Simultaneous Delivery of Doxorubicin and Protease Inhibitor Derivative to Solid Tumors via Star-Shaped Polymer Nanomedicines Overcomes P-gp- and STAT3-Mediated Chemoresistance.

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.

Bloodstream Stability Predetermines the Antitumor Efficacy of Micellar Polymer-Doxorubicin Drug Conjugates with pH-Triggered Drug Release.

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.

Evaluation of linear versus star-like polymer anti-cancer nanomedicines in mouse models.

Nanomedicines are considered next generation therapeutics with advanced therapeutic properties and reduced side effects. Herein, we introduce tailored linear and star-like water-soluble nanosystems as stimuli-sensitive nanomedicines for the treatment of solid tumors or hematological malignancies. The polymer carrier and drug pharmacokinetics were independently evaluated to elucidate the relationship between the nanosystem structure and its distribution in the body. Positron emission tomography and optical imaging demonstrated enhanced tumor accumulation of the polymer carriers in 4T1-bearing mice with increased tumor-to-blood and tumor-to-muscle ratios. Additionally, there was a significant accumulation of doxorubicin bound to various polymer carriers in EL4 tumors, as well as excellent in vivo therapeutic activity in EL4 lymphoma and moderate efficacy in 4T1 breast carcinoma. The linear nanomedicine showed at least comparable pharmacologic properties to the star-like nanomedicines regarding doxorubicin transport. Therefore, if multiple parameters are considered such as its optimized structure and simple and reproducible synthesis, this polymer carrier system is the most promising for further preclinical and clinical investigations.

Dimerization of an immunoactivating peptide derived from mycobacterial hsp65 using N-hydroxysuccinimide based bifunctional reagents is critical for its antitumor properties.

We have shown previously that a short pentapeptide derived from the mycobacterial heat shock protein hsp65 can be highly activating for the immune system based on its strong reactivity with the early activation antigen of lymphocytes CD69. Here, we investigated an optimal form of presentation of this antigen to the cells of the immune system. Four different forms of the dimerized heptapeptide LELTEGY, and of the control inactive dimerized heptapeptide LELLEGY that both contained an extra UV active glycine-tyrosine sequence, were prepared using dihydroxysuccinimidyl oxalate (DSO), dihydroxysuccinimidyl tartarate (DST), dihydroxysuccinimidyl glutarate (DSG), and dihydroxysuccinimidyl suberate (DSS), respectively. Heptapeptides dimerized through DST and DSG linkers had optimal activity in CD69 precipitation assay. Moreover, dimerization of active heptapeptide resulted in a remarkable increase in its proliferation activity and production of cytokines in vitro. Furthermore, while DST and DSG dimerized heptapeptides both significantly enhanced the cytotoxicity of natural killer cells in vitro, only the DSG dimerized compound was active in suppressing growth of melanoma tumors in mice and in enhancing the cytotoxic activity of tumor infiltrating lymphocytes ex vivo. Thus, while the dimerization of the immunoactive peptide caused a dramatic increase in its immunoactivating properties, its in vivo anticancer properties were influenced by the chemical nature of linker used for its dimerization.

SOT101 induces NK cell cytotoxicity and potentiates antibody-dependent cell cytotoxicity and anti-tumor activity.

SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.

Glycopolymers Decorated with 3-O-Substituted Thiodigalactosides as Potent Multivalent Inhibitors of Galectin-3.

Galectin-3 (Gal-3) participates in many cancer-related metabolic processes. The inhibition of overexpressed Gal-3 by, e.g., ß-galactoside-derived inhibitors is hence promising for cancer treatment. The multivalent presentation of such inhibitors on a suitable biocompatible carrier can enhance the overall affinity to Gal-3 and favorably modify the interaction with Gal-3-overexpressing cells. We synthesized a library of C-3 aryl-substituted thiodigalactoside inhibitors and their multivalent N-(2-hydroxypropyl)methacrylamide (HPMA)-based counterparts with two different glycomimetic contents. Glycopolymers with a higher content of glycomimetic exhibited a higher affinity to Gal-3 as assessed by ELISA and biolayer interferometry. Among them, four candidates (with 4-acetophenyl, 4-cyanophenyl, 4-fluorophenyl, and thiophen-3-yl substitution) were selected for further evaluation in cancer-related experiments in cell cultures. These glycopolymers inhibited Gal-3-induced processes in cancer cells. The cyanophenyl-substituted glycopolymer exhibited the strongest antiproliferative, antimigratory, antiangiogenic, and immunoprotective properties. The prepared glycopolymers appear to be prospective modulators of the tumor microenvironment applicable in the therapy of Gal-3-associated cancers.

