Polymers containing enzymatically degradable bonds, 3. Poly[N-(2-hydroxypropyl)methacrylamide] chains connected by oligopeptide sequences cleavable by trypsin

Copolymers ( 12–20 ) of N-(2-hydroxypropyl)methacrylamide ( 1 ) with p-nitrophenyl esters of N-methacryloylated oligopeptides ( 2–10 ) were prepared. These copolymers were crosslinked below the gel-point with N,N′-bis(N^ε-Boc-lysine)hexamethylenediamine ( 11 ). The crosslinks connecting poly[N-(2-hydroxypropyl)methacrylamide] chains contained an oligopeptide sequence of 2–4 amino acids: -Gly-N^ε-Boc-Lys-; -Gly-X-N^ε-Boc-Lys- (X…Gly, Val, Leu, Phe, D -Phe); -Ala-Gly-Val-N^ε-Boc-Lys; -Gly-Gly-Y-N^ε-Boc-Lys-(Y…Phe, Val). After removal of the Boc blockade of lysine, copolymers degradable by trypsin were obtained. Changes in the molecular weight distribution of the polymers studied after incubation with trypsin allowed us to evaluate the influence of steric effects and of subsite interactions on the degradability of polymeric substrates.     

Polymers containing enzymatically degradable bonds, 8. Degradation of oligopeptide sequences in N-(2-hydroxypropyl)methacrylamide copolymers by bovine spleen cathepsin B

Three types of copolymers of N-(2-hydroxypropyl)methacrylamide(HPMA) were prepared which contain oligopeptide sequences: (a) HPMA copolymers containing oligopeptide side-chains terminated with p-nitroaniline; (b) soluble HPMA copolymers containing oligopeptide sequences in crosslinks connecting two poly(HPMA) chains; (c) a hydrophilic gel, i.e. a three dimensional copolymer of HPMA containing an oligopeptide sequence in the crosslinks. These polymeric substrates (suitable as drug carriers) containing potentially degradable oligopeptide sequences were incubated with an intracellular proteolytic enzyme, bovine spleen cathespin B. The degradation process of the substrates made it possible to reveal the relationship between the structure of oligopeptide sequences and their degradability. The results suggest an important role played by cathepsin B in the degradation of polymeric substrates investigated in this study under physiological conditions.     

Polymers containing enzymatically degradable bonds, 7. Design of oligopeptide side-chains in poly[N-(2-hydroxypropyl)methacrylamide] copolymers to promote efficient degradation by lysosomal enzymes

N-(2-Hydroxypropyl)methacrylamide copolymers are considered to be a potential drug delivery system. To fulfil this role the drug-polymer linkage must be susceptible to intralysosomal hydrolysis. Taking p-nitroanilide as a drug analogue, copolymers were synthesized bearing oligopeptidyl-p-nitroanilide side-chains designed to match known specificities of the lysosomal enzymes cathepsin L or cathepsin D. Degradation of side-chains by rat liver lysosomal enzymes (measured by monitoring terminal p-nitroaniline release) occurred only in the presence of reduced glutathione (5 mmol/l) and was effectively inhibited by leupeptin, indicating the involvement of thiol-proteinases in every case. Depending on side-chain composition, between 20 and more than 50% of the terminal p-nitroaniline residues were liberated during a 5 h incubation. It has also been shown that 1) a polymer molecule may contain side-chains of a single type that are nevertheless differentially susceptible to lysosomal hydrolysis; 2) two of the side-chains studied liberate only a p-nitroaniline residue, whereas the others also release amino-acyl-p-nitroanilides; 3) the cleavage of all side-chains displays a broad pH optimum pH 5 to pH 7; 4) the Michaelis-Menten constant K_m for side-chain cleavage varied between 26,1 and 143,2 mg/ml, depending on the amino acid sequence of the side-chain.     

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.     

