NZ720748B2 - Ezrin-derived peptides and pharmaceutical compositions thereof - Google Patents

Abstract

The present invention concerns ezrin-derived peptides, in particular, a peptide comprising an amino acid sequence of general formula (I) X1 EKKRRETVERE X2X3 (SEQ ID. No:5), wherein each X represents a non-polar amino acid residue and wherein the amino acid sequence is 14 to 25 residues in length. The invention further includes the use of these ezrin-derived peptides as immunostimulatory agents, and more specifically, for use in treating and preventing antiviral and treatment of diseases of the GI tract, in particular ulcerative disorders of the GI tract. The present invention also relates to pharmaceutical compositions comprising the peptides. Further, the invention relates to methods of treatment of infection and ulcerative diseases of the GI tract comprising administering the peptides to patients in need thereof.

Description

Ezrin-Derived Peptides and Pharmaceutical Compositions Thereof The present invention s to the field of ne, ically, to the field of chemical and pharmaceutical industry and concerns ezrin—derived peptides, in particular, a peptide comprising an amino acid sequence of general formula (I) X] EKKRRETVERE X2X3, wherein each X represents a non—polar amino acid e. The use of the peptides as immunostimulatory , and more specifically, for use in treating and preventing antiviral, antibacterial and antifungal infections, and treatment of diseases of the GI tract, in particular ulcerative disorders of the GI tract. The present invention also relates to pharmaceutical compositions comprising the es. Further, the invention relates to s of treatment of ion and ulcerative diseases of the GI tract sing administering the es to patients in need thereof.

Ezrin protein, also known as cytovillin or villin-Z, is a protein encoded in humans by the EZR gene.

Peptides derived from ezrin protein, having biological ty, are well-known. The closest ue to the peptide of the invention is a ceutical ecapeptide NH3_Thr—Glu—Lys— Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu_COOH, comprising 14 amino acid residues, which is known as HEP—1 peptide or human ezrin peptide one (sequence ID No. 2: TEKKRRETVEREKE) and which was developed for the treatment of HIV—infection [R.D.Holms, AIDS prophylactics. PCT/GB95/001285, 02.06.95, WO 95/33768] and, further, for the treatment of a wide range of bacterial, fungal, and viral infections.

Lyophilized pharmaceutical product GEPON® (marketing authorization number: P N000015/ 010911), comprising about 96% of HEP-l peptide, is widely used for medical proposes in the Russian Federation (Kladova, O.V., mova, F.S., Shcherhakova, A.A., Leg/(ova, T.P, Feldfix, L.l., skaya, A.A., Ovchinnikova, G.S., Uchaikin, V.F. The first experience of Hepon intranasal application in children with respiratory infections. // Pediatrics, 2002, No. 2, pp. 86-88), (Polyakova T.O., Magomedov, M.M., Arten'zyev, M.E., Surikov, E. V., Palchun, V. T. A new approach in the treatment of chronic diseases of the pharynx / Attending Doctor, 2002, No. 4, pp.

, (Novokshonov, A.A., Uchaikin, V.F., Sokolova, N.V., Tikhonova, 0.N., Portnykh, 0.Yu.

Biocenosis—protecting therapy of intestinal infections in children. // Russian l Journal, special supplement "Diseases of the Digestive System 2004, volume 6, No. I), (Pctljfe110\i, A]. The activator of the local immunity Hepon in the complex treatment of dysbiotic disorders of the intestine. // Experin’zental and Clinical Gastroenterology, 2003, N93, pp. 66—69),. (Tishchenko, AL.

A new approach in the treatment of recurrent urogenital candidiasis. // Attending Doctor, 2002, No. 3, pp. 46—47), (Telunts, AV. Treatn’zent of candidiasis in infants. // Questions of Gynecology, rics, and Perinatology, 2004, vol. 3, No. 4, pp. 89—90), (Ataullakhanov, R.I., Holms, R.D., Katlinsky, A.V., Pichugin A.V., Papuashvili, M.N., Shishkova, N.M. Treatment with Hepon ininiLmomodulator ses the efficacy of the immune control of unistic ii’tfections in HIV— infected patients. / / Allergy, Asthn’za, and Clinical Immunology, 2002, No. 10, pp. 3—11), (Cherednichenko, T.V., Uchaikin, V.F., Chaplygina, G.V., Kurbanova, GM. A new exicient treatment of viral hepatitis.// Attending Doctor, 2003, No. 3, pp. 82—83), rets, I.P., Voronkova, N. V., Lopatina, T.V., Ivanovskaya, V.N., Braginsky, D.M., Blokhina, N.P., Ma/yshev.

NA. The combined use of Hepon drug product and recombinant ii’tteiyferon—alpha in the patients with chronic hepatitis C increases the e?iciency of antiviral treatment and reduces side (y’j‘ects of the therapy.// Hepatology, 2003, No. 4, pp. 23—28), (Lazehnik, L.B., Zvenigorodskaya, L.A., Firsakova, V. Yu., in, A. V., Atardlakhanov, RI. The application ofHepon immunomodulator in the treatment of erosive deemus lesions of gastroduodenal / Experimental and Clinical Gastroenterology, 2003, No. 3, pp. 17—20), (Mala/(how, N.S., Pichugin, A. V., Khalif, l.L., Ataullakhanov, RI. The application ofHepon immunomodulator for the ent of nonspecific ]] , ulcerative colitis.// Farmateka, 2005, No. 6 [ pp. 105-108). DLulchenko, M.A., Lysenko, B.F., Chelishvili, A.L., Katlinsky, A.V., Ataullalchanov, RR. Complex treatment of trophic .// Attending Doctor, 2002, N0. 10, pp. 72—75), chev, M.S. Treatment l ion injuries with the activator of local immunity.// Russian Medical Journal, 2003, volume I I, No. l l (183), pp. 646—647), Salamov G., R.Holms, W. r, R. Ataullakhanov. Treatment of hepatitis C virus infection with human ezrin peptide one (HEPI) in HIV infected patients.// Arzneimittel—Forschung (Drug Research) 2007; 57(7): 497—504].

HEP—l has anti—viral hepatitis C biological activity and can be used for the treatment of the patients with hepatitis C [R.D.Holms. R.I.Ataullakhanov. HCV combination therapy.

PCT/GBZ()04/()()()330, 27.01.2004, WO 7024 A2].

HEP-l has antiulcer ical activity and can be used for the treatment of ulcer diseases of the gastrointestinal tract [R.D.Holms, R.l.Ataullakhanov. The use of peptides in anti-ulcer y. , 23.1 1.2006, WO/2007/060440].

The t inventors have developed a novel peptide which was prepared tically and which has high immunostimulatory activity. As biological tests have shown, the peptide of the invention as described and pharmaceutical compositions thereof have high immunostimulatory activity, exceeding the activity of HEP—l peptide, which is the main component of GEPON® drug product.

In a first aspect of the invention, there is provided a peptide comprising an amino acid sequence of general formula (I) XIEKKRRETVEREXZXx wherein each of X1, X3 and X3 represent a non—polar amino acid residue (SEQ ID No: 5).

Non—polar amino acid residues can be independently selected from the group consisting of e, alanine, valine, leucine, methionine, isoleucine, proline, alanine and tryptophan, and/ combinations thereof.

In some embodiments, X]_ X: and/ X; can be independently selected from non—polar amino acids or or having small R groups, in particular e, alanine and/ valine, and/ combinations thereof.

Thus, in some embodiments of the invention, each of X1, X3 and X; are ndently selected from the group consisting of glycine, alanine and valine.

