GB2191694A - Antifungal compositions - Google Patents

Description

SPECIFICATION Antifungal compositions There are provided pharmaceutical antimycotic compositions for use as antifungal agents against a wide variety of medically important fungi. The active ingredients of the compositions are compounds which may be isolated from the roots of alfalfa, which can also be prepared synthetically, and derivatives of these. The active principles of the compositions of the invention are highly efficient at low concentrations and can be used in human and veterinary medicine to inhibit growth of fungi and to kill same. The occurrence of mycosis as a primary or secondary disease is increasingly rapid, due to the widespread use of immunosuppressive drugs and broad spectrum antibiotics. The development of new antifungal agents has therefore become essential in modern medicine. This development, however, has been very slow, primarily because fungi are eucaryotes and it has been difficult to develop agents that specifically affectthe fungus without affecting host cells. Only a relatively small number of drugs are currently available for the treatment of fungal infections and most of these are toxic. The most important antimycotic drugs are the polyene macrolides and the more recently discovered imidazole derivatives, both of which affectthe cell membrane. Other antifungal agents are 5-fluorocytosine which affects nucleic acid synthesis, and griseofulvin which inhibits microtubularfunction and cell division. There are many problems associated with the antimycotic drugs in current usage. Among these are the poor solubility, stability and absorption of polyene antibiotics and their high toxicity, mainly forthe kidney. Recently, fungal resistance to amphotericin B has been reported. The imidazoles are well absorbed from the gastrointestinal tract but prolonged administration produces hepatic and adrenal changes and dysfunction, and they are inefficient in treating systemic mycoses. The therapeutic use of fluorocytosine is limited because of its narrow antimycotic spectrum and the high degree of fungal resistance to the drug. Griseofulvin, which is very potent against dermatophytes, has little or no effect on other fungi.All these problems, compounded by the increasing occurrence of systemic fungal infections, particularly in the compromised host, add impetus to the search for a new group of anti-mycotic agents. Saponins are glycosidesthat occur in a wide variety of plants and have the potential to affect resistance to plant diseases. It was found that in the legume alfalfa, saponins have selective toxicity against: Sclerotium rolfsii, Alternaria solani and Sclerotinia sclerotiourum which are known plant pathogen fungi. They are concentrated mostly in the cortex of the mature roots protecting them from fungal invasion. There has been demonstrated the activity of medicagenic acid, an aglycone constituent of some alfalfa saponins, in fungal growth inhibition. There exists a preliminary report (B. Gestetner, Y. Assa and M. Rotman, Experientia 29, 529-530,1973) describing the activity of medicagenic acid and some glycoside derivatives thereof against a single plant pathogen, Sclerotium rolfsii at one (10-4M) concentration of the preparation.No data were published on fungal diseases of mammals.

SUMMARY OF THE INVENTION: The present invention relates to pharmaceutical and veterinary antifungal compositions for use in the treatment of a wide scope of fungal infections. The compositions can be administered by the oral route, by injection or they can be used for topical or local use. The active ingredients are medicagenic acid or derivatives of same of the general formula I

wherein R1 is hydrogen or Ac,

R3 is hydrogen or C1-C6 alkyl and functional derivatives of these. Medicagenic acid and the substances defined herein as Compounds G, G2 and F can be purified from alfalfa. The invention also relates to such compounds in chemically pure form and to novel derivatives of these, as above defined. The novel compositions have a low hemolytic effect and are effective in very small concentrations of the active ingredient. Three of the active substances can be purified from alfalfa. These exhibit selective toxicity towards a wide variety of clinically important yeasts. There were prepared substances, designated as G, G2 and F, G2 being the more potent one. G2 is obtained by the further purification of G. The invention relates to antimycotic compositions containing any of the compounds defined above. The antifungal agents G and G2 have a poor solubility in water and thus they must be provided in a suitable solvent or carrier for adequately high concentrations. Solutions in polyethylene glycol (0.1 %) with aqueous sodium hydroxide (2 mM) resulted in the solubilization ofthese. When tested against 11 of the most important yeasts, which have considerable clinical significance, it was established that the compositions of the invention exhibit a pronounced inhibitory effect at low concentrations. MIC values obtained by both agar and broth dilution methods ranged between 2 and 15ug/ ml when G2 was used. Cryptococcus neoformans was the most susceptible yeast and the modal MIC value was 2 pg/ml. G2 exhibits fungicidal activity at low concentrations and there were obtained MFC values of between 4 and 24 Microg/ml with G2. G2 is a relatively stable compound and thus can be used as active ingredient in fungicidal preparations for use against a wide variety of fungal afflictions. The activity of G is lower by a factor of about 15, and also this compound can be used as active ingredient. Also the other substances have a high antimycotic activity. In view of the above it is clear that the novel compositions are of considerable pharmaceutical value for the treatment of mycotic infections in human and veterinary medicine. The essentially pure substances of

