COMPOSITION AND TREATMENT WITH BIOLOGICALLY ACTIVE PEPTIDES AND ANTIBIOTICS WHICH INHIBIT DNA GYRASE

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 antibiotic which inhibits DNA gyrase. The biologically active amphiphilic peptide and the antibiotic which inhibits DNA gyrase may be administered in amounts effective to inhibit growth of a target cell such as a bacterium.

Description: -1- COMPOSITION AND TREATMENT WITH BIOLOGICALLY ACTIVE PEPTIDES AND ANTIBIOTICS WHICH INHIBIT DNA GYRASE This invention relates to biologically active peptides and protein and more particularly to compositions and uses involving biologica active peptides or proteins and an antibiotic which inhibits DNA gyras and in particular quinolone antibiotics such as ciprofloxacin . In accordance with an aspect of the present invention, there provided a composition which includes includes at least one biological active amphiphilic peptide and/or biologically active protein ; and antibiotic which inhibits DNA gyrase. In accordance with another aspect of the present invention , there provided a process wherein there is administered to a host at least o biologically active amphiphilic peptide which is an ion channel formi peptide and/or biologically active protein; and an antibiotic which inhibi DNA gyrase. An ion channel- forming peptide or protein or ionophore is a pepti or protein which increases the permeability for ions across a natural synthetic lipid membrane. B . Christensen et al. PNAS Vol. 85 Pg 5072-76 (July, 1988) describes methodology which indicates whether or n a peptide or protein has ion channel-forming properties and is therefo an ionophore. As used herein an ion channel- forming peptide or io channel forming protein is a peptide or protein which has io channel- forming properties as determined by the method of Christensen al. An amphiphilic peptide is a peptide which includes both hydrophobi and hydrophilic peptide regions. In accordance with an aspect of the present invention wherein th biologically active peptide or protein, and an antibiotic which inhibit DNA gyrase are administered to a host, such biologically active peptide o protein and the antibiotic which inhibits DNA gyrase may be administere as a single composition or in separate compositions, and the single o separate compositions may include additional materials, actives and/o inactives, in addition to the peptide and/or protein and antibiotic whic inhibits DNA gyrase. The ion channel- forming peptides employed in the present inventio are generally water soluble to a concentration of at least 20 mg/ml 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 peptid molecule. When the peptide is placed in water, It does not assume a amphiphilic structure. When the peptide encounters an oily surface o membrane, the peptide chain folds upon itself into a rod- like structure. In general, such peptides have at least 16 a ino acids, an preferably at least 20 amino acids. In most cases, such peptides do n have in excess of 40 amino acids. DNA gyrase is an enzyme which is involved in the formation bonds between individual coiling strands of replicating bacterial DNA Thus, DNA gyrase is necessary for the normal replication of bacteri DNA, and, therefore, antibiotics which inhibit DNA gyrase inhibit t normal replication of bacterial DNA. 4453 Examples of antibiotics which inhibit DNA gyrase include nalid acid, oxolinic acid, cinoxacin, and quinolone antibiotics which incl ciprofloxacin , norfloxacin, ofloxacin, enoxacin, pefloxacin, lomefloxa fleroxacin, tosulfloxacin, temafloxacin, and rufloxacin. The following structural formulae of representative examples of antibiotics which inh DNA gyrase. Nalidixic acid has the following structure: Oxolinic acid has the following structure: Of the antibiotics which inhibit DNA gyrase which are also quinol antibiotics, the following are representative structural formulae. Ciprofloxacin has the following structure: Norfloxacin has the following structure: ^ C0ftV 4 Antibiotics which inhibit DNA gyrase are further described i Clinical and Infectious Diseases. W. B . Saunders Co . (1987) . In preferred embodiment, the antibiotic which inhibits DNA gyrase is quinolone antibiotic, and most preferably, the quinolone antibiotic ciprofloxacin . In employing both an ion channel-forming biologically acti amphiphilic peptide or an ion channel- forming protein , and an antibiot which inhibits DNA gyrase, whether administered or prepared in a sing composition, or in separate compositions, the peptide or protein and t antibiotic which inhibits DNA gyrase