Combination chemotherapy and photodynamic therapy of targetable N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin/mesochlorin e(6)-OV-TL 16 antibody immunoconjugates.

The aim of this study was to evaluate the combination chemotherapy and photodynamic therapy of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin (DOX) and mesochlorin e(6) (Mce(6)) targeted with an OV-TL 16 monoclonal antibody (P-DOX-Ab and P-Mce(6)-Ab, respectively) in nude mice bearing human ovarian OVCAR-3 carcinoma xenografts. P-DOX-Ab and P-Mce(6)-Ab were synthesized by first conjugating DOX or Mce(6) to an HPMA copolymer precursor (Mw=21000), then reacting with OV-TL 16 antibody. The immunoconjugates were purified by size exclusion chromatography on Superose 6 column and analyzed. The Mce(6) concentration in tissues was determined by a fluorescence assay. Eighteen hours after administration, the tumors received a light dose of 220 J/cm(2) from a KTP 650-nm dye-laser. P-DOX-Ab and P-Mce(6)-Ab had polymer:drug:protein weight ratios of 32:3:62 and 26:2:72, corresponding to polymer:drug:protein molecular ratios of approximately 4:14:1 and 3:8:1, respectively. The biodistribution results indicated that the percentage of total administered dose of Mce(6) in tumors reached approximately 1% for the nontargeted conjugate at 18 h after administration, while that of P-Mce(6)-Ab was approximately 13 times higher. Nude mice bearing OVCAR-3 xenografts that received one i.v. dose of P-DOX-Ab (2.2 mg/kg DOX equivalent) and P-Mce(6)-Ab (1.5 mg/kg Mce(6) equivalent) with light irradiation achieved a xenograft cure rate of more than 60%. The incorporation of OV-TL 16 antibody dramatically enhanced the accumulation in tumors with a concomitant increase in the therapeutic efficacy of P-DOX-Ab and P-Mce(6)-Ab in combination therapy, which may probably be attributed to both antibody targeting and enhanced permeability and retention (EPR) effects.

Preparation and biological evaluation of polymerizable antibody Fab' fragment targeted polymeric drug delivery system.

A new polymerizable antibody Fab' fragment with a PEG spacer (MA-PEG-Fab') was prepared from OV-TL 16 antibody, specific against the OA-3 antigen expressed on most human ovarian carcinomas. The MA-PEG-Fab' possessed a higher reactivity in the copolymerization with N-(2-hydroxypropyl)methacrylamide (HPMA) than the polymerizable Fab' fragment MA-Fab' with a short spacer. The MA-PEG-Fab' was copolymerized with HPMA and MA-Gly-Phe-Leu-Gly-Mce(6) producing an Fab' targeted HPMA copolymer-Mce(6) conjugate. The number and weight average molecular weights of the copolymer were 164000 and 271000 Da, respectively. About two MA-PEG-Fab' fragments per chain were incorporated in the copolymer conjugates. Preliminary in vivo antitumor studies indicated that the Fab' targeted conjugates showed a higher efficacy of tumor growth inhibition in nude mice than the non-targeted conjugate.

Biodistribution and antitumour efficacy of long-circulating N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates in nude mice.

The aim of this study was to evaluate the influence of the molecular weight (mol. wt) of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (DOX) conjugates (P-DOX) on biodistribution and therapeutic efficacy in nu/nu mice bearing human ovarian carcinoma OVCAR-3 xenografts. Copolymerisation of HPMA, a polymerisable derivative of DOX (N-methacryloylglycylphenylalanylleucylglycyl doxorubicin) and a newly designed crosslinking agent, N(2),N(5)-bis(N-methacryloylglycylphenylalanyl-leucylglycyl)ornithine methyl ester monomers resulted in novel, high mol. wt, branched, water-soluble P-DOX containing lysosomally degradable oligopeptide sequences as crosslinks and side-chains terminated in DOX. Four conjugates with mol. wt of 22, 160, 895 and 1230 kDa were prepared. The results indicated that the half-life in blood and the elimination rate from the tumour were up to 28 times longer and 25 times slower, respectively, for P-DOX (mol. wt=1230 kDa) than for free DOX. Treatment with P-DOX (mol. wt > or = 160 kDa) inhibited tumour growth more efficiently than that of 22 kDa P-DOX or free DOX (P<0.02) at a 2.2 mg/kg DOX equivalent dose. In conclusion, the administration of long circulating P-DOX resulted in enhanced tumour accumulation with a concomitant increase in therapeutic efficacy.

