The present composition combines an RNAIII-inhibiting peptide (RIP) with an antimicrobial peptide, such as a cathelicidin, that is capable of binding and neutralizing lipidic and polyanionic components of bacterial cell envelope. In another embodiment, the RIP is combined with an antibiotic, with or without an antimicrobial peptide. The present composition is advantageously used in a method of treatment of bacterial sepsis.

Description of the Invention

SUMMARY

The present invention provides a therapeutic composition comprising a RIP and an antimicrobial peptide to meet the ongoing need for treating diseases associated with bacterial infection, particularly staphylococcal sepsis. RIP by itself inhibits LTA-induced production of TNF-.alpha. and NO, and RIP and a cathelicidin antimicrobial peptide synergistically inhibit LTA-induced production of TNF-.alpha. and NO. When administered in vivo, RIP by itself reduces mortality and bacteremia, and RIP and a cathelicidin antimicrobial peptides act synergistically in vivo to reduce mortality and bacteremia. While the present composition can be used in combination with conventional antibiotic chemotherapy, the present composition advantageously is effective against antibiotic resistant bacteria and may be used as an alternative to convention chemotherapy.

According to a first aspect of the invention, a composition comprises a RIP and a polycationic antimicrobial peptide that is capable of binding and neutralizing a lipidic and polyanionic component of a bacterial cell envelope, such as LTA or LPS. Antimicrobial peptides that are useful in the present composition include a cathelicidin, such as a human cathelicidin, or BMAP-28.

The composition further may comprise conventional antibiotics or other pharmaceutically acceptable agents, such as agents that assist or delay adsorption of the composition by the host. Pharmaceutical agents, e.g., liposomes or nanoparticles, may be included to assist in delivering or targeting the composition to a desired location or cell type. The composition may be formulated for administration by any acceptable method, such as topical application, ingestion, or parenteral administration or as a coating on a medical device.

The RIP may comprise five contiguous amino acids of the sequence YX.sub.2PX.sub.1TNF (SEQ ID NO: 2), where X.sub.1 is C, W, I or a modified amino acid, and X.sub.2 is K or S; or amino acids having a sequence that differs from the sequence YX.sub.2PX.sub.1TNF (SEQ ID NO: 2) by two substitutions or deletions, where X.sub.1 is C, W, I or a modified amino acid, and X.sub.2 is K or S. In one embodiment, the RIP does not consist of the sequence YSPX.sub.1TNF (SEQ ID NO: 3), where X.sub.1 is C, W, I or a modified amino acid. Alternatively, the RIP may comprise amino acids having a sequence that differs from the sequence YX.sub.2PX.sub.1TNF (SEQ ID NO: 2) by one substitution or deletion, where X.sub.1 is C, W, I or a modified amino acid, and X.sub.2 is K or S. In various other embodiments, the RIP comprises the amino acid sequences YKPX.sub.1TNF (SEQ ID NO: 4), where X.sub.1 is C, W, I or a modified amino acid; the amino acid sequence IKKYX.sub.2PX.sub.1TNF (SEQ ID NO: 1), where X.sub.1 is C, W, I or a modified amino acid and X.sub.2 is K or S; or one of the sequences PCTNF (SEQ ID NO: 6), YKPITNF (SEQ ID NO: 7), or YKPWTNF (SEQ ID NO: 8). The RIP may be ten amino acids in length and may comprise about 0.1% to 50% by weight of the composition, or about 2% to 20% by weight of the composition.

According to a second aspect of the invention, a method of treating a disease associated with a bacterial infection comprises administering a composition comprising a RIP and an antimicrobial peptide that is capable of binding and neutralizing a lipidic and polyanionic component of a bacterial cell envelope, such as LTA or LPS, to a mammalian individual. The method of the invention is particularly advantageous in treating or reducing the risk of a bacterial infection that comprises an inflammatory response caused by a lipidic and polyanionic component of a bacterial cell envelope, such as bacterial sepsis. The method may be used to treat a systemic bacterial infection, or an infection localized to particular tissue, skin or region of the body. The infection also may be associated with other diseases, such as cellulitis, keratitis, osteomyelitis, septic arthritis or mastitis. The administering may be by a topical, oral, intravenous, intraperitoneal, intramuscular, transdermal, nasally, or iontophoretic route, such as by a depot-style system, an encapsulated form, or an implant.

The present method also is useful in the treatment of bacterial infection associated with biofilms, or in reducing the risk of a disease associated with biofilms, particularly those whose pathologies involve an inflammatory response caused by a lipidic and polyanionic component of a bacterial cell envelope. For example, the present composition may be used to coat devices inserted into an individual to reduce the risk that the implanted device will develop a biofilm.

