Combination for ophthalmology

Abstract

The present invention relates to combinations of nerve growth factor and sesamin and their use in the treatment of ophthalmic conditions.

Description

Technical Field

[0001] The present invention relates to a combination of nerve growth factor and sesamin and use thereof in treating ophthalmic disorders. Background Art

[0002] Nerve growth factor (NGF) is a member of the evolutionarily well-conserved neurotrophic growth factor family, which is required for the development and survival of specific neuronal populations and also includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and NT4 / 5.

[0003] NGF exerts its activity by interacting with two structurally unrelated cell surface receptors: the high-affinity receptor tyrosine kinase A (TrKA) and the low-affinity p75 neurotrophin receptor (p75 NTR ).

[0004] NGF, TrKA and p75 NTR It is widely expressed in the visual system and plays a vital role in the nutrition of both the anterior and posterior segments of the eye.

[0005] For example, NGF regulates the development and differentiation of, and promotes survival and recovery after injury to, retinal ganglion cells, photoreceptors, optic nerve, and visual cortex.

[0006] (Carmignoto et al, J Neurosci. 1989, 9: 1263-72; Siliprandi et al, Invest Ophthalmol Vis Sci 1993, 34: 3232-43; Lambiase et al, Graefes Archive for Clinical and Experimental Ophthalmology 1997, 235: 780-5, Lambiase et al. al,Invest.Ophthalmol.Vis.Sci., 2002, 43(7):2334-2340, Garcia et al., Cytokine&GrowthFactors Reviews 2017,34:43-57; Sornelli et al., Molecular Vision 2010;16:1439-1447; Di Girolamo et al., J Cell Mol.Med 2008, 12(6B):2799-2811).

[0007] In the anterior segment, TrKA and p75 NTR Constitutively expressed in the basal epithelial cells and stroma of the conjunctiva, and in the epithelial and endothelial cells of the cornea. Nerve growth factor (NGF) has been shown to maintain ocular surface homeostasis by exerting effects on epithelial cell health, limbal stem cell differentiation, immune regulation, and tear production in vitro, ex vivo, and animal models.

[0008] (Lambaase et al, Curr Opin Ophthalmol. 2012, 23(4): 296-302; Lambiase et al, Arch Ital Biol. 2011, 149(2): 283-292).

[0009] The therapeutic efficacy of NGF in various conditions of the cornea, conjunctiva, and sclera has been demonstrated in animal models and clinical trials, especially in phototoxic keratopathy.

[0010] (Rocco ML et al., Graefes Archive for Clinical and ExperimentalOphthalmology 2018, 256: 729-738),

[0011] Iatrogenic, immune, and neurotrophic corneal epithelial defects and ulcers, scleritis, neurotrophic keratopathy

[0012] (Lambiase, et al., N Engl J Med 1998, 338: 1174-1180; Lambiase et al., Invest Ophthalmol Vis Sci 2000, 41(5): 1063-1069; Lambiase et al, ArchOphthalmol. 2000, 118(10): 1446-1449; Bonini et al. al., Ophthalmology 2000, 107: 1347-1351; Bonini et al, Ophthalmology 2018, 125(9): 1332-1343),

[0013] Neuropathic corneal pain

[0014] (Kenyon et al. Investigative Ophthalmology&Visual Science 2021, 62: 842),

[0015] Keratoconjunctivitis sicca

[0016] (Coassin et al., Graefes Arch Clin Exp Ophthalmol Albrecht Von GraefesArch Klin Exp Ophthalmol 2005, 243(2): 151-155, Sacchetti et al, Br.J. Ophthalmol 2020, 104: 127-135)

[0017] and Sjögren's syndrome-related dry eye ( syndrome-associated dry eye) (https: / / clinicaltrials.gov / ct2 / show / NCT05133180).

