Benzophenothiazine and Benzoporphyrin Derivative Combination Phototherapy Effectively Eradicates Large Murine Sarcomas

Abstract— The tumoricidal effects of photochemotherapy with two photosensitizers, 5-ethylamino-9-diethylaminobenzo[ a ] phenothiazinium chloride (EtNBS) and benzoporphyrin derivative monoacid ring A (BPD-MA), were evaluated separately and in combination against the EMT-6 fibrosarcoma implanted subcutaneously in BALB/c mice. Animals carrying tumors 8-10 mm in diameter were divided into eight different groups (∼20/group) and subjected to various photoirradiation and drug conditions. The tumor response to photodynamic therapy (PDT) was measured as the mean tumor wet weight 2 weeks post-PDT. The combination treatment with 5.25 mg/kg EtNBS and 2.5 mg/kg BPD-MA followed by photoirradiation with 100 J/cm² at 652 nm and then by 100 J/cm² at 690 nm resulted in a 95% reduction in the average tumor weights compared to controls (no light, no drugs) with 76% of the mice being tumor free 2 weeks post-PDT. Because treatment with EtNBS or BPD-MA at twice the light dose and drug concentration resulted in either no significant reduction in tumor weights or increased the lethality of treatment, respectively, the data suggest that the enhanced PDT effect observed with the combination of drugs is synergistic rather than additive. Histology of tumors 24 h post-PDT with the combination of drugs showed nearly complete destruction of the tumor mass with little or no damage to the vasculature and no extravasation of red blood cells. There was no damage to the normal skin adjacent to the tumor. Fluorescence microscopy of EMT-6 cells incubated in vitro with the two photosensitizers revealed that they were localized to different intracellular compartments. The fluorescence pattern from frozen tumor tissue slices following the in vivo administration of the photosensitizers indicated a greater intracellular localization for EtNBS vs BPD-MA.     

Measurement of Tumor Vascular Damage in Mice with 99m Tc-MIBI Following Photodynamic Therapy

Abstract— The clinical perfusion agent ^99mTc-MIBI was used to monitor changes in tumor vascular perfusion (TVP) induced by Photofrin® (Pll)-mediated photodynamic therapy (PDT). BALB/c mice bearing an EMT-6 tumor on each hind thigh were given an intravenous injection of 1, 2 or 5 mg kg⁻¹ PII. Twenty-four hours later, one tumor was illuminated (600–650 run, 200 mW cm⁻² 400 J cm⁻²) while the other served as a control. At various time intervals after PDT (0, 2 and 24 h) mice received an intravenous injection of ^99mTc-hexakismethoxy(sobutyusonitri-le (MIBI) (0.18 MBq g⁻¹) and were sacrificed 2 min later. The light-treated and the untreated tumors were then dissected, the radioactivity was counted and the percentage of the injected dose per gram of tumor (%ID g⁻¹) was calculated as a measure of TVP. We observed that TVP is drug dose dependent, develops progressively with time post-PDT and is inversely related to PDT efficacy. Our data show that early tumor retention of ^99mMIBI is a simple method to assess TVP and vascular damage induced by PDT.     

Combination of 5-Fluorouracil with Photodynamic Therapy: Enhancement of Innate and Adaptive Immune Responses in a Murine Model of Actinic Keratosis†

We previously showed that a combination of differentiation-inducing agents (5-fluorouracil [5FU], vitamin D3 or methotrexate) and aminolevulinate-based photodynamic therapy (PDT) improves clinical responses by enhancing protoporphyrin IX (PpIX) photosensitizer levels and cell death. Here, we show that in addition to its previously known effects, 5FU enhances PDT-induced tumor-regressing immunity. Murine actinic keratoses were treated with topical 5FU or vehicle for 3 days prior to aminolevulinic acid application, followed by blue light illumination (~417 nm). Lesions were harvested for time-course analyses of innate immune cell recruitment into lesions, i.e. neutrophils (Ly6G+) and macrophages (F4/80+), which peaked at 72 h and 1 week post-PDT, respectively, and were greater in 5FU-treated lesions. Enhanced infiltration of activated T cells (CD3+) throughout the time course, and of cytotoxic T cells (CD8+) at 1–2 weeks post-PDT, also occurred in 5FU-treated lesions. 5FU pretreatment reduced the presence of cells expressing the immune checkpoint marker PD-1 at ~72 h post-PDT, favoring cytotoxic T cell activity. A combination of 5FU and PDT, each individually known to induce long-term tumor-targeting immune responses in addition to their more immediate effects on cancer cells, may synergize to provide better management of squamous precancers.     

