METHODS FOR DETECTING, TREATING, AND PREVENTING GPR68 -MEDIATED OCULAR DISEASES, DISORDERS, AND CONDITIONS

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional Application Serial No. 63/203,212, filed July 13, 2021, which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, or drawings.

FIELD OF THE INVENTION

The present invention concerns the detection of ocular disorders based on the presence and/or activity of ovarian cancer G protein-coupled receptor 1 (OGRI), also referred to as GPR68, and methods for treating and preventing GPR68-mediated ocular disorders by modulating GPR68 in ocular tissues and fluids.

BACKGROUND OF THE INVENTION

Ovarian cancer G protein-coupled receptor 1 (OGR1), also referred to GPR68, is a G protein-coupled receptor that maps to chromosome 14 and was first described 1996[1], GPR68 is the receptor for sphingosylphosphorylcholine [2], but is also a sentinel for proton concentration, enabling cells to respond to the surrounding pH [3, 4], It has been shown to be involved in various processes in tumor biology, including tumorigenesis, tumor growth, and metastasis [5], The activation of GPR68 leads to the modification of various cell functions and is associated with the regulation of inflammation and fibrosis. However, there is currently no evidence of its involvement in ocular pathology or physiology; therefore, it is not currently a target of therapy or treatment for the eye.

The lack of interest in GPR68 as a target of therapy or treatment of ocular disorders may be at least partially explained by the fact that proton- sensitive receptors are activated via a decrease in pH or an acidic environment; there is no reason to expect any importance of the receptor in the eye because ocular tissues and fluids have a higher pH and a good buffer function. One publication investigated the expression of some GPRs, including GPR68, in the context of retinal degeneration, but no increase of retinal GPR68 expression was observed in mouse models of inherited and induced retinal degeneration, thus revealing no evidence for participation of GPR68 in the pathophysiology of the disease [6], GPR68 is still currently referred to as an “orphan” receptor; that is, belongs to the large number of known receptors for which specific endogenous ligands are largely unknown [7] and is still considered GPR68 “pharmacological dark matter” [7, 8],

BRIEF SUMMARY OF THE INVENTION

To the inventor’s knowledge, there are no published models of ocular physiology or pathophysiology implicating ovarian cancer G protein-coupled receptor 1 (OGR1 or GPR68) as an important protein, ft is against this backdrop that the inventor proposes that GPR68 plays an important role for normal ocular physiology and in various diseases and disorders of the eye.

The inventor has detected GRP68 in some ocular tissues, as shown in Figures 1 A, IB, 2, and 3, using a GPR68 recombinant rabbit monoclonal antibody (16H23L16), Invitrogen®, Thermo Fisher Scientific, Waltham, MA USA). This is the first observation of its kind and may change our model of ocular (patho-) physiology. This detection in ocular structures suggests a potential influence of important cell functions and regulatory mechanisms in the eye and ocular structures. Against current models that completely disregard or ignore GPR68 as a protein of importance, the inventor proposes its presence is consequential in ocular physiology and ocular pathology. Without being limited by theory of a proposed mechanism of action, the inventor proposes that regulation of GPR68’s activity (e.g., modulating an increase or decrease in GPR68 activity, or maintaining a desired level of GPR68 activity) can be an important mechanism to maintain or reconstitute tissue homeostasis in ocular tissues, and may contribute in the regulation of inflammation and fibrosis.

The present invention concerns the specific modulation (increase or decrease) of GPR68 expression and/or function in the eye (e.g., ocular tissues, at the ocular surface, and/or in ocular fluids), thereby treating, preventing, or delaying the onset of an ocular disease, ocular disorder, or ocular condition that is mediated by GPR68. Thus, for example, the method of the invention may be used to reduce or delay the onset of irritation, inflammation, scar formation, and/or an immune response, or by enhancing the effects of GPR68 (such as neuroprotection).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

Figures 1A and IB. GPR68 in human corneal epithelium (bright red staining). Figure 1 A shows GPR68 in human corneal epithelium of a melanoma patient, at approximately 400x magnification. GPR68-specific staining was observed in all samples throughout the epithelial layers of the corneal epithelium. The most prominent staining was in the area of the wing cells (yellow arrow to the right in Figure 1A). However, there was also intense staining for GPR68 observed at the basement membrane level (white arrow to the left in Figure 1A). In conjunctival tissues, GPR68 was detected in a similar pattern as in the corneal epithelium such that even the superficial cell layers occasionally stained positive (yellow arrow in Figure IB).

Figure 2. GPR68 in mouse retina (bright red staining), at approximately lOOx magnification.

Figure 3. GPR68 in human optic nerve (bright red staining) in a melanoma patient, at approximately 400x magnification.

Figure 4. Specific staining for GPR68 was also found in the basal layers of the conjunctiva (yellow and blue arrows of Figure 4) of the fornix but decreased toward the conjunctival of the lids (Figure 4).

Figure 5. GPR68 in human lacrimal gland (bright red staining). The distribution and intensity of staining over the field of observation was uneven (see arrow).

Figure 6. GPR68 in human lacrimal gland (bright red staining). In some of the acini, there was intense staining for GPR68 (see arrows).

Figure 7. GPR68 in human lacrimal gland (bright red staining). The most profound staining was detected in the epithelium lining the excretory ducts of the lacrimal gland (see arrow).

Figure 8. GPR68 in human lacrimal gland (bright red staining). The epithelium lining the lumen of even larger ducts of the lacrimal gland stained positive for GPR68 (see arrow). DETAILED DESCRIPTION OF THE INVENTION

An aspect of the invention includes a method for treating, preventing, or delaying the onset of an ocular disease, ocular disorder, or ocular condition by modulating the expression and/or activity of GPR68 in the eye of a human or non-human animal subject. In some embodiments, the ocular disease, disorder, or condition is selected from among an irritation, inflammation, immune response, or combination of two or more of the foregoing, in ocular tissues, to/at the ocular surface and/or in ocular fluids. In some embodiments, GPR68 expression and/or activity in the eye is modulated by administering one or more modulators (one or more inducers or inhibitors) of GPR68 expression and/or GPR68 expression activity to the eye of the human or non-human animal subject. Some embodiments disclosed herein provide modulators of GPR68 expression and/or activity in the manufacture of a medicament for the treatment of an ocular disease, ocular disorder, or ocular condition in a human or nonhuman animal subject in need thereof.

For example, GPR68 modulators may be topically administered by drops, or be administered by direct injection into tissues or cavities of the eye or its adnexa, or be systemically administered to the human or non-human animal subject. In some embodiments, the GPR68 modulator is administered to the ocular surface, anterior chamber fluid, vitreous fluid/body, or any other space in the eye. Optionally, the GPR68 modulator may be administered in a composition that further includes a pharmaceutically acceptable carrier or diluent. The GPR68 modulator may be the only active ingredient in the composition, or the composition may include one or more additional active ingredients.

Potent GPR68 modulators exist such as allosteric derivatives of its activator, ogerin (Ml· : C ₁₇ H ₁₇ N ₅ O, MW: 307.35g/mol, InChIKey: MDGIEDNDSFMSLP-UHFFFAOYSA-N, IUPAC Name: [2-[4~ammo-6~(benzylamino)-1,3,5-triazin-2-y1]phenyl]methano l, source pubchem.ncbi.nlm.nih.gov/#query=Ogerin) [9], of which some ogerin-based allosteric derivatives have shown promising results [8, 10] and divalent metal ions [11], which are incorporated herein by reference in their entireties. Such modulators have been shown to reduce GPR68-mediated intestinal inflammation in mice, and can be used in the invention to modulate GPR68 in the eye. The invention also includes the activation or induction of GPR68 receptors in the eye in order to maximize the positive effects that have been reported in other tissues such as neuroprotection [12]

In some embodiments, in the ocular tissue or fluid of the subject to which a GPR68 modulator is administered, there is no activation of GPR68 or inflammation present, and the GPR68 modulator is intended to prevent or delay onset or recurrence of such activation. This can be prior to an imminent anticipated stimulus for the GPR68 such as mechanical forces or alterations of the pH in the tissues of the eye or the ocular surface [13], such as enhanced friction, attrition, or events such as trauma or surgery.

This can be after or during a presumed or known stimulus for the GPR68 activation such as mechanical forces or alterations of the pH in the tissues of the eye or the ocular surface [13], such as enhanced friction, attrition or events such during and after trauma or surgery [14] An “ocular condition” includes any event that activates GPR68 or elevates GPR68 activity above a normal, healthy state, or inhibits GPR68 or decreases GPR68 below a normal, healthy state, and is inclusive of ocular diseases and ocular disorders that are mediated by GPR68 activity.

In some embodiments, the ocular tissue or fluid of the subject to which inhibitor or modulator of GPR68 is administered, no activation of GPR68 or inflammation needs to be present as the administration is intended to enhance or regulate such activation. Amongst the indications such modulation is the imminent present ischemic condition of the eye compartments such as glaucoma with elevated pressure or circulatory insufficiencies of optical nerve perfusion (such as papillary edema). Similarly, in conditions that include local or generalized edema of the retinal layers inside (macula, posterior pole) or outside (peripheral retina) of the central region of the retina.

The inhibitor or modulator of GPR68 may be administered to the ocular surface, in ocular tissues, to/at the ocular surface and/or in ocular fluids the ocular surface of one or both eyes of the subject by any topical administration method, even and specifically injections or lavages. For example, the inhibitor or modulator of GPR68 may be also administered as one or more drops from a dropper.

The GPR68 modulator may be administered prophylactically, before the ocular disease, disorder, or condition exists, to reduce the severity of the ocular disease, disorder, or condition and/or delay its onset; or the GPR68 modulator may be administered therapeutically, after the ocular disease, disorder, or condition exists, in order to reduce the severity of the ocular disease, disorder, or condition. In some embodiments, onset of the ocular disease, disorder, or condition is delayed indefinitely (i.e., prevented). In some embodiments, one or more symptoms of the ocular disease, disorder, or condition are alleviated or eliminated. In some embodiments, all symptoms of the ocular disease, disorder, or condition are alleviated or eliminated.

In cases in which the ocular disease, disorder, or condition exists at the time of the administration of the GPR68 modulator is administered therapeutically, optionally, the method further comprises the step of identifying the subject as having the ocular disease, disorder, or condition prior to administering the GPR68 modulator.

The ocular disease, disorder, or condition may be caused by various stimuli - external, internal, or both In some embodiments, the ocular disease, disorder, or condition is caused by an external stimulus resulting in a disruption of the smoothness and/or integrity of the ocular surface ( e.g ., ocular surgery, non-surgical trauma, contact lens wearing, microbial infection, allergen, hapten, toxic agent, or irritative substance). In some embodiments, the ocular disease, disorder, or condition is caused by an internal stimulus (e.g., rheumatic disease, epithelial-mesenchymal transition (EMT), or autoimmune disease).

The ocular disease, disorder, or condition may be caused by a wound of the eye. In some embodiments, the wound is caused by physical trauma, chemical trauma, or radiation (radiation injury). In some embodiments, the wound is caused by an ocular surgery. Examples of ocular surgeries include but are not limited to natural or artificial corneal transplantation, corneal implantation (e.g, intracorneal rings (ICRs), and keratoprosthesis), glaucoma surgery, cataract surgery (e.g, phacoemulsification, extrapsular cataract surgery, or intracapsular surgery), refractive surgery (e.g, radial keratotomy or refractive corneal incision), retinal surgery, squint (strabismus) surgery, corrective laser eye surgery (e.g, laser- assisted in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK, LASER, LASIK, fs-Lasik)), and cross-linking surgery.

