Oceanic Meteorological Conditions Influence Incidence of Aneurysmal Subarachnoid Hemorrhage.

OBJECTIVE: Publications concerning the weather pattern of occurrence of the subarachnoid hemorrhage have produced controversial results. We chose to study subarachnoid hemorrhage occurring in oceanic climate with deep variations focusing on partial oxygen volume (pO2) and patient history. METHODS: Seventy-one patients had been successively recruited from a single center 45 km from the Atlantic shore. Climate conditions had been analyzed from 72 hours before subarachnoid hemorrhage to 24 hours after. According to Dalton's law, climate conditions influence pO2, recalculated with Dupré's formula, and patient history analyzed and scored according to the induced oxidative stress. RESULTS: Subarachnoid hemorrhage risk is highest during spring and autumn, lowest between midnight and 6:00 a.m. Risk is highest after a period of atmospheric pressure higher than 1010 hPa (83%) and high pO2 and lowest for atmospheric pressure lower than 990 hPa and pO2 lower than 20.6. According to the medical history, 2 groups of patients could be identified: patients without history (22%), women (62%), high atmospheric pressure, and relatively lower pO2; and patients with a medical history, relatively lower atmospheric pressure, and higher pO2. Atmospheric pressure decreased significantly before disruption (994 hPa) but with a constant pO2. Subarachnoid hemorrhages during high atmospheric pressure were preceded by a decrease of pO2 despite a highly stable period of high atmospheric pressure. DISCUSSION: Atmospheric O2 changes and the subsequent oxidative stress could be the local ultimate trigger of subarachnoid hemorrhage that could result in the "ideal" fit of patient's health conditions with the meteorological environment.

Evidence for antioxidants consumption in the coronary blood of patients with an acute myocardial infarction.

Blood flow arrest and reperfusion during myocardial infarction (MI) cause myocyte and endothelium injury through oxidative stress and inflammation, both of which involve reactive oxygen species (ROS) and peroxides that consume antioxidant defenses. The aim of this study was to determine whether serum from the occluded coronary vessel has impaired anti-oxidative defenses as compared to serum from aortic blood or from the periphery of healthy controls. Forty-seven patients (44 men) were included for study. Inclusion criteria were chest pain, ST elevation, and cardiac troponin increase. A photoreaction producing a standardized amount of singlet oxygen ((1)O2), an excited form of oxygen, was performed in serum samples obtained during primary percutaneous coronary intervention (PCI). Immediately after laser light delivery to 5% sera containing 5 µg/mL rose bengal, dichloro-dihydro-fluorescein (DCFH) was added and its post-oxidation transformation into the fluorescent DCF, was recorded. At least 5 h after the start of symptoms, the mean secondary ROS production after (1)O2 delivery was increased in coronary sera (p < 0.001), but in aortic blood remained similar to that of healthy controls. The peak troponin value correlated with DCF fluorescence throughout the interval between symptoms onset and PCI. A high fluorescence was associated with a higher risk of MACE. These results show that oxidants secondary to (1)O2 are increased in occluded vessels during AMI in parallel to c-troponin, demonstrating that antioxidants are consumed. A O2 increase during reperfusion would thus extend the damage resulting from IDM necrosis. The effect of conditioning during PCI could be studied using the described method.

Antiproliferative activity of violaxanthin isolated from bioguided fractionation of Dunaliella tertiolecta extracts.

Dunaliella tertiolecta (DT) was chemically investigated to isolate molecules inhibiting cancer cell proliferation and inducing apoptosis in vitro. The potency to inhibit cell growth was used for the bio-guided fractionation and isolation of active compounds using chromatographic techniques. The DT dichloromethane extract exhibited a strong anti-proliferative activity on MCF-7 and LNCaP cells, and was further fractionated and sub-fractionated by RP-HPLC. High resolution mass spectrometry and spectrophotometric analysis unequivocally identified violaxanthin as the most antiproliferative molecule present in DT DCM extract. Violaxanthin purified from DT induced MCF-7 dose-dependent growth inhibition in continuous and discontinuous treatments, at concentrations as low as 0.1 µg·mL⁻¹ (0.17 µM). Phosphatidylserine exposure, typical of early apoptosis, was observed after 48 h treatment at 8 µg·mL⁻¹ (13.3 µM) but no DNA fragmentation, characteristic of late apoptosis steps, could be detected even after 72 h treatment at 40 µg·mL⁻¹ (66.7 µM). Taken together, our results demonstrate the strong antiproliferative activity of violaxanthin on one human mammary cancer cell line, and suggest that studying the pharmacology of violaxanthin and pharmacomodulated derivatives on cancer cells may allow potent antiproliferative drugs to be obtained.

