phytochlorin and Ovarian-Neoplasms

PEGylated chlorin e(6) photosensitizers were synthesized with tri(ethylene glycol) attached at the ester bond(s) for a 1:1 conjugate at the 17(3)-position, a 2:1 conjugate at the 15(2)- and 17(3)-positions, and a 3:1 conjugate at the 13(1)-, 15(2)-, and 17(3)-positions. These chlorin sensitizers were studied for hydrolytic stability and solubility, as well as ovarian OVCAR-5 cancer cell uptake, localization, and phototoxicity. Increasing numbers of the PEG groups in the mono-, di-, and tri-PEG chlorin conjugates increased the water solubility and sensitivity to hydrolysis and uptake into the ovarian cancer cells. The PEG chlorin conjugates accumulated in the cytoplasm and mitrochondria, but not in lysosomes. Higher phototoxicity was roughly correlated with higher numbers of PEG groups, with the tri-PEG chlorin conjugate showing the best overall ovarian cancer cell photokilling of the series. Singlet oxygen lifetimes, solvent deuteration, and the effects of additives azide ion and d-mannitol were examined to help clarify the photokilling mechanisms. A Type-II (singlet oxygen) photosensitized mechanism is suggested for the di- and tri-PEG chlorin conjugates; however, a more complicated process based in part on a Type-I (radicals or radical ions) mechanism is suggested for the parent chlorin e(6) and the mono-PEG chlorin conjugate.

Topics: Chlorophyllides; Female; Humans; Ovarian Neoplasms; Photochemotherapy; Polyethylene Glycols; Porphyrins; Radiation-Sensitizing Agents

Photodynamic therapy is emerging as a viable modality for the treatment of many cancers. A limiting factor in its use against intracavity tumors such as disseminated ovarian cancer is insufficient selectivity of the photosensitizer for tumor compared with normal tissue. We report on an approach to improve tumor targeting by exploiting differences between cell types and by chemical modification of a photosensitizer conjugate. Attachment of polyethylene glycol (pegylation) to a polyacetylated conjugate between poly-l-lysine and chlorin(e6) increased the relative phototoxicity in vitro toward an ovarian cancer cell line (OVCAR-5) while reducing it toward a macrophage cell line (J774), compared with the nonpegylated conjugate. Surprisingly, the increased phototoxicity of the pegylated conjugate correlated with reduced oxygen consumption. Pegylation also reduced the tendency of the conjugate to aggregate and reduced the consumption of oxygen when the conjugates were illuminated in solution in serum containing medium, suggesting a switch in photochemical mechanism from type II (singlet oxygen) to type I (radicals or electron transfer). Pegylation led to more mitochondrial localization as shown by confocal fluorescence microscopy in OVCAR-5 cells, and, on illumination, produced a switch in cell death mechanism toward apoptosis not seen with J774 cells. Conjugates were injected i.p. into nude mice bearing i.p. OVCAR-5 tumors, and the pegylated conjugate gave higher amounts of photosensitizer in tumor and higher tumor:normal tissue ratios and increased the depth to which the chlorin(e6) penetrated into the peritoneal wall. Taken together, these results suggest that pegylation of a polymer-photosensitizer conjugate improves tumor-targeting and may increase the efficacy of photodynamic therapy for ovarian cancer.

Topics: Animals; Chlorophyllides; Female; Humans; Macrophages; Mice; Mice, Nude; Ovarian Neoplasms; Oxygen Consumption; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols; Polylysine; Porphyrins; Subcellular Fractions; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The objective of this study was to compare the efficacy of photoimmunoconjugates with cationic and anionic molecular charges on intraperitoneal photoimmunotherapy of ovarian cancer xenografts in nude mice.. The photosensitizer chlorin(e6) (c(e6)) was conjugated via a poly-l-lysine linker to the F(ab')(2) fragment of the murine anti-ovarian cancer monoclonal antibody OC125, resulting in a photoimmunoconjugate with a pronounced cationic charge. Alternatively, by succinylating the poly-l-lysine conjugate, a photoimmunoconjugate with a pronounced anionic charge was obtained. A murine model of ovarian cancer derived from intraperitoneal inoculation of NIH:OVCAR-5 cells was employed. The conjugate was injected intraperitoneally followed after 3 h by red light delivered through a fiber into the peritoneal cavity. These photoimmunotherapy treatments were repeated three times, and the results obtained with the anionic and cationic photoimmunoconjugates were compared with those obtained with free c(e6) and control. The extent of residual macroscopic disease and death from disease were the evaluable outcomes for tumoricidal and survival studies, respectively.. In contrast to other intraperitoneal photosensitizers, mice showed no systemic toxicity or morbidity from the treatment. In this initial study the mean residual tumor weights in all treatment groups ranged from 33 to 73 mg, as compared with 330 mg in untreated controls (P < 0.0001), and the response to the cationic conjugate was significantly better than that to the anionic conjugate or free c(e6) (P < 0.005). The median survival for mice treated with cationic photoimmunoconjugate was 41 days, compared with 35 days in controls (P = 0.009).. Photoimmunotherapy with a cationic photoimmunoconjugate produces results superior to those obtained with an anionic conjugate, and further optimization of the treatment regimen may lead to a potential treatment for advanced ovarian cancer.

