Therapeutic modulation of ocular surface lubrication

Description

FIELD OF THE INVENTION [0001 The present invention relates to regulating the hydration of the eye surface. In particular, the present invention relates to pharmaceutical compositions for use in the treatment of an eye surface moisturizing deficiency or related symptoms.

BACKGROUND OF THE INVENTION [0002] The gene encoding proteoglycan 4 (prg4) encodes highly glycosylated proteins called megakaryocyte stimulating factor (MSF), lubrycin and surface zone protein (SZP) (1). These molecules are collectively referred to as PRG4 or PRG4 proteins. PRG4 is present in the synovial fluid and on the surface of the synovial membrane (2), tendon (3) and meniscus (4) and is suspected to be an important component of healthy synovial joints. See e.g. (5), (6).

[0003] In tissues such as synovial joints, physicochemical lubrication methods are classified as fluid film moisturizing or border lubrication. The operational methods of lubrication depend on normal and tangential forces on articulated tissues, on the relative speed of the tangential movement between these surfaces and on the history of both load and movement. The friction coefficient μ provides a quantitative measurement and is defined as the ratio of the tangential friction force to the normal force. One type of fluid-mediated lubrication method is the hydrostatic type. Due to the biphasic nature of the tissue, at the beginning of the load and usually for a prolonged period, pressure builds up in the interstitial fluid within the cartilage; fluid may also be forced into the roughness between the joint surfaces through the filtration mechanism. Interstitial fluid under pressure and trapped lubricant tanks can thus contribute significantly to the transfer of normal load at low shear resistances, thus facilitating a very small μ. Also at the beginning of loading and / or movement there is lubrication of a fluid film of the extruded film type, hydrodynamic and elastohydrodynamic, with pressure generation, movement and deformation with the effect of introducing a viscous glazing agent with and / or by a gap between two surfaces moving relative to each other.

[0004] The appropriate range in which pressure / film wetting occurs relative to boundary lubrication classically depends on a number of factors (13). When the lubricant film can flow between the corresponding sliding surfaces that can deform flexibly, elastohydrodynamic moisturizing occurs. Pressure, surface roughness and relative sliding speed determine when the interruption of full fluid lubrication begins and lubrication enters new schemes. As the speed decreases further, lubricant films adhering to articulated surfaces begin to take part and a mixed lubrication pattern occurs. If the speed decreases even further and only the ultra-thin lubricant layer of several molecules remains, border lubrication occurs. The boundary lubrication is therefore indicated by the coefficient of friction (the ratio of the measured friction force between two contacting surfaces in relative movement to the applied normal force) during constant sliding, which does not change with factors that affect the formation of the fluid film, such as the relative sliding speed and axial load (14). In the case of articular cartilage, borderline lubrication has been found to occur for sure, although supplemented with fluid pressure and other mechanisms (15-18).

[0005] In boundary lubrication, the load is supported by surface-to-surface contact and the associated friction properties are determined by the lubricant's surface particles. It has been proposed that this method is important because the opposing layers of cartilage contact in more than ~ 10% of the total surface and this may be where most of the friction occurs (19). In addition, with increasing load times and the dispersion of hydrostatic pressure, the surfaces coated with a lubricant transfer an increasing part of the load relative to the fluid under pressure and, as a consequence, this method is becoming more and more dominant (13, 20). In fact, boundary lubrication reduces friction vibrations (13), and thus manifests itself as reduced drag during steady state motion and when the motion begins. The last situation is appropriate for articulated surfaces carrying load after prolonged loading under pressure (e.g. sitting or in vivo condition) (21). Typical cartilage surface wear patterns (22) also suggest that border lubrication of articular cartilage is critical to protecting and maintaining articular surface structure.

[0006] With increasing load time and the dispersion of hydrostatic pressure, surfaces coated with a lubricant carry an increasing proportion of the load relative to the fluid under pressure and, as a consequence, μ may become increasingly dominated by this lubrication method. Boundary lubrication is indicated by μ values ​​during constant sliding, which do not change with factors that affect fluid film formation, such as relative sliding speed and axial load. In fact, boundary lubrication reduces friction vibrations, and thus manifests itself as reduced drag both during steady state motion and when motion begins.

[0007] Precision boundary lubrication mechanisms on biological contact surfaces are not currently known. However, proteoglycan 4 (PRG4) may play a significant role as a border lubricant in joint joints. It is believed that this secreted glycoprotein protects cartilage surfaces against frictional forces, cell adhesion and protein deposition. Various native and recombinant lubrycin proteins and isoforms have been isolated and characterized. For example, in US patents No. 5,326558; 6433142; 7030223 and 7361738 disclose a family of human megakaryocyte stimulating factors (MSFs) and pharmaceutical compositions containing one or more of such MSFs for the treatment of disease states or disorders such as platelet deficiency. In US Patent Descriptions No. 6960562 and 6743774 also disclose a moisturizing polypeptide, a tribonectin, comprising a substantially pure MSF fragment, and methods of moisturizing the joints by administering the tribonectin systemically or directly to tissues.

[0008] The challenge for border lubrication is the presence of inflammation in surrounding tissues, as well as increased levels of protease in the synovial fluid. The loss of the ability to lubricate border synovial fluid after injury is associated with damage to the articular cartilage matrix. This can be attributed to inflammatory processes resulting from trauma, especially in the early stages. Another challenge for border lubrication is the imbalance of sex steroids, especially in joint diseases such as rheumatoid arthritis. Sex steroids are involved in the pathogenesis and regulation of inflammation in rheumatoid arthritis, a disease characterized by chronic inflammation that is synovitis. Androgens inhibit inflammatory processes, while estrogens support them. Therefore, the relative levels of androgens and estrogens in the synovial environment are extremely important in determining the progression of inflammation (7, 8, 23). Various androgen compounds reduce the amount of lymphocyte penetration in the lacrimal tissue. See, e.g., US Patent Nos. 5,629,921, 5,688,765, 5,629,921 and 6,107,289.

SUMMARY OF THE INVENTION [0009] The present invention provides, in various forms, pharmaceutical compositions for use in regulating the hydration of the eye surface, including therapeutic supplementation or enrichment of border spreads on the eye surface.

[0010] The present invention is based on the finding that PRG4 mRNA is expressed in human corneal and conjunctival epithelial cells as well as in the mouse lacrimal and thyroid glands, indicating that the PRG4 protein is present in these tissues on the surface of the eye. In addition, the role of the PRG4 protein is to protect the cornea and conjunctiva from significant shear forces generated during eyelid blinking, wearing contact lenses and other undesirable conditions. The effect of tear film, including the effect of inflammation, proinflammatory cytokines, imbalance of sex steroids and proteases on the composition and function of films ensure the course of therapy for ocular tissues that promote border lubrication.

[0011] In some embodiments, the present invention provides a pharmaceutical composition for use for treating ocular surface hydration deficiency or related symptoms for topical application to the eye surface, comprising a PRG4 inducing compound in combination with a therapeutically effective concentration of PRG4. The PRG4 inducing compounds used in the present invention are androgen, an androgen analogue, wherein said androgen analogue is selected from the group consisting of 17a-methyl-17e-hydroxy-2-oxa-5a-androstan-3-one, 4,5a-dihydrotestosterone and 19- nortestosterone or a selective androgen receptor modulator. In some cases of the present invention, it has been observed that PRG4 expression in corneal and conjunctival epithelial cells is increased by PRG4 inducing compounds, as discussed above, thereby providing synergistic PRG4 protection of the cornea and conjunctiva against significant shear forces.

[0012] Androgen analogues are 17a-methyl-17e-hydroxy-2-oxa-5a-androstan-3-one, 4,5a-dihydrotestosterone or 19-nortestosterone.

[0013] In some embodiments, selective androgen receptor modulators (SARMs) include an aryl propionamide compound such as S-3- (4-acetylaminophenoxy) -2-hydroxy-2-methyl-N- (4-nitro-3-trifluoromethylphenyl) -propionamide [ S-4] or S-3- (4-fluoro-phenoxy) -2-hydroxy-2-methyl-N- (4-nitro-3-trifluoromethyl-phenyl) -propionamide [S-1]).

[0014] In some embodiments, the pharmaceutical composition for use in the present invention comprises a therapeutically effective concentration of PRG4 inducer, as defined in the appended claims, in the range of about 0.0001-0.1% w / v. in combination with a therapeutically effective concentration of PRG4 in the range of 100-300 pg / ml. In some embodiments, the pharmaceutical composition for use in the present invention further comprises a therapeutically effective concentration of one or more hyaluronic acid or a salt thereof, in the range of about 10-100000 pg / ml. In some embodiments, the pharmaceutical composition for use in the present invention further comprises a therapeutically effective concentration of one or more surface active phospholipids, such as La-dipalmitoyl phosphatidylcholine (DPPC), phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (Sp), neutral or polar lipids, in the range of about 10-10000 pg / ml. The present invention ensures that combinations of the PRG4 inducing compound as defined in the appended claims and PRG4 and other modulators or moisturizing agent molecules border allow direct transport of PRG4 and other border lubricants to the eye surface cells, where PRG4 and boundary spread molecules tend to accumulate and provide a pharmaceutically effective carrier for the cornea and conjunctiva to efficiently regulate boundary lubrication.

