(12) United States Patent (10) Patent No.: US 9.220,672 B2 Lang (45) Date of Patent: Dec

US009220672B2

(12) United States Patent (10) Patent No.: US 9.220,672 B2 Lang (45) Date of Patent: Dec. 29, 2015

(54) PEPTIDE FOR USE IN THE TREATMENT OF OTHER PUBLICATIONS SKIN CONDITIONS Definition of derivative, from http://cancerweb.ncl.ac.uk/cgi-bin/ omd?query-derivative, pp. 1-5, accessed Jul. 7, 2005.* (75) Inventor: Christine Lang, Berlin (DE) Definition of moiety, from http://dictionary reference.com/browse/ moiety, pp. 1-3. Accessed Aug. 26, 2010.* (73) Assignee: ORGANOBALANCE GMBH, Berlin Water, from http://www.biology-online.org/dictionary Water, pp. (DE) 1-3, accessed Apr. 24, 2014.* Rudinger, Peptide Hormones, JA Parsons, Ed., 1976, pp. 1-7.* (*) Notice: Subject to any disclaimer, the term of this SIGMA, 2004, pp. 1-2.* patent is extended or adjusted under 35 Berendsen, A Glimpae of the Holy Grail?, Science, 1998, 282, pp. U.S.C. 154(b) by 0 days. 642-643. Voet et al. Biochemistry, John Wiley & Sons Inc., 1995, pp. 235 (21) Appl. No.: 13/984,120 241.* Ngo et al. Computational Complexity, Protein Structure Protection, (22) PCT Filed: Feb. 3, 2012 and the Levinthal Paradox, 1994, pp. 491-494.* Bradley et al., Limits of Cooperativity in a Structurally Modular (86). PCT No.: PCT/EP2012/OOO697 Protein: Response of the Notch Ankyrin Domain to Analogous Alanine Substitutions in Each Repeat, J. Mol. BIoL (2002) 324. S371 (c)(1), 373-386. (2), (4) Date: Oct. 15, 2013 Vippagunta et al. Crystalline Solids, Advanced Drug Delivery Reviews, 2001, 48, pp. 3-26.* (87) PCT Pub. No.: WO2012/107244 Magnet etal, Specificity of L.D-Transpeptidases from Gram-positive Bacteria Producing Different Peptidoglycan Chemotypes, The Jour PCT Pub. Date: Aug. 16, 2012 nal of Biological Chemistry, 2007, 282, pp. 13151-13159.* (65) Prior Publication Data Types of Inflammation, from http://quizlet.com/580 1283/types-of inflammation-flash-cards/alphabetical, pp. 1-2, accessed Nov. 5, US 2014/OO5785O A1 Feb. 27, 2014 2014. Dermatitis-Symptoms diagnosis and treatment, from http://www. (30) Foreign Application Priority Data webmdboots.com/skin-problems-and-treatments/guide? dermatitis?print=true, pp. 1-3, accessed Nov. 5, 2014.* Feb. 9, 2011 (EP) ...... 1 1153771 What is inflammation and What causes inflammation, from http:// www.medicalnewstoday.com/articles/248423.php, pp. 1-14, (51) Int. Cl. accessed Nov. 5, 2014.* A6 IK38/07 (2006.01) Bobrova et al., “Synthesis and biological activity of branched C07K 5/037 (2006.01) enkephal in analogues'. European Journal of Medicine Cheistry, A61O 19/00 (2006.01) Editions Scientifique Elsevier, Paris, FR, vol.33, No. 4, Apr. 1, 1998, A6 IK 8/64 (2006.01) pp. 255-266. Rollan et al., International Journal of Food Microbiology 70 2001. C07K 5/02 (2006.01) 303-307. C07K 5/09 (2006.01) Matsuduchi et aI.Clinical and Diagnostic Laboratory Immunology, A61 K38/00 (2006.01) Mar. 2003, p. 259-266 vol. 10, No. 2. A61O 5/02 (2006.01) Stentz et al., Applied and Environmental Microbiology, Oct. 2000, p. A61 O 19/10 (2006.01) 4272-4278 vol. 66, No. 10. (52) U.S. Cl. Varmanen et al., Journal of Bacteriology, Jan. 2000, p. 146-154 vol. CPC ...... A61K 8/64 (2013.01); A61O 19/007 182, No. 1 in J. Bacteriology 182 (2000), 146-154. (2013.01); C07K 5/0215 (2013.01); C07K 5/0815 (2013.01); A61 K 38/00 (2013.01); A61 O * cited by examiner 5/02 (2013.01); A61 O 19/10 (2013.01) (58) Field of Classification Search CPC ...... A61K 8/64; A61K 38/00; CO7K 5/0215; Primary Examiner — Julie Ha C07K5/0815; A61O5/02; A61O 19/007; Assistant Examiner — Li Ni Komatsu A61Q 19/10 (74) Attorney, Agent, or Firm — Ann Wieczorek; Mayer & See application file for complete search history. Williams PC (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS The present invention relates to a peptide of the following 7,056,942 B2 * 6/2006 Hildesheim et al...... 514,411 formula (I) X (X)-X-X or a salt or solvate thereof, wherein is an amino acid comprising two or more NH-functionalities, FOREIGN PATENT DOCUMENTS and X, X, and X are, independently from one another, an EP 1997826 12/2008 amino acid residue, and wherein the Salt or Solvate is prefer GB 1523812 9, 1978 ably a physiologically acceptable Salt or Solvate. WO 01.09173 2, 2001 WO 2005/048968 A1 6, 2005 WO 2006,136420 A2 12/2006 33 Claims, 3 Drawing Sheets U.S. Patent Dec. 29, 2015 Sheet 1 of 3 US 9.220,672 B2

Fig. 1 a

Fig. 1b

Fig. 1c U.S. Patent Dec. 29, 2015 Sheet 2 of 3 US 9.220,672 B2

is: MXumssm ------S-Xaaraas)&a-X-Xaa-a-tax-r--a------&-au-3-a-a-a-ram--aura-Sax-a-a-ra i .C. S. epidermidis + 1 mg/ml peptide, pH 6.5 - C - - B. s. epidermidis + 0.2 mg/ml --S-a--- peptide, pH 6.5

1. incubation time h

Fig. 2b -A. S. epidermidis (control) B. S. epidermidis + 1 mg/ml amino acid mix, pH 6.5

incubation time (h) U.S. Patent Dec. 29, 2015 Sheet 3 of 3 US 9.220,672 B2

-A. S. aureus (control) 1,50 .. -B. S. aureus + 1 mg/ml peptide, pH 6.5 1.25 - 100