Inhibitory Effect of Anti-HER-2 Anti-CD3 Bi-specific Antibody on the Growth of Gastric Carcinoma

Introduction Gastriccancerisoneofthemostcommonlyen counteredmalignantdiseasesworldwide,especiallyin AsiaandAfrica[1].Thecombinationofoperation,chemotherapyandradiotherapyisusedfortreatinggas triccarcinoma.However,the5yearsurvivalrateof patientsofgastricca…     

Two-signal blockade with anti-CD45RB and anti-CD154 monoclonal antibodies inhibits graft rejection via CD4-dependent mechanisms in allogeneic skin transplantation

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low) and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low) T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two- signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.     

The immunosuppressive effects of phthalocyanine photodynamic therapy in mice are mediated by CD4+ and CD8+ T cells and can be adoptively transferred to naive recipients

Photodynamic therapy (PDT) is a promising treatment modality for malignant tumors but it is also immunosuppressive which may reduce its therapeutic efficacy. The purpose of our study was to elucidate the role of CD4+ and CD8+ T cells in PDT immunosuppression. Using silicon phthalocyanine 4 (Pc4) as photosensitizer, nontumor-bearing CD4 knockout (CD4-/-) mice and their wild type (WT) counterparts were subjected to Pc4-PDT in a manner identical to that used for tumor regression (1 cm spot size, 0.5 mg kg(-1) Pc4, 110 J cm(-2) light) to assess the effect of Pc4-PDT on cell-mediated immunity. There was a decrease in immunosuppression in CD4-/- mice compared with WT mice. We next examined the role of CD8+ T cells in Pc4-PDT-induced immunosuppression using CD8-/- mice following the same treatment regimen used for CD4-/- mice. Similar to CD4-/- mice, CD8-/- mice exhibited less immunosuppression than WT mice. Pc4-PDT-induced immunosuppression could be adoptively transferred with spleen cells from Pc4-PDT treated donor mice to syngenic naive recipients (P < 0.05) and was mediated primarily by T cells, although macrophages were also found to play a role. Procedures that limit PDT-induced immunosuppression but do not affect PDT-induced regression of tumors may prove superior to PDT alone in promoting long-term antitumor responses.     

Apoptosis is an Early Event During Phthalocyanine Photodynamic Therapy-Induced Ablation of Chemically Induced Squamous Papillomas in Mouse Skin

Abstract— Photodynamic therapy (PDT) is a promising new modality to treat malignant neoplasms including superficial skin cancers. In our search for an ideal photosensitizer for PDT, Pc 4, a silicon phthalocyanine, has shown promising results both in in vitro assays and in implanted tumors. In this study we assessed the efficacy of Pc 4 PDT in the ablation of murine skin tumors; and the evidence for apoptosis during tumor ablation was also obtained. The Pc 4 was administered through tail vein injection to SENCAR mice bearing chemically induced squamous papillomas, and 24 h later the lesions were illuminated with an argon ion-pumped dye laser tuned at 675 nm for a total light dose of 135 J/cm². Within 72-96 h, almost complete tumor shrinkage occurred; no tumor regrowth was observed up to 90 days post-PDT. As evident by nucleosome-size DNA fragmentation, appearance of apoptotic bodies in hematoxylin and eosin staining and direct immunoperoxidase detection of digoxigenin-labeled genomic DNA in sections, apoptosis was clearly evident 6 h post-PDT at which time tumor shrinkage was less than 30%. The apoptotic bodies, as evident by the condensation of chromatin material around the periphery of the nucleus and increased vacuolization of the cytoplasm, were also observed in electron microscopic studies of the tumor tissues following Pc 4 PDT. The extent of apoptosis was greater at 15 h than at 6 and 10 h post-PDT. Taken together, our results clearly show that Pc 4 may be an effective photosensitizer for PDT of nonmelanoma skin cancer, and that apoptosis is an early event during this process.     

