Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-12-30
2001-08-21
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C514S002600, C514S014800, C424S192100, C530S350000, C435S069100, C435S071100
Reexamination Certificate
active
06277821
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention provides bactericidal/permeability-increasing protein (BPI) dimer products characterized by enhanced in vivo biological activity and stable pharmaceutical compositions containing the same.
Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria and consists of serotype-specific O-side-chain polysaccharides linked to a conserved region of core oligosaccharide and lipid A. Raetz.
Ann. Rev. Biochem.,
59:129-170 (1990). LPS is an important mediator in the pathogenesis of gram-negative septic shock, one of the major causes of death in intensive-care units in the United States. Morrison, et al.,
Ann. Rev. Med.
38:417-432 (1987).
LPS-binding proteins have been identified in various mammalian tissues Morrison,
Microb. Pathol.,
7:389-398 (1989); Roeder, et al.,
Infect., Immun.,
57:1054-1058 (1989). Among the most extensively studied of the LPS-binding proteins is bactericidal/permeability-increasing protein (BPI), a basic protein found in the azurophilic granules of polymorphonuclear leukocytes. Human BPI protein has been isolated from polymorphonuclear neutrophils by acid extraction combined with either ion exchange chromatography [Elsbach,
J. Biol. Chem.,
254:11000 (1979)] or
E. coli
affinity chromatography [Weiss, et al.,
Blood,
69:652 (1987)] and has potent bactericidal activity against a broad spectrum of gram-negative bacteria.
The amino acid sequence of the entire human BPI protein, as well as the DNA encoding the protein, have been elucidated in
FIG. 1
of Gray, et al.,
J. Biol. Chem.,
264:9505 (1989), incorporated herein by reference (SEQ ID NOS: 1 and 2). The Gray et al. publication discloses the isolation of human BPI-encoding cDNA from a cDNA library derived from DMSO-induced cells of the human promyelocytic leukemia HL-60 cell line (ATTC CCL 240). Multiple PCR amplifications of DNA from CDNA library derived from such DMSO-induced HL-60 cells as well as DNA from normal human blood and bone marrow cell have revealed the existence of human BPI-encoding cDNAs wherein the codon specifying valine at amino acid position 151 is either GTC (as set out in SEQ ID No: 1) or GTG. Moreover, cDNA species employing GTG to specify valine at position 151 have also been found to specify either lysine (AAG) for the position 185 amino acid (as in SEQ ID Nos: 1 and 2) or a glutamic acid residue (GAG) at that position.
A proteolytic fragment corresponding to the N-terminal portion of human BPI holoprotein possesses the antibacterial activity of the naturally-derived 55 kDa human BPI holoprotein. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity. Ooi, et al.,
J. Exp. Med.,
174:649 (1991). A BPI N-terminal fragment designated rBPI
23
, Gazzano-Santoro et al.,
Infect. Immun.
60:4754-4761 (1992), and comprising approximately the first 199 amino acids of the human BPI holoprotein, has been produced by recombinant means as a 23 kD protein.
The bactericidal effect of BPI has been shown to be highly specific for sensitive gram-negative species. The precise mechanism by which BPI kills bacteria is not yet completely elucidated, but it is known that BPI must first attach to the surface of susceptible gram-negative bacteria. This initial binding of BPI to the bacteria involves electrostatic and hydrophobic interactions between the basic BPI protein and negatively charged sites on LPS. LPS has been referred to as “endotoxin” because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, the most toxic and most biologically active component of LPS.
In susceptible bacteria. BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Osbach and Weiss,
Inflammation: Basic Principles and Clinical Correlates,
eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992)]. BPI is thought to act in two stages. The first is a sublethal stage that is characterized by immediate growth arrest, permeabilization of the outer membrane and selective activation of bacterial enzymes that hydrolyze phospholipids and peptidoglycan. Bacteria at this stage can be rescued by growth in serum albumin supplemented media but not by growth in whole blood, plasma or serum. The second stage, defined by growth inhibition that cannot be reversed by serum albumin, occurs after prolonged exposure of the bacteria to BPI and is characterized by extensive physiologic and structural changes, including penetration of the cytoplasmic membrane.
BPI-induced permeabilization of the bacterial cell envelope to hydrophobic probes such as actinomycin D is rapid and depends upon the initial binding of BPI to LPS, leading to organizational changes which probably result from binding to the anionic groups in the KDO region of LPS, normally responsible for stabilizing the outer membrane through binding of Mg
++
and Ca
++
. Binding of BPI and subsequent bacterial killing depends, at least in part, upon the LPS polysaccharide chain length, with long 0 chain bearing organisms being more resistant to BPI bactericidal effects than short, “rough” organisms (Weiss et al.,
J. Clin. Invest.
65: 619-628 (1980). This first stage of BPI action is reversible upon dissociation of the BPI, a process requiring synthesis of new LPS and the presence of divalent cations (Weiss et al.,
J. Immunol.
132: 3109-3115 (1984). Loss of bactericidal viability, however, is not reversed by processes which restore the membrane integrity, suggesting that the bactericidal action is mediated by additional lesions induced in the target organism and which may be situated at the cytoplasmic membrane (Mannion et al.,
J. Clin. Invest.
86: 631-641 (1990)). Specific investigation of this possibility has shown that, on a molar basis, BPI is at least as inhibitory of cytoplasmic membrane vesicle function as polymyxin B (In't Veld et al.,
Infection and Immunity
56:1203-1208 (1988)) but the exact mechanism has not yet been elucidated.
BPI is also capable of neutralizing the endotoxic properties of LPS to which it binds. Because of its bactericidal properties for gram-negative organisms and its ability to neutralize LPS, BPI can be utilized for the treatment of mammals suffering from diseases caused by gram-negative bacteria, such as bacteremia or sepsis.
U.S. Pat. No. 5,198,541 (PCT/US88/02700) the disclosures of which are hereby incorporated by reference, describe recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI. They also describe the use of N-terminal fragments of BPI protein for co-treatment with certain antibiotics, specifically penicillin. cephalosporins, rifampicin and actinomycin D.
Gram-negative bacteria include bacteria from the following species: Acidaminococcus, Acinetobacter, Aeromonas, Alcaligenes, Bacteroides, Borderella, Branhamella, Brucella, Calymmawobacterium, Campylobacier, Cardiobactenium, Chromobacterium, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Flavobacterium, Francisella, Fusobacierium, Haemophilus, Klebsiella, Legionella, Moraxella, Morganella, Neisseria, Pasturella, Plesiomonas, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Streptobacillus, Veillonella, Vibrio, and Yersinia species.
Antibiotics are natural chemical substances of relatively low molecular weight produced by various species of microorganisms, such as bacteria (including Bacillus species), actinomycetes (including Streptomyces) and fungi, that inhibit growth of or destroy other microorganisms. Substances of similar structure and mode of action may be synthesized chemically, or natural compounds may be modified
Ammons William Steve
Little Roger G.
Carlson Karen Cochrane
Marshall O'Toole Gerstein Murray & Borun
Robinson Patricia
Xoma Corporation
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