WO2013148155A1

WO2013148155A1 - Compositions and methods for the treatment of dry eye disease

Info

Publication number: WO2013148155A1
Application number: PCT/US2013/030397
Authority: WO
Inventors: Javier Dotor De Las Herrerias
Original assignee: Digna Biotech Usa, Llc
Priority date: 2012-03-26
Filing date: 2013-03-12
Publication date: 2013-10-03

Abstract

The invention relates to compositions and methods for the prevention and/or treatment of dry eye. More specifically, the invention relates to compositions and methods for the prevention and/or treatment of dry eye, comprising a transforming growth factor βΐ (TGF-βΙ) inhibitor peptide, as an active ingredient.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF DRY EYE

DISEASE

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional patent application: 61/615,593 filed March 26, 2012, the contents of which are incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for the prevention and/or treatment of dry eye disease. More specifically, the invention relates to compositions and methods for the prevention and/or treatment of dry eye disease, comprising a transforming growth factor βΐ (TGF-βΙ) inhibitor peptide, as an active ingredient.

BACKGROUND OF THE INVENTION

Dry eye disease

Dry eye disease (DED) also known as keratoconjunctivitis sicca or dysfunctional tear syndrome, is currently understood as a multifunctional disorder or abnormality of the tear film and of the ocular surface which results in ocular discomfort, visual disturbance, and often even in ocular surface damage caused by tear film instability. In general, dry eye disease is characterized by symptoms such as a sandy-gritty feeling in the eye, burning, irritation, or a foreign-body sensation that worsens during the day. Patients suffering from dry eye disease complain of mild to severe symptoms, and those with severe symptoms may experience constant and disabling eye irritation, and develop ocular surface epithelial disease and sight-threatening sterile or microbial corneal ulceration.

The main physiological function of the tear film is the lubrication of the ocular surface and the inner eyelid. In addition, it supplies the ocular surface with the nutrients which it requires, provides a smooth and regular optical surface for the eye. Moreover, it protects the ocular surface against pathogens by various mechanisms, including mechanical removal of foreign particles but also through antimicrobial substances which it contains.

The tear film is composed of a mucous component, an aqueous component, and a lipid component. The inner layer of the tear film is the mucous layer or component, which is bound to the ocular epithelium via the interaction of mucin molecules which are produced by conjunctival goblet cells and by stratified squamous cells of the conjunctiva and the cornea. The lubricating effect of the tear film is substantially based on the mucous layer and its composition.

On top of the mucous layer is the aqueous layer which is produced by the main and accessory lacrimal glands. Its primary function is to hydrate the mucous component and contribute to the transport of nutrients, electrolytes, antibacterial compounds, and oxygen to the ocular surface. The aqueous component contains water, electrolytes, lysozyme, lactoferrin, immunoglobulins (in particular IgA), retinol, hepatocyte growth factor, epidermal growth factor as its important constituents.

The lipid layer which covers the aqueous layer is produced by the tarsal glands which are positioned at the tarsal plates of the eyelids, and to some degree also by the glands of Zeis which open into the eyelash follicles. Its functions include the enhancement of the spreading of the tear film, decrease of water loss from the aqueous layer by reducing evaporation, and preventing tear film contamination.

It is today acknowledged that DED is a complex, multifunctional disorder involving several interacting pathophysiological mechanisms which are only beginning to be understood (H. D. Perry, Am. J. Man. Care 13;3, S79-S87, 2008). The two mechanisms that are being discussed as pivotal in the etiology of the disease and which also appear to reinforce each other mutually are tear hyperosmolarity and tear film instability. Hyperosmolar tear fluid can result from excessive tear film evaporation or reduced aqueous flow. It activates an inflammatory cascade and causes the release of inflammatory mediators into the tear fluid, with multiple pathophysiological effects eventually leading to increased tear film evaporation and tear film instability. Thus, tear film instability can be a consequence of hyperosmolarity. Alternatively, it can develop as the original etiological pathway, e.g. via abnormalities of the lipid layer composition, such as in tarsal gland disease.

Once DED has become manifest, inflammation is one of the key processes that maintain and potentially progress the disease. Depending on the severity of the condition, patients often develop a reversible squamous metaphase and punctate erosions of the ocular epithelium. Secondary diseases whose development may be triggered by DED include filamentary keratitis, microbial keratitis, corneal neo vascularis ation, and ocular surface keratinisation.

The management of DED relies on both non-pharmacological and pharmacological approaches and the therapeutic options depend significantly on the severity of the disease state (Lemp, Am. J. Man. Care 14:3, S88-S101, 2008). Non- pharmacological approaches may be used initially when only mild symptoms occur, or as palliative measures to support medical interventions. They include the avoidance of exacerbating factors such as dry air, wind and drafts, tobacco smoke, modification of working habits; eye lid hygiene; tear supplementation; and physical tear retention by punctual plugs or therapeutic contact lenses.

The mainstay of non-pharmacological DED treatment is the use of artificial tears for tear substitution. Most of the available products are designed as lubricants. In addition, they may function as carriers for nutrients and electrolytes (importantly, potassium and bicarbonate), and some products attempt to correct physical parameters such as an increased osmolality in certain forms of DED. The major functional component of artificial tear compositions is an agent which increases or adjusts the viscosity and which at the same time exhibits lubricant functionality. Common compounds used for this purpose include carboxymethylcellulose and its sodium salt (CMC, carmellose), polyvinyl alcohol, hydroxypropyl methylcellulose (HPMC, hypromellose), hyaluronic acid and its sodium salt, and hydroxypropyl guar gum. However, compositions with a relatively high viscosity, and in particular gel-type formulations, have a tendency to cause visual blurring.

Some artificial tears comprise lipids to substitute for the lipid component of the natural tear film. Unfortunately, the commonly used lipids are physically and biochemically poorly related to native lipid compositions: they are based on castor oil or even mineral oil. It is intended to thereby decrease the rate of tear fluid evaporation. The same effect may perhaps also be achieved by hydrocolloids which exhibit some degree of bioadhesiveness, such as hydroxypropyl guar gum or hyaluronic acid.

For moderate to severe forms of DED, non-pharmacological approaches are not normally sufficient to manage the symptoms adequately. However, there are presently not many pharmacological therapies available which have proven to be effective and/or which have been authorised by the regulatory agencies.

Cholinergic agents such as muscarinic acetylcholine receptor antagonists may be used in aqueous deficient patient as secretagogues to stimulate tear production. An agent that has been tested successfully in several clinical studies with Sjogren syndrome patients is pilocarpine. The drug given orally at doses of 5 to 7.5 mg QID (Lemp, cited supra) significantly improved DED symptoms. However, the product has not been approved by any major regulatory agencies for the use in DED, neither as an oral formulation nor in the form of eye drops as they are available for the treatment of glaucoma. Cevimeline is another parasympathomimetic drug and muscarinic agonist. It acts particularly on muscarinic M3 receptors. It is available in a few countries as an oral formulation and used in the treatment of dry mouth associated with Sjogren's syndrome. Clinical studies indicate that it is also effective in the management of symptoms associated with DED of the Sjogren type.

Anti-inflammatory agents may be used to intervene in the viscous circle of symptoms causing inflammatory response which in turn increase symptom severity. The rationale of using such agents is not restricted to aqueous deficient or even Sjogren syndrome patients. Both topical corticosteroids and topical non-steroidal antiinflammatory (NSAID) compounds have been proposed as treatment options.

From the clinical studies that have been conducted so far (Lemp, cited supra) it appears that corticosteroids such as loteprednol etabonate and prednisolone acetate are more effective in the control of several DED symptoms than NSAIDs such as diclofenac and ketorolac. However, they are generally recommended only for short-term use. In the long term, they may cause or support the development of ocular infections, glaucoma, and cataracts. Both loteprednol etabonate and prednisolone acetate are poorly water- soluble and thus formulated as a suspension, which may be considered a disadvantage in view of the symptoms of DED. Further, the steroid drugs elevate intraocular pressure and the prolonged use thereof in chronic diseases and disorders may undesirably cause cataract and glaucoma complications.

Moreover, clinical studies with, and the off-label use of, oral tetracyclines such as doxycyclin, minocycline and oxytetracycline for DED have been reported (Lemp, cited supra). It is assumed that they are not primarily effective on the basis of their antibacterial properties, but due to their anti-inflammatory activity.

