JP2001510164A - A bioactive peptide having reduced toxicity to animals and a method for preparing the same. - Google Patents

Abstract

(57) Summary The present invention relates to bioactive peptides with reduced toxicity and methods for preparing them. The peptide of the present invention is an unsubstituted peptide having the formula (I) or an N-terminal substituted peptide, wherein X is a biologically active / amphiphilic ion channel forming peptide or protein, and T is a lipophilic group or Is hydrogen and W is T or hydrogen. Preferably, T is of the formula (II), wherein R is a hydrocarbon having at least 2 and no more than 10 carbon atoms (alkyl, aromatic or alkylaromatic). T is preferably an octanoyl group. The peptides or proteins of the invention have improved antimicrobial and antitumor activity while exhibiting reduced toxicity. A preferred method of reducing toxicity consists of forming the relevant methanesulfonic acid derivative or analog. In addition, the compounds of the invention are used for treating pulmonary infections such as those that occur in sepsis, septic shock, and cystic fibrosis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to bioactive peptides. More particularly, the present invention relates to bioactive peptides with reduced toxicity. Furthermore, the present invention relates to methods for modifying bioactive peptides, which are substituted or unsubstituted at the N-terminus (amino terminus), to reduce their toxicity to animals. RELATED APPLICATION DATA This application is filed on June 1, 1992, and has been abandoned US patent application Ser.
US Patent Application Serial No. 08 / 184,462, filed January 18, 1994, which is a continuation-in-part of 7 / 891,201, is a divisional application of US Patent Application Serial No. 08 / 184,462.
U.S. patent application Ser. No. 08/893, filed Jul. 15, 1997, which is a continuation-in-part of U.S. patent application Ser. No. 08 / 465,330 filed Jun. 5, 1995.
No. 006 is a continuation-in-part application. Both of these U.S. patents and patent applications are:
Which is fully incorporated herein by reference.

[0002]

[Means for Solving the Problems]

According to one aspect of the present invention, there is provided an unsubstituted bioactive peptide or protein. In another aspect, the following formula: Where X is a biologically active peptide or protein and N is a nitrogen atom
An N-terminally substituted peptide or protein having the formula: The peptide or protein is preferably an ion channel forming peptide or protein. T is a lipophilic group and W is another T group or hydrogen.

[0003]

Embodiment

As used herein, "lipophilic" means a lipophilic group that enhances the interaction of a peptide or protein with a lipid membrane, such as a cell membrane. Lipophilic groups that can be used include, but are not limited to, any group that can be placed at the N-terminus of a peptide by a condensation reaction with nitrogen. The lipophilic group T is, for example,
It is a carboxylic acid, phosphoric acid, preferably alkylphosphoric acid, phosphonic acid, preferably alkylphosphonic acid, sulfonic acid, preferably alkylsulfonic acid, or an alkyl group. Preferably, T is Wherein R is a hydrocarbon group having at least 2 and not more than 16 carbon atoms.

[0004] In certain embodiments, R is an alkyl group. The alkyl group is a straight or branched chain alkyl group or a cycloalkyl group. For example, R is CH ₃ (
CH _{2) n} - is (wherein, n is 1 to 14). Preferably, n is from 3 to 12, more preferably from 4 to 11, even more preferably from 6 to 11, and most preferably n is 6, where T is an octanoyl group.

[0005] In another embodiment, R is an aromatic (including phenyl and naphthyl) or an alkyl aromatic group. For example, R is Where z is from 0 to 6. Preferably, z is 1 or 2.

In another embodiment, R is (Where n is from 1 to 5). Preferably, n is 1, where R
Is an ibu profile group.

In yet another embodiment, T is: Where X is from 1 to 14. Preferably, X is 2 and T is a succinyl group.

[0008] In another embodiment, T is: Where Y is from 1 to 14. Preferably, Y is 12, where T is a sphingosine group.

In yet another embodiment, T is: Wherein X and Y are as described above. Preferably, X is 2 and Y is 12. In certain embodiments, W is hydrogen.

[0010] When the biologically active peptide is substituted at the N-terminus as described above, we compare such a peptide with an unsubstituted peptide or a peptide with an N-terminally substituted acetyl group. Have been found to have enhanced biological activity against target cells, viruses, and virus-infected cells. Applicants have further found that the N-terminal substitutions described above significantly enhance the biological activity of "short" peptides, ie, peptides having 14 or fewer amino acid residues. In another embodiment of the present invention, the bioactive peptide is not an N-terminal substitution and has a broad spectrum of antitumor and antimicrobial activity.

As already mentioned, the bioactive peptides or proteins of the present invention are preferably ion channel forming peptides. An ion channel forming peptide or protein or ion permeable carrier refers to a peptide or protein that enhances the permeability of ions through a natural or synthetic lipid membrane. Christensen et al. (
B. Christensen et al. , PNAS, Vol. 85, p. 5
072-5076 (July 1988)) describes a methodology to indicate whether a peptide or protein has ion channel forming properties and is, therefore, an ionophore. As used herein, an ion channel forming peptide or an ion channel forming protein refers to a peptide or protein having an ion channel forming property measured by the method of Christensen et al. This Christensen article is fully incorporated herein by reference.

An amphipathic peptide or protein is a peptide or protein that contains both hydrophobic and hydrophilic peptide or protein regions.

The ion channel forming peptides used in the present invention are generally water soluble at a neutral pH in water at a concentration of at least 20 mg / ml. In addition, the structure of such peptides provides the flexibility of the peptide molecule. Such peptides can form an α-helical structure. When this peptide is placed in water, it does not adopt an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain itself folds into a rodlike structure.

Generally, such peptides will have at least 7 amino acids, and often will have at least 20 amino acids. In most cases, such a peptide would have 4
It has no more than zero amino acids.

[0015] The peptide and / or analog or derivative thereof is administered to a host, eg, a human or non-human animal, in an amount effective to inhibit the growth of target cells, viruses, or virus-infected cells. Thus, for example, the peptides and / or analogs or derivatives thereof can be used as antimicrobial, antiviral, antibacterial, antitumor, antiparasitic, spermicidal, and other agents that exhibit biological function.

The term “antimicrobial” as used herein means that the polypeptide or protein of the present invention inhibits, prevents or destroys the growth or proliferation of microorganisms such as bacteria, fungi, and viruses. As used herein, the term “antibacterial” refers to the normal activity of a bacterium, including the death of a bacterium or the destruction and protection of its growth or proliferation when the peptide or protein used in the present invention comes into contact with the peptide or protein. Has the effect of countering biological functions. As used herein, the term "antibiotic" refers to a peptide or protein used in the present invention that kills or prevents the growth or proliferation of a non-host cell, tissue or organ when contacted with the peptide or protein. Means to reverse the normal biological function of the non-host cell, tissue or organ. As used herein, "spermicide" means that the peptide or protein used in the present invention suppresses, prevents, or destroys sperm motility. As used herein, "antifungal" means that the peptide or protein used in the present invention suppresses, prevents, or destroys fungal growth or proliferation. As used herein, "anti-virus" means that the peptide or protein used in the present invention suppresses, prevents, or destroys the growth or proliferation of the virus or cells infected with the virus. As used herein, the term “anti-tumor” means that the peptide or protein of the present invention suppresses or destroys the growth of tumors including cancerous tumors. The term “anti-parasite” as used herein means that the peptide or protein used in the present invention suppresses, prevents, or destroys the growth or proliferation of a parasite.

The peptides or proteins of the present invention have broad and potent antibiotic activity against multiple tumor types, Gram-positive and Gram-negative bacteria, fungi, protozoa, and the like, as well as microorganisms, including parasites. The peptides or proteins of the present invention provide a method for treating or controlling tumor growth and microbial infection caused by an organism susceptible to the peptide or protein. Such treatment comprises administering to a host organ or tissue susceptible to or infected by a microbial infection an antitumor or antimicrobial amount of at least one peptide or protein. In another embodiment of the present invention, there is provided a method of reducing the toxicity of an unmodified or N-terminally modified peptide to a host without reducing the antitumor or antimicrobial activity of the peptide. This method has the following formula: Wherein X is a bioactive peptide or protein, wherein the peptide is an ion channel forming peptide or protein, and T is a lipophilic group or hydrogen, or a methanesulfonic acid of an N-terminally substituted peptide having Forming a derivative or analog. The antitumor or antimicrobial activity of the methanesulfonic acid derivative or analog of the unsubstituted or N-terminally substituted peptide is not reduced or significantly reduced as compared to the corresponding peptide not induced by the methanesulfonic acid group. The term "not significantly reduced", as used herein, means that the methanesulfonic acid derivative or analog retains the anti-tumor or anti-microbial activity of the non-induced compound. Preferably, the methanesulfonic acid derivative or analog retains at least 50%, preferably 75%, of the antitumor or antimicrobial activity of the non-induced compound.

In one embodiment of the present invention, the methanesulfonic acid derivative is prepared by suspending the free base of an unsubstituted or N-terminal substituted peptide in water, and then suspending the suspended peptide and each free amino group of the peptide. At least 0.5 equivalents of sodium bisulfite-formaldehyde complex (or other suitable sodium bisulfite-formaldehyde complex). The reaction proceeds for a period of 10 minutes or more to produce a methanesulfonic acid derivative or analog of the peptide. The amount of the bisulfite-formaldehyde complex can be varied without departing from the invention. The suspended free base is preferably mixed with 0.5 to 10 equivalents of bisulfite-formaldehyde complex for each free amino group of the peptide. 1
The use of from 1 to 5 equivalents is preferred, with 1.1 to 3 equivalents being particularly preferred.

In the process according to the embodiment of the present invention, the mixing period can be greatly changed. Preferably, the peptide free base and complex are mixed for a period of 10 minutes to 2 hours, or 10 minutes to 1 hour. Mixing periods in the range from 15 minutes to 30 minutes are particularly preferred.
The starting free base of an unsubstituted or N-terminally substituted peptide can be prepared by neutralizing the peptide salt. It can be neutralized by treating the salt with a basic solution such as a sodium carbonate solution. After neutralization, the free base peptide probably precipitated,
Can be separated before suspending in water. After the reaction procedure is completed, the methanesulfonic acid derivative or analog of the peptide product can be recovered, for example, by filtration. Further, the product can be lyophilized, if desired.

One method according to an embodiment of the present invention can be summarized by the following reaction scheme: Wherein X is a biologically active peptide or protein, N is a nitrogen atom,
T is a lipophilic group or hydrogen). Preferably, the peptide or protein is an ion channel forming peptide or protein. In another embodiment of the present invention, the methanesulfonic acid derivative is prepared by mixing the peptide salt with 12.5 equivalents of a 30% formaldehyde solution and 6.25 equivalents of a 1 M sodium bicarbonate solution for each amino group of the peptide. It is formed by doing.
The adduct of the peptide with formaldehyde precipitates and can be recovered by centrifugation or filtration. The sodium methanesulfonate derivative is then formed by mixing the formaldehyde adduct with sodium metabisulfite. It is clear that the methanesulfonic acid analog of the peptide has reduced toxicity in vivo, but its antitumor and antimicrobial activity is retained. Due to the antibiotic, antimicrobial, antiviral and antibacterial properties of the peptides or proteins, they can also be used as preservatives or as sterilants or disinfectants for substances susceptible to microbial or viral contamination.

The peptide or protein and / or derivative or analog thereof can also be administered in combination with a non-toxic pharmaceutical carrier or vehicle, such as an excipient, a non-toxic buffer, or saline solution. Such pharmaceutical compositions can be used topically or systemically and can be liquids, solids, semi-solids, injections, tablets, ointments, lotions,
It may be in any suitable form, such as a paste, capsule, or the like. Peptides or proteins may be used as adjuvants, protease inhibitors, if such combinations are deemed desirable or advantageous in controlling infections caused by harmful microorganisms, including protozoa, viruses, etc., as well as parasites. Alternatively, they can be used in combination with compatible drugs.

[0022] The peptide or protein of the present invention may be an antibiotic and / or an antitumor and / or
Or an antifungal and / or antiviral and / or antimicrobial and / or antibacterial and / or antiparasitic and / or spermicidally effective amount administered to a host, especially a human or non-human animal. Depending on the intended use, the composition of the invention may have an antimicrobial effective amount and / or a spermicidally effective amount and / or a fungicidally effective amount and / or an antiviral effective amount and / or an antitumor effective amount and / or Or an antiparasitic and / or antibiotic effective amount of a peptide or protein of the invention having such activity.
Include one or more. The peptide or protein may be administered directly to the target cell or virus or virus infected cell, or may be applied indirectly by systemic administration. The peptides or proteins of the invention can also be used to promote or stimulate the healing of host wounds.

As used herein, “wound healing” includes various aspects of the wound healing process. These aspects include increased wound contraction, for example, increased connective tissue deposition as evidenced by increased deposition of collagen on the wound, and increased wound tensile strength (ie, peptide or protein is (Increasing the breaking strength). The peptides or proteins of the invention can also be used to counteract the inhibition of wound healing caused by conditions that suppress or compromise the immune system.

The peptides or proteins of the present invention can be used for treating external burns, and for treating and / or preventing skin and burn infections. In particular, peptides or proteins are P. aeruginosa and S. aeruginosa. Aureus can be used to treat skin and burn infections caused by organisms such as, but not limited to.

The peptide or protein is also useful for preventing or treating infections of the eye. Such infections are described in aeruginosa, S .; aureus and N.A. go
bacteria such as, but not limited to, C. norrhoeae; albic
and ans. fungi, such as, but not limited to, A. fumigatus; caused by parasites, such as, but not limited to, castellani, or viruses.

The peptides or proteins are also useful for killing cysts, spores, or vegetative forms of organisms that cause infection. Such organisms may be vegetative or cyst-forming. C. canthamoeba, C. spores. albicans and also A. albicans that form spores. fumigatus, but is not limited thereto. The peptides or proteins of the present invention can also be used for treating tumors. In general, they are active against tumors arising in tissues and organs such as, but not limited to, breast, lung, colon, rectum, cervix, ovary, prostate, stomach, and melanoma, and leukemia. More specifically, they are active against non-small cell lung cancer and adenocarcinoma of the breast, cervix, prostate, lung, colon, rectum, stomach and ovary, for example. Further, the peptides or proteins of the present invention are active against tumors that are resistant to other anti-tumor agents. The clear reason for resistance, the removal of antineoplastic agents by efflux pumps, does not appear to apply to the peptides and proteins of the invention, which are thought to act by a unique mechanism of forming ion channels.

The peptides or proteins can also be administered to plants in an antimicrobial or antiviral or antiparasitic effective amount to prevent or treat plant contamination by microorganisms or viruses or parasites. The peptides or proteins of the invention can also be used to treat severe lung infections. In particular, the use of these peptides or proteins can include, but is not limited to, P. cystic fibrosis patients. Pulmonary infections caused by microorganisms such as areruginosa can be treated. Generally, in treating such infections, the peptide or protein will be administered to a patient in need of treatment by inhalation. For example, the active peptide or protein component may be nebulized, metered dose inhaler (aspirator) (
(MDI), or by inhalation using dry powder inhaler (DPI). When using such a configuration by inhalation, the active peptide or protein is present in the following amounts. That is, a solution between 5 and 200 mg / ml for a nebulizer, and between 0.5 and 45 mg for an MDI or DPI. The peptides or proteins can also be administered to patients to treat sepsis, septic shock, and other related disorders, as such peptides neutralize bacterial endotoxins. Generally, peptides or proteins are positively charged, while bacterial endotoxins are generally negatively charged. Peptides or proteins are particularly useful in that such compounds neutralize bacterial endotoxins without neutralizing essential proteins in plasma, such as heparin.

The terms “treat,” “treated,” or “treatment,” as used herein, may mean the complete elimination of a disease, chronic illness, or symptom, May mean reducing, controlling or ameliorating the severity of a chronic disease or condition. When used as a topical composition, the peptide or protein is generally present at at least 0.1% by weight. In most cases, it is not necessary to use more than 2.0% by weight.