Without being limited by theory, the inventor proposes that GPR68’s function(s) in ocular tissues include, but are not limited to:

• Optic nerve and neuronal tissues such as retina in the eye: Prevent damage from ischemic lesion such as ocular pressure and circulation deficits (venous or arterial vessel occlusions);

• Mediator of effects caused by enhanced friction and attrition; • Mediator of ocular pain during and after surgery;

• Mediator of ocular pain and discomfort due to lubrication issues such as dry eye diseases;

• Regulation and mediation of pain and inflammation in retinal tissues in any kind of disease;

• Decreasing undesired response (such as, but not limited to, inflammation) to increased fluid flow during any kind of ocular surgery, both in the anterior and posterior part of the eye; and

• Receptor being sensitive to hypoxic conditions.

Based on the proposed function(s) of GPR68 in ocular tissues, several ocular diseases, disorders, and conditions may be treated, prevented, or onset delayed, by inhibiting or downregulating the receptor in the eye. These include, but are not limited to:

Postoperative inflammation following surgery;

Ocular surface inflammation as result of lubrication deficits (dry eye);

Edema of retinal layers and localized swellings such as in cystoid macular edema; Preventing scarformation as result of inflammation may it be short lasting or long lasting, even recurrent inflammation;

Enhancing tissue resilience to the effects of manipulation during surgery such as pressure, fluid flow and direct mechanical forces a well as radiation;

Enhanced tolerance to acidification of the tissue of fluids as response to stimuli, infection or inflammation by reducing the release of PGD E2 as pain mediator [15]; Counteracting enhanced endoplasmatic reticulum stress caused by pH changes and associated activation of GPR68 [16];

Decrease sensations of pain and discomfort in dry eye disease as G-protein-coupled receptors could sensitize TRPV1 [17], and pH induced activation of the GPR68 could hence also affect TRPVl. Re-establishing the pH in dry eye disease could hence be a very effective way to stabilize proinflammatory proteins present in corneal epithelium and reduce pain;

Pretreatment with benzodiazepines (such as sulfazepam or lorazeparn) prior to any kind of treatment or surgery could lead to downregu!ation of GPR-68 receptor desensitization [18] and hence decrease possible reactions to altered pH or mechanical stimulation; • Similarly like in the pathophysiology of asthma [19] inhibition of GPR-68 on dendritic cells in the cornea may lead to decreased goblet cell metaplasia, inhibition of Th2 cytokine and IgE production and be part of the treatment of allergic keratoconjunctivitis and even keratoconus;

• Treatment with oxygen or increased availability of oxygen could counteract the known hypoxia induced expression of GPR68 [20], Hypoxia is known to be one of the main reasons for ocular neovascularization in all ocular tissues both in the cornea such as during contact lens wear leading to formation of a pannus as well as in diabetic retinopathy, vessel occlusions such as CVO, BRVO and others; and

• Similar inhibition of the GPR68 in these conditions or prior to their occurrence (hypoxia) could help to reduce the GPR68 controlled or enhanced effects in the ocular tissues foreseeably suffering oxygen deprivation.

Based on the proposed function(s), other ocular disorders/conditions that can be treated, prevented, or onset delayed by activating or inducing GPR68 in the eye:

• Conditions in which an enhanced scarformation or inflammation is not desired for example suppression of IL-22 and IL-1Q secretion by T cells, activation of GPR68 with the lorazepam derivative ogerin can be used having with no effect on IL-17 [21];

• In conditions in which neuroprotective effects such as is corneal neuropathy and disease of the optic nerve activation of GPR-68 could enhance the recently suggested neurotrophic effects [12, 22];

• In conditions with poor attachment of cells to the ECM such as in chronic corneal erosions and ulcers with problems to reepithelialize GPR68 activation could be desired as GPR-68 cell adhesion to the extracellular matrix [23];

• treatment with lenalidomide could upregulate GPR68 expression levels [24];

• lenalidomide mediates degradation of IKZF1, leading to derepression of GPR68[24]; and

• inhibiting foreseeable activation of GPR68 due to hypoxia [20] as during vascular failure or malfunction such as stenosis all sorts like in diabetic retinopathy, fundus hypertonicus (i.e. vessel changes as result of high blood pressure), impaired optic nerve perfusion, even and especially as due to elevated intraocular pressure (glaucoma), impaired retinal perfusion in the presence of glaucoma as well as during enhanced intraocular pressure during and after operations such as retinal operations with expanding gas such as sulfur hexafluoride (SF6) and perfluoroethane (C2F6) used for intraocular gas tamponade [25, 26] inhibition of foreseeable decrease of blood flow in the eye, blood pressure and/or a combination of all elevated intraocular pressure during abdominal and any other surgery with head down position (Trendelenburg position) which is known to cause elevated pressure in the eye [27-29]

As the nucleic acid sequence is known, also gene therapy could potentially be used to up-regulate the receptor, as well as could potentially nucleic acid-based inhibitors, such as antisense or interfering RNA or gene editing, could be used to down-regulate the receptor.

With the availability of a specific antibody [30] the production of interfering antibodies to the receptor is an option.

Most recently, lenalidomide, an immunomodulatory drug, interacts with cereblon (CRBN), a component of the CRL4CRBN E3 ubiquitin ligase complex, leading to ubiquitination and subsequent degradation of substrates, such as transcription factor Ikaros (Ikaros family zinc finger 1, IKZF1). IKZF1 functions as a transcription repressor for GPR68 [24] and could hence be used to repress GPR68 effects.

Inhibitors of the NF-kappaB pathway can be administered to a subject to counteract upregulation of GPR-68 as result of TNF mediated inflammation [31] As TNF is a major actor/mediator in ocular inflammation specific inhibitors target its activity [32] but have side effects [33] Examples of NF-kappaB inhibitors that may be used in the invention include those disclosed in Gupta SC et al ., “Inhibiting NF-KB Activation by Small Molecules As a Therapeutic Strategy”, Biochim Biophys Acta. 2010 Oct-Dec; 1799(10-12): 775-787, particularly in Table 1, which is incorporated herein by reference in its entirety, and in Ramadass V et al., “Small Molecule NF-KB Pathway Inhibitors in Clinic”, Int J Mol Sci. 2020 Jul; 21(14): 5164, particularly in Tables 1-5, which are incorporated herein by reference in their entirety. In some embodiments, the NF-kappaB inhibitor is selected from among an inhibitor of a cell membrane receptor in the NF-kappaB pathway, or inhibitor of a cellular receptor adaptor protein in the NF-kappaB pathway, or inhibitor targeting the IKK complex of the NF-kappaB pathway, or an inhibitor targeting the ubiquitin-proteasome system (UPS) in the NF-kappaB pathway, or an inhibitor targeting nuclear translocation, DNA binding, and/or transcriptional activation of NF-kappaB.

Optionally, the GPR68 modulator is administered to the subject in a composition that further includes one or more bioactive agents ( e.g ., a hydrophobic active ingredient), or the GPR68 modulator can be administered to the subject in combination with one or more bioactive agents in separate compositions, simultaneously or consecutively in any order.

The bioactive agent may be any substance that has an effect on the human or non human animal subject when administered. The bioactive agent may be any class of substance such as a small molecule ( e.g ., drug) or biologic (e.g, polypeptide, immunoglobulin, nucleic acid), may be natural products or artificially produced, and may act by any mechanism such as mechanical, pharmacological, immunological, or metabolic. Examples of classes of bioactive agents include substances that modify the pressure of the eye (e.g, enzyme inhibitors) and anti-angiogenic agents. Some specific examples of bioactive agents include tacrolimus, plasmin activator, anti-plasmin, hyaluronic acid, and cyclosporine A. In some embodiments, the bioactive agent is a steroid to treat inflammation or antibiotic to treat or prevent eye infection; glaucoma drug such as prostaglandin analog, beta blocker, alpha agonist, or carbonic anhydrase inhibitor; agent for allergy eye relief such as histamine antagonist or non-steroidal anti-inflammatory drug; or mydriatic agent.

In some embodiments, the GPR68 modulator is administered to the subject in combination with one or more agents, together in a single composition, or separately in separate compositions administered simultaneously or consecutively in any order, wherein the one or more agents influence lubrication of the ocular surface such as, but not limited to, hyaluronic acid. In some embodiments, the hyaluronic acid is high molecular weight hyaluronic acid.

In some embodiments, the composition comprising the GPR68 modulator contains no other bioactive agent (e.g, no hydrophobic active ingredient).

In some circumstances, it may be desirable to include one or more preservatives or detergents within the composition. Often, such preservatives and detergents are irritative or damaging to the eye. Such side effects may need the additional application of substances that alleviate such side effects.

In some embodiments, the GPR68 modulator is administered to the subject before, during, or after administration of another composition comprising a bioactive agent to the subject. The other composition may be in any form and administered by any route (e.g, local or systemic). In some embodiments, the other composition is administered to the eye, e.g, topically or by injection.

In some embodiments, the other composition is topically administered to the ocular surface. In some embodiments, in addition to one or more bioactive agents, the other composition that is topically administered to the ocular surface includes a preservative or detergent or oxidative preservative that is irritative or damaging to the eye.

In some embodiments, the preservative or detergent is one that kills susceptible microbial cells by disrupting the lipid structure of the microbial cell membrane, thereby increasing microbial cell membrane permeability.

In some embodiments, the preservative or detergent is one that causes damage to the corneal endothelium or other cell structures in the eye or its adnexa.

In some embodiments, the preservative or detergent is selected from the group consisting of quaternary ammonium preservative ( e.g ., benzalkonium chloride (BAR)), chlorobutanol, edetate disodium (EDTA), polyquatemarium-1 (e.g., Polyquad™ preservative), stabilized oxidizing agent (e.g, stabilized oxychloro complex (e.g, Purite™ preservative)), ionic-buffered preservative (e.g, sofZia™ preservative), polyhexamethylene biguanide (PHMB), sodium perborate (e.g, GenAqua™ preservative), and sorbate.

In some embodiments, the composition comprising the GPR68 modulator may contain various additives in order to enhance the desired effect by simultaneously preconditioning tissue or surfaces exposed to the GPR68 modulator.

In some embodiments, the composition comprising a GPR68 modulator includes a preservative. In other embodiments, the composition does not include a preservative (i.e., the fluid is preservative-free).

In some embodiments, the composition comprising a GPR68 modulator further includes one or more glycosaminoglycans (GAGs); electrolyte (e.g, sodium chloride); buffer (e.g, potassium buffer); or a combination of two or more of the foregoing.

The subject may or may not have dry eye syndrome (the aqueous tear deficiency type or qualitative dry eye type) at the time the GPR68 modulator is administered to the eye of the subject. In some embodiments, the eye of the subject to which the GPR68 modulator is administered does not have aqueous tear deficiency (ATD) at the time of administering the fluid (i.e., in the absence of ATD). In some embodiments, the eye of the subject to which the GPR68 modulator is administered does not have qualitative dry eye at the time of administering the GPR68 modulator (i.e., in the absence of qualitative dry eye). In some embodiments, the eye of the subject to which the GPR68 modulator is administered does not have dry eye syndrome at the time of administering the GPR68 modulator (i.e., in the absence of aqueous tear deficiency or qualitative dry eye). The GPR68 modulator may or may not be used in conjunction with a bandage contact lens depending on the interaction of the GPR68 modulator and the contact lens material and the condition of the ocular surface of the patient.

