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V Subr

Publications and source records attributed to V Subr.

At least 19 recordsLinked to original sources
PubMed2005

Thermoresponsive polymers as promising new materials for local radiotherapy.

We describe a novel thermoresponsive polymeric drug delivery system based on poly(N-isopropylacrylamide) with isotopically labellable end groups [l-tyrosinamide or diethylenetriaminepentaacetic acid (DTPA)] designed for local radiotherapy. The polymers are readily soluble in isotonic aqueous sodium chloride at room temperature and the phase separation is complete at body temperature as proved by DSC measurements. Sufficent binding capacity for radionuclides and chemical stability are demonstrated on 125I and 90Y-labelled polymers.

Acrylic Resins↗
PubMed2005

Induction of systemic antitumour resistance with targeted polymers.

Conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) represent a new generation of antibody-targeted polymeric anticancer drugs with both cytotoxic and immunoprotecting/immunomobilizing activity. 20-90% of mice that are cured of EL4 mouse T-cell lymphoma, BCL1 mouse B-cell leukaemia and 38C13 mouse B-cell lymphoma by injection of doxorubicin-HPMA conjugate develop a long-lasting memory and systemic antitumour resistance. It is suggested that the main activity of the polymeric drug, directly after application is - due to the high level of the drug - of cytotoxic and cytostatic nature. Thereafter, long-term conjugates persist at low concentration in the circulation, which are capable of mobilizing the defence mechanisms of the host. Until now, seven patients with generalized carcinoma were treated with doxorubicin-HPMA-human-Ig conjugate. Disease stabilization, lasting from 6 to more than 18 months, was recorded.

Animals↗
PubMed2004

HPMA copolymers containing doxorubicin bound by a proteolytically or hydrolytically cleavable bond: comparison of biological properties in vitro.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin bound either by a proteolytically degradable bond (non-targeted PK1 or targeted with alpha-CD71 mAb) or by a hydrolytically degradable bond were synthesised and tested in vivo for various biological properties. Mouse 38C13 B-cell lympoma was used as a well established and defined cell line for this study. 38C13 cells are sensitive to free doxorubicin and IC50 was very low, about 0.014 microM. PK1 showed a strongly decreased cytostatic effect, IC50 being 12.6 microM. alpha-CD71 targeted conjugate, which can be considered as an antibody-targeted form of PK1, had IC50 0.358 microM. HPMA copolymer with doxorubicin bound via a hydrolytically sensitive bond (HYD conjugate) showed a high cytostatic effect with IC50 about 0.052 microM. We demonstrated that HYD conjugate inhibited DNA synthesis and induced p21(Waf1/Cip1) protein expression (p21(Waf1/Cip1) is cyclin-dependent kinase inhibitor which blocks cell cycle progression) as quickly as free doxorubicin, whereas PK1 acted much more slowly. Similarly, apoptosis induction measured by Annexin V binding and Caspase 3 activity was detected later after incubation of cells with PK1 or alpha-CD71 targeted conjugate. Apoptosis was manifested by elevation of bax and bad mRNA levels, which was much more rapid and intense in the case of free doxorubicin and HYD conjugate. Expression of antiapoptotic genes as well as cyclin-dependent kinases was surprisingly not affected.

Acrylamides↗
PubMed2002

Differences in the intracellular fate of free and polymer-bound doxorubicin.

Internalization and subcellular fate of free doxorubicin or its polymeric conjugates based on poly N-(2-hydroxypropyl)methacrylamide (pHPMA), either non-targeted or targeted with anti-Thy1.2 or anti-CD71 monoclonal antibody was tested on EL-4 mouse T-cell lymphoma, SW620 human colorectal carcinoma and OVCAR-3 human ovarian adenocarcinoma. Doxorubicin fluorescence allowed us to follow the internalization and intracellular distribution of tested conjugates by laser scanning confocal microscopy and/or by fluorescent microscopy. Whereas free doxorubicin was always detectable only in the nuclei of treated cells, detectable fluorescence of doxorubicin bound to a polymeric carrier, targeted or non-targeted, was detectable up to 3 days of incubation only in the cytoplasmatic structures. While free doxorubicin causes apoptosis in the populations of tested cancer cell lines, significant number of apoptotic cells was never found in cell cultures exposed to targeted or non-targeted polymeric conjugates. In contrast to free doxorubicin, which is a strong inducer of p53 expression, increased p53 expression was never observed after the treatment with the polymeric drug. High-performance liquid chromatographic analysis shows that the percentage of cleaved doxorubicin is very low even after 48 h of incubation of tested cells with the polymeric conjugate, and cannot be the only reason for the toxicity of the conjugate. We suggest that: (a) after the treatment with pHPMA-bound drug, the cells die by necrosis and (b) the toxicity of pHPMA-based conjugates is a combination of the toxic effect of released doxorubicin and the toxic effect of doxorubicin in polymer-bound form directed against cell membranes.