0.75 -r 0.50 0.25

0.00 it

Incubation time h

Fig. 4

2. 3. A. 5. No treatment (LB stimulans (LPS LB stimulans (LB stimulans+LPS -- -- - w No treatment No treatment No treatment LPS (LB stimulans 24h pre-treatment as 24h treatment US 9.220,672 B2 1. 2 PEPTIDE FOR USE IN THE TREATMENT OF transient pathogenic micro flora, as well as to the use of Such SKIN CONDITIONS microorganisms in cosmetic and pharmaceutical composi tions, in particular for the treatment of dermatitis. CROSS REFERENCE TO RELATED There however remains the need for a compound or com APPLICATIONS position which does not depend on cell cultures requiring special care and handling, and which are thus limited with This application is a 371 of International Application No. respect to their application. In particular, the need exists for a PCT/EP12/00697, filed Feb. 3, 2012 which claims benefit and compound or composition which may be easily produced in a priority to European Application No. 11153771.8, filed Feb. cost-effective manner. Furthermore there is a need for a com 9, 2011 herein incorporated by reference in their entirety. 10 pound or composition which displays a low effective dose with respect to its beneficial effects, not only in view of TECHNICAL FIELD providing more cost-effective cosmetic formulation, but also for opening new perspectives with respect to new applications The present invention relates to a peptide and to composi in the field of both cosmetics and pharmaceuticals. tions comprising a peptide, and to their respective non-thera 15 peutic use in skin care. Furthermore the present invention SUMMARY OF THE INVENTION relates to an anti-inflammatory drug, in particular for use in the treatment and prophylaxis of inflammation of epithelial The present invention concerns a peptide of the following tissues. formula (I) BACKGROUND OF THE INVENTION or a salt or Solvate thereof, wherein X is an amino acid The human skin is populated by a large variety of micro comprising two or more NH-functionalities, and X, X, and organisms that mainly live as commensals in a relatively X are, independently from one another, an amino acid resi stable composition on the surface of the skin. This normal 25 due, and wherein the salt or Solvate is preferably a physiologi skin flora is termed “resident skin flora’ and supports the cally acceptable salt or Solvate. healthy appearance of the skin, which is shiny and Sufficiently Furthermore, the present invention relates to a peptide moist. In addition to creating a natural barrier for retaining according to the present invention as a medicament, and moisture within the epidermis, the normal skin flora protects preferably as an anti-inflammatory drug. Furthermore, the the skin against the intrusion of potentially pathogenic micro 30 present invention equally relates to a method for treating of a organisms. pro-inflammatory condition or disease in a Subject, said Among the microorganisms constituting the resident method comprising administering to a subject suffering from microbial flora of healthy skin, often more than ninety percent the aforementioned condition or disease a therapeutically thereofare Staphylococcus epidermidis (coagulase negative), effective dose of the peptide of the invention, wherein the Micrococcus spec. Diphteroids and propionibacteria. A typi 35 condition or disease being treated is preferably an inflamma cal imbalance of the skin microflora is for example observed tion of epithelial tissues, allergies, allergic reactions, rash, when Staphylococcus aureus populates the skin microflora to and/or rheumatoid arthritis, more preferably inflammation, an increasing extent, as a result of which the skin is increas allergies, and/or allergic reactions of the epidermis, more ingly prone to dryness. In this condition, the epidermis dis preferably dermatitis, and even more preferably atopic der plays cracks and tends to show red areas as a result of inflam 40 matitis, seborrhoeic dermatitis, psoriasis, poison-ivy derma mation. The danger of colonization by pathogenic titis, eczema herpeticum, kerion, diaper rash, or scabies. In microorganisms increases drastically in the case of Small addition to this, the present invention also relates to a method lesions or other damages on the Surface of the skin, especially for prophylaxis of a pro-inflammatory condition or disease in when the normal skin flora is diminished as a result of fre a subject, said method comprising administering to an appar quent washing, make-up, UV-light, or by antibiotics. 45 ently healthy subject a prophylactically effective dose of the Thus, the microbial skin flora affects several factors of the peptide of the invention, wherein the method of prophylaxis skin that are of cosmetic relevance, namely the pH value of of a pro-inflammatory condition or disease in a subject pref the skin, the barrier function and the skin's lipid content. In erably involves the prophylaxis of inflammation of epithelial this respect, S. epidermidis assists the skin in fighting against tissues, allergies, allergic reactions, rash, and/or rheumatoid pathogenic microorganisms by lowering the pH to values at 50 arthritis, more preferably inflammation, allergies, and/or which pathogens are not able to effectively grow. The water allergic reactions of the epidermis, more preferably dermati barrier function and the lipid content of the skin, on the other tis, and even more preferably atopic dermatitis, Seborrhoeic hand, depend on the ceramide content of the horny layers. dermatitis, psoriasis, poison-ivy dermatitis, eczema herpeti Lowering of the ceramide content leads to a dried out and cum, kerion, diaper rash, or scabies. cracked skin condition. In this respect, a study conducted on 55 In addition to these, the present invention concerns both a atopical dermatitis patients having Such a skin condition cosmetic composition and a pharmaceutical composition, revealed that Staphylococcus aureus may dominate the said compositions respectively comprising the peptide microbial skin flora under these circumstances. In particular, according to the present invention. it was found that as opposed to normal commensals of the The present invention also concerns the use of the inventive skin, this pathogen displays a very high ceramidase activity. 60 peptide or of a composition of the invention for non-thera Under these conditions, the loss of humidity and lipids leads peutic skin care, preferably for the treatment and/or prophy to the generation of inflammation of the epidermis. laxis of dry skin condition. In addition to this, the present WO 2006/136420 A2 relates to microorganisms, and in invention equally relates to a method of using the inventive particular to microorganisms belonging to the genus Lacto peptide or of a composition of the invention for non-thera bacillus, which are able to stimulate the growth of one or 65 peutic skin care, preferably for the treatment and/or prophy more microorganisms of the resident skin microbial flora and laxis of dry skin condition, preferably dry facial skin and/or which does not stimulate the growth of microorganisms of the dry scalp, and even more preferably dry facial skin, said US 9.220,672 B2 3 4 method comprising administering to a subject a cosmetically mutants or derivatives thereof, and combinations of two or effective dose of the peptide of the invention. more thereof, wherein more preferably the Lactobacillus spe Furthermore, the present invention preferably relates to the cies is L. brevis, more preferably selected from the group use of the inventive peptide or compositions for stimulating consisting of L. brevis having DSMZaccession number DSM the growth of healthy normal resident skin microflora, pref 17247, DSM 17250, and mutants or derivatives thereof, and erably for stimulating the growth of healthy normal resident wherein even more preferably the Lactobacillus species is L. skin microflora belonging to the Staphylococcus genus. In brevis having DSMZ accession number DSM 17250 and/or addition to this the present invention equally relates to a mutants or derivatives thereof. method of using the inventive peptide or compositions for stimulating the growth of healthy normal resident skin micro 10 DETAILED DESCRIPTION flora, preferably for stimulating the growth of healthy normal resident skin microflora belonging to the Staphylococcus Thus, it has been Surprisingly been found that a simple genus, more preferably for stimulating the growth of S. epi compound which is easily accessible may be provided for use dermidis, and even more preferably for stimulating the in both cosmetic and pharmaceutical applications, in particu growth of S. epidermidis without stimulating the growth of 15 lar with respect to cosmetic skin care as well a with regard to pathogenic microorganisms, in particular of S. aureus, said the treatment and/or prophylaxis of both non-pathological method comprising administering an effective dose of the and pathological skin conditions. Furthermore, it has quite peptide of the invention to the subject. unexpectedly been found that a simple compound may be Additionally, the present invention further preferably provided for use as a medicament, in particular as an anti relates to the use of the inventive peptide or compositions for inflammatory drug. inhibiting the growth of transient pathogenic skin microflora, Therefore, in a first aspect, the present invention concerns preferably for inhibiting the growth of transient pathogenic a peptide of the following formula (I) skin microflora belonging to the Staphylococcus genus. In addition to this the present invention equally relates to a (I) method of using the inventive peptide or compositions for 25 or a salt or solvate thereof, wherein inhibiting the growth of transient pathogenic skin microflora, X is an amino acid residue comprising two or more NH preferably for inhibiting the growth of transient pathogenic functionalities, and skin microflora belonging to the Staphylococcus genus, more X, X, and X are, independently from one another, an amino preferably for inhibiting the growth of S. aureus, and even acid residue, and more preferably for inhibiting the growth of S. aureus without 30 wherein the salt or solvate is preferably a physiologically inhibiting the growth of beneficial microorganisms, in par acceptable salt or Solvate. ticular of S. epidermidis, said method comprising administer Throughout this specification and the claims which follow, ing an effective dose of the peptide of the invention to the unless the context requires otherwise, the word “comprise’, subject, wherein said effective dose may be any one of a and variations such as "comprises' and "comprising, will be cosmetically effective dose, a prophylactically effective dose, 35 understood to imply the inclusion of a stated integer or step or or a therapeutically effective dose. constituent, or group of integers or steps or constituents, but Also, the present invention concerns the use of a microor not the exclusion of any other integer or step or constituent, or ganism for the production of a peptide of the present inven group of integers or steps or constituents. tion, wherein the microorganism is preferably a prokaryote It must be noted that as used herein and in the appended microorganism, more preferably one or more strains of bac 40 claims, the singular forms “a”, “an', and “the’, include plural teria, more preferably one or more lactic acid bacteria, and referents unless the context clearly indicates otherwise. Thus, more preferably one or more bacteria of the genus Lactoba for example, reference to “a reagent' includes one or more of cillus. In addition to this the present invention equally relates such different reagents, and reference to “the method” to a method of using a microorganism for the production of a includes reference to equivalent steps and methods known to peptide of the present invention, in particular by isolating the 45 those of ordinary skill in the art that could be modified or peptide of the invention or a precursor thereoffrom the micro substituted for the methods described herein. organism, wherein the microorganism is preferably a According to the present invention, the peptide of formula prokaryote microorganism, more preferably one or more (I) comprises a tripeptide sequence X-X-X, wherein X strains of bacteria, more preferably one or more lactic acid constitutes the N-terminus, and X constitutes the C-termi bacteria, more preferably one or more bacteria of the genus 50 nus. Furthermore, the peptide contains an amino acid residue Lactobacillus, wherein more preferably the bacteria is X, which is covalently bound to the side-chain of the amino selected from the group consisting of the Lactobacillus spe acid residue X. In principle, there is no restriction according cies L. acidophilus, L. plantarum, L. reuteri, L. Casei, L. to the present invention regarding the covalent bond formed paracasei, L. brevis, L. fermentum, L. buchneri, L. delbrickii. between X and X, provided that X is not covalently bound preferably L. delbrickii ssp. delbrickii, and combinations of 55 to the N-terminus of X. Consequently, the peptide of formula two or more thereof, wherein more preferably the Lactoba I does not, for example, comprise tetrapeptides or a tetrapep cillus species is selected from the group consisting of L. tide motif of the sequence X-X-X-X, wherein X would brevis, preferably L. brevis having DSMZ accession number be covalently bound to the N-terminal group of the N-termi DSM 17247, DSM 17250, and mutants orderivatives thereof, S. L. paracasei, preferably L. paracasei having DSMZ acces 60 Within the meaning of the present invention, the terms sion number DSM 17248 and mutants or derivatives thereof, “amino acid and “amino acid residue” are not particularly L. fermentum, preferably L. fermentum having DSMZ acces restricted, and in general designate any conceivable com sion number DSM 17249 and mutants or derivatives thereof, pound containing one or more amino moieties and one or L. delbrickii ssp. delbrickii, preferably L. delbrickii ssp. more carboxyl moieties, such as to be able to form peptidic delbrickii having DSMZ accession number DSM 18006 and 65 bonds with one another. In particular, the terms “amino acid mutants or derivatives thereof, L. buchneri, preferably L. and "amino acid residue' as employed herein includes and buchneri having DSMZ accession number DSM 18007 and encompasses all of the naturally occurringamino acids, either US 9.220,672 B2 5 6 in the D-, L-, allo-, or other stereoisomeric configurations if dine (Aze), beta-Alanine (Bal), 4-Amino-3-hydroxybutanoic optically active, as well as any known or conceivable non acid (Bux), gamma-Amino-beta-hydroxycyclohexanepen native, synthetic, and modified amino acids. According to tanoic acid (Cap), 3-Cyclohexylalanine (Cha), 3-Sulfoala preferred embodiments of the present invention, the optically nine (Cya), S-Ethylthiocysteine (Edc), Pyroglutamic acid active amino acids and amino acid residues are preferably in 5 (Glp), Homocysteine (Hey), Homophenylalanine (Hph), the D- and/or L-configuration, including racemic mixtures, Homoserine (Hse), 4-Hydroxyproline (Hyp), 3-Hydroxypro wherein it is more preferred that the optically active amino line (3Hyp), alpha-Amino-2-indanacetic acid (Igl), Isovaline acid and amino acid residues are in the L-configuration. (Iva), 3-Hydroxy-4-methylproline (Mhp), 3-Naphthylala According to preferred embodiments of the present inven nine (Nal), Norleucine (Me), N-Benzylglycine (Nphe), tion wherein the amino acid in X is an alpha-amino acid, the 10 Nortyrosine (Nty), Norvaline (Nva), 2-Carboxyoctahydroin N-terminus is accordingly constituted by the alpha-amino dole (Oic), Penicillamine (Pen), 2-Phenylglycine (Phg), group thereof. According to alternatively preferred embodi 2-Carboxypiperidine (Pip), Phospho-L-serine (pSer), Phos ments of the present invention wherein the amino acid in X pho-L-threonine (pThr), Phospho-L-tyrosine (pTyr), Sar is not an alpha-amino acid, and wherein X contains two or cosine (Sar), 1-Amino-1-carboxycyclopentane (Spg), Statine more amino groups, there is no general restriction as to which 15 (4-amino-3-hydroxy-6-methylheptanoic acid) (Sta), 3-Thie of the two or more amino groups constitutes the N-terminus, nylalanine (Thi), 3-Carboxylsoquinoline (Tic), 3-Methylva and is therefore not covalently bound to X. According to line (Tle), epsilon-N-Trimethyllysine (Tml), 3-Thiazolylala specific embodiments of said alternatively preferred embodi nine (Tza), alpha-Amino-2,4-dioxopyrimidinepropanoic ments, wherein X further contains one or more side-chains, acid (Wil), and derivatives thereof. and the second and/or further amino group(s) is/are Within the meaning of the present invention, a "derivative' covalently bound to said one or more side-chains, it is pre of any amino acid or the "derivatization thereof refers to any ferred that the N-terminus is not an amino group which is conceivable chemical modification thereof, provided that the present on a side-chain. derivative is capable of forming the same peptidic bonds as Thus, it has quite Surprisingly been found that a peptide for the underivatized amino acid residue. According to pre according to formula (I) containing a sequence of three amino 25 ferred embodiments the term "derivative' refers to chemi acid residues, wherein a fourth amino acid is covalently cally modified amino acid residues having the same carbon bound to the amino acid of the N-terminus other than by the chain(s) as the underivatized amino acid, and wherein and the N-terminal group thereof, affords a peptide which may be amino and carbonyl functionalities present in the underiva advantageously employed in both cosmetic and pharmaceu tized amino acid may be found at the same positions of the tical applications. In particular, as will be shown in the illus 30 carbon chain(s) in the derivatized residues. trative inventive examples below, it has quite unexpectedly With respect to the types of chemical modifications which been found that such a peptide may advantageously be used may be present in the amino acid derivatives, these preferably for both cosmetic and pharmaceutical treatment of the skin. concern the replacement of hydrogen in C H and/or N H Furthermore, the inventive peptide Surprisingly shows an moieties and/or the replacement of terminal oXo, —OH, and/ anti-inflammatory activity, Such that it also proves to be 35 or —OR moieties, one or more of which are present in the advantageous for a variety of potential applications, in par underivatized amino acid residue. In particular, according to ticular as a medicament in the prophylaxis and/or treatment of preferred derivatives of the amino acid residues wherein one inflammatory conditions in addition to a variety of inflamma or more hydrogen atoms in C-H moieties of the underiva tion-related conditions and diseases. Thus, although a variety tized amino acid have been chemically modified, it is pre of Smaller peptide sequences are known to be of a certain 40 ferred that independently from one another the one or more benefit in predominantly cosmetic applications such as e.g. hydrogenatoms are replaced with halogen, OH, alkyl, alkoxy, disclosed in WO 2005/048968 A1, WO 00/43417 A1, U.S. NH, alkylamino, dialkylamino, thiol, thioether, formyl, acyl, Pat. No. 6,620,419 B1, and U.S. Pat. No. 6,492,326 B1, it has alkoxycarbonyl, or a derivative thereof, more preferably with quite unexpectedly been found that the specific binding pat F, Cl, Br, OH, (C-C)alkyl, (C-C)alkoxy, NH, (C-C) tern in a peptide according to the present invention Surpris 45 alkylamino, di(C-C)alkylamino, thiol, (C-C)thioether, ingly leads to an unprecedented activity of a highly advanta formyl, (C-C)acyl, (C-C)alkoxycarbonyl, or a derivative geous use and potential not only in non-therapeutic cosmetic thereof. Furthermore, according to preferred derivatives of applications but also as a medicament for the effective pro the amino acid residues wherein one or more hydrogenatoms phylaxis and/or treatment of numerous conditions and dis in N H moieties of the underivatized amino acid have been CaSCS. 50 chemically modified, it is preferred that independently from According to preferred embodiments of the present inven one another the one or more hydrogen atoms are replaced tion, the term "amino acid designates one or more amino with halogen, alkyl, formyl, acyl, alkoxycarbonyl, or a acids selected from the group consisting of Arg, His, LyS, derivative thereof, more preferably with F. (C-C)alkyl, 3,6-Diaminohexanoic acid (Bly), N5-Aminocarbonylorni formyl, (C-C)acyl, (C-C)alkoxycarbonyl, or a derivative thine (Cit), 2,4-Diaminobutanoic acid (Dab), 2,3-Diamino 55 thereof. Furthermore, according to preferred derivatives of propanoic acid (Dpr), 2,7-DiaminoSuberic acid (DSu), the amino acid residues wherein one or more —OH, and/or Homoarginine (Har), Homohistidine (Hhs), 5-Hydroxyl —OR moieties of the underivatized amino acid have been ysine (Hyl), Ornithine (Orn), Pyrrolysine (Pyl), Asn., Asp, chemically modified, it is preferred that independently from Gln, Glu, 2-Aminoadipic acid (Aad), C.-Asparagine (Aan), one another these have been modified to halogen, OH, alkyl, 2-Aminocapric acid (Aca), alpha-Glutamine (Agn), 2-Ami 60 alkoxy, NH, alkylamino, dialkylamino, thiol, thioether, nopimelic acid (Apn), gamma-Amino-beta-hydroxyben formyl, acyl, alkoxycarbonyl, or a derivative thereof, more Zenepentanoic acid (App), 2-AminoSuberic acid (ASu), beta preferably with F, Cl, Br, OH, (C-C)alkyl, (C-C)alkoxy, Aspartic acid (Bas), Diaminopimelic acid (Dpm), gamma NH. (C-C)alkylamino, di(C-C)alkylamino, thiol, (C- Glutamic acid (Ggu), gamma-Carboxyglutamic acid (Gla), Cl)thioether, formyl, (C-C)acyl, (C-C)alkoxycarbonyl, Ala, Cys, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, 65 or a derivative thereof. Furthermore, according to preferred Scy, 2-Aminobutanoic acid (Abu), 2-Aminoheptanoic acid derivatives of the amino acid residues wherein one or more (Ahe), alpha-Aminoisobutyric acid (Aib), 2-Carboxyazeti terminal oXo ligands of the underivatized amino acid have US 9.220,672 B2 7 8 been chemically modified, it is preferred that terminal oxo is (Glu)-, Lys(Glu)-, Bly (Glu)-, Cit(Glu)-, Dab(Glu)-, Dpr chemically modified to terminal thio, acetal, thioacetal, hemi (Glu)-, DSu(Glu)-, Har(Glu)-, Hhs(Glu)-, Hyl(Glu)-, Orn acetal, or a derivative thereof. (Glu)-, Pyl (Glu)-, Arg(Aad)-. His(Aad)-, Lys(Aad)- Bly According to the present invention, the peptide may be in (Aad)- Cit(Aad)-, Dab(Aad)-, Dpr(Aad)-, DSu(Aad)-, Har any conceivable form, such as for example in the form of a 5 (Aad)- Hhs(Aad)-, Hyl(Aad)-, Orn(Aad)-, Pyl(Aad)-, Arg neutral or Zwitterionic compound, or in the form of a salt or (Aan)-. His(Aan)-, LyS(Aan)-, Bly(Aan)- Cit(Aan)-, Dab Solvate, wherein there is no particular restriction as to the type (Aan)-, Dpr(Aan)-, DSu(Aan)-, Har(Aan)-. Hhs(Aan)-, Hyl of salt or solvate which the peptide may be present. Within a (Aan)-, Orn(Aan)-, Pyl(Aan)-, Arg(Aca)-. His(Aca)-. Lys preferred meaning of the present invention, the expression (Aca)-. Bly(Aca)-. Cit(Aca)-. Dab(Aca)-. Dpr(Aca)-, DSu 'salt' is understood as meaning both salts of carboxyl groups 10 (Aca)-. Har(Aca)-. Hhs(Aca)-. Hyl(Aca)-. Orn(Aca)-, Pyl and also acid addition salts of amino groups of the peptide (Aca)-. Arg(Agn)-. His(Agn)-, LyS(Agn)-, Bly(Agn)-, Cit according to the invention. Salts of carboxyl groups prefer (Agn)-, Dab(Agn)-, Dpr(Agn)-, DSu(Agn)-, Har(Agn)-. Hhs ably comprise inorganic salts, such as, for example, Sodium, (Agn)-, Hyl(Agn)-, Orn(Agn)-, Pyl(Agn)-, Arg(Apn)-. His calcium, ammonium, iron and Zinc salts, and/or salts with (Apn)-, Lys(Apn)-, Bly(Apn)-. Cit(Apn)-, Dab(Apn)-. organic bases, such as, for example, amines Such as trietha- 15 Dpr(Apn)-, DSu(Apn)-, Har(Apn)-, Hhs(Apn)-, Hyl nolamine, arginine, lysine, piperidine and the like. Further (Apn)-, Orn(Apn)-, Pyl(Apn)-, Arg(App)-. His(App)-, Lys more, the salts of the present invention include acid addition (App)-, Bly (App)-, Cit(App)-. Dab(App)-, Dpr(App)-, DSu salts, such as, for example, salts with mineral acids, such as (App)-, Har(App)-, Hhs(App)-, Hyl(App)-, Orn(App)-, Pyl hydrochloric acid or Sulfuric acid and salts with organic acids, (App)-, Arg(ASu)-. His(ASu)-, Lys(ASu)-, Bly(ASu)-. Cit Such as acetic acid and oxalic acid. The expression “solvate (Asu)-, Dab(Asu)-, Dpr(Asu)-, DSu(Asu)-, Har(Asu)-. Hhs on the other hand refers to any conceivable polar or non-polar (Asu)-, Hyl(Asu)-, Orn(Asu)-, Pyl(Asu)-, Arg(Bas)-. His solvent provided that it may form a solvate with the peptide of (Bas)-, Lys(Bas)-, Bly(Bas)-, Cit(Bas)-, Dab(Bas)-, Dpr the present invention, including salts thereof. According to (Bas)-, DSu(Bas)-, Har(Bas)-, Hhs(Bas)-, Hyl(Bas)-, Orn preferred embodiments, the solvate is selected from the group (Bas)-, Pyl(Bas)-, Arg(Cit)-. His(Cit)-, Lys(Cit)-, Bly (Cit)-. consisting of water, acetonitrile, dimethyl Sulfoxide, metha 25 Cit(Cit)-, Dab(Cit)-, Dpr(Cit)-, DSu(Cit)-, Har(Cit)-, Hhs nol, propylene carbonate, ammonia, ethanol, and acetone, (Cit)-, Hyl (Cit)-, Orn(Cit)-, Pyl (Cit)-, Arg(Dpm)-. His and mixtures of two or more thereof, more preferably from (Dpm)- Lys(Dpm)- Bly(Dpm)- Cit(Dpm)- Dab(Dpm)- the group consisting of water, dimethyl Sulfoxide, propylene Dpr(Dpm)- DSu(Dpm)- Har(Dpm)- Hhs(Dpm)- Hyl carbonate, ethanol, and mixtures of two or more thereof. (Dpm)-, Orn(Dpm)-, Pyl(Dpm)- Arg(DSu)-. His(DSu)-, Lys It is, however, particularly preferred that in embodiments 30 (Dsu)-, Bly (Dsu)-. Cit(DSu)-, Dab(Dsu)-, Dpr(DSu)-, DSu of the present invention in which the peptide is in the form of (Dsu)-, Har(Dsu)-, Hhs(DSu)-, Hyl(DSu)-, Orn(Dsu)-, Pyl a salt or solvate, said salt or solvate is physiologically accept (DSu)-, Arg(Ggu)-. His(Ggu)-, Lys(Ggu)-, Bly(Ggu)-, Cit able, in particular with respect to the systemic and/or topical (Ggu)-, Dab(Ggu)-, Dpr(Ggu)-, DSu(Ggu)-, Har(Ggu)-. Hhs application thereof, and even more preferably with respect to (Ggu)-, Hyl(Ggu)-, Orn(Ggu)-, Pyl(Ggu)-, Arg(Gla)-. His the topical application thereof. 