Fungicidal compositions and method

Novel fungicidal compositions comprising a 14.alpha.-methyldemethylase inhibiting azole compound and a .beta.-lactone compound and a method for controlling mycotic infections is disclosed.

BACKGROUND OF THE INVENTION 
Fungal diseases or mycoses may be superficial, affecting primarily skin, 
hair and mucous membrane, or may be deep or systemic, affecting lungs and 
other internal organs. The superficial mycotic infections which are caused 
by organisms referred to as dermatophytes are generally considered more 
annoying than serious. The deep or systemic mycotic infections which are 
caused generally by different organisms are quite serious, frequently 
resulting in death. 
Antifungal agents considered with specific reference to deep or systemic 
fungal infections caused by organisms such as Candida species, 
Cryptococcus neoformans, Histoplasma capsulatum and the like are found for 
the most part to be fungistatic, i.e., merely inhibit the growth of the 
fungal organism without effecting a kill. A few fungicidal agents are 
known. Amphotericin B and other polyenes are known to damage membranes 
that contain ergosterol and therefore are effectively fungicidal. However, 
their use is normally precluded because of a number of severe side 
effects. Other possibly fungicidal drugs, e.g. 5-fluorocytosine, have side 
effects or may be limited by the scope of their spectrum. 5-Fluorocytosine 
is further limited by the ease with which an organism develops resistance 
to it. In the search for antifungal drugs for treating systemic 
infections, it is desirable to find a drug or a combination of drugs which 
is effective at low concentration levels thereby minimizing side effects. 
It is particularly desirable to find a drug or a combination of drugs in 
which the resultant drug is fungicidal. 
STATEMENT OF THE INVENTION 
The present invention concerns an improved method for the treatment of deep 
or system mycotic infections made possible by the discovery than when 
certain fungistatic agents namely, a 14 .alpha.-methyldemethylase 
inhibiting azole compound and a .beta.-lactone compound, are employed in 
combination, a synergistic antifungal combination is obtained. It has been 
found further than certain combinations are able further to cause 
irreversible damage to the fungi resulting in a killing or cidal effect on 
the fungi. The .beta.-lactone compound as a component is especially 
desirable because the high effectiveness of the .beta.-lactone compound 
itself is such as to render the combination effective at very lose doses. 
The invention also concerns fungicidal compositions which are suitable for 
use in the treatment of system mycotic infections. 
DESCRIPTION OF THE INVENTION 
The fungicidal composition of the present invention comprises a 
.beta.-lactone compound and a 14 .alpha.-methyldemethylase inhibiting 
compound. 
The .beta.-lactone component is a compound having the formula 
##STR1## 
or a pharmaceutically acceptable salt thereof. The compound is named 
11-(3-hydroxymethyl-4-oxo-2-oxetanyl)-7-methyl-2,4-undecadienoic acid and 
may be produced by fungi; it is also known as Antibiotic 1233A reported by 
Aldridge et al, Chem. Comm., 1970, p. 639 and in J. Chem. Soc (c), 1971, 
pp. 3888-3890 (1972). The antifungal properties of the compound against 
fungi such as Trichophyton sp., Cryptococcus sp., Hormodendrum sp., 
Geotrichum sp., and Candida sp. are disclosed in the U.S. Ser. No. 825,496 
filed Feb. 3, 1986, now abandoned; published by EPO under 0234752, Sept. 
2, 1987. The teachings of the foregoing are incorporated herein by 
reference. 
The pharmaceutically acceptable salts of the .beta.-lactone component of 
this invention include those formed from cations such as sodium, 
potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases 
such as ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, 
ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, 
diethanolamine, procaine, N-benzylphenethylamine, diethylamine, 
piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium 
hydroxide. 
