Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-02-05
2002-06-04
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S008100, C514S012200, C530S324000, C530S300000
Reexamination Certificate
active
06399570
ABSTRACT:
BACKGROUND OF THE INVENTION
Although humans are at continuous risk of infection by microbial pathogens, most survive these repeated onslaughts by mounting rapid responses that utilize a variety of antimicrobial proteins and small polypeptides. This branch of the human innate immune system represents a more fundamental host defense mechanism than the slower acting clonal systems since antimicrobial polypeptides are also used by primitive animals, insects, and even plants (H. G. Boman, J. Marsh and J. A. Goode, Eds.,
Antimicrobial Peptides,
(John Wiley & Sons Ltd., New York, N.Y., 1994); Hoffmann et al.,
Curr. Opin. Immunol.
8: 8-13 (1996)).
In addition to inhibiting the growth of microbial pathogens, the immune system also neutralizes a variety of toxins produced by invading microbes. One particularly toxic product, produced by Gram-negative bacteria, is endotoxin. Endotoxin (lipopolysaccharide; LPS) is a constitutive component of the outer membrane of Gram-negative bacteria and is released when the bacteria die or multiply (Rietschel et al.,
Immunobiology.
187: 169-190 (1993)). It is estimated that approximately 400,000 patients annually in the United States present with bacterial sepsis, of which 100,000 ultimately die of septic shock and about half of these cases are caused by Gram-negative bacteria (Parrillo, J. E., Shock syndromes related to sepsis. In Cecil Textbook of Medicine (20th edition). J. C. Bennett and F. Plum, editors. W. B. Saunders Company, Philadelphia. 496-501 (1996)). Gram-negative sepsis and septic shock primarily results from endotoxin-induced excessive production and release of inflammatory cytokines by cells of the immune system, particularly macrophages (Beutler, B., and A. Cerami,
Annu. Rev. Biochem.
57: 505-518 (1988); Rosenstreich, D. L., and S. Vogel, Central role of macrophages in the host response to endotoxin. p. 11-15. In D. Schlessinger (ed.), Microbiology. American Society for Microbiology. Washington, D.C. (1980)). TNF-&agr; is the primary mediator of the systemic toxicity of endotoxin (Beutler, B., and A. Cerami,
Annu. Rev. Biochem.
57: 505-518 (1988); Heumann et al.,
J. Endotoxin Res.
3: 87-92 (1996)).
Lipid A is the toxic portion of endotoxin (Rietschel et al.,
Immunobiology.
187: 169-190 (1993)). Monoclonal anti-lipid A antibodies have been tested for treating Gram-negative sepsis and septic shock, but their clinical efficacy has not been demonstrated consistently (Verhoef et al.,
J. Antimicrob. Chemother.
38: 167-182 (1996)), probably due to their poor ability to bind and neutralize endotoxin (Warren et al.,
J. Exp. Med.
177: 89-97 (1993)). Newer developments include identification of synthetic anti-endotoxin polypeptides mimicking polymyxin B (Rustici et al.,
Science
259: 361-365 (1993)) and a number of cationic anti-endotoxin polypeptides derived from host defense proteins. These include a recombinant 23 kDa fragment derived from bactericidal/permeability-increasing protein (Fisher et al.,
Crit. Care Med.
22: 553-558 (1994); Marra et al.,
Crit. Care Med.
22: 559-565 (1994)), a 28-mer peptide derived from bee melittin (Gough et al.,
Infect. Immun.
64: 4922-4927 (1996)), a 33-mer peptide derived from an 18 kDa cationic antibacterial protein (Larrick et al.,
Infect. Immun.
63: 1291-1297 (1995)), and synthetic polypeptides based on the crystal structure of Limulus anti-LPS factor (Reid et al.,
J. Biol. Chem.
271: 28120-28127 (1996)).
Lactoferrin (LF) is an 80 kDa iron-binding glycoprotein that is synthesized exclusively by neutrophils and mucosal epithelium and released extracellularly upon their activation by inflammatory stimuli (Sanchez et al.,
Arch. Dis. Child.
67: 657-61 (1992); P. F. Levay and M. Viljoen,
Haematologica
80: 252-67 (1995); B. Lonnerdal and S. Iyer,
Annu. Rev. Nutr.
