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Background: The lipophilic photosensitizer, TONS 501, is a novel porphyrin-derived methyl ester that was developed for photodynamic antimicrobial chemotherapy. This study developed a hydrophilic and anionic porphyrin salt of this compound (TONS 501-Na) for use in photodynamic therapy (PDT). This chlorin derivative is synthesized from the protoporphyrin IX dimethyl ester., Materials and Methods: We investigated the in vitro cytotoxic effects of TONS 501-Na-mediated PDT on EMT6 mouse breast cancer cells. EMT6 cells were incubated with 0-100 μg/ml TONS 501-Na for 24 h prior to replacing the culture medium and exposing the cells to 6 mW/cm 2 diode laser irradiation at 0-13 J/cm 2 to induce PDT. Morphological changes and cell viability were evaluated 24 h after PDT. The percentages of apoptotic cells were evaluated 4 h and 24 h after PDT., Results: The concentrations of TONS 501-Na that killed 50% of EMT6 cells after exposure to light doses of 0, 0.4, 3, 6, or 13 J/cm 2 were 84.6, 33.2, 18, 8.2, and 2.2 μg/ml, respectively. Tumor cells exposed to PDT showed chromatin condensation and fragmentation. The percentages of apoptotic cells increased in a TONS 501-Na concentration-dependent manner in the PDT group, and were significantly higher than those in the control group or in cells treated with TONS 501-Na or laser irradiation alone., Conclusion: TONS 501-Na-mediated PDT induced mouse breast cancer cell death in a concentration-dependent manner. Future studies should evaluate the in vivo pharmacokinetics, tissue distribution, and photodynamic effects of TONS 501-Na., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

New disodium mercaptoundecahydro-closo-dodecaborate (BSH)-conjugated chlorin derivatives 11, 12, 16 and 20 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized. The in vivo biodistribution and clearance of 11, 12, 16 and 20 were investigated in tumor-bearing mice. Compounds 12 and 16 showed good tumor-selective accumulation among the four derivatives. The time to maximum accumulation of compound 16 in tumor tissue was one-fourth of that of compound 12, and clearance from normal tissues of compound 16 was similar to that of compound 12. The in vivo therapeutic efficacy of PDT using 16, which has twice as many boron atoms as 12, was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 6h after injection of 16. Tumor growth was significantly inhibited by PDT using 16. These results suggested that 16 is a good candidate for both PDT and BNCT of cancer., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Numerous reports indicate therapeutic efficacy of photodynamic therapy (PDT) against skin tumors, acne and for skin rejuvenation. However, few reports exist regarding its efficacy for fungal skin diseases. In order to determine the antifungal effect, PDT was applied on Malassezia furfur. M. furfur was cultured in the presence of a novel cationic photosensitizer, TONS504, and was irradiated with a 670-nm diode laser. TONS504-PDT showed a significant antifungal effect against M. furfur. The effect was irradiation dose- and TONS504 concentration-dependent and the maximal effect was observed at 100 J/cm2 and 1 μg/mL, respectively. In conclusion, TONS504-PDT showed antifungal effect against M. furfur in vitro, and may be a new therapeutic modality for M. furfur-related skin disorders., (© 2014 Japanese Dermatological Association.)

