Lysophosphatidic acid receptor (LPAR) modulators for treating ocular surface disorders

Abstract

Disclosed herein are methods for treating an ocular surface disorder by administering a therapeutically effective amount of at least one lysophosphatidic acid receptor (LPAR) modulator to a subject.

Description

LYSOPHOSPHATIDIC ACID RECEPTOR (LPAR) MODULATORS FOR TREATING OCULAR SURFACE DISORDERSSTATEMENT OF GOVERNMENT INTEREST

[0001] This invention was made with government support under Grant Numbers K08 EY033859, K12 EY031372, R01 EY036139 and R01 DK126070 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0002] Ocular surface disorders are a multi-billion-dollar market. For example, dry eye disease (DED) affects approximately 7% of adults in USA, with approximately 50% global prevalence. The USA spends about $55 billion annually for DED.

[0003] The tear film is a liquid barrier that lubricates and protects the ocular surface (cornea and conjunctiva) from the external environment (Ref. [1]). Dysregulation of the tear film can lead to DED, which is characterized by aqueous tear deficiency, tear film instability, tear hyperosmolarity, and inflammation (Ref. [2]). DED is a multifactorial disease that drives tear film instability and hyperosmolarity, directly damaging the ocular surface epithelium. DED affects about 8% of adults in the US, with a global prevalence as high as 50% — making it one of the top three reasons for seeking eye care (Ref. [3]).

[0004] There are currently seven FDA-approved therapies for DED: five of them are topical eye drops that target inflammation (cyclosporine 0.05%, cyclosporine 0.09%, cyclosporine 0.1%, lifitegrast 5%, and loteprednol 0.25%), one of them is a topical eye drop that targets meibominan gland disease (perfluorohexyloctane), and one of them is a nasal spray that stimulates tear secretion (varenicline 0.03 mg).

[0005] Ocular surface hydration, one of the main regulators of DED, is maintained by the secretion and absorption of ions in the ocular surface epithelia. Ion channels such as cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) regulate fluid secretion and absorption, respectively (Refs. [4] & [5]). In addition to CFTR, Ca2+-activated CF channels (CaCCs) also secrete CF into the tear film, facilitating ocular surface hydration (Ref. [6]). As a result, ion channels represent an attractive drug target for anti-absorptive and prosecretory DED therapies (Refs. [4] & [6]).

[0006] A need exists to discover compounds and methods for treating ocular surface disorders, including dry eye disease.BRIEF SUMMARY

[0007] Provided herein are methods for treating ocular surface disorders, such as neuropathic ocular pain, dry eye disease (DED), ocular inflammation, and various ocular wounds, by topically administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising at least one lysophosphatidic acid receptor (LPAR) modulator. In some embodiments the ocular surface disorder is a neuropathic ocular pain, a dry eye disease (DED), an ocular inflammation, an ocular wound, or any combination thereof.

[0008] Another embodiment provides for the use of at least one LPAR modulator, or the use of a pharmaceutical composition comprising at least one LPAR modulator, in the preparation of a medicament for treating a subject with an ocular surface disorder.

[0009] Another embodiment provides at least one LPAR modulator, or a pharmaceutical composition comprising at least one LPAR modulator, which is used to treat an ocular surface disorder.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0010] Figure 1 shows ocular surface potential difference (OSPD) traces in mice with linoleoyl LPA (10 pM).

[0011] Figure 2 shows OSPD traces in mice with linoleoyl LPA (10 pM) pretreated with Ki 16425 (10 pM), a selective LPAR1 / 3 antagonist.

[0012] Figure 3 shows a summary of the OSPD data in Figures 1 and 2.

[0013] Figure 4 shows OSPD changes (A OSPD) induced by linoleoyl LPA (10 pM) and linoleoyl LPA (10 pM) pretreated with Ki 16425 (10 pM).

[0014] Figure 5 shows OSPD traces in mice induced by linoleoyl LPA (10 pM), LPAR1 selective agonist UCM-05194 (10 pM), LPAR2 selective agonist DB IBB (10 pM), LPAR3 selective agonist 2S-OMPT (10 pM), LPAR1 / 3 / 6 selective agonist Alkyl-OMPT (10 pM) + LPAR1 / 3 selective antagonist Ki 16425 (10 pM) (LPAR6), and LPAR1 / 3 / 6 agonist Alkyl-OMPT (10 pM).

[0015] Figure 6 shows changes in OSPD (A OSPD) in mice induced by linoleoyl LPA (10 pM), LPAR1 selective agonist UCM-05194 (10 pM), LPAR2 selective agonist DBIBB (10 pM), LPAR3 selective agonist 2S-OMPT (10 pM), LPAR1 / 3 / 6 selective agonist Alkyl-OMPT (10 pM) + LPAR1 / 3 selective antagonist Ki 16425 (10 pM) (LPAR6), and LPAR1 / 3 / 6 agonist Alkyl-OMPT (10 pM).

[0016] Figure 7 shows corneal, conjunctival, and lacrimal gland sections obtained from 12-week-old BALB / c mice with immunofluorescence staining with Anti-LPAR3 antibody (1:200 dilution) versus negative controls with no primary antibody.

[0017] Figure 8 shows immunofluorescence staining with Anti-LPAR3 antibody (1:200 dilution), anti-NKCCl antibody (1:400), a ductal cell marker in the lacrimal gland obtained from 12-week-old BALB / c mice.

[0018] Figure 9 shows Anti-LPAR3 antibody (1:200 dilution) immunofluorescence staining in the corneal and conjunctival epithelia sections obtained from a human donor.

[0019] Figure 10 shows immortalized HCECs with Anti-LPAR3 antibody (1 :500 dilution) immunofluorescence staining and intracellular Ca2+under three test conditions with linoleoyl LPA (10 pM, black), linoleoyl LPA with Ki 16425 (10 pM, blue), and linoleoyl LPA with U-73122 (10 pM, red).

[0020] Figure 11 shows primary HCECs with Anti-LPAR3 antibody (1:500 dilution) immunofluorescence staining and intracellular Ca2+under three test conditions with linoleoyl LPA (10 pM, black), LPA with Ki 16425 (1 pM, blue), and LPA with a PLC inhibitor, U-73122 (10 pM, red).

[0021] Figure 12 illustrates a mechanism for prosecretory effects of LPAR modulators via LPAR3 activation and increased intracellular calcium via the Gq / PLC pathway.DETAILED DESCRIPTION

[0022] Lysophosphatidic acid receptors (LPARs) are G-protein coupled receptors (GPCRs) responsible for Ca2+and cAMP mobilization via PLC and adenylyl cyclase activation. LPAR is a family of GPCRs expressed widely across the body that play a major role in calcium mobilization, cellular proliferation, wound healing, and pain signaling. LPAR signaling is involved in cell proliferation and wound healing in human corneal epithelial cells (HCECs) as well as in neuropathic pain.

[0023] Lysophosphatidic acid (LPA) is a small bioactive lipid involved in cell proliferation and wound healing in human corneal epithelial cells (HCECs) as well as in neuropathic pain (Ref. [7]). LPA interacts with six G-protein coupled receptors (GPCRs), denoted LPAR1-6, as well as GPR87, P2Y10, and TRPV1 (Refs. [7]-

[0010] ). LPAR1-6 are widely expressed throughout the body and are coupled to one or more G-proteins: Gs, Gi, Gq, and G12 / 13. The coupling between LPARs and G-proteins Gq and Gs triggers Ca2+mobilization via PLC activation and cAMP mobilization via adenylyl cyclase activation, respectively (Ref.

[0010] ).As a result, the downstream products of LPAR activation have the potential to activate ion channels such as CaCCs (Ca2+) and CFTR (cAMP). This would be consistent with the discovery that LPA activates Cl’ channels in keratocytes, maintains the integrity of the normal cornea, and promotes cellular regeneration during corneal wound healing in rabbits (Refs.

[0011] &

[0012] ).

[0024] Although the relationship between LPAR, CaCCs, and ocular surface hydration in the ocular surface epithelium is definitively studied, LPA was found to elevate intracellular Ca2+(a regulator of CaCCs) in rabbit corneal endothelial cells, which were partially inhibited by an LPAR3 selective antagonist, DGPP (Ref.

[0013] ). CaCCs are the indirect target of diquafosol, a DED therapeutic approved in Japan that agonizes P2Y2, a purinergic receptor coupled to the Gq / PLC pathway that increases intracellular Ca2+(Refs. [6] &

[0014] ).