Polymers containing enzymatically degradable bonds, 1. Chymotrypsin catalyzed hydrolysis of p-nitroanilides of phenylalanine and tyrosine attached to side-chains of copolymers of N-(2-hydroxypropyl)methacrylamide

A series of copolymers of N-(2-hydroxypropyl)methacrylamide were prepared, which contained side chains of the general formula -Gly-X-Y-NAp, where Gly ¨ glycine; X ¨ glycine, alanine, β-alanine, valine, leucine, isoleucine, phenylalanine; Y ¨ phenylalanine or tyrosine; NAp ¨ p-nitroanilide, the latter modelling biologically active compounds. The rates of chymotrypsin-catalyzed hydrolysis of p-nitroanilide groups at pH = 8,0 and 25°C were determined over a range of substrate concentrations to derive values for k_cat and K_M. The results allowed us to determine the influence of the structure of side chains on the rate of cleavage of Y-NAp. The increase in the susceptibility to chymotrypsin attack with an increasing spacing of the Y-NAp residue from the backbone of the polymer chains is demonstrated by comparing the kinetic data of copolymers containing -Gly-Gly-Phe-Phe-NAp, -Gly-Gly-Phe-NAp and -Gly-Phe-NAp side chains. Results obtained with α-chymotrypsin were compared with the cleavage of the above polymer substrates with chymotrypsin covalently bound to a copolymer of N-(2-hydroxypropyl)methacrylamide.     

Stability in rat plasma and serum of lysosomally degradable oligopeptide sequences in N-(2-hydroxypropyl) methacrylamide copolymers

Soluble copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) were prepared containing either oligopeptide side chains terminating in rho-nitroaniline, or oligopeptide sequences forming crosslinks between polymer chains. Such copolymers have potential as targetable drug carriers and already it has been shown that oligopeptide side chains and oligopeptide crosslinks are degraded intracellularly by lysosomal enzymes. The susceptibility of these oligopeptide sequences to degradation on incubation with rat plasma or rat serum was evaluated by monitoring either the liberation of rho-nitroaniline or, with the crosslinked polymers, the change in molecular weight distribution. Release of rho-nitroaniline from some of the polymers was not detectable, and from others proceeded very slowly, the maximum rate being from the side chain Gly-Gly-Phe-Leu-Gly-Phe-NAp where 5.1% of the bound rho-nitroaniline was released by rat serum over a 5 h incubation period. No cleavage of crosslinked HPMA copolymers by plasma or serum was detectable even after a 24 h incubation period.     

Solution properties of drug carriers based on poly[N-(2-hydroxypropyl)methacrylamide] containing biodegradable bonds

The solution properties of water-soluble drug carriers based on copolymers of N-(2-hydroxypropyl)methacrylamide containing p-nitroaniline (drug model) attached to the copolymer by enzymatically degradable oligopeptide chains, were studied using light scattering, GPC and sedimentation methods. It was found that these polymers associate in water forming micelles with p-nitroaniline being inside and hydrophilic polymer chains outside. The association number and compactness of micelles both depend on the content of hydrophobic side chains, polymer concentration and temperature. Micellar shells hinder the penetration of enzymes into the micellar core, thus reducing the rate of enzyme-catalyzed release of p-nitroaniline.     

Polymers containing enzymatically degradable bonds V. Hydrophilic polymers degradable by papain

Copolymers of N-(2-hydroxypropyl)methacrylamide were prepared, in which synthetic polymer chains are joined by crosslinks containing oligopeptidic sequences degradable with papain, with the general structure P-(Gly)_nX-Y-NH-(CH₂)₆6-NH-Y-X-(Gly)_n-P (P is the polymer chain, N = 1, 2; X…Phe, Val, Gly;Y... Lys, Gly, Tyr, Ala, Phe). The relationship between the structure of these polymeric substrates and their degradability with papain was investigated viscometrically. It was shown that -Phe-Lys- was the most suitable -X-Y- sequence. Extension of the oligopeptidic sequence by one amino acid residue causes a pronounced rise in the rate of cleavage of the polymeric substrates.     