35 (Gla)-. Lys(Gla)-. Bly (Gla)-. Cit(Gla)-. Dab(Gla)-. Dpr According to a preferred embodiment of the present inven (Gla)-, DSu(Gla)-. Har(Gla)-. Hhs(Gla)-. Hyl(Gla)-. Orn tion, X is an amino acid residue selected from the group (Gla)-, Pyl(Gla)-, and derivatives thereof. consisting of Arg, His, Lys, Bly, Cit, Dab, Dpr, DSu, Har, Hhs, More preferably, the fragment X (X)- of formula I is a Hyl, Orn, Pyl, and derivatives thereof, more preferably from combination of two amino acid residues selected from the the group consisting of Dpr, Dab, Orn, Lys, DSu, and deriva 40 group consisting of Lys(Asp)-, Dab(Asp)-, Dpr(Asp)-, DSu tives thereof, more preferably from the group consisting of (Asp)-, Orn(Asp)- Lys(Glu)-, Dab(Glu)-, Dpr(Glu)-, DSu Dpr, Dab, Orn, Lys, and derivatives thereof, more preferably (Glu)-, Orn(Glu)-, Lys(Aad)-, Dab(Aad)-, Dpr(Aad)-, DSu from the group consisting of Orn, Lys, and derivatives (Aad)-, Orn(Aad)-, Lys(Apn)-, Dab(Apn)-, Dpr(Apn)-. thereof, and wherein even more preferably X is Lys or a DSu(Apm)- Orn(Apn)-, and derivatives thereof, more pref derivative thereof. 45 erably from the group consisting of Lys(Asp)-, Dab(Asp)-. Alternatively or in addition to the aforementioned pre Dpr(Asp)-, Orn(Asp)- Lys(Glu)-, Dab(Glu)-, Dpr(Glu)- ferred amino acid residues for X, and preferably in addition Orn(Glu)-, Lys(Aad)-, Dab(Aad)- Dpr(Aad)-, Orn(Aad)- to the aforementioned preferred amino acid residues for X, it LyS(Apn)-, Dab(Apn)-. Dpr(Apn)-, Orn(Apn)-, and is further preferred according to the present invention that X derivatives thereof, more preferably from the group consist is an amino acid residue selected from the group consisting of 50 ing of Lys(Asp)-, Orn(Asp)-, LyS(Glu)-, Orn(Glu)-, Lys ASn, Asp, Gln, Glu, Aad, Aan, Aca, Agn, Apm, App. ASu, Bas, (Aad)-, Orn(Aad)-, LyS(Apn)-, Orn(Apn)-, and derivatives Cit, Dpm, DSu, Ggu, Gla, and derivatives thereof, more pref thereof, and even more preferably from the group consisting erably from the group consisting of Asp, Glu, Aad, Apm, and of LyS(Asp)-, Orn(Asp)-, LyS(Glu)-, Orn(Glu)-, and deriva derivatives thereof, more preferably from the group consist tives thereof. ing of Asp, Glu, and derivatives thereof, and wherein even 55 Alternatively or in addition to the aforementioned pre more preferably X is Asp or a derivative thereof. ferred amino acid residues for X and/or X, and preferably in Thus, according to the present invention it is preferred that addition to the aforementioned preferred amino acid residues the fragment X (X)- of formula I is a combination of two for X and/or X, it is further preferred according to the amino acid residues selected from the group consisting of present invention that X is an amino acid residue selected Arg(ASn)-. His(ASn)-, Lys(Asn)-, Bly(ASn)-, Cit(ASn)-, Dab 60 from the group consisting of Ala, Cys, Gly, Ile, Leu, Met, Phe, (ASn)-. Dpr(ASn)-, DSu(ASn)-, Har(ASn)-. Hhs(ASn)-, Hyl Pro, Ser, Thr, Trp, Tyr, Val, Scy, Abu, Ahe, Aib, Aze, Bal, Bux, (ASn)-, Orn(ASn)-, Pyl(ASn)-, Arg(Asp)-. His(Asp)-, Lys Cap, Cha, Cya, Edc, Glp, Hcy, Hph, Hse, Hyp, 3Hyp, Igl, Iva, (Asp)-, Bly (Asp)- Cit(Asp)-, Dab(Asp)-, Dpr(Asp)-, DSu Mhp, NaI, Nle, Nphe, Nty, Nva, Oic, Pen, Phg, Pip, pSer, (Asp)-, Har(Asp)- Hhs(Asp)-, Hyl(Asp)-, Orn(Asp)-, Pyl pThr, pTyr, Sar, Spg, Sta. Thi, Tic, Tie, Tml, Tza, Wil, and (Asp)-, Arg(Gln)-. His(Gln)- Lys(Gln)-, Bly (Gln)- Cit 65 derivatives thereof, more preferably from the group consist (Gln)-, Dab(Gln)-, Dpr(Gln)-, DSu(Gln)-, Har(Gln)-; Hhs ing of Ala, Aib, Abu, Nva, Nile, Ahe, and derivatives thereof, (Gln)-, Hyl(Gln), Orn(Gln)-, Pyl(Gln)-; Arg(Glu)-. His more preferably from the group consisting of Ala, Aib, Abu, US 9.220,672 B2 10 Nva, and derivatives thereof, and wherein even more prefer Therefore, according to preferred embodiments of the ably X is Ala or a derivative thereof. present invention, X and X of the inventive peptide are Alternatively or in addition to the aforementioned pre bound to one another via an amide bond, wherein the amide ferred amino acid residues for X and/or X, and preferably in bond is preferably formed by an N-moiety of the X amino addition to the aforementioned preferred amino acid residues 5 acid residue and a carbonyl-moiety of the X amino acid for X and/or X, it is further preferred according to the residue. present invention that X is an amino acid residue selected According to embodiments of the present invention which from the group consisting of ASn, Asp, Gln, Glu, Aad, Aan, are further preferred, X is covalently bound to X neither by Aca, Agn, Apm, App. ASu, Bas, Cit. Dpm, DSu, Gigu, Gla, and the N-terminal amino group, nor by the C-terminal carboxyl derivatives thereof, more preferably from the group consist 10 ing of Asp, Glu, Aad, Apm, and derivatives thereof, more or carbonyl group thereof, i.e. of the amino acid residue X, preferably from the group consisting of Asp, Glu, Aad, and respectively. Thus, according to preferred embodiments derivatives thereof, and wherein even more preferably X is wherein X and X are bound by an amide bond formed by an Glu or a derivative thereof. N-moiety of the X amino acid residue and a carbonyl-moiety Therefore, according to preferred embodiments of the 15 of the X amino acid residue, it is further preferred that the inventive peptide, X is an amino acid residue selected from carbonyl moiety of the X amino acid moiety is not a carbonyl the group consisting of Arg, His, Lys, Bly, Cit. Dab, Dpr, DSu, moiety of the C-terminal group thereof. Furthermore, accord Har, Hhs, Hyl, Orn, Pyl, and derivatives thereof, ing to preferred embodiments thereof, wherein the amino acid and/or, preferably and in X is an O-amino acid, the C-terminus is accordingly con wherein X and X are, independently from one another, an stituted by the C-carboxyl or -carbonyl group thereof. amino acid residue selected from the group consisting of ASn, According to alternatively preferred embodiments of the Asp, Gln, Glu, Aad, Aan, Aca, Agn, Apm, App. ASu, Bas, Cit. present invention wherein the amino acid in X is not an Dpm, DSu, Ggu, Gla, and derivatives thereof, C.-amino acid, and wherein X contains two or more carboxyl and/or, preferably and and/or carbonyl groups, there is no general restriction as to wherein X is an amino acid residue selected from the group 25 which of the two or more carboxyl and/or carbonyl groups consisting of Ala, Cys, Gly, Ile, Leu, Met, Phe, Pro, Ser. Thr, constitutes the C-terminus, and is therefore not covalently Trp, Tyr, Val, Scy, Abu, Ahe, Aib, Aze, Bal, Bux, Cap, Cha, bound to X in accordance with the preferred embodiments of Cya, Edc, Glp, Hcy, Hph, Hse, Hyp, 3Hyp, Igl, Iva, Mhp, NaI, the present invention. According to specific embodiments of Nle, Nphe, Nty, Nva, Oic, Pen, Phg. Pip, pSer, pThr, pTyr, Sar, said alternatively preferred embodiments, whereinX further Spg, Sta. Thi, Tic, Tle, Tml, Tza, Wil, and derivatives thereof. 30 contains one or more side-chains, and the second and/or As mentioned in the foregoing, there is no particular further carboxyl and/or carbonyl group(s) is/are covalently restriction regarding the covalent bond formed between X bound to said one or more side-chains, it is preferred that the and X, provided that the N-terminal group of the N-terminus C-terminus is not a carboxyl or carbonyl group which is present in X does not engage in a covalent bond with X. present on a side-chain. Thus, apart from said restriction, X and X may be bound to 35 Thus, according to further preferred embodiments, the one another via one or more covalent bonds, wherein the term invention relates to a peptide having the formula (II): “covalent bond' includes single, double, and triple bonds, wherein according to the present invention it is preferred that (II) the term “covalent bond designates a single or double bond, wherein X is covalently bound to X neither by the N-termi and even more preferably designates a single covalent bond. 40 nal amino group, nor by the C-terminal carboxyl or carbonyl Furthermore, with respect to the one or more covalent bonds group thereof, i.e. of the amino acid residue X, respectively, formed between X and X, there is no particular restriction as and wherein RandR represent the optional derivatization of to the types of atoms contained in X and X which engage in the N- and C-terminal groups, respectively. said one or more covalent bonds, provided that this does not In principle, RandR represent any suitable group which include the specific nitrogen atom contained in the N-termi 45 may be present at the N- and C-terminus of the amino acid nal group of the N-terminus of X. Thus, by way of example, residue X, wherein it is preferred that R' is H or a physi the one or more covalent bonds include C C, C N, C-O, ologically acceptable N-terminal blocking group, and R is and C S bonds, wherein the one or more covalent bonds OH or a physiologically acceptable C-terminal blocking formed between X and X is preferably one or more of a group. In particular, according to further preferred embodi C. C. C. N. or C Obond, more preferably of a C C or 50 ments, R' is selected from the group consisting of H, alkyl, C N bond, and wherein the one or more covalent bonds formyl, acyl, alkoxycarbonyl, and derivatives thereof, preferably include one or more C N bonds. wherein more preferably R' is H. (C-C)alkyl, formyl, (C- According to further preferred embodiments of the present Cs)acyl, (C-C)alkoxycarbonyl or a derivative thereof, more invention, X and X are covalently bound by one or more preferably H, (C-C)alkyl, or a derivative thereof, and C N bonds, more preferably by one C N bond, wherein 55 wherein even more preferably R' is H. even more preferably the C-atom of the C N bond is a Furthermore, according to preferred embodiments of the C-atom belonging to the X amino acid residue, and the present invention wherein the inventive peptide has the for N-atom belongs to the one or more NH-functionalities of X mula II, it is further preferred that R is selected from the which is not the N-atom of the N-terminal group of the N-ter group consisting of OH, alkyl, alkoxy, NH, alkylamino, minus of X. According to particularly preferred embodi 60 dialkylamino, thiol, thioether, and derivatives thereof, ments of the present invention, X and X are covalently wherein more preferably R is OH, (C-C)alkoxy, NH. (C- bound by one or more amide bonds, preferably by one amide C.)alkylamino, di(C-C)alkylamino, thiol, (C-C)thioet bond, wherein even more preferably the C-atom of the her, or a derivative thereof, more preferably OH, (C-C) C(O)—N bond is a C-atom belonging to a carbonyl group of alkoxy, NH. (C-C)alkylamino, di(C-C)alkylamino, or a the X amino acid residue, and the N-atom belongs to the one 65 derivative thereof, more preferably OH, NH, or a derivative or more NH-functionalities of X which is not the N-atom of thereof, more preferably OH or NH, and wherein even more the N-terminal group of the N-terminus of X. preferably R is NH. US 9.220,672 B2 11 12 Alternatively to or in addition to and preferably in addition tive peptide are selected among the group consisting of amino to the aforementioned optional derivatization of the N-termi acids naturally occurring in proteins and derivatives thereof, nus and the preferred substitution of the C-terminus in X, and in particular among the proteinogenic amino acids coded respectively, the N-terminal group of the N-terminus in X for in the genetic code. In particular, it is preferred that the may also be derivatized. In principle, the N-terminal group in amino acid residues X, X2, X, and X of the inventive X may optionally be derivatized in any conceivable fashion, peptide are selected from the group consisting of Arg, ASn, wherein it is preferred that the optionally derivatized N-ter Ala, His, Asp, Cys, Lys, Gln, Gly, Glu, Ile, Leu, Met, Phe, Pro, minal group contains a physiologically acceptable N-termi Ser. Thr, Trp, Tyr, Val, Scy, and derivatives thereof. nal blocking group. In particular, according to further pre According to a preferred embodiment of the present inven ferred embodiments, the N-terminal amino group is 10 tion wherein the amino acid residues are selected from the underivatized, i.e. is NH, or one or independently from one group consisting of proteinogenic amino acids and deriva another both of the hydrogen of the N-terminal amino tives thereof, X is an amino acid residue selected from the group are Substituted by alkyl, formyl, acyl, alkoxycarbonyl, group consisting of Arg, His, Lys, and derivatives thereof, or a derivative thereof, more preferably by (C-C)alkyl, wherein even more preferably X is Lys or a derivative formyl, (C-C)acyl, (C-C)alkoxycarbonyl or a derivative 15 thereof. Alternatively or in addition to the aforementioned thereof, and even more preferably by (C-C)alkyl or a preferred amino acid residues for X, and preferably in addi derivative thereof. According to particularly preferred tion to the aforementioned preferred amino acid residues for embodiments of the present invention, however, the N-termi X, it is further preferred according to the present invention nus in X is underivatized, i.e. is NH2. that X is an amino acid residue selected from the group Furthermore, alternatively to or in addition to and prefer consisting of ASn, Asp, Gln, Glu, and derivatives thereof, ably in addition to the aforementioned optional derivatization wherein even more preferably X is Asp or a derivative of the N-terminus and the preferred substitution of the C-ter thereof. minus in X, respectively, the C-terminal group of the C-ter Alternatively or in addition to the aforementioned pre minus in X may also be derivatized. In principle, the C-ter ferred amino acid residues for X and/or X, and preferably in minal group in X may optionally be derivatized in any 25 addition to the aforementioned preferred amino acid residues conceivable fashion, wherein it is preferred that the optionally for X and/or X, it is further preferred according to the derivatized C-terminal group contains a physiologically present invention that X is an amino acid residue selected acceptable C-terminal blocking group. In particular, accord from the group consisting of Ala, Cys, Gly, Ile, Leu, Met, Phe, ing to further preferred embodiments, the C-terminal car Pro, Ser. Thr, Trp, Tyr, Val, Scy, and derivatives thereof, more boxyl group is underivatized, i.e. is C(O)—OH, or the 30 preferably from the group consisting of Gly, Ala, Val, Leu, Ile, hydroxyl moiety of the C-terminal carboxyl group is substi Met, Pro, Phe, Trp, and derivatives thereof, more preferably tuted by alkyl, alkoxy, NH, alkylamino, dialkylamino, thiol, from the group consisting of Gly, Ala, Val, Leu, Ile, and thioether, or a derivative thereof, more preferably by (C-C) derivatives thereof, more preferably from the group consist alkoxy, NH, (C-C)alkylamino, di(C-C)alkylamino, ing of Gly, Ala, Val, and derivatives thereof, and derivatives thiol, (C-C)thioether, or a derivative thereof, more prefer 35 thereof, and wherein even more preferably X is Ala or a ably by (C-C)alkoxy, NH, (C-C)alkylamino, di(C-C) derivative thereof. alkylamino, or a derivative thereof, wherein even more pref Alternatively or in addition to the aforementioned pre erably the hydroxyl moiety of the C-terminal carboxyl group ferred amino acid residues for X and/or X, and preferably in of X is substituted by NH or a derivative thereof, more addition to the aforementioned preferred amino acid residues preferably by NH 40 for X and/or X, it is further preferred according to the Therefore, according to embodiments of the present inven present invention that X is an amino acid residue selected tion which are further preferred, the peptide has the formula from the group consisting of Ala, Cys, Gly, Ile, Leu, Met, Phe, (II): Pro, Ser. Thr, Trp, Tyr, Val, Scy, and derivatives thereof, more preferably from the group consisting of Asp, ASn, Glu, Gln, 45 and derivatives thereof, more preferably from the group con wherein R' is H or a physiologically acceptable N-terminal sisting of Asp, Glu, Gln, and derivatives thereof, more pref blocking group, and wherein R' is preferably H, alkyl, erably from the group consisting of Glu, Gln, and derivatives formyl, acyl, alkoxycarbonyl, or a derivative thereof, more thereof, and wherein even more preferably X is Glu or a preferably H., (C-C)alkyl, formyl, (C-C)acyl, (C-C) derivative thereof. alkoxycarbonyl or a derivative thereof, more preferably H, 50 Therefore, according to preferred embodiments of the (C-C)alkyl, or a derivative thereof, and wherein even more inventive peptide, X is an amino acid residue selected from preferably R' is H, and/or, preferably and the group consisting of Arg, His, Lys, and derivatives thereof, wherein R is OH or a physiologically acceptable C-terminal X preferably being Lys or a derivative thereof, blocking group, wherein preferably R is OH, alkyl, alkoxy, and/or, preferably and NH, alkylamino, dialkylamino, thiol, thioether, or a deriva 55 X is an amino acid residue selected from the group consist tive thereof, more preferably OH, (C-C)alkoxy, NH. (C- ing of Asn., Asp, Gln, Glu, and derivatives thereof, X prefer C.)alkylamino, di(C-C)alkylamino, thiol, (C-C)thioet ably being Asp or a derivative thereof her, or a derivative thereof, more preferably OH, (C-C) and/or, preferably and alkoxy, NH. (C-C)alkylamino, di(C-C)alkylamino, or a X is an amino acid residue selected from the group consist derivative thereof, more preferably OH, NH, or a derivative 60 ing of Ala, Cys, Gly, Ile, Leu, Met, Phe, Pro, Ser. Thr, Trp, Tyr, thereof, more preferably OH or NH, and wherein even more Val, Scy, and derivatives thereof, preferably from the group preferably R is NH, consisting of Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp, and and/or, preferably and derivatives thereof, more preferably from the group consist wherein the C-terminal carboxyl group of the peptide in X is ing of Gly, Ala, Val, Leu, Ile, and derivatives thereof, more functionalized to an amide, preferably to X. NH2. 65 preferably from the group consisting of Gly, Ala, Val, and According to the present invention it is further preferred derivatives thereof, and wherein X is even more preferably that the amino acid residues X, X2, X, and X of the inven Ala or a derivative thereof, US 9.220,672 B2 13 14 and/or, preferably and Therefore, according to a particularly preferred embodi X is an amino acid residue selected from the group consist ment of the present invention, the peptide has the following ing of Ala, Cys, Gly, Ile, Leu, Met, Phe, Pro, Ser. Thr, Trp, Tyr, formula (III): Val, Scy, and derivatives thereof, preferably from the group consisting of Asp, ASn, Glu, Gln, and derivatives thereof, Lys(H-Asp-R)-Ala-Glu (III) more preferably from the group consisting of Asp, Glu, Gln, wherein R is OH or NH, and preferably NH and derivatives thereof, more preferably from the group con As mentioned in the foregoing with respect to preferred sisting of Glu, Gln, and derivatives thereof, and whereinX is embodiments of the present invention, wherein the amino even more preferably Glu or a derivative thereof. 10 acid residues are selected from the group consisting of amino With respect to the preferred embodiments of the present acids naturally occurring in proteins and derivatives thereof, invention wherein the amino acid residues X, X, X, and X the particularly preferred amino acid sequence motif accord of the inventive peptide are selected from the group consisting ing to formula III may also be linked via the N-terminus of of amino acids naturally occurring in proteins and derivatives Lys and/or via the C-terminus of Glu to one or more further thereof, and in particular among the proteinogenic amino 15 proteinogenic amino acids and/or to one or more peptide or acids and derivatives thereof, said proteins also comprise peptide moieties as described with respect to the embodi embodiments wherein the protein is contained as a sequence ments and preferred embodiments in the foregoing. motif in a larger protein or protein fragment or derivative According to the present invention it is however preferred thereof. Thus, according to said preferred embodiments, the that the peptide according to formula III is a tetrapeptide inventive peptide may be linked via the N-terminus of X which is not further contained as a sequence motif in a larger and/or via the C-terminus of X to one or more further pro polypeptide or protein, whereinfurthermore the C- and N-ter teinogenic amino acids and/or to one or more peptide or minal groups of the tetrapeptide in Glu and Lys may be peptide moieties. In general, according to said preferred underivatized or derivatized according to any of the afore embodiments of the present invention, there is no particular mentioned embodiments and preferred embodiments relating restriction as to the number of further proteinogenic amino 25 to the derivatization of the C- and N-termini. In particular, the acids to which the inventive peptide may be linked or to the N-terminal amino group in Lys may optionally be derivatized type and size of the peptide or peptide moieties to which it to contain a physiologically acceptable N-terminal blocking may be further bound via C- and/or N-terminal peptide bonds. group, wherein preferably or one or independently from Thus, by way of example, the inventive peptide may be linked one another both of the hydrogen of the N-terminal NH to or comprised in a larger polypeptide or protein, such that 30 group are Substituted by alkyl, formyl, acyl, alkoxycarbonyl, the resulting polypeptide or protein may contain anywhere or a derivative thereof, more preferably by (C-C)alkyl, from 5 to 200 amino acid residues, wherein it is preferred formyl, (C-C)acyl, (C-C)alkoxycarbonyl or a derivative according to said embodiments that the larger polypeptide or thereof, and even more preferably by (C-C)alkyl or a protein in which the inventive peptide is bound contains from 35 derivative thereof. More specifically, the N-terminal amino 5 to 100 amino acid residues, more preferably from 5 to 50 group of the peptide in Lys is preferably NRR, wherein R amino acid residues, more preferably from 5 to 20 amino acid and Rare independently from one another Horalkyl, pref residues, more preferably from 5 to 10 amino acid residues, erably H or (C-C)alkyl. According to particularly preferred and even more preferably from 5 to 7 amino acid residues. embodiments of the present invention, however, the N-termi Alternatively, according to preferred embodiments of the 40 nus in Lys is underivatized. Furthermore, the C-terminal car present invention wherein the amino acid residues X1,X2, X, boxyl group in Glu may also be optionally derivatized to and X of the inventive peptide are selected from the group contain a physiologically acceptable C-terminal blocking consisting of amino acids naturally occurring