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Biomedical subjects

J Kopecek

Publications and source records attributed to J Kopecek.

At least 91 records · Page 5Linked to original sources
PubMed1987

Effect of molecular weight (Mw) of N-(2-hydroxypropyl)methacrylamide copolymers on body distribution and rate of excretion after subcutaneous, intraperitoneal, and intravenous administration to rats.

A copolymer of N-(2-hydroxypropyl) methacrylamide (HPMA) and N-methacryloyltyrosinamide was prepared and fractionated using Sepharose 4B/6B (1:1) chromatography to produce eight HPMA copolymer fractions of narrow polydispersity and mean molecular weight (Mw) ranging from 12 to 778 kD. These fractions were radioiodinated and injected intravenously, subcutaneously, and intraperitoneally into rats. Their bloodstream-concentration profiles were monitored and rates of excretion assessed. Following intravenous administration the circulating blood volume available to the copolymers was not molecular-weight-dependent. A molecular-weight threshold limiting glomerular filtration was identified at approximately 45 kD, and preparations greater than this threshold were lost from the bloodstream only slowly by extravasation. Molecular weight did not influence the movement of copolymers from the peritoneal compartment to the bloodstream after intraperitoneal injection. The transfer rates observed could be accounted for by bulk phase lymphatic drainage alone, no transcapillary routes being implicated. Following subcutaneous administration the largest HPMA copolymer fraction (Mw = 778 kD, diameter approximately 30 nm) showed increased retention at the site of the injection, approximately 20% of the dose remaining there after 21 days. This could result from physical restriction of movement or from internalization into local phagocytic cells. The smaller copolymer fractions moved readily into the bloodstream whence they were either lost in the urine or they gradually penetrated into other tissues and organs. Long-term (21 days) body distribution of copolymers following both intraperitoneal and subcutaneous administration showed size-dependent accumulation in organs of the reticuloendothelial system.

Acrylamides↗
PubMed1987

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro.

During recent years N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been developed as targetable drug carriers. These soluble synthetic polymers are internalized by cells by pinocytosis and they can be tailor-made to include peptidyl side-chains degradable intracellularly by specific lysosomal enzymes. Thus they provide the opportunity fo achieve controlled intracellular delivery of anticancer agents. The anthracycline antibiotic daunomycin, and protein synthesis inhibitor puromycin, were bound to HPMA copolymers via several different peptide side-chains, including Gly-Gly, Gly-Phe-Leu-Gly and Gly-Phe-Phe-Leu. Incubation of polymer-drug conjugates with isolated lysosomal enzymes (either a mixture of rat liver lysosomal enzymes or purified thiol-dependent lysosomal proteinases, cathepsins L and B) showed that significant release of drug occurred over 20 h, more than 20% of daunomycin and more than 80% of puromycin being liberated. To test their pharmacological activity conjugates were incubated with either the mouse leukaemia L1210, or the human lymphoblastoid leukaemia CCRF in vitro. The conjugates tested were all less effective than free daunomycin, but they showed differential toxicity against L1210 depending on the aminoacid sequence of their drug-polymer linkage. Inclusion of fucosylamine-terminating side-chains into the HPMA copolymer structure increased the affinity of conjugates for the L1210 cell membrane and resulted in increased toxicity. In contrast HPMA-daunomycin conjugates with or without fucosylamine affected CCRF cells equally, but this cell line was more sensitive than the mouse leukaemia to both free and polymer-bound daunomycin. Incubation of L1210 cells in polymer-bound daunomycin for 72 h, followed by plating cells out in low density in drug-free medium, showed that a concentration of polymer-bound drug (184 micrograms ml-1) could be selected to achieve a cytotoxic effect.

Acrylates↗
PubMed1986

Bioaffinity therapy with antibodies and drugs bound to soluble synthetic polymers.

