EA046208B1 - PEPTIDE COMPOUNDS AND THEIR THERAPEUTIC USE - Google Patents

Description

Related applications

This invention claims priority to Israeli Patent Application No. 260555, filed July 11, 2018, the contents of which are incorporated herein by reference in their entirety.

Presentation of the list of sequences

The ASCII file entitled 77809 Sequence Listing.txt, created on July 7, 2019, containing 8,069,120 bytes, submitted contemporaneously with the filing of this invention, is incorporated herein by reference.

Field of the present invention and background of the present invention

The present invention, in some embodiments, relates to compositions and methods of using them for the treatment of inflammatory and autoimmune diseases.

There is an unmet need for new compositions that can serve to attenuate the cellular and immune response to stress in normal tissue in a specific, safe and effective manner, thereby reducing the severity of stress-related degenerative diseases and stress-induced inflammation.

LPPLPYP peptide (SEQ ID NO: 2, also known as Stressin-1 and IPL344) is a short 7 amino acid peptide that protects various cell types from proapoptotic pressure and activates the Akt signaling system. The structure of IPL344 resembles the binding sites of adapter proteins. A mechanism of action has been proposed that involves mimicking such proteins and activating cellular defense processes through Akt and possibly other pathways.

International patent application publications WO 2006/021954 and WO 2012/160563 disclose the use of the peptide LPPLPYP (SEQ ID NO: 2) for the treatment of diseases such as ALS.

Brief Disclosure of the Present Invention

According to an aspect of the present invention, there is provided an isolated peptide consisting of more than ten amino acids, which contains the amino acid sequence represented by the formula

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ (SEQ ID NO: 17), where (i) X1 is proline or an analog or derivative thereof;

(ii) X ₂ is proline or an analogue thereof;

(iii) X ₃ is selected from the group consisting of alanine, valine, leucine, cysteine, isoleucine, methionine and a derivative or analogue thereof;

(iv) X4 is selected from the group consisting of alanine, valine, serine and a derivative or analogue thereof;

(v) X5 is any amino acid;

(vi) X6 is proline or an analogue or derivative thereof and (vii) the peptide is capable of reducing the amount of dexamethasone-induced spleen and/or thymus weight loss in mice.

According to an aspect of the present invention, there is provided an isolated peptide, the length of which does not exceed six amino acids, which contains the amino acid sequence represented by the formula

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ (SEQ ID NO: 1), where (i) X ₁ is proline or an analogue or derivative thereof;

(ii) X ₂ is proline or an analogue or derivative thereof;

(iii) X3 is selected from the group consisting of alanine, valine, leucine, cysteine, isoleucine, methionine and a derivative or analogue thereof;

(iv) X4 is selected from the group consisting of alanine, valine, serine, proline and a derivative or analogue thereof;

(v) X5 is any amino acid and (vi) the peptide is capable of reducing the amount of dexamethasone-induced spleen and/or thymus weight loss in mice.

According to an aspect of the present invention, there is provided an isolated peptide consisting of five amino acids, which consists of the amino acid sequence represented by the formula

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ (SEQ ID NO: 5), where (i) X ₁ and X ₃ are any amino acids;

(ii) X ₂ is proline or an analogue or derivative thereof;

(iii) X4 is not proline;

(iv) the peptide is able to reduce the amount of dexamethasone-induced loss of spleen and/or thymus mass in mice.

According to an aspect of the present invention, there is provided an isolated seven amino acid peptide which consists of the amino acid sequence represented by the formula

X ₁ -X ₂ -X ₃ -X ₄ -X ₅ -X ₆ -X ₇ (SEQ ID NO: 6), where (i) X1, X ₅ and X ₆ are any amino acids;

- 1 046208 (ii) X ₂ , X3 and X7 are proline or an analogue or derivative thereof;

(iii) X ₄ is not proline and (iv) the peptide is able to reduce the amount of dexamethasone-induced spleen and/or thymus weight loss in mice.

According to an aspect of the present invention, there is provided a pharmaceutical composition comprising a peptide according to any one of claims 1 to 27 as an active agent and a physiologically acceptable carrier.

According to embodiments of the present invention, the proline derivative is selected from the group consisting of N-methylproline, alpha-methylproline and α-aminobutyric acid.

According to embodiments of the present invention, the length of the peptide is 6 amino acids.

In embodiments of the present invention, the peptide contains an amino acid that is attached to the N-terminus of X ₁ , wherein the amino acid is selected from the group consisting of alanine, valine, leucine, cysteine, isoleucine, methionine, and a derivative or analogue thereof.

In embodiments of the present invention, the amino acid that is attached to the N-terminus of X1 is leucine or a derivative or analog thereof.

According to embodiments of the present invention, the amino acid that is attached to the N-terminus of X1 is a D-amino acid.

According to embodiments of the present invention, the length of the peptide is 7 amino acids.

In embodiments of the present invention, X ₄ is alanine or an analog or derivative thereof.

According to embodiments of the present invention, X5 is selected from the group consisting of tyrosine, phenylalanine, tryptophan, and a derivative or analogue thereof.

In embodiments of the present invention, X5 is tyrosine.

In embodiments of the present invention, the isolated peptide contains the amino acid sequence set forth in SEQ ID NO: 4.

In embodiments of the present invention, the isolated peptide consists of the amino acid sequence set forth in SEQ ID NO: 4.

In embodiments of the present invention, the peptide contains the amino acid sequence set forth in SEQ ID NO: 18.

In embodiments of the present invention, the peptide consists of the amino acid sequence set forth in SEQ ID NO: 18.

According to embodiments of the present invention, the isolated peptide consists of 5 amino acids.

In embodiments of the present invention, X4 is selected from the group consisting of alanine, proline, an alanine derivative or analogue, and a proline derivative or analogue.

In embodiments of the present invention, the proline derivative or analogue is selected from the group consisting of N-methylproline, alpha-methylproline, and α-aminobutyric acid.

According to embodiments of the present invention, the amino acid sequence includes the sequence shown in SEQ ID NO: 3.

In embodiments of the present invention, the isolated peptide consists of the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 18.

In embodiments of the present invention, X ₃ is leucine.

In embodiments of the present invention, X ₁ and/or X ₃ are D-amino acids.

In embodiments of the present invention, the peptide is a staple peptide.

In embodiments of the present invention, the peptide is a cyclic peptide.

In embodiments of the present invention, the sequence order is reversed and all amino acids are D-type.

In embodiments of the present invention, the peptide is attached to a cell penetrating moiety.

In embodiments of the present invention, the cell penetrating portion is attached to the N-terminus of the peptide.

In embodiments of the present invention, the peptide is for treating an inflammatory or degenerative disease.

According to embodiments of the present invention, the disease associated with apoptosis is an inflammatory or degenerative disease.

According to embodiments of the present invention, the inflammatory disease is an autoimmune disease.

According to embodiments of the present invention, the degenerative disease is a neurodegenerative disease.

- 2 046208

In embodiments of the present invention, the apoptosis-related disease is selected from the group consisting of age-related macular degeneration (AMD), retinitis pigmentosa, stroke, and myocardial infarction.

Unless otherwise specified, all technical and/or scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the present invention, exemplary methods and/or materials are described below. In the event of a conflict, the specification of the patent, including its definitions, will control. Moreover, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Brief description of graphic materials

Certain embodiments of the present invention are described herein by way of example only with reference to the accompanying drawings. Turning now to the specific detailed drawings, it should be emphasized that the details are presented by way of example and for purposes of illustrative discussion of embodiments of the present invention. In this regard, the description of the drawings will make it apparent to those skilled in the art how embodiments of the present invention may be practiced.

