COMPOSITION AND TREATMENT WITH BIOLOGICALLY ACTIVE PEPTIDES AND ANTI-PARASITIC AGENTS OR ANTI-FUNGAL AGENTS

Abstract: A composition comprising at least one biologically active amphiphilic peptide or protein, said peptide or protein being an ion channel-forming peptide or protein, and an anti-parasitic agent or an anti-fungal agent. The biologically active amphiphilic peptide and the anti-parasitic agent or anti-fungal agent may be administered in amounts effective to inhibit growth of a parasite or fungus.

Description: COMPOSITION AND TREATMENT WITH BIOLOGICALLY ACTIVE PEPTIDES AND ANTI-PARASITIC AGENTS OR ANTI-FUNGAL AGENTS This invention relates to biologically active peptides and proteins, and more particularly to compositions and uses involving biologically active peptides or proteins and anti-parasitic agents or anti-fungal agents, in particular pentamidine isethionate, propamidine isethionate (Brolene) , and ketoconazole. In accordance with an aspect of the present invention, there is provided a composition which includes includes at least one biologically active amphiphilic peptide and/or biologically active protein; and an anti-parasitic agent or an antifungal agent. In accordance with another aspect of the present invention, there is provided a process wherein there is administered to a host at least one biologically active amphiphilic peptide which is an ion channel forming peptide and/or biologically active protein; and an anti-parasitic agents or an anti-fungal agent. An ion channel- forming peptide or protein or ionophore is a peptide or protein which increases the permeability for ions across a natural or synthetic lipid membrane. B . Christensen et al. PNAS Vol. 85 Pgs . 5072-76 (July, 1988) describes methodology which indicates whether or not a peptide or protein has ion channel- forming properties or influences membrane ' ion conductance. As used herein an ion channel- forming peptide or ion channel forming protein is a peptide or protein which has ion channel-forming properties or influences membrane ion channel conductance as determined by the method of Christensen et al. An amphiphilic peptide is a peptide which includes both hydrophobia and hydrophilic peptide regions. In accordance with an aspect of the present invention wherein the biologically active peptide or protein, and anti-parasitic agent or anti- fungal agent are administered to a host, such biologically active peptide or protein and the anti-parasitic agent or anti-fungal agent may be administered as a single composition or in separate compositions, and the single or separate compositions may include additional materials, actives and/or inactives, in addition to the peptide and/or protein and anti-parasitic agent or anti-fungal agent. The ion channel- forming peptides employed in the present invention are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water. In addition, such peptides are non-hemolytic; i. e. , they will not rupture blood cells at effective concentrations. In addition, the structure of such peptide provides for flexibility of the peptide molecule. When the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself into a rod-like structure. In general, such peptides have at least IS amino acids, and preferably at least 20 amino acids. In most cases, such peptides do not have in excess of 40 amino acids. Anti-parasitic agents which may be employed include, but are not limited to, anti-protozoan agents. Examples of specific anti-parasitic agents which may be employed include, but are not limited to, pentamidine isethionate and propamidine isethionate (Brolene) . Anti-fungal agents which may be employed include, but are not limited to, ketocanazole. It is also to be understood that certain anti-parasitic agents may also have anti-fungal activity, and that certai anti-fungal agents may also have anti-parasitic activity. In employing both an ion channel- forming biologically activ amphiphilic peptide or an ion channel-forming protein, and a anti-parasitic agent or an anti-fungal agent, whether administered o prepared in a single composition, or in separate compositions, the peptid or protein and the anti-parasitic agent or anti-fungal agent are employe in amounts effective to inhibit and/or prevent and/or destroy the growt of a parasite or fungus. In effect, the anti-parasitic agent or anti-funga agent potentiates the action of the peptide or protein, and the peptide o protein potentiates the action of the anti-parasitic agent or anti-funga agent. The term "potentiate," as employed herein, means that the amoun of anti-parasitic agent or antifungal agent is effective to reduce the minimum effective concentration of the peptide or protein for inhibiting growth