Immunological activity for a peptide of the limulus anti-LPS...

Details Immunological activity for a peptide of the limulus anti-LPS... Immunological activity for a peptide of the limulus anti-LPS...

1998-09-25

2001-02-20

Borin, Michael (Department: 1654)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S012200

Reexamination Certificate

active

06191114

ABSTRACT:

Despite aggressive management, septic shock arising from Gram-negative sepsis continues to be a leading cause of death in both surgical and medical patients. Death in such patients usually results from cardiovascular collapse and/or multiple organ system failure. One of the main components of Gram-negative bacterial thought to play an integral role in causing septic shock is an outer wall constituent, endotoxin (LPS).
Endotoxins are high molecular weight complexes, associated with the outer membrane of Gram-negative bacteria that produce pyrogenic reactions upon intravenous administration. Endotoxin is shed from living bacteria and is also released into the environment when bacteria die and decompose.
Bacterial endotoxin is a complex consisting of lipid, carbohydrate and protein. It is characterized by an overall negative charge, heat stability and high molecular weight. Highly purified endotoxin does not contain protein, and is a lipopolysaccharide (LPS).
Bacterial endotoxins are known to have profound biological effects in animals and humans, and to cause severe medical problems when present. Symptoms include induction of high fever, activation of complement and cytokine cascade and hypotension. It is critical to avoid endotoxin contamination in any pharmaceutical product or medical device, which come into contact with body fluids. High endotoxin levels in sera due to bacterial diseases, such as septicemia, are not easily treated. Antibiotic treatment of the infection only kills the bacteria, leaving the endotoxin from their cell walls free to cause fever.
LPS is an important mediator in the pathogenesis of septic shock and is one of the major causes of death in intensive-care units in the United States. It has been observed that exposure to LPS during sepsis stimulates an immune response in monocytes and macrophages that results in a toxic cascade resulting in the production of TNF-&agr; and other proinflammatory cytokines. Morrison and Ulevitch, Am. J. Pathol, 93:527 (1978). Endothelial damage in sepsis probably results from persistent and repetitive inflammatory insults. Bone,
Annals Int. Med.
115:457 (1991).
Several proteins have been investigated for their ability to bind and neutralize LPS and the potential use in the septic shock treatment. Among the most extensively studied of the LPS-binding proteins is bactericidal/permeability-increasing protein (BPI), a basic protein found in the azurophillic granules of polymorphonuclear leukocytes. The BPI protein from human PMNs has potent bactericidal activity against a broad spectrum of Gram-negative bacteria. This antibacterial activity appears to be associated with the amino terminal region (amino acids residues 1-199) of the isolated human BPI protein. Recently it has been shown that the N-terminal fragment of BPI (rBPI
25
) neutralizes endotoxin activities and inhibits LPS-induced events in neutrophiles and macrophages. Helene et al.,
Infection and Immunity
. 62:1185 (1994). In addition to its bactericidal effects, BPI has been shown to neutralize the toxic and cytokine-inducing effects of LPS to which it binds. Xoma Corporation is building a portfolio of therapeutic products based on BPI protein. The company's lead BPI-derived product, Neuprex, is in clinical efficacy trials for four indications: [1] Meningococcemia, [2] Hemorrhagic trauma, [3] Partial hepatectomy, [4] Severe intro-abdominal infections. XOMA Corporation Jul. 30, 1996.
Lipopolysaccharide binding protein (LBP) is a 60 kD glycoprotein synthesized in the liver, which shows significant structural homology with BPI. Shumann et al. Disclose the amino acid sequences and encoding cDNA of both human and rabbit. Like BPI, LBP has a binding site for lipid A and binds to the LPS from rough and smooth form bacterial. Unlike BPI, LBP does not possess significant bactericidal activity, and it enhances (rather than inhibits) LPS-induced TNF production. Schumann et al., Science, 249:1429 (1990).
