Biomedical subjects

M Jelínková

Publications and source records attributed to M Jelínková.

At least 19 recordsLinked to original sources

PubMed2004

Antibody-targeted polymer-doxorubicin conjugates with pH-controlled activation.

The paper is dealing with the synthesis and properties of new non-targeted or antibody-targeted polymer drug conjugates, bearing doxorubicin (DOX) attached via a spacer susceptible to pH-controlled hydrolysis (hydrazone conjugates), designed as anticancer drugs facilitating site-specific therapy. These conjugates are stable in a pH 7.4 buffer, modeling conditions during transport in the body, but release DOX and activate it inside target cells as a result of pH changes when going from outside to inside the cells. Conjugates containing an antibody directed against T lymphocytes bind effectively and specifically T cell lymphoma EL 4 cells. Cytotoxicity of the hydrazone conjugates is higher than that of classic conjugates, depending on the detailed structure of the polymer, the spacer between the drug and polymer carrier and method of antibody conjugation. Cytotoxicity of some of the conjugates is comparable even with that of the free drug. In both protective and therapeutic regimes of drug administration, the in vivo anti-tumor activity of the conjugates containing DOX was enhanced with long-term survivors (T-cell lymphoma EL 4, C57BL/6 mice) in comparison with much less effective free DOX or a classic P(N-(2-hydroxypropyl)methacrylamide)HPMA-DOX conjugate (already clinically tested).

Animals↗

PubMed2003

HPMA copolymers with pH-controlled release of doxorubicin: in vitro cytotoxicity and in vivo antitumor activity.

Data on the synthesis, physicochemical characterisation and in vitro and in vivo biological properties of the new, nontargeted or antibody-targeted polymer-doxorubicin conjugates designed as anticancer drugs are presented. In the conjugates, the anticancer drug doxorubicin (DOX) is attached to the polymer carrier via a simple hydrolytically labile spacer containing either a hydrazone bond or cis-aconitic acid residue. In vitro incubation of the conjugates in buffers led to a fast DOX release from the polymer at pH 5 (modelling intracellular environment) while at pH 7.4 (modelling blood) the conjugates are relatively stable. Cytotoxicity of the conjugates to T cell lymphoma EL4 depended on the detailed structure of the spacer and the method used for antibody attachment and was much higher compared with the effect of similar classic conjugates (DOX attached to the polymer via enzymatically degradable spacer). In both protective and therapeutic regimes of drug administration, the in vivo anti-tumor activity of the hydrazone conjugates containing only DOX was significantly enhanced (T cell lymphoma EL4, C57BL/10 mice) in comparison with free DOX or classic PK1, the PHPMA-DOX conjugate clinically tested at present. Increasing the molecular weight of the polymer carrier resulted in a more pronounced in vivo antitumor effect. Antibody-targeted conjugates with DOX bound via hydrazone bond exhibited even more extensive inhibition of the tumor growth with some long-term survivors. No survivors were observed after treatment of mice with free DOX or the nontargeted PHPMA-DOX conjugate.

Animals↗

PubMed2002

Acquired and specific immunological mechanisms co-responsible for efficacy of polymer-bound drugs.

We present data providing new evidence that poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA)-bound drugs, unlike free drugs, have both cytostatic and immunomobilizing activity (CIA). Immediately after injection, due to the high level of the drug, the main activity of the polymeric conjugate is cytotoxic and cytostatic. Later on, long-term circulating PHPMA-bound drug, at concentrations lower than its minimal inhibitory levels, mobilizes the defense mechanisms of the host. Cytotoxic and cytostatic effects of drug-PHPMA were repeatedly confirmed. The following data support the concept of the immunomobilizing activity of the N-(2-hydroxypropyl)methacrylamide (HPMA) conjugates: (a) pre-treatment with free drugs (doxorubicin, cyclosporin A) accelerates the appearance of EL4 mouse T-cell lymphoma while a similar pre-treatment with doxorubicin-PHPMA induces limited but definitive mobilization of the host's defense mechanisms; (b) mice cured of EL4 mouse T-cell lymphoma, BCL1 mouse B-cell leukemia and 38C13 mouse B-cell lymphoma by injection of doxorubicin-PHPMA conjugate targeted with monoclonal antibodies (anti-Thy 1.2 for EL4, anti-B1 for BCL1 and anti-CD71 for 38C13) and re-transplanted with a lethal dose of the same cancer cells survive without any treatment considerably longer than control mice; (c) increased NK activity and anti-cancer antibody was detected only in animals treated with doxorubicin-PHPMA conjugate; and (d) considerably increased NK and LAK activity was seen in a human patient treated for generalized breast carcinoma with doxorubicin-PHPMA-IgG.

