WO2000018798A1 - Antifungal and antibacterial peptide - Google Patents

Abstract

The present invention relates generally to a novel peptide, XMP.445, derived from or based on Domain III (amino acids 142-169) of bactericidal/permeability-increasing protein (BPI) and therapeutic uses of this peptide.

Description

ANTIFUNGAL AND ANTIBAKTERIAL PEPTIDE

This application claims priority based on U.S. Provisional Application Serial No. 60/109,896 filed November 25, 1998 and U.S. Provisional Application Serial No. 60/101,958 filed September 25, 1998.

BACKGROUND OF THE INVENTION

The present invention relates generally to peptides derived from or based on Domain HI (amino acids 142-169) of bactericidal/permeability-increasing protein (BPI) and therapeutic uses of such peptides. 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. The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein (SEQ ID NOS: 2 and 3) have been reported in Figure 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. Recombinant human BPI holoprotein has also been produced in which valine at position 151 is specified by GTG rather than GTC, residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG) and residue 417 is alanine (specified by GCT) rather than valine (specified by GTT). See U.S. Patent No. 5,627,153. Fungi are eukaryotic cells that may reproduce sexually or asexually and may be dimorphic. Fungi are not only important human and animal pathogens, but they are also among the most common causes of plant disease. Fungal infections (mycoses) are becoming a major concern for a number of reasons, including the limited number of antifiingal agents available, the increasing incidence of species resistant to known antifiingal agents, and the growing population of immunocompromised patients at risk for opportunistic fungal infections, such as organ transplant patients, cancer patients undergoing chemotherapy, burn patients, AIDS patients, or patients with diabetic ketoacidosis. The incidence of systemic fungal infections increased 600% in teaching hospitals and 220% in non-teaching hospitals during the 1980's. The most common clinical isolate is Candida albicans (comprising about 19% of all isolates). In one study, nearly 40% of all deaths from hospital-acquired infections were due to fungi. [Sternberg, Science, 266: 1632-1634 (1994).].

Newly explored as antifiingal agents are a class of products related to bactericidal/permeability-increasing protein (BPI). BPI protein products are described in U.S. Patent No. 5,627,153 and corresponding International Publication No. WO 95/19179

(PCT/US95/00498), all of which are incorporated by reference herein, to have antifiingal activity. BPI-derived antifiingal peptides are described in co-owned, co-pending U.S. Application Serial No. 08/621,259 filed March 21, 1996, now U.S. Patent No. 5,858,974, which is in turn a continuation-in-part of U.S. Application Serial No. 08/504,841 filed July 20, 1994 and corresponding International Publication Nos. WO 96/08509

(PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), all of which are incorporated by reference herein. Other peptides with antifiingal activity are described in U.S. Patent No. 5,652,332 [corresponding to International Publication No. WO 95/19372 (PCT/US94/10427)], and in U.S. Patent Nos. 5,763,567 and 5,733,872 [corresponding to International Publication No. WO 94/20532 (PCT/US94/02465)], which is a continuation-in-part of U.S. Patent Application Serial No. 08/183,222 filed January 14, 1994, which is a continuation-in-part of U.S. Patent Application Serial No. 08/093,202 filed July 15, 1993 [corresponding to International Publication No. WO 94/20128 (PCT/US94/02401)], which is a continuation-in-part of U.S. Patent Application Ser. No. 08/030,644 filed March 12, 1993, now U.S. Patent No. 5,348,942, the disclosures of all of which are incorporated herein by reference.

Resistance of bacteria and other pathogenic organisms to antimicrobial agents 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).]

Gram-positive bacteria have a typical lipid bilayer cytoplasmic membrane surrounded by a rigid cell wall that gives the organisms their characteristic shape, differentiates them from eukaryotic cells, and allows them to survive in osmotically unfavorable environments. This cell wall is composed mainly of peptidoglycan, a polymer of N-acetylglucosamine and N-acetylmuramic acid. In addition, the cell walls of gram- positive bacteria contain teichoic acids which are anchored to the cytoplasmic membrane through lipid tails, giving rise to lipoteichoic acids. The various substituents on teichoic acids are often responsible for the biologic and immunologic properties associated with disease due to pathogenic gram-positive bacteria. Most pathogenic gram-positive bacteria have additional extracellular structures, including surface polysaccharides, capsular polysaccharides, surface proteins and polypeptide capsules.

