WO1991015121A1 - Method for treating fungal infection - Google Patents

Abstract

Aspartic acid proteinases are useful in a method for treating fungal disease.

Description

METHOD FOR TREATING FUNGAL INFECTION

Field of the Invention

This invention relates to a method for treating fungal infection. More particularily, this invention relates to the use of proteinase inhibitors in the manufacture of

medicaments having anti-fungal activity and to a method for treating fungal infection by administering proteinase inhibitors.

Background

Fungi are unicellular and/or multicellular eukaryotic protista of which only a limited number are important

mammalian pathogens. These organisms range from those whose habitat is soil, but yet are capable of infecting normal healthy humans, to commensal fungi which, under conditions where the host's defense mechanisms are impaired (i.e., immunosuppressive or antibiotic therapy; immunodeficiency- related diseases such as AIDS, leukemia; diabetes), cause life-threatening diseases.

Most human mycoses are the result of infection by members of the Trichophyton (cutaneous infections) or Candida (cutaneous, mucosal and systemic infections) genera. Candida is unique among opportunistic pathogens because it is an ubiquitous fungus commonly found in normal human flora of the mouth, oropharynx, intestines, vagina and skin and has the ability to sustain a variety of disease pathologies including superficial, locally invasive and deep/systemic forms.

Fungal infections have traditionally been difficult to treat by intervention with pharmaceutical agents because fungi, like their human hosts, are eukaryotic organisms.

Thus, one reason for this difficulty, is the lack of

biochemical targets which may produce toxicity in the fungal cell without effecting toxicity in mammalian cells. Typical agents for the treatment of fungal infections include the polyene antibiotics, 5-flucytosine, miconazole, ketoconazole, clotrimazole, fluconazole, itraconazole and griseofulvin. The polyene antibiotics, such as amphotericin B and nystatin, are believed to exert their effect by binding to sterols in the fungal cell membrane causing leakage of cell constituents with subsequent cell death. Imidazole derivatives, such as clotrimazole, miconazole, ketoconazole, fluconazole and itraconazole have a variety of inhibitory effects on cell membrane-associated functions in sensitive organisms as a result of impairment of membrane sterol synthesis. Most of these compounds exhibit only fungistatic effects at

clinically achievable concentrations. The modes of action of 5-flucytosine and griseofulvin are believed to involve nucleic acids and nuclear division, respectively. The activities of clotrimazole, miconazole and griseofulvin are limited to cutaneous infections only. There is a need for therapy which may intervene in other biochemical processes which are critical to fungal infection.

The existence of extracellular proteolytic activity by C. albicans was reported in 1965 [Staib, F., Sabouraudia, 4, 187 (1965)]. Subsequently, Remold et al., Biochim. Biophys. Acta, 167, 399 (1968), disclosed the isolation, purification and characterization of the responsible enzyme. The

proteolytic enzyme has been classified as an aspartic proteinase based upon its inhibition by pepstatin and its optimal activity at acidic pH, although it is resistant to the aspartic proteinase inhibitors diazoacetylnorleucine methyl ester and epoxy (p-nitrophenoxy) propane [Ruchel et al., Sabouraudia 20, 233 (1982)].

Secretion of a proteinase in vitro is a ubiquitous property of C. albicans isolates, common in C. tropicalis and occasional in C. parapsilosis . Proteinases from different species have similar activities. The enzyme has a broad protein substrate specificity, including albumin, hemoglobin, collagen, fibronectin, casein, epidermal keratin of skin and nail and immunoglobulins. In addition, two major proteinase scavengers of human plasma α-1-antitrypsin and α-2- macroglobulin, can also be digested by the proteinase.

A gene similar to the proteinase gene of C. albicans is also present in S. cerevisiae (85% amino acid homology).

[Lott, et al., Nuc. Acids Res. 17, 1779 (1989).] An aspartic proteinase in S. cerevisiae has been shown to be an

intracellular post-translational activator of a number of essential vacuolar hydrolases.

An association between species/strain proteolytic ability and pathogenicity has been suggested by Staib,

Mycopathol. Mycol. Appl., 37, 345 (1969) and Ghannoum et al.,

J. Med. Vet. Mycol., 24, 407. (1986), based upon a

correlation between gross proteinase activity and strain virulence and adherence. The enzyme has been shown by immunofluorescence to be synthesized in infected tissues in vivo and antibodies to the purified enzyme are detected both in sera of animals experimentally infected with C. albicans and in sera from patients with visceral Candida infections

[Macdonald, et al., J. Med. Microbiol. 13, 423. (1980).]

Specific examples of compounds of this invention are broadly disclosed as inhibitors of HIV-1 protease in EP-A 0

352 000 and EP-A 0 337 714, and as renin inhibitors in U.S.

Patent 4,661,473, U.S. Patent 4,882,420 and U.S. Patent

4,864,017. Summary of the Invention

Aspartic acid proteinase inhibitors and, in particular, compounds of formula (I), as herein defined, are useful in the inhibition of a proteinases secreted by certain fungi, the prevention of infection by fungi and the treatment of fungal infection. Methods of treating fungal infection and the use of proteinase inhibitors in the manufacture of medicaments for treating fungal infection are also disclosed.

Detailed Description of the Invention

This invention is concerned with the use of aspartic acid proteinase inhibitors, especially compounds of formula (I), and pharmaceutical compositions thereof, in the

manufacture of a medicament for the inhibition and/or

treatment of fungal infection. This invention is also a method for treating or preventing fungal infection by

administering an effective amount of a proteinase inhibitor sufficient to inhibit/treat the fungal infection. Broadly considered within the context of this invention, aspartic acid proteinase inhibitors effective for the treatment of fungal infection are those compounds which are capable of inhibiting an acid protease which is critical for the

viability or pathogenicity of the fungus. Examples of such compounds are disclosed in the following patents and patent applications: U.S. Patent 4,864,017, U.S. Patent 4,880,781, U.S. Patent 4,882,420, U.S. Patent 4,661,473, U.S. Patent 4,782,043, U.S. Patent 4,812,442, U.S. Patent 5,755,592, U.S Patent 4,812,555, U.S. Patent 4,721,776, U.S. Patent

