# Patent

## Title
Use of antimicrobial proteins and peptides for the treatment of otitis media and paranasal sinusitis

## Abstract

Disclosed herein is a composition and a method for the treatment of otitis media and paranasal sinusitis using human defensins, lysozyme and/or lactoferrin as a new class of non-antibiotic antimicrobials. From studies of otitis media and paranasal sinusitis, it was observed that certain innate immune modulators were important in the bodies response to the infection. Therefore, these innate immune modulators, lysozyme, lactoferrin, and defensins were tested for use as a non-antibiotic treatment for infection, particularly infections such as otitis media and sinusitis.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 09/998,547 filed Nov. 27, 2001, now U.S. Pat. No. 6,716,813, which claims priority of the U.S. Provisional Application 60/253,492, filed Nov. 20, 2000, both of which are herein incorporated by reference in their entireties.


This invention was made with U.S. Government support under grant NIH NIDCD R01 DC05025. The U.S. Government has certain rights in the invention.


FIELD OF THE INVENTION
The invention relates generally to the use of human beta defensins, lysozyme, and lactoferrin as a new class of non-antibiotic antimicrobials. More specifically, the invention relates to the use of these antimicrobials for the treatment of otitis media and paranasal sinusitis.
BACKGROUND OF THE INVENTION
The rapid worldwide increase in antibiotic resistance among pathogens has given rise to an urgent need to develop new and innovative non-antibiotic approaches to prevent and manage disease. Otitis media and sinusitis are two very common infections which are difficult to treat for a number of reasons, including antibiotic resistance. Otitis media (OM) is the most prevalent infectious disease affecting young children, and the major cause of conductive hearing loss among this group. OM is also the leading indication for antibiotic therapy. OM results in 31 million annual visits to physicians' offices and is estimated to have a yearly cost exceeding $5 billion.
Most cases of OM are caused by one of three major pathogens, Streptococcus pneumoniae (S. pneumoniae) (30–40%), non-typeable Haemphilus influenzae (NTHi) (30%) and Moraxella catarrhalis (M. catarrhalis) (20%). Typically, these infections are treated with antibiotics. In the past three decades, there has been a dramatic worldwide increase in antibiotic resistance in OM pathogens. This has resulted in a reduction of the number of effective antibiotics for this disease and begun to pose a major public health threat. Thus, the use of antibiotics is becoming more complicated as resistance increases, necessitating the testing of microbes before treatment, which can sometimes fatally delay the necessary treatment. In addition, wide antibiotic use is further contributing to the problem of resistance. The need to identify new antibiotics is causing the price of these substances to be so high as to be prohibitive in some cases. Therefore, there is a need for new, innovative, and cost-effective approaches to prevent and manage these diseases. It is believed that the defenses of the eustachian tube and the middle ear (tubotympanum) help to maintain the sterility of the middle ear under normal conditions. To this end, an understanding of the mechanisms that protect the tubotympanum (the middle ear and eustachian tube) from invading organisms and determine the role that they play in the pathogenesis of OM could prove useful in identifying a new treatment.
SUMMARY OF THE INVENTION
A method and an antimicrobial composition for the treatment of ear and sinus infections is disclosed which is effective, reasonably priced, and does not contribute to the problem of antibiotic resistance.
Thus, one embodiment is a pharmaceutical preparation for the treatment of otitis and sinusitis, which has at least one component selected from the group consisting of: lactoferrins, lysozyme, and defensins in an amount effective to reduce the growth of microbes, and a pharmaceutically acceptable carrier. In one embodiment, the defensins are alpha defensins or beta defensins, particularly beta defensin 1 or beta defensin 2 and preferably beta defensin 2. In one embodiment, the dose is from about 0.1 to 100 mg/kg/dose, alternatively from about 0.5 to 50 mg/kg/dose or 0.1 to 1000 mg/kg/day. In one embodiment, the pharmaceutical preparation includes β-defensin-2, lysozyme, and β-defensin-1 in an excipient formulated for treatment of and administration to the middle ear.
A further embodiment is a method for the treatment of microbial infections of the ear and sinuses in a mammal, by administering to the mammal a pharmaceutical preparation comprising at least one component selected from the group consisting of lactoferrins, lysozyme, and defensins in an amount effective for the reduction in numbers of the causative infective agents. In one embodiment, the microbial infections of the ear and sinus are otitis media and paranasal sinusitis. In a further embodiment, the administration is orally, intranasally, by aerosolization or into the ear canal. In one embodiment, the beta defensins are beta defensin 1 or beta defensin 2, preferably beta defensin 2. The pharmaceutical may be administered at a dose of about 0.1 to 100 mg/kg/dose, alternatively, 0.5 to 50 mg/kg/dose, 0.1 to 1000 mg/kg/day, or about 0.8 to 800 mg/kg/day. The pharmaceutical composition may be administered 1 to 8 times per day as a pill, a solution, or a suspension. In one embodiment, the pharmaceutical composition includes a salt chelator. In a further embodiment, the pharmaceutical composition includes both lysozyme and lactoferrin. In a further embodiment, the pharmaceutical composition includes both lysozyme and a defensin, preferably β-defensin-2. In a further embodiment, the pharmaceutical composition includes lysozyme, beta defensin 1 and beta defensin 2. The mammal may be a human, a dog, a cat, a horse, a ferret, a mouse, a rat or a cow.
In a further embodiment, the Otitis media is caused by a microbe selected from the group consisting of NTHi strain 12, M. catarrhalis, S. pneumoniae serotype 3, and S. pneumoniae serotype 6B.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing will be provided by the U.S. Patent and Trademark Office upon request and payment of necessary fee.
FIG. 1 is a gel overlay assay for the anti-microbial activity of rat middle ear effusion proteins resolved by acid-urea gel electrophoresis. Lane 1, 0.5 mg protegrin; Lane 2, 1 mg human milk lysozyme and human β-defensin 2; Lane 3, PBS wash of normal middle ear; Lane 4, effusion from middle ear treated with 1.0 mg S. typhimurium LPS.
FIG. 2 shows the fold change of MRNA levels for human lysozyme, lactoferrin, β-defensin 1 (HBD1) and β-defensin 2 (HBD2) from real-time PCR analysis of the expression of these molecules in normal and inflamed middle ear tissue (black bars). The β-actin gene served as the internal standard. RNAs from one sample of normal and one sample of inflamed middle ear tissue were reverse transcribed and subjected real-time PCR. The ordinate is log10 of the fold difference of MRNA levels for each antimicrobial molecule between normal and inflamed middle ear mucosa samples. The results are based on the differences of threshold cycle for each molecule in the normal and inflamed samples.
FIG. 3 is a representative radial inhibition assay for the measurement of the activity of innate immune molecules against nontypeable Haemophilus influenzae strain 12 (NTHi12), Moraxella catarrhalis strain 035E (M cat), Streptococcus pneumoniae serotype 3 (Sp3) and Streptococcus pneumoniae serotype 6b (Sp 6b) Three doses (4 μg, 10 μg and 40 μg) of human lysozyme (Lz) and lactoferrin (Lf) were tested against the bacteria. For human β-defensin-1 (hBD-1) and β-defensin-2 (hBD-2), two doses (4 μg and 10 μg) were tested. Each dose was delivered in a total volume of 4 μl. The control well received only solvent (0.01% acetic acid). The diameter of the inhibition zone was measured and averaged in three separate experiments.
FIG. 4 is a graph demonstrating the inhibition of NTHi growth by beta defensins 1 and 2, lysozyme and lactoferrin in a colony formation assay.
FIG. 5 shows the effect on the ultrastructure of NHTi after treatment with natural antibacterial agents. A) No treatment showing normal appearance of NTHi. An inner membrane encloses the nucleic acid compartment. The outer surface membrane encloses a compartment filled with amorphous appearing material. Bar=100 nm. B) Treatment with lysozyme (100 μg/ml). Few completely lysed bacteria were observed. Bar=0.5 micron. Bar=0.5
FIG. 6 is an ultra-structural analysis of the effect of human β-defensin-2 and lysozyme on NTHi showing the damage caused to the bacteria by the treatment. Untreated NTHi are shown in panel A. The bacteria were treated for three hours with β-defensin-2 (10 μg/ml) (panel B), or with 1 mg/ml human milk lysozyme (panel C). Bar=0.2 micron.
FIG. 7 is an ultra-structural analysis of the effect of human β-defensin-2 and lysozyme on S. pneumoniae serotype 3 showing the damage caused to the bacteria by the treatment. Untreated bacteria are shown in panel A. The bacteria were treated for three hours with human β-defensin-2 (10 μg/ml) (panel B), or with 1 mg/ml human milk lysozyme (panel C). Untreated NTHi are shown in panel B. Bar=0.5 micron.
FIG. 8 is an ultra-structural analysis of the effect of human β-defensin-2 on S. pneumoniae serotype 6B showing the damage caused to the bacteria by the treatment. Untreated bacteria are shown in panel A. The bacteria were treated for three hours with human β-defensin-2 (10 μg/ml) are shown in panel B. Bar=0.5 micron.
FIG. 9 is an ultra-structural analysis of the effect β-defensin-2 and lysozyme on M. catarrhalis. Untreated bacteria are shown in panel A. Results of a 1-minute treatment of the bacteria with lysozyme are shown in panel B and those of a 30-minute incubation with β-defensin-2 are shown in panel C. Bar=0.5 micron.
FIG. 10 shows immunolabeling of archival temporal bone sections showing no change in β-defensin 1 between OM and control. However, human β-defensin 2 is expressed at high levels in middle ear epithelium from OM patients (A), but not in normal subjects (B).