The GPR68 modulator can be a molecule that changes the activity or the expression of the GPR68 receptor. The GPR68 modulator can be any pH-altering chemical component such as a buffer.

Any GPR68 modulator can be combined with any other GPR68 modulator and administered to a subject, whether administered within the same composition, or administered in separate compositions.

The term “GPR68 polypeptide” is used to refer collectively to all naturally occurring isoforms of a GPR68 protein of any species, or a variant thereof. For example, a “GPR68 polypeptide” can be a human GPR68 polypeptide, a mouse GPR68 polypeptide, a rat GPR68 polypeptide, or a variant thereof. In some embodiments, a GPR68 polypeptide can be a protein having the amino acid sequence of SEQ ID NO: 1, or a variant thereof.

A GPR68 variant can differ from a naturally occurring GPR68 protein by, for example, a modification (e.g, substitution, deletion, or insertion) of one or more amino acid residues in the naturally occurring GPR68 protein, but retains the biological activities of GPR68, e.g., pH-sensing activity, endothelial shear stress sensing activity, or mechano- sensing activity, or a combination of two or more of its activities. The GPR68 variant can have one or more conservative or nonconservative amino acid substitution.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g, lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains(e.g ^· ., threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine).

In some embodiments, the GPR68 variant includes one or more mutations (e.g., substitutions (e.g, conservative substitutions or substitutions), insertions, or deletions) of non-essential amino acids relative to a naturally occurring GPR68 protein. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of GPR68 protein without abolishing or more preferably, without substantially altering a biological activity, such as pH sensor activity, endothelial shear stress sensor activity, mechano-sensor activity, or a combination of two or more of its activities,, whereas changing an “essential” amino acid residue results in a substantial loss of biological activity,

A GPR68 variant may have at least one, two, three, or four, and no more than 10, 9, 8, 7, 6, or 5 mutations (e.g, substitutions (e.g, conservative substitutions or substitutions of non-essential amino acids), insertions, or deletions) relative to a naturally occurring GPR68 protein. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect biological properties, such as fusogenic activity, can be predicted, e.g., by evaluating whether the mutation is conservative or by an activity assay.

In some embodiments, a GPR68 variant can have about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a naturally occurring GPR68 protein. For example, a human GPR68 variant can have about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to SEQ ID NO: 1. A mouse GPR68 variant can have about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the dominant mouse GPR68 protein sequence (NCBI accession no. NP_001171145).

In some embodiments, the GPR68 modulator decreases or increases the level of a GPR68 gene product. It will be understood by one skilled in the art, based upon the disclosure provided herein, that a decrease or increase in the level of a GPR68 gene product encompasses the decrease or increase in the expression, including DNA transcription, mRNA translation, mRNA stability, protein stability or any ail of their combinations. The skilled artisan will also appreciate, once armed with the teachings of the present invention, that a decrease or increase in the level of a GPR68 gene product includes a decrease or increase in the activity of GPR68, e.g., pH sensor activity, endothelial shear stress sensor activity, mechano-sensor activity, or a combination of two or more of its activities. Thus, decrease or increase in the level or activity of GPR68 includes, but is not limited to, decreasing or increasing the amount of polypeptide of GPR68, and decreasing or increasing transcription, translation, or both, of a nucleic acid encoding GPR68; and it also includes decreasing or increasing any activity of GPR68, e.g., pH-sensing activity, endothelial shear stress sensing activity, mechano-sensing activity, or a combination of two or more of its activities.

In some embodiments, the modulator of a GPR68 gene product, e.g., an agonist or antagonist of a GPR68 gene product, may reduce flow-mediated dilation (FMD) response and/or flow-mediated outward remodeling (FMR) of small-diameter arteries, also known as resistance arteries, in a subject (see International Application Publication No. W02019/150309 (Hammack et al., “Modulators of GPR68 and Uses Thereof for Treating and Preventing Disease”, Novartis AG and Scripps Research Institute, published August 8, 2019), For example, the modulator of a GPR68 gene product, e.g., an agonist or antagonist of a GPR68 gene product, may reduce the FMR response and/or FAIR of small-diameter arteries in a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more.

In some embodiments, the modulator of a GPR68 gene product, e.g., an agonist or antagonist of a GPR68 gene product, may reduce the systemic vascular resistance (SVR) and/or the left ventricle afterload of a subject (see International Application Publication No. W02019/150309 (Hammack et al). For example, the modulator of a GPR68 gene product, e.g., an agonist or antagonist of a GPR68 gene product, may reduce the SVR and/or the left ventricle afterload of a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more.

Examples of GPR68-targeting sequences for nucleic acid-based inhibitors are disclosed in International Application Publication No. W02019/150309 (Hammack et al), which are incorporated herein by reference in their entirety.

The GPR68 inhibitor may be a blocking or interfering antibody that binds to GPR68 (anti-GPR68 antibody). In some embodiments, the antibody is a human or humanized antibody. The antibody may be a whole antibody or an antigen-binding fragment thereof. The antibody may be monoclonal or polyclonal.

The term “antibody” as referred to herein includes whole antibodies and any antigen binding fragment ( i.e ., “antigen-binding portion”) or single chains thereof. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system ( e.g ., effector cells) and the first component (Clq) of the classical complement system.

An antibody of the invention may be a monoclonal antibody or a polyclonal antibody, and will typically be a monoclonal antibody. An antibody of the invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanised antibody or an antigen-binding portion of any thereof. For the production of both monoclonal and polyclonal antibodies, the experimental animal is typically a non-human mammal such as a goat, rabbit, rat or mouse but the antibody may also be raised in other species.

Polyclonal antibodies may be produced by routine methods such as immunisation of a suitable animal, with the antigen of interest. Blood may be subsequently removed from the animal and the IgG fraction purified.

Antibodies against GPR68 may be obtained, where immunisation of an animal is necessary, by administering the polypeptides to an animal, e.g. a non-human animal, using well-known and routine protocols, see for example Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows, camels or pigs may be immunized. However, mice, rabbits, pigs and rats are generally most suitable.

Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al ., 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp 77-96, Alan R Liss, Inc., 1985).

Antibodies of the invention may also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies by for example the methods described by Babcook, J. et al. , 1996, Proc. Natl. Acad. Sci. USA 93(15): 7843- 78481; WO92/02551; W02004/051268 and W02004/106377.

The antibodies of the present invention can also be generated using various phage display methods known in the art and include those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177- 186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

Fully human antibodies are those antibodies in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, but not necessarily from the same antibody. Examples of fully human antibodies may include antibodies produced, for example by the phage display methods described above and antibodies produced by mice in which the murine immunoglobulin variable and optionally the constant region genes have been replaced by their human counterparts e.g. as described in general terms in EP 0546073, U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, EP 0438474, and EP 0463151.

Alternatively, an antibody according to the invention may be produced by a method comprising immunising a non-human mammal with a GPR68 immunogen; obtaining an antibody preparation from the mammal; and deriving therefrom monoclonal antibodies that recognize GPR68.

The antibody molecules of the present invention may comprise a complete antibody molecule having full length heavy and light chains or a fragment or antigen-binding portion thereof. The term “antigen-binding portion” or “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to selectively bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibodies and fragments and antigen binding portions thereof may be, but are not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews— Online 2(3), 209-217). The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in International patent applications WO 2005/003169, WO 2005/003170 and WO 2005/003171 and Fab-dAb fragments described in International patent application W02009/040562. Multi-valent antibodies may comprise multiple specificities or may be monospecific (see for example WO 92/22853 and WO 05/113605). These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

The constant region domains of the antibody molecule of the present invention, if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains may be used, especially of the IgGl and IgG3 isotypes when the antibody molecule is intended for therapeutic uses and antibody effector functions are required. Alternatively, IgG2 and IgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required.

An antibody of the invention may be prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal ( e.g ., a mouse) that is transgenic or transchromosomal for the immunoglobulin genes of interest or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody of interest, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.

An antibody of the invention may be a human antibody or a humanised antibody. The term “human antibody”, as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

Such a human antibody may be a human monoclonal antibody. Such a human monoclonal antibody may be produced by a hybridoma that includes a B cell obtained from a transgenic nonhuman animal, e.g, a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

Human antibodies may be prepared by in vitro immunisation of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus. The term “human antibody derivatives” refers to any modified form of the human antibody, e.g. , a conjugate of the antibody and another agent or antibody.

The term “humanized antibody” is intended to refer to CDR-grafted antibody molecules in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

As used herein, the term “CDR-grafted antibody molecule” refers to an antibody molecule wherein the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g. a murine or rat monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody). For a review, see Vaughan el al , Nature Biotechnology, 16, 535-539, 1998. In one embodiment rather than the entire CDR being transferred, only one or more of the specificity determining residues from any one of the CDRs described herein above are transferred to the human antibody framework (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment only the specificity determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework. In another embodiment only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework.

When the CDRs or specificity determining residues are grafted, any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions. Suitably, the CDR-grafted antibody according to the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs or specificity determining residues described above. Thus, provided in one embodiment is a neutralising CDR-grafted antibody wherein the variable domain comprises human acceptor framework regions and non-human donor CDRs. Detecting GPR68

Another aspect of the invention concerns a method for detecting GPR68 in an ocular sample. The GPR68 may be a GPR68 polypeptide or a nucleic acid encoding a GPR68 polypeptide. In some embodiments, the level of GPR68 is measured quantitatively. GPR68 may be used as a biomarker by itself, or in combination with other biomarkers, to assess the presence or status of an ocular disease, ocular disorder, or ocular condition, that is mediated at least in part by aberrant GPR68 activity.

In accordance with the detection methods of the invention, an antibody to GPR68 may be used to identify and/or localize inflammation in one or more anatomical sites in or around the eye selected from among: the retina, one or more layers of the retina, cornea, one or more layers of the cornea, conjunctiva, one or more layers of the conjunctiva, different parts of the lacrimal system including but not restricted to lacrimal glands with all its components, lacrimal path ways in compromised expiratory duct system of the lacrimal gland, the different parts of the tear duct system including but not restricted to the punctum lacrimale, and the tear ducts themselves and all their different layers, sites around punctum plugs for the determination of how punctum plugs are positioned, the different layers of conjunctiva, sclera, and cornea in response to glaucoma surgery, different parts of the anterior chamber angle in response to glaucoma surgery, in ocular tissues and structures as response to ocular surgery. In some embodiments, the inflammation is ocular surgery-induced inflammation, such as in response to changed ocular pressures prior to, during, or after ocular surgery, or in response to changed flow of fluids prior to, during, or after ocular surgery, or in response to mechanical pressure or traction prior to, during, or after ocular surgery.