The 14-.alpha.-methyldemethylase inhibiting azole compounds are generally 
well-known for treating human mycotic infections, and the more important 
compounds imidazoles and triazoles. Many of these compounds are in use 
clinically as fungistats or are being developed for such purpose. The 
generic drug names for those compounds already developed or being 
developed have the suffix "conazole." In subsequent discussions, the 
compounds will sometimes will be referred to as "conazole compounds," even 
though some may not have a generic name. The foremost compound is 
ketoconazole which is cis-1-acetyl- 
4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazole-1-ylmethyl)-1,3-dioxolan-4-y 
l]methoxy]-phenyl]piperazine. Other fungistatic conazole compounds which 
are 14 .alpha.-methyldemethylase inhibitors and which are either in 
clinical use or in development include fluconazole, 
.alpha.-(2,4-difluorophenyl)-.alpha.-(1H-1,2,4-triazol-a-ylmethyl)-1H-1,2, 
4,-triazole-1-ethanol; miconazole, 
1-[2,4-dichloro-.beta.-(2,4-dichlorobenzyloxy) phenethyl]imidazole as 
nitrate; econazole, 1-[2-(2,4-dichlorophenyl)-2-(4-chlorobenzyloxy) 
ethyl]imidazole; isoconazole, 1-[2,4-dichloro-.beta.(2,6- 
dichlorobenzyloxy)phenethyl]imidazole as nitrate; terconazole, 
cis-1,4,2-(2,4-dichlorophenyl)-2-(1-ylmethyl)-1,3-dioxolan-4-yl-methoxy-ph 
enyl-4-(methyl-ethyl)piperazine; tioconazole, 
1-[2-[(2-choloro-3-thienyl)methoxy]-2-(2-choloro-3-thienyl)methoxy]-2-(2,4 
-dichloro-phenyl) ethyl]-1H-imidazole;bifonazole, 
1-[(4-biphenyl)phenyl-methyl]-1H-imidazole. Still other azoles include 
ICI-153066 (ICI Pharmaceutical Division), 
[(R,S)-1-(2,4-dichlorophenyl)-1-(4- 
fluorophenyl)-2-(1,2,4-triazol-1-yl)ethanol]; Bay-n-7133 (Bayer AG, West 
Germany), 1-(4-chlorophenoxy)-3,3'- 
dimethyl-2-(1,2,4-triazol-1-yl)-methylbutan-2-ol; 
(E)-1-(5-chlorothien-2-yl)-2-(1H-imidazole-1-yl)ethanone-2, 
6-dichlorophenylhydrazone hydrochloride; SM-4770 (Sumitomo Chemical Co., 
Ltd.), 
(R)-3-(n-butylthio)-2-(2,4-dichloro-phenyl)-1-(imidazole-1-yl)-2-propanol 
hydrochloride; oriconazole or itraconazole, 
(+)-cis-4-[4-[4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-1,2-4-triazol-1-ylmethyl 
)-1,3-dioxolan-4-yl]-methoxy] phenyl]- 
1-piperazinyl]phenyl]-2,4-dihydro-2-(1-methylpropyl)-3H-1,2,4-triazol-3-on 
e; fenticonazole, 
.alpha.-(2,4-dichlorophenyl)-.beta.,N-imidazolylethyl-4-phenyl-thiobenzyle 
ther nitrate; 
oxiconazole,(Z)-[2,4-dichloro-2-imidazole-1-yl)acetophenone]-0-(2,4-di-chl 
orobenzyl)oxime; 
omoconazole(E)-1-[2,4-chloro-.beta.-[2-(p-chloro-phenoxy)ethoxy]-.alpha.-m 
ethylstyryl]imidazole ;aliconazole. Still other imidazole antifungal 
compounds which may be employed include 
methyl-4-[3-2-methyl-5-nitro-1H-imidazole-1-yl)propyl]piperazine, 
5-nitro-(1-methylimidazolyl-t-butyl)(2-hydroxy-5-methoxyphenyl)carbinol, 
Z-1-[2-(2,4-dichlorophenyl)-3-methyl-1-pentenyl]-1H-imidazole 
hydrochloride, 
cis-3-(2-chloro-3-thienylmethyloxy)-2,3-dihydro-5-fluoro-2-(1-imidazolymet 
hyl)benzo[b]thiophene. 
The azole compounds may have a basic nitrogen and therefore may be present 
as an acid addition salt. Pharmaceutically acceptable salts suitable as 
acid addition salts include those from acids such as hydrochloric, 
hydrobromic, hydroiodic, phosphoric, sulfuric, trifluoroacetic, 
trichloracetic, oxalic, maleic, pyruvic, malonic, succinic, citric, 
mandelic, benzoic, cinnamic, methanesulfonic, ethanesulfonic, 
trifluoromethanesulfonic and the like. Reference to conazole compounds is 
intended to embrace both forms. 