Tumor Oxygenation Changes Post-Photodynamic Therapy

Abstract— Tumor oxygenation after a photodynamic therapy (PDT) treatment is a critical factor for understanding the post-treatment metabolic pathway of the tumor. It also provides important information for designing combination therapy of PDT and other oxygen-dependent anticancer modalities. In this study, mammary carcinoma in flank and hind leg of C3H mice were subjected to PDT at either subcurative or curative level (12.5 mg/kg Photofrin; 200 or 600 J/cm², respectively). The before and post-PDT tumor oxygenation was measured with an oxygen-sensitive microelectrode. The data revealed that tumor oxygenation at the time of PDT has a profound effect on posttreatment tumor oxygenation, which may largely be due to an interplay between direct PDT cytotoxicity and PDT damage to the tumor microvasculature. Transient reoxygenation occurred after PDT, which may provide a window for improved combination therapy for other oxygen-dependent modalities.     

Combination therapy with antiangiogenic treatment and photodynamic therapy for the nude mouse bearing U87 glioblastoma

The objective of this study was to evaluate the effects of combination therapy with photodynamic therapy (PDT) and a novel antiangiogenic regimen using monoclonal antibodies against both vascular endothelial growth factor receptors (VEGFR)-1 (MF1) and VEGFR-2 (DC101) on intracranial glioblastoma xenografts in nude mice. Nude mice bearing intracerebral U87 glioblastoma were treated with PDT and the antiangiogenic regimen (MF1 and DC101) either alone or in combination, while those left untreated served as tumor controls. Tumor volume and animal survival time were analyzed to evaluate the outcome of different treatment modalities. In addition, the immunohistochemical expression of VEGF in the brain adjacent to the tumor, von Willebrand factor (vWF), apoptotic, and proliferative markers in the tumor area were examined. PDT or MF1 + DC101 alone significantly reduced the tumor volume and prolonged the survival time of glioma-implanted animals. Combined therapy markedly reduced tumor volume and increased survival time with significantly better outcomes than both monotherapies. Both vWF and VEGF levels significantly increased after PDT while they both significantly decreased after antiangiogenic treatment, compared with no treatment. PDT plus antiangiogenic treatment led to significant decreases in both vWF and VEGF expression, compared with PDT alone. Either PDT or antiangiogenic treatment alone significantly increased tumor cell apoptosis compared with no treatment, while combination therapy resulted in further augmentation of apoptosis. Antiangiogenic treatment with or without PDT significantly decreased tumor cell proliferation, compared with either no treatment or PDT alone. In summary, we demonstrate both significant inhibition of tumor growth and extended survival of mice treated by the combination therapy with PDT and antiangiogenic agents, compared with each single treatment, suggesting that the combination therapy may be a promising strategy to improve clinical outcomes in glioblastoma.     

Phthalocyanine 4 (Pc 4) photodynamic therapy of human OVCAR-3 tumor xenografts

Photodynamic therapy (PDT) is a cancer treatment modality utilizing a photosensitizer, light and oxygen. Photodynamic therapy with Photofrin has been approved by the U.S. Food and Drug Administration for treatment of advanced esophageal and early lung cancer. Because of certain drawbacks associated with the use of Photofrin, there is a need to identify new photosensitizers for human use. The photosensitizer Pc 4 (HOSiPc-OSi[CH3]2[CH2]3N[CH3]2) has yielded promising PDT effects in many in vitro and in vivo systems. The aim of this study was to assess the usefulness of Pc 4 as a PDT photosensitizer for a human tumor grown as a xenograft in athymic nude mice. The ovarian epithelial carcinoma (OVCAR-3) was heterotransplanted subcutaneously in athymic nude mice. Sixty mice bearing OVCAR-3 tumors (approximately 80-130 mm3) were divided into six groups of 10 animals each, three for controls and three for treatment. The Pc 4 was given by tail vein injection, and 48 h later a 1 cm area encompassing the tumor was irradiated with light from a diode laser coupled to a fiberoptic terminating in a microlens (lambda = 672 nm, 150 J/cm2, 150 mW/cm2). Tumors of control animals receiving no treatment, light alone or Pc 4 alone continued to grow. Of animals receiving 0.4 mg/kg Pc 4 and light, one (10%) had a complete response and was cured (no regrowth up to 90 days post-PDT), while all others (90%) had a partial response and were delayed in regrowth. Of animals receiving 0.6 mg/kg Pc 4 and light, eight (80%) had a complete response, and two of these were cured. Of animals receiving 1.0 mg/kg Pc 4 and light, six (60%) had a complete response, and two of these were cured. In additional experiments, tumors from animals treated with Pc 4 (1 mg/kg) and light were removed 15, 30, 60 and 180 min post-PDT, and from these tumors DNA and protein were extracted. Agarose gel electrophoresis revealed the presence of apoptotic DNA fragmentation as early as 15 min post-PDT. Western blotting showed the cleavage of the 116 kDa native poly(ADP-ribose) polymerase (PARP) into fragments of approximately 90 kDa, another indication of apoptosis, and the presence of p21/WAF1/CIP1 (p21) in all PDT-treated tumors. These changes did not occur in control tumors. Pc 4 appears to be an effective photosensitizer for PDT of human tumors grown as xenografts in nude mice. Early apoptosis, as revealed by PARP cleavage, DNA fragmentation and p21 overexpression, may be responsible for the excellent Pc 4-PDT response. Clinical trials of Pc 4-PDT are warranted.     