[0018] In the posterior segment of the eye, NGF is expressed in the retinal pigment epithelium, Müller cells, and retinal ganglion cells, while NGF receptors are present in the retinal pigment epithelium, photoreceptors, Müller cells, and retinal ganglion cells. NGF receptors have also been shown to be expressed in oligodendrocytes of the optic nerve, and several studies have shown that NGF has a neuroprotective effect on neurons in the optic nerve under pathological conditions.

[0019] The therapeutic efficacy of NGF in retinal and optic nerve disorders has been widely demonstrated, such as in the following: retinitis pigmentosa

[0020] (Lenzi et al, Vision Res. 2005, 45(12): 1491-1500; Sacchetti et al, 2017, Current Eye Research, 42: 7, 1064-1068),

[0021] Retinal detachment (Sun et al., Ophthalmologica 2008, 222: 58-61), diabetic retinopathy (Ali et al., Diabetes, 2008, 57: 889-898), post-ischemic retinal degeneration (Xien et al., Exp Eye Res 2014, 125: 156-63), phototoxic retinopathy

[0022] (Rocco et al., Graefes Arch Clin Exp Ophthalmol 2018, 256: 729-738; Garcia et al., J Neurochem 2014, 131(3): 303-13),

[0023] Epiretinal membrane (Minchiotti et al., Retina 2008, 28(4): 628-37), macular hole (Zhang et al., BMC Ophthalmol 2019, 19(1): 130), macular degeneration (Lambiase et al., Ann lst Super Sanità 2009, 45(4): 439-442), optic neuropathy

[0024] (Mesentier-Louro et al., Mol Neurobiol. 2019, 56(2): 1056-1069; Guo et al Sci Rep 2020, 10: 3375), optic glioma (Falsini et al, Brain J Neurol 2016, 139(Pt2): 404-414), and glaucoma

[0025] (Lambiase et al, Proc Natl Acad Sci USA 2009, 106(32): 13469-13474; Lambiase et al, Graefes Arch Clin Exp Ophthalmol Albrecht Von Graefes ArchKlin Exp Ophthalmol 1997, 235(12): 780-785).

[0026] Pharmacokinetic studies have shown that after topical administration of NGF at a concentration of 200 μg / mL, high levels of the protein were detected in all ocular tissues, including the retina and optic nerve, with a peak increase 6 hours after administration (Lambiase et al, IOVS 2005. Vol. 46(10): 3800-3806). Therefore, this route of administration is also suitable for treating pathological conditions of the retina and optic nerve.

[0027] Topical administration of NGF on the ocular surface has been shown to be safe in clinical trials. However, the main side effects reported by patients following topical ocular administration of NGF in clinical trials or use of NGF for approved indications are ocular discomfort, including eye pain, eye or eyelid irritation, eye itching, and abnormal sensations in the eye. This side effect is thought to be at least partially related to the therapeutic activity of NGF, as restoration of ocular innervation and sensitivity may be associated with an increase in ocular surface symptoms. However, this has not been fully investigated.

[0028] Although the above-mentioned side effects are mild and transient, in a few cases, they lead to cessation of treatment and cause suffering to the patient. In addition, they may interfere with the evaluation of the treatment of ocular conditions characterized by similar symptoms, such as neuropathic corneal pain, dry eyes and ocular autoimmune diseases, in which patients experience eye pain, burning and stinging as pathological symptoms. In these cases, the therapeutic effect of NGF may be masked or significantly weakened by the above-mentioned side effects. In addition, this side effect is particularly enhanced when topical NGF is used to treat pathological conditions in the posterior segment of the eye and therefore high concentrations of NGF are used.

[0029] Therefore, there is an urgent need to develop NGF ophthalmic compositions that do not cause eye discomfort.

[0030] Sesamin, whose IUPAC name is 5,5'-[(1S,3aR,4S,6aR)-tetrahydro-1H,3H-furo[3,4-c]furan-1,4-diyl]bis(2H-1,3-benzodioxole), is a lignan abundant in sesame seeds and sesame oil that has been described to have many different physiological effects and is widely used as a dietary supplement, especially in Asia. For example, sesamin has been described to exert hypoglycemic effects, reactive oxygen species (ROS) scavenging activity, antioxidant and anti-inflammatory activities, anxiolytic effects, and to inhibit diabetes-related cognitive decline.