One of the normal host effector mechanisms for clearance of bacteria involves the binding to and subsequent phagocytosis by neutrophils and monocytes. As part of this process, bacteria are exposed to bactericidal and bacteriostatic factor, including oxygen radicals, lysosomal enzymes, lactoferrin and various cationic proteins. LBP opsonizes LPS-bearing particles and intact Gram-negative bacteria, mediating attachment of these LPB-coated particles to macrophages. Wright et al,
J. Exp. Med.
170: 1231 (1989). The attachment appears to be through the CD14 receptor of monocytes, which bind complexes of LPS and LBP, Wright et al.
Science
249: 1431 (1991). Interaction of CD14, which is present on the surface of polymorphonuclear leukocytes as well as monocytes, with LPS in the presence of LBP has been shown to increase the adhesive activity of neutrophils. Wright et al.,
J. Exp. Med.
173: 1281 (1991), Worthen et al.,
J. Clin. Invest.
90: 2526 (1992). Thus, while BPI has been shown to be cytotoxic to bacteria and to inhibit proinflammatory cytokine production stimulated by bacteria, LBP promotes bacterial binding to and activation of monocytes through a CD14-dependent mechanism. Novel biologically active lipopolysaccharide binding protein (LBP) derivatives including LBP derivative hybrid proteins which are characterized by the ability to bind to and neutralize LPS and which lack the CD14-mediated immunostimulatory properties of holo-LBP. Gazzano-Santoro, WO 95/00641. Moreover, peptides corresponding to residues 91-108 of LBP protein were identified that specifically bound the lipid A with high affinity. The peptides inhibited binding of LPS to LBP, inhibited the chromogenic Limulus amebocyte lysate reaction, and blocked release of TNF following LPS challenge both in vitro and in vivo, Taylor
24
et al., J. of Biol. Chem. 270: 17934, [1995].
Another LPS-binding proteins capable to bind and neutralize the endotoxin have been isolated from a horseshoe crab such as
Limulus polyphemus
and Tachypleus antilipopolysaccharide factor (TALF) isolated from
Tachypleus tridentatus
, Kloczewiak
29
et al., J. Infect. Dis. 170: 1490-7 (1994). The cells from their hemolymph (amebocytes) undergo a complex series of biochemical reactions resulting in clot formation, analogous to mammalian blood coagulation. This phenomenon has been exploited in the form of bioassays sensitive to very low endotoxin levels.
Currently, a bioassay of this type is the method of choice for monitoring pharmaceutical manufacturing and is termed Limulus Amebocyte Lysate (LAL). Wainwright et al., WO 92/20715 relates the invention on pharmaceutical utility of the endotoxin binding
eutralizing protein and disclose the use of the endotoxin binding
eutralizing protein for an endotoxin assay. It is yet another object of it invention to provide pharmaceutical compositions capable of binding and neutralizing endotoxin in vivo and containing therein an endotoxin binding
eutralizing protein corresponding at least to part of the endotoxin binding and neutralizing domain of the endotoxin binding
eutralizing protein isolated from a horseshoe crab in accordance with the invention.
Limulus anti-LPS factor (LALF) have been investigated for use in sepsis. Warren et al.,
Infect. Immun.
60: 2506-2513 (1992) and Garcia
25
et al., Crit. Care. Med. 22: 1211 [1994]. This protein is almost certain to sufer the disadvantages associated with other foreign proteins for human therapy, it is immunogenic and has only a short half-life in circulation. These factors will reduce its clinical potential. None of these substances have been proven to be effective for the treatment of the serious conditions associated with Gram-negative infection in humans.
The solution to the above technical problem was achieved by providing substances, which relate to peptides, which bind tightly to LPS, and therefore have utility in the diagnosis and treatment of Gram-negative and other septic conditions. Battafaraono et al., synthesized three peptides from BPI, LALF, LBP (each 27 amino acid in le

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