[0015] In certain embodiments, the pharmaceutical compositions for use as described herein comprise a residence time extender that extends the residence time of the PRG4 inducer on the surface of the eye. In some embodiments, the residence time extender is present in an amount such that when the pharmaceutical composition is administered to the surface of the subject's eye, a therapeutically effective amount of a PRG4 inducing compound described herein is retained on the surface of the eye. In some embodiments, the residence time extender is selected and / or present in an amount such that a therapeutically effective amount of a PRG4 inducer is retained on the surface of the eye for a therapeutically effective period of time of at least 1 minute, at least 2 minutes, at least minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour or more. In some embodiments, ophthalmically acceptable residence time enhancers or mucoadhesive agents may include, as non-limiting examples, hydroxypropyl methylcellulose, carboxymethylcellulose, carbomer (acrylic acid polymer), polymethylmethacrylate, polyacrylamide, polycarbophyll, polyethylene oxide, butylacrylate acrylic acid sodium, dextran or combinations thereof. The present invention includes any high molecular weight polymers that would increase the time during which the PRG4 inducing compound remains on the surface of the eye.

[0016] The pharmaceutical composition for use in the present invention may also contain one or more ophthalmically acceptable agents selected from the group consisting of an ophthalmically acceptable soothing agent, an ophthalmically acceptable excipient, an ophthalmically acceptable astringent, an ophthalmically acceptable vasoconstrictor and an ophthalmically acceptable emollient.

[0017] Exemplary ophthalmically acceptable soothing agents contemplated for use in the present invention include, but are not limited to, sodium carboxymethyl cellulose (e.g., about 0.2 to 2.5% w / v), hydroxyethyl cellulose (e.g., about 0.2 to 2.5% w / v), hypromellose (e.g. about 0.2 to 2.5% w / v), methyl cellulose (e.g. about 0.2 to 2.5% w / v) , dextran 70 (e.g., about 0.1% w / v), gelatin (e.g., about 0.01% w / v), glycerin (e.g. about 0.2 to 1% w / v), polyethylene glycol 300 (e.g. about 0.2 to 1% w / v), polyethylene glycol 400 (e.g. about 0.2 to 1% w / v) vol.), polysorbate 80 (e.g., about 0.2 to 1% w / v), propylene glycol (e.g., about 0.2 to 1% w / v), polyvinyl alcohol) (e.g., about 0 , 1 to 4% w / v), povidone (e.g. about 0.1 to 2% w / v). Exemplary ophthalmically acceptable excipients / softeners contemplated by the present invention include, but are not limited to, anhydrous lanolin (e.g. about 1 to 10% w / v), lanolin (e.g. about 1 to 10% w / v), light mineral oil (e.g. <about 50% w / v), mineral oil (e.g. <about 50% w / v), paraffin (e.g. <about 5% w / v), petroleum jelly (e.g. <about 100% w / v), wax ointment (e.g. <about 100% w / v), white petrolatum (e.g. <about 100% w / v), white wax (e.g. <about 5% w / v), yellow wax (e.g. <about 5% w / w) / vol.). Exemplary ophthalmically acceptable astringent contemplated by the present invention includes, but is not limited to, zinc sulfate (e.g., about 0.25% w / v). Exemplary ophthalmically acceptable vasoconstrictors contemplated by the present invention include, but are not limited to, ephedrine hydrochloride (e.g., about 0.123% w / v), naphazoline hydrochloride (e.g., about 0.01 to about 0.03% w / v .), phenylephrine hydrochloride (e.g. 0.08 to 0.2% w / v) and tetrahydrozoline hydrochloride (e.g., 0.01 to 0.05% w / v).

[0018] In some of these forms, soothing agents, excipients, astringents, vasoconstrictors, emollients and electrolytes provide means to deliver the PRG4 inducing compound and PRG4 protein in an ophthalmically acceptable manner. Ophthalmically acceptable compositions are suitable for topical application to the eye surface if, after application, they do not exhibit unacceptable eye toxicity, burning, itching, stickiness, blurred vision, etc.

[0019] The present disclosure relates to a pharmaceutical composition for use for treating ocular surface moisture deficiency or related symptoms for topical application to the eye surface, comprising a therapeutically effective concentration of PRG4 inducer and PRG4 protein suspended in phosphate buffered saline or ophthalmically acceptable saline balanced salt solution containing tear electrolytes, which include, but are not limited to, sodium chloride (e.g. about 44% -54% mole fraction), potassium chloride (e.g. about 8% -14% mole fraction), sodium bicarbonate (e.g. about 8% -18% mole fraction), potassium bicarbonate (e.g. about 0% -4% mole fraction), calcium chloride (e.g. about 0% -4% mole fraction), magnesium chloride (e.g. about 0% -4% mole fraction), sodium citrate (e.g. about 0% -4 % mole fraction), hydrochloric acid (e.g. about 0% -20% mole fraction) or sodium hydroxide (e.g. about 0% -20% mole fraction). In one embodiment, the carrier can be prepared to form an aqueous electrolyte solution in the range of 150-200 mM.

[0020] The present disclosure relates to a pharmaceutical composition for use for treating ocular surface deficiency or associated symptoms for topical administration to the eye surface, comprising a therapeutically effective concentration of PRG4 and PRG4 inducing compound suspended in an ophthalmically acceptable balanced salt solution containing at least three electrolytes, including, but not limited to, sodium chloride (NaCl) 0.64%, potassium chloride (KCl) 0.075%, calcium chloride dihydrate (CaCl2 * 2H2O) 0.048%, magnesium chloride hexahydrate (MgCl2 * 6H2O) 0.03%, sodium acetate trihydrate (C2H3NaO2 * 3H2O) 0.39%, sodium citrate dihydrate (C6HSNa3O7 * 2H2O) 0.17%, sodium hydroxide and / or hydrochloric acid (to adjust the pH to about 7.5) with an osmolality of about 300 milliosmol / L.

[0021] The present disclosure relates to a pharmaceutical composition for use for treating ocular surface moisture deficiency or related symptoms for topical application to the eye surface, which composition comprises a therapeutically effective concentration of PRG4 and PRG4 inducing compound suspended in an ophthalmically acceptable balanced saline solution from sodium (Na +) at a concentration of about 128 mM, potassium (K +) at a concentration of about 24 mM, chloride (Cl-) at a concentration of about 113 mM, calcium (Ca2 +) at a concentration of about 0.4 mM, magnesium (Mg2 +) at a concentration of about 0.3 mM, HCO3- at a concentration of about 5 mM, citrate at a concentration of about 1 mM, about 14 mM phosphate, about 15 mM acetate, and sodium hydroxide and / or hydrochloric acid (to adjust the pH to about 7.5) with an osmolality of about 300 milliosmol / L.

[0022] The present invention further provides a pharmaceutical composition for use in a method of treatment of an eye surface moisturizing deficiency or related symptoms in an individual in need thereof. The method includes topical administration to the surface of an eye in need thereof of any pharmaceutical composition described herein. The pharmaceutical composition is a composition comprising a therapeutically effective concentration of the PRG4 inducer of the present invention and the PRG4 protein. In some embodiments, a pharmaceutical composition, e.g. a composition comprising a PRG4 inducing compound and a PRG4 protein is administered in combination with an ophthalmically acceptable formulation containing one or more ophthalmically acceptable agents selected from the group consisting of an ophthalmically acceptable soothing agent, an ophthalmically acceptable excipient, an ophthalmically acceptable astringent, an ophthalmically acceptable vasoconstrictor and an ophthalmically acceptable emollient.

[0023] In some embodiments, a pharmaceutical composition comprising an inducing PRG4 as defined in the appended claims and an ophthalmically acceptable mucoadhesive agent and a PRG4 protein is administered in combination with an ophthalmically acceptable solution containing a therapeutically effective concentration of sodium hyaluronate or hyaluronic acid or surface-active phospholipid as discussed above. In certain embodiments, a pharmaceutical composition comprising a PRG4 inducing compound and a PRG4 protein is administered in combination with a phosphate buffered saline solution or an ophthalmically acceptable balanced saline solution containing one or more electrolytes, as discussed above.

[0024] In some embodiments, the present invention provides a pharmaceutical composition for use in a method of treating eye surface moisturizing deficiency or related symptoms (e.g., dry eye) that are caused by loss of tears or an unstable tear film in an ocular border loop, such as androgen deficiency, Sjogren's syndrome and dry keratoconjunctivitis (KCS). Such a method involves topically administering the pharmaceutical composition used in the present invention to the surface of the eye of the subject in need thereof.

[0025] The disclosure further relates to a pharmaceutical composition for use in a method of solving and treating conditions associated with eye surface moisturizing, including adverse or insufficient eye surface moisturizing. Exemplary conditions include, but are not limited to, dry eye disease associated with water layer deficiency or excessive tear evaporation, Sjogren's syndrome, dry keratoconjunctivitis, androgen deficiency, thyroid gland disease, estrogen replacement therapy, wearing contact lenses, refractive surgery, reduced tear film interruption time, allergy, eye surface disorders, increased levels of proteases in the tear film and on the eye surface, chronic inflammation, hyperosmolemia and aging.

[0026] The present disclosure also relates to a pharmaceutical composition for use in a method of topically inducing PRG4 on the surface of an eye, comprising topically administering to the surface of the eye of the subject in need thereof a therapeutically effective amount of any of the pharmaceutical compositions described herein, e.g. mucoadhesive. In some embodiments, a pharmaceutical composition is provided for use in a method of topically inducing PRG4 on the surface of an eye, comprising topically administering to the surface of the eye of the subject in need thereof a therapeutically effective amount of any of the pharmaceutical compositions described herein, wherein the therapeutically effective amount of the PRG4 inducer is therapeutically effective period of time.

