The cysteine protease cathepsin K is a target for the treatment of diseases associated with high bone turnover. Cathepsin K is mainly expressed in osteoclasts and responsible for the destruction of the proteinaceous components of the bone matrix. We designed various fluorescent activity-based probes (ABPs) and their precursors that bind to and inactivate cathepsin K. ABP 25 exhibited extraordinary potency (kinac/Ki = 35,300 M-1s-1) and selectivity for human cathepsin K. Crystal structures of cathepsin K in complex with ABP 25 and its nonfluorescent precursor 21 were determined to characterize the binding mode of this new type of acrylamide-based Michael acceptor with the particular orientation of the dibenzylamine moiety to the primed subsite region. The cyanine-5 containing probe 25 allowed for sensitive detection of cathepsin K, selective visualization in complex proteomes, and live cell imaging of a human osteosarcoma cell line, underlining its applicability in a pathophysiological environment.
- MeSH
- akrylamidy chemická syntéza chemie metabolismus MeSH
- fluorescenční barviva chemická syntéza chemie metabolismus MeSH
- fluorescenční mikroskopie MeSH
- inhibitory cysteinových proteinas chemická syntéza chemie metabolismus MeSH
- katalytická doména MeSH
- kathepsin K antagonisté a inhibitory chemie metabolismus MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- racionální návrh léčiv MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamidy MeSH
- CTSK protein, human MeSH Prohlížeč
- fluorescenční barviva MeSH
- inhibitory cysteinových proteinas MeSH
- kathepsin K MeSH
Here, we describe innovative synthesis of well-defined biocompatible N-(2-hydroxypropyl) methacrylamide (HPMA)-based polymer carriers and their drug conjugates with pirarubicin intended for controlled drug delivery and pH-triggered drug activation in tumor tissue. Polymer carrier synthesis was optimized to obtain well-defined linear HPMA-based polymer precursor with dispersity close to 1 and molar mass close to renal threshold with minimal synthesis steps. The developed synthesis enables preparation of tailored polymer nanomedicines with highly enhanced biological behavior in vivo, especially the biodistribution, urine elimination, tumor accumulation and anticancer activity. STATEMENT OF SIGNIFICANCE: The manuscript reports on novel synthesis and detailed physicochemical characterization and in vivo evaluation of well-defined biocompatible hydrophilic copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) and their drug conjugates with pirarubicin enabling controlled drug delivery and pH-triggered drug activation in tumor tissue. Polymer carrier synthesis was optimized to obtain well-defined linear HPMA-based polymer precursor with minimal synthesis steps using controlled polymerization. Compared to previously published HPMA-based polymer drug conjugates whose polymer carriers were prepared by classical route via free radical polymerization, the newly prepared polymer drug conjugates exhibited enhanced biological behavior in vivo, especially the prolonged blood circulation, urine elimination, tumor accumulation and excellent anticancer activity. We believe that the newly prepared well-defined polymer conjugates could significantly enhance tumor therapy in humans.
- Klíčová slova
- Anti-tumor therapy, HPMA polymer conjugate, Pirarubicin, RAFT polymerization, pH-sensitive release, EPR effect,
- MeSH
- akrylamidy chemická syntéza farmakokinetika terapeutické užití MeSH
- antitumorózní látky chemická syntéza farmakokinetika terapeutické užití MeSH
- doxorubicin analogy a deriváty chemická syntéza farmakokinetika terapeutické užití MeSH
- experimentální sarkom farmakoterapie MeSH
- kapronáty chemická syntéza farmakokinetika terapeutické užití MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nanomedicína metody MeSH
- polymerizace MeSH
- systémy cílené aplikace léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 6-methacrylamidohexanoic acid MeSH Prohlížeč
- akrylamidy MeSH
- antitumorózní látky MeSH
- doxorubicin MeSH
- kapronáty MeSH
- N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
- pirarubicin MeSH Prohlížeč
The effects of novel polymeric therapeutics based on water-soluble N-(2-hydroxypropyl)methacrylamide copolymers (P(HPMA)) bearing the anticancer drug doxorubicin (Dox), an inhibitor of ABC transporters, or both, on the viability and the proliferation of the murine monocytic leukemia cell line P388 (parental cell line) and its doxorubicin-resistant subline P388/MDR were studied in vitro. The inhibitor derivatives 5-methyl-4-oxohexanoyl reversin 121 (MeOHe-R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe-RIT), showing the highest inhibitory activities, were conjugated to the P(HPMA) via the biodegradable pH-sensitive hydrazone bond, and the ability of these conjugates to block the ATP driven P-glycoprotein (P-gp) efflux pump was tested. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121 showed a dose-dependent increase in the ability to sensitize the P388/MDR cells to Dox from 1.5 to 24 μM, and achieved an approximately 50-fold increase in sensitization at 24 μM. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-RIT showed moderate activity at 6 μM (∼10 times higher sensitization) and increased sensitization by 50-fold at 12 μM. The cytostatic activity of the P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121(Dox) containing Dox and the P-gp inhibitor MeOHe-R121, both bound via hydrazone bonds to the P(HPMA) carrier, was almost 30 times higher than that of the conjugate P-Ahx-NH-N═Dox toward the P388/MDR cells in vitro. A similar result was observed for P-Ahx-NH-N═MeOHe-RIT(Dox), which exhibited almost 10 times higher cytostatic activity than P-Ahx-NH-N═Dox.