In some embodiments, X1. X2 and/or X; can all be the same amino acid. In a preferred embodiment, at least one of X1, X3 and X3 is glycine. In a more preferred embodiment, at least 2 of X1, X2 and X3 is glycine. In the most preferred embodiment, each of X1 ‘X2 and X3 is glycine. In one embodiment, the peptide comprises an amino acid sequence of general formula (I) X] EKKRRETVERE X2X3, wherein each of X1, X2 and X; are non-polar, and further n at least one, or at least two, of X1, X2 and X; are e. In a preferred embodiment, the peptide has the sequence GEKKRRETVEREGG.

In the present disclosure, the one letter codes have been used to designate the various amino acids.

Using the three letter and one letter codes the amino acids may also be ed to as follows: glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg), histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N or Asn), glutamine (Q or Gln), cysteine (C or Cys), methionine (M or Met), serine (S or Ser) and threonine (T or Thr). Where a e may be aspartic acid or asparagine, the symbols Asx or B may be used. Where a residue may be glutamic acid or glutamine, the symbols Glx or Z may be used.

References to aspartic acid include aspartate, and glutamic acid include glutamate, unless the context specifies otherwise.

The term peptide may include compositions sing the amino acid sequences disclosed herein.

For example, peptides may be modified (for example at the C or N terminals) to t them from degradation or to increase their bioavailability and/ biocompatibility, as deemed suitable or required by the skilled person. It is noted that features ng to "peptide of the invention" apply equally to the amino acid sequence specified in the general formulae.

The peptide of the invention may be of any length provided the amino acid sequence comprises the l formula (I) XIEKKRRETVEREX3X3, wherein each X represents a non—polar amino acid residue, as described herein.

In some embodiments, the peptide is 1 4 to 25 residues in length, for example 1 4 to 20 residues in length, or 1 to 18 residues in length and comprises the amino acid sequence of general a (I) (or further defined embodiments thereof). In some ments, the peptide is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 residues in length and comprises amino acid sequence of general formula (I) (or further defined embodiments thereof). In further embodiments, the peptide is 1 4 to residues in length, for example 1 4 to 20 residues in length, or 1 4 to 18 residues in length and comprises the sequence of SEQ ID No: l (GEKKRRETVEREGG). In some ments, the peptide is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 residues in length and comprises the sequence of SEQ ID No: 1 (GEKKRRETVEREGG). In one embodiment, the peptide is between 1 4 and 18 residues in length and comprises an amino acid sequence of general formula (I) X1 EKKRRETVERE X2X3, wherein each of X1, X3 and X; are non—polar, and further n at least two of X1, X2 and X; are glycine. In preferred embodiments, the peptide of the ion is at least 14 amino acids in length and ses SEQ ID No: 1 (GEKKRRETVEREGG).

Preferably, the peptide of the invention is 14 amino acid residues in length. In the most preferred embodiment of the invention, the peptide has the sequence of NH2_Gly—Glu—Lys—Lys—Arg—Arg— Glu—Thr—Val—Glu—Arg—Glu—Gly—Gly_COOH (sequence ID No.1: GEKKRRETVEREGG) n referred to as HP-V2).

The objective of the present invention is the extension of the range of peptide compounds having enhanced immunostimulatory activity. Moreover, the objective of the t invention is the preparation of a pharmaceutical composition with the highest immunostimulatory activity and with a wide range of action on the basis of the claimed peptide and known Ezrin peptides. Generally, the es of the ion have a higher immunostimulatory profile and/or a higher antiviral activity when compared to peptides comprising or consisting of SEQ ID No: 2.

Immunostimulation and ral activity may be measured by any method known to a skilled person. For example, the immunological activity of the e can be assessed according to the effect on the synthesis of cytokine mRNA in J—96 cells (for example: IFN—u, IL—IB and IL—6).

Antiviral ty can be measured according to the cytotoxic effect on Encephalomyocarditis Virus (ECM) and/or according to the ICSO for the peptide in inhibiting EMC Virus replication. Other s would be apparent to the skilled person.

The desired objective can be ed with the proposed novel peptide comprising the sequence of formula (I), and derivatives thereof as discussed herein, and more particularly the sequence of SEQ ID No. 1: GEKKRRETVEREGG. These peptides of the invention are prepared synthetically and have high immunostimulatory activity. A search of the peptide sequences in the database http://research.bioinformatics.udel.edu/peptidematch/index.jsp and a search of the protein sequences in the database http://www.uniprot.org/blast/ has confirmed the novelty of the peptide (HP—V2), comprising 1 amino acid residues (sequence ID No. l: GEKKRRETVEREGG).

As biological tests have shown, the peptide of the invention and ceutical compositions thereof have high immunostimulatory activity, exceeding the activity of HEP—l peptide, which is the main component of GEPON® drug product.

Known ezrin peptide HEP—l (sequence ID No. 2: TEKKRRETVEREKE), and two smaller peptides: HPl—S peptide of the formula NHgiThr—Glu—Lys—Lys—Arg?COOH (sequence ID No. 3: TEKKR) hereinafter defined as HPl—S, and HP6—l4 peptide of the formula NHLArg—Glu—Thr—Val— Glu—Arg—Glu—Lys—GluiCOOH (sequence ID No. 4: RETVEREKE) hereinafter defined as HP6—l4, were used for the preparation of pharmaceutical compositions with the peptide of the ion.

The latter two peptides are cleavage products of HEP] e (sequence ID No. 2: TEKKRRETVEREKE) and have sequences, which are identical to those described previously [R.D.Holms. Regulatory/unfolding peptides of ezrin. PCT/GB00/03566, 2000, WO 01/025275]. HPl—5 peptide and HP6—l4 peptide are two satellite peptides, which were found in GEPON® drug product as impurities.

In some embodiments, the peptide of the invention can be combined with HEP-l (sequence ID No. 2: TEKKRRETVEREKE), or with HPl—5 peptide of the formula NHgiThr—Glu—Lys—Lys— Arg_COOH (sequence ID No. 3: TEKKR) hereafter defined as HPl—5, or with HP6—l4 peptide of the formula NH3_Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu_COOH (sequence ID No. 4: RETVEREKE) ter d as HP6-l4.

Peptides of the invention and combinations of es as bed herein may be useful in medicine, for example for use in the treatment and prevention of viral, ial and fungal infections, as well as for the treatment of ulcer diseases of mucous membranes and soft tissues.

In a second aspect of the invention, there is provided a peptide of the ion (such as a peptide of general formula (I), in particular a peptide comprising the amino acid sequence of SEQ ID No: l), for use in medicine. Particular medical uses include the treatment or prevention of infection.

The infection can be a viral, bacterial or fungal infection. Further embodiments of the invention include treatment or prevention of ulceration of the mucous membranes of the gut, such as ulcers of the h, large intestine, duodenum or small intestine, and the treatment or prevention of inflammatory bowel diseases, including diseases and/or disorders related to ulcers of the stomach, large ine, um or small intestine, and irritable bowel me (IBS). The peptides of the invention are also ed for use in the treatment or prevention of ulcerative colitis and Crohn’s disease. In preferred embodiments, the peptides of the invention are provided for use in the treatment or prevention of the lower gut inflammation and/or ulceration.

There is also provided a peptide of the invention (such as a peptide of l a (I), in particular a peptide comprising the amino acid sequence of SEQ ID No: l), for use in stimulating an immune response in a subject. In some embodiments of the invention, the peptides can be used to stimulate endogenous production of interferon in a subject. The es may also be used to to stimulate activation of MAPK/ signaling pathway.