BRIEF DESCRIPTION OF THE DRAWINGS [0027]

Figure 1 shows feedback loops within boundary lubrication of the eye surface.

Figure 2 shows the expression of PRG4 mRNA in human corneal epithelial cells. Human corneal epithelial cells were isolated from corneal-scleral rings from men and women as donors. The amplified samples were screened for the presence of PRG4 products using an Agilent 2100 Bioanalyzer device. Vertical paths include: L. Marker MW; 1. Control without matrix; 2. Corneal tissue from a 33-year-old woman; 4. Cultured corneal epithelial cells from a 70-year-old woman; 6. Cultured corneal epithelial cells from a 53-year-old male.

Figure 3 shows the expression of PRG4 mRNA in human conjunctival epithelial cells. Human corneal epithelial cells were isolated from corneal scleral rings from men and women as donors. The amplified samples were screened for the presence of PRG4 products using agarose gel electrophoresis. Vertical paths include: 1. Marker MW; 2. Control without matrix; 4. Human conjunctiva of women; 5. Human conjunctiva of men.

Figure 4 shows the expression of PRG4 mRNA in human specimens of human conjunctiva. Conjunctival cytological specimens were isolated to men and women as donors. The amplified samples were screened for the presence of PRG4 products using an Agilent 2100 Bioanalyzer device. Vertical paths include: L. Marker MW; 1-9. Conjunctival cytological specimens; 10. Repetition with human conjunctival epithelial cells (lane 4 in figure 3).

Figure 5 shows the expression of PRG4 mRNA in human corneal scleral ring tissue samples. L. Human corneal epithelial cells were isolated from corneal scleral rings from men and women as donors. / amplified samples were screened for the presence of PRG4 products using an Agilent 2100 Bioanalyzer device. Vertical paths include: Marker MW; 1. Human liver cDNA pattern; 2. Corneal scleral ring tissue from a 24-year-old woman; 3. Corneal scleral ring tissue from a 51-year-old woman; 4. Human conjunctival epithelial cells.

Figure 6 shows the time course of the relative increase in PRG4 mRNA in primary corneal epithelial cells under the influence of 10 nM dihydrotestosterone. Cells were cultured in serum-free keratinocyte medium until approximately 80% confluency was reached. Cells (n = 3 wells / treatment / experiment) were then incubated with vehicle or 10 nM dihydrotestosterone (DHT) for up to 5 days. At designated times, cells were processed for total RNA isolation and PRG4 mRNA mRNA analysis by RT-PCR. The results show that DHT induces a marked increase in PRG4 mRNA levels in primary human corneal epithelial cells. This androgen effect, when compared to control levels on Day 0, became apparent after 3 (10.3-fold increase), 4 (3.6-fold increase) and 5 (2.8-fold increase) days of hormone treatment. This DHT effect on PRG4 mRNA expression in primary human corneal epithelial cells was confirmed in another experiment. Treatment of cells for 5 days with DHT resulted in a 46-fold increase in PRG4 mRNA content, compared to content in vehicle-treated control groups.

DETAILED DESCRIPTION OF THE INVENTION [0028] In some embodiments, compositions are provided herein for use in methods of treating an eye surface moisturizing deficiency (e.g. (lack of border lubrication of the eye surface) or related symptoms in a subject in need thereof, comprising topically administering to the surface of the eye of the subject a pharmaceutical composition comprising a therapeutically effective concentration of a PRG4 inducing compound as defined in the appended claims in combination with PRG4. In further embodiments, the PRG4 inducing compound is combined with an ophthalmically acceptable residence time enhancer (e.g. an agent that prolongs the period during which the PRG4 and / or PRG4 inducer remains therapeutically available in the eye). Pharmaceutical compositions provided herein in specific embodiments containing the PRG4 and PRG4 inducing compound for use as described herein are in an ophthalmically acceptable formulation. A pharmaceutical composition suitable for topical application to the eye surface is described herein comprising a therapeutically effective concentration of PRG4 inducer in combination with PRG4 suspended in phosphate buffered solution or in an ophthalmically acceptable balanced salt solution, and may also be combined with one or more ophthalmically acceptable agents or carriers selected from the group consisting of an ophthalmically acceptable soothing agent, an ophthalmically acceptable excipient, an ophthalmically acceptable astringent, an ophthalmically acceptable vasoconstrictor, an ophthalmologically acceptable emollient, hyaluronic acid, sodium hyaluronate and surface active phospholipids as described herein.

[0029] In some embodiments, pharmaceutical compositions are provided herein for use in the treatment of an eye surface hydration deficiency (e.g., deficiency, such as reduced or undesirable border lubrication of the eye surface). The pharmaceutical composition for use in some embodiments of the present description comprises a PRG4 inducing compound in combination with an isolated or purified PRG4 protein suspended in a phosphate buffered solution or ophthalmically acceptable balanced salt solution, and may also be combined with one or more selected ophthalmic agents from the group consisting of soothing agent, excipient, astringent, vasoconstrictor and emollient. In some embodiments, any pharmaceutical composition for use as described herein may further comprise one or more additional therapeutic agents selected from the group consisting of sodium hyaluronate, surfactant phospholipids and electrolytes in a pharmaceutically acceptable topical carrier.

[0030] In some embodiments, the present invention provides a pharmaceutical composition for regulating reduced borderline lubrication of the eye surface by increasing expression of the borderline lubricant on the eye surface. In some embodiments, the pharmaceutical composition increases PRG4 production by a PRG4 inducer. as defined in the appended claims.

[0031] In some cases, the increase in PRG4 expression is specifically localized on the eye surface (acting on the conjunctival and corneal epithelium, goblet cells, etc.) and does not require modification of other ocular tissues such as the lacrimal or thyroid gland.

[0032] As used herein, "PRG4 inducer" or "PRG4 inducer" refers to a compound that increases PRG4 concentration, e.g., a compound that is capable of increasing PRG4 expression, promoting PRG4 biosynthesis, inhibiting PRG4 degradation, or similar. The PRG4 inducing compounds used in the present invention are an androgen or androgen analogue as defined in the appended claims or a selective androgen receptor modulator.

[0033] The androgen or androgen analogue is selected from the group consisting of I7u-metvio173-hydroxy-2-oxa-5tt-androstan-3-one, 4,5u-dihydrotestosterone or 19-nortestosterone.

[0034] In another preferred embodiment, the selective androgen receptor modulators (SARMs) are selected from the group consisting of arylpropionamide (e.g. S-3- (4-acetylaminophenoxy) -2-hydroxy-2-methyl-N- (4-nitro-3 -trifluoromethylphenyl) propionamide [S-4] or S-3- (4-fluoro-phenoxy) -2-hydroxy-2-methyl-N- (4-nitro-3-trifluoromethyl-phenyl) -propionamide [S-1]).

[0035] Another embodiment of the present invention provides the pharmaceutical compositions mentioned above for regulating reduced borderline lubrication of the eye surface by modulating hyperosmolarity on the eye surface. By interfering with feedback mechanisms that prevent reduction of friction coefficients and reduction of shear stress, the present invention includes pharmaceutical compositions for regulating reduced boundary lubrication by modifying the osmolality on the eye surface.

[0036] In another embodiment, the present invention also provides a therapeutic composition for use for regulating and alleviating undesirable conditions of border surface lubrication of the eye by compensating for changes in expression of sex steroids on the eye surface. Androgens, when their concentration is high enough, inhibit aromatization in synovial cells. Testosterone antagonizes the effects of IL-1 on both the loss of proteoglycan and the synthesis of proteoglycan in cartilage. Dehydroepiandrosterone, an androgen precursor, reduces knee swelling during acute and chronic antigen-induced arthritis (AIA), as well as histological signs of inflammation and joint damage during chronic AIA (9). Androgens also appear to protect cartilage against decay caused by inflammation. This finding confirms the pathogenic role of hypoandrogenism in rheumatoid arthritis and suggests that long-term androgen replacement can help prevent joint damage and disability. Thus, in one embodiment of the invention, by the administration of androgens, it is possible to counteract the pro-inflammatory effects of cytokines on the imbalance of sex steroids and associated inflammations on the surface of the eye. [0037] Certain embodiments of the present disclosure relate to therapeutic compositions for use for regulating and ameliorating undesirable borderline lubrication conditions on the eye surface, such as chronic inflammation and hyperosmolarity, which result from androgen deficiency, estrogen replacement therapy, contact lens wear, allergy, aging, eye surface diseases and increased levels of protease in the tear film and on the eye surface. In one embodiment, modification of PRG4 regulation on the eye surface promotes favorable conditions for proper boundary lubrication by breaking the central positive feedback loop by reducing shear stress on the eye surface.

[0038] It should be noted that the importance and mechanism of border lubrication of the eye surface has not been recognized in the ophthalmic community so far. Over the years, a scientific consensus in the orthopedic research community was that hydrodynamic hydration was definitely the dominant way of hydrating cartilage, and that border lubrication was simply a complement. In addition, researchers investigating boundary lubrication on cartilage surfaces suggest that boundary lubrication is probably the only one important at "high load and low speed" that is opposite to surface conditions where there are relatively low axial loads and relatively high sliding speeds. See, e.g. (10). In addition, border lubrication involving corneal glycocalyx has not yet been considered. Jay et al. they compared the purified moisturizing factor of bovine synovial fluid with "mucous glycoprotein from human submandibular saliva and stimulated tears" and found that "mucin secreted by the lacrimal gland did not cause hydration", not paying attention to the possibility that the corneal epithelium was the source of the moisturizing agent or that border lubrication was an important factor on the eye surface. See, e.g. (11). In the latest mathematical models of tear film dynamics, the possibility of border lubrication is also not taken into account, providing a "moisturizing approximation" to the height of the tear film, so that "the mucus layer on the cornea can be scooped providing a non-slip surface for the water film", and that "should be noted that the model only predicts development before reaching the [tear film] thickness of some critical low value at which the model breaks down. " See, e.g. (12).