[0025] LPAR modulation thus represents a novel pro-secretory mechanism of action via calcium activated chloride channels. Additionally, because LPAR modulators have been shown to play a role in cellular proliferation, wound healing, and pain signaling, LPAR modulators could also potentially be used therapeutically to target multiple pathways in addition to tear secretion, representing a major advancement over current FDA-approved options.

[0026] One example of an LPAR modulator is (lS,3S)-3-[2-Methyl-6-[l-methyl-5-[[methyl(propyl)carbamoyl]oxymethyl]tri azol-4-yl]pyri din-3 -yl]oxy cy cl ohexane-1 -carboxylic acid (BMS-986278), an investigational LPAR1 antagonist for the treatment of progressive pulmonary fibrosis. Other suitable LPAR modulators are described herein in more details.

[0027] Ocular surface disorder,” as used herein, refer to conditions that affect and damage the surface layers of the eyes, include cornea and conjunctiva. Symptoms of the ocular surface disorder includes, for example, corneal epithelial atrophy, corneal pseudomicrocysts, punctate erosion, eye pain, blurry vision, mononuclear cell infiltration, dry eye, and the like.

[0028] “Subject” and “subject in need thereof’ refer to a patient suffering from or prone to suffering from an ocular surface disorder.

[0029] The terms “treating,” or “treatment” refer to any indicia of success in the treatment or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; or improving a patient’s physical well-being. The treatment of symptoms can be based on objective or subjective parameters, including the results of a physical examination. The term “treating” includes prevention of an injury, pathology, condition, or disease. “Treating” in reference to treating a symptom of an ocular surface disorder refers to, for example, reducingcorneal epithelial atrophy, reducing corneal pseudomicrocysts, reducing punctate erosion, reducing eye pain, reducing blurry vision, reducing mononuclear cell infiltration, reducing dry eye disease (DED) and / or reducing ocular inflammation.

[0030] A “therapeutically effective amount” is an amount of the active agent sufficient to accomplish a stated purpose, e.g., achieve the effect for which it is administered ( / .< ., reducing dry eye disease (DED)) in a patient. A “therapeutically effective amount” is an amount of the active agent sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In embodiments, the “therapeutically effective amount” is the amount described herein.

[0031] As discussed in detail herein, the LPAR modulators, or compositions comprising the same, may be administered once or as frequently as the symptoms occur.

[0032] The LPAR modulators and compositions described herein can be used in combination with one or more other drugs known to be useful in treating ocular surface disorders. The LPAR modulators and compositions described herein can be used with adjunctive agents that may not be effective alone, but may contribute to the efficacy of the LPAR modulators. Thus, the LPAR modulators described herein may be co-administered with one or more other drugs that are useful to treat ocular surface disorders in patients. Exemplary drugs used to treat ocular surface disorders include vasoconstricting agents, epithelial sodium channel inhibitors, lymphocyte function-associated antigen-1 antagonists, anti-inflammatory agents, cholinergic agonists, steroids, antibiotics, and the like. Steroids that may be used in combination with LPAR modulators of the present disclosure include, for example, prednisolone phosphate, prednisolone acetate, fluoromethoIone acetate, dexamethasone, and the like.

[0033] By “co-administer” it is meant that LPAR modulators or compositions described herein are administered at the same time, prior to (e.g., minutes or hours), or after (e.g., minutes or hours) the administration of one or more additional therapies. The LPAR modulators described herein can be administered alone or can be co-administered to the patient. Coadministration is meant to include simultaneous or sequential administration of the LPARmodulators individually or in combination. Thus, the preparations can also be combined, when desired, with other active substances.

[0034] Co-administration includes administering one LPAR modulator(s) within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of a second pharmaceutical compound (e.g., anti-dry eye agents). Also contemplated herein, are embodiments, where co-administration includes administering one LPAR modulator within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of another pharmaceutical compound. Co-administration includes administering the LPAR modulator(s) and at least one other pharmaceutical compound simultaneously, approximately simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. Co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition including both the LPAR modulator(s) and the other pharmaceutical compound. In other embodiments, the LPAR modulator(s) and other pharmaceutical compound can be formulated separately.

[0035] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of LPAR modulators to and absorption by a patient and can be included in the compositions described herein. In particular, the pharmaceutically acceptable excipient or carrier is suitable for ophthalmic uses, including topical or ocular surface administration. Exemplary pharmaceutically acceptable excipients include stabilizers, co-solvents, and the like. Other non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with other pharmaceutically acceptable excipients such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like.

[0036] A “stabilizer” refers to a pharmaceutically acceptable excipient that maintains the properties of the LPAR modulators described herein and / or that delays or prevents physical or chemical degradation of the LPAR modulators described herein. Exemplary stabilizers include microcrystalline cellulose, carboxymethyl cellulose, hydromellose, dextran, and the like.

[0037] Co-solvent” refers to pharmaceutically acceptable excipients that can increase, maintain, or prolong the solubility of the LPAR modulators. Exemplary co-solvents includesorbitol, glycerol, propylene glycol, polyethylene glycol, polyvinyl alcohol, polysorbate, and the like.

[0038] Pharmaceutical compositions used in the treatment methods herein are generally administered topically. “Topically administering” means topical application of the LPAR modulators or compositions described herein to one or both eyes of a patient. In embodiments, the topical administration is topical administration to the cornea of the eye. The active agents and compositions described herein can be delivered topically as a liquid formulation, e.g., as eye drops. In embodiments, the topical liquid formulation is a solution. In embodiments, the topical liquid formulation is an aqueous solution. In embodiments, the topical liquid formulation is a suspension. In embodiments, the topical liquid formulation is an emulsion. In embodiments, the topical liquid formulation is an ointment.

[0039] In other embodiments, the LPAR modulators or compositions comprising the same may be delivered through an implantable device in the eyes, including fluid-eluting contact lens. The implantable device includes a reservoir containing the pharmaceutical composition described herein and may further include means that allows the active agent to elute onto the ocular surface in a sustained manner. See, e.g., U.S. 2020 / 0409177.

[0040] The term “linoleoyl LPA” refers to the compound shown below, i.e., 1-linoleoyl-2-hydroxy-sw-glycero-3 -phosphate sodium salt.(linoleoyl LPA)

[0041] The term “Alkyl-OMPT” refers to the compound shown below, i.e., [(2S)-2-methoxy-3-[(9Z)-octadec-9-en-l-yloxy]propoxy](sulfanylidene)phosphonous acid.(Alkyl-OMPT)

[0042] The term “Ki 16425” refers to the compound shown below, i.e., 3-[[[4-[4-[[[l-(2- Chlorophenyl)ethoxy]carbonyl]amino]-3-methyl-5-isoxazolyl]phenyl]methyl]thio]-propanoic acid.(Ki 16425)

[0043] The term “2S-OMPT” refers to the compound shown below, i.e., (2S)-1-(9Z-octadecenoyl)-2-O-methyl-glycero-3 -phosphothionate ammonium salt.<(2S-OMPT)

[0044] The term “UCM-05194” refers to the compound shown below, i.e., (2S)-1- Bromo-3-(phosphonooxy)propan-2-yl 10-Phenyldecanoate ammonium salt.(UCB-05194)

[0045] The term “DBIBB” refers to the compound shown below, i.e., 2-[[[4-(l,3-dioxo-lH-benz[de]isoquinolin-2(3H)-yl)butyl]amino]sulfonyl]-benzoic acid.(DBIBB)

[0046] The term “U73122” refers to the compound shown below, i.e., 1 -[6-[[( 17|3)-3 -methoxyestra-1, 3, 5(10)-trien-17-yl]amino]hexyl]-lH-pyrrole-2, 5-dione.(U73122)

[0047] As used herein with respect to LPAR selectivity, the term “selective” means ICso or ECso of 10 micromolar or lower for LPAR.

[0048] The term “pharmaceutically acceptable” refers an agent that has been approved for human consumption and is generally non-toxic. For example, the term “pharmaceutically acceptable salt” refers to nontoxic inorganic or organic acid and / or base addition salts (Ref.

[0015] ).Methods of Treatment

[0049] In various embodiments, disclosed herein are methods for treating ocular surface disorders, such as neuropathic ocular pain, dry eye disease (DED), ocular inflammation, and various ocular wounds, by topically administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising at least one lysophosphatidic acid receptor (LPAR) modulator.

