Marek Kovář

Doxorubicin bound to a HPMA copolymer carrier through hydrazone bond is effective also in a cancer cell line with a limited content of lysosomes

We have synthesized conjugates containing doxorubicin (DOX) bound to oligopeptide side chains (GlyGly or GlyPheLeuGly) of a water-soluble copolymer carrier based on poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) either through proteolytically (PK1 conjugates) [Synthetic polymeric drugs. U.S. Patent 5,037,883 (1991)] or hydrolytically cleavable bond (HC conjugates). Pharmacological efficacy of PK1 and HC conjugates was compared in vitro on murine: T-cell lymphoma EL4, B-cell leukemia BCL1, B-cell lymphoma 38C13, leukemia P388 and Con A-stimulated A/Ph splenocytes and on human: primary (SW480) and metastatic (SW620) colorectal cancer cell lines parent and transfected with Thy 1.2 gene [2] and on erythromyeloid leukemia cell line K 562. Inhibition of proliferation determined by 3[H]-thymidine incorporation revealed that the cytostatic effect of HC conjugates is up to two orders of magnitude higher compared to PK1 conjugates. In some cancer cell lines (SW 620/T, SW 480) the pharmacological activity of HC conjugates is in vitro comparable with the activity of the free drug. Unlike PK1 conjugates, HC conjugates with a lysosomally degradable spacer (GlyPheLeuGly) are less effective compared to HC conjugates containing lysosomally non-degradable spacer (GlyGly). Moreover, HC conjugates exert pronounced anti-proliferative activity also in erythroblastoid leukemia cell line K 562 with a limited content of lysosomes.

Cytostatic and immunomobilizing activities of polymer-bound drugs: experimental and first clinical data

An N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing doxorubicin and human immunoglobulin as an actively/passively targeting moiety was used in four patients with generalized breast cancer resistant to standard cytotoxic chemotherapy. The dose and time schedule were deduced from a Phase I clinical trial in which doxorubicin bound to HPMA copolymer carrier (PK1) was tested. It was confirmed that the Dox-HPMA-HuIg conjugate is stable and doxorubicin remains in the peripheral blood with a small amount also in the urine, mostly in its polymer-bound form. More than 116 biochemical, immunological and hematological parameters were determined for blood samples taken from patients 24 h, 48 h, 72 h and 1 to 11 weeks after treatment. Depending on the patient, some parameters decreased permanently or temporarily to the normal level (CRP, C3, CA 72-4, beta(2)-microglobulin, ferritin, CEA, CA 125, CD4, CD8, CE19, CD16(+)56(+), leu, ery) and some moved markedly towards physiological values (AST, ALT, ALP, GMT, CA 15-3, NSE, AFP). While the number of peripheral blood reticulocytes was significantly decreased after treatment with the classical free drug, their number was not affected or was even elevated after treatment with Dox-HPMA-HuIg. Increased absolute numbers of CD16(+)56(+) and CD4(+) cells in the peripheral blood and activation of NK and LAK cells in all patients support data obtained in experimental animals, pointing to a dual, i.e. cytostatic and immunomobilizing character of Dox-HPMA conjugates containing a targeting immunoglobulin moiety.

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.

Acquired and specific immunological mechanisms co-responsible for efficacy of polymer-bound drugs

We present data providing new evidence that poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA)-bound drugs, unlike free drugs, have both cytostatic and immunomobilizing activity (CIA). Immediately after injection, due to the high level of the drug, the main activity of the polymeric conjugate is cytotoxic and cytostatic. Later on, long-term circulating PHPMA-bound drug, at concentrations lower than its minimal inhibitory levels, mobilizes the defense mechanisms of the host. Cytotoxic and cytostatic effects of drug-PHPMA were repeatedly confirmed. The following data support the concept of the immunomobilizing activity of the N-(2-hydroxypropyl)methacrylamide (HPMA) conjugates: (a) pre-treatment with free drugs (doxorubicin, cyclosporin A) accelerates the appearance of EL4 mouse T-cell lymphoma while a similar pre-treatment with doxorubicin-PHPMA induces limited but definitive mobilization of the hosts defense mechanisms; (b) mice cured of EL4 mouse T-cell lymphoma, BCL1 mouse B-cell leukemia and 38C13 mouse B-cell lymphoma by injection of doxorubicin-PHPMA conjugate targeted with monoclonal antibodies (anti-Thy 1.2 for EL4, anti-B1 for BCL1 and anti-CD71 for 38C13) and re-transplanted with a lethal dose of the same cancer cells survive without any treatment considerably longer than control mice; (c) increased NK activity and anti-cancer antibody was detected only in animals treated with doxorubicin-PHPMA conjugate; and (d) considerably increased NK and LAK activity was seen in a human patient treated for generalized breast carcinoma with doxorubicin-PHPMA-IgG.