New boron-containing chlorin derivatives 9 and 13 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized from photoprotoporphyrin IX dimethyl ester (2) and L-4-boronophenylalanine-related compounds. The in vivo biodistribution and clearance of 9 and 13 were investigated in tumor-bearing mice. The time to maximum accumulation of compound 13 in tumor tissue was one-fourth of that of compound 9, and compound 13 showed rapid clearance from normal tissues within 24h after injection. The in vivo therapeutic efficacy of PDT using 13 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3h after injection of 13. Tumor growth was significantly inhibited by PDT using 13. These results suggested that 13 might be a good candidate for both PDT and BNCT of cancer., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Three new water-soluble chlorin derivatives 3, 5 and 8 for potential use as photosensitizers in photodynamic therapy (PDT) for cancer were synthesized from photoprotoporphyrin IX dimethyl ester (1). The in vivo biodistribution and clearance of chlorin derivatives 3, 5 and 8 were investigated in tumor-bearing mice. Iminodiacetic acid derivative 8 showed the greatest tumor-selective accumulation among the new chlorin derivatives with maximum accumulation in tumor tissue at 3h after intravenous injection and rapid clearance from normal tissues within 24h after injection. The in vivo therapeutic efficacy of PDT using 8 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3h after injection of 8. Tumor growth was significantly inhibited by PDT using 8. These results indicate that iminodiacetic acid derivative 8 is useful as a new photosensitizer to overcome the disadvantages of photosensitizers that are currently in clinical use., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Although topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is applied for skin tumors including actinic keratosis, Bowen disease, and squamous cell carcinoma, there are no approved photosensitizers in dermatological field in Japan. TONS501 and TONS504 are novel hydrophobic photosensitizers with anionic and cationic chemical characteristics, respectively., Objective: Using 7, 12-dimethylbenz[a]anthracene (DMBA) and 12-ο-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin papilloma model, we compared the efficacy of ALA-, TONS501-, and TONS504-PDT on the skin tumor regression., Methods: Following application of ALA, TONS501, TONS504 ointment or TONS501 lotion on DMBA- and TPA-induced mouse papillomas, 670 nm laser irradiation by LD670-05 diode laser was performed. Then tumor regression rate was calculated at the indicated time., Results: The anti-tumor effect of ALA, TONS501, and TONS504 ointment was detected at 24 h and the maximal response was observed at 3 day following the PDT treatment. The maximal response was observed at 150 J/cm(2) irradiation in all 3 photosensitizers. Although both ALA, TONS501 (ointment)-PDT showed more potent anti-tumor effect compared with that of TONS504 ointment or TONS501 lotion, no significant difference was detected between ALA ointment and TONS501 ointment., Conclusion: A novel TONS501ointment-PDT shows potent anti-tumor effect on DMBA- and TPA-induced mouse skin papilloma and might be more useful for the clinical applications., (Copyright © 2012. Published by Elsevier Ireland Ltd.)

Background: Ultrasonically induced cell damage and active oxygen generation with a novel porphyrin derivative DCPH-P-Na(I), were compared in the same in vitro insonation setup., Materials and Methods: Sarcoma 180 cells suspended in air-saturated PBS were exposed to ultrasound at 2 MHz for up to 60 s in the presence and absence of DCPH-P-Na(I). Cell viability was determined with the trypan blue exclusion test. Lipid peroxidation in cell membranes was estimated by measuring the amount of reactive substance produced immediately following the addition of thiobarbituric acid., Results: Significant enhancement of the rates of both ultrasonically induced cell damage and lipid peroxidation was observed in the presence of 2-8 muM DCPH-P-Na(I). Both rates correlated very well., Conclusion: The enhancement of both rates with DCPH-P-Na(I) was suppressed by 10 mM histidine. These results suggest that ultrasonically generated active oxygen plays a primary role in the ultrasonically induced cell damage in the presence of DCPH-P-Na(I).

To improve the efficacy of sonodynamic therapy of cancer using photosensitizers, we developed a novel porphyrin derivative designated DCPH-P-Na(I) and investigated its photochemical characteristics and sonotoxicity on tumor cells. DCPH-P-Na(I) exhibited a minimum fluorescent emission by excitation with light, compared with a strong emission from ATX-70, which is known to reveal both photo- and sonotoxicity. According to this observation, when human tumor cells were exposed to light in the presence of DCPH-P-Na(I) in vitro, the least phototoxicity was observed, in contrast to the strong phototoxicity of ATX-70. However, DCPH-P-Na(I) exhibited a potent sonotoxicity on tumor cells by irradiation with ultrasound in vitro. This sonotoxicity was reduced by the addition of L-histidine, but not D-mannitol, thus suggesting that singlet oxygen may be responsible for the sonotoxicity of DCPH-P-Na(I). DCPH-P-Na(I) demonstrated significant sonotoxicity against a variety of cancer cell lines derived from different tissues. In addition, in a mouse xenograft model, a potent growth inhibition of the tumor was observed using sonication after the administration of DCPH-P-Na(I) to the mouse. These results suggest that sonodynamic therapy with DCPH-P-Na(I) may therefore be a useful clinical treatment for cancers located deep in the human body without inducing skin sensitivity, which tends to be a major side-effect of photosensitizers.