[0031] (Farbood et al, Life Sciences 2019, 230:169-177; Guo et la, Nutritional Neuroscience 2016:19(6):231-236). Summary of the invention

[0032] The present inventors have now surprisingly found that sesamin, when administered in combination with nerve growth factor (NGF), is able to counteract the above-mentioned side effects associated with topical administration on the ocular surface.

[0033] Therefore, a first object of the present invention is a combination of nerve growth factor (NGF) and sesamin for use in the treatment of ocular disorders by topical administration.

[0034] A second object of the present invention is an ophthalmic composition comprising nerve growth factor (NGF), sesamin and at least one ophthalmologically acceptable excipient.

[0035] A third object of the present invention is a kit comprising a) an ophthalmic composition containing nerve growth factor (NGF) and b) an ophthalmic composition containing sesamin.

[0036] A fourth object of the present invention is a method for treating an ophthalmic disorder comprising topically administering the above combination or ophthalmic composition to a patient in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Shown are the levels of Atg5 (Panel A), Beclin (Panel B), LC3-I (Panel C), LC3-II (Panel D), and P62 (Panel E) in control cells (CTR) and DRG after treatment with NGF (NGF) or a combination of NGF and sesamin (NGF+DF24437), measured as described in Example 1 and expressed as relative optical units.

[0038] Figure 2 Shown are the number of eye rubs following ocular administration of Vehicle 1 (Veh 1), Vehicle 2 (Veh 2), rhNGF in Vehicle 1 (rhNGF), and rhNGF and sesamin in Vehicle 2 (sesamin+rhNGF), as described in Example 2. Data are shown as mean ± SEM (n=4 to 8). *** p<0.001 vs vehicle 1 and vehicle 2 groups; ○○○ p<0.001vs rhNGF group. DETAILED DESCRIPTION

[0039] The present invention is based on the observation that sesamin has the ability to alleviate ocular discomfort induced by administration of nerve growth factor to the ocular surface.

[0040] Therefore, a first object of the present invention is a combination of nerve growth factor (NGF) and sesamin for use in the treatment of ocular disorders.

[0041] Preferably, the ocular disorder is an ocular disorder known to be treatable by NGF.

[0042] Preferably, the combination is administered topically, more preferably topically to the eye.

[0043] According to one embodiment, the combination is contained in a single ophthalmic composition.

[0044] Therefore, a second object of the present invention is an ophthalmic composition comprising nerve growth factor (NGF) and sesamin.

[0045] The ophthalmic compositions according to the present invention are conveniently administered topically to the eye.

[0046] According to an alternative embodiment, the combination is a combination of two ophthalmic compositions, one comprising nerve growth factor (NGF) and the other comprising sesamin, which are administered simultaneously or sequentially.

[0047] Therefore, a third object of the present invention is a kit comprising:

[0048] a) an ophthalmic composition comprising nerve growth factor (NGF), and

[0049] b) An ophthalmic composition comprising sesamin.

[0050] Preferably, the nerve growth factor (NGF) is human nerve growth factor (hNGF).

[0051] Preferably, the NGF has the following amino acid sequence of SEQ.ID NO.1:

[0052] SEQ.ID NO.1:

[0053] SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR

[0054] Alternatively, the NGF has the following amino acid sequence of SEQ ID NO.2:

[0055] SEQ ID NO.2:

[0056] SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA

[0057] Alternatively, the NGF is a mixture of NGFs having the sequences of SEQ ID NO.1 and SEQ ID NO.2.

[0058] Preferably, the NGF is recombinant hNGF (rhNGF) produced by recombinant DNA technology. Methods for producing rhNGF are known to those skilled in the art, such as those described in WO0022119A1 and WO2013092776A1.

[0059] Preferably, the purity of the NGF is higher than 70%, more preferably higher than 80%, higher than 90%, higher than 95%, higher than 98% or higher than 99%. The purity of NGF can be determined by conventional means known to those skilled in the art, such as by HPLC analysis.