In accordance with the detection methods of the invention, an antibody to GPR68 may be used to identify and/or localize friction in one or more anatomical sites in or around the eye selected from among: the retina, one or more layers of the retina, cornea, one or more layers of the cornea, conjunctiva, one or more layers of the conjunctiva, any other tissue of or in proximity of tissues of the eye, including but not restricted to the orbital tissues with muscles and nerves including the optical nerve, different parts of the lacrimal system including but not restricted to lacrimal glands with all its components, lacrimal pathways here in compromised expiratory duct system of the lacrimal gland, different parts of the tear duct system including but not restricted to the punctum lacrimale, and the tear ducts themselves and all their different layers, sites around punctum plugs for the determination of how punctum plugs are positioned, or any other tissue of or in proximity of tissues of the eye, including but not restricted to the orbital tissues with muscles and nerves including the optical nerve, different layers conjunctiva, sclera and cornea in response to glaucoma surgery, different parts of the anterior chamber angle in response to glaucoma surgery, in ocular tissues and structures as response to ocular surgery. In some embodiments, the friction is ocular surgery-induced friction, such as in response to changed ocular pressures prior to, during, or after ocular surgery, or in response to changed flow of fluids prior to, during, or after ocular surgery, or in response to mechanical pressure or traction prior to, during, or after ocular surgery.

In accordance with the detection methods of the invention, an antibody to GPR68 may be used to identify the presence and/or intensity of attrition in one or more anatomical sites in or around the eye selected from among: the retina, one or more layers of the retina, cornea, one or more layers of the cornea, conjunctiva, one or more layers of the conjunctiva, different parts of the lacrimal system including but not restricted to lacrimal glands with all its components, lacrimal path ways in compromised expiratory duct system of the lacrimal gland, the different parts of the tear duct system including but not restricted to the punctum lacrimale, and the tear ducts themselves and all their different layers, sites around punctum plugs for the determination of how punctum plugs are positioned, the different layers of conjunctiva, sclera, and cornea in response to glaucoma surgery, different parts of the anterior chamber angle in response to glaucoma surgery, in ocular tissues and structures as response to ocular surgery. In some embodiments, the attrition is ocular surgery-induced attrition, such as in response to changed ocular pressures prior to, during, or after ocular surgery, or in response to changed flow of fluids prior to, during, or after ocular surgery, or in response to mechanical pressure or traction prior to, during, or after ocular surgery.

In accordance with the detection methods of the invention, an antibody to GPR68 may be used to localize fibrotic processes in different parts of the lacrimal pathways.

Optionally, the GPR68 may then be modulated positively or negatively in accordance with the invention by administering a GPR68 inducer or inhibitor, respectively. The detected level of GPR68 may be used by itself, or with other biomarkers, prognostically or to gauge the effectiveness (success or failure) of a treatment, such as a GPR68 modulatory treatment or other treatment of the ocular surface or ocular tissues.

GPR68 polypeptides and nucleic acids may be detected in an ocular sample by any technique effective for measuring polypeptides, nucleic acids encoding polypeptides, such as liquid chromatography-mass spectroscopy (LC-MS), antibody-based detection method, multiplex protein detection method, or mRNA detection method.

The detection methods described herein encompass the use of GPR68 as a biomarker, singly or in combination with other biomarkers, utilizing a triple quadrupole LC-MS/MS platform, which may be carried out at a centralized laboratory testing facility. The ocular samples collected from individuals can be shipped to the testing facility in this embodiment. The identified GPR68 polypeptides and their subsequent proteolytic fragments may be used for quantitative analysis of diagnostic peptides produced in the triple quad. A threshold value or a relative or actual value in terms of GPR68 polypeptide concentration can be defined or ocular samples can be compared directly to normal or healthy controls, and/or compared to a relative or actual value that is consistent with presence of the ocular disease, disorder, or condition. The quantitative information in report form can be provided to health care providers to help in making decisions regarding the pathway of patient care. Subjects can base treatment decisions on these results and the final step can include administration of an appropriate treatment to the subject, such as a positive or negative GPR68 modulator.

In other embodiments, the polypeptides selected from can be detected by implementing binding agents, for example antibodies, peptoids, or coated surfaces, and reagents that accommodate a binding interaction specific to these proteins to produce a reaction that can be quantitated based on production of a detectable signal such as florescence, color change, or UV absorbance. Implementing these components in a cartridge with a partnering reading instrument, such as a point-of-care device that can be used at point of care is also provided. Binding agents for these proteins and polypeptides can also be used for detection in a lateral flow device. Thus, methods of detecting the level of protein expression in the samples using a binding partner such as an antibody can be used to detect the markers provided herein in an immunoassay.

The immunoassay typically includes contacting a test sample ( e.g ., an ocular sample) with an antibody or antigen that specifically binds to, or otherwise recognizes a biomarker (e.g., GPR68), and detecting the presence of a complex of the antibody or antigen bound to the biomarker in the sample. Anti-GPR68 antibodies as GPR68 inhibitors, and techniques for their production, are described above and may be used for detection methods as well. The immunoassay procedure can be selected from a wide variety of immunoassay procedures known in the art involving recognition of antibody/antigen complexes, including enzyme- linked immunosorbent assays (ELISA), radioimmunoassay (RIA), and Western blots, and use of multiplex assays, including use of antibody arrays, wherein several desired antibodies are placed on a support, such as a glass bead or plate, and reacted or otherwise contacted with the test sample. Such assays are well-known to the skilled artisan.

The antibody or antigen-binding fragment can be bound to a solid support or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art. (For a general discussion of conjugation of fluorescent or enzymatic moieties, see Johnstone & Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, Oxford, 1982.) The binding of antibodies to a solid support is also well known in the art (see for a general discussion Harlow & Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Publications, New York, 1988 and Borrebaeck, Antibody Engineering— A Practical Guide, W.H. Freeman and Co., 1992). The detectable moieties contemplated with the present invention include radiolabels and enzymes, for example. Specific examples of detectable labels include, but are not limited to, fluorescent labels, metallic labels, gold, ferritin, alkaline phosphatase, beta-galactosidase, peroxidase ( e.g ., horse radish peroxidase), urease, fluorescein, rhodamine, tritium, and iodination. Other types of detectable labels include chemical moieties such as biotin, which may be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin.

The antibodies and antigen-binding fragments of the present invention can be used to detect and quantitate (e.g, by use of a standard curve) the presence of GPR68 in ocular samples.

The presence of GPR68 in ocular sample can be analyzed at a high sensitivity and precision and with a high specificity in a simple manner by the use of antibodies and antigen binding fragments of the invention in conventional immunoassay formats, such as enzymatic immunoassays EIA), enzyme-linked immunosorbent assays (ELISA), immunohistochemistry (IHC), immunoprecipitation, immunoelectrophoresis, dipstick (antibody, antigen-binding fragment, or immunoconjugate coupled to a solid support), radioimmunometric assays (RIA), immunoturbidimetric assays, or others known in the art. Because the antibodies of the present invention react with GPR68 with specificity they are useful for the determination of GPR68 in ocular samples by immunoassay, thus enabling screening for various ocular diseases, disorders, and conditions associated with elevated levels of GPR68 or low levels of GPR68. Thus, the present invention further provides immunoassay methods for determining the presence or amount of GPR68 in an ocular sample using the antibodies or antigen-binding fragment of the invention. The assay comprises immunochemical reagents for forming an immunoreaction product whose presence or amount relates, either directly or indirectly, to the presence or amount of GPR68 in the ocular sample. Those skilled in the art will appreciate that there are numerous well known clinical diagnostic procedures in which the immunochemical reagents of this invention can be used to form an immunoreaction product whose presence and/or amount relates to the presence and/or amount of GPR68 present in a sample. While exemplary assay methods are exemplified herein, the invention is not limited to these. Various heterogeneous and homogenous protocols, either competitive or noncompetitive, can be employed in performing an assay of this invention.

Furthermore, the antibodies and antigen-binding fragments of the present invention can be used in a quantitative immunohistochemical analysis of cells and tissues. For example, cells or tissue sections can be immobilized on glass slides or other supports. Analysis can be conducted with, for example, fluorescently or otherwise labeled GPR68-specific antibodies or antigen-binding fragments of the antibodies under conditions where the fluorescence intensity of the stained sections is proportional to the amount of GPR68 present in the ocular sample.

In other embodiments, the GPR68-specific antibodies or antigen-binding fragments can be immobilized and used to absorb or capture soluble antigen from known amounts of ocular samples such as cells, tissue, and fluids. This can be used as a concentration step prior to elution and a detection and quantitation step using other methodologies. In addition, a detection and quantitation step involving inhibition of antibody or antibody fragment binding to antigen as discussed below could be applied.

Furthermore, soluble antigen present in known amounts of samples can be detected and quantitated either directly or after an initial concentration step by determining the amount of this material required to provide inhibition of antibody binding to immobilized antigen. In these procedures, the specimen would be combined with antibody or antibody fragment of the present invention and incubated for a period of time sufficient to allow antibody or antibody fragment complexes to form with the soluble antigen. The resulting mixture would be incubated with immobilized antigen and the amount of antibody or antibody fragment binding to the immobilized antigen determined. The concentration of antigen present in the specimen would be determined by comparison to the effect with known amounts of GPR68- containing soluble fractions in either single determinations or in serial dilutions of the specimen. The dilution state required to relieve the inhibition of binding to the immobilized antigen to a proscribed level would be proportional to the concentration of GPR68 present in the specimen. In another embodiment, the immunoassay utilized is the surface plasmon resonance (SPR) assay (see, for example, Mullett WM el al ., “Surface plasmon resonance-based immunoassays,” Methods , 2000, 22(1):77-91, which his incorporated herein by reference in its entirety).

In another illustrative embodiment, a double antibody or “sandwich” immunoassay format may be employed comprising the steps of (a) forming a first immunoreaction admixture by admixing a sample with a first antibody or antigen-binding fragment thereof, e.g ., a monoclonal antibody, wherein the antibody or fragment and GPR68 present in the ocular sample are capable of forming a first immunoreaction product (the first antibody or fragment can be coupled to a solid matrix); (b) maintaining the first immunoreaction admixture so formed under biological assay conditions for a time period sufficient to form the first immunoreaction product (the first immunoreaction product can then be separated from the sample); (c) forming a second immunoreaction admixture by admixing the first immunoreaction product with a second antibody or fragment, monoclonal or polyclonal, which recognizes GPR68; (d) maintaining the second immunoreaction admixture so formed under biological assay conditions for a period sufficient to form the second or “sandwich” immunoreaction product; and (e) determining the presence and, optionally, the amount of second immunoreaction product formed, and thereby the presence and, optionally, the amount of GPR68 in the ocular sample. Preferably, the second antibody is labeled, such as with an enzyme, and thus the second immunoreaction product formed will be a labeled product to facilitate determination of the second immunoreaction product.

In preferred double antibody assay methods, the amount of immunoreaction product determined is related to the amount of immunoreaction product similarly formed and determined using a standard sample in place of the ocular sample wherein the standard sample contains a known amount of GPR68 in accordance with this invention. Alternatively, a synthetic secondary standard can be used.

It is also preferred that the second antibody or antibody fragment be directed to a site on the GPR68 which is not the same as the site to which the first antibody or antibody fragment is directed. For example, the first antibody or antibody fragment can be directed to a site other than that which reacts with the antibodies of the present invention.