Recent study revealed that photodynamic therapy (PDT) with a novel photosensitizer (ATX-S10(Na)) shows more potent effects for various skin diseases than ALA-PDT. The effect of ATX-S10(Na)-PDT on dermal fibroblasts is still unknown. Using dermal fibroblasts derived from normal and scleroderma patients, and mouse skin in vivo, we compared the effects of ATX-S10(Na)-PDT and ALA-PDT. Fibroblasts from normal, scleroderma patients or mice skin were treated with ATX-S10(Na)-PDT or ALA-PDT. After the PDT treatments, the expression of matrix metalloproteinases (MMPs) Tissue inhibitors of metalloproteinases (TIMPs) and collagen synthesis was assayed using ELISA and reverse transcription-PCR (RT-PCR). The expression of MMP-1 and MMP-3 was slightly decreased and collagen I mRNA was significantly increased in scleroderma fibroblasts compared with normal fibroblasts. Both ATX-S10(Na)-PDT and ALA-PDT increased the expression of MMP-1 and MMP-3 in protein and mRNA levels in both normal and scleroderma fibroblasts with more potent effect by ATX-S10(N)-PDT. Collagen I synthesis was markedly decreased by ATX-S10(Na)-PDT and by ALA-PDT again with more potent effect by ATX-S10(Na)-PDT in both normal and scleroderma fibroblasts. In mice skin the effect of PDT for MMPs and collagen I was also detected and the effect was more potent in ATX-S10(Na)-PDT. In contrast, MMP-2, TIMP-1, TIMP-2, and collagen III expression was not affected by the ATX-S10(Na)-PDT or ALA-PDT treatment. ATX-S10(Na)-PDT is more potent modulator for dermal matrix components than ALA-PDT and might be useful for scleroderma patients.

Background: Photodynamic therapy (PDT) is available for the treatment of various skin tumours and other skin diseases. Ultraviolet (UV) irradiation induces DNA damage, cyclobutane pyrimidine dimers (CPD) (6-4) photoproducts (6-4PP) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), all of which are carcinogenic for the skin. However, effects of PDT on DNA damage and carcinogenesis are unclear., Objectives: To compare the production of photoproducts and the induction of skin tumours in mouse epidermis treated with UVB or PDT., Methods: We performed UVB irradiation or ATX-S10(Na)-PDT on the skin of 20 hairless mice, in each case, and analysed DNA damage and tumour induction., Results: After a single irradiation of UVB on mouse skin, CPD, 6-4PP and 8-OHdG were detected in the nuclei of keratinocytes. In contrast, PDT-treated mouse keratinocytes showed induction of 8-OHdG, but not of CPD or 6-4PP. Skin tumours induced by UVB irradiation (3 kJ m(-2) three times weekly) were observed following 15 weeks of irradiation (mean +/- SEM tumour incidence 3.2 +/- 1.8%; tumour number 3.2 +/- 1.6 per mouse) and increased depending on irradiation times and doses. Following 30 weeks of UVB irradiation (3 kJ m(-2) three times weekly), mean +/- SEM tumour incidence and tumour number were 28.7 +/- 4.8% and 14.2 +/- 2.8% per mouse, respectively. Although skin tumours were also detected in PDT-treated mouse skin following 80 weeks of treatment (mean +/- SEM tumour incidence 9.1 +/- 1.8%; tumour number 12.2 +/- 2.3 per mouse), the number of tumours was not statistically different from untreated mouse skin (mean +/- SEM tumour incidence 4.1 +/- 3.8%; tumour number 5.2 +/- 3.3 per mouse)., Conclusions: PDT induced 8-OHDG but not CPD or 6-4PP, and was shown to be a relatively safe modality following multiple applications to mouse skin.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is available for the treatment of actinic keratosis (AK). Recently, we developed a new PDT photosensitizer, ATX-S10(Na), and have shown that ATX-S10(Na) PDT is effective for the treatment of various human skin diseases, such as squamous cell carcinoma, Bowen's disease, basal cell carcinoma, and psoriasis. In the present study, we compared the effects of ATX-S10(Na) PDT and ALA PDT on hyperproliferative skin induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), on the squamous cell carcinoma cell line, SCC15, in vitro, and on UVB-induced skin tumors in vivo. TPA treatment induced epidermal acanthosis, which was more markedly suppressed by ATX-S10(Na) PDT than by ALA PDT. ATX-S10(Na) PDT more effectively eliminated UVB-induced AK and squamous cell carcinoma (SCC) than ALA PDT. Furthermore, both ATX-S10(Na) PDT and ALA PDT induced the death of SCC15 cells, and the effect of ATX-S10(Na) PDT was greater than that of ALA PDT. Our results indicate that ATX-S10(Na) PDT might be more effective than ALA PDT for the treatment of various skin diseases.

