US 5,523,288 AGrant
Method of Treating Gram-Negative Bacterial Infection by Administration of Bactericidal/Permeability-Increasing (BPI) Protein Product and Antibiotic
Issue Date:1996-06-04
•37 Claims
•22 Drawing Sheets
Abstract
The present invention relates to methods and compositions for treating gram-negative bacterial infections, using BPI protein products. Co-treatment, or concurrent administration, of BPI protein product with an antibiotic in treatment of gram-negative bacterial infections improves the therapeutic effectiveness of the antibiotic, including increasing antibiotic susceptibility of gram-negative bacteria and reversing resistance of the bacteria to antibiotics.
Metadata
Assignee
- Xoma Corporation
Inventors
- Jonathan Cohen
- Ada H. C. Kung
- Lewis H. Lambert, Jr.
- Roger G. Little, II
Application Information
Application Number:US 3116115
Filing Date:1994-09-22
Priority Date:1993-09-22
Art Unit:185
Classifications
IPC:
A61K 3817C07G 1100C07H 15234C07K 1447
Field of Search:
51442453653054055212;152;192114;4057.2;13.1;13.2;13.3;13.4;13.5;13.7;13.9;16.9;14;13.6;8.8;16.1;16.2;16.8319;350226;304;314;342;335;341202;204;205
Patent Drawings (22 sheets)
Description
BACKGROUND OF THE INVENTION
The present invention relates generally to methods and compositions for treating gram-negative bacterial infections, and more specifically to the use of bactericidal/permeability-increasing protein (BPI) protein products for co-treatment of such infections with an antibiotic substance. Co-treatment with BPI protein products can improve the therapeutic effectiveness of antibiotics in gram-negative bacterial infections, increase the susceptibility of gram-negative organisms to antibiotics, and reverse resistance of gram-negative organisms to antibiotics.
BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652 (1987)]. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in FIG. 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Gray et al. amino acid sequence is set out in SEQ ID NO: 69 hereto.
BPI is a strongly cationic protein. The N-terminal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of -3. [Elsbach and Weiss (1981), supra.] A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD has an amphipathic character, containing alternating hydrophobic and hydrophilic regions. This N-termina fragment of human BPI possesses the anti-bacteria efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi et at., J. Bio. Chem., 262:14891-14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms. [Ooi et at., J. Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately 23 kD, referred to as "rBPI.sub.23," has been produced by recombinant means and also retains anti-bacterial activity against gram-negative organisms. Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).
The bactericidal effect of BPI has been reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et at., Chapter 30, Raven Press, Ltd. (1992). BPI is commonly thought to be non-toxic for other microorganisms, including yeast, and for higher eukaryotic cells. Elsbach and Weiss (1992), supra, reported that BPI exhibits anti-bacterial activity towards a broad range of gram-negative bacteria at concentrations as low as 10.sup.-8 to 10.sup.-9 M, but that 100- to 1,000-fold higher concentrations of BPI were non-toxic to all of the gram-positive bacterial species, yeasts, and higher eukaryotic cells tested at that time. It was also reported that BPI at a concentration of 10-6M or 160 .mu.g/ml had no toxic effect, when tested at a pH of either 7.0 or 5.5, on the gram-positive organisms Staphylococcus aureus (four strains), Staphylococcus epidermidis, Streptococcus faecalis, Bacillus subtills, Micrococcus lysodeikticus, and Listeria monocytogenes. BPI at 10.sup.-6 M reportedly had no toxic effect on the fungi Candida albicans and Candida parapsilosis at pH 7.0 or 5.5, and was non-toxic to higher eukaryotic cells such as human, robbit and sheep red blood cells and several human rumor cell lines. See also Elsbach and Weiss, Advances in Inflammation Research, ed. G. Weissmann, Vol. 2, pages 95-113 Raven Press (1981 ). This reported target cell specificity was believed to be the result of the strong attraction of BPI for lipopolysaccharide (LPS), which is unique to the outer membrane (or envelope) of gram-negative organisms.
The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on LPS. LPS has been referred to as "endotoxin" because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, reported to be the most toxic and most biologically active component of LPS.
In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992), supra]. BPI is thought to act in two stages. The first is a sublethal stage that is characterized by immediate growth arrest, permeabilization of the outer membrane and selective activation of bacterial enzymes that hydrolyze phospholipids and peptidoglycans. Bacteria at this stage can be rescued by growth in serum albumin supplemented media [Mannion et al., J. Clin. Invest., 85:853-860 (1990)]. The second stage, deftned by growth inhibition that cannot be reversed by serum albumin, occurs after prolonged exposure of the bacteria to BPI and is characterized by extensive physiologic and structural changes, including apparent damage to the inner cytoplasmic membrane.
Initial binding of BPI to LPS leads to organizational changes that probably result from binding to the anionic groups in the KDO region of LPS, which normally stabilize the outer membrane through binding of Mg.sup.++ and Ca.sup.++. Attachment of BPI to the outer membrane of gram-negative bacteria produces rapid permeabitization of the outer membrane to hydrophobic agents such as actinomycin D. Binding of BPI and subsequent gram-negative bacterial killing depends, at least in part, upon the LPS polysacchafide chain length, with long O-chain bearing, "smooth" organisms being more resistant to BPI bactericidal effects than short O-chain beating, "rough" organisms [Weiss et al., J. Clin. Invest. 65:619-628 (1980)]. This first stage of BPI action, permeabilization of the gram-negative outer envelope, is reversible upon dissociation of the BPI, a process requiting the presence of divalent cations and synthesis of new LPS [Weiss et al., J. Immunol. 132:3109-3115 (1984)]. Loss of gram-negative bacterial viability, however, is not reversed by processes which restore the envelope integrity. suggesting that the bactericidal action is mediated by additional lesions induced in the target organism and which may be situated at the cytoplasmic membrane (Mannion et at., J. Clin. Invest. 86:631-64 1 (1990)). Specific investigation of this possibility has shown that on a molar basis BPI is at least as inhibitory of cytoplasmic membrane vesicle function as polymyxin B (In't Veld et at., Infection and Immunity 56:1203-1208 (1988)) but the exact mechanism as well as the relevance of such vesicles to studies of intact organisms has not yet been elucidated.
BPI is also capable of neutralizing the endotoxic properties of LPS to which it binds. Because of its bactericidal properties for gram-negative organisms and its ability to neutralize LPS, BPI can be utilized for the treatment of mammals suffering from diseases caused by gram-negative bacteria, such as bacteremia or sepsis.
U.S. Pat. No. 5,198,541 discloses recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI. It also describes the use of N-terminal fragments of BPI protein for co-treatment with certain antibiotics, specifically penicillin, cephalosporins, rifampicin and actinomycin D.
Gram-negative bacteria include bacteria from the following species: Acidarninococcus, Acinetobacter, Aeromonas, Alcaligenes, Bacteroides, Bordetella, Branhamella, Brucella, Calymmatobacterium, Carnpylobacter, Cardiobacterium, Chromobacterium, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Flavobacterium, Francisella, Fusobacterium, Haermophilus, Klebsiella, Legionella, Moraxella, Morganella, Neisseria, Pasturella, Plesiornonas, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Streptobacillus, Veillonella, Vibrio, and Yersinia species.
Antibiotics are natural chemical substances of relatively low molecular weight produced by various species of microorganisms, such as bacteria (including Bacillus species), actinomycetes (including Streptomyces) and fungi, that inhibit growth of or destroy other microorganisms. Substances of similar structure and mode of action may be synthesized chemically, or natural compounds may be modified to produce semi-synthetic antibiotics. These biosynthetic and semi-synthetic derivatives are also effective as antibiotics. The major classes of antibiotics are (1) the .beta.-lactams, including the penicillins, cephalosporins and monobactams; (2) the aminoglycosides, e.g., gentamicin, tobramycin, netilmycin, and amikacin; (3) the tetracyclines; (4) the sulfonamides and trimethoprim; (5) the fluoroquinolones, e.g., ciprofloxacin, norfloxacin, and ofloxacin; (6) vancomycin; (7) the macrolides, which include for example, erythromycin, azithromycin, and clarithromycin; and (8) other antibiotics, e.g., the polymyxins, chloramphenicol and the lincosamides.
Antibiotics accomplish their anti-bacterial effect through several mechanisms of action Which can be generally grouped as follows: (1) agents acting on the bacterial cell wall such as bacitracin, the cephalosporins, cycloserine, fosfomycin, the penicillins, ristocetin, and vancomycin; (2) agents affecting the cell membrane or exerting a detergent effect, such as colistin, novobiocin and polymyxins; (3) agents affecting cellular mechanisms of replication, information transfer, and protein synthesis by their effects on ribosomes, e.g., the aminoglycosides, the tetracyclines, chloramphenicol, clindamycin, cycloheximide, fucidin, lincomycin, puromycin, rifampicin, other streptomycins, and the macrolide antibiotics such as erythromycin and oleandomycin; (4) agents affecting nucleic acid metabolism, e.g., the fluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine, griseofulvin, rifamycins; and (5) drugs affecting intermediary metabolism, such as the sulfonamides, trimethoprim, and the tuberculostatic agents isoniazid and para-aminosalicylic acid. Some agents may have more than one primary mechanism of action, especially at high concentrations. In addition, secondary changes in the structure or metabolism of the bacterial cell often occur after the primary effect of the antimicrobial drug.
The penicillins have a characteristic double-ring system composed of a .beta.-lactam ring, which provides the antibacterial activity, and a thiazolidene ring. The penicillins are differentiated by a single side chain that is unique for each penicillin. The compounds are bactericidal and act by inhibiting bacterial transpeptidase, an enzyme involved in synthesis of the bacterial cell wall. Because of their mechanism of action, penicillins are generally active against growing, but not resting, cells. Penicillins, especially penicillin G, have largely gram-positive activity; the relative insensitivity of gram-negative rods to penicillin G and several other penicillins is probably due to the permeability barrier of the outer membrane of gram-negative bacteria. Ampicillin, carbenicillin, ticarcillin, and some other penicillins are active against gram-negative bacteria because they can pass through this outer membrane. Penicillins have relatively few adverse effects, the most important of which are the hypersensitivity (allergic) reactions. These compounds are widely distributed in the body, but do not enter cells and do not usually accumulate in CSF.
Bacterial resistance to the penicillins is by production of the enzyme .beta.-lactamase, which catalyzes hydrolysis of the .beta.-lactam ring. The percentage of bacteria resistant to penicillin has risen to about 80%. Several penicillins, including methicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin, are not affected by the .beta.-lactamase of staphylococci. These antibiotics are useful against most .beta.-lactamase-producing species of Staphylococcus. However, a small number of species are resistant even to these penicillins. Some penicillins, amoxicillin and ticarcillin, are marketed in combination with clavulanic acid, which is a .beta.-lactamase inhibitor that covalently binds to the enzyme and prevents it from hydrolyzing the antibiotics. Another inhibitor, sulbactam, is marketed in combination with ampicillin.
The cephalosporins are characterized by a .beta.-lactam ring, like the penicillins, but have an adjacent dihydrothiazine ring instead of a thiazolidene ring. For convenience, these compounds are generally classified by generations. The first generation includes cephalothin, cephapirin, cefazolin, cephalexin, cephradine and cefadroxil. These drugs generally have excellent gram-positive activity except for enterococci and methicillin-resistant staphylococci, and have only modest gram-negative coverage. The second generation includes cefamandole, cefoxitin, ceforanide, cefuroxime, cefuroxime axetil, cefaclor, cefonicid and cefotetan. This generation generally loses some gram-positive activity by weight and gains limited gram-negative coverage. The third generation includes cefotaxime, moxalactam, ceftizoxime, ceftriaxone, cefoperazone and ceftazidime. These compounds generally sacrifice further gram-positive activity by weight but gain substantial gram-negative coverage against Enterobacter and sometimes are active against Pseudornonas. The cephalosporins bind to penicillin-binding proteins with varying affinity. Once binding occurs, protein synthesis is inhibited. Cephalosporins are usually well tolerated; adverse effects include hypersensitivity reactions and gastrointestinal effects. Cephalosporins may interact with nephrotoxic drags. particularly aminoglycosides. to increase toxicity. Resistance to cephalosporins is mediated by several mechanisms, including production of .beta.-lactamase, although some strains that do not produce .beta.-lactamase are nevertheless resistant.
Imipenem is a N-formimidoyl derivative of the mold product thienamycin. It contains a .beta.-lactam ring and somewhat resembles penicillin except for differences in the second ring. It has activity against both gram-positive and gram-negative organisms and is resistant to most .beta.-lactamases, although not those from Pseudomonas. It is marketed in combination with cilastin, a compound that inhibits inactivation of imipenem in the kidney by renal dihydropeptidase I enzyme. Cilastin increases the concentration of imipenem in urine, although not in blood.
Aztreonam is the first of a new group of antibiotics referred to as the monobactams. These agents have a .beta.-lactam ring but lack the second ring characteristic of the penicillins and cephalosporins. It acts by binding to penicillin-binding proteins, and produces long, filamentous bacterial shapes that eventually lyse. Aztreonam is active only against aerobic gram-negative bacteria, is susceptible to inactivation by some .beta.-lactamases, and has few adverse effects.
The aminoglycosides contain amino sugars linked to an aminocyclitol ring by glycosidic bonds. They have similar mechanisms of action and properties, but differ somewhat in spectrum of action, toxicity, and susceptibility to bacterial resistance. The compounds are bactericidal, with activity against both gram-positive and gram-negative organisms, and act by binding to proteins on the 30S ribosome of bacteria and inhibiting protein synthesis. The aminoglycosides also bind to isolated LPS and have a very weak outer membrane permeabilizing effect. [Taber et al., Microbiological Reviews 53:439-457 (1987)); Kadurugamuwa et al., Antimicrobial Agents and Chemotherapy, 37:715-721 (1993); Vaara, Microbiological Reviews 56: 395-411 (1992)]. This class of antibiotics includes amikacin, gentamicin, kanamycin, neomycin, netilmycin, paromomycin and tobramycin. The aminoglycosides are usually reserved for more serious infections because of severe adverse effects including ototoxicity and nephrotoxicity. There is a narrow therapeutic window between the concentration required to produce a therapeutic effect, e.g., 8 .mu.g/ml for gentamicin, and the concentration that produces a toxic effect, e.g., 12 .mu.g/ml for gentamicin. Neomycin in particular is highly toxic and is never administered parenterally.
Tetracyclines have a common four-ring structure and are closely congeneric derivatives of the polycyclic naphthacenecarboxamide. The compounds are bacteriostatic, and inhibit protein synthesis by binding to the 30S subunit of microbial ribosomes and interfering with attachment of aminoacyl tRNA. The compounds have some activity against both gram-positive and gram-negative bacteria; however, their use is limited because many species are now relatively resistant. Adverse effects include gastrointestinal effects, hepatotoxicity with large doses, and nephrotoxicity in some patients. This antibiotic class includes tetracycline, chlortetracycline, demeclocycline, doxycycline, methacycline, minocycline and oxytetracycline.
The sulfonamides are derivatives of sulfanilamide, a compound similar in structure to para-aminobenzoic acid (PABA), which is an essential precursor for bacterial synthesis of folic acid. The compounds are generally bactefiostatic, and act by competitively inhibiting incorporation of PABA into tetrahydrofolic acid, which is a required cofactor in the synthesis of thymidines, purines and DNA. Sulfonamides have a wide range of activity against gram-positive and gram-negative bacteria, but their usefulness has diminished with increasingly high prevalence of bacterial resistance. The sulfonamide class of antibiotics includes sulfacytine, sulfadiazine, sulfamethizole, sulfisoxazole, sulfamethoxazole, sulfabenzamide and sulfacetamide. Adverse effects include hypersensitivity reactions and occasional hematological toxicity.
Trimethoprim is an inhibitor of the dihydrofolate reductase enzyme, which converts dihydrofolic to tetrahydrofolic acid, a required factor for DNA synthesis. Adverse effects include gastrointestinal distress and rare hematological toxicity. Trimethoprim is also available in combination with sulfamethoxazole (also known as co-trimoxazole). The combination is usually bactericidal, although each agent singly is usually bacteriostatic. The combination is the drug of choice for Salmonella infections, some Shigella infections, E. coli traveler's diarrhea and Pneumocysas carinii pneumonia.
The fluoroquinolones and quinolones are derivatives of nalidixic acid, a naphthyridine derivative. These compounds are bactericidal, and impair DNA replication, transcription and repair by binding to the DNA and interfering with DNA gyrase, an enzyme which catalyzes negative supercoiling of DNA. The fluoroquinolones, which include norfloxacin, ciprofioxacin, and ofloxacin, and the quinolones, which include cinoxacin, have a broad spectrum of antimicrobial activity against gram-negative and gram-positive organisms. These compounds distribute widely through extravascular tissue sites, have a long serum half-life, and present few adverse effects. Because of their effect on DNA, the drugs are contraindicated in pregnant patients and in children whose skeletal growth is incomplete.