are employed in amounts effective inhibit and/or prevent and/or destroy the growth of * a target cell. I effect, the quinolone antibiotic potentiates the action of the peptide protein, and the peptide or protein potentiates the action of the antibiot which inhibits DNA gyrase. The term "potentiate, " as employed herei means that the amount of antibiotic which inhibits DNA gyrase is effecti to reduce the minimum effective concentration of the peptide or protei for inhibiting growth of a target cell and the amount of peptide or prote is effective to reduce the minimum effective concentration of the antibiot which inhibits DNA gyrase for inhibiting growth of a target cell. In general, the peptide or protein is administered topically at concentration of from .05% to 10%. When administered systemically, t peptide or protein is employed to provide peptide or protein dosages from lmg to 500mg per kilogram of host weight. The antibiotic which inhibits DNA gyrase, in general, is us topically at a concentration of from 0.05% to 10%. When used systemicall US91/04453 the antibiotic which inhibits DNA gyrase is generally employed in amount of from 1.25 to 45mg per kilogram of host weight per day. The use of a combination of peptide or protein and antibiotic w inhibits DNA gyrase, in accordance with the present invention is effec as an antibiotic, and may be employed to inhibit, prevent or destroy growth or proliferation of microbes, such as bacteria. The compositions have a broad range of potent antibiotic acti against a plurality of microorganisms, including Gram- positive Gram -negative bacteria. Such compositions may be employed for treat or controlling microbial infection caused by organisms which are sensit to such compositions. The treatment may comprise administering to a h organism or tissues acceptable to or affiliated with a microbial infection an ti- microbial amount of such peptide or protein and antibiotic wh inhibits DNA gyrase. The compositions may also be used as preservatives or sterilants materials susceptible to microbial contamination . The compositions of the present invention may also be used in t treatment of external burns and to treat and/or prevent skin and bu infections. In particular, the compositions may be used to treat skin a burn infections caused by organisms such as, but not limited P . aeruginosa and S . aureus . Such compositions may also be used in the prevention or treatment eye infections. Such infections may be caused by bacteria such as , b not limited to, P. aeruginosa. S .auerus. and N . gonorrhoeae . In accordance with a preferred embodiment, the peptide used conjunction with the antibiotic which inhibits DNA gyrase is a ba (positively charged) polypeptide having at least sixteen amino ac wherein the polypeptide includes at least eight hydrophobic amino a and at least eight hydrophilic amino acids. Still more particularly, hydrophobic amino acids are in groups of two adjacent amino acids, each group of two hydrophobic amino acids is spaced from another gr of two hydrophobic amino acids by at least one amino acid other tha hydrophobic amino acid (preferably at least two amino acids) generally by no greater than four amino acids , and the amino ac between pairs of hydrophobic amino acids may or may not be hydrophil The hydrophilic amino acids are generally also in groups of adjacent amino acids in which at least one of the two amino acids i basic hydrophilic amino acid, with such groups of two hydrophilic a acids being spaced from each other by at least one amino acid other t a hydrophilic amino acid (preferably at least two amino acids) generally no greater than four amino acids, and the amino acids betw pairs of hydrophilic amino acids may or may not be hydrophobic. In accordance with a particularly preferred embodiment, polypeptide comprises a chain of at least four groups of amino acids, each group consisting of four amino acids. Two of the four amino a in each group are hydrophobic amino acids, and two of the four a acids in each group are hydrophilic, with at least one of the hydrop amino acids in each group being a basic hydrophilic amino acid and other being a basic or neutral hydrophilic amino acid. The hydrophobic amino acids may be selected from the c consisting of Ala, Cys, Phe, Gly, He, Leu, Met, Val, Trp, and T The neutral hydrophilic amino acids may be selected from the c consisting of Asn, Gin, Ser, and Thr. The basic hydrophilic amino a 453 may be selected from the class consisting of Lys, Arg, His and ornit (0) . Each of the groups of four amino acids may be of the