WO1998031706A1

WO1998031706A1 - METHODS OF TREATING LUNG INFECTIONS WITH HUMAN TRACHEAL ANTIMICROBIAL PEPTIDE (hTAP)

Info

Publication number: WO1998031706A1
Application number: PCT/US1998/000638
Authority: WO
Inventors: Young Hee Ko; Michael Delannoy; Peter L. Pedersen
Original assignee: Johns Hopkins University
Priority date: 1997-01-15
Filing date: 1998-01-15
Publication date: 1998-07-23

Abstract

A method of treating or preventing lung diseases caused by bacterial infections, including Pseudomonas aeruginosa in a person with a lung disease such as cystic fibrosis, pneumonia, tuberculosis, emphysema, AIDS, and the like comprising: administering to the person an effective dose of a pharmeceutical composition comprising at least one active ingredient selected from the group consisting of human antimicrobial peptide (hTAP); a derivative thereof having amino acid partial substitutions or additions which do not abolish the antimicrobial properties; pharmaceutically acceptable salts thereof; and combinations of these in admixture with a pharmaceutically acceptable dilutent or carrier wherein the composition is administered in a dosage of about 0.1 to about 200 mg of active ingredient per day.

Description

METHODS OF TREATING LUNG INFECTIONS WITH HUMAN TRACHEAL ANTIMICROBIAL PEPTIDE (hTAP)

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of co-pending U.S. provisional application serial number 60/033,763, filed on January 15, 1997, and which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel therapy for the treatment or prevention of bacterial infections such as Pseudomonas aeruginosa in patients with lung diseases such as cystic fibrosis, pneumonia, tuberculosis, emphysema, AIDS, and the like, which comprises administering human tracheal antimicrobial peptide (hTAP) alone or in combination with other antimicrobial peptides, bacterial inhibitors, or combination of these to patients in need of this therapy.

2. Background

Lung diseases pose a serious health problem and are responsible for untold pain and suffering. Research is continually on going to determine the cause of diseases such as cystic fibrosis, pneumonia, tuberculosis, emphysema. AIDS and similar maladies which effect the lungs of the victims diagnosed with these diseases. Various publications, which contain more detailed disclosures of the state of this art are referred to herein. All of which are incorporated by reference. Cystic fibrosis, for example is a common hereditary disease that appears usually in early childhood and is marked, inter alia, by difficulty in breathing. It is an autosomal recessive genetic disease caused by mutations in the protein CFTR (cystic fibrosis transmembrane conductance regulator). The most common mutation, the deletion of phenylalanine 508, results in infections by Pseudomonas aeruginosa and other organisms in the lungs of CF patients.

The CFTR protein is known to function as a chloride ion channel as disclosed by C. Cliff et al., in Am. J. PhysioL, 262:C1154-C1160 (1992). There has been much recent work and investigation of the first nucleotide binding domain of the CFTR protein in which the ΔF508 mutation resides as evidenced by the following publications: P. Thomas et al, J. Biol. Chem., 267:5727-5730 (1992); Y. Ko et al., J. Biol.Chem., 268:24330-24338 (1993); Y. Ko et al., J. Biol. Chem., 270:22093-22096 (1995); J. Hartman et al, J. Biol. Chem., 267:6455-6458 (1992); and M. Anderson et al., Cell, 67:775-784 (1991).

There has also been much investigation of the pathogenesis of CF by L. Saiman et al., J. Clin. Invest., 92:1875-1880 (1993); L. Imundo et al., Proc. Natl. Acad. Sci. USA 92:3019-3022 (1995); G. Pier et al., Science, 271:64-67 (1996); and J. Smith et al., Cell, 85:229-236 (1996)). However, despite this detailed work and investigation, the mechanism by which CF patients acquire chronic lung infections is unclear.

Pseudomonas aeruginosa infections are rarely eradicated and continue to be a leading cause of death in cystic fibrosis patients. Although this infection has been recognized for years, a satisfactory treatment had not previously been found. Other lung diseases also continue to take their toll and some, such as tuberculosis, are on the rise and have now proven to be resistant to previous effective treatment.

Anti-inflammatory agents have been tried in the therapy of Pseudomonas aeruginosa infections in CF patients. Efforts have been made to decrease the inflammatory response in the CF lung by use of a systemic steroidal anti- inflammatory therapy using prednisone by H. Auerback et al. as described in 'Alternative-day Prednisone Reduces Morbidity and Improves Pulmonary Function in Cystic Fibrosis", Lancet, pp. 686-688 (1985). Unfortunately, prednisone therapy includes such risks as growth retardation, glucose intolerance and development of cataracts.

U.S. Patent No. 5,441,938 discloses another therapy to prevent or combat Pseudomonas aeruginosa infections based on the administration of a glucose or mannose containing composition. The phagocytosis of nonopsonized Pseudomonas aeruginosa by macrophages is reportedly dependent upon the presence of glucose. However, critics of this therapy contend that bacteria grows profusely in glucose. It is a bacterial nutrient. Furthermore, this therapy involves a second line defense. Multiple defense mechanisms have also been identified which protect the respiratory tract. There have also been many isolations and characterizations of antimicrobial peptides in a variety of species and tissues. U.S. Patent No. 5,202,420, and 5,432,270 for example, disclose tracheal antimicrobial peptides isolated from bovine trachea which may be used systemically or topically for treating microbial infections, bacterial or fungal, such as acne, burns and eye infection or as a contact disinfectant in, for example, mouth wash or deodorant, or as a topical fungicide.

U.S. Patent No. 5,459,235 discloses a new family of cysteine-rich antimicrobial peptides isolated from bovine blood neutrophils called beta defensins. These are reportedly useful in human and veterinary medicine and as agents in agricultural, food science and industrial applications.