In some embodiments of the invention, the peptide of the invention can be ed with HEP—l (sequence ID No. 2: TEKKRRETVEREKE), or with HPl—S peptide of the formula NHLThr—Glu— Lys-Lys-Arg_COOH (sequence ID No. 3: TEKKR) hereinafter defined as HPl-S, or with HP6-l4 peptide of the formula NHLArg-Glu-Thr-Val-Glu-Arg-Glu-Lys-GluiCOOH (sequence ID No. 4: RETVEREKE) hereinafter defined as HP6—l4 For example, a peptide of general formula (I), in particular a peptide comprising the amino acid sequence of SEQ ID No: 1, can be combined with one or more of SEQ ID No: 2, 3 or 4. Such combinations are useful in the medical aspects of the invention, including treatment or prevention of infection. The infection can be a viral, bacterial or fungal infection. The combinations of peptides are also useful in the ent or tion of ulceration of the mucous membranes of the gut such as ulcers of the stomach, large intestine, um or small intestine, and the treatment or tion of in?ammatory bowel diseases, including diseases and/ disorders related to ulcers of the stomach. large intestine, duodenum or small intestine, and irritable bowel syndrome (IBS). The peptides of the invention are also provided for use in the treatment or prevention of ulcerative colitis and s e. In preferred embodiments, the peptide combinations of the invention are provided for use in the treatment or prevention of the lower gut inflammation and/or ulceration.

Embodiments of the invention further extend to methods of treating or preventing infections, ulcers of the stomach, large intestine, duodenum or small intestine, diseases and/or disorders d to ulcers of the stomach, large intestine, duodenum or small intestine, irritable bowel me (IBS), ulcerative colitis and Crohn’s disease comprising administering the peptide of the invention (such as a e of general formula (I), in particular a peptide sing the amino acid sequence of SEQ ID No: l) to a patient in need thereof. The infection may be a viral, bacterial or fungal infection. The peptides of the invention are administered in a therapeutically effective amount and may be administered with one or more other ceutically active compounds. In particular, the es of the invention may be administered in combination with one or more of the peptides of SEQ ID No: 2, 3 and/or 4.

Thus there is also provided the combination of a peptide of the invention (such as a peptide of general formula (I), in particular a peptide sing an amino acid sequence of SEQ ID No: l) and a peptide comprising the amino acid sequence of SEQ ID No: 2, 3, or 4, for use in medicine.

Such medical uses include treating or preventing infections (such as viral, bacterial or fungal infections), tion of the mucous membranes of the gut (such as stomach ulcers, large intestine ulcers, duodenal ulcers and small ine ulcers), or inflammatory bowel disease or a disease or disorder associated with in?ammatory bowel disease (such as IBS, ulcerative colitis or s disease). In red embodiments, the peptides of the invention are provided for use in the treatment or prevention of the lower gut inflammation and/or ulceration. Such medical uses also include ating an immune response in a subject.

In preferred embodiments, when the peptides of the invention are used in combination with HEP-l, HPl-S or HPo-l4, the onal peptide consists of the amino acid sequence of SEQ ID No: 2, 3, or 4.

There is also provided a method of stimulating an immune response in a subject, comprising administering a therapeutically effective amount of a e of the invention (such as a peptide of general formula (I), in particular a peptide comprising the amino acid sequence of SEQ ID No: l) to a patient in need thereof.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising the peptide of the invention and a pharmaceutically acceptable carrier or . In some embodiments of the invention, the pharmaceutical composition may further se an additional pharmaceutically active compound. In particular, the pharmaceutical composition may further se HEP-l (sequence ID No. 2: TEKKRRETVEREKE), HPl-S nce ID No. 3: TEKKR), or HP6—l4 (sequence ID No. 4: RETVEREKE).

Accordingly, in one embodiment, the invention provides a pharmaceutical composition comprising a peptide of between 14 and 18 residues in length, the peptide comprising an amino acid sequence having the sequence X] EKKRRETVERE X2X3, wherein each of X, X: and X; are non—polar, and further wherein at least two of X1, X2 and X; are glycine, and a pharmaceutically acceptable carrier or filler.

Pharmaceutical compositions of the invention may comprise other pharmaceutically active substances, such as anti—Viral, acterial, ungal, sic and/or anti—inflammatory substances. The pharmaceutical composition may be formulated using any convenient adjuvant and/ physiologically acceptable diluents.

Fillers, carriers, preservatives, and stabilizers, which are usually used by persons skilled in drug delivery technology, may be used as an acceptable carrier or filler for preparation of the provided ceutical compositions. For injections, led water or logic saline are predominantly used.

The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical ding buccal, sublingual or transdermal), vaginal or parenteral ding aneous, intramuscular, intravenous or intradermal) route. Such itions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the r(s) or excipient(s) under e conditions. In particular embodiments, the pharmaceutical composition of the invention comprising the peptides of the invention is in the form of a tablet for oral administration, such as a glucose tablet. In some embodiments, the table is dissolvable.

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non- aqueous liquids; or as edible foams or whips; or as emulsions). In preferred embodiments, the pharmaceutical composition of the ion comprising the peptides of the invention is in the form of a tablet for oral administration, such as a glucose table. In some embodiments, the tablet is dissolvable.

In some embodiments of the ion, the peptides are provided in the form of lyophilised powder or granules.

Suitable excipients for tablets or hard gelatine es include lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatine capsules include for e vegetable oils, waxes, fats, olid, or liquid polyols etc.

For the preparation of solutions and syrups, excipients which may be used include for example water, polyols and sugars. For the preparation of suspensions oils (e.g. vegetable oils) may be used to provide oil—in—water or water in oil suspensions.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in lemzmremical Research, 3 (6), page 318 (1986).

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, s, powders, solutions, pastes, gels, sprays, aerosols or oils. For infections of the eye or other al tissues, for example mouth and skin, the compositions are suitably applied as a topical ointment or cream. When ated in an ointment, the active ingredient may be employed with either a paraffinic or a water—miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil—in—water cream base or a water—in—oil base. Pharmaceutical compositions adapted for l administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical compositions d for administration by inhalation include fine le dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.

Pharmaceutical itions adapted for vaginal administration may be ted as pessaries, tampons, , gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administration include s and non—aqueous sterile injection solution which may contain anti—oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and s and non—aqueous sterile suspensions which may include ding agents and thickening agents.

Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The itions may be presented in unit—dose or multi—dose ners, for example sealed ampoules and vials, and may be stored in a freeze—dried ilized) condition requiring only the addition of the sterile liquid carried, for example water for ions, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

The pharmaceutical compositions may n preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, nts, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the substance of the present ion.

Dosages of the pharmaceutical compositions of the t invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

Such compositions may be ated for human or for veterinary medicine. The present application should be interpreted as applying equally to humans as well as to animals, unless the context clearly implies otherwise.

In one embodiment, the invention provides a ceutical composition in the form of a tablet for oral administration, in particular a glucose , comprising a e of between 14 and 18 residues in length, the e comprising an amino acid ce having the sequence X] EKKRRETVERE Xng, wherein each of X1, X2 and X; are non—polar, and further wherein at least two of X1, X3 and X; are e, and a pharmaceutically acceptable carrier or filler. In a more preferred embodiment, the peptide has the sequence GEKKRRETVEREGG.