Animals↗
PubMed2001

Poly[N-(2-hydroxypropyl)methacrylamide] polymers diffuse in brain extracellular space with same tortuosity as small molecules.

Integrative optical imaging was used to show that long-chain synthetic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) polymers in a range of molecular weights from 7.8 to 1057 kDa were able to diffuse through the extracellular space in rat neocortical slices. Tortuosity (square root of ratio of diffusion coefficient in aqueous medium to that in brain) measured with such polymers averaged 1.57, a value similar to that obtained previously with tetramethylammonium, a small cation. When PHPMA was conjugated with bovine serum albumin (BSA) to make a bulky polymer with molecular weight 176 kDa, the tortuosity rose to 2.27, a value similar to that obtained previously with BSA alone and with 70-kDa dextran. The method of image analysis was justified with diffusion models involving spherical and nonspherical initial distributions of the molecules.

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↗
PubMed2000

Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin.

Binding of serum proteins to polyelectrolyte gene delivery complexes is thought to be an important factor limiting bloodstream circulation and restricting access to target tissues. Protein binding can also inhibit transfection activity in vitro. In this study a multivalent reactive hydrophilic polymer has been used to inhibit protein binding. This polymer is based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA) bearing pendent oligopeptide (Gly-Phe-Leu-Gly) side chains terminated in reactive 4-nitrophenoxy groups (8.6 mol%). The polymer reacts with the primary amino groups of poly(L-lysine) (pLL) and produces a hydrophilic coating on the surface of pLL.DNA complexes (as measured by fluorescamine). The resulting pHPMA-coated complexes show a decreased surface charge (from +14 mV for pLL.DNA complexes to -25 mV for pHPMA-modified complexes) as measured by zeta potential analysis. The pHPMA-coated complexes also show a slightly increased average diameter (approximately 90 nm compared with 60 nm for pLL. DNA complexes) as viewed by atomic force and transmission electron microscopy and around 100 nm as viewed by photon correlation spectroscopy. They are completely resistant to protein interaction, as determined by turbidometry and SDS-polyacrylamide gel electrophoresis analysis of complexes isolated from plasma, and show significantly decreased nonspecific uptake into cells in vitro. Spare reactive ester groups can be used to conjugate targeting ligands (e.g. transferrin) on to the surface of the complex to provide a means of tissue-specific targeting and transfection. The properties of these complexes therefore make them promising candidates for targeted gene delivery, both in vitro and potentially in vivo.

Acrylic Resins↗
PubMed2000

A versatile system for receptor-mediated gene delivery permits increased entry of DNA into target cells, enhanced delivery to the nucleus and elevated rates of transgene expression.

We have developed a method for stabilisation of polyelectrolyte gene delivery vectors by crosslinking their surfaces with biodegradable multivalent copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA). The resulting nanoparticulate vectors resist attack by serum proteins and can be modified for cell-specific delivery by incorporation of targeting ligands onto the polymer coating. Here we show that vascular endothelial growth factor (VEGF), transferrin and basic fibroblast growth factor (bFGF) can each be linked to polyHPMA-coated poly(L-lysine)/DNA complexes. All ligand-targeted complexes demonstrated increased uptake into receptor-positive cells (measured using plasmids containing 32P-dCTP), that could be antagonised with excess free ligand. Targeted complexes also showed increased transfection, resistant to inhibition by serum, suggesting the possibility of effective application in vivo. Analysis using fluorescence microscopy confirmed enhanced uptake of ligand-targeted complexes (using Texas Red-labelled plasmid DNA), although VEGF- and transferrin-targeted complexes were restricted to cytoplasmic or perinuclear distributions. In contrast, bFGF-targeted complexes showed efficient delivery into the nucleus, with accumulation of more than 100000 plasmids per cell within distinct intranuclear compartments. This method permits versatile targeting of genes to selected cells and may also permit manipulation of intracellular trafficking. It should find several important applications in gene delivery systems both in vitro and in vivo.