Further aspects, features and advantages of this invention will become apparent from the detailed description of the preferred embodiments which follow.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A method and a composition for the treatment of ear and sinus infections is disclosed. The method and composition take advantage of the fact that some of the body's innate defenses, lysozyme, lactoferrin and the β-defensins, are typically expressed by middle mucosal ear epithelial cells upon infection. The method and composition also take advantage of the fact that lysozyme and the β-defensins can cause damage to and inhibit the growth of nontypeable Haemophilus influenzae strain 12 (NTHi12), Moraxella catarrhalis strain 035E, and Streptococcus pneumoniae serotypes 3, and 6B, three bacteria most commonly found to cause OM. The studies herein show that the innate immune system may be one of the primary lines of defense against middle ear infections and, thus, future therapies based on these molecules may hold promise for the treatment of otitis media and sinusitis.
Surface epithelia, including the epithelium of the middle ear, form the first barrier against pathogen invasion. Innate immune molecules produced by the epithelial cells provide the host with a constitutive or immediately inducible defense system that is capable of effectively dealing with the continuous attacks of a variety of pathogens at the mucosal epithelial surfaces. Like that of the lung, the epithelium of the nasopharyngeal tract is constantly exposed to a multitude of microorganisms. Although the nasopharynx is connected to the middle ear cavity via the eustachian tube, giving pathogens potential access to this site, under normal conditions, the middle ear of humans and laboratory animals remains sterile. Furthermore, non-inflamed tubal and middle ear mucosa have been shown to contain relatively few immunocytes. These findings suggest that the components of the innate immune system may be important in protecting the tubotympanum (middle ear and eustachian tube) and may be playing the role of the first line of defense, prior to the activation of adaptive immunity, against otitis media pathogens.
The pathogenesis of OM is multi-factorial, with risk factors such as upper respiratory viral infection, poor tubal function, delayed development of the immune system, as well as other environmental and genetic factors, play an important role, depending on the age of the infected individual. Available evidence suggests that the mucociliary system, systemic immunity (humoral and cell mediated) and the complement system play important roles in the protection of the tubotympanum. Recent evidence, however, suggests that other biological defense systems—particularly the innate (natural) immune system—may also be playing a role in the protection of the middle ear against OM pathogens. It is now evident that homeostasis of the nasopharyngeal tract, eustachian tube and the middle ear is maintained in part by anti-microbial proteins and peptides, including lysozyme, lactoferrin, and the defensins.
In addition, there is a need for non-antibiotic treatment of infections in view of the problems associated with decades of antibiotic use and misuse. The incidence of antibiotic resistance is increasing rapidly to the point where some microbes are resistant to all of the present antibiotics known. This requires a careful choice of treatment as well as reducing the speed of treatment, because it may require testing to identify which antibiotic will be effective for treating the specific microbe. In addition, wide antibiotic use is further contributing to the problem of resistance. Thus, the need to identify new antibiotics is causing the price of these substances to be so high as to be prohibitive in some cases.
Thus, one embodiment is a method for the treatment of infection in the middle ear and sinuses by administering one or more antimicrobial peptides to the middle ear and/or sinuses to inhibit the growth of pathogenic microorganisms. This method is based on an antimicrobial mixture for which one or more components are selected from the group consisting of defensins, lysozyme and lactoferrin, particularly the beta defensins.
Because molecules of the innate immune system including lysozyme, lactoferrin, and the defensins form the first line of defense of the tubotympanum and paranasal sinuses during otitis media and paranasal sinusitis, these molecules present an option to be used alone against otitis media and paranasal sinusitis or to be used in combination with other known methods of treatment, such as antibiotics. These innate mediators mount a rapid response against the invading pathogens, before adaptive immune mechanisms are mobilized in vivo. As such, these molecules represent ideal therapeutic candidates to replace antibiotics as the primary treatment modalities for otitis media, sinusitis, and other types of infections.
The present invention uses these innate immune antibiotic molecules to treat infections. This treatment offers a number of advantages. Unlike existing antibiotics, these molecules are unlikely to induce antibiotic resistance. A further advantage is that these molecules, because they are produced by the host, will not induce allergic reactions. Lastly, the method of the present invention is more cost effective then that of antibiotic treatment.
Lysozyme (Lz)
Lysozyme also known as muramidase, is an important component of innate immunity against pathogens at mucosal surfaces. It catalyzes the hydrolysis of 1–4-glycosidic bonds between N-acetylmuraminic acid and N-acetyl-D-glucosamine, which are constituents of the cell walls of most bacteria. Lz has potent antibacterial properties and is thus an important participant in host defense at mucosal surfaces, pleural fluid and in leukocytes. Recent work has shown that inhibition of cationic antimicrobial proteins such as Lz predisposes the airway epithelium to infection. At mucosal surfaces, Lz expression is confined to specialized epithelial cells, including those of the serous glands of the respiratory epithelium and the Paneth's cell of the gastrointestinal epithelium. Lz is also produced by macrophages and cells of the granulocyte lineages.
Chronic middle ear effusions contain high levels of Lz, which is produced by secretory cells of the middle ear mucosal epithelia, as well as by PMNs and macrophages. Hence, chronic middle ear effusions of humans contain high levels of this protein. The fraction of Lz produced by middle ear epithelial cells, has been shown to account for 50% to 80% of the variation of Lz levels seen in middle ear effusions. The presence of Lz in serous cells of the tubal glands and the secretory epithelial cells suggests a role for this molecule in the defense of the middle ear and eustachian tube against pathogens. The developmental course of Lz secretion in the murine tubotympanum suggests that its maturation follows that of secretory components and that it occurs postnatally.
In the middle ear, lysozyme is produced by the secretory cells of the epithelium, as well as by the PMNs and macrophages. Hence, chronic middle ear effusions of humans contain high levels of this protein. The presence of lysozyme in serous cells of the tubal glands and the secretory epithelial cells suggests a role for this molecule in the defense of the tubotympanum against pathogens.
Lactoferrin (Lf)
Lactoferrin is an iron-binding glycoprotein found in the milk and exocrine secretions of mammals and which is released from neutrophilic granules during inflammation. Lf receptors have been detected on activated T and B cells, monocytes, intestinal brush border cells, platelets and neoplastic cells. Ingested microorganisms are destroyed in cellular phagosomes containing proteolytic enzymes, including Lf. Arnold and colleagues demonstrated a bacterial killing property of Lf on a variety of bacteria. It also has been shown that Lf synergistically interacts with immunoglobulins, complement, and neutrophil cationic proteins against Gram-negative bacteria. Moreover, Lf can damage the outer membrane of Gram-negative bacteria. It has been well established that breast milk fed babies are more resistant to otitis media than babies fed cow milk. Antibacterial lysozyme and Lf in human milk may contribute to the protection of breast fed babies from otitis media.
It was recently shown that, in addition to its iron-binding properties, Lf possessed proteolytic activity. This activity was first discovered when strains of NTHi were cultured in the presence of human milk whey. The only substrates identified for the proteolytic activity of Lf to date are two related proteins of NTHi, the non-pilus Hap adhesin and the IgA protease precursor. Other outer membrane proteins of NTHi, such as P2, P5 and P6 are unaffected. The proteolysis of pilus Hap adhesin and the IgA protease precursor yields several large fragments, suggesting that Lf attacks specific peptide bonds in these proteins. Furthermore, although the proteolytic activity of Lf causes Hap-positive NTHi strains to lose their ability to adhere to Chang epithelial cells and removes most of the IgA protease inhibitor from the outer membrane of these bacteria, it-does not affect the viability of the bacteria. The biological importance of the Lf hydrolysis of NTHi is presently unknown, but the effect may be to attenuate the pathogenicity of this microorganism.
Lf belongs to the gene family that includes transferrin (Tf) and the melanoma surface antigen P97, and shows a high level of homology in both its sequence and genomic organization with transferrin. To date, Tfs and Lfs from a variety of sources and cells have been cloned. Pierce and colleagues screened a lactating mammary gland cDNA library with rat liver Tf as probe as well as with a human Lf cDNA in order to pull out the rat Tf and Lf clones. Their results suggest however, that Lf is either not expressed by the rat mammary gland or that it is expressed at undetectably low levels. Previous results suggest that a similar situation may exist in the rat tubotympanum. Previous results herein were not able to demonstrate expression of Lf in rat tubotympanal epithelial cells or in the tissue. Without being restricted to the following theory, it is possible that Lf is absent in rat and Tf may have replaced its function. Lf has also been detected in middle ear effusion, and has been localized to the serous cell of the eustachian tube glands and to the cuboidal epithelium area (containing the serous cells) of the transition zone of the middle ear. These data suggest that Lf may play a role against bacterial infection of the middle ear and eustachian tube.
Defensins
