Biologically active peptides with reduced toxicity in...

Details Biologically active peptides with reduced toxicity in... Biologically active peptides with reduced toxicity in...

1998-07-15

2002-02-19

Davenport, Avis M. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S324000, C530S333000, C530S334000

Reexamination Certificate

active

06348445

ABSTRACT:

BACKGROUND AND SUMMARY OF THE INVENTION
This invention relates to biologically active peptides. More particularly, this invention relates to biologically active peptides with reduced toxicity. Further, this invention relates to a method of modifying biologically active peptides, that are either N-terminally (amino-terminal) substituted or unsubstituted, to reduce their toxicity in animals.
In accordance with an aspect of the present invention, there is provided an unsubstituted biologically active peptide or protein. In another aspect, there is provided a N-terminal substituted peptide or protein having the formula:
wherein X is a biologically active peptide or protein, and N is a nitrogen atom. The peptide or protein is preferably an ion channel-forming peptide or protein. T is a lipophilic moiety, and W is another T group or hydrogen.
The term “lipophilic,” as used herein, means that the lipophilic moiety enhances the interaction of the peptide or protein with a lipid membrane, such as, for example, a cell membrane.
Lipophilic moieties which may be employed, include, but are not limited to, any moiety which may be placed on the N-terminal of the peptide through a condensation reaction with nitrogen. The lipophilic moiety T may be, for example, a carboxylic acid, a phosphoric acid, preferably an alkylphosphoric acid, a phosphonic acid, preferably an alkylphosphonic acid, a sulfonic acid, preferably an alkylsulfonic acid, or an alkyl group. Preferably T is:
wherein R is a hydrocarbon having at least two and no more than 16 carbon atoms.
In one embodiment, R is an alkyl group. The alkyl group may be a straight chain or branched chain alkyl group, or a cycloalkyl group. For example, R may be CH
3
(CH
2
)
n
—, wherein n is from 1 to 14. Preferably, n is from 3 to 12, more preferably from 4 to 11, still more preferably from 6 to 11, and most preferably n is 6, whereby T is an octanoyl group.
In another embodiment, R is an aromatic (including phenyl and naphthyl), or an alkyl aromatic group. For example, R may be
wherein z is from 0 to 6. Preferably, z is 1 or 2.
In another embodiment, R is
wherein n is from 1 to 5. Preferably n is 1, whereby R is an ibuprofyl group.
In yet another embodiment, T is:
wherein x is from 1 to 14. Preferably, x is 2, and T is a succinyl group.
In another embodiment, T is:
wherein y is from 1 to 14. Preferably, y is 12, whereby T is a sphingosine group.
In yet another embodiment T is
wherein x and y are hereinabove described. Preferably, x is 2, and y is 12.
In one embodiment, W is hydrogen.
Applicant has found that when biologically active peptides have substitutions at the N-terminal such as those hereinabove described, such peptides have increased biological activity against target cells, viruses, and virally-infected cells, as compared with unsubstituted peptides or peptides substituted at the N-terminal with an acetyl group. Applicant also has found that the N-terminal substitutions hereinabove described significantly increase the biological activity of “short” peptides, i.e., peptides having no more than 14 amino acid residues.
In another embodiment of this invention the biologically active peptides are not N-terminally substituted and have broad spectrum anti-tumor and anti-microbial activity.
As hereinabove stated, the biologically active peptides or proteins of the present invention are preferably ion channel-forming peptides. An ion channel-forming peptide or protein or ionophore is a peptide or protein which increases the permeability for ions across a natural or synthetic lipid membrane. B. Christensen, et al.,
PNAS
Vol. 85, pgs. 5072-5076 (July 1988) describes a methodology which indicates whether or not a peptide or protein has ion channel-forming properties and is therefore an ionophore. As used herein, an ion channel-forming peptide or ion channel-forming protein is a peptide or protein which has ion channel-forming properties as determined by the method of Christensen, et al. This Christensen article is entirely incorporated herein by reference.
An amphiphilic peptide or protein is a peptide or protein which includes both hydrophobic and hydrophilic peptide or protein regions.
The ion channel-forming peptides employed in the present invention are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water. In addition, the structure of such peptides provides for flexibility of the peptide molecule. Such peptides are capable of forming an alphahelical structure. When the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself into a rodlike structure.
In general, such peptides have at least 7 amino acids, and in many cases have at least 20 amino acids. In most cases, such peptides do not have in excess of 40 amino acids.
The peptides and/or analogues or derivatives thereof may be administered to a host, for example a human or non-human animal, in an amount effective to inhibit growth of a target cell, virus, or virally-infected cell. Thus, for example, the peptides and/or analogues or derivatives thereof may be used as anti-microbial agents, anti-viral agents, anti-bacterial agents, anti-tumor agents, anti-parasitic agents, and spermicides, as well as agents exhibiting other bioactive functions.
The term “anti-microbial” as used herein means that the peptides or proteins of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes, such as bacteria, fungi, viruses, or the like.
The term “anti-bacterial” as used herein means that the peptides or proteins employed in the present invention produce effects adverse to the normal biological functions of bacteria, including death, destruction, or prevention of the growth or proliferation of the bacteria when contacted with the peptides or proteins.
The term “antibiotic” as used herein means that the peptides or proteins employed in the present invention produce effects adverse to the normal biological functions of the non-host cell, tissue, or organism, including death, destruction, or prevention of the growth or proliferation of the non-host cell, tissue, or organism when contacted with the peptides or proteins.
The term “spermicidal” as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the motility of sperm.
The term “anti-fungal” as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of fungi.
The term “anti-viral” as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of viruses, or of virally-infected cells.
The term “anti-tumor” as used herein means that the peptides or proteins inhibit the growth of or destroy tumors, including cancerous tumors.
The term “anti-parasitic” as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of parasites.
The peptides or proteins of the present invention have a broad range of potent anti-tumor and antibiotic activity against a plurality of tumor types and microorganisms, including gram-positive and gram-negative bacteria, fungi, protozoa, and the like, as well as parasites. The peptides or proteins of the present invention allow a method for treating or controlling tumor growth and microbial infection caused by organisms which are sensitive to the peptides or proteins. Such treatment may comprise administering to a host organism or tissue susceptible to or affiliated with a microbial infection an anti-tumor or anti-microbial amount of at least one of the peptides or proteins.
In another embodiment of this invention, methods are provided for reducing the toxicity of unmodified peptides or of N-terminally modified peptides in a host without reducing the anti-tumor or anti-microbial activity of the peptides. This method includes forming a methane sulfonate derivativ

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