Method for treating dry eye disease

Abstract

Methods for treating dry eye disease in a subject and for slowing, inhibiting, or delaying the progression of dry eye disease are provided. The methods include administering a composition comprising a therapeutically effective amount of a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor. In embodiments, the compound is a protein or a peptide.

Description

Technical Field

[0001] Reference to a Sequence Listing, Table, or Computer Program

[0001] This application includes a Sequence Listing electronically submitted in XML format. The Sequence Listing XML is incorporated herein by reference. The XML file created on June 7, 2023, is named 113665-0038-8001WO00_SEQ.xml and is 186,910 bytes.

[0002] Technical Field

[0002] The subject matter described herein relates to methods for treating dry eye disease and for slowing, inhibiting, or delaying the progression of dry eye disease in a subject by administering a composition comprising a compound that inhibits the binding of a therapeutically effective amount of an inflammatory interleukin-17 (IL-17) cytokine to an IL-17 receptor.

Background Art

[0003] Background

[0003] Dry eye is a multifactorial, inflammatory, immune-mediated disease of the ocular surface that results in discomfort, visual disturbances, and symptoms of tear film instability and epithelial cell damage on the ocular surface. The normal tear film is a relatively stable thin film composed of an aqueous layer mixed with a surface lipid layer and a mucin gel layer that is partially adherent to the corneal and conjunctival surface epithelia. The natural tear film is important for lubrication and maintenance of the refractive surface of the eye. Dry eye disease is a complex disorder characterized by dysfunction of one or more components of the tear film, resulting in a decrease in tear film homeostasis or stability, hyperosmotic changes in the tear film osmotic balance, and / or an inappropriate amount of body fluid on the ocular surface. This is characterized by rapid disruption of the tear film and a number of symptoms including dry eye, eye pain, burning / stinging, foreign body sensation, itching, and photophobia.

Summary of the Invention

Means for Solving the Problems

[0004] Summary

[0004] The following aspects and embodiments described and illustrated below are intended to be representative and exemplary without limiting the scope.

[0005]

[0005] In one aspect, a method for treating dry eye disease in a subject diagnosed with dry eye disease is provided. The method includes directly administering to the eye of a subject in need thereof a composition comprising a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor in a therapeutically effective amount.

[0006]

[0006] In another aspect, a method for slowing, inhibiting, or delaying the progression of dry eye disease in a subject is provided. The method includes directly administering to the eye of a subject in need thereof a composition comprising a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor in a therapeutically effective amount.

[0007]

[0007] In another aspect, a method for reversing the progression of dry eye disease in a subject is provided. The method includes directly administering to the eye of a subject in need thereof a composition comprising a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor in a therapeutically effective amount.

[0008]

[0008] In another aspect, a method for restoring the homeostasis of the tear film on the surface of the eye is provided. The method includes directly administering to the eye of a subject in need thereof a composition comprising a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor in a therapeutically effective amount.

[0009]

[0009] In one embodiment, the method includes identifying a subject having mild or moderate dry eye disease.

[0010]

[0010] In one embodiment, the method includes identifying a subject having mild, moderate, or severe dry eye disease.

[0011]

[0011] In one embodiment, identifying the subject includes using a questionnaire.

[0012]

[0012] In one embodiment, the questionnaire is selected from a Visual Analogue Scale (VAS), an Ocular Surface Disease Index (OSDI) questionnaire, a Symptom Assessment Questionnaire iN Dry Eye (SANDE), a Dry Eye Questionnaire (DEQ), and a Standard Patient Evaluation of Eye Dryness Questionnaire (SPEED).

[0013]

[0013] In one embodiment, the method includes identifying a subject having mild or moderate dry eye disease and monitoring the progression of the dry eye disease.

[0014]

[0014] In certain embodiments, the progression of the dry eye disease is monitored using a visual analogue scale of eye dryness, such as VAS or SANDE.

[0015]

[0015] In one embodiment, the composition to be administered includes a compound selected from a biomolecule or an organic synthetic compound.

[0016]

[0016] In one embodiment, the organic synthetic compound is a spirocyclic indane compound or a spirocyclic oxoindoline compound.

[0017]

[0017] In one embodiment, the biocompound is an antibody having binding affinity for an IL-17 cytokine or an antibody having binding affinity for an IL-17 receptor.

[0018]

[0018] In one embodiment, the antibody is a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a humanized, recombinant antibody, a chimeric antibody, or an antibody fragment.

[0019]

[0019] In one embodiment, the antibody is selected from the group consisting of afasevikumab, bimekizumab, brodalumab, ixekizumab, izokibep netakimab, perakizumab, secukinumab, sonelokizumab, tibulizumab, bunakizumab, ABY-035, CJM-112, CNTO-6785, DC-806 / S-011806, FPP-003, GR-1501, HB-0017, IMU-035, LZM-012, QX-002N, BH-1657, HB-0043, HT-0017, ILCT-1001, IQ-001, LEO153339 / LP0200, LP-0200, MT-6194, MYMD-1, ND-016, SCT-650A, SM-17, YBL-004, ABM-60, ETI-1023, LQ-025, LQ-026, ABBV-257, AFB-035, ANB-004, BCD-121, COVA-322, CYT-017-IL17Qb, DLX-2882, DLC-2907, DLX-2909, DLX-3003, E-34935, E-35018, E-35762, E-36041, EBI-006, HEISCO-III-002, IL-17-RC, MEDI-571, MOR-106, MP-0230, PRS-190, SCH-900117, Y-320, ABT-122, BITS-7201A, CDP-435, EBI-028, IL-17E, JNJ-6118104, KHK-4827, and RG7624.

[0020]

[0020] In one embodiment, the biological compound is a protein or peptide that specifically inhibits the binding of interleukin 17A to the interleukin 17A receptor.

[0021]

[0021] In one embodiment, the peptide is a contiguous sequence of 10 to 25, 12 to 20, 13 to 18, 13 to 16, 14 to 16, or 15 amino acid residues, and consists of a contiguous sequence having at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of Formula I. In another embodiment, the peptide is a contiguous sequence of 12 to 18 amino acid residues and consists of a contiguous sequence having at least about 70% sequence identity with SEQ ID NO: 1. In another embodiment, the peptide is a contiguous sequence of 12 to 18 amino acid residues and consists of a contiguous sequence having at least about 70% sequence identity with any one of the sequences identified herein as SEQ ID NOs: 1 to 166. In another embodiment, the peptide is a contiguous sequence of 12 to 18 amino acid residues and consists of a contiguous sequence having at least about 70% sequence identity with any one of the sequences identified herein as SEQ ID NOs: 1 to 214. In another embodiment, the peptide is a contiguous sequence of 12 to 18 amino acid residues and consists of a contiguous sequence having at least about 70% sequence identity with any one of the sequences identified herein as SEQ ID NOs: 1 to 166, provided that the peptide is not SEQ ID NOs: 167 to 214.

[0022]

[0022] In one embodiment, the peptide is of Formula I: X1-X2-X3-X4X5-X6-X7-X8-X9-X 10 -X1I-X 12 -X 13 -X 14 -X 15 Formula (I) (wherein, X1 is I, V or L; X2 is H, M, R, K or E; X3 is V, F or I; X4 is T, Q, S, Y or N; X5 is I, F or V; X6 is P or G; X7 is A, Q, or L; X8 is D, E, or Q; X9 is L, W, F, V or I; X10 is W, Y or F; X 11 is D, E or N,; X 12 is W or F; X 13 is I, V, F or L; X 14 is N, R, Q or E; and X 15 is K, R, H or E) comprises the amino acid sequence of.

[0023]

[0023] In one embodiment, the sequence is not IHVTIPADLWDWINK (SEQ ID NO: 167).

[0024]

[0024] In one embodiment, in the amino acid sequence of Formula I, (a) X1 is I or V, X2 is H, M or R, X3 is V or F; X4 is T or Q; X5 is I, F or V; X6 is P or G; X7 is A or Q; X8 is D or E; X9 is L; X 10 is W or Y; X is D or E; X 12 is W; X 13 is I or V; X 14 is N, R or E; and X 15 is K, R or E; or (b) X1 is I or V; X2 is H or M, X3 is V; X4 is T; X5 is I; X6 is P; X7 is A; X8 is D; X9 is L, W, F, V or I; X 10 is W or Y; X 11 is D or E; X 12 is W; X 13 is I or V; X 14 is N, R or E; and X 15 is K, R or E.

[0025]

[0025] In one embodiment, the sequence is not IHVTIPADLWDWINK (SEQ ID NO: 167), HVTIPADLWDWIN (SEQ ID NO: 168), IHVTIPADLWDWI (SEQ ID NO: 169) or IHVTIPADLWDW (SEQ ID NO: 170).

[0026]

[0026] In one embodiment, the peptide binds its C-terminus and / or N-terminus to a protective cap group, where the protective cap group bound to the C-terminus is selected from the group consisting of amide, aldehyde, ester, p-nitroanilide, 7-amino-4-methylcoumarin. In one embodiment, the protective group cap bound to the N-terminus is selected from the group consisting of acetyl, formyl, pyroglutamyl, fatty acid, urea, carbamate sulfonamide, and alkylamine.

[0027]

[0027] In one embodiment, in the form of a dimer peptide formed by two peptides, each peptide is a peptide of Formula I.

[0028]

[0028] In one embodiment, the two peptides in the dimer are linked by a polyethylene spacer.

[0029]

[0029] In one embodiment, the peptide is in the form of a bioconjugate comprising a peptide of formula I and a biomolecule, where the biomolecule is attached to the N-terminus and / or C-terminus of the peptide.

[0030]

[0030] In one embodiment, the biomolecule is selected from the group consisting of capric acid, caproic acid, ascorbic acid, NAG-NAM, NAG, NAM, hyaluronic acid, alginic acid, chitin, (GalNAc)2, Gal-alpha1,3-GalNAc, and trigalacturonic acid.

[0031]

[0031] In one embodiment, the peptide is present in a pharmaceutically acceptable composition comprising at least one pharmaceutically acceptable excipient.

[0032]

[0032] In one embodiment, administration comprises instilling onto the ocular surface.

[0033]

[0033] In one embodiment, administration comprises instilling into the conjunctival sac.

[0034]

[0034] In one embodiment, administration comprises once-daily administration.

[0035]

[0035] In one embodiment, a therapeutically effective dose of the peptide is between about 0.01 - 1,000 μM or between about 0.05 - 750 μM or between about 0.05 - 500 μM, or at least about 500 μM.

[0036]

[0036] In one embodiment, the dose is administered once daily.

[0037]

[0037] In one embodiment, administration is carried out over a period of at least about 3 weeks, where the signs and / or symptoms of dry eye disease are resolved over the disease-free period in the 3rd week of said administration. In another embodiment, administration is carried out over a period of at least about 3 weeks, where the signs of dry eye disease are resolved over the disease-free period in the 3rd week of said administration.

[0038]

[0038] In another aspect, a method for restoring the function and / or density of goblet cells in an eye having signs and / or symptoms of dry eye disease and / or preventing goblet cell depletion in the eye of a person having dry eye disease is provided. The method includes topically administering to the eye of a subject in need thereof a therapeutically effective amount of a compound that inhibits the binding of IL-17A to the interleukin 17A receptor.