The invention extends to methods of manufacture of suitable pharmaceutical compositions, as well as the use of a peptide of the present invention in the manufacture of a medicament for use in medicine, for use in any of the uses specified herein.

In a further aspect of the invention, there is provided a nucleic acid ce encoding a peptide of the invention, in particular a peptide of general formula I, such as a peptide comprising the amino acid sequence of SEQ ID No: l or SEQ ID No: 5.

The nucleic acid may be DNA, cDNA or RNA such as mRNA obtained by cloning or produced by chemical synthesis. The DNA may be single or double stranded. Single stranded DNA may be the coding sense strand, or it may be the non—coding or anti—sense strand.

In a still further aspect of the invention there is provided a vector comprising a nucleic acid of the invention. In yet a further aspect of the invention there is provided a host cell comprising the vector of the t invention. Methods of manufacture or obtaining of such nucleic acids, vectors and host cells are also ed in the present invention and are known in the art.

The peptide of the present ion can be synthesized by e synthetic chemistry, for example the peptide of the invention can be synthesized by —phase synthesis (Cattzbinatorial Chemistry: A Practical Approach, ed. Hicham Fenniri, Oxford University Press (2000)) using standard procedure or by solid—phase synthesis , for example "Fmoc" or "Bmoc" synthesis, (Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Practical Approach 8.), ed.s W. Chan &, Peter White, Oxford University Press (2000)). When solid—phase synthesis is employed, then a solid phase is used, such as polystyrene resin or ide resin, or PEG hybrid polystyrene resin, or resin based on PEG. Different protective groups are used during the synthesis, for example, N- terminal protecting groups, t-Boc or FMOC protective groups. er, benzyloxycarbonyl (Z) groups or allyloxycarbonyl (Alloc) protective groups, or photoremovable (lithographic) protective groups, or side group protection technique may be employed. Peptide products are purified by HPLC separation or by any other purification method. e structure is confirmed by amino acid analysis, mass spectrometry, and high performance liquid chromatography data.

In some instances, fragments may be synthesised using solid-state methods and then coupled er in solution. Peptides can be synthesized from the carbonyl group side to amino group side of the amino acid chain in this method, although peptides are synthesized in the opposite direction in cells. In such methods, an amino-protected amino acid is bound to a substrate bead (i.e. a resin bead), forming a covalent bond between the carbonyl group and the resin. The amino group is then de-protected and d with the carbonyl group of the next amino-protected amino acid. The cycle is repeated as often as required in order to form the desired peptide chain. The synthesized peptide is then cleaved from the bead at the end of the procedure. The protecting groups for the amino groups mostly used in this peptide sis are 9— fluorenylmethyloxycarbonyl group ("Fmoc") and t—butyloxycarbonyl ("Boc"). The Fmoc group is removed from the amino us with base while the Boc group is removed with acid.

HEP—l peptide, HPl—S peptide, and HP6—l4 peptide, used in the present invention for the preparation of the pharmaceutical compositions, were prepared by liquid—phase synthesis method by the licensed manufacturer "Immapharma", LLC (Moscow).

In a further aspect of the invention, there is provided a method of making the peptides of the invention, in particular peptides according to general formula 1, such as es comprising the amino acid sequence of SEQ ID No: l or SEQ ID No: 5. The method may comprise synthesizing the peptides by liquid—phase synthesis or solid—phase synthesis.

In one embodiment of the invention, there is provide a peptide of 1 amino acids in length, wherein the amino acid sequence is GEKKRRETVEREGG. The peptide is useful in medicine, in particular in treating lower gut in?ammation and/ tion. The peptide may be provided in the form of a tablet for oral administration, in particular a glucose tablet. red features for the second and uent aspects of the invention are as for the first aspect mututis m Lila/1011‘s.

The present invention will now be described by way of reference to the following Examples which are present for the purposes of reference only and are not to be construed as being limiting on the invention. In the Examples, reference is made to a number of drawings in which: Figure 1. High mance liquid chromatography of HEP-l peptide. Luna Cl 8 (2) column, 4.6 x 250 mm, 5.0 pm particles. Mobile phase: A — water, 5% ACN, 0,l% TFA; B — 0,l% TFA; program —25% ACN for 20 min. Flow rate: 1 ml/ X—axis: time, Y—axis: UV absorption (mV, upper line, with peaks), pressure (lower, horizontal line).

Figure 2. High performance liquid chromatography of the mixture of HEP—l peptide and HP 1—5 e. Luna C18 (2) column, 4 x 250 mm, 5.0 pm les. Mobile phase: A — water, 5% ACN, 0,1 % TFA; B — 0,1 % TFA; m 5-25% ACN for 20 min. Flow rate: 1 ml/min. X-axis: time, Y- axis: UV absorption (mV, upper line, with peaks), pressure (lower, ntal line).

Figure 3. High performance liquid chromatography of the mixture of HEP-l peptide and HP 6-14 e. Luna C18 (2) column, 4 x 250 mm, 5.0 pm particles. Mobile phase: A — water, 5% ACN, 0,l% TFA: B — O,l% TFA; program 5—25% ACN for 20 min. Flow rate: 1 ml/ X—axis: time, Y— axis: UV absorption (mV, upper line, with peaks), pressure (lower, horizontal line).

Figure 4. High performance liquid chromatography of the mixture of HEP—l peptide and HP—V2 peptide. Luna C18 (2) column, 4.6 x 250 mm, 5.0 pm particles. Mobile phase: A — water, 5% ACN, 0,1% TFA; B — (),1% TFA: program 5—25% ACN for 20 min. Flow rate: 1 ml/min. X—axis: time, Y— axis: UV absorption (mV, upper line, with peaks), pressure (lower, horizontal line).

Figure 5. High performance liquid tography of the mixture of HP—VZ peptide. Luna C18 (2) column, 4 x 250 mm, 5.0 pm particles. Mobile phase: A — water, 5% ACN,0,1% TFA; B — 0,1 % TFA: program 5—25% ACN for 20 min. Flow rate: 1 ml/ X—axis: time, Y—axis: UV absorption (mV, upper line, with peaks), pressure (lower, ntal line).

Figure 6. MALDI TOF/ spectrum of the mixture of HEP—l peptide and HP—VZ peptide.

EXAMPLES The method of high performance liquid chromatography was used for accurate assessment of each component concentration during ation of the ceutical compositions. Results of the study demonstrated that it was impossible to distinguish HP—V2 peptide, having r amino acid number with HEP—l peptide, in the high performance liquid chromatography (HPLC) conditions, and this fact does not allow to access the tative t of the compositions based on HP—VZ and HEP-l (Example 4; Fig. I, Fig. 4, Fig. 5). Meanwhile the quantitative composition of the claimed HP-V2 e and satellite peptides HPl-S and HP6-l4 may be readily calculated based on the chromatograms as a uence of their good separation (Examples 2, 3; Fig. 2, Fig. 3).

However MALDI TOF mass spectrometry clearly detects each peptide in the mixture of two peptides, comprising 1 4 amino acid residues, in particular, in the mixture of HP—V2 peptide (sequence ID No. l: GEKKRRETVEREGG), which is defined as HP-V2, and HEP-l peptide (sequence ID No. 2: TEKKRRETVEREKE). which is defined as HEP-l (Example 5; Fig. 6).

Example 1. Preparation of HP-V2 peptide.