Cell Nucleus↗
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↗
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↗
PubMed1995

Intracerebral implantation of hydrogel-coupled adhesion peptides: tissue reaction.

Arg-Gly-Asp peptides (RGD) were synthesized and chemically coupled to the bulk of N-(2-hydroxypropyl) methacrylamide-based polymer hydrogels. Fourier Transform Infrared Spectroscopy (FTIR) and amino acid analysis confirmed the peptide coupling to the polymer. Activated and control (unmodified) polymer matrices were stereotaxically implanted in the striata of rat brains, and two months later the brains were processed for immunohistochemistry using antibodies for glial acidic fibrillary protein (GFAP), laminin and neurofilaments. RGD-containing polymer matrices promoted stronger adhesion to the host tissue than the unmodified polymer matrices. In addition, the RGD-grafted polymer implants promoted and supported the growth and spread of GFAP-positive glial tissue onto and into the hydrogels. Neurofilament-positive fibers were also seen running along the surface of the polymer and, in some instances, penetrating the matrix. These findings are discussed in the context of using bioactive polymers as a new approach for promoting tissue repair and axonal regeneration of damaged structures of the central nervous system.

Adhesives↗
PubMed1994

Tumour tropism and anti-cancer efficacy of polymer-based doxorubicin prodrugs in the treatment of subcutaneous murine B16F10 melanoma.

Doxorubicin (5 mg kg-1) was administered intravenously to C57 mice bearing subcutaneous B16F10 melanomas, distributing into the tumour with an area under the concentration-time curve (0-48 h; AUC) of 8.7 micrograms h g-1. Injection of doxorubicin-N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugate, containing 5 mg of doxorubicin equivalent per kg, mediated an AUC for free doxorubicin (i.e. doxorubicin released from the conjugate) of 15.2 micrograms h g-1 and for total doxorubicin (i.e. free plus conjugated) of 149.1 micrograms h g-1. An increased dose of doxorubicin-HPMA copolymer conjugate (18 mg of doxorubicin equivalent per kg) produced AUC values of 40.1 micrograms h g-1 and 671.7 micrograms h g-1 for free and total doxorubicin respectively. Hence administration of doxorubicin-HPMA copolymer conjugate achieved rises of 1.7- to 4.6-fold in tumour AUC (free doxorubicin) and 17.19 to 77.0-fold in tumour AUC (total doxorubicin). HPMA copolymers bearing fluorescein isothiocyanate accumulated in vascularised stromal regions, particularly in new growth sites at the tumour periphery. Treatment of mice with doxorubicin-HPMA copolymer conjugate achieved treated/control lifespans up to 320% (three doses of 27 mg of doxorubicin equivalent per kg) compared with only 133% using aggressive regimens of free doxorubicin (3 x 5 mg kg-1).

Animals↗
PubMed1993

Synthetic polymer derivatives as substrata for neuronal adhesion and growth.

The adhesion and viability of dissociated neurons of rat cerebral hemispheres onto methacrylate and methacrylamide hydrogels, either unmodified or containing collagen, basement membrane proteins, and glucosamine, were measured in vitro. The degree of cell adhesion was affected by properties of the polymers such as hydrophilicity, hydrophobicity, presence of reactive chemical groups, and incorporation of biological molecules. Adhesion was promoted by attachment of glucosamine to the polymer backbone. Viability was enhanced by the presence of basement membrane proteins within the polymer network. Morphological studies of cells seeded, both onto and within the polymeric matrices, demonstrated the capacity of such substrates to support neuritic outgrowth. The potential of these in vitro assays in the design of polymeric matrices as neural tissue repair promoter substrate is discussed.