Many of the conazole compounds are established antifungal compounds. 
Ketoconazole is one of the preferred antifungal compounds for its broad 
spectrum and substantial absence of side effects. The combination of 
ketoconazole and 
11-(3-hydroxy-methyl-4-oxo-2-oxetanyl)-7-methyl-2,4-undecadienoic acid 
hereinafter (".beta.-lactone compound") represents a preferred embodiment 
of the present invention. 
The synergistic antifungal and fungicidal combinations of the present 
invention are effective in the the treatment of mycotic infections caused 
by such fungal organisms as Candida species, for example, C. albicans, C. 
tropicalis, and C. stellatoidea. 
The efficacy of the combination of the present invention in producing a 
synergistic antifungal as well as fungicidal effect may be seen in the in 
vitro interaction studies for the determination of activity and 
determination of viable cells. Synergistic antifungal properties have been 
demonstrated with ketoconazole and the .beta.-lactone compound in tests 
against a representative fungal organism known to be the causative agent 
of mycotic infections, such as Candida albicans. Representative 
synergistic antifungal and fungicidal properties of combinations of the 
.beta.-lactone compound and various conazole compounds are demonstrated 
against Candida albicans as seen in the following examples. 
Minimum Inhibitory Concentration of .beta.-Lactone Compound 
11-(3-hydroxymethyl-4-oxo-2-oxetanyl)-7-methyl-2,4-undecadienoic acid 
(.beta.-lactone compound), was solubilized in 100 percent 
dimethylsulfoxide (DMSO). Twofold dilutions were made with DMSO to obtain 
final drug concentrations in the broth dilution assay tubes ranging from 
0.625 to 100 .mu.g/ml. 
The Candida albicans, MY 1055, yeast culture maintained in yeast nitrogen 
base/glucose (1/2 percent), YNB/G, was transferred to fresh medium and 
incubated 7 hours at 37.degree. with shaking at 250 rpm. After incubation, 
each culture was diluted to A.sub.600 =0.0004 U/ml which was previously 
determined to be equal to 3000 cfu/ml (colony forming units per 
milliliter). 
1 milliliter of YNB/G inoculated with yeast culture was added to sterile 
test tubes. The tubes were incubated at 250 rpm, 37.degree. C. for 17 hr. 
The minimum inhibitory concentrations (MIC) was recorded as the lowest 
concentration of drug showing visible growth. 
The minimum inhibitory concentration, against Candida albicans MY 1055, was 
determined to be a 2.5 .mu.g/ml. 
Minimum Fungicidal Concentration of .beta.-Lactone Compound 
In the manner above described for the determination of minimum inhibitory 
concentration, broth dilution assay tubes were prepared ranging from 0.625 
to 100 .mu.g/ml and 1 milliliter of YNB/G inoculated with Candida albicans 
MY 1055 sterile test tubes. The tubes were incubated at 250 rpm at 
37.degree. C. for 17 hours. 
The minimum fungicidal concentration (MFC) was determined by serially 
diluting samples of MIC tubes in 0.9% saline. Aliquots were plated on 
Sabouraud dextrose agar. The plates were incubated at 37.degree. for 48 
hr. and the colonies counted. From the counts obtained, the number of 
cfu/ml in the undiluted drug-culture tube was calculated. The MFC is 
defined as the minimum amount of drug required to reduce the number of 
viable cells initially present in the drug-culture tubes greater than or 
equal to 95%. The MFC was 10 .mu.g/ml. 
Synergistic & Fungicidal Effect .beta.-Lactone and Ketoconazole 
Synergistic and fungicidal effects were determined by treating exponential 
phase Candida albicans cultures with .beta.-Lactone at MIC and MFC levels 
and ketoconazole at 0.1 .mu.g/ml or a 1 .mu.g/ml alone or in combination. 