of a parasitie or fungus and the amount of peptide or protein is effective to reduce the minimum effective concentration of the anti-parasitic agent or anti-fungal agent for inhibiting growth of a parasite or fungus. In addition, the interaction between the peptide or protein and the anti-parasitic agent or anti-fungal agent may allow one of the components to produce a result that it could not achieve alone. In general, the peptide or protein is administered topically at a concentration of from .05% to 5%. The anti-parasitic agent or anti-fungal agent, in general, is used topically at a concentration of from 0.05% to 10%. The use of a combination of peptide or protein and anti-fungal agent or anti-parasitic agent in accordance with the present invention may be employed to inhibit, prevent or destroy the growth or proliferation of parasites and/or fungi. The compositions may also be used as preservatives, disinfectants, or sterilants for materials susceptible to contamination by parasites or fungi. Such compositions are especially useful in the prevention or treatment of eye and skin infections caused by parasites or fungi. Such infections may be caused by fungi such as but not limited to C.albicans and A.fumigatus. and by parasites such as but not limited to A. castellan! . Applicants have found that significant synergistic effects against such parasites or fungi may be obtained when the ion-channel-forming peptides or proteins are employed in conjunction with an anti-fungal agent or anti-parasitic agent. Such compositions may also be effective in killing cysts, spores, or trophozoites of infection- causing organisms. Such organisms include, but are not limited to Acanthamoeba which forms trophozoites or cysts and is a causative agent of amebic keratitis, C. albicans. which forms spores, and A. fumigatus, which forms spores as well. In accordance with a preferred embodiment, the peptide used in conjunction with the anti-fungal agent or anti-parasitic agent is a basic (positively charged) polypeptide having at least sixteen amino acids wherein the polypeptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids. Still more particularly, the hydrophobic amino acids are in groups of two adjacent amino acids, and each group of two hydrophobic amino acids is spaced from another group of two hydrophobic amino aci s -y «t least one amino aeid other than a hydrophobic aαinc acid (preferably at least two amino »»ωc) and generally by no greater than four amino acids, and the amino acid between pairs of hydrophobic amino acids may or may not be hydrophilic. The hydrophilic amino acids are generally also in groups of tw adjacent amino acids in which at least one of the two amino acids is basic hydrophilic amino acid, with such groups of two hydrophilic amin acids being spaced from each other by at least one amino acid other tha a hydrophilic amino acid (preferably at least two amino acids) an generally no greater than four amino adds, and the amino acids betwee pairs of hydrophilic amino acids may or may not be hydrophobic. In accordance with a particularly preferred embodiment, th polypeptide comprises a chain of at least four groups of amino acids, wit each group consisting of four amino acids. Two of the four amino acids in each group are hydrophobic amino acids, and two of the four amino acids in each group are hydrophilic, with at least one of the hydrophilic amino acids in each group being a basic hydrophilic amino acid and the other being a basic or neutral hydrophilic amino acid. The hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, He, Leu, Met, Val, Trp, and Tyr. The neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gin, Ser, and Thr. The basic hydrophilic amino acids may be selected from the class consisting of Lys, Ar«r, His and ornithine (0) . Each of the groups of four amino acids may be of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C or D is a bas hydrophilic amino acid, and the other of C or D is a ββic or neu tral hydrophilic amino acid and may be the same or different. In a preferred embodiment, the polypeptide chain may comprise 5 or 6 groups of this sequence. In each group, each of A, B, C and D may be the same in some or all of the groups or may be different in some or all of the groups. The polypeptide chain preferably has at least 20 amino acids, and no greater than 50 amino acids. It is to be understood, however, that the polypeptide does not have to consist entirely of the groups described above. The polypeptide may have amino acids extending from either or both ends of the noted groups forming the polypeptide chain and/or there may be amino acids between one or more of the at least four groups and still remain within the