Jiří Strohalm

Biocompatibility of N-(2-hydroxypropyl) methacrylamide copolymers containing adriamycin: Immunogenicity, and effect on haematopoietic stem cells in bone marrow in vivo and mouse splenocytes and human peripheral blood lymphocytes in vitro

N-(2-hydroxypropyl)methacrylamide polymeric prodrugs containing adriamycin bound to polymers via glycylphenylalanylleucylglycine side chains and, in one case, galactosamine bound via the same sequence, were tested for immunogenicity after intravenous, subcutaneous and oral application in two inbred strains of mice. The serum antibody level was determined by enzyme-linked immunoassay on the 3rd and 6th day after the last treatment. It was found that antibodies were only produced in very small amounts. In some experimental groups, the antibody titres measured following administration of copolymer conjugate were comparable with those present in non-treated controls. Attachment of adriamycin to N-(2-hydroxypropyl)methacrylamide copolymer considerably decreased its toxicity against haematopoietic precursors in bone marrow as measured by the in vivo colony-forming unit-spleen assay and its ability to inhibit [3H] thymidine incorporation by mouse splenocytes and human peripheral blood lymphocytes measured in vitro.

Preclinical evaluation of polymer-bound doxorubicin

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers (Mw approximately 20 000) were synthesised to contain doxorubicin (DOX) ( approximately 7 wt% ) attached to the polymer backbone via biodegradable Gly-Phe-Leu-Gly peptidyl spacers (polymer 1) and optionally in addition (polymer 2) the carbohydrate moiety galactosamine (approximately 4 mol%) to facilitate liver targeting. Both polymeric DOX conjugates were degraded by isolated rat lysosomal enzymes in vitro and within rat liver following intravenous (i.v.) administration, to liberate free DOX in a time-dependent fashion. Total release of DOX occurred over 24–48 h. When administered either intraperitoneally (i.p.) at doses 2.5–50 mg DOX/kg (triplicate doses) or i.v. (single dose, 13–100 mg DOX/kg) to mice bearing L1210 leukaemia inoculated i.p., both polymers 1 and 2 showed good antitumour activity. The highest T/C seen for conjugates after i.p. treatment was > 762 compared with a T/C of 214 seen for free Dox. At 30 mg/kg given i.p. polymer 2 produced a large number of long-term survivors 720. A variety of dosing schedules were used to test the activity of polymer-DOX against a number of solid tumour models; M5076, P388, B16 melanoma, Walker sarcoma and the xenograft LS174T. In each case treatment with polymer-bound DOX produced an increase in survival time greater than seen with free DOX and experiments with P388 and Walker sarcoma showed remarkable tumour regression (in some cases tumours completely disappeared). B16 melanoma and M5076 were used as a liver metastatic model in particular to study the antitumour activity of the liver-targeted polymer 2. Although polymer 2 did decrease tumour burden attributable to liver metastases it was not significantly better than polymer 1 or free DOX. In these studies HPMA copolymer-DOX conjugates expressed a wide range of antitumour activities, but their overall mechanism of action is still unclear. Certainly these macromolecular produgs are subject to intracellular lysosomal activation, they have the ability to concentrate drug in solid tumours, and with incorporation of targeting residues can promote organ-specific or tumour-specific uptake. In addition covalent conjugation markedly reduces all aspects of DOX-associated toxicity.

Antibody-directed affinity therapy applied to the immune system: In vivo effectiveness and limited toxicity of daunomycin conjugated to HPMA copolymers and targeting antibody

The applicability of targeting therapy intervention in lymphatic tissue was studied. The effect was measured as the inhibition of anti-sheep red blood cell antibody response expressed in plaque-forming cells. Daunomycin was used as the effective drug and polyclonal and monoclonal anti-Thy 1.2 or anti-Iak antibody served for targeting. Both components were coupled to a soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer with oligopeptidic side sequences which permitted a controlled release of the drug in the target tissue. HPMA copolymer conjugates with side sequences Gly-Phe-Leu-Gly cleavable by lysosomal enzymes decreased in vivo the antibody reaction by 60-85%. A comparable amount of free targeting antibody was without a significant effect. Injection of targeted daunomycin decreased the toxicity of the drug against hematopoietic precursors in bone marrow colony-forming unit-spleen 80 times compared to the same amount of free drug. The in vivo effectiveness of targeted daunomycin was confirmed morphologically. Application of free daunomycin lead to a significant irritation of Kupffer cells in liver while none of the daunomycin-antibody-copolymer conjugate had such an effect.