Antitumor activity of IL-2/anti-IL-2 mAb immunocomplexes exerts synergism with that of N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugate due to its low immunosuppressive activity.

Interleukin (IL)-2 has been approved for treatment of metastatic renal cancer and malignant melanoma. However, its unfavorable pharmacologic properties, severe side effects and the negative role of IL-2 in maintaining T regulatory cells are severe drawbacks. It has been shown that immunocomplexes of IL-2 and certain anti-IL-2 mAbs possess selective and high stimulatory activity in vivo. Here, we show that IL-2/S4B6 mAb immunocomplexes expand not only CD122(high) subsets and newly activated CD8(+) T cells but also natural killer T cells and γδ T cells. Further, we demonstrate that natural killer (NK) cells expanded by IL-2/S4B6 mAb immunocomplexes in vivo have high cytolytic activity, which can be further increased by coadministration of IL-12. We also demonstrate that IL-2/S4B6 mAb immunocomplexes possess noticeable antitumor activity in two syngeneic mouse tumor models, namely BCL1 leukemia and B16F10 melanoma, but only if administered early in tumor progression. To effectively treat established tumors, we administered the tumor-bearing mice first with N-(2-hydroxypropyl)methacrylamide copolymer-bound doxorubicin conjugate, and subsequently with IL-2/S4B6 mAb immunocomplexes alone or with IL-12 to induce an efficient antitumor immune response. Importantly, we show that the conjugate has significantly lower immunosuppressive activity than free doxorubicin when using dosage with comparable antitumor activity, thus eliminating the majority of tumor cells while leaving the immune system mostly unimpaired for stimulation with IL-2/S4B6 mAb immunocomplexes. Indeed, we demonstrate that the conjugate and IL-2/S4B6 mAb immunocomplexes together have synergistic antitumor activity.

HPMA-Based Copolymers Carrying STAT3 Inhibitor Cucurbitacin-D as Stimulus-Sensitive Nanomedicines for Oncotherapy.

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.

Polymer-ritonavir derivate nanomedicine with pH-sensitive activation possesses potent anti-tumor activity in vivo via inhibition of proteasome and STAT3 signaling.

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.

HPMA copolymer conjugates of paclitaxel and docetaxel with pH-controlled drug release.

In this paper we describe the synthesis, physicochemical characteristics and data on the biological activity of polymer prodrugs based on the anticancer drugs paclitaxel (PTX) and docetaxel (DTX) conjugated with a water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer drug carrier. The drugs were derivatized and then attached to the polymer backbone via a spacer that is stable under physiological conditions (pH 7.4) and hydrolytically degradable in mild acidic environments (e.g., endosomes, pH approximately 5). Polymer-drug conjugates were designed to achieve prolonged blood circulation and release of the active compound in target cells. Six types of conjugates differing in the structure of the keto acid (levulic, 3-(acetyl)acrylic acid) and 4-(2-oxopropyl)benzoic acid-containing spacer or in the amount of drug bound to the HPMA copolymer were synthesized. In all the conjugates, the linkage susceptible to hydrolytic cleavage was formed by the reaction of the carbonyl group of a drug derivative with the hydrazide group-terminated side chains of the polymer. In vitro incubation of the conjugates in buffers resulted in much faster release of drugs or their derivatives from the polymer at pH 5 than at pH 7.4 with the rate depending on the detailed structure of the spacer. Conjugates containing drugs acylated with levulic acid were tested for their anticancer activity in vivo using two murine models. The PTX-containing conjugate showed better antitumor efficacy in the 4T1 model of mammary carcinoma than the parent drug and its derivative. The DTX-containing conjugate demonstrated high activity in treating EL4 T cell lymphoma. The treatment with the polymer conjugates was devoid of side toxicity. In both models, we achieved complete regression of established tumors accompanied by a durable tumor resistance in most of the cured animals.

