Polypeptides with therapeutic activity and methods of use Number:7,144,982 from the United States Patent and Trademark Office (PTO) owispatent Polypeptides and methods of use including treating bacterial infection and/or endotoxemia, decreasing the amount of TNF-.alpha., inhibiting endothelial cell proliferation, inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation, and inhibiting angiogenesis.<H Polypeptides, therapeutic, Polypeptides, wi, 7144982.html, Patent, pto, 7144982

Title: Polypeptides with therapeutic activity and methods of use

Abstract: Polypeptides and methods of use including treating bacterial infection and/or endotoxemia, decreasing the amount of TNF-.alpha., inhibiting endothelial cell proliferation, inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation, and inhibiting angiogenesis.

Patent Number: 7,144,982 Issued on 12/05/2006 to Mayo

Inventors: Mayo; Kevin H. (Minnetonka, MN)
Assignee: University of Minnesota (Minneapolis, MN)

Appl. No.: 09/766,353

Filed: January 19, 2001

Current U.S. Class: 530/327 ; 530/300

Current International Class: A61K 38/04 (20060101); A61K 38/10 (20060101); C07K 7/08 (20060101)

Field of Search: 530/300,327 514/14,15

References Cited [Referenced By]

U.S. Patent Documents


4938949	July 1990	Borch et al.
5595887	January 1997	Coolidge et al.
5786324	July 1998	Gray et al.
5830860	November 1998	Gray et al.
5837678	November 1998	Little, II
5854214	December 1998	Little, II
5856302	January 1999	Ammons et al.
5955577	September 1999	Mayo

Foreign Patent Documents


0 955 312	Nov., 1999	EP
WO 99/17616	Apr., 1999	WO
WO 01/53335	Jul., 2001	WO

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Primary Examiner: Borin; Michael
Attorney, Agent or Firm: Mueting, Raasch & Gebhardt, P.A.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to Provisional Patent Application Ser. Nos. 60/177,255, filed on Jan. 20, 2000 and 60/210,297, filed Jun. 8, 2000, both of which are incorporated herein by reference.

Claims

What is claimed is:

1. A polypeptide selected from the group consisting of those represented by SEQ ID NOs:1 6 and 9 17 and active analogs thereof, wherein X is an amino acid, wherein an active analog thereof demonstrates at least one of bactericidal activity, endotoxin neutralizing activity, decreasing the amount of TNF-.alpha. in vitro, inhibiting endothelial cell proliferation in vitro, inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation in vitro, inhibiting angiogenesis in vitro, and inhibiting tumorigenesis in vitro; and wherein the active analog is selected from the group consisting of deletion of one amino acid, one or two additional amino acids, conservative amino acid substitutions, chemical modifications, and enzymatic modifications.

2. A polypeptide selected from the group consisting of those represented by SEQ ID NOs:1 6.

3. A polypeptide selected from the group consisting of those represented by SEQ ID NOs:9 17, wherein X is an amino acid.

4. The polypeptide of claim 3 wherein X is norleucine.

5. The polypeptide of claim 1 which is active for the treatment of bacterial infection and/or endotoxemia.

6. A polypeptide having an amphipathic .alpha.-helical or 3.sub.10 helical structure having one surface comprising positively charged amino acid residues and an opposing surface comprising hydrophobic amino acid residues, wherein the positively charged amino acid residues and the opposing hydrophobic amino acid residues identify a surface active domain, wherein the polypeptide has 12, 13, or 14 amino acid residues, wherein the surface active domain comprises amino acids K1, K4, K8, and R5, wherein amino acid K1 is N-terminal, and wherein the polypeptide demonstrates at least one of bactericidal activity, endotoxin neutralizing activity, decreasing the amount of TNF-.alpha. in vitro, inhibiting endothelial cell proliferation in vitro, inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation in vitro, inhibiting angiogenesis in vitro, and inhibiting tumorigenesis in vitro.

7. The polypeptide of claim 6 wherein the surface active domain comprises atoms 1 24, 64 109, and 146 167 listed in Table 5.

8. The polypeptide of claim 6 having the structure coordinates listed in Table 5.

9. The polypeptide of claim 6 having the sequence KLFKRHLKWKII (SEQ ID NO:4).

10. The polypeptide of claim 6 having at least one hydrophobic residue at the C-terminal position.

11. The polypeptide of claim 6 having 12 amino acid residues.

12. The polypeptide of claim 6 having the sequence KLFKRHLKWXII (SEQ ID NO:15), wherein X is an amino acid.

13. The polypeptide of claim 12 wherein X is norleucine.

14. The polypeptide of claim 6 having 14 amino acid residues.

15. A polypeptide selected from the group consisting of those represented by SEQ ID NOs:1 6 and 9 17 and active analogs thereof, wherein X is an amino acid, wherein an active analog thereof demonstrates at least one of bactericidal activity, endotoxin neutralizing activity, decreasing the amount of TNF-.alpha. in vitro, inhibiting endothelial cell proliferation in vitro, inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation in vitro, inhibiting angiogenesis in vitro, and inhibiting tumorigenesis in vitro; and wherein the active analog is selected from the group consisting of deletion of one amino acid, one or two additional amino acids, and conservative amino acid substitutions.