The method further may be practiced on an individual at risk of having or suspected of having an infection caused by bacteria, such as an individual who is suffering from burns, trauma, etc. Alternatively, the composition may be administered to treat an ongoing infection, delay the onset of symptoms of bacterial infection, or reduce the risk of developing an infection.

In one embodiment, the individual receiving the composition is infected or at risk of infection by Gram-positive bacteria, such as Streptococcus ssp, including S. aureus and S. epidermidis, or an antibiotic resistant strain thereof. In other embodiments, the pathogen may be Listeria spp, including L. innocua, and L. monoctogenes, Lactococcus spp, Enterococcus spp, Escherichia coli, Clostridium acetobtylicum, and Bacillus spp, including B. subtilus, B. anthracis, and B. cereus or an antibiotic resistant strain thereof. The method may comprise administering the composition by any pharmacologically acceptable means, such as topical application, ingestion, parenteral administration, or as a coating on a medical device.

According to a third aspect of the invention, a method of treating a disease associated with a bacterial infection comprises administering a composition comprising a RIP and an antibiotic that is an aminoglycoside, beta-lactam, caphalosoprin or vancomycin in an amount effective to treat or reduce the risk of bacterial infection in a mammalian individual, e.g., a human, receiving the composition. In particular, the antibiotic may be imipenem or vancomycin. The composition may further comprise an antimicrobial peptide. In a fourth aspect of the invention, a method of treating or reducing the risk of bacterial infection in a mammalian individual, e.g., a human, comprises administering this same composition to the individual.

DETAILED DESCRIPTION

The present composition combines an RNAIII-inhibiting peptide with a polycationic antimicrobial peptide that is capable of binding and neutralizing a lipidic and polyanionic component of a bacterial cell envelope, such as LTA or LPS. The antimicrobial peptide may be a cathelicidin. RNAIII-inhibiting peptides of the invention generally are those that are able to inhibit RNAIII activity, decrease the phosphorylation TRAP, inhibit production of cytokines or NO in an in vitro model, or display related activities. Recognizing the importance of cell wall components and exotoxin production in the pathology of bacterial sepsis, the present composition is advantageously used in a method of treatment of bacterial sepsis or a similar condition in which bacterial pathology is related to a lipidic and polyanionic component of a bacterial cell envelope, such as LTA or LPS.

The present composition alternatively or additionally combines a RIP with a conventional antibiotic, such as a beta-lactam, an aminoglycoside, cephalosporin or vancomycin. Such a composition is particularly advantageously when the infected individual is infected with, or is at risk of being infected with, antibiotic resistant bacteria, since RIP exerts its antibacterial effects by a mechanism separate from such conventional antibiotics. For example, such a composition may be particularly useful for treating or reducing the risk of infections associated with biofilms, which tend to be recalcitrant to chemotherapy with conventional antibiotics. This recalcitrance necessitates the prolonged use of antibiotics in the affected individual, which promotes the rise of resistant bacteria. Resistance to some antibiotics, e.g., antibiotics of the penicillin family and more recently vancomyicn, has become so widespread that the use of these antibiotics is severely restricted. It is perceived that use of the present compositions comprising RIP will revive the use of these antibiotics, however, because of the ability of RIP to eliminate or reduce biofilms, thereby reducing an obstacle to prolonged antibiotic therapy and overcoming some of the resistance developed to these antibiotics.

In one embodiment, the method may be used to treat or reduce the risk of infection by Gram-positive bacteria. The method of the invention also is useful to treat diseases like cellulitis, keratitis, osteomyelitis, septic arthritis or mastitis. The present composition may be administered in an amount effective to treat an infection by Staphylococcus in a host individual, but the composition also is useful in treating infections caused by Listeria spp, including L. innocua, and L. monoclogenes, Lactococcus spp, Enterococcus spp, Escherichia coli, Clostridium acetobtylicum, and Bacillus spp., including B. subtilus, B. anthracis, and B. cereus or an antibiotic resistant strain thereof.

RNAIII-Inhibiting Peptides of the Invention

The quorum-sensing inhibitor RIP does not affect bacterial growth but reduces the pathogenic potential of the bacteria by interfering with the signal transduction that leads to production of exotoxins. RIP blocks toxin production by inhibiting the phosphorylation of its target molecule TRAP, which is an upstream activator of the agr locus. By contrast, the mechanism of action of antimicrobial peptides comprises disrupting the bacterial outer membrane barrier and perturbing the cytoplasmic membrane. In addition, polycationic antimicrobial peptides bind and neutralize lipidic and polyanionic components of the bacterial cell envelope, like LPS and LTA. Because RIP and antimicrobial peptides act by different mechanisms, the two can act synergistically to treat bacterial infections.

