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Biomedical subjects

Jindrich Kopecek

Publications and source records attributed to Jindrich Kopecek.

At least 19 recordsLinked to original sources
PubMed2006

Biodistribution and pharmacokinetics of colon-specific HPMA copolymer--9-aminocamptothecin conjugate in mice.

A water soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-9-aminocamptothecin (9-AC) conjugate was designed for oral colon-specific drug delivery in the treatment of colon cancer. Comparative studies between the polymer conjugate and free drug have been performed to assess their biodistribution and pharmacokinetics in mice. After oral administration of equal doses of the polymer conjugate or free 9-AC, the drug concentrations in major organs at fixed time points were determined using an HPLC-fluorescence assay. Only 2+/-1% of 9-AC released from the polymer conjugate was detected in the small intestine (SI), and the mean peak concentration of free 9-AC was 45-fold higher than that from released drug. Colon-specific release of 9-AC produced high local concentrations. The mean peak concentration of released 9-AC in cecal contents, feces, cecal tissue, and colon tissue were, respectively, 3.2-fold, 3.5-fold, 2.2-fold and 1.6-fold higher than that using free 9-AC. In plasma, the high and sharp drug concentration profile from free drug was in contrast to the relatively low and flat pharmacokinetic profile obtained from drug released from the HPMA copolymer. There was no significant difference between released and free drug for the area under the concentration-time curve (AUC) and bioavailability values. As a consequence of the colon-specific release of unmodified 9-AC from the polymer conjugate, antitumor efficacy can be anticipated to be enhanced due to prolonged colon tumor exposure to higher and more localized drug concentrations.

Administration, Oral↗
PubMed2006

Hybrid hydrogels self-assembled from HPMA copolymers containing peptide grafts.

Graft copolymers were designed that self-assemble into hydrogels mediated by the interaction of coiled-coil peptide domains. A linear hydrophilic polymer of HPMA was chosen as the backbone, and coiled-coil forming peptides, covalently attached to the backbone, formed the grafts. Microrheology was used to evaluate the self-assembly of graft copolymers into hydrogels. The results revealed that the length and the number of coiled-coil grafts per chain had a significant influence on the gelation process. At least 4 heptads were needed to achieve the association of graft copolymers into hydrogels. CD spectra of the copolymer containing 5 heptad grafts further suggested that coiled-coil formation may contribute to the self-assembly. Gelation of graft copolymers containing CC4 peptides indicated that a threshold amount of grafts per macromolecule is needed to form a three-dimensional structure. These studies demonstrated a potential of the graft copolymers to create self-assembling hydrogels with desirable and controllable structures.

Amino Acid Sequence↗
PubMed2006

Refolding hydrogels self-assembled from N-(2-hydroxypropyl)methacrylamide graft copolymers by antiparallel coiled-coil formation.

A novel hybrid hydrogel system based on N-(2-hydroxypropyl)methacrylamide copolymers was proposed. It consisted of the hydrophilic polymer backbone and a pair of oppositely charged peptide grafts. Two distinct pentaheptad peptides (CCE and CCK) were anticipated to create a dimerization motif and serve as physical cross-linkers. Consequently, the graft copolymers CCE-P and CCK-P self-assembled into hybrid hydrogels in situ; the process was modulated by the formation of antiparallel heterodimeric coiled-coils. This approach possesses an advantage to decrease the steric hindrance of the polymer backbone on the "in-register" alignment of peptide grafts. Indeed, equimolar mixtures of the graft copolymers, CCE-P/CCK-P, have been observed to self-assemble into hydrogels in PBS solution at neutral pH at concentrations as low as 0.1 wt %. Circular dichroism spectroscopy, sedimentation equilibrium experiments, and microrheology revealed that the self-assembly process corresponded to the two-stranded alpha-helical coiled-coil formation between CCE and CCK. Moreover, the formation of hybrid hydrogels was reversible. Denaturation of the coiled-coil domains with guanidine hydrochloride (GdnHCl) solutions resulted in disassembly of the hydrogels. Removal of GdnHCl by dialysis caused coiled-coil refolding and hydrogel reassembly. Scanning electron microscopy results demonstrated that the concentration of the graft copolymers had a significant impact on the structure and morphology of self-assembled hydrogels.