When such compositions are used systemically (intramuscularly, intravenously, intraperitoneally), the active peptide or protein is present in an amount that achieves a serum concentration of at least about 5 μg / ml peptide. Generally, the serum concentration of the peptide or protein is 50
It need not exceed 0 μg / ml. A preferred serum concentration is about 100 μg / ml. Such serum concentrations may range from 1 to about 10 mg / kg in the composition to be administered systemically.
Is achieved by including a peptide or protein at a dosage of. In general,
The peptide or protein need not be administered at a dose greater than 100 mg / kg.

[0030] The peptide or protein is produced using known methods and is obtained substantially purely. For example, peptides are synthesized on an automatic peptide synthesizer. Journal
al of the American Chemical Society,
Vol. 85, p. 2149-54 (1963), which is fully incorporated herein by reference. Such a peptide or protein can also be produced by genetic engineering techniques. Codons encoding particular amino acids are known to those of skill in the art, and thus DNA encoding the peptide can be constructed by any suitable method, and such DNA can be cloned into an appropriate expression vehicle (eg, a plasmid), This can be transfected into an appropriate organism to express the peptide or protein.

In one embodiment of the present invention, after producing or synthesizing a peptide or protein, the N-terminus (NH ₂ or amino terminus) of the peptide is reacted to form the N-terminus of the peptide.
A lipophilic group is attached to the end. For example, this reaction is a condensation reaction with an amine. Lipophilic group T is Wherein the N-terminus is reacted with a carboxylic acid of the formula R-COOH, wherein R is a hydrocarbon having at least 2 carbon atoms. The reaction is carried out in the presence of a coupling agent such as DCC or DIC, and HOBT, or in the presence of an acid chloride. Such a reaction results in the formation of an N-terminal substituted peptide or protein having the above structural formula.

In one embodiment, X is a basic (X) having at least 16 amino acids.
(Positively charged) polypeptide, wherein the polypeptide has at least 8 hydrophobic amino acids and at least 8 hydrophilic amino acids. More specifically,
The hydrophobic amino acids are in groups of two adjacent amino acids, each group of two hydrophobic amino acids being at least one amino acid other than the hydrophobic amino acids from the other group of two hydrophobic amino acids. Separated by (preferably at least two amino acids) and generally by no more than four amino acids, the amino acids between a pair of hydrophobic amino acids may or may not be hydrophilic.

[0033] Hydrophilic amino acids also generally fall into the group of two adjacent amino acids, at least one of the two amino acids being a basic hydrophilic amino acid, The group of hydrophilic amino acids is at least one other than the hydrophilic amino acids.
Separated by two amino acids (preferably at least two amino acids), and generally by four or fewer amino acids, the amino acids between groups of hydrophilic amino acids may or may not be hydrophobic. According to a particularly preferred embodiment, the polypeptide consists of at least four groups of amino acid chains, 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 amino acids, wherein the hydrophilic amino acids in each group are hydrophilic amino acids. At least one of them is a basic hydrophilic amino acid and the other is a basic or neutral hydrophilic amino acid.

The hydrophobic amino acids include Ala, Cys, Phe, Gly, Ile, Leu, Me
t, Pro, Val, Trp, Tyr, norleucine (Nle), norvaline (
Nva) and cyclohexylalanine (Cha). Neutral hydrophilic amino acids are selected from the class consisting of Asn, Gln, Ser, Thr and homoserine (Hse). The basic hydrophilic amino acid is Ly
s, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu) and p-aminophenylalanine. Each of the groups of four amino acids has the sequence ABCD, BCDA, CDAB
Or DABC (where A and B are each a hydrophobic amino acid and may be the same or different, and one of C or D is a basic hydrophilic amino acid and the other of C or D Is a basic or neutral hydrophilic amino acid, which may be the same or different). In certain embodiments, the polypeptide chain consists of 5 or 6 groups of this sequence. Within each group, each of A, B, C and D may be the same in some or all groups;
Alternatively, it may be different in some or all groups.

The polypeptide chain preferably has at least 20 and no more than 50 amino acids. It should be understood that the polypeptide need not consist solely of the above groups. The polypeptide may have amino acids extending from one or both ends of any of the above groups forming a polypeptide chain, and / or may have amino acids between one or more of the at least four groups. It is included in the scope of the present invention. The amino acid group may be a repeat of the amino acid group, or the amino acids in each group have two hydrophobic amino acids and two hydrophilic amino acids as described above in each group of at least four amino acids. The condition may be different.

Thus, a biologically active polypeptide consists of a chain comprising at least four groups of amino acids, each comprising four amino acids. Two of the four amino acids in each group are hydrophobic, at least one amino acid is basic hydrophilic, and the other one is basic or neutral hydrophilic; The polypeptide chain preferably has at least 20 and no more than 50 amino acids.

In certain embodiments, each of the at least four groups of amino acids in the peptide chain is
, The sequence ABCD, BCDA, CDAB or DABC
Wherein A and B are hydrophobic amino acids, and one of C and D is a basic hydrophilic amino acid.
And the other of C or D is a basic or neutral hydrophilic amino acid
Acid). Thus, the resulting polypeptide chain has the following sequence:
There is one: (X₁)_a(A-B-C-D)_n(Y₁)_b  (X₂)_a(B-C-D-A)_n(Y₂)_b  (X₃)_a(CDAB)_n(Y₃)_b  (X₄)_a(DABC)_n(Y₄)_b  (Where X₁Is D, CD- or BCD-; Y₁Is -A or -AB or -ABC; X₂Is A-, DA- or CDA-; Y is₂Is -B, -BC or BCD; X₃Is B-, AB-, DABB-; Y₃Is -C, -CD, -CDA; X₄Is C-, BC-, ABC-; Y₄Is -D, -DA, -DAB; a is 0 or 1; b is 0 or 1; and n is at least 4). In the peptide chain, the spacing between the groups and the charge on the amino acid are amphipathic and positive.
Does not change the properties of the peptide chain, which has a negative effect on the folding of the chain.
The above groups of 4 amino acids are not separated from each other, provided that they are not removed
Amino acids may be included between the above four groups of amino acids so as to be significantly different from
Should be understood.

The following are typical examples of such peptides. The peptide can have amino acids extending from either end of the chain.
For example, a chain can have a Ser-Lys sequence before the "Ala" end and / or have an Ala-Phe sequence after the "Lys" end. Alternative amino acid sequences can also be added at the “Ala” and / or “Lys” ends. Similarly, in any of the polypeptide chains having at least four groups of amino acids of the above sequence, the chains can have, for example, a CD sequence before the first ABCD group. Alternatively, another amino acid sequence may be substituted for one of these polypeptide chains "
It can be attached to the "A" and / or "D" ends. There may also be amino acids in the chain that separate one or more of the four amino acids from one another.

According to another embodiment, X is a magatinin peptide.
You. A magainin peptide is a magainin such as magainin I, II or III.
Or an analog or derivative thereof. Magainin peptides are preferably the following salts
Basic peptide structure X₁₂Including: --R₁₁-R₁₁-R₁₂-R₁₃-R₁₁-R₁₄-R₁₂-R₁₁-R_1
4− R₁₂-R₁₁-R₁₁-R₁₁-R_14a− (R_Fifteen)_n-R_14a-R₁₄
--- (wherein, R₁₁Is a hydrophobic amino acid; R₁₂Is a basic hydrophilic amino acid
R;₁₃Is a hydrophobic, neutral hydrophilic, or basic hydrophilic amino acid;
₁₄And R_14aIs a hydrophobic or basic hydrophilic amino acid;_FifteenIs
Glutamic or aspartic acid; and n is 0 or 1). Like
In a preferred embodiment, R₁₃Is a hydrophobic or neutral hydrophilic amino acid
, R_14aIs a hydrophobic amino acid and R_FifteenIs glutamic acid or Aspa
Laginic acid. Thus, for example, magainin peptides include those of the following structure: -Y₁₂-X₁₂− [(Where X₁₂Is a basic peptide structure as described above;₁₂Is (i) R₁₂  (Ii) R_14a-R₁₂  (Iii) R₁₁-R_14a-R₁₂  (Iv) R₁₄-R₁₁-R_14a-R₁₂  (Where R₁₁, R₁₂, R₁₄And R_14aIs as defined above)
Is]. Magainin peptides also include those of the following structures: -X₁₂-Z₁₂Where [X₁₂Is as defined above, and Z₁₂Is (i) R₁₆(Where R₁₆Is a basic hydrophilic amino acid or asparagine
Or glutamine) (ii) R₁₆-R₁₇(Where R₁₇Are neutral hydrophilic amino acids, hydrophobic amino acids
Acid or basic hydrophilic amino acids. Preferably R₁₇Is basic hydrophilic
Is a sex amino acid)]. Magainin peptides also include those of the following structures: (Y₁₂)_a-X₁₂− (Z₁₂)_b  (Where X₁₂, Y₁₂And Z₁₂Is as defined above, and a is 0
Or 1 and b is 0 or 1.).

Representative examples of such peptides include the following sequences: (SEQ ID NO: 154) (OH) or (NH₂) (SEQ ID NO: 155) (OH) or (NH₂The magainin peptide has the following basic peptide structure X₁₃Including: --R₁₄-R₁₁-R_14a-R₁₂-R₁₁-R₁₁-R₁₂-R₁₃− R₁₁-R₁₄-R₁₂-R₁₁-R₁₁-R₁₂-Where R₁₁, R₁₂, R₁₃, R₁₄And R_14aIs the above amino acid
). Magainin peptides also include those of the following structures: -X₁₃-Z₁₃Where [X₁₃Is the basic peptide structure described above, and Z₁₃Is (R₁₁)_n-(R₁₁)_n-(R₁₁)_n-(R_14a)_n-(R_Fifteen)_n-
(R_14a)_n-(R₁₄)_n-  (R₁₆)_n-(R₁₇)_n  (Where R₁₁, R₁₄, R_14a, R_Fifteen, R₁₆, And R₁₇Is defined first
And n is 0 or 1, each n being the same or different
Good). Magainin peptides generally contain at least 14 amino acids, up to 40
Amino acids. The magainin peptide is preferably 22 or
Contains 23 amino acids. Therefore, the basic marker of the above-mentioned magainin peptide
The peptide structure contains additional amino acids at the amino or carboxy terminus or at both ends.
You may go out.

Representative examples of these magainin peptides include peptides having the following primary structure, as shown in the attached sequence listing, and suitable analogs and derivatives thereof: (a) (SEQ ID NO: 6) (OH ) or _(NH 2) (magainin I) (b) (SEQ ID NO: 7) (OH) or _(NH 2) (magainin II) (c) (SEQ ID NO: 8) (OH) or _(NH 2) (magainin III) the following are examples of peptide derivatives or analogs of the basic structure: (d) (SEQ ID NO: 9) (OH) or _(NH 2) _{(e) (SEQ} ID NO: 10) (OH) or _(NH 2) (F) (SEQ ID NO: 11) (OH) or (NH ₂ ) magainin peptide was obtained from Proc. Natl. Acad. Sci. Vol. 8
4, p. 5449-53 (August 1987), which is hereby incorporated by reference in its entirety. As used herein, “magainin peptide” refers to a basic magainin structure, and derivatives and analogs thereof, including, but not limited to, representative derivatives or analogs.

[0042] In another embodiment, X is a PGLa peptide or an XPF peptide.
You. The PGLa peptide is PGLa or an analog or derivative thereof. P
The GLa peptide preferably has the following basic peptide structure X₁₄Including: -R₁₁-R₁₇-R₁₂-R₁₁-R₁₄-R₁₄-R₁₁− R₁₁-R₁₄-R₁₂-R₁₁-R₁₁-R₁₂-R₁₁− R₁₁-R₁₁-R₁₂-Where R₁₁, R₁₂, R₁₄, And R₁₇Is as defined above). PGLa peptides generally contain at least 17 amino acids and can contain as many as 40 amino acids. Therefore, the above PGLa peptide
The basic peptide structure for amino or carboxy terminal or amino or
And amino acids at both ends of the carboxy terminus. Thus, for example, PGLa peptides include those of the following structure: -Y₁₄-X₁₄Where [X₁₄Is as defined above, and Y₁₄Is (i) R₁₁Or (ii) R₁₄-R₁₁  (Where R₁₁And R₁₄Is as defined above). For example, PGLa peptides also include those of the following structures: -X₁₄-Z₁₄Where [X₁₄Is as defined above, and Z₁₄Is (i) R₁₁Or (ii) R₁₁-R₁₁  (Where R₁₁Is as defined above).

PGLa peptides also include those of the following structures: (Y₁₄)_a-X₁₄− (Z₁₄)_b  (Where X₁₄, Y₁₄And Z₁₄Is as defined above, and a is 0
Or 1 and b is 0 or 1.). The XPF peptide is XPF or an analog or derivative thereof. XPF pen
The peptide preferably has the following basic peptide structure X₁₆Including: --R₁₁-R₁₇-R₁₂-R₁₁-R₁₄-R₁₈-R₁₇− R₁₁-R₁₄-R₁₂-R₁₁-R₁₁-R₁₂− R₁₁-R₁₁-R₁₁-R₁₂− (R_Fifteen)_n-R₁₁--- (wherein, R₁₁, R₁₂, R₁₄, R_FifteenAnd R₁₇Is as defined above
R₁₈Is glutamine or asparagine or basic hydrophilic or hydrophobic
And n is 0 or 1). XPF peptides generally contain at least 19 amino acids and up to 40 amino acids.
It can include a mino acid. Therefore, the base for the XPF peptide described above
The amino acid or carboxy terminus or amino and carboxyl
It may further contain amino acids at both ends of the c-terminus. Thus, for example, XPF peptides include those of the following structure: -Y₁₆-X₁₆Where [X₁₆Is as defined above, and Y₁₆Is (i) R₁₁Or (ii) R₁₄-R₁₁  (Where R₁₁And R₁₄Is as defined above). XPF peptides also include those of the following structures: -X₁₆-Z₁₆Where [X₁₆Is as defined above, and Z₁₆Is (i) R₁₁(Ii) R₁₁-R₁₈(Iii) R₁₁-R₁₈-Proline; or (iv) R₁₁-R₁₈-Proline-R₁₂(Where R₁₁, R₁₂And R
₁₈Is as defined above). XPF peptides also include those of the following structures: (Y₁₆)_a-X₁₆− (Z₁₆)_b  (Where X₁₆, Y₁₆And Z₁₆Is as defined above, and a is 0
Or 1 and b is 0 or 1.). Preferred are characterized by the following primary amino acid sequences shown in the attached sequence listing
PGLa: (SEQ ID NO: 12) (NH₂) XPF: (SEQ ID NO: 13)

A review on XPF and PGLa can be found in Hoffman et al. , EM
BOJ. 2: 711-714, 1983; Andreu et al. , J. et al.
Biochem. 149: 531-535, 1985; Gibson et a
l. , J. et al. Biol. Chem. 261: 5341-5349, 1986; and Giovannini et al. Bochem. J. 243: 113-
120, 1987. Each of these documents is fully incorporated herein by reference.

According to yet another embodiment, X is a CPF peptide or a suitable analog or derivative thereof. CPF peptides and analogs and derivatives thereof are collectively referred to herein as CPF peptides. CPF peptide is intended to include the following basic peptide structure _{X 20: --R 21 -R 21 -R} 22 -R 22 -R 21 -R 21 -R 23 -R 21 - -R 21 -R 21 -R 23 _{-R 21 -R 21 -R 24 -R 25} -R 21 - ( _{wherein, R 21} is a hydrophobic amino acid; _{R 22} is a hydrophobic amino acid or a basic hydrophilic amino acid; _{R 23} is a base R ₂₄ is a hydrophobic or neutral hydrophilic amino acid; and R ₂₅ is a basic or neutral hydrophilic amino acid). The above basic structure represented by the symbol of Hereafter X _20. Hydrophobic amino acids include Ala, Cys, Phe, Gly, Ile, Leu, Me
t, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva)
And cyclohexylalanine (Cha). Neutral hydrophilic amino acids are Asn, Gln, Ser, Thr and homoserine (Hse). Basic hydrophilic amino acids are Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu) and p-aminophenylalanine. The CPF peptide may contain only the above amino acids, or may contain additional amino acids at the amino and / or carboxy termini or at both the amino and carboxy termini. Generally, peptides do not contain more than 40 amino acids.

