Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving glucose or galactose
Reexamination Certificate
2001-08-10
2004-06-01
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving glucose or galactose
C435S233000, C424S725000, C424S780000, C424S195170
Reexamination Certificate
active
06743598
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides methods for simultaneously assessing microbial phosphoglucose isomerase, ketol-isomerase and glucosamine-6-phosphate acetyltransferase activities, by measuring the production of Coenzyme A (CoA). The present invention finds use in the isolation of new classes of antifungal drugs, wherein the compounds have the ability to inhibit fungal glucose utilization.
BACKGROUND OF THE INVENTION
During the last three decades there has been a dramatic increase in the frequency of fungal infections, especially disseminated systemic mycoses in immunocompromised patients (Cox and Perfect,
Curr. Opin. Infect. Dis.
6:422-426 [1993]; and Fox,
ASM News
59:515-518 [1993]). Human pathogenic fungi of particular importance include:
Candida sp.
(
C. albicans, C. glabrata, C. krusei
and
C. parapsilosis
),
Aspergillus fumigatus
, and
Cryptococcus neoformans. C. albicans
and
A. fumigatus
cause most opportunistic mycoses. At present, treatments for fungal infections are limited to few options. Amphotericin B (a polyene) is fungicidal, but is toxic to humans. Azoles (fluconazole, itraconazole, and others) are safer than amphotericin B, but are only fungistatic. In addition, resistance to azoles has become a major clinical concern. Some azoles (Sheehan,
Clin. Microbiol. Rev.
12:40-79 [1999]) and a new class of (1,3)&bgr;-glucan synthase inhibitors, echinocandins (Denning,
J. Antimicrob. Chemother.
40:611-614 [1997]), are now in clinical and pre-clinical trials. Recently, the FDA approved caspofungin, a (1,3)&bgr;-glucan synthase inhibitor, as a “salvage treatment” for aspergillosis. In spite of the new azoles and the (1,3)&bgr;-glucan synthase inhibitors, new classes of antifungal drugs are needed for therapy of infections caused by drug-resistant mutants (and species) or for preventing the emergence of drug-resistant mutants.
Fungal Infections and Drug Resistance
Fungal organisms have become increasingly significant pathogens in immunocompromised patients, especially those who because of cancer, organ transplantation, chemotherapy, pregnancy, age, diabetes, complications following extensive surgery, and various immune system dysfunctions, are at risk of experiencing life-threatening diseases caused by microorganisms which do not ordinarily pose a threat to normal, immunocompetent people. Other risk factors for deeply invasive fungal infections include protracted treatment using broad spectrum antimicrobials, corticosteroids, and vascular catheters.
Indeed, immunocompromised patients provide a significant challenge to modern health care delivery. For example, fungal infections have become one of the leading factors contributing to morbidity and mortality in cancer patients, and fungi account for 4-12% of nosocomial pathogens in leukemia patients (Anaissie,
Clin. Infect. Dis.,
14[Suppl. 1]:S43 [1992]). The incidence of nosocomial bloodstream infections with fungi such as Candida spp. (“candidemia”) has increased in recent years and has been reported to account for 5.6% of all primary bloodstream infections. There are an estimated 200,000 patients/year who acquire nosocomial fungal infections, with bloodstream infections having a mean mortality rate of 55% (See e.g., Beck-Sague et al.,
J. Infect. Dis.,
167:1247 [1993]; and the Centers for Disease Control website at www.cdc.gov
cidod/publications/brochures/hip.html). Fungal infections in non-humans, such as livestock, is also of significant health and economic concern. The most common fungal pathogens in humans are the opportunistic yeast,
Candida albicans
and the filamentous mold,
Aspergillus fumigatus
(See, Bow,
Br. J. Haematol.,
101:1 [1998]; and Warnock,
J. Antimicrob. Chemother.,
41:95 [1998]).