[0039] There is a need to regulate the hydration of the eye surface and to protect the cornea and conjunctiva from significant shear forces generated by the undesirable conditions described herein, including, as a non-limiting example, dry eye disease in the form associated with water layer deficiency or excessive tear evaporation, Sjogren's syndrome, dry conjunctivitis, androgen deficiency, thyroid gland disease, estrogen replacement therapy, wearing contact lenses, refractive surgery, shortened time to tear film interruption, allergies, eye surface disorders, increased levels of tear film and ocular surface proteases, chronic inflammation, hyperosmolemia and aging.

[0040] In some cases, the load on the cornea and conjunctiva is probably dominated by shear forces. In some cases, eyelid blinking, as well as wearing contact lenses, creates significant stresses on the epithelial cells of the eye surface, and this is particularly the case in the presence of a disturbed tear film. As shown in Figure 1, it is suggested that increased shear stress leads to instability of the tear film, evaporating loss of tears, hyperosmolarity, pressure changes resulting in swelling and a return increase in shear stress. In some cases, it is also believed that increased shear stress causes inflammation, androgen deficiency, and reduced expression of proteoglycans. In some cases, increased shear stress and its consequences may, over time, lead to a loss of boundary lubrication on the eye surface.

[0041] Deficiency in eye surface moisturizing and related symptoms can be determined by any suitable method. In some cases, the lack of hydration of the eye surface and the symptoms associated with it are determined either qualitatively (e.g. feeling of poor moisturizing, dry eye, discomfort, etc.) or quantitatively (e.g. measured by mechanical, biochemical, electrical, optical or other quantitative methods of testing) ).

[0042] In some cases, in undesirable conditions for border lubrication of the eye surface, such as those resulting from dry eye disease in the form associated with water layer deficiency or excessive evaporation of tears, Sjogren's syndrome, dry keratoconjunctivitis, androgen deficiency, thyroid gland disease , estrogen replacement therapy, wearing contact lenses, refractive surgery, reduced tear film break time, allergies, eye surface disorders, increased levels of proteases in the tear film and on the surface of the eye, chronic inflammation, hyperosmolemia and aging, there will be a disturbed tear film. In some of these situations, increased evaporation may exclude effective fluid film moisturizing, but allows for boundary lubrication and a molecular protective mechanism to reduce shear stress on the cell surface. Certain embodiments of the present invention ensure that the therapeutic regulation, supplementation and enrichment of boundary spread agent molecules on the eye surface disrupts the feedback loop through which adverse conditions associated with a lack of hydration of the eye surface endanger the eye surface.

[0043] In some cases, as provided herein, the PRG4 protein plays a critical role in the eye as a border spread. In some cases, this secreted glycoprotein protects the eye surface, protecting the cornea and conjunctiva from significant shear forces generated during eyelid blinking, wearing contact lenses and any other unwanted border lubrication of the eye surface caused by chronic inflammation and hyperosmolality that result from dry eye disease, deficiency androgens, estrogen replacement therapy, allergies, aging, eye surface diseases and increased levels of proteases in the tear film and on the surface of the eye.

[0044] Any pharmaceutical composition used in the present disclosure (e.g. a composition containing a PRG4 inducer and a PRG4 protein suspended in a phosphate buffered solution or ophthalmically acceptable balanced solution) is applied topically to the eye surface, where the PRG4 inducer increases the expression and location of the PRG4 protein on the eye surface, where PRG4 binds, binds and acts as a membrane-bound receptor that can interact with endogenous proteins and proteoglycans within the tear film, setting a protective mechanism to reduce friction when blinking the eyelid on the surface of the eye, prevent protein adsorption on the surface of the eye, and reduce dry spots caused by the instability of tear film.

[0045] In another embodiment of the present invention, any pharmaceutical composition for use as described herein (e.g., a composition comprising a PRG4 inducer and a PRG4 protein) may also be combined with one or more hyaluronic acid and phospholipid products. In some cases of this form, PRG4 acts as a membrane-bound receptor that interacts with exogenously delivered hyaluronic acid and / or phospholipids, establishing a protective mechanism to reduce friction when blinking the eyelid on the eye surface, preventing protein adsorption on the eye surface, and reducing dry spots caused by the impermanence of the tear film. In this embodiment, the hyaluronic acid and phospholipid constructs are dissociated from PRG4 during the shear event. In yet another embodiment, the entire construct detaches during a shear event to prevent shear stresses from reaching the epithelium.

[0046] In yet another embodiment, functional fragments, multimers (e.g., dimers, trimers, tetramers, etc.), PRG4 homologs or orthologs act as a surface receptor and / or gel forming constructs in a sacrificial mechanism. Functional fragments and homologues of PRG4 include those with fewer repeats within the central mucin-like domain with KEPAPTT repeats, glycosylated and non-glycosylated forms of the protein, splice variants, recombinant forms and the like. The PRG4 moisturizing fragment exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% ophthalmic moisturizing effect of human PRG4 as measured qualitatively, mechanically, optically, electrically or in a test biochemical.

[0047] As used herein, the terms "PRG4", "PRG4 protein", "proleoglycan" and "moisturizing agent" are used interchangeably. PRG4 as used herein also includes the term megakaryocyte stimulating factor (MSF) which has been accepted for the UCL / HGNC / HUGO Human Gene Nomenclature database and surface zone protein (SZP). As used herein, the term PRG4 protein or lubrycin (used interchangeably herein with the proteoglycan lubrycin) refers to any isolated or purified native or recombinant lubrycin protein, homologues, functional fragments or motifs, isoforms and / or their mutated forms. In some embodiments, an isolated or purified PRG4 protein comprises the amino acid sequence of human native or recombinant lubricin protein. In other embodiments, an isolated or purified PRG4 protein comprises the amino acid sequence encoded by exons of genuprg4 that encode the primary full-length PRG4 protein structures or isoforms. The proteoglycan 4 (prg4) gene contains 12 exons. The PRG4 protein used herein comprises the amino acid sequence encoded by exup 1-12 genuprg4, more preferably exons 6-12, and most preferably exons 912.

[0048] The term PRG4 protein as used herein includes any PRG4 protein currently known or described later. In some embodiments, a preferred amino acid sequence of the PRG4 protein is given as SEQ ID NO: 1. The PRG4 protein has the same primary amino acid structure as any known PRG4 protein or isoform with at least 60% homology, preferably 75% homology, more preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology. In some embodiments, a preferred PRG4 protein has an average molar mass between 50 kDa and 400 kDa, comprising one or more biologically active parts of the PRG4 protein or functional fragments, such as a moisturizing fragment or homologue thereof.

[0049] The term PRG4 protein as used herein includes the biologically active portion of the protein. As used herein, the term "biologically active portion" of a PRG4 protein includes a functional protein fragment comprising amino acid sequences that are sufficiently homologous or obtained from an amino acid sequence of the protein that has fewer amino acid residues than the full length protein and has at least one full length protein activity. Typically, the biologically active portion includes a functional domain or motif with at least one protein activity. The biologically active part of the protein may be a polypeptide that has a length, for example, 10, 25, 50, 100, 200 or more amino acid residues. In one embodiment, the biologically active portion of the PRG4 protein can be used as a therapeutic agent alone or in combination with other therapeutic agents for the treatment of unwanted or reduced border surface lubrication.

[0050] Nucleic and amino acid sequences of several native and recombinant PRG4 or lubrycin proteins and the characteristics of PRG4 proteins and various isoforms are disclosed, for example, in US Patent Nos. 5,326558; 6433142; 7030223; 7361738, Turner et al., And in US Patent Nos. 6,743,774 and 6,960,562, Jay et al. In US Publication No. 20070191268, Flannery et al. also disclosed are recombinant PRG4 or lubrycin molecules useful in the present invention.

[0051] Methods for isolating, purifying and recombinantly expressing a PRG4 protein are well known in the art. In some embodiments, the method is started by cloning and isolating mRNA and cDNA encoding PRG4 proteins or isoforms using standard molecular biology techniques such as PCR or RT-PCR. The isolated cDNA encoding the PRG4 protein or isoform is then cloned into an expression vector and then transformed and expressed in a host cell to produce a recombinant PRG4 protein.

[0052] The term "recombinant" as used herein refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., "recombinant polynucleotide"), methods for using recombinant polynucleotides to produce gene products in cells or other biological systems, or an encoded polypeptide ("recombinant protein") by a recombinant polynucleotide. "Recombinant" also includes ligation of nucleic acids having different coding regions or domains or promoter sequences from various sources into an expression cassette or expression vector, e.g., with inducible or constitutive expression of a fusion protein comprising the active domain of the PRG4 gene, and a nucleic acid sequence amplified using a starter according to the invention.

[0053] In some embodiments, the PRG4 protein encoding the nucleic acid may contain one or more mutations, deletions or insertions. In such embodiments, the nucleic acid encoding the PRG4 protein has at least 60% homology, preferably 75% homology, more preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the nucleic acid encoding the protein Wild type PRG4.