RIP comprises the general formula YX.sub.2PX.sub.1TNF (SEQ ID NO: 2), where X.sub.1 is C, W, I or a modified amino acid and X.sub.2 is K or S. Specific RIP sequences are disclosed in U.S. Pat. No. 6,291,431 and Gov et al., Peptides 22:1609-20 (2001), incorporated herein by reference. RIP sequences include polypeptides comprising the amino acid sequence KKYX.sub.2PX.sub.1TN (SEQ ID NO: 9), where X.sub.1 is C, W, I or a modified amino acid and X.sub.2 is K or S. RIP sequences also include polypeptides comprising YSPX.sub.1TNF (SEQ ID NO: 10), where X.sub.1 is C or W, and YKPITN (SEQ ID NO: 11). In one embodiment, the RIP comprising the general formula YX.sub.2PX.sub.1TNF (SEQ ID NO: 2) above is further modified by one or two amino acid substitutions, deletions, and other modifications, provided the RIP exhibits activity.

Assay Systems for Determining Activity of RIP and RIP Formulations

The mechanism through which RIP inhibits quorum-sensing mechanisms, as discussed above, involves inhibition of the phosphorylation of TRAP. There is evidence of the presence of TRAP and TRAP phosphorylation in S. epidermidis, indicating that there is a similar quorum sensing mechanisms both in S. aureus and in S. epidermidis and the potential for RIP to interfere with biofilm formation and infections caused by both species. In addition, there is evidence that TRAP is conserved among all staphylococcal strains and species; therefore, RIP should be effective against any type of Staphylococcus. Further, other infection-causing bacteria appear to have proteins with sequence similarity to TRAP, including Bacillus subtilus, Bacillus anthracis, Bacillus cereus, Listeria innocua, and Listeria monoctogenes. Moreover, RAP is an ortholog of the ribosomal protein L2, encoded by the rplB gene. See Korem et al., FEMS Microbiol. Lett. 223: 167-75 (2003), which is incorporated by reference herein with regard to its description of RAP orthologs encoded by the rplB gene. L2 is highly conserved among bacteria, including Streptococcus ssp, Listeria spp, Lactococcus spp, Enterococcus spp, Escherichia coli, Clostridium acetobtylicum, and Bacillus spp. This finding indicates that treatment aimed at disturbing the function of RAP in S. aureus also will be effective in treating L2-synthesizing bacteria as well.

RNAIII-inhibiting peptides according to the invention exhibit activity, which can be assayed using a number of routine screens. For example, RIPs are capable of inhibiting production of RNAIII or TRAP phosphorylation in vitro using the assay methods described in Balaban et al., Peptides 21:1301-11 (2000), incorporated herein by reference. RIP activity includes inhibiting staphylococcal infections. RIP inhibits Staphylococci from adhering and from producing toxins by interfering with the known function of a staphylococcal quorum-sensing system. RIP competes with RAP induction of TRAP phosphorylation, thus leading to inhibition of the phosphorylation of TRAP. See Balaban et al., J Biol. Chem. 276: 2658-67 (2001). This leads to a decrease in cell adhesion and biofilm formation, to inhibition of RNAIII synthesis and to suppression of the virulence phenotype. See Balaban et al., Science 280: 438-40 (1998). The amide form of a synthetic RIP analogue YSPWTNF(-NH.sub.2) (SEQ ID NO: 12) effectively inhibits RNAIII in vitro and suppresses S. aureus infections in vivo, including cellulitis (tested in mice against S. aureus Smith Diffuse), septic arthritis (tested in mice against S. aureus LS-1), keratitis (tested in rabbits against S. aureus 8325-4), osteomyelitis (tested in rabbits against S. aureus MS), and mastitis (tested in cows against S. aureus Newbould 305, AE-1, and environmental infections). See Balaban et al., Peptides 21:1301-11 (2000) and Table 1 (see Original Patent). These findings demonstrate the range of RIP activities and screens available to assay for RIP activity and further indicate that RIP can serve as a useful therapeutic molecule to prevent and suppress staphylococcal infections.