APOPTOSIS DURING PHOTODYNAMIC THERAPY-INDUCED ABLATION OF RIF-1 TUMORS IN C3H MICE: ELECTRON MICROSCOPIC, HISTOPATHOLOGIC AND BIOCHEMICAL EVIDENCE

Abstract Very little is known about the applicability of the metabolic and biochemical events observed in cell culture systems to in vivo tumor shrinkage following photodynamic therapy (PDT). The purpose of this study was to assess whether PDT induces apoptosis during tumor ablation in vivo . We treated radiation-induced fibrosarcoma (RIF-1) tumors grown in C3H/HeN mice with PDT employing three photosensitizers, Photofrin-II, chloroaluminum phthalocyanine tetrasulfonate, or Pc IV (a promising phthalocyanine developed in this laboratory). Each photosensitizer was injected intraperitoneally and 24 h later the tumors were irradiated with an appropriate wavelength of red light using an argon-pumped dye laser. During the course of tumor shrinkage, the tumors were removed at 1, 2, 4 and 10 h post-PDT for DNA fragmentation, histopathologic, and electron microscopic studies. Markers of apoptosis, viz . the ladder of nucleosome-size DNA fragments, increased apoptotic bodies, and condensation of chromatin material around the periphery of the nucleus, were evident in tumor tissue even 1 h post-PDT; the extent of these changes increased during the later stages of tumor ablation. No changes were observed in tumors given photosensitizer alone or irradiation alone. Our data suggest that the damage produced by in vivo PDT may activate endonucleolysis and chromatin condensation, and that apoptosis is an early event in tumor shrinkage following PDT.     

PHOTODYNAMIC THERAPY OF A MURINE TUMOR FOLLOWING SENSITISATION WITH CHLORO ALUMINUM SULFONATED PHTHALOCYANINE

Abstract Syngeneic mice bearing a colorectal carcinoma (Colo 26) growing subcutaneously in the flank region received photodynamic therapy (PDT) when the tumor was 7-12 mm diameter. Light (emission at 675 nm from an argon ion pumped dye laser, laser energy 100 J, power 50 mW) was delivered to the tumor 24-28 h after the i.v. injection of a single dose of chloro aluminum sulfonated phthalocyanine (ClAlSPc) at 5 mg kg¹ body weight. Control tumor-bearing animals received (1) phosphate buffered saline (PBS) but not PDT, (2) ClAlSPc but not PDT, or (3) PBS injection plus PDT. Five days later PDT tumors of the ClAlSPc + PDT group were markedly reduced in size (mean weight 0.075 ± 0.027 g) as compared with those from control groups 1 (0.408 ± 0.167 g), 2 (0.475 ± 0.143 g) or 3 (0.376 ± 0.153 g). Histological examination revealed that ClAlSPc + PDT induced severe necrosis and cytotoxicity of neoplastic cells with viable tumor limited to a small peripheral margin. Animals in the ClAlSPc + PDT group in a repeat experiment survived significantly longer than animals in the three control groups suggesting that ClAlSPc may be a useful photosensitiser for PDT of cancer.     

