Isocyclosporine A for the topical treatment of ocular diseases

Abstract

The present invention relates to isocyclosporine A, its salts, and ophthalmic compositions thereof for topical use in the eye in the prevention or treatment in an individual of an inflammatory and / or autoimmune disease of the eye, preferably selected from inflammatory and / or autoimmune diseases of the cornea and ocular surface and inflammatory diseases of the eyelid margin.

Description

[Technical field]

[0001] The present invention relates to isocyclosporin A, its salts and pharmaceutical compositions, and their use in the prevention or treatment of inflammatory and / or autoimmune eye diseases in individuals. [Background technology]

[0002] Cyclosporin A is a neutral lipophilic cyclic undecapeptide obtained from the fungus Tolypocladium Inflatum that contains seven N-methyl amino acid residues and an unusual amino acid, (4R)-4-([E]-2-butenyl)-4-N-methyl-(L)-threonine (MeBmt), at position 1.

[0003] Cyclosporine was originally approved as an immunosuppressant to prevent organ rejection of transplanted organs. In ophthalmology, cyclosporine A was initially investigated for administration after corneal transplantation, but was later shown to be effective in several ocular inflammatory diseases, such as uveitis, vernal keratoconjunctivitis (VKC), akrokeratoconjunctivitis (AKC), and dry eye disease (DED) (Feroze et al., J Ophthalmol 2019, 31:182-190; Prabhu et al., British Journal of Ophthalmology 2016, 100:345-347).

[0004] Due to the hydrophobic nature of the molecule, it was initially administered by systemic routes such as intravenous injection or oral administration, even for ophthalmic indications. These routes of administration allowed therapeutic concentrations of the active ingredient to reach the intraocular fluids (aqueous humor or vitreous humor) and the extraocular organs or adnexa (cornea, conjunctiva and lacrimal gland). However, serious systemic side effects such as nephrotoxicity and hypertension occurred, which prompted the search for suitable formulations for the local administration of the molecule (Lallemand et al., European Journal of Pharmaceutics and Biopharmaceutics 2003,56:307-318).

[0005] The development of eye drop formulations of cyclosporine A for topical use in the eye has been hindered by the hydrophobic nature of the molecule and its extremely low aqueous solubility (6.6 mg / ml), which does not allow for formulations based on commonly used aqueous ophthalmic vehicles.

[0006] Cyclosporine A was initially formulated in the form of solutions in oily solvents such as castor oil or corn oil, but these were later replaced by oil-in-water emulsions and micelle-based solutions (de Oliveira et al., Clinical Ophthalmology 2019:13 1115-1122).

[0007] However, the presence of surfactants required for the latter formulation was found to have adverse effects on the cornea, resulting in blurred vision. Furthermore, although emulsions have the advantage of rapid diffusion into the eye upon application, the amount of active ingredient available in the already harsh aqueous intraocular environment is still limited due to the low bioavailability of lipophilic drugs from emulsions (Jerkins et al., Clinical Ophthalmology 2020:14 481-489).

[0008] The poor solubility of cyclosporine and the presence of surfactants in most currently available ophthalmic formulations that are harmful to the cornea are major limitations to its use from an ophthalmic therapeutic standpoint and probably the cause of the poor tolerability and highly variable responses reported in clinical trials.

[0009] Isocyclosporin A is an isomer of cyclosporin A and differs from it in that the residue at position 1 (N-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)threonyl) is attached to the residue at position 11 via the 3'-O-atom rather than the N-atom. Isocyclosporin A was initially identified as a rearranged degradation product formed by acid treatment of cyclosporin A. Isocyclosporin A has been described as having no biological activity of its own and being converted very slowly in vivo to the corresponding active cyclosporin form when administered systemically (WO1993017039A1, pages 5 and 15). In particular, it has been shown that the molecule is quantitatively converted to cyclosporin A at a pH higher than 5. Specifically, the isomerization rate of isocyclosporine is greatest at pH 8-10 (Oliyai R, Stella VJ, Pharm Res, 1992, May;9(5):617-22), while the slowest conversion rate is observed at pH 6-8. In particular, conversion is minimal at pH 7.4, with a conversion half-life of 21.7 hours (see Bundgaard et al., International Journal of Pharmaceutics, 82(1992):85-90).

[0010] Given its ability for slow conversion to cyclosporine A and its high solubility in water, isocyclosporine A has been proposed for use as a prodrug of cyclosporine A for oral administration to improve stability and avoid side effects associated with peak blood concentrations of cyclosporine.

[0011] In this regard, WO1993017039A1 describes acid addition salts of isocyclosporines, including isocyclosporine A, which offer improved galenical characteristics such as improved solubility and stability. The document discloses that these salts are particularly useful as oral prodrugs of cyclosporines, since when administered orally they release the active molecule at a slow rate and maintain a constant concentration of cyclosporine in the blood.