Antitumor activity of N-(2-hydroxypropyl) methacrylamide copolymer-Mesochlorine e6 and adriamycin conjugates in combination treatments.

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-Mce6 conjugates was the most effective regimen.

Influence of pH on aggregation and photoproperties of n-(2-hydroxypropyl)methacrylamide copolymer-meso-chlorin e6 conjugates.

The influence of pH on the aggregation and photoproperties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing meso-chlorin e(6) monoethylenediamine (Mce(6)) attached to the copolymer via either nonbiodegradable G or biodegradable GFLG side chains was studied. Dynamic light scattering, UVIVIS and fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and fluorescence quenching techniques were used. The photosensitizing efficiencies of these conjugates were also determined. The dynamic light-scattering data indicate that the intermolecular aggregation of Mce(6) species within the copolymer conjugates is not significant and is not affected by pH or loading of Mce(6) to copolymer at 5 x 10(-4) g/mL of copolymer conjugate concentration. However, intramolecular aggregation of the Mce(6) species within the copolymer conjugates does occur in aqueous buffers, as demonstrated by absorption and fluorescence measurements in ethanol-buffer mixtures. The fluorescence lifetime of excited Mce(6) was influenced by aggregation, mainly attributed to the pH and copolymer side-chain hydrophobicity. The Stern-Volmer collisional quenching constant, K(sv) iodide anion with Mce(6) species was found to be a function of pH, reflecting both the electrostatic repulsion between negatively charged Mce(6) species and iodide anions and the intramolecular aggregation of Mce(6) moieties. The extent of aggregation was found to be a function of solvent pH, loading of Mce(6) to copolymer, and side-chain hydrophobicity. The photosensitizing efficiency of the copolymer bound Mce(6), as determined through the photooxidation of furfuryl alcohol, was dominated by Mce(6) loading to copolymer and side-chain hydrophobicity, but was only slightly pH dependent. Evidently, the Mce(6) aggregation only weakly influenced the charge transfer in the process of oxygen generation.

Intracellularly biorecognizable derivatives of 5-fluorouracil. Implications for site-specific delivery in the human condition.

The release of 5-fluorouracil from polymer-based conjugates can be influenced by the type of linkages used to bind the drug to the polymer carrier. The use of specific oligopeptide sequences designed to be biorecognizable by intracellular enzymes is a promising approach for increasing the site-specific release of 5-fluorouracil from polymer-based conjugates. In this study, we investigated the biorecognizability of specific oligopeptide sequences linking 5-fluorouracil to a water-soluble copolymer carrier based on N-(2-hydroxypropyl) methacrylamide by human cathepsin B (EC 3.4.22.1), cathepsin H(EC 3.4.22.6), and a homogenate of the human colon adenocarcinoma cell line SW 480. The cathepsins were chosen based on the hypothesis that they were two principal lysosomal enzymes responsible for the release of 5-fluorouracil from these conjugates. Our results support this hypothesis; however, these two enzymes may not be the only lysosomal enzymes responsible for the release kinetics observed. While the results for cathepsin B corresponded well to our hypothesis, the cleavage via cathepsin H was lower than predicted, suggesting the presence of additional lysosomal enzymes with catalytic activity toward these 5-fluorouracil derivatives.

Combination chemotherapy and photodynamic therapy with N-(2-hydroxypropyl) methacrylamide copolymer-bound anticancer drugs inhibit human ovarian carcinoma heterotransplanted in nude mice.