[0039]

[0039] In another aspect, a method for improving the balance of the functions of the lacrimal gland, Meibomian gland and conjunctival goblet cells and restoring the homeostasis of the tear film in a subject is provided. The method includes topically administering to the eye of a subject in need thereof a therapeutically effective amount of a compound that inhibits the binding of IL-17A to the interleukin 17A receptor.

[0040]

[0040] In another aspect, a method for increasing basal tear production in a human subject is provided. The method includes topically administering to the eye of a subject in need thereof a therapeutically effective amount of a compound that inhibits the binding of IL-17A to the interleukin 17A receptor.

[0041]

[0041] In one embodiment, the subject is a subject at least about 50 years old (an “elderly” subject). In certain embodiments, the elderly subject has hormonal changes and / or is over 50, 55 or 60 years old.

[0042]

[0042] In one embodiment, the compound for use in any of the methods is not any one or more of DLSAVCWAFPWDPECH (SEQ ID NO: 171), DSSAVCWAFPHHPLCHMKAT (SEQ ID NO: 172), ADADMCWFFPTSPWCH (SEQ ID NO: 173), DSSAVCWAFPYLPECH (SEQ ID NO: 174), DISAVCWAFPFDPECH (SEQ ID NO: 175), AYECPRLEYDMFGALHCLPS (SEQ ID NO: 176), CPRLEYDMFGALHCL (SEQ ID NO: 177), CLDLQYDPWGALHCI (SEQ ID NO: 178), CFDLQYDPWGALHCI (SEQ ID NO: 179), CLDLQYDMFGALHCV (SEQ ID NO: 180), CLDLVYDPWGALHCI (SEQ ID NO: 181), CWVLEYDMFGALHCR (SEQ ID NO: 182), CWALEYDMFGYLHCR (SEQ ID NO: 183), CWVLEYDMFGFLHCR (SEQ ID NO: 184), CWVLEYDMFGYLHCR (SEQ ID NO: 185), and GPYYFDSSGYLYYYYGLDV (SEQ ID NO: 186).

[0043]

[0043] In addition to the above exemplary aspects and embodiments, further aspects and embodiments will become apparent by reference to the drawings and by examination of the following description.

[0044]

[0044] Additional embodiments of the present method and the like will be apparent from the following description, drawings, examples, and claims. As can be understood from the foregoing and the following description, all features described herein, and any combination of two or more such features, are included within the scope of the present disclosure, provided that the features included in such combinations are not mutually inconsistent. Further, any feature or combination of features may be specifically excluded from any embodiment of the present disclosure. Additional aspects and advantages of the present disclosure are described in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings.

Brief Description of the Drawings

[0045] Brief Description of the Drawings

Figure 1

[0045] Figure 1 is a graph of corneal fluorescein staining (CFS) scored using the National Eye Institute Industry Workshop Scale of 0 - 15 as a function of time over several days in mice in which dry eye disease was induced by placing the mice in a controlled environmental chamber (relative humidity: <20%, dry airflow: 15 L / min, temperature: 21 - 23 °C) for 14 days. Corneal epithelial disease was evaluated using corneal fluorescein staining (CFS) and scored using the National Eye Institute Industry Workshop Scale of 0 - 15 on day 0 (normal baseline), day 4 (before topical therapy instillation), day 7, day 10, and day 14 in mice treated by topical application to the eye of anti - IL - 17A antibody once daily (black circles) or twice daily (black squares), lifitegrast twice daily (XIIDRA®) (inverted triangles), cyclosporine twice daily (RESTASIS®) (triangles), or saline twice daily (diamonds).

Figure 2A

[0046] Figure 2A is a bar graph showing the effect of the indicated treatments on improving the immunoregulatory function of Treg cells as measured by the expression of FoxP3 in the lymph nodes of the mice from the tests in Figure 1 and Example 1.

Figure 2B

[0046] Figure 2B is a bar graph showing the effect of the indicated treatments on suppressing the differentiation of naive T cells into pathogenic Th17 cells in the lymph nodes of the mice from the tests in Figure 1 and Example 1.

Figure 3A

[0047] Figure 3A is an image of conjunctival tissue showing the frequency of goblet cells (large black dots in the upper cell layer) from healthy mice without dry eye disease.

Figure 3B

[0047] Figure 3B is an image of conjunctival tissue showing the frequency of goblet cells (large black dots in the upper cell layer) from mice in which dry eye disease was induced by placing the mice in a controlled environmental chamber and treated twice daily with saline as a control.

Figure 3C

[0047] Figure 3C is an image of conjunctival tissue showing the frequency of goblet cells (large black dots in the upper cell layer) from mice in which dry eye disease was induced by placing the mice in a controlled environmental chamber and treated twice daily with cyclosporine ophthalmic emulsion (RESTASIS®).

Figure 3D

[0047] Figure 3D is an image of conjunctival tissue showing the frequency of goblet cells (large black dots in the upper cell layer) from mice in which dry eye disease was induced by placing the mice in a controlled environmental chamber and treated twice daily with lifitegrast ophthalmic solution (XIIDRA®).

Figure 3E

[0047] Figure 3E is an image of conjunctival tissue showing the frequency of goblet cells (large black dots in the upper cell layer) from mice in which dry eye disease was induced by placing the mice in a controlled environmental chamber and treated twice daily with an anti-IL-17A antibody.

Figure 3F

[0048] Figure 3F is a bar graph of the percentage of goblet cells for normal (healthy) mice and for mice in which dry eye disease was induced by placing the mice in a controlled environmental chamber and treated with saline, cyclosporine, lifitegrast, or an IL-17A inhibitor.

Figure 4A

[0049] Figure 4A is an image of a microscopic examination of human corneal epithelial cells.

Figure 4B

[0049] Figure 4B is an image of a microscopic examination of human corneal epithelial cells under co-culture with Th17 cells treated with a saline vehicle.

Figure 4C

[0049] Figure 4C is an image of a microscopic examination of human corneal epithelial cells under co-culture with Th17 cells treated with an IL-17 inhibitor, the IL-17A antibody.

Figure 5A

[0050] Figure 5A is an image from a microscopic examination (40x) of human corneal epithelial cells under bright light.

Figure 5B

[0050] Figure 5B is an image from a microscopic examination (40x) of human corneal epithelial cells under bright light under co-culture with Th17 cells treated with a saline vehicle.

Figure 5C

[0050] Figure 5C is an image from a bright-field microscopy (40x) of human corneal epithelial cells under co-culture with Th17 cells treated with an IL-17 inhibitor (SEQ ID NO: 1 (VHVTIPADLWDWINK)) at a dose of 0.1 μM.

Figure 5D

[0050] Figure 5D is an image from a bright-field microscopy (40x) of human corneal epithelial cells under co-culture with Th17 cells treated with an IL-17 inhibitor (SEQ ID NO: 1 (VHVTIPADLWDWINK)) at a dose of 1 μM.

Figure 5E

[0050] Figure 5E is an image from a bright-field microscopy (40x) of human corneal epithelial cells under co-culture with Th17 cells treated with an IL-17 inhibitor (SEQ ID NO: 1 (VHVTIPADLWDWINK)) at a dose of 10 μM.

Figure 5F

[0050] Figure 5F is an image from a bright-field microscopy (40x) of human corneal epithelial cells under co-culture with Th17 cells treated with an IL-17 inhibitor (SEQ ID NO: 1 (VHVTIPADLWDWINK)) at a dose of 100 μM.

Figure 6A

[0051] Figure 6A is a graph of corneal fluorescein staining (CFS) scored using the National Eye Institute Industry Workshop Scale of 0 - 15, on day 0 (normal baseline), day 4 (before topical treatment instillation), and during treatment with an anti-IL-17A antibody at a dose of 1% of an IL-17 inhibitor administered twice daily from day 4 to day 12 (inverted triangles), or during treatment with an IL-17 inhibitor (SEQ ID NO: 1 (VHVTIPADLWDWINK)) at either a dose of 0.05 wt% (circles) or 0.1 wt% (diamonds) in phosphate-buffered saline; the control group received a saline vehicle twice daily from day 4 to day 12 (squares).

Figure 6B

[0052] Figure 6B is a bar graph of the percentage of Th17 cells in the draining lymph nodes of the same mice from Figure 6A, including mice without dry eye disease and mice treated with physiological saline (control), or an IL-17A antibody at a dose of 1 wt% (10 mg / mL) or an IL-17 inhibitor (SEQ ID NO: 1 (VHVTIPADLWDWINK)) at either a dose of 0.05 wt% or 0.1 wt% in phosphate buffered saline, which were induced to have dry eye disease by placement in a controlled environmental chamber and administered twice daily for 9 days.

Figure 7

[0053] Figure 7 is a graph of corneal fluorescein staining (CFS) scored using the National Eye Institute Industry Workshop Scale of 0 - 15 as a function of time over several days during the treatment phase of a study on mice with dry eye disease and treated with an IL-17 inhibitor (SEQ ID NO: 1 (VHVTIPADLWDWINK)) formulated in phosphate buffered saline at a concentration of 50 μM (administered twice daily, inverted triangles), 250 μM (administered once daily (diamonds) and twice daily (black squares) to separate groups), and 500 μM (administered once daily, circles), and a physiological saline vehicle (white squares) twice daily.

Figure 8

[0054] Figure 8 is a graph of corneal fluorescein staining (CFS) scored using the National Eye Institute Industry Workshop Scale of 0 - 15 as a function of time over several days for three groups of mice during treatment with an IL-17 inhibitor (SEQ ID NO: 1 (VHVTIPADLWDWINK)) formulated in phosphate buffered saline at a concentration of 250 μM and administered twice daily for 7 days (triangles) or 21 days (squares), and a vehicle (control, white squares). DETAILED DESCRIPTION OF THE INVENTION

[0046] Detailed Description I. Definitions

[0055] Here, various aspects will be described in more detail hereinafter. However, such aspects can be embodied in many different forms and should not be construed as limited to the embodiments described herein; rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope thereof to those skilled in the art.

[0047]

[0056] For convenience, certain terms used in the specification, examples, and claims are gathered here. Unless otherwise specifically defined, all scientific and technical terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0048]

[0057] When various values are provided, each intervening value between the upper and lower limits of that range and any other stated value or intervening value within the stated range is intended to be encompassed within the disclosure. For example, when a range of 1 μm to 8 μm is recited, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm, as well as ranges of values greater than or equal to 1 μm and less than or equal to 8 μm, are also intended to be explicitly disclosed.

[0049]

[0058] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymer" includes a single polymer and two or more of the same or different polymers, and reference to "an excipient" includes a single excipient and two or more of the same or different excipients, and so on.

[0050]

[0059] The term "about" as related to a given quantity means including a deviation of ±5 percent.

[0051]

[0060] "Amino acid" refers to all naturally occurring L-amino acids and includes D-amino acids. Amino acids are specified by either one-letter or three-letter designations.