Gram-negative bacteria also have a cytoplasmic membrane and a peptidoglycan layer similar to but reduced from that found in gram-positive organisms. However, gram-negative bacteria have an additional outer membrane that is covalently linked to the tetrapeptides of the peptidoglycan layer by a lipoprotein; this protein also contains a special lipid substituent on the terminal cysteine that embeds the lipoprotein in the outer membrane. The outer layer of the outer membrane contains the lipopolysaccharide (LPS) constituent. BPI protein products are also described to have antibacterial activities in

U.S. Patent Nos. 5,198,541 and 5,523,288 and International Publication No. WO 95/08344 (PCT/US94/11255), all of which are incorporated by reference herein, disclosing activity against gram-negative bacteria, and U.S. Patent Nos. 5,578,572 and 5,783,561 and International Publication No. WO 95/19180 (PCT/US95/00656), all of which are incorporated by reference herein, disclosing activity against gram-positive bacteria and mycoplasma, and co-owned, co-pending U.S. Application Serial No. 08/626,646, which is in turn a continuation of U.S. Application Serial No. 08/285,803, which is in turn a continuation-in-part of U.S. Application Serial No. 08/031,145 and corresponding International Publication No. WO 94/20129 (PCT/US94/02463), all of which are incorporated by reference herein, disclosing activity against mycobacteria.

BPI protein products have been shown to have additional antimicrobial activities. For example, U.S. Patent No. 5,646,114 and International Publication No. WO 96/01647 (PCT/US95/08624), all of which are incorporated by reference herein, disclose activity of BPI protein products against protozoa. There continues to exist a need in the art for new antimicrobial methods and materials, including, for example, those that target animal and plant pathogens. In particular, effective antifiingal therapy for systemic mycoses is limited. Products and methods responsive to this need would ideally involve substantially non-toxic compounds available in large quantities by means of synthetic or recombinant methods. Ideal therapeutic compounds would have a rapid effect upon systemic and/or oral administration, high potency, low toxicity, and a broad spectrum of activity against a variety of different microbes when administered or applied as the sole agent. Ideal compounds would also be useful in combinative therapies with other antimicrobial agents, particularly where these activities would reduce the amount of other antimicrobial agent required for therapeutic effectiveness, enhance the effect of such agents, or limit potential toxic responses and high cost of treatment.

SUMMARY OF THE INVENTION

Three separate functional domains within the recombinant 23 kD N-terminal BPI sequence have been discovered (Little et al, 1994, J. Biol. Chem. 269: 1865). These functional domains of BPI designate regions of the amino acid sequence of

BPI that contributes to the total biological activity of the protein and were essentially defined by the activities of proteolytic cleavage fragments, overlapping 15-mer peptides and other synthetic peptides. Domain I is defined as the amino acid sequence of BPI comprising from about amino acid 17 to about amino acid 45. Initial peptides based on this domain were moderately active in both the inhibition of LPS-induced LAL activity and in heparin binding assays, and did not exhibit significant bactericidal activity. Domain II is defined as the amino acid sequence of BPI comprising from about amino acid 65 to about amino acid 99. Initial peptides based on this domain exhibited high LPS and heparin binding capacity and exhibited significant antibacterial activity. Domain III is defined as the amino acid sequence of BPI comprising from about amino acid 142 to about amino acid 169. Initial peptides based on this domain exhibited high LPS and heparin binding activity and exhibited surprising antimicrobial activity, including antifiingal and antibacterial (including, e.g., anti-gram-positive and anti-gram-negative) activity. The biological activities of peptides derived from or based on these functional domains (i.e., functional domain peptides) may include LPS binding, LPS neutralization, heparin binding, heparin neutralization or antimicrobial activity. The present invention provides a novel peptide, designated XMP.445, derived from or based on Domain III (amino acids 142-169) of bactericidal/permeability- increasing protein (BPI) and therapeutic uses of this peptide, especially as an antifiingal agent. The sequence of XMP.445 is set forth in SEQ ID NO: 1. The peptide, or pharmaceutical compositions comprising the peptide and suitable diluents, adjuvants or carriers, may be administered alone or concurrently with other known antimicrobial (particularly antifiingal) agents. When used as adjunctive therapy, the peptide may reduce the amount of the other agent needed for effective therapy, enhance the effect of such other agent, accelerate the effect of such other agent, or reverse (e.g., overcome) resistance of the pathogenic organism to such other agent.