In any of the illustrative assays, the ocular sample can be provided as a known or unknown quantity of material. In some embodiments, a cell/tissue specimen is examined by tissue microarrays (TMA). For example, multiple tissue samples may be taken from multiple such tissue specimens, and the multiple samples from a particular specimen are similarly placed at corresponding positions in the multiple supports. Each of the resulting supports contains an array of tissue samples from multiple specimens, in which corresponding positions in each of the arrays represent tissue samples from the same tissue specimen. In particular examples, each support is then sectioned into multiple similar sections with samples from the same tissue specimen at corresponding positions of the sequential sections. The different sections may then be subjected to different reactions, such as exposure to different histological stains or molecular markers, so that the multiple “copies” of the tissue microarrays can be compared for the presence of reactants of interest, such as GPR68. The large number of tissue samples, which are repeated in each of a potentially large number of sections of multiple substrates, can be exposed to as many different reactions as there are sections. For example, about 100,000 array sections may be obtained from a set of 1000 tissue specimens measuring 15x15x3 mm. This approach provides for high-throughput techniques, including rapid parallel analysis of many different tissue specimens.

In one embodiment, a sample can be processed by exposing different cut sections on the array to different biological reagents (such as standard stains, or immunohistochemical or genetic markers, oligonucleotides probes/primers, peptides, polypeptides, ligands, and small molecules, hormones, lipids, carbohydrates, lectins, etc) that recognize biological structures in the cut sections. An imager then obtains an image of the cut processed sections, and an image processor identifies regions of the cut sections that contain images of biological interest (such as evidence of gene copy numbers), and stores images of the cut sections. If desired, quantities of biological reagents such as GPR68 can be detected to quantify reactions, or to determine the distribution of the reagent in the sample.

The term “complex” as used herein refers to the product of a specific binding reaction such as an antibody-antigen, antibody fragment-antigen, or receptor-ligand reaction. Exemplary complexes are immunoreaction products.

As used herein, the terms “label”, “detectable label”, “labeling agent” in their various grammatical forms refers to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating agent can be linked to or incorporated in an expressed protein, peptide, or antibody molecule that is part of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well known in the diagnostic art.

The label can be a fluorescent labeling agent that chemically binds to antibodies, antibody fragments, or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-diethylamine-l- natpthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, “Immunofluorescence Analysis,” Antibody As a Tool , Marchalonis et al., Eds., John Wiley & Sons, Ltd., pp. 189- 231 (1982).

The indicating group may also be an enzyme such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principle indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to indicate that a receptor- ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2,-azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents and may be used in practicing the present invention. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as ¹²⁴ I, ¹²⁵ I, ¹²⁸ I, ¹³² I and 'Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Another group of useful labeling agents are those elements such as ³ C, ¹⁸ F, ¹⁵ 0 and ¹³ N which themselves emit positrons. Also useful is a beta emitter, such as ^U1 indium or 3H.

The linking of labels, i.e., labeling of peptides and proteins, is well known in the art. For instance, monoclonal antibodies produced by a hybridoma, or antigen-binding fragments of such antibodies, can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et al., Meth. Enzymol, 73:3-46 (1981). The techniques of protein conjugation or coupling through activated functional groups are also applicable. See, for example, Aurameas et al, Scand. J Immunol., 8(7):7-23 (1978); Rodwell et al, Biotech., 3:889-894 (1984); and U.S. Patent No. 4,493,795. The detection of GPR68 described herein in an ocular sample can be performed in a variety of other ways. In one embodiment, the method provides the reverse-transcription of complementary DNAs from mRNAs obtained from the sample. Fluorescent dye-labeled complementary RNAs can be transcribed from complementary DNAs that are then hybridized to the arrays of oligonucleotide probes. The fluorescent color generated by hybridization can be read by machine, such as a SureScan microarray scanner (Agilent Technologies) and data obtained and processed using software, such as Agilent Feature Extraction Software (9.1). Such array -based methods include microarray analysis to develop a gene expression profile. As used herein, the term “gene expression profile” refers to the expression levels of mRNAs or proteins of a panel of genes in the subject. As used herein, the term “diagnostic panel” refers to a panel of genes, peptides or proteins with an expression level that can be relied on to diagnose or predict the status of the ocular disease, disorder, or condition.

In other embodiments, complementary DNAs are reverse-transcribed from mRNAs obtained from the ocular sample, amplified, and simultaneously quantified by real-time PCR, thereby enabling both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific gene product in the complementary DNA sample as well as the original mRNA sample.

The detection methods of invention include detecting GPR68 in an ocular sample. However, any number of additional biomarkers can be detected. In some embodiments, at least two biomarkers are detected in the analysis. However, it is realized that three, four, or more, including all, of the biomarkers described herein can be utilized in the analysis.

Thus, not only can GPR68 be detected, any number or combination of markers can be used in detection, including biomarkers not herein described, to aid in the diagnosis of an ocular disease, disorder, or condition.

GPR68 can increase or decrease at least, for example, 1.5 fold, 2 fold, 4 fold, 5 fold, 8 fold, 10 fold or more, relative to the level of the marker in a control sample. The control sample can be a sample from a subject that does not have the ocular disease, disorder, or condition, a pooled sample from subjects that do not have the ocular disease, disorder, or condition, or can be a control or baseline expression level known to be the average expression level of subjects without the ocular disease, disorder, or condition.

As provided herein, “mass spectrometry” or “MS” refers to an analytical technique generating electrical or magnetic fields to determine mass-to-charge ratio of peptides and chemical compounds in order to identify or determine peptide sequence, such as GPR68, and chemical structures. LC-MS/MS spectrometry refers to an analytical technique combining the separation capabilities of high-performance liquid chromatography (HPLC) with the mass analysis of mass spectrometry. Triple quadrupole mass spectrometry refers to a tandem mass spectrometer with three ionizing chambers (Ql, Q2, & Q3). This technique allows for target detection of molecules of interest such as GPR68. Ion pairs refers to a parent peptide detected in Ql in its doubly or triply charged form and a resulting y or b ion as generated by Q2 and detected in Q3 of a triple quadrupole mass spectrometry instrument. SIS internal peptide refers to a synthesized isotopically-labeled peptide with the same sequence as the peptide to be monitored in Ql and used as an internal standard for reference to quantify the peptide of interest, such as a GPR68 peptide. The -y ion refers to an ion generated from the c-terminal of a peptide fragment. The -b ion refers to an ion generated from the n-terminal of a peptide fragment. Quantitative ion refers to the selected highest intensity y or b ion used to determine the quantity of its parent protein in a biological sample such as an ocular sample. Qualitative Ion refers to ion/ions chosen to ensure the integrity of the Qualitative Ion to selected protein of interest and labeled peptide to selected standards.

“Point-of-care device” refers to an instrument or cartridge available at the location of patient and physician care, which contains binding agents to a biomarker such as GPR68, or series of biomarkers of interest that include GPR68, and which can generate information on the presence, absence, and in some cases concentrations of detected biomarkers. Analyte refers to any measurable biomarker, which can be protein, peptide, macromolecule, metabolite, small molecule, or autoantibody. In the present invention, the analyte refers to a GPR68 polypeptide or discernible fragment thereof. “Biomarker” refers to any substance (e.g. protein, peptide, metabolite, polynucleotide sequence) the concentration level of which changes in the body, for example increased or decreased, as a result of a disease or condition. Marker and biomarker can be used interchangeably used herein.

“Lateral flow” test refers to a device that measures the presence of an analyte such as GPR68 in a biological fluid such as an ocular sample using porous paper of sintered polymer. ELISA refers to Enzyme-linked immunosorbent assay, which utilizes antibodies or antigens to detect the presence and concentration of an analyte of interest. Diagnostic panel refers to a group of molecules for example proteins or peptides, the combined concentrations of which are used to diagnose a state of ocular disease, ocular disorder, or ocular condition. In addition to being useful to diagnose an ocular disease, disorder, or condition, and in particular a GPR68-mediated ocular disease, disorder, or condition, in a subject, kits and methods provided herein can be used to monitor treatment or recurrence of the ocular disease, disorder, or condition in an individual previously diagnosed with the ocular disease, disorder, or condition.

Specific embodiments of the detection method of the invention include, but are not limited to:

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation in the retina.

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation in the different layers of the retina.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction in the retina.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction the different layers of the retina.

• The use of antibodies primers to GPR68 for the identification and/or localization of inflammation in the cornea.

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation in the different layers of the cornea.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction in the cornea.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction the different layers of the cornea.

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation in the conjunctiva.

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation in the different layers of the conjunctiva.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction in the conjunctiva.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction the different layers of the conjunctiva. • The use of antibodies or primers to GPR68 for the identification and/or localization of friction in any other tissue of or in proximity of tissues of the eye, including but not restricted to the orbital tissues with muscles and nerves including the optical nerve.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction the different parts of the lacrimal system including but not restricted to lacrimal glands with all its components, lacrimal path ways here in compromised expiratory duct system of the lacrimal gland.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction the different parts of the tear duct system including but not restricted to the punctum lacrimale, and the tear ducts themselves and all their different layers.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction for the determination of how punctum plugs are positioned.

• The use of antibodies or primers to GPR68 for the purpose to localize fibrotic processes in different parts of the lacrimal pathways.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction in any other tissue of or in proximity of tissues of the eye, including but not restricted to the orbital tissues with muscles and nerves including the optical nerve.

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation the different parts of the lacrimal system including but not restricted to lacrimal glands with all its components, lacrimal path ways here in compromised expiratory duct system of the lacrimal gland.

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation the different parts of the tear duct system including but not restricted to the punctum lacrimale, and the tear ducts themselves and all their different layers.

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation for the determination of how punctum plugs are positioned.

• The use of antibodies or primers to GPR68 for the purpose to localize fibrotic processes in different parts of the lacrimal pathways.

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation the different layers conjunctiva, sclera, and cornea in response to glaucoma surgery. • The use of antibodies or primers to GPR68 for the identification and/or localization of friction of the different layers conjunctiva, sclera and cornea in response to glaucoma surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation in the different parts of the anterior chamber angle in response to glaucoma surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction in the different parts of the anterior chamber angle in response to glaucoma surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of inflammation in ocular tissues and structures as response to ocular surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of friction in ocular tissues and structures as response to ocular surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of surgically induced inflammation in ocular tissues and structures as response to ocular surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of surgically induced friction in ocular tissues and structures as response to ocular surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of surgically induced inflammation in ocular tissues and structures as response to changed ocular pressures prior to, during, or after ocular surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of surgically induced friction in ocular tissues and structures as response to response to changed ocular pressures prior to, during, or after ocular surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of surgically induced inflammation in ocular tissues and structures as response to changed flow of fluids prior to, during, or after ocular surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of surgically-induced friction in ocular tissues and structures as response to changed flow of fluids prior to, during, or after ocular surgery. • The use of antibodies or primers to GPR68 for the identification and/or localization of surgically induced inflammation in ocular tissues and structures as response to mechanical pressure or traction prior to, during, or after ocular surgery.

• The use of antibodies or primers to GPR68 for the identification and/or localization of surgically induced-friction in ocular tissues and structures as response to response mechanical pressure or traction prior to, during, or after ocular surgery.

