WO2009137217A2 - Therapeutic replenishment and enrichment of ocular surface lubrication - Google Patents

Abstract

A pharmaceutical composition, and methods of use thereof, for treating ocular boundary deficiency, symptoms associated therewith, or undesired condition is disclosed The composition comprises a human PRG4 protein, a lubricant fragment, homolog, or isoform thereof, and may optionally one or more agents selected from the group consisting of a demulcent, excipient, astringent, vasoconstrictor, emollient, sodium hyaluronate, hyaluronic acid, or surface active phospholipids

Description

THERAPEUTIC REPLENISHMENT AND ENRICHMENT OF OCULAR SURFACE LUBRICATION

CROSS-REFERENCE TO RELATED APPLICATIONS |ooi l This patent application claims pπoπn benefit of U S Pro\ ιsional Application No 61/051 , 1 12 filed May 7, 2008, which is incorporated herein b\ reference

FIELD OF THE INVENTION

|002| The present inv ention relates to the management of ocular lubrication In particular, the present inv ention relates to pharmaceutical compositions, and method of use thereof, for treating diseases associated with compromised lubrication at the corneal and conjunctn al surfaces

BACKGROUND

[003] The proteoglycan 4 (prg4) gene encodes for highly glycos} lated proteins termed megakar\ oc\ te stimulating factor (MSF), lubπcin. and superficial /one protein (SZP) (/)) Lubπcin was first isolated from synovial fluid and demonstrated lubricating abilih m vitro similar to s> novιal fluid at a cartilage-glass interface (2) Lubricin was later identified as a product of s> novial fibroblasts (3) and also shown to possess boundary lubricating ability at a latex -glass interface b> Ja\ el al (3-9) O-linked β(l-3)Gal- GaINAc oligosaccharides within a large mucin like domain of 940 amino acids (]ϋ). encoded for b> e\on 6. were subsequently shown to mediate, in part, this boundan lubricating ability (tf) SZP was first localized at the surface of explant cartilage from the superficial /one and isolated from conditioned medium (I J) SZP also demonstrated lubricating ability at a cartilage-glass interface (12) These molecules are collectι\ el> referred to as PRG4 PRG4 was also shown to be present at the surface of s\ novιum (58), tendon (13). and meniscus (N) In addition. PRG4 has been shown to contribute, both at physiological and pathophλ siological concentrations, to the boundan lubrication of apposing articular cartilage surfaces (59)

|004| The functional importance of prg4 was shown by mutations that cause the camptodacU l> -arthropathλ -co\a \ ara-peπcardιlis (CACP) disease sx ndrome in humans CACP is manifest by camptodactyh , noninflammatory arthropathy and

8^ 14832 1 hypertrophic synovitis, u ilh co\a \ ara deformit\ , pericarditis, and pleural effusion (15) Also, in PRG4-null mice, cartilage deteπoration and subsequent joint failure were observed (16) Therefore, PRG4 expression is a necessary component of health\ s> no\ ιal joints [0051 PRG4 is a member of the mucin family, which are generalK abundant on epithelial linings and prowde many functions, including lubrication and protection from im ading microorganisms (17) The functional properties of mucins are generalK determined bv specialized gl\ cosylation patterns and their ability to form multimers through intermolecular disulfide bonds (/<"?). both of which are altered in chronic diseases (e g cvstic fibrosis asthma) (17) Biochemical characteπ/ation of PRG4 isolated from synovial fluid (2 19) showed molecular heterogeneitv in O-gh cosvlation. which appears to influence lubricating properties (<*?) Recently. PRG4 from bovine s\ no\ ιal fluid has been shown to exist as disulfide-bonded dimers, in addition to the monomeric forms, as suggested b\ the consen ed cvsteine-πch domains at both N- and C-terminals. along with an unpaired cysteine at the C-terminal (20)

[006| In tissues such as synoual joints. ph\ sicochemical modes of lubrication ha\ e been classified as fluid film or boundan The operative lubrication modes depend on the normal and tangential forces on the articulating tissues, on the relative rate of tangential motion between these surfaces, and on the time history of both loading and motion The friction coefficient, μ, provides a quantitati\ e measure, and is defined as the ratio of tangential friction force to the normal force One t) pe of fluid-mediated lubrication mode is h\ drostatic At the onset of loading and t> picallv for a prolonged duration, the interstitial fluid within cartilage becomes pressuπ/ed. due to the biphasic nature of the tissue, fluid ma\ also be forced into the aspeπties between articular surfaces through a weeping mechanism Pressuπ/ed interstitial fluid and trapped lubricant pools max therefore contribute significantly to the bearing of normal load with little resistance to shear force, facilitating a very low μ Also, at the onset of loading and/or motion, squeeze film. rr> drodvnamic, and elastohydrodynarruc t> pes of fluid film lubrication occur, with pressuπzation. motion, and deformation acting to dπ\ e viscous lubricant from and/or through the gap between two surfaces in relative motion

8^ 14812 I |007| The relevant extent to which fluid pressure/film versus boundan lubrication occurs classicalK depends on a number of factors (31) When lubricant film can flow between the conforming sliding surfaces, which can deform elasticallv, elastoh) drodynamic lubrication occurs Pressure, surface roughness, and relative sliding velocih determine when full fluid lubrication begins to break down and the lubrication enters new regimes As velocity decreases further, lubricant films adherent to the articulating surfaces begin to contribute and a mixed regime of lubrication occurs If the \elocity decreases ev en further and onl> an ultra-thin lubricant la\ er composed of a few molecules remain, boundan' lubrication occurs A boundan' mode of lubrication is therefore indicated by a friction coefficient (ratio of the measured fπctional force between two contacting surfaces in relativ e motion to the applied normal force) duπng steady sliding being invariant with factors that influence formation of a fluid film, such as relative sliding v elocity and axial load (35) For articular cartilage, it has been concluded boundan lubrication is certain to occur, although complemented b> fluid pressuπ/ation and other mechanisms (36-39)

|()08| In boundan lubrication. load is supported by surface-to-surface contact, and the associated fπctional properties are determined bv lubricant surface molecules This mode has been proposed to be important because the opposing cartilage la> ers make contact ov er ~10% of the total area and this ma¹* be where most of the friction occurs (30) Furthermore with increasing loading time and dissipation of h\ drostatic pressure, lubricant-coated surfaces bear an increasingly higher portion of the load relative to pressun/ed fluid, and consequently, this mode can become increasingly dominant (31 32) Boundan' lubrication, in essence, mitigates stick-slip (31), and is therefore manifest as decreased resistance both to stead\ motion and the start-up of motion The latter situation is relevant to load bearing articulating surfaces after prolonged compressn e loading (e g . sitting or standing in vivo) (33) T\ pical wear patterns of cartilage surfaces (34) also suggest that boundan' lubrication of articular cartilage is critical to the protection and maintenance of the articular surface structure

|009| With increasing loading time and dissipation of hy drostatic pressure, lubπcant- coated surfaces bear an increasingly higher portion of the load relative to pressurised fluid, and consequently, μ can become increasingly dominated by this mode of lubrication A boundan' mode of lubrication is indicated by values of μ duπng steady

8M48T2 I sliding being invariant with factors that influence formation of a fluid film, such as relative sliding velocity and axial load. Boundary lubrication, in essence, mitigates stickslip. and is therefore manifest as decreased resistance both to steady motion and the start-up of motion.

5 |ooio] The accumulation of PRG4 within synovial fluid and at the articular surface, are likely key functional determinants of PRG4^"s boundary lubricating ability. Recently, it was. demonstrated that a significant, threefold secretion of PRG4 resulted from the dynamic shear loading of cultured cartilage explants. as compared to free-swelling or statically compressed cultures (27). This PRG4 synthesis and secretion by chondrocytes

K) could significantly contribute to the concentration of PRG4 within synovial fluid, in both homeostatic and pathological conditions where physiological regulators are present (25). Although the amount of PRG4 bound to the surface does not appear to correlate with secretion rates, previous studies suggest surface bound PRG4 can exchange with endogenous PRG4 in synovial fluid (25). especially under the influence

15 of mechanical perturbation (26. 27). Clarification of the spatial and temporal aspects of PRG4 metabolism within the joint, particularly at the articular surface, would further the understanding of PRG4's contribution to the low-friction properties of articular cartilage, and possibly lead to treatments to prevent loss of this function (40. 41). More remains to be determined about the processing, and the potentially additional or 0 alternative functions of various PRG4 molecules of different molecular weight ( 10, 27. 28, 61). Moreover, the combination of chemical and mechanical factors to stimulate PRG4 expression in chondrocytes near the articular surface may be useful for creating tissue engineered cartilage from isolated sub-populations (29) with a surface that is bioactive and functional in lubrication. 5 [ooi l ] The precise mechanisms of boundary lubrication at biological interfaces are currently unknown. However, proteoglycan 4 (PRG4) may play a critical role as a boundary lubricant in articulating joints. This secreted glycoprotein is thought to protect cartilaginous surfaces against frictional forces, cell adhesion and protein deposition. Various native and recombinant lubricin proteins and isoforms have been 0 isolated and characterized. For instance, U.S. Patent Nos. 5,326,558; 6,433, 142; 7.030223, and 7.361.738 disclose a family of human megakaryocyte stimulating factors (MSFs) and pharmaceutical compositions containing one or more such MSFs for

S? MX? 2.1 treating disease states or disorders, such as a deficiency of platelets U S Patent Nos 6,960,562 and 6,743,774 also disclose a lubricating polypeptide, tπbonectin, compπsing a substantially pure fragments of MSF. and methods of lubricating joints or other tissues b> administering tπbonectin systemicallv or directly to tissues SUMMARY OF THE INV ENTlON

[00i2| The present im ention provides, in \ arious embodiments, pharmaceutical compositions, and methods of use thereof, for managing ocular lubrication, including the therapeutic replenishment and enrichment of boundary lubricant molecules at the ocular surface Described in certain embodiments of the present im ention is the observation that PRG4 mRNA is expressed in human corneal and conjunctival epithelial cells, as well as in mouse lacrimal and meibomian glands, indicating that PRG4 protein is presented in these tissues on the ocular surface Described in certain instances of the present invention is the observation that the role PRG4 protein sen es on the ocular surface is to protect the cornea and con|uncti\ a against significant shear forces generated during an e> elid blink, contact lens wear, and other undesirable conditions The impact of the tear film, including the impact of inflammation, proinflammatory cy tokines. se\ steroid imbalance and proteases on the composition and function of the films, suggest a course of therapy for ocular tissues which promotes boundan lubrication |0013| In certain embodiments, the present invention proudes a pharmaceutical composition suitable for topical application to an ocular surface comprising a therapeuticallv effective concentration of a PRG4 protein suspended in an ophthalmically acceptable balanced salt solution The pharmaceutical composition of the present invention may also comprise one or more ophthalmically acceptable agents selected from the group consisting of an ophthalmically acceptable demulcent, ophthalmicaJK acceptable exαpient, ophlhalmically acceptable astringent, ophthalmically acceptable vasoconstrictor, and ophthalmicalK acceptable emollient