4,743,584, U.S. Patent 4,826,958, U.S. Patent 4,743,585, U.S. Patent 4,818,748, U.S. Patent 4,424,207, U.S. Patent

4,650,661, U.S. Patent 4,638,047, U.S. Patent 4,713,445, U.S. Patent 4,729,985, W/O 89/10920, W/O 88/05050, W/O 88/04324, W/0 87/02986, EP-A 229 667, EP-A 209 897, EP-A 312 157, EP-A 337 714, EP-A 342 541, EP-A 346 847, EP-A 402 646, and EP-A 352 000, all of which are incorporated herein by reference as if fully set forth. Generally, compounds which contain the partial

structure: -NR'CH (R₄) -T, wherein T is -Q-CHR₅C=0-, -Q-CHR₅-Y or U, and R₄, Q, R₅, U and Y are as defined for formula (I), are useful and within the scope of this invention.

In one embodiment, compounds of formula (I) are

considered useful for practicing this invention. Compounds of formula (I) are defined as:

A-B-D-D-E-M-X-X-Y

(I)

wherein:

A is H or R₁CO-, R₁OCO-,R₁OCH (R₁,) CO-, R₁NHCH (R₁,) CO-,

R₁SCH(R₁,)CO-, R₁SO₂- or R₁SO;

R₁ and R₁' are H, C_1-5alkyl, C_3-7cycloalky, aryl or aryl substituted by one or two C_1-5alkyl, trifluoromethyl,

hydroxy, C_1-5alkoxy or halogen groups; B is absent, β-alanine or one or two natural amino acids; D is absent prolyl or -NR'CH (R₂) CO-; R₂ is (CH₂)_m-W; R' is H, C_1-5alkyl or benzyl;

W is C_1-5alkyl, CH₂OR', CO₂NHR', CO₂R', CH₂SR', CH₂NHR', guanidyl, imidazolyl, indolyl, aryl or aryl substituted by one or two C_1-5alkyl, trifluoromethyl, hydroxy, C_1-5alkoxy or halogen groups; m is 0 to 4; E is absent or -NR'CH(R₃) CO-;

R₃ is (CH₂)_m-V, wherein m is as above; V is C_1-5alkyl, CH₂OR', CO₂NHR', CH₂SR', imidazolyl, indolyl, aryl or aryl substituted by one or two C_1-5alkyl, trifluoromethyl, hydroxy, C_1-5alkoxy or halogen groups; M is -NR'CH(R₄)-Q-CHR₅CO- or -NR" CH (R₄) -U;

R₄ is C_1-6alkyl, C_3-7cycloalkyl, (CH₂)_qOR', (CH₂)_qSR', aryl or aryl substituted by one or two C_1-5alkyl,

trifluoromethyl, hydroxy, nitro, amino, C_1-5alkoxy or halogen groups;

Q is -CH₂CH₂-, -CONR'-, -CH₂NR'-, -CH₂S(O)_p-, -COCH₂-, - CHOH-, -CH(OH)CHR'-, -CH (OH) CH (OH) -, -CH (NHR' ) CH₂-, - COCF₂CH₂-, -P=O(OR')CH₂- or -CH=CR'-;

R₅ is H, -CH(R₄)₂ or chosen from the same group as R₄; X is absent or -NR'CH (R₂) CO-, wherein R' and R₂ are as above; n is 1 or 2; p is 0, 1 or 2; q is 1 or 2;

Y is -NR'CH(R₂)-Z, -(CH₂)_r-R", -O (CH₂)_r-R", -NR' (CH₂)_r-R" R₆

I

or -NH(CH)_q-R"; r is O to 5;

Z is H, CO₂R', CONR'R", CO₂(CH₂)_rR", CO(CH₂)₂R", CH₂O-A, CH₂NR'-A or CH₂NR' (CH₂)_qR" R₆ is CO₂R ' or CON (R ' ) ₂ ; and

R" is H, O-A, C_1-5alkyl, C_3-7cycloalkyl, amino, mono-, di- or tri-C_1-5alkylamino, aryl, C_3-7Cycloalkylaryl,

heterocycle or guanidyl, or C_3-7cycloalkyl, heteroaryl or aryl substituted by one or two C_1-5alkyl, C_1-4alkoxy, hydroxy, halogen, trifluoromethyl, carboxy, carboalkoxy, carboxamide, guanidinyl, phenylC_1-4alkylene, guanidinyl, guanidinylC_1-5alkylene, carboxyC_1-5alkylene,

carboalkoxyC_1-5alkylene, carboxamideC_1-5alkylene,

carboxyC_1-5alkyloxy carboalkoxyC_1-5alkyloxy,

carboxamideC_1-5alkyloxy, amino, mono-C_1-5alkylamino, di- C_1-5alkylamino or aminoC_1-5alkylene; and pharmaceutically acceptable salts thereof.

In the peptides of formula (I), the constituent residues defined by B, D, E, M, X and Y may have asymmetric center and occur as racemates and as diastereomers. All isomeric forms are included in the present invention, although, as will be apparent, certain of the nonracemic residues are preferred.

Suitably, in the moiety Q-CHR₅C=O- in the M residue, Q is -CH(OH)-, -CH(OH)CH₂-, -COCF₂CH₂- or -P=O (OR') CH₂- .

Suitably, in the moiety Q-U- in the M residue, Q is -

CH(OH)- or -P=O(OR')-

Suitably the residues the residue E and X are present and D is present at least once, such as the partial sequence

-D-E-M-X-X.

