Oligopeptide Side Chains Research Articles

Release of p‐nitroaniline from oligopeptide side chains attached to N‐(2‐hydroxypropyl)methacrylamide copolymers during incubation with rat intestinal brush border enzymes

Die Makromolekulare Chemie, Rapid CommunicationsVolume 12, Issue 2 p. 101-106 Article Release of p-nitroaniline from oligopeptide side chains attached to N-(2-hydroxypropyl)methacrylamide copolymers during incubation with rat intestinal brush border enzymes Pavla Kopec̆ková, Pavla Kopec̆ková Department of Pharmaceutics/CCCD, University of Utah, Salt Lake City, Utah 84112, U.S.A.Search for more papers by this authorMark A. Longer, Mark A. Longer CRC Polymer-Controlled Drug Delivery Research Group, University of Keele, Keele, Staffordshire ST5 5BG, EnglandSearch for more papers by this authorJohn F. Woodley, John F. Woodley CRC Polymer-Controlled Drug Delivery Research Group, University of Keele, Keele, Staffordshire ST5 5BG, EnglandSearch for more papers by this authorRuth Duncan, Ruth Duncan CRC Polymer-Controlled Drug Delivery Research Group, University of Keele, Keele, Staffordshire ST5 5BG, EnglandSearch for more papers by this authorJindřich Kopec̆ek, Jindřich Kopec̆ek Department of Pharmaceutics/CCCD, University of Utah, Salt Lake City, Utah 84112, U.S.A. Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112, U.S.A.Search for more papers by this author Pavla Kopec̆ková, Pavla Kopec̆ková Department of Pharmaceutics/CCCD, University of Utah, Salt Lake City, Utah 84112, U.S.A.Search for more papers by this authorMark A. Longer, Mark A. Longer CRC Polymer-Controlled Drug Delivery Research Group, University of Keele, Keele, Staffordshire ST5 5BG, EnglandSearch for more papers by this authorJohn F. Woodley, John F. Woodley CRC Polymer-Controlled Drug Delivery Research Group, University of Keele, Keele, Staffordshire ST5 5BG, EnglandSearch for more papers by this authorRuth Duncan, Ruth Duncan CRC Polymer-Controlled Drug Delivery Research Group, University of Keele, Keele, Staffordshire ST5 5BG, EnglandSearch for more papers by this authorJindřich Kopec̆ek, Jindřich Kopec̆ek Department of Pharmaceutics/CCCD, University of Utah, Salt Lake City, Utah 84112, U.S.A. Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112, U.S.A.Search for more papers by this author First published: February 1991 https://doi.org/10.1002/marc.1991.030120206Citations: 7AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume12, Issue2February 1991Pages 101-106 RelatedInformation

Selectivity of Antibody-Targeted Anthracycline Antibiotics on T Lymphocytes

Targeted polymeric prodrugs based on N-(2-hydroxypropyl) methacrylamide were tested on human peripheral blood lymphocytes or mouse splenocytes triggered in vitro to proliferation by T cell specific (Con A) or B cell specific (S. aureus Cowan 1) mitogens. Only a selective inhibition of 3H- thymidine incorporation by T lymphocytes was observed after in vitro incuba tion with prodrugs prepared by covalent attachment of daunomycin or adriamycin and antibody (anti-CD3; anti Thy 1.2) to biodegradable oligopeptide side chains of soluble synthetic copolymer HPMA. The in vitro results were confirmed in vivo by inhibition of antibody response to thymus dependent (ARS-BGG) or thymus independent (TNP-E. coli) antigens in normal Balb/c mice and in athymic nu/nu Balb/c mice. After administration of biocompatible, antibody targeted prodrugs only the antibody response against ARS-BGG was considerably reduced.

The potential of water-soluble polymeric carriers in targeted and site-specific drug delivery