seque ABCD, BCDA, CDAB, or DABC , wherein A and B are each hydroph amino acids and may be the same or different, one of C or D is a b hydrophilic amino acid, and the other of C or D is a basic or neu hydrophilic amino acid and may be the same or different. In a prefer embodiment, the polypeptide chain may comprise 5 or 6 groups of t sequence . In each group, each of A, B, C and D may be the same some or all of the groups or may be different in some or all of groups . The polypeptide chain preferably has at least 20 amino acids , and greater than 50 amino acids . It is to be understood, however, that polypeptide does not have to consist entirely of the groups descri above. The polypeptide may have amino acids extending from either both ends of the noted groups forming the polypeptide chain and/or th may be amino acids between one or more of the at least four groups a still remain within the scope of the invention. The groups of amino acids may be repeating groups of amino aci or the amino acids in the various groups may vary provided that in ea group of the at least four groups of amino acids there are t hydrophobic and two hydrophilic amino acids as hereinabove noted. Thus, in a preferred embodiment, the biologically active polypepti comprises a chain including at least four groups of amino acids, ea containing four amino acids. Two of the four amino acids in each gro are hydrophobic, at least one amino acid is basic hydrophilic, and t remaining one is basic or neutral hydrophilic, with the polypeptide ch preferably having at least 20 amino acids but no greater than 50 am acids . In one embodiment, each of the at least four groups of amino ac which are in the peptide chain is of the sequence A-B-C-D, B-C-D- C-D-A-B or D-A-B-C wherein A and B are hydrophobic amino acids, of C or D is a basic hydrophilic amino acid, and the other of C or D basic or neutral hydrophilic amino acid. The resulting polypeptide cha therefore, may have one of the following sequences: (X 1 ) a (A-B-C-D) n (Y 1 ) b (X 2 ) a (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- Is D; C-D- or B-C-D- , Y. is -A or -A-B or -A-B-C X- 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- 3 is -C , -C-D, -C-D-A X 4 is C- , B-C- , A-B-C- 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 ae between the hereinabove noted groups of four amino acids provided t the spacing between such groups and the charge on the amino acids d not change the characteristics of the peptide chain which prov amphiphilicity and a positive charge and do not adversely affect folding characteristics of the chain to that which is significantly differ from one in which the hereinabove noted group of four amino acids not spaced from each other. As representative examples of peptides in accordance with 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 chain. For example, the chains may have a Ser-Lys sequence before "Ala" end, and/or an Ala-Phe sequence after the "Lys" end. Other am acid sequences may also be attached to the "Ala" and/or the "lys" end. Similarly, in any polypeptide chain having at least four groups amino acids of the sequence as described above, the chain may have , example, a C-D sequence before the first A-B-C-D group . Also ot amino acid sequences may be attached to the "A" and/or the "D" end one of these polypeptide chains. Also there may be amino acids in 10 chain which space one or more groups of the hereinabove noted fou amino acids from each other. The peptides may be produced by known techniques and obtained i substantially pure form. For example, the peptides may be synthesize on an automatic synthesizer. Journal of American Chemical Society, Vol 85 Pages 2149-54(1963) . It is also possible to produce such peptides b genetic engineering techniques. In accordance with another preferred embodiment, the peptid employed in conjunction with an antibiotic which inhibits DNA gyrase ma be a magainin peptide. A magainin peptide is either a magainin such as magainin I, II or II or an analogue or derivative thereof . The magainin peptides preferabl include the following basic peptide structure X. - " R I R IΓ R I2 " R I3 " R IΓ R I4 " R I2 " R I R 14 *R 12 " R ll " R ir R ir R 14a " (R 15 ) n "R 14a "R 14 " wherein R., , is a hydrophobic amino acid, R- 2 is a basic hydrophili amino acid; R- 3 is a hydrophobic, neutral hydrophilic, or basi hydrophilic amino acid; R j . and R^ 4fl are hydrophobic or basi hydrophilic amino acids; R χg is glutamic acid or aspartic acid, or hydrophobic or a basic hydrophilic amino acid, and n is 0 or 1. In preferred embodiment, R 13 is a hydrophobic or neutral hydrophilic amin acid, R χ4 is a hydrophobic amino acid, and R ιg is glutamic acid o aspartic acid. Thus, for example, a magainin peptide may include the followin structure: "Y 12 "X 12 " 3 11 where X.. Is the hereinabove described basic peptide structure a 12 is (i) R 12 W R 14a- R 12 ( iv ) Rι 4 - R i R l4a- R 12 where R.., - 2 , R.. and 14a 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 1.