Particular compounds of this invention are:

1. (4S,5S)-5-(t-butyloxycarbonyl-serylalanylalanyl) amino-4- hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl ester; 2. (5S)-4-[t-butyloxycarbonyl-seryl(O-benzyl)

alanylalanyl]amino-2,2-difluoro-1,3-oxo-5-phenylpentyl-valyl valine methyl esterr 3. (5S)-4-[t-benzyloxycarbonyl-seryl(O-benzyl)

alanylalanyl]amino-2,2-difluoro-1,3-oxo-5-phenylpentyl- benzyl)alanylalanyl]amino-2,2-difluoro-1,3-oxo-5- phenylpentyl-valyl valine, methyl ester;

4. 2-[hydroxy[[(2-phenyl-1-serylalanylalanyl)amino] - ethyl]phosphinyl]cyclopentylcarbonylvalylvaline methyl ester;

5. (4S,5S)-5-(benzyloxycarbonly-alanylalanyl) amino-4- hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl ester;

6. (4S,5S)-5-(benzyloxycarbonyl-alanyl)amino-4- hydroxy-1- oxo-6-phenylhexyl-valyl valine methyl ester; 7. (4S,5S)-5-(t-butyloxycarbonyl-alanylalanyl)amino-4- hydroxy-6-(4-hydroxyphenyl)-1-oxo-hexyl-valyl valine methyl ester;

8. (4S,5S)-5-(methoxycarbonyl-alanylalanyl)amino -4-hydroxy- 1-oxo-6-phenylhexyl-valyl valine benzyl ester;

9. (4S,5S)-5-(methoxycarbonyl-alanylalanyl)amino -4-hydroxy- 1-oxo-6-phenylhexyl-valyl valine; 10. 2-(-1-hydroxy-3-phenyl-2-(serylalanylalanyl) amino- propyl)cyclopentylcarbonylvalylvaline methyl ester;

11. (3R,4S,5S)-5-(benzyloxycarbonyl-alanylalanyl) amino-3- methyl-4-hydroxy-1-oxo-6-ρhenylhexyl-valyl valine methyl ester; and

12. (3R,4S,5S)-5-(benzyloxycarbonyl-alanyl)amino-3- methyl- 4-hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl ester; and pharmaceutically acceptable salts thereof.

The abbreviations used herein are common to the art of chemistry and include, for the natural amino acids, the following representations: 3 1 3 1

Amino Acid letter letter Amino Acid letter letter

code code code code

Alanine Ala A Leucine Leu L

Arginine Arg R Lysine Lys K

Asparagine Asn N Methionine Met M

Aspartic Acid Asp D Phenylalanine Phe F

Cysteine Cys C Proline Pro P

Glutamine Gin Q Serine Ser S

Glutamic Acid Glu E Threonine Thr T

Glycine Gly G Tryptophan Trp w

Histidine His H Tyrosine Tyr Y

Isoleucine He I Valine Val V

Asparagine or Aspartic Acid Asx B

Glutamine or Glutamic Acid Glx Z

In accordance with conventional representation, the amino terminus is on the left and the carboxy terminus is on the right. Unless specified otherwise, all of the above chiral amino acids are assumed to be of the L absolute configuration. β-Ala refers to 3-amino propanoic acid. Boc refers to the t-butyloxycarbonyl radical, Cbz refers to the carbobenzyloxy radical, Bzl refers to the benzyl radical, Ac refers to acetyl. Ph refers to phenyl.

C_1-5alkyl refers to a saturated or unsaturated, straight or branched hydrocarbon chain containing the indicated number of carbon atoms. As applied herein this is meant to include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl, pentyl, isopentyl, ethylene, propylene, isopropylene, butylene, isobutylene and the like. C_1-5alkoxy refers to a C_1-5alkyl group connected through an oxygen bridge. C_3-7cycloalkyl refers to a saturated aliphatic hydrocarbon ring of the indicated number of carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl . Carboxamide refer to the amide group -CONH₂. Carboalkoxy refers to an ester group such as -CO₂R^f, wherein R^f is C_1-5alkyl as defined above. CarboxyC_1-5alkylene, carboalkoxyC_1-5alkylene and carboxamideC_1-5alkylene, refer to a carboxy, carboalkoxy or carboxamide group connected through a straight or branched, saturated or unsaturated alkyl chain of the indicated number of carbons. CarboxyC_1-5alkyloxy, carboalkoxyC_1-5alkyloxy and carboxamideC_1-5alkyloxy indicate a like structure connected through an oxygen bridge. Mono- C_1-5alkylamino, di-C_1-5alkylamino and tri-C_1-5alkylamino refer to an amino group which is substituted with the one, two or three C_1-5alkyl groups. AminoC_1-5alkylene refers to an amino group which is connected through an alkylene chain of the indicated number of carbons. In like manner,

guanidinylC_1-5alkylene and phenylC_1-4alkylene refer to a guanidinyl or phenyl group which is connected through an alkylene chain of the indicated number of carbons. Aryl refers to phenyl or naphthyl, which may optionally be

independently substituted by one or two C_1-5alkyl,

C_1-4alkoxy, hydroxy, halogen, trifluoromethyl, carboxy, carboalkoxy, carboxamide, phenylC_1-4alkylene

carboxyC_1-5alkylene, carboalkoxyC_1-5alkylene,

carboxamideC_1-5alkylene, carboxyC_1-5alkyloxy,

carboalkoxyC_1-5alkyloxy, carboxamideC_1-5alkyloxy, amino, mono-C_1-5alkylamino, di-C_1-5alkylamino, aminoC_1-5alkylene, guanidinyl or guanidinylC_1-5alkylene groups. The term heterocycle as used herein refers to any stable 5-7 membered ring which is either saturated or unsaturated and consists of carbon atoms and from one to three heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur.

Representative examples of heterocycles are pyridyl, thienyl, furanyl, imidazolyl, imidazolinyl, imidazolidinyl, thiazolyl, thiazolidinyl, pyrazinyl, piperidinyl, piperidyl,

piperazinyl, pyrryl, pyrrolinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, tetrazolyl and morpholinyl. The heterocycles may optionally be substituted by one or two C_1-5alkyl C_1-4alkoxy, hydroxy, halogen, trifluoromethyl, carboxy, carboalkoxy, carboxamide, phenylC_1-4alkylene, carboxyC_1-5alkylene, carboalkoxyC_1-5alkylene,

carboxamideC_1-5alkylene, carboxyC_1-5alkyloxy,

carboalkoxyC_1-5alkyloxy, carboxamideC_1-5alkyloxy, amino, mono-C_1-5alkylarmino, di-C_1-5alkylamino, aminoC_1-5alkylene, guanidinyl or guanidinylC_1-5alkylene groups.