[0012] However, topical use of isocyclosporine A in the eye is considered impractical given the slow conversion rate of this molecule in that environment. In fact, the rate of interconversion of isocyclosporine A to cyclosporine A is so slow that virtually no cyclosporine A is formed during the residence of the molecule on the ocular surface. As mentioned above, the conversion rate is slowest at pH values ​​between 6 and 8, and it has been reported that the time required for 50% conversion of isocyclosporine A to cyclosporine A is 21.7 hours at pH 7.4 (Bundgaard et al., International Journal of Pharmaceutics, 82 (1992): 85-90).

[0013] In view of the above, to the applicant's knowledge, isocyclosporine A has never been considered a viable alternative to cyclosporine A for topical ophthalmic application, as it was not expected to exert any pharmacological activity. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Patent Publication No. 1993 / 017039 (WO1993017039A1) [Non-patent literature]

[0015] [Non-Patent Document 1] Feroze et al, J Ophthalmol 2019, 31: 182-190 [Non-Patent Document 2] Prabhu et al, British Journal of Ophthalmology 2016, 100: 345-347 [Non-Patent Document 3] Lallemand et al, European Journal of Pharmaceutics and Biopharmaceutics 2003, 56: 307-318 [Non-Patent Document 4] de Oliveira et al, Clinical Ophthalmology 2019:13 1115-1122 [Non-Patent Document 5] Jerkins et al, Clinical Ophthalmology 2020:14 481-489 [Non-Patent Document 6] Oliyai R, Stella VJ, Pharm Res, 1992, May;9(5):617-22 [Non-Patent Document 7] Bundgaard et al, International Journal of Pharmaceutics, 82 (1992):85-90 Summary of the Invention

[0016] Applicants have now surprisingly found that isocyclosporine A, when administered topically to the eye, unexpectedly exhibits biological activity that is independent of conversion to cyclosporine A. As noted above, this finding is entirely unexpected on the basis of the background literature.

[0017] Even more surprisingly, as shown by in vivo studies carried out in relevant animal models of inflammatory ocular surface disease in the experimental section, the Applicant observed that treatment with isocyclosporine A exhibited higher efficacy in several pharmacologically relevant parameters compared to cyclosporine A.

[0018] The inventors have also found that isocyclosporin A can inhibit the activity of TRPV3 and TRPML2 ion channels, which have been described in the literature to be involved in the symptoms of inflammatory and allergic diseases (Takahiro Yamada et al., Exp Eye Res. 2010 Jan; 90(1): 121-129; MP Cuajungco et al., Pflugers Arch. 2016 Feb; 468(2): 177-92; and Eva Plesch et al., eLife. 2018; 7: e39720). Moreover, unlike cyclosporin A, isocyclosporin A does not stimulate the hyperproliferation of epithelial cells induced by the activation of the TRPC4 channel, which is undesirable in inflammatory and allergic eye diseases.

[0019] Thus, a first object of the present invention is isocyclosporin A or an ophthalmologically acceptable salt thereof for topical use in the eye for the prevention or treatment of inflammatory and / or autoimmune eye diseases.

[0020] Preferably, said inflammatory and / or autoimmune eye disease is selected from inflammatory and / or autoimmune diseases of the cornea and ocular surface and inflammatory diseases of the eyelid margin. A second object of the invention is an ophthalmic formulation comprising isocyclosporin A or an ophthalmologically acceptable salt thereof and at least one ophthalmologically acceptable excipient or diluent.

[0021] A further object of the present invention is a method for the prevention or treatment of inflammatory and / or autoimmune eye diseases, preferably selected from inflammatory and / or autoimmune diseases of the cornea and ocular surface and inflammatory diseases of the eyelid margin, comprising topically administering to an individual in need thereof an effective amount of isocyclosporine A or an ophthalmologically acceptable salt thereof and / or an ophthalmic formulation thereof. [Brief description of the drawings]

[0022] [Figure 1]FIG. 1 shows the percentage of fluorescence, reported as the mean value, measured in each group on days 0, 9, 11, 17, 22 and 25 after scopolamine treatment, as described in Example 1. [Diagram 2] FIG. 2 shows tear production (expressed in mm) measured in the three experimental groups on days 22 (top) and 25 (bottom) after scopolamine treatment, as described in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] A first object of the present invention is isocyclosporin A or an ophthalmologically acceptable salt thereof for topical use in the eye in the prevention or treatment in individuals of inflammatory and / or autoimmune eye diseases.

[0024] Preferably, said inflammatory and / or autoimmune diseases are selected from inflammatory and / or autoimmune diseases of the cornea and ocular surface and inflammatory diseases of the eyelid margin. Inflammatory and / or autoimmune diseases of the cornea and ocular surface are diseases characterized by damage to the epithelium covering the cornea and conjunctiva, induced by local inflammatory conditions.