[0054] As used herein, the term "cDNA" includes DNA that is complementary to mRNA molecules present in the cell or mRNA in the body that can be converted to cDNA using an enzyme such as reverse transcriptase. In some embodiments, the cDNA encoding the PRG4 protein is isolated from PRG4 mRNA expressed in human corneal or conjunctival epithelial cells using an RT-PCR method well known in the art.

[0055] As used herein, the terms "polynucleotide", "nucleic acid / nucleotide" and "oligonucleotide" are used interchangeably and include polymeric forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: gene or gene fragment, exons, introns, matrix RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, DNA, cDNA, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, any DNA of any kind sequences, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can be naturally occurring, synthetic, recombinant or any combination thereof.

[0056] A polynucleotide may include modified nucleotides such as methylated nucleotides and nucleotide analogues. If present, modifications of the nucleotide structure may be violated before or after polymer assembly. Non-nucleotide components may be present as members in the nucleotide sequence. The polynucleotide can also be modified after polymerization, such as by coupling to a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, any form of this invention that is a polynucleotide includes both a double-stranded form and each of two complementary single-stranded forms known or anticipated to form the double-stranded form.

[0057] The term "polynucleotide sequence" as used herein means an alphabetical representation of the polynucleotide molecule. A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) in place of thymine when the polynucleotide is RNA instead of DNA. This alphabetic representation can be inserted into computer databases and used for bioinformatics applications such as functional genomics and homology search, for example.

[0058] The term "isolated polynucleotide / cDNA" as used herein includes polynucleotide molecules that have been separated from other polynucleotide molecules that are present in a natural polynucleotide source. For example, when referring to genomic DNA, the term "isolated" includes polynucleotide molecules that have been separated from the chromosome to which the genomic DNA is naturally associated. Preferably, the "isolated" polynucleotide is free of sequences that 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 was derived. For example, in various embodiments, an isolated polynucleotide molecule encoding a PRG4 protein used in the invention may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank a polynucleotide molecule in the genomic DNA of the cell from which the polynucleotide was derived. In addition, an "isolated" polynucleotide molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium, if produced by recombinant techniques, or substantially free of chemical precursors or other chemical agents when chemically synthesized.

[0059] The term "gene" as used herein includes a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after transcription and translation. Any of the polynucleotide sequences described herein can also be used to identify larger fragments or full-length coding sequences of the gene to which they are associated. Methods for isolating larger sequence fragments are known to those skilled in the art. As used herein, the term "native or naturally occurring" polynucleotide molecule includes, for example, an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encoding a natural protein).

[0060] As used herein, the terms "polypeptide" or "protein" are interchangeable and include the association of two or more amino acid subunits, amino acid analogs, or peptidomimetics. The subunits can bind via peptide bonds. In other forms, the subunit may bind through other bonds, e.g., ester, ether, etc. The term "amino acid" as used herein includes natural and / or unnatural or synthetic amino acids, including glycine and optical D and L isomers, as well as amino acid analogs and peptidomimetics. A peptide with three or more amino acids is commonly called an oligopeptide. Peptide chains larger than three or more amino acids are called a polypeptide or protein.

[0061] In some embodiments, the PRG4 protein used herein refers to PRG4 proteins or their various homologues or isoforms that are expressed naturally or recombinantly in humans or in other host cells. The term "express" or "expression" as used herein includes the process by which polynucleotides are transcribed into RNA and / or translated into polypeptides. If the polynucleotide was derived from genomic DNA, expression may include RNA splicing if the appropriate eukaryotic host was selected. The regulatory elements required for expression include promoter sequences for binding RNA polymerase and transcription initiation sequences for ribosome binding. For example, the bacterial expression vector contains a promoter, such as a lac promoter, and a Shine-Dalgarno sequence and AUG start codon for transcription initiation. Similarly, the eukaryotic expression vector includes a heterologous or homologous promoter for RNA II polymerase, the following polyadenylation signal, the AUG start codon and the ribonome termination codon. Such vectors can be obtained commercially or assembled by the sequences described in methods well known in the art, for example, methods described below for constructing vectors in general. The term "vector" as used herein includes a self-replicating nucleic acid molecule that carries the inserted polynucleotide into and / or between host cells. The term is intended to include vectors whose function is primarily to introduce the nucleic acid molecule into the cell, replication vectors whose function is primarily to replicate the nucleic acid, and expression vectors whose function is to transcribe and / or translate DNA or RNA . Vectors that provide one or more of the above functions are also included.

[0062] The term "host cell" as used herein is intended to include any single cell or cell culture that may or may be the recipient of vectors or the inclusion of exogenous polynucleotides and / or polypeptides. It is also intended to include single cell offspring. The offspring need not necessarily be completely identical (in morphology or genomic or complete complementary DNA) to the primary parent cell due to natural, accidental or intentional mutation. Cells may be prokaryotic or eukaryotic and may include, but are not limited to, bacterial cells, yeast cells, insect cells, animal cells and mammalian cells, including, but not limited to, mouse, rat, monkey or human cells. The term "host cell" as used herein also includes genetically modified cells. The term "genetically modified cells" includes cells containing and / or expressing a foreign or exogenous gene or polynucleotide sequence, which in turn modifies the genotype or phenotype of the cell or its progeny. "Genetically modified" also includes a cell containing or expressing a gene or polynucleotide sequence that has been introduced into the cell. For example, in this form, a gene has been introduced into the genetically modified cell, which gene is also endogenous to the cell. The term "genetically modified" also includes any addition, deletion or disruption of endogenous nucleotides of a cell. As used herein, the "host cell" may be any cells that express the human PRG4 protein.

[0063] The term "homologs" as used herein is defined herein as two nucleic acids or peptides that have similar or substantially identical nucleic acids or amino acid sequences, respectively. The term "homologue" further includes nucleic acid molecules that differ from one of the nucleotide sequences due to the degeneracy of the genetic code and thus encode the same amino acid sequences. In one preferred embodiment, the homologues include allelic variants, orthologs, paralogs, agonists and antagonists of nucleic acids encoding the PRG4 protein (e.g., SEQ ID NO: 1).

[0064] The term "orthologs" as used herein refers to two nucleic acids from different species, but which have evolved from a common ancestor gene through speciation. Normally, orthologs encode peptides having the same or similar functions. In particular, orthologs according to the invention generally exhibit at least 80-85%, preferably 85-90% or 90-95%, and most preferably 95%, 96%, 97%, 98% or even 99% identity or 100% sequence identity , with all or part of the amino acid sequence of any of the known PRG4 proteins (e.g., SEQ ID NO: 1), isoforms or analogs thereof, and exhibit a similar function to these peptides. As used herein, the term "paralogs" refers to two nucleic acids that are bound by duplication within the genome. Paralogs usually have different functions, but these functions can be related.

[0065] To determine the percent sequence identity of two amino acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be inserted into the sequence of one polypeptide for optimal alignment with another polypeptide or nucleic acid). The amino acid residues are then compared at the corresponding amino acid positions. When a position in one sequence is occupied by the same amino acid residue as the corresponding position in the other sequence, then the molecules are identical in that position. The same type of comparison can be made between two nucleic acid sequences. The percent sequence identity between two sequences is a function of the number of identical positions common to those sequences (i.e., percent sequence identity = number of identical positions / total number of positions x 100). Preferably, the isolated amino acid homologues included in the present invention are at least about 30-60%, 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 preferably at least about 96%, 97%, 98%, 99% or more identical to the entire amino acid sequence of any known PRG4 protein (e.g., SEQ ID NO: 1).

[0066] In some embodiments, an isolated nucleic acid homologue encoding a PRG4 protein comprises a nucleotide sequence that is at least about 40-60%, preferably at least about 60-70%, more preferably at least about 70-75% , 75-80%, 8085%, 85-90% or 90-95%, and more preferably at least about 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence coding amino acid sequences of such a PRG4 protein (e.g., SEQ ID NO: 1).

[0067] Determining the percent sequence identity between two nucleic acid or peptide sequences is well known in the art. For example, the Vector NTI 6.0 (PC) software package (InforMax, Bethesda, MD) can be used to determine the percentage sequence identity between two nucleic acid or peptide sequences. In this method, a penalty for introducing a gap of 15 and a penalty for extending the gap of 6.66 are used to determine the percent identity of two nucleic acids. To determine the percent identity of two polypeptides, a gap penalty of 10 and a gap penalty of 0.1 are used. All other parameters are set to the default settings. For multiple alignment purposes (Clustal W algorithm), the penalty for entering a gap is 10, and the penalty for extending the gap is 0.05 with the blosum62 matrix. It should be understood that for purposes of determining sequence identity when comparing DNA sequences with RNA sequences, the thymidine nucleotide is equivalent to the uracil nucleotide.

[0068] In addition, the PRG4 protein used herein includes a PRG4 protein encoded by a polynucleotide that hybridizes to a polynucleotide encoding the PRG4 protein under stringent conditions. The term "hybridization" as used herein includes a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between bases of nucleotide residues. Hydrogen bonding can occur by base pairing according to the principle of complementarity ( Watson-Crick base pairing), Hoogstein-type binding or any other sequence-specific manner. The complex may include two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand and any combination thereof. The hybridization reaction can be a step in a more extensive process, such as initiating PCR or enzymatic digestion of a polynucleotide by ribozyme.