The purification of G, G2 and F from alfalfa root is set out in the enclosed Scheme I. The pharmaceutical compositions of the invention are suitable for topical application in a suitable carrier or diluent. They can also be administered orally or by injection. Among suitable pharmaceutical composition forms there may be mentioned oral tablets, lotions, creams, ointments, powders for topical application; lyophilized powders for dilution and injection, oral gel formulations, vaginal suppositories, powder for aerosol or suspensions for aerosols. In the topical preparations the active ingredient will generally comprise from about 0.2 to about 2 per cent by weight. The unit dosage forms will generally provide about 0.1 to about 2 mg of the active substance per kg weight of the patient per day.

MATERIALS AND METHODS Strains, media and chemicals Yeasts used were isolated from clinical specimens submitted to different body sites of patients before treatment. Only one isolate per patient was studied. The yeasts were identified to species by current conventional methods. The isolates were maintained on Sabouraud dextrose agar (SDA) until tested. Extraction and Purification of the antimycotic agent

TLC analyses were carried out on Merck silica 60 F254 plates, developed with EtOAc-H20-AcOH (7:2:2, c/ v). HPTLC analyses were performed on Merck RP-18 F254 plates, developed with MeOH-H20 (4: 1, v/v). Staining in all cases involved spraying with H2SO4 (5%, v/v in EtOH) and heating for 10 min at 100[deg]C. Mesh sizes stated are ASTM sieve sizes.

Alfalfa (Medicago sativa L. Gilboa variety from the RAM ranch, Beer-Sheva) root flour (200 g) was extracted with EtOH 80% (v/v, 2.5 L) in water for 16 h at 60[deg]C. The solid was then filtered off and the ethanol removed under reduced pressure. The resulting aqueous solution was thrice extracted with ether (400 mL portions) which was subsequently removed under reduced pressure while adsorbing the extract onto silica gel (Merck, 70-230 mesh, 6 g), applied and chromatographed (2 g portions) on the same adsorbent (150 g) with EtOAc-H20-AcOH (7:2:2:, v/v, 350 mL). Fractions containing the typical blue spot on TLC (Rf 0.75) were pooled, solvents were evaporated and the residue was resuspended in water (5 mL) and lyophilized to yield about 300 mg of G. Alternatively fractions containing G and F from the first chromatographic step (CI) were adsorbed onto silica gel (Merck, 70-230 mesh, 2 g) and were further purified to yield G2 and F by applying C1 onto a flash chromatography column (Merck, silica gel 60, 230 400 mesh, 15 g) eluted successively with EtOAc-MeOH 4:1, 2:1, 1:11 (v/v, each fraction 250 ml) at a low rate of 10 mUmin. Fractions containing G or F (TLC, Rf 0.75) were pooled and applied (20-40 mg samples) to a preparation HPLC column eluted with MeOH-H20 gradient ranging from 70% to 85% over 60 min. Fractions (4 mL each) were monitored by TLC, those containing G1 were further monitored by HPTLC. Fractions containing a sole blue spot at Rf 0.28 after staining were pooled, concentrated under pressure and lyophilized to yield a white amorphous solid - G2:

glucopyranoside (IV), (Lit. mp 253-4[deg]C; [a]D = +70[deg]). In a similar manner, fractions containing F, (Rf = 0.60 (TLC); 0.42 (HPTLC), were concentrated, lyophilized to yield amorphous F which was shown to be medicagenic acid 3-O-beta -D maltoside (VI). The structures of compounds G2 and F were assigned: The sugar moieties following chemical degradation and enzymic hydrolysis. Compound G2 was a substrate of P-glucosidase but not of a-glucosidase, while compound F was a substrate of a-glucosidase but not (3-glucosidase; digestion of compound F with both enzymes afforded the aglycone (table 1). The aglycone, its dimethyl ester and dimethyl ester diacetate cochromatographed and had H-NMR spectra identical to derivatives of an authentic sample of medicagenic acid (I, II and III, respectively). Moreover, in the case of compound G2, CNMR completed the elucidation of the structure establishing inter alia the glucosidic substitution on C-3 rather than on C-2 of the aglycone.