[0060] Preferably, the sesamin is in the form of a purified compound, preferably with a purity higher than 70%, more preferably higher than 80%, higher than 90% or higher than 95%.

[0061] When referring to the percentage purity of a protein or compound in this application, it means the mass of the pure protein or compound divided by the total mass of the protein or compound sample multiplied by 100%.

[0062] Therefore, all purity percentages in this application are expressed as w / w. The purity of NGF or sesamin can be determined by conventional means known to those skilled in the art, such as by HPLC analysis.

[0063] According to an alternative embodiment, preferably, the sesamin is in an unpurified form as a component of sesame oil.

[0064] Preferably, the ophthalmic composition according to the second object of the present invention or the ophthalmic composition of the kit according to the third object of the present invention further comprises at least one ophthalmologically acceptable excipient.

[0065] Preferably, the ophthalmologically acceptable excipient is selected from the following: ophthalmologically acceptable viscosity enhancers, permeation enhancers, buffers, osmotic regulators, preservatives, antioxidants, surfactants, emulsifiers and polyethylene glycol.

[0066] The viscosity enhancer is preferably selected from: polyvinyl pyrrolidone; cellulose ethers, preferably hydroxymethylcellulose, hydroxyethylcellulose (HEC), hydroxypropylmethylcellulose (HPMC) and methylcellulose; and gelling agents, preferably gellan gum, xanthan gum and carbopol-974. The preferred viscosity enhancer according to the present invention is hydroxypropylmethylcellulose.

[0067] The penetration enhancer is preferably selected from cyclodextrins, chelating agents, crown ethers, bile acids and bile salts.

[0068] Buffers are compounds capable of providing and maintaining a pH of the ophthalmic composition compatible for use in the eye, preferably a pH of 6.5 to 8. The preferred buffer is a phosphate buffer, but other buffers suitable for ophthalmic use that are capable of maintaining the pH in the desired range are also included.

[0069] Osmolarity modifiers are salts that can make ophthalmic compositions isotonic with ocular fluids. The preferred salt is sodium chloride (NaCl), but other ophthalmologically acceptable salts such as potassium chloride (KCl), calcium chloride (CaCl2), and magnesium chloride (MgCl2) and mixtures thereof may also be used.

[0070] The preservative is preferably selected from quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0071] The surfactant and emulsifier are preferably selected from the group consisting of sodium linoleic acid, Kolliphor HS15, polysorbates (preferably Tween 80) and poloxamers (preferably Kolliphor P 188).

[0072] The term "Kolliphor P188" as used herein refers to the block copolymer Poloxamer 188, which is a synthetic copolymer of ethylene oxide and propylene oxide represented by the following chemical structure:

[0073]

[0074] where the a and b blocks have the following values:

[0075]

[0076] As used herein, "Kolliphor HS15", also known as polyethylene glycol (15)-hydroxystearate, refers to a pH-independent nonionic surfactant having the following structure: polyethylene glycol (15)-hydroxystearate.

[0077] Preferably, the ophthalmic composition according to the second object of the invention or the ophthalmic composition of the kit according to the third object of the invention comprises one or more excipients selected from the group consisting of trehalose, mannitol, cellulose ethers, buffers, surfactants, emulsifiers, polyethylene glycols and antioxidant compounds.

[0078] According to a preferred embodiment, the ophthalmic composition according to the second object of the present invention or the ophthalmic composition of the kit according to the third object of the present invention comprises trehalose, mannitol, cellulose ether, buffer and polyethylene glycol as excipients.

[0079] According to another preferred embodiment, the sesamin-containing ophthalmic composition of the kit according to the third object of the present invention comprises a buffer and polyethylene glycol as excipients.

[0080] The combination according to the first object of the invention, the ophthalmic composition according to the second object of the invention or the ophthalmic composition of the kit according to the third object of the invention is in a form suitable for topical administration to the ocular surface.