[0050] In more specific embodiments the at least one LPAR modulator comprises an LPAR agonist. In yet other embodiments the at least one LPAR modulator comprises an LPAR antagonist. In some embodiments the at least one LPAR modulator comprises both an LPAR agonist and an LPAR antagonist.

[0051] In more specific embodiments the at least one LPAR modulator comprises an LPARi selective modulator, an LPAR2 selective modulator, an LPAR3 selective modulator, an LPAR4 selective modulator, an LPARs selective modulator, an LPARe selective modulator, or any combination thereof.

[0052] In some embodiments the pharmaceutical composition comprises at least one LPARi selective modulator. In some embodiments the pharmaceutical composition comprises at least one LPAR2 selective modulator. In some embodiments the pharmaceutical composition comprises at least one LPAR3 selective modulator. In some embodiments the pharmaceutical composition comprises at least one LPAR4 selective modulator. In some embodiments the pharmaceutical composition comprises at least one LPAR5 selective modulator. In some embodiments the pharmaceutical composition comprises at least one LPARe selective modulator.

[0053] In more specific embodiments the at least one LPAR modulator comprises an LPARi selective agonist, an LPAR2 selective agonist, an LPAR3 selective agonist, an LPAR4 selective agonist, an LPARs selective agonist, an LPARe selective agonist, or any combination thereof.

[0054] In some embodiments the pharmaceutical composition comprises at least one LPARi selective agonist. In some embodiments the pharmaceutical composition comprises at least one LPAR2 selective agonist. In some embodiments the pharmaceutical compositioncomprises at least one LPAR3 selective agonist. In some embodiments the pharmaceutical composition comprises at least one LPAR4 selective agonist. In some embodiments the pharmaceutical composition comprises at least one LPAR5 selective agonist. In some embodiments the pharmaceutical composition comprises at least one LPARe selective agonist.

[0055] In more specific embodiments the at least one LPAR modulator comprises an LPARi selective antagonist, an LPAR2 selective antagonist, an LPAR3 selective antagonist, an LPAR4 selective antagonist, an LPARs selective antagonist, an LPARe selective antagonist, or any combination thereof.

[0056] In some embodiments the pharmaceutical composition comprises at least one LPARi selective antagonist. In some embodiments the pharmaceutical composition comprises at least one LPAR2 selective antagonist. In some embodiments the pharmaceutical composition comprises at least one LPAR3 selective antagonist. In some embodiments the pharmaceutical composition comprises at least one LPAR4 selective antagonist. In some embodiments the pharmaceutical composition comprises at least one LPAR5 selective antagonist. In some embodiments the pharmaceutical composition comprises at least one LPARe selective antagonist.

[0057] In more specific embodiments the at least one LPAR modulator comprises at least one LPAR modulator that is selective for a plurality of LPAR subtypes. For example, in some embodiments the at least one LPAR modulator includes an LPAR modulator that is selective for at least two of LPARi, LPAR2, LPAR3, LPAR4, LPARs and LPARe. In some embodiments the at least one LPAR modulator includes an LPAR modulator that is selective for at least three of LPARi, LPAR2, LPAR3, LPAR4, LPARs and LPARe. In some embodiments the at least one LPAR modulator includes an LPAR modulator that is selective for at least four of LPARi, LPAR2, LPAR3, LPAR4, LPARs and LPARe. For example, in some embodiments the at least one LPAR modulator comprises an LPAR1 / 3 selective modulator, an LPAR1 / 3 / 6 selective modulator, or any combination thereof.

[0058] In some embodiments the pharmaceutical composition comprises an LPARi selective agonist. In some embodiments the pharmaceutical composition comprises LPAR2 selective agonist. In some embodiments the pharmaceutical composition comprises an LPAR3 selective agonist. In some embodiments the pharmaceutical composition comprises LPAR1 / 3 selective antagonist. In some embodiments the pharmaceutical composition comprises an LPAR1 / 3 / 6 selective agonist.

[0059] In more specific embodiments the at least one LPAR modulator comprises linoleoyl LPA, Alkyl-OMPT, Ki 16425, 2S-OMPT, UCM-05194, DBIBB, or any combinationthereof. In some embodiments the at least one LPAR modulator comprises linoleoyl LPA. In some embodiments the at least one LPAR modulator comprises Alkyl-OMPT. In some embodiments the at least one LPAR modulator comprises Ki 16425. In some embodiments the at least one LPAR modulator comprises 2S-OMPT. In some embodiments the at least one LPAR modulator comprises UCM-05194. In some embodiments the at least one LPAR modulator comprises DBIBB.

[0060] In more specific embodiments the pharmaceutical composition is administered to an eye of the subject. For example, in some embodiments the pharmaceutical composition is administered to the cornea or conjunctiva of the subject. In some embodiments the method for treating the ocular surface disorder comprises administering from about 2 nanomoles to about 200 nanomoles of the at least one LPAR modulator to the eye of the subject. The 2-200 nanomoles independently relates to each individual LPAR modulator, such that when the pharmaceutical composition contains two LPAR modulators the amount of each LPAR modulator administered to the eye if the subject independently ranges from 2-200 nanomoles. In some embodiments the method for treating the ocular surface disorder comprises administering from about 2 nanomoles to about 10 nanomoles, or from about 10 nanomoles to about 50 nanomoles, or from about 50 nanomoles to about 75 nanomoles, or from about 75 nanomoles to about 125 nanomoles, or from about 125 nanomoles to about 200 nanomoles of the at least one LPAR modulator to the eye of the subject.

[0061] In more specific embodiments the therapeutically effective amount of the pharmaceutical composition provides a concentration of about 100 nM to about 1,000 mM of the at least one LPAR modulator in the eye of the subject after a period of about 1-3 hours following administration. The about 100 nM to about 1,000 nM independently relates to each individual LPAR modulator, such that when the pharmaceutical composition contains two LPAR modulators the concentration of each LPAR modulator administered to the eye if the subject independently ranges from about 100 nM to about 1,000 mM.

[0062] In some embodiments the pharmaceutical composition is administered to the subject in the form of a solution or dispersion having a concentration ranging from about 1% (m / v) ( / .< ., ~1 gram per 100 mL) to about 10% (m / v) ( / .< ., ~10 grams per 100 mL). For example, in some embodiments the pharmaceutical composition is administered to the subject in the form of a solution or dispersion having a concentration ranging from about 1.0% (m / v) to about 1.5% (m / v), or from about 1.5% (m / v) to about 2.0% (m / v), or from about 2.0% (m / v) to about 2.5% (m / v), or from about 2.5% (m / v) to about 3.0% (m / v), or from about 3.0% (m / v) toabout 3.5% (m / v), or from about 3.5% (m / v) to about 4.0% (m / v), or from about 4.0% (m / v) to about 4.5% (m / v), or from about 4.5% (m / v) to about 5.0% (m / v), or from about 5.0% (m / v) to about 5.5% (m / v), or from about 5.5% (m / v) to about 6.0% (m / v), or from about 6.0% (m / v) to about 6.5% (m / v), or from about 6.5% (m / v) to about 7.0% (m / v), or from about 7.0% (m / v) to about 7.5% (m / v), or from about 7.5% (m / v) to about 8.0% (m / v), or from about 8.0% (m / v) to about 8.5% (m / v), or from about 8.5% (m / v) to about 9.0% (m / v), or from about 9.0% (m / v) to about 9.5% (m / v), or from about 9.5% (m / v) to about 10.0% (m / v). In a specific embodiment, the concentration is 4%. In some embodiments the pharmaceutical composition is administered to the subject in the form of an aqueous solution or aqueous dispersion; in other embodiments the pharmaceutical composition is administered to the subject in the form of a non-aqueous solution or non-aqueous dispersion.

[0063] In more specific embodiments the pharmaceutical composition further comprises at least one additional therapeutic agent. For example, in some embodiments the method for treating the ocular surface disorder further comprises co-administering at least one additional therapeutic agent to the subject. In some embodiments the at least one additional therapeutic agent is administered in an amount effective to enhance a therapeutic effect of the at least one LPAR modulator. In some embodiments the at least one additional therapeutic agent comprises a vasoconstricting agent, an epithelial sodium channel inhibitor, a lymphocyte function-associated antigen- 1 antagonist, an anti-inflammatory agent, a cholinergic agonist, a steroid, an antibiotic, or any combination thereof. In more specific embodiments the at least one additional therapeutic agent comprises a steroid selected from the group consisting of prednisolone phosphate, prednisolone acetate, fluorometholone acetate, dexamethasone, and any combination thereof.

