AU2006233251B2 - Methods for Treating Disorders of the Eye and Surrounding Tissue with Thymosin Beta4 (TBeta4), Analogues, Isoforms and Other Derivatives - Google Patents

Description

P/00/011 28/5/91 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Name of Applicant: Actual Inventor Address for service is: Regenerex Biopharmaceuticals, Inc.

Allan L Goldstein WRAY ASSOCIATES Level 4, The Quadrant 1 William Street Perth, WA 6000 Attorney code: WR Invention Title: Methods for Treating Disorders of the Eye and Surrounding Tissue with Thymosin 34 (T34), Analogues, Isoforms and other Derivatives This application is a divisional application of Australian Patent Application 2002255736 by virtue of Section 79B.

The following statement is a full description of this invention, including the best method of performing it known to me:-

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SMETHODS OF TREATING DISORDERS OF THE EYE AND SURROUNDING

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CIWITH THYMOSIN 34 (T34), ANALOGUES, ISOFORMS AND OTHER DERIVATIVES O BACKGROUND OF THE INVENTION CROSS-REFERENCE TO RELATED APPLICATION The present application claims the benefit of U.S. Provisional Application Serial tn No. 60/275,645, filed March 15, 2001.

Cc, 1. FIELD OF THE INVENTION IND The present invention relates to the field of the treatment of eye disorders such as "dry eye syndrome." 2. DESCRIPTION OF THE BACKGROUND ART The phenomenon called "dry eye syndrome" may occur not only with advancing age due to normal aging of the glands of the eye, but also due to other degenerative changes and environmental factors and can occur at any age. Dry eye syndrome results from deleterious changes in the physiological, biochemical and immunological properties of the eye.

Certain patients experience constant pain from eye irritation caused from the decline of the quality or quantity of tears. Such patients have a sandy or gritty sensation that, if untreated, can lead to scarring or ulceration of the cornea, and thus loss of vision. In many cases, dry eye results from disorders of the various glands which work together to produce normal tears. Tears themselves are a complex combination of substances which form three layers on the eye. The very thin outer layer contains lipids from the Meibomian glands in the eyelid, to reduce evaporation. The lacrimal glands produce the middle watery layer that keeps the salinity and the acidity of the tears at proper levels. This middle layer also carries antibodies and other immune defense agents to defend the eye against infection. The inner mucous layer helps the tear film "stick" to the cornea and stay intact.

There may be many causes of dry eye syndrome. The normal aging of tear glands, as well as specific diseases and disorders, may cause changes in the amount and condition of tears produced. For example, Sjbgren's syndrome is an immune system disorder characterized by inflammation and dryness of the mouth, eyes, and other mucous membranes, damages the lacrimal glands, and this damage affects tear production.

Decreased sensitivity of the cornea can also lead to insufficient production of tears. This lack of sensitivity can be brought on by a disease known as "neurotrophic keratitis" as well as by some types of contact lens wear. Excessive evaporation of tears can also cause dry 1/2

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O eye syndrome. Such evaporation may be caused by "meibomitis," which results from infection and inflammation of the meibomian glands in the eyelids. People with unusually O large eyes, as well as those who suffer from thyroid disease, may also experience dry eye syndrome caused by excessive evaporation. Dry eye can also result from unusual facial c anatomy or irregularities in the cornea, resulting in uneven or inadequate tear coverage of the eye. Some patients suffer from dry eye as a result of medications such as antibiotics, VI) antihistamines, diuretics, and anti-diarrheals, which can dry up the mucous membranes.

Cc Hormonal changes, such as may be associated with menopause and the aging process, can C also affect secretions of TP4 from the tear glands and result in dry eyes and inflamation of the eye.

c A number of approaches have been reported to delay and/or to decrease such eye disorders. Dry eyes are typically treated by applying artificial tears and ointments. These give temporary relief, but usually do not arrest or reverse damage to the eye., Eye drops which are aimed at restoring the electrolyte balance of the tears and promoted healing of the cornea are in development. There is also evidence that dry eye may be treated with hormone therapy or antibodies. In addition, Some forms of dry eye benefit from the placement of tiny plugs in the ducts that drain tears from the eye. For severe forms of dry eye, special goggles called "moisture-chamber spectacles" can be worn.

There remains a need in the art for improved methods and compositions for the treatment of dry eye disorders.

