WO1990008552A1

WO1990008552A1 - Composition and treatment with peptide combinations

Info

Publication number: WO1990008552A1
Application number: PCT/US1990/000460
Authority: WO
Inventors: Michael Zasloff
Original assignee: Magainin Sciences Inc.
Priority date: 1989-01-30
Filing date: 1990-01-25
Publication date: 1990-08-09
Also published as: EP0455719A4; JPH04506056A; EP0455719A1; CA2007913A1; AU5042890A

Abstract

A composition comprising a magainin peptide or an analogue or derivative thereof, and at least one member selected from the group consisting of a PGLa peptide or analogue or derivative thereof, and an XPF peptide or analogue or derivative thereof. The composition is employed as a pharmaceutical.

Description

COMPOSITION AND TREATMENT WITH

PEPTIDE COMBINATIONS

This invention relates to biologically active peptides, and more particularly to compositions and uses involving combinations of biologically active peptides.

In accordance with an aspect of the present invention, there is provided a composition which includes (a) a magainin peptide or analogue or derivative thereof; and (b) at least one member selected from the group consisting of (i) a PGLa peptide or analogue or derivative thereof and (ii) an XPF peptide or analogue or derivative thereof. The magainin peptide or analogue or derivative thereof, as well as the PGLa peptide or analogue or derivative thereof and the XPF peptide or analogue or derivative thereof, may be amide terminated or

carboxy-terminated.

In accordance with another aspect of the present invention, there is provided a process which

comprises administering to a host both (a) a magainin peptide or analogue or derivative thereof and (b) at least one member selected from the group consisting of (i) an XPF peptide or analogue or derivative thereof and (ii) a PGLa peptide or analogue or derivative thereof. In one embodiment, components (a) and (b) may be administered in separate

compositions. In another embodiment, components (a) and (b) may be administered in a single composition. In addition, components (a) and (b) may be

administered in amounts effective to inhibit growth of a target.

Although the present invention is not to be limited by any theoretical reasoning, it is believed that the combination of the magainin peptide with the PGLa peptide or XPF peptide provides a synergistic effect in the inhibition of growth of a target. The target, for example, may be bacteria, fungi,

protozoa, virally infected cells, malignant cells, or sperm cells as compared to normal host cells. The synergism may be due to association of the peptides to a novel multimeric complex, such as a dimer, which possesses markedly increased membrane affinity for the target cells. The complex formed between the two peptides is believed to possess potent membrane disruptive properties. It is believed that the peptides have the capacity to organize within target cell membranes and to disturb cellular functions.

In general, the magainin peptide or analogue or derivative thereof is employed in a dosage of from about lmg to about 100mg per kilogram of host weight, when administered systemically. When administered topically, the magainin peptide is used in a

concentration of from about .05% to about .50%.

The PGLa peptide or analogue or derivative thereof, when administered systemically, is

administered in a dosage of from about lmg to about 100mg per kilogram of host weight. When administered topically, the PGLa peptide is administered in a concentration of from about 0.5% to about .50%.

The XPF peptide or analogue or derivative thereof, when administered systemically, is

administered in a dosage of from about 1mg to about 100mg per kilogram of host weight. When administered topically, the XPF peptide or analogue or derivative thereof is administered in a concentration of from about .05% to about .50%.

The use of this combination of peptides in accordance with the present invention is effective as an antibiotic, and may be employed to inhibit, prevent or destroy the growth or proliferation of microbes, such as bacteria, fungi, viruses or the like. Similarly, such compositions may be employed as an anti-viral composition to inhibit, prevent or destroy the growth or proliferation of viruses.

Such compositions may also be employed as a spermicide to inhibit, prevent or destroy the

motility of sperm.

Such compositions may also be employed as anti-tumor agents to inhibit the growth of or destroy tumors.

The compositions have a broad range of potent antibiotic activity against a plurality of

microorganisms, including gram-positive and

gram-negative bacteria, fungi, protozoa and the like. Such compositions may be employed for treating or controlling microbial infection caused by organisms which are sensitive to such composition. The

treatment may comprise administering to a host organism or tissues acceptable to or affiliated with a microbial infection an anti-microbial amount of magainin peptide or analog or derivative thereof and of PGLa peptide or XPF peptide. The compositions may also be used as

preservatives or sterilants for materials susceptible to microbial contamination.