[0064] In more specific embodiments the ocular surface disorder is a neuropathic ocular pain, a dry eye disease (DED), an ocular inflammation, an ocular wound, or any combination thereof. For example, in some embodiments the ocular surface disorder is a dry eye disease (DED).

[0065] In some embodiments treating the ocular surface disorder comprises reducing corneal epithelial atrophy, reducing corneal pseudomicrocysts, reducing punctate erosion, reducing eye pain, reducing blurry vision, and / or reducing mononuclear cell infiltration. In some embodiments the ocular surface disorder is ADC-induced corneal toxicity.

[0066] In some embodiments the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier.

[0067] In some embodiments the pharmaceutical composition is administered once per day. In some embodiments the pharmaceutical composition is administered twice per day. In some embodiments pharmaceutical composition is administered for about 14 days. In some embodiments the pharmaceutical composition is administered for about one month. In some embodiments the pharmaceutical composition is administered for more than one month. In some embodiments the pharmaceutical composition is administered for at least one year. In some embodiments the pharmaceutical composition is administered for more than one year. In some embodiments the pharmaceutical composition is administered indefinitely.

[0068] In some embodiments the therapeutically effective amount provides a concentration of the at least on LPAR modulator in an amount of about 100 pM or more in the tear fluid of the eye about 1 hour to about 12 hours after administration. In some embodiments the therapeutically effective amount provides a concentration of the at least on LPAR modulator in an amount of (i) about 200 pM or more in the tear fluid of the eye about 30 minutes to about 3 hours after administration, or (ii) about 100 pM or more in the tear fluid of the eye about 4 hours to about to about 12 hours after administration, or (iii) both (i) and (ii). In some embodiments the therapeutically effective amount provides a concentration of the at least on LPAR modulator in an amount of (i) about 100 pM to about 200 pM about 1 hour to about 3 hours after administration, or (ii) about 50 pM to about 150 pM about 4 hours to about 8 hours after administration, or (iii) both (i) and (ii). In some embodiments the therapeutically effective amount provides a concentration of the at least on LPAR modulator in an amount of (i) about 150 pM to about 200 pM about 1 hour to about 3 hours after administration, or (ii) about 100 pM to about 150 pM about 4 hours to about 8 hours after administration, or (iii) both (i) and (ii). In some embodiments the therapeutically effective amount provides a concentration of the at least on LPAR modulator in an amount of (i) about 150 pM to about 250 pM about 1 hour to about 3 hours after administration, or (ii) about 50 pM to about 200 pM about 5 hours to about 7 hours after administration, or (iii) both (i) and (ii). In some embodiments the therapeutically effective amount provides a concentration of the at least on LPAR modulator in an amount of (i) about 200 pM to about 300 pM about 1 hour to about 3 hours after administration, or (ii) about 100 pM to about 200 pM about 5 hours to about 7 hours after administration, or (iii) both (i) and (ii). In some embodiments the therapeutically effective amount provides a concentration of the at least on LPAR modulator in an amount of (i) about 175 pM about 2 hours after administration, or (ii) about 50 pM about 6 hours after administration, or (iii) both (i) and (ii).

[0069] In some embodiments the pharmaceutical composition administered orally such that the therapeutically effective amount provides an oral dose of the at least one LPAR modulator ranging from about 1 mg / day to about 100 mg / day. In some embodiments the oral dose ranges from about 1 mg / day to about 5 mg / day, or from about 5 mg / day to about 10 mg / day, or from about 10 mg / day to about 15 mg / day, or from about 15 mg / day to about 20 mg / day, or from about 20 mg / day to about 25 mg / day, or from about 25 mg / day to about 30 mg / day, or from about 30 mg / day to about 35 mg / day, or from about 35 mg / day to about 40 mg / day, or from about 40 mg / day to about 45 mg / day, or from about 45 mg / day to about 50 mg / day, or from about 50 mg / day to about 55 mg / day, or from about 55 mg / day to about 60 mg / day, or from about 60 mg / day to about 65 mg / day, or from about 65 mg / day to about 70 mg / day, or from about 75 mg / day to about 80 mg / day, or from about 80 mg / day to about 85 mg / day, or from about 85 mg / day to about 90 mg / day, or from about 90 mg / day to about 95 mg / day, or from about 95 mg / day to about 100 mg / day.

[0070] In some embodiments the pharmaceutical composition is administered orally in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup) — such that the unit dose ranges from about 1 mg / dose to about 10 mg / dose. In some embodiments the unit dose ranges from about 1 mg / dose to about 2 mg / dose, or from about 2 mg / dose to about 3 mg / dose, or from about 3 mg / dose to about 4 mg / dose, or from about 4 mg / dose to about 5 mg / dose, or from about 5 mg / dose to about 6 mg / dose, or from about 6 mg / dose to about 7 mg / dose, or from about 7 mg / dose to about 8 mg / dose, or from about 8 mg / dose to about 9 mg / dose, or from about 9 mg / dose to about 10 mg / dose.

[0071] Any eye dropper known in the art can be used to topically administer the LPAR modulators and compositions described herein. In embodiments, the eye dropper has a volume sufficient to house from about 1 drop to about 50 drops of the pharmaceutical compositions described herein. In embodiments, the eye dropper has a volume sufficient to house from about 1 drop to about 25 drops of the pharmaceutical compositions described herein. In embodiments, the eye dropper has a volume sufficient to house from about 1 drop to about 20 drops of the pharmaceutical compositions described herein. In embodiments, the eye dropper has a volume sufficient to house from about 1 drop to about 15 drops of the pharmaceutical compositions described herein. In embodiments, the eye dropper has a volume sufficient to house from about 1 drop to about 10 drops of the pharmaceutical compositions described herein. In embodiments, the eye dropper has a volume sufficient to house 1 to 5 drops of the composition. In embodiments, the eye dropper has a volume sufficient to house 1 to 4 drops of the composition.In embodiments, the eye dropper has a volume sufficient to house 1 to 3 drops of the composition. In embodiments, the eye dropper has a volume sufficient to house 1 or 2 drops of the composition.

[0072] A “drop” will be a volume of the pharmaceutical composition described herein that can provide a therapeutically effective amount of the LPAR modulators described herein when administered at the doses (e.g., 1 microgram or more; 5 nanomoles or more) and dosing regimen (e.g., one or twice per day) described herein. In embodiments, a drop has a volume from about 10 pL to about 100 pL. In embodiments, a drop has a volume from about 20 pL to about 90 pL. In embodiments, a drop has a volume from about 30 pL to about 80 pL. In embodiments, a drop has a volume from about 40 pL to about 70 pL. In embodiments, a drop has a volume from about 50 pL to about 85 pL. In embodiments, a drop has a volume from about 30 pL to about 65 pL. In embodiments, a drop has a volume from about 40 pL to about 60 pL. In embodiments, a drop has a volume from about 55 pL to about 65 pL.

[0073] The disclosure provides kits comprising the eye droppers described herein. The kit can contain any number of eye droppers that can conveniently be used by the patient for administration of the compositions described herein. Generally, the kit will contain an amount of eye droppers to meet the frequency of the dosing regimen. In embodiments, the kit will contain one eye dropper that can be re-used for the duration of the treatment regimen. In embodiments, the kit will contain seven eye droppers, sufficient to provide single use eye droppers for one week of treatment. In embodiments, the kit will contain fourteen eye droppers. In embodiments, the kit will contain twenty-eight eye droppers. In embodiments, the kit will contain fifty-six eye droppers.

[0074] The disclosure provides kits comprising an eye dropper container which comprises a topical pharmaceutical composition comprising the pharmaceutical composition. The kit can contain any number of eye droppers and any number of containers housing the pharmaceutical compositions that can conveniently be used by the patient for administration of the compositions described herein. Generally, the kit will contain an amount of eye droppers and containers to meet the frequency of the dosing regimen. In embodiments, the kit will comprise one eye dropper and one container, where the container comprises one dose of the composition. In embodiments, the kit will comprise two eye droppers and one container; wherein the container comprises two doses of the composition. In embodiments, the kit will comprise two eye droppers and two containers; wherein each container comprises one dose of the composition. In embodiments, the kit will comprise seven eye droppers and seven containers; wherein eachcontainer comprises one dose of the composition. In embodiments, the kit will comprise fourteen eye droppers and seven containers; wherein each container comprises two doses of the composition. In embodiments, the kit will comprise fourteen eye droppers and fourteen containers; wherein each container comprises one dose of the composition.