[0014] Exemplary ophthalmicallx acceptable demulcents contemplated in the present im ention include, but are not limited to, carbo\\ meth\ icellulose sodium (e g . about 0 2 to 2 5% w/v), h> droxyeth\ 1 cellulose (e g , about 0 2 to 2 5% w/v), h> promellose

(e g . about 0 2 to 2 5% w/\ ). meth> lcellulose (e g . about 0 2 to 2 5% wΛ ), dextran 70

8* 14812 I (e g . about 0 1 % w/v), gelatin (e g . about 0 01 % \v/v), glycerin (e g . about 0 2 to 1% w/v), pol\ ethylene gh col 300 (e g . about 0 2 to 1 % w/v), poh ethy lene glycol 400 (e g , about 0 2 to 1 % w/v). poK sorbate 80 (e g . about 0 2 to 1% w/v). propylene gh col (e g . about 0 2 to 1 % \\/\ ). pol\ \ in\ l alcohol (e g . about 0 1 to 4% w/v). povidone (e g , about 0 1 to 2% w/v) Exemplary ophthalmicalK acceptable excipients/emollients contemplated in the present in\ention include, but are not limited to. anrn drous lanolin (e g . about 1 to 10% w/v). lanolin (e g , about 1 to 10% w/\ ), light mineral oil (e g . < about 50% \v/\ ), mineral oil (e g , < about 50% w/v). paraffin (e g . < about 5% w/v), petrolatum (e g . < about 100% w/\ ). white ointment (e g . < about 100% w/\ ). white petrolatum (e g . < about 100% w/v), white wax (e g . < about 5% w/v). yellow wax (e g . < about 5% w/v) An exemplar) ophthalmicalh acceptable astringent contemplated in the present im enlion includes, but is not limited to. /inc sulfate (e g , about 0 25% w/\ ) Exemplar) ophthalmicalh acceptable vasoconstrictors contemplated in the present invention include, but are not limited to ephedπne hy drochloride (e g , about 0 123% w/v). naplwolme hydrochloride (e g . about 0 01 to about 0 03% w/\ ). phenylephrine hydrochloride (e g . about 0 08 to about 0 2% w/\ ), and tetrahydro/ohne hydrochloride (e g . about 0 01 to about 0 05% w/v)

[0015] In some of these embodiments, the demulcents, excipients, astringents, vasoconstrictors, emollients and electroly tes provide a means to deln er the PRG4 protein in an ophthalmicall\ acceptable manner Ophthalmically acceptable compositions are suitable for topical application to the ocular surface if the\ lack unacceptable eye toxicity, burning, itchiness, viscosity, blurred v ision, etc upon application

|00l6| In certain embodiments, the pharmaceutical composition of the present invention fuither comprises a iherapeuticaJh effectiv e concentration of one or more additional therapeutic agents, including but not limited to, sodium h> aluronate, h> aluronic acid, and phospholipid Exemplary' phospholipid includes, but is not limited to, L-α-dipalmiloylphosphalidylchohne, ' phosphatidylcholine, phosphatidλ lethanolamine and sphingorm elm |00i7[ In certain embodiments, the present invention proudes a pharmaceutical composition suitable for topical application to an ocular surface comprising a therapeutically effective concentration of PRG4 protein suspended in an ophthalmicalh

8M4812 1 acceptable balanced salt solution comprising at least three electrolytes, including but not limited to. sodium chloride (NaCl) 0 64%. potassium chloride (KCl) 0 075%, calcium chloride dirn drate (CaC12«2H2O) 0 048%, magnesium chloride hexahydrate (MgC12^«6H2O) 0 03%, sodium acetate tπhydrate (C2H3NaO2^«3H2O) 0 39%, sodium citrate dehydrate (C6H5Na3O7«2H2O) 0 17%. sodium hydroxide and/or hx drochloric acid (to adjust pH to approximateh 7 5) with an osmolarity of approximateh 300 mOsms/L

10018] In certain embodiments, the present im ention proudes a pharmaceutical composition suitable for topical application to an ocular surface comprising a therapeutically effective concentration of PRG4 protein suspended in an ophthalmicallv acceptable balanced salt solution, comprised of sodium (Na+) of approximateh 128 mM. potassium (K+) of approximateh 24 mM chloride (Cl-) of approximateh 1 13 mM. calcium (Ca2+) of approximateh 0 4 mM. magnesium (Mg2+) of approximateh 0 3 mM, HCO3- of approximately 5 mM. citrate of approximateh 1 mM. phosphate of approximateh 14 mM, acetate of approximateh 15 mM, and sodium hydroxide and/or hydrochloric acid (to adjust pH to approximateh 7 5) with an osmolaπtv of approximateh 300 mOsms/L

[0019] The present invention further provides a method for treating ocular lubrication deficiency, or s\ mptoms associated therewith, in an indn idual in need The method compπses topically administering to the ocular surface of the individual in need a pharmaceutical composition compπsing a therapeutically effective concentration of a PRG4 protein In certain embodiments, the pharmaceutical composition compπsing the PRG4 protein is administered in combination with an ophlhalmicalh acceptable formulation comprising one or more ophthalmicalh acceptable agents selected from the group consisting of an ophthalmicalh acceptable demulcent ophthalmicalh acceptable excipient ophthalmicalh acceptable astringent ophthalmicalh acceptable v asoconstrictor and ophthalmicalh acceptable emollient

[0020] In some embodiments, the pharmaceutical composition comprising the PRG4 protein is administered in combination with an ophthalmically acceptable solution compπsing a therapeutical h effective concentration of sodium

aluronate or hyaluronic acid, or a surface active phospholipid, as discussed above In vet certain embodiments, the pharmaceutical composition comprising the PRG4 protein is

8λ |4)H2 1 administered in combination with a phosphate buffered saline solution or an ophthalmically acceptable balanced salt solution comprising one or more electroK tes. as discussed abo\ e

|002l| The present invention provides a method for treating a deficiencx in ocular lubrication or symptoms associated therewith, that due to tear loss or unstable tear film in the ocular boundary loop, such as androgen deficiencx , Sjogren^'s

ndrome and keratoconjunctiv itis sicca (KCS) Such method comprises topicalh administering to the ocular surface of a patient in need the pharmaceutical composition of the present invention |0022i In certain embodiments, the present invention further provides a method for addressing and treating the conditions associated with unfa\ orable or deficient ocular lubrication Exemplary conditions include, but are not limited to aqueous or e\ aporati\ e dr\ eye disease. Sjogren^'s s-s ndrome. keratoconjunctmtis sicca, androgen deficiency, meibomian gland disease, estrogen replacement therapy, contact lens wear. refractive surger> . allergy , reduced tear film breakup time, allergy, ocular surface disorders, increased protease levels in the tear film and at the ocular surface, chronic inflammation.

perosmolarm , and aging

BRIEF DESCRIPTION OF THE DRAWINGS

|0023] Figure 1 represents feedback loops within ocular surface boundary lubrication |0024| Figure 2 illustrates PRG4 mRNA expression in human corneal epithelial cells Human corneal epithelial cells were isolated from the corneoscleral rims of male and female donors Amplified samples were screened for the presence of PRG4 products b\ using an Agilent 2100 Bioanalwer Vertical lanes contain L MW ladder. 1 No template control. 2 Corneal tissue from a 33-year female. 4 Cultured corneal epithelial cells from a 70-year female, 6 Cultured corneal epithelial cells from a 53-year male

|0025l Figure 3 illustrates PRG4 mRNA expression in human conjunctiv al epithelial cells Human corneal epithelial cells were isolated from the corneoscleral rims of male and female donors Amplified samples were screened for the presence of PRG4 products by using agarose gel electrophoresis Vertical lanes contain 1 MW ladder. 2 No template control. 4 Human female conjunctiva. 5 Human male conjunctiva

Sϊ 148 '2 I |0026] Figure 4 illustrates PRG4 mRNA expression in human corneoscleral rim tissue samples L Human corneal epithelial cells were isolated from the corneoscleral rims of male and female donors Amplified samples were screened for the presence of PRG4 products tn using an Agilent 2100 Bioanal\ /er Vertical lanes contain MW ladder. 1 Human liver cDNA standard. 2 Corneoscleral rim tissue from a 24-\ ear female 3 Corneoscleral rim tissue from a 51 -year female. 4 Human conjunctival epithelial cells

|0027| Figure 5 illustrates PRG4 mRNA expression in human conjunctival impression c\ tolog> samples Conjunctival impression cuology samples were isolated from male and female donors Amplified samples were screened for the presence of PRG4 products bλ using an Agilent 2100 Bioanalv/er Vertical lanes contain L MW ladder 1-9 Conjunctiv al impression c\ tologv samples. 10 Repeat of human conjunctival epithelial cells (Lane 4 in Figure 3)

|0028] Figure 6 illustrates a friction test schematic The corneal ocular surface (605) was fastened to the spherical end of an inert non-permeable semi-rigid rubber plug c\ linder (603) (radius r=6 mm) The plug c\ linder (603) was attached to the rotational actuator of the mechanical testing machine (Bose ELF 3200) forming the bottom articular surface An annulus (601 ) (outer radius=3 2 mm. inner radius=l 5 mm) was punched from the eyelid (604) The annulus (601) was attached to the linear actuator coupled w ith an axial load (N) and torsion (τ) load cells, forming the upper articulating surface Lubπcant bath (602) was formed b\ securing an inert tube around the plug o, linder (603) ω is the angular frequencv

[0029| Figure 7 illustrates the reduction of in vitro lid/cornea kinetic friction w ith addition of PRG4 protein (lubncin)