The magainin peptide may be, for example, 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 X₁₂

-- R₁₁ -R₁₁ -R₁₂ -R₁₃ -R₁₁ -R₁₄ -R₁₂ -R₁₁ - ^R14 ^-R12 ^-R11 ^-R11 ^-R11 ^-R14a ^-(R15⁾n ^-R14a ^-R14 - wherein R₁₁ is a hydrophobic amino acid, R₁₂ is a basic hydrophilic amino acid; R₁₃ is a hydrophobic, neutral hydrophilic or basic hydrophilic amino acid;R₁₄ and R_14a are hydrophobic or basic hydrophilic amino acids; R₁₅ is glutamic or aspartic acid, or a hydrophobic or basic hydrophilic amino acid, and n is

0 or 1. In a preferred embodiment, R₁₃ is a

hydrophobic or neutral hydrophilic amino acid, R_14a is a hydrophobic amino acid, and R₁₅ is glutamic acid or aspartic acid.

The hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, Ile, Leu, Met, Val, Trp, and Tyr. The neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser, and Thr. The basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, and His, and ornithine (0).

The magainin peptides generally include at least seventeen amino acids and may also include up to forty amino acids. Accordingly, the hereinabove described basic peptide structure of a magainin peptide may include additional amino acids at the amino end or at the carboxyl end, or at both ends.

Thus, for example, a magainin peptide may include the following structure: -Y₁₂ -X₁₂- where X₁₂ is the hereinabove described basic

peptide structure and Y₁₂ is

(i) R₁₂

(ii) R_14a -R₁₂

(iii) R₁₁ -R_14a -R₁₂

(iv) R₁₄ -R₁₁ -R_14a -R₁₂

wherein R₁₁, R₁₂, R₁₄ and R_14a are as previously defined.

A magainin peptide may also have the following structure

- X₁₂ -Z₁₂- wherin X₁₂ is as previously defined and Z₁₂ is:

(i) R₁₆ where R₁₆ is a basic hydrophilic amino acid or asparagine or glutamine.

(ii) R₁₆ -R₁₇ where R₁₇ is a neutral

hydrophilic amino acid , a hydrophobic amino acid, or a basic hydrophilic amino acid. Preferably R₁₇ is a neutral hydrophilic amino acid.

A magainin peptide may also have the following structure:

(Y₁₂) a-^X12^{- ( Z}12⁾ _b where X₁₂, Y₁₂ and Z₁₂ 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 X₁₃ :

-R₁₄-R₁₁-R_14a-R₁₂-R₁₁-R₁₁-R₁₂-R₁₃-

R₁₁-R₁₄-R₁₂-R₁₁-R₁₁-R₁₂-, wherein R₁₁,R₁₂,R₁₃, R₁₄, and R_14a are amino acids as hereinabove described.

The magainin peptide may also include the

following structure X₁₃-Z₁₃; wherein X₁₃ is the

hereinabove described basic peptide structure and Z₁₃ is

(R₁₁)n-(R₁₁)n-(R₁₁)n-(R_14a)_n-(R₁₅)_n-(R_14a)_n-(R₁₄)_n-(R₁₆)_n- (R₁₇)_n wherein R₁₁, R₁₄, R_14a, R₁₅, R₁₆, and R₁₇ 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 following primary sequence (expressed as a single letter code) as well as appropriate analogues and deriatives thereof:

(a) (NH₂) GIGKFLHSAGKFGKAFVGEIMKS (OH) or (NH₂) (Magainin I)

(b) (NH₂) GIGKFLHSAKKFGKAFVGEIMNS (OH) or (NH₂) (Magainin II)

(c) (NH₂) GIGKFLHSAKKFGKAFVGEIMN (OH) or (NH₂) (Magainin III)

The following are examples of peptide

derivatives or analogs of the basic structure:

(d)(NH₂) IGKFLHSAKKFGKAFVGEIMNS (OH) or (NH₂) (e)(NH₂) GKFLHSAKKFGKAFVGEIMNS (OH) or (NH₂) (f)(NH₂) KFLHSAKKFGKAFVGEIMNS (OH) or (NH₂) 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.

The peptide employed in conjunction with the magainin peptide is 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 structure X₁₄:

^R11 ^-R17 ^-R12 ^-R11 ^-R14 ^-R14 ^-R11 - ^R11 ^-R14 ^-R12 ^-11l ^-R11 ^-R12 ^-R11 - ^R11 ^-R11 ^-R12 - where R₁₁, R₁₂, R₁₄, and R₁₇ 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 sturcture 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 sturcture:

-Y₁₄ -X₁₄- where X₁₄, is as previously defined and

Y₁₄ is

(i) R₁₁;

(ii) R₁₄ -R₁₁

where R₁₁ and R₁₄ are as previously defined. For example, a PGLa peptide may also have the following structure:

-X₁₄ -Z₁₄- where X₁₄ is as previously defined; and Z₁₄ is

(i) R₁₁; or

(ii) R₁₁-R₁₁, where R₁₁ is as previously defined.