The CPF peptide containing the above basic structure is preferably one to four at the amino terminus.
With additional amino acids. Accordingly, such a preferred peptide is represented by the following structural formula: Y₂₀-X₂₀Where [X₂₀Is a basic peptide structure as described above;₂₀Is (i) R₂₅-; (Ii) R₂₂-R₂₅-; (Iii) R₂₁-R₂₂-R₂₅(Iv) R₂₂-R₂₁-R₂₂-R₂₅Or preferably (v) Gly-R₂₁-R₂₂-R₂₅  (Where R₂₁, R₂₂And R₂₅Is as defined above). The carboxy terminus of the basic peptide structure also has 1 to 13 additional amino acids.
It may be. In a preferred embodiment, the basic structure has 1 to 7 carboxy termini.
It may have additional amino acids and is represented as:₂₀-Z₂₀  [Where X₂₀Is the basic peptide structure described above, and Z₂₀Is (i) R₂₁-; (Ii) R₂₁-R₂₁-; (Iii) R₂₁-R₂₁-R₂₄(Iv) R₂₁-R₂₁-R₂₄-R₂₄(V) R₂₁-R₂₁-R₂₄-R₂₄-R₂₆(Vi) R₂₁-R₂₁-R₂₄-R₂₄-R₂₆-Gln; or (vii) R₂₁-R₂₁-R₂₄-R₂₄-R₂₆-Gln-Gln (wherein, R₂₁And R₂₄Is as defined above, and R₂₆Is a professional
Phosphorus or hydrophobic amino acids)].

Preferred peptides have the following structural formula: (Y₂₀)_a-X₂₀− (Z₂₀)_b  (Where X₂₀, Y₂₀And Z₂₀Is as defined above, and a is 0
Or 1 and b is 0 or 1.). Some representative examples of CPF peptides that can be used are described in the literature,
(SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 20) 21) (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 25) (SEQ ID NO: 26).

For a review on CPF peptides, see Richter, K .; Egger, R .; ,
and Kreil (1986) J. Am. Biol. Chem. 261,3676-
3680; Wakabayashi, T .; Kato, H .; , And Tach
ibaba, S .; (1985) Nucleic Acids Research
13, 1817-1828; Gibson, B .; W. , Poulter, L .; ,
Williams, D.M. H. , And Maggio, J .; E. FIG. (1986) J
. Biol. Chem. 261, 5341-5349. Each of these documents is fully incorporated herein by reference.

In yet another embodiment, X is a basic structure X₃₁X to X₃₇
X is a peptide comprising any of the following:₃₁Is-[R₃₁-R₃₂-R₃₂-R₃₃-R₃₁-R₃₂-R₃₂]_n
-; X₃₂Is-[R₃₂-R₃₂-R₃₃-R₃₁-R₃₂-R₃₂-R₃₁]_n
-; X₃₃Is-[R₃₂-R₃₃-R₃₁-R₃₂-R₃₂-R₃₁-R₃₂]_n
-; X₃₄Is-[R₃₃-R₃₁-R₃₂-R₃₂-R₃₁-R₃₂-R₃₂]_n
-; X₃₅Is-[R₃₁-R₃₂-R₃₂-R₃₁-R₃₂-R₃₂-R₃₃]_n
-; X₃₆Is-[R₃₂-R₃₂-R₃₁-R₃₂-R₃₂-R₃₃-R₃₁]_n
-; And X₃₇Is-[R₃₂-R₃₁-R₃₂-R₃₂-R₃₃-R₃₁-R₃₂]-
_n(Wherein, R₃₁Is a basic hydrophilic amino acid;₃₂Is a hydrophobic amino acid
R₃₃Is a neutral, basic, or hydrophobic amino acid,
And n is 1 to 5). Basic hydrophilic amino acids include Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu) and p-amino acid.
It is selected from the group consisting of phenylalanine. Hydrophobic amino acids include Ala, Cys, Phe, Gly, Ile, Leu, Met, Pro, Val, Trp and Tyr, norleucine (Nle
), Norvaline (Nva) and cyclohexylalanine (Cha)
More choice. Neutral hydrophilic amino acids include Asn, Gln, Ser, Thr and homoserine.
(Hse). According to one embodiment, the peptide has the structure X₃₁When containing, the peptide is
The structure can include: Y₃₁-X₃₁Where [X₃₁Is as defined above, and Y₃₁Is (i) R₃₂(Ii) R₃₂-R₃₂(Iii) R₃₁-R₃₂-R₃₂(Iv) R₃₃-R₃₁-R₃₂-R₃₂(V) R₃₂-R₃₃-R₃₁-R₃₂-R₃₂Or (vi) R₃₂-R₃₂-R₃₃-R₃₁-R₃₂-R₃₂  (Where R₃₁, R₃₂And R₃₃Is as defined above).

In another embodiment, the peptide has the structure X₃₁When the peptide contains
The following structures can be included: X₃₁-Z₃₁  [Where X₃₁Is as defined above, and Z₃₁Is (i) R₃₁(Ii) R₃₁-R₃₂(Iii) R₃₁-R₃₂-R₃₂(Iv) R₃₁-R₃₂-R₃₂-R₃₃(V) R₃₁-R₃₂-R₃₂-R₃₃-R₃₁Or (vi) R₃₁-R₃₂-R₃₂-R₃₃-R₃₁-R₃₂(Where R₃₁,
R₃₂, And R₃₃Is as described above).

In yet another embodiment, the peptide can include the structure:
: (Y₃₁)_a-X₃₁− (Z₃₁)_b  (Where Y₃₁And Z₃₁Is as defined above, and a is 0 or 1
And b is 0 or 1.) The peptide has the structure X₃₂The peptide can include the following structure:
: Y₃₂-X₃₂  [Where X₃₂Is as defined above, and Y₃₂Is (i) R₃₁(Ii) R₃₂-R₃₁(Iii) R₃₂-R₃₂-R₃₁(Iv) R₃₁-R₃₂-R₃₂-R₃₁(V) R₃₃-R₃₁-R₃₂-R₃₂-R₃₁Or (vi) R₃₂-R₃₃-R₃₁-R₃₂-R₃₂-R₃₁(Where R₃₁,
R₃₂, And R₃₃Is as described above).

According to another embodiment, the peptide has the structure X₃₂When containing, the peptide is
The structure can include: X₃₂-Z₃₂  [Where X₃₂Is as defined above, and Z₃₂Is (i) R₃₂(Ii) R₃₂-R₃₂(Iii) R₃₂-R₃₂-R₃₃(Iv) R₃₂-R₃₂-R₃₃-R₃₁(V) R₃₂-R₃₂-R₃₃-R₃₁-R₃₂Or (vi) R₃₂-R₃₂-R₃₃-R₃₁-R₃₂-R₃₂  Is].

[0053] In yet another embodiment, the peptide can include the structure:
: (Y₃₂)_a-X₃₂− (Z₃₂)_b  (Where Y₃₂And Z₃₂Is as defined above, and a is 0 or 1
And b is 0 or 1.) According to another embodiment, the peptide has the structure X₃₃When containing, the peptide is
The structure can include: Y₃₃-X₃₃  [Where X₃₃Is as defined above, and Y₃₃Is (i) R₃₂(Ii) R₃₁-R₃₂(Iii) R₃₂-R₃₁-R₃₂(Iv) R₃₂-R₃₂-R₃₁-R₃₂(V) R₃₁-R₃₂-R₃₂-R₃₁-R₃₂Or (vi) R₃₃-R₃₁-R₃₂-R₃₂-R₃₁-R₃₂  (Where R₃₁, R₃₂And R₃₃Is as defined above).

In another embodiment, the peptide has the structure X₃₃When the peptide contains
The following structures can be included: X₃₃-Z₃₃  [Where X₃₃Is as defined above, and Z₃₃Is (i) R₃₂(Ii) R₃₂-R₃₃(Iii) R₃₂-R₃₃-R₃₁(Iv) R₃₂-R₃₃-R₃₁-R₃₂(V) R₃₂-R₃₃-R₃₁-R₃₂-R₃₂Or (vi) R₃₂-R₃₃-R₃₁-R₃₂-R₃₂-R₃₁  Is].

In yet another embodiment, the peptide can include the structure:
: (Y₃₃)_a-X₃₃− (Z₃₃)_b  (Where X₃₃Y₃₃And Z₃₃Is as defined above, and a is 0 or
Or b is 0 or 1). According to another embodiment, the peptide has the structure X₃₄When containing, the peptide is
The structure can include: Y₃₄-X₃₄  [Where X₃₄Is as defined above, and Y₃₄Is (i) R₃₂(Ii) R₃₂-R₃₂(Iii) R₃₁-R₃₂-R₃₂(Iv) R₃₂-R₃₁-R₃₂-R₃₂(V) R₃₂-R₃₂-R₃₁-R₃₂-R₃₂Or (vi) R₃₁-R₃₂-R₃₂-R₃₁-R₃₂-R₃₂(Where R₃₁,
R₃₂And R₃₃Is as described above).

In another embodiment, the peptide has the structure X₃₄When the peptide contains
The following structures can be included: X₃₄-Z₃₄  [Where X₃₄Is as defined above, and Z₃₄Is (i) R₃₃(Ii) R₃₃-R₃₁(Iii) R₃₃-R₃₁-R₃₂(Iv) R₃₃-R₃₁-R₃₂-R₃₂(V) R₃₃-R₃₁-R₃₂-R₃₂-R₃₁Or (vi) R₃₃-R₃₁-R₃₂-R₃₂-R₃₁-R₃₂(Where R₃₁,
R₃₂, And R₃₃Is as described above).

In yet another embodiment, the peptide can include the structure:
: (Y₃₄)_a-X₃₄− (Z₃₄)_b  (Where X₃₄, Y₃₄And Z₃₄Is as defined above, and a is 0
Or 1 and b is 0 or 1.). According to yet another embodiment, the peptide has the structure X₃₅Containing a peptide
Can include the following structure: Y₃₅-X₃₅  [Where X₃₅Is as defined above, and Y₃₅Is (i) R₃₃(Ii) R₃₂-R₃₃(Iii) R₃₂-R₃₂-R₃₃(Iv) R₃₁-R₃₂-R₃₂-R₃₃(V) R₃₂-R₃₁-R₃₂-R₃₂-R₃₃Or (vi) R₃₂-R₃₂-R₃₁-R₃₂-R₃₂-R₃₃  (Where R₃₁, R₃₂And R₃₃Is as defined above).

In another embodiment, the peptide has the structure X₃₅When the peptide contains
The following structures can be included: X₃₅-Z₃₅  [Where X₃₅Is as defined above, and Z₃₅Is (i) R₃₁(Ii) R₃₁-R₃₂(Iii) R₃₁-R₃₂-R₃₂(Iv) R₃₁-R₃₂-R₃₂-R₃₁(V) R₃₁-R₃₂-R₃₂-R₃₁-R₃₂Or (vi) R₃₁-R₃₂-R₃₂-R₃₁-R₃₂-R₃₂(Where R₃₁You
And R₃₂Is as described above).

[0059] In yet another embodiment, the peptide can include the structure:
: (Y₃₅)_a-X₃₅− (Z₃₅)_b  (Where X₃₅, Y₃₅And Z₃₅Is as defined above, and a is 0
Or 1 and b is 0 or 1.). According to yet another embodiment, the peptide has the structure X₃₆Containing a peptide
Can include the following structure: Y₃₆-X₃₆  [Where X₃₆Is as defined above, and Y₃₆Is (i) R₃₁(Ii) R₃₃-R₃₁(Iii) R₃₂-R₃₃-R₃₁(Iv) R₃₂-R₃₂-R₃₃-R₃₁(V) R₃₁-R₃₂-R₃₂-R₃₃-R₃₁Or (vi) R₃₂-R₃₁-R₃₂-R₃₂-R₃₃-R₃₁  (Where R₃₁, R₃₂And R₃₃Is as defined above).

In another embodiment, the peptide has the structure X₃₆When the peptide contains
The following structures can be included: X₃₆-Z₃₆  [Where X₃₆Is as defined above, and Z₃₆Is (i) R₃₂(Ii) R₃₂-R₃₂(Iii) R₃₂-R₃₂-R₃₁(Iv) R₃₂-R₃₂-R₃₁-R₃₂(V) R₃₂-R₃₂-R₃₁-R₃₂-R₃₂Or (vi) R₃₂-R₃₂-R₃₁-R₃₂-R₃₂-R₃₃(Where R₃₁,
R₃₂, And R₃₃Is as described above).

[0061] In yet another embodiment, the peptide may comprise the following _{structure: (Y 36) a -X 36} - (Z 36) b ( _wherein, X _{36, Y 36} and _{Z 36} is As defined above, and a is 0
Or 1 and b is 0 or 1).

According to yet another embodiment, the peptide has the structure X₃₇Containing a peptide
Can include the following structure: Y₃₇-X₃₇  [Where X₃₇Is as defined above, and Y₃₇Is (i) R₃₂(Ii) R₃₁-R₃₂(Iii) R₃₃-R₃₁-R₃₂(Iv) R₃₂-R₃₃-R₃₁-R₃₂(V) R₃₂-R₃₂-R₃₃-R₃₁-R₃₂Or (vi) R₃₁-R₃₂-R₃₂-R₃₃-R₃₁-R₃₂  (Where R₃₁, R₃₂And R₃₃Is as defined above).

In another embodiment, the peptide has the structure X₃₇When the peptide contains
The following structures can be included: X₃₇-Z₃₇  [Where X₃₇Is as defined above, and Z₃₇Is (i) R₃₂(Ii) R₃₂-R₃₁(Iii) R₃₂-R₃₁-R₃₂(Iv) R₃₂-R₃₁-R₃₂-R₃₂(V) R₃₂-R₃₁-R₃₂-R₃₂-R₃₃Or (vi) R₃₂-R₃₁-R₃₂-R₃₂-R₃₃-R₃₁(Where R₃₁,
R₃₂, And R₃₃Is as described above).

In yet another embodiment, the peptide can include the following structure:
: (Y₃₇)_a-X₃₇− (Z₃₇)_b  (Where X₃₇, Y₃₇And Z₃₇Is as defined above, and a is 0
Or 1 and b is 0 or 1.).

In a preferred embodiment of the peptides according to formulas X _{31 to} X ₃₇ , n is 3
And most preferably the peptide has any of the following structures as shown in the attached sequence listing: In (SEQ ID NO: 67) and (SEQ ID NO: 68), Xaa is p-aminophenylalanine.

According to another embodiment, X has the basic structure X₄₀Is a peptide containing
: R₃₁-R₃₂-R₃₂-R₃₃-R₃₄-R₃₂-R₃₂-R₃₁-R₃₂−
R₃₂-R₃₂-R₃₄-R₃₂-R₃₂  (Where R₃₁, R₃₂And R₃₃Is as defined above, and R₃₄
Is a basic hydrophilic or hydrophobic amino acid). According to one embodiment, the peptide can include the structure: Y₄₀-X₄₀  [Where X₄₀Is as defined above, and Y₄₀Is (i) R₃₂(Ii) R₃₂-R₃₂(Iii) R₃₄-R₃₂-R₃₂(Iv) R₃₃-R₃₄-R₃₂-R₃₂(V) R₃₂-R₃₃-R₃₄-R₃₂-R₃₂(Vi) R₃₂-R₃₂-R₃₃-R₃₄-R₃₂-R₃₂Or (vii) R₃₁-R₃₂-R₃₂-R₃₃-R₃₄-R₃₂-R₃₂  (Where R₃₁, R₃₂, R₃₃And R₃₄Is as defined above).

In another embodiment, X is a peptide comprising the structure: X₄₀-Z₄₀  [Where X₄₀Is as defined above, and Z₄₀Is (i) R₃₁(Ii) R₃₁-R₃₂(Iii) R₃₁-R₃₂-R₃₂(Iv) R₃₁-R₃₂-R₃₂-R₃₃(V) R₃₁-R₃₂-R₃₂-R₃₃-R₃₄(Vi) R₃₁-R₃₂-R₃₂-R₃₃-R₃₄-R₃₂Or (vii) R₃₁-R₃₂-R₃₂-R₃₃-R₃₄-R₃₂-R_{32 (Where R 31} , R₃₂, R₃₃And R₃₄Is as defined above).

In yet another embodiment, the peptide can include the structure:
: (Y₄₀)_a-X₄₀− (Z₄₀)_b  (Where X₄₀, Y₄₀And Z₄₀Is as defined above, and a is 0
Or 1 and b is 0 or 1.).