On graphic materials:

Fig. 1 is a graph illustrating the effect of exemplary peptides on spleen weight, spleen cell count, thymus weight, and thymus cell count.

The peptides used in the screening were as follows:

PPLPY - SEQ ID NO: 3

LPPLAYP - SEQ ID NO: 4

PLPYP - SEQ ID NO: 9

PPL - SEQ ID NO: 10

PLP-SEQIDNO: 11

PYP - SEQ ID NO: 12

LPGLPYP - SEQ ID NO: 13

LPPLGYP - SEQ ID NO: 14

LAPLPYP - SEQ ID NO: 15

LPALPYP - SEQ ID NO: 16

Fig. 2 is a graph illustrating the effect of exemplary peptides on spleen weight, spleen cell count, thymus weight, and thymus cell count.

The peptides used in the screening were as follows:

LPPLPYP - SEQ ID NO: 2 (control)

PPLAYP - SEQ ID NO: 18

LPPLPY- SEQ ID NO: 7

LPPLAYP - SEQ ID NO: 4

PPLPY-NH2 - SEQ ID NO: 19

PPLPY - SEQ ID NO: 3

Description of Specific Embodiments of the Present Invention

The present invention, in some embodiments, relates to compositions and methods of using them for the treatment of inflammatory and autoimmune diseases.

Before explaining at least one embodiment of the present invention in detail, it should be understood that the present invention is not necessarily limited in its application to the details set forth in the following description or illustrated examples. The present invention may be embodied in other embodiments or practiced in various ways.

Peptide bonds almost always occur in the trans conformation with an omega torsion angle close to 180°, whereas only a very limited proportion is observed in the cis conformation with an omega angle of approximately 0°. The energy barrier between trans/cis configurations is approximately 20 [kcal/mol], and the energy barrier for the trans isomer is ~2.5 [kcal/mol]. Among the 20 canonical amino acids, proline plays a special role. The peptide bond of proline to the preceding amino acid residue (Xaa-Pro, Xaa stands for any amino acid) is devoid of amide hydrogen, so that the peptide bond cannot act as a hydrogen bond donor, and the energy barrier as well as the energy pass is significantly reduced to ~13 and ~0 .5 [kcal/mol] respectively [2], shifting the trans/cis equilibrium towards the cis configuration. The strong changes in omega angle associated with the process of trans/cis isomerization of proline (from 0 to 180) are modulated by both intra- and intermolecular

- 3 046208 interactions and have a tremendous impact on the ratio of the structural activity of the polypeptide chain. Thus, proline isomerization becomes an important component in controlling the activity of many biological processes. This is especially true for short peptides, in which the cis configuration can occupy more than 30% of the conformational population, depending on the preceding residue.

In addition, it is known that the multiconformational effect of proline is manifested in proline residues located as the second residue from the N-terminus of the peptide (penultimate proline). Truncation of the first N-terminal residue in a peptide containing the penultimate proline usually results in a conformational change in the peptide. In a data set of 58 peptides, it was found that almost 80% of the peptides exhibited this effect [Glover, M.S., et al., 2014, J. Am. Soc. Mass. Spectrom. 26(3): p. 444-5].

Multiproline peptide LPPLPYP (SEQ ID NO: 2), also known as IPL344 and Stressin-1, is a short 7 amino acid peptide that protects various cell types from proapoptotic pressure and activates the Akt signaling system. Suitable for the treatment of inflammatory and autoimmune diseases.

Given the delicate conformational nature of prolines, we set out to determine whether the trans-proline configuration dominates the functionality of all four proline residues in the LPPLPYP peptide (SEQ ID NO: 2). The present inventors unexpectedly discovered that replacing the penultimate proline with alanine results in a peptide (SEQ ID NO: 4) that significantly reduces the amount of dexamethasone-induced spleen and/or thymus weight loss in mice (FIG. 1). In contrast, replacing the penultimate proline with glycine resulted in a peptide lacking this activity.

Upon further practice of the present invention, the present inventors discovered that deletion of the first and last amino acids of SEQ ID NO: 2 also resulted in a peptide (having the sequence shown in SEQ ID NO: 3) that significantly reduced the amount of dexamethasone-induced spleen weight loss and/or thymus in mice.

The present inventors have concluded that short peptides based on SEQ ID NO: 3 and 4 are promising for the treatment of inflammatory and autoimmune diseases.

Upon further practice of the present invention, the present inventors discovered that an additional peptide (SEQ ID NO: 18), which corresponded to the formula given in SEQ ID NO: 17, showed significant improvement in reducing the amount of dexamethasone-induced loss of spleen and/or thymus mass in mice (see Fig. 2).

The present inventors have also shown that exemplary peptides that fall within any of the disclosed general formulas exhibit significant improvement over the main peptide (SEQ ID NO: 2) in reducing the amount of dexamethasone-induced splenic and/or thymic mass loss in mice.

As used herein, the term peptide refers to a polymer of natural or synthetic amino acids, including native peptides (degradants, synthetically synthesized polypeptides, or recombinant polypeptides) and peptidomimetics (typically synthetically synthesized peptides), as well as peptoids and hemeptoids, which represent are analogues of polypeptides, which may have, for example, modifications that make the peptides even more stable in the body or more able to penetrate cells.

The present invention also covers derivatives (with modification and/or addition of chemical function to the amino acid side chain, without chemical change of the peptide backbone) and analogs (with modification and/or addition of chemical function to the peptide backbone, e.g., modification of the N-terminus or C-terminus , modification of a peptide bond, modification of an amino acid not defined as a derivative), complexes with other species such as a metal ion (for example, copper, zinc, manganese, magnesium and others).

Such modifications include, but are not limited to, N-terminal modification, C-terminal modification, polypeptide bond modification including, but not limited to, CH2-NH, CH2-S, CH ₂ -S=O, O=C-NH , CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modifications and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are described, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if set forth in its entirety in this document. More information on this is provided below.

Polypeptide bonds (-CO-NH-) within a polypeptide can be replaced, for example, by N-methylated bonds (-N(CH ₃ )-CO-), ester bonds (-C(R)HCOOC(R)-N-), ketomethylene bonds (-CO-CH ₂ -), α-aza bonds (-NH-N(R)-CO-), where R represents any alkyl, for example methyl, carba bonds (-CH2-NH-), hydroxyethylene bonds (-CH(OH)-CH ₂ -), thioamide bonds (-CS-NH-), olefinic double bonds (-CH=CH-), retroamide bonds (-NH-CO-), polypeptide derivatives (-N(R )-CH2-CO-), where R represents the normal side

- 4 046208 chain naturally present on the carbon atom.

These modifications can occur in any of the bonds along the polypeptide chain and even in several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, can be replaced by synthetic non-natural acids such as phenylglycine analogue, TIC, naphthylamine (Nol), ring methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

Proline can be replaced by synthetic, non-natural acids such as derivatives of N-methylproline, α-methylproline and α-aminobutyric acid analogues.

Other unnatural amino acids are given in table. 2 below.

In addition to the foregoing, the polypeptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.).

The term amino acid or amino acids as used herein in the description and claims section includes the 20 naturally occurring amino acids; those amino acids that are frequently post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine and ornithine. Additionally, the term amino acid includes both D- and L-amino acids (stereoisomers).

In table 1 and 2 list naturally occurring amino acids (Table 1) and non-traditional or modified amino acids (Table 2) that can be used with the present invention.