Cross-Linking Reagents↗
PubMed2006

Semitelechelic HPMA copolymers functionalized with triphenylphosphonium as drug carriers for membrane transduction and mitochondrial localization.

Semitelechelic HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers possessing a single terminal lipophilic triphenylphosphonium (TPP) cation and fluorescent labels were synthesized to determine how the attached cation affected cellular uptake and intracellular trafficking. In vitro mitochondrial uptake fluorescence quenching assays using isolated mouse liver mitochondria indicated that only lower molecular weight (<5 kDa) BODIPY FL-labeled TPP-semitelechelic HPMA copolymers exhibited significant organelle localization or uptake. In vitro cellular uptake and intracellular trafficking was evaluated using cultured human ovarian carcinoma cells. Cells incubated with all types of TPP copolymers used in the study appeared to internalize the polymer by endocytosis only, and all of the internalized copolymer was confined to the lysosomal compartment after 24 h. Endocytotic uptake of the TPP-HPMA copolymer conjugates was rapid, suggesting that they were internalized by adsorptive endocytosis, rather than fluid-phase pinocytosis. Low-molecular weight (<5 kDa) and high-molecular weight (>5 kDa) semitelechelic copolymers, microinjected into cultured cells indicated that the TPP moiety did not significantly localize the polymers to mitochondria.

Biological Transport↗
PubMed2006

Two-step fluorescence screening of CD21-binding peptides with one-bead one-compound library and investigation of binding properties of N-(2-hydroxypropyl)methacrylamide copolymer-peptide conjugates.

Using the one-bead one-compound (OBOC) combinatorial method, four heptapeptide ligands of CD21 receptor, a cell surface marker of malignant B cell lymphoma, were identified with an innovative two-step fluorescence screening method to overcome the limitation caused by autofluorescence of TentaGel resin. The binding affinities of selected peptides, YILIHRN (B1), PTLDPLP (B2), and LVLLTRE (B3), were in the micromolar region as determined by a fluorescence quenching assay. Peptide B1 was conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via spacers of different lengths, composed of one to four repeats of the 8-amino-3,6-dioxaoctanoic acid (A) group. The evaluation of the biorecognizability of HPMA copolymer-B1 conjugates by the CD21 receptor revealed that increasing the number of repeats of A in the spacer from one to three resulted in continuous improvements in the biorecognition by the CD21 receptor; the increase from three to four repeats showed no significant effect. This work showed the potential of the OBOC combinatorial approach to select peptide ligands as targeting moieties for CD21 specific polymeric drug carriers.

Acrylamides↗
PubMed2006

Water-soluble HPMA copolymer--prostaglandin E1 conjugates containing a cathepsin K sensitive spacer.

A novel bone targeting, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer based, prostaglandin E1 (PGE1) delivery system was designed, synthesized and characterized. PGE1 was bound to the polymer backbone via a spacer, composed of a cathepsin K sensitive tetrapeptide (Gly-Gly-Pro-Nle) and a self-eliminating 4-aminobenzyl alcohol structure. The HPMA copolymer conjugates were prepared by photo-initiated free radical copolymerization of HPMA, PGE1-containing macromonomer, and optionally a comonomer containing a reactive p-nitrophenyl ester group. The latter group was used as attachment points for the D-aspartic acid octapeptide targeting moieties. Incubation of the PGE1-containing macromonomer and HPMA copolymer-PGE1 conjugates with cathepsin K resulted in release of unmodified PGE1. The rate of release depended on the composition of the conjugate. The higher the PGE1 content in the conjugate, the slower the PGE1 release. This appeared to be the result of association of hydrophobic side-chains in aqueous media, which rendered the formation of the enzyme substrate complex more difficult. The data seems to indicate that HPMA copolymer-PGE1 conjugates have a potential in the treatment of osteoporosis and other bone diseases.

Acrylamides↗
PubMed2005

Synthesis and characterization of novel aromatic azo bond-containing pH-sensitive and hydrolytically cleavable IPN hydrogels.