Soluble synthetic copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) containing different oligopeptide side-sequences were tested as transport molecules for drugs and anti-Thy 1.2 antibodies in affinity therapy. As target cells, T lymphocytes were studied. (1) HPMA copolymers containing targeting anti-Thy 1.2 antibodies are 70 times more cytotoxic against T lymphocytes than HPMA copolymers with non-specific immunoglobulin. (2) Daunomycin conjugated to a biodegradable side-sequence (Gly-Phe-Leu-Gly) is effective in a concentration 100 times lower than daunomycin conjugated to a non-cleavable sequence (Gly-Gly). (3) HPMA copolymers containing drug and targeting antibodies are effective both in vitro and in vivo.

Animals↗
PubMed1986

Separation of human lymphoid cells by affinity chromatography and cell surface labelling by hydroxyethyl methacrylate particles using monoclonal antibodies.

The hydroxyalkyl methacrylate gel Separon, Sepharose 6MB and polystyrene dishes with bound antibodies were used for affinity chromatography of cells isolated from human tonsils and peripheral blood. Enrichment of T cells by filtration through anti-human immunoglobulin Separon or Sepharose 6MB columns was comparable to that achieved by "panning" on dishes or by Nylon wool column fractionation. High enrichment of B cells was obtained when cells pretreated with monoclonal anti-T cell antibodies were fractionated on columns or dishes where anti-mouse antibody was bound. Hydroxyethyl methacrylate copolymer microspheres were conjugated with monoclonal antibodies directed against surface markers of human lymphocytes or with anti-mouse immunoglobulin antibodies and used for surface labelling of cells. The results obtained by immunofluorescence were in good correlation with labelling by hydroxyethyl methacrylate immunobeads.

Animals↗
PubMed1986

Fate of N-(2-hydroxypropyl)methacrylamide copolymers with pendent galactosamine residues after intravenous administration to rats.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers bearing galactosamine residues accumulate in the liver after intravenous administration to rats (Duncan, R., Kopecek, J., Rejmanová, P. and Lloyd, J.B. (1983) Biochim. Biophys. Acta 755, 518-521). In this study HPMA copolymers bearing pendent galactosamine residues (1.0-11.6 mol%) were injected intravenously into rats and their rates of blood clearance and liver accumulation were measured. A level of substitution of 4 mol% was found to be sufficient to cause substantial deposition in the liver 30 min after administration. The most highly substituted polymer (11.6 mol%) was directed rapidly to the liver, 80-90% being recovered there less than 10 min after administration. Separation of liver into hepatocytes and non-parenchymal cells indicated that polymer was largely associated with the hepatocytes, and density-gradient subcellular fractionation of liver at various times after administration confirmed that polymer was internalized by liver cells and transported, with time, into the secondary lysosomes. Experiments using isolated rat hepatocytes indicated that HPMA copolymers with high galactosamine content have higher affinity for the hepatocyte plasma membrane. HPMA copolymers containing galactosamine and in addition glycylglycyltyrosinamide side-chains were used to demonstrate release of a drug analogue across the lysosomal membrane. These polymers were radioiodinated and, following intravenous administration to rats, the liver lysosomes were isolated and incubated at 37 degrees C in 0.25 M sucrose. Radioactivity was released from the lysosomes faster than the lysosomal enzyme arylsulphatase, an observation that indicates intralysosomal hydrolysis of the copolymer side-chain with subsequent passage of low molecular weight degradation product across the lysosomal membrane.

Acrylates↗
PubMed1986

Co-expression of different types of Fc receptors on murine peritoneal macrophages.

Simultaneous expression of particular immunoglobulin Fc receptors (FcR) was studied on the plasma membranes of murine peritoneal macrophages. This was facilitated by the use of sheep red blood cells (SRBC) and/or synthetic microspheres coated with monoclonal antibodies of different isotypes. It was concluded that a majority of macrophages bear more than one type of FcR; macrophages bearing at least three types of FcR were present in the peritoneal cavity; macrophages bearing Fc mu R did not bind IgE, IgA or IgG; all macrophages bearing Fc alpha R also expressed Fc gamma 2bR, Fc gamma 3R and Fc epsilon R; all macrophages bearing Fc epsilon R also expressed Fc gamma 2bR and Fc alpha R. Except for Fc alpha R, essentially equivalent numbers of FcR-bearing macrophages were detected when antibody-coated SRBC or polymeric microspheres were used. Simultaneous applications of these reagents permitted the most detailed and direct investigations yet performed of multiple FcR expression on individual cells.