Table 1

Amino acid Three letter abbreviation Single letter character Alanin Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gin Q Glutamic acid Glu E Glycine Gly G G istidine His H Isoleucine He I Leucine Leu L Lysine Lys TO Methionine Met M Phenylalanine Phe F Proline Pro P Serin Ser S Threonine Thr T Tryptophan Trp w Tyrosine Tyr Y Valin Vai V Any amino acid as above Xaa X

table 2

Unconventional amino acid Code Unconventional amino acid Code ornithine From hydroxyproline Nur α-aminobutyric acid Abu aminonorbornylcarboxylate Norb D-alanine Dala aminocyclopropanecarboxylate Srgo D-arginine Darg I-(3-guanidinopropyl)glycine Narg D-asparagine Dasn I-(carbamylmethyl)glycine Nasn D-aspartic acid Dasp I-(carboxymethyl)glycine Nasp D-cysteine Days N-(thiomethyl)glycine Neys D-glutamine Dgln I-(2-carbamylethyl)glycine Ngln D-glutamic acid Dglu I-(2-carboxyethyl)glycine Nglu D-histidine Dhis I-(imidazolylethyl)glycine Nhis D-isoleucine Dile N-(1-methylpropyl)glycine Nile D-leucine Dleu I-(2-methylpropyl)glycine Nleu D-lysine Dlys N-(4-aminobutyl)glycine Nlys D-methionine Dmet N-(2-methylthioethyl)glycine Nmet D-ornithine Dorn N-(3-aminopropyl)glycine Norn D-phenylalanine Dphe N-benzylglycine Nphe D-proline Dpro M-(hydroxymethyl)glycine Nser D-serine Dser N-(l-hydroxyethyl)glycine Nthr

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D-threonine Dthr M-(3-indolylethyl)glycine Nhtrp D-tryptophan Dtrp M-(p-hydroxyphenyl)glycine Ntyr D-tyrosine Dtyr M-(1-methylethyl)glycine Nval D-valine Dval N-methyl glycine Nmgly D-N-methylalanine Dnmala L-N-methylalanine Nmala D-N-methyl arginine Dnmarg L-N-methylarginine Nmarg D-N-methylasparagine Dnmasn L-N-methylasparagine Nmasn D-N-methyl aspartate Dnmasp L-N-methylaspartic acid Nmasp D-N-methyl cysteine Dnmcys L-N-methyl cysteine Nmcys D-N-methyl glutamine Dnmgln L-N-methyl glutamine Nmgln D-N-methylglutamate Dnmglu L-N-methylglutamic acid Nmglu D-N-methyl histidine Dnmhis L-N-methyl histidine Nmhis D-N-methylisoleucine Dnmile L-N-methylisoleucine Nmile D-N-methylleucine Dnmleu L-N-methylleucine Nmleu D-N-methyllysine Dnmlys L-N-methyllysine Nmlys D-N-methyl methionine Dnmmet L-N-methyl methionine Nmmet D-N-methyl or nitine Dnmorn L-N-methylornithine Nmorn D-N-methylphenylalanine Dnmphe L-N-methyl phenyl al anine Nmphe D-N-methylproline Dnmpro L-N-methylproline Nmpro D-N-methylserine Dnmser L-N-methylserine Nmser D-N-methyltreonine Dnmthr L-N-methylthreonine Nmthr D-N-methyltryptophan Dnmtrp L-N-methyltryptophan Nmtrp D-N-methyltyrosine Dnmtyr L-N-methyltyrosine Nmtyr D-N-methylvaline Dnmval L-N-methylvaline Nmval L-norleucine Nie L-N-methylnorleucine Nmnle L-norvaline Nva L-N-methylnorvaline Nmnva L-ethylglycine Etg L-N-methyl-ethyl glycine Nmetg L-tert-butylglycine Tbug L-N-methyl-tert-butylglycine Nmtbug L-homophenylalanine Hphe L-N-methyl-homophenyl al-anine Nmhphe a-naphthylalanine Anap N-methyl-a-naphthylalanine Nmanap penicillamine Pen N - methyl penicillamine Nmpen γ-aminobutyric acid Gabu N-methyl-y-aminobutyrate Nmgabu cyclohexylalanine Chexa Ν - methyl ring ohexyl al anine Nmchexa cyclopentylalanine Cpen N-methyl-cyclopentylalanine Nmcpen α-amino-a-methylbutyrate Aabu N-methyl-a-amino-amylbutyrate Nmaabu a-aminoisobutyric acid Aib N-methyl-a-aminoisobutyrate Nmaib D-a-methylarginine Dmarg L-α-methylar ginine Marg D-a-methylasparagine Dmasn L-a-methylasparagine Masn D-a-methylaspartate Dmasp L-a-methylaspartate Masp D-a-methylcysteine Dmcys L-a-methylcysteine Meys D-a-methylglutamine Dmgln L-a-methylglutamine Mgln D-a-methylglutamic acid Dmglu L-a-methylglutamate Mglu D-a-methylhistidine Dmhis L-a-methylhistidine Mhis D-a-methylisoleucine Dmile L-a-methylisoleucine Mile D-a-methylleucine Dmleu L-a-methylleucine Mleu

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D-a-methyllysine Dmlys L-a-methyllysine Mlys D-a-methylmethionine Dmmet L-a-methylmethionine Mmet D-a-methylornithine Dmorn L-a-methyl ornithine Mom D-a-methylphenylalanine Dmphe L-a-methyl phenyl al anine Mphe D-a-methylproline Dmpro L-α-methylproline Mpro D-a-methylserine Dmser L-a-methylserine Mser D-a-methylthreonine Dmthr L-a-methylthreonine Mthr D-a-methyltryptophan Dmtrp L-a-methyltryptophan Mtrp D-a-methyltyrosine Dmtyr L-a-methyltyrosine Mtyr D-a-methylvaline Dmval L-a-methylvaline Mval N-cyclobutylglycine Ncbut L-a-methylnorvaline Mnva N-cycloheptylglycine Nchep L-a-methylethylglycine Metg N-cyclohexylglycine Nchex L-a-methyl-tert-butylglycine Mtbug N-cyclodecylglycine Ncdec L-a-methyl-homophenyl alanine Mhphe N-cyclododecylglycine Ncdod a-methyl-a-naphthylalanine Manap N-cyclooctylglycine Ncoct a-methylpenicillamine Mpen N-cyclopropylglycine Ncpro a-methyl-a-aminobutyrate Mgabu N-cycloundecylglycine Ncund a-methyl-cyclohexylalanine Mchexa N-(2-aminoethyl)glycine Naeg a-methyl-cyclopentylalanine Mcpen N-(2,2- diphenylethyl)glycine Nbhm N-(N-(2,2- diphenylethyl)carbamylmethylglycine Nnbhm N-(3,3- diphenylpropyl)glycine Nbhe N-(N-(3,3- diphenylpropyl)carbamylmethyl l-glycine Nnbhe 1-carboxy-1-(2,2diphenylethylamino) oppropane ring Nmbc 1,2,3,4-tetrahydroisoquinoline3-carboxylic acid Tic phosphoserine pSer phosphothreonine pThr phosphotyrosine pTyr O-methyl-tyrosine 2-Aminoadipic acid hydroxylysine

The amino acids of the peptides of the present invention can be replaced conservatively or non-conservatively.

As used herein, the term conservative substitution refers to the replacement of an amino acid present in the native sequence in a peptide with naturally occurring or non-naturally occurring amino or peptidomimetics having similar steric properties. If the side chain of the native amino acid to be replaced is polar or hydrophobic, the conservative replacement must be with a native amino acid, a non-natural amino acid, or a peptidomimetic moiety that is also polar or hydrophobic (in addition to having the same steric properties as the side chain of the replaced amino acid ).