Vancomycin is a glycopeptide, with a molecular weight of about 1500, produced by a fungus. It is primarily active against gram-positive bacteria. The drug inhibits one of the final steps in synthesis of the bacterial cell wall, and is thus effective only against growing organisms. It is used to treat serious infections due to gram-positive cocci when penicillin G is not useful because of bacterial resistance or patient allergies. Vancomycin has two major adverse effects, ototoxicity and nephrotoxicity. These toxicities can be potentiated by concurrent administration of another drug with the same adverse effect, such as an aminoglycoside.
The macrolides are bacteriostatic and act by binding to the 50S subunit of 70S ribosomes, resulting in inhibition of protein synthesis. They have a broad spectrum of activity against gram-positive and gram-negative bacteria and may be bacteriostatic or bactericidal, depending on the concentration achieved at sites of infection. The compounds distribute widely in body fluids. Adverse effects include gastrointestinal distress and rare hypersensitivity reactions. The most common macrofide used is erythromycin, but the class includes other compounds such as clarithromycin and azithromycin.
The polymyxins are a group of closely related antibiotic substances produced by strains of Bacillus polymyxa. These drugs, which are cationic detergents, are relatively simple, basic peptides with molecular weights of about 1000. Their antimicrobial activity is restricted to gram-negative bacteria. They interact strongly with phospholipids and act by penetrating into and disrupting the structure of cell membranes. Polymyxin B also binds to the lipid A portion of endotoxin and neutralizes the toxic effects of this molecule. Polymyxin B has severe adverse effects, including nephrotoxicity and neurotoxicity, and should not be administered concurrently with other nephrotoxic or neurotoxic drugs. The drug thus has limited use as a therapeutic agent because of high systemic toxicity, but may be used for severe infections, such as Pseudomonas aeruginosa meningitis, that respond poorly to other antibiotics.
Chloramphenicol inhibits protein synthesis by binding to the 50S ribosomal subunit and preventing binding of aminoacyl tRNA. It has a fairly wide spectrum of antimicrobial activity, but is only reserved for serious infections, such as meningitis, typhus, typhoid fever, and Rocky Mountain spotted fever, because of its severe and fatal adverse hematological effects. It is primarily bacteriostatic, although it may be bactericidal to certain species.
Lincomycin and clindamycin are lincosamide antimicrobials. They consist of an amino acid linked to an amino sugar. Both inhibit protein synthesis by binding to the 50S ribosomal subunit. They compete with erythromycin and chloramphenicol for the same binding site but in an overlapping fashion. They may be bacteriostatic or bactericidal, depending on relative concentration and susceptibility. Gastrointestinal distress is the most common side effect. Other adverse reactions include cutaneous hypersensitivity, transient hematological abnormalities, and minor elevations of hepatic enzymes. Clindamycin is often the drug of choice for infections caused by anaerobic bacteria or mixed aerobic/anaerobic infections, and can also be used for susceptible aerobic gram-positive cocci.
Some drugs, e.g. aminoglycosides, have a small therapeutic window. For example, 2 to 4 .mu.g/ml of gentamicin or tobramycin may be required for inhibition of bacterial growth, but peak concentrations in plasma above 6 to 10 .mu.g/ml may result in ototoxicity or nephrotoxicity. These agents are more difficult to administer because the ratio of toxic to therapeutic concentrations is very low. Antimicrobial agents that have toxic effects on the kidneys and that are also eliminated primarily by the kidneys, such as the aminoglycosides or vancomycin, require particular caution because reduced elimination can lead to increased plasma 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 function. Similarly, dosages of drugs that are metabolized or excreted by the liver, such as erythromycin, chloramphenicol, or clindamycin, must be reduced in patients with decreased hepatic function.
Antibiotic resistance in bacteria is an increasingly troublesome problem. The accelerating development of antibiotic-resistant bacteria, intensified by the widespread use of antibiotics in farm animals and overprescription of antibiotics by physicians, has been accompanied by declining research into new antibiotics with different modes of action. [Science, 264:360-374 (1994).] Antibiotic resistance, once acquired, can be rapidly spread to other bacteria, including bacteria of a different species. There are some species of bacteria that are resistant to all but one antibiotic; it may be only a matter of time before the appearance of bacterial strains that are resistant to all antibiotics.
Bacteria acquire resistance to antibiotics through several mechanisms: (1) production of enzymes that destroy or inactivate the antibiotic [Davies, Science, 264:375-381 (1994)]; (2) synthesis of new or altered target sites on or within the cell that axe not recognized by the antibiotic [Spratt, Science, 264:388-393 (1994)]; (3) low permeability to antibiotics, which can be reduced even further by altering cell wall proteins, thus restricting access of antibiotics to the bacterial cytoplasmic machinery; (4) reduced intracellular transport of the drug; and (5) increased removal of antibiotics from the cell via membrane-associated pumps [Nikaido, Science, 264:382-387 (1994)].
The susceptibility of a bacterial species to an antibiotic is generally determined by two microbiological methods. A rapid but crude procedure uses commercially available filter paper disks that have been impregnated with a specific quantity of the antibiotic drug. These disks are placed on the surface of agar plates that have been streaked with a culture of the organism being tested, and the plates are observed for zones of growth inhibition. A more accurate technique, the broth dilution susceptibility test, involves preparing test tubes containing serial dilutions of the drug in liquid culture media, then inoculating the organism being tested into the tubes. The lowest concentration of drug that inhibits growth of the bacteria after a suitable period of incubation is reported as the minimum inhibitory concentration.
The resistance or susceptibility of an organism to an antibiotic is determined on the basis of clinical outcome, i.e., whether administration of that antibiotic to a subject infected by that organism will successfully cure the subject. While an organism may literally be susceptible to a high concentration of an antibiotic in vitro, the organism may in fact be resistant to that antibiotic at physiologically realistic concentrations. If the concentration of drug required to inhibit growth of or kill the organism is greater than the concentration that can safely be achieved without toxicity to the subject, the microorganism is considered to be resistant to the antibiotic. To facilitate the identification of antibiotic resistance or susceptibility using in vitro test results, the National Committee for Clinical Laboratory Standards (NCCLS) has formulated standards for antibiotic susceptibility that correlate clinical outcome to in vitro determinations of the minimum inhibitory concentration of antibiotic.
Thus, there exists a desire in the an for agents that could act as adjuncts to conventional antibiotic therapy and that could act to improve the therapeutic effectiveness of antibiotics.
SUMMARY OF THE INVENTION
The present invention generally provides methods and compositions for prophylaxis or treatment of gram-negative bacterial infections, using BPI protein products. The methods and compositions, in addition to being useful for treatment, are useful for prophylaxis of patients at high risk of gram-negative bacterial infection, e.g., patients who will undergo abdominal or genitourinary surgery, or trauma victims. Specifically, the present invention provides, in a method for treating a gram-negative bacterial infection with an antibiotic, the improvement comprising the step of concurrently administering BPI protein product in an amount effective to improve the therapeutic effectiveness of the antibiotic.
The present invention is based upon the finding that BPI protein product is useful as adjunct therapy with conventional antibiotics, and specifically the finding that concurrent administration, or co-treatment, of a BPI protein product and an antibiotic or combination of antibiotics can improve the therapeutic effectiveness of the antibiotic or combination of antibiotics. BPI protein product may improve the therapeutic effectiveness of antibiotics in a variety of ways, including by increasing susceptibility of gram-negative bacteria to a reduced dosage of antibiotics, by effectively reversing resistance of gram-negative bacteria to antibiotics, by providing synergistic or potentiating effects beyond the individual or additive effects of the BPI protein product or antibiotic alone, or by neutralizing endotoxin released by bacteria killed by antibiotics. Concurrent administration of BPI protein product and antibiotic provides unexpectedly superior therapeutic effects in vivo than either agent provides when administered alone. Concurrent administration of BPI protein product according to this improved method of treatment is effective even when the gram-negative bacteria involved are considered to be resistant to the bactericidal effects of BPI protein product alone and/or antibiotic alone.
The present invention provides a use of a BPI protein product for the manufacture of a medicament for the co-treatment with an antibiotic of a gram-negative bacterial infection in mammals. This aspect of the invention contemplates co-treatment with any antibiotic or combinations of antibiotics, including .beta.-lactam antibiotics with and without .beta.-lactamase inhibitors, aminoglycosides, tetracyclines, suffonamides and trimethoprim, vancomycin, macrofides, fluoroquinolones and quinolones, polymyxins and other antibiotics.
This aspect of the invention also provides the use of a BPI protein product for the manufacture of a medicament for improving the therapeutic effectiveness of antibiotics in a gram-negative bacterial infection, use of a BPI protein product for the manufacture of a medicament for increasing the susceptibility to an antibiotic of gram-negative bacteria involved in the gram-negative bacterial infection, and use of a BPI protein product for the manufacture of a medicament for reversing resistance to an antibiotic of gram-negative bacteria involved in the gram-negative bacterial infection.
The invention utilizes any of the large variety of BPI protein products known to the art including natural BPI protein, recombinant BPI protein, BPI fragments, BPI analogs, BPI variants, and BPI peptides. Concurrent administration of BPI protein product with any antibiotic or combination of antibiotics is contemplated, including .beta.-lactam antibiotics with or without .beta.-lactamase inhibitors, aminoglycosides, tetracyclines, sulfonamides and trimethoprim, vancomycin, macrolides, fluoroquinolones and quinolones, polymyxins, and other antibiotics.
Either the BPI protein product or the antibiotic may be administered systemically or topically to a subject suffering from a suspected or confirmed gramnegative bacterial infection. The BPI protein product and antibiotic may be administered by different routes and may be administered simultaneously or sequentially.
The invention also provides pharmaceutical compositions for treatment of gram-negative bacterial infections, comprising an antibiotic and a BPI protein product in an amount effective to improve the therapeutic effectiveness of the antibiotic. Such compositions optionally comprise pharmaceutically acceptable diluents, adjuvants or carriers. The compositions may be formulated for systemic or topical administration to subjects. In addition, compositions comprising BPI protein product and an antibiotic can be used in a variety of in vitro uses, such as use as a bactericide to decontaminate fluids and surfaces and to sterilize surgical and other medical equipment and implantable devices, including prosthetic joints.
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently preferred embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts survival data after treatment with rBPI.sub.21 and cefamandole, separately or in combination, in an E. coli O 111:B4 mouse peritonitis assay.
FIGS. 2-4 relate to results from an E. coli O111:B4 mouse peritonitis assay with rBPI.sub.21 and cefamandole, separately or in combination. FIG. 2 depicts survival data; FIG. 3 shows bacterial counts from peritoneal lavage fluid; and FIG. 4 shows bacterial counts in blood.
FIGS. 5A and 5B display results from two trials of treatment with rBPI.sub.21 and cefamandole, separately or in combination, in an E. coli O7:K1 mouse peritonitis assay.
FIGS. 6-16 relate to results, including cardiovascular and metabolic findings, from an E. coli O7:K1 rabbit bacteremia assay with rBPI.sub.21 and cefamandole, separately or in combination. FIG. 6 depicts survival data; FIG. 7 shows bacterial count in blood, which FIG. 8 displays as percentage of bacterial dose; FIG. 9 shows blood endotoxin levels; FIGS. 10 shows mean arterial blood pressure; FIG. 11, cardiac index; FIG. 12, total peripheral resistance; FIG. 13, arterial oxygen tension; FIG. 14, alveolar-artefial oxygen gradient; FIG. 15, respiration rate; and FIG. 16, arterial blood pH.
FIG. 17 depicts survival data after treatment with rBPI.sub.21 and gentamicin, separately or in combination, in an E. coli O7:K1 mouse peritonitis assay.
FIG. 18 shows the bactericidal effect of rBPI.sub.21 and cefifiaxone, separately or in combination, on growth of ceftfiaxone-resistant E. coli.
FIGS. 19-25 relate to the synergistic effect of rBPI.sub.21 on antibiotic killing curves for selected gram-negative organisms. FIG. 19 shows the bactericidal effect of rBPI.sub.21 alone. FIG. 20 shows the effect of rBPI.sub.21 in combination with trimethoprim/suffamethoxazole; FIG. 21, rBPI.sub.21 with ciprofloxacin; FIG. 22, rBPI.sub.21 with piperacillin; FIG. 23, rBPI.sub.21 with cefotaxime; FIG. 24, rBPI.sub.21 with cefuroxime; and FIG. 25, rBPI.sub.21 with amikacin.
DETAILED DESCRIPTION
The present invention relates to methods and compositions for treating a gram-negative bacterial infection, using a BPI protein product. The invention is based on the unexpected discovery that, when treating a gram-negative bacterial infection with an antibiotic, the concurrent administration of BPI protein product with the antibiotic improves the therapeutic effectiveness of the antibiotic, even at doses at which the BPI protein product alone or antibiotic alone may be inactive. BPI protein product by itself typically has an antibacterial potency less than that of conventional antibiotics. However, because its administration unexpectedly improves the therapeutic effectiveness of conventional antibiotic therapy. BPI protein product is useful as adjunct therapy with conventional antibiotic therapy for the treatment of gram-negative bacterial infections.
"Gram-negative bacterial infection," as used herein, encompasses conditions associated with or resulting from gram-negative bacterial infection (e.g., sequelae). These conditions include gram-negative sepsis, endotoxin-related hypotension and shock, and one or more of the conditions associated therewith, including fever, metabolic acidosis, disseminated intravascular coagulation and related clotting disorders, anemia, thrombocytopenia, leukopenia, adult respiratory distress syndrome and related pulmonary disorders, renal failure and related renal disorders, hepatobiliary disease and central nervous system disorders. These conditions also include translocation of bacteria from the intestines and concomitant release of endotoxin.
BPI protein product may improve the therapeutic effectiveness of the antibiotic in a variety of ways, including by increasing susceptibility of gram-negative bacteria to a reduced dosage of antibiotics, by effectively reversing resistance of gram-negative bacteria to antibiotics, by providing synergistic or potentiating effects beyond the individual or additive effects of the BPI protein product or antibiotic alone, or by neutralizing endotoxin released by bacteria killed by antibiotics. Concurrent administration of BPI protein product and antibiotic is expected to provide more effective treatment of gram-negative bacterial infection. Concurrent administration of the two agents may provide greater therapeutic effects in vivo than either agent provides when administered singly. It may permit a reduction in the dosage of one or both agents with achievement of a similar therapeutic effect. Alternatively, the concurrent administration may produce a more rapid or complete bactericidal/bacteriostatic effect than could be achieved with either agent alone.
Therapeutic effectiveness is based on a successful clinical outcome, and does not require that the antimicrobial agent or agents kill 100% of the organisms involved in the infection. Success depends on achieving a level of antibacterial activity at the site of infection that is sufficient to inhibit the bacteria in a manner that tips the balance in favor of the host. When host defenses are maximally effective, the antibacterial effect required may be minimal. Reducing organism load by even one log (a factor of 10) may permit the host's own defenses to control the infection. In addition, augmenting an early bactericidal/baeteriostatic effect can be more important than long-term bactericidal/bacteriostatic effect. These early events are a significant and critical part of therapeutic success, because they allow time for host defense mechanisms to activate. Increasing the bactericidal rate may be particularly important for infections such as meningitis, bone or joint infections. [Stratton, Antibiotics in Laboratory Medicine, 3rd ed. (Lorian, V., Ed.) pp. 849-879, Williams and Wilkins, Baltimore Md. (1991)].
The effect of BPI protein product to improve the therapeutic effectiveness of antibiotics in vivo may be demonstrated in in vivo animal models, or may be predicted on the basis of a variety of in vitro tests, including (1) determinations of the minimum inhibitory concentration (MIC) of an antibiotic required to inhibit growth of a gram-negative organism for 24 hours, (2) determinations of the effect of an antibiotic on the kinetic growth curve of a gram-negative organism, and (3) checkerboard assays of the MIC of serial dilutions of antibiotic alone or in combination with serial dilutions of BPI protein product. Exemplary models or tests are described in Eliopoulos and Moellering In Antibiotics in Laboratory Medicine, 3rd ed. (Lorian, V., Ed.) pp. 432-492, Williams and Wilkins, Baltimore Md. (1991 ).