[0081] Thus, the combination according to the first object of the invention, the ophthalmic composition according to the second object of the invention or the ophthalmic composition of the kit according to the third object of the invention is conveniently administered topically to the eye.

[0082] The ophthalmic composition according to the second object of the present invention or the ophthalmic composition of the kit according to the third object of the present invention is preferably in the form of a ready-to-use liquid ophthalmic preparation or a powder or granular preparation to be dissolved in a suitable carrier to form a liquid ophthalmic preparation when used, preferably in the form of a lyophilized powder. According to a preferred embodiment, the ophthalmic composition according to the second object of the present invention is in the form of a liquid ophthalmic preparation.

[0083] According to an alternative preferred embodiment, the ophthalmic composition according to the second object of the present invention is in the form of a powder formulation, preferably a freeze-dried powder formulation.

[0084] According to a preferred embodiment, in the kit according to the invention, at least one of the ophthalmic compositions a) and b) is in the form of a liquid ophthalmic preparation.

[0085] Preferably, in the kit according to the present invention, both the ophthalmic composition comprising nerve growth factor (NGF) and the ophthalmic composition comprising sesamin are in the form of liquid ophthalmic preparations.

[0086] Preferably, in the kit according to the present invention, one of the ophthalmic composition comprising nerve growth factor (NGF) and the ophthalmic composition comprising sesamin is in the form of a liquid ophthalmic preparation and the other is in the form of a powder preparation, preferably a freeze-dried powder preparation.

[0087] More preferably, the ophthalmic composition a) comprising nerve growth factor (NGF) is in the form of a powder preparation (preferably a lyophilized powder preparation), and the ophthalmic composition b) comprising sesamin is in the form of a liquid ophthalmic preparation.

[0088] Even more preferably, said liquid ophthalmic composition b) comprising sesamin is a sesamin-containing reconstitution solvent of said powder (eg lyophilized powder) ophthalmic composition a) comprising nerve growth factor (NGF).

[0089] According to an alternative preferred embodiment, in the kit according to the present invention, both the ophthalmic composition comprising nerve growth factor (NGF) and the ophthalmic composition comprising sesamin are in the form of powder preparations.

[0090] "Liquid ophthalmic preparation" according to the present invention means a preparation suitable for administration on the ocular surface and dispensed from a dropper in small volume units (eg drops).

[0091] Preferably, the liquid ophthalmic formulation comprises NGF and / or sesamin and at least one of the ophthalmologically acceptable excipients, as described above, dissolved or suspended in a liquid carrier, preferably an aqueous liquid carrier.

[0092] Preferably, the osmolarity of the liquid ophthalmic formulation is from 200 to 380 mOsmol / kg, more preferably from 250 to 330 mOsmol / kg, even more preferably from 280 to 320 mOsm / Kg.

[0093] Preferably, the pH of the liquid ophthalmic formulation is from 6.5 to 8, more preferably from 6.8 to 7.5, even more preferably from 7 to 7.4.

[0094] When the liquid ophthalmic preparation comprises NGF, as described above, NGF is present in the liquid ophthalmic preparation at a concentration of preferably 5 μg / ml to 300 μg / ml.

[0095] When the ophthalmic preparation is used to treat ocular conditions affecting the anterior part of the eye, preferably the cornea, conjunctiva and sclera, the NGF is present in the liquid ophthalmic preparation at a concentration of 5 μg / ml to 50 μg / ml, more preferably 10 μg / ml to 50 μg / ml, even more preferably 10, 20, 30 or 40 μg / ml.

[0096] Alternatively, when the ophthalmic preparation is used to treat an ophthalmic condition affecting the back of the eye, preferably the retina or optic nerve, the NGF is present in the liquid ophthalmic preparation at a concentration of preferably 50 μg / ml to 300 μg / ml, more preferably 100 μg / ml to 250 μg / ml, even more preferably 150 or 200 μg / ml.

[0097] When the liquid ophthalmic preparation contains sesamin, sesamin is present in the liquid ophthalmic preparation at a concentration of preferably 0.5 to 20 μg / ml.