[003U) Figure 8 illustrates the reduction of in vitro lid/cornea kinetic fπction measured 1 minute after the addition of PRG4 protein (lubncin)

|003i i Figure 9 illustrates the reduction of //? vitro lid/cornea kinetic fπction measured 5 minutes after the addition of PRG4 protein (lubncin)

|0032| Figure 10 illustrates the reduction of in vitro lid/cornea kinetic friction over lime following addition of PRG4 protein (lubncin)

fn i4iπ2 i DETAILED DESCRIPTION OF THE INVENTION

|()033| Prov ided in certain embodiments herein, is a method for treating ocular lubrication defiαenc\ (e g . ocular boundan lubrication deficiency ), or symptoms associated therew ith, in an indmdual in need thereof comprising topicalK administering to the ocular surface of the individual a pharmaceutical composition comprising a therapeutically effectiv e amount of PRG4 protein Also prov ided in some embodiments herein are pharmaceutical compositions comprising PRG4 protein in an ophthalmically acceptable formulation In specific embodiments, provided herein is a pharmaceutical composition suitable for topical application to an ocular surface comprising a therapeutically effectι\ e amount of PRG4 suspended in an ophthalmicallv acceptable balanced salt solution, and ma\ also be in combination w ith one or more ophthalmicalK acceptable agents selected from the group consisting of an ophthalmically acceptable demulcent, an ophthalmicallv acceptable excipient, an ophthalmically acceptable astπngent. an ophthalmicalK acceptable vasoconstrictor, and an ophthalmically acceptable emollient

|0034] Provided in some embodiments herein are pharmaceutical compositions, and methods of use thereof, for treating a deficiency in ocular lubrication at the ocular surface (e g . a deficiency of. such as decreased or undesirable, ocular boundan lubrication) A pharmaceutical composition of certain embodiments of the present invention compπses an isolated or purified PRG4 protein suspended in an ophthalmicalK acceptable balanced salt solution in combination with one or more ophthalmic agents selected from the group consisting of an ophthalmic demulcent, excipient, astπngent, vasoconslructor, and emollient In some embodiments, any pharmaceutical composition provided herein further comprises one or more additional therapeutic agents selected from the group consisting of sodium h> aluronate. surface active phospholipids, and electrolytes in a pharmaceutically acceptable carrier for topical administration

|0035| The present invention prov ides, in certain embodiments, a novel approach to manage ocular lubrication, including the therapeutic replenishment and enrichment of boundarv lubricant molecules at the ocular surface It should be noted that the importance and the mechanism of ocular boundarv lubrication has not heretofore been recogni/ed w ithin the ophthalmic communih For \ ears, the scientific consensus within

8^ 14812 1 I O the orthopaedic research communiu was that h\ drod\ namic lubrication was by far the dominant mode of lubrication for articular cartilage, and that boundary lubrication w as simply an afterthought Moreover, those researchers studying boundars lubrication at cartilage surfaces suggest that boundary lubrication is likely only important under "high load and low v elocih ," which are opposite to the conditions at the ocular surface, where there are relativ ely low axial loads and relativel\ fast sliding velocities See. e g , (54) Moreo\er, boundan lubrication involving the corneal glyocalyx has not heretofore been considered Jay et al compared purified lubricating factor from bovine s\ novial fluid to "mucinous gly coprotein from human submandibular saliva and stimulated tears.^"' and concluded "mucin secreted by the lacrimal gland did not lubricate.^"' overlooking the possibility that the corneal epithelium was a source of lubricant or that boundary lubrication was an important contributor at the ocular surface See, e g . (55) The most recent mathematical models of tear film d\ namics also ignore the possibiliU of boundary lubrication, claiming a "lubrication approximation^" for the height of the tear film such that "the mucus layer on the cornea can be taken to prov ide a no-slip surface for the aqueous film" and that "it should be noted that the model only predicts the ev olution prior to the |tear film| thickness reaching some cπticalK thin \ alue al which the model breaks down ^" See. e g . (57)

[0036| There is a need to manage ocular lubrication and protect the cornea and conjunctiv a against significant shear forces generated from the ^'undesirable conditions described herein, including, bv wa\ of non-limiting example, aqueous or evaporative eve disease, Sjogren^'s s\ ndrome. keratoconjunctivitis sicca, androgen deficiency. meibomian gland disease, estrogen replacement therap\ , contact lens wear, refractive surgerv. allergy, reduced tear film breakup time, allergy, ocular surface disorders, increased protease levels in the tear film and at the ocular surface, chronic inflammation. h\ perosmolaπty. and aging

|0037| In some instances, the loading of cornea and conjunctiv a is likeK dominated b\ shear forces In certain instances. e\ elid blinking, as well as contact lens wear, generates significant stress upon ocular surface epithelial cells, and this is especialh true in the presence of a compromised tear film As shown in Figure 1 , it is suggested that incieased sheai stress leads to tear film instability, evaporativ e tear loss. hyperosmolaπty, changes in swelling pressure and a feedback elevation in shear stress

8314832 1 | | In some instances, increased shear stress is also thought to promote inflammation, androgen deficiency and decreased expression of proteogh cans In certain instances increased shear stress and its sequelae may. over time, lead to a loss of boundan lubrication at the ocular surface (00J8| A deficienc> in ocular lubrication and s\ mptoms associated therewith can be determine by am suitable method In some instances, a deficiency in ocular lubrication and symptoms associated therewith is defined either qualitatnely (e g , a feeling of low lubrication, dry eye, discomfort, etc ) or quantitatively (e g , measured through mechanical, biochemical, electrical, optical or other methods of quantitative assays)

[0039| In certain instances, in undesirable conditions for ocular boundan lubrication, such those resulting from aqueous or e\ aporatn e drv eye disease. Sjogren^'s s> ndrome, keratoconjunctiutis sicca, androgen deficienc} . meibomian gland disease, estrogen replacement therapy, contact lens wear, refiactive surgery, allergv reduced tear film breakup time, allergy, ocular surface disorders, increased protease lex els in the tear film and at the ocular surface, chronic inflammation, hyperosmolar^ , and aging, a compromised tear film will exist In some of these situations, increased e\ aporation may preclude efficient fluid film lubrication, but allow boundan' lubrication and a molecular sacrificial mechanism to reduce shear stress at the cell surface Certain embodiments of the present invention provide that therapeutic replenishment and enrichment of boundan lubricant molecules at the ocular surface would interrupt the feedback loop through which the unfa\ orable conditions associated w ith a deficiency in ocular lubrication promote ocular surface distress

[0040] In certain instances, and as provided herein, PRG4 protein plays a critical role in the eye as a boundan lubricant In some instances, this secreted glycoprotein protects the ocular surface to protect the cornea and conjunctiva against significant shear forces generated during an ex elid blink, contact lens wear, and am other undesirable ocular boundary lubrication caused by chronic inflammation and hyperosmolar!!} that result from dr> eye disease, androgen deficiency, estrogen replacement therapx , compromised tear film, allergλ . aging, ocular surface diseases, and increased protease levels in the tear film and at the ocular surface Given the relationship between osmotic pressure and the electromechanical interactions within charged molecules, the present invention provides, in some embodiments, a

8^ 14812 1 1 2 pharmaceutical composition for managing a deficiency in ocular lubrication bv modulating hyperosmolaπty or osmolaπt} at the ocular surface via interrupting the feedback mechanisms that pres ent secreted components from reducing friction coefficients and mitigating shear stress (0(Mt] In another exemplars embodiment, the present invention features a sacrificial mechanism for ocular boundars lubrication, whereto surface bound receptors re\ersibly bind one or more gel forming or surfactant constructs In some instances, the gel forming or surfactant constructs detach during a shear event, thereto preventing the shear stress from reaching (or reducing the shear stress reaching) the epithelial surface In certain embodiments, following the transient shearing event, the gel forming and surfactant constructs, allowed to return to their undisturbed equilibrium, rebind to the surface bound receptors In some embodiments, the entire construct can detach during shear One could imagine, in certain instances that the thermod\ namics of this equilibrium would increase the probabilih of release from the receptor w ith increasing shear amplitude, but that anv one association is easilv re\ ersible

(00-12] In one embodiment of the current im ention. the pharmaceutical composition comprising a PRG4 protein suspended in an ophthalmically acceptable balanced solution is applied topicalh to the ocular surface, where the PRG4 protein associates or binds to In certain instances of this embodiment. PRG4 acts as the surface bound receptor that is allowed to interact with endogenous proteins and proteoglvcans within the tear film to establish a sacrificial mechanism to reduce the friction during

ehd blinks at the ocular surface pres ent protein adsorption at the ocular surface, and reduce dr\ spots caused b\ tear film instabilih

|0(M3| In another embodiment of the current im ention. PRG4 is applied lopicalh and associates or binds to the ocular surface in combination with one or more of

aluronic acid and phospholipid constiucts In certain instances of this emodimenl. PRG4 acts as the suiface bound receptor that interacts w ith the exogenously supplied hyaluronic acid and/or phospholipids to establish the sacrificial mechanism to reduce the friction during e> elid blinks at the ocular sui face, prevent protein adsorption at the ocular surface, and reduce drv spots caused by tear film instabiliU In this embodiment, the hyaluronic acid and phospholipid constructs disassociate from the PRG4 during a shear ev ent In \ et

8" 14812 I | 3 another embodiment, the entire construct detaches during a shear event to prevent the shear stress from reaching the epithelium.