Differential roles of T-cell subsets in regulation of ultraviolet radiation induced cutaneous photocarcinogenesis

Ultraviolet (UV) radiation, in particular the midwavelength range (UVB; 290-320 nm), is one of the most significant risk factors for the development of nonmelanoma skin cancer. UVB radiation-induced immunosuppression, which occurs in both humans and laboratory animals, contributes to their pathogenesis. However, there are conflicting reports on the relative role of CD4(+) and CD8(+) T cells in UVB induced skin cancer. The purpose of this study was to delineate the contribution of these two cell subpopulations to UVB induced immunosuppression and tumor development using C3H/HeN (WT), CD4 knockout (CD4(-/-) ) and CD8 knockout (CD8(-/-) ) mice. We observed that UVB induced skin carcinogenesis was retarded in terms of number of tumors per group, tumor volume and percentage of mice with tumors, in mice deficient in CD4(+) T cells compared with wild-type mice, whereas significantly greater (P < 0.05) numbers of tumors occurred in CD8(-/-) mice. These results indicate that, CD4(+) T cells promote tumor development while CD8(+) T cells have the opposite effect. Further, we found that CD4(+) T cells from tumor-bearing mice produced interleukin (IL)-4, IL-10, and IL-17 whereas CD8(+) T cells produced interferon-γ. Manipulation of T-cell subpopulations that are induced by UVB radiation could be a means of preventing skin cancers caused by this agent.     

Exploitation of Immune Response-eliciting Properties of Hypocrellin Photosensitizer SL052-based Photodynamic Therapy for Eradication of Malignant Tumors

A diaminophenyl derivative of hypocrellin B (SL052) has been developed as a photosensitizer for use in photodynamic therapy (PDT) of solid tumors. Testing SL052-PDT on mouse carcinoma and fibrosarcoma models revealed a typical response seen with clinically established photosensitizers featuring initial rapid tumor ablation with ensuing recurrence at rates dependent on photosensitizer/light doses. Elevated numbers of immune cells were found in lymph nodes draining SCCVII mouse squamous cell carcinomas treated by SL052-PDT (in particular T cells), and the accumulation of degranulating cytotoxic T cells was detected at the tumor-treated site. This indicates that a significant contribution to tumor cures is elicited by an antitumor adaptive immune response. Two different immunotherapy agents, γ-interferon and antibody blocking inhibitory FcγRIIB receptor, were both found to be highly effective in potentiating the curative effect of SL052-PDT with SCCVII tumors. Combining SL052-PDT with FcγRIIB-blocking antibody treatment caused a further increase in the number of cells in tumor-draining lymph nodes and in degranulating CD8⁺ cells, suggesting the amplification of the immune response induced by PDT. Vaccines consisting of SCCVII cells treated with SL052-PDT in vitro were effective in reducing growth of established subcutaneous SCCVII tumors. In conclusion, PDT mediated by SL052 is suitable to be integrated with various immunotherapy protocols.     

In vivo resistance to photofrin-mediated photodynamic therapy in radiation-induced fibrosarcoma cells resistant to in vitro Photofrin-mediated photodynamic therapy.

The effects of Photofrin-mediated photodynamic therapy (PDT) on the in vitro cell survival and in vivo tumor growth of murine radiation-induced fibrosarcoma (RIF) cell tumors have been examined following in vivo PDT treatment of tumors. The response to in vivo PDT is examined in tumors derived from RIF-1 mouse fibrosarcoma cells and in tumors derived from RIF-8A cells, which show in vitro resistance to PDT. A significant reduction in tumor volume is observed over the first three days following in vivo PDT treatment of either 5 or 10 mg/ kg. The reduction in tumor volume is greater for a 10 compared to a 5 mg/ml dose and occurs to a similar extent for both RIF-1 and RIF-8A tumors. The re-growth is significantly delayed for RIF-1 compared to RIF-8A tumors, indicating a greater response for RIF-1 tumors compared to RIF-8A tumors following PDT. A reduced response of the RIF-8A compared to the RIF-1 tumor cells is also observed in the clonogenic survival of cells from tumors that were excised and explanted in vitro immediately following in vivo PDT treatment. These data indicate that the intrinsic cell sensitivity to PDT is an important component in the mechanism that leads to tumor response following in vivo photodynamic therapy.     

Photodynamic properties of naphthosulfobenzoporphyrazines, novel asymmetric, amphiphilic phthalocyanine derivatives.