Antimicrobial peptides have been identified as key elements in the innate host defense against infection. The defensins are cationic (polar) molecules with antimicrobial activity and have spatially separated hydrophobic and charged regions which have antimicrobial activity. In vitro, defensins (at micromolar concentrations) have a broad spectrum of antimicrobial activity against bacteria, fungi, and even some enveloped viruses. In mammals and birds, defensins are among the most abundant polypeptides secreted by phagocytic white blood cells involved in host defense against bacteria and fungi. During phagocytosis, ingested microbes are exposed to very high concentrations of defensins. The defensins may also have roles in protecting the host, based on their capacity to chemoattract T cells, to promote host immunity, and to activate the classical complement pathway.
In humans and other vertebrates, the defensins can be divided into the alpha- and beta-defensin subfamilies on the basis of the position and bonding of six, conserved cysteine residues. The alpha-defensins are produced by neutrophils and intestinal Paneth's cells. The precursors of alpha-defensin peptides require activation for bactericidal activity. In mouse small intestine, a metalloproteinase, matrilysin, found in the Paneth cell granules, is capable of cleaving the pro segment from alpha-defensin (cryptidin) precursors.
The beta-defensins, on the other hand, are mainly produced by epithelial cells of the skin, kidneys, and trachea-bronchial lining of nearly all vertebrates. There are no reports of the expression of beta defensins in middle ear or eustachian tube epithelial cells, although previous data suggests that the beta defensins are expressed in these tissues. Because beta-defensins are released upon microbial invasion and are located at the host-environment interface, such as mucosal surfaces and skin, they may also function to alert the adaptive immune system of vertebrates. Human beta-defensin 2 (hBD-2), produced by epithelial cells, exhibits potent antimicrobial activity against Gram-negative bacteria and Candida, but is not as effective against Gram-positive Staphylococcus aureus. HBD-2 represents the first human defensin that is produced following stimulation of epithelial cells by contact with microorganisms such as Pseudomonas aeruginosa or cytokines such as TNF-alpha and IL-1 beta. Human beta defensin 2 functions as an NF-kB target gene in the intestinal epithelium and blocking NF-kB activation inhibits the up-regulated expression of hBD-2 in response to IL-1 alpha stimulation or bacterial infection. The HBD-2 gene and protein are locally expressed in keratinocytes associated with inflammatory skin lesions such as psoriasis as well as in the infected lung epithelia of patients with cystic fibrosis.
HBD-1 however, is not affected by IL-1 alpha and other proinflammatory stimuli, thus suggesting that unlike the inducible HBD-2 protein, HBD-1 may serve as a defense in the absence of inflammation. The presence of HBD-1 has been reported in the pars tensa and pars flaccida of the tympanic membrane and in the meatal skin. In situ hybridization studies localized the HBD-1 mRNA to the epidermal layer of these tissues. The HBD-1 transcripts were also evident in the sebaceous glands and in hair follicles of the meatal skin. In contrast, HBD-1 MRNA was not detected in the tympanal epithelium of the eardrum.
Defensins are normally sequestered in cytoplasmic granules with their primary site of action in phagolysosomes, although some peptide is released into the circulation during the course of infection or inflammation. Developmental regulation of the alpha defensins (cryptidins) has been studied in mice, where it has been shown that cryptdin-6 is the most abundant enteric defensin mRNA in the newborn. Paneth cell mRNAs, including cryptdins-4 and -5, lysozyme, matrilysin, and defensin-related sequences, also were detected in RNA from P1 mouse intestine. Unlike adult mice, where only Paneth cells are immunopositive for cryptdin, cryptdin-containing cells were distributed throughout the newborn intestinal epithelium and not in association with rudimentary crypts. In studies of human newborns, it was found that their neutrophils were not deficient in defensins although they were deficient in bactericidal/permeability-increasing protein (BPI), a 55 kD polypeptide that binds with high affinity to bacterial lipopolysaccharides and kills gram-negative bacteria. The human enteric defensins, on the other hand, are present at sub-adult levels in the developing embryo and it is postulated that levels of enteric defensin expression in the fetus may be characteristic of an immaturity of local defense, which is thought to predispose infants born prematurely to infection from intestinal microorganisms.
In vitro, the defensins (at micromolar concentrations) have a broad spectrum of antimicrobial activity against bacteria, fungi, and even some enveloped viruses. In mammals and birds, defensins are among the most abundant polypeptides secreted by phagocytic leukocytes and epithelial cells involved in host defense. During phagocytosis, ingested microbes are exposed to very high concentrations of defensins. The defensins may also have roles in protecting the host, by their capacity to chemoattract T cells, to promote host immunity, and to activate the classical complement pathway.
Surface epithelia, including the epithelium of the middle ear, form the first barrier against pathogen invasion. Innate immune molecules produced by the epithelial cells provide the host with a constitutive or immediately inducible defense system that is capable of effectively dealing with the continuous attacks of a variety of pathogens at the mucosal epithelial surfaces. Like that of the lung, the epithelium of the nasopharyngeal tract is constantly exposed to a multitude of microorganisms. Although the nasopharynx is connected to the middle ear cavity via the eustachian tube, giving pathogens potential access to this site, under normal conditions, the middle ear of humans and laboratory animals remains sterile. Furthermore, non-inflamed tubal and middle ear mucosa have been shown to contain relatively few immunocytes. These findings suggest that the components of the innate immune system may be important in protecting the tubotympanum (middle ear and eustachian tube) and may be playing the role of the first line of defense, prior to the activation of adaptive immunity, against otitis media pathogens.
Active Variants and Homologues
Lactoferrin, lysozyme, and the defensins are well-known molecules which have previously been analyzed in detail. Thus, the regions necessary for activity of these molecules are easily identified. Active variants which possess truncations, single or multiple nucleotide transitions and transversions, and additions may be easily identified and produced with little to no loss of activity. In one embodiment, the active variants which are produced contain at least about 60% of the activity of the non-mutagenized protein, including, but not limited to: 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% of the activity.
For example, any mammalian lysozyme may be cloned using methods known to one of skill in the art, for example, by PCR or by isolating or purchasing a full-length cDNA from a library (also see Kikuchi, et al. U.S. Pat. No. 4,945,051, issued Jun. 24, 1987 for a method of producing recombinant human lysozyme—both of which are herein incorporated by reference). Using the information in the literature which identifies regions necessary for lysozyme activity, variants can be produced by deleting, truncating, or mutating regions which are not necessary. For example, Conneely, et al. U.S. Pat. No. 6,111,081, issued May 30, 1997 and U.S. Pat. No. 5,571,691, issued Oct. 28, 1993 (both herein incorporated by reference) provide a method for cloning and mutagenizing lactoferrin, without affecting its iron binding activity. A number of variants are disclosed.
The β-defensins are peptides which are generally between about 38 and 42 amino acids which make up a chain having a net charge of +4 to +10. They are further characterized by their content of half-cysteine residues which are distributed in the peptide chain separated by 6 intervening residues. The third and fourth half-cysteines are separated by 4 intervening residues; the second and third half-cysteines are separated by 9 intervening residues. The fourth and fifth half-cysteines are separated by 6 intervening residues; and the fifth and sixth half-cysteines are adjacent. Furthermore, the cysteine residues are paired via disulfide bonds in a characteristic manner: the first cysteine to the fifth cysteine; the second cysteine to the fourth cysteine, and the third cysteine to the sixth cysteine. Some β-defensins are characterized by a pyroglutamate residue at the amino terminus which makes these molecules resistant to most aminopeptidases. Thus, one of skill in the art, having this detailed information about β-defensins as well as that in the literature could identify mutations which would have the least affect on the antimicrobial activity (see also Selsted, et al, U.S. Pat. No. 6,211,148, issued Dec. 11, 1997 herein incorporated by reference).
However, in all cases, the antimicrobial activity of the mutants may be tested in a radial assay, such as that detailed in Example 1 or any other assay known to one of skill in the art.
Homologues of these molecules may be identified in all mammals. Thus, homologues may be identified which may be used for the treatment of infections in other mammals, including, but not limited to: dogs, cats, horses, monkeys, apes, cows, sheep, pigs, and a variety of zoo animals. One of skill in the art may identify homologues by searching known public and private databases. It is envisioned that most homologues have already been identified. However, if a homologue has not yet been identified, one of skill in the art may identify a homologue using known techniques.
For example, should one of skill in the art wish to identify the rhinoceros lactoferrin homologue, he or she may produce probes or primers which are specific to conserved regions of the lactoferrin proteins. The skilled artisan may then purchase or produce a rhinoceros cDNA library and, using the probes or primers, isolate the transferrin homologue. Other methods known to one of skill in the art may also be used to isolate and/or identify homologues.
Anti-Microbial Composition
The proteins or active variants of the anti-microbial compounds herein may be purified from a natural source, such as, but not limited to, a body fluid, milk, or neutrophils (see Selsted, et al, U.S. Pat. No. 6,211,148, issued Dec. 11, 1997) Alternatively, they may be expressed recombinantly and purified by any method known to one of skill in the art. The proteins or active variants are said to be “substantially free of natural contaminants” if preparations which contain them are substantially free of materials with which these products are normally and naturally found. Active variants may be produced using methods known to those of skill in the art. However, typically, the genes coding for the proteins are cloned and mutagenesis is performed on the gene which is then expressed and the mutagenized protein isolated. Natural active variants may also be purified from a mammal which naturally produces such variants.