scope of the invention. The groups of amino acids may be repeating groups of amino acids, or the amino acids in the various groups may vary provided that in each group of the at least four groups of amino acids there are two hydrophobic and two hydrophilic amino acids as hereinabove noted. Thus, in a preferred embodiment, the biologically active polypeptide comprises a chain including at least four groups of amino acids, each containing four amino acids. Two of the four amino acids in each group are hydrophobic, at least one amino acid is basic hydrophilic, and the remaining one is basic or neutral hydrophilic, with the polypeptide chain preferably having at least 20 amino acids but no greater than 50 amino acids. In one embodiment, each of the at least four groups of amino acids which are in the peptide chain is of the sequence A-B-C-D, B-C-D-A, C-D-A-B or D-A-B-C wherein A and B are hydrophobic amino acids, one of C or D is basic hydrophilic amino acid, and the other of C or D is basic or neutral hydrophilic amino acid. The resulting polypeptide chain , therefore, may have one of the following sequences: (X 1 ) a (A-B-C-D) B (Y 1 ) b (X 2 ) (B-C-D-A) n (Y 2 ) b (X 3 ) a (C-D-A-B) n (Y 3 ) b (X 4 ) a (D-A-B-C) n (Y 4 ) b wherein X χ is D; C-D- or B-C-D- , Y 1 is -A or -A-B or -A-B-C X 2 is A- , D-A- or C-D-A- Y 2 is -B, -B-C or B-C-D X 3 is B- , A-B- , D-A-B- Y 3 is -C, -C-D, -C-D-A X 4 is C- , B-C- , A-B-C- Y 4 is -D, -D-A, -D-A-B a is o or 1; b is o or 1 and n is at least 4 It is to be understood that the peptide chain may include amino acids between the hereinabove noted groups of four amino acids provided that the spacing between such groups and the charge on the amino acids does not change the characteristics of the peptide chain which provide amphiphilicity and a positive charge and do not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted group of four amino acids are not spaced from each other. As representative examples of peptides in accordance with the present invention, there may be mentioned. I Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys II Ala-Phe- Ser-Lys- Ala-Phe- Ser-Lys- Ala-Phe- Ser- Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe- Ser-Lys. III Phe-Ser-Lys-Ala-Phe- Ser- Lys- Ala-Phe- Ser-Lys- Ala- Phe- Ser-Lys- Ala- IV Ser-Lys -Ala-Phe- Ser-Lys- Ala- Phe- Ser-Lys- Ala-Phe- Ser-Lys- Ala- Phe- Ser-Lys- Ala-Phe- V Lys-Ala-Phe-Ser-Lys-Ala-Phe- Ser-Lys- Ala-Phe- Ser- Lys- Ala-Phe- Ser The peptide, may have amino acids extending from either end of the chain. For example, the chains may have a Ser-Lys sequence before the "Ala" end, and/or an Ala-Phe sequence after the "Lys" end. Other amino acid sequences may also be attached to the "Ala" and or the "lys" end. Similarly, in any polypeptide chain having at least four groups of amino acids of the sequence as described above, the chain may have, for example, a C-D sequence before the first A-B-C-D group. Also other amino acid sequences may be attached to the "A" and/or the "D" end of one of these polypeptide chains. Also there may be amino acids in the chain which space one or more groups of the hereinabove noted four amino acids from each other. The peptides may be produced by known techniques and obtained in substantially pure form. For example, the peptides may be synthesized on an automatic synthesizer. Journal of American Chemical Society, Vol. 85 Pages 2149-54(1963) . It is also possible to produce such peptides by genetic engineering techniques. In accordance with another preferred embodiment, the peptide employed in conjunction with an anti-fungal agent or anti-parasitic agent may be a magainin peptide. A magainin peptide is either a magainin such as magainin I, II or III or an analogue or derivative thereof. The magainin peptides preferably include the following basic peptide structure X. k 12 ^ " ^^ll^H^ll^Ha ' ^lS^^Ha^H " wherein R.. is a hydrophobic amino acid, R. 2 is a basic hydrophilic amino acid; R-, , is a hydrophobic, neutral hydrophilic, or basic hydrophilic amino acid; R« 4 and * . are hydrophobic or basic hydrophilic amino acids; R. s is glutamic acid or aspartic acid, or a hydrophobic or a basic hydrophilic amino acid, and n is 0 or 1. In a preferred embodiment, R, g is a hydrophobic or neutral hydrophilic amino acid, R 1 a is a hydrophobic amino acid, and R. -. is glutamic acid or aspartic acid. Thus, for example, a magainin peptide may include the following structure: "Y 12 "X 12 " where X 12 is the hereinabove described basic peptide structure and 12 is (i) R 12 (iii) R n -R 14a -R 12 where R χι , R 12 , R χ4 and R χ4a are as previously defined. A magainin peptide may also have the following structure: "X 12 "Z 12 " wherein X- 2 is as previously defined