[0069] Hybridization reactions can be carried out under various stringent conditions. The present invention includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably under stringent conditions, and most preferably under very stringent conditions, with polynucleotides encoding the PRG4 protein described herein. The term "stringent conditions" as used herein refers to hybridization overnight at 60 ° C in 10 x Denhart solution, 6xSSC, 0.5% SDS and 100 mg / ml denatured salmon sperm DNA. Blots are washed successively at 62 ° C for 30 minutes each in 3xSSC / 0.1% SDS, then 1xSSC / 0.1% SDS and finally 0.1xSSC / 0.1% SDS. Also used herein, in some embodiments, the term "stringent conditions" refers to hybridization in a 6xSSC solution at 65 ° C. In other embodiments, the term "very stringent conditions" refers to hybridization overnight at 65 ° C in 10x Denhart solution, 6xSSC, 0.5% SDS and 100 mg / ml denatured salmon sperm DNA. Blots are washed sequentially at 65 ° C for 30 minutes each in 3xSSC / 0.1% SDS, then 1xSSC / 0.1% SDS and finally 0.1xSSC / 0.1% SDS. Methods for hybridizing nucleic acids are well known in the art. Accordingly, the PRG4 proteins encoded by the nucleic acids used herein include nucleic acids having at least 60% homology, preferably 75% homology, more preferably 83%, more preferably 90%, most preferably 95%, 96%, 97%, 98%, 99% homology to a polynucleotide sequence that encodes a human PRG4 protein (e.g., SEQ ID NO: 1) or a specific isoform or homolog thereof.

[0070] In addition, the PRG4 proteins used herein may also be a chimeric or fusion protein. The term "chimeric protein" or "fusion protein" as used herein includes a first polypeptide operably linked to a second polypeptide. Chimeric proteins may optionally include a third, fourth or fifth polypeptide operably linked to the first or second polypeptide. Chimeric proteins can include two or more different polypeptides. Chimeric proteins can include multiple copies of the same polypeptide. Chimeric proteins can also include one or more mutations in one or more polypeptides. Methods for producing chimeric proteins are well known in the art. In certain embodiments of the present invention, the chimeric protein is a chimeric PRG4 protein with other isoforms of the PRG4 protein.

[0071] As used herein, the term "isolated" or "purified" protein, polynucleotide or molecule means that they are removed from the environment in which they naturally occur, or substantially free of cellular material, such as other contaminating proteins from a cellular or tissue source, from which a protein, polynucleotide or molecule is obtained, or substantially free of chemical precursors or other chemical substances when chemically synthesized. The phrase "substantially free of cellular material" includes preparations separated from cellular components of the cells from which it was isolated or recombinantly produced or synthesized. In some embodiments, the phrase "substantially free of cellular material" includes PRG4 protein preparations having less than about 30% (dry weight) of other proteins (also referred to herein as "contaminating protein"), more preferably less than about 20%, even more preferably less than about 10%, and most preferably less than about 5% of other proteins. When the protein or polynucleotide is produced recombinantly, they are also preferably substantially free of culture medium, i.e. the culture medium constitutes less than about 20%, more preferably less than about 10%, and most preferably less than about 5% by volume of the protein preparation of interest.

[0072] In some embodiments, the present invention provides a pharmaceutical composition for use in the treatment of an eye surface moisturizing deficiency or related symptoms for topical administration to the eye surface of a subject in need of a pharmaceutically effective concentration of a PRG4 inducer, as defined in the appended claims. optional mucoadhesive and PRG4 suspended in an ophthalmically acceptable balanced salt solution and in combination with one or more ophthalmically acceptable agents. Ophthalmically acceptable agents can be selected from the group consisting of an ophthalmically acceptable soothing agent, excipient, astringent, vasoconstrictor and emollient. The term "effective concentration or amount" or "therapeutically effective concentration or amount" as used herein is intended to mean a non-toxic but sufficient concentration or amount of an PRG4 inducer, PRG4 or other therapeutic agent to provide the desired therapeutic effects. The concentration or amount that is effective will vary between individuals, depending on the age, general condition of the individual, individual agents, and the like. Thus, it is not always possible to determine the exact effective concentration or amount. However, the appropriate effective concentration or amount in any individual case may be determined by one skilled in the art using routine experimentation. Furthermore, the exact effective concentration or amount of PRG4 in this compound, PRG4 protein or other therapeutic agents included in the composition or dosage form of the present invention is not critical as long as the concentration is in a range sufficient to allow for easy application of the solution or preparation to provide the amount PRG4 including a compound, PRG4 protein or other active agents that is in a therapeutically effective range.

[0073] In some embodiments, the pharmaceutically effective concentration of PRG4 inducer, as defined in the appended claims, is in the range of 0.0001-0.1% w / v, and the pharmaceutically effective concentration of PRG4 protein is in the range of 100-300 pg / ml. Ophthalmically acceptable agents used herein include ophthalmically acceptable soothing agents, excipients, astringents, vasoconstrictors and emollients are fully defined in Code of Federal Regulations 21CFR349.

[0074] In some embodiments, the pharmaceutical compositions for use as described herein comprise a residence time extender that increases the residence time of the PRG4 inducer on the surface of the eye. In some embodiments, the residence time extender is present in an amount such that when the pharmaceutical composition is administered to the surface of the subject's eye, a therapeutically effective amount of a PRG4 inducing compound described herein is retained on the surface of the eye. In some embodiments, the residence time extender is selected and / or present in an amount such that a therapeutically effective amount of a PRG4 inducer is retained on the surface of the eye for at least a therapeutically effective period of time, at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour or more. In some embodiments, ophthalmically acceptable residence time enhancers or mucoadhesive agents may include, as non-limiting examples, hydroxypropyl methylcellulose, carboxymethylcellulose, carbomer (acrylic acid polymer), polymethylmethacrylate, polyacrylamide, polycarbophil, polyethylene oxide, butylacrylate acrylic acid sodium, dextran or combinations thereof. The present invention includes any high molecular weight polymers that would increase the time during which the PRG4 inducing compound remains on the surface of the eye.

[0075] The term "topical administration" as used herein is used in its usual sense and means delivery of a composition comprising a PRG4 protein and one or more ophthalmically acceptable agents to the eye. Generally, topical administration is accomplished using a liquid eye drop or rinse formulation and provides a local effect.

[0076] In certain embodiments, any pharmaceutical composition for use as described herein comprises, or the above-mentioned ophthalmically acceptable agents are or may be combined with one or more of sodium carboxymethyl cellulose (e.g., about 0.2 to about 2.5 % w / v), hydroxyethyl 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), dextran 70 (e.g. 0.1% w / v), gelatin (e.g. 0.01% w / v), glycerin (e.g. 0.2 to 1% w / v), poly (glycol ethylene) 300 (e.g. about 0.2 to about 1% w / v), polyethylene glycol 400 (e.g. about 0.2 to about 1% w / v), polysorbate 80 (e.g. about 0.2 to about 1% w / v), propylene glycol (e.g. about 0.2 to about 1% w / v), poly (vinyl alcohol) (e.g. about 0.1 to about 4 % w / v), povidone (e.g. about 0.1 to about 2% w / v), zinc sulfate (e.g. 0.25% w / v), anhydrous lanolin (e.g., about 1 to about 10% w / v); lanolin (e.g. about 1 to about 10% w / v), light mineral oil (e.g. <about 50% w / v), mineral oil (e.g. <about 50% w / v), paraffin (e.g. <about 5% w / v), petroleum jelly (e.g. <about 100% w / v), wax ointment (e.g. <about 100% w / v), white petrolatum (e.g. <about 100% w / v), white wax (e.g. <about 5% w / v), yellow wax (e.g. <about 5% w / v), ephedrine hydrochloride (e.g. about 0.123% w / v), naphazoline hydrochloride (e.g. about 0.01 to about 0.03% w / v), phenylephrine hydrochloride (e.g., about 0.08 to about 0.2% w / v) and tetrahydrozoline hydrochloride (e.g., about 0.01 to about 0.05% w / v). In some cases, the percentages used herein are amounts in percent by weight.

[0077] In further embodiments, any pharmaceutical composition for use in the present disclosure (e.g., a composition comprising a PRG4 and PRG4 inducer) may further comprise a therapeutically effective concentration of hyaluronic acid or sodium hyaluronate in the range of 10-100,000 pg / ml, preferably 500-5000 pg / ml. In addition, the pharmaceutical composition for use in the present invention may further contain one or more surface-active phospholipids in the range of 10-10000 pg / ml, such surface-active phospholipids include, but are not limited to, L-dipalmitoyl phosphatidylcholine (DPPC), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (Sp) or other neutral and polar lipids. In this embodiment, the combination of more hydrophobic modulators with amphiphilic border spreads molecules allows the direct transport of therapeutically effective molecules to the surface cells of the eye, where border spreads tend to aggregate, and provides a pharmaceutically effective carrier for therapeutic compounds of the corneal epithelium and conjunctiva effective border lubrication. For example, solubilization of hydrophobic androgens in DPPC, followed by complexation of DPPC, HA and PRG4 in a pharmaceutically acceptable carrier, transports and concentrates androgens in the surface cells of the eye, where they can most effectively increase the expression of the border spread.

