The derivative of protease inhibitor ritonavir (5-methyl-4-oxohexanoic acid ritonavir ester; RD) was recently recognized as a potent P-gp inhibitor and cancerostatic drug inhibiting the proteasome and STAT3 signaling. Therefore, we designed high-molecular-weight HPMA copolymer conjugates with a PAMAM dendrimer core bearing both doxorubicin (Dox) and RD (Star-RD + Dox) to increase the circulation half-life to maximize simultaneous delivery of Dox and RD into the tumor. Star-RD inhibited P-gp activity, potently sensitizing both low- and high-P-gp-expressing cancer cells to the cytostatic and proapoptotic activity of Dox in vitro. Star-RD + Dox possessed higher cytostatic and proapoptotic activities compared to Star-Dox and the equivalent mixture of Star-Dox and Star-RD in vitro. Star-RD + Dox efficiently inhibited STAT3 signaling and induced caspase-3 activation and DNA fragmentation in cancer cells in vivo. Importantly, Star-RD + Dox was found to have superior antitumor activity in terms of tumor growth inhibition and increased survival of mice bearing P-gp-expressing tumors.

• MeSH
• chemorezistence MeSH
• cytostatické látky * MeSH
• doxorubicin farmakologie   MeSH
• inhibitory proteas farmakologie   MeSH
• myši MeSH
• nádory * MeSH
• nanomedicína MeSH
• polymery MeSH
• ritonavir MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytostatické látky * MeSH
• doxorubicin MeSH
• inhibitory proteas MeSH
• polymery MeSH
• ritonavir MeSH

Drug repurposing is a promising strategy for identifying new applications for approved drugs. Here, we describe a polymer biomaterial composed of the antiretroviral drug ritonavir derivative (5-methyl-4-oxohexanoic acid ritonavir ester; RD), covalently bound to HPMA copolymer carrier via a pH-sensitive hydrazone bond (P-RD). Apart from being more potent inhibitor of P-glycoprotein in comparison to ritonavir, we found RD to have considerable cytostatic activity in six mice (IC50 ~ 2.3-17.4 μM) and six human (IC50 ~ 4.3-8.7 μM) cancer cell lines, and that RD inhibits the migration and invasiveness of cancer cells in vitro. Importantly, RD inhibits STAT3 phosphorylation in CT26 cells in vitro and in vivo, and expression of the NF-κB p65 subunit, Bcl-2 and Mcl-1 in vitro. RD also dampens chymotrypsin-like and trypsin-like proteasome activity and induces ER stress as documented by induction of PERK phosphorylation and expression of ATF4 and CHOP. P-RD nanomedicine showed powerful antitumor activity in CT26 and B16F10 tumor-bearing mice, which, moreover, synergized with IL-2-based immunotherapy. P-RD proved very promising therapeutic activity also in human FaDu xenografts and negligible toxicity predetermining these nanomedicines as side-effect free nanosystem. The therapeutic potential could be highly increased using the fine-tuned combination with other drugs, i.e. doxorubicin, attached to the same polymer system. Finally, we summarize that described polymer nanomedicines fulfilled all the requirements as potential candidates for deep preclinical investigation.

• Klíčová slova
• Antitumor activity, Polymer carrier, Proteasome inhibition, Ritonavir derivate, STAT3 signaling inhibition, pH-controlled release,
• MeSH
• antitumorózní látky * MeSH
• doxorubicin MeSH
• koncentrace vodíkových iontů MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nanomedicína MeSH
• polymery * MeSH
• proteasomový endopeptidasový komplex MeSH
• ritonavir MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antitumorózní látky * MeSH
• doxorubicin MeSH
• polymery * MeSH
• proteasomový endopeptidasový komplex MeSH
• ritonavir MeSH

