IL-17A binding peptides and medical uses thereof

Abstract

The invention relates to IL-17A binding peptides, inhibitors of the interaction of IL-17A with the receptor IL-17RA, and to bioconjugates, dimers, pharmaceutical compositions and medical use thereof.

Claims

1. A peptide that specifically inhibits IL-17A binding to ILRA, comprising the amino acid sequence of Formula (I), wherein Formula (I) comprises: TABLE-US-00015 (I) (SEQ ID NO: 258) X.sub.1-X.sub.2-X.sub.3-X.sub.4 X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15 wherein in Formula (I): (a) X.sub.1 is I, V or L; X.sub.2 is H, M, R, K or E; X.sub.3 is V, F or I; X.sub.4 is T, Q, S, Y or N; X.sub.5 is I, F or V; X.sub.6 is P; X.sub.7 is A, Q, or L; X.sub.8 is D, E, or Q; X.sub.9 is L, W, F, V or I; X.sub.10 is W, Y or F; X.sub.11 is D, E or N; X.sub.12 is W or F; X.sub.13 is I, V, F or L; X.sub.14 is N, R, Q or E; X.sub.15 is K, R, H or E; with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO:1); or (b) X.sub.1 is I or V, X.sub.2 is H, M or R, X.sub.3 is V or F; X.sub.4 is T or Q; X.sub.5 is I, F or V; X.sub.6 is P or G; X.sub.7 is A or Q; X.sub.8 is D or E; X.sub.9 is L; X.sub.10 is W or Y; X.sub.11 is D or E; X.sub.12 is W; X.sub.13 is I or V; X.sub.14 is N, R or E; X.sub.15 is K, R or E; with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO: 1) or (c) X.sub.1 is I or V; X.sub.2 is H or M, X.sub.3 is V; X.sub.4 is T; X.sub.5 is I; X.sub.6 is P; X.sub.7 is A; X.sub.8 is D; X.sub.9 is L, W, F, V or I; X.sub.10 is W or Y; X.sub.11 is D or E; X.sub.12 is W; X.sub.13 is I or V; X.sub.14 is N, R or E; X.sub.15 is K, R or E; with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO: 1), HVTIPADLWDWIN (SEQ ID NO:259), IHVTIPADLWDWI (SEQ ID NO:260) or IHVTIPADLWDW (SEQ ID NO:261).

2. The peptide of claim 1, wherein in formula (I) X.sub.1 is V.

3. The peptide of claim 1, having an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 42, SEQ ID SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 171, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 187, SEQ ID NO: 193, SEQ ID NO: 198, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 254, and SEQ ID NO: 256.

4. The peptide of claim 1, further comprising sequence (A).sub.m at the C- and/or N-terminal of the peptide, wherein A is an amino acid selected from the group consisting of R, K, G, E, Q and A, and m is an integer between 1 and 10.

5. A peptide as claimed in claim 4, having sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 43.

6. The peptide of claim 1, wherein a protective cap group is bound at the C- and/or N-terminal, wherein the protective cap group is selected from the group consisting of amides, aldehydes, esters, p-Nitroanilide, 7-Amino-4-methylcoumarin and the protective group cap bound to a N-terminal being selected from acetyl, formyl, pyroglutamyl, fatty acids, urea, carbamate sulfonamide, and alkylamine.

7. A dimer formed by two peptides, wherein each peptide is a peptide of claim 1.

8. The dimer of claim 7, wherein the peptides are linked by a polyethylene spacer.

9. A bioconjugate comprising the peptide of claim 1 and a biomolecule, wherein the biomolecule is bound to the N- and/or C-terminal of the peptide.

10. The bioconjugate of claim 9, wherein the biomolecule is selected from the group consisting of capric acid, capronic acid, ascorbic acid, NAG-NAM, NAG, NAM, hyaluronic acid, alginic acid, chitin, (GaINAc)2, Ga1-alpha1,3-GaINAc and trigalacturonic acid.

11. A pharmaceutical composition comprising the peptide of claim 1 and at least one pharmaceutically acceptable excipient.

12. A method of treating a subject having an inflammatory and autoimmune disease comprising administering to the subject an effective amount of the peptide of claim 1.

13. The method of claim 12, wherein the inflammatory and autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, Crohn's disease, systemic lupus erythematosus, asthma, Behçet's disease, hyper IgE syndrome, ankylosing spondylitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, keratoconjunctivitis sicca, vernal keratoconjunctivitis, stromal herpetic keratitis, corneal allograft rejection, corneal infections, herpes virus or Pseudomonas aeruginosa keratitis and dry eye disease, preferably psoriasis and dry eye disease.

14. A method of producing the peptide of claim 1, comprising (a) synthesizing the peptide; and (b) purifying the peptide.

15. The peptide of claim 1, comprising an amino acid sequence of SEQ ID NO: 2.

16. The peptide of claim 1, having a 15 amino acid sequence.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows some dose-response curves obtained in the IL-17RA-IL17RC dimerization assay described in Example 5 of with representative IL-17A binding peptides according to the invention. The percentage of inhibition of IL-17RA/IL17RC dimerization induced by IL-17A obtained by treatment with different peptides is reported.

(2) FIG. 2 shows the activity of the IL-17A binding peptide of SEQ ID NO:18 on secretion of IL6, IL-17A and IL23 by human differentiated and activated TH17 cells, measured as described in Example 6. The concentration of the different cytokines in the supernatant of TH17 cells with or without (control) peptide of SEQ ID NO:18 is reported.

(3) FIG. 3 shows the activity of the IL-17A binding peptide of SEQ ID NO:18 on secretion of Metalloproteinase 3 by human differentiated and activated TH17 cells, measured as described in Example 6. The concentration of Metalloproteinase 3 in the supernatant of TH17 cells with or without (CONTROL) peptide of SEQ ID NO:18 is reported.

(4) FIG. 4 shows viability of human TH17 cells after 24 h, in the presence and absence (control) of the peptide of SEQ ID NO: 18, as described in Example 6.

(5) FIG. 5 shows the binding site on IL-17A of the IL-17A peptides according to the invention.

(6) FIG. 6 shows the sensograms obtained in the experiment of Example 9 with the peptide of SEQ ID NO: 2 (upper panel) or SEQ ID NO: 1 (lower panel).

(7) FIG. 7 shows the value of the affinity constant at different concentrations of the peptide of SEQ ID NO: 2 (upper panel) or SEQ ID NO: 1 (lower panel), obtained in the experiment of Example 9.

(8) FIGS. 8A-8B show the level of expression of IL-8 measured by real-time PCR as described in Example 10 in HCEC cells not treated (vehicle) or treated with the peptide of SEQ ID NO: 2 (FIG. 8A) or with the peptide HAP (SEQ ID NO: 1) (FIG. 8B). The error bars represent the standard deviation. T-test was performed as statistical analysis. P-value *<0.05, **<0.005, ***<0,0005.

(9) FIGS. 9A-9B show the amount of IL-6 measured by real-time PCR as described in Example 10 in HCEC cells not treated (vehicle) or treated with the peptide of SEQ ID NO: 2 (FIG. 9A) or with the peptide HAP (SEQ ID NO: 1) (FIG. 9B). The error bars represent the standard deviation. T-test was performed as statistical analysis. P-value *<0.05, **<0.005, ***<0,0005.

(10) FIGS. 10A-10B show the amount of TNF-α measured by real-time PCR as described in Example 10, in HCEC cells not treated (vehicle) or treated with the peptide of SEQ ID NO: 2 (FIG. 10A) or with the peptide HAP (SEQ ID NO: 1) (FIG. 10B). The error bars represent the standard deviation. T-test was performed as statistical analysis. P-value *<0.05, **<0.005, ***<0,0005.

DEFINITIONS

(11) Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those persons skilled in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference; thus, the inclusion of such definitions herein should not be construed to represent a substantial difference over what is generally understood in the art.

(12) The term “pharmaceutically acceptable excipient” refer to substances other than the active pharmaceutical ingredient (API) that have been appropriately evaluated for safety and are intentionally included in a drug delivery system. Pharmaceutically acceptable excipients are well known in the prior art and are disclosed, for example in the Handbook of Pharmaceutical Excipients, seventh edition 2013, which is included here for reference (Rowe, Sheskey et al. 2012).

(13) Excipients are normally classified according to the function that they have in the final pharmaceutical composition. Preferably, suitable excipients according to the present invention are for example diluent, adsorbent, glidant, binder, lubricant, surfactant, disintegrating, preservatives, antioxidant or mixtures thereof.

(14) The terms “approximately” and “about” herein refers to the range of the experimental error, which may occur in a measurement.

(15) The terms “comprising”, “having”, “including” and “containing” are to be construed open-ended terms (i.e. meaning “including, but not limited to”) and are to be considered as providing support also for terms as “consist essentially of”, “consisting essentially of”, “consist of” or “consisting of”.

(16) The terms “consist essentially of”, “consisting essentially of” are to be construed as semi-closed terms, meaning that no other ingredients which materially affects the basic and novel characteristics of the invention are included (optional excipients may thus be included).

(17) The terms “consists of”, “consisting of” are to be construed as closed terms.

(18) The term “bioconjugate” as used herein refers to a conjugate formed by a stable covalent link between different molecules, preferably two molecules, optionally linked by means of a spacer, at least one of which is a biomolecule.

(19) The term “biomolecule” as used herein refers to molecules of biological origin. The term includes macromolecules, such as carbohydrates, lipids and proteins or small natural products. For the purpose of the present invention “biomolecules” are preferably selected from ascorbic acid, capric acid, capronic acid, N-Acetyl-Glucosamine (also referred to as NAG), N-Acetylmuramic acid (also referred to as NAM), hyaluronic acid, alginic acid, chitin, (GaINAc).sub.2, GaI-alpha1,3-GaINAc or trigalacturonic acid

(20) The definition “conservative substitution” herein refers to a conservative replacement (also called a conservative mutation or a conservative substitution) is an amino acid replacement that changes a given amino acid to a different amino acid with similar biochemical properties (Simon French 1983).

DESCRIPTION OF THE INVENTION

(21) The present inventors have identified a number of peptides that are able to bind with high affinity to IL-17A and inhibit its interaction with the ILRA receptor. These compounds act as inhibitors of IL-17A signaling.

(22) Accordingly, a first object of the present invention is a peptide, able to inhibit IL-17A binding to ILRA, having the aminoacid sequence of either:

(23) formula (I):

(24) TABLE-US-00001 (I) (SEQ ID NO: 258) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15

(25) wherein, independently from each other:

(26) X.sub.1 is I, V, D, K, W, A, G, L or P;

(27) X.sub.2 is H, M, K, N, R, E, Q, W or Y;

(28) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(29) X.sub.4 is T, Q, H, S, N or Y;

(30) X.sub.5 is I, F, A, G, L, P, V or Y;

(31) X.sub.6 is P, G, A, L, V, I or N;

(32) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(33) X.sub.8 is D, E, N, Q or Y;

(34) X.sub.6 is L, V, F, W, A, G, P, I or H;

(35) X.sub.10 is W, Y, F or Q or it is absent;

(36) X.sub.11 is D, E or N or it is absent;

(37) X.sub.12 is W, F, V, H or Y or it is absent;

(38) X.sub.13 is I, V, F, E, K, A, G, L, P or Y or its absent;

(39) X.sub.14 is N, R, E, F, Q or D or it is absent;

(40) X.sub.15 is K, R, E, F, V, W, H or D or it is absent;

(41) with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO: 1) or a 12-14 aminoacid long C-terminal truncated sequence thereof IHVTIPADLWDWIN (SEQ ID NO: 259), IHVTIPADLWDWI (SEQ ID NO: 260), or IHVTIPADLWDW (SEQ ID NO: 261);

(42) or formula (II):

(43) TABLE-US-00002 (II) a.sub.n-DLSAVCWAFPWDPECH-b.sub.n, (an-SEQ ID NO: 41-bn′

(44) wherein, independently from each other:

(45) a, b are selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y or V;

(46) n=integer from 0 to 3;

(47) n′=integer from 0 to 3.

(48) Preferably, in formula (II) each of the aminoacids different from a or b can be replaced with a conservative substitution.

(49) A preferred peptide of formula (II) is DLSAVCWAFPWDPECH (SEQ ID NO: 41).

(50) Particularly preferred are the peptides of formula (I).

(51) According to a preferred embodiment, in said peptide of formula (I):

(52) X.sub.1 is I, V, D, K or W;

(53) X.sub.2 is H, M, K or N;

(54) X.sub.3 is V or F;

(55) X.sub.4 is T, Q, H, V or Y;

(56) X.sub.5 is I or F;

(57) X.sub.6 is P or G;

(58) X.sub.7 is A or Q;

(59) X.sub.8 is D, E, N;

(60) X.sub.9 is L, V, F or W;

(61) X.sub.10 is W, Y, F or it is absent;

(62) X.sub.11 is D, E, N or it is absent;

(63) X.sub.12 is W, F, V or it is absent;

(64) X.sub.13 is I, V, F or it is absent;

(65) X.sub.14 is N, R, E, F or it is absent;

(66) X.sub.15 is K, R, E, F, V, W or it is absent;

(67) with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO: 1) or a 12-14 aminoacid long C-terminal truncated sequence thereof IHVTIPADLWDWIN (SEQ ID NO: 259), IHVTIPADLWDWI (SEQ ID NO: 260), or IHVTIPADLWDW (SEQ ID NO: 261).

(68) According to an alternative preferred embodiment, in said peptide of formula (I):

(69) X.sub.1 is I, V, A, G, L, or P;

(70) X.sub.2 is H, M, K, R, E, Q, W or Y;

(71) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(72) X.sub.4 is T, Q, S, N or Y;

(73) X.sub.5 is I, F, A, G, L, P, V or Y;

(74) X.sub.6 is P, G, A, L, V, I or N;

(75) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(76) X.sub.8 is D, E, N, Q or Y;

(77) X.sub.9 is L, V, F, W, A, G, P, I or H;

(78) X.sub.10 is W, Y, F or Q or it is absent;

(79) X.sub.11 is D, E or N or it is absent;

(80) X.sub.12 is W, F, H or Y or it is absent;

(81) X.sub.13 is I, V, F, E, K, A, G, L, P or Y or its absent;

(82) X.sub.14 is N, R, E, Q or D or it is absent;

(83) X.sub.15 is K, R, E, V, W, H or D or it is absent;

(84) with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO: 1) or a 12-14 aminoacid long C-terminal truncated sequence thereof IHVTIPADLWDWIN (SEQ ID NO:259), IHVTIPADLWDWI (SEQ ID NO:260), or IHVTIPADLWDW (SEQ ID NO:261).

(85) Preferably, in the above peptide of formula (I) all aminoacids X.sub.1 to X.sub.15 are present and the above described peptides of formula (I) have a 15 aminoacid sequence. Preferably, in the peptides of formula (I) having a sequence of less than 15 aminoacids, when one aminoacid is absent, also all aminoacids at the N terminal of said aminoacid are absent. For example, if aminoacid X.sub.10 is absent, also aminoacids X.sub.11 to X.sub.15 are absent.

(86) Particularly preferred peptides of formula (I) are those wherein X.sub.1 is V.

(87) A particular preferred peptide having X.sub.1 as V is the peptide of SEQ ID NO: 2.

(88) As will be demonstrated in the experimental section, the present inventors have found that the peptide of SEQ ID NO: 2, where the first aminoacid Isoleucin in the peptide HAP of SEQ ID NO:1 has been replaced by a Valine, surprisingly shows strikingly different functional and chemical-physical properties as well as improved tolerability compared to HAP.

(89) In particular, as demonstrated in the Examples, the peptide shows improved chemical-physical properties, that are particularly advantageous for topical and ophtalmic use, a better permeability and an increased affinity for IL-17A compared to the corresponding peptide of SEQ ID NO: 1. Also, the inventors have found that the peptide HAP, independently from its activity on IL-17A signaling, exerts a direct toxicity effect on corneal cells by inducing the expression of inflammatory cytokines. Surprisingly, the peptide of SEQ ID NO: 2 does not show this effect and therefore is characterized by an improved tolerability compared to HAP.