When any variable substituent occurs more than once within any residue or among residues, its definition at each occurrence is independent of every other occurrence. This pertains in particular to R', R", R₁ , R₂, R₃, R₄, n, p, q, r, and aryl and to representations such as -CH(R₄)₂.

Combination of substituents and variables as indicated in the formulae herein are permissible only if such combinations result in stable compounds.

The peptides of this invention are generally

octapeptides or smaller. However, longer peptides which encompass the residues defined herein as -E-M-X-, as given in formula (I), are also considered within the scope of this invention.

The peptides of this invention are prepared by the solid phase technique of Merrifield (J. Am. Chem. Soc, 85, 2149 (1964)), although solution methods known to the art may be successfully employed. A combination of solid phase and solution synthesis may be used, as in a convergent synthesis in which di-, tri-, tetra-, or penta-peptide fragments may be prepared by solid phase synthesis and either coupled or further modified by solution synthesis. The methods of peptide synthesis generally set forth in J. M. Stewart and J. D. Young, "Solid Phase Peptide Synthesis", Pierce Chemical Company, Rockford, II (1984) or M. Bodansky, Y. A. Klauser and M. A Ondetti, "Peptide Synthesis", John Wiley & Sons, Inc., New York, N.Y. (1976) may be used to produce the peptides of this invention and are incorporated herein by reference.

Each amino acid or peptide is suitably protected as known in the peptide art. For example, the Boc or

carbobenzyloxy group is preferred for protection of the amino group, especially at the a position. A benzyl group or suitably substituted benzyl group is used to protect the mercapto group of cysteine, or other thiol containing amino acids; or the hydroxy of serine or threonine. The tosyl or nitro group may be used for protection of the guanidine of Arg or the imidazole of His, and a suitably substituted carbobenzyloxy group or benzyl group may be used for the hydroxyl group of Tyr, Ser or Thur, or the e-amino group of lysine. Suitable substitution of the carbobenzyloxy or benzyl protecting groups is ortho and/or para substitution with chloro, bromo, nitro or methyl, and is used to modify the reactivity of the protective group. Cysteine and other sulfur-containing amino acids may also be protected by formation of a disulfide with a thioalkyl or thioaryl group. Except for the Boc group, the protective groups are, most conveniently, those which are not removed by mild acid treatment. These protective groups are removed by such methods as catalytic hydrogenation, sodium in liquid ammonia of HF treatment as known in the art. The selection of protecting groups is generally dictated by the amino acids an peptide fragments involved in the reaction and the particular coupling conditions employed.

Amide coupling are performed by the carbodiimide, acid chloride, mixed anhydride or activated ester methods as generally known in the art and also set forth in EP 0 352 000 and U.S. Patent 4,661,473.

The M residues of the present invention are also

prepared as described therein and by other methods common to the art. For instance, the preparation of residues such as H₂N-CH(R₄)CH(OH)CH₂-CO₂H are disclosed by Rich et al., J.

Org. Chem., 43, 3624 (1978) and Rich et al., J. Org. Chem., 23, 27 (1980). The preparation of residues such as

H₂N-CH(R₄)CH(OH)CH₂CH(R₅)-CO₂H are disclosed by Evans et al.,

J. Org. Chem., 50, 4615 (1985), Kempf, D.J., J. Org. Chem., 51, 3921 (1986), Fray et al., J. Org. Chem., 51, 4828 (1986) and Boger et al., J. Med. Chem., 28, 1779 (1985). The preparation of residues such as H₂N-CH(R₄) CH(NH₂) CH(R₅) -CO₂H and H₂N-CH(R₄)CH(NH₂)CH₂CH(R₅)-CO₂H are disclosed in EP O 334

239. The preparation of residues such as

H₂N-CH(R₄)CH(OH)CH(OH)CH(R₅)-CO₂H are disclosed in U.S.

Patent 4,864,017. The preparation of residues such as

H₂N-CH(R₄CH(OH)CF₂CH(R₅)-CO₂H and its corresponding ketone are disclosed in U.S. Patent 4,882,420 and Thaisrivongs et al., J., Med. Chem., 29, 2080 (1986). The preparation of residues such as H₂N-CH(R₄) CH(OH) CH₂CH₂-O(CH₂)_r-R" and

H₂N-CH(R₄)CH(OH)CH₂CH₂-N(CH₂)_r-R" are disclosed in EP 0 209

897. The preparation of peptides containing residues such as H₂N-CH(R₄)CH(OH)CH=CH-CO₂H are disclosed in U.S. Patent 4,743,585. The preparation of residues corresponding to H₂N-CH(R₄)CH(OH)-U- are disclosed in EP-A 0 352 000.

If the peptide bears a basic functional group,

pharmaceutically acceptable acid addition salts of the peptides are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, hydriodic, sulfuric, phosphoric, acetic, maleic, succinic, methanesulfonic, adipate, alginic, aspartic, benzoic, benzenesulfonic,

butyric, citric, camphoric, digluconic, dodecylsulfuric, fumaric, hexanoic, lactic, tartaric, toluenesulfonic and undecanoic acid. The acetate salt form is especially useful. Certain of the compounds form inner salts or zwitterions which may be acceptable.

If the peptide contains an acid group, pharmaceutically acceptable cationic salts may be prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the

appropriate cation; or with an appropriate organic amine.

Cations of the alkali metals or alkaline earth metals are acceptable. Cations such as Li+, Na+, K+, Ca++, Mg++ and NH₄+ are specific examples of cations present in

pharmaceutically acceptable salts. In addition, basic nitrogen containing groups can be quaternized with such agents as lower alkyl halides, such as methyl ethyl, propyl and butyl chloride, bromide and iodide; long chain alkyl halides, such as decyl, lauryl, myristyl and stearyl

chloride, bromide and iodide; or aralkyl halides such as benzyl and phenethyl bromide.

In accordance with this invention, pharmaceutical compositions comprising a pharmaceutical carrier and an aspartic acid inhibitor, such as that given by formula (I), are provided. Pharmaceutical compositions of the peptides of this invention may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water, Ringer's solution, or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxycellulose, acacia, polyethylene glycol, dimethylsulfoxide, formamide, mannitol, sodium chloride or sodium citrate.