Poly(ethylene glycol)s containing enzymatically degradable bonds

The possibility to use poly(ethylene glycol) (PEG) as a synthetic, water-soluble polymeric carrier of biologically active compounds was investigated. The relationship between structure of oligopeptide (amino acid)-PEG conjugates and their hydrolysis catalyzed by chymotrypsin was studied. Two types of derivatives were synthesized: Derivatives of PEG 2000, containing an oligopeptide sequence terminating in p-nitroaniline (drug model) and higher molecular weight derivatives of PEG 2000 containing in the main chain enzymatically and hydrolytically degradable bonds. The rates of chymotrypsin catalyzed release of p-nitroaniline at pH 8,0 and 25°C were determined over a range of substrate concentrations to derive values for k_cat and K_M. The influence of the length and detailed structure of the oligopeptide sequence on the rate of hydrolysis was demonstrated. Comparison with copolymers of N-(2-hydroxypropyl)methacrylamide and poly(maleic anhydride-co-vinylpyrrolidone) showed that the sterical hindrance of the formation of the enzyme-substrate complex is much less pronounced in the PEG-oligopeptide conjugates. The latter conclusion is also valid in the case of cleavage of PEG derivatives which contain enzymatically degradable bonds in the main chain. The incorporation of a single amino acid residue (phenylalanine) into the main PEG chain is sufficient to make the polymer susceptible to chymotrypsin catalyzed hydrolysis.     

Polymers containing enzymatically degradable bonds, 4. Preliminary experiments in vivo

Copolymers of N-(2-hydroxypropyl)methacrylamide containing oligopeptidic sequences either in side chains or as part of crosslinks were prepared. These polymers were applied intravenously to rats, and spectrophotometric analysis (oligopeptidic side chains) or gel permeation chromatography of urine (oligopeptidic crosslinks) have shown that both types of polymers are cleavable in vivo.     

Biorecognition of sugar containing N-(2-hydroxypropyl)methacrylamide copolymers by immobilized lectin

The interactions of sugar containing N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with Tetragonolobus purpureas (Lotus tetragonolobus) lectin immobilized on 4% beaded agarose have been studied. HPMA copolymers containing side-chains terminating in N-acylated amino sugars, namely, fucosylamine, galactosamine, glucosamine and mannosamine were synthesized and their biorecognition evaluated. As expected, only fucosylamine containing HPMA copolymers showed specific binding to the lectin. The dissociation constants (K_d) for HPMA copolymers were determined by frontal affinity chromatography. The binding constant was dependent on the amount of fucosylamine residues per macromolecule. An increase in fucosylamine content from 9,9 to 33,2 mol-% resulted in a decrease of K_d by one order of magnitude. Incorporation of hydrophobic side-chains such as glycylglycyl-5-[4-(2-aminoethylcarbamoyl)phenylazo]salicylic acid, 1′-(β-hydroxyethyl)-3′,3′-dimethyl-6-nitrospiro(2H-1-benzopyran-2,2′-indoline) (SP), or 1′-(β-glycylglycyloxyethyl)-3′,3′-dimethyl-6-nitrospiro(2H-1-benzopyran-2,2′-indoline) (GG-SP) into the copolymers increased the non-specific binding to the lectin. Photoinduced isomerization of bound spiropyrans to merocyanines was accompanied by changes in the conformation and biorecognition of HPMA copolymers. The results obtained demonstrated the impact of the structure and solution conformation of sugar containing HPMA copolymers on their biorecognition.     

Cleavage of oligopeptide p-nitroanilides attached to N-(2-hydroxypropyl)methacrylamide copolymers by guinea pig intestinal enzymes

N-(2-Hydroxypropyl)methacrylamide copolymers containing oligopeptide side chains terminated with p-nitroaniline, and low-molecular-weight oligopeptide p-nitroanilides, were synthesized. They were used as substrates to evaluate the enzymatic activity in the gastrointestinal tract of guinea pigs. Brush border membrane enzymes and luminal enzymes (supernatant and pellet) were isolated from guinea pig small intestine and colon, and the cleavage of the substrates was studied in vitro. The results indicate the presence of endopeptidase activity in both small intestine and colon brush border. Phosphoramidon, a potent inhibitor of endopeptidase-24.11, was effective in the cleavage of low-molecular-weight substrates, but ineffective in the cleavage of polymeric substrates, indicating the participation of a different endopeptidase in the cleavage of the latter. Luminal enzymes were more active in the cleavage of polymeric substrates (when compared to low-molecular-weight ones) in accordance with the physiological function of the gastrointestinal tract. The enzymatic activity, both luminal and brush border, was considerably lower in the colon than in the small intestine. This suggests that the colon might be a suitable site for the oral delivery of peptides and proteins.     

