Calreticulin peptidomimetic inhibitors and prodrugs

Abstract

The invention relates to peptidomimetic inhibitors of calreticulin, to their prodrugs and to pharmaceutical compositions comprising them. Specifically provided are cyclic peptidomimetic s and their prodrugs having a lipophilic charge mask, thus metabolically stable and highly bioavailable. Methods for treatment of autoimmune diseases and other conditions associated with calreticulin are also provided.

Description

[0001] CALRETICULIN PEPTIDOMIMETIC INHIBITORS AND PRODRUGS

[0002] FIELD OF THE INVENTION

[0003] The present invention is in the field of medicinal chemistry and provides pep tidomime tics and prodrugs thereof for the treatment of autoimmune diseases.

[0004] BACKGROUND OF THE INVENTION

[0005] Uveitis encompasses a group of potentially blinding inflammatory eye diseases which, in the majority of cases, are idiopathic. The eye has a special relationship with the immune system known as "ocular immune privilege". This term is used to describe certain sites in the body which are able to tolerate the introduction of antigens without eliciting an inflammatory immune response. Ocular immune privilege protects vital structures of the eye from the potentially damaging effects of an inflammatory immune response.

[0006] T lymphocytes undergo a process of "education" in the thymus in order to ignore antigens that compose our own tissue, among them retinal antigens such as, interphotoreceptor retinoid binding protein (IRBP). However, thymus education is not fully efficient, and self-reactive cells that should have been eliminated exit from the thymus to the periphery. A damaged ocular immune privilege may lead to uveitis by impeding establishment of peripheral tolerance to retinal antigens. When T lymphocytes from peripheral blood respond to antigenic proteins expressed in the retina, the responses play a central role in the pathogenesis of autoimmune uveitis (R. Caspi, “Understanding autoimmune uveitis through animal models: The friedenwald lecture,” Investig. Ophthalmol. Vis. Sci., vol. 52, no. 3, pp. 1873-1879, 2011).

[0007] The clinical severity of uveitis depends on whether the anterior, intermediate, or posterior part of the uvea is involved and may range from nearly asymptomatic to rapidly sight- threatening forms. Race, genetic background and environmental factors can influence the clinical picture. Uveitis can lead to blindness and affects all age groups. However, the highest incidence is in the age group of 20 to 50. It is estimated that uveitis causes 10% to 15% of all cases of total blindness in the USA (A. Rothova, “The possible impact of uveitis in blindness: a literature survey,” Br J Ophthalmol, vol. 400, pp. 844-848, 1996). Pediatric uveitis affects around 10% of children with juvenile idiopathic arthritis, with a strong prevalence among girls (80%) (T. elik, “Pediatric uveitis,” Guncel Pediatr., vol. 16, no. 2, pp. 230-246, 2018). Therefore, this disease raises severe individual and socio-economic issues.

[0008] Uveitis carries the risk of being sight- threatening: in posterior uveitis, irreversible retinal and optic disc damage may occur resulting in severe visual morbidity, this is encountered in conditions such as Behget disease, sarcoidosis and lupus (Tugal-Tutkun et al., “Uveitis in Behget disease: An analysis of 880 patients,” Am. J. Ophthalmol., vol. 138, no. 3, pp. 373-380, 2004, and A. Matsou and K. T. Tsaousis, “Management of chronic ocular sarcoidosis: Challenges and solutions,” Clin. Ophthalmol., vol. 12, pp. 519-532, 2018). Chronic anterior uveitis in children may as well lead to severe visual morbidity due to the asymptomatic smoldering nature of the disease and the associated complications such as cataract, glaucoma, band keratopathy and maculopathy (J. Choi et al., “An update on the modem management of paediatric uveitis,” Br J Ophthalmol. 2019 Dec;103(12):1685-1689.

[0009] Experimental Autoimmune Uveoretinitis (EAU) is a robust mouse model for human uveitis. EAU is an eye-specific, T-cell-mediated disease that is characterized by inflammation and subsequent destruction of the neural retina and adjacent tissues mimicking the human uveitis pathology. The process of lymphocytes gaining access into the posterior segment of the eye in the experimental models of autoimmune uveitis is governed by the activation of inflammatory hematopoietic cells including T lymphocytes. Recruitment of these cells is a fundamental step in the development of the disease. In general, lymphocyte extravasation from the blood vessels is governed by a variety of factors, such as the inflammatory state of the lymphocyte and the repertoire of pro-inflammatory cytokines released. Lymphocyte trafficking into the retina, however, is controlled also by calreticulin (CRT), a lymphocyte calcium-regulating protein which is overexpressed in inflammatory conditions.

[0010] Calreticulin (CRT) is a highly conserved calcium-binding protein comprising a C- terminal endoplasmic reticulum (ER) tetrapeptide target sequence KDEL (SEQ ID NO: 3), and lectin-like properties. CRT is considered to be an endoplasmic reticulum (ER) chaperone involved in the assembly and folding of nascent glycoproteins in the hematopoietic cells. Interaction between CRT and the endothelium stimulates the release of nitric oxide and inhibits clot formation. In many autoimmune diseases, CRT undergoes cleavage of the C- terminal tetrapeptide, followed by migration of CRT from the ER to the cell surface (CS- CRT) which regulates lymphocyte inflammatory activity. CRT is expressed on the cell surface of activated human peripheral blood T lymphocytes (e.g., CD8+ and CD4+) where it is physically associated with a pool of unfolded MHC class I molecules (K.-H. Krause and M. Michalak, Calreticulin Meeting Review, Cell, vol. 88, pp. 439-443, 1997; and F. A. Arosa et al., Calreticulin Is Expressed on the Cell Surface of Activated Human Peripheral Blood T Lymphocytes in Association with Major Histocompatibility Complex Class I Molecules, vol. 274, no. 24, pp. 16917-16922, 1999). CS-CRT undergoes interaction with various mutated proteins on the surface of immune cells (e.g., HLA-DRB 1). This also contributes to inflammation associated with autoimmune diseases.

[0011] Calreticulin has been identified in sera from patients suffering from several autoimmune diseases including systemic lupus erythematosus (SLE), autoimmune myelofibrosis, celiac disease, thyroid autoimmunity disease, Graves' disease, Hashimoto's disease, systemic sclerosis, spondyloarthritis, Sjogren's syndrome, dermatitis herpetiformis, rheumatic disease and various parasitic diseases, which implies a pathological role of calreticulin in these diseases (Eggleton P, Llewellyn DH. Pathophysiological roles of calreticulin in autoimmune disease. Scand J. Immunol. 1999; 49(5):466-73). Calreticulin was also recently suggested as a therapeutic target in erosive arthritis.

[0012] CRT is involved in several pathological pathways. CRT is a major Ca2+binding (storage) protein in the cytoplasm, lumen of the endoplasmic reticulum (ER) and the nuclear envelope of peripheral lymphocytes, regulating T cell immune responses. CRT-dependent Ca2+signaling critically contributes to the inflammatory properties of the T lymphocytes. In the nucleus of lymphocyte, CRT changes gene transcription by binding and modulating the DNA-binding domain of the superfamily of the glucocorticoid receptor and preventing the receptor from binding to its specific promoter -glucocorticoid response element (GRE). CRT enhances leukocyte cell adhesion and migration and infiltration to an inflamed tissue by binding activated integrin a subunits (ITGAs). The recruitment of lymphocytes to areas of inflammation is a crucial process for the inflammatory pathogenesis. It is well known that the activated integrin a subunits (ITGAs) expressed on the surface of multiple lymphocyte types, become the key mediators of infiltration into the diseased tissue. CRT binds to the intracellular COOH-terminal region of ITGAs, which contains a highly conserved amino acid sequence., increasing leukocyte cell adhesion and migration (M. Ohkuro et al., Calreticulin and integrin alpha dissociation induces anti-inflammatory programming in animal models of inflammatory bowel disease, 2018).

[0013] While uveitis is a heterogeneous disease with polygenic and environmental influences, most forms of immune-inflammatory-mediated uveitis are thought to be due to an imbalance between regulatory mechanisms that inhibit the immune system and inflammatory mechanisms. The management of uveitis remains a challenge for clinicians, in particular because of the lack of specific eye targeted anti-inflammatory drugs. Despite the multitude of causes, corticosteroids are the first-line and the mainstay of therapy for patients with active uveitis. Therapeutics targeting common inflammatory pathways are also used. These treatments, including corticosteroids and immunomodulatory agents, although often effective, have many untoward side effects, limiting their utility. In many patients, the severity of the disease, the presence of corticosteroid side effects, or the requirement for high doses of systemic corticosteroids are highly likely to result in complications and significant adverse effects. Although, the TNF-a, IL-6 and IL-1 blockers have been also provided for treatment, a large armamentarium to treat uveitis, ongoing challenges, patients' severe intolerance and cases of recalcitrant uveitis remain, posing a challenge to physicians.

[0014] Peptides and polypeptides are hallmarks in a variety of physiological and pathological processes and play important roles in modulating various cell functions. However, as drugs, the pharmacokinetics and pharmacodynamics properties of peptides are challenging, especially when administered orally. Since peptides undergo rapid metabolism and are exposed to enzymatic digestion, they suffer from poor bioavailability and low membrane permeability. Thus, therapeutic peptides and polypeptides are limited to parenteral administration. However, parenteral administration does not entirely solve the permeability challenge since intestinal epithelial cells also constitute a physical barrier to peptides with low membrane permeability.

[0015] Previous publications have disclosed the cyclic peptide denoted HS(4-4)c Trp bridged by a urea bond, having the structure:

[0016] HS(4-4)c Trp This peptide was found to have antagonistic effect to the shared epitope in the DRP- chain of the HLA-DRB 1 protein. The shared epitope is a signal transduction ligand that interacts with CS-CRT and accelerates osteoclast-mediated bone damage in collagen-induced arthritis (CIA). HS(4-4)c Trp inhibits CRT, thereby conferring an anti-inflammatory effect in picogram doses in mice with CIA (S. Ling, Y. Liu, J. Fu, A. Colletta, C. Gilon, and J. Holoshitz, “Shared Epitope-Antagonistic Ligands: A New Therapeutic Strategy in Mice With Erosive Arthritis,” Arthritis Rheumatol., vol. 67, no. 8, pp. 2061-2070, 2015; J. Fu et al., “A Small Shared Epitope - Mimetic Compound Potently Accelerates Osteoclast-Mediated Bone Damage in Autoimmune Arthritis,” 2016).

[0017] WO 2019 / 058367 discloses processes for the preparation of peptide-based prodrug. Reducing the net charge of a parent peptide by masking groups provides more lipophilic prodrugs with enhanced bioavailability.

[0018] WO 2019 / 058365 discloses methods for improving passive membrane permeability of somatostatin analogs by providing their prodrugs comprising masked ionizable (charged) functional groups.

[0019] WO 2019 / 058374 discloses A-methylated cyclic peptides having high metabolic stability and high affinity to integrin avP3. The peptides are suitable for oral administration when formulated as a prodrug.

[0020] There remains an unmet clinical need for novel and effective immunomodulatory agents for treatment of uveitis and other autoimmune diseases. These agents should be bioavailable, safe, with less adverse effects and thus with higher compliance potential.

[0021] SUMMARY OF THE INVENTION

[0022] The present invention provides peptidomimetics capable of inhibiting or blocking the activity of the protein calreticulin (CRT) as well as prodrugs of these peptidomimetics, having improved permeability and stability. A particular cyclic peptidomimetic disclosed, clarstatin, is shown to modulate calreticulin related immune responses in experimental autoimmune uveoretinitis (EAU), an animal model for uveitis.

[0023] The prodrugs of the peptidomimetics of the present invention comprise, according to some embodiments, moieties that mask some or all charged groups of the peptide sequence, thereby increasing their hydrophobicity and permeability through biological membranes, in particular transcellular permeability. Advantageously, the increased stability and bioavailability of the peptidomimetics allows for oral administration, without losing activity or specificity.

[0024] The masking moieties of the present invention are for example hydrophobic or lipophilic moieties. According to some embodiments of the present invention, one or more of the masking moieties are connected to the termini and / or side chain of the peptidomimetic via a cleavable linkage that allows for selective removal and release of the active drug in circulation or in a target tissue. According to other embodiments, the linkage between one or more masking moieties and the peptidomimetic is uncleavable under physiologic conditions.

[0025] Clarstatin, an exemplary cyclic peptidomimetic of the present invention, containing a thiourea bridge, was found to be metabolically stable in sera, and safe, with no apparent cytotoxicity in an array of tested organs, predicting its safety in future therapeutic applications. Clarstatin inhibited the development of uveitis and its clinical effects on the retina of EAU model mice and exerted its therapeutic effect in a dose dependent manner. Clarstatin significantly reduced infiltration of CD45+leukocytes to the retina and showed a remarkable preservation of the outer retinal structure of EAU-induced mice. Similarly, clarstatin significantly reduced infiltration of CD4+T cells to the spleen of the EAU mice. In particular, clarstatin had a remarkable therapeutic and anti-inflammatory effect on the fundus of mice induced with experimental uveitis. Clinical observations showed that clarstatin exhibited an anti-inflammatory effect (e.g., reduced edema and pathological damage) correlating with the retinal architecture, and significantly decreased cytokines and chemokines expression levels.

[0026] The prodrugs of the peptidomimetics of the present invention have high permeability coefficient value, remarkably permeates through enterocyte monolayer indicating remarkable transcellular permeability. The prodrugs were therapeutically active (e.g., anti-inflammatory effects and reduced clinical signs of uveitis) following oral administration. Thus, indicating that the lipophilic masking groups protected it from peptidases metabolic degradation while enabling it to penetrate and exert its therapeutic effect in target tissues.

[0027] In one aspect of the present invention, a peptidomimetic according to Formula I is provided:

[0028] Formula I wherein Ri and R2 are each independently a side chain of an amino acid and R3 represents the carboxy terminus of the peptide, which may be a modified carboxy terminus. In one embodiment of the present invention, Ri and R2 are each hydrogen.

[0029] In another embodiment, Ri and R2 are each hydrogen and the peptidomimetic comprises the sequence Trp-*Gly-Asp-Lys-Ser-*Gly-Ala (WGDKSGA, SEQ ID NO: 1), wherein each *Gly residue is an Na-co-functionalized derivative residue (building unit, BU), used to connect the other building unit to form a bridge. According to some embodiments, the bridge is a thiourea bridge. In another embodiment, R3 is selected from an amidated, carboxylated, esterified or alcohol carboxy terminus. In another embodiment, Ri and R2 are each hydrogen and R3 is an amidated carboxy terminus.

[0030] In a specific embodiment, the peptidomimetic is a compound according to Formula II, herein denoted clarstatin:

[0031] Formula II. In another aspect, the present invention provides a prodrug comprising a peptidomimetic according to Formula I or Formula II.

[0032] In another embodiment the prodrug is represented by Formula III:

[0033] Formula III wherein R4 is independently at each position H or a masking moiety selected from the group consisting of hexyloxycarbonyl (Hoc), propyloxy carbonyl (Poc), methoxy ethyloxycarbonyl (MEoc), hydroxy ethyloxycarbonyl (HEoc), and (5-Methyl-2-oxo- l,3-dioxol-4-yl)methyloxycarbonyl (ODOL-oc);

[0034] Rs is H or a masking moiety selected from ethyl (Et) and methyl (Me); and

[0035] R3 represents the carboxy terminus of the peptide, which may be a modified carboxy terminus.

[0036] According to some embodiments, RHs identical in both positions.

[0037] In another embodiment, R4 is independently at each position Hoc or MEoc. In another embodiment, R5 is Et. In another embodiment, R3 is selected from an amidated, carboxylated, esterified or alcohol carboxy terminus. In another embodiment, R4 is independently at each position Hoc or MEoc and R3 is an amidated carboxy terminus. In another embodiment, each one of R4 is Hoc or MEoc, R5 is Et or H, and R3 is an amidated carboxy terminus. According to some embodiments, the peptidomimetic is according to Formula IV:

[0038] Formula IV wherein R4is independently at each position H or a masking moiety selected from the group consisting of hexyloxycarbonyl (Hoc), propyloxy carbonyl (Poc), methoxy ethyloxycarbonyl (MEoc), hydroxy ethyloxycarbonyl (HEoc), and (5-Methyl-2-oxo- l,3-dioxol-4-yl)methyloxycarbonyl (ODOL-oc); and wherein Rs is H or a masking moiety selected from ethyl (Et) and methyl (Me). Each option represents a separate embodiment of the present invention.

[0039] In another embodiment of the present invention, the prodrug is represented by Formula V:

[0040] Formula V (clarstatin-P(Hoc2). In yet another embodiment of the present invention, the prodrug is represented by Formula VI:

[0041] Formula VI (clarstatin-P(MEoc2)Et).

[0042] In another embodiment, the peptidomimetic is capable of inhibiting at least one activity of calreticulin (CRT).

