WO1998037898A1

WO1998037898A1 - Synergistically acting anti-fungal agents that are inhibitors of folate metabolism, especially methotrexate and sulfamethoxazole

Info

Publication number: WO1998037898A1
Application number: PCT/CA1998/000152
Authority: WO
Inventors: Barry J. Barclay
Original assignee: Barclay Barry J
Priority date: 1997-02-24
Filing date: 1998-02-24
Publication date: 1998-09-03
Also published as: AU6203098A

Abstract

The invention provides an anti-fungal method comprising exposing the fungal organisms to two anti-folate drugs, methotrexate and sulfamethoxazole (or their analogues), in combination. The drugs act synergistically in their anti-fungal effect, so that relatively low doses of each drug are required. The method is effective against several pathogenic species of Candida including C. albicans, C. krusei and C. glabrata which are common in patients whose immunity has been suppressed as a result of HIV or chemotherapy.

Description

SYNERGISΗCALLY ACTING ANTI-FUNGAL AGENTS THAT ARE INHIBITORS OF FOLATE METABOLISM, ESPECIALLY METHOTREXATE AND SULFAMETHOXAZOLE

2 FIELD OF THE INVENTION

3 The invention relates to anti-fungal agents, and particularly to those agents

4 which are active against Candida species. 5

6 BACKGROUND OF THE INVENTION

7 The number of reported cases of systemic fungal infection (also known as

8 systemic mycoses) in major U.S. medical centres has increased by almost 500

9 percent over the period from 1982 to 1992. These mycoses are often life-

10 threatening, especially in patients whose immune system has been compromised

11 by HIV infection, or by administration of chemotherapeutic or immunosuppressive

12 drugs. A genus of fungus known as Candida is responsible for approximately 10 -

13 15 percent of serious bloodstream fungal infections. For patients with systemic

14 infections caused by a species of Candida known as Candida albicans, the

15 mortality rate approaches 50 percent. The mortality rate can be even higher for

16 other Candida species.

17 Unlike bacterial bloodstream infections, which usually respond to antibiotic

18 therapy, fungal infections are notoriously difficult to treat. Because yeast cells are

19 eucaryotic, they are more similar to human cells biochemically and physiologically

20 than are bacterial cells. As a consequence, drugs designed to inhibit the growth of

21 various fungi often interfere with normal human physiology and metabolism,

22 resulting in serious toxicity in the host.