Topics: Animals; Anions; Antibodies, Monoclonal; Cations; Chlorophyllides; Female; Immunoconjugates; Immunotherapy; Mice; Mice, Nude; Neoplasm Transplantation; Ovarian Neoplasms; Photosensitizing Agents; Phototherapy; Porphyrins; Tumor Cells, Cultured

Covalent conjugation of a photosensitizer to a ligand that specifically recognized and internalized by a cell-surface receptor may be a way of improving the selectivity of photodynamic therapy (PDT). The class A Type-I scavenger receptor of macrophages, which among other ligands recognizes maleylated serum albumin and has a high capacity is a good candidate for testing this approach. Chlorin(e6) was covalently attached to bovine serum albumin to give conjugates with molar substitution ratios of 1:1 and 3:1 (dye to protein), and these conjugates could then be further modified by maleylation. A novel way of purifying the conjugates by acetone precipitation was developed in order to remove traces of unbound dye that could not be accomplished by size-exclusion chromatography. Conjugates were characterized by polyacrylamide gel electrophoresis and thin-layer chromatography. Photosensitizer uptake was measured by target J774 murine macrophage-like cells and nontarget OVCAR-5 human ovarian cancer cells, and phototoxicity was examined after illumination by a 660 nm diode laser by a tetrazolium assay. All of the purified conjugates were taken up by and after illumination killed J774 cells while there was only small uptake and no phototoxicity toward OVCAR-5 cells. The higher dye:protein ratio and maleylation of the conjugates both produced higher uptakes and lower survival ratios in J774 cells. The uptake and phototoxicity by J774 cells were decreased after incubation at 4 degrees C demonstrating internalization, and confocal microscopy with organelle-specific green fluorescent probes showed largely lysosomal localization. Uptake and phototoxicity by J774 cells could both be competed by addition of the scavenger receptor ligand maleylated albumin. These data show that scavenger receptor-targeted PDT gives a high degree of specificity toward macrophages and may have applications in the treatment of tumors and atherosclerosis.

Topics: Animals; Cell Line; Chlorophyllides; Drug Carriers; Female; Humans; Macrophages; Mice; Ovarian Neoplasms; Photochemotherapy; Porphyrins; Radiation-Sensitizing Agents; Receptors, Immunologic; Receptors, Scavenger; Scavenger Receptors, Class A; Serum Albumin, Bovine; Tumor Cells, Cultured

