John B. Lloyd

Evidence that desferrioxamine cannot enter cells by passive diffusion

Accumulation of [14C]desferrioxamine by rat visceral yolk sac in vitro has been compared with that of [14C]sucrose, a probe for fluid-phase pinocytosis. Kinetic parameters for both substrates are closely similar, as are the effects of inhibitors. It is concluded from these data, and from theoretical considerations, that desferrioxamine cannot enter cells other than by pinocytosis and that, once internalized, it will remain in the lysosomes. The results indicate the need for a re-evaluation of the pharmacokinetic mechanisms traditionally accepted for the drugs ability to deplete iron from cells and tissues.

Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro

Both phagocytosis (of particles) and pinocytosis (of solutes) occur in macrophages. It is not known, however, whether particles, if they are small enough, can enter by pinocytosis, nor whether there is a minimum size of particle capable of triggering phagocytic uptake. These questions have been investigated by studying, in vitro, the uptake by rat peritoneal macrophages of particles ranging in diameter from 30 nm to 1100 nm. Percoll (30 nm diameter) and polystyrene beads (100, 300, 600, 800 or 1100 nm diameter) were 125I-iodinated and their uptake by macrophages was measured in the absence or presence of metabolic and cytoskeletal inhibitors. Since uptake, expressed as an Endocytic Index (microliter/10(6) cells per h), increased steadily with the duration of incubation and was inhibited by low temperature or metabolic inhibitors, it was concluded that true endocytosis, and not a superficial cell-association, was being measured. Rates of clearance increased with increasing particle diameter. The rate of uptake of Percoll was 10-times, and of 100 nm polystyrene beads 100-times, the rate of fluid-phase pinocytosis, as measured by the uptake of 125I-labelled polyvinylpyrrolidone. Polystyrene beads of 1100 nm diameter were captured at 700-times this rate. The differential effects of colchicine and cytochalasin B on the uptake of 125I-labelled polyvinylpyrrolidone and of 1100 nm polystyrene beads were taken as indicators of their effects on pinocytosis and phagocytosis respectively. It is concluded that Percoll, although particulate, is captured by pinocytosis. The pattern of inhibition of uptake of polystyrene particles suggests that there is no radical discontinuity between pinocytic and phagocytic uptake, but that the contribution of phagocytosis steadily increases with increasing particle diameter. The results are discussed.

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro.

During recent years N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been developed as targetable drug carriers. These soluble synthetic polymers are internalized by cells by pinocytosis and they can be tailor-made to include peptidyl side-chains degradable intracellularly by specific lysosomal enzymes. Thus they provide the opportunity fo achieve controlled intracellular delivery of anticancer agents. The anthracycline antibiotic daunomycin, and protein synthesis inhibitor puromycin, were bound to HPMA copolymers via several different peptide side-chains, including Gly-Gly, Gly-Phe-Leu-Gly and Gly-Phe-Phe-Leu. Incubation of polymer-drug conjugates with isolated lysosomal enzymes (either a mixture of rat liver lysosomal enzymes or purified thiol-dependent lysosomal proteinases, cathepsins L and B) showed that significant release of drug occurred over 20 h, more than 20% of daunomycin and more than 80% of puromycin being liberated. To test their pharmacological activity conjugates were incubated with either the mouse leukaemia L1210, or the human lymphoblastoid leukaemia CCRF in vitro. The conjugates tested were all less effective than free daunomycin, but they showed differential toxicity against L1210 depending on the aminoacid sequence of their drug-polymer linkage. Inclusion of fucosylamine-terminating side-chains into the HPMA copolymer structure increased the affinity of conjugates for the L1210 cell membrane and resulted in increased toxicity. In contrast HPMA-daunomycin conjugates with or without fucosylamine affected CCRF cells equally, but this cell line was more sensitive than the mouse leukaemia to both free and polymer-bound daunomycin. Incubation of L1210 cells in polymer-bound daunomycin for 72 h, followed by plating cells out in low density in drug-free medium, showed that a concentration of polymer-bound drug (184 micrograms ml-1) could be selected to achieve a cytotoxic effect.

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.

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

DBA2 mice were inoculated i.p. with 10(5)L1210 cells. Animals subsequently treated with daunomycin (single i.p. dose, 0.25-5.0 mg kg-1) all died. The maximum increase in mean survival time observed was approximately 135%. Animals treated with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers conjugated to daunomycin (DNM) showed a significant increase in mean survival time when the polymer-drug linkage was biodegradable (i.e., Gly-Phe-Leu-Gly). Such treatment also produced a number of long term survivors (greater than 50 days). In contrast, HPMA copolymer conjugated to DNM via a non-degradable linkage (Gly-Gly) produced no increase in survival time relative to untreated control animals. The effect observed with biodegradable HPMA copolymer-DNM conjugates was dependent on the concentration of conjugated drug administered (optimum greater than 5 mg kg-1); the frequency of administration (multiple doses were more effective than single); the timing of administration (single doses given on days 1 and 3 were most effective); and the site of tumour inoculation and route of drug administration. Biodegradable HPMA copolymer-DNM conjugates administered i.p. were active against L1210 inoculated s.c. at higher doses than required to curb a peritoneal tumour. Under certain experimental conditions polymer-DNM conjugates containing fucosylamine or galactosamine proved more active than conjugates without the carbohydrate moeity. The mechanism of drug-conjugate action in vivo is at present unclear. Radioiodination of polymer showed approximately 75% of polymer-drug conjugate to be excreted 24 h after i.p. administration. Synthesis of HPMA conjugates containing [3H]DNM showed that polymer containing Gly-Gly-[3H]DNM was excreted (60% of radioactivity in the urine, 24 h) in macromolecular form. In contrast polymer containing Gly-Phe-Leu-Gly-[3H]DNM was largely excreted in the form of low molecular weight species.

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.

Cellular uptake and release of two contrasting iron chelators.

Desferrioxamine and CP94 (1,2‐diethyl‐3‐hydroxypyridin‐4‐one) are metal chelators used or proposed for use in the clinical treatment of iron overload. Recent data on their capacity to deplete intracellular iron led to the conjecture that the differences observed arose from the different membrane‐penetration properties of the two compounds.