Animals↗

PubMed2001

Doxorubicin bound to a HPMA copolymer carrier through hydrazone bond is effective also in a cancer cell line with a limited content of lysosomes.

We have synthesized conjugates containing doxorubicin (DOX) bound to oligopeptide side chains (GlyGly or GlyPheLeuGly) of a water-soluble copolymer carrier based on poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) either through proteolytically (PK1 conjugates) [Synthetic polymeric drugs. U.S. Patent 5,037,883 (1991)] or hydrolytically cleavable bond (HC conjugates). Pharmacological efficacy of PK1 and HC conjugates was compared in vitro on murine: T-cell lymphoma EL4, B-cell leukemia BCL1, B-cell lymphoma 38C13, leukemia P388 and Con A-stimulated A/Ph splenocytes and on human: primary (SW480) and metastatic (SW620) colorectal cancer cell lines parent and transfected with Thy 1.2 gene [2] and on erythromyeloid leukemia cell line K 562. Inhibition of proliferation determined by 3[H]-thymidine incorporation revealed that the cytostatic effect of HC conjugates is up to two orders of magnitude higher compared to PK1 conjugates. In some cancer cell lines (SW 620/T, SW 480) the pharmacological activity of HC conjugates is in vitro comparable with the activity of the free drug. Unlike PK1 conjugates, HC conjugates with a lysosomally degradable spacer (GlyPheLeuGly) are less effective compared to HC conjugates containing lysosomally non-degradable spacer (GlyGly). Moreover, HC conjugates exert pronounced anti-proliferative activity also in erythroblastoid leukemia cell line K 562 with a limited content of lysosomes.

Animals↗

PubMed2001

New HPMA copolymers containing doxorubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro and in vivo biological properties.

In this paper we describe the synthesis, physico-chemical characteristics and results of tests of biological activity of polymer drugs based on conjugates of anti-cancer drug doxorubicin (Dox) with water-soluble polymer drug carriers, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In the conjugates the drug is attached to the polymer backbone via a spacer stable under physiological conditions (pH 7.4) and hydrolytically degradable in mild acidic environment (e.g., endosomes, pH approximately 5). This enables designing polymer drugs with long blood circulation and release and specific activation of the active compound in endosomes of target cells. Two types of Dox conjugates differing in the length and structure of the oligopeptide spacer were synthesised (GG and GFLG). In both types, the linkage susceptible to hydrolytic cleavage was formed by the reaction of the carbonyl group of Dox with the hydrazide group terminating the oligopeptide side chains of the polymer. In vitro incubation of conjugates in buffers resulted in much faster release of Dox from the polymer at pH 5 than at pH 7.4 (more than 10 times) the rate being higher for the conjugate containing GG spacer. The presence of cathepsin B in incubation media increased the rate of Dox release from the conjugate with GFLG spacer, Dox release from conjugate with GG spacer remained unchanged. Cytotoxicity of conjugates for T-splenocytes and mouse EL-4 T cell lymphoma cells was much higher compared with the effect of similar 'classic' conjugates bearing Dox attached via amide bond. In vivo anti-tumor activity of conjugates containing hydrolytically sensitive linkage was also significantly improved in mouse EL4 T cell lymphoma.

Animals↗

PubMed2000

Polymeric drugs based on conjugates of synthetic and natural macromolecules. II. Anti-cancer activity of antibody or (Fab')(2)-targeted conjugates and combined therapy with immunomodulators.