and Z-,„ is: (i) R 16 where R lg is a basic hydrophilic amino acid or asparagine or glutamine. (ii) R j g-R j where R-, ? is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid. Preferably, R 17 is a neutral hydrophilic amino acid. A magainin peptide may also have the following structure: where X 12 , Y 12 and Z 12 are as previously defined and a is 0 or 1 and b is 0 or 1. The magainin peptides may also include the following basic peptide structure X.,: " R 14~ R ll" R 14a" R 12~ R ll" R ll" R 12" R 13" i i ~ i4~ i2 ~R ll ~R ll~ R l2~ ' wherein Rι ι » Ri2' R 13' R 14* an< ^ R 14a are amino acids as hereinabove described. The magainin peptide may also include the following structure X 13~ Z 13' wherein X. -. is the hereinabove described basic peptide structure and Z. , is ^ll)n-( R ll)n-( R ll)n-( R 14a)n-( R 15>n-( R 14a>n-( R 14>n-( R 16)n- (R-.) wherein R.. , R« 4 , R-**^. R 15 » R j g. and R 17 are as hereinabove described, and n is 0 or 1, and each n may be the same or different. The magainin peptides generally include at least fourteen amino acids and may include up to forty amino acids. A magainin peptide preferably has 22 or 23 amino acids. Accordingly, the hereinabove described basic peptide structures of a magainin peptide may include additional amino acids at the amino end or at the carboxyl end, or at both ends. As representative examples of such magainin peptides, there may be mentioned peptides having the foUowing primary sequence (expressed as a single letter code) as well as appropriate analogues and derivatives thereof: (a) (NH 2 ) GIGKFLHSAGKFGKAFVGEIMKS(OH) or (NH 2 ) (Magainin I) (b) (NH 2 ) GIGKFLHSAKKFGKAFVGEIMNS(OH) or (NH 2 ) (Magainin II) (c) (NH 2 ) GIGKFLHSAKKFGKAFVGEIMN(OH) or (NH 2 ) (Magainin III) The following are examples of peptide derivatives or analogs of the basic structure: (d) (NH 2 ) IGKFLHSAKKFGKAFVGEIMNS(OH) or (NH 2 ) (e) (NH 2 ) GKFLHSAKKFGKAFVGEIMNS(OH) or (NH 2 ) (f) (NH 2 ) KFLHSAKKFGKAFVGEIMNS(OH) or (NH 2 ) Magainin peptides are described in Proc. Natl. Acad Sci. Vol. 84 pp. 5449-53 (Aug. 87) . The term "magainin peptides" as used herein refers to the basic magainin structure as well as derivatives and analogs thereof, including but not limited to the representative derivatives or analogs. In accordance with a further embodiment, the peptide employed in conjunction with an anti-fungal agent or anti-parasitic agent may be a PGLa peptide or an XPF peptide. A PGLa peptide is either PGLa or an analogue or derivative thereof. The PGLa peptides preferably include the following basic peptide structure X- 4 : - R 11 -R 17 -R 12 - R ιι- R i4 "R i4 "R ιι " R ll "R 14 "R 12 "R ll "R ll "R 12 "R ll " R 11 -R 11 -R 12 - where R.^, R 12 , R 14 , and R 17 are as previously defined. The PGLa peptides generally include at least seventeen amino acids and may include as many as forty amino acids. Accordingly, the hereinabove described basic peptide structure for a PGLa peptide may include additional amino acids at the amino end or at the carboxyl end or at both the amino and carboxyl end. Thus, for example, a PGLa peptide may have the following structure: ~ Y 14~ X 14~ where X< 4 is as previously defined and Y 14 is (i) R n ; (ii) R 14 -R u where R, . and R« 4 are as previously defined. For example, a PGLa peptide may also have the following structure: "X 14 "Z 14 " where X, 4 is as previously defined; and Z 14 Is: (i) R u ; or (ii) R ι R n where R... is as previously defined. A PGLa peptide may also have the following structure: ( Y 14>a "Xl4" (Zl4) b where X 14 ; Y 14 and Z 14 are as previously defined, a is 0 or 1 an b is 0 or 1. An XPF peptide is either XPF or an analogue or derivative thereof The XPF peptides preferably include the following basic peptide structur X 16 : "R ll~ R 17 'R 12 "R ll 'R 14 "R 18 "R 17 " R ll 'R 14 "R 12 "R ll "R ll "R 12 " R n - R n - hereIn R ll' R 12' R 14' R 15 an< ^ R 17 are as P rev * ously defined and R. g i glutamine or asparagine or a basic hydrophilic, or hydrophobic amino aci and, n is O or 1. The XPF peptides generally include at least nineteen amino acids an may include up to forty amino acids. Accordingly, the hereinabov described basic peptide structure of XPF may include additional amin acids at the amino end, or at the carboxyl end or at both the amino an carboxyl ends. Thus, for example, an XPF peptide may include the followin structure: where R.. and R 14 are as previously defined. An XPF peptide may include the following structure: "X lβ 'Z 16 " where X. fi is as previously defined and Z- fi is (iv) R n -R 18 -Proline-R 12 An XPF peptide may also have the following structure: < Y 16>a "Xlβ(Zlβ) b where lg , Y lg and Z lg are as previously defined: a is 0 or 1 and b is 0 or 1. Preferred are XPF or PGLa peptides, which are characterized by the following primary amino acid sequence (single letter amino acid code) : PGLa : GMASKAGAIAGKIAKVALKAL (NH 2 ) XPF : GWASKIGQTLGKIAKVGLKELIQPK A review of XPF and PGLa can be found in Hoffman et al, EMBO J. 2: 711-714, 1983; Andreu et al, J. Biochem. 