[0075] As used herein, the term “pharmaceutical composition” refers to a composition containing at least one of the LPAR modulators described herein, or a pharmaceutically acceptable isomer, racemate, hydrate, solvate, isotope or salt thereof, formulated with a pharmaceutically acceptable carrier, which can also include other additives, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); for administration to a pediatric subject (e.g., solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible / effervescent tablet, chewable tablet, lollipop, freezer pops, troches, oral thin strips, orally disintegrating tablet, orally disintegrating strip, and sprinkle oral powder or granules); or in any other formulation described herein. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005) and in The United States Pharmacopeia: The National Formulary (USP 36 NF31), published in 2013.

[0076] A “hydrate” is a compound that exists in combination with water molecules. The combination can include water in stoichiometric quantities, such as a monohydrate or a dihydrate, or can include water in random amounts. As the term is used herein a “hydrate” refers to a solid form; that is, a compound in a water solution, while it may be hydrated, is not a hydrate as the term is used herein.

[0077] “Isomer” is used herein to encompass all chiral, diastereomeric or racemic forms of a structure (also referred to as a stereoisomer, as opposed to a structural or positional isomer), unless a particular stereochemistry or isomeric form is specifically indicated. Such compounds can be enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions, at any degree of enrichment. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these are all within the scope of certainembodiments of the disclosure. The isomers resulting from the presence of a chiral center comprise a pair of nonsuperimposable-isomers that are called “enantiomers.” Single enantiomers of a pure compound are optically active ( / .< ., they are capable of rotating the plane of plane polarized light and designated R or S).

[0078] “Isotope” refers to atoms with the same number of protons but a different number of neutrons, and an isotope of a compound of structure (I) includes any such compound wherein one or more atoms are replaced by an isotope of that atom. For example, carbon 12, the most common form of carbon, has six protons and six neutrons, whereas carbon 13 has six protons and seven neutrons, and carbon 14 has six protons and eight neutrons. Hydrogen has two stable isotopes, deuterium (one proton and one neutron) and tritium (one proton and two neutrons). While fluorine has a number of isotopes, fluorine- 19 is longest-lived. Thus, an isotope of a compound having the structure of structure (I) includes, but not limited to, compounds of structure (I) wherein one or more carbon 12 atoms are replaced by carbon-13 and / or carbon-14 atoms, wherein one or more hydrogen atoms are replaced with deuterium and / or tritium, and / or wherein one or more fluorine atoms are replaced by fluorine-19.

[0079] A “solvate” is similar to a hydrate except that a solvent other that water is present. For example, methanol or ethanol can form an “alcoholate,” which can again be stoichiometric or non-stoichiometric. As the term is used herein a “solvate” refers to a solid form; that is, a compound in a solvent solution, while it may be solvated, is not a solvate as the term is used herein.

[0080] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the detailed description is exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0081] The phrase “at least one” or “at least one of’ when followed by a list of items or elements refers to an open-ended set of one or more of the elements in the list, which may, but does not necessarily, include more than one of the elements.

[0082] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 100 pL” means “about 100 pL” and also “100 pL.” In some embodiments, about means within 5% of the value. Hence, “about 100 pL” means 95-105 pL. In some embodiments, about means within 4% of the value. In some embodiments, about means within 3% of the value. In some embodiments, about means within 2% of the value. In some embodiments, about means within 1% of the value. Generally, the term “about” includes an amount that would be expected to be within experimental error.

[0083] Where values are described as ranges, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.EXAMPLES

[0084] The following examples are for purposes of illustration and are not intended to limit the spirit or scope of the disclosure or claims.Chemicals

[0085] Linoleoyl LPA, a general LPAR agonist, and Alkyl-OMPT, an LPAR1 / 3 / 6 selective agonist, were purchased from Fisher Scientific (Hanover Park, IL, USA). Ki 16425, an LPAR1 / 3 selective antagonist, was purchased from Tocris Biosciences (Minneapolis, MN, USA). 2S-OMPT, an LPAR3 selective agonist, was purchased from Echelon Biosciences (Salt Lake City, UT, USA). UCM-05194, an LPAR1 selective agonist, was purchased from Axon Medchem LLC (Reston, VA, USA). DBIBB, an LPAR2 selective agonist, was purchased from MedChemExpress (Monmouth Junction, NJ, USA). All other chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). Hoechst 33258 is a DNA staining dye comprising 4-[6-(4-methyl-l-piperazinyl)[2,6'-bi-lH-benzimidazol]-2'-yl]-phenol, trihydrochloride.Animals

[0086] BALB / c mice (female and male, 8-20 weeks old) were bred in UCSF Laboratory Animal Resource Center. The experimental protocols were approved by the UCSF Institutional Animal Care and Use Committee. Animal experiments are done in adherence with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.Ocular surface potential difference (OSPD) measurements

[0087] OSPD provides insight into ion transport in ocular surface epithelia. Notably, the effect of LPA on Ca2+levels in HCECs was more prolonged than the transient effect on OSPD voltage, which likely is due to the nonlinear dependence of OSPD. We focused on LPAR1, LPAR2, LPAR3, and LPAR6 since these have the highest gene expression in both human cornea and conjunctiva (Ref.

[0016] ).

[0088] Isoosmolar (310 mOsm / kg H2O) perfusion solutions with previously standardized compositions were used in these studies (Refs.

[0001] &

[0020] ). High C1-, amiloride, and low Cl-solutions were made in 1 -liter batches, pH balanced to 7.4, filtered in a sterile environment, and used within 3 months. High Cl- solution contained IL Ringers injection with (in mM) 147 NaCl, 2 CaCh, 4 KC1, 2.4 K2HPO4, 0.4 KH2PO4, and 1.2 MgCh. 100 pM Amiloride was added to the high Cl- solution to create the amiloride solution. Low Cl- solution replaced NaCl with 174 mM sodium gluconate. LPAR modulators were freshly added to the low Cl- solution right before the experiments.

[0089] For the OSPD measurements, mice were anesthetized with isoflurane and body temperature was maintained at 37 °C via a heating pad. As described previously (Ref. [4]), opencircuit transepithelial potential differences were measured in response to serial perfusions at room temperature. Solutions were perfused over the ocular surface at 5-10 mL / min via a single perfusion catheter. The measuring electrode connected with the perfusion catheter and the multiport tubing via a three-way stopcock. The perfusion catheter was carefully positioned using a three-axis micromanipulator directly above, but not touching, the mouse ocular surface. A 23-gauge butterfly needle connected to the reference electrode was inserted subcutaneously in the mouse’s back.

[0090] Measuring and reference electrodes consisted of Ag / AgCl electrodes with 3 M KC1 agar bridges, and both electrodes were connected to an ISO-Z headstage, BMA-200 high-impedance amplifier / voltmeter, and a PowerLab analog-to-digital converter (ADInstruments; Colorado Springs, CO, USA) connected to a computer. Solutions were individually perfused onto the ocular surface using a gravity perfusion system (ALA Scientific; Farmingdale, NY, USA) for 1-3 minutes until a stable OSPD reading was obtained. OSPD values after each perfusion solution were calculated as the mean value of a 10-second interval at the end of the solution perfusion, as standardized in human OSPD and nasal potential difference measurements (Refs. [4],

[0017] &

[0018] ).Human tissue

[0091] Fresh human whole eye globes with no corneal epithelial defect or opacities were provided as a gift from Sierra Donor Services Eye Bank (West Sacramento, CA). These tissues came from deidentified donors with no history of visual or ocular disease (age 18-70 years) and death- to-preservation time less than 24 hours.Immunofluorescence staining

[0092] Sections were obtained from WT BALB / c mouse eyes and lacrimal glands, and from human eye donors with no history of visual or ocular disease. Mouse and human eyes were fixed in 4% paraformaldehyde (PF A), embedded in optical cutting temperature (OCT) compound, and cut into 15 pm thick cryosections using a cryostat microtome (LEICA CM1860, Leica Biosystems; Deer Park, IL, USA). The cryosections were mounted onto Fisherbrand Superfrost Plus Microscope Slides (Fisher Scientific) and stored at -80 °C. For immunostaining, cryosections were brought to room temperature, washed with phosphate-buffered saline (PBS) three times (5 minutes for each wash), and incubated with block buffer (50mM Tris pH 7.4, 100 mMNaCl, 1440.1% TX100, 3% Normal Goat Serum, 0.1% BSA, and deionized water) for 1 hour at room temperature in a humidity chamber. The room temperature sections were subsequently treated with Anti-LPAR3 antibody (Fisher Scientific) dissolved in block buffer (1 :200) overnight at 4 °C. Controls were treated with block buffer absent of any primary antibody. The following day, slides were rinsed and washed in washing solution (50mM Tris pH 7.4, 100 mM NaCl, 0.1% TX100, and deionized water) 4 times for 5 minutes each. Secondary antibody Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch; West Grove, PA, USA) was diluted in block buffer (1 :500) and added for 2 hours at room temperature in a humidity chamber. The slides were then rinsed and washed 4 times in washing solution for 5 minutes and once in PBS for 5 minutes. The slides were then incubated for 5 minutes with Hoechst 33258 (nucleus marker) diluted in deionized water (1 : 10,000) and were then rinsed in deionized water. The slides were then mounted with Fluoromount-G 124 (Fisher Scientific) and covered with Corning Cover Glass 24 mm x 50 mm (Coming Inc; Corning, NY, USA). Controls were obtained using secondary antibody in the absence of primary antibody.