We provide data on in vivo targeting of the Thy 1.2 (CDw90) cell surface receptor expressed on neoplastic T cells, mouse EL4 T cell lymphoma. The targeting antibody and the anticancer drug, doxorubicin (DOX) were conjugated to a water-soluble copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA) acting as a carrier responsible for controlled intracellular release of the conjugated drug. The in vivo therapeutic efficacy of HPMA copolymer-bound DOX targeted with anti-EL4 antibody, polyclonal anti-thymocyte globulin (ATG), monoclonal anti-Thy 1.2 antibody or its F(ab')(2) fragment was compared with the efficacy of DOX conjugated to HPMA copolymer containing nonspecific IgG or bovine serum albumin (BSA). Anti-EL4 antibody-targeted conjugate caused a significant retardation of tumor growth and an extension of the life span of treated mice. The effect was comparable with that of HPMA copolymer-bound DOX targeted with ATG, anti-Thy 1.2 antibody or its F(ab')(2) fragment. However, considerable antitumor effect was seen also in conjugates targeted instead of specific antibodies with syngeneic nonspecific IgG or BSA. Patients with advanced cancer are often immunocompromised due to dysfunction of their immune system induced by cancer and cytotoxic drugs. A significant decrease of unwanted side-effects of targeted drugs against a number of vital organs was already documented. In this study we have compared immunotoxic effects of free DOX with those of its antibody-targeted form on NK cells and cytolytic T lymphocytes (CTLs) isolated from C57BL/10 mice bearing EL4 T cell lymphoma. In the same model we have tested the combination therapy with immunomodulators (beta-glucan or AM-2) injected together with targeted daunomycin. We have observed a significant protective effect of targeted DOX against NK cells and CTLs. Moreover, the data revealed that combination therapy considerably enhances antitumor efficacy of the targeted anticancer drug.

Adjuvants, Immunologic↗

PubMed2000

Polymeric drugs based on conjugates of synthetic and natural macromolecules. I. Synthesis and physico-chemical characterisation.

This paper describes the synthesis, physico-chemical characteristics and results of selected biological tests of conjugates of antibodies or proteins with poly(HPMA) or with poly(HPMA) carriers of anti-cancer drug doxorubicin, designed for targeted cancer therapy. Two types of conjugates differing in the method of conjugation of polymer with protein were synthesized. In the first, protein is attached to the polymer via an oligopeptide sequence in the side chain of the polymer backbone and, in the second, the polymer is attached to protein via its end-chain functional group. Conjugation of an antibody with poly(HPMA) does not influence the binding activity of the antibody for cell surface antigen. The physico-chemical characteristics and biological activity of both systems depend on the detailed structure of the polymer, the type of antibody or protein moiety and the structure of the whole system.

Amino Acid Sequence↗

PubMed1999

Brain lesion-induced alteration of selected phenotypic properties of spleen macrophages and their partial restoration in the course of foreign body reaction against intraperitoneally implanted polymers.

A lesion in the dorsoposterior part of the rat brain septum is known to exert an inhibitory effect on the delayed skin hypersensitivity and incorporation of radiolabeled thymidine into the lymphoid organs. To determine whether distinct properties of macrophages will also be modulated by this type of injury, we have focused upon the monitoring of expression of sugar receptors (lectins). In this study we show a reduction in the number of macrophages expressing carbohydrate-binding sites for asialoglycoproteins (beta-D-galactoside), alpha-D-mannoside and alpha-D-mannoside-6-phosphate in spleen macrophages after the lesion of the dorsoposterior septum of the brain in the rat. The number of ED-1+ macrophages was not influenced. The intraperitoneal injection of beads prepared from the copolymer of 2-hydroxyethyl methacrylate with dimethyl aminoethyl methacrylate (30 wt %) elevated significantly the number of ED-1+ spleen macrophages and number of macrophages with binding site(s) recognizing asialoglycoproteins and alpha-D-mannoside-6-phosphate, respectively. These results indicate that a foreign-body reaction appears to be able to mediate a phenotypic restoration of lectin expression by spleen macrophages altered by the brain lesion. It can be suggested that, for example, a probable production of cytokines by the inflammatory cells colonizing the implanted beads plays a role in this process.

Journal Article↗

PubMed1998

Targeting of human and mouse T-lymphocytes by monoclonal antibody-HPMA copolymer-doxorubicin conjugates directed against different T-cell surface antigens.