Synthesis of HP-V2 peptide was med by solid-phase method using the alkoxybenzyl polymer. Couplings were performed employing the method based on the usage of either: 1) tetrafluoroborate O-(benzotriazol-l-yl)-N,N,N’,N’- tetramethyluronium (TBTU); or 2) l- hydroxybenzotriazole (HOBT) and N,N’— diisopropylcarbodiimide (DIPC). Attachment of the sequential Fmoc—protected amino acid residues was performed singly, except the cases when unreacted amino groups were found on the growing yl—polymer after the coupling on.

Control over the unreacted amino group content in the peptidyl—polymer was held by means of ninhydrin test. Trifluoroacetic acid with addition of thioanisole, phenol, ethanedithiol, triisopropylsilane, and water was used for separation of the e and polymeric carrier and for final unblocking. Structure and homogeneity of the desired product was confirmed by amino acid analysis, mass spectrometry, and high performance liquid chromatography data.

The following pharmaceutical compositions based on provided HP—VZ peptide nce ID N 0. 1: GEKKRRETVEREGG), as well as on the mixtures thereof with the following peptides were HEP—l (sequence ID N 0. 2: TEKKRRETVEREKE); HPl—S (sequence ID No. 3: TEKKR); HP6—l4 (sequence ID No. 4: RETVEREKE). ceutical compositions: The pharmaceutical composition, comprising an effective amount of the peptide according to the sequence ID No. l: GEKKRRETVEREGG, and a pharmaceutically acceptable carrier or filler — the others.

The pharmaceutical composition, comprising an effective amount of the peptide according to the sequence ID No. l: GEKKRRETVEREGG, the peptide according to the sequence ID No. 2: ETVEREKE, and a pharmaceutically acceptable carrier or filler — the .

The pharmaceutical ition, comprising an effective amount of the peptide according to the sequence ID No. l: GEKKRRETVEREGG, the e according to the sequence ID No. 3: TEKKR, and a ceutically acceptable carrier or filler — the others.

The ceutical composition, comprising an effective amount of the peptide according to the sequence ID No. l: GEKKRRETVEREGG, the peptide according to the sequence ID No. 4: RETVEREKE, and a pharmaceutically acceptable carrier or filler.

The specific mixtures, comprising HP—V2 peptides and HEP—l, HPl—S, HP6—l4, which were used for preparation of the pharmaceutical compositions, are ed in the Example 6.

HP—VZ peptide or mixtures thereof with abovementioned peptides in the amount of 0.01 mg to 1000 mg is dissolved in the volume of 1 ml to 10 ml of sterile water. Dosage forms may be prepared on the basis of prepared solution and may be used orally, anally, or vaginally, or intranasally as drops, or as spray for inhalation.

HP—VZ peptide or combination of peptides in the amount of 0.01 mg to 1000 mg is placed in a tablet or a capsule, or suppositories, or a gel, or an ointment formulation in combination with appropriate s, carriers, preservatives, and stabilizers, which are y used by persons skilled in drug delivery technology.

HP—V2 peptide or combination of peptides in the abovementioned pharmaceutical compositions may be employed for the ation of dosage forms, which may be used orally, anally, or vaginally, or which may be locally applied. Sterile solution, containing 0.01 mg to 1000 mg of HP— V2 peptide or combination of peptides, dissolved in the volume of 1 ml to 10 ml of water for injection or any physiologic saline, is administrated as injection by subcutaneous, intramuscular or intravenous route. e 2. Separation of HEP-1 and HP 1-5 peptides by HPLC.

High performance liquid tography (HPLC) was used for separation of the peptides based on the retention times thereof. Stock solutions of HEP-1 and HP 1-5 peptides were prepared by means of solubilization thereof in deionized water at a concentration of 1—2 mg/ml ed by filtration sterilization through Millipore filters with pore size 0.2 um. Composition, containing 80% of HEP- ] peptide and 20% of HP 1—5 peptide, was ed by mixing of the ponding stock ons in appropriate volumes.

HPLC was conducted on Luna C18 (2) column, 4.6 x 250 mm by size, filled with 5 um particles.

Mobile phase was prepared by program mixing of phases A and B, n A contained water, 5% acetonitrile (ACN), 0.1% TFA, and B contained 0.1% TFA. Programmed gradient 5 to 25% acetonitrile (ACN) was formed during 20 minutes. The sample volume was 20 u]; the flow rate was 1 ml/min. The peaks were recorded automatically by UV-absorption thereof at different retention times.

Fig. 1 illustrates HPLC peak of HEP—l peptide with its typical retention time between 9 and 1— minutes. Fig. 2 rates the clear separation of two peaks, HEP—l (RT: 9.822 min) and HPl—5 (RT: 3.666 min), by HPLC.

Example 3. Separation of HEP-1 and HP6-14 peptides by HPLC.

Stock solutions of HEP—l and HP6—l4 peptides and mixtures thereof were prepared as described in the Example 2. Analysis of the peptides and mixtures thereof by HPLC was conducted on Luna C18 (2) column, 4.6 x 250 mm by size, filled with 5 pm particles as described in the Example 1.

In HPLC conditions used, the mixture of HEP—l and HP6—l4 peptides was clearly separated into 2 peaks (Fig. 3), one of which had the retention time, which was l for HP6—l4 peptide (RT29.495 min), and the other one had the ion time, which was typical for HEP—1 peptide (RT: 9.894 min).

Example 4. Analysis of the e of HEP-l and HP-V2 es by HPLC.

Stock solutions of HEP—l and HP—V2 peptides, as well as mixtures thereof were prepared as described in the Example 2. HPLC analysis of the peptides and the mixtures was conducted on Luna C18 (2) column, 4.6 x 250 mm by size, filled with 5 pm particles as described in the Example In HPLC conditions used, the mixture of HEP-l and HP-V2 peptides was eluted from the column as one peak (Fig. 4) with the retention time, which was typical for HEP-l peptide. HPLC-analysis of HP—V2 peptide solution alone has shown (Fig. 5) that the retention time of this peptide, indeed, was the same as the retention time of HEP-1 peptide. It means that it is impossible to distinguish HP—V2 and HEP—l peptides in this HPLC conditions.

Example 5. The mass spectrometry of the mixture of HEP-l and HP-V2 peptides.

Stock solutions of HEP—l and HP—V2 peptides, as well as mixtures thereof, were prepared as described in the Example 2. MALDI TOF spectra were reordered on the Bruker Ultra?ex F mass spectrometer using 2,5-dihydroxybenzoic acid as a matrix.

The mass spectrum of the mixture of HEP-1 and HP-V2 peptides (Figure 6) clearly reveals both peptides with their molecular ions 1818.0726 (MW) for HEP-1 and 1630.9385 (MW) for HP-V2.

Introduction to Examples 6 — 8.

The following Examples 6—8 trate ical activity of all the peptides used and mixtures thereof. These peptides induce interferon production and t different types of human cells from death caused by infection of cytopathic encephalomyocarditis virus at a dose of 100 TCIDSO/ml in the cell cultures in vim).

Peptide ) (sequence ID No. 1: GEKKRRETVEREGG), comprising 14 amino acid es, and compositions thereof with HEP—l peptide (sequence ID N o. 2: TEKKRRETVEREKE), or with HPl—S peptide (sequence ID No. 3: TEKKR), or with HP6—l4 peptide (sequence ID No. 4: RETVEREKE) were studied for their ability to induce antiviral response with production of the l type interferon in the cultures of different cells. We used the conventional methodology of antiviral (interferon—inducing) activity testing of the compounds in the culture in virm, which is widely used for screening of immunostimulatory, antiviral drugs, and interferon inducers.