Acrylamides↗
PubMed1991

Synthetic polymers conjugated to monoclonal antibodies: vehicles for tumour-targeted drug delivery.

(N-(2-Hydroxypropyl)methacrylamide (HPMA)) copolymers have seen extensive development as sophisticated lysosomotropic drug carriers. They can be used for site-specific drug delivery by incorporation of appropriate targeting groups and here we report their conjugation to antitumour monoclonal antibodies (the murine IgG, antibody B72.3 and its Fab' and Fab'2 fragments) and assessment as vehicles for tumour-specific drug delivery. Conjugates were synthesised containing an average 5 copolymer units (Mw 20kD) per antibody molecule. Kinetics of elimination and body distribution of radiolabelled conjugates in mice were substantially modified compared with native antibody and fragments, showing prolonged circulation in the bloodstream. Notably, the half-time for bloodclearance of the Fab' fragment (35min) was extended ten-fold following conjugation (6h). The conjugates provoked only a low immune response in A/J mice, following three injections in adjuvant (IgG titre-1 less than 100), and were resistant (up to 50%) to proteolytic degradation by preparations of rat liver lysosomal enzymes. The parent antibody targeted efficiently to human colorectal carcinoma (LS174T) xenografts in nude mice (up to 25%/g); the conjugates, however, showed no tumour-targeting, probably due to masking by polymer chains (which are attached by non-specific aminolysis). Conjugates designed to maintain immunoreactivity following linkage through oxidised carbohydrates are currently being synthesised. Nevertheless, the conjugates display increased rates of extravasation, compared with proteins of the same hydrodynamic size, and the decreased charge is anticipated to accelerate diffusion through tumour interstitium.

Acrylamides↗
PubMed1990

Release of macromolecules and daunomycin from hydrophilic gels containing enzymatically degradable bonds.

The synthesis of hydrophilic gels based on N-(2-hydroxypropyl)methacrylamide copolymers crosslinked via degradable oligopeptide sequences is described. The influence of the conditions of preparation and of the gel structure on the equilibrium degree of swelling (network density) was determined. To evaluate the potential of such gels for controlled delivery of macromolecules and drugs, the release of FITC-dextrans of different molecular weights was studied and the rate of release was found to depend mainly on the equilibrium degree of swelling and not on the structure of the crosslinks. However, the degradation of the gels by a mixture of lysosomal enzymes isolated from rat liver (Tritosomes) or chymotrypsin was dependent on both the equilibrium degree of swelling and the structure of the crosslinks (length of the oligopeptide sequence and structure of the amino acid residues). The release of the anticancer drug daunomycin imbibed in gels was pH-dependent, the rate of release being higher at lower pH. In addition, a gel was synthesized which contained a pentapeptide in the crosslinks and daunomycin bound via a tetrapeptide side-chain, and in this case, incubation with Tritosomes led to degradation with simultaneous release of the drug.

Amino Acid Sequence↗
PubMedDate not supplied

Poly(ethylene glycol) multiblock copolymer as a carrier of anti-cancer drug doxorubicin.

The synthesis of a novel water-soluble polymer drug carrier system based on biodegradable poly(ethylene glycol) block copolymer is described in this paper. The copolymer consisting of PEG blocks of molecular weight 2000 linked by means of an oligopeptide with amino end groups was prepared by interfacial polycondensation of the diamine and PEG bis(succinimidyl carbonate). The structure of the oligopeptide diamine consisting of glutamic acid and lysine residues was designed as a substrate for cathepsin B, a lysosomal enzyme, which was assumed to be one of the enzymes responsible for the degradation of the polymer carrier in vivo. Each of the oligopeptide blocks incorporated in the carrier contained three carboxylic groups of which some were used for attachment of an anti-cancer drug, doxorubicin (Dox), via a tetrapeptide spacer Gly-Phe-Leu-Gly. This tetrapeptide spacer is susceptible to enzymatic hydrolysis. In vitro release of Dox and the degradation of the polymer chain by cathepsin B as well as preliminary evaluation of in vivo anti-cancer activity of the conjugate are also demonstrated.

Animals↗