Exponential phase cultures were prepared by diluting an overnight culture 
1:50 or 1:1000 in YNB/G. After incubating the diluted cells 7 or 17 hrs. 
at 37.degree. C., the exponential phase cells were diluted in YNB/G to 
A.sub.600 =0.0004 u/ml to obtain 3000 cfu/ml. 
10 microliters (.mu.l) of .beta.-lactone compound or ketoconazole prepared 
in DMSO was added to 1 ml of diluted exponential phase cells. The tubes 
were incubated at 37.degree. C. at 250 rpm for 27 hours. Periodically, 
aliquots were diluted in 0.9% saline and plated on Sabouraud dextrose agar 
plates to determine the number of cfu/ml. 
A. Synergistic Effect 
The result for the .beta.-lactone compound at minimum inhibitory 
concentration of 2.5 .mu.g/ml with and without 1 .mu.g/ml of ketoconazole 
are seen in Table 1 and FIG. 1. 
TABLE 1 
______________________________________ 
Fungal Growth (CFU/ML) 
.beta.-Lactone 
Compound 
.beta.-Lactone 
Keto- (2.5 .mu.g/ml) + 
Time No Compound conazole 
Ketoconazole 
(Hours) Drug (2.5 .mu.g/ml) 
(1 .mu.g/ml) 
(1 .mu.g/ml) 
______________________________________ 
0 2.95 .times. 10.sup.3 
2.95 .times. 10.sup.3 
2 3.85 .times. 10.sup.3 
3.85 .times. 10.sup.3 
5.1 .times. 10.sup.3 
9.20 .times. 10.sup.3 
4.5 1.30 .times. 10.sup.4 
8.80 .times. 10.sup.3 
1.09 .times. 10.sup.4 
8.50 .times. 10.sup.3 
7 6.65z10.sup.4 
1.12 .times. 10.sup.4 
2.40 .times. 10.sup.4 
6.75 .times. 10.sup.3 
11 9.45 .times. 10.sup.5 
1.28 .times. 10.sup.4 
6.90 .times. 10.sup.4 
7.00 .times. 10.sup.3 
17 4.05 .times. 10.sup.7 
9.35 .times. 10.sup.3 
1.36 .times. 10.sup.5 
5.90 .times. 10.sup.2 
22 7.30 .times. 10.sup.7 
1.23 .times. 10.sup.4 
3.30 .times. 10.sup.5 
9.00.10.sup.1 
27 7.00 .times. 10.sup. 7 
1.80 .times. 10.sup.4 
7.20 .times. 10.sup.5 
2.50 .times. 10.sup.2 
______________________________________ 
The results show that after 17 hours, there is a definite synergistic 
effect of the combination of the .beta.-lactone compound and ketoconazole. 
B. Fungicidal Effect 
The results for the .beta.-lactone compound at 4 times the minimum 
inhibitory concentration with and without 1 .mu.g/ml of ketoconazole are 
seen in Table 2 and FIG. 2. 
TABLE 2 
______________________________________ 
Fungal Growth (CFU/ML) 
.beta.-Lactone 
Compound 
.beta.-Lactone 
Keto- (10 .mu.g/ml) + 
Time No Compound conazole 
Ketoconazole 
(Hours) Drug (10 .mu.g/ml) 
(1 .mu.g/ml) 
(1 .mu.g/ml) 
______________________________________ 
0 2.95 .times. 10.sup.3 
2.95 .times. 10.sup.3 
2 3.85 .times. 10.sup.3 
6.50 .times. 10.sup.3 
5.1 .times. 10.sup.3 
5.20 .times. 10.sup.3 
4.5 1.30 .times. 10.sup.4 
7.50 .times. 10.sup.3 
1.09 .times. 10.sup.4 
3.95 .times. 10.sup.3 
7 6.65 .times. 10.sup.4 
3.35 .times. 10.sup.3 
2.40 .times. 10.sup.4 
3.85 .times. 10.sup.3 
11 9.45 .times. 10.sup.5 
8.70 .times. 10.sup.2 
6.90 .times. 10.sup.4 
7.00 .times. 10.sup.1 
17 4.05 .times. 10.sup.7 
5.00 .times. 10.sup.1 
1.36 .times. 10.sup.5 
0 
22 7.30 .times. 10.sup.7 
6.50 .times. 10.sup.1 
3.30 .times. 10.sup.5 
5 
27 7.00 .times. 10.sup.7 
7.50 .times. 10.sup.1 
7.20 .times. 10.sup.5 
0 
______________________________________ 
The results show that after 17 hours, complete kill of the microorganism is 
effected by the combination of the .beta.-lactone at MFC (10 .mu.g/ml) and 
ketoconazole at 1 .mu.g/ml. 