Nevertheless, the major pharmacological treatment option for moderate to severe DED is ciclosporin (i.e., ciclosporin A, also known as cyclosporine A), which is an approved medicine in the form of an ophthalmic emulsion (Restasis®) for increasing tear production in patients whose tear production is presumed to be suppressed due to ocular inflammation associated with DED. According to the evidence that is available, topical ciclosporin is probably disease-modifying rather than only palliative. It acts as an antagonist in various inflammatory processes and cascades. For example, it reduces conjunctival interleukin 6 (IL-6) levels, decreases activated lymphocytes in the conjunctiva, suppresses other conjunctival inflammatory and apoptotic markers, and increases the number of goblet cells in the conjunctiva (Lemp, cited supra).

Ciclosporin [(E)-14,17,26,32-tetrabutyl-5-ethyl-8-(l-hydroxy-2-methylhex-4- enyl)-l ,3,9,12,15, 18,20,23,27-nonamethyl-l l ,29-dipropyl-l ,3,6,9,12,15, 18,21 ,24,27,30- undecaazacyclodotriacontan-2,4,7, 10, 13, 16,19,22,25, 28, 31-undecaone] is a cyclic non- ribosomal peptide of 11 amino acids. It is an immunosuppressant drug widely used in post-allergenic organ transplant to reduce the activity of the patient's immune system and, so, the risk of organ rejection.

Ciclosporin is thought to bind to the cytosolic protein cyclophilin

(immunophilin) of immunocompetent lymphocytes, especially T-lymphocytes. This complex of ciclosporin and cyclophilin inhibits calcineurin, which, under normal circumstances, is responsible for activating the transcription of interleukin 2 (IL-2). It also inhibits lymphokine production and interleukin release and, therefore, leads to a reduced function of effector T-cells. Other immunosuppressant drugs with similar activity include tacrolimus, pimecrolimus, everolimus, sirolimus, deforolimus, temsirolimus, and zotarolimus, abetimus, gusperimus, and mycophenolic acid. Based on pharmacological considerations, it is presumed that these compounds would also be beneficial in the management of diseases or symptoms which are controlled by ciclosporin, such as DED.

Macrolide immunosuppressants such as ciclosporin, tacrolimus, sirolimus, everolimus and the like, while being highly active once they have been effectively delivered into the organism or to the target tissue, are challenging compounds to formulate and deliver to the site of action, in particular due to their extremely poor solubility and relatively large molecular size. In fact, Restasis®, which comprises ciclosporin at a concentration of 0.05%, is formulated as a sterile, preservative-free oil- in-water (o/w) emulsion. The formulation is white opaque to slightly translucent presented in single-use LDPE (low-density polyethylene) vials filled with 0.4 mL liquid. As inactive ingredients, it contains glycerine, castor oil, polysorbate 80, carbomer 1342, purified water and sodium hydroxide to adjust the pH to 6.5 to 8.0. The active ingredient is dissolved in the dispersed oily phase of the emulsion consisting of castor oil. It is assumed that the amphiphilic polysorbate 80 and probably also the carbomer act as stabilisers of the emulsion. The major adverse effects of Restasis® include ocular burning and stinging, occurring in a phase III trial at a frequency of 14.7% and 3.4%, respectively. Other events reported in 1% to 5% of the patients include conjunctival hyperaemia, discharge, epiphora, eye pain, foreign body sensation, pruritus, and visual disturbance which is typically blurring.

Recently, some antibodies have been postulated as potentially useful therapeutical agents for treating DED such as anti-IL-17 antibodies (US 2011/0223169) and anti-KLK-13 antibodies (US 2011/0223170).

Although there are some therapeutic agents for treating DED, most of said agents involve treatments for symptomatic relief and therefore they do not present definitive solutions to the treatment of DED. Thus, in order to maximize the number of options available to the physician and the patient, a new and effective treatment for DED is highly desirable.

Therefore, there is a need in the art for the development of an alternative drug which is capable of treating and/or preventing DED and overcoming at least one of the limitations or disadvantages associated with prior art formulations for treating DED. Advantageously, said alternative drug should be easily formulated in a pharmaceutically composition to be topically administered in the eye. In this connection, the inventors of the present application have discovered that some TGF-βΙ inhibitor peptides are capable of treating and/or preventing DED. Based on these findings, a composition for prevention and/or treatment of DED comprising a TGF-βΙ inhibitor peptide as an active ingredient is provided.

Transforming growth factor βΐ (TGF-βΙ)

TGF-βΙ is a glycoprotein belonging to a superfamily of structurally related regulatory proteins (cytokines) included within one of the three isoforms described in mammals (TGF-β 1, 2 and 3). The most abundant isoform is TGF-βΙ, which consists of a 25 kDa homodimer composed of two subunits joined by a disulfide bond. The amino acid sequence of human TGF-βΙ has been described by authors such as Derynck K et al., "Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells". Nature 316 (6030), 701-705 (1985).

TGF-βΙ presents a highly conserved protein sequence in evolutive terms. Although it was originally defined by its capacity to induce adhesion independent of proliferation and morphological changes in rat fibroblasts, subsequent investigations have shown that TGF-βΙ is a general inhibitor of proliferation of a broad range of cell types. The molecule is produced by a great variety of cell types and in different tissues during all phases of cell differentiation. It has a large series of biological effects, with the generation of potent and very often opposite effects in relation to development, physiology and immune response. An excessive or deregulated expression of TGF-βΙ is associated with some diseases, for example, liver fibrosis, pulmonary fibrosis, corneal fibrosis and haze.

Experimental evidence demonstrates that conjunctival and corneal cells secrete TGF-βΙ and that TGF-βΙ stimulates the production of some matrix metaloproteins (MMPs), such as gelatinase (MMP-9), collagenases (MMP-1 and MMP-13) and stromelysins (MMP-3, MMP-10 and MMP-11) by human corneal epithelial cells (Hyun-Seung Kim et al., Experimental Eye Research 79 (2004):263-274). Additional experimental evidences (Enriquez de Salamanca et al., Poster entitled "In vitro TGF- Beta Effect on Cytokine/Cheinokine and Metalloproteinases (MMPs) Secretion by Human Ocular Surface Epithelial Cells", ARVO Meeting Abstracts April 11, 2008; 49:60-41 ) cytokine/chemokine and metalloproteinases (MMPs) secretion by ocular surface epithelial cells was modified after TGF-β treatment. Different effects were observed in some molecules depending on the cell type (corneal or conjunctival), time of exposure (24 h or 48 h) and type of TGF-β (TGF-βΙ or ΤΟΡ-β2). GM-CSF secretion was significantly upregulated in both cell types after 24 h (human corneal epithelial cells or 48h (human corneal epithelial cells and human conjunctival epithelial cells) of TGF-βΙ or ΤΟΕ-β2 exposure. IL-8 secretion upon TGF-βΙ (24 h) and upon ΤΟΕ-β2 (24 h and 48 h) exposure was increased in human conjunctival epithelial cells. IL-10 secretion upon 24 h or 48 h of TGF-βΙ or ΤΟΕ-β2 exposure was increased in human corneal epithelial cells. After 48h, ΤΟΕ-β2 significantly increased interferon gamma (IFN-γ) and IL-2 secretion by human corneal epithelial cells. IL-10 secretion was significantly downregulated in human conjunctival epithelial cells after 24 h or 48 h of TGF-βΙ or TGF- 2 exposure. RANTES secretion was significantly downregulated in human conjunctival epithelial cells after 24 h of TGF-βΙ or TGF- 2 exposure. MMP-3 and -9 secretion was significantly increased in human corneal epithelial cells upon 24 h and 48 h of TGF-βΙ or TGF- 2 treatment, whereas no significant effect in MMPs secretion was detected in human conjunctival epithelial cells.

In ocular surface diseases, TGF-β is increased in the conjunctival epithelium of vernal keratoconjunctivitis, a chronic immune-mediated severe disease showing collagen deposition and tissue remodeling (Leonardi A, et al., Invest Ophthalmol Vis Sci. 2000; 41:4175-4181; Abu El-Asrar AM, et al., Eye. 2006; 20:71-79), in pterygium, a fibrovascular proliferative tissue growing over the cornea (Kria L et al., Acta Histochem. 1996; 98: 195-201) and more recently in DED, an inflammatory disease affecting the quantity/quali of tears, causing damage to ocular surface (Chotikavanich S, et al. Invest Ophthalmol Vis Sci. 2009; 50:3203-3209; De Paiva et al., Mucosal Immunol. 2009 May;2(3):243-53. Epub 2009 Feb 25).

Inhibition of the biological activity of TGF-βΙ has been proposed for treating some ocular diseases. By illustrative, antisense oligonucleotides targeting TFG-βΙ have been proposed for reducing post-surgical scarring in glaucoma surgery (Cordeiro et al., Gene Therapy. 2003; 10:59-71) and antibodies to TGF-βΙ are potentially useful for topically treating corneal fibrosis (Jester et al., Cornea. 1997; 16(2): 177-87).