SUMMARY OF THE INVENTION In accordance with the present invention, a method of treatment for promoting reversal of or inhibiting'eye degeneration, such as may be associated with dry eye syndrome, comprises administering to a subject in need of such treatment an effective amount of a composition comprising-aneye degeneration-inhibitingpolypeptidecomprising amino acid sequence LKKTET, or a conservative variant thereof having eye degenerationinhibiting activity.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on a discovery that actin-sequestering peptides such as thymosin P4 (Tp4) and other actin-sequestering peptides containing amino acid sequence LKKTET or conservative variants thereof, promote reversal of or inhibit eye degeneration such as may be associated with or result from dry eye syndrome. The

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potential clinical applications might include disorders due to inflammatory conditions dry eyes, uveitis, iritis, post.operative cataract surgery, LASIK or PRK, corneal melts due to 0 rheumatoid arthritis, systemic lupus erythematosus, Mooren's ulcers, Sjbgrens syndrome,

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etc. Other applications could be keratitis due to bacterial, viral, mycobacterial or fungal S pathogens. Still other applications could be due to metabolic diseases of the eye such as caused by diabetes (keratopathy and retinopathy) or as a result of chemical injury, trauma and abrasions.

C Thymosin 34 was initially identified as a protein that is up regulated during S endothelial cell migration and differentiation in vitro. Thymosin p4 was originally isolated ID from the thymus and is a 43 amino acid, 4.9 kDa ubiquitous polypeptide identified in a 0 variety of tissues. Several roles have been ascribed to this protein including a role. in a endothelial cell differentiation and migration, T cell differentiation, actin sequestration and vascularization.

In accordance with one embodiment, the invention is a method of treatment for promoting reversal of or inhibiting dry eye syndrome comprising administering to a subject in need of such treatment an effective amount of a composition comprising an agent that stimulates production of an eye degeneration-inhibiting polypeptide comprising amino acid sequence LKKTET, or a conservative variant thereof having eye degeneration-inhibiting activity, preferably Thymosin p4, an isoform of Thymosin 04, oxidized Thymosin p4 or an antagonist of Thymosin p4.

The present invention promotes the healing and reversal of inflammatory, degenerative, immunological and other disorders of the eye and surrounding tissue.

Compositions which may be used in accordance with the present invention include Thymosin 34 (T14), T4 isoforms, oxidized TP4, polypeptides comprising the amino acid sequence LKKTET'or conservative variants thereof having eye degeneration-inhibiting activity. International Application Serial No. PCT/US99/17282, incorporated herein by reference, discloses isoforms of Tp4 which may be useful in accordance with the present invention as well as amino acid sequence LKKTET and conservative variants thereof having eye degeneration-inhibiting activity, which may be utilized with the present invention.

International Application Serial No. PCT/GB99/00833 (WO 99/49883), incorporated herein by reference, discloses oxidized Thymosin p4 which may be utilized in accordance with the present invention. Although the present invention is described primarily hereinafter with respect to Tp4 and Tp4 isoforms, it is to be understood that the following description is intended to be equally applicable to amino acid sequence LKKTET, conservative variants thereof having eye degeneration-inhibiting activity, as well as oxidized Thymosin 34.

00 O In one embodiment, the invention provides a method of treatment for promoting reversal of or inhibiting eye degeneration, such as may be associated with dry eye syndrome, comprising administering to a subject in Ineed of such treatment an effective amount of a composition comprising an eye degeneration-inhibiting polypeptide comprising amino acid sequence SLKKTET, or a conservative variant thereof having eye degeneration-inhibiting activity. The contacting may be topically or systemically. Examples of topical administration include, for example, contacting the eye with a solution, lotion, 1 salve, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, or 10 oil comprising Tp4. Systemic administration includes, for example, intravenous, intraperitoneal, intramuscular injections of a composition containing T34 or a Tp4 isoform. A subject may be any mammal, preferably human.

The invention further provides a composition for reducing vacuolization between cell layers of a cornea in an eye of a subject due to LASIK or PRK, comprising an effective amount of at least one polypeptide containing amino acid sequence LKKTET (SEQ ID NO:1), a conservative variant of a polypeptide containing amino acid sequence LKKTET (SEQ ID NO:1), thymosin 34 (Tp4), an isoform of T04, oxidized Tp4, T4 a a Tp9, Tp10, T1 1, Tp12, Tp13, Tp14 or Tr15, gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, capping protein, p-actinin, I actobindin or acumentin so as to reduce vacuolization between cell layers of said cornea.

A composition in accordance with the present invention can be administered daily, every other day, etc., with a single application or multiple applications per day of administration, such as applications 2, 3, 4 or more times per day of administration.

Tp4 isoforms have been identified and have about 70%, or about or about 80% or more homology to the known amino acid sequence of Tp4.

