Jan Pohl

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.

Amino acid sequence of CAP37, a human neutrophil granule-derived antibacterial and monocyte-specific chemotactic glycoprotein structurally similar to neutrophil elastase.

We report the amino acid sequence of CAP37, a human neutrophil granule protein with antibacterial and monocyte‐specific chemotactic activity. CAP37 is a single‐chain protein consisting of 222 amino acid residues. It has three N‐glycosylation sites, at Asn residues 100, 114 and 145. Some species of CAP37 are glycosylated at all three sites; some at Asn‐114 alone, others at Asn‐114 and Asn‐110 or Asn‐145. CAP37 has 45% sequence identity to human neutrophil elastase, and 30–37% identity to several other granule serine proteinases. Despite these similarities, CAP37 is not a serine proteinase because the active site residues serine and histidine are replaced.

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.

Chromophoric and fluorophoric peptide substrates cleaved through the dipeptidyl carboxypeptidase activity of cathepsin B

The action of bovine spleen cathepsin B as a dipeptidyl carboxypeptidase on newly synthesized substrates of the type peptidyl-X-p-nitrophenylalanyl (Phe(NO2))-Y (X,Y = amino acid residue) or 5-dimethylaminonaphthalene-1-sulfonyl (Dns)-peptidyl-X-Phe(NO2)-Y was investigated. The kinetic parameters of hydrolysis of the X-Phe(NO2) bond were determined by difference spectrophotometry (delta epsilon 310 = 1600 M-1 cm-1) or by spectrofluorometry by following the five- to eightfold increase of Dns-group fluorescence with excitation at 350 nm and emission at 535 nm. The substrates were moderately sensitive to cathepsin B; kcat varied from 0.7 to 4 s-1 at pH 5 and 25 degrees C; Km varied from 6 to 240 microM. The very acidic optima of pH 4-5 are characteristic for dipeptidyl carboxypeptidase activity of cathepsin B. Bovine spleen cathepsins S and H had little and no activity, respectively, when assayed with Pro-Glu-Ala-Phe(NO2)-Gly. These peptides should be a valuable tool for routine assays and for mechanistic studies on cathepsin B.

Purification of pepsins and cathepsin D by affinity chromatography on Sepharose 4B with an immobilized synthetic inhibitor.

Val-D-Leu-Pro-Phe-Phe-Val-D-Leu, a specific inhibitor of aspartate proteinases of the pepsin type, was synthesized. Its bonding to activated 6-aminohexanoic acid-Sepharose 4B afforded an affinity support suitable for the purification of human, porcine, and chicken pepsin, human gastricsin, and bovine cathepsin D. These enzymes bind to the support over the pH range 2-5 at 0-1.5 M concentration of NaCl. A buffer at pH greater than or equal to 6, low ionic strength, and containing 20% dioxane can serve as a general desorption agent. The proteinases were isolated from the crude extracts by a single-step procedure in a high degree of purity and in yields exceeding 70%; human pepsin, however, was not separated from human gastricsin. The support does not show any binding capacity for rat plasma renin at pH 7.4 and for some cysteine endopeptidases (cathepsin B, H, and L) at pH 3-5. The cathepsin D preparations isolated by affinity chromatography on the new support and on pepstatin-Sepharose were of the same degree of purity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequences, and specific activity.

Identification of the active site cysteine and of the disulfide bonds in the N-terminal part of the molecule of bovine spleen cathepsin B.

Cathepsin B (CB), the most intensively studied lysozomal thiol endopeptidase, plays an important role in intracellular protein catabolism [1,2]. Pig liver CB exists in the single-chain (M) and two-chain form. The latter consists of a light (L-) and a heavy (H-) chain and most likely is the result of proteolytic cleavage in the N-terminal part of the molecule of the M-form [3,4]. The active cysteine is localized in the L-chain. Rat liver CB exists in the two-chain form only. The primary structure of its L-chain and of the N-terminal part of its H-chain have been determined [5]. The enzymatic characteristics of bovine spleen CB, its interaction with protein inhibitors, and its similarity with papain have been studied [6-9]. We have found that spleen CB exists in both forms. This paper describes the determination of the primary structure of the L-chain and the identification of active cysteine. The behavior of 3 other half-cystines present in the structure of the L-chain as well as the manner in which the Land H-chain are joined one to another have been elucidated. The similarity between CB and papain is discussed.

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: A novel assay

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (delta epsilon = 860 M-1 cm-1) and by ninhydrin colorimetry (substrate I, epsilon 570 = 2.31 X 10(4) M-1 cm-1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5-6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.