[0044| In yet another embodiment, functional fragments., mullimers (e g , dimers. tπmers. tetramers. etc.). homologs or orthologs of PRG4 act as the surface receptor and/or gel forming constructs in the sacrificial mechanism Functional fragments and homologs of PRG4 include those with a fewer repeats within the central mucin-like

KEPAPTT-repeat domain, glycosylated and non-glycosylated forms of the protein, splice variants, recombinant forms, and the like. A lubricating fragment of PRG4 exhibits at least 20%. 30%. 40%. 50%. 60%. 70%. 80%. 90%. or 95% of the ophthalmic lubricating effect of human PRG4. as measured qualitatively, mechanically. optically, electrically, or by biochemical assa\

[0045] As used herein, the term "PRG4^". "PRG4 protein^" or "proteoglycan 4^" protein, is used interchangeably with the term " lubricin^" protein PRG4 is used herein also to encompass the term megakaryocyte stimulating factor (MSF). that has been accepted for the UCL/HGNC/HUGO Human Gene Nomenclature data base, and superficial zone protein (SZP). The PRG4 or lubricin protein as used herein refers to any isolated or purified native or recombinant lubπcm proteins, homologs, functional fragments or motifs, isoforms, and/or mutants thereof. In certain embodiments, the isolated or purified PRG4 protein comprises an amino acid sequence for a human native or recombinant lubricin protein. In other embodiments, the isolated or purified PRG4 protein comprises an amino acid sequence encoded by prg-tgene exons that encode the full length PRG4 protein or isoforms^' primary structures The proteoglycan 4 (prg4) gene contains 12 exons The PRG4 protein used herein comprises an amino acid sequence encoded by prg-Jgene exons 1-12. more preferably, exons 6- 12. and most preferably, exons 9- 12.

|0046| As used herein, the PRG4 protein includes any PRG4 proteins now known, or later described. In certain embodiments, a preferred PRG4 protein amino acid sequence is prouded in SEQ ID NO: I The PRG4 protein shares the primary amino acid structure of any known PRG4 proteins or isoforms with at least 60% homology. preferably 75% homology, more preferably 85%, 90%, 95%. 96%. 97%, 98%. 99% or

• more homology In certain embodiments, a preferred PRG4 protein has an average molar mass of between 50 kDa and 400 kDa. compπsing one or more biological active

8? 14832 1 14 portions of the PRG4 protein, or functional fragments, such as a lubricating fragment, or a homolog thereof

|0047] As used herein, the PRG4 protein comprises a biological active portion of the protein As used herein, a "biologicalh actn e portion^"' of the PRG4 protein includes a functional fragment of a protein comprising amino acid sequences sufficientK homologous to, or deriv ed from, the amino acid sequence of the protein, which includes fewer amino acids than the full length protein, and exhibits at least one acimh of the full-length protein Typically a biologicalK active portion comprises a functional domain or motif with at least one activih of the protein A bιologιcall\ active portion of a protein can be a poly peptide which is. for example, K), 25. 50. 100. 200. or more amino acids in length In one embodiment, a biologically actn e portion of the PRG4 protein can be used as a therapeutic agent alone or in combination w ith other therapeutic agents for treating undesirable or decreased ocular boundar> lubrication

|0(M8] The nucleic acid and amino acid sequences of several nativ e and recombinant PRG4 or lubπcin proteins, and characteπ/ation of the PRG4 proteins and various isoforms are disclosed in. for instance. U S Patent Nos 5.326.558. 6 433.142. 7,030.223. 7.361.738 to Turner et al . and U S Patent Nos 6.743.774 and 6.960.562 to Jaλ et al U S Publication No 20070191268 to Flannery et al also discloses recombinant PRG4 or lubricin molecules useful in the present invention |0(M9) Methods for isolation, purification, and recombinant expression of a PRG4 protein are well known in the art In certain embodiments, the method starts with cloning and isolating mRNA and cDNA encoding PRG4 proteins or isoforms using standard molecular biolog\ techniques, such as PCR or RT-PCR The isolated cDNA encoding the PRG4 protein or isoform is then cloned into an expression vector, and further transformed and expressed in a host cell for producing iecombinant PRG4 protein

|ooso] As used herein, "recombinant^" refers to a poly nucleotide s> nthesi/ed or otherwise manipulated in vitro (e g , '"recombinant polynucleotide^"), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a poh peptide ("recombinant protein") encoded b> a recombinant polynucleotide "Recombinant^" also encompasses the ligation of nucleic acids having

8M48^2 I | 5 \ aπous coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of. e g . inducible or constitutiv e expression of a fusion protein comprising an active domain of the PRG4 gene and a nucleic acid sequence amplified using a primer of the invention

5 (0051] In certain embodiments, the PRG4 protein encoding nucleic acid ma\ contain one or more mutations, deletions, or insertions In such embodiments, the PRG4 protein encoding nucleic acid is at least 60% homology , preferabh 75% homology more preferabh 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology , to a wild t> pe PRG4 protein encoding nucleic acid

K) [0052) As used herein, the term "cDNAs^" includes DNA that is complementary to mRNA molecules present in a cell or organism mRNA that can be convened into cDNA w ith an en/yme such as reλ erse transcriptase In certain embodiments, the cDNA encoding PRG4 protein is isolated from PRG4 mRNA expressed in human corneal or coniunctn al epithelial cells using an RT-PCR method well known in the art

15 |0053] As used herein, the terms "poh nucleotide.^" "nucleic acid/nucleotide.^" and "oligonucleotide^"" are used interchangeably, and include poh meπc forms of nucleotides of anv length, either deoxvπbonucleotides or ribonucleotides, or analogs thereof Pol> nucleotides ma\ have any three-dimensional structure, and ma\. perform anv function, known or unknown The following are non-limiting examples of 0 polynucleotides a gene or gene fragment, exons introns. messenger RNA (mRNA),

. transfer RNA. πbosomal RNA. πbo/) mes. DNA. cDNA, genomic DNA. recombinant polynucleotides, branched pol> nucleotides, plasmids. vectors, isolated DNA of any sequence, isolated RNA of am sequence, nucleic acid probes, and pπmers

PoK nucleotides ma> be naturally-occurring, synthetic, recombinant or am combination 5 thereof

[0054] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs If present, modifications to the nucleotide structure may be imparted before or after assembh of the poK mer The sequence of nucleotides mav be interrupted b> non-nucleolide components A polynucleotide ma\ be further 0 modified after pol\ meπ/ation. such as b\ conjugation with a labeling component The term also includes both double- and single-stranded molecules Unless otherwise

8^ I48^"«2 I K, specified or required, any embodimenl of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single- stranded forms known or predicted to make up the double-stranded form.

|0055] As used herein, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cylosine (C); guanine (G): thymine (T); and uracil (LJ) in place of thymine when the polynucleotide is RNA, instead of DNA. This alphabetical representation can be inputted into databases in a computer and used for bioinformatics applications such as. for example, functional genomics and homology searching.

[0056] As used herein, the term "isolated polynucleotide/cDNA" includes polynucleotide molecules which are separated from other polynucleotide molecules which are present in the natural source of the polynucleotide. For example, with regard to genomic DNA. the term "isolated^" includes polynucleotide molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated^" polynucleotide is free of sequences which naturally flank the polynucleotide (i.e.. sequences located at the 5^" and 3' ends of the polynucleotide of interest) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide molecule encoding the PRG4 protein used in the invention can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, I kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the polynucleotide molecule in genomic DNA of the cell from which the polynucleotide is derived. Moreover, an "isolated" polynucleotide molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

|0057| As used herein, a "gene^" includes a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotide sequences described herein may also be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art. As used herein, a "native or naturally-occurring"

8314832.1 I₇ pol\ nucleotide molecule includes, for example, an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e g , encodes a natural protein)

|()058) As used herein, the term "poh peptide" or "protein" is interchangeable, and includes a compound of two or more subunit amino acids, ammo acid analogs, or peptidomimetics The subunits ma\ be linked bv peptide bonds In another embodiment, the subunit mav be linked b> other bonds, e g , ester, ether, etc As used herein, the term "amino acid^"' includes either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics A peptide of three or more amino acids is commonK referred to as an oligopeptide Peptide chains of greater than three or more amino acids are referred to as a polypeptide or a protein

|()059) In certain embodiments, the PRG4 protein used herein refers to PRG4 proteins or \aπous homologs or isoforms thereof, that are naturalK or recombinanth expiessed in humans or other host cells As used herein, "express^" or "expression^" includes the process b\ which polynucleotides are transcribed into RNA and/or translated into polypeptides If the polynucleotide is derπ ed from genomic DNA, expression ma> include splicing of the RNA, if an appropriate eukaryotic host is selected Regulator elements required for expression include promoter sequences to bind RNA poh merase and transcription initiation sequences for πbosome binding For example, a bacterial expression \ector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG Similarh , a eukan otic expression vector includes a heterologous or homologous promoter for RNA poh merase II. a dow nstream poh adem lation signal, the start codon AUG. and a termination codon for detachment of the πbosome Such vectors can be obtained commercially or assembled b\ the sequences descπbed in methods well known in the art for example, the methods described below for constructing \ectors in general As used herein, the term "vector^" includes a self-replicaling nucleic acid molecule that transfers an inserted polynucleotide into and/or between host cells The term is intended to include vectors that function pπmaπh for insertion of a nucleic acid molecule into a cell, replication \ ectors that function pπmaπh for the replication of nucleic acid and expiession vectois that function foi transcription and/oι tianslation of the DNA oi RNA Also intended are \eclors that pro\ ide more than one of the above function

8M4812 I I S |0060] As used herein, a "host cell^" is intended to include any individual cell or cell culture which can be. or has been, a recipient for vectors or for the incorporation of exogenous pol\ nucleotides and/or poK peptides It is also intended to include piogem of a single cell The progem ma\ not necessarily be complete!) identical (in morpholog\ or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation The cells ma\ be prokaryotic or eukaryotic, and include but are not limited to bacterial cells. > east cells, insect cells, animal cells, and mammalian cells, including but not limited to murine, rat. simian or human cells As used herein, a "host cell'^" also includes genetically modified cells T he term "geneticall) modified cells^" includes cells containing and/or expressing a foreign or exogenous gene or poh nucleotide sequence which in turn modifies the genot> pe oi phenoK pe of the cell or its progem "Genelicalh modified^" also includes a cell containing or expressing a gene or polynucleotide sequence which has been introduced into the cell For example, in this embodiment, a geneticalh modified cell has had introduced a gene which gene is also endogenous to the cell The term geneticalh modified^" also includes am addition, deletion, or disruption to a cell^'s endogenous nucleotides As used herein, a "host cell^" can be any cells that express a human PRG4 protein