TABLE 1

Determination of the sugar moiety in Compounds G2 and F

One should note that although compound G2 is isolated quite readily and in acceptable yield (0.12%, Scheme) from alfalfa root flour, the proportion of compound F is much lower and is subject to variation. There are instances where only trace amounts of F are present; as a result, compound F is currently synthesized from a medicagenic acid derivative by the Koenigs-Knorr synthesis. Drug Preparation: The antifungal agent has a low solubility in water. Therefore a solution of 0.1 % (w/v) polyethyleneglycol (MW 20,000, Sigma, St. Louis, Mo) in 2 mM sodium hydroxide was used to solubilize compounds G and G2. Appropriate drug-free solvent controls were included in each test method. The concentration of the drug in the stock solution was 2.5 mg per mL. Since the drug is heat resistant it was sterilized by autoclaving for 15 min at 121[deg]C, dilutions of the drug were prepared in sterile distilled water. Hemagglutination and hemolysis test Red blood cells (RBC) from different sources were resuspended with citrate as an anticoagulant and washed fourtimes with phosphate buffer saline (PBS) pH 7.2 in a clinical centrifuge and finally resuspended in the same buffer to give a concentration of 2% (v/v). The antimycotic agent was serially diluted in PBS using an automatic dilutor. Dilutions were performed in microtitration plates. An equal volume of RBC was then added to each well, containing fifty microliters of each dilution. Hemolysis and hemagglutination were recorded macroscopically after 1 h incubation at 37[deg]C and further incubation for 24 h in the cold. Partial hemolysis was considered as the end-point. Susceptibility test by agar dilution method Yeast suspensions were prepared from overnight growth at 30[deg]C on SDA and diluted in sterile distilled waterto a concentration of 105 cells per mL as measured by hemacytometer count. Appropriate dilutions of the drug were made in sterile distilled water in a final volume of 1 mL. Each 1 mL of drug solution was added to 3 mL of 3% sterile molten Noble Agar (Difco Laboratories, Detroit, Michigan) supplemented, after cooling to 50[deg]C, with 0.5 mL of 10 x strength unbuffered YNB sterile broth (Difco) and 0.5 mL of 20% glucose. The final pH was 5.1. After vortex mixing the agar medium was dispensed into 60 x 15 mm sterile petri dishes.The plates were dried uncovered in a laminarflow hood for 15 min and could be stored at4[deg]C for 2 weeks. 10 pL of the yeast suspension was added by micropipet to the agar plates containing varying concentrations of the drug to give a final inoculum of 10 cells per 10 MicroL drop. Seven drops could be added on a single plate without interference. The inoculated plates were incubated at 30[deg]C. The test included a pair of drug free controls, one contained the solubilising solution of the drug and one was used as a growth control. Positive growth responses had to be obtained on both control plates for results to be valid with any isolate. In addition, an appropriate control organism (Candida tropicalis 908) of known susceptibility was included in each plate. Time of incubation was controlled by appearance of growth.Results were recorded after 24 and 48 h of incubation at 30[deg]C. Each test was performed at least twice on different occasions. The MIC was defined as the lowest concentration of the drug preventing macroscopic growth of colonies. Hazy responses were regarded as negative. In experiments where the pH effect was studied the following buffers were added to the unbuffered YNB medium: 20 mM KH2P04 pH7 to give final pH of 7, 10 mM KH2PO4 pH7 to give pH 6.5 and 20 mM MES pH 6.1 to give pH 6.0. Susceptibility test by broth dilution method Yeast isolates were grown overnight on SDA at 30[deg]C. These cultures were resuspended and diluted to yield a final concentration of 10 , 104 and 105 cells per mL as measured by hemacytometer count. The dilutions were prepared in YNB broth (Difco) supplemented with glucose and buffered to a final pH of 6.5 with 10 mM KH2P04 in pH 7. After overnight growth in a shaker at 30[deg]C, cultures in log phase growth (less than 25% transmission at 530 nm) were used for the susceptibility test. The test was performed in test tubes containing different concentrations of the antimycotic agent in 3 mL of unbuffered YNB broth (pH 5.1) supplemented with 2% glucose. The inoculum varied from 10 105 cells per mL, from a log phase culture dependent on the strain. Two tubes containing drug-free medium and inoculum were used as controls.All the tubes were incubated - loosely capped at 30[deg]C in a shaker. Susceptibility was estimated after overnight growth only if the culture in the control tubes did not give less than 25% transmission at 530 nm. The MIC was determined visually as the lowest drug concentration that remained clear. The inhibitory concentration (IC50) was calculated spectrophotometrically as the lowest drug concentration that met the criterion: % T , % T control + 0.5 (100% - % T control), where % T = percent transmission at 530 nm and control = drug-free tube. The minimum fungicidal concentration (MFC) was determined by assaying the colony forming units (CFU) on SDA. Three aliquots of 50 Microl obtained immediately after vortex mixing from the initial inoculum in the control tubes, and just after determining the MIC from tubes showing no growth, were subcultured on SDA. The MFC was established at the lowest concentration of drug from which subcultures were negative. Killing kinetics The time killing studies were performed in 10 mL of unbuffered YNB broth supplemented with glucose and containing the appropriate drug concentration. The yeast inoculum was prepared as described for the susceptibility test by broth dilution method, to yield a final concentration of 105 cells per mL. The experiment was carried out at 30[deg]C in a shaking water bath. Samples of 0.4 mL were removed periodically. Three aliquots of 50 Microl of appropriate dilutions were inoculated on SDAfor assaying the CFU. In other experiments, the samples for CFU determination were washed twice with the YNB broth medium in order to remove the antimycotic agent, before planting for residual arowth.