The peptide may be effective for treating animals (e.g., mammals) in vivo, for treating plants, and for a variety of in vitro uses such as to decontaminate fluids and surfaces and to sterilize surgical and other medical equipment and implantable devices, including prosthetic joints and indwelling invasive devices. A further aspect of the invention involves use of the peptide for the manufacture of a medicament for treatment of microbial infection, e.g., fungal or bacterial infection. The medicament may additionally include other chemotherapeutic agents such as antimicrobial agents.

Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon considering the following detailed description of the invention, which describes the presently preferred embodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel peptide designated XMP.445 (SEQ ID NO: 1) which consists of amino acid residues 150 through 161 (Lys-Val-Gly-Trp-Leu-

Ile-Gln-Leu-Phe-His-Lys-Lys) of human BPI (SEQ ID NOS: 2 and 3), but in which the two N-terminal amino acid residues (at positions 150 and 151) have been changed to D- amino acids, the two C-terminal amino acid residues (at position 160 and 161) have been changed to D-amino acids, and the remainder are L-amino acids. XMP.445 has been demonstrated to possess antifiingal activity in a variety of in vitro killing assays and in vivo models of fungal infection, including, for example, by measuring improved host survival or a reduction of colony-forming units in organs after fungal challenge. Other properties of XMP.445, such as serum stability, oral absorption, and effect on fungal cells treated with a membrane potential indicator dye that localizes to mitochondria, were also determined. XMP.445 was observed to exhibit a spectrum of superior properties, including high potency and a broader therapeutic range in vivo, in one or more of the preceding assays in comparison to other peptides derived from or based on Domain III of BPI ("Domain III derived peptides").

The invention also provides methods of using XMP.445 for treating a subject suffering from infection (including fungal, bacterial, or other microbial infection), especially mammalian subjects such as humans, but also including farm animals such as cows, sheep, pigs, horses, goats and poultry (e.g., chickens, turkeys, ducks and geese), companion animals such as dogs and cats, exotic and/or zoo animals, and laboratory animals including mice, rats, rabbits, guinea pigs, and hamsters. Immunocompromised or immuno suppressed subjects, e.g., subjects suffering from cancer, subjects undergoing radiation therapy and/or cytotoxic chemotherapy, subjects being treated with immunosuppressive drugs, and subjects suffering from natural or acquired immune deficiencies such as AIDS, may be treated according to this aspect of the invention. Treatment of infection of plants is also contemplated. "Treatment" as used herein encompasses both prophylactic and therapeutic treatment, and may be accompanied by concurrent administration of other antimicrobial agents, including any of the agents discussed herein.

Fungal infection may be caused by a variety of fungal species including, e.g., species of Candida (including C. albicans, C. tropicalis, C. parapsilosis, C. stellatoidea, C. krusei, C. parakrusei, C. lusitanae, C. pseudotropicalis, C. guilliermondi and C. glabrata), Aspergillus (including A. fumigatus, A. flavus, A. niger, A. nidulans,

A. terreus, A. sydowi, A. flavatus, and A. glaucus), Cryptococcus, Histoplasma, Coccidioides, Paracoccidioides, Blastomyces, Basidiobolus, Conidiobolus, Rhizopus, Rhizomucor, Mucor, Absidia, Mortierella, Cunninghamella, Saksenaea, Pseudallescheria, Sporotrichosis, Fusarium, Trichophyton, Trichosporon, Microsporum, Epidermophyton, Scytalidium, Malassezia, Actinomycetes, Sporothrix, Penicillium,