The screening assay can be a binding assay, wherein one or more of the molecules may be joined to a label that provides a detectable signal. Purified RIP may be used to determine a three-dimensional crystal structure, which can be used for modeling intermolecular interactions. Alternatively, the screening assay can determine the effect of a candidate RIP on RNAIII production and/or virulence factor production. For example, the effect of the candidate peptide on rnaiii transcription in Staphylococcus can be measured. Such screening assays can utilize recombinant host cells containing reporter gene systems such as CAT (chloramphenicol acetyltransferase), .beta.-galactosidase, and the like, according to well-known procedures in the art. Alternatively, the screening assay can detect rnaiii or virulence factor transcription using hybridization techniques that also are well known in the art.

In Vitro High Throughput Analysis of RIP Formulations

The following screening assay for RIP compositions exemplifies the types of assays that may be used to determine whether a particular RIP or RIP composition or formulation exhibits the desired level of biological activity. In this assay system, agr expression is tested in a high throughput assay using an RNAIII reporter gene assay, which is confirmed by Northern blotting. S. aureus cells in early exponential growth (2.times.10.sup.7 colony forming units (CFU)) containing the rnaiii::blaZ fusion construct are grown with increasing concentrations of the test RIP formulations in 96 well plates at 37.degree. C. with shaking for 2.5-5 hrs. In this assay, .beta.-lactamase acts as a reporter gene for RNAIII. Bacterial viability is tested by determining OD 650 nm and further by plating to determine CFU. .beta.-lactamase activity is measured by adding nitrocefin, a substrate for .beta.-lactamase. Hydrolysis of nitrocefin by .beta.-lactamase is indicated by a change in relative adsorption at 490 nm and 650 nm, where yellow color indicates no RNAIII synthesis, and pink color indicates RNAIII synthesis.

Formulations showing efficacy in the high throughput assay are further confirmed by Northern blotting. Bacteria are similarly grown with candidate RIP formulations. Cells are then collected by centrifugation, and total RNA is extracted and separated by agarose gel electrophoresis and Northern blotted. RNAIII is detected by hybridization to radiolabeled RNAIII-specific DNA produced by PCR, for example. Control formulations, containing, for example, random peptides, are tested at 0-10 .mu.g/10.sup.7 bacteria.

In Vivo Analysis of RIP Formulations

Candidate peptides also can be assayed for activity in vivo, for example by screening for an effect on Staphylococcus virulence factor production in a non-human animal model. The candidate agent is administered to an animal that has been infected with Staphylococcus or that has received an infectious dose of Staphylococcus in conjunction with the candidate agent. The candidate agent can be administered in any manner appropriate for a desired result. For example, the candidate agent can be administered by injection intravenously, intramuscularly, subcutaneously, or directly into the tissue in which the desired affect is to be achieved, or the candidate can be delivered topically, orally, etc. The agent can be used to coat a device that will then be implanted into the animal. The effect of agent can be monitored by any suitable method, such as assessing the number and size of Staphylococcus-associated lesions, microbiological evidence of infection, overall health, etc.

The selected animal model will vary with a number of factors known in the art, including the particular pathogenic strain of Staphylococcus or targeted disease against which candidate agents are to be screened. For example, when assessing the ability of the RIP formulation to suppress infections associated with toxin production, a mouse sepsis/cellulitis model is particularly useful. Balaban et al., Science 280: 438-40 (1998). This model is particularly preferred when, for example, the formulation comprises a RIP and a polycationic antimicrobial peptide that is capable of binding and neutralizing bacterial exotoxins and toxic cell wall components, which otherwise may induce an inflammatory response and toxic shock syndrome.

In the mouse sepsis cellulitis model, hairless immunocompetent mice (n=10) typically are challenged by a subcutaneous injection with 100 .mu.L saline containing 5.times.10.sup.8 CFU S. aureus strain Smith diffuse together with cytodex beads. Formulated RIP is administered by intravenous administration or orally by gavage at 10 times the i.v. dose. A typical i.v. dose will be <10 mg RIP/kg host body weight. Animals are observed for the five days and lesions are measured. It is expected that some of the animals will die of sepsis within the first 48 hrs due to the infection and others will develop lesions of various sizes.

A rat graft model is especially useful because it can be used to assess the ability of a formulation to suppress infections associated with biofilm formation. Giacometti et al., Antimicrob. Agents Chemother. 47: 1979-83 (2003); Cirioni et al., Circulation 108: 767-71 (2003); Balaban et al., J. Infect. Dis. 187: 625-30 (2003). This model is highly relevant to the clinical setting because it provides a time interval between bacterial challenge and biofilm infection, typically within 72 hours, allowing testing of the optimal route of administration and dose of the RIP formulation. This model provides a more challenging test of activity because biofilms are known to be extremely resistant to antibiotics.