TABLE 2: Comparison of antimycotic activity of compound G and G2(a)

(')The data are for the mean of MlCs(Microg/mL) as measured by the agar dilution method.

TABLE 3:

Hemolytic and hemagglutination properties of the antimycotic agent Gs(a)

(a)Drug concentrations (ug/mL) are the lowest that showed hemolysis and hemagglutination.

TABLE 4:

TABLE 5: Hemolytic properties of some derivatives of medicagenic acida

MICs of the antifungal agent G2 against various species of yeasts(a)

(a)MICs were determined by the agar dilution method after 24 h with inoculum size of 105 cell per ml; n1, total number of isolates in each species; n2, number of isolates showing mode MIC.

TABLE 6: IC50, MIC and MFC of the antimycotic agent G2 against various species of yeasts(a)

(a)IC50, MIC and MFC were determined by the broth dilution method after 17-22 h. (')An initial inoculum of 105 cells per mi. (')An initial inoculum of 10 cells per ml. (')An initial inoculum of 104 cells per ml. In the following, Compounds I to VI are the compounds of Formula I in which (I) R1 =R2=R3=H, (II)

Glcp, R3=CH3; and (VI) R1=R3=H, R2=a-Glcp (1- 4) (3-Glcp (Compound F). The clinical use of saponins may be limited due to their hemolytic activity. The purification method used in this study, however, markedly decreased the hemolytic and hemaglutination activities to such a level that no hemolysis or hemaglutination was observed in vitro with compound IV in the range of concentrations needed for complete growth inhibition of the 11 medically most important yeasts illustrated in Table 5. Compound IV was non-toxic to mice at concentrations up to 500 Microg/ml and no hemolytic activity was observed in mice treated with this dose - indicating that this compound is of practical significance. Additional hemolysis tests leading to similar results (and even to improved results in the case of compound F) were obtained with other medicagenic acid derivatives (Table 7). Yeasts pathogenic to humans and animals obviously are of particular interest. Exploratory experiments conducted on single isolates of medically important yeasts indicate cases (C. tropicalis, C. albicans and C. parapsilosis, data not shown), where the conversion of compounds I and IV to their methyl esters (compounds II and V, respectively) made very little difference. However, in the case of T. glabrata (the second most common yeast from urine isolates), compound II was more potent than compound I at a pH range where deprotonation of medicagenic acid takes place. In addition, among the four compounds used, compound IV was most effective on the most serious pathogen, C. neoformans, at lower pH (Table 7). Compound G2 was examined also on clinical isolates of dermatophytes (human and animal pathogens) on potato dextrose agar (PDA). The results (Table 2) are indicative of the potential usefulness of compound G2 as a fungistat possibly complementary to griseofulyin a fungistat in regular use against all forms of dermatophyte infections in animals and man.