Since naturally occurring amino acids are generally grouped according to their properties, conservative substitutions with naturally occurring amino acids can be easily identified given the fact that the present invention contemplates replacing charged amino acids with sterically similar uncharged amino acids as conservative substitutions .

To make conservative substitutions with non-naturally occurring amino acids, amino acid analogs (synthetic amino acids) well known in the art can also be used. A naturally occurring amino acid peptidomimetic is well described in the literature known to the skilled practitioner.

When conservative substitutions are used, the replacement amino acid must contain the same or similar functional group on the side chain as the original amino acid.

As used herein, the phrase non-conservative substitutions refers to the replacement of an amino acid present in the original sequence with another natural or non-natural amino acid having different electrochemical and/or steric properties. Thus, the side chain of a replacement amino acid may be significantly larger (or smaller) than the side chain of the native amino acid being replaced, and/or may contain functional groups with significantly different electronic properties than the amino acid being replaced. Examples of non-conservative substitutions of this type include replacing phenylalanine or cyclohexylmethylglycine with alanine, isoleucine with glycine, or -NH-CH[(-CH2)5-COOH]-CO- with aspartic acid. Those non-conservative substitutions that fall within the scope of the present invention are substitutions that still constitute a peptide having antibacterial properties.

As mentioned, the N- and C-termini of the peptides of the present invention may be protected with functional groups. Suitable functional groups are described in Green and Wuts, Protecting Groups in Organic Synthesis, John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups are those that facilitate the transport of the compound attached to them into the cell, for example, by reducing

- 7 046208 reducing the hydrophilicity and increasing the lipophilicity of compounds.

These fragments can be degraded in vivo either by hydrolysis or enzymatically within the cell. Hydroxyl protecting groups include esters, carbonates and carbamate protecting groups. Amine protecting groups include alkoxy and aryloxycarbonyl groups, as described above for N-terminal protecting groups. Carboxylic acid protecting groups include aliphatic, benzyl and aryl esters, as described above for C-terminal protecting groups. In one embodiment, the carboxylic acid group on the side chain of one or more glutamic acid or aspartic acid residues in the peptide of the present invention is preferably protected with a methyl, ethyl, benzyl or substituted benzyl ester.

Examples of N-terminal protecting groups include acyl groups (-CO-R ₁ ) and alkoxycarbonyl or aryloxycarbonyl groups (-CO-O-R 1 ), where R 1 is aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or substituted aromatic group. Specific examples of acyl groups include acetyl, (ethyl-CO-, n-propyl-CO-, isopropyl-CO-, n-butyl-CO-, sec-butyl-CO-, tert-butyl-CO-, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoylphenyl-CO-, substituted phenyl-CO-, benzyl-CO- and (substituted benzyl)-CO-.Examples of alkoxycarbonyl and aryloxycarbonyl groups include CH3-O-CO-, (ethyl) -O-CO-, n-propyl-O-CO-, isopropyl-O-CO-, n-butyl-O-CO-, sec-butyl-O-CO-, tert-butyl-O-CO-, phenyl -O-CO-, substituted phenyl-O-CO- and benzyl-O-CO-, (substituted benzyl)-O-CO-, adamantane, naphthalene, myristoleyl, toluene, biphenyl, cinnamoyl, nitrobenzene, toluoyl, furoyl, benzoyl , cyclohexane, norbornane, Z-caproic acid To facilitate N-acylation, one to four glycine residues may be present at the N-terminus of the molecule.

The carboxyl group at the C-terminus of the compound may be protected, for example, by an amide (i.e. the hydroxyl group at the C-terminus is replaced by -NH2, -NHR2 and -NR2R3) or an ester (i.e. the hydroxyl group at the C-terminus replaced by -OR2). R2 and R3 independently represent an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group. In addition, taken together with a nitrogen atom, R ₂ and R ₃ can form a heterocyclic ring from C ₄ to C ₈ with about 0-2 additional heteroatoms such as nitrogen, oxygen or sulfur. Examples of suitable heterocyclic rings include piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples of C-terminal protecting groups include _-NH2 , -NHCH3, -NHCH3, -CN(ethyl), -N(ethyl) ₂ , -N(methyl)(ethyl), -CN(benzyl), -^C-C^alkylChbenzyl), -CN(phenyl), -^C-C^alkylChphenyl), -OCH3, -O-(ethyl), -O-(n-propyl), -O-(n-butyl ), -O-(isopropyl), -O-(sec-butyl), -O-(tert-butyl), -O-benzyl and -O-phenyl.

The peptides of the present invention may also contain non-amino acid moieties, such as, for example, hydrophobic moieties (various linear, branched, cyclic, polycyclic or heterocyclic hydrocarbons and hydrocarbon derivatives) attached to the peptides; agents that do not penetrate peptides; various protecting groups, especially if the compound is linear, which are attached to the ends of the compound to reduce degradation. Chemical (non-amino acid) groups present in the compound can be included to improve various physiological properties such as; reduced degradation or clearance; reduction of repulsion by various cellular pumps, improvement of immunogenic activity, improvement of various routes of administration (such as the addition of various sequences that allow penetration through various barriers, through the intestine, etc.); increased specificity, increased affinity, reduced toxicity, etc.

The attachment of an amino acid sequence component of the peptides of the present invention to other non-amino acid agents may occur by covalent binding, forming a non-covalent complex, for example, complexing with a hydrophobic polymer that can be broken down or cleaved to form a sustained release compound; by entrapping the amino acid portion of the peptide into liposomes or micelles to produce the final peptide of the present invention. The coupling may occur by entrapment of the amino acid sequence in another component (liposome, micelle) or impregnation of the amino acid sequence into a polymer to produce the final peptide of the present invention.

In a specific embodiment, the peptide is attached to a cell penetrating moiety.

As used herein, the term cell-penetrating moiety refers to a moiety (eg, a lipid, eg, palmitic acid) that enhances the translocation of the attached peptide across the cell membrane. In a specific embodiment, the cell penetrating fragment is not a peptide fragment. The fragment may be attached to the N- or C-terminus.

The peptides of the present invention may be linear or cyclic (cyclization may improve stability). Cyclization can occur by any means known in the art.

- 8 046208 worthwhile area of technology. If the compound consists predominantly of amino acids, cyclization can occur through N- to C-terminus, N-terminus to side chain and N-terminus to main chain, C-terminus to side chain, C-terminus to main chain, side chain to main chain chains and side chain to side chain, as well as backbone to backbone cyclization. Cyclization of a peptide can also occur through non-amino acid organic moieties contained in the peptide.

The present inventors also consider staple peptides.

The term staple peptide as used herein refers to a peptide having a selected number of standard or non-standard amino acids and, in addition, having at least two moieties capable of reacting to promote the formation of a carbon-carbon bond, which has been contacted with a reagent to create at least at least one cross-linking agent between the at least two fragments, which modulates, for example, the stability of the peptide.

As used herein, the term "stacking" introduces into a peptide at least two moieties capable of reacting to form a carbon-carbon bond that can contact a reagent to form at least one cross-linker between the at least two moieties. Stacking imposes restrictions on secondary structure, such as α-helix structure. The length and geometry of the crosslinker can be optimized to improve the yield of the desired secondary structure content. The constraint provided may, for example, prevent the secondary structure from unfolding and/or may enhance the shape of the secondary structure. For example, a secondary structure that cannot unfold is more stable.