Using the rat graft model, RIP was shown to reduce infection by four orders of magnitude when grafts were soaked with 20 .mu.g/mL RIP for 20 minutes or when RIP was injected by an intraperitoneal route at 10 mg RIP/kg body weight. These results with the rat graft model will be repeated with the most promising RIP formulations as determined by the in vitro assays described above, using higher or lower RIP concentrations than used with RIP alone. That is, formulation efficacy can be compared to intraperitoneal RIP administration at doses known to be effective. Administering RIP locally and parenterally at the time of surgery is 100% effective in preventing infection in this model system. Dell'Acqua et al., J. Infect. Dis. 190: 318-20 (2004). RIP formulations of the invention thus preferably can be carried out under the same or similar conditions. RIP formulation can be administered daily before and/or after biofilm formation for 0-6 days after bacterial challenge.

In a typical experiment, Wistar adult male rats (n=10) are anesthetized, and a subcutaneous pocket is made on each side of the median line by a 1.5 cm incision. 1-cm.sup.2 sterile collagen-sealed double velour knitted polyethylene terephthalate (Dacron) grafts ALBOGRAFT.TM., Italy) are soaked with saline, RIP, or a RIP formulation and implanted into the pockets. Pockets are closed with skin clips, and 2.times.10.sup.7 CFU/mL bacteria are inoculated onto the graft surface using a tuberculin syringe to create a subcutaneous fluid-filled pocket. The animals are returned to individual cages and examined daily. Animals receive an intravenous or oral administration of RIP or a RIP formulation 0-6 days after the graft infection. Free RIP is administered via an intraperitoneal route as a positive control. Grafts are explanted at 7 days following implantation and CFU are according to known procedures, e.g., Giacometti et al. (2003). The explanted grafts are placed in sterile tubes, washed in sterile saline solution, placed in tubes containing 10 mL of phosphate-buffered saline solution, and sonicated for 5 minutes to remove the adherent bacteria from the grafts. After sonication, grafts are microscopically checked to verify that all bacteria are removed. Quantification of viable bacteria is performed by culturing serial dilutions (0.1 mL) of the bacterial suspension on blood agar plates. All plates are incubated at 37.degree. C. for 48 hours and evaluated for number of CFUs per plate. Of note is that no significant differences in cell viability (CFU/mL) were present upon testing the effect of sonication for up to 10 minutes on either antibiotic sensitive or antibiotic resistant bacteria. The limit of detection for this method is approximately 10 CFU/mL.

Antimicrobial Peptides of the Invention

Antimicrobial peptides useful for the present invention have the ability to bind and neutralize lipidic and polyanionic components of the bacterial cell envelope, like LPS and LTA. The lipidic and polyanionic component may be embedded in the bacterial cell envelop or in soluble form. The antimicrobial peptide in either case binds the component and prevents or inhibits its ability to provoke an inflammatory response in the host. For Gram-negative organisms, cationic antimicrobial peptides may bind LPS, thereby detoxifying its endotoxic activity. See Scott et al., Infect. Immun. 67: 2005-09 (1999). Similarly, for Gram-positive bacteria, cationic antimicrobial peptides may bind and neutralize LTA. See Scott (2001). In one embodiment of the invention, the antimicrobial peptide binds LTA or teichoic acid of Gram-positive bacteria.

Antimicrobial peptides have a broad spectrum of activities, killing or neutralizing both gram-negative and gram-positive bacteria, including antibiotic-resistant strains. See Hancock, Lancet Infect. Dis. 1: 156-64 (2001). Wang, University of Nebraska Medical Center, Antimicrobial Peptide Database, at aps.unmc.edu/AP/main.php (last modified Mar. 5, 2005), which is incorporated herein by reference in its entirety, provides a database of about 500 antimicrobial peptides with antibacterial activity that potentially are useful for the present invention. Antimicrobial peptides usually are made up of between 12 and 50 amino acid residues and are polycationic. Usually about 50% of their amino acids are hydrophobic, and they are generally amphipathic, where their primary amino acid sequence comprises alternating hydrophobic and polar residues. Antimicrobial peptides fit into one of four structural categories: (i) .beta.-sheet structures that are stabilized by multiple disulfide bonds (e.g., human defensin-1), (ii) covalently stabilized loop structures (e.g., bactenecin), (iii) tryptophan (Trp)-rich, extended helical peptides (e.g., indolicidin), and (iv) amphipathic .alpha.-helices (e.g., the magainins and cecropins). See Hwang et al., Biochem. Cell Biol. 76: 235-46 (1998); Stark et al., Antimicrob. Agents Chemother 46: 3585-90 (2002).

