The effective treatment of inflammatory diseases, particularly their chronic forms, is a key task of modern medicine. Herein, we report the synthesis and evaluation of biocompatible polymer conjugates based on N-2-(hydroxypropyl)methacrylamide copolymers enabling the controlled release of acetylsalicylic acid (ASA)-based anti-inflammatory drugs under specific stimuli. All polymer nanotherapeutics were proposed as water-soluble drug delivery systems with a hydrodynamic size below 10 nm ensuring suitability for the parenteral application and preventing opsonization by the reticuloendothelial system. The nanotherapeutics bearing an ester-bound ASA exhibited long-term release of the ASA/salicylic acid mixture, while the nanotherapeutics carrying salicylic acid hydrazide (SAH) ensured the selective release of SAH in the acidic inflammatory environment thanks to the pH-sensitive hydrazone bond between the polymer carrier and SAH. The ASA- and SAH-containing nanotherapeutics inhibited both cyclooxygenase isoforms and/or the production of pro-inflammatory mediators. Thanks to their favorable design, they can preferentially accumulate in the inflamed tissue, resulting in reduced side effects and lower dosage, and thus more effective and safer treatment.
- MeSH
- akrylamidy chemie farmakologie aplikace a dávkování MeSH
- antiflogistika farmakologie aplikace a dávkování chemie MeSH
- Aspirin * aplikace a dávkování farmakologie chemie MeSH
- cyklooxygenasy metabolismus MeSH
- inhibitory cyklooxygenasy farmakologie aplikace a dávkování chemie MeSH
- léky s prodlouženým účinkem * MeSH
- mediátory zánětu metabolismus MeSH
- myši MeSH
- nanočástice * chemie MeSH
- nosiče léků chemie MeSH
- polymery * chemie aplikace a dávkování MeSH
- uvolňování léčiv MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Klíčová slova
- osimertinib,
- MeSH
- akrylamidy farmakologie terapeutické užití MeSH
- analýza přežití MeSH
- aniliny farmakologie terapeutické užití MeSH
- inhibitory proteinkinas farmakologie terapeutické užití MeSH
- lidé MeSH
- nemalobuněčný karcinom plic * farmakoterapie MeSH
- protinádorové látky farmakologie terapeutické užití MeSH
- protokoly protinádorové léčby MeSH
- randomizované kontrolované studie jako téma MeSH
- Check Tag
- lidé MeSH
Real-time surveillance of photodynamic therapy (PDT) has been desired by the research community for a long time. The impact of the treatment is encoded in the phosphorescence kinetics of its main mediator: singlet oxygen. We report successful in vivo measurements of these weak kinetics through the skin of living mice after systemic drug application. Using special high transmission optics centered around 1200, 1270 and 1340 nm, singlet oxygen phosphorescence can be clearly discriminated from other signals. N-(2-Hydroxypropyl)methacrylamide copolymers conjugated with pyropheophorbide-a exhibit highly selective accumulation in tumors. Signals of this drug in tumors were compared to those in normal tissue. In both places, the major part of the signal could be identified as arising from drug still circulating in the bloodstream. Despite high concentrations of extravasated drug in the tumors due to the EPR effect, nearly no signal could be detected from these photosensitizers in vivo, contradicting in vitro experiments. We propose that the reason for this discrepancy is oxygen depletion in tumor tissue in vivo, even at moderate (at PDT scale) illumination intensities, soon after the start of the illumination. These results underline the importance of singlet oxygen surveillance during PDT treatment.
- MeSH
- akrylamidy chemie farmakologie MeSH
- fotochemoterapie * MeSH
- fotosenzibilizující látky chemie farmakologie MeSH
- hypoxie * MeSH
- kinetika MeSH
- luminiscence MeSH
- molekulární struktura MeSH
- myši MeSH
- nádory * farmakoterapie metabolismus MeSH
- protinádorové látky chemie farmakologie MeSH
- singletový kyslík * analýza metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and regulation of NAMPT's nuclear transport are not known. Here, we constructed a GFP-NAMPT fusion protein to study NAMPT's subcellular trafficking. We observed that in unsynchronized 3T3-L1 preadipocytes, 25% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 62% had higher GFP-NAMPT fluorescence in the nucleus. In HepG2 hepatocytes, 6% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 84% had higher GFP-NAMPT fluorescence in the nucleus. In both 3T3-L1 and HepG2 cells, GFP-NAMPT was excluded from the nucleus immediately after mitosis and migrated back into it as the cell cycle progressed. In HepG2 cells, endogenous, untagged NAMPT displayed similar changes with the cell cycle, and in nonmitotic cells, GFP-NAMPT accumulated in the nucleus. Similarly, genotoxic, oxidative, or dicarbonyl stress also caused nuclear NAMPT localization. These interventions also increased poly(ADP-ribosyl) polymerase and sirtuin activity, suggesting an increased cellular demand for NAD. We identified a nuclear localization signal in NAMPT and amino acid substitution in this sequence (424RSKK to ASGA), which did not affect its enzymatic activity, blocked nuclear NAMPT transport, slowed cell growth, and increased histone H3 acetylation. These results suggest that NAMPT is transported into the nucleus where it presumably increases NAD synthesis required for cell proliferation. We conclude that specific inhibition of NAMPT transport into the nucleus might be a potential avenue for managing cancer.