(90) Preferably, in the above peptide of formula (I):

(91) X.sub.1 is I, V or L;

(92) X.sub.2 is H, M, R, K or E;

(93) X.sub.3 is V, F or I;

(94) X.sub.4 is T, Q, S, Y or N;

(95) X.sub.5 is I, F or V;

(96) X.sub.6 is P;

(97) X.sub.7 is A, Q, or L;

(98) X.sub.9 is D, E, or Q;

(99) X.sub.9 is L, W, F, V or I;

(100) X.sub.10 is W, Y or F;

(101) X.sub.11 is D, E or N;

(102) X.sub.12 is W or F;

(103) X.sub.13 is I, V, F or L;

(104) X.sub.14 is N, R, Q or E;

(105) X.sub.15 is K, R, H or E; with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO: 1);

(106) or

(107) X.sub.1 is I or V;

(108) X.sub.2 is H, M or R,

(109) X.sub.3 is V or F;

(110) X.sub.4 is T or Q;

(111) X.sub.5 is I, F or V;

(112) X.sub.6 is P or G;

(113) X.sub.7 is A or Q;

(114) X.sub.8 D or E;

(115) X.sub.9 is L;

(116) X.sub.10 is W or Y;

(117) X.sub.11 is D or E;

(118) X.sub.12 is W;

(119) X.sub.13 is I or V;

(120) X.sub.14 is N, R or E;

(121) X.sub.15 is K, R or E;

(122) with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO: 1);

(123) or

(124) X.sub.1 is I or V;

(125) X.sub.2 is H or M,

(126) X.sub.3 is V;

(127) X.sub.4 is T;

(128) X.sub.5 is I;

(129) X.sub.6 is P;

(130) X.sub.7 is A;

(131) X.sub.8 is D;

(132) X.sub.9 is L, W, F, V or I;

(133) X.sub.10 is W or Y;

(134) X.sub.11 is D or E;

(135) X.sub.12 is W;

(136) X.sub.13 is I or V;

(137) X.sub.14 is N, R or E;

(138) X.sub.15 is K, R or E;

(139) with the proviso that said sequence is not IHVTIPADLWDWINK (SEQ ID NO: 1);

(140) or

(141) X.sub.1 is I, V, A, G, L, or P;

(142) X.sub.2 is M, K, R, E, Q, W or Y;

(143) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(144) X.sub.4 is T, Q, S, N or Y;

(145) X.sub.5 is I, F, A, G, L, P, V or Y;

(146) X.sub.8 is P, G, A, L, V, I or N;

(147) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(148) X.sub.8 is D, E, N, Q or Y;

(149) X.sub.9 is L, V, F, W, A, G, P, I or H;

(150) X.sub.10 is Y, F or Q;

(151) X.sub.11 is D, E or N;

(152) X.sub.12 is W, F, H or Y;

(153) X.sub.13 is I, V, F, E, K, A, G, L, P;

(154) X.sub.14 is N, R, E, Q or D;

(155) X.sub.15 is K, R, E, V, W, H or D;

(156) or

(157) X.sub.1 is V, A, G, L, or P;

(158) X.sub.2 is H, M, K, R, E, Q, W or Y;

(159) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(160) X.sub.4 is T, Q, S, N or Y;

(161) X.sub.5 is I, F, A, G, L, P, V or Y;

(162) X.sub.8 is P, G, A, L, V, I or N;

(163) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(164) X.sub.8 is D, E, N, Q or Y;

(165) X.sub.9 is L, V, F, W, A, G, P, I or H;

(166) X.sub.10 is Y, F or Q;

(167) X.sub.11 is D, E or N;

(168) X.sub.12 is W, F, H or Y;

(169) X.sub.13 is I, V, F, E, K, A, G, L, P or Y;

(170) X.sub.14 is N, R, E, Q or D;

(171) X.sub.15 is K, R, E, V, W, H or D;

(172) or

(173) X.sub.1 is V, A, G, L, or P;

(174) X.sub.2 is M, K, R, E, Q, W or Y;

(175) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(176) X.sub.4 is T, Q, S, N or Y;

(177) X.sub.5 is I, F, A, G, L, P, V or Y;

(178) X.sub.6 is P, G, A, L, V, I or N;

(179) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(180) X.sub.8 is D, E, N, Q or Y;

(181) X.sub.9 is L, V, F, W, A, G, P, I or H;

(182) X.sub.10 is Y, F or Q;

(183) X.sub.11 is D, E or N;

(184) X.sub.12 is W, F, H or Y;

(185) X.sub.13 is I, V, F, E, K, A, G, L, P or Y;

(186) X.sub.14 is N, R, E, Q or D;

(187) X.sub.15 is K, R, E, V, W, H or D;

(188) or

(189) X.sub.1 is V, A, G, L, or P;

(190) X.sub.2 is H, M, K, R, E, Q, W or Y;

(191) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(192) X.sub.4 is T, Q, S, N or Y;

(193) X.sub.5 is I, F, A, G, L, P, V or Y;

(194) X.sub.6 is P, G, A, L, V, I or N;

(195) X.sub.7 is A, O, G, L, P, V, I, N or E;

(196) X.sub.8 is D, E, N, Q or Y;

(197) X.sub.9 is L, V, F, W, A, G, P, I or H;

(198) X.sub.10 is W, Y, F or Q;

(199) X.sub.11 is D, E or N;

(200) X.sub.12 is W, F, H or Y;

(201) X.sub.13 is V, F, E, K, A, G, L, P or Y;

(202) X.sub.14 is N, R, E, Q or D;

(203) X.sub.15 is K, R, E, V, W, H or D;

(204) or

(205) X.sub.1 is I, V, A, G, L, or P;

(206) X.sub.2 is M, K, R, E, Q, W or Y;

(207) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(208) X.sub.4 is T, Q, S, N or Y;

(209) X.sub.5 is I, F, A, G, L, P, V or Y;

(210) X.sub.6 is P, G, A, L, V, I or N;

(211) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(212) X.sub.8 is D, E, N, Q or Y;

(213) X.sub.9 is L, V, F, W, A, G, P, I or H;

(214) X.sub.10 is W, Y, F or Q;

(215) X.sub.11 is D, E or N;

(216) X.sub.12 is W, F, H or Y;

(217) X.sub.13 is V, F, E, K, A, G, L, P or Y;

(218) X.sub.14 is N, R, E, Q or D;

(219) X.sub.15 is K, R, E, V, W, H or D;

(220) or

(221) X.sub.1 is I, V, A, G, L, or P;

(222) X.sub.2 is H, M, K, R, E, Q, W or Y;

(223) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(224) X.sub.4 is T, Q, S, N or Y;

(225) X.sub.5 is I, F, A, G, L, P, V or Y;

(226) X.sub.6 is P, G, A, L, V, I or N;

(227) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(228) X.sub.8 is D, E, N, Q or Y;

(229) X.sub.9 is L, V, F, W, A, G, P, I or H;

(230) X.sub.10 is Y, F or Q;

(231) X.sub.11 is D, E or N;

(232) X.sub.12 is W, F, H or Y;

(233) X.sub.13 is V, F, E, K, A, G, L, P or Y;

(234) X.sub.14 is N, R, E, Q or D;

(235) X.sub.15 is K, R, E, V, W, H or D;

(236) or

(237) X.sub.1 is I, V, A, G, L, or P;

(238) X.sub.2 is M, K, R, E, Q, W or Y;

(239) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(240) X.sub.4 is T, Q, S, N or Y;

(241) X.sub.5 is I, F, A, G, L, P, V or Y;

(242) X.sub.6 is P, G, A, L, V, I or N;

(243) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(244) X.sub.8 is D, E, N, Q or Y;

(245) X.sub.9 is L, V, F, W, A, G, P, I or H;

(246) X.sub.10 is Y, F or Q;

(247) X.sub.11 is D, E or N;

(248) X.sub.12 is W, F, H or Y;

(249) X.sub.13 is V, F, E, K, A, G, L, P or Y;

(250) X.sub.14 is N, R, E, Q or D;

(251) X.sub.15 is K, R, E, V, W, H or D;

(252) or

(253) X.sub.1 is V, A, G, L, or P;

(254) X.sub.2 is H, M, K, R, E, Q, W or Y;

(255) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(256) X.sub.4 is T, Q, S, N or Y;

(257) X.sub.5 is I, F, A, G, L, P, V or Y;

(258) X.sub.6 is P, G, A, L, V, I or N;

(259) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(260) X.sub.8 is D, E, N, Q or Y;

(261) X.sub.9 is L, V, F, W, A, G, P, I or H;

(262) X.sub.10 is Y, F or Q;

(263) X.sub.11 is D, E or N;

(264) X.sub.12 is W, F, H or Y;

(265) X.sub.13 is V, F, E, K, A, G, L, P or Y;

(266) X.sub.14 is N, R, E, Q or D;

(267) X.sub.15 is K, R, E, V, W, H or D;

(268) or

(269) X.sub.1 is V, A, G, L, or P;

(270) X.sub.2 is M, K, R, E, Q, W or Y;

(271) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(272) X.sub.4 is T, Q, S, N or Y;

(273) X.sub.5 is I, F, A, G, L, P, V or Y;

(274) X.sub.6 is P, G, A, L, V, I or N;

(275) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(276) X.sub.8 is D, E, N, Q or Y;

(277) X.sub.9 is L, V, F, W, A, G, P, I or H;

(278) X.sub.10 is Y, F or Q;

(279) X.sub.11 is D, E or N;

(280) X.sub.12 is W, F, H or Y;

(281) X.sub.13 is V, F, E, K, A, G, L, P or Y;

(282) X.sub.14 is N, R, E, Q or D;

(283) X.sub.15 is K, R, E, V, W, H or D;

(284) or

(285) X.sub.1 is I, V, A, G, L or P;

(286) X.sub.2 is M, K, N, R, E, Q, W or Y;

(287) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(288) X.sub.4 is T, Q, S, Nor Y;

(289) X.sub.5 is I, F, A, G, L, P, V or Y;

(290) X.sub.6 is P, G, A, L, V, I or N;

(291) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(292) X.sub.8 is D, E, N, Q or Y;

(293) X.sub.9 is L, V, F, W, A, G, P, I or H;

(294) X.sub.10 is W, Y, F or Q;

(295) X.sub.11 is E or N;

(296) X.sub.12 is W, F, H or Y;

(297) X.sub.13 is I, V, F, E, K, A, G, L, P or Y;

(298) X.sub.14 is N, R, E, Q or D;

(299) X.sub.15 is K, R, E, V, W, H or D;

(300) or

(301) X.sub.1 is I, V, A, G, L or P;

(302) X.sub.2 is H, M, K, N, R, E, Q, W or Y;

(303) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(304) X.sub.4 is T, Q, S, N or Y;

(305) X.sub.5 is I, F, A, G, L, P, V or Y;

(306) X.sub.6 is P, G, A, L, V, I or N;

(307) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(308) X.sub.8 is D, E, N, Q or Y;

(309) X.sub.9 is L, V, F, W, A, G, P, I or H;

(310) X.sub.10 is W, Y, F or O;

(311) X.sub.11 is E or N;

(312) X.sub.12 is W, F, H or Y;

(313) X.sub.13 is I, V, F, E, K, A, G, L, P or Y;

(314) X.sub.14 is N, R, E, Q or D;

(315) X.sub.15 is K, R, E, V, W, H or D;

(316) or

(317) X.sub.1 is V, A, G, L or P;

(318) X.sub.2 is M, K, N, R, E, Q, W or Y;

(319) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(320) X.sub.4 is T, O, S, N or Y;

(321) X.sub.5 is I, F, A, G, L, P, V or Y;

(322) X.sub.6 is P, G, A, L, V, I or N;

(323) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(324) X.sub.8 is D, E, N, Q or Y;

(325) X.sub.9 is L, V, F, W, A, G, P, I or H;

(326) X.sub.10 is W, Y, F or Q;

(327) X.sub.11 is E or N;

(328) X.sub.12 is W, F, H or Y;

(329) X.sub.13 is I, V, F, E, K, A, G, L, P or Y;

(330) X.sub.14 is N, R, E, Q or D;

(331) X.sub.15 is K, R, E, V, W, H or D;

(332) or

(333) X.sub.1 is I, V, A, G, L, or P;

(334) X.sub.2 is H, M, K, R, E, Q, W or Y;

(335) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(336) X.sub.4 is T, Q, S, Nor Y;

(337) X.sub.5 is I, F, A, G, L, P, V or Y;

(338) X.sub.6 is P, G, A, L, V, I or N;

(339) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(340) X.sub.5 is D, E, N, Q or Y;

(341) X.sub.9 is L, V, F, W, A, G, P, I or H;

(342) X.sub.10 is Y, F or Q;

(343) X.sub.11 is E or N;

(344) X.sub.12 is W, F, H or Y;

(345) X.sub.13 is I, V, F, E, K, A, G, L, P or Y;

(346) X.sub.14 is N, R, E, Q or D;

(347) X.sub.15 is K, R, E, V, W, H or D;

(348) or

(349) X.sub.1 is I, V, A, G, L, or P;

(350) X.sub.2 is H, M, K, R, E, Q, W or Y;

(351) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(352) X.sub.4 is T, Q, S, N or Y;

(353) X.sub.5 is I, F, A, G, L, P, V or Y;

(354) X.sub.6 is P, G, A, L, V, I or N;

(355) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(356) X.sub.8 is D, E, N, Q or Y;

(357) X.sub.9 is L, V, F, W, A, G, P, I or H;

(358) X.sub.10 is W, Y, F or Q;

(359) X.sub.11 is E or N;

(360) X.sub.12 is W, F, H or Y;

(361) X.sub.13 is V, F, E, K, A, G, L, P or Y;

(362) X.sub.14 is N, R, E, Q or D;

(363) X.sub.15 is K, R, E, V, W, H or D;

(364) or

(365) X.sub.1 is I, V, A, G, L, or P;

(366) X.sub.2 is H, M, K, R, E, Q, W or Y;

(367) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(368) X.sub.4 is T, Q, S, N or Y;

(369) X.sub.5 is I, F, A, G, L, P, V or Y;

(370) X.sub.6 is P, G, A, L, V, I or N;

(371) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(372) X.sub.8 is D, E, N, Q or Y;

(373) X.sub.9 is L, V, F, W, A, G, P, I or H;

(374) X.sub.10 is Y, F or Q;

(375) X.sub.11 is E or N;

(376) X.sub.12 is W, F, H or Y;

(377) X.sub.13 is V, F, E, K, A, G, L, P or Y;

(378) X.sub.14 is N, R, E, Q or D;

(379) X.sub.15 is K, R, E, V, W, H or D;

(380) or

(381) X.sub.1 is I, V, A, G, L, or P;

(382) X.sub.2 is H, M, K, R, E, Q, W or Y;

(383) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(384) X.sub.4 is T, Q, S, N or Y;

(385) X.sub.5 is I, F, A, G, L, P, V or Y;

(386) X.sub.6 is P, G, A, L, V, I or N;

(387) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(388) X.sub.8 is D, E, N, Q or Y;

(389) X.sub.9 is L, V, F, W, A, G, P, I or H;

(390) X.sub.10 is W, Y, F or Q;

(391) X.sub.11 is D, E or N;

(392) X.sub.12 is W, F, H or Y;

(393) X.sub.13 is I, V, F, E, K, A, G, L, P or Y;

(394) X.sub.14 is R, E, Q or D;

(395) X.sub.15 is R, E, V, W, H or D;

(396) or

(397) X.sub.1 is V, A, G, L, or P;

(398) X.sub.2 is H, M, K, R, E, Q, W or Y;

(399) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(400) X.sub.4 is T, Q, S, Nor Y;

(401) X.sub.5 is I, F, A, G, L, P, V or Y;

(402) X.sub.6 is P, G, A, L, V, I or N;

(403) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(404) X.sub.8 is D, E, N, Q or Y;

(405) X.sub.9 is L, V, F, W, A, G, P, I or H;

(406) X.sub.10 is W, Y, F or Q;

(407) X.sub.11 is D, E or N;

(408) X.sub.12 is W, F, H or Y;

(409) X.sub.13 is I, V, F, E, K, A, G, L, P or Y;

(410) X.sub.14 is R, E, Q or D;

(411) X.sub.15 is R, E, V, W, H or D;

(412) or

(413) X.sub.1 is I, V, A, G, L, or P;

(414) X.sub.2 is M, K, R, E, Q, W or Y;

(415) X.sub.3 is V, F, A, G, L, P, I, Y or W;

(416) X.sub.4 is T, Q, S, N or Y;

(417) X.sub.5 is I, F, A, G, L, P, V or Y;

(418) X.sub.6 is P, G, A, L, V, I or N;

(419) X.sub.7 is A, Q, G, L, P, V, I, N or E;

(420) X.sub.8 is D, E, N, Q or Y;

(421) X.sub.9 is L, V, F, W, A, G, P, I or H;

(422) X.sub.10 is W, Y, F or Q;

(423) X.sub.11 is D, E or N;

(424) X.sub.12 is W, F, H or Y;

(425) X.sub.13 is I, V, F, E, K, A, G, L, P or Y;

(426) X.sub.14 is R, E, Q or D;

(427) X.sub.15 is R, E, V, W, H or D.