[0052]

[0061] The term "amino acid sequence variant" refers to a molecule that has some differences in its amino acid sequence as compared to the sequences according to the present disclosure. For example, an amino acid sequence variant of a polypeptide according to the present disclosure of a specific sequence retains the ability to bind to a specific IL-17 cytokine or receptor, such as human IL-17A, or inhibits, for example, the binding of IL-17A to its receptor. A substitution variant is one in which at least one amino acid residue has been removed and a different amino acid has been inserted in its place at the same position in a polypeptide according to the present disclosure of a specific sequence. These substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. An insertion variant is one in which one or more amino acids have been inserted immediately adjacent to an amino acid at a specific position in a polypeptide, for example of a specific sequence. Immediately adjacent to an amino acid means linked to either the α-carboxy or α-amino functional group of the amino acid. A deletion variant is one in which one or more amino acids have been removed from a polypeptide of a specific sequence.

[0053]

[0062] "Identity" with respect to a native polypeptide and functional derivatives thereof is defined as the percentage of amino acid residues in the candidate sequence that are identical with the residues of the corresponding native polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent identity, and any conservative substitutions are not considered part of the sequence identity. It is to be understood that no N-terminal or C-terminal extensions or insertions are made that would reduce the identity. Methods and computer programs for alignment are well known. Percent identity can be determined by standard alignment algorithms such as the Basic Local Alignment Search Tool (BLAST) described by Altshul et al. ((1990) J. Mol. Biol., 215:403-410); the algorithm of Needleman et al. ((1970) J. Mol. Biol., 48:444-453); or the algorithm of Meyers et al. ((1988) Comput. Appl. Biosci., 4:11-17). A set of parameters may be the Blosum 62 scoring matrix with a 12 gap penalty, a 4 gap extension penalty, and a 5 frameshift gap penalty. Also, percent identity between two amino acid sequences or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) incorporated in the ALIGN program (version 2.0) using the PAM120 weight residue table, a 12 gap length penalty, and a 4 gap penalty.

[0054]

[0063] The term "IL-17" refers to an IL-17 cytokine such as IL-17A, IL-17B, IL-17C, IL-17D or IL-17E.

[0055]

[0064] "IL-17A" is intended to mean cytokines previously known as CTLA8 and includes wild-type IL-17A, polymorphic variants of IL-17A, and functional equivalents of IL-17A from various species (e.g., human, mouse, and monkey). Functional equivalents of IL-17A have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity with wild-type IL-17A (e.g., human IL-17A), and in certain embodiments, the functional fragment substantially retains the ability to induce IL-6 production by human skin fibroblasts.

[0056]

[0065] "IL-17 inhibitor" refers to a compound that binds to an IL-17 cytokine and / or an IL-17 receptor (e.g., IL-17RA, RB, RC, RD, or RE) to inhibit the interaction between the IL-17 cytokine and its receptor.

[0057]

[0066] "IL-17 cytokine inhibitor" refers to a compound that binds to an IL-17 cytokine and inhibits its interaction with its receptor. In embodiments, the IL-17 cytokine inhibitor specifically binds to a particular IL-17 cytokine and is referred to according to the cytokine to which it binds; e.g., an "IL-17A cytokine inhibitor" refers to a compound that specifically binds to the IL-17A cytokine and inhibits its binding or its interaction with the IL-17A receptor, thereby inhibiting the formation of the IL-17A / IL-17RA / IL17RC complex.

[0058]

[0067] A compound that "inhibits" one or more of the functional properties of IL-17 (such as biochemical activity, immunochemical activity, cellular activity, physiological activity, or other biological activity) is determined according to the methods described herein, which are known in the art, and achieves a statistically significant decrease relative to the activity observed in the absence of the compound (or in the presence of a control compound with irrelevant specificity) in a particular activity. A compound that is an IL-17 inhibitor or an IL-17 cytokine inhibitor provides, for example, a statistically significant decrease of at least 10%, at least 50%, 80%, or 90% of the measured parameter, and in certain embodiments, can inhibit more than 95%, 98%, or 99% of the functional activity of IL-17.

[0059]

[0068] As used herein, the term "pharmaceutically acceptable" refers to compounds, salts, compositions, dosage forms, etc. that are suitable for use in contact with human and / or other mammalian tissues, are without undue toxicity, irritation, allergic response, or other problems or complications, and are within the "scope of sound medical judgment" such that they have a reasonable risk-benefit ratio. In some aspects, "pharmaceutically acceptable" means approved by a federal or state government regulatory authority or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in mammals (e.g., animals), and more specifically in humans.

[0060]

[0069] As used herein, the terms "subject" and "patient" include any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0061]

[0070] The term "substantially identical" means that the relevant amino acid or nucleotide sequence is identical to or has non-substantial differences (e.g., through conservative amino acid substitutions) when compared to a specific reference sequence. Non-substantial differences include minor amino acid changes, for example, one or two substitutions within a 5-amino acid sequence in a specified region. In the case of antibodies, the secondary antibody has the same specificity and at least 50% of its affinity. Also, sequences that are substantially identical to the sequences disclosed herein (e.g., having at least about 85% sequence identity) are part of this application. In some embodiments, the sequence identity can be about 90% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0062]

[0071] As used herein, "therapeutically effective amount" refers to the amount of IL-17 inhibition that is effective upon single or multiple dose administration to a subject (such as a human patient) to slow, inhibit, or delay the progression of dry eye disease and / or to treat, restore, or cure dry eye disease.

[0063]

[0072] The term "treatment" or "treating" refers to a curative treatment or a disease-modifying treatment in a patient diagnosed with dry eye disease.

[0064]

[0073] The compositions of the present disclosure can contain, consist essentially of, or consist of the disclosed components.

[0065]

[0074] All percentages, parts, and ratios are based on the total weight of the composition, and all measurements are made at about 25°C unless otherwise specified.

[0066]

[0075] The right is reserved to claim, with a proviso out or exclude, any individual member of any such group, including any sub-range or combination of sub-ranges within the group, that can be claimed according to a range or in any similar way, so that, for any reason, what does not meet the full measure of this disclosure can be claimed. Further, the right is reserved to claim, with a proviso out or exclude, any individual substituent, analog, compound, ligand, structure, or group thereof, or any member of the claimed group, so that, for any reason, what does not meet the full measure of this disclosure can be claimed.

[0067]

[0076] Throughout this disclosure, various patents, patent applications, and publications are referenced. The entire disclosure of these patents, patent applications, and publications is incorporated herein by reference to more fully describe the prior art that is known to those of ordinary skill in the art as of the date of this disclosure. This disclosure will govern in the event of any conflict between the cited patents, patent applications, and publications and this disclosure.

[0068] II. Method

[0077] Methods for treating dry eye disease and for slowing, inhibiting, or delaying the progression of dry eye disease in a subject are provided. The methods include administering a composition comprising a therapeutically effective amount of a compound that inhibits the binding of the inflammatory interleukin-17 (IL-17) cytokine, referred to as an IL-17 inhibitor, to the IL-17 receptor. The tests conducted in support of the methods are described in connection with Examples 1-7.

[0069]

[0078] In the first test described in the Examples, the treatment of dry eye disease with an IL-17 cytokine inhibitor, lifitegrast ophthalmic solution (XIIDRA®), and cyclosporine ophthalmic emulsion (RESTASIS®) was compared. The IL-17 cytokine inhibitor was an anti-IL-17A antibody formulated at 1 wt% in phosphate buffered saline. Dry eye disease was induced in mice before treatment, and after induction of dry eye disease, the mice were separated into groups for treatment with (i) anti-IL-17A antibody once daily; (ii) 1 wt% anti-IL-17A antibody twice daily; (iii) 5 wt% lifitegrast twice daily; (iv) 0.05 wt% cyclosporine twice daily; or (v) saline twice daily as a control. The test compounds were topically applied to the eyes for 9 days (from day 4 to day 12), and disease severity was evaluated using corneal fluorescein staining (CFS) and scored using the National Eye Institute Industry Workshop Scale of 0 - 15 on days 4, 7, 10, and 12.

[0070]

[0079] Figure 1 is a graph of dry eye disease scores as a function of time over several days in subjects with dry eye disease treated by topical application to the eye of an anti-IL-17A antibody once daily (black circles) or twice daily (black squares), lifitegrast twice daily (XIIDRA®, 5 wt%) (inverted triangles), cyclosporine twice daily (RESTASIS®, 0.05 wt%) (triangles), or saline twice daily (diamonds). In the treatment of dry eye disease, topical treatment of the eye with an IL-17 cytokine inhibitor was more effective than lifitegrast or cyclosporine when evaluated by disease score. Subjects treated with cyclosporine and having a dry eye disease score of about 8 on the 4th day of the trial had a dry eye disease score that remained at about 8 over the treatment period with little reduction in disease severity in its presence. Subjects treated with lifitegrast and having a dry eye disease score of about 8 on the 4th day of the trial showed no improvement in the reduction of disease severity during the first 6 days of treatment and then showed an improved disease score on the 12th day. In subjects treated with an IL-17 cytokine inhibitor, improvement in the severity of dry eye disease was reported within 3 days of treatment and continuous improvement in the severity of dry eye disease over the remaining treatment period.

[0071]

[0080] The data in Figure 1 also show that the IL-17 cytokine inhibitor was effective in treating dry eye disease when administered once daily. No statistical difference was observed in the reduction of disease scores in the test cohort treated once or twice daily with the IL-17 cytokine inhibitor. This finding is also supported by the data shown in Figure 2A when the presence of protective immunoregulatory T cells (Tregs) was restored by once-daily or twice-daily treatment with an IL-17 cytokine inhibitor. Figure 2B shows that both once-daily and twice-daily treatment with an IL-17 cytokine inhibitor were effective in reducing the percentage of pathogenic Th17 immune cells in draining lymph nodes.

[0072]

[0081] Accordingly, provided is a method for treating dry eye disease when a subject having dry eye disease is treated with an IL-17 cytokine inhibitor. In one embodiment, the IL-17 cytokine inhibitor is administered to the eye once a day. In one embodiment, the IL-17 cytokine inhibitor is administered to the eye twice a day.

[0073]

[0082] Continuing to refer to the study of Example 1, eye tissues were collected from mice and tissue sections were prepared for microscopic analysis. Cross-sections of the conjunctiva were stained and images were examined to quantify goblet cell density. Goblet cells within the conjunctival epithelium secrete mucin onto the surface of the eye. FIG. 3A is an image of the conjunctiva from a healthy mouse without dry eye disease, and goblet cells can be visualized as darkly stained, bulging cells. FIGS. 3B-3E are images of the conjunctiva obtained from subjects treated twice a day with saline (FIG. 3B), cyclosporine ophthalmic emulsion (RESTASIS®; FIG. 3C), lifitegrast eye drops (XIIDRA®) (FIG. 3D), or an anti-IL-17A antibody (FIG. 3E) as a control in animals having dry eye disease. The number of goblet cells was counted, and FIG. 3F is a bar graph of the percentage of normal goblet cells for normal (healthy) mice and mice having dry eye disease treated with saline, cyclosporine, lifitegrast, or an IL-17A inhibitor. Subjects treated with the IL-17A inhibitor did not have a decrease in the number of goblet cells, and the IL-17A inhibitor prevented the decrease in goblet cells in the conjunctival tissue. Subjects having dry eye disease treated with cyclosporine or lifitegrast had approximately 60% of the normal number of goblet cells. The maintenance and / or restoration of goblet cell function and / or density is a disease-modifying therapy for dry eye disease in which the IL-17A inhibitor treats the disease itself rather than simply relieving the symptoms of dry eye disease, and demonstrates the restoration of tear film homeostasis.