in proteins and group, wherein preferably the hydroxyl moiety of the C-ter minal carboxyl group is substituted by alkyl, alkoxy, NH2, derivatives thereof, and in particular among the proteinogenic alkylamino, dialkylamino, thiol, thioether, or a derivative amino acids and derivatives thereof, the inventive peptide is a 45 thereof, more preferably by (C-C)alkoxy, NH. (C-C) tetrapeptide which is not further contained as a sequence alkylamino, di(C-C)alkylamino, thiol, (C-C)thioether, or motif in a larger polypeptide or protein, wherein the C- and a derivative thereof, more preferably by (C-C)alkoxy, NH, N-terminal groups of the tetrapeptide in X and X may be (C-C)alkylamino, di(C-C)alkylamino, or a derivative underivatized or derivatized according to any of the afore thereof, and wherein even more preferably the hydroxyl moi mentioned embodiments and preferred embodiments relating 50 ety of the C-terminal carboxyl group of X is substituted by to the derivatization of the C- and N-termini. NH, or a derivative thereof. More specifically, Glu is prefer According to a particularly preferred embodiment of the ably functionalized to an amide Glu-NR'R'', wherein Rand present invention, X is Lys or a derivative thereof, X is Asp Rare independently from one another Horalkyl, preferably or a derivative thereof, X is Ala or a derivative thereof, and 55 H or (C-C)alkyl, wherein even more preferably R and R' X is Glu or a derivative thereof, wherein more preferably X are H. is Lys, X is Asp, X is Ala, and X is Glu. According to said Therefore, according to further preferred embodiments of embodiments it is further preferred that Asp and Lys are the present invention, the inventive peptide has the formula bound via their respective side chains in the form of an (III): isopeptide bond, wherein the beta-carboxyl moiety of Asp 60 forms a peptidic bond with the e-amino moiety of Lys via Lys(H-Asp-R)-Ala-Glu (III) condensation thereof. Furthermore, according to said pre wherein R is OH or NH, and preferably NH, and ferred embodiment it is yet further preferred that the C-ter wherein the C-terminal carboxyl group of the peptide in Glu minal carboxyl group of Asp is underivatized or amidated, is preferably functionalized to an amide Glu-NR'R''. wherein the C-terminus is preferably amidated, wherein even 65 wherein R and Rare independently from one another Hor more preferably the –OH moiety of the C-terminal carboxyl alkyl, preferably H or (C-C)alkyl, wherein even more pref group is substituted by NH. erably Rand Rare H, US 9.220,672 B2 15 16 and/or, preferably and taxonomical point of view divided up into the subdivisions of wherein the N-terminal amino group of the peptide in Lys is Streptococcus, Leuconostoc, Pediococcus, Lactococcus and preferably NRR, Lactobacillus. The microorganism of the present invention is wherein Rand Rare independently from one another Hor preferably a Lactobacillus species. Members of the lactic acid alkyl, preferably H or (C-C)alkyl, wherein even more pref bacteria group normally lack porphyrins and cytochromes, do erably Rand Rare H. not carry out electron-transport phosphorylation and hence In particular, according to a particularly preferred embodi obtain energy only by Substrate-level phosphorylation. I.e. in ment thereof, the peptide has the formula IV: lactic acid bacteria ATP is synthesized through fermentation of carbohydrates. All of the lactic acid bacteria grow anaero 10 bically, however, unlike many anaerobes, most lactic acid Accordingly, a tetrapeptide having the following chemical bacteria are not sensitive to oxygen and can thus grow in its structure (V) is particularly preferred according to the present presence as well as in its absence. Accordingly, the bacteria invention: preferably used in the present invention are preferably aero tolerant anaerobic lactic acid bacteria, preferably belonging 15 to the genus of Lactobacillus. It is further preferred according to the present invention (V) that the microorganism used is a probiotic Lactobacillus spe HO O cies. The term “probiotic' in the context of the present inven tion means that the microorganism has a beneficial effect on NH2 O NH2 O health if it is topically applied to the skin. Preferably, a “pro H biotic microorganism is a live microorganism which, when N N topically applied to the skin, is beneficial for health of this --- H ~. N H NH2 tissue. NH2 O O In a preferred embodiment the microorganism used in the 25 production of the inventive protein belongs to the species of In general, the peptide of the present invention may be selected from the group consisting of the Lactobacillus spe obtained by any conceivable means using any of the methods cies L. acidophilus, L. plantarum, L. reuteri, L. Casei, L. well known in the art such as by chemical synthesis, wherein paracasei, L. brevis, L. fermentum, L. buchneri, L. delbrickii. said synthesis may in part or entirely rely on biotechnological However, the Lactobacillus species are not limited thereto. methods using recombinant strains or cultures. In particular, 30 According to a particularly preferred embodiment of the the small size of the inventive peptides is particularly advan present invention, the microorganism which is employed is tageous since it allows for the use of cost-effective industrial selected from the group of Lactobacillus species consisting of synthetic methodologies. Their demonstrated high activity L. brevis, L. paracasei, L. fermentum, L. delbrickii ssp. del permits the commercial use in a large number of cosmetic or brickii, L. buchneri, and combinations of two or more (dermo)pharmaceutical products in a cost-efficient manner. 35 thereof. In particular, the group of Lactobacillus species pref The peptide can however also be obtained by fermentation of erably consists of L. brevis having DSMZ accession number one or more strains of bacteria, modified or not by genetic DSM 17247 and/or DSM 17250, L. paracasei having DSMZ engineering, to produce the sought sequences or at least frag accession number DSM 17248, L. fermentum having DSMZ ments thereof. accession number DSM 17249, L. delbrickii ssp. delbrickii In addition to these production methods, the peptide can 40 having DSMZ accession number DSM 18006, L. buchneri also be obtained by extraction of proteins of animal or Veg having DSMZ accession number DSM 18007, including etable origin, preferably vegetable, adapted to contain these mutants or derivatives thereof. Even more preferably, the sequences in their structure, which may be followed by con Lactobacillus species is L. brevis, more preferably selected trolled hydrolysis, enzymatic or not, which frees the desired from the group consisting of L. brevis having DSMZ acces peptide fragment. To carry out the invention, it is possible but 45 sion number DSM 17247, DSM 17250, and mutants or not necessary, either to extract the proteins in question first derivatives thereof, wherein even more preferably the Lacto and then to hydrolyze them, or to carry out hydrolysis first on bacillus species is L. brevis having DSMZaccession number a raw extract and to purify the peptide fragments. There can DSM 17250 and/or mutants orderivatives thereof. The afore also be used the hydrolysate without extracting from it the mentioned deposits were performed at the Deutsche Sam peptide fragments in question, by ensuring however the stop 50 mlung für Mikroorganismen and Zellkulturen (DSMZ) pur ping of the enzynamic hydrolysis reaction timewise and suant to the terms of the Budapest treaty on the international determining the presence of the peptides in question by Suit recognition of the deposit of microorganisms for the purposes able analytic means such as radioactivity tracing, immunof of patent procedures. Furthermore, within the meaning of the luorescence or immunoprecipitation with specific antibodies, present invention, a mutant or derivative of the above-men and the like. 55 tioned deposited Lactobacillus strains designates any con According to a preferred embodiment of the present inven ceivable mutants or derivatives having retained their capabil tion, the inventive peptide is obtained from a microorganism, ity to be used in the production of the inventive peptide. preferably from a prokaryote, and in particular from one or Therefore, the present invention further relates to the use of more strains of bacteria. According to the present invention it a microorganism for the production of the inventive peptide is particularly preferred that the microorganism used for pro 60 according to embodiments and preferred embodiments ducing the inventive peptide is a microorganism belonging to described in the foregoing, wherein the microorganism is the group of lactic acid bacteria. The term “microorganism preferably a prokaryote microorganism, more preferably one belonging to the group of lactic acid bacteria” encompasses or more strains of bacteria, more preferably one or more lactic (a) microorganism(s) which belong(s) to bacteria, in particu acid bacteria, more preferably one or more bacteria of the lar belonging togram-positive fermentative eubacteria, more 65 genus Lactobacillus, wherein more preferably the bacteria is particularly belonging to the family of lactobacteriaceae selected from the group consisting of the Lactobacillus spe including lactic acid bacteria. Lactic acid bacteria are from a cies L. acidophilus, L. plantarum, L. reuteri, L. Casei, L. US 9.220,672 B2 17 18 paracasei, L. brevis, L. fermentum, L. buchneri, L. delbrickii. bination of an enzyme other than the proteolytic enzyme, an preferably L. delbrickii ssp. delbrickii, and combinations of acid, a base and the like may also be utilized. For example, the two or more thereof, cells of the microorganisms preferably used in the present wherein more preferably the Lactobacillus species is selected invention are lysed by freezing and thawing, more preferably from the group consisting of L. brevis, preferably L. brevis freezing at temperatures of at most -70° C. and thawing at having DSMZ accession number DSM 17247, DSM 17250, temperatures of at least 30°C., particularly freezing is pre and mutants or derivatives thereof, L. paracasei, preferably L. ferred at temperatures of at most -75° C. and thawing is paracasei having DSMZ accession number DSM 17248 and preferred attemperatures of at least 35° C. and most preferred mutants or derivatives thereof, L. fermentum, preferably L. are temperatures for freezing of at most-80° C. and tempera fermentum having DSMZaccession number DSM 17249 and 10 tures for thawing of at least 37°C. It is also preferred that said mutants or derivatives thereof, L. delbrickii ssp. delbrickii, freezing/thawing is repeated one or more times, more prefer preferably L. delbrickii ssp. delbrickii having DSMZ acces ably two or more times, and even more preferred three or sion number DSM 18006 and mutants or derivatives thereof, more times, wherein particularly preferably the procedure is L. buchneri, preferably L. buchneri having DSMZ accession repeated 4 or more times and even more preferably 5 or more number DSM 18007 and mutants or derivatives thereof, and 15 times. combinations of two or more thereof, Preferably, the aqueous medium used for the lysates as wherein more preferably the Lactobacillus species is L. described is water, physiological saline, or a buffer solution. brevis, more preferably selected from the group consisting of An advantage of a bacterial cell lysate is that it can be easily L. brevis having DSMZaccession number DSM 17247, DSM produced and stored cost efficiently since less technical facili 17250, and mutants or derivatives thereof, and ties are needed. wherein even more preferably the Lactobacillus species is L. According to the invention, lysates are also preparations of brevis having DSMZ accession number DSM 17250 and/or fractions of molecules from the above-mentioned lysates. mutants or derivatives thereof. These fractions can be obtained by methods known to those In general, regarding the inventive use of a microorganism skilled in the art, e.g., chromatography, including, e.g., affin for the production of the inventive peptide, there is no par 25 ity chromatography, ion-exchange chromatography, size-ex ticular restriction as to the method of obtaining the inventive clusion chromatography, reversed phase-chromatography, peptide from the microorganism, provided that the peptide or and chromatography with other chromatographic material in a precursor thereof is obtained in a form in a form in which the column or batch methods, other fractionation methods, e.g., inventive peptide may be employed for achieving the effects filtration methods, e.g., ultrafiltration, dialysis, dialysis and described herein, in particular with respect to its use in cos 30 concentration with size-exclusion in centrifugation, centrifu metic and pharmaceutical applications. In particular, the use gation in density-gradients or step matrices, precipitation, ofa microorganism for the production of the inventive peptide e.g., affinity precipitations, salting-in or salting-out (ammo preferably involves its effective isolation from the microor niumsulfate-precipitation), alcoholic precipitations or other ganism by any suitable means known to the skilled person. proteinchemical, molecular biological, biochemical, immu According to a preferred embodiment, a lysate of the 35 nological, chemical or physical methods to separate above microorganism is first obtained, after which the inventive components of the lysates. peptide is won from the lysate in a subsequent step. Within the The proteins of the present invention may also suitably be meaning of the present invention, the term “lysate' designates produced from a filtrate of the microorganisms used in the a solution or Suspension in an aqueous medium of cells of the present invention, preferably of the Lactobacillus species one or more microorganisms which may be used according to 40 disclosed herein. Within the meaning of the present invention, the present invention which have been suitably severed or an the term “filtrate” means a cell-free solution or suspension of extract thereof. However, the term should not be construed in the microorganisms used in the present invention which has any limiting way. In particular, in addition to the inventive been obtained as Supernatant of a centrifugation procedure of peptide or to one or more suitable precursors thereof, the cell a culture of the microorganisms used in the present invention lysate regularly further comprises, e.g., macromolecules, like 45 in any appropriate liquid, medium or buffer known to the DNA, RNA, other proteins and peptides, carbohydrates, lip person skilled in the art. However, the term should not be ids and the like and/or micromolecules such as amino acids, construed in any limiting way. In particular, a filtrate regularly sugars, lipid acids and the like, or fractions thereof. Further further comprises, e.g., macromolecules, like DNA, RNA, more, said lysate regularly comprises cell debris which may proteins, peptides, carbohydrates, lipids and the like and/or be of Smooth or granular structure. 50 micromolecules, like amino acids, Sugars, lipid acids and the Methods for preparing cell lysates of microorganism are like, or fractions of it. Methods for preparing filtrates of known in the art, for example, by employing French press, microorganism are known in the art. In addition, “filtrate' cells mill using glass or ironbeads or enzymatic cell lysis and relates to various methods known in theart. The exact method the like. In addition, lysing cells relates to various methods is not essential and any method that can achieve filtration of known in the art for opening/destroying cells. The method for 55 the cells of the microorganism used in the production of the lysing a cell is not essential to the present invention and thus inventive peptide may be employed. any method that can achieve lysis of the cells of the microor Furthermore, according to the present invention also any ganisms which are preferably used in the present invention part of the cells of the microorganisms used in the present may be employed. Thus, by way of example, the opening/ invention may be used for producing the inventive peptide, destruction of cells can be done enzymatically, chemically or 60 wherein preferably a membrane fraction is employed as physically. Non-limiting examples for enzymes and enzyme obtained by membrane-preparation. Membrane preparations cocktails are proteases, like proteinase K, lipases or glycosi of microorganisms, and in particular of microorganisms dases; non-limiting examples for chemicals are ionophores, belonging to the genus of Lactobacillus can be obtained by detergents, like Sodium dodecyl sulfate, acids or bases; and methods known in the art, for example, by employing the non-limiting examples of physical means are high pressure, 65 method described by Rollanetal. in Int. J. Food Microbiol. 70 like French-pressing, osmolarity, temperature, like heat or (2001), 303-307, by Matsuduchi et al. in Clin. Diagn. Lab. cold. Additionally, a method employing an appropriate com Immunol. 10 (2003), 259-266, by Stentz et al. in Appl. Envi US 9.220,672 B2 19 20 ron. Microbiol. 66 (2000), 4272-4278, or by Varmanen et al. tide of the invention, and shall be biocompatible upon in J. Bacteriology 182 (2000), 146-154. Alternatively, also a administration to a subject, in particular, non-toxic and/or whole cell preparation may be used. non-immunogenic. The present invention also provides the peptide of the Moreover, the pharmaceutical composition could com present invention for use as a medicament. prise further ingredients such as Stabilizers, Sustained release The term “medicament” as used herein refers to a formu agents, further drugs, and the like. Preferred stabilizers are lation of the peptide of the invention or a composition com protein stabilizers (e.g., human serum albumin (HSA)) or prising it wherein said peptide is the pharmaceutical active non-protein stabilizers. Sustained release agents are well compound. The peptide may be formulated in liquid, dry, known to the art and, preferably, include glyceryl mono gaseous or gel form. If the peptide is formulated in dry form, 10 stearate or glyceryl distearate alone or with a wax. Further a lyophilized form is preferably envisaged. If a liquid formu ingredients also encompass preservatives, perfumes, anti lation is envisaged, the peptide is preferably a component of foams, dyes, pigments, thickeners, Surface-active Substances, a pharmaceutical composition which in addition to the pep emulsifiers, emollients, finishing agents, fats, oils, waxes or tide comprises a Suitable solvent as a liquid pharmaceutical other customary constituents of a dermatological formula acceptable carrier. In gaseous form, the peptide can be 15 tion, such as alcohols, polyols, polymers, foam stabilizers, included into an aerosol. solubility promoters, electrolytes, organic acids, organic Sol The term “pharmaceutically acceptable carrier” refers to a vents, or silicone derivatives. diluent, adjuvant, excipient, or vehicle with which the peptide The medicament according to the invention may comprise is administered. Such a carrier is pharmaceutically accept emollients. Emollients may be used in amounts which are able, i.e. is non-toxic to a recipient at the dosage and concen effective to prevent or relieve dryness. Useful emollients tration employed. It is preferably isotonic, hypotonic or include, without limitation: hydrocarbon oils and waxes; sili weakly hypertonic and has a relatively low ionic strength, cone oils; triglyceride esters; acetoglyceride esters; ethoxy Such as provided by a Sucrose solution. Such pharmaceutical lated glyceride; alkyl esters; alkenyl esters; fatty acids; fatty carriers can be sterile liquids, such as water and oils, includ alcohols; fatty alcohol ethers; etheresters; lanolin and deriva ing those of petroleum, animal, vegetable or synthetic origin, 25 tives; polyhydric alcohols (polyols) and polyether deriva Such as peanut oil, soybean oil, mineral oil, sesame oil and the tives; polyhydric alcohol (polyol) esters; wax esters; beeswax like. Saline solutions and aqueous dextrose and glycerol solu derivatives; vegetable waxes; phospholipids; sterols; and tions can also be employed as liquid carriers. Suitable phar amides. Thus, for example, typical emollients include min maceutical excipients include starch, glucose. Sucrose, gela eral oil, especially mineral oils having a viscosity in the range tin, malt, rice, flour, chalk, silica gel, Sodium Stearate, 30 of 50 to 500 SUS (Saybolt Universal second), lanolin oil, glycerol monoStearate, talc, sodium ion, dried skim milk, mink oil, coconut oil, cocoa butter, olive oil, almond oil, glycerol, propylene, glycol, water, ethanol and the like. The macadamianut oil, aloa extract, jojoba oil, safflower oil, corn composition, if desired, can also contain minor amounts of oil, liquid lanolin, cottonseed oil, peanut oil, purcellin oil, wetting or emulsifying agents, or pH buffering agents. These perhydrosqualene (squalene), caster oil, polybutene, odorless compositions can take the form of, e.g., Solutions, Suspen 35 mineral spirits, Sweet almond oil, avocado oil, calophyllum sions, emulsion, powders, Sustained-release formulations and oil, ricin oil, vitamin E acetate, olive oil, mineral spirits, the like. Examples of suitable pharmaceutical carriers are cetearyl alcohol (mixture of fatty alcohols consisting pre described in “Remington’s Pharmaceutical Sciences” by E. dominantly of cetyl and Stearyl alcohols), linolenic alcohol, W. Martin. Some other examples of substances which can oleyl alcohol, octyl dodecanol, the oil of cereal germs such as serve as pharmaceutical carriers are Sugars, such as glucose 40 the oil of wheat germ cetearyl octanoate (ester of cetearyl and Sucrose; starches such as corn starch and potato starch; alcohol and 2-ethylhexanoic acid), cetyl palmitate, diisopro cellulose and its derivatives such as Sodium carboxymethyl pyl adipate, isopropyl palmitate, octyl palmitate, isopropyl cellulose, ethylcellulose and cellulose acetates; powdered myristate, butyl myristate, glyceryl Stearate, hexadecyl Stear tragacanth; malt, gelatin; talc.; Stearic acids; magnesium ate, isocetyl Stearate, octyl Stearate, octylhydroxy Stearate, Stearate, calcium sulfate; calcium carbonate; vegetable oils, 45 propylene glycol Stearate, butyl Stearate, decyl oleate, glyc Such as peanut oils, cotton seed oil, sesame oil, olive oil, corn eryl oleate, acetylglycerides, the octanoates and benzoates of oil and oil of theobroma; polyols such as propylene glycol, (C12-C15) alcohols, the octanoates and decanoates of alco glycerine, Sorbitol, mannitol, and polyethylene glycol, agar, hols and polyalcohols such as those of glycol and glycerol, alginic acids; pyrogen-free water, isotonic saline; cranberry and ricin-oleates of alcohols and poly alcohols such as those extracts and phosphate buffer solution; skim milk powder, as 50 of isopropyl adipate, hexyl laurate, octyl dodecanoate, dime well as other non-toxic compatible Substances used in phar thicone copolyol, dimethiconol, lanolin, lanolin alcohol, maceutical formulations such as Vitamin C, estrogen and lanolin wax, hydrogenated lanolin, hydroxylated lanolin, echinacea, for example. Wetting agents and lubricants such as acetylated lanolin, petrolatum, isopropyl lanolate, cetyl Sodium lauryl Sulfate, as well as coloring agents, flavoring myristate, glyceryl myristate, myristyl myristate, myristyl agents, lubricants, excipients, tabletting agents, stabilizers, 55 lactate, cetyl alcohol, isostearyl alcohol Stearyl alcohol, and anti-oxidants and preservatives, can also be present. It is also isocetyl lanolate, and the like. advantageous to administer the active ingredients in encap Moreover, the medicament according to the invention may Sulated form, e.g. as cellulose encapsulation, in gelatine, with also comprise emulsifiers. Emulsifiers (i.e., emulsifying polyamides, niosomes, wax matrices, with cyclodextrins or agents) are preferably used in amounts effective to provide liposomally encapsulated. Preferred pharmaceutical accept 60 uniform blending of ingredients of the composition. Useful able carriers are also lactose, terra alba, Sucrose, talc, gelatin, emulsifiers include (i) anionics such as fatty acid soaps, e.g., agar, pectin, acacia, magnesium Stearate, Stearic acid and the potassium Stearate, sodium Stearate, ammonium Stearate, and