The design of targetable water-soluble polymeric drug carriers based on N- (2-hydroxypropyl)methacrylamide (HPMA) copolymers is described. Three types of conjugates have been studied: (a) HPMA copolymers containing oligopeptide side-chains terminated in anticancer drugs (daunomycin, adriamycin); (b) conjugates of HPMA copolymers with chlorin e 6 (photoactivatable drug); and (c) HPMA copolymers for site-specific oral delivery of 5-aminosalicylic acid (5-ASA). Polymer-bound drugs (a) and (b) are lysosomotropic. When conjugated to a targeting moiety (galactosamine or antibody) their concentration in the target tissue is increased. It was shown that binding drugs to HPMA copolymers decreases their toxicity and immunogenicity . Two ways of drug activation in the lysosomal compartment are discussed: release by lysosomal cysteine proteinases (daunomycin, adriamycin) and activation by light (chlorin e 6). Conjugates of chlorin e 6 with HPMA copolymers and optionally galactosamine were synthesized and their photodynamic activity was tested in vitro on a human hepatoma cell line (PLC/PRF/5 cells). The conjugate containing galactosamine as the targeting moiety was more active compared to the conjugate without the targeting moiety. A new concept of oral drug delivery was proposed based on a combination of site-specific delivery of 5-ASA to the colon with bioadhesive properties of the carrier. HPMA copolymers containing saccharide units complementary to mucosal lectins of the GI tract are used as carriers. They also contain side-chains terminated in salicylic acid bound via an azo bond. Cleavage experiments were carried out using an isolated strain of bacteria commonly found in the colon. When incubated with Streptococcum faecium in vitro 5-ASA is released. Body distributions in guinea pigs after oral administration have shown that HPMA copolymers containing fucosylamine associate with the colon. Further experiments are necessary to verify the therapeutical importance of these observations and to develop water soluble polymers as carriers for site-specific oral delivery.

Action of polymeric prodrugs based on N-(2-hydroxypropyl)-methacrylamide copolymers. II. Body distribution and T-cell accumulation of free and polymer-bound [ 125i]daunomycin

N-(2-hydroxypropyl)methacrylamide copolymers containing oligopeptide side-chains terminated in [ 125I]daunomycin (DNM), and targeting moieties [anti-Thy 1.2 antibodies or nonspecific rabbit gammaglobulin (RGG)], were synthesized. The body distribution of these copolymers after i.v. administration was studied in vivo in an inbred strain of mice (C57L/J). Covalent binding of [ 125I]DNM to an HPMA copolymer containing anti-Thy 1.2 antibodies increased its level in blood 5–20 times and decreased its rate of elimination compared to the free drug. The maximal organ accumulation of [ 125I]DNM bound to the targetable conjugate in the spleen, thymus and liver was detected after 2 hours. Liver accumulation was observed only when the specific anti-Thy 1.2 antibody was replaced with nonspecific RGG in the conjugates. Similar results were obtained when using i.p. administration of the copolymers studied. The accumulation of HPMA copolymer conjugates in thymocytes was studied in vitro. Intracellular radioactivity of thymocytes cultivated in the presence of [ 125I]DNM-HPMA copolymer conjugates with anti-Thy 1.2 antibodies was maximal after 2 hours while the maximum accumulation of free drug was observed after 1 hour. Comparing HPMA copolymers without targeting moieties, the copolymer with biodegradable oligopeptide side-chains (Gly-Phe-Leu-Gly) demonstrated a higher binding and accumulation of radioactivity in the T cells than the copolymer containing nonbiodegradable sidechains (Gly-Gly). The higher hydrophobicity of the former may contribute to the observed phenomenon.

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. 3. Evaluation of adriamycin conjugates against mouse leukaemia L1210 in vivo

N-(2-Hydroxypropyl)methyocrylamide (HPMA) copolymers were synthesized to contain adriamycin (ADR) and in certain cases fucosylamine or galactosamine residues. Drug was attached to polymer via biodegradable (-Gly-Phe-Leu-Gly) or non-biodegradable (-Gly-Gly) oligopeptide side-chains. Fucosylamine and galactosamine were included to promote conjugate targeting to L1210 cells and hepatocytes, respectively. Although free ADR (5 mg/kg) can increase the mean life span of DBA 2 mice bearing L1210 leukaemia (up to 24%), animals do not survive beyond this time. Treatment with P-Gly-Phe-Leu-Gly-ADR (5 mg/kg) consistently increased mean survival time, and in addition produced survivors at 50 days (up to 80% surviving). Polymers containing in addition galactosamine or fucosylamine were equally effective. Degradation of the drug-polymer linkage was shown to be a prerequisite for pharmacological activity, P-Gly-Gly-ADR was totally ineffective. Conjugation of ADR limited toxicity, a > 10 fold increase in dose could be given in the polymer-bound form without obvious ill effect. Measurement of the pharmacokinetics of 125I-labelled HPMA copolymer-ADR conjugates showed a marked alteration from the pattern of distribution reported previously for free ADR, and the levels of radioactivity detected in the heart were extremely low. The latter observation supports the observed decrease in toxicity seen for conjugated drug.

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

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.

