Inhibitors of anthrax lethal factor activity

Details Inhibitors of anthrax lethal factor activity Inhibitors of anthrax lethal factor activity

2001-03-26

2002-08-20

Rose, Shep K. (Department: 1614)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Heterocyclic carbon compounds containing a hetero ring...

C514S235800, C514S235200, C514S234500, C514S235500, C514S238500

Reexamination Certificate

active

06436933

ABSTRACT:

GOVERNMENT SUPPORT
This invention was made with the support of the United States Government and the United States Government has certain rights in the invention.
TECHNICAL FIELD
The present invention relates to the prophylaxis and treatment of anthrax infections and, more particularly, to compounds that act as specific inhibitors of Anthrax Lethal Factor activity, methods and means for making such inhibitors and their use as pharmaceuticals.
BACKGROUND OF THE INVENTION
Anthrax is a zoonotic illness recognized since antiquity. In the 1870s, Robert Koch demonstrated for the first time the bacterial origin of a specific disease, with his studies on experimental anthrax, and also discovered the spore stage that allows persistence of the organism in the environment. Shortly afterward,
Bacillus anthracis
was recognized as the cause of woolsorter disease (inhalational anthrax). William Greenfield's successful immunization of livestock against anthrax soon followed in 1880, although Louis Pasteur's 1881 trial of a heat-cured anthrax vaccine in sheep is usually remembered as the initial use of a live vaccine.
Human cases of anthrax are invariably zoonotic in origin, with no convincing data to suggest that human-to-human transmission has ever taken place. Primary disease takes one of three forms: (1) Cutaneous, the most common, results from contact with an infected animal or animal products; (2) Inhalational is much less common and a result of spore deposition in the lungs, while (3) Gastrointestinal is due to ingestion of infected meat. Most literature cites cutaneous disease as constituting the large majority (up to 95%) of cases.
Bacillus anthracis
is a large, gram-positive, sporulating rod, with square or concave ends. Growing readily on sheep blood agar,
B. anthracis
forms rough, gray-white colonies of four to five mm, with characteristic comma-shaped or “comet-tail” protrusions. Several tests are helpful in differentiating
B. anthracis
from other Bacillus species.
Bacillus anthracis
is characterized by an absence of the following: Hemolysis, motility, growth on phenylethyl alcohol blood agar, gelatin hydrolysis, and salicin fermentation.
Bacillus anthracis
may also be identified by the API-20E and API-50CHB systems used in conjunction with the previously mentioned biochemical tests. Definitive identification is based on immunological demonstration of the production of protein toxin components and the poly-D-glutamic acid capsule, susceptibility to a specific bacteriophage, and virulence for mice and guinea pigs. The virulence of
B anthracis
is dependent on two toxins, lethal toxin and edema toxin, as well as on the bacterial capsule. The importance of a toxin in pathogenesis was demonstrated in the early 1950s, when sterile plasma from anthrax-infected guinea pigs caused disease when injected into other animals (Smith, H. and J. Keppie,
Nature
173:869-870 (1954)). It has since been shown that the anthrax toxins are composed of three entities, which in concert lead to some of the clinical effects of anthrax (Stanley, J. L. and H. Smith,
J. Gen Microbiol
26:49-66 (1961); Beall, F. A. et al.,
J. Bacteriol
83:1274-1280 (1962)). The first of these, protective antigen, is an 83 kd protein so named because it is the main protective constituent of anthrax vaccines. The protective antigen binds to target cell receptors and is then proteolytically cleaved of a 20 kd fragment. A second binding domain is then exposed on the 63 kd remnant, which combines with either edema factor, an 89 kd protein, to form edema toxin, or lethal factor, a 90 kd protein, to form lethal toxin (Leppla, S. H. et al.,
Salisbury Med Bull Suppl
., 68:41-43 (1990)). The respective toxins are then transported across the cell membrane, and the factors are released into the cytosol where they exert their effects. Edema factor, a calmodulin-dependent adenylate cyclase, acts by converting adenosine triphosphate to cyclic adenosine monophosphate. Intracellular cyclic adenosine monophosphate levels are thereby increased, leading to the edema characteristic of the disease (Leppla, S. H.,