The peptides of the present invention can be synthesized biochemically, for example, using standard solid phase technologies. These methods include exclusively solid-phase synthesis, methods of partial solid-phase synthesis, condensation of fragments, and classical synthesis from solutions. Solid phase polypeptide synthesis procedures are well known in the art and are further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses ( ^2nd Ed., Pierce Chemical Company, 1984).

Large-scale peptide synthesis is described in Andersson Biopolymers, 2000; 55(3):227-50.

Synthetic peptides can be purified using preparative high performance liquid chromatography [Creighton T. (1983), Proteins, structures and molecular principles. W. H. Freeman and Co. N.Y.], the composition of which can be confirmed using amino acid sequencing.

Recombinant technologies can also be used to produce the peptides of the present invention. To produce the peptide of the present invention using recombinant technology, a polynucleotide encoding a peptide of the present invention is ligated into a nucleic acid expression vector that contains a polynucleotide sequence under the transcriptional control of a cis-regulatory sequence (e.g., a promoter sequence) suitable for controlling constitutive, tissue-specific or inducible transcription of the polypeptides of the present invention in host cells.

In addition to the fact that the peptides of the present invention can be synthesized in host cells, they can also be synthesized using in vitro expression systems. These methods are well known in the art, and the components of the system are commercially available.

The peptides described herein are capable of reducing the amount of dexamethasone-induced loss of spleen and/or thymus mass in mice, for example, following injection (IP) of 100 μg of dexamethasone.

In another embodiment, the peptides described herein are capable of interfering with and blocking the secretion of both TNF-α and IL-6 by macrophage cells in response to innate activators such as lipopolysaccharide (LPS) and CpG oligonucleotides.

Additionally or alternatively, the peptides described herein are capable of reducing, preventing, or inhibiting apoptosis in eukaryotic cells. Regardless of the mechanism by which the peptides of the present invention mediate stress responses, and without wishing to be bound by any theory or mechanism of action, it is postulated that the peptides may be able to bind p53, thereby preventing p53 from binding to damaged DNA. The peptide can be tested by analyzing its ability to inhibit the hyperthermia response of the L12 cell line (which lacks endogenous p53 activity and has been stably transfected with the p53 gene or control vector). In these cells, p53 activity causes cell growth arrest and survival rather than apoptosis in response to hyperthermia (as described in WO 2012/160563, the contents of which are incorporated herein by reference).

Methods for measuring apoptosis.

Apoptosis is an active gene-directed process of cell self-destruction associated with characteristic morphological and biochemical changes. Nuclear and cytoplasmic condensation and fragmentation of the dying cell into membrane-bound apoptotic bodies are typical characteristics of apoptosis. Another feature of apoptotic cell death

- 9 046208 is the degradation of chromosomal DNA into oligonucleosomal fragments after activation by specific nucleases.

By inhibition of apoptosis or suppression of apoptotic activity is meant any reduction in the number of cells that undergo apoptosis compared to untreated control (ie, cells not exposed to the peptides of the present invention). Preferably the reduction is at least 25%, more preferably the reduction is at least 50%, and most preferably the reduction is at least one factor.

Flow cytometry offers a wide range of options for measuring apoptosis. Various methods have been developed and implemented, some of which stain the surface of cells and some of which stain intracellularly.

One of the first approaches was, in addition to observing that apoptotic cells shrink and have higher intracellular granularity, to stain with DNA-specific fluorochromes (eg, propidium iodide [PI], ethidium bromide [EtBr]). Once the fatal blow is dealt, the DNA begins to change its profile. Apoptotic DNA not only consists of fragmented DNA (visualized as shorter bands, called DNA ladders, in an agarose gel), but is also partially degraded into individual nucleotides, so that fluorochromes such as PI or EtBr have less DNA to stain ( Nicoletti et al., 1991). This is typically observed by a leftward shift called a sub-G^ peak on a specific fluorochrome detection channel in FACScan™ (from Becton Dickinson, USA).

Another method is terminal deoxynucleotidyl transferase (TdT)-mediated DNA break end labeling (TUNEL). The TUNEL method detects DNA strand breaks in cells undergoing apoptosis. TdT is an enzyme that catalyzes the addition of deoxyribonucleotide triphosphate to the 3'-OH ends of double- or single-stranded DNA. Unlike normal cells, the nuclei of apoptotic cells contain exogenous nucleotides (dUTP)-DIG in the presence of TdT. A fluorochrome-conjugated anti-DIG antibody fragment allows visualization of apoptotic cells. An increase in the number of apoptotic cells causes more DNA fragments and therefore brighter fluorescence. The advantage of this method is its very high specificity (Gavrieli et al., 1992). The disadvantage of this method is that it is expensive and can only be used for a small set of samples as it is time consuming. Therefore, it is not applicable for large screening programs.

The loss of cell membrane polarity and the presentation of increased amounts of phosphatidylserine (PS) on the outer side of the cell membrane during the early phase of apoptosis has led to another new approach. Annexin V is a calcium-dependent phospholipid binding protein with high affinity for PS. The integrity of the cell membrane is maintained during the early and intermediate phases of apoptosis. Early and intermediate apoptotic cells show increased annexin-FITC binding and are generally negative for PI staining. Late stages of apoptosis and necrotic cells become double positive due to surface presentation of PS and intracellular nucleic acid staining of PI due to membrane breakdown. This method is also expensive and labor-intensive.

Other methods for measuring apoptosis in vivo and in vitro are disclosed in US Patent Nos. 6,726,895 and 6,723,567.

Thus, according to the first aspect of the present invention, there is provided an isolated peptide, the length of which does not exceed six amino acids, which contains the amino acid sequence represented by the formula

X ₁ -X ₂ -X ₃ -X4-X ₅ (SEQ ID NO: 1), where (i) X1 is proline or an analogue or derivative thereof;

(ii) X ₂ is proline or an analogue or derivative thereof;

(iii) X3 is selected from the group consisting of alanine, valine, leucine, cysteine, isoleucine, methionine and a derivative or analogue thereof;

(iv) X ₄ is selected from the group consisting of alanine, valine, serine, proline and a derivative or analogue thereof;

(v) X ₅ is any amino acid and (vi) the peptide is capable of reducing the amount of dexamethasone-induced spleen and/or thymus weight loss in mice.

The peptide according to this aspect of the present invention may be five or six amino acids in length.

Preferably, the amino acids are selected such that the peptide bond between X1 and X2 (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) is in cis -configurations, i.e. the equilibrium was shifted towards the cis configuration; and a peptide bond between X3 and X4 (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even

- 10 046208

90% of the time) was in the trans configuration, i.e. the balance was shifted towards the trans configuration.

Methods for testing the cis/trans configuration of a peptide bond are known in the art and include, for example, NMR.

In one embodiment, X ₄ is selected from the group consisting of alanine, proline, an alanine derivative or analogue, and a proline derivative or analogue.

In a specific embodiment, the proline derivative or analogue is N-methylproline, α-methylproline, or α-aminobutyric acid.

In one embodiment, the peptide contains the sequence set forth in SEQ ID NO: 3 (PPLPY).

In another embodiment, the peptide consists of the sequence set forth in SEQ ID NO: 3 (PPLPY).

In another embodiment, the peptide consists of the sequence set forth in SEQ ID NO: 7 (LPPLPY).

In a specific embodiment, X ₁ and/or X ₃ may be D-amino acids, such as the peptide set forth in SEQ ID NO: 20 (d-LPPLPY).