[0025] Inflammatory diseases of the eyelid margin are conditions characterized by generalized and persistent inflammation of the eyelid. In particular, according to the invention, said inflammatory and / or autoimmune eye disease is selected from vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC), allergic conjunctivitis, ocular rosacea, uveitis, dry eye disease (DED), ocular pemphigoid (OCP), ocular graft versus host disease (GVHD), immune corneal ulcer, inflammatory pterygium and chronic blepharitis.

[0026] According to the present invention, the isocyclosporin A or a salt thereof is administered topically to the eye of the individual. According to the present invention, the term "prevention" refers to the partial or complete prevention of a disorder or pathological event before it is established or occurs.

[0027] According to one aspect of the invention, the prevention is a complete prevention, in which the disorder or pathological event is completely averted. According to an alternative aspect of the invention, the prevention is partial prevention, delaying the onset and / or reducing the severity of the disorder or pathological event.

[0028] According to the present invention, the term "treatment" refers to completely reversing or reducing the severity or progression of a disorder or pathological event after it has already been established or has occurred. According to the present invention, the term "individual" refers to a human or an animal, preferably a human.

[0029] Preferably, said inflammatory and / or autoimmune eye diseases treatable with isocyclosporine A or an ophthalmologically acceptable salt thereof are eye diseases that show improvement or remission upon treatment with cortisone and similar corticosteroids.

[0030] The inflammatory and / or autoimmune diseases of the cornea and ocular surface which can be treated with isocyclosporine A or an ophthalmologically acceptable salt thereof are preferably selected from vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC), allergic conjunctivitis, ocular rosacea, uveitis, dry eye disease (DED), ocular pemphigoid (OCP), ocular graft versus host disease (GVHD) and immune corneal ulcer. Preferably, said allergic conjunctivitis is seasonal or perennial allergic conjunctivitis.

[0031] Inflammatory diseases of the eyelid margin which can be treated with isocyclosporin A or an ophthalmologically acceptable salt thereof are, for example, inflammatory pterygium and chronic blepharitis. According to the present invention, preferably, said isocyclosporin A or an ophthalmologically acceptable salt thereof can be used for the prevention and treatment of one, two or more of the above-mentioned inflammatory and / or autoimmune eye diseases.

[0032] According to the invention, the isocyclosporine A or the salt thereof is topically applied to the ocular surface or eyelid margin of the individual's eye. In a preferred embodiment, the isocyclosporin A or the salt thereof is topically applied to the ocular surface of the individual's eye via an ophthalmic liquid or semi-liquid formulation as described below.

[0033] Preferably, said isocyclosporin A or said salt thereof is administered once, twice, three times, four or more times daily depending on the medical condition and the severity of the disease being treated. According to a preferred embodiment of the invention, the ophthalmologically acceptable salt is an acid addition salt of isocyclosporin A.

[0034] Preferably, the ophthalmically acceptable salt of isocyclosporin A is an acetate, adipate, ascorbate, benzoate, benzenesulfonate, bicarbonate, hydrogensulfate, butyrate, camphorate, camphorsulfonate, citrate, cyclohexylsulfamate, ethanesulfonate, fumarate, glutamate, glycolate, hemisulfate, 2-hydroxyethylsulfonate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, or the like salt of isocyclosporin A. The salt is selected from the group consisting of peroxidase, hydroxymaleate, lactate, malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nitrate, oxalate, pamoate, persulfate, phenylacetate, phosphate, diphosphate, picrate, pivalate, propionate, quinate, salicylate, stearate, succinate, sulfamate, sulfanilate, sulfate, tartrate, tosylate (p-toluenesulfonate), trifluoroacetate, and undecanoate.

[0035] More preferably, the salt is the hydrochloride or trifluoroacetate salt of isocyclosporin A. The acid addition salts of the present invention are preferably formed at the alpha-N atom of the residue at position 1 of isocyclosporin A.

[0036] Methods for the preparation of the salts according to the invention are well known and are described, for example, in patent application WO1993017039A1. A second object of the invention is an ophthalmic formulation comprising, as active ingredient, isocyclosporin A or an ophthalmologically acceptable salt thereof as defined above, and at least one ophthalmologically acceptable excipient or diluent.

[0037] According to a preferred embodiment, the ophthalmic formulation is a liquid ophthalmic formulation, preferably a liquid eye drop formulation. The ophthalmic liquid formulation may be a monophasic liquid formulation, preferably a solution, more preferably an aqueous solution, or a biphasic liquid formulation, preferably an emulsion, more preferably a microemulsion.

[0038] The liquid ophthalmic formulation preferably contains saline as a vehicle. According to another preferred aspect, said ophthalmic formulation is a semi-solid ophthalmic formulation, preferably a cream, ointment or gel.