Additionally, De Paiva CS et al., (PLOS ONE, vol. 6, no. 12, December 2011 , page e29017) disclose that disruption of TGF-β causes improvement of DED. The authors achieved their results by using a negative dominant mouse model for the TGF beta receptor type II (CD4-DNTGFbetaRII) to which DED was induced by subcutaneous injection of scopolamine hydrobromide for 5 to 10 consecutive days and placed in a cage with a perforated plastic screen on one side to allow air flow from a fan for 16 h / day. However, it is not the most appropriate model for evaluating the possible effectiveness of a treatment for DED because:

as the authors themselves recognize it presents some limitations because the expression of TGF-β is ubiquitous, and therefore, strategies based on gene deletions in mice are not the most suitable for studying its blockade due to autoimmunity and death events (De Paiva et al., cited supra, page 6, right- hand column); the transgenic mice (knockout mice) deficient for TGF-βΙ [TGF-βΙ (-/-)] die within weeks after birth by uncontrolled, progressive and generalized inflammatory processes (Christ M, et al. J Immunol. 1994 Sep 1 ;153(5): 1936-46). That KO mouse has a problem during maturation of the immune system, there is no control on the maturation and generate immune responses and autoimmune processes. But it is not equivalent to what happens when TGF-β is locked in an adult mouse, as its immune system matures properly and the role of TGF-β varies in intensity and direction depending on the inflammatory and temporal context. In this regard, mice with CD4 lymphocytes lacking TGF-β receptor type II develop the same phenotype; thus it is a partial model because it is a selective and very artificial blockade; for it blocks TGF-β signaling in mouse CD4 lymphocytes from the appearance of cell type in the embryo and neonate;

CD4-DNTGFbRII mice spontaneously develop DED but those receiving treatment to induce DED have improved (De Pavia et al., cited supra, abstract); thus, a role of TGF-β in maturation and regulation of immune system is appreciated; however, in order to conclude that TGF-β inhibition may be therapeutic in DED, inhibition must be present only after having specific inflammatory processes of the ocular surface in motion, because the absence of TGF-β signaling in CD4 lymphocytes may be altering the maturation and efficacy of specific immune response (John J. Letterio and Anita B. Roberts, Annual Review of Immunology Vol. 16: 137-161 (Volume publication date April 1998));

further, the animal model used by De Paiva et al. (cited supra), is a model in which TGF-β signaling pathway is interrupted from birth and, subsequently, the disease is induced; that type of model does not provide reliable or representative results because the model has been able to use alternative mechanisms to compensate the absence of the TGF-beta receptor type 2 (TGFbetaRII); and

on the other hand, the receptor TGFbetaRII interacts with several ligands, not only TGF-βΙ, such as for example, apolipoprotein J, cyclin B2, AP2B1, endoglin, HSP 90kDa alpha (cytosolic) and STRAP, so that it cannot be ruled out that the effect of the improvement in DED is due to inhibition of the interaction between a ligand other than TGF-βΙ and receptor TGFbetaRII; and, what is relevant, TGFbetaRII receptor interacts with the 3 isoforms 1 , 2 and 3 of TGF-β and may have a different, even opposite, regulatory role in the development of immune responses (information on evidences of the different origin, targets and effects of the different TGF-β isoforms in the immune system is provided by Bottoms SE et al., PLoS One. 2010 Mar 12;5(3):e9674. doi: 10.1371/journal.pone.0009674 and Matejuk A et al. Cytokine. 2004 Jan 21 ;25(2):45- 51). On the other hand, it has been suggested that two TGF-βΙ inhibitor peptides, identified as P144 and P17, which downregulated TGF^l-estimulated MMP-13 secretion but did not inhibit TGF^l-estimulated MMP-9, could be a potential topical therapy for ocular surface inflammatory pathology secretion (Enriquez de Salamanca et al., Poster entitled "In vitro P144 and P17 anti-TGF-β peptides effect in TGF-β- stimulated corneal epithelial cells", ARVO Meeting Abstracts April 11, 2009; 50:6289), although the eventual use of said TGF-βΙ inhibitor peptides (PI 44 and PI 7) as eventual therapeutic agents for treating DED has not been mentioned or suggested. Said peptides (P144 and P17) have shown to be potentially useful in the treatment of liver fibrosis, lung fibrosis and corneal fibrosis and haze. Peptide PI 44 is disclosed, for example, in WO 00/31135, whereas peptide P17 is disclosed in WO 2005/019244).

SUMMARY OF THE INVENTION

The solution provided by the present invention to the confronted problem is based on the fact that the inventors have identified that peptides P144 and P17, which are capable of inhibiting the biological activity of TGF-βΙ through direct binding to said factor, can be used in the treatment of DED, as shown in Examples 1 and 2.

Thus, in an aspect, the invention relates to a peptide selected from the group consisting of peptide P144, peptide P17, a fragment of said peptide P17 having the capacity to inhibit the biological activity of TGF-βΙ comprising between 9 and 14 consecutive amino acids, and any combination thereof, and their pharmaceutically acceptable salts, for use in the prevention and/or treatment of DED. Alternatively, the invention relates to the use of a peptide selected from the group consisting of peptide P144, peptide P17, a fragment of said peptide P17 having the capacity to inhibit the biological activity of TGF-βΙ comprising between 9 and 14 consecutive amino acids, and any combination thereof, and their pharmaceutically acceptable salts, in the manufacture of a pharmaceutical composition for the prevention and/or treatment of DED.

In another aspect, the invention relates to a method for the prevention and/or treatment of dry eye disease which comprises topically administering to a patient in need thereof a pharmaceutical composition comprising a peptide selected from the group consisting of peptide P144, peptide P17, a fragment of said peptide P17 having the capacity to inhibit the biological activity of TGF-βΙ comprising between 9 and 14 consecutive amino acids, and any combination thereof, and their pharmaceutically acceptable salts.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically shows the evaluation of anti-TGF-bl PI 7 peptide in the C57BL/6 Mouse Model of Dry Eye with Adoptive Transfer.

FIG. 2 is a bar diagram which shows that PI 7 is able to significantly recover the tear production in mice after stress desiccation, comparing with animals treated with vehicle (for details, see Example 1).

FIG. 3 is a bar diagram that shows that animals receiving adoptive lymphocytes transfer from dry eye induced animals increased pro-inflammatory cytokines tear content. Treatment with P17 is able to block the increasing levels of IFN-γ, IL-17 and IL-12. P17 strongly and significantly decreases the IL-2 tear content, even quite below normal levels (for details, see Example 1).

FIG. 4 shows that PI 7 significantly decreases ocular surface infiltrating capacity of donor T cells after dry eye induction. These results clearly show the antiinflammatory properties of this compound in a dry eye animal model (for details, see Example 1).

FIG. 5 shows that topical treatment with PI 7 significantly increases the number of goblet cells in recipient lymphocytes transfer mice. One of the most important changes that occur in dry eye is a reduction in the number of conjunctival goblet cells on the surface of the eye. There are a high number of mucus-containing cells called "goblet cells" in the surface of the eye which are responsible of secreting mucin, which dissolves in water to form mucus (responsible of lubrication and anti-desiccation function). Protective or/and stimulation effect over mantainance in goblet cell number and activity is a key factor in dry eye pathologies (for details, see Example 1). FIG. 6 schematically shows the evaluation of anti-TGF-bl PI 44 peptide in the C57BL/6 Mouse Model of Dry Eye with Adoptive Transfer.

FIG. 7 shows that PI 44 topical eye surface treatment maintains goblet cell number in donor mice and reflects a better behavior in comparison with the reference treatment (topical cyclosporine, Restasis®) (for details, see Example 2).

FIG. 8 shows that PI 44 significantly decreases ocular surface infiltrating capacity of donor T cells after dry eye induction. These results clearly show the potent anti-inflammatory properties of this compound in a dry eye animal model. This effect is in the range of the reference treatment in use (Restasis®) and closer to the conjunctiva T cell count in healthy mice (for details, see Example 2).