[0078] The pharmaceutical composition for use in the present invention may further comprise one or more pharmaceutically acceptable carriers or excipients comprising any acceptable materials and / or any one or more additives known in the art. The term "carrier" or "vehicle" as used herein refers to carrier materials suitable for topical drug administration. Carriers and substrates useful herein include any such materials known in the art that are not toxic and do not interact with other components of the composition in a detrimental manner. Various additives known to those skilled in the art can be included in the composition. For example, solvents, including relatively small amounts of alcohol, can be used to dissolve certain drug substances. Other optional additives include opacifiers, antioxidants, fragrance, coloring agent, gelling agents, thickeners, stabilizers, surfactants and the like. Other agents, such as antimicrobials, may also be added to prevent spoilage during storage, i.e. to inhibit the growth of microorganisms such as yeasts and molds. Suitable antimicrobials are usually selected from the group consisting of methyl and propyl esters of p-hydroxybenzoic acid (i.e. methyl and propylparaben), sodium benzoate, sorbic acid, imidourea and combinations thereof. Penetration enhancers and / or irritation suppressants may also be included in the pharmaceutical composition of the present invention.

[0079] In some embodiments, the pharmaceutical composition for use in the present invention is prepared in a pharmaceutically acceptable carrier, such as phosphate buffered saline or osmotically balanced tear electrolyte salt solution, including one or more of sodium chloride at a molar fraction of about 44 % to about 54%, potassium chloride in a mole fraction of about 8% to about 14%, sodium bicarbonate in a mole fraction of about 8% to about 18%, potassium bicarbonate in a mole fraction of about 0% to about 4%, calcium chloride in a mole fraction of about 0% to about 4%, magnesium chloride in a mole fraction of about 0% to about 4%, trisodium citrate in a mole fraction of about 0% to about 4 % and hydrochloric acid in a mole fraction of about 0% to about 20% or sodium hydroxide in a mole fraction of about 0% to about 20%. In some embodiments, a pharmaceutical carrier may be prepared to form an aqueous electrolyte solution in the range of about 150-200 nM. Other suitable formulations, such as ointments, creams, gels, pastes and the like, suitable for topical administration, are also contemplated by the present invention. In some embodiments, the electrolytes provide a proper osmotic balance when combined with the PRG4 inducing compound and optionally PRG4, rendering the solution ophthalmically acceptable.

[0080] The present disclosure further relates to a pharmaceutical composition for use in a method of treating reduced or unwanted border surface lubrication of the eye, symptoms associated with it, or a condition that is associated with or causes a lack of moisturizing of the eye surface in a subject in need thereof, comprising topical administration to the surface eye that needs this individual, a pharmaceutical composition containing a therapeutically effective amount of a PRG4 inducer in combination with PRG4. In one embodiment, the method of the present invention comprises the topical administration of a pharmaceutical composition comprising a therapeutically effective amount of a PRG4 inducing compound in combination with PRG4, which is suspended in a phosphate buffered saline solution or in an ophthalmically acceptable balanced salt solution containing one or more tear electrolytes. In yet another embodiment, the present disclosure includes topical administration of a pharmaceutical composition comprising a PRG4 inducing compound as defined in the appended claims and PRG4, which is prepared in an ophthalmically acceptable formulation containing one or more additional ophthalmically acceptable agents as discussed above.

[0081] The term "treat or treatment" as used herein refers to reducing the severity and / or frequency of symptoms, removing symptoms and / or underlying causes, preventing the occurrence of symptoms and / or underlying causes, and reducing or repairing damage. The term "treat or treat" also includes both preventing the disorder in a predisposed subject and treating the disorder in the subject with clinical symptoms.

[0082] In some embodiments, reduced border lubrication of the eye surface results in increased loss of tears by evaporation or unstable tear film in the ocular border loop. Such reduced or undesirable borderline lubrication of the eye surface is associated with dry eye disease resulting from watery component deficiency or excessive evaporation, Sjogren's syndrome, dry keratoconjunctivitis (KCS), androgen deficiency, thyroid gland disease, estrogen replacement therapy, wearing contact lenses , refractive surgery, allergies, shortened time to tear film interruption, eye surface disorders, increased levels of proteases in the tear film and on the surface of the eye, chronic inflammation, hyperosmolemia and aging. As discussed above, increased shear stresses lead to tear film instability, loss of tears by evaporation, hyperosmolarity, edema pressure changes, and a reversal of shear stresses. Increased shear stress also causes inflammation, androgen deficiency, and reduced expression of proteoglycans. Over time, increased shear stresses and their consequences lead to a loss of border lubrication on the eye surface. Accordingly, the present invention provides for reduction of shear stress by supplementing and enriching expression of proteoglycans such as the PRG4 protein on the eye surface, using the PRG4 inducing compound as disclosed above, so as to prevent or increase border lubrication of the eye surface.

[0083] The invention is further illustrated by the following examples.

EXAMPLES

EXAMPLE 1

Expression of PRG4 mRNA in human corneal and conjunctival epithelial cells [0084] Human corneal epithelial cells were isolated from the corneal scleral rings of men and women as donors. Cells were processed directly (n = 8) or first grown in serum-free and phenol red keratinocyte medium (n = 2). Conjunctival conjunctiva (n = 2), conjunctival cytological specimens (n ​​= 9), immortalized human conjunctival epithelial cells after culture (n = 1), tear glands of NOD mice (n = 5 adult mice / sex, 10 glands / sample) and BALB / c mouse thyroid glands (n = 7 adult mice / gender, 28 eyelid glands / sample) were obtained during surgical procedures. These samples were processed for PRG4 mRNA analysis using first RT-PCR (n = 18 people, all mice) and Affymetrix GeneChips (n = 4 human corneas). PRG4 primers for PCR included 1 kb of intron sequences to inhibit the amplification of contaminating chromosomal DNA (Table 1). The amplified samples were screened for the presence of PRG4 products using agarose gel electrophoresis and an Agilent 2100 Bioanalyzer device. To confirm the identity of the amplicons, PCR products from corneal samples (n = 2), conjunctival epithelial cells (n = 1) and human liver standard (n = 1) were sequenced using a 3100 Genetic Analyzer at Massachusetts Eye and Ear Infirmary DNA Sequencing Center for Vision Research (Boston, MA) and the data obtained was analyzed using BLASTn searches of the GenBank databases.

Table 1. Oligonucleotide primers designed for PRG4 mRNA analysis by RT-PCR

Species Orientation Nucleotide sequence (5 '- 3') Exons Amplification size (bp)

Meaningful Man GATGCAGGGTACCCCAAA 9-12 526 (SEQ ID NO: 2)

Antisense CAGACTTTGGATAAGGTCTGCC (SEQ ID NO: 3) [0085] PRG4 mRNA has been shown to be present in all human corneal and conjunctival epithelial cells and cytological imprint samples. The identity of PRG4 PCR products was confirmed by DNA sequence analysis (Table 2). The results show that PRG4 is transcribed in human corneal and conjunctival epithelial cells.

Table 2. Identification of the amplicon sequence from samples of the human cornea, conjunctiva and liver. Direction of alignment of the pair with total pairs Identity according to the search sequencing with human PRG4 orchard from the amplicon of swinging BLASTING

Pattern from human liver

<td>AND</td><td>Forward</td><td> 495</td><td> 500</td><td>Human PRG4</td>

<td>AND</td><td>Backwards</td><td> 488</td><td> 491</td><td>Human PRG4</td>

<td>B</td><td>Forward</td><td> 496</td><td> 499</td><td>Human PRG4</td>

<td>B</td><td>Backwards</td><td> 498</td><td> 500</td><td>Human PRG4</td>

Human cornea (24-year-old woman)

<td>AND</td><td>Forward</td><td> 497</td><td> 499</td><td>Human PRG4</td>

<td>AND</td><td>Backwards</td><td> 490</td><td> 492</td><td>Human PRG4</td>

<td>B</td><td>Forward</td><td> 500</td><td> 504</td><td>Human PRG4</td>

<td>B</td><td>Backwards</td><td> 498</td><td> 501</td><td>Human PRG4</td>

Human cornea (51-year-old woman)

<td>AND</td><td>Forward</td><td> 498</td><td> 499</td><td>Human PRG4</td>

<td>AND</td><td>Backwards</td><td> 474</td><td> 489</td><td>Human PRG4</td>

<td>B</td><td>Forward</td><td> 496</td><td> 498</td><td>Human PRG4</td>

<td>B</td><td>Backwards</td><td> 490</td><td> 491</td><td>Human PRG4</td>

Human conjunctival epithelial cells

<td>AND</td><td>Forward</td><td> 496</td><td> 499</td><td>Human PRG4</td>

<td>AND</td><td>Backwards</td><td> 490</td><td> 492</td><td>Human PRG4</td>

<td>B</td><td>Forward</td><td> 495</td><td> 499</td><td>Human PRG4</td>

<td>B</td><td>Backwards</td><td> 474</td><td> 491</td><td>Human PRG4</td>

Two different samples (A and B) from each preparation were sequenced forward and backward. Samples of the human conjunctiva were epithelial cells from the corneo-scleral rings from women as donors. The gene access number for human PRG4 is NM_005807.

EXAMPLE 2

In vitro regulation of PRG4 expression in vitro [0086] Androgen treatment increases PRG4 mRNA expression in primary human corneal epithelial cells. Ways. Cells were cultured in serum-free keratinocyte medium until approximately 80% confluency was reached. Cells (n = 3 wells / treatment / experiment) were then incubated with vehicle or 10 nM dihydrotestosterone (DHT) for up to 5 days. At designated times, cells were processed for total RNA isolation and PRG4 mRNA mRNA analysis by RT-PCR. Results. The results show that DHT induces a marked increase in PRG4 mRNA levels in primary human corneal epithelial cells (Figure 6). This androgenic effect, when compared to control levels on Day 0, became apparent after 3 (10.3-fold increase), 4 (3.6-fold increase) and 5 (2.8-fold increase) days of exposure to hormones. This effect of DHT on PRG4 mRNA expression in primary human corneal epithelial cells was confirmed in another experiment. Treatment of cells for 5 days with DHT resulted in a 46-fold increase in PRG4 mRNA content, compared to content in vehicle-treated control groups.

