||||||||| US005523288A United States Patent (19) 11 Patent Number: 5,523,288 Cohen et al. 45) Date of Patent: Jun. 4, 1996

(54) METHOD OF TREATING GRAM-NEGATIVE 5,348,942 9/1994 Little, II et al...... 514/12 BACTERIAL INFECTION BY ADMINISTRATION OF FOREIGN PATENT DOCUMENTS BACTERICDAL/PERMEABILITY-NCREASING WO89/01486 2/1989 WIPO (BPI) PROTEIN PRODUCT AND ANTIBIOTIC 8912644 12/1989 WIPO. WO92/03535 3/1992 WIPO 75) Inventors: Jonathan Cohen, London, United WO92/0962 6/1992 WIPO. Kingdom; Ada H. C. Kung, Woodside, WO93/06228 4/1993 WIPO WO93/05797 4/1993 WIPO Calif.; Lewis H. Lambert, Jr., WO93/23540 11/1993 WIPO. Fremont, Calif.; Roger G. Little, II, WO93/23434 1/1993 WIPO. Benicia, Calif. WO94/18323 8/1994 WIPO WO94/17819 8/1994 WIPO. 73 Assignee: Xoma Corporation, Berkeley, Calif. WO94/20129 9/1994 WIPO WO94/20128 9/1994 WIPO (21) Appl. No.: 311,611 OTHER PUBLICATIONS 22 Filed: Sep. 22, 1994 Davies, "Inactivation of Antibiotics and the Dissemination of Resistance Genes', Science, 264:375–381 (Apr. 15, Related U.S. Application Data 1994). 63) Continuation-in-part of Ser. No. 273,401, Jul. 11, 1994, Eliopoulos and Moellering, "Antimicrobial Combinations", abandoned, which is a continuation-in-part of Ser. No. in Antibiotics in Laboratory Medicine, 3rd ed., pp. 432-492, 125,651, Sep. 22, 1993, abandoned. (Lorian ed., Baltimore, MD) (1991). (51) Int. Cl...... A61K 38/17; C07G 11/00; Elsbach, 'Antibiotics from Within: Antibacterials from C07H 15/234; C07K 14/47 Human and Animal Sources', Trends. Biotech., 8(1):26-30 52 U.S. Cl...... 514/12; 514/152; 514/192; (Jan. 1990). 424/114; 530/319; 530/350, 536/13.6; 536/16.8 (List continued on next page.) 58) Field of Search ...... 514/12, 152, 192; 424/114, 405; 536/7.2, 13.1, 13.2, 13.3, Primary Examiner-Mindy Fleisher 13.4, 13.5, 13.7, 13.9, 16.9, 14, 13.6, 8.8, Assistant Examiner-Nancy J. Degen 16.1, 16.2, 16.8; 530/319, 350; 540/226, Attorney, Agent, or Firm-Marshall, O'Toole, Gerstein, 304,314, 342, 335, 341,552/202, 204, Murray & Borun 205 57 ABSTRACT 56) References Cited The present invention relates to methods and compositions U.S. PATENT DOCUMENTS for treating gram-negative bacterial infections, using BPI protein products. Co-treatment, or concurrent administra 4,863,727 8/1989 Zimmerman et al...... 424/85.2 tion, of BPI protein product with an antibiotic in treatment 5,000,958 3/1991 Fountain et al...... 424/450 5,089,274 2/1992 Marra et al. ... 504/38 of gram-negative bacterial infections improves the therapeu 5,156,665. 10/1992 Sherba et al...... 514/12 tic effectiveness of the antibiotic, including increasing anti 5,171,739 12/1992 Scott ...... 435/69.1 biotic susceptibility of gram-negative bacteria and reversing 5, 198,541 3/1993 Elsbach et al...... 514/21 resistance of the bacteria to antibiotics.

5,234,912 8/1993 Marra et al...... 514/12 5,308,834 5/1994 Scott et al...... 514/12 5,334.584 8/1994 Scott et al...... 514/12 37 Claims, 22 Drawing Sheets

Gentamicin 20 rBPI 0.1 mg/kg Gentamicin (p<0.001 vs vehicle p: 0.05 vs reP+0.03 Genlankcin)

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OTHER PUBLICATIONS Ooi et al., "Endotoxin-neutralizing Properties of the 25 kD Elsbach and Weiss, "Oxygen-Independent Bactericidal Sys N-Terminal Fragment and a Newly Isolated 30 kD C-Ter tems of Polymorphonuclear Leukocytes', in Advances in minal Fragment of the 55-60 kD Bactericidal/Permeabili Inflammation Research, vol. 2, pp. 95-113 (Weissmann ed., ty-Increasing Protein of Human Neutrophils', J. Exp. Med., Raven Press, Ltd.) (1981). 174:649-655 (Sep. 1991). Elsbach and Weiss, "Oxygen-Independent Antimicrobial Ooi et al., “Isolation of Two Isoforms of a Novel 15-kDa Systems of Phagocytes," in Inflammation: Basic Principles Protein from Rabit Polymorphonuclear Leukocytes that and Clincial Correlates, pp. 603-636, (Gallin et al. eds., Modulate the Antibacterial Actions of Other Leukocyte Raven Press, Ltd.) (1992). Proteins”, J. Biol. Chem., 265(26): 15956-15962 (Sep. 15, Elsbach et al., "Separation and Purification of a Potent 1990). Bactericidal/Permeability-Increasing Protein and a Closely Spratt, "Resistance to Antibiotics Mediated by Target Alter Associated Phospholipase A from Rabbit Polymorpho ations', Science, 264:388-393 (Apr. 15, 1994). nuclear Leukocytes', J. Biol. Chem., 254(21):11000-11009 Stratton, "In Vitro Testing: Correlations Between Bacterial (Nov. 10, 1979). Susceptibility, Body Fluid Levels and Effectiveness of Anti Gabay, "Ubiquitous Natural Antibiotics”, Science, bacterial Therapy', in Antibiotics in Laboratory Medicine, 264:373-374 (Apr. 15, 1994). pp. 849-879 (Lorian ed., Williams & Wilkins) (1991). Gazzano-Santoro et al., "High-Affinity Binding of the Bac tericidal/Permeability-Increasing Protein and a Recombi Taber et al., "Bacterial Uptake of Aminoglycoside Antibi nant Amino-Terminal Fragment to the Lipid A Region of otics', Microbiological Reviews, 51(4):439–457 (Dec. Lipopolysaccharide', Infect. Immun., 60(11):4754-4761 1987). (Nov. 1992). Travis, "Reviving the Antibiotic Miracle', Science, Gray et al., "Cloning of the cDNA of a Human Neutrophil 264:360-362 (Apr. 15, 1994). Bactericidal Protein, J. Biol. Chem., 264(16):9505-9509 Vaara, "Agents that Increase the Permeability of the Outer (Jun. 5, 1989). Membrane", Microbiological Reviews, 56(3):395-411 (Sep. Kadurugamuwa et al., “Interaction of Gentamicin with the A 1992). Band and B Band Lipopolysaccharides of Pseudomonas Veld et al., "Effects of the Bactericidal/Permeability-In aeruginosa and Its Possible Lethal Effect', Antimicrobial creasing Protein of Polymorphonuclear Leukocytes on Iso Agents and Chemotherapy, 37(4):715-721 (Apr. 1993). lated Bacterial Cytoplasmic Membrane Vesicles', Infect. Kingman, "Resistance a European Problem, Too', Science, Immun., 56(5):1203–1208 (May 1988). 264:363-365 (Apr. 15, 1994). Levy et al., "Antibacterial 15-kDa Protein Isoforms (p15s) Weiss and Olsson, "Cellular and Subcellular Localization of are Members of a Novel Family of Leukocyte Proteins', J. the Bactericidal/Permeability-Increasing Protein of Neutro Biol. Chenn., 268(8):6058-6068 (Mar. 16, 1993). phils", Blood, 69(2):652-659 (Feb. 1987). Mannion et al., “Preferential Binding of the Neutrophil Weiss et al., "Human Bactericidal/Permeability-Increasing Cytoplasmic Granule-Derived Bactericidal/Permeability Protein and a Recombinant NH. Terminal Fragment Cause Increasing Protein to Target Bacteria”, J. Clin. Invest., Killing of Serum-resistant Gram-negative Bacteria in 142(8):2807-2812 (Apr. 15, 1989). Whole Blood and Inhibit Tumor Necrosis Factor Release Mannion et al., "Separation of Sublethal and Lethal Effects Induced by the Bacteria”, J. Clin. Invest., 90:1122-1130 of Polymorphonuclear Leukocytes on Escherichia coli", J. (Sep. 1992). Clin. Invest., 86:631-641 (Aug. 1990). Weiss et al., "Resistance of Gram-negative Bacteria to Mannion et al., "Separation of Sublethal and Lethal Effects Purified Bactericidal Leukocyte Proteins”, J. Clin. Invest., of the Bactericidal/Permeability Increasing Protein on 65:619-628 (Mar. 1980). Escherichia coli'', J. Clin. Invest, 85:853-860 (Mar. 1990). Nikaido, "Prevention of Drug Access to Bacterial Targets: Weiss et al., "The Role of Lipopolysaccharides in the Action Permeability Barriers and Active Efflux', Science, of the Bactericidal/Permeability-Increasing Neutrophil Pro 264:382–387 (Apr. 15, 1994). tein on the Bacterial Envelope', J. Immunol., Ooi et al., “A 25-kDa NH2-terminal Fragment Carries All 132(6):3109-3115 (Jun. 1984). the Antibacterial Activities of the Human Neutrophil Cross et al. "Choice of Bacteria in Animal Models of Sepsis' 60-kDa Bactericidal/Permeability-Increasing Protein', J. Infection & Immunity 61 (7):2741-2747 1993. Biol. Chem., 262(31): 14891-14894 (1987). Dictionary of Drugs 1990 pp. 900, 886, 282. U.S. Patent Jun. 4, 1996 Sheet 1 of 22 5,523,288

