Effects of TONS504-photodynamic therapy on mouse mammary tumor cells.

In the present study, TONS504 (C51H58N8O5I2; molecular weight, 1,116.9), a novel cationic hydrophilic photosensitizer, was synthesized from protoporphyrin IX dimethyl ester through a five-step process according to a patented method for use in photodynamic therapy (PDT). The subcellular localization of TONS504 and the cytotoxic effects of TONS504-mediated PDT in the mouse mammary tumor EMT6 cell line were investigated. TONS504 was localized primarily in the lysosomes and partially in the mitochondria. The cytotoxic effects of TONS504-mediated PDT in the mouse mammary tumor EMT6 cell line were investigated using a WST8 assay and an Oxidative Stress kit. The cell viability values following treatment with 10 µg/ml TONS504 at light energies of 0, 1, 5 and 10 J/cm2 were 92.5, 101.8, 27.7 and 1.8%, respectively. The percentages of reactive oxygen species (ROS)(+) cells following the same treatment were 8.6, 8.5, 29.2 and 70.1%, respectively, whereas the percentages of apoptotic cells were 7.1, 5.6, 24.8 and 48.7%, respectively. The percentages of ROS(+) and apoptotic cells in the group subjected to TONS504-mediated PDT increased in a manner dependent on the TONS504 concentration and light energy. Further studies are required to evaluate the in vivo pharmacokinetics, tissue distribution and photodynamic effects of TONS504.

Observation of Zn-photoprotoporphyrin red Autofluorescence in human bronchial cancer using color-fluorescence endoscopy.

BACKGROUND: We observed red autofluorescence emanating from bronchial cancer lesions using a sensitive color-fluorescence endoscopy system. We investigated to clarify the origin of the red autofluorescence. METHODS: The wavelengths of the red autofluorescence emanating from lesions were measured in eight patients using a spectrum analyzer and compared based on pathologic findings. Red autofluorescence at 617.3, 617.4, 619.0, and 617.1 nm was emitted by normal bronchus, inflamed tissue, tissue exhibiting mild dysplasia, and malignant lesions, respectively. Protoporphyrin, uroporphyrin, and coproporphyrin, the major porphyrin derivatives in human blood, were purchased to determine which porphyrin derivative is the source of red fluorescence when acquired de novo. We synthesized photoporphyrin, Zn-protoporphyrin and Zn-photoprotoporphyrin from protoporphyrin. RESULTS: Coproporphyrin and uroporphyrin emitted only weak fluorescence. Fluorescence was emitted by our synthesized Zn-photoprotoporphyrin at 625.5 nm and by photoprotoporphyrin at 664.0 nm. CONCLUSIONS: From these results, we conclude that Zn-photoprotoporphyrin was the source of the red autofluorescence observed in bronchial lesions. Zn-protoporphyrin is converted to Zn-photoprotoporphyrin by radiation with excitation light. Our results suggest that red autofluorescence emanating from Zn-photoprotoporphyrin in human tissues could interfere with photodynamic diagnosis using porphyrin derivatives such as Photofrin® and Lazerphyrin® with a sensitive endoscopy system, because color cameras cannot differentiate Zn-photoprotoporphyrin red fluorescence from that of other porphyrin derivatives.

Photodynamic Therapy Mediated by a Novel Chlorin Derivative, TONS 501-Na, in EMT6 cells.

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/cm2 diode laser irradiation at 0-13 J/cm2 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/cm2 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.

Antifungal effect of TONS504-photodynamic therapy on Malassezia furfur.

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.

Photodynamic therapy using a novel irradiation source, LED lamp, is similarly effective to photodynamic therapy using diode laser or metal-halide lamp on DMBA- and TPA-induced mouse skin papillomas.

Photodynamic therapy (PDT) is useful for superficial skin tumors such as actinic keratosis and Bowen disease. Although PDT is non-surgical and easily-performed treatment modality, irradiation apparatus is large and expensive. Using 7, 12-dimethylbenz[a]anthracene (DMBA) and 12-ο-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin papilloma model, we compared the efficacy of TONS501- and ALA-PDT with a LED lamp, a diode laser lamp or a metal-halide lamp on the skin tumor regression. TONS501-PDT using 660 nm LED lamp showed anti-tumor effect at 1 day following the irradiation and the maximal anti-tumor effect was observed at 3 days following the irradiation. There was no significant difference in the anti-tumor effects among TONS501-PDT using LED, TONS501-PDT using diode laser, and 5-aminolevulinic acid hydrochloride (ALA)-PDT using metal-halide lamp. Potent anti-tumor effect on DMBA- and TPA-induced mouse skin papilloma was observed by TONS501-PDT using 660 nm LED, which might be more useful for clinical applications.

Synthesis and biological evaluation of new BSH-conjugated chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer.

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.

Synthesis and biological evaluation of new boron-containing chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer.

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.

Capturing by self-assembled block copolymer thin films: transfer printing of metal nanostructures on textured surfaces.