like as solid carriers and phosphate buffered saline solution, triethanolamine Stearate; polyol fatty acid monoesters con syrup, oil, water, emulsions, various types of wetting agents, taining fatty acid soaps, e.g., glycerol monostearate contain and the like as liquid carriers. The carriers shall be pharma 65 ing either potassium or Sodium salt; Sulfuric esters (sodium ceutically acceptable in that they shall not affect the biologi salts), e.g., sodium lauryl 5 sulfate, and Sodium cetyl sulfate; cal properties of other ingredients and, in particular, the pep and polyol fatty acid monoesters containing Sulfuric esters, US 9.220,672 B2 21 22 e.g., glyceryl monostearate containing Sodium lauryl Surfate; glycerin; honey; maltitol; methylgluceth-10; methylgluceth (ii) cationics chloride Such as N(Stearoyl colamino formylm 20; propylene glycol; sodium lactate; sucrose; and the like. ethyl) pyridium; N-soya-N-ethyl morpholinium ethosulfate: Of course, the medicament of the present invention can alkyl dimethyl benzyl ammonium chloride; diisobutylphe also comprise a preservative. Preservatives according to cer noxytheoxyethyl dimethylbenzyl ammonium chloride; and 5 tain compositions of the invention include, without limita cetyl pyridium chloride; and (iii) nonionics such as polyoxy tion: butylparaben; ethylparaben; imidazolidinyl urea; meth ethylene fatty alcohol ethers, e.g., monostearate; polyoxyeth ylparaben; O-phenylphenol; propylparaben; quaternium-14: ylene lauryl alcohol; polyoxypropylene fatty alcohol ethers, quaternium-15; Sodium dehydroacetate; Zinc pyrithione; and e.g., propoxylated oleyl alcohol; polyoxyethylene fatty acid the like. The preservatives are used in amounts effective to esters, e.g., polyoxyethylene Stearate; polyoxyethylene Sor 10 prevent or retard microbial growth. Generally, the preserva bitan fatty acid esters, e.g., polyoxyethylene Sorbitan tives are used in amounts of about 0.1% to about 1% by monostearate; Sorbitan fatty acid esters, e.g., Sorbitan; poly weight of the total composition with about 0.1% to about oxyethylene glycol fatty acid esters, e.g., polyoxyethylene 0.8% being preferred, and about 0.1% to about 0.5% being glycol monostearate; and polyol fatty acid esters, e.g., glyc most preferred. eryl monostearate and propylene glycol monostearate; and 15 The medicament according to the invention may also com ethoxylated lanolin derivatives, e.g., ethoxylated lanolins, prise a perfume. Perfumes (fragrance components) and colo ethoxylatedlanolin alcohols and ethoxylated cholesterol. The rants (coloring agents) well known to those skilled in the art selection of emulsifiers is exemplarily described in Schrader, may be used in effective amounts to impart the desired fra Grundlagen and Rezepturen der Kosmetika, Hüthig Buch grance and color to the compositions of the invention. Verlag, Heidelberg, 2" edition, 1989, 3' part. Furthermore, the medicament according to the present The medicament according to the invention may also invention may also comprise a wax. Suitable waxes which are include a surfactant. Suitable Surfactants may include, for useful in accord with the invention include: animal waxes, example, those Surfactants generally grouped as cleansing Such as beeswax, spermaceti, or wool wax (lanolin); plant agents, emulsifying agents, foam boosters, hydrotropes, solu waxes, such as carnauba or candelilla; mineral waxes, such as bilizing agents, Suspending agents and nonsurfactants (facili 25 montan wax or oZokerite; and petroleum waxes. Such as par tates the dispersion of Solids in liquids). The Surfactants are affin wax and microcrystalline wax (a high molecular weight usually classified as amphoteric, anionic, cationic and non petroleum wax). Animal, plant, and some mineral waxes are ionic Surfactants. Amphoteric Surfactants include acylamino primarily esters of a high molecular weight fatty alcohol with acids and derivatives and N-alkylamino acids. Anionic Sur a high molecular weight fatty acid. For example, the hexade factants include: acylamino acids and salts, such as, acyl 30 canoic acid ester of tricontanol is commonly reported to be a glutamates, acylpeptides, acylsarcosinates, and acyltaurates; major component of beeswax. Other Suitable waxes accord carboxylic acids and salts, such as, alkanoic acids, ester car ing to the invention include the synthetic waxes including boxylic acids, and ether carboxylic acids; Sulfonic acids and polyethylene polyoxyethylene and hydrocarbon waxes salts, such as, acyl isethionates, alkylaryl Sulfonates, alkyl derived from carbon monoxide and hydrogen. Representative Sulfonates, and SulfoSuccinates; Sulfuric acid esters, such as, 35 waxes also include: cerosin; cetyl esters; hydrogenatedjojoba alkyl ether sulfates and alkyl sulfates. Cationic surfactants oil; hydrogenated jojoba wax, hydrogenated rice bran wax: include: alkylamines, alkyl imidazolines, ethoxylated Japan wax, jojoba butter, jojoba oil; jojoba wax: munk wax: amines, and quaternaries (such as, alkylbenzyldimethylam montan acid wax; ouricury wax, rice bran wax; shellac wax: monium salts, alkyl betaines, heterocyclic ammonium salts, Sulfurized jojoba oil; synthetic beeswax, synthetic jojoba and tetra alkylammonium salts). And nonionic Surfactants 40 oils; trihydroxy Stearin; cetyl alcohol; Stearyl alcohol; cocoa include: alcohols, such as primary alcohols containing 8 to 18 butter, fatty acids of lanolin; mono-, di- and 25 triglycerides carbon atoms; alkanolamides such as alkanolamine derived which are solid at 25°C., e.g., glyceryl tribehenate (a triester amides and ethoxylated amides; amine oxides; esters such as of behenic acid and glycerine) and C19-C36 acid triglyceride ethoxylated carboxylic acids, ethoxylated glycerides, glycol (a mixture of triesters of C19-C36 carboxylic acids and glyc esters and derivatives, monoglycerides, polyglyceryl esters, 45 erine) available from Croda, Inc., New York, N.Y. under the polyhydric alcohol esters and ethers, sorbitan/sorbitol esters, tradenames Syncrowax. HRC and Syncrowax HGL-C, and triesters of phosphoric acid; and ethers such as ethoxy respectively; fatty esters which are solid at 25°C.: silicone lated alcohols, ethoxylated lanolin, ethoxylated polysilox waxes such as methyloctadecaneoxypolysiloxane and poly anes, and propoxylated polyoxyethylene ethers. (dimethylsiloxy) Stearoxysiloxane; Stearyl mono- and dietha Furthermore, the medicament according to the invention 50 nolamide; rosin and its derivatives such as the abietates of may also comprise a film former. Suitable film formers which glycol and glycerol: hydrogenated oils Solid at 25°C.; and are used in accord with the invention keep the composition Sucroglycerides. Thickeners (viscosity control agents) which Smooth and even and include, without limitation: acrylamide/ may be used in effective amounts inacqueous systems include: Sodium acrylate copolymer, ammonium acrylates copoly algin; carbomers such as carbomer 934, 934P 940 and 941; mer; Balsam Peru; cellulose gum; ethylene/maleic anhydride 55 cellulose gum, cetearyl alcohol, cocamide DEA, dextrin; copolymer; hydroxyethylcellulose; hydroxypropylcellulose; gelatin: hydroxyethylcellulose; hydroxypropylcellulose; polyacrylamide: polyethylene; polyvinyl alcohol; pVm/MA hydroxypropyl methylcellulose; magnesium aluminum sili copolymer (polyvinyl methylether/maleic anhydride); PVP cate; myristyl alcohol; oat flour, oleamide DEA, oleyl alco (polyvinylpyrrolidone); maleic anhydride copolymer Such as hol; PEG-7M; PEG-14.M.; PEG-90M: stearamide DEA: PA-18 available from Gulf Science and Technology; PVP/ 60 Stearamide MEA, Stearyl alcohol; tragacanth gum; wheat hexadecene copolymer such as Ganex V-216 available from starch; Xanthan gum; and the like in the above list of thick GAF Corporation; acryliclacrylate copolymer; and the like. eners, DEA is diethanolamine, and MEA is monoethanola Generally, film formers can be used in amounts of about 0.1% mine. Thickeners (viscosity control agents) which may be to about 10% by weight of the total composition with about used in effective amounts in nonaqueous systems include 1% to about 8% being preferred and about 0.1% to about 5% 65 aluminum Stearates; beeswax, candelilla wax, carnauba; cer being most preferred. Humectants can also be used in effec esin: cetearyl alcohol; cetyl alcohol; cholesterol: hydrated tive amounts, including: fructose; glucose, glulamic acid; silica; hydrogenated castor oil; hydrogenated cottonseed oil; US 9.220,672 B2 23 24 hydrogenated soybean oil; hydrogenated tallow glyceride; The medicament according to the invention can be formu hydrogenated vegetable oil; hydroxypropyl cellulose; lanolin lated as neutral or salt forms. Pharmaceutically acceptable alcohol; myristyl alcohol; octytdodecyl stearoylsulfate; oleyl salts include those formed with anions such as those derived alcohol; oZokerite; microcystalline wax, paraffin, pentaeryth from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, rity1 tetraoctanoate; polyacrylamide; polybutene; polyethyl etc., and those formed with cations such as those derived from ene; propylene glycol dicaprylate; propylene glycol dipelar Sodium, potassium, ammonium, calcium, ferric hydroxides, gonate; Stearalkonium hectorite: Stearyl alcohol; Stearyl isopropylamine, triethylamine, 2-ethylamino ethanol, histi Stearate; synthetic beeswax, trihydroxy Stearin; trilinolein; dine, procaine, etc. Forms Suitable for topical application tristearin: Zinc Stearate; and the like. Customary native and include, e.g., a paste, an ointment, a lotion, a cream, a gel or synthetic thickeners or gel formers in formulations are 10 a transdermal patch. A Suitable paste comprises, e.g., petro crosslinked polyacrylic acids and derivatives thereof, leum, Soft white paraffin, yellow petroleum jelly and glycerol. polysaccharides, such as Xanthane gum or alginates, car A Suitable ointment comprises the peptide as active ingredi boxymethylcellulose or hydroxycarboxymethylcellulose, ent Suspended or dissolved in a carrier Such as, one or more of hydrocolloids such as gum Arabic or montmorillonite miner 15 glycerol, mineral oil, liquid oil, liquid petroleum, white petro als. Such as bentonites or fatty alcohols, polyvinyl alcohol and leum, yellow petroleum jelly, propylene glycol, alcohols, polyvinlypyrrolidone. triglycerides, fatty acid esters such as cetyl ester, polyoxyeth Other ingredients which can be added or used in medica ylene polyoxypropylene compound, waxes such as white wax ment according to the invention in amounts effective for their and yellow beeswax, fatty acid alcohols such as cetyl alcohol, intended use, include: biological additives to enhance perfor Stearyl alcohol and cetylstearylalcohol, fatty acids such as mance or consumer appeal Such as amino acids, proteins, Stearic acid, cetyl Stearate, lanolin, magnesium hydroxide, vanilla, aloe extract, bioflavinoids, and the like; buffering kaolin and water. Alternatively, the medicament may also be agents, chelating agents such as EDTA; emulsion stabilizers; formulated a lotion or cream comprising the active compo pH adjusters; opacifying agents; and propellants such as nents suspended or dissolved in a carrier. Such carriers butane carbon dioxide, ethane, hydrochlorofluorocarbons 22 25 include, but are not limited to, one or more of mineral oil such and 142b, hydrofluorocarbon 152a, isobutane, isopentane, as paraffin, vegetable oils such as castor oil, castor seed oil nitrogen, nitrous oxide, pentane, propane, and the like. and hydrogenated castor oil, Sorbitan monostearat, polysor Furthermore, the medicament according to the invention bat, fatty acid esters such as cetyl ester, wax, fatty acid alco may also comprise compounds which have an antioxidative, hols such as cetyl alcohol, Stearyl alcohol, 2-octyldodecanol, free-radical scavenger, skin moisturizing or moisture-retain 30 benzyl alcohol, alcohols, triglycerides and water. Alterna ing, antierythematous, antiinflammatory or antiallergic tively, the medicament may also be formulated with a suitable action, in order to supplement or enhance their action. In gel comprising the active components suspended or dissolved particular, these compounds can be chosen from the group of in a carrier. Such carriers include, but are not limited to, one Vitamins, plant extracts, alpha- and beta-hydroxy acids, cera or more of water, glycerol, propyleneglycol, liquid paraffin, mides, antiinflammatory, antimicrobial or UV-filtering sub 35 polyethylene, fatty oils, cellulose derivatives, bentonite and stances, and derivatives thereof and mixtures thereof. Advan colloidal silicon dioxide. tageously, preparations according to the invention can also It is to be understood that the formulation of a medicament comprise substances which absorb UV radiation in the UV-B preferably takes place under GMP standardized conditions in and/or UV-A region. The lipid phase is advantageously cho order to ensure quality, pharmaceutical security, and effec Sen from the group of Substances of mineral oils, mineral 40 tiveness of the medicament. Further criteria for an ingredient waxes, branched and/or unbranched hydrocarbons and being pharmaceutically acceptable can be derived from hydrocarbon waxes, triglycerides of Saturated and/or unsat approval regulations by a regulatory agency or other gener urated, branched and/or unbranched C8-C24-alkanecarboxy ally recognized pharmacopoeias. lic acids; they can be chosen from synthetic, semisynthetic or The medicament referred to herein is, preferably, admin natural oils, such as olive oil, palm oil, almond oil or mix 45 istered topically. However, a systemic administration is also tures; oils, fats or waxes, esters of Saturated and/or unsatur feasible. For systemic administration, the medicament can be ated, branched and/or unbranched C3-C30-alkane carboxylic applied intra-muscular, Subcutaneous or intravenous. Other acids and Saturated and/or unsaturated, branched and/or routes of administration are, however, also possible. unbranched C3-C30-alcohols, from aromatic carboxylic The medicament shall, preferably, provide to a subject in acids and Saturated and/or unsaturated, branched and/or 50 need thereofatherapeutically effective dose of the peptide of unbranched C3-C30-alcohols, for example isopropyl the invention. A therapeutically or prophylactically effective myristate, isopropyl Stearate, hexyldecyl Stearate, oleyl ole dose refers to an amount of the peptide which in a subject in ate; and also synthetic, semisynthetic and natural mixtures of need thereof prevents, ameliorates or treats the symptoms Suchesters, such as jojoba oil, alkylbenzoates or silicone oils, accompanying a disease or condition referred to in this speci Such as, for example, cyclomethicone, dimethylpolysiloxane, 55 fication. Therapeutic or prophylactic efficacy and toxicity of diethylpolysiloxane, octamethylcyclo-tetrasiloxane and mix the compound can be determined by standard pharmaceutical tures thereof or dialkyl ethers. procedures in cell cultures or experimental animals, e.g., The medicament may be formulated dependent on the ED50 (the dose therapeutically effective in 50% of the popu desired mode of administration. These procedures may lation) and LD50 (the dose lethal to 50% of the population). involve mixing, granulating, and compression, or dissolving 60 The dose ratio between therapeutic and toxic effects is the the ingredients as appropriate to the desired preparation. It therapeutic index, and it can be expressed as the ratio, LD50/ will be appreciated that the form and character of the phar ED50. A therapeutically or prophylactically effective dose maceutical acceptable carrier is dictated by the amount of can be provided at once, i.e., as a single bolus or by repeated active ingredient with which it is to be combined, the route of administration of the medicament. The dosages are, prefer administration, and other well-known variables. Details are 65 ably, given once a week, more preferably 2 times, 3 times, 4 well known in the art, see, e.g., Remington's Pharmaceutical times, 5 times or 6 times a week and most preferably daily and Sciences, Mack Publishing Company, Easton, Pa. even more preferably, 2 times a day or more often. US 9.220,672 B2 25 26 The dosage regimen for the medicament referred to herein disorders within a prophylactic time window for a statistically will be determined by an attending physician and clinical significant portion of Subjects to be subjected to the prophy factors. AS is well known in the medical arts, dosages for an laxis. Maintaining health with respect to the diseases or dis individual depend upon many factors, including the patients orders referred to herein can be assessed by monitoring a size, body Surface area, age, the particular compound to be Subject for the presence or absence of symptoms accompa administered, sex, time and route of administration, general nying the diseases or disorders and/or clinical parameter health, and other drugs being administered concurrently. shifts indicative for the diseases or disorders during the pro Dosage recommendations shall be indicated in the prescrib phylactic time window. Whether a portion is statistically sig ers or users instructions in order to anticipate dose adjust nificant can be determined as specified elsewhere herein. ments depending on the considered recipient. 10 Preferred prophylactic time windows as used herein are up to Preferably, the medicament is administered directly or in 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, up to 2 combination with an adjuvant. Adjuvants may be selected weeks, up to 1 week, up to 2 days, or at least 1 day. from the group consisting of a chloroquine, protic polar com A subject as referred to herein refers to an animal, prefer pounds, Such as propylene glycol, polyethylene glycol, glyc ably a mammal, more preferably a human, farming animal, erol, EtOH, 1-methyl L-2-pyrrolidone or their derivatives, or 15 Such as a cow, pig, horse, sheep, or goat or a pet. Such as a cat aprotic polar compounds such as dimethylsulfoxide or dog. Most preferably, the Subject is a human. (DMSO), diethylsulfoxide, di-n-propylsulfoxide, dimethyl The present invention also contemplates following from Sulfone, Sulfolane, dimethylformamide, dimethylacetamide, the above a method for treating of a pro-inflammatory con tetramethylurea, acetonitrile or their derivatives. These com dition or disease in a Subject, said method comprising admin pounds are added in conditions respecting pH limitations. istering to a subject Suffering from the aforementioned con The present invention in particular provides a pharmaceu dition or disease a therapeutically effective dose of the tical composition comprising the peptide of the invention and, peptide of the invention. preferably, one or more pharmaceutically acceptable carriers Moreover, contemplated by the present invention is a or excipients. Preferably, said composition comprises the method for prophylaxis of a pro-inflammatory condition or peptide in a concentration of from 0.01 to 100 mg/ml, pref 25 disease in a Subject, said method comprising administering to erably of from 0.05 to 50 mg/ml, more preferably of from 0.1 an apparently healthy Subject a prophylactically effective to 10 mg/ml, more preferably of from 0.2 to 5 mg/ml, and dose of the peptide of the invention. even more preferably of from 0.5 to 2 mg/ml. In a preferred embodiment of these methods referred to It has been found in the studies underlying the present above, the pro-inflammatory condition or disease is inflam invention that the peptide of the invention effectively inhibits 30 mation of epithelial tissues, allergies, allergic reactions, rash, the secretion of pro-inflammatory substances, preferably of and/or rheumatoid arthritis, more preferably for the treatment pro-inflammatory cytokines, more preferably of Interleu and/or prophylaxis of inflammation, allergies, and/or allergic kin-1 (IL-1), and even more preferably of Interleukin-1 alpha reactions of the epidermis, more preferably for the treatment (IL 1-alpha). Preferably, the secretion of such pro-inflamma and/or prophylaxis of dermatitis, and even more preferably tory Substances is inhibited in epithelial cells and, in particu 35 for treatment and/or prophylaxis of atopic dermatitis, Sebor lar, in the keratinocytes of the skin. rhoeic dermatitis, psoriasis, poison-ivy dermatitis, eczema Accordingly, the peptide orpharmaceutical composition of herpeticum, kerion, diaper rash, or scabies. the invention is to be used, preferably, as a medicament for the It will be understood that the methods are, moreover, appli treatment and/or prophylaxis of inflammation of epithelial cable to both human therapy and veterinary applications. tissues, allergies, allergic reactions, rash, and/or rheumatoid 40 The present invention, furthermore, contemplates a arthritis, more preferably for the treatment and/or prophy method for the manufacture of a medicament comprising the laxis of inflammation, allergies, and/or allergic reactions of step of formulating the peptide of the invention in a pharma the epidermis, more preferably for the treatment and/or pro ceutically acceptable form. phylaxis of dermatitis, and even more preferably for treat Details on how the peptide can be formulated in a pharma ment and/or prophylaxis of atopic dermatitis, Seborrhoeic 45 ceutically acceptable form are described elsewhere herein in dermatitis, psoriasis, poison-ivy dermatitis, eczema herpeti detail. cum, kerion, diaper rash, or scabies. It has been, furthermore, found in accordance with the The aforementioned diseases and disorders are well known present invention that the peptide of the invention is capable to the skilled artisan and the symptoms and clinical param of affecting the growth of microorganisms which are part of eters accompanying them are described in standard textbooks 50 the microflora on skin. Specifically, the growth of pathogenic of medicine such as Stedman or Pschyrembl. bacteria was found to be inhibited specifically while normal The term “treatment as used herein refers to ameliorating healthy microorganisms which may be present on skin were the symptoms and/or clinical parameters accompanying a stimulated in growth. This effect of the peptide of the inven disease or disorder referred to above in a statistically signifi tion is a direct effect on the bacteria of the microflora and shall cant portion of the subject to be treated by the medicament 55 be independent of any effects mediated by the skin of a and to an extent which becomes apparent in that the improve subject which harbors the said microflora. Thus, by directly ment of the symptoms or clinical parameters is statistically affecting the microflora the peptide of the present invention significant, too. Preferably, the term also encompasses the elicits a probiotic effect in that it increases the probiotic entire restoration of health in a subject to be treated with bacteria in the microflora at the expense of the pathogenic respect to the aforementioned diseases or disorders. How to 60 bacteria. As a consequence, the overall well being and health determine whether a statistically significant portion can be ofa Subject will improve due to the generation of a essentially Successfully treated or whetheran improvement of symptoms probiotic microflora on the skin. or clinical parameters is statistically significant can be deter Therefore, the peptide of the invention is also to be used for mined by the skilled person without furtherado by applying inhibiting the growth of transient pathogenic skin microflora, standard statistics such as Student's t-test and the like. 65 preferably for inhibiting the growth of transient pathogenic The term “prophylaxis' as used herein refers to maintain skin microflora belonging to the Staphylococcus genus, and ing health with respect to the aforementioned diseases or preferably for inhibiting the growth of S. aureus. Further, it is US 9.220,672 B2 27 28 to be used for stimulating the growth of healthy normal resi provided for non-therapeutic use, wherein it is preferably dent skin microflora, preferably for stimulating the growth of administered to a subject for cosmetic purposes. healthy normal resident skin microflora belonging to the Sta The term "cosmetic composition” as used in accordance phylococcus genus, and preferably for stimulating the growth with the present invention, relates to one or more composi of S. epidermidis. tions which comprise at least one tetrapeptide of the present The term “transient pathogenic skin microflora’ as used invention as described above. It is envisaged that the compo herein refers to microorganisms which are transiently present sitions of the present invention which are described herein on the Surface of the skin. The presence of said microorgan below comprise the one or more further ingredients to the isms on the skin shall be harmful for the subject in that the inventive peptide in any combination. Preferably, the cos microorganisms itselfelicit pathogenic process in the Subject 10 metic compositions of the present invention may comprise at or destroy beneficial microorganism Such as microorganisms least one further ingredient suitable for stimulating the of a probiotic microflora referred to elsewhere herein. Pref growth of healthy normal resident skin microflora and/or for erably, the microorganisms are bacteria. More preferably, the protecting the skin against pathogenic microorganisms, and said bacteria are of the Staphylococcus genus and, even more preferably for inhibiting the growth of transient pathogenic preferably, S. aureus. 