Binding of HPMA copolymer-conjugated doxorubicin targeted with monoclonal antibodies directed against various T-cell surface receptors, i.e. Thy1.2 (CDw90), I-A (MHC class II. glycoprotein), L3T4 (CD4), IL-2R (CD25) and CD3, is considerably increased in Con A stimulated T-lymphocytes. FACS analysis showed that the binding is most intensive with anti-Thy1.2 and anti-L3T4 targeted derivatives and it is proportional to the antiproliferative effect of the antibody-targeted drug. No binding and no antiproliferative capacity was observed after in vitro incubation of mouse T-cells with a nonspecific mouse IgG-HPMA-DOX conjugate. [3H]-TdR incorporation was inhibited considerably more in Con A stimulated T-cell culture and in EL4 mouse T-cell lymphoma as compared with the culture of nonactivated T-lymphocytes. This proves that intensively proliferating cells are more susceptible to the inhibitory action of an antibody-targeted drug. The cytotoxic efficacy of HPMA copolymer with GlyPheLeuGly or GlyLeuPheGly side-chains to which the drug is conjugated was superior to HPMA copolymer with GlyPheGly or GlyLeuGly side-chains. However, there is no direct correlation between the rate of in vitro drug release and the in vitro cytotoxicity of the respective conjugates. This suggests that the rate of drug release from the conjugate is only one factor responsible for the pharmacological efficacy of the preparation. Furthermore, we detected substantial and prolonged inhibition of proliferation of Con A activated T-cells only if doxorubicin was injected in vivo in the form of an anti-Thy1.2-targeted conjugate.

Amino Acid Sequence↗

PubMed1998

Analysis of the leuB gene from Corynebacterium glutamicum.

The leuB gene of Corynebacterium glutamicum was found to be present on a 2.2-kb BamHI-SacI chromosomal fragment which complemented the leuB mutation of Escherichia coli. The activity of 3-isopropylmalate dehydrogenase (EC 1.1.1.85), encoded by the leuB gene, was significantly increased in C. glutamicum cells harbouring a plasmid containing the 2.2-kb fragment. The nucleotide sequence of the C. glutamicum leuB coding region (an open reading frame, ORF, of 1020 bp encoding a polypeptide of 340 amino acids with M(r) of 36 144) was determined. The deduced amino acid sequence of the product of this ORF is highly homologous to those of 3-isopropylmalate dehydrogenases from three species of mycobacteria. The transcriptional start site of the leuB gene was localized 35 bp upstream of its translational start; a functional terminator was detected in the 3' flanking region. Northern hybridization analysis showed that the C. glutamicum leuB gene is transcribed as a single monocistronic RNA (approximately 1.2 kb in size). Activity of the leuB promoter was significantly reduced when leucine was present in the growth medium. This suggests the negative regulation of the leuB expression on the transcriptional level in C. glutamicum cells.

3-Isopropylmalate Dehydrogenase↗

PubMed1997

HPMA-based biodegradable hydrogels containing different forms of doxorubicin. Antitumor effects and biocompatibility.

Novel hydrogels based on N-(2-hydroxypropyl)methacrylamide (HPMA) and N,O-dimethacryloylhydroxylamine containing either doxorubicin (DOX) or water-soluble HPMA carrier-bound doxorubicin (P-GlyPheLeuGly-DOX; HPMA-DOX) were synthesized. The cross-linkages are susceptible to hydrolytic cleavage at physiological pH 7.4. Hydrogels in the form of rods or discs loaded with DOX or P-GlyLeuGly-DOX were implanted subcutaneously on the back of C57BL/10 mice on day 1 or on day 9 after inoculation with EL4 mouse T-cell lymphoma. The implanted hydrogels varied in the total load of DOX and rate of hydrolysis, which is dependent on the crosslinking density of the gels. The effect of HPMA based hydrogels containing DOX or HPMA carrier-bound DOX on tumor growth, animal life span, leukocyte populations in peripheral blood and bone marrow function evaluated by reticulocyte count was investigated. It was shown that: a) DOX and HPMA carrier-bound DOX administered in the form of HPMA-based hydrogels has better antitumor activity against experimental EL4 mouse T-cell lymphoma than soluble forms of the drug, b) hydrogels with shorter degradation rate (16-17 h) show better antitumor activity than hydrogels with longer duration time (48-52 h), c) the therapeutic effect of hydrogels with rate 16-17 h is directly related to the doxorubicin content; the higher the doxorubicin content, the better antitumor activity, d) the gel containing free doxorubicin showed significant antitumor activity even when implanted on day 9, i.e., in the time when tumor growth is already established, e) the hydrogel matrix without drug does not induce release of IL-1 or IL-6 into peripheral blood, does not induce formation of antibodies, and it is not mitogenic. Use of doxorubicin in the form of HPMA-based hydrogels allows a several-fold increase in the administered dose compared to soluble forms without detectable serious toxic side-effects.

