Toru Hirano

Macular Carotenoid Levels of Normal Subjects and Age-Related Maculopathy Patients in a Japanese Population

PURPOSE Macular carotenoid pigments composed of lutein and zeaxanthin are thought to affect the development of age-related maculopathy (ARM). Macular carotenoid levels were measured in normal Japanese subjects and Japanese patients with ARM. DESIGN Observational case-control series. PARTICIPANTS One hundred normal eyes of 100 normal subjects and 187 eyes of 97 patients with ARM; all were Japanese. The definitions of early ARM and late ARM (exudative age-related macular degeneration [AMD] and dry AMD) were used according to an accepted international classification system. METHODS Macular carotenoid levels were measured using resonance Raman spectroscopy. MAIN OUTCOME MEASURE Raman signal intensity generated from carbon-carbon double bond vibrations of lutein and zeaxanthin. RESULTS The mean (+/-standard deviation [SD]) macular carotenoid level in normal subjects was 1471+/-540 Raman counts. The macular carotenoid levels in normal subjects declined with age. The mean macular carotenoid level was 620+/-204 (+/-SD) in eyes with early ARM and 427+/-283 (+/-SD) in eyes with late ARM (equal to AMD). The macular carotenoid levels of early ARM and AMD were significantly lower than those in normal subjects older than 60 years (1100+/-340 [+/-SD]). No difference was revealed in carotenoid levels by the severity for ARM, type of AMD (exudative, atrophic, and disciform scar), or types of choroidal neovascularization (classic, minimally classic, occult, polypoidal choroidal vasculopathy), although small numbers in some groups weakened statistical power. Macular carotenoid levels were affected by the severity of macular disease in the opposing eye. The average for normal eyes where AMD was found in the opposite eye was significantly lower than that of normal eyes in the absence of AMD in the opposite eye (i.e., healthy volunteers older than 60 years). CONCLUSIONS Macular carotenoids decreased even in older healthy individuals. The ARM patients showed lower macular carotenoid levels than healthy people. Low macular carotenoid levels may be one of the risk factors of progression in ARM.

Monitoring of singlet oxygen is useful for predicting the photodynamic effects in the treatment for experimental glioma.

Purpose: Singlet oxygen (1O2) generated in photodynamic therapy (PDT) plays a very important role in killing tumor cells. Using a new near-IR photomultiplier tube system, we monitored the real-time production of 1O2 during PDT and thus investigated the relationship between the 1O2 production and photodynamic effects. Experimental Design: We did PDT in 9L gliosarcoma cells in vitro and in an experimental tumor model in vivo using 5-aminolevulinic acid and nanosecond-pulsed dye laser. During this time, we monitored 1O2 using this system. Moreover, based on the 1O2 monitoring, we set the different conditions of laser exposure and investigated whether they could affect the tumor cell death. Results: We could observe the temporal changes of 1O2 production during PDT in detail. At a low fluence rate the 1O2 signal gradually decreased with a low peak, whereas at a high fluence rate it decreased immediately with a high peak. Consequently, the cumulative 1O2 at a low fluence rate was higher, which thus induced a strong photodynamic effect. The proportion of apoptosis to necrosis might therefore be dependent on the peak and duration of the 1O2 signal. A low fluence rate tended to induce apoptotic change, whereas a high fluence rate tended to induce necrotic change. Conclusions: The results of this study suggested that the monitoring of 1O2 enables us to predict the photodynamic effect, allowing us to select the optimal laser conditions for each patient.

The Microenvironment of DNA Switches the Activity of Singlet Oxygen Generation Photosensitized by Berberine and Palmatine

The effect of the interaction between DNA and the photosensitizer on photosensitized singlet oxygen (1O2) generation was investigated using DNA‐binding alkaloids, berberine and palmatine. These photosensitizers were bound to DNA by electrostatic force. Near‐infrared luminescence measurement demonstrated that the photoexcited alkaloids can generate 1O2 only when the photosensitizers are bound to DNA. A fluorescence decay study showed significant enhancement of the lifetime of their photoexcited state with the DNA binding. A calculation study suggested that the electrostatic interaction with DNA inhibits the quenching of the photoexcited state of these alkaloids via intramolecular electron transfer, leading to the prolongation of the lifetime of their excited state. This effect should enhance their intersystem crossing and the yield of energy transfer to molecular oxygen. The results show that the electrostatic interaction with DNA significantly affects the 1O2 generation activity of a photosensitizer. In addition, this interaction may be applied to the control and the design of photosensitizers for medical applications such as photodynamic therapy.