23 There are few drugs available to treat fungal infections, all of which have

24 serious side effects. These are described below. Fluconazole is the most commonly used drug for the treatment of systemic fungal infections of Candida species in humans. The cellular target of this drug (as well as other azole derivatives) is cytochrome P-450, a protein required for the biosynthesis of ergosterol. Fluconazole use has become increasingly widespread for the treatment of oropharyngeal Candidiasis in AIDS. Yeast isolates which are resistant to the drug have begun to appear. This drug is also associated with liver damage. The drug Amphotericin B is commonly used for Candidemia, especially in life-threatening systemic infections. Serious toxicity occurs frequently. Renal damage results from drug-induced vasoconstriction, impaired acid excretion, and direct tubular damage. Bone marrow suppression usually occurs. The severe toxicity of the drug limits its usefulness, especially in critically ill patients. The drug 5-Fluorocytosine (5-FC) is used against a variety of Candida species. It is converted by fungal metabolism to halogenated uracil derivatives which impair both RNA and DNA synthesis in target cells. Natural and acquired drug resistance are the main limitations of this drug. To summarize, the currently available treatments for human fungal infections are problematic in that they are both highly toxic at effective dosages, and subject to natural and acquired drug resistance. There is an urgent need for methods of combating fungi which are less harmful at effective dosages. SUMMARY OF THE INVENTION The primary object of the invention is to provide methods of inhibiting the growth of pathogenic fungi which are less toxic than drug regimens currently available. A further object of the invention is to provide antifungal agents for which the side effects in mammalian hosts can be ameliorated by the co-administration of compounds which "rescue" mammalian cells from toxicity, thereby allowing the anti-fungal agents to act preferentially on the fungal cells. A further object of the invention is to provide methods of inhibiting the growth of pathogenic fungi which will not tend to generate drug-resistant fungal clones. The invention is based on two novel observations. The first is that two drugs, methotrexate and sulfamethoxazole, both known inhibitors of folate metabolism, act synergistically to inhibit fungal growth. The second is that this treatment is fungicidal. The invention provides a method of inhibiting the growth of fungi utilizing a combination of the two types of anti-folate compounds, namely: (1) compounds which are structural analogues of dihydrofolate, folate and folic acid; and (2) compounds which are structural analogues of para-aminobenzoate and para-aminobenzoic acid. Structural analogues of dihydrofolate, folate and folic acid will be referred to as "DHF analogues" herein. Structural analogues of para-amino-benzoate and para- aminobenzoic acid will be referred to as "PAB analogues". The synergistic combination of the DHF analogue methotrexate and the PAB analogue sulfamethoxazole is particularly effective in reducing fungal growth. Either compound used alone has at most a marginal anti-fungal effect even at high concentrations (up to 500 μg/ml). Used in combination, low concentrations (in the range of 50 μg/ml) of the compounds profoundly inhibit fungal growth. Methotrexate has been widely used in the treatment of human cancer. It was previously believed to be ineffective against protozoal cells, due to their lack of a membrane transport system for the compound. Sulfamethoxazole has been used for many years in the treatment of bacterial and other protozoal infections. The discovery, described herein, that these two drugs synergize at therapeutically useful levels to inhibit the proliferation of pathogenic yeast cells provides the basis for a new approach to anti-fungal therapy in humans. The synergy between the two classes of compounds reduces the effective dose of each compound which is required for inhibition of yeast growth. Broadly stated, in one aspect, the invention is a method for inhibiting the growth of fungi, comprising contacting the fungi with effective concentrations of at least one member from each of two groups of compounds: (1) DHF analogues and (2) PAB analogues. In another aspect, the invention is a synergistic drug combination which inhibits fungal growth, comprising at least one DHF analogue and at least one PAB analogue. DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the synergy between methotrexate (MTX) and sulfamethoxazole (SMZ) in the growth inhibition of Candida albicans and Candida krusei. Yeast cells were cultured in liquid Saboroud's medium containing 50 μg/ml of methotrexate, and, in addition, varying concentrations of sulfamethoxazole, as indicated on the x-axis. The number of yeast colony forming units (CFU) in these cultures was assayed by plating samples of the liquid culture onto agar plates. The number of CFU per ml of culture is indicated on the y-axis. Figure 2 is a photograph showing the pattern of Candida glaborata growth on agar plates inset with wells containing either 200 μg methotrexate (MTX), 200 μg sulfamethoxazole (SMZ) or 200 μg of both drugs in combination (MTX/SMZ). The growth of C. albicans is inhibited around the well containing methotrexate and sulfamethoxazole in combination, but not around the wells containing either drug alone. Figure 3 shows the structure of folic acid, para-aminobenzoic acid and some analogues of each.

DESCRIPTION OF THE INVENTION The invention is based on the novel observation, described herein, that two classes of anti-folate compounds act synergistically to inhibit the proliferation of a number of species of the genus Candida. The nature of these compounds, and the current understanding of their mechanism of action will be described below. In both eucaryots and procaryots, a cellular enzyme known as dihydrofolate reductase (DHFR) catalyzes the sequential reduction of the metabolite, folate, to dihydrofolate and subsequently to tetrahydrofolate. This series of reactions, is crucial in the biosynthetic pathway of purines, pyrimidines and certain amino acids. Agents which interfere with the folate pathway are toxic to growing cells, and generally prevent cells from proliferating. Such agents are referred to as anti- folates. They exert their effect by mimicking normal folate metabolites. DHFR and the folate pathway have been studied extensively because they are targets for the action of a number of clinically useful anti-metabolites. An anti- metabolite is a synthetic compound which interferes with the cellular utilization of a normal, or physiological, metabolite. Many anti-metabolites are structural analogues of the normal metabolite. A structural analogue is a member of a group of chemical compounds which are similar in structure, but different in the precise composition of elements.