FIG. 9 shows that animals receiving adoptive lymphocytes transfer from dry eye induced animals decrease pro-inflamatory cytokines tear content. Treatment with PI 44 is able to block the increasing levels of IL-6, Rantes, IP-10 and IL-10. This effect is similar to that obtained with Restasis® and higher in the case of RANTES and IP-10. CCL5/RANTES level increases in the tears of dry eye patients and correlates with various tear film and ocular surface parameters (for details, see Example 2).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that the topical administration of TGF-βΙ inhibitor peptide PI 7 to an experimental animal model of dry eye disease (DED) surprisingly allows to (i) recover tear production in said animal model, (ii) decrease the content of pro-inflammatory cytokines (IFN-γ, IL-17, IL-12 and IL-2) in animals receiving adoptive lymphocytes transfer from dry eye induced animals, (iii) decrease the ocular surface infiltrating capacity of donor T cells after dry eye induction, and (iv) increase the number of goblet cells in recipient lymphocytes transfer mice. Further, inventors have also discovered that the topical administration of TGF-βΙ inhibitor peptide PI 44 to an experimental animal model of dry eye disease (DED) surprisingly allows to (i) decrease the ocular surface infiltrating capacity of donor T cells after dry eye induction, and (ii) decrease the content of pro-inflammatory cytokines (IL-6, Rantes, IP-10 and IL-10) in animals receiving adoptive lymphocytes transfer from dry eye induced animals. These results show that P17 and P144 are potentially useful in the treatment of DED thus providing a new therapeutic window for the prevention and/or treatment of DED. Therefore, in an aspect, the invention relates to a peptide selected from the group consisting of peptide PI 44, peptide P17, a fragment of said peptide PI 7 having the capacity to inhibit the biological activity of TGF-βΙ comprising between 9 and 14 consecutive amino acids, and any combination thereof, and their pharmaceutically acceptable salts, for use in the prevention and/or treatment of dry eye disease (DED). Alternatively drafted, the invention relates to the use of a peptide selected from the group consisting of peptide P144, peptide P17, a fragment of said peptide P17 having the capacity to inhibit the biological activity of TGF-βΙ comprising between 9 and 14 consecutive amino acids, and any combination thereof, and their pharmaceutically acceptable salts, in the manufacture of a pharmaceutical composition for the prevention and/or treatment of DED. Nevertheless, the skilled person in the art will understand that said TGF-βΙ inhibitor peptides can be presented in multiple forms not only as a peptide, but also as, for example, a fusion protein, a polynucleotide, a vector, a cell, etc.

Peptides PI 44, PI 7 and fragments of peptide P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, are TGF-βΙ inhibitor peptides.

In the present invention, the term "TGF-βΙ inhibitor peptide" refers to a peptide having the ability to inhibit a biological activity of TGF-βΙ; the inhibition of the biological activity of TGF-βΙ can be achieved, for example, by interacting the inhibitor peptide with the active form of TGF-βΙ; thus, in a particular embodiment, the TGF-βΙ inhibitor peptide binds directly to TGF-βΙ and inhibits a biological activity of said TGF-βΙ.

TGF-βΙ is a glycoprotein belonging to a superfamily of structurally related regulatory proteins (cytokines) included within one of the three isoforms described in mammals (TGF-βΙ, ΤΟΕ-β2 and ΤΟΕ-β3). The most abundant isoform is TGF-βΙ, which consists of a 25 kDa homodimer composed of two subunits joined by a disulfide bond. The sequences of said growth factors are well known for several animal species. The amino acid sequence of human TGF-βΙ has been disclosed (Derynck K et al., Nature (1985). 316 (6030):701-705). The GenBank accession No. for the human sequences correspond to: TGF-βΙ and TGF-βΙ precursor: NP _ 000651 (protein) and NM _ 000660 (cDNA), ΤΟΕ-β2 and ΤΟΕ-β2 precursor: NP _ 003229 (protein) and NM - 003238 (cDNA) and ΤΟΕ-β3 and ΤΟΕ-β3 precursor: NP-003230 (protein) and NM _ 003239 (cDNA). A further amino acid sequence of TGF-βΙ is shown under accession number GenBank: AAL27646.2. TGF-βΙ is a molecule with a highly preserved sequence in evolutive terms. Although it was originally defined by its capacity to induce adhesion independent of proliferation and morphological changes in rat fibroblasts, subsequent investigations have shown that TGF-βΙ is a general inhibitor of proliferation of a broad range of cell types. The molecule is produced by a great variety of cell types and in different tissues during all phases of cell differentiation. It has a large series of biological effects, with the generation of potent and very often opposite effects in relation to development, physiology and immune response. Many diseases or pathological alterations are associated with excessive or deregulated expression of TGF-βΙ, e.g., fibrosis associated to organ or tissue function loss, or surgical or esthetic complications, for example liver fibrosis or pulmonary fibrosis.

In a particular embodiment, the peptide for use in the prevention and/or treatment of DED is peptide PI 44. Peptide PI 44 (SEQ ID NO: 1) was firstly disclosed in WO 00/31135. In another particular embodiment, the peptide for use in the prevention and/or treatment of DED is peptide P17. Peptide P17 (SEQ ID NO: 2) was firstly disclosed in WO 2005/019244 as well as some fragments of the peptide P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ which have been further disclosed by Dotor et al. (Dotor J et al., Cytokine (2007); 39: 106-115). In a particular embodiment, said fragment is a fragment comprising 9, 10, 11, 12, 13, or 14 consecutive amino acid residues of the peptide P17 and maintains the capacity to inhibit the biological activity of TGF-βΙ.

Illustrative examples of the TGF-βΙ inhibitor peptides which can be used within the context of the present invention are listed in Table 1.

Table 1

TGF-βΙ inhibitor peptides for use in prevention and/or treatment of DED

P17(3-l l) 5 IWFIPRSSW

P17(4-12) 6 WFIPRSSWY

P17(5-13) 7 FIPRSSWYE

P17(6-14) 8 IPRSSWYER

P17(7-15) 9 PRSSWYERA

P17(l-10) 10 KRIWFIPRSS

P17(2-l l) 11 RIWFIPRSSW

P17(3-12) 12 IWFIPRSSWY

P17(4-13) 13 WFIPRSSWYE

P17(5-14) 14 FIPRSSWYER

P17(6-15) 15 IPRSSWYERA

P17(l-l l) 16 KRIWFIPRSSW

P17(2-12) 17 RIWFIPRSSWY

P17(3-13) 18 IWFIPRSSWYE

P17(4-14) 19 WFIPRSSWYER

P17(5-15) 20 FIPRS S W YERA

P17(l-12) 21 KRIWFIPRSSWY

P17(2-13) 22 RIWFIPRSSWYE

P17(3-14) 23 IWFIPRSSWYER

P17(4-15) 24 WFIPRSSWYERA

P17(l-13) 25 KRIWFIPRSSWYE

P17(2-14) 26 RIWFIPRSSWYER

P17(3-15) 27 rWFIPRS S WYERA

P17(l-14) 28 KRIWFIPRSSWYER

P17(2-15) 29 RIWFIPRSSWYERA

The TGF-βΙ inhibitor peptides for use within the present invention may be obtained by conventional techniques; for example, they may be synthesized by chemical synthetic methods, including but not limited to, solid phase peptide synthesis (see, for example, WO 00/31135 or WO 2005/019244), and, if desired, purified with high performance liquid chromatography (HPLC), and analyzed by conventional techniques such as for example sequencing and mass spectrometry, amino acid analysis, nuclear magnetic resonance techniques, etc. Alternatively, said peptides may be obtained by means of the recombinant DNA technology from a variety of cell sources that synthesize said peptides including, for example, cells transfected with recombinant DNA molecules capable of directing the synthesis or secretion of the peptides. The nucleotide sequence encoding said peptide can easily be deduced from the amino acid sequence of the peptide. Further, said peptides may be also commercially available, for instance PI 44 may be supplied by Sigma-Genosys, Ltd. (Cambridge, UK).

In a particular embodiment, the peptide to be used in the treatment of DED is selected from peptide P144 (SEQ ID NO: 1), peptide P17 (SEQ ID NO: 2), peptide P17(l-l l) (SEQ ID NO: 16), peptide P17(l-12) (SEQ ID NO: 21), peptide P17Q-14) (SEQ ID NO: 28), and any combination thereof.