U.S. Patent 5,424,396 also discloses antimicrobial peptides which may be used in a variety of products including medicinal products for eye, foot, mastitis or diarrhea medications and non-medicinal products such as antiperspirants and cosmetics. Unfortunately despite investigations of the defense mechanisms which protect the respiratory tract, there are currently no treatments that have resulted in the complete eradication or prevention of Pseudomonas aeruginosa infections in patients with lung disease, such as cystic fibrosis. Antimicrobial therapy using antibiotics has been used. However, complications have been observed including the fact that patients with CF dispose of antimicrobial agents more rapidly than do normal individuals. Therefore, higher doses are required than those normally recommended. Strains of Pseudomonas aeruginosa also dissociate into multiple phenotypic forms and often with different antimicrobial susceptibility patterns. Furthermore, resistance to multiple antimicrobial agents develops frequently. Finally, allergy to certain antibiotics (such as betalactam) renders this type of therapy with antibiotics difficult in some patients.

Therefore, there is a need to provide a therapy for the prevention and eradication of bacterial infections such as Pseudomonas aeruginosa in patients with lung diseases such as cystic fibrosis. Because of development of resistances to drugs and adverse drug side effects, therapy based on classical drugs is being reconsidered. Immunotherapies which involve inducing a natural immune response to the pathogen are presently being investigated since these should provide an effective treatment without adverse side effects. SUMMARY OF THE INVENTION

It is a purpose of the present invention to provide a novel method of treating or preventing lung diseases caused by bacterial infections including Pseudomonas aeruginosa which comprises administering to a person in need of such treatment an effective amount of a pharmaceutical composition comprising at least one active ingredient selected from the group consisting of human antimicrobial peptide (hTAP), a derivative thereof having amino acid partial substitutions or additions which do not abolish the antimicrobial properties, pharmaceutically acceptable salts thereof, and combinations of these in admixture with a pharmaceutically acceptable diluent or carrier. The composition may further comprise other bacterial inhibitors and/or medications.

The present inventors have discovered that normal human tracheal epithelial cells express human tracheal antimicrobial peptide (hTAP) and have demonstrated that normal tracheal epithelial cells exhibit the capacity to kill Pseudomonas aeruginosa bacteria and are able to resist the bacterial attack. The inventors therefore have discovered that the first line of defense against Pseudomonas aeruginosa may involve secretion of one or more antimicrobial peptides and have utilized this determination to provide a needed therapy for the prevention and eradication of Pseudomonas aeruginosa in patients with lung diseases such as cystic fibrosis.

The present inventors have demonstrated that the lungs cells of healthy people, unlike those of CF patients, are protected against, bacterial infections. Transmission electron microscopy (TEN) on vertical as well as horizontal cut thin sections of several thousand human tracheal epithelial cells infected with bacteria were studied in order to understand how healthy lungs provide a defense and to visualize their interaction. These studies revealed that at 104mM NaCl Pseudomonas aeruginosa do not multiply when planted onto cells from healthy humans, whereas the bacteria grow profusely on cells from ΔF508 CF patients. Significantly, these studies also revealed, for the first time, that Pseudomonas aeruginosa infectious bacteria gain entrance into CF cells. The inventors further report the novel finding that normal human tracheal epithelial cells express a small (~4 kDa) antimicrobial peptide (hTAP). BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A depicts the DNA sequence of CFTR near the F508 region from N505 through S511 (SEQ ID NO: 1) wherein the ΔF508 mutation, the deletion of C TT, is represented as a bar across the sequence.

Figure IB depicts normal human bronchial epithelial primary cells (NHBE) containing the predicted normal type CFTR DNA sequence near the F508 region (SEQ ID NO: 1). Figure 1C depicts normal human tracheal epithelial cells containing the predicted normal type CFTR DNA sequence near the F508 region (SEQ ID NO: 2).

Figure ID depicts cystic fibrosis tracheal epithelial (CFTE) cells wherein the deletion of C TT, the ΔF508 mutation, is shown without generation of a frame shift (SEQ ID NO: 3). Figure IE depicts cystic fibrosis pancreatic cells wherein the deletion of C TT, the ΔF508 mutation, is shown without generation of a frame shift (SEQ ID NO: 4) In Figure IE, "N" refers to an unknown nucleotide.

Figure 2 A depicts ultrastructural analysis by TEM of normal HTE cells after 10 hours exposure to Pseudomonas aeruginosa. The scale bar represents lOμm. The inset depicts Pseudomonas aeruginosa observed after 10 hours in the absence of HTE cells but the scale bar represents 1 μm.

Figure 2B depicts ultrastructural analysis by TEM of CFTE cells infected with Pseudomonas aeruginosa. The scale bar represents lOμm. In Figures 2A and 2B, "N" designates nucleus. Figure 2C depicts ultrastructural analysis by TEM of CFTE cells infected with

Pseudomonas aeruginosa. The scale bar represents lμm.

Figure 2D ultrastructural analysis by TEM of CFTE cells infected with Pseudomonas aeruginosa. The scale bar represents lμm.

Figure 2E depicts ultrastructural analysis by TEM of CFPAC after infection with Pseudomonas aeruginosa. The scale bar is lOμm.

Figure 2F depicts ultrastructural analysis by TEM of CFPAC after infection with Pseudomonas aeruginosa. The scale bar is 1 μm. Figure 3A depicts an agarose gel showing the RT-PCR amplified product of hTAP.

Figure 3B depicts an agarose gel showing the RT-PCR amplified product of hTAP. Figure 3C depicts a nucleotide (SEQ ID NO: 5) and a predicted amino acid sequence (SEQ ID NO: 6) of the RT-PCR amplified 220 bp product hTAP and bTAP.

