V. Subr

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.

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.

Novel biodegradable hydrogels prepared using the divinylic crosslinking agent N,O-dimethacryloylhydroxylamine. 1. Synthesis and characterisation of rates of gel degradation, and rate of release of model drugs, in vitro and in vivo

Abstract Hydrophilic gels based on copolymers of N-hydroxypropy1) methacrylamide and N,O-dimethacryloylhydroxylamine were synthesized, and the hydrolytic stability of these gels was studied in buffers with a pH range 2–8. The hydrogels were stable at acidic pH (below 5), but were hydrolysed at neutral and mild alkaline pH. The rate of hydrolysis depended on the pH of the solution and on the crosslinking density of the gel. The in vivo rate of gel hydrolysis correlated with in vitro data. The potential of hydrogels as novel drug release systems was investigated with methotrexate, 5-fluorouracil, doxorubicin and polymer-bound doxorubicin as model drugs. The rate of drug release was studied in vitro and data obtained for targetable and nontargetable polymeric conjugates of doxorubicin in vitro was compared with the kinetics of release measured in vivo in mice.

Macromolecular prodrugs for use in targeted cancer chemotherapy: melphalan covalently coupled to N- (2-hydroxypropyl) methacrylamide copolymers

Abstract Although there are approximately thirty antitumour agents in clinical use recent progress has been relatively slow in the identification of novel compounds with improved therapeutic index. Macromolecular drug-carriers afford the potential to permit controlled release and site-specific delivery of those antitumour agents whose activity may be limited by their inherent toxicity and/or lack of tumourspecific deposition. In this study N -(2-hydroxypropyl) methacrylamide (HPMA) copolymers were synthesised to contain melphalan (ME) or sarcolysin (SE) (1–10 wt%) linked to the polymer backbone (P) via peptide side-chains e.g. P-Gly-ME, P-Gly-Gly-ME, P-Gly-Leu-Gly-ME, P-Gly-Phe-Gly-ME and P-Gly-Phe-Leu-Gly-ME. ME release during incubation with isolated rat liver lysosomal enzymes was dependent on the amino acid composition of the side-chain used. Cytotoxicity measured in vitro towards Walker sarcoma was dependent on the peptide spacer and ME content of the polymer, and likewise those conjugates which showed the fastest rates of cleavage by lysosomal enzymes displayed greatest antitumour activity against Walker sarcoma in vivo. Administration (i.p.) of biodegradable polymer-SE conjugates on day 1 (5 mg/kg) after inoculation of 10 6 Walker cells (s.c.) reduced or prevented establishment of tumours. Similarly, treatment of established Walker sarcoma (approximately 200 mm 2 in area) with polymer conjugate of SE or ME caused a reduction in tumour size. However, it was found that free drug at equivalent dose was also effective against Walker sarcoma in vivo and the therapeutic index of polymer conjugated drug was not appreciably greater than that of the free drug and was dependent on the dosing regime. The pharmacokinetics of [ 3 H] melphalan and 125 I-labelled HPMA copolymer-ME was followed in animals bearing established Walker tumours. Macromolecular conjugate delivered substantially more radioactivity to the tumour than free drug. Although this altered pharmacokinetics must contribute to tumour specific drug activity, the precise mechanism of action of polymer-bound melphalan remains unclear. Preliminary experiments showed a significant increase in the number of macrophages and T and B lymphocytes found in tumours taken from animals 4 days after administration of polymer conjugate when compared to those from untreated or melphalan treated animals.

Release of macromolecules and daunomycin from hydrophilic gels containing enzymatically degradable bonds

The synthesis of hydrophilic gels based on N-(2-hydroxypropyl)methacrylamide copolymers crosslinked via degradable oligopeptide sequences is described. The influence of the conditions of preparation and of the gel structure on the equilibrium degree of swelling (network density) was determined. To evaluate the potential of such gels for controlled delivery of macromolecules and drugs, the release of FITC-dextrans of different molecular weights was studied and the rate of release was found to depend mainly on the equilibrium degree of swelling and not on the structure of the crosslinks. However, the degradation of the gels by a mixture of lysosomal enzymes isolated from rat liver (Tritosomes) or chymotrypsin was dependent on both the equilibrium degree of swelling and the structure of the crosslinks (length of the oligopeptide sequence and structure of the amino acid residues). The release of the anticancer drug daunomycin imbibed in gels was pH-dependent, the rate of release being higher at lower pH. In addition, a gel was synthesized which contained a pentapeptide in the crosslinks and daunomycin bound via a tetrapeptide side-chain, and in this case, incubation with Tritosomes led to degradation with simultaneous release of the drug.

