice under exposure to blue light showed that after 8-24 h of continuous application of ALA, the whole mouse was fluorescent, whereas in the case of ALA-Me, ALA-Hex and ALA-Oct the fluorescence of PpIX was located only at the area of initial cream application. The amount of the active compound in the applied cream necessary to induce 90% of the maximal amount of PpIX was determined for normal mouse skin. Optimal PpIX fluorescence can be attained using around 5% ALA, 10% ALA-Me and 5% ALA-Hex creams during short application times (2-4 h). Topical application of ALA-Oct may not gain optimal PpIX accumulation for short applications (

Photodynamic therapy (PDT) is a recently approved treatment modality that involves the sequential administration of a photosensitizer or its precursor and light to generate singlet oxygen for treating diseased tissue. The use of topical aminolevulinic acid (ALA) and blue light for nonhypertrophic actinic keratoses currently represents the only approved dermatologic application for PDT in the U.S.A. ALA is a photosensitizer precursor that is metabolized by cells into protoporphyrin IX (PpIX), which can be subsequently activated by visible light. PDT with topical ALA has been shown to improve psoriasis, but post-treatment hyperpigmentation as well as inconsistent clinical responses despite repeated PDT sessions have limited the development of this treatment approach for psoriasis. Furthermore the use of topical PDT photosensitizers becomes somewhat impractical for treating larger body surface areas in patients with extensive psoriasis. We have recently shown that oral administration of ALA induces preferential accumulation of PpIX in psoriatic plaques. The objectives of this study were to evaluate the effects of PDT with blue light on psoriatic plaques after systemic ALA administration as well as to determine whether systemic ALA-PDT induces apoptosis in lesional T lymphocytes. It has been suggested that induction of apoptosis in lesional T lymphocytes may be indicative of longer remission time following treatment of psoriasis.

Accumulation of protoporphyrin IX (PpIX) was investigated in normal skin and UV-induced tumours in hairless mice after topical application of a cream containing 2, 8 or 16% of 5-aminolevulinic acid methyl ester (ALA-Me). Higher levels of PpIX were measured in tumours compared to normal skin. The maximal amount of PpIX was reached at 1.5, 3 and 4 h after 2, 8 and 16% ALA-Me application, respectively. Higher tumour to normal skin PpIX fluorescence ratios were measured after application of 8 and 16% ALA-Me than after application of 2%. After irradiation with a broad spectrum of visible light from a slide projector, more than 90% of PpIX was bleached by fluences of 36 and 48 J/cm2, at fluence rates of 10 and 40 mW/cm2 respectively. At these fluences, the PpIX photobleaching rate was significantly higher (P

The skin of nude mice was exposed to erythemogenic doses of UV radiation, which resulted in erythema with edema. An ointment containing 5-aminolevulinic acid (ALA) was topically applied on mouse and human skin. Differences in the kinetics of protoporphyrin accumulation were investigated in normal and UV-exposed skin. At 24 and 48 h after UV exposure, skin produced significantly less protoporphyrin IX (PpIX) than skin unexposed to UV. Human skin on body sites frequently exposed to solar radiation (the lower arm) also produced less PpIX than skin exposed more rarely to the sun (the upper arm). It is concluded that UV radiation introduces persisting changes in the skin, relevant to its capability of producing PpIX from ALA. The observed differences in ALA-induced PpIX fluorescence may be the result of altered penetration of ALA through the stratum corneum or altered metabolizing ability of normal and UV-exposed skin (or both).

Photobleaching and phototransformation of protoporphyrin IX (PpIX) was investigated in normal mouse skin. The PpIX was induced by topical application of 5-aminolaevulinic acid (ALA). Exposure to laser light (635 nm) caused photobleaching of PpIX fluorescence and formation of fluorescent products. Analysis of the fluorescence spectra revealed appearance of new fluorescent photoproducts during light exposure. The main photoproduct, supposedly chlorin-type photoprotoporphyrin (PPp), exhibited fluorescence with an emission maximum at 675 nm. The other products exhibited main fluorescence peaks at around 588 and 623 nm that can presumably be attributed to an endogenous metallo-porphyrin and water-soluble porphyrin(s), respectively. Our results indicate that light exposure causes alterations in the enzymatic pathway of PpIX synthesis from ALA and leads to accumulation of intermediate water-soluble porphyrins. ALA-induced porphyrins are transported away from the treated area and partly deposited in remote skin sites.

Photostability studies of porphyrin-type sensitizers performed in aqueous solutions, model systems (sensitizers bound to human serum albumin or in a suspension of released erythrocyte ghosts) and in tumour tissue reveal that similar photomodification takes place in all investigated environments: photobleaching during exposure to light is followed by the formation of red-absorbing photoproducts. Photoproducts are not formed in the solutions of the less photostable sensitizer chlorin e 6 and the most photostable sensitizer aluminium phthalocyanine tetrasulphonate. Interaction with photooxidizible biomolecules increases the photobleaching rate of porphyrin-type sensitizers. This should be taken into account in the estimation of optimal photodynamic doses. The modified formula including photodestruction of sensitizer and formation of photoproduct is suggested.

Normal skin of nude mice (Balb/c) was treated topically with 5-aminolevulinic acid (ALA) and its methyl ester (ALA-Me) for 24 hours. Approximately 0.1 gram of freshly prepared cream was applied to a spot of 1 cm2 on the flank of the mice, which was then covered with a transparent dressing. The ALA induced protoporphyrin IX (PpIX) was studied by means of a noninvasive fiber-optic fluorescence probe connected to a luminescence spectrometer. The excitation wavelength was 407 nm, and the emission wavelength was 637 nm. For the first hour a slight lag in PpIX production was observed for the mice treated with ALA-Me compared to the mice treated with ALA. After approximately 12 hours the ALA and the ALA-Me treated mice showed the same PpIX fluorescence intensity. From 12 hours until 24 hours the PpIX fluorescence intensity decreased for both treatment modalities, even though ALA and ALA-Me were continuously present. At 24 hours ALA-Me-treated mice had less than half the amount of PpIX in their skin compared with ALA- treated mice.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The clearance of protoporphyrin IX (PpIX) from the skin of hairless BALB/c mice after topical application of 5- aminolevulinic acid (ALA) and its methyl ester (ALA-Me) was investigated. Creams containing 2 or 20% of ALA or ALA-Me were topically applied on spots of approximately 1 cm2 for 12 hours. The PpIX fluorescence was detected by the means of a Perkin Elmer LS50B luminescence spectrometer equipped with a fiber-optic probe. The emission spectrum was identical with that of cell-bound PpIX. After 12 hours application of ALA and ALA-Me similar amounts of PpIX were found. After creme removal the ALA-induced PpIX fluorescence decayed with a half-life of about 20 hours (20% ALA cream). The ALA-Me-induced PpIX was faster cleared from the skin than ALA-induced PpIX, and had a half-life of about 7 hours (20% ALA-Me cream).© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.