[0039] According to yet another preferred embodiment, the ophthalmic formulation is a solid ophthalmic formulation for extemporaneous preparation of said liquid or semi-solid ophthalmic formulation by adding an ophthalmologically acceptable diluent prior to administration.

[0040] Preferably, the solid ophthalmic formulation is in the form of a powder, more preferably, it is in the form of a lyophilized powder. Preferably, said liquid or semi-solid ophthalmic formulation has a pH comprised between 6 and 8, more preferably between 6.4 and 7.8, and even more preferably between 6.5 and 7.5. Indeed, under these conditions, the rate of interconversion of isocyclosporin A to cyclosporin A is known to be minimal, ensuring the stability of the final product (at pH 7.4 and 37° C., 50% conversion takes 21.7 hours; Bundgaard et al., International Journal of Pharmaceutics, 82 (1992): 85-90).

[0041] Preferably, the liquid or semi-solid ophthalmic formulation contains a buffer capable of maintaining the formulation at a desired pH. The buffer is preferably selected from, but not limited to, acetate, carbonate, citrate, phosphate and borate buffers. Acids or bases can also be used to adjust the pH of these formulations, if necessary.

[0042] Preferably, the ophthalmic formulations according to the present invention also contain one or more excipients selected from ophthalmologically acceptable surfactants, preservatives, stabilizers, tonicity adjusting agents, viscosity adjusting agents, antioxidants and chelating agents.

[0043] The surfactant(s) can be used to aid in dissolving excipients or active agents, dispersing solids or liquids in the composition, enhancing wettability (wetting), modifying droplet size, or for some other purpose.

[0044] Preferably, the surfactants contained in the ophthalmic formulations according to the invention are selected from alcohols, amine oxides, block polymers, carboxylated alcohol or alkylphenol ethoxylates, carboxylic acids / fatty acids, ethoxylated alcohols, ethoxylated alkylphenols, ethoxylated arylphenols, ethoxylated fatty acids, ethoxylated fatty acid esters or oils (animal and vegetable), fatty acid esters, fatty acid methyl ester ethoxylates, glycerol esters, glycol esters, lanolin based derivatives, lecithin and lecithin derivatives, lignin and lignin derivatives, methyl esters, monoglycerides and derivatives, polymeric surfactants, propoxylated and ethoxylated fatty acids, alkylphenols, protein based surfactants, sarcosine derivatives, sorbitan derivatives, sucrose and glucose esters and derivatives.

[0045] Preferably, the preservatives contained in the ophthalmic formulations according to the invention are selected from cationic preservatives, preferably quaternary ammonium compounds, more preferably benzalkonium chloride or polyquad chloride; guanidine-based preservatives, preferably PHMB or chlorhexidine; chlorobutanol; mercurial preservatives, preferably selected from thimerosal, phenylmercuric acetate and phenylmercuric nitrate; oxidizing preservatives, preferably stabilized oxychloro complexes, more preferably stabilized chlorine dioxide, such as the commercial product Purite®; parabens, such as methylparaben and polypropylparaben.

[0046] According to one embodiment, the ophthalmic formulation according to the present invention does not contain a preservative. Preferably, the tonicity adjusting agent contained in the ophthalmic preparation according to the invention is selected from salts, preferably sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sorbitol, trehalose, mannitol and glycerin.

[0047] Preferably, the viscosity of the ophthalmic preparation according to the present invention at room temperature is 25 to 50 cps. A viscosity adjuster may be added to the preparation to obtain the desired viscosity. Preferably, the viscosity modifier contained in the liquid ophthalmic formulation according to the present invention is selected from polyvinyl alcohol, poloxamer, hyaluronic acid, carbomer, acrylate, cellulose derivative, dextran, polyacrylic acid, povidone, polyethylene glycol, propylene glycol, chitosan, gellan gum, and xanthan gum. Preferably, the cellulose derivative is selected from hydroxypropylmethylcellulose, carboxymethylcellulose, and hydroxyethylcellulose.

[0048] Preferably, the antioxidant contained in the ophthalmic preparation according to the invention is selected from citrate, L-methionine, cysteine, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.

[0049] Preferably, the chelating agent contained in the ophthalmic preparation according to the present invention is edetate disodium. Preferably, when the liquid ophthalmic formulation is an emulsion, the formulation further comprises one or more oils.

[0050] Preferably, the oil is selected from anise oil, castor oil, clove oil, cassia oil, cinnamon oil, almond oil, corn oil, groundnut oil, cottonseed oil, safflower oil, maize oil, linseed oil, rapeseed oil, soybean oil, olive oil, caraway oil, rosemary oil, peanut oil, peppermint oil, sunflower oil, eucalyptus oil, and sesame oil.