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O N M ru N 5,523,288 1. 2 METHOD OF TREATING GRAM-NEGATIVE exhibits anti-bacterial activity towards a broad range of BACTERAL INFECTION BY gram-negative bacteria at concentrations as low as 10 to ADMINISTRATION OF 10M, but that 100- to 1,000-fold higher concentrations of BACTERICEDAL/PERMEABILITY-INCREASING BPI were non-toxic to all of the gram-positive bacterial (BPI) PROTEIN PRODUCT AND ANTIBIOTIC 5 species, yeasts, and higher eukaryotic cells tested at that time. It was also reported that BPI at a concentration of This is a continuation-in-part of U.S. application Ser. No. 10-6M or 160 ug/ml had no toxic effect, when tested at a pH of either 7.0 or 5.5, on the gram-positive organisms Staphy 08/273.401 filed Jul. 11, 1994, now abandoned, which is a lococcus aureus (four strains), Staphylococcus epidermidis, continuation-in-part of U.S. application Ser. No. 08/125,651 Streptococcus faecalis, Bacillus subtills, Micrococcus lyso filed Sep. 22, 1993, now abandoned. O deikticus, and Listeria monocytogenes. BPI at 10M report edly had no toxic effect on the fungi Candida albicans and BACKGROUND OF THE INVENTION Candida parapsilosis at pH 7.0 or 5.5, and was non-toxic to higher eukaryotic cells such as human, robbit and sheep red The present invention relates generally to methods and blood cells and several human rumor cell lines. See also compositions for treating gram-negative bacterial infections, 15 Elsbach and Weiss, Advances in Inflammation Research, ed. and more specifically to the use of bactericidal/permeability G. Weissmann, Vol. 2, pages 95-113 Raven Press (1981). increasing protein (BPI) protein products for co-treatment of This reported target cell specificity was believed to be the such infections with an antibiotic substance. Co-treatment result of the strong attraction of BPI for lipopolysaccharide with BPI protein products can improve the therapeutic (LPS), which is unique to the outer membrane (or envelope) effectiveness of antibiotics in gram-negative bacterial infec 20 of gram-negative organisms. tions, increase the susceptibility of gram-negative organisms The precise mechanism by which BPI kills gram-negative to antibiotics, and reverse resistance of gram-negative bacteria is not yet completely elucidated, but it is believed organisms to antibiotics. that BPI must first bind to the surface of the bacteria through BPI is a protein isolated from the granules of mammalian electrostatic and hydrophobic interactions between the cat polymorphonuclear leukocytes (PMNs or neutrophils), 25 ionic BPI protein and negatively charged sites on LPS. LPS which are blood cells essential in the defense against invad has been referred to as "endotoxin' because of the potent ing microorganisms. Human BPI protein has been isolated inflammatory response that it stimulates, i.e., the release of from PMNs by acid extraction combined with either ion mediators by host inflammatory cells which may ultimately exchange chromatography Elsbach, J. Biol. Chem., result in irreversible endotoxic shock. BPI binds to lipid A, 254:11000 (1979) or E. coli affinity chromatography 30 reported to be the most toxic and most biologically active Weiss, et al., Blood, 69:652 (1987). BPI obtained in such component of LPS. a manner is referred to herein as natural BPI and has been In susceptible gram-negative bacteria, BPI binding is shown to have potent bactericidal activity against a broad thought to disrupt LPS structure, leading to activation of spectrum of gram-negative bacteria. The molecular weight 35 bacterial enzymes that degrade phospholipids and pepti of human BPI is approximately 55,000 daltons (55 kD). The doglycans, altering the permeability of the cell's outer amino acid sequence of the entire human BPI protein and the membrane, and initiating events that ultimately lead to cell nucleic acid sequence of DNA encoding the protein have death. Elsbach and Weiss (1992), supra). BPI is thought to been reported in FIG. 1 of Gray et al., J. Biol. Chem., act in two stages. The first is a sublethal stage that is 264:9505 (1989), incorporated herein by reference. The 40 characterized by immediate growth arrest, permeabilization Gray et al. amino acid sequence is set out in SEQID NO: of the outer membrane and selective activation of bacterial 69 hereto. enzymes that hydrolyze phospholipids and peptidoglycans. BPI is a strongly cationic protein. The N-terminal half of Bacteria at this stage can be rescued by growth in serum BPI accounts for the high net positive charge; the C-terminal albumin supplemented media Mannion et al., J. Clin. half of the molecule has a net charge of -3. Elsbach and 45 Invest, 85:853-860 (1990)). The second stage, defined by Weiss (1981), supra.) A proteolytic N-terminal fragment of growth inhibition that cannot be reversed by serum albumin, BPI having a molecular weight of about 25 kD has an occurs after prolonged exposure of the bacteria to BPI and amphipathic character, containing alternating hydrophobic is characterized by extensive physiologic and structural and hydrophilic regions. This N-termina fragment of human changes, including apparent damage to the inner cytoplas BPI possesses the anti-bacteria efficacy of the naturally SO mic membrane. derived 55 kD human BPI holoprotein. Ooi et at., J. Bio. Initial binding of BPI to LPS leads to organizational Chenn., 262:14891-14894 (1987). In contrast to the N-ter changes that probably result from binding to the anionic minal portion, the C-terminal region of the isolated human groups in the KDO region of LPS, which normally stabilize BPI protein displays only slightly detectable anti-bacterial the outer membrane through binding of Mg" and Ca". activity against gram-negative organisms. Ooi et at., J. Exp. 55 Attachment of BPI to the outer membrane of gram-negative Med., 174:649 (1991).) An N-terminal BPI fragment of bacteria produces rapid permeabitization of the outer mem approximately 23 kD, referred to as "rBPI2,” has been brane to hydrophobic agents such as actinomycin D. Bind produced by recombinant means and also retains anti ing of BPI and subsequent gram-negative bacterial killing bacterial activity against gram-negative organisms. Gaz depends, at least in part, upon the LPS polysacchafide chain zano-Santoro et al., Infect. Immun. 60:4754-4761 (1992). 60 length, with long O-chain bearing, "smooth' organisms The bactericidal effect of BPI has been reported to be being more resistant to BPI bactericidal effects than short highly specific togram-negative species, e.g., in Elsbach and O-chain beating, "rough' organisms Weiss et al., J. Clin. Weiss, Inflammation: Basic Principles and Clinical Corre Invest. 65:619-628 (1980)). This first stage of BPI action, lates, eds. Gallin et at., Chapter 30, Raven Press, Ltd. permeabilization of the gram-negative outer envelope, is (1992). BPI is commonly thought to be non-toxic for other 65 reversible upon dissociation of the BPI, a process requiting microorganisms, including yeast, and for higher eukaryotic the presence of divalent cations and synthesis of new LPS cells. Elsbach and Weiss (1992), supra, reported that BPI Weiss et al., J. Immunol. 132:3109-3115 (1984). Loss of 5,523,288 3 4. gram-negative bacterial viability, however, is not reversed the macrollide antibiotics such as erythromycin and olean by processes which restore the envelope integrity. suggest domycin; (4) agents affecting nucleic acid metabolism, e.g., ing that the bactericidal action is mediated by additional the fluoroquinolones, actinomycin, ethambutol, 5-fluorocy lesions induced in the target organism and which may be tosine, griseofulvin, rifamycins; and (5) drugs affecting situated at the cytoplasmic membrane (Mannion et at., J. intermediary metabolism, such as the sulfonamides, trime Clin. Invest. 86:631-64. 1 (1990)). Specific investigation of thoprim, and the tuberculostatic agents isoniazid and para aminosalicylic acid. Some agents may have more than one this possibility has shown that on a molar basis BPI is at least primary mechanism of action, especially at high concentra as inhibitory of cytoplasmic membrane vesicle function as tions. In addition, secondary changes in the structure or polymyxin B (In’t Veld et at., Infection and Immunity metabolism of the bacterial cell often occur after the primary 56:1203-1208 (1988)) but the exact mechanism as well as O effect of the antimicrobial drug. the relevance of such vesicles to studies of intact organisms The penicillins have a characteristic double-ring system has not yet been elucidated. composed of a 3-lactam ring, which provides the antibac BPI is also capable of neutralizing the endotoxic proper terial activity, and a thiazolidene ring. The penicillins are ties of LPS to which it binds. Because of its bactericidal differentiated by a single side chain that is unique for each properties for gram-negative organisms and its ability to 15 penicillin. The compounds are bactericidal and act by inhib neutralize LPS, BPI can be utilized for the treatment of iting bacterial transpeptidase, an enzyme involved in syn mammals suffering from diseases caused by gram-negative thesis of the bacterial cell wall. Because of their mechanism bacteria, such as bacteremia or sepsis. of action, penicillins are generally active against growing, U.S. Pat. No. 5,198,541 discloses recombinant genes but not resting, cells. Penicillins, especially penicillin G, encoding and methods for expression of BPI proteins, 20 have largely gram-positive activity; the relative insensitivity including BPI holoprotein and fragments of BPI. It also of gram-negative rods to penicillin G and several other describes the use of N-terminal fragments of BPI protein for penicillins is probably due to the permeability barrier of the co-treatment with certain antibiotics, specifically penicillin, outer membrane of gram-negative bacteria. Ampicillin, car cephalosporins, rifampicin and actinomycin D. benicillin, ticarcillin, and some other penicillins are active Gram-negative bacteria include bacteria from the follow 25 against gram-negative bacteria because they can pass ing species: Acidarninococcus, Acinetobacter, Aeromonas, through this outer membrane. Penicillins have relatively few Alcaligenes, Bacteroides, Bordetella, Branhamella, Bru adverse effects, the most important of which are the hyper cella, Calymmatobacterium, Carnpylobacter, Cardiobacte sensitivity (allergic) reactions. These compounds are widely rium, Chromobacterium, Citrobacter, Edwardsiella, Entero 30 distributed in the body, but do not enter cells and do not bacter, Escherichia, Flavobacterium, Francisella, usually accumulate in CSF. Fusobacterium, Haermophilus, Klebsiella, Legionella, Bacterial resistance to the penicillins is by production of Moraxella, Morganella, Neisseria, Pasturella, Plesiornonas, the enzyme f-lactamase, which catalyzes hydrolysis of the Proteus, Providencia, Pseudomonas, Salmonella, Serratia, B-lactam ring. The percentage of bacteria resistant to peni Shigella, Streptobacillus, Veillonella, Vibrio, and Yersinia 35 cillin has risen to about 80%. Several penicillins, including species. methicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin, Antibiotics are natural chemical substances of relatively are not affected by the B-lactamase of staphylococci. These low molecular weight produced by various species of micro antibiotics are useful against most B-lactamase-producing organisms, such as bacteria (including Bacillus species), species of Staphylococcus. However, a small number of actinomycetes (including Streptomyces) and fungi, that 40 species are resistant even to these penicillins. Some peni inhibit growth of or destroy other microorganisms. Sub cillins, amoxicillin and ticarcillin, are marketed in combi stances of similar structure and mode of action may be nation with clavulanic acid, which is a f-lactamase inhibitor Synthesized chemically, or natural compounds may be modi that covalently binds to the enzyme and prevents it from fied to produce semi-synthetic antibiotics. These biosyn hydrolyzing the antibiotics. Another inhibitor, sulbactam, is thetic and semi-synthetic derivatives are also effective as 45 marketed in combination with ampicillin. antibiotics. The major classes of antibiotics are (1) the The cephalosporins are characterized by a B-lactam ring, B-lactams, including the penicillins, cephalosporins and like the penicillins, but have an adjacent dihydrothiazine monobactams; (2) the aminoglycosides, e.g., gentamicin, ring instead of a thiazolidene ring. For convenience, these tobramycin, netilmycin, and amikacin; (3) the tetracyclines; compounds are generally classified by generations. The first (4) the sulfonamides and trimethoprim; (5) the fluoroqui 50 generation includes cephalothin, cephapirin, cefazolin, nolones, e.g., ciprofloxacin, norfloxacin, and ofloxacin; (6) cephalexin, cephradine and cefadroxil. These drugs gener vancomycin; (7) the macrollides, which include for example, ally have excellent gram-positive activity except for entero erythromycin, azithromycin, and clarithromycin; and (8) cocci and methicillin-resistant staphylococci, and have only other antibiotics, e.g., the polymyxins, chloramphenicol and modest gram-negative coverage. The second generation the lincosamides. 55 includes cefamandole, cefoxitin, ceforanide, cefuroxime, Antibiotics accomplish their anti-bacterial effect through cefuroxime axetil, cefaclor, cefonicid and cefotetan. This several mechanisms of action Which can be generally generation generally loses some gram-positive activity by grouped as follows: (1) agents acting on the bacterial cell weight and gains limited gram-negative coverage. The third wall such as bacitracin, the cephalosporins, cycloserine, generation includes cefotaxime, moxalactam, ceftizoxime, fosfomycin, the penicillins, ristocetin, and vancomycin; (2) 60 ceftriaxone, cefoperazone and ceftazidime. These com agents affecting the cell membrane or exerting a detergent pounds generally sacrifice further gram-positive activity by effect, such as colistin, novobiocin and polymyxins; (3) weight but gain substantial gram-negative coverage against agents affecting cellular mechanisms of replication, infor Enterobacter and sometimes are active against Pseudor mation transfer, and protein synthesis by their effects on nonas. The cephalosporins bind to penicillin-binding pro ribosomes, e.g., the aminoglycosides, the tetracyclines, 65 teins with varying affinity. Once binding occurs, protein chloramphenicol, clindamycin, cycloheximide, fucidin, lin synthesis is inhibited. Cephalosporins are usually well tol comycin, puromycin, rifampicin, other streptomycins, and erated; adverse effects include hypersensitivity reactions and 5,523.288 5 6 gastrointestinal effects. Cephalosporins may interact with PABA into tetrahydrofolic acid, which is a required cofactor nephrotoxic drags. particularly aminoglycosides. to increase in the synthesis of thymidines, purines and DNA. Sulfona toxicity. Resistance to cephalosporins is mediated by several mides have a wide range of activity against gram-positive mechanisms, including production of B-lactamase, although and gram-negative bacteria, but their usefulness has dimin some strains that do not produce f-lactamase are neverthe ished with increasingly high prevalence of bacterial resis less resistant. tance. The sulfonamide class of antibiotics includes sulfa Imipenem is a N-formimidoyl derivative of the mold cytine, sulfadiazine, sulfamethizole, sulfisoxazole, product thienamycin. It contains a B-lactam ring and some sulfamethoxazole, sulfabenzamide and sulfacetamide. what resembles penicillin except for differences in the Adverse effects include hypersensitivity reactions and occa second ring. It has activity against both gram-positive and 10 sional hematological toxicity. gram-negative organisms and is resistant to most B-lacta Trimethoprim is an inhibitor of the dihydrofolate reduc mases, although not those from Pseudomonas. It is marketed tase enzyme, which converts dihydrofolic to tetrahydrofolic in combination with cilastin, a compound that inhibits acid, a required factor for DNA synthesis. Adverse effects inactivation of imipenem in the kidney by renal dihydro include gastrointestinal distress and rare hematological tox peptidase I enzyme. Cilastin increases the concentration of 15 icity. Trimethoprim is also available in combination with imipenem in urine, although not in blood. sulfamethoxazole (also known as co-trimoxazole). The con Aztreonam is the first of a new group of antibiotics bination is usually bactericidal, although each agent singly referred to as the monobactams. These agents have a B-lac is usually bacteriostatic. The combination is the drug of tam ring but lack the second ring characteristic of the choice for Salmonella infections, some Shigella infections, penicillins and cephalosporins. It acts by binding to peni 20 E. coli traveler's diarrhea and Pneumocysas Carinii pneu cillin-binding proteins, and produces long, filamentous bac monia. terial shapes that eventually lyse. Aztreonam is active only The fluoroquinolones and quinolones are derivatives of against aerobic gram-negative bacteria, is susceptible to nalidixic acid, a naphthyridine derivative. These compounds inactivation by some 3-lactamases, and has few adverse are bactericidal, and impair DNA replication, transcription effects. 25 and repair by binding to the DNA and interfering with DNA The aminoglycosides contain amino sugars linked to an gyrase, an enzyme which catalyzes negative supercoiling of aminocyclitol ring by glycosidic bonds. They have similar DNA. The fluoroquinolones, which include norfloxacin, mechanisms of action and properties, but differ somewhat in ciprofioxacin, and ofioxacin, and the quinolones, which spectrum of action, toxicity, and susceptibility to bacterial include cinoxacin, have a broad spectrum of antimicrobial resistance. The compounds are bactericidal, with activity 30 activity against gram-negative and gram-positive organisms. against both gram-positive and gram-negative organisms, These compounds distribute widely through extravascular and act by binding to proteins on the 30S ribosome of tissue sites, have a long serum half-life, and present few bacteria and inhibiting protein synthesis. The aminoglyco adverse effects. Because of their effect on DNA, the drugs sides also bind to isolated LPS and have a very weak outer are contraindicated in pregnant patients and in children membrane permeabilizing effect. Taber et al., Microbio 35 whose skeletal growth is incomplete. logical Reviews 53:439-457 (1987)); Kadurugamuwa et al., Vancomycin is a glycopeptide, with a molecular weight of Antimicrobial Agents and Chemotherapy, 37:715–721 about 1500, produced by a fungus. It is primarily active (1993); Vaara, Microbiological Reviews 56: 395-411 against gram-positive bacteria. The drug inhibits one of the (1992)). This class of antibiotics includes amikacin, gen final steps in synthesis of the bacterial cell wall, and is thus tamicin, kanamycin, neomycin, netilmycin, paronomycin 40 effective only against growing organisms. It is used to treat and tobramycin. The aminoglycosides are usually reserved serious infections due to gram-positive cocci when penicillin for more serious infections because of severe adverse effects G is not useful because of bacterial resistance or patient including ototoxicity and nephrotoxicity. There is a narrow allergies. Vancomycin has two major adverse effects, oto therapeutic window between the concentration required to 45 toxicity and nephrotoxicity. These toxicities can be poten produce a therapeutic effect, e.g., 8 uglml for gentamicin, tiated by concurrent administration of another drug with the and the concentration that produces a toxic effect, e.g., 12 same adverse effect, such as an aminoglycoside. g/ml for gentamicin. Neomycin in particular is highly toxic The macrollides are bacteriostatic and act by binding to the and is never administered parenterally. 50S subunit of 70S ribosomes, resulting in inhibition of Tetracyclines have a common four-ring structure and are 50 protein synthesis. They have a broad spectrum of activity closely congeneric derivatives of the polycyclic naphthacen against gram-positive and gram-negative bacteria and may ecarboxamide. The compounds are bacteriostatic, and be bacteriostatic or bactericidal, depending on the concen inhibit protein synthesis by binding to the 30S subunit of tration achieved at sites of infection. The compounds dis microbial ribosomes and interfering with attachment of tribute widely in body fluids. Adverse effects include gas aminoacyl tRNA. The compounds have some activity 55 trointestinal distress and rare hypersensitivity reactions. The against both gram-positive and gram-negative bacteria; most common macrofide used is erythromycin, but the class however, their use is limited because many species are now includes other compounds such as clarithromycin and relatively resistant. Adverse effects include gastrointestinal azithromycin. effects, hepatotoxicity with large doses, and nephrotoxicity The polymyxins are a group of closely related antibiotic in some patients. This antibiotic class includes tetracycline, 60 substances produced by strains of Bacillus polymyxa. These chlortetracycline, demeclocycline, doxycycline, methacy drugs, which are cationic detergents, are relatively simple, cline, minocycline and oxytetracycline. basic peptides with molecular weights of about 1000. Their The sulfonamides are derivatives of sulfanilamide, a antimicrobial activity is restricted togram-negative bacteria. compound similar in structure to para-aminobenzoic acid They interact strongly with phospholipids and act by pen (PABA), which is an essential precursor for bacterial syn 65 etrating into and disrupting the structure of cell membranes. thesis of folic acid. The compounds are generally bactefio Polymyxin B also binds to the lipid A portion of endotoxin static, and act by competitively inhibiting incorporation of and neutralizes the toxic effects of this molecule. Polymyxin 5,523,288 7 8 B has severe adverse effects, including nephrotoxicity and cell wall proteins, thus restricting access of antibiotics to the neurotoxicity, and should not be administered concurrently bacterial cytoplasmic machinery; (4) reduced intracellular with other nephrotoxic or neurotoxic drugs. The drug thus transport of the drug; and (5) increased removal of antibi has limited use as a therapeutic agent because of high otics from the cell via membrane-associated pumps Ni systemic toxicity, but may be used for severe infections, kaido, Science, 264:382–387 (1994). Such as Pseudomonas aeruginosa meningitis, that respond The susceptibility of a bacterial species to an antibiotic is poorly to other antibiotics. generally determined by two microbiological methods. A Chloramphenicol inhibits protein synthesis by binding to rapid but crude procedure uses commercially available filter the 50S ribosomal subunit and preventing binding of ami paper disks that have been impregnated with a specific noacyl tRNA. It has a fairly wide spectrum of antimicrobial 10 quantity of the antibiotic drug. These disks are placed on the activity, but is only reserved for serious infections, such as surface of agar plates that have been streaked with a culture meningitis, typhus, typhoid fever, and Rocky Mountain of the organism being tested, and the plates are observed for spotted fever, because of its severe and fatal adverse hema zones of growth inhibition. A more accurate technique, the tological effects. It is primarily bacteriostatic, although it broth dilution susceptibility test, involves preparing test may be bactericidal to certain species. 15 tubes containing serial dilutions of the drug in liquid culture Lincomycin and clindamycin are lincosamide antimicro media, then inoculating the organism being tested into the bials. They consist of an amino acid linked to an amino tubes. The lowest concentration of drug that inhibits growth sugar. Both inhibit protein synthesis by binding to the 50S of the bacteria after a suitable period of incubation is ribosomal subunit. They compete with erythromycin and reported as the minimum inhibitory concentration. chloramphenicol for the same binding site but in an over 20 The resistance or susceptibility of an organism to an lapping fashion. They may be bacteriostatic or bactericidal, antibiotic is determined on the basis of clinical outcome, i.e., depending on relative concentration and susceptibility. Gas whether administration of that antibiotic to a subject infected trointestinal distress is the most common side effect. Other by that organism will successfully cure the subject. While an adverse reactions include cutaneous hypersensitivity, tran organism may literally be susceptible to a high concentration sient hematological abnormalities, and minor elevations of 25 of an antibiotic in vitro, the organism may in fact be resistant hepatic enzymes. Clindamycin is often the drug of choice for to that antibiotic at physiologically realistic concentrations. infections caused by anaerobic bacteria or mixed aerobic? If the concentration of drug required to inhibit growth of or anaerobic infections, and can also be used for susceptible kill the organism is greater than the concentration that can aerobic gram-positive cocci. safely be achieved without toxicity to the subject, the Some drugs, e.g. aminoglycosides, have a small thera 30 microorganism is considered to be resistant to the antibiotic. peutic window. For example, 2 to 4 g/ml of gentamicin or To facilitate the identification of antibiotic resistance or tobramycin may be required for inhibition of bacterial susceptibility using in vitro test results, the National Com growth, but peak concentrations in plasma above 6 to 10 mittee for Clinical Laboratory Standards (NCCLS) has ug/ml may result in ototoxicity or nephrotoxicity. These formulated standards for antibiotic susceptibility that corre agents are more difficult to administer because the ratio of 35 late clinical outcome to in vitro determinations of the toxic to therapeutic concentrations is very low. Antimicro minimum inhibitory concentration of antibiotic. bial agents that have toxic effects on the kidneys and that are Thus, there exists a desire in the an for agents that could also eliminated primarily by the kidneys, such as the ami act as adjuncts to conventional antibiotic therapy and that noglycosides or Vancomycin, require particular caution 40 could act to improve the therapeutic effectiveness of anti because reduced elimination can lead to increased plasma biotics. concentrations. which in turn may cause increased toxicity. Doses of antimicrobial agents that are eliminated by the kidneys must be reduced in patients with impaired renal SUMMARY OF THE INVENTION function. Similarly, dosages of drugs that are metabolized or 45 The present invention generally provides methods and excreted by the liver, such as erythromycin, chlorampheni compositions for prophylaxis or treatment of gram-negative col, or clindamycin, must be reduced in patients with bacterial infections, using BPI protein products. The meth decreased hepatic function. ods and compositions, in addition to being useful for treat Antibiotic resistance in bacteria is an increasingly trouble ment, are useful for prophylaxis of patients at high risk of some problem. The accelerating development of antibiotic 50 gram-negative bacterial infection, e.g., patients who will resistant bacteria, intensified by the widespread use of undergo abdominal or genitourinary surgery, or trauma antibiotics in farm animals and overprescription of antibi victims. Specifically, the present invention provides, in a otics by physicians, has been accompanied by declining method for treating a gram-negative bacterial infection with research into new antibiotics with different modes of action. an antibiotic, the improvement comprising the step of con Science, 264:360-374 (1994). Antibiotic resistance, once 55 currently administering BPI protein product in an amount acquired, can be rapidly spread to other bacteria, including effective to improve the therapeutic effectiveness of the bacteria of a different species. There are some species of antibiotic. bacteria that are resistant to all but one antibiotic; it may be The present invention is based upon the finding that BPI only a matter of time before the appearance of bacterial protein product is useful as adjunct therapy with conven strains that are resistant to all antibiotics. 60 tional antibiotics, and specifically the finding that concurrent Bacteria acquire resistance to antibiotics through several administration, or co-treatment, of a BPI protein product and mechanisms: (1) production of enzymes that destroy or an antibiotic or combination of antibiotics can improve the inactivate the antibiotic Davies, Science, 264:375-381 therapeutic effectiveness of the antibiotic or combination of (1994); (2) synthesis of new or altered target sites on or antibiotics. BPI protein product may improve the therapeutic within the cell that axe not recognized by the antibiotic 65 effectiveness of antibiotics in a variety of ways, including by Spratt, Science, 264:388-393 (1994); (3) low permeability increasing susceptibility of gram-negative bacteria to a to antibiotics, which can be reduced even further by altering reduced dosage of antibiotics, by effectively reversing resis 5,523,288 9 10 tance of gram-negative bacteria to antibiotics, by providing BRIEF DESCRIPTION OF THE DRAWINGS synergistic or potentiating effects beyond the individual or additive effects of the BPI protein product or antibiotic FIG. 1 depicts survival data after treatment with rBPI alone, or by neutralizing endotoxin released by bacteria and cefamandole, separately or in combination, in an E. coli killed by antibiotics. Concurrent administration of BPI pro O 111:B4 mouse peritonitis assay. tein product and antibiotic provides unexpectedly superior FIGS. 2-4 relate to results from an E. coli O111:B4 mouse therapeutic effects in vivo than either agent provides when peritonitis assay with rBPI and cefamandole, separately or administered alone. Concurrent administration of BPI pro in combination. FIG. 2 depicts survival data; FIG. 3 shows tein product according to this improved method of treatment bacterial counts from peritoneal lavage fluid; and FIG. 4 is effective even when the gram-negative bacteria involved 10 shows bacterial counts in blood. are considered to be resistant to the bactericidal effects of FIGS. 5A and 5B display results from two trials of BPI protein product alone and/or antibiotic alone. treatment with rBPI2 and cefamandole, separately or in The present invention provides a use of a BPI protein combination, in an E. coli O7:K1 mouse peritonitis assay. product for the manufacture of a medicament for the co FIGS. 6-16 relate to results, including cardiovascular and treatment with an antibiotic of a gram-negative bacterial 5 metabolic findings, from an E. coli O7:K1 rabbit bacteremia infection in mammals. This aspect of the invention contem assay with rBPI2 and cefamandole, separately or in com plates co-treatment with any antibiotic or combinations of bination. FIG. 6 depicts survival data; FIG.7 shows bacterial antibiotics, including B-lactam antibiotics with and without count in blood, which FIG. 8 displays as percentage of B-lactamase inhibitors, aminoglycosides, tetracyclines, suf bacterial dose, FIG. 9 shows blood endotoxin levels; FIGS. fonamides and trimethoprim, Vancomycin, macrofides, fluo 20 10 shows mean arterial blood pressure; FIG. 11, cardiac roquinolones and quinolones, polymyxins and other antibi index; FIG. 12, total peripheral resistance; FIG. 13, arterial otics. oxygen tension; FIG. 14, alveolar-artefial oxygen gradient; This aspect of the invention also provides the use of a BPI FIG. 15, respiration rate; and FIG. 16, arterial blood pH. protein product for the manufacture of a medicament for FIG. 17 depicts survival data after treatment with rBPI improving the therapeutic effectiveness of antibiotics in a 25 and gentamicin, separately or in combination, in an E. coli gram-negative bacterial infection, use of a BPI protein O7:Ki mouse peritonitis assay. product for the manufacture of a medicament for increasing FIG. 18 shows the bactericidal effect of rBPI and the susceptibility to an antibiotic of gram-negative bacteria cefifiaxone, separately or in combination, on growth of involved in the gram-negative bacterial infection, and use of ceftfiaxone-resistant E. coli. a BPI protein product for the manufacture of a medicament 30 for reversing resistance to an antibiotic of gram-negative FIGS. 19-25 relate to the synergistic effect of rBPI on bacteria involved in the gram-negative bacterial infection. antibiotic killing curves for selected gram-negative organ The invention utilizes any of the large variety of BPI isms. FIG. 19 shows the bactericidal effect of rBPI alone. protein products known to the art including natural BPI FIG. 20 shows the effect of rBPI in combination with protein, recombinant BPI protein, BPI fragments, BPI ana 35 trimethoprim/suffamethoxazole; FIG. 21, rBPI with cipro logs, BPI variants, and BPI peptides. Concurrent adminis floxacin; FIG. 22, rBPI with piperacillin; FIG. 23, rBPI tration of BPI protein product with any antibiotic or com with cefotaxime; FIG. 24, rBPI with cefuroxime; and FIG. bination of antibiotics is contemplated, including 3-lactam 25, rBPI2 with amikacin. antibiotics with or without B-lactamase inhibitors, ami noglycosides, tetracyclines, sulfonamides and trimethoprim, 40 DETAILED DESCRIPTION Vancomycin, macrollides, fluoroquinolones and quinolones, The present invention relates to methods and composi polymyxins, and other antibiotics. tions for treating a gram-negative bacterial infection, using Either the BPI protein product or the antibiotic may be a BPI protein product. The invention is based on the administered systemically or topically to a subject suffering 45 unexpected discovery that, when treating a gram-negative from a suspected or confirmed gramnegative bacterial infec bacterial infection with an antibiotic, the concurrent admin tion. The BPI protein product and antibiotic may be admin istration of BPI protein product with the antibiotic improves istered by different routes and may be administered simul the therapeutic effectiveness of the antibiotic, even at doses taneously or sequentially. at which the BPI protein product alone or antibiotic alone The invention also provides pharmaceutical compositions 50 may be inactive. BPI protein product by itself typically has for treatment of gram-negative bacterial infections, compris an antibacterial potency less than that of conventional anti ing an antibiotic and a BPI protein product in an amount biotics. However, because its administration unexpectedly effective to improve the therapeutic effectiveness of the improves the therapeutic effectiveness of conventional anti antibiotic. Such compositions optionally comprise pharma biotic therapy. BPI protein product is useful as adjunct ceutically acceptable diluents, adjuvants or carriers. The 55 therapy with conventional antibiotic therapy for the treat compositions may be formulated for systemic or topical ment of gram-negative bacterial infections. administration to subjects. In addition, compositions com "Gram-negative bacterial infection,' as used herein, prising BPI protein product and an antibiotic can be used in encompasses conditions associated with or resulting from a variety of in vitro uses, such as use as a bactericide to gram-negative bacterial infection (e.g., sequelae). These decontaminate fluids and surfaces and to sterilize surgical 60 conditions include gram-negative sepsis, endotoxin-related and other medical equipment and implantable devices, hypotension and shock, and one or more of the conditions including prosthetic joints. associated therewith, including fever, metabolic acidosis, Numerous additional aspects and advantages of the inven disseminated intravascular coagulation and related clotting tion will become apparent to those skilled in the art upon disorders, anemia, thrombocytopenia, leukopenia, adult res consideration of the following detailed description of the 65 piratory distress syndrome and related pulmonary disorders, invention which describes presently preferred embodiments renal failure and related renal disorders, hepatobiliary dis thereof. ease and central nervous system disorders. These conditions 5,523.288 11 12 also include translocation of bacteria from the intestines and By measuring the effect of antibiotics on the in vitro concomitant release of endotoxin. growth curves of gram-negative organisms, in the presence BPI protein product may improve the therapeutic effec or absence of a BPI protein product, the BPI protein product tiveness of the antibiotic in a variety of ways, including by may be shown to enhance the early antibacterial effect of increasing Susceptibility of gram-negative bacteria to a 5 antibiotics at 0-24 hours. Enhancement of early bactericidal/ reduced dosage of antibiotics, by effectively reversing resis growth inhibitory effects is important in determining thera tance of gram-negative bacteria to antibiotics, by providing peutic outcome. synergistic or potentiating effects beyond the individual or The BPI protein product and antibiotic may also be shown additive effects of the BPI protein product or antibiotic to have synergistic or potentiating effects beyond the indi alone, or by neutralizing endotoxin released by bacteria 10 vidual effects of each agent alone or the additive effects of killed by antibiotics. Concurrent administration of BPI pro the agents together. In a checkerboard assay, the combina tein product and antibiotic is expected to provide more tion of BPI protein product with antibiotics may be shown effective treatment of gram-negative bacterial infection. to result in a "synergistic' fractional inhibitory concentra Concurrent administration of the two agents may provide tion index (FIC). The checkerboard method is based on greater therapeutic effects in vivo than either agent provides additivity, which assumes that the result observed with when administered singly. It may permit a reduction in the 15 multiple drugs is the sum of the separate effects of the drugs dosage of one or both agents with achievement of a similar being tested; according to this system a FIC of less than 0.5 therapeutic effect. Alternatively, the concurrent administra is scored as synergy, 1 is scored as additive, and greater than tion may produce a more rapid or complete bactericidal/ 1 but less than 2 is scored as indifferent. In contrast, kinetic bacteriostatic effect than could be achieved with either agent assays are based on the idea that only one metabolic pathway alone. 20 at a time can be growth rate-limiting for an organism; Therapeutic effectiveness is based on a successful clinical according to this system, the combined effect of drugs that outcome, and does not require that the antimicrobial agent or do not interact with one another (autonomous or indifferent) agents kill 100% of the organisms involved in the infection. is simply the effect of the most active drug alone. Success depends on achieving a level of antibacterial activ Concurrent administration of BPI protein products and ity at the site of infection that is sufficient to inhibit the 25 antibiotics is shown herein to lower MICs of a variety of bacteria in a manner that tips the balance in favor of the host. antibiotics for a variety of gram-negative organisms. It is When host defenses are maximally effective, the antibacte also shown to reverse resistance of a variety of gmrn rial effect required may be minimal. Reducing organism load negative organisms to antibiotics. In some cases where BPI by even one log (a factor of 10) may permit the host's own protein product does not affect the MIC of antibiotic at 24 defenses to control the infection. In addition, augmenting an 30 hours, BPI protein product is shown herein to enhance the early bactericidal/baeteriostatic effect can be more important early bactericidal effect of antibiotics on growth curves at than long-term bactericidal/bacteriostatic effect. These early 0-7 or 7-24 hours. The BPI protein products exert these events are a significant and critical part of therapeutic effects even on gram-negative organisms that are not con success, because they allow time for host defense mecha sidered susceptible to the direct bactericidal or growth nisms to activate. Increasing the bactericidal rate may be 35 inhibitory effects of BPI protein product alone. It is also particularly important for infections such as meningitis, shown herein that the concurrent administration of BPI bone or joint infections. Stratton, Antibiotics in Laboratory protein products with antibiotics in vivo allows a reduction Medicine, 3rd ed. (Lorian, V., Ed.) pp. 849-879, Williams in the dosages of both agents to amounts that, if adminis and Wilkins, Baltimore Md. (1991). 40 tered alone, would be insufficient to exert the same clinical The effect of BPI protein product to improve the thera effect. peutic effectiveness of antibiotics in vivo may be demon Either the BPI protein product or the antibiotic, or both, strated in in vivo animal models, or may be predicted on the may be administered at levels at which neither would alone basis of a variety of in vitro tests, including (1) determina be therapeutically effective against a gram-negative bacte tions of the minimum inhibitory concentration (MIC) of an 45 rial infection. Alternatively, according to a preferred antibiotic required to inhibit growth of a gram-negative method, the antibiotic and BPI protein product can be organism for 24 hours, (2) determinations of the effect of an administered in amounts where each would alone be thera antibiotic on the kinetic growth curve of a gram-negative peutically effective against a gram-negative bacterial infec organism, and (3) checkerboard assays of the MIC of serial tion but wherein the combination of the two antibiotics dilutions of antibiotic alone or in combination with serial 50 provides even more potent effects. The BPI protein product dilutions of BPI protein product. Exemplary models or tests may be administered in an amount which increases suscep are described in Eliopoulos and Moellering In Antibiotics in tibility of gram-negative bacteria to reduced antibiotic dos Laboratory Medicine, 3rd ed. (Lorian, V., Ed.) pp. 432–492, age, or in an amount which reverses resistance of the Williams and Wilkins, Baltimore Md. (1991). gram-negative bacteria to an antibiotic. Using in vitro determinations of antibiotic MIC at 24 55 BPI protein product is thought to interact with a variety of hours, a BPI protein product may be shown to reduce the host defense elements present in whole blood or serum, MIC of the antibiotic. With this result, it is expected that including complement, p15 and LBP, and other cells and concurrent administration of the BPI protein product in vivo components of the immune system. Such interactions may will increase susceptibility of the gram-negative organism to result in potentiation of the activities of BPI protein product. the antibiotic. A BPI protein product may also be shown to 60 Because of these interactions, BPI protein products can be reduce the MIC of an antibiotic from the range in which the expected to exert even greater activity in vivo than in vitro. organism is considered clinically resistant to a range in Thus, while in vitro tests are predictive of in vivo utility, which the organism is considered clinically susceptible. absence of activity in vitro does not necessarily indicate With this result, it is expected that concurrent administration absence of activity in vivo. For example, BPI has been in vivo of the BPI protein product with the antibiotic will 65 observed to display a greater bactericidal effect on gram reverse resistance and effectively convert the antibiotic negative bacteria in whole blood or plasma assays than in resistant organism into an antibiotic-susceptible organism. assays using conventional media. Weiss et al., J. Clin. 5,523,288 13 14 Invest. 90:1122–1130 (1992)). This may be because con be used in a variety of in vitro uses such as use as a ventional in vitro systems lack the blood elements that bacteficide to alecontaminate fluids and surfaces and to facilitate or potentiate BPI's function in vivo, or because sterilize surgical and other medical equipment and implant conventional media contain higher than physiological con able devices, including prosthetic joints. The invention also centrations of magnesium and calcium, which are typically provides improved methods of in vitro decontamination of inhibitors of the antibacterial activity of BPI protein prod fluids and surfaces comprising administering a BPI protein ucts. Furthermore, in the host, BPI protein product is avail product in combination with an antibiotic. able to neutralize endotoxin released during antibiotic kill Either the BPI protein product or the antibiotics may be ing of bacteria, a further clinical benefit not seen in or administered systemically or topically. Systemic routes of predicted by in vitro tests. 10 administration include oral, intravenous, intramuscular or It is also contemplated that the BPI protein product be Subcutaneous injection, intrathecal, intraperitoneal (e.g. by administered with other products that potentiate the bacte intraperitoneal lavage), transpulmonary using aerosolized or ricidal activity of BPI protein products. For example, serum nebulized drug, or transdermal. Topical routes include complement potentiates the gram-negative bactericidal administration in the form of salves, ophthalmic drops, ear activity of BPI protein products; the combination of BPI 5 drops, or irrigation fluids (for, e.g., irrigation of wounds). protein product and serum complement provides synergistic "Concurrent administration,' or co-treatment, as used bactericidal/growth inhibitory effects. See, e.g., Ooi et al. J. herein includes administration of the agents together, or Biol. Chem., 265:15956 (1990) and Levy et al. J. Biol. before or after each other. The BPI protein product and Chem., 268: 6038-6083 (1993) which address naturally antibiotics may be administered by different routes. For occurring 15 kD proteins potentiating BPI antibacterial example, the BPI protein product may be administered activity. See also co-owned, co-pending PCT Application intravenously while the antibiotics are administered intra No. US94/07834 filed Jul. 13, 1994, which corresponds to muscularly, intravenously, subcutaneously, orally or intrap U.S. patent application Ser. No. 08/274,303 filed Jul. 11, eritoneally. Alternatively, the BPI protein product may be 1994 as a continuation-in-part of U.S. patent application Ser. administered intraperitoneally while the antibiotics are No. 08/093,201, now abandoned, filed Jul. 14, 1993. These administered intraperitoneally or intravenously, or the BPI applications, which are all incorporated herein by reference, 25 protein product may be administered in an aerosolized or describe methods for potenfiating gram-negative bacteri nebulized form while the antibiotics are administered, e.g., cidal activity of BPI protein products by administering intravenously. The BPI protein product and antibiotics are lipopolysaccharide binding protein (LBP) and LBP protein preferably both administered intravenously. The BPI protein products. LBP protein derivatives and derivative hybrids 30 product and antibiotics may be given sequentially in the which lack CD-14 immunostimulatory properties are same intravenous line, after an intermediate flush, or may be described in PCT Application No. US94/06931 filed Jun. 17, given in different intravenous lines. The BPI protein product 1994, which corresponds to co-owned, co-pending U.S. and antibiotics may be administered simultaneously or patent application Ser. No. 08/261,660, filed Jun. 17, 1994 as sequentially, as long as they are given in a manner sufficient a continuation-in-part of U.S. patent application Ser. No. 35 to allow both agents to achieve effective concentrations at 08/079,510, now abandoned, filed Jun. 17, 1993, the disclo the site of infection. sures of all of which are hereby incorporated by reference. As used herein, "BPI protein product” includes naturally An advantage provided by the present invention is the and recombinantly produced BPI protein; natural, synthetic, ability to provide more effective treatment of gram-negative and recombinant biologically active polypeptide fragments bacterial infection by virtue of the improved therapeutic 40 of BPI protein, biologically active polypeptide variants of effectiveness of the antibiotic treatment. Another advantage BPI protein or fragments thereof, including hybrid fusion is the ability to treat gram-negative organisms that are proteins and dimers; and biologically active polypeptide normally resistant to one or more antibiotics. Yet another analogs of BPI protein or fragments or variants thereof, advantage is the ability to accelerate the killing of gram including cysteine-substituted analogs. The BPI protein negative organisms by antibiotics. An additional advantage 45 products administered according to this invention may be is the ability to neutralize endotoxin released during antibi generated and/or isolated by any means known in the art. otic killing of bacteria. A further advantage is the ability to U.S. Pat. No. 5, 198,541, the disclosure of which is incor use lower concentrations of toxic antibiotics such as gen porated herein by reference, discloses recombinant genes tamicin and polymyxin B, or expensive antibiotics such as encoding and methods for expression of BPI proteins includ vancomycin. Because the use of some antibiotics is limited 50 ing recombinant BPI holoprotein, referred to as rBPIs and by their systemic toxicity or prohibitive cost, lowering the recombinant fragments of BPI. Co-owned, copending U.S. concentration of antibiotic required for therapeutic effec patent application Ser. No. 07/885,501, now abandoned, and tiveness reduces toxicity and/or cost of treatment, and thus a continuation-in-part thereof, U.S. patent application Ser. allows wider use of the antibiotic. The present invention No. 08/072,063, now U.S. Pat. No. 5,439,807, filed May 19, may also provide quality of life benefits due to, e.g., 55 1993 and corresponding PCT Application No. 93/04752 decreased duration of therapy, reduced stay in intensive care filed May 19, 1993, which are all incorporated herein by units or overall in the hospital, with the concomitant reduced reference, disclose novel methods for the purification of risk of serious nosocomial (hospital-acquired) infections. recombinant BPI protein products expressed in and secreted The invention further provides pharmaceutical composi from genetically transformed mammalian host cells in cul tions for treatment of gram-negative bacterial infections, 60 ture and discloses how one may produce large quantities of comprising an antibiotic and a BPI protein product in an recombinant BPI products suitable for incorporation into amount effective to improve the therapeutic effectiveness of stable, homogeneous pharmaceutical preparations. the antibiotic. Such compositions optionally comprise phar Biologically active fragments of BPI (BPI fragments) maceutically acceptable diluents, adjuvants or carriers. The include biologically active molecules that have the same or compositions may be formulated for systemic or topical 65 similar amino acid sequence as a natural human BPI holo administration to subjects. In addition. antiseptic composi protein, except that the fragment molecule lacks amino tions comprising BPI protein product and an antibiotic can terminal amino acids, internal amino acids, and/or caxboxy 5,523,288 15 16 terminal amino acids of the holoprotein. Nonlimiting 11, 1994, and corresponding PCT Application No. PCT/ examples of such fragments include a N-terminal fragment US95/03125 filed Mar. 13, 1995, the disclosures of which of natural human BPI of approximately 25 kD, described in are incorporated herein by reference. Ooi et al., J. Exp. Med., 174:649 (1991), and the recombi Other BPI protein products useful according to the meth nant expression product of DNA encoding N-terminal amino ods of the invention are peptides derived from or based on acids from 1 to about 193 or 199 of natural human BPI, BPI produced by recombinant or synthetic means (BPI described in Gazzano-Santoro et al., Infect. Immun. derived peptides), such as those described in co-owned and 60:4754-4761 (1992), and referred to as rBPI. In that copending PCT Application No. PCT/US94/10427 filed publication, an expression vector was used as a source of Sep. 15, 1994, which corresponds to U.S. patent application DNA encoding a recombinant expression product (rBPI) 10 Ser. No. 08/306,473 filed Sep. 15, 1994, and PCT Applica having the 31-residue signal sequence and the first 199 tion No. US94/O2465 filed Mar. 11, 1994, which corre amino acids of the N-terminus of the mature human BPI, as sponds to U.S. patent application Ser. No. 08/209,762, filed set out in FIG. 1 of Gray et al., supra, except that valine at Mar, 11, 1994, which is a continuation-in-part of U.S. patent position 151 is specified by GTG rather than GTC and application Ser. No. 08/183,222, filed Jan. 14, 1994, now residue 185 is glutamic acid (specified by GAG) rather than abandoned, which is a continuation-in-part of U.S. patent lysine (specified by AAG). Recombinantholoprotein (rBPI) 15 application Ser. No. 08/093,202, now abandoned, filed Jul. has also been produced having the sequence (SEQID NOS: 15, 1993 (for which the corresponding international appli 145 and 146) set out in FIG. 1 of Gray et al., supra, with the cation is PCT Application No. US94/02401 filed Mar. 11, exceptions noted for rBPI and with the exception that 1994), which is a continuation-in-part of U.S. patent appli residue 417 is alaninc (specified by GCT) rather than valine cation Ser. No. 08/030,644 filed March 12, 1993, now U.S. (specified by GTT). Other examples include dimeric forms 20 Pat. No. 5,348,942, the disclosures of all of which are of BPI fragments, as described in co-owned and co-pending incorporated herein by reference. U.S. patent application Ser. No. 08/212,132, now U.S. Pat. Presently preferred BPI protein products include recom No. 5,447,913, filed Mar. 11, 1994, and corresponding PCT binantly-produced N-terminal fragments of BPI, especially Application No. PCT/US95/03125 filed Mar. 13, 1995, the 25 those having a molecular weight of approximately between disclosures of which are incorporated herein by reference. 21 to 25 kD such as rBPI or rBPI, or dimeric forms of Preferred dimeric products include dimeric BPI protein these N-terminal fragments (e.g., rBPI dimer). Addition products wherein the monomers are amino-terminal BPI ally, preferred BPI protein products include rBPIs and fragments having the N-terminal residues from about 1 to BPI-derived peptides. 175 to about 1 to 199 of BPI holoprotein. A particularly 30 The administration of BPI protein products is preferably preferred dimeric product is the dimeric form of the BPI accomplished with a pharmaceutical composition compris fragment having N-terminal residues 1 through 193, desig ing a BPI protein product and a pharmaceutically acceptable nated rBPI dimer. diluent, adjuvant, or carrier. The BPI protein product may be Biologically active variants of BPI (BPI variants) include administered without or in conjunction with known surfac but are not limited to recombinant hybrid fusion proteins, 35 tants, other chemothempeutic agents or additional known comprising BPI holoprotein or biologically active fragment anti-microbial agents. A preferred pharmaceutical composi thereof and at least a portion of at least one other polypep tion containing BPI protein products (e.g., rBPIs, rBPI) tide, and dimeric forms of BPI variants. Examples of such comprises the BPI protein product at a concentration of 1 hybrid fusion proteins and dimeric forms are described by mg/ml in citmte buffered saline (5 or 20 mM citrate, 150 mM Theofan et al. in co-owned, copending U.S. patent applica 40 NaCl, pH 5.0) comprising 0.1% by weight of poloxamer 188 tion Ser. No. 07/885,911, now abandoned, and a continua (Pluronic F-68, BASF Wyandotte, Parsippany, N.J.) and tion-in-part application thereof, U.S. patent application Ser. 0.002% by weight of polysorbate 80 (Tween 80, ICI Ameri No. 08/064,693 filed May 19, 1993 and corresponding PCT cas Inc., Wilmington, Del.). Another preferred pharmaceu Application No. US93/04754 filed May 19, 1993, which are tical composition containing BPI protein products (e.g., all incorporated herein by reference and include hybrid 45 rBPI) comprises the BPI protein product at a concentration fusion proteins comprising, at the amino-terminal end, a BPI of 2 mg/ml in 5 mM citrate, 150 mMNaCl, 0.2% poloxamer protein or a biologically active fragment thereof and, at the 188 and 0.002% polysorbate 80. Such preferred combina carboxy-terminal end, at least one constant domain of an tions are described in co-owned, co-pending PCT Applica immunoglobulin heavy chain or allelic variant thereof. tion No. US94/O1239 filed Feb. 2, 1994, which corresponds Biologically active analogs of BPI (BPI analogs) include 50 to U.S. patent application Ser. No. 08/190,869 filed Feb. 2, but are not limited to BPI protein products wherein one or 1994, now U.S. Pat. No. 5,488,034, and U.S. patent appli more amino acid residues have been replaced by a different cation Ser. No. 08/012,360 filed Feb. 2, 1993, now aban amino acid. For example, co-owned, copending U.S. patent doned, the disclosures of all of which are incorporated application Ser. No. 08/013,801 filed Feb. 2, 1993, now U.S. herein by reference. Pat. No. 5,420,019, and corresponding PCT Application No. 55 Suitable antibiotics, and therapeutically effective concen US94/O1235 filed Feb. 2, 1994, the disclosures of which are trations thereof when administered with BPI protein prod incorporated herein by reference, discloses polypeptide ana ucts, may be determined in in vivo models or according to logs of BPI and BPI fragments wherein a cysteine residue is in vitro tests, for example, the in vitro minimum inhibitory replaced by a different amino acid. A preferred BPI protein concentration (MIC) and in vivo mouse peritonitis or rabbit product described by this application is the expression 60 bacteremia assays taught herein. Suitable antibiotics are product of DNA encoding from amino acid 1 to approxi antibiotics that act on the bacterial cell wall, cell membrane, mately 193 or 199 of the N-terminal amino acids of BPI protein metabolism or nucleic acid metabolism. These holoprotein, but wherein the cysteine at residue number 132 would include antibiotics or combinations of antibiotics is substituted with alanine and is designated rBPI Acys or from the following classes: B-lactam antibiotics with or rBPI. Other examples include dimeric forms of BPI ana 65 without B-lactamase inhibitors, aminoglycosides, tetracy logs; e.g. co-owned and co-pending U.S. patent application clines, sulfonamides and trimethoprim, Vancomycin, mac Ser. No. 08/212,132, now U.S. Pat. No. 5,497,913, filed Mar. rotides, fluoroquinolones and quinolones, polymyxins, and 5,523,288 17 18 other antibiotics. Dosage and administration of suitable given in 6 equally divided doses every 4 hours. For children, antibiotics are known in the art, and briefly summarized the preferred parenteral dose of mezlocillin is 150 to 300 below. mg/kg per day. Nafcillin is preferably administered intravenously to PENCILLINS adults in doses of 3 grams per day, given in 6 equally divided doses every 4 hours, with doubled doses for very severe When a BPI protein product is concurrently administered infections. In conventional administration, it is effective with a penicillin, for treatment of a gram-negative bacterial largely against gram-positive organisms. In children, the infection, the BPI protein product is generally given preferred parenteral dose is 20 to 50 mg/kg per day, in 2 parenterally in doses ranging from 1 ug/kg to 100 mg/kg O daily, and preferably at doses ranging from 1 mg/kg to 20 equally divided doses every 12 hours. The preferred oral mg/kg daily. The penicillin is generally given in doses dose for na?cillin ranges from 1 gram per day to 6 grams per ranging from 1 mg/kg to 750 mg/kg daily, preferably not to day in 4 to 6 divided doses. exceed 24 grams daily for adults (or 600 mg/kg daily for Oxacillin is preferably administered parenterally to adults children), and is preferably administered as follows: in doses of 2 to 12 grams per day, in 4 to 6 equally divided 5 doses. In conventional administration, it is effective largely Penicillin G is preferably administered parenterally to against gram-positive organisms. In children, oxacillin is adults in doses ranging from 600,000 to 1,000,000 units per day. In conventional administration, it is effective largely preferably administered in doses of 100 to 300 mg/kg per against gram-positive organisms. For treatment of pneumo day. coccal meningitis, penicillin G is administered in doses of 20 Piperacillin is preferably administered parenterally to 20–24 million units daily, in divided doses every 2 or 3 adults in doses ranging from 100 mg/kg, or 6 grams per day, hours. For children, the preferred parenteral dose of peni in 2 to 4 equally divided doses, up to a maximum of 24 cillin G is 300,000 to 1,000,000 units per day. One unit of grams per day, in 4 to 6 equally divided doses. Higher doses penicillin G contains 0.6 ug of pure sodium penicillin G (i.e., have been used without serious adverse effects. 1 mg is 1667 units). 25 Ticarcillin is preferably administered parenterally to Amoxicillin may be administered parenterally to adults in adults in doses ranging from 4 grams per day to 18 grams per doses ranging from 750 mg to 1.5 grams per day, in 3 equally day administered in 4 to 6 equally divided doses. The usual divided doses. For children, preferred parenteral doses of dose is 200 to 300 mg/kg per day. For children, the preferred amoxicillin range from 20 to 40 mg/kg per day in 3 equally parenteral dose of ticarcillin ranges from 50 mg/kg per day divided doses. Amoxicillin is also available in combination 30 to 300 mg/kg per day, given in 3, 4 or 6 equally divided with clavulanic acid, a 3-lactamase inhibitor. A 250 mg dose doses. The combination ticarcillin/clavulanate is preferably of the combination drug amoxicillin/clavulanate will contain administered parenterally to adults in doses of 200 to 300 250 mg of amoxicillin and either 125 or 62.5 mg of mg/kg per day (based on ticarcillin content), in 4 to 6 equally clavulanic acid. The combination is preferably administered divided doses. For adults, the usual dose is 3.1 grams (which to adults orally in doses of 750 mg per day divided into 3 35 contains 3 grams of ticarcillin and 100 mg of clavulanic equal doses every 8 hours, with a preferred dose of 1.5 acid) every 4 to 6 hours. The combination is also available grams per day for severe infections, given in 3 equally in a dose of 3.2 grams, which contains 3 grams ofticarcillin divided doses. In children, the preferred oral dose is 20 to 40 and 200 mg of clavulanic acid. mg/kg per day in 3 equally divided doses. In general, it is desirable to limit each intramuscular Ampicillin is preferably administered parenterally to 40 injection of a penicillin or cephalosporin to 2 grams; larger adults in doses of 6 to 12 grams per day for severeinfections, doses should be administered by multiple injections in in 3 to 4 equally divided doses. In children, the preferred different large muscle masses. parenteral dose of ampicillin is 50 to 200 mg/kg per day in 3 to 4 equally divided doses. Larger doses of up to 400 CEPHALOSPORNS mg/kg per day, for children, or 12 grams per day, for adults, 45 When a BPI protein product is concurrently administered may be administered parenterally for treatment of meningi with a cephalosporin, for treatment of a gram-negative tis. Ampicillin is also available in combination with sulbac bacterial infection, the BPI protein product is generally tam, a 3-lactamase inhibitor. Each 1.5 gram dose of ampi given parenterally in doses ranging from 1/g/kg to 100 cillin/sulbactam contains 1 gram of ampicillin and 0.5 grams mg/kg daily, and preferably at doses ranging from 1 mg/kg of Sulbactan. The combination is preferably administered 50 to 20 mg/kg daily. The cephalosporin is generally given in parenterally to adults in doses of 6 to 12 grams per day doses ranging from 1 ug/kg to 500 mg/kg daily, preferably divided into 4 equal doses every 6 hours, not to exceed a not to exceed 16 grams daily, and is preferably administered total of 12 grams per day. as follows: Azlocillin is preferably administered parenterally to 55 Cefamandole is preferably administered parenterally to adults in doses of 8 to 18 grams per day, given in 4 to 6 adults in doses ranging from 1.5 grams per day, given in 3 equally divided doses. equally divided doses every 8 hours, to 12 grams per day for Carbenicillin is preferably administered parenterally to life-threatening infections, given in 6 equally divided doses adults in doses of 30 to 40 grams per flay, given by every 4 hours. In children, cefamandole is preferably admin continuous infusion or in 4 to 6 equally divided doses. Daily 60 istered in doses ranging from 50 to 150 mg/kg per day, in 3 doses of up to 600 mg/kg have been used to treat children to 6 equally divided doses, not to exceed a total of 12 grams with life-threatening infections. per day. Mezlocillin is preferably administered to adults parenter Cefazolin is preferably administered parenterally to adults ally in doses of 100 to 300 mg/kg per day, given in 4 to 6 in doses of 750 mg per day, given in 3 equally divided doses equally divided doses. The usual dose is 16 to 18 grams per 65 every 8 hours. In severe, life-threatening infections, it may day; for life threatening infections, 350 mg/kg per day may be administered at doses of 6 grams per day divided into 4 be administered, but in doses not to exceed 24 grams per day equal doses every 6 hours; in rare instances, up to 12 grams 5,523,288 19 20 per day have been used. In children, the preferred parenteral from 1 to 4 grams per day in 2 to 4 equally divided doses. dose of cefazolin is 20 to 50 mg/kg per day, divided into 3 For children, the preferred dose is 20 to 50 mg/kg per day or 4 equal doses, with 100 mg/kg per day administered for in divided doses, with doses being doubled for severe severe infections. infections. Cefonicid is preferably administered parenterally to Cephalothin is usually administered parenterally to adults adults in doses ranging from 500 mg once daily, to 2 grams in doses of 8 to 12 grams per day. once daily for life-threatening infections. For intramuscular administration, a 2 gram dose should be divided into two 1-gram injections. OTHER BETA-LACTAMS 10 Cefoperazone is preferably administered parenterally to When a BPI protein product is concurrently administered adults in doses ranging from 2 grams per day, given in 2 with an imipenem antibiotic, for treatment of a gram equally divided doses every 12 hours, to 12 grams per day negative bacterial infection, the BPI protein product is for severe infections, given in 2, 3 or 4 equally divided generally given parenterally in doses ranging from 1 lug/kg doses. Doses up to 16 grams per day have been administered to 100 mg/kg daily, and preferably at doses ranging from 1 without complications. 5 mg/kg to 20 mg/kg daily. The imipenem is generally given Cefotetan is preferably administered parenterally to adults in doses ranging from 1 ug/kg to 100 mg/kg daily, and is in doses of 1 to 4 grams per day, in 2 equally divided doses preferably administered as follows: every 12 hours. Cefotetan may be administered in higher Imipenem is available in combination with cilastatin, an doses for life-threatening infections, not to exceed a total 20 inhibitor of the renal dipeptidase enzyme that rapidly inac dose of 6 grams per day. tivates imipenem. The combination is preferably adminis Cefotaxime is preferably administered parenterally to tered intramuscularly to adults in doses of 1 to 1.5 grams per adults in doses ranging from 1 to 12 grams per day, not to day, given in 2 equally divided doses every 12 hours. exceed 12 grams per day (2 grams every 4 hours) for Intramuscular doses exceeding 1.5 grams per day are not life-threatening infections. In children, the parenteral dose of 25 recommended. The combination is preferably administered cefotaxirne is preferably 50 to 180 mg/kg, divided into 4 to intravenously in doses ranging from 1 to 4 grams per day, in 6 equal doses. 4 equally divided doses every 6 hours; doses exceeding 50 Cefoxitin is preferably administered parenterally to adults mg/kg per day, or 4 grams per day, are not recommended. in doses ranging from 3 to 12 grams per day, given in 3, 4, When a BPI protein product is concurrently administered or 6 equally divided doses. In children, cefoxitin is prefer 30 with a monobactam antibiotic, for treatment of a gram ably administered parenterally in doses of 80 to 160 mg/kg negative bacterial infection, the BPI protein product is per day, given in 4 or 6 equally divided doses, not to exceed generally given parenterally in doses ranging from 1 g/kg a total dose of 12 grams per day. to 100 mg/kg daily, and preferably at doses ranging from 1 Ceftazidime is preferably administered parenterally to mg/kg to 20 mg/kg daily. The monobactam is generally adults in doses ranging from 500 mg per day, given in 2 to 35 given in doses ranging from 1 g/kg to 200 mg/kg daily, and 3 equally divided doses (every 8 or 12 hours), up to a is preferably administered as follows: maximum of 6 grams per day. In children, ceftazidime is Aztreonam is preferably administered parenterally to preferably administered intravenously in doses of 30 to 50 adults in doses ranging from 1 gram per day, given in 2 mg/kg, to a maximum of 6 grams per day. equally divided doses every 12 hours, up to a maximum Ceftizoxime is preferably administered parenterally to 40 recommended dose of 8 grams per day in cases of life adults in doses ranging from 1 gram per day, given in 2 threatening infection, given in 3 or 4 equally divided doses. equally divided doses every 12 hours, to 12 grams per day for life-threatening infections, given in 3 equally divided AMINOGLYCOSIDES doses every 8 hours. The usual adult dose is 1 to 2 grams 45 every 8 or 12 hours. For children, the preferred parenteral When a BPI protein product is concurrently administered dose is 50 mg/kg every 6 or 8 hours, for a total daily dose with an aminoglycoside, for treatment of a gram-negative of 200 mg/kg. bacterial infection, the BPI protein product is generally Ceftriaxone is preferably administered parenterally to given parenterally in doses ranging from 1 g/kg to 100 adults in doses ranging from 1 to 2 grams per day, given in 50 mg/kg daily, and preferably at doses ranging from 1 mg/kg 2 equally divided doses every 12 hours. It may be given in to 20 mg/kg daily. The aminoglycoside is generally given in higher doses, not to exceed a total of 4 grams per day. In doses ranging from 1 g/kg to 20 mg/kg daily, preferably not children, the preferred parenteral dose of ceftriaxone is 50 to to exceed 15 mg/kg daily, and is preferably administered as 75 mg/kg per day, not to exceed 2 grams per day. In follows: meningitis, ceftriaxone may be administered in doses of 100 55 When administering aminoglycosides, it is desirable to mg/kg per day, not to exceed 4 grams per day. measure serum peak and trough concentrations to ensure the Cefuroxime is preferably administered parenterally to adequacy and safety of the dosage. Dosages should gener adults in doses ranging from 2.25 to 4.5 grams per day, in 3 ally be adjusted to avoid toxic peak and trough concentra equally divided doses every 8 hours. For life-threatening tions. Amikacin is preferably administered parenterally to infections, 6 grams per day may be administered in 4 equally 60 adults and children in doses of 15 mg/kg per day, divided divided doses every 6 hours, and for meningitis, 9 grams per into two or three equal doses every 8 or 12 hours, and not day may be administered in 3 equally divided doses every 8 to exceed a total dose of 1.5 grams per day. For uncompli hours. For children, the preferred parenteral dose of cated infections, a dose of 500 mg amikacin per day, in 2 cefuroxime is 50 to 150 mg/kg per day in 3 to 4 equally equally divided doses, may be administered. Dosages should divided doses, or 240 mg/kg per day for meningitis. 65 be adjusted to avoid prolonged serum peak concentrations of Cephalexin is formulated for oral administration, and is amikacin above 35 g/ml and prolonged trough concentra preferably administered orally to adults in doses ranging tions greater than 10 g/ml. 5,523,288 21 22 Gentamicin is preferably administered parenterally to 960 mg trimethoprim/4.8g suffamethoxazole daily, and is adults in doses of 3 mg/kg per day, in three equally divided preferably administered as follows: doses every 8 hours. For life-threatening infections, up to 5 The combination trimethoprim/sulfamethoxazole is avail mg/kg per day in 3 to 4 equally divided doses may be able in a formulation containing a 1:5 ratio of t?imethoprim administered, but this dosage should be reduced to 3 mg/kg and sulfamethoxazole (e.g., 16 mg trimethoprim and 80 mg per day as soon as clinically indicated. For children, gen sulfamethoxazole). The combination is preferably adminis tamicin is preferably administered parenterally in doses of 6 tered intravenously to adults or children in doses of 8 to 10 to 7.5 mg/kg per day. Dosages should be adjusted to avoid mg/kg (based on the weight of the trimethoprim component) prolonged serum peak concentrations of gentamicin above per day, in 2 to 4 equally divided doses. For Pneumocysas 12 g/ml and prolonged trough concentrations greater than 10 carini infection, the combination can be administered in 2 g/ml. doses of 20 mg/kg (based on the weight of the trimethoprim Netilmicin may be administered parenterally to adults in component) per day, in 3-4 equally divided doses, to a doses ranging from 3 mg/kg per day, in 2 equally divided maximum recommended dose of 960 mg trimethoprim/4.8g doses every 12 hours, to 6.5 mg/kg per day for serious suffamethoxazole per day. Trimethoprim alone is preferably systemic infection, in 2 or 3 equally divided doses. In 15 administered orally to adults in doses of 200 mg per day. children, the preferred parenteral dose is 5.5 to 8 mg/kg per Sulfamethoxazole alone is preferably administered orally to day, in 2 or 3 equally divided doses. Dosages should be adults in doses of 2 to 3 grams per day, and to children orally adjusted to avoid prolonged serum peak concentrations of in doses of 50 to 60 mg/kg per day. netilmicin above 16 g/ml and prolonged serum trough concentrations above 4 pug/ml. 20 FLUOROQUINOLONES Tobramycin is preferably administered parenterally to When a BPI protein product is concurrently administered adults in doses of 3 mg/kg per day, given in three equally with a fiuoroquinolone or quinolone, for treatment of a divided doses every 8 hours. For lifethreatening infections, gram-negative bacterial infection, the BPI protein product is tobramycin may be administered in doses up to 5 mg/kg per generally given parenterally in doses ranging from 1 ug/kg day, in 3 or 4 equally divided doses, but this dosage should 25 to 100 mg/kg daily, and preferably at doses ranging from 1 be reduced to 3 mg/kg per day as soon as clinically indi mg/kg to 20 mg/kg daily. The fluoroquinolone or quinolone cated. In children, tobramycin is preferably administered is generally given in doses ranging from 1 g/kg to 50 mg/kg parenterally in doses of 6 to 7.5 mg/kg per day. Prolonged daily, preferably not to exceed 1 gram daily, and is prefer serum concentrations of tobramycin above 12 ug/ml should be avoided, and rising trough levels above 2 pg/ml may 30 ably administered as follows: indicate tissue accumulation, which may contribute to tox Norfloxacin is preferably administered orally to adults in icity. doses from 400 to 800 mg daily, divided into two doses every 12 hours. Cinoxacin is preferably administered orally Concurrent administration of BPI protein product with the to adults in doses of 1 gram per day, given in 2 or 4 equally aminoglycosides, including amikacin, gentamicin, netilmi 35 divided doses. Ciprofloxacin is preferably administered to cin and tobramycin, may permit a lowering of the dose of adults intravenously in doses from 400 to 800 mg daily, or these toxic antibiotics necessary to achieve a therapeutic orally in doses from 500 to 1500 mg daily, divided into two effect. doses every 12 hours. Ofioxacin is preferably administered TETRACYCLINES to adults intravenously in doses from 400 to 800 mg daily, 40 or orally in doses from 400 to 800 mg daily, divided into two When a BPI protein product is concurrently administered doses every 12 hours. with a tetracycline, for treatment of a gram-negative bacte rial infection, the BPI protein product is generally given VANCOMYCIN parenterally in doses ranging from 1 ug/kg to 100 mg/kg When a BPI protein product is concurrently administered daily, and preferably at doses ranging from 1 mg/kg to 20 45 with vancomycin, for treatment of a gram-negative bacterial mg/kg daily. The tetracycline is generally given in doses infection, the BPI protein product is generally given ranging from 1 ug/kg to 50 mg/kg daily, and is preferably parenterally in doses ranging from 1 ug/kg to 100 mg/kg administered as follows: daily, and preferably at doses ranging from 1 mg/kg to 20 The tetracycline antibiotics are generally administered to mg/kg daily. The Vancomycin is generally given in doses adults in doses of 1 to 2 grams per day. An exception is 50 ranging from 1 mg/kg to 50 mg/kg daily, and is preferably doxycycline, which is preferably administered intravenously administered parenterally to adults in doses of 2 grams per to adults in doses of 100 to 200 mg per day, and to children day, divided into 2 or 4 doses every 6 or 12 hours. In children in doses of 2 mg/lb per day. Tetracycline may be adminis it is preferably administered in doses of 40 mg/kg, given in tered parenterally to adults in doses of 0.5 to 2 grams per 4 equally divided doses every 6 hours. In conventional day, in 2 equally divided doses, and to children in doses of 55 administration, vancomycin is effective largely against 10 to 20 mg/kg per day. gram-positive organisms. SULFONAMIDES MACROLIDES When a BPI protein product is concurrently administered 60 When a BPI protein product is concurrently administered with a sulfonamide or trimethoprim, for treatment of a with a macrollide, for treatment of a gram-negative bacterial gram-negative bacterial infection, the BPI protein product is infection, the BPI protein product is generally given generally given parenterally in doses ranging from 1 ug/kg parenterally in doses ranging from 1 ug/kg to 100 mg/kg to 100 mg/kg daily, and preferably at doses ranging from 1 daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg to 20 mg/kg daily. The sulfonamide or trimethoprim 65 mg/kg daily. The macrollide is generally given in doses is generally given in doses ranging from 1 ug?kg to 150 ranging from 1 g/kg to 100 mg/kg daily, and is preferably mg/kg daily, preferably not to exceed a combination dose of administered as follows: 5,523,288 23 24 Erythromycin is preferably administered intravenously to of E. coli. Example 4 addresses the bactericidal effect of adults and children in doses of 15 to 20 mg/kg per day, given polymyxin B and BPI for a number of other gram-negative by continuous infusion or in 4 equally divided doses every strains. Example 5 addresses the effect of a BPI protein 6 hours. Erythromycin can be administered at doses up to 4 product with a cephalosporin antibiotic in an in vivo mouse grams per day in cases of very severe infection. 5 peritonitis E. coli O111:B4 challenge model. Example 6 also Clarithromycin is preferably administered orally to adults relates to the effect of a BPI protein product with a cepha in doses of 500 mg to 1 gram daily, in equally divided doses losporin antibiotic in an in vivo mouse peritonitis E. coli every 12 hours. O111: B4 challenge model. Example 7 relates to the effect Azithromycin is preferably administered orally to adults of a BPI protein product with a cephalosporin antibiotic in at a dose of 500 mg on the first day of treatment followed by an in vivo mouse peritonitis E. coli O7:K1 challenge model. 250 mg once daily for 4 days, for a total dose of 1.5 grams. Example 8 addresses the effect of a BPI protein product with a cephalosporin antibiotic in an in vivo rabbit bacteremia E. coli O7:K1 challenge model. Example 9 addresses the effect OTHERS of a BPI protein product with an aminoglycoside antibiotic When a BPI protein product is concurrently administered 5 in an in vivo mouse peritonitis E. coli O7:K1 challenge with other antibiotics, for treatment of a gram-negative model. Example 10 relates to the effect of a BPI protein bacterial infection, the BPI protein product is generally product in vitro on the antibiotic susceptibility of ceftriax given parenterally in doses ranging from 1 ug/kg to 100 one-resistant gram-negative organisms. Examples 11-19 mg/kg daily, and preferably at doses ranging from 1 mg/kg address large-scale screening of the antibiotic susceptibility to 20 mg/kg daily. 20 increasing effect of a BPI protein product on a variety of Polymyxin B is generally given in doses ranging from 1 gram-negative organisms: Pseudomonas aeruginosa and unit/kg to 45,000 units/kg daily, and is preferably adminis other Pseudomonas species (Example 11), E. coli (Example tered intravenously to adults and children in doses of 15,000 12), Citrobacter (Example 13), Klebsiella (Example 14), to 25,000 units/kg per day, divided into 2 equal doses every Enterobacter (Example 15), Serratia (Example 16), Proteus 12 hours. It may be administered intramuscularly in doses of (Example 17), Providencia (Example 18), Morganella 25,000 to 30,000 units/kg per day, although these injections (Example 19), Acinetobacter (Example 20), and Salmonella are very painful. Doses of polymyxin B as high as 45,000 and Shigella (Example 21). Example 22 examines the early units/kg per day have been used in limited clinical studies to in vitro bactericidal effect of BPI protein product and treat neonates for Pseudomonas aeruginosa sepsis. Poly selected antibiotics on E. coli J5, E. coli O7:K1, Entero myxin B is the treatment of choice for P. aeruginosa 30 bacter cloacae and Klebsiella pneumoniae. Example 23 meningitis, and is preferably administered intrathecally to examines the effect of a variety of BPI protein products on adults and older children in doses of 50,000 units once daily several representative organisms, Acinetobacter anitratus, for 3 to 4 days, followed by 50,000 units every other day; in Enterobacter cloacae, and two strains of E. coli. Example 24 children under two years old, it is administered intrathecally relates to the screening of BPI peptides for antibacterial in doses of 20,000 daily for 3 to 4 days, followed by 25,000 35 activity against E. coli. Example 25 addresses the effect on units every other day. E. coli O111:B4 of concurrent administration of BPI protein Chloramphenicol is preferably administered intrave product with tetracycline or gentamicin. nously to adults in doses of 50 mg/kg per day, in 4 equally divided doses; in exceptional cases, it can be administered in EXAMPLE 1. doses up to 100 mg/kg per day. In children, chloramphenicol 40 is preferably administered intravenously in doses of 25 mg/kg per day, although up to 100 mg/kg per day can be SYNERGISTIC BACTERICIDAL EFFECTS OF administered in cases of severe infection. GENTAMCIN AND BPI. ADMENISTION TO E. Clindamycin is preferably administered parenterally to COL AND E. COLI 011:B4 GRAM-NEGATIVE adults in doses ranging from 600 mg to 4.8 grams per day, 45 ORGANISMS given in 2, 3 or 4 equally divided doses. It is recommended In this example, a micro dilution plate minimum inhibi that the dose in each intramuscular injection not exceed 600 tory concentration (MIC) assay was conducted to determine mg. For children, clindamycin is preferably administered the sensitivity of E. coli organisms to the bactericidal effects parenterally in doses of 15-40 mg/kg per day, given in 3 or 50 of BPI protein products concurrently administered with the 4 equally divided doses. antibiotic gentamicin. The assays were conducted against Dosages of all antimicrobial agents should be adjusted in the BPI sensitive organism E. coli J5 (an Rc rough mutant patients with renal impairment or hepatic insufficiency, due of E. coli 0111:B4), and a BPI resistant organism E. coli to the reduced metabolism and/or excretion of the drugs in O111:B4. patients with these conditions. Doses in children should also 55 Specifically, organisms were grown overnight on blood be reduced, generally according to body weight. Those agar plates at 37 C. in air, single colonies were then skilled in the art can readily optimize effective dosages and sub-cultured in 100 mL of nutrient broth No. 2 (Oxoid administration regimens for the BPI protein product and the CM67) and incubated with gentle agitation on an orbital antibiotics in concurrent administration. shaker for 54 hours until in log phase. Fifty mL of the Other aspects and advantages of the present invention will 60 bacterial suspension was then spun down in a Dency be understood upon consideration of the following illustra BR401 bench centrifuge at 4000 rpm for 15 minutes and the tive examples. Example 1 addresses the bactericidal effect of pellet was resuspended and washed twice using sterile gentamicin and BPI when administered to two different normal saline. Bacteria were then resuspended in saline such strains of E. coli. Example 2 addresses the bactericidal effect that a 1:10 dilution had an optical density of 0.9 (+/-0.01) of gentamicin and BPI for a number of other gram-negative 65 at 325 nm (corresponding to approximately 4x10 cells per strains. Example 3 addresses the bactericidal effect of poly mL) and diluted to give a final concentration of 4x10 cells myxin B and BPI when administered to two different strains per mL in "BPI media' (described below). 5,523,288 25 26 All assays and dilutions of BPI protein products and gentamicin were performed using "BPI media' consisting of 50% peptone water (Oxoid L37, Lot: 25851279) with 0.1M MOPS (Sigma M-1254) buffered to pH 6.00 with sodium hydroxide. This provides a nutritive media with a low 5 protein and divalent cation concentration adjusted to a pH which is not inhibitory to bacterial growth and allows for readily measurable (though not optimal) BPI activity. rBPI and gentamicin (Sigma G-1264, Lot: 91H00325) were diluted in BPI media such that 100 uL of diluted BH, 10 50 L of diluted gentamicin and 50 ul of bacterial suspen sion in the final volume of 200 L per well, gave concen trations in serial dilutions from 1000 nM (25 ug per mL) BPI and 32 ug per mL of gentamicin with a fixed concentration of 10 cells/mL. Checkerboards were then constructed in 15 round boUomed 96 well microtitre plates (Greiner No. 650180) and incubated with non-sealing lids at 37° C. in air for 18 hours. Plates were then read by eye and in an automatic plate reader (Titretek Multiscan plus) at 580 nm. with visible growth corresponding to an optical density of 20 approximately 0.1 (see Table 1 for E. coli J5, and Table 3 for E. coli O111:134). Viable counts were made from the wells adjacent to the cutoff of visible growth for the two E. coli plates (see Table 2 for E. coli J5, and Table 4 for E. coli O 111:B4) by dilution of 10 ulin 990 Lof Nutrient Broth No. 25 2 and spread plates were prepared with 25ul on blood agar. The inhibitory activity of BPI protein product with antibiotic was evaluated by the method of Eliopoulos and Moellering In Antibiotics in Laboratory Medicine, 3rd ed. (Lorian, V., Ed.) pp. 432–492, Williams and Wilkins, Baltimore Md. 30 (1991), wherein a fractional inhibitory concentration index (FIC) of less than 0.5 was scored as synergy, 1 was scored as additive and greater than 1 but less than 2 was scored as indifferent. A positive synergistic interaction was demonstrated 35 between rBPI2 and gentamicin using the BPI sensitive E. coli J5 with gentamicin at a concentration of 0.25ug per mL, reducing the MIC of BPI by approximately eight-fold from 500 nM (12.5ug per mL) to 62.5 nM (1.56 ug per mL) with similar reductions of the minimum bactericidal concentra 40 tion (MBC).