- MeSH
- ABC transportéry antagonisté a inhibitory MeSH
- akrylamidy chemická syntéza MeSH
- antibiotika antitumorózní farmakologie MeSH
- chemorezistence * MeSH
- doxorubicin farmakologie MeSH
- hydrazony chemie MeSH
- koncentrace vodíkových iontů MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- P-glykoprotein metabolismus MeSH
- systémy cílené aplikace léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ABC transportéry MeSH
- akrylamidy MeSH
- antibiotika antitumorózní MeSH
- doxorubicin MeSH
- hydrazony MeSH
- N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
- P-glykoprotein MeSH
The specificity of polymer conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) bearing cytostatic drugs for cancer cells could be significantly increased by the incorporation of a suitable targeting ligand, such as a monoclonal antibody (mAb). However, direct binding of the protein to the polymer carrier could cause considerable problems, such as decreasing the binding capacity of mAb to its target. Here, we introduce a novel strategy of joining a targeting moiety to a polymeric conjugate with cytostatic drug. The scFv of B1 mAb (specific for BCL1 leukemia cells) was tagged with peptide K ((VAALKEK)4). Peptide E ((VAALEKE)4), which forms a stable coiled coil structure heterodimer with peptide K, was assembled with the HPMA copolymers bearing doxorubicin. Such targeted polymeric conjugates possess very selective and high binding activity toward BCL1 cells. Similarly, targeted polymeric conjugates exert approximately 100 times higher cytostatic activity toward BCL1 cells in comparison to nontargeted conjugates in vitro. At the same time, the conjugates have comparable and rather low cytostatic activity for 38C13 cells, which are used as a negative control, in vitro.
- MeSH
- akrylamidy chemická syntéza farmakologie MeSH
- antibiotika antitumorózní chemie farmakologie MeSH
- biokompatibilní materiály chemická syntéza farmakologie MeSH
- cytostatické látky chemie farmakologie MeSH
- doxorubicin chemie farmakologie MeSH
- leukemie farmakoterapie MeSH
- methakryláty chemie MeSH
- monoklonální protilátky chemie MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nosiče léků chemie MeSH
- polymery chemie farmakologie MeSH
- proliferace buněk MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamidy MeSH
- antibiotika antitumorózní MeSH
- biokompatibilní materiály MeSH
- cytostatické látky MeSH
- doxorubicin MeSH
- hydroxypropyl methacrylate MeSH Prohlížeč
- methakryláty MeSH
- monoklonální protilátky MeSH
- N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
- nosiče léků MeSH
- polymery MeSH
- rekombinantní fúzní proteiny MeSH
Five poly(betaine) brushes were prepared, and their resistance to blood plasma fouling was studied. Two carboxybetaines monomers were copolymerized with 2-hydroxyethyl methacrylate (HEMA) to prepare novel hydrogels. By increasing the content of the zwitterionic comonomer, a 4-fold increase in the water content could be achieved while retaining mechanical properties close to the widely used poly(HEMA) hydrogels. All hydrogels showed an unprecedentedly low fouling from blood plasma. Remarkably, by copolymerization with 10 mol % of carboxybetaine acrylamide, hydrogels fully resistant to blood plasma were prepared.