Metallo naphthosulfobenzoporphyrazines sulfonated to different degrees (M-NSBP) were prepared, and their potential as photosensitizers for the photodynamic therapy (PDT) of cancer was evaluated. M-NSBP can be viewed as hybrid molecules between sulfophthalocyanines and naphthalocyanines resulting in distinct differences in the absorption spectra between the mono-through tetrasulfonated derivatives. This feature greatly facilited their purification. Using V-79 Chinese hamster cells in vitro, the disulfonated derivatives were found slightly more photoactive than the hydrophilic trisulfonated derivatives while the monosulfonated derivative was inactive, in spite of a sixfold higher cell uptake. In the case of the di- and trisulfonated derivatives, differences in phototoxicity correlated well with their relative cell uptake. Substitution of Al for Zn had little effect on the extent of phototoxicity of the M-NSBP. In vitro PDT of the EMT-6 cells after in vivo dye administration, revealed a similar potency for direct cell killing between the di- and trisulfonated AlOH-NSBP, while the monosulfonated analog was inactive. PDT with the amphiphilic disulfonated AlOH-NSBP on the EMT-6 mammary tumor in BALB/c mice induced a significant tumor response, while the monosulfonated derivative was much less active.     

Bombyx batryticatus Protein-Rich Extract Induces Maturation of Dendritic Cells and Th1 Polarization: A Potential Immunological Adjuvant for Cancer Vaccine

Bombyx batryticatus, a protein-rich edible insect, is widely used as a traditional medicine in China. Several pharmacological studies have reported the anticancer activity of B. batryticatus extracts; however, the capacity of B. batryticatus extracts as immune potentiators for increasing the efficacy of cancer immunotherapy is still unverified. In the present study, we investigated the immunomodulatory role of B. batryticatus protein-rich extract (BBPE) in bone marrow-derived dendritic cells (BMDCs) and DC vaccine-immunized mice. BBPE-treated BMDCs displayed characteristics of mature immune status, including high expression of surface molecules (CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II), increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-12p70), enhanced antigen-presenting ability, and reduced endocytosis. BBPE-treated BMDCs promoted naive CD4⁺ and CD8⁺ T-cell proliferation and activation. Furthermore, BBPE/ovalbumin (OVA)-pulsed DC-immunized mice showed a stronger OVA-specific multifunctional T-cell response in CD4⁺ and CD8⁺ T cells and a stronger Th1 antibody response than mice receiving differently treated DCs, which showed the enhanced protective effect against tumor growth in E.G7 tumor-bearing mice. Our data demonstrate that BBPE can be a novel immune potentiator for a DC-based vaccine in anticancer therapy.     

PHOTODYNAMIC ACTIVITIES and SKIN PHOTOSENSITIVITY OF THE bis(DIMETHYLTHEXYLSILOXY)SILICON 2,3-NAPHTHALOCYANINE IN MICE

The photodynamic therapy (PDT) activity of the bis(dimethylthexylsiloxy)silicon 2,3-na-phthalocyanine (SiNc 8 ) was evaluated against the EMT-6 tumor implanted intradermally in BALB/c mice. The SiNc 8 was formulated in aqueous emulsions based on Cremophor EL or Solutol HS 15. The formulation was shown to affect plasma clearance and overall pharmacokinetics. Compared to Cremophor, Solutol promoted rapid plasma clearance and high liver retention of the dye, combined with a slight increase of dye tumor concentrations. The PDT action spectrum for tumor response of SiNc 8 in Cremophor (190 mW cm², 200 J cm², 24 h postinjection [p.i.] of 1 (jimol kg¹) showed a maximum at 780 nm, which corresponds to the absorption maximum of the monomelic dye as well as the in vivo maximum change in the “diffuse optical density” produced by the dye. The extent of tumor necrosis increased with augmented dye and light doses. Regardless of the formulation, at 1 h p.i. of 0.1 μmol kg^?! SiNc 8 , PDT efficiency (190 mW cm'², 400 J cm²) was high but accompanied by severe damage to normal tissues, at 24 h PDT resulted in complete tumor regression in 80% of the animals without adverse effects to adjacent tissues, while at 72 h p.i. PDT induced no tumor response with Cremophor and only a partial response with Solutol. At the latter time point, plasma dye clearance was nearly complete while tumor tissue levels remained high, suggesting that tumor response correlates with plasma rather than tumor dye levels. Skin sensitivity of SKhl mice to solar-simulated radiation was lower with SiNc 8 as compared to Photofrin®. Our data suggest the potential of SiNc 8 as a far-red absorbing photosensitizer in clinical PDT.     