The skilled person in the art will recognize that functionally equivalent derivatives of peptides P144, P17 and fragments of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, are also included within the context of the invention. The term "derivative" as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included within the scope of the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the biological activity of the peptides as described above (i.e., the ability to inhibit the biological activity of TGF-βΙ) and do not confer toxic properties on compositions containing it. Derivatives may have chemical moieties, such as carbohydrate or phosphate residues, provided such a derivative retains the biological activity of the TGF-βΙ inhibitor peptide and remains pharmaceutically acceptable. For example, derivatives may include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives or free amino groups of the amino acid residues formed with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups), or O- acyl derivatives of free hydroxyl group (e.g., that of seryl or threonyl residues) formed with acyl moieties. Such derivatives may also include for example, polyethylene glycol side-chains, which may mask antigenic sites and extend the residence of the molecule in body fluids. The capacity of a peptide to inhibit the biological activity of TGF-βΙ may be determined by any suitable conventional bioassay for measuring TGF-βΙ activity, for example by the assays described by Meager (Meager A. Journal of Immunological Methods (1991) 141 : 1-14]. Amongst these methods, the Mv-l-Lu cell growth inhibition assay is particularly suitable. Mv-l-Lu cell line is a cell line derived from mink lung epithelium, whose proliferation is inhibited by TGF-βΙ. A description of said Mv-l-Lu cell growth inhibition assay is provided in WO 2005/019244. Further, the capacity of a peptide to in vivo inhibit the biological activity of TGF-βΙ can be evaluated and, if desired, quantified by testing in an animal model of acute liver damage induced for example by the administration of carbon tetrachloride (CC1₄) as disclosed in WO 2005/019244, or by any similar assay known by the skilled person in the art.

The skilled person in the art will understand that also within the scope of this invention are the pharmaceutically acceptable salts of the peptides PI 44, P17 and fragments of PI 7 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ. The term "salt" herein refers to both salts of carboxyl groups and acid addition salts of amino groups of the peptides. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric, or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine, lysine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids, such as, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc., and salts with organic acids, such as, for example, acetic acid, citric acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, methanesulfonic acid, benzenesulfonic acid, maleic acid, etc. For therapeutic use, salts of TGF-βΙ inhibitor peptides are those wherein the counter- ion is pharmaceutically acceptable. The nature of the pharmaceutically acceptable salt is not a critical consideration, provided it is pharmaceutically acceptable.

The peptides P144, P17 and fragments of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, and their pharmaceutically acceptable salts, may be in the form of a composition, wherein said composition may comprise one or more of said peptides. Thus, in a particular embodiment, the invention provides a composition comprising at least one peptide selected from the group consisting of peptides P144, P17, fragments of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, and any combination thereof, or their pharmaceutically acceptable salts. In another particular embodiment, the invention provides a composition comprising a combination of two or more peptides, wherein said peptides are selected from the group consisting of peptides PI 44, PI 7 and fragments of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, or their pharmaceutically acceptable salts.

The peptides P144, P17 and fragments of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, can be used in the prevention and/or treatment of DED. The person skilled in the art will understand that, for any of said peptides to be able to be used for therapeutic purposes, it must be suitably formulated for its administration.

Therefore, in a particular embodiment, the composition provided by the instant invention is a pharmaceutical composition for use in the treatment of DED and comprises a peptide selected from the group consisting of peptides PI 44, P17 and fragments of PI 7 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, or their pharmaceutically acceptable salts, and any combination thereof, and a pharmaceutically acceptable vehicle. As used herein, the term "pharmaceutically acceptable vehicle" refers to any carrier which is safe and provides an effective delivery of an effective amount of at least one product of the invention to the target tissue.

In a particular embodiment, said pharmaceutical composition comprises just one peptide selected from the group consisting of peptides PI 44, PI 7 and fragments of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, or their pharmaceutically acceptable salts.

In another particular embodiment, said pharmaceutical composition comprises two or more peptides, said peptides being selected from the group consisting of peptides P144, P17 and fragments of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, or their pharmaceutically acceptable salts.

In another particular embodiment, said pharmaceutical composition further comprises, in addition to at least a peptide selected from the group consisting of peptides P144, P17 and fragments of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, or their pharmaceutically acceptable salts, a further therapeutic agent aimed at the treatment of DED. Illustrative, non-limitative, examples of therapeutic agents suitable for the treatment of DED includes compounds which increase or adjust the viscosity and which at the same time exhibit lubricant functionality such as carboxymethylcellulose and its sodium salt (CMC, carmellose), polyvinyl alcohol, hydroxypropyl methylcellulose (HPMC, hypromellose), hyaluronic acid and its sodium salt, hydroxypropyl guar gum, etc.; cholinergic agents such as muscarinic acetylcholine receptor antagonists that may be used in aqueous deficient patient as secretagogues to stimulate tear production, e.g., pilocarpine, cevimeline, etc.; anti-inflammatory agents, for example, topical corticosteroids e.g., loteprednol etabonate, prednisolone acetate, etc., or topical nonsteroidal anti-inflammatory (NSAID) compounds, e.g., diclofenac, ketorolac, etc.; tetracyclines such as doxycyclin, minocycline, oxytetracycline, etc.; immune- suppressant drugs such as ciclosporin (i.e., ciclosporin A or cyclosporine A), tacrolimus, pimecrolimus, everolimus, sirolimus, deforolimus, temsirolimus, and zotarolimus, abetimus, gusperimus, mycophenolic acid, etc.; antibodies, such as anti-IL- 17 antibodies, anti-KLK-13 antibodies, etc. Also, the pharmaceutical composition provided by the instant invention may comprise one or more TGF-βΙ inhibitor peptides, other than peptides P144, P17 and fragments of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ.

The active ingredient in the pharmaceutical composition provided by the invention, i.e., the peptide P144, P17 or a fragment of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, or any combination of said peptides, will be present ins said pharmaceutical composition in a therapeutically effective amount. In the present invention, a "therapeutically effective amount" is understood as the amount of active ingredient which is sufficient to delay, reduce or eliminate the symptoms associated with DED or the severity thereof.

Said active ingredients may be contained in various types of pharmaceutical compositions in accordance with formulation techniques known to those skilled in the art. The route of administration (e.g., topical) and the dosage regimen will be determined by skilled clinicians, based on factors such as the exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, the particular product of the invention used, the pharmacokinetic properties of the individual and so on. A review of the different processes for administering active ingredients, the excipients to be used and the processes for preparing them can be found in Tratado de Farmacia Galenica, C. Fauli i Trillo, Luzan 5, S.A. de Editions, 1993 and in Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 20^th edition, Williams & Wilkins PA, USA. (2000).

The pharmaceutical composition provided by the instant invention is an ophthalmic pharmaceutical composition, i.e., a pharmaceutical composition comprising a therapeutically effective amount of an active ingredient as provided by the present invention and a pharmaceutically acceptable vehicle for said compound(s) wherein said pharmaceutically acceptable vehicle is a vehicle suitable for ophthalmic administration. As used herein, the term "ophthalmic pharmaceutical composition" denotes a composition intended for application in the eye or intended for treating a device to be placed in contact with the eye such as a contact lens.

Pharmaceutical compositions provided by the present invention may be prepared for storage by mixing the active ingredient with optional physiologically acceptable carriers, excipients or stabilizers, as mentioned above, in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as ethylenediaminotetraacetic acid (EDTA); sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as polysorbates (e.g., TWEEN®), poloxamers (e.g., PLURONICS®), or polyethylene glycol (PEG).

In a particular embodiment, the pharmaceutical composition provided by this invention will be formulated for, and administered by, topical application. Topical formulations are generally aqueous in nature, buffered to a physiological acceptable pH and typically preserved for multi-dispensing. Thus, in a particular embodiment, the pharmaceutical composition is a topical ophthalmic pharmaceutical composition comprising a therapeutically effective amount of an active ingredient provided by the present invention (i.e., peptides PI 44, PI 7 or a fragment of PI 7 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ, or any combination of said peptides) and a pharmaceutically acceptable vehicle for said compound(s) wherein said pharmaceutically acceptable vehicle is a vehicle suitable for topical ophthalmic administration. As it is well-known by the skilled person in the art, various types of vehicles may be utilized. The vehicles will generally be aqueous in nature. Aqueous solutions are generally preferred, based on ease of formulation, as well as patients' ability to easily administer such compositions by means of instilling one to two drops of the solutions in the affected eyes. However, the active ingredients provided by the invention may also be readily incorporated into other types of compositions, such as suspensions, viscous or semi-viscous gels or other types of solid or semi-solid compositions. Suspensions may be preferred for active ingredients which are relatively insoluble in water. The ophthalmic compositions of the present invention may also include various other ingredients, such as buffers, preservatives, co- solvents and viscosity building agents.

In relationship to any of the compositions described herein, it is preferable that an effective amount of buffer be included to maintain the pH from about 6 to about 8, preferably about 7. Buffers used are those known to those skilled in the art, and, while not intending to be limiting, some examples are acetate, ascorbate, borate, bicarbonate, carbonate, citrate, and phosphate buffers. Preferably, the buffer comprises borate. An effective amount of buffer necessary for the purposes of this invention can be readily determined by a person skilled in the art without undue experimentation. In cases where the buffer comprises borate, it is preferable that the concentration of the borate buffer be about 0.6%.