[0074]

[0083] Dry eye disease is characterized by a decrease in tear volume, rapid disruption of the tear film, and / or an increase in the evaporative properties of the tear film layer. Tears are made up of three layers: an outer oily or lipid layer, an intermediate aqueous layer, and an inner mucin layer. The production of a healthy tear film involves the meibomian glands, lacrimal glands, and goblet cells. The outer lipid layer of the tear film is produced by the meibomian glands and functions to maintain the tear film on the surface of the eye by preventing it from evaporating too quickly. The lacrimal glands produce the intermediate aqueous layer, which contains a large volume and flow of tears. This layer is an aqueous mixture containing proteins, enzymes, antibodies, and growth factors that are cytoprotective, anti-inflammatory, and antimicrobial. The aqueous layer nourishes the cornea and conjunctiva that cover the entire front of the eye and the inner surface of the eyelids. The innermost mucin layer is produced by goblet cells and functions to bind to the water from the aqueous layer to ensure the maintenance of eye hydration. Dry eye disease is associated with a decrease in the function and / or density of conjunctival goblet cells, as is apparent from the data in FIGS. 3B and 3F. Thus, in one embodiment, a method is provided for restoring the function and / or density of goblet cells in an eye having signs or symptoms of dry eye disease. In another embodiment, a method is provided for preventing goblet cell depletion in the eyes of a person having dry eye disease. The method includes topically administering to the eye of a subject in need thereof a therapeutically effective amount of a compound that inhibits the binding of an IL-17 cytokine to an IL-17 receptor. In one embodiment, the compound inhibits the binding of IL-17A to the IL-17A receptor.

[0075]

[0084] In another embodiment, a method is provided for improving the balance of the functions of the lacrimal glands, meibomian glands, and conjunctival goblet cells to restore the homeostasis of the tear film in a subject. The method includes topically administering to the eye of a subject in need thereof a therapeutically effective amount of a compound that inhibits the binding of an IL-17 cytokine to an IL-17 receptor. In one embodiment, the compound inhibits the binding of IL-17A to the IL-17A receptor. In certain embodiments, the subject in need is a person having dry eye disease.

[0076]

[0085] In another embodiment, a method for increasing basal tear production in an elderly subject is provided. The method includes topically administering to the eye of a subject in need thereof a therapeutically effective amount of a compound that inhibits the binding of an IL-17 cytokine to an IL-17 receptor. In one embodiment, the compound inhibits the binding of IL-17A to the IL-17A receptor. In certain embodiments, the subject in need thereof is a person having dry eye disease. In one embodiment, the elderly subject has a hormonal change or is over 50 years old.

[0077]

[0086] In another test described in Example 2, a co-culture of human corneal epithelial cells and Th17 cells was prepared to recapitulate the in vivo interaction between Th17 cells and corneal tissue in dry eye disease. The therapeutic effect of an IL-17 inhibitor in the form of an antibody that inhibits the binding of IL-17A to its receptor to protect human corneal epithelial cells from Th17-induced damage was tested. Under co-culture, the density and morphology of human corneal epithelial cells in the presence and absence of an IL-17 inhibitor were observed under an inverted microscope under bright light equipped with a camera. Microscopic images of the cells are shown in FIGS. 4A-4C, where the image in FIG. 4A is an image of human corneal epithelial cells without Th17 cells or IL-17 treatment as a control. FIG. 4B is an image of a co-culture of human corneal epithelial cells and Th17 cells treated with a saline medium, and FIG. 4C is an image of a co-culture of human corneal epithelial cells and Th17 cells treated with an IL-17 inhibitor. The IL-17 inhibitor protected human corneal epithelial cells from Th17-mediated corneal damage, as is evident from the comparable cell density and morphology of human corneal epithelial cells under co-culture (FIG. 4C) and control cultures of human corneal epithelial cells (FIG. 4A).

[0078]

[0087] A similar test using co - culture of human corneal epithelial cells and Th17 cells was carried out to test the therapeutic effect of the IL - 17 inhibitor identified as SEQ ID NO: 1 (VHVTIPADLWDWINK) in the form of a peptide that inhibits the binding of IL - 17A to the IL - 17A receptor, and to evaluate whether the peptide protects human corneal epithelial cells from Th17 - induced damage. As described in Example 3, the IL - 17 inhibitory peptide was formulated using phosphate - buffered saline at an appropriate concentration to obtain compositions having the IL - 17 inhibitor at concentrations of 0.1 μM, 1 μM, 10 μM and 100 μM. After 24 - hour co - culture in the presence of each of the treatment doses of the IL - 17 inhibitor, the density and morphology of human corneal epithelial cells were observed under an inverted microscope under bright light equipped with a camera. Representative images are shown in FIGS. 5A - 5F. The image in FIG. 5A is an image of human corneal epithelial cells in culture without Th17 cells or IL - 17 treatment as a control. The image in FIG. 5B is an image of co - culture of human corneal epithelial cells and Th17 cells treated with a physiological saline medium. The images in FIGS. 5C - 5F are images of co - culture of human corneal epithelial cells and Th17 cells treated with IL - 17 inhibitors at concentrations of 0.1 μM, 1 μM, 10 μM and 100 μM, respectively. After 24 hours, significant death of human corneal epithelial cells was observed in cells treated solely with the medium (FIG. 5B). In contrast, co - cultures treated with IL - 17 inhibitors at all concentrations protected human corneal epithelial cells from Th17 - mediated corneal damage, as evidenced by cell density and morphology equivalent to those of the human corneal epithelial cell culture in the control (FIG. 5A) (FIGS. 5C - 5F). Also, the images of human corneal epithelial cells in FIGS. 5C - 5F indicate that the IL - 17 inhibitor was not toxic to human corneal epithelial cells.

[0079]

[0088] Example 4 describes a test demonstrating that an IL-17 inhibitor is effective in slowing, inhibiting, and / or delaying the progression of dry eye disease. Four days after induction of dry eye disease, the mice were treated with an IL-17 inhibitor. The IL-17 inhibitor was either a 1 wt% dose of an anti-IL-17A antibody in phosphate buffered saline or a peptide that inhibits the binding of IL-17A to the IL-17A receptor, identified as SEQ ID NO: 1 (VHVTIPADLWDWINK). The IL-17A peptide inhibitor was formulated in phosphate buffered saline at doses of 0.05 wt% and 0.1 wt%. Nine days after treatment with the IL-17 inhibitory antibody or peptide, ocular surface damage was evaluated using corneal fluorescein staining (CFS) and scored using the National Eye Institute Industry Workshop Scale of 0 - 15 at day 0 (normal baseline), day 4 (before instillation of topical treatment), day 7, day 10, and day 12.

[0080]

[0089] The results are shown in Figure 6A, where corneal fluorescein staining (CFS) scored using the National Eye Institute Industry Workshop Scale of 0 - 15 as a function of time at day 0 (normal baseline) is shown. The CFS scores were determined at day 4 (before instillation of topical treatment) and during treatment with the IL-17 inhibitor. The data show that administration of both the IL-17 inhibitory peptide (SEQ ID NO: 1, 0.05 wt% (circles) and 0.1 wt% (diamonds)) and the IL-17 inhibitory antibody (1 wt%, inverted triangles) effectively alleviated the progression of dry eye disease compared to the vehicle treatment cohort (open squares). In the absence of treatment with the IL-17 inhibitor, untreated subjects (open squares) had a CFS score close to 8 at day 4 of the study, and the CFS score remained at approximately 8 over the duration of the study, indicating that the progression of dry eye disease continued in the absence of treatment. In subjects treated with the IL-17 inhibitor, the CFS score was reduced by formulations containing an IL-17 inhibitor that reduced the severity of dry eye disease and inhibited its progression at the time of treatment with the IL-17 inhibitor.

[0081]

[0090] The lymph nodes draining the eye were collected at the end of the treatment, and the frequency of IL-17+CD4+Th17 cells was determined in the tissue. The results are shown in FIG. 6B. The percentage of Th17 cells in the draining lymph nodes of mice without dry eye disease and mice with dry eye disease treated with a saline vehicle (control) or an IL-17 inhibitor is shown. The IL-17 inhibitor suppressed pathogenic Th17 cells in the eye tissue.

[0082]

[0091] Thus, in one embodiment, a method is provided for slowing, inhibiting, or delaying the progression of dry eye disease in a subject. The method comprises directly administering to the eye of a subject in need thereof a composition comprising a therapeutically effective amount of a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor. In another embodiment, a method is provided for reversing the progression of dry eye disease in a subject, wherein a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor is directly administered to the eye of a subject in need thereof.

[0083]

[0092] Additional tests were conducted to further evaluate the IL-17A peptide inhibitor. Example 5 describes tests in which mice with dry eye disease were treated with an IL-17A peptide inhibitor (SEQ ID NO: 1 (VHVTIPADLWDWINK)) at several doses (50 μM, 250 μM, and 500 μM) once or twice daily. FIG. 7 shows the results for the treatment groups over the first 7 days of the treatment period. The data in FIG. 7 show that treatment of dry eye disease with twice-daily doses of 50 μM (inverted triangles) and once-daily doses of 250 μM (diamonds) of the IL-17A peptide inhibitor did not result in improvement or transient improvement compared to treatment with twice-daily saline (white squares). Subjects treated with twice-daily doses of 250 μM (black squares) and once-daily doses of 500 μM (circles) of the IL-17A inhibitor improved the disease, as evidenced by a significant decrease in the CFS score.

[0084]

[0093] In another experiment, as described in Example 5, dry eye disease was induced in mice. Mice with similar severities of dry eye disease were randomly divided into three treatment groups (n = 6) for treatment after induction of dry eye disease with a dose of 250 μM of the IL-17A peptide inhibitor (SEQ ID NO: 1) twice a day, where one group of mice was treated until day 7 of the study and then with vehicle, and another group of mice was treated with the IL-17 inhibitor for 21 days. The third group was a control group treated with saline vehicle for 21 days. All subjects in the cohort were evaluated by corneal fluorescein staining (CFS) on days 3, 7, 10, 14, 17, and 21, and the results scored using the National Eye Institute Industry Workshop Scale of 0 - 15 are shown in FIG. 8. Subjects in the vehicle (control) treatment group (squares) had a continuous progression and worsening of dry eye disease, as evidenced by the increase in CFS scores over the 21-day study. Subjects treated with the IL-17 inhibitor experienced a decrease in CFS scores (squares, triangles), indicating an improvement in the condition. A continuous improvement in disease severity and inhibition of disease progression were observed for animals treated over the 21-day study (squares), while subjects treated for 7 days and then left untreated (triangles) showed a recurrence of disease severity and continuous progression of the disease. Treatment of dry eye disease with the IL-17 inhibitor blocked and / or reduced the progression of dry eye disease.