Figures 4A and 4B depict working models showing how differently normal and CF epithelial cells interact with Pseudomonas aeruginosa. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT As discussed, the present invention features novel methods for treatment and prevention of lung disease. In particular, therapeutic methods are disclosed that include administering a therapeutically effective amount of hTAP (alone or in combination with another agent) to a subject, preferably a mammal and more preferably a primate such as a human in need of such treatment. To gain greater insight into the mechanism by which CF patients acquire chronic lung infections, the present inventors studied a normal human tracheal epithelial cell line (HTE) and two cell lines from humans afflicted with CF, i.e., CFTE cells from the trachea and CFPAC cells from the pancreas. The ΔF508 genotype of CFTE and CFPAC cells was confirmed (Figures ID and IE, respectively) by use of PCR and automated DNA sequencing. Specific information is detailed below and further details can be found in the following publications: W. Ansorge et al., Nud. Acids. Res., 15:4593-4602 (1987); C. Connel et al., Biotechniques, 5:342-348 (1987); L. Middendorf et al., Electrophoresis, 13:487-494. (1992). The DNA sequence of CFTR in the HTE cell line and in NHBE (normal human bronchial epithelial) primary cells, also used in this study, was normal in the F508 region (Figures IB and 1 C).

Confirmation of the genotypes of the cells employed by DNA sequencing near the

ΔF508 region. Figures 1A, IB, 1C, ID and IE The confirmation of the genotypes of the cells employed by DNA sequencing near the ΔF508 region is supported as referenced in Figures 1A, IB, 1C, ID and IE. In Figure 1A the DNA sequence of CFTR near the F508 region (from N505 through S511) is shown to depict the ΔF508 mutation resulting from deleting C TT (represented as a bar across the sequence). NHBE (B) and HTE (C) cells contain the predicted normal type CFTR DNA sequence, near the F508 region; whereas, CFTE (D) and CFPAC (E) cells show that C TT has been deleted without generating a frame shift, thus, resulting in the ΔF508 mutation. The antisense DNA is sequenced in HTE cells and the arrow indicates the direction of the sense DNA. NHBE cells were from Clonetics, HTE and CFTE cell lines (1996 stocks) were gifts from Dr. D. Gruenert of USCSF, and the CFPAC cell line was obtained from the American Type Culture Collection (ATCC). Method

DNA templates for the automated DNA sequencing reaction were obtained after purifying the PCR amplified products from 3% low melting point NuSieve GTG agarose gels. For NHBE, HTE, and CFPAC cells, total RNA was isolated from these cells and respective cDNAs were synthesized for RT-PCR according to the method described under Figure 3. In the case of the CFTE cells, the genomic DNA was prepared from the cell lysate by use of Gentra's Puregene DNA Isolation Kit which was used as the template for PCR synthesis of the F508 region of CFTR. The sequence of the primers employed for the PCR were as follows: 5' primer: base G1548-base T1565 (18 mer), 3' primer: base G1685-C1708 (24 mer). The nucleotide sequence of the CFTR gene has been reported. See e.g., Riordan et al., Science, 245:1066-1073 (1989) and references cited therein

Following the establishment of cell genotypes, bacterial infection experiments were conducted to determine how normal and CF. cells interact with Pseudomonas aeruginosa. Cells were grown on a permeable filter support (Snapwell polycarbonate membrane) to improve cell differentiation. Subsequently, they were exposed to

Pseudomonas aeruginosa from the apical side for 10 hours at 37°C. Bacteria were diluted in BEGM (bronchial epithelial growth medium), a modified version of LHC8 medium having a NaCl concentration of 104mM, considered to be close to the normal concentration in the airways. See: J. Smith et al., Cell, 85:229-236 (1996). Inspection of both horizontal and vertical cut thin sections of the normal cells by TEM after a 10 hour exposure to Pseudomonas aeruginosa, as shown in Figure 2A, did not reveal any free floating bacteria, although in companion experiments conducted in the absence of epithelial cells numerous bacteria were present (Figure 2A, Inset). Furthermore, some normal cells were seen to continue with mitosis.

These experiments demonstrate that normal tracheal epithelial cells exhibit the capacity to clear or kill these bacteria during a 10 hour incubation period while maintaining growth. Even when the infection ratio of Pseudomonas aeruginosa to normal cells was increased from 1:300 (Figure 2A) to 1:1, these cells were able to resist the bacterial attack and completely clear the surrounding bacteria.

In sharp contrast, the CF ΔF508 cells (CFTR) were unable to clear the surrounding bacteria. Consequently, these bacteria multiplied substantially and became bound to the epithelial cells, interacting with the microvilli, (Figures 2B to 2D. Significantly, many bacteria gained entrance to the cells during a 10 hour infection period. Similarly, cells from the other CF ΔF508 line (CFPAC) were also unable to mount a defense against invading bacteria during a 10 hour incubation period. Again, Pseudomonas aeruginosa multiplied, interacted with, and entered many of the mutant cells (Figures 2E and 2F).

Ultrastructural Analysis by TEM Figures 2A, 2B, 2C, 2D, 2E and 2F Figure 2 A discloses ultrastructural analysis by TEM of normal (HTE) cells after a 10 hour exposure to Pseudomonas aeruginosa. The bacterial infection has cleared and no free floating or bound bacteria are observed. N and PM designate the nucleus and plasma membrane, respectively. The inset discloses Pseudomonas aeruginosa observed after 10 hour in the absence of HTE cells. The scale bar represents 1 Oμm in (A) and 1 μm in the inset.

Figures 2B to 2D disclose ultrastructural analysis by TEM of CFTE cells (ΔF508 CF tracheal cells) infected with Pseudomonas aeruginosa. Binding of the bacteria to CFTE cells (single arrows) and their growth on or near the microvilli of these cells is clearly observed in Figures 2B and 2C. CFTE cells in which Pseudomonas aeruginosa gained entrance are shown in (C) and (D) as indicated by double arrows. The bar corresponds to lOμm in (B) and to lμm in(C) and (D). Figures 2E to 2F disclose ultrastructural analysis by TEN of CFPAC cells

(ΔF508 CF pancreatic cells) after infection with Pseudomonas aeruginosa. CFPAC cells were unable to clear the bacterial infection. Rather, Pseudomonas aeruginosa were found to bind and enter these cells. The scale bar is lOμm in length (E). Higher magnification (F, scale bar = lμm), clearly shows that Pseudomonas aeruginosa both bind (single arrows) and enter (double arrows) CFPAC cells. The infection ratio of Pseudomonas aeruginosa to CFPAC cells was 1 :30. Method

The trypsinized epithelial cells (~10⁶ cells) were seeded onto polycarbonate Snapwell membrane (growth area = 1 cm ) and cultivated for 5 days in a 37°C incubator supplemented with 5% C0 . Then, freshly grown Pseudomonas aeruginosa (A₆₀₀nm = 0.5) were diluted with BEGM (modified version of LHC8 medium containing 104mM NaCl, from Clonetics) to provide bacteria to epithelial cell ratios of 1 :300, 1 :30, or 1:1 in lOOμl. The apical sides of epithelial cells was exposed to bacteria for 10 hours.