[0087] Combination treatment with 17β-estradiol and progesterone reduces PRG4 mRNA expression in mouse tear tissue. Age-matched and young adult BALB / c mice that had their ovaries removed at 8 weeks of age were purchased from Taconic Laboratories (Germantown, NY). The animals were kept in constant temperature rooms with a set light / dark period of 12 hours. Ten days after surgery, ovariectomized mice were implanted subcutaneously with granules containing vehicle (cholesterol, methylcellulose, lactose) or ββ-estradiol (0.5 mg) plus progesterone (10 mg). Granules were obtained from the Innovative Research of America (Sarasota, Florida) and were designed for continuous release of placebo or physiological amounts of sex steroid (i.e. as in pregnancy) for 3 weeks. After 14 days of treatment, the mice (n = 7 mice / condition / experiment) were sacrificed by CO2 inhalation and the exorbital tear glands were removed, combined according to the group (n = 14 glands / sample) and processed for molecular biological procedures.

[0088] Total RNA was isolated from tissues using TRIzol reagent (Invitrogen Corp., Carlsbad, CA). When indicated, the samples were also exposed to RNase-free DNase (Invitrogen), examined spectrophotometrically at 260 nm to determine the concentration and evaluated on 6.7% formaldehyde / 1.3% agarose gels (Gibco / BRL, Grand Island, NY ) to verify RNA integrity. RNA samples were further purified using RNAqueous centrifugation columns (Ambion, Austin, Tx), and the integrity of these preparations was assessed using an RNA 6000 Nano LabChip chip with an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, California). RNA samples were then processed for hybridization with CodeLink Bioarray. Briefly, cDNA was synthesized from RNA (2 Lig) using the CodeLink Expression Reagent Reagent Kit (Amersham, Piscataway, NJ) and purified using the QIAquick Purification Kit (Qiagen, Valencia, California). After drying the sample, cRNA was generated using the CodeLink expression reagent kit (Amersham), recovered using the RNeasy kit (Qiagen) and quantified using a UV spectrophotometer. The fragmented, biotin labeled cRNA was then incubated and shaken (shaking at 300 rpm) on a CodeLink Bioarray matrix at 37 ° C for 18 hours. The Bioarray matrix was washed, exposed to streptavidin-Alexa 647 and scanned using ScanArray Express software and ScanArray Express HT (Packard BioScience, Meriden, CT) with the laser set at 635 nm, laser power at 100% and photoelectron multiplier voltage at 60% . Files with scanned images were evaluated using CodeLink image and data analysis software (Amersham), which generated both raw and normalized hybridization signal intensities for each point on the matrix. Point intensities (~ 10,000) on the microarray image were standardized to median 1. Standardized data, with signal intensities exceeding 0.75, was analyzed using GeneSifter.Net software (VizX Labs LLC, Seattle, WA, vizxlabs.com). Statistical analysis of data on the expression of individual genes was carried out using the Student's t test (two-sided, unpaired).

[0089] The data show that combined treatment with estradiol and progesterone, compared to placebo treatment, results in a significant (p = 0.020) 1.6-fold decrease in PRG4 gene expression in the mouse tear gland.

EXAMPLE 3

Treatment of borderline lubrication of the eye surface in vivo with androgen and PRG4 [0090] A patient complaining of eye surface irritation was examined for eye surface hydration or conditions associated with eye surface hydration deficiency by measuring symptoms of more than 2 positive questionnaire responses

McMonnies, score greater than 5 in the Ocular Surface Disease Index (OSDI) or by signs of some symptoms on a visual analog scale, in combination with objective signs including one or more of the shortened tear film interruption time (less than 10 seconds), lateral osmolality lower tear meniscus greater than 308 milliosmoles / l, low value for the Schirmer belt (less than • 10 mm), sodium fluorescein staining of the cornea or conjunctiva (results&gt; 0 with many large dots), significant remains obtained from the cytological imprint, freely determined thyroid gland dysfunction, reduction of the contact lens displacement speed after blinking, changes in the spatio-temporal transfer function of the contact lens after application a series of pressure pulses, reducing the tear film relaxation index after blinking in an interferometric test, increase in proinflammatory cytokines, decrease in lactoferrin or lysozyme, or increase in the decoherence index of the blink point function.

[0091] 1 to 2 drops of a solution containing 4,5? -Dihydrotestosterone and 200 pg / ml PGR4 protein suspended in an ophthalmically acceptable balanced salt solution are administered to the patient's surface. The patient was instructed to close his eyes for 10 seconds.

[0092] During follow-up visits, a reduction in lateral lower tear osmolality, an increased tear film break time, or other previously mentioned symptoms may be traced. In particular, if the osmolality of the tear film has decreased from an abnormal value (maybe 330 milliosmoles / L) to a more normal value (maybe 304 milliosmoles / L), the therapeutic modification and supplementation of eye surface moisturizing is considered effective.

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LIST OF SEQUENCES [0094]

SEQ ID NO: 1 MAWK'n.PIYLLLLLSVFVIQQVSSQDLSSCAGR.CGEGYSRDAT € NCDYNCQHYM EC-CPDFKRVCTAELSCKGRCFESFERGRECDCDAQCKKYDKCCPDYESFCAEVHN PTSPPSSKKA PPPSG.A SQTlKSlTFKRSPKPPNKKKTKKYIESE EITEEHSY SEN QESSS SSSSSSSSST1RKIKSSKNSAANRELQKKLKVKDNKK.NRTKKKPTPKPPWDEAGS GLQNGDFKVTTPDTSrrQHNKVSTrePKlTTAKPINPRPSLPPNSDTSKETSLTVNKE TTVETKETTTTOK.QmDGKEKTnrSAKim SIEKTSAKDLAPTSK.Vlj ^ \ KPTPKAET TrKGPALTIPK.EPTPrFPKEPASITPKliPTPTTIKSAPTTFKEPAPTITKSAPnPKEP

AP Π TK EPA PΓΓΡΚΕΡΑΡTI1KEPAPTF1KSAPITPKEPAP ΠΡΚΚΡΑΡΓΓΡΚΕΡΑΡ T ΤΡΚΕΡ'ΓΡΤΓΡΚΕΡΑΡ'Π'ΚΕΡΑΡ'Π'ΡΚΕΡΑΡΓΑΡΚΚΡΑΡ'Π'ΡΚΕΡΛΡ'ΪΤΡΚΕΡΑΡΤΓΓΚ EPSPTTPKEPA PTTTKSA PTTTKEPAPTTTKSAPTTP KEPSPTTTKEPAPTTPKEPAPT ΤΡΚΚΡΑΡΤΤΡΚΕΡΑΡΤΤΡΚΕΡΑΡΤΤΤΚΚΡΑΡΤΤΡΚΕΡΑΡΓΤΡΚΕΤΑΡΤΓΡΚΚΕΤΡΤΤΡ EKLAPTTPEKPAPTTPEELAPTTPEEPTPTTPEEPAPTTPKAAAPNTPKEPAPTTPKE PAPTTPKEPAPTTPKETAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKEPTSTT αΐΚΡΑΡΓΓΡΚΟΤΑΡ1ΤΡΚΕΡΑΡΤΓΡΚΕΡΑΡΤΓΡΚΟΓΑΡΓΕΕΚΕΡ.ΑΡΤΓΡΚΚΡΑΡΚΕΕ ΑΡΤΓΓΚΟΡΤδΊΊ'δΟΚΡΑΡ'ΠΡΚΕΤΑί'ΤΓΡΚΕΡΑΓΓΓΡΚΚΡΑΡΤΪ'Ρ.ΕΤΡΡΡΓΓδΕνδΤΡ TnKEFniHKSPDESTPELSAEP ^ KAŁENSPKEPGYPTrKWAATKPEhfnTAKD KTTERDLRTFPETTTAAPKMTKETATTTEKTTESK1TATTTQVTSTTTQDTFPFK1T TLKTTTLAPKVTTTKKTITTTEIMNKPEETAKPKDRATNSKATTPKPQKPTKAPKK PTSTKKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRIAEAMLQTTTRPNQTPNSK lTEVNPKSEDAGGAEGErPHMl, LRPH.VFMPEVTPOMDYLPRVPNQGlllNPMI.SD ETNlCNGKPVDGi; n'LRNGTLVAFRGPn'FWMLSPFS.PPSPARRLTEVWGIPSPlD1A 'FTRCNCEGKTFFFKDSQYWRFTNDIKDAGYPKPIFKGFGGLTOQIVAALSTAKYK WPESVYFPKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGEUQVRRRRFERA1 GPSQTHTTR1QYSPARLAYQDKGVLHNEVKVSILWRGLPNVVTSAISLPNIRKPDG YDYYAFSKDQ ¥ YNIDVPSRTARAITTRSGQTLSKVWYNCP

SEQ ID NO: 2: GATGCAGGGTACCCCAAA (human, sensible)

SEQ ID NO: 3: CAGACTTTGGATAAGGTCTGCC (human, antisense)