[0085]

[0094] In one embodiment, a CFS score using the criteria of about 2 - 3 of the NEI / Industry Workshop correlates with mild dry eye disease, about 4 - 7 correlates with mild dry eye disease, about 8 - 10 correlates with moderate dry eye disease, and about 11 - 15 correlates with severe dry eye disease. The subjects in the trial had an initial CFS score of about 8 or had moderate dry eye disease. In another embodiment, a CFS score using the VAS scale indicates mild dry eye disease for scores of about 10 - 20, mild dry eye disease for scores of about 30 - 40, moderate dry eye disease for scores of about 50 - 70, and severe dry eye disease for scores of about 80 - 100. Regarding the trial in Example 5 when the NEI / Industry Workshop scale was used for CFS scoring, treatment with an IL - 17 inhibitor for 7 days improved dry eye disease, and the CFS score on day 7 was 4.5 - 5. In subjects treated with an IL - 17 inhibitor until day 21, the CFS score continued to decrease, and the CFS score was about 2.5 - 3 on days 18 - 21, which correlates with treatment when the disease is mild. However, as a result of suspending treatment on day 7, the disease progressed from mild (CFS score of 4.5 on day 7) to the upper range of mild and approached moderate (CFS score of 7 on day 14). Thus, the trial shows that treatment of dry eye disease with an IL - 17 inhibitor is effective in preventing, delaying, inhibiting, or reducing the progression of the disease. In one embodiment, the treatment is effective in preventing, delaying, inhibiting, or reducing the progression from mild dry eye disease to moderate dry eye disease. In one embodiment, the treatment is effective in preventing, delaying, inhibiting, or reducing the progression from mild dry eye disease to severe dry eye disease. In one embodiment, the treatment is effective in preventing, delaying, inhibiting, or reducing the progression from moderate dry eye disease to severe dry eye disease.

[0086]

[0095] The data in FIG. 8 also indicate that the treatment of dry eye disease with an IL-17 inhibitor is a disease-modifying therapy. Subjects without dry eye disease have an average baseline CFS score of about 2. In the treatment of subjects with dry eye disease with an IL-17 inhibitor, daily administration was carried out, the condition recovered, and disease recovery was almost achieved. Subjects with dry eye disease had an initial CFS score of about 8, and within 9 days of treatment with an IL-17 inhibitor, the CFS score was approaching that of subjects without dry eye disease. This result indicates that the treatment method is a disease-modifying therapy for achieving almost or complete disease recovery. In one embodiment, subjects with dry eye disease are treated at least daily for a period of 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months or more to substantially recover from the disease.

[0087]

[0096] Methods for restoring the homeostasis of the tear film on the surface of the eye are also contemplated. Homeostasis refers to a relatively stable equilibrium state among interdependent elements, particularly as maintained by physiological processes. With respect to the tear film, it is understood that the tear film composition is dynamic, in a steady flow state, and responds to environmental conditions to maintain the homeostasis of the ocular surface. Traditionally, the tear film has been described as being composed of three separate and distinct layers, such as the mucin layer, the aqueous layer, and the lipid layer. However, it is now understood that mixing occurs between the mucin layer and the aqueous layer, creating a decreasing mucin concentration gradient into the aqueous layer. This aqueous-mucin layer forms a hydrated gel by complex biology and is then covered by the lipid layer, which has its own highly ordered structure. Treatment with an IL-17 inhibitor restores the homeostasis of the tear film, such that the tear film in those with dry eye disease is more similar to that of those without dry eye disease. By way of example, a measure of the homeostasis of the tear film via the tear film break-up time is described in Example 6. The homeostasis of the tear film can also be inferred from CFS scoring or the Schirmer test. The tear film break-up time and ocular stating can be used to evaluate the balance of the functions of the lacrimal gland, Meibomian glands, and conjunctival goblet cells, and to restore the homeostasis of the tear film in a subject. Methods for restoring the homeostasis of the immunomodulatory function of regulatory T cells in the eye are also contemplated. Dry eye disease is at least in part an immunological response resulting from an imbalance in protective immunomodulation against Th17-mediated inflammatory pathways on the ocular surface. Chronic dry eye disease involves a cycle of inflammatory responses perpetuated by the resistance of pathogenic IL-17 cytokine-secreting Th17 cells to suppression by regulatory T cells (Tregs). Unchecked Th17 activity perpetuates dry eye disease, while blockade of IL-17 reduces inflammation on the ocular surface, reduces the transport of immune cells from the ocular surface to lymphoid tissue, and restores the immunomodulatory function of Tregs. Administration of an IL-17 inhibitor to the ocular region, and particularly to the ocular surface, reduces inflammation in the eye (ye) by blocking the pathogenic inflammatory cytokine IL-17 of dry eye disease.Blocking IL-17 causes downregulation of inflammatory cytokines including TNF-α, IL-1, IL-6, IL-8, IL-23 and MMP-9. Also, blocking IL-17 restores the immunomodulatory function of regulatory T cells (Tregs), disrupting the dry eye disease cycle. Thereafter, other non-IL-17 related contributors to inflammation are suppressed by Tregs, reducing the transport of immune cells from the ocular surface to lymphoid tissue.

[0088] Composition components

[0097] The methods described herein include administering to the eye a composition comprising a therapeutically effective amount of a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to an IL-17 receptor. Exemplary compounds and other composition components are described herein.

[0089]

[0098] IL-17 is a potent pro-inflammatory cytokine produced by a new lineage of CD4 T cells (Th17). IL-17 signals through a heteromeric receptor complex composed of IL-17RA and IL-17RC. IL-17 has pleiotropic effects on several immune and non-immune cells, leading to the association between T cell activation and the inflammatory response. Furthermore, IL-17 cooperates additively or synergistically with other pro-inflammatory cytokines such as TNFα, IL1β or IL6, causing amplification of the inflammatory process. IL-17, also known as IL-17A, is part of a larger family that includes six cytokines designated IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. All members of this family share a common protein structure. Among these family members, IL-17A and IL-­17F are most frequently expressed within immune cells. In the methods described herein, one or more of these family members are targeted by an antagonist to inhibit or modify their activity.

[0090]

[0099] In one embodiment, the IL-17 inhibitor is a synthetic organic or inorganic compound. Examples of macrocyclic compounds that inhibit the interaction of IL-17 cytokine with its receptor include, for example, the macrocycles described in Wang et al., MedChemComm, 9(1):22-26(2018), and spirocycline indane compounds, such as those described in International Publication No. WO 2020 / 0011731, which is incorporated herein by reference, or spirocyclic oxindoles, such as those described in International Publication No. WO 2019 / 229079, which is incorporated herein by reference.

[0091]

[0100] Embodiments in which the compound that inhibits the binding of IL-17 cytokine to the IL-17 receptor is a protein or peptide include administering a purified and / or isolated protein or peptide. As used herein, an "isolated" or "purified" polypeptide or protein or peptide is substantially free of other cellular material, or of the medium in the case of being produced by recombinant techniques, or of chemical precursors or other chemicals in the case of being chemically synthesized. A purified compound is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest. Purity is measured by any suitable standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0092]

[0101] In one embodiment, the IL-17 inhibitor is a biological compound such as an antibody or a peptide. Antibodies having specific binding to the IL-17 cytokine or the IL-17 receptor are described, for example, in U.S. Patent No. 9,872,901, which is incorporated herein by reference. The antibody can be monoclonal or polyclonal. The contemplated antibody binds to one or more sequences in the IL-17 or IL-17 receptor polypeptide. In some embodiments, the antibody includes a single-chain antibody, a humanized antibody, a recombinant antibody, or a chimeric antibody. In other embodiments, the IL-17 inhibitor is an antibody fragment including, but not limited to, Fab, F(ab’)2, Fab’Fv, single-chain Fv, etc. Antibodies against IL-17 can be a reformulation or humanized derivative of a human IL-17 affinity-purified polyclonal antibody, a human IL-17 allophycocyanin monoclonal antibody, a human IL-17 biotinylated affinity-purified polyclonal antibody, a human IL-17 monoclonal antibody, a human IL-17 monoclonal antibody, a human IL-17 phycoerythrin monoclonal antibody, a mouse IL-17 affinity-purified polyclonal antibody, a mouse IL-17 biotinylated affinity-purified polyclonal antibody, a mouse IL-17 monoclonal antibody, or a mouse IL-17 monoclonal antibody (each commercially available from, for example, R&D Systems), or can bind to its epitope. Preferably, a neutralizing or function-blocking antibody against IL-17 can be a monoclonal anti-human IL-17 antibody, an anti-human IL-17 antibody, a polyclonal produced in goats, or a reformulation or humanized derivative of a recombinant human IL-17R / Fc chimera, or can bind to its epitope.Antibodies against the interleukin-17 receptor (IL-17R) can be reformulated or humanized derivatives of a human IL-17R affinity-purified polyclonal antibody, a human IL-17R allophycocyanin monoclonal antibody, a human IL-17R biotinylated affinity-purified polyclonal antibody, a human IL-17R fluorescein monoclonal antibody, a human IL-17R monoclonal antibody, a human IL-17R monoclonal antibody, a human IL-17R phycoerythrin monoclonal antibody, a mouse IL-17R affinity-purified polyclonal antibody, a mouse IL-17R biotinylated affinity-purified polyclonal antibody, or a mouse IL-17R monoclonal antibody (each commercially available from R&D Systems), or can bind to an epitope thereof.

[0093]

[0102] In certain embodiments, the IL-17 inhibitor is an antibody selected from secukinumab, ixekizumab, brodalumab, ABT-122, KHK4827, perakizumab, RG7624, ANB004, or COVA322. Secukinumab is a fully human monoclonal antibody specific for human IL-17A derived from the human IgG1 kappa isotype, and ixekizumab is a humanized IgG4 antibody. Both secukinumab and ixekizumab are specific for IL-17A homodimers and IL-17A. ABT-122 is a bispecific variable domain immunoglobulin that targets both TNF and IL-17A. Brodalumab is a human mAb that neutralizes IL-17RA with high affinity and can block the biological activity of IL-17, IL-17F, the heterodimer composed of 17A / 17F, or 17E. In another embodiment, the antibody is described in U.S. Patent No. 9,765,140, which is incorporated herein by reference.