Animals↗

PubMed1996

Influence of intraperitoneal injection of three types of hydrogel beads on expression of carbohydrate-binding sites in spleen macrophages.

The influence of the chemical structure of polymer implants on selected characteristics of macrophages was studied to improve our understanding of the mechanisms of non-self recognition of synthetic materials. Three types of polymers differing in net charge were prepared to compare in vivo responses. Beads from preparation of poly(2-hydroxyethyl methacrylate), a copolymer of 2-hydroxyethyl methacrylate with sodium methacrylate, and a copolymer of 2-hydroxyethyl methacrylate and dimethylaminoethyl methacrylate were injected intraperitoneally into rats and harvested 48 h later. The effects of these polymers on the presence of inflammatory cells in the peritoneal exudate, on the adhesion of macrophages to individual batches of the different types of beads and on distinct molecular aspects of macrophages in the red pulp of spleen were evaluated. Beads from both types of copolymer caused an elevation in the number of macrophages in the exudate, in contrast to the situation in rats treated with poly(2-hydroxyethyl methacrylate) beads and physiological saline solution as control. The molecular design of the implant had no significant influence on the extent of macrophage adhesion to beads or on the expression of selected carbohydrate-binding sites. Since important cellular functions such as cell adhesion and glycoprotein routing depend on the sugar part of glycoconjugates, labelled neoglycoproteins were employed to analyse this aspect of macrophages in the tested animals. The beads of the copolymer of 2-hydroxyethyl methacrylate with dimethylaminoethyl methacrylate clearly led to an elevation of the expression of specific binding sites for beta-galactoside-terminating structures which are presented by asialofetuin, for mannose, fucose, sialic acid and N-acetylgalactosamine, which had been used as the ligand parts of biotinylated neoglycoproteins, in spleen macrophages whereas the levels of sites which recognize mannose-6-phosphate were unaffected. Expression of sites with specificity to N-acetylglucosamine was lessened. The effect of beads from the copolymer of 2-hydroxyethyl methacrylate with sodium methacrylate on the measured glycobiological features in the splenic macrophages was only negligible. These results suggest the possibility of systemic effects of implanted polymers on the distinct recognitive functions of macrophages.

Animals↗

PubMed1996

Cultivation of keratinocytes on poly HEMA and their migration after inversion.

This study demonstrates how the pretreatment of poly HEMA disks with bovine serum influences the adhesion of human keratinocytes in vitro. The ability of these cells to migrate from poly HEMA disks to a polystyrene petri dish surface colonized with irradiated 3T3 cells is also evaluated. The stimulatory effect of 24-h preincubation of the poly HEMA disks with bovine serum on the keratinocyte adhesion and growth was dependent on the concentration of the serum used. The keratinocytes migrated from the poly HEMA disks to polystyrene colonized with irradiated 3T3 cells. The numerous cells which migrated from the poly HEMA were positive for cytokeratin 10 (suprabasal layers) after 3 weeks and for cytokeratin 1 (cornified layer) after 1 month. These results seem to be encouraging for further experiments with respect to clinical application.

Animals↗

PubMed1995

Mapping of endogenous lectins in macrophages colonizing an implanted polymer surface--effect of polymer structure.

The design of synthetic polymers may affect structural features of inflammatory cells, for example the expression of endogenous sugar receptor molecules like lectins in macrophages and foreign body giant multinucleate cells. This characteristic was studied histochemically by use of biotinylated (neo)glycoproteins. The results demonstrate the influence of polymer structure on the phenotypic expression of molecules recognizing carbohydrates in these cells. Very strong activity was observed for carrier-immobilized beta-galactose in cells colonizing hydrophobic polystyrene implants. Sialic acid moieties were not recognized by cells located on the surface of all types of polymeric implant. This study encourages investigation of the influence of polymer design on the differentiation of macrophages.