(428) Particularly preferred peptides of formula (I) among those described above are those wherein

(429) X.sub.1 is V.

(430) As will be demonstrated in the experimental section, when the first aminoacid in the peptide of formula (I) is a valine, the peptide surprisingly shows chemical-physical properties particularly advantageous for topical and ophthalmic use and an increased affinity for IL-17A.

(431) Preferably, in the above peptide of formula (I) all aminoacids X.sub.1 to X.sub.15 are present. Preferred individual peptides of formula (I) according to the present invention are listed in Table 1 below.

(432) TABLE-US-00003 TABLE 1 SEQ ID NO: Sequence SEQ ID NO: 2 VHVTIPADLWDWINK SEQ ID NO: 3 IMVTIPADLWDWINK SEQ ID NO: 4 VMVTIPADLWDWINK SEQ ID NO: 5 IHVTIPAELWDWINK SEQ ID NO: 6 IHVTIPADLYDWINK SEQ ID NO: 7 IHVTIPADLYEWINK SEQ ID NO: 8 IHVTIPADLWDWVNK SEQ ID NO: 9 IHVTIPADLWDWIRK SEQ ID NO: 10 IHVTIPADLWDWINR SEQ ID NO: 11 IHVTIPADLWDWIEK SEQ ID NO: 12 IHVTIPADLWDWINE SEQ ID NO: 13 IHVTIPADLYEWINK SEQ ID NO: 14 IHVTIPADLWDWVRR SEQ ID NO: 15 IHVTIPADLWDWVEE SEQ ID NO: 16 VMVTIPADLYEWINK SEQ ID NO: 17 VMVTIPADLYEWIRR SEQ ID NO: 18 VMVTIPADLYEWIEE SEQ ID NO: 42 IMVTIPADLYEWIEE SEQ ID NO: 111 VHVTIPAELWEWVRR SEQ ID NO: 112 VHFTIPADLWEWVRR SEQ ID NO: 113 VHVQIPADLWEWVRR SEQ ID NO: 114 VHVTFPADLWEWVRR SEQ ID NO: 115 VHVTIPQDLWEWVRR SEQ ID NO: 116 VHVTIPANLWEWVRR SEQ ID NO: 117 VHVTIPADFWEWVRR SEQ ID NO: 118 VHVTIPADLYEWVRR SEQ ID NO: 119 VHVTIPADLWNWVRR SEQ ID NO: 120 VHVTIPADLWEFVRR SEQ ID NO: 121 VHVTIPADLWEWFRR SEQ ID NO: 122 VHVYIPAELWEWVRR SEQ ID NO: 123 VHVTIPAEWWEWVRR SEQ ID NO: 124 VHFTFPQDLWEWVRR SEQ ID NO: 125 VHFTFPQDFWEWVRR SEQ ID NO: 126 VHFTIPQDLYEWVRR SEQ ID NO: 127 VHFTFPQDLWNWVRR SEQ ID NO: 128 VHFTFPQDLWEFVRR SEQ ID NO: 129 VHFTFPQDLWEWFRR SEQ ID NO: 130 VHFQFPADLWEWVRR SEQ ID NO: 131 VHFQFPADFWEWVRR SEQ ID NO: 132 VHFQFPADLYEWVRR SEQ ID NO: 133 VHFQFPADLWNWVRR SEQ ID NO: 134 VHFQFPADLWEFVRR SEQ ID NO: 135 VHFQFPADLWEWFRR SEQ ID NO: 136 VHFQFPQDWWEWVRR SEQ ID NO: 137 VHFQIPQDWWEWVRR SEQ ID NO: 138 VHFQFPQDWYEWVRR SEQ ID NO: 139 VHFQFPQDWWNWVRR SEQ ID NO: 140 VHFQFPQDLWEFVRR SEQ ID NO: 141 VHFQFPQDWWEWFRR SEQ ID NO: 142 VHFTIPADFWEWFRR SEQ ID NO: 143 VHVQIPADFWEWFRR SEQ ID NO: 144 VHVTFPADLWEWFRR SEQ ID NO: 145 VHVTIPQDFWEWFRR SEQ ID NO: 146 VHFTIPQDWWEWVRR SEQ ID NO: 147 VHFTFPQDLYNWVRR SEQ ID NO: 148 VHFTFPQDLYNFVRR SEQ ID NO: 149 VHVTIPADLYNFFRR SEQ ID NO: 150 VHFQFPQDLWEWVRR SEQ ID NO: 151 VHFTIPQDLYNWRR SEQ ID NO: 152 VHFTIPADLYNFVRR SEQ ID NO: 153 VHFQIPQDLYNFFRR SEQ ID NO: 154 VHFQFPQEWYNWFRR SEQ ID NO: 155 VRFQFGQEWYNFFRR SEQ ID NO: 158 AHVTIPADLWDWINK SEQ ID NO: 159 GHVTIPADLWDWINK SEQ ID NO: 160 LHVTIPADLWDWINK SEQ ID NO: 161 PHVTIPADLWDWINK SEQ ID NO: 162 IRVTIPADLWDWINK SEQ ID NO: 163 IKVTIPADLWDWINK SEQ ID NO: 164 IEVTIPADLWDWINK SEQ ID NO: 165 IQVTIPADLWDWINK SEQ ID NO: 166 IYVTIPADLWDWINK SEQ ID NO: 167 IHATIPADLWDWINK SEQ ID NO: 168 IHGTIPADLWDWINK SEQ ID NO: 169 IHLTIPADLWDWINK SEQ ID NO: 170 IHPTIPADLWDWINK SEQ ID NO: 171 IHITIPADLWDWINK SEQ ID NO: 172 IHYTIPADLWDWINK SEQ ID NO: 173 IHWTIPADLWDWINK SEQ ID NO: 174 IHFTIPADLWDWINK SEQ ID NO: 175 IHVSIPADLWDWINK SEQ ID NO: 176 IHVYIPADLWDWINK SEQ ID NO: 177 IHVNIPADLWDWINK SEQ ID NO: 178 IHVQIPADLWDWINK SEQ ID NO: 179 IHVTAPADLWDWINK SEQ ID NO: 180 IHVTGPADLWDWINK SEQ ID NO: 181 IHVTLPADLWDWINK SEQ ID NO: 182 IHVTPPADLWDWINK SEQ ID NO: 183 IHVTVPADLWDWINK SEQ ID NO: 184 IHVTFPADLWDWINK SEQ ID NO: 185 IHVTYPADLWDWINK SEQ ID NO: 186 IHVTIAADLWDWINK SEQ ID NO: 187 IHVTIGADLWDWINK SEQ ID NO: 188 IHVTILADLWDWINK SEQ ID NO: 189 IHVTIVADLWDWINK SEQ ID NO: 190 IHVTIIADLWDWINK SEQ ID NO: 191 IHVTINADLWDWINK SEQ ID NO: 192 IHVTIPGDLWDWINK SEQ ID NO: 193 IHVTIPLDLWDWINK SEQ ID NO: 194 IHVTIPPDLWDWINK SEQ ID NO: 195 IHVTIPVDLWDWINK SEQ ID NO: 196 IHVTIPIDLWDWINK SEQ ID NO: 197 IHVTIPNDLWDWINK SEQ ID NO: 198 IHVTIPQDLWDWINK SEQ ID NO: 199 IHVTIPAQLWDWINK SEQ ID NO: 200 IHVTIPAYLWDWINK SEQ ID NO: 201 IHVTIPADAWDWINK SEQ ID NO: 202 IHVTIPADGWDWINK SEQ ID NO: 203 IHVTIPADPWDWINK SEQ ID NO: 204 IHVTIPADVWDWINK SEQ ID NO: 205 IHVTIPADIWDWINK SEQ ID NO: 206 IHVTIPADLHDWINK SEQ ID NO: 207 IHVTIPADLFDWINK SEQ ID NO: 208 IHVTIPADLWNWINK SEQ ID NO: 209 IHVTIPADLWDHINK SEQ ID NO: 210 IHVTIPADLWDFINK SEQ ID NO: 211 IHVTIPADLWDYINK SEQ ID NO: 212 IHVTIPADLWDWENK SEQ ID NO: 213 IHVTIPADLWDWKNK SEQ ID NO: 214 IHVTIPADLWDWANK SEQ ID NO: 215 IHVTIPADLWDWGNK SEQ ID NO: 216 IHVTIPADLWDWLNK SEQ ID NO: 217 IHVTIPADLWDWPNK SEQ ID NO: 218 IHVTIPADLWDWFNK SEQ ID NO: 219 IHVTIPADLWDWYNK SEQ ID NO: 220 IHVTIPADLWDWIQK SEQ ID NO: 221 IHVTIPADLWDWIDK SEQ ID NO: 222 IHVTIPADLWDWINH SEQ ID NO: 223 IHVTIPADLWDWIND SEQ ID NO: 224 VHVTVPQELWEWVRR SEQ ID NO: 225 VHVTVPQELFEWVRR SEQ ID NO: 226 VHVTVPQELYEWVRR SEQ ID NO: 227 VHVTVPQELWEWVEE SEQ ID NO: 228 VHVTVPQELFEWVEE SEQ ID NO: 229 VHVTVPQELYEWVEE SEQ ID NO: 230 VHVSIPQELWEWVRR SEQ ID NO: 231 VHVSVPQELWEWVRR SEQ ID NO: 232 VHVSVPQELYEWVRR SEQ ID NO: 233 VRVTIPQELWEWVRR SEQ ID NO: 234 VRVTVPQELYEWVRR SEQ ID NO: 235 VRVTVPQELWEWVRR SEQ ID NO: 236 VHVTVPQEIYEWVRR SEQ ID NO: 237 VHVTIPQEIWEWVRR SEQ ID NO: 238 VHFTVPQELYEWVRR SEQ ID NO: 239 VKISVPADLWDWINK SEQ ID NO: 240 LRISVPADLWDWINK SEQ ID NO: 241 LRIYVPADLWDWINK SEQ ID NO: 242 VRGYVPADLWDWINK SEQ ID NO: 243 VRAYVPADLWDWINK SEQ ID NO: 244 VRLYVPADLWDWINK SEQ ID NO: 245 VRIYLPADLWDWINK SEQ ID NO: 246 IHVTIPLEIFEWLQH SEQ ID NO: 247 IHVTIPLEIFEWAQH SEQ ID NO: 248 IHVTIPLEIFEWLQR SEQ ID NO: 249 IHVTIPLEVFEWLQH SEQ ID NO: 250 IHVTIPGEIFEWLQH SEQ ID NO: 251 VRFSVPQEIYEWVRR SEQ ID NO: 252 LRISVPLEIFEWLQH SEQ ID NO: 253 LRGSVPLEIFEWLQH SEQ ID NO: 254 VKISVPLEIFEWLQH SEQ ID NO: 255 VEFNFPQQVYEWFDD SEQ ID NO: 256 VEFNFPQQVYEWVRR SEQ ID NO: 257 TVVYVFNEQHQEYVRK

(433) Other peptides inhibitor of IL-17A according to the invention are those of Table 1A

(434) TABLE-US-00004 TABLE 1A SEQ ID NO: 156 VPGAGVPGAGIHVTI SEQ ID NO: 157 VPGAGVPGAGIHVTIPA

(435) Further aminoacid sequences may be added at the N- or C-terminal of the peptides according to the first object of the invention.

(436) According to a preferred embodiment, the above described peptides of formula (I) or (II) are bound at their N- and/or C-terminal to a further aminoacid sequence, Sequence A, having the aminoacid sequence of formula (III)
Y.sub.1-Y.sub.2-Y.sub.3-Y.sub.4−Y.sub.5-Y.sub.6-Y.sub.7-Y.sub.8-Y.sub.9-Y.sub.10-Y.sub.11-Y.sub.12-Y.sub.13-Y.sub.14-Y.sub.15-Y.sub.16  (III)
wherein:

(437) Y.sub.1 is A, T, V, K, R, I, L, X or G;

(438) Y.sub.2 is R, W, P, E, Q or A;

(439) Y.sub.3 is K, W, G, T, I, R or P;

(440) Y.sub.4 is K, T, E, W, A, R, D, G, X or F;

(441) Y.sub.5 is A, E, T, G, W, I, R, P or V;

(442) Y.sub.6 is A, W, E, R, G, P or absent;

(443) Y.sub.7 is K, S, W, T, F, R, V, G or absent;

(444) Y.sub.8 is A, Q, W, R, G or absent;

(445) Y.sub.9 is N, G or absent;

(446) Y.sub.10 is R or absent;

(447) Y.sub.11 is R or absent;

(448) Y.sub.12 is M or absent;

(449) Y.sub.13 is K or absent;

(450) Y.sub.14 is W or absent;

(451) Y.sub.15 is K or absent;

(452) Y.sub.16 is K or absent.

(453) Preferably, in the sequence of formula (III), when an aminoacid is absent, also the aminoacids at the N terminal of said aminoacids are absent. For example, if aminoacid Y.sub.6 is absent, also aminoacids Y.sub.7 to Y.sub.16 are absent.

(454) Preferred sequences of formula (III) according to the present invention are listed in Table 2 below.

(455) TABLE-US-00005 TABLE 2 SEQUENCE ID Sequence SEQ ID NO: 44 ARKKAAKA SEQ ID NO: 45 TWWTEWSQ SEQ ID NO: 46 TWWETWW SEQ ID NO: 47 VPGWG SEQ ID NO: 48 KETWWETW SEQ ID NO: 49 VPGKG SEQ ID NO: 50 VPGAG SEQ ID NO: 51 RQIKIWFQNRRMKWKK SEQ ID NO: 52 RRRRRRRR SEQ ID NO: 53 VPGDG SEQ ID NO: 54 VPGXG SEQ ID NO: 55 IPGG SEQ ID NO: 56 AVGVP SEQ ID NO: 57 IPGXG SEQ ID NO: 58 IPGVG SEQ ID NO: 59 LPGXG SEQ ID NO: 60 LPGVG SEQ ID NO: 61 VAPGVG SEQ ID NO: 62 XPGVG SEQ ID NO: 63 GVGVPGVG SEQ ID NO: 64 VPGFGVGAG SEQ ID NO: 65 VPGGVPGG

(456) Accordingly, a second object of the invention is a peptide having an aminoacid sequence comprising, preferably consisting of, the sequence of the peptide of formula (I) or (II) according to the first object of the invention and at the C- and/or N-terminal of said sequence a Sequence A as above described.