Preclinical evaluation of linear HPMA-doxorubicin conjugates with pH-sensitive drug release: efficacy, safety, and immunomodulating activity in murine model.

PURPOSE: In vivo efficacy and safety of HPMA-based copolymers armed with doxorubicin via a spacer containing pH-sensitive linkage that can be prepared within a broad range of attached drug contents (1) was tested in murine tumor models. METHODS: Mice bearing T cell lymphoma EL4 or B cell lymphoma 38C13 were treated with a single dose of the conjugate (15, 25, and 75 mg Dox eq./kg i.v.) in a therapeutic regime. Anti-tumor resistance of the cured animals was proved by a second challenge with a lethal dose of tumor cells without additional treatment. RESULTS: The content of drug bound to the polymer is an important parameter in relation to the conjugate therapeutic efficacy. The best anti-tumor effects were produced by conjugates with 10 - 13 wt% of bound doxorubicin. Free doxorubicin up to 4.6% relative to total drug content had no impact on the treatment efficacy and acute toxicity. The conjugates induced a complete cure of mice and regular treatment-dependent development of specific anti-tumor resistance. No myelosuppression or organ damage was observed. CONCLUSIONS: A well-defined HPMA copolymer-doxorubicin conjugate with pH-sensitive drug release is a good candidate for clinical trials as it has remarkable anti-tumor efficacy and a favorable safety profile.

Polymer nanomedicines based on micelle-forming amphiphilic or water-soluble polymer-doxorubicin conjugates: Comparative study of in vitro and in vivo properties related to the polymer carrier structure, composition, and hydrodynamic properties.

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.

HPMA-based star polymer biomaterials with tuneable structure and biodegradability tailored for advanced drug delivery to solid tumours.

Design, controlled synthesis, physico-chemical and biological characteristics of novel well-defined biodegradable star-shaped copolymers intended for advanced drug delivery is described. These new biocompatible star copolymers were synthesised by grafting monodispersed semitelechelic linear (sL) N-(2-hydroxypropyl)methacrylamide copolymers onto a 2,2-bis(hydroxymethyl)propionic acid (bisMPA)-based polyester dendritic core of various structures. The hydrodynamic diameter of the star copolymer biomaterials can be tuned from 13 to 31 nm and could be adjusted to a given purpose by proper selection of the bisMPA dendritic core type and generation and by considering the sL copolymer molecular weight and polymer-to-core molar ratio. The hydrolytic degradation was proved for both the star copolymers containing either dendron or dendrimer core, showing the spontaneous hydrolysis in duration of few weeks. Finally, it was shown that the therapy with the biodegradable star conjugate with attached doxorubicin strongly suppresses the tumour growth in mice and is fully curative in most of the treated animals at dose corresponding approximately to one fourth of maximum tolerated dose (MTD) value. Both new biodegradable systems show superior efficacy and tumour accumulation over the first generation of star copolymers containing non-degradable PAMAM core.

Micelle-Forming Block Copolymers Tailored for Inhibition of P-gp-Mediated Multidrug Resistance: Structure to Activity Relationship.

Multidrug resistance (MDR) is often caused by the overexpression of efflux pumps, such as ABC transporters, in particular, P-glycoprotein (P-gp). Here, we investigate the di- and tri- block amphiphilic polymer systems based on polypropylene glycol (PPO) and copolymers of (N-(2-hydroxypropyl)methacrylamide) (PHPMA) as potential macromolecular inhibitors of P-gp, and concurrently, carriers of drugs, passively targeting solid tumors by the enhanced permeability and retention (EPR) effect. Interestingly, there were significant differences between the effects of di- and tri- block polymer-based micelles, with the former being significantly more thermodynamically stable and showing much higher P-gp inhibition ability. The presence of Boc-protected hydrazide groups or the Boc-deprotection method did not affect the physico-chemical or biological properties of the block copolymers. Moreover, diblock polymer micelles could be loaded with free PPO containing 5-40 wt % of free PPO, which showed increased P-gp inhibition in comparison to the unloaded micelles. Loaded polymer micelles containing more than 20 wt % free PPO showed a significant increase in toxicity; thus, loaded diblock polymer micelles containing 5-15 wt % free PPO are potential candidates for in vitro and in vivo application as potent MDR inhibitors and drug carriers.