Novel interpenetrating network (IPN) hydrogels, composed of pH-sensitive, aromatic azo group containing network as one component (Network A), and a hydrolyzable network as the other (Network B), were prepared by a sequential process. The first network was formed by crosslinking of a reactive polymer precursor (copolymer of N,N-dimethylacrylamide, acrylic acid, N-tert.butylacrylamide, and N-methacryloylglycylglycine p-nitrophenyl ester) with an aromatic azo group containing diamine ((N,N'-epsilon-aminocaproyl)-4,4'-diaminoazobenzene). The second network was formed by radical crosslinking copolymerization of N-(2-hydroxypropyl)methacrylamide with N,O-dimethacryloylhydroxylamine. The composition of the hydrogels was manipulated to determine the influence of hydrogel composition on the equilibrium degree of swelling, modulus of elasticity in compression, and on the rate of degradation of Network B. Modeling of network structure was accomplished using the statistical branching theory. The major advantage of IPN hydrogels, when compared to traditional pH-sensitive networks, is the linear swelling profile following abrupt change of pH from 2 to 7.4. This indicates the suitability of IPN as carriers for oral drug delivery.

Absorption↗
PubMed2005

Bone-targeting macromolecular therapeutics.

Musculoskeletal diseases such as osteoporosis are recognized as major public health problems worldwide. Many novel therapeutic agents have been identified for the treatment of these diseases. However, the majority of them are not specific to hard tissue, resulting significant toxicity. Bone-targeting drug delivery systems based on water-soluble polymers can specifically direct candidate drugs to bone thereby reducing side effects due to non-specific tissue interactions. Incorporation of a targeting moiety, a drug release mechanism, drug selection and optimization of the polymer carrier are all essential elements in the development of bone-targeting macromolecular therapeutics. Successful clinical application of this approach can significantly contribute to the development of treatments for many musculoskeletal diseases.

Animals↗
PubMed2005

Biopolymer-based delivery systems for advanced imaging and skeletal tissue-specific therapeutics.

There is considerable advantage in developing tissue-specific delivery systems for therapeutic and diagnostic applications. Synthetic water-soluble polymeric delivery systems have been developed to allow selective delivery of therapeutic and imaging agents to musculoskeletal tissues. For mineralized tissues, bone-targeting agents such as aspartic acid octapeptide could concentrate the polymer conjugates to bone surfaces including resorption sites, which was demonstrated with routine bone histomorphometry. For bone-associated soft tissues, other targeting approaches based on pathophysiological properties unique to the local tissue environment, such as the leaky vasculature in arthritic joints, were utilized to achieve the selective deposition of the polymeric delivery systems to the desired sites. For this study, magnetic resonance imaging (MRI) was used to assess real-time pharmacokinetics and biodistribution of the MRI contrast agent conjugated polymer in major organs including skeletal tissues. The MRI data were then correlated with other standard imaging methods such as pQCT and DXA as well as routine histopathology and skeletal histomorphometry. Clearly, biopolymeric delivery systems may be used to improve the pharmacological and pharmacokinetic properties of different therapeutic agents for musculoskeletal diseases such as osteoporosis and arthritis. In addition, this or related technologies may also be useful to improve diagnosis and medical imaging with positron emission tomography, gamma scintigraphy, or other technologies.

Animals↗
PubMed2005

Confocal microscopy studies of a model oligoribonucleotide HIV inhibitor.

Previous work has shown that novel amphipalhic oligo and polyribonucleotides are potent inhibitors of HIV. It was hypothesized that the mechanism(s) of action for these compounds might be inhibition of retroviral reverse transcriptase (RT) and/or viral uptake by cells. A fluorescent oligonucleotide analog was prepared, and confocal microscopy studies were undertaken in order to examine oligonucleotide-cell interactions.

Anti-HIV Agents↗
PubMed2004

Intracellular targeting of polymer-bound drugs for cancer chemotherapy.

Macromolecules have been traditionally employed as drug carriers due to their ability to selectively accumulate in malignant tissues compared to healthy tissues by either passive or active targeting, thus precluding undesirable side effects generated by free drug. The therapeutic activity proffered by such conjugates requires that the drug concentrate at its specific subcellular target such as the nucleus. Thus, the suitability of macromolecules as carriers also extends to their propensity to deliver the drug to a predetermined intracellular location. As binding a macromolecule to a drug facilitates cellular uptake by endocytosis, various approaches have been employed to either guide the drug to targets different from endosomal/lysosomal compartments by mediating vesicular escape, or to directly accomplish intracellular (cytoplasmic and nuclear) localization. This review discusses the utility of macromolecules in drug delivery and describes the numerous modalities (with a focus on cell-penetrating peptides) currently available for achieving effective intracellular drug delivery.