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. 3. Evaluation of adriamycin conjugates against mouse leukaemia L1210 in vivo

Abstract N -(2-Hydroxypropyl)methyocrylamide (HPMA) copolymers were synthesized to contain adriamycin (ADR) and in certain cases fucosylamine or galactosamine residues. Drug was attached to polymer via biodegradable (-Gly-Phe-Leu-Gly) or non-biodegradable (-Gly-Gly) oligopeptide side-chains. Fucosylamine and galactosamine were included to promote conjugate targeting to L1210 cells and hepatocytes, respectively. Although free ADR (5 mg/kg) can increase the mean life span of DBA 2 mice bearing L1210 leukaemia (up to 24%), animals do not survive beyond this time. Treatment with P-Gly-Phe-Leu-Gly-ADR (5 mg/kg) consistently increased mean survival time, and in addition produced survivors at 50 days (up to 80% surviving). Polymers containing in addition galactosamine or fucosylamine were equally effective. Degradation of the drug-polymer linkage was shown to be a prerequisite for pharmacological activity, P-Gly-Gly-ADR was totally ineffective. Conjugation of ADR limited toxicity, a > 10 fold increase in dose could be given in the polymer-bound form without obvious ill effect. Measurement of the pharmacokinetics of 125 I-labelled HPMA copolymer-ADR conjugates showed a marked alteration from the pattern of distribution reported previously for free ADR, and the levels of radioactivity detected in the heart were extremely low. The latter observation supports the observed decrease in toxicity seen for conjugated drug.

Polymers containing enzymatically degradable bonds, XII. Effect of spacer structure on the rate of release of daunomycin and adriamycin from poly [N-(2-hydroxypropyl)-methacrylamide] copolymer drag carriers in vitro and antitumour activity measured in vivo

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing the anticancer agents adriamycin (ADR) and daunomycin (DNM) were synthesised to include different peptidyl spacers, triand tetra-peptides, linking drug to the polymeric carrier. The relationship between the length and detailed structure of the oligopeptide spacer and the rate of drug release was studied by incubation of polymer with the plant thiol-proteinase papain (as a model enzyme) or alternatively a mixture of lysosomal enzymes isolated from rat liver (tritosomes). The drug conjugate would be exposed to lysosomal enzymes following pinocytic inlernalizalion by cells. Tetrapeptide spacers released both anthracyclines more effectively than tripeptide spacers, Gly-Leu-Phe-Gly being cleaved the most effectively throughout (up to > 90% drug released over 48 h). In addition the rate of drug liberation was measured during incubation of polymer conjugates with human serum, and all the conjugates were almost completely stable ( < 5% drug released over 24 h ). To determine the relationship between polymer structure and antitumour activity, conjugates containing ADR were tested against the mouse leukemia L1210 both in vitro and in vivo. Cytotoxicity measured in vitro did not correlate with the rate of drug release seen during incubation with papain and tritosomes, the conjugate containing the spacer Gly-Phe-Leu-Gly-ADR being more cytotoxic than the more readily degradable conjugate containing Gly-Leu-Phe-Gly-ADR side-chains. Those conjugates containing tetrapeptide spacers showed greatest ability to prevent appearance of intraperitoneally inoculated L1210 tumours, (when administered intraperitoneally to DBA2 mice at an ADR dose of 3 × 5 mg/kg on days 1,2 and 3 ). However, although the tumours appeared more quickly following administration of those polymeric carriers releasing ADR more slowly, these tumours did not progress rapidly (in fact some subsequently disappeared) and indeed the slow releasing ADR conjugates containing tripeptide spacers were most effective in increasing animal lifespan.

Polymers containing enzymatically degradable bonds. VI. Hydrophilic gels cleavable by chymotrypsin.

New types of hydrophilic gels based on N-(2-hydroxypropyl)methacrylamide which contain oligopeptide sequences in the crosslinks were prepared. These gels are enzymatically degradable by chymotrypsin. The rate of their degradation may be varied within a broad range by changes in the length and detailestructure of the oligopeptide sequence in the crosslinks and by changing their network density.