Animals↗

PubMed1995

Identification of the minimal replicon of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica.

The actinomycete Amycolatopsis methanolica contains a 13.3 kb plasmid (pMEA300), capable of enhancing the spontaneous mutation frequency of its host. Depending on the growth medium pMEA300 is not only maintained as an integrated element but can additionally be present as a multicopy, autonomously replicating plasmid. The minimal replicon of pMEA300 was identified. Two unlinked DNA fragments of 2.6 kb and 0.8 kb were required for pMEA300 maintenance. Sequence analysis of the 2.6 kb fragment revealed at least two open reading frames, orfA and orfB, encoding putative proteins of 170 amino acids (18,373 Da) and 416 amino acids (45,260 Da), respectively. No clear similarities were found between the deduced amino acid sequences of the putative orfA and orfB products of pMEA300 and replication proteins identified for various Streptomyces plasmids. The pMEA300 proteins of A. methanolica thus may represent unfamiliar types. The 0.8 kb fragment contained a single complete open reading frame (korA), encoding a protein of 118 amino acids (12,917 Da). The putative KorA protein of pMEA300 shows sequence similarity with various other Streptomyces plasmid-encoded Kor proteins which may belong to the GntR family of transcriptional repressor proteins. The data provide preliminary evidence for the possible involvement of a kilkor system in autonomous replication of pMEA300.

Actinobacteria↗

PubMed1994

Immunolocalization of cyclophilin in normal and cyclosporin A-treated human lymphocytes.

A polyclonal rabbit antibody against a protein fraction (10-30 kDa) of human thymuses with a high CsA-binding activity of dominant protein cyclophilin (CPH) was prepared and characterized. In immunoblotting with the cell lysate from JURKAT T cell line, this antibody specifically reacted with 18-kDa protein corresponding to CPH. In indirect immunofluorescence the antibody visualized granular structures in JURKAT cells and human peripheral blood lymphocytes. In JURKAT cells cultivated with 1-2 micrograms of CsA per ml for 7 days a much weaker reaction of the antibody was found, compared with non-treated cells. In some CsA-treated cells the antibody visualized various 'star-like' or filamentous structures. A similar staining pattern has also been obtained in lymphocytes of the patients receiving CsA therapy. Complementary staining with rhodamine-tagged phalloidin revealed changes in F-actin distribution of CsA-treated JURKAT cells. In conclusion, the treatment with CsA induces dramatic changes of CPH cellular distribution, which may take part in the final therapeutic effect of the drug.

Actins↗

PubMed1994

[The effect of antibiotics on the development of enterotoxemia-- experimental study].

When testing antibiotics in rabbits Dalacin was shown to be the most effective in inducing enterotoxaemia in rabbits. Autopsy disclosed signs of enterotoxaemia even in some animals without previous symptoms. Histopathological lesions were found even in intestines macroscopically normal. Experiments proved that after application of tested antibiotics spread through intestinal mucosa and produced a loss of microbial balance. Rabbit is to be considered suitable for modelling antibiotic-induced enterotoxaemia.

Animals↗

PubMed1991

Influence of mobile phase composition on evaluation of lipophilicity by partition chromatography.

The problems of the concentration dependence of retention indices and the applicability of extrapolated values in the evaluation of lipophilicity were studied. The reversed-phase high-performance liquid chromatography of arylalkanoic acids were carried out with experimental data for substituted estra-1,3,5 (10)-trienes, benzodiazepines, dermorphine derivatives and dansylamides selected from the literature for this purpose. Fair linear relationships between slopes of concentration dependences and extrapolated and non-extrapolated values of RM and log k' were found. Equivalence of these indices in the evaluation of lipophilicity can be inferred. Statistically significant dependences of log P (sigma pi) values on concentration slopes make it possible to use them as new parameters of lipophilicity. The goodness of fit of these relationships increases when the values of ET(30), as a measure of the solvatochromic solvent polarity of mobile phases, are used instead of the change in modifier concentration.

Acetates↗