15 skin microflora. According to a particularly preferred The term “healthy normal resident skin microflora’ as used embodiment, the term “ingredients suitable for protecting the herein refers to microorganisms which are resident on the skin against pathogenic microorganisms' designates com surface of the skin of a subject and which are not harmful for pounds or compositions and/or combinations thereof which the subject. Preferably, the microorganisms are probiotic lower the pH and/or maintain a low pH level of the skin, microorganisms which protect the Subject from pathogenic wherein the term “low pH level of the skin' preferably refers microorganisms or which actively improve health and well to a pH level of between 6 and 7. being of the subject. Preferably, the microorganisms are bac In general, there is no particular restriction as to the amount teria. More preferably, the said bacteria are of the Staphylo of inventive peptide which may be contained in the cosmetic coccus genus and, even more preferably, S. epidermidis. composition of the present invention. Thus, by way of The term “inhibiting the growth' as used herein refers to a 25 example, the cosmetic composition may contain the inventive reduction of the growth rate of the bacteria. Preferably, the peptide in a concentration ranging anywhere from 0.005 to 20 growth is statistically significant inhibited. Preferably, a mg/ml, wherein according to preferred embodiments, the reduction of the growth rate envisaged by the present inven content thereofranges 0.01 to 10 mg/ml, preferably from 0.05 tion is a reduction of at least 20%, at least 30%, at least 40%, to 5 mg/ml, more preferably from 0.1 to 2 mg/ml, and even at least 50%, at least 75%, at least 80% or at least 90%. The 30 more preferably from 0.2 to 1 mg/ml. Within the meaning of growth rate for bacteria can be determined by the skilled the present invention the concentration given in mg/ml indi person using methods well known in the art without further cates the content of the inventive peptide in the composition, ado. wherein according to preferred embodiments wherein the The term “stimulating the growth refers to an increase in peptide is comprised in a larger polypeptide or protein the the growth rate. Preferably, said increase is statistically sig 35 content is based on the weight of the peptide of the present nificant. Preferably, a stimulation of growth is characterized invention contained therein and not on the weight of the by an increase in the growth rate of at least 20%, at least 30%, polypeptide or protein in which it is contained. at least 40%, at least 50%, at least 75%, at least 80%, at least In addition, the present invention relates to the use of a 90% or at least 100%. peptide as described above for the preparation of a cosmetic The peptide is administered in accordance with the afore 40 composition. In particular, the present invention provides a mentioned uses to the skin of a subject comprising pathogenic method for the production of a cosmetic composition com skin microflora or Suspected to comprise Such a microflora. prising the steps of formulating a tetrapeptide as described Dosages for the peptide which are effective for inhibiting the above with one or more cosmetically acceptable carriers or growth of pathogenic microflora or which stimulate the excipients. growth of healthy normal resident microflora can be deter 45 Therefore, the present invention also relates to a cosmetic mined as described elsewhere herein. Preferably, the peptide composition which further comprises one or more cosmeti is to be administered once a week, more preferably 2 times, 3 cally and/or pharmaceutically acceptable carriers or excipi times, 4 times, 5 times or 6 times a week and most preferably entS. daily and even more preferably, 2 times a day or more often. In general, the cosmetic compositions of the invention may In particular, it may be preferable to give a dosage each time 50 comprise any further components which are suitable for their after a disturbance of the resident skin flora occurred, e.g. by cosmetic application. In particular, said compositions may washing. However, during progression of the treatment the comprise one or more of the aforementioned pharmaceuti dosages can be given in much longer time intervals and in cally acceptable carriers, stabilizers, Sustained release agents, need can be given in much shorter time intervals, e.g., several emollients, emulsifiers, Surfactants, film formers, preserva times a day. In a preferred case the immune response is 55 tives, perfumes, waxes, and other ingredients including bio monitored using herein described methods and further meth logical additives to enhance performance or consumer ods known to those skilled in the art and dosages are opti appeal, buffering agents, chelating agents such as EDTA, mized, e.g., in time, amount and/or composition. Progress can emulsion stabilizers, pH adjusters, opacifying agents, and be monitored by periodic assessment. It is also envisaged that propellants, as described in the foregoing with respect to the pharmaceutical compositions are employed in co-therapy 60 embodiments and preferred embodiments of the medicament approaches, i.e. in co-administration with other medicaments and pharmaceutical composition of the present invention. or drugs, for example other drugs for protecting skin against In addition thereto, the cosmetic composition of the present pathogenic microorganisms. invention may also comprise further auxiliaries as are cus In addition to the aforementioned, the present invention tomarily used in Such preparations such as one or more of further relates to a cosmetic composition comprising the pep 65 antifoams, dyes, pigments, thickeners, Surface-active Sub tide of the invention. In particular, according to these embodi stances, finishing agents, fats, oils, and other customary con ments of the present invention, the cosmetic composition is stituents, of a cosmetic or dermatological formulation, Such US 9.220,672 B2 29 30 as alcohols, polyols, polymers, foam stabilizers, Solubility more of water, glycerol, propyleneglycol, liquid paraffin, promoters, electrolytes, organic acids, organic solvents, or polyethylene, fatty oils, cellulose derivatives, bentonite and silicone derivatives. According to a particularly preferred colloidal silicon dioxide. embodiment, said compositions are in the form of emulsions, According to preferred embodiments, the cosmetic com e.g. oil in water or water in oil emulsions, in the form of 5 position further comprises emollients. Emollients may be ointments or in the form of micro-capsules and/or liposomes. used in amounts which are effective to prevent or relieve In general, the cosmetic and pharmaceutical compositions dryness. More preferably, emollients used in the cosmetic may be provided in any form suitable for their respective compositions of the present invention include one or more of application. Thus, by way of example, the compositions may hydrocarbon oils and waxes; silicone oils; triglyceride esters; 10 acetoglyceride esters; ethoxylated glyceride; alkyl esters; be in Solid, liquid or gaseous form and may be, interalia, in alkenyl esters; fatty acids; fatty alcohols; fatty alcohol ethers: the form of (a) powder(s), (a) solution(s) (an) aerosol(s), etheresters; lanolin and derivatives; polyhydric alcohols Suspensions, emulsions, liquids, elixirs, extracts, tincture or (polyols) and polyether derivatives; polyhydric alcohol fluid extracts. According to preferred embodiments, the com (polyol) esters; wax esters; beeswax derivatives; vegetable positions of the invention are in a form which is suitable for 15 waxes; phospholipids; sterols; and amides. topical administration. Forms suitable for topical application Thus, preferred emollients include mineral oil, especially include, a paste, an ointment, a lotion, a cream, a gel or a mineral oils having a viscosity in the range of 50 to 500 SUS transdermal patch. (Saybolt Universal Second), lanolin oil, mink oil, coconut oil, Preferably, the composition of the present invention is a cocoa butter, olive oil, almond oil, macadamia nut oil, aloa cosmetic composition further comprising a cosmetically extract, jojoba oil, safflower oil, corn oil, liquid lanolin, cot acceptable carrier or excipient. More preferably, said cos tonseed oil, peanut oil, purcellin oil, perhydrosqualene metic composition is a paste, an ointment, a lotion, a cream or (squalene), caster oil, polybutene, odorless mineral spirits, a gel. In particular, the cosmetic composition of the present Sweet almond oil, avocado oil, calophyllum oil, ricin oil, invention comprises the inventive peptide in connection with Vitamin E acetate, olive oil, mineral spirits, cetearyl alcohol the composition of the invention and further a cosmetically 25 (mixture of fatty alcohols consisting predominantly of cetyl acceptable carrier. and Stearyl alcohols), linolenic alcohol, oleyl alcohol, octyl Within the meaning of the present invention, the term “cos dodecanol, the oil of cereal germs such as the oil of wheat metically acceptable carrier” means any Suitable vehicle, germ cetearyl octanoate (ester of cetearyl alcohol and 2-eth which can be used to apply the present compositions to the ylhexanoic acid), cetylpalmitate, diisopropyladipate, isopro 30 pyl palmitate, octyl palmitate, isopropyl myristate, butyl skin in a safe and effective manner. Such vehicle may include myristate, glyceryl Stearate, hexadecyl Stearate, isocetyl materials such as emulsions, e.g. oil in water or water in oil Stearate, octyl stearate, octylhydroxy stearate, propylene gly emulsions, ointments or micro capsules, it is also advanta col Stearate, butyl Stearate, decyl oleate, glyceryl oleate, geous to administer the active ingredients in encapsulated acetylglycerides, the octanoates and benzoates of (C12-C15) form, e.g. as cellulose encapsulation, in gelatine, with polya 35 alcohols, the octanoates and decanoates of alcohols and poly mides, niosomes, wax matrices, with cyclodextrins or lipo alcohols such as those of glycol and glycerol, and ricin somally encapsulated. The term "safe and effective amount oleates of alcohols and poly alcohols such as those ofisopro as used herein preferably means a Sufficient amount to stimu pyl adipate, hexyl laurate, octyl dodecanoate, dimethicone late growth of at least one microorganism of the resident skin copolyol, dimethiconol, lanolin, lanolin alcohol, lanolin wax, microbial flora. 40 hydrogenated lanolin, hydroxylated lanolin, acetylated lano According to the present invention, a suitable paste as a lin, petrolatum, isopropyllanolate, cetyl myristate, glyceryl carrier comprises the inventive peptide Suspended in a carrier. myristate, myristyl myristate, myristyl lactate, cetyl alcohol, Such carriers include, but are not limited to, petroleum, soft isostearyl alcohol Stearyl alcohol, and isocetyl lanolate, and white paraffin, yellow petroleum jelly and glycerol. the like. The cosmetic composition may also be formulated with a 45 According to further preferred embodiments, the cosmetic Suitable ointment comprising the active components sus composition according to the invention comprises one or pended or dissolved in a carrier. Such carriers include, but are more emulsifiers. In particular, the emulsifiers (i.e., emulsi not limited to, one or more of glycerol, mineral oil, liquid oil, fying agents) are preferably used in amounts effective to liquid petroleum, white petroleum, yellow petroleum jelly, provide uniform blending of ingredients of the composition. propylene glycol, alcohols, triglycerides, fatty acid esters 50 Preferred emulsifiers include one or more of (i) anionics such Such as cetyl ester, polyoxyethylene polyoxypropylene com as fatty acid soaps, e.g., potassium Stearate, Sodium Stearate, pound, waxes such as white wax and yellow beeswax, fatty ammonium Stearate, and triethanolamine Stearate; polyol acid alcohols such as cetyl alcohol, Stearyl alcohol and cetyl fatty acid monoesters containing fatty acid soaps, e.g., glyc Stearylalcohol, fatty acids such as Stearic acid, cetyl Stearate, erol monostearate containing either potassium or Sodium salt; lanolin, magnesium hydroxide, kaolin and water. Alterna 55 Sulfuric esters (sodium salts), e.g., sodium lauryl 5 sulfate, tively, the cosmetic composition may also be formulated with and Sodium cetyl Sulfate; and polyol fatty acid monoesters a Suitable lotion or cream comprising the active components containing Sulfuric esters, e.g., glyceryl monostearate con suspended or dissolved in a carrier. Such carriers include, but taining sodium lauryl Surfate; (ii) cationics chloride Such as are not limited to, one or more of mineral oil such as paraffin, N(stearoyl colamino formylmethyl) pyridium; N-soya-N- Vegetable oils such as castor oil, castor seed oil and hydroge 60 ethyl morpholinium ethosulfate; alkyl dimethyl benzyl nated castor oil, Sorbitan monostearat, polysorbat, fatty acid ammonium chloride; diisobutylphenoxytheoxyethyl dim esters such ascetyl ester, wax, fatty acid alcohols such as cetyl ethylbenzyl ammonium chloride; and cetyl pyridium chlo alcohol, Stearyl alcohol, 2-octyldodecanol, benzyl alcohol, ride; and (iii) nonionics such as polyoxyethylene fatty alcohol alcohols, triglycerides and water. Alternatively, the cosmetic ethers, e.g., monostearate; polyoxyethylene lauryl alcohol; composition may also be formulated with a suitable gelcom 65 polyoxypropylene fatty alcohol ethers, e.g., propoxylated prising the active components Suspended or dissolved in a oleyl alcohol; polyoxyethylene fatty acid esters, e.g., poly carrier. Such carriers include, but are not limited to, one or oxyethylene Stearate; polyoxyethylene Sorbitan fatty acid US 9.220,672 B2 31 32 esters, e.g., polyoxyethylene Sorbitan monostearate; Sorbitan embodiments, one or more humectants are comprised in the fatty acid esters, e.g., Sorbitan; polyoxyethylene glycol fatty inventive cosmetic composition, wherein said humectants are acid esters, e.g., polyoxyethylene glycol monostearate; and preferably selected from the group consisting of fructose; polyol fatty acid esters, e.g., glyceryl monostearate and pro glucose:glulamic acid; glycerin; honey; maltitol; methylglu pylene glycol monostearate; and ethoxylated lanolin deriva- 5 ceth-10; methyl gluceth-20; propylene glycol; sodium lac tives, e.g., ethoxylated lanolins, ethoxylated lanolin alcohols tate. Sucrose; and combinations thereof. and ethoxylated cholesterol. The selection of emulsifiers is It is further preferred according to the invention that the exemplarly described in Schrader, Grundlagen and Rezep cosmetic composition comprises one or more preservatives. turen der Kosmetika, Hüthig Buch Verlag, Heidelberg, 2" More preferably, the one or more preservatives comprise one edition, 1989, 3" part. 10 or more of butylparaben; ethylparaben; imidazolidinyl urea; According to further preferred embodiments of the present methylparaben; O-phenylphenol; propylparaben; quater invention, the cosmetic composition comprises a Surfactant. nium-14; quaternium-15; Sodium dehydroacetate; and Zinc Suitable surfactants include, for example, those Surfactants pyrithione. generally grouped as cleansing agents, emulsifying agents, In general, the preservatives may be used in any amount foam boosters, hydrotropes, Solubilizing agents, Suspending 15 which is effective to prevent or retard microbial growth. Pref agents and nonSurfactants (facilitates the dispersion of solids erably, the preservatives are used in an amount ranging from in liquids). 0.1% to 1% by weight of the total composition, more prefer In general, Surfactants are usually classified as amphoteric, ably from 0.1% to 0.8% by weight, and even more preferably anionic, cationic and nonionic Surfactants, wherein one or from 0.1% to 0.5% by weight. more of said classes of surfactants are preferably used in the 20 The cosmetic composition according to the invention may cosmetic composition of the invention. In particular, pre also further comprise a perfume. Perfumes (fragrance com ferred amphoteric Surfactants include one or more acylamino ponents) and colorants (coloring agents) well known to those acids and derivatives and N-alkylamino acids. Furthermore, skilled in the art may be used in effective amounts to impart preferred anionic surfactants include one or more of the fol the desired fragrance and color to the compositions of the lowing: acylamino acids and salts, such as, acylglutamates, 25 invention. acylpeptides, acylsarcosinates, and acyltaurates; carboxylic According to embodiments of the present invention which acids and salts, such as, alkanoic acids, ester carboxylic acids, are further preferred, the cosmetic composition comprises and ether carboxylic acids; Sulfonic acids and salts, such as, one or more waxes. Preferred waxes include one or more of acyl isethionates, alkylaryl Sulfonates, alkyl Sulfonates, and the following: animal waxes, such as beeswax, and preferably SulfoSuccinates; Sulfuric acid esters, such as, alkyl ether Sul- 30 hexadecanoic acid ester of tricontanol contained therein, fates and alkyl sulfates. Furthermore, preferred cationic sur spermaceti, or wool wax (lanolin); plant waxes. Such as car factants include one or more of the following: alkylamines, nauba or candelilla; mineral waxes, such as montan wax or alkyl imidazolines, ethoxylated amines, and quaternaries oZokerite; and petroleum waxes, such as paraffin wax and (such as, alkylbenzyldimethylammonium salts, alkyl microcrystalline wax (a high molecular weight petroleum betaines, heterocyclic ammonium salts, and tetra alkylammo- 35 wax). Alternatively or in addition to these, one or more Syn nium salts). In addition to these, preferred nonionic Surfac thetic waxes may be used in the cosmetic composition, tants include one or more of the following: alcohols, such as wherein said one or more synthetic waxes preferably include primary alcohols containing 8 to 18 carbon atoms; alkanola polyethylene, polyoxyethylene, and hydrocarbon waxes mides such as alkanolamine derived amides and ethoxylated derived from carbon monoxide and hydrogen, and combina amides; amine oxides; esters such as ethoxylated carboxylic 40 tions of two or more thereof. acids, ethoxylated glycerides, glycol esters and derivatives, In particular, preferred waxes which may be used in the monoglycerides, polyglyceryl esters, polyhydric alcohol cosmetic composition of the present invention include one or esters and ethers, sorbitan/sorbitol esters, and triesters of more of cerosin; cetyl esters; hydrogenatedjojoba oil; hydro phosphoric acid; and ethers such as ethoxylated alcohols, genatedjojoba wax, hydrogenated rice bran wax; Japan wax: ethoxylatedlanolin, ethoxylated polysiloxanes, and propoxy- 45 jojoba butter, jojoba oil; jojoba wax: munk wax: montan acid lated polyoxyethylene ethers. wax; ouricury wax, rice bran wax; shellac wax; Sulfurized Furthermore, a cosmetic composition is preferred accord jojoba oil; synthetic beeswax, synthetic jojoba oils; trihy ing to the invention which comprises a film former. In general, droxyStearin; cetyl alcohol; Stearyl alcohol; cocoa butter; film formers which are used in accord with the invention fatty acids of lanolin; mono-, di- and triglycerides which are preferably keep the composition Smooth and even. According 50 solid at 25°C., e.g., glyceryl tribehenate (a triester of behenic to the present invention, preferred film formers include one or acid and glycerine) and C19-C36 acid triglyceride (a mixture more of the following: acrylamide/sodium acrylate copoly of triesters of C19-C36 carboxylic acids and glycerine) avail mer, ammonium acrylates copolymer; Balsam Peru; cellu able from Croda, Inc., New York, N.Y. under the tradenames lose gum, ethylene/maleic anhydride copolymer; hydroxy Syncrowax. HRC and Syncrowax HGL-C, respectively; fatty ethylcellulose; hydroxypropylcellulose; polyacrylamide; 55 esters which are solid at 25°C.: silicone waxes such as methy polyethylene; polyvinyl alcohol; pVm/MA copolymer (poly loctadecaneoxypolysiloxane and poly(dimethylsiloxy) Stear vinyl methylether/maleic anhydride); PVP (polyvinylpyrroli oxysiloxane; Stearyl mono- and diethanolamide; rosin and its done); maleic anhydride copolymer such as PA-18 available derivatives such as the abietates of glycol and glycerol; from Gulf Science and Technology; PVP/hexadecene hydrogenated oils solid at 25°C.; and Sucroglycerides. copolymer such as Ganex V-216 available from GAF Corpo- 60 Furthermore, cosmetic compositions are preferred accord ration; and acryliclacrylate copolymer. ing to the present invention which comprise one or more Generally, the film formers can be used in any suitable thickeners (viscosity control agents). Preferred thickeners amount. By way of example, the film formers may be used in used in cosmetic compositions of the present invention which the inventive cosmetic composition in amounts ranging any are in the form of a solution comprise one or more of algin; where from 0.1% to 10% by weight of the total composition, 65 carbomers such as carbomer 934, 934P 940 and 941; cellu wherein preferably 1% to 8% are contained therein, more lose gum, cetearyl alcohol, cocamide DEA, dextrin; gelatin: preferably 0.1% to 5%. According to further preferred hydroxyethylcellulose; hydroxypropylcellulose; hydrox US 9.220,672 B2 33 34 ypropyl methylcellulose; magnesium aluminum silicate; selected from the group consisting of olive oil, palm oil, myristyl alcohol; oat flour; oleamide DEA. oleyl alcohol: almond oil; fats or waxes, esters of Saturated and/or unsatur PEG-7M; PEG-14M, PEG-9OM: stearamide DEA; Steara ated, branched and/or unbranched C3-C30-alkane carboxylic mide MEA: Stearyl alcohol; tragacanth gum, wheat starch; acids and Saturated and/or unsaturated, branched and/or xanthan gum; wherein DEA is diethanolamine, and MEA is unbranched C3-C30-alcohols, from aromatic carboxylic monoethanolamine. Alternatively or in addition thereto, acids and Saturated and/or unsaturated, branched and/or thickeners used in cosmetic compositions of the present unbranched C3-C30-alcohols, for example isopropyl invention which are in the form of a nonaqueous system myristate, isopropyl Stearate, hexyldecyl Stearate, oleyl ole preferably comprise one or more of aluminum Stearates; ate; and also synthetic, semisynthetic and natural mixtures of beeswax, candelilla wax, carnauba; ceresin: cetearyl alcohol; 10 Suchesters, such as jojoba oil, alkylbenzoates or silicone oils, cetyl alcohol; cholesterol: hydrated silica; hydrogenated cas Such as, for example, cyclomethicone, dimethylpolysiloxane, tor oil; hydrogenated cottonseed oil; hydrogenated Soybean diethylpolysiloxane, octamethylcyclo-tetrasiloxane and mix oil, hydrogenated tallow glyceride; hydrogenated vegetable tures thereof or dialkyl ethers. oil, hydroxypropyl cellulose; lanolin alcohol; myristyl alco In general, the cosmetic compositions of the invention may hol; octytdodecyl stearoyl sulfate; colleyl alcohol; ozokerite: 15 be formulated and provided in any suitable form which is microcystalline wax, paraffin, pentaerythrity1 tetraoctanoate; advantageous and effective for consumer use. In this respect, polyacrylamide; polybutene; polyethylene; propylene glycol considering the advantageous effects of the inventive peptide dicaprylate; propylene glycol dipelargonate; Stearalkonium and compositions containing the same, in particular in non hectorite; Stearyl alcohol; Stearyl Stearate; synthetic beeswax: therapeutic cosmetic applications, it is preferred that inven trihydroxy Stearin; trilinolein; tristearin, Zinc Stearate; and the tive peptide and cosmetic compositions containing the same like. Furthermore, it is preferred according to the present be formulated as or contained in compositions for the cleans invention that the cosmetic composition comprises custom ing of the skin, Such as bar Soaps, toilet Soaps, curd Soaps, ary native and synthetic thickeners or gel formers in formu transparent Soaps, luxury Soaps, deodorizing Soaps, cream lations which preferably comprise one or more crosslinked Soaps, baby Soaps, skin protection soaps, abrasive Soaps, Syn polyacrylic acids and derivatives thereof, polysaccharides, 25 dets, liquid Soaps, pasty Soaps, soft Soaps, washing pastes, Such as Xanthane gum or alginates, carboxymethylcellulose liquid washing, showering and bath preparations, e.g. wash or hydroxycarboxymethylcellulose, hydrocolloids such as ing lotions, shower preparations, shower gels, foam baths, gum Arabic or montmorillonite minerals, such as bentonites cream foam baths, oil baths, bath extracts, Scrub preparations, or fatty alcohols, polyvinyl alcohol and polyvinlypyrroli in-situ products, shaving foams, shaving lotions, shaving done, and combinations of two or more thereof. 