[0061] As used herein, "homologs^" are defined herein as two nucleic acids or peptides that have similar, or substantialh identical, nucleic acids or amino acid sequences, respectively The term '"homolog^" further encompasses nucleic acid molecules that differ from one of the nucleotide sequences due to degenerac} of the genetic code and thus encodes the same amino acid sequences In one of the preferred embodiments. homologs include allelic \ ariants. orthologs. paialogs. agonists, and antagonists of nucleic acids encoding the PRG4 protein (e g . SEQ ID NO 1 )

|0062| As used herein, the term " orthologs^" refers to two nucleic acids from different species, but that have ev olved from a common ancestral gene by speciation Normally, orlhologs encode peptides having the same or similar functions In particular, orthologs of the invention will generally exhibit at least 80-85%, more preferabl) 85-90% or 90- 95%, and most preferably 95%. 96%. 97%, 98%, or even 99% identity, or 100% sequence identiK . with all or part of the amino acid sequence of anv known PRG4 proteins (e g , SEQ ID NO 1 ), isoforms, or analogs thereof, and will exhibit a function similar to these peptides As also used herein, the term ""paralogs^" refers to two nucleic acids that are related by duplication within a genome Paralogs usually ha\ e different functions, but these functions may be related

|0063| To determine the percent sequence identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e g . gaps can be introduced in the sequence of one pol\ peptide for optimal alignment w ith the other pol\ peptide or nucleic acid) The amino acid residues at corresponding amino acid positions are then compared When a position in one sequence is occupied b\ the same amino acid residue as the corresponding position in the other sequence, then the molecules are identical at that position The same U pe of comparison can be made betw een two nucleic acid sequences The percent sequence identity between the two sequences is a function of the number of identical positions shared by the sequences (i e , percent sequence identit} = numbers of identical positions/total numbers of positions \ 100) Preferabty . the isolated amino acid homologs included in the present im ention are at least about 50-60%. preferably at least about 60-70%, and more preferably at least about 70-75%. 75-80%. 80-85%. 85-90%, or 90-95%, and most preferabh at least about 96%. 97%. 98%. 99%, or more identical to an entire ammo acid sequence of any known PRG4 protein (e g , SEQ ID NO 1)

|0064) In certain embodiments, an isolated nucleic acid homolog encoding the PRG4 protein comprises a nucleotide sequence which is at least about 40-60% preferably at least about 60-70%. more preferabh at least about 70-75%. 75-80%, 80-85%. 85-90%. or 90-95%. and e\ en more preferabh at least about 95%. 96%. 97%. 98%. 99%. or more identical to a nucleotide sequence encoding amino acid sequences of such PRG4 protein (e g . SEQ ID NO 1 ) |0t)65| The determination of the percent sequence identity between two nucleic acid or peptide sequences is well known in the art For instance, the Vector NTl 6 0 (PC) software package (inforMαx, Bethesda. MD) to determine the percent sequence identity between two nucleic acid or peptide sequences can be used In this method, a gap opening penalty of 15 and a gap extension penalty of 6 66 are used for determining the percent identitv of two nucleic acids A gap opening penalty of I O and a gap extension penalty of 0 1 are used for determining the percent identity of two polypeptides All other parameters are set at the default settings For purposes of a multiple alignment

8^ J4»%2 I 20 (Clustal W algorithm), the gap opening penalty is 10, and the gap extension penalu is O 05 with blosum62 matrix It is to be understood that for the purposes of determining sequence identitx when comparing a DNA sequence to an RNA sequence, a tin midine nucleotide is equivalent to a uracil nucleotide [0066| Furthermore, the PRG4 protein used herein includes PRG4 protein encoded b\ a poh nucleotide that hybridizes to the poh nucleotide encoding PRG4 protein under stringent conditions As used herein, "h\ bπdi/ation'^" includes a reaction in which one or more polynucleotides react to form a complex that is stabilized via hx drogen bonding between the bases of the nucleotide residues The h\ drogen bonding ma\ occur b} Watson-Crick base pairing. Hoogstein binding, or in any other sequence- specific manner The complex may compπse h\ o strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-h\ bπdι/ιng strand, or am combination of these A hvbπdi/ation reaction may constitute a step in a more extensn e process, such as the initiation of a PCR reaction, or the en/\ malic cleavage of a poh nucleotide b> a πbo/yme

[0067| H> bπdi/atιon reactions can be performed under different stringent conditions The present inv ention includes poh nucleotides capable of h) bπdi/ing under reduced stringency conditions, more preferabh stringent conditions, and most pieferabh highh stringent conditions, to poh nucleotides encoding PRG4 protein described herein As used herein, the term "stringent conditions^" refers to h\ bπdi/ation o\ ernight at 60⁰C in 1Ox Denhart's solution. 6xSSC. 0 5% SDS, and 100 mg/ml denatured salmon sperm DNA Blots are washed sequentially at 62°C for 30 minutes each time in 3xSSC/0 1 % SDS. followed b> lxSSC/0 1% SDS, and finalh 0 IxSSC/0 1% SDS As also used herein, in certain embodiments, the phrase "stringent conditions^" refers to hybπdi/ation in a 6xSSC solution at 65°C In other embodiments, "highly stringent conditions^"' refer to hybridization overnight at 65°C in lOxDenhart^'s solution, 6xSSC, 0 5% SDS and 100 mg/ml denatured salmon sperm DNA Blots are washed sequentially at 65°C for 30 minutes each time in 3xSSC/0 1% SDS. followed by l xSSC/0 1% SDS, and finalh 0 IxSSC/O 1% SDS Methods for nucleic acid h\ bπdizalions are well known in the art Accordingh , the PRG4 proteins encoded by nucleic acids used herein include nucleic acid having at least 60% homolog> , piefeiably 75% homology, more prefeiably 85%. more preferably 90%, most preferably 95%. 96%. 97%, 98%, 99% homolog> to a

8M4812 1 21 polynucleotide sequence that encodes a human PRG4 protein (e g . SEQ ID NO 1) or a specific isoform or homolog thereof

|0068] Moreover, the PRG4 proteins used herein can also be chimeric protein or fusion protein As used herein, a "chimeric protein^" or "fusion protein^" comprises a first polypeptide operatι\ ely linked to a second polypeptide Chimeric proteins may optionally comprise a third, fourth or fifth or other polypeptide operativelj linked to a first or second poh peptide Chimeric proteins ma\ comprise two or more different poh peptides Chimeric proteins may comprise multiple copies of the same polypeptide Chimeric proteins ma> also comprise one or more mutations in one or more of the poh peptides Methods for making chimeric proteins are well known in the art In certain embodiments of the present inv ention, the chimeric protein is a chimera of PRG4 protein w ith other PRG4 protein isoforms

|0069) As used herein, an "isolated^" or "purified^" protein, polynucleotide or molecule means removed from the environment in which thev naturally occur, or substantially free of cellular material, such as other contaminating proteins from the cell or tissue source from which the protein poh nucleotide or molecule is derived, or substantialh free from chemical precursors or other chemicals when chemicalh synthesized The language "substantialh free of cellular material^" includes preparations sepaiated from cellular components of the cells from v\hιch it is isolated or recombinanth produced or s\ nlhesi/ed In certain embodiments, the language "substantialh free of cellular material^"' includes preparations of a PRG4 protein having less than about 30% (b> dr> weight) of other proteins (also referred to herein as a "contaminating protein^"), more preferably less than about 20%. still more preferabh less than about 10%, and most preferabh less than about 5% of other proteins When the protein or polynucleotide is recombinantly produced, it is also preferably substantially free of culture medium. / e . culture medium represents less than about 20%. more preferablv less than about 10%. and most preferably less than about 5% of the volume of the pieparalion of the protein of interest

|0070| In certain embodiments, the present inv ention provides a pharmaceutical composition suitable for topical administration to an ocular surface of an individual in need a pharmaceutically effective concentration of PRG4 protein suspended in an ophthalmically acceptable balanced salt solution, and in combination with one or more ophthalmically acceptable agents. The ophthalmically acceptable agents can be selected from the group consisting of an ophthalmically acceptable demulcent, excipient, astringent, vasoconstrictor, and emollient. As used herein, the term "effective concentration or amount'^: or ' therapeutically effective concentration or amount^" is intended to mean a nontoxic but sufficient concentration or amount of a PRG4 protein or other therapeutic agents to provide the desired therapeutic effects. The concentration or amount that is effective will vary from subject to subject, depending on the age and general condition of the individual, the particular agents, and the like. Thus, it is not always possible to specify an exact effective concentration or amount. However, an appropriate effective concentration or amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Furthermore, the exact effective concentration or amount of a PRG4 protein and other therapeutic agent incorporated into a composition or dosage form of the present invention is not critical, so long as the concentration is within a range sufficient to permit ready application of the solution or formulation so as to deliver an amount of the PRG4 protein and other active agents that is within a therapeutically effective range.

|007i] In certain embodiments, the pharmaceutically effective concentration of PRG4 protein is in a range of 10-10,000 μg/mL, preferably 50-5,00 mg/mL, and more preferably 100-300 mg/mL. As used herein, the ophthalmically acceptable agents comprising the ophthalmically acceptable demulcents, excipients, astringents, vasoconstrictors, and emollients that are fully defined in the Code of Federal Regulations 21 CFR349.

|0072| As used herein, the term "topical administration" is used in its conventional sense to mean delivery of the composition comprising the PRG4 protein and one or more ophthalmically acceptable agents to the eye. In general, topical administration is achieved through a liquid formulation for eye drops or lavage and provides a local effect. μ>073j In certain embodiments, any pharmaceutical composition described herein comprise or the aforementioned ophthalmically acceptable agents are or can be combined with one or more of carboxymethylcellulose sodium (e.g., about 0.2 to about 2.5% w/v), hydroxy ethyl cellulose (e.g., about 0.2 to about 2.5% w/v), hypromellose (e.g., about 0.2 to about 2.5% w/v). methylcellulose (e.g., about 0.2 to about 2.5% w/v),

8314832.1 23 dextran 70 (e g , about 0 1% \v/\). gelatin (e g , about 0 01% w/v). gl> ceπn (e g , about 0 2 to about 1 % w/v). polyethvlene glycol 300 (e g , about 0 2 to about 1% w/\ ), pol\ ethylene glycol 400 (e g , about 0 2 to about 1% w/v), polysorbate 80 (e g , about 0 2 to about 1% w/v ). prop\ lene gK col (eg . about 0 2 to about 1% w/\ ). pol> \ ιn\ l alcohol (e g , about 0 1 to about 4%