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 and 75 mg/kg; 1.5 mg/kg and 8.7 mg/kg of Mce6 equivalent, respectively) with light resulted in tumor volumes that were significantly less than control tumor volumes and the tumor volumes of mice receiving either P-A (30 mg/kg, 2.2 mg/kg Adriamycin equivalent) or P-C with light (12.5 or 75 mg/kg. 1.5 or 8.7 mg/kg Mce6 equivalent, respectively) alone (all P < 0.02). P-C (75 mg/kg, 8.7 mg/kg Mce6 equivalent) added to P-A (30 mg/kg, 2.2 mg/kg Adriamycin equivalent) resulted in complete tumor ablation. Free Mce6 demonstrates a narrow margin of safety, which is extended by incorporation into HPMA copolymers. P-A demonstrates safety and efficacy in vivo. The combined chemotherapy and photodynamic therapy of P-A (30 mg/kg, 2.2 mg/kg Adriamycin equivalent) with P-C and light (12.5 and 75 mg/kg 1.5 and 8.7 mg/kg Mce6 equivalent, respectively) was nontoxic and allowed us to attain a significant improvement in tumor cures than those obtained by P-A or P-C with light alone.

HPMA copolymer-anticancer drug-OV-TL16 antibody conjugates. 1. influence of the method of synthesis on the binding affinity to OVCAR-3 ovarian carcinoma cells in vitro.

The influence of different methods of binding the OV-TL16 antibody and its Fab' fragment to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer--drug (adriamycin [ADR] or meso chlorin e6 mono(N-2-aminoethylamide) (Mce6)) conjugates on the affinity of conjugates to an ovarian carcinoma (OVCAR-3) cell associated antigen was investigated. The binding of the antibody to HPMA copolymer--drug (ADR or Mce6) conjugates via amino groups resulted in conjugates which were heterogeneous in their antigen binding. Coupling, the HPMA copolymer--Mce6 conjugate to the carbohydrate region of the antibody resulted in conjugates with a more homogeneous distribution of affinity constants than conjugates prepared by linking the antibody to the polymer via amino groups. However, both methods resulted in a decrease in the affinity constant compared to the native antibody. Conjugates prepared with the Fab' frgment of the OV-TL16 antibody demonstrated a more homogenous affinity than either conjugate prepared with the whole antibody. To verify the hypothesis that the changes in the binding affinity and homogeneity are a consequence of conformational changes in the antibody structure, a series of physiocochemical methods were employed to characterize the conjugates. The excitation energy transfer between OV-TL16 antibody and drugs (ADR and Mce6) and the spectral properties of Mce6 were used to monitor the interactions between the antibody and drugs. The quenching of the intrinsic fluorescence of the antibody was also employed to study its conformational changes. An attempt has been made to correlate the biorecognition at the cellular surface with the interactions of drug with the antibody molecule and with the changes in antibody conformation.

Isobolographic assessment of the interaction between adriamycin and photodynamic therapy with meso-chlorin e6 monoethylene diamine in human epithelial ovarian carcinoma (OVCAR-3) in vitro.

OBJECTIVE: Considering the differing mechanisms of cytotoxicity produced by adriamycin and the photosensitizer meso-chlorin e6 monoethylene diamine (Mce6) with light, the interaction of these agents in combination on human ovarian epithelial carcinoma (OVCAR-3 in vitro) was evaluated by dose and effect addition isobole analysis. METHODS: Mitochondrial respiration via the 3-(4,5-dimethyl thiazol-2 yl)-2,5-diphenyl tetrazolium bromide cleavage assay (MTT) and reproductive capacity via the tritiated thymidine incorporation assay (TI) were assessed 72 and 144 hours after exposure to adriamycin, Mce6, and light (650 nm), and to their combinations, in OVCAR-3 cells grown in vitro (20,000 cells per well). RESULTS: In the majority of assays, reproductive capacity was more sensitive to the drug(s) than was mitochondrial respiration (2-10x). Dose-addition isobole analysis showed synergy for the combination of 50% median effective dose (ED50) adriamycin with 50% ED50 Mce6/light in all assays (all P < or = .027). Antagonism was noted with the combination 25% ED50 adriamycin with 75% ED50 Mce6/light. Additivity and synergy were the predominant interactions for 75% ED50 adriamycin with 25% ED50 Mce6/light by dose-addition isobole analyses. Effect-addition isoboles showed a predominance of synergy, particularly for the combination 50% ED50 adriamycin with 50% ED50 Mce6/light. CONCLUSION: Synergy and additivity are the primary in vitro interactions for the combination of adriamycin and Mce6/light in the dosage range tested. Reproductive capacity is more sensitive to these agents than is mitochondrial respiration.