The cathelicidins, a recently described class of antimicrobial peptides occurring at least in humans, cows, sheep, rabbits, mice, and pigs, utilize all of these structural motifs. See Ganz et al., Curr. Opinion Hematol. 4: 53-58 (1997). The cathelicidins share a highly conserved N-terminal propeptide segment of approximately 100 amino acids and a C-terminal domain that encodes the antimicrobial peptide motif. See Hwang et al., Biochem. Cell Biol. 76: 235-46 (1998). In humans, neutrophil activation leads to elastase-mediated endoproteolytic cleavage and generation of the C-terminal antimicrobial peptide. The human cathelicidin, referred to alternatively as FALL-39, hCAP18, LL-37, or CAMP, in its active processed form is a 37-amino acid amphiphilic .alpha.-helical cationic peptide. See Zanetti et al., FEBS Lett. 374: 1-5(1995). Expression of LL-37 has been detected in human neutrophils, testicular cells, respiratory epithelia, and in keratinocytes at sites of inflammation.

The amphipathic cationic peptides of the .alpha.-helical class typically demonstrate minimal bactericidal concentrations in the .mu.g/mL range, which is comparable to other antimicrobial agents. Amphipathic cationic peptides are able to kill a broad range of gram-negative and gram-positive bacterial pathogens, including those that are highly resistant to multiple antibiotics. See Hancock, Drugs 57: 469-73 (1999). These peptides kill bacteria first by binding the negatively charged bacterial surface and then inserting into the bacterial membrane, disrupting its structural integrity. The hallmark of amphipathic cationic .alpha.-helical antimicrobial peptides is their capacity to fold into an amphipathic secondary structure that presents a hydrophilic face with a net positive charge of at least +2. A number of different amino acid sequence combinations allow a peptide to achieve this characteristic structure. Consequently, hundreds of host-derived amphipathic cationic .alpha.-helical peptides have been described to date all showing limited sequence homology at the level of primary sequence comparison. See Hwang et al., Biochem. Cell Biol. 76: 235-46 (1998). The screening assays described above for RIPs also may be used to screen antimicrobial peptides for activity, especially in the form of a composition comprising both a RIP and an antimicrobial peptide.

The terms "protein," "polypeptide," or "peptide," as used herein with reference to both RIP and antimicrobial peptides, include modified sequences (e.g., glycosylated, PEG-ylated, containing conservative amino acid substitutions, containing protective groups, including 5-oxoprolyl, amidation, D-amino acids, etc.). Amino acid substitutions include conservative substitutions, which are typically within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. The skilled artisan appreciates that antimicrobial peptides do not include conventional antibiotics.

Proteins, polypeptides and peptides of the invention may be naturally occurring or produced recombinantly or by chemical synthesis according to methods well known in the art. The artisan skilled in this art is aware of various methods of recombinantly producing antimicrobial peptides in a bacterial host, despite the toxicity of the native peptides to bacteria. U.S. Pat. Nos. 5,589,364 and 5,789,377, incorporated herein by reference in its entirety, provide two examples of disclosures of suitable methods of recombinant production of amphiphilic peptides with biologically and therapeutically significant activities. For example, E. coli protease-deficient K-12 cells are transformed with a vector that expresses a cleavable fusion protein comprising at least part of a carbohydrate binding protein and an amphiphilic antimicrobial peptide. The fusion protein is expressed in the cell, the carbohydrate binding portion facilitates purification of the expressed fusion protein, and the fusion protein is then cleaved to obtain the amphiphilic peptide substantially free of carbohydrate binding protein residues. The biologically active amphiphilic peptide so produced may be further treated chemically or enzymatically to obtain a chemically distinct amphiphilic antimicrobial peptide with desired biological and therapeutic properties. In one embodiment, a DNA encoding a RIP may be co-expressed with a DNA encoding an antimicrobial peptide, so that recombinant expression produces both a RIP and an antimicrobial peptide. For example, the encoding DNAs may be contained on the same genetic construct under the operable control on the same promoter. In another embodiment, the reading frames of the encoding DNAs are fused in-frame, so that the construct expresses a fusion protein containing both RIP and antimicrobial peptide sequences. See Balaban et al., Antimicrob. Agents Chemother. 48: 2544-50 (2004).