Animals↗
PubMed1986

Pinocytic capture and exocytosis of rat immunoglobulin IgG-N-(2-hydroxypropyl)methacrylamide copolymer conjugates by rat visceral yolk sacs cultured in vitro.

Rat immunoglobulin (IgG) was covalently bound to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via glycylglycyl spacer. The resultant conjugate, free IgG and HPMA copolymer (containing a low percentage of tyrosinamide to facilitate radiolabelling) were radioiodinated, and their rates of pinocytic uptake, intracellular degradation and exocytic release by rat visceral yolk sacs cultured in vitro were determined. Free IgG was pinocytosed rapidly by the yolk sac and some IgG was subject to intracellular proteolysis. In comparison the IgG-HPMA copolymer conjugate was captured more slowly, but faster than unmodified HPMA. IgG was also exocytosed rapidly by the yolk sac following pinocytic capture and similarly IgG-HPMA copolymer had a much higher rate of release than unmodified HPMA. Measurement of tissue accumulation of 125I-labelled IgG-HPMA copolymer in the presence of increasing concentrations of non-radiolabelled IgG showed competition for membrane binding sites between the free, and polymer-bound immunoglobulin. These experiments indicate that immunoglobulins can be covalently bound to a soluble polymer developed as a drug-carrier in such a way that they can still interact with specific membrane receptors and they are subsequently subjected to specific cellular transport mechanisms.

Animals↗
PubMed1986

Effect of a synthetic poly N-(2-hydroxypropyl)methacrylamide (Duxon) on haemopoiesis and graft-versus-host reaction.

A synthetic polymer, Duxon, was developed and tested as a substitute of blood plasma for transfusion purposes. Tests of this preparation included a test for its influence on the haemopoietic stem cells and the graft-versus-host reaction (GVHR). A single dose or repeated doses of Duxon did not reduce the number of pluripotent haemopoietic colonies (CFU-S) in mice and short-term incubation of cells from haemopoietic organs of mice with Duxon resulted in a slight, yet significant, increase in the number of CFU-S. Injection of Duxon non-significantly diminished the manifestations of the regional GVHR. Incubation of spleen cells with Duxon significantly reduced their GVH reactivity in the regional test. The systemic GVHR was moderately inhibited by repeated doses of Duxon in sublethally irradiated mice. A significant delay in deaths of animals from GVHR was seen in lethally irradiated mice receiving a single dose of Duxon on day 4 after GVHR induction. Survival of lethally and sublethally irradiated mice was prolonged following injection of cells incubated with Duxon. Possible uses of Duxon, a preparation with mild immunosuppressive properties, not impairing haemopoiesis, are discussed.

Animals↗
PubMed1985

Stability in rat plasma and serum of lysosomally degradable oligopeptide sequences in N-(2-hydroxypropyl) methacrylamide copolymers.

Soluble copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) were prepared containing either oligopeptide side chains terminating in rho-nitroaniline, or oligopeptide sequences forming crosslinks between polymer chains. Such copolymers have potential as targetable drug carriers and already it has been shown that oligopeptide side chains and oligopeptide crosslinks are degraded intracellularly by lysosomal enzymes. The susceptibility of these oligopeptide sequences to degradation on incubation with rat plasma or rat serum was evaluated by monitoring either the liberation of rho-nitroaniline or, with the crosslinked polymers, the change in molecular weight distribution. Release of rho-nitroaniline from some of the polymers was not detectable, and from others proceeded very slowly, the maximum rate being from the side chain Gly-Gly-Phe-Leu-Gly-Phe-NAp where 5.1% of the bound rho-nitroaniline was released by rat serum over a 5 h incubation period. No cleavage of crosslinked HPMA copolymers by plasma or serum was detectable even after a 24 h incubation period.

Acrylamides↗
PubMed1985

Properties of macrophages from low- and high-responder strains of mice. I. Effect of antigenic stimulation.