Using in vitro determinations of antibiotic MIC at 24 hours, a BPI protein product may be shown to reduce the MIC of the antibiotic. With this result, it is expected that concurrent administration of the BPI protein product in vivo will increase susceptibility of the gram-negative organism to the antibiotic. A BPI protein product may also be shown to reduce the MIC of an antibiotic from the range in which the organism is considered clinically resistant to a range in which the organism is considered clinically susceptible. With this result, it is expected that concurrent administration in vivo of the BPI protein product with the antibiotic will reverse resistance and effectively convert the antibiotic-resistant organism into an antibiotic-susceptible organism.
By measuring the effect of antibiotics on the in vitro growth curves of gram-negative organisms, in the presence or absence of a BPI protein product, the BPI protein product may be shown to enhance the early antibacterial effect of antibiotics at 0-24 hours. Enhancement of early bactericidal/growth inhibitory effects is important in determining therapeutic outcome.
The BPI protein product and antibiotic may also be shown to have synergistic or potentiating effects beyond the individual effects of each agent alone or the additive effects of the agents together. In a checkerboard assay, the combination of BPI protein product with antibiotics may be shown to result in a "synergistic" fractional inhibitory concentration index (FIC). The checkerboard method is based on additivity, which assumes that the result observed with multiple drugs is the sum of the separate effects of the drugs being tested; according to this system a FIC of less than 0.5 is scored as synergy, 1 is scored as additive, and greater than 1 but less than 2 is scored as indifferent. In contrast, kinetic assays are based on the idea that only one metabolic pathway at a time can be growth rate-limiting for an organism; according to this system, the combined effect of drugs that do not interact with one another (autonomous or indifferent) is simply the effect of the most active drug alone.
Concurrent administration of BPI protein products and antibiotics is shown herein to lower MICs of a variety of antibiotics for a variety of gram-negative organisms. It is also shown to reverse resistance of a variety of gmrn-negative organisms to antibiotics. In some cases where BPI protein product does not affect the MIC of antibiotic at 24 hours, BPI protein product is shown herein to enhance the early bactericidal effect of antibiotics on growth curves at 0-7 or 7-24 hours. The BPI protein products exert these effects even on gram-negative organisms that are not considered susceptible to the direct bactericidal or growth inhibitory effects of BPI protein product alone. It is also shown herein that the concurrent administration of BPI protein products with antibiotics in vivo allows a reduction in the dosages of both agents to amounts that, if administered alone, would be insufficient to exert the same clinical effect.
Either the BPI protein product or the antibiotic, or both, may be administered at levels at which neither would alone be therapeutically effective against a gram-negative bacterial infection. Alternatively, according to a preferred method, the antibiotic and BPI protein product can be administered in amounts where each would alone be therapeutically effective against a gram-negative bacterial infection but wherein the combination of the two antibiotics provides even more potent effects. The BPI protein product may be administered in an amount which increases susceptibility of gram-negative bacteria to reduced antibiotic dosage, or in an amount which reverses resistance of the gram-negative bacteria to an antibiotic.
BPI protein product is thought to interact with a variety of host defense elements present in whole blood or serum, including complement, p15 and LBP, and other cells and components of the immune system. Such interactions may result in potentiation of the activities of BPI protein product. Because of these interactions, BPI protein products can be expected to exert even greater activity in vivo than in vitro. Thus, while in vitro tests are predictive of in vivo utility, absence of activity in vitro does not necessarily indicate absence of activity in vivo. For example, BPI has been observed to display a greater bactericidal effect on gram-negative bacteria in whole blood or plasma assays than in assays using conventional media. [Weiss et al., J. Clin. Invest. 90:1122-1130 (1992)]. This may be because conventional in vitro systems lack the blood elements that facilitate or potentiate BPI's function in vivo, or because conventional media contain higher than physiological concentrations of magnesium and calcium, which are typically inhibitors of the antibacterial activity of BPI protein products. Furthermore, in the host, BPI protein product is available to neutralize endotoxin released during antibiotic killing of bacteria, a further clinical benefit not seen in or predicted by in vitro tests.
It is also contemplated that the BPI protein product be administered with other products that potentiate the bactericidal activity of BPI protein products. For example, serum complement potentiates the gram-negative bactericidal activity of BPI protein products; the combination of BPI protein product and serum complement provides synergistic bactericidal/growth inhibitory effects. See, e.g., Ooi et al. J. Biol. Chem., 265:15956 (1990) and Levy et al. J. Biol. Chem., 268: 6038-6083 (1993) which address naturally-occurring 15 kD proteins potentiating BPI antibacterial activity. See also co-owned, co-pending PCT Application No. US94/07834 filed Jul. 13, 1994, which corresponds to U.S. patent application Ser. No. 08/274,303 filed Jul. 11, 1994 as a continuation-in-part of U.S. patent application Ser. No. 08/093,201, now abandoned, filed Jul. 14, 1993. These applications, which are all incorporated herein by reference, describe methods for potenfiating gram-negative bactericidal activity of BPI protein products by administering lipopolysaccharide binding protein (LBP) and LBP protein products. LBP protein derivatives and derivative hybrids which lack CD-14 immunostimulatory properties are described in PCT Application No. US94/06931 filed Jun. 17, 1994, which corresponds to co-owned, co-pending U.S. patent application Ser. No. 08/261,660, filed Jun. 17, 1994 as a continuation-in-part of U.S. patent application Ser. No. 08/079,510, now abandoned, filed Jun. 17, 1993, the disclosures of all of which are hereby incorporated by reference.
An advantage provided by the present invention is the ability to provide more effective treatment of gram-negative bacterial infection by virtue of the improved therapeutic effectiveness of the antibiotic treatment. Another advantage is the ability to treat gram-negative organisms that are normally resistant to one or more antibiotics. Yet another advantage is the ability to accelerate the killing of gram-negative organisms by antibiotics. An additional advantage is the ability to neutralize endotoxin released during antibiotic killing of bacteria. A further advantage is the ability to use lower concentrations of toxic antibiotics such as gentamicin and polymyxin B, or expensive antibiotics such as vancomycin. Because the use of some antibiotics is limited by their systemic toxicity or prohibitive cost, lowering the concentration of antibiotic required for therapeutic effectiveness reduces toxicity and/or cost of treatment, and thus allows wider use of the antibiotic. The present invention may also provide quality of life benefits due to, e.g., decreased duration of therapy, reduced stay in intensive care units or overall in the hospital, with the concomitant reduced risk of serious nosocomial (hospital-acquired) infections.
The invention further provides pharmaceutical compositions for treatment of gram-negative bacterial infections, comprising an antibiotic and a BPI protein product in an amount effective to improve the therapeutic effectiveness of the antibiotic. Such compositions optionally comprise pharmaceutically acceptable diluents, adjuvants or carriers. The compositions may be formulated for systemic or topical administration to subjects. In addition. antiseptic compositions comprising BPI protein product and an antibiotic can be used in a variety of in vitro uses such as use as a bacteficide to alecontaminate fluids and surfaces and to sterilize surgical and other medical equipment and implantable devices, including prosthetic joints. The invention also provides improved methods of in vitro decontamination of fluids and surfaces comprising administering a BPI protein product in combination with an antibiotic.
Either the BPI protein product or the antibiotics may be administered systemically or topically. Systemic routes of administration include oral, intravenous, intramuscular or subcutaneous injection, intrathecal, intraperitoneal (e.g. by intraperitoneal lavage), transpulmonary using aerosolized or nebulized drug, or transdermal. Topical routes include administration in the form of salves, ophthalmic drops, ear drops, or irrigation fluids (for, e.g., irrigation of wounds).
"Concurrent administration," or co-treatment, as used herein includes administration of the agents together, or before or after each other. The BPI protein product and antibiotics may be administered by different routes. For example, the BPI protein product may be administered intravenously while the antibiotics are administered intramuscularly, intravenously, subcutaneously, orally or intraperitoneally. Alternatively, the BPI protein product may be administered intraperitoneally while the antibiotics are administered intraperitoneally or intravenously, or the BPI protein product may be administered in an aerosolized or nebulized form while the antibiotics are administered, e.g., intravenously. The BPI protein product and antibiotics are preferably both administered intravenously. The BPI protein product and antibiotics may be given sequentially in the same intravenous line, after an intermediate flush, or may be given in different intravenous lines. The BPI protein product and antibiotics may be administered simultaneously or sequentially, as long as they are given in a manner sufficient to allow both agents to achieve effective concentrations at the site of infection.
As used herein, "BPI protein product" includes naturally and recombinantly produced BPI protein; natural, synthetic, and recombinant biologically active polypeptide fragments of BPI protein; biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins and dimers; and biologically active polypeptide analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs. The BPI protein products administered according to this invention may be generated and/or isolated by any means known in the art. U.S. Pat. No. 5,198,541, the disclosure of which is incorporated herein by reference, discloses recombinant genes encoding and methods for expression of BPI proteins including recombinant BPI holoprotein, referred to as rBPI.sub.50 and recombinant fragments of BPI. Co-owned, copending U.S. patent application Ser. No. 07/885,501, now abandoned, and a continuation-in-part thereof, U.S. patent application Ser. No. 08/072,063, now U.S. Pat. No. 5,439,807, filed May 19, 1993 and corresponding PCT Application No. 93/04752 filed May 19, 1993, which are all incorporated herein by reference, disclose novel methods for the purification of recombinant BPI protein products expressed in and secreted from genetically transformed mammalian host cells in culture and discloses how one may produce large quantities of recombinant BPI products suitable for incorporation into stable, homogeneous pharmaceutical preparations.
Biologically active fragments of BPI (BPI fragments) include biologically active molecules that have the same or similar amino acid sequence as a natural human BPI holoprotein, except that the fragment molecule lacks amino-terminal amino acids, internal amino acids, and/or caxboxy-terminal amino acids of the holoprotein. Nonlimiting examples of such fragments include a N-terminal fragment of natural human BPI of approximately 25 kD, described in Ooi et al., J. Exp. Med., 174:649 (1991), and the recombinant expression product of DNA encoding N-terminal amino acids from 1 to about 193 or 199 of natural human BPI, described in Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992), and referred to as rBPI.sub.23. In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI.sub.23) having the 31-residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI, as set out in FIG. 1 of Gray et al., supra, except that valine at position 151 is specified by GTG rather than GTC and residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG). Recombinant holoprotein (rBPI) has also been produced having the sequence (SEQ ID NOS: 145 and 146) set out in FIG. 1 of Gray et al., supra, with the exceptions noted for rBPI.sub.23 and with the exception that residue 417 is alaninc (specified by GCT) rather than valine (specified by GTT). Other examples include dimeric forms of BPI fragments, as described in co-owned and co-pending U.S. patent application Ser. No. 08/212,132, now U.S. Pat. No. 5,447,913, filed Mar. 11, 1994, and corresponding PCT Application No. PCT/US95/03125 filed Mar. 13, 1995, the disclosures of which are incorporated herein by reference. Preferred dimeric products include dimeric BPI protein products wherein the monomers are amino-terminal BPI fragments having the N-terminal residues from about 1 to 175 to about 1 to 199 of BPI holoprotein. A particularly preferred dimeric product is the dimeric form of the BPI fragment having N-terminal residues 1 through 193, designated rBPI.sub.42 dimer.
Biologically active variants of BPI (BPI variants) include but are not limited to recombinant hybrid fusion proteins, comprising BPI holoprotein or biologically active fragment thereof and at least a portion of at least one other polypeptide, and dimeric forms of BPI variants. Examples of such hybrid fusion proteins and dimeric forms are described by Theofan et al. in co-owned, copending U.S. patent application Ser. No. 07/885,911, now abandoned, and a continuation-in-part application thereof, U.S. patent application Ser. No. 08/064,693 filed May 19, 1993 and corresponding PCT Application No. US93/04754 filed May 19, 1993, which are all incorporated herein by reference and include hybrid fusion proteins comprising, at the amino-terminal end, a BPI protein or a biologically active fragment thereof and, at the carboxy-terminal end, at least one constant domain of an immunoglobulin heavy chain or allelic variant thereof.
Biologically active analogs of BPI (BPI analogs) include but are not limited to BPI protein products wherein one or more amino acid residues have been replaced by a different amino acid. For example, co-owned, copending U.S. patent application Ser. No. 08/013,801 filed Feb. 2, 1993, now U.S. Pat. No. 5,420,019, and corresponding PCT Application No. US94/01235 filed Feb. 2, 1994, the disclosures of which are incorporated herein by reference, discloses polypeptide analogs of BPI and BPI fragments wherein a cysteine residue is replaced by a different amino acid. A preferred BPI protein product described by this application is the expression product of DNA encoding from amino acid 1 to approximately 193 or 199 of the N-terminal amino acids of BPI holoprotein, but wherein the cysteine at residue number 132 is substituted with alanine and is designated rBPI.sub.21 .DELTA.cys or rBPI.sub.21. Other examples include dimeric forms of BPI analogs; e.g. co-owned and co-pending U.S. patent application Ser. No. 08/212,132, now U.S. Pat. No. 5,497,913, filed Mar. 11, 1994, and corresponding PCT Application No. PCT/US95/03125 filed Mar. 13, 1995, the disclosures of which are incorporated herein by reference.
Other BPI protein products useful according to the methods of the invention are peptides derived from or based on BPI produced by recombinant or synthetic means (BPI-derived peptides), such as those described in co-owned and copending PCT Application No. PCT/US94/10427 filed Sep. 15, 1994, which corresponds to U.S. patent application Ser. No. 08/306,473 filed Sep. 15, 1994, and PCT Application No. US94/02465 filed Mar. 11, 1994, which corresponds to U.S. patent application Ser. No. 08/209,762, filed Mar. 11, 1994, which is a continuation-in-part of U.S. patent application Ser. No. 08/183,222, filed Jan. 14, 1994, now abandoned, which is a continuation-in-part of U.S. patent application Ser. No. 08/093,202, now abandoned, filed Jul. 15, 1993 (for which the corresponding international application is PCT Application No. US94/02401 filed Mar. 11, 1994), which is a continuation-in-part of U.S. patent application Ser. No. 08/030,644 filed March 12, 1993, now U.S. Pat. No. 5,348,942, the disclosures of all of which are incorporated herein by reference.
Presently preferred BPI protein products include recombinantly-produced N-terminal fragments of BPI, especially those having a molecular weight of approximately between 21 to 25 kD such as rBPI.sub.23 or rBPI.sub.21, or dimeric forms of these N-terminal fragments (e.g., rBPI.sub.42 dimer). Additionally, preferred BPI protein products include rBPI.sub.50 and BPI-derived peptides.
The administration of BPI protein products is preferably accomplished with a pharmaceutical composition comprising a BPI protein product and a pharmaceutically acceptable diluent, adjuvant, or carrier. The BPI protein product may be administered without or in conjunction with known surfactants, other chemothempeutic agents or additional known anti-microbial agents. A preferred pharmaceutical composition containing BPI protein products (e.g., rBPI.sub.50, rBPI.sub.23) comprises the BPI protein product at a concentration of 1 mg/ml in citmte buffered saline (5 or 20 mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1% by weight of poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, N.J.) and 0.002% by weight of polysorbate 80 (Tween 80, ICI Americas Inc., Wilmington, Del.). Another preferred pharmaceutical composition containing BPI protein products (e.g., rBPI.sub.21) comprises the BPI protein product at a concentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer 188 and 0.002% polysorbate 80. Such preferred combinations are described in co-owned, co-pending PCT Application No. US94/01239 filed Feb. 2, 1994, which corresponds to U.S. patent application Ser. No. 08/190,869 filed Feb. 2, 1994, now U.S. Pat. No. 5,488,034, and U.S. patent application Ser. No. 08/012,360 filed Feb. 2, 1993, now abandoned, the disclosures of all of which are incorporated herein by reference.
Suitable antibiotics, and therapeutically effective concentrations thereof when administered with BPI protein products, may be determined in in vivo models or according to in vitro tests, for example, the in vitro minimum inhibitory concentration (MIC) and in vivo mouse peritonitis or rabbit bacteremia assays taught herein. Suitable antibiotics are antibiotics that act on the bacterial cell wall, cell membrane, protein metabolism or nucleic acid metabolism. These would include antibiotics or combinations of antibiotics from the following classes: .beta.-lactam antibiotics with or without .beta.-lactamase inhibitors, aminoglycosides, tetracyclines, sulfonamides and trimethoprim, vancomycin, macrotides, fluoroquinolones and quinolones, polymyxins, and other antibiotics. Dosage and administration of suitable antibiotics are known in the art, and briefly summarized below.