[0098] According to a preferred embodiment, the sesamin is present in the liquid ophthalmic preparation at a concentration of 2 to 20 μg / ml, preferably 2 to 15 μg / ml, more preferably 3 to 5 μg / ml.

[0099] According to another preferred embodiment, said sesamin is present in said liquid ophthalmic preparation in a concentration of 0.5 to 10 μg / ml, preferably 0.5 to 8 μg / ml, even more preferably 0.5 to 2 μg / ml, most preferably 1 to 2 μg / ml, conveniently 1.5 to 2 μg / ml.

[0100] Thus, when sesamin is present in the formulation in an unpurified form in sesame oil, the amount of sesame oil present in the liquid ophthalmic formulation will be, for example, an amount that results in the above-mentioned concentration of sesamin.

[0101] Preferably, when the liquid ophthalmic formulation comprises sesamin, the vehicle is an aqueous vehicle further comprising an ophthalmologically acceptable organic solvent, preferably dimethyl sulfoxide (DMSO).

[0102] Preferably, the DMSO is present in the liquid ophthalmic formulation in a concentration of 0.1% to 1.2% v / v, more preferably 0.2% to 0.9% v / v, even more preferably 0.2% or 0.6% v / v.

[0103] Preferably, the liquid ophthalmic formulation of the present invention comprises one or more excipients selected from the group consisting of trehalose, mannitol, cellulose ethers, buffers, polyethylene glycols, surfactants, emulsifiers and optionally antioxidant compounds.

[0104] According to one embodiment, the liquid ophthalmic formulation of the present invention comprises trehalose, mannitol, a cellulose ether, a buffer, polyethylene glycol and optionally an antioxidant compound.

[0105] According to another embodiment, the liquid ophthalmic preparation of the present invention comprises a buffer and polyethylene glycol. This embodiment is particularly preferred for the sesamin-containing composition of the kit according to the third object of the present invention.

[0106] According to another embodiment, the liquid ophthalmic formulation of the present invention comprises trehalose, mannitol, a buffer, one or more surfactants and optionally an antioxidant compound.

[0107] Preferably, the molecular weight of the polyethylene glycol is 4000 to 8000 g / mol, more preferably 5000 to 7000 g / mol, more preferably 5500 to 6500 g / mol, even more preferably 6000 g / mol. Preferably, when the molecular weight of the polyethylene glycol is 6000 g / mol (PEG6000), it is present in the liquid ophthalmic preparation at a concentration of 5 to 15 mg / ml, preferably 8 to 12 mg / ml.

[0108] Preferably, the antioxidant compound is selected from L-methionine and cysteine.

[0109] Preferably, the antioxidant compound L-methionine is present in the liquid ophthalmic formulation in a concentration of 0.005 to 0.02 mg / ml, more preferably 0.01 mg / ml.

[0110] Preferably, trehalose is present in the liquid ophthalmic formulation in a concentration of 30 to 60 mg / ml, more preferably 40 to 55 mg / ml, more preferably 45 to 53 mg / ml, even more preferably 45 mg / ml and 50 mg / ml.

[0111] Preferably, mannitol is present in the liquid ophthalmic formulation in a concentration of 5 to 20 mg / ml, more preferably 10 to 15 mg / ml.

[0112] Preferably, the concentration of the cellulose ether in the liquid ophthalmic preparation is, for example, a concentration to obtain a kinematic viscosity of about 1 to 25 cSt as determined using a capillary viscometer at 25°C.

[0113] Preferably, the cellulose ether is hydroxypropyl methylcellulose, more preferably in a concentration of 0.5 to 2 mg / ml, preferably 0.8 to 1.2 mg / ml.

[0114] Preferably, the buffer in the liquid ophthalmic preparation has a composition and concentration, for example, to obtain a liquid ophthalmic preparation pH of 6.5 to 8, more preferably 6.8 to 7.5, even more preferably 7 to 7.4. Preferably, the buffer is a phosphate buffer and / or sodium hydroxide.