Background/purpose: Photodynamic therapy (PDT) is a promising treatment for various skin tumors and other skin diseases. We investigated the potential therapeutic effects of PDT using ATX-S10(Na) ointment and a diode laser in mouse skin models of experimental skin tumors as well as transplanted human samples of superficial skin tumors and lesional psoriatic skin., Methods: ATX-S10(Na) ointment (1% w/v) was introduced into tape-stripped mouse skin, transplanted squamous cell carcinoma (SCC) samples and human skin diseases after topical application, then PDT was performed., Results: ATX-S10(Na) ointment (1% w/v) was introduced effectively into tape-stripped mouse skin and transplanted SCC samples after topical application, but was not detected after 48 h, as assessed by fluorescence microscopy. PDT, using 1% ATX-S10(Na) ointment and diode laser (50 J/cm(2)), was found to decrease epidermal thickness in 12-0-tetradecanoylphorbol-13-acetate (TPA)-treated mouse skin by 6 days. PDT with 1% ATX-S10(Na) ointment and diode laser (150 J/cm(2)) was also effective for transplanted SCC, and tumors were eliminated by 6 weeks. PDT against Bowen disease, basal-cell carcinoma, and psoriasis xenografts onto SCID mice also showed marked suppression of tumor growth and cell proliferation, respectively., Conclusion: Our results indicate that ATX-S10(Na)-PDT is an effective treatment for various skin tumors and psoriasis in experimental mouse models.

Intermolecular interactions of human serum proteins with a hydrophilic nonmetalloporphyrin, 13,17-bis(1-carboxypropionyl)carbomoylethyl-8-ethenyl-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetramethylporphyrin sodium salt (ATX-S10 (Na)), or a hydrophilic gallium-metalloporphyrin, diethylenetriamine pentaacetic acid ester of 2-[1-(2-hydroxy-ethoxy)ethyl]-4-vinyl-deuteroporphyrin (IX) Ga complex (ATN-2), were investigated using spectrophotometry. ATX-S10 (Na) caused a bathochromic shift with albumin, high-density lipoprotein and low-density lipoprotein, but little or no shift was observed with hemopexin, transferrin and immunoglobulin G. In contrast, ATN-2 displayed a bathochromic shift only with hemopexin. These results suggest that the association energy of ATX-S10 (Na) with albumin might be slightly greater than that with lipoproteins and that of ATN-2 with hemopexin might be greater than that with other serum proteins.

We describe here the detection by fluorescence of a new photosensitizer, PAD-S31, in tumours in dogs and cats and the effect of photodynamic therapy (PDT) by using PAD-S31 for skin tumours in two dogs and one cat. PAD-S31 is a hydrophilic photosensitizer that has two peaks at absorption wavelengths 406 and 665 nm in distilled water. In a preliminary experiment in mice transplanted with SCCVII and colon 26, PAD-S31 was retained in tumour tissues rather than in other organs. The tumours resected from dogs and cats after intravenous administration of PAD-S31 at a dose of 15 mg/kg emitted strong red fluorescence under light illumination of 402 nm wavelength. Animals given PAD-S31 showed no cutaneous photosensitivity under room light illumination. Irradiation at laser light 670 nm wavelength (fluence rate 150 mW/cm2 and total light dosage 150 J/cm2) on cutaneous mast cell tumours in dogs ( n=2 ) and a cutaneous basal cell tumour in a cat induced complete remission. These results suggest PAD-S31 could be a promising photosensitizer for use in a small animal veterinary practice.

