T. Etrych

HPMA copolymers with pH-controlled release of doxorubicin: in vitro cytotoxicity and in vivo antitumor activity.

Data on the synthesis, physicochemical characterisation and in vitro and in vivo biological properties of the new, nontargeted or antibody-targeted polymer-doxorubicin conjugates designed as anticancer drugs are presented. In the conjugates, the anticancer drug doxorubicin (DOX) is attached to the polymer carrier via a simple hydrolytically labile spacer containing either a hydrazone bond or cis-aconitic acid residue. In vitro incubation of the conjugates in buffers led to a fast DOX release from the polymer at pH 5 (modelling intracellular environment) while at pH 7.4 (modelling blood) the conjugates are relatively stable. Cytotoxicity of the conjugates to T cell lymphoma EL4 depended on the detailed structure of the spacer and the method used for antibody attachment and was much higher compared with the effect of similar classic conjugates (DOX attached to the polymer via enzymatically degradable spacer). In both protective and therapeutic regimes of drug administration, the in vivo anti-tumor activity of the hydrazone conjugates containing only DOX was significantly enhanced (T cell lymphoma EL4, C57BL/10 mice) in comparison with free DOX or classic PK1, the PHPMA-DOX conjugate clinically tested at present. Increasing the molecular weight of the polymer carrier resulted in a more pronounced in vivo antitumor effect. Antibody-targeted conjugates with DOX bound via hydrazone bond exhibited even more extensive inhibition of the tumor growth with some long-term survivors. No survivors were observed after treatment of mice with free DOX or the nontargeted PHPMA-DOX conjugate.

Antibody-targeted Polymer–doxorubicin Conjugates with pH-controlled Activation

The paper is dealing with the synthesis and properties of new non-targeted or antibody-targeted polymer drug conjugates, bearing doxorubicin (DOX) attached via a spacer susceptible to pH-controlled hydrolysis (hydrazone conjugates), designed as anticancer drugs facilitating site-specific therapy. These conjugates are stable in a pH 7.4 buffer, modeling conditions during transport in the body, but release DOX and activate it inside target cells as a result of pH changes when going from outside to inside the cells. Conjugates containing an antibody directed against T lymphocytes bind effectively and specifically T cell lymphoma EL 4 cells. Cytotoxicity of the hydrazone conjugates is higher than that of classic conjugates, depending on the detailed structure of the polymer, the spacer between the drug and polymer carrier and method of antibody conjugation. Cytotoxicity of some of the conjugates is comparable even with that of the free drug. In both protective and therapeutic regimes of drug administration, the in vivo anti-tumor activity of the conjugates containing DOX was enhanced with long-term survivors (T-cell lymphoma EL 4, C57BL/6 mice) in comparison with much less effective free DOX or a classic P(N-(2-hydroxypropyl)methacrylamide)HPMA–DOX conjugate (already clinically tested).

New HPMA copolymer-based drug carriers with covalently bound hydrophobic substituents for solid tumour targeting

Various conjugates of anticancer drug doxorubicin (Dox) covalently bound by the hydrolytically degradable hydrazone bond to the drug carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesised. Structure of the conjugates differed in the type and the content of hydrophobic substituent (dodecyl, oleic acid and cholesterol moieties) introduced into the polymer structure. In aqueous solutions the conjugates self-assembled into high-molecular-weight supramolecular structures, such as polymeric micelles or stable hydrophilic nanoparticles 13-37 nm in diameter, depending on the type and the content of hydrophobic substituents. Treatment of mice bearing EL-4 T cell lymphoma with the conjugates in the therapeutic regime of drug administration (i.v.) resulted in significant tumour regression with up to 100% of long-term survivors, depending on the dose and the detailed structure of the carrier. The nanoparticles formed by the conjugate bearing cholesterol moiety exhibited prolonged blood circulation and enhanced tumour accumulation indicating an important role of the EPR effect in excellent anticancer activity of the conjugate.

HPMA copolymer-bound doxorubicin targeted to tumor-specific antigen of BCL1 mouse B cell leukemia.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin and targeted with B1 monoclonal antibody (mAb) to BCL1 leukemia cells was synthesised and tested in vitro and in vivo. BCL1 leukemia growing in syngenic Balb/c mice was selected as a tumor model system. B1 mAb recognising the idiotype of surface IgM on BCL1 cells was used as a targeting moiety. Both B1 mAb and doxorubicin were conjugated to HPMA copolymer carrier by aminolysis through a tetrapeptidic Gly-Phe(D,L)-Leu-Gly spacer to ensure the intracellular delivery and controlled release of the drug. B1 mAb-targeted conjugate was shown to possess strictly tumor-specific binding capacity to target BCL1 cells in vitro. A similar conjugate, but containing human nonspecific Ig (HuIg) instead of B1 mAb, failed to bind to BCL1 cells. In vitro, B1 mAb-targeted conjugate demonstrated 40-fold higher cytotoxic effect than nontargeted or human nonspecific Ig-containing HPMA copolymer-bound doxorubicin. Conjugate targeted with B1 mAb was also shown to bind to target BCL1 cells in vivo. B1 mAb-targeted conjugate was shown to be more efficient in the treatment of established BCL1 leukemia than free doxorubicin, nontargeted and human nonspecific Ig-containing conjugate. Antibody-targeted polymeric drugs are thus promising conjugates for cancer treatment.