The two types of anti-folates which have been identified in the present study to synergize to inhibit the growth of yeast cells are: • structural analogues of dihydrofolate, folate and folic acid (called DHF analogues), and • structural analogues of para-aminobenzoate and para-aminobenzoic acid (called PAB analogues). Both DHF analogues and PAB analogues are known to exert their inhibitory effect via the folate pathway, but they act at different points therein. DHF is a physiological substrate for the enzyme DHFR. It is well established that a number of its analogues interfere with the folate pathway by binding to DHFR, and thereby preventing it from binding to its physiological substrates, folate and dihydrofolate. PAB is a precursor for the synthesis of folate. It is known that several PAB analogues interfere with the folate pathway by inhibiting the enzyme which incorporates PAB into folate, and thus prevent the synthesis of folate. There are many known analogues of both DHF and PAB. The chemical structures of many such compounds are found in the following references: Merck Index; Chemistry and Biology of Pteridine and Folates edited by J. Ayling, M. G. Nair and C. M. Baugh Plenum Press, New York; J. R. Piper et al., "Studies on analogues of classical antifolates bearing the napthoyl group in place of benzoyl in the side chain" J. Med. Chem. 36:4161(1993); C. H. Takimoto, "Antifolates in Clinical development", Semin. Oncol. 24: S18 (1997); Y. L. Hong, "Inhibition of recombinant Pheumocystis carinii dihydropteroate synethesis by sulfa drugs", Antimicrob Agents Chemother. 39:1756 (1995). DHF analogues include aminopterin and amethopterin (also called methotrexate). Methotrexate has been used clinically as a chemotherapeutic agent for leukemia and other cancers, as a treatment for some autoimmune diseases, and as an immuno-supressive drug to prevent graft rejection. Until the present study was conducted, it was believed that methotrexate was ineffective against protozoa because some types of protozoal cells failed to transport the drug across the cell membrane (for example, see U.S. Patent No. 4,694,007 to Allegra et al.). PAB analogues, also known as sulfa drugs, have been widely used in the treatment of bacterial infections. PAB analogues include the well known anti-bacterial agents sulfamethoxazole and sulfanilamide. The chemical structures of folic acid, para-aminobenzoic acid, and two analogues of each is shown in Figure 3. The anti-fungal synergy between a DHF analogue and a PAB analogue is demonstrated in the Examples, in which methotrexate and sulfamethoxazole are used in combination to inhibit the growth of cells of the species of yeast known as Candida. In the Examples, the following three types of tests were used to confirm the anti-fungal synergy between methotrexate and sulfamethoxazole: 1. direct inhibition of Candida proliferation in liquid culture, 2. reduction in colony forming units by Candida grown in liquid culture, and 3. inhibition of Candida growth on agar plates. In all three tests, methotrexate alone or sulfamethoxazole alone exerted only a minimal anti-fungal effect, whereas in combination, at relatively low doses, they markedly reduced Candida proliferation. Example 1

This Example demonstrates the synergy between the anti-folates methotrexate and sulfamethoxazole as inhibitors of fungal proliferation in two species of Candida: C. albicans and C. /cruse/. Cells of C. albicans and C. krusei (clinical isolates originally obtained from the American Tissue Culture Collection and denoted as ATCC 18644 and ATCC 6258, respectively) were grown on agar plates containing Saboraud's medium (SAB), a well known commercially available medium used to grow yeast. Single clones of yeast were picked and used to inoculate 10 ml cultures in SAB in 15 X 125 mm test tubes. The cells were grown overnight with vigorous shaking in a gyrotary water bath at a temperature of 35° C. The cells were then collected by centrifugation and resuspended in similar test tubes containing SAB and various concentrations (ranging from 0 to 500 μg/ml) of either or both of methotrexate (Cyanamid) and sulfamethoxazole (Sigma). The cultures were placed in a gyrotary water bath at 35° C for 72 hours. The growth of yeast in the cultures was monitored by determining the optical density (OD 600 nm) of an aliquot of the cultures using an LKB Ultraspec II spectrophotometer. The results of the test are shown in Table I. There was a synergistic interaction between methotrexate and sulfamethoxazole with respect to growth inhibition of both Candida species. Methotrexate alone at high concentration inhibited both Candida species to some extent. Table 1

Candida albicans

Candida krusei

Microscopic examination of the cells in all inhibited cultures exhibited extremely abherrant morphology. Cells were swollen in appearance, elongated and distorted, as well as being severely clumped. This was especially true of the C. krusei strain. It is likely, therefore, that optical density alone is not a reliable measure of the true extent of growth inhibition.