Alternately, these peptides may be encapsulated,

tableted or prepared in an emulsion or syrup for oral

administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the

composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit.

The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the

preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly per ossum or filled into a soft gelatin capsule.

For rectal administration, the peptides of this

invention may also be combined with suitable non-irritating excipients such as cocoa butter, glycerin, gelatin, synthetic glyceride esters or polyethylene glycols and molded into a suppository. For topical application, the peptides of this invention may be formulated as a suitable ointment, cream or lotion. For instance, the drug, in a finely milled form, may be incorporated into a base of petrolatum, waxes, emulsifiers, oils or fatty acids or alcohols as it known in the art.

Cetyl esters, parafin and beeswax are suitable waxes.

Lanolin, cholesterol and the like are suitable emulsifiers. Mineral oil, olive oil and rose oil are suitable oils.

Oleic, stearic, myristic and palmitic acid are suitable fatty acids. Anti-oxidants, such as α-tocopherol and parabens, may be used as preservatives.

The pulverized powders may also be compounded with an oily preparation, gel, cream or emulsion, buffered or

unbuffered, and administered through a patch.

It is also contemplated that the peptides of this invention may be combined with one or more anti-fungal agents, which is a compound which is fungicidal or

fungistatic, for the treatment of fungal infection. For instance, the compounds of this invention may be combined an agent such as miconazole, ketoconazole, clotrimazole,

fluconazole, itraconazole, griseofulvin, 5-flucytosine, amphotericin B or nystatin. The therapeutic efficacy of generally fungistatic agents, such as the imidazole

derivatives, may be enhanced by (1) the presence of fungal adherence-inhibiting agents which would delay fungal

attachment and penetration of host tissue (which makes them less accessible to drug) and extend therapeutic contact time and (2) through a synergistic effect whereby the aspartic proteinase inhibitors prevent fungal proteolytic nutrient acquirement thus resulting in a metabolically less active and more susceptible target for static agents.

In accordance with this invention there is provided a method for treating fungal infection by administering an aspartic acid proteinase inhibitor. The method of treatment comprises the administration topically, orally, parenterally, buccally, trans-dermally, rectally or by insufflation, of an effective quantity of the chosen compound, preferably

dispersed in a pharmaceutical carrier in a manner consistent with the condition of the patient. Indications for such therapy include infection by a fungus which produces a proteinase which is critical for its viability, such as an intracellular processing or extracellular proteolytic

proteinase or its pathogenicity. In particular, infection by fungi which are dependent upon a secreted proteinase is a suitable indication. A preferred indication is infection by Candida species which produces a secreted proteinase. Such infections usually occur in the keratinous or moist cutaneous areas of the body, but they may be systemic. Infections most commonly involve the skin, oral and vaginal mucous membranes, and gastrointestinal and respiratory tracts.

Cases of mucosal and systemic Candidiasis are a

recognized hazard in immunocompromised patients (underlying disease process, immunosuppressive therapy, surgical

transplants, etc) and those with in-dwelling catheters. In patients with AIDS, esophageal Candidiasis has emerged as the predominant fungal infection. In these cases, the potential exists that the secreted proteinase may act as an active virulence factor by enhancing the organism's ability to attach to, degrade, or penetrate a host surface . In

cutaneous candidiasis, the digestion of insoluble keratin is likely in order for Candida to penetrate and multiply within host stratum corneum. Thus, the compounds of this invention are believed to function by inhibiting the ability of the proteinase to degrade the host surface and thereby effect the adherence and penetration which is required for infection. In addition, the ability of certain species to secrete an extracellular acid proteinase is dependent upon intracellular processing of the proteinase by secondary intracellular proteinases. Certain of the compounds of this invention are believed to be effective in inhibiting this processing and thereby inhibiting secretion of the extracellular proteinase. Nevertheless, having set forth the proposed mechanism of action, the method of this invention is not intended to be limited by any particular mechanism.

Acid proteinases are most effective in acidic

environments. It has been demonstrated that C. albicans and C. tropicalis excrete organic acids in response to

carbohydrate exposure. Thus, these organisms may have the capacity to provide their own acidified microenvironment suitable for acid proteinase activity upon invasion of and growth in areas over a wide pH range. It has been shown that areas of the kidney (target organ of systemic disease) and foci of organisms/damaged tissue have acid pH values. Low pH environments are also found in certain parts of the mouth (under dentures) and in the vagina and these are two of the most frequent sites of superficial Candida infection.

In all of these disease states, an anti-proteinase therapeutic agent is useful. The efficacy of the agent when used upon diagnosis, as opposed to prophylactically, would depend primarily upon the extent of dissemination and

severity of the disease. For patient populations at risk for mucosal and systemic disease, prophylactic therapy would be recommended. Administration of drug orally or parenterally would probably be equally effective in these disease states except, perhaps, in the case of oral/vaginal thrush, where topical application would be more efficacious.

Dosage units of the active ingredient are selected from the range of .05 to 15 mg/kg. These dosage units may be administered one to five times daily. For critical cases of systemic infection, parenteral administration is preferred. An intravenous infusion of the peptide in 5% dextrose in water or normal saline is most effective, optionally with a solubilizing agent, although an intramuscular bolus injection may be suitable. For less critical states, an oral dosage form may be suitable. For cutaneous or mucosal infections application of an ointment containing the drug is suitable.