In a separate experiment, the volume of the infection solution containing Pseudomonas aeruginosa was 500μl, sufficient to cover the entire area of the membrane (1 cm²). After a 10 hour infection period at 37°C, cells on the membrane were rinsed with 1 X PBS (phosphate buffered saline solution, Gibco BRL) and fixed with 2ml of paraformaldehyde plus 2% glutaldehyde in 1 X PBS, pH 7.4, with the top plate covered. The cells in the fixative above were placed in a water bath inside a microwave oven (Pelco, Model 3400) and samples were microwave pulsed according to Giberson et al, Microsc. Res. Techn., 32:246-254 (1995); G. Paton, J. Histochem. Cytochem., 43:731-733 (1995) with a 10 second pulse/20 second rest/10 second pulse, maintaining 100% power at each step. The fixative temperature did not exceed 30°C. Cells were allowed to remain in the fixative for an additional 5 minutes and briefly rinsed in D-PBS, followed by a rinse with 0.1M sodium cacodylate. Samples were microwave-postfixed as before in K₃Fe(CN)₆ reduced in 1% osmium tetraoxide, rinsed in distilled water, and stained in filtered 1 % aqueous uranyl acetate for 15 minutes. Treated cells on the membrane were quickly dehydrated in a graded series of ethanol washes, infiltrated with eponate (Pella), and cured first in an oven for 3 days at 37°C and then overnight at 60°C. Ultrathin sections (80nm) were cut on a low angle Diatome diamond knife and collected on 200 mesh copper grids. Sections were additionally stained with 2% aqueous uranyl acetate for 25 minutes, rinsed, and allowed to dry. Samples were viewed on a Zeiss 10 B Transmission Electron Microscope operating at 60kV.

In order to assure the validity of the present conclusions, the relative capacities of several thousand normal and CF cells to bind and permit bacterial entry were monitored and quantified as shown in Table 1.

TABLE 1 Quantification of epithelial cells having bound and entered P. aeruginosa.

Cells were infected with Pseudomonas aeruginosa exactly as described in Figure 2. Thin sections of epithelial cells on 200 mesh copper grids (Pelco) were scored by viewing with a Zeiss 10B transmission electron microscope (TEM). Of the total number of epithelial cells examined (first column), the percentage observed to have bacteria bound (fourth column) plus the percentage observed to have bacteria entered (fifth column), was 20% (CFTE cells) and 50% (CFPAC cells). An epithelial cell having either one or many bound bacteria was scored as one (fourth column).

Significantly, multiple bacteria tend to surround or target particular CF epithelial cells at any one time rather than making a concerted attack on the whole population of cells.

These extensive studies clearly demonstrate a dramatic difference between the capacity of normal and CF cells to resist Pseudomonas aeruginosa. Thus, binding and entry of these bacteria to CF cells was significant (20% in CFTE cells and 50% in CFPAC cells), in sharp contrast to normal cells where bacterial binding could not be detected in ~4000 cells examined, although 2 cells showed bacterial entry.

It is interesting to note that multiple bacteria tend to surround or target particular CF epithelial cells at any one time rather than making a concerted attack on the whole population of cells. Although it is generally believed that Pseudomonas aeruginosa do not enter epithelial cells, the present inventors clearly demonstrate that this organism can enter ΔF508 CF cells in culture. This novel finding of bacteria inside CF cells may reflect either the cells final line of defense in a "last ditch stand" to fight back against an overwhelming microbial attack, or their surrender to the invading bacteria. In normal cells, the primary "first line defense" against Pseudomonas aeruginosa may involve secretion of one or more antimicrobial peptides.

Significantly, the present inventors have shown by RT-PCR analysis (Figure 3A) that human tracheal epithelial cells express a mRNA that corresponds closely to that of bovine TAP (bTAP) G. Diamond et al, in Proc. Natl. Acad. Sci. USA, 88:3952-3956 (1991). The expression of bTAP is reported to be specific to bovine airway mucosa, but not to several other tissues according to G. Diamond et al., in the Proc. Natl.

Acad. Sci. USA, 90:4596-4600 (1993).

In humans, as in the bovine, the expression of the gene may be specific to the airway cells, as the present inventors have found it to be absent in the colon carcinoma cell line, T84 (Figure 3B) where bactericidal activity is not necessary.

Expression of hTAP in NHBE cells Figure 3 A depicts an agarose gel (1.4%) demonstrating the RT-PCR amplified product of hTAP (220 bp from total RNA, lane 3) and from mRNA (lane 4). Control experiments were performed in the absence of templates (lanes 5 and 7) and in the presence of the template and the primers (lane 6) that were provided by Perkin Elmer.

The PCR amplified product having the expected size (308 bp) was observed (lane 6).

Molecular weight standards of λ-BstE II and φxl74-Hae III are shown in lanes 1 and

2, respectively.

Figure 3B depicts the RT-PCR amplified product of hTAP from total RNA of T84 cells (lane 1) or from total RNA of NHBE cells (lane 2) was analyzed by staining the 1.4% agarose gel with ethidium bromide solution (0.5μg/ml). Lane 3 corresponds to the DNA molecular weight standard, φxl74-Hae III digest.