(457) The aminoacid sequence Sequence A may be bound to both the N- and C-terminal of the peptide of formula (I) or (II). In such a case, the two aminoacid sequences Sequence A bound to the N- and to C-terminal of said peptide may be the same or different.

(458) Alternatively, the aminoacid sequence Sequence A may be bound either to the N- or to the C-terminal of the peptide.

(459) Preferred individual peptides according to this object of the invention are listed in Table 3 below.

(460) TABLE-US-00006 TABLE 3 SEQ ID NO: Sequence SEQ ID NO: 21 VMVTIPADLYEWIEEARKKAAKA SEQ ID NO: 22 VMVTIPADLYEWIEEGGTWWTEWSQ SEQ ID NO: 23 VMVTIPADLYEWIEETWWETWW SEQ ID NO: 24 VMVTIPADLYEWIEEVPGWG SEQ ID NO: 25 VMVTIPADLYEWIEEGGKETWWETW SEQ ID NO: 26 VMVTIPADLYEWIEEVPGKG SEQ ID NO: 27 VMVTIPADLYEWIEEVPGAG SEQ ID NO: 28 VMVTIPADLYEWIEERQIKIWFQNRRMKWKK SEQ ID NO: 31 ARKKAAKAVMVTIPADLYEWIEE SEQ ID NO: 32 GGTWWTEWSQVMVTIPADLYEWIEE SEQ ID NO: 33 GGKETWWETWVMVTIPADLYEWIEE SEQ ID NO: 34 VPGWGVMVTIPADLYEWIEE SEQ ID NO: 35 VPGAGVMVTIPADLYEWIEE SEQ ID NO: 36 VPGKGVMVTIPADLYEWIEE SEQ ID NO: 37 VMVTIPADLYEWIEEVPGAGVPGAG SEQ ID NO: 38 VPGAGVPGAGVMVTIPADLYEWIEE SEQ ID NO: 39 VPGDGVMVTIPADLYEWIEE SEQ ID NO: 40 VMVTIPADLYEWIEEVPGDG

(461) In an embodiment alternative to the above, said peptides of formula (I) or (II) according to the first object of the invention may be bound at their N- and/or C-terminal to an aminoacid sequence (A).sub.m, wherein A is an aminoacid selected from R, K, G, E, Q or A, preferably R, K, G, E or A, and m is an integer between 1 and 10.

(462) The aminoacid sequence (A).sub.m may be bound to both the N- and C-terminal of the peptide of formula (I) or (II). In such a case, the two aminoacid sequences (A).sub.m attached to the N- and to the C-terminal of said peptide have may be the same or different.

(463) Alternatively, the aminoacid sequence (A).sub.m may be attached either to the N- or the C-terminal of said peptide.

(464) Accordingly, a third object of the invention is a peptide having an aminoacid sequence that comprises, preferably consists of, the sequence of the peptide according to the first object of the invention and at the C- and/or N-terminal of said sequence a sequence (A).sub.m as above defined.

(465) Preferred individual peptides according to this embodiment of the invention are listed in Table 4 below.

(466) TABLE-US-00007 TABLE 4 SEQ ID NO: Sequence SEQ ID NO: 19 VMVTIPADLYEWIEERRRRRR SEQ ID NO: 20 VMVTIPADLYEWIEERRR SEQ ID NO: 29 RRRRRRRRVMVTIPADLYEWIEE SEQ ID NO: 30 VMVTIPADLYEWIEERRRRRRRR SEQ ID NO: 43 IMVTIPADLYEWIEEQ

(467) In a particular embodiment, said peptides of formula (I) or (II) are bound to both a sequence Sequence A and a sequence (A).sub.m. In this case, said peptides are bound at their N- and/or C-terminal to an aminoacid sequence (A).sub.m, as defined above, which is bound at its N- and/or C-terminal to an aminoacid sequence Sequence A, as described above.

(468) Accordingly, a fourth object of the invention is a peptide having an aminoacid sequence that comprises, preferably consists of, the sequence of the peptide according to the first object of the invention, at the C- and/or N-terminal of said sequence a sequence (A).sub.m as above described and at the C- and/or N-terminal of said sequence (A).sub.m a Sequence A as above described.

(469) According to a preferred embodiment of the peptide according to any of the above objects of the invention, said peptide is bound at the C- and/or N-terminal to a protective cap group, able to prevent degradation.

(470) Any protective cap group customary in the art may be used.

(471) Preferably, the protective cap group bound to a C-terminal is selected from amides, preferably N-alkyl amides, aldehydes, esters, preferably methyl and ethyl esters, p-Nitroanilide, 7-Amino-4-methylcoumarin.

(472) Preferably, the protective group cap bound to a N-terminal is selected from acetyl, formyl, pyroglutamyl, fatty acids, urea, carbamate sulfonamide, alkylamine.

(473) Preferably, the above peptides are bound to the protective cap group at the N-terminal only. More preferably said peptides are bound to the protective cap group at the N-terminal only and the protective cap group is acetyl.

(474) It is particularly advantageous to prepare dimers of the above described peptides according any of the different objects of the invention, preferably of the peptides of formula (I) or (II) according to the first object of the invention, in order to increase binding affinity and inhibitor activity.

(475) In accordance with the above, preferably the peptides according to the different objects of the invention above described, preferably of the peptides of formula (I) or (II) according to the first object of the invention, more preferably the peptides of formula (I) according to the first object of the invention, are present in form of dimers.

(476) Accordingly, a fifth object of the present invention is a dimer formed by two peptides according to any of the above objects of the invention. Preferably said peptides are peptides of formula (I) or (II) according to the first object of the invention, more preferably they are peptides of formula (I) according to the first object of the invention.

(477) Preferably, said dimer is a homodimer, wherein the two peptides forming the dimer have an identical sequence. Preferably, in said dimer, the peptides are linked by means of a spacer molecule, preferably a polyethylene glycol spacer.

(478) Preferably, according to this embodiment, polyethylene glycol is linked to the N-terminal of the peptides according to the invention or to the aminoacids X.sub.4, X.sub.7 or X.sub.14 of two peptides of formula (I).

(479) According to a further embodiment, in the absence of a protective group cap, the peptides according to the different objects of the invention are bound, preferably at their C- and/or N-terminal, to a biomolecule to form a bioconjugate. Preferably, said biomolecule is selected from capric acid, capronic acid, ascorbic acid, NAG-NAM, NAG, NAM, hyaluronic acid, alginic acid, chitin, (GaINAc).sub.2, GaI-alpha1,3-GaINAc and trigalacturonic acid.

(480) The biomolecule is bound to the peptide either to add a specific function to the peptide or to modulate the physical chemical properties of the peptide. For example, ascorbic acid provides an antioxidant activity to the peptide, capronic acid favors the anchoring of the peptide to the cell membrane.

(481) Accordingly, a sixth object of the invention is a bioconjugate comprising, preferably consisting of, a peptide according to the different objects of the invention, preferably a peptide of formula (I) or (II) according to the first object of the invention, more preferably a peptide of formula (I) according to the first object of the invention, and at least a biomolecule, as above described.

(482) Preferably, in said bioconjugate, said biomolecule is bound to the N- and/or C-terminal of said peptide.

(483) Preferred bioconjugates according to this embodiment are listed in Table 5 below.

(484) TABLE-US-00008 TABLE 5 Bioconjugate name Sequence Bio-1 NAM-IMVTIPADLYEWIEE (NAM plus SEQ ID NO: 42) Bio-2 IMVTIPADLYEWIEEQ-NAG (SEQ ID NO: 42 plus NAG) Bio-3 NAG-NAM- IMVTIPADLYEWIEE (NAG-NAM plus SEQ ID NO: 42) Bio-4 capric acid- IMVTIPADLYEWIEE (capric acid plus SEQ ID NO: 42) Bio-5 Ascorbic acid IMVTIPADLYEWIEE (Ascorbic acid plus SEQ ID NO: 42)

(485) According to one embodiment, in the above bioconjugate the above biomolecule or protective cap group are bound directly to the N and/or C-terminal of the peptide.

(486) According to one embodiment, said biomolecule is bound to both the N- and C-terminal of the peptide. In such a case, the two biomolecules bound to the N and to the C terminal of said peptide may be the same or different.

(487) According to an alternative embodiment, said biomolecule is bound to either the N- or the C-terminal of the peptide.

(488) According to an alternative embodiment, the above biomolecule or protective cap group are linked to the N- and/or C terminal of the peptide by means of a linker. Preferably, said linker is selected from 4-aminobutyric acid, beta-alanine, 2-aminoethoxy-acetic acid, 5-aminovaleric acid, 6-aminocaproic acid, 8-Amino-3,6-dioxaoctanoic acid, 12-amino-4,7,10-trioxadodecanoic acid, 15-amino-4,7,10,13-tetraoxapenta-decanoic acid and trioxatridecan-succinamic acid. Preferably, when the linker is attached on one amino acid Lysine, said linker is selected from NHS-ester, isocyanates, benzoyl fluorides or carbamates.

(489) In accordance with to the different embodiments described above, the present invention relates to a compound comprising or consisting of the IL-17A binding peptide of formula (I) or (II) above described, and having the following formula (IV):
[Biomolecule or CAP].sub.a-[Linker].sub.b-(Sequence A).sub.n-A.sub.m-[Peptide]-A′.sub.m′-(Sequence A).sub.n′-[Linker].sub.b′-[Biomolecule or CAP].sub.a′  (IV)
wherein, independently from each other:

(490) a=0 or 1;

(491) b=0 or 1;

(492) a′=0 or 1;

(493) b′=0 or 1;

(494) m=0 to 10;

(495) m′=0 to 10;

(496) n=0 or 5;

(497) n′=0 or 5,

(498) and

(499) Peptide is the polypeptide according to formula (I) or formula (II);

(500) A or A′ is one amino acid selected from R, K, G, E or A repeated m or m′ times;

(501) and Sequence A comprises the sequence of formula (III) above described;

(502) Biomolecule is, independently from each other, capronic acid, ascorbic acid, NAG-NAM, NAG, NAM, hyaluronic acid, alginic acid, chitin, (GaINAc).sub.2, GaI-alpha1,3-GaINAc or trigalacturonic acid;

(503) CAP is, independently from each other, amide, aldehyde, ester, p-Nitroanilide, 7-Amino-4-Methylcoumarin, acetyl, formyl, pyroglutamyl or a fatty acid;

(504) Linker is, independently from each other, 4-aminobutyric acid, beta-alanine, 2-aminoethoxy-acetic acid, 5-aminovaleric acid, 6-aminocaproic acid, 8-Amino-3,6-dioxaoctanoic acid, 12-amino-4,7,10-trioxadodecanoic acid, 15-amino-4,7,10,13-tetraoxapenta-decanoic acid or trioxatridecan-succinamic acid.

(505) According to a preferred embodiment, said Sequence A is attached to the N-terminal or to the C-terminal of the peptide of formula (I) or (II).

(506) Some examples of the possible compounds according to the present invention are reported here below, but all the other possible combination comprised in the formula (IV) can be present: [Biomolecule or Cap]-[Linker]-(Sequence A)-A-[Peptide]-; [Biomolecule or Cap]-(Sequence A)-A-[Peptide]-; [Biomolecule or Cap]-A-[Peptide]-; [Biomolecule or Cap]-[Peptide]-; -[Peptide]-A.sub.m′-(Sequence A).sub.n-[Biomolecule or Cap].sub.a′; -[Peptide]-A.sub.m[Biomolecule or Cap].sub.a′; -[Peptide]-[Biomolecule or Cap].sub.a′;

(507) According to a preferred embodiment, the biomolecule in formula (IV) of the present invention is selected from ascorbic acid, capronic acid, NAG or NAM.

(508) According to a further preferred embodiment the Cap according to the present invention is a C-ter modification selected from amides, preferably N-alkyl amides, aldehydes, esters, preferably methyl and ethyl esters, p-Nitroanilide, 7-Amino-4-Methylcoumarin or N-ter modification selected from acetyl, formyl, pyroglutamyl, fatty acids, preferably capronic acid, urea, carbamate, sulfonamide or alkylamine, preferably said Cap is selected from amides, fatty acids as capronic acid and acetyl.

(509) According to the present invention, said linker, biomolecule or Cap in formula (IV) can be the same or different each other.

(510) In a further preferred embodiment of the present invention the Linker of formula (IV) is selected from 4-aminobutyric acid, beta-alaninine, 2-aminoethoxy-acetic acid, 5-aminovaleric acid or trioxatridecan-succinamic acid.

(511) Preferably, when the linker is attached on one amino acid Lysine, said linker is selected from NHS-ester, isocyanates, benzoyl fluorides or carbamates.

(512) A seventh object of the present invention is a pharmaceutical composition comprising said peptide, dimer or a bioconjugate according to the objects of the invention, as described above, and at least one pharmaceutically acceptable excipient.

(513) The pharmaceutical composition of the present invention can be formulated in a form that is suitable for topical or ophthalmic administration.

(514) Preferably, when the administration of the pharmaceutical composition of the invention is performed by topical route, the pharmaceutical form is selected from cream, ointment, gel, salve, solution, washing, suspension, drops, buffer (buffer solution), suspension, eye drops, drops, spray, wipe, or powder, preferably it is selected from cream, gel, spray, or ointment. Among the ophthalmic administration, the pharmaceutical form is preferably selected from eye drops, ophthalmic gels, ointments, wash, wipe, spray or cream.

(515) According to a particular embodiment the peptides or bioconjugates of the present invention are administered locally by using microparticles or nanoparticles.

(516) According to the invention, the pharmaceutical composition of the present invention can be administered to animals and humans, defined as adults and as “paediatric population”, wherein with the term “pediatric population” is indicated the part of the population from birth to eighteen years of age.

(517) An eighth object of the present invention is the above described peptide, dimer or bioconjugate according to the invention for use in the treatment and/or prevention of an inflammatory and autoimmune disease.

(518) A ninth object of the invention is a method for the treatment and/or prevention of an inflammatory and autoimmune disease, comprising administering to a patient in need thereof a therapeutically effective amount of the above described peptide, dimer or bioconjugate according to the invention.

(519) Preferably, said inflammatory and autoimmune disease is selected from rheumatoid arthritis, multiple sclerosis, Crohn's disease, systemic lupus erythematosus, asthma, Behçet's disease, hyper IgE syndrome, ankylosing spondylitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, keratoconjunctivitis sicca, vernal keratoconjunctivitis, stromal herpetic keratitis, corneal allograft rejection, corneal infections, preferably herpes virus and Pseudomonas aeruginosa keratitis, and dry eye disease. More preferably said disease is an autoimmune ophthalmic or dermatological disease, even more preferably it is dry eye disease or psoriasis.

(520) All of the above specified diseases and medical conditions have in common that their origin and/or symptoms are IL-17A and/or Th-17-related.

(521) More specifically for the latter, dry eye disease (DED), a highly prevalent condition that includes a wide spectrum of ocular surface disorders, ocular mucosal inflammation is a peculiar characteristic potentially leading to vision loss if uncontrolled, due to inflammation-induced corneal ulceration and scarring. In the progression of DED, pathogenic immune cells, predominantly Th17 cells continuously migrate to the ocular mucosal surface and secrete pro-inflammatory mediators, including IL17, causing ocular surface inflammation and epitheliopathy.

(522) The peptides, dimers or bioconjugates of the invention are conveniently and preferably administered topically as eye drops or ophthalmic gels and ointments.

(523) In a further embodiment, the peptide, dimer or bioconjugate for use according to the present invention, are administered as the only active principle or in combination with further active principles, and/or in combination with medical devices for the symptomatic treatment of ophthalmic conditions including but not limited to DED, e.g. ocular lubricants or “artificial tears”, topical re-epithelizing agents, therapeutic contacts lenses and punctum plugs.