Such isoforms include, for example, TP4 ala Tp9, T310, Tp11, Tp12, Tp13, Tp14 and Tp15. Similar to Tp4, the Tp10 and T315 isoforms have been shown to sequester actin. Tp4, Tp10 and Tp15, as well as these other 00 O isoforms share an amino acid sequence, LKKTET, that appears to be c involved in mediating actin sequestration or binding. Although not wishing to be bound to any particular theory, the activity of Tp4 isoforms may be due, in part, to the ability to polymerize actin. For example, Tp4 can modulate actin polymerization in the eye P.thymosins appear to depolymerize F-actin by sequestering free G-actin). Tp4's ability to modulate actin polymerization may therefore be due to all, or in part, its ability to bind to or sequester actin via the LKKTET sequence. Thus, as with T04, other proteins which bind or sequester C actin, or modulate actin polymerization, including Tp4 isoforms having the 0 10 amino acid sequence LKKTET, are likely to reduce dry eye syndrome, alone CN or in a combination with Tp4, as set forth herein.

Thus, it is specifically contemplated that known Tp4 isoforms, such as Tp4 ala T39, T310, Tp11, Tp12, Tp13, T314 and T315, as well as Tp4 isoforms not yet identified, will be useful in the methods of the invention. As such Tp4 isoforms are useful in the methods of the invention, including the methods practiced in a subject. The invention therefore further provides pharmaceutical compositions comprising Tp4, as well as Tp4 isoforms Tp4 a Tp9, Tp10, Tp11, Tp12, T313, Tp14 and Tp15, and a pharmaceutically acceptable carrier.

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In addition, other proteins having actin sequestering or binding capability, or that can C mobilize actin or modulate actin polymerization, as demonstrated in an appropriate Ssequestering, binding, mobilization or polymerization assay, or identified by the presence of an amino acid sequence that mediates actin binding, such as LKKTET, for example, can S similarly be employed in the methods of the invention. Such proteins include gelsolin, -vitamin D binding protein (DBP), profilin,.cofilin, depactin, Dnasel, vilin, fragmin, severin, S capping protein, P-actinin, actobindin and acumentin, for example. As such methods include M those practiced in a subject, the invention further provides pharmaceutical compositions N comprising gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, cappinj protein, P-actinin, actobindin and acumentin as set forth herein.

Thus, the invention includes the use of a dry eye syndrome reducing polypeptide comprising the amino acid sequence LKKTET and conservative variants thereof.

As used herejp,:.theterm "conservative variant" or grammatical variations thereof denotes the replacement of an amino acid residue by another, biologically similar residue.

Examples of conservative variations include the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.

Tp4 has been localized to a number of tissue and cell types and thus, agents which stimulate the production of Tp4 can be added to or comprise a composition to effect Tp4 production from a tissue and/or a cell. Such agents include members of the family of growth factors, such as insulin-like growth factor (IGF-1), platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor beta basic fibroblast growth factor (bFGF), thymosin al (Tal) and vascular endothelial growth factor (VEGF).

More preferably, the agent is transforming growth factor beta (TGF-) or other members of the TGF-p superfamily. Tp4 compositions of the invention may reduce dry eye syndrome by effectuating growth of the connective tissue through extracellular matrix deposition, cellular migration and downregulation of inflammatory cytokines.

Additionally, agents that assist or stimulate dry eye syndrome reduction may be added to a composition along with Tp4 or a Tp4 isoform. Such agents include angiogenic agents, growth factors, agents that direct differentiation of cells, agents that promote migration of cells and agents that stimulate the provision of extracellular matrix material in the eye. For example, and not by way of limitation, Tp4 or a Tp4 isoform alone or in combination can be added in combination with any one or more of the following agents:

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VEGF, KGF, FGF, PDGF, TGFP, IGF-1, IGF-2, IL-1, prothymosin a and thymosin al in an effective amount.

o The invention also includes a pharmaceutical composition comprising a therapeutically effective amount of T4 or a Tp4 isoform in a pharmaceutically acceptable S carrier. Such carriers include those listed above with reference to parenteral administration.

The actual dosage or reagent, formulation or composition that inhibits or promotes S reversal of dry eye syndrome may depend on many factors, including the size and health of S a subject. However, persons of ordinary skill in the art can use teachings describing the methods and techniques for determining clinical dosages as disclosed in PCT/US99/17282, N supra, and the references cited therein, to determine the appropriate dosage to use. Tp4, or its analogues, isoforms or derivatives, may be administered in any suitable amount which are effective for the treatment of dry eye or similar disorders. For example, TP4 may be administered in dosages within the range of about 0.1-50 microgrdrihs of Tp4, more preferably in amounts of about 1-25 micrograms Tp4. The TP4 may be administered as a one-time treatment, or may be administered daily, twice per day, three times per day, etc., or on alternate days and the like, until the desired results are obtained.