Characterisation of the binding interaction between poly(L‐lysine) and DNA using the fluorescamine assay in the preparation of non‐viral gene delivery vectors

A major factor limiting the development of non‐viral gene delivery systems is the poor characterisation of polyelectrolyte complexes formed between cationic polymers and DNA. The present study uses the fluorescamine reagent to improve characterisation of poly(L‐lysine) (pLL)/DNA complexes post‐modified with a multivalent hydrophilic polymer by determining the availability of free amino groups. The results show that the fluorescamine reagent can be used to monitor the self‐assembly reaction between pLL and DNA and the degree of surface modification of the resultant complexes with a hydrophilic polymer. This experimental approach should enable the preparation of fully defined complexes whose properties can be better related to their biological activity.

Novel star HPMA-based polymer conjugates for passive targeting to solid tumors

Novel star polymer-doxorubicin conjugates designed for passive tumor targeting have been developed and their potential for treatment of cancer has been investigated. In the present study the synthesis, physico-chemical characterization, drug release, bio-distribution and preliminary data of in vivo efficacy of the conjugates are described. In the water-soluble conjugates the core of a molecule formed by poly(amido amine) (PAMAM) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing doxorubicin (Dox) attached by hydrazone bonds enabling intracellular pH-controlled hydrolytic drug release, or by GFLG sequence susceptible to enzymatic degradation. The controlled synthesis utilizing semitelechelic copolymer precursors facilitated preparation of polymer conjugates in a broad range of molecular weights (1.1–3.0·105 g/mol). In contrast to free drug or linear conjugates the star polymer-Dox conjugates exhibited prolonged blood circulation and enhanced tumor accumulation in tumor-bearing mice indicating important role of the EPR effect. The star polymer-Dox conjugates showed significantly higher anti-tumor activity in vivo than Dox·HCl or its linear or graft polymer conjugates, if treated with a single dose 15 or 5 mg Dox eq./kg. Method of tumor initialization (acute or chronic experimental tumor models) significantly influenced effectiveness of the treatment with much lower success in treatment of mice bearing chronic tumors.

Doxorubicin release is not a prerequisite for the in vitro cytotoxicity of HPMA-based pharmaceuticals: In vitro effect of extra drug-free GlyPheLeuGly sequences

A systematic study was designed to elucidate differences in cytostatic activity in vitro between HPMA-based doxorubicin conjugates synthesized using different polymerization techniques and differing in peptidyl side chain. A polymer-drug conjugate containing doxorubicin (DOX) bound to HPMA copolymer backbone through the enzymaticaly non-cleavable sequence GlyGly shows low but significant cytotoxicity in vitro in seven cancer cell lines of mouse (EL4, 38C13, 3T3, BCL1) and human (SW620, Raji, Jurkat) origin. The low cytotoxicity can be considerably increased by the presence of additional drug-free GlyPheLeuGly side chains. P1 conjugate, i.e. non-targeted HPMA copolymer bearing doxorubicin bound via a biodegradable GlyPheLeuGly sequence, synthesized by direct copolymerization of HPMA with monomeric doxorubicin and thus without additional drug-free GlyPheLeuGly sequences is less effective compared to PK1 synthesized by polymer analogous reaction and thus containing extra drug-free GlyPheLeuGly sequences. Significant activity-enhancing effect was not seen with other amino acid/oligopeptide sequences (e.g., Gly or GlyGly). The activity-enhancing effect of GlyPheLeuGly sequences is more obvious in the conjugate containing doxorubicin bound to HPMA through GlyGly sequence. Derivatization of the terminal carboxyl group of the extra GlyPheLeuGly side chains (amide, N-substituted amide, free carboxyl) does not significantly influence the cytotoxicity of the conjugates. The presence of the GlyPheLeuGly sequence in the conjugate structure increases its rate of intracellular accumulation. Normal cells (Balb/c splenocytes) accumulate less polymer-doxorubicin conjugate compared to cancer cells (T cell lymphoma EL4, B cell lymphoma Raji and T cell leukemia JURKAT).

High-molecular weight star conjugates containing docetaxel with high anti-tumor activity and low systemic toxicity in vivo

Here we present the polymer conjugates where the core formed by poly(amido amine) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing docetaxel (DTX) attached by a pH-sensitive hydrazone bond. DTX was derivatized with three different keto acids prior to attachment to the polymer carrier to introduce reactive keto groups into the drug. The therapeutic efficacy of such high-molecular-weight star conjugates is based on: (a) the enhanced permeability and retention (EPR) effect facilitating selective accumulation within solid tumors; (b) pH-controlled release of the drug, thus ensuring faster DTX release in the mildly acidic tumor microenvironment. The star DTX conjugate had a remarkably higher maximum tolerated dose in comparison with free DTX when administered as a single i.v. injection (∼160 mg kg−1vs. 40 mg kg−1 of DTX) in C57BL/6 mice. The star DTX conjugate showed significantly higher antitumor activity than free drug in the EL4 T cell lymphoma growing in syngeneic C57BL/6 mice even when given at the same dose (20 mg kg−1 of DTX eq.). Thus, the star DTX conjugates exert a much higher therapeutic activity and yet a lower systemic toxicity than free DTX.