Proc Natl Acad Sci USA
79:3162-3166 (1982)). The action of lethal factor, believed to be a metalloprotease, is less well understood. Lethal toxin has been demonstrated to lyse macrophages at high concentration, while inducing the release of tumor necrosis factor and interleukin 1 at lower concentrations (Hanna, P. C. et al.,
Proc Natl Acad Sci USA
90:10198-10201 (1993); Freidlander, A. M.,
J Biol Chem
. 261:7123-7126 (1986)).
It has been shown that a combination of antibodies to interleukin 1 and tumor necrosis factor was protective against a lethal challenge of anthrax toxin in mice, as was the human interleukin 1 receptor antagonist (Hanna, P. C. et al.,
Proc Natl Acad Sci USA
90:10198-10201 (1993)). Macrophage-depleted mice were shown to resist lethal toxin challenge, but to succumb when macrophages were reconstituted. The genes for both the toxin and the capsule are carried by plasmids, designated pX01[33] and pX02, respectively (Green, B. D. et al.,
Bacillus anthracis Infect Immunol
. 49:291-297 (1985); Uchida, I. Et al.,
J Gen Microbiol
. 131:363-367 (1985)).
Disease occurs when spores enter the body, germinate to the bacillary form, and multiply. In cutaneous disease, spores gain entry through cuts, abrasions, or in some cases through certain species of biting flies. Germination is thought to take place in macrophages, and toxin release results in edema and tissue necrosis but little or no purulence, probably because of inhibitory effects of the toxins on leukocytes. Generally, cutaneous disease remains localized, although if untreated it may become systemic in up to 20% of cases, with dissemination via the lymphatics. In the gastrointestinal form,
B. anthracis
is ingested in spore-contaminated meat, and may invade anywhere in the gastrointestinal tract. Transport to mesenteric or other regional lymph nodes and replication occur, resulting in dissemination, bacteremia, and a high mortality rate. As in other forms of anthrax, involved nodes show an impressive degree of hemorrhage and necrosis.
The pathogenesis of inhalational anthrax is more fully studied and understood. Inhaled spores are ingested by pulmonary macrophages and carried to hilar and mediastinal lymph nodes, where they germinate and multiply, elaborating toxins and overwhelming the clearance ability of the regional nodes. Bacteremia occurs, and death soon follows. Penicillin remains the drug of choice for treatment of susceptible strains of anthrax, with ciprofloxacin and doxycycline employed as suitable alternatives. Some data in experimental models of infection suggest that the addition of streptomycin to penicillin may also be helpful. Penicillin resistance remains extremely rare in naturally occurring strains; however, the possibility of resistance should be suspected in a biological warfare attack. Cutaneous anthrax may be treated orally, while gastrointestinal or inhalational disease ordinarily should receive high doses of intravenous antibiotics (penicillin G, 4 million units every 4 hours; ciprofloxacin, 400 mg every 12 hours; or doxycycline hyclate, 100 mg every 12 hours). The more severe forms require intensive supportive care and have a high mortality rate despite optimal therapy. The use of anti-anthrax serum, while no longer available for human use except in the former Soviet Union, was thought to be of some use in the preantibiotic era, although no controlled studies were performed.
Although anthrax vaccination dates to the early studies of Greenfield and Pasteur, the “modern” era of anthrax vaccine development began with a toxin-producing, unencapsulated (attenuated) strain in the 1930s. Administered to livestock as a single dose with a yearly booster, the vaccine was highly immunogenic and well tolerated in most species, although somewhat virulent in goats and llamas. This preparation is essentially the same as that administered to livestock around the world today. The first human vaccine was developed in t

Affiliated with

Also associated with

Rating

Inhibitors of anthrax lethal factor activity does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Inhibitors of anthrax lethal factor activity, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Inhibitors of anthrax lethal factor activity will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2901010