TABLE 1. Visible Growth (bold line) of E. coli J5 and ODS (580 nm) Gentamicin (ug per ml)

BPI (nM): 32 16 8 4. 2 0.5 0.25 1.25 06 03 O 1000 07 067 06906 06807 073072 loss 068062.06s soo lossosol 052 lossos ossos. 059 10054 ossosa 250 0560s Os3 057 058.0570s 0600s 05419, 20 125 058 0530s 0530s 062-064.06 06021s 258 236 625 losososososososos in 1962s2 2s. 31 057 0580520s 056 loss 0633s 20625 259,286 16 057 059 || 057 058 059 0208148 240 262 29, 327 o ogo os3 060057054.058 on is 252 28s 330 326

65 5,523,288 27 28

TABLE 2 Visible Growth (bold line) of E. coli J5 and Viable Counts Gentamicin (ug per ml)

BPI (nM): 32 16 8 4 2 1 0.5 O.25 125 06 03 O 1000 Nc Inc NcNc Nc Nc Nc 0 || 0 || 0 || 0 || 0 500 Nc Nc Nc Nc Nc Nc o o 147 2 0 || 0 250 NC Nc Nc Nc Nc o o o o oo inc 125 Nc Nc Nc Nc o 0 || 0 || 0 || 0 Nc Nc 625 Nc Nc Nc Nc 0 || 0 | 12 Nc Nc Nc 31 NC NC Nc Nc 0 || 0 is Nc Nc Nc Nc 16 Nc Nc NC Nc o O 43s 438 Nc Nc Nc Inc O Nc Nc Nc Nc o O 267 - Nc Nc Nc Nc Counts expressed as counts per ml, NC = not counted, - E too eros to cont.