Suitable topical formulations include Tp4 or a Tp4 isoform at a concentration within the range of about 0.001 10% by weight, more preferably within the range of about 0.01 0.1% by weight, most preferably about 0.05% by weight.

The therapeutic approaches described herein involve various routes of administration or delivery of reagents or compositions comprising the Tp4 or other compounds of the invention, including any conventional administration techniques (for example, but not limited to, topical administration, local administration, or systemic administration), to a subject. The methods and compositions using or containing Tp4 or other compounds of the invention may be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable non-toxic excipients or carriers.

The invention includes use of antibodies which interact with Tp4 peptide or functional fragments thereof. Antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art as disclosed in PCT/US99/17282, supra.

The term antibody as used in this invention is meant to include monoclonal and polyclonal antibodies.

In yet another embodiment, the invention provides a method of treating a subject by administering an effective amount of an agent which modulates Tp4 gene expression. The

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Sterm "modulate" refers to inhibition or suppression of Tp4 expression when T(34 is over S expressed, and induction of expression when Tp4 is under expressed. The term "effective o) amount" means that amount of Tp4 agent which is effective in modulating Tp4 gene 0 expression resulting in reducing the symptoms of the Tp4 associated dry eye syndrome. An c agent which modulates TB4 or Tp4 isoform gene expression may be a polynucleotide for example. The polynucleotide may be an antisense, a triplex agent, or a ribozyme. For itr example, an antisense directed to the structural gene region or to the promoter region of q Tp4 may be utilized.

SIn another embodiment, the invention provides a method for utilizing compounds that modulate Tp4 activity. Compounds that affect Tp4 activity antagonists and agonists) Sinclude peptides, peptidomimetics, polypeptides, chemical compounds, minerals such as zincs, and biological agents.

While not be bound to any particular theory, it is believed that the present invention may promote reversal of or inhibit eye degeneration associated with dry eye syndrome by inducing terminal deoxynucleotidyl transferase (a non-template directed DNA polymerase), to decrease the levels of one or more inflammatory cytokines, and to act as a chemotactic factor for endothelial cells, and thereby inhibit or promote reversal of degenerative changes in the eyes brought about by aging or other degenerative or environmental factors.

Example 1 Tears from healthy young people under the age of 40 and older people over the age of 40 were examined for levels of Tp4. It was found that Tp4 is present at highest levels in tears of healthy young people, and that Tp4 in tears decreases significantly with age and menopause. Thus, dry eye syndrome and inflammation of eyes may be due to deficiency of TP4 in tears. Therefore, administering Tp4 may reduce inflammation, promote healing of inflamed eyes and mucosa, and stimulate production of tears via healing of the glands of the eye responsible for tear production.

Example 2 Disks of Whatman TM filter paper (size 50) were cut with a 2 mm diameter trephine.

The disks were soaked in 1.0 N NaOH and applied to the central cornea of isoflouraneanesthetized mice for 30 seconds. The eyes then were irrigated with 10 ml of PBS and subsequently treated with either Tp4 (5 mg-5 ml) or a similar volume of PBS (as control) topically twice daily for seven days. After seven days, marked differences between the PBS-treated and the TP4-treated eyes were noted. The PBS-treated eyes exhibit markedly 00 edematous and inflamed corneas and the anterior chamber contained marked hyphema and an intense inflammatory cell infiltrate. In contrast, the T34treated corneas showed decreased stromal edema and more regularly I arranged stromal lamellae. The overall anatomically integrity of the anterior segment of the T134-treated as compared to PBS-treated eyes was markedly more normal in appearance.

1 Transmission electron microscopic analysis also was done at day 7 after ¢q treatment with PBS and T34. Corneas treated with T34 revealed a more Sregular alignment of epithelial intercullar junctions and less vacuolization between cell layers. Similarly, the PBS-treated corneas demonstrated a marked inflammatory infiltrate in areas of stromal digestion and edema, whereas the stroma of the T34-treated corneas appeared intact with more regularly spaced collagen lamellae.

Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The discussion of the background art is included exclusively for the purpose of providing a context for the present invention. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was common general knowledge in the field relevant to the present invention in Australia or elsewhere before the priority date.

