Pavla Rejmanová

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.

Fate of N-(2-hydroxypropyl)methacrylamide copolymers with pendent galactosamine residues after intravenous administration to rats

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers bearing galactosamine residues accumulate in the liver after intravenous administration to rats (Duncan, R., Kopecek, J., Rejmanová, P. and Lloyd, J.B. (1983) Biochim. Biophys. Acta 755, 518-521). In this study HPMA copolymers bearing pendent galactosamine residues (1.0-11.6 mol%) were injected intravenously into rats and their rates of blood clearance and liver accumulation were measured. A level of substitution of 4 mol% was found to be sufficient to cause substantial deposition in the liver 30 min after administration. The most highly substituted polymer (11.6 mol%) was directed rapidly to the liver, 80-90% being recovered there less than 10 min after administration. Separation of liver into hepatocytes and non-parenchymal cells indicated that polymer was largely associated with the hepatocytes, and density-gradient subcellular fractionation of liver at various times after administration confirmed that polymer was internalized by liver cells and transported, with time, into the secondary lysosomes. Experiments using isolated rat hepatocytes indicated that HPMA copolymers with high galactosamine content have higher affinity for the hepatocyte plasma membrane. HPMA copolymers containing galactosamine and in addition glycylglycyltyrosinamide side-chains were used to demonstrate release of a drug analogue across the lysosomal membrane. These polymers were radioiodinated and, following intravenous administration to rats, the liver lysosomes were isolated and incubated at 37 degrees C in 0.25 M sucrose. Radioactivity was released from the lysosomes faster than the lysosomal enzyme arylsulphatase, an observation that indicates intralysosomal hydrolysis of the copolymer side-chain with subsequent passage of low molecular weight degradation product across the lysosomal membrane.

Targeting of N - (2-hydroxypropyl) methacrylamide copolymers to liver by incorporation of galactose residues.

Soluble synthetic polymers have potential as targetable carriers of pharmacological agents. Here we report that incorporation into poly[N-(2-hydroxypropyl)methacrylamide)] of an oligopeptide side-chain terminating in galactose enhanced the polymers pinocytic uptake from the rat bloodstream by the liver. Within the liver lysosomes enzymic digestion led to the intracellular release of a drug analogue also bound to oligopeptide side-chains of the polymer.

Degradation of side-chains of N-(2-hydroxypropyl)methacrylamide copolymers by lysosomal thiol-proteinases

N-(2-Hydroxypropyl)methacrylamide copolymers bearing oligopeptide side-chains terminating in p-nitroaniline (NAp) were incubated with rat liver lysosomal enzymes in the presence of the thiol glutathione, and the rate of p-nitroaniline release was measured. Twelve of the 16 side-chains investigated were hydrolysed to release p-nitroaniline and in all but one case degradation was partially or totally inhibited by leupeptin. The effect of substrate concentration on the degradation of the most readily cleaved side-chain, -Ala-Gly-Val-Phe-NAp, was measured.

Pinocytic uptake and intracellular degradation of N-(2-hydroxypropyl)methacrylamide copolymers a potential drug delivery system

Synthetic 125I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [125I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.

Tyrosinamide residues enhance pinocytic capture of N-(2-hydroxypropyl)methacrylamide copolymers.

N-(2-Hydroxypropyl)methacrylamide ( HPMA ) copolymers have been proposed as a potential lysosomotropic drug delivery system. HPMA copolymers bearing tyrosinamide residues, bound either directly to the polymer backbone or via a glycylglycine spacer, were radiolabelled with [125I]iodide and the effect of tyrosinamide content on their rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro was measured. Incorporation of tyrosinamide enhanced uptake of the copolymer, most markedly at substitutions above 10 mol%. 2,4-Dinitrophenol, an inhibitor of pinocytosis, was used to confirm that tissue association of 125I-radiolabelled copolymer was due to pinocytic uptake. The side-chain -Gly-Gly-Tyr-NH2 was degraded following the internalization of copolymers containing this spacer and degradation was partially sensitive to the lysosomal thiol-proteinase inhibitor leupeptin. It is postulated that the effect of tyrosinamide residues is to increase the hydrophobicity of poly( HPMA ) and thus to increase its capacity for nonspecific adsorptive pinocytosis.

Soluble, crosslinked N-(2-hydroxypropyl)methacrylamide copolymers as potential drug carriers: Pinocytosis by rat visceral yolk sacs and rat intestine cultured in vitro. Effect of molecular weight on uptake and intracellular degradation

Abstract N- (2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing oligopeptide side-chains terminating in a p- nitrophenyl ester were crosslinked with dityrosylhexamethylene-diamine to a level below the gel point. The resultant soluble crosslinked HPMA copolymer sample was then fractionated to produce five samples of mean molecular weights (M w ) between 34,000 and >400,000. These fractions were radiolabelled with [ 125 I] iodide and used to study uptake by rat visceral yolk sacs and adult rat small intestine cultured in vitro. Rat visceral yolk sacs captured these HPMA copolymers at a rate consistent with uptake by fluid-phase pinocytosis. The oligopeptide crosslinks of the internalized copolymers were degraded intracellularly, resulting in the release of [ 125 I] iodotyrosine back into the culture medium. The rate of uptake of copolymer by the yolk sac decreased markedly with increasing molecular weight, but molecular weight had no effect on the extent of intracellular degradation of the different HPMA copolymer fractions. The effect of 2,4-dinitrophenol (a known inhibitor of pinocytosis) and leupeptin (a lysosomal thiol-pro-teinase inhibitor) confirmed that accumulation of copolymers was by pinocytosis and indicated the importance of lysosomal thiol-proteinases in oligopeptide crosslink degradation. Incubation of copolymer with purified cathepsin B confirmed that this enzyme is able to cleave oligopeptide crosslinks. Crosslinked HPMA copolymers were also found to be pinocytosed by rat intestine and also translocated across to the serosal side. Uptake and translocation were both inhibited by low temperature (15°C) and once again pinocytosis was shown to be size-dependent. However, in this case the rate of uptake increased with increasing polymer size .

Targeting and Lysosomal Handling of Polymethacrylamide-Oligopeptide Conjugates

In the previous volume in this series, Trouet et al., (1982) explained the concept of the lysosomotropic drug-carrier conjugate and prescribed the necessary conditions for this approach to chemotherapy to succeed. We are engaged in the preparation and testing of such a conjugate, and here present a progress report on our work.