Use of nikkomycin compounds to treat infections of dimorphic, highly chitinous fungi

Method of treating an infection of dimorphic, highly chitinous fungi, the method comprising administering therapeutically effective amounts of a nikkomycin or derivative thereof. Treatment is especially suited for infections of medically significant fungi having cell walls with about 10-20% by weight chitin in the parasitic phase. Such fungi include Coccidiodes immitis, Histoplasma capsulatum, Blastomyces dermatitidis, and Paracoccidiodes brasiliensis.

BACKGROUND OF THE INVENTION 
1. Field 
This disclosure is concerned generally with the use of antibiotics to treat 
infections of medically significant fungi. Specifically, the disclosure is 
concerned with the use of a class of antibiotics known as nikkomycins to 
treat infections of dimorphic and highly chitinous (more than 10% by 
weight chitin) fungi in mammals. 
2. Prior Art 
Compounds inhibitory to the synthesis of fungal cell wall material 
(synthase inhibitors) have been reported recently to have demonstrable 
effects against fungi of agricultural importance (U.S. Pat. Nos. 4,315,922 
and 4,158,608). See also U.S. Pat. Nos. 4,585,761 and 4,552,954 for 
descriptions of the preparation and purification of such compounds. The 
agents mentioned in the cited patents, nikkomycins, together with similar 
agents known as polyoxins, are known to act by interfering with the 
synthesis of chitin in the cell walls of fungi. Because fungi of medical 
importance to humans also have varying amounts of chitin in their cell 
walls, experiments have been conducted to determine if the chitin synthase 
inhibitors are capable of inhibiting the growth of such fungi (Hector and 
Pappagianis, J. Bacteriol. 154:488-498, 1983, and Hector and Braun, 
Antimicrobial Agents Chemother, 29:389-394, 1986). In earlier work, 
certain fungi such as Candida albicans were reported to be insensitive to 
chitin synthase inhibitors (see Naider et al, Antimicrobial Agents 
Chemother. 24:787-796, 1983). Subsequently, C. albicans was found to be 
more sensitive to nikkomycins than polyoxins (see Yadan et al, J. 
Bacteriol. 160:884-888, 1984) but the concentrations necessary for killing 
that yeast would (for toxicity reasons) preclude their use as 
chemotherapeutic agents for yeast infections (see Hector and Braun, 
Antimicrobial Agents Chemother., 29:389-394, 1986). 
Quite surprisingly, I have now found that nikkomycin compounds are 
efficacious in treating fungal infections if the fungal agents are highly 
chitinous in the parasitic phase. Although the results described below 
were surprising, it is thought that they may be based on the difference in 
chitin content in the cell walls of the fungi (see, for example, Chattaway 
et al, J. Gen. Microbiol. 51:367-376, 1967, where C. albicans is reported 
to contain less than 1% by weight chitin in the cell wall). 
As reported below, data from in vivo and in vitro studies using 
Coccidioides immitis dimorphic fungi show that nikkomycin compounds are 
efficacious in treating infections of such fungi. 
SUMMARY OF INVENTION 
Nikkomycin compounds have been found effective in treating a mammal having 
an infection of a dimorphic, highly chitinous fungus. The compounds are 
especially suited for treating medically significant fungi having cell 
walls comprising at least 10% by weight chitin in the parasitic phase. 
Such fungi include Coccidioides immitis, Histoplasma capsulatum, 
Blastomyces dermatitides, and Paracoccidioides brasiliensis. In one 
embodiment the nikkomycin is nikkomycin X or nikkomycin Z administered in 
an amount sufficient to treat infection of the dimorphic, highly chitinous 
fungi in a mammal (such as the mouse). In another embodiment, the amount 
of nikkomycin or a derivative thereof is sufficient to inhibit the enzyme 
chitin synthase. It is thought that the treatment with nikkomycin will be 
especially useful against C. immitis infections, the causative agent of 
what is commonly referred to as valley fever. As used herein, the term 
highly chitinous means that the chitin must be at least 10% by weight of 
the cell wall of the organism. The term dimorphic means having two 
distinct growth phases (i.e. saprophytic and parasitic phases). 
Therapeutically effective amount (of nikkomycin or a nikkomycin 
derivative) means an amount (commonly expressed as mg/kg body weight) 
sufficient to result in clinical improvement in the signs and symptoms of 
disease and/or prevention of mortality in the more critically ill. 
Pharmaceutically acceptable vehicle means a carrier suitable for delivering 
safe and efficacious amounts of the nikkomycin or a derivative thereof. 
As can be appreciated, the above microorganisms are known as primary 
pulmonary pathogens (cf. opportunistic pathogens) and it is thought that 
the nikkomycin compounds are especially useful in treating infections of 
that class of pathogens. 