149:531-535, 1985; Gibson et al J. Biol. Chem. 261:5341-5349, 1986; and Giovannini et al, Biochem J. 243:113-120, 1987. In accordance with yet another embodiment, the peptide employed in conjunction with an anti-fungal agent or anti-parasitic agent may be a CPF peptide or appropriate analogue or derviative thereof. CPF peptides as well as analogues and derivatives thereof are herein sometimes referred to collectively as CPF peptides. The CPF peptide is preferably one which includes the following peptide structure 3Q : _R 2l "R 2l "R 22 "R 22 "R 2l "R 2l "R 23 "R 2l ' "R 2l "R 2l "R 23 'R 2l 'R 2l "R 24 "R 25 "R 2l " wherein R 21 is a hydrophobic amino a d; R 22 is a hydrophobic amino acid or a basic hydrophilic amino acid; R,„ is a basic hydrophilic amino acid; and R_ . is a hydrophobic or neutral hydrophilic amino acid; and R 25 is a basic or neutral hydrophilic amino acid. The hereinabove basic structure is hereinafter symbolically indicate as X 3Q . The hydrophobic amino acids are Ala, Cys, Phe, Gly, He, Leu, Met Val, Trp, and Tyr. The neutral hydrophilic amino acids are Asn, Gin, Ser, and Thr. The basic hydrophilic amino acids are Lys, Arg, His and ornithine. The CPF peptide may include only the hereinabove noted amino acid or may include additional amino acids at the amino end or carboxyl end o both the amino and carboxyl end. In general, the peptide does no include more than 40 amino acids. The CPF peptides including the above basic peptide structure ma have from 1 to 4 additional amino acids at the amino end. Accordingly, such preferred peptides may be represented by the structural formula: Y 30 "X 30 " wherein X- 0 is the hereinabove described basic peptide structur and Y 3Q is (i) R 25 - , or (ii) R 22 _R 25 ; or (iϋ) R 21 -R 22 -R 25 ; or (iv) 22 'R 2 R 22 _R 25 ; P referabl y Glycine -R 2 R 22 "R 25 ' wherein R 21 , R 22 , and R 2g are as previously defined. The carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13 additional amino acids . In a preferred embodiment, the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows: - χ 30 _Z 30 wherein X 30 is the hereinabove defined basic peptide structure and Z, 0 is ( V ) R 2l "R 2l "R 24 "R 24 "R 26 ; (vi) R 21 -R 21 -R 24 -R 24 -R 2β -Gln; or (vii) R 21 -R 21 -R 24 -R 24 -R 26 -Gln-Gln, wherein R 21 and R 24 are as previously defined, and R 26 is proline or a hydrophobic amino acid. Preferred peptides may be represented by the following structural formula: < Y 30>a "X30 ~ (Z3θ) b wherein X gQ , Yg Q and Z 3Q are as previously defined and a is 0 or 1 and b is 0 or 1. Representative examples of CPF peptides which are useful in the present invention some of which have been described in the literature and comprise the following sequences (single letter amino acid code) : (1) GFGSFLGLALKAALKIGANALGGAP Q (2) GLASFLGKALKAGLKIGAHLLGGAPQQ (3) GLASLLGKALKAGLKIGTHFLGGAPQ (4) GLASLLGKALKATLKIGTHFLGGAP Q (5) GFASFLGKALKAALKIGANMLGGTPQQ (6) GFGSFLGKALKAALKIGANALGGAP Q (7) GFGSFLGKALKAALKIGANALGGSPQQ (8) GFASFLGKALKAALKIGANLLGGTPQQ A review of the CPF peptides can be found in Richter, K. , Egger, R. , and Kreil (1986) J. Biol. Chem. 261, 3676-3680; Wakabayashi, T. Kato, H. , and Tachibaba, S. (1985) Nucleic Acids Research 13, 1817-1828; Gibson, B.W. , Poulter, L. , Williams, D.H. , and Maggio, J.E. (1986) J. Biol. Chem. 261, 5341-5349. CPF peptides which may be employed in the present invention are represented by the following (single letter amino acid code) : G12S3LG4ALKA5LKIG678LGG9(10) Q Where: 1 = F, L 2 = G, A 3 F, L 4 K, L 5 - A, G, T 6 = A, T 7 = H, N 8 - A, M, F, L 9 - ■ A. S, T 10 = P, The numbered amino acids may be employed as described in any combination to provide either a basic CPF peptide structure or an analogue or derivative. The term CPF peptide includes the basic peptide structure as well as analogs or derivatives thereof. In accordance with still another embodiment, the biologically active peptide may include the following basic strucutre X..: 40 t R 4 R 42 "R 42 "R 43 ~R 4l "R 42 ~R 42V wherein R 4 χ is a baslc hydrophilic amino acid, R^ is a hydrophobic amino acid, R 43 is a neutral hydrophilic or hydrophobic amino acid, and n is from 2 to 5. In one embodiment, such peptide may include the following structure: 4θ" X 40 * nβreul x 4 o ^ as hereinabove described, and Y 40 is: (i) R^; (Hi) R^-R^- ^; (v) 42 -R 3- R 4 ι-R 4 2 " R 4 2 ; or (vi) R 4 2-R 4 2 " R 4 3 ' R 4 i " R 4 2 "R 42' hereln R 4 i » K _> > and R 43 are as hereinabove described In accordance with another embodiment, such peptide may include the foUowing structure: X.