30 creams. In addition, they are Suitable for skin cosmetic prepa Other ingredients which are preferably comprised in the rations, such as W/O or O/W skin and body creams, day and inventive cosmetic composition include one or more of bio night creams, light protection compositions, aftersun prod logical additives to enhance performance or consumer appeal ucts, hand care products, face creams, multiple emulsions, Such as amino acids, proteins, Vanilla, aloe extract, bioflavi gelees, microemulsions, liposome preparations, niosome noids, and the like; buffering agents, chelating agents such as 35 preparations, antiwrinkle creams, face oils, lipogels, sport EDTA, emulsion stabilizers; pH adjusters; opacifying agents; gels, moisturizing creams, bleaching creams, vitamin creams, and propellants such as butane carbon dioxide, ethane, hydro skin lotions, care lotions, ampoules, aftershave lotions, pre chlorofluorocarbons 22 and 142b, hydrofluorocarbon 152a, shaves, humectant lotions, tanning lotions, cellulite creams, isobutane, isopentane, nitrogen, nitrous oxide, pentane, pro depigmentation compositions, massage preparations, body pane, and the like. 40 powders, face tonics, deodorants, antiperspirants, nose strips, Alternatively or in addition to these, the cosmetic compo antiacne compositions, repellents and others. sitions of the present invention preferably further comprise Therefore, according to preferred embodiments of the one or more compounds which have an antioxidative, free present invention, the inventive peptide or the inventive cos radical scavenger, skin moisturizing or moisture-retaining, metic and/or pharmaceutical composition comprising the antierythematous, antiinflammatory and/or antiallergic 45 same is contained in a cleansing agent and/or formulation for action, in order to Supplement or enhance their cosmetic application to the epidermis, preferably to the facial skin action by non-therapeutic means. According to preferred and/or to the scalp, and even more preferably to the facial embodiments thereof, said compounds comprise one or more skin, of Vitamins, plant extracts, alpha- and beta-hydroxy acids, wherein the cleansing agentis preferably a soap and/or lotion, ceramides, antiinflammatory, antimicrobial or UV-filtering 50 more preferably a facial lotion and/or a shampoo, and Substances, including derivatives and mixtures thereof. Fur wherein the formulation for application to the epidermis is thermore, the cosmetic compositions of the present invention preferably a liquid or semisolid solution, lotion, cream, gel, or preferably further comprise one or more substance which ointment. absorb UV radiation in the UV-B and/or UV-A region. In According to preferred embodiments of the present inven embodiments of the invention wherein the cosmetic compo 55 tion wherein the peptide or a composition comprising the sition further comprises one or more of the aforementioned same is for topical application, the peptide or composition is compounds, it is further preferred that the composition com preferably formulated with a suitable paste, ointment, lotion, prises a lipid phase, wherein said lipid phase preferably com cream, gel or as a transdermal patch. Depending on the field prises one or more substances selected from the group con ofuse, however, the means of application may preferably be sisting of mineral oils, mineral waxes, branched and/or 60 in the form of a spray (pump spray or aerosol), foam, gel unbranched hydrocarbons and hydrocarbon waxes, triglycer spray, mousse, Suspensions or powders. According to the ides of Saturated and/or unsaturated, branched and/or aforementioned formulations and modes of applications it unbranched C8-C24-alkanecarboxylic acids, and combina therefore preferable that the cosmetic and or pharmaceutical tions of two or more thereof, wherein the oils include syn composition comprises a suitable propellant for the afore thetic, semisynthetic as well as natural oils. More specifically, 65 mentioned types of formulations, and in particular with the lipid phase of the cosmetic composition according to said respect to modes of application involving an aerosol. In gen embodiments preferably comprises one or more components eral, any conceivable propellant may be comprised in said US 9.220,672 B2 35 36 specific cosmetic and pharmaceutical composition, wherein In principle, there is no restriction according to the present according to the invention the use of a propellant comprising invention as to the regions of the skin which may be subject to one or more of propane, butane, pentane, and mixtures skin-care using the inventive peptide or composition com thereof is preferred. prising the same. Preferably, however, the inventive peptide Therefore, according to certain embodiments of the inven and composition is preferably used for skin-care of those tive peptide or the inventive cosmetic and/or pharmaceutical regions of the skin which are prone to a dry-skin condition. composition comprising the same, it is further preferred that More preferably, the skin-care is directed to those regions of the cleansing agent and/or formulation for application to the the skin which may easily become dry as a result of predis epidermis is contained in and/or applied as an aerosol. position to dryness and/or environmental and seasonal factors The present invention further concerns the use of the inven 10 and/or general hygiene involving the frequent washing of tive peptide for non-therapeutic skin care. In particular, the those regions of the skin. Thus, according to preferred present invention further concerns the use of the inventive embodiments of the present invention, the inventive protein peptide or of a composition comprising the same for the or composition is employed for the skin-care of the hands, of treatment and/or prophylaxis of a dry skin condition. Within 15 the facial skin, and/or of the scalp, more preferably of the the meaning of the present invention, the term “dry skin hands and/or of the facial skin, and even more preferably of condition' generally designates any condition of the epider the facial skin. According to certain embodiments of the misfor which increasing or maintaining the moisture content present invention which are preferred, the inventive protein or thereof is beneficial to its cosmetic appearance and/or to the composition is specifically used for skin-care of the hands well-being of the subject to which the inventive peptide or and/or lips, more preferably for the skin-care of the lips. composition is applied. In particular, the term does not des Therefore, the present invention further relates to the use of ignate a pathological condition of the skin necessitating the inventive peptide or of the inventive composition com therapeutic action. prising the same for non-therapeutic skin care, preferably for The term “skin', on the other hand, generally refers to the the treatment and/or prophylaxis of dry skin condition, pref body's outer covering, as known to the person skilled in the 25 erably dry facial skin and/or dry scalp, and even more pref art. Preferably the term relates to three layers: epidermis, erably dry facial skin. dermis, and Subcutaneous fatty tissue. The epidermis is the According to further embodiments of the present inven outermost layer of the skin. It typically forms the waterproof, tion, the inventive peptide or composition comprising the protective wrap over the body's surface and is made up of same is used for improving and Sustaining the healthy normal stratified squamous epithelium with an underlying basal 30 resident microflora present on the skin, preferably the healthy lamina. It usually contains no blood vessels, and is nourished normal resident microflora present on the facial skin and/or by diffusion from the dermis. The main types of cells which scalp, and more preferably the healthy normal resident micro make up the epidermis are keratinocytes, with melanocytes flora present on the facial skin. In particular, according to said and Langerhans cells also present. The epidermis is divided embodiments and preferred embodiments, the inventive pep into several layers where cells are formed through mitosis at 35 tide or composition is used for stimulating the growth of the innermost layers. They move up the strata changing shape healthy microflora which normally resident on the skin. In and composition as they differentiate and become filled with general, with respect to said embodiments and preferred keratin. They eventually reach the top layer called stratum embodiments of the present invention, there is no particular corneum and become sloughed off, or descquamated. The restriction as to the specific components of the healthy normal outermost layer of the epidermis consists of 25 to 30 layers of 40 resident skin microflora which may be stimulated by the dead cells. Conventionally, the epidermis is divided into 5 inventive protein or composition comprising the same, pro Sublayers or strata (from Superficial to deep): the stratum vided that the overall healthy normal resident skin microflora corneum, the stratum lucidum, the stratum granulosum, the is improved or Sustained. According to preferred embodi stratum spinosum and the Stratum germinativum or stratum ments thereof, the components of the healthy normal resident basale. Typically, the interface between the epidermis and 45 skin microflora of which the growth is stimulated comprises dermis is irregular and consists of a succession of papillae, or healthy normal resident skin microflora belonging to the Sta fingerlike projections, which are Smallest where the skin is phylococcus genus, wherein more preferably the growth S. thin and longest in the skin of the palms and soles. Typically, epidermidis is stimulated. the papillae of the palms and soles are associated with eleva Generally speaking, many different microorganisms exist tions of the epidermis, which produce ridges. Subcutaneous 50 on the skin. Some belong to the normal (resident) flora of the fatty tissue is the deepest layer of the skin. A characteristic of skin and are harmless commensals and some are potential this layer is that it is composed of connective tissue, blood pathogens. vessels, and fat cells. Typically, this layer binds the skin to More specifically, organisms on the skin can be classified underlying structures, insulates the body from cold, and into two categories: stores energy in the form of fat. In general the skin forms a 55 1. Resident organisms: resident organisms are permanent protective barrier against the action of physical, chemical, inhabitants of the skin which colonise on the surface of the and bacterial agents on the deeper tissues. This means that skin, the stratum corneum and within the outer layer of the tissues belonging, e.g. to the oral cavity or the vaginal region epidermis and the deeper crevices of the skin and hair or mucous membranes do not belong to the skin. In a pre follicles. These microorganisms of the resident microbial ferred embodiment the term "skin' relates to the outermost 60 skin flora can grow and multiply on the skin without invad layer of the body's covering, i.e. the epidermis. In a more ing or damaging the skin tissue. Washing does not easily preferred embodiment the term “skin' relates to the stratum remove these organisms in deeper skin regions. Resident corneum of the epidermis. In an even more preferred embodi microorganisms are harmless commensals. ment the term skin relates to the outermost 25 to 30 layers of 2. Transient organisms: transient organisms are microorgan dead cells of the epidermis. In the most preferred embodiment 65 isms which are deposited on the skin but do not multiply the term "skin' relates to the outermost 10 layers of dead cell there or contaminants which multiply on the skin and per of the epidermis. sist for short periods. They cannot settle permanently on US 9.220,672 B2 37 38 healthy skin whose microenvironment is heavily deter after different time intervals of incubation and can be com mined by the resident micro flora. Transient organisms are pared with a control which does not contain a microorganism potentially pathogenic. according to the invention, thereby allowing to determine Thus, the term “resident skin microbial flora' relates to the whether there is an increase in growth. The determination of microorganisms which can normally be found on healthy 5 the growth may be effected by available means and methods skin, preferably human skin, and which constitute the major for determining the number of cells and/or colonies, such as ity of the microorganisms found on the skin. In particular, the by staining with an appropriate dye and/or optical means Such term “resident skin microbial flora' relates to microorgan as densitometry and counting the cells/colonies under the isms which are permanent inhabitants on the Surface of the microscope. skin, the stratum corneum and within the outer layer of the 10 epidermis and the deeper crevices of the skin and hair fol Preferably, the stimulation of growth of the microorgan licles. These microorganisms are characterized in that they ism(s) of the resident skin microbial flora is determined using can grow and multiply on the skin without invading or dam in an in situ skin assay. By way of example, an in situ skin aging the skin tissue. A characteristic of these microorgan assay may be conceived to comprise the following steps: isms is that washing does not easily remove them in deeper 15 cultivation of at least one microorganism of the resident skin skin regions. The microorganisms of the resident skin micro microbial flora and evenly spreading it on an area of skin of a bial flora are harmless commensals. test individual; applying an aliquot of a peptide or composi More specifically, the term “resident skin microbial flora' tion according to the invention in a punctual area within the preferably relates to a flora of aerobic and anaerobic micro area on which the microorganism(s) of the resident skin organisms which can be found on skin, preferably human microbial flora has/have been spread; incubating the skin for skin. More preferably, it relates to a flora of microorganisms an amount of time sufficient to allow growth of the microor which comprises Staphylococcus epidermidis (coagulase ganism(s) of the resident skin microbial flora; transferring the negative), Micrococcus spec, Diphteroids and propioni bac upper skin layers, including the microorganisms comprised in teria. Typically, about 90% of the aerobic resident microbial these, to an agar plate containing an appropriate growth skin flora consists of Staphylococcus epidermidis. The 25 medium; incubation of the agarplates for a period of time and remaining about 10% are composed of mainly Micrococcus under conditions allowing the growth of the microorgan spec. (80%. Micrococcus luteus) and Diphteroids (13%). The ism(s) of the resident skin microbial flora; determining the term “Diphtheroid denotes a wide range of bacteria belong growth of the microorganism(s) of the resident skin microbial ing to the genus Corynebacterium. For convenience, cutane flora Surrounding the area at which the peptide or composi ous diphtheroids have been categorized into the following 30 tion according to the invention was applied and comparing it four groups: lipophilic or nonlipophilic diphtheroids; anaero to the growth of the microorganism(s) in a control in which no bic diphtheroids; diphtheroids producing porphyrins. Major peptide of the invention was applied. representatives (90%) of the anaerobic microbial skin flora The area of skin used for this assay may be any Suitable area are propionibacteria; especially Propionibacterium acnes, P of skin of an individual, preferably of a human individual. In granulosum and Pavidum can be isolated from the skin. The 35 a preferred embodiment it is an area of skin on the forearm of anaerobic flora accounts for approximately 4% of the total a human individual. The size of the area of the skin used for resident skin flora. testing preferably preferably ranges from 1 to 40 cm, more More preferably, more than 90% of the microorganisms of preferably from 5 to 20 cm, even more preferably from 5 to the microbial flora belong to Staphylococcus epidermidis, 10 cm. Micrococcus spec, Diphteroids and propioni bacteria. Even 40 According to an exemplary mode of conducting the in situ more preferably, the healthy resident skin microbial flora is skin assay, microorganism(s) of the resident skin microbial characterized in that its major constituent is Staphylococcus flora are evenly distributed on the area, preferably in a density epidermidis. of approximately 10 cfu/cm-10 cfu/cm. The microorgan The term “stimulates' in connection with the growth of ism(s) spread on the skin are air dried and an aliquot of a microorganisms of the resident skin microbial flora means 45 peptide or composition according to the invention is applied that the growth of one or more of these microorganisms is in a punctual manner within the area. This can be achieved by increased when contacted with a peptide or composition means known to the person skilled in the art. according to the invention. An increased growth means pref The subsequent incubation of the skin preferably takes erably an increase in proliferation, i.e. cell divisions per time place at room temperature for, e.g., two hours. The transfer of unit. Alternatively, the term “stimulates' also refers to an 50 the upper skin layers, including the microorganisms com increase in size of individual cells. Bacterial cell size can be prised therein, may, e.g., be effected with the help of an assessed by flow cytometry (e.g. Becton-Dickinson FACSort adhesive tape Stripe. The agar plates to which the upper skin flow cytometer, San Jose, Calif.) after staining with the stain layers have been transferred are incubated at a temperature SYBR Green I (Molecular Probes, USA). Bacteria cell size is allowing growth of the microorganism(s) or the resident skin assessed in Side-Angle Light Scatter (SSC) mode. Accord- 55 microbial flora to be tested and contain a growth medium ingly, within the meaning of the present invention, an known to Support growth of this (these) microorganism(s). increased growth preferably means an increase in biomass The incubation typically takes place for about 24 hours. The production per time unit. growth of the microorganism(s) can be detected by methods The stimulation of growth of the microorganism(s) of the known to the person skilled in the art. Preferably, it is deter resident skin microbial flora can be determined by any suit- 60 mined by densitometry or by counting the colonies formed in able means known in the art, wherein the stimulation is pref the neighborhood of the point at which an aliquot of the erably observed in vitro, more preferably in an assay in which peptide or composition of the invention was applied. Bacte a peptide or composition according to the invention is con rial cell size can be assessed by flow cytometry (e.g. Becton tacted with one or more microorganisms of the resident skin Dickinson FACSort flow cytometer, San Jose, Calif.) after microbial flora and the growth of the(se) microorganism(s) of 65 staining with the stain SYBR Green I (Molecular Probes, the resident skin microbial flora is determined. The growth USA). Bacteria cell size is assessed in Side-Angle Light can be determined by counting the numbers of cells/colonies Scatter (SSC) mode. US 9.220,672 B2 39 40 According to the present invention, the inventive peptide or pathogenic skin micro flora if it leads to a decrease of growth a composition comprising the same is preferably regarded to of Such a microorganism of the transient pathogenic skin stimulate the growth of one or more microorganisms of the micro flora when contacted with it. The term “inhibits the resident skin microbial flora if it leads to an increase of growth of microorganisms of the transient pathogenic skin growth of at least one Such microorganism in an in vitro hole microflora' preferably means that the peptide or composition plate assay of at least 5%, preferably of at least 10%, 20%, of the invention decreases the growth of at least one, prefer 30%, 40%, 50%, 60%, or 70%, more preferably of at least ably of more than one, preferably of more than two, more 75% and more preferably of at least 80% and even more preferably of more than five and particularly preferred of any preferably of at least 85% in comparison to a control to which of the microorganisms of the transient pathogenic flora. In a no inventive peptide or composition comprising the same has 10 further preferred embodiment, the peptide or composition of been added. More preferably, the inventive peptide or a com the present invention inhibits the growth of the major repre position comprising the same is regarded as stimulating the sentative of the transient pathogenic skin micro flora, i.e. growth of one or more microorganisms of the resident skin Staphylococcus aureus. In a further preferred embodiment, microbial flora if it leads to an increase of growth of at least the peptide of the present invention specifically inhibits the one such microorganism in an in situ skin assay of at least 5%, 15 growth of Staphylococcus aureus. “Specifically' preferably preferably of at least 10%, 20%, 30%, 40%, 50%, 60%, or means that it inhibits the growth of Staphylococcus aureus, 70%, more preferably of at least 75%, even more preferably but does not significantly or only to a minor degree inhibit the of at least 80% and most preferably of at least 85%. growth of other microorganisms, in particular of those micro According to a preferred embodiment of the presenting organisms which belong to the resident skin micro flora. invention, in addition to stimulating the growth of S. epider More preferably, the term “specifically” means that the midis, the peptide or composition of the present invention also degree of inhibition on Staphylococcus is much higher than stimulates the growth of Micrococcus spec, preferably of the degree of inhibition on another microorganism, in par Micrococcus luteus. In a further preferred embodiment, also ticular a microorganism of the resident skin micro flora. Par the growth of Diphteroids, preferably ofbacteria belonging to ticularly preferably, the term “specifically’ means that in a the genus Corynebacterium is stimulated. In a particularly 25 Suitable growth assay known to the person skilled in the art preferred embodiment the peptide according to the invention the proliferation of Staphylococcus aureus in the presence of stimulates the growth of the majority of the microorganisms the peptide or composition of the present invention is at the of the resident skin microbial flora, preferably 60% or more of most 50% of the proliferation of another microorganism, in the healthy normal resident skin microflora population, more particular another microorganism of the resident skin micro preferably 70% or more, more preferably 80% or more, more 30 flora in the presence of the peptide or composition of the preferably 90% or more, and even more preferably 95% or present invention. Preferably, the proliferation of Staphylo Oe. coccus aureus is 40% or less, 30% or less, 20% or less, 10% Furthermore, according to the embodiments and preferred or less, more preferably 5% or less and most preferably 1% or embodiments of the present invention relating to the use of the less of the proliferation of another microorganism, in particu inventive peptide or composition for stimulating the growth 35 lar another microorganism of the resident skin micro flora, in of healthy normal resident skin microflora, it is further pre the presence of a peptide or composition of the present inven ferred that said use does not additionally stimulate the growth tion. In a preferred embodiment the peptide or composition of of transient pathogenic microorganisms. In general, there is the present invention inhibits the growth of Staphylococcus no particular restriction as to the specific types of transient aureus but does not inhibit the growth of Micrococcus luteus pathogenic microorganisms of which the growth is not addi 40 and/or Escherichia coli. According to a particularly preferred tionally stimulated by the use of the inventive peptide or embodiment, the specific inhibition of Staphylococcus composition, provided that the overall healthy normal resi aureus can be detected when culture conditions are used dent skin microflora is improved or Sustained. It is, however, which include glycerol. preferred that the transient pathogenic microorganisms of Furthermore, a decreased growth means preferably a which the growth is not additionally stimulated comprises S. 45 decrease in proliferation, i.e. in cell divisions per unit. Alter aureus. Within the meaning of the present invention, the natively, the term “inhibits' preferably refers to a decrease in growth of a microorganism is not stimulated by the inventive size of individual cells. Bacterial cell size can be assessed by peptide or composition if no stimulation of its growth may be flow cytometry (e.g. Becton-Dickinson FACSort flow cytom detected when compared to a control sample not containing eter, San Jose, Calif.) after staining with the stain SYBR the inventive peptide or composition. 