TABLE 3 Visible Growth (bold line) of E. coli 0.11:B4 and ODS (580 nm) Gentamicin (ug per ml)

BPI (nM): 32 16 8 4 2 1 0.5 0.25 .25 06 03 O 100 06 064.05705, Ios Ios I 0.19 is 32s 30s so 500 05405049050so losol 0479 24s 2227s 342 250 ossos ossos ossos osa is 2s 2s2 2s. 34s 125 04.05090470490so 109 19839 267 26, 328 625 062 ossosos os os2 059 is 2s2 2s 287 as 31 059 .054.054.0520s os os 19 so 2s. 278 357 16 06 0540s 06 052054.048 is 26 28s 284 349 0 054.058 0509,050 05309 is 2030932. 34

50 5,523.288 29

TABLE 4 Visible Growth (bold line) of E. coli 0.11:B4 and viable counts Gentamicin (ug per ml)

BPI (nM): 32 16 8 4. 2 l 0.5 0.25 125 .06 03 O 1000 NC Nc Nc Nc o o 6 NC NC NC NC soo Nc Nc Nc Nc o o o Nc Nc Nc Inc. 250 Nc Nc Nc inco o o NcNc NcNc 125 NC NC NC NC 0 || 0 || 0 NCNc NC NC NC 62.5 Nc Nc Nc Nc 0 || 0 || 0 c Nc Nc 3 Nc Nc Inc inco o 16 Nc NC Nc NC 0 || 0 || 0 o Nc Nc Nc Nc o o 287 + Counts expressed as count per ml, NC = not counted, -- are too Illinerous to cont. 25 EXAMPLE 2 goviae (11434), P. aeruginosa (10332) and P. aeruginosa (10662). The concurrent administration of gentamicin and SYNERGISTIC BACTERICEDAL EFFECTS OF GENTAMCIN AND BPI: ADMINISTRATION TO rBPI had only additive, indifferent or indeterminate effects ADDITIONAL GRAM-NEGATIVE ORGANISMS 30 against the other tested gram-negative bacteria. In this example, micro dilution plate MIC assays were conducted according to the method of Example 1 to deter mine the sensitivity of a variety of gram-negative organisms to the cytotoxic effects of BPI protein products concurrently 35 administered with gentamicin antibiotic. The results of those assays axe shown in Table 5 below. Positive synergistic bactericidal interactions were observed against E. coli J5, E. coli 01:K1, E. coli (S2252), K. oxytoca, E. tarda, Salmonella typhimurium (S2136) and Salmonella arizonae. Possible 40 synergistic bactericidal interactions (indicated as 'additive (+)") were observed against E. cloacae (10005), E. ger

TABLE 5 MIC Values and Results of Checkerboard Susceptibility Testing With BPI and Gentamicin MIC of Single MIC of Single Lowest MIC of the Agent BPI Agent Gentamicin Agents Together Interpretation Organism (NCTC) (nM) (ug/ml) rBPI/Gentamicin (Gentainicin) E. coli J5 250 1.0 62.5/0.125 synergy E. coli 01:Kl >2000 2.0 3.10.5 synergy E. coli (S2252) NCTC 10418 500 0.5 250/0.25 additive/synergy E. coli 01:B4 >2000 1.0 >2000.0 indifferent E. colt (S2216) >500 1.0 >500/10 indifferent E. coli H262 >500 - - indifferent E. dispar (S2162) >500 - - indifferent E. alcalescens (S2196) >2000 - - indifferent K. oxytoca >2000 1.0 1000/0.25 synergy K. pneumoniae >2000 0.5 >2000/0.5 indifferent E. cloacae (10005) >2000 1.0 1000/0.5 additive (+) E. gergoviae (11434) 2000 2.0 2000/0.5 additive (+) S. marcescens (10211) >2000 40 >2000/40 indifferent Prettgeri (S2253) >500 2.0 1000/10 indifferent P. vulgaris (4175) 500 0.25 2500.125 additive P. morgani (S2161) >2000 2.0 2000/20 indifferent P. aeruginosa (10332) 2000 0.5 31/0.25 additive (+) P. aeruginosa (840P) >2000 1.0 >2000/1.0 indifferent P. aeruginosa (10662) >2000 0.25 2000/0.125 additive (+) P. aeruginosa (U600) >2000 0.5 62.5/0.25 indifferent 5,523,288 31 32

TABLE 5-continued MIC Values and Results of Checkerboard Susceptibility Testing With BPI and Gentamicin MIC of Single MIC of Single Lowest MIC of the Agent BPI Agent Gentamicin Agents Together Interpretation Organism (NCTC) (nM) (ug/ml) rBPI/Gentamicin (Gentamicin) K. aerogenes NCTC 9496 >2000 1.0 2000/0.5 indifferent Shigella dysenteriae NCTC 4837 >2000 .0 2000.0 indifferent E. tarda 500 O 310.125 synergy K. rhinoscleromatis 500 10 s5000 indifferent Salmonella choleresuis >2000 --- indifferent Saimonella typhimurium (S2185) 500 0.25 >500,0.25 indifferent Salmonella typhimurium (S2136) >2000 >4.0 31f4.0 synergy Salmonella arizonae s500 10 synergy E. aerogenes (S2164) s2000 10 62.511.0 indifferent C. freundi >2000 -- indifferent (+) possibility of synergy but FIC incalculable because rBPI MIC outside tested concentration range.

EXAMPLE 3 20 SYNERGISTIC BACTERICIDAL EFFECTS OF POLYMYXIN BAND BPI: ADMINISTATION TO E. COLIJ5 AND E. COLI 01.11:B4 GRAM-NEGATIVE ORGANISMS 25 In this example, micro dilution plate MIC assays were conducted according to the method of Example 1 to deter mine the sensitivity of E. coli J5 and E. coli 01 11:B4 to the cytotoxic effects of BPI protein products concurrently administered with the antibiotic polymyxin B. Polymyxin B 30 solution with an activity of 10240 units/mL was prepared by preparing a solution of 1.595 mg of polymyxin B sulfate stock powder per mL, and diluting it in sterile water for injection as 20 pug in 12.54 mL. A positive synergistic interaction was demonstrated 35 between rBPI and polymyxin Busing E. coli 0111:B4 (see Tables 8 and 9) but the administration of polymyxin B with BPI did not have synergistic bactericidal effects with BPI when applied to E. coli J5 as illustrated by the results shown in Tables 6 and 7.

TABLE 6 Visible Growth (bold line) of E. coli J5 and ODS (580 nm) Polymyxin B (gfml)

BPI (nM): 5 2.5 1.25 6 3 15 O75 O75 O

2000 07 06 01 01 02001 01102.06 100 090030204 000 oooooo, soo loss 0001800 007 or or loos 250 049,000 || 0 || 012026025 013.06 as or loosansion isoloist 62.5 04.00204 13.14164.141.124 is 31 04001094 160 16s 184 issues 238 0 046 002123 230 254 28227024s 307 5,523,288 33

TABLE T Visible Growth (bold line) of E. coli J5 and Viable Counts Polymyxin B (ig/ml)

BPI (nM): 2000 1000 500 250 125 62.5 31

0.

Counts expressed as counts per ml, NC = not counted, --- r too numerous to Coult.

TABLE 8 Visible Growth (bold line) of E. coli 01.11:B4 and ODS (580 nM) Polymyxin B (ig/ml)

BPI (nM): 10 5 2.5 1.25 6 3 15 O75 O 2000 o ososo on on 29s 379 sco 100 o ososs io9 as 2.726.30s as soo o osos as 16280 23s. 292 2s2 250 o 06 on 146 26 26s as 294 260 125 o os to 19322s 269,233,298 269 625 o oso on 22 20 277 246 28s 28 31 o 0.054,245 223 260 234 29, 27 o o 0.144 267 265 295 on 309| 290 5,523.288 35 36 TABLE 9 Visible Growth (bold line) of E. coli O111:B4 and Viable Counts

BPI (nM): 2000

1000 500

250

125

Counts expressed as counts per ml, NC = not counted, -- E to eros to cont. 25 EXAMPLE 4 EXAMPLE 5

SYNERGISTIC BACTERICIDAL EFFECTS OF SYNERGISTC EFFECTS OF CEFAMANDOLE POLYMYXN BAND BPI. ADMINISTRATION AND BPI PROTEIN PRODUCT IN VIVO IN TO ADDITIONAL GRAM-NEGATIVE 30 MICE CHALLENGED INTRAPERITONEALLY ORGANISMS WITH LIVE E. COLI O111:B4 BACTERIA: In this example, micro dilution MIC assays were con EFFECT ON SURVIVAL ducted according to the method of Example 1 to determine In this example, the protective effect of cefamandole the sensitivity of a variety of gram-negative organisms to the nafate antibiotic (MANDOLCE), Lilly) a semisynthetic broad cytotoxic effects of BPI protein products concurrently 35 spectrum cephalosporin antibiotic with and without a BPI administered with polymyxin B antibiotic. The results of protein product was evaluated by means of challenging ICR those assays are shown in Table 10 below. mice with an LDoooo dose level of live E. coli 01.11:B4 These assay results show additive or synergistic effects bacteria, a strain that is not susceptible to the bactericidal/ with the use of polymyxin B at concentrations of 0.3 g/mL, 40 growth inhibitory effects of BPI protein product. Specifi a level of which is 10-20 times lower than when that cally, four groups of 15 ICR mice were treated such that each antibiotic is used conventionally. ICR mouse received an injection of bacteria (1.8x10 CFU/

TABLE 10 MIC Values and Results of Checkerboard Susceptibility Testing With BPI and Polymyxin B (PB) Lowest MIC of the MIC of Single MIC of Single Agents Together Interpretation Organism (NCTC) Agent BPI (nM) Agent PB (ug/ml) rBPI/PB (Polymyxin B) E. coli J5 250 2.5 25012.5 indifferent E. coli (S2252) NCTC 10418 500 5.0 25012.5 indifferent E. coli O111:B4 s2000 5.0 250/25 Synergy E. cloacae (10005) >500 0.5 5000.5 indifferent E. gergoviae (11434) >2000 5.0 12512.5 additive (+) S. marcescens (10211) >2000 >100 >2000.10.0 indifferent Prettgeri (S2253) >2000 2.5 1251.25 synergy P. aeruginosa (10332) 2000 1.25 250,0.6 synergy P. aeruginosa (840P) 2000 1.25 1000/06 synergy P. aeruginosa (10662) 2000 25 25,0.6 synergy P. aeruginosa (U600) >2000 1.25 2000/0.3 synergy E. aerogenes (S2164) >2000 >10.0 >2000.10.0 indifferent

mouse) intraperitoneally; an intraperitoneal injection of cefamandole nafate (MANDOLE), 100 mg/kg) or saline; and 65 then an intrapefitoneal injection of rBPI (500 g/mouse) or BPI buffer. Survival of the mice was then evaluated over a period of 7 days with the results illustrated in FIG. 1. 5,523.288 37 38 Concurrent administration of BPI protein product with that achieved by cefamandole alone (p<0.01 of concurrent cefamandole, or administration of cefamandole alone, administration vs. cefamandole only) at the 2 and 6 hour showed significant protection in mice challenged with E. time points, and entirely eliminated counts after 24 hours. coli O111:B4 when compared to the buffer control (ps0.001 and ps0.05 respectively). The protective effect of the BPI Blood of mice that received concurrent administration of protein product when administered without cefamandole rBPI and cefamandole was completely free of bacteria at was not evident. When compared to treatment with cefa all time points. mandole alone, concurrent administration of BPI protein Thus, the concurrent administration of a BPI protein product with cefamandole showed improved protection at product, rBPI2, with a suboptimal dose of a cephalosporin ps 0.1. These results indicate that the concurrent adminis 10 antibiotic, cefamandole, resulted in a superior therapeutic tration of cefamandole and a BPI protein product has effect. The data indicates that BPI protein products and synergistic therapeutic effects against E. coli 01 11:B4. cephalosporin antibiotics produce a synergistic therapeutic EXAMPLE 6 effect. Since cefamandole alone reduced counts by approxi 15 mately two orders of magnitude compared to vehicle-treated SYNERGISTIC EFFECTS OF CEFAMANDOLE animals, another experiment was conducted to determine if AND BPI PROTEIN PRODUCT IN VIVO IN rBPI2 alone reduced bacterial counts when the inoculum MICE CHALLENGED INTRAPERITONEALLY was reduced to 10 CFU. Data from this experiment showed WITH LIVE E. COLI 01.11:B4 BACTERIA: that rBPI (500 g) did not significantly reduce bacterial EFFECT ON SURVIVAL AND ON BACTERIAL 20 counts in blood or peritoneal lavage fluid after a challenge CLEARANCE FROM BLOOD AND of 10 CFU. This suggests that an antibiotic-mediated reduc PERITONEAL LAVAGE FLUID tion in the magnitude of the bacterial count cannot, in itself, The protective effects of a cephalosporin antibiotic and a explain the protection associated with concurrent adminis BPI protein product were evaluated in mice challenged tration of BPI protein product and antibiotic. intrapefitoneally with E. coli 0111:B4, a strain that is resis 25 tant to the bactericidal effects of BPI protein product. The assay was conducted using the following procedure. Male EXAMPLE 7 ICR mice (Simonsen Laboratories, Gilroy Calif.), 5–7 weeks old, were housed under controlled climate and dark/ light cycles and were allowed free access to food and water. 30 Mice received an intraperitoneal injection of 0.5 ml of SYNERGISTIC EFFECTS OF CEFAMANDOLE bacteria in doses near an LDoo (2x10 CFU/mouse). Imme AND BPI PROTEIN PRODUCT IN VIVO IN diately after bacterial challenge the animals received an MICE CHALLENGED INTRAPERITONEALLY intraperitoneal injection of (1) vehicle only, (2) 500 WITH LIVE E. COLI O7:K1 BACTERIA: uglimouse rBPI2 and vehicle, (3) 100 mg/kg cefamandole 35 EFFECT ON SURVIVAL nafate (Mandolce) in phosphate buffered saline; Eli Lilly, Indianapolis, Ind.) and vehicle, or (4) 500 ug rBPI2 and 100 The protective effects of a cephalosporin antibiotic and a mg/kg cefamandole. BPI protein product were evaluated in mice challenged Survival of the four groups was monitored for 7 days. 40 intraperitoneally with E. coli O7:K1 (ATCC Accession No. Survival data was statistically analyzed using the Chi-square 23503), a strain that is susceptible to the bactericidal effects test. In a separate experiment using a bacterial challenge of of BPI protein product. The general procedure described 2.5x10 CFU E. coli O 111:B4, blood and peritoneal lavage above in Example 5 was followed. Four groups of 20 mice fluid were collected for culture at different time points were challenged intraperitoneally with 2x10' E. coli O7:Kl following bacterial challenge. Blood was obtained from the 45 bacteria and then treated with (1) vehicle, (2) 50 ug rBPI retro-orbital sinus. At least 1 ml of peritoneal lavage fluid only, (3) 20 mg/kg cefamandole only, or (4) both 50 ug was obtained after intraperitoneal injection of 3 ml of rBPI and 20 mg/kg cefamandole. Survival of the mice was phosphate buffered saline. Bacterial counts (expressed as followed over a period of 7 days; results of two trials are CFU/ml) were determined by inoculating trypticase soy agar displayed in FIGS. 5A and 5B. plates with 10-fold dilutions of blood or peritoneal lavage samples, incubating the plates overnight at 37 C., and 50 In one trial, rBPI alone protected 11% of the survivors counting the colonies. Statistical comparisons of this data compared to vehicle controls. cefamandole protected 47% were performed with the analysis of variance. of the survivors compared to vehicle controls (p<0.05 vs. vehicle), and the concurrent administration of rBPI and FIG. 2 shows effects on survival of cefamandole (100 cefamandole protected 100% of the survivors compared to mg/kg), rBPI2 (500 ug) or the concurrent administration of 55 the two agents. The BPI protein product alone had no effect, vehicle controls (p<0.00 i vs. vehicle, p<0.01 vs. cefaman while cefamandole treatment alone increased survival, dole alone). In the second trial, rBPI alone protected 0% although not significantly. In contrast, the concurrent admin of the survivors compared to vehicle, cefamandole protected istration of rBPI with cefamandole resulted in a significant 12% compared to vehicle, and the concurrent administration increase in survival (p<0.05) above that achieved by either 60 of rBPI and cefamandole protected 59% compared to treatment alone. FIGS. 3 and 4 show that rBPI alone failed vehicle (p<0.01 vs. vehicle, p<0.05 vs. cefamandole alone). to reduce counts in either peritoneal lavage fluid or blood In both trials, the increase in survival associated with the after challenge with bacteria, while cefamandole treatment concurrent administration of rBPI2 and cefamandole was alone significantly reduced counts in both (p<0.01 vs. greater than the sum of the increases in survival due to the vehicle). However, the concurrent administration of rBPI 65 individual therapies. Thus, there appears to be synergy (a and cefamandole reduced bacterial counts in the peritoneal greater than additive effect) between cefamandole and BPI lavage fluid by more than two orders of magnitude below protein product in this model. 5,523,288 39 40 EXAMPLE 8 Blood samples for blood gas determinations were drawn from the femoral artery catheter every 30 minutes through SYNERGISTIC EFFECTS OF CEFAMANDOLE out the study. Blood gases were measured with a Ciba AND BPI PROTEIN PRODUCT IN VIVO IN Coming Blood Gas System, Model 278 (Ciba-Corning RABBITS CHALLENGED INTRAVENOUSLY Diagnostics Corp., Medfield, Mass.). The blood gas system WITH LIVE E. COLI O7:K1 BACTERIA: directly measures blood pH, partial pressure of pCO2, and EFFECT ON BACTERIAL CLEARANCE AND partial pressure of p0. Other parameters including the ON CARDIOVASCULAR, RESPIRATORY AND alveolar-arterial oxygen gradient, arterial oxygen content, METABOLIC PARAMETERS estimated oxygen saturation, standard bicarbonate, and in vivo base excess were calculated using the formulas pro Adult male New Zealand White rabbits (Charles River 10 vided by Ciba-Coming Diagnostic Corp. Plasma levels of Laboratories, St. Constant, Canada) weighing between 1.8 glucose and lactate were determined using a Glucose/L- and 2.3 kilograms were fasted for 24 hours before the lactate Analyzer (2300 STAT, YSI, Yellow Springs, Ohio). experiment. Each rabbit was anesthetized with an intramus Survival data is shown in FIG. 6. Two of the four animals cular injection of 80/4 mg/kg Ketamine/xylazine. The left (50%) treated with vehicle alone died before the end of the femoral artery was catheterized for blood pressure determi 15 experiment. None of the animals concurrently treated with nations and blood sample collection. A catheter was placed rBPI and cefamandole died. Bacterial counts in blood, adjacent the right atrium via the right jugular vein and a expressed as CFU/ml or percent bacteria dose/ml, are shown thermistor-tipped catheter was placed in the aortic arch via in FIGS. 7 and 8, respectively. In FIG. 7, the squares the right carotid artery. The rabbits were allowed to stabilize represent treatment with vehicle alone, the diamonds repre for 90-120 minutes following catheterization to normalize 20 sent cefamandole alone, the circles represent rBPI2 alone, hemodynamic and blood gas parameters. and the triangles represent the concurrent administration of rBPI and cefamandole. In FIG. 8, the bar with horizontal The rabbits were divided into four treatment groups with hatching indicates treatment with cefamandole alone, the 4 animals per group: (1) vehicle alone, (2) cefamandole and hollow bar indicates the concurrent administration of rBPI2 vehicle, (3) rBPI and vehicle, and (4) cefamandole and 25 and cefamandole, the bar with vertical hatching indicates rBPI. The rabbits were administered cefamandole (Man rBPI alone, and the solid bar indicates buffer alone. The dol(E); Eli Lilly, Indianapolis, Ind.) or vehicle intravenously group concurrently treated with rBPI and cefamandole 5 minutes before the start of the bacterial infusion (consid demonstrated a higher clearance of bacteria compared to the ered to be T-0). At T=0, 2x10'CFU/rabbit of E. coli O7:K1 groups treated with either rBPI alone or cefamandole was infused intravenously over 10 minutes into the ear vein. 30 alone. There appears to be a synergistic effect at 30 and 60 Simultaneously (at T=0), 10 mg/kg rBPI or vehicle was minutes; at 30 minutes, the concurrent administration of infused over 10 minutes via the right jugular catheter. After rBPI and cefamandole resulted in a higher percentage the ten minute infusion, rBPI was slowly infused at 10 clearance of bacteria than the sum of the separate treatments. mg/kg/hr for 2 hours (resulting in a total dose of 30 mg/kg Endotoxin levels are displayed in FIG. 9. The open rBPI). 35 diamonds indicate treatment with vehicle alone, filled dia Arterial blood samples for determination of bacterial monds indicate rBPI alone, the filled squares indicate counts and endotoxin levels were collected at the end of the cefamandole alone, and the open squares indicate concurrent 10 minute bacterial infusion and at 30 minutes, 1, 2, 3 and administration of rBPI and cefamandole. Animals admin 4 hours. The whole blood was 10-fold serially diluted in istered cefamandole alone have a much higher LPS level sterile PBS and aliquots were plated onto tryptic soy agar 40 than animals treated with vehicle alone, due to release of plates, incubated at 37° C. overnight, and the plates were LPS as cefamandole kills bacteria. The concurrent admin counted for colony forming units (CFU). The results were istration of rBPI2 and cefamandole produced a dramatic expressed as CFU/ml blood and percent bacteria dose per ml decrease in LPS levels compared to cefamandole therapy blood. The remaining portion of the blood was centrifuged, alone. the plasma removed and passed through a 0.2 micron 45 Cardiovascular/pulmonary parameters (MABP, CI, TPR, Whatman syringe filter to remove the bacteria. The endot arterial oxygen tension. alveolar-arterial O. gradient, respi oxin levels were determined using a modified limulus amoe ration rate, and arterial blood pH) are shown in FIGS. 10-16, bocyte lysate assay (Pyrochrome LAL Assay, Associates of respectively. These figures only display results for the Cape Cod, Woods Hole, Mass.). These results were groups treated with cefamandole alone (indicated by open expressed as ng LPS per ml plasma. 50 squares) and concurrent treatment with rBPI and cefaman Cardiovascular, respiratory and metabolic parameters dole (indicated by filled squares). Single stars indicate that were measured every 30 minutes. Mean arterial blood pres the concurrent administration of both agents provided sta sure (MABP) and heart rate were monitored continuously tistically significant (p<0.05) improvement over antibiotic throughout the experiments and displayed on a cardiac alone, while two stars indicate p<0.01. Cefamandole alone output computer (Columbus Instruments Cardiomax II) or 55 or rBPI2 alone failed to protect the animals; cardiovascular on a chart recorder. Heart rate was derived from the arterial and respiratory dysfunction began du-ring the bacterial pressure wave. Cardiac output was determined in duplicate infusion and the animals were in circulatory shock by the with the thermodilution technique: Changes in blood tem end of the infusion. Cardiovascular shocklasted for the rest perature resulting from injection of 900 pil of room tempera of the experiment. Arterial blood pH began to decrease at 60 ture PBS were recorded with the thermistor-tipped catheter 60 minutes in the group treated with cefamandole alone, and in the aortic arch. The cardiac output computer then gener was at its lowest level by the end of the experiment. In ated thermodilution curves that were visualized on the chart contrast, concurrent administration of rBPI2 and cefaman recorder, and derived cardiac output from the temperature dole preserved cardiopulmonary function and prevented time curves. Cardiac index (CI) was then calculated as septic shock. Thus, the concurrent administration of BPI cardiac output per kg body weight. In addition, total periph 65 protein product with antibiotic protected the animals against eral resistance (TPR) was determined by dividing blood the lethal effect of bacteremia and preserved cardiopulmo pressure by cardiac output. nary function when antibiotic alone failed to do so. 5,523,288 41 42 EXAMPLE 9 Ceftriaxone were prepared using a multichannel pipetting instrument, inoculated with organisms diluted to approxi SYNERGISTIC EFFECTS OF GENTAMICIN mately 2.5x10 CFU/ml, and incubated overnight at 37° C. AND BPI PROTEIN PRODUCT IN VIVO IN Media controls containing no organism and growth controls containing organisms but no ceftriaxone or rBPI were also MICE CHALLENGED INTRAPERITONEALLY 5 prepared. The MIC was determined as the lowest drag WITH LIVE E. COLI O7:K1 BACTERIA: concentration (ug/mL) that inhibited bacterial growth. Agar EFFECT ON SURVIVAL MIC studies were performed as follows: The organism was The synergistic effects of an aminoglycoside antibiotic grown in Mueller-Hinton broth (Difco Laboratories) over and a BPI protein product were evaluated in mice challenged night at 37°C., transferred and grown to logarithmic phase, intraperitoneally with E. coli O7:K1 (ATCC Accession No. 10 counted by optical densitometry, diluted with buffer and 23503), a smooth encapsulated strain that is susceptible to rBPI2 and ce?triaxone, and incubated for 30 minutes at 37 the bactericidal effects of BPI protein product. The general C. After the 30 minutes of incubation, samples were serially procedure described above in Example 4 was followed. Six diluted in sterile saline and plated onto TSA agar for groups of 20 mice were challenged intraperitoneally with bacterial counts after overnight incubation at 37° C. 2x10' E. coli O7:K1 bacteria and treated immediately with 15 A variety of different bacterial species were tested, includ (l) vehicle, (2) 0.03 mg/kg gentamicin, (3) 0.1 mg/kg of ing Pseudomonas, Enterobacter, Citrobacter, Klebsiella, and gentamicin, (4) 50 g of rBPI, (5) 0.03 mg/kg gentamicin Escherichia species. A summary of the results is displayed in followed by 50 ug of rBPI, or (6) 0.1 mg/kg of gentamicin Table 11 below, which reports the minimum inhibitory followed by 50 ug of rBPI. Survival was followed over 7 concentration (MIC) of BPI protein product alone, the MIC days. Results are shown in FIG. 17. Neither antibiotic alone 20 of ceftriaxone alone, and the MIC of both agents together (in nor rBPI2 alone had any effect on mortality other than to a fixed 1:1 proportion of rBPI and ceftriaxone). The slightly retard the death rate. However, the concurrent experiments were replicated, and each number given in the administration of rBPI2 with low-dose gentamicin signifi table represents the highest, or worst-case, MIC for the cantly increased survival (p<0.5 vs. vehicle). Concurrent group. administration of rBPI with high-dose gentamicin dra matically increased survival (p<0.001 vs. vehicle, and 25 TABLE 11 p<0.05 vs. 0.03 mg/kg gentamicin with rBPI) protecting Synergistic Effect of rBPI2 and Ceftriaxone all but two of the mice from the lethal effects of bacterial on Ceftriaxone-Resistant Organisms challenge. The results clearly indicate synergism between gentamicin and BPI protein product. The greater synergistic MIC of MIC of MIC of 30 BPI alone Ceftriaxone alone BP - Cefriaxone effect of the concurrent administration of BPI protein prod Organism (g/mL) (gfml) (ugln) uct with gentamicin, compared to the effect of the concurrent administration of BPI protein product with cefamandole, PA 589 64 >64 4. may be related to the fact that aminoglycosides, which PA 631 >64 >64 4 inhibit protein synthesis, have a different mechanism of PA 672 32 32 2 35 PA 677 >64 >64 4. action than BPI protein product. EA 658 >64 >64 4. CF 595 4. >64 1 EXAMPLE 10 CF 596 4. >64 1. CF 597 >64 >64 8 CF 598 8 32 2 EFFECT OF BPI PROTEIN PRODUCT IN CF 642 4 16 8 VITRO ON ANTIBIOTIC SUSCEFHBILITY OF 40 CF 661 32 6 CEFTRIAXONE-RESISTANT GRAM-NEGATIVE KP 60 64 >64 8 ORGANISMS EC 004 >64 >64 <0. EC 600 >64 32 8 EC 664 64 >64 32 The ability of BPI protein product, BPI, to reverse the ECL 03 16 >64 16 resistance of a variety of gram-negative organisms to ceftri 45 ECL 05 I6 >64 6 aXOne (Roche Laboratories) was evaluated in vitro. ECL 07 >64 64 16 The Strain of gram-negative bacteria to be tested was ECL 13 >64 >64 16 ECL 14 16 >64 32 grown overnight at 37° C. on Trypticase soy agar (TSA) ECL 15 >64 >64 32 plates. Colonies from the plate were then inoculated into ECL 19 >64 >64 32 nutrient broth (or triethanolamine-buffered minimal salts 50 PA 001 >64 >64 8 medium), grown overnight to stationary growth phase, PA 003 >64 >64 8 diluted 1:10 in fresh medium, and grown to mid-late loga PA 004 >64 64 16 rithmic growth phase (3 to 4 hours at 3° C) to an approxi PAO05 >64 >64 64 mate concentration of 6 to 10x10 organisms/mL. Organism PA O12 >64 64 6 counts were performed by making serial dilutions, plating in PA 014 >64 >64 8 55 PA 017 >64 >64 32 triplicate on TSA plates, incubating at 37° C. oventight, and PA O23 >64 >64 32 counting colonies by visual inspection. Following develop PA 026 64 64 8 ment of a standard curve, counts were made by measuring PA O27 64 64 8 ODao and confirming by plating. The bacteria were sedi PA O28 >64 >64 16 mented by centrifugation at 6,000 g for 10 min. and resus PA = Pseudomonas aeruginosa pended in sterile saline to the desired concentration. 60 EA = Enterobacter aerogenes Ceftriaxone solutions were prepared from standard pow CF = Citrobacter freundi der (Roche Laboratories). Solutions of BPI protein product KP = Klebsiella pneumoniae were prepared from rBPI, Hanks solution, vitamin-free EC = Escherichia coli casamino acid, and TRIS-HCl buffer, pH 7.0. Broth MIC ECL = Enterobacter cloacae studies were performed as follows: U-bottom, disposable, 65 Checkerboard synergy studies on selected strains were microtiter array plates (Dynatech) containing 100 L/well of performed as follows. Microplates were prepared using the broth and serial dilutions of 1:1 proportion rBPI and foBowing organization: Column 1, control organisms 5,523,288 43 44 (growth control); Columns 2 through 9, serial dilutions of EXAMPLE 11 cefiriaxone; Column 10, cefiriaxone alone; Column il, rBPI alone; Column 12, control media; Rows 1 through 8, serial dilutions of rBPI. Columns 2 through 9 thus con tained a serial array of various proportions of ceftriaxone EFFECTS OF BPI PROTEIN PRODUCT AND and BPI concentrations. All webs, except the media control, 5 ANTIBIOTICS IN VITRO ON PSEUDOMONAS were inoculated with suspended organisms and incubated SPECIES Sight at 37° C. Turbidity was recorded at 24 and 48 The effects of a BPI protein product, rBPI, on the Results of a representative synergy study of the effects of to EEGEGRE the concurrent administration of rBPI, and cefiriaxone on Microscan?) library, Sacramento, Calif.) was evaluated cefiriaxone-resistant E. coli are shown in the checkerboard using Microscan?) panel plates (Baxter Diagnostics, Inc., incefiriaxone Table 12 below.were each In this serially checkerboard diluted assay,to concentrations rBPI and Deerfield,eerneia, Ill.)). thatat allowa simultaneousimultan determinatidetermination o f ranging from 100 pug/ml to 0.8 ug/ml. These results show minimum inhibitory concentrations for a number of different that the concurrent administration of both agents is syner- antibiotics. Control assays confirmed that the formulation gistic. There was uniform growth in all growth control wells buffer for rBPI had no effect on the antibiotic susceptibility (containing bacteria, no rBPI and no antibiotic), while of various organisms. there was no growth in the media control wells (containing The antimicrobial susceptibility tests performed on the no bacteria). There was uniform growth in all ceftriaxone Microscan?& panel plates are miniaturizations of the broth control webs (bacteria with ceftriaxone alone). In the BPI 20 dilution susceptibility test. Antimicrobial agents are serially control wells (bacteria with rBPI, alone) there was growth diluted in Mueller-Hinton broth (supplemented with calcium in the wells with a concentration of 6.2 pg/ml rBPI or less, and magnesium, or with sodium chloride for oxacillin, or s g growth at higher concentrations of BPI protein with thymidine phosphorylase for trimethoprim, sul product. famethoxazole and trimethoprim/sulfamethoxazole) to con Survival (or kill) curve studies were performed as fol- 25 centrations bridging the A. of clinical interest. One well lows. Tubes containing (1) media alone, (2) media plus on the 96-well Microscano plate is a growth control well ce?triaxone, (3) media plus rBPI21, and (4) media plus that contains dehydrated broth only. The remaining wells Cefiriaxone and rBPI21, were prepared, inoculated with the contain dehydrated broth and antibiotic (or broth and bio desired organism, and incubated as described above. At 0, 1, chemical reagent indicator), which is rehydrated to the 2, 4, 8 and 24 hours, diluted aliquots from each tube were 30 desired cocation by inoculation of a standardized sus plated, incubated and counted as described above. Growth pension of test organism. The chromogenic biochemical werecurves constructed over 24 hours to demonstrate (plots of logo the dynamics CFU/mL ofversus antibiotic- time) agentt indicatindicators are usedd to identiiden fy a d characterizeCataCZee th BPI protein product interactions on bacterial growth and species of bacteria based on detection of pH changes and Survival. 35 substrate utilization. After incubation overnight, the w Results of a representative killing curve study of the inhibitory cont (MIC) of an als R effects of the concurrent administration of rBPI and ceftri- organism is determined by observing the well with the axone on ceftriaxone-resistant E. coli are shown in FIG. 18. OWest COICentration of the antibiotic that shows inhibition The filled square is the control (neither rBPI, nor cefiriax- of growth. Gram-negative organisms were tested using Neg one), the open square is rBPI2 alone, the fried diamond is Combo Type 16, MIC Plus. Type 2, or Neg Breakpoint cefiriaxone alone, and the open diamond is the concurrent " Combo Type 9 panel plates (MicroScanG), Baxter Diagnos administration of cefiriaxone and rBPI. Cefiriaxone or tics, Inc., Deerfield, Ill.). The concentrations of antibiotics rBPI alone have some early bactericidal effect at 4-8 tested in these panel plates are shown below in Tables 13, 14 hours, but organism growth for both almost reaches that of and 15, respectively. The antibiotic susceptibility standards the control curve by 24 hours (about a 1 log difference from (interpretation of an MIC as resistant, intermediate or sus control). In contrast, the concurrent administration of rBPI2 ceptible according to MicroscanG)'s NCCLS-derived stan and cefiriaxone produces a significantly greater bactericidal dards) applicable to the gram-negative organisms tested in effect that is sustained at 24 hours (a more than 6 log each panel plate appear in Tables 13A, 14A and 15A, difference from control). respectively.