16. The polypeptide of claim 15 further modified by chemical and/or enzymatic derivatization.

17. A method for treating bacterial infection and/or endoxtoxemia comprising administering to a patient an amount of a pharmaceutical composition effective to inhibit the bacterial infection and/or neutralize endotoxin, wherein the pharmaceutical composition comprises one or more polypeptides of claim 1, 6 or 15.

18. The method of claim 17 wherein the polypeptide neutralizes endotoxin.

19. The method of claim 17 wherein the polypeptide is bactericidal.

20. The method of claim 17 wherein the polypeptide is both bactericidal and capable of neutralizing endotoxin.

21. The method of claim 17 wherein the composition comprises a polypeptide having endotoxin neutralizing activity and is selected from the group consisting of those represented by SEQ ID NOs:1 6; and combinations thereof.

22. The method of claim 17 wherein the composition comprises a polypeptide having bactericidal activity and is selected from the group consisting of those represented by SEQ ID NOs:1 6; and combinations thereof.

23. The method of claim 17 wherein the composition comprises KLFKRHLKWKII (SEQ ID NO:4).

24. A method for inhibiting bacterial infection and/or endotoxemia in vitro, the method comprising contacting cells with an amount of a composition effective to inhibit the bacterial infection and/or to neutralize endotoxin, wherein the composition comprises one or more polypeptides of claim 1, 6 or 15.

Description

BACKGROUND

A series of designed peptide 33mers (.beta.pep peptides) has been reported to be bactericidal and to be capable of neutralizing the bacterial endotoxin lipopolysaccharide (LPS) (Mayo et al., Biochim. Biophys. Acta 1425, 81 92 (1998)). CD and NMR conformational analyses indicate that .beta.pep peptides form .beta.-sheets (Mayo et al., Protein Sci. 5, 1301 1315 (1996)), and one of these, .beta.pep-4, folds compactly as anti-parallel .beta.-sheet sandwich (Ilyina et al., Biochemistry 36, 5245 5250 (1997)). .beta.pep-19 is potently bactericidal in the 10.sup.-8 M range (Mayo et al., Biochim. Biophys. Acta 1425, 81 92 (1998)). .beta.pep peptides function like Limulus anti-LPS factor (LALF) and the homologous bactericidal/permeability increasing protein (B/PI) (Hoess et al., EMBO J. 12, 3351 3356 (1993)) in that all appear to express activity through an amphipathic .beta.-sheet structural motif having a cationic .beta.-sheet face (Kelly et al., Surgery 114, 140 146 (1993); Siefferman et al., Infect. Immun. 59, 2152 2157 (1991); Beamer et al., Science 276, 1861 1864 (1997); and Gray et al., Biochim. Biophys. Acta 1244, 185 190 (1995)).

Numerous studies on bactericidal peptides indicate the functional importance of a net positive charge and high hydrophobicity in the context of an amphipathic, usually helical, structure (Maloy et al., Biopolymers 37, 105 122 (1995)). The net positive charge promotes interaction with the negatively charged surface of bacterial membranes (Matsuzaki et al., Biochemistry 36, 2104 2111(1997)), whereas structure-activity relationships demonstrate that the amphipathic conformation of the peptide promotes bacterial cell lysis (Andreu et al., Biochemistry 24, 1683 1688 (1985)). Bactericidal peptides such as cecropins (Lee et al., Proc. Natl. Acad. Sci. USA 86, 9159 9162 (1989)), magainins (Zasloff, Proc. Natl. Acad. Sci. USA, 84, 5449 5453 (1987)), proline-arginine-rich peptides (Agerberth et al., Eur. J. Biochem. 202, 849 854 (1991)), and sapecin (Matsuyama et al., J. Biol. Chem. 263, 17112 17116 (1988)), like .beta.pep peptides, all have a net positive charge and considerable hydrophobic character. The cecropins and magainins are helix-forming peptides (Holak et al., Biochemistry 27, 7620 7629 (1988); and Marion et al., FEBs Lett. 227, 21 26 (1988)), whereas the sapacins contain both .alpha.-helix and .beta.-sheet segments (Hanzawa et al., FEBs Lett. 269, 413 420 (1990)). Structures for the proline-arginine-rich peptides are unknown. Tachyplesin, a bactericidal and endotoxin neutralizing peptide isolated from hemocytes of the horseshoe crab (Kawano et al., J. Bio. Chem. 265, 15365 15367 (1990)), as well as anti-bacterial peptide defensins (Selsted et al., J Biol. Chem. 264, 4003 4007 (1989); and Lebrer et al., Cell 64, 229 230 (1991)), form dimeric .beta.-sheets which are stabilized by three intramolecular disulfide bridges (Hill et al., Science 251, 1481 1485 (1991)). In addition, a number of small, antibiotic peptides based on the structure of the anti-LPS, cyclic peptide polymyxin B (Morrison et al., Immunochem. 13, 813 818 (1976)) have been designed as short .beta.-hairpins constrained by a disulfide bridge (Rustici et al., Science 259, 361 365 (1993)).