(524) Preferably, said further active principle is an adjuvant, immunosuppressive agent, immunomodulating agent or anti-inflammatory agent.

(525) For example, the IL-17A binding peptide of the present invention may be used in combination with DMSO.

(526) According to a preferred embodiment, the IL-17A binding peptide of the present invention may be used in combination with immunosuppressive monoclonal antibodies, such as monoclonal antibodies with affinity to leukocyte receptors, selected from MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds, preferably a recombinant binding molecule having at least a portion of the extracellular domain of CTLA4 or a mutant thereof, an at least extracellular portion of CTLA4 or a mutant thereof joined to a non-CTLA4 protein sequence, e.g. (e.g. CTLA4Ig, designated ATCC 68629) or a mutant thereof, e.g. LEA29Y; adhesion molecule inhibitors, LFA-I antagonists, ICAM-I or -3 antagonists, VCAM-4 antagonists or VLA-4 antagonists.

(527) In a further preferred embodiment the IL-17A binding peptide of the present invention is used in combination with DMARD (disease-modifying antirheumatic drug), preferably Gold salts, sulphasalazine, anti-malarias, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, cyclosporine A, tacrolimus, sirolimus, minocycline, leflunomide, glucocorticoids; a calcineurin inhibitor, preferably cyclosporin A or FK 506; a modulator of lymphocyte recirculation, preferably FTY720 and FTY720 analogs; a mTOR inhibitor, preferably rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; an ascomycin having immuno-suppressive properties, preferably ABT-281, ASM981; corticosteroids; cyclophosphamide; azathioprene; methotrexate; leflunomide; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or an immunosuppressive homologue, analogue or derivative thereof; or a chemotherapeutic agent, preferably pacli-taxel, gemcitabine, cisplatinum, doxorubicin or 5-fluorouracil; anti-TNF agents, preferably monoclonal antibodies to TNF, preferably infliximab, adaiimumab, CDP870, or receptor constructs to TNF-RI or TNF-RII, preferably Etanercept, PEG-TNF-RI; blockers of proinflammatory cytokines, IL-1 blockers, preferably Anakinra or IL-1 trap, AAL160, ACZ 885, IL-6 blockers; inhibitors or activators of proteases, preferably metalloproteases, anti-IL-15 antibodies, anti-IL-6 antibodies, anti-IL-23 antibodies, anti-IL-22 antibodies, anti-IL-21 antibodies, anti-IL-12 antibodies, anti-IFN-gamma antibodies, anti-IFN-alpha antibodies, anti-CD20 antibodies, anti-inflammatory agents, preferably aspirin or an anti-infectious agent. Naturally, this list of agents for co-administration is not limiting nor complete.

(528) A further object of the present invention is one the peptides listed in Table 6 for use in the treatment and/or prevention of an inflammatory and autoimmune disease described above.

(529) TABLE-US-00009 TABLE 6 SEQ ID NO: SEQUENCE SEQ ID NO: 1 IHVTIPADLWDWINK SEQ ID NO: 66 IVVTMPADLWDWIKA SEQ ID NO: 67 IVVTMPADLWDWIRA SEQ ID NO: 68 IVVTMPADLWDWIRK SEQ ID NO: 69 IVVTMPADLWDWIAA SEQ ID NO: 70 IVVTMPADLWDWARA SEQ ID NO: 71 IVVTMPADLWAWIRA SEQ ID NO: 72 IVVTMPADLADWIRA SEQ ID NO: 73 IVVTMPADAWDWIRA SEQ ID NO: 74 IVVTMPADLWDWIRA SEQ ID NO: 75 IVVTAPADLWDWIRA SEQ ID NO: 76 IVVAMPADLWDWIRA SEQ ID NO: 77 IAVTMPADLWDWIRA SEQ ID NO: 78 AVVTMPADLWDWIRA SEQ ID NO: 79 IHVTMPADLWDWIRA SEQ ID NO: 80 IQVTMPADLWDWIRA SEQ ID NO: 81 IRVTMPADLWDWIRA SEQ ID NO: 82 ITVTMPADLWDWIRA SEQ ID NO: 83 IWVTMPADLWDWIRA SEQ ID NO: 84 IYVTMPADLWDWIRA SEQ ID NO: 85 IVVTIPADLWDWIRA SEQ ID NO: 86 IVVTLPADLWDWIRA SEQ ID NO: 87 IVVTVPADLWDWIRA SEQ ID NO: 88 IVVTMPADLWDWIMA SEQ ID NO: 89 IVVTMPADLWDWINA SEQ ID NO: 90 IVVTMPADLWDWIQA SEQ ID NO: 91 IVVTIPADLWDWIRA SEQ ID NO: 92 IVVTLPADLWDWIRA SEQ ID NO: 93 IHVTIPADLWDWINK SEQ ID NO: 94 IHVTIPADLWDWIN SEQ ID NO: 95 IHVTIPADLWDWI SEQ ID NO: 96 DSSAVCWAFPHHPLCHMKAT SEQ ID NO: 97 ADADMCWFFPTSPWCH SEQ ID NO: 98 DLSAVCWAFPWDPECHM SEQ ID NO: 99 DSSAVCWAFPYLPECH SEQ ID NO: 100 DISAVCWAFPFDPECH SEQ ID NO: 101 AYECPRLEYDMFGALHCLPS SEQ ID NO: 102 CPRLEYDMFGALHCL SEQ ID NO: 103 CLDLQYDPWGALHCI SEQ ID NO: 104 CFDLQYDPWGALHCI SEQ ID NO: 105 CLDLQYDMFGALHCV SEQ ID NO: 106 CLDLVYDPWGALHCI SEQ ID NO: 107 CWVLEYDMFGALHCR SEQ ID NO: 108 CWALEYDMFGYLHCR SEQ ID NO: 109 CWVLEYDMFGFLHCR SEQ ID NO: 110 CWVLEYDMFGYLHCR

(530) The amount and mode of administration of the peptides according to the present invention will vary depending upon the particular peptide inhibitor of the invention, the individual patient group or patient, the presence of further medically active compounds and the nature and severity of the condition being treated.

(531) According to a preferred embodiment, the prophylactic and/or therapeutic use dosages is of about 5-50 μg/ml, preferably of about 10-25 μg/ml Preferably, the frequency of administration for prophylactic and/or therapeutic uses lies in the range of about once to twice daily applications, preferably once.

(532) The invention is further described by way of illustration in the following examples, none of which are to be interpreted as limiting the scope of the invention as outlined in the appended claims.

EXAMPLES

Example 1 Peptides Synthesis & Purification

(533) We have developed a homology model of IL-17A by use of crystallographic data available (PDB 5h13, PDB 5vb9 and PDB 4hsa) (Liu et al. Nat Commun 2013, 4, 1888, Liu et al. Sci Rep 2016, 6: 30859, Liu et al. Sci Rep 2016, 6, 26071, Ting, Tung et al. Plos One 2018, 13(1): e01908502018). In our models we have also recreated the loops unresolved in the crystals and optimized models through a molecular dynamics simulation performed with Desmond software, as implemented in Schrodinger Maestro macromodel suite. All molecular simulations were run for 1 microsecond to guarantee the system stability.

(534) Based on structure and properties of IL-17A α-pocket, different peptide sequences have been designed so as to obtain i) a stable secondary and tertiary conformation that engages IL-17A with an optimized protein binding site occupancy, and ii) physical-chemical properties suitable for topical use in ophthalmic and dermatological pathologies (Liu et al. Nat Commun 2013, 4, 1888, Espada et al J Med Chem 2016, 59(5), 2255-2260).

(535) Additionally, we optimized the interactions ligand-IL-17A by the introduction of hydrogen bonds and optimization of hydrophobic interactions.

(536) a. Fmoc-Based Solid-Phase Peptide Synthesis

(537) All the chemicals were purchased and used without further purification.

(538) Peptides of SEQ ID NO: 1 to 43 and 111-257 of Table 1 were prepared by manual or automatic solid-phase peptide synthesis, on Rink Amide-MBNA resin as the solid support, as described below and with N-terminal amine capped with acetyl group.

(539) The Fmoc (9-fluorenylmethoxycarbonyl) group used for Na-protection was cleaved by 8 min treatment with 20% piperidine in dimethylformamide (DMF) followed by a further treatment with the same reagent for 10 min. After Fmoc cleavage, the peptide-resin complex was washed with DMF (×6). The next residue was then incorporated through the use of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU)/N,N-diisopropylamine (DIPEA) coupling protocol [Fmoc-amino acid (3 equiv), HATU (3 equiv), and DIPEA (6 equiv)]. After gentle stirring (1 hr) and washing with DMF (×6), part of the peptide-resin was analyzed by Kaiser test. On completion of the assembly, the peptide-resin was washed with DMF (×3), dichloromethane (DCM) (×4) and then dried in vacuo.

(540) Cleavage and Deprotection

(541) The peptide was cleaved from the resin and, when necessary, deprotected by using as cleavage solution a mixture of trifluoroacetic acid/1,2-ethanedithiol/thioanisole/phenol/H2O/triisopropylsilane in the ratio 68.5/10/10/5/3.5/1, v/v. 3 ml of cleavage solution was used per 100 mg of resin. Complete deprotection was achieved under stirring for 4 hours at 30° C. Following the cleavage reaction, the peptide in the mixture was precipitated in cold diethyl ether and dried in vacuo at 50° C.

(542) b. Disulfide Bond Formation in Peptide 41

(543) Disulfide bond formation in peptide of SEQ ID NO: 41 of Table 1 was obtained by incubation of the peptide in a mixture DMSO/H2O (20/80, v/v), which was monitored by RP-HPLC, MS and free sulfhydryl detection (DTNB method).

(544) c. Purification by Preparative RP-HPLC and Purity Assessment by Analytical HPLC

(545) The crude peptide samples were purified by preparative RP-HPLC (Agilent) using a C18 column (10 μm, 100 Å, 50×250 mm). A solvent system consisting of solvent A (0.1% TFA, 2% CH3CN in water) and solvent B (90% CH3CN/H2O) at a flow rate of 25 mL/min was used for elution, and the absorbance was detected at 220 nm. The solvent was removed by lyophilization. The final products were characterized by MALDI-TOF-MS, and the purity of the material assessed by analytical RP-HPLC (C18-250 mm×4.6 mm I.D., flow rate of 1 mL/min), and the absorbance was detected at 220 nm.

Example 2: Preparation of NAM and/or NAG Bioconjugated Peptides BIO-1, BIO-2 and BIO-3

(546) The bioconjugated peptides BIO-1, BIO-2 and BIO-3 were prepared by a conjugation reaction of peptide 42 or 43 according the following procedures.

(547) 1. Preparation of Donor and Acceptor

(548) All reactions were performed under N.sub.2 atmosphere. NMR spectra were run on a Brucker 400 MHz instrument. HPLC-UV analysis were obtained on an Agilent 1260 Infinity System, equipped with a G1311B Agilent 1260 quaternary pump, G1329B Agilent 1260 autosampler, G1315C Agilent 1260 diode array detector, and G1316A Agilent 1260 column thermostat modules. A Phenomenex GEMINI C18 150×4.6 mm2 (5 μm) column was used. The mobile phase A was MilliQ water with 0.05% TFA, and B was HPLC grade acetonitrile with 0.05% TFA at a flow rate of 1.0 mL/min. The HPLC system was coupled to an Agilent Quadrupole 6120 LC/MS mass spectrometer operated in positive ion mode. Ions were generated using an electrospray ionization ion source. Acquisition data were processed with Agilent Chemstation Software.

a. Synthesis of 1-(2,2,2-Trichloroacetimine)2-Deoxy-3-O-Acetyl-4,6-Obenzylidene-2-(2,2,2-Tri-Chloroethoxycarbonylamino)-a-D-Glucopyranoside (Donor)

(549) Trichloroethoxycarbonyl chloride (2.1 mL, 15.3 mmol) was added dropwise at room temperature to a vigorously stirred solution of D-glucosamine hydrochloride (3 g, 13.9 mmol) and NaHCO.sub.3, (3.5 g, 41.7 mmol) in water (30 mL). The mixture was stirred for 2 h, then neutralized with 1 M HCl, concentrated, and dried in vacuo. 2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyranoside (intermediate A) was obtained.

(550) The crude product obtained was directly used for the next reaction step without further purification.

(551) In details, zinc chloride (1.9 g, 13.9 mmol) was added to a solution of intermediate A (4.9 g, 13.9 mmol) in benzaldehyde (24 ml) and molecular sieves, 4 Å (4 Å MS, 600 mg). After stirring overnight at room temperature, saturated aq NaHCO.sub.3 (30 mL) and diethyl ether (90 mL) were added and the reaction mixture was stirred for 15 min. The formed precipitate was filtered, washed with water, diethyl ether, and dried. The residue was dissolved in pyridine (13 mL) cooled to 0° C., treated with acetic anhydride (6.4 mL, 68 mmol) and stirred overnight at room temperature, then the solution was concentrated with toluene, cooled to 0° C. and extracted with DCM and saturated aq NaHCO.sub.3 (3×), then washed with a saturated NaCl solution. The combined organic layers were dried, concentrated, and the crude purified by Isolera (EtPet/EtOAc) to give 1,3-di-O-acetyl 2-deoxy-4,6-O-benzylidene-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyranoside (intermediate B), as a white solid (3.05 g, 41.7% yield).

(552) Morpholine (1.2 mL, 13.9 mmol) was then added to a solution of intermediate B (3.05 g, 5.8 mmol) in dry ethyl acetate (EtOAc) (12.2 mL). After stirring overnight at room temperature the reaction mixture was quenched with 3N HCl solution (3.5 mL) and then stirred for 20 min.

(553) Extracted with EtOAc and washed with water, saturated aq NaHCO.sub.3 and a saturated NaCl solution saturated NaCl solution, dried, and concentrated. The crude was purified by Isolera (EtPet/EtOAc) to give 2-deoxy-3-O-acetyl-4,6-O-benzylidene-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyranoside (intermediate C) as a white solid (2.2 g, 78.4%). To a solution of intermediate C (2.2 g, 4.54 mmol) in dry DCM (44 ml), Cs.sub.2CO.sub.3 (680 mg, 2.1 mmol) and CCl.sub.3CN (4.4 mL, 43.8 mmol) were added. The reaction mixture was stirred for 2 h at room temperature, then filtered over celite and concentrated to give 1-(2,2,2-Trichloroacetimine)2-deoxy-3-O-acetyl-4,6-Obenzylidene-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyranoside (Donor) as a light yellow solid (2.2 g, 77%).

b. Synthesis of 1,6-Di-O-benzyl 2-deoxy-3-O-((R)-10-ethoxycarbonylethyl)-2-(2,2,2-trichloroetho-xycarbonylamino)-a-D-glucopyranoside (Acceptor)

(554) Allyl chloroformate (6.6 mL, 62.2 mmol) was added dropwise at room temperature to a vigorously stirred solution of D-glucosamine hydrochloride (12.2 g, 56.6 mmol) and NaHCO.sub.3, (14.3 g, 169.7 mmol) in water (61 mL). The mixture was stirred for 2 h, then neutralized with 1M HCl, concentrated, and dried in vacuo. Crude 2-deoxy-2-(allyloxycarbonylamino)-a-D-glucopyranoside (Intermediate D) was obtained. The product was used for next reaction step without further purification, Intermediate D (14.9 g, 56.6 mmol) was dissolved in benzyl alcohol (89 mL, and acetyl chloride (15.4 mL, 215.2 mmol) was added dropwise at 0° C. After stirring for 3 h at 80° C. the reaction mixture was quenched with cold saturated aq NaHCO.sup.3 (20 ml) and stirred for additional 30 min. Cold water and diethyl ether were added and the reaction mixture was stirred for 30 min. The two phases were separated, and the aqueous phase was concentrated, and dried in vacuo. 30 ml of water and 200 ml of ethyl ether were added until the formation of a precipitate that was filtered and washed several times with cold diethyl ether (until no traces of benzyl alcohol were detected. Benzyl 2-deoxy-2-(allyloxycarbonylamino)-a-D-glucopyranoside (intermediate E) was obtained as a white solid (11 g, 55%) and directly used for next reaction step without further purification. Zinc chloride (4.2 g, 31.1 mmol) was added to a solution of intermediate E (11 g, 31.1 mmol) in benzaldehyde (55 mL) and 4 Å MS (4.9 g). After 2 h, more zinc chloride (4.2 g, 31.1 mmol) was added. After stirring overnight at room temperature, the reaction mixture was treated with saturated aq NaHCO.sub.3 (70 mL), petroleum ether (420 mL), and stirred for 10 min. The precipitate was filtered, washed with petroleum ether and dissolved in DCM. The organic solution was extracted with saturated aq NaHCO.sub.3, water, and a saturated NaCl solution dried and concentrated. The crude was purified by Isolera (DCM/EtOAc) to give Benzyl 2-deoxy-4,6-O-benzylidene-2-(allyloxycarbonylamino)-a-D-glucopyranoside (intermediate F) as a white solid (2.75 g, 20%).