- is: (i) R. β where R. g is a basic hydrophilic amino acid or asparagi or glutamine. (ii) Ri 6 "R* i 7 where R.„ is a neutral hydrophilic amino acid, hydrophobic amino acid, or a basic hydrophilic amino acid. Preferabl R 17 is a neutral hydrophilic amino acid. A magainin peptide may also have the following structure: where X 12 , Y 12 and Z χ2 are as previously defined and a is 0 OF and b is 0 or 1. The magainin peptides may also include the following basic peptid structure < 3 : "R 14 "R ir R 14a ~R 12 "R lf R lf R 12 "R 13 ~ R i R 14 'R 12- R i R H "R 12 " ' Wherβin R 11' R 12' R 13' R 14' and R 14a ar amino acids as hereinabove described. 12 The magainin peptide may also include the following structu X 13~ Z 13' wnerein x i3 is the hereinabove described basic pepti structure and Z 13 is R ll)n-( R ll^n-( R ll n-( R 14a)n^ R 15)n^ R 14a)n-< R 14)n-( R 16)n- (R 17 ) n wherein R.^, R 14 , B- 14a , R 15 R lg , and R j - are as hereinabo 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 aci and may include up to forty amino acids. A magainin peptide prefera has 22 or 23 amino acids. Accordingly, the hereinabove described ba peptide structures of a magainin peptide may include additional ami acids at the amino end or at the carboxyl end, or at both ends . As representative examples of such magainin peptides, there may mentioned peptides having the following primary sequence (expressed as single letter code) as well as appropriate analogues and derivativ thereo : (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 t 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 ) 13 Magainin peptides are described in Proc . Natl. Acad Sci. Vol. 84 p 5449-53 (Aug. 87) . The term "magainin peptides" as used herein refe to the basic magainin structure as well as derivatives and analo thereof , including but not limited to the representative derivatives analogs . In accordance with a further embodiment, the peptide employed i conjunction with an antibiotic which inhibits DNA gyrase may be a PG peptide or an XPF peptide . A PGLa peptide is either PGLa or an analogue or derivative thereo The PGLa peptides preferably include the following basic pepti structure Λ ' - R 11 -R 17 -R 12 - R I R I4- R I4- R I R ιι "R i4 ~R i2 "R ι R n "R i2 "R ι R 11 ' R 11 ' R 12 ' where R- , , R 12 , R j . , and R 1? are as previously defined. The PGLa peptides generally include at least seventeen amino acid and may include as many as forty amino acids. Accordingly, th hereinabove described basic peptide structure for a PGLa peptide ma include additional amino acids at the amino end or at the carboxyl end o at both the amino and carboxyl end. Thus, for example, a PGLa peptide may have the followin structure: ~ Y 14~ X 14 " where X« 4 is as previously defined and 14 where R- . and R« . are as previously defined. For example, a PGLa peptide may also have the following structure: ~X 14 ~ Z 14 ~ where X 14 is as previously defined; and Z. . is: (I) R u ; or (ii) R n- R n where R.. . is as previously defined. A PGLa peptide may also have the following structure: fY 1 ~X 14 " ^ 14) ^ x 14 a ~ - ~ - b where X- . ; Y- 4 and Z. . 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 lβ : -R 11 -R 17 -R 12 -R n -R 14 -R 18 -R 17 - R ll " R 14 "R 12 "R i R ir R 12 " R n -R n - Rn-Ru-CRiβ 11 !! " " ' Wherβin R ll' R 12 ' R 14' R 15 and R 17 are as P revlous* *y defined and R lg 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. 1 4453 15 Thus, for example, an XPF peptide may include the followi structure : "Y 16 ~X 16 ~ where X lg is as previously defined and Y lg is (i) R n or (ii) R 14 - n where R-- and R-. are as previously defined. An XPF peptide may include the following structure: "X 16 "Z 16 " where X^ g is as previously defined and Z. g is (i) R n ; or (ii) R n "R i8 ; or (iii) R- j^ -R.g-Proline; or (iv) R 11 -R lg -Proline-R 12 An XPF peptide may also have the following structure: (Y lg ) a - X 16< Z 16> b where X. g , Y- g and Z. g are as previously defined: a is 0 or 1 an b is 0 or 1. Preferred are XPF or PGLa peptides, which are characterized by th following primary amino acid sequence (single letter amino acid code) : PGLa: GMASKAGAIAGKIAKVALKAL (NH 2 ) XPF : GWASKIG TLGKIAKVGLKELIQPK 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 e al J. Biol. Chem. 261:5341-5349, 1986; and Giovannini et al, Biochem J 243:113-120, 1987. 