In any of the compositions related described herein related to this invention, it is preferable for a tonicity agent to be used. Tonicity agents are used in ophthalmic compositions to adjust the concentration of dissolved material to the desired isotonic range. Tonicity agents are known to those skilled in the ophthalmic art, and, while not intending to be limiting, some examples include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes. Preferably, the tonicity agent is sodium chloride.

Ophthalmic formulations are typically packaged in multidose form.

Preservatives are thus required to prevent microbial contamination during use. There may also be reasons to use a preservative in single use compositions depending on the individual circumstances. The term "preservative" has the meaning commonly understood in the ophthalmic art. Preservatives are used to prevent bacterial contamination in multiple-use ophthalmic preparations, and, while not intending to be limiting, examples include benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium- 1 , stabilized oxychloro complexes (otherwise known as Purite®)), phenylmercuric acetate, chlorobutanol, benzyl alcohol, or other agents known to those skilled in the art. Such preservatives are typically employed at a level of from 0.001 to 1.0 percent by weight, based on the total weight of the composition (wt. %).

Under certain circumstances, a surfactant or other appropriate co-solvent might be used in any of the compositions provided by this invention which are described herein. The term "surfactant" and "co-solvent" used herein has the meaning commonly understood in the art. Surfactants are used to help solubilize the therapeutically active agent or other insoluble components of the composition, and may serve other purposes as well. Illustrative, non-limitative, examples of said compounds include polyethoxylated castor oils, Polysorbate 20, 60 and 80; Pluronic® F-68, F-84 and P-103 (BASF Corp., Parsippany N.J., USA); cyclodextrins; or other agents known to those skilled in the art. Anionic, cationic, amphoteric, zwitterionic, and non-ionic surfactants may all be used in this invention. For the purposes of this invention, it is preferable that a non-ionic surfactant, such as polysorbates, poloxamers, alcohol ethoxylates, ethylene glycol-propylene glycol block copolymers, fatty acid amides, alkylphenol ethoxylates, or phospholipids, is used in situations where it is desirable to use a surfactant. Such surtactants or co-solvents are typically employed at a level of from 0.01 to 2 wt. %.

Viscosity greater than that of simple aqueous solutions may be desirable to increase ocular absorption of the active ingredient, to decrease variability in dispensing the formulations, to decrease physical separation of components of a suspension or emulsion of formulation and/or otherwise to improve the ophthalmic formulation. Such viscosity building agents include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose or other agents known to those skilled in the art. Such agents are typically employed at a level of from 0.01 to 2 wt. %.

Another type of compound that might be used in any composition of this invention described herein is a chelating agent. The term chelating agent refers to a compound that is capable of complexing a metal, as understood by those of ordinary skill in the chemical art. Chelating agents are used in ophthalmic compositions to enhance preservative effectiveness. While not intending to be limiting, some useful chelating agents for the purposes of this invention are edetate salts, like edetate disodium, edetate calcium disodium, edetate sodium, edetate trisodium, and edetate dipotassium.

In a particular embodiment, the active ingredient is administered in the form of a topical pharmaceutical composition suitable for ophthalmic application, such as eyedrops, ointments, creams, etc.

Compositions of the invention are administered topically to the eye. Depending on the type and severity of the disease, about 1 μg/kg to about 50 mg/kg (e.g., 0.1-20 mg/kg) of active ingredient is an initial candidate dosage for administration to the patient. A typical daily or weekly dosage might range from about 1 μg/kg to about 20 mg/kg or more. The doses utilized for any of the above-described purposes of topical administration will generally be from about 0.01 to about 100 mg per kilogram of body weight (mg/kg), administered one to several, e.g., four, six, eight or even more, times per day.

In a particular embodiment, the pharmaceutical composition provided by the present invention comprises two or more active ingredients, wherein at least one of them is the peptide P144, P17 or a fragment of P17 comprising between 9 and 14 consecutive amino acids and having the capacity to inhibit the biological activity of TGF-βΙ; in this embodiment, said components can be formulated for their separate, simultaneous or successive use.

The pharmaceutical composition provided by the invention can be in the form of a kit. In the present invention, a "kit" is understood as a product containing the active ingredient(s) provided by the present invention and/or the additional therapeutic compounds forming the packaged composition such that the transport, storage and simultaneous or successive administration thereof is allowed. Therefore, the kits of the invention can contain one or more suspensions, syringes, and the like which contain the active ingredients of the invention and which can be prepared in a single dose or as multiple doses. The kit can additionally contain a vehicle suitable for solubilizing the active ingredients such as aqueous media such as saline solution, Ringer's solution, dextrose and sodium chloride, water-soluble media such as alcohol, polyethylene glycol, propylethylene glycol and water- insoluble vehicles if necessary. Another component which may be present in the kit is a package which allows maintaining the compositions of the invention within determined limits. Materials suitable for preparing such packages include glass, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, vials, paper, sachets and the like.

The kit of the invention can additionally contain instructions for the simultaneous, successive or separate administration of the different pharmaceutical formulations present in the kit. Therefore, the kit of the invention can further comprise instructions for the simultaneous, successive or separate administration of the different components. Said instructions can be in the form of printed material or in the form of an electronic support which can store the instructions such that they can be read by a subject, such as electronic storage media (magnetic disks, tapes and the like), optical media (CD-ROM, DVD) and the like. The media can additionally or alternatively contain Internet webpages providing said instructions.

As it has been indicated above, the findings described in the present invention are useful for the prevention and/or treatment of DED. In the present invention, "dry eye disease" or "DED", also known as keratoconjunctivitis sicca or dysfunctional tear syndrome, is understood as a multifunctional disorder or abnormality of the tear film and of the ocular surface which results in ocular discomfort, visual disturbance, and often even in ocular surface damage caused by tear film instability. In general, dry eye disease is characterized by symptoms such as a sandy-gritty feeling in the eye, burning, irritation, or a foreign-body sensation that worsens during the day. Patients suffering from dry eye disease complain of mild to severe symptoms, and those with severe symptoms may experience constant and disabling eye irritation, and develop ocular surface epithelial disease and sight-threatening sterile or microbial corneal ulceration. It is today acknowledged that DED is a complex, multifunctional disorder involving several interacting pathophysiological mechanisms which are only beginning to be understood (Perry HD, Am. J. Man. Care. (2008). 13;3, S79-S87). The two mechanisms that are being discussed as pivotal in the etiology of the disease and which also appear to reinforce each other mutually are tear hyperosmolarity and tear film instability. Hyperosmolar tear fluid can result from excessive tear film evaporation or reduced aqueous flow. It activates an inflammatory cascade and causes the release of inflammatory mediators into the tear fluid, with multiple pathophysiological effects eventually leading to increased tear film evaporation and tear film instability. Thus, tear film instability can be a consequence of hyperosmolarity. Alternatively, it can develop as the original etiological pathway, e.g. via abnormalities of the lipid layer composition, such as in tarsal gland disease. Once DED has become manifest, inflammation is one of the key processes that maintain and potentially progress the disease. Depending on the severity of the condition, patients often develop a reversible squamous metaphase and punctate erosions of the ocular epithelium. Secondary diseases whose development may be triggered by DED include filamentary keratitis, microbial keratitis, corneal neo vascularis ation, and ocular surface keratinisation.

Two major categories of DED are distinguished today, namely, aqueous- deficient DED and evaporative DED. Within the class of aqueous -deficient forms of DED, two major subtypes are differentiated, Sjogren and non-Sjogren. Sjogren syndrome patients suffer from autoimmune disorders in which the lacrimal glands are invaded by activated T-cells, which lead not only to DED but also to a dry mouth condition. The Sjogren syndrome can be a primary disease or result from other autoimmune diseases such as systemic lupus erythrematosus or rheumathroid arthritis. Non-Sjogren patients suffering from an aqueous -deficient DED usually have a lacrimal gland insufficiency, lacrimal duct obstruction or reflex hyposecretion. The second major class, evaporative DED, is also somewhat heterogeneous and can develop as a result of diverse root causes. One of the major causes is meibomian gland disease, eyelid aperture disorders, blink disorders (as in Parkinson disease) or ocular surface disorders (as in allergic conjunctivitis). Among the many risk factors for DED that are currently known, some of the best studied ones are advanced age and female sex. It appears that in particular post-menopausal women have a reduced tear production, probably related to hormonal effects which are not very well understood as yet. Further risk factors include diets with low omega-3-fatty acids, occupational factors (e.g. associated with reduced blink frequency), environmental conditions, contact lens wearing, certain systemic (anticholinergics, beta-blockers, isotretinoin, interferons, hormones) and ophthalmic medications (any frequently administered eye drops including artificial tears; especially formulations comprising preservatives), and a number of primary diseases such as Parkinson disease, hepatitis C, HIV infection, and diabetes mellitus.

