Biomedical subjects

J Kopecek

Publications and source records attributed to J Kopecek.

At least 37 records · Page 2Linked to original sources

PubMed2000

Biorecognition of HPMA copolymer-lectin conjugates as an indicator of differentiation of cell-surface glycoproteins in development, maturation, and diseases of human and rodent gastrointestinal tissues.

Lectins are proteins that bind glycoproteins; binding patterns are altered with changes in glycoprotein expression accompanying maturation or disease. Binding of two lectins, wheat germ agglutinin (WGA) and peanut agglutinin (PNA), in human and rodent colon were previously examined. Normal tissue showed intense WGA binding; PNA binding was minimal. Diseased tissues showed increased PNA binding. We hypothesized that N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-lectin-drug conjugates could deliver therapeutic agents to diseased tissues by targeting colonic glycoproteins. We examined biorecognition of free and HPMA copolymer-conjugated WGA and PNA and anti-Thomsen-Friedenreich (TF) antigen antibody binding in normal neonatal, adult, and diseased rodent tissues, human specimens of inflammation, and Barrett's esophagus. Neonatal WGA binding was comparable to the adult, with additional luminal columnar cell binding. PNA binding was more prevalent; luminal columnar cell binding existed during the first 2.5 weeks of life. WGA binding was strong in both normal and diseased adult tissues; a slight decrease was noted in disease. PNA binding was minimal in normal tissues; increases were seen in disease. Anti-TF antigen antibody studies showed that PNA did not bind to the antigen. The results suggest that HPMA copolymer-lectin-drug conjugates may provide site-specific treatment of conditions such as colitis and Barrett's esophagus.

Animals↗

PubMed2000

Efficacy of the chemotherapeutic action of HPMA copolymer-bound doxorubicin in a solid tumor model of ovarian carcinoma.

Anticancer activity and main mechanisms of action of free doxorubicin (DOX) and HPMA copolymer-bound DOX (P(GFLG)-DOX) were studied in solid tumor mice models of DOX sensitive and resistant human ovarian carcinoma. Free DOX was effective only in sensitive tumors decreasing the tumor size about three times, whereas P(GFLG)-DOX decreased the tumor size 28 and 18 times in the sensitive and resistant tumors. An enhanced accumulation of P(GFLG)-DOX in the tumor was observed, whereas only low concentrations of DOX were detected in other organs following P(GFLG)-DOX administration. This effect was dependent on the high permeability of blood vessels in untreated tumors. After treatment with P(GFLG)-DOX the permeability decreased concomitantly with the downregulation of VEGF gene expression. P(GFLG)-DOX effectively killed both types of tumors inducing apoptosis and necrosis through the activation of p53, Apaf-1, caspase 9, c-fos, or c-jun pathways, and the downregulation of the bcl-2 gene. HPMA copolymer-bound DOX preserved its activity inside cells, inhibited detoxification and defensive mechanisms encoded by GST-pi, BUDP, and HSP-70 genes, and limited DNA repair, replication, and biosynthesis by downregulation of Topo-IIalpha,beta, and TK1 genes. P(GFLG)-DOX also produced tumor tissue hypoxia and significantly activated lipid peroxidation in tumors. No damage to other organs after exposure to P(GFLG)-DOX was detectable. On the other hand, free DOX activated lipid peroxidation and led to tissue hypoxia in many organs. All data relevant to the mechanism of anticancer action of P(GFLG)-DOX indicated a higher antitumor activity and lower systemic toxicity of HPMA copolymer-bound DOX when compared with free DOX.

Animals↗

PubMed2000

Chronic exposure of human ovarian carcinoma cells to free or HPMA copolymer-bound mesochlorin e6 does not induce P-glycoprotein-mediated multidrug resistance.

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

HPMA copolymer-anticancer drug conjugates: design, activity, and mechanism of action.

The design, synthesis and properties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers as carriers of anticancer drugs are reviewed. Macromolecular therapeutics based on HPMA copolymers are biocompatible, preferentially accumulate in tumors, and possess a higher anticancer efficacy than low molecular weight drugs. Novel designs of HPMA copolymer carriers resulted in long-circulating conjugates and gene and oligonucleotide delivery systems. HPMA copolymer based macromolecular therapeutics were active against numerous cancer models and are in clinical trials. The data obtained indicated that macromolecular therapeutics activated different signaling pathways and possessed a different mechanism of action than free drugs. This bodes well for the success of future research aimed at identification of new intracellular molecular targets as a basis for the design of the second generation of macromolecular therapeutics.