Photodynamic treatment of culture medium containing serum induces long-lasting toxicity in vitro.

Photodynamic therapy (PDT) produces singlet oxygen and reactive oxygen species (ROS) that damage tumor cells and the vasculature. The resulting effect is a balance between photo-oxidations through primary or secondary ROS and scavenging activity. Sensitizers are distributed in the extracellular space before and during cell sensitization, suggesting that PDT could act directly on cell structures and on extracellular compartments, including sera. In this study we endeavored to determine whether the application of PDT to culture medium could affect cell survival. Culture medium [RPMI 1640 supplemented with fetal calf serum (FCS)] was incubated with Rose Bengal and irradiated before being added to cells for various contact times as a replacement for untreated medium. Cells were then kept in darkness until the survival assay. Treated medium reduced cell survival by up to 40% after 30 min of contact for 10 microg/ml of Rose Bengal and 20 J/cm(2). Rose Bengal or m-THPC alone or irradiated in water had no effect. This effect was dependent on the doses of Rose Bengal and light and decreased when FCS was replaced by human serum mixed with FCS. The reduction in survival observed with treated medium was more pronounced when the cell doubling time was shorter. Analysis of ROS or peroxide production in treated medium by DCFH added at the end of irradiation of Rose Bengal in serum-containing medium revealed a long-lasting oxidizing activity. Our findings support the hypothesis of an ROS- or peroxide-mediated, PDT-induced, long-lasting cell toxicity.

Secondary oxidants in human serum exposed to singlet oxygen: the influence of hemolysis.

Singlet oxygen (1O2) is produced by leucocytes during inflammatory reactions, various biochemical reactions and during photoreactions. It deactivates by reacting with a number of targets to produce reactive oxygen species (ROS) and peroxides (that in turn produce ROS). To verify whether serum had the same capability to deactivate secondary oxidants after exposure to 1O2, we provoked a photoreaction using rose bengal added to sera of 53 healthy donors and, after light delivery, reduced 2',7'-dichlorofluorescein (DCFH) was added at the end of irradiation and fluorescence of the oxidized derivative (DCF) was recorded. To avoid optical artifacts, we analyzed the influence of hemolysis. Deactivation capability of secondary oxidants after exposure to (1)O(2) was stable over a long period of time, slightly different between men and women, but standard biochemistry parameters had little influence. Hemolysis, age and platelet number reduced deactivation of 1O2-induced secondary oxidants. Addition of lysed cancer cells had no influence. Blood sampling in clot act tubes gave a better signal than in heparinized tubes. Red blood cells (RBCs) loaded with antioxidants strongly decreased deactivation of secondary oxidants. Assays are in progress to evaluate the clinical implications of these findings.

Photodynamic effects of SIM01, a new sensitizer, on experimental brain tumors in rats.

BACKGROUND: Glioblastomas are the third most common cause of cancer death in patients between 15 and 35 years old. Literature suggests that PDT could represent a promising treatment, providing that sensitizers could accumulate within the cancer tissues despite the blood-brain barrier. METHODS: Distribution and PDT effect of SIM01, a promising photosensitizer, have been evaluated on orthotopic C6 tumor model in rats by comparison with HPD and m-THPC. Pharmacokinetics had been analyzed with fluorescence and ROS. Photodynamic treatment was done using a 630-nm light with an energy density of 100 J cm(-2) for HPD and a 652-nm light with an energy density of 20 J cm(-2) for m-THPC and SIM01. RESULTS: The correlation between fluorescence and ROS dosimetry was found to be excellent. An optimal concentration was found after 12 hours for SIM01 (4 mg/kg), 24 hours for HPD (10 mg/kg), and 48 hours for m-THPC (4 mg/kg). The best normal tissue/cancer ratio of concentration had been found after 12 hours for SIM01 and 48 hours for HPD and m-THPC. Pathological examinations after PDT showed that the criteria for histology of glioblastic origin were absent in SIM01-treated rats 12 hours after injection but were present in 50% of rats treated 24 hours after injection and in all after a 48-hour delay. Mean survival of rats treated 12 or 24 hours after SIM01 injection was significantly improved compared with controls, HPD-, or m-THPC-treated groups. Survival of rats treated 12 or 24 hours after SIM01 injection reached 20 days but decreased for longer delays. On the contrary, survival reached 18 days at the maximum for rats treated 48 hours after m-THPC or HPD injection. CONCLUSIONS: Our results confirm that PDT is a promising treatment for glioblastomas. SIM01 efficacy is as efficient as m-THPC but with much more favorable pharmacokinetics.