TABLE 7'-

MICs of medicagenic acid derivatives against C. neoformans and T. glabrataa

aMICs determined on single isolates of yeasts by agar dilution after 24h.

TABLE 8: MICs of G2 against various species of dermatophytes

MICs were determined by the agar dilution method after 7 days at 26[deg]C with an inoculum size of 104 cells per mL. Initial results applying medicagenic acid to dermatophytes were obtained using once again PDA medium (Table 7). The only instance where medicagenic acid appeared to be more active than compound G2 is T. violaceum (one strain only).

TABLE 9:

Antimycotic activity of medicagenic acid on some dermatophytes

There was estabished an animal model of cryptococcosis. The drug used was the compound designated herein as G-2, and this has an adequate solubility in water for the concentrations used in this study. The dilutions were in water to the required concentrations used for the agar dilution test. The minimum inhibitory values (MIC) were determined using 1:2 dilution steps on Yeast Nitrogen Base agar (YNB). The in vivo activity of G-2 was established in the model for murine cryptococcosis. 8.106 cryptococcus neoformans cells were injected intravenously into male albino mice of about 20 g weight. Untreated control animals died within a week. In the animal model a dose of 100 mg/kg given i.p. in one dose proved to be highly toxic and all animals died within 24 hours. A dosage of 10 mg/kg proved to be inactive. A dosage of 20 mg/kg was effective and resulted in a very substantial prolongation of life duration of the thus treated animals. A further study was carried out with Compound F and the MIC for Cryptococcus neoformans was found to be 20 ug/ml.

Claims (17)

1. An antifungal composition for use in human and veterinary medicine comprising an antifungally active quantity of a compound of the formula

wherein R1 is hydrogen or Ac,

R3 is hydrogen or C1-C6 alkyl and functional derivatives of these, in combination with a suitable carrier or diluent.

2. A composition as claimed in Claim 1, R1, R2 and R3 are hydrogen.

3. A composition as claimed in Claim 1, wherein R1 and R2 are each hydrogen and R3 is methyl.

4. A composition as claimed in Claim 1, wherein R1 and R2 are each Ac and R3 is methyl.

5. A composition as claimed in Claim 1, wherein R1 and R3 are hydrogen and R2 is P-Glcp.

6. A composition as claimed in Claim 1, wherein R1 is hydrogen, R2 is beta -Glcp and R3 is methyl.

7. A composition as claimed in Claim 1, wherein R1 and R3 are hydrogen and R2 is a-Glcp (1 4) (3-Glcp.

8. A composition as claimed in Claim 1, where the active substance is a purified material obtained from alfalfa roots and designated herein as G, G2 or F.

9. A composition as claimed in any one of the preceding claims, in unit dosage form for oral administration or for administration by injection.

10. A composition as claimed in any one of Claims 1 to 8, in a form for topical or local administration.

11. A composition as claimed in any one of Claims 1 to 8, in a form for administration as an aerosol.

12. A composition as claimed in any one of Claims 1 to 11, for use as fungicidal or fungistatic composition against a yeast selected from: Cryptococcus neoformans, Rhodotorula glutinis, Candida tropicalis, Candida pseodotropicalis, Candida krusei, Candida albicans, Torulopsis glabrata, Candida parapsilosis, Torulopisis candida, Candida guilliermodii, Greotrichum candidum, Trichophyton rubrum, Trichophyton metagrophytes, Microsporum canis.

13. An antifungal composition comprising a compound defined herein as G or as G2, or an equivalent thereof, substantially as hereinbefore described.

14. The essentially pure compounds G2 and F as described herein whenever obtained by a process as hereinbefore described.

15. Fungicidal and fungistatic compositions for use in human and veterinary medicine, containing a compound as defined in Claim 1, and substantially as described herein.

16. A compound as defined in any one of Claims 1 to 8 for use in the treatment of fungal infections.

17. The use of a compound as defined in any one of Claims 1 to 8 for the manufacture of a medicament for the treatment of fungal infections.