15: 93-110 (1995); R. T. Ellison,
Adv. Exp. Med. Biol.
357: 71-90 (1994)). It is thought to be a mammalian host defense protein whose mechanism of protection is poorly understood. In vivo LF provides an antibacterial prophylactic effect (Trumpler et al.,
Eur. J. Clin. Microbiol. Infect. Dis.
8: 310-3 (1989)). LF treatment in vivo has been reported to lower the incidence of Gram-negative bacteremia (Trumpler et al.,
Eur. J. Clin. Microbiol. Infect. Dis.
8: 310-313 (1989)). In vitro it has been shown to inhibit the growth of a variety of microbes by chelating iron (J. D. Oram and B. Reiter,
Biochim. Biophys. Acta
170: 351-65 (1968); A. Bezkorovainy,
Adv. Exp. Med. Biol.
135: 139-54 (1981)).
LF contains a strongly basic region close to its N-terminus and binds to a variety of anionic biological molecules including lipid A (Appelmelk et al.,
Infect. Immun.
62: 2628-2632 (1994)) and glycosaminoglycans which occur on the surface of most cells and in most extracellular matrices (Mann et al.,
J. Biol. Chem.
269: 23661-7 (1994)). Lactoferricin H (residues 1-47) and lactoferricin B (residue 17-41) are released by pepsinolysis of human or bovine LF, respectively, and may have more potent antibacterial activity than the native proteins (Bellamy et al.,
Biochim. Biophys. Acta.
1121: 130-136 (1992)). A region composed of residues 28-34 is reported to contribute to the high affinity binding of human LF and lactoferricin H to endotoxin (Elass-Rochard et al.,
Biochem. J.
312: 839-845 (1995)). LF and lactoferricin B have been shown to inhibit the endotoxin-induced interleukin-6 response in human monocytic cells (Mattsby-Baltzer et al.,
Pediatr. Res.
40: 257-262 (1996)). Previously identified fragments of LF which exhibit antimicrobial activity were isolated from pepsin hydrolysates of LF (Tomita et al., (1993) U.S. Pat. No. 5,214,028; Tomita et al., (1994) U.S. Pat. No. 5,304,633; Tomita et al., (1994) U.S. Pat. No. 5,317,084; Tomita et al., (1997) U.S. Pat. No. 5,656,591).
Previous studies have established that the N-terminal 33 residues of human LF represent the minimal sequence that mediates binding of the protein to anionic polysaccharides such as glycosaminoglycans (Mann et al.,
J. Biol. Chem.
269: 23661-7 (1994)). This sequence contains a cationic head (residues 1-6) and tail (residues 28-33) which combine to form the glycosaminoglycan-binding site. However, these studies provided no evidence which indicated that this polypeptide had antimicrobial or endotoxin-neutralizing activity.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a 6 kDa host-defense polypeptide which is generated by proteolytic digestion of the lactoferrin molecule. The 6 kDa host-defense polypeptide has antimicrobial activity and also endotoxin-neutralizing activity. The present invention also relates to functional variants of the 6 kDa host defense polypeptide, which include N-terminal and C-terminal truncations of the 6 kDa polypeptide, and other modifications of the polypeptide, such as amino acid substitutions which preserve or enhance the activity of the polypeptide.
In another aspect, the present invention relates to a therapeutic method for treating or preventing a disease resulting from a microbial infection of an individual comprising administering a therapeutic amount of the antimicrobial polypeptide or functional variant thereof to the individual. This method is useful in treating bacterial infections. This method can also be used to treat diseases which resulting from infections caused by a mycobacterium, such as tuberculosis or leprosy. This method is also useful in treating bacterial infections which cause bacterial sepsis in the infected individual. This method can also be used to treat infections caused by other microbes, such as fungal infections. The present invention can also be used to potentiate the therapeutic action of an antimicrobial drug in a patient, by administering the polypeptide of the present invention with the antimicrobial drug.
In another aspect, the present invention relates to a method for neutralizing circulating endotoxin in a patient by administering the endotoxin-neutralizing polypeptide or functional variant thereof of the present invention to the patient. Similar methods of u
Agennix Inc.
Halluin Albert P.
Howrey Simon Arnold & White
Portner Ginny Allen
Smith Lynette R. F.
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