PENICILLINS
When a BPI protein product is concurrently administered with a penicillin, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily. The penicillin is generally given in doses ranging from 1 mg/kg to 750 mg/kg daily, preferably not to exceed 24 grams daily for adults (or 600 mg/kg daily for children), and is preferably administered as follows:
Penicillin G is preferably administered parenterally to adults in doses ranging from 600,000 to 1,000,000 units per day. In conventional administration, it is effective largely against gram-positive organisms. For treatment of pneumococcal meningitis, penicillin G is administered in doses of 20-24 million units daily, in divided doses every 2 or 3 hours. For children, the preferred parenteral dose of penicillin G is 300,000 to 1,000,000 units per day. One unit of penicillin G contains 0.6 .mu.g of pure sodium penicillin G (i.e., 1 mg is 1667 units).
Amoxicillin may be administered parenterally to adults in doses ranging from 750 mg to 1.5 grams per day, in 3 equally divided doses. For children, preferred parenteral doses of amoxicillin range from 20 to 40 mg/kg per day in 3 equally divided doses. Amoxicillin is also available in combination with clavulanic acid, a .beta.-lactamase inhibitor. A 250 mg dose of the combination drug amoxicillin/clavulanate will contain 250 mg of amoxicillin and either 125 or 62.5 mg of clavulanic acid. The combination is preferably administered to adults orally in doses of 750 mg per day divided into 3 equal doses every 8 hours, with a preferred dose of 1.5 grams per day for severe infections, given in 3 equally divided doses. In children, the preferred oral dose is 20 to 40 mg/kg per day in 3 equally divided doses.
Ampicillin is preferably administered parenterally to adults in doses of 6 to 12 grams per day for severe infections, in 3 to 4 equally divided doses. In children, the preferred 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 mg/kg per day, for children, or 12 grams per day, for adults, may be administered parenterally for treatment of meningitis. Ampicillin is also available in combination with sulbactam, a .beta.-lactamase inhibitor. Each 1.5 gram dose of ampicillin/sulbactam contains 1 gram of ampicillin and 0.5 grams of sulbactam. The combination is preferably administered parenterally to adults in doses of 6 to 12 grams per day divided into 4 equal doses every 6 hours, not to exceed a total of 12 grams per day.
Azlocillin is preferably administered parenterally to adults in doses of 8 to 18 grams per day, given in 4 to 6 equally divided doses.
Carbenicillin is preferably administered parenterally to adults in doses of 30 to 40 grams per flay, given by continuous infusion or in 4 to 6 equally divided doses. Daily doses of up to 600 mg/kg have been used to treat children with life-threatening infections.
Mezlocillin is preferably administered to adults parenterally in doses of 100 to 300 mg/kg per day, given in 4 to 6 equally divided doses. The usual dose is 16 to 18 grams per day; for life threatening infections, 350 mg/kg per day may be administered, but in doses not to exceed 24 grams per day given in 6 equally divided doses every 4 hours. For children, the preferred parenteral dose of mezlocillin is 150 to 300 mg/kg per day.
Nafcillin is preferably administered intravenously to adults in doses of 3 grams per day, given in 6 equally divided doses every 4 hours, with doubled doses for very severe infections. In conventional administration, it is effective largely against gram-positive organisms. In children, the preferred parenteral dose is 20 to 50 mg/kg per day, in 2 equally divided doses every 12 hours. The preferred oral dose for nafcillin ranges from 1 gram per day to 6 grams per day in 4 to 6 divided doses.
Oxacillin is preferably administered parenterally to adults in doses of 2 to 12 grams per day, in 4 to 6 equally divided doses. In conventional administration, it is effective largely against gram-positive organisms. In children, oxacillin is preferably administered in doses of 100 to 300 mg/kg per day.
Piperacillin is preferably administered parenterally to adults in doses ranging from 100 mg/kg, or 6 grams per day, in 2 to 4 equally divided doses, up to a maximum of 24 grams per day, in 4 to 6 equally divided doses. Higher doses have been used without serious adverse effects.
Ticarcillin is preferably administered parenterally to adults in doses ranging from 4 grams per day to 18 grams per day administered in 4 to 6 equally divided doses. The usual dose is 200 to 300 mg/kg per day. For children, the preferred parenteral dose of ticarcillin ranges from 50 mg/kg per day to 300 mg/kg per day, given in 3, 4 or 6 equally divided doses. The combination ticarcillin/clavulanate is preferably administered parenterally to adults in doses of 200 to 300 mg/kg per day (based on ticarcillin content), in 4 to 6 equally divided doses. For adults, the usual dose is 3.1 grams (which contains 3 grams of ticarcillin and 100 mg of clavulanic acid) every 4 to 6 hours. The combination is also available in a dose of 3.2 grams, which contains 3 grams of ticarcillin and 200 mg of clavulanic acid.
In general, it is desirable to limit each intramuscular injection of a penicillin or cephalosporin to 2 grams; larger doses should be administered by multiple injections in different large muscle masses.
CEPHALOSPORINS
When a BPI protein product is concurrently administered with a cephalosporin, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1/ .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily. The cephalosporin is generally given in doses ranging from 1 .mu.g/kg to 500 mg/kg daily, preferably not to exceed 16 grams daily, and is preferably administered as follows:
Cefamandole is preferably administered parenterally to adults in doses ranging from 1.5 grams per day, given in 3 equally divided doses every 8 hours, to 12 grams per day for life-threatening infections, given in 6 equally divided doses every 4 hours. In children, cefamandole is preferably administered in doses ranging from 50 to 150 mg/kg per day, in 3 to 6 equally divided doses, not to exceed a total of 12 grams per day.
Cefazolin is preferably administered parenterally to adults in doses of 750 mg per day, given in 3 equally divided doses every 8 hours. In severe, life-threatening infections, it may be administered at doses of 6 grams per day divided into 4 equal doses every 6 hours; in rare instances, up to 12 grams per day have been used. In children, the preferred parenteral dose of cefazolin is 20 to 50 mg/kg per day, divided into 3 or 4 equal doses, with 100 mg/kg per day administered for severe infections.
Cefonicid is preferably administered parenterally to adults in doses ranging from 500 mg once daily, to 2 grams once daily for life-threatening infections. For intramuscular administration, a 2 gram dose should be divided into two 1-gram injections.
Cefoperazone is preferably administered parenterally to adults in doses ranging from 2 grams per day, given in 2 equally divided doses every 12 hours, to 12 grams per day for severe infections, given in 2, 3 or 4 equally divided doses. Doses up to 16 grams per day have been administered without complications.
Cefotetan is preferably administered parenterally to adults in doses of 1 to 4 grams per day, in 2 equally divided doses every 12 hours. Cefotetan may be administered in higher doses for life-threatening infections, not to exceed a total dose of 6 grams per day.
Cefotaxime is preferably administered parenterally to adults in doses ranging from 1 to 12 grams per day, not to exceed 12 grams per day (2 grams every 4 hours) for life-threatening infections. In children, the parenteral dose of cefotaxirne is preferably 50 to 180 mg/kg, divided into 4 to 6 equal doses.
Cefoxitin is preferably administered parenterally to adults in doses ranging from 3 to 12 grams per day, given in 3, 4, or 6 equally divided doses. In children, cefoxitin is preferably administered parenterally in doses of 80 to 160 mg/kg per day, given in 4 or 6 equally divided doses, not to exceed a total dose of 12 grams per day.
Ceftazidime is preferably administered parenterally to adults in doses ranging from 500 mg per day, given in 2 to 3 equally divided doses (every 8 or 12 hours), up to a maximum of 6 grams per day. In children, ceftazidime is preferably administered intravenously in doses of 30 to 50 mg/kg, to a maximum of 6 grams per day.
Ceftizoxime is preferably administered parenterally to adults in doses ranging from 1 gram per day, given in 2 equally divided doses every 12 hours, to 12 grams per day for life-threatening infections, given in 3 equally divided doses every 8 hours. The usual adult dose is 1 to 2 grams every 8 or 12 hours. For children, the preferred parenteral dose is 50 mg/kg every 6 or 8 hours, for a total daily dose of 200 mg/kg.
Ceftriaxone is preferably administered parenterally to adults in doses ranging from 1 to 2 grams per day, given in 2 equally divided doses every 12 hours. It may be given in higher doses, not to exceed a total of 4 grams per day. In children, the preferred parenteral dose of ceftriaxone is 50 to 75 mg/kg per day, not to exceed 2 grams per day. In meningitis, ceftriaxone may be administered in doses of 100 mg/kg per day, not to exceed 4 grams per day.
Cefuroxime is preferably administered parenterally to adults in doses ranging from 2.25 to 4.5 grams per day, in 3 equally divided doses every 8 hours. For life-threatening infections, 6 grams per day may be administered in 4 equally divided doses every 6 hours, and for meningitis, 9 grams per day may be administered in 3 equally divided doses every 8 hours. For children, the preferred parenteral dose of cefuroxime is 50 to 150 mg/kg per day in 3 to 4 equally divided doses, or 240 mg/kg per day for meningitis.
Cephalexin is formulated for oral administration, and is preferably administered orally to adults in doses ranging from 1 to 4 grams per day in 2 to 4 equally divided doses. For children, the preferred dose is 20 to 50 mg/kg per day in divided doses, with doses being doubled for severe infections.
Cephalothin is usually administered parenterally to adults in doses of 8 to 12 grams per day.
OTHER BETA-LACTAMS
When a BPI protein product is concurrently administered with an imipenem antibiotic, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily. The imipenem is generally given in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and is preferably administered as follows:
Imipenem is available in combination with cilastatin, an inhibitor of the renal dipeptidase enzyme that rapidly inactivates imipenem. The combination is preferably administered intramuscularly to adults in doses of 1 to 1.5 grams per day, given in 2 equally divided doses every 12 hours. Intramuscular doses exceeding 1.5 grams per day are not recommended. The combination is preferably administered intravenously in doses ranging from 1 to 4 grams per day, in 4 equally divided doses every 6 hours; doses exceeding 50 mg/kg per day, or 4 grams per day, are not recommended.
When a BPI protein product is concurrently administered with a monobactam antibiotic, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily. The monobactam is generally given in doses ranging from 1 .mu.g/kg to 200 mg/kg daily, and is preferably administered as follows:
Aztreonam is preferably administered parenterally to adults in doses ranging from 1 gram per day, given in 2 equally divided doses every 12 hours, up to a maximum recommended dose of 8 grams per day in cases of life-threatening infection, given in 3 or 4 equally divided doses.
AMINOGLYCOSIDES
When a BPI protein product is concurrently administered with an aminoglycoside, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily. The aminoglycoside is generally given in doses ranging from 1 .mu.g/kg to 20 mg/kg daily, preferably not to exceed 15 mg/kg daily, and is preferably administered as follows:
When administering aminoglycosides, it is desirable to measure serum peak and trough concentrations to ensure the adequacy and safety of the dosage. Dosages should generally be adjusted to avoid toxic peak and trough concentrations. Amikacin is preferably administered parenterally to adults and children in doses of 15 mg/kg per day, divided into two or three equal doses every 8 or 12 hours, and not to exceed a total dose of 1.5 grams per day. For uncomplicated infections, a dose of 500 mg amikacin per day, in 2 equally divided doses, may be administered. Dosages should be adjusted to avoid prolonged serum peak concentrations of amikacin above 35 .mu.g/ml and prolonged trough concentrations greater than 10 .mu.g/ml.
Gentamicin is preferably administered parenterally to adults in doses of 3 mg/kg per day, in three equally divided doses every 8 hours. For life-threatening infections, up to 5 mg/kg per day in 3 to 4 equally divided doses may be administered, but this dosage should be reduced to 3 mg/kg per day as soon as clinically indicated. For children, gentamicin is preferably administered parenterally in doses of 6 to 7.5 mg/kg per day. Dosages should be adjusted to avoid prolonged serum peak concentrations of gentamicin above 12 .mu.g/ml and prolonged trough concentrations greater than 2 .mu.g/ml.
Netilmicin may be administered parenterally to adults in doses ranging from 3 mg/kg per day, in 2 equally divided doses every 12 hours, to 6.5 mg/kg per day for serious systemic infection, in 2 or 3 equally divided doses. In children, the preferred parenteral dose is 5.5 to 8 mg/kg per day, in 2 or 3 equally divided doses. Dosages should be adjusted to avoid prolonged serum peak concentrations of netilmicin above 16 .mu.g/ml and prolonged serum trough concentrations above 4 .mu.g/ml.
Tobramycin is preferably administered parenterally to adults in doses of 3 mg/kg per day, given in three equally divided doses every 8 hours. For lifethreatening infections, tobramycin may be administered in doses up to 5 mg/kg per day, in 3 or 4 equally divided doses, but this dosage should be reduced to 3 mg/kg per day as soon as clinically indicated. In children, tobramycin is preferably administered parenterally in doses of 6 to 7.5 mg/kg per day. Prolonged serum concentrations of tobramycin above 12 .mu.g/ml should be avoided, and rising trough levels above 2 .mu.g/ml may indicate tissue accumulation, which may contribute to toxicity.
Concurrent administration of BPI protein product with the aminoglycosides, including amikacin, gentamicin, netilmicin and tobramycin, may permit a lowering of the dose of these toxic antibiotics necessary to achieve a therapeutic effect.
TETRACYCLINES
When a BPI protein product is concurrently administered with a tetracycline, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily. The tetracycline is generally given in doses ranging from 1 .mu.g/kg to 50 mg/kg daily, and is preferably administered as follows:
The tetracycline antibiotics are generally administered to adults in doses of 1 to 2 grams per day. An exception is doxycycline, which is preferably administered intravenously to adults in doses of 100 to 200 mg per day, and to children in doses of 2 mg/lb per day. Tetracycline may be administered parenterally to adults in doses of 0.5 to 2 grams per day, in 2 equally divided doses, and to children in doses of 10 to 20 mg/kg per day.
SULFONAMIDES
When a BPI protein product is concurrently administered with a sulfonamide or trimethoprim, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily. The sulfonamide or trimethoprim is generally given in doses ranging from 1 .mu.g/kg to 150 mg/kg daily, preferably not to exceed a combination dose of 960 mg trimethoprim/4.8g suffamethoxazole daily, and is preferably administered as follows:
The combination trimethoprim/sulfamethoxazole is available in a formulation containing a 1:5 ratio of tfimethoprim and sulfamethoxazole (e.g., 16 mg trimethoprim and 80 mg sulfamethoxazole). The combination is preferably administered intravenously to adults or children in doses of 8 to 10 mg/kg (based on the weight of the trimethoprim component) per day, in 2 to 4 equally divided doses. For Pneumocysas carinii infection, the combination can be administered in doses of 20 mg/kg (based on the weight of the trimethoprim component) per day, in 3-4 equally divided doses, to a maximum recommended dose of 960 mg trimethoprim/4.8 g suffamethoxazole per day. Trimethoprim alone is preferably administered orally to adults in doses of 200 mg per day. Sulfamethoxazole alone is preferably administered orally to adults in doses of 2 to 3 grams per day, and to children orally in doses of 50 to 60 mg/kg per day.
FLUOROQUINOLONES
When a BPI protein product is concurrently administered with a fiuoroquinolone or quinolone, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily. The fluoroquinolone or quinolone is generally given in doses ranging from 1 .mu.g/kg to 50 mg/kg daily, preferably not to exceed 1 gram daily, and is preferably administered as follows:
Norfloxacin is preferably administered orally to adults in doses from 400 to 800 mg daily, divided into two doses every 12 hours. Cinoxacin is preferably administered orally to adults in doses of 1 gram per day, given in 2 or 4 equally divided doses. Ciprofloxacin is preferably administered to adults intravenously in doses from 400 to 800 mg daily, or orally in doses from 500 to 1500 mg daily, divided into two doses every 12 hours. Ofioxacin is preferably administered to adults intravenously in doses from 400 to 800 mg daily, or orally in doses from 400 to 800 mg daily, divided into two doses every 12 hours.
VANCOMYCIN
When a BPI protein product is concurrently administered with vancomycin, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily. The vancomycin is generally given in doses ranging from 1 mg/kg to 50 mg/kg daily, and is preferably administered parenterally to adults in doses of 2 grams per day, divided into 2 or 4 doses every 6 or 12 hours. In children it is preferably administered in doses of 40 mg/kg, given in 4 equally divided doses every 6 hours. In conventional administration, vancomycin is effective largely against gram-positive organisms.
MACROLIDES
When a BPI protein product is concurrently administered with a macrolide, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily. The macrolide is generally given in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and is preferably administered as follows:
Erythromycin is preferably administered intravenously to adults and children in doses of 15 to 20 mg/kg per day, given by continuous infusion or in 4 equally divided doses every 6 hours. Erythromycin can be administered at doses up to 4 grams per day in cases of very severe infection.
Clarithromycin is preferably administered orally to adults in doses of 500 mg to 1 gram daily, in equally divided doses every 12 hours.