Claims (7)

17. Sep. 2008 9:04 No. 3965 P. 4 oO 0 Claims- C 1. A method of treatment for reducing vacuolization between cell layers of a Scornea in an eye of a subject in need of such treatment, comprising administering to the subject an effective amount of a composition comprising Sat least one of a polypeptide containing amino acid sequence LKKTET (SEQ ID NO:1), a conservative variant of a polypeptide containing amino acid sequence LKKTET (SEQ ID NO:1), thymosin 134 (Tp4), an isoform of Tp4, C( oxidized Tp4, T14a, T39, TP10, TP11, Tp12, T313, TP14, T315, gelsolin, n vitamin D binding protein (DBP), profilin, cofilin, depactin, Dnasel, vilin, IN fragmin, severin, capping protein, p-actininm actobindin or acrumentin, so as to reduce vacuolization between cell layers of said cornea. 2. The method of claim 1 wherein said polypeptide comprises at least one of Thymosin 34 (Tp4), or an isoform of Tp4. 3. The method of claim 1 wherein said polypeptide comprises Thymosin 14, (Tp4). 4. The method of claim 1 wherein said polypeptide comprises at least on of Tp4a a Tp9, Tp10, Tp11, T312. T313, T314 or The method of claim 1 wherein said composition is administered systematically. 6. The method of claim 1 wherein said composition is administered topically. 7. The method of claim 1 wherein said polypeptide is present in said composition at a concentration in range of about 0.001-10% by weight. 8. The method of claim 7 wherein said range is about 0.01-0.1% by weight. 9. The method of claim 8 wherein said concentration is about 0.05% by weight. method of claim 1 wherein said polypeptide is administered to said subject at a dosage within a range of about 0.1-50 micrograms. 11.The method of claim 10 wherein said range is about 1-25 micrograms. 12.The method of claim 1 wherein said vacuolization is due to LASIK or PRK. 9 COMS ID No: ARCS-206461 Received by IP Australia: Time 11:06 Date 2008-09-17 17, Sep. 2008 9:04 No. 8965 P. 0 13.A method of treatment for decreasing comeal stromal edema in a subject in O need of such treatment, comprising topically administering to a comea of a Ssubject an effective amount of a composition comprising a pharmaceutically acceptable carrier and a polypeptide comprising at least one of thymosin P4 (Tp4), an isoform of Tp4, TI4a l a TP9, Tp10, T311, TP12, Tl13, Tp14, gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, capping protein, 3-actininm actobindin or acrumentin so in as to decrease comeal stromal edema in said patient. Cci en 14. The method of claim 13 wherein said polypeptide comprises at least one of N0 Thymosin 04 (Tp4), or an isoform of Tp4. 0 15.The method of claim 13 wherein said polypeptide comprises Thymosin P4 (Tp4). 16.The method of claim 13 wherein said polypeptide comprises at least one of TP4 a la Tp9, Tp10, To11, T012, Tp13, T314 or 17.The method of claim 13 wherein said composition is administered topically.

18.The method of claim 13 wherein said composition is present in said composition at a concentration within a range of about 0.001-10% by weight.

19. The method of claim 13 wherein said composition is in a form of a solution, gel, cr6me, paste, lotion, spray, suspension, dispersion, salve, hydrogel or ointment. The method of claim 13 wherein said polypeptide is recombinant or synthetic.

21.The method of claim 18 wherein said range is about 0.01-0.1% by weight.

22.The method of claim 21 wherein said concentration is about 0.05% by weight.

23. The method of claim 13 wherein said polypeptide is administered to said subject at a dosage within a range of about 0.1-50 micrograms.

24.The method of claim 23 wherein said range is about 1-25 micrograms. COMS ID No: ARCS-206461 Received by IP Australia: Time 11:06 Date 2008-09-17 17. Sep. 2008 9:04 No. 8965 P. 6 00 o 25.The use of at least one of a polypeptide containing amino acid sequence 0 LKKTET (SEQ ID NO:1), a conservative variant of a polypeptide containing 0 amino acid sequence LKKTET (SEQ ID NO:1), thymosin 34 (Ti4), an isoform Cr. of T34, oxidized Tp4, Tp4 al a T39, T10, Tp311, T312, Ti13, T314, gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, capping protein, p-actininm actobindin or acrumentin in _the manufacture of a medication for reducing vacuolization between cell l n layers of a cornea in an eye of a subject. Cci e 26.The use of a pharmaceutically acceptable carrier and a polypeptide ID comprising at least one of thymosin 14 (TI4), an isoform of T34, TIB4 T39, o T310, T 311, TI12, T313, T314, Tp15, gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, capping protein, p-actininm actobindin or acrumentin in the manufacture of a medication for decreasing coreal stromal edema in a subject. 11 COMS ID No: ARCS-206461 Received by IP Australia: Time 11:06 Date 2008-09-17