Synergistic Effect of .beta.-Lactone Compound and Different Conazoles 
The effectiveness of the combination of the .beta.-lactone compound with 
various conazole compounds may be illustrated with ketoconazole, 
fluconazole and itraconazole. 
The .beta.-lactone compound and the following conazole compounds: 
cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-1H-imidazole-1-ylmethyl)-1,3- 
dioxolan-4-yl]-methoxyl]phenyl]piperazine (ketoconazole), 
.alpha.-(2,4-difluorophenyl)-.alpha.-(1H-1,4-triazol-1-ylmethyl)1H-1,2-4-t 
riazole-1-ethanol (fluconazole and 
cis-4-[4-[4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazole-1-ylmethyl)-1 
,3-dioxolan-4-yl]-methoxy] 
phenyl]-1-piperazinyl]phenyl-2,4-dihydro-2-(1-methylpropyl)-3H-1,2,4-triaz 
ol-3-one (itraconazole), were dissolved in DMSO and serially diluted in the 
manner previously described. 
Assay tubes were prepared in a manner similar to that previously described 
and in operations carried out in a manner similar to that previously 
described, the effect of various conazole compounds on the MIC of the 
.beta.-lactone compound were determined. The results are summarized in 
Table 3. 
TABLE 3 
______________________________________ 
Effect of Azole Antifungals on the MIC'S of the 
.beta.-Lactone Compound Against Candida albicans MY 1055 
POTENTIA- 
MIC'S OF .beta.-LACTONE COMPOUND 
TOR FLUCON- ITRACONAZOLE KETOCON- 
(.mu.g/ml) 
AZOLE (.mu.g/ml) AZOLE 
______________________________________ 
0 2.5 2.5 2.5 
0.3125 1.25 2.5 0.625 
1.25 0.625 1.25 0.625 
5.0 0.625 1.25 0.625 
______________________________________ 
Synergistic Effect of .beta.-Lactone Compound and Ketoconazole Against 
Fungal Panel 
In a manner similar to that above described for effect against Candida 
albicans the synergistic effect of the combination of the .beta.-lactone 
compound and ketoconazole against a fungal panel was determined by plating 
on potato dextrose agar. 
First the MIC for the .beta.-lactone compound and the MIC for the 
ketoconazole were determined against an array of organisms. Thereafter, 
the effects on the MIC of adding 0.031, 0.125, 0.5 and 2 .mu.g/ml of 
ketoconazole were determined. The results are seen in Table 4. 
TABLE 4 
______________________________________ 
Minimum Inhibitory Concentration (MIC) 
Keto- 
.beta.-Lactone Compound in 
cona- 
Presence of Ketoconazole 
zole 
.mu.g/ml of Ketoconazole 
Alone 
Organism 0 0.031 0.123 0.5 2 .mu.g/ml 
______________________________________ 
A. niger &gt;50 &gt;50 &gt;50 &gt;50 &gt;50 &gt;2 
C. miyabeamis 
0.78 0.39 0.78 0.195 
N.G. 2 
F. oxysporium 
&gt;50 &gt;50 &gt;50 &gt;50 &gt;50 &gt;2 
U. zeae 3.125 3.125 0.78 0.19 0.19 &gt;2 
C. neoformans 
12.5 12.5 3.125 
0.19 0.19 &gt;2 
C. albicans 
3.125 0.78 0.78 0.19 0.19 &gt;2 
(Y1055) 
C. albicans 
3.125 0.78 0.195 
0.19 0.19 &gt;2 
(Y1750) 
______________________________________ 
From the foregoing test results and from known dosage ranges of the 
"conazole compound" as applied to man, it is determined that generally 
from about 2.85 to about 4.75 mg/kg of body weight of the conazole 
compound and about 2.85 to about 4.75 mg/kg of body weight of the 
.beta.-lactone compound is to be employed while considering patient's 
health, weight, age and other factors which influence response to a drug 
as well as the particular drug to be employed. These amounts when 
expressed as doses suitable for man are in the range of from about 200 to 
about 400 mg of each active ingredient given BID by oral or parenteral 
route. 