Cleavage of oligopeptide side-chains in N-(2-hydroxypropyl)meth-acrylamide copolymers by mixtures of lysosomal enzymes☆

Abstract N-(2- Hydroxypropyl ) methacrylamide copolymers containing oligopeptide side-chains terminating in drug have been developed as lysosomotropic drug carriers. To obtain more information on the degradation process of such copolymers inside the lysosomes, two polymeric prodrug models have been synthesized: P-Ala-Gly-Phe-Gly-NAp and P-Ala-Gly-Phe-Leu-NAp ( P = polymeric backbone ; NAp = p - nitroanilide ). These substrates were incubated with lysosomal enzymes isolated from bovine spleen — cathepsin B and mixtures of cathepsin B with cathepsin C or H. The products of cleavage (peptide fragments) were quantitatively determined by gel permeation chromatography on a calibrated Sephadex G-15 column and the time course of cleavage followed for 48 hours.The inhibition of cathepsin C by one of the products of cleavage (Phe-Gly) was observed.

Immunogenicity of protein-N-(2-hydroxypropyl)methacrylamide copolymer conjugates in A/J and B10 mice

Two proteins (model targeting residues) human immunoglobulin fraction (IgG) and human transferrin have been conjugated to N-(2-hydroxy propyl)methacrylamide (HPMA) copolymer and the antibody titer elicited, after subcutaneous or intraperitoneal administration to A/J and B10 mice of free and conjugated protein, was measured using the ELISA technique. The measured IgG titer against protein-HPMA copolymer conjugates was always higher than the IgM titer. Also, the titer (IgG) measured against native protein was up to 250-fold greater than that raised against protein-HPMA copolymer conjugates. This reduction in antibody titer against conjugate had a limited de pendence on its molecular weight.

Synthetic polymer derivatives as substrata for neuronal adhesion and growth

The adhesion and viability of dissociated neurons of rat cerebral hemispheres onto methacrylate and methacrylamide hydrogels, either unmodified or containing collagen, basement membrane proteins, and glucosamine, were measured in vitro. The degree of cell adhesion was affected by properties of the polymers such as hydrophilicity, hydrophobicity, presence of reactive chemical groups, and incorporation of biological molecules. Adhesion was promoted by attachment of glucosamine to the polymer backbone. Viability was enhanced by the presence of basement membrane proteins within the polymer network. Morphological studies of cells seeded, both onto and within the polymeric matrices, demonstrated the capacity of such substrates to support neuritic outgrowth. The potential of these in vitro assays in the design of polymeric matrices as neural tissue repair promoter substrate is discussed.

Evaluation of protein-N-(2-hydroxypropyl) methacrylamide copolymer conjugates as targetable drug-carriers. 2. Body distribution of conjugates containing transferrin, antitransferrin receptor antibody or anti-Thy 1.2 antibody and effectiveness of transferrin-containing daunomycin conjugates against mouse L1210 leukaemia in vivo

Abstract The body distribution (5 h) of protein-N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates (Mw∼-15000) was evaluated in DBA2 mice, using either the transferrin receptor, or the Thy-1.2 antigen as specific targets. Radioiodinated HPMA copolymer (25 μg), proteins (anti-Thy 1.2, monoclonal antibody B3/25 specific for the transferrin receptor, non-specific IgG, or diferric transferrin; 10 /gmg) and protein-HPMA copolymer conjugates (10 /gmg protein) were injected intraperitoneally (i.p.) or intravenously (i.v.). Administration of protein conjugates via either route resulted in an up to 4.7-fold increase in blood-associated radioactivity (compared with free protein) together with a 2-fold decrease in the rate of excretion via the kidney. However, the organ distribution of radioactivity following administration of protein-conjugates was essentially similar to that seen following administration of free protein, but generally greater than that of the parent copolymer. For example, a maximum 11-fold and 3.5-fold increase in thymus localisation of both the anti-Thy 1.2 conjugate and the transferrin conjugate was observed when compared with that of either free polymer or non-specific IgG, respectively. However, it should be noted that this thymus-localised radioactivity accounted only fora small proportion (approximately 1%) of the dose administered. Conjugates containing transferrin, B3/25 or anti-Thy 1.2 accumulated at sites of rapid cell division namely bone marrow, intestine and skin. In a preliminary experiment to assess anti-tumour activity of transferrin-containing conjugates, HPMA copolymers were synthesised to contain additionally daunomycin (DNM) bound to the copolymer via degradable (Gly-Phe-Leu-Gly) or non-degradable (Gly-Gly) peptidc spacers. These were administered (DNM, 10 mg/kg) i.p. to DBA2 mice bearing an i.p. L1210 ascites (5 X 105 cells).