[0051] According to a preferred embodiment, the formulation according to the invention does not contain ethanol. The ophthalmic formulations according to the invention may further comprise one or more drugs used in the prevention or treatment of inflammatory and / or autoimmune eye diseases and / or cyclosporine.

[0052] A further object of the invention is a formulation as described above for topical use in the eye in the prevention or treatment of an individual with an inflammatory and / or autoimmune eye disease selected from inflammatory and / or autoimmune diseases of the cornea and ocular surface and inflammatory diseases of the eyelid margin, as defined above.

[0053] The present disclosure also relates to a method for the prevention or treatment of at least one inflammatory and / or autoimmune eye disease selected from inflammatory and / or autoimmune diseases of the cornea and ocular surface and inflammatory diseases of the eyelid margin, the method comprising topically administering to an individual in need thereof an effective amount of isocyclosporine A or an ophthalmologically acceptable salt thereof and / or an ophthalmic formulation as described above.

[0054] The inflammatory and / or autoimmune diseases of the cornea and ocular surface which can be treated with isocyclosporine A or an ophthalmologically acceptable salt thereof are preferably selected from vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC), allergic conjunctivitis, ocular rosacea, uveitis, dry eye disease (DED), ocular pemphigoid (OCP), ocular graft versus host disease (GVHD) and immune corneal ulcer.

[0055] Preferably, said allergic conjunctivitis is seasonal or perennial allergic conjunctivitis. Inflammatory diseases of the eyelid margin which can be treated with isocyclosporine A or an ophthalmologically acceptable salt thereof are inflammatory pterygium and chronic blepharitis.

[0056] According to the present invention, a method of treatment comprises topically administering to the eye isocyclosporine A or an ophthalmologically acceptable salt thereof and / or an ophthalmic formulation as described above for the prevention and treatment of one, two or more of the inflammatory and / or autoimmune eye diseases described above.

[0057] Preferably, said isocyclosporine A or said salt thereof, or ophthalmic formulation is applied topically to the ocular surface or eyelid margin of the eye of said individual. In a preferred embodiment, the isocyclosporin A or the salt thereof is topically applied to the ocular surface of the individual's eye by means of an ophthalmic liquid or semi-liquid formulation.

[0058] Preferably, said isocyclosporin A or said salt thereof is administered once, twice, three times, four or more times daily depending on the medical condition and the severity of the disease being treated. According to one embodiment, the treatment method comprises topical administration of isocyclosporine A or an ophthalmologically acceptable salt thereof and / or an ophthalmic formulation as described above, together with administration of one or more drugs used in the prevention or treatment of inflammatory and / or autoimmune eye diseases.

[0059] Drugs commonly used to treat inflammatory and / or autoimmune eye diseases are corticosteroids and other anti-inflammatory, immunosuppressant and / or immunomodulatory drugs known in the art.

[0060] In one embodiment, the treatment comprises administration of cyclosporine in conjunction with administration of isocyclosporine A or an ophthalmologically acceptable salt thereof and / or an ophthalmic formulation as described above. Topical administration of isocyclosporine A or an ophthalmic acceptable salt or ophthalmic formulation thereof to the eye may be prior to, simultaneous with, or subsequent to administration of one or more drugs commonly used to treat inflammatory and / or autoimmune eye diseases.

[0061] In a particularly preferred embodiment, the inflammatory and / or autoimmune eye diseases treatable with isocyclosporine A or its ophthalmologically acceptable salts and / or ophthalmic formulations thereof are eye diseases that show improvement or remission upon treatment with cortisone and similar corticosteroids.

[0062] Thus, in accordance with this preferred embodiment, isocyclosporine A, or an ophthalmic salt thereof, or an ophthalmic formulation comprising isocyclosporine A and at least one ophthalmically acceptable excipient or diluent, is administered prior to, simultaneously with, or after treatment with cortisone and similar corticosteroids. EXAMPLES

[0063] Experimental section Example 1 - Corneal fluorescein staining The efficacy of an ophthalmic formulation of isocyclosporine A was evaluated in a mouse model of corneal epithelial injury induced by scopolamine.

[0064] Specifically, 15 mice (N=5 / group) were randomly assigned to three groups prior to treatment (baseline) and treated with a subcutaneous injection protocol of 0.5 mg / 0.2 ml scopolamine hydrobromide to induce dry eye as previously described (Yeh S et al. Invest Ophthalmol Vis Sci, 2003, 44(1):124-9).

[0065] The experimental groups consisted of a control group ("CTRL") that did not receive any eye drop treatment, and two treatment groups, one treated with a conventional cyclosporine A trifluoroacetate ophthalmic suspension containing 0.05% cyclosporine A trifluoroacetate in phosphate buffer with 2% Tween 80 ("Old" group), and the other treated with the corresponding isocyclosporine A trifluoroacetate ophthalmic solution containing 0.05% isocyclosporine A trifluoroacetate in phosphate buffer with 2% Tween 80 ("New" group).