), pov idone (e g . about 0 1 to about 2% \\/\ ). /me sulfate (e g , about 0 25% w/v), anhydrous lanolin (e g , about I to about 10% w/v), lanolin (e g . about 1 to about 10% w/v). light mineral oil (e g . < about 50% w/\ ), mineral oil (<? g . < about 50% w/v). paraffin (e g , < about 5% w/v), petrolatum (e g , < about 100% w/v). white ointment (e g . < about 100% w/v), white petrolatum (e g , < about 100% w/\). white wax (e g , < about 5% w/v). \, ellow wax (e g . < about 5% w/v). ephedπne hydrochloride (e g , about 0 123% w/v), napha/oline hydrochloride (e g . about 0 01 to about 0 03% w/v), phen> lephrine hv drochloride (c g , about 0 08 to about 0 2% w/v), and tetrahydrozohne h\, drochloπde (e g . about 0 01 to about 0 05% w/v) In certain instances, percent amounts utili/ed herein are percent amounts b\ weight

|0074| In further embodiments, the pharmaceutical composition of the present inv ention comprising a PRG4 protein in combination w ith one or more ophthalmicalh acceptable agents discussed abov e further comprises a therapeutically effectn e concentration of h\ aluronic acid or sodium rn aluronate in the range of 10-100.000 μg/mL, preferabh 500-5.000 tng/mL Furthermore, the pharmaceutical composition of the present invention further comprises one or more surface active phospholipids in the range of 10- 10.000 μg/mL such surface activ e phospholipids include, but are not limited to. L-α-dψalmitoylphosphatιd\ lcholtne (DPPC). phosphatidylcholine (PC), phosphatid> lethanolamine (PE) and sphingoid elin (Sp). or other neutral and polar lipids

|0()75l The pharmaceutical composition of the present inv ention ma> further comprise one or more pharmaceutically acceptable earners or vehicles comprising am acceptable materials, and/or an\ one or more additiv es known in the art As used herein, the term "earners^" or "v ehicle'^" refer to carrier materials suitable for topical drug administration Carriers and vehicles useful herein include any such materials known in the art. which aie nontoxic and do not interact with other components of the composition in a deleterious manner Various additives, known to those skilled in the art. ma\ be

HT 14X12 I 24 included in the composition For example, solvents, including relatively small amounts of alcohol, may be used to solubih/e certain drug substances Other optional additives include opacifiers. antioxidants, fragrance, colorant, gelling agents, thickening agents, stabilizers, surfactants, and the like Other agents may also be added, such as antimicrobial agents, to prevent spoilage upon storage, / e . to inhibit growth of microbes such as \ easts and molds Suitable antimicrobial agents are t> picalh selected from the group consisting of the methvl and prop} I esters of p-h\ droxyben/oic acid (ι e , methyl and prop> l paraben). sodium ben/oate, sorbic acid, imidurea. and combinations thereof Permeation enhancers and/or irritation-mitigating addiln es ma> also be included in the pharmaceutical composition of the present invention

|0076) In certain embodiments, the pharmaceutical composition of the present invention is prepared in a pharmaceutically acceptable earner, such as a phosphate buffered saline or an osmoticalh balanced salt solution of tear eleclroh tes, including one or more of sodium chloride in about 44% to about 54% mole fraction, potassium chloride in about 8% to about 14% mole fraction, sodium bicarbonate in about 8% to about 18% mole fraction, potassium bicarbonate in about 0% to about 4% mole fraction, calcium chloride in about 0% to about 4% mole fraction, magnesium chloπde in about 0% to about 4% mole fraction, tπsodium citrate in about 0% to about 4% mole fraction, and hydrochloric acid in about 0% to about 20% mole fraction or sodium hydroxide in about 0% to about 20% mole fraction In certain embodiments, the pharmaceutical earner can be formulated to generate an aqueous electrol> te solution in about 150-200 mM range Other suitable formulations, such as ointments, creams, gels, pastes, and the like, suitable for topical administration, are also contemplated in the present in\enlion In certain embodiments, electrolytes provide proper osmotic balance when combined w ith PRG4 to make a solution ophthalmically acceptable

|0077| The present invention further provides a method for treating decreased or undesired ocular boundary lubrication, sx mptoms associated therewith, or a condition that is associated with or causes a deficienc\ in ocular lubrication, in an individual in need thereof, comprising topically administering to the ocular surface of the indiv idual in need a pharmaceutical composition comprising a therapeutically effective amount of PRG4 protein In one embodiment, the method of the present invention compiises topically administering a pharmaceutical composition comprising the therapeutically

8Ϊ 14812 I 25 effective amount of the PRG4 protein that is suspended in a phosphate buffered saline solution or an ophthalmically acceptable balanced salt solution comprising one or more electrolytes In yet other embodiment, the method of the present invention comprising topically administering a pharmaceutical composition comprising the PRG4 protein formulated in an ophthalmicalh acceptable formulation comprising one or more additional ophthalmically acceptable agent as discussed above

|0078| As used herein, the term "treating or treatment^" refers to reduction in se\ eπt\ and/or frequency of s> mptoms, elimination of sy mptoms and/or underK ing cause, prev ention of the occurrence of s> mptoms and/or their underlying cause, and improvement or remediation of damage The term "treating or treatment^" also encompasses both prev ention of a disorder in a predisposed indiv idual and treatment of the disorder in a clinicalh symptomatic individual

|0079j In certain embodiments the decreased ocular boundar\ lubrication is caused b\ increased ev aporative tear loss or unstable tear film in the ocular boundary loop Such decreased or undesired ocular boundars lubrication is associated with aqueous or ev aporative dr\ eye disease. Sjogren's s> ndrome. keratoconjunctiv itis sicca (KCS), androgen deficiency, meibomian gland disease, estrogen replacement therapv . contact lens wear, refractive surgerv, allergy, reduced tear film breakup time, compromised tear film, ocular surface disorders, increased protease lev els in the tear film and at the ocular surface, chronic inflammation. h\ peros molarity, and aging As discussed abov e, the increased shear stress leads to tear film instability evaporative tear loss. h\ perosmolaπtλ . changes in swelling pressure and a feedback elevation in shear stress Increased shear stress also promotes inflammation, androgen deficient and decreased expression of proteogK cans Over time, increased shear stress and its sequelae leads to a loss of boundary lubrication at the ocular surface Accordingly, the present invention prov ides a method for reducing shear stress b\ replenishing and enriching the expression of proteogK cans, such as PRG4 protein at the ocular surface, so as to prevent or increase ocular boundary lubrication

[0080] Throughout this application, various publications are referenced The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fulK describe the state of the art to which this invention pertains

8M48U I 2(> |008i J It should also be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the an without departing from the spirit of the present invention and/or the scope of the appended claims.

|()082| Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims. These and many other variations and embodiments of the invention will be apparent to one of skill in the art upon a review of the appende description and examples.

EXAMPLES EXAMPLE I

PRG4 mRNA Expression in Human Corneal and Conjunctival Epithelial Cells

[0083| Human corneal epithelial cells were isolated from the corneoscleral rims of male and female donors. Cells were processed either directly (n = 8), or first cultured in phenol red-free keratinocyte serum free media (n = 2). Bulbar conjunctivae (n = 2). conjunctival impression cytology samples (n = 9). immortalized human conjunctival epithelial cells after culture (n = I ), NOD mouse lacrimal glands (n = 5 adult mice/sex. 10 glands/sample), and BALB/c mouse meibomian glands (n = 7 adult mice/sex, glands from 28 lids/sample) were obtained during surgical procedures. These samples were processed for the analysis of PRG4 mRNA by using primarily RT-PCR (n = 18 human, all mouse) and Affymetrix GeneChips (n = 4 human corneas). The PRG4 primers for PCR spanned over 1 kbp of intron sequences, in order to suppress amplification of contaminating chromosomal DNA (Table 1). Amplified samples were screened for the presence of PRG4 products by using agarose gel electrophoresis and an Agilent 2100 Bioanaly/.er. To confirm the identity of amplicons, PCR products from cornea samples (n = 2), conjunctival epithelial cells (n = 1 ) and a human liver standard (n = 1 ) were sequenced with a 3100 Genetic Analyzer at the Massachusetts Eye and Ear Infirmary

»714X32.1 27 DNA Sequencing Center for Vision Research (Boston, MA) and resulting data were anal\ zed with BLASTn searches of Gen Bank databases

Table 1 Oligonucleotide primers designed for RT- PCR anah sis of PRG4 mRNA

Species Orientation Nucleotide sequence (5^" - 3^") E\ons Amplicon

Size (bp) Human Sense GATGCAGGGTACCCCAAA (SEQ ID NO 2) 9-12 526

Antisense C AG ACTTTGG ATA AGGTCTGCC (SEQ ID NO 3)

[0084] It was demonstrated that PRG4 mRNA is present in all human corneal and conjunctival epithelial cell and impression cytology samples The identity of PRG4 PCR products was confirmed b\ DNA sequence anaK sis (Table 2) The results show that PRG4 is transcribed in human corneal and conjunctival epithelial cells

Table 2 Identification of amplicon sequences from human cornea, conjunctn al and liver samples

Sequencing Aligned Base Pairs Total Base Pairs BLASTn

Search

Direction To Human PRG4 from Amplicon Identitλ

Human Liver Standard

A Forward 495 500 Human

PRG4

A Re^ erse 488 491 Human

PRG4

B For\\ ard 496 499 Human

PRG4

B Re\ erse 498 500 Human

PRG4

Human Cornea (24 \ i ear old female)

A Forward 497 499 Human

PRG4

A Reλ erse 490 492 Human

PRG4

B Forward 500 504 Human

PRG4

B Reverse 498 501 Human

PRG4

Human Cornea (51 year old female)

8M4812 I 2S A Forward 498 499 Human

PRG4

A Reverse 474 489 Human

PRG4

B Forward 496 498 Human

PRG4

B Reverse 490 491 Human

PRG4 Human Conjunctival Epithelial Cells

A Forward 496 499 Human

PRG4

A Reverse 490 492 Human

PRG4

B Forward 495 499 Human

PRG4

B Reverse 474 491 Human

PRG4 Two different samples (Λ & B) of each preparation were seqiienced in forward and reverse directions. The human cυmυu samples were epithelial cells from the corneoscleral rims of female donors. The gene accession number for human PRG4 is NM_005807.

EXAMPLE 2

Reduction of friction in vitro with the addition of PRG4 (lubricin)

[0085| An in vitro friction test with clinically relevant interfaces, such as an ocular surface-eyelid and ocular surface-contact lens interface is described below. Clinically relevant methods capable of quantitatively assessing the lubricating ability of artificial tears are currently lacking. Friction tests with synthetic (e.g. latex and glass) or non- ocular 'native^' surfaces (e.g. umbilical cord vein segments) may facilitate some, but likely not all of the molecular interactions that occur during articulation/blinking. Indeed, the relevance of data obtained with non-tissue interfaces is unclear.