Figure 3C shows an illustrative example of a nucleotide and amino acid sequence in accord with the invention. More specifically, Figure 3C shows the nucleotide (SEQ ID NO: 5) and predicted amino acid sequence (SEQ ID NO: 6) of the RT-PCR amplified 220 bp product hTAP and shows its identity with bTAP. The predicted normal human TAP amino acid sequence is illustrated with 3 letter codes and an asterisk (termination) below the bTAP DNA sequence. The 5' and the 3' primer sequences used for the RT-PCR experiment are underlined in Figure 3C. The 5' primer sequence is

5'-CGCGCGAATTCCAGCATGAGGCTCCATCACCTGCTC-3' (SEQ ID NO: 7) and the 3 ' primer sequence is

5'-GCGCCTCGAGTTACTTCTTTCTACAGCATTTTACTGC-3' (37 mer, SEQ ID NO: 8).

Preferred hTAP amino acid sequences in accord with the invention will have substantial sequence identity to the sequence shown in Figure 3C (SEQ ID NO: 6). More particularly, preferred hTAP amino acid sequences include those that have at least about 60 percent homology (sequence identity) to SEQ ID NO: 6, more preferably about 70 percent or more homology to SEQ ID NO: 6, still more preferably about 85, 90 or 95 percent or more homology to SEQ ID NO: 6.

The term "homology" as used herein in reference to an amino acid sequence refers to the extent of amino acid sequence identity between polypeptides. When a first amino acid sequence is identical to a second amino acid sequence, then the first and second amino acid sequences exhibit 100% homology. The homology between any two polypeptides is a direct function of the total number of matching amino acids at a given position in either sequence, e.g., if half of the total number of amino acids in either of the two sequences are the same then the two sequences are said to exhibit 50% homology.

Other contemplated hTAP proteins in accord with the invention will include at least one amino acid deleted from the amino acid sequence shown in Figure 3C (SEQ ID NO: 6) up to a deletion of about 2, 5, 10, 20, 35, 40, 50 or 60 amino acids. Such deletions are sometimes referenced to herein as "fragments". The deleted amino acids in the hTAP sequence can be contiguous or non-contiguous and can begin, e.g., at the C- or N-end (or both ends) of the hTAP sequence essentially up to about 60 amino acids or more of the full-length sequence. For example, a deletion can be made in the sequence shown in Figure 3C (SEQ ID NO: 6) extending from amino acid 1 (Met) up to about amino acid 26 (Gly) or 27 (Asn).

Further examples of hTAP proteins in accord with the invention include those with one or more amino acid substitutions with respect to the hTAP sequence shown in Figure 3C (SEQ ID NO: 6). In particular, the amino acid substitutions can be contiguous and include about 1, 2, 5, 10, 20, 50, or 60 amino acid substitutions. Noncontiguous amino acid additions or substitutions encompassing the same size range are also contemplated. Preferred amino acid substitutions can be conservative or non- conservative amino acid substitutions. Thus, a tyrosine amino acid substituted with a phenylalanine will be an example of a conservative amino acid substitution, whereas an arginine replaced with an alanine would represent a non-conservative amino acid substitution.

Still further examples of hTAP proteins in accord with the invention include those with one or more amino acid additions to the hTAP sequence shown in Figure 3C (SEQ ID NO: 6). In particular, the amino acid additions can be contiguous and include about 1, 2, 5, 10, 20, 50 or 100 amino acid additions. Preferred amino acid additions include neutral or hydrophilic amino acids added to the C- or N-terminus (or both terminii). In particular, an hTAP protein may include a tag, e.g., 6 X His, EE or Myc tag to aid purification.

Additionally, fusion proteins comprising a heterologous protein and all or part of the amino acid sequence shown in Figure 3C (SEQ ID NO: 6) are within the scope of the present invention. In general, the fusion protein will include covalently linked in sequence (C to N terminus) the heterologous protein and the hTAP protein or fragment thereof.

Preferred hTAP proteins (including protein fusions) in accord with the invention will exhibit significant activity in a conventional anti-microbial assay. A variety of such assays are known in the field and include those that measure activity against bacteria, e.g., E. coli, B. subtilis, S. aureus, E.faecalis and P. aeruginosa; fungi, e.g., A. fumigatus; and yeast, e.g., C. albicans. The assays are typically formatted to measure capacity to kill or inhibit growth of a desired microbe (or a combination of microbes).

In particular, a preferred hTAP protein in accord with the invention will exhibit about 24 to about a 100 hour minimum inhibitory concentration (MIC) values of between about 10 to 300μg/ml or less, preferably about 0.1 to 1 μg/ml or less in a standard anti-microbial assay. A variety of conventional anti-microbial assays are suitable for detecting hTAP activity against bacteria. In one approach sometimes referred to herein as "a standard in vitro assay" or similar term, E. coli (e.g., strain D31) are grown in a suitable broth such as LB (Luria) up to an OD₆oo of about 0.8. That optical density typically represents about 10⁹ colony-forming units/ml. Approximately, 10⁶ bacteria are added to about 8mls of 0.7% agarose in LB broth and poured over a 150mm Petri dish containing 50ml of 1.5% agarose in LB broth. Anti-bacterial activity can be assayed e.g., by suppression of bacterial growth dependent on application of fractions containing hTAP protein to the top agar surface. That suppression can be quantitated by a variety of means including visual inspection of colonies in treated and control plates. See generally T. Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y) (1982); H. Boman et al, Infec. Immun., 10:136-145 (1974).

Particularly preferred hTAP proteins in accord with the invention will exhibit about a 24 hour MIC value of between about 10 to 150μg/ml or less, preferably about 1 μg/ml or less in the standard in vitro assay described above.