Polymer donors of nitric oxide improve the treatment of experimental solid tumours with nanosized polymer therapeutics.

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.

The structure of polymer carriers controls the efficacy of the experimental combination treatment of tumors with HPMA copolymer conjugates carrying doxorubicin and docetaxel.

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.

Tumor-targeted micelle-forming block copolymers for overcoming of multidrug resistance.

New amphiphilic diblock polymer nanotherapeutics serving simultaneously as a drug delivery system and an inhibitor of multidrug resistance were designed, synthesized, and evaluated for their physico-chemical and biological characteristics. The amphiphilic character of the diblock polymer, containing a hydrophilic block based on the N-(2-hydroxypropyl)methacrylamide copolymer and a hydrophobic poly(propylene oxide) block (PPO), caused self-assembly into polymer micelles with an increased hydrodynamic radius (Rh of approximately 15nm) in aqueous solutions. Doxorubicin (Dox), as a cytostatic drug, was bound to the diblock polymer through a pH-sensitive hydrazone bond, enabling prolonged circulation in blood, the delivery of Dox into a solid tumor and the subsequent stimuli-sensitive controlled release within the tumor mass and tumor cells at a decreased pH. The applicability of micellar nanotherapeutics as drug carriers was confirmed by an in vivo evaluation using EL4 lymphoma-bearing C57BL/6 mice. We observed significantly higher accumulation of micellar conjugates in a solid tumor because of the EPR effect compared with similar polymer-drug conjugates that do not form micellar structures or with the parent free drug. In addition, highly increased anti-tumor efficacy of the micellar polymer nanotherapeutics, even at a sub-optimal dose, was observed. The presence of PPO in the structure of the diblock polymer ensured, during in vitro tests on human and mouse drug-sensitive and resistant cancer cell lines, the inhibition of P-glycoprotein, one of the most frequently expressed ATP-dependent efflux pump that causes multidrug resistance. In addition, we observed highly increased rate of the uptake of the diblock polymer nanotherapeutics within the cells. We suppose that combination of unique properties based on MDR inhibition, stimuli sensitiveness (pH sensitive activation of drug), improved pharmacokinetics and increased uptake into the cells made the described polymer micelle a good candidate for investigation as potential drug delivery system.

Properties of HPMA copolymer-doxorubicin conjugates with pH-controlled activation: effect of polymer chain modification.

Various conjugates of anticancer drug doxorubicin (DOX) covalently attached via hydrolytically degradable hydrazone bond to water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer drug carriers were synthesized. Three types of precursors containing either positively or negatively charged groups or a hydrophobic substituent were employed. In vitro incubation of the conjugates in buffers showed relative stability at pH 7.4 (modelling blood) and a fast DOX release at pH 5 (modelling intracellular environment). The presence of carboxylic groups in the copolymer structure resulted in an increase in the DOX release rate of 15-20% while no effect of the introduction of positively charged groups was observed if compared with the unmodified conjugate. Self-assembling of the oleoyl groups-containing conjugate led into formation of polymeric micelles with high apparent molecular weight (M(w)=170,000) in aqueous solution and resulted in a decrease in the DOX release rate of approximately 20%. The cytostatic activity of the conjugates tested on several cancer cell lines was comparable with that of free DOX.HCl, depending on the sensitivity of a particular cell line to DOX. All the conjugates showed a much higher antitumour activity in vivo than the free drug tested in mice bearing EL4 T-cell lymphoma and treated using the therapeutic regime of drug administration. The highest activity (100% long-term survivors) exhibited polymer-DOX conjugate containing negatively charged GFLG sequences.

The structure-dependent toxicity, pharmacokinetics and anti-tumour activity of HPMA copolymer conjugates in the treatment of solid tumours and leukaemia.

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.