The following compounds were prepared in the manner explicitly set forth in EP-A 0 352 000, which is incorporated herein by reference:

1. (4S,5S)-5-(t-butyloxycarbonyl-serylalanylalanyl)amino-4- hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl ester;

2. (5S)-4-[t-butyloxycarbonyl-seryl(O-benzyl)alanylalanyl] amino-2-,2-difluoro-1,3-oxo-5-phenylpentyl-valyl valine methyl ester; 3. (5S)-4-[t-benzyloxycarbonyl-seryl(O-benzyl)alanylalanyl] amino-2,2-difluoro-1,3-oxo-5-phenylpentyl-valyl valine methyl ester;

4. 2-[hydroxy[[(2-phenyl-1-serylalanylalanyl)amino]-ethyl] phosphinyl]cyclopentylcarbonylvalylvaline methyl ester;

5. (4S,5S)-5-(benzyloxycarbonyl-alanylalanyl)amino-4- hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl ester;

6. (4S, 5S)-5-(benzyloxycarbonyl-alanyl) amino-4- hydroxy-1- oxo-6-phenylhexyl-valyl valine methyl ester;

7. (4S,5S)-5-(t-butyloxycarbonyl-alanylalanyl)amino-4- hydroxy-6-(4-hydroxyphenyl)-1-oxo-hexyl-valyl valine methyl ester;

8. (4S,5S)-5-(methoxycarbonyl-alanylalanyl)amino-4- hydroxy- l-oxo-6-phenylhexyl-valyl valine benzyl ester;

9. (4S,5S)-5-(methoxycarbonyl-alanylalanyl)amino-4- hydroxy- 1-oxo-6-phenylhexyl-valyl valine;

10. 2-(-1-hydroxy-3-phenyl-2-(serylalanylalanyl)amino- propyl)cyclopentylcarbonylvalylvaline methyl ester;

11. (3R,4S,5S)-5-(benzyloxycarbonyl-alanylalanyl)amino-3- methyl-4-hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl ester; and

12. (3R,4S,5S)-5-(benzyloxycarbonyl-alanyl)amino-3-methyl-4- hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl ester.

The pharmacological activity of the peptides was assessed by the following tests. These tests may also be used to assess the suitability of a particular proteinase to inhibit the fungal proteinase in order to provide effective treatment. Example A

Effect of Inhibition of Extracellular Proteinase Activity: Cultures of Candida albicans 28366 and 44505 were grown in yeast carbon base media supplemented with 0.2% bovine hemoglobin (YCB-Hb) or bovine serum albumin (YCB-BSA) at 37°C and 125 rpm for specified times to induce production and secretion of extracellular aspartic proteinase. The number of cells/mL were obtained visually by hemocytometer counts following brief sonication and culture supernatants were harvested by centrifugation and filtered through a 0.2 μ filter. To determine extracellular proteinase activity, 5 μL aliquots of supernatant were incubated with pre-warmed (37°C, 10 min) 50 μL aliquots of ¹⁴C-methemoglobin substrate

(2 mg/mL bovine methemoglobin, 0.2 μCi/mL ¹⁴C-methemoglobin,

0.02 mM sodium citrate (pH 3.1) and 0.015 N HCl) at 37°C for 20 min. Following incubation, 100 μL of cold 10% TCA was added to each tube, the contents mixed well and incubated on ice for 5 min. The tubes were microfuged at 16,000 x g for 5 min and 100 μL of supernatant removed to 10 mL scintillation fluid for counting. To determine background activity, the supernatant samples were preincubated with 10 μM pepstatin for 30 min at 37°C prior to addition to the radiolabelled substrate. Standards to determine amount of hemoglobin hydrolyzed were determined by counting 50 μL aliquots of serially diluted radiolabelled substrate (final

concentrations = 0-100 μg hemoglobin). All tests were run in duplicate. Data are calculated based on total μg of

hemoglobin hydrolyzed/50 mL sample or standardized to μg hemoglobin hydrolysed/10⁶ cells.

To assay the direct effects of inhibitors on cell-free enzymatic activity, duplicate aliquots of culture

supernatants (40 hr) were incubated with inhibitor or 0.1% DMSO (vehicle control) a 37°C for 30 min. Following

incubation, 50 μL of pre-warmed radiolabelled substrate was added to each sample and incubation continued for an

additional 20 min. The samples were processed and data determined as described above. The amount of reduction in enzymatic activity due to the inhibitors as compared to the DMSO control was calculated. Data illustrating the

extracellular protease activity of the compounds of this invention are given in Table 1.

Preliminary studies utilizing cells grown under non- proteinase producing conditions in yeast nitrogen base medium (containing 0.15% asparagine and 2% glucose; YNB) indicated no direct effects of the proteinase inhibitors on cell growth or viability.

Effect of Inhibitors on Concommitant Growth and Extracellular

Proteinase Activity: Aliquots from cultures of C. albicans

(25 mL), grown in YCB-Hb or YCB-BSA (37°C, 125 rpm) in the presence of 10 μM inhibitors or 0.1% DMSO, were removed at specified times for the determination of viable cells/mL (dye exclusion/hemocytometer counts) and extracellular proteinase activity (as described above, data standardized to μg hemoglobin/10⁶ cells). Alternatively, some assays ere conducted in a stationary microtiter format by the following procedure: 10⁵ cells/mL were incubated with 10 μM inhibitor in a final volume of 150 μL/well in 96-well flat-bottom microtiter dishes at 37°C. At timed intervals, an OD₅₄₀ reading was obtained on each well, the well contents collected and microfuged and the resulting supernatant assayed for enzymatic activity.

In some cases, the presence of proteinase in culture filtrates and cell lysates was analyzed on Western blots (murine anti-C. albicans extracellular proteinase polyclonal antibody; courtesy of Dr. C. Morrison, Center for Disease Control) of SDS-PAGE preparations (comparing supernatants from equivalent numbers of cells) of dialysed and lyophilized culture supernatants following 40 h of incubation in the presence or absence of inhibitors. Data illustrative of the inhibitory effect of the proteinase inhibitors on the production of active extracellular proteinase resulting in a subsequent reduction in cell viability are given in Table 2.