The standard in vitro assay described above can be modified, if desired, to include other microbes such as those specified. Additionally, growth in liquid culture is readily accommodated by the assay. More specifically, if liquid growth is desired, fractions which include a desired hTAP protein an be added to about lOOμl of a suspension of a desired microbe, that suspension is typically diluted from about a mid logarithmic-phase liquid culture to a final concentration of about 10 cells per ml in a suitable growth broth. For example, one broth that is suitable for several, bacterial strains is TSB (Baltimore Biological Laboratory). The TSB can be adjusted to a pH 7.5 with, e.g., NaOH prior to autoclaving. After incubation at 37°C for about 4 hours, an OD₆oo measurement can be taken.

A "polypeptide" refers to any polymer consisting essentially of any of the 20 amino acids regardless of its size. Although the term "protein" is often used in reference to relatively large proteins, and "peptide" is often used in reference to small polypeptides, use of these terms in the field often overlaps. The term "polypeptide" refers generally to proteins, polypeptides, and peptides unless otherwise noted. A nucleic acid encoding an hTAP protein in accord with the invention preferably will have a length sufficient (preferable at least about 15, 20, 50, 100, 150, 200, 220 or greater base pairs up to about 350 or 400 base pairs) to bind to the sequence shown in Figure 3C (SEQ ID NO: 5) under the following moderately stringent conditions (referred to herein as "normal stringency" conditions): use of a hybridization buffer comprising 20% formamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing once with the SSC buffer at 37°C.

More preferably, the nucleic acid (preferably at least about 15, 20, 50, 100, 150, 200, 220 or greater base pairs up to about 350 or 400 base pairs) will bind to the sequence of SEQ ID NO: 5 under the following highly stringent conditions (referred to herein as "high stringency" conditions): use of a hybridization buffer comprising 40% formamide in 0.9M saline/0.09M sodium citrate (SSC) buffer at a temperature of 42°C and remaining bound when subject to washing twice with the SSC buffer at 42°C.

Additionally, the nucleic acid will include at least about 15, 20, 25 or 30 base pairs, more preferably at least about 50 base pairs, and still more preferably a nucleic acid of the invention comprises at least about 100, 150, 200, or 220 base pairs. The nucleic acids may be cloned or subcloned using any method known in the field (See, for example, J. Sambrook et al., Molecular Cloning, Cold Spring Harbor Press, New York, 1989), the entire contents of which are incorporated herein by reference. In particular, nucleic acids in accord with the invention may be cloned into any of a large variety of vectors. Possible vectors include, but are not limited to, cosmids, plasmids, phagemids or modified viruses, although the vector system must be compatible with the host cell used for expression. Viral vectors include, but are not limited to, lambda, simian virus, bovine papillomavirus, Epstein-Barr virus, and vaccinia virus. Viral vectors also include retroviral vectors, such as Amphatrophic Murine Retrovirus (see Miller et al., Biotechniques, 7:980-990 (1984)), incorporated herein by reference). Plasmids include, but are not limited to, pBR, PUC, pGEM (Promega), and Bluescript Registered TM (Stratagene) plasmid derivatives. Introduction into and expression in host cells is done for example by, transformation, transfection, infection, biolistic transfer or electroporation. Generally preferred nucleic acids of the invention will express an hTAP that exhibits the preferred anti-microbial properties disclosed herein.

As noted, preferred nucleic acids of the invention also will have substantial sequence identity of DNA sequence shown in Figure 3C (SEQ ID NO: 5). More particularly, preferred nucleic acids will comprise a sequence that has at least about 70 percent homology (sequence identity) to SEQ ID NO: 4, more preferably about 80 percent or more homology to SEQ ID NO: 4, still more preferably about 85, 90 or 95 percent or more homology to SEQ ID NO: 4.

Nucleic acids of the invention are isolated, usually constituting at least about 0.5%, preferable at least 2%, and more preferably at least about 5% by weight of total nucleic acid present in a given fraction. A partially pure nucleic acid constitutes at least 10%, preferably at least 30%, and more preferably at least about 60% by weight of total nucleic acid present in a given fraction. A pure nucleic acid constitutes at least about 80%, preferably at least about 90%, and more preferably at least about 95% by weight of total nucleic acid present in a given fraction.

Method Total RNA or mRNA was prepared from NHBE and T84 cells that were grown to confluency on T-75 flasks by employing Triazol (GibcoBRL) and Oligotex Direct mRNA Purification Kit (Qiagen), respectively. According to the protocol provided by Perkin Elmer's RNA PCR kit, cDNAs were synthesized from the RNA prepared as above and were used as templates for RT-PCR under the following conditions: reverse transcription reaction: 10 minutes at 25°C (ambient temperature), 15 minutes at 42°C, 5 minutes at 99°C, and 5 minutes at 4°C, PCR: 2 minutes at 95°C, one cycle; followed by 35 cycles of: 1 minute at 95°C, 1 minute at 55°C, and 3 minutes at 72°C; finally, 10 minutes at 72°C, one cycle.

The RT-PCR amplified products were then analyzed by agarose gel (1.4%) electrophoresis, after which the bands having the expected molecular mass of 220 bp were excised from the gel, purified by the method of Qiagen's Qiaquick Gel Extraction Kit, and finally ligated into TA cloning sites of the TA cloning Kit Vector pCR™ II. TA Cloning® One Shot competent cells were used and transformed by the ligation mix above. The resultant recombinant plasmid was purified by use of Qiagen's Plasmid Miniprep Kit and 3μg was subjected to automated DNA sequencing analysis with the T7 promoter sequence as the sequencing primer.

Recently, it was reported by G. Pier et al, in Science, 271:64-67 (1996), that cultured human airway epithelial cells expressing the ΔF508 allele of CFTR are defective in uptake of Pseudomonas aeruginosa. Unfortunately, these investigators provided no electron micragraphs as visual support for their conclusions. In a more recent study by J. Smith et al., in Cell, 85:229-236 (1996), although the preferential bactericidal action of normal airway epithelial cells relative to CF cells is noted, a low NaCl concentration (near lOOmM) was implicated as a key factor for the bactericidal action. Thus, in an experiment supported by visual examination of two CF cells, the presence of Pseudomonas aeruginosa was observed at a high Cl^" concentration (182mM), but not at a low concentration. However, in the extensive study by the present inventors reported here, where -4000 CF cells were examined ultrastructurally after infection with Pseudomonas aeruginosa at a low NaCl concentration (104mM), the capacity of these bacteria to bind and enter the diseased cells was unimpaired (Table 1, Figures 2B to 2F).