A method to fabricate metal nanostructures by transfer printing, applicable to textured surfaces, is described. The key is the use of self-assembled polystyrene-block-poly-2-vinylpyridine thin films as binding layers. The plasmonic properties of the obtained metal (Ag) nanostructures showed the potential of this method in the design of novel devices.

Synthesis and biological evaluation of new chlorin derivatives as potential photosensitizers for photodynamic therapy.

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.

Photodynamic therapy using a novel photosensitizer, TONS501, is similarly effective to ALA and EC036 photodynamic therapy on DMBA-and TPA-induced mouse skin papilloma.

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.

Sonodynamically induced cell damage and membrane lipid peroxidation by novel porphyrin derivative, DCPH-P-Na(I).

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).

Sonodynamic therapy of cancer using a novel porphyrin derivative, DCPH-P-Na(I), which is devoid of photosensitivity.

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.

Synthesis of amphiphilic derivatives of rose bengal and their tumor accumulation.

It is known that the combination of laser light and its sensitizer is effective for noninvasive tumor treatment, referred to as photodynamic therapy. Using the combination of ultrasound and its sensitizer has also been suggested for a similar kind of tumor treatment, referred to as sonodynamic therapy. The purpose of this paper is to obtain such sensitizers accumulating selectively in tumors. Amphiphilic derivatives of rose bengal (RB) were synthesized to add a tumor-accumulating property to RB. One type of the synthesized RB derivatives (RBD3), having an alkyl chain with a branching carboxyl group, was found to be superior in amphiphilicity to the other types. Tumor tissue distribution of the synthesized derivatives in mice bearing colon 26 carcinoma was evaluated. It was found that RBD3s with carbon chain lengths of 12, 14, and 16 had higher concentrations in the tumor tissue than RB by more than 1 order of magnitude, several hours after administration. The concentrations correlated well with their water/1-octanol partition coefficients. Since RB is known to induce in vitro cell damage in combination with either laser light or ultrasound, the newly synthesized amphiphilic RB derivatives may be potentially useful as a tumor-selective sensitizer for both light and ultrasound.

ATX-S10(Na)-PDT shows more potent effect on collagen metabolism of human normal and scleroderma dermal fibroblasts than ALA-PDT.

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.

Photodynamic therapy using a novel photosensitizer, ATX-S10(Na): comparative effect with 5-aminolevulinic acid on squamous cell carcinoma cell line, SCC15, ultraviolet B-induced skin tumor, and phorbol ester-induced hyperproliferative 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.

A novel ATX-S10(Na) photodynamic therapy for human skin tumors and benign hyperproliferative skin.

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.

Tumor enhancement using Mn-metalloporphyrin in mice: magnetic resonance imaging and histopathologic correlation.

PURPOSE: To determine the signal enhancement characteristics of tumors after administration of a metalloporphyrin derivative, HOP-9P (13, 17-bis (1-carboxypropionyl) carbamoylethyl-3, 8-bis (1-phenylpropyloxyethyl)-2, 7, 12, 18-tetramethyl-porphyrinato manganese (III)) and to determine whether HOP-9P is tumor-necrosis specific. MATERIALS AND METHODS: Ten C3H/He mice bearing a SCC VII tumor in the right flank were examined using T1-weighted conventional spin echo magnetic resonance (MR) imaging before contrast injection, and five minutes, one hour, and 24 hours after intravenous administration of 0.1 mmol/kg of HOP-9P. Following the imaging schedule, the mice were sacrificed, and sectioned in the same axial planes as the MR images. Based on an MR imaging-histopathologic correlation, mean signal intensities were measured, and signal-to-noise ratios (SNR) were calculated for both pure viable component and admixture of necrotic and viable component of the tumor. RESULTS: Mean SNR of the pure viable component peaked at one hour (35.0 +/- 3.8) and maintained that level until 24 hours (34.6 +/- 3.6). Mean SNR of the admixture of necrotic and viable component peaked at 24 hours (44.3 +/- 12.1). CONCLUSION: Although different enhancement patterns were seen between the pure viable component and the admixture of necrotic and viable component, HOP-9P enhanced both of the two components.

Tissue retention and adverse reaction after intravenous injection of hematoporphyrin derivatives in dogs.

We evaluated changes in hematology and chemical profile, and the tissue retention of hematoporphyrin derivative (HpD) following the intravenous injection in dogs. HpD at concentrations of 1, 5, 10, and 15 mg/kg was intravenously injected to 16 dogs (n=4 each) and complete blood count (CBC) and blood chemistry were performed on days 1, 3, 5, and 7 after the injection. To examine tissue retention, HpD (5 mg/kg) was administered to 15 dogs and 3 dogs were euthanized on days 1, 2, 7, 14, and 28 after the injection, respectively, to collect the skin, muscle, small intestine, spleen, kidney and liver as tissue samples. There were no significant changes in CBC and blood chemical profile except for an increase in LDH concentrations in dogs given 10 and 15 mg/kg of HpD at day 3. The levels of HpD retention in the tissues were ranked in the following order: liver > kidney > spleen > intestine > muscle > skin.