SUMMARY OF THE INVENTION

The present invention provides a polypeptide selected from the group consisting of: ANIKLSVQMKLF (SEQ ID NO:1); KLSVQMKLFKRH (SEQ ID NO:2); VQMKLFKRHLKW (SEQ ID NO:3); KLFKRHLKWKII (SEQ ID NO:4); KRHLKWKIIVKL (SEQ ID NO:5); LKWKIIVKLNDG (SEQ ID NO:6); KIIVKLNDGREL (SEQ ID NO:7); VKLNDGRELSLD (SEQ ID NO:8); QMKLFKRHLKWK (SEQ ID NO:9); MKLFKRHLKWKI (SEQ ID NO:10); MKLFKRHLKWKIIV (SEQ ID NO:11); XLFKRHLKWKII (SEQ ID NO:12); KLFXRHLKWKII (SEQ ID NO:13); KLFKRHLXWKII (SEQ ID NO:14); KLFKRHLKWXII (SEQ ID NO:15); KLFKKHLKWKII (SEQ ID NO:16); KLFKXHLKWKII (SEQ ID NO:17); analogs thereof (preferably that are active for the treatment of bacterial infection and/or endotoxemia); and combinations thereof; wherein X is an amino acid.

The present invention also provides a method for treating bacterial infection and/or endotoxemia. This includes administering to a patient an amount of a pharmaceutical composition effective to inhibit the bacterial infection and/or neutralize endotoxin, wherein the pharmaceutical composition includes a polypeptide (i.e., one or more polypeptide) listed above. A method for inhibiting bacterial infection and/or endotoxemia in vitro is also provided. The method includes contacting cells with an amount of a composition effective to inhibit the bacterial infection and/or to neutralize endotoxin, wherein the composition includes a polypeptide listed above.

The present invention also provides a method for decreasing the amount of TNF-.alpha. in a patient. The method includes administering to the patient a therapeutically effective amount of a pharmaceutical composition including a polypeptide listed above. A method for decreasing the amount of TNF-.alpha. in vitro is also provided. The method includes incubating cells with an effective amount of a composition comprising a polypeptide selected listed above. Other polypeptides suitable for these methods include analogs of those listed above that are active for decreasing the amount of TNF-.alpha..

The present invention also provides a method for inhibiting endothelial cell proliferation in a patient. The method involves administering to the patient a therapeutically effective amount of a composition including a polypeptide listed above. A method for inhibiting endothelial cell proliferation in vitro is also provided. The method involves contracting cells with an effective amount of a composition including a polypeptide listed above. Other polypeptides suitable for these methods include analogs of those listed above that are active for inhibiting endothelial cell proliferation.

The present invention also provides a method for inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation in a patient. The method includes administering to the patient a therapeutically effective amount of a composition including a polypeptide listed above. A method for inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation in vitro is also provided. The method includes contacting cells with an effective amount of a composition including a polypeptide listed above. Other polypeptides suitable for these methods include analogs of those listed above that are active for inhibiting antiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation.

The present invention also provides a method for inhibiting angiogenesis in a patient. The method includes administering to the patient a therapeutically effective amount of a composition including a polypeptide listed above. A method for inhibiting angiogenesis in vitro is also provided. The method includes contacting cells with an effective amount of a composition including a polypeptide listed above. Other polypeptides suitable for these methods include analogs of those listed above that are active for inhibiting angiogenesis.

The present invention also provides a method for inhibiting tumorigenesis in a patient. The method includes administering to the patient a therapeutically effective amount of a composition including a polypeptide listed above. Other polypeptides suitable for this method includes analogs of those listed above that are active for inhibiting tumorigenesis.

The present invention also provides the three-dimensional structure of certain of the polypeptides using nuclear magnetic resonance (NMR) spectroscopy (e.g., one- and two-dimensional NMR) and circular dichroism (CD) spectroscopy. This information is of significant utility in fields such as drug discovery. Thus, the present invention also provides methods of using such structural information.