SPECIFIC EMBODIMENTS 
Strains and conditions of culture. Coccidioides immitis strain Silveira was 
employed in illustrative studies. For growth of arthrospores, slants of 
glucose-yeast extract agar were inoculated with an endospore suspension 
and allowed to grow for several weeks at room temperature. The 
arthrospores were collected by gently disrupting the mycelial growth under 
liquid, the liquid decanted, centrifuged, ad the pellet washed and 
resuspended. The spherule phase of the fungus was maintained by repeated 
passage of endospores in liquid modified Converse medium (Trans. N.Y. 
Acad. Sci. 20:436-449, 1960) incubated at 37.degree. C. with shaking. 
In vitro experiments. 
Susceptibility testing was performed by subjecting suspensions of 15 h 
immature spherules (final 0.D. of 0.2 at 440 nm) to concentrations of 
either nikkomycin X or nikkomycin Z of 0.04, 0.1, or 0.4 mM (final 
concentration diluted in modified Converse medium) with continued 
incubation for intervals of 8 and 24 h. Inhibition of growth was to be 
determined visually by light microscopy and by staining with calcofluor 
white, a fluorchrome with known affinity for chitin, followed by 
epifluorescent microscopy. 
In vivo studies. 
A murine model of coccidioidomycosis was used to assess the compounds in 
vivo. Outbred Swiss-Webster mice, 25 g. average weight, were anesthetized, 
then inected intranasally by placing 0.05 ml of a suspension containing 
500 arthrospores of C. immitis on the nares of each animal, and allowing 
them to inhale the suspension. A total of 30 animals were infected, then 
divided into groups of 10 each for testing. The following day therapy was 
begun. Nikkomycin X and Z were prepared by suspending the powders in 
sterile water to achieve a dose of 100 mg/kg body weight delivered in 0.1 
ml. The liquids were aliquoted into 1 ml portions and held at -70.degree. 
C. until needed. Animals were treated with nikkomycin X, nikkomycin Z, or 
given sterile water starting the day after infection. Doses were given 
orally three times a day for a total of nine days. Nine days after the 
cessation of therapy, 5-6 animals from each of the three groups were 
sacrificed and their lungs, livers, and spleens removed for quantitative 
cultures for the enumeration of viable fungus. Organ homogenates were 
cultured on Sabourauds agar and held for two weeks before being considered 
negative. 
Results 
In vitro studies. 
Examination of cultures at the 8 h interval revealed that the two highest 
concentrations of nikkomycin X (NX) and nikkomycin Z (NZ) resulted in the 
complete cessation of growth of the fungus. In general, only small 
spherules with no evidence of endosporulation were present. At the lowest 
concentration, it was evident that endosporulation had been arrested by 
both nikkomycin X and Z, but a small percentage of swollen cells, 
indicating osmotic sensitivity and a weakened cell wall, were seen with 
both compounds. In general, NZ appeared slightly more effective than NX. 
In contrast, the control culture showed normal patterns of maturation, 
i.e., endosporulation followed by the rupture of the spherule to release 
the progeny endospores. Results of epifluorescent microscopy confirmed the 
absence of cleavage planes and indicated a reduction of chitin content in 
the cell walls. 
In vivo studies. 
Results of the cultures demonstrated that the control animals were heavily 
infected, with 5.766 log10 colony forming units of fungus in lungs, 2.256 
log10 units in the livers, and 1.594 log10 units in the spleens as average 
values for 6 sacrificed animals. In marked contrast, none of the cultures 
from treated animals were positive for fungus, indicating a complete 
sterilization of the organs by the nikkomycins. These results are 
summarized in the Table below. 
TABLE 
______________________________________ 
Use of Nikkomycin X and Z 
in Murine Coccidioidomycosis* 
Geometric Mean Log CFU/GM Tissue 
Group** Lung Liver Spleen 
______________________________________ 
Control 5.766 2.256 1.594 
Nikkomycin X 
Neg Neg Neg 
Nikkomycin Z 
Neg Neg Neg 
______________________________________ 
*5-6 Animals/group infected intranasally with 500 arthrospores of C. 
Immitis. 
**Animals given 100 mg/kg nikkomycin orally T.I.D. starting 18 hours afte 
infection; controls received water. 
The findings of the in vivo experiments in which treated animals were 
devoid of viable fungus while the control animals were heavily infected in 
the lungs with evidence of dissemination were unexpected. As has been 
previously reported (Yadan et al, J. Bacteriol. 160:884-888, 1984) the 
portal of entry for the nikkomycins into the fungal cells is via the 
peptide transport system. Given that in an animal host the infecting 
fungus (in the organs or bloodstream) is constantly bathed in a millieu 
rich in peptides, it was reasonable to assume that competition for 
available transport sites could have significantly reduced the amount of 
active substance transported into the cell, thus reducing efficacy of the 
drugs. This situation would not manifest itself in agricultural 
applications of the nikkomycins because most phytopathic fungi and insects 
exist as ectoparasites. Thus, the findings reported herein represent a 
unique and unexpected application. 