Proteins, polypeptides and peptides of the invention can be purified or isolated. "Purified" refers to a compound that is substantially free, e.g., about 60% free, about 75% free, or about 90% free, from components that normally accompany the compound as found in its native state. An "isolated" compound is in an environment different from that in which the compound naturally occurs.

Pharmaceutical Compositions and Treatment Modalities

The term "treatment" or "treating" means any therapeutic intervention in an individual animal, e.g. a mammal, preferably a human. Treatment includes (i) "prevention," causing the clinical symptoms not to develop, e.g., preventing infection from occurring and/or developing to a harmful state; (ii) "inhibition," arresting the development of clinical symptoms, e.g., stopping an ongoing infection so that the infection is eliminated completely or to the degree that it is no longer harmful; and (iii) "relief," causing the regression of clinical symptoms, e.g., causing a relief of fever and/or inflammation caused by an infection. Treatment may comprise the prevention, inhibition, or relief of biofilm formation. Administration to an individual "at risk" of having a bacterial infection means that the individual has not necessarily been diagnosed with a bacterial infection, but the individual's circumstances place the individual at higher than normal risk for infection of infection, e.g. the individual is a burn victim. Administration to an individual "suspected" of having a bacterial infection means the individual is showing some initial signs of infection, e.g. elevated fever, but a diagnosis has not yet been made or confirmed.

The term "effective amount" means a dosage sufficient to provide treatment. The quantities of active ingredients necessary for effective therapy will depend on many different factors, including means of administration, target site, physiological state of the patient, and other medicaments administered; therefore, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in vivo administration of the active ingredients. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. The concentration of the active ingredients in the pharmaceutical formulations typically vary from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Various appropriate considerations are described, for example, in Goodman and Gilman, "The Pharmacological Basis of Therapeutics," Hardman et al., eds., 10.sup.th ed., McGraw-Hill, (2001) and "Remington: The Science and Practice of Pharmacy," University of the Sciences in Philadelphia, 21.sup.st ed., Mack Publishing Co., Easton Pa. (2005), both of which are herein incorporated by reference in their entirety. Methods for administration are discussed therein, including administration by oral, intravenous, intraperitoneal, intramuscular, transdermal, nasal, and iontophoretic routes, and the like.

The compositions of the invention may be administered in a variety of unit dosage forms depending on the method of administration. For example, unit dosage forms suitable for oral administration include solid dosage forms such as powder, tablets, pills, and capsules, and liquid dosage forms, such as elixirs, syrups, and suspensions. The active ingredients may also be administered parenterally in sterile liquid dosage forms. Gelatin capsules contain the active ingredient and as inactive ingredients powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like.

Examples of inactive ingredients that may be added to the composition of the invention include agents that provide desirable color, taste, stability, buffering capacity, dispersion or other features, such as red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

The compositions of the invention may also be administered via liposomes, including emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the composition of the invention to be delivered may be incorporated as part of the liposome, alone or in conjunction with a targeting molecule, such as antibody, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired composition of the invention of the invention can delivered systemically or can be directed to a tissue of interest.

Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by the desired liposome size, acid lability and stability in the blood stream. A variety of methods are available for preparing liposomes as described in Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, which are incorporated herein by reference. A liposome suspension containing a composition of the invention may be administered intravenously, locally, topically, etc. in a dose which varies according to the manner of administration, the composition of the invention being delivered, and the stage of the disease being treated, among other things.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, and more preferably at a concentration of 25%-75%. The constructs of the invention can additionally be delivered in a depot-type system, an encapsulated form, or an implant by techniques well-known in the art. Similarly, the constructs can be delivered via a pump, e.g. an osmotic pump, to a tissue of interest.

For aerosol administration, the compositions of the invention are preferably supplied in finely divided form along with a surfactant and propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

For the purpose of the invention, "administration of a composition" includes the administration of separate formulations of the RNAIII-inhibiting peptide and the antimicrobial peptide(s) and/or antibiotic(s) to the same individual at or around the same point in time, such that therapeutic concentrations of each active ingredient are achieved at the same time in the individual. The term also includes administering an antibiotic(s) to the individual in the same formulation that comprises the RIP and antimicrobial peptide, or administering the antibiotic(s) as a separate formulation at or around the same time as the RIP and antimicrobial peptide are administered. For example, the present method comprises oral co-administration of separate pills containing RIP, an antimicrobial peptide and an antibiotic. Useful antibiotics include aminoglycosides (e.g., gentamycin), beta-lactams (e.g., penicillin), cephalosporin or vancomycin. Administration of the RIP and antimicrobial peptide may occur within about 48 hours and preferably within about 2-8 hours, and most preferably, substantially concurrently with administration of the antibiotic.