[0093] The above LPAR3 staining protocol was also performed in immortalized and primary human comeal epithelial cells (HCECs). HCECs were treated with anti-LPAR3 antibody (1:500) diluted in blocking solution overnight at 4 °C. Subsequently, secondaryantibody (1 :500) diluted in blocking solution was incubated overnight at 4°C. Nuclear staining was achieved with DAPI.

[0094] To study LPAR3 expression on lacrimal gland ductal epithelial cells, co-staining with chicken anti-NKCCl primary antibody (1:400) (Jackson ImmunoResearch) and a Cy3 donkey anti-chicken IgG secondary antibody (1:300) (Santa Cruz Biotechnology; Dallas, TX, USA) was performed on mouse lacrimal gland.Corneal cell culture and Ca2+assay

[0095] To isolate primary HCECs, donor corneoscleral buttons were digested overnight at 4 °C in Dispase II enzyme (15 mg / mL, Thermo Fisher Scientific) supplemented with D-sorbitol (100 mM, Sigma-Aldrich). Primary HCECs were cultured in corneal epithelial cell basal medium plus corneal epithelial cell growth kit (ATCC; Manassas, VA, USA) for approximately 2 weeks in a Matrigel matrix (1:30 169 dilution in DPBS, Coming) pre-coated T25 flask until 70-80% confluence. Primary HCECs were sub-cultured (passage 2) at a 171 density of 30,000 cells per well into Matrigel matrix pre-coated 96-well, black-walled microplates (Corning). Telomerase-immortalized HCECs were generously gifted from Dr. James V. Jester at University of California, Irvine (Ref.

[0018] ).

[0096] Confluent HCECs were loaded with Ca2+indicator Fluo4-NW (Invitrogen) according to the manufacturer's instructions. Cells were assayed for intracellular Ca2+release using a FLUOstar Omega multimode microplate reader (BMG Labtech; Cary, NC, USA) at excitation / emission wavelengths of 485 nm / 520 nm with temperature and atmosphere-controlled settings. Fluorescence was continuously measured in each well after cells were injected with either general LPAR agonist, linoleoyl LPA (10 pM), or LPAR3 selective agonist, 2S-OMPT (10 pM). Cells were pretreated with a phospholipase C inhibitor (PLCinh), U73122 (10 pM), or LPAR1 / 3 antagonist, Ki 16425 (1 pM), for 5 minutes prior to linoleoyl LPA or 2S-OMPT addition.Statistical analysis

[0097] Experiments with two groups were analyzed using two-tailed un-paired Student’s t-test; for three or more groups, analysis was done with one-way analysis of variance (ANOVA) and post hoc Tukey’s multiple comparisons test using GraphPad Prism (GraphPad Software; Boston, MA, USA). Data are expressed as mean ± SEM. In all analyses, p < 0.05 was considered as statistically significant.EXAMPLE 1:LPAR1 / 3 SELECTIVE ANTAGONIST SUPPRESSES LPA-INDUCED CL- SECRETIONIN MOUSE OCULAR SURFACE

[0098] OSPD uses pharmacological activators and inhibitors to study the activities of ion channels. Baseline OSPD is established by a high Cl- solution that mimics the tear fluid.Amiloride (ENaC inhibitor) induces OSPD changes that suppresses Na+ transport via ENaC. Ca2+-agonist ATP induces a rapid transient hyperpolarization in OSPD due to Cl- secretion mediated by CaCCs (Ref.

[0017] ).

[0099] To test the role of LPAR on the ocular surface epithelia, we tested the effect of a non-selective LPAR agonist, linoleoyl LPA, after ENaC inhibition. Linoleoyl LPA (10 pM) produced a rapid transient hyperpolarization in OSPD (-20.0 ± 3.1 mV), suggesting that LPA increases intracellular Ca2+and stimulates Cl- secretion via CaCCs (Fig. 1). Pretreatment with Ki 16425 (10 pM), a selective LPAR1 / 3 antagonist, abolished the OSPD hyperpolarization (-0.2 ± 0.1 mV, p=0.007) (Figs. 1-4). These results suggest that LPAR1 and / or LPAR3 are responsible for the LPA-induced Cl secretion on the ocular surface.EXAMPLE 2:LPAR3 SELECTIVE AGONIST PRODUCES ROBUST CL- SECRETIONIN MOUSE OCULAR SURFACE

[0100] LPAR1, LPAR2, LPAR3, and LPAR6 are the mostly highly expressed LPAR isoforms in the human cornea and conjunctiva (Ref.

[0016] ). We studied OSPD measurements from selective agonists and antagonists for each LPAR isoform to understand their relative contributions to ocular surface ion transport (Figs. 5 and 6).

[0101] General LPAR agonist linoleoyl LPA (10 pM), LPAR3 selective agonist 2S-OMPT (10 pM), and LPAR1 / 3 / 6 selective agonist Alkyl-OMPT (10 pM) each induced a robust OSPD hyperpolarization (-20.0 ± 3.1 mV, -17.9 ±1.9 mV, and -23.6 ± 3.0 mV, respectively). The OSPD changes induced by 2S-OMPT and Alkyl-OMPT were similar to linoleoyl LPA (p=0.6 and p=0.4, respectively). Conversely, LPAR1 selective agonist UCM-05194 (10 M) and LPAR2 selective agonist DBIBB (10 pM) produced minimal OSPD hyperpolarization compared to linoleoyl LPA (-0.4 ± 0.1 mV, p=0.007 and -2.3 ± 0.7 mV, p=0.008 respectively). Cotreatment of LPAR1 / 3 / 6 selective agonist Alkyl-OMPT (10 pM) with LPAR1 / 3 selective antagonist Ki 16425 (10 pM) was performed to isolate the activity of LPAR6, which induced minimal OSPD hyperpolarization compared to linoleoyl LPA (-2.5 ± 0.6 mV, p=0.009).Together, these results suggest LPAR3 is the key LPAR isoform mediating Cl- secretion in mouse ocular surface.EXAMPLE 3:LPAR3 IS EXPRESSED IN MOUSE AND HUMAN OCULAR SURFACEAND MOUSE LACRIMAL GLAND

[0102] Immunofluorescence staining in mouse cornea and conjunctiva showed prominent expression of LPAR3 in both epithelia with no expression in the corneal stroma or the conjunctival fibrous layer (Fig. 7). LPAR3 was also heavily expressed in mouse lacrimal gland, specifically in ductal epithelial cells, which facilitate tear fluid secretion (Figs. 7 and 8) (Ref.

[0019] ).

[0103] To investigate roles of LPAR3 in a clinically relevant setting and to determine potential translational relevance of our findings, LPAR3 expression was also studied in human ocular surface by immunofluorescence staining. Similar to mouse eye, LPAR3 was heavily expressed in the epithelia of the human cornea and conjunctiva which directly face the tear film (Fig. 9).EXAMPLE 4:LPA ACTIVATES THE GQ / PLC INTRACELLULAR CA2+SIGNALING PATHWAY IN HCECS

[0104] LPAR3 expression in vitro was assessed through immunofluorescence in immortalized and primary HCECs. HCECs showed positive expression for LPAR3 in both cell types (Figs. 10 and 11).