The present invention provides a polypeptide having an amphipathic structure having one surface with positively charged amino acid residues and an opposing surface with hydrophobic amino acid residues, wherein these define a surface active domain. Preferably, the surface active domain includes amino acids K1, K4, K8, and R5 shown in FIG. 6B, and more preferably, atoms 1 24, 64 109, and 146 167 listed in Table 5. More preferably, the polypeptide has the structure coordinates listed in Table 5 and most preferably has the sequence KLFKRHLKWKII (SEQ ID NO:4).

One specific method of the present invention involves evaluating a candidate compound for structural similarity to that of KLFKRHLKWKII (SEQ ID NO:4) by: supplying a three-dimensional structure of KLFKRHLKWKII (SEQ ID NO:4) or a portion thereof; supplying a three-dimensional structure of a candidate compound; and comparing the three-dimensional structure of the candidate compound with the three-dimensional structure of KLFKRHLKWKII (SEQ ID NO:4) or a portion thereof.

As used herein, "a" or "an" refers to one or more of the term modified. Thus, the compositions of the present invention include one or more polypeptides.

"Amino acid" is used herein to refer to a chemical compound with the general formula: NH.sub.2--CRH--COOH, where R, the side chain, is H or an organic group. Where R is an organic group, R can vary and is either polar or nonpolar (i.e., hydrophobic). The amino acids of this invention can be naturally occurring or synthetic (often referred to as nonproteinogenic). As used herein, an organic group is a hydrocarbon group that is classified as an aliphatic group, a cyclic group or combination of aliphatic and cyclic groups. The term "aliphatic group" means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example. The term "cyclic group" means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group. The term "alicyclic group" means a cyclic hydrocarbon group having properties resembling those of aliphatic groups. The term "aromatic group" refers to mono- or polycyclic aromatic hydrocarbon groups. As used herein, an organic group can be substituted or unsubstituted.

The terms "polypeptide" and "peptide" as used herein, are used interchangeably and refer to a polymer of amino acids. These terms do not connote a specific length of a polymer of amino acids. Thus, for example, the terms oligopeptide, protein, and enzyme are included within the definition of polypeptide or peptide, whether produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. This term also includes polypeptides that have been modified or derivatized, such as by glycosylation, acetylation, phosphorylation, and the like.

The following abbreviations are used throughout the application:

TABLE-US-00001 A = Ala = Alanine T = Thr = Threonine V = Val = Valine C = Cys = Cysteine L = Leu = Leucine Y = Tyr = Tyrosine I = Ile = Isoleucine N = Asn = Asparagine P = Pro = Proline Q = Gln = Glutamine F = Phe = Phenylalanine D = Asp = Aspartic Acid W = Trp = Tryptophan E = Glu = Glutamic Acid M = Met = Methionine K = Lys = Lysine G = Gly = Glycine R = Arg = Arginine S = Ser = Serine H = His = Histidine

Other abbreviations used throughout include: B/PI, bactericidal/permeability increasing protein; LALF, Limulus anti-lipopolysaccharide factor; LPS, lipopolysaccharide; PF4, platelet factor 4; NMR, nuclear magnetic resonance spectroscopy; NOE, nuclear Overhauser effect; rf, radio frequency; FID, free induction decay; CD, circular dichroism; HPLC, high performance liquid chromatography; PBS, phosphate buffered saline; FCS, fetal calf serum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Peptide sequences: peptide sequences are shown for .beta.pep-19 (SEQ ID NO: 18) and .beta.pep-25 (SEQ ID NO: 19), along with those of eight dodecapeptides which "walk through" the sequence of .beta.pep-25. The dodecapeptides are referred to as SC peptides. The --NH.sub.2 at the right of each sequence indicates amidation of the C-terminal carboxylate group.

FIGS. 2A through 2C. Bactericidal Dose Response Curves for Peptides. A select number of dose response curves are shown in these figures as percent bacteria killed versus the log concentration of peptide. Data are provided for three strains of bacteria: (2A) rough strain E. coli J5; (2B) smooth strain E. coli IA2; and (2C) a strain of Gram-positive S. aureus MNHO. Dose response curves were acquired as described in the Methods Section.

FIG. 3. Dose Response Curves for Endotoxin Neutralization. Dose response curves, as described in the Methods Section, are shown as percent LPS neutralization versus log concentration of peptide. The top panel illustrates results for SC peptides and for .beta.pep-25, and the bottom panel illustrates results for various variants of SC-4 as described in the text.