According to another aspect of the present invention, there is provided an isolated peptide consisting of no more than ten amino acids, which contains the amino acid sequence represented by the formula

X ₁ -X ₂ -X ₃ -X ₄ -X5-X ₆ (SEQ ID NO: 17), where (i) X1 is proline or an analogue or derivative thereof;

(ii) X ₂ is proline or an analogue thereof;

(iii) X3 is selected from the group consisting of alanine, valine, leucine, cysteine, isoleucine, methionine and a derivative or analogue thereof;

(iv) X4 is selected from the group consisting of alanine, valine, serine and a derivative or analogue thereof;

(v) X5 is any amino acid;

(vi) X6 is proline or an analogue or derivative thereof and (vii) the peptide is capable of reducing the amount of dexamethasone-induced spleen and/or thymus weight loss in mice.

Preferably, the amino acids are selected such that the peptide bond between X1 and _X2 (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) is in cis configuration, i.e. the equilibrium was shifted towards the cis configuration; and the peptide bond between X ₃ and X ₄ (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) was in the trans configuration, i.e. .e. the equilibrium was shifted towards the trans configuration; and the peptide bond between X5 and X6 (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) was in the cis configuration, i.e. . the equilibrium was shifted towards the cis configuration.

In one embodiment, the peptide of this aspect of the present invention is 6 amino acids in length.

An example of such a peptide is the peptide shown in SEQ ID NO: 18 (PPLAYP).

Preferred unnatural amino acids that can be used to replace proline include, but are not limited to, N-methylproline, α-methylproline and α-aminobutyric acid.

The present inventors contemplate an additional amino acid attached to the N-terminus of X1 SEQ ID NO: 17.

The additional amino acid is preferably selected such that the peptide bond between it and X ₁ (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) is in cis configuration, i.e. the equilibrium was shifted towards the cis configuration.

Candidate amino acids include, but are not limited to, alanine, valine, leucine, cysteine, isoleucine, methionine, and a derivative or analogue thereof.

In a specific embodiment, the additional amino acid attached to the N-terminus of X1 is leucine or a derivative or analog thereof.

In a further embodiment, the additional amino acid attached to the N-terminus of X1 is a D-amino acid.

Thus, the present inventors provide peptides containing 7 amino acids and having the formula given in SEQ ID NO: 17.

In a specific embodiment, X4 is alanine or an analogue or derivative thereof.

In yet another embodiment, X5 is selected from the group consisting of tyrosine, phenylalanine, tryptophan, and a derivative or analogue thereof.

- 11 046208

An example of an amino acid that is considered an X5 is tyrosine.

An example of an amino acid that is considered an X3 is leucine.

The peptide of this aspect of the present invention may comprise the amino acid sequence set forth in SEQ ID NO: 4 (LPPLAYP). Such a peptide may consist of 7, 8, 9 or 10 amino acids.

The peptide of this aspect of the present invention may consist of the amino acid sequence set forth in SEQ ID NO: 4.

For peptides of this aspect of the present invention, X1 and/or X3 and/or X5 may be D-amino acids, such as, for example, d-LPPLAYP (SEQ ID NO: 21).

For any of the peptides described herein, the present invention also contemplates retro-inverse peptides. Such peptides are resistant to proteases and consist of D-amino acids in reverse order, which leads to a change in the peptide backbone, but the orientation of the side chains remains unchanged.

According to another aspect of the present invention, another isolated five amino acid peptide is provided, which consists of the amino acid sequence represented by the formula

X ₁ -X ₂ -X3-X ₄ -X5 (SEQ ID NO: 5), where (i) X1 and X3 are any amino acids;

(ii) X ₂ is proline or an analogue or derivative thereof;

(iii) X ₄ is not proline;

(iv) the peptide is able to reduce the amount of dexamethasone-induced loss of spleen and/or thymus mass in mice.

Preferably, the amino acids are selected such that the peptide bond between X1-X2 (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) is in cis- configurations, i.e. the equilibrium was shifted towards the cis configuration; and the peptide bond between X3-X4 (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) was in the trans configuration, i.e. . the balance was shifted towards the trans configuration.

According to another aspect of the present invention, there is provided an isolated peptide consisting of seven amino acids, which consists of the amino acid sequence represented by the formula

1-X ₂ -X3-X ₄ -X ₅ -X ₆ -X ₇ (SEQ ID NO: 6), where (i) X1, X ₅ and X ₆ are any amino acids;

(ii) X2, X3 and X7 are proline or an analogue or derivative thereof;

(iii) X4 is not a proline and (iv) the peptide is able to reduce the amount of dexamethasone-induced spleen and/or thymus weight loss in mice.

Preferably, the amino acids are selected such that the peptide bond between X ₁ -X ₂ (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) is in cis configuration, i.e. the equilibrium was shifted towards the cis configuration; the peptide bond between X2-X3 (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) was in the cis configuration, i.e. the equilibrium was shifted towards the cis configuration; the peptide bond between X6-X7 (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) was in the cis configuration, i.e. the equilibrium was shifted towards the cis configuration; and the peptide bond between X4-X5 (at least 50% of the time, at least 60% of the time, at least 70% of the time, at least 80% of the time, or even 90% of the time) was in the trans configuration, i.e. . the equilibrium was shifted towards the trans configuration.

The peptides described herein can be used to treat a variety of diseases, including those associated with stress-associated responses. These include pathological conditions such as neurodegenerative diseases (for example, stroke, Parkinson's disease and Alzheimer's disease), myocardial infarction, exposure to radiation or chemotherapy, inflammation, trauma (for example, burns and damage to the central nervous system), cellular senescence, hyperthermia, convulsions, hypoxia (eg, ischemia and stroke), as well as in graft tissues and organs prior to transplantation.

These conditions also include autoimmune diseases, characterized by a state of immunization of a person against at least one of the normal components of the body. These phenomena are observed in particular in pathologies including, but not limited to, infections associated with SLE (systemic lupus erythematosus), Huger-Sjögren syndrome (or Sjögren's disease) and rheumatoid polyarthritis, as well as in pathologies such as sarcoidosis and osteopenia, spondyloarthritis, scleroderma, multiple sclerosis, amyotrophic lateral sclerosis (ALS), hyperthyroidism, AD

- 12 046208 dysona, autoimmune hemolytic anemia, Crohn's disease, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, idiopathic purpura, hemorrhage, insulin-dependent diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, post-streptococcal glomerulonephritis, psoriasis and spontaneous infertility, as well as immediate or delayed effects observed during graft rejection and graft-versus-host disease. In one specific embodiment, the peptides of the present invention are useful for treating multiple sclerosis. In another embodiment, the peptides of the present invention are useful for the treatment of myocardial ischemia or infarction.

Other diseases contemplated by the present invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, secondary degeneration after trauma, stroke, CNS toxicity, glaucoma, macular degeneration, type 1 diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, autoimmune uveitis , graft versus host disease, graft rejection, arthritis, systemic inflammatory response syndrome (SIRS), inflammatory bowel disease (IBD), adult respiratory distress syndrome (ARDS), psoriasis, atherosclerosis, myocardial infarction, radiation sickness, hyperthermia, hypoxia, fulminant toxic liver, kidney failure, infertility and many others.

The phenomenon of transplant rejection is a state of immunization of a person against foreign components (biological fluids such as blood, cerebrospinal fluid, etc., cells, tissues, organs, antibodies, etc.) intentionally implanted into the patient.

As used herein, the terms degenerative disorder, degenerative disease and degenerative condition refer to any disorder, disease or condition characterized by inappropriate cell proliferation or inappropriate cell death, or in some cases both, or aberrant or unregulated apoptosis. These conditions also include those in which excessive apoptosis, although appropriate and regulated at the individual cell level, is associated with organ dysfunction or failure.

