meso-chlorin-e(6)-monoethylene-diamine and Ovarian-Neoplasms

It is essential to understand cellular responses on photodynamic therapy (PDT) to design delivery systems that maximize cytotoxic effects coupled with minimal induction of side effects or protective mechanisms (or both). Here, we investigated mechanisms of toxicity in human ovarian carcinoma A2780 cells treated with structurally diverse N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (P)-mesochlorin e6 monoethylenediamine (Mce6) conjugates that possessed differential subcellular accumulation or covalent attachments of photosensitizers (or both). Apoptosis and necrosis were observed after photoactivation, with increased apoptotic responses observed in cells exposed to conjugates possessing Mce6 linkage via a lysosomally degradable tetrapeptide spacer (HPMA copolymer-Mce6 conjugates containing Mce6 bound via glycylphenylalanylleucylglycine [GFLG] linker [P-GFLG-Mce6], HPMA copolymer-Mce6 conjugates containing Mce6 bound via a GFLG spacer and containing nuclear localization sequence, PKKKRKV132K(FITC)C [NLS(fluorescein-5-isothiocyanate [FITC])] bound via a thioether linkage [P-NLS(FITC)-GFLG-Mce6]). Furthermore, the induction of necrosis was more pronounced in cells exposed to conjugates containing both a nuclear localization sequence (NLS) and Mce6 bound by a degradable linker (P-NLS(FITC)-GFLG-Mce6). Caspase-independent mechanisms of cell death were identified in cells treated with nuclear-targeted conjugates possessing Mce6 attached using a nondegradable tether (HPMA copolymer-Mce6 conjugates containing Mce6 bound via a GG spacer and containing NLS(FITC) bound via a thioether linkage [P-NLS(FITC)-GG-Mce6]), whereas low levels of apoptosis and necrosis were detected in cells exposed to photoactivated nontargeted HPMA copolymer-Mce6 conjugates containing Mce6 coupled through a nondegradable spacer (HPMA copolymer-Mce6 conjugates containing Mce6 bound via GG linker [P-GG-Mce6]). Variations in gene expression were observed in cells on PDT. Specifically, HSP70 expression was solely detected in cells treated with P-GFLG-Mce6, whereas the loss of detection of several genes were observed in cells treated with P-NLS(FITC)-GFLG-Mce6. Variations in cellular responses on PDT using different HPMA copolymer-Mce6 conjugates will prove useful in the design of optimal HPMA copolymer PDT delivery systems.

Topics: Apoptosis; Caspase Inhibitors; Caspases; Cell Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Mesoporphyrins; Methacrylates; Molecular Structure; Necrosis; Ovarian Neoplasms; Photochemotherapy; Photosensitizing Agents; Porphyrins; Protein Biosynthesis; RNA, Messenger; Tumor Cells, Cultured

Photosensitizers, light-sensitive compounds, become activated upon illumination with a specific wavelength of light generating cytotoxic oxygen species. Due to the short half-life of singlet oxygen, the subcellular site of localization and excitation affects the type of cellular damage produced as well as cellular responses to different types of photodamage created within the cell. Here, we investigated the effects of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-mesochlorin e(6) monoethylenediamine (Mce(6)) conjugates localized to different subcellular compartments. Temperature was utilized to achieve subcellular localization of conjugates and subcellular fractionation was performed to confirm localization patterns of HPMA copolymer-Mce(6) conjugates. Cytotoxicity studies suggest plasma membrane and late endosomes were more sensitive to photodamage than lysosomal compartments as observed by an approximate 2-fold decrease in the IC(50) compared to lysosomally accumulated conjugate. Releasing Mce(6) from the polymer backbone within lysosomal compartments significantly lowered the IC(50) when compared to HPMA copolymer conjugates with Mce6 bound via a nondegradable linkage. These differences will prove useful in the future design of HPMA copolymer-Mce(6) conjugates for the treatment of ovarian cancer.