TABLE 12 Checkerboard Synergy Study for Ceftriaxone-Resistant E. coli Using Ceftriaxone and rBPI 0.8 uglmL 1.5 g/mL 3.1 ugmL 6.2 g/mL 12.5 g/mL 25 g/mL 50 g/mL 100 pg/mL Ceftriaxone Ceftriaxone Ceftriaxone Ceftriaxone Ceftriaxone Ceftriaxone Ceftriaxone Ceftriaxole 0.8 g/mL BPI 1.5 lug/mL BPI 3.1 g/mL BPI 6.2 g/mL BPI 12.5 g/mL BPI 25 g/mL BPI 50 g/mL BPI 100 glimL BPI KEY: -- Growth ONo Growth 5,523,288 45 46 TABLE 13 TABLE 14-continued ANTIBIOTIC CONCENTRATIONSTESTED IN ANTIBIOTIC CONCENTRATIONSTESTED IN NEG COMBOTYPE 16 PANEL, PLATE MIC PLUS TYPE 2 PANEL, PLATE Two-Fold Serial Two-Fold Serial Antibiotic Dilutions Tested ug/ml Antibiotic Dilutions Tested (ug/ml) Amikacin 2-16 Cefotetan 4-32 Ampicillin 2-16 Ceftazidime 1-32 Ampicillin/Sulbactam 814-1618 10 Ceftizoxime 2-32 Aztreonam 8-16 Ceftriaxone 2-64 Cefazolin 2-16 Chloramphenicol 2-16 Cefotaxime 4-32 Ciprofloxacin 0.25-4 Cefoxitin 2-16 Imipenem 0.5-16 Ceftazidime 2-16 Mezlocillin 16-128 Ceftriaxone 4-32 Netilmicin 2-16 Cefuroxime 2-16 15 Ticarcillin 16-128 Ciprofloxacin 1-2 Ticarcillin/K Clavulanate 16-128 Gentamicin 1-4.6 Imipenern 4-8 Ofloxacin 2-4 Piperacillin 8-64 TABLE 4A Ticarcillin 8-64 20 Tobramycin 1-4,6 MICROSCANMIC PLUS TYPE 2 ANTIBIOTIC Trimethoprin/Sulfamethoxazole 0.5/9.5, 2/38 SUSCEPTIBILITYRANGES FOR GRAM-NEGATIVE BACTERIA MIC (ughml) TABLE 13A 25 MICROSCAN NEG COMBO PANEL 16 Antibiotic Resistant Intermediate Susceptible ANTIBIOTICSUSCEPTIBILITYRANGES Anoxicillin/K- 232/6 1618 s8/4 FOR GRAM-NEGATIVE BACTERIA Clavulanate MIC (ug/ml) Ampicillin/Sulbactam 232116 1618 s3f4 30 Azlocillin >64 s64 Aztreonan 232 16 s8 Antibiotic Resistant Intermediate Susceptible Carbenicillin 264 32 s16 Amikacin >16 316 Carbenicillin 128 s28 Ampicillin >16 16 s8 Cefanandole 232 16 s8 Ampicillin/Sulbactam >16/8 1618 38/4 Cefonicid >16 16 s8 Aztreonan >16 16 s8 35 Cefoperazone >32 32 s16 Cefazolin >16 16 s8 Cefotaxime 264 16-32 s8 Cefotaxine >32 16-32 s8 Cefotetan s32 32 s16 Cefoxitin >6 16 s8 Ceftazidime 232 16 s8 Ceftazidime >16 16 s8 Ceftizoxime s32 16-32 s8 Ceftriaxone 264 16-32 s8 Ceftriaxone >32 16-32 s8 Chloramphenicol >16 16 s8 Cefuroxime >16 16 s8 40 Ciprofloxacin e4 2 s1 Ciprofloxacin >2 2 s1 imipenem 216 8 s4 Gentamicin >6 6 s4 Mezlocillin 2128 32-64 s16 Imipenem >8 8 s4 Mezlocillin 2128 sé4 Ofioxacin >4 4 s2 Netilmicin >6 16 s Piperacillin >64 32-64 s16 Ticarcillin 2128 32-64 s16 Piperacillin >64 s64 45 Ticarcillin >64 32-64 s16 Ticarcillin 228 s64 Ticarcillin >64 s64 Ticarcillin/K- 2128 32-64 s16 Tobranycin >6 6 s4 Clavulanate Trimethoprim/Sulfameth- >2/38 s2/38 Ticarcillin/K- 228 s64 oxazole Clavulanate Enterobacteriaceae only 50 Enterobacteriaceae only Pseudomonas only Pseudomonas only

TABLE 4 TABLE 1.5 ANTIBIOTIC CONCENTRATIONSTESTED IN 55 ANTIBIOTIC CONCENTRATIONSTESTED IN MIC PLUS TYPE 2 PANEL, PLATE NEG BREAKPOINT COMBOTYPE 9 PANEL, PLATE Two-Fold Serial Antibiotic Dilutions Tested (ug/ml) Antibiotic Dilutions Tested (ughinl) Nitrofurantoin 32 & 64 Amoxicillin/K Clavulanate /0.5-32/16 Cephalothin 8 & 16 Ampicillin/Sulbactam 1/0.5-32/16 60 Ampicillin 8 & 16 Azlocillin 64 Oftoxacin 2 & 4 Azetreonam 1-32 Ticarcillin 16 & 64 Carbenicillin 16-128 Piperacillin 6 & 64 Cefamandole 4-32 Mezlocillin 16 & 64 Cefonicid 2-16 Tetracycline 4 & 8 Cefoperazone 4-32 65 AImpicillin/Sulbactan 814 & 16/8 Cefotaxine 2-64 Anoxicillin/K Clavulanate 8/4 & 16/8 5,523,288 47 48

TABLE 15-continued ANTIBIOTIC CONCENTRATIONSTESTED IN TABLE 15A-continued NEG BREAKPOINT COMBOTYPE 9 PANEL, PLATE MICROSCAN NEG BREAKPOINT COMBOTYPE 9 ANTIBIOTC SUSCEPTIBILITYRANGES Antibiotic Dilutions Tested (ug/ml) FOR GRAM-NEGATIVE BACTERIA Ticarcillin/K Clavulanate 6 & 64 Gentamicin 4 & 8 MIC (ug/ml) Tobramycin 4 & 8 Amikacin 16 & 32 10 Antibiotic Resistant Intermediate Susceptible Ciprofloxacin 1 & 2 Imipenen 4 & 8 Cinoxacin >16 sió Cefazolin 8 & 16 Trimethoprim >8 s8 Cefamandole 8 & 16 Sulfamethoxazole >256 s256 Cefuroxime 8 & 16 Cefotetan 16 & 32 15 Enterobacteriacaeae only Cefoxiitin 8 & 16 Pseudomonas only Aztreonam 8 & 16 Ceftriaxone 8 & 32 For each experimental run, the following procedure was Ceftazidime 8 & 16 performed: The organism was streaked onto 5% sheep blood Cefoperazone 16 & 32 agar plates (Remel, Lenexa, Kansas) and incubated for Cefotaxine 8 & 32 20 18-24 hours overnight. Well-isolated colonies from the Chloramphenicol 8 & 16 Trimethoprim/Sulfamethoxazole 238 & 8,152 plates were emulsified in 3 ml of sterile Inoculum Water Norfloxacin 4 & 8 (catalog no. B1015-2, MicroScanG) system, Baxter Diag Cinoxacin 6 nostics, Inc., Deerfield, Ill.) to a final turbidity equivalent to Trimethoprim 8 0.5 McFarland Barium Sulfate standard. This cell suspen Sulfamethoxazole 256 sion was vortexed for 2 to 3 seconds and 100 ul was 25 transferred to glass tubes containing 25 ml of Inoculum Water with Pluronic-D (catalog no. B1015-7, MicroScanG) system, Baxter Diagnostics, Inc., Deerfield, Ill.) (hereinafter "Pluronic Inoculum Water'), or 25 ml of Pluronic Inoculum TABLE 5A Water into which rBPI (in formulation buffer) had been 30 MICROSCAN NEG BREAKPOINT COMBOTYPE 9 diluted to the desired concentration between 0.5 to 64 g/ml ANTIBIOTIC SUSCEPTIBILITYRANGES rBPI. FOR GRAM-NEGATIVE BACTERIA The 25 ml of this inoculum containing rBPI was mixed by inversion and poured into a tray. The inoculum was MIC (ig/ml) drawn up into a manual 96-well pipetting system Antibiotic Resistant Intermediate Susceptible 35 (RENOKTM rehydrator-inoculator system, Baxter Health Care Corporation, West Sacramento, Calif.) designed for use Nitrofurantoin >64 64 332 Cephalothin >16 16 s8 with the Microscan(E) panel plates, and 110 ul of the inocu Ampicillin >16 16 s8 lum was delivered to each well of a Microscan?E) Neg Ofloxacin >4 4 s2 Combo Type 16 panel plate. When added to the wells, this Ticarcillin >64 64 s6 40 inoculum achieves a final bacterial concentration of 4x10 to Ticarcillin >64 s64 Piperacillin >64 64 s16 7x10 CFU/ml. The panel plates were then incubated at 35° Piperacillin >64 s64 C. for 15-24 hours and read visually for cell growth. Mezlocillin >64 64 16 No growth was defined as a slight whiteness in the well Mezlocillin >64 s64 Tetracycline >8 8 S4 45 or a clear broth. Growth appeared as turbidity which could Ampicillin/Sulbactam >1678 1618 s8f4 take the form of a white haze throughout the well, a white Amoxicillin/K- s168 1618 s8.4 button in the center of the well, or a fine granule growth Clavulanate >1678 1618 38.4 throughout the well. All wells were read against a black Ticarcilin/K- >64 64 s16 indirectly lighted background. Visual results of the bio Clavulanate chemical reactions were read into a database for bacterial Ticarcilin/K- >64 só4 50 Clavulanate identification. The MICs for each antibiotic tested were Gentamicin >8 8 s4 determined by identifying the lowest concentration of anti Tobramycin >8 8 s4 Amikacin >32 32 S16 biotic which inhibited visible growth. Ciprofloxacin >2 2 s1 The clinical isolates of Pseudomonas aeruginosa and Imipenem >8 8 s4 other Pseudomonas species were tested using the Neg Cefazolin >16 16 s8 55 Cefamandole >16 16 38 Combo Type 16 panel plate. Tables 16, 17 and 18 below Cefuroxime >16 16 s8 display a summary of the results of the antibiotic screening Cefotetan >32 32 s6 panels, reported for each strain tested as the MIC of the Cefoxitin >6 16 s8 tested antibiotics at the various concentrations of rBPI2 Aztreonam >16 16 s8 Ceftriaxone >32 32 a8 60 indicated. Results are reported for each strain tested, but Ceftazidime >16 16 S8 susceptibility data is listed for only those antibiotics for Cefoperazone >32 32 s16 which BPI protein product altered susceptibility. The anti Cefotaxine >32 32 s8 biotic susceptibility standards (interpretation of an MIC as Chloramphenicol >16 16 s8 Trimethoprin/Sulfameth- s8,152 8/152 s238 resistant, intermediate or susceptible according to oxazole 65 Microscan?&'s NCCLS-derived standards) applicable to the Norfloxacin >8 8 s4 organism tested appear in Table 13A. Stars after the antibi otic name in the 'antibiotic tested' column indicate whether 5,523,288 49 50 rBPI2 reversed the resistance of that organism to the antibiotic tested (two stars) or converted an indifferent MIC TABLE 16-continued into a susceptible MIC (one star). These data show that BPI EFFECTS OF rBPI, it ANTIBIOTICS ON Pseudomonas protein product reversed resistance to ticarcillin, cefazolin, aeruginosa cefoxitin, cefuroxime, ofloxacin, aztreonam, piperacillin, Minimum and amikacin for some strains of P. aeruginosa and Inhibitory Concentration increased the susceptibility of some P. aeruginosa strains to of Antibiotic (g/mL) ticarcillin, aztreonam, imipenem, piperacillin, of oxacin, ceftazidime, amikacin, ceftriaxone, cefotaxime, cefuroxime, Microscan With With With tobramycin, ciprofloxacin, trimethoprim/sulfamethoxazole, 10 Library Antibiotic 0 g/mL 8 g/mL 32 pg/mL gentamicin, and cefazolin. BPI protein product reversed ID No. Tested rBPI21 rBPI21 rBPI resistance of some P. cepacia strains to cefazolin, cefoxitin, Ceftriaxone 32 16 16 cefuroxime, ceftriaxone, ticarcillin, and increased the sus Gentamicin 4 16 <2 <2 lin, piperacillin, cefazolin, cefoxitin, cefuroxime, ceftriax 15 19660 BPI G G NG one, amplicillin/sulbactam, cefotaxine, gentamicin, Trimethoprim/ >2 2 - tobramycin, and amikacin. BPI protein product reversed Sulfamethoxazole Ceftriaxone 16 8 ------resistance of Xanthamonas maltophilia to trimethoprim/ Cefotaxine I6 16 - sulfamethoxazole, piperacillin and amikacin, and increased Opal BPI G G G susceptibility to ciprofloxacin. 20 Ampicillin' >16 >6 4. Trimethoprim/ >2 >2 2 Tables 16, 17 and 18 also show the presence or absence Sulfamethoxazole of bacterial growth in the growth control wells, which Ticarcillin 16 16 <8 contained varying concentrations of rBPI alone without Cefazolin >16 >16 <2 antibiotic. "G” indicates growth, while "NG" indicates no Cefoxiitin >16 >16 <2 25 Cefuroxide >16 >16 <2 growth. These results indicate that rBPI at a concentration Ceftriaxone 16 8 <4 of 32 pg/ml has direct bactericidal/growth inhibitory effects Ampicillin/ >16 >16 <8 on Some of the tested Pseudomonas isolates. Sulbactam" Cefotaxime 16 8 <4 TABLE 16 30 "The Microscan () worksheet did not supply antibiotic susceptibility ranges, EFFECTS OF rBPI, + ANTIBIOTICS ON Pseudomonas but there was reversal of antibiotic indifference according to NCCLS stan dards, Publication M7-A3, Table 2 (1993). aeruginosa Strain 12.4.4, provided by S. M. Opal, Brown University, Providence, RI. Minimum Inhibitory Concentration TABLE 17 of Antibiotic (ughmii) 35 Microscan With With With EFFECTS OF rBPI, + ANTIBIOTICS ON Pseudomonas Library Antibiotic 0 ug/mL 8 pg/mL 32 uglmL aeruginosa ID No. Tested rBPI21 rBPI2 rBPI Minimum 19610 BPI G G G Inhibitory Concentration Ticarcillin >64 64 64 40 of Antibiotic (pg/mL) Aztreonam 16 <8 <8 Microscan With With With Piperacillin 332 16 <8 Ofloxacin >4 4 <2 Library Antibiotic 0 uglm 4 ug/mL 16 g/mL Ceftazidime 8 8 4 ID No. Tested rBPI2 rBPI rBPI Amikacin 16 8 8 45 N113-100 BP G G G 18433 BPI G G NG Ofloxacin 4 4 <2 Azetreonan 16 <8 - Gentamicin >6 6 6 Ceftriaxone >32 32 - Tobramycin 2 2 < Cefotaxime >32 32 - N113-101 BPI G G G Cefuroxime 16 4. - Aztreonam >16 >16 <8 Tobramycin 2 <1 - Ofioxacin 4 4 <2 Amikacin 16 8 - 50 Gentamicin >6 6 6 12892 BPI G G G Tobramycin 4 2 2 >2 2 N113-102 BPI G G G Oflaxacin >4 ">4 4. Piperacillin 64 <8 <8 Ceftazidime 4 4 <2 Ceftriaxone 32 16 8 Cefotaxime >32 >32 32 Cefotaxine 32 16 16 Amikacin 16 16 8 55 Gentamicin 4. 4 <1 19054 BPI G G G Anikacin 8 8 <2 Trimethoprim/ >2 >2 2 N113-103 BP G G G Sulfamethoxazole Ticarcillin 16 16 32 Ticarcillin 64 16 16 Amikacin 4 8 4 Ceftriaxone 16 32 8 N113-104 BPI G G G Gentamicin 4 4. 2 60 Ceftazidine <2 <2 4 Amikacin 16 8 4 Ceftriaxone 16 8 8 Cefazolin >6 >16 16 N113-105 BPI G G G 19672 BPI G G G Ticarcillin 64 32 32 Tobramycin >6 >6 6 Aztreonam 16 <8 <8 19551 BPI G G G Ceftriaxone 32 32 16 Aztreonian >16 <8 <8 Cefotaxime 32 32 16 Piperacillin'* >64 <8 <8 65 5,523,288 51 52 evaluated in the Microscance) antibiotic susceptibility TABLE 7-continued screening assay of Example 11 using the Neg Combo Type EFFECTS OF rBPI + ANTIBIOTICS ON Pseudomonas 16 panel plate. The direct growth inhibitory effect of rBPI2 aeruginosa on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of E. coli (from Baxter Minimum Inhibitory Concentration MicroscanG) library, Sacramento, Calif.). of Antibiotic (ug?mL) A summary of the results of the antibiotic screening panels, reported as MICs (ug/ml) of the antibiotic tested, is Microscan With With With shown in Table 19 below. Results are reported for each strain Library Antibiotic 0 ug/mL 4 ug/mL 16 g/mL 10 tested, but susceptibility data is listed for only those antibi ID No. Tested rBPI21 rBPI rBPI otics for which BPI protein product altered susceptibility. Gentamicin 2 2 <1. The antibiotic susceptibility standards (interpretation of an N113-106 BPI G G G Ticarcillin 16 16 <8 MIC as resistant, intermediate or susceptible according to Ceftriaxone 32 16 8 Microscan?)'s NCCLS-derived standards) applicable to the Cefotaxime 6 16 8 15 organism tested appear in Table 13A. These results show Gentamicin 4 2 2 that BPI protein product reversed the resistance of some Amikacin 8 4 4 N113-107 BPI G G G strains to cefazolin and increased the susceptibility of other Ticarcillin >64 >64 64 strains to ampicillin, cefuroxime, cefazolin, amikacin, and Ciproflaxacin 2 2 <1. cefoxitin. Ceftazidime 8 4. 4 20 Ceftriaxone 32 32 >32 TABLE 19 Imipenem >8 8 >8 EFFECTS OF rBPI it ANTIBIOTICS ON Escherichia coli Minimurn TABLE 18 25 Inhibitory Concentration of Antibiotic (ug/mL) EFFECTS OF rBPI + ANTIBIOTICS ON Pseudomonas SPECIES Microscan With With With Library Antibiotic 0 pg/mL 4 g/mL 16 pg/mL Minimum ID No. Tested rBPI21 rBPI rBPI Inhibitory Concentration 30 of Antibiotic (ugfmL) 19536 Ampicillin' 16 16 8 Cefuroxime 4. 8 4 Microscan With With With F101-309 Cefoxiitiin 4 <2 4 Library Antibiotic Oughm 8 g/mL 32 g/mL Cefuroxime 8 4. 4 ID No. Tested rBPI2 rBPI rBPI 19612 Cefazolin >16 >16 8 Amikacin 16 8 8 31142 BPI G G G 35 Cefoxitin <2 4 <2 (P. cepacia) Ampicillin >16 16 - 17164 Ampicillin 8 8 4. Ticarcilin 32 <8 - Cefoxitin 8 8 4. Piperacillin 32 <8 - 19522 Cefazolin 8 8 4. Cefazolin >16 8 - Cefoxiitin >16 >16 16 Cefoxiitin * >16 <2 - Cefuroxime >6 <2 - 40 The Microscan () worksheet did not supply antibiotic susceptibility ranges Ceftriaxone >32 8 - for this organism, but there was reversal of antibiotic indifference according Ampicillin/ 16 <8 - to NCCLS standards, Publication M7-A3, Table 2 (1993). Sulbactam" Cefotaxime 32 <4 - Gentamicin 6 <1 - EXAMPLE 13 Tobramycin 2 <1 - 45 Amikacin 8 <2 - 12122 BPI G G G EFFECTS OF BPI PROTEIN PRODUCT AND (P. cepacia) Ticarcillin' >64 64 32 ANTIBIOTICS IN VETRO ONCITROBACTER Piperacillin 16 <8 <8 SPECIES Cefuroxime 4. <2 <2 Amikacin 4. 4 <2 17211 BPI G G NG 50 The effect of a BPI protein product, rBPI, on the anti (Xantha- Trimethoprim/ >2 2 - biotic susceptibility of various Citrobacter species was monas Sulfa evaluated in the Microscance) antibiotic susceptibility maltophilia) methoxazole' screening assay of Example 11 using the Neg Combo Type Piperacillin'* >64 16 - Amikacin >16 16 - 16 panel plate. The direct growth inhibitory effect of rBPI2 Ciprofloxacin 2 2 - 55 on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of Citrobacter species "The Microscan (R) worksheet did not supply antibiotic susceptibility ranges (from Baxter MicroscanCE) library, Sacramento, Calif.). for this organism, but there was reversal of antibiotic indifference according A summary of the results of the antibiotic screening to NCCLS standards, Publication MT-A3, Table 2 (1993). panels, reported as MICs (ug/ml) of the antibiotic tested, is 60 shown in Table 20 below. Results are reported for each strain EXAMPLE 12 tested, but susceptibility data is listed for only those antibi EFFECTS OF BPI PROTEIN PRODUCT AND otics for which BPI protein product altered susceptibility. ANTIBIOTICS IN VITRO ON STRANS OF E. The antibiotic susceptibility standards (interpretation of an COLI MIC as resistant, intermediate or susceptible according to 65 MicroscanG)'s NCCLS-derived standards) applicable to the The effect of a BPI protein product, rBPI, on the organism tested appear in Table 13A. These results show antibiotic susceptibility of various strains of E. coli was that BPI protein product increased the susceptibility of the 5,523.288 S3 54 tested Citrobacter species to aztreonam, cefotaxime, tobra mycin, amikacin, cefuroxime, amplicillin, ticarcillin, pipen TABLE 21 cillin, and cefuroxime. EFFECTS OF rBPI, + ANTIBIOTICS ON Klebsiella SPECIES 5 TABLE 20 Mininuin EFFECTS OF rBPI, + ANTIBIOTICS ON Citrobacter Inhibitory Concentration SPECIES of Antibiotic (ug/mL) Mininum Microscan With With With Inhibitory Concentration O Library Antibiotic 0 ug/mL 4 ug/mL 16 ug?mL of Antibiotic (ugfml) ID No. Tested rBPI21 rBPI rBPI Microscan With With With 1964.5 Cefoxitin 8 k2 4. Library Antibiotic 0 uglimL 4 g/mL 16 g/mL (K. Cefoxitin 8 <2 4. DN0. Tested rBPI2 rBPI rBPI pneumoniae) Ampicillin? >16 >16 >16 15 Sulbactam 18419 Aztreonam >16 >16 16 18427 Cefazolin <2 4 <2 (C. freundii) Cefotaxime >32 32 32 (K. Ampicillin/ 16 16 <8 Tobramycin 4 2 2 >2 (0.5 18420 Cefuroxime >16 >6 16 (K. Sulfa (C. freundii) Amikacin >2 >2 >2 20 pneumoniae) methoxazole F-052-007 Ampicillin >16 >16 16 Cefazolin 16 16 8 (C. diversits). Ticarcillin >64 >64 64 30434 Cefazolin 16 16 8 Piperacillin' 32 16 <8 (K. oxytoca) Cefoxitin 4 <2 4 Cefuroxime 8 4 4 Cefuroxime 8 4 8 Ampicillin/ <8 16 <8 The Microscan (8) worksheet did not supply antibiotic susceptibility ranges Sulbactan for this organism, but there was reversal of antibiotic indifference according "The Microscan (8) worksheet did not supply antibiotic susceptibility ranges to NCCLS standards, Publication M7-A3, Table 2 (1993). for this organism, but there was reversal of antibiotic indifference according to NCCs standards, Publication M7-A3, Table 2 (1993).

EXAMPLE 14 30 EXAMPLE 1.5

EFFECTS OF BPI PROTEIN PRODUCT AND ANTBIOTICS IN VITRO ON KLEBSIELLA 35 EFFECTS OF BPI PROTEIN PRODUCT AND SPECIES ANTIBIOTICS IN VITRO ON ENTEROBACTER SPECIES The effect of a BPI protein product, rBPI, on the antibiotic susceptibility of various Klebsiella species was 40 The effect of a BPI protein product, rBPI, on the antibiotic susceptibility of various Enterobacter species was evaluated in the Microscan?E) antibiotic susceptibility evaluated in the Microscan?) antibiotic susceptibility screening assay of Example 11 using the Neg Combo Type screening assay of Example 11 using the Neg Combo Type 16 panel plate. The direct growth inhibitory effect of rBPI2 16 panel plate. The direct growth inhibitory effect of rBPI on these strains was also evaluated in the same assay. Assays as on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of Klebsiella species were conducted on clinical isolates of Enterobacter species (from Baxter Microscan?R) library, Sacramento, Calif.). (from Baxter Microscan?E) library, Sacramento, Calif.). A summary of the results of the antibiotic screening A summary of the results of the antibiotic screening panels, reported as MICs (ug/ml) of the antibiotic tested, is 50 panels, reported as MICs (ug/ml) of the antibiotic tested, is shown in Table 22 below. Results are reported for each strain shown in Table 21 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibi tested, but susceptibility data is listed for only those antibi otics for which BPI protein product altered susceptibility. otics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to MIC as resistant, intermediate or susceptible according to Microscan?E)'s NCCLS-derived standards) applicable to the Microscan(E)'s NCCLS-derived standards) applicable to the organism tested appear in Table 13A. These results show organism tested appear in Table 13A. These results show that BPI protein product reversed the resistance of one strain that BPI protein product reversed the resistance of one strain of E. cloacae to ticarcillin, cefuroxime, ceftazidime and of K. pneumoniae to trimethoprim/suffamethoxazole and cefotaxime. BPI protein product also increased the suscep tibility of some Enterobacter species to ticarcillin, aztre increased the susceptibility of the tested species to cefoxitin, onam, piperacillin, ciprofloxacin, cefotaxime, trimethoprim/ ampicillin/sulbactam, trimethoprim/sulfamethoxazole, cefa sulfamethoxazole, cefuroxime, ceftazidime, ceftriaxone, Zolin, and cefuroxime. 65 and ampicillin/sulbactam. 5,523,288 SS 56 lin/sulbactam, and ampicillin. TABLE 22 TABLE 23 EFFECTS OF rBPI, it ANTIBIOTICS ON Enterobacter SPECIES EFFECTS OF rBPI, + ANTIBIOTICS ON Serratia Rese Minimum Inhibitory Concentration Minimum of Antibiotic (ug/mL) Inhibitory Concentration of Antibiotic (ug/mL) Microscan With With With Library Antibiotic 0 pg/mL 4 g/mL 16 g/mL 10 Microscan With With With ID No. Tested rBPI21 rBPI rBPI Library Antibiotic 0 uglmL 8 g/mL 32 g/mL ID No. Tested rBPI2 rBPI rBPI 19565 Ticarciin 64 >64 64 (E. aerogenes) Aztreonam 6 <8 <8 19646 Piperacillin >64 >64 32 Piperacillin >64 >64 64 Cefoxitin 16 >6 8 Ciprofloxacin 2 K1 <1 5 Ceftazidime >6 >16 8 Cefotaxine 16 8 8 Ceftriaxone 32 16 8 19626 Piperacillin >64 64 64 Cefotaxine >32 32 <4 (E. aerogenes) Ticarcillin >64 >64 >64 Tobramycin 6 6 4. 19625 Trimethoprim/ 2 k0.5 a0.5 19647 Piperacillin >64 64 16 (E. aerogenes) Sulfa Cefoxiitin 8 8 4. methoxazole Ceftazidime 16 16 4 Piperacillin >64 >64 >64 20 Ceftriaxone 8 <4 <4 1968O Ticarcillin >64 >64 <8 Ampicillin/ >16 >16 16 (E. cloacae) Aztrenam >16 16 k8 Sulbactan Piperacillin 64 32 k8 Cefotaxime 8 C4 <4 Cefuroxime s16 >6 8 Tobramycin 2 2 <1 Ceftazidime: >32 32 <4 18443 Ampicillin 16 6 8 Ceftriaxone >16 >16 16 25 Ampicillin >16 >6 16 Ampicillin/ >16 >16 16 Sulbactan Sulbactam Cefotaxine >32 >32 <4 "The Microscan (8 worksheet did not supply antibiotic susceptibility ranges 19686 Ceftriaxone 32 32 16 for this organism, but there was reversal of antibiotic indifference according (E. cloacae) Piperacillin >64 >64 >64 to NCCLS standards, Publication M7-A3, Table 2 (1993). 30 EXAMPLE 16 EXAMPLE 7 EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON SERRATIA EFFECTS OF BPI PROTEIN PRODUCT AND MARCESCENS ANTIBIOTICS IN WITRO ON PROTEUS 35 MIRABILIS The effect of a BPI protein product, rBPI, on the antibiotic susceptibility of Serratia marcescens was evalu The effect of a BPI protein product, rBPI, on the ated in the Microscance) antibiotic susceptibility screening antibiotic susceptibility of Proteus mirabilis was evaluated assay of Example 11 using the Neg Combo Type 16 panel in the Microscan?) antibiotic susceptibility screening assay plate. The direct growth inhibitory effect of rBPI on these 40 of Example 11 using the Neg Combo Type 16 panel plate. strains was also evaluated in the same assay. Assays were The direct growth inhibitory effect of rBPI on these strains conducted on clinical isolates of Serratia marcescens (from was also evaluated in the same assay. Assays were con Baxter Microscan?E) library, Sacramento, Calif.). ducted on clinical isolates of Proteus mirabilis (from Baxter A summary of the results of the antibiotic screening Microscan?E) library, Sacramento, Calif.). panels, reported as MICs (ig/ml) of the antibiotic tested, is 45 A summary of the results of the antibiotic screening shown in Table 23 below. Results are reported for each strain panels, reported as MICs (ug/ml) of the antibiotic tested, is tested, but susceptibility data is listed for only those antibi shown in Table 24 below. Results are reported for each strain otics for which BPI protein product altered susceptibility. tested, but susceptibility data is listed for only those antibi The antibiotic susceptibility standards (interpretation of an otics for which BPI protein product altered susceptibility. MIC as resistant, intermediate or susceptible according to 50 The antibiotic susceptibility standards (interpretation of an Microscan?E's NCCLS-defived standards) applicable to the MIC as resistant, intermediate or susceptible according to organism tested appear in Table 13A. These results show Microscan?&'s NCCLS-derived standards) applicable to the that BPI protein product reversed the resistance of some organism tested appear in Table 13A. These results show strains to ceftazidime and cefotaxime, and increased the that BPI protein productincreased the susceptibility of some susceptibility of other strains to piperacillin, cefoxitin, 55 strains to trimethoprim/sulfamethoxazole, cefazolin, cefox ceftazidime, ceftfiaxone, cefotaxime, tobramycin, ampicil itin, imipenem, tobramycin, and amikacin. 5,523.288 57 S8