Targetable polymeric prodrugs

N- (2-Hydroxypropyl) methacrylamide (HPMA) copolymers containing oligopeptide side-chains terminating in anticancer drugs (daunomycin, adriamycin) have been synthesized. The bond between the drug and the carrier was stable in the bloodstream, but was cleaved intracellularly on exposure to the lysosomal cysteine proteinases. HPMA copolymers have also been modified with targeting moieties: galactosamine, which targets the conjugate to hepatocytes; anti θ antibodies recognizing θ alloantigen expressed on immunocompetent lymphocytes; and fucosylamine, since there is a receptor on mouse leukemia L1210 cells that recognizes and binds this carbohydrate moiety. In vitro and in vivo experiments demonstrated preferential interaction of modified HPMA copolymers with the respective target cells. Subsequent experiments were performed to test the pharmacological activity of anticancer polymeric prodrugs in vivo against L1210 leukemia in DBA 2 mice. Two localizations of tumor were chosen — intraperitoneal and subcutaneous. In both cases experimental animals were treated intraperitoneally with free drug or drug-HPMA copolymer conjugates. HPMA copolymers containing anticancer drugs have shown therapeutic effect only when the oligopeptide sequence between the drug and the polymeric carrier was biodegradable . Polymeric products produced increased life span and an increased number of long term survivors depending on the structure of the conjugate (i.e. presence of biodegradable side-chains or targeting moieties), timing of administration and number of doses. From the data presented it can be concluded that targetable anticancer polymeric prodrugs may be useful clinically.

Soluble, crosslinked N-(2-hydroxypropyl)methacrylamide copolymers as potential drug carriers : 3. Targeting by incorporation of galactosamine residues. Effect of route of administration

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers with oligopeptide side-chains (GlyGly) terminating in ester-linked p-nitrophenol were crosslinked with di(phenylalanyl)-hexamethylenediamine to a level below the gel point. A sample was also prepared with galactosamine residues bound to the crosslinked polymer via the glycylglycyl side chains. The polymer also contained tyrosinamide residues enabling radiolabelling with [ 125I]iodide and thus monitoring of polymer fate in vivo. Following intravenous administration, the galactosamine-containing HPMA copolymer was cleared from the circulation more rapidly than the unsubstituted copolymer and this was accompanied by efficient deposition in the liver (60% after 1 h). Targeting to the liver was also observed after intraperitoneal and subcutaneous administration. After intraperitoneal injection 52% of the galactosamine-containing copolymer recovered after 24 h was found in the liver, whereas only 4% of the unmodified control copolymer was recovered there, 67% being found in the urine at this time. After subcutaneous administration the rate of migration of HPMA copolymer away from the injection site was comparatively slow and with the galactosamine-containing polymer, the level of radioactivity detected in the liver never exceeded 25% of the total radioactivity recovered. There was no significant transfer of crosslinked HPMA copolymer into the tissues following oral administration. Incorporation of galactosamine, did, however, accelerate polymer transfer from the stomach into the small intestine.

Soluble, crosslinked N-(2-hydroxypropyl)methacrylamide copolymers as potential drug carriers : 2. Effect of molecular weight on blood clearance and body distribution in the rat after intravenous administration. Distribution of unfractionated copolymer after intraperitoneal, subcutaneous or oral administration

Preparation and fractionation of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers containing oligopeptide side-chains crosslinked with dityrosylhexamethylenediamine to a level below the gel point has been described previously [1]. Fractions of mean molecular weight ( M ̄ w) from 34,000 to > 400,000 were radiolabelled with 125I and used to study the effect of molecular weight on the fate of polymer in vivo. The blood clearance and body distribution (60 min) of each molecular weight fraction was measured after intravenous administration to rats. Blood clearance was clearly related to molecular weight. In addition the body distribution of unfractionated HPMA copolymer ( M ̄ w = 190,000) was monitored at various times after intraperitoneal, subcutaneous or oral administration to rats. Copolymer administered intraperitoneally progressively moved out of the peritoneal cavity into the bloodstream, and after 24 h most radioactivity was recovered in the urine. Sepharose 4B/6B column chromatography of urine samples showed that most radioactivity represented low molecular weight polymer but low molecular weight degradation products were also present. After subcutaneous administration polymer persisted at the injection site, gradually moving away over 24 h. Material entering the body was recovered principally from the bloodstream, eventually being excreted in the urine and faeces. Oral administration led to rapid transfer of radioactivity along the gastrointestinal tract, after 24 h most radioactivity being recovered in the urine and faeces. Sepharose 4B/6B chromatography of radioactivity recovered from the caecum showed that extensive degradation of copolymer had taken place. Incubation of 126I-labelled HPMA copolymers in rat plasma and a mixture of rat liver lysosomal enzymes, followed by analysis with Sepharose 4B/6B or Sephadex G-15 chromatography, showed that the copolymers used in this study were relatively stable in plasma but degradable by lysosomal enzymes.

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

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 (M̄ w) between 34,000 and >400,000. These fractions were radiolabelled with [ 125I] 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 [ 125I] 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 .