Cell Membrane↗
PubMed2004

The arthrotropism of macromolecules in adjuvant-induced arthritis rat model: a preliminary study.

PURPOSE: To study the accumulation of macromolecules into the arthritic joints and the possible applications of such phenomenon. METHODS: The accumulation of plasma albumin in the joints of adjuvant-induced arthritis (AIA) rat model was first visualized with Evans blue injection. A N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer contrast agent was then synthesized and injected into the AIA rats to allow qualitative examination of biodistribution and pharmacokinetics of the injected macromolecule with magnetic resonance imaging (MRI). Vital organs and the diseased joints were isolated and examined histologically to correlate with the MRI findings. RESULTS: Deep blue color developed around the arthritic joints of the AIA rat a few hours after the injection of Evans blue. MR imaging of the AIA rats injected with polymer contrast agent demonstrated a gradual but very strong accumulation of the injected polymer in the arthritic joints, which lasted for 1-2 days. Observed differences in the concentration of the injected polymer in the joints correlated with disease severity as assessed histologically. CONCLUSIONS: Profound arthrotropism of macromolecules in the AIA rat model was demonstrated with various imaging tools. These observations should help in the conceptual and practical design of novel macromolecular delivery systems for the imaging and treatment of rheumatoid arthritis.

Animals↗
PubMed2003

Free and N-(2-hydroxypropyl)methacrylamide copolymer-bound geldanamycin derivative induce different stress responses in A2780 human ovarian carcinoma cells.

The effects of geldanamycin (GA), 17-(3-aminopropylamino)-17-demethoxygeldanamycin (AP-GA), and N-(2-hydroxypropyl)methacrylamide copolymer-AP-GA conjugate [P(AP-GA)] on A2780 human ovarian carcinoma cells at an equitoxic dose (2x IC(50)) were compared by the gene expression array analysis. All treatments resulted in similar gene expression profiles up to 12 h (e.g., down-regulation of CDK4 and up-regulation of APAF-1), although P(AP-GA)-treated cells showed delayed gene expression because of time-dependent internalization of the conjugate and intracellular drug release from P(AP-GA). However, AP-GA-treated cells showed elevated expression of HSP70 and HSP27 after 6 h compared with that observed by GA and P(AP-GA) treatments. Depletion of C-Raf, an HSP90 client protein, was observed in all treatments up to 12 h. Confocal microscopy using mesochlorin e(6) as a model drug revealed that drug release caused by the lysosomal cleavage of glycylphenylalanylleucylglycine oligopeptide spacer, used as GA derivative copolymer attachment/release point, was moderately fast. These results suggested that AP-GA treatment may activate stress-response pathways, whereas P(AP-GA) treatment may suppress them and trigger signaling pathways essential to cell growth arrest and death by inducing an HSP90-active factor. Although GA and P(AP-GA) treatments induced a time-dependent increase in HSP70 and HSP27 protein expression (detected by Western blotting analysis), AP-GA treatment resulted in more rapid and more intense expression of both proteins. Our results suggest that conjugation of AP-GA to N-(2-hydroxypropyl)methacrylamide copolymer may be able to modulate the cell stress responses induced by AP-GA because of differences in its internalization mechanism, subcellular localization, and intracellular concentration gradients.

Antibiotics, Antineoplastic↗
PubMed2003

Subcellular trafficking of HPMA copolymer-Tat conjugates in human ovarian carcinoma cells.

One of the main obstacles to efficient intracellular delivery of therapeutic macromolecules is the barrier posed by the plasma membrane. In this study, the cell penetrating peptide Tat was conjugated to a synthetic macromolecule based on N-(2-hydroxypropyl)methacrylamide (HPMA) and its subcellular distribution in human ovarian carcinoma cell lines was studied. The Tat peptide mediated uptake resulted in cytoplasmic and nuclear localization and was found to be energy independent. Time and concentration studies verified the rapidity and dependence of the transport process on these parameters. Enhanced uptake of a polymer bound anticancer drug doxorubicin was also demonstrated. These results were corroborated independently by subcellular fractionation.

