Biomedical subjects

J Kopecek

Publications and source records attributed to J Kopecek.

At least 19 recordsLinked to original sources

PubMed2001

Improved synthesis and evaluation of 17-substituted aminoalkylgeldanamycin derivatives applicable to drug delivery systems.

The 17-methoxy group of geldanamycin was substituted with 1,3-diaminopropane and 1,3-diamino-2-hydroxypropane to introduce a primary amino group useful for conjugation with targeting moieties and drug carriers. We have developed a procedure that has provided improved yield and reproducibility of the syntheses. Both geldanamycin derivatives demonstrated antiproliferative activity towards the human ovarian carcinoma cell line, A2780.

Antibiotics, Antineoplastic↗

PubMed2001

Biorecognizable HPMA copolymer-drug conjugates for colon-specific delivery of 9-aminocamptothecin.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates for colon-specific delivery of 9-aminocamptothecin (9-AC) were designed. They hold 9-AC bound via spacers containing amino acid residues and aromatic azo bonds. In vitro release profiles of 9-AC from HPMA copolymer conjugates were evaluated under artificial conditions that simulated large intestinal azoreductase and peptidase activities. The studies indicated that the azo bond was reduced first, followed by the release of unmodified 9-AC from the 9-AC containing fragment by peptidases. Release profiles depended on the chemical structure of the peptide part of the spacer. Conjugates containing leucylalanine showed high colon-specific release of 9-AC when compared to alanine containing conjugates. It appears that the studied conjugates are suitable as colon-specific drug delivery systems.

Animals↗

PubMed2001

Synthesis and characterization of HPMA copolymer-aminopropylgeldanamycin conjugates.

Geldanamycin (GDM) is a benzoquinone ansamycin antibiotic with anticancer activity. The use of drug delivery systems based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing lysosomally degradable oligopeptide (GFLG) spacers results in an increased therapeutic efficacy of anticancer drugs. The objective of this study was to synthesize HPMA copolymer-GDM conjugates with anticancer activity and reduced toxic side-effect of the compound. 17-(3-Aminopropylamino)-17-demethoxygeldanamycin (AP-GDM) was synthesized and converted into a polymerizable GDM derivative, N-methacryloylglycylphenylalanylglycyl-17-(3-aminopropylamino)-17-demethoxygeldanamycin [MA-GFLG-(AP-GDM)]. The structures of AP-GDM and MA-GFLG-(AP-GDM) were validated by mass spectroscopy, elemental analysis, and two-dimensional nuclear magnetic resonance. MA-GFLG-(AP-GDM) was copolymerized with HPMA and N-methacryloyglycylglycine p-nitrophenylester by radical precipitation polymerization. Water-soluble HPMA copolymer-AP-GDM conjugates (M(r)=16 kDa) were obtained. Monoclonal antibody OV-TL16, which recognizes the OA-3 antigen expressed on the OVCAR-3 human ovarian carcinoma cell line, was optionally attached to the HPMA copolymer-AP-GDM conjugate. Cytotoxicity of polymer-bound AP-GDM (both targeted and non-targeted) was determined using OVCAR-3 and another human ovarian carcinoma cell line, A2780. The HPMA copolymer-AP-GDM conjugate was cytotoxic toward A2780 cells. Attachment of OV-TL16 antibody enhanced cytotoxicity of the conjugate toward OVCAR-3 cells.

Antibiotics, Antineoplastic↗

PubMed2001

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.

Animals↗

PubMed2001

Water soluble polymers in tumor targeted delivery.

The rationales for the use of water soluble polymers for anticancer drug delivery include: the potential to overcome some forms of multidrug resistance, preferential accumulation in solid tumors due to enhanced permeability and retention (EPR) effect, biorecognizability, and targetability. The utility of a novel paradigm for the treatment of ovarian carcinoma in an experimental animal model, which combines chemotherapy and photodynamic therapy with polymer-bound anticancer drugs is explained. Research and clinical applications as well as directions for the future development of macromolecular therapeutics are discussed.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

The effects of subcellular localization of N-(2-hydroxypropyl)methacrylamide copolymer-Mce(6) conjugates in a human ovarian carcinoma.

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.

Antineoplastic Agents↗

PubMed2001

Potential of lectin-N-(2-hydroxypropyl)methacrylamide copolymer-drug conjugates for the treatment of pre-cancerous conditions.