[0066] In the treatment group, 10 microliters of the test formulation was instilled into both eyes three times daily for 25 days, starting on day 1 after the first scopolamine injection. Treatments were coded and group assignments were blinded to the technicians administering the treatments and to the investigators assessing the outcomes of the experiment. Group identification was revealed at the end of the analysis.

[0067] In vivo fluorescein staining was performed in each group on days 0, 9, 11, 17, 22, and 25 after scopolamine treatment to confirm the presence of corneal epithelial defects. Corneal fluorescein staining is a valuable clinical tool to assess epithelial viability.

[0068] Digital images were acquired at 10x magnification using cobalt blue light under a slit lamp and analyzed semi-automatically for fluorescein staining using ImageJ software. As shown in Figure 1 , less epithelial damage was observed in the group treated with isocyclosporine A ophthalmic solution (new) compared to the group treated with cyclosporine A (old) or the untreated group (as shown by corneal fluorescein staining).

[0069] Interestingly, this effect was large over the first 17 days of the study, reaching statistical significance on day 9, suggesting a direct effect of the isocyclosporine formulation on corneal epithelial healing (independent of long-term ocular lubrication (eye drops)).

[0070] Tear secretion was also measured in all animals on days 22 and 25 by a modified Schirmer test as described in Rossi S. et al. Arch Ital Biol. 2012 Mar;150(1):15-21.

[0071] As can be seen from FIG. 2, significant differences were also observed in tear secretion at both time points between the different treatment groups, with significantly higher levels in the isocyclosporine-treated group. The levels of cyclosporine and isocyclosporine absorbed into ocular tissues were also assessed in sample animals. Biopsies of relevant tissues were performed in a subgroup of 3 mice / group at three different time points: day 1 (5 min after the first treatment), day 2 (5 min after the first treatment), and day 7 (5 min after the first treatment).

[0072] The tissue was centrifuged, homogenized, and lyophilized. 1 mg of lyophilized tissue was extracted with EtOAc (0.5 ml) at room temperature, and the organic solution was analyzed by HPLC to assess tissue concentrations and relative amounts of cyclosporine A and isocyclosporine A.

[0073] The chromatographic conditions for the HPLC analysis are shown in Tables A and B below. Table A

[0074] [Table A]

[0075] Table B: Mobile Phase (Gradient)

[0076] [Table B]

[0077] Mobile phase A: Dissolve 0.5 ml of trifluoroacetic acid (TFA) in 1000 ml of water. Mobile phase B: Dissolve 0.5 ml of trifluoroacetic acid (TFA) in 1000 ml of acetonitrile.

[0078] The data obtained showed that isocyclosporine A showed a higher biodistribution to the ocular surface and intraocular tissues (>10-fold) compared to cyclosporine A, with significantly less variability between different animals receiving the same treatment.

[0079] The mean detection levels (ng / mg) of isocyclosporine A and cyclosporine A in the cornea, conjunctiva, and intraocular tissues (sclera and uvea) are reported in Table C below. Table C Biodistribution of isocyclosporine A and cyclosporine A in cornea, conjunctiva, and intraocular tissues.

[0080] [Table C]

[0081] The biodistribution of isocyclosporine A and cyclosporine A in intraocular tissues was assessed in the sclera and uvea, which were not separated at the time of dissection. Thus, the term "combined" in Table C above refers to an assessment of biodistribution levels in the sclera and uvea, which were not separated at the time of dissection.

[0082] The time course of conversion of isocyclosporine A to cyclosporine A in ocular tissues was also measured and, as expected, was minimal after both single and repeated dosing. The data obtained indicate that the observed efficacy is clearly attributable to a direct effect of isocyclosporine A, thus leading to a novel and independent mechanism of action.

[0083] Example 2 -Anti-inflammatory properties of topically applied isocyclosporine A eye drops in a rat model of ocular alkali burn The isocyclosporine A trifluoroacetate formulation of Example 1 was also tested in an acute rat model of alkaline ocular burn. Burns were induced by applying a filter paper disk soaked in 1N NaOH to the cornea for 10 seconds.

[0084] The experimental groups (n=6 per group) consisted of a control group ("CTRL") that did not receive any eye drop treatment, and a group ("New") that was treated with the above-mentioned isocyclosporine ophthalmic solution (10 microliters of the test formulation was instilled into each mouse three times over a 24-hour period).

[0085] Conjunctival inflammatory infiltrate was then measured at the end of treatment. A significant reduction in inflammatory infiltrate was observed in the treated group compared to the untreated group, further confirming the active anti-inflammatory properties of topically applied isocyclosporine eye drops.

[0086] Furthermore, the conversion of isocyclosporine A to cyclosporine A as described above was also evaluated in these animals, and no significant interconversion was observed in either the ocular surface and / or intraocular tissues.