Carcinoma↗
PubMed2003

Cytoplasmic delivery and nuclear targeting of synthetic macromolecules.

Delivery of macromolecular drugs (e.g. antisense oligonucleotides, polymer-drug conjugates, etc.) designed to work in specific sites inside cells is complicated as macromolecules typically have access to fewer biological compartments than small molecules. To better understand the fate of macromolecules in cells and begin to alter that fate, we investigated the internalization and subcellular fate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers and HPMA copolymer-drug conjugates in Hep G2 and A2780 cells. The subcellular fate of fluorescently labeled polymers was monitored by confocal microscopy and subcellular fractionation. Initially, the HPMA copolymers and HPMA copolymer-drug conjugates were internalized by endocytosis and remained in endosomes/lysosomes. At longer incubation times (>8 h), small amounts of the HPMA copolymers were observed to enter the cytoplasm and accumulate in the nucleus of the cells. Nuclear accumulation was confirmed after cytoplasmic microinjection. Oligonucleotides conjugated via lysosomally degradable spacers entered into the cytoplasm and nucleus of the cells faster than the polymers. The effect of the subcellular location was correlated to the toxicity of the photosensitizer, mesochlorin e(6) (Mce(6))-HPMA copolymer conjugates. The plasma membrane and late endosomes were more sensitive to damage by Mce(6). Targeting the polymer conjugates to the nucleus with the nuclear localization sequence (NLS) as well as conjugating the Mce(6) via a degradable spacer increased cell adhesion and uptake, promoted their entry into the cytoplasm and nucleus of the cells, and increased their toxicity. To further promote entry of the polymers into the cytoplasm and nucleus of the cells, the protein transduction domain, Tat peptide, was conjugated to the HPMA copolymers. This resulted in high binding to the cell membrane, but also facilitated rapid (<5 min) entry of the macromolecules into the cytoplasm and nucleus of cells. These results will prove valuable in the future design of macromolecular therapeutics.

Animals↗
PubMed2003

HPMA copolymer delivery of chemotherapy and photodynamic therapy in ovarian cancer.

Our studies document a unique and unexpected advantage of the combination of HPMA copolymer bound doxorubicin with mesochlorin e6/photodynamic therapy in the treatment of ovarian cancer. Each drug's activity is individually enhanced when compared with free (low molecular weight) drugs, furthermore, in combination these HPMA copolymer bound agents act synergistically to create an unexpected biological effect. Figure 8 depicts the known activities of each agent which may play synergistic roles. HPMA copolymer-doxorubicin has been widely evaluated in preclinical and clinical studies. It demonstrates marked advantages over free doxorubicin: control of biodistribution and accumulation via molecular weight restrictions, biodegradability, minimal immunogenicity, subcellular localization, anticancer activity, enhanced permeability and retention, increased apoptosis, lipid peroxidation, DNA damage, and reduced nonspecific toxicity. Recent clinical trials in the UK provide "proof of principle" of the "enhanced permeability and retention effect" for solid tumors and the unique advantages of this novel drug delivery system for the treatment of ovarian cancer. With regards to photodynamic therapy using the photosensitizer mesochlorin e6, the preclinical evaluations thus far document: control of biodistribution and accumulation via molecular weight restrictions, biodegradability, subcellular localization, anticancer activity, enhanced permeability and retention, and reduced nonspecific toxicity. Ongoing microarray studies document unique cellular pathways and new pharmaceutical properties which are initiated by the HPMA copolymer delivery delivery of these agents, and predict an exciting future for this novel drug delivery system.

Antibiotics, Antineoplastic↗
PubMed2003

Smart and genetically engineered biomaterials and drug delivery systems.

The design, synthesis, and properties of novel stimuli-sensitive and genetically engineered biomaterials and drug delivery systems are reviewed. Two approaches to their engineering are presented. One approach is to improve the traditional methods of synthesis, as demonstrated by the example of controlled copolymerization of alpha-amino acid N-carboxyanhydrides. The other approach, discussed in more detail, uses genetic engineering methods. The design of hybrid hydrogel systems whose components derive from at least two distinct classes of molecules, e.g., synthetic macromolecules and protein domains, is assessed. The design of self-assembling block copolymers is discussed in detail. Finally, the pharmaceutics related applications of these materials are presented.

Biocompatible Materials↗