16 In accordance with yet another embodiment, the peptide employed i conjunction with an antibiotic which inhibits DNA gyrase may be a CP peptide or appropriate analogue or derviative thereof . CPF peptides as well as analogues and derivatives thereof are herei sometimes referred to collectively as CPF peptides. The CPF peptide is preferably one which includes the followin peptide structure X 3Q : ~ R 2l "R 2l "R 22 "R 22 " R 2l "R 2l " R 23 " R 2l " "R 2 R 2l " R 23 "R 2 R 2l "R 24 "R 25 " R 2r wherein R 21 is a hydrophobic amino acid; R- 2 is a hydrophobic amino acid or a basic hydrophilic amino acid; R 23 is a basic hydrophilic amino acid; and R„ - is a hydrophobic or neutral hydrophilic amino acid; and R„- 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, an 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 . 17 The CPF peptides including the above basic peptide structure m have from 1 to 4 additional amino acids at the amino end. Accordingl such preferred peptides may be represented by the structural formula: v -~ ~ *30 Λ 30 wherein X k 3» n 0 is the hereinabove described basic peptide structu and Y 3Q is (i) R 25 - , or W R 22 " R 25 ; or (iv) R 22 " R 2l " R 22 " R 25* P referablv Glycine - R 21 - 22 - R 25 - wherein R 21 > R 22 , and R 25 are as previously defined. The carboxyl end of the basic peptide structure may also ha additional amino acids which may range from 1 to 13 additional ami acids . In a preferred embodiment, the basic structure may have from 1 to additional amino acids at the carboxyl end, which may be represented follows: "X 30 " Z 30 herein X «0 is the hereinabove defined basic peptide structure and Z 3Q is 18 (vii) R 21 -R 21 -R 24 -R 24 -R 2g -Gln-Gln, wherein R 21 and R 24 are as previously defined, and R 2g is proli or a hydrophobic amino acid. Preferred peptides may be represented by the following structur formula: ( Y 30>a "X30" ( Z3θ) b wherein X 3Q , Y 3Q and Z 3Q are as previously defined and a is 0 or and b is 0 or 1. Representative examples of CPF peptides which are useful in t present invention some of which have been described in the literature a comprise the following sequences (single letter amino acid code) : ( 1 ) GFGSFLGLALKAALKIG AN ALGGAPQQ (2) GLASFLGKALKAGLKIGAHLLGGAP Q (3) GLASLLGKALKAGLKIGTHFLGGAPQQ (4) GLASLLGKALKATLKIGTHFLGGAP (5) GFASFLGKALKAALKIGANMLGGTPQQ (6) GFGSFLGKALKAALKIGANALGGAPQQ (7) GFGSFLGKALKAALKIGANALGGSPQQ (8) GFASFLGKALKAALKIGANLLGGTPQQ A review of the CPF peptides can be found in Richter, K . , Egge R. , and Kreil (1986) J. Biol. Chem. 261, 3676-3680; Wakabayashl, Kato, H. , and Tachibaba, S . (1985) Nucleic Acids Research 1 1817-1828; Gibson, B .W. , Poulter, L. , Williams, D.H. , and Maggio, J. (1986) J. Biol. Chem. 261, 5341-5349. CPF peptides which may be employed in the present invention a represented by the following (single letter amino acid code) : 19 G12S3LG4ALKA5LKIG678LGG9(10)QQ 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, L The numbered amino acids may be employed as described in a combination to provide either a basic CPF peptide structure or analogue or derivative. The term CPF peptide includes the basic pepti structure as well as analogs or derivatives thereof . In accordance with still another embodiment, the biologically acti peptide may include the following basic strucutre X 4Q : [R 41 -R 42 -R 42 -R 43 -R 41 -R 42 -R 42 ] n , wherein R 41 is a basic hydrophil amino acid, R 42 is a hydrophobic amino acid, R 43 is a neutral hydrophil or hydrophobic amino acid, and n is from 2 to 5. In one embodiment, such peptide may include the followi structure : Y 4(J -X 40 , wherein X 4Q is as hereinabove described, and Y 4Q is: (*> R 42 ; (ii) R 42 -R 42 ; (ill) R 41 - R 42 - R 42 ; 20 (iv) R 43 -R 41 -R 42 -R 42 ; ( V) R 42- R 43- R 4r R 42 "R 42 ; 0r (vi) R 42 "R 2 - R 43 - R 41 - R - R 2 » wherein R 4χ , R 42 , and R 43 are hereinabove described In accordance with another embodiment, such peptide may inclu the following structure: "40 Z "4. n 0,' wherein X4. n 0 is as hereinabove described, and Z 4.0- is: (i) R 41 ; (ii) R 41 -R 42 ; (ill) R 41 - 42 - R 42 ; (iv) R 1 - R 42 - R 2 - R 43 ; (v) R 4i- R 42 ' R 42 " R 43 "R 41 ; 0r (vi) R 41 -R 4 2- R 42- R 43 " R 4 R 42 - In accordance another embodiment, such peptide may include t following structure: (Y 40'a -X 40 -(Z 40'b, wherein Y and Z are as previously