In this methodology in vitm, we pretreated different types of cells with the studied es, and then the cells were infected with a dose of 100 LDSO 0f encephalomyocarditis virus. 24 hours after the infection the cytopathic effect of the virus was assessed in order to evaluate protective ty of the tested compound if the latter has the ability to bring the cells to a state. resistant to the virus, which is deadly for cells.

As it is well known, in the vast majority of cases such protective activity of the compounds is ated upon erons induction (the term «interferon» means the compound, which is produced by cell and which prevents replication of the Virus). It was possible to evaluate antiviral (interferon-inducing) activity of these compounds by using of different concentrations of said peptides and compositions thereof in virro.

The lower is the concentration which protects 50% of cells from death due to the infection at a dose of 100 LD50 of encephalomyocarditis Virus, the higher is antiviral (interferon—inducing) activity of the tested compound.

Peptide ) (sequence ID No. l: GEKKRRETVEREGG), comprising 1 amino acid residues. and combinations thereof with HEP—l peptide (sequence ID N0. 2: TEKKRRETVEREKE), or with HPl-S peptide (sequence ID No. 3: TEKKR), or with HP6-l4 peptide (sequence ID No. 4: RETVEREKE) were tested with respect to the immunostimulatory (antiviral, interferon-inducing) activity.

Example 6. Study of antiviral (interferon-inducing) activity of the peptides in the culture of human hepatoma cell line.

PLC/PRF/S (Alexander) human hepatoma cell line was obtained from Research Institute of gy named after Ivanovskiy (Moscow). Complete medium for cells ing was ed on the basis of MEM Eagle medium mented with 10% fetal calf serum (FCS), L—glutamine (300 ug/ml), and penicillin (100 U/ml).

The ing peptides were tested: HEP—l (sequence ID No. 2: TEKKRRETVEREKE); HPl—S (sequence ID No. 3: TEKKR); HP6—l4 (sequence ID No. 4: RETVEREKE); HP—VZ (sequence ID No. 1: GEKKRRETVEREGG); Mixtures of peptides: MXHP l —5+IIEP—l — the mixture of 90% HEP—l peptide (sequence ID N0. 2: TEKKRRETVEREKE) and 10% of HPl—S e (sequence ID N0. 3: TEKKR): MXHP6—l HEP—1 — the mixture of 90% HEP—l peptide (sequence ID N0. 2: TEKKRRETVEREKE) and 10% of HP6—l4 peptide (sequence ID No. 4: RETVEREKE); MXHP—V2+HP1—5 — the mixture of 90% HP—V2 peptide (sequence ID No. l: GEKKRRETVEREGG) and 10% of HPl—S peptide (sequence ID No. 3: TEKKR); MXHP—V2+HP6—l4 — the mixture of 90% HP—VZ peptide (sequence ID No. l: GEKKRRETVEREGG) and 10% of HP6—14 peptide (sequence ID No. 4: RETVEREKE).

MXHP-V2+HEP-l — the mixture of 95% HP-V2 peptide (sequence ID No. l: GEKKRRETVEREGG) and 5% of HEP-l e (sequence ID No. 2: ETVEREKE).

The peptides were dissolved in distilled water. and then sterilized by passing through s with pore size 0.2 pm to obtain the stock solutions of 1—2 mg/ml. On day 0, cells were seeded in the wells of 96—well plate in complete culture medium with cell y of 200 thousands of cells in 1 ml. On day 1. serial dilutions of each tested sample were prepared (24 serial dilutions in increments of 2) in triplets in the wells of the 96-well plate. On day 3. all the cultures were infected with a dose of 100 TCIDsO/ml of encephalomyocarditis Virus strain "Columbia SK—Col—SK". Finally, on day 4, cytopathic effect of the virus was assessed by using Leitz inverted microscope in the presence of different concentrations of the tested peptide or in the cultures without peptide ol).

Antiviral effect of the drug product was assessed based on minimal concentration thereof protecting 50% of the cells from death caused by encephalomyocarditis virus at a dose of 100 TCIDso/ml. Interferon titre (U/ml) was ated as a value, e to the maximal dilution of the drug product, which protected 50% of the cells from death caused by encephalomyocarditis Virus at a dose of 100 TCID50/ml.

The data obtained are presented in the Table 1. It is evident that all the tested peptides and combinations thereof prevent the replication of encephalomyocarditis virus in human hepatoma cells. The peptides have protected hepatoma cells from cytopathic effect of the Virus by inducing interferon production. Efficacy of the es and compositions thereof was ent. The highest level of ral (interferon—inducing) ty was registered with HP—VZ peptide and its composition with HPl —5 peptide.

PRF/ S Table l. Antiviral (interferon—inducing) activity of the peptides in the culture of PLC/ (Alexander) human hepatoma cell line.

The maximal Titre of induced Antiviral efficacy Compound concentration tested interferon (pg/ml) (U/ml) :1: HEP— 1 100 3 .0 320 HPl—S 100 1.6 6 HP6—14 100 0.78 1280 MXHP1—5+ HEP-1 200 62.5 16 MXHP6—1 HEP—1 200 62.5 16 MXHP-V2+HP1-5 100 0.1 10240 MXHP—V2+HP6— 1 4 100 3 .0 320 MXHP-V2+HEP1 100 0.78 1280 HP-V2 100 0.78 1280 Notes: '1' The antiviral efficacy is shown as the minimal compound concentration, protecting 50% of the cells from death as a result of infecting of 100 LD50 of encephalomyocarditis virus :The titre of induced interferon was calculated as a value. e to the maximal dilution of the compound, which protected 50% of the cells from death as a result of ing of 100 LDSO of encephalomyocarditis virus.

Example 7. Study of ral (interferon-inducing) activity of the peptides in the culture of human cervical carcinoma cell line.

HELA human cervical carcinoma cell line was obtained from Research Institute of Virology named after lvanovskiy (Moscow). Complete medium for cells culturing, es and compositions thereof, as well as assessment method for their antiviral (interferon—inducing) activity were the same as described in the Example 5.

The data ed are presented in the Table 2. It is evident that all the tested peptides and the itions prevent the pment of encephalomyocarditis virus infection in the human cervical carcinoma cells. The peptides have protected cervical carcinoma cells from cytopathic effect of the virus by inducing interferon production. The peptides and compositions thereof had different activity. The maximal antiviral (interferon—inducing) activity was detected when using HPl—5 peptide nce ID No. 3: TEKKR) and HP 6—1 peptide (sequence ID No. 4: RETVEREKE), as well as combinations thereof with HP—V2 peptide.

Table 2. Antiviral (interferon—inducing) activity of the peptides in the culture of HELA human cervical carcinoma cell line.

Titre of The maximal Antiviral efficacy d nd concentration tested (pg/ml) ’1‘ interferon (rig/ml) (U/ml) :1: HEP—1 1000 3.9 256 4 9 48 HP1—5 1000 0. 20 HP6-14 1000 0.49 2048 MXHPl -5+ HEP-1 2000 7.8 256 4+ 4 1 HEP—1 2000 1.95 102 MXHP—V2+HP1—5 1000 0.97 102 4 4 9 48 MXHP—V2+HP6— 1 1000 0. 20 HP—V2 1000 7.8 128 Notes: t The ral efficacy is shown as the minimal compound concentration, protecting 50% of the cells from death as a result of infecting of 100 LD50 of encephalomyocarditis virus.

:{:The titre of induced interferon was calculated as a value, inverse to the l dilution of the compound, which protected 50% of the cells from death as a result of infecting of 100 LD50 of encephalomyocarditis virus.