Animals↗

PubMed2000

HPMA copolymer-anticancer drug-OV-TL16 antibody conjugates. 3. The effect of free and polymer-bound adriamycin on the expression of some genes in the OVCAR-3 human ovarian carcinoma cell line.

The effect of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-Adriamycin-OV-TLl6 antibody conjugate [P(GFLG)-ADR-Ab] on OVCAR-3 human ovarian carcinoma cells was studied. A nontargeted HPMA copolymer-ADR conjugate (P(GFLG)-ADR) and free ADR were the controls. The IC(50) doses were 0.65, 3.0, and 65 microM for free ADR, targeted P(GFLG)-ADR-Ab conjugate, and nontargeted P(GFLG)-ADR conjugate, respectively. These differences reflect the different mechanisms of cell entry of the compounds evaluated. Free ADR and HPMA copolymer-ADR conjugates had different impacts on the expression of MDR1, MRP, c-fos, c-jun, and bcl-2 genes which encode the P-glycoprotein (MDR1) and the multidrug resistance-associated protein (MRP) efflux pumps, and play an important role in cell death signaling pathways (c-fos, c-jun, and bcl-2). Whereas high doses of free ADR induced MDR1 gene expression, HPMA copolymer-bound ADR appeared to be without effect. On the contrary, expression of the MRP gene was not influenced by free ADR, whereas HPMA copolymer-ADR conjugates seemed to suppress the gene expression in a concentration-dependent manner. There were differences in the expression of c-fos, c-jun, and bcl-2 genes after the incubation of OVCAR-3 cells with free and HPMA copolymer-bound ADR indicating differences in activation of cell death signaling pathways.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Synthesis of starlike N-(2-hydroxypropyl)methacrylamide copolymers: potential drug carriers.

Starlike HPMA copolymers were synthesized by conjugating semitelechelic poly[N-(2-hydroxypropyl)-methacrylamide] macromolecules (ST-PHPMA, arm) with PAMAM dendrimers (core: G2, G3, G4). ST-PHPMA was synthesized by chain transfer free radical polymerization, and the terminal -COOH was activated with N-hydroxysuccinimide. Doxorubicin (DOX) was introduced into the starlike HPMA copolymer to evaluate its potential as a drug delivery system. The polymers were characterized with SEC, NMR, and UV. Cytotoxicity of the DOX containing starlike HPMA copolymer was determined on an A2780 human ovarian carcinoma cell line and compared with DOX-containing linear HPMA copolymers. The rate of in vitro DOX release from polymer--DOX conjugates in the presence of cathepsin B (CP-B, lysosomal cysteine proteinase) was determined and correlated with cytotoxicity results.

Antibiotics, Antineoplastic↗

PubMed2000

The influence of cytotoxicity of macromolecules and of VEGF gene modulated vascular permeability on the enhanced permeability and retention effect in resistant solid tumors.

PURPOSE: To study the influence of cytotoxicity of macromolecules, VEGF gene expression, and vascular permeability on the enhanced permeability and retention (EPR) effect. METHODS: Mice bearing xenografts of A2780 multidrug resistant human ovarian carcinoma were treated by free doxorubicin (DOX) and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound DOX (P(GFLG)-DOX), Texas Red (P-TR), and FITC (P-FITC). Antitumor activity, drug distribution in tumor, vascular permeability, VEGF gene expression, and DNA fragmentation were studied. RESULTS: The accumulation of free DOX led to the VEGF gene overexpression and increased the vascular permeability, which in turn enhanced the drug accumulation in the same location. This positive feedback loop led to a highly inhomogeneous distribution of the drug within the tumor. In contrast, P(GFLG)-DOX down-regulated the VEGF gene and decreased vascular permeability. This negative feedback seemed to prevent additional drug accumulation in dead necrotic tissue, resulting in a more uniform drug distribution and enhanced the antitumor activity P(GFLG)-DOX. CONCLUSIONS: The EPR effect significantly differed for macromolecules containing DOX when compared to macromolecules without drug. The cytotoxicity of P(GFLG)-DOX amplified the EPR effect, led to a more homogenous distribution of the drug, increased the average drug concentration in tumor and augmented its efficacy.

Animals↗

PubMed2000

HPMA copolymer-modified avidin: immune response.