According to the present invention, the synergistic antifungal or 
fungicidal composition may be formulated for injection and may be present 
in unit dosage form in ampoules or in multidose containers, if necessary, 
with an added preservative. The compositions may also take such forms as 
suspensions, solutions or emulsions in oily or aqueous vehicles, and may 
contain formulating agents such as suspending, stabilizing and/or 
dispersing agents. Alternatively, the active ingredients may be in powder 
form for reconstituting with a suitable vehicle prior to parenteral or 
oral administration. 
The compounds also may be prepared in tablet or capsule form as well as in 
liquid form for oral administration. These also may be in unit dosage 
form. 
For parenteral applications the drugs may be formulated in conventional 
parenteral solutions such as 0.85 percent sodium chloride or 5 percent 
dextrose in water, or other pharmaceutically acceptable compositions. 
The outstanding properties are most effectively utilized when the conazole 
compound and the .beta.-lactone compound are formulated into novel 
pharmaceutical composition with a pharmaceutically acceptable carrier 
according to conventional pharmaceutical compounding techniques. 
In preparing the compositions in oral dosage form, the component drugs are 
intimately admixed with any of the usual pharmaceutical media, including 
for liquid preparations, liquid carriers such as water, glycols, oils, 
alcohols, and the like, and for solid preparations such as capsules and 
tablets, solid carriers such as starches, sugars, kaolin, ethyl cellulose, 
generally with a lubricant such as calcium stearate, together with 
binders, disintegrating agents and the like. Because of their ease in 
administration, tablets and capsules represent the most advantageous oral 
dosage form. It is especially advantageous to formulate the compositions 
in unit dosage form for ease of administration and uniformity of dosage. 
Compositions in unit dosage form constitutes an aspect of the present 
invention. 
The term "unit dosage form" as used in the specification and claims refer 
to physically discrete units, each unit containing a predetermined 
quantity of active ingredient calculated to produce the desired 
therapeutic effect in association with the pharmaceutical carrier. 
Examples of such unit dosage forms are tablets, capsules, pills, powder 
packets, wafers, measured units in ampoules or in multidose containers and 
the like. A unit dosage of the present invention will generally contain 
from 200 to 400 milligrams of each of the component drugs. 
The following examples illustrate novel compositions useful in the practice 
of the present invention, but are not to be construed as limiting:

EXAMPLE I 
1000 compressed tablets each containing 200 milligrams of ketoconazole and 
300 milligrams of .beta.-lactone compound are prepared from the following 
formulation: 
______________________________________ 
Grams 
______________________________________ 
Ketoconazole 200 
.beta.-Lactone compound 
300 
Starch 750 
Dibasic calcium phosphate hydrous 
5000 
Calcium stearate 2.5 
______________________________________ 
The finely powered ingredients are mixed well and granulated with 10 
percent starch paste. The granulation is dried and compressed into 
tablets. 
EXAMPLE II 
1000 hard gelatin capsules, each containing 210 milligrams of ketoconazole 
and 290 milligrams of .beta.-lactone compound are prepared from the 
following formulation: 
______________________________________ 
Amount 
______________________________________ 
Ketoconazole 210 grams 
.beta.-Lactone compound 
290 grams 
Starch 250 grams 
Lactose 750 grams 
Talc 250 grams 
Calcium stearate 10 grams 
______________________________________ 
A uniform mixture of the ingredients is prepared by blending and used to 
fill two-piece hard gelatin capsules. 
EXAMPLE III 
250 milliliters of an injectable solution are prepared by conventional 
procedures having the following formulation: 
______________________________________ 
Amount 
______________________________________ 
Dextrose 12.5 grams 
Water 250 milliliters 
Ketoconazole 200 milligrams 
.beta.-Lactone compound 
200 milligrams 
______________________________________ 
The ingredients are blended and thereafter sterilized for use.