defined, a 0 or 1, and b is 0 or 1. In one embodiment, n is 3, and most preferably the peptide is of t following structure as indicated by the single letter amino acid code: [KIAGKIA] 3 . In another embodiment, n is 2, and the peptide preferably is of t following structure as indicated by the single letter amino acid code: KIA( KIAGKIA) 2 KIAG . In accordance with yet another embodiment, the biologically acti amphiphilic peptide may be a biologically active amphiphilic pepti including the following basic structure Xg Q : 21 R 4 R 42 "R 42 'R 43 "R 4l "R 42 "R 42 "R 4 R 42 "R 42 "R 42 "R 4 R 42 "R 42' wherein R 41 , R 42 and R 43 are as hereinabove described. In accordance with one embodiment, such peptide may include t following structure: 50 _ 50' w *- ιerem Xςn - s s hereinabove described, and Y 5Q is: (i) R 42 ; (ii) R 42 -R 42 ; (iii) R 41 -R 42 -R 42 ; (iv) R 43 "R 4i "R 42 "R 42 ; ( V ) R 42 "R 43 'R 4l "R 42 "R 42 ; (vi) R 42 - R 42 - R 43- R 4 R 42 - R 42 » or (vϋ) R 41 -R 42 -R 42 -R 43 -R 41 -R 42 -R 42 , wherein R 4r R 42 and R 43 a as hereinabove described. In accordance with another embodiment, such peptide may inclu the following structure: X 5θ" Z 50' wnerein X 5o s - as hereinabove described and Z 5Q is: (i) R 41 ; (iii) R 41 -R 42 - 42 ; (v) R 4i "R 42- R 42 "R 43 "R 41 ; (vi) R 41 - R 42 - R 42 - 43 - R 4 R 42 Ϊ or (vii) R 41 - R 42 - R 42 - R 4 3 - R 4 R 42- R 42' Wherein R 41' R 42 and R 43 a as hereinabove described. In accordance with yet another embodiment the peptide may inclu the following structure: 22 wherein x ------ γ are as previously defined, a 0 or 1, and b is 0 or 1. In one embodiment, the peptide is of t following structural formula as indicated by the single letter amino ac code : KLASKAGKIAGKIAKVALKAL . In another embodiment, the peptide is of the following structu formula as indicated by the single letter amino acid code: KIAGKIAKIAGOIAKIAGKIA . In still another embodiment, the peptide employed in conjunction wi an antibiotic which inhibits DNA gyrase is a cecropin. The cecroptns a analogs and derivatives thereof are described in Ann. Rev. Microbiol 19 Vol. 41 pages 103-26 , in particular p . 108 and Christensen at al PN Vol. 85 p. 5072-76, which are hereby incorporated by reference. The term cecropins includes the basic structure as well as analogu and derivatives. In yet another embodiment, the peptide employed in conjunction wi an antibiotic which inhibits DNA gyrase is a sarcotoxin . The sarcotoxi and analogs and derivatives thereof are described in Molecular Entomolo pages 369-78 in particular p. 375 Alan R. Liss Inc . (1987) , which hereby incorporated by reference. The term sarcotoxin includes the basic materials as well as analogu and derivatives. It is also contemplated that within the scope of the prese invention, that each of the amino acid residues of the biologically acti amphiphilic peptide structures hereinabove described is a D- mino a residue or a glycine residue. In another embodiment, an ion channel -forming protein may be use in conjunction with an antibiotic which inhibits DNA gyrase . Io channel- forming proteins which may be employed include defensins, als known as human neutrophil antimicrobial peptides (HNP) , major basi protein (MBP) of eosinophils, bactericidal permeability -increasing protei (BPI) , and a pore-forming cytotoxin called 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 ar 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 b reference . The term ion channel -forming proteins includes the basic structure of the ion -forming proteins as well as analogues and derivatives. The present invention will be further described with respect to th following example; however, the scope of the invention is not to be limited thereby. Example 1 Approximately 1 - 5 x 10 colony forming units (CFU's) of P. aeruginosa strain 27853 or of P. aeruginosa strain 107 (which is gentamicin - resistant) dispersed in 100 ul of trypticase soy broth (TSB) were added to each of a series of test wells. Either Peptide 1, Peptide 2, or Peptide 3 was added to each test well in increasing amounts from 0.25 to 256 μg/ml in absence of or in the presence of 20% of the minimal inhibitory concentration (MIC) of ciprofloxacin. For purposes of this 24 example, Peptide 1 is amide- terminated Magainin II, Peptide 2 is of th following structural formula: [KIAGKIA] 3 ; and Peptide 3 is of the following structural formula: KLASKAGKIAGKIAKVALKAL . The MIC of ciprofloxacin alone against P. aeruginosa strain 27853 wa lμg/ml, and against P. aeruginosa strain 107 was 2 μg/ml. The MI values