FIGS. 4A through 4C. CD Spectra for SC Peptides. Far-ultraviolet circular dichroic spectra for two SC peptides, SC-5 and SC-7, are shown in FIGS. 4A and 4B, respectively, as mean residue ellipticity versus wavelength (nm). Peptide concentration was 40 .mu.M in 20 mM potassium phosphate, pH 6.3. The temperature was set at 20.degree. C. Other experimental conditions are discussed in the Methods Section. CD traces shown were acquired as a function of TFE concentration from 0% to 70% (volume/volume). FIG. 4C shows the change in (.THETA.)222 versus the concentration of trifluoroethanol (TFE).

FIGS. 5A and 5B. NOESY Spectra for SC-4. 600 MHz .sup.1H NMR spectra are shown for SC-4 in the presence (5A) and absence (5B) of 30% trifluoroethanol/70% water. Peptide concentration was 6.3 mM in 10 mM potassium phosphate, pH 5.5 and 25.degree. C. Spectra were accumulated with 8 k data points over 6000 Hz sweep width and were processed with 1 Hz line broadening. Only spectral regions downfield from the HDO resonance are shown and some resonance assignments are indicated.

FIGS. 6A and 6B. NOE-Derived Structures of SC-4. For dodecapeptide SC-4, 14 final NOE-derived structures have been superimposed in FIG. 6A and structural statistics are given in Table 3. The average structure is shown in FIG. 6B, and four positively charged residues, K1, K4, R5, and K8, which lie on one surface of the amphipathic helix, are shown in the space-filling mode.

FIG. 7. Helical wheel projections for SC peptides. SC peptide sequences are shown in helical wheel projections as discussed in the text.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

This invention contributes to the development of agents in combating the ever-recurring problem of drug-resistant microorganisms. It involves the discovery of polypeptides effective in the treatment of bacterial infection and/or bacterial endotoxemia, as well as other disorders.

The compositions comprising the polypeptides of this invention can be added to cells in culture or used to treat patients, such as mammals. Where the polypeptides are used to treat a patient, the polypeptide is preferably combined in a pharmaceutical composition with a pharmaceutically acceptible carrier such as a larger molecule to promote polypeptide stability or a pharmaceutically acceptible buffer that serves as a carrier for the polypeptide.

Treatment can be prophylactic or therapeutic. Thus, treatment can be initiated before, during, or after the development of the condition (e.g., bacterial infection or endotoxemia). As such, the phrases "inhibition of" or "effective to inhibit" a condition such as bacterial infection and/or endotoxemia, for example, includes both prophylactic and therapeutic treatment (i.e., prevention and/or reversal of the condition).

The present invention provides a method for treating bacterial infection and/or endotoxemia in a patient (e.g., a mammal such as a human). This involves administering to a patient an amount of a pharmaceutical composition effective to inhibit the bacterial infection and/or to neutralize endotoxin, wherein the pharmaceutical composition includes one or more polypeptides described herein. Analogously, the present invention provides a method for inhibiting bacterial infection and/or endotoxemia in vitro (e.g., in a cell culture). This method involves contacting cells with an amount of a composition effective to inhibit the bacterial infection and/or to neutralize endotoxin, wherein the composition includes one or more polypeptides described herein.

More than 100 years ago, it was determined that heat stable extracts of gram-negative enteric bacteria were highly toxic. Assuming that these toxins were released from the interior of the bacterium upon its death, the investigators termed these toxins "endotoxins." Subsequent chemical studies showed that these endotoxins are actually lipopolysaccharide (LPS) components of the outer membrane of enteric bacteria. Toxic LPS is composed of three structural units--an outer polysaccharide component, a core oligosaccharide region, and the inner portion, lipid A, which affords the molecule its proinflammatory activities. Toxic LPS is released from the bacterium when it dies or during periods of rapid bacterial growth.

In many species, for instance humans, cows, rabbits, mice, and rats, toxic LPS in serum binds rapidly to a plasma polypeptide named lipopolysaccharide binding protein (LBP). LBP, which is synthesized by hepatocytes as part of the acute phase response of inflammation, has a strong affinity for the lipid A portion of endotoxin.

Activation of a cell by a toxic LPS-containing complex results in the synthesis, release, or activation of cell-derived proinflammatory mediators, which can include cytokines (e.g., interleukin-1, interleukin-6, interleukin-8, and tumor necrosis factor-.alpha.), platelet activating factor, nitric oxide, complement (e.g., C5a and C3a), prostagladins, leukotrienes, the kinin system, oxygen metabolites, catecholamines and endorphines. The mediators can impact organ systems including the heart, vascular system, coagulation system, lungs, liver, kidney and the central nervous system. These factors can lead to endotoxemia, also referred to as endotoxic shock, septic shock, circulatory shock, and septicemia, a progressive disease that can lead to death.

Endotoxemia is typically caused by toxic LPS from Gram-negative bacteria, but can also be caused by Gram-positive bacteria, and occasionally, by fungi. Components released by Gram-positive bacteria that can cause endotoxemia include peptidoglycan and lipoteichoic acid, and lipoarabinomannan from the cell wall of Mycobacterium spp.