Azithromycin is preferably administered orally to adults at a dose of 500 mg on the first day of treatment followed by 250 mg once daily for 4 days, for a total dose of 1.5 grams.
OTHERS
When a BPI protein product is concurrently administered with other antibiotics, for treatment of a gram-negative bacterial infection, the BPI protein product is generally given parenterally in doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20 mg/kg daily.
Polymyxin B is generally given in doses ranging from 1 unit/kg to 45,000 units/kg daily, and is preferably administered intravenously to adults and children in doses of 15,000 to 25,000 units/kg per day, divided into 2 equal doses every 12 hours. It may be administered intramuscularly in doses of 25,000 to 30,000 units/kg per day, although these injections are very painful. Doses of polymyxin B as high as 45,000 units/kg per day have been used in limited clinical studies to treat neonates for Pseudomonas aeruginosa sepsis. Polymyxin B is the treatment of choice for P. aeruginosa meningitis, and is preferably administered intrathecally to adults and older children in doses of 50,000 units once daily for 3 to 4 days, followed by 50,000 units every other day; in children under two years old, it is administered intrathecally in doses of 20,000 daily for 3 to 4 days, followed by 25,000 units every other day.
Chloramphenicol is preferably administered intravenously to adults in doses of 50 mg/kg per day, in 4 equally divided doses; in exceptional cases, it can be administered in doses up to 100 mg/kg per day. In children, chloramphenicol is preferably administered intravenously in doses of 25 mg/kg per day, although up to 100 mg/kg per day can be administered in cases of severe infection.
Clindamycin is preferably administered parenterally to adults in doses ranging from 600 mg to 4.8 grams per day, given in 2, 3 or 4 equally divided doses. It is recommended that the dose in each intramuscular injection not exceed 600 mg. For children, clindamycin is preferably administered parenterally in doses of 15-40 mg/kg per day, given in 3 or 4 equally divided doses.
Dosages of all antimicrobial agents should be adjusted in patients with renal impairment or hepatic insufficiency, due to the reduced metabolism and/or excretion of the drugs in patients with these conditions. Doses in children should also be reduced, generally according to body weight. Those skilled in the art can readily optimize effective dosages and administration regimens for the BPI protein product and the antibiotics in concurrent administration.
Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples. Example 1 addresses the bactericidal effect of gentamicin and BPI when administered to two different strains of E. coli. Example 2 addresses the bactericidal effect of gentamicin and BPI for a number of other gram-negative strains. Example 3 addresses the bactericidal effect of polymyxin B and BPI when administered to two different strains of E. coli. Example 4 addresses the bactericidal effect of polymyxin B and BPI for a number of other gram-negative strains. Example 5 addresses the effect of a BPI protein product with a cephalosporin antibiotic in an in vivo mouse peritonitis E. coli O111:B4 challenge model. Example 6 also relates to the effect of a BPI protein product with a cephalosporin antibiotic in an in vivo mouse peritonitis E. coli O111: B4 challenge model. Example 7 relates to the effect of a BPI protein product with a cephalosporin antibiotic in an in vivo mouse peritonitis E. coli O7:K1 challenge model. 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 of a BPI protein product with an aminoglycoside antibiotic in an in vivo mouse peritonitis E. coli O7:K1 challenge model. Example 10 relates to the effect of a BPI protein product in vitro on the antibiotic susceptibility of ceftriaxone-resistant gram-negative organisms. Examples 11-19 address large-scale screening of the antibiotic susceptibility-increasing effect of a BPI protein product on a variety of gram-negative organisms: Pseudomonas aeruginosa and other Pseudomonas species (Example 11), E. coli (Example 12), Citrobacter (Example 13), Klebsiella (Example 14), Enterobacter (Example 15), Serratia (Example 16), Proteus (Example 17), Providencia (Example 18), Morganella (Example 19), Acinetobacter (Example 20), and Salmonella and Shigella (Example 21). Example 22 examines the early in vitro bactericidal effect of BPI protein product and selected antibiotics on E. coli J5, E. coli O7:K1, Enterobacter cloacae and Klebsiella pneumoniae. Example 23 examines the effect of a variety of BPI protein products on several representative organisms, Acinetobacter anitratus, Enterobacter cloacae, and two strains of E. coli. Example 24 relates to the screening of BPI peptides for antibacterial activity against E. coli. Example 25 addresses the effect on E. coli O111:B4 of concurrent administration of BPI protein product with tetracycline or gentamicin.
EXAMPLE 1
SYNERGISTIC BACTERICIDAL EFFECTS OF GENTAMICIN AND BPI: ADMENISTION TO E. COLI AND E. COLI 0111:B4 GRAM-NEGATIVE ORGANISMS
In this example, a micro dilution plate minimum inhibitory concentration (MIC) assay was conducted to determine the sensitivity of E. coli organisms to the bactericidal effects of BPI protein products concurrently administered with the antibiotic gentamicin. The assays were conducted against the BPI sensitive organism E. coli J5 (an Rc rough mutant of E. coli 0111:B4), and a BPI resistant organism E. coli 0111:B4.
Specifically, organisms were grown overnight on blood agar plates at 37.degree. C. in air, single colonies were then sub-cultured in 100 mL of nutrient broth No. 2 (Oxoid CM67) and incubated with gentle agitation on an orbital shaker for 5 1/4 hours until in log phase. Fifty mL of the bacterial suspension was then spun down in a DenIcy BR401 bench centrifuge at 4000 rpm for 15 minutes and the pellet was resuspended and washed twice using sterile normal saline. Bacteria were then resuspended in saline such that a 1:10 dilution had an optical density of 0.9 (+/-0.01) at 325 nm (corresponding to approximately 4.times.10.sup.9 cells per mL) and diluted to give a final concentration of 4.times.10.sup.6 cells per mL in "BPI media" (described below).
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 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.sub.23 and gentamicin (Sigma G-1264, Lot: 91H00325) were diluted in BPI media such that 100 .mu.L of diluted BH, 50 .mu.L of diluted gentamicin and 50 .mu.L of bacterial suspension in the final volume of 200 .mu.L per well, gave concentrations in serial dilutions from 1000 nM (25 .mu.g per mL) BPI and 32 .mu.g per mL of gentamicin with a fixed concentration of 10.sup.6 cells/mL. Checkerboards were then constructed in round boUomed 96 well microtitre plates (Greiner No. 650180) and incubated with non-sealing lids at 37.degree. 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 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 .mu.L in 990 .mu.L of Nutrient Broth No. 2 and spread plates were prepared with 25 .mu.L 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. (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 between rBPI.sub.23 and gentamicin using the BPI sensitive E. coli J5 with gentamicin at a concentration of 0.25 .mu.g per mL, reducing the MIC of BPI by approximately eight-fold from 500 nM (12.5 .mu.g per mL) to 62.5 nM (1.56 .mu.g per mL) with similar reductions of the minimum bactericidal concentration (MBC).
EXAMPLE 2
SYNERGISTIC BACTERICIDAL EFFECTS OF GENTAMICIN AND BPI: ADMINISTRATION TO ADDITIONAL GRAM-NEGATIVE ORGANISMS
In this example, micro dilution plate MIC assays were conducted according to the method of Example 1 to determine the sensitivity of a variety of gram-negative organisms to the cytotoxic effects of BPI protein products concurrently 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 synergistic bactericidal interactions (indicated as "additive (+)") were observed against E. cloacae (10005), E. gergoviae (11434), P. aeruginosa (10332) and P. aeruginosa (10662). The concurrent administration of gentamicin and rBPI.sub.23 had only additive, indifferent or indeterminate effects against the other tested gram-negative bacteria.
EXAMPLE 3
SYNERGISTIC BACTERICIDAL EFFECTS OF POLYMYXIN B AND BPI: ADMINISTATION TO E. COLI J5 AND E. COLI 0111:B4 GRAM-NEGATIVE ORGANISMS
In this example, micro dilution plate MIC assays were conducted according to the method of Example 1 to determine the sensitivity of E. coli J5 and E. coli 0111:B4 to the cytotoxic effects of BPI protein products concurrently administered with the antibiotic polymyxin B. Polymyxin B 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 .mu.g in 12.54 mL.
A positive synergistic interaction was demonstrated between rBPI.sub.23 and polymyxin B using 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.
EXAMPLE 4
SYNERGISTIC BACTERICIDAL EFFECTS OF POLYMYXIN B AND BPI: ADMINISTRATION TO ADDITIONAL GRAM-NEGATIVE ORGANISMS
In this example, micro dilution MIC assays were conducted according to the method of Example 1 to determine the sensitivity of a variety of gram-negative organisms to the cytotoxic effects of BPI protein products concurrently administered with polymyxin B antibiotic. The results of those assays are shown in Table 10 below.
These assay results show additive or synergistic effects with the use of polymyxin B at concentrations of 0.3 .mu.g/mL, a level of which is 10-20 times lower than when that antibiotic is used conventionally.
EXAMPLE 5
SYNERGISTIC EFFECTS OF CEFAMANDOLE AND BPI PROTEIN PRODUCT IN VIVO IN MICE CHALLENGED INTRAPERITONEALLY WITH LIVE E. COLI 0111:B4 BACTERIA: EFFECT ON SURVIVAL
In this example, the protective effect of cefamandole nafate antibiotic (MANDOL.RTM., Lilly) a semisynthetic broad-spectrum cephalosporin antibiotic with and without a BPI protein product was evaluated by means of challenging ICR mice with an LD.sub.90-100 dose level of live E. coli 0111:B4 bacteria, a strain that is not susceptible to the bactericidal/growth inhibitory effects of BPI protein product. Specifically, four groups of 15 ICR mice were treated such that each ICR mouse received an injection of bacteria (1.8.times.10.sup.9 CFU/mouse) intraperitoneally; an intraperitoneal injection of cefamandole nafate (MANDOL.RTM., 100 mg/kg) or saline; and then an intrapefitoneal injection of rBPI.sub.21 (500 .mu.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.
Concurrent administration of BPI protein product with cefamandole, or administration of cefamandole alone, showed significant protection in mice challenged with E. coli 0111:B4 when compared to the buffer control (p.ltoreq.0.001 and p.ltoreq.0.05 respectively). The protective effect of the BPI protein product when administered without cefamandole was not evident. When compared to treatment with cefamandole alone, concurrent administration of BPI protein product with cefamandole showed improved protection at p.ltoreq.0.1. These results indicate that the concurrent administration of cefamandole and a BPI protein product has synergistic therapeutic effects against E. coli 0111:B4.
EXAMPLE 6
SYNERGISTIC EFFECTS OF CEFAMANDOLE AND BPI PROTEIN PRODUCT IN VIVO IN MICE CHALLENGED INTRAPERITONEALLY WITH LIVE E. COLI 0111:B4 BACTERIA: EFFECT ON SURVIVAL AND ON BACTERIAL CLEARANCE FROM BLOOD AND PERITONEAL LAVAGE FLUID
The protective effects of a cephalosporin antibiotic and a BPI protein product were evaluated in mice challenged intrapefitoneally with E. coli 0111:B4, a strain that is resistant to the bactericidal effects of BPI protein product. The assay was conducted using the following procedure. Male 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. Mice received an intraperitoneal injection of 0.5 ml of bacteria in doses near an LD.sub.90 (2.times.10.sup.9 CFU/mouse). Immediately after bacterial challenge the animals received an intraperitoneal injection of (1) vehicle only, (2) 500 .mu.g/mouse rBPI.sub.21 and vehicle, (3) 100 mg/kg cefamandole nafate (Mandol.RTM. in phosphate buffered saline; Eli Lilly, Indianapolis, Ind.) and vehicle, or (4) 500 .mu.g rBPI.sub.21 and 100 mg/kg cefamandole.
Survival of the four groups was monitored for 7 days. Survival data was statistically analyzed using the Chi-square test. In a separate experiment using a bacterial challenge of 2.5.times.10.sup.9 CFU E. coli O 111:B4, blood and peritoneal lavage fluid were collected for culture at different time points following bacterial challenge. Blood was obtained from the retro-orbital sinus. At least 1 ml of peritoneal lavage fluid was obtained after intraperitoneal injection of 3 ml of phosphate buffered saline. Bacterial counts (expressed as CFU/ml) were determined by inoculating trypticase soy agar plates with 10-fold dilutions of blood or peritoneal lavage samples, incubating the plates overnight at 37.degree. C., and counting the colonies. Statistical comparisons of this data were performed with the analysis of variance.
FIG. 2 shows effects on survival of cefamandole (100 mg/kg), rBPI.sub.21 (500 .mu.g) or the concurrent administration of the two agents. The BPI protein product alone had no effect, while cefamandole treatment alone increased survival, although not significantly. In contrast, the concurrent administration of rBPI.sub.21 with cefamandole resulted in a significant increase in survival (p<0.05) above that achieved by either treatment alone. FIGS. 3 and 4 show that rBPI.sub.21 alone failed to reduce counts in either peritoneal lavage fluid or blood after challenge with bacteria, while cefamandole treatment alone significantly reduced counts in both (p<0.01 vs. vehicle). However, the concurrent administration of rBPI.sub.21 and cefamandole reduced bacterial counts in the peritoneal lavage fluid by more than two orders of magnitude below that achieved by cefamandole alone (p<0.01 of concurrent administration vs. cefamandole only) at the 2 and 6 hour time points, and entirely eliminated counts after 24 hours. Blood of mice that received concurrent administration of rBPI.sub.21 and cefamandole was completely free of bacteria at all time points.
Thus, the concurrent administration of a BPI protein product, rBPI.sub.21, with a suboptimal dose of a cephalosporin antibiotic, cefamandole, resulted in a superior therapeutic effect. The data indicates that BPI protein products and cephalosporin antibiotics produce a synergistic therapeutic effect. Since cefamandole alone reduced counts by approximately two orders of magnitude compared to vehicle-treated animals, another experiment was conducted to determine if rBPI.sub.21 alone reduced bacterial counts when the inoculum was reduced to 10.sup.7 CFU. Data from this experiment showed that rBPI.sub.21 (500 .mu.g) did not significantly reduce bacterial counts in blood or peritoneal lavage fluid after a challenge of 10.sup.7 CFU. This suggests that an antibiotic-mediated reduction in the magnitude of the bacterial count cannot, in itself, explain the protection associated with concurrent administration of BPI protein product and antibiotic.
EXAMPLE 7
SYNERGISTIC EFFECTS OF CEFAMANDOLE AND BPI PROTEIN PRODUCT IN VIVO IN MICE CHALLENGED INTRAPERITONEALLY WITH LIVE E. COLI O7:K1 BACTERIA: EFFECT ON SURVIVAL
The protective effects of a cephalosporin antibiotic and a BPI protein product were evaluated in mice challenged intraperitoneally with E. coli O7:K1 (ATCC Accession No. 23503), a strain that is susceptible to the bactericidal effects of BPI protein product. The general procedure described above in Example 5 was followed. Four groups of 20 mice were challenged intraperitoneally with 2.times.10.sup.7 E. coli O7:K1 bacteria and then treated with (1) vehicle, (2) 50 .mu.g rBPI.sub.21 only, (3) 20 mg/kg cefamandole only, or (4) both 50 .mu.g rBPI.sub.21 and 20 mg/kg cefamandole. Survival of the mice was followed over a period of 7 days; results of two trials are displayed in FIGS. 5A and 5B.
In one trial, rBPI.sub.21 alone protected 11% of the survivors compared to vehicle controls. cefamandole protected 47 % of the survivors compared to vehicle controls (p<0.05 vs. vehicle), and the concurrent administration of rBPI.sub.21 and cefamandole protected 100% of the survivors compared to vehicle controls (p<0.00 1 vs. vehicle, p<0.01 vs. cefamandole alone). In the second trial, rBPI.sub.21 alone protected 0% of the survivors compared to vehicle, cefamandole protected 12% compared to vehicle, and the concurrent administration of rBPI.sub.21 and cefamandole protected 59% compared to vehicle (p<0.01 vs. vehicle, p<0.05 vs. cefamandole alone). In both trials, the increase in survival associated with the concurrent administration of rBPI.sub.21 and cefamandole was greater than the sum of the increases in survival due to the individual therapies. Thus, there appears to be synergy (a greater than additive effect) between cefamandole and BPI protein product in this model.