[0094]

[0103] In one embodiment, the IL-17 inhibitor is a peptide capable of inhibiting the binding of IL-17A to IL-17RA. In one embodiment, the peptide binds to IL-17A with high affinity and inhibits its interaction with the ILRA receptor, thereby inhibiting the signaling of IL-17A. Exemplary peptides are described in International Publication No. WO 2020 / 021103, which is incorporated herein by reference. In one embodiment, the peptide comprises the amino acid sequence of Formula I, provided that it is not IHVTIPADLWDWINK (SEQ ID NO: 167): X1-X2-X3-X4X5-X6-X7-X8-X9-X 10 -X1I-X 12 -X 13 -X 14 -X 15 Formula (I), (wherein, X1 is I, V or L; X2 is H, M, R, K or E; X3 is V, F or I; X4 is T, Q, S, Y or N; X5 is I, F or V; X6 is P or G; X7 is A, Q, or L; X8 is D, E, or Q; X9 is L, W, F, V or I; X 10 is W, Y or F; X 11 is D, E or N,; X 12 is W or F; X 13 is I, V, F or L; X 14 is N, R, Q or E; X 15 is K, R, H or E)

[0095]

[0104] In another embodiment, Formula I is (a) provided that it is not IHVTIPADLWDWINK (SEQ ID NO: 167), X1 is I or V, X2 is H, M or R, X3 is V or F; X4 is T or Q; X5 is I, F or V; X6 is P or G; X7 is A or Q; X8 is D or E; X9 is L; X 10 is W or Y; X is D or E; X 12 is W; X 13 is I or V; X 14 is N, R or E; X 15 is K, R or E; or (b) on condition that it is not IHVTIPADLWDWINK (SEQ ID NO: 167), HVTIPADLWDWIN (SEQ ID NO: 168), IHVTIPADLWDWI (SEQ ID NO: 169) or IHVTIPADLWDW (SEQ ID NO: 170), X1 is I or V; X2 is H or M, X3 is V; X4 is T; X5 is I; X6 is P; X7 is A; X8 is D; X9 is L, W, F, V or I; X 10 is W or Y; X 11 is D or E; X 12 is W; X 13 is I or V; X 14 is N, R or E; X 15 is K, R or E; is selected from.

[0096]

[0105] In certain embodiments, the peptide has its C-terminus and / or N-terminus attached to a protecting cap group. Exemplary protecting groups that can be attached to the C-terminus include amides, aldehydes, esters, p-nitroanilides, and 7-amino-4-methylcoumarin. Exemplary protecting groups that can be attached to the N-terminus are selected from the group consisting of acetyl, formyl, pyroglutamyl, fatty acids, ureas, carbamates, sulfonamides, and alkylamines.

[0097]

[0106] In another embodiment, one or more amino acid residues can be added to the N-terminus or C-terminus of the peptide. In one embodiment, any of the peptides described herein can have amino acid substitutions that are conservative substitutions, also referred to in the art as conservative mutations or conservative replacements. An amino acid substitution that changes a given amino acid to a different amino acid provides a mutant peptide having biochemical properties similar to those of a peptide that does not have a conservative substitution.

[0098]

[0107] In certain embodiments, the peptide consists of an adjacent sequence of 8 to 50, 8 to 40, 8 to 35, 8 to 30, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, or 12 to 18 amino acid residues. In certain embodiments, the peptide has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, or 99% sequence identity with the peptide of Formula I. In another embodiment, the peptide has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, or 99% sequence identity with the peptides of SEQ ID NOs: 1-166. In another embodiment, the peptide has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, or 99% sequence identity with the peptides of SEQ ID NOs: 1-214.

[0099]

[0108] The IL-17 inhibitor can also be a bioconjugate consisting of a dimer of two such peptides or an IL-17 inhibitory peptide attached to a biomolecule.

[0100]

[0109] In one embodiment, in the peptide in the form of a dimer formed by two peptides, each peptide is a peptide of Formula I. In one embodiment, the two peptides in the dimer are linked by a polyethylene spacer.

[0101]

[0110] In another embodiment, in the peptide in the form of a bioconjugate comprising a peptide of Formula I and a biomolecule, the biomolecule is attached to the N-terminus and / or C-terminus of the peptide. The bioconjugate may optionally include a linker between the peptide and the biomolecule. Exemplary biomolecules include macromolecules such as carbohydrates, lipids and proteins or small natural products. Other exemplary biomolecules are ascorbic acid, capric acid, caproic acid, N-acetyl-glucosamine (NAG), N-acetylmuramic acid (NAM), NAG-NAM, hyaluronic acid, alginic acid, chitin, (GalNAc)2, Gal-alpha1,3-GalNAc and trigalacturonic acid.

[0102]

[0111] An IL-17 inhibitor can be formulated into a composition for administration to the eye. In embodiments, the composition comprises physiological saline or another ophthalmically acceptable buffer. In other embodiments, the composition comprises one or more ophthalmically acceptable excipients, such as alcohols (ethanol, propanol, and nonanol), fatty alcohols (lauryl alcohol), fatty acids (valeric acid, caproic acid, and capric acid), fatty acid esters (isopropyl myristate and isopropyl n-hexanoate), alkyl esters (ethyl acetate and butyl acetate), polyols (propylene glycol, propanedione, and hexanetriol), sulfoxides (dimethyl sulfoxide and decyl methyl sulfoxide), amides (urea, dimethylacetamide, and pyrrolidone derivatives), surfactants (sodium lauryl sulfate, cetyltrimethylammonium bromide, polaxamer, span, tween, bile salts, and lecithin), terpenes (d-limonene, α-terpineol, 1,8-cineole, and menthone), or alkanones (N-heptane and N-nonane).

[0103]

[0112] Optionally, the composition further contains a compound selected from the group consisting of physiologically acceptable salts, poloxamer analogs having carbopol, carbopol / hydroxypropylmethylcellulose (HPMC), carbopol-methylcellulose, carboxymethylcellulose (CMC), hyaluronic acid, cyclodextrin, and petroleum. Bioadhesive compositions, such as those containing polymers, are also contemplated. Exemplary mucoadhesive polyanionic natural or semi-synthetic polymers include, but are not limited to, polygalacturonic acid, hyaluronic acid, carboxymethyl amylose, carboxymethyl chitin, chondroitin sulfate, heparin sulfate, and mesoglycan. Suitable hydrogels include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids, such as polypeptides, particularly poly(lysine), polyesters, such as polyhydroxybutyric acid and poly-ε-caprolactone, polyanhydrides; polyphosphazenes, poly(vinyl alcohol), poly(alkylene oxides), particularly poly(ethylene oxide), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly(vinyl pyrrolidone), and graft copolymers including the above copolymers. In another embodiment, synthetic polymers and naturally occurring polymers, such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels are contemplated.

[0104]

[0113] The composition is administered to the eye. In certain embodiments, the composition is administered directly by topical application to the ocular surface. In another embodiment, the composition is administered directly by topical application to the conjunctival sac. In another embodiment, the composition is administered by dropping the composition into or onto the eye. In certain embodiments, the composition is dropped as one or more droplets into the conjunctival sac or onto the ocular surface.

[0105]

[0114] The dosage of the IL-17 inhibitor varies depending on the compound, its potency, and other factors. In certain embodiments, the composition provides a therapeutically effective amount of the IL-17 inhibitor. By way of example, when the compound is an IL-17A peptide inhibitor, the therapeutically effective amount is typically about 0.01 - 1,000 μM or about 0.05 - 750 μM or about 0.05 - 500 μM, or at least about 500 μM or 1,000 μM as the total daily dose.

[0106] Dry eye questionnaire

[0115] In certain embodiments, the method includes identifying a subject having dry eye disease and / or quantifying the severity of dry eye disease. In these embodiments, a questionnaire can be used to identify a subject having dry eye disease or to quantify the severity of dry eye disease. In another embodiment, the progression of dry eye disease, and more particularly, the progression of mild or moderate dry eye disease to moderate or severe dry eye disease, is evaluated using a questionnaire to determine if treatment is delayed, slowed, or inhibited.

[0107]

[0116] An exemplary questionnaire is the Visual Analog Scale (VAS) symptom index. The VAS questionnaire asks questions regarding eye discomfort, and the subject is asked to subjectively rank the symptoms of each eye by placing a vertical mark on a horizontal line to indicate the level of discomfort (where 0% corresponds to no discomfort and 100% corresponds to maximum discomfort). The eye symptoms queried are burning / stinging pain, itching, foreign body sensation, eye discomfort, dry eye, photophobia, and pain.

[0108]

[0117] Another exemplary questionnaire is the Ocular Surface Disease Index (OSDI; Allergan Inc., Irvine, CA; Walt J.G. et al., Drug Inf J., 1997; 31:1436), which is a means frequently used to evaluate dry eye disease (Amparo, F., et al., Ophthalmology, 2015:122(7):1498-1503). The questionnaire consists of 12 questions and evaluates the frequency of symptoms over the preceding week. The score ranges from 0 to 100, and based on the score, the patient's symptoms can be classified as normal (0-12), mild dry eye (13-22), moderate dry eye (23-32), or severe dry eye (33-100) (Schiffman R.M. et al., Arch Ophthalmol., 2000; 118:615-21; Miller K.L., Arch Ophthalmol., 2010; 128:94-101).

[0109]

[0118] The Dry Eye Symptom Assessment Questionnaire (SANDE) is a questionnaire based on a visual analog scale that quantifies both the severity and frequency of dry eye symptoms. SANDE consists of two questions, and for each question, a 100-mm horizontal linear visual analog scale is used. The measurement of symptom frequency ranges from "rarely" to "always," and symptom severity ranges from "very mild" to "very severe" (Gulati A. et al., Am J Ophthalmol., 2006;142:125-31; Schaumberg D.A. et al., Ocul Surf., 2007;5:50-7). Both tests are reliable and valid measures of dry eye symptoms (Schiffman R.M. et al., Arch Ophthalmol., 2000;118:615-21; Gulati A. et al., Am J Ophthalmol., 2006;142:125-31). The first question in the SANDE questionnaire explores the frequency of symptoms by asking the subject to place an "x" on a line ranging from "rarely" to "always," indicating on average how often the individual's eyes feel dry and / or irritated. The second question assesses the severity of symptoms by asking the subject to place an "x" on a line ranging from "very mild" to "very severe," indicating on average how severely the individual feels the symptoms of dryness and / or irritation.

[0110]

[0119] In the Dry Eye Questionnaire (DEQ), dry eye disease is diagnosed and its severity level is quantified. In this questionnaire, several symptoms are measured using four variables: degree of irritation, frequency, intensity in the morning, and intensity in the late afternoon (Chalmers R.L. et al., Contact Lens and Anterior Eye, 2010;33(2):55-60). The DEQ enables the discrimination of ophthalmic states: dry eye patients versus non-dry eye patients, Sjogren's syndrome and dry keratoconjunctivitis patients versus non-Sjogren's syndrome and dry keratoconjunctivitis patients, and controls versus Sjogren's syndrome and dry keratoconjunctivitis patients and non-Sjogren's syndrome and dry keratoconjunctivitis patients (Begley C.G. et al., Cornea, 2002;21(7):664-67.).

[0111]

[0120] The SPEED questionnaire was designed by Korb and Blackie (Blackie, C. et al., Ocular Surgery News, Europe Edition 2012) to rapidly track the progression of dry eye symptoms over time. The SPEED questionnaire gives a score from 0 to 28 as a result of evaluating the frequency and severity of symptoms. The symptoms to be evaluated include a feeling of dryness, gritty or stinging sensations; a burning or stinging sensation; burning pain or tearing; and eye fatigue. In the questionnaire, the frequency of each symptom is measured by asking whether the symptom occurs never (0), sometimes (1), often (2), or always (3). The severity is evaluated by asking whether these symptoms were not a problem (0), tolerable (1), uncomfortable (2), troublesome (3), or intolerable (4), where tolerable is defined as "not perfect, but not uncomfortable"; uncomfortable is "annoying, but does not interfere with my daily life", troublesome is "annoying and interferes with my daily life", and intolerable is "unable to perform my daily work".

