Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-02-05
2003-05-20
Brumback, Brenda (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S015800, C514S016700, C530S326000, C530S327000, C530S328000, C530S329000
Reexamination Certificate
active
06566334
ABSTRACT:
This invention pertains to amphipathic peptides having activity against bacteria and intracellular pathogens.
Bacterial resistance has hampered antibiotic therapy since the discovery of penicillin. The efficacy of current antibiotics is declining at an alarming rate due to the increase of multi-drug resistant bacteria. The lack of effectiveness arises from current antibiotics'reliance on few unique mechanisms of action. There is an unfilled need for new drugs with novel mechanisms of action upon bacterial infections.
Intracellular pathogens are especially difficult to control because they are sequestered within host cells. For example,
Brucella abortus
is an intracellular pathogen that lives and replicates in host macrophages. The ability to survive in macrophages allows Brucella to quickly establish a chronic infection. Treatment of chronic brucellosis is difficult because the sequestered bacteria are not exposed to the body's immune response system: complement cascade, neutrophils, Brucella-specific antibodies, and the host's cellular immune response. See Araya et al., “Temporal Development or Protective Cell-Mediated and Humoral Immunity in BALB/c Mice Infected with
Brucella abortus
,” Journal of Immunology, vol. 53, pp. 3330-3337 (1989).
Brucella are short, non-motile, non-sporulating, non-encapsulated, Gram-negative aerobic rods. Brucella are important veterinary pathogens. The bacteria localize in reproductive organs, mammary glands, supramammary lymph nodes, and other reticuloendothelial tissues, leading to abortion and infertility.
The zoonotic bacterial disease brucellosis has a significant impact on human health worldwide. In humans, Brucella infection results in a chronic debilitating disease known as undulant fever. Humans are exposed through direct contact with infected animals or infected animal products. In the United States, human brucellosis is an occupational hazard for veterinarians, abattoir (slaughterhouse) workers, animal handlers, and laboratory workers. Due to the highly infectious nature of the Brucella species via aerosolization, several members of the genus are candidates for biological weapons, placing military personnel at risk for infections.
Human brucellosis is characterized by malaise, fever, anorexia, muscular weakness, arthritis, and dementia. Cardiac and neurologic disorders may occur and, if untreated, may result in a mortality rate as high as 10%. Lengthy antibiotic therapy with one or multiple drugs for up to 30-45 days is required to treat brucellosis, but relapses of infection often occur after treatment is stopped. Unfortunately, antibiotic therapy does not relieve the symptoms of malaise, depression, and occasional severe dementia associated with the disease. Currently there are no vaccines available for humans; the live vaccines used in the eradication of animal brucellosis are virulent for man.
The BALB/c mouse model has been widely used for Brucella pathogenesis and vaccine efficacy studies. Inoculation of mice with virulent Brucella results in colonization of the liver and spleen. The immune response of mice to Brucella is similar to that observed in the natural hosts and humans. Cell-mediated immune (CMI) responses that aid in the clearance of infections have been defined in the murine model (Araya, et al., 1989). In general, vaccine strains that induce protective immunity in the natural hosts demonstrate abbreviated colonization profiles and also confer protection in mice.
Another example of an intracellular pathogen is
Mycobacterium tuberculosis,
an acid-fast Gram-positive bacterium that is the main cause of tuberculosis in humans. Tuberculosis is the leading cause of human deaths due to an infectious organism; estimates are that a third of the world's population is currently infected. Approximately 3 million people die from tuberculosis annually, and it is expected that this number will steadily increase over the next decade as drug resistant strains proliferate.
Organisms have many defense mechanisms against invasion by pathogens, including the cellular release of defense peptides. Some defense peptides perturb the barrier function of the membrane of either the invading pathogen or of infected host cells. Although the mechanism is not completely understood, it is thought that the defense peptide forms a transmembrane channel that allows irregular ion transport across the membrane, resulting in cell lysis or death due to a loss of osmotic integrity. See Saberwal et al., “Cell-Lytic and Antibacterial Peptides that Act by Perturbing the Barrier Function of Membranes: Facets of their Conformational Features, Structure-Function Correlations and Membrane-Perturbing Abilities,” Biochimica et Biophysica Acta, vol. 1197, pp. 109-131 (1994).
Many antimicrobial peptides selectively inhibit and kill bacterial cells while maintaining low cytotoxicity for normal mammalian cells. The selectivity for pathogens has been attributed to a difference between bacterial and mammalian cell membranes. The exterior membranes of bacteria are negatively charged, whereas mammalian cell exterior membranes are generally neutral. Antimicrobial peptides are positively charged and therefore may preferentially bind to bacterial membranes. The cholesterol in mammalian cell membranes has also been suggested as the basis for the selectivity of antimicrobial peptides. See Maloy et al., “Structure-Activity Studies on Magainins and Other Host Defense Peptides,” Biopolymers (Peptide Science), vol. 37, 105-122 (1995). Membrane disruption by the antimicrobial peptides could be inhibited by cholesterol. Finally the lower membrane potential across mammalian cells, or some combination of the above factors, could be responsible for the observed selectivity of the antimicrobial peptides between bacteria and normal mammalian cells.
The specificity of various antimicrobial peptides differs. For example, melittin, a component of honeybee venom, is not selective. The minimum bactericidal concentration of melittin also damages normal mammalian cells. By contrast, the naturally occurring magainins and cecropins exhibit substantial bactericidal activity at concentrations that are not lethal to normal mammalian cells. It has been found that sequence homology is not a prerequisite for biological activity. (Saberwal et al., 1994) Many natural antimicrobial peptides of widely varying sequences have been isolated. One consistent structural feature is the presence of an amphipathic helical domain. Synthetic analogs of native peptides with amino acid-substitutions expected to enhance amphipathicity and helicity have shown increased biological activity. However, most analogs with increased antimicrobial activity unfortunately also show increased cytotoxicity against normal mammalian cells. Notable exceptions are the melittin-cecropin hybrids, which are more bacteriostatic than cecropins and less cytotoxic than melittin. Melittin, a 26-residue peptide, is cytotoxic and has broad spectrum antimicrobial activity at micromolar concentrations. There are a number of other natural amphipathic peptides that are much less cytotoxic than melittin, but that have comparable broad spectrum antimicrobial activity. Magainins and cecropins exhibit bacteriostatic and bactericidal activity at concentrations that are not cytotoxic toward normal mammalian cells. These peptides are unstructured in dilute aqueous solution, but become helical in amphipathic media such as micelles, synthetic bilayers, and cell membranes.
Some defense peptides have been reported to selectively attack host cells infected with an intracellular pathogen, while not affecting normal mammalian cells. See Barr et al., “Activity of Lytic Peptides Against Intracellular
Trypanosoma cruzi
Amastigotes in vitro and Parasitemias in Mice,” Journal of Parasitology, vol. 81, no. 6, pp. 974-978 (1995). Such selectivity has not been previously reported for Brucella infection. There have been no previous studies on in vivo activity of a peptide against an intracellular pathogen. The mechanism for peptide selectivity again
Elzer Philip H.
Enright Frederick M.
Hammer Robert P.
McLaughlin Mark L.
Yokum Thomas S.
Board of Supervisors of Louisiana State University and Agricultu
Brumback Brenda
Davis Bonnie J.
Gupta Anish
Runnels John H.
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