Background/purpose: Photodynamic therapy (PDT) is a potent treatment for skin tumors. Although the therapeutic effect of PDT is supposed to be due to cellular cytotoxicity, the precise mechanism is still unknown. ATX-S10(Na) [13,17-bis(1-carboxypropionyl)carbamoylethyl-8-ethenyl-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetramethylporphyrin sodium salt], a novel hydrophilic chlorin photosensitizer, shows good accumulation in tumors and is suitable for use in PDT. In this study, we investigated the mechanism of PDT-induced cell death using ATX-S10(Na)., Methods: . Following ATX-S10(Na) treatment for 12 h, normal human keratinocytes (NHK) were irradiated using a diode laser. PDT-induced cell death and the activity of various caspases were measured. Activation of Fas antigen was also determined by immunoprecipitation analysis. The expression of Bax, cytochrome c, and apoptosis-inducing factor (AIF) was determined by Western blotting., Results: ATX-S10(Na)-PDT had induced apoptosis of NHK by 2 h and the maximal effect was observed at 6 h following irradiation. The effect was suppressed by pretreatment of NHK with inhibitors of caspases 3, 6, 8 and 9. A caspase activity assay revealed the sequential activation of caspases 8, 3 and 6, and caspases 9, 3 and 6, respectively. Immunoprecipitation analysis indicated multimerization of Fas antigen without Fas ligand binding in ATX-S10(Na)-PDT-treated NHK. Western blotting revealed cytosolic release of cytochrome c and AIF accompanied by decreased Bax expression in the cytosol., Conclusions: ATX-S10(Na)-PDT induces apoptosis of NHK, and this was mediated by sequential activation of two caspase cascades, caspases 8, 3 and 6, and caspases 9, 3 and 6. This was accompanied by multimerization of Fas antigen and cytosolic release of cytochrome c and AIF.

Ultrasonically induced cell damage and active oxygen generation with 4-formyloximeetylidene-3-hydroxyl-2-vinyl-deuterio-porphynyl(IX)-6-7-diaspartic acid (ATX-S10) were compared in the same in vitro insonation setup. Sarcoma 180 cells suspended in air-saturated PBS were exposed to ultrasound at 2 MHz for up to 60 s in the presence and absence of ATX-S10. The viability was determined by Trypan blue exclusion test. Ultrasonically induced active oxygen generation in the presence and absence of ATX-S10 in air-saturated aqueous solutions of 50 mM 2,2,6,6-tetramethyl-4-piperidone was detected by electron spin resonance (ESR). Significant enhancement of the rates of both ultrasonically induced cell damage and nitroxide generation was demonstrated with 40-160 microM ATX-S10. Both rates correlated very well. The enhancement of both rates with ATX-S10 was suppressed by 10 mM histidine. These results suggest that ultrasonically generated active oxygen plays a primary role in the ultrasonically induced cell damage in the presence of ATX-S10.

Background and Objectives: Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of photosensitizer and visible light. We examined the effect of PDT using a new photosensitizer, PAD-S31, and the 670-nm diode laser in human oral squamous cell carcinomas (SCC)., Study Design/materials and Methods: SAS and HSC-4 cell lines were used in all the experiments. Cell viability was determined by a modified MTT assay. Two methods were used for the determination of apoptosis in human oral SCC cells: TUNEL assay and detection of fragmented mono- and oligo-nucleosomes by ELISA. Xenografts of human oral SCC cells were generated in KSN S1c nude mice., Results: In vitro PDT using PAD-S31 and the 670-nm diode laser showed cytotoxicity that was a function of laser energy, drug concentration, and time to the SAS and HSC-4 cell lines. On the other hand, PAD-S31 without irradiation had no effect on cell viability. The combinated use of PAD-S31 and the laser irradiation showed excellent anti-tumor activity against tumor xenografts without severe side effects. PDT-mediated cell death occurred predominantly by apoptosis in vitro and in vivo., Conclusions: The present study demonstrates that PAD-S31 may serve as a potent photosensitizer for PDT. Furthermore, it is expected that this therapy will be clinically useful for the treatment of patients with oral carcinoma., (Copyright 2003 Wiley-Liss, Inc.)