N-(2-Hydroxypropyl)methacrylamide (HPMA)-lectin (wheat germ agglutinin (WGA), peanut agglutinin (PNA)) drug conjugates for treatment of the pre-cancerous conditions ulcerative colitis and Barrett's esophagus are being developed. Cell-surface glycoproteins that are altered in disease and development bind lectins. PNA binds alpha-lactose and the Thomsen-Friedenreich (TF) antigen, a disease- and development-associated glycoprotein. PNA incorporation in conjugates may allow for preferential delivery to diseased over healthy tissues. Conjugates were prepared by attaching lectins to HPMA copolymers via an amide linkage. Frontal affinity chromatography was used to measure dissociation constants (K(d)) of free and conjugated lectins. Animal models of colitis (DSS, TNBS/EtOH) were developed. Human biopsy specimens were obtained. Free and HPMA copolymer-conjugated FITC-labeled lectin and anti-TF antigen antibody binding patterns were examined in normal neonatal, adult and diseased rodent tissues and normal and diseased human tissues. K(d) values of free and conjugated lectins were similar ( approximately 10(-5) M(-1)). Free and conjugated lectins had comparable binding patterns. In health, strong WGA binding was seen in goblet cells; PNA binding was minimal, occurring only in the supranuclear goblet cell region. In disease, WGA binding was not altered, but PNA binding was increased in both human and rodent tissues; entire goblets bound the lectin. Anti-TF antigen antibody binding was minimal, but did overlap with PNA binding patterns both in normal and diseased tissues. Conjugation of lectins to HPMA copolymers does not affect binding affinity. Alterations in glycoprotein structures in development and disease resulted in modified lectin binding patterns. In development and disease, the PNA binding seen was to the TF antigen and other lactose-containing glycoproteins. The results suggest that site-specific delivery of therapeutic agents such as cyclosporin A (CsA) for ulcerative colitis and mesochlorin e(6) for Barrett's esophagus may be achieved. P(HPMA)-lectin-CsA conjugates have been prepared and preliminary in vivo studies are underway.

Algorithms↗

PubMed2001

Water-soluble HPMA copolymer-wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo.

Phosphoinositide kinases and ATM-related genes play a central role in many physiological processes. Activation of phosphoinositide 3-kinase (PI 3-kinase) is essential for signal transduction by many growth factors and oncogenes and may contribute to tumor progression. In the nanomolar range, Wortmannin (WM), a fungal metabolite, is a potent inhibitor of type I PI 3-kinase; it covalently modifies its catalytic subunit. Because WM is soluble only in organic solvents and unstable in water, there are difficulties in its use in vivo. To generate a water-soluble WM derivative, we used a conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and 11-O-desacetylwortmannin (DAWM), which has a slightly lower inhibitory activity than WM. We covalently attached DAWM to HPMA copolymer containing oligopeptide (GFLG) side-chains. The final product had an estimated molecular mass of 20 kDa and contained 2 wt.% of DAWM. The HPMA copolymer (PHPMA)-DAWM conjugate inhibited type I PI 3-kinase activity in vitro and growth factor-stimulated activation of Akt in vivo; it possessed approximately 50% of the inhibitory activity of DMSO solubilized WM. The specificity and stability of the PHPMA-DAWM conjugate is currently under investigation. The new water-soluble form of WM may be useful in investigations of the role of PI 3-kinase in tumor progression and other cellular biological functions in vivo.

Androstadienes↗

PubMed2001

The coiled coils in the design of protein-based constructs: hybrid hydrogels and epitope displays.

Recombinant DNA technology provides a powerful tool for producing protein-based biomaterials. Genetically engineered coiled coils have been used as a structural module for the construction of a variety of bio-based systems useful in drug delivery studies. Two of such approaches developed in the authors' laboratory were described here. One approach was to assemble hybrid hydrogels from coiled coil protein domains and synthetic polymers. Preliminary results showed that temperature-sensitive volume transition of the hybrid hydrogels could be triggered by the thermal unfolding of the engineered coiled coil protein domains. The other approach, discussed in detail, was to construct an epitope display model system based on a coiled coil stem loop peptide self-assembled on a solid substrate. This model construct displayed a constrained nonapeptide sequence, which was found to mediate specific binding with immunocompetent cells bearing complementary surface receptors. These novel approaches will likely find important applications in the rational design of more effective drug delivery systems.

Amino Acid Sequence↗

PubMed2001

Preliminary evaluation of caspases-dependent apoptosis signaling pathways of free and HPMA copolymer-bound doxorubicin in human ovarian carcinoma cells.

The purpose of the study was to examine the role of caspases in signaling pathways of apoptosis induced by free doxorubicin (DOX) and HPMA copolymer-bound DOX (P(GFLG)-DOX) in human ovarian carcinoma cells. Sensitive A2780 and DOX resistant A2780/AD cells were exposed to different doses of drugs within 12, 18, 24 and 36 h. Caspase activity, expression of genes encoding human caspases 1-10, Apaf-1 and bcl-2 proteins and apoptosis were studied. In sensitive cells both free and P(GFLG)-DOX activated caspases 3, 7 and 9. In addition, P(GFLG)-DOX activated caspases 6 and 8. In resistant cells apoptosis induced by free DOX depended on the activation of caspases 2, 7 and 9, while caspase 3 was not involved; this explains the low degree of apoptosis induced by free DOX in resistant cells. P(GFLG)-DOX triggered the additional caspases 3, 6 and 8. A more pronounced degree of caspase activation and apoptosis after the action of P(GFLG)-DOX depended on the inhibition of bcl-2-encoded cellular defensive mechanisms and a more significant activation of Apaf-1. It was concluded that HPMA copolymer-bound DOX induced additional caspase-dependent apoptosis signaling pathways and the degree of the induction was higher, which led to more pronounced apoptosis when compared to free DOX.