The influence of storage conditions on delta amino levulinic acid induced toxicity and phototoxicity in vitro.

Delta amino levulinic acid photodynamic therapy (ALA-PDT) represents one of the most prominent advances in PDT. ALA itself or its derivatives are marketed for a variety of clinical indications. Despite the development of clinical applications, experimental ALA results are very heterogeneous and experimentally used parameters are still not standardized. This suggests that some problems remain unsolved that are likely to impair experiments to be performed but also that clinical results obtained could be greatly improved. Frequently unmentioned or imprecise data concern solvents, pH of ALA solutions, storage time, ALA degradation or ALA efficacy. In addition, diversity of experimental model is huge while capabilities of ALA transformation into PpIX are known to vary from one cell to the other. Thus, the aim of the present paper was to quantify the level of ALA degradation or changes in ALA efficacy using one single cell line without presuming of the mechanisms and determine the conditions of storage inducing the best transformation into PpIX and/or cell phototoxicity. We added ALA diluted in water, PBS or RPMI to C6 cells, a murine brain tumour cell line that can be used in vivo as an orthotopic graft. We measured in cells used as tools for final bio efficacy estimation, both the induced fluorescence and phototoxicity in various conditions of storage before use chosen to be as close as possible to the real lab conditions. Water had been found to better preserve ALA than, respectively, PBS and RPMI and this for any temperature or storage durations. The lowest temperature and the shortest duration for storage used had also been shown to better preserve ALA-induced fluorescence and phototoxicity. The fact that these properties were found to be better preserved in 7.4 buffered solvent could be in relationship with a fast ALA condensation occurring at neutral or lightly acidic pH modifying its availability for an optimal transformation into PpIX.

PDT in clinics: indications, results, and markets.

Photodynamic therapy (PDT) is based on the selective light activation of an exogenously given drug to patients. PDT acts mainly on cell membranes either of neovascular endothelial cells or of cancer cells leading to cancer cell death. Six drugs are now marketed based on clinical assays in various indications, which showed a clear cost efficiency as compared to other classical procedures. PDT is easy to handle and can be performed in medical installations fitting the conditions of health care in developing countries. Its cost effectiveness could represent an appropriate solution to the increasing number of cancers of various origin. However despite all the clinical results now available, PDT development remains slow. The reasons for this situation include cost of development, intellectual property, and competition between pharmaceutical companies.

Influence of Vitamins on Secondary Reactive Oxygen Species Production in Sera of Patients with Resectable NSCLC.

BACKGROUND: Singlet oxygen (¹O2) oxidizes targets through the production of secondary reactive oxygen species (SOS). Cancers induce oxidative stress changing with progression, the resulting antioxidant status differing from one patient to the other. The aim of this study was to determine the oxidative status of patients with resectable Non-Small cell lung cancers (NSCLC) and the potential influence of antioxidants, compared to sera from healthy donors. MATERIALS AND METHODS: Serum samples from 10 women and 28 men, 19 adenocarcinomas (ADK), 15 patients N1 or M1 were submitted to a photoreaction producing ¹O2. Then, samples were supplemented with vitamins (Vit C, Vit E), or glutathione (GSH). RESULTS: Squamous cell carcinomas (SCC) and metastatic SCCs induced a lower SOS rate. While Vit C increased SOS in controls as in patients with metastases, Vit E or the combination of Vit E and C strongly reduced SOS. GSH alone lightly decreased SOS in controls but had no effect in patients either alone or combined with Vit C. CONCLUSION: In "early" lung cancers, SOS are comparable or lower than for healthy persons. The role of Vitamins varies with gender, cancer type, and metastases. This suggests that an eventual supplementation should be performed on a per-patient basis to evidence any effect.