HPMA-hydrogels containing cytostatic drugs: Kinetics of the drug release and in vivo efficacy

This study proposes a strategy to generate new anticancer therapy using hydrogel-based drug delivery systems to improve drug bioavailability and increase the therapeutic efficacy. We have synthesized biodegradable hydrogels based on N-(2-hydroxypropyl)methacrylamide (HPMA) with prolonged drug release. Pharmacokinetic data from in vitro studies showed that the in vitro release of hydrophilic drugs (doxorubicin, vinblastine) from HPMA-hydrogels is affected mainly by drug diffusion and only partially by hydrogel degradation. The release of hydrophobic drugs (cyclosporine A, CsA) actually copies the process of degradation and therefore it is slower. Hydrogels with degradation time of 50 h released the doxorubicin over a period of at least 96 h after s.c. implantation. Drug concentration at pharmacologically active levels was maintained in the bloodstream over a period of at least 4 days, ranging between 0.1 and 1 microg/ml. The therapeutic potential of HPMA-hydrogels in vivo was studied in Bcl1 leukemia. HPMA-hydrogels containing DOX were significantly more effective in inhibition of Bcl1 leukemia in comparison with free DOX or non-targeted polymeric drug (PK1). The efficacy of therapeutic combination using unspecific, hydrogel-based therapy with specific, antibody-targeted therapy at late stages of Bcl1 leukemia was also tested. In contrast to application of DOX alone, a cocktail of DOX with CsA as a blocker of P-glycoprotein (Pgp) incorporated into HPMA-hydrogel blocked the proliferation of Pgp-overexpressing multidrug resistant cell lines in vitro by induction of apoptosis.

Immunoprotective therapy with targeted anticancer drugs

Doxorubicin or mitomycin C bonded to poly[N 5 -(2-hydroxyethyl)-L-glutamine]-graft-poly(ethylene glycol) or poly[N-(2-hydroxypropyl)methacryl-amide], non-targeted or targeted with monoclonal antibodies, do not induce expression of FasL on selected cancer cells (human colorectal cancer cell line SW 620) thus protecting effector cells of the immune system against Fas-counterattack. The pre-treatment with PHPMA-bound DOX does not only protect but in fact mobilizes defense mechanisms of the tumor-bearing hosts. The treatment with selected monoclonal antibody-targeted PHPMA-bound DOX causes a rapid and complete rejection of established tumors (mouse B cell leukemia BCL1, mouse B cell lymphoma 38C13 and mouse T cell lymphoma EL4) and generates prolonged systemic anti-tumor immunity.

Cytotoxicity and immunostimulation: double attack on cancer cells with polymeric therapeutics.

The successful treatment of cancer with conventional drugs is frequently complicated by the resistance of tumor cells to such a non-specific therapy. Over the last few years, immunotherapy has gained attention as a tumor-specific approach. Recent findings demonstrated that some conventional cytostatics stimulate local anticancer responses. New anticancer drugs, including their polymeric derivatives, are currently being developed with the aim of destroying tumors more effectively and more specifically. Among these, the water-soluble conjugates of doxorubicin with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carrier have emerged as efficient therapeutics because they are able to not only directly destroy cancer cells but also elicit systemic tumor-specific anticancer responses. Here, we discuss new insights into their mechanisms of immune surveillance, which could suggest novel approaches to cancer therapy.

Drug-HPMA-HuIg Conjugates Effective Against Human Solid Cancer

N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA)-bound doxorubicin conjugated with human Ig as a targeting moiety was used for the first time in the setting of metastatic breast cancer (patients E. G., J. K., K. R. and K.H.) and angiosarcoma (patient D. H) resistant to conventional cytotoxic chemotherapy. It was confirmed that Dox-PHPMA-Hulg conjugate is stable and doxorubicin remains in the peripheral blood mostly in its polymer-bound form. In patients E. G., J. K., K.R. and K.H. more than 116 biochemical and immunological parameters were tested in blood samples taken from the patients 24 h, 48 h, 72 h, 7 d, 14 d and 21, 28 and eight weeks after the treatment. In the patient E. G., 15 parameters had pathological values before the treatment. During the treatment, seven parameters dropped permanently or temporarily to a normal level and seven moved markedly towards the physiological value. While the number of peripheral blood reticulocytes was significantly decreased after the treatment with free doxorubicin, their number was stable or elevated after the treatment with Dox-PHPMA-Hulg conjugate. Increased absolute number of CD16+56+ and CD4+ cells in the peripheral blood and activation of NK and LAX cells in a human patients support the data previously obtained in experimental animals pointing to a dual role, i.e. the cytotoxic and immunomobilizing character of doxorubicin-PHPMA conjugates containing a targeting immunoglobulin moiety.

Polymer therapeutics with a coiled coil motif targeted against murine bcl1 leukemia.

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.

HPMA Copolymer Conjugates of DOX and Mitomycin C for Combination Therapy: Physicochemical Characterization, Cytotoxic Effects, Combination Index Analysis, and Anti-Tumor Efficacy

The synthesis, characterization and results of evaluation of the biological behavior of HPMA copolymer conjugates bearing anti-cancer drugs doxorubicin and mitomycin C are described. Two HPMA copolymer carrier types were synthesized: the linear copolymer and the biodegradable high-molecular-weight diblock copolymer containing a degradable disulfide bond. The polymer-drug conjugates incubated in buffers modeling the intracellular environment released the drugs more rapidly than those incubated in bloodstream conditions. Significant in vitro and in vivo antitumor synergistic activity of the conjugates in the treatment of EL-4 T-cell demonstrates their high potential for solid tumor treatment.