[0105] To understand the downstream pathway of LPAR activation in HCECs, we measured intracellular Ca2+changes induced by a general LPAR agonist, linoleoyl LPA, and LPAR3 selective agonist, 2S-OMPT, using Fluo-4 NW fluorescence (Figs. 10 and 11). In immortalized HCECs, linoleoyl LPA (10 pM) elevated intracellularly Ca2+by 63% ± 6.9% which was significantly inhibited by 5 minutes of pretreatment with Ki 16425 (1 pM) and PLC inhibitor U73122 (10 pM) (pO.OOOl) (Fig. 10).

[0106] In primary HCECs, extracellular addition of linoleoyl LPA (10 pM) and 2S-OMPT (10 pM) significantly elevated intracellular Ca2+by 48.8 ± 2.8% and 41.4 ± 3.7%, respectively (Fig. 11). Pretreatment of HCECs with Ki 16425 (1 pM) for 5 minutes largely reversed those elevations, reducing Ca2+levels in LPA and 2S-OMPT groups (p<0.0001 andp=0.0009, respectively). Similarly, pretreatment with the PLC inhibitor U73122 (10 pM) for 5 minutes essentially abolished the Ca2+response (p<0.0001 and p=0.001, respectively).

[0107] Our results suggest that LPA activated the G -coupled LPAR3 in HCECs, inducing a significant intracellular Ca2+elevation through Gq- PLC signaling pathway (Fig. 12).Summary

[0108] As illustrated in Examples 1-4, the presence of LPAR3 in mouse and human ocular surface epithelia and ductal epithelial cells of mouse lacrimal gland has been confirmed (see, e.g., Figures 7-9). LPAR activation, specifically LPAR3, was also shown to trigger Cl-secretion via CaCCs in mouse ocular surface epithelia through activation of the Gq / PLC pathway (see, e.g, Figures 1-6 and 10-12). Together, these findings suggest potential therapeutic utility of LPAR3 activators as novel drug candidates for ocular surface disorders, such as DED.EMBODIMENTS

[0109] Embodiment [1] relates to a method for treating an ocular surface disorder in a subject in need thereof, the method comprising topically administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one lysophosphatidic acid receptor (LPAR) modulator.

[0110] Embodiment [2] relates to Embodiment [1], wherein the at least one LPAR modulator comprises an LPAR agonist.

[0111] Embodiment [3] relates to Embodiments [l]-[2], or a combination thereof, wherein the at least one LPAR modulator comprises an LPAR antagonist.

[0112] Embodiment [4] relates to Embodiments [l]-[3], or any combination thereof, wherein the at least one LPAR modulator comprises an LPARi selective modulator, an LPAR2 selective modulator, an LPAR3 selective modulator, an LPAR4 selective modulator, an LPARs selective modulator, an LPARe selective modulator, or any combination thereof.

[0113] Embodiment [5] relates to Embodiments [l]-[4], or any combination thereof, wherein the at least one LPAR modulator comprises an LPARi selective agonist, an LPAR2 selective agonist, an LPAR3 selective agonist, an LPAR4 selective agonist, an LPARs selective agonist, an LPARe selective agonist, or any combination thereof.

[0114] Embodiment [6] relates to Embodiments [l]-[5], or any combination thereof, wherein the at least one LPAR modulator comprises an LPARi selective antagonist, an LPAR2selective antagonist, an LPAR3 selective antagonist, an LPAR4 selective antagonist, an LPARs selective antagonist, an LPARe selective antagonist, or any combination thereof.

[0115] Embodiment [7] relates to Embodiments [l]-[6], or any combination thereof, wherein the at least one LPAR modulator comprises an LPARi selective agonist, an LPAR2 selective agonist, an LPAR3 selective agonist, an LPAR1 / 3 selective antagonist, an LPAR1 / 3 / 6 selective agonist, or any combination thereof.

[0116] Embodiment [8] relates to Embodiments [l]-[7], or any combination thereof, wherein the at least one LPAR modulator comprises linoleoyl LPA, Alkyl-OMPT, Ki 16425, 2S-OMPT, UCM-05194, DBIBB, or any combination thereof.

[0117] Embodiment [9] relates to Embodiments [l]-[8], or any combination thereof, wherein the pharmaceutical composition is administered to an eye of the subject.

[0118] Embodiment

[0010] relates to Embodiments [l]-[9], or any combination thereof, wherein the pharmaceutical composition is administered to the cornea or conjunctiva of the subject.

[0119] Embodiment

[0011] relates to Embodiments [l]-

[0010] , or any combination thereof, wherein the method comprises administering from about 2 nanomoles to about 200 nanomoles of the at least one LPAR modulator to the eye of the subject.

[0120] Embodiment

[0012] relates to Embodiments [1]-[H], or any combination thereof wherein the therapeutically effective amount of the pharmaceutical composition provides a concentration of about 100 nM to about 1,000 mM of the at least one LPAR modulator in the eye of the subject after a period of about 1-3 hours following administration.

[0121] Embodiment

[0013] relates to Embodiments [1]-

[0012] , or any combination thereof, wherein the pharmaceutical composition further comprises at least one additional therapeutic agent.

[0122] Embodiment

[0014] relates to Embodiments [1]-

[0013] , or any combination thereof, wherein the method further comprises co-administering at least one additional therapeutic agent to the subject.

[0123] Embodiment

[0015] relates to Embodiments

[0013] -

[0014] , or any combination thereof wherein the at least one additional therapeutic agent is administered in an amount effective to enhance a therapeutic effect of the at least one LPAR modulator.

[0124] Embodiment

[0016] relates to Embodiments

[0013] -

[0015] , or any combination thereof, wherein the at least one additional therapeutic agent comprises a vasoconstricting agent, an epithelial sodium channel inhibitor, a lymphocyte function-associated antigen-1 antagonist, ananti-inflammatory agent, a cholinergic agonist, a steroid, an antibiotic, or any combination thereof.

[0125] Embodiment

[0017] relates to Embodiments

[0013] -

[0015] , or any combination thereof, wherein the at least one additional therapeutic agent comprises a steroid selected from the group consisting of prednisolone phosphate, prednisolone acetate, fluoromethoIone acetate, dexamethasone, and any combination thereof.

[0126] Embodiment

[0018] relates to Embodiments [1]-

[0017] , or any combination thereof, wherein the ocular surface disorder is a neuropathic ocular pain, a dry eye disease (DED), an ocular inflammation, an ocular wound, or any combination thereof.

[0127] Embodiment

[0019] relates to Embodiments [1]-

[0018] , or any combination thereof, wherein the ocular surface disorder is a dry eye disease (DED).

[0128] Embodiment

[0020] relates to Embodiments [1]-

[0019] , or any combination thereof, wherein the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier.REFERENCES

[0129] The following references, referred to above, are incorporated herein by reference.

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[0131] [2] Craig, J. P., K. K. Nichols, E. K. Akpek, B. Caffery, H. S. Dua, C. K. Joo, Z. Liu, J. D. Nelson, J. J. Nichols, K. Tsubota, and F. Stapleton “TFOS DEWS II Definition and Classification Report,” Ocul. Surf., 2017, 15(3), 276-283 (https: / / pubmed.ncbi.nlm.nih.gov / 28736335 / ).

[0132] [3] Chan, C., S. Ziai, V. Myageri, J. G. Burns, and C. L. Prokopich “Economic burden and loss of quality of life from dry eye disease in Canada,” BMJ Open Ophth, 2021, 6, e000709 (https: / / bmjophth.bmj.eom / content / 6 / l / e000709).

[0133] [4] Pasricha, N. D., E. S. Lindgren, R. Yan, Y. M. Kuo, M. Chan, A. S.Verkman, T. Chu, P. Yottasan, L. de Souza Goncalves, and O. Cil “Calcium-sensing receptor (CaSR) modulates ocular surface chloride transport and its inhibition promotes ocular surface hydration,” Ocul Surf, 2024, 34: 30-37 (https: / / doi.Org / 10.1016 / j.jtos.2024.06.002).

[0134] [5] Levin, M. H., and A. S. Verkman “Aquaporins and CFTR in ocular epithelial fluid transport,” JMembr Biol, 2006, 210(2): 105-15 (https: / / doi.org / 10.1007 / s00232-005-0849-1).

[0135] [6] Lindgren, Ethan S., Onur Cil, Alan S. Verkman, and Neel D. Pasricha “Ocular Surface Ion Transport and Dry Eye Disease.” Current Ophthalmology Reports, 2022, 10(4): 188-197 (https: / / doi.org / 10.1007 / s40135-022-00295-3).