[0115] Preferably, the one or more surfactants are Kolliphor P188 (Poloxamer 188) and / or Kollihor HS15 as defined above; most preferably, each of the one or more surfactants is present in the liquid ophthalmic formulation in a concentration of 1 to 10 mg / ml, more preferably 1.5 to 6 mg / ml, more preferably 1.5 to 5 mg / ml.

[0116] Preferably, when the ophthalmic composition according to the second object of the present invention or the ophthalmic composition of the kit according to the third object of the present invention is in powder form, it can be obtained by freeze-drying the above-mentioned liquid ophthalmic preparation, more preferably, it is obtained by freeze-drying the above-mentioned liquid ophthalmic preparation.

[0117] According to one embodiment, in the kit according to the present invention, the ophthalmic composition a) comprising nerve growth factor (NGF) is in the form of a powder preparation, preferably a lyophilized powder preparation, and the ophthalmic composition b) comprising sesamin is in the form of a liquid ophthalmic preparation.

[0118] According to this specific embodiment, the ophthalmic composition a) comprises NGF and at least one or more excipients selected from the group consisting of trehalose, mannitol, cellulose ethers, buffers, polyethylene glycols, surfactants, emulsifiers and optionally antioxidant compounds.

[0119] Preferably, the ophthalmic composition a) comprises NGF according to the concentrations defined above as well as trehalose, mannitol, a buffer, one or more surfactants and optionally an antioxidant compound.

[0120] More preferably, the ophthalmic composition a) comprises NGF as well as trehalose, mannitol, a buffer, Kolliphor P188 and optionally an antioxidant compound, according to the concentrations defined above.

[0121] Advantageously, the ophthalmic composition a) comprising nerve growth factor (NGF) in the form of a powder formulation, preferably a lyophilized powder formulation, can be used as a component of a kit according to the present invention to provide a convenient and economical method for providing stable lyophilized NGF, wherein the lyophilized NGF is in a form that can be quickly and easily reconstituted in an appropriate reconstitution carrier containing sesamin for administering NGF and sesamin to patients in need of treatment.

[0122] Advantageously, the ophthalmic composition b) containing sesamin in the form of a liquid ophthalmic preparation of the kit according to the present invention can be the sesamin-containing reconstitution solvent of the powder (e.g., lyophilized powder) ophthalmic composition a) containing nerve growth factor (NGF) of the kit according to the present invention.

[0123] Thus, the ophthalmic composition b) comprises sesamin, an ophthalmologically acceptable organic solvent and one or more ophthalmologically acceptable excipients, as described above.

[0124] Preferably, the ophthalmic composition b) comprises sesamin, an ophthalmologically acceptable organic solvent, an osmotic regulator and optionally a surfactant.

[0125] Preferably, the sesamin is present in the ophthalmic composition b) at a concentration of 0.25 to 2.5 μg / ml, more preferably 0.5 to 2 μg / ml.

[0126] Preferably, the ophthalmically acceptable organic solvent is dimethyl sulfoxide (DMSO) and is present in the liquid ophthalmic composition b) in a concentration of 0.1% to 1.2% v / v, more preferably 0.2% to 0.9% v / v, even more preferably 0.2% or 0.6% v / v.

[0127] Preferably, the osmolarity adjusting agent is sodium chloride (NaCl) and is present in the ophthalmic composition b) in a concentration of 0.5 to 3 mg / ml, more preferably 1 to 2 mg / ml, even more preferably 1.5 mg / ml.

[0128] Preferably, the surfactant is Kolliphor HS15 and is present in the ophthalmic composition b) in a concentration of 0.10% w / v to 0.20% w / v, more preferably 0.15% w / v.

[0129] Preferably, the ophthalmic composition b) according to the present invention is an aqueous composition.

[0130] Surprisingly, as shown in the following examples, when an ophthalmic composition a) comprising nerve growth factor (NGF) in the form of a powder formulation, preferably a lyophilized powder formulation, according