PDT for Barrett's esophagus: Status and unsolved problems.

PDT had been proposed in gastroenterology for various indications and the esophageal cancer treatment had been among the very first having been approved. However, PDT failed to be a real breakthrough. One reason for it was that although it had been approved for the palliative treatment of advanced tumors, PDT only has by nature a limited in-depth efficacy fitting better to the treatment and often the cure of "early cancers". For this reason PDT has also been proposed for the treatment of Barrett's esophagus (BE) with high-grade dysplasias. Barrett's mucosa (BM) is a field of a specialized metaplastic columnar epithelium replacing the normal stratified squamous epithelium or mucosa lining the distal esophagus. In this case, PDT has to destroy an area of thin tissues spread eventually over a wide area instead of a mass of tissues. Something important is that existing treatments allow the treatment of foci of dysplastic tissues but not the regression of the whole BM. BE is thus an unsolved medical problem having medical as well as economic consequences as BM being likely to transform into a cancer has to be carefully surveyed. The esophageal cancer, an adenocarcinoma, has to be surgically removed when it is possible something pretty heavy with a high morbidity. Economic burnt is also important with high survey costs independently to the additional surgical costs in case of diagnosed cancer. Treatments proposed for non or mild dysplastic BM regression have in common to have an inhomogenous impact on the target. Treatments for high-grade dysplasia (HGD, the ultimate pathological step before cancer) are based on mucosectomy and are limited to small areas of tissues. Recently circumferential mucosectomy had been proposed but at a higher risk making it suitable only to highly experienced hands in infrequent indications.

DNA damages after SIM01 photodynamic treatment.

Photodynamic therapy (PDT) is a new approach to cancer treatment for a variety of malignant tumors. A new photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01), had been evaluated for its genotoxic effects on glioma cells (C6). Comet assay had been used to evaluate the potential genotoxic effect induced by SIM01 on the C6 cells. When SIM01 had been shown to be a powerful sensitizer no DNA strand break was detected in the absence of light. SIM01 localized in cytoplasm but not in the nucleus of the tumors cells, which supported the finding of undetectable DNA damage under darkness and low photodynamic dose. Cell exposure to 20Jcm(-2) after an incubation time of 2h with 0, 0.25, 0.5, 2 or 4µgmL(-1) induced less than 25% of cell death but significant Tail Moment changes. If DNA damage intensity increased according to SIM01 doses under light exposure, importance of repair seemed to increase proportionally to PDT-induced damage. Positive controls consisted of doxorubicin-treated C6 cells this mutagen being known to induce genetic damage. Whatever the conditions used SIM01 appeared to be less deleterious than doxorubicin. As the comet assay can not give us the certitude that no mutation, photoadducts or oxidative damage had been developed under light exposure this point will have to be verified with another mutagenicity assay. SIM01 appears to be safe from a mutagenic point of view something of importance as tumors of small volume in patients with a long lifespan are at first indicated for PDT.

Indirect detection of photosensitizer ex vivo.

Photodynamic therapy induces the production of reactive oxygen species (ROS) within tissues exposed to laser light after administration of a sensitizer. In the context of continuing clinical and commercial development of chemicals with sensitizing properties, a minimally invasive assay is needed to determine the tissue kinetics of fluorescent or non-fluorescent photoreactive drugs. The level of ROS was determined ex vivo from 1 mm3 biopsy samples using 2'-7' dichlorofluorescin diacetate (DCFH-DA), a fluorescent probe which was converted into highly fluorescent dichlorofluorescein (DCF) in the presence of ROS. This assay was tested on meta(tetrahydroxyphenyl)chlorin (m-THPC, FOSCAN), a powerful and fluorescent sensitizer, and bacteriochlorophyll derivative WST09 (TOOKAD), a near-infrared absorbing sensitizer that is only slightly fluorescent. In conjunction with the ROS assay, the tissue accumulation of m-THPC was determined on biopsy samples using an optic fibre spectrofluorometer (OFS). DCF fluorescence was proportional to the level of oxidation induced by horseradish peroxidase used as a control and to the concentration (range: 0-5 microg x ml(-1)) of both selected photosensitizers irradiated in a tube together with DCFH. Regardless of the organ studied, an excellent correlation was found between fluorescence measurement by OFS and ROS determination for m-THPC. m-THPC (2 mg x kg(-1) iv) accumulation in tumour tissues was best after 48 h, and the best signal was obtained in liver. With non-fluorescent WST09 (2 mg x kg(-1)), ROS determination showed the best tumour uptake 48 h after injection, with a tumour/muscle ratio of 5.4. The ROS assay appears to be feasible for determining sensitizer concentration in regular grip biopsy tissue samples.