[0069] In a preferred embodiment, the peptide has the following structural formula as shown in the attached sequence listing: (SEQ ID NO: 69) In another preferred embodiment, the peptide has the following structural formula shown in the attached sequence listing: (SEQ ID NO: 70) According to a further embodiment, the peptide has any of the following structural formulas as shown in the attached sequence listing: (SEQ ID NO: 71) (SEQ ID NO: 72) (SEQ ID NO: 73) (SEQ ID NO: 74) (SEQ ID NO: 75) (SEQ ID NO: 76) (SEQ ID NO: 77) (SEQ ID NO: 78) (SEQ ID NO: 79) (SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) (SEQ ID NO: 85) ).

According to another embodiment, X is a peptide comprising any of the following structural formulas
: (I)-(Lys Ile Ala Lys Lys Ile Ala)_n−,
(Ii)-(Lys Phe Ala Lys Lys Phe Ala)_n−
, And (iii)-(Lys Phe Ala Lys Lys Ile Ala)_n
-(Where n is 2 to 5). Preferably, n is 3 and the peptide is
With any of the structural formulas: (Lys Ile Ala Lys Lys Ile Ala)₃  (SEQ ID NO: 86) (Lys Phe Ala Lys Lys Phe Ala)₃  (SEQ ID NO: 87) (Lys Phe Ala Lys Lys Ile Ala)₃  (SEQ ID NO: 88) According to another embodiment, X consists of the following structural formula shown in the attached sequence listing
Peptides selected from the group: (SEQ ID NO: 89) (SEQ ID NO: 90) (SEQ ID NO: 91) (SEQ ID NO: 92).

According to yet another embodiment, X is cecropin or sarcotoxin. "Secropin" includes basic structures and analogs and derivatives thereof. Cecropin and its analogs and derivatives are described in Ann. Rev .. Microbi
ol. 1987, Vol. 41, p. 103-26, especially p. 108, and C
Christensen et al. , PNAS Vol. 85, p. 5072
-76, which are incorporated herein by reference. "Sarcotoxin" includes basic substances and analogs and derivatives thereof. Sarcotoxins and their analogs and derivatives are available from Molecular E
ntomology, p. 369-78, especially p. 375, Alan R. Li
ss, Inc. (1987), which is incorporated herein by reference.

According to another embodiment, X is melittin or an analogue or derivative thereof. Melittin is an amphipathic peptide consisting of 26 amino acid residues, and was isolated from honeybee (Apis mellifera) venom. Ha
bermann et al. , Hoppe-Seyler's Zeitsc
lift Physiol. Chem. , Vol. 348, p. 37-50 (
1987), which is fully incorporated herein by reference. Melittin has the following structural formula, represented by three letter amino acid abbreviations: (SEQ ID NO: 93).

In another embodiment, X is a cecropin peptide and a melittin peptide or a
Hybrid amphipathic peptides with analogs. Such hybrid pen
Peptides are described in US Pat. No. 5,714,467, which is incorporated herein by reference.
Be incorporated. Examples of this peptide are shown below. (SEQ ID NO: 156) (-OH or -NH2) In another embodiment, X is the following basic structure X₅₀Amphiphilic pep containing
Is a tide: R₄₁-R₄₂-R₄₂-R₄₁-R₄₂-R₄₂-R₄₁-R₄₁-R₄₂−
R₄₁-R₄₁  (Where R₄₁Is a hydrophobic amino acid and R₄₂Is basic hydrophilic or
A neutral hydrophilic amino acid).

In one embodiment, the peptide comprises the following basic structure: Y₅₀-X₅₀  [Where X₅₀Is as defined above, and Y₅₀Is (i) R₄₁(Ii) R₄₂-R₄₁Or (iii) R₄₂-R₄₂-R₄₁  (Where R₄₁And R₄₂Is as defined above).

In some embodiments, R₄₁Is leucine. In another embodiment,
R₄₂Is ricin. Representative examples of peptides in this aspect of the invention include:
Including those with the structure: (SEQ ID NO: 94) (SEQ ID NO: 95) (SEQ ID NO: 96) (SEQ ID NO: 97). According to another embodiment, X is a basic structure X₅₂Amphiphilic pepti containing
Is: R₄₂-R₄₁-R₄₂-R₄₂-R₄₁-R₄₁-R₄₂-R₄₂-R₄₁−
R₄₂-R₄₂  (Where R₄₁Is a hydrophobic amino acid and R₄₂Is basic hydrophilic or
A neutral hydrophilic amino acid). In some embodiments, R₄₁Is leucine. In another embodiment,
R₄₂Is ricin.

In one embodiment, the peptide comprises the following basic structure: Y₅₂-X₅₂  [Where X₅₂Is as defined above, and Y₅₂Is (i) R₄₂(Ii) R₄₁-R₄₂(Iii) R₄₁-R₄₁-R₄₂(Iv) R₄₂-R₄₁-R₄₁-R₄₂Or (v) R₄₂-R₄₂-R₄₁-R₄₁-R₄₂(Where R₄₁And R₄₂
Is as described above). In one embodiment, the peptide has the following structure: (SEQ ID NO: 98). In another embodiment, the peptide comprises the following basic structure: X₅₂-Z₅₂  [Where X₅₂Is as defined above, and Z₅₂Is (i) R₄₁(Ii) R₄₁-R₄₁(Iii) R₄₁-R₄₁-R₄₂(Iv) R₄₁-R₄₁-R₄₂-R₄₂Or (v) R₄₁-R₄₁-R₄₂-R₄₂-R₄₁(Where R₄₁And R₄₂
Is as described above).

In one embodiment, the peptide has the structure: (SEQ ID NO: 99). In another embodiment, the peptide comprises the structure: (Y₅₂)_a-X₅₂− (Z₅₂)_b (Where X₅₂, Y₅₂And Z₅₂Is as defined above, and a is 0
Or 1 and b is 0 or 1.). In another embodiment, X is the basic structure X₅₄Biologically active both
Amphiphilic peptide: R₄₁-R₄₂-R₄₂-R₄₁-R₄₁-R₄₂-R₄₂-R₄₁-R₄₂
-R₄₂-R₄₁-R₄₁-R₄₂-R₄₂-R₄₃-R₄₃  (Where R₄₁And R₄₂Is as defined above, and R₄₃Is neutral
Hydrophilic amino acids).

In one embodiment, the peptide has the structure: (SEQ ID NO: 100) In another embodiment, the peptide has the following structure: (SEQ ID NO: 101). In another embodiment, X is a basic structure X₅₆Bioactive amphiphilic containing
Is a peptide: R₄₁-R₄₂-R₄₂-R₄₁-R₄₁-R₄₂-R₄₂-R₄₁-R₄₁
-R₄₂-R₄₂-R₄₄  (Where R₄₁And R₄₂Is as defined above, and R₄₄Is neutral
Hydrophilic amino acids or proline). In one embodiment, the peptide comprises the following basic structure: X₅₆-Z₅₆  [Where X₅₆Is as defined above, and Z₅₆Is (i) -R₄₂(Ii) -R₄₂-R₄₂(Iii) -R₄₂-R₄₂-R₄₁(Iv) -R₄₂-R₄₂-R₄₁-R₄₁(V) -R₄₂-R₄₂-R₄₁-R₄₁-R₄₂(Vi) -R₄₂-R₄₂-R₄₁-R₄₁-R₄₂-R₄₂Or (vii) -R₄₂-R₄₂-R₄₁-R₄₁-R₄₂-R₄₂-R₄₁(formula
Medium, R₄₁And R₄₂Is as described above).

In a preferred embodiment, the peptide has the following structure: (SEQ ID NO: 102); or (SEQ ID NO: 103). In another embodiment, X is a basic structure X₅₈Bioactive amphiphilic containing
Is a peptide: R₄₁-R₄₁-R₄₂-R₄₂-R₄₁-R₄₂-R₄₂-R₄₁-R₄₁
-R₄₂-R₄₂-R₄₁-R₄₃  (Where R₄₁, R₄₂And R₄₃Is as defined above). In one embodiment, the peptide comprises the structure: X₅₈-Z₅₈  [Where X₅₈Is as defined above, and Z₅₈Is (i) -R₄₁(Ii) -R₄₁-R₄₅(Iii) -R₄₁-R₄₅-R₄₅(Iv) -R₄₁-R₄₅-R₄₅-R₄₃(V) -R₄₁-R₄₅-R₄₅-R₄₃-R₄₁(Vi) -R₄₁-R₄₅-R₄₅-R₄₃-R₄₁-R₄₃(Vii) -R₄₁-R₄₅-R₄₅-R₄₃-R₄₁-R₄₃-R₄₃(Viii) -R₄₁-R₄₅-R₄₅-R₄₃-R₄₁-R₄₃-R₄₃−
R₄₅Or (ix) -R₄₁-R₄₅-R₄₅-R₄₃-R₄₁-R₄₃-R₄₃-R_4
5-R₄₃  (Where R₄₁And R₄₃Is as defined above, and R₄₅Is a professional
Phosphorus)).

In one embodiment, the peptide has the following structure: (SEQ ID NO: 104) In another embodiment, X is is a biologically active amphiphilic peptide comprising the following basic structure _{X 60: R 41 -R 41 -R} 43 -R 42 -R 41 -R 41 -R 41 -R ₄₁ -R ₄₁ -
_{R 41 -R 42 - R 41 -R} 41 -R 42 -R 42 -R 41 -R 41 -R 42 -R 42 -R 41 -
(Wherein R ₄₁ , R ₄₂ and R ₄₃ are as defined above). In one embodiment, the peptide has the following structure: (SEQ ID NO: 105)

In another embodiment, X is a basic structure X₆₂Is a peptide comprising: -R₄₁-R₄₂-R₄₂-R₄₁-R₄₂-R₄₂-R₄₁-Where R₄₁And R₄₂Is as defined above). In one embodiment, the peptide comprises the structure: Y₆₂-X₆₂  [Where X₆₂Is as defined above, and Y₆₂Is (i) R₄₁(Ii) R₄₂-R₄₁(Iii) R₄₂-R₄₂-R₄₁Or (iv) R₄₁-R₄₂-R₄₂-R₄₁(Where R₄₁, R₄₂Is the above
Is).

[0082] Representative examples of such peptides include the following, the sequences of which are shown in the attached Sequence Listing: (SEQ ID NO: 106) (SEQ ID NO: 107) (SEQ ID NO: 108) (SEQ ID NO: 109) ( (SEQ ID NO: 110) (SEQ ID NO: 111) (SEQ ID NO: 141) (SEQ ID NO: 142) (SEQ ID NO: 143) (SEQ ID NO: 144) (SEQ ID NO: 145) (SEQ ID NO: 146)

In one embodiment, the peptide comprises the structure: X₆₂-Z₆₂  [Where X₆₂Is as defined above, and Z₆₂Is (i) R₄₁(Ii) R₄₁-R₄₂(Iii) R₄₁-R₄₂-R₄₂Or (iv) R₄₁-R₄₂-R₄₂-R₄₁  (Where R₄₁And R₄₂Is as defined above). Representative examples of the peptides are the following peptides shown in the following structural formula and attached sequence listing
Including: (SEQ ID NO: 112). In another embodiment, the peptide has the structure: (Y₆₂)_a-X₆₂− (Z₆₂)_b  (Where X₆₂, Y₆₂And Z₆₂Is as defined above, and a is 0
Or 1 and b is 0 or 1.).

[0084] Representative examples of such peptides include the following, the sequences of which are shown in the attached Sequence Listing: (SEQ ID NO: 113) (SEQ ID NO: 114) (SEQ ID NO: 115) (SEQ ID NO: 116). In another embodiment, X is a peptide having the structural formula: (SEQ ID NO: 117).

In another embodiment, X is a basic structure X₆₄Biologically active amphiphilic pen containing
Is a peptide: -R₄₂-R₄₂-R₄₂-R₄₁-R₄₁-R₄₂-R₄₂-R₄₁-Where R₄₁And R₄₂Is as described above). In one embodiment, the peptide has the structure Y₆₄-X₆₄[Wherein
, X₆₄Is as described above, and Y₆₄Is: (i) R₄₁Or (ii) R₄₂-R₄₁(Where R₄₁And R₄₂Is as above)
is there]. In another embodiment, the peptide has the structure X₆₄-Z₆₄[Wherein
X₆₄Is as described above, and Z₆₄Is: (i) R₄₂(Ii) R₄₂-R₄₂Or (iii) R₄₂-R₄₂-R₄₁(Where R₄₁And R₄₂Is as above
Is). In yet another embodiment, the peptide has the structure: (Y₆₄)_a-X₆₄− (Z₆₄)_b(Where X₆₄, Y₆₄, And Z₆₄
Is as described above, a is 0 or 1, and b is 0 or 1.) Representative examples of such peptides include: (SEQ ID NO: 127) (SEQ ID NO: 128) (SEQ ID NO: 129).

In yet another embodiment, X is a basic structure X₆₆Biologically active, including
Amphiphilic peptide: R₄₁-R₄₂-R₄₂-R₄₁-R₄₁-R₄₆-R₄₂-R₄₁-R_4
2-R₄₂-R₄₁(Where R₄₁  And R₄₂Is as described above, and R₄₆Is glutamic acid). this
A representative example of such a peptide is: (SEQ ID NO: 130).

[0087] In yet another embodiment, X is is biologically active amphiphilic peptide comprising the following basic structure _{X 68: -R 42 -R 42 -R} 41 -R 41 -R 42 -R 46 - R ₄₁ -R ₄₂ -R _4
2 -R ₄₁ - _(wherein, R _{41, R 42,} and _{R 46} are as described above). In one embodiment, the peptide includes the following basic structure _Y 68 _{-X 68} [
Where X ₆₈ is as described above and Y ₆₈ is R ₄₁ (defined above).

[0088] Representative examples of such peptides include: (SEQ ID NO: 131) (SEQ ID NO: 132). In another embodiment, X is is biologically active amphiphilic peptide comprising the following basic structure _{X 70: -R 41 -R 42 -R} 42 -R 41 -R 41 -R 42 -R 42 -R ₄₁ -R
₄₂ -R ₄₂ -R ₄₁ -R _{41 (wherein, R 41} and _{R 42} are as described above). A representative example of such a peptide has the following structure: (SEQ ID NO: 133). In another embodiment, X is is biologically active amphiphilic peptide comprising the following basic structure _{X 72: -R 42 -R 42 -R} 41 -R 41 -R 42 -R 47 -R 41 -R ₄₂ -R _4
2 -R ₄₁ - _{(wherein, R 41} and _{R 42} are as _{above, R 47} is aspartic acid). A representative example of such a peptide has the following structure: (SEQ ID NO: 134).

In yet another embodiment, X is a bioactive amphipathic peptide having the structure:
(SEQ ID NO: 135). In yet another embodiment, X has the structure X₇₄Biologically active amphiphilic pen containing
Is a peptide: R₄₂-R₄₁-R₄₂-R₄₁-R₄₁-R₄₂-R₄₂-R₄₁-R₄₆
-R₄₂-R₄₁(Where R₄₁, R₄₂, And R₄₆Is as described above)
. A representative example of such a peptide has the following structure: (SEQ ID NO: 136)

[0090] In another embodiment, X is is biologically active amphiphilic peptide comprising the following structure _{X 76: -R 41 -R 42 -R} 42 -R 41 -R 41 -R 42 - ( wherein , R ₄₁ and R ₄₂ are as described above).

In another embodiment, the peptide has the structure Y₇₆-X₇₆Wherein X is₇₆
Is as described above, and Y₇₆Is: (i) -R₄₂(Ii) -R₄₂-R₄₂(Iii) -R₄₁-R₄₂-R₄₂(Iv) -R₄₁-R₄₁-R₄₂-R₄₂(V) -R₄₂-R₄₁-R₄₁-R₄₂-R₄₂Or (vi) -R₄₂-R₄₂-R₄₁-R₄₁-R₄₂-R₄₂(Where R₄₁
And R₄₂Is as described above)]. In another embodiment, the peptide has the structure X₇₆-Z₇₆Wherein X₇₆Is
As above and Z₇₆Is: (i) R₄₈(Ii) R₄₈-R₄₁Or (iii) R₄₈-R₄₁-R₄₂(Where R₄₁And R₄₂Is as above
And R₄₈  Is a basic hydrophilic, neutral hydrophilic or hydrophobic amino acid)].