The study describes the synthesis, physicochemical properties, and biological evaluation of polymer therapeutics based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers intended for a tumor-targeted immuno-oncotherapy. Water-soluble linear and cholesterol-containing HPMA precursors were synthesized using controlled reversible addition-fragmentation chain transfer polymerization to reach molecular weight Mn about 2 × 104 g·mol-1 and low dispersity. These linear or self-assembled micellar conjugates, containing immunomodulatory agent cucurbitacin-D (CuD) or the anticancer drug doxorubicin (Dox) covalently bound by the hydrolytically degradable hydrazone bond, showed a hydrodynamic size of 10-30 nm in aqueous solutions. The CuD-containing conjugates were stable in conditions mimicking blood. Importantly, a massive release of active CuD in buffer mimicking the acidic tumor environment was observed. In vitro, both the linear (LP-CuD) and the micellar (MP-CuD) conjugates carrying CuD showed cytostatic/cytotoxic activity against several cancer cell lines. In a murine metastatic and difficult-to-treat 4T1 mammary carcinoma, only LP-CuD showed an anticancer effect. Indeed, the co-treatment with Dox-containing micellar polymer conjugate and LP-CuD showed potentiation of the anticancer effect. The results indicate that the binding of CuD, characterized by prominent hydrophobic nature and low bioavailability, to the polymer carrier allows a safe and effective delivery. Therefore, the conjugate could serve as a potential component of immuno-oncotherapy schemes within the next preclinical evaluation.

• Klíčová slova
• N-(2-hydroxypropyl) methacrylamide (HPMA), cucurbitacin-D, immuno-oncotherapy, pH-controlled release, signal transducer and activator of transcription 3 (STAT3) inhibition,
• Publikační typ
• časopisecké články MeSH

The study compared the physico-chemical and biological properties of a water-soluble star-like polymer nanomedicine with three micellar nanomedicines formed by self-assembly of amphiphilic copolymers differing in their hydrophobic part (statistical, block and thermosensitive block copolymers). All nanomedicines showed a pH-responsive release of the drug, independent on polymer structure. Significant penetration of all polymer nanomedicines into tumor cells in vitro was demonstrated, where the most pronounced effect was observed for statistical- or diblock copolymer-based micellar systems. Tumor accumulation in vivo was dependent on the stability of the nanomedicines in solution, being the highest for the star-like system, followed by the most stable micellar nanomedicines. The star-like polymer nanomedicine showed a superior therapeutic effect. Since the micellar systems exhibited slightly lower systemic toxicity, they may exhibit the same efficacy as the star-like soluble system when administered at equitoxic doses. In conclusion, treatment efficacy of studied nanomedicines was directly controlled by the drug pharmacokinetics, namely by their ability to circulate in the bloodstream for the time needed for effective accumulation in the tumor due to the enhanced permeability and retention (EPR) effect. Easy and scalable synthesis together with the direct reconstitution possibility for nanomedicine application made these nanomedicines excellent candidates for further clinical evaluation.

• Klíčová slova
• Anti-cancer therapy, Doxorubicin, Drug delivery, EPR effect, Polymer micelles, Star-like copolymers, pH-responsive release,
• MeSH
• doxorubicin * chemie   MeSH
• hydrodynamika MeSH
• micely * MeSH
• nanomedicína * MeSH
• nosiče léků MeSH
• polymery * MeSH
• voda MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• doxorubicin * MeSH
• micely * MeSH
• nosiče léků MeSH
• polymery * MeSH
• voda MeSH

Herein, the biodegradable micelle-forming amphiphilic N-(2-hydroxypropyl) methacrylamide (HPMA)-based polymer conjugates with the anticancer drug doxorubicin (Dox) designed for enhanced tumor accumulation were investigated, and the influence of their stability in the bloodstream on biodistribution, namely, tumor uptake, and in vivo antitumor efficacy were evaluated in detail. Dox was attached to the polymer carrier by a hydrazone bond enabling pH-controlled drug release. While the polymer-drug conjugates were stable in a buffer at pH 7.4 (mimicking bloodstream environment), Dox was released in a buffer under mild acidic conditions modeling the tumor microenvironment or cells. The amphiphilic polymer carriers differed in the structure of hydrophobic cholesterol derivative moieties bound to the HPMA copolymers via a hydrolyzable hydrazone bond, exhibiting different rates of micellar structure disintegration at various pH values. Considerable dependence of the studied polymer-drug conjugate biodistribution on the stability of the micellar structure was observed in neutral, bloodstream-mimicking, environment, showing that a faster rate of the micelle disintegration in pH 7.4 increased the conjugate blood clearance, decreased tumor accumulation, and significantly reduced the tumor:blood and tumor:muscle ratios. Similarly, the final therapeutic outcome was strongly affected by the stability of the micellar structure because the most stable micellar conjugate showed an almost similar therapeutic outcome as the water-soluble, nondegradable, high-molecular-weight starlike HPMA copolymer-Dox conjugate, which was highly efficient in the treatment of solid tumors in mice. Based on the results, we conclude that the bloodstream stability of micellar polymer-anticancer drug conjugates, in addition to their low side toxicity, is a crucial parameter for their efficient solid tumor accumulation and high in vivo antitumor activity.