• The use of antibodies or primers to GPR68 for the identification of the absence of GPR68 as indicative of various diseases, such as malignant diseases such as tumors and cancers, especially malignant melanoma.

• The use of antibodies or primers to GPR68 to evaluate the effectiveness (success or failure) of an agent for the treatment, prevention, or delay of onset of an ocular disease, disorder, or condition. The agent may be administered to the ocular surface or other tissues of the eye or its adnexa and the effects of the agent on GPR68 level and/or activity can be determined by comparing GPR68 levels and/or activity before and after administration of the agent and comparing.

• The use of antibodies or primers to GPR68 to identify the presence and intensity of attrition.

Kits

Kits for performing detection methods described herein are also provided. Kits can contain binding agents for GPR68 such as antibodies with specific binding affinity for a GPR68 polypeptide, or primer pairs capable amplifying GPR68 nucleic acid sequences, and optionally, an ocular sample collection platform, a tube for collection and extraction that can include a protease inhibitor or other protein-stabilizing agent, sample extraction reagents and testing apparatus.

Also provided herein are methods and kits to collect ocular samples, such as lacrimal sections, for use in determining the GPR68 activity and/or expression levels of GPR68 polypeptides or nucleic acids. The use of collection strips and tubes, optionally containing protease inhibitor or protein stabilizing agents is contemplated. Kits further may include buffers or reagents for the elution of GPR68 polypeptides and nucleic acids from the collection strips that have been in contact with an eye to collect ocular samples such as lacrimal secretions. The design of devices to collect polypeptides and nucleic acids from the ocular cavity, as well as the packaging of this device with a container to house the collection device and elution buffers, is contemplated.

A kit for performing detection methods described herein may comprise a collection tube, optionally, and a protease inhibitor, or other protein stabilizing agent.

The kit for performing detection methods of the invention may include an antibody capable of selectively binding to a GPR68 polypeptide. In some embodiments, the antibody is immobilized on a collection tube or other ocular sample collecting platform.

The kit for performing detection methods of the invention may include a primer pair capable of amplifying at least one GPR68 nucleic acid, e.g ., for polymerase chain reaction (PCR).

The invention is described only exemplarily by the embodiments in the description and is not limited thereto but rather includes all variations, modifications, substitutions, and combinations the expert may take from the complete documents of this application under consideration of and/or combination with his specific knowledge.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

Definitions

The term “a,” “an,” “the” and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. Thus, for example, reference “a cell” or “an agent” should be construed to cover both a singular cell or singular agent and a plurality of cells and a plurality of agents unless indicated otherwise or clearly contradicted by the context.

As used herein, the term “administration” is intended to include, but is not limited to, the following delivery methods: topical, oral, parenteral, subcutaneous, transdermal, transbuccal, intravascular ( e.g ., intravenous or intra-arterial), intramuscular, subcutaneous, intranasal, and intra-ocular administration. Administration can be local at a particular anatomical site, such as the eye or adnexa, or systemic. In some embodiments, administration is topical administration to the ocular surface, optionally using iontophoresis, electroporation, or other delivery-assistance mechanisms. In some embodiments, topical administration to the ocular surface, or other administration methods into the eye, may be facilitated by co-administration of a permeation-enhancing agent before, during, or after administration of the GPR68 modulator.

As used herein, the term “attrition” in the context of the eye refers to the individual and collective effects of mechanical forces constantly and repeatedly challenging tissues at the ocular surfaces with friction, stretching, and compression, as described in van Setten et al. [14], which is incorporated herein by reference in its entirety. The process of attrition is a highly dynamic process leading to pain and neurogenic inflammation. Attrition is enhanced by the thinning corneal epithelium in severe dry eye disease.

The terms “compounds of the invention” or “compounds of the present invention” (unless specifically identified otherwise), and grammatical variations thereof, refer to GPR68 modulatory agents, including pharmaceutically acceptable salts of such agents, as well as all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties (e.g., polymorphs, solvates and/or hydrates).

The terms “comprising”, “including”, “having”, and “containing” (and grammatical variations of these) are interchangeable, and are open (inclusive) terms that do not exclude the presence of one or more additional elements, ingredients or process steps that are not explicitly mentioned, while the term “consisting of’ and grammatical variations thereof are closed terms that exclude the presence of any other additional element, step or ingredient that is not explicitly stated. The term “essentially consisting of’ and grammatical variations thereof are partially open terms that do not exclude the presence of one or more additional elements, ingredients or steps as long as these additional elements, ingredients or steps do not essentially affect the basic and novel properties of the invention. Consequently, the transitional term “comprising” (or grammatical variations such as “comprises/comprise”) includes the terms “consisting of’, as well as the terms “essentially consisting of’, and grammatical variations thereof. The transitional terms/phrases (and any grammatical variations thereof) “comprising”, “comprises”, “comprise”, “consisting essentially of’, “consists essentially of’, “consisting of’ and “consists of’ can be used interchangeably to attach the specific meaning associated with each term.

As used herein, the term “contacting” in the context of contacting a cell with at least one GPR68 modulator in vitro or in vivo means bringing the modulator into contact with the cell, or vice-versa, or any other manner of causing the modulator and the cell to come into contact. In those embodiments of the method for inhibiting GPR68-mediated ocular disease, disorder, or condition cells in vitro or in vivo , when a cell is contacted with a modulator in vivo , the modulator is administered to a subject, and the administration may occur by any route ( e.g ., topical, oral, parenteral, subcutaneous, transdermal, transbuccal, intravascular ( e.g ., intravenous or intra-arterial), intramuscular, subcutaneous, intranasal, topical administration to the ocular surface, and intra-ocular administration).

The phrase “effective amount”, in the context of a subject, means an amount of at least one GPR68 modulator that (i) treats or prevents the particular ocular disease, ocular condition, or ocular disorder in a subject, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular ocular disease, ocular condition, or ocular disorder in a subject, or (iii) prevents or delays the onset of one or more symptoms of the particular ocular disease, ocular condition, or ocular disorder described herein in a subject.

The phrase “effective amount”, in the context of a cell in vitro or in vivo , means an amount of at least one GPR68 modulator that (i) treats or prevents the particular ocular disease, ocular condition, or ocular disorder in a cell, (ii) attenuates, ameliorates, or eliminates one or more effects of the particular ocular disease, ocular condition, or ocular disorder a cell, or (iii) prevents or delays the onset of one or more effects of the particular ocular disease, ocular condition, or ocular disorder described herein in a subject.

The terms “GPR68 activity”, “GPR68 function”, and grammatical variations thereof, refer to its receptor activities, such as pH-sensing activity, endothelial shear stress sensing activity, mechano-sensing activity, or a combination of two or more of its activities as may he detected and assessed in vitro, in vivo , or both [34], [35], [36], and [37], which are incorporated herein by reference in their entireties A GPR68 -mediated ocular disease, disorder, or condition includes one that is characterized by, caused by, propagated by, and/or or worsened by, aberrant GPR68 activity (e.g., elevated or diminished receptor levels or activity such as those noted above) in the relevant ocular tissue or fluid relative to that of a normal, healthy individual.

As used herein, the term “inhibit”, “inhibition” or “inhibiting” refers to the reduction or suppression of a given ocular condition, ocular symptom, or ocular disorder, or ocular disease, such as one that is mediated by GPR68, or a significant decrease in the baseline activity of a biological activity or process (e.g., inhibits or suppresses GPR68 activation, or inhibits or suppresses GPR68 expression, or inhibits or suppresses GPR68 expression). Thus, in some embodiments, the term “inhibit”, means to suppress GPR68 activity or function, for example, about ten percent relative to a control value. Preferably, the activity is suppressed y 50% compared to a control value, more preferably by 75%, and even more preferably by 95%. “Inhibit,” as used herein, also means to reduce the level of a molecule (e.g, GPR68), a reaction, an interaction, a gene, an mRNA, and/or a protein's expression, stability, function or activity by a measurable amount or to prevent entirely. Inhibitors are compounds that, e.g, bind to, partially or totally inhibit activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g, antagonists.

As used herein, a subject is “in need of’ a treatment if such human or non-human animal subject would benefit biologically, medically or in quality of life from such treatment (preferably, a human). In some embodiments, the subject has an ocular disease, condition, or disorder and is in need of therapy. In other embodiments, the subject does not have the ocular disease, ocular condition, or ocular disorder, and is in need of prophylaxis. In some embodiments, the subject in need of prophylaxis is at risk of developing the ocular disease, ocular condition, or ocular disorder. In some embodiments, the subject is at increased risk of developing the ocular disease, ocular condition, or ocular disorder, relative to others in the population.

The term “isolated,” when used as a modifier of a composition, means that the compositions are made by the hand of man or are separated from their naturally occurring in vivo environment. Generally, compositions so separated are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane. A “substantially pure” molecule can be combined with one or more other molecules. Thus, the term “substantially pure” does not exclude combinations of compositions. Substantial purity can be at least about 60% or more of the molecule by mass. Purity can also be about 70% or 80% or more, and can be greater, for example, 90% or more. Purity can be determined by any appropriate method, including, for example, UV spectroscopy, chromatography ( e.g ., HPLC, gas phase), gel electrophoresis (e.g., silver or coomassie staining) and sequence analysis (for nucleic acid and peptide).

The present invention includes derivatives of compounds identified herein, also referred to herein as pharmaceutically active derivatives. “Pharmaceutically active derivative” refers to any compound that upon administration to the subject or cell, is capable of providing directly or indirectly, the GPR68 modulatory activity disclosed herein. The term “indirectly” also encompasses prodrugs which may be converted to the active form of the drug via endogenous enzymes or metabolism. The prodrug is a derivative of the compounds according to the invention and presenting GPR68 modulatory activity that has a chemically or metabolically decomposable group, and a compound that may be converted into a pharmaceutically active compound according to the invention in vivo by solvolysis under physiological conditions. The prodrug is converted into a compound according to the present invention by a reaction with an enzyme, gastric acid or the like under a physiological condition in the living body, e.g, by oxidation, reduction, hydrolysis or the like, each of which is carried out enzymatically. These compounds can be produced from compounds of the invention according to well-known methods. The term “indirectly” also encompasses metabolites of compounds according to the invention. Chemical reactions, reactants, and reagents useful for making derivatives can be found, for example, in March ’s Advanced Organic Chemistry, 7 ^th edition, 2013, Michael B. Smith, which is incorporated herein by reference in its entirety.

The term “metabolite” refers to all molecules derived from any of the compounds according to the invention in a cell or organism, preferably mammal. Pharmaceutically active metabolites of the compounds of the invention may be administered to a subject or contacted with a cell in vitro or in vivo.

As used herein, the term “modulating”, and grammatical variations thereof, in the context of GPR68 in the invention means changing, adjusting, or varying the expression and/or activity of GPR68 in the eye. A “GPR68 modulator” or “GPR68 modulatory agent” is an agent that directly or indirectly causes or contributes to positive or negative modulation of GPR68 expression and/or activity in the eye. Thus, modulation is intended to encompass antagonism, agonism, partial antagonist, and/or partial agonism of an activity associated with a GPR68 gene product. Examples of GPR68 modulators are described in International Application Publication No. W02019/150309 (Hammack et al., “Modulators of GPR68 and Uses Thereof for Treating and Preventing Disease”, Novartis AG and Scripps Research Institute, published August 8, 2019), which is incorporated herein by reference in its entirety.