[0043] In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one peptidomimetic or prodrug thereof described herein, and optionally, a pharmaceutically acceptable carrier, excipient or diluent. According to some embodiments, the pharmaceutical composition comprises a peptidomimetic according to Formula I or Formula II, or a prodrug according to Formula III, Formula IV, Formula V, or Formula VI.

[0044] According to some embodiments, the pharmaceutical composition is formulated for administration by a route selected from oral, intraocular, intravitreal, juxtascleral, subconjunctival, intracameral, retrobulbar, submucosal, intranasal, topical, intravenous, intramuscular, intraperitoneal, subcutaneous, intra-dermal, intra-arterial, intraarticular, and intralesional. Each option represents a separate embodiment of the invention.

[0045] According to some embodiments, the pharmaceutical composition is formulated for administration by a route selected from oral, ophthalmic, submucosal, intranasal, and topical. Each option represents a separate embodiment of the invention.

[0046] In a more specific embodiment, the pharmaceutical composition is formulated for oral administration. In another embodiment, the pharmaceutical composition is formulated for topical administration. In yet another specific embodiment, the pharmaceutical composition is formulated for ophthalmic administration. According to some embodiments, the ophthalmic administration is by a route selected from topical, intraocular, intravitreal, juxtascleral, subconjunctival, intracameral, and retrobulbar. Each option represents a separate embodiment of the invention. According to some embodiments, the pharmaceutical composition is formulated for administration in a dosage form selected from eye drops, ointment, ophthalmic solution, suspension, gel, and contact lenses.

[0047] In another embodiment, the pharmaceutical composition is for use in inhibiting at least one activity of calreticulin (CRT). In another embodiment, the pharmaceutical composition is for use in the treatment of a CRT-related disease, disorder or condition. In another embodiment, the CRT -related disease is an autoimmune disease. In another embodiment the autoimmune disease is selected from autoimmune uveitis, systemic lupus erythematosus, autoimmune myelofibrosis, celiac disease, thyroid autoimmunity disease, Graves' disease, Hashimoto's disease, systemic sclerosis, spondyloarthritis, Sjogren's syndrome, dermatitis herpetiformis, rheumatic disease and erosive arthritis. In another embodiment, the CRT- related disease is uveitis.

[0048] In another aspect, the present invention provides, a method of treating an autoimmune disease or condition, comprising administering to a subject in need thereof the pharmaceutical composition.

[0049] In some embodiments, the autoimmune disease is selected from a group consisting of autoimmune uveitis, systemic lupus erythematosus, autoimmune myelofibrosis, celiac disease, thyroid autoimmunity disease, Graves' disease, Hashimoto's disease, systemic sclerosis, spondyloarthritis, Sjogren's syndrome, dermatitis herpetiformis, rheumatic disease and erosive arthritis.

[0050] In another aspect, the present invention provides a method of treating uveitis, comprising administering to a subject in need the pharmaceutical composition.

[0051] In another embodiment, the pharmaceutical composition is administered via a route selected from oral, ophthalmic, submucosal, intranasal, topical, intravenous, intramuscular, intraperitoneal, subcutaneous, intra-dermal, intra-arterial, intraarticular, and intralesional. In another embodiment, the pharmaceutical composition is administered orally. Each option represents a separate embodiment of the invention. In another embodiment, the pharmaceutical composition administered topically. In yet another specific embodiment, treatment is by ophthalmic administration. According to some embodiments, the ophthalmic administration is selected from topical, intraocular, intravitreal, juxtascleral, subconjunctival, intracameral, and retrobulbar. Each option represents a separate embodiment of the invention. According to some embodiments, the treatment is in a dosage form selected from eye drops, ointment, ophthalmic solution, suspension, gel, and contact lenses.

[0052] Also included within the scope of the present invention are peptidomimetics and prodrugs thereof comprising a substitution, addition, or deletion of 1-3 amino acid residues in the above-sequence as long as the resulting peptidomimetic retains or even improves the ability of inhibiting human CRT.

[0053] BRIEF DESCRIPTION OF THE FIGURES

[0054] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0055] Fig. 1 presents the analytical HPLC analysis of clarstatin.

[0056] Fig. 2 presents the analytical HPLC analysis of the prodrug clarstatin-P(Hoc2).

[0057] Fig. 3 presents the mass spectrometry analysis of clarstatin.

[0058] Fig. 4 presents the spectrometry analysis of the prodrug clarstatin-P(Hoc2).

[0059] Fig. 5 presents the apparent permeability coefficient (Papp) apical to basolateral (A-B) of clarstatin in a Caco-2 model. Atenolol, the paracellular permeability marker and metoprolol, the transcellular permeability marker, were used as controls.

[0060] Figs. 6A-6C present calibration curves of the standard (control) molecules atenolol and metoprolol and the pro-drug clarstatin-P(Hoc2) in basolateral buffer containing 4% w / v BSA. The internal standard was metoprolol for clarstatin-P and propanolol for the Caco-2 model. Fig. 6A Atenolol as a paracellular permeability marker, Fig. 6B Metoprolol, as a transcellular permeability marker, and Fig. 6C the prodrug clarstatin-P(Hoc2). Fig. 7 presents the intestinal permeability of the prodrug clarstatin-P(Hoc2) evaluated in the Caco-2 model. Atenolol, the paracellular permeability marker and Metoprolol, the transcellular permeability marker, were used as controls.

[0061] Fig. 8 presents the intestinal permeability of the prodrug clarstatin-P(MEoc2)Et evaluated in the Caco-2 model, basolateral to apical permeability (BA) in gray and apical to basolateral (AB) in black. Atenolol, and metoprolol were used as controls. Final concentrations were 10 pg / ml for clarstatin-P(MEoc2)Et atenolol and metoprolol.

[0062] Fig. 9 presents clarstatin stability evaluation by HPLC-MS as area under the curve / area under the curve of the internal standard (IS) in rat sera over time.

[0063] Fig. 10 presents an HPLC-based calibration curve using different concentrations of clarstatin (ng / mL) in the mobile phase.

[0064] Fig. 11 presents the pharmacokinetic profile of clarstatin, evaluated by HPLC, of a plasma sample taken from each of 4 tested rats following an intravenous dose of 1 mg / kg of the peptidomimetic.

[0065] Fig. 12A presents the profile of clarstatin concentration in rat plasma vs. time following intravenous bolus administration of 1 mg / kg of clarstatin (n = 4 animals, mean).

[0066] Fig. 12B, presents a semi-logarithmic plot of clarstatin concentration in rat plasma vs. time following intravenous bolus administration of 1 mg / kg of clarstatin.

[0067] Fig. 13 presents sections of ten different organs of one of the clarstatin-treated mice (injected IV, n=5), stained with Hematoxylin & Eosin (H&E). A naive mouse which did not receive clarstatin was used as a control.

[0068] Figs. 14A-14C represent H&E-stained histopathological sections of the right eye of female C57BL / 6 mice 36 days following immunization with interphotoreceptor retinoid binding protein (IRBP1-20), Freund’s complete adjuvant (CFA), purified Bordetella pertussis toxin (PTX) and Mycobacterium tuberculosis H37RA (herein after H37RA), to induce Experimental Autoimmune Uveoretinitis (EAU model). Fig. 14A - a non-immunized and non-treated female C57BL / 6 mouse, wild type, used as a control for a healthy retinal structure (WT). Fig. 14B -a female EAU mouse treated with phosphate buffer saline (PBS) twice a week for a total of 5 treatments showing active uveitis with vitreous cells (black arrow) and several foci of retinal infiltrates and vasculitis (white arrows). Fig. 14C - EAU-induced female mouse that was treated with clarstatin (3.6pg / kg mice, 50 pl, IP , twice per week for a total of 5 treatments). Figs. 15 presents the therapeutic effect of clarstatin as quantified by an EAU score, on a scale from 0 to 4 according to the extent of inflammation and tissue damage of the histopathological sections of female mice. Healthy - non-immunized C57BL / 6 mice; Untreated - EAU-induced mice; EAU+clarstatin - EAU mice treated with clarstatin as described for Fig. 14C. Each point represents one mouse, (p<0.05).

[0069] Figs. 16A-16C depict immunofluorescence images of a retina of an EAU mouse stained with anti-mCD45 in red and anti-calreticulin in green, 36 days following immunization with IRBP1-20, CFA, PTX and H37RA. Contrast staining of the cell nucleus by DAPI, is marked in blue. Fig. 16A, a non-immunized and untreated C57BL / 6 mouse (healthy) control. Fig. 16B, an EAU mouse, treated with PBS twice a week for a total of 5 treatments. Fig. 16C, an EAU mouse treated with clarstatin (0.036 mg / kg, i.p.), twice per week for a total of 5 treatments. GCL- ganglion cell layer.

[0070] Fig. 17 shows a graphic presentation of the percentile of CD45+expressing cells in the retina of EAU mice (Untreated), and EAU mice treated with clarstatin (CL) and the wild type (healthy) control as described for Figs. 16A-16C *p < 0.05, p***<0.001.

[0071] Figs. 18A-18E depict exemplary photographs of retinal fundus taken by a Micron III camera, 4 weeks after induction of EAU in the different dose regimen test groups. Fig. 18A, Healthy, untreated, non-EAU induced mouse used as a control. Fig. 18B, EAU-induced mouse (untreated). Foci of retinal infiltrates are indicated by i-arrows. Fig. 18C, EAU mouse, treated with 180 pg / kg of clarstatin in a volume of 50 pl intraperitoneally once a week. The c-arrow points to perivascular cuffing, indicative of retinal vasculitis, Fig. 18D, EAU mouse, treated with 180 pg / kg of clarstatin in a volume of 50 pl intraperitoneally three times a week. Fig. 18E, EAU mouse treated with methylprednisolone sodium succinate salt, 20 mg / kg in a volume of 50 pl i.p. three times a week, as a positive control.

[0072] Fig. 19 presents the quantitation of the therapeutic effect of different dose regimen of clarstatin based on clinical EAU score, assigned on a scale from 0 to 4 according to the extent of inflammation and tissue damage as apparent on funduscopy. The test groups are as described for Figs. 18A-18E. CL - clarstatin.

[0073] Figs. 20A-20E depict the therapeutic effect of different dose regimen of clarstatin in H&E- stained histological exemplary retinal sections of mice from the different test groups: Fig. 20A: healthy, non-EAU induced mice. Fig. 20B: untreated EAU mice (Untreated), Fig. 20C:EAU mice treated with clarstatin once a week. Fig. 20D: EAU mice treated with clarstatin three times a week. Fig. 20E: mice treated with methylprednisolone three times a week. As shown on the images, L-lens; Vit-vitreous; R-retina; Rf-retinal fold; V-vasculitis; (Scale bars: 200 pm).

[0074] Fig. 21 depicts the corresponding EAU histological score evaluated from the histopathological sections (as detailed for Figs. 20A-20E) as a quantitative score for the therapeutic effect of dose regimen of clarstatin. Each point represents one mouse, (*p<0.05, ** p<0.01, *** p<0.001, ns (not significant) p > 0.05). CL - clarstatin.

[0075] Fig. 22 presents fluorescence-activated cell sorting (FACS) analysis for the percentile of CD4+expressing cells. The tested groups are: healthy, non-EAU induced mice (Healthy), untreated EAU mice (Untreated), EAU mice treated with clarstatin three times a week, EAU mice treated with clarstatin once a week, and mice treated with methylprednisolone three times a week (MP), respectively. Each point represents one mouse, (*p<0.05, ** p<0.01).

[0076] Fig. 23 presents FACS analysis for the percentile of macrophage common antigen F4 / 80+expressing cells. The tested groups are: healthy, non-EAU induced mice (Healthy), untreated EAU mice (Untreated), EAU mice treated with clarstatin three times a week, EAU mice treated with clarstatin once a week, and mice treated with methylprednisolone three times a week (MP), respectively. Each point present one mouse, (*p<0.05, ** p<0.01).

[0077] Figs. 24A-24F show photographic evaluation of dose dependent therapeutic effect of clarstatin on the fundus of mice, taken 18 days post EAU induction. Fig. 24A, WT, untreated, non-EAU induced mice used as a control. Fig. 24B, untreated EAU-induced mice (EAU). Fig. 24C, CLAR 3.6*1, EAU mice treated IP with 3.6 pg / kg of clarstatin once a week. Fig. 24D, CLAR 36*1, EAU mice treated IP with 36 pg / kg of clarstatin once a week. Fig. 24E, CLAR 360*1, EAU mice treated IP with 360 pg / kg of clarstatin once a week. Fig. 24F, PRED*3, EAU induced mice treated three times a week with 20 mg / kg methylprednisolone.

[0078] Figs. 25A-25F present photograph of the fundus taken 30 days post EAU induction of mice of each of the tested groups as described for Figs. 24A-24F: WT, EAU, CLAR 3.6*1, CLAR 36*1, CLAR 360*1, PRED*3, respectively.

[0079] Fig. 26 presents the EAU score of the different tested groups as quantified from H&E histopathological sections. The tested groups are as described for Figs. 24A-24F: WT, EAU, CLARSTATIN 3.6*1, CLARSTATIN 36*1, CLARSTATIN 360*1, PRED*3. p* < 0.05, **p < 0.01, *** p < 0.001, ns (not significant) p > 0.05. Each point represents one mouse. Fig. 27 presents the quantitation of the therapeutic effect of clarstatin and its prodrug clarstatin-P(MEoc2)Et based on EAU score from histopathological sections of the right eyes of the tested mice. Groups are as follows: mice treated with clarstatin (clarstatin IP 0.18 mg / kg), mice treated with clarstatin-P(MEoc2)Et (clarstatin-P(MEoc2)Et oral 1.8 mg / kg), positive control mice treated with methylprednisolone (PRED oral (20 mg / kg), untreated EAU mice (untreated) and wild-type mice, untreated mice, which were not induced with EAU (WT). Each point represents one mouse (*p<0.05, ** p<0.01).

[0080] Fig. 28 presents IL-6 expression levels (pg / mg), measured by Enzyme-Linked Immunosorbent Assay (ELISA), in the different test groups as described for Fig. 27. Each point represents one mouse (*p<0.05, ns - not statistically significant).

[0081] Fig. 29 presents monocyte chemoattractant protein- 1 (MCP-1) expression levels (pg / mg), measured by ELISA, in the different test groups as described for Fig. 27. Each point represents one mouse (*p<0.05, ns - not Statistically Significant).

[0082] Figs. 30A-B present graphs indicating that Ca2+signaling was reduced in Jurkat cells upon clarstatin treatment. Figs. 30A is a graph indicating changes with time of Ca2+signaling in Jurkat cells in response to LPS and Fig. 30B is a graph indicating changes with time of Ca2+signaling in Jurkat cells in response to clarstatin (Fig. 30B).

[0083] Fig. 31 presents necrotic cell death measured by lactate dehydrogenase release in Jurkat cells exposed for 48 h to different clarstatin concentrations with H2O2 treatment used as a positive cytotoxic control. Values expressed as mean ± SEM (n = 18). p < 0.05 for H2O2 vs. control are percent of LDH released in the medium out of the total culture LDH.

[0084] Figs. 32A-B present clarstatin ameliorated ocular pathology and reduced retinal infiltration of CD45+ leukocytes in a EAU B10RIII severe mice model. The tested groups consisted of untreated, naive mice (healthy), severe EAU-diseased untreated mice (control), or clarstatin- treated mice (CL; 0.36 mg / kg and 1 mg / kg, i.p.) or methylprednisolone-treated mice (MP, 20 mg / kg) three times a week. Fig. 32A shows quantitation of the histopathological scores in individual mouse eye sections, 14 days after EAU induction indicating the therapeutic effect of clarstatin (CL). Each data point represents an eye and the mean score per treatment group is indicated with error bars computed as SEM from three independent experiments; *p < 0.05, **p < 0.01, *** p < 0.001, ns (not significant) p > 0.05. Fig. 32B shows quantitation of the therapeutic effect of clarstatin based on FACS analysis for the percentage of CD45+ expressing cells on the retina. Results are shown as scatter graphs of the percentage of cells per sample indicated by the symbol, mean of all samples indicated by the bar and error bars indicate SEM from three independent experiments *p < 0.05, **p < 0.01, ***p < 0.001, ns = p > 0.05.

[0085] DETAILED DESCRIPTION OF THE INVENTION

[0086] The present invention provides, according to some embodiments, novel pep tidomime tics and prodrugs thereof, for the treatment of calreticulin-related diseases, e.g., autoimmune diseases. In one embodiment the autoimmune disease is uveitis.

[0087] Advantageously, the peptidomimetics and prodrugs of the present invention induce immunomodulation that is similar to the action of corticosteroids but without the long-term side effects caused by corticosteroid treatment. Importantly, the peptidomimetic prodrugs of the present invention are highly efficacious when administered orally, thereby allowing for improved patient adherence and ultimately, improved treatment outcomes.

[0088] Without wishing to be bound by theory or a mechanism of action, it is proposed that the peptidomimetics and prodrugs of the present invention target the three pathological pathways of calreticulin by inhibiting it: (i) preventing lymphocyte calcium oscillation, thus, providing an immunosuppressive effect, (ii) inhibiting CRT-induced change in gene expression; and (iii) inhibiting CRT binding to the cytosolic tail of ITGAs thus, providing an anti-inflammatory effect.