Topics: Antineoplastic Agents; Cytotoxicity Tests, Immunologic; Female; Humans; Mesoporphyrins; Methacrylates; Ovarian Neoplasms; Photosensitizing Agents; Porphyrins; Reactive Oxygen Species; Subcellular Fractions; Temperature; Tumor Cells, Cultured

This study demonstrates the selective tumor targeting and the antitumor efficacy of the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound mesochlorin e6 monoethylenediamine (Mce6) and HPMA copolymer-bound Adriamycin (ADR) in combination photodynamic therapy (PDT) and chemotherapy against human ovarian OVCAR-3 carcinoma xenografted in female athynmic mice. The concentrations of Mce6 and ADR in blood and tissues, in free or HPMA copolymer-bound form, were determined by fluorescence and high-performance liquid chromatography fluorescence assays, respectively. Xenograft responses to single and combination therapies were recorded. The peak concentration of HPMA copolymer-Mce6 conjugate in tumor was achieved 18 h after administration. For HPMA copolymer-bound drugs, the concentration ratios of liver and spleen versus muscle were significantly higher than those of free drugs. The HPMA copolymer-bound drugs demonstrated selective targeting and accumulation in the tumor, probably attributed to the enhanced permeability and retention effect. In vivo studies revealed that all tumors in the treatment groups showed significant responses after receiving any of the various types of therapy as compared with controls (P < 0.001). PDT with HPMA copolymer-Mce6 conjugate (PDTMC) at a dose of 13.4 mg/kg (1.5 mg/kg of Mce6 equivalent) and light doses of 110 J/cm2 at 12 and 18 h, respectively, resulted in significant suppression of the growth of OVCAR-3 tumors. Three courses of chemotherapy using 35 mg/kg (2.2 mg/kg of ADR equivalent) of HPMA copolymer-ADR conjugate (CHEMO) were effective in suppressing the growth of tumors. Single PDTMC plus multiple CHEMO exhibited significantly greater therapeutic efficacy than multiple CHEMO. In the group of mice receiving multiple PDTMC, tumor recurrence became obvious after day 20. However, 10 of 12 tumors exhibited complete responses in the group of mice receiving multiple PDTMC plus multiple CHEMO. The least to most effective treatments were ranked as follows: multiple CHEMO < single PDTMC plus multiple CHEMO < multiple PDTMC < multiple PDTMC plus multiple CHEMO. The results clearly demonstrate that: (a) HPMA copolymer-bound drugs exhibited selective tumor accumulation contrary to free drugs; (b) PDT using HPMA copolymer-Mce6 conjugate with multiple light irradiations was a better therapy than that with single light irradiation; and (c) combination chemotherapy and photodynamic therapy with HPMA copolymer-ADR and HPMA copolymer-M

Topics: Acrylamides; Animals; Antineoplastic Agents; Combined Modality Therapy; Doxorubicin; Drug Therapy, Combination; Female; Humans; Injections, Intravenous; Mesoporphyrins; Mice; Mice, Nude; Neoplasm Transplantation; Ovarian Neoplasms; Photochemotherapy; Photosensitizing Agents; Tissue Distribution; Transplantation, Heterologous; Treatment Outcome; Tumor Cells, Cultured; Weight Loss

The acquisition of multidrug resistance in human ovarian carcinoma A2780 cells was investigated after chronic exposure to free mesochlorin e6 monoethylenediamine (Mce6) and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound Mce6 (P(GG)-Mce6). The dose that inhibits growth by 50% (IC50) was determined for free Mce6 (2.09 +/- 0.32 microM) and P(GG)-Mce6 (204.15 +/- 28.97 microM) to utilize similar effective doses of drug. A total of 14 drug exposures were performed over a period of 78 days. Cells were characterized by IC50 dose, MDR1 gene expression and anti-human P-glycoprotein (P-gp) antibody binding after each drug exposure. At the conclusion of the experiment, neither the A2780 cells habitually exposed to free Mce6 or P(GG)-Mce6 were significantly different than the control A2780 cells indicating cells did not acquire a MDR phenotype. The doxorubicin (DOX)-resistant A2780/AD cells served as a positive control.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, MDR; Humans; Immunoglobulin G; Mesoporphyrins; Methacrylates; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Ovarian Neoplasms; Pharmaceutical Vehicles; Phenotype; Porphyrins; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