--Z.-, wherein X 4Q is as hereinabove described, and Z 4Q is: or (vi) R 41 -R 4 2-R 42 " R 3 "R 41 "R 42 - In accordance another embodiment, such peptide may include the foUowing structure: , wherein Y and Z are as previously defined, a is 0 or 1, and b is 0 or 1. In one embodiment, n is 3, and most preferably the peptide is of the foUowing structure as indicated by the single letter amino acid code: [KIAGKIAJg. In another embodiment, n is 2 , and the peptide preferably is of the foUowing structure as indicated by the single letter amino acid code: KIA(KIAGKIA) 2 KIAG . In accordance with yet another embodiment, the biologicaUy active amphiphiUc peptide may be a biologicaUy active amphiphiUc peptide including the foUowing basic structure X go : R 4l "R 42 "R 42 " R43 "R 4l "R 42 "R 42 "R 4l "R 42 "R 42 " R42 "R 4l "R 42 "R 42' wherein R., , R- 2 and R43 are as hereinabove described. In accordance with one embodiment, such peptide may include the foUowing structure: Y 50" X 50' nereul X 50 k as hereinabove described, and Y 50 is: (Hi) R^-R^-R^; (v) R 2 - R 43 "R 41 "R 42 "R 42 ; vi) R 42 - R 42- R 43- R 4 R 42 "R 42 ' OΓ (vH) R 41 -R 42 -R 4 2-R 4 3 "R 4l "R 42 "R 42' herein R 41 ' R 42 and R 43 are as hereinabove described. In accordance with another embodiment, such peptide may include the foUowing structure: X 50~ Z 50 * wnereuι X 5o is as hereinabove described and Z 50 is: (i) R 41 ; (H) R^-R^; (Hi) R 41 -R 42 -R 42 ; ( V ) R4 1 -R 4 2-R.i2- R 43- R 4 1 ; (vU) R4 1 -R42 ' R42 "R 43 " R4l " R42 "R 42 ' nereuι R 4 ι» R 42 and R 43 are as hereinabove described. In accordance with yet another embodiment the peptide may include the foUowing structure: (Y 50 )a-X 50 -(Zg 0 )b, wherein X and Y are as previously defined, a is 0 or 1, and b is 0 or 1. In one embodiment, the peptide is of the foUowing structural formula as indicated by the single letter amino acid code: KLASKAGKIAGKIAKVALKAL . In another embodiment, the peptide is of the foUowing structura formula as indicated by the single letter amino acid code: KIAGKIAKIAGOIAKIAGKIA. In stiU another embodiment, the peptide employed in conjunction wit an anti-fungal agent or anti-parasitic agent is a cecropin. The cecropin and analogs and derivatives thereof are described in Ann. Rev. Microbio 1987 Vol. 41 pages 103-26, in particular p. 108 and Christensen at a PNAS Vol. 85 p. 5072-76, which are hereby incorporated by reference. The term cecropins includes the basic structure as weU as analogue and derivatives. In yet another embodiment, the peptide employed in conjunction wit an anti-fungal agent or anti-parasitic agent is a sarcotoxin. The sarcotoxins and analogs and derivatives thereof are described in Molecular Entomology pages 369-78 in particular p. 375 Alan R. Liss Inc. (1987) , which is hereby incorporated by reference. The term sarcotoxin includes the basic materials as weU as analogues and derivatives. It is also contemplated that within the scope of the present invention, that each of the amino acid residues of the biologicaUy active amphiphiUc peptide structures hereinabove described is a D-amino acid residue or a glycine residue. In another embodiment, an ion channel-forming protein may be used in conjunction with an anti-parasitic agent or an anti-fungal agent. Ion channel-forming proteins which may be employed include defensins, also known as human neutrophU antimicrobial peptides (HNP) , major basic protein (MBP) of eosinophUs, bactericidal permeabUity-increasing protein (BPI) , and a pore-forming cytotoxin caUed variously perforin, cytolysin, or pore-forming protein. Defensins are described in Selsted, et al. , -J^ Clin. Invest. , Vol. 76, pgs. 1436-1439 (1985) . MBP proteins are described in Wasmoen, et al. , J. Biol. Chem. . Vol. 263, pgs 12559-12563. (1988) . BPI proteins are described in Ooi, et al, J. Biol. Chem. . Vol. 262, pgs. 14891-14894 (1987) . Perforin is described in Henkart, et al. , J. Exp. Med. , 160: 75 (1984) , and in Podack, et al. , J. Exp. Med. , 160:695 (1984) . The above articles are hereby incoroporated by reference. The term ion channel-forming proteins includes the basic structures of the ion-forming proteins as weU as analogues and derivatives. The present invention wHl be further described with respect to the foUowing example; however, the scope of the invention is not to be limited thereby. Example For purposes of this example, Peptide 1 is of the foUowing structure: GIGKFLKKAKKFGKAFVKIMKK ; and Peptide 2 is of the foUowing structure: [KIAGKIA] 3 Logarithmic phase axenic cultures of Acanthamoeba polyphaga at 30° C in fluid ppyg medium (2% proteose-peptone; 0.5% yeast extract; 0.5% glucose; pH7.2) . Cells were counted using a Coulter counter, and added to fresh ppyg medium to give a ceU concentration of approximately 10,000 amoebas/ml. CeU suspensions were then transferred to control flasks or to C „orni .ng _ tissue cul ,t_ure f •l.ask. s (,,2,5.