[0087] Example 3 -Draize eye irritation test A typical Draize eye irritation test was performed in the right eyes of six healthy rabbits (3 / group) to compare the tolerability of topical ophthalmic formulations of isocyclosporine A over cyclosporine A (Draize, JH, et al, Journal of Pharmacology and Experimental Therapeutics (1944), 82:377-390).

[0088] No behavioral responses such as eye rubbing or signs of eye irritation (i.e., conjunctival reaction, swelling and / or discharge) were observed in either group. However, because an increased blink rate was observed in cyclosporine A-treated animals, we also performed the rabbit blink method described by Li et al. (Li et al., Int. J Pharm, 2008;363(1-2):177-182): 10 s after administration of the eye drops, the blink frequency of the rabbits was recorded for 2 min.

[0089] The frequency was higher in the cyclosporine A group (9 ± 1) compared with the isocyclosporine A group (5 ± 1), p < 0.05. Example 4 -FLIPR assay The ability of isocyclosporin A and cyclosporin A to modulate the activation of ion channels involved in several inflammatory and / or allergic eye diseases was tested in the FLIPR assay.

[0090] The FLIPR assay is used to screen ion channel targets using membrane-permeable fluorescent dyes. In particular, ion channel targets with significant Ca permeability lead to an increase in intracellular calcium, which can be measured using calcium-sensitive dyes (Michelle R. Arkin et al. FLIPR TMAssays for GPCR and Ion Channel Targets. Assay Guidance Manual [Internet]. Bethesda, MD: Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004).

[0091] Specifically, the above formulations containing isocyclosporine A or cyclosporine A at a concentration of 30 μM were tested in the FLIPR assay for their ability to activate or inhibit the ion channels TRPV3, TRPML2, or TRPC4. TRPV3 is an ion channel that plays an important role in itch and inflammation, two characteristic aspects of ocular surface diseases such as VKC and AKC.

[0092] TRPML2 is an ion channel that plays an important role in immune and inflammatory responses. TRPC4 is an ion channel expressed by the corneal epithelium and plays a role in epithelial cell proliferation and migration.

[0093] Specifically, human TRP cells are trypsinized, counted, and seeded into black, clear-bottom 96-well plates at a density of 50,000 cells per well and incubated overnight. The next day, media is removed from the cell plates and 25 μl of assay buffer is added. Red membrane potential dye or Calcium 5 dye solution (10 μl) is added to the wells and incubated at 37° C. for 1 hour. The membrane potential dyes are used to test TRPV3, TRPML2, and TRPC4.

[0094] Dye solutions are prepared in assay buffer. Compound dilutions are performed in 100% DMSO or dH2O and then briefly (<10 min) transferred to intermediate dilutions (5% DMSO / assay buffer or 5% dH2O / assay buffer) just prior to addition to the cell plate.

[0095] For agonist testing: After incubation with the dye, the plate is placed in the FLIPR and fluorescence is monitored every 1.52 seconds. After 20 seconds, IsoCsA, CsA or standard agonists are added to the wells and fluorescence is monitored for 5 minutes at ex / em (excitation / emission wavelengths): 488 nm / 510-570 nm.

[0096] For antagonist testing: After incubation with the dye, add isocyclosporine A or cyclosporine A using the manual multichannel and incubate for 10 minutes at room temperature. The plate is placed in the FLIPR and fluorescence is monitored every 1.52 seconds. After 20 seconds, 10 μl of the appropriate standard agonist is added and fluorescence is monitored for 5 minutes at ex / em: 488 nm / 510-570 nm.

[0097] To validate the assay, reference agonists and antagonists are tested for each channel and values ​​are checked to be within acceptable ranges. The test compounds and reference compounds were evaluated for percent activation and percent inhibition values.

[0098] Although no significant agonist activity was detected for isocyclosporine A compared to cyclosporine A, a significant inhibitory effect was observed. The results of the inhibition assays are reported in Tables 1-3 below.

[0099] Table 1 reports the percentage inhibition of TRPV3 by isocyclosporin A, cyclosporin A and reference compounds. As can be seen from the data below, isocyclosporine A (IsoCsA in the table) has four times more antagonistic ability to this channel than cyclosporine A (CsA in the table).

[0100] Table 1-TRPV3 Inhibition Rate

[0101] [Table 1]

[0102] In the table, 2-APB is 2-aminoethoxydiphenylborate (borinate) (agonist) and RR is ruthenium red (antagonist). 2-APB is the reference standard agonist, i.e. it activates the receptor and causes a biological response, so its inhibition rate of TRPV3 is considered to be equal to 0%.

[0103] Table 2 reports the percentage inhibition of TRPML2 by isocyclosporine A, cyclosporine A and reference compounds. As can be seen from the data below, isocyclosporine A (Iso-CsA in the table) has more than twice the ability to antagonize this channel as cyclosporine A (CsA in the table).