GPR68 is a Gq/n-coupled receptor, and GPR68 activation may lead to the cleavage of PIP2 into IP3 and DAG by phospholipase C (PLC), and induce calcium release from the store, GPR68 senses flow and is essential for vascular physiology [34], In endothelial cells, activation of GPR68 may trigger several acute signaling pathways, such as activation of Ca ²⁺ - gated channels (OraM, Kc _a 2.3, Kc _a 3.1) or synthesis of NO by NOS or release of EDHF. Activation of GPR68 may also activate KCNK channels and lead to the hyperpolarization of the cells. Activation of GPR68 may lead to hyperpolarization of smooth muscles surrounding the vessels, causing them to relax and therefore dilate the vessels. The inventor proposes there are similar effects in ocular tissues, such as the choroid and other retinal tissues.

A GPR68 modulator may be a negative GPR68 modulator, also referred to herein as a “GPR68 inhibitor”, which reduces or inhibits GPR68 expression and/or GPR68 activity in the eye, or a GPR68 modulator may be a positive GPR68 modulator, also referred to herein as a “GPR68 inducer”, which increases GPR68 expression and/or activates or increases GPR68 receptor activity in the eye. Thus, the term “GPR68 modulator” or “GPR68 modulatory agent” is intended to encompass an agent that is an agonist, antagonist, partial agonist, or partial antagonist of an activity associated with a GPR68 gene product. In various embodiments, a GPR68 modulator may inhibit or stimulate GPR68 expression and/or activity.

A GPR68 modulator (positive or negative modulator) may be any class of substance, such as a small molecule (e.g, a drug) or a biomolecule, such as a polypeptide or nucleic acid. For example, a GPR68 inhibitor may be a small molecule, anti-GPR68 blocking or interfering antibody or antigen-binding fragment thereof, or a nucleic acid molecule such as an antisense oligonucleotide, triplex molecule, catalytic RNA, ribozyme, RNA interference molecule (e.g, modified or unmodified small interfering RNAs (siRNA) or short hairpin RNA (shRNA)), or single guide RNA for a gene editing enzyme (e.g, Cas9).

Therapeutic approaches based on RNA interference involve the introduction of an RNA interference molecule into the target cells to elicit RNA interference (RNAi), thereby inhibiting the expression of a specific messenger RNA (mRNA) to produce a gene silencing effect. In contrast, miRNA-based therapeutics comprise two approaches: miRNA inhibition and miRNA replacement. The former approach resembles antisense therapy, with synthetic single stranded RNAs acting as miRNA antagonists (also known as antagomirs or anti-miRs) to inhibit the action of the endogenous miRNAs. In the replacement approach, synthetic miRNAs (also known as miRNA mimics) are used to mimic the function of the endogenous miRNAs. It thus leads to mRNA degradation/inhibition, and produces a gene silencing effect (Lam JKW et al., “siRNA Versus miRNA as Therapeutics for Gene Silencing”, Molecular Therapy Nucleic Acids, 2015, Volume 4:E252).

For example, a GPR68 inducer may be a small molecule, a polypeptide, such as a GPR68 polypeptide, which may be administered as “protein therapy”, or a nucleic acid encoding a GPR68 polypeptide, which may be administered as “gene therapy”. The GPR68 polypeptide may optionally include one or more heterologous coding or non-coding sequences. For example, the GPR68 polypeptide may be a fusion polypeptide comprising the GPR68 sequence and different polypeptides or multiple copies of the GPR68 sequence (i.e., a multimer).

The amino acid sequence of human GPR68 and its encoding nucleic acid sequence are known (NCBI Accession No. NP_001171147; Uniprot Accession no. Q15743), presented below as SEQ ID NO: l and SEQ ID NO:2, respectively.

MGNIT ADN S SM S CTIDHTIHQTL AP V V Y VT VL V V GFP AN CL SL YF GYLQIK AR NELGVYLCNLTVADLFYICSLPFWLQYVLQHDNWSHGDLSCQVCGILLYENIYISVG FLC Cl S VDRYL A V AHPFRFHQFRTLK A A V GV S V VIW AKELLT SI YFLMHEE VIEDEN Q HRVCFEHYPIQAWQRAINYYRFLVGFLFPICLLLASYQGILRAVRRSHGTQKSRKDQI QRLVLSTVVIFLACFLPYHVLLLVRSVWEASCDFAKGVFNAYHFSLLLTSFNCVADP VLYCFVSETTHRDLARLRGACLAFLTCSRTGRAREAYPLGAPEASGKSGAQGEEPEL LTKLHPAFQTPNSPGSGGFPTGRLA (SEQ ID NO: 1)

ATGGGGAACATCACTGCAGACAACTCCTCGATGAGCTGTACCATCGACCA

TACCATCCACCAGACGCTGGCCCCGGTGGTCTATGTTACCGTGCTGGTGGTGGGC

TTCCCGGCCAACTGCCTGTCCCTCTACTTCGGCTACCTGCAGATCAAGGCCCGGA

ACGAGCTGGGCGTGTACCTGTGCAACCTGACGGTGGCCGACCTCTTCTACATCTG

CTCGCTGCCCTTCTGGCTGCAGTACGTGCTGCAGCACGACAACTGGTCTCACGGC

GACCTGTCCTGCCAGGTGTGCGGCATCCTCCTGTACGAGAACATCTACATCAGCG

TGGGCTTCCTCTGCTGCATCTCCGTGGACCGCTACCTGGCTGTGGCCCATCCCTTC CGCTTCCACCAGTTCCGGACCCTGAAGGCGGCCGTCGGCGTCAGCGTGGTCATCT

GGGCCAAGGAGCTGCTGACCAGCATCTACTTCCTGATGCACGAGGAGGTCATCG

AGGACGAGAACCAGCACCGCGTGTGCTTTGAGCACTACCCCATCCAGGCATGGC

AGCGCGCCATCAACTACTACCGCTTCCTGGTGGGCTTCCTCTTCCCCATCTGCCTG

CTGCTGGCGTCCTACCAGGGCATCCTGCGCGCCGTGCGCCGGAGCCACGGCACC

CAGAAGAGCCGCAAGGACCAGATCCAGCGGCTGGTGCTCAGCACCGTGGTCATC

TTCCTGGCCTGCTTCCTGCCCTACCACGTGTTGCTGCTGGTGCGCAGCGTCTGGGA

GGCCAGCTGCGACTTCGCCAAGGGCGTTTTCAACGCCTACCACTTCTCCCTCCTG

CTCACCAGCTTCAACTGCGTCGCCGACCCCGTGCTCTACTGCTTCGTCAGCGAGA

CCACCCACCGGGACCTGGCCCGCCTCCGCGGGGCCTGCCTGGCCTTCCTCACCTG

CTCCAGGACCGGCCGGGCCAGGGAGGCCTACCCGCTGGGTGCCCCCGAGGCCTC

CGGGAAAAGCGGGGCCCAGGGTGAGGAGCCCGAGCTGTTGACCAAGCTCCACCC

GGCCTTCCAGACCCCTAACTCGCCAGGGTCGGGCGGGTTCCCCACGGGCAGGTT

GGCCTAG (SEQ ID NO:2)

The GPR68 polypeptide encompasses the dominant isoform for the species of subject, as well as alternative isoforms or transcript variants for the species of subject.

Thus, using known amino acid sequence and nucleic acid sequence information, GPR68 polypeptides may be administered to the eye, or nucleic acids encoding the GPR68 polypeptides (optionally carried by a viral or non-viral vector) may be administered to the eye as GPR68 inducers. Likewise, antisense oligonucleotides, catalytic RNA, ribozymes, RNAi molecules, triplex molecules, miRNAs, single guide RNA for gene editing, or other nucleic acid-based GPR68 inhibitor may be administered to the eye.

As used herein, the term “ocular sample” refers to an isolated cell, tissue, or fluid sample collected from the eye or adnexa of a subject. Ocular samples include, but are not limited to, surface cornea, conjunctiva, retina, optic nerve, tears, and vitreous body. For example, the ocular sample may be an ocular wash, or a tear sample which may include shedded cells and secretions from the lacrimal gland and other tissues that connect with the lymphatic system.

The term “prodrug” refers to a chemical compound that can be converted by the body (/. ., biotransformed) to another chemical compound that has pharmacological activity. The prodrug may itself have pharmacological activity before conversion, or be inactive before conversion and activated upon conversion. Active prodrugs or inactive prodrugs of compounds of the invention may be administered to a subject or contacted with a cell in vitro or in vivo. Instead of administering a drug directly, a prodrug may be used instead to improve how a drug is absorbed, distributed, metabolized, and excreted (ADME). For example, a prodrug may be used to improve bioavailability when a drug itself is poorly absorbed from the gastrointestinal tract, or to improve how selectively the drug interacts with cells or processes that are not its intended target, which can reduce adverse or unintended effects of a drug. Major types of prodrugs include, but are not limited to, type I prodrugs, which are biotransformed inside cells (intracellularly), and type II prodrugs, which are biotransformed outside cells (extracellularly), such as in digestive fluids or in the body’s circulatory system. These types can be further categorized into subtypes based on factors such as whether the intracellular bioactivation location is also a site of therapeutic action, or whether or not bioactivation occurs in the gastrointestinal fluids or in the circulation system (Wu, Kuei-Meng, “A New Classification of Prodrugs: Regulatory Perspectives, Pharmaceuticals , 2009, 2(3):77-81, which is incorporated by reference herein in its entirety).

Pharmaceutical formulations include “pharmaceutically acceptable” and “physiologically acceptable” carriers, diluents or excipients. As used herein the terms “pharmaceutically acceptable” and “physiologically acceptable” include solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration. Such formulations can be contained in a liquid; emulsion, suspension, syrup or elixir, or solid form; tablet (coated or uncoated), capsule (hard or soft), powder, granule, crystal, or microbead. Supplementary compounds ( e.g ., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions. The phrase “pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.

As used herein, the term “nucleic acid” means any DNA-based or RNA-based molecule, and may be a GPR68 modulator of the invention. The term is inclusive of polynucleotides and oligonucleotides. The term is inclusive of synthetic or semi -synthetic, recombinant molecules which are optionally amplified or cloned in vectors, and chemically modified, comprising unnatural bases or modified nucleotides comprising, for example, a modified bond, a modified purine or pyrimidine base, or a modified sugar. The nucleic acid may be in the form of single-stranded or double-stranded DNA and/or RNA. The nucleic acid may be a synthesized molecule, or isolated using recombinant techniques well-known to those skilled in the art. The nucleic acid may encode a polypeptide of any length, such as a GPR68 polypeptide (a GPR68 inducer), or the nucleic acid may be a non-coding nucleic acid. The nucleic acid may be a messenger RNA (mRNA). The nucleic acid may be a morpholino oligomer. For nucleic acids encoding polypeptides such as GPR68 polypeptides, the nucleic acid sequence may be deduced from the sequence of the polypeptide and the codon usage may be adjusted according to the host cell in which the nucleic acid is to be transcribed. DNA encoding a polypeptide such as GPR68 polypeptides optionally includes a promoter sequence operably linked to the encoding DNA for expression ( e.g ., expression of a coding sequence).