A PGLa peptide may also have the following structure: (Y₁₄)_a-X₁₄-(Z₁₄)_b

where X₁₄; Y₁₄ and Z₁₄ are as previously defined, a is 0 or 1 and b is 0 or 1. An XPF peptide is either XPF or an analogue or derivative thereof. The XPF like peptides preferably include the following basis peptide structure X, 16 :

R₁₁-R₁₇-R₁₂-R₁₁-R₁₄-R₁₈-R₁₇-

R₁₁-R₁₄-R₁₂-R₁₁-R₁₁-R₁₂-

R₁₁-R₁₁-R₁₁-R₁₂-(R₁₅)_n-R11--,

wherein R₁₁, R₁₂, R₁₄, R₁₅ and R₁₇ are as previously defined and R₁₈ is glutamine or

asparagine, or a basic hydrophilic, or hydrophobic amino acid, and n is 0 or 1.

The XPF peptides generally include at least nineteen amino acids and may include up to forty amino acids. Accordingly, the hereinabove described basic peptide structure of XPF may include additional amino acids at the amino end, or at the carboxyl end or at both the amino and carboxyl ends.

Thus, for example, an XPF peptide may include the following structure:

-Y₁₆ -X₁₆- where X₁₆ is as previously defined and Y₁₆ is

(i) R₁₁ or

(ii) R₁₄ -R₁₁

where R₁₁ and R₁₄ are as previously defined.

An XPF peptide may include the follwing

structure:

-X₁₆ - Z₁₆- where X₁₆ is as previously defined and Z₁₆ is

(i) R₁₁; or

(ϋ) R₁₁ -R₁₈; or

(iϋ) R₁₁ -R₁₈ -Proline; or

(iv) R₁₁ -R₁₈ -Proline -R₁₂

An XPF peptide may also have the followingstructure:

(Y₁₆)_a -X₁₆ (Z₁₆)_b where X_16, Y₁₆ and Z₁₆ 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 (NH₂)

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 Giovanni et al,

Biochem J. 241:113-120, 1987.

The present invention will be further described with respect to the following examples, however, the scope of the present invention is not to be limited thereby.

EXAMPLE 1

About 10 bacteria/ml were added to a small volume of Luria broth. Magainin 2 (MGN2), PGLa peptide, or a mixture of MGN2 and PGLa in a 1:1 molar ratio was added to the broth in increasing

concentration. The minimal inhibitory concentration (MIC), which inhibits microbial growth completely at 24 hours is noted below in Table 1.

TABLE 1

ANTIBACTERIAL ACTIVITY OF COMBINATION OF PGLa AND MAGAININ 2

ORGANISM MINIMAL INHIBITORY CONCENTRATION (μg/ml)

MGN2 PGLa PGLa/MGN2

(1:1 molar ratio)

S. aureus >500 >500 10

P. aeruginosa 250 250 10

C. albicans 250 240 10

Micrococcus 125 125 10

Diphtheroids 125 125 10

E. coli 50 50 10

The above results indicate an increase in antibacterial activity when a combination of Magainin 2 and PGLa peptides is added to a culture broth of bacteria in a 1:1 molar ratio of Magainin 2 to PGLa over either peptide added alone.

EXAMPLE 2

In this example, the concentration of S. aureus killed by 100 μg/ml of preparations of Magainin 2, PGLa, and a preparation of Magainin 2 and PGLa in a 1:1 molar ratio is noted. It was found that a 100 μg/ml preparation of Magainin 2 does not completely kill S. aureus at concentrations of bacteria at less than 10 bacterial/ml. Similar results were also obtained for PGLa. The 100 μg/ml preparation of the equimolar miture of Magainin 2 and PGLa, however, achieved complete killing of a concentration of 10⁵ bacteria/ml. Thus, the equimolar mixture of Magainin 2 and PGLa achieved an increase in bactericidal potency over either peptide by greater than 10⁵.

EXAMPLE 3

Approximately 10³ S. aureus bacteria were added per/ml of Luria broth. Peptide preparations

containing varying molar ratio amounts of PGLa to

Magainin 2 (mole PGLa/mole MGN2) were added to the broths to increasing concentration of peptide

(μg/ml). The microbial inhibitory concentration

(MIC), at which point no growth was evident after 24 hours was measured for each peptide preparation. The results are given in Table 2 below.