In summary, the results of the present study using ultrastructural analysis by TEM clearly demonstrate that human tracheal epithelial cells containing normal type CFTR interact differently with Pseudomonas aeruginosa than ΔF508 CF cells, in which the bacteria bind and enter the diseased cells. These findings suggest a plausible working model depicted in Figure 4 to account, at least in part, for how normal tracheal cells may clear Pseudomonas aeruginosa; ΔF508 CF cells are unable to mount an effective defense.

In Figure 4A, the normal cells (a) are shown preparing their first line of defense against invasion by Pseudomonas aeruginosa (b). The antimicrobial peptide, hTAP for destroying the microbial organism may be secreted by a mechanism dependent on the normal function(s) of CFTR (b) and may function also inside the cells against the entering Pseudomonas aeruginosa (c). Consequently, infected cells finally clear the bacteria (a). In Figure 4B, CF cells having defective CFTR (a) are infected with Pseudomonas aeruginosa. In this case, secretion of hTAP, a first line defense weapon, may be impaired or compromised (b). Thus, the efficiency of bacterial clearance by CF cells is much less than that of normal cells resulting in increased bacterial binding, multiplication, and entrance into cells (c), where killing by internally located TAP cannot keep pace with the rate of bacterial entry. Thus, the antimicrobial peptide, hTAP, may play a CFTR dependent role as part of a first line defense, functioning outside and on the surface of the tracheal epithelial cells. This role may be dramatically impaired or compromised in diseased (ΔF508) cells.

Based on this theory, the present inventors propose a method for treating lung diseases which involves exploiting a natural immune response to pathogens which provides an effective treatment without adverse side effects. The present invention comprises methods of treating lung diseases caused by bacterial infection including Pseudomonas aeruginosa, comprising administering to a patient in need of such treatment an antimicrobially effective amount of hTAP. The term "antimicrobial" as used herein refers to killing microorganisms or suppressing their multiplication or growth. The term "anti-microbial effective amount or dose" suitably denotes an amount or dose of a composition that includes an hTAP protein exhibiting the preferred MIC values described herein.

For use as an antimicrobial agent, hTAP can be formulated into pharmacological compositions containing an effective amount of hTAP and a usual nontoxic carrier, such carriers being known to those skilled in the art. The composition can be given via a route of administration suited to the form of the composition. Such compositions are, for example, in the form of usual liquid preparations including solutions, suspensions, emulsions and the like which can be administered via inhalation using aerosol or other effective forms of delivery.

As used herein, "hTAP" refers to a protein having at least in part or in whole substantially the same amino acid sequence and at least the same antimicrobial activity as the protein defined in Figure 3C (SEQ ID NO: 6). The hTAP protein of the invention, depending on the pH of its environment, if suspended or in solution, or of its environment when crystallized or precipitated, if in solid form, may be in the form of pharmaceutically acceptable salts or may be in neutral form. The free amino groups of the protein are, of course, capable of forming acid addition salts with, for example, inorganic acids such a hydrochloric, phosphoric, or sulfuric acid; or with organic acids such as, for example, acetic, glycolic, succinic, or mandelic acid. The free carboxyl groups are capable of forming salts with bases, including inorganic basis such as sodium, potassium, or calcium hydroxides, and such organic bases as piperidine, glucosamine, trimethylamine, choline, and caffeine. In addition, the protein may be modified by combination with other biological materials such as lipids and saccarides, or by side chain modification such a acetylation of amino groups, phosphorylation of hydroxyl side chains, or oxidation or sulfhydryl groups. Also hTAP protein may be modified enzymatically or lipophylically.

As noted, modifications of hTAP (e.g., amino acid substitutions, deletions etc.) are included within the scope of the definition, so long as the antimicrobial activity as described herein is retained particularly with respect to the MIC values disclosed above. It is understood that minor modifications of hTAP may result in proteins which have substantially equivalent or enhanced antimicrobial activity as compared to the sequence set forth in Figure 3C (SEQ ID NO: 6). These modification may be deliberate, as through site-directed mutagenesis, or may be accidental such as through mutation in hosts which are hTAP producers. All of these modifications are included as long as the antimicrobial activity, is retained.

Having described the amino acids of hTAP in Figure 3C, it is believed this peptide or modifications thereof can be routinely synthesized in substantially pure form by standard techniques well known in the art, such as commercially available peptide synthesizers and the like. Additionally, it is believed hTAP can be efficiently prepared using any of numerous well known recombinant techniques. Most of the techniques which are used to transform cells, construct vectors, extract messenger RNA, prepare cDNA libraries, and the like are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. See e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989), Sambrook et al. infra.

The present invention, therefore, provides a method of using a therapeutic antimicrobial composition comprising at least one active ingredient selected from hTAP, a derivative thereof having amino acid partial substitutions or additions which do not abolish the antimicrobial properties, pharmaceutically acceptable salts thereof, and combinations of those in admixture with a pharmaceutically acceptable diluent or carrier to treat or prevent lung diseases caused by bacterial infections including Pseudomonas aeruginosa. The composition may further comprise other bacterial inhibitors and/or medications such as one or more antibiotic, bactericidal, or bacteriostatic agents. Additionally, other anti-microbial agents may be used such as those effective against yeast or fungi.

The amounts of active ingredients in the compositions may vary depending on the nature of the other components, the degree of protection required, and the intended use of the composition. These antimicrobial compositions are intended to be used prophylactically, that is, to prevent infection, as well as to treat patients having chronic infections.