Patients with ovarian cancer that is clinically resistant to cisplatin-based chemotherapy have little hope of a cure of their disease. Photoimmunotherapy, which involves the antibody-targeted delivery of a nontoxic photosensitizer that is activated to a cytotoxic state with visible light, may offer a new treatment option. Photoimmunotherapy may be applied intraperitoneally to target disseminated tumor. We tested the hypothesis that this treatment in combination with cisplatin potentiates cytotoxicity in ovarian cancer cell lines and primary cultures of human tumors.. Five human cancer cell lines (ovarian and breast) and 19 primary cultures were studied. The primary cultures were from solid and ascites tumor samples obtained from 14 patients with ovarian cancer who were undergoing primary surgery. The photosensitizer chlorin e(6) was conjugated to the F(ab')(2) fragment of the murine monoclonal antibody OC-125, which is directed against the antigen CA 125. Cytotoxicity was measured by the microculture tetrazolium assay. Treatments consisted of cisplatin alone, photoimmunotherapy alone, and photoimmunotherapy followed by cisplatin. The fractional product method was used to assess synergy in treatment effects. Ex vivo cultured human cells exhibiting 80% or greater survival at cisplatin concentrations of 10 microM for 24 hours were defined as cisplatin resistant for this study.. When all cell types (cisplatin sensitive and cisplatin resistant) were considered together, combination treatment yielded cytotoxicity that was, on average, 6.9 times (95% confidence interval = 1.86-11.94) greater than that of cisplatin alone (two-sided P =.023). Cisplatin-resistant cells showed a synergistic effect of the two treatments (two-sided P =.044), while cisplatin-sensitive cells showed an additive effect.. These ex vivo data suggest that platinum resistance in human ovarian cancer cells may be reversible by pretreatment with OC-125-targeted photoimmunotherapy. Further studies are required to confirm the efficacy of this approach in vivo.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Breast Neoplasms; CA-125 Antigen; Chlorophyllides; Cisplatin; Combined Modality Therapy; Drug Resistance, Neoplasm; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunotherapy; Ovarian Neoplasms; Photosensitizing Agents; Phototherapy; Porphyrins; Treatment Outcome; Tumor Cells, Cultured

This study characterizes the efficacy and toxicity of: (a) free Adriamycin and N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-Adriamycin conjugate (P-A); (b) free and HPMA copolymer-meso-chlorin e6 monoethylene diamine disodium salt (Mce6) conjugate (P-C) and light-induced photodynamic therapy; and (c) combinations of the HPMA copolymer conjugates (P-A and P-C) in the destruction of human epithelial ovarian carcinoma heterotransplanted in the nude mouse (OVCAR-3). Eight-week-old female nu/nu mice were injected in both flanks with 0.04-0.05 cm3 OVCAR-3 solid tumor dispersed in media. When bilateral tumors reached a minimum volume of 0.18 cm3 (one axis, 2.0-mm minimum) and demonstrated consistent growth, the experiments were initiated. Drugs were given i.v. unless otherwise noted. Tumor-bearing mice were allocated to the following protocols: (a) Adriamycin at 1 mg/kg, P-A at 30 mg/kg (2.2 mg/kg Adriamycin equivalent), and controls (n = 6 each); (b) Mce6 and light (2 h after administration: 650 nm light for 15 min to deliver 220 J/cm2) at 1.25, 2.5, 5, and 10 mg/kg (n = 6 each), 2.5 mg/kg i.p. (n = 4), and controls (n = 6); (c) P-C at 12.5, 25, and 75 mg/kg (1.5, 2.9, and 8.7 mg/kg Mce6 equivalent, respectively with light (18 h after administration; 650 nm light for 15 min to deliver 220 J/cm2), P-C at 25 mg/kg (2.9 mg/kg Mce6 equivalent) with no light administration, and controls (n = 7 each); and (d) a combination of P-A (30 mg/kg, 2.2 mg/kg adriamycin equivalent) and P-C (12.5 and 75 mg/kg, 1.5 mg/kg and 8.7 mg/kg Mce6 equivalent, respectively) with and without light (n = 7 each; 18 h after administration; 650 nm light for 15 min to deliver 220 J/cm2) and controls (n = 12). Tumor volumes and animals weights were assessed for significant differences from the treated and controls groups by Student's t test. Adriamycin (1 mg/kg) and P-A (30 mg/kg. 2.2 mg/kg Adriamycin equivalent) caused less than a 10% weight loss, and treated tumor volumes (day 10-32) were significantly less than those of controls (all P < 0.045). Mce6 (2.5-10 mg/kg i.v.), caused tumor regression in 80% of tumors and a shock syndrome in 17-83%. i.p. dosing (2.5 mg/kg) was uniformly fatal. Mce6 at 1.25 mg/kg did not show reproducible efficacy. P-C with light (25 and 75 mg/kg, 2.9 and 8.7 mg/kg Mce6 equivalent, respectively) demonstrated significant tumor destruction (P < 0.003) but not complete ablation. The combinations of P-A (30 mg/kg, 2.2 mg/kg Adriamycin equivalent) plus P-C (12.5

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Chlorophyllides; Doxorubicin; Drug Carriers; Female; Humans; Methacrylates; Mice; Mice, Nude; Neoplasm Transplantation; Ovarian Neoplasms; Photochemotherapy; Porphyrins; Radiation-Sensitizing Agents; Transplantation, Heterologous