EXAMPLE 8
SYNERGISTIC EFFECTS OF CEFAMANDOLE AND BPI PROTEIN PRODUCT IN VIVO IN RABBITS CHALLENGED INTRAVENOUSLY WITH LIVE E. COLI O7:K1 BACTERIA: EFFECT ON BACTERIAL CLEARANCE AND ON CARDIOVASCULAR, RESPIRATORY AND METABOLIC PARAMETERS
Adult male New Zealand White rabbits (Charles River Laboratories, St. Constant, Canada) weighing between 1.8 and 2.3 kilograms were fasted for 24 hours before the experiment. Each rabbit was anesthetized with an intramuscular injection of 80/4 mg/kg Ketamine/xylazine. The left femoral artery was catheterized for blood pressure determinations and blood sample collection. A catheter was placed adjacent the right atrium via the right jugular vein and a thermistor-tipped catheter was placed in the aortic arch via the right carotid artery. The rabbits were allowed to stabilize for 90-120 minutes following catheterization to normalize hemodynamic and blood gas parameters.
The rabbits were divided into four treatment groups with 4 animals per group: (1) vehicle alone, (2) cefamandole and vehicle, (3) rBPI.sub.21 and vehicle, and (4) cefamandole and rBPI.sub.21. The rabbits were administered cefamandole (Mandol.RTM.; Eli Lilly, Indianapolis, Ind.) or vehicle intravenously 5 minutes before the start of the bacterial infusion (considered to be T=0). At T=0, 2.times.10.sup.10 CFU/rabbit of E. coli O7:K1 was infused intravenously over 10 minutes into the ear vein. Simultaneously (at T=0), 10 mg/kg rBPI.sub.21 or vehicle was infused over 10 minutes via the right jugular catheter. After the ten minute infusion, rBPI.sub.21 was slowly infused at 10 mg/kg/hr for 2 hours (resulting in a total dose of 30 mg/kg rBPI.sub.21).
Arterial blood samples for determination of bacterial counts and endotoxin levels were collected at the end of the 10 minute bacterial infusion and at 30 minutes, 1, 2, 3 and 4 hours. The whole blood was 10-fold serially diluted in sterile PBS and aliquots were plated onto tryptic soy agar plates, incubated at 37.degree. C. overnight, and the plates were counted for colony forming units (CFU). The results were expressed as CFU/ml blood and percent bacteria dose per ml blood. The remaining portion of the blood was centrifuged, the plasma removed and passed through a 0.2 micron Whatman syringe filter to remove the bacteria. The endotoxin levels were determined using a modified limulus amoebocyte lysate assay (Pyrochrome LAL Assay, Associates of Cape Cod, Woods Hole, Mass.). These results were expressed as ng LPS per ml plasma.
Cardiovascular, respiratory and metabolic parameters were measured every 30 minutes. Mean arterial blood pressure (MABP) and heart rate were monitored continuously throughout the experiments and displayed on a cardiac output computer (Columbus Instruments Cardiomax II) or on a chart recorder. Heart rate was derived from the arterial pressure wave. Cardiac output was determined in duplicate with the thermodilution technique: Changes in blood temperature resulting from injection of 900 .mu.l of room temperature PBS were recorded with the thermistor-tipped catheter in the aortic arch. The cardiac output computer then generated thermodilution curves that were visualized on the chart recorder, and derived cardiac output from the temperature-time curves. Cardiac index (CI) was then calculated as cardiac output per kg body weight. In addition, total peripheral resistance (TPR) was determined by dividing blood pressure by cardiac output.
Blood samples for blood gas determinations were drawn from the femoral artery catheter every 30 minutes throughout the study. Blood gases were measured with a Ciba-Coming Blood Gas System, Model 278 (Ciba-Corning Diagnostics Corp., Medfield, Mass.). The blood gas system directly measures blood pH, partial pressure of pCO.sub.2, and partial pressure of pO.sub.2. Other parameters including the alveolar-arterial oxygen gradient, arterial oxygen content, estimated oxygen saturation, standard bicarbonate, and in vivo base excess were calculated using the formulas provided by Ciba-Coming Diagnostic Corp. Plasma levels of glucose and lactate were determined using a Glucose/L-lactate Analyzer (2300 STAT, YSI, Yellow Springs, Ohio).
Survival data is shown in FIG. 6. Two of the four animals (50%) treated with vehicle alone died before the end of the experiment. None of the animals concurrently treated with rBPI.sub.21 and cefamandole died. Bacterial counts in blood, expressed as CFU/ml or percent bacteria dose/ml, are shown in FIGS. 7 and 8, respectively. In FIG. 7, the squares represent treatment with vehicle alone, the diamonds represent cefamandole alone, the circles represent rBPI.sub.21 alone, and the triangles represent the concurrent administration of rBPI.sub.21 and cefamandole. In FIG. 8, the bar with horizontal hatching indicates treatment with cefamandole alone, the hollow bar indicates the concurrent administration of rBPI.sub.21 and cefamandole, the bar with vertical hatching indicates rBPI.sub.21 alone, and the solid bar indicates buffer alone. The group concurrently treated with rBPI.sub.21 and cefamandole demonstrated a higher clearance of bacteria compared to the groups treated with either rBPI.sub.21 alone or cefamandole alone. There appears to be a synergistic effect at 30 and 60 minutes; at 30 minutes, the concurrent administration of rBPI.sub.21 and cefamandole resulted in a higher percentage clearance of bacteria than the sum of the separate treatments.
Endotoxin levels are displayed in FIG. 9. The open diamonds indicate treatment with vehicle alone, filled diamonds indicate rBPI.sub.21 alone, the filled squares indicate cefamandole alone, and the open squares indicate concurrent administration of rBPI.sub.21 and cefamandole. Animals administered cefamandole alone have a much higher LPS level than animals treated with vehicle alone, due to release of LPS as cefamandole kills bacteria. The concurrent administration of rBPI.sub.21 and cefamandole produced a dramatic decrease in LPS levels compared to cefamandole therapy alone.
Cardiovascular/pulmonary parameters (MABP, CI, TPR, arterial oxygen tension. alveolar-arterial O.sub.2 gradient, respiration rate, and arterial blood pH) are shown in FIGS. 10-16, respectively. These figures only display results for the groups treated with cefamandole alone (indicated by open squares) and concurrent treatment with rBPI.sub.21 and cefamandole (indicated by filled squares). Single stars indicate that the concurrent administration of both agents provided statistically significant (p<0.05) improvement over antibiotic alone, while two stars indicate p<0.01. Cefamandole alone or rBPI.sub.21 alone failed to protect the animals; cardiovascular and respiratory dysfunction began du-ring the bacterial infusion and the animals were in circulatory shock by the end of the infusion. Cardiovascular shock lasted for the rest of the experiment. Arterial blood pH began to decrease at 60 minutes in the group treated with cefamandole alone, and was at its lowest level by the end of the experiment. In contrast, concurrent administration of rBPI.sub.21 and cefamandole preserved cardiopulmonary function and prevented septic shock. Thus, the concurrent administration of BPI protein product with antibiotic protected the animals against the lethal effect of bacteremia and preserved cardiopulmonary function when antibiotic alone failed to do so.
EXAMPLE 9
SYNERGISTIC EFFECTS OF GENTAMICIN AND BPI PROTEIN PRODUCT IN VIVO IN MICE CHALLENGED INTRAPERITONEALLY WITH LIVE E. COLI O7:K1 BACTERIA: EFFECT ON SURVIVAL
The synergistic effects of an aminoglycoside antibiotic and a BPI protein product were evaluated in mice challenged intraperitoneally with E. coli O7:K1 (ATCC Accession No. 23503), a smooth encapsulated strain that is susceptible to the bactericidal effects of BPI protein product. The general procedure described above in Example 4 was followed. Six groups of 20 mice were challenged intraperitoneally with 2.times.10.sup.7 E. coli O7:K1 bacteria and treated immediately with (1) vehicle, (2) 0.03 mg/kg gentamicin, (3) 0.1 mg/kg of gentamicin, (4) 50 .mu.g of rBPI.sub.21, (5) 0.03 mg/kg gentamicin followed by 50 .mu.g of rBPI.sub.21, or (6) 0.1 mg/kg of gentamicin followed by 50 .mu.g of rBPI.sub.21. Survival was followed over 7 days. Results are shown in FIG. 17. Neither antibiotic alone nor rBPI.sub.21 alone had any effect on mortality other than to slightly retard the death rate. However, the concurrent administration of rBPI.sub.21 with low-dose gentamicin significantly increased survival (p<0.5 vs. vehicle). Concurrent administration of rBPI.sub.21 with high-dose gentamicin dramatically increased survival (p<0.001 vs. vehicle, and p<0.05 vs. 0.03 mg/kg gentamicin with rBPI.sub.21) protecting all but two of the mice from the lethal effects of bacterial challenge. The results clearly indicate synergism between gentamicin and BPI protein product. The greater synergistic effect of the concurrent administration of BPI protein product with gentamicin, compared to the effect of the concurrent administration of BPI protein product with cefamandole, may be related to the fact that aminoglycosides, which inhibit protein synthesis, have a different mechanism of action than BPI protein product.
EXAMPLE 10
EFFECT OF BPI PROTEIN PRODUCT IN VITRO ON ANTIBIOTIC SUSCEFHBILITY OF CEFTRIAXONE-RESISTANT GRAM-NEGATIVE ORGANISMS
The ability of BPI protein product, BPI.sub.21, to reverse the resistance of a variety of gram-negative organisms to ceftriaxone (Roche Laboratories) was evaluated in vitro.
The strain of gram-negative bacteria to be tested was grown overnight at 37.degree. C. on Trypticase soy agar (TSA) plates. Colonies from the plate were then inoculated into nutrient broth (or triethanolamine-buffered minimal salts medium), grown overnight to stationary growth phase, diluted 1:10 in fresh medium, and grown to mid-late logarithmic growth phase (3 to 4 hours at 3.degree. C.) to an approximate concentration of 6 to 10.times.10.sup.8 organisms/mL. Organism counts were performed by making serial dilutions, plating in triplicate on TSA plates, incubating at 37.degree. C. oventight, and counting colonies by visual inspection. Following development of a standard curve, counts were made by measuring OD.sub.540 and confirming by plating. The bacteria were sedimented by centrifugation at 6,000 g for 10 min. and resuspended in sterile saline to the desired concentration.
Ceftriaxone solutions were prepared from standard powder (Roche Laboratories). Solutions of BPI protein product were prepared from rBPI.sub.21, Hanks solution, vitamin-free casamino acid, and TRIS-HCl buffer, pH 7.0. Broth MIC studies were performed as follows: U-bottom, disposable, microtiter array plates (Dynatech) containing 100 .mu.L/well of broth and serial dilutions of 1:1 proportion rBPI.sub.21 and ceftriaxone were prepared using a multichannel pipetting instrument, inoculated with organisms diluted to approximately 2.5.times.10.sup.5 CFU/ml, and incubated overnight at 37.degree. C. Media controls containing no organism and growth controls containing organisms but no ceftriaxone or rBPI.sub.21 were also prepared. The MIC was determined as the lowest drag concentration (.mu.g/mL) that inhibited bacterial growth. Agar MIC studies were performed as follows: The organism was grown in Mueller-Hinton broth (Difco Laboratories) overnight at 37.degree. C., transferred and grown to logarithmic phase, counted by optical densitometry, diluted with buffer and rBPI.sub.21 and ceftriaxone, and incubated for 30 minutes at 37.degree. C. After the 30 minutes of incubation, samples were serially diluted in sterile saline and plated onto TSA agar for bacterial counts after overnight incubation at 37.degree. C.
A variety of different bacterial species were tested, including Pseudomonas, Enterobacter, Citrobacter, Klebsiella, and Escherichia species. A summary of the results is displayed in Table 11 below, which reports the minimum inhibitory concentration (MIC) of BPI protein product alone, the MIC of ceftriaxone alone, and the MIC of both agents together (in a fixed 1:1 proportion of rBPI.sub.21 and ceftriaxone). The experiments were replicated, and each number given in the table represents the highest, or worst-case, MIC for the group.
Checkerboard synergy studies on selected strains were performed as follows. Microplates were prepared using the foBowing organization: Column 1, control organisms (growth control); Columns 2 through 9, serial dilutions of cefiriaxone; Column 10, cefiriaxone alone; Column 11, rBPI.sub.21 alone; Column 12, control media; Rows 1 through 8, serial dilutions of rBPI.sub.21. Columns 2 through 9 thus contained a serial array of various proportions of ceftriaxone and BPI concentrations. All webs, except the media control, were inoculated with suspended organisms and incubated oventight at 37.degree. C. Turbidity was recorded at 24 and 48 hours.
Results of a representative synergy study of the effects of the concurrent administration of rBPI.sub.21 and cefiriaxone on cefiriaxone-resistant E. coli are shown in the checkerboard in Table 12 below. In this checkerboard assay, rBPI.sub.21 and cefiriaxone were each serially diluted to concentrations ranging from 100 .mu.g/ml to 0.8 .mu.g/ml. These results show that the concurrent administration of both agents is synergistic. There was uniform growth in all growth control wells (containing bacteria, no rBPI.sub.21 and no antibiotic), while there was no growth in the media control wells (containing no bacteria). There was uniform growth in all ceftriaxone control webs (bacteria with ceftriaxone alone). In the BPI control wells (bacteria with rBPI.sub.21 alone) there was growth in the wells with a concentration of 6.2 .mu.g/ml rBPI.sub.21 or less, and no growth at higher concentrations of BPI protein product.
Survival (or kill) curve studies were performed as follows. Tubes containing (1) media alone, (2) media plus ceftriaxone, (3) media plus rBPI.sub.21, and (4) media plus cefiriaxone and rBPI.sub.21, were prepared, inoculated with the desired organism, and incubated as described above. At 0, 1, 2, 4, 8 and 24 hours, diluted aliquots from each tube were plated, incubated and counted as described above. Growth curves over 24 hours (plots of log.sub.10 CFU/mL versus time) were constructed to demonstrate the dynamics of antibiotic-BPI protein product interactions on bacterial growth and survival.
Results of a representative killing curve study of the effects of the concurrent administration of rBPI.sub.21 and ceftriaxone on ceftriaxone-resistant E. coli are shown in FIG. 18. The filled square is the control (neither rBPI.sub.21 nor cefiriaxone), the open square is rBPI.sub.21 alone, the fried diamond is cefiriaxone alone, and the open diamond is the concurrent administration of cefiriaxone and rBPI.sub.21. Cefiriaxone or rBPI.sub.21 alone have some early bactericidal effect at 4-8 hours, but organism growth for both almost reaches that of the control curve by 24 hours (about a 1 log difference from control). In contrast, the concurrent administration of rBPI.sub.21 and cefiriaxone produces a significantly greater bactericidal effect that is sustained at 24 hours (a more than 6 log difference from control).
EXAMPLE 11
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON PSEUDOMONAS SPECIES
The effects of a BPI protein product, rBPI.sub.21, on the antibiotic susceptibility of clinical isolates of Pseudornonas aeruginosa and other Pseudornonas species (from Baxter Microscan.RTM. library, Sacramento, Calif.) was evaluated using Microscan.RTM. panel plates (Baxter Diagnostics, Inc., Deerfield, Ill.) that allow simultaneous determination of minimum inhibitory concentrations for a number of different antibiotics. Control assays confirmed that the formulation buffer for rBPI.sub.21 had no effect on the antibiotic susceptibility of various organisms.
The antimicrobial susceptibility tests performed on the Microscan.RTM. panel plates are miniaturizations of the broth dilution susceptibility test. Antimicrobial agents are serially diluted in Mueller-Hinton broth (supplemented with calcium and magnesium, or with sodium chloride for oxacillin, or with thymidine phosphorylase for trimethoprim, sulfamethoxazole and trimethoprim/sulfamethoxazole) to concentrations bridging the range of clinical interest. One well on the 96-well Microscan.RTM. plate is a growth control well that contains dehydrated broth only. The remaining wells contain dehydrated broth and antibiotic (or broth and biochemical reagent indicator), which is rehydrated to the desired concentration by inoculation of a standardized suspension of test organism. The chromogenic biochemical agent indicators are used to identify and characterize the species of bacteria based on detection of pH changes and substrate utilization. After incubation overnight, the minimum inhibitory concentration (MIC) of an antibiotic for the test organism is determined by observing the well with the lowest concentration of the antibiotic that shows inhibition of growth. Gram-negative organisms were tested using Neg Combo Type 16, MIC Plus Type 2, or Neg Breakpoint Combo Type 9 panel plates (Microscan.RTM., Baxter Diagnostics, Inc., Deerfield, Ill.). The concentrations of antibiotics tested in these panel plates are shown below in Tables 13, 14 and 15, respectively. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-derived standards) applicable to the gram-negative organisms tested in each panel plate appear in Tables 13A, 14A and 15A, respectively.
