Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2002-10-24
2004-09-14
Lilling, Herbert J. (Department: 1651)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C435S118000, C435S125000, C435S254100
Reexamination Certificate
active
06790968
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel FKI-0076 substance having potentiation activity for azole antifungal agents and production thereof.
2. Description of Related Art
Azoles specifically used for treatment of diseases such as deep-seated mycosis, for example, miconazole {1-[2,4-dichlorobenzyloxy]-2-(2-,4-dichlorophenyl)ethyl} imidazole: Sigma Inc.}, fluconazole [2,4-difluoro- &agr;, &agr;-bis(1H,1,2,4-triazol-1-yl-methyl) benzyl alcohol: ICN Pharmaceuticals Inc.] and itraconazole ((±)-1-Sec-butyl-4-[P-[4-[P-[[2R,4S]-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl-methyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-piperazinyl]phenyl}-&Dgr;
2
-1,2-,4-triazolin-5-one: Kyowa Hakko Kogyo K. K.), are highly safe as compared with the most frequently used polyenes, amphotericin B, for treatment of such diseases (Anaissie E. J. et al., Clinical Infectious Diseases, 23: 964-972, 1996). However, recently, the appearance of resistant strains of microbes caused by long-term or repetitive administration of these azole antimicrobial agents has become a problem. As a result, the development of drugs with a high level of safety and a low level of resistance exhibited by microorganisms is urgently necessary.
SUMMARY OF THE INVENTION
In diseases accompanied with immunocompromised conditions such as HIV infection and hematologic disease, the compromised host condition is induced, and the risk of developing a fungal infection is increased as an opportunistic infection. Diseases with these immunocompromised conditions are frequently serious, and long-term therapy is required. Consequently, at present the most frequently used azole antifungal agents may not be used to present inducing drug resistance, since the long term chemotherapy of fungal infection is required in many cases.
A mechanism of resistance against azole antifungal agents is known as follows. In
Candida albicans
, the target enzyme, P-450, i.e. excess expression of 14-&agr;-demethylase or decrease in affinity with drug as a result of amino acid mutation (Vanden Bossche, H. et al. Antimicrobial Agents and Chemotherapy, 36, 2602-2610, 1992; Sanglard, D. et al., ibid., 42, 241-153, 1998) and decrease in intracellular drug concentration by the multiple drug excretion transporter (Fing, M. E. et al., Mol. Gen. Gent., 227, 318-329, 1991; Sanglard, D. et al., Microbiology, 143, 405-416, 1997) are known. In
Saccharomyces cerevisiae
, it has reported that MDR (Multiple Drug Resistant) genes, PDR16 and PDR17, could change the lipid metabolism to acquire the resistance against azole compounds (H. Bart van den Hazel, et al., J. Biol. Chem., 274, 1934-1941, 1999).
Consequently, the drugs, which can increase activity of azole antifungal agents, may be expected to decrease the frequency of appearance of the resistant microorganisms by reducing the amount of the administration of the drug as well as shortening the administration term. Further, as a result of use in combination with drugs having two different skeletal structures, the resistance against azole antifungal agents can be overcome.
In such condition, to provide drugs having enhanced activity of azole antifungal agents is useful for the treatment of various fungal infections such as deep-seated mycosis.
We have found that, as a result of studies on metabolites produced by various microorganisms, a substance having properties to activate azole antifungal agents was produced in a cultured mass of the strain FKI-0076 which was newly isolated from soil. We have also found, as a result of isolating and purifying the active substance from the cultures mass, the substance having the chemical structure shown in the formula [I] hereinafter and designated as FKI-0076 substance due to its unknown chemical structure.
The present invention was obtained as a result of these findings. The present invention relates to FKI-0076 substance indicated by the chemical structure [I] hereinbelow.
The present invention also relates to the FKI-0076 substance having the following physicochemical properties.
(1) Nature: yellow oily substance.
(2) Molecular weight: 418 (fast atom bombardment mass spectrometry)
(3) Molecular formula: C
21
H
22
O
9
.