Compositions for use in the methods herein may contain one or more active proteins or variants selected from the group consisting of lysozyme, lactoferrin, alpha defensins, and beta defensins. In one embodiment, the composition contains only one of these proteins. In a further embodiment more than one of these proteins is included in the composition, including but not limited to two, three, and four.
In one embodiment, other treatments are included in the composition. The other treatments may be any treatment which may enhance the anti-microbial properties, reduce the side-effects, enhance uptake, and increase the comfort of the patient. For example, it may be possible to include substances which reduce the drying effect on the membranes, or increase healing of the membranes in the area in which it is to be administered.
Vectors Expressing Proteins or Active Variants
It can be envisioned that one method of administering the lysozyme, lactoferrin, alpha defensins, and beta defensins uses expression vectors which express these proteins. The expression vectors may be targeted to the tissue or cell which is infected or which is near the infected cells. The vectors may be any vectors known to one of skill in the art including but not limited to: viral vectors, plasmid vectors, and naked DNA. Expression from these vectors may be constitutive or may be under the control of a specific promoter, such as a eukaryotic promoter, or an inducible promoter.
One advantage of this method is that for those patients who experience chronic otitis or sinusitis, the presence of a vector may allow the effectiveness of the treatment to last for a longer time.
Method of Admistration and Dosage
It is envisioned that the antimicrobial mixture can be administered to any type of infection, by injection, topically, or even orally. The method will depend on the type of infection being treated. For the treatment of otitis media and sinusitis, the antimicrobial mixture may be administered to the ear or the sinuses. The administration may be from the outer ear to the middle ear, e.g. to a patient whose ear drum is pierced and a grommet inserted. Alternatively, if the infection is otitis externa, the administration may be using ear drops. If the infection is of the middle ear, the ear drops may contain a substance which allows permeabilization of the antimicrobial molecules across the ear drum. In a further embodiment, the drug may be administered orally or intranasally where the antimicrobial mixture will act as an antimicrobial agent. Alternatively, the antimicrobial mixture may be administered orally, intravenously, intramuscularly, in the tear ducts, or by inhalation.
Substances which may be used to permeabilize the ear drum and allow entry of the antimicrobial molecules may be any substance which increases the permeability of membranes, such as those which are used to permeabilize skin in dermatology. Examples of such substances include, but are not limited to dimethylsulfoxide (DMSO), dimethylacetamide, methyldecyl sulfoxide, cotton seed oil, caster oil derivatives, fatty acid esters, glycerol, vesicles, liposomes, silicone vesicles (see Hill, et al. U.S. Pat. No. 5,364,633, issued Mar. 14, 1994, herein incorporated by reference), anionic surfactants, preparations such as those in Miyazawa, et al. U.S. Pat. No. 5,500,416, issued Sep. 10, 1993 (herein incorporated by reference),
A composition is said to be “pharmacologically acceptable” if its administration can be tolerated by the recipient patient. Such an agent is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. Alternatively, the amount may be analyzed by the effect. For example if the chosen amount produces a reduction in the number of microbes.
The dosage of the protein components of the antimicrobial mixture to be administered may vary with the method of administration and the severity of the condition to be treated. In general, however, a dosage of from about 0.1 to 100 mg/kg/dose, and more preferably 0.5 to 50 mg/kg/dose of the drug administered 1 to 8 times a day by the intranasal route, or from 1 to 10 drops of a solution or suspension administered from 1 to 10 and preferably 1 to 6 times a day, to each ear. In a further embodiment, from about 0.01 mg/ml to about 100 mg/ml, including, but not limited to 0.1 mg/ml, 1 mg/ml, 2, mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, and 90 mg/ml is administered to the ear, sinuses, or upper respiratory tract at least one time per day. Local administration is preferable because it reduces that chances of unwanted side-effects. However, for systemic administration, a dose of from about 0.01 mg/ml to about 1 g/ml may be administered at least one time per day for at least and including one day.
The composition for administration may additionally include additives, excipients, thickeners, and other substances which allow for more effective administration. Examples include oils, emolients, or other substances which increase the effectiveness and comfort of ear drops, nasal sprays, and inhalable compositions. This may also include substances which enhance the smell or taste.
Additional pharmaceutical methods may be employed to control the duration. Controlled release preparations may be achieved through the use of polymers to complex or adsorb the composition. Alternatively, it is possible to entrap the composition into microcapsules, vesicles, or comparable molecules.
The inhibitory effect of salt is not envisioned to be a problem as long as high concentrations of the antimicrobial molecules are administered. However, to minimize the effect, in one embodiment, the pharmaceutical composition is administered in a salt-free vehicle, including but not limited to: water or acidified water. In a further embodiment, the pharmaceutical preparation is administered with a salt chelator. In a further embodiment, the pharmaceutical preparation is protected from salt by administration within liposomes, microcapsule, or any other method known to one of skill in the art.
The expression of lysozyme (LZ), lactoferrin (LF), and the human beta defensins (HBD) was analyzed in the middle ear and tissue in normal and diseased ears in the Examples below. The effect of these substances on OM pathogens morphologically and as antimicrobials was then tested. From these results a method and composition for the treatment of OM and sinusitis was developed. Selected embodiments of this invention are illustrated in the Examples below:
EXAMPLES
The examples below demonstrate that lysozyme, lactoferrin, β-defensin-1 and β-defensin-2 are expressed in middle ear mucosa and that are effective against the tested pathogens. The expression of β-defensin-2, lysozyme and lactoferrin mRNAs were higher in the inflamed tissue, while the levels of β-defensin-1 showed minimal change. The PCR results for β-defensins 1 and 2 were confirmed by immnunohistochemical analysis. Previous results are consistent with the PCR results, showing that β-defensin 2 protein was expressed at a higher level in inflamed mucosa, whereas β-defensin-1 showed no change.
The bacteria used in the following examples were cultured as follows: Stocks of NTHi12 and M. catarrhalis 035E (generous gift of Dr. Xin-Xing Gu) were maintained at −80° C. For experiments, the bacteria were plated on chocolate agar and incubated overnight at 37° C. in 5% CO2. A single colony was then picked and transferred to 10 ml of brain heart infusion (BHI, Becton Dickinson, Cockeysville, Md.), supplemented with hemin (10 μg/ml, Sigma, St. Louis, Mo.) and NAD (10 μg/ml, Sigma, St. Louis, Mo.), and allowed to grow overnight. In the morning, 1/10 volume of the overnight culture was transferred to fresh medium and incubated for 3-hours. The subculture was then washed twice with 10 mM sodium phosphate and the O.D. at 620 nm was determined. For the S. pneumoniae serotypes 3 and 6B (generous gift of Dr. Xin-Xing Gu), a 50 μl aliquot of the −80° C. stock was thawed on ice, added to 10 ml of Todd-Hewitt broth (THB, Becton Dickinson, Cockeysville, Md.) and cultured overnight. One tenth of the volume of the overnight culture was then transferred to fresh medium and incubated for 3-hours. The subculture was then washed twice with 10 mM sodium phosphate and the O.D. at 620 nm was determined.
Example 1
Expression of Innate Immune Molecules: Lysozyme, Lactoferrin, and Human Beta Defensins in the Middle Ear and the Middle Ear Mucosa Normally and in Response to Pathogens
As shown in FIG. 1, a gel overlay assay was performed to show that experimentally induced effusion in rats contained molecules electrophoretically consistent with the molecules of innate immunity and which had anti-microbial activity. Rats were inoculated with 1.0 mg of S. typhimurium endotoxin in 100 μl of sterile saline. Animals were sacrificed after 48 hours and the effusion was collected. The microbicidal activity of the samples was then evaluated using the gel over-lay assay. The radial assay-method of Lehrer and coworkers was used, with minor modifications (Lehrer et al., 1991). The subcultured bacteria (4×106 CFU/10 ml underlay gel) were mixed with melted underlay gel at 42° C. (0.1× culture broth—BHI for NTHi and Moraxella and THB for S. pneumoniae-10 mM sodium phosphate, 0.8% low electroendosmosis (EEO)-type agarose) and poured into 8 cm×8 cm square petri plates. The gel was allowed to solidify in the petri plates and wells were punched out with a calibrated 200 μl micropipette tip, cut off at the 50 μl mark (approximately 3 mm). The antimicrobial peptides and proteins were dissolved in 0.01% acetic acid and 4 μl was added to each well. The plates were then incubated for 3 hours at 37° C. in the 5% CO2 incubator. They were next overlaid with the overlay gel (0.5× culture broth, 10 mM sodium phosphate, 0.8% low EEO-type agarose) and covered. The plates were then allowed to incubate overnight at 37° C. in a 5% CO2. The diameter of the zones of inhibition produced by each of the antimicrobial molecules against the OM pathogens (NTHi, M. catarrhalis and S. pneumoniae) was then measured in three separate experiments and the average and standard deviation were calculated. As shown in FIG. 1 lane 4, the areas corresponding to low molecular weight peptides or proteins (consistent with β-defensins) as well as those corresponding to larger proteins (consistent with lysozyme, lactoferrin, SP-A and SP-D) display anti-microbial activity in this assay (FIG. 1).
Next, normal and inflamed human middle ear tissues derived from biopsies, was analyzed by real-time PCR for lysozyme, lactoferrin, β-defensin-1 and β-defensin-2. Real-time PCR was performed as follows: Human middle ear mucosa was collected from patients undergoing surgery. Total RNA was isolated by using the TRIzol reagent (Gibco BRL, Rockville, Md.), according to the manufacturer's protocol. cDNA was generated by reverse transcription (RT), using the Superscript II-kit (Gibco BRL, Rockville, Md.), according to the manufacturer's protocol. Real-time PCR was carried out using human lysozyme, lactoferrin, β-defensin-1, β-defensin 2 and β-actin primers. The primer sequences are as follows:










human lysozyme (1223–1395) (XM_006858),




5′- ACT TTT TGT TGG GCA AT-3′ (forward),



(SEQ ID NO: 1)



 



5′- AGG CTC ATC TGC CTC AG-3′ (reverse);



(SEQ ID NO: 2)



 



human lactoferrin (947–1282) (AF332168),



5′- TCT CCG CCA GGC ACA -3′ (forward),



(SEQ ID NO: 3)



 



5′- GGA GGC CGA GGA GCA -3′ (reverse);



(SEQ ID NO: 4)



 



human beta-defensin-1 (68–321) (XM_005297),



5′- CGC CAT GAG AAC TTC CTA CCT T -3′ (forward),



(SEQ ID NO: 5)



 



5′- AGT TCA TTT CAC TTC TGC GTC ATT T -3′



(reverse);



(SEQ ID NO: 6)



 



human beta-defensin-2 (41–336) (XM_005029),



5′- GGG TCT TGT ATC TCC TCT TCT CGT T -3′



(forward),



(SEQ ID NO: 7)



 



5′- TGC GTA TCT TTG GAC ACC ATA GTT T -3′



(reverse);



(SEQ ID NO: 8)



 



human β-actin (696–964) (BC_002409)



5′-CGG GAA ATC GTG CGT GAC AT-3′ (forward),



(SEQ ID NO: 9)



 



5′-GCG TAC AGG TCT TTG CGG ATG -3′(reverse).



(SEQ ID NO: 10)






The PCR mixture contained 200 μM of each primer, 2 μl of template cDNA, and 12.5 μl of SYBR Green master mix (Applied Biosystems, Foster City, Calif.) in a final volume 25 μl of total volume. PCR was carried out in a ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.) using the following thermal cycling program: 2 minutes at 50° C., 10 minutes at 95° C., followed by 50 cycles of 15 seconds at 95° C. and 1 minute at 60° C. A real-time amplification curve was constructed for each amplification reaction by relating the fluorescence signal intensity (Rn) to the cycle number. The Rn value corresponded to the variation in the reporter fluorescence intensity before and after PCR, normalized to the fluorescence of an internal passive reference present in the buffer solution (6-carboxy-x-rhodamine, a rhodamine derivative). The Ct value (cycle threshold; corresponding to the cycle number at which a significant increase in the fluorescence signal was first detected) was determined for each reaction and served as the basis for comparison of the relative amount of molecule-specific cDNA in each reaction. The Ct for β-actin was subtracted from the Ct values for each of the other amplified molecules, in order to correct for differences in the amount of starting material. The corrected Ct values for each molecule were compared between the normal and inflamed samples and the cycle difference CD was used to calculate the difference (2CD) for each molecule between the normal and inflamed samples. Human lung cDNA was used as a positive control and for the negative controls, no template was included. PCR products were also analyzed by electrophoresis on a 2% agarose gel containing ethidium bromide in order to ensure that only the specific product was amplified. The results showed that the transcripts for lysozyme, lactoferrin and β-defensin-2 were present at elevated levels in the inflamed tissues. The β-actin adjusted threshold cycle differences between normal and inflamed samples for lysozyme, lactoferrin, β-defensin-1 and β-defensin-2, were 3.48, 7.38, −0.89 and 10.16, respectively, corresponding to an increase of 11 and 166, and 1150 fold for lysozyme, lactoferrin, and β-defensin-2 and a decrease of 0.54 fold for β-defensin-1 (FIG. 2 and Table 1).