In one embodiment, the peptides are useful for preventing cell death in non-cancerous tissue or cells in a subject suffering from a neoplastic disease and undergoing chemotherapy and/or radiation therapy to treat cancer.

As used herein, the terms inflammatory disease and inflammatory condition mean any disease or condition in which an excessive or unregulated inflammatory response results in excessive inflammatory symptoms, host tissue damage, or loss of tissue function.

In one embodiment, the inflammatory disease or condition is an autoimmune disease.

In another embodiment, the inflammatory disease or condition is characterized by an etiology related to the production of at least one pro-inflammatory cytokine selected from IL-6 and TNF-α.

In another embodiment, the disease or condition is selected from the group consisting of Alzheimer's disease, Parkinson's disease, secondary degeneration after trauma, stroke, central nervous system toxicity, glaucoma, macular degeneration, myocardial infarction, radiation sickness, hyperthermia, hypoxia, fulminant toxic liver, renal failure and infertility.

In yet another embodiment, the disease includes retinitis pigmentosa and macular degeneration.

In another embodiment, the disease includes a stroke or myocardial infarction.

The peptides may be provided by themselves or as part of a pharmaceutical composition where they are mixed with suitable carriers or excipients.

As used herein, the term pharmaceutical composition refers to a preparation of one or more active ingredients described herein with other chemical components, such as physiologically appropriate carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound into the body.

As used herein, the term active ingredient refers to the peptides responsible for the biological effect.

As used herein and hereinafter, the phrases physiologically acceptable carrier and pharmaceutically acceptable carrier, which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to the body and does not interfere with the biological activity and properties of the compound administered. An auxiliary is included below these phrases.

The preparation of pharmaceutical compositions containing peptides or polypeptides as active ingredients is well known in the art. Typically, such compositions are prepared for their intended purpose as either liquid solutions or suspensions, however solid forms can also be prepared which can be suspended or solubilized before ingestion.

- 13 046208 section. The drug can also be emulsified. The active therapeutic ingredient is mixed with inorganic and/or organic carriers that are pharmaceutically acceptable and compatible with the active ingredient. Carriers are pharmaceutically acceptable excipients (excipients) containing more or less inert substances when added to a pharmaceutical composition to give the composition a suitable consistency or form. Suitable carriers include, for example, water, saline, dextrose, glycerol, ethanol and the like. and their combinations. In addition, if desired, the composition may contain minor amounts of auxiliary agents, such as wetting or emulsifying agents and pH buffering agents, which increase the effectiveness of the active ingredient.

The toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, by determining the IC50 (concentration producing 50% inhibition) and _LD50 (lethal dose causing death in 50% of the animals tested) for the test animal. connections. Data obtained from these cell culture assays and animal studies can be used to determine dose ranges for use in humans. The dosage may vary depending on the dosage form used and the route of administration used. The exact composition, route of administration and dosage can be selected by the individual physician taking into account the patient's condition. (See, for example, Fingl et al., 1975).

The amount of active agent used in the composition for administration of the present invention is an amount effective to achieve the purpose of the particular active agent for the target indication. The amount of active agent in the compositions is generally a pharmacologically, biologically, therapeutically or chemically effective amount. However, this amount may be less than that amount when the composition is administered in a unit dosage form, since the unit dosage form may contain multiple compounds or active agents in a single composition or may contain separate pharmacologically, biologically, therapeutically or chemically effective amounts. The total effective amount can then be administered in cumulative units containing a total effective amount of the active agent.

A therapeutically effective amount of a peptide of the present invention is an amount that, when administered to a patient, is capable of exhibiting anti-apoptotic activity and/or anti-inflammatory activity. Assays for detecting the antiapoptotic activity of the peptide of the present invention include, but are not limited to, DNA staining with specific fluorochromes such as propidium iodide and ethidium bromide, Annexin V assays, TUNEL assays, and the like, some non-limiting examples of such assays are presented in the examples below . Assays for determining the anti-inflammatory activity of peptides are also well known in the art.

Although the suitable dosage of the peptide of the present invention varies depending on the route of administration, age, body weight, gender or condition of the patient and should be determined by the physician at the end, the dose suitable for adult humans may generally be approximately 0.2-2000 mg/day. kg body weight, preferably about 2-200 mg/kg.

The pharmaceutical compositions of the present invention contain one or more compounds of the present invention and one or more excipients or diluents, in one embodiment, one or more compounds, or solvates, or salts of these compounds.

As used herein, the term pharmaceutically acceptable salt refers to salts that are substantially non-toxic to living organisms. Exemplary pharmaceutically acceptable salts include those obtained by reacting the compounds of the present invention with a pharmaceutically acceptable mineral or organic acid. Such salts are also known as acid addition salts.

Compositions containing compounds and active agents are useful for delivering active agents to selected biological systems and for increasing or improving the bioavailability of the active agent compared to administration of the active agent without the delivery vehicle. Delivery can be improved by delivering more of the active agent over a period of time, or by delivering the active agent at a specific time period (eg, to provide faster or delayed delivery) or over a period of time (eg, extended delivery).

Thus, pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers containing excipients and excipients that facilitate the processing of the active compounds into preparations that can be used in pharmaceuticals. The correct composition depends on the chosen route of administration.

The pharmaceutical compositions can be administered topically or systemically by any conventional and

- 14 046208 by walking route, including, but not limited to, oral, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, transdermal, intrathecal, topical, rectal, buccal, inhalation or intranasal.

For injection, the compounds of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer. For mucosal administration, the formulation uses penetration agents suitable for a permeable barrier. Such penetration agents, for example DMSO or polyethylene glycol, are generally known in the art.

Pharmaceutical compositions that can be administered orally include hard capsules made from gelatin, as well as soft sealed capsules made from gelatin and a plasticizer such as glycerin or sorbitol. Hard capsules may contain the active ingredients in admixture with excipients such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and optionally stabilizers.

In soft capsules, the active compounds may be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration must be in dosages appropriate for the chosen route of administration.

Alternatively, the compounds of the present invention may be included in liquid preparations for oral administration, such as aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, for example. Moreover, formulations containing these compounds may be presented as a dry product for mixing with water or other suitable carrier before use. Such liquid preparations may contain conventional additives such as suspending agents such as sorbitol syrup, methylcellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and hydrogenated edible fats; emulsifying agents such as lecithin, sorbitan monooleate or gum arabic; non-aqueous excipients (which may include edible oils) such as almond oil, fractionated coconut oil, oily esters, propylene glycol and ethyl alcohol; and preservatives such as methyl or propyl p-hydroxybenzoate and sorbic acid.

For administration by inhalation, the peptides for use in accordance with the present invention are conveniently delivered in the form of an aerosol spray from a pressurized package or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be defined by providing a valve to deliver the metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mixture of the peptide and a suitable powder base such as lactose or starch.

The pharmaceutical compositions of the present invention are also useful for topical and intralesional application. As used herein, the term topical means relating to a specific surface area, such as the skin and mucous membranes, and a topical applied to a specific surface area will only affect the area to which it is applied. The peptide/peptide analog formulations may be administered topically as a gel, ointment, cream, emulsion, sustained release formulation, including a transdermal patch, and may contain liposomes and any other pharmaceutically acceptable carrier suitable for topical administration of the drug. The pharmaceutical compositions described herein may also include suitable solid carriers or gel phase excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.