The purpose of this study was to determine the interaction between free (unbound) and N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer bound adriamycin and meso-chlorin e6 monoethylene diamine (Mce6) induced photodynamic therapy in combination in their cytotoxic activities against human ovarian epithelial carcinoma (OVCAR-3) in vitro. The effects of each agent (free drugs and HPMA copolymer bound) alone and in combination were measured simultaneously utilizing two measures of cell viability: a) mitochondrial respiration via the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide reduction (MTT) assay; and b) thymidine incorporation via the tritiated thymidine incorporation (TI) assay. These were performed at 72 and 144 h after drug exposure. Forty-eight hours from time zero (24 h after drug addition), the cells treated with Mce6 (free and HPMA copolymer bound) and controls were exposed to 650 nm light (13 min at 15 mW/cm2, 11.7 J/cm2). The calculated ED50 values by the MTT 72 h assay for adriamycin (A) and Mce6/light (C) were 1.5 microg/ml and 209 ng/ml, respectively. Adriamycin demonstrated progressive cellular toxicity over time in both assays. Mce6/light demonstrated initial damage at 72 h by MTT and TI which recovered by 144 h. Adriamycin and Mce6/light acted cooperatively to increase the percentage of cells inhibited. In combination, 21.3+/-1.5% MTT reduction activity was observed by free adriamycin and Mce6/light compared to the expected 27+/-5% (p<0. 0001) based on additivity. Twice the ED50 of adriamycin (2A=3 microg/ml) or Mce6/light (2C=418 ng/ml) resulted in only 42+/-3.6% and 39.2+/-2.0% activity, respectively (both p<0.0001 vs. combination). When Mce6/light at 10x ED50 (10C) was combined with 1x ED50 of adriamycin (1A), or the reciprocal combination, additional cooperativity was demonstrated. Compared to free drugs, both HPMA copolymer bound adriamycin (P-A) and HPMA copolymer bound Mce6/light (P-C) required a 10-fold increase in drug concentration to show equivalency with free drugs (A or C). Dose response curves demonstrated a reduced slope compared to free drugs in the same dose ranges. When P-A was added (1-10x free adriamycin ED50) to an effective concentration of P-C (10P-C: equivalent to 10x free Mce6 ED50) an improved long-term inhibition of OVCAR-3 cell multiplication was noted in both the MTT and TI 144 h assays. P-C (1-10x free Mce6 ED50) added to an effective concentration of P-A (10P-A: equivalent to 10x free adriam

Topics: Antineoplastic Agents; Carcinoma; Doxorubicin; Drug Carriers; Drug Evaluation; Drug Synergism; Endocytosis; Female; Humans; Lysosomes; Mesoporphyrins; Methacrylates; Ovarian Neoplasms; Photochemotherapy; Photosensitizing Agents; Tumor Cells, Cultured