-- cm 3) ' to which had been added one of the foUowing preparations: (a) Peptide 1 (20 μg/ml) (b) Peptide 1 (20 μg/ml) and propamidine isethionate (Brolene, l%) (c) Peptide 1 (20 μg/ml) and ketoconazole. (d) Peptide 2 (20 μg/ml) (e) Peptide 2 (20 μg/ml) and Brolene (1%) (f) Peptide 2 (20 μg/ml) and ketoconazole (g) Brolene (1%) ; or (h) Ketoconazole Each flask contained 10 ml of medium. The flasks were incubated at 30° C . 0.5 ml samples were removed from the flasks, generaUy over a 5-day or β -day period, for counting in a Coulter counter. The number of amoebas growing out of the control flasks as weU as the flasks containing preparations (a) through (h) , is shown in Figures 1A, IB, 2A, and 2B. It was found that the combination of Peptide 1 plus either Brolene or ketoconazole was effective in inhibiting growth. Amoebas grew out from the inhibited populations contacted with preparations (a) , (b) , or (g) . Amebas did not grow out from the population contacted with preparation (c) ; such preparation appeared to be amoebicidal. It was also found that amoebas did not grow out from populations treated with preparations (d) , (e) or, (f) , nor did amoebas grow out from the population contacted with ketoconazole alone. The peptide or protein and anti-parasitic agent or anti-fungal agent, as hereinabove described, may be employed for treating a wide variety of hosts. In accordance with a preferred embodiment, a host is an animal, and such animal may be a human or non-human animal. The peptide or protein and the anti-parasitic agent or anti-fungal agent may be employed together in a single composition, or in separate compositions. Moreover, the anti-parasitic agent or anti-fungal agent and the peptide or protein may be deUvered or administered in dtfferent forms, or by different routes; for example, the anti-parasitic agent or anti-fungal agent may be administered systemicaJly, whUe the peptide or protein may be administered topicaUy. The peptide or protein and/or anti-parasitic agent or anti-fungal agent may be employed in a wide variety of pharmaceutical compositions in combination with a non-toxic pharmaceutical carrier or vehicle such as a fiUer, non- toxic buffer, or physiological saline solution. Such pharmaceutical compositions may be used topicaUy or systemicaUy and may be in any suitable form such as a Uquid, soUd, semi- solid, injectable solution, tablet, ointment, lotion, paste, capsule, or the like. The peptide or protein and/or anti-parasitic agent or anti-fungal agent may also be used in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful parasites, including parasitic, protozoa, and fungi. The peptide(s) or protein of the present invention may be administered to a host; in particular an animal, in an effective anti-fungal and/or anti-parasitic amount in conjunction with anti-parasitic agent or anti-fungal agent for potentiating the activity of the peptide or protein, or the peptide or protein may potentiate the anti-fungal agent or anti-parasitic agent. As representative examples of administering the peptide or protein and anti-parasitic agent or anti-fungal agent for topical or local administration, the peptide could be administered in an amount of up to about 1% weight to weight and the anti-parasitic or anti-fungal agent deUvered in an amount of about 50 mM (about 0.1%) . Alternatively, the anti-parasitic agent or anti-fungal agent could be administered topicaUy in conjunction with systemic administration of the peptide and/or protein. For example, the peptide or protein may be administered IV or IP to achieve a serum dose of 100 micrograms per milUUter (10 miUigrams per kilogram) in conjunction with a topical dose of anti-parasitic agent or anti-fungal agent of from about 4 μg/ml to about 100 μg/ml. Numerous modifications and variations of the present invention are possible in Ught of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