Antibiotics, Antineoplastic↗

PubMed2001

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.

Animals↗

PubMed2001

Hybrid hydrogels cross-linked by genetically engineered coiled-coil block proteins.

Hybrid hydrogels of hydrophilic synthetic polymers cross-linked by protein modules undergo externally triggered volume transitions as a result of protein conformational changes. To investigate the influence of coiled-coil protein structure and stability on hydrogel volume transition, a series of block proteins containing interspersed naturally derived recombinant coiled-coils was synthesized. Proteins were characterized using circular dichroism, size exclusion chromatography, gel electrophoresis, and analytical ultracentrifugation. The block proteins formed self-associating oligomers and displayed thermal unfolding profiles indicative of a hierarchic higher-order structure. Hybrid hydrogels were assembled from an N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer and His-tagged block proteins through metal complexation. A temperature-induced decrease in hydrogel swelling was observed, and the onset temperature of the volume transition corresponded to the onset temperature of protein unfolding. We conclude that stimuli-responsive properties of hybrid hydrogels can be tailored by engineering the structure and properties of protein cross-links.

Amino Acid Sequence↗

PubMed2001

The cytoplasmic escape and nuclear accumulation of endocytosed and microinjected HPMA copolymers and a basic kinetic study in Hep G2 cells.

The development of macromolecules as drugs and drug carriers requires knowledge of their fate in cells. To this end, we studied the internalization and subcellular Fate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers in Hep G2 (human hepatocellular carcinoma) cells. Semiquantitative fluorometry confirmed that galactose was an effective ligand for receptor-mediated endocytosis for Hep G2 cells. The rate of internalization of a galactose-targeted copolymer was almost 2 orders of magnitude larger than that of the nontargeted copolymer. Confocal fluorescent microscopy of both fixed and live cells revealed that the polymer entered the cells by endocytosis. After longer incubation times (typically >8 hours), polymer escaped from small vesicles and distributed throughout the cytoplasm and nuclei of the cells. Polymer that entered the cytoplasm tended to accumulate in the nucleus. Microinjection of the HPMA copolymers into cells' cytoplasm and nuclei indicated that the polymers partitioned to the nucleus. The data from fixed cells was confirmed by microscopy of live, viable cells. To examine the effect of the fluorescent dye on the intracellular fate, polymers with fluorescein, Oregon Green 488, Lissamine rhodamine B, and doxorubicin were tested; no significant differences were observed.

Acrylamides↗

PubMed2001

The influence of a colonic microbiota on HPMA copolymer lectin conjugates binding in rodent intestine.

Germ-free (GF) animals lack a colonic microflora like that seen in conventional (CV) animals. Bacterial presence plays a role in the development of glycoproteins in the gastrointestinal (GI) tract; the absence of a microbiota has been seen to suppress the production of certain glycoproteins and glycolipids. Binding patterns of lectins are modified when glycoprotein structures are altered (e.g., during development or disease). Little information on lectin binding patterns in mature GF animals is available. We examined the binding of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-conjugated fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA) [P(HPMA)-(WGA-FITC)] and FITC-labeled peanut agglutinin (PNA) [P(HPMA)-(PNA-FITC)] in CV and GF mouse colon with and without neuraminidase pretreatment. Anti-Thomsen-Friedenreich (TF) antigen (a development and disease-related glycoprotein) antibody binding was also examined in these tissues. Subtle differences were seen in the binding patterns between CV and GF animals. CV animals showed strong P(HPMA)-(WGA-FITC) binding in goblet cells, but minimal P(HPMA)-(PNA-FITC) binding was visible. In GF animals, luminal surface binding of P(HPMA)-(WGA-FITC) was visible, and goblet cell binding of P(HPMA)-(PNA-FITC) was seen. These subtle changes suggest that altered glycoprotein expression occurred under GF conditions.

Animals↗

PubMed2000

Time- and concentration-dependent apoptosis and necrosis induced by free and HPMA copolymer-bound doxorubicin in human ovarian carcinoma cells.

A2780 sensitive and A2780/AD doxorubicin (DOX) resistant human ovarian carcinoma cells were exposed to different concentrations (0.25, 0.5, 1, 5 and 10xIC(50)) of free and HPMA copolymer-bound DOX for 12, 24, 36, 48, 60 and 72 h. Apoptosis and necrosis were evaluated using the FITC-conjugated annexin V and propidium iodide staining. The data obtained showed that the induction of apoptosis and necrosis by both free DOX and HPMA copolymer-bound DOX were time- and concentration-dependent. The data also showed significant differences between the drugs. It was found that: (i) under the action of HPMA copolymer-bound doxorubicin the alterations in the plasma membrane permeability preceded disturbances in cellular metabolism; (ii) HPMA copolymer-bound doxorubicin kills the cells mainly by necrosis; (iii) HPMA copolymer-bound doxorubicin is a more effective anticancer drug than free doxorubicin.

Adult↗

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↗