Secondary reactive oxygen species production after PDT during pulmonary tumor growth in sera of nude mice.

Photodynamic therapy (PDT), mediated by a sensitizer exposed to light to produce singlet oxygen ((1)O2), induces tumor responses varying from one person to another. Cancer growth induces oxidative stress at any step of its development from induction to treatment, which could also modify response to PDT. After the initial amount of (1)O2 delivered, secondary oxidative species (SOS) are also generated inducing additional damages. Using an in vitro assay we saw variations among mice strains concerning their serum capability to generate SOS after (1)O2 production. Nude mice had a higher capability to generate SOS as compared to the non mutated strain. Capability to generate SOS evolved during growth of orthotopically-grafted pulmonary cancers (A549), with either values corrected for hemolysis or not. Immediately after graft SOS production decreased, then increased again, reaching a plateau phase after 10 days which lasted for 20 days and finally increased steeply during the last phase of tumor growth, preceding cachexia and death. This profile differed profoundly from the one observed after heterotopic tumor grafts for which hemolysis induced artifacts masking important variations in SOS production. Our results demonstrate experimentally a relationship between the general health status of an individual, cancer progression and serum capability to generate SOS during PDT. These findings could explain some PDT failures as well as some unexpected successes on large tumors and should be taken into account when determining treatment parameters. They may also explain why different effects are observed on different experimental models with similar sensitizers.

Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke.

Oxidative stress is a leading cause of neuronal damage in ischemic stroke. Melatonin may play a role in the antioxidant response. Melatonin and its metabolites may be involved in the modulation of oxidative stress in human acute stroke. No data are available in humans to establish this relationship. In this context, on the first and the fifth days post-stroke, we assessed serum total antioxidant capacity (TAC) and urine levels of melatonin, 6-sulfatoxymelatonin (aMT6S), and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), the last compound being produced in the brain after reaction of melatonin with reactive oxygen species. Compared to controls' values, TAC and levels of melatonin and aMT6S were reduced, without difference between the first and the fifth days post-stroke, whereas AFMK levels remained in the normal range at both time points. Melatonin catabolism might be speeded up in acute ischemic stroke in order to increase the antioxidant response.

Increased singlet oxygen-induced secondary ROS production in the serum of cancer patients.

Photodynamic therapy (PDT) generates singlet oxygen ((1)O(2)) and Reactive Oxygen Species (ROS) that are counteracted by patient's defenses. As cancer treatments are among the most important PDT applications the aim of this pilot study was to determine whether the serum of cancer patients produces more or less secondary ROS or peroxides after a photoreaction as compared to healthy persons. Fifty-three volunteers and 105 cancer patients were recruited. The capacity of (1)O(2) or secondary oxidant production was found to be increased in 6 healthy donors and 36 cancer patients (23/69 women and 13/31 men p<0.007 and p<0.04) with a mean value of 1.52 as compared to 1.29 in the healthy subjects (p<0.05) when considering values higher than the normal range (norm=1±10%) or 1.1 vs. 0.85 (p<0.01) in the whole cohort. This increase correlated with a poor prognosis, TNM and SBR classification. Serum (1)O(2) deactivation capacity was impaired and secondary ROS were more produced during cancer progression. Although it is currently unclear whether this is the cause or effect of cancer, this finding may hold interest as a potential marker of cancer severity. It would also support the interest of PDT as an adjuvant for cancer treatment, even for aggressive tumors particularly when associated to surgery for bulk removal.

Influence of animal species on secondary oxidant activation in serum exposed to singlet oxygen.