Studies have shown that serum concentrations of the cytokine tumor necrosis factor (TNF) increase after onset of endotoxemia, and serum TNF activity is directly associated with the onset of signs of abdominal pain and fever, for example. Thus, endotoxin activity can also be measured by determining the amount of release of tumor necrosis factor alpha (TNF-.alpha.) from a macrophage cell line or by evaluating the symptoms of shock in animals. Production of TNF-.alpha. can be assayed as described by Mosmann (J. Immunological Methods 65:55 63 1983).

In both the in vivo and in vitro methods, "inhibiting" a bacterial infection includes preventing as well as reversing or reducing the growth of bacteria in a patient or a cellular sample, and "neutralizing" endotoxin includes binding LPS and thereby removing it from the system of a patient or a cellular sample. The level of bacterial infection can be determined according to the bactericidal assay described in the Examples Section. The level of endotoxemia can be determined according to the LPS neutralization assay described in the Examples Section. These assays can be used to determine the effectiveness of a polypeptide, whether used in vivo or in vitro. To determine the effectiveness of the treatment of a patient having a bacterial infection, a blood sample can be taken, a culture developed, and the amount of live bacteria determined according to the bactericidal assay described in the Examples Section. To determine the effectiveness of the treatment of a patient having endotoxemia, a blood sample can be taken, a culture developed, and the amount of cytokines (e.g., TNF-.alpha., IL-1) can be determined using methods known to one of skill in the art. For example, the WEHI assay can be used for the detection of TNF-.alpha. (Battafarano et al., Surgery 118, 318 324 (1995)).

The effective amount of a peptide for treating a bacterial infection will depend on the bacterial infection, the location of the infection and the peptide. An effective amount of the peptide for treating bacterial infection is that amount that diminishes the number of bacteria in the animal and that diminishes the symptoms associated with bacterial infection such as fever, pain and other associated symptoms of the bacterial infection. The effective amount of a peptide can be determined by standard dose response methods in vitro and an amount of peptide that is effective to kill at least about 50% to about 100% of the bacteria (LD.sub.50) and more preferably about 60% to about 100% of the bacteria would be considered an effective amount. Preferably, the peptide has an effective dose at a concentration of about 1.times.10.sup.-4 M to about 1.times.10.sup.-10M, and more preferably at a concentration of about 1.times.10.sup.-7 M to about 1.times.10.sup.-9 M. Peptides that are considered to be bactericidal kill at least one organism selected from the group of P. aeruginosa, P. cepacia, E. coli B, Salmonella, Proteus mirabilis, and Staphylococcus aureus at concentrations of about 10.sup.-10 M or greater under physiological conditions (e.g., at a pH of 5.6).

Alternatively, an effective amount of the peptide for treating a bacterial infection can be determined in an animal system such as a mouse. Acute peritonitis can be induced in mice such as outbred Swiss webster mice by intraperitoneal injection with bacteria such as P. aeruginosa as described by Dunn et al. (Surgery, 98:283 290 1985; Cody et al. (J. Int. Surg. Res., 52:314 319 1992). Different amounts of peptide can be injected at one hour intravenously prior to the injection of the bacteria. The percentage of viable bacteria in blood, spleen, and liver can be determined in the presence and absence of the peptide or other antibiotics. While not meant to limit the invention, it is believed that bactericidal peptide could also enhance the effectiveness of other antibiotics such as erythromycin, and the like.

Bactericidal activity can be evaluated against a variety of bacteria, preferably Gram-negative bacteria, but the types of bacteria can include Pseudomonas spp including P. aeruginosa and P. cepacia, E. coli strains, including E. coli B, Salmonella, Proteus mirabilis and Staphylococcus strains such as Staphylococcus aureus. A preferred organism is Pseudomonas aeruginosa.

Peptides with endotoxin neutralizing activity can be used to treat mammals infected with Gram-negative bacteria systemically and that exhibit symptoms of endotoxin shock such as fever, shock, and TNF-.alpha. release. The animals are typically infected with one or more Gram-negative bacteria such as Pseudomonas spp., rough strains of E. coli, encapsulated E. coli and smooth strain E. coli. The endotoxin neutralizing peptide can be combined with other agents that are known and used to treat endotoxin shock.