Saccharomyces and Pneumocystis. Bacterial infection may be caused by gram-negative bacterial species, including Acidaminococcus, Acinetobacter, Aeromonas, Alcaligenes, Bacteroides, Bordetella, Branhamella, Brucell , Calymmatobacterium, Campy lobacter, Cardiobacterium, Chromobacteήum, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Flavobacterium, Francisella, Fusobacterium, Haemophilus, Klebsiella, Legionella, Moraxella, Morganella, Neisseria, Pasturella, Plesiomonas, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Streptobacillus, Veillonella, Vibrio, and Yersinia species, or gram-positive bacterial species, including Staphylococcus, Streptococcus, Micrococcus, Peptococcus, Peptostreptococcus, Enterococcus, Bacillus, Clostridium, Lactobacillus, Listeria, Erysipeloihrix, Propionibacterium, Eubacterium, and Corynebacteriwn species, or mycoplasma or mycobacteria. Other microbial infections may be caused by, e.g., protozoa, including Plasmodia, Toxoplasma, Leishmania, Trypanosoma, Acanthamoeba, Nagleria, and Pneumocystis species. Other therapeutic uses of XMP.445 according to the invention include methods of treating conditions associated with endotoxin, such as exposure to gram- negative bacterial endotoxin in circulation, endotoxemia, bacterial and/or endotoxin- related shock and one or more conditions associated therewith, including a systemic inflammatory response, cytokine overstimulation, complement activation, disseminated intravascular coagulation, increased vascular permeability, anemia, thrombocytopema, leukopenia, pulmonary edema, adult respiratory distress syndrome, renal insufficiency and failure, hypotension, fever, tachycardia, tachypnea, and metabolic acidosis. Thus, not only gram-negative bacterial infection but also its sequelae associated with exposure to gram-negative bacterial endotoxin may be ameliorated through endotoxin-binding or endotoxin-neutralizing activities of XMP .445.

Therapeutic compositions of the peptide may include a pharmaceutically acceptable diluent, adjuvant, or carrier. The BPI protein product may be administered without or in conjunction with known surfactants, other chemotherapeutic agents or additional known antimicrobial agents. As described in U.S. Application Serial No. 08/586,133 filed January 12, 1996, which is in turn a continuation-in-part of U.S.

Application Serial No. 08/530,599 filed September 19, 1995, which is in turn a continuation-in-part of U.S. Application Serial No. 08/372,104 filed January 13, 1995, and corresponding International Publication No. WO96/21436 (PCT/US96/01095), all of which are incorporated herein by reference, other poloxamer formulations of BPI protein products with enhanced activity may be utilized. Therapeutic compositions may be administered systemically or topically.

Systemic routes of administration include oral, intravenous, intramuscular or subcutaneous injection (including into depots for long-term release), intraocular or retrobulbar, intrathecal, intraperitoneal (e.g. by intraperitoneal lavage), intrapulmonary (using powdered drug, or an aerosolized or nebulized drug solution), or transdermal. Topical routes include administration in the form of salves, creams, jellies, ophthalmic drops or opthalmic ointments, ear drops, suppositories, such as vaginal suppositories, or irrigation fluids (for, e.g., irrigation of wounds).

Suitable dosages include doses ranging from 1 μg/kg to 100 mg/kg per day and doses ranging from 0.1 mg/kg to 20 mg/kg per day. When given parenterally, BPI protein product compositions are generally injected in doses ranging from 1 mg/kg to 100 mg/kg per day, preferably at doses ranging from 0.1 mg kg to 20 mg/kg per day, and more preferably at doses ranging from 1 to 20 mg/kg/day. The treatment may continue by continuous infusion or intermittent injection or infusion, or a combination thereof, at the same, reduced or increased dose per day for as long as determined by the treating physician. When given topically, BPI protein product compositions are generally applied in unit doses ranging from 1 mg/mL to 1 gm/mL, and preferably in doses ranging from 1 mg/mL to 100 mg/mL. Those skilled in the art can readily optimize effective dosages and administration regimens for therapeutic compositions as determined by good medical practice and the clinical condition of the individual subject. "Concurrent administration," or "co-administration," as used herein includes administration of one or more agents, in conjunction or combination, together, or before or after each other. The agents may be administered by the same or by different routes. If administered via the same route, the agents may be given simultaneously or sequentially, as long as they are given in a manner sufficient to allow all agents to achieve effective concentrations at the site of action. For example, a BPI protein product may be administered intravenously while the second agent(s) is(are) administered intravenously, intramuscularly, subcutaneously, orally or intraperitoneally. A BPI protein product and a second agent(s) may be given sequentially in the same intravenous line or may be given in different intravenous lines. Alternatively, a BPI protein product may be administered in a special form for gastric delivery, while the second agent(s) is(are) administered, e.g., orally.