[0089] The terms “calreticulin” and “CRT” refer to the mammalian protein, also termed CRP55, Calregulin, Endoplasmic reticulum resident protein 60 (ERp60), HACBP and grp60. CRT according to the present invention, encompasses any mammalian variant of the protein. According to particular embodiments, CRT protein is from human, encoded by the gene CALR (also denoted CRTC), and identified, e.g., by UniProt accession number P27797.

[0090] The CRT inhibitors of the present invention were developed using the cycloscan method (O. Ovadia, S. Greenberg, B. Laufer, C. Gilon, A. Hoffman, and H. Kessler, Improvement of drug-like properties of peptides: the somatostatin paradigm, Expert Opin. Drug Discov., vol. 5, no. 7, pp. 655-671, 2010). Briefly, this method is based on the selection of backbone cyclic peptide(s) from rationally designed combinatorial library with conformational diversity. Subsequently, a library of clarstatin prodrugs (clarstatin-P) molecules was synthesized. A Lipophilic Prodrug Charge Masking approach (Schumacher- Klinger, A., et al., 2018) was used to design the prodrugs with enhanced oral bioavailability. In this approach, charged side-chains (e.g. Asp, Glu, carboxy terminus, Lys, Arg, and / or amino terminus) are masked by lipophilic pro-moieties with a range of lipophilicities, forming esters or carbamates to generate a prodrug. The prodrug, following intestinal permeation and absorption, is optionally hydrolyzed by esterases to generate the parent drug.

[0091] The term “prodrug” as used herein, may be an active form of the active agent, or an inactive or relatively less active form that becomes the active form through one or more metabolic processes in a subject. Accordingly, a “prodrug” may be a precursor substance that is chemically or biochemically metabolized after administration, or can function as an effective drug. Specifically, the prodrugs of the present invention are modified compounds containing clarstatin, that, in some embodiments may undergo an in vivo biotransformation through chemical or enzymatic cleavage that releases clarstatin. Thus, clarstatin is delivered to its target, thereby overcoming pharmacokinetic, pharmacodynamic and toxicology challenges without permanently altering the pharmacological properties of the parental drug (J. M. Clancy, J. F. Gilmer, and U. N. De Lisboa, Prodrugs for Amines, Molecules, vol. 13, pp. 519-547, 2008).

[0092] The present invention provides, according to some embodiments, CRT inhibitors that are cyclic pep tidomime tics comprising the general sequence Trp-*X-Asp-Lys-Ser-*X-Ala (WXDKSXA, SEQ ID NO: 2), wherein each X is an Na-co-functionalized derivative residue (building unit, BU), used to connect the other building unit X to form a bridge, preferably a thiourea bridge. According to some embodiments, the sequence is Trp-*Gly-Asp-Lys-Ser- *Gly-Ala (WGDKSGA, SEQ ID NO: 1), wherein each glycine (Gly) residue is an Na-co- functionalized derivative residue (Gly BU). According to further embodiments, the two Gly BUs are covalently connected by a thiourea bond.

[0093] The length of the bridge, defined by the type and number of the connecting groups, (typically methylene groups), can vary. According to some embodiments, each BU comprises 1-6 methylene groups. According to some embodiments, each BU comprises 4 methylene groups, thus, in some places herein, the cyclic peptidomimetic is denoted (c4-4). According to further embodiments, each Gly BU comprises 4 methylene groups.

[0094] The term “peptidomimetic” as used herein refers to a compound comprising at least one unnatural residue, bridge or bond, that mimics a natural peptide or protein in 3D space and which has the ability to interact with the biological target and produces the same biological effect as the corresponding natural peptide or protein. According to some embodiments, the peptidomimetic is a cyclic peptidomimetic.

[0095] The term "cyclic peptide" or “cyclic peptidomimetic” as used herein refers to a peptide having an intramolecular bond between two amino acids of the peptide. The cyclization can be affected through a covalent or non-covalent bond, or bridge. Intramolecular bridges include, but are not limited to, backbone to backbone bridge, side-chain to backbone bridge and side-chain to side-chain bridge. The terms "cyclic peptide-prodrug" and "cyclic peptidomimetic prodrug" are interchangeable and refer to a prodrug variation of a peptide or peptidomimetic as termed herein. According to some embodiments of the present invention, the peptidomimetic has a thiourea bridge.

[0096] A "building unit" (BU) indicates a N“-co-functionalized derivative of amino acids. Use of such building units permits different length and type of linkers and different types of moieties to be attached to the scaffold. This enables flexible design and easiness of production using conventional and modified solid-phase peptide synthesis methods known in the art.

[0097] A building unit (BU) according to some embodiments of the invention designates a N“- co-functionalized amino acid residue (N-BU) of the formula:

[0098] Formula VII

[0099] According to some embodiments, the peptide sequence is cyclized by covalently connecting one N ’-a-functionalizcd derivative of amino acid residue added to the sequence, or substituted an amino acid residue in the sequence, with another N ’-a-functionalizcd derivative of amino acid residue in the sequence.

[0100] Any covalent bond may be used to connect the anchoring positions of the peptide sequence using backbone cyclization. According to some embodiments, the building units are connected by a bond selected from the group consisting of: thiourea bond, urea bond, amide bond, disulfide bond and guanidino group, namely the cyclization bridge is selected from the group consisting of: thiourea bridge, urea bridge, and guanidino bridge. According to some particular embodiments, the bond used for cyclization is a thiourea bond. According to some embodiments, the backbone cyclic peptide comprises at least one modified terminal, including but not limited to an amidated C-terminus and an acylated N- terminus.

[0101] According to some embodiments, there is provided a peptidomimetic or prodrug as disclosed herein comprising a substitution, addition or deletion of 1 or 2 amino acid residues, wherein the resulting peptidomimetic retains or improves the ability of inhibiting human CRT. Each possibility represents a separate embodiment of the invention. According to some embodiments, there is provided a peptidomimetic or a prodrug as disclosed herein comprising a sequence of between 5 and 10 amino acids.

[0102] According to some embodiments, the peptidomimetic is according to Formula I:

[0103] Formula I wherein Ri and R2 are each independently an amino acid side chain, and R3 designates the peptide’s carboxy terminus (C-terminus), which may be modified.

[0104] As used herein, the term “an amino acid side chain” refers to a side chain of the twenty DNA-encoded amino acids as well as those of Selenocysteine and Pyrrolysine. In addition, the scope of the present invention includes naturally-occurring amino acids that are not incorporated into proteins, such as norvaline, ornithine, and homocysteine. Unnatural amino acid side chains also fall within the scope of the present invention.

[0105] According to some embodiments, the C-terminus is an amidated, carboxylated, esterified or alcohol C-terminus. Each option represents a separate embodiment of the present invention.

[0106] According to some embodiment, a prodrug of the peptidomimetic of Formula I is provided. According to some embodiments, the peptidomimetic is according to Formula II:

[0107] Formula II.

[0108] According to some embodiment, a prodrug of the peptidomimetic of Formula II is provided.

[0109] According to some embodiments, the prodrug is according to Formula III:

[0110] Formula III wherein R4 is independently at each position H or a masking moiety selected from Hexyloxycarbonyl (Hoc), propyloxycarbonyl (Poc), Methoxyethyloxycarbonyl (MEoc), Hydroxyethyloxycarbonyl (HEoc), and (5-Methyl-2-oxo-l,3-dioxol-4-yl)methyloxycarbonyl (ODOL-oc);

[0111] Rs is H or a masking moiety selected from ethyl (Et) and methyl (Me); and R3 designates the carboxy terminus of the peptide (C-terminus), which may be modified. Each option represents a separate embodiment of the present invention. The structures of the selected masking groups of R4 are as shown in Table 1:

[0112] Table 1.

[0113] According to some embodiments, the C-terminus is an amidated, carboxylated, esterified or alcohol C-terminus. Each option represents a separate embodiment of the present invention.

[0114] According to some embodiments, R3 designates an amidated carboxy terminus.

[0115] According to some embodiments, the peptidomimetic is according to Formula IV: Formula IV wherein R4 is independently at each position H or a masking moiety selected from Hexyloxycarbonyl (Hoc), propyloxycarbonyl (Poc), Methoxyethyloxycarbonyl (MEoc), Hydroxyethyloxycarbonyl (HEoc), and (5-Methyl-2-oxo-l,3-dioxol-4-yl)methyloxycarbonyl (ODOL-oc); and wherein R5 is H or a masking moiety selected from ethyl (Et) and methyl (Me). Each option represents a separate embodiment of the present invention.

[0116] According to some embodiments, Rus identical in both positions.

[0117] In another embodiment, R4 is independently at each position Hoc or MEoc. In another embodiment, R5 is Et or Me. In another embodiment, R5 is Et. In another embodiment, R3 is selected from an amidated, carboxylated, esterified or alcohol carboxy terminus. In yet another embodiment, R3 is an amidated carboxy terminus. In another embodiment, R4 is independently at each position Hoc or MEoc and R3 is an amidated carboxy terminus. In another embodiment, each one of R4 is Hoc or MEoc, R5 is Et or H, and R3 is an amidated carboxy terminus. In another embodiment, each one of R4 is Hoc, R5 is H, and R3 is an amidated carboxy terminus. In yet another embodiment, each one of R4 is MEoc, R5 is Et, and R3 is an amidated carboxy terminus.

[0118] According to some specific embodiments, the peptidomimetic prodrug is according to Formula IV, wherein R4 is Hoc and R5 is H; or R4 is Hoc and R5 is Et; or R4 is Poc and R5 is Et; or R4 is MEoc and R5 is Et; or R4 is MEoc and R5 is Et; or R4 is ODOL-oc and R5 is Et. Each option represents a separate embodiment of the present invention.

[0119] According to yet specific embodiments, the prodrug is clarstatin-P(Hoc2) according to Formula V :

[0120] Formula V. According to yet specific embodiments, the prodrug is clarstatin-P(MEoc2)Et according to Formula VI:

[0121] Formula VI.

[0122] Backbone-cyclized peptidomimetics

[0123] There have been tremendous advances in chemistry which have led to the development of very promising approaches aimed to develop peptide-based therapeutics, overcoming their natural low enzymatic stability and oral bioavailability while retaining, or even improving, their biological activity. Some of the chemical modifications utilized for preparation of such peptidomimetics are N-methylation and cyclization, which may be for example head-to-tail- cyclization, side chain to side chain, or to terminal cyclization, and backbone cyclization (BC).

[0124] Backbone cyclization (Gilon C, et al., Backbone cyclization: A new method for conferring conformational constraint on peptides. Biopolymers, 1991;31:745-50) is a general method by which conformational constraint is imposed on peptides. In backbone cyclization, a peptidomimetic is formed by covalently interconnecting atoms in the backbone (N and / or C) of a target linear peptide to form a ring.

[0125] In general, the procedures utilized to construct backbone cyclic molecules and their building units rely on the known principles of peptide synthesis and peptidomimetic synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis. Some of the methods used for producing backbone cyclized peptides and their building units are disclosed in US Patent Nos.: 5,811,392; 5,874,529; 5,883,293; 6,051,554; 6,117,974; 6,265,375, 6,355613, 6,407059, 6,512092 and international applications WO 95 / 33765; WO 97 / 09344; WO 98 / 04583; WO 99 / 31121; WO 99 / 65508; WO 00 / 02898; WO 00 / 65467 and WO 02 / 062819.

[0126] The advantage of backbone cyclization over other modes of peptide cyclization is that cyclization is achieved mainly using backbone atoms rather than side chains that are essential for biological activity. This method has been shown to dramatically enhance the metabolic stability of peptides in serum (Ovadia et al. The effect of backbone cyclization on PK / PD properties of bioactive peptide-peptoid hybrids: the melanocortin agonist paradigm. Bioorg. Med. Chem. 2010;18:580-9).

[0127] The method described overcomes the two major disadvantages of the standard cyclization strategies that result in loss of biological activity: (1) pharmacophoric side chains are often used for cyclization and / or these side chains must be replaced by residues that allow cyclization; and (2) restriction of the conformational space of linear peptide by cyclization leads to many inactive cyclic peptides because they are unable to attain the proper bioactive conformation. Using these methods, multitude of peptides and active regions of proteins were successfully converted into metabolically stable, highly active and specific peptidomimetics (e.g. WO 2014 / 130949). However, the intestinal absorption was still insufficient as these approaches did not affect the permeability rate of the backbone cyclic peptides.

[0128] Prodrugs

[0129] A prodrug is a poorly active or inactive modified form of an active, parental compound that undergoes in vivo biotransformation through chemical or enzymatic cleavage, enabling the delivery of the active compound in an effective manner and amount; thus overcoming pharmacokinetic, pharmacodynamic and toxicology challenges while maintaining the pharmacological properties of the parental drug. Prodrugs can be designed to serve several objectives. Carrier-linked prodrugs can be targeted to a specific site of action. For example, in cancer treatment studies, the parent drug is conjugated to a specific anti-tumor monoclonal antibody that targets certain cells. Additionally, a prodrug can be designed to target specific tissues by drug activation of enzymes that are unique or present at a higher concentration in those tissues. For example, glycosidase and azo reductase activity of the colonic microflora offers an opportunity to design a colon- specific drug delivery system.

[0130] Importantly, the prodrug approach can also be used to modify the physicochemical properties of peptide drugs to improve their passive membrane permeability, thereby improving the oral bioavailability of the peptide. Peptides are key players in a variety of physiological and pathological processes and play important roles in modulating various cell functions. Thus, the pharmaceutical industry has shown tremendous interest in exploiting these properties by developing peptide-based drugs. While parenterally-administered peptide- based drugs have been developed, peptide-based drugs for oral administration are challenging to develop due to the physico-chemical properties of peptides, such as high polarity, electrical charge, and high hydrogen bond potential, that limit their absorption through membranes. Indeed, orally-administered drugs are absorbed by intestinal epithelial cells that constitute about 80-90% of the cells in the absorptive surface of the intestinal track (K. Fosgerau and T. Hoffmann, Peptide therapeutics: current status and future directions, Drug Discov. Today, vol. 20, no. 1, pp. 122-8, 2015). As such, peptides are often too large and polar to pass this barrier and thus, cannot penetrate the intestine (B. C. Doak, et al., Oral druggable space beyond the rule of 5: Insights from drugs and clinical candidates, Chem. Biol., vol. 21, no. 9, pp. 1115— 1142, 2014.).

[0131] To improve the oral bioavailability of the peptidomimetics disclosed herein, a Lipophilic Prodrug Charge Masking (LPCM) method was implemented (WO 2019 / 058367). LPCM is directed to various synthetic processes for the preparation of prodrugs of peptides drugs. Prodrugs developed using the LPCM method are generally characterized by the following features: (a) reduction or omission of electrically charged atoms in the peptide skeleton, e.g., through charge masking of charged amino acid residues (e.g., Arg, Asp) and terminal amino and carboxylate moieties via a masking moiety; (b) improved lipophilicity provided through introduction of lipophilic groups; (c) optional lability in the presence of cellular enzymes, which transform the prodrugs into the charged biologically active peptide drugs; and (d) permeability through the enterocyte layer in the intestines. Following its arrival to the blood circulation, the bond connecting the masking moiety to the peptidomimetic is optionally cleaved, and the active peptide is released.

[0132] The term "peptidomimetic prodrug" refers to a prodrug variation of a peptidomimetic.

[0133] The term “masking moiety” as used herein refers to a chemical moiety used to modify the peptidomimetic to form the prodrug. According to some embodiments, the masking moiety reduces the net electric charge of the peptide. Non-limiting examples of masking moieties or masking moieties when coupled to the peptidomimetic include hexyloxycarbonyl (Hoc), ethyl ester (OEt), methyl ester (OMe), hydroxy ethyloxy carbonyl (HEoc), propyloxycarbonyl (Poc), (5-Methyl-2-oxo-l,3-dioxol-4-yl)methyloxycarbonyl (ODOL-oc), and methoxy ethyloxycarbonyl (MEoc). According to some embodiments, the masking moiety is selected from the group consisting of hexyloxycarbonyl (Hoc), ethyl (Et), methyl (Me) propyloxycarbonyl (Poc), (5-Methyl-2-oxo-l,3-dioxol-4-yl)methyloxycarbonyl (ODOL-oc), hydroxy ethyloxycarbonyl (HEoc), and methoxy ethyloxycarbonyl (MEoc). Each possibility represents a separate embodiment of the invention.

[0134] According to some embodiments, the present invention provides a prodrug according to Formula III, Formula IV, Formula V, or Formula VI. Each possibility represents a separate embodiment of the invention. According to some embodiments, the present invention provides a prodrug according to Formula III. According to some embodiments, the present invention provides a prodrug according to Formula IV. According to some embodiments, the present invention provides a prodrug according to Formula V. According to some embodiments, the present invention provides a prodrug according to Formula VI.

[0135] Amino acids

[0136] The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and either sequential, divergent or convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions.