TABLE I









Average Threshold Cycles















Inflamed





Normal
(adjusted to β-actin)
Normal − Inflamed
















hLz
27.37
23.89
3.48



hLf
30.83
23.45
7.38



HBD-1
27.91
28.80
−0.89



HBD-2
42.46
32.30
10.16







Threshold cycles for human lysozyme (hLz), lactoferrin (hLf), β-defensin 1 (HBD-1) and β-defensin 2 (HBD-2), compared between normal and inflamed middle ear mucosa. cDNAs derived from one sample of normal and one sample of inflamed middle ear tissue were subjected to real-time PCR for thedetection of the antimicrobial molecules using SYBER Green I dye on a ABI Prism 7700 Sequence Detection system. The average of three real-time PCR runs is given above for each cDNA. The threshold cycle for β-actin was 21.94 for normal tissue and 19.72 for inflamed tissue (2.22 cycle difference).In order to control for differences in the amount of starting material, this difference of 2.22 cycles was added to the threshold cycles for the antimicrobial molecules to arrive at the adjusted values.






Example 2
The Effect of Lysozyme, Lactoferrin and Human Beta-Defensins on OM Pathogens
The radial assay method of Lehrer and coworkers was used, with minor modifications (Lehrer et al., 1991). The subcultured bacteria (4×106 CFU/10 ml underlay gel) were mixed with melted underlay gel at 42° C. (0.1× culture broth—BHI for NTHi and Moraxella and THB for S. pneumoniae-10 mM sodium phosphate, 0.8% low electroendosmosis (EEO)-type agarose) and poured into 8 cm×8 cm square petri plates. The gel was allowed to solidify in the petri plates and wells were punched out with a calibrated 200 μl micropipette tip, cut off at the 50 μl mark (approximately 3 mm). The antimicrobial peptides and proteins were dissolved in 0.01% acetic acid and 4 μl was added to each well. The plates were then incubated for 3 hours at 37° C. in the 5% CO2 incubator. They were next overlaid with the overlay gel (0.5× culture broth, 10 mM sodium phosphate, 0.8% low EEO-type agarose) and covered. The plates were then allowed to incubate overnight at 37° C. in a 5% CO2. The diameter of the zones of inhibition produced by each of the antimicrobial molecules against the OM pathogens (NTHi, M. catarrhalis and S. pneumoniae) was then measured in three separate experiments and the average and standard deviation were calculated.
After confirming the expression of the innate immune molecules in the middle ear mucosa, the bacteriostatic/bactericidal effects of these molecules on four otitis media pathogens, NTHi strain12, M. catarrhalis strain 035E, S. pneumoniae serotype 3, and S. pneumoniae serotype 6B was determined. Treatment of the bacteria with lysozyme resulted in a slight but visible inhibition of the growth of M. catarrhalis and S. pneumoniae serotype 6B (FIG. 3 and Tables 2 and 3), This effect was seen with 40 μg of peptide per well, equivalent to a concentration of 10 mg/ml. Lysozyme, however, had no effect on NTHi and appeared to enhance the growth of S. pneumoniae serotype 3 (FIG. 3 and Table 2). Treatment with β-defensin-1 was effective only against M. catarrhalis and only at the high dose (10 μg—equivalent to 2.5 mg/ml). In contrast β-defensin-2, at both concentrations (4 and 10 μg—equivalent to 1 and 2.5 mg/ml respectively), significantly inhibited the growth of all four bacterial types (FIG. 3 and Tables 2 and 3). M. catarrhalis strain 035E was most susceptible to β-defensin-2, followed by NTHi strain12, S. pneumoniae serotype 3 and S. pneumoniae serotype 6B. Lactoferrin treatment, conversely, not only did not inhibit bacterial growth, but appeared to result in an enhancement of the growth of S. pneumoniae serotype 3 (10 μg—equivalent to 2.5 mg/ml), and S. pneumoniae serotype 6B (10 and 40 μg—equivalent to 2.5 and 10 mg/ml respectively) (FIG. 3 and Tables 2 and 3).










TABLE II









Total amount of
Bacteria













protein/peptide



S.



added (in a total

M.
S.
pneumoniae


Molecule
volume of 4 μl)
NTHi
catarrhalis
pneumoniae 3
6B





hLz
 4 μg
—
—
—
—



10 μg
—
—
—
—



40 μg
—
  4 ± 1.7 (P)
—
  5 ± 1.7 (P)


hLf
 4 μg
—
—
—
—



10 μg
E
—
E
—



40 μg
E
E
E
E


HBD-1
 4 μg
—
—
—
—



10 μg
—
 7.7 ± 0.6 (P)
—
—



40 μg
ND
ND
ND
ND


HBD-2
 4 μg
 9.7 ± 2.5 (C)
11.7 ± 0.6 (C)
8.7 ± 1.5 (C)
 8.7 ± 1.2 (C)



10 μg
12.3 ± 2.9 (C)
14.7 ± 0.6 (C)
 10 ± 1.3 (C)
10.7 ± 1.5 (C)



40 μg
ND
ND
ND
ND





Measurements of the effect of human lysozyme (hLz), lactoferrin (hLf), β-defensin 1 (HBD-1) and β-defensin 2 (HBD-2) on the growth of otitis media pathogens using the radial inhibition assay. Diameter (mm) of the inhibition zone (average of 3 separate experiments ± SD) caused by the different concentrations of the antimicrobial molecules is given in the column below each pathogen.


—: No effect;


E: Enhanced growth;


C: Complete inhibition;


P: Partial inhibition;


ND: Not done






The ability of a few of these innate immune mediators: human milk lysozyme (Lz), human β-defensins (HBD) 1 and 2 and human milk lactoferrin (LF) to inhibit the growth of non-typeable Haemophilus influenzae (NTHi) at other concentrations was also investigated using a different type of assay, a colony formation assay. The molecules were tested separately, as well as in combination, and the effect on NTHi growth was determined using a colony formation assay. Incubation of cultures of NTHi with 100 μg/ml human milk lysozyme for 3 hours, resulted in an 80% reduction of the number of viable bacteria (FIG. 4). In previous studies, it was shown that the concentration of lysozyme in middle ear effusion can be as high as 3.7 mg/ml and thus the concentrations used in these studies are well within the physiological range. Incubating the bacteria with 10 μg/ml of either human β-defensin 1 or 2 produced an even more pronounced effect, with close to a 95% reduction in the number of colonies. Interestingly, incubation of the bacteria with a combination of lysozyme and the β-defensins, with or without lactoferrin (1 mg /ml), did not result in a further decrease in the number of viable bacteria. Although these results are slightly different from those produced by the radial assay, without being restricted to the following theory, this may be due to the assay conditions or to the concentration tested. For example, the bacteria may be more sensitive to the antimicrobials in a liquid assay, such as the colony forming assay.
Example 3
Dose Response Analysis
A dose response analysis is performed for each molecule in order to determine the concentration range where the best effect is observable as well as a synergistic effect of different combinations. Thus, various concentrations of lactoferrin with various concentrations of defensins are used as in Example 1 and the concentrations at which maximum effectiveness of the combination occurs are used in further treatments.
Example 4
Effect of NaCl
Physiologically, salt concentrations vary depending on the site. However, typically they can-be as high as 150 mM. Thus, the salt sensitivity of the antimicrobial molecules was tested. NaCl was added to the gel in Example 2 to obtain a final concentration of 100 mM. As shown in Table III, 100 mM salt completely blocked the growth inhibitory effect of lysozyme, lactoferrin and β-defensin-1 against all bacteria tested. It also blocked the effect of β-defensin-2 against S. pneumoniae serotype 3 and S. pneumoniae serotype 6B. High salt concentration, however did not inhibit the activity of β-defensin-2 against NTHi and M. catarrhalis, although it did result in a reduction of this effect.