TABLE 24 TABLE 25-continued EFFECTS OF rBPI it ANTIBIOTICS ON Proteus EFFECTS OF rBPI it ANTIBIOTICS ON Providencia mirabilis SPECIES

Minimum Minimum Inhibitory Concentration Inhibitory Concentration of Antibiotic (ug/mL) of Antibiotic (ug/mL) Microscan With With With Microscan With With With Library Antibiotic 0 uglm 8 g/mL 32 giml 10 Library Antibiotic 0 uglm 8 uglmL 32 pg/mL ID No. Tested rBPI21 rBPI rBPI I) No. Tested rBPI2 rBPI rBPI 19593 Trimethoprim/ 2 <0.5 <0.5 Sulbactam Cefazolin 4. <2 4 Innipenem 8 <4 <4 Cefoxiitin 8 2 k2 Amikacin 16 16 8 Imipenem 8 <4 <4 Tobramycin 2 2 <1 15 "The Microscan (8 worksheet did not supply antibiotic susceptibility ranges Amikacin 8 4. 4. for this organism, but there was reversal of antibiotic indifference according F23-129 Cefoxiitiin 4 <2 4. to NCCLS standards, Publication M7-A3, Table 2 (1993). Innipenem 8 <4 <4 Amikacin 4. <2 <2 EXAMPLE 19 EXAMPLE 1.8 EFFECTS OF BPI PROTEIN PRODUCT AND EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN WITRO ON MORGANELLA MORGANII ANTIBIOTICS IN WITRO ON PROVIDENCIA 25 SPECIES The effect of a BPI protein product, rBPI, on the The effect of a BPI protein product, rBPI, on the antibiotic susceptibility of Morganella morgani was evalu antibiotic susceptibility of various Providencia species was ated in the Microscan(E) antibiotic susceptibility screening evaluated in the Microscan(E) antibiotic susceptibility assay of Example 11 using the Neg Combo Type 16 panel screening assay of Example 11 using the Neg Combo Type plate. The direct growth inhibitory effect of rBPI on these 16 panel plate. The direct growth inhibitory effect of rBPI StrainS was also evaluated in the same assay. Assays were on these Strains was also evaluated in the same assay. Assays conducted on clinical isolates of Morganella morgani (from were conducted on clinical isolates of Providencia species Baxter MicroscanG) library, Sacramento, Calif.). (from Baxter Microscan?E) library, Sacramento, Calif.). A summary of the results of the antibiotic screening A summary of the results of the antibiotic screening 35 panels, reported as MICs (ug/ml) of the antibiotic tested, is panels, reported as MICs (ug/ml) of the antibiotic tested, is shown in Table 26 below. Results are reported for each strain shown in Table 25 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibi tested, but susceptibility data is listed for only those antibi otics for which BPI protein product altered susceptibility. otics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to MIC as resistant, intermediate or susceptible according to Microscan(E)'s NCCLS-derived standards) applicable to the MicroscanG)'s NCCLS-defived standards) applicable to the organism tested appear in Table 13A. These results show organism tested appear in Table 13A. These results show that BPI protein product increased the susceptibility of the that BPI protein product reversed the resistance of Pstuartii tested strains to ampicillin/sulbactam, amikacin and piper to cefazolin and cefuroxime, and increased its susceptibility 45 acillin. to piperacillin, ceftazidime, ampicillin/sulbactam, ini penem, and amikacin. BPI protein product also increased TABLE 26 susceptibility of Prettgeri to cefoxitin and cefuroxime. EFFECTS OF rBPI it ANTIBIOTICS ON Morganella morgani TABLE 25 50 Minimum EFFECTS OF rBPI, + ANTIBIOTICS ON Providencia Inhibitory Concentration SPECIES of Antibiotic (g/mL) Minimum Microscan With With With Inhibitory Concentration 55 Library Antibiotic OugfmL 8 ug/mL 32 uglmL of Antibiotic (ughnL) ID No. Tested rBPI2 rBPI rBPI Microscan With With With F19-004 Ampicillin? >16 16 16 Library Antibiotic 0 ugmL 8 uglmL 32 uglmL Sulbactan ID No. Tested rBPI21 rBPI rBPI Amikacin 4. <2 4. 60 F19-005 Ampicillin? s6 <8 a8 19614 Cefoxiin >16 8 >16 Sulbactan" (P retigeri) Cefuroxime" 16 8 <2 Amikacin 4. <2 <2 Amikacin 16 16 16 F19–006 Piperaciliin >64 64 64 18435 Piperacillin >64 32 16 Amikacin k2 <2 4 (P. Stuarii) Cefazolin >16 16 8 Cefuroxime >6 >16 8 65 "The Microscan (8 worksheet did not supply antibiotic susceptiblity ranges Ceftazidine 4 <2 <2 for this organism, but there was reversal of antibiotic indifference according Ampicillind >16 16 16 to NCCLS standards, Publication MT-A3, Table 2 (1993). 5,523.288 59 60 EXAMPLE 2.0 TABLE 27 EFFECTS OF rBPI + ANTIBIOTICS ON Acinetobacter EFFECTS OF BPI PROTEIN PRODUCT AND SPECIES 5 ANTIBIOTICS IN VITRO ON Minimum ACINETOBACTER SPECIES inhibitory Concentration The effect of a BPI protein product, rBPI, on the of Antibiotic (uglmL) antibiotic susceptibility of various Acinetobacter species Microscan With With With was evaluated in the Microscan?) antibiotic susceptibility 10 Library Antibiotic 0 g/mL 4 uglmL 16 uglmL screening assay of Example 11 using the Neg Combo Type ID No. Tested rBPI2 rBPI rBPI, 16 and MIC Plus Type 2 panel plate. The direct growth NO11-002 BPI G G G (A. anitratus) Ampicillin >16 16 16 inhibitory effect of rBPI on these strains was also evalu Amikacin 4 <2 <2 ated in the same assay. Assays were conducted on clinical 15 N011-003 BPI G G G isolates of Acinetobacter species (from Baxter Microscan?) (A. anitratus) Ampicillin 4 <2 4 Ticarcillin 16 <8 <8 library, Sacramento, Calif.). Different production lots of Cefazolin >16 >16 8 rBPI2 that had been formulated with surfactant or unfor Cefoxitin >16 8 8 mulated (without surfactant) were tested, but no difference Cefuroxime 8 4. 8 NO11-070 BPI G Reduced NG was seen in results for formulated or unformulated rBPI2. 20 (A. anitratus) Ampicillin 8 8 <2 A summary of the results of the antibiotic screening Trimeth/ >2 2 <0.5 panels, reported as MICs (ug/ml) of the antibiotic tested, is Sulfaks Cefazolin >16 >16 C2 shown in Tables 27 and 28 below. Results are reported for Cefoxitin 16 16 <2 each strain tested, but susceptibility data is listed for only Cefuroxime 16 8 <2 those antibiotics for which BPI protein product altered 25 Ceftazidime 4 <2 <2 NO11-071 BPI G G Very susceptibility. The antibiotic susceptibility standards (inter (A. anitratus) reduced pretation of an MIC as resistant, intermediate or susceptible Ticarcillin 32 <8 <8 according to Microscan?)'s NCCLS-derived standards) Aztreonam >16 16 16 Piperacillin' 32 16 <8 applicable to the organism tested appear in Tables 13A and 30 Ciprofloxacin' >2 2 <1 14A. For interpretation purposes, when antibiotic suscepti Ofloxacin 4. <2 <2 bility standards were given only for Enterobacter or Ceftazidime 8 8 4 Ceftriazone 32 16 16 Pseudomonas, the standards for Acinetobacter were consid Cefotaxime 32 16 16 ered to be the same as for Enterobacter. Gentamicin >6 >6 2 These results show that BPI protein product reversed 35 Tobramycin 6 4 4 Amikacin >16 16 8 resistance of A. anitratus strains to amoxicillin/K clavulan NO11-072 BPI G Reduced NG ate, ampicillin/sulbactam, aztreonam, carbenlcillin, cefa (A. anitratus) Ampicillin 16 16 <2 mandole, cefazolin, cefonicid, cefoperazone, cefotaxime, Trinleth >2 >2 <0.5 Sulfaks cefotetan, cefoxitin, ceftazidime, ceftizoxime, ceftriaxone, Cefazolin >16 >16 <2 chloramphenicol, ciprofloxacin, gentamicin, mezlocillin, 40 Cefoxitin 16 16 <2 netilmicin, ticarcillin, ticarcillin/K clavulanate, and t?ime Cefuroxime 8 8 <2 Gentamicin 6 4 <1 thopfim/sulfamethoxazole, and increased susceptibility of Tobramycin 2 C2 <2 some A. anitratus strains to amikacin, amoxicillin/K clavu Amikacin 8 4 <2 lanate, ampicillin. ampicillin/sulbactam, azlocillin, aztre N012-001 BPI G G G 45 (A. lwafii) Aztreonam' >16 >6 <8 onam, carbenicillin, cefamandole, cefazolin, cefonicid, Cefazolin >6 8 <2 cefoperazone, cefotaxime, cefotetan, cefoxitin, ceftazidime, Cefuroxime >16 4 <2 cefiizoxime, ceftriaxone, cefuroxime, chloramphenicol, Ceftazidime >16 4. <2 ciprofloxacin, gentamicin, imipenem, mezlocillin, netfirmi Ampicill/ >16 >6 6 Sulbact cin, ofloxacin, piperacillin, ticarcfilin, ticarcillin/K clavulan 50 Cefotaxine 8 <4 <4 ate, tobramycin and trimethoprim/suffamethoxazole. N012-002 BPI G NG NG (A. lowfii) Ampicillin 4 <2 <2 BPI protein product reversed resistance of A. lwofi Trimeth/ >2 2 <0.5 strains to aztreonam, cefazolin, cefuroxime, ceftazidime, Sulfakk cefoxitin, trimethoprim/suffamethoxazole and piperacillin, Aztreonam >16 k8 C8 55 Cefazoli >16 <2 k2 and increased susceptibility of A. lwofistrains to amplicillin, Cefoxiitin >16 <2 <2 ampicillin/sulbactam, aztreonam, cefazolin, cefotaxime, Cefuroxime 8 <2 <2 cefoxitin, ceftazidime, cefiriaxone, cefuroxime, pipemcillin, Ceftazidime 4. <2 <2 ticarcillin and trimethoprim/suffamethoxazole. N012-003 BPI G G NG (A. twoffii) Ampicillin' >16 4 <2 These results also show that rBPI at a concentration of Trimeth/ 2 <0.5 <0.5 16 ug/ml has direct bactericidal/growth inhibitory effects on 60 Sulfa some of the tested Acinetobacter isolates. 5,523,288 61 62 TABLE 27-continued TABLE 28-continued EFFECTS OF rBPI + ANTIBIOTICS ON Acinetobacter EFFECTS OF rBPI, it ANTIBIOTICS ON Acinetobacter SPECIES SPECIES

Minimum Minimum Inhibitory Concentration Inhibitory Concentration of Antibiotic (pg/mL) of Antibiotic (ig/mL) Microscan With With With Microscan With With With Library Antibiotic 0 uglinL 4 ughmL 16 g/mL 10 Library Antibiotic 0 ug/mL 4 uglmL 16 uglmL ID No. Tested rBPI2 rBPI2 rBPI2 ID No. Tested rBPI21 rBPI rBPI Ticarcillin 64 <8 <8 Cefotetan s32 Aztreolam >16 K8 C8 Netilmicin 16 Piperacillin' >64 <8 C8 Cefamandole s32 Cefazoli >16 >16 <2 Chlor >16 Cefoxitin' >16 8 <2 15 amphenicol Cefuroxime >16 <2 <2 Ticarcillin <16 Ceftazidime 16 <2 <2 Azlocilin Ceftriazone 16 <4 <4 Imipenem Cefotaxime 8 <4 <4 AmplSulbact 2 2 NO12-004 BPI G G G Aztreonan 8 8 (A. twoffii) Ampicillin 4. <2 4. 20 AmoxiK 16 16 Trinleth/ 2 2 <0.5 Clavulanate Sulfa Aiprofloxacin' Cefazolin >16 16 16 TicariK Cefoxitin 16 K2 4. Clavulante Cefuroxime 4 <2 <2 Mezlocillikk 128 N012-005 BPI G G NG 25 Carbenicillin <16 (A. iwoffii) Ampicillin 4 <2 <2 12487 Ceftizoxime Trimeth/ 2 <0.5 <0.5 (A. anitratus) Ceftazidime Sulfa Cefotaxine * Cefazolin' s16 >16 4 Ceftriaxone Cefoxitin 16 8 4 Cefoperazone' Cefuroxime 4 4 <2 30 Cefonicid Cefotetan Netilmicin Cefalandole TABLE 28 Chor amphenicol* EFFECTS OF rBPI, it ANTIBIOTICS ON Acinetobacter Ticarcink 35 128 SPECIES Azlocillin Imipenem Minimum AmplSulbact Inhibitory Concentration Aztreonam 16 of Antibiotic (ug/mL) Amox/K 32 <2 <2 Carbeniciini <16 (A. anitratus) Ceftazidime' >32 32 <1 19687 Ceftizoxime s32 Cefotaxine >64 8 <2 45 (A. anitratus) Ceftazidime s32 CeftriaxOne' >64 <2 <2 Cefotaxine Cefoperazone"* >32 8 <4 Ceftriaxone Cefonicid >16 16 <2 Cefoperazone Cefotetan >32 32 <4 Cefonicid Netilmicin >16 <2 <2 Cefotetan Cefamandole' >32 <4 <4 50 Netilmicin Chloram- >16 <2 <2 Cefamandole phenicol'* Chlor Ticarcillin 64 32 <16 amphenicol' Azlocillin >64 <64 <64 Ticarcillin Imipenem 4 <0.5 <0.5 Azocillin AmplSulbact" 32 4 32 Clavulanate AmoxiK 16 Ciprofloxacin'" >4 1. <25 Clavulanate Tica/K 64 <16 <16 Ciprofloxacin'" Clavulanate Ticar/K 28 Mezlocilin >128 <16 <16 60 Clavulanate Carbenicillin >28 K16 <16 Mezlocillin 32 12300 Ceftizoxime >32 >32 <8 Carbenicilink K6 (A. anitratus) Ceftazidine 8 8 2 19693 Ceftizoxime Cefotaxime 64 64 <2 (A. anitratus) Ceftazidime s32 Ceftriaxone 32 32 <2 Cefotaxime 32 Cefoperazone >32 >32 32 65 Ceftriaxone 16 Cefonicid >16 >16 >16 Cefoperazone 5,523,288 63 64 organisms. Microscan?) panel plates were prepared for E. TABLE 28-continued coli J5, E. coli O7:K1, Enterobacter cloacae (Microscan EFFECTS OF rBPI it ANTIBIOTICS ON Acinetobacter library ID no. 19680) and Klebsiella pneumoniae (Micros SPECIES can library ID no. 16135), according to Example 11. Cell suspensions were added to 25 ml Pluronic Inoculum Water Minimum Inhibitory Concentration containing 0 or 16 ug/ml rBPI. After inoculation, the panel of Antibiotic (ughmL) plates were incubated at 35° C. for 24 hours. At 0, 4, 7 and 24 hours after inoculation, 5 pil samples were removed from Microscan With With With each growth control well (containing culture media without Library Antibiotic 0 g/mL 4 g/mL 16 uglm 10 antibiotic) and from each well containing: 2/38 ug/ml tri ID No. Tested rBPI2 rBPI rBPI2 methoprim/sulfamethoxazole, 2 g/ml ciprofloxacin, 64 Cefonicid >16 >16 <2 ug/ml piperacillin, 32 ug/ml cefotaxine, 6 ug/ml Cefotetan * >32 >32 <4 cefuroxime, and 16 g/ml amikacin. These 5 ul samples Netilmicin >16 >16 >16 Cefamandole >32 >32 16 were diluted in sterile water and inoculated onto Trypticase Chlo- >16 >16 <2 15 Soyagar plates (Remel, Lenexa, Kansas). After 48 hours of amphenicol'* incubation at 35°C., the plates were counted and the number Ticarcilink >128 >28 <16 of colony forming units of bacteria in the well was calcu Azocillin >64 >64 <64 lated. Imipenem 2 2 <0.5 Amp/Sulbact 32 16 4 >4 >4 rBPI, is indicated for: E. coli J5 (a filled square): E. coli Ticar/K >128 32 <16 O7:K1 (a filled diamond); E. cloacae (a filled triangle); and Clavulanate K. pneumoniae (an “X”). Also, in all figures, the growth in Mezocillin >128 128 <16 the presence of antibiotic with rBPI' is indicated for E. coli Carbenicillin >128 >128 <16 25 19694 Ceftizoxine >32 >32 32 J5 (an open square); E. coli O7:K1 (an open diamond); E. (A. anitratus) Ceftazidime 16 16 8 cloacae (an open triangle); and K. pneumoniae (a star). Cefotaxine 64 64 64 FIG. 19 shows the kinetic growth curve of organisms with Ceftriaxone 64 64 32 Cefoperazone >32 >32 >32 rBPI2 (and without antibiotic) and without rBPI (and Cefolicid >16 >16 8 30 without antibiotic). In FIG. 19, the growth curves for E. coli Cefotetan >32 >32 8 J5 without rBPI (filled squares), E. coli O7:K1 with rBPI Netilmicin >16 >16 4 (open diamonds), E. coli O7:K1 without rBPI (filled Cefamandole 32 >32 >32 Chlor- >16 >16 16 diamonds), and K. pneumoraae without rBPI ("X's) over amphenicol lap substantially, while the growth curves for E. coli J5 with Ticarcillin >128 >128 32 rBPI (open squares) and E. cloacae with rBPI2 (open Azlocillin >64 >64 >64 35 triangles) overlap at 0–7 hours but diverge by 24 hours. FIG. Imipenem 1 1 <0.5 AmplSulbact 32 32 16 19 demonstrates that BPI protein product alone has a bac Aztreonam 8 8 8 tericidal effect at 0-7 hours on E. coli J5 and K. pneumoniae, Amox/K 32 8 4 and a bactericidal effect on E. cloacae throughout the 24 Clavulanate hour period studied. Ciprofloxacin >4 >4 >4 40 Ticar?k >128 <16 <16 In FIG. 20, the growth curves for E. coli J5 with rBPI Clavulanate (open squares), E. cloacae with rBPI (open triangles) and Mezocillin >128 >128 64 K. pneumoniae with rBPI (stars) overlap. FIG. 20 shows Carbenicillin >128 >128 <16 that rBPI2 enhanced the bactericidal effect of trimethoprim/ sulfamethoxazole on E. cloacae and K. pneumoniae at 0-24 45 hours, and slightly enhanced the antibiotics' effect on E. coli EXAMPLE 21 J5 and E. coli O7:K1. EFFECTS OF BPI PROTEIN PRODUCT AND In FIG. 21, the growth curves for E. coli J5 with rBPI ANTIBIOTICS IN VITRO ON SALMONELLA (open squares), E. coli J5 without rBPI (filled squares), E. AND SHIGELLA SPECIES 50 coli O7:K1 with rBPI (open diamonds), E. coil O7:K1 Ten clinical isolates of Salmonella (F270-001 through without rBPI (filled diamonds), E. cloacae with rBPI -010) and 10 clinical isolates of Shigella (F321-010 and (open triangles), E. cloacae without rBH (filled triangles), F325-002 through -010) (all isolates from Baxter Micros and K. pneumoniae with rBPI (stars) overlap. FIG. 21 cance library, Sacramento, Calif.), were evaluated in the shows that rBPI2 reversed resistance of K. pneumoniae to MicroscanG) antibiotic susceptibility screening assay of 55 ciprofioxacin at 0-24 hours; the other organisms were Example 11 using the Neg Breakpoint Combo Type 9 panel already very susceptible to ciprofloxacin. plate. Essentially no effect was seen at rBPI concentrations In FIG. 22, the growth curves for E. coli O7:K1 without of 0, 4 and 16 g/mL. rBPI (filled diamonds) and E. cloacae without rBPI (filled triangles) overlap, while the growth curves for E. coli EXAMPLE 22 60 J5 with rBPI2 (open squares), E. coli J5 without rBPI EFFECTS OF BPI PROTEIN PRODUCT ON (filled squares), E. coli O7:K1 with rBPI (open diamonds), ANTIBIOTIC KILLING CURVES FOR E. COLI E. cloacae with rBPI2 (open triangles), K. pneumoniae J5, E. COLI O7:K1, ENTEROBACTER CLOACAE, without rBPI ("X's) and K. pneumoniae with rBPI AND KLEBSIELLA PNEUMONIAE (stars) overlap. FIG. 22 shows that rBPI, enhanced the 65 bactericidal effect of piperacillin on E. coli O7:K1 and E. The effect of a BPI protein product, rBPI, on the killing cloacae; the other organisms were already susceptible to the curves of selected antibiotics was determined for selected drug. 5,523,288 65 In FIG. 23, the growth curves for E. coli J5 with rBPI2 (open squares), E. coli O7:K1 with rBPI2 (open diamonds), E. coli O7:K1 without rBPI (filled diamonds), K. pneu moniae without rBPI ("X's) and K. pneumoniae with rBPI (stars) overlap. FIG. 23 shows that rBPI, enhanced 5 the bactericidal effect of cefotaxime on J5 and E. cloacae, the other organisms were susceptible to the drug. In FIG. 24, all of the curves overlap. FIG. 24 shows the effect of rBPI and cefuroxime; addition of rBPI had no effect on the killing curves because all of the tested species 10 were very susceptible to the antibiotic. In FIG. 25, all of the curves overlap. FIG. 25 shows the effect of rBPI2 and amikacin; again, all tested species were very susceptible to the antibiotic. 15 EXAMPLE 23 EFFECTS OF A VARIETY OF BPI PROTEIN PRODUCTS AND ANTIBIOTICS IN VITRO ON REPRESENTATIVE GRAM-NEGATIVE 20 ORGANISMS The effects of a variety of BPI protein products, rBPI, rBPI2, rBPIs and rBPI2 dimer on the antibiotic suscepti bility of various representative gram-negative organisms 25 was evaluated in the Microscan?E) antibiotic susceptibility screening assay of Example 11 using the MIC Plus Type 2 panel plate. Assays were conducted on clinical isolates of Acinetobacter anitratus and Enterobacter cloacae (from Baxter MicroscanG) library, Sacramento, Calif.), and on E. 30 coli J5-L and O7:K1. The results, reported as MICs (ug/ml) of the antibiotic tested at the various concentrations of BPI protein product indicated, are shown in Tables 29, 30, 31 and 32 below.

TABLE 29 EFFECTS OF BPI PROTEIN PRODUCTS ANTIBIOTICS ON A. anitratus (Microscan ID No. 12292) Minimun Inhibitory Concentration of Antibiotic (ug/ml) With: 0. 4 4 4 4 pg/mL 16 16 16 16 g/mL ANTIBIOTIC uglmL g/mL ug/mL uglml BPI uglimi Hg/mL Hg/mL BPI-12ml TESTED BP BPI BPI. BPIso dimer BPI. BPI. BPIso dinner Ceftizoxime >32 >32 >32 >32 >32 >32 <32 <32 >32 Ceftazidime >32 16 >32 >32 >32 32 >32 >32 32 Cefotaxine >64 >64 >64 >64 >64 16 >64 >64 >64 Ceftriaxone >64 8 >64 >64 >64 4 >64 >64 >64 Cefoperazone >32 >32 >32 >32 >32 32 >32 >32 >32 Cefonicid >16 >16 >16 >16 >16 8 >16 >16 >16 Cefotetan >32 32 >32 >32 >32 <4 >32 >32 >32 Netilmicin >6 <2 >16 >16 >16 >6 >16 >6 >16 Cefamandole >32 8 >32 >32 >32 <4 >32 >32 >32 Chloramphenicol >6 <2 >16 16 16 <2 16 16 16 Ticarcillin 128 <16 128 64 64 <16 64 32 64 Azlocillin >64 >64 >64 >64 >64 <64 >64 >64 >64 Imipenem 4. <0.5 2 2 2 <0.5 2 2 2 Ampicillin? 32 4 16 32 16 <1 8 16 8 Sulbactam Aztreonam 32 16 32 32 32 4 32 32 32 Amoxicillin/K 16 2 16 16 8 4 8 8 4 Clavulanate Ciprofloxacin 4. 2 4 4 4. 2 4 4 2 Ticarcillin/K 64 (16 64 64 32 32 32 32 32 Clavulanate Mezlocillin >28 128 >128 >28 >128 <16 >128 >128 >128 Carbeniciin 128 k16 128 128 64 k6 64 64 64

65

5,523.288 69 70 TABLE 32 EFFECTS OF BPI PROTEIN PRODUCTS - ANTIBIOTICS ON E. coli O7:K1 Minimum Inhibitory Concentration of Antibiotic (ug/ml) With: O 4 4 4 4 ughmL 16 6 16 16 g/mL ANTIBIOTIC uglmL g/mL g/mL ag/mL BP4. g/mL Hg/mL g/mL BP42nt TESTED BPI BPI. BPI. BPIs dimer BPI. BPI. BPIso dimer Ceftizoxine <2 <2 <2 <2 <2 <2 <2 <2 <2 Ceftazidime <

EXAMPLE 24 30 The entire contents of such tubes were then poured into a

GRAM-NEGATIVEOF BPI BACTERICIDAL PEPTIDES ACTIVITY EEE"E.C. garOS C Seconds and had a uniform thickness of about 1 mm. A series BPI peptides were produced according to co-owned and 35 of wells were then punched into the hardened agarose using copending PCT Application No. PCT US94/10427 filed Sep. 15, 1994, which corresponds to U.S. patent application Ser. a sterile 3 mm punch attached to a vacuum apparatus. The No. 08/306,473 filed Sep. 15, 1994, and PCT Application punch was sterilized with 100% alcohol and allowed to air No. US94/02465 filed Mar. 11, 1994, which corresponds to dry prior to use to avoid contaminating the bacterial culture. U.S. patent application Ser. No. 08/209,762, filed Mar. 11, 5 or 10 puL of each of the BPI peptides were carefully 1994, which is a continuation-in-pan of U.S. patent appli pipetted into each well. As a negative control, dilution buffer cation Ser. No. 08/183,222, filed Jan. 14, 1994, which is a (pH 8.3) was added to a separate well, and rBPI at continuation-in-part of U.S. patent application Ser. No. concentrations of 5ug/mL and 1 ug/mL were also added as 08/093,202 filed Jul. 15, 1993 (for which the corresponding positive controls. Each plate was incubated at 37° C. for 3 international application is PCT Application No. US94/ 45 hours, and then 10 mL of molten overlayer agarose (at 02401 filed Mar. 11, 1994), which is a continuation-in-part approximately 45° C) was added into the level petri dish, of U.S. patent application Ser. No. 08/030,644 filed Mar. 12, allowed to harden and incubated overnight at 37° C. The 1993, the disclosures of all of which are incorporated herein next day, a clear Zone was seen against the lawn of bacteria by reference. in those wells having bactericidal activity. In order to The BPI peptides were screened for bactericidal effects on 50 visually enhance this zone, a dilute Coomassie solution E. coli J5 and E. Coli O111: B4 bacteria in a radial diffusion (consisting of 0.002% Coomassie Brilliant Blue, 27% assay. Specifically, an overnight culture of the bacteria was methanol. 15% formaldehyde (37% stock solution) and diluted 1:50 into fresh tryptic soy broth and incubated for 3 water) was poured over the agar and allowed to stain for 24 hours at 3 C. to attain log phase growth of the culture. hours. The bacterial zones were measured with a microme Bacteria were then pelleted at 3,000 rpm for 5 minutes in a 55 Sorval RT6000B centrifuge (Sorval Instruments, Newton, ter. The assay results for exemplary peptides (BPI. 1 through TC).5 mL of 10 mM sodium phosphate buffer (pH 74) was BPI. 169) are summarized in Table 33 for the Gram-negative added and the preparation was re-pelleted. The supernatant bacteria E. coli J5 (rough) and E. coli O113 (smooth). The was decanted and 5 mL of fresh buffer was added, the bactericidal activities are expressed as the amount of peptide bacteria were resuspended and their concentration was 60 (pmol/well and ug?well) required to generate a 30 mm determined by measurement of absorbance at 590 nm (an bactericidal zone. Additional exemplary BPI peptides Absorbance value of 1.00 at this wavelength equals a include BPI.221 through BPI.281. BPI peptides which retain concentration of about 1.25x10 CFU/mL in suspension). antibacterial activity are expected to improve the therapeutic The bacteria were diluted to 4x10 CFU/mL in 10 mL of effectiveness of antibiotics when concurrently administered molten underlayer agarose (at approximately 45 C.) and 65 therewith. The peptides are screened for such activity in an inverted repeatedly to mix in 15 mL polypropylene tubes in vivo model or according to in vitro tests, including models conventionally used for this purpose. and tests described herein.