Singlet oxygen ((1)O(2)) produced during inflammatory reactions and during photodynamic therapy deactivates by producing in tissues secondary reactive oxygen species and peroxides (SOS) as well as other degradation products. We investigated the influence of animal species on SOS production secondary to standardized (1)O(2) production by performing in vitro experiments with rose bengal as the (1)O(2) producer, human serum (HS) as a control, sera derived from various animal species, and dichloro-dihydro-fluorescein as a nonspecific marker that becomes fluorescent when oxidized. The overall SOS production in HS from a presumed healthy cohort of 53 donors (31 males and 22 females) gave a mean "normal" value of 0.91 compared with a previous pool of 75 male sera samples. SOS production after a photo-reaction was two or four times lower in HS than in fetal calf serum or mice sera, respectively. In mice, the "nude" characteristic increased even more than in the SOS production. In the Aves order, this production appeared to be distributed randomly according to the number of branches after the appearance of Amniotas. For primates, SOS production appeared to decrease linearly with the number of branches (R(2) = 0.98). Adding hemolysates from complete bloods to the corresponding sera induced an increase in SOS production in all species, proportional to the production in sera. These findings should be kept in mind when interpreting results from studies of secondary reactive oxygen species-induced pathways following (1)O(2) production, regardless of its origin.

Biophysical parameters influencing secondary oxidants activation in human serum exposed to singlet oxygen.

Singlet oxygen (¹O2), produced during photodynamic therapy, deactivates during its interaction with tissues by producing reactive oxygen species (ROS) and peroxides as well as other degradation products. Here we investigated the role of parameters of light delivery, O(2), and temperature on the ROS and peroxides production, secondary to ¹O2. A series of simple in vitro experiments has been performed with Rose Bengal (RB) as a ¹O2 producer, human serum (HS) as a target and dichlorofluorescein (DCFH) as a nonspecific marker, becoming fluorescent when oxidized. The overall secondary production of ROS and peroxides in HS had also been compared to fetal calf serum (FCS) or mice sera. Increasing power but with a same delivered energy decreased secondary ROS and peroxides when increasing power with a same duration for light delivery increased them. Increasing delivered energy increased linearly secondary ROS or peroxides. Delivering O2 by bubbling before light delivery increased secondary ROS or peroxides, when Ar decreased them. Delivering gases after light delivery had no influence on secondary ROS or peroxides production. Increasing temperature from 20 to 40 °C increased secondary ROS or peroxides production but freezing after light delivery either before or after measurement had only a mild influence. Secondary ROS or peroxides production was 2 or 4 times lower in HS than in FCS or nude mice sera respectively. PDT seems to consist of two subsequent phases, both linked but developing independently. The intensity of photo-reactions varied with the model, human sera producing less secondary ROS than fetal calf or mouse sera. Search for new sensitizers should consider secondary ROS-induced pathways in addition to ¹O2 production.

Serum resistance to singlet oxygen in patients with diabetes mellitus in comparison to healthy donors.

Diabetes mellitus causes endothelial injury through oxidative stress involving reactive oxygen species and peroxides as well as inflammation, both of which consume antioxidant defenses. Singlet oxygen ((1)O(2)) is produced by leukocytes during inflammatory and biochemical reactions and deactivated by producing reactive oxygen species and peroxides. To determine whether serum was capable of deactivating (1)O(2), we triggered a photo reaction in sera from 53 healthy donors and 52 diabetic patients. Immediately after light delivery, dichlorofluorescein was added and then its fluorescence was recorded. The mean capacity of (1)O(2) or secondary oxidant deactivation was reduced in patients with diabetes mellitus. Hemolysis reduced deactivation of (1)O(2)-induced secondary oxidants in both healthy and diabetic patients. Body mass index, age, platelet counts, and blood cell numbers exerted a nonlinear influence. High levels of glycated hemoglobin were associated with an increased deactivation of oxidative species, whereas high-density lipoprotein cholesterol, total cholesterol, and the total cholesterol to high-density lipoprotein cholesterol ratio decreased the serum deactivation capacity. Oral antidiabetics bore no influence on deactivation, which was restored by insulin in women. Deactivation capacity was lower in women, who had half the complications found in men, suggesting that, with more severe diabetes mellitus, protection was maintained against complications. Resistance to (1)O(2) should be considered during the monitoring of diabetes mellitus.

In vitro evaluation of Radachlorin sensitizer for photodynamic therapy.