Polymeric Drug-Carriers Containing Doxorubicin and Melanocyte-Stimulating Hormone: In Vitro and In Vivo Evaluation Against Murine Melanoma

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing doxorubicin (DOX, approximately 8% by weight) bound via the lysosomally degradable spacer Gly-Phe-Leu-Gly and, in certain cases, also melanocyte-stimulating hormone (MSH, 5-10% by weight) were synthesized with the aim of developing a drug conjugate for site-specific delivery to malignant melanoma. Polymer-bound MSH, like free MSH, was able to stimulate tyrosinase activity in B16F10 cells in vitro, confirming the ability of conjugated hormone to interact with the MSH receptor. Similarly, a 125I-labelled conjugate containing MSH was captured by B16F10 cells in vitro more rapidly than a similar polymer without the targeting moiety. HPMA copolymers containing DOX bound via the lysosomally degradable Gly-Phe-Leu-Gly linkage were cytotoxic to a mouse melanoma cell line (M3 S91) in vitro, the MSH-containing conjugate being more active than that without (although the difference in the ID50 was not significant). When administered intraperitoneally or intravenously to C57BL/6J mice bearing intraperitoneal B16F10 tumours, HPMA copolymers containing DOX linked via this biodegradable spacer (with or without MSH) significantly increased animal survival, the maximum ratio of the mean survival of the test group (T) to that of the untreated control (C) T/C observed (approximately 200) over the dose range 5-20 mg DOX/kg being similar to that seen for free DOX. In contrast, neither polymer conjugates containing DOX bound via a non-degradable linkage (Gly-Gly) nor free MSH showed antitumour activity. In mice bearing established subcutaneous B16F10 tumours, biodegradable polymer-bound DOX conjugates given intraperitoneally were more effective than free DOX (which was virtually inactive in this system); conjugates containing MSH were significantly more effective than those without, the maximum T/C being approximately 148 and 324 respectively. Preliminary pharmacokinetic experiments showed evidence of selective MSH targeting of polymer conjugates to subcutaneous B16F10.

Action of polymeric prodrugs based on N-(2-hydroxypropyl)-methacrylamide copolymers. II. Body distribution and T-cell accumulation of free and polymer-bound [125i]daunomycin

Abstract N -(2-hydroxypropyl)methacrylamide copolymers containing oligopeptide side-chains terminated in [ 125 I]daunomycin (DNM), and targeting moieties [anti-Thy 1.2 antibodies or nonspecific rabbit gammaglobulin (RGG)], were synthesized. The body distribution of these copolymers after i.v. administration was studied in vivo in an inbred strain of mice (C57L/J). Covalent binding of [ 125 I]DNM to an HPMA copolymer containing anti-Thy 1.2 antibodies increased its level in blood 5–20 times and decreased its rate of elimination compared to the free drug. The maximal organ accumulation of [ 125 I]DNM bound to the targetable conjugate in the spleen, thymus and liver was detected after 2 hours. Liver accumulation was observed only when the specific anti-Thy 1.2 antibody was replaced with nonspecific RGG in the conjugates. Similar results were obtained when using i.p. administration of the copolymers studied. The accumulation of HPMA copolymer conjugates in thymocytes was studied in vitro . Intracellular radioactivity of thymocytes cultivated in the presence of [ 125 I]DNM-HPMA copolymer conjugates with anti-Thy 1.2 antibodies was maximal after 2 hours while the maximum accumulation of free drug was observed after 1 hour. Comparing HPMA copolymers without targeting moieties, the copolymer with biodegradable oligopeptide side-chains (Gly-Phe-Leu-Gly) demonstrated a higher binding and accumulation of radioactivity in the T cells than the copolymer containing nonbiodegradable sidechains (Gly-Gly). The higher hydrophobicity of the former may contribute to the observed phenomenon.

iBodies: Modular Synthetic Antibody Mimetics Based on Hydrophilic Polymers Decorated with Functional Moieties

Abstract Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named “iBodies”, consist of an HPMA copolymer decorated with low‐molecular‐weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live‐cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand.

Osmotic opening of the blood-brain barrier permeability to N-(2-hydroxypropyl)methacrylamide copolymers. Effect of polymer -Mw charge and hydrophobicity

Abstract N -(2-Hydroxypropyl)methacrylamide (HPMA) copolymers were detected in the ipsilateral cerebral hemisphere following intracarotid infusion of l -(+)-arabinose (1.8 M ) in the rat. The measured cerebrovascular permeability-surface area product (PA) values (× 10 6 s −1 ) varied from 12.7 to 141.1, the PA value being dependent on the molecular weight average, and chemical characteristics, of the HPMA copolymer preparation used. Higher PA values were observed for HPMA copolymers of lowest molecular weight, and for copolymers containing cationic and hydrophobie substituent groups. Although less than 1% of the administered HPMA copolymer was transferred into the brain (within 10 min), this should be sufficient to permit delivery of a therapeutic dose of anticancer agent.