Endotoxin neutralizing activity can be measure by determining the molar concentration at which the peptide completely inhibits the action of lipopolysaccharide in an assay such as the Limulus amoebocyte lysate assay (LAL, Sigma Chemicals, St. Louis, Mo.) or the chromogenic LAL 1000 test (Biowhittacker, Walkersville, Md.). Endotoxin neutralizing activity can also be measured by calculating an inhibitory dose 50 (LD.sub.50) using standard dose response methods. An inhibitory dose 50 is that amount of peptide that can inhibit 50% of the activity of endotoxin. Peptides preferably neutralized endotoxin at a molar concentration of about 1.times.10.sup.-4 M to about 10.sup.-8 M, more preferably about 10.sup.-5 M to about 10.sup.-6 M. Peptides considered to not have endotoxin neutralizing activity do not neutralize endotoxin at a molar concentration of about 10.sup.-4 M or less.

The present invention also provides a method for decreasing the amount of TNF-.alpha. in a patient (e.g., a mammal such as a human). This involves administering to a patient an amount of a pharmaceutical composition effective to decrease the amount of TNF-.alpha. in a patient's system as determined by evaluating serum levels of TNF-.alpha., wherein the pharmaceutical composition includes one or more polypeptides described herein. Analogously, the present invention provides a method for decreasing the amount of TNF-.alpha. in vitro (e.g., in a cell culture). This method involves incubating cells with an amount of a composition effective to decrease TNF-.alpha. amounts in the cell culture, wherein the composition includes one or more polypeptides described herein. For both in vivo and in vitro methods, the WEHI assay can be used for the detection of TNF-.alpha. (Battafarano et al., Surgery 118, 318 324 (1995)) in cell culture or in serum from a patient. Alternatively, the amount of TNF-.alpha. in a sample can be assayed using an anti-TNF-.alpha. antibody. A polypeptide "active" for decreasing TNF-.alpha. can be evaluated using an in vitro test, and preferably shows an at least 10% decrease in the amount of TNF-.alpha..

Angiogenesis is crucial to numerous biological functions in the body, from normal processes like embryogenesis and wound healing to abnormal processes like tumor growth, arthritis, restenosis and diabetic retinopathy. The use of agents that can inhibit angiogenesis in vitro and in vivo, particularly in anti-tumor research, has indicated that anti-angiogenic therapy will be a promising therapeutic modality in the future. The search for angiogenic inhibitors has been focused on controlling two of the processes that promote angiogenesis: endothelial cell (EC) growth and adhesion primarily because ECs are more accessible than are other cells to pharmacologic agents delivered via the blood and ECs are genetically stable and are not easily mutated into drug resistant variants. Most anti-angiogenic agents have been discovered by identifying endogenous molecules, primarily proteins, which inhibit EC growth. This traditional approach has produced a number of anti-angiogenics, such as platelet factor-4 (PF4), thrombospondin, tumor necrosis factor (TNF), interferon-.gamma. inducible protein-10, angiostatin, endostatin, vasostatin, and bactericidal-permeability increasing (BPI) protein. In toto, about forty anti-angiogenic agents, identified using various approaches, are currently known.

It has also been postulated that tumor growth can be controlled by deprivation of vascularization (Folkman J. natl. Cancer. Inst. 82, 4 6 (1990); Folkman et al., J. biol. Chem. 267, 10931 10934 (1992)). A growing number of endogenous inhibitors of angiogenesis such as platelet factor-4 (PF4), interferon-.gamma. inducible protein-10 (IP-10), thrombospondin-1 (TSP-1), angiostatin, as well as synthetic agents, e.g., thalidomide, TNP-470, and metalloproteinase inhibitors have been described. Some of these agents are currently being tested in phase I/II clinical trials. Previous research described in Griffioen et al., Blood 88, 667 673 (1996), and Griffioen et al., Cancer Res. 56, 1111 1117 (1996) has shown that pro-angiogenic factors in tumors induce down-regulation of adhesion molecules on endothelial cells in the tumor vasculature and induce anergy to inflammatory signals such as tumor necrosis factor .alpha. (TNF.alpha.), interleukin-1, and interferon-.gamma.. EC exposed to vascular endothelial cell growth factor (VEGF) (Griffioen et al., Blood 88, 667 673 (1996)) and basic fibroblast growth factor (bFGF) (Griffioen et al., Blood 88, 667 673 (1996); and Melder et al., Nature Med. 2, 992 997 (1996)) have a severely hampered up-regulation of intercellular adhesion molecule-1 (ICAM-1) and induction of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. This phenomenon, which was named tumor-induced EC anergy, is one way in which tumors with an angiogenic phenotype may escape infiltration by cytotoxic leukocytes.

Because angiogenesis-mediated down-regulation of endothelial adhesion molecules (EAM) may promote tumor outgrowh by avoiding the immune response (Griffioen et al., Blood 88, 667 673 (1996); Kitayama et al., Cancer. Res. 54 4729 4733 (1994); and Piali et al., J. exp. Med. 181, 811 816 (1995)), it is believed that inhibition of angiogenesis would overcome the down-regulation of adhesion molecules and the unresponsiveness to inflammatory signals. In support of this hypothesis, a relation between E-selectin up-regulation and the angiostatic agent AGM-1470 has been reported (Budson et al., Biochem. Biophys. Res. Comm. 225, 141 145 (1996)). It has also been shown that inhibition of angiogenesis by PF4 up-regulates ICAM-1 on bFGF-simulated EC. In addition, inhibition of angio-genesis by PF4 overcomes the angiogenesis-associated EC anergy to inflammatory signals.