The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment, prevention, inhibition or relief of pathogenic bacterial infection. The therapeutic agents may be administered in a variety of ways, such as orally, topically, parenterally, intraperitoneally, intravascularly, intrapulmonary (i.e., inhalation), etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways.

The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt. %. The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the therapeutic situation. Human dosage levels for treating infections are known and generally include a daily dose from about 0.1 to 500 mg/kg of body weight per day, preferably about 6 to 200 mg/kg, and most preferably about 12 to 100 mg/kg. The amount of formulation administered will, of course, be dependent on the subject and the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician. Generally, serum concentrations should be maintained at levels sufficient to treat infection in less than 10 days, although an advantage offered by the present invention is the ability to extend treatment for longer than 10 days at relatively low levels of the composition because of the decreased likelihood that bacteria will develop resistance to the present composition over treatment.

The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents. The compositions may include other pharmaceutical excipients, carriers, etc. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like. Methods of preparing pharmaceutical compositions are well known to those skilled in the art. See, for example, "Remington: The Science and Practice of Pharmacy," University of the Sciences in Philadelphia, 21.sup.st ed., Mack Publishing Co., Easton Pa. (2005).

The present composition is useful in reducing the overall pathology or delaying the onset of disease symptoms in various diseases caused by bacterial infection in addition to bacterial sepsis, including bacterial-induced systemic inflammatory syndrome (SIRS), cellulitis, keratitis, osteomyelitis, septic arthritis, mastitis, skin infections, pneumonia, endocarditis, meningitis, post-operative wound infections, device-associated infections and toxic shock syndrome.

Treatment of Biofilm-Related Infections

Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. See Costerton et al., Science 284: 1318-22 (1999). Biofilms develop preferentially on inert surfaces, or on dead tissue, and occur commonly on medical devices and fragments of dead tissue such as sequestra of dead bone; they can also form on living tissues, as in the case of endocarditis. Biofilms grow slowly, in one or more locations, and biofilm infections are often slow to produce overt symptoms. Sessile bacterial cells release antigens and stimulate the production of antibodies, but the antibodies are not effective in killing bacteria within biofilms and may cause immune complex damage to surrounding tissues. Even in individuals with excellent cellular and humoral immune reactions, biofilm infections are rarely resolved by the host defense mechanisms. Antibiotic therapy typically reverses the symptoms caused by planktonic cells released from the biofilm, but fails to kill the biofilm. For this reason biofilm infections typically show recurring symptoms after cycles of antibiotic therapy, until the sessile population is surgically removed from the body. It is therefore preferable to prevent biofilm formation rather than to try to eradicate biofilms once they have formed.

The compositions and methods of the present invention are useful in the treatment of bacterial infection associated with biofilms, or in reducing the risk of a disease associated with biofilms, particularly biofilms caused by bacteria whose pathogenicity is related to a lipidic and polyanionic components of the bacterial cell envelope. For example, a composition comprising a RIP and an antibiotic, such as an aminoglycoside, a beta-lactam, cephalosporin or vancomycin, may be used to treat or reduce the risk of biofilms. In another embodiment, the RIP is combined with an antimicrobial peptide in addition to, or instead of, a conventional antibiotic to treat or reduce the risk of an infection associated with a biofilm.

The present composition may be used to coat devices that are inserted into an individual, e.g., a surgical device, catheter, prosthetic or other implant, to reduce the risk that the implanted device will develop a biofilm. Alternatively, the composition may be implanted to provide a high, localized concentration of the composition in the treatment of a localized infection. In this embodiment, the composition may be provided in a depot and formulated for sustained release. Table 2 (see Original Patent) provides a partial list of nosocomial infections, for which the present composition and method are expected to be useful.
 

Claim 1 of 12 Claims

1. A method of treating or reducing the risk of bacterial infection in a mammalian individual, comprising administering to said individual a composition comprising an amount of an isolated RNAIII-inhibiting peptide (RIP) and an antimicrobial peptide capable of binding and neutralizing a lipidic and polyanionic component of a bacterial cell envelope, where the RIP and antimicrobial peptide are present in an amount effective to treat or reduce the risk of a bacterial infection in said individual, where the bacterial infection is caused by Staphylococcus spp Bacillus spp., B. subtilus, B. cereus, B. anthracis, Listeria spp., L. innocua, L. monocytogenes, Streptococcus pyogenes, Lactococcus lactis, Enterococcus faecalis, Escherichia coli, or Clostridium acetobtylicum.

 

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