• Klíčová slova
• HPMA copolymer, degradation, pH-controlled release, polymer micelles, solid tumor treatment,
• MeSH
• antitumorózní látky chemie   farmakokinetika   terapeutické užití   MeSH
• doxorubicin chemie   farmakokinetika   terapeutické užití   MeSH
• hydrofobní a hydrofilní interakce MeSH
• koncentrace vodíkových iontů MeSH
• lymfom krev   farmakoterapie   MeSH
• micely MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nosiče léků chemie   MeSH
• polymery chemie   MeSH
• uvolňování léčiv MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antitumorózní látky MeSH
• doxorubicin MeSH
• micely MeSH
• nosiče léků MeSH
• polymery MeSH

Polymer carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with incorporated organic nitrates as nitric oxide (NO) donors were designed with the aim to localise NO generation in solid tumours, thus highly increasing the enhanced permeability and retention (EPR) effect. The NO donors were coupled to the polymer carrier either through a stable bond or through a hydrolytically degradable, pH sensitive, bond. In vivo, the co-administration of the polymer NO donor and HPMA copolymer-bound cytotoxic drug (doxorubicin; Dox) resulted in an improvement in the treatment of murine EL4 T-cell lymphoma. The polymer NO donors neither potentiated the in vitro toxicity of the cytotoxic drug nor exerted any effect on in vivo model without the EPR effect, such as BCL1 leukaemia. Thus, an increase in passive accumulation of the nanomedicine carrying cytotoxic drug via NO-enhanced EPR effect was the operative mechanism of action. The most significant improvement in the therapy was observed in a combination treatment with such a polymer conjugate of Dox, which is characterised by increased circulation in the blood and efficient accumulation in solid tumours. Notably, the combination treatment enabled the development of an anti-tumour immune response, which was previously demonstrated as an important feature of HPMA-based polymer cytotoxic drugs.

• Klíčová slova
• Drug delivery, EL4 lymphoma, HPMA copolymers, anti-tumour immune response, enhanced EPR effect, polymer NO donor, polymer cytotoxic drugs, solid tumour treatment,
• MeSH
• antitumorózní látky aplikace a dávkování   metabolismus   MeSH
• donory oxidu dusnatého aplikace a dávkování   metabolismus   MeSH
• experimentální nádory farmakoterapie   metabolismus   patologie   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nanočástice aplikace a dávkování   metabolismus   MeSH
• nosiče léků aplikace a dávkování   metabolismus   MeSH
• oxid dusnatý metabolismus   MeSH
• polymery aplikace a dávkování   metabolismus   MeSH
• tumor burden účinky léků   fyziologie   MeSH
• výsledek terapie MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antitumorózní látky MeSH
• donory oxidu dusnatého MeSH
• nosiče léků MeSH
• oxid dusnatý MeSH
• polymery MeSH