The binding, internalization, subcellular trafficking and in vitro cytotoxicity of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anti-cancer drug-OV-TL16 antibody (Ab) conjugates in the ovarian carcinoma OVCAR-3 cell line have been investigated. Adriamycin (ADR) and meso chlorin e6 mono(N-2-aminoethylamide) (Mce6) photosensitizer were used as anti-cancer drugs. Targeted (Ab-containing) conjugates were compared with non-targeted HPMA copolymer-drug conjugates and with free drugs. Targeted conjugates were taken up rapidly by cells and detected within lysosomes by confocal fluorescence microscopy. The ADR attached to polymer chains via a degradable GFLG spacer was released from the conjugate, diffused via the lysosomal membrane into the cytoplasm and ultimately accumulated in the cell nuclei. In contrast, conjugates containing ADR bound via the GG spacer accumulated in the lysosomes, but no fluorescence could be detected in the cell nuclei. Binding the drugs to a non-targeted HPMA copolymer decreased their cytotoxicity in vitro. The IC50 dose increased from 2 microM for free ADR to 150 microM for P(GFLG)-ADR (P is the HPMA copolymer backbone) and from 0.34 microM for free Mce6 (with light) to 290 microM for P-(GG)-Mce6. However, attachment of OV-TL16 Abs rendered HPMA copolymer-drug conjugates biorecognizable by OVCAR-3 cells and markedly increased their cytotoxicity. The IC50 doses were 4.4 and 0.38 microM for the targeted conjugates P(GFLG)-ADR-Ab and P(GG)-Mce6-Ab (with light), respectively. Biorecognition was shown to be specific by inhibition experiments with free Ab. The findings indicate the potential of these conjugates as effective agents in the treatment of ovarian cancer.

Topics: Antibodies, Monoclonal; Biological Transport; Cell Compartmentation; Cell Survival; Cells, Cultured; Doxorubicin; Endocytosis; Female; Humans; Immunotoxins; Lysosomes; Mesoporphyrins; Methacrylates; Microscopy, Confocal; Microscopy, Fluorescence; Ovarian Neoplasms; Pharmaceutical Vehicles; Photosensitizing Agents; Tumor Cells, Cultured

In photodynamic therapy (PDT), photosensitisers accumulate somewhat preferentially in malignant tissues; photoactivation with appropriate wavelength of light release toxic molecular species which lead to tumour tissue death. In order to target ovarian cancer with increased specificity, a chlorin-based photosensitiser (chlorin e6 monoethylendiamine monoamide) was conjugated to OC125, a monoclonal antibody recognising an antigen expressed in 80% of non-mucinous ovarian cancers. In previous work, this immunoconjugate (IC) was shown to be selectively phototoxic to cancer cells from ovarian cancer patients ex vivo and to localise preferentially in ovarian cancer tissue in vivo. In this study we report results from in vivo phototoxicology and photodynamic treatment studies using this IC in a murine model for ovarian cancer. A comparison of single vs multiple treatments was also made. For in vivo experimentation, Balb C nude mice were injected with 30 x 10(6) NIH:OVCAR 3 cancer cells to create an ascitic tumour model. Animals were then given intraperitoneal injections of the immunoconjugate (0.5 mg kg-1). Twenty-four hours later the intraperitoneal surfaces were exposed to 656 nm light from an argon-ion pumped-dye laser (50 mW, 656 nm), using a cylindrical diffusing tip fibre. The overall treatment was given either once or multiply. No animals died from treatment complications. Twenty-four hours following one and three PDT treatments, the percentage of viable tumour cells in the ascites of the treated animals analysed ex vivo was 34% and 5% of control for one and three treatments respectively. With respect to survival, all control mice (n = 18) died between 30 and 50 days. However, for those treated three times (n = 10), 40% were still alive after 50 days, and for those treated four times (n = 12) 58% were alive after 50 days. Evaluation with log-rank test revealed a significant survival with intraperitoneal PDT compared with controls (P = 0.0006). These preliminary results suggest that PDT with an OC125 immunoconjugate may be an effective therapy for the management of advanced ovarian cancer. Clinical application of this therapy needs to be further optimised and may require multiple treatments, similar to fractionated radiation therapy and cyclic chemotherapy, in order to control malignant disease with acceptable toxicity to normal tissue.

Topics: Animals; Antibodies, Monoclonal; Combined Modality Therapy; Disease Models, Animal; Female; Immunoconjugates; Immunotherapy; Mesoporphyrins; Mice; Mice, Inbred BALB C; Mice, Nude; Ovarian Neoplasms; Photochemotherapy; Photosensitizing Agents; Tissue Distribution