In yet another embodiment, the peptide has the structural formula: (Y₇₆)_a-X₇₆− (Z₇₆)_b(Where X₇₆, Y₇₆, And Z₇₆
Is as described above, a is 0 or 1, and b is 0 or 1.) Representative examples of such peptides include: (SEQ ID NO: 137) (SEQ ID NO: 138) (SEQ ID NO: 139).

[0093] In yet another embodiment, X is is biologically active amphiphilic peptide comprising the following structural formula _{X 78: -R 41 -R 42 -R} 41 -R 41 -R 42 -R 42 -R ₄₁ -R ₄₂ -R
₄₂ -R _{41 (wherein, R 41} and _{R 42} are as described above). A representative example of such a peptide has the following structure: (SEQ ID NO: 140). In another embodiment, X has the following structure: (SEQ ID NO: 149). In another embodiment, X is is biologically active amphiphilic peptide comprising the following structural formula _{X 80: -R 41 -R 42 -R} 42 -R 41 -R 41 -R 42 -R 46 -R 41 -R
₄₁ -R ₄₂ -R ₄₁ - _(wherein, R _{41, R 42,} and _{R 46} are as described above). A representative example of such a peptide has the following structure: (SEQ ID NO: 151). Further, according to another embodiment, X is an ion channel forming peptide or protein.

The ion channel forming peptides or proteins used are defensins, also known as human neutrophil antimicrobial peptides (HNP), eosinophil major basic protein (MBP), enhanced bacterial permeability Protein (BPI) and perforin
, A pore-forming cytotoxin, variously referred to as cytolysin or a pore-forming protein. Defensins are Celsted et
al. , J. et al. Clin. Invest. , Vol. 76, p. 1436-14
39 (1985). MBP proteins are described in Wasmoen et al. , J. et al. Biol. Chem. , Vol. 263, p. 12559-12
563 (1988). BPI protein is available from Ooi et al.
. , J. et al. Biol. Chem. , Vol. 262, p. 14891-14894
(1987). Perforin is described in Henkart et al.
, J. et al. Exp. Med. , 160: 75 (1984) and Podack et al. , J. et al. Exp. Med. 160: 695 (1984)
)It is described in. The above references are incorporated herein by reference.

“Ion channel forming protein” includes basic structures and analogs and derivatives of ion channel forming protein. According to yet another embodiment, the amino acid residues of the peptide or protein may be D-amino acids or glycine, respectively. Although the scope of this particular embodiment is not limited to any theoretical inference, the peptides or proteins described above, when exclusively composed of D-amino acids or glycine residues, will be proteolytically retained while retaining their activity. It is thought that resistance to the enzyme has increased.
Thus, such peptides can be administered orally. According to another embodiment, all of the amino acid residues may be D-amino acids or glycine residues, or L-amino acids or glycine residues.

It will also be appreciated that the peptides or proteins may be administered in combination with one another. According to another embodiment, the N-terminally substituted peptide or protein of the invention comprises:
It can be used in combination with ions having pharmacological action for the above purpose. A pharmacological ion is an ion that, when introduced into a target cell, virus or virus-infected cell, inhibits and / or prevents and / or destroys the growth of the target cell, virus or virus-infected cell. .

In the absence of an ion channel-forming peptide, such pharmacologically active ions pass through a natural or synthetic lipid membrane (particularly a cell membrane or virus membrane) in an amount sufficient to cause a harmful effect on cells or viruses. Can not do it. The pharmacologically active ion with the peptide or protein can be administered as a single composition or a separate composition, and the single or separate composition, in addition to the pharmacologically active ion with the peptide or protein, Additional active and / or inert materials may be included. Representative examples of pharmacological ions that can be used include fluoride, peroxide, bicarbonate, silver, zinc, mercury, arsenic, copper,
Platinum, antimony, gold, thallium, nickel, selenium, bismuth, and cadmium ions can be mentioned.

[0098] The peptide or protein and the ion having a pharmacological effect, whether administered or prepared as a single composition or as a separate composition, enhance the growth of target cells, viruses or virus-infected cells. It is used in an amount effective to inhibit and / or interfere and / or destroy. In practice, ions enhance the action of the peptide. That is, the amount of ions is an amount effective to reduce the maximum effective concentration of a peptide or protein for suppressing the growth of target cells, viruses or virus-infected cells.

The pharmacologically acting ions are generally used at a concentration of 0.05 to 2.0% for topical use. When used systemically, generally 1 to 1 kg of host body weight
An amount of 10 mg of ion is used. The dosage of the peptide or protein can be in the ranges described above. Further, it will be appreciated that the peptide or protein as well as the pharmacologically active ions may be administered by different routes. For example, ions may be administered orally while peptides may be administered IV or IP. As a representative example of topical administration of peptides or proteins and ions, peptides could be administered in amounts up to about 1% by weight and ions in amounts of about 50 mM (about 0.1%). Alternatively, ions in the form of a salt, such as sodium fluoride, could be administered orally, along with systemic administration of the peptide or protein. For example, 100 or 100 administration by IV or IP of a peptide or protein
A serum dose of micrograms / milliliter (10 milligrams / kilogram) is achieved, while an oral dose of 10 meq / kilogram ions (especially sodium fluoride) is achieved.

According to another embodiment, the peptides or proteins of the invention are bacitracins, gramicidin, polymyxin, vancomycin, teicoplanin, aminoglycosides, hydrophobic antibiotics, penicillins, monobactams, or derivatives or analogs thereof. It can be administered to a host together with an antibiotic selected from the body class. Vacitracins, gramicidin, polymyxin, vancomycin, teicoplanin, and their derivatives and analogs are a group of polypeptide antibiotics. As the bacitracin, bacitracin A is preferable. Aminoglycoside antibiotics include tobramycin, kanamycin, amikacin, gentamicins (eg, gentamicin C ₁ , gentamicin C ₂ , gentamicin C _1a ), netilmycin, and derivatives and analogs thereof. Preferred aminoglycoside antibiotics are tobramycin and gentamicins. Aminoglycosides and the above-mentioned bacitracins are hydrophilic and easily soluble in water.

Examples of usable penicillins include benzylpenicillin, ampicillin, methyl
Syrin (dimethoxyphenyl penicillin), ticaricillin, penicillin V (fe
Nonoxymethylpenicillin), oxacillin, cloxacillin, dicloxacillin
, Flucloxacillin, amoxicillin, and amidinocillin
Yes, but not limited to these. Preferred penicillins are benzylpenicillin and ampicillin.
Sirin. A preferred monobactam that can be used is aztreonam. A typical example of hydrophobic antibiotics that can be used in the present invention is a macrolide system.
For example, erythromycin, roxithromycin, clarithroma
9-N-alkyl derivatives of erythromycin; midecamycin ace
Tate; azithromycin; flurithromycin; rifabutin;
6-O-methylerythromycin A, known as TE-031 (Taisho);
Rifapentine; CGP-7040, CGP-5909, CGP-279353
Benzylpiperazinyl rifamycins such as (Ciba-Geigy); A
-62514 (Abbott)₁₁/ C₁₂
An erythromycin A derivative having a cyclic carbamate fused at the position; AC-
7230 (Toyo Brewery); benzoxazinolifamycin; dirithromycin; F
3-N-piperidino known as CE-22250 (Farmitalia)
Methylzinomethylrifamycin SV; M-119-a (Kirin Brewery); A
6-O-methyl-l-4 "-O-, known as -63075 (Abbott)
Carbamoyl erythromycin; CGP-27557 and CGP-2986 (
3-form having a diazabicycloalkyl side chain such as Ciba-Geigy)
Milrifamycin SV-hydrazones; 3-O-α-L-clazinosyldepo
16 member having a 3-O-α-L-clazinosyl moiety such as xylosalamycin
Macrolides: tyrosine and acyl demisinosyl tyrosine.

In addition to the macrolides mentioned above, rifamycin, carbenicillin and naphcillin can be used as well. Other usable antibiotics (whether hydrophobic or not) include antibiotics that are 50-S ribosome inhibitors such as lincomycin, clindamycin, chloramphenicol; large lipids such as mystatin, pimaricin There are antibiotics with a similar lactone ring. Peptides or proteins and antibiotics can be administered by direct administration to target cells, or by systemic or local administration to a host containing target cells, to prevent, destroy or suppress the growth of target cells. Target cells whose growth can be prevented, destroyed or suppressed by administration of peptides and antibiotics include Gram-positive bacteria, Gram-negative bacteria, and fungal cells. Antibiotics or derivatives or analogs thereof as described above are generally used at a concentration of about 0.1 to 10% (wt%) for topical use. When used systemically, antibiotics or derivatives or analogs thereof are generally used at about 1.25 per day.
Used in an amount of ~ 45 mg / kg (body weight of the host). The dose of the peptide or protein is as described above.

As a representative example of the topical administration of a peptide or protein and an antibiotic, the peptide or protein is administered in an amount of about 0.1 to 10% by weight, and
. It could be administered in an amount of 1 to 10% by weight. According to another embodiment, the peptides or proteins of the present invention can be administered in combination with an antiparasitic or antifungal agent.

[0104] Antiparasitic agents that can be used include, but are not limited to, antiprotozoal agents. Examples of specific antiparasitic agents that can be used include, but are not limited to, pentamidine isethionate and propamidine isethionate (Brlone). Antifungal agents that can be used include, but are not limited to, ketoconazole. It will be appreciated that certain antiparasitic agents also have antifungal activity, and certain antifungal agents also have antiparasitic activity. According to another embodiment, a peptide or protein of the invention can be administered in combination with an antibiotic that inhibits DNA gyrase, wherein the DNA gyrase binds between the individual helical strands of the replicating bacterial DNA. It is an enzyme involved in the formation of Thus, DNA gyrase is an enzyme required for normal replication of bacterial DNA, and therefore, antibiotics that inhibit DNA gyrase will inhibit normal replication of bacterial DNA.

Examples of antibiotics that inhibit DNA gyrase include nalidixic acid, oxophosphoric acid, sinoxacin, and quinolone antibiotics (ciprofloxacin, norfloxacin, ofloxacin, enoxacin, pefloxacin, lomefloxacin, fleroxacin, tosulofloxacin, The invention is further illustrated by the following specific examples, which are to be considered as illustrative of the invention and considered as limiting the same. should not be done.

【Example】

Example 1 Table I below shows the S.P. aureus strain ATCC 25923 (S)
, P. aeruginosa strain ATCC 27853 (P); E. coli strain A
TCC 25922 (E), and C.I. The minimum inhibitory concentration (MIC) for Albicans (CA) is shown in μg / ml. "D" indicates that each amino acid residue is a D amino acid residue or a glycine residue. Whether the peptide is unsubstituted at the N-terminus, or substituted at the N-terminus with an acetyl group as shown by Ac-,
Whether the N-terminus is substituted with an octanoyl group as shown by Oct-,
As shown by-, substituted by a succinyl group as shown by Suc-, substituted by a hexanoyl group as shown by Hex-, or as shown by Hep- Substituted with a heptanoyl group, substituted with a valeryl group as shown by Val-, substituted with a myristoryl group as shown by Myr-, or by an ibuprofile group as shown by Ibu-. Has been replaced.

The procedure for the antimicrobial assay is described in the National Committee for C
linear Laboratory Standards, Documen
t M7-T2, Volume 8, No. 8, 1988, which is hereby incorporated by reference in its entirety. Stock solutions of peptides with or without corresponding substitutions were prepared in sterile deionized distilled water at a concentration of 512 μg / ml and stored at -70 ° C. Serial dilution of peptide stock solution into wells of microtiter plate (1
: 2) and the final concentration of peptide in the wells was 0.25, 0.50, 1, 2
, 4, 8, 16, 32, 64, 128 and 256 μg / ml. S. of 1~5 × ¹⁰ 5 CFU / ml aureus strain ATCC 25923;
coli strain ATCC 25922; aeruginosa strain ATCC 2785
3, or C.I. albicans was added from mid-log culture to each well in full strength Mueller Hinton broth (BBL 11443). The inoculum was normalized by spectrophotometer at 600 nm and confirmed by colony count. Plates were incubated at 37 ° C for 16-20 hours and the minimum inhibitory concentration (MIC) was determined for each peptide. The minimum inhibitory concentration is defined as the lowest concentration of peptide that results in a clear well in a microtiter plate. The minimum inhibitory concentrations of each of the peptides with and / or without corresponding substitutions are shown in Table I below. From the above results, it can be seen that when a biologically active peptide is substituted with the lipophilic group of the present invention, the peptide has enhanced biological activity against various microorganisms.

Example 2 gingivalis, S .; mutans or A.M. Viscosus stock cultures were prepared using hemin and vitamin K ₁ (BBL, Cockeysville).
, MD) on a Brucella blood agar plate and at 37 ° C for 8 hours.
Under anaerobic conditions with an atmosphere of 0% N ₂ -10% H ₂ -10% CO ₂ (Coy
Anaerobic Chamber, Ann Arbor, MI). The experimental culture was Brain Heart Infusion (BHI) broth (BBL)
, Cockeysville, HD) + hemin (2.5 mg / l) (Sigma Chemical Co., St. Louis, IL) + vitamin K1 (0.
25 mg / l) (Sigma Chemical Co., St. Louis,
MO). For the susceptibility test, cultures were harvested from pros cultures the whole day (24 hours), diluted with fresh BHI broth (+ hemin + vitamin K1) and 1 × 10 ⁶ colony forming units in each microtiter test well. (CFU) / ml was supplied.

The antimicrobial susceptibility test was performed using the National Committee for
Clinical Laboratory Standards (NCCLS)
(Document M11-T2, 1989 is incorporated herein by reference). Microtiter plates (Corning, Corning, NY) are available from Beckman Biomek.
1000 robotic device (Beckman Instruments, Palo
(Alto, CA) and aseptically filled with BHI broth (+ hemin + vitamin K ₁ ) to a volume of 100 μl. The peptide was 100 μl of 1.024 m
Tested in duplicate lanes by manually adding g / ml aqueous peptide solution (W / V) to the top well of a microtiter plate lane. The peptide is
Beckman Biomek 1000 (Beckman Instrument)
(nts, Palo Alto, Calif.), serially diluted 1: 2 by mixing and moving from the upper well of the lane to the lower well below. The last 100 μl from the lower well was discarded. To obtain the final peptide dilution from 0.25 μl / ml, 100 microtiter bacteria were added to each test well in BHI (+ hemin + vitamin K ₁ ). Plates are 24-48 at 37 ° C
Time, incubated in anaerobic chamber. After incubation
The minimum inhibitory concentration (MIC) as the lowest concentration of peptide that inhibited growth was determined by visual inspection and optical density read at 630 nm on a Dynatech MR5000 microtiter plate reader. The results are shown in Table II below.

Example 3 CD-1 male mice (average body weight 22.8 g) were treated with live E.coli mice. coli strain 21
915-1 (2.3 × 10 ⁵ CFU mice) were inoculated by intraperitoneal injection.
Next, oct- (SEQ ID NO: 143) -NH _2, were intravenously injected from the tail vein after 1 hour and after 5 hours of inoculation. Control mice were inoculated and treated with 0.9% saline. Each different treatment group had 10 mice per group. All control mice died. Oct- treatment dose of (SEQ ID NO: 143) -NH ₂ as a whole in 1, 5, 10 and 20 mg / kg, 20% respectively, the survival rate at six days after inoculation, 40%, 90% and 90% Met.

Example 4 E. coli serotype 0111: Lipopolysaccharide from B4 (0.1 μg or 0.1 μg).
5 [mu] g / mouse) and a solution of galactosamine (8 mg / mouse) of about 2 minutes prior to injection into the abdominal cavity, oct- (SEQ ID NO: 143) -NH ₂ is male C57BL
/ 6J mice (average body weight 20.1 g) were injected intravenously. Oct- treatment dose of (SEQ ID NO: 143) -NH ₂ is 0.5,7.5,10,12.5 or 15m
g / kg (10 mice / group), when administered prior to 0.5 μg of lipopolysaccharide / mouse, the survival rate 5 days after lipopolysaccharide administration was 10%, respectively.
0%, 30%, 0%, 50% and 60%. 0.1 μg of these doses
When performed prior to administration of lipopolysaccharide / mouse, the results were 40%, 90%, 100%, 100% and 10%, respectively, 5 days after lipopolysaccharide administration.
0% survival rate.