5,523,288 75 76

TABLE 33-continued Bacteridical Activity BPI Sequence E. coli J5 E. coli O11:B4 Peptide ID No. (pmol/well) (ug?well) (pmol/well) (ugwell) BPI.158 154 N.T. N.T. N.T. N.T. BPI.159 155 765.43 2.41 >1589.88 >5 BPI.160 156 288.78 0.81 1781.59 >5 BPI.161 157 20179 2.00 w BPI.62 58 N.T. N.T. N.T. N.T. BPI.63 159 N.T. N.T. N.T. N.T. BPI.164 60 N.T. N.T. N.T. N.T. BPI.65 161 N.T. N.T. N.T. N.T. BPI.66 162 54.00 0.83 >3078.72 >5 BPI.67 163 >4585.73 >5 BPI.68 64 1460.98 2.87 s1948.48 >5 BPI.169 165 4893.83 >5 4974.43 >5 BPI. 170 227 3693.06 >5 am MAP1* 06 0.82 552.79 427 MAP2: >690.9 >5 >690.9 >5 "Amount added to well to achieve a 30 mm hole as determined by PROBIT analysis as described in Examples 15 and 16. No detectable activity up to 5 pig?well. N.T. = not tested *MAP1 = B-Ala-No, Ne-(No.Ne(BPI.2).LysLys **MAP2 = B-Ala-NoNe-No.Ne(BPI.13)Lys) Lys

EXAMPLE 25 TABLE 34 EFFECTS OF CONCURRENT EFFECTS OF BP PROTEIN PRODUCT ADMINISTRATION OF BPI PROTEIN 30 ANTIBIOTIC WHEN CONCURRENTLY ADMINISTER PRODUCT AND TETRACYCLINE OR CLASS ED WITH ANTIBIOTICS WITHIN CLASS GENTAMICIN ON E. COLI O111:B4 3-lactams: reversed resistance of Pseudomonas aeruginosa, Additional MIC assays were performed to determine the penicillins and other Pseudomonas, Xanthalmonas, E. coli, cephalosporins Citrobacter, Klebsiella, Enterobacter, sensitivity of E. coli 01 11:B4 (as described in Example 1) to 35 Serratia, Providencia, Acinetobacter the effects of BPI protein products concurrently adminis increased susceptibility of Pseudomonas tered with the antibiotic tetracycline or with the antibiotic aeruginosa, other Pseudomonas, E. coli, gentamicin. Citrobacter, Klebsiella, Enterobacter, Serratia, roteus, Providencia, Morganella, For these experiments, organisms were grown overnight Acinetobacter at 37° C. in 5 mL of Mueller-Hinton broth. This overnight 40 enchanced early bctericidal effect for E. coli, culture was diluted 1:50 into 5 mL of fresh broth and Enterobacter in killing curves incubated for an additional 3 hours at 37° C. to attain in vivo synergy shown for treatment of E. coli infection in mouse and rabbit models log-phase growth. Bacteria were pelleted for 5 minutes at B-lactams aztreoal: 1500 x g and resuspended in fresh broth to give a final other than reversed resistance of Pseudomonas aeruginosa, concentration of 2x10 cells per mL. 45 penicillins and Acinetobacter m increased susceptibility of Acinetobacter rBPI2 and antibiotic (either tetracycline or gentamicin) poor. increased susceptibility of Pseudomonas were diluted such that 100 ul bacterial suspension, 50 il imipenem aeruginosa, Citrobacter, Enterobacter imipenem: antibiotic and 50 pil diluted rBPI gave concentrations in increased susceptibility of Pseudomonas serial dilutions from 10 ug/mL tetracycline or 2.5 lug/mL aeruginosa, Proteus, Providencia, gentamicin and from 30 ug/mL rBPI with a fixed concen- 50 Acinetobacter tration of 10 cells/mL. Incubation was carried out in flat aminoglycosides reversed resistance of Pseudomonas aeruginosa, bottom 96 well microliter plates for 18 hours at 37° C., and Xanthamonas, Acinetobacter increased susceptibility of Pseudomonas the plates were read in an automatic plate reader (Titretek aeruginosa, other Pseudomonas, E. coli, Multiscan) at 590 nm. Citrobacter, Serratia, Proteus, Providencia, A 4-6 fold decrease in absorbance was observed with 55 Morganella, Acinetobacter checkerboard synergy (FIC < 0.5) for E. coli, tetracycline or gentamicin with certain concentrations of Salmonella, Klebsiella, Edwardsiella, greater BPI protein product. The MIC of tetracycline without rBPI than additive interaction (FIC s 1.0) for of 10 g/mL and was reduced by rBPI to 5 ug/mL. The Pseudomonas aeruginosa, Enterobacter MIC of gentamicin without rBPI was 0.6 ug/mL and was in vivo synergy shown for treatment of E. coli infection in mouse model reduced by rBPI to 0.3 ug/mL. 60 sulfonamides reversed resistance of Pseudomonas aeruginosa, A partial summary of the data described in the foregoing and Xanthamonas, Klebsiella, Acinetobacter examples, grouped by general classes of antibiotics, appears trimethoprim increased susceptibility of Enterobacter, below in Table 34 and is displayed as the effects of BPI Proteus, Acinetobacter protein products on the therapeutic effectiveness of antibi enhanced early bactericidal effect for otics for various gram-negative organisms. 5,523,288 77 78 TABLE 34-continued TABLE 34-continued EFFECTS OF BPI PROTEIN PRODUCT EFFECTS OF BPI PROTEIN PRODUCT ANTIBIOTIC WHEN CONCURRENTLY ADMINISTER. ANTIBIOTIC WHEN CONCURRENTLY ADMINISTER CLASS ED WITH ANTIBIOTICS WITH IN CLASS 5 CLASS ED WITH ANTIBIOTICS WITHIN CLASS Klebsiella, Enterobacter in killing curves; chloramphenicol reversed resistance and increased susceptibility slight early enhancement for E. coli for Acinetobacter fluoroquinolones reversed resistance of Pseudomonas aeruginosa, and quinolones Acinetobacter increased susceptibility of Pseudomonas 10 aeruginosa, Xanthamonas, Enterobacter, Numerous modifications and variations in the practice of Acinetobacter the invention are expected to occur to those skilled in the art reversed resistance of Klebsiella in antibiotic killing curves upon consideration of the foregoing description of the polymyxins checkerboard synergy (FIC < 0.5) for presently preferred embodiments thereof. Consequently, the Pseudomonas aeruginosa, E. coli, Providencia, only limitations which should be placed upon the scope of additive interaction (FIC s 1.0) for 15 Enterobacter the present invention are those which appear in the appended claims.

SEQUENCE LISTING

( 1) GENERAL INFORMATION: ( i i i ) NUMBER OF SEQUENCES: 227

( 2) INFORMATION FOR SEQ LD NO:l: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH:29 ainino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "Domain ( xi ) SEQUENCE DESCRIPTION: SEQID NO:1: A a Sc r G in G 1 in G 1 y T h r A a A 1 a Lic u G 1 m Lys G 1 u Lc u Lys Arg Il c 5 1 0 5 Ly is I c Pro A s p Ty r S c r A s p S c r Phc Lys I 1 c L y s H is 2 O 25

( 2) INFORMATION FOR SEQ ID NO:2: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 amino acids (B) TYPE; amino acid ( D TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature ( D ) OTHER INFORMATION: "BPI.14" ( xi ) SEQUENCE DESCRIPTION: SEQID NO:2: G l y Th r A 1 a. A la L. c u G l n Il y s G 1 u Lic u Lys A rig I c L y s I l c Pro As p 1. 5 1 O 15 Ty r S c r A s p S c r Phc Lys I 1 c L y s H is Le u Gil y Lys G 1 y H is 20 25 3 0

( 2) INFORMATION FOR SEQED NO:3: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 5,523,288 79 80 -continued

( i i ) MOLECULETYPE: peptide ( i x ) FEATURE; (A) NAME/KEY: Inisc feature (D) OTHER INFORMATION: "BPI.4.” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:3:

L. c u G l n Lys G 1 u L. c u Lys le As p Ty r S c. r As p S. c. r 1 5 O 15 Phc Lys I c L y s H is L. c u 2 O

( 2) INFORMATION FOR SEQID NO:4: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid ( D TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE; (A) NAMEIKEY: Inisc feature ( D ) OTHER INFORMATION: "BPI.1.” ( x i ) SEQUENCE DESCRIPTION: SEQ D NO:4:

G l n G 1 n G y T h r A la A 1 a L clu G In Ly is G 1 u L c u Lys A rig I l c L y S 5 1 O 1 5

( 2) INFORMATION FOR SEQID NO:5: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptidc ( i x FEATURE: (A) NAMEIKEY: misc feature (D) OTHER INFORMATION: "BPI.54" ( x i ). SEQUENCE DESCRIPTION: SEQID NO:5:

G 1 y Th r A 1 a. A 1 a L c u G 1 in L y S Glu L. c u Lys A rig I c Lys I c P to 5 1 O 15

( 2) INFORMATION FOR SEQ LD NO:6: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 amino acids (B) TYPE: amino acid ( D.) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAMEIKEY: misc featurc ( D.) OTHER INFORMATION: "Domain II” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:6:

Sc I S. c G n 1 c S c Mc Wa P ro As in Wal Gly L clu Ly is P c S e r I e 1 5 O 15

Sc r A s in A 1 a. As n I lic Lys I l c See G l y Lys T r p Lys A a G In Lys A rig 20 25 3 O Ph c L eu Lys 35

( 2) INFORMATION FOR SEQIP No.:7; 5,523,288 81 82 -continued

( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: ( A ) NAME/KEY: misc feature ( D OTHER INFORMATION: "BPI.2" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Il c L y is I c Sc r Gil y Lly s Trip Lys Al a G 1 in Lys Arg Phc L clu S 1. 5 0.

( 2) INFORMATION FOR SEQID NO:8: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: Inisc ?cature ( D.) OTHER INFORMATION: “BPI.8” ( x i ). SEQUENCE DESCRIPTION: SEQID NO:8: Lys Trip Lys Al a G n Lys Arg Phc L c u Lys 5 1 O

( 2) INFORMATION FOR SEQID NO;9: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE; (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.58" ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:9: Cys l l c L y s I c Sc r Gil y Lys Trip Lys Al a G 1 in Lys Arg Phc L c u Lys 5 1 O 1 5

( 2) INFORMATION FOR SEQ ID NO:10: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: pcptide ( i x ) FEATURE; (A) NAME/KEY: misc ?calure (D) OTHER INFORMATION: “BPI.65 oxidized' ( xi ) SEQUENCE DESCRIPTION: SEQID NO:10: Cys I l c L y s I lic Sc r G y Lly s Trip Lys A a G l n Lys Arg Phc Lc u Lys 1. 5 1 0 5 Cy is

( 2) INFORMATION FOR SEQID NO:11: ( i ) SEQUENCE CHARACTERISTICS: 5,523.288

-continued (A) LENGTH: 27 amino acids (B) TYPE: amino acid ( D TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: ( A ) NAME/KEY: misc feature ( D.) OTHER INFORMATION: "BPI.3" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:11: As n V a l G 1 y L. c u Lys Ph. c Sc r I 1 c Sc r A sin A 1 a. As n I c L y s I Sc I 5 1 O 1 5 G l y Lys Trip Lys A l a G l n Lys A rig Phc L. c u Lys 2 O 25

( 2) INFORMATION FOR SEQID NO:12: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "Domain III ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:12: V a l H is V a 1 H is I 1 c Sc r Lys Sc r Lys V a G 1 y Trip L. c u I 1 c G 1 n L clu 1. 5 1 0 5 Ph c H is Ly S L y S I 1 c G 1 u S c r A a L c u A rig As n Lys 20 25

( 2) INFORMATION FOR SEQID NO:13: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAMEKEY: Inisc featurc (D) OTHER INFORMATION: "BP.11” ( x i ). SEQUENCE DESCRIPTION: SEQID NO:13: L y S S C T L y S V a l T r p L. c u I 1 c G 1 in L c u Ph c H is Lys Lys 5 1 0

( 2) INFORMATION FOR SEQID NO:14: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: misc feature ( D OTHER INFORMATION: “BPI.12' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:14: S C T V a I H is V a l H is I lic Sc r Lys Sc r Lys v a 1 G 1 y Trip L clu. I l G 1 a 1. 5 O 15 L. c u Ph C H is Lys Lys I 1 c G 1 u S c r A la L c u A rig As n Lys 20 25 5,523,288 85 86 -continued

( 2) INFORMATION FOR SEQ ID NO:15: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE:amino acid ( D ). TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: Inisc ?cature ( D ) OTHER INFORMATION: "BPI.13' ( xi ) SEQUENCE DESCRIPTION: SEQID NO:15: Ly s Sc r Lys V a 1 G l y T r p L. c u I 1 c G 1 in Lc u Ph c H is Lys Lys 5 1 0

( 2) INFORMATION FOR SEQID NO:16: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.15" ( xi ) SEQUENCE DESCRIPTION: SEQID NO:16: A la Lys I c Sc r G i y Lys T r p Lys Al a Gil n Lys Arg Phc L. c u Lys l 5 0. 1 5

( 2) INFORMATION FOR SEQ ID NO:17: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: pcptidc ( i x ) FEATURE: (A) NAMEKEY: misc feature (D) OTHER INFORMATION: “BPI.16" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:17: I 1 c A la I c Sc r Gil y Llys Trip Lys A a Gl in Lys A g Phc L. c u Lys 5 1 O 15

(2) INFORMATION FOR SEQ ID NO:18: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptidc ( i x ) FEATURE: (A) NAME/KEY: misc feature ( D ) OTHER INFORMATION: "BPI.1.7" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:18: I 1 c L y s Al a Sc r G 1 y Ly S T r p Lys A 1 a G l n Lys A rig Phc Lc u Lys 1. 5 1 0 5

(2) INFORMATION FOR SEQD NO:19: 5,523,288

-continued ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: pcptide ( i x ) FEATURE; (A) NAMEKEY: misc cature ( D ) OTHER INFORMATION: "BPI.18" ( x i ). SEQUENCE DESCRIPTION: SEQID NO:19: I c L y S I 1 C A a G 1 y Ly S T rip Lys A la G. l n Lys Arg Phc L cu Lys 5 1 0 5

( 2) INFORMATION FOR SEQ ID NO:20: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: misc caturc ( D.) OTHER INFORMATION: "BPI.19" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:20: I l c L y S I 1 c Sc r A la L y S T r p Lys Al a G l n Lys Arg Phc Le u Lys 5 1 O 5

( 2) INFORMATION FOR SEQ ID NO:21: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid ( D TOPOLOGY: incar ( i i ) MOLECULETYPE: pcptide ( i x ) FEATURE: (A) NAMEIKEY: misc feature (D) OTHER INFORMATION: “BPI.20" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:21: I lic Lys I l c Sc r G l y A la T r p Lys Al a G 1 in Lys Arg Phc Lc u Lys 5 0 1 5

( 2) INFORMATION FOR SEQ ID NO:22: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: pcptide ( i x ) FEATURE: (A) NAMEKEY: misc featurc ( D ) OTHER INFORMATION: "BPI21” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:22: I l c L y S I lic S c 1 Gil y Lys A a Lys Al a G in Lys Arg Phc Lc u Lys 5 1 O 5

( 2) INFORMATION FOR SEQID NO:23; ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid ( D TOPOLOGY: lincar 5,523,288 89 90 -continued

( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: Inisc ?cature ( D ) OTHER INFORMATION: “BPI22” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:23:

I c L y S I c Sc r G 1 y Lys T r p A a A a G Ly is A T g Ph. c. L C u. L y S 1. 5 O 15

( 2) INFORMATION FOR SEQ LD NO:24: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid CD TOPOLOGY: Iincar ( i i ) MOLECULE TYPE: pcptide

( i x ) FEATURE: (A) NAMEKEY: Lisc featurc ( D ) OTHER INFORMATION: “BPI.23' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:24:

I 1 c L y S I lic Sc r G 1 y Lys Tr p Lys A 1 a A 1 a Lys A rig P c L C u Lys l 5 15

( 2) INFORMATION FOR SEQID NO:25: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature ( D ) OTHER INFORMATION: “BPI.24 ( x i ) SEQUENCE DESCRIPTION: SEQID NO:25:

I 1 c L y s I c S c r G 1 y Lys T r p L y S A a G 1 I A 1 a A rig P c L. c u Ly S 5 1 O 5

( 2) INFORMATION FOR SEQ LD NO:26: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc cature ( D.) OTHER INFORMATION: "BPI.25" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:26:

I 1 c L y S I 1 c Sc r G y Lys T r p Ly is A 1 a G I L y S A a Ph. c. L clu Lys 5 1 O 5

( 2) INFORMATION FOR SEQ ID NO:27: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i MOLECULE TYPE: peptide ( i x ) FEATURE: 5,523,288

-continued (A) NAME/KEY: misc feature (D) OTHER INFORMATION: “BPI.26 ( x i ) SEQUENCE DESCRIPTION: SEQID NO:27: I c L y S I lic Sc r G l y Lys Trip Lys A l a G 1 in Lys A rig A 1 a L cu Lys 5 0 5

( 2) INFORMATION FOR SEQID NO:28: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: pcptidic ( i x ) FEATURE: (A) NAMEIKEY: misc feature ( D.) OTHER INFORMATION: "BPI.27' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:28: I 1 C L y S I lic S c I Gil y Lys T r p Lys A a G l n Lys A rig Phc A la Lys 5 1 O 15

( 2) INFORMATION FOR SEQ ID NO:29: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE; (A) NAMEKEY: misc ?cature (D) OTHER INFORMATION: "BPI.28' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:29: I l c L y S I l C S c r G l y Ly S T r p Lys A 1 a G 1 n Lys Arg Phc Lc u A a 5 1 O 15

( 2) INFORMATION FOR SEQI NO:30: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: Inisc feature ( D ) OTHER INFORMATION: "BPI.59" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:30: I I c L y S I lic Sc r G 1 y A la T r p A a A 1 a G 1 n Lys Arg Phc Lc u Lys 5 0 5

( 2) INFORMATION FOR SEQ ID NO:31: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE; (A) NAMEKEY: misc feature (D) OTHER INFORMATION: "BPI.45 ( x i ). SEQUENCE DESCRIPTION: SEQID NO:31: 5,523,288 93 94 -continued

I l c L y s I 1 c Sc r G 1 y Lys T r p Lys A a A 1 a. A 1 a. A rig Ph c L c u Lys 1. 5 0 5

( 2) INFORMATION FOR SEQID NO:32: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: pcptide ( i x ) FEATURE: (A) NAMEKEY: Inisc feature ( D ) OTHER INFORMATION: "BPI.60” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:32: I c. A 1 a I l c Sc r Gil y Lys Trip Lys A la G 1 n Lys Arg Phc Lc u A 1 a l 5 1 0 1 5

( 2) INFORMATION FOR SEQ ID NO:33: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc featurc ( D ) OTHER INFORMATION: "BPI.31” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:33: A la Sc r Lys V a G 1 y Trip L. c u I 1 c G l n Lc u Ph c H is Lys Lys 1 5 1 O

( 2) INFORMATION FOR SEQID NO:34: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: pcptide ( i x ) FEATURE; (A) NAMEKEY: misc feature ( D ) OTHER INFORMATION: "BPI.32” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:34: Lys A a Lys V a l G y Trip L. c u I c G 1 n L c u Ph e H is Lys Lys 1. 5 1 O

( 2) INFORMATION FOR SEQ ID NO:35: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI33" ( x i ) SEQUENCE DESCRIPTION: SEQ LD NO:35: L y S S c I. A 1 a V a G l y Trip L. c u I l c G l n L c u Ph c H is Lys Lys 1 5 1 0 5,523,288 95 96 -continued

( 2) INFORMATION FOR SEQ ID NO:36: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 anino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: pcptide ( i x ) FEATURE; (A) NAMEKEY: misc feature ( D.) OTHER INFORMATION: "BPI.34” ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Ly is S c r Lys A l a G l y T r p L clu G I L clu Phc H is Ly S Lys 5 0

( 2) INFORMATION FOR SEQID NO:37; ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid ( D TOPOLOGY: incar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE; (A) NAME/KEY: misc feature ( D ) OTHER INFORMATION: "BPI.35” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:37:

Lys S c r Lys V a l A a T r p cu c G 1 in I. c u P H is Ly S Lys 1 5 O

( 2) INFORMATION FOR SEQ ID NO:38: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: misc ?cature (D) OTHER INFORMATION: “BPI.36 ( x i ) SEQUENCE DESCRIPTION: SEQID NO:38:

Lys S c r Lys V a 1 Gil y A 1 a L C u I c G 1 a L clu Ph H is Lys Ly is 5 1 O

( 2) INFORMATION FOR SEQ ID NO:39: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature ( D ) OTHER INFORMATION: "BPI,37” ( x i ) SEQUENCE DESCRIPTION: SEQDD NO:39:

Ly is S c r Lys V a 1 G i y Tr p A l a c G In Lic u P H is Lys ... ys 5 O

( 2) INFORMATION FOR SEQID No:40: ( i) SEQUENCE CHARACTERISTICS: 5,523,288 97 98 -continued (A) LENGTH: 14 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc ?cature (D OTHER INFORMATION: "BPI.38" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:40: Ly S S c r Lys V a G l y T r p L c u A a G 1 n Lc u Ph c His Lys Lys 1. 5 1 0

( 2) INFORMATION FOR SEQID NO:41: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE:amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: pcptidc ( i x ) FEATURE: (A) NAME/KEY: misc ?cature ( D OTHER INFORMATION: “BPI.39" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:41: Lys Sc r Lys V a G y Trip L. c u I lic A i a L c u Ph c H is Lys Lys 1. 5 1 0

( 2) INFORMATION FOR SEQID NO:42: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptidc ( i x ) FEATURE: (A) NAMEKEY: misc feature ( D.) OTHER INFORMATION: “BPI.40' ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:42: Ly is S c r Lys V a G 1 y Trip L. c u I l c G | n A l a Ph. c H is Lys Lys 1. 5 1 0

( 2) INFORMATION FOR SEQ ID NO:43: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: Inisc ?caturc ( D ) OTHER INFORMATION: "BPI.4.1" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:43: Lys Sc r Lys V a l G l y T r p L. c u I l c Gil n Lc u A la His Lys Lys 1. 5 0

( 2) INFORMATION FOR SEQ ID NO:44: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar 5,523,288 99 100 -continued ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAMEIKEY: Inisc feature (D) OTHER INFORMATION: "BPI.42' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:44: Lys Sc r Lys V a l G l y Trip L. c u I c G n L cu Ph c A 1 a Lys Lys 1. 5 1 0

( 2) INFORMATION FOR SEQID NO:45: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid ( D.) TOPOLOGY: incar ( i i ) MOLECULETYPE: pcptide ( i x FEATURE: (A) NAME/KEY: Inisc feature ( D.) OTHER INFORMATION: "BPI.43' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:45: Lys Sc r Lys V a l G 1 y T r p L. c u I 1 c G in L cu Ph c H is A la Lys 1. 5 0

( 2) INFORMATION FOR SEQ ID NO:46: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature ( D OTHER INFORMATION: "BPI.44” ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:46: Lys Sc r Lys V a l G 1 y Trip L. c u I 1 c G 1 in Lc u Ph c H is Lys . A 1 a 5 1 O

( 2) INFORMATION FOR SEQID NO:47: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.56 ( x i ) SEQUENCE DESCRIPTION: SEQID NO:47: I lic Lys I 1 c S c r G I y Lly s Trip Lys A 1 a Lys Gl in Arg Phc Le u Lys 5 0 15

( 2) INFORMATION FOR SEQID NO:48: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: misc feature 5,523,288

-continued ( D.) OTHER INFORMATION: "BPI.61" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:48: Il c L y s I le S c r G 1 y Lys Phc Lys Al a G l n Lys A g Phc Lc u Lys 1 5 1 0 15

( 2) INFORMATION FOR SEQ ID NO:49: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptidc ( i x ) FEATURE: (A) NAMEIKEY: misc ?cature (D) OTHER INFORMATION: "BPI.66" ( i x ) FEATURE: (A) NAMEKEY: Modified-site (B) LOCATION: 7 ( D.) OTHER INFORMATION: label-D-Trp f notc="The amino acid at position 7 is D- tryptophan' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:49: I 1 c L y s 1 c Sc r G 1 y Lys Trip Lys A a G in Lys Arg Phc L cu Lys 5 1 0 1 5

( 2) INFORMATION FOR SEQID NO:50: ( i ) SEQUENCE CHARACTERISTICS: ( A ) LENGTH; 15 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE; (A) NAMEIKEY: misc feature ( D.) OTHER INFORMATION: "BPI.67" ( i x ) FEATURE: (A) NAME/KEY: Modified-sic (B) LOCATION: 6.8 ( D ) OTHER INFORMATION: flabcl=Substitulcd-Ala I note="The alaninc at position 7 is bcla-l-naphthyl-substitut.cd" ( xi ) SEQUENCE DESCRIPTION: SEQID NO:50: I 1 c L y s I 1 c Sc r G 1 y Lys A 1 a Lys A 1 a G l n Lys Arg Phc L. c u Lys 5 1 0 15

( 2) INFORMATION FOR SEQID NO:51: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: anino acid (D) TOPOLOGY: lincar ( i ; ) MOLECULETYPE: pcptide ( i x ) FEATURE: (A) NAMEIKEY: misc feature ( D.) OTHER INFORMATION: "BPI.9" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:51: Lys Arg Phc L c u Lys Lys Trip Lys A i a G l n Lys Arg Phc Lc u Lys 1. 5 1 0 5

( 2) INFORMATION FOR SEQ ID NO:52: 5,523,288 103 104 -continued

( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: pcptidc ( i x ) FEATURE; (A) NAMEIKEY: misc ?cature (D) OTHER INFORMATION: "BPI.30" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:52: Ly is Trip Lys A 1 a G in Lys A rig Phc L clu L. y S Lys S r Lys Wa G 1 y Tr p 5 1 0 15

Lic I c G l n L c u Ph e H is Lys Ly is 20

( 2) INFORMATION FOR SEQ DD NO:53: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 amino acids (B) TYPE: amino acid (D TOPOLOGY: incar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.63" ( x i ). SEQUENCE DESCRIPTION: SEOD NO:53:

I l c L y s I 1 c S c r G y Lys Tr p Lys A 1 a G 1 In Lys A rig P c L. c u Lys L. y S 5 O 5

Sc r Lys V a 1 Gil y Trip L clu I c G 1 in E. c u P he H is L. y S I y S 20 25

( 2) INFORMATION FOR SEQ DD NO:54: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: “BPI.7" ( xi ) SEQUENCE DESCRIPTION: SEQID NO:54:

Lys Trip Lys A 1 a G 1 in Lys A rig P c Lic u Lys Lys T r p Ly is A l a G 1 in Lys 1. 5 1 0 15 A rig Phc L. eu Lys 20

( 2) INFORMATION FOR SEQID No:55: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: misc feature (D) OTHER INFORMATION: "BPI.10.1 ( xi ) SEQUENCE DESCRIPTION: SEQID NO:55: 5,523.288

-continued Lys A rig Ph c L c u Lys Ly s Trip Lys Al a G in Lys A rig Phc L. c u Lys Lys 5 0 15 Trip Lys A a G 1 in Lys Arg Phc Lc u Lys 2 O 25

( 2) INFORMATION FOR SEQ ID NO:56: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B) TYPE:amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: Inisc feature (D) OTHER INFORMATION: “BPI.29" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:56: Ly is S c r Lys V a G l y T r p L c u I l c G l n L c u Ph c H is Lys Lys Lys S c r 5 0 5 Lys Wa i G l y Trip L. c u I 1 c G l n L c u Ph c His Lys Lys 2 0 25

( 2) INFORMATION FOR SEQED NO:57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptidc ( i x ) FEATURE: (A) NAMEIKEY: Inisc ?cature (D) OTHER INFORMATION: "BPI.46 ( x i ) SEQUENCE DESCRIPTION: SEQID NO:57: Ly is Trip Lys A 1 a. A la A a A rig Phc Le u Lys Lys T r p Lys A I a G 1 n Lys 1. 5 1 0 15 A rig Phc L c u Lys 20

( 2) INFORMATION FOR SEQ ID NO:58: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar { i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.47' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:58: Ly S T rip Lys A la G 1 in Lys Arg Phc Le u Lys Lys Trip Lys A la A la A 1 a 1. 5 1 0 5 A rig Phc L. c u Lys 20

( 2) INFORMATION FOR SEQ LD NO:59: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 anino acids (B) TYPE: amino acid (D) TOPOLOGY: incar 5,523,288 107 108 -continued ( i i ) MOLECULE TYPE: pcptide ( i x ) FEATURE; (A) NAMEIKEY: misc cature ( D ) OTHER INFORMATION: "BPI.48" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:59: Ly S Trip Lys A 1 a. A la A la A rig Phc L c u Lys y s Trip Lys A la A a A 1 a 5 1 0 5 A rig Phc Lc u Lys 20

( 2) INFORMATION FOR SEQ ID NO:60: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.69 ( x i ) SEQUENCE DESCRIPTION: SEQID NO:60: Ly S T I p Lys A la A la A la A rig Phc L c u L y s Ly s Trip Lys A la A la Al a 5 1 O 1 5 A rig Ph. c L c u I y S L y S T r p Lys A la A la A a Arg Phc L cu Lys 2 O 25 3 O

( 2) INFORMATION FOR SEQID NO:61: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE:amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: misc featurc (D) OTHER INFORMATION: "BPI 55" ( x i ). SEQUENCE DESCRIPTION: SEQID NO:61: G y Trip L. c u I 1 c G 1 in L. c u Ph c H is Lys Lys I I e G 1 u S c r A a L c u A rig 1 5 1 O 5 As n Lys Mc As n Sc r 20

( 2) INFORMATION FOR SEQ ID NO:62: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: misc featurc (D) OTHER INFORMATION: "BPI.73' ( x i ) SEQUENCE DESCRIPTION: SEQID No.62: I l c L y S I 1 c Sc r Gil y Ly s Trip Lys Al a G in Phc Arg Phc Lc u Lys 5 1 O 15

( 2) INFORMATION FOR SEQID NO:63: 5,523,288 109 110 -continued ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i MOLECULETYPE: pcptide ( i x ) FEATURE; (A) NAMEIKEY: Inisc featurc ( D ) OTHER INFORMATION: “BPI.70" ( i x ) FEATURE: (A) NAME/KEY: Modifica-sitc (B) LOCATION: 8.10 (D) OTHER INFORMATION: Fiabcl-Substitulcd-Ala | notc="The alaninc at position 7 is bcla-3-pyridyl-substituted" ( x i ) SEQUENCE DESCRIPTION: SEQID No.63: I 1 c L y s I l c Sc r G 1 y Lys A 1 a Lys Al a G in Lys Arg Phc Lc u Lys l 5 1 0 15

( 2) INFORMATION FOR SEQ ID NO:64: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptidc ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.71' ( i x ) FEATURE; (A) NAME/KEY: Modifica-sic (B) LOCATION: 13.15 (D) OTHER INFORMATION:/label=Substituted-Ala I notc="The alaninc al position 13 is bcta-3-pyridyl-substitut.cd" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:64: I c L y s Il c Sc r G 1 y Lys TT p Lys A l a G l n Lys A rig A 1 a L c u Lys 5 1 O 5

( 2) INFORMATION FOR SEQID NO:65: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc ?cature (D) OTHER INFORMATION: "BPI.10.2” ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:65: G 1 in Lys A rig Phc L. c u L y S L y s Trip Lys Al a G l n Lys A rig Phc Lc u Lys l 5 1 0 15 Lys Trip Lys A I a G n Lys A rig Phc L. c u Lys 20 25

( 2) INFORMATION FOR SEQ LD NO:66: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide 5,523.288 111 112 -continued

( i x ) FEATURE; (A) NAMEIKEY: misc ?cature ( D.) OTHER INFORMATION: "BPI.72" ( i x ) FEATURE: (A) NAMEIKEY: Modificd-site (B) LOCATION: 1.3 (D) OTHER INFORMATION: labc=D-alanine f notice"Thc position 1 and position 2 alaninc residucs arc both D-alaninc'

( x i ) SEQUENCE DESCRIPTION: SEQID NO:66: A 1 a. A la I l c L y s I l c Sc r G y Lys Trip Lys A 1 a G 1 in Lys Arg Phc L clu 1. 5 1 O 1 5 Lys

( 2) INFORMATION FOR SEQID NO:67: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: Inisc feature (D) OTHER INFORMATION: "BPI.5" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:67: V a l H is V a H is I c Sc r Lys S c r Lys V a 1 G1 y Trip L. c u I c G 1 n Lc u 5 1 0 1 5 Ph c H is Lys Lys I 1 c G. lu 2 O

( 2) INFORMATION FOR SEQID NO:68: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i) MOLECULE TYPE: peptide ( i x ) FEATURE; (A) NAMEIKEY: misc feature (D) OTHER INFORMATION: “BPI.65 reduccd' ( i x ) FEATURE; (A) NAMEIKEY: Disulfidc-bond (B) LOCATION: 1.17 ( x i ) SEQUENCE DESCRIPTION: SEQID NO:68: Cy s I 1 c L y s I 1 c Sc r G y Lys Trip Lys A 1 a G 1 n Lys Arg Phc Le u Lys 5 O 5 Cys

( 2) INFORMATION FOR SEQID NO:69: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 487 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: procin ( i x ) FEATURE; (A) NAMEIKEY: misc feature ( D.) OTHER INFORMATION: "rBPI

5,523,288 115 116 -continued

L c u G 1 u Lc u Lys H is S c r A s in I c G 1 y P P c P I Wa G L. c. l. L clu 3 90 395 40 0

Gl in A s p I 1 c Me As in Ty r I ic Wa P r I c L clu Wa 1 L C u P A rig Wa 4 O 5 4 0 4 15

As n G 1 u Lys I c u G in Lys Gly P c P r L c u P ro Thr Pr 0 A l a A rig Wa 4 25 43 O.