This paper reports the evaluation of a new photosensitizer, Radachlorin in comparison with one of its well known components but used solely, Chlorin e(6). The photodynamic properties and cell uptake and localisation of the two drugs were compared. In vitro studies were conducted on human adenocarcinoma cells (HT29) and lung carcinoma cell line (A549). Both dyes showed an absorption maximum between 640 and 650 nm, but those absorption peaks are enhanced by interactions with serum, with a shifted maximum at 661 and 664 nm, and much higher absorbance. As Radachlorin is constituted of different products and as photoreactivity is dependent on absorbed light energy, we chose to adapt concentrations so that both drugs had the same absorption at the irradiation wavelength (664 nm) for photoreactivity tests, and express concentrations in optical density at 664 nm. The capacity of the two drugs to generate Reactive Oxygen Species was identical, but on HT29 cells, Radachlorin reaches its optimal LD50 sooner than Chlorin e(6). Radachlorin LD50 on HT29 cells was 0.0251 OD(664 nm) after 2 h and 0.0672 OD(664 nm) for Chlorin e(6) for a 20 J cm(-2) irradiation. Radachlorin gave very similar results on A549 cells, LD50 being 0.05 for 5 J irradiation, and 0.026 for 10 and 20 J cm(-2). Pharmacokinetics using fluorescence showed that, even if Radachlorin quickly crossed HT29 (a human colonic cancer line) cell membrane, cellular distribution evolved from a diffuse cytoplasmic repartition 1 hour after Radachlorin addition to a delimited localisation into organelles all around the nucleus. Radachlorin intracellular fluorescence decreased after 4 h, whereas we did not observe a decrease of Chlorin e(6) intracellular fluorescence for times up to 24 h. In both case, a quick decline was observed as soon as the culture medium was replaced with a drug-free one. Radachlorin appears to be an excellent photosensitizer, with similar phototoxicity to Chlorin e(6) on cell cultures, but with quicker kinetics, which could be an improvement if confirmed on further in vivo studies.

In vitro and in vivo evaluation of Radachlorin(R) sensitizer for photodynamic therapy.

This paper reports the evaluation of a new photosensitizer, Radachlorin in comparison with one of its well known components but used solely, Chlorin e6. The photodynamic properties, cell uptake and localisation of the 2 drugs were compared. In vitro studies were conducted on human adenocarcinoma cells (HT-29) and lung carcinoma cell line (A549). Both dyes showed an absorption maximum between 640 and 650 nm, that were enhanced by serum, with a shifted maximum at 661 nm. In vitro, phototoxicities of Radachlorin and Chlorin e6 were nearly identical for HT29 and A549 cells. However, Radachlorin reached its optimal LD50 sooner (0.59 microg ml(-1) for 3 h incubation followed by 20 J cm(-2) of 664 nm light (0.02 W cm(-2))) than Chlorin e6 (0.60 microg ml(-1) for 4 h incubation). For in vivo studies, Swiss athymic mice were grafted with human lung carcinoma of the line A549 15 days before intravenous photosensitizer injection. Fluorescence was recorded through an optical fibre spectrofluorimeter using the 666 nm peak for detection. Maximum Radachlorin fluorescence in tumor was observed 2 h after injection (1412 +/- 313 AU). Selectivity was expressed by the calculated tumor-to-skin and tumor-to-muscle ratios. Maximum ratios (1.45 +/- 0.14 for tumor-to-skin and 1.95 +/- 0.29 tumor-to-muscle) were observed 7 h after injection with Radachlorin. Maximal Chlorin e6 fluorescence was observed 1 h (shortest time interval measured) after injection in all organs and highest tumor-to-muscle ratio (2.56 +/- 0.97) 8 h after injection. Chlorin e6 fluorescence in skin was always at least equivalent to tumor fluorescence. Complete response of grafted tumor was achieved (no recurrence observed during 15 days) after 20 mg kg(-1) IV injection and 200 J cm(-2) irradiation (0.3 W cm(-2)) with both drugs. Optimal delays between injection and light delivery were between 1 and 7 h with Radachlorin and 3 h for Chlorin e6 but severe adverse effects were noted for both drugs when drug-light intervals were shorter than 3 h. This suggests that clinical use would be easier with Radachlorin than Chlorin e6.