The tissue distribution of a gallium-porphyrin photo- and sono-sensitizer, 7,12-bis(1-decyloxyethyl)-Ga(III)-3,8,13,17-tetramethylporphyrin-2,18-dipropionyldiaspartic acid, ATX-70, was pharmacokinetically examined in tumor-bearing mice. The drug was administered intravenously to CDF(1) mice implanted with Colon 26 carcinoma. Blood and tissue samples were collected for up to 72 h after administration. The drug concentration was determined by high-performance liquid chromatography (HPLC) with fluorescence detection. ATX-70 was found to accumulate in tumors at a relatively high concentration that peaked between 2 h and 6 h after administration. However, modest concentrations of ATX-70 also remained in healthy tissues for up to 6 h. We examined the distribution of ATX-70 in the tumor in comparison with other tissues from the viewpoint of minimizing possible side effects of laser or ultrasound exposure while maintaining the treatment effect. About 24 h after administration, the tumor / plasma concentration ratio peaked, and relatively high tumor / skin and tumor / muscle concentration ratios were seen.

We examined the simultaneous uptake of porphyrin and (LDL) by four established cell lines of glioma and normal fibroblasts using flow cytometry (FCM). The results indicated porphyrin and LDL showed competitive conjugation with the LDL receptor. These results support the theory of the porphyrin uptake via the LDL receptor.

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 microg / 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 degrees 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 microg / ml ATX-S10(Na) soon after laser irradiation at 50 J / cm(2), and tumor necrosis was rapidly induced. This result indicated that ATX-S10(Na) was widely distributed within the cell.

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 / cm(2) 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 / cm(2) 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.

During size exclusion HPLC, ATN-2 binding protein separated from human and mouse sera, SCCVII and colon 26 tumor tissues were found in fraction 13 (A: estimated molecular weight 70,000). Fraction 13(A) of human sera was exclusively reactive to the human hemopexin antibody. During two-dimensional electrophoresis and amino acid sequence analysis, Fraction 13(A) of C3H/He mouse sera was found to have partial homology with the mouse hemopexin precursor. Glycoprotein with the same domain structure of hemopexin has been proposed to be an important carrier protein that forms the tumor-localizing activity of water-soluble porphyrin.

The sonodynamically induced antitumor effect of 4-formyloximethylidene-3-hydroxy-2-vinyl-deuterio-porphyn yl(IX)-6,7-diaspartic acid (ATX-S10) was investigated. Both in vitro and in vivo antitumor effects were tested in combination with ultrasound at 2 MHz. The rate of ultrasonically induced damage to isolated sarcoma 180 cells in air-saturated suspension was enhanced two-fold with 80 microM ATX-S10. This enhancement was significantly inhibited by histidine, which may suggest that it was mediated by ultrasonically induced oxidation. The coadministraion of 25 mg/kg ATX-S10 followed by ultrasonic exposure at 2 MHz stopped the growth of implanted colon 26 tumors at an intensity at which ultrasound alone showed only a slight antitumor effect.

We used a two-color analysis system to assess the porphyrin uptake and DNA content in four established cell lines of glioma employing flow cytometry (FCM). The FCM study revealed porphyrin uptake in all cells, regardless of the phase of the cell cycle they were in. However, those in the G0/G1 phase showed moderate uptake of porphyrin and those in the G2/M phase showed a higher uptake. These results indicated the advantage of using porphyrin as the carrier of tumor targeting agents as a therapeutic strategy for malignant tumors.

We examined the effect of interstitial photodynamic therapy (PDT) with a new photosensitizer ATX-S10(Na). This photosensitizer showed the strongest therapeutic effect 2-4 h after administration and was rapidly excreted from individual organs except tumor and liver 24 h after i.v. injection. Microscopic histofluorescent imaging showed fluorescence of ATX-S10(Na) in the cytoplasm of the tumor cells, but not in nuclei and in the vascular wall. Irradiation of Liniac 30 Gly+20 Gly slightly reduced the tumor size, but all mice died of relapse within 60 days after irradiation. In the PDT group, all tumors became non-palpable and healing was achieved in 50% of mice 120 days after PDT.