50 Green I (Molecular Probes, USA). Bacteria cell size is Therefore, the present invention further relates to the use of assessed in Side-Angle Light Scatter (SSC) mode. A the inventive peptide or of the inventive composition com decreased growth thus means a decrease in biomass produc prising the same for stimulating the growth of healthy normal tion per time unit. resident skin microflora, preferably for stimulating the The stimulation of growth of the microorganism(s) of the growth of healthy normal resident skin microflora belonging 55 transient pathogenic skin micro flora can be determined by to the Staphylococcus genus, more preferably for stimulating any Suitable means known in the art, wherein the stimulation the growth of S. epidermidis, and even more preferably for is preferably observed in vitro, more preferably in an assay in stimulating the growth of S. epidermidis without stimulating which one or more microorganisms of the transient patho the growth of transient pathogenic microorganisms, in par genic skin micro flora is contacted with a peptide or compo ticular of S. aureus. 60 sition according to the invention and the growth of the(se) According to yet further embodiments of the present inven microorganism(s) of the transient pathogenic skin micro flora tion, the inventive peptide or composition comprising the is determined. The growth can be determined by counting the same is used for inhibiting the growth or transient pathogenic numbers of cells/colonies after different time intervals of skin microflora. incubation and can be compared with a control which does Within the meaning of the present invention, the inventive 65 not contain a peptide or composition according to the inven peptide or a composition comprising the same is regarded as tion, thereby allowing to determine whether there is an inhibiting the growth of a microorganism of the transient increase or decrease in growth. More preferably, the inhibi US 9.220,672 B2 41 42 tion of growth of the microorganism(s) of the transient patho growth of Such a microorganism of the resident skin micro genic skin micro flora can be determined in an “in vitro liquid flora when contacted with it. The inhibition of growth or its assay”. By way of example, Such an assay is described in absence can be tested in vitro or in situ as described above in Examples 2 and 3 below. connection with the property of a peptide or composition of Even more preferably, the inhibition of growth of the the invention to inhibit the growth of at least one microorgan microorganism(s) of the transient pathogenic skin micro flora ism of the transient pathogenic skin micro flora. Most pref can also be observed in an “in situ skin assay'. In particular, erably the test for determining inhibition or its absence takes said assay corresponds to the “in situ skin assay” described in place by carrying out an “in vitro hole plate assay” and/or "in the foregoing with respect to the monitoring of the stimula vitro liquid assay” and/or an “in situ skin assay, preferably tion of the growth of the microorganism(s) of the resident skin 10 with a microorganism of the resident skin micro flora as microbial flora, with the difference that the agar plates to explained herein. which the upper skin layers have been transferred are incu Alternatively, a peptide or composition of the invention is bated at a temperature allowing growth of the microorgan regarded as not altering the growth of beneficial microorgan ism(s) or the transient pathogenic skin micro flora to be tested isms preferably belonging the resident skin micro flora if the and contain a growth medium known to Support growth of this 15 growth of the latter microorganism is not decreased or only (these) microorganism(s). slightly decreased when contacted with the former peptide or According to the present invention, the inventive peptide or composition of the invention. “Slightly decreased preferably a composition comprising the same is preferably regarded to means that the growth is decreased not more than by 5% when inhibit the growth of one or more microorganisms of the compared to the control, more preferably not more than 2% pathogenic transient micro flora if it leads to a decrease of when compared to the control. The term “not decreased growth of at least one such microorganism in an “in vitro hole means that no statistically relevant difference can be found plate assay” of at least 5%, preferably of at least 10%, 20%, between the growth of the microorganism of the resident skin 30%, 40%, 50%, 60%, or 70%, 80%, more preferably of at micro flora contacted with a peptide or composition of the least 90% and more preferably of at least 95% and even more invention when compared to the control where no peptide or preferably of at least 99% in comparison to a control to which 25 composition of the invention is present. The term “not no inventive peptide or composition comprising the same has decreased in a preferred embodiment also includes those been added. More preferably, the inventive peptide or a com cases where a peptide actually leads to an increase of the position comprising the same is regarded to inhibit the growth growth of a microorganism of the resident skin micro flora, of one or more microorganisms of the pathogenic transient i.e. where it stimulates the growth of Such a microorganism. micro flora if it leads to a decrease of growth of at least one 30 According to particularly preferred embodiments, the ben Such microorganism in an “in vitro liquid assay of at least eficial microorganisms of which the growth is not addition 5%, preferably of at least 10%, 20%, 30%, 40%, 50%, 60%, ally inhibited comprise one or more of the microorganisms or 70%, 80%, more preferably of at least 90% more prefer constituting the healthy normal resident skin microflora, ably of at least 95% and even more preferably of at least 99% wherein said one or more microorganisms preferably com in comparison to the control. Even more preferably, a peptide 35 prise S. epidermidis. or composition of the invention is regarded as inhibiting the Therefore, the present invention further relates to the use of growth of one or more microorganisms of the transient patho the inventive peptide or of the inventive composition com genic skin micro flora if it leads to an decrease of growth of at prising the same for inhibiting the growth of transient patho least one such microorganism in an in situ skin assay of at genic skin microflora, preferably for inhibiting the growth of least 5%, preferably of at least 10%, 20%, 30%, 40%, 50%, 40 transient pathogenic skin microflora belonging to the Staphy 60%, or 70%, 80%, more preferably of at least 90%, more lococcus genus, more preferably for inhibiting the growth of preferably of at least 95% and even more preferably of at least S. aureus, and even more preferably for inhibiting the growth 99%. of S. aureus without inhibiting the growth of beneficial micro Furthermore, according to the embodiments and preferred organisms, in particular of S. epidermidis. embodiments of the present invention relating to the use of the 45 The present invention is illustrated by the figures and inventive peptide or composition for inhibiting the growth of examples as described hereinbelow. transient pathogenic skin microflora, it is further preferred that said use does not also inhibit the growth of microorgan DESCRIPTION OF THE FIGURES isms which are beneficial, in particular which are beneficial to the skin, and in particular does not inhibit the growth of the 50 FIG. 1a shows results from the in-vitro-hole? well plate healthy normal resident skin micro flora. In general, there is assay according to Example 1, wherein the stimulation of the no particular restriction as to the specific types of beneficial growth of S. epdermidis having been treated with the tet microorganisms of which the growth is not equally inhibited rapeptide H-Lys(H-Asp-NH)-Ala-Glu-NH may be by the use of the inventive peptide or composition. observed in view of the formation of a black ring around the The term “not inhibit in connection with the growth of 55 well. beneficial microorganisms, and in particular microorganisms FIG. 1b shows results from the in-vitro-hole? well plate of the resident skin micro flora preferably means that the assay according to Example 1, wherein the stimulation of the growth of at least one, preferably of more than one, preferably growth of S. epdermidis having been treated with the tet of more than two, more preferably of more than five and rapeptide H-Lys(H-Asp-NH)-Ala-Glu-NH may be particularly preferred of any of said beneficial microorgan 60 observed in view of the formation of a black ring around the isms preferably belonging to the resident skin micro flora is well. not altered when contacted with a peptide or composition FIG. 1 c shows the result from the control assay in Example according to the invention. A not altered growth means pref 1 containing water. erably an unchanged proliferation, i.e. cell divisions per time FIG. 2a shows results from the bioassay according to unit. A peptide or composition of the invention is thus 65 Example 2, displaying the stimulation of the growth of S. regarded as not altering the growth of a microorganism of the epidermidis having been treated with the tetrapeptide H-Lys resident skin micro flora if it does not lead to a decreased (H-Asp-NH)-Ala-Glu-NH, wherein the y-axis represents US 9.220,672 B2 43 44 the optical density ODoo, measured for the respective SCD-bouillon (1:2). The samples used in the experiment samples and the X-axis represents the incubation time in further contained the inventive tetrapeptide H-Lys(H-Asp hours. Line A shows the results for the untreated sample NH)-Ala-Glu-NH in respective concentrations of 1 mg/ml (control), line B shows the results for the sample treated with and 0.2 mg/ml. As control assay, Samples containing 1 mg/ml 0.2 mg/ml of the inventive tetrapeptide, and line C shows the 5 of a mixture containing Alanine, Glutamine, Lysine, and results for the sample treated with 1 mg/ml of the inventive Asparagine in equal parts and samples containing no further tetrapeptide. components in addition to S. epidermidis were respectively FIG. 2b shows results from the bioassay according to employed. The pH of all of the samples was 6.5. Example 2, displaying the influence of an mixture of four Results from the bioassay are shown in FIGS. 2a and 2b. In amino acids on the growth of S. epidermidis, wherein the 10 particular, as may be taken from FIG. 2a, the samples con y-axis represents the optical density ODoo, measured for taining the inventive tetrapeptide (see lines B and C) clearly the respective samples and the X-axis represents the incuba stimulates the growth of S. epidermidis compared to the con tion time in hours. Line A shows the results for the untreated trol sample devoid of an additive (see line A), wherein the sample (control) and line B shows the results for the sample stimulation is already apparent at a concentration of 0.2 treated with a mixture of Alanine, Glutamine, Lysine, and 15 mg/ml (see line B). In particular, as may be taken from the Asparagine in equal parts (“amino acid mix”) at a total con results displayed in FIG. 2b for the sample of S. epidermidis centration of 1 mg/ml in the sample. containing a mixture of 4 amino acids (see line B), the same FIG. 3 shows results from the bioassay according to effect may not be observed for the constituent amino acid Example 3, displaying the inhibition of the growth of S. residues of the inventive tetrapeptide by themselves, said aureus having been treated with the tetrapeptide H-Lys(H- sample showing a progression of the S. epidermis population Asp-NH)-Ala-Glu-NH, wherein the y-axis represents the which is comparable to the control sample devoid of an addi optical density ODoo, measured for the respective samples tive (see line A). and the X-axis represents the incubation time in hours. Line A Example 3 shows the results for the untreated sample (control), and line 25 B shows the results for the sample treated with 1 mg/ml of the An S. aureus in vitro liquid assay was performed on a inventive tetrapeptide. Microtiter plate having wells with a respective volume of 200 FIG. 4 shows results from the bioassay of Example 4, ul. To this effect, a 24h cell culture of S. aureus was diluted displaying the secretion of IL-1alpha by NHEK after 24 h of 1:500 thus affording an optical density ODoo of 0.05 in pre-treatment and 24 h of treatment. 1. Is the negative control, SCD-bouillon (1:2). One of the samples used in the experi 2. and 3. show the pro-inflammatory effects of Lactobacillus 30 ment further contained the inventive tetrapeptide H-Lys(H- DSM 17250 and LPS, respectively, 4. the precautionary anti Asp-NH)-Ala-Glu-NH in a concentration of 1 mg/ml. The inflammatory effect of Lactobacillus DSM 17250, and 5... the pH of the samples was 6.5. protective anti-inflammatory effect of Lactobacillus DSM The results from the bioassay of Example 3 are shown in 17250. FIG.3, wherein it is apparent that the growth of S. aureus was 35 clearly inhibited in the sample containing the inventive tet EXAMPLES rapeptide (see line B) compared the control sample devoid of Example 1 an additive (see line A). Example 4 The tetrapeptide H-Lys(H-Asp-NH)-Ala-Glu-NH, was 40 found to be able to stimulate the growth of S. epidermidis on Monolayer cultures of normal human epidermal kerati agar plates in an in-vitro hole plate assay. To this effect, an S. nocytes (NHEK) were cultivated for a 48 h-period consisting epidermidis growth simulation assay was employed, using in a 24h pre-treatment followed by a 24 h treatment with or potassium tellurite as the growth indicator, observed in the without Lactobacillus DSM 17250 (200 ug/ml) producing a assay as brown Zones. An S. epidermidis log phase culture at 45 peptide according to the present invention. 1:200 (containing approximately 1x10 cfu/ml of bacteria) At the end of the incubation, the effect of the Lactobacillus was used as the working culture in the experiment. producing a peptide according to the present invention on the The working culture was plated onto tellurite-TSA, after secretion of pro-inflammatory cytokine IL-1C. was evaluated which 5 mm diameter holes were respectively punched into by quantification using a commercially available ELISA kit. the agar for forming the testing wells. 40 ul of a solution 50 The different culture conditions are summarized in Table 1. containing the tetrapeptide at a concentration of 1 mg/ml were then added to a first and second well, and the same TABLE 1 Volume of water was added as the control to a third well. The Table 1: Culture protocol and properties Petri dish was then incubated at 35° C. for 24 h. evaluated. LPS: Lipopolysaccharides The results of the assay are displayed in FIGS. 1 and 2. 55 which respectively show the S. epidermidis culture treated Pre-treatment Treatment Property evaluated with the tetrapeptide (FIGS. 1a and 1b) and the control assay 1 None (culture None (culture Untreated control (negative with water (FIG. 1c). In particular, it is apparent from the medium alone) medium alone) control) assays containing the inventive tetrapeptide that the growth of 2 Lactobacilius None (culture Pro-inflammatory effect of DSM 172SO medium alone) Lactobacilius DSM 17250 S. epidermidis is clearly stimulated. 60 3 LPS None (culture Pro-inflammatory effect of medium alone) LPS (positive control) Example 2 4 Lactobacilius LPS Precautionary anti-inflammatory DSM 172SO effect of Laciobacilius DSM 17250 5 Lactobacilius Lactobacilius Protective anti-inflammatory A Microtiter plate having wells with a respective volume of DSM 172SO - DSM 172SO effect of Laciobacilius DSM 17250 200 ul was used for the S. epidermidis in vitro liquid assay. To 65 LPS this effect, a 24h cell culture of S. epidermidis was diluted 1:200 thus affording an optical density ODoo of 0.06 in US 9.220,672 B2 45 46 The results from the determination of IL-1C. in the bioassay 12. The peptide of claim 1, wherein said peptide is con are shown in FIG. 4. It can be concluded from the results that tained in a formulation for application to the scalp, wherein untreated keratinocytes do not secrete pro-inflammatory the formulation is a liquid or semisolid solution, lotion, IL-1C. (1, negative control) while LPS-treated keratinocytes cream, gel, or ointment. secrete large quantities of IL-1C. (3, positive control). 13. The peptide of claim 1, wherein said peptide is con The cells which were pretreated with Lactobacillus DSM tained in a formulation for application to the facial skin, 17250 do not secrete IL-1C. Therefore, it can be concluded wherein the formulation is a liquid or semisolid solution, that the lactobacillus has no pro-inflammatory effect (2). lotion, cream, gel, or ointment. Pre-treatment with Lactobacillus DSM 17250 dramati 14. A cosmetic composition comprising a peptide accord cally reduces IL-1C. production after treatment with LPS (4). 10 Therefore, a precautionary anti-inflammatory effect of Lac ing to claim 1. tobacillus DSM 17250 can be concluded. 15. The cosmetic composition of claim 14, wherein said The addition of Lactobacillus DSM 17250 to LPS as pre composition comprises the peptide in a concentration of from treatment before treatment with the lactobacillus reduces the 0.005 to 20 mg/ml. inflammatory effect of LPS. Therefore, a protective anti 15 16. The cosmetic composition of claim 14, wherein said inflammatory effect by the lactobacillus can also be con composition comprises the peptide in a concentration of from cluded (5). 0.01 to 10 mg/ml. 17. The cosmetic composition of claim 14, wherein said CITED PRIOR ART DOCUMENTS composition comprises the peptide in a concentration of from 0.05 to 5 mg/ml. WO 2006/13642O A2 18. The cosmetic composition of claim 14, wherein said WO 2005/0489.68A1 composition comprises the peptide in a concentration of from WOOOf 43417 A1 0.1 to 2 mg/ml. U.S. Pat. No. 6,620,419 B1 19. The cosmetic composition of claim 14, wherein said U.S. Pat. No. 6,492,326 B1 25 composition comprises the peptide in a concentration of from Rollanet al., Int. J. Food Microbiol. 70 (2001), 303-307 0.2 to 1 mg/ml. Matsuduchi et al., Clin. Diagn. Lab. Immunol. 10 (2003), 20. The composition of claim 14, wherein said composi 259-266 tion further comprises one or more cosmetically acceptable Stentzet al., Appl. Environ. Microbiol. 66 (2000), 4272-4278 carriers or excipients. Varmanen et al., J. Bacteriology 182 (2000), 146-154 30 21. The composition of claim 14, wherein said composi Schrader, Grundlagen and Rezepturender Kosmetika, Hüthig tion is contained in a cleansing agent for application to the BuchVerlag, Heidelberg, 2" edition, 1989, 3" part epidermis, wherein the cleansing agent is a soap or lotion. The invention claimed is: 22. The composition of claim 14, wherein said composi 1. A peptide of the following formula (III): tion is contained in a cleansing agent for application to the 35 Scalp, wherein the cleansing agent is a shampoo. Lys(H-Asp-R)-Ala-Glu (III) 23. The composition of claim 14, wherein said composi or a salt thereof; tion is contained in a cleansing agent for application to the wherein R is OH or NH: facial skin, wherein the cleansing agent is a facial lotion. wherein the C-terminal carboxyl group in Glu is optionally 24. The composition of claim 14, wherein said composi an amide Glu-NR'R'', and Rand Rare independently 40 tion is contained in a formulation for application to the epi from one another H or alkyl: dermis, wherein the formulation is a liquid or semisolid solu and wherein the N-terminal amino group in Lys is option tion, lotion, cream, gel, or ointment. ally NRR, and Rand Rare independently from one 25. The composition of claim 14, wherein said composi another H or alkyl. tion is contained in a formulation for application to the scalp. 2. The peptide of claim 1, wherein Rand Rare indepen 45 wherein the formulation is a liquid or semisolid solution, dently H or (C1-C3)alkyl. lotion, cream, gel, or ointment. 3. The peptide of claim 1, wherein Rand Rare H. 26. The composition of claim 14, wherein said composi 4. The peptide of claim 1, wherein Rand Rare indepen tion is contained in a formulation for application to the facial dently H or (C1-C3)alkyl. skin, wherein the formulation is a liquid or semisolid solu 5. The peptide of claim 1, wherein Rand Rare H. 50 tion, lotion, cream, gel, or ointment. 6. The peptide of claim 1, wherein said peptide is contained 27. A pharmaceutical composition comprising a peptide in a cleansing agent for application to the epidermis, according to claim 1. wherein the cleansing agent is a Soap or lotion. 28. The pharmaceutical composition of claim 27, wherein 7. The peptide of claim 6, wherein the cleansing agent is said composition comprises the peptide in a concentration of contained in or applied as an aerosol. 55 from 0.01 to 100 mg/ml. 8. The peptide of claim 1, wherein said peptide is contained 29. The pharmaceutical composition of claim 27, wherein in a cleansing agent for application to the scalp, wherein the said composition comprises the peptide in a concentration of cleansing agent is a shampoo. from 0.05 to 50 mg/ml. 9. The peptide of claim 1, wherein said peptide is contained 30. The pharmaceutical composition of claim 27, wherein in a cleansing agent for application to the facial skin, wherein 60 said composition comprises the peptide in a concentration of the cleansing agent is a Soap or lotion. from 0.1 to 10 mg/ml. 10. The peptide of claim 1, wherein said peptide is con 31. The pharmaceutical composition of claim 27, wherein tained in a formulation for application to the epidermis, said composition comprises the peptide in a concentration of wherein the formulation is a liquid or semisolid solution, from 0.2 to 5 mg/ml. lotion, cream, gel, or ointment. 65 32. The pharmaceutical composition of claim 27, wherein 11. The peptide of claim 10, wherein the formulation is said composition comprises the peptide in a concentration of contained in or applied as an aerosol. from 0.5 to 2 mg/ml. US 9.220,672 B2 47 48 33. The composition of claim 27, wherein said composi tion further comprises one or more pharmaceutically accept able carriers or excipients. k k k k k