Susumu Nakajima

Combination effect of photodynamic and sonodynamic therapy on experimental skin squamous cell carcinoma in C3H/HeN mice

We studied a combination of photodynamic therapy (PDT) and sonodynamic therapy (SDT) for improving tumoricidal effects in a transplantable mouse squamous cell carcinoma (SCC) model. Two sensitizers were utilized: the pheophorbide‐a derivative PH‐1126, which is a newly developed photosensitizer, and the gallium porphyrin analogue ATX‐70, a commonly used sonosensitizer. Mice were injected with either PH‐1126 or ATX‐70 i.p. at doses of 5 or 10 mg/kg.bw. At 24 (ATX‐70) or 36 hr (PH‐1126) (time of optimum drug concentration in the tumor) after injection, SCCs underwent laser light irradiation (88 J/cm2 of 575 nm for ATX‐70; 44 J/cm2 of 650 nm for PH‐1126) (PDT), ultrasound irradiation (0.51 W/cm2 at 1.0 MHz for 10 minutes) (SDT), or a combination of the two treatments. The combination of PDT and SDT using either PH‐1126 or ATX‐70 as a sensitizer resulted in significantly improved inhibition of tumor growth (92–98%) (additive effect) as compared to either single treatment (27–77%). The combination using PH‐1126 resulted in 25% of the treated mice being tumor free at 20 days after treatment. Moreover, the median survival period (from irradiation to death) of PDT + SDT‐treated mice (>120 days) was significantly greater than that in single treatment groups (77–95 days). Histological changes revealed that combination therapy could induce tumor necrosis 2–3 times as deep as in either of the single modalities. The combination of PDT and SDT could be very useful for treatment of non‐superficial or nodular tumors.

CRITICAL IMPORTANCE OF THE TRIPLET LIFETIME OF PHOTOSENSITIZER IN PHOTODYNAMIC THERAPY OF TUMOR

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), 610 |xs (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 102 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.

Sonodynamically Induced Antitumor Effect of 4-Formyloximethylidene-3-Hydroxy-2-vinyl-deuterio-porphynyl(IX)-6,7-diaspartic Acid (ATX-S10)

The sonodynamically induced antitumor effect of 4‐formyloximethylidene‐3‐hydroxy‐2‐vinyl‐deuterio‐porphynyl(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 μM 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.

The tumour-localizing properties of porphyrin derivatives

The tumour-localizing abilities of various kinds of porphyrin derivatives in tumour-bearing hamsters were assessed by nitrogen-pulsed laser spectrofluorometry (N2-PLS). On examination of porphine derivatives (from haemoglobin), it was found that the dimer and acetylated and amidated compounds had a high affinity for tumour tissue; the dimer and hydroxylated compound of phorbine derivatives (from chlorophyll) also showed a high affinity. Furthermore, of the metalloporphines (gallium, zinc and indium complexes), those which contained hydrophilic groups showed a high affinity for tumour tissue; of the metallophorbines (gallium, zinc and indium complexes), those which contained hydrophobic groups showed a high affinity. A correlation was found between the side-chain structure of the porphyrins and metalloporphyrins and their affinity for tumour tissue.

Tumor-localizing activity of porphyrin and its affinity to LDL, transferrin

We spectrochemically analyzed 12 Ga-Metal porphyrins (Ga-Cn-P) with different degrees of hydrophobicity, and observed a close association between the in vivo Ga-Cn-P localization in the tumor and its affinity to transferrin and LDL. Image analysis of tumors with 99m-Tc-STA-R12, a porphyrin-scintigraphic agent, revealed STA-R12 localization in the tumor tissue alone about 1 h after intravenous infusion. Our results suggest the importance of endocytosis mediated by transferrin as well as LDL that have been reported as a mechanism of the tumor-localizing activity of porphyrin.

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.

Treatment of experimental tumors with a combination of a pulsing magnetic field and an antitumor drug

We investigated the effects of a combination treatment involving a pulsing magnetic field (PMF) and an antitumor drug, mitomycin C (MMC), on two experimental tumors (fibrosarcoma KMT‐17 and hepatocellular carcinoma KDH‐8) in WKA rats, paying attention to possible potentiation of the therapeutic effect of the antitumor drug. PMF was obtained using a system generating a pulsed current in a solenoid coil. On day 7 after tumor implantation into the right thighs of rats, the region of the tumor was exposed to PMF (frequency 200 Hz, mean magnetic flux density 40 gauss) for 1 h immediately after iv injection of MMC at a dose of 1 mg/kg. Survival rates at day 90 of KMT‐17 implanted rats were 0% (0/10) in the non‐treated group, 34% (4/12) in the MMC‐treated group, 47% (6/13) in the PMF‐treated group and 77% (10/13) in the MMC/PMF combination group. The increase of life span (ILS) of KDH‐8‐implanted rats in the combination therapy group was significantly prolonged (%ILS 17.6%) compared with that in the MMC‐treated (%ILS 3.4%) and PMF‐treated (%ILS 7.6%) groups. By using cultured cells of the above two lines of tumor, the therapeutic effects of MMC and PMF were also determined from the cell colony‐forming efficiency in soft agar. The colony‐forming efficiencies of both cell lines were significantly suppressed in the combination therapy group compared with those in the other single therapy groups. The present results indicate that PMF exhibited a potentiation of the antitumor effect of mitomycin C.

Selective occlusion of choroidal neovascularization by photodynamic therapy with a water-soluble photosensitizer, ATX-S10

To determine the optimal treatment parameters for selective occlusion of choroidal neovascularization (CNV) by photodynamic therapy (PDT) by using the photosensitizer ATX‐S10 and a diode laser (wavelength = 670 nm).

Therapeutic effect of interstitial photodynamic therapy using ATX-S10(Na) and a diode laser on radio-resistant SCCVII tumors of C3H/He mice.

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.

Pharmacokinetic Study of a Gallium-porphyrin Photo- and Sono-sensitizer, ATX-70, in Tumor-bearing Mice

The tissue distribution of a gallium‐porphyrin photo‐ and sono‐sensitizer, 7,12‐bis(l‐decyloxy‐ethyl)‐Ga(III)‐3,8,13,17‐tetramethylporphyrin‐2,18‐dipropionyldiaspartic acid, ATX‐70, was phar‐niacokinetically examined in tumor‐bearing mice. The drug was administered intravenously to CDF1mice 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.