Photodynamic Therapy for Experimental Tumors Using ATX‐S10(Na), a Hydrophilic Chlorin Photosensitizer, and Diode Laser

ATX‐S10(Na), a hydrophilic chlorin photosensitizer having an absorption maximum at 670 nm, is a candidate second‐generation photosensitizer for use in photodynamic therapy (PDT) for cancer treatment. The effectiveness of PDT using ATX‐S10(Na) and a diode laser for experimental tumors was evaluated in vitro and in vivo. In‐vitro PDT using ATX‐S10(Na) and the diode laser showed drug concentration‐, laser dose‐ and drug exposure time‐dependent cytotoxicity to various human and mouse tumor cell lines. In Meth‐A sarcoma‐implanted mice, optimal PDT conditions were found where tumors were completely eliminated without any toxicity. Against human tumor xenografts in nude mice, the combined use of 5 mg/kg ATX‐S10(Na) and 200 J/cm2 laser irradiation 3 h after ATX‐S10(Na) administration showed excellent anti‐tumor activity, and its efficacy was almost the same as that of PDT using 20 mg/kg porfimer sodium and a 100 J/cm2 excimer dye laser 48 h after porfimer sodium injection. Microscopic observation of tumor tissues revealed that PDT using ATX‐S10(Na) and the diode laser induced congestion, thrombus and degeneration of endothelial cells in tumor vessels, indicating that a vascular shutdown effect plays an important role in the anti‐tumor activity of PDT using ATX‐S10(Na) and the diode laser.

Dynamics of singlet oxygen generation by DNA-binding photosensitizers.

The dynamics of photosensitized singlet oxygen generation in a DNA microenvironment were examined using the DNA-binding photosensitizers berberine and palmatine. These photosensitizers generate singlet oxygen only under interaction with DNA because the singlet excited state deactivates rapidly in a nonbinding environment. A kinetic study demonstrated the reaction process whereby singlet oxygen is generated through energy transfer from the triplet excited state of DNA-binding berberine (or palmatine) to molecular oxygen. The guanine-containing sequence of DNA slightly deactivated the singlet excited state of the photosensitizers, resulting in a decrease of the singlet oxygen yield. By the steric hindrance of the DNA strand, the rate constant of the singlet oxygen generation became smaller than that of the other water-soluble photosensitizer.

Liposomal photofrin enhances therapeutic efficacy of photodynamic therapy against the human gastric cancer.

Photodynamic therapy (PDT) has been established as a potent and less invasive treatment for gastrointestinal tumors. The aim of the present study was to investigate whether or not liposomalization of the photosensitizer enhanced the therapeutic efficacy of PDT. Photofrin (PF) was entrapped in multilammelar liposomes. Mice implanted with a human gastric cancer xenograft, were divided into a PF group and a liposomal photofrin (LPF) group and intravenously administered 10 mg/kg of PF or LPF (as a dose of PF), respectively. At 8 h after injection PF level in tumor tissue in the LPF group was significantly higher level by 2.4-fold of that in the PF group, whereas the PF levels in the skin were almost equal. Irradiation was performed with the excimer dye laser at 150 mW/cm(2), total dose 40 J, at 8 h after PF or LPF administration. The results revealed that the volume of necrotic tumor tissue was significantly higher in the LPF group than in the PF group. The apoptotic index of the tumor was also significantly higher in the LPF group. In conclusion, the liposomalization of the photosensitizer increased its tumor accumulation, with a resulting enhancement of the therapeutic effect of PDT.

In vitro Plasma Protein Binding and Cellular Uptake of ATX‐S10(Na), a Hydrophilic Chlorin Photosensitizer