for Peptides 1 , 2 and 3 , either alone or in combination with 20% o the MIC of ciprofloxacin are given in Table I below. Table I 1. Peptide 1 alone 2. Peptide 1 plus 20% MIC of ciprofloxacin 3. Peptide 2 alone 4. Peptide 2 plus 20% MIC of ciprofloxacin 5. Peptide 3 alone 6. Peptide 3 plus 20% MIC of ciprofloxacin Example 2 For Examples 2 through 4, microorganisms employed in the assay were grown according to the procedure described in Stutman, et al. Antimicrobial Agents in Chemotherapy. Vol. 34, July 1990. t "4c In Examples 2 through 4, $ * organisms were subcultured on aga plates, and then grown in trypticase soy broth (TSB) , or Mueller -Hin to broth. The final assays were then conducted in microtiter plates containing Mueller- Hin ton broth 10 organisms were added to each well. The minimal inhibitory concentrations (MIC's) of ciprofloxacin alone, of Peptide 1, as hereinabove described in Example 1 , alone, of ciprofloxacin when Peptide 1 was added (ciprofloxacin + Peptide 1) , and of Peptide 1 when ciprofloxacin was added, (Peptide 1 + ciprofloxacin) were tested against various isolates of strain MR- PSA * Pseudomonas aeruginosa . The second compound (or "->-" compound) dose to establish synergy was the lowest dose of the synerglzing drug which alone lacked antibacterial effect and was at least a 50% lower dose than the MIC for the synerglzing agent alone. The results are given below in Table 2. Table 2 MIC f ug/ml) Ciprofloxacin Ciprofloxacin Peptide 1 Peptide 1 Isolate Alone +Peptide 1 Alone * Ciprofloxacin The minimal inhibitory concentrations, (MIC's) , according to the procedure of Example 2 , were tested against various isolates of strain MR- PSA, of Pseudomonas aeruginosa, except that Peptide 4 replaces Peptide 1. Peptide 4 has the following structural formula: GIGKFLKSAKKFGKAFVKIMNS . The results are given below in Table 3. Table 3 MIC f ug/ml) Ciprofloxacin Ciprofloxacin Peptide 4 Peptide 4 Isolate* Alone ÷ Peptide 4 Alone "--ci p rofloxacin 26 The minimal inhibitory concentrations (MIC's) of Peptide 4 and ciprofloxacin, either alone or in combination with each other, according to the procedure described in Example 3, were tested against various isolates of Staphylococcus aureus . The results are given in Table 4 below . Table 4 The peptide or protein and antibiotic which inhibits DNA gyrase, 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 antibiotic which inhibits DNA gyrase may be employed together in a single composition, or in separate compositions . Moreover, the antibiotic which inhibits DNA gyrase and the peptide or protein may be delivered or administered in different forms, for example, the antibiotic which inhibits DNA gyrase may be administered systemically while the peptide or protein may be administered topically. The peptide or protein and/or antibiotic which inhibits DNA gyras may be employed in a wide variety of pharmaceutical compositions i combination with a non- toxic pharmaceutical carrier or vehicle such as filler, non -toxic buffer, or physiological saline solution . Suc pharmaceutical compositions may be used topically or systemically and ma be in any suitable form such as a liquid, solid, semi-solid, injectabl solution, tablet, ointment, lotion, paste, capsule, or the like. Th peptide or protein and/or antibiotic which inhibits DNA gyrase may als be used in combination with adjuvants, protease inhibitors, or compatibl drugs where such a combination is seen to be desirable or advantageou in controlling infection caused by harmful microorganisms, in particula bacteria . The peptide(s) or protein(s) of the present invention may be administered to a host; in particular an animal, in an effective anti-microbial, in particular in an anti-bacterial amount, in conjunction with an antibiotic which inhibits DNA gyrase, for potentiating the activity of the peptide or protein. As representative examples of administering the peptide or protein and antibiotic which inhibits DNA gyrase for topical or local administration, the peptide could be administered in an amount of up to about 1% weight to weight and the antibiotic which inhibits DNA gyrase delivered in an amount of about 50 mM (about 0.1%) . Alternatively, the antibiotic which inhibits DNA gyrase could be administered topically 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 milliliter (10 milligrams per kilogram) in conjunction with a topical dose of antibiotic which inhibits DNA gyrase of from about 4 μg/ml to about 100 μg/ml. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced other than as particularly described.