¹ as compared to DMSO control; measured after 48 h of

incubation

2 strain 28366 tested in YCB-BSA shaking flask cultures (vol = 25 mL) started at 10⁶/mL

³ strain 44505 tested in YCB-Hb and YCB-BSA stationary microtiter cultures (vol= .150 mL) started at 10⁵/mL Example C

Effect of Inhibitors on Adherence of C. albicans to

Endothelial Cells: Endothelial cells (CPAE; bovine pulmonary artery endothelium, ATCC CCL 209) were grown to confluency in 96-well microtiter dishes in Earle's minimal essential medium/20% heat-inactivated fetal bovine serum (EMEM/FBS; 3 days, 37°C, 5% CO₂). ¹⁴C-glucose radiolabelled C. albicans were prepared by incubating washed, YCB-Hb-grown cells (24 hrs, 37°C) with ¹⁴C-glucose (2 μCi/10⁶ viable cells) in YNB

(without glucose) for 75 min at 30°C. Following incubation, the cells were briefly sonicated to produce single cell suspension, washed three times and resuspended to 10⁶

cells/mL in Dulbecco's phosphate buffered saline (DPBS).

Aliquots (100 μL) of radiolabelled cells containing

proteinase inhibitors (10μM, 100 μM) or 0.1% DMSO were added to washed CPAE monolayers (quadruplicate wells/treatment). Viability of uninfected CPAE monolayers treated with test compounds for 1 hr was verified by trypan blue dye exclusion. Following incubation at 37°C for 1 hr, the wells were washed 3 times with warm DPBS and the contents were collected by trypsinization (100μL 0.25% trypsin, 30 min, 37°C) and counted in 10 mL scintillation fluid. Data illustrating the inhibition of the ability of the fungal cells to adhere to endothelial cells is illustrated in Table 3.

TABLE 3

THE EFFECT OF PROTEASE INHIBITORS (100 μM) ON THE ADHERENCE OF CANDIDA ALBICANS* TO ENDOTHELIAL CELLS

C. albicans strains 28366 and 44505

Many variations of the principles of this invention will be apparent to those skilled in the art. However, this invention is limited only by the scope of the claims which follow.

Claims

What is claimed is:

1. A method of treating fungal infection in a mammal by administering an effective amount of an aspartic acid

proteinase inhibitor.

2. A method of treating fungal infection in a mammal by administering an amount of an aspartic acid protease

inhibitor effective to inhibit the proteolytic activity of an extracellular proteinase of the fungus.

3. A method of treating fungal infection in a mammal by administering an amount of an aspartyl protease inhibitor effective to inhibit the secretion of an extracellular protease of the fungus.

4. A method according to claim 1 for treating candidiasis.

5. A method according to claim 1 in which the compound has the partial structure:

-NR'CH(R₄)-T

wherein:

T is -Q-CHR₅C=O-, -Q-CHR₅-Y or -U-; R₄ is C_1-6alkyl, C_3-7Cycloalkyl, (CH₂)_qOR', (CH₂)_qSR', aryl or aryl substituted by one or two C_1-5alkyl,

trifluoromethyl, hydroxy, nitro, amino, C_1-5alkoxy or halogen groups;

Q is -CH₂CH₂-, -CONR'-, -CH₂NR'-, -CH₂S (O) p-, -COCH₂-, -CHOH-, -CH(OH)CHR'-, -CH(OH)CH(OH)-, -CH(NHR')CH₂-,

-COCF₂CH₂-, -P=O(OR')CH₂- or -CH=CR'-;

R₅ is H, -CH(R₄)₂ or chosen from the same group as R₄, Y is -NR'CH(R₂)-Z, -(CH₂)_r-R", -O(CH₂)_r-R",

R₆

I

-NR' (CH₂)_r-R" or -NH(CH)_q-R";

R₂ is (CH₂)_m-W;

W is C_1-5alkyl, CH₂OR', CO₂NHR', CO₂R', CH₂SR', CH₂NHR', guanidyl, imidazolyl, indolyl, aryl or aryl substituted by one or two C_1-5alkyl, trifluoromethyl, hydroxy, C_1-5alkoxy or halogen groups;

Z is H, CO₂R', CONR'R", CO₂(CH₂)_rR", CO(CH₂)_rR", CH₂O-A, CH₂NR'-A or CH₂NR' (CH₂)_qR";

A is H or R₁CO-, R₁OCO-, R₁OCH (R₁,) CO-, R₁NHCH (R₁,) CO-, R₁SCH(R₁,)CO-, R₁SO₂- or R₁SO; R₁ and R₁, are H, C_1-5alkyl, C_3-7cycloalkyl, aryl or aryl substituted by one or two C_1-5alkyl, trifluoromethyl, hydroxy, C_1-5alkoxy or halogen groups;

R₆ is CO₂R' or CON(R')₂; m is 0 to 4; n is 1 or 2; p is 0, 1 or 2; q is 1 or 2; r is 0 to 5;

R' is H, C_1-5alkyl or benzyl; and R" is H, O-A, C_1-5alkyl, C_3-7Cycloalkyl, amino, mono- di- or tri-C_1-5alkylamino, aryl, C_3-7cycloalkylaryl,

heterocycle or guanidyl, or C_3-7cycloalkyl, heteroaryl or aryl substituted by one or two C_1-5alkyl, C_1-4alkoxy,

hydroxy, halogen, trifluoromethyl, carboxy, carboalkoxy, carboxamide, guanidinyl, phenylC_1-5alkylene, guanidinyl, guanidinylC_1-5alkylene, carboxyC_1-5alkylene,

carboalkoxyC_1-5alkylene, carboxamideC_1-5alkylene,

carboxyC_1-5alkyloxy, carboalkoxyC_1-5alkyloxy,

carboxamideC_1-5alkyloxy, amino, mono-C_1-5alkylamino, di- C_1-5alkylamino or aminoC_1-5alkylene.