The tumor-specific targeting of cancerostatics using polymer drug carriers represents a potential strategy to achieve an effective treatment with reduced side toxicity. Synthetic water-soluble copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) are carriers with tunable architecture and drug loading, tumor-specific accumulation of the drug, and its controlled release. We describe a combination treatment of murine EL4 T cell lymphoma with HPMA-based star conjugates (Mw 250,000gmol-1) of doxorubicin (Dox) or docetaxel (Dtx) designed for enhanced tumor accumulation and combination therapy. Although the combination of linear conjugates (Mw=28,000gmol-1) containing Dox or Dtx resulted in an additive effect in the treatment of the lymphoma, the opposite was observed in the combination of two star conjugates with Dox or Dtx, as the star Dtx conjugate decreased the treatment efficacy of the star Dox conjugate. The Dtx conjugate alone was virtually ineffective in the reduction of tumor growth or survival time extension; thus, a curative effect could be solely attributed to the Dox-containing conjugate. When Dtx was delivered to the tumor on the same polymer carrier as Dox, the efficacy of the Dox-induced treatment was reduced to a lesser extent. No reduction was found when Dtx was delivered by a linear polymer or applied as a free drug. The phenomenon was strictly related to the enhanced permeability and retention (EPR) effect, as it was not observed in BCL1 leukemia, a model without EPR. The diminished treatment outcome in the combination therapy with the two star conjugates was underlined by the significantly decreased accumulation of Dox in the tumor. The use of the drug-free polymer carrier instead of the Dtx-containing star conjugate did not reduce the treatment efficacy of the Dox conjugate. Thus, the physicochemical characteristics of the polymer carrier designed for tumor-specific drug delivery systems control the activity of the respective drug, leading to changes within the tumor microenvironment that can determine ultimate efficacy of the combination therapy.

• Klíčová slova
• Docetaxel, Doxorubicin, Drug delivery, EPR effect, HPMA, Tumor microenvironment,
• MeSH
• akrylamidy chemie   MeSH
• antitumorózní látky aplikace a dávkování   chemie   farmakokinetika   terapeutické užití   MeSH
• docetaxel MeSH
• doxorubicin aplikace a dávkování   chemie   farmakokinetika   terapeutické užití   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory farmakoterapie   patologie   MeSH
• nosiče léků chemie   MeSH
• taxoidy aplikace a dávkování   chemie   farmakokinetika   terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• akrylamidy MeSH
• antitumorózní látky MeSH
• docetaxel MeSH
• doxorubicin MeSH
• N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
• nosiče léků MeSH
• taxoidy MeSH

Multidrug resistance (MDR) is a common cause of failure in chemotherapy for malignant diseases. MDR is either acquired as a result of previous repeated exposure to cytostatic drugs (P388/MDR cells) or naturally, as some tumors are congenitally resistant to chemotherapy (CT26 cells). One of the most common mechanisms of MDR is upregulation of P-glycoprotein (P-gp) expression. Here, we used HPMA copolymer conjugates, whereby the cytostatic drug doxorubicin (Dox) or the derivative of the P-gp inhibitor reversin 121 (R121) or both were covalently bound through a degradable pH-sensitive hydrazone bond. We proved that R121, when bound to a polymeric carrier, is capable of inhibiting P-gp in P388/MDR cells and sensitizing them in relation to the cytostatic activity of Dox. Conjugate bearing both Dox and R121 was found to be far more potent in P388/MDR cells than conjugate bearing Dox alone or a mixture of conjugates bearing either Dox or R121 when cytostatic activity in vitro, cell cycle arrest, accumulation of Dox in cells and induction of apoptosis were determined. Importantly, conjugate bearing R121 is also effective in vivo as it inhibits P-gp in P388/MDR tumors after intraperitoneal administration, while both the conjugate bearing Dox and R121 induces apoptosis in P388/MDR tumors more effectively than conjugate bearing Dox alone. Only conjugate bearing Dox and R121 significantly inhibited P388/MDR tumor growth and led to the prolonged survival of treated mice. However, the most dramatic antitumor activity of this conjugate was found in the CT26 tumor model where it completely cured six out of eight experimental mice, while conjugate bearing Dox alone cured no mice.