As used herein, the terms “protein”, “polypeptide”, and “peptide” are used interchangeably to refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, natural amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term “polypeptide” includes full-length proteins and fragments or subunits of proteins. For example, in the case of enzymes, the polypeptide may be the full-length enzyme or an enzymatically active subunit or portion of the enzyme. The term “polypeptide” includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N- terminal methionine residues; immunologically tagged proteins; and the like. The term “polypeptide” includes polypeptides comprising one or more of a fatty acid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety. The term “polypeptides” includes post- translationally modified polypeptides. In some embodiments, the polypeptide is a GPR68 polypeptide, such as the amino acid sequence of SEQ ID NO:l, or an isoform or transcript variant thereof.

The GPR68 modulators can be formulated into pharmaceutically-acceptable salt forms. Pharmaceutically-acceptable salts of the compounds of the invention can be prepared using conventional techniques. “Pharmaceutically acceptable salt” includes both acid and base addition salts. A pharmaceutically acceptable salt of any one of the compounds described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

“Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and. aromatic sulfonic acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenyl acetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S. M. et al ., “Pharmaceutical Salts,” Journal of Pharmaceutical Science , 66:1-19 (1997), which is hereby incorporated by reference in its entirety). Acid addition salts of basic compounds may be prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.

“Pharmaceutically acceptable base addition salt” refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. See Berge et al, supra.

As used herein, the term “small molecule” refers to compounds, preferably organic compounds, with a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Small organic molecules can range in size up to about 5000 Da, e.g., up to about 4000, in some embodiments up to 3000 Da, in some embodiments up to 2000 Da, in some embodiments up to about 1000 Da, e.g, up to about 900, 800, 700, 600 or up to about 500 Da.

As used herein, the terms “subject”, “patient”, and “individual” refer to a human or non-human animal having one or more eyes. Typically, the animal is a mammal. A subject also refers to for example, primates (e.g, humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human. The subject may be any age or gender.

As used herein, the term “treat”, “treating” or “treatment” of any ocular disease, ocular condition, or ocular disorder refers in one embodiment, to ameliorating the ocular disease, condition, or disorder (i.e., slowing or arresting or reducing the development of the disease, condition, or disorder, or at least one of the clinical symptoms thereof). In another embodiment “treat”, “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the subject. In yet another embodiment, “treat”, “treating” or “treatment” refers to reducing the severity of the ocular disease, condition, or disorder, either physically, (e.g, stabilization of a discernible symptom), physiologically, (e.g, stabilization of a physical parameter), or both. In yet another embodiment, “treat”, “treating” or “treatment” refers to prophylaxis (preventing or delaying the onset or development or progression of the ocular disease, condition, or disorder).

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

MATERIALS AND METHODS FOR EXAMPLE 1

Samples and sample preparation. Study samples of de-identified healthy human corneas and anterior segment sections were investigated. All samples derived from ocular surgery in which cornea or anterior segment was not the target tissue. Immunohistochemical staining for if G protein-coupled receptor GPR-68 was performed with a well established protocol using GPR68 Recombinant Rabbit Monoclonal Antibody (Fisher Scientific™ part of Thermo Fisher Scientific™ , Gothenburg, Sweden). The histological steps have been reviewed and illustrated [38] Basically the tissue samples were obtained from de-identified donor tissues from patients and were kindly provided by the histology biobank of St Eriks Eye Hospital . This research has been approved by the Swedish Ethical Review Authority (Dmo. 2021-06724-01) and is in accordance to the WMA Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects, in its current version of 2018 (https://www.wma.net/policies-post/wma-declaration-of-helsin ki-ethical-principles-for medical-research-involving-human-subjects) [39] The tissues had been initially fixed in of 4% paraformaldehyde (PFA) in and were then subsequently processed for paraffin embedding. After deparaffmization, 4 pm sections were cut and submitted to immunohi stochemi stry .

Immunohi stochemi stry . After Heat Induced Epitope Retrieval (HIER) using EDTA buffer (20 minutes) and peroxiblock, primary antibodies were added in a dilution of 1:200 (GPR68 Recombinant Rabbit Monoclonal Antibody; Fisher Scientific™ part of Thermo Fisher Scientific™ , Gothenburg, Sweden). Then a secondary antibody with the chromogene fast red was used. All steps of immunohi stochemi stry from deparaffmization to hematoxycilin staining were completely automized and performed in a Bond III robotic system (Leica Biosystems, Newcastle, UK). Images were acquired on an Axioskop 2 plus (Carl Zeiss, Germany). Considering the presence of tear film deficiencies on the lubrication issues, i.e., the clinical history as to dry eye diagnostics, the secretion stage of the tissues investigated at the time surgery is unknown but did not constitute the main diagnosis (mainly uveal melanoma). Example 1 - GPR68 in Human Corneal and Conjunctival Epithelium

Attrition and osmotic stress have been identified as major forces in dry eye pathophysiology. Impaired tolerance to mechano-transduction in presence of lubrication deficiencies has been associated with disturbance of ocular surface homeostasis and enhanced nudging of inflammatory reactions challenging the regulating coping mechanisms. In spite of the probable link between enhanced attrition and secondary inflammation, the key mediators driving the vicious circle of severe dry eye disease are not yet identified. The aim of this study was therefore to investigate human corneal and conjunctival epithelium for the presence of G-protein-coupled receptor 68 (GPR68). This protein had most recently been shown to be not only chemically activated but also mechanically, in that possibly by attrition.

De-identified sections of human cornea and conjunctiva were stained for the presence of G protein-coupled receptor 68 with specific antibodies using immunohistochemistry.

Specific staining for GPR68 was observed in all samples of the cornea throughout the epithelial layers of the corneal epithelium, most prominently in the area of the wing cells and the basement membrane level. Even in the conjunctival specific staining for GPR68 was found.

G-protein-coupled receptor 68 (GPR68) specific staining was observed in all samples throughout the epithelial layers of the corneal epithelium. The most prominent staining was in the area of the wing cells (yellow arrow, Figure 1A). However also at the basement membrane level intense staining for GPR-68 was observed (white arrow, Figure 1 A).

In conjunctival tissues G-protein-coupled receptor 68 (GPR68) was detected in a similar pattern as in the corneal epithelium that here even the superficial cell layers occasionally stained positive (yellow arrow, Figure IB), especially close to the limbal area (green arrow, Figure 4). Specific staining for GPR-68 was also found in the basal layers of the conjunctiva (yellow and blue arrow, Figure 4) the fornix but decreased towards the conjunctival of the lids (Figure 4).

Without being limited by theory' of mechanism of action, the mechanical activation of GPR68 in situations with enhanced friction and attrition could modify various cell functions and, possibly jeopardize normal inflammatory homeostasis at the ocular surface. Accordingly decreased lubrication in dry eye disease could result activation of GP68. This could lead to secondary inflammation initially in the epithelium and surrounding stroma. Continuous mechanical stress could result in chronic inflammation, reaching also deeper structures of the cornea, possibly making GPR-68 an important actor in the vicious circle of dry eye disease.

GPR68, sensitive to flow and mechanic stimulation, is present in the human corneal epithelium and conjunctiva. Decreased lubrication and increased attrition, accompanied by sensations typical for dry eye, might lead to local inflammation. It is possible that subtle signs of conjunctival and later corneal surface damage in context with sensations could be a better indicator for need and success of therapy than the clinical signs of dry eye disease alone, at least in the early stages of the disease. The inhibition of GPR68 could mark a new area in the treatment of dry eye disease.

MATERIALS AND METHODS FOR EXAMPLE 2

Samples and sample preparation. Study samples of de-identified healthy human lacrimal glands were investigated. All samples derived from orbital surgery in which the lacrimal gland tissue was not the target tissue. The histological steps have been reviewed and illustrated [38] Basically tissue samples were obtained from de-identified donor tissues from patients kindly provided by the histology biobank of St Eriks Eye Hospital in accordance with the declaration of Helsinki [39] and referring to the rules of the ethical committee as outlined by the Swedish Ethical Review Authority. The tissues had been initially fixed in of 4% paraformaldehyde (PFA) in and were then subsequently processed for paraffin embedding. After deparaffmization, 4 pm sections were cut and submitted to immunohi stochemi stry .

Immunohi stochemi stry . After (HER) Heat Induced Epitope Retrieval with EDTA buffer (20 minutes) and peroxiblock, primary antibodies were added in a dilution of 1:200 (GPR68 Recombinant Rabbit Monoclonal Antibody; FISHER SCIENTIFIC part of THERMO FISHER SCIENTEIC, Gothenburg, Sweden). Then a secondary antibody with the chromogene fast red was used. All steps of immunohi stochemi stry from deparaffmization to hematoxycilin staining were completely automized and performed in a Bond III robotic system (Leica Biosystems, Newcastle, UK). Images were acquired on an Axioskop 2 plus (Carl Zeiss, Germany). Example 2 - GPR68 in the Human Lacrimal Gland

The equivalent of dry eye as ocular surface disease often is a dysfunctionality of the lacrimal gland apparatus. The functionality of the lacrimal gland is of major importance for maintenance of ocular surface integrity and health even in conditions of enhanced stimulation and secretion requirements. Such enhanced secretion demands can push the lacrimal gland to the edge of its productive capacity with maximized tear fluid secretion with increased flow through the ducts of the lacrimal gland.

The aim of this study was to investigate if G protein-coupled receptor GPR-68 is present in the lacrimal gland as this protein had most recently been shown to be sensitive for flow and osmolarity. For this purpose, de-identified sections of human lacrimal gland tissues were stained for the presence of G protein-coupled receptor 68 with specific antibodies using immunohi stochemi stry .

Specific staining was detected in the acini and ducts of human lacrimal gland. In the ducts the specific staining was found around the lumen of the ducts. In the acini, the specific staining was observed more towards the lumen but also intercellularly between the acinar cells.

GPR68 was detected in the human lacrimal gland (Figure 5). The distribution, the intensity of staining over the field of observation was uneven (Figure 5). In some of the acini there was intense staining for GPR68 (Figure 6). The most profound staining was detected in the epithelium lining the excretory ducts of the lacrimal gland (Figure 7, arrow). Also, the epithelium lining the lumen of even larger ducts of the lacrimal gland stained positive for GPR-68 (Figure 8, arrow).

It was hence shown that GPR68 does occur in the lacrimal glands of humans. GPR68 specific staining is found in the acini as well as in the epithelium lining smaller and larger ducts of the gland. Whereas the staining in the ducts was seen in the majority of all samples investigated the intensity of specific staining for GPR68 various considerably between the acini.

Without being limited by theory of mechanism of action, the activation of GPR68 leads to the modification of various cell functions and is associated with the regulation of inflammation. Accordingly, enhanced, secretion induced, augmentation of flow could exert fluid flow stress to the ducts and acini. This could lead to temporary and localized activation of GPR68 and secondary inflammation within the gland. Depending on the intensity, continuity or repetitive character of the stimuli, exhaustion of the lacrimal gland secretion capacity could follow, and chronification of the inflammation in the parenchyme as well as around the ducts could be the consequence.

GPR68, sensitive to flow, is present in the human lacrimal gland. Increased flow, triggered by sensations even those typical for dry eye, might lead to local inflammation. It is possible that sensations could be a better indicator for need and success of therapy than the clinical signs of dry eye disease, at least in the early stages of the disease.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

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