In another aspect, the invention relates to a method for the prevention and/or treatment of dry eye disease which comprises topically administering to a patient in need thereof a pharmaceutical composition comprising a peptide selected from the group consisting of peptide PI 44, peptide P17, and a fragment of said peptide PI 7 having the capacity to inhibit the biological activity of TGF-βΙ comprising between 9 and 14 consecutive amino acids, and their pharmaceutically acceptable salts.

As used herein, "treatment", "treat" or "treating" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, disease or disorder even if the treatment is ultimately unsuccessful. Those in need of treatment may include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. In particular, the active ingredients provided by the instant invention are suitable for treating DED, i.e., to deal with medically, and includes administering said active ingredients to alleviate symptoms of DED, such as inflammation and dryness, as well as to correct the physiological changes associated with dry eye disease, such as increased pro-inflammatory cytokine expression, inflammatory cell infiltration, decreased tear production and goblet cell number.

In a particular embodiment, said peptide is in the form of a composition, wherein said composition comprises one or more of said peptides. In another particular embodiment, said composition further comprises a pharmaceutically acceptable vehicle and/or a therapeutic compound aimed at the treatment of DED, or a TGF-βΙ inhibitor peptide other than peptides PI 44, P17, or a fragment of P17 having the capacity to inhibit the biological activity of TGF-βΙ comprising between 9 and 14 consecutive amino acids.

In a particular embodiment, the peptide is a peptide selected from the group of peptides whose sequence is shown in SEQ ID NO: 1-29, or any combination of said peptides, i.e., a peptide whose amino acid sequence is shown in SEQ ID NO: 1, or in SEQ ID NO: 2, or in SEQ ID NO: 3, or in SEQ ID NO: 4, or in SEQ ID NO: 5, or in SEQ ID NO: 6, or in SEQ ID NO: 7, or in SEQ ID NO: 8, or in SEQ ID NO: 9, or in SEQ ID NO: 10, or in SEQ ID NO: 11, or in SEQ ID NO: 12, or in SEQ ID NO: 13, or in SEQ ID NO: 14, or in SEQ ID NO: 15, or in SEQ ID NO: 16, or in SEQ ID NO: 17, or in SEQ ID NO: 18, or in SEQ ID NO: 19, or in SEQ ID NO: 20, or in SEQ ID NO: 21, or in SEQ ID NO: 22, or in SEQ ID NO: 23, or in SEQ ID NO: 24, or in SEQ ID NO: 25, or in SEQ ID NO: 26, or in SEQ ID NO: 27, or in SEQ ID NO: 28, or in SEQ ID NO: 29. In another particular embodiment, a combination of 2 or more of the above identified peptides is selected for use in the prevention and/or treatment of DED in accordance with the invention. In a preferred embodiment, at least one of the peptides P144 (SEQ ID NO: 1), P17 (SEQ ID NO: 2), or a fragment of P17 having the capacity to inhibit the biological activity of TGF-βΙ comprising between 9 and 14 consecutive amino acids, is present in said combination.In another particular and preferred embodiment, the peptide is a peptide selected from the group of peptides PI 44 (SEQ ID NO: 1), P17 (SEQ ID NO: 2), P17(l-l l) (SEQ ID NO: 16), P17(l-12) (SEQ ID NO: 21), P17(l-14) (SEQ ID NO: 28), and any combination thereof.

The following examples illustrate the invention and should not be taken to reflect limitations to the latter.

EXAMPLE 1

Evaluation of anti-TGF-βΙ P17 peptide in the C57BL/6 Mouse Model of Dry Eve with Adoptive Transfer In order to evaluate the effect of anti-TGF-βΙ P17 peptide in the C57BL/6 Mouse Model of Dry Eye with Adoptive Transfer, the inventors performed the following experiment. A schematic diagram of this experiment is shown in FIG. 1.

1. Materials & Methods

Mice

Female C57BL/6 mice were purchased from Taconic Inc. (Germantown, NY). Mice were used at 6 to 10 weeks of age. Animal studies approval was obtained from the Allergan Animal Care and Use Committee. All studies adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Dessicating stress (OS)

Desiccating Stress (DS) was induced by treating mice with subcutaneous (SC) injections of scopolamine hydrobromide (0.5 mg/0.2 mL; Sigma-Aldrich, St. Louis, MO) three times a day (TID) alternating between the left and right flanks. Up to 4 mice were placed in a cage containing perforated plastic screens on 2 sides of the cage to permit airflow from fans (one fan on each side of the cage) for 16 h/d in a hood (AirClean Systems, Raleigh, NC). Room humidity was kept below 40%. DS was administered for 10 consecutive days (Niederkorn et al., J Immunol. 2006; 176:3950- 3957; Dusun et al., Invest Ophthalmol Vis. Sci. 2002; 43:632-638). This model of dry eye disease (DED) has been used previously with no discernible ill effects from the scopolamine treatment or low humidity.

Groups

Three donor groups were made:

- PI 7 treated group: This group of mice was treated topically with an eye drops formulation containing 2 mg/ml of P17 in isotonic NaCl, esterilized by filtration (0.2 μιη) in vial, starting 3 days before exposure to DS, TID;

- Vehicle group: This group of mice was treated topically with an eye drops formulation containing only isotonic NaCl, esterilized by filtration (0.2 μιη) in vial, at the same conditions as the P17 treated group; and

- Control group: This further group of mice was not treated with any formulation.

On day 10, mice were sacrificed and tears were collected for determining tear production from baseline in the donor groups.

Adoptive transfer On day 10, mice were sacrificed and CD4+ T cells were also isolated from the spleen and superficial cervical lymph nodes of mice exposed to 10 days of DS in the presence or absence of PI 7 and adoptively transferred to syngeneic T cell-deficient nude recipient mice that were not administered P17. On day 3 post-adoptive transfer, tears and ocular surface tissues were collected from nude recipient mice for analysis of cytokine levels and histopathology.

Tear collection

1.5 μL· of phosphate buffered saline (PBS) was placed on each eye, and then 1 μL· of tear was collected from both eyes and placed in 8 μL· of cytokine assay buffer (Beadlyte; Millipore, Billerica, MA). Buffer and tear fluid were collected by capillary action using a 1 volume glass capillary tube (Drummond Scientific, Broomhall, PA) that was placed in the tear meniscus of the lateral canthus. The samples were then frozen at -80°C until the time of the assay. The production of tears was quantified by conventional methods.

Determination of cytokine levels

Cytokine levels in recipient tears, namely interleukin 2 (IL-2), interleukin 12 (IL- 12), interleukin 17 (IL- 17) and interferon gamma (IFN-γ) were analyzed by using the corresponding cytokine pairs (Beadmate; Millipore). For the bioassay (Luminex Corp.), a 96-well filter plate (Millipore) was prewetted with 25 μL· of cytokine assay buffer (Beadlyte; Millipore). A vacuum manifold (Millipore) was used to aspirate the buffer from the wells. For tears, 10 μL· of diluted (2 μL· tears to 8 μL· Beadlyte buffer) tear sample was placed in each well. The beads (25 μί) were pipetted into the wells. Standard curves for each cytokine were generated in duplicate by placing 10 μL· of the appropriate dilution of standards purchased from Upstate (Lake Placid, NY). The plate was incubated overnight with gentle shaking in the dark at 4°C. The plate was washed with cytokine assay buffer and wash buffer was eliminated by using a vacuum manifold (Millipore). Twenty-five microliters of the appropriate biotin-conjugated secondary antibody (Upstate) was added to each well for 90 minutes at room temperature with gentle shaking. The beads were incubated with streptavidin-phycoerythrin (1 :25 dilution in Beadlyte assay buffer) for 30 minutes at room temperature with gentle shaking. The beads were washed, resuspended in 125 μL· of cytokine assay buffer, and analyzed (system 100; Luminex Corp.). The mean fluorescence intensities obtained from 50 beads per cytokine minimum were analyzed (Beadview software; Upstate). Standard curves were generated (eight data points including a zero standard run in duplicate) using a four- or five-parametric logistic curve. R² was between 0.99 and 1. Data are expressed in picograms or nanograms per millilitre.

Measuring globet cell density

The density of goblet cells in recipient conjunctiva was estimated in sections stained by conventional periodic acid - Schiff (PAS) histochemistry.

2. Results

FIG. 2 shows that PI 7 is able to significantly recover the tear production in mice of the "PI 7 treated group" after DS (DS + Top PI 7) in comparison with the mice of the "vehicle group" (Ds + Top Veh Donor).