[0086] An annulus-on-disk rotational test configuration has been shown to be ideal for studying boundary lubrication at an articular cartilage-cartilage interface. A boundary mode of lubrication is indicated by kinetic friction being invariant with factors that influence formation of a fluid film, including sliding velocity and axial load. This is because surface-to-surface contact is occurring, and surface bound molecules contribute to lubrication (by decreasing friction and wear). Boundary lubrication has been discovered to be a critical and operative mechanism at the ocular surface, like it is at

, 8314832.1 29 the articular cartilage surface. Therefore, the in vitro friction test previously developed and characterized to study boundary lubrication at an articular cartilage-cartilage interface was modified for the study of ocular surface-eye lid and ocular surface- contact lens interfaces. |0087| To determine the test conditions in which boundary lubrication is dominant at the ocular surface-eyelid and ocular surface-contact lens interfaces, the dependence of frictional properties on axial load and sliding velocity was examined. Normal fresh human ocular surfaces (resected corneas with ~3mm of sclera) were obtained from the Lions Eye Bank of Alberta. The resected corneas were stored in Optisol-GS at 4°C and used w ithin 2 weeks Eyelids (age 60-80 years old) were obtained from the University of Calgary Body Donation Program within 1 -3 days after death and used immediately or stored at -2O⁰C in saline for at most 2 weeks until use. Comparative lubricants consisted of Lens Plus Sterile Saline Solution (Advanced Medical Optics) as a negative control: Systane* Lubricant Eye Drops (Alcon Laboratories), Refresh Tears Lubricant Eve Drops (Allergan), Aquify* Long Lasting Comfort Drops (CIBA Vision) and Blink^* Tears Lubricant Eye Drops (Advanced Medical Optics) as test lubricants.

[0088] The friction test schematic is shown in Figure 6. The corneal ocular surface (605) was fastened to the spherical end of an inert non-permeable semi-rigid rubber plug cylinder (603) (radius r=6mm) by applying super glue to the sclera. This plug cylinder (603) was attached to the rotational actuator of the mechanical testing machine (BoseELF 3200) thus forming the bottom articular surface. An annulus (601) (outer radius=3.2mm, inner radius=1.5mm) was punched from the eyelid (604). and was attached to the linear actuator coupled with an axial load (N) and torsion (τ) load cell, thus forming the upper articulating surface. Lubricant bath 602 was formed by securing an inert tube around the plug cylinder (603).

|0089| Samples were first tested in saline, then in one of the three (3) test lubricants. The lubricant bath was filled with -0.3 ml, and the articulating surfaces allowed to equilibrate with the lubricant. The sample surfaces were slowly (0.05mm/s) brought into contact and compressed until the spherical plug flattened out and the entire annular eyelid surface was in contact with the cornea (605). The resulting normal stress (calculated from axial load as, in units of MPa, as N/(π| router - run_ner!) can be varied by using different stiffness rubber plugs to mimic physiological stresses - 5kPa. The test

8? 14X32.1 3() sequence was initiated b> preconditioning the sample by rotating +4 re\ olutions (re\ ) and reset with -4 revolutions at a ph\ siologically relevant effective linear sliding \ elocitv \ eff = 30 mm/s (where \ eff =ωReff, ω is the angular frequency, and Reff=2 4mm is the effecti\ e radius calculated bv integrating the shear stress distribution over the annular contact area) Samples were then tested by rotating +4 re\ olutιons, immediately followed by -4 reset revolutions at veff = 30. 10. I. 0 3 and then 30 mm/s. with a dwell time of 12 second between each revolution. The test sequence was then be repealed in the opposite direction of rotation

[0090] To evaluate the lubrication properties of the ocular surface, h\ o friction coefficients (μ) of the form μ=τ/(RuiN) ) where is torque, Rui is effectn e radius, and N is axial load, described above A static friction coefficient, which reflects the resistance to the onset of motion. μ,uu._<. was calculated as the peak \ alue of μ just after (within

-10°) the start of rotation An a\ erage kinetic friction coefficient, which reflects the resistance to stead} stale motion. <μi_ιiπct₁(.^> w as calculated from μ averaged during the third and fourth complete test revolution Both μ_Matιc and <μkmetic^> were averaged for the

+ and - re\ olutιons in each test to account for potential directional effects on τ measurements Data was collected at a frequence of 20 H/

[0091] The results of lubricin (PRG4) added to the corneal surface at a concentration in the range of 100-300 ug/mL are shown in Figure 7 Lubricin had a fπction lowering effect at the evelid interface, both in terms of kinetic and static fπction. at all velocities Al a concentration 1/l Olh of lhal of pin siological h\ aluronic acid, lubricin was similar to Blink* Tears Lubricant Eye Drops, which contains h> aluronic acid In combination, the two lubricants are better than either alone

[0092] Figure 8 demonstrates the reduction of in vitro comea/lid kinetic friction measured during the first minute after the addition of lubricin. as compared to Aquif> ^*' e> e drops Lubricants were lhoroughl> washed from lhe ocular surface using saline between tests Λ s> nergistιc effect (reduced μkmuic over either alone) was βλ ident when

Aquify* (with hy aluronic acid) was combined with lubricin The saline repeat w as lower than the oπginal saline control This showed a retention of lubπcm^"s effect even after washing with saline, suggesting that the molecules were binding to the ocular surface, and that lubricin demonstrated superior retention time as compared to sodium hyaluronate alone

8^ 148^2 1 T l [0093] Figure 9 demonstrates the reduction of in vitro cornea/lid kinetic friction measured during the 5th minute after the addition of lubricin. as compared to Aquify* e> e drops A synergistic effect (reduced μkmetic over either alone) was eudent when Aquif\ * (with hy aluronic acid) was combined with lubπcin The fπction coefficient of Aquify*' had returned to statistical equivalence to saline after 5 minutes, whereas lubπcin remains lower, as did the combination of lubricin and hyaluronic acid

1009-4] Figure 10 shows the reduction of kinetic friction coefficient Oλ er time, follow ing addition of lubricin Again, the continual reduction suggested binding to the ocular surface

K)

EXAMPLE 3 Treatment of Deficient Ocular Boundan Lubrication in Vivo

[0095] A patient complaining of ocular surface irritation is examined for ocular 15 lubrication or conditions associated w ith a deficienc} in ocular lubrication b> measuring symptoms greater than 2 positive responses on the McMonnies questionnaire, greater than a score of 5 on the Ocular Surface Disease Index (OSDI). or through evidence of some symptoms on the Visual Analog Scale, m combination w ith objectiv e signs including one or more of a reduced tear film breakup time (less than 0 =10 seconds), inferior lateral tear meniscus osmolarm greater than 308 mOsms/L. low Schirmer strip v alue (less than -IO mm), sodium fluorescein corneal or conjunctiv al staining (scores > 0 w ith multiple macropunctates). significant debris resulting from impression c\ tolog\ . meibomian gland d\ sfunctιon however determined, a decrease in the rate of post-blink displacement of a contact lens, a change in the spatiotemporal 5 transfer function of a contact lens following application of a series of pressure impulses, a decrease in the rate of post-blink interferometric tear film relaxation, an increase in the concentration of proinflammatory cUokines, a reduced concentration of lactoferπn or l\ soτ> me. or an increase in the rate of post-blink point spread function decoherence

8^ l4tn2 l -?2 |()«96] The patient administers 1 to 2 drops on the surface of each eye a solution containing 200 μg/mL PGR4 protein suspended in an ophthalmically acceptable balanced salt solution. The patient is instructed to close their eyes for 10 seconds.

|0097) Follow-up visits may track a reduction in inferior lateral tear osmolality. increased tear film breakup time, or the other aforementioned signs. In particular if the tear film osmolality is reduced from an abnormal value (perhaps 330 mOsms/L) to a more normal value (perhaps 304 mOsms/L), the therapeutic modulation and replenishment of the ocular surface lubrication would be deemed successful.

8.-U4832. I 33 REFERENCES

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25 Nugent-Derfus GE. Chan AH. Schumacher BL. Sah RL PRG4 exchange between the articular cartilage surface and s\ novial fluid J Orthop Res 2007 Oct.25( 10) 1269-76

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27 Nugent GE, Aneloski NM, Schmidt TA. Schumacher BL. Voegtline MS. Sah RL Dynamic shear stimulation of box me cartilage bios\ nthesis of proteoglycan 4 Arthritis Rheum 2006 Jun.54(6) 1888-96

28 D K Rhee et al . J Biol Chem 280. 31325 (2005)

29 T J Klein et al . Osteoarthritis Cartilage 1 1. 595 (2003)

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38 K C Morell. W A Hodge. D E Krebs. R W Mann. Pioc Natl Acad Sci U S A 102. 14819 (Oct 1 1. 2005)

39 S A V Sw anson. in Adult Articular Cartilage M A R Freeman. Ed (Pitman Medical. Tunbπdge Wells. England. 1979) pp 415-460

8* 14812 1 35 40 Elsaid KA, Jay GD, Chichester CO Reduced expression and proteol> tic susceptibility of lubricin/superficial /one protein may explain earl} elevation in the coelTicient of friction in the joints of rats with antigen-induced arthritis Arthritis Rheum 2007.56 108-1 16 41 Elsaid KA. Ja\ GD, Warman ML, Rhee DK, Chichester CO Association of articular cartilage degradation and loss of boundary -lubricating abilih of s\ novιal fluid following injun. and ιnflammator\ arthritis Arthritis Rheum 2005.52 1746- 1755

42 Cutolo M. Capellino S. SuIIi A. Seπoh B. Secchi ME. Villaggio B. Straub RH Estrogens and autoimmune diseases Ann N Y Acad Sci 2006.1089 538-547 43 Cutolo M, Sulli A. Capellino S, Villaggio B, Montagna P. P\7/om\ C, Paolino S. Seπolo B, Felli L. Straub RH Anti-TNF and sex hormones Ann N Y Acad Sci 2006.1069 391-400

44 Schmidt M, Naumann H. Weidler C. Schellenberg M. Anders S. Straub RH Inflammation and se\ hormone metabolism Ann N Y Acad Sci 2006.1069 236-246 45 Ront/sch A. Thoss K, Petrow PK. Hen/gen S. Brauer R Amelioration of murine antigen-induced arthritis b\ deh\ droepiandrosterone (DHEA) lnflamm Res 2004,53 189- 198

46 Zierhut M. Dana MR. Stern ME. Sullivan DA Immunology of the Lacπmal Gland and Ocular Tear Film Trends Immunol 2002,23 333-335 47 Stem ME. Gao J. Siemasko KF. Beuerman RW. Pflugfelder SC The role of the lacπmal functional unit in the pathophλ siologj of dr> e> e E\p

e Res 2004.78 409- 416

48 Tomlinson A. Khanal S. Ramaesh K, Diaper C. McFad> en A Tear film osmolaπty determination of a referent for dr> e_\ e diagnosis Imest Ophthalmol Vis Sci 2006.47 4309-4315