Example 5 1.68 mg of (1-ethyl-2- (3 [1-ethylnaphthol (1,2-d
) -Thiazoline-2-ylidene] -2-methylpropenyl) naphthol- (1
, 2-d) -thiazolium bromide (Signa E-7762) in 5 ml of 200
In addition to proof methanol, a 0.6 mM dye stock solution (10 ×) was prepared. 1 ml of this solution is added to 9 ml of ethanol to add 0.06 mM of dye (60
μM dye) was obtained. E. FIG. A stock solution of lipopolysaccharide (LPS) from E. coli serotype 0111: B4 was prepared at 1.5 mg / ml. 400 μl of this solution was mixed with 4.6 ml of pyrogen-free water to obtain a 120 μg / ml solution.

Rows 1 and 3 to 12 of the microtiter plate were filled with 100 μl of pyrogen-free water or 10 mg / ml bovine serum albumin. Next, 200 μl of peptide was added to the second row of the microtiter plate at a concentration of 1 mg / ml. Add 200 μl of pyrogen-free water to two lanes (dye and LPS
With no peptide or with dye but without LPS or peptide) was added to each of the control wells. Next, 100 μl were serially diluted from the second row to the twelfth row of the microtiter plate. 50 μl of PBS (P
H7.4) and 50 μl of LPS solution were added to the first row (blank well) of the plate. Mix equal volumes of LPS solution, dye, and PBS (PH 7.4, approximately 150 mM) to form a die buffer LPS mixture with a final concentration of 40 μg / ml LPS and a final concentration of 20M dye. The dye buffer LPS mixture was then incubated for 10 minutes at room temperature in the dark.

Next, 100 μl of the die LPS buffer mixture was added to all wells of the microtiter plate except for blank wells and to control lanes without LPS or peptide. Plates are incubated for 10 minutes at room temperature in the dark and 460
The absorbance at nm and 510 nm were read. From these absorbances, the concentration of the peptide in mg / ml needed to prevent 50% binding of the lipopolysaccharide to the dye
LPS50 value was calculated.

The above procedure was performed on the peptides listed in Table III below, but demonstrates that N-terminally modified antimicrobial peptides also block LPS binding to hydrophobic dyes.

Example 6 General Method for Preparing Sodium Methanesulfonate Derivatives of N-Terminal-Modified Peptides The free base of N-terminal modified peptides was prepared by neutralizing the acetate or trifluoroacetate salt with saturated sodium carbonate solution. Generated. The precipitated peptide was separated by centrifugation or filtration. The formaldehyde-sodium bisulfite compound was prepared by dissolving 5 g of sodium bisulfite in a mixture of 75 ml of water and 4 ml of 35-40% formaldehyde by stirring. The solution was cooled and ethanol was added. The resulting precipitate was filtered, washed with water and dried. The product was recrystallized from water-ethanol. The peptide free base is suspended in water and the reaction mixture is stirred while the formaldehyde-sodium bisulfite complex (Aldrich # 11,270-4; Milwaukee)
, WI) (1.1-3 equivalents for each free amino group) was added slowly. About 1
After stirring for 5-30 minutes, the solution was filtered and lyophilized.

Example 7 The in vitro antimicrobial activity of the methanesulfonic acid derivative was measured according to the method described in Examples 1 and 2. S. aureus, E.A. coli, P.E. ae
ruginosa, and C.I. Albicans activity against the ATCC strain and red blood cell hemolysis were compared to the parent compound (Table IV). P. isolated from a patient with cystic fibrosis aeruginosa against a clinical strain of compound 843 (
Oct-SEQ ID NO: 143) The activity of methanesulfonate was compared to the parent compound (Table V). P. The activity of compound 469 (Oct-SEQ ID NO: 27) against gingivalis was compared to the parent compound (Table VI). These results indicate that the methanesulfonic acid analog has antimicrobial activity comparable to the parent compound.

Example 8 Effect of the Addition of Methanesulfonate on the Maximum Tolerated Dosage of Compound 843 (Oct-SEQ ID NO: 143) in Mice Summary Mice were treated with a single dose of Compound 1324, a methanesulfonic acid analog of Compound 843. They received intravenous dosing and were monitored for survival for at least 4 days. 180mg
There were no deaths at doses less than or equal to / kg. This indicates that chemical modification of compound 843 to produce compound 1324 increases safety by a factor of nine. Objective To estimate the 50% lethal dose (LD50) of compound 1324 sodium salt by measuring the maximum tolerated dose (MTD) when a single intravenous dose was administered to mice. Materials and Methods Animals: At the beginning of the study upon arrival, the average weight of three groups of 37 male CD-1 mice (Charles River Lab) was 39, 28 and 28 g, respectively. Materials: Test article: Compound 1324 Active part 47% Vehicle: 0.9% saline Solution preparation: 120 mg / ml of active compound 1324 (active part corrected) was made in saline. All concentrations from 5 to 80 mg / ml diluted the 120 mg / ml solution with saline. The highest concentration used for intravenous administration was 20 mg / ml. Protocol: Mice were randomly assigned to one of 10 groups (4 mice per group, but only the highest dose group was one). Mice are 0, 50, 60, 70
, 80, 140, 160, 180, 200 mg / ml for 10 ml /
kg of 0 (saline), 5, 6, 7, 8, 14, 16, 18, or 20 mg / kg
ml solution was administered intravenously. One mouse received a 14.1 ml / kg 20 mg / ml solution versus a 282 mg / kg dose. Mice were monitored for survival for at least 4 days post-dose. Results: See Table VII showing viability. All mice receiving intravenous administration of compound 1324 at a dose of 180 mg / kg or less survived. Three of the four mice receiving 200 mg / kg survived. One mouse receiving 200 mg / kg and one mouse receiving 282 mg / kg died.

[0119]

Example 9 Alternative Method for Preparing Sodium Methanesulfonate Derivatives of Cationic Peptides Using this method, sodium methanesulfonate derivatives of SEQ ID NOs: 154-156 were prepared. Peptide salts (hydrochloride, acetate or trifluoroacetate) are taken up in water and more than 30% of a neutral formaldehyde solution (up to 12.5 equivalents for each amino group of the peptide) and an excess of 1M sodium bicarbonate solution (Up to 6.25 equivalents for each amino group of the peptide). The precipitated peptide adduct of formaldehyde was separated by centrifugation or filtration, washed with water and dried. The formaldehyde adduct was suspended in water and excess sodium metabisulfite was added (up to 1.9 equivalents for each amino group of the peptide) to form the methanesulfonic acid derivative. The clear solution was filtered through a 0.2 μm filter and lyophilized. Elemental analysis, mass spectra and antimicrobial activity were determined for these derivatives.

Example 10 In Vitro Antitumor Activity of Peptide Methanesulfonic Acid Derivatives Compared to Non-Induced Peptide Cell Lines: A-549 (ATCC), a human lung cancer cell, and MDA-4, a human breast cancer cell
35 (from Sandoz) refers to Ham's F supplemented with 10% fetal bovine serum.
Cultured in 12K medium (GIBCO-BRL). K-562 (ATCC), a human chronic myeloid leukemia cell, contains RP supplemented with 10% fetal bovine serum.
Maintained in MT1640 medium (GIBCO-BRL). The human melanoma cell line WM1617 (from Wistar Institute) was maintained in 2% tumor culture.

Cytotoxicity Assay Cytotoxicity assay was performed according to the manufacturer's instructions, CytoLite (Packard Instrument Company).
Was performed on the above cell lines. Briefly, 2 × 10 ⁴ cells / well in black 96-well ViewPla in 100 μl / well culture medium
tes (Packard Instrument Company). Next, a serial dilution of the Magainin peptide was added to the cells and the plates were incubated for 24 hours at 37 ° C. in a humidified 5% CO ₂ atmosphere.
After the incubation, 25 μl / well of the CytoLite activator solution was added, followed by 125 μl / well of the amplifier solution.
) was added. TopCount Microplate Sin
tillation and Luminescence counter (Pack
ard Instrument Company) at 25 ° C.
Luminescence was measured using a one second count time in the (single photon count) mode. All assays were performed in triplicate. Peptides are tested in this assay, MSI-78 (SEQ ID NO: _{154 (-NH 2)), MSI} -1858 ( SEQ ID NO: 154 (-NH ₂ methanesulfonate)), MSI-344 (SEQ ID NO: 15
4 (-OH)) and MSI-1857 (SEQ ID NO: 154 (-OH methanesulfonate)). The results of these assays are shown in Figure 2-9, the corresponding IC ₅
The values of ₀ are shown in Table VIII. In all cases, the activity of methane sulfonated peptide, as is clear from both the survival curve and an IC ₅₀ value, was good in the parent peptide greater than or equal.

Thymidine Uptake Assay 2 × 10 ⁴ cells / well were seeded in 96-well tissue culture plates in 100 μl / well medium and left overnight at 35 ° C. in a humidified 5% CO ₂ atmosphere. You. Next, a serial dilution of the magainin peptide is added to the cells and the plate is incubated for 18 hours. After incubation, 0.2 [mu] Ci / well of [< ³ > H] -thymidine is added to the plate and further incubated at 37 [deg.] C for 6 hours. Cells were harvested ^{[3 H]} - thymidine binding is measured by liquid scintillation counting. All assays are performed in triplicate.

[0124]

Example 11 MSI-344 (SEQ ID NO: 154 (-OH)) and MSI-18 in mice
Investigation of the maximum tolerated dose (MTD) of a single dose (intravenous or intraperitoneal) of 57 (SEQ ID NO: 154 (-OH methanesulfonate)) Summary MSI- in 100 CD-1 ^* BR mice 344 or MSI-1857
The maximum tolerated dose (MTD) of a single intravenous (iv) or intraperitoneal (ip) administration of was measured. A single intravenous (iv) dose is given by MSI-
It was 10 mg / kg for 344 and 60 mg / kg for MSI-1857. The MTD for a single intraperitoneal (ip) dose was less than 20 mg / kg for MSI-344 and between 50 and 100 mg / kg for MSI-1857. The estimated LD50 for intravenous administration (the dose that causes lethality in 50% of the injected animals) is the MSI-
344 and MSI-1857 were 12.5 and 73 mg / kg, respectively. LD
50i. p. Is 23.5 for MSI-344 and MSI-1857, respectively.
And 120 mg / kg. These data indicate that methanesulfonation of MSI-344 makes the compound administered in vivo about 5-fold safer than MSI-344.
Indicates an increase. Objective MSI-344 and MSI- administered once intravenously or intraperitoneally to mice
Determining the MTD and LD50 estimates of 1857. Materials and Methods Animals: 100 male CD-1 ^* BR mice (Charles River)
Lab) arrived in two groups, and body weights at the start of the study ranged from 20 to 26.3 g. Solution Preparation: A 5 mg / ml solution of MSI-344 was prepared in saline (0.9% sodium chloride injec) corrected for peptide components.
Tion USP, Abbott, North Chicago, IL). MSI-344 at concentrations of 4, 3, 2, 1.5, 1, and 0.5 mg / ml
Was made by diluting the 5 mg / ml solution using saline as diluent. (Because there was negligence in the solution preparation of the 4, 3, and 2 mg / ml solutions,
Groups of animals that received these solutions, ie groups 12, 13 and 1
Results from 4 were excluded from the survey analysis). A 20 mg / ml solution of MSI-1857 was made in saline. M at concentrations of 15, 10, 8, 6 and 5 mg / ml
A solution of SI-1857 was made by diluting the 20 mg / ml solution using saline as diluent. Another 5 mg / ml solution of MSI-344 was made in saline corrected for peptide components. 4, 3 and 2 mg / ml solutions were made by diluting the 5 mg / ml solution using saline as diluent. A 15 mg / ml solution of MSI-1857 in saline was made corrected for the peptide component. The 10 mg / ml solution was made by diluting the 15 mg / ml solution with saline. Protocol mice (4 mice per treatment group) were prepared at the appropriate solution concentration of 10 ml / k
MSI-344 or MSI-1857 was administered in a single dose intravenously or intraperitoneally using an infusion of g. If the first two mice in one group died, no other mice were infused with this dose of the compound (for moral reasons). Survival numbers were monitored daily for at least 8 days post-dose. The study plan is shown in Tables IX and X. Toxicity Survey # 262 is a supplement to Toxicity Survey # 261.

[0126]

[0129] Results See Table XI for survivor count results. MS of 10 mg / kg or less
All animals that received I-344 intravenously survived, and all animals that received 60 mmg / kg MSI-1857 survived. 50 mg / kg MS
All animals that received I-1857 intraperitoneally survived. From the dose response curve (FIG. 10), the LD50i. v.
Was found to be about 12.5 and 73 mg / kg, respectively. Dose response curve (
FIG. 11) shows that LD50i. p
. Was found to be about 23.5 and 120 mg / kg, respectively.

[0128] Conclusions MSI-344 and MSI-1857 (methanesulfonated MSI-34)
The maximum tolerated dose (MTD) for intravenous administration of 4) was 10 and 60 mg / kg in mice, respectively. The MTD for intraperitoneal administration of MSI-344 and MSI-1857 was <20 and 50 mg / kg, respectively. For intravenous administration of MSI-344 and MSI-1857 in mice, the approximate dose that resulted in lethality (LD50) in 50% of the recipient animals was 1
2.5 and 73 mg / kg. The LD50 for intraperitoneal administration of MSI-344 and MSI-1857 were approximately 23.5 and 120 mg / kg, respectively. These results indicate that methane sulfonation of MSI-344 results in a significant improvement (about a 5-fold increase) in the safety profile compared to MSI-344.

Example 12 MSI-344 (SEQ ID NO: 154 (-OH)) and MSI-1 in mice
Investigation of the maximum tolerated dose (MTD) of repeated intraperitoneal administration of 857 (SEQ ID NO: 154 (-OH methanesulfonate)) Summary Repeated intraperitoneal injection of MSI-344 and MSI-1857 in 60 SCID mice The measurement of the maximum tolerated dose (MTD) of ip) administration was performed. Mice were injected twice or three times weekly with various doses of MSI-344 or MSI-1857. Survival numbers and body weight were monitored. The twice-weekly dosing schedule includes MSI
-344 and MSI-1857 have an MTD of 12 and 134 mg / k, respectively.
g / week. This indicates that MSI-1857 has an 11-fold increase in safety index over MSI-344. In the three-weekly dosing schedule, no deaths occurred in any group, so MSI-344 and MSI-1857
High dose groups received 90 mg / kg / week and 16 mg / kg / week, respectively. Transient weight loss was noted in most MSI-344 and MSI-1857 groups after the initial dose. On a three-weekly dosing schedule, none of the gpurs produced more than 6% weight loss. From this result, MSI-3
It can be seen that the methanesulfonation of 44 improves the safety profile compared to MSI-344 in a long-term dosing regime.

Objectives MSI-344 when SCID mice were administered intraperitoneally in a multiple dose regimen.
And determining an estimate of the MTD of MSI-1857. The selection of SCID mice was based on a future plan to use this strain as a host in a xenograft human potency animal model and was a prerequisite for determining the MTD of long-term dosing used in that potency model . Materials and Methods Animals: Two groups of 60 SCID male mice (Fox Chase
C. B-17 / IcrTac-scidfDf, Taconic Labs) at the start of the study ranged from 19.6 to 25.8 g. SCID mice were rendered immunodeficient and therefore housed in a sterile environment and tested. Mice were fed sterile water and a sterile diet ad libitum. Solution preparation: A 1 mg / ml solution of MSI-344 was prepared in saline (0.9% sodium chloride inject) corrected for peptide components.
ion USP, Abbott, North Chicago, IL). Solutions of MSI-344 at concentrations of 0.6, 0.3 and 0.1 mg / ml were
It was made by diluting its 1 mg / ml with brine as diluent. MSI-1
A 10 mg / ml solution of 857 was made in brine. Solutions of MSI-1857 at concentrations of 6.7, 3.3, 3, 2, and 1 mg / ml were made by diluting the 10 mg / ml solution with brine as diluent. A 4.5 mg / ml aqueous solution of metabisulfite (Aldrich Co.) was prepared. The solution was updated on a weekly basis.