(555) To a solution of intermediate F (2.75 g, 6.2 mmol) in dry DCM (40.6 mL), cooled to 0° C., sodium hydride (NaH, 348.8 mg, 8.7 mmol, 60% oil dispersion) was added, and the mixture stirred for 30 min at room temperature. The mixture was then treated with neat (−)-ethyl (S)-2-trifluromethanesulfonyl propionate dropwise and stirred for 2 h at room temperature. The reaction mixture was quenched by addition of ice, and extracted with DCM. The organic solution was washed with saturated aq NaHCO.sub.3, a saturated NaCl solution, dried and concentrated. The residue was purified by Isolera (DCM/EtOAc) to give benzyl 2-deoxy-3-O—((R)-10-ethoxycarbonylethyl)-4,6-Obenzyl idene-2-(allyloxy-carbonylamino)-a-D-glucopyranoside (intermediate G) as a white solid (2.35 g, 69.7%).

(556) Tetrakis(triphenylphosphine) palladium (1.5 g, 1.3 mmol) and acetic acid (AcOH, 0.385 mL, 6,725 mmol) were added to a solution of the intermediate G (2.35 g, 4.34 mmol) in dry DCM (42.7 mL). The reaction mixture was stirred at room temperature for 15 min and then 2,2,2-trichloroethyl chloroformate (TrocCl, 1.224 mL, 8,895 mmol) was added dropwise, and the resulting solution stirred for 1 h at room temperature. The reaction mixture was quenched with saturated aq NaHCO.sub.3, extracted with DCM, washed with H.sub.2O and a saturated NaCl solution. After concentration in vacuo, the residue was dissolved in diethyl ether (100 ml) and insoluble materials filtered off. The organic phase was dried, concentrated, and purified by Isolera (Cyclohexane/AcOEt) to give benzyl 2-deoxy-3-O—((R)-10-ethoxycarbonylethyl)-4,6-O-benzylidene-2-(2,2,2-tri-chloroethoxycarbonylamino)-a-D-glucopyranoside (intermediate H) as a white solid (2.3 g, 83.7%).

(557) To a solution of intermediate H (2.4 g, 3.8 mmol) in dry CH3CN (38 mL) at 0° C. a solution of Me3N—BH.sub.3 (332 mg, 4.55 mmol) in CH.sub.3CN (2.2 mL) was added, followed by a solution of BF.sub.3-OEt.sub.2 (2.89 mL, 23.4 mmol) in CH3CN (8 mL) added dropwise. After stirring for 3 h at 0° C., the mixture was quenched with cold saturated aq NaHCO.sub.3 (30 mL), diluted with EtOAc (350 ml), and washed with saturated aq NaHCO.sub.3 (100 ml), 5% citric acid (4×50 ml), saturated aq NaHCO.sub.3 (50 mL), and a saturated NaCl solution (40 mL). The organic layer was dried, concentrated, and the crude was purified by Isolera (Cyclohexane/AcOEt) to give 1,6-di-O-benzyl 2-deoxy-3-O—((R)-10-ethoxycarbonylethyl)-2-(2,2,2-trichloroetho-xycarbonylamino)-a-D-glucopyranoside (Acceptor), as a colorless foam (1.2 g, 49.8%).

(558) 2. Synthesis of NAG-NAM Conjugated Peptide BIO-3

(559) Donor (1.6 g, 2.6 mmol) and Acceptor (1.1 g, 1.73 mmol), suspended in dry DCM (55 mL) with 4 Å molecular sieves (200 mg), were treated with Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 188 μL, 1.04 mmol) at −15° C. After stirring for 20 min, more 0.75 eq of Donor and 0.3 eq of TMSOTf were added. After stirring for 20 min, more 0.75 eq of Donor and 0.3 eq of TMSOTf were added. The mixture was then quenched with cold saturated aq solution of NaHCO.sub.3 (15 mL) and extracted with DCM (60 mL). The organic layer was washed with saturated aq NaHCO.sub.3 and a saturated NaCl solution, dried and concentrated. The residue was purified by Isolera reverse phase (H2O/ACN neutral phases) to give benzyl 6-O-benzyl-4-O-[2-deoxy-3-O-acetyl-4,6-Obenzylidene-2-(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyranosyl]-2-deoxy-3-O—[(R)-1′-ethoxycarbonyl ethyl]-2-(2,2,2-trichloroethoxycarbonylamino)-a-D-glucopyranoside (intermediate L), as a white foam (1.2 g, 62.9%).

(560) A solution of intermediate L (1.2 g, 1.09 mmol) and zinc-copper couple (3.12 g, 24.2 mmol) in a mixture AcOH/Ac2O/THF 1:1:1 (12 mL) was stirred for 4 h at room temperature. The reaction mixture was filtered over celite, washed with EtOAc, and concentrated. The crude was concentrated and purified by column chromatography (Cyclohexane/AcOEt) to give benzyl 6-O-benzyl-4-O-[2-deoxy-3-O-acetyl-4,6-Obenzylidene-2-acetylamino-b-D-glucopyranosyl]-2-deoxy-3-O—[(R)-10-ethoxycarbonylethyl]-2-acetylamino-a-D-glucopyranoside (intermediate M) as a white solid (560 mg, 0.67 mmol, 61.6%). HPLC-MS (ESI+) m/z: C.sub.44H.sub.54N.sub.2O.sub.14+Na: 857.3472. Found: 857.6124.

(561) To a solution of intermediate M (91 mg, 0.109 mmol) in THF/1,4-dioxane/H.sub.2O 4:2:1 (2.8 mL) was added LiOH.H.sub.2O (56 mg, 1.34 mmol). After stirring for 2 h at room temperature, the reaction mixture was filtered over Dowex H+ (freshly activated with 1N HCl). The residue was purified by diaion HP-20 column chromatography (2×7 cm) previously washed with water, MeOH and water. Column was eluted first with H.sub.2O (50 mL) and then with MeOH (30 mL). The alcohol fractions were concentrated to give benzyl 6-O-benzyl-4-O-[2-deoxy-4,6-O-benzylidene-2-acetylamino-b-D-glucopyranosyl]-2-deoxy-3-O—[(R)-10-ethoxycarbonylethyl]-2-acetylamino-a-D-glucopyranoside (intermediate N), as a whitish solid (57.4 mg, 69%). Mp 102-105° C., 1HNMR (600 MHz, CD.sub.3OD) δH 7.51-7.27 (m, 15H, ArH), 5.59 (s, 1H, CHPh), 5.36 (d, J 3.1 Hz, H−1), 4.85 (m) H.sub.2O, H−10, ½ CH.sub.2Ph), 4.66-4.57 (m, 3H, J 12.1 Hz, CHCH.sub.3, CH.sub.2-Ph), 4.46 (d, 1H, J 12.2 Hz, ½ CH.sub.2Ph), 4.29 (dd, 1H, J 10.3 Hz, J 5.0 Hz, H-60b), 4.07 (t, 1H, J 9.6 Hz, H−30), 3.95 (t, 1H, J 9.1 Hz, H−4), 3.82-3.77 (m, 2H, H−6b, H−3), 3.72-3.62 (m, 3H, H−60a, H−5, H−6a), 3.55-3.40 (m, 3H, H−20, H−40, H−2), 3.30-3.27 (m, 1H, H−50), 1.98 (s, 3H, COCH.sub.3), 1.96 (s, 3H, COCH.sub.3), 1.37 (d, J 6.9 Hz, 3H, CHCH.sub.3). HPLC-MS (ESI+) m/z: C.sub.40H.sub.48N.sub.2O.sub.13+Na: 787.3054 Found: 787.7830.

(562) The peptide of SEQ ID NO: 42 was coupled to compound N in the conditions used for aminoacid coupling and the resulting bioconjugate was cleaved from the solid support by treatment with trifluoroacetic acid (1% solution in DCM). The resulting bioconjugate (0.026 mmol) was dissolved in acetic acid (8 mL), and Pd(OH)2/C (58 mg) was added. The resulting mixture was stirred at room temperature for 6 h under hydrogen atmosphere. The mixture was filtered over celite and concentrated to give the bioconjugate B10-3 in quantitative yield. The bioconjugate was cleaved from the resin and the last residue deprotected by using as cleavage solution a mixture of trifluoroacetic acid/1,2-ethanedithiol/thioanisole/phenol/H2O/triisopropylsilane in a ratio of 68.5/10/10/5/3.5/1, v/v. 3 ml of cleavage solution was used per 100 mg of resin. Complete deprotection was achieved after 4 hours at 30° C. Following the cleavage reaction, the crude peptide was precipitated by addition of cold diethyl ether and dried in vacuo at 50° C.

(563) The crude bioconjugate was purified by preparative RP-HPLC (Agilent) using a C18 column (10 μm, 100 Å, 50×250 mm). A solvent mixture consisting of solvent A (0.1% TFA, 2 CH.sub.3CN in H.sub.2O) and solvent B (90% CH.sub.3CN/H.sub.2O) at a flow rate of 25 mL/min was used for elution, and the absorbance detected at 220 nm. The solvent was removed by lyophilization and the final products characterized by MALDI-TOF-MS. The purity of the purified material was assessed by analytical RP-HPLC (C18-250 mm×4.6 mm I.D., flow rate of 1 mL/min), and the absorbance was detected at 220 nm.

(564) 3. Synthesis of NAG and NAM Bioconjugates B10-1 and BIO-2

(565) Synthesis of NAM-Peptide and Peptide-NAG were performed starting from the peptide anchored resin (Example 1), wherein the peptide was the peptide of SEQ ID NO: 42 (for BIO-1) or the peptide of SEQ ID NO: 43 (for BIO-2), respectively, and Donor and Acceptor were the molecules described above according the procedure described in Swaminathan et al., Proc Natl Acad Sci USA. 2006, 17; 103 (3):684-9.

Example 3: Synthesis of Capric Acid Bioconjugate BIO-4

(566) Peptides capped with Capric Acid were synthetized according the procedure described in Example 1 by using a N-terminal amine capped with Capric Acid.

Example 4: Synthesis of Ascorbic Acid Bioconjugate BIO-5

(567) 5,6-O-isopropylidene-L-ascorbic acid (iASA) was synthesized by Jung's method (Jung, M. E.; Shaw, T. J. J. Am. Chem. Soc. 1980, 102, 6304). For the activation, CDI (10 eq.) was added to the peptide anchored resin for 2 h. Activation of N-terminal amine of peptide, after cleavage from the resin, was confirmed by RP-HPLC using the following conditions: A to B (A: 0.1% TFA in H.sub.2O, B: 0.1% TFA in CH.sub.3CN; from 0% to 60% B over 30 min, at a flow rate of 1.0 mL/min); detection, UV 230 nm. Next, iASA was introduced to the activated peptide anchored resin. The product was separated from the resin by treating with 50% TFA/DCM (v/v) for 1 h. The resin was filtered, the filtrate was concentrated in high vacuum, and precipitated with cold diethyl ether. The structure of the resulting bioconjugate BIO-5 was confirmed by LC/MS using 0.1% formic acid/methanol as eluent at a flow rate of 250 μL/min over 30 min, and monitored at 230 nm.

Example 5: IL-17RA/IL17RC Dimerization Assay in Response to IL-17A

(568) The peptides of Table 7 were tested for their ability to inhibit the binding of IL-17A to its receptor and the subsequent interaction of IL-17RA and IL17RC.

(569) Interleukin receptor RA and RC dimerization assay (DiscoverX) was used to measure the interaction of the receptor chains IL-17RA and IL17RC upon activation by IL-17A in the absence or presence of the peptides of Table 7.

(570) In the PathHunter® Cytokine Receptor Assay, one cytokine receptor chain is tagged with a small peptide epitope ProLink (PK) and the other chain is tagged with Enzyme Acceptor (EA). Ligand binding induces dimerization of the two receptors, facilitating complementation of PK and EA fragments. This interaction generates an active unit of b-galactosidase, which is detected using a chemiluminescent substrate. PathHunter cell lines were expanded from freezer stocks according to standard procedures. Cells were seeded in a total volume of 20 μL into white walled, 384-well microplates and incubated for the appropriate time prior to testing. For agonist activity inhibition, cells were incubated with sample to induce response. Intermediate dilution of sample stocks was performed to generate 5× sample in assay buffer. 5 μL of 5× sample was added to cells and incubated at 37° C. or room temperature for 60 minutes. Vehicle concentration was 1%. 5 μL of 6×EC.sub.80 agonist (IL-17A) in assay buffer was added to the cells and incubated at 37° C. for 6 to 16 hours depending on the assay. 1. Assay signal was generated through a single addition of 12.5 or 15 μL (50% v/v) of PathHunter Detection reagent cocktail for agonist (IL-17A) and (peptides of table 7) assays respectively, followed by a one hour incubation at room temperature. For some assays, activity was detected using a high sensitivity detection reagent (PathHunter Flash Kit) to improve assay performance. In these assays, an equal volume of detection reagent (25 or 30 uL) was added to the wells, followed by a one hour incubation at room temperature. Microplates were read following signal generation with a PerkinElmer Envision™ instrument for chemiluminescent signal detection. Compound activity was analyzed using CBIS data analysis suite (ChemInnovation, CA). % Inhibition=100%×(1−(mean RLU of test sample−mean RLU of vehicle control)/(mean RLU of EC80 control−mean RLU of vehicle control)).

(571) As reported in Table 7 below, all tested molecules were found active in inhibiting IL-17RA/IL17RC dimerization thus inhibiting the IL-17A pathway activation showing a IC50 value in the mid nanomolar range.