Known antifiingal agents include polyene derivatives, such as amphotericin B (including lipid or liposomal formulations thereof) and the structurally related compounds nystatin and pimaricin; flucytosine (5-fluorocytosine); azole derivatives (including ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, tioconazole, terconazole, fluconazole, itraconazole, voriconazole [Pfizer] and

SCH56592 [Schering-Plough]); allylamines-thiocarbamates (including tolnaftate, naftifine and terbinafine); griseofulvin; ciclopirox; haloprogin; echinocandins (including MK-0991 [Merck]); and nikkomycins. Recently discovered as antifiingal agents are a class of products related to bactericidal/permeability-increasing protein (BPI), described in U.S. Patent Nos. 5,627,153, 5,858,974, 5,652,332, 5,763,567 and 5,733,872, the disclosures of all of which are incorporated herein by reference.

Known antibacterial agents include antibiotics, which 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 β-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.

BPI protein products are also described to have antibacterial activities in U.S. Patent Nos. 5,198,541 and 5,523,288 and International Publication No. WO 95/08344 (PCT/US94/11255), all of which are incorporated by reference herein, disclosing activity against gram-negative bacteria, and U.S. Patent Nos. 5,578,572 and

5,783,561 and International Publication No. WO 95/19180 (PCT/US95/00656), all of which are incorporated by reference herein, disclosing activity against gram-positive bacteria and mycoplasma, and co-owned, co-pending U.S. Application Serial No. 08/626,646, which is in turn a continuation of U.S. Application Serial No. 08/285,803, which is in turn a continuation-in-part of U.S. Application Serial No. 08/031,145 and corresponding International Publication No. WO 94/20129 (PCT/US94/02463), all of which are incorporated by reference herein, disclosing activity against mycobacteria.

BPI protein products have been shown to have additional antimicrobial activities. For example, U.S. Patent No. 5,646,114 and International Publication No. WO 96/01647 (PCT/US95/08624), all of which are incorporated by reference herein, disclose activity of BPI protein products against protozoa. Concurrent administration of the XMP.445 peptide for adjunctive therapy with other antimicrobial agents (particularly antifiingal agents) is expected to improve the therapeutic effectiveness of the antimicrobial agents. This may occur through reducing the concentration of antimicrobial agent required to eradicate or inhibit target cell growth, e.g., replication. Because the use of some antimicrobial agents is limited by their systemic toxicity or prohibitive cost, lowering the concentration of antimicrobial agent required for therapeutic effectiveness reduces toxicity and/or cost of treatment, and thus allows wider use of the agent. For example, concurrent administration of XMP.445 peptide and another antifiingal agent may produce a more rapid or complete fungicidal or fungistatic effect than could be achieved with either agent alone. Administration of XMP.445 peptide may reverse the resistance of fungi to antifiingal agents or may convert a fungistatic agent into a fungicidal agent. Similar results may be observed upon concurrent administration of XMP.445 with other antimicrobial agents.

Therapeutic effectiveness in vivo 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 antimicrobial activity at the site of infection that is sufficient to inhibit growth or replication of the pathogenic organism in a manner that tips the balance in favor of the host. When host defenses are maximally effective, the antimicrobial 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 microbicidal/microbistatic effect can be more important than a long-term effect. These early events are a significant and critical part of therapeutic success, because they allow time for host defense mechanisms to activate.