Example 8. Study of antiviral (interferon-inducing) activity of the peptides in the culture of Girardi Heart human epithelial cell line.

The culture of Girardi Heart human epithelial cell line was obtained from Research Institute of gy named after Ivanovskiy (Moscow). Complete medium for cells culturing, peptides and compositions thereof, as well as ment method for their antiviral (interferon—inducing) activity were the same as described in the Example 5.

The results obtained are presented in the Table 3. It is evident from the ted data that all the tested peptides and compositions prevent the development of encephalomyocarditis virus infection in the cells of Girardi Heart human epithelial cell line. The tested peptides and compositions f were highly effective in the ng of interferons production and in the protection of Girardi Heart lial cell line from cytopathic effect of the Virus. The maximal antiviral (interferon—inducing) activity was detected when using of HEP—l peptide (sequence ID No. 2: TEKKRRETVEREKE) with HPl—5 peptide (sequence ID No. 3: TEKKR) and HP6—l4 peptide (sequence ID No. 4: RETVEREKE) combinations, as well as the combination of HP—V2 peptide (sequence ID No. l: GEKKRRETVEREGG) with HPl—5 peptide (sequence ID No. 3: TEKKR) and with HEPl peptide (sequence ID No. 2: TEKKRRETVEREKE).

Table 3 Antiviral feron—inducing) activity of the peptides in the culture of Girardi Heart epithelial cell line The maximal Titre of induced Antiviral efficacy Compound concentration tested interferon (pg/m1) T (pg/ml) (U/ml) i HEP-1 1000 0.0015 655 360 HP1—5 1000 0.0015 655 360 HP6-14 1000 0.006 163 840 MXHP1-5+ HEP-1 2000 0.0005 4 194 304 4+ 4 l HEP—l 2000 0.0005 4 19 304 MXHP—V2+HP1-5 1000 0.0002 5 242 880 MXHP—V2+HP6—l4 1000 0.006 163 8 MXHP—V2+HEP1 1000 0.0008 1 310 720 HP—VZ 1000 0.0015 655 360 Notes: f The antiviral efficacy is shown as the minimal nd tration, protecting 50% of the cells from death as a result of infecting of 100 LD50 of encephalomyocarditis virus :{:The titre of induced eron was calculated as a value, e to the maximal dilution of the compound, which protected 50% of the cells from death as a result of infecting of 100 LDSO of encephalomyocarditis virus As the provided examples with description of biological tests and the tables demonstrate, the proposed novel HP—V2 peptide possesses high immunostimulatory activity, which is the same (Table 3) or which is 4 times higher (Table 1) when comparing with the activity of known HEP—l peptide. Moreover, the pharmaceutical compositions with the highest immunostimulatory activity and with a wide range of action (Tables 1—3), which are in several times higher than those known for HEP—l peptide and compositions thereof, were obtained based on the claimed HP—VZ peptide and known Ezrin peptides. e 9: Study to show in?uence of peptide on molecular mechanism in tissue repair and cell proliferation The following study provides evidence that the e of the invention (HP—V2) is involved in the molecular mechanism of tissue repair and cell proliferation. It is known that compounds which influence the molecular mechanism of cell proliferation, for example regulation of the TGFB expression is associated with repair of the gut (Monteleone er ((1,, "Mongersen, an Oral SMAD7 Antisense Oligonucleotide, and Crohn’s Disease", New England Journal ochdicine, 372: l 10 l 1 13). In addition, Akita 91011., "Basic Fibroblast Growth Factor in Scarless Wound Healing", Adv.

Wound Care, 2013, 2(2):44-49 discusses the benefit and role of basic fibroblast growth factor (bFGF) in scarless wound healing in clinical application and basic mechanism. bFGF is a glycoprotein which is widely used in treating wounds and ulcers. bFGF is easily applicable to any type of wound and leads to a better outcome in color, texture, and firmness. Chen etc/1., "NGF Accelerates Cutaneous Wound Healing by Promoting the Migration of Dermal lasts via the PI3K/Akt-Racl -JNK and ERK Pathways". BioMed Research [aremaria/ml, Volume 2014, Article ID 547187) showed that NGF significantly accelerated the healing of skin onal wounds in rats and the fibroblast migration d by NGF may contribute to this healing process. This also showed that the activation of PI3K/Akt, Racl, JNK, and ERK were all involved in the regulation of NGF-induced last migration. Further, Raffetto cf (11., "Mitogen-activated protein kinase pathway regulates cell proliferation in venous ulcer fibroblasts", Vasc. Endomscular Surg., 2006, 59-66 showed that MAPK ERK pathway is important for cell proliferation in venous ulcer fibroblasts.

Hence, the peptides of the ion are useful in the prevention and treatment of lower gut mation and ulceration.

It is shown that peptide ETVEREGG (SEQ ID No: l) induces activation of fibroblasts which are the main cell type responsible for tissue ration, as well as healing of wounds and ulcers. This example trates direct activating influence of peptide GEKKRRETVEREGG (SEQ ID No: l) on mouse fibroblasts, revealing quick activation signature within the cells on the level of MAPK—ERK signaling pathway.

Peptide GEKKRRETVEREGG (SEQ ID No: 1) was obtained as described in Example 1, BALB/ mouse fibroblast were purchased from American Type Culture Collection and propagated in a complete medium consisting of high glucose DMEM mented with 10% fetal calf serum, 1 mM sodium pyruvate, 1:50 MEM non—essential amino acids, 2 mM amine, 10 ug/ml gentamicin and 50 uM aptoethanol. Cells were grown in 10 cm culture dishes at 5% CO2 and 370C. Cell passages were made at the 50—60% con?uency. For cell detachment, 0.05% n/EDTA was used with following centrifugation (470 x g, 15 min) in10—fold volume of washing medium ning 10% FCS to quench trypsin. Supernatant was discarded and the cell pellet was suspended in 10 m1 of complete culture medium, then cell numbers were counted using hemocytometer before cells were used for experiments.

Fibroblasts were cultured in 23—well culture plates, the examined peptide or cytokine TGF—Bl ive control) solution, or equivalent volume of culture medium (negative control) were added into respective triplicate cultures. Cells were incubated in presence of the peptide GEKKRRETVEREGG SEQ ID No: 1 (10 ug/ml) or TGF—Bl, or without any effector compound during 1 or Ghours at 5% CO2 and 370C. At the time points indicated, cell were harvested, washed twice with PBS and lyzed using the cell extraction buffer in the presence of protease inhibitors during 30 min at 4 Extracts were cleared by centrifugation (14,000 x g, 10 min, 40C). Protein concentration was measured using protein assay reagent (Pierce, 23225). Appropriate dilutions of extracts (10 ug n per a track) were then fractionated in 8% SDS-PAGE and then erred onto PVDF—membrane (Amersham) for immune blotting. Targeted proteins were detected using antibodies specific to phospho—p44/42 MAPK (Cell Signaling, 4370) and GAPDH (Abcam ab8245). Protein bands were visualized and then ed their intensities using Image] soft-ware.

Data were expressed as a value (pixels per a band) of ERKl, ERK2 or phospho-ERK] and phospho-ERK2 normalized to a value of respective GAPDH-bands. Mean values + SD were calculated using the data of 3 independent runs.