Protein-polymer conjugates to be used in the pretargeted delivery of a photosensitizer to cells were synthesized and characterized. Avidin was modified by N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing the photosensitizer, mesochlorin e6 mono(N-2-aminoethylamide) (Mce6). Synthesis of HPMA copolymer-avidin-Mce6 conjugates was carried out so that either predominantly single point attachment or multipoint attachment of copolymer chains to avidin would result. HPMA copolymer-avidin conjugates were used which retained specific binding activity to a lower affinity biotin analog. Antigen specific anti-avidin immune response was shown to be reduced six-fold in some HPMA copolymer-avidin conjugates when compared to immune response to unmodified avidin. HPMA copolymer itself was shown to elicit a very low (IgM) immune response.

Animals↗

PubMed2000

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.

Acrylamides↗

PubMed1999

Biodistribution of free and N-(2-hydroxypropyl)methacrylamide copolymer-bound mesochlorin e(6) and adriamycin in nude mice bearing human ovarian carcinoma OVCAR-3 xenografts.

The purpose of this study was to examine the biodistribution of the photosensitizing drug, mesochlorin e(6) monoethylenediamine (Mce(6)), and the antineoplastic agent, adriamycin (ADR), as well as their N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates in female nu/nu athymic mice bearing human ovarian carcinoma OVCAR-3 xenografts. The levels of Mce(6) and HPMA copolymer-bound Mce(6) in tissues were assayed spectrophotometrically, while the levels of ADR and HPMA copolymer-bound ADR were determined using high-performance liquid chromatography. It appeared that the circulation lifetimes of HPMA copolymer-bound Mce(6) and ADR were three times more than those of the drugs in the free form. The concentrations of the HPMA copolymer-conjugated drugs in tumor reached maximum levels 18 h post injection. Intravenous injection routinely gave higher tissue levels of the drugs than intraperitoneal administration at time intervals less than 24 h. The biodistribution of the HPMA copolymer-bound drugs in tumor-bearing mice was significantly different from that of the free drugs, which is important in optimizing the treatment protocols. In particular, the HPMA copolymer-conjugated drugs accumulated at significantly higher levels in tumor tissues. This effect is attributed to the increased vascular permeability and reduced lymphatic drainage characteristic of tumor tissues [enhanced permeability and retention (EPR) effect].

Animals↗

PubMed1999

High-molecular weight HPMA copolymer-adriamycin conjugates.

High-molecular weight (branched) water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing lysosomally degradable oligopeptide crosslinks were synthesized by radical copolymerization of HPMA and newly designed crosslinking agents, N(2), N(5)-bis(N-methacryloylglycylphenylalanylleucylglycyl)ornithine s with different modification of the carboxy group. The length of the primary chain was controlled by the addition of a chain transfer agent, 3-mercaptopropionic acid. A polymerizable derivative of the anticancer drug adriamycin (ADR), N-methacryloylglycylphenylalanylleucylglycyl adriamycin, was added to some polymerization mixtures. This resulted in high-molecular weight, branched, water-soluble HPMA copolymers containing oligopeptide sequences in the crosslinks as well as in side-chains terminated in ADR. The degradability of the crosslinks as well as the release of ADR by lysosomal enzymes isolated from rat liver were investigated.

3-Mercaptopropionic Acid↗

PubMed1999

Chronic exposure to HPMA copolymer-bound adriamycin does not induce multidrug resistance in a human ovarian carcinoma cell line.

The influence of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound adriamycin (ADR) on the induction of multidrug resistance in the A2780 human ovarian carcinoma cell line was studied in vitro. It was found that chronic exposure to free ADR led to an increase in resistance to ADR and Taxol and overexpression of the MDR1 gene. No significant changes in the expression of the MRP gene were found during adaptation to free ADR. In addition to MDR1 gene-encoded multidrug resistance, a significant increase in the resistance against ADR was found before the overexpression of the MDR1 gene was measurable. This non-P-glycoprotein resistance does not appear to be connected with MRP gene-encoded resistance. During adaptation to free ADR, changes in cellular metabolism such as increased rate of glucose uptake, oxidation and glycolysis were detected. Adapted sensitive A2780 cells expressed the MDR1 gene and possessed almost the same decreased sensitivity toward ADR as the ADR-resistant human ovarian carcinoma A2780/AD cells. However, they significantly differed in proliferation rate, cellular metabolism and MRP gene expression. On the contrary, multidrug resistance was not induced after repeated exposure of sensitive A2780 cells to HPMA copolymer-bound adriamycin. The cells did not express the MDR1 gene, the expression of the MRP gene was partially inhibited, and the resistance against Taxol was decreased. Differences were also observed in metabolic changes. In summary, the data indicate that, contrary to free ADR, HPMA copolymer-bound ADR does not induce multidrug resistance in A2780 cell culture after repeated exposure.