In addition, the invention provides a method of killing or inhibiting growth of pathogenic organisms (particularly fungi) comprising contacting the organism with

XMP.445, optionally in conjunction with other antimicrobial agents. This method can be practiced in vivo or in a variety of in vitro uses such as to decontaminate fluids and surfaces or to sterilize surgical and other medical equipment and implantable devices, including prostheses and intrauterine devices. These methods can also be used for in situ sterilization of indwelling invasive devices such as intravenous lines and catheters, which are often foci of infection. A further aspect of the invention involves use of XMP.445 for the manufacture of a medicament for treatment of microbial infection (e.g., fungal or bacterial infection) or a medicament for concurrent administration with another agent for treatment of microbial infection. The medicament may optionally comprise a pharmaceutically acceptable diluent, adjuvant or carrier and also may include, in addition to XMP.445, other chemotherapeutic agents.

Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples. Example 1 addresses preparation and purification of XMP.445. Example 2 addresses in vitro antifiingal activity testing of XMP.445. Example 3 addresses in vivo antifiingal activity testing of XMP.445.

Example 4 addresses testing of XMP.445 for oral availability. Example 5 addresses testing of other properties of XMP.445, including serum stability and activity on cells treated with a membrane potential indicator dye that localizes to mitochondria.

Example 1

Preparation of XMP.445

Preparation and purification of Domain HI derived peptides, analysis of the properties of these peptides in a number of in vitro and in vivo assays, and therapeutic uses of these peptides are described generally in U.S. Patent No. 5,858,974 and corresponding International Publication No. WO 97/04008 (PCT/US96/03845), both of which are incorporated by reference herein. XMP.445 was synthesized and purified generally according to Example 1 of U.S. Patent No. 5,858,974.

Example 2 In Vitro Antifungal Activity

XMP.445 was tested for in vitro antifungal activity against Candida albicans, Candida tropicalis, Aspergillus fumigatus, Cryptococcus neoformans, Fusarium solani, Fusarium oxysporum and Scytalidium hyalinum in a broth assay generally according to Example 2 of U.S. Patent No. 5,858,974. XMP.445 was also tested for in vitro antifungal activity in radial diffusion assays against C. albicans SLU-1 generally according to Examples 2 and 3 of U.S. Patent No. 5,858,974. The minimum fiingicidal concentration of XMP.391, XMP.342 and XMP.445 against various strains of fungi are shown in Tables 1 and 2 below (which include data from Tables 3 and 4 of U.S. Patent No. 5,858,974).

Table 1

NT=not tested

Table 2

NT=not tested

The structures of XMP.391 (amino acid residues 150-161 of BPI; SEQ ID NO:4) and XMP.342 (amino acid residues 153-157 of BPI with an additional two D- lysines at the N-terminus and an additional two D-lysines at the C-terminus; SEQ ID NO:5) are described in U.S. Patent No. 5,858,974. Additional testing of XMP.445 against various species of Fusarium using the National Committee for Clinical Laboratory Standards macrobroth dilution method, M-27A, showed that XMP.445 exhibited potent activity against all tested species. The minimal fungal growth inhibitory concentrations (MICs) of XMP.445 ranged from 2 to 8 μg/mL for F. solani, 4 to 32 μg/mL for F. oxysporium, 2 to 4 μg/mL for F. moniliforme, 2 to A μg/mL for F. dimerum and about 4 μg/mL for F. chlamydosporium.

Example 3 In Vivo Antifungal Activity

XMP.445 was tested for in vivo antifungal activity in mice with systemic C. albicans infection, as measured by effect on mortality, generally according to Example

4 of U.S. Patent No. 5,858,974.

Results of testing peptides XMP.391, XMP.342 and XMP.445 in the mouse models showed that: when XMP.342 was tested at doses of 0.5 and 5 mg/kg, a statistically significant (p=0.02) reduction in mortality (compared to saline control) was observed at the 5 mg/kg dose; when XMP.391 was tested at doses of 0.5 and 5 mg/kg, a significant (p=0.05) reduction in mortality (vs. saline control) was observed at the 5 mg/kg dose; when XMP.445 was tested in multiple experiments at doses ranging from 0.5 to 5 mg/kg, a statistically significant (p<0.05) reduction in mortality at a 0.5 mg/kg dose was observed in two of four separate experiments in which that dose was tested (in a q.d. or q.o.d. regimen), a statistically significant (p<0.05) reduction in mortality at a 2 mg/kg or 2.5 mg/kg dose was observed in two of three experiments in which those doses were tested (in a q.d. or q.o.d. regimen), and no statistically significant reduction in mortality for the 5 mg/kg dose was seen in two experiments in which that dose was tested.