Claims: WHAT IS CLAIMED IS: 1. A process, comrpising: administering to a host at least one biologicaUy active amphiphiUc peptide and/or biologicaUy active protein, said peptide or protein being an ion channel- forming peptide or protein; and an antiparasitic agent, said components being administered to inhibit growth of a parasite in a host. 2. The process of Claim 1 wherein the peptide is a magainin peptide. 3. The process of Claim 1 wherein the peptide includes the foUowing basic structure X. Q : [R 4l "R 42 "R 42 "R 43 "R 4l "R 42 "R 42 ] nl wherein R 41 is a basic hydophUic amino acid, R« 2 is a hydrophobic amino acid, R 4g is a neutral hydrophUic or hydrophobic amino acid, and n is from 2 to 5. 4. The process of Claim 1 wherein the peptide inlcudes the foUowing basic structure X 50 : R 4l "R 42 "R 42 "R 43 'R 4l "R 42 "R 42 'R 4l "R 42 "R 42 "R 42 " 41~ R 42~ R 42' n erein R-. is a basic hydrophUic amino acid, R. 2 is a hydrophobic amino acid, and R 43 is a neutral hydrophUic or hydrophobic amino acid. 5. The process of Claim 1 wherein said antiparasitic agent is selected from the group consisting of pentamidine isethionate and propamidine isethionate. 6. A process, comprising: administering to a host at least one biologicaUy active amphiphiUc peptide and/or biologicaUy active protein, said peptide or protein being an ion channel- forming peptide or protein; and an anti-fungal agent, said components being administered in an amount to inhibit growth of a fungus in a host. 7. The process of Claim 6 wherein the peptide is a magainin peptide. 8. The process of Claim 15 wherein the peptide includes the foUowing basic structure X. fl : [R 4l "R 42 "R 42 "R 43 "R 4l "R 42 'R 42 ] n, wherein R 41 is a basic hydrophUic amino acid, R. 2 is a hydrophobic amino acid, R 43 is a neutral hydrophUic or hydrophobic amino acid, and n is from 2 to 5. 9. The process of Claim 6 wherein the peptide includes the foUowing basic strcture X--: R 4l 'R 42 'R 42 "R 43 "R 4l 'R 42 "R 42 "R 4 R 42 "R 42 "R 42 "R 4l 'R 42 'R 42' wherein R.~ is a basic hydrophUic amino acid, R- 2 is a hydrophobic amino acid, and R 43 is a neutral hydrophiUc or hydrophobic amino acid. 10. The process of Claim 6 wherein said anti-fungal agent is ketoconazole. 11. A composition, comprising: (a) at least one biologicaUy active amphiphiUc peptide and/or biologicaUy active protein, said peptide or protein being an ion channel-forming peptide or protein; and . (b) an anti-parasitic agent. 12. The composition of Claim 11 wherein said components (a) and (b) are present in an amount to inhibit growth of a parasite. 13. The composition of Claim 11 wherein the peptide is a magainin peptide. 14. The composition of Claim 11 wherein the peptide includes the foUowing basic strucutre X.-.: [ R 4l "R 42 "R 42 'R 43 "R 4l "R 42 "R 42 ] n wherein R 41 is a basic hydrophiUc amino acid, R 42 is a hydrophobic amino acid, R 43 is a neutral hydrophiUc or hydrophobic amino acid, and n is from 2 to 5. 15. The composition of Claim 11 wherein the peptide inlcudes the foUowing basic structure X- Q : R 4 R 42 ~R 42~ R 43~ R 4 R 42 "R 42 "R 4 R 42 ~R 42 ~R 42 ~R 4r R 42~ R 42' wherein R 41 is a basic hydrophUic amino acid, R^ is a hydrophobic amino acid and R 4 , is a neutral hydrophUic or hydrophobic amino acid. 16. The composition of Claim 11 wherein said anti-parasitic agent is selected from group consisting of pentamidine isethionate and propamidine isethionate. 17. A composition, comprising: (a) at least one biologicaUy active amphiphiUc peptide and/or biologicaUy active protein, said peptide or protein being an ion channel-forming peptide or protein; and (b) an anti-fungal agent. 18. The composition of Claim 17 wherein said components (a) and (b) are present in an amount to inhibit growth of a fungus. 19. The composition of Claim 17 wherein the peptide is a magainin peptide. 20. The composition of Claim 17 wherein the peptide includes the foUowing basic strucutre X 4Q : [R 4l "R 2 "R 42 "R 43 "R 4l "R 42 'R 42 ] n wherein R 41 is a basic hydrophUic amino acid, R 42 is a hydrophobic amino acid, R 43 is a neutral hydrophUic or hydrophobic amino acid, and n is from 2 to 5. 21. The composition of Claim 17 wherein the peptide inlcudes the foUowing basic structure X- Q : R 4l "R 42 "R 42 "R 43 "R 4l "R 42 "R 42 "R 4l "R 42 "R 42 "R 42 "R 4l "R 42 "R 42' wherein R 4 - is a basic hydrophUic amino acid, R 42 is a hydrophobic amino acid and R 4g is a neutral hydrophUic or hydrophobic amino acid. 22. The composition of Claim 17 wherein the anti-fungal agent is ketoconazole. 23. The composition of Claim 13 wherein said magainin peptide is of the foUowing structure: GIGKFLKKAKKFGKAFVKIMKK . 24. The composition of Claim 19 wherein said magainin peptide is of the foUowing structure: GIGKFLKKAKKFGKAFVKIMKK . 25. The composition of Claim 14 wherein said peptide is of the foUowing sturcture: [KIAGKIA] 3 . 26. The composition of Claim 20 wherein said peptide is of the foUowing structure: [KIAGKIA] 3 .

Inventors: BERKOWITZ BARRY, JACOB LEONARD, BERKOWITZ, BARRY, JACOB, LEONARD
Applicants: MAGAININ SCIENCES INC, MAGAININ SCIENCES INC.
Application year: 1991
Main classification: C07K7
IPC codes: A61K38, A61P31, A61P33