In vitro and in vivo efficacy of photofrin and pheophorbide a, a bacteriochlorin, in photodynamic therapy of colonic cancer cells

This study was designed to investigate the efficacy of photodynamic therapy (PDT) in treating colonic cancer in a preclinical study. Photofrin, a porphyrin mixture, and pheophorbide a (Ph a), a bacteriochlorin, were tested on HT29 human colonic tumor cells in culture and xenografted into athymic mice. Their pharmacokinetics were investigated in vitro, and the PDT efficacy at increasing concentrations was determined with proliferative, cytotoxic and apoptotic assessments. The in vivo distribution and pharmacokinetics of these dyes (30 mg/kg, intraperitoneal) were investigated on HT29 tumor-bearing nude mice. The inhibition of tumor growth after a single 100 J/cm2 PDT session was measured by the changes in tumor volume and by histological analysis of tumor necrosis. PDT inhibited HT29 cell growth in culture. The cell photodamage occurred since the time the concentrations of Ph a and Photofrin reached 5.10(-7) M (or 0.3 microg/mL) and 10 microg/mL, respectively. A photosensitizer dose-dependent DNA fragmentation was observed linked to a cleavage of poly(ADP-ribose) polymerase and associated with an increased expression of mutant-type p53 protein. PDT induced a 3-week delay in tumor growth in vivo. The tumor injury was corroborated by histological observation of necrosis 48 h after treatment, with a correlated loss of specific enzyme expression in most of the tumor cells. In conclusion, PDT has the ability to destroy human colonic tumor cells in vitro and in vivo. This tumoricidal effect is likely associated with a p53-independent apoptosis, as HT29 cells express only mutated p53. The current study suggests a preferential use of Photofrin in PDT of colonic cancer because it should be more effective in vivo than Ph a as a consequence of better tumor uptake.     

Potentiation of Photodynamic Therapy Antitumor Activity in Mice by Nitric Oxide Synthase Inhibition Is Fluence Rate Dependent

The effects of systemic administration of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) in combination with photodynamic therapy (PDT) on tumor response, tumor oxygenation and tumor and normal skin perfusion were studied in C3H mice bearing subcutaneous radiation-induced fibrosarcoma tumors. Photodynamic therapy was carried out using the photosensitizer Photofrin (5 mg/kg) in conjunction with a low fluence rate (30 mW/cm2) and a high fluence rate (150 mW/cm2) protocol at a total fluence of 100 J/cm2. Low fluence rate PDT produced approximately 15% tumor cures, a response not significantly altered by administration of 20 mg/kg L-NNA either 5 min before or after PDT. In contrast, high fluence rate PDT produced no tumor cures by itself, but addition of L-NNA either pre- or post-PDT resulted in approximately 30% and approximately 10% tumor cures, respectively. The L-NNA by itself tended to decrease tumor pO2 levels and perfusion, but statistically significant differences were reached only at one time point (1 h) with one of the oxygenation parameters measured (% values < 2 mm Hg). Photodynamic therapy by itself decreased tumor oxygenation and perfusion more significantly. Addition of L-NNA before PDT further potentiated this effect. The L-NNA exerted its most striking effects on the PDT response of the normal skin microvasculature. Low fluence rate PDT caused severe and lasting shut-down of skin microvascular perfusion. With high fluence rate PDT, skin perfusion was initially decreased but recovered to persistent normal levels within 1 h of treatment. Administration of L-NNA reversed this response, converting it to complete and lasting vascular shut-down identical to that achieved with low fluence rate PDT. This effect was somewhat L-NNA dose dependent but was still marked at a dose of 1 mg/kg. It occurred whether L-NNA was given before or after PDT. The L-NNA did not alter the long-term vascular response of skin to low fluence rate PDT. The ability of L-NNA to correspondingly improve tumor response and severely limit skin vascular perfusion following high fluence rate PDT, while providing no benefit for the low fluence rate protocol, suggests that vascular changes in the tumor surrounding normal tissue contribute to the enhanced tumor curability with adjuvant L-NNA treatment.