XMP.445 was also tested for in vivo antifungal activity in cyclosporin A- immunosuppressed mice with systemic C. albicans infection generally according to

Example 4 of U.S. Patent No. 5,858,974. The cyclosporin A injections were administered daily for 8 days (day -1 to day 7) and XMP.445 was administered at 10 mg/kg once daily for 14 days (day 0 to day 14) via intraperitoneal injection. A reduction in mortality was observed, although the results did not reach statistical significance. Further testing of XMP.445 in 5-fluorouracil-treated mice with systemic C. albicans infection (wherein 150 mg/kg 5-FU was administered once intravenously at day -1 and XMP.445 was administered at 5 mg/kg twice daily for 14 days via intraperitoneal injection) resulted in a statistically significant reduction in mortality compared to saline control (p=0.0012 or 0.0056).

Additional testing of XMP.445 in mice infected with an Aspergillus inoculum equivalent to approximately LD₈₀ over 28 days (about 2 x 10⁶ spores in 0.25 ml) showed that administration of XMP.445 (7 doses q.o.d. starting at day 0) provided a trend towards survival benefit, although the results did not reach statistical significance.

XMP.445 was also tested in mice infected with Fusarium solani (clinical isolate #99-6, for which the MFC of XMP.445 was previously determined to be approximately 2 to 4 μg/mL at 24 to 48 hours). Groups of 10 mice were given 150 mg/kg

5-FU on day -1 to render them neutropenic, infected with 5 x 10⁶ CFU F. solani IV on day 0, and treated with saline IP or with XMP.445 at doses of 5 mg/kg IP daily, or 2.5 mg/kg IP twice daily (bid.), or 1 mg/kg IP daily, or 0.5 mg/kg IP b.i.d. on days 1-7 post- challenge. Mice were observed for 30 days for survival. A survival benefit was seen at doses of 2.5 mg/kg b.i.d. and 5 mg kg, and statistical significance (p< 0.05) was achieved at the dose of 5 mg/kg XMP.445.

Increasing doses of various BPI-derived peptides were also administered to healthy mice via intravenous injection into the tail vein to determine the maximum tolerated dose of peptide that could be administered without causing observable symptoms such as alterations in respiratory rate and loss of righting reflex. When administered as an intravenous bolus, doses of XMP.342 higher than 5 mg/kg were not well tolerated, doses of XMP.391 higher than 10 mg/kg were not well tolerated, and doses of XMP.445 higher than 5 mg/kg were not well tolerated. Comparison of maximum tolerated doses (MTDs) for these peptides to the minimum therapeutically effective dose (MEDs) for the same peptides in the mouse model of C. albicans infection showed that XMP.445 exhibited the broadest range of therapeutic doses without adverse effects. The minimum effective dose (MED) for XMP.342 was 5 mg/kg (the same as the MTD); the MED for XMP.391 was 5 mg/kg (about half the MTD); the MED for XMP.445 was 0.5 mg/kg (about one-tenth the MTD).

Example 4

Oral Availability of XMP.445

Various BPI-derived peptides were screened for oral absorption in in vitro screening assays using CACO-2 and MDCK cells. Cultured monolayers of CACO-2 (Human colon carcinoma) [Audus, K.L., et al. Pharm. Res., 1: 435-451 (1990)] or Madin- Derby canine kidney epithelial (MDCK) cells (ATCC Accession No. CCL34) were grown upon collagen-coated, permeable-filter supports (Becton Dickenson, Mountainview, CA). The cells were grown to confluency and allowed to differentiate. The integrity of the monolayers was determined by measuring the transepithelial resistance. The cells were incubated with peptide on the apical side for 2.5 hours in MDCK screening or 4 hours for CACO-2 screening. The transepithelial transport of the peptide was measured by quantitative HPLC analysis of the incubation media on the basolateral side of the cells. Radiolabelled mannitol and cortisone were used as positive controls.