Discussion 
The data indicate that nikkomycins X and Z can serve as efficacious agents 
against dimorphic, highly chitinous fungi such as C. immitis. The 
compounds appear to work by interfering with normal cell wall synthesis, a 
necessary component of endosporulation in the fungus. The animal 
experiment indicated that animals infected with a potentially lethal 
challenge with C. immitis can be protected by administration of the 
compounds orally for as short a period as 9 days. Therefore, it is thought 
that these agents should be considered as antifungal drugs in human use 
for the described fungus as well as other medically important fungi in 
which chitin can be demonstrated to be present in the cell wall in 
relatively high amounts (i.e. more than 10% by weight or in the range of 
10-20% by weight).

FURTHER EXAMPLES 
The previous in vivo experiments demonstrated the ability of orally 
administered doses of nikkomycin X and Z to sterilize the organs of mice 
infected with a potentially, though not acutely, lethal number of 
arthrocondia of Coccidioides immitis via the pulmonary route. Two 
additional experiments were done; one to test of ability of these two 
compounds to prolong survival in animals infected with a more lethal 
challenge of this fungus, and a second to assess the pathology of lungs 
from mice infected with a massive challenge and treated on a short-term 
basis with nikkomycin X or Z. 
Materials and Methods 
Survival Experiment: Female Swiss-Webster mice (Charles River Labs.), 25 
grams average weight, were anesthetized and then infected via the 
intranasal route with 4800 arthrocondia of C. immitis strain Silveira. 
Fifty-two hours after infection, groups of 9-10 animals animals each were 
begun on a 9 day B.I.D. regimen of 75 mg/kg of nikkomycin X or Z delivered 
orally, with two groups of mice held without treatment. Upon the first 
death in one of the untreated groups, the remaining survivors of that 
group were initiated on an oral regimen of nikkomycin X, given T.I.D. at 
75 mg/kg for a total of 9 days. Additionally, because of the low 
mortalities seen in the two initial treatment groups, five animals each 
from the nikkomycin X and Z groups were sacrificed and autopsied. Four 
animals were processed for organ cultures of lungs, livers, and spleens, 
and the lungs from the fifth animals were processed for histological 
examination. The remaining animals were held for observation for a total 
of 30 days, then survivors posted for organ cultures. 
Pathology Studies: Six female Swiss-Webster mice were infected intranasally 
with 10,000 arthrocondida of C. immitis silveira. After a delay of 52 
hours, two mice each were begun on an oral regimen of nikkomycin X or Z at 
75 mg/kg given B.I.D. for 4 days, with two mice held as controls. The day 
following cessation of treatment, all mice were sacrificed, and lungs, 
livers, and kidneys were removed for histology. 
Results: Deaths in the control group had rapid, onset, reaching 80% 
mortality by day 11. Therapy with nikkomycin Z was completely successful 
in preventing death, while there was a single death in animals receiving 
nikkomycin X in the initial group. In those animals given nikkomycin X 
after the first death there were 4 survivors, indicating therapy with this 
compound was effective in reducing death in moribund animals, with a 
survival rate of 50% (4/8) in animals receiving therapy. Data from the 
organ cultures taken at days 18 and 30, shows that both compounds are able 
to greatly reduce the amount of viable fungus in the lungs and prevent the 
dissemination of the disease to two key organs of the reticuloendothelial 
system. 
Histology: Examination of the lung histological preparations from the 
treated mice of the survival experiment failed to reveal any recognizable 
fungal elements or evidence of significant pathology. In the experiment 
conducted exclusively for the purposes of histology, the lung preparation 
from control animals showed large numbers of parasitic phase C. immitis 
and pathology consistent with early inflammatory disease and granuloma 
formation described for coccidioidomyciosis. In marked contrast, lung 
preparations from nikkomycin Z-treated mice showed no evidence of fungus 
and only limited pathology (i.e., infrequent foci of inflammation). 
Examination of the nikkomycin X lung preparations showed no evidence of 
intact fungal cells, though there existed less than 10 cells per slide 
with an abberant morphology which could have been fungal in origin. 
Summary: Data indicates that orally administered nikkomycins X and Z are 
markedly effective in preventing mortality and morbidity in the described 
animal model of pulmonary coccidioidomuyciosis. In marked contrast to 
previous reports of drugs effective in reducing disease in this model, the 
duration of therapy was short, yet the results are superior. Also in 
contrast to other human use antifungal agents of record, the nikkomycin X 
and Z appear to be potentially fungicidal, as demonstrated by the lack of 
microscopic evidence of intact fungi in the histologic preparations from 
treated animals. As such, this confirms and extends the findings 
documented in the earlier examples. 
Given the above disclosure, it is thought variations (such as ideal 
dosages, vehicles, etc.) will occur to those skilled in the art. For 
example, it is thought that the nikkomycins may also be effective against 
infections in mammals by chitinous organisms other than the dimorphic 
fungi of this disclosure. One such organism is the parasite Pneumocystis 
carinii which causes pneumonia in certain immuno-compromised individuals. 
Accordingly, it is intended that the above examples should be limited only 
by the following claims.