Claims: WHAT IS CLAIMED IS : 1. A process, comprising: administering to a host at least one biologically active amphiphilic peptide and/or biologically active protein, said peptide or protein being an ion channel- forming peptide or protein; and a antibiotic which inhibits DNA gyrase, said components being administered to inhibit growth of a target cell 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 following basic structure X 4 _ : [ R 4r R 42 " R 42 ~R 43 ~ R 4r R 42 ~ R 42 ] n' wherein R 41 is a basic hydophillc amino acid, 42 is a hydrophobic amino acid, R 43 is a neutral hydrophilic or hydrophobic amino acid, and n is from 2 to 5. 4. The process of Claim 1 wherein the peptide inlcudes the following basic structure X. Q : R 4 R 42 "R 42 "R 43 "R 4l " R 42 "R 42 "R 4l "R 42 " R 42 " R 42 " R 41~ R 42~ R 42' hereln R 4i ls a baslc hydrophilic amino acid, R 42 is a hydrophobic amino acid, and R 43 is a neutral hydrophilic or hydrophobic amino acid. 5. The process of Claim 1 wherein said antibiotic which inhibits DNA gyrase is a quinolone antibiotic. 6. The process of Claim 5 wherein said quinolone antibiotic is ciprofloxacin . 7. The process of Claim 1 wherein said antibiotic which inhibits DNA gyrase is nalidixic acid. 8. The process of Claim 1 wherein said antibiotic which inhibits DNA gyrase is oxolinic acid. 9. The process of Claim 1 wherein said antibiotic which inhibits DNA gyrase is cinoxacin. 10. A composition, comprising: (a) at least one biologically active amphiphilic peptide and/or biologically active protein, said peptide or protein being an ion channel -forming peptide or protein; and (b) an antibiotic which inhibits DNA gyrase. 11. The composition of Claim 10 wherein said components (a) and (b) are present in an amount to inhibit growth of a target cell. 12. The composition of Claim 10 wherein the peptide is a magainin peptide. 13. The composition of Claim 10 wherein the peptide includes the following basic strucutre 40 : t R 4l "R 42 "R 42 "R 43 "R 4l "R 42 'R 42 J n wherein R 41 is a basic hydrophilic amino acid, R 42 is a hydrophobic amino acid, R.» is a neutral hydrophilic or hydrophobic amino acid, and n is from 2 to 5. 14. The composition of Claim 10 wherein the peptide inlcudes the following basic structure X gQ : R 4 R 42~ R 42~ R 43~ R 4 R 42 ~R 42~ R 4 R 42~ R 42 ~R 42 ~R 4 R 42 ~R 42' wherein R 41 is a basic hydrophilic amino acid, R 42 is a hydrophobic amino acid and R 43 is a neutral hydrophilic or hydrophobic amino acid. 15. The composition of Claim 10 wherein said antibiotic which inhibits DNA gyrase is a quinolone antibiotic. 16. The composition of Claim 15 wherein said quinolone antibiotic is ciprofloxacin . 17. The composition of Claim 10 wherein said antibiotic which inhibits DNA gyrase is nalidixic acid. 18. The composition of Claim 10 wherein said antibiotic which inhibits DNA gyrase is oxolinic acid. 19. The composition of Claim 10 wherein said antibiotic which inhibits DNA gyrase is cinoxacin . 20. The composition of Claim 13 wherein said peptide is of the following sturcture: [KIAGKIA] 3 . 21. The composition of Claim 14 wherein said peptide is of the following structure: KLASKAGKIAGKIAKVALKAL 22. The composition of Claim 12 wherein said magainin peptide is Magainin II.

Inventors: BERKOWITZ BARRY, BERKOWITZ, BARRY
Applicants: MAGAININ SCIENCES INC, MAGAININ SCIENCES INC.
Application year: 1991
Main classification: C07K7
IPC codes: A61K31, A61K38, A61P31