TABLE III









Total amount of
Bacteria













protein/peptide



S.



added (in a total

M.
S.
pneumoniae


Molecule
volume of 4 μl)
NTHi
catarrhalis
pneumoniae 3
6B





hLz
 4 μg
—
—
—
—



10 μg
—
—
—
—



40 μg
—
—
—
—


hLf
 4 μg
—
—
—
—



10 μg
—
—
—
—



40 μg
—
—
—
—


HBD-1
 4 μg
—
—
—
—



10 μg
—
—
—
—



40 μg
ND
ND
ND
ND


HBD-2
 4 μg
 8.8 ± 1.0(C)
 9.0 ± 0.7 (C)
—
—



10 μg
11.3 ± 0.3 (C)
11.0 ± 0.7 (C)
—
—



40 μg
ND
ND
ND
ND





Measurements of the effect of 100 mM NaCl on the inhibition of the growth of otitis media pathogens by human lysozyme (hLz), lactoferrin (hLf), β-defensin 1 (HBD-1) and β-defensin 2 (HBD-2), using the radial inhibition assay. Diameter (mm) of the inhibition zone (average of 3 separate experiments ± SD) caused by the different concentrations of the antimicrobial molecules is given in the column below each pathogen.


—: No effect;


ND: Not done






Example 5
Morphological Changes in Response to the Action of Lactoferrin, Lysozyme, and the Human Defensins.
Molecules of innate immunity can damage the cell wall and membranes of the OM microbes. Thus, in order to determine what the effect of these molecules was, electron microscopy was performed as follows:
Electron microscopy was performed by treating the bacterial cultures with antimicrobial protein or peptide for 3 hours, mixing with an equal volume of 5% buffered glutaradehyde (pH 7.4) and centrifuging (5000×g, for 20 minutes). The bacterial pellets were left in the fixative at 4° C. for 2 hr, post-fixed in 2% osmium tetroxide, dehydrated in ethanol and embedded in Eponate 12 (Ted Pella, Redding, Calif.). Sections were mounted in coated specimen grids, contrasted with uranyl acetate and lead citrate and examined in a transmission electron microscope (CM120 BioTwin, FEI-Philips, Hillsboro, Oreg.) operating at 80 kV. Images were recorded on photographic film and were subsequently digitized.
In order to determine if inhibition of NTHi growth was due to disruption of the bacterial cell wall and membranes, ultrastructural analysis was performed. As shown in FIG. 5 (3 hour), incubation of NTHi with lysozyme 100 μg/ml human milk lysozyme caused sufficient damage to the bacterial membranes that extrusion of the outer membrane could clearly be seen. Morphological changes were also apparent in bacteria treated for 3 hours with 10 μg/ml human β-defensins 1 and 2, or a combination of lysozyme (100 μg /ml), human milk lactoferrin (1 mg/ml). Thus these immune mediators function very well as antibiotics in the above studies.
Ultrastructural changes were analyzed in NTHi after treatment with lysozyme and β-defensin-2: Ultrastructural analysis of NTHi, S. pneumoniae serotype 3, S. pneumoniae serotype 6B and M. catarrhalis treated with the antimicrobial molecules revealed that significant changes occurred in the bacteria following exposure to β-defensin-2 and lysozyme. The morphology of the untreated NTHi is shown in FIG. 6A. FIGS. 6B and 6C show the morphology of NTHi treated with 10 μg/ml β-defensin-2 for 3 hours, and 1 mg/ml of human milk lysozyme for 3 hour, respectively. The results showed that in the presence of β-defensin-2 and lysozyme, NTHi show blebbing of the membranes with extrusion of the cytoplasmic contents into the blebs (FIGS. 6B and 6C). Moreover, none of the untreated bacteria showed any evidence of blebbing suggesting that the observed changes were not fixation artifacts. From examination of over 100 EM fields, it can be envisioned that with either of the antimicrobial molecules, at least 30% of the bacteria showed signs of membrane damage. At the time points measured, there did not appear to be any ruptured NTHi. A one hour treatment of the bacteria with β-defensin-2 also showed similar results, suggesting that the effect on the bacteria was occurring relatively quickly (data not shown). Furthermore, the morphology and the proportion of affected to non affected cells were similar in NTHi treated with lysozyme for the one hour, as compared to those treated with this molecule for 3 hours.
Treatment of S. pneumoniae serotype 3 with β-defensin-2 and lysozyme also resulted in damage to the bacteria, although the former molecule appears to be much more potent. The untreated bacteria are shown in FIG. 7A, while FIGS. 7B and 7C show the effect of a three-hour treatment with β-defensin-2 and lysozyme, respectively. As seen in FIG. 7B, treatment with β-defensin-2 results in disappearance of the capsule in certain regions and the lysis of the bacteria. Although lysozyme has a similar effect, many fewer bacteria appear to be lysed, consistent with the results of the radial assay.
Human β-defensin-2 showed activity against S. pneumoniae serotype 9B as well. As shown in FIG. 8A, these bacteria are capsulated with a distinct cell wall. A three-hour treatment with β-defensin-2, however resulted in damage to the capsule and condensation of the cytoplasmic material (FIG. 8B). The effect of lysozyme was not tested on this bacterium.
The effect of β-defensin-2 and lysozyme on M. catarrhalis was also tested. As shown in FIG. 9A, untreated M. catarrhalis have a distinct lobulated capsule. Results of a 1-minute treatment of the bacteria with lysozyme are shown in FIG. 9B and suggest that this treatment can result in the formation of blebs. Samples from later time points, however, showed no evidence of blebs and, as expected, contained more lysed bacteria. As shown in FIG. 9C, a 30-minute incubation of M. catarrhalis with β-defensin-2 resulted in substantial damage to the cells.
Of the four molecules tested in the radial inhibition assay, β-defensin-2 displayed the highest potency and was effective against all four pathogens tested. NTHi and M. catarrhalis are both Gram negative bacteria and the effect of β-defensin-2 on their viability is consistent with other studies. None of the treated NTHi showed any evidence of the presence of mesosome-like structures such as those seen in S. aureus treated with defensins. It is interesting, however, that treatment of the NTHi with lysozyme, although effective at the ultrastructural level, had no effect on the viability of the bacteria. Similar results were obtained by Shimoda and coworkers, when they exposed S. aureus to defensins (Shimoda et al., 1995). The effect of β-defensin-2 on the viability of a Gram positive bacteria, S. pneumoniae, however, was unexpected and is an unexpected finding.
The observed activity of β-defensin-1 against M. catarrhalis is also consistent with previous observations of the antimicrobial activities of this molecule against Gram negative bacteria. None of the other pathogens, including NTHi, were affected by β-defensin-1 treatment suggesting that this defensin molecule is less active than β-defensin-2 against OM pathogens. As expected, both molecules showed salt sensitivity and were inhibited by 100 mM salt
Lysozyme caused no detectable inhibition in the growth of S. pneumoniae serotype 3 and NTHi, while it showed only minimal activity against M. catarrhalis and S. pneumoniae serotype 6b. Lysozyme is a muramidase that cleaves the glycan backbone by catalyzing the hydrolysis of 1–4-glycosidic bonds between N-acetylmuraminic acid and N-acetyl-D-glucosamine, which are constituents of the cell walls of most bacteria. Studies of the cell wall of S. pneumoniae, however, have shown that a very high proportion of the hexosamine units (greater than 80% of the glucosamine and 10% of the muramic acid residues) are not N-acetylated, thus rendering the peptidoglycan of these bacteria to be resistant to the hydrolytic action of lysozyme. It is of interest that the growth of and S. pneumoniae serotype 6b is slightly inhibited by lysozyme, while that of S. pneumoniae serotype 3 is unaffected. The results suggest that even minor changes in the cell wall of bacteria, such as those that may exist between the different serotypes of S. pneumoniae, may have a profound effect on the resistance of the bacteria to this innate immune molecule. Consistent with previous observations, lysozyme also displayed salt sensitivity and its effect was inhibited by 100 mM salt.
