Albumin microspheres as carrier of an inhibitor of leukocyte elastase: potential therapeutic agent for emphysema.

Abstract

Methods are described for the covalent attachment of succinoyl-Ala-Ala-Pro-ValCH2Cl, an active sitedirected inhibitor of human leukocyte elastase (EC 3.4.21.11), to microspheres of human albumin. (EC 3.4.21.11), an enzyme that has been implicated in lung tissue injury resulting in the development of emphysema (6, 7). Although these inhibitors would appear to offer promise for the treatment of emphysema, their therapeutic effectiveness would be considerably enhanced if they could be targeted directly to the lungs. As a step in this direction we report here the manner in which the succinoyl peptide chloromethyl ketone (SPCK) succinoyl (Suc)-AlaAla-Pro-ValCH2Cl, which has been shown to be one of the more potent inhibitors of human leukocyte elastase (5), may be covalently attached to HAM with the retention of significant inhibitory activity towards this enzyme. Also reported here are the results of preliminary experiments in vio that show that the carrier-borne inhibitor is in fact targeted primarily to the lungs, where about half of the injected dose has a half-life of approximately 17 days. (8) and is currently available in kit form as a carrier of radioactive tracers for diagnostic lung scanning (2-4), circulatory studies (9), and cardiac output (10). SPCK was synthesized as described (5). Ethylenediamine, 5,5'-dithiobis-(2-nitrobenzoic acid), and hexanediamine were purchased from Aldrich; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-acetylglycine from Sigma; ethyleneimine from Pierce; N-acetyl-DL-homocysteine thiolactone from Schwarz/Mann; and iodo[1-'4C]acetic acid and Na125I (carrier-free) from New England Nuclear. All other chemicals were reagent grade available from commercial sources. All aqueous reagents which were used during the course of the derivatization of HAM contained 0.025% Pluronic F-68 (BASF, Wyandotte, MI) in order to prevent the adherence of microspheres to the sides of reaction vessels and to facilitate uniform suspension. MATERIALS AND METHODSThe sequence of reactions involved in the covalent attachment of SPCK to HAM with extended side chains is described in detail in the legend accompanying Fig. 1. Briefly, the exposed carboxyl groups of HAM were first aminoethylated (step 1), and the amino groups so introduced (I), plus any amino groups already exposed on the surface of HAM, were thiolated with N-acetyl-DL-homocysteine thiolactone (step 2). The thiolated derivative (II) was again aminoethylated with ethyleneimine (step 3) to give the amino derivative (III), or with iodoacetate (step 4) to give the carboxyl derivative (IV). The side chain of IV could be further elongated by condensation with hexanediamine (step 5), yielding V. By virtue of the succinoyl group at the NH2 terminus of the peptide, the latter could be condensed with the amino groups of I, III, and V (step 6), thus yielding HAM-inhibitor derivatives denoted as Ii, IIIi, and Vi, respectively.Assay for Enzyme Activity. Human leukocyte elastase was prepared from purulent sputum as described (12) Step 1: 1...

Inhibition of elastase by a synthetic cotton‐bound serine protease inhibitor: in vitro kinetics and inhibitor release

“…In the present study we have applied them as a model to show release from cotton fibers and enzymatic inhibition of elastase for wound healing. Previous studies showing their ability to inhibit elastase in vivo as a covalently bonded conjugate of albumin microspheres 32 suggest the potential of CMK as an inhibitory sequestrant of elastase if it is covalently attached to the fibers of a wound dressing.…”

Acidic derivatives of homocysteine thiolactone: utility as anionic linkers

“…(d-Maleimidopropionyl)-L-arginyl-L-arginyl-Larginine (9). To a solution of L-arginyl-L-arginyl-Larginine (triarginine) triacetate (18 mg, 0.027 mmol) in 1 mL of 60% acetonitrile-water was added 0.324 mL of 0.5 M sodium bicarbonate (6 equiv) followed by /3-maleimidopropionic acid IV-hydroxysuccinimide ester (9 mg, 0.034 mmol).…”

One-Pot Double Modification of Polymers Based on Thiolactone Chemistry

“…This thiolation consists of aminolysis of the water-soluble N-acetylhomocysteinethiolactone 2 by the ε-NH 2 groups of lysine residues [92][93][94]. Thiolation of a large variety of macromolecular biochemical systems has been reported [80,[94][95][96][97][98][99][100][101][102][103][104][105][106].…”