[0112]

[0121] The symptoms of dry eye disease can be measured by various methods including staining (e.g., corneal fluorescein staining and lissamine green conjunctival staining), biomarker analysis, tear film break-up time, Schirmer test (with or without anesthesia), or conjunctival hyperemia. Staining criteria for the evaluation of dry eye disease include, for example, corneal staining with fluorescein or lissamine, and staining grading using a system such as the National Eye Institute (NEI) grading system or another scale. An exemplary NEI scale is a fluorescein staining scale using a standardized grading system from 0 to 3 for each of 5 regions on each cornea. In an exemplary NEI system of the standard, a 0-15 point scale is used for fluorescein staining; in another NEI system, a 0-18 point scale and lissamine green conjunctival staining are used. The methods described herein may additionally include measuring, monitoring, or determining dry eye disease and the symptoms of dry eye disease using one or more of these metrics. The methods described herein may additionally include identifying a subject having dry eye disease using one or more of these metrics.

[0113]

[0122] In one embodiment, the subject having dry eye disease is a subject who does not have dry eye disease caused by Sjogren's syndrome, meibomian gland dysfunction, uveitis, intraocular pathology, or inflammation of the internal tissues of the eye. In other embodiments, neither the IL-17 inhibitor nor the IL-17 cytokine inhibitor is a peptide having the sequences identified herein as SEQ ID NOs: 167-214.

Example

[0114] III. Example

[0123] The following examples are illustrative in nature and are in no way intended to be limiting.

[0115] Example 1 Treatment of Dry Eye Disease

[0124] A study was conducted to compare the treatment of dry eye disease with an IL-17 cytokine inhibitor, lifitegrast ophthalmic solution (XIIDRA®), and cyclosporine ophthalmic emulsion (RESTASIS®). The IL-17 cytokine inhibitor was an anti-IL-17A antibody (R&D Systems, Inc. clone 50105) formulated at 1 wt% in phosphate buffered saline. Female C57BL / 6 mice, 6 - 8 weeks old, were obtained (Charles River Laboratories). Dry eye disease was induced by placing the mice in a controlled environmental chamber with <20% relative humidity, a 15 L / min airflow, and a constant temperature of 21 - 23 °C for 14 days. Corneal epithelial disease was evaluated using corneal fluorescein staining (CFS) and scored on days 0 (normal baseline), 4 (before topical treatment instillation), 7, 10, and 14 using the National Eye Institute Industry Workshop Scale of 0 - 15.

[0116]

[0125] After induction of dry eye disease, the mice were randomly assigned to cohorts for treatment with (i) anti-IL-17A antibody once daily; (ii) anti-IL-17A antibody twice daily; (iii) lifitegrast twice daily; (iv) cyclosporine twice daily; or (v) twice-daily saline as a control. The test compounds were topically applied to the eyes for 12 days, and the disease severity was evaluated daily using the National Eye Institute Industry Workshop Scale. The results are shown in Figure 1.

[0117]

[0126] The draining submandibular and cervical lymph nodes were collected from the mice and a single cell suspension was prepared. The suspension was analyzed for CD4+CD25+Foxp3+ Tregs and total CD4+ T cells as described by Chauhan, S. et al., J. Immunology, 182:1247 (2009). The results are shown in Figures 2A - 2B.

[0118]

[0127] In addition, eye tissues were collected from mice, tissue sections were prepared, and they were prepared for microscopic analysis. Cross-sectional images of conjunctival tissues of healthy mice without dry eye disease (Figure 3A) and physiological saline as a control (Figure 3B), cyclosporine ophthalmic emulsion (RESTASIS®; Figure 3C), lifitegrast eye drops (XIIDRA®) (Figure 3D), or anti-IL-17A antibody (Figure 3E) treated twice a day in mice with dry eye disease are shown in Figures 3A to 3E. Figure 3F is a bar graph of the percentage of goblet cells in normal (healthy) mice and mice with dry eye disease treated with physiological saline, cyclosporine, lifitegrast, or an IL-17A inhibitor.

[0119] Example 2 Treatment of Dry Eye Disease

[0128] Co-cultures of human corneal epithelial cells and Th17 cells were prepared to reproduce the in vivo interaction between Th17 cells and corneal tissue in dry eye disease. The therapeutic effect of an IL-17 inhibitor on protecting human corneal epithelial cells from Th17-induced injury was tested. Immortalized human corneal epithelial cells were obtained. To obtain human Th17 cells, first naive CD4+ T cells were purified from primary human peripheral blood mononuclear cells (StemCell) by negative selection using MACS separation, and then an in vitro polarization into Th17 cells was performed in X-VIVO 15 medium (Lonza) using a human Th17 cell differentiation kit (RnD Systems). After confirming the successful differentiation of human Th17 cells by flow cytometry, co-cultures were performed. At approximately 80% confluence of the human corneal epithelial cell culture, 1×10 5 individual Th17 cells were added in the presence and absence of an IL-17 inhibitor. After 24-hour co-culture in the presence of the IL-17 inhibitor, the density and morphology of human corneal epithelial cells were observed under an inverted microscope with bright light equipped with a camera. Representative images are shown in Figures 4A to 4C.

[0120] Example 3 Treatment of Dry Eye Disease

[0129] Co - cultures of human corneal epithelial cells and Th17 cells were prepared to reproduce the in - vivo interaction between Th17 cells and corneal tissue in dry - eye disease. The therapeutic effect of an IL - 17 inhibitor for protecting human corneal epithelial cells from Th17 - induced injury was tested. Immortalized human corneal epithelial cells were obtained. To obtain human Th17 cells, first naive CD4+ T cells were purified from primary human peripheral blood mononuclear cells (StemCell) by negative selection using MACS separation, and then an in - vitro polarization into Th17 cells was performed in X - VIVO 15 medium (Lonza) using a human Th17 cell differentiation kit (RnD Systems). After confirming the successful differentiation of human Th17 cells by flow cytometry, co - cultures were performed. At approximately 80% confluence of the human corneal epithelial cell culture, 1×10 5 cells of Th17 were added in the presence and absence of an IL - 17 inhibitor. The IL - 17 inhibitor was a 15 - amino - acid peptide that inhibits the binding of IL - 17A to the IL - 17A receptor, identified as SEQ ID NO: 1 (VHVTIPADLWDWINK). The IL - 17 inhibitory peptide was formulated using phosphate - buffered saline at an appropriate concentration to obtain compositions having the IL - 17 inhibitor at concentrations of 0.1 μM, 1 μM, 10 μM, and 100 μM.

[0121]

[0130] After 24 - hour co - culture in the presence of the IL - 17 inhibitor at each of the treatment doses, the density and morphology of human corneal epithelial cells were observed under an inverted microscope with bright light equipped with a camera. Representative images are shown in FIGS. 5A - 5F.

[0122] Example 4 Slow progression of dry - eye disease

[0131] Six- to eight-week-old female C57BL / 6 mice were used in this study, and dry eye disease was induced as described in Example 1. Four days after induction of dry eye disease, the mice were randomized into four treatment cohorts (n = 10) for treatment with saline or an IL-17 inhibitor. Two IL-17 inhibitors were tested: an anti-IL-17A antibody (R&D Systems, Inc. clone 50105) formulated at 1 wt% in phosphate-buffered saline and a 15-amino acid peptide identified as SEQ ID NO: 1 (VHVTIPADLWDWINK) that inhibits the binding of IL-17A to the IL-17A receptor, formulated at doses of 0.05 wt% or 0.1 wt% in phosphate-buffered saline. The treatment compositions, and the saline control, were administered topically by twice-daily instillation of 3 μL into the eyes from day 4 to day 12. Corneal epithelial disease was evaluated using corneal fluorescein staining (CFS) and scored using the National Eye Institute Industry Workshop Scale of 0 - 15 at day 0 (normal baseline), day 4 (before topical treatment instillation), day 7, day 10, and day 12. The results are shown in Figure 6A.

[0123]

[0132] The draining lymph nodes of the eyes were harvested at the end of treatment, and the frequency of IL-17+CD4+ Th17 cells was analyzed by flow cytometry. The percentage of Th17 cells in the draining lymph nodes for the treatment cohorts is shown in Figure 6B.

[0124] Example 5 Slow progression of dry eye disease

[0133] Female C57BL / 6 mice at 6 - 8 weeks of age were used in this study, and dry eye disease was induced as described in Example 1. Four days after the induction of dry eye disease, the mice were randomized into treatment cohorts (n = 6) for treatment with saline or an IL - 17 inhibitor. The IL - 17 inhibitor was a 15 - amino acid peptide identified as SEQ ID NO: 1 (VHVTIPADLWDWINK) that inhibits the binding of IL - 17A to the IL - 17A receptor, formulated at concentrations of 50 μM, 250 μM, and 500 μM in phosphate - buffered saline. Formulations with 250 μM of the IL - 17A inhibitor were administered once daily to one treatment cohort and twice daily to a second treatment cohort. Formulations with 50 μM of the IL - 17A inhibitor were administered twice daily to one treatment cohort, and formulations with 500 μM were administered once daily to one cohort. The treatment compositions, and the saline control, were administered topically by instilling 3 μL into both eyes of the subject. Table 5 - 1 shows an overview of the treatment cohorts and the study design.

[0125]

Table 1

[0126]

[0134] Corneal epithelial disease was evaluated using corneal fluorescein staining (CFS) and scored using the National Eye Institute Industry Workshop Scale of 0 - 15 at day 0 (normal baseline), day 3 (before topical treatment instillation), day 7, and day 10. The cohort treated with the formulation with 250 μM of the IL - 17A inhibitor was treated with CFS for 21 days, and scoring was performed at day 3, day 7, day 10, day 14, day 17, and day 21. The results are shown in Figures 7 and 8.

[0127] Example 6 Restoration of Tear Film Homeostasis

[0135] Human subjects with moderate to severe dry eye disease are enrolled in a study for treatment with an IL - 17 inhibitor. The IL - 17 inhibitor is administered directly to the subject's eyes daily for one month. Before the first administration and then weekly thereafter, an evaluation of the ocular surface is performed.

[0128]

[0136] Use a case report form using the National Eye Institute Industry Workshop Scales to evaluate and record ocular surface staining using fluorescein and lissamine green in a schematic of 5 corneal regions and 6 conjunctival regions per eye. Include a tabulated, explanatory grading scale (grades 0 - 15 or 0 - 18) on the case report form. 1. Evaluate corneal staining using a 1.0 mg fluorescein sodium strip. 2. After wetting the end of the strip with a single drop of buffered saline, discard the excess by shaking with a sharp flick. 3. Next, gently apply the flat end of the tip to the inferior tarsal conjunctiva with the intention of instilling a very small volume of dye without inducing reflex tearing while pulling down the lower eyelid. 4. Instruct the patient to blink naturally several times without forced closure of the eyelids to distribute the fluorescein. 5. After allowing the fluorescein to remain in the eye for at least 1 minute, grade the 5 corneal regions by maximizing the fluorescent field using a yellow (Wratten #12) barrier filter in combination with a cobalt (blue) filter. Lift the upper eyelid slightly to grade the entire corneal surface. Position the yellow barrier filter in the path of the reflected light (not in the path of the incident light) to enhance the contrast.