[0137] Conservative substitution of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, affinity to the target protein, metabolic stability, penetration into the central nervous system or to immune privileged organ or structure, targeting to specific cell populations and the like. One of skill will recognize that individual substitutions, deletions or additions to peptide sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0138] The following six groups each contain amino acids that are conservative substitutions for one another:

[0139] 1) Alanine (A), Serine (S), Threonine (T);

[0140] 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q);

[0141] 4) Arginine (R), Lysine (K);

[0142] 5) Isoleucine (I), Leucine (L), methionine (M), Valine (V); and

[0143] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0144] Also included within the scope of the present invention are salts of the peptidomimetic and the prodrug, analogs, and chemical derivatives of the peptides of the invention. As used herein the term “salts” refers to both salts of carboxyl groups and to acid addition salts of amino or guanidino groups of the peptide molecule.

[0145] The peptides of the present invention may be produced by any method known in the art, including recombinant (when possible) and synthetic methods. Synthetic methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. Solid phase peptide synthesis (SPPS) procedures are well known to one skilled in the art and described, for example by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). In some embodiments, synthetic peptides are purified by preparative high-performance liquid chromatography (Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.).

[0146] Solid phase synthesis

[0147] Solid phase peptide synthesis procedures are well known in the art and further described in "Solid-Phase Synthesis: A Practical Guide" , Ed. Steven A. Kates and Fernando Albericio, CRC Press; 1st Edition (2000). A skilled artesian may synthesize any of the peptides of the present invention by using an automated peptide synthesizer using standard chemistry such as, for example, t-Boc or Fmoc chemistry. The methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, and classical solution synthesis.

[0148] Coupling of the amino acids in solid phase peptide chemistry can be achieved by means of a coupling agent such as but not limited to dicyclohexycarbodiimide (DCC), bis(2- oxo-3 “'-oxazolidinyl) phosphinic chloride (BOP-CI), benzotriazolyl-N-oxytrisdimethyl- aminophosphonium hexafluoro phosphate (BOP), 1 -oxo- 1 -chlorophospholane (Cpt-Cl), hydroxybenzotriazole (HOBT), or mixtures thereof.

[0149] The use of additional coupling reagents including, but not limited to: coupling reagents such as PyBOP (Benzotriazole-l-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), PyBrOP (Bromo-tris-pyrrolidino-phosphonium hexafluoro- phosphate), HBTU (2-(lH-Benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluoro- phosphate), TBTU (2-( IH-Benzotriazole- 1 -yl)- 1 , 1 ,3 ,3 -tetramethyluronium tetrafluoroborate), may be also utilized for synthesizing the peptide compounds of the present invention.

[0150] Additional coupling chemistries may be used, such as pre-formed urethane-protected N-carboxy anhydrides (UNCAs), pre-formed acyl halides, and most preferably, acyl chlorides.

[0151] Such coupling may take place at room temperature and also at elevated temperatures, in solvents such as toluene, DCM (dichloromethane), DMF (dimethylformamide), DMA (dimethylacetamide), NMP (N-methyl pyrrolidinone), dioxane, tetrahydrofuran, diglyme and 1,3 dichloropropane, or mixtures of the above.

[0152] Synthesized peptides can be purified by preparative high-performance liquid chromatography, and the compositions of which can be confirmed via amino acid sequencing.

[0153] The term "solid phase resin", "solid support resin" and "solid support" as used herein are interchangeable and intended to mean an insoluble polymeric matrix whereupon a molecule, e.g., a ligand in the form of a polypeptide, can be synthesized or coupled with or without a linker or spacer in-between, solid support resins are typically used in peptide synthesis. These polymers are generally employed in the form of beads. Polymer resins preferred for peptide synthesis are polystyrenes, polyacrylamides and the like, specifically copolymers of styrene and divinylbenzene. Prior to the coupling with the first amino acid, the solid support resin contains surface functionality or can be derivatized to contain surface functionality which can interact with an amine group of an amino acid (or peptide) so as to attach the amino acid (or peptide) to the support directly or indirectly through the amine group of the peptide. Solid phase resin, as used herein is not limited to the parent commercial derivatized resins, in their form prior the first coupling of amino acid or peptide. Rather, after the first coupling of amino acid and during the peptide synthesis, while the resin is coupled to a growing peptide, the resin is still considered a solid phase resin. According to some embodiments, the solid phase resin is coupled to at least one amino acid. According to some embodiments, the solid phase resin is not coupled to amino acids. Autoimmune diseases

[0154] Many autoimmune diseases are CRT-related (Eggleton, and Llewellyn, (1999), Pathophysiological Roles of Calreticulin in Autoimmune Disease. Scandinavian Journal of Immunology, 49: 466-473) and therefore are eligible for being treated with the peptidomimetic inhibitors of the present invention.

[0155] The term “calreticulin related disease” (CRT-related disease) as used herein refers to any disease, disorder or condition in which CRT is involved with the pathological initiation, severity or progression of the disease. The terms “calreticulin related disease”, “calreticulin related diseases” or “calreticulin related condition” are interchangeable.

[0156] According to some embodiments, the calreticulin related disease is an autoimmune disease. According to some embodiments, the calreticulin related disease is an autoimmune disease selected from the group consisting of uveitis, systemic lupus erythematosus, autoimmune myelofibrosis, celiac disease, thyroid autoimmunity disease, Graves' disease, Hashimoto's disease, systemic sclerosis, spondyloarthritis, Sjogren's syndrome, dermatitis herpetiformis, rheumatic disease and erosive arthritis. Each possibility represents a separate embodiment of the invention. According to some embodiments, the calreticulin related disease is uveitis.

[0157] Autoimmune diseases particularly eligible for treatment by the peptidomimetic s and prodrug of the present invention include but are not limited to: uveitis, systemic lupus erythematosus, autoimmune myelofibrosis, celiac disease, thyroid autoimmunity disease, Graves' disease, Hashimoto's disease, systemic sclerosis, spondyloarthritis, Sjogren's syndrome, dermatitis herpetiformis, rheumatic disease and erosive arthritis.

[0158] Pharmaceutical compositions

[0159] According to an aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient a peptidomimetic or a prodrug as disclosed above. According to some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

[0160] According to some embodiments, the composition is formulated for administration by a route selected from oral, intraocular, intravitreal, juxtascleral, subconjunctival, intracameral, retrobulbar, submucosal, intranasal, topical, intravenous, intramuscular, intraperitoneal, subcutaneous, intra-dermal, intra-arterial, intraarticular, and intralesional. According to some embodiments, the composition is formulated for administration by a route selected from oral, ophthalmic, submucosal, intranasal, and topical.

[0161] According to some embodiments, the pharmaceutical composition is formulated for ophthalmic administration. According to some embodiments, the pharmaceutical composition is formulated for ophthalmic administration by a route selected from topical, intraocular, intravitreal, juxtascleral, subconjunctival, intracameral, and retrobulbar. According to some embodiments, the composition is formulated for oral administration. According to some embodiments, the composition is formulated for topical administration. According to some embodiments, the composition is formulated for intraocular administration.

[0162] According to some embodiments, pharmaceutical composition is formulated in a dosage form appropriate for ophthalmic delivery of the peptidomimetics or prodrugs thereof of the present invention. Ocular dosage forms may be liquid, semi-solid, solid, or mixed. Liquid dosage forms include, but are not limited to, eye drops, suspensions, emulsions, and injectables.

[0163] According to some embodiments, the pharmaceutical composition is formulated for topical administration in a dosage form selected from eye drops, ointment, ophthalmic solution, suspension, gel, and contact lenses.

[0164] According to some embodiments, the dosage form is formulated for immediate, slow, delayed or sustained -release characteristics.

[0165] According to some embodiments, the pharmaceutical composition is formulated for oral administration as a liquid or solid dosage form for immediate, slow, delayed or sustained- release characteristics. According to some embodiments, the pharmaceutical composition, which comprises the prodrug as the active ingredient, is formulated for oral administration as a liquid or solid dosage form for immediate, slow, delayed or sustained-release characteristics.

[0166] In another aspect, the present invention provides a pharmaceutical composition for use in treating autoimmune diseases, the pharmaceutical composition comprises as an active ingredient the peptidomimetic or the prodrugs according to the invention.

[0167] In another aspect, the present invention provides a method of treating calreticulin- related condition or disease, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the pharmaceutical composition described hereinabove. In another aspect, the present invention provides a method of treating autoimmune disease, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the pharmaceutical composition described herein, thereby treating autoimmune disease in the subject. According to some embodiments, the autoimmune disease is selected from uveitis, systemic lupus erythematosus, autoimmune myelofibrosis, celiac disease, thyroid autoimmunity disease, Graves' disease, Hashimoto's disease, systemic sclerosis, spondyloarthritis, Sjogren's syndrome, dermatitis herpetiformis, rheumatic disease and erosive arthritis. Each possibility represents a separate embodiment of the invention.

[0168] According to some embodiments, the autoimmune disease is uveitis.

[0169] Any administration mode can be used for administering the pharmaceutical compositions of the present invention, including parenteral and enteral routes.

[0170] Any administration route suitable for delivery of peptides and their prodrugs may be used with the compositions and methods of the present invention and the compositions administered are formulated according to the specific administration mode.

[0171] According to some embodiments, the treatment is by a route selected from intravenous, intraocular, intravitreal, juxtascleral, subconjunctival, intracameral, retrobulbar, intraocular, intramuscular, intraperitoneal, subcutaneous, intra-dermal, intra-arterial, intraarticular, intralesional, submucosal, intranasal, oral, and topical. Each possibility represents a separate embodiment of the invention.

[0172] According to some embodiments, the pharmaceutical composition is administered orally.

[0173] According to some embodiments, the pharmaceutical composition is administered topically.

[0174] According to some embodiments, the pharmaceutical composition is administered intraocularly.

[0175] According to some embodiments, the peptidomimetic reduces CRT activity.

[0176] The pharmaceutical composition according to the present invention may be administered as a standalone treatment or in addition to a treatment with any other known treatments for the disease or disorder, e.g., for treatment of autoimmune disease.

[0177] The pharmaceutical compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0178] The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.

[0179] The compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the peptidomimetic or prodrugs according to the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0180] The amount of the peptidomimetic or prodrugs which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disease or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the severity of the disease or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances.

[0181] It will be apparent to those of ordinary skill in the art that the therapeutically effective amount of the peptidomimetic or prodrugs molecules according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered and the judgment of the treating physician. As used herein, a “therapeutically effective amount” refers to the amount of a molecule required to alleviate one or more symptoms associated with a disease or condition being treated over a period of time.

[0182] Although an appropriate dosage of a molecule of the invention varies depending on the administration route, type of molecule, age, body weight, sex, or conditions of the patient, it will be determined by the physician in the end. The dosage can be administered, for example, in daily, weekly, biweekly, monthly or bimonthly regimens. Various considerations in arriving at an effective amount are described, e.g., in Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990.

[0183] According to some embodiments, the peptidomimetic or prodrug is administered in combination with at least one immunosuppressive agent. The peptidomimetic or prodrugs and the immunosuppressive agent can be administered according to an overlapping schedule. According to some embodiments, the administering of the peptidomimetic or prodrugs and of the at least one immunosuppressive agent is carried out substantially simultaneously, concurrently, alternately, sequentially or successively.

[0184] The peptidomimetics and prodrugs of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.

[0185] Toxicity and therapeutic efficacy of the peptidomimetic and prodrugs described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the LD50 (lethal dose causing death in 50 % of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0186] The preferred doses for administration of such pharmaceutical compositions range from about 0.1 pg / kg to about 20 mg / kg body weight, of the active ingredient. Preferably, the amount of the active ingredient is in the range of from about 10 to 5000 pg / kg.

[0187] In some embodiments, the peptidomimetics or prodrugs of the invention are administered at a dose ranging from about 20 ng / kg to about 100 ng / kg of the subject weight. In other embodiments, the peptide of the invention is administered at a dose ranging from about 0.1 mg / kg to about 10 mg / kg of the subject weight. In yet other embodiments, the peptide of the invention is administered at a dose ranging from 0.1, 1, 10, 20, 30, 50, 100, 200, 400, 500, 700, 900 or 1000 ng / kg of the subject weight, to about 100, 200, 400, 500, 700, 900, 1000, 1200, 1400, 1700, or 2000 ng / kg of the subject weight. Each possibility represents a separate embodiment of the invention. In yet other embodiments, the peptide of the invention is administered at a dose ranging from about 0.01, 0.05, 0.1, 0.5, 0.7, 1, or 2 mg / kg of the subject weight, to about 0.05, 0.1, 0.5, 0.7, 1, 2, 5, 10, 15, 20, 50, 100, 250, or 500 mg / kg of the subject weight. Each possibility represents a separate embodiment of the invention.

[0188] All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

[0189] The terms “a,” “an,” and “the” are used herein interchangeably and mean one or more.

[0190] The term “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes, (A and B) and (A or B).

[0191] The term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination if the combination is not mutually exclusive.

[0192] The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)," are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated or listed. The term “consisting essentially of’ means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

[0193] As used herein, the term “about”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / - 10%, or + / - 5%, + / -1%, or even + / -0.1% from the specified value.

[0194] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0195] MATERIALS AND METHODS

[0196] Materials

[0197] 2-(lH-Benzotriazole-l-yl)-l,l,3,3-tetramethylaminium tetrafluoroborate (TBTU), N- [(lH-benzotriazol-l-yl)(dimethylamino)methylene]-N-methyl-methanaminium hexafluorophosphate N-oxide (HOBT); and 9-fluorenylmethyloxycarbonyl-Na-protected amino acids (Fmoc-Na-AA-OH) were purchased from Chem-Impex International Inc. (Wood Dale, IL, U.S.A.). Fmoc-Rink- Amide methylbenzhydrylamine (MB HA) resin (200-400 mesh, 0.66 mmol / g resin) was purchased from Iris Biotech GmbH (Marktredwitz, Germany).

[0198] 1,2-Diaminoethane, l,4diaminobutane, 1,6-diaminohexane, 2,4,6- trimethylpyridine (collidine), bis(trichloromethyl) carbonate (BTC), tetrakis (triphenylphosphine) palladium(O), diethyl ether, bromoacetic acid, acetic anhydride (AC2O), piperidine, trifluoroacetic acid (TFA), diisopropylethylamine (DIEA), methanol (MeOH), triethylamine (EtsN), triisopropylsilane (TIS), dibromomethane (DBM), dimethyl sulfoxide (DMSO), N-methyl morpholin, and other organic materials were purchased from ACROS ORGANICS N.V (Geel, Belgium).

[0199] Organic solvents for solid phase peptide synthesis (SPPS) and for high performance liquid chromatography (HPLC) including: N-Methyl-2-pyrrolidone (NMP), dichloromethane (DCM), N,N-dimethylformamide (DMF), and acetonitrile (ACN) were purchased from J. T. Baker (NJ, U.S.A.).

[0200] Human IRBP1-20 (GPTHLFQPSLVLDMAKVLLD (SEQ ID NO: 4)) and IRBPiei-iso (SGIPYIISYLHPGNTILHVD (SEQ ID NO: 5)), (purity >98%), were purchased from Adar Biotech Co. (Rehovot, Israel). Pertussis toxin (PTX) was received from List biological laboratories (Campbell, CA, USA). A heat-Mycobacterium tuberculosis strain H37Ra and complete Freund’s adjuvant (CFA) were obtained from BD (Bethesda, MD, USA). DMSO (D2650), protease inhibitor cocktail (P8340), and deoxyribonuclease I from bovine (D5025) were purchased from Sigma-Aldrich, (St. Louis MO, USA). Collagenase D (50-100-3282) was obtained from Roche Diagnostics (Mannheim, Germany). Methylprednisolone (Solu- medrol 125 mg / 2ml, Pfizer, New York, NY, USA) and eye drops (cyclopentolate hydrochloride 1%, oxybuprocaine hydrochloride 0.4%, tropicamide 0.5% and phenylephrine hydrochloride 2.5%) were purchased from the pharmacy of the Hadassah Medical Center (Jerusalem, Israel). HBSS (02-016-1A), DMEM (01-055-1A), fetal bovine serum FBS (04- 001-1 A), L-glutamine (03-020- IB), penicillin G sodium and streptomycin sulfate (03-031- 1B) were obtained from Biological Industries (Beit-Haemek, Afula, Israel). The monoclonal antibodies anti-mouse CD45 (FITC, 11-0451-82) and anti-mouse CD4 (APC-eFlour 780, 47- 0042-82), used for flow cytometric (FACS) analysis were sourced from eBioscience (San Diego, CA, USA). The anti-mouse calreticulin monoclonal antibody (ab22683), rabbit antimouse CD45 monoclonal antibody (ab208022), and the secondary polyclonal antibodies goat anti-mouse (Alexa Fluor 488, abl50117) and goat anti-rabbit (Alexa Fluor 594, abl50080) and mounting medium with DAPI (abl04139) used for immunofluorescence were sourced from Abeam (Cambridge, UK). The cytokines and chemokines were measured with the

[0201] Milliplex MAP Mouse Cytokine / Chemokine (MCYTOMAG-70K) ELISA kit purchased from Millipore Co. (Billerica, MA USA).