Example 2 Therefore, further tests were conducted to measure the number of viable yeast cells with proliferative capacity after the cells had been cultured with methotrexate, sulfamethoxazole or both drugs. This was done by measuring the Colony Forming Units (CFU) in drug-treated yeast cultures. When yeast are spread at low density on an agar containing growth medium, each normal yeast cell with proliferative capacity replicates until it forms a visible colony or clone on the agar plate. The number of such colonies which can be grown from cultured yeast cells provides a reliable measure of the number of viable cells with proliferative capacity within such a culture, or the CFU. C. albicans and C. /cruse/ cells were cultured in liquid SAB in 125 ml Erlenmeyer flasks with vigorous shaking at 35° C for 48 hours. The cultures contained 50 μg/ml methotrexate (MTX), or 50 μg/ml methotrexate with the addition of various concentrations of sulfamethoxazole (SMZ), ranging from 0 to 50 μg/ml. The CFU in each culture were quantitated by plating serial dilutions on SAB-containing agar plates, and incubating the plates for 2 days at 35° C. The results, shown in Figure 1 , indicate a clear synergy between methotrexate and sulfamethoxazole in CFU reduction. In addition, there is a clear species difference in the susceptibility of the two strains, C. /cruse/ being more susceptible than C. albicans. The minimum fungicidal concentration of sulfamethoxazole required for 90% killing (MFCso) was 7.5 μg/ml and 40 μg/ml for the two strains respectively.

Example 3 In this example, the susceptibility of Candida glabrata to the combination of methotrexate and sulfamethoxazole was demonstrated by a third type of test, inhibition of growth on agar plates. Yeast were uniformly spread on SAB agar plates. Three "wells" were cored out of the agar in each plate. The wells were filled with 200 μl of a 1 mg/mi solution of methotrexate (MTX), sulfamethoxazole (SMZ), or MTX and SMZ in combination. The drugs diffused out of the wells, into the agar over the course of the experiment, contacting the yeast cells in the vicinity of the wells. The plates were incubated for 2 days at 35° C, and then scored for drug sensitivity. The results are shown in Figure 2. The growth of yeast in the area around the wells containing SMZ alone and MTX alone was fairly uniform, and didn't differ noticeably from the background growth away from the wells. However, there was a "halo" which was completely devoid of viable yeast cells around the wells containing SMZ and MTX in combination. Similar results have been obtained for other Candida species. Examples 1-3 establish that the drug combination of methotrexate and sulfamethoxazole, at concentrations of 50 μg/ml each or lower, is effective in inhibiting the growth of yeast cells. Similar results have been obtained using the drug combination of aminopterin and sulfamilamide in other yeast strains. It is anticipated that other combinations of structural analogues of DHF and PAB will also act synergistically to inhibit fungal growth, and the invention includes such combinations. The invention anticipates that the following DHF and PAB analogues will be useful: DHF analogues: denopterin, edatrexate, methotrexate, 5-deaza- methotrexate, 5-methyl-5-deaza-methotrexate, piritrexim, pteropterin, raltritrexed, trimetrexate, aminopterin, 5-deaza-aminopterin, 5-methyl-5-deaza-aminopterin, 5,8-dideaza-aminopterin PAB analogues: sulfanilamide, sulfacetamide, sulfadiazine, sulfamerazine, sulfamethoxazole, sulfathiazole, sulfachlorpyridazine, sulfathiourea, sulfisoxazole. Additional synergistic pairs or combinations of DHF and PAB analogues can be rapidly identified using a screening test such as the well diffusion test described in Example 3. The synergistic drug combination of the invention was chosen in part to minimize toxicity to the host. PAB analogues such as sulfamethoxazole are not highly toxic to mammalian cells, which do not require PAB for survival. (Mammals take up folate from the diet, rather than synthesizing it dejπo o.) However, methotrexate is readily transported into mammalian cells. The toxic side effects of the drug in humans is well established. It is anticipated that methotrexate toxicity can be amielorated by co-administering leucovorin, and/or mammalian folate- dependent one carbon metabolites, such as glycine, hypoxanthine and thymidine. Leucovorin is a physiological folate that is normally transported into mammalian cells by means of a membrane transport system. Protozoal cells are generally believed to lack this membrane transport system. The ability of leucovorin to prevent the cellular toxicity of anti-folate drugs is well-documented. Therefore, co-administration of leucovorin with DHF analogues such as methotrexate can be used to protect mammalian cells, while not diminishing the toxic effect on fungal cells. Because both methotrexate and sulfamethoxazole have been extensively used in the treatment of human diseases, there is much available data on dosages (for example, see the Merck Manual, Index Medicus. and The Pharmacological Basis of Therapeutics, edited by Goodman and Gilman). In view of the synergy between the two drugs documented herein, it is anticipated that minimum inhibitory concentration of each drug required for anti-fungal treatment in vivo will be lower than the concentrations that have heretofor been used for treatment of other conditions. By extrapolating from the effective anti-fungal concentrations in vitro, it will be possible to design a test protocol for in vivo use. Pharmeceutically acceptable carriers for the drugs are already used clinically. The advantages of the synergistic drug combination and methods of the invention over existing anti-fungal agents and methods can be summarized as follows: 1. Because two synergistic agents are combined, lower concentrations of each agent are required for an anti-fungal effect. 2. Because two agents are used, the likelihood of generating resistant fungal variants may be much lower than when a single agent is used. 3. In vivo toxicity might be minimized by co-administration of leucovorin, other physiological folates, or folate-dependent one carbon metabolites such as glycine hypoxanthine and thymidine.