Lactoferrin is an iron-sequestering glycoprotein that predominates in mucosal secretions. The presence of molecules such as lactoferrin allows free extracellular iron to be kept at levels (10−18 M) that do not allow bacterial growth and thus represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. Although this function of lactoferrin plays an important role in the protection of mucosal surfaces, bacteria have evolved mechanisms that allow them to use lactoferrin's iron-sequestering to their benefit. Thus, unlike the results obtained with lysozyme and the β-defensins, treatment of the four OM pathogens with lactoferrin appeared to have a growth enhancing effect. All four bacteria tested had a positive growth response to lactoferrin, with NTHi12 and S. pneumoniae serotype 3 showing a higher sensitivity than the other two. This result is, however, not surprising, as M. catarrhalis has been shown to be able to use lactoferrin as an iron source for growth in vitro and Streptococcus pneumoniae has been shown to specifically recognize and bind human lactoferrin. Moreover, other bacteria have also been shown to be able to use lactoferrin for growth. When grown under iron starvation, Neisseria meningitidis expresses receptors for transferrin and lactoferrin in the outer membrane. Iron transport mutants of Helicobacter pylori have also been shown to be able grow in the presence of lactoferrin and transferrin. Lactoferrin saturated with iron has been shown to enhance the intracellular growth of Legionella pneumophila in HeLa cells. However, it is possible that the effect of lactoferrin in enhancing the growth of these microbes may be restricted to the in vitro environment and when administered in vivo, lactoferrin may exhibit an inhibitory role. In addition, not all bacteria have evolved methods to use lactoferrin, thus, lactoferrin may be more useful against bacteria other than those currently found most commonly in OM. It is envisioned that the bacteria found most commonly in OM may change in the future with other bacteria becoming more predominant as causative agents of OM. Thus, antimicrobials such as lactoferrin may become more useful.
The results of the electron microscope studies suggest that β-defensin-2 and lysozyme have a profound effect on structural integrity of NTHi, S. pneumoniae serotype 3, S. pneumoniae serotype 6B and M. catarrhalis. β-defensin-2 is a cationic peptide known to damage bacterial outer membranes, while lysozyme is known to disrupt the bacterial cell wall. These findings appear to be consistent with earlier observations of the effect of these molecules on bacterial integrity.
The following U.S. patents contain related subject matter and are herein incorporated by reference: U.S. Pat. Nos: 5,242,902; 4,268,519; 5,240,909; 5,834,424; 4,961,927; 5,962,410; and 5,910,479.
Thus, in one embodiment of the invention, the pharmaceutical preparation herein comprises β-defensin-2. In a further embodiment, the pharmaceutical preparation herein comprises β-defensin-2 and lysozyme. In a further embodiment, the pharmaceutical preparation herein comprises β-defensin-2, lysozyme, and β-defensin-1. It is envisioned that for the treatment of some microbes, lactoferrin is not used due to the fact that these microbes have developed ways of using lactoferrin. In a further embodiment, a pharmaceutical preparation of β-defensin-2, lysozyme, and β-defensin-1 is used to treat an OM infection caused by M. catarrhalis. In a further embodiment, a pharmaceutical preparation comprising β-defensin-2, and lysozyme is used to treat OM caused by S. Pneumonia serotype 6B. In a further embodiment, a pharmaceutical preparation of β-defensin-2, lysozyme, and β-defensin-1 is used to treat an OM infection caused by NTHi strain 12. In a further embodiment, a pharmaceutical preparation of β-defensin-2 is used to treat an OM infection caused by M. catarrhalis. However, it is envisioned that a pharmaceutical preparation comprising β-defensin-2, lysozyme, and β-defensin-1 at different concentrations may be useful for the treatment of any OM infection.
Example 6
Expression of Changes in Response to the Action of Lactoferrin, Lysozyme, and the Human Defensins
Immunohistochemistry was performed using Celloidin embedded 20 μM thick sections of normal and inflamed mucosa, from archival temporal bone specimens were obtained from the House Ear Institute's Temporal Bone Collection. Following decelloidination with ether and absolute ethanol, non-specific binding sites on the slides were blocked using normal goat serum. The sections were then subjected to antigen retrieval (Vector Laboratories, Burlingame, Calif.) and incubated with polyclonal antibodies against human β-defensin 2–1:500 dilution. Signals were detected by the avidin/biotin complex (ABC) method (Vector Laboratories). As shown in FIGS. 10A and B (lower panel) β-defensin 2 was expressed in tissue from the diseased, but not the normal middle ear. These results are consistent with studies of human β-defensin 2 that have shown this molecule to be highly inducible by inflammatory stimuli, with minimal expression in non-challenged tissue or cells. The expression of β-defensin 1 was also examined in these sample, but, as expected, there was little to no difference in levels of this molecule in normal versus diseased epithelium (FIGS. 10A and B, upper panel).
Example 7
Treatment of an OM Infection Caused by M. catarrhalis 
A pharmaceutical preparation comprising Lysozyme, β-defensin-1, and β-defensin-2 at a concentration of 10 mg/ml Lysozyme, 10 mg/ml β-defensin-1, and 1 mg/ml β-defensin-2 is produced for administration to the middle ear of a patient whose ear drum is pierced. A grommet is inserted into the outer ear up to the middle ear and the pharmaceutical preparation is applied. Application is repeated 2× per day for 2 weeks. If the infection returns, the treatment is repeated.
Thus, the results herein demonstrate that innate immune molecules such as lysozyme, lactoferrin, β-defensin-1 and β-defensin-2 can be used to treat bacterial infections of the middle ear and sinuses, and are particularly effective against OM pathogens. Resulting in a new, innovative, and cost-effective approach to prevent and treat these diseases.
Although the present invention has been described in terms of certain preferred embodiments, other embodiments of the invention will become apparent to those of skill in the art in view of the disclosure herein. Thus, obvious changes and modifications may be made without departing from the spirit and scope of the invention. Accordingly, the scope of the invention is not intended to be limited by the foregoing, but rather to be defined only by the claims which follow.

## Claims

1. A pharmaceutical preparation for the treatment of otitis and sinusitis in a human, comprising at least one component selected from the group consisting of: lactoferrins, lysozyme, and defensins in an amount effective to reduce the growth of microbes, wherein said microbes comprise a bacteria selected from the group consisting of: Streptococcus pneumoniae, non-typeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis, said preparation further comprising a pharmaceutically acceptable carrier, wherein said carrier has a concentration of sodium chloride salt below 100 mM.

2. The pharmaceutical preparation of claim 1 wherein said defensins are alpha defensins or beta defensins.

3. The pharmaceutical preparation of claim 2 wherein said beta defensins are beta defensin 1 or beta defensin 2.

4. The pharmaceutical preparation of claim 1 wherein said pharmaceutical preparation is at a dose of about 0.1 to 100 mg/kg/dose.

5. The pharmaceutical preparation of claim 1 wherein said pharmaceutical preparation is at a dose of about 0.5 to 50 mg/kg/dose.

6. The pharmaceutical preparation of claim 1 wherein said pharmaceutical preparation is at a dose of about 0.1 to 1000 mg/kg/day.

7. The pharmaceutical preparation of claim 1 wherein said pharmaceutical preparation is at a dose of about 0.8 to 800 mg/kg/day.

8. The pharmaceutical preparation of claim 1 comprising β-defensin-2, lysozyme, and β-defensin-1 in an excipient formulated for treatment of and administration to the middle ear.