Intestinal absorption screening of peptides XMP.365, XMP.391 and XMP.445 identified XMP.445 as a potential orally available compound. XMP.445 was further tested for activity upon oral administration (oral activity) in a 28-day comparative survival efficacy study in mice systemically infected with Candida albicans. Specifically, male DBA/2 mice (Charles River Laboratories) six weeks of age were dosed with 7.9 x 10⁴ Candida albicans, SLU-1 in 100 μ intravenously via the tail vein in a single dosage on day 0. Treatment began immediately thereafter with 400 μl oral gavage of either 0.5% dextrose, or XMP.445 in 0.5% dextrose at levels of either

10 mg/kg or 20 mg/kg every other day for a total of eight times. Amphotericin B

® (Fungizone ) was administered intravenously at 0.5 mg/kg as a positive control every other day for a total of eight times. Twice-day monitoring (once daily on weekends and holidays) for mortality was performed. The animals treated with XMP.445 showed improvements in mortality compared with the dextrose-treated controls, and the animals treated with XMP.445 at 10 mg/kg showed significant improvement (p-value of 0.025). The results of this study show that XMP.445 has oral antifungal activity.

XMP.445 was also tested for in vivo oral antifungal activity in cyclosporin A-immunosuppressed mice with systemic C. albicans infection generally according to Example 4 of U.S. Patent No. 5,858,974. The cyclosporin A injections were administered daily for 8 days (day -1 to day 7) and XMP.445 was administered at 10 mg/kg once daily for 14 days (day 0 to day 14) via oral gavage. A reduction in mortality was observed, although the results did not reach statistical significance.

Example 5

Other Properties of XMP.445

XMP.445 was also tested for serum stability using a bioassay generally according to Example 5 of U.S. Serial No. 08/621,259, wherein the peptide is incubated with serum for varying amounts of time and the resulting serum-treated product is tested for antifungal activity against C. albicans SLU-1 in a radial diffusion assay. XMP.445 was further tested for its effect on fiingal cells treated with a membrane potential indicator dye that localizes to mitochondria, as described in co-owned U.S. Provisional Application Serial Nos. 60/101,778 filed September 25, 1998, 60/109,905 filed November 25, 1998, and 60/143,485 filed July 12, 1999, and in co-owned concurrently filed U.S. Application Serial No. [Attorney Docket No. 27129/36271], all of which are incorporated by reference herein.

Numerous modifications and variations of the above-described invention are expected to occur to those of skill in the art. Accordingly, only such limitations as appear in the appended claims should be placed thereon.

Claims

WHAT IS CLAIMED IS:

1. An antifungal peptide XMP.445 having the sequence set forth in SEQ ID NO: 1.

2. A composition comprising an antifungal peptide XMP.445 having the sequence set forth in SEQ ID NO: 1 and another antifungal agent.

3. A method for treating a subject suffering from fungal infection comprising administering a therapeutically effective amount of XMP.445 having the sequence set forth in SEQ ID NO: 1.

4. The method of claim 3 wherein the XMP.445 is administered orally.

5. The method of claim 3 wherein the XMP.445 is administered intravenously.

6. The method of claim 3 wherein the fungal infection involves a fungal species selected from the group consisting oϊ Candida species, Aspergillus species and Fusarium species.

7. The method of claim 3 further comprising the step of administering another antifungal agent.

8. A method for killing or inhibiting growth of fungi in vitro comprising contacting the fungi with an effective amount of XMP.445 having the sequence set forth in SEQ ID NO: 1.

9. The method of claim 8 further comprising the step of contacting the fungi with another antifungal agent.

10. A method for treating a subject suffering from bacterial infection or sequelae thereof comprising administering a therapeutically effective amount of XMP.445 having the sequence set forth in SEQ ID NO: 1.

11. The method of claim 10 further comprising the step of administering another antibacterial agent.

12. A method for killing or inhibiting growth of bacteria in vitro comprising contacting the bacteria with an effective amount of XMP.445 having the sequence set forth in SEQ ID NO: 1.

13. The method of claim 12 further comprising the step of contacting the bacteria with another antibacterial agent.

14. Use of XMP.445 in preparation of a medicament for treatment of fungal infection.

15. Use of XMP.445 in preparation of a medicament for treatment of bacterial infection.