[0129]

[0137] Use a slit lamp biomicroscope following the steps below to evaluate the tear film break-up time (TFBUT): 1. Set the slit lamp to a magnification of approximately 10x. 2. Place sufficient fluorescein (preferably using a DET strip), and the subject is asked to stare straight ahead without blinking until otherwise noted. The test needs to be performed indoors without directing air directly at the patient's face. 3. Use a stopwatch to record the time between the first appearance of growing micelles indicating the final complete blink and tear film break-up. If the patient blinks prematurely before the break-up of mires occurs, the examiner needs to continue trying to obtain a reading. 4. Once TFBUT is observed, instruct the patient to blink freely. Next, this test needs to be repeated twice in the same eye. 5. If the difference between the first reading and the second reading is different by more than 2 seconds, a third measurement needs to be performed and recorded. 6. Next, this procedure is performed on the other eye. 7. It is recommended that TFBUT be performed indoors at a temperature of about 18°C and a humidity of about 50%.

[0130]

[0138] Ocular surface staining evaluation is completed with lissamine green conjunctival staining. 1. The lissamine green eye drop strip needs to be moistened with buffered saline and applied to the inferior tarsal conjunctiva. Care should be taken to instill sufficient dye. 2. After allowing lissamine green to remain in the eye for 1 minute, grade the six nasal and temporal conjunctival areas. 3. To grade the temporal zone, the subject needs to observe the nasal side; to grade the nasal zone, the subject needs to be instructed to observe the temporal side. 4. Next, this procedure needs to be performed on the other eye.

[0131]

[0139] The test shows that TFBUT, which indicates the restoration of tear film homeostasis, is improved by chronic treatment with an IL-17 inhibitor.

[0132] Example 7 Method for treating dry eye disease

[0140] Human subjects having moderate to severe dry eye disease are enrolled in a study for treatment with an IL-17 inhibitor. The IL-17 inhibitor is a spirocycline indane compound and is administered directly to the eyes of the subjects daily for one month. Before the first dose and then weekly thereafter, ocular surface and tear film evaluations are performed as described in Example 6.

[0133]

[0141] While some exemplary aspects and embodiments have been discussed above, those skilled in the art will appreciate certain modifications, permutations, additions and partial combinations thereof. Accordingly, the following appended claims and the claims hereafter introduced are intended to be construed to include all such modifications, permutations, additions and partial combinations within their true spirit and scope.

Claims

**Claim 1** A method for treating dry eye disease in a subject diagnosed with dry eye disease, comprising: directly administering to the eye of a subject in need thereof a composition comprising a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor in a therapeutically effective amount A method comprising. **Claim 2** A method for slowing, inhibiting, or delaying the progression of dry eye disease in a subject, comprising: directly administering to the eye of a subject in need thereof a composition comprising a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor in a therapeutically effective amount A method comprising. **Claim 3** A method for reversing the progression of dry eye disease in a subject, comprising: directly administering to the eye of a subject in need thereof a composition comprising a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor in a therapeutically effective amount A method comprising. **Claim 4** A method for restoring the homeostasis of the tear film on the surface of the eye, comprising: directly administering to the eye of a subject in need thereof a composition comprising a compound that inhibits the binding of an inflammatory interleukin-17 (IL-17) cytokine to the IL-17 receptor in a therapeutically effective amount A method comprising. **Claim 5** The method according to any one of claims 1 to 4, further comprising identifying a subject having mild or moderate dry eye disease. **Claim 6** The method according to any one of claims 1 to 4, further comprising identifying a subject having mild, moderate or severe dry eye disease. **Claim 7** The method according to claim 5 or claim 6, wherein said identifying comprises the use of a questionnaire. **Claim 8** The method according to claim 7, wherein said questionnaire is selected from a Visual Analogue Scale (VAS), an Ocular Surface Disease Index (OSDI) questionnaire, a Symptom Assessment Questionnaire iN Dry Eye (SANDE), a Dry Eye Questionnaire (DEQ), and a Standard Patient Evaluation of Eye Dryness Questionnaire (SPEED). **Claim 9** identifying a subject having mild or moderate dry eye disease and monitoring the progression of dry eye disease using the Standard Patient Evaluation of Eye Dryness Questionnaire (SPEED), further comprising the method according to any one of claims 2. **Claim 10** The method according to claim 9, wherein identifying comprises using a questionnaire. **Claim 11** The method according to any one of claims 1 to 10, wherein administering comprises administering a composition comprising a compound selected from a biomolecule or an organic synthetic compound. **Claim 12** The method according to claim 11, wherein the organic synthetic compound is a spirocyclic indane compound or a spirocyclic oxoindoline compound. **Claim 13** The method according to claim 11, wherein the biomolecule is an antibody having binding affinity for an IL-17 cytokine or an antibody having binding affinity for an IL-17 receptor. **Claim 14** The method according to claim 13, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a humanized, recombinant antibody, a chimeric antibody, or an antibody fragment. **Claim 15** The method according to any one of claims 13 to 14, wherein the antibody is selected from the group consisting of afasevikumab, bimekizumab, brodalumab, ixekizumab, izokibep netakimab, perakizumab, secukinumab, sonelokizumab, tildrakizumab, bunakizumab, ABY-035, CJM-112, CNTO-6785, DC-806 / S-011806, FPP-003, GR-1501, HB-0017, IMU-035, LZM-012, QX-002N, BH-1657, HB-0043, HT-0017, ILCT-1001, IQ-001, LEO153339 / LP0200, LP-0200, MT-6194, MYMD-1, ND-016, SCT-650A, SM-17, YBL-004, ABM-60, ETI-1023, LQ-025, LQ-026, AbbV-257, AFB-035, ANB-004, BCD-121, COVA-322, CYT-017-IL17Qb, DLX-2882, DLC-2907, DLX-2909, DLX-3003, E-34935, E-35018, E-35762, E-36041, EBI-006, HEISCO-III-002, IL-17-RC, MEDI-571, MOR-106, MP-0230, PRS-190, SCH-900117, Y-320, ABT-122, BITS-7201A, CDP-435, EBI-028, IL-17E, JNJ-6118104, KHK-4827, and RG7624.

16. The method according to claim 11, wherein the biological compound is a protein or peptide that specifically inhibits the binding of interleukin 17A to the interleukin 17A receptor.

17. The method according to claim 16, wherein the peptide is a contiguous sequence of 12 to 18 amino acid residues and consists of a contiguous sequence having at least about 70% sequence identity with SEQ ID NO:

1.

18. The peptide according to claim 16 has the formula I: X １ -X ２ -X ３ -X ４ X ５ -X ６ -X ７ -X ８ -X ９ -X １０ -X １ I - X １２ -X １３ -X １４ -X １５ Formula (I) (wherein X １ is I, V, or L; X ２ is H, M, R, K or E; X ３ is V, F, or I; X ４ is T, Q, S, Y or N,; X ５ is I, F or V; X ６ is P or G; X ７ is A, Q, or L; X ８ is D, E, or Q; X ９ is L, W, F, V or I; X １０ is W, Y, or F; X １１ is D, E or N,; X １２ is either W or F; X １３ is I, V, F or L; X １４ is N, R, Q, or E; and X １５ is K, R, H or E) contains the amino acid sequence of, the method according to claim 16.

19. (a) X １ is I or V, X ２ is H, M, or R, X ３ is V or F; X ４ is T or Q; X ５ is I, F or V; X ６ is P or G; X ７ is either A or Q; X ８ is D or E; X ９ is L; X １０ is either W or Y; X is D or E; X １２ is W; X １３ is either I or V; X １４ is N, R or E; and X １５ is K, R or E; or (b) X １ is I or V; X ２ is H or M, X ３ is V; X ４ is T; X ５ is I; X ６ is P; X ７ is A; X ８ is D; X ９ is L, W, F, V or I; X １０ is W or Y; X １１ is D or E; X １２ is W; X １３ is I or V; X １４ is N, R, or E; and X １５ is K, R or E, The method according to claim 18.

20. Said administering comprises administering said peptide wherein its C-terminus and / or N-terminus is linked to a protecting cap group, wherein the protecting cap group linked to the C-terminus is selected from the group consisting of amide, aldehyde, ester, p-nitroanilide, 7-amino-4-methylcoumarin, and the protecting cap linked to the N-terminus is selected from the group consisting of acetyl, formyl, pyroglutamyl, fatty acid, urea, carbamate sulfonamide, and alkylamine, the method according to any one of claims 17 to 19.

21. Said administering comprises administering said peptide in the form of a dimer formed by two peptides, each peptide being a peptide of formula I, the method according to any one of claims 17 to 20.

22. The method according to claim 21, wherein the two peptides in the dimer are linked by a polyethylene spacer.

23. Said administering comprises administering said peptide in the form of a bioconjugate comprising a peptide of formula I and a biomolecule, wherein the biomolecule is linked to the N-terminus and / or C-terminus of the peptide, the method according to any one of claims 17 to 22.

24. The biomolecule is selected from the group consisting of capric acid, caproic acid, ascorbic acid, NAG-NAM, NAG, NAM, hyaluronic acid, alginic acid, chitin, (GalNAc) ２ , Gal-alpha1,3-GalNAc and trigalacturonic acid, the method according to claim 23.

25. Said administering comprises administering said peptide in a pharmaceutically acceptable composition comprising at least one pharmaceutically acceptable excipient, the method according to any one of claims 17 to 24.

26. The method according to any one of claims 1 to 25, wherein administering comprises instilling onto the ocular surface.

27. The method according to any one of claims 1 to 26, wherein administering comprises instilling into the conjunctival sac.

28. The method according to any one of claims 1 to 27, wherein administering comprises administering once a day.

29. The method according to any one of claims 17 to 28, wherein the therapeutically effective dose of said peptide is at least about 500 micromoles.

30. The method according to claim 29, wherein the dose is administered once a day.

31. The method according to any one of claims 1 to 30, wherein said administering is carried out over a period of at least about 3 weeks, wherein the signs and symptoms of dry eye disease are resolved over the disease-free period in the 3rd week of said administering.

32. A method for restoring the function and / or density of goblet cells in an eye having signs or symptoms of dry eye disease or for preventing goblet cell reduction in an eye of a person having dry eye disease, comprising locally administering to the eye of a subject in need thereof a therapeutically effective amount of a compound that inhibits the binding of IL-17A to the interleukin 17A receptor.

33. A method for improving the balance of the functions of the lacrimal gland, Meibomian glands and conjunctival goblet cells and restoring the homeostasis of the tear film in a subject, comprising locally administering to the eye of a subject in need thereof a therapeutically effective amount of a compound that inhibits the binding of IL-17A to the interleukin 17A receptor.

34. A method for increasing basal tear production in an elderly subject, comprising locally administering to the eye of a subject in need thereof a therapeutically effective amount of a compound that inhibits the binding of IL-17A to the interleukin 17A receptor.

35. The method according to claim 34, wherein the elderly subject has hormonal changes or is over 50 years old.

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