TABLE 2

Mole PGLa/

Mole MGN2 MIC vs. S. aureus (μg/ml)

10/0 >500

10/1 25

7.5/2.5 15

5/5 8

2.5/7.5 15

1/10 25

0/10 >500

The above results indicate that maximum

bactericidal activity is achieved when an equimolar mixture of PGLa and Magainin 2 is added to the S.

aureus culture. It is also shown, however, that preparations containing both peptides in any molar ratio achieved greater bactericidal activity than either peptide alone.

The peptide combinations, in accordance with the present invention, may be employed for treating a wide variety of hosts. In accordance with a

preferred embodiment, a host may be an animal, and such animal may be a human or non-human animal. The magainin peptide and the PGLa and/or XPF peptide may be employed together in a single composition, or in separate compositions.

The magainin peptide and PGLa and/or XPF peptide may be employed in a wide variety of pharmaceutical compositions in combination with a non-toxic

pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution. Such pharmaceutical compositions may be used

topically or systemically and may be in any suitable form such as a liquid, solid, semi-solid, injectable solution, tablet, ointment, lotion, paste, capsule, or the like. It is also contemplated that the magainin peptide and the PGLa and/or XPF peptide may be delivered or administered in different forms. The magainin peptide and PGLa and/or XPF peptide 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

microorganisms including protozoa viruses, and the like.

The peptide(s) of the present invention may be administered to a host; in particular an animal, in an effective antibiotic and/or anti-tumor and/or anti-viral and/or anti-microbial and/or a spermicidal amount.

Numerous modifications and variations of the present invention are possible in light of the above teachings, and, therefore, within the scope of the accompanying claims, the invention may be practiced other than as particularly described.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising:

(a) a magainin peptide or analogue or

derivative thereof; and

(b) at least one member selected from the group consisting of (i) a PGLa peptide or analogue or derivative thereof and (ii) an XPF peptide or analogue or derivative thereof.

2. The composition of Claim 1 wherein

components (a) and (b) are present in an amount to inhibit growth of a target cell.

3. The composition of Claim 1 wherein

component (b) is a PGLa peptide or analogue or derivative thereof.

4. The composition of Claim 1 wherein

component (b) is an XPF peptide or analogue or derivative thereof.

5. A process, comprising:

administering to a host both (a) a magainin peptide or analogue or derivative thereof and (b) at least one member selected from the group consisting of (i) an XPF peptide or analogue or derivative thereof and (ii) a PGLa peptide or analogue or derivative thereof.

6. The process of Claim 5 wherein components

(a) and (b) are administered in separate

compositions.

7. The the process of Claim 5 wherein (a) and

(b) are administered in a single composition.

8. The process of Claim 5 wherein (a) and (b) are administered in amounts effective to inhibit growth of a target.

9. The process of Claim 8 wherein the magainin peptide or analogue or derivative thereof is

administered systemically.

10. The process of Claim 9 wherein the magainin peptide or analogue or derivative thereof is

administered in an amount of from about lmg to about 100mg per kilogram of host body weight.

11. The process of Claim 8 wherein the magainin peptide or analogue or derivative thereof is

administered topically.

12. The process of Claim 11 wherein the

magainin peptide or analogue or derivative thereof is administered in a concentration of from about .05% to about .50%.

13. The process of Claim 8 wherein component (b) is a PGLa peptide or analogue or derivative thereof.

14. The process of Claim 13 wherein the PGLa peptide or analogue or derivative thereof is

administered systemically.

15. The process of Claim 14 wherein the PGLa peptide or analogue or derivative thereof is

16. The process of Claim 13 wherein the PGLa peptide or analogue or derivative thereof is

administered topically.

17. The process of Claim 16 wherein the PGLa peptide or analogue or derivative thereof is

administered in a concentration of from about .05% to about .50%.

18. The process of Claim 8 wherein component (b) is an XPF peptide or analogue or derivative thereof.

19. The process of Claim 18 wherein the XPF peptide is administered systemically.

20. The process of Claim 19 wherein the XPF peptide or analogue or derivative thereof is administered in an amount of from about 1mg to about 100mg per kilogram of host body weight.

21. The process of Claim 18 wherein the XPF peptide or analogue or derivative thereof is

administered topically.

22. The process of Claim 21 wherein the XPF peptide or analogue or derivative thereof is

administered in a concentration of from about .05% to about .50%.

23. The process of Claim 8 wherein said target is a bacterium.

24. The process of Claim 8 wherein said target is a fungus.

25. The process of Claim 8 wherein said targetis a protozoan.

26. The process of Claim 7 wherein said targetis a tumor cell.

27. The process of Claim 8 wherein said targetis a virally-infected cell.

28. The process of Claim 8 wherein said targetis a sperm cell.