Immunogenicity of N-(2-hydroxypropyl) methacrylamide copolymers

The homopolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) and copolymers of HPMA differing in oligopeptide side chains (-Gly-Gly-OH;-Acap-Phe-OH;-Acap-Leu-HMDA and -Gly-Phe-Tyr-OH) or in their content (1.0, 3.5 and 8.4 mol. % of -Gly-Gly-OH side chains)1 Abbreviations see appropriate Tables and Figures. were investigated with respect to their ability to induce antibody formation in inbred strains of mice. The dependence on the antigen dose on structure of the side chains and on the genetic background of the immunized organism was defined. It was demonstrated that the specificity of the antibody formed is predominantly directed against oligopeptide side chains though some part of the produced antibody is against hydroxypropyl groups. Haptens, such as arsanilic acid and fluorescein isothiocyanate, modifying the side chains, were used as drug models, and their influence on immunogenicity of HPMA copolymers was tested. The HPMA copolymers can be considered as a possible targetable drug delivery system as conditions were found under which the specific antibodies attached to them can damage the target site.

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

Soluble copolymers of -(2-hydroxypropyl)methacrylamide (HPMA) were prepared containing either oligopeptide side chains terminating in p-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 p-nitroaniline or, with the crosslinked polymers, the change in molecular weight distribution. Release of p-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-Glv-Phe-NAp where 5.1% of the bound p-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.

Effect of the chemical structure of N- (2- hydroxypropyl) methacrylajnide copolymers on their ability to induce antibody formation in inbred strains of mice

The homopolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) and copolymers of HPMA differing in oligopeptide side chains (-Gly-Gly-OH; -Acap-Phe-OH; -Acap-Leu-HMDA and -Gly-Phe-Tyr-OH) or in their content (1%, 3,5% and 8,4% mole of-Gly-Gly-OH side chains) were investigated with respect to their ability to induce antibody formation and mitogenic reaction in inbred strains of mice. The dependence on the antigen dose, on composition of the side chain and on the genetic background of the immunized organism was defined. It was demonstrated that the specificity of the antibody formed is predominantly directed against oligopeptide side chains, though some part of the antibody is also produced against hydroxypropyl chains. Neither the homopolymer nor the copolymers behave in the tissue culture as mitogens.

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)

AbstractCopolymers 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.

Controlled biodegradability of polymers — a key to drug delivery systems

By combining oligopeptide sequences with synthetic polymeric chains, carriers of drug models can be prepared. The drug model ( p-nitroaniline) is cleaved from the carrier in the lysosomal compartment of the cell. By changing the length and structure of the oligopeptide sequence, it is possible to regulate the rate of cleavage of drug model by individual thiol proteinases (cathepsins B, H and L) which are the most important as regards cleavage of the substrates studied. By connecting synthetic polymeric chains via oligopeptide bridges, it is possible to regulate the molecular weight as well as the biodegradability of the carrier molecule. Molecular weight also influences other biological properties e.g., elimination from the organism, rate of pinocytic uptake and biological activity. Homopolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) is nonimmunogenic in rats. Attachment of oligopeptide side chains gives rise to a macromolecule possessing immunogenic activity. The degree of antibody response depends on the detailed structure of the copolymer.

Soluble synthetic polymers as targetable agents for intracellular drug release

Soluble synthetic polymers have potential as targetable drug delivery and release agents. Copolymers of 2-hydroxypropylmethacrylamide with oligopeptide side-chains have been used to demonstrate the attachment of drug analogues by covalent linkages susceptible to hydrolysis by lysosomal enzymes but stable in plasma. Targeting of polymers to particular sites has been illustrated by making use of the substrate specificity of adsorptive pinocytosis.

Two new monobactam antibiotics produced by a Flexibacter sp. I. Taxonomy, fermentation, isolation and biological properties.

Two new beta-lactam antibiotics, namely SQ 28,502 and SQ 28,503, have been isolated from fermentations of a Flexibacter sp. They are demethoxy monobactams with oligopeptide side chains and have molecular weights of 1,462 and 1,446, respectively. These beta-lactams show a high degree of stability to a variety of beta-lactamases and act as potent irreversible inactivators of P99 beta-lactamase from Enterobacter cloacae. They exhibit weak antibacterial activity.

Formation of the cysteinyl form of slow reacting substance (leukotriene E 4) in human plasma

The two major species of slow reacting substance (SRS) contain either a glutathionyl or cysteinyl-glycyl side chain. Incubation of these SRS's with undiluted or diluted (usually 1:10 or 1:50) human plasma at 37°C resulted in marked losses of smooth muscle contracting activity due primarily to conversion of their oligopeptide side chains to cysteine.