- MeSH
- akrylamidy farmakologie MeSH
- aktivní transport - buněčné jádro MeSH
- buněčné jádro metabolismus MeSH
- buňky 3T3-L1 MeSH
- buňky Hep G2 MeSH
- cytoplazma metabolismus MeSH
- histony metabolismus MeSH
- kontrolní body buněčného cyklu MeSH
- lidé MeSH
- mutageneze cílená MeSH
- myši MeSH
- NAD metabolismus MeSH
- nikotinamidfosforibosyltransferasa chemie genetika metabolismus MeSH
- oxidační stres MeSH
- piperidiny farmakologie MeSH
- poly(ADP-ribosa)polymerasy metabolismus MeSH
- proliferace buněk MeSH
- rekombinantní fúzní proteiny chemie genetika metabolismus MeSH
- sirtuiny metabolismus MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Polymer prodrugs can considerably improve the treatment of tumors with multidrug resistance, often caused by overexpression of P-glycoprotein (P-gp). Here, we present the effect of the N-(2-hydroxypropyl) methacrylamide-based polymer conjugate with P-gp inhibitor ritonavir (RIT) on the increase of free doxorubicin (DOX) and polymer-bound DOX cytotoxicity in the human neuroblastoma 4 cell line and its resistant clones to different cytostatics. The increase in cytotoxicity after polymer-RIT conjugate pretreatment was higher for the lines overexpressing P-gp and less pronounced for those with decreased P-gp levels. Moreover, the effect of polymer conjugate containing inhibitor and DOX on the same polymer chain was lower than that of two individual polymer conjugates used sequentially. In conclusion, the polymer-RIT conjugate can significantly increase the cytotoxicity of free DOX and polymer-DOX conjugates in cells with various multidrug resistance origins and can thus be considered a suitable therapeutic enhancer of polymer prodrugs.
- MeSH
- akrylamidy aplikace a dávkování farmakologie MeSH
- chemorezistence MeSH
- doxorubicin aplikace a dávkování farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- neuroblastom farmakoterapie metabolismus MeSH
- P-glykoprotein antagonisté a inhibitory biosyntéza MeSH
- protokoly protinádorové kombinované chemoterapie farmakologie MeSH
- ritonavir aplikace a dávkování farmakologie MeSH
- screeningové testy protinádorových léčiv MeSH
- synergismus léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The enzymatic release of a model drug from a polymer carrier inside a tumor using multispectral optical imaging in vivo in nude mice bearing colorectal carcinomas HT-29 and DLD-1 is demonstrated. Much higher release rate in vivo from a linear (30 kDa) (N-2-hydroxypropyl)methacrylamide-based polymer compared with a high molecular weight branched (170 kDa) polymer conjugate is observed, probably due to steric hindrance of the cleavable spacer of the latter polymer to proteolytic enzymes. There is no significant difference in the relative biodistribution of the two polymers, but the branched polymer circulates much longer. Both polymers are labeled with two different fluorophores. Dyomics-676 as a drug model is attached to the polymer via an enzymatically cleavable Gly-Phe-Leu-Gly spacer; Dyomics 782 is bound to the same polymer via a nondegradable amide bond, enabling the tracking of the polymer carrier after i.v. application to mice.