C. albicans
is the most common fungal pathogen in humans, with other Candida species becoming increasingly important in fungal disease in humans and other animals (See, Walsh and Dixon, “Spectrum of Mycoses,” in Baron [ed.],
Medical Microbiology,
4th ed, University of Texas Medical Branch, Galveston, Tex. [1996], pp. 919-925). Approximately 200 Candida species are recognized, with approximately seven of those species isolated with significant frequency from human specimens (See, Warren and Hazen, Ch. 95, pp. 1184-1199, “Candida, Cryptococcus, and Other Yeasts of Medical Importance,” in Murray et al., [eds.],
Manual of Clinical Microbiology,
7th ed., ASM Press, Washington, D.C. [1999]; and Mitchell, in
Zinsser Microbiology
, Joklik et al., [eds], Appleton, Century-Crofts, Norwalk, Conn., pp. 1183-1190 [1984]).
The clinical manifestations of Candida infections and disease are many and varied, as Candida species are known to invade most organ systems of the body. Superficial candidiasis may involve the epidermal and mucosal surfaces (e.g., the oral cavity, pharynx, esophagus, intestines, urinary bladder, and vagina). In deep candidiasis, the gastrointestinal tract and intravascular catheters are the two major portals of entry, with the kidneys, liver, spleen, brain, eyes, heart, and other tissues being the major sites involved.
The major difficulties in treating Candida infections are encountered in cases of systemic disease. Chronic mucocutaneous, pulmonary candidiasis, endocarditis, and fungemia must be diagnosed early in order to avoid fatality. The incidence of candidiasis in certain patient populations is striking. Up to 30% of leukemia patients acquire systemic candidiasis (Anaissie,
Clin. Infect. Dis.,
14[Suppl. 1]:S43 [1992]). This is of great significance, as some reports indicate that the fatality rate for disseminated candidiasis in cancer patients is as high as 80% (Meunier, et al.,
Clin. Infect. Dis.,
14[Suppl. 1]:S120 [1992]).
Aspergillus species are the second most common isolate, after Candida species, in patients with positive fungal cultures (See, Sigler and Kennedy, Ch. 97, “Aspergillus, Fusarium, and Other Opportunistic Moniliaceous Fungi,” in Murray et al., (eds),
Manual of Clinical Microbiology,
7th ed., ASM Press, Washington, D.C. [1999], pp. 1213-1241; and Goodwin et al.,
J. Med. Vet. Mycol.,
30:153 [1992]). A large number of species of the genus Aspergillus have clinical relevance, although
A. fumigatus, A. niger
and
A. flavus
are most commonly isolated. Of these isolates,
A. fumigatus
is the most common human pathogen. Three main types of disease have been associated with
A. fumigatus
, namely allergic asthma, aspergilloma, and invasive aspergillosis (See e.g., Lortholary et al.,
Amer. J. Med.,
95:177-187 [1993]).
Allergic pulmonary asthma due to
A. fumigatus
exposure affects an estimated 50,000 individuals in the United States alone. Aspergillomas are formed when fungal spores germinate in situ in tissue such as the lungs and form fungus balls. There is typically no invasion of underlying tissues, and in most cases treatment involves the simple surgical removal of the aspergilloma. However, invasive aspergillosis involves the invasion of host tissues, and is most commonly observed in patients with predisposing conditions (e.g., immunosuppressive drugs, neutropenia, chemotherapy, AIDS). Transplant (e.g., bone marrow or organ) and chemotherapy patients are at the greatest risk for this form of aspergillosis (See e.g., Denning et al.,
New Eng. J. Med.,
324:654-662 [1992]; and Miller et al.,
Chest
105:37-44 [1994]). The prognosis for patients with invasive aspergillosis is particularly grave, with mortality rates greater than 50% (See e.g., Polis et al., “Fungal Infections in Patients with the Acquired Immunodeficiency Syndrome,” in DeVita et al. (eds),
AIDS: Biology, Diagnosis, Treatment, and Prevention,
4th ed., Lippincott-Raven, [1997]), due to the lack of a rapid diagnostic method to confirm
A. fumigatus
infection, and the lack of safe antifungal drugs.
The development of effectiv
Nakata Mitsunori
Selitrennikoff Claude P.
Medlen & Carroll LLP
Mycologics, Inc.
Prats Francisco
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