Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Patent
1994-09-27
1997-09-09
Fleisher, Mindy
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
514 12, 530324, 530351, 530402, 930141, A61K 3820, A61K 3819, C07K 1452, C07K 1454
Patent
active
056653468
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to the human cytokine, interleukin-8.
BACKGROUND OF THE INVENTION
A human cytokine that promotes the recruitment and activation of neutrophil leukocytes has been identified as one of several endogenous mediators of the acute inflammatory response. In the past it was variously termed neutrophil-activating factor, monocyte-derived neutrophil chemotactic factor, interleukin-8 (IL-8), and neutrophil-activating peptide-1. IL-8 appears to have gained the widest acceptance and the term will be used herein.
The most abundant naturally occurring form of the IL-8 monomer is a 72-residue protein apparently derived by processing of a 99-residue precursor. Other proteins with related sequences, including neutrophil-activating peptide-2 and GRO.alpha. (with melanoma growth stimulatory activity) are IL-8 homologues which have neutrophil-activating properties.
The in vitro effects of IL-8 on neutrophils are similar to those of other chemotactic agonists such as C5a and fMet-Leu-Phe and include induction of a transient rise in cytosolic free calcium, the release of granules containing degradative enzymes such as elastase, the respiratory H.sub.2 O.sub.2 burst, neutrophil shape change, and chemotaxis. IL-8 appears to bind to at least one class of receptor sites on neutrophils with a frequency of approximately 64,000/cell and a K.sub.4 of 0.2 nM.
The three-dimensional structure of IL-8 is known by two-dimensional NMR and x-ray diffraction techniques. The IL-8 monomer has antiparallel .beta. strands followed by a single overlying COOH-terminal .alpha. helix. Two disulfide bridges, between cysteines 7 and 34, and between cysteines 9 and 50 seem to stabilize the tertiary structure. Residues 1-6 and the loop residues 7-18 seem to have little defined secondary structure. In solution, IL-8 is a noncovalent homodimer which is stabilized primarily by interactions between the .beta. strands of the two monomers.
Examination of the three-dimensional structure indicates that following the cysteine at position 50, the residues form a type 1 .beta. turn (at residues 51 to 55) followed by an amphipathic .alpha. helix (at residues 55 to 72) that transverses the .beta. sheet. The hydrophobic face of the .alpha. helix interacts with and stabilizes the hydrophobic face of the .beta. sheet. Some of the interactions are between the two subunits of the dimeric molecule.
As it is established that IL-8 is a key mediator of inflammatory diseases, it would be desirable to identify substances capable of blocking or interrupting the activity of IL-8 for use in anti-inflammatory compositions. Such compositions may prove to be advantageous over presently available steroid based anti-inflammatory drugs which often have severe side-effects with the continued usage that is required for chronic inflammatory diseases. It would also be desirable to identify IL-8 analogs having an increased inflammatory activity for medical research applications.
The investigation described herein arose as a result of an investigation of the functioning of the IL-8 cytokine carried out by production of structural analogs of IL-8.
IL-8 has been previously produced through chemical synthesis (for example see: Clark-Lewis, et al "Chemical Synthesis, Purification, and Characterization of Two Inflammatory Proteins; Neutrophil-Activating Peptide-1 (Interleukin-8) and Neutrophil-Activating Peptide-2" (1991) Biochemistry 30: 3128-3135) and by recombinant DNA methods (for example see: Hebert, et al "Scanning Mutagenesis of Interleukin-8 Identifies A Cluster of Residues Required for Receptor Binding" (1991) J. Biol. Chem. 286: 18989-18994). Such methods of synthesis make it possible to produce analogs of IL-8 in order to investigate such aspects of the cytokine as the receptor binding site(s). In addition, it is known that IL-8 exists in several forms that vary at the NH.sub.2 -terminus, which have been detected in preparations purified from natural sources. These variations correspond to the predominant 72-residue form (which is generally consi
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Clark-Lewis Ian
Moser Bernhard
Degen Nancy J.
Fleisher Mindy
Research Corporation Technologies Inc.
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