[0202] Methods

[0203] Acute toxicity in Mice Female ICR mice, (n = 5) were injected intravenously with 0.2 ml clarstatin at a maximal tolerated dosage of 10 mg / kg. Brain, liver, kidneys, spleen, heart, lungs, small intestine and large intestine, stomach and thymus from 5 mice were harvested after 48h and fixed in 4% formaldehyde. Next, the tissues were trimmed, put in embedding cassettes and processed routinely for paraffin embedding. Six cassettes were prepared per animal for the 8 organs. Paraffin sections (4 microns thick) were cut, put on glass slides and stained with Hematoxylin & Eosin (H&E) for general histology. Pictures were taken, using Olympus microscope and microscope's Camera (Olympus DP73, serial NO. OH05504) at objective magnification of X10 and X4. The H&E-stained slides were examined and scored by a pathologist, using a semi-quantitative grading scale, of 5-point scale, for the severity of the histopathological changes: Grade 0 - The tissue appears normal, without any changes at all; Grade 1 - Minimal pathological findings; Grade 2 - Mild pathological findings. Grade 3 - Moderate pathological findings. Grade 4 - Severe pathological findings. The histopathological evaluation included a comparison between treated and naive animals.

[0204] Animal model for human uveitis Experimental Autoimmune Uveoretinitis (EAU) is a robust mouse model for human uveitis. EAU is an organ (eye)-specific, T-cell-mediated disease that is characterized by inflammation and subsequent destruction of the neural retina and adjacent tissues mimicking the human uveitis pathology. EAU can be induced in rodents by immunization with one of several retinal peptide antigens (I. Gery, et al., Chapter 3 Retinal specific antigens and immunopathogenic processes they provoke, Prog. Retin. Res., vol. 5, no. C, pp. 75-109, 1986). It is usually induced by immunization of susceptible animals with a retinal antigen such as inter-photoreceptor retinoid binding protein (IRBP) and retinal arrestin (retinal soluble Ag, S-Ag) (R. K. Agarwal, Rodent models of experimental autoimmune uveitis, Methods Mol. Med., vol. 102, pp. 395-420, 2004). Both proteins are highly evolutionarily conserved and constitute major components of the photoreceptor cell layer. EAU resembles some human posterior uveoretinitis syndromes, including sympathetic ophthalmia, Vogt-Koyanagi-Harada disease (VKH), sarcoidosis, Behget’s disease, and birdshot retinochoroidopathy (J. Forrester, Uveitis: pathogenesis, Lancet, vol. 338, no. 8781, pp. 1498-1501, 1991). The pathologic and clinical courses of EAU in the mouse model closely resemble those in human uveitis of putative autoimmune and inflammatory etiology. The process of lymphocytes gaining access into the posterior segment of the eye in the experimental models of autoimmune uveitis is governed by the activation of inflammatory hematopoietic cells including T lymphocytes. Recruitment of these cells is a fundamental step in the development of the disease. The lymphocyte population that migrates to the eye is constituted by both retinal antigen- specific lymphocytes and nonspecific cells. In general, lymphocyte extravasation from the blood vessels is governed by a variety of factors, such as the inflammatory state of the lymphocyte and the repertoire of pro-inflammatory cytokines released. Lymphocyte trafficking into the retina, however, is controlled not only by these factors but also by calreticulin (CRT), a lymphocyte calcium-regulating protein which is overexpressed in inflammatory conditions. Because of the potential involvement of calreticulin in ocular inflammatory disorders, its blockade could be beneficial for the development of newer treatment modalities.

[0205] Induction of EAU Uveitis model: The therapeutic effect of clarstatin and clarstatin-P was evaluated in C57BL / 6 mice using an experimental autoimmune uveitis EAU mouse model. Female, C57BL / 6 mice were maintained in the Specific-Pathogen-Free (SPF) unit and all experiments were approved by the Hebrew University-Hadassah Institutional Animal Care and Use Committee. Mice were immunized subcutaneously (SC) with 500 pg IRBP (interphotoreceptor retinoid-binding protein, IRBP1-20, GPTHLFQPSLVLDMAKVLLD, (SEQ ID NO: 4)) (Adar Biotech, Rehovot, Israel) emulsified with an equal volume of Freund’s complete adjuvant (CFA, Sigma, St. Louis, MO, USA) in a total volume of 200 pl. The mixture contained also 2.5 mg / ml Mycobacterium tuberculosis H37RA (BD, MD, USA) (herein H37RA). An additional intraperitoneal injection of 1 pg of purified Bordetella pertussis toxin (PTX, List biological laboratories, Campbell, CA, USA) was also applied to each mouse. Control mice were immunized with the same volume of PBS instead of IRBP1-20 in CFA and PTX.

[0206] Induction of EAU in B 10RIII severe model: 41 B 10RIII female mice were immunized subcutaneously (s.c.) with 50 pg IRBPiei-iso, SGIPYIISYLHPGNTILHVD (SEQ ID NO: 5) emulsified with CFA, and 2.5 mg / ml Mycobacterium tuberculosis H37RA. An intraperitoneal (i.p.) injection of 1 pg of PTX was administrated to each mouse. Control mice received PBS. Mice were sacrificed on day 14 post-immunization. One eye was collected for histopathological analysis while the retina of the other eye was used for immunological characterization by FACS. The intraocular levels of the inflammatory mediators were measured by multiplex ELISA.

[0207] Clinical evaluations - The clinical appearance and progression of the disease was followed by fundus examination. Prior to clinical examination, mice were anesthetized systemically by IP of a mixture of ketamine (85 mg / kg) and xylazine (15mg / kg). Pupil dilation and local anesthesia of the eyes were performed using cyclopentolate hydrochloride 1%. In some cases, eye drops containing Oxybuprocaine hydrochloride 0.4%, Tropicamide 0.5% and phenylephrine hydrochloride 2.5% were used. Clinical score of ocular inflammation was evaluated using Micron-III small animal retina imaging system (Phoenix Research Laboratories, INC), on a scale of 0-4 based on the number, type, and size of lesions and the extent of inflammation. Pre-clinical evaluations- Mice were sacrificed at day 35-40 after primary immunization. Eyes and spleen were isolated. For histopathological analyses, eyes were collected immediately after exitus and prefixed for 24 hours in Davidson solution. Next, the fixed eyes were dehydrated in alcohol with concentration gradients and embedded in paraffin. Then, tissue sections (3-6 pm) were stained with hematoxylin and eosin. The intensity of uveitis was evaluated histologically and graded in a masked fashion. This grading system, detailed in Table 2, permits a semiquantitative assessment of the severity and extent of both infiltrative and structural / morphological changes of the uveitis at various points throughout the course of EAU.

[0208] Table 2. Masked fashion grading system of uveitis severity Isolation of retinal-infiltrating cells - mice eyes were enucleated and the retinas of each animal were dissected microscopically and washed. The retinas were cut up into small pieces and digested in 1 mL wash medium, supplemented with 0.5 mg / mL collagenase D. and 750 U / mL DNase I for 20 minutes at 37°C. After that, additional 0.5 mg collagenase D and 750 U DNase was added, and the specimens were incubated for a further 10 minutes at 37°C. Next, the cells suspensions are forced through a 40-pm cell strainer (BD Falcon, Bedford, MA) by using a syringe plunger and then stained for flow cytometry.

[0209] Isolation of spleen-infiltrating cells - to isolate immune cells from the surgically removed spleens, the spleens were mechanically mashed and suspended in PBS. Optionally the suspension was treated with collagenase and passed through a cotton wool column to filter out debris. The cells were analyzed by FACS or used for auto-antigen- specific proliferation or cytokine release assays.

[0210] Evaluation of intraocular levels of cytokines - intraocular levels of cytokines were measured by specific ELISA. Preparation of ocular extracts: left or right or both eyes were enucleated from C57BL / 6 mice on day 36. Each eye was incised at the equator and the intraocular contents were carefully scraped into a 0.5 ml Eppendorf tube containing 50 pl Ringer's Sol. (KREBS) with proteinase inhibitor mixture (Sigma, 1:100) on ice. Each sample was mechanically homogenized with 50 pl Ringer's Sol. with proteinase inhibitor mixture. The tissues were briefly sonicated using an ultrasonic disrupter for 30 one-second pulses. The soluble fraction was collected after high-speed 10,000 rpm centrifugation for 10 min at 4°C and stored at 80°C for cytokine assay by multiplex ELISA. Supernatants were used to assay for VEGF A, interferon-y (IFN- y), tumor necrosis factor-a (TNF-a), interleukin- la (IL-la), interleukin- lb (IL- lb) interleukin-6 (IL-6), interleukin- 10 (IL- 10), interleukin- 12 (IL- 12), interleukin- 17 (IL- 17) and MCP-1 (Monocyte chemotactic protein- 1) by sandwich ELISA immunoassay according to the manufacturer’s instructions.

[0211] Immunofluorescence staining - For deparaffinization, the paraffin sections were heated to 60°C for 15 min, then incubated in xylene at room temperature (RT) for 15 min. The sections were sequentially transferred into 100%, 95%, 70%, and 50% ethanol for 4 min each, at RT. Afterward, the sections were rinsed in deionized water and stored in PBS. Antigen retrieval was performed using a buffer containing 10 mM citrate (pH 6.2), 2 mM EDTA, and 0.05 % Tween-20. The samples were incubated overnight at 4°C with the goat anti-mouse calreticulin and rabbit anti-mouse CD45 monoclonal antibodies. After washing, the slides were treated for one hour, at RT, with goat anti-mouse (Alexa Fluor 488) (green) and goat anti-rabbit (Alexa Fluor 594) (red) secondary polyclonal antibodies at 1 / 1000 dilution. DAPI was used to stain the cell nuclei (blue). Imaging was conducted using a Zeiss LSM 710 confocal laser scanning system (Zeiss, Oberkochen, Germany) and Zeiss Axiovert 135M microscope, equipped with a Plan-Apochromat Zeiss 63X lens.

[0212] EXAMPLES

[0213] Example 1. Synthesis of clarstatin, clarstatin-P(Hoc2) and Clarstatin-P(MEoc2)Et

[0214] Clarstatin was synthesized using solid phase peptide synthesis (SPPS). Typically, reactions performed on solid support utilized fluorenylmethoxycarbonyl (Fmoc) protecting group for the Na protection. The reactions were shaken using Bigger Bill orbital shaker. Preactivation tubes were stirred over a vortex. Excluding the cleavage procedure, all other reactions on the solid support were performed under basic conditions of pH 8-9. The process of the reactions was monitored by High Performance Liquid Chromatography Mass Spectrometry (HPLC / MS) following a “small-scale cleavage” procedure, described hereinbelow.

[0215] The synthesis via solid support was performed in a vessel equipped with a sintered glass bottom. Fmoc Rink Amide methylbenzhydrylamine (Fmoc Rink Amide-MBHA) resin (loading capacity 0.66 mmol / g resin) was used as the solid support. The equivalents of all reagents used were calculated in respect to the resin loading capacity and weight. The volume of the solvents in all the reactions was fixed to 30 mL to maintain constant concentration of reagents. After cleavage from the resin, the crude final peptides were lyophilized, dissolved in triple-distilled water / Acetonitrile (TDW / ACN) 1:1 mixture, and analyzed by HPLC / MS.

[0216] Fmoc deprotection protocol: The Fmoc protected peptidyl-resin was treated with a solution of 20% piperidine in DMF for 15 minutes two times. The resin was washed three times with DMF.

[0217] Coupling protocol: A solution of Fmoc-Na-AA-OH (3 equiv.) and DIEA (6 equiv.) in DMF was prepared and cooled down to 0°C. TBTU (3 equiv.) was then added for preactivation of the amino acid prior to reaction with the peptidyl-resin, and the solution was shaken at 0°C for 3 minutes. The pre-activated solution was added to the peptidyl-resin and the mixture was shaken for 60 minutes. The procedure was repeated twice. The resin beads were washed with 30 mL DMF (3 x 2 min). Allyloxycarbonyl (Alloc) removal: For the removal of Alloc protecting group the peptidyl-resin was added to a saturated argon solution composed of acetic acid (5%), N- methyl morpholin (2.5%), and DCM (92.5%) and the mixture was shaken for 5 minutes, after which tetrakis(triphenylphosphine)palladium(0) Pd(PPh3)4 (0.1 equiv.) was added. The reaction mixture was shaken vigorously in the dark and under argon for three hours, after which the peptidyl-resin beads were washed with 30 mL solution of 0.5% v / v DIEA in Dimethylformamide (DMF) (5 times for 2 min), 0.5% sodium diethyldithiocarbamate trihydrate salt in DMF (5 times for 2 min), and DCM (5 times for 2 min).

[0218] Formation of isothiocyanate: The peptidyl-resin was suspended in DMF (30mL). Carbon disulfide (3 equivalent) was added, followed by HBTU (3 equivalent) and DIEA (3 equivalent). The peptidyl-resin was agitated at room temperature for 30 minutes. Afterward, the solvent was removed by suction, and the resin was washed with DCM (10 times), DMF (5 times), and then nitrogen was circulated through it for 10 minutes to eliminate any residual carbon disulfide.

[0219] Mtt deprotection: the Mtt protecting group on the second Gly building unit was performed by subjecting the resin to six 20-minute treatments with a mixture of DCM / TFE / AcOH (7 / 2 / 1), followed by washes with DMF (3 X 2 min), MeOH (3 X 2 min), and DCM (3 X 2 min), DMF (3 X 2 min).

[0220] Thiourea cyclization: DIEA (3 equivalent) was added to the isothiocyanate peptidyl- resin in DMF and the resin was shaken for 5 hours at room temperature. Following this, the solvent was removed by suction, and the resin was washed with DMF (5 times) and DCM (5 times).

[0221] Small-scale cleavage: A small amount of the peptidyl-resin (few visible beads) was treated with a pre-cooled mixture of TFA (2 mL), TDW (1 drop) and TIS (1 drop) for 30 minutes in an Eppendorf. The resin beads were removed by filtration. TFA was fully evaporated with a stream of nitrogen. The residual was dissolved in an ACN: TDW (1:1) and examined by HPLC / MS.

[0222] Cleavage protocol: a simultaneous removal of the synthesized peptides from the solid support together with the acid labile protecting groups on their side chains was performed by the following procedure: a 30 ml pre-cooled solution (at 0°C) composed of trifluoroacetic acid (TFA) (95%), TDW (2.5%), and triisopropylsilane (TIS) (2.5%) was added to a dried and desiccated 2.1 g peptide-resin beads. The reaction mixture was kept standing at 0°C for 30 minutes after which it was shaken for 150 minutes at room temperature. The TFA solution containing the cleaved peptide was then separated from the resin beads via filtration and the TFA was partially evaporated by a stream of nitrogen. Cold diethyl ether was added to the remaining volume of TFA, and the mixture was centrifuged to separate scavengers and other hydrophobic impurities from the precipitated peptide. Diethyl ether was then removed from the precipitate by decanting. The cycle of precipitation, centrifugation, and decanting was repeated three times. The precipitate was dissolved in 10 mL ACN / TDW (1:1) (relative to 2.1g peptide-resin beads) and the solution was lyophilized overnight prior to purification via preparative HPLC. The lyophilized crude product was obtained as fluffy white solid.

[0223] The solid phase synthesis of clarstatin synthesis is illustrated in the scheme below:

[0224] Fmoc-Ala

[0225] Clarstatin

[0226] = Rink Amide MBHA resina’c?= peptide elongation

[0227] Reagents: (a) piperidine / DMF 20%; (b) coupling of Fmoc-[N-(Mtt)6-aminobutyl]glycine building unit: TBTU / DIEA / DMF;(c) FmocAa+TBTU / DIEA / DMF; (d) coupling of Fmoc- [N-(alloc)6-aminobutyl]glycine building unit: TBTU / DIEA / DMF; (e)(PPh3)4Pd(0) / N- methylmorpholine / AcOH; (f) isothiocyanate formation: CS2 / HBTU / DIEA / DMF (g) DCM / TFE / AcOH (7 / 2 / 1); (h) DIEA / DMF; (i) TFA / TIS / H2O (95 / 2.5 / 2.5). Preparative high performance liquid chromatography (HPLC): The crude peptides were dissolved in TDW / ACN 1:1 mixture, filtered through a 0.45 pm polytetrafluoroethylene (PTFE) filters and injected in 5- 10 mL volumes to a reversed phase preparative HPLC column of Vydac (Cl 8, 22 x 250 mm, 10 pm). The analysis utilized a Merck- Hitachi L-6200A pump and L-7400 variable wavelength detector recording at 220 nm at room temperature. The gradient of the mobile phase consisted of solution A, TDW (0.1% v / v TFA) and solution B, ACN (0.085% v / v TFA). First the column was equilibrated for 5 minutes at 95% solution A, then linear gradient was applied from 5 to 40 minutes to reach 95% solution B. The mobile phase remained for 5 minutes at 95% solution B for column equilibration. The gradient was returned back to the starting conditions (95% solution A and 5% solution B) within 5 minutes, and kept at this point for additional 5 minutes for column equilibration. The flow rate of the mobile phase was 9 mL / min. The collected fractions were analyzed by mass spectrometer (MS), lyophilized, and injected to an analytical HPLC column to determine the degree of purity.