Cell Division↗

PubMed1999

Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains.

Stimuli-sensitive polymer hydrogels, which swell or shrink in response to changes in the environmental conditions, have been extensively investigated and used as 'smart' biomaterials and drug-delivery systems. Most of these responsive hydrogels are prepared from a limited number of synthetic polymers and their derivatives, such as copolymers of (meth)acrylic acid, acrylamide and N-isopropyl acrylamide. Water-soluble synthetic polymers have also been crosslinked with molecules of biological origin, such as oligopeptides and oligodeoxyribonucleotides, or with intact native proteins. Very often there are several factors influencing the relationship between structure and properties in these systems, making it difficult to engineer hydrogels with specified responses to particular stimuli. Here we report a hybrid hydrogel system assembled from water-soluble synthetic polymers and a well-defined protein-folding motif, the coiled coil. These hydrogels undergo temperature-induced collapse owing to the cooperative conformational transition of the coiled-coil protein domain. This system shows that well-characterized water-soluble synthetic polymers can be combined with well-defined folding motifs of proteins in hydrogels with engineered volume-change properties.

Acrylamides↗

PubMed1999

Degradation and aggregation of human calcitonin in vitro.

PURPOSE: To investigate the degradation of human calcitonin (hCT) by enzymes or mucosa from different gastrointestinal (GI) compartments and evaluate the stabilization effect of a synthetic ionizable copolymer on the stability of hCT in an aqueous solution. These data are a prerequisite for the development of a hydrogel based colon-specific hCT delivery system. METHODS: Luminal and brush border membrane (BBM) enzymes from the colon and small intestine (SI) of the rabbit were isolated and their enzymatic activity toward hCT in vitro was evaluated. Human fecalase was used to mimic the luminal enzymatic activity in the human colon and its degradation ability was assessed. Excised intact rabbit intestinal tissues from both the colon and the SI were used to study the degradation patterns of hCT by intact mucosa. Detection of intact human calcitonin was performed using gradient elution, reverse phase high-pressure liquid chromatography (RP-HPLC). The structure of the hCT fragments was determined by Matrix Assisted Laser Desorption/lonization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) analysis. UV/VIS and fluorescence spectroscopy methods were used to evaluate the influence of a copolymer, possessing the same structure as the primary chains in hydrogels degradable in the colon, on the fibrillation process of hCT. RESULTS: In vitro results showed that isolated luminal enzymes and BBM enzymes from the SI were more potent in degrading intact hCT, as expected. Moreover, BBM enzymes were far more abundant in the SI than in the colon. Compared with rabbit colonic luminal enzymes, the degradation potency of human fecalase was further abated. Intact mucosal studies revealed extensive degradation by the SI mucosa but not by the colonic mucosa. The primary structures of the peptide fragments were identified by MALDI-TOF MS analysis. Fibrillation studies of hCT indicated that acrylic acid-containing polymeric materials were able to decrease the aggregation of hCT in aqueous solutions. CONCLUSIONS: Reduced proteolytic activity suggests that the colon is an advantageous site for peptide delivery. The structures of hCT degradation products were identified and the participation of particular enzymes in the degradation process was suggested. In addition, it was determined that an acrylic acid-containing copolymer improved the physical stability of hCT in aqueous solution.

Amino Acid Sequence↗

PubMed1999

Comparison of the anticancer effect of free and HPMA copolymer-bound adriamycin in human ovarian carcinoma cells.

PURPOSE: To study peculiarities and the mechanism of the anticancer effect of free and HPMA copolymer-bound ADR in sensitive and resistant human ovarian carcinoma cells. METHODS: Sensitive A2780 and ADR resistant A2780/AD cells were exposed to different doses of drugs during 12, 24, 36, 48, 60, and 72 hours. Cell viability, drug accumulation, apoptosis, cellular metabolism, lipid peroxidation, DNA content and gene expression were studied. RESULTS: HPMA copolymer-bound ADR (P(GFLG)-ADR) possessed a comparable cytotoxicity to free ADR when comparison was based on intracellular concentrations. While free ADR up-regulated genes encoding ATP driven efflux pumps (MDR1, MRP), P(GFLG)-ADR overcame existing pumps and down regulated the MRP gene. Free ADR also activated cell metabolism and expression of genes responsible for detoxification and DNA repair. P(GFLG)-ADR down-regulated HSP-70, GST-pi, BUDP, Topo-IIalpha, beta, and TK-1 genes. Apoptosis, lipid peroxidation and DNA damage were significantly higher after exposure to P(GFLG)-ADR, as reflected by simultaneous activation of p53, c-fos in A2780 cells) or c-jun (A2780/AD) signaling pathways and inhibition of the bcl-2 gene. Differences between free ADR and P(GFLG)-ADR increased with the time of incubation and drug concentration. CONCLUSIONS: P(GFLG)-ADR overcame drug efflux pumps, more significantly induced apoptosis and lipid peroxidation, inhibited DNA repair, replication, and biosynthesis when compared to free ADR.