Thus, the present invention provides a method for inhibiting endothelial cell proliferation in a patient (e.g., a mammal such as a human). This involves administering to a patient an amount of a pharmaceutical composition effective to inhibit the growth or endothelial cells, wherein the pharmaceutical composition includes one or more polypeptides described herein. Analogously, the present invention provides a method for inhibiting endothelial cell proliferation in vitro (e.g., in a cell culture). This method involves contacting cells with an amount of a composition effective to prevent and/or reduce the growth of endothelial cells, wherein the composition includes one or more polypeptides described herein.

For determining the amount of endothelial cell proliferation in vivo, various methods known to one of skill in the art could be used. For example, for evaluation of endothelial cell growth in tumors, tissue sections can be appropriately stained to quantify vessel density. For determining the amount of endothelial cell proliferation in vitro, the EC Proliferation Assay described in the Examples Section can be used, which involves the uptake of tritiated thymidine by cells in cell culture. A polypeptide that is "active" for inhibiting endothelial cell proliferation is preferably one that causes an at least 10% reduction in endothelial cell proliferation at a concentration lower than 10.sup.-4 M.

The present invention also provides a method for inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation in a patient (e.g., a mammal such as a human). This involves administering to a patient an amount of a pharmaceutical composition effective to prevent and/or reduce the amount of ICAM expression down-regulation, wherein the pharmaceutical composition includes one or more polypeptides described herein. Analogously, the present invention provides a method for inhibiting angiogenic-factor mediated inter-cellular adhesion molecule expression down-regulation in vitro (e.g., in a cell culture). This method involves contacting cells with an amount of a composition effective to prevent and/or reduce the amount of ICAM expression down-regulation, wherein the composition includes one or more polypeptides described herein.

The present invention provides a method for inhibiting angiogenesis (i.e., new blood vessel formation) in a patient (e.g., a mammal such as a human). This involves administering to a patient an amount of a pharmaceutical composition effective to prevent and/or reduce angiogenesis, wherein the pharmaceutical composition includes one or more polypeptides described herein. Analogously, the present invention provides a method for inhibiting angiogenesis in vitro (e.g., in a cell culture). This method involves contacting cells with an amount of a composition effective to prevent and/or reduce angiogenesis, wherein the composition includes one or more polypeptides described herein.

For determining the amount of angiogenesis in vivo, various methods known to one of skill in the art could be used. For example, for evaluation of angiogenesis in tumors, tissue sections can be appropriately stained to quantify vessel density. For determining the amount of angiogenesis in vitro, the In vitro Angiogenesis Assay (i.e., EC Tube Formation Assay) described in the Examples Section can be used, which involves the disappearance of EC sprouting in cell culture. A polypeptide that is "active" for angiogenesis inhibition is preferably one that causes an at least 10% reduction in endothelial cell sprouting at a concentration lower than 10.sup.-4 M.

The present invention provides a method for inhibiting tumorigenesis in a patient (e.g., a mammal such as a human). This involves administering to a patient an amount of a pharmaceutical composition effective to prevent and/or reduce tumor growth, wherein the pharmaceutical composition includes one or more polypeptides described herein. Methods of determining the inhibition of tumorigenesis are well known to those of skill in the art, including evaluation of tumor shrinkage, survival, etc.

A preferred polypeptide is selected from the group consisting of: ANIKLSVQMKLF (SEQ ID NO:1); KLSVQMKLFKRH (SEQ ID NO:2); VQMKLFKRHLKW (SEQ ID NO:3); KLFKRHLKWKII (SEQ ID NO:4); KRHLKWKIIVKL (SEQ ID NO:5); LKWKIIVKLNDG (SEQ ID NO:6); KIIVKLNDGREL (SEQ ID NO:7); VKLNDGRELSLD (SEQ ID NO:8); and analogs thereof.

An alternative polypeptide is selected from the group consisting of: QMKLFKRHLKWK (SEQ ID NO:9); MKLFKRHLKWKI (SEQ ID NO:10); MKLFKRHLKWKIIV (SEQ ID NO:11); XLFKRHLKWKII (SEQ ID NO:12); KLFXRHLKWKII (SEQ ID NO:13); KLFKRHLXWKII (SEQ ID NO:14); KLFKRHLKWXII (SEQ ID NO: 15); KLFKKHLKWKII (SEQ ID N