[0228] Analytical high performance liquid chromatography (HPLC): All samples were dissolved in TDW / ACN 1:1 mixture, filtered through a 0.45 pm PTFE filters and injected to a reversed phase analytical HPLC column of Vydac (Cl 8, 4.6x250 mm, 10 pm). The analysis utilized a Merck- Hitachi L-7100 pump and L-7400 variable wavelength detector recording at 220 nm at room temperature. The gradient of the mobile phase consisted of solution A, TDW (0.1% v / v TFA) and solution B, ACN (0.085% v / v TFA). First, the column was equilibrated for 5 minutes at 95% solution A, and then linear gradient was applied from 5 to 20 minutes to reach 95 % solution B . The mobile phase remained for 5 minutes at 95 % solution B for column equilibration. The gradient was returned back to the starting conditions (95% solution A, 5% solution B) within 5 minutes, and kept at this point for additional 5 minutes for column equilibration. The flow rate of the mobile phase was 1 mL / min. The collected fractions were further analyzed by MS. Figs. 1-2 present the HPLC analytical results (arbitrary units ((AU) / minutes) of clarstatin and clarstatin-P(Hoc2), respectively.

[0229] Mass spectrometry (MS): Mass spectra were obtained on LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific) utilizing electrospray ionization. Figs. 3-4 present the Mass spectrometry analysis of clarstatin and clarstatin-P(Hoc2), respectively.

[0230] Synthesis of clarstatin-P(Hoc2) and Clarstatin-P(MEoc2)Et:

[0231] Clarstatin-P(Hoc2) was synthesized as follows: 10 mg of clarstatin were dissolved in

[0232] ACN (0.5 mL). Following the addition of triethylamine (4 eq), hexylchloroformate (2 eq) was added. The reaction mixture was stirred overnight, and the advance of the reaction was followed by MS. At the end of the reaction, the ACN was evaporated, and the residue was purified by preparative HPLC.

[0233] Clarstatin-P(MEoc2)Et was prepared in an analogous manner to Clarstatin-P(Hoc2), however, in place of clarstatin, clarstatin ethyl ester was used as the starting material and reacted with 2-methoxyethyl chloroformate. Clarstatin ethyl ester was synthesized according to the scheme shown above for the SPPS of clarstatin, however, in step c of the peptide elongation, instead of coupling of Fmoc-Asp(OtBu), Fmoc Asp(OEt) was coupled.

[0234] Example 2. Evaluation of clarstatin permeability in Caco-2 model

[0235] The intestinal permeability of clarstatin and its prodrugs clarstatin-P(Hoc2) and clarstatin-P(MEoc2)Et was evaluated using in the Caco-2 model. The culture of colorectal adenocarcinoma 2 (Caco-2) model is a widely used tool in both the academic research and pharmaceutical industry to evaluate and predict compounds' permeability mechanism. The Caco-2 system consists of human colon cancer cells that multiply and grow to create a monolayer that emulate the human small intestinal mucosa (I. Hubatsch, E. G. E. Ragnarsson, and P. Artursson, Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers, vol. 2, no. 9, pp. 2111-2120, 2007).

[0236] Caco-2 cells (passage 30-60, ATCC, Manassas, VA, USA) were grown in 75 cm2flasks with approximately 0.5 x 106cells / flask (Thermo-Fischer, Waltham, MA, USA) at 37°C in a 5% CO2 atmosphere and at a relative humidity of 95%. The culture growth medium consisted of DMEM supplemented with 10% heat-inactivated FBS, 1% MEM-NEAA, 2 mM Lglutamine, 1 mM sodium pyruvate, 50,000 units of penicillin G sodium, and 50 mg of streptomycin sulfate (Biological Industries, Israel). The medium was replaced every other day.

[0237] Next, cells were seeded at density of 25 x 105cells / cm2on untreated culture inserts of polycarbonate membrane with 0.4 pm pores and surface area of 1.1 cm2. Culture inserts containing Caco-2 monolayer were placed in 12 mm transwell plates (Corning Inc., Corning, NY, USA). Culture medium was replaced every other day. Transepithelial Electrical Resistance (TEER) values were measured by Millicell ERS-2 System (Millipore, Burlington, MA, USA) a week after seeding up to experiment day (21-23 days) to ensure proliferation and differentiation of the cells. When the cells were fully differentiated and TEER values became stable (200- 500 (1 cm2), the TEER values were compared to control inserts containing only the medium. To measure in vitro permeability, the experiment was initiated by replacing the medium from both sides by apical (600 pL) and basolateral (1500 pL) buffers, both warmed to 37 °C. The cells were incubated with the buffer solutions for 30 min at 37 °C on a shaker (100 cycle / min). For the apical to basolateral (A to B) evaluation, the apical buffer was replaced by an apical buffer containing 10 pg / mL of peptide derivative. Samples (50 pL) were taken from the apical side immediately at the beginning of the experiment, resulting in a 550 pL apical volume during the experiment. Samples of 200 pL at fixed time points (30, 60, 90, 120 and 150 min) from the basolateral side were replaced with the same volume of fresh basolateral buffer to maintain a constant volume. Two drugs, atenolol and metoprolol (10 pg / mL), as the paracellular and transcellular permeability markers respectively were used as controls. For the basolateral to apical (B to A) evaluation, compounds were placed in the basolateral chamber, followed by sampling the apical side similar to the A to B protocol. For the quantification of clarstatin, samples were spiked with metoprolol (10 pg / mL) as an internal standard. Acetonitrile (ACN) was added to each sample (2:1) and vortex-mixed for 1 min. The samples were then centrifuged (14,635g, 10 min) and the supernatant was transferred to fresh glass tubes and evaporated to dryness (Vacuum Evaporation System, Labconco, Kansas City, MO, USA). Then, the glass tubes were reconstituted in 80 pL of mobile phase and centrifuged a second time (14,635g, 10 min). The amount of the compounds was determined using high-performance liquid chromatography mass spectrometry (HPLC-MS) (Waters 2695 Separation Module), equipped with a mass spectrometer (Waters Micromass ZQ, Waters Corporation, Milford, MA, USA).

[0238] The permeability coefficient (Papp) for each compound was calculated from the linear plot of drug accumulated versus time, using the following equation: dq n > dt aPP - Co x A

[0239] Where dq / dt is the steady state appearance rate of the compound on the receiver side, Co is the initial concentration of the drug on the donor side, and A is the exposed tissue surface area (1.1 cm2).

[0240] Intestinal permeability was examined and compared with the two standards, metoprolol, a marker for high transcellular permeability and atenolol, a marker for paracellular pathway permeability. Fig. 5 presents the PappAB (apical to basolateral) and PappBA (basolateral to apical) of clarstatin in Caco-2 model, final concentration of lOpg of clarstatin (n=3), atenolol, and metoprolol. As can be seen in Fig. 5, atenolol, the marker for paracellular permeability, had a permeability coefficient value of 0.03 cm / s xlO6. Metoprolol, the marker for transcellular permeability, had a permeability coefficient value of 8.49+0.35 cm / s xlO6. However, the Pappvalue for clarstatin in the Caco-2 model was below the detection threshold, indicating that clarstatin in vitro intestinal permeability is very low.

[0241] Clarstatin is a hydrophilic molecule having a CLogP value of -3.4 and a strong positive charge on the side chain of the Lysine (K) residue. Without wishing to be bound by any theory or mechanism of action, this strong positive charge is a major contributor to the lack of Caco- 2 permeability and the increased membrane permeability of the tested prodrugs involved the masking of the strong positive charge.

[0242] Example 3. Evaluation of Clarstatin-P(Hoc2) permeability in Caco-2 model

[0243] Lipophilic prodrug charge masking (LPCM) method was chosen to enhance clarstatin membrane permeability. A prodrug was synthesized, clarstatin-P with two Hexyloxycarbonyl (Hoc) masking groups on Lys and N-terminus as described in Example 1. The resulting prodrug, denoted clarstatin-P(Hoc2), is a lipophilic molecule which has a CLogP value of 5.2. The apparent permeability (Papp) of highly lipophilic compounds in Caco-2 cell could be underestimated due to considerable retention by the Caco-2 monolayer and nonspecific binding to transwell surface. A general approach for the determination of permeability of lipophilic compounds such as clarstatin-P(Hoc2) was utilized. 1% DMSO was added to the donor side and 4% bovine serum albumin (BSA) to the acceptor side of the Caco-2 model. BSA provided sink conditions similar to in vivo situation. For the apical to basolateral (A-B) direction permeability evaluation, clarstatin-P(Hoc2) was added to the apical side of the monolayer and 1.5 mL of basolateral buffer containing 4% w / v bovine serum albumin was added to the receiver compartment on the basolateral side of the monolayer. For the basolateral to apical (B-A) direction evaluation, 1.5 mL of basolateral buffer with 1% v / v DMSO containing 20 pg / mL clarstatin-P was added to the basolateral side of the monolayer, and 0.6 mL of apical buffer containing 4% w / v bovine serum albumin was added to the receiver compartment on the apical side of the monolayer.

[0244] The TEER values, as measured both in the beginning and at the end of the experiment are shown in Table 3. Table 3. TEER values ( ± SEM) of clarstatin-P(Hoc2) in Caco-2 monolayers.

[0245] The TEER measurements throughout the Caco-2 cells have been used as indicators for tight junctions and tissue integrity. Also, clarstatin-P(Hoc2) and the standards (e.g., metoprolol and atenolol) were added in different concentrations to basolateral buffer with 4% BSA. Calibration curve was prepared by spiking the samples with internal standard (10 pg / mL), metoprolol for clarstatin-P and propranolol for the standards, atenolol and metoprolol. The quantification of clarstatin-P(Hoc2) was performed as described in Example 2. Figs. 6A-6C present the calibration curves of atenolol, metoprolol and clarstatin-P(Hoc2) (denoted clarstatin-P in the figure), respectively. Fig. 7 presents the intestinal permeability of clarstatin-P(Hoc2) evaluated in the Caco-2 model. Atenolol, and metoprolol were used as controls. Final concentrations were 20 pg / ml for clarstatin-P(Hoc2), and 10 pg / ml for atenolol and metoprolol.

[0246] As can be seen in Fig. 7, the PappA-B and B-A values of clarstatin-P(Hoc2) were 26.01+16.00 and 2.86+0.94cm / s xlO6, respectively. Clarstatin-P(Hoc2) permeates through enterocyte monolayer, with a permeability coefficient value higher than Metoprolol (9.66+2.18 cm / s xlO6), the marker for transcellular permeability. It is concluded that the incorporation of the two Hoc masking groups to form clarstatin-P(Hoc2) resulted in a remarkable transcellular permeability.

[0247] Example 4. Evaluation of clarstatin-P(MEoc2)Et permeability in Caco-2 model

[0248] Next, the permeability of another clarstatin prodrug clarstatin-P(MEoc2)Et (Formula VI), was evaluated. This prodrug contains three masking groups on the side chains of the Lys and Asp residues and the N-terminus. Clarstatin-P(MEoc2)Et is a lipophilic molecule which has a CLogP value of 1.4. The apparent permeability (Papp) of highly lipophilic compounds in Caco-2 cell could be underestimated because of considerable retention by the Caco-2 monolayer and nonspecific binding to transwell surface. A general approach for the determination of permeability of lipophilic compounds such as clarstatin-P(MEoc2)Et was utilized. 1% DMSO was added to the donor side and 4% bovine serum albumin (BSA) in the acceptor side of the Caco-2 model. BSA provides sink conditions similar to in vivo situation. Apical buffer with 1% v / v DMSO (0.6 mL) containing 10 pg / mL. For the apical to basolateral (A-B) direction permeability evaluation, clarstatin-P(MEoc2)Et was added to the apical side of the monolayer and 1.5 mL of basolateral buffer containing 4% w / v bovine serum albumin was added to the receiver compartment on the basolateral side of the monolayer. For the basolateral to apical (B-A) direction evaluation, 1.5 mL of basolateral buffer with 1% v / v DMSO containing 10 pg / mL clarstatin-P(MEoc2)Et was added to the basolateral side of the monolayer, and 0.6 mL of apical buffer containing 4% w / v bovine serum albumin was added to the receiver compartment on the apical side of the monolayer. The quantification of clarstatin-P(MEoc2)Et was performed as described in Example 2.

[0249] Fig. 8 presents the intestinal permeability of clarstatin-P(MEoc2)Et evaluated in the Caco-2 model, basolateral to apical permeability (BA) in gray and apical to basolateral (AB) in black. Atenolol, and metoprolol were used as controls. Final concentrations were 10 pg / ml for clarstatin-P(MEoc2)Et, atenolol and metoprolol.

[0250] As can be seen in Fig. 8, the Papp A-B and B-A values of clarstatin-P(MEoc2)Et were 18.02±16.00 and 5.84±2.77 cm / s xlO6, respectively. clarstatin-P(MEoc2)Et permeates substantially through enterocyte monolayer, with permeability coefficient value higher than Metoprolol (9.66±2.18 cm / s xlO6), the marker for transcellular permeability.

[0251] Example 5. Metabolic stability in rat sera

[0252] Metabolic instability is one of the major drawbacks of linear peptides that are rapidly degraded by intestinal and plasma peptidases and are not orally bioavailable due to metabolizing enzymes in the intestinal lumen. To characterize the metabolic stability of clarstatin, 2pM stock solutions of clarstatin were diluted with fresh plasma from male Wistar rats (Harlan, Israel) to a final concentration of 0.5 pM and incubated for up to 6 hours at 37°C. During incubation, samples were taken at fixed time points. The enzymatic reaction was stopped by adding 2: 1 v / v of ice-cold acetonitrile (ACN) or methanol and centrifuged (4000g, lOmin) before analysis. The amount of clarstatin was determined using HPLC-MS. Fig. 9. presents the results as area under the curve / signal intensity, wherein the signal intensity is given as area / time.

[0253] Clarstatin was found to be stable with above 90% from initial concentration following 120 min of incubation with rat serum. Without wising to be bound by any theory or mechanism of action, the backbone cyclization of clarstatin takes advantage of the naturally selected structure of the peptide, while providing a ring element that decreases conformational freedom and sensitivity to metabolizing enzymes.

[0254] Example 6. Pharmacokinetic (PK) and bioavailability analysis of clarstatin

[0255] Pharmacokinetics evaluations were performed in the freely moving rat model. All procedures were performed on 275-300 g male Wistar rats (n=4) (Envigo, Jerusalem, Israel). Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC). Animals were anesthetized by 1 mL / kg of 9:1 ketamine: xylazine solution IP, and temperature was controlled using a 37 °C heated surface (Harvard Apparatus Inc., Holliston, MA). For blood sampling, an indwelling cannula was implanted in the right jugular vein. Animals were transferred to cages to recover overnight (12-18 h) before the pharmacokinetic experiment to allow full recovery from the surgical procedure. For pharmacokinetic evaluation, clarstatin was dissolved in water and administered at 1 mg / kg i.v., and blood was sampled (with heparin, 15 U / ml) at 5 min pre-dose and 5, 20, 40, and 60, 90, 120 and 240 min post-dose. For clarstatin-P pharmacokinetic evaluation, the prodrug was prepared similarly to clarstatin and was administered orally (PO).

[0256] Clarstatin amount was determined using a HPEC-MS system (Waters 2695 Separation Module with Micro-mass ZQ, Waters Corporation, Milford, MA). Injection volume was 10 gE. The HPEC-MS conditions were as follows: Kinetex 2.6 pm HILIC 100 A, 100 mm x 2.1 mm column (Phenomenex, Torrance, CA, USA), an isocratic mobile phase, and an acetonitrile:water:ammonium acetate buffer 50 mM pH 4 (75:15:10, NININ), with flow rate of 0.2 mL / min at 20 °C. The detection mass (m / z) of clarstatin was 903.3. The concentrations used were 0, 10 ng, 100 ng, 1 pg and 10 pg / mL. The limit of quantification was 100 ng / mL.

[0257] For the HPLC-MS calibration curve, 1 mg of clarstatin was weighted and dissolved in 100 pF of DMSO, to concentration of 10 pg / mL. The compound dissolved completely to a clear solution. The stock solution was diluted in methanol (MeOH), mobile phase, to the following concentrations: 0, 10,100, 1000 and 10000 ng / mL. Fig. 10 presents the calibration curve of clarstatin in mobile phase as area under the curve / signal intensity (area / SI) vs. the different tested concentrations (ng / mL) of clarstatin.