Antineoplastic Agents↗

PubMed1999

Biorecognition of HPMA copolymer-adriamycin conjugates by lymphocytes mediated by synthetic receptor binding epitopes.

PURPOSE: The EDPGFFNVE nonapeptide (NP) was recognized as the CD21 (CR2) binding epitope of the Epstein-Barr virus (EBV) gp350/ 220 envelope glycoprotein which mediates the virus attachment to human B lymphocytes (Nemerow et al., Cell 56:369-377, 1989). Here we evaluated the targeting potential of a synthetic receptor binding epitope (NP) covalently attached to a water-soluble polymeric drug carrier. In particular, the biorecognition of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-NP conjugates by B- and T-cells and the cytotoxicity of HPMA copolymer-NP-adriamycin (ADR) conjugates toward B-cells, T-cells, and peripheral blood lymphocytes (PBL) were evaluated. METHODS: HPMA copolymer-NP and optionally ADR conjugates varying in the NP density and mode of NP attachment were incubated with Raji B-cells (human Burkitt's lymphoma), CCRF-CEM T-cells (acute human lymphoblastic leukemia), and CCRF-HSB-2 T-cells (human lymphoblastic leukemia). The kinetics of binding was studied, the Langmuir adsorption isotherms analyzed, binding constants calculated, and IC50 doses determined. RESULTS: Flow cytometry studies revealed that binding was homogeneous to both cell types. The apparent binding constants to T-cells were about two times higher when compared to B-cells. The binding and cytotoxicity increased with increased amount of epitopes per polymer chain. Attachment of the NP via a GFLG spacer resulted in increased biorecognition when compared with conjugates containing NP bound via a GG spacer. HPMA copolymer-NP-ADR conjugates possessed specific cytotoxicity to T- and B-malignant cells. Concentrations, which were lethal to the latter, were not toxic for PBL. CONCLUSIONS: The data obtained seem to indicate the potential of the HPMA copolymer-NP conjugates as polymer anticancer drug carriers targetable to immunocompetent cells.

B-Lymphocytes↗

PubMed1999

Polymerizable Fab' antibody fragments for targeting of anticancer drugs.

We have designed a new pathway for the synthesis of targeted polymeric drug delivery systems, using polymerizable antibody Fab' fragments (MA-Fab'). The targeted systems can be directly prepared by copolymerization of the MA-Fab', N-(2-hydroxypropyl)methacrylamide (HPMA) and drug-containing monomers. Both MA-Fab' and the Fab'-targeted copolymers can effectively bind to target cells. An MA-Fab' (from OV-TL 16 Ab) targeted HPMA copolymer containing mesochlorin e6 (Mce6) was synthesized by copolymerization of MA-Fab', HPMA, and MA-GFLG-Mce6. The targeted copolymer exhibited a higher cytotoxicity toward OVCAR-3 human ovarian carcinoma cells than the nontargeted Mce6-containing copolymer or free Mce6. The targeted copolymer was internalized more efficiently by OVCAR-3 cells than the nontargeted copolymer.

Antineoplastic Agents↗

PubMed1999

Cooperativity between free and N-(2-hydroxypropyl) methacrylamide copolymer bound adriamycin and meso-chlorin e6 monoethylene diamine induced photodynamic therapy in human epithelial ovarian carcinoma in vitro.

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 adriamycin ED50) did not appear to significantly improve the efficacy profile of P-A. A and C in vitro appear to act independently and are cooperative in their combined toxicity against the human ovarian epithelial carcinoma cell line OVCAR-3. HPMA copolymer-adriamycin and Mce6 conjugates (P-A and P-C, respectively) inhibited growth of OVCAR-3 in vitro. HPMA copolymer-adriamycin added to HPMA copolymer-Mce6 improved the efficacy of HPMA copolymer-Mce6.

Antineoplastic Agents↗