Claims

I CLAIM: 1. A method of inhibiting the growth of fungi, comprising: contacting said fungi with effective amounts of at least one member selected from each of two groups of anti-folate compounds, the first group consisting of analogues of dihydrofolate, folate and folic acid (DHF analogues), and the second group consisting of analogues of para-aminobenzoate and para-aminobenzoic acid (PAB analogues), wherein the amounts of said selected compounds are effective, in combination, to inhibit the growth of said fungi.

2. The method of claim 1 , wherein the group of DHF analogues consists of: denopterin, edatrexate, methotrexate, 5-deaza-methotrexate, 5-methyl-5-deaza- methotrexate, piritrexim, pteropterin, raltritrexed, trimetrexate, aminopterin, 5- deaza-aminopterin, 5-methyl-5-deaza-aminopterin, 5,8-dideaza-aminopterin,. and the group of PAB analogues consists of sulfanilamide, sulfacetamide, sulfadiazine, sulfamerazine, sulfamethoxazole, sulfathiazole, sulfachlorpyridazine, sulfathiourea, sulfisoxazole.

3. The method of Claim 1 , wherein the member selected from the group consisting of DHF analogues is methotrexate.

4. The method of Claim 1 wherein the member selected from the group consisting of PAB analogues is sulfamethoxazole.

5. The method of Claim 1 , wherein the member selected from the group consisting of DHF analogues is methotrexate, and the member selected from the group consisting of PAB analogues is sulfamethoxazole.

6. The method of Claim 1 , wherein the member selected from the group consisting of DHF analogues is aminopterin and the member selected from the group consisting of PAB analogues is sulfanilamide. 7. A composition for inhibiting the growth of fungi, comprising a mixture of at least one DHF analogue, at least one PAB analogue and a pharmaceutically acceptable carrier. 8. The composition of Claim 7, wherein the DHF analogue is methotrexate. 9. The composition of Claim 7, wherein the PAB analogue is sulfamethoxazole. 10. The composition of Claim 7, wherein the DHF analogue is methotrexate and the PAB analogue is sulfamethoxazole. 11. The composition of claim 7 wherein the DHF analogue is aminopterin and the PAB analogue is sulfanilamide. 12. The method of Claim 1 , wherein said amounts of said members of said two classes of anti-folate compounds are administered to a patient infected with a fungus which is effective to inhibit the growth of said fungus. 13. The method of Claim 12, wherein said members of said two classes of anti-folate compounds are administered simultaneously. 14. The method of Claim 12, wherein said members of said two classes of anti-folate compounds are administered sequentially. 15. The method of Claim 12, wherein an amount of a physiological folate is administered which is effective to reduce the toxicity of the DHF analogue. 16. The method of Claim 12, wherein the physiological folate is leucovorin.

7. The method of Claim 1 , wherein the fungi belong to the genus Candida.