[0258] As seen in Fig. 10, the linearity was reserved while increasing compound concentrations. Non-compartmental PK analysis was performed with WinNonlin software. Maximum plasma concentrations (Cmax) and time at which Cmax will occur (tmax) were determined; terminal elimination rate constant (Iz) was calculated by logarithmic linear regression of the plasma concentration-time terminal curve, and half-life (ti / 2) was calculated as In 2 / lz. The area under the curve (AUC) was calculated by non-compartmental analysis to the final measurable sample using the linear-log trapezoidal method. For the pharmacokinetic analysis, plasma samples were spiked with metoprolol (1.5 pg / mL) as an internal standard. Acetonitrile (ACN) was added to each sample (2:1) and samples were vortexed and evaporated to dryness (Vacuum Evaporation System, Labconco, Kansas City, MO, USA). The tubes were reconstituted with 80 pL of mobile phase, centrifuged and transferred to HPLC vials. Fig. 11 present the pharmacokinetic profile of each rat (n=4) following intravenous dose of 1 mg / kg clarstatin.

[0259] Figs. 12A-12B present the profile of plasma concentration of clarstatin vs time. Fig. 12A, profile of plasma concentration in rats following intravenous bolus administration of 1 mg / kg of clarstatin. Fig. 12B, a semi-logarithmic plot of plasma concentration of clarstatin Vs. time (n = 4 animals, mean).

[0260] Table 4 summarizes the pharmacokinetic parameters of clarstatin. Following intravenous administration, clarstatin exposure was with a mean plasma area under the curve (AUC) value of 37783.3 ng*min / mU. The mean clearance (CU) value was 26.4 mU / min / kg, and the volume of distribution was 0.91 U / kg. The mean half-life (ti / 2) was 23.9 min.

[0261] Table 4. The half-life, AUC, clearance (CU) and volume of distribution (Vd) of clarstatin following intravenous administration of 1 mg / kg (n=4).

[0262] Overall, the pharmacokinetic profile of clarstatin is the typical profile of peptides following intravenous administration. The apparent short elimination half-life indicates that the peptide is rapidly cleared from the plasma. The apparent very small volume of distribution implies that the peptide has minimal distribution into the tissues. The oral bioavailability (F) of clarstatin-P was calculated as the ratio of AUCp.o. / AUCi.v.

[0263] Example 7. Acute toxicity evaluation of clarstatin Acute toxicity assay was conducted to determine the short-term (48 hours) adverse pathological effects of clarstatin on major mice organs when administered in a single dose, as described in the Methods section above. One group of 5 mice received clarstatin by intravenous injection in a volume of 0.2 ml / mouse at a dose of 0.25 mg / mice (about 10 mg / kg). As a control, one naive mouse did not receive the peptide. After 48 hours exposure, the animals were sacrificed, and organs were harvest for pathology analyses. Fig. 13 presents the pathological changes in ten different organs of one of the clarstatin treated mice compared to the control mouse, as appear in Hematoxylin & Eosin (H&E) stained sections. Table 5 details the severity grading of the mice.

[0264] Table 5. semi-quantitative analysis of the histopathological effects of clarstatin administration on different mice organs. As can be seen, the H&E stained sections showed pathological changes only in the liver of all animals, including the untreated animals. In the rest of the organs, no pathological changes were observed.

[0265] The livers of all tested animals (control and clarstatin treated mice) showed lipidosis of hepatocytes in zones 1 and 2 of the liver (according to the Rapapport acinus liver definition). These hepatocytes were swollen, pale and contained many micro-vacuoles in the cytoplasm. This finding was present in both, the tested group, and the control group, although less pronounced in the control (score 1.6, compared to 1 in the control). This finding of a mild to moderate liver lipidosis is most likely due to a stress condition. All other tested organs, including brain, kidneys, spleen, heart, lungs, small intestine and large intestine, stomach and thymus appeared normal without any pathological or cytotoxic changes. This information on the lack of acute toxicity of clarstatin may predict the safety of this compound in future therapeutic evaluation.

[0266] Example 8. The therapeutic effect of clarstatin in EAU mice model

[0267] An EAU model was established in mice. To this end, female, C57BL / 6 mice were immunized as described in the Methods section. Briefly, C57BL / 6 mice were subcutaneously (SC) injected with 500 pg IRBP1-20, 1 pg of PTX, 2.5 mg / ml H37RA and CFA in a total volume of 200 pl. Each mouse in the treatment group received 3.6 pg / kg of clarstatin in a volume of 50 pl, intraperitoneally (IP), twice per week, with the first dose given concurrently with EAU induction. Mice in the negative control group received PBS IP at the same volume. Mice were sacrificed at day 36 in which the eyes were collected. One eye was collected and embedded in paraffin for histopathological analysis. Wild type mice, non-immunized and untreated were used as a control for healthy retinal histological section.

[0268] Figs. 14A-14C present H&E staining of retinal histological sections of the right eye of C57BL / 6 mice 36 days following immunization (EAU induction. Fig. 14A presents the retinal histological sections of the WT, not immunized with IRBP1-20, control group (healthy), for a normal retinal structure. Fig. 14B presents the retinal histological sections of the negative control group, EAU-induced mice immunized with IRBP1-20, and treated only with PBS, showing active uveitis with vitreous cells (black arrow) and several foci of retinal infiltrates and vasculitis (white arrow). Fig. 14C presents the retinal histological sections of the tested group, EAU-induced mice immunized with IRBP1-20, and treated with clarstatin. Each point represents one mouse. As can be seen from Figs. 14A-14C the histology sections of mice in the treatment group with clarstatin showed no signs of active uveitis, compared to the EAU negative control mice treated with PBS only. The PBS treated EAU mice showed signs of active disease such as vitreous cells and several foci of retinal infiltrates and vasculitis. These results indicate that the ocular inflammatory response was ameliorated in mice treated with clarstatin, (p<0.05).

[0269] Next, the therapeutic effect of clarstatin on the retina of the EAU-induced female C57BL / 6 mice was quantified according to the masked fashion grading system as described in Table 2 described in the above Methods section. A scale from 0 to 4 according to extent of inflammation and tissue damage. Fig. 15 demonstrates the therapeutic effect of clarstatin based on the EAU score from histopathological sections. Control, non-immunized mice (Healthy); EAU-induced IRBP1-20 immunized mice (Untreated); EAU+Clarstatin, IRBP1-20 immunized treated with clarstatin as described for Fig. 14C (EAU+Clarstatin). Each point represents one mouse, (p<0.05).

[0270] As can be seen in Fig. 15, EAU-induced mice, immunized with PBS (Untreated), developed EAU score at an average value of 1.4. Healthy control mice which were not immunized with IRBP1-20 developed EAU score of 0. However, EAU-induced mice that were treated with clarstatin (EAU+Clarstatin) developed EAU score at an average score of 0.75. Thus, clarstatin reduced the EAU score in the treated group from 1.4 to 0.75.

[0271] Example 9. Leukocytes infiltration to the retina of EAU model mice and the therapeutic effect of clarstatin

[0272] The retina from the left eye of the female C57BL / 6 mice from the different groups of Example 8 (i.e., healthy control, Untreated, EAU+Clarstatin) were collected for immunological characterization. CD45 (leukocyte common antigen), a receptor-linked protein tyrosine phosphatase expressed on all leukocytes, was stained with a specific monoclonal antibody. Immunofluorescence images were compared and the percentile of cells expressing CD45 was detected by flow cytometry. Fig. 16A provides an immunofluorescence image of a retina of a healthy, non-EAU induced mouse. Fig. 16B provides an immunofluorescence image of a retina of an untreated EAU model mouse. Fig. 16C provides an immunofluorescence image of a retina of an EAU model mouse treated with clarstatin. Staining was performed with anti-mCD45 in red and anti-calreticulin in green.

[0273] As can be seen in Figs. 16B-16C, in the EAU-induced untreated mice, CD45+cells and calreticulin infiltrate the inner and outer nuclear layers as well as the ganglion cell layer. However, in EAU-induced mice treated with clarstatin, there was a remarkable decrease in infiltration of CD45+cells and a preservation of the outer retinal structure, namely the inner and outer segments of the photoreceptor layer.

[0274] Fig. 17 presents the percentile of CD45+in the retina of the different tested groups. Unimmunized mice (Healthy), EAU induced mice immunized with IRBP1-20 and treated with PBS (Untreated), and EAU-induced mice immunized with IRBP1-20 and treated with clarstatin (CL). As can be seen, clarstatin significantly reduced, by 30% CD45+(from 24.52+3.77 to 17.54+2.05) infiltrating leukocytes in the retina compared to untreated mice. The results clearly demonstrated that a dose of 3.6 pg / kg clarstatin delivered IP, twice a week, in EAU- induced mice reduced by 50% the inflammatory response, measured by EAU retinal score, reflecting a significant decrease of 30% in lymphocyte eye infiltration._*p<0.01,***p<0.001.

[0275] Example 10. Therapeutic effect of different regiments of clarstatin in EAU-induced mice

[0276] C57BL / 6 female mice were immunized subcutaneously (SC) with 500 pg IRBP1-20, 1 pg of PTX, 2.5 mg / ml H37RA and CFA in a total volume of 200 pl intraperitoneally (IP). Each mouse in the treatment groups received 180 pg / kg of clarstatin IP in a volume of 50 pl. The mice treated with clarstatin were divided into two groups, each tested group received clarstatin with a different regimen. One group received clarstatin once a week (CL 1 / week), and the other group three times a week, each treatment with 180 pg / kg (CL 3 / week). The first dose was administered 48hr after EAU induction. The clarstatin treated groups were compared to an untreated EAU-induced mouse (Untreated). As a positive control group, mice received methylprednisolone (as sodium succinate) 20 mg / kg in a volume of 50 pl, via IP injection three times a week (MP). As a negative control group (Healthy), for a healthy retinal structure, non-immunized mice received IP PBS with the same volume. The retinal fundus from the left eye of mice was photographed 2 and 4 weeks after induction of EAU. Mice were sacrificed at day 35 in which the eyes and spleen were isolated and collected. One eye was embedded in paraffin for histopathological analysis.

[0277] Retinal fundus

[0278] Figs. 18A-18E contain exemplary photographs of retinal fundus taken by Micron III camera, 4 weeks after induction of EAU in the different tested groups described hereinabove: non-immunized mice (healthy), untreated EAU-induced, , clarstatin once a week, clarstatin 3 times a week, and methylprednisolone 3 times a week, respectively. Fig. 18A demonstrates the fundus of a WT mouse in which EAU was not induced. Fig. 18B demonstrates the fundus of an EAU mouse that sustained a full-blown inflammation with multiple, diffusely scattered white chorioretinal infiltrates and perivascular sheathing. Figs. 18C and D demonstrate the fundi of EAU mice treated with clarstatin; few foci of chorioretinal infiltrates and focal perivascular sheathing can be observed. Fig. 18E demonstrates the fundus of an EAU mouse treated with methylprednisolone; mild inflammation is observed similar to mice treated with clarstatin.

[0279] The therapeutic effect was quantified providing an EAU clinical score based on the masked fashion grading system as described in Table 2 in the Methods section. Fig. 19 presents the quantification of the EAU clinical score of all the different tested groups described hereinabove: non-immunized mice (Healthy), untreated EAU-induced (Untreated), clarstatin once a week (CL 1 / week), clarstatin 3 times a week (CL 3 / week), and methylprednisolone 3 times a week (MP). As can be seen in Fig. 19, the EAU score quantified from the exemplary photographs of the retinal fundus of the mice in the different test groups indicates that untreated EAU (Untreated) mice developed EAU clinical score at an average score of 2.9 while wild-type (Healthy) mice, which were not immunized with IRBP1-20 did not develop EAU (score of 0)., the two groups of mice treated with clarstatin showed a remarkable reduction of the EAU score, indicating a reduced development of EAU. Notwithstanding, there was no significant difference between the groups which were treated with clarstatin once a week (average score of 2.0) or three times a week (score of 1.8). EAU mice treated with methylprednisolone three times a week developed EAU at an average score of 1.5. In the group which received clarstatin three times a week, the score was reduced from 2.9 to 1.8, (p<0.01). In the group which received clarstatin once a week, the score was reduced from 2.9 to 2.0 (p<0.05).

[0280] Figs. 20A-20E depict the therapeutic effect of clarstatin in H&E-stained histological exemplary retinal sections (right eye) of mice from the different test groups described hereinabove: non-immunized mice (Healthy) (Fig. 20A), untreated EAU-induced (Fig. 20B), clarstatin once a week (Fig. 20C), clarstatin 3 times a week (Fig. 20D), and methylprednisolone 3 times a week (Fig. 20E). As can be seen from the histological section, both untreated mice show active uveitis with vitreous cells, several foci of retinal infiltrates and retinal vasculitis. The histological sections of clarstatin treated mice either three times or once a week, show mild uveitis with some retinal infiltrates and retinal vasculitis or very mild uveitis with a single focus of retinal inflammation and retinal vasculitis, respectively. The histological section of the retina of an EAU mouse treated with methylprednisolone three times a week showed the same effect as the EAU mouse treated with clarstatin. Treatment with clarstatin surprisingly reduced signs of active uveitis as treatment with methylprednisolone.

[0281] Fig. 21 depicts the corresponding EAU clinical score evaluated from the histological sections (shown in Figs. 20A-20E) as a quantitative score for the therapeutic effect of clarstatin. The tested groups are as described above for Fig. 19: Healthy, Untreated, CE 1 / week, CL 3 / week, and MP. Each point present one mouse, (p*<0.05, **p < 0.01, *** p < 0.001, ns (not significant) p > 0.05).

[0282] The quantitation of the therapeutic effect of clarstatin based on EAU score from histopathological sections showed that untreated EAU mice developed EAU at an average score of 1.5 while wild-type mice, which were not immunized with IRBP1-20 did not develop EAU (score of 0). There was no significant difference between the groups which were treated with clarstatin in different regimens: EAU mice treated with clarstatin once a week developed EAU at an average score of 0.8 while EAU mice treated with clarstatin three times a week developed EAU at an average score of 0.7. EAU mice treated with methylprednisolone three times a week developed EAU at an average score of 0.5. In the group which received clarstatin three times a week, the EAU score was reduced from 1.5 to 0.7, (p<0.05). In the treatment group which received clarstatin once a week, the EAU score was reduced from 1.5 to 0.75 (p=0.05). Thus, markedly reduced scores were noted in EAU-treated mice treated with clarstatin of 50 % and 53 %, respectively, when compared to untreated, EAU-diseased group.

[0283] Spleen

[0284] Spleens from the mice were collected, as described in the Methods section, for immunological characterization by flow cytometry. The spleens were stained by monoclonal antibodies directed to CD45, CD4 (helper T cell common antigen) and F4 / 80 (macrophage common antigen). The percentile of cells expressing CD4 and F4 / 80+in each of the tested groups was analyzed by FACS, as shown in Figs. 22-23. The tested groups were as described in the retinal fundus section hereinabove: Healthy, Untreated, clarstatin 3 times a week, clarstatin once a week, and methylprednisolone 3 times a week (MP). Each point present one mouse, (*p<0.05, ** p<0.01).

[0285] As can be seen from Figs. 22-23, clarstatin significantly reduced, by 50%, CD4+infiltrating cells in the spleen of treated mice three times a week compared to EAU-induced, untreated mice (56.96+19.12 to 28.19+13.17, p<0.01). Additionally, clarstatin significantly reduced by 38% CD4+infiltrating cells in the spleen of mice treated once a week compared to untreated mice (56.96+19.12 to 35.17+20.84, p<0.05). There was no significant difference between the two clarstatin treatment groups with regard to the percentage of CD4+infiltrating cells in the spleen. The positive control group that was treated with methylprednisolone three times a week had 50% less CD4+infiltrating cells in the spleens compared to untreated mice (56.96+19.12 to 28.67+5.58, p<0.01). Clarstatin showed similar reduction of CD4+infiltration, compared to methylprednisolone. However, clarstatin had no effect on the percentage of F4 / 80+infiltrating cells. Without wishing to be bound by any theory or mechanism of action, clarstatin may have less effect on cells of innate immunity and more targeted effect against lymphocytes.

[0286] In this study, similar to the preliminary experiment, it was found that clarstatin administered IP, reduced by 50% the inflammatory response, as measured by the histological EAU score and by quantitation of the CD4+infiltrating cells in the spleens. Additionally, as the results did not show difference between administering clarstatin once a week or three times a week, a dose dependent effect of clarstatin treatment given once a week was further evaluated.

[0287] Example 11. Dose dependent effect of clarstatin in E AU-induced mice

[0288] Following the dose regimen evaluation, a clarstatin dose dependent effect was examined in EAU mice model. To this end, C57BL / 6 female mice were immunized subcutaneously with 500 pg IRBP1-20, 1 pg of PTX, 2.5 mg / ml H37RA and CFA in a total volume of 200 pl, IP. Each mouse in the treatment groups received different doses of clarstatin, specifically, 3.6, 36, or 360 pg / kg, once a week in a volume of 50 pl, IP. The first dose was administered 48hr after EAU induction. Mice in the positive control group received methylprednisolone (as sodium succinate) 20 mg / kg in a volume of 50, pl, IP, three times a week. Mice in the negative control group (denoted WT) received IP the same volume of PBS. EAU-induced, IRBP1-20 immunized untreated mice (denoted EAU in the figure).