For each experimental run, the following procedure was performed: The organism was streaked onto 5% sheep blood agar plates (Remel, Lenexa, Kansas) and incubated for 18-24 hours overnight. Well-isolated colonies from the plates were emulsified in 3 ml of sterile Inoculum Water (catalog no. B1015-2, MicroScan.RTM. system, Baxter Diagnostics, Inc., Deerfield, Ill.) to a final turbidity equivalent to 0.5 McFarland Barium Sulfate standard. This cell suspension was vortexed for 2 to 3 seconds and 100 .mu.l was transferred to glass tubes containing 25 ml of Inoculum Water with Pluronic-D (catalog no. B1015-7, MicroScan.RTM. system, Baxter Diagnostics, Inc., Deerfield, Ill.) (hereinafter "Pluronic Inoculum Water"), or 25 ml of Pluronic Inoculum Water into which rBPI.sub.21 (in formulation buffer) had been diluted to the desired concentration between 0.5 to 64 .mu.g/ml rBPI.sub.21.
The 25 ml of this inoculum containing rBPI.sub.21 was mixed by inversion and poured into a tray. The inoculum was drawn up into a manual 96-well pipetting system (RENOK.TM. rehydrator-inoculator system, Baxter Health Care Corporation, West Sacramento, Calif.) designed for use with the Microscan.RTM. panel plates, and 110 .mu.l of the inoculum was delivered to each well of a Microscan.RTM. Neg Combo Type 16 panel plate. When added to the wells, this inoculum achieves a final bacterial concentration of 4.times.10.sup.5 to 7.times.10.sup.5 CFU/ml. The panel plates were then incubated at 35.degree. C. for 15-24 hours and read visually for cell growth.
No growth was defined as a slight whiteness in the well or a clear broth. Growth appeared as turbidity which could take the form of a white haze throughout the well, a white button in the center of the well, or a fine granule growth throughout the well. All wells were read against a black indirectly lighted background. Visual results of the biochemical reactions were read into a database for bacterial identification. The MICs for each antibiotic tested were determined by identifying the lowest concentration of antibiotic which inhibited visible growth.
The clinical isolates of Pseudomonas aeruginosa and other Pseudomonas species were tested using the Neg Combo Type 16 panel plate. Tables 16, 17 and 18 below display a summary of the results of the antibiotic screening panels, reported for each strain tested as the MIC of the tested antibiotics at the various concentrations of rBPI.sub.21 indicated. Results are reported for each strain tested, but susceptibility data is listed for only those antibiotics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-derived standards) applicable to the organism tested appear in Table 13A. Stars after the antibiotic name in the "antibiotic tested" column indicate whether rBPI.sub.21 reversed the resistance of that organism to the antibiotic tested (two stars) or converted an indifferent MIC into a susceptible MIC (one star). These data show that BPI protein product reversed resistance to ticarcillin, cefazolin, cefoxitin, cefuroxime, ofloxacin, aztreonam, piperacillin, and amikacin for some strains of P. aeruginosa and increased the susceptibility of some P. aeruginosa strains to ticarcillin, aztreonam, imipenem, piperacillin, ofloxacin, ceftazidime, amikacin, ceftriaxone, cefotaxime, cefuroxime, tobramycin, ciprofloxacin, trimethoprim/sulfamethoxazole, gentamicin, and cefazolin. BPI protein product reversed resistance of some P. cepacia strains to cefazolin, cefoxitin, cefuroxime, ceftriaxone, ticarcillin, and increased the susceptibility of some P. cepacia strains to ampicillin, ticarcillin, piperacillin, cefazolin, cefoxitin, cefuroxime, ceftriaxone, ampicillin/sulbactam, cefotaxime, gentamicin, tobramycin, and amikacin. BPI protein product reversed resistance of Xanthamonas maltophilia to trimethoprim/sulfamethoxazole, piperacillin and amikacin, and increased susceptibility to ciprofloxacin.
Tables 16, 17 and 18 also show the presence or absence of bacterial growth in the growth control wells, which contained varying concentrations of rBPI.sub.21 alone without antibiotic. "G" indicates growth, while "NG" indicates no growth. These results indicate that rBPI.sub.21 at a concentration of 32 .mu.g/ml has direct bactericidal/growth inhibitory effects on some of the tested Pseudomonas isolates.
EXAMPLE 12
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON STRAINS OF E. COLI
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic susceptibility of various strains of E. coli was evaluated in the Microscan.RTM. antibiotic susceptibility screening assay of Example 11 using the Neg Combo Type 16 panel plate. The direct growth inhibitory effect of rBPI.sub.21 on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of E. coli (from Baxter Microscan.RTM. library, Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 19 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibiotics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-derived standards) applicable to the organism tested appear in Table 13A. These results show that BPI protein product reversed the resistance of some strains to cefazolin and increased the susceptibility of other strains to ampicillin, cefuroxime, cefazolin, amikacin, and cefoxitin.
EXAMPLE 13
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON CITROBACTER SPECIES
The effect of a BPI protein product, rBPI.sub.2, on the antibiotic susceptibility of various Citrobacter species was evaluated in the Microscan.RTM. antibiotic susceptibility screening assay of Example 11 using the Neg Combo Type 16 panel plate. The direct growth inhibitory effect of rBPI.sub.21 on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of Citrobacter species (from Baxter Microscan.RTM. library, Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 20 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibiotics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-derived standards) applicable to the organism tested appear in Table 13A. These results show that BPI protein product increased the susceptibility of the tested Citrobacter species to aztreonam, cefotaxime, tobramycin, amikacin, cefuroxime, ampicillin, ticarcillin, pipemcillin, and cefuroxime.
EXAMPLE 14
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON KLEBSIELLA SPECIES
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic susceptibility of various Klebsiella species was evaluated in the Microscan.RTM. antibiotic susceptibility screening assay of Example 11 using the Neg Combo Type 16 panel plate. The direct growth inhibitory effect of rBPI.sub.21 on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of Klebsiella species (from Baxter Microscan.RTM. library, Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 21 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibiotics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-derived standards) applicable to the organism tested appear in Table 13A. These results show that BPI protein product reversed the resistance of one strain of K. pneumoniae to trimethoprim/suffamethoxazole and increased the susceptibility of the tested species to cefoxitin, ampicillin/sulbactam, trimethoprim/sulfamethoxazole, cefazolin, and cefuroxime.
EXAMPLE 15
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON ENTEROBACTER SPECIES
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic susceptibility of various Enterobacter species was evaluated in the Microscan.RTM. antibiotic susceptibility screening assay of Example 11 using the Neg Combo Type 16 panel plate. The direct growth inhibitory effect of rBPI.sub.21 on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of Enterobacter species (from Baxter Microscan.RTM. library, Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 22 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibiotics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-derived standards) applicable to the organism tested appear in Table 13A. These results show that BPI protein product reversed the resistance of one strain of E. cloacae to ticarcillin, cefuroxime, ceftazidime and cefotaxime. BPI protein product also increased the susceptibility of some Enterobacter species to ticarcillin, aztreonam, piperacillin, ciprofloxacin, cefotaxime, trimethoprim/sulfamethoxazole, cefuroxime, ceftazidime, ceftriaxone, and ampicillin/sulbactam.
EXAMPLE 16
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON SERRATIA MARCESCENS
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic susceptibility of Serratia marcescens was evaluated in the Microscan.RTM. antibiotic susceptibility screening assay of Example 11 using the Neg Combo Type 16 panel plate. The direct growth inhibitory effect of rBPI.sub.21 on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of Serratia marcescens (from Baxter Microscan.RTM. library, Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 23 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibiotics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-defived standards) applicable to the organism tested appear in Table 13A. These results show that BPI protein product reversed the resistance of some strains to ceftazidime and cefotaxime, and increased the susceptibility of other strains to piperacillin, cefoxitin, ceftazidime, ceftfiaxone, cefotaxime, tobramycin, ampicillin/sulbactam, and ampicillin.
EXAMPLE 17
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON PROTEUS MIRABILlS
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic susceptibility of Proteus mirabilis was evaluated in the Microscan.RTM. antibiotic susceptibility screening assay of Example 11 using the Neg Combo Type 16 panel plate. The direct growth inhibitory effect of rBPI.sub.21 on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of Proteus mirabilis (from Baxter Microscan.RTM. library, Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 24 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibiotics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-derived standards) applicable to the organism tested appear in Table 13A. These results show that BPI protein product increased the susceptibility of some strains to trimethoprim/sulfamethoxazole, cefazolin, cefoxitin, imipenem, tobramycin, and amikacin.
EXAMPLE 18
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON PROVIDENCIA SPECIES
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic susceptibility of various Providencia species was evaluated in the Microscan.RTM. antibiotic susceptibility screening assay of Example 11 using the Neg Combo Type 16 panel plate. The direct growth inhibitory effect of rBPI.sub.21 on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of Providencia species (from Baxter Microscan.RTM. library, Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 25 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibiotics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-defived standards) applicable to the organism tested appear in Table 13A. These results show that BPI protein product reversed the resistance of P. stuartii to cefazolin and cefuroxime, and increased its susceptibility to piperacillin, ceftazidime, ampicillin/sulbactam, imipenem, and amikacin. BPI protein product also increased susceptibility of P. rettgeri to cefoxitin and cefuroxime.
EXAMPLE 19
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON MORGANELlA MORGANII
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic susceptibility of Morganella morganii was evaluated in the Microscan.RTM. antibiotic susceptibility screening assay of Example 11 using the Neg Combo Type 16 panel plate. The direct growth inhibitory effect of rBPI.sub.21 on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of Morganella morganii (from Baxter Microscan.RTM. library, Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 26 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibiotics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-derived standards) applicable to the organism tested appear in Table 13A. These results show that BPI protein product increased the susceptibility of the tested strains to ampicillin/sulbactam, amikacin and piperacillin.
EXAMPLE 20
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON ACINETOBACTER SPECIES
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic susceptibility of various Acinetobacter species was evaluated in the Microscan.RTM. antibiotic susceptibility screening assay of Example 11 using the Neg Combo Type 16 and MIC Plus Type 2 panel plate. The direct growth inhibitory effect of rBPI.sub.21 on these strains was also evaluated in the same assay. Assays were conducted on clinical isolates of Acinetobacter species (from Baxter Microscan.RTM. library, Sacramento, Calif.). Different production lots of rBPI.sub.21 that had been formulated with surfactant or unformulated (without surfactant) were tested, but no difference was seen in results for formulated or unformulated rBPI.sub.22.
A summary of the results of the antibiotic screening panels, reported as MICs (.mu.g/ml) of the antibiotic tested, is shown in Tables 27 and 28 below. Results are reported for each strain tested, but susceptibility data is listed for only those antibiotics for which BPI protein product altered susceptibility. The antibiotic susceptibility standards (interpretation of an MIC as resistant, intermediate or susceptible according to Microscan.RTM.'s NCCLS-derived standards) applicable to the organism tested appear in Tables 13A and 14A. For interpretation purposes, when antibiotic susceptibility standards were given only for Enterobacter or Pseudomonas, the standards for Acinetobacter were considered to be the same as for Enterobacter.
These results show that BPI protein product reversed resistance of A. anitratus strains to amoxicillin/K clavulanate, ampicillin/sulbactam, aztreonam, carbenlcillin, cefamandole, cefazolin, cefonicid, cefoperazone, cefotaxime, cefotetan, cefoxitin, ceftazidime, ceftizoxime, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, mezlocillin, netilmicin, ticarcillin, ticarcillin/K clavulanate, and tfimethopfim/sulfamethoxazole, and increased susceptibility of some A. anitratus strains to amikacin, amoxicillin/K clavulanate, ampicillin. ampicillin/sulbactam, azlocillin, aztreonam, carbenicillin, cefamandole, cefazolin, cefonicid, cefoperazone, cefotaxime, cefotetan, cefoxitin, ceftazidime, cefiizoxime, ceftriaxone, cefuroxime, chloramphenicol, ciprofloxacin, gentamicin, imipenem, mezlocillin, netfirnicin, ofloxacin, piperacillin, ticarcfilin, ticarcillin/K clavulanate, tobramycin and trimethoprim/suffamethoxazole.
BPI protein product reversed resistance of A. lwoffii strains to aztreonam, cefazolin, cefuroxime, ceftazidime, cefoxitin, trimethoprim/suffamethoxazole and piperacillin, and increased susceptibility of A. lwoffii strains to ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, cefotaxime, cefoxitin, ceftazidime, cefiriaxone, cefuroxime, pipemcillin, ticarcillin and trimethoprim/suffamethoxazole.
These results also show that rBPI.sub.21 at a concentration of 16 .mu.g/ml has direct bactericidal/growth inhibitory effects on some of the tested Acinetobacter isolates.
EXAMPLE 21
EFFECTS OF BPI PROTEIN PRODUCT AND ANTIBIOTICS IN VITRO ON SALMONELLA AND SHIGELLA SPECIES
Ten clinical isolates of Salmonella (F270-001 through -010) and 10 clinical isolates of Shigella (F321-010 and F325-002 through -010) (all isolates from Baxter Microscan.RTM. library, Sacramento, Calif.), were evaluated in the Microscan.RTM. antibiotic susceptibility screening assay of Example 11 using the Neg Breakpoint Combo Type 9 panel plate. Essentially no effect was seen at rBPI.sub.21 concentrations of 0, 4 and 16 .mu.g/mL.
EXAMPLE 22
EFFECTS OF BPI PROTEIN PRODUCT ON ANTIBIOTIC KILLING CURVES FOR E. COLI J5, E. COLI O7:K1, ENTEROBACTER CLOACAE, AND KLEBSIELLA PNEUMONIAE
The effect of a BPI protein product, rBPI.sub.21, on the killing curves of selected antibiotics was determined for selected organisms. Microscan.RTM. panel plates were prepared for E. coli J5, E. coli O7:K1, Enterobacter cloacae (Microscan library ID no. 19680) and Klebsiella pneumoniae (Microscan library ID no. 16135), according to Example 11. Cell suspensions were added to 25 ml Pluronic Inoculum Water containing 0 or 16 .mu.g/ml rBPI.sub.21. After inoculation, the panel plates were incubated at 35.degree. C. for 24 hours. At 0, 4, 7 and 24 hours after inoculation, 5 .mu.l samples were removed from each growth control well (containing culture media without antibiotic) and from each well containing: 2/38 .mu.g/ml trimethoprim/sulfamethoxazole, 2 .mu.g/ml ciprofloxacin, 64 .mu.g/ml piperacillin, 32 .mu.g/ml cefotaxime, 6 .mu.g/ml cefuroxime, and 16 .mu.g/ml amikacin. These 5 .mu.l samples were diluted in sterile water and inoculated onto Trypticase Soy agar plates (Remel, Lenexa, Kansas). After 48 hours of incubation at 35.degree. C., the plates were counted and the number of colony forming units of bacteria in the well was calculated.
The results are shown below in FIGS. 19-25. In all of the figures the growth, in the presence of antibiotic but without rBPI.sub.21, is indicated for: E. coli J5 (a filled square): E. coli O7:K1 (a filled diamond); E. cloacae (a filled triangle); and K. pneumoniae (an "X"). Also, in all figures, the growth in the presence of antibiotic with rBPI.sup.21 is indicated for E. coli J5 (an open square); E. coli O7:K1 (an open diamond); E. cloacae (an open triangle); and K. pneumoniae (a star).
FIG. 19 shows the kinetic growth curve of organisms with rBPI.sub.21 (and without antibiotic) and without rBPI.sub.21 (and without antibiotic). In FIG. 19, the growth curves for E. coli J5 without rBPI.sub.21 (filled squares), E. coli O7:K1 with rBPI.sub.21 (open diamonds), E. coli O7:K1 without rBPI.sub.21 (filled diamonds), and K. pneumoraae without rBPI.sub.21 ("X"s) overlap substantially, while the growth curves for E. coli J5 with rBPI.sub.21 (open squares) and E. cloacae with rBPI.sub.21 (open triangles) overlap at 0-7 hours but diverge by 24 hours. FIG. 19 demonstrates that BPI protein product alone has a bactericidal effect at 0-7 hours on E. coli J5 and K. pneumoniae, and a bactericidal effect on E. cloacae throughout the 24 hour period studied.
In FIG. 20, the growth curves for E. coli J5 with rBPI.sub.21 (open squares), E. cloacae with rBPI.sub.21 (open triangles) and K. pneumoniae with rBPI.sub.21 (stars) overlap. FIG. 20 shows that rBPI.sub.21 enhanced the bactericidal effect of trimethoprim/sulfamethoxazole on E. cloacae and K. pneumoniae at 0-24 hours, and slightly enhanced the antibiotics' effect on E. coli J5 and E. coli O7:K1.