6. A method according to claim 1 in which the compound has the formula (I):

A-B-D-D-E-M-X-X-Y

(I)

wherein:

A is H or R₁CO-, R₁OCO-, R₁OCH (R₁,) CO- R₁NHCH (R₁,) CO-,

R₁SCH (R₁,) CO-, R₁SO₂- or R₁SO;

R₁ and R₁, are H, C_1-5alkyl, C_3-7cycloalkyl, aryl or aryl substituted by one or two C_1-5alkyl, trifluoromethyl, hydroxy, C_1-5alkoxy or halogen groups;

B is absent, β-alanine or one or two natural amino acids;

D is absent prolyl or -NR'CH (R₂)CO-; R₂ is (CH₂)_m-W;

R' is H, C_1-5alkyl or benzyl;

W is C_1-5alkyl, CH₂OR', CO₂NHR', CO₂R', CH₂SR', CH₂NHR', guanidyl, imidazolyl, indolyl, aryl or aryl substituted by one or two C_1-5alkyl, trifluoromethyl, hydroxy, C_1-5alkoxy or halogen groups; m is 0 to 4; E is absent or -NR'CH (R₃) CO-; R₃ is (CH₂)_m-V, wherein m is as above;

V is C_1-5alkyl, CH₂OR', CO₂NHR', CH₂SR', imidazolyl, indolyl, aryl or aryl substituted by one or two C_1-5alkyl, trifluoromethyl, hydroxy, C_1-5alkoxy or halogen groups;

M is -NR'CH(R₄)-Q-CHR₅CO- or -NR' CH (R₄) -U-;

R₄ is C_1-6alkyl, C_3-7Cycloalkyl, (CH₂)_qOR', (CH₂)_qSR', aryl or aryl substituted by one or two C_1-5alkyl,

trifluoromethyl, hydroxy, nitro, amino, C_1-5alkoxy or halogen groups;

Q is -CH₂CH₂-, -CONR'-, -CH₂NR'-, -CH₂S(O)_p-, -COCH₂-, -CHOH-, -CH(OH)CHR'-, -CH (OH) CH (OH) -, -CH (NHR' ) CH₂-,

-COCF₂CH₂-, -P=O(OR')CH₂- or -CH=CR'-;

R₅ is H, -CH(R₄)₂ or chosen from the same group as R₄; X is absent or -NR'CH(R₂) CO-, wherein R' and R₂ are as above; n is 1 or 2; p is 0, 1 or 2; q is 1 or 2; Y is -NR'CH(R₂)-Z, -(CH₂)_r-R", -O(CH₂)_r-R", -NR' (CH₂) _r-R" or R₆

I

-NH (CH) q-R" ; r is 0 to 5;

Z is H, CO₂R', CONR'R", CO₂(CH₂)_rR", CO(CH₂)_rR", CH₂O-A, CH₂NR'-A or CH₂NR' (CH₂) _qR"; R₆ is CO₂R' or CON(R')₂; and

R" is H, O-A, C_1-5alkyl, C_3-7Cycloalkyl, amino, mono- di- or tri-C_1-5alkylamino, aryl, C_3-7cycloalkylaryl,

heterocycle or guanidyl, or C_3-7cycloalkyl, heteroaryl or aryl substituted by one or two C_1-5alkyl, C_1-4alkoxy,

hydroxy, halogen, trifluoromethyl, carboxy, carboalkoxy, carboxamide, guanidinyl, phenylC_1-4alkylene, guanidinyl, guanidinylC_1-5alkylene, carboxyC_1-5alkylene,

carboalkoxyC_1-5alkylene, carboxamideC_1-5alkylene, carboxy- C_1-5alkyloxy, carboalkoxyC_1-5alkyloxy, carboxamideC_1-

₅alkyloxy, amino, mono-C_1-5alkylamino, di-C_1-5alkylamino or aminoC_1-5alkylene; and pharmaceutically acceptable salts thereof.

7. A method according to claim 6 in which the residues E and X are present and D is present at least once.

8. A method according to claim 6 in which the M residue is -NHCH(R₄)-Q-CHR₅C=0, and Q is -CH(OH)-, -CH(OH)CH₂-, -

COCF₂CH₂- or -P=O(OR')CH₂-.

9. A method according to claim 6 in which E is Ala and X is Val.

10. A method according to claim 6 wherein the compound is chosen from the group consisting of: (4S,5S)-5-(t-butyloxycarbonyl-serylalanylalanyl)amino-4- hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl ester;

(5S)-4-[t-butyloxycarbonyl-seryl(O-benzyl)alanylalanyl]- Omino-2,2-difluoro-1,3-oxo-5-phenylpentyl-valyl valine methyl ester;

(5S)-4-[t-benzyloxycarbonyl-seryl(O-benzyl)alanylalanyl]amino-2,2-difluoro-1,3-oxo-5-phenylpentyl-valyl valine, methyl ester;

2- [hydroxy[[(2-phenyl-1-serylalanylalanyl)amino]ethyl]- phosphinyl]cyclopentylcarbonylvalylvaline methyl ester; (4S,5S)-5-(benzyloxycarbonyl-alanylalanyl)amino-4- hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl ester;

(4S,5S)-5-(benzyloxycarbonyl-alanyl)amino-4-hydroxy-1- oxo-6-phenylhexyl-valyl valine methyl ester;

(4S,5S)-5-(t-butyloxycarbonyl-alanylalanyl)amino-4- hydroxy-6-(4-hydroxyphenyl)-1-oxo-hexyl-valyl valine methyl ester; (4S,5S)-5-(methoxycarbonyl-alanylalanyl)amino-4-hydroxy-1-oxo-6-phenylhexyl-valyl valine benzyl ester;

(4S,5S)-5-(methoxycarbonyl-alanylalanyl)amino-4-hydroxy- 1-oxo-6-phenylhexyl-valyl valine;

2-(-1-hydroxy-3-phenyl-2-(serylalanylalanyl) amino- propyl)cyclopentylcarbonylvalylvaline methyl ester;

(3R,4S,5S)-5-(benzyloxycarbonyl-alanylalanyl)amino-3- methyl-4-hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl estee; and (3R,4S,5S)-5-benzyloxycarbonyl-alanyl)amino-3-methyl-4- hydroxy-1-oxo-6-phenylhexyl-valyl valine methyl ester; and pharmaceutically acceptable salts thereof.

11. A pharmaceutical composition comprising an aspartic acid proteinase inhibitor, an anti-fungal agent and a carrier.

12. A pharmaceutical composition comprising an aspartic acid proteinase inhibitor and a carrier suitable for topical application.

13. The use of a proteinase inhibitor in the manufacture of a medicament having anti-fungal activity.

14. The use of a proteinase inhibitor in the manufacture of a medicament having anti-candidal activity.

15. The use of a compound, as defined in claims 5-10, in the manufacture of a medicament having anti-fungal activity.