[0289] The different treatments, dosage, group denotation and number of animals in each group (n) are detailed in Table 6. Table 6. the different tested groups and treatment details.

[0290] Photographs of retinal fundus from the left eye of mice were taken a by Micron III camera, at days 18 and 30 after induction of EAU. Mice were sacrificed at day 35 in which the eyes were collected and embedded in paraffin for histopathological analysis. Figs. 24A- 24F show photographs of the retinal fundus taken 18 days post EAU induction of mice in each one of the tested groups as described in Table 6: WT, EAU, CLAR 3.6* 1, CLAR 36*1, CLAR 360*1, PRED*3, respectively. Figs. 25A-25F present photograph of the retinal fundus taken 30 days post EAU induction of mice in each of the tested groups.

[0291] As can be seen from Figs. 24A-24F and 25A-25F, mice treated with 360 pg / kg of clarstatin showed similar therapeutic effect to that of methylprednisolone already at 18 days post EAU induction. Mice treated with 36 pg / kg of clarstatin showed a mild therapeutic effect, while mice treated with 3.6 pg / kg of clarstatin showed no therapeutic effect.

[0292] The clarstatin dose dependent therapeutic effect was quantified providing an EAU clinical score from the photographs of the retinal fundus of mice 18- and 30-days post EAU induction, based on the masked fashion grading system described in Table 2 in the Methods section. Fig. 26 presents the EAU score of the different tested groups as quantified from H&E histopathological sections of mice 35 days post EAU induction. The tested groups are as described in Table 6. Each point represents one mouse. The results show that untreated EAU mice developed EAU at an average score of 1.5 compared to score of 0 of WT mice, which were not immunized and did not develop EAU. EAU-induced mice treated with clarstatin dose of 3.6 pg / kg developed EAU at an average score of 1.3, EAU mice treated with clarstatin dose of 36 pg / kg developed EAU at an average score of 0.8, and EAU mice treated with clarstatin dose of 360 g / kg developed EAU at an average score of 0.6. EAU mice treated with methylprednisolone (three times a week) developed EAU at an average score of 0.3. Clarstatin reduced the EAU score in all treated groups. The significant effect is seen in the group which received 360 pg / kg of clarstatin. In that treatment group, the EAU score was reduced from 1.5 to 0.6, (*p<0.05). In the treatment group which received clarstatin dose of 36 pg / kg, the EAU score was reduced from 1.5 to 0.8 (p=0.07).

[0293] Example 12. intravitreal administration of clarstatin

[0294] EAU mice injected intravitreally with clarstatin were analyzed by ocular fundoscopy examination after receiving a drop of phenylephrine 10% and tropicamide 1% in each eye. Five minutes after administration of the mydriatic drugs, animals were anesthetized, and examination of the eye fundus was performed with a stereoscope under 20X magnification.

[0295] Clarstatin had a remarkable therapeutic effect as was observed in histopathology of the eyes of this group of mice compared to the EAU-induced mice. As observed by a clinician, mice treated with clarstatin exhibited a clear trend of an anti-inflammatory effect expressed by reduced edema and pathological damage as was also showed by histopathology analyzes indicating a correction of the retinal architecture. On the contrary, control untreated EAU mice group showed retinal disorganization and presence of inflammatory cells in the retina, retinal vasculitis and granuloma formation.

[0296] Example 13. Systemic administration of clarstatin-P(MEoc2)Et and clarstatin in EAU mice model

[0297] EAU was induced in C57BL / 6 female mice, as described in the Methods section. The mice were divided into two treatment groups. Each mouse in the first treatment group received 0.18 mg / kg of clarstatin, IP, in a volume of 50 pl. Each mouse in the second treatment group received, orally by gavage, 1.8 mg / kg of clarstatin-P(MEoc2)OEt in a volume of 100 pl. Both of the groups were treated three times a week, the 1st dose administered 24 hours after EAU induction. Mice in the positive control group received methylprednisolone (as the sodium succinate salt) 20 mg / kg in a volume of 100 pl, orally by gavage three times a week. Mice were sacrificed at day 35 in which the eyes were collected. Eyes and spleen were isolated. The right eye embedded in paraffin for histopathological analysis. The intraocular levels of cytokines were measured from the left eye by specific ELISA. Fig. 27, demonstrate quantitation of the therapeutic effect of clarstatin and clarstatin-P(MEoc2)OEt based on EAU score from histopathological sections of the right eyes of the tested mice. Mice treated with clarstatin (clarstatin IP 0.18 mg / kg), mice treated with clarstatin-P(MEoc2)OEt) (clarstatin-P oral 1.8 mg / kg), positive control mice treated with methylprednisolone (PRED oral 20 mg / kg), untreated EAU mice (untreated) and WT untreated mice, which were not induced with EAU (WT). Each point represents one mouse (*p<0.05, ** p<0.01).

[0298] As can be seen in Fig. 27, untreated EAU mice developed EAU score at an average score of 1.07 while WT mice, which were not immunized with IRBP1-20 did not develop EAU (score of 0). EAU mice treated with clarstatin 0.18 mg / kg three times a week IP developed EAU at an average score of 0.35, thus, the EAU score was reduced from 1.07 to 0.35, (*p<0.05). EAU mice treated with clarstatin-P(MEoc2)Et 1.8 mg / kg three times a week orally developed EAU at an average score of 0.25, thus, the EAU score was reduced from 1.07 to 0.25 (*p<0.01). EAU mice treated with methylprednisolone three times a week orally developed EAU at an average score of 0.5. The reduction of EAU score by clarstatin correspond with results of previous experiments.

[0299] Additionally, the therapeutic effect of clarstatin and clarstatin-P(MEoc2)OEt was quantified based on the intraocular expression levels of the pro-inflammatory cytokine IL-6 and the monocyte chemoattractant protein- 1 (MCP-1). MCP-1 is a key chemokine in the regulation of migration and infiltration of monocytes / macrophages. The expression levels of IL-6 and MCP-1 were measured by specific ELISA. Figs. 28-29 present IL-6 and MCP-1 expression levels (pg / mg), in the different test groups as described for Fig. 27. Each point represents one mouse (*p<0.05, ns - not statistically significant).

[0300] As can be seen in Figs. 28-29, treatment with clarstatin or clarstatin-P(MEoc2)OEt significantly decreased IL-6 expression levels compared to untreated EAU mice. Clarstatin and clarstatin-P(MEoc2)OEt reduced the IL-6 expression levels, substantially to similar levels of the WT. Methylprednisolone treatment, showed a smaller reduction of IL-6 expression levels compared to clarstatin and clarstatin-P(MEoc2)Et . Compared to the untreated EAU mice, clarstatin and clarstatin-P(MEoc2)OEt treatments, significantly reduced MCP-1 expression levels as well. The reduction of MCP-1 expression levels by clarstatin and clarstatin-P(MEoc2)Et were substantially similar to the reduction achieved by methylprednisolone treatment. Example 14. Ca2+signaling reduction in Jurkat cells upon clarstatin treatment

[0301] Multiple inflammatory stimuli converge on Ca2+signaling in immune cells. The effect of clarstatin on Ca2+signaling in Jurkat cells was characterized by comparing it to the inflammatory lipopolysaccharide bacterial endotoxin (LPS). Cytosolic intracellular Ca2+content was measured in Jurkat cells loaded with Fura-2AM, in the presence of EGTA to prevent extracellular influx by calcium channels.

[0302] Specifically, Jurkat cells were attached to polylysine-coated glass coverslips for Ca2+imaging conducted in Ringer’s solution composed of 126 mM NaCl, 5.4 mM KC1, 0.8 mM MgCh, 20 mM HEPES, 1.8 mM CaCh, and 15 mM glucose. The pH of the solution was adjusted to 7.4 using NaOH. Prior to imaging, the cells were loaded with Fura 2 AM from Teflabs, Jackson Springs, NC, USA. Intracellular Ca2+responses were observed in the presence of 2 mM EGTA. Illumination of cells was accomplished using a 175W xenon arc lamp, and the excitation wavelengths of 340 / 380 nm were selected via a Lambda DG-4 monochromatic wavelength changer from Sutter Instrument, Novato, CA, USA. The intracellular Ca2+concentration was quantified using digital video microfluorometry employing a frontilluminated interline CCD camera (Exi Blue; Qlmaging, Surrey, BC, Canada) alongside MetaFluor Fluorescence Ratio Imaging Software (Meta Imaging Series 6.1) from Molecular Devices, Sunnyvale, CA, USA. Dual images (340 and 380 nm excitation, 510 nm emission) were captured, and pseudocolor ratio-metric images were recorded every 2 s throughout the experiment, all performed at room temperature.

[0303] As expected, intracellular Ca2+levels significantly decreased upon LPS treatment (Fig. 30A). In the presence of clarstatin intracellular Ca2+content was also significantly attenuated (Fig. 30B). Overall calcium level, calculated as the area under the curve (AUC), was reduced by 65% in LPS -treated cells and by 15% in clarstatin treated cells compared to control untreated cells. Overall, the reduced calcium levels remained significantly decreased for about 500 seconds. ic effects of clarstatin

[0304] To evaluate a potential cytotoxic effect, Jurkat cells were incubated with 0.3, 36, and 100 pg / mL clarstatin for 48 h and thereafter the necrotic cell death was measured by the release of lactate dehydrogenase (LDH), using an LDH reagent. H2O2-treated cells were used as a positive control. LDH activity was determined spectrophotometrically at 340 nm by following the rate of conversion of oxidized NAD+ to the reduced form of NAD (NADH). LDH release was expressed as the optical density units and calculated as a percentage of total LDH. Each experiment was performed three times in six replicates (n = 18). As shown in Fig. 31, in all concentrations, clarstatin did not significantly increase cell death compared to control-untreated cells, stressing the cellular safety of the Jurkat cells treated with clarstatin and indicating that the effect of clarstatin on the intracellular Ca2+content (Example 14) was not due to cytotoxicity.

[0305] Example 16. Clarstatin ameliorated ocular pathology and reduced retinal infiltration of CD45+ leukocytes in EAU B10RIII severe mice model

[0306] B10RIII female mice were immunized with IRBPiei-isoand PTX, thereby inducing an acute, severe EAU disease, as described in the Methods section. The tested groups consisted of wild-type mice (healthy), severe E AU-diseased untreated mice, clarstatin-treated mice (by i.p. injection with 0.36 and 1 mg / kg, three times a week for two weeks), or methylprednisolone-treated mice (20 mg / kg, three times a week for 2 weeks). Quantitation of the therapeutic effects of clarstatin was based on EAU score from the histopathological sections (Fig. 32A). Severe EAU mice developed the disease with an average score of 3.7 (p<0.001) (Untreated) while wild-type mice presented a score of zero (Healthy). The histopathological analysis indicated improvement in clarstatin-treated (CL) (2.4; p<0.05) and methylprednisolone treated mice (MP) (1.9; p<0.01) respectively, with a significant decrease in the histopathological scores by 36 %, and 48 %, respectively, when compared to EAU- diseased, untreated mice.

[0307] To evaluate the impact of clarstatin on the inflammatory CD45+ cell infiltrate, retinas were collected on day 14 for immunological characterization by FACS (Fig. 32B). Clarstatin (CL) at doses of 0.36 mg / kg and 1 mg / kg, significantly reduced the CD45+ cell levels in the retinas of treated mice, compared to untreated mice (untreated), by 48 % (p<0.05). There was no significant difference between the two clarstatin treatment groups regarding the percentage of CD45+ cell levels in the retina, and clarstatin effect was comparable to methylprednisolone (MP) that reduced CD45+ retinal cell levels by 58 % (p<0.05). Cumulatively, these results indicate that clarstatin significantly inhibited leukocyte retinal CD45+ cell infiltration and the resultant structural damage in the severe EAU model of B10RIII mice.

[0308] Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

CLAIMS1. A peptidomimetic of Formula I:Formula I wherein Ri and R2 are each independently a side chain of an amino acid and R3 represents the carboxy terminus of the peptide, which may be a modified carboxy terminus.

2. The peptidomimetic according to claim 1, wherein Ri and R2 are each hydrogen.

3. The peptidomimetic according to claim 1, wherein R3 is selected from an amidated, carboxylated, esterified or alcohol carboxy terminus.

4. The peptidomimetic according to claim 1, wherein Ri and R2 are each hydrogen and R3 is an amidated carboxy terminus, wherein the peptidomimetic is represented by Formula II:Formula II.

5. A prodrug comprising a peptidomimetic according to claim 1.

6. The prodrug according to claim 5, represented by Formula III:Formula III wherein R4 is independently at each position H or a masking moiety selected from the group consisting of hexyloxycarbonyl (Hoc), propyloxycarbonyl (Poc), methoxyethyloxycarbonyl (MEoc), hydroxyethyloxycarbonyl (HEoc), and (5-Methyl-2- oxo- 1 ,3-dioxol-4-yl)methyloxycarbonyl (ODOL-oc);Rs is H or a masking moiety selected from ethyl (Et) and methyl (Me); andR3 represents the carboxy terminus of the peptide, which may be a modified carboxy terminus.

7. The prodrug according to claim 6, wherein R4 in both positions is identical.

8. The prodrug according to claim 6, wherein R4 is independently at each position Hoc or MEoc.

9. The prodrug according to any one of claims 5 to 8, wherein R3 is selected from an amidated, carboxylated, esterified or alcohol carboxy terminus.

10. The prodrug according to any one of claims 6 to 9, wherein R4 is independently at each position Hoc or MEoc and R3 is an amidated carboxy terminus.

11. The prodrug according to claim 5, represented by Formula V:Formula V.

12. The prodrug according to claim 5, represented by Formula VI:Formula VI.

13. The peptidomimetic according to any one of claims 1 to 12 capable of inhibiting at least one activity of calreticulin (CRT).

14. A pharmaceutical composition comprising a therapeutically effective amount of the peptidomimetic or prodrug according to any one of claims 1 to 13, and optionally, a pharmaceutically acceptable carrier, excipient or diluent.

15. The pharmaceutical composition according to claim 14, wherein the prodrug is according to Formula III, Formula IV, Formula V, or Formula VI.

16. The pharmaceutical composition according to claim 14 or 15, wherein the pharmaceutical composition is formulated for administration by a route selected from oral, intraocular, intravitreal, juxtascleral, subconjunctival, intracameral, retrobulbar, submucosal, intranasal, topical, intravenous, intramuscular, intraperitoneal, subcutaneous, intra-dermal, intra-arterial, intraarticular, and intralesional.

17. The pharmaceutical composition according to any one of claims 14 to 15, wherein the pharmaceutical composition is formulated for administration by a route selected from oral, ophthalmic, submucosal, intranasal, and topical.

18. The pharmaceutical composition according to claim 17, wherein the ophthalmic administration is selected from topical, intraocular, intravitreal, juxtascleral, subconjunctival, intracameral, and retrobulbar.

19. The pharmaceutical composition according to any one of claims 14 to 17, wherein the pharmaceutical composition is formulated for oral administration.

20. The pharmaceutical composition according to any one of claims 14 to 19, for use in inhibiting at least one activity of calreticulin (CRT).

21. The pharmaceutical composition according to any one of claims 14 to 19, for use in the treatment of a CRT -related disease, disorder or condition.

22. The pharmaceutical composition for use according to claim 21, wherein the CRT-related disease is an autoimmune disease.

23. The pharmaceutical composition for use according to claim 22, wherein the autoimmune disease is selected from autoimmune uveitis, systemic lupus erythematosus, autoimmune myelofibrosis, celiac disease, thyroid autoimmunity disease, Graves' disease, Hashimoto's disease, systemic sclerosis, spondyloarthritis, Sjogren's syndrome, dermatitis herpetiformis, rheumatic disease and erosive arthritis.

24. The pharmaceutical composition for use according to claim 21 or 22 wherein the CRT- related disease is uveitis.

25. A method of treating an autoimmune disease or condition, comprising administering to a subject in need thereof a pharmaceutical composition according to any one of claims 14 to 19.

26. The method of claim 25, wherein the autoimmune disease is selected from a group consisting of autoimmune uveitis, systemic lupus erythematosus, autoimmunemyelofibrosis, celiac disease, thyroid autoimmunity disease, Graves' disease, Hashimoto's disease, systemic sclerosis, spondyloarthritis, Sjogren's syndrome, dermatitis herpetiformis, rheumatic disease and erosive arthritis.

27. A method of treating uveitis, comprising administering to a subject in need, a pharmaceutical composition according to any one of claims 14 to 19.

28. The method according to any one of claims 25-27, wherein the pharmaceutical composition is administered via a route selected from ophthalmic, oral, intraocular, submucosal, intranasal, topical, intravenous, intramuscular, intraperitoneal, subcutaneous, intra-dermal, intra-arterial, intraarticular, and intralesional.

29. The method according to any one of claims 25 to 28, wherein the pharmaceutical composition is administered orally.

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