(572) TABLE-US-00010 TABLE 7 IC.sub.50 in IL-17RA/IL7RC dimerization SEQUENCE NAME assay SEQ ID NO: 1 <0.2 μM SEQ ID NO: 2 <0.2 μM SEQ ID NO: 3 <0.4 μM SEQ ID NO: 4 <0.1 μM SEQ ID NO: 5 <0.1 μM SEQ ID NO: 6 <0.4 μM SEQ ID NO: 7 <0.1 μM SEQ ID NO: 8 <0.2 μM SEQ ID NO: 9 <0.3 μM SEQ ID NO: 10 <0.4 μM SEQ ID NO: 11 <0.4 μM SEQ ID NO: 12 <0.2 μM SEQ ID NO: 13 <0.4 μM SEQ ID NO: 14 <0.4 μM SEQ ID NO: 15 <0.4 μM SEQ ID NO: 16 <0.4 μM SEQ ID NO: 17 <0.4 μM SEQ ID NO: 18 <0.4 μM SEQ ID NO: 19 <1 μM SEQ ID NO: 20 <1 μM SEQ ID NO: 21 <1 μM SEQ ID NO: 22 <1 μM SEQ ID NO: 23 <1 μM SEQ ID NO: 24 <1 μM SEQ ID NO: 25 <1 μM SEQ ID NO: 26 <1 μM SEQ ID NO: 27 <1 μM SEQ ID NO: 28 <1 μM SEQ ID NO: 29 <1 μM SEQ ID NO: 30 <1 μM SEQ ID NO: 31 <1 μM SEQ ID NO: 32 <1 μM SEQ ID NO: 33 <1 μM SEQ ID NO: 34 <1 μM SEQ ID NO: 35 <1 μM SEQ ID NO: 36 <1 μM SEQ ID NO: 37 <0.2 μM SEQ ID NO: 38 <0.2 μM SEQ ID NO: 39 <0.2 μM SEQ ID NO: 40 <0.2 μM SEQ ID NO: 41 <1 μM SEQ ID NO: 111 <0.5 μM SEQ ID NO: 112 <0.5 μM SEQ ID NO: 113 <0.5 μM SEQ ID NO: 114 <0.5 μM SEQ ID NO: 115 <0.5 μM SEQ ID NO: 116 <0.5 μM SEQ ID NO: 117 <0.5 μM SEQ ID NO: 118 <0.5 μM SEQ ID NO: 119 <0.5 μM SEQ ID NO: 120 <0.5 μM SEQ ID NO: 121 <0.5 μM SEQ ID NO: 122 <0.5 μM SEQ ID NO: 123 <0.5 μM SEQ ID NO: 124 <0.5 μM SEQ ID NO: 125 <0.5 μM SEQ ID NO: 126 <0.5 μM SEQ ID NO: 127 <0.5 μM SEQ ID NO: 128 <0.5 μM SEQ ID NO: 129 <0.5 μM SEQ ID NO: 130 <0.5 μM SEQ ID NO: 131 <0.5 μM SEQ ID NO: 132 <0.5 μM SEQ ID NO: 133 <0.5 μM SEQ ID NO: 134 <0.5 μM SEQ ID NO: 135 <0.5 μM SEQ ID NO: 136 <0.5 μM SEQ ID NO: 137 <0.5 μM SEQ ID NO: 138 <0.5 μM SEQ ID NO: 139 <0.5 μM SEQ ID NO: 140 <0.5 μM SEQ ID NO: 141 <0.5 μM SEQ ID NO: 142 <0.5 μM SEQ ID NO: 143 <0.5 μM SEQ ID NO: 144 <0.5 μM SEQ ID NO: 145 <0.5 μM SEQ ID NO: 146 <0.5 μM SEQ ID NO: 147 <0.5 μM SEQ ID NO: 148 <0.5 μM SEQ ID NO: 149 <0.5 μM SEQ ID NO: 150 <0.5 μM SEQ ID NO: 151 <0.5 μM SEQ ID NO: 152 <0.5 μM SEQ ID NO: 153 <0.5 μM SEQ ID NO: 154 <0.5 μM SEQ ID NO: 155 <0.5 μM SEQ ID NO: 156 <0.5 μM SEQ ID NO: 157 <0.5 μM NAM-IMVTIPADLYEWIEE BIO-1 <1 μM (NAM plus SEQ ID NO: 42) IMVTIPADLYEWIEEQ-NAG BIO-2 <1 μM (SEQ ID NO: 42 plus NAG) NAG-NAM-IMVTIPADLYEWIEE BIO-3 <1 μM (NAG-NAM plus SEQ ID NO: 42) capric acid-IMVTIPADLYEWIEE BIO-4 <1 μM (capric acid plus SEQ ID NO: 42) Ascorbic acid-IMVTIPADLYEWIEE BIO-5 <1 μM (Ascorbic acid plus SEQ ID NO: 42) SEQ ID NO: 158 <10 μM SEQ ID NO: 159 <10 μM SEQ ID NO: 160 <10 μM SEQ ID NO: 161 <10 μM SEQ ID NO: 162 <10 μM SEQ ID NO: 163 <10 μM SEQ ID NO: 164 <10 μM SEQ ID NO: 165 <10 μM SEQ ID NO: 166 <10 μM SEQ ID NO: 167 <10 μM SEQ ID NO: 168 <10 μM SEQ ID NO: 169 <10 μM SEQ ID NO: 170 <10 μM SEQ ID NO: 171 <10 μM SEQ ID NO: 172 <10 μM SEQ ID NO: 173 <10 μM SEQ ID NO: 174 <10 μM SEQ ID NO: 175 <10 μM SEQ ID NO: 176 <10 μM SEQ ID NO: 177 <10 μM SEQ ID NO: 178 <10 μM SEQ ID NO: 179 <10 μM SEQ ID NO: 180 <10 μM SEQ ID NO: 181 <10 μM SEQ ID NO: 182 <10 μM SEQ ID NO: 183 <10 μM SEQ ID NO: 184 <10 μM SEQ ID NO: 185 <10 μM SEQ ID NO: 186 <10 μM SEQ ID NO: 187 <10 μM SEQ ID NO: 188 <10 μM SEQ ID NO: 189 <10 μM SEQ ID NO: 190 <10 μM SEQ ID NO: 191 <10 μM SEQ ID NO: 192 <10 μM SEQ ID NO: 193 <10 μM SEQ ID NO: 194 <10 μM SEQ ID NO: 195 <10 μM SEQ ID NO: 196 <10 μM SEQ ID NO: 197 <10 μM SEQ ID NO: 198 <10 μM SEQ ID NO: 199 <10 μM SEQ ID NO: 200 <10 μM SEQ ID NO: 201 <10 μM SEQ ID NO: 202 <10 μM SEQ ID NO: 203 <10 μM SEQ ID NO: 204 <10 μM SEQ ID NO: 205 <10 μM SEQ ID NO: 206 <10 μM SEQ ID NO: 207 <10 μM SEQ ID NO: 208 <10 μM SEQ ID NO: 209 <10 μM SEQ ID NO: 210 <10 μM SEQ ID NO: 211 <10 μM SEQ ID NO: 212 <10 μM SEQ ID NO: 213 <10 μM SEQ ID NO: 214 <10 μM SEQ ID NO: 215 <10 μM SEQ ID NO: 216 <10 μM SEQ ID NO: 217 <10 μM SEQ ID NO: 218 <10 μM SEQ ID NO: 219 <10 μM SEQ ID NO: 220 <10 μM SEQ ID NO: 221 <10 μM SEQ ID NO: 222 <10 μM SEQ ID NO: 223 <10 μM SEQ ID NO: 224 <10 μM SEQ ID NO: 225 <10 μM SEQ ID NO: 226 <10 μM SEQ ID NO: 227 <10 μM SEQ ID NO: 228 <10 μM SEQ ID NO: 229 <10 μM SEQ ID NO: 230 <10 μM SEQ ID NO: 231 <10 μM SEQ ID NO: 232 <10 μM SEQ ID NO: 233 <10 μM SEQ ID NO: 234 <10 μM SEQ ID NO: 235 <10 μM SEQ ID NO: 236 <10 μM SEQ ID NO: 237 <10 μM SEQ ID NO: 238 <10 μM SEQ ID NO: 239 <10 μM SEQ ID NO: 240 <10 μM SEQ ID NO: 241 <10 μM SEQ ID NO: 242 <10 μM SEQ ID NO: 243 <10 μM SEQ ID NO: 244 <10 μM SEQ ID NO: 245 <10 μM SEQ ID NO: 246 <10 μM SEQ ID NO: 247 <10 μM SEQ ID NO: 248 <10 μM SEQ ID NO: 249 <10 μM SEQ ID NO: 250 <10 μM SEQ ID NO: 251 <10 μM SEQ ID NO: 252 <10 μM SEQ ID NO: 253 <10 μM SEQ ID NO: 254 <10 μM SEQ ID NO: 255 <10 μM SEQ ID NO: 256 <10 μM SEQ ID NO: 257 <10 μM

(573) In FIG. 1, a dose-response profile for exemplary peptides is reported.

Example 6: Phenotipic Assay in Human Differentiated TH17 Cells and Viability Assay

(574) To evaluate the effect of the peptides of the invention on the target cells responsible for inflammation in ophtalmic or dermatological pathologies, we tested the ability of the peptide of SEQ ID NO: 18 to inhibit in vitro pro-inflammatory cytokines and metalloproteinases secretion by human Th17 cells.

(575) Human primary CD4 T-cells were differentiated into Th17 cells in vitro by culturing the cells in Th17 differentiation medium for 10 days. In details, purified human peripheral blood CD4 T-cells were cultured in Th-17 differentiation medium (CellXVivo Human Th17, Tocris) per manufacturer's instructions. Briefly: 96-well tissue culture plates were coated with anti-CD3 and CD28 agonistic antibodies. Cells were counted via trypan blue exclusion and suspended at a concentration of 1E5 cells/ml in Th-17 differentiation medium. 0.2 mL of this suspension was plated into individual wells of a 96-well plate. Cells were incubated at 37° C. with 5% CO2 for 10 days, with media replaced as needed. Following 10-day Cell Differentiation, the peptides synthetized in Example 1, such as the peptide of Seq. ID No 18, were diluted to the appropriate concentration in CGM (cell growth medium, XVivo-15, Lonza). Cell plate was centrifuged at 300×g for 5 min, and Th17 differentiation medium was removed.

(576) Following differentiation, the cells were stimulated with both an activation cocktail containing Phorbol 12-myristate 13-acetate and lonomycin in the presence of varying concentrations of test articles.

(577) At 24 h following stimulation, culture supernatants were analyzed for expression of IL-17, IL-6, IL-23, MMP3 and examined for viability using the fluorescence viability dye, Alamar blue. The CD4 cells secreted high levels of IL-17A in response to stimulation with an activation cocktail, indicating successful differentiation into Th17 cells. Additionally, stimulated cells secreted IL-6, IL23 and MMP3.

(578) Supernatant was removed and replaced with 200 μL of diluted peptide or vehicle (CGM+DMSO diluted as with TI concentration) in appropriate groups. Cells were incubated with peptide test items for 1 hour before stimulation. The following stimulation solutions were generated: 10× Cell Activation Cocktail: Diluted from 500× to 10× working solution in CGM. 22 μL of appropriate stimulants were added to wells 1 hr after TI addition. Finally, 25 μL of Alamar blue reagent (10×) was added to each well 20 hours after stimulant (4 hrs prior to 24 hr serum collection) to determine cell viability. 24 hrs After Stimulant, Alamar blue fluorescence was measured per manufacturer's instructions. Remaining Supernatants were collected for subsequent multiplex protein analyses. Expression levels of IL-6, IL-17, and IL-23 as well as MMP3 in 24 hour stimulated culture supernatants was evaluated using a Luminex Multiplex Platform on a MAGPIX instrument.

(579) FIGS. 2 and 3 show the effect of the peptide of SEQ ID NO: 18. As can be seen, this peptide is able to significantly reduce the secretion of IL-17A, IL-6 and IL-23 as well as the secretion of MMP3. As shown in FIG. 4 the inhibition of cytokines and metalloproteinases secretion is not due to cell viability issues since cell treatment with the peptide of SEQ. ID N. 18 for eleven days did not impair cell viability.

Example 7: Physical-Chemical Analysis of Peptides

(580) The physical-chemical properties of the prior art peptide HAP and of some peptides according to the invention (see table 8) have been tested as described below.

(581) i. Methods

(582) a. Isoelectric Poitn IP Log D and log P

(583) The main physicochemical properties (Isoelectric point (IP), log D (7.4) and log P) of the peptides of Table 8 below were determined by using the SiriusT3 apparatus (Sirius Analytical Instruments Ltd., East Sussex, UK) equipped with an Ag/AgCl double junction reference pH electrode, a Sirius D-PAS spectrometer and a turbidity sensing device. The pH electrode was calibrated titrimetrically in the pH range 1.8-12.2. An overhead stirrer was used and a temperature probe (Peltier controller) monitored the temperature during the course of the titration. The titration experiments were conducted in 0.15 M KCl Ionic Strength Adjusted solution (ISA water) under a nitrogen atmosphere at a temperature of 25±1° C. All tests were performed using standardized 0.5 M KOH and 0.5 M HCl as titration reagents, for the partition coefficient tests a saturated Octanol in ISA water (5% of ISA water) was used as partition solvent. The pKas were determined by potentiometric method by pH-metric titration. The powder (around 0.5 mg) was dissolved in 1.5 ml of ISA water and the titration was performed in triplicate in the pH range 2.0−12.0. Each Log P were performed in triplicate by dissolving the powder (around 1 mg) in 1 ml of ISA water followed by pH metric titration in three different percentages of octanol (in general 50%, 60%, 70%).

(584) b. Water Solubility and Stability

(585) General Procedure for the Preparation of the Stock Solutions

(586) Each lyophilized peptide was weighed in an amber vial. A calculated volume of phosphate buffered solution at pH 7.8 was added in order to directly obtain 10 mM Stock Solutions. The recovered suspensions were stirred for 30 minutes by orbital shaker incubator at 37° C. Subsequent dilutions were performed to obtain the more diluted stock solutions for the less soluble compounds.

(587) In order to evaluate the stability and the solubility, all the stock solutions were stored at 25° C. The title was evaluated by UHPLC-MS analysis in comparison with the standard solutions, at 3 time points: T0, 3 days, 1 week.

(588) The chromatographic profile was evaluated for the quantitative assay while the MS spectra were detected as support in the stability studies.

(589) General Procedure for the Standard Solution Preparation

(590) Each peptide was weighed and directly diluted at 100 μM with the phosphate buffer at pH 7.8. The solutions were stirred for 5 minutes by vortex and heated at 37° C. for 20 minutes. The obtained standard solution was stored at 25° C. in the dark under controlled temperature.

(591) General Procedure for the Sample Preparation

(592) A portion of the stock solutions was collected in an amber vial and diluted to 100 μM by phosphate buffer at pH 7.8 (dilution factor, 1:100).

(593) Instrument Method

(594) Reversed-phase UHPLC was performed operating at a flow rate of 0.3 ml/min with a Luna Omega Polar C18 column (pore size 1.6 μm, 2.1×100 mm). Solvent A was 0.5% Formic Acid in water and solvent B was 0.5% Formic Acid in acetonitrile. First, samples were loaded onto the column and washed isocratically at 10% B for 3 min. Then, a gradient of 10-95% B was run over 22 min, held at 95% for 2 min, and then decreased to 10% over 1 min and held, for the pressure stabilization, for 5 min. UV Absorption was monitored by DAD (220-400 nm). ESI-MS ion trap was used for the monitoring of TIC.

(595) ii. Results

(596) All peptides tested showed stability at 25° C. up to 1 week since no significant variations were found in the chromatographic profile and in the mass spectra.