The compositions of the present invention may, if desired, be presented in a package or dosing device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient. The packaging may, for example, comprise metal or plastic foil, such as a blister pack. Instructions for use may be included with the package or dispenser. The package or dispenser may also bear a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the composition form or administration to a human or animal. Such a notice may, for example, relate to FDA-approved labeling for prescription drugs or to an approved product insert. Compositions containing a drug of the present invention formulated in a compatible pharmaceutical carrier may also be formulated, placed in an appropriate container and labeled for the treatment of a specified condition, as further described in detail above.

- 15 046208

As used herein, the term approximately refers to ±10%.

The terms contains, containing, includes, including, having and related words by root and meaning mean including, without limitation.

The term consisting of means including and limited.

The term consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not significantly change the essential and novel characteristics of the claimed composition , method or structure.

As used herein, the singular forms include plural references unless the context clearly dictates otherwise. For example, the term compound or at least one compound may include a plurality of compounds, including mixtures thereof.

Herein, various embodiments of the present invention may be represented in a range format. It should be understood that the description in range format is provided merely for convenience and brevity and should not be construed as strictly limiting the scope of the present invention. Accordingly, the range description should be considered to specifically disclose all possible subranges as well as the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6 etc., as well as individual numbers within that range, such as 1, 2, 3, 4, 5 and 6. This applies regardless of the width of the range.

Whenever a numerical range is specified herein, it is intended to include any quoted number (fractional or integer) within the stated range. The phrases range/ranges between the first specified number and the second specified number and range/ranges from the first specified number to the second specified number are used interchangeably herein and are intended to include the first and second specified numbers and all fractional and integer numbers between them.

As used herein, the term method refers to methods, means, technologies and procedures for performing a given task, including, without limitation, those methods, means, technologies and procedures that are either known or readily developed from known methods, means, technologies and procedures by practitioners in chemistry, pharmacology, biology, biochemistry and medicine.

As used herein, the term treatment includes stopping, substantially suppressing, slowing or reversing the progression of a condition, substantially alleviating clinical or aesthetic symptoms of a condition, or substantially preventing the onset of clinical or aesthetic symptoms of a condition.

It will be understood that certain features of the present invention, which for clarity are described in the context of individual embodiments, may also be provided in combination according to one embodiment. Conversely, various features of the present invention, which for brevity are described in the context of one embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the present invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment operates without those features.

Various embodiments and aspects of the present invention, as described above and claimed in the claims section, find experimental support in the following examples.

Examples

Reference is made to the following examples, which together with the above descriptions illustrate some embodiments of the present invention without limitation.

Generally, the nomenclature used herein and laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such methods are described in detail in the literature. See for example:

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Molecular Cloning: A laboratory

Manual Sambrook et al., (1989); Current Protocols in Molecular Biology Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al. (eds) Genome Analysis: A Laboratory Manual Series, Vols. 14, Cold Spring Harbor Laboratory Press, New York (1998); methodologies set forth in US Pat. No. 4,666,828; 4683202; 4801531; 5192659 and 5272057; Cell Biology: A Laboratory Handbook, Volumes I-III Cellis, J. E., ed. (1994); Culture of Animal Cells - A Manual of Basic Technique by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; Current

Protocols in Immunology Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W. H. Freeman and Co., New York (1980); available immunoassays are described in detail in the patent and scientific literature, see, for example, US patent No. 3,791,932; 3839153; 3850752; 3850578; 3853987; 3867517; 3879262; 3901654; 3935074; 3984533; 3996345; 4034074; 4098876; 4879219;

5011771 and 5281521; Oligonucleotide Synthesis Gait, M. J., ed. (1984); “Nucleic Acid Hybridization Hames, B. D., and Higgins S. J., eds. (1985); Transcription and Translation Hames, B. D., and Higgins S. J., eds. (1984); Animal Cell Culture Freshney, R. I., ed. (1986); Immobilized Cells and Enzymes IRL Press, (1986); A Practical Guide to Molecular Cloning Perbal, B., (1984) and Methods in Enzymology Vol. 1-317, Academic Press; PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, CA (1990); Marshak et al., Strategies for Protein Purification and Characterization - A Laboratory Course Manual

CSHL Press (1996);

all of which are incorporated by reference as if they were fully set forth herein. Other general references are provided in this document. It is assumed that the procedures described herein are well known in the art and are provided for the convenience of the reader. All information contained herein is incorporated herein by reference.

Example 1.

Dexamethasone is a corticosteroid drug that causes apoptosis of immune cells and lymphomyeloid tissues. BALB/c mice were used to study the ability of candidate peptides to rescue lymphocyte cells from apoptosis. Mice were intraperitoneally injected with 100 μg of dexamethasone. Dexamethasone-treated mice received an intravenous injection of candidate peptides (250 or 400 μg peptide/mouse) immediately after dexamethasone treatment and 24 h thereafter. Mice were sacrificed 48 h after the first exposure. The spleen and thymus were weighed and a complete cell count of both organs was performed.

The peptides used in the screening were the following:

PPLPY - SEQ ID NO: 3

LPPLAYP - SEQ ID NO: 4

PLPYP - SEQ ID NO: 9

PPL - SEQ ID NO: 10 PLP-SEQIDNO: 11 PYP - SEQ ID NO: 12 LPGLPYP - SEQ ID NO: 13 LPPLGYP - SEQ ID NO: 14 LAPLPYP - SEQ ID NO: 15 LPALPYP - SEQ ID NO: 16 Results.

Dexamethasone caused loss of spleen and thymus mass and a decrease in spleen cell number by approximately 50%. The number of thymus cells was reduced by almost 80% compared to normal mice. Two peptides had a significant effect on reducing spleen and thymus weight loss, as well as cell number reduction, SEQ ID NO: 3 and SEQ ID NO: 4 (see FIG. 1). Did not have

- 17 046208 difference between two exposure doses (250 or 400 μg/mouse).

Example 2.

BALB/c mice were used to study the ability of candidate peptides to rescue lymphocyte cells from apoptosis. Mice were intraperitoneally injected with 100 μg of dexamethasone. Dexamethasone-treated mice received an intravenous injection of candidate peptides (200 μg peptide/mouse) immediately after dexamethasone treatment and 24 h thereafter. Mice were sacrificed 48 h after the first exposure. The spleen and thymus were weighed and a complete cell count of both organs was performed. The peptides used in the screening were the following:

LPPLPYP - SEQ ID NO: 2 (control)

PPLAYP - SEQ ID NO: 18

LPPLPY- SEQ ID NO: 7

LPPLAYP - SEQ ID NO: 4

PPLPY - SEQ ID NO: 3

PPLPY-NH2 - SEQ ID NO: 19

Results.

The results are presented in Fig. 2. Each of the peptides tested showed an improvement over the control peptide (SEQ ID NO: 2).

Although the present invention has been described in connection with its specific embodiments, it will be appreciated that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to cover all such alternatives, modifications and variations that come within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned herein are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent or patent application had been specifically and individually indicated to be incorporated herein by reference. . Moreover, the citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art for the present invention. To the extent that section headings are used, they should not be construed as mandatory limitations.

In addition, the priority application document for this invention is incorporated herein by reference in its entirety.

Claims (4)

1. An isolated peptide consisting of no more than ten amino acids, which contains the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 18 or SEQ ID NO: 21. 2. Isolated peptide according to claim 1, consisting of 6 or 7 amino acids. 3. The isolated peptide according to claim 1, consisting of the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 18 or SEQ ID NO: 21. 4. A pharmaceutical composition containing the peptide according to any one of claims 1-3 as an active agent and a physiologically acceptable carrier.