It is necessary to administer these antimicrobial compositions in a form that effectively reaches the airways of the patient. As noted above, delivery of these therapeutic agents can be effected by using an aerosol formulation that is administered by inhalation. The preferred mode of administration is therefore by inhalation of an aerosol preparation containing the antimicrobial composition. These aerolized compositions can be readily prepared by methods known in the art. The composition can be administered in an antimicrobially effective amount.

Generally a dose of about 0.1 to about 200mg/day, preferably from about 10 to about 150mg/day and more preferably from about 20 to about lOOmg/day of active ingredient, is expected to be useful. The dosage should be somewhat smaller when used prophylactically as a preventive measure for people with high risk of lung infections, or for those who are genetical disposed for lung disease, than the dosage for treatment of patients who are diagnosed with chronic bacterial lung infections. The preferred prophylactic dosage is from about 0.1 to about lmg/day. The most preferred prophylactic dosage is about 0.25 to 0.5.mg taken each morning.

Accordingly, a pharmacological (antimicrobial) composition according to the invention will include an anti-microbial effective amount of at least one hTAP protein described herein (including modified hTAP proteins), preferably one of hTAP protein, particularly the hTAP protein shown in Figure 3C (SEQ ID NO: 6). As noted, the composition will typically further include one or more suitable vehicles for the administration route chosen (e.g., aerosol or intravenous routes) along with one or more optional agents such as another hTAP protein in accord with the invention or anti-microbial agent (e.g., antibiotic, bactericidal, bacteriostatic, fungistatic or fungicidal agent). Such as pharmacological treatment compositions of the invention preferably also will be pharmaceutically acceptable, e.g. sterile and otherwise suitable for administration to a subject. Such pharmacological compositions of the invention suitably will be stored in a sealed (preferably, hermetically sealed) container prior to use.

All documents mentioned herein are incorporated herein by reference.

The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modification and improvements within the spirit and scope of the invention as set forth in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing lung diseases caused by bacterial infections, comprising: administering to a person in need of such treatment an antimicrobially effective dose of a pharmaceutical composition comprising at least one active ingredient selected from a group consisting of human tracheal antimicrobial peptide (hTAP); a derivative thereof having amino acid partial substitutions or additions which do not abolish the antimicrobial properties; pharmaceutically acceptable salts thereof and combinations of these in admixture with a pharmaceutically acceptable dilutent or carrier.

2. The method according to claim 1 wherein the lung disease is cystic fibrosis.

3. The method according to claim 1 wherein the bacterial infection is Pseudomonas aeruginosa.

4. The method according to claim 1 wherein the composition further comprises other bacterial inhibitors or medications.

5. The method according to claim 1 wherein the composition is administered in a dosage of about 0.1 to about 200mg of active ingredient per day.

6. The method of claim 1 wherein the composition is administered in a dosage of about 10 to about 150mg of active ingredient per day.

7. The method of claim 1 wherein the composition is administered in a dosage of about 20 to about lOOmg of active ingredient per day.

8. The method of claim 1 wherein the composition is administered in a dosage of about 0.1 to about lmg of active ingredient per day.

9. The method of claim 1 wherein the composition is administered in a dosage of about 0.25 to 0.5mg of active ingredient per day.

10. The method of claim 5 wherein the composition further comprises other bacterial inhibitors or medications.

11. The method according to claim 1 wherein the administration is by inhalation.

12. The method according to claim 11 wherein the inhalation method is dispensed from a pressurized container.

13. The method according to claim 12 wherein the pressurized container is an aerosol.

14. The method according to claim 5 wherein the bacterial infection is Pseudomonas aeruginosa, and the lung disease is cystic fibrosis.

15. The method according to claim 14 wherein the administration is by inhalation of an aerosol preparation.

16. The method according to claim 5 which is a prophylactic measure for people with high risk of lung infections or who are genetically disposed for lung disease.

17. The method according to claim 5 which is for treatment of patients who have chronic lung infections.

18. The method according to claim 10 which is a prophylactic measure for people with high risk of lung infections or who are genetically disposed for lung disease.

19. The method according to claim 10 which is for treatment of patients who have chronic lung infections.

20. A method of killing or preventing Pseudomonas aeruginosa bacterial infections in cystic fibrosis patients, comprising: contacting the Pseudomonas aeruginosa bacteria with an antimicrobial effective amount of a composition comprising at least one active ingredient selected from the group consisting of human antimicrobial peptide (hTAP), a derivative thereof having amino acid partial substitutions or additions which do not abolish the antimicrobial properties, pharmaceutically acceptable salts thereof, and combinations of these.

21. A method of detecting a bacterial infection in a respiratory system of a patient, the method comprising isolating lung or lung airway cells from the patient and visualizing bacteria inside the cells as indicative of the bacterial infection in the patient.

22. The method of claim 21, wherein the patient is suffering from or suspected of suffering from cystic fibrosis and the bacteria is Pseudomonas aeruginosa.

23. The method of claim 22, wherein the visualization comprises transmission electron microscopy (TEM).

24. A pharmacological composition comprising at least one human tracheal antimicrobial peptide (hTAP).

25. The pharmacological composition of claim 24, wherein the pharmacological composition comprises a human tracheal antimicrobial peptide (hTAP) and the peptide has a minimum inhibitory concentration (MIC) value of between about 1 to lOO╬╝g/ml in a standard in vitro assay.

26. The pharmacological composition of claim 24, wherein the pharmacological composition comprises one human tracheal antimicrobial peptide (hTAP) and the peptide is at least 60% homologous to SEQ ID NO: 6.

27. The pharmacological composition of claim 26, wherein the human tracheal antimicrobial peptide (hTAP) comprises at least one amino acid deleted from or added to the amino acid sequence shown in Figure 3C (SEQ ID NO: 6).

28. The pharmacological composition of claim 25, wherein the composition further comprises at least one agent selected from the group consisting of bactericidal, bacteriostatic, or fungicidal agents.

29. The pharmacological composition of claim 28, wherein the agent is an antibiotic.

30. The pharmacological composition of claim 24 packaged in a container suitable for administering the composition as an aerosol.