In FIG. 21, the growth curves for E. coli J5 with rBPI.sub.21 (open squares), E. coli J5 without rBPI.sub.21 (filled squares), E. coli O7:K1 with rBPI.sub.21 (open diamonds), E. coil O7:K1 without rBPI.sub.21 (filled diamonds), E. cloacae with rBPI.sub.21 (open triangles), E. cloacae without rBH.sub.21 (filled triangles), and K. pneumoniae with rBPI.sub.21 (stars) overlap. FIG. 21 shows that rBPI.sub.21 reversed resistance of K. pneumoniae to ciprofioxacin at 0-24 hours; the other organisms were already very susceptible to ciprofloxacin.
In FIG. 22, the growth curves for E. coli O7:K1 without rBPI.sub.21 (filled diamonds) and E. cloacae without rBPI.sub.21 (filled triangles) overlap, while the growth curves for E. coli J5 with rBPI.sub.21 (open squares), E. coli J5 without rBPI.sub.21 (filled squares), E. coli O7:K1 with rBPI.sub.21 (open diamonds), E. cloacae with rBPI.sub.21 (open triangles), K. pneumoniae without rBPI.sub.21 ("X"s) and K. pneumoniae with rBPI.sub.21 (stars) overlap. FIG. 22 shows that rBPI.sub.21 enhanced the bactericidal effect of piperacillin on E. coli O7:K1 and E. cloacae; the other organisms were already susceptible to the drug.
In FIG. 23, the growth curves for E. coli J5 with rBPI.sub.21 (open squares), E. coli O7:K1 with rBPI.sub.21 (open diamonds), E. coli O7:K1 without rBPI.sub.21 (filled diamonds), K. pneumoniae without rBPI.sub.21 ("X"s) and K. pneumoniae with rBPI.sub.21 (stars) overlap. FIG. 23 shows that rBPI.sub.21 enhanced 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.sub.2 and cefuroxime; addition of rBPI.sub.21 had no effect on the killing curves because all of the tested species were very susceptible to the antibiotic.
In FIG. 25, all of the curves overlap. FIG. 25 shows the effect of rBPI.sub.21 and amikacin; again, all tested species were very susceptible to the antibiotic.
EXAMPLE 23
EFFECTS OF A VARIETY OF BPI PROTEIN PRODUCTS AND ANTIBIOTICS IN VITRO ON REPRESENTATIVE GRAM-NEGATIVE ORGANISMS
The effects of a variety of BPI protein products, rBPI.sub.21, rBPI.sub.23, rBPI.sub.50 and rBPI.sub.42 dimer on the antibiotic susceptibility of various representative gram-negative organisms was evaluated in the Microscan.RTM. 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 Microscan.RTM. library, Sacramento, Calif.), and on E. coli J5-L and O7:K1.
The results, reported as MICs (.mu.g/ml) of the antibiotic tested at the various concentrations of BPI protein product indicated, are shown in Tables 29, 30, 31 and 32 below.
EXAMPLE 24
GRAM-NEGATIVE BACTERICIDAL ACTIVITY OF BPI PEPTIDES
BPI peptides were produced according to co-owned and copending PCT Application No. PCT US94/10427 filed Sep. 15, 1994, which corresponds to U.S. patent application Ser. No. 08/306,473 filed Sep. 15, 1994, and PCT Application No. US94/02465 filed Mar. 11, 1994, which corresponds to U.S. patent application Ser. No. 08/209,762, filed Mar. 11, 1994, which is a continuation-in-pan of U.S. patent application Ser. No. 08/183,222, filed Jan. 14, 1994, which is a continuation-in-part of U.S. patent application Ser. No. 08/093,202 filed Jul. 15, 1993 (for which the corresponding international application is PCT Application No. US94/02401 filed Mar. 11, 1994), which is a continuation-in-part of U.S. patent application Ser. No. 08/030,644 filed Mar. 12, 1993, the disclosures of all of which are incorporated herein by reference.
The BPI peptides were screened for bactericidal effects on E. coli J5 and E. Coli 0111:B4 bacteria in a radial diffusion assay. Specifically, an overnight culture of the bacteria was diluted 1:50 into fresh tryptic soy broth and incubated for 3 hours at 3.degree. C. to attain log phase growth of the culture. Bacteria were then pelleted at 3,000 rpm for 5 minutes in a Sorvall RT6000B centrifuge (Sorvall Instruments, Newton, TC). 5 mL of 10 mM sodium phosphate buffer (pH 7.4) was added and the preparation was re-pelleted. The supernatant was decanted and 5 mL of fresh buffer was added, the bacteria were resuspended and their concentration was determined by measurement of absorbance at 590 nm (an Absorbance value of 1.00 at this wavelength equals a concentration of about 1.25.times.10.sup.9 CFU/mL in suspension). The bacteria were diluted to 4.times.10.sup.6 CFU/mL in 10 mL of molten underlayer agarose (at approximately 45.degree. C.) and inverted repeatedly to mix in 15 mL polypropylene tubes conventionally used for this purpose.
The entire contents of such tubes were then poured into a level square petri dish and distributed evenly by rocking the dish side-to-side. The agarose hardened in less than 30 seconds and had a uniform thickness of about 1 mm. A series of wells were then punched into the hardened agarose using a sterile 3 mm punch attached to a vacuum apparatus. The punch was sterilized with 100% alcohol and allowed to air dry prior to use to avoid contaminating the bacterial culture.
5 or 10 .mu.L of each of the BPI peptides were carefully pipetted into each well. As a negative control, dilution buffer (pH 8.3) was added to a separate well, and rBPI.sub.23 at concentrations of 5 .mu.g/mL and 1 .mu.g/mL were also added as positive controls. Each plate was incubated at 37.degree. C. for 3 hours, and then 10 mL of molten overlayer agarose (at approximately 45.degree. C.) was added into the level petri dish, allowed to harden and incubated overnight at 37.degree. C. The next day, a clear zone was seen against the lawn of bacteria in those wells having bactericidal activity. In order to visually enhance this zone, a dilute Coomassie solution (consisting of 0.002% Coomassie Brilliant Blue, 27% methanol. 15% formaldehyde (37% stock solution) and water) was poured over the agar and allowed to stain for 24 hours. The bacterial zones were measured with a micrometer. The assay results for exemplary peptides (BPI. 1 through BPI. 169) are summarized in Table 33 for the Gram-negative bacteria E. coli J5 (rough) and E. coli O113 (smooth). The bactericidal activities are expressed as the amount of peptide (pmol/well and .mu.g/well) required to generate a 30 mm.sup.2 bactericidal zone. Additional exemplary BPI peptides include BPI.221 through BPI.281. BPI peptides which retain antibacterial activity are expected to improve the therapeutic effectiveness of antibiotics when concurrently administered therewith. The peptides are screened for such activity in an in vivo model or according to in vitro tests, including models and tests described herein.
EXAMPLE 25
EFFECTS OF CONCURRENT ADMINISTRATION OF BPI PROTEIN PRODUCT AND TETRACYCLINE OR GENTAMICIN ON E. COLI O111:B4
Additional MIC assays were performed to determine the sensitivity of E. coli 0111:B4 (as described in Example 1) to the effects of BPI protein products concurrently administered with the antibiotic tetracycline or with the antibiotic gentamicin.
For these experiments, organisms were grown overnight at 37.degree. C. in 5 mL of Mueller-Hinton broth. This overnight culture was diluted 1:50 into 5 mL of fresh broth and incubated for an additional 3 hours at 37.degree. C. to attain log-phase growth. Bacteria were pelleted for 5 minutes at 1500 .times. g and resuspended in fresh broth to give a final concentration of 2.times.10.sup.6 cells per mL.
rBPI.sub.23 and antibiotic (either tetracycline or gentamicin) were diluted such that 100 .mu.l bacterial suspension, 50 .mu.l antibiotic and 50 .mu.l diluted rBPI.sub.23, gave concentrations in serial dilutions from 10 .mu.g/mL tetracycline or 2.5 .mu.g/mL gentamicin and from 30 .mu.g/mL rBPI.sub.23 with a fixed concentration of 10.sup.6 cells/mL. Incubation was carried out in flat bottom 96 well microliter plates for 18 hours at 37.degree. C., and the plates were read in an automatic plate reader (Titretek Multiscan) at 590 nm.
A 4-6 fold decrease in absorbance was observed with tetracycline or gentamicin with certain concentrations of BPI protein product. The MIC of tetracycline without rBPI.sub.23 of 10 .mu.g/mL and was reduced by rBPI.sub.23 to 5 .mu.g/mL. The MIC of gentamicin without rBPI.sub.23 was 0.6 .mu.g/mL and was reduced by rBPI.sub.23 to 0.3 .mu.g/mL.
A partial summary of the data described in the foregoing examples, grouped by general classes of antibiotics, appears below in Table 34 and is displayed as the effects of BPI protein products on the therapeutic effectiveness of antibiotics for various gram-negative organisms.
Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description of the presently preferred embodiments thereof. Consequently, the only limitations which should be placed upon the scope of the present invention are those which appear in the appended claims.
Claims
What is claimed is:
1. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and polymyxin B in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Escherichia, Providencia, and Pseudomonas species.
2. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and ciprofloxacin in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Pseudomonas, Enterobacter, and Acinetobacter species.
3. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and ofloxacin in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Pseudomonas and Acinetobacter species.
4. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and aztreonam in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Pseudomonas, Citrobacter, Enterobacter and Acinetobacter species.
5. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and imipenem in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Enterobacter, Proteus, Providencia and Acinetobacter species.
6. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and gentamicin in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Escherichia, Klebsiella, Edwardsiella, Salmonella, Pseudotnonas and Acinetobacter species.
7. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and amikacin in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Pseudomonas, Xanthamonas, Escherichia, Proteus, Providencia, Morganella and Acinetobacter species.
8. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and tobramycin in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Pseudomonas, Citrobacter, Serratia, Proteus and Acinetobacter species.
9. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and netilmicin in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Acinetobacter species.
10. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and trimethoprim/sulfamethoxazole in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Pseudomonas, Xanthamonas, Klebsiella, Enterobacter, Proteus and Acinetobacter species.
11. In a method for enhancing the effect of antibiotic treatment of a patient infected with gram-negative bacteria by concurrently administering bactericidal/permeability-increasing (BPI) protein and an antibiotic, the improvement comprising concurrently administering BPI protein product and chloramphenicol in the treatment of a patient infected with a gram-negative bacteria selected from the group consisting of Enterobacter and Acinetobacter species.
12. A method of treating a gram-negative bacterial infection in a subject comprising administering a BPI protein product and an antibiotic in synergistically effective amounts, wherein the antibiotic is selected from the group consisting of gentamicin, polymyxin B, cefamandole and ceftriaxone.
13. The method of claim 12 wherein said antibiotic and said BPI protein product are administered in amounts where each would alone be bactericidally effective against a gram negative bacterial infection.
14. The method of claim 12 wherein the antibiotic is gentarnicin and the gram-negative bacterial infection involves gram-negative bacteria selected from the group consisting of Escherichia, Klebsiella, Edwardsiella and Salmonella species.
15. The method of claim 12 wherein the antibiotic is polymyxin B and the gram-negative bacterial infection involves gram-negative bacteria selected from the group consisting of Escherichia, Providencia and Pseudomonas species.
16. The method of claim 12 wherein the antibiotic is cefamandole and the gram-negative bacterial infection involves Escherichia species.
17. The method of claim 12 wherein the antibiotic is ceftriaxone and the gram-negative bacterial infection involves gram-negative bacteria selected from the group consisting of Pseudomonas, Enterobacter, Citrobacter, Klebsiella and Escherichia species.
18. A method of killing gram-negative bacteria comprising administering a BPI protein product and an antibiotic in synergistically effective amounts, wherein the antibiotic is selected from the group consisting of gentamicin, polymyxin B, cefamandole and ceftriaxone.
19. The method of claim 18 wherein the antibiotic is gentamicin and the gram-negative bacterial infection involves gram-negative bacteria selected from the group consisting of Escherichia, Klebsiella, Edwardsiella and Salmonella species.
20. The method of claim 18 wherein the antibiotic is polymyxin B and the gram-negative bacterial infection involves gram-negative bacteria selected from the group consisting of Escherichia, Providencia and Pseudomonas species.
21. The method of claim 18 wherein the antibiotic is cefamandole and the gram-negative bacterial infection involves Escherichia species.
22. The method of claim 18 wherein the antibiotic is ceftriaxone and the gram-negative bacterial infection involves gram-negative bacteria selected from the group consisting of Pseudomonas, Enterobacter, Citrobacter, Klebsiella and Escherichia species.
23. The method of claim 18 wherein said BPI protein product and said antibiotic are administered in vivo.
24. The method of claim 18 wherein said BPI protein product and said antibiotic are administered in vitro.
25. A pharmaceutical composition for treatment of gram-negative bacterial infection comprising a BPI protein product and an antibiotic in synergistically effective amounts, wherein the antibiotic is selected from the group consisting of gentamicin, polymyxin B, cefamandole and ceftriaxone.
26. The pharmaceutical composition of claim 25 comprising a pharmaceutically-acceptable diluent, adjuvant, or carder.
27. In a method for treating a patient infected with a Pseudomonas gram-negative bacterial species that is resistant to an antibiotic selected from the group consisting of amikacin, aztreonam, cefazolin, cefoxitin, ceftriaxone, cefuroxime, ofloxacin, piperacillin and ticarcillin, the improvement comprising the step of concurrently administering the antibiotic and an amount of BPI protein product effective to reverse the resistance of the gram-negative bacterial species to the antibiotic.
28. In a method for treating a patient infected with a Xanthamonas gram-negative bacterial species that is resistant to an antibiotic selected from the group consisting of amikacin, piperacillin and trimethoprim/sulfamethoxazole, the improvement comprising the step of concurrently administering the antibiotic and an amount of BPI protein product effective to reverse the resistance of the gram-negative bacterial species to the antibiotic.
29. In a method for treating a patient infected with a Escherichia gram-negative bacterial species that is resistant to an antibiotic selected from the group consisting of cefazolin and ceftriaxone, the improvement comprising the step of concurrently administering the antibiotic and an amount of BPI protein product effective to reverse the resistance of the gram-negative bacterial species to the antibiotic.
30. In a method for treating a patient infected with a Klebsiella gram-negative bacterial species that is resistant to an antibiotic selected from the group consisting of trimethoprim/suifamethoxazole and ceftriaxone, the improvement comprising the step of concurrently administering the antibiotic and an amount of BPI protein product effective to reverse the resistance of the gram-negative bacterial species to the antibiotic.
31. In a method for treating a patient infected with a Citrobacter gram-negative bacterial species that is resistant to ceftriaxone antibiotic, the improvement comprising the step of concurrently administering the antibiotic and an amount of BPI protein product effective to reverse the resistance of the gram-negative bacterial species to the antibiotic.
32. In a method for treating a patient infected with a Enterobacter gram-negative bacterial species that is resistant to an antibiotic selected from the group consisting of ampicillin, aztreonam, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, piperacillin, ticarcillin, azlocillin, carbenicillin, cefamandole and cefonicid, the improvement comprising the step of concurrently administering the antibiotic and an amount of BPI protein product effective to reverse the resistance of the gram-negative bacterial species to the antibiotic.
33. In a method for treating a patient infected with a Serratia gram-negative bacterial species that is resistant to an antibiotic selected from the group consisting of cefotaxime and ceftazidime, the improvement comprising the step of concurrently administering the antibiotic and an amount of BPI protein product effective to reverse the resistance of the gram-negative bacterial species to the antibiotic.
34. In a method for treating a patient infected with a Providencia gram-negative bacterial species that is resistant to an antibiotic selected from the group consisting of cefazolin and cefuroxime, the improvement comprising the step of concurrently administering the antibiotic and an amount of BPI protein product effective to reverse the resistance of the gram-negative bacterial species to the antibiotic.
35. In a method for treating a patient infected with a Acinetobacter gram-negative bacterial species that is resistant to an antibiotic selected from the group consisting of amikacin, ampicillin, aztreonam, cefazolin, cefotaxime, cefoxitin, cefiazidime, ceftriaxone, cefuroxime, ciprofloxacin, gentamicin, piperacillin, ticarcillin, trimethoprim/sulfamethoxazole, amoxicillin, carbenicillin, cefamandole, cefonicid, cefoperazone, cefotetan, ceftizoxime, chloramphenicol, mezlocillin and netilmicin, the improvement comprising the step of concurrently administering the antibiotic and an amount of BPI protein product effective to reverse the resistance of the gram-negative bacterial species to the antibiotic.
36. The method of any one of claim 1 through 35 wherein the BPI protein product is an N-terminal fragment with a molecular weight of approximately 21 to 25 kD, or a dimeric form thereof.
37. The method of any one of claim 1 through 35 wherein the BPI protein product is rBPI.sub.23, rBPI.sub.21, or rBPI.sub.50.
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