49 Sulln an DA. SuIIn an BD, Evans JE. Schirra F. Yamagami H. Liu M. Richards SM. Su/uki T, Schaumberg DA. Sulli\ an RM, Dana MR Androgen deficιenc\ , meibomian gland dysfunction and e\ aporatn e dry eye Ann NY Acad Sci 2002.966 21 1 -222 ^' 50 Sulln an DA Tearful relationships'? Sev hormones and aqueous-deficient dr\ e\ e Ocular Surface 2004.2 92- 123

51 Schaumberg DA. Buπng JE, SuIIn an DA. Dana MR Hormone replacement therap\ and dr\ e> e syndrome JAMA 2001.286 21 14-21 19

52 de Sou/a GA, Godoy LM. Mann M Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors Genome

Biol 2006.7 R72 Epub 2006

53 Schirra F, Su/uki T, Dickinson DP, Townsend DJ, Gipson IK. Sullivan DA Identification of steroidogenic en/yme mRNAs in the human lacrimal gland, meibomian gland, cornea and conjunctiva Cornea 2006:25 438-42

8^ 14812 1 3(> 54 Schwar/ IM. Hills BA. Br J Rheum 1998.37 21 -26

55 Ja> GD. Hong BS Connect Tissue Res. 1992. 28(1-2) 89-98

56 Matnelh F. Argueso P. Curr Opin Allergy Clin Immuno, 2008. 8(5) 477-483

57 Jones MB et al Mathematical Medicine and Biologλ 2005, 22, 265 58 Schumacher BL. Hughes CE. Kuettner KE, Calerson B. A> delotte MB Immunodetection and partial cDNA sequence of the proteogh can. superficial /one protein, s\ nthesι/ed by cells lining sy novial joints J Orthop Res 1999 Jan, 17(1 ) I 10-

59 Schmidt TA, Gastelum NS, Ngu> en QT. Schumacher BL. Sah RL Boundary lubrication of articular cartilage role of synovial fluid constituents Arthritis Rheum

2007 Mar.56(3) 882-91

60 Schmidt TA, Plaas AH, Sandv JD Disul fide-bonded multimers of proteogl> can 4 (PRG4) are present in normal s> no\ ial fluids Biochim Biophys Acta 2009 Mar 27

61 Sullivan DA The definition and classification of dr\ ev e disease report of the Definition and Classification Subcommittee of the International Dr\ Ev e Workshop

(2007) Ocular Surface 2007 Apr.5(2) 75-92

XM-UO 2 I VI

Claims

What is claimed is

1 A pharmaceutical composition suitable for topical application to an ocular surface comprising a therapeutical I) effective concentration of PRG4 suspended in an ophthalmicallv acceptable balanced salt solution 2 The pharmaceutical composition of claim I. comprising one or more ophthalmicallv -acceptable agents selected from the group consisting of an ophthalmicall\ acceptable demulcent, an ophthalmicalh acceptable excipient. an ophthalmicallv acceptable astringent, an ophthalmicalh acceptable \ asoconstnctor. and an ophthalmicalh acceptable emollient 3 The pharmaceutical composition of claim 1. wherein the pharmaceutical composition comprises the PRG4 in the therapeutically effective concentration of 10- 10,000 μg/mL

4 The pharmaceutical composition of claim I . wherein the pharmaceutical composition comprises the PRG4 in the therapeutically effective concentration of 50- 500 μg/mL

5 The pharmaceutical composition of claim 1 , compπsing a therapeutically effective concentration of sodium hyaluronate or hyaluronic acid

6 The pharmaceutical composition of claim 5, wherein the pharmaceutical composition comprises sodium hj aluronate or h> aluronic acid in the therapeutically effective concentration of 10- 100,000 μg/mL

7 The pharmaceutical composition of claim 5. wherein the pharmaceutical composition comprises sodium hyaluronate or h> aluronic acid in the therapeutically effectn e concentration of 500-5.000 μg/mL

8 The pharmaceutical composition of claim I . comprising a therapeuticallv effectn e concentration of surface actn e phospholipid selected from the group consisting of I -α-dιpalmito\ I phosphatidylcholine. phosphatid\ lcholine. phosphatid> lethanolamine and sphingorm elin

8M4!H2 I • 39 9 The pharmaceutical composition of claim 8. wherein the pharmaceutical composition comprises the surface activ e phospholipid in the therapeuticalK effectiv e concentration of 10- 10,000 μg/mL

10 The pharmaceutical composition of claim 1. wherein the ophthalmically acceptable balanced salt solution comprises at least three different electrolytes selected from the group consisting of sodium phosphate, sodium chloride, potassium chloride, sodium bicarbonate, potassium bicarbonate, calcium chloride, magnesium chloride, sodium acetate, sodium citrate, hydrochloric acid, and sodium hydroxide

1 1 The pharmaceutical composition of claim 1. wherein the PRG4 has an average molar mass of between 50 kDa and 400 kDa

12 The pharmaceutical composition of claim 1. wherein the PRG4 comprises a lubricating fragment multimer or a homolog thereof

13 The pharmaceutical composition of claim I . wherein the PRG4 is a recombinant PRG4 protein or functional fragment thereof

14 The pharmaceutical composition of claim I wherein the PRG4 is a purified naturalK occurred PRG4 protein

15 A method for treating ocular lubrication deficiencv or svmptoms associated therewith, in an indn idual in need thereof comprising topicalK administering to the ocular surface of the individual in need a pharmaceutical composition comprising a therapeutically effective concentration of PRG4

16 The method of claim 15, wherein the pharmaceutical composition comprising the PRG4 is administered in combination with an ophthalmically acceptable formulation comprising one or more ophthalmically acceptable agents selected from the gioup consisting of an ophthalinicalK acceptable demulcent, an ophthalmicalh acceptable excipienl. an ophthalmicalK acceptable astringent an ophthalmicalK acceptable v asoconstrictor, an ophthalmicalK acceptable emollient and an ophthalmicalK acceptable electroh te

17 The method of claim 15. wherein the pharmaceutical composition comprising the PRG4 is administered in combination with an ophthalmically acceptable solution

8^ 14812 1 4(J compπsing a therapeutically effective concentration of sodium h> aluronate or hyaluronic acid

18 The method of claim 17, wherein the ophthalmically acceptable solution compπses sodium h> aluronate or hyaluronic acid in the therapeuticall> acceptable concentration of 10- 100.000 μg/mL

19 The method of claim 17. wherein the ophthalmicallv acceptable solution comprises sodium

aluronate or h\ aluronic acid in the therapeuticalK acceptable concentration of 500-5,000 μg/mL

20 The method of claim 15. wherein the pharmaceutical composition compπsing the PRG4 is administered in combination with an ophthalmically acceptable solution compπsing a therapeutically effective concentration of a surface active phospholipid selected from the group consisting of L-α-dipalmitθ} lphosphatιd> lcholine. phosphatid\ lcholine. phosphatid\ lethanolamine and sphingomyelin

21 The method of claim 20. wherein the ophthalmically acceptable solution comprises the surface active phospholipid in the therapeutically effecln e concentration of 10-10.000 μg/mL

22 The method of claim 15. wherein the pharmaceutical composition comprising the PRG4 is administered in combination with a phosphate buffered saline solution comprising at least sodium phosphate and sodium chloride 23 The method of claim 15. w herein the pharmaceutical composition compπsing the PRG4 is administered in combination w ith an ophthalmically acceptable balanced salt solution compπsing one or more electroK tes selected from the group consisting of potassium chloride, sodium bicarbonate, potassium bicarbonate, calcium chloπde. magnesium chloride, tπsodium citrate. h\ drochloπc acid, and sodium hydroxide 24 The method of claim 15. wherein the PRG4 is a recombinant PRG4 protein, or functional fragment thereof

25 The method of claim 15. wherein the PRG4 is a purified naturally occurred PRG4 protein

8T 14812 I ₄ | 26 The method of claim 15. wherein the pharmaceutical composition comprising the PRG4 is administered in combination with an ophthalmicalh acceptable solution comprising a therapeutically effectn e concentration of sodium h> aluronate or hyaluronic acid, and a surface active phospholipid selected from the group consisting of L-α-dipalmiloylphosphatidj Icholrne, phosphatidylcholine, phosphatidylethanolamine and sphingomyelin

27 The method of claim 15, wherein the individual in need thereof has a deficιenc\ in ocular boundaπ. lubrication that is associated with aqueous or evaporati\ e dry e\ e disease, Sjogren^'s SMidrome. keratoconjunctivitis sicca, androgen deficient meibomian gland disease, estrogen replacement therap> . contact lens wear, refractive surgen . allergy, reduced tear film breakup time, compromised tear film, allergy . ocular surface disorders, increased protease levels in the tear film and at the ocular surface, chronic inflammation. h\ perosmolaπty aging, or a combination thereof

28 A method for treating a condition that is associated with or causes a deficiency in ocular lubrication, or the symptoms thereof, in an indiv ldual, the method compπsing topically administering to the ocular surface of the indn lduaJ a pharmaceutical composition comprising a therapeutical K effective concentration of PRG4

29 The method of claim 28, wherein the pharmaceutical composition comprising the PRG4 is administered in combination w ith an ophthalmicall) acceptable formulation comprising one or more ophthalmically acceptable agents selected from the group consisting of an ophthalmically acceptable demulcent, an ophthalmicalh acceptable excipient. an ophthalmically acceptable astringent, an ophthalmicalh acceptable vasoconstrictor, an ophthalmically acceptable emollient, an ophthalmically acceptable electrolyte, an ophthalmicallv acceptable sodium h\ aluronate or hyaluronic acid, and an ophathalmicall} acceptable surface active phospholipid

30 The method of claim 28. wherein the condition is selected from the group consisting of aqueous or e\ aporative dr> eye disease. Sjogren^'s syndrome, keratoconjuncimtis sicca androgen deficiency . meibomian gland disease estrogen replacement therapy, contact lens wear, refractive surgen , allergy, reduced tear film breakup time, compromised tear film, allergv. ocular surface disorders, increased

8" 14812 I 42 protease levels in the tear film and at the ocular surface, chronic inflammation, hyperosmolarity, aging, and combinations thereof.

8314832.1 4.1