(597) The physchemical properties of the tested peptides are displayed in Table 8 below

(598) TABLE-US-00011 TABLE 8 PEPTIDE IP Log D (7.4) logP SOLUBILITY SEQ ID NO: 1 5.14 −3.68 −5.98 >10 mM SEQ ID NO: 2 5.14 −0.56 0.99 >10 mM SEQ ID NO: 3 3.90 −1.87 1.33 10 μM SEQ ID NO: 4 3.90 −1.32 1.88 20 μM SEQ ID NO: 5 5.19 −1.26 −0.51 1 mM SEQ ID NO: 6 5.14 −3.65 −0.37 >10 mM SEQ ID NO: 7 5.19 −1.41 −0.86 >10 mM SEQ ID NO: 8 5.14 −4.06 −5.12 >10 mM SEQ ID NO: 9 8.28 −1.08 −0.53 >10 mM SEQ ID NO: 10 5.14 −3.66 0.09 >10 mM SEQ ID NO: 11 4.25 −2.92 1.05 >10 mM SEQ ID NO: 12 3.66 −5.24 1.31 10 μM SEQ ID NO: 13 6.06 1.15 −0.49 >10 mM SEQ ID NO: 14 8.99 −2.80 −5.40 >10 mM SEQ ID NO: 15 3.51 1.88 1.66 50 μM SEQ ID NO: 16 5.19 0.24 0.13 >10 mM SEQ ID NO: 17 8.05 −3.68 −0.44 >10 mM SEQ ID NO: 18 3.55 −3.79 1.89 50 μM SEQ ID NO: 41 3.54 −1.94 1.24 >10 mM SEQ ID NO: 19 12.49 −3.94 −8 100 μM SEQ ID NO: 20 11.29 −3.69 −6.79 100 μM SEQ ID NO: 21 10.6 −0.55 −4.99 >10 mM SEQ ID NO: 22 4.26 −3.18 0.4 >10 mM SEQ ID NO: 23 4.26 −2.24 1.01 50 μM SEQ ID NO: 24 5.14 2.15 2.45 <50 μM SEQ ID NO: 25 4.66 −5.64 0.4 100 μM SEQ ID NO: 26 8.13 0.148 0.69 >10 mM SEQ ID NO: 27 5.14 0.148 0.69 >10 mM SEQ ID NO: 28 12.25 −6.44 −6.36 100 μM SEQ ID NO: 29 12.25 −6.44 −6.39 >10 mM SEQ ID NO: 30 12.96 −3.89 −7.44 100 μM SEQ ID NO: 31 12.5 −3.59 −4.9 >10 mM SEQ ID NO: 32 na −3.59 −10 <50 μM SEQ ID NO: 33 4.46 −1.06 1.99 100 μM SEQ ID NO: 34 5.14 −3.38 −5.43 <50 μM SEQ ID NO: 35 5.14 −3.52 −5.43 >10 mM SEQ ID NO: 36 8.13 −1.24 −1.09 100 μM SEQ ID NO: 37 5.14 −3.9 −6.89 >10 mM SEQ ID NO: 38 5.14 −3.46 −5.88 >10 mM SEQ ID NO: 39 4.2 −2.86 −5.36 >10 mM SEQ ID NO: 40 4.21 −3.5 −4.93 >10 mM SEQ ID NO: 111 8.99 −0.93 −0.38 <50 μM SEQ ID NO: 112 8.99 −7.295 −4.76 <50 μM SEQ ID NO: 113 8.99 −3.472 −0.13 <50 μM SEQ ID NO: 114 8.99 −3.591 −0.21 <50 μM SEQ ID NO: 115 8.99 −3.914 −5.94 <50 μM SEQ ID NO: 121 8.99 −1.474 −0.51 <50 μM

(599) As can be seen from the above, most of the peptides according to the invention have optimal physchem properties, showing a good balance between solubility and lipophilicity. The mean of Log D of the peptides was −2.64±2.06, while for most of the peptides, the solubility observed in water was very good (>10 mM). The properties observed make them particularly suitable to be used for ophthalmic and dermatological applications, since both hydrophobic and hydrophilic components are present in the derma and eye surface. They also explain the good permeability observed by peptides (see Example 8).

Example 8: Permeability Test

(600) The peptides tested were the peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115 and SEQ ID NO: 121. They were dissolved in Cell Culture Grade Water (Corning, Manassas, Va., USA) in order to have 10 mM stock solution.

(601) i. Methods

(602) In Vitro Model

(603) The I-HCEC, Immortalized Human Corneal Epithelial cell line was derived from Primary Human Comeal Epithelial Cells (>99% purity). These cells are suitable for studies of human cornea in health and disease. The cells were cultured following the protocol and using the media suggested by Innoprot, Bizkaia, Spain (IM-Corneal Epithelial Cell Medium ref. P60131).

(604) Immunofluorescence

(605) Qualitative and quantitative techniques were developed to first confirm and quantify the barrier integrity before proceeding with drug testing. First, we performed immunofluorescence for a protein characteristic of tight junctions (Z0-1) (Sugrue S P et al., (1997) Exp Eye Res. January; 64 (1): 11-20). The cells were seeded in coverslip coated with Collagen I 50 μg/ml (stock solution 3 mg/ml from Gibco, NY, USA) at a density of 1*104 cells/cm2 and cultured for 14 DIV and post-confluency the media were refreshed every day. Cells were fixed in methanol at −20° C. for 10 min, rinsed with phosphate buffer solution (PBS), incubated for 10 min at room temperature (RT) in PBS containing 4% bovine serum albumin (BSA; Sigma-Aldrich), and successively incubated with the primary and secondary antibodies for 60 min each at RT. Primary antibody was goat anti-human Z0-1 (1:100, Thermo Fisher) overnight at 4° C. followed by anti-goat Alexafluor 488 (1:10000). Cells were finally counterstained with DAPI (0.5 mg/ml) (Sigma-Aldrich) for 5 min at room temperature, mounted with Vectashield Mounting Medium (Vector Laboratories Inc, Burlingame, Calif.) and observed at confocal laser scanning Microscope (Leica TCS SP5, Wetzlar, Germany).

(606) MTS Assay

(607) Cell viability was determined at 24 h using Cell Titer One Solution Cell Proliferation Assay (Promega Corporation Madison, Wis., USA) a colorimetric method based on 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenii)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). The quantity of formazan formed, as a function of viability, was measured at 492 nm using an ELISA plate reader, Infinite F200 (Tecan, Mannedorf, Swiss). The assay was performed at 50 and 100 μM (N=9). The results were expressed as absorbance at 492 nm.

(608) TEER Assay

(609) The barrier integrity of corneal epithelial cultures was verified using measurement of TEER (TransEpithelial/TransEndothelial Electrical Resistance)) and permeability of paracellular permeants (e.g., mannitol, 6-carboxyfluorescein) (Ronkko S et al., (2016) Drug Deliv Transl Res 6: 660-675). TEER is a widely accepted quantitative technique to measure integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. TEER values are indicators of the integrity of the cellular barriers before they are evaluated for transport of drugs or chemicals. TEER measurements can be performed in real-time without cell damage and generally are based on measuring osmic resistance or measuring impedance across a wide spectrum of frequencies (Snirivasan et al., 2015 J Lab Autom. 20(2): 107-126). To perform this assay Millicell ERS-2, Electrical Resistence System by EMD Millipore (Burlington, Mass., USA), was used following manufacturer's instructions. The assay was performed at 50 and 100 μM (N=9). Results were expressed as Resistance (Q/cm2).

(610) Permeability Test

(611) The objective of this study was to evaluate the apparent permeability coefficient (Papp) value of the peptides across stratified human corneal epithelial cells. All peptides were tested at 50 μM in duplicate.

(612) In particular, human corneal epithelial cells (hCEPIC) were stratified on Transwell cell culture inserts (Corning, N.Y., USA). The permeation experiments were performed taking samples from receiver chambers at 5, 30, 60 minutes time points and from donor chambers at the same time points. Each aliquot collected was analyzed by HPLC instrument (Knauer) equipped with C18 Vertex Plus Column (Knauer) thermostatically controlled at 40° C. The rate (apparent permeability coefficient, Papp) of the study compounds was calculated and compared to the Papp values of the low and high permeability markers, 6-carboxyfluorescein and Rhodamine B, respectively.

(613) The apparent permeability coefficient (Papp) was calculated according to Equation:
Papp=(I/A*C0)(dM/dt)

(614) where dM/dt is the flux (nanomoles per second) across the cell layers;

(615) A (square centimeters) is the exposed surface area of the insert membrane;

(616) C0 is the initial drug concentration (micromolar) in the donor compartment at t=0 (Xiang et al., Drug Metab Dispos. 2009 May; 37(5):992-8.

(617) Statistics

(618) All groups (studied compound and rhodamine) were compared to low permeability control. All the other experiments were expressed as the meant S.E. and the significance were calculated by Student's t test versus control cells. Difference were considered to be statistically significant (p<0.05) as determined by Student's t test.

(619) ii. Results

(620) MTS and TEER Assays

(621) In our experimental conditions, the peptides considered did not significantly affect cell viability or TEER at 50 μM and 100 μM test concentration.

(622) Permeability Test

(623) The results obtained for each peptide are shown in Table 9 below.

(624) TABLE-US-00012 TABLE 9 Permeability Peptide N/Name Sequence (10.sup.−6 cm/sec) SEQ ID NO: 1 IHVTIPADLWDWINK 19.333 SEQ ID NO: 7 IHVTIPADLWEWINK 24.252 SEQ ID NO: 11 IHVTIPADLWDWIEK 28.020 SEQ ID NO: 2 VHVTIPADLWDWINK 28.862 SEQ ID NO: 14 IHVTIPADLWDWVRR 28.873 SEQ ID NO: 38 VPGAGVPGAGIHVTI 30.417 PADLWDWINK SEQ ID NO: 121 VHVTIPADLWEWFRR 47.083 SEQ ID NO: 115 VHVTIPQDLWEWVRR 68.800 SEQ ID NO: 113 VHVQIPADLWEWVRR 76.797 SEQ ID NO: 111 VHVTIPAELWEWVRR 83.698 Rhodamine B 51.26 6-carboxyfluorescein 0.61

(625) To verify that the HCE cell culture was useful in screening the peptides, the permeability of a lipophilic transcellular marker rhodamine B was determined. As expected, rhodamine B exhibited high permeability of the HCE cell culture (51.26±5.44 10.sup.−6 cm/sec). Then, the permeability of 6-carboxyfluorescein was assayed and in agree with the literature (0.61±0.33 10.sup.−6 cm/sec). This marker has a very low lipophilicity, and it permeates biological membranes only through the intercellular space.

(626) The tested peptides showed a very good permeability towards the Immortalized Human Corneal Epithelial compared to high permeability standard, Rhodamine B. Obviously, the HCE permeability model does not include stroma or endothelium, but these layers are not critical barriers to corneal drug absorption. We found peptides with a good permeability (SEQ ID NO: 2) and peptides with a higher permeability compared to Rhodamine B (SEQ ID NO: 115, SEQ ID NO: 113 and SEQ ID NO: 111).

Example 9: Peptide-IL-17A Affinity Test

(627) The peptide of the prior art HAP (SEQ ID NO:1) and peptide of SEQ ID NO:2 were tested for affinity for mIL-17A, as described below.

(628) a. Immobilization of mIL-17A

(629) For the immobilization of mouse IL-17A protein, amine coupling chemistry was used. On the senorchip surface the dextran matrix was first activated with a mixture of 1-ethyl-3-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in order to obtain reactive succinimido esters. 10 ug/ml of IL17 was diluted in 10 mM acetate buffer at pH=5.0 and then the ligand passed over the surface to allow the esters to react spontaneously with uncharged amino groups, in this way the ligand is covalently linked to dextran. Covalent immobilization of mIL17 results in stable attachment of 3000 RU of ligand to the surface.

(630) b. Characterization of the Peptides/IL17 Interaction

(631) The peptides of SEQ ID NO: 1 (HAP) and SEQ ID NO: 2 were analyzed for their ability to bind the immobilized mIL-17A. The molecules were diluted at different concentrations (from 62.5 to 1000 nM) in HBS-EP buffer and injected on the sensorchip for 4 minutes and allowed to dissociate for 5 minutes. After each run, the sensorchip was regenerated by 2 M NaCl injection. Based on the sensorgrams (FIG. 1, panel A) and using the BIAevaluation software, the Scatchard analysis of the RU values at equilibrium allowed to determine an affinity constant as a function of the analyte concentration in solution (FIG. 6, panel B). The results obtained are shown in Table 10 below

(632) TABLE-US-00013 TABLE 10 Compound Kdea (nM) SEQ ID NO: 1 3700 SEQ ID NO: 2 130

(633) As shown in Table 8, the peptide of SEQ ID NO: 2 binds to IL-17A with a value of affinity equal to 130 nM, while peptide of SEQ ID NO: 1 binds to IL17-A with a very low affinity value (3700 nM).

(634) In view of the results obtained in Example 8 and 9, the modification of the Isoleucin in position 1 in the HAP peptide of the prior art with a Valine, as in the peptide of SEQ ID NO: 2, leads to a striking difference regarding the physchemical properties, permeability and affinity for the ligand.

(635) We summarize herein below the properties of the two peptides:

(636) TABLE-US-00014 TABLE 11 SEQ ID NO: 1 Property (HAP) SEQ ID NO: 2 Sequence IHVTIPADLWDWINK VHVTIPADLWDWINK logP −5.98 0.99 logD (7.4) −3.68 −0.56 Permeability 19.3 28.86 (Papp ± SE) SPR Kdeq (nM) 3700 130

(637) Table 11 shows the comparison data between peptide SEQ ID NO: 1 and SEQ ID NO: 2. We found great differences in term of log P and log D (6-log and 3-log difference, respectively): this explained the higher permeable observed with SEQ ID NO: 2 compared to SEQ ID NO: 1 (28.8 vs 19.3). The Kd (nM) measured in the Surface plasmon resonance (SPR) assay performed on mouse IL-17A, showed that SEQ ID NO: 2 was 28-fold more affine than SEQ ID NO: 1.

(638) Furthermore, molecular dynamics simulations studies were also carried out, which indicate that Valine (SEQ ID NO: 2) instead of the Isoleucine (HAP) greatly stabilizes the folded structure: SEQ ID NO: 2 was predicted to be 80% more stable.

Example 10: In Vitro Evaluation of Expression of Inflammatory Cytokines

(639) The peptide HAP (SEQ ID NO:1) and the peptide of SEQ ID NO:2 were tested in vitro for evaluation of their direct influence on expression of inflammatory cytokines in the cornea. The tests were conducted on Immortalized Human Corneal Epithelial cell line (I-HCEC) (Innoprot, Bizkaia, Spain, Ref. P10871). This is derived from Primary Human Corneal Epithelial Cells (>99% purity) and is suitable for studies of human cornea in health and disease. The cells were cultured following the protocol and using the media suggested by Innoprot (IM-Corneal Epithelial Cell Medium ref. P60131). The cell medium was supplemented with 5% Fetal Bovine Serum (FBS).

(640) Cells were seeded at 320000 cells/well 6 plate and 15000 cells/well 96 plate. When the cells reached 80% of confluence were treated with SEQ ID NO: 1 and SEQ ID NO: 2 compounds in dose-curve (0.01-500 μM) for 24 hours.

(641) Total RNA was isolated from 6 wells plate using Quick-Start RNeasy Mini Kit (Qiagen, cat. No 74104) according to the manufacturer's instructions. Retro-transcription was performed using SuperScript Vilo (ref. 11755050, Life technologies) and real-time PCR using TaqMan protocol (ref. 4444557, Applied Biosystem) adapted for CFX96 Real-Time System. The experiments were performed three times, each time in duplicate. The used probes from Thermo Fisher Scientific are: human CXCL8 (Hs00174103_m1), human IL6 (Hs00174131_m1), human TNFα (Hs99999043_m1). As housekeeping gene was used GAPDH (probe Hs02758991_g1). IL-8, IL-6 and TNF-α expression in I-HCEC was evaluated after treatment for 24 hours with different concentration of the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 (0.01, 0.1, 1, 10, 100, 500 μM). Untreated I-HCEC were used as negative control (Vehicle).

(642) The results show that the compound SEQ ID NO: 2 does not change the expression of any of the measured inflammatory cytokines (FIGS. 8A, 9A and 10A), while the treatments with 500 μM of the compound SEQ ID NO: 1 significantly induce the expression of the two inflammatory cytokines IL-6 and TNFα (FIGS. 9B and 10B).

(643) The data obtained demonstrate that peptide of SEQ ID NO: 1 has a direct pro-inflammatory activity and induces significantly the expression of IL-6 and TNF-α inflammatory cytokines by HCEC, with an increase of 10 and 8 fold, respectively. On the contrary, the peptide of SEQ ID NO: 2 does not show any effect on the expression of cytokines.

(644) In view of the above, it can be concluded that the peptide of SEQ ID NO: 2 surprisingly shows significantly better tolerability and lack of toxicity compared to the peptide HAP of SEQ ID NO: 1.