[0136] [7] Geraldo, Luiz Henrique Medeiros, Tania Cristina Leite de Sampaio Spohr, Rackele Ferreira do Amaral, Anna Carolina Carvalho da Fonseca, Celina Garcia, Fabio de Almeida Mendes, Catarina Freitas, Marcos Fabio dosSantos, and Flavia Regina Souza Lima “Role of lysophosphatidic acid and its receptors in health and disease: novel therapeutic strategies.” Signal Transduction and Targeted Therapy, 2021, 6(1): 45(https : / / doi. org / 10.1038 / s41392-020-00367-5).

[0137] [8] Murakami, M., A. Shiraishi, K. Tabata, and N. Fujita “Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine- 1 -phosphate and lysophosphatidic acid receptor.” Biochem Biophys Res Commun, 2008, 371 (4): 707-12 (https: / / doi.org / 10.1016 / j.bbrc.2008.04.145).

[0138] [9] Tabata, K., K. Baba, A. Shiraishi, M. Ito, and N. Fujita “The orphan GPCR GPR87 was deorphanized and shown to be a lysophosphatidic acid receptor.” Biochem Biophys Res Commun, 2007, 363 (3): 861-6 (https: / / doi.Org / 10.1016 / j.bbrc.2007.09.063).

[0139]

[0010] Yung, Y. C., N. C. Stoddard, and J. Chun “LPA receptor signaling: pharmacology, physiology, and pathophysiology.” J Lipid Res, 2014, 55 (7): 1192-214 (https: / / doi.org / 10.1194 / jlr.R046458).

[0140]

[0011] Watsky, M.A. “Lysophosphatic acid, serum, and hyposmolarity activate Cl- currents in corneal keratocytes,” Am. J. Physiol., 1995, 269(6 ptl), C1385-98 (https: / / pubmed.ncbi.nlm.nih.gov / 8572167).

[0141]

[0012] Liliom, K., Z. Guan, J. L. Tseng, D. M. Desiderio, G. Tigyi, and M. A. Watsky “Growth factor-like phospholipids generated after corneal injury,” Am J. Physiol. , 1998, 274(4), C1065-74 (https: / / pubmed.ncbi.nlm.nih.gov / 9575804).

[0142]

[0013] Wang, D. A., H. Du, J. H. Jaggar, D. N. Brindley, G. J. Tigyi, and M. A. Watsky “Injury-elicited differential transcriptional regulation of phospholipid growth factor receptors in the cornea.” Am J Physiol Cell Physiol, 2002, 283(6): C1646-54 (https: / / doi.org / 10.1152 / ajpcell.00323.2002).

[0143]

[0014] Lau, O. C., C. Samarawickrama, and S. E. Skalicky “P2Y2 receptor agonists for the treatment of dry eye disease: a review,” 2014, 8, 327-334 (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3915022).

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[0015] Philip L. Gould, “Salt Selection for Basic Drugs,” hit. J. Pharm., 33, 201- 217, 1986 (https: / / doi.org / 10.1016 / 0378-5173(86)90055-4).

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[0016] Wolf et al. “The Human Eye Transcriptome Atlas: A searchable comparative transcriptome database for healthy and diseased human eye tissue,” Genomics, 2022, 114(2): 110286 (https: / / pubmed.ncbi.nlm.nih.gov / 35124170).

[0146]

[0017] Pasricha, N. D., A. J. Smith, M. H. Levin, J. M. Schallhorn, and A. S. Verkman. 2020. “Ocular Surface Potential Difference Measured in Human Subjects to Study Ocular Surface Ion Transport.” Transl Vis Sci Technol, 2020, 9(11): 20 (https: / / doi.org / 10.1167 / tvst.9.11.20).

[0147]

[0018] Robertson, D. M., L. Li, S. Fisher, V. P. Pearce, J. W. Shay, W. E. Wright, H. D. Cavanagh, and J. V. Jester. 2005. “Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line.” Invest Ophthalmol Vis Sci, 2005, 46(2): 470-8 (https: / / doi.org / 10.1167 / iovs.04-0528).

[0148]

[0019] Galletti, Jeremias G., and Cintia S. de Paiva “The ocular surface immune system through the eyes of aging.” The Ocular Surface, 2021, 20: 139-162 (https: / / d0i.0rg / https: / / doi.org / 10.1016 / j.jtos.2021.02.007).

[0149]

[0020] Solomon, G. M., I. Bronsveld, K. Hayes, M. Wilschanski, P. Melotti, S. M. Rowe, and I. Sermet-Gaudelus “Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD).” J Vis Exp 2018, (139) (https: / / doi.org / 10.3791 / 57006).

[0150] This application claims the benefit of priority to U.S. Provisional Application No.63 / 742,747 filed January 7, 2025, the entirety of which is incorporated by reference herein.

[0151] These and other changes can be made to the embodiments in light of the abovedetailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

CLAIMS1. A method for treating an ocular surface disorder in a subject in need thereof, the method comprising topically administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one lysophosphatidic acid receptor (LPAR) modulator.

2. The method of claim 1, wherein the at least one LPAR modulator comprises an LPAR agonist.

3. The method of claim 1 or 2, wherein the at least one LPAR modulator comprises an LPAR antagonist.

4. The method of any one of claims 1-3, wherein the at least one LPAR modulator comprises an LPARi selective modulator, an LPAR2 selective modulator, an LPAR3 selective modulator, an LPAR4 selective modulator, an LPARs selective modulator, an LPARe selective modulator, or any combination thereof.

5. The method of any one of claims 1-4, wherein the at least one LPAR modulator comprises an LPARi selective agonist, an LPAR2 selective agonist, an LPAR3 selective agonist, an LPAR4 selective agonist, an LPARs selective agonist, an LPARe selective agonist, or any combination thereof.

6. The method of any one of claims 1-5, wherein the at least one LPAR modulator comprises an LPARi selective antagonist, an LPAR2 selective antagonist, an LPAR3 selective antagonist, an LPAR4 selective antagonist, an LPARs selective antagonist, an LPARe selective antagonist, or any combination thereof.

7. The method of any one of claims 1-6, wherein the at least one LPAR modulator comprises an LPARi selective agonist, an LPAR2 selective agonist, an LPAR3 selective agonist, an LPAR1 / 3 selective antagonist, an LPAR1 / 3 / 6 selective agonist, or any combination thereof.

8. The method of any one of claims 1-7, wherein the at least one LPAR modulator comprises linoleoyl LPA, Alkyl-OMPT, Ki 16425, 2S-OMPT, UCM-05194, DBIBB, or any combination thereof.

9. The method of any one of claims 1-8, wherein the pharmaceutical composition is administered to an eye of the subject.

10. The method of any one of claims 1-9, wherein the pharmaceutical composition is administered to the cornea or conjunctiva of the subject.

11. The method of any one of claims 1-10, comprising administering from about 2 nanomoles to about 200 nanomoles of the at least one LPAR modulator to the eye of the subject.

12. The method of any one of claims 1-11, wherein the therapeutically effective amount of the pharmaceutical composition provides a concentration of about 100 nM to about 1,000 mM of the at least one LPAR modulator in the eye of the subject after a period of about 1-3 hours following administration.

13. The method of any one of claims 1-12, wherein the pharmaceutical composition further comprises at least one additional therapeutic agent.

14. The method of any one of claims 1-12, further comprising co-administering at least one additional therapeutic agent to the subject.

15. The method of claim 13 or 14, wherein the at least one additional therapeutic agent is administered in an amount effective to enhance a therapeutic effect of the at least one LPAR modulator.

16. The method of at least one of claims 13-15, wherein the at least one additional therapeutic agent comprises a vasoconstricting agent, an epithelial sodium channel inhibitor, a lymphocyte function-associated antigen- 1 antagonist, an anti-inflammatory agent, a cholinergic agonist, a steroid, an antibiotic, or any combination thereof.

17. The method of at least one of claims 13-15, wherein the at least one additional therapeutic agent comprises a steroid selected from the group consisting of prednisolone phosphate, prednisolone acetate, fluorometholone acetate, dexamethasone, and any combination thereof.

18. The method of any one of claims 1-17, wherein the ocular surface disorder is a neuropathic ocular pain, a dry eye disease (DED), an ocular inflammation, an ocular wound, or any combination thereof.

19. The method of any one of claims 1-18, wherein the ocular surface disorder is a dry eye disease (DED).

20. The method of any one of claims 1-19, wherein the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable excipient or a pharmaceutically acceptable carrier.

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