Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-04-19
2002-12-10
Horlick, Kenneth R. (Department: 1637)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S300000, C530S326000
Reexamination Certificate
active
06492328
ABSTRACT:
INTRODUCTION
Background
The development of effective antimicrobial agents was once seen as a definitive cure for bacterial diseases. But even before the development of the first antibiotics, bacteria had demonstrated an ability to adapt to stress in the environment, resulting in the development of resistance. In recent years, the variety of antimicrobial agents has increased substantially, along with a parallel increase in resistant pathogenic microorganisms. Resistance is now recognized against all clinically available antimicrobial agents. The response to antimicrobial resistance in the medical community has been to use new or alternative antibiotics not previously used against the resistant bacteria. This approach has required the continuous development of new antibiotics, either as modifications of currently existing compounds or as combinations of compounds that may inhibit or bypass the bacterial resistance mechanisms.
Natural polycationic antibiotic peptides have been found in many different species of animals and insects and shown to have broad antimicrobial activity. In mammals, these antimicrobial peptides are mainly represented by two families, the defensins and the cathelicidins. Nearly all of these peptides have membrane affinity, and can permeate and permeabilize bacterial membranes, resulting in injury, lysis, and/or death to the microbes. For example, the human peptides termed alpha-defensins are produced by neutrophils and intestinal Paneth cells. In three-dimensions, defensins manifest an amphiphilic, largely beta-sheet structure, with a polar face formed largely by its arginines and with N- and C-terminal residues playing an important role in defining antimicrobial potency and spectrum. (See Gudmundsson et al. (1999)
J Immunol Methods
232(1-2):45-54.) Antimicrobial peptides are reviewed by Hancock and Lehrer (1998)
Trends in Biotechnology
16:82.
Cystic fibrosis (CF) is an inherited disorder that occurs in one of every 3,300 U.S. newborns. It affects some 30,000 Americans today. The median survival age of patients with CF is only 31.3 years, making CF the most common life-shortening inherited disease in the U.S. Most CF patients die from pulmonary failure that results from chronic, progressive infection by
Pseudomonas aeruginosa
—a Gram-negative bacterium that is widely distributed throughout the environment.
P. aeruginosa
has limited ability to infect normal individuals, but can be a devastating secondary invader in immunocompromised, severely burned, or antibiotic-treated persons. Because
Pseudomonas aeruginosa
strains frequently are or become resistant to conventional antibiotics, infections caused by them are often difficult to eradicate.
The in vitro activity of antimicrobial peptides, including cecropin P1, indolicidin, magainin II, nisin and ranalexin has been tested against clinical isolates of
P. aeruginosa
. The peptides were found to have a varied range of inhibitory values, and showed some synergy when combined with conventional antibiotics (Giacometti et al (1999)
J Antimicrob Chemother
. 44(5):641-5)
There is a clinical need for novel antibiotic agents that are active against drug resistant Gram-negative bacteria, and which have low toxicity against mammalian cells. The present invention addresses this need.
Relevant Literature
Saiman et al. (1999) Pediatr Pulmonol, Suppl. 17:320 report that drug resistant organisms from CF patients are inhibited by cathelicidin peptides; and Brogden et al. (1999) Pediatr Pulmonol, Suppl. 17:320 report on the efficacy of SMAP29 in an ovine model of pulmonary infection and its potential for treating
P. aeruginosa
infection in patients with cystic fibrosis (CF).
SUMMARY OF THE INVENTION
Methods and compositions are provided for the use of novispirin peptides. Novispirin peptides are small antimicrobial agents with potent activity against Gram-negative bacteria, including
Chlamydia trachomatis, Pseudomonas aeruginosa, Eschelichia coli
and
Stenotrophomonas maltophilia
. The peptides are nonhemolytic, exhibit reduced in vitro cytotoxicity relative to other antimicrobial peptides, and are well-tolerated in vivo after intravenous injection. Novispirins are equally effective against growing and stationary phase
P. aeruginosa
, and they retain activity in the presence of high concentrations of salt or human serum. Novispirins also bind lipopolysaccharide (LPS), a property that may mitigate symptoms associated with Gram-negative bacterial infection.
A pharmaceutical composition comprising novispirin as an active agent is administered to a patient suffering from a microbial infection, particularly bacterial infections. The protein is also effective at killing a variety of microbial organisms in vitro. Novispirin may be administered alone, or in combination with other bacteriocidal agents, e.g. antibiotics, as a cocktail of effective peptides, etc. Novispirin mediated killing of microbes is also useful for modeling and screening novel antibiotics.
REFERENCES:
Mahoney, M. M. et al., “Molecular analysis of the sheep cathelin family reveals a novel antimicrobial peptide”, FEBS Letters, vol. 377, pp. 519-522 (1995).*
SwissProt accession No. P49928, Cathelin-related peptide SC5 precursor 1, Oct. 1, 1996.*
SwissProt accession No. P49929, Cathelin-related SC5 precursor 2, Oct. 1, 1996.*
Brogden et al. (1998), “Detection of Small, Anionic Antimicrobial Peptides in Bronchoalveolar Lavage Fluid and Respiratory Epithelium of Patients with and without Cystic Fibrosis-1998 Cystic Fibrosis Conference.”Pediatric Pulmonolgy, Supplement 17, Abstract No. 587.
Giacometti et al. (1999), “In-Vitro Activity of Cationic Peptides Alone and in Combination with Clinically Used Antimicrobial Agents Against Pseudomonas Aeruginosa.”Journal of Antimicrobial Chemotherapy, vol. 44:641-645.
Gudmundsson et al. (1999), “Neutrophil Antibacterial Peptides, Multifunctional Effector Molecules in the Mammalian Immune System.”Journal of Immunological Methods, vol. 232:45-54.
Hancock et al. (Feb. 1998), “Cationic Peptides: a New Source of Antibiotics.”Tibtech, vol. 16:82-88.
Jia et al. (Sep. 1999), “Molecular Cloning and Characterization of Rat Genes Encoding Homologues of Human-Defensins.”Infection and Immunity, vol. 67(9):4827-4833.
Lehrer et al. (1999), “Defensins and Other Antimicrobial Peptides”Mucosal Immunology, Chapter 6:89-99.
Takemura et al. (Oct. 1996), “Evaluation of Susceptibility of Gram-Positive and -Negative Bacteria to Human Defensins by Using Radial Diffusion Assay.”Antimicrobial Agents and Chemotherapy, vol. 40(10):2280-2284.
Turner et al. (Sep. 1998), “Activities of LL-37, a Cathelin-Associated Antimicrobial Peptide of Human Neutrophils”Antimicrobial Agents and Chemotherapy, vol. 42(9):2206-2214.
Lehrer Robert I.
Tack Brian F.
Waring Alan J.
Bozicevic Field & Francis LLP
Horlick Kenneth R.
Sherwood Pamela J.
Strzelecka Teresa
The University of Iowa Research Foundation
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