In the high-responder A/J mice a lower percentage of peritoneal macrophages express Fc receptors and accordingly phagocytize less intensely via these receptors than the macrophages of the low-IgG-responder C57BL/10ScSn strain. Similarly, the phagocytosis mediated by the receptors for foreign substances tested by CdCO3 microcrystals is lower in the A/J strain. Pinocytic activity was the same in both mouse strains. Intraperitoneal immunization with sheep red blood cells led to a prompt increase in Fc receptor expression and in both phagocytic and pinocytic activities in A/J mice, while in C57BL/10ScSn mice these activities remained unchanged. A similar effect was found when spontaneous and induced rosette formation with sheep red blood cells was studied. The opposite was true when the phagocytic activity of peripheral leucocytes to non-antigenic synthetic microspheres was studied; after immunization with sheep red blood cells a strong increase was observed not in the A/J but C57BL/10ScSn strain. The importance of these results for the regulatory role of macrophages in immune responses in the low- and high-IgG-responder strains of mice is discussed.

Animals↗
PubMed1984

Tyrosinamide residues enhance pinocytic capture of N-(2-hydroxypropyl)methacrylamide copolymers.

N-(2-Hydroxypropyl)methacrylamide ( HPMA ) copolymers have been proposed as a potential lysosomotropic drug delivery system. HPMA copolymers bearing tyrosinamide residues, bound either directly to the polymer backbone or via a glycylglycine spacer, were radiolabelled with [125I]iodide and the effect of tyrosinamide content on their rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro was measured. Incorporation of tyrosinamide enhanced uptake of the copolymer, most markedly at substitutions above 10 mol%. 2,4-Dinitrophenol, an inhibitor of pinocytosis, was used to confirm that tissue association of 125I-radiolabelled copolymer was due to pinocytic uptake. The side-chain -Gly-Gly-Tyr-NH2 was degraded following the internalization of copolymers containing this spacer and degradation was partially sensitive to the lysosomal thiol-proteinase inhibitor leupeptin. It is postulated that the effect of tyrosinamide residues is to increase the hydrophobicity of poly( HPMA ) and thus to increase its capacity for nonspecific adsorptive pinocytosis.

2,4-Dinitrophenol↗
PubMed1984

Effect of the chemical structure of N-(2-hydroxypropyl)methacrylamide copolymers on their ability to induce antibody formation in inbred strains of mice.

The homopolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) and copolymers of HPMA differing in oligopeptide side chains (-Gly-Gly-OH; -Acap-Phe-OH; -Acap-Leu-HMDA and -Gly-Phe-Tyr-OH) or in their content (1%, 3.5% and 8.4% mole of -Gly-Gly-OH side chains) were investigated with respect to their ability to induce antibody formation and mitogenic reaction in inbred strains of mice. The dependence on the antigen dose, on composition of the side chain and on the genetic background of the immunized organism was defined. It was demonstrated that the specificity of the antibody formed is predominantly directed against oligopeptide side chains, though some part of the antibody is also produced against hydroxypropyl chains. Neither the homopolymer nor the copolymers behave in the tissue culture as mitogens.

Acrylamides↗
PubMed1984

Controlled biodegradability of polymers--a key to drug delivery systems.

By combining oligopeptide sequences with synthetic polymeric chains, carriers of drug models can be prepared. The drug model (p-nitroaniline) is cleaved from the carrier in the lysosomal compartment of the cell. By changing the length and structure of the oligopeptide sequence, it is possible to regulate the rate of cleavage of drug model by individual thiol proteinases (cathepsins B, H and L) which are the most important as regards cleavage of the substrates studied. By connecting synthetic polymeric chains via oligopeptide bridges, it is possible to regulate the molecular weight as well as the biodegradability of the carrier molecule. Molecular weight also influences other biological properties, e.g., elimination from the organism, rate of pinocytic uptake and biological activity. Homopolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) is nonimmunogenic in rats. Attachment of oligopeptide side chains gives rise to a macromolecule possessing immunogenic activity. The degree of antibody response depends on the detailed structure of the copolymer.

Animals↗