Protocol mice (4 mice per dose group) were dosed twice weekly (groups 1 to 7) or three times weekly (groups 8 to 15) for 20 weeks each week at the appropriate solution concentration. MSI-344 or MS using an injection volume of 10 ml / kg
I-1857 was administered intraperitoneally. Survivors were monitored daily and body weight was recorded three times a week. A preliminary analysis is performed in the third week and the results of this preliminary analysis are presented in this report. The study plan is shown in Table XII.

[0133]

Results See FIG. 12 for survival results for the twice weekly dosing schedule. All animals in all groups dosed three times a week survived. The maximum tolerated dose of MSI-1857 is 134 mg / kg / week on a twice weekly dosing schedule (ie, twice weekly injection x 67 m
g / kg / injection). With the same schedule, the MTD of MSI-344 is 12m
g / kg / week. The MTD for the MSI-1857 and MSI-344 three-weekly dosing schedules were ≧ 90 and 18 mg / kg / week, respectively. The average weight is shown in FIGS. In the twice weekly dosing schedule, there was a temporary loss of body weight in all groups compared to saline after the first dose, but recovered in the medium and low dose groups. In the three weekly dosing schedule, there was a temporary loss of body weight in the medium and high dose groups, but the weight gain recovered after the first week of treatment. Both regimens had a dose dependent effect on body weight. Sodium metabisulfite is 135 mg / kg on a three-weekly infusion schedule
/ Week. This dose is 90 mg / kg / week administered MSI-18.
Equivalent to the amount of sodium metabisulfite used in the preparation of MSI-1857 in 57 groups. There were no fatalities in this group and there was no effect of sodium metabisulfite on body weight.

Conclusions A twice weekly intraperitoneal dosing schedule for three consecutive weeks indicated that MSI-1857 (13
The maximum tolerated dose (MTD) at 4 mg / kg / week was MSI-3 in SCID mice.
11 times greater than 44 (12 mg / kg / week). On a three-week intraperitoneal dosing schedule, the MTD of MSI-1857 is 90 mg.
/ Kg / week greater than or equal to, the MTD of MSI-344 is 18 mg / kg / week.
Weeks were good or big. Initial doses of MSI-344 or MSI-1857 resulted in dose-dependent transient weight loss in all surviving groups except the low dose group on a three-weekly dosing schedule. In the three-weekly dosing schedule, the maximum weight loss for all groups was less than 6% of the initial weight. Sodium metabisulfite was administered in an amount corresponding to the metabisulfite included in the MSI-1857 group administered at 90 mg / kg / week (the dose was corrected for the peptide component). . The toxicity or change observed in the MSI-1857 group may have been due to administration of the peptide or, if any free bound metabisulfite was in solution, due to administration of metabisulfite. Maybe. If metabisulfite had no effect on mortality or body weight, this would mean that the temporary weight loss observed in the MSI-1857 group was not due to free metabisulfite. Would. Furthermore, complete release of bound metabisulfite from the high dose group will not have any deleterious effect on mortality or weight loss. The overall results from this study indicate that methanesulfonation of MSI-344 significantly improved the safety profile (11-fold increase) during long-term administration compared to MSI-344.
Indicates that

Example 13 Pharmacokinetic Study of a Single Administration of MSI-1324 (Oct-SEQ ID NO: 143-NH ₂ Methanesulfonate) in Mice Summary Drug of MSI-1324 in mice using a single administration A dynamic study was conducted.
Thirty-two CD-1 male mice were injected with MSI-1324 sodium at a dose of 140 mg / kg (corrected for the peptide component). Mortality blood samples were collected from 2 to 4 mice at 0 (pre-dose), 5, 15, 30, 60 minutes and 2, 4, 6, 24 and 48 hours post-dose. Plasma concentrations were estimated by bioassay. The peak activity was observed 5 minutes after administration and was below the detection limit of the assay at 60 minutes. Objective To determine the pharmacokinetic profile of MSI-1324 after intravenous administration in mice. Materials and Methods Animals: Forty CD-1 ^* BR mice (Charles River Lab) with an average weight of 25.6 g at the start of the study were used. Solution preparation: A 14 mg / ml solution of MSI-1324 was prepared in saline (0.9% sodium chloride inje) corrected for peptide components.
(Cable USP, Baxter). Experimental design: 36 mice, MSI at a dose of 140 mg / kg corrected for the peptide component using a 10 mg / kg injection of a 14 mg / ml solution
-1324 was administered once intravenously. Four mice received no infusion and received pre-dose blood samples. Blood samples at death were 0 (before administration), 5, 1
Halocein anesthetized mice were harvested by cardiac puncture at 5, 30, 60 minutes and 2, 4, 6, 24 and 48 hours. Two to four mice were bled periodically at each time point. Blood samples were centrifuged and plasma was transferred to separate tubes for bioassay analysis. The bioassay used was antimicrobial zone clearing (disk diffusion). Results: FIG. 15 shows the E. coli of MSI-1324 used in the bioassay. col
3 shows a standard curve of i-zone clearing. FIG. 16 shows the plasma concentration of mice at each time point after administration of 140 mg / kg (estimated from bioassay). Five minutes after administration (first sampling time after administration), a peak concentration of about 600 μg / ml was observed. At 60 minutes post-dose, the concentration is the limit of detection of the bioassay (about 1
50 μg / ml) or less. The inset graph in the figure indicates that there is no detectable level at a later point in time. Conclusion: MSI-1324 showed a peak concentration 5 minutes after 140 mg / kg iv in mice. Plasma levels were below the limit of detection after 60 minutes.

Example 14 In Vivo Human Tumor Xenograft Model For a human lung tumor model, a female nude mouse weighing about 20 g was treated with the nude mouse host S.D. C. A strip of SK-MES human lung tumor taken from a growing tumor is implanted with a trocar, and for a human breast tumor model, 1EE6 MDA-MB-435 cells are injected from the culture medium into the mammary fat pad (mpf) of a mouse. .
When the tumors reach about 5 mm x 5 mm (about 10-14 days after inoculation), the animals are grouped in pairs into a treatment group and a control group. Each group contains 10 tumor-bearing mice, each of which is individually tagged and ear-tagged throughout the duration of the experiment. Drug or vehicle administration begins when the animals are grouped in pairs (day 1). Mice are administered intraperitoneally twice or three times weekly.

Mice are weighed twice weekly and tumor measurements are taken twice weekly with calipers starting on day 1. These tumor measurements are converted to mg tumor weight by the well-known formula L5 x W / 2. The experiment showed that the control tumor was 1 gram in breast tumor,
For lung tumors, it ends when it reaches 2 grams in size. After termination, all mice are weighed, sacrificed and the tumor removed. Tumor weights are measured and the average tumor weight per group is calculated. In this model, tumor growth inhibition for each group is determined by subtracting the mean treated tumor weight / mean control tumor weight x 100 (T / C) from 100%. Some drugs cause tumor shrinkage in this human tumor xenograft model. For these drugs, the final weight of a given tumor is subtracted from its own weight at the start of treatment on day 1. This difference divided by the tumor weight is the% shrinkage. Mean% shrinkage can be calculated from data from mice in the group where tumor regression occurred.
If the tumor has completely disappeared from a mouse, this is considered complete regression or complete tumor shrinkage. If desired, partially or completely regressed mice can be kept alive after termination to determine if they survive to long-term, tumor-free survival.

Statistical processing of data is mainly performed using a log-rank p-value test. The peptides or proteins of the present invention may be administered alone or in combination with drugs, such as ions having pharmacological properties, antibiotics, or other bioactive peptides or proteins described herein. Alternatively, it can be used in a variety of pharmaceutical compositions in combination with excipients, non-toxic buffers, or non-toxic pharmaceutical carriers such as saline, or vehicles. Such pharmaceutical compositions can be used topically or systemically and can be liquids, solids, semi-solids, injectables, tablets, ointments,
It can be used in any suitable form, such as a lotion, paste, capsule or the like. The peptides or proteins and / or the above agents may be used in combination with adjuvants or protease inhibitors, or the combination may control infections caused by harmful microorganisms including protozoa, viruses, parasites, fungi and the like. Can also be used in combination with affinity agents that are considered desirable or advantageous.

The peptide or protein may be administered to a host, especially an animal, in an antibacterial and / or antitumor and / or antiviral and / or antimicrobial and / or spermicidally effective amount and / or an antifungal amount. Administered in an effective amount and / or an antiparasitic effective amount, in an amount effective to promote host wound healing, or in an amount effective to treat host sepsis or septic shock. Can be. The peptide or protein may be administered alone, or may be administered with ions having pharmacological properties, antibiotics, or the ion channel forming peptide or protein described above. When the peptide or protein is administered together with ions having pharmacological properties, the activity of the peptide or protein is enhanced. When the peptide or protein is administered together with the above-mentioned drug, the peptide and the drug can be administered in separate forms. For example, the agent can be administered systemically and the peptide or protein can be administered locally. When administering the peptide or protein topically, it may be administered with a water-soluble vehicle in the form of an ointment, cream, lotion, paste or the like. Examples of water-soluble vehicles used include, but are not limited to, glycols such as polyethylene glycol, hydroxycellulose, and KY jelly. Preferably, the water-soluble vehicle does not contain an oily substance. The peptides or proteins may also be used in the form of oral compositions for oral hygiene, alone or with ions having the above-mentioned pharmacological properties. Such compositions can be incorporated into various compositions and materials used for oral hygiene, including but not limited to toothpastes, mouthwashes, gel-like toothpastes, and toothpastes. Accordingly, such compositions can be used to treat or prevent periodontal disease, prevent or reduce plaque or gingivitis, and / or prevent, treat or reduce caries. Peptides and ions with pharmacological properties are Streptococcus associated with caries and periodontal disease
It may be used to suppress, prevent, or destroy mutans growth.

However, it is understood that the scope of the present invention should not be limited to the particular embodiments described above. The invention may be practiced other than in the detailed description, which is also within the scope of the appended claims.

[Sequence list]

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be more completely understood when considering the following specific examples with reference to the accompanying drawings, in which: FIG.

1 is a comparison of the maximum tolerated dose of compound 843 and its methanesulfonic acid derivative, compound 1324 (see Example 8, Table VII).

FIG. 2: Survival curve of A-549 human lung cancer cells treated with MSI-78 (SEQ ID NO: 154 (—NH ₂ )) and MSI-1858 (SEQ ID NO: 154 (—NH ₂ methanesulfonate)) FIG.

FIG. 3 depicts the survival curve of A-549 human lung cancer cells treated with MSI-344 (SEQ ID NO: 154 (-OH)) and MSI-1857 (SEQ ID NO: 154 (-OH methanesulfonate)). FIG.

FIG. 4 depicts the survival curve of MDA-435 human breast cancer treated with MSI-78 (SEQ ID NO: 154 (—NH ₂ )) and MSI-1858 (SEQ ID NO: 154 (—NH ₂ methanesulfonate)). FIG.

FIG. 5 shows the survival curve of MDA-435 human breast cancer cells treated with MSI-344 (SEQ ID NO: 154-OH) and MSI-1857 (SEQ ID NO: 154 (—OH methanesulfonate)). FIG.

[6] MSI-78 (SEQ ID NO: 154 _(-NH 2)) and MSI-1858 (SEQ ID NO: 154 (-NH ₂ methanesulfonate)) and de-treated in K-562 human chronic myelogenous leukemia cells It is a figure showing a survival rate curve.

FIG. 7: MSI-344 (SEQ ID NO: 154-OH) and MSI-1857 (SEQ ID NO: 15)
4 (-OH methanesulfonate)) and K-562 human chronic myeloid leukemia cells.

FIG. 8: MSI-78 (SEQ ID NO: 154-NH ₂ ) and MSI-1858 (SEQ ID NO: 15)
4 is a diagram showing a survival analysis of the (-NH ₂ methanesulfonate)) and de-treated WM1617 human melanoma cells.

FIG. 9: MSI-344 (SEQ ID NO: 154-OH) and MSI-1857 (SEQ ID NO: 15)
4 (-OH methanesulfonate)) with WM1617 human melanoma cells.

FIG. 10 shows the survival curve of CD-1 mice that received a single iv dose of MSI-344 (SEQ ID NO: 154-OH) or MSI-1857 (SEQ ID NO: 154 (—OH methanesulfonate)). FIG.

FIG. 11 shows the survival curves of CD-1 mice that received a single ip administration of MSI-344 (SEQ ID NO: 154-OH) or MSI-1857 (SEQ ID NO: 154 (—OH methanesulfonate)). FIG.

Figure 12 SC administered twice weekly with either MSI-344 (SEQ ID NO: 154-OH) or MSI-1857 (SEQ ID NO: 154 (-OH methanesulfonate))
It is a figure showing the survival rate curve of ID mouse.

Figure 13 SC administered three times a week with either MSI-344 (SEQ ID NO: 154-OH) or MSI-1857 (SEQ ID NO: 154 (-OH methanesulfonate))
It is a figure showing the weight curve of an ID mouse.

Figure 14: S administered twice weekly with either MSI-344 (SEQ ID NO: 154-OH) or MSI-1857 (SEQ ID NO: 154 (-OH methanesulfonate)).
It is a figure showing the weight curve of a CID mouse.

FIG. 15 shows a standard curve relating antimicrobial activity (zone diameter) to concentration of MSI-1324 (OCT-SEQ ID NO: 143 (—NH ₂ methanesulfonate)).

FIG. 16: MSI-1324 (OCT-SEQ ID NO: 143) in plasma after a single iv dose
It is a graph showing the concentration of -NH ₂ methanesulfonate)).

────────────────────────────────────────────────── ─── Continued on the front page (72) Williams, Taffy, J. United States, Pennsylvania, Lansdale, Colwin Terrace 103 (72) Inventor MacLaine, Michael United States of America, Pennsylvania, Bluebell, Ramsgate Court 64 F term (reference) 4C084 AA02 AA06 BA01 BA17 BA18 BA31 DA42 MA66 ZA362 ZB352 4H045 AA30 BA14 BA15 BA16 BA17 BA18 BA55 EA26 EA28 EA29

Claims (61)