In order to develop tumor-localizing fluorescent diagnostic agents without phototoxicity, various heterodimers linked by some spacers between a chlorine derivative and its Mn or Cu complex were synthesized. The representative agent of them was named HAT-DO1 and has a molecular formula of m-phthalyl-([13,17-bispropanoic acid-3-ethenyl-8-formylethylidene-7-hydroxy-2,7,12,18-tetramethyl- porphyrinate]-manganese (III))-[3'-ethenyl-8'-formylethylidene-7'-hydroxy-2',7',12',18'-te tramethyl- porphine-13',17'-bispropanoyl aspartic acid]-bishydrazone.

Chemiluminescence (CL) caused by photosensitization of porphyrins in phosphate buffered saline (PBS) solution containing 3% human serum albumin (HSA) was observed for the first time. Irrespective of porphyrins concerned, CL shows a spectrum ranging from 380 to 520 nm with a peak near 450 nm and decays almost single-exponentially with a lifetime of about 15 s. The intensity of CL depends on concentrations of porphyrins and HSA in PBS solution. We have examined a number of porphyrins and observed CL for the compounds with triplet lifetimes longer than 0.1 ms. The appearance and quenching of CL by photosensitization of porphyrin-HSA systems indicate that type II reaction by singlet oxygen occurs significantly in photodynamic therapy resulting in hypoxic regions in environments surrounding the sensitizer.

A carrier for a new tumor imaging agent, a bifunctional chelating agent (BCA)-coupled porphyrin (ATN-2), has been synthesized from protoporphyrin dimethyl ester in 4 steps. At first, the hydrobromination of protoporphyrin dimethyl ester is carried out to obtain a monobromo derivative. The derivative is treated with ethylene glycol. The resulting porphyrin has an ether group at the either 7- or 12-position of the ring. Metallation with GaCl3 of the porphyrin having a ethylene glycol residue affords Ga-metalloporphyrin. Final condensation of the metalloporphyrin with diethylenetriaminepentaacetic acid (DT-PA) gave BCA-coupled porphyrin (ATN-2). The chelation of ATN-2 with 111InCl3 easily afforded [111In]ATN-2. This agent was used for imaging transplantable pancreatic carcinoma in Syrian golden hamster at 72 h after postinjection. The efficacy of the new agent was compared with that of [67Ga]citrate. The images with [111In]ATN-2 were found to be clearer than those with [67Ga]citrate. Therefore, ATN-2, a carrier for 111InCl3, seems to be more useful for tumor imaging agents.

We have synthesized a new tumor imaging agent, 111In-labeled porphyrin (111In-ATN-2). In order to image transplantable pancreatic carcinoma in Syrian golden hamsters, we investigated the biodistribution of 111In-ATN-2 72 hr after injecting the agent by means of whole-body autoradiography. The efficacy of the agent was compared with that of 67Ga citrate. The images with 111In-ATN-2 were found to be clearer than those with 67Ga citrate. Tumor-to-tissue radiodistribution ratios of the former were higher than those of the latter. Thus, 111In-ATN-2 seems to be more useful for tumor diagnosis.

The relation between the lifetimes of the triplet states of various porphyrins and their photosensitizing effects on the photodynamic therapy (PDT) of tumor has been examined. Diethylene-triamine pentaacetic acid ester of 4-[1-(2-hydroxy-ethyloxy)ethyl]-2-vinyl deuteroporphyrin-IX gallium (III) complex (Ga-DP), zinc (II) complex (Zn-DP), and manganese (III) complex (Mn-DP) and Photofrin II (PII) are used as the photosensitizer. The triplet lifetimes have been measured for the samples adsorbed on filter paper (FP) and found to be 57 ms (Ga-DP), 26 ms (Zn-DP), less than or equal to 10 microseconds (Mn-DP) and 9 ms (PII). The phosphorescence of Ga-DP in tumor-bearing golden hamsters are measured both in tumor tissue and in liver. They show bi-exponential decay with the lifetimes of about 5 and 20 ms. From the values, the generation rate, kct[3O2], of singlet molecular oxygen in living animal tissue may be estimated to be an order of 10(2) s-1. The PDT effects have been quantitatively investigated for in vitro experiments; upon irradiation the growth inhibitions of mouse p388 leukemia cells are obtained as a function of concentration of Ga-DP, Zn-DP, Mn-DP and PII. The experimental results indicate that the PDT effects depend essentially on the triplet lifetimes of the photosensitizers.