ATX‐S10(Na), a hydrophilic chlorin photosensitizer having an absorption maximum at 670 nm, is a candidate second‐generation photosensitizer for photodynamic therapy (PDT) for cancer treatment. In this study, we examined plasma protein binding, cellular uptake and subcellular targets of ATX‐S10(Na) in vitro. Protein binding ratios of 50 μg/ml ATX‐S10(Na) in rat, dog and human plasma were 73.0%, 87.2% and 97.7%, respectively. Gel filtration chromatography revealed that 1 mg/ml ATX‐S10(Na) bound mainly to high‐density lipoprotein (HDL) and serum albumin at the protein concentration of 0.4%, with binding ratios of 46% and 36%, respectively. The free form of ATX‐S10(Na) was mostly incorporated into T.Tn cells, and its cellular uptake was partially but significantly inhibited by endocytosis inhibitors such as phenylarsine oxide, chloroquine, monensin and phenylglyoxal, and by chilling the cells to 4°C. However, ouabain, harmaline, sodium cyanide, probenecid and aspartic acid did not influence the uptake of ATX‐S10(Na), suggesting that cellular uptake of ATX‐S10(Na) was not related to sodium‐potassium pump activity, sodium‐dependent transporter activity, mitochondrial oxidative respiration, organic anion transporter activity or aspartic acid transporter activity. By fluorescence microscopy, lysosomal localization of ATX‐S10(Na) was observed in T.Tn cells. However, electron microscopic observation revealed that many subcellular organelles such as mitochondria, endoplasmic reticulum, ribosomes, Golgi complex and plasma membrane were damaged by PDT using 25 μg/ml ATX‐S10(Na) soon after laser irradiation at 50 J/cm2, and tumor necrosis was rapidly induced. This result indicated that ATX‐S10(Na) was widely distributed within the cell.

Interstitial photodynamic therapy with rotating and reciprocating optical fibers

Photodynamic therapy (PDT) is an effective treatment modality that allows selective destruction of malignant tumor cells. However, because of the difficulty in exposing deeper areas of tumors, the modality has strictly limited indications. In this study, the authors introduce a new method for delivering laser light to a three‐dimensional, wide area with the purpose of improving the therapeutic value of PDT.

Structure Effect on Antioxidant Activity of Catecholamines toward Singlet Oxygen and Other Reactive Oxygen Species in vitro

The reactivity of catecholamine neurotransmitters and the related metabolites were precisely investigated toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and reactive oxygen species. Catecholamines reacted immediately with DPPH radicals, their reactivity being stronger than that of ascorbic acid as a reference. Superoxide scavenging activities of catecholamines determined by WST-1 and electron spin resonance (ESR) spin trapping methods were also high. Whereas tyrosine, the dopamine precursor showed no reactivity toward superoxide. The reactivity toward singlet oxygen was evaluated by observing specific photon emission from singlet oxygen. The results revealed that reactivity of catecholamines was markedly higher than that of sodium azide, and catechin as catechol reference. The reaction of catecholamines and singlet oxygen was further studied by ESR using 55-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping reagent and rose bengal as photosensitizer. DMPO-OH signal of epinephrine was significantly small compared to other catecholamines, catechin, and 4-methylcatechol as a reference compound and was as small as that of tyrosine. The signal formation was totally dependent on singlet oxygen, and the presence of catechol compounds. These results indicated that epinephrine is the most potent singlet oxygen quencher than other catecholamines, and the secondary amino group in its alkyl side chain could play a role in unique singlet oxygen quenching property of epinephrine.

Phototoxicity of coproporphyrin as a novel photodynamic therapy was enhanced by liposomalization

This study attempted the liposomalization of coproporphyrin I (CPI) with hydrophobic properties. Liposomalization of CPI was not successful at any pH when using lactate buffer. In contrast, when using 9% sucrose/10mM phosphate buffer (pH 7.8), CPI liposomes (Lipo-CPI) and polyethyleneglycol (PEG) modified liposomes (PEG-CPI) were prepared with a high entrapment ratio of CPI and small particle size. Plasma CPI concentration at 6h after PEG-CPI injection were 6.5-fold greater than that after the injection of Lipo-CPI. In tumors, the CPI concentration was higher after PEG-CPI injection than after Lipo-CPI or CPI solution. Therefore, PEG-CPI was likely to increase blood circulation and achieve greater accumulation of CPI in the tumor. When loaded into tumor cells, photosensitizers generate singlet oxygen during laser irradiation, resulting in the induction of necrosis in the cells. The order of magnitude of CPI tumor cells uptake was PEG-CPI>Lipo-CPI>CPI solution. Thus, the PEG modification of CPI liposomes improved its tumor cell uptake. Furthermore, it is likely that the order of the ability to produce singlet oxygen was PEG-CPI [symbol: see text] Lipo-CPI>CPI solution. The cytotoxicity of PEG-CPI was significantly greater than the other formulations, suggesting that the cytotoxicity reflected the CPI concentration in tumor cells. In conclusion, PEG-CPI was confirmed to show effective tissue distribution, elevated CPI concentration in the tumor cells, to produce singlet oxygen, and cytotoxicity by PDT.