[0104] Table 2 - Inhibition rate of TRPML2

[0105] [Table 2]

[0106] In the table, CaCl2 is the agonist and gadolinium Gd3 is the antagonist. Since CaCl2 acts as an agonist, i.e., activates the receptor and elicits a biological response, its inhibition of TRPML2 is considered to be equal to 0%.

[0107] TRPC4 is an ion channel expressed by the corneal epithelium and plays a role in epithelial cell proliferation and migration (Hua Yang et al, J Biol Chem.2005 Sep 16;280(37):32230-7). Vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) are characterized by hyperproliferative phenomena of the ocular surface. Therefore, molecules that can promote epithelial proliferation and migration in these pathologies may be detrimental.

[0108] Table 3 reports the activation rates of TRPC4 by isocyclosporin A, cyclosporin A and reference compounds. The data below show that isocyclosporine A (Iso-CsA in the table) has no effect on the activation of this channel, but cyclosporine A (CsA in the table) acts as an agonist and its activation leads to undesirable side effects.

[0109] Table 3-TRPC4 activation rate

[0110] [Table 3]

[0111] In the table, Englerin A is the agonist and gadolinium GD3 is the antagonist. Since GD3 acts as an antagonist, i.e. inhibits the receptor and causes a biological response, its activation rate of TRPC4 is considered to be equal to 0%.

Claims

1. A formulation for topical use in the eye in the prevention or treatment of inflammatory and / or autoimmune eye disease in individuals, comprising isocyclosporine A or an ophthalmologically acceptable salt thereof.

2. 2. The formulation of claim 1, wherein the inflammatory and / or autoimmune eye disease is selected from inflammatory and / or autoimmune diseases of the cornea and ocular surface and inflammatory diseases of the eyelid margin.

3. 3. The formulation of claim 2, wherein the inflammatory and / or autoimmune disease of the cornea and ocular surface is selected from vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC), allergic conjunctivitis, ocular rosacea, uveitis, dry eye disease (DED), ocular pemphigoid (OCP), ocular graft-versus-host disease (GVHD), and immune-mediated corneal ulcer.

4. 3. The formulation of claim 2, wherein the inflammatory condition of the eyelid margin is selected from inflammatory pterygium and chronic blepharitis.

5. The salts may be acetate, adipate, ascorbate, benzoate, benzenesulfonate, bicarbonate, hydrogensulfate, butyrate, camphorate, camphorsulfonate, citrate, cyclohexylsulfamate, ethanesulfonate, fumarate, glutamate, glycolate, hemisulfate, 2-hydroxyethylsulfonate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxymaleate, lactate, malic acid salt, or the like of isocyclosporin A. The formulation according to any one of claims 1 to 4, wherein the compound is selected from the group consisting of benzoyl acetate, benzoyl phosphate, maleate, methanesulfonate, 2-naphthalenesulfonate, nitrate, oxalate, pamoate, persulfate, phenylacetate, phosphate, diphosphate, picrate, pivalate, propionate, quinate, salicylate, stearate, succinate, sulfamate, sulfanilate, sulfate, tartrate, tosylate (p-toluenesulfonate), trifluoroacetate, and undecanoate.

6. An ophthalmic formulation comprising isocyclosporin A or an ophthalmologically acceptable salt thereof and at least one ophthalmologically acceptable excipient or diluent.

7. 7. The ophthalmic formulation of claim 6, wherein the ophthalmic formulation is a liquid ophthalmic formulation, preferably a liquid eye drop formulation.

8. 7. The ophthalmic formulation of claim 6, wherein the ophthalmic formulation is a semi-solid ophthalmic formulation, preferably a cream, ointment or gel.

9. 9. The ophthalmic formulation of claim 6, wherein the ophthalmic formulation is a solid ophthalmic formulation for extemporaneous preparation of a liquid or semi-solid ophthalmic formulation of claim 7 or 8 by adding an ophthalmologically acceptable diluent before administration.

10. 9. The ophthalmic formulation according to any one of claims 6 to 8, having a pH comprised between 6 and 8, preferably between 6.4 and 7.8, more preferably between 6.5 and 7.

5.

11. An ophthalmic preparation according to any one of claims 6 to 8 for use in the prevention or treatment of an inflammatory and / or autoimmune eye disease in an individual.

12. 12. The ophthalmic preparation of claim 11, wherein the inflammatory and / or autoimmune eye disease is selected from inflammatory and / or autoimmune diseases of the cornea and ocular surface and inflammatory diseases of the eyelid margin.

13. An ophthalmic preparation according to any one of claims 6 to 8, further comprising at least one drug suitable for the treatment of inflammatory and / or autoimmune eye diseases, preferably a corticosteroid.

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