FIG. 3 shows that animals receiving adoptive lymphocytes transfer from dry eye induced animals decreased the content in tear of pro-inflammatory cytokines such as IFN-γ, IL-2, IL-12, and IL-17; in particular, P17 strongly and significantly decreases the content of IL-2 in tear, even quite below normal levels.

Further, PI 7 significantly decreased the ocular surface infiltrating capacity of donor T cells after dry eye induction (FIG. 4), thus clearly showing the antiinflammatory properties of P17 in a dry eye animal model.

Finally, topical treatment with PI 7 significantly increased the number of goblet cells in recipient lymphocytes transfer mice (FIG. 5). One of the most important changes that occur in dry eye disease is a reduction in the number of conjunctival goblet cells on the surface of the eye. There are a high number of mucus -containing cells called "goblet cells" in the eye surface which are in charge of secreting mucin, which dissolves in water to form mucus (responsible of the lubrication and anti-desiccation function). A protective or/and stimulation effect over maintenance in goblet cell number and activity is a key factor in dry eye pathologies.

Overall these results show that PI 7 is potentially useful in the treatment of dry eye disease.

EXAMPLE 2

Evaluation of anti-TGF-βΙ P144 peptide in the C57BL/6 Mouse Model of Dry Eve with Adoptive Transfer In order to evaluate the effect of anti-TGF-βΙ PI 44 peptide in the C57BL/6 Mouse Model of Dry Eye with Adoptive Transfer, the inventors performed the following experiment. A schematic diagram of this assay is shown in Figure 6.

1. Materials & Methods

Mice

Dessicating stress (OS)

Groups

Five donor groups were made:

- PI 44 treated group: This group of mice was treated topically with an eye drops formulation containing 2 mg/ml of P144 and 0.075% Ultrez 10 (pH adjusted to 7.2), TID, starting 3 days before exposure to DS, TID.

- Restasis treated group: This group of mice was treated topically with Restasis® (cyclosporine A), TID, starting 3 days before exposure to DS, TID.

- 10 DPS group: This group of mice corresponds to the group of donor mice subjected to 10 days DS;

- Vehicle group: This group of mice was treated topically with an eye drops formulation containing only the vehicle (0.075% Ultrez 10 (pH adjusted to 7.2)), at the same conditions as the PI 44 treated group; and Control group: This group of mice was not treated with any formulation.

On day 10, mice were sacrificed and tears, blood and lacrimal glands of both eyes and were collected for further assays, as well as spleen and superficial cervical lymph nodes for adoptive transfer.

Adoptive transfer

CD4+ T cells isolated from the spleen and superficial cervical lymph nodes of the sacrificed mice exposed to 10 days of DS in the presence or absence of PI 44 or Restasis® were adoptively transferred to syngeneic T cell-deficient nude recipient mice that were not administered P144 or Restatin®. On day 3 post-adoptive transfer, tears, blood and ocular surface tissues were collected from nude recipient mice for analysis of cytokine levels and histopathology.

Tear collection

1.5 of PBS was placed on each eye, and then 1 μL· of tear was collected from both eyes and placed in 8 μL· of cytokine assay buffer (Beadlyte; Millipore, Billerica, MA). Buffer and tear fluid were collected by capillary action using a 1 volume glass capillary tube (Drummond Scientific, Broomhall, PA) that was placed in the tear meniscus of the lateral canthus. Samples were frozen at -80°C until the time of the assay. The production of tears was quantified by conventional methods.

Determination of cytokine levels

Cytokine levels in recipient tears, namely interleukin 6 (IL-6), interleukin 10

(IL-10), IFN-y-induced protein 10 kDa (IP- 10) and RANTES (CCL5) were analyzed by using the corresponding cytokine pairs (Beadmate; Millipore). For the bioassay (Luminex Corp.), a 96-well filter plate (Millipore) was prewetted with 25 μL· of cytokine assay buffer (Beadlyte; Millipore). A vacuum manifold (Millipore) was used to aspirate the buffer from the wells. For tears, 10 μL· of diluted (2 μL· tears to 8 μL· Beadlyte buffer) tear sample was placed in each well. The beads (25 μί) were pipetted into the wells. Standard curves for each cytokine were generated in duplicate by placing 10 μL· of the appropriate dilution of standards purchased from Upstate (Lake Placid, NY). The plate was incubated overnight with gentle shaking in the dark at 4°C. The plate was washed with cytokine assay buffer and wash buffer was eliminated by using a vacuum manifold (Millipore). Twenty-five microliters of the appropriate biotin- conjugated secondary antibody (Upstate) was added to each well for 90 minutes at room temperature with gentle shaking. The beads were incubated with streptavidin- phycoerythrin (1:25 dilution in Beadlyte assay buffer) for 30 minutes at room temperature with gentle shaking. The beads were washed, resuspended in 125 μL· of cytokine assay buffer, and analyzed (system 100; Luminex Corp.). The mean fluorescence intensities obtained from 50 beads per cytokine minimum were analyzed (Beadview software; Upstate). Standard curves were generated (eight data points including a zero standard run in duplicate) using a four- or five-parametric logistic curve. R² was between 0.99 and 1. Data are expressed in picograms or nanograms per millilitre.

Measuring globet cell density

2. Results

Figure 7 shows that PI 44 topical eye surface treatment maintains goblet cell number in donor mice, this tendency is not significant but reflects a better behavior in comparison with the treatment with topical cyclosporine A (Restasis®).

Figure 8 shows that PI 44 significantly decreases the ocular surface infiltrating capacity of donor T cells after dry eye induction. These results clearly show the potent anti-inflammatory properties of PI 44 in a dry eye animal model. This effect is in the range of the reference treatment (Restasis®) and closer to the conjunctiva T cell count in healthy mice.

Finally, Figure 9 shows that animals receiving adoptive lymphocytes transfer from dry eye induced animals decreased the content in tear of pro-inflamatory cytokines. In fact, treatment with P144 is able to block the increasing levels of IL-6, Rantes, IP-10 and IL-10. This effect is similar to that obtained with Restasis® and higher in the case of RANTES and IP-10. RANTES level increases in the tears of dry eye disease patients and correlates with various tear film and ocular surface parameters.

Overall these results show that PI 44 is potentially useful in the treatment of dry eye disease.

Claims

WHAT IS CLAIMED IS:

1. A method for the prevention and/or treatment of dry eye disease which comprises topically administering to a patient in need thereof a pharmaceutical composition comprising a peptide selected from the group consisting of peptide PI 44 (SEQ ID NO: 1), peptide P17 (SEQ ID NO: 2), a fragment of said peptide P17 having the capacity to inhibit the biological activity of TGF-βΙ and comprising between 9 and 14 consecutive amino acids, and any combination thereof, and their pharmaceutically acceptable salts.

2. Method according to claim 1, wherein said peptide is selected from the group consisting of peptides whose amino acid sequence is shown in SEQ ID NO: 1-29 and any combination thereof.

3. Method according to claim 1, wherein said peptide is selected from the group consisting of peptides P144 (SEQ ID NO: 1), P17 (SEQ ID NO: 2), P17(l-l l) (SEQ ID NO: 16), P17(l-12) (SEQ ID NO: 21), P17(l-14) (SEQ ID NO: 28), and any combination thereof.

4. A peptide selected from the group consisting of peptide PI 44 (SEQ ID

NO: 1), peptide P17 (SEQ ID NO: 2), a fragment of said peptide P17 having the capacity to inhibit the biological activity of TGF-βΙ and comprising between 9 and 14 consecutive amino acids, and any combination thereof, and their pharmaceutically acceptable salts, for use in the prevention and/or treatment of eye dry disease.

5. Peptide, or combination of peptides, for use according to claim 4, wherein said peptide, or combination of peptides, is selected from the group consisting of peptides whose amino acid sequence is shown in SEQ ID NO: 1-29 and any combination thereof.

6. Peptide for use according to claim 4, wherein said peptide is selected from the group consisting of peptides PI 44 (SEQ ID NO: 1), P17 (SEQ ID NO: 2), P17(l-l l) (SEQ ID NO: 16), P17Q-12) (SEQ ID NO: 21), P17(l-14) (SEQ ID NO: 28), and any combination thereof.

7. Use of a peptide selected from the group consisting of peptide P144 (SEQ ID NO: 1), peptide P17 (SEQ ID NO: 2), a fragment of said peptide P17 having the capacity to inhibit the biological activity of TGF-β Ι and comprising between 9 and 14 consecutive amino acids, and any combination thereof, and their pharmaceutically acceptable salts, in the manufacture of a pharmaceutical composition for the prevention and/or treatment of eye dry disease.