[Claims] [1] The host has the following formula: Where X is a biologically active amphipathic peptide or protein, the peptide is an ion channel forming peptide or protein, T is a lipophilic group, and W is T or hydrogen. A method for treating sepsis or septic shock in a host, comprising administering an effective amount of an N-terminally substituted peptide having the same, a pharmaceutically acceptable salt thereof, or a methanesulfonic acid derivative or analog thereof. 2. The N-terminally substituted peptide has the structure: Oct-SEQ ID NO: 1.
The method of claim 1 having 43. Wherein X is a biologically active peptide, the peptide is an ion channel forming peptide, X has the following structure _{X 50: -R 41 -R 42 -R} 42 -R 41 -R 42 -R 42 - R ₄₁ -R ₄₁ -R _4
2 -R ₄₁ -R ₄₁ - _{(wherein, R 41} is a hydrophobic amino _{acid, R 42} is a hydrophilic amino acid of the basic hydrophilic or neutral) a; and T is a lipophilic group Yes, T The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. Wherein X comprises formula _Y 50 _{-X 50, wherein, Y 50 is: (i) R 41; (} ii) R 42 -R 41; or _(iii) R _{42 -R 42 -R 4.} The method of claim 3, wherein the number is ₄₁ . Wherein X is a biologically active peptide, the peptide is an ion channel forming peptide, X has the following structure _{X 52: -R 42 -R 41 -R} 42 -R 42 -R 41 -R 41 - R ₄₂ -R ₄₂ -R _4
1 -R ₄₂ -R ₄₂ - _{(wherein, R 41} is a hydrophobic amino _{acid, R 42} is a hydrophilic amino acid of the basic hydrophilic or neutral) a; and T is a lipophilic group Yes, T The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. 6. X comprises the structural formula Y ₅₂ -X ₅₂ , wherein Y ₅₂ is: (i) R ₄₂ ; (ii) R ₄₁ -R ₄₂ ; (iii) R ₄₁ -R ₄₁ -R _{42. ; (iv) R 42 -R 41} -R 41 -R 42; a or _{(v) R 42 -R 42 -R} 41 -R 41 -R 42, a method according to claim 5. 7. X comprises the structural formula Y ₅₂ -Z ₅₂ , wherein Z ₅₂ is: (i) R ₄₁ ; (ii) R ₄₁ -R ₄₁ ; (iii) R ₄₁ -R ₄₁ -R ₄₂ ; _{(iv) R 41 -R 41 -R} 42 -R 42; a or _{(v) R 41 -R 41 -R} 42 -R 42 -R 41, a method according to claim 5. 8. X is a biologically active peptide, the peptide is an ion channel forming peptide, X has the following structure _{X 62: -R 41 -R 42 -R} 42 -R 41 -R 42 -R 42 - R _{41 -} (wherein, R ₄₁ is a hydrophobic amino acid, R ₄₂ is a is a hydrophilic amino acid of the basic hydrophilic or neutral) a; and T is a lipophilic group, T is The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. 9. X comprises the structure Y ₆₂ -X ₆₂ , wherein Y ₆₂ is: (i) R ₄₁ ; (ii) R ₄₂ -R ₄₁ ; (iii) R ₄₃ -R ₄₂ -R ₄₁ ; _(iv) a _{R 41 -R 42 -R 42 -R 41} , a method according to claim 8. 10. The method of claim 1, wherein X is a biologically active peptide having the following structure: (SEQ ID NO: 143). 11. X comprises the structure X ₆₂ -Z ₆₂ , wherein Z ₆₂ is: (i) R ₄₁ ; (ii) R ₄₁ -R ₄₂ ; (iii) R ₄₁ -R ₄₂ -R ₄₂ ; _(iv) a _{R 41 -R 42 -R 42 -R 41} , a method according to claim 8. 12. X have the structural formula _{(Y 62) a -X 62 -} (Z 62) comprises a _b, wherein _{Y 62 is: (i) R 41; (} ii) R 42 -R 41; (iii) R ₄₂ -R ₄₂ -R _41; or _(iv) a _{R 41 -R 42 -R 42 -R 41} , Z 62 _{is: (i) R 41; (} ii) R 41 -R 42; (iii) R ₄₁ -R ₄₂ -R _42; or _(iv) a _{R 41 -R 42 -R 42 -R 41} , a is 0 or 1, b is 0 or 1, the method of claim 8. 13. X is a biologically active, amphiphilic peptides, the peptide is an ion channel forming peptide, X has the following structure _{X 64: -R 42 -R 42 -R} 42 -R 41 -R 41 - R ₄₂ -R ₄₂ -R _41-wherein R ₄₁ is a hydrophobic amino acid and R ₄₂ is a basic hydrophilic or neutral hydrophilic amino acid; and T is a lipophilic group. Yes, T The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. 14. The method of claim 13, wherein X comprises the structure Y ₆₄ -X ₆₄ , wherein Y ₆₄ is: (i) R ₄₁ ; or (ii) R ₄₂ -R ₄₁ . 15. X comprises the structure _X 64 _{-Z 64, wherein, Z 64 is: (i) R 42; (} ii) R 42 -R 42; or _{(iii) R 42 -R 42 -R} 41 14. The method of claim 13, wherein 16. X comprises the structural formula: (Y ₆₄ ) _a -X _64- (Z ₆₄ ) _b , wherein Y ₆₄ is: (i) R ₄₁ ; or (ii) R ₄₂ -R ₄₁ , Z ₆₄ is: (i) R ₄₂ ; (ii) R ₄₂ -R ₄₂ ; or (iii) R ₄₂ -R ₄₂ -R ₄₁ , a is 0 or 1 and b is 0 or 1 14. The method of claim 13. 17. X is a biologically active amphipathic peptide, said peptide being
X is an on-channel forming peptide, and X has the following structure X₆₆: -R₄₁-R₄₂-R₄₂-R₄₁-R₄₁-R₄₆-R₄₂-R₄₁-R_4
2-R₄₂-R₄₁  (Where R₄₁Is a hydrophobic amino acid and R₄₂Is basic hydrophilic or neutral
A hydrophilic amino acid and R₄₆Is glutamic acid); and T is a lipophilic group;Wherein R is a hydrocarbon having at least 2 carbon atoms.
The described method. 18. X is a biologically active amphipathic peptide, wherein said peptide is
X is an on-channel forming peptide, and X has the following structure X₆₆: -R₄₂-R₄₂-R₄₁-R₄₁-R₄₂-R₄₆-R₄₁-R₄₂-R_4
2-R₄₁  (Where R₄₁Is a hydrophobic amino acid and R₄₂Is basic hydrophilic or neutral
A hydrophilic amino acid and R₄₆Is glutamic acid); and T is a lipophilic group;Wherein R is a hydrocarbon having at least 2 carbon atoms.
The described method. 19. X comprises the structure Y ₆₈ -X ₆₈ , wherein Y ₆₈ is R ₄
19. The method of claim 18, wherein the value is ₁ . 20. X is a biologically active amphipathic peptide, said peptide is an ion channel forming peptide and X has the following structure X ₇₀ : -R ₄₁ -R ₄₂ -R ₄₂ -R ₄₁ -R ₄₁ -R ₄₂ -R ₄₂ -R ₄₁ -R _{4
2 -R 42 -R 41 -R 41 -} ( _{wherein, R 41} is a hydrophobic amino _{acid, R 42} is a is a hydrophilic amino acid of the basic hydrophilic or neutral) a; and T is lipophilic And T is The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. 21. X is a biologically active amphipathic peptide, said peptide is an ion channel forming peptide, and X has the following structure X ₇₂ : -R ₄₂ -R ₄₂ -R ₄₁ -R ₄₁ -R ₄₂ -R ₄₇ -R ₄₁ -R ₄₂ -R _4
2 -R ₄₁ - _{(wherein, R 41} is a hydrophobic amino _{acid, R 42} is a hydrophilic amino acid basic hydrophilic or neutral, and _{R 47} is aspartic acid) a; and T Is a lipophilic group, and T is The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. 22. X is a biologically active peptide, the peptide is an ion channel forming peptide, X has the following structure _{X 76: -R 41 -R 42 -R} 42 -R 41 -R 41 -R 42 - Wherein R ₄₁ is a hydrophobic amino acid and R ₄₂ is a basic hydrophilic or neutral hydrophilic amino acid; and T is a lipophilic group and T is The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. 23. X comprises the structure Y ₇₆ -X ₇₆ , wherein Y ₇₆ is: (i) R ₄₂ ; (ii) R ₄₂ -R ₄₂ ; (iii) R ₄₁ -R ₄₂ -R ₄₂ ; _{iv) R 41 -R 41 -R 42} -R 42; (v) R 42 -R 41 -R 41 -R 42 -R 42; or _{(vi) R 42 -R 42 -R} 41 -R 41 -R 42 is -R _42, claim 2
3. The method according to 2. 24. X comprises the structure X ₇₆ -Z ₇₆ , wherein Z ₇₆ is: (i) R ₄₈ ; (ii) R ₄₈ -R ₄₁ ; or (iii) R ₄₈ -R ₄₁ -R _42. 23. The method of claim 22, wherein _R48 is a base hydrophilic, neutral hydrophilic, or hydrophobic amino acid. 25. X comprises the structural formula (Y ₇₆ ) _a -X _76- (Z ₇₆ ) _b , wherein Y ₇₆ is: (i) R ₄₂ ; (ii) R ₄₂ -R ₄₂ ; (iii) _{R 41 -R 42 -R 42; (} iv) R 41 -R 41 -R 42 -R 42; (v) R 42 -R 41 -R 42 -R 42 -R 42; or _{(vi) R} 42 -R ₄₂ is _{-R 41 -R 41 -R 42 -R 42} , Z 76 _{is: (i) R 48; (} ii) R 48 -R 41; or _{(iii) R 48 -R 42 -R} 41 ( wherein , _R48 is a basic hydrophilic, neutral hydrophilic, or hydrophobic amino acid); a is 0 or 1; and b is 0 or 1. 26. X is a biologically active, amphipathic peptide, which is an ion channel forming peptide, and X has the following structural formula X ₇₈ : -R ₄₂ -R ₄₂ -R ₄₁ -R ₄₁ -R _{42 -R 42 -R 42 -R 42 -R} 4
₂ -R 41 _- (wherein, R ₄₁ is a hydrophobic amino acid, R ₄₂ is a hydrophilic amino acid basic hydrophilic amino acid or neutral) a; and T is a lipophilic group, T is The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. 27. X is a biologically active amphipathic peptide, said peptide is an ion channel forming peptide, and X has the following structural formula X ₈₀ : -R ₄₁ -R ₄₂ -R ₄₂ -R ₄₁ -R _{41 -R 42 -R 46 -R 41 -R} 4
₁ -R ₄₂ -R ₄₁ - _{(wherein, R 41} is a hydrophobic amino _{acid, R 42} is a hydrophilic amino acid basic hydrophilic amino acid or neutral, and _{R 46} is glutamic acid) include ;
And T is a lipophilic group, and T is The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. 28. X is a biologically active peptide, the peptide is an ion channel forming peptide, X has the following structure _{X 54: -R 41 -R 42 -R} 42 -R 41 -R 41 -R 42 - R ₄₂ -R ₄₁ -R _{4
2 -R 42 -R 41 -R 41 -R} 42 -R 42 -R 42 -R 43 - ( _{wherein, R 41} is a hydrophobic amino _{acid, R 42} is a hydrophilic basic hydrophilic or neutral Amino acids and R ₄₃ is a neutral hydrophilic amino acid);
And T is a lipophilic group, and T is The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. 29. X is a biologically active peptide, wherein said peptide is an ion channel forming peptide, X comprises the following structure X ₅₆ -Z ₅₆ , wherein X _5
6 is: -R ₄₂ -R ₄₂ -R ₄₂ -R ₄₁ -R ₄₁ -R ₄₂ -R ₄₂ -R ₄₁ -R _4
1 -R ₄₂ -R ₄₂ -R ₄₄ - is and and _{Z 56 is: (i) R 42; (} ii) R 42 -R 42; (iii) R 42 -R 42 -R 41; (iv) R _{42 -R 42 -R 41 -R 41;} (v) R 42 -R 42 -R 41 -R 41 -R 42; or _{(vi) R 42 -R 42 -R} 41 -R 41 -R 42 -R 42 -R ₄₁ ,
Wherein R ₄₁ is a hydrophobic amino acid, R ₄₂ is a basic hydrophilic or neutral hydrophilic amino acid, and R ₄₄ is a neutral hydrophilic amino acid or protein); Is a lipophilic group, and T is The method of claim 1, wherein, if R is a hydrocarbon having at least 2 carbon atoms. 30. The following formula: Wherein X is a bioactive peptide or protein, wherein the peptide is an ion channel forming peptide or protein, and T is a lipophilic group or hydrogen, or a methanesulfone of an N-terminally substituted peptide having A method of preparing an acid derivative or analog, comprising suspending the free base of the peptide in water; and at least 0.5 equivalent of bisulfite based on the number of amino groups in the suspended peptide and the peptide Mixing the salt-formaldehyde complex for a period of 10 minutes or more to form a methanesulfonic acid derivative or analog of the peptide. 31. The free base of the suspended peptide is mixed with 1 to 5 equivalents of a bisulfite-formaldehyde complex based on the number of amino groups of the peptide,
A method according to claim 30. 32. The method of claim 30, wherein the free base of the suspended peptide is mixed with 1.1 to 3 equivalents of a bisulfite-formaldehyde complex based on the number of amino groups in the peptide. 33. The method of claim 30, wherein said mixing period ranges from 10 minutes to 2 hours. 34. The mixing period ranges from 10 minutes to 1 hour.
A method according to claim 30. 35. The mixing period ranges from 15 minutes to 30 minutes.
A method according to claim 30. 36. The method of claim 30, further comprising neutralizing a salt of said peptide to produce a free base of said peptide. 37. The method of claim 36, wherein said peptide salt is neutralized by treatment with a basic solution. 38. The method according to claim 37, wherein said basic solution is a sodium carbonate solution. 39. The method of claim 36, wherein said free basic peptide precipitates upon neutralization. 40. The method of claim 39, further comprising separating the precipitated free basic peptide before suspending in water. 41. The method of claim 30, further comprising recovering the methanesulfonic acid derivative or analog of the peptide prior to mixing. 42. The method of claim 41, wherein the methanesulfonic acid derivative or analog of the peptide is recovered by filtration. 43. The method of claim 41, further comprising lyophilizing the recovered methanesulfonic acid derivative or analog of the peptide. 44. The following formula: Where X is a bioactive peptide or protein, the peptide is an ion channel forming peptide or protein, and T is a lipophilic group or hydrogen. Forming a methanesulfonic acid derivative or analog of the peptide. 45. The formation comprises suspending the free base of the unsubstituted peptide or the N-terminally substituted peptide in water; and adding 10% of the suspended peptide and at least 0.5 equivalents of bisulfite-formaldehyde complex. 45. The method of claim 44, comprising mixing for a period of at least one minute to produce a methanesulfonic acid derivative or analog of the peptide. 46. After the formation of the methanesulfonic acid derivative or analog, the antimicrobial activity of the unsubstituted peptide or the N-terminally substituted peptide is increased by an unsubstituted peptide or a N-terminally substituted peptide that does not contain a methanesulfonic acid group. 46. The method of claim 44, wherein the method does not reduce or does not significantly reduce antimicrobial activity. 47. The peptide of the formula: Wherein X is a biologically active amphipathic peptide or protein, the peptide is an ion channel forming peptide or protein, and T is a lipophilic group or hydrogen. Alternatively, a method for treating a microbial infection, comprising administering to a patient an effective amount of a methanesulfonic acid analog or derivative of an N-terminally substituted peptide. 48. The method of claim 47, wherein said microbial infection is a pulmonary infection. 49. The pulmonary infection is characterized by the microorganism P. 49. The method of claim 48, wherein the method is caused by A. cruginosa. 50. The method of claim 48, wherein said pulmonary infection occurs in a cystic fibrosis patient. 51. The peptide of claim 47, wherein said peptide is administered by inhalation.
The method described in. 52. After the formation of the methanesulfonic acid derivative or analog, the antitumor activity of the unsubstituted peptide or the N-terminal substituted peptide shows that the unsubstituted peptide or N-terminal substituted peptide does not contain a methanesulfonic acid group 45. The method of claim 44, wherein the method does not reduce or does not significantly decrease the antitumor activity of 53. The following formula: Wherein X is a bioactive peptide or protein, wherein the peptide is an ion channel forming peptide or protein, and T is a lipophilic group or hydrogen, or the methane sulfone of an N-terminally substituted peptide. A method for preparing an acid derivative or analog, comprising: (a) dissolving a salt of the peptide with water; (b) more than 1 mole of 3 based on the number of amino acids in the peptide salt and the peptide.
Mixing a 0% neutral formaldehyde solution and more than 1 mole of a base to form a formaldehyde adduct; and (c) converting the formaldehyde adduct to more than 1 mole of meta based on the number of amino groups of the peptide Mixing with sodium bisulfite to form a methanesulfonic acid derivative or analog of the peptide. 54. The method of claim 53, wherein said greater than 1 molar 30% neutral formaldehyde solution is 1 to 25 equivalents. 55. The method of claim 53, wherein said more than 1 molar 30% neutral formaldehyde solution is from 1.1 to 12.5 equivalents. 56. The method of claim 53, wherein said more than 1 mole of base is from 1.1 to 12.5 equivalents. 57. The method of claim 53, wherein said more than 1 mole of base is from 1.1 to 6.25 equivalents. 58. The method of claim 53, wherein said base is sodium bicarbonate. 59. The method of claim 53, wherein said more than 1 mole of sodium metabisulfite is 1 to 5 equivalents. 60. The composition of claim 1, wherein said sodium metabisulfite exceeds 1.1 moles.
54. The method of claim 53, wherein the method is between 3 and 3 equivalents. 61. The peptide having the formula: Wherein X is a biologically active amphipathic peptide or protein, wherein the peptide is an ion channel forming peptide or protein, and T is a lipophilic group or hydrogen, or an N-terminal. A method of treating a tumor, comprising administering to a patient an effective amount of a methanesulfonic acid analog or derivative of a substituted peptide.