Results of our ments show that both the peptide GEKKRRETVEREGG (SEQ ID No: l) and the positive l TGF—Bl induce a rapid activation of MAPK/ERK signaling pathway in fibroblasts. In 1 hour after inoculation of these effector compounds a concentration of orylated ERKl (44kD) and ERK2 (42 kD) was increased approximately 3—5—fold. Thus, 1 hour. after activation of fibroblasts with the GEKKRRETVEREGG (SEQ ID No: 1) peptide (10 ug/ml) or TGF—Bl (5 ng/ml), pospho—ERKl normalized values were 0.4+0.02 (p< 0.01) and 02 (p< 0.01), respectively, as compared to 0.1+0.002 in the negative control, and phospho— ERK2 values were 1.3+0.05 (p< 0.01) and 1.9+0.1 (p< 0.01) as compared to 0,4+0.01 in the negative control. Later, 6 hours after activation, the values of pospho—ERK] in fibroblasts ted with the peptide ETVEREGG (SEQ ID No: 1) or TGF—Bl were 0.1+0.006 (p> 0.1) and 0.3 +0.02 (p< 0.05), respectively, while 0.1+0.01 in the negative control cultures. Pospho—ERKZ values at the 6 hour time point were 0.1 + 0.01 (p> 0.1), 0.35+0.02 (p< 0.01) and 0.1+0.007 in the peptide, TGF—Bl and non—activated cultures, respectively.

The above study has shown that the activity of HP—VZ les the activity of TGF [5 (the positive control), and thus the potential for use in the tion and treatment of lower gut in?ammation and ulceration.

Claims (14)

1. A peptide comprising an amino acid ce of general formula (I) X1EKKRRETVEREX2X3 (SEQ ID. No:5) 5 wherein each of X1, X2 and X3 represent a non-polar amino acid residue and wherein the amino acid sequence is 14 to 25 residues in length.

2. The peptide as claimed in claim 1, wherein the non-polar amino acid is independently selected from the group consisting of glycine, alanine, valine, leucine, methionine, 10 isoleucine, proline, alanine, tryptophan , and/or combinations thereof.

3. The peptide as claimed in claim 1 or claim 2, wherein X1X2 and/or X3 is glycine.

4. The peptide as d in claim 3, wherein the amino acid ce is 15 GEKKRRETVEREGG (SEQ ID. No: 1).

5. A ceutical ition, comprising an effective amount of the peptide as claimed in any one of claims 1 to 4, and a pharmaceutically able carrier or filler. 20

6. The ceutical composition as claimed in claim 5, further comprising an effective amount of the peptide according to SEQ ID. No: 2 (TEKKRRETVEREKE).

7. The pharmaceutical ition as claimed in claim 5, further comprising an effective amount of the peptide according to the SEQ ID No.3 (TEKKR).

8. The pharmaceutical composition as claimed in claim 5, further comprising an effective amount of the peptide according to the SEQ ID No. 4 (RETVEREKE).

9. Use of the peptide as claimed in any one of claims 1 to 4, or the pharmaceutical composition 30 of any one of claims 5 to 8, in the manufacture of a medicament for stimulating an immune response in a subject.

10. Use of the peptide as claimed in any one of claims 1 to 4, or the pharmaceutical composition of any one of claims 5 to 8, in the manufacture of a medicament for the treatment or 35 prevention of a viral infection.

11. Use of the peptide as claimed in any one of claims 1 to 4, or the pharmaceutical composition of any one of claims 5 to 8, in the manufacture of a medicament for the treatment or prevention of tion of the mucous nes of the gut. 5

12. The use of the peptide or pharmaceutical composition according to claim 11, wherein the ulceration of the mucous membrane of the gut is a stomach ulcer, large intestine ulcer, duodenal ulcer, or an ulcer of the small intestine.

13. Use of the peptide as claimed in any one of claims 1 to 4, or the ceutical composition 10 of any one of claims 5 to 8, in the manufacture of a medicament for the ent or prevention of an inflammatory bowel disease or a disease or disorder ated with inflammatory bowel disease.

14. The use of the peptide or pharmaceutical composition according to claim 13, wherein the 15 disease or disorder associated with inflammatory bowel disease is selected from the group consisting of irritable bowel syndrome (IBS), ulcerative colitis and Crohn’s disease. 3726577_1.txt CE LISTING <110> Nearmedic Internationa1 Limited <120> Ezrin—Derived Peptides and Pharmaceutica] Compositions Thereof <130> P103771 <150> RU2015120667 5 1> 5—06—01 <1 201 <1 6 70> n <1 P atentI version 3.5 <21 1> 1 <21 2> 1 4 <21 P R <213> Synthetic e — HP—V2 peptide <400> 1 u u r u u G1y G1 Lys Lys Arg Arg GW Th Va1 GW Arg G1 61y G1y 1 5 1 0 <21 1> 2 <21 2> 1 4 <21 3> P R <21 Fragment of human ezrin protein — HEP—1 <400> 2 r u u r u u u Th G1 Lys Lys Arg Arg GW Th Va1 GW Arg G1 Lys G1 1 5 1 0 <21 1> 3 <21 2> 5 <21 3> P R 1 ite —S <21 Sate1 peptide of human ezrin protein — HPl <400> 3 r u Th G1 Lys Lys Arg 1 S <21 1 > 4 <21 2> 9 <21 3> P R 1 ite 14 <21 Sate1 peptide of human ezrin protein — HP6— <400> 4 Arg G1u Th Va1 GWu Arg GWu Lys GWu 1 5 <210> 5 <211> 14 2> <21 PRT <213> synthetic fragment of human ezrin protein — 1 formu1a I <220> <221> Variant <222> (1)..(1) Page 1 3726577_1.txt <223> /rep1ace="Giy" /rep1ace="A1a" /rep1ace="va1" /rep1ace="Leu" /rep1ace="Met" ce="I1e" /rep1ace="Pro" /rep1ace="Phe" /rep1ace="Trp" <220>1> <22 Variant <222> (13)..(13) <223> /rep1ace="Giy" /rep1ace="A1a" /rep1ace="va1" /rep1ace="Leu" /rep1ace="Met" /rep1ace="I1e" /rep1ace="Pro" /rep1ace="Phe" /rep1ace="Trp" <220>1> <22 Variant <222> (14)..(14) <223> /rep1ace="Giy" /rep1ace="A1a" /rep1ace="Va1" /rep1ace="Leu" ce="Met" /rep1ace="I1e" /rep1ace="Pro" /rep1ace="Phe" /rep1ace="Trp" <400> 5 u u r u u aa X aa Gi Lys Lys Arg Arg Gi Th Vai Gi Arg Gi X aa X l 5 1 0 <21 1 > 6 <21 2> l l <21 3> P R 12 <21 nt of human ezrin protein — HEP—l residues 2— <400> 6 u u r u u Gi Lys Lys Arg Arg Gi Th Vai Gi Arg Gi l 5 1 0 Page 2 mV 1 4 00‘ 1 00- U V e 0 | 234 5| | | | 71M i _‘| Hi I“ WKIHH¥IH 1 01 7 910111213140“n etention P eak R Area, Area, P eak o. ime ( sec) * seC entification N T mV % Id 4 - 1 1 569. 4 966.50 1 00.00 ‘ H EP 1 900 4 966.50 1 00.00 1 FI 1 600 400 . . O 1 2 3 4 5 6 7 8 9 10 11 12 13 14min Peak Retention Height Area, Conc., Conc., Peak No. Time (min) mV mV*sec mg/ml % fication 1 3.666 344.77 1269.78 0.4545 18.98 H P 1-5 2 9.822 637.76 5112.29 1.94 81.02 H EP 2 15 982.52 6382.07 2.395 100.00