Tumor targeting by pH-sensitive,biodegradable, cross-linked N-(2-hydroxypropyl) methacrylamide copolymer micelles

Increasing the molecular weight of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers by using micellar structures could result in more pronounced enhanced permeability and retention effect, thus increase the tumor accumulation of drug. However, most micellar formulations are relatively unstable and release their drug non-specifically. To improve on these disadvantages, we developed a micellar drug delivery system based on self-assembly of HPMA copolymers. Amphiphilic conjugates were synthesized by conjugating the hydrophobic drug doxorubicin and hydrophobic β-sitosterol to the hydrophilic HPMA polymer backbone via pH-sensitive hydrazone linkages. This linkage is quite stable at physiological pH but hydrolyzes easily at acidic pH. After conjugates self-assembly into micelles, HPMA copolymer side chains were cross-linked through the hydrazone linkages to ensure micelle stability in the blood. Using this approach, cross-linked micelles were obtained with molecular weight of 1030 KD and diameter of 10–20 nm. These micelles remained stable with undetectable doxorubicin release at pH 7.4 or mouse plasma, whereas collapsed quickly with 80% of the drug released at pH 5 which corresponds to the pH of lyso/endosome compartments of tumor cells. Both cross-linked and non-cross-linked micelles displayed similar in vitro anti-tumor activity as linear copolymer conjugates in Hep G2 and A549 cancer cell lines with internalization mechanism by caveolin, clathrin, and giant macropinocytosis. In vivo studies in an H22 mouse xenograft model of hepatocarcinoma showed the tumor accumulation (1633 μCi/L*h) and anti-tumor rate (71.8%) of cross-linked micelles were significantly higher than non-cross-linked ones (698 μCi/L*h, 64.3%). Neither type of micelle showed significant toxicity in heart, lung, liver, spleen or kidney. These results suggest that cross-linked HPMA copolymer micelles with pH-sensitivity and biodegradability show excellent potential as carriers of anti-cancer drugs.     

Activation of poly[N-(2-hydroxypropyl)methacrylamide] for the binding of bioactive molecules, 2. Activation with cyanogen bromide

The amount of chymotrypsinogen linked to poly[N-(2-hydroxypropyl)methacrylamide], which was activated by cyanogen bromide, under various conditions was determined. The optimal activation time was found to be indicated by the beginning of an invariable base consumption during the reaction of the polymer with cyanogen bromide. The degree of activation increased with the concentration of cyanogen bromide and with the pH, and the yield of coupling can be increased by variation of the temperature, concentration of the reaction components, and pH. At room temperature and pH 9,3, the coupling reaction was completed in less than 24 h. The stability of the activated polymer depends strongly on temperature and pH. The analysis of the active moieties on the polymer carrier revealed that the application of the generally accepted mechanism for cyanogen bromide activation of polysaccharide carriers to the activation of poly[N-(2-hydroxypropyl)methacrylamide] remains to be proved.     

Synthesis of N-(2-hydroxypropyl)methacrylamide copolymers with antimicrobial activity

Copolymers of N-(2-hydroxypropyl)methacrylamide which contained up to 20 mol % of p-nitrophenyl esters of N-methacryloylated oligopeptides, and of N-methacryloylaminophenoxyacetic acids (o-, m-, p-) have been prepared. The aminolyses of these polymers with tert. butylamine, ampicillin and 6-aminopenicillanic acid were kinetically characterized. Based on these results polymer bound ampicillin and polymer bound 6-aminopenicillanic acid were prepared. These preparations possessed antimicrobial activity; they inhibited the growth of Staphylococcus aureus 209 P.     

Bis(2-chloroethyl)amine bound to copolymers of N-(2-hydroxypropyl)methacrylamide and methacryloylated oligopeptides via biodegradable bonds

Drug carriers were prepared by combining oligopeptidic sequences with synthetic polymeric chains. Soluble copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) were synthesized containing oligopeptidic side chains terminating in a drug model (p-nitroaniline). The susceptibility of these oligopeptidic sequences to degradation on incubation with lysosomal enzymes and human serum was evaluated by monitoring the p-nitroaniline liberation. It was shown that the drug model can be attached to the polymer carrier by means of an oligopeptidic spacer stable in blood serum and degradable after the polymer has been in contact with intracellular lysosomal enzymes. The studied polymer carriers were used for binding an anticancer drug, bis(2-chloroethyl)amine.     