- MeSH
- akrylamidy chemická syntéza MeSH
- betain chemie MeSH
- biokompatibilní materiály chemie MeSH
- hydrogely chemická syntéza MeSH
- krevní plazma chemie MeSH
- lidé MeSH
- methakryláty chemická syntéza MeSH
- polymerizace MeSH
- polymery chemie MeSH
- povrchová plasmonová rezonance MeSH
- povrchové vlastnosti MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- voda chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamidy MeSH
- betain MeSH
- biokompatibilní materiály MeSH
- hydrogely MeSH
- hydroxyethyl methacrylate MeSH Prohlížeč
- methakryláty MeSH
- polymery MeSH
- voda MeSH
Among the class of zwitterionic polymers poly(carboxybetaine)s (poly(CB)s) are unique, emerging as the only ultra-low fouling materials known allowing the preparation of biosensors, fouling resistant nanoparticles, and non-adhesive surfaces for bacteria. Poly(carboxybetaine methacrylate) and poly(carboxybetaine acrylamide) have been prepared via atom transfer radical polymerization (ATRP), however a polymerization with living characteristics has not been achieved yet. Herein, the first successful living/controlled reversible addition fragmentation transfer (RAFT) polymerization of (3-methacryloylamino-propyl)-(2-carboxy-ethyl)-dimethyl-ammonium (carboxybetaine methacrylamide) (CBMAA-3) in acetate buffer (pH 5.2) at 70 and 37 °C is reported. The polymerization afforded very high molecular weight polymers (determined by absolute size exclusion chromatography, close to 250,000 g·mol(-1) in less than 6 h) with low PDI (<1.3) at 70 °C. The polymerization was additionally carried out at 37 °C allowing to achieve yet lower PDIs (1.06 ≤ PDI ≤ 1.15) even at 90% conversion, demonstrating the suitability of the polymerization conditions for bioconjugate grafting. The living character of the polymerization is additionally evidenced by chain extending poly(CBMAA-3) at 70 and 37 °C. Block copolymerization from biologically relevant poly[N-(2-hydroxypropyl)methacrylamide] macroCTAs was additionally performed.
- MeSH
- akrylamidy chemická syntéza chemie MeSH
- chemie organická metody MeSH
- molekulová hmotnost MeSH
- polymerizace MeSH
- polymery chemická syntéza chemie MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamidy MeSH
- methacrylamide MeSH Prohlížeč
- poly(carboxybetaine acrylamide) MeSH Prohlížeč
- polymery MeSH
Brachytherapy is of increasing popularity in clinical oncology for the local therapy of solid tumors due to high radiation doses delivered to malignant tissue while keeping the whole-body radiation burden low. Pronounced dose-dependent tumor growth reduction was achieved by single dose of injectable intratumoral brachytherapy with iodine-131-labeled thermoresponsive polymer [poly(N-isopropyl acrylamide)] in murine xenograft model (PC3 human prostate adenocarcinoma). The two doses of radionuclide were used, 2 MBq/mouse and 25 MBq/mouse. The higher dose caused gradual tumor volume reduction and 2 of 6 mice from this group were cured. The lower dose caused tumor growth retardation only. In both cases there were no signs of inflammation. The effects of both doses were statistically significant compared to untreated controls. Such injectable system should keep advantages of brachytherapy while making system administration easier and less invasive (injection instead of implantation), patient-tailored (splitting of doses into several depoes) and bioerodable.
- MeSH
- akrylamidy chemická syntéza chemie MeSH
- akrylové pryskyřice MeSH
- brachyterapie metody MeSH
- dávka záření MeSH
- injekce do léze MeSH
- lidé MeSH
- myši nahé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory prostaty patologie radioterapie MeSH
- nosiče léků chemická syntéza chemie MeSH
- polymery chemická syntéza chemie MeSH
- radiofarmaka aplikace a dávkování chemická syntéza chemie terapeutické užití MeSH
- radioizotopy jodu aplikace a dávkování chemie terapeutické užití MeSH
- rozpustnost MeSH
- teplota * MeSH
- vztah dávky záření a odpovědi MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamidy MeSH
- akrylové pryskyřice MeSH
- nosiče léků MeSH
- poly-N-isopropylacrylamide MeSH Prohlížeč
- polymery MeSH
- radiofarmaka MeSH
- radioizotopy jodu MeSH
Synthesis, physicochemical behavior, tumor accumulation and preliminary anticancer activity of a new biodegradable graft copolymer-doxorubicin (DOX) conjugates designed for passive tumor targeting were investigated. In the graft high-molecular-weight conjugates the multivalent N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer was grafted with a similar but semitelechelic HPMA copolymer; both types of polymer chains were bearing doxorubicin attached by hydrazone bonds enabling intracellular pH-controlled drug release. The polymer grafts were attached to the main chain through spacers, degradable enzymatically or reductively, facilitating, after the drug release, intracellular degradation of the graft polymer carrier to short fragments excretable from the organism by glomerular filtration. The graft polymer-DOX conjugate exhibited prolonged blood circulation and enhanced tumor accumulation in tumor-bearing mice indicating the important role of the EPR effect in the anticancer activity. The graft polymer-DOX conjugates showed a significantly higher antitumor activity in vivo than DOX.HCl or the linear polymer conjugate when tested in mice bearing 38C13 B-cell or EL4 T-cell lymphoma, with a significant number of long-term-surviving (LTS) mice with EL4 T-cell lymphoma treated with a single dose 15 mg DOX equiv./kg on day 10.
- MeSH
- akrylamidy aplikace a dávkování chemická syntéza farmakokinetika farmakologie MeSH
- antibiotika antitumorózní aplikace a dávkování chemická syntéza farmakokinetika farmakologie MeSH
- biologický transport MeSH
- buněčné linie MeSH
- chemie farmaceutická MeSH
- doxorubicin aplikace a dávkování chemická syntéza farmakokinetika farmakologie MeSH
- hydrolýza MeSH
- injekce intravenózní MeSH
- koncentrace vodíkových iontů MeSH
- léky s prodlouženým účinkem MeSH
- lymfom farmakoterapie metabolismus patologie MeSH
- molekulová hmotnost MeSH
- myši inbrední C3H MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nosiče léků * MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamidy MeSH
- antibiotika antitumorózní MeSH
- doxorubicin MeSH
- léky s prodlouženým účinkem MeSH
- N-(2-hydroxypropyl)methacrylamide co-polymer-doxorubicin conjugate MeSH Prohlížeč
- nosiče léků * MeSH
- MeSH
- akrylamidy chemická syntéza chemie MeSH
- disulfidy chemická syntéza chemie MeSH
- doxorubicin chemická syntéza chemie MeSH
- oligopeptidy chemická syntéza chemie MeSH
- technika přenosu genů * MeSH
- thiazolidiny chemická syntéza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 2-mercaptothiazoline MeSH Prohlížeč
- akrylamidy MeSH
- disulfidy MeSH
- doxorubicin MeSH
- N-(2-hydroxypropyl)methacrylamide co-polymer-doxorubicin conjugate MeSH Prohlížeč
- N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
- oligopeptidy MeSH
- thiazolidiny MeSH
A novel class of anti-cancer therapeutics - polymeric conjugates of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers and doxorubicin with pH-controlled release of the drug - is highly efficient in killing tumor cells in vitro and is potent in eradicating growing tumors in vivo. Moreover, in comparison with low-molecular-weight drugs, the macromolecular therapeutics show decreased acute as well as delayed adverse side-toxicity. More importantly, the polymeric conjugates trigger the onset of specific anti-tumor immune response and this anti-tumor immunity can be transferred with splenocytes to naïve recipients. In other terms, chemotherapy based on conjugates of HPMA copolymer with doxorubicin possesses immunomodulating properties. This finding might also have wider implications for the management of relapsing tumors in human patients.
- MeSH
- akrylamidy chemická syntéza farmakologie MeSH
- antitumorózní látky chemická syntéza farmakologie MeSH
- doxorubicin analogy a deriváty chemická syntéza farmakologie MeSH
- experimentální nádory imunologie prevence a kontrola MeSH
- galaktosamin chemická syntéza farmakologie MeSH
- imunoterapie adoptivní * MeSH
- inhibiční koncentrace 50 MeSH
- injekce subkutánní MeSH
- koncentrace vodíkových iontů MeSH
- kyseliny polymethakrylové chemická syntéza farmakologie MeSH
- léky s prodlouženým účinkem MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- proliferace buněk účinky léků MeSH
- slezina cytologie imunologie transplantace MeSH
- transplantace nádorů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamidy MeSH
- antitumorózní látky MeSH
- doxorubicin-N-(2-hydroxypropyl)methacrylamide copolymer conjugate MeSH Prohlížeč
- doxorubicin MeSH
- galaktosamin MeSH
- HPMA coploymer-doxorubicin-galactosamine MeSH Prohlížeč
- kyseliny polymethakrylové MeSH
- léky s prodlouženým účinkem MeSH