(4) Optical rotation: [&agr;]
D
25
=+5.2° (c=0.23, methanol)
(5) DV spectrum: Ultraviolet absorption spectrum measured in methanol as shown in
FIG. 1
, and has a specific absorption maximum at 208 nm (&egr;=34900), 246 nm (&egr;=11000) and 314 nm (&egr;=3950).
(6) IR spectrum: Infrared absorption spectrum measured in KBr tablet as shown in FIG.
2
and has specific absorption bands at 3410, 1735, 1724, 1683, 1658, 1602 and 1579 cm
−1
.
(7)
1
H-NMR spectrum: As shown in
FIG. 3
(measured by using NRM spectrometer, Varian Japan, in CDCl
3
).
(8)
13
C-NMR spectrum: As shown in
FIG. 4
(measured by using NRM spectrometer, Varian Japan, in CDCl
3
).
(9) Solubility in solvents: Soluble in methanol, chloroform and ethyl acetate.
Slightly soluble in hexane.
(10) Color reaction: Positive in molybdic acid
(11) Differentiation in acidic, neutral and alkaline nature: Neutral substance.
The present invention further relates to a process for production of FKI-0076 substance comprising culturing a microorganism belonging to
Talaromyces flavus
capable of producing FKI-0076 substance, accumulating FKI-0076 substance in the cultured mass and isolating FKI-0076 substance from the cultured mass.
The present invention further relates to the process for producing FKI-0076 substance wherein the microorganism is capable of producing FKI-0076 substance is
Talaromyces flavus
FKI-0076 FERM BP-7037. The present invention further relates to the microorganism capable of producing FKI-0076 substance wherein the microorganism is
Talaromyces flavus
FKI-0076 FERM BP-7037.
A preferable example of the microorganism strain of the present invention used for production of FKI-0076 substance is the strain of
Talaromyces flavus
FKI-0076, which was newly isolated from soil by the inventors of the present invention.
Taxonomical properties of the microorganism strain are shown as follows.
1. Morphological Properties:
Good growth is observed in Czapek-yeast extract agar medium, oatmeal agar medium, malt extract agar medium and cornmeal agar medium and large numbers of ascocarps covered with yellow to orange colored soft hyphae are observed. Many penicili are observed on cornmeal agar. On the other hand, on 25% glycerol nitrate agar medium, growth is suppresed without observing ascocarps and only penicili are observed. On day 7, growth is more rapid at 37° C. in the Czapek-yeast extract agar medium than at 25° C., but no matured ascocarps are observed.
Ascocarps are spherical with diameter of 300-800 &mgr;m and are matured within 10 days. Ascocarp primordium is grown with the ascogone coiling around the club-shaped antheridium. The shape of the ascus is spherical to aspherical (8.8-11.0×7.5-10.0 &mgr;m) and disappears with maturation. The ascospore is ellipsoidal (3.5-4.5×2.5-3.5 &mgr;m) with spiny spikes covering the whole surface.
The conidiophore of penicili grow from substrate mycelium and aerial mycelium with longer conidiophore of the length of 180-250 &mgr;m grown from substrate mycelium, on the other hand, the length of the conidiophore is shorter with a length of 50-120 &mgr;m. The penicili forms monoverticillate to biverticillate. Metula is 12.5-17.5×2.5-3.0 &mgr;m with growing 2-4metulae in colonies. A pen point shaped phialide has a length of 11.0-15.0×1.8-3.0 &mgr;m with 3-6 verticillates in the metulae. The conida were elliptical with a length of 2.0-3.0×1.5-2.5 &mgr;m and the surface is smooth.
2. Cultured Properties on Various Agar Medium
Results of macroscopic observation cultured on various agar media at 25° C. for 14 days are shown in Table 1.
TABLE 1
Medium
(diameter of colony)
Color of surface
Color of reverse
Growth on medium
of colony
s
Arai Masayoshi
Masuma Rokuro
Omura Satoshi
Tomoda Hiroshi
Lilling Herbert J.
The Kitasato Institute
Young & Thompson
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