G l n L. c u Ty r A sm W a V a L clu G 1 in P H is G 1 in As in Phc L. c u Lic u Phc 4 35 4 4 O 4 45 G l y A 1 a. A s p V a l Wa i Ty r Lys 45 O 455

( 2) INFORMATION FOR SEQED NO:70: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAMEIKEY: misc feature (D) OTHER INFORMATION: “BPI.74' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:70: Lys Sc r Lys V a l Gil y Tr p I c L. c u H is Ly is Lys Lys T r p 5 1 O 15

Lys Al a G l n Lys A g Ph c L clu Lys 20

( 2) INFORMATION FOR SEQID NO:71: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: misc feature ( D ) OTHER INFORMATION: "BPI.76" ( i x ) FEATURE: (A) NAMEIKEY: Modified-sitc (B) LOCATION: 10.12 ( D ) OTHER INFORMATION: Ilabcl-D- Pic f nolc="The amino acid at position 11 is D- phenylalanine' ( x i ) SEQUENCE DESCRIPTION: SEQID NO;71:

Il c L y s I c S c T G 1 y Lys T r p Lys A a G in P A rig P c L c u Lys 1. 5 1 O 1 5

( 2) INFORMATION FOR SEQ LD NO:72: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i MOLECULETYPE; pcptide ( i x ) FEATURE: (A) NAMEIKEY: misc feature (D) OTHER INFORMATION: “BPI.77 ( x i ). SEQUENCE DESCRIPTION: SEQID No.:72:

l c L y s 1 c Sc r G 1 y Lys T r p Lys A l a G Tr p A rig P c L. c u Lys 5,523,288

-continued

5 1 0 5

( 2) INFORMATION FOR SEQ ID NO:73: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptidc ( i x ) FEATURE; (A) NAME/KEY: misc-?caturc ( D ) OTHER INFORMATION: "BPI.79” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:73: I c L y s I lic Sc r G y Lly s Trip Lys Al a Lys Lys Arg Phc L c u Lys 5 0 15

( 2) INFORMATION FOR SEQED NO:74: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: anino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: Inisc feature ( D.) OTHER INFORMATION: "BPI.80” ( i x ) FEATURE: (A) NAME/KEY: Modified-sic (B) LOCATION: 10.12 (D) OTHER INFORMATION: Fiabcl-Substitut.cd-Ala f notc="Thc alaninc at position 11 is bcta-i-naphthyl-substitut.cd" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:74: I 1 c L y s I 1 c Ser G 1 y Lys Trip Lys Al a G n A 1 a. A 1 g Phc L. c u Lys 1 5 1 0 1 5

(2) INFORMATION FOR SEQID NO:75: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptidc

( i x ) FEATURE: (A) NAME/KEY: misc feature ( D ) OTHER INFORMATION: "BPI.81" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:75: I c L y is I lic Ser G i y Lys Trip Lys A a Phc Lys Arg Phc Le u Lys 5 10 1 5

( 2) INFORMATION FOR SEQID NO:76: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE; peptide

( i x ) FEATURE: (A) NAME/KEY: misc feature ( D.) OTHER INFORMATION: "BPI.82" 5,523.288 119 120 -continued ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:76: Lys Sc r Lys Wall Gil y Trip L. c u I 1 c G 1 in c u T r p H is Lys Lys 5 O

( 2) INFORMATION FOR SEQ ID NO:77: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 14 amino acids (B) TYPE: amino acid ( D TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptidc ( i x ) FEATURE: (A) NAMEIKEY: misc feature ( D ) OTHER INFORMATION: “BPI.83" ( i x ) FEATURE; (A) NAME/KEY: Modificd-sitc (B) LOCATION: 10.12 (D) OTHER INFORMATION: label=Substituted-Ala f noLe="Thc alaninc alposition 6 is bcta-1-naphthyl-substitulcd'

( x i ) SEQUENCE DESCRIPTION: SEQID NO:77: Ly is Sc r Lys V a l G 1 y A la Lys I 1 c G 1 in L c u Ph c H is Lys Lys 1 5 1 O

( 2) INFORMATION FOR SEQ D NO:78: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: pcpidc ( i x ) FEATURE; (A) NAMEIKEY: misc feature ( D ) OTHER INFORMATION: "BPI.84” ( i x FEATURE: (A) NAMEEKEY: Modificd-sic (B) LOCATION: 6.8 (D) OTHER INFORMATION: Flabel=Substituted-Ala A noc="The alanine at position 7 is bca-1-naphthyl-substituted' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:78: I c L y s I l c Sc r G 1 y Lys A 1 a Lys A a G in Phc Arg Phc L c u Lys 5 1 O 5

( 2) INFORMATION FOR SEQ ID NO:79: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: pcptide ( i x ) FEATURE; (A) NAMEKEY: misc feature ( D OTHER INFORMATION: "BPI.85" ( x i ) SEQUENCE DESCRIPTION: SEQ D NO:79: L y S S c r Lys V a l L c u T r p L. c u I l c G | n L c u Ph c H is Lys Lys 1. 5 1 O

( 2) INFORMATION FOR SEQ ID NO:80: ( i) SEQUENCE CHARACTERISTICS: 5,523,288 121 122 -continued

(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: pcptide

( i x ) FEATURE; (A) NAMEIKEY: Inisc feature ( D ) OTHER INFORMATION: "BPI.86" ( x i ) SEQUENCE DESCRIPTION: SEQED NO:80: Lys S c Lys V a G 1 y T 1 p L c u 1 c L cu Lc u Ph c H is Ly S Lys 5 1 0

( 2) INFORMATION FOR SEQ ID NO:81: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid ( D TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: misc feature ( D ) OTHER INFORMATION: “BPE,87” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:81: Ly is S c Il y s V a 1 Gil y Trip L. c u I c G l n Lc u Ph c L c u Lys Lys 5 O

( 2) INFORMATION FOR SEQID NO:82; ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature ( D OTHER INFORMATION: "BPI.88” ( xi ) SEQUENCE DESCRIPTION: SEQID NO:32: 1 c L y s I 1 c Sc r G 1 y Lys Trip Lys A a Ph. c Ph. c. A rig Phi e L cu Lys 5 1 O 5

( 2) INFORMATION FOR SEQID NO:83: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH; 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide

( i x ) FEATURE: (A) NAMEIKEY: misc feature ( D.) OTHER INFORMATION: “BPI.98 ( i x ) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2 ( D.) OTHER INFORMATION: flabcl=Substituted-Tip f nolc="The alaninc at position 2 is beta-1-naphthyl-substitutcd'

( x i ) SEQUENCE DESCRIPTION: SEQ LD NO:33: L y S T r p Lys A a G 1 in Ph. c. A rig Phc L. c u Lys Lys Sc r Lys V a 1 G1 y T r p 1 5 O 5 L c u I l c Ph. c L c u Ph c H is Lys Lys 5,523,288

-continued

20

( 2) INFORMATION FOR SEQID NO:84: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptidc

( i x ) FEATURE: (A) NAME/KEY: misc-?calurc (D) OTHER INFORMATION: "BPI,89” ( i x ) FEATURE; (A) NAME/KEY: Modifica-sic (B) LOCATION: 6.8 (D) OTHER INFORMATION: ?labcl=Substitutcd-Ala 1 notc="Thc alaninc at position 7 is bcla-1-naphthyl-substitut.cd" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:84: I 1 c L y s I c Sc r Gil y Lys A 1 a Lys A 1 a Ph. c. Lys Arg Phc L. c u Lys 1. 5 1 0 15

( 2) INFORMATION FOR SEQ ID NO:85: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: pcpidc ( i x ) FEATURE: (A) NAME/KEY: misc featurc ( D.) OTHER INFORMATION: "BPI-90” ( i x ) FEATURE; (A) NAME/KEY: Modifica-sic (B) LOCATION: 6.8 (D) OTHER INFORMATION: labcl-Substitut.cd-Ala. f notc="Thc alaninc at position 7 is bcta-1-naphthyl-substitulcd' ( xi ) SEQUENCE DESCRIPTION: SEQID NO:85: I 1 c L y s I 1 c Sc r Gil y Lys A la Lys Al a Phc Ph. c. A rig Phc Lc u Lys 5 1 0 1 5

( 2) INFORMATION FOR SEQID NO:86: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptidc ( i x ) FEATURE; (A) NAME/KEY: misc-fcature (D) OTHER INFORMATION: "BPI.91" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:86: Lys Sc r Lys V a 1 G1 y Trip L. c u I 1 c Ph. c L c u Ph c H is Lys Lys 1. 5 1 0

( 2) INFORMATION FOR SEQID NO:87: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar 5,523,288 12S 126 -continued

( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: ( A ) NAMEIKEY: misc.?caturc ( D ) OTHER INFORMATION: "BPI.92” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:87: Lys S c r Lys V a l G 1 y Trip L. c u I c L y s L. c u Ph c H is Lys Lys 5 0

( 2) INFORMATION FOR SEQ DD NO:88: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: pcptidc ( i x ) FEATURE; (A) NAMEIKEY: misc feature ( D ) OTHER INFORMATION: "BPI.93” ( i x ) FEATURE: (A) NAME/KEY: Modifica-sic (B) LOCATION: 6.8 (D) OTHER INFORMATION: flabcl=Substitut.cd-Ala f notc="The alanine all position 7 is bcla-1-naphthyl-substituted” ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:38: I l c L y s I 1 c Sc r G 1 y Lys A 1 a Lys A 1 a Gil n Ph. c. A rig Phc Lc u Lys Lys 1. 5 1 0 5 S c r Lys V a l G y T r p L c u I 1 c G n L c u Ph c H is Lys Lys 20 25

( 2) INFORMATION FOR SEQ ID NO:89: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE:amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: misc ?cature (D) OTHER INFORMATION: “BPI.94 ( x i ) SEQUENCE DESCRIPTION: SEQID NO:89: Ly s Sc I Lys V a l G 1 y T r p L c u I 1 c G l n Lc u Ph c Phc Lys Lys 1. 5 0

( 2) INFORMATION FOR SEQID NO:90: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE:amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: misc feature ( D.) OTHER INFORMATION: "BPI.95” ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:90: Lys Sc r Lys V a Ph. c T r p L. c u I l c G l n Lc u Ph c H is Lys Lys 5 1 0 5,523,288 127 128 -continued ( 2) INFORMATION FOR SEQD NO:91: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: misc-?calure (D) OTHER INFORMATION: "BPI.96" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:91: Ly is S c r Lys V a 1 Gil y Trip L. c u I l c G 1 n L c u Ph c H is Lys Ph. c. l 5 1 O

( 2) INFORMATION FOR SEQ LD NO:92: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAMEKEY: Inisc ?cature ( D ) OTHER INFORMATION: "BPI.97" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:92: Ly is S c r Lys V a Lys Trip L. c u I 1 c G 1 in L cu Ph c H is Lys Lys l 5 1 0

( 2) INFORMATION FOR SEQ ID NO:93: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH:30 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.99" ( x i ). SEQUENCE DESCRIPTION: SEQ ID NO:93: Lys Trip Lys Al a G 1 in Trip A rig Phc L. c u Lys Lys Trip Lys Al a G 1 n Trip 1. 5 1 O 15 A rig Phc L. c u Lys Lys Trip Lys Al a G l n Trip A rig Phc Lc u Lys 20 25 3 0

( 2) INFORMATION FOR SEQ ID NO:94: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid ( D TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: Inisc featurc (D) OTHER INFORMATION: "BPI.100" ( xi ) SEQUENCE DESCRIPTION: SEQID NO:94: Ly S S c r Lys V a l Lys Trip L. c u 1 c L y s L. c u Ph c His Lys Lys 1 5 1 O.

(2) INFORMATION FOR SEQID NO:95: 5,523.288 129 130 -continued

( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc. ?cature (D) OTHER INFORMATION: “BPI.101." ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:95: Lys Sc r Lys V a l Ly s Trip L. c u I l c L y s L. c u Ph c P b c L y s Ph. c L y s Sc r 5 1 0 15 Ly is V a l Lys T r p L. c u I l c L y S L cu Ph c Phc Lys Ph. c 2 0 25

( 2) INFORMATION FOR SEQ ID NO:96: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: Inisc feature ( D ) OTHER INFORMATION: “BPI.102 ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:96: Ly S T rip Lys A la G 1 in Phc Arg Phc Lc u Lys Ly S S c r Lys V a G l y Tr p 5 0 5 L c u I c L c u L c u Ph c H is Lys Lys 20

(2) INFORMATION FOR SEQ D NO:97: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 1443 basc pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: DNA ( i x ) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1.1443 ( i x ) FEATURE: (A) NAME/KEY: mat peptidc (B) LOCATION: 76.1443 ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: “rLBP" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:97:

A T G GGG GCC TT G GCC AGA G C C C T G CCG TCC ATA CT G C T G G CA TT G CTG 4 8 Mc i G y Al a L c u A a A rig A a L c u Pro Sc r I 1 c L c u Lc u A la L c u L clu - 25 - 20 - 15 - 0.

CTT ACG T C C A C C C C A GAG GCT. CTG G G T G C C A A C C C C G GC TT G GT C GCC 9 6 Lic u Th r S c r Thr Pro G 1 u A 1 a L c u G 1 y A la As n Pro G y Lc u Val Al a - 5 1. 5

A GG AT C A C C GAC AAG G GA CT G CAG T A T G CG GCC CAG GAG GGG CTA TT G 1 4 4 A rig I lic Th r A s p Lys Gl y L. c u Gl in Ty r A la Al a G 1 in G 1 u Gil y L. c u Le u 0 15 2 O

G C T C T G CAG AG T G A G C T G C T C A G G AT C A CG C T G CCT GAC TT C A C C GGG 19 2.

5,523,288 137 138 -continued ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: misc ?caturc ( D ) OTHER INFORMATION: "BPI.75" ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:100: I 1 c L y s Lys A rig A 1 a I I c S c r Ph. c L c u G y Lys Ly S T r p G 1 in Lys 5 0 5

(2) INFORMATION FOR SEQ ID NO:101: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAME/KEY: misc feature ( D ) OTHER INFORMATION: "BPI282" ( x i ) SEQUENCE DESCRIPTION: SEQID NO:101: Ly is T r p Lys Al a Ph. c. Ph c Arg Phc L c u Lys Ly is T r p Lys A a Ph. c. Ph c l 5 1 O 15

A r g Ph c L c u Lys 20

( 2) INFORMATION FOR SEQ ID NO:102: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: pcptidc

( i x ) FEATURE: (A) NAME/KEY: misc feature ( D ) OTHER INFORMATION: "BPI.103" ( x i ) SEQUENCE DESCRIPTION: SEQED NO:102: I 1 c L y S I C S c r G 1 y Lys Trip Lys A a T r p Lys A rig Phc L c u Lys Lys 5 1 0 1 5

( 2) INFORMATION FOR SEQ ID NO:103: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.104” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:103: L y S S c r Lys V a l G y T 1 p L c u I c S c r L. c u Ph c H is Lys Ly is 5 1 O

(2) INFORMATION FOR SEQID NO:104: ( i ) SEQUENCE CHARACTERISTICS: 5,523,288 139 140 -continued (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: misc feature ( D.) OTHER INFORMATION: "BPI.105” ( i x ) FEATURE: (A) NAMEIKEY: Modifica-sic (B) LOCATION: 13 (C) OTHER INFORMATION:/labcl=Substitulcd-Ala 1 notc="The alaninc alposition 13 is bcla-1- naphthyl- substitulcd." ( x i ) SEQUENCE DESCRIPTION: SEQID NO:104: I 1 c L y s I 1 c Sc r G 1 y Lys T r p Lys A la T r p Lys A rig A 1 a L c u Lys Lys 5 1 O 15

(2) INFORMATION FOR SEQID NO:105: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptidc ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.106" ( xi ) SEQUENCE DESCRIPTION: SEQID NO:105: L y S S c r Lys V a 1 Gil y Trip L. c u I 1 c Th T L c u Ph c H is Lys Lys 1. 5 1 0

( 2) INFORMATION FOR SEQ ID NO:106: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptidc ( i x ) FEATURE: (A) NAME/KEY: misc ?caturc (D) OTHER INFORMATION: "BPI.107" ( xi ) SEQUENCE DESCRIPTION: SEQID NO:106: Lys S c r Lys V a l G l y Trip L. c u I l c G l n Lc u Ph c T r p Lys Lys 1 5 1 0

( 2) INFORMATION FOR SEQID NO:107: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc ?cature (D) OTHER INFORMATION: “BPI.108” ( x i ) SEQUENCE DESCRIPTION: SEQID NO:107: Lys S c r Lys V a 1 G l y Trip L. c u I 1 c G l n L. c u Ph c H is Lys Trip 1. 5 1 0 5,523,288 141 142 -continued ( 2) INFORMATION FOR SEQ ID No:108: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: pcptide ( i x ) FEATURE: (A) NAMEIKEY: Inisc feature ( D.) OTHER INFORMATION: "BPI.109 ( i x ) FEATURE: (A) NAME/KEY: Modifica-sic (B) LOCATION: 11 (C) OTHER INFORMATION: flabcl-Substitutcd-Ala 1 notc="The alaninc at position 11 is bcta-l- naphthyl- substituted.” ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:108: Ly is S c r Lys V a 1 Gil y Trip L. c u I 1 c G 1 in L. c u A 1 a His Lys Lys l 5 0

( 2) INFORMATION FOR SEQ ID NO:109: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: Inisc ?cature (D) OTHER INFORMATION: "BPI, 110” ( i x ) FEATURE: (A) NAME/KEY: Modifica-sitc ( B, ) LOCATION: 12 (C) OTHER INFORMATION: label=Substitulcd-Ala 1 notc="Thc alaninc at position 12 is bcla-i- naphthyl- substituted." ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:109: Lys S e r Lys V a 1 Gil y Trip L. c u I l c G l n L cu Ph c All a Lys Lys l 5 0

( 2) INFORMATION FOR SEQID NO:110: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc featurc ( D ) OTHER INFORMATION: "BPI.111” ( i x ) FEATURE; (A) NAME/KEY: Modifica-sitc (B) LOCATION: 14 (C) OTHER INFORMATION: ?labcl=Substituted-Ala I notc="The alanine at position 14 is beta-1- naphthyl- substitutcd.' ( xi ) SEQUENCE DESCRIPTION: SEQ LD NO:110: Lys S c r Lys V a 1 G y Trip L. c u I lic G l n Lc u Ph c His Lys A 1 a 1 5 1 O

( 2) INFORMATION FOR SEQID NO:111: 5,523,288 143 144 -continued ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptidc i x ) FEATURE; (A) NAMEKEY: misc ?caturc ( D.) OTHER INFORMATION: "BPI.112" ( i x ) FEATURE: (A) NAME/KEY: Modifica-sic (B) LOCATION: 7 (C) OTHER INFORMATION: flabel=Substituted-Ala f notc="Thc alaninc at position 7 is bcla-l- naphthyl- substitulcd." ( i x FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 11 (C) OTHER INFORMATION: labcl=Substituted-Ala I note="The alaninc a position 11 is bcta-1- naphthyl- substituted.' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:11: I c L y s I 1 c Sc r G y Lys A 1 a Lys A a G 1 m A la A rig Phc L. c u Lys 5 0 5

( 2) INFORMATION FOR SEQID NO:112: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE; (A) NAME/KEY: Inisc feature ( D.) OTHER INFORMATION: "BPI.113” ( xi ) SEQUENCE DESCRIPTION: SEQID NO:112: Ly is Sc r Lys V a G 1 y T r p it c u I 1 c G n Phc Phc H is Lys Lys 5 1 0

( 2) INFORMATION FOR SEQID NO:113: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: pcpidc ( i x ) FEATURE: (A) NAME/KEY: misc feature (D) OTHER INFORMATION: "BPI.114' ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:113: Lys Tr p G 1 in L clu. A rig Sc r Lys G 1 y Lys I 1 c L y s I 1 e Phc Lys A 1 a 5 0 5

( 2) INFORMATION FOR SEQ LD NO:114: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: incar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: ( A ) NAMEIKEY: misc feature 5,523.288 145 146 -continued ( D.) OTHER INFORMATION: “BPI.116” ( i x ) FEATURE: (A) NAME/KEY: Modifica-site (B) LOCATION: 6 (C) OTHER INFORMATION: fiabcl=Substitulcd-Ala f nolca"The alaninc at position 6 is beta-1- Inaphthyl- substilulcd.” ( xi ) SEQUENCE DESCRIPTION: SEQID NO:114; Ly S S c r Lys V a l Lys Al a L c u I I c G l n L cu Ph c H is Lys Lys l 5 1 0

( 2) INFORMATION FOR SEQ ID NO:15: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:amino acid (D TOPOLOGY: lincar ( i i ) MOLECULE TYPE: pcptide ( i x ) FEATURE: (A) NAME/KEY: Inisc feature ( D.) OTHER INFORMATION: “BPI.1.19" ( i x ) FEATURE: (A) NAME/KEY: Modifica-sic (B) LOCATION: 7 (C) OTHER INFORMATION: flabcl=Substitutcd-Ala 1 notc="Thc alanine at position 7 is bcla-l- naphthyl- substituted.” ( i x ) FEATURE: (A) NAME/KEY: Modifica-sile (B) LOCATION: 10 (C) OTHER INFORMATION: Flabcl=Substituted-Ala | notc="The alaninc alposition 10 is bcta-l- naphthyl- substitulcd.' ( x i ) SEQUENCE DESCRIPTION: SEQID No:115: I I c L y S I lic S c r GI y Lys A la Lys A la A la Lys Arg Phc Lc u Lys 1 5 0 5

( 2) INFORMATION FOR SEQID NO:l 16: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 annino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: misc feature ( D.) OTHER INFORMATION: “BPI.120” ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:116: i l c L y S I lic S c 1 Gil y Lly s Trip Lys A 1 a G 1 in Lys A rig Lys L. c u Lys 5 1 O 1 5

( 2) INFORMATION FOR SEQID NO:117: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAME/KEY: Inisc feature ( D.) OTHER INFORMATION: "BPI.121" 5,523,288 147 148 -continued ( i x ) FEATURE: (A) NAMEIKEY: Modificd-sic ( B) LOCATION: 10 (C) OTHER INFORMATION: label=Substitulcd-Ala f nolc="Thc alaninc a position 10 is bca-1- naphthyl-substituted.”

( i x ) FEATURE: (A) NAMEIKEY: Modifica-sic (B) LOCATION: 11 (C) OTHER INFORMATION: Fiabcl=Substituted-Ala f notics"Thc alaninc al position 11 is bcla-l- naphthyl- substituted." ( x i ) SEQUENCE DESCRIPTION: SEQID NO:117: I l c L y S I 1 c Sc r Gil y Lys Trip Lys A 1 a. A la A a Arg Phc Lc u Lys 5 0 1 5

( 2) INFORMATION FOR SEQ D NO:118: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: misc cature ( D OTHER INFORMATION: "BPI.1.22' ( i x ) FEATURE; (A) NAME/KEY: Modific-site (B) LOCATION: 7 (C) OTHER INFORMATION: labels.Substituticid-Ala I nolc="The alanine at position 7 is bcla-l- naphthyl- substituted.' ( i x ) FEATURE: (A) NAMEIKEY: Modified-sitc (B) LOCATION: 10 (C) OTHER INFORMATION: labcl-Substituted-Ala f notc="Thc alanine at position 10 is bcla-1- naphthyl- substitulcd.' ( i x ) FEATURE; (A) NAMEKEY: Modifica-sic (B) LOCATION: 11 (C) OTHER INFORMATION: flabel=Substituted-Ala f nolc="The alanine a position 11 is beta-1- naphthyl- substituted.” ( x i ) SEQUENCE DESCRIPTION: SEQD NO:118: I c L y S I 1 c S c r G y Lys A I a Lys A a A la A 1 a. A rig Phc Lc u Lys 5 1 O 1 5

( 2) INFORMATION FOR SEQID No:119. ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE; (A) NAMEIKEY misc feature ( D.) OTHER INFORMATION: "BPI.123” ( i x ) FEATURE: (A) NAMEKEY: Modified-site (B) LOCATION: 9 (C) OTHER INFORMATION: labcl=Substituted-Phc f note="The phenylalaninc at position 9 is p-amino-substituted. 5,523,288 149 150 -continued ( x i ) SEQUENCE DESCRIPTION: SEQID NO:119: Ly is S c 1 Lys V a G l y T r p L c u I l c Ph c L cu Ph c H is Lys Lys 1. 5 1 0

( 2) INFORMATION FOR SEQ LD NO:120: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE; amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: pcptide ( i x ) FEATURE; (A) NAME/KEY: misc featurc ( D.) OTHER INFORMATION: "BPI.124' ( x i ). SEQUENCE DESCRIPTION: SEQID NO:120: L y S S c r Lys V a l Lys T r p L. c u I c G l n L c u T 1 p H is Lys Lys 5 1 0

( 2) INFORMATION FOR SEQ ID NO:121: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: Inisc ?cature (D) OTHER INFORMATION: “BPI.125" ( x i ) SEQUENCE DESCRIPTION: SEQ LD NO:12: Ly is S c r Lys V a l G l y Trip L. c u I l c Ty I L c u Ph c H is Lys Lys 1 5 1 0

( 2) INFORMATION FOR SEQ ID NO:122: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE:amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULE TYPE: pcptidc ( i x ) FEATURE: (A) NAME/KEY: misc featurc (D) OTHER INFORMATION: "BPI.126” ( i x ) FEATURE: (A) NAMEIKEY: Modificd-site (B) LOCATION: 6 (C) OTHER INFORMATION: ?label=D-Trp f notc="The amino acid a position 6 is D- tryptophan." ( x i ) SEQUENCE DESCRIPTION: SEQID NO:122: Ly is S c r Lys V a l G l y Trip L. c u I l c G | n Lc u Ph c H is Lys Lys i 5 1 0

( 2) INFORMATION FOR SEQID No:123: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lincar ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: 5,523,288 151 152 -continued (A) NAMEIKEY: misc feature ( D.) OTHER INFORMATION: “BPI.127' ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:123; Lys Sc r Lys V a Gil y Ph. c L c u I l c G. l n Lc u Ph c H is Lys Lys 5 1 O

( 2) INFORMATION FOR SEQ ID NO.124: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE:amino acid ( D TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: misc feature ( D. OTHER INFORMATION: "BPI.128' ( i x ) FEATURE: (A) NAMEKEY: Modificd-sitc (B) LOCATION: 6 (C) OTHER INFORMATION: Fiabcl-D- Phc f nolc="The amino acid a position 6 is D-phcnylalaninc.'

( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:124: Lys Sc r Lys V a G 1 y Ph c L c u I 1 c G l n L. c u Pro H is Lys Lys 5 1 O

( 2) INFORMATION FOR SEQID NO:125: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 14 amino acids (B) TYPE: amino acid ( D TOPOLOGY: linear ( i i ) MOLECULETYPE: peptide ( i x ) FEATURE: (A) NAMEKEY: misc feature ( D ) OTHER INFORMATION: “BPI.129 ( i x ) FEATURE; (A) NAMEKEY: Modific-site (B) LOCATION: 6 (C) OTHER INFORMATION: label=Substituted-Ala f note="The alaninc at position 6 is D-1-bcla-1-naphthyl substituted.'

( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:125: L. y S S c r Lys V a l G 1 y A 1 a L c u I l c G l n L c u Ph c H is Lys Lys 5 1 O

( 2) INFORMATION FOR SEQ ID NO:126: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: peptide ( i x ) FEATURE: (A) NAMEIKEY: misc featurc ( D.) OTHER INFORMATION: “BPI.130” ( i x ) FEATURE: (A) NAMEEKEY: Modifica-site (B) LOCATION: 6 (C) OTHER INFORMATION: labels Substituted-Ala f note="Thc alanine at position 6 is