Activation of poly[N-(2-hydroxypropyl)methacrylamide] for the binding of bioactive molecules, 1. Activation with 4-nitrophenyl chloroformate

Proper reaction conditions were determined for the activation of water-soluble, biocompatible poly[N-(2-hydroxypropyl)methacrylamide] ( 1 ) by esterification with 4-nitrophenyl chloroformate ( 2 ). The rate constants of hydrolysis and aminolysis of the 4-nitrophenyl ester groups in resulting polymer 3 were compared. It was shown that at pH 8 – 9 the rate of aminolysis in water solutions is high enough for binding bioactive molecules via ? NH₂ groups. Cobra venom acetylcholinesterase was bound to the activated polymer 3 and thermostability and immunogenicity of this product were studied.     

Polymers containing enzymatically degradable bonds, 9. Chymotrypsm catalyzed hydrolysis of a p-nitroanilide drug model,p bound via oligopeptides onto poly(vinylpyrrolidone-co-maleic anhydride)

Copolymers of 1-vinyl-2-pyrrolidone with maleic anhydride which contain oligopeptide sidechains with l-phenylalanine p-nitroanilide (Phe-NAp) end groups were synthesized. The chymotrypsin catalyzed hydrolysis of these polymers was studied. The rate of p-nitroaniline release was found to depend on the structure of the oligopeptied sequences: ? Gly? Gly? Val? Phe? NAp > ? Gly? Val? Phe? NAp > ? Gly? Gly? Phe? NAp. From a comparison with similar substrates based on poly[N-(2-hydroxypropyl)methacrylamide] the influence of the structure of the polymeric backbone on the rate of cleavage of bonds at the ends of oligopeptide side-chains was estimated.     

Steric effects on excimer formation in 2-amino-1,3-di(2-naphtyl)propane luminiscent markers covalently bonded to poly [N-(2-hydroxypropyl)methacrylamide]

The fluorescence spectra of poly[N-(2-hydroxypropyl)methacrylamide] (poly[1-(2-hydroxypropylaminocarbonyl)-1-methylethylene]) with covalently bonded luminiscent markers (2-amino-1, 3-di(2-naphthyl)propane (ADNP)) dissolved in methanol were measured over the temperature range 190–290 K. The effect of the spacer length on intramolecular excimer formation in ADNP was investigated. The ratio of the fluorescence intensities of the dimer I_D and the monomer I_M follows an Arrhenius temperature dependence in the temperature range 190–250 K with an activation energy independent of the spacer length. The numerical value of I_D/I_M at constant temperature increased with increasing spacer length reaching quickly a limiting value. The effect is explained by increasing hindrance to intramolecular excimer formation which is due—particularly for the shortest spacers—to increased steric interactions of the marker with the polymer chain.     

Release of 5-aminosalicylic acid from bioadhesive N-(2-hydroxypropyl)methacrylamide copolymers by azoreductases in vitro

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers ( 11a, b ) containing side chains terminating in 5-aminosalicylic acid (5-ASA) and fucosylamine ( 10 ) were synthesized. A reactive polymeric precursor ( 9 ), a copolymer of HPMA ( 7 ) and N-methacryloylglycylglycine p-nitrophenyl ester ( 8 ), was consecutively aminolyzed with 5-[4-(2-aminoethylcarbamoyl)phenylazo]-salicylic acid ( 6 ) and fucosylamine ( 10 ). The latter renders the copolymers bioadhesive in the gastrointestinal tract. The release of 5-ASA from 11a, b was studied in vitro by incubation with cell-free extracts isolated from rat cecum, and factors influencing the degradation were determined. The rate of azo bond cleavage of 11a, b was similar to the rate of cleavage of low-molecular-weight substrates, methyl orange and compound 6 . Addition of benzyl viologen, a low potential electron carrier, increased the rate of cleavage. At optimal benzyl viologen and substrate concentration ratios, a zero order release was observed. At higher substrate concentration, the rate of 5-ASA release decreased.