• Klíčová slova
• Doxorubicin, HPMA copolymer carrier, Multidrug resistance, P-glycoprotein, Polymer-drug conjugate, Reversin 121,
• MeSH
• chemorezistence MeSH
• cytostatické látky aplikace a dávkování   MeSH
• doxorubicin aplikace a dávkování   MeSH
• experimentální nádory farmakoterapie   patologie   MeSH
• inbrední kmeny myší MeSH
• methakryláty chemie   MeSH
• mnohočetná léková rezistence MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nanokapsle aplikace a dávkování   chemie   MeSH
• nanokonjugáty aplikace a dávkování   chemie   MeSH
• oligopeptidy aplikace a dávkování   MeSH
• P-glykoprotein antagonisté a inhibitory   MeSH
• protokoly antitumorózní kombinované chemoterapie aplikace a dávkování   MeSH
• výsledek terapie MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytostatické látky MeSH
• doxorubicin MeSH
• hydroxypropyl methacrylate MeSH Prohlížeč
• methakryláty MeSH
• nanokapsle MeSH
• nanokonjugáty MeSH
• oligopeptidy MeSH
• P-glykoprotein MeSH
• reversin 121 MeSH Prohlížeč

Polymer drug carriers that are based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been widely used in the development and synthesis of high-molecular-weight (HMW) drug delivery systems for cancer therapy. In this study, we compared linear (Mw ~27kDa, Rh ~4nm) and non-degradable star (Mw ~250kDa, Rh ~13nm) HPMA copolymer conjugates bearing anthracycline antibiotic doxorubicin (DOX) bound via pH-sensitive hydrazone bond. We determined the in vitro and in vivo toxicity of both conjugates and their maximum tolerated dose (MTD). We also compared their anti-tumour activity in mouse B-cell leukaemia (BCL1) and a mouse T-cell lymphoma (EL4) model. We found that MTD was higher for the linear conjugate (85mgDOX/kg) and lower for the star conjugate (22.5mgDOX/kg). An evaluation of the intestinal barrier integrity using FITC-dextran as a gut permeability tracer proved that no pathology was caused by the MTD of either conjugate. However, free DOX showed some damage to the gut barrier. The therapy of BCL1 leukaemia by both of the polymeric conjugates using the MTD or its fraction (i.e., equitoxic dosage) showed better results in the case of the star conjugate. On the other hand, treatment of EL4 lymphoma seemed to be more efficient when the linear conjugate was used. We suppose that the anti-cancer treatment of solid tumours and leukaemias requires different types of drug conjugates. We hypothesise that the most suitable HPMA copolymer-DOX conjugate for the treatment of solid tumours should have an HMW structure with increased Rh that would be stable for three to four days after the conjugate administration and then rapidly disintegrate in the short polymer chains, which are excretable from the body by glomerular filtration. On the other hand, the treatment of leukaemia requires a drug conjugate with a long circulation half-life. This would provide an active drug, whilst slowly degrading to excretable fragments.

• Klíčová slova
• Anti-tumour activity, Doxorubicin, HPMA, Structure, Toxicity,
• MeSH
• akrylamidy chemie   farmakokinetika   terapeutické užití   toxicita   MeSH
• antibiotika antitumorózní * chemie   farmakokinetika   terapeutické užití   toxicita   MeSH
• dendrimery chemie   farmakokinetika   terapeutické užití   toxicita   MeSH
• doxorubicin chemie   farmakokinetika   terapeutické užití   toxicita   MeSH
• játra účinky léků   patologie   MeSH
• kostní dřeň účinky léků   patologie   MeSH
• maximální tolerovaná dávka MeSH
• molekulová hmotnost MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory krev   farmakoterapie   metabolismus   MeSH
• nosiče léků * chemie   farmakokinetika   terapeutické užití   toxicita   MeSH
• slezina účinky léků   patologie   MeSH
• střevní sliznice metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akrylamidy MeSH
• antibiotika antitumorózní * MeSH
• dendrimery MeSH
• doxorubicin MeSH
• N-(2-hydroxypropyl)methacrylamide co-polymer-doxorubicin conjugate MeSH Prohlížeč
• nosiče léků * MeSH
• PAMAM Starburst MeSH Prohlížeč

• Publikační typ
• časopisecké články MeSH