- MeSH
- akrylamidy * chemie farmakokinetika farmakologie MeSH
- fluorescenční barviva * chemie farmakokinetika farmakologie MeSH
- kolorektální nádory * farmakoterapie metabolismus patologie MeSH
- lidé MeSH
- myši nahé MeSH
- myši MeSH
- nosiče léků * chemie farmakokinetika farmakologie MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- 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
- protinádorová antibiotika chemie farmakologie MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
gamma-Fe2O3 nanoparticles obtained by coprecipitation of Fe(II) and Fe(III) chlorides with a base and subsequent oxidation were coated with a shell of hydrophilic biocompatible poly(N,N-dimethylacrylamide) (PDMAAm). Various initiators were attached to the iron oxide surface to enable the use of the "grafting-from" approach for immobilization of PDMAAm. They included 2,2'-azobis(2-methylpropanimidamide) dihydrochloride (AMPA), 2,2'-azobis(N-hydroxy-2-methylpropanimidamide) dihydrochloride (ABHA) and 4-cyano-4-{[1-cyano-3-(N-hydroxycarbamoyl)-1-methylpropyl]azo}pentanoic acid (CCHPA). Engulfment of PDMAAm-coated y-Fe2O3 nanoparticles by murine J774.2 macrophages was investigated. Only some nanoparticles were engulfed by the macrophages. PDMAAm-AMPA-gamma-Fe2O3 and PDMAAm-ABHA-y-Fe2O3 nanoparticles were rapidly engulfed by the cells. In contrast, neat y-Fe2O3 and PDMAAm-CCHPA-gamma-Fe2O3 particles induced formation of transparent vacuoles indicating toxicity of the particles. Thus, PDMAAm-coated AMPA- and ABHA-gamma-Fe2O3 nanoparticles can be recommended as non-toxic labels for mammalian cells.
- MeSH
- akrylamidy chemie farmakologie MeSH
- buněčné linie MeSH
- fluorescenční mikroskopie MeSH
- hydrofobní a hydrofilní interakce účinky léků MeSH
- magnetické jevy MeSH
- magnety * MeSH
- makrofágy cytologie účinky léků metabolismus MeSH
- myši MeSH
- nanočástice chemie ultrastruktura MeSH
- polymerizace účinky léků MeSH
- radiační rozptyl MeSH
- savci metabolismus MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- světlo MeSH
- velikost částic MeSH
- železité sloučeniny farmakologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Interaction of nine forms of human hepatic cytochromes P450 (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) with two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based doxorubicin (DOX) conjugates designed for passive tumor targeting was studied using pooled human microsomes. The compounds used in this study were two high-molecular-weight HPMA copolymers bearing doxorubicin attached to the polymeric carrier by 1) hydrazone bond enabling intracellular pH-controlled drug release or 2) amide bond through enzymatically cleavable tetrapeptide GlyPheLeuGly spacer. Both polymeric conjugates differing in mechanism of their antitumor activity and the free doxorubicin as the control were tested for potential inhibition activity. Among nine cytochrome P450 forms studied, no HPMA copolymer with bound DOX caused an inhibition of potential clinical significance. The extent of inhibition of enzymatic activities of the cytochrome P450 forms studied was negligible with the exception of CYP2B6 and was apparently caused by DOX as no inhibition was observed with polymers alone, and the extent of inhibition by the complex corresponded to this of the free DOX at the same concentration. In conclusion, the polymers and their conjugates with DOX seem to be relatively safe, at least in this respect, i.e., of inhibition of the liver microsomal drug-metabolizing enzymes.
- MeSH
- akrylamidy chemie farmakologie MeSH
- amidy chemie MeSH
- doxorubicin analogy a deriváty farmakologie MeSH
- hydrazony chemie MeSH
- inhibitory cytochromu P450 MeSH
- jaterní mikrozomy účinky léků enzymologie metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- léky s prodlouženým účinkem MeSH
- lidé MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A set of single-tryptophan mutants of the Na(+)/K(+)-ATPase isolated, large cytoplasmic loop connecting transmembrane helices M4 and M5 (C45) was prepared to monitor effects of the natural cytoplasmic ligands (i.e., Mg(2+) and/or ATP) binding. We introduced a novel method for the monitoring of the changes in the electrostatic surface potential (ESP) induced by ligand binding, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. This approach opens a new way to understanding the interactions within the proteins. Our experiments revealed that the C45 conformation in the presence of the ATP (without magnesium) substantially differed from the conformation in the presence of Mg(2+) or MgATP or in the absence of any ligand not only in the sense of geometry but also in the sense of the ESP. Notably, the set of ESP-sensitive residues was different from the set of geometry-sensitive residues. Moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. This property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain.
- MeSH
- adenosintrifosfát farmakologie metabolismus MeSH
- akrylamidy farmakologie metabolismus MeSH
- cytoplazma metabolismus MeSH
- fluorescence MeSH
- hořčík farmakologie metabolismus MeSH
- jodidy farmakologie metabolismus MeSH
- konformace proteinů účinky léků MeSH
- ligandy MeSH
- molekulární modely MeSH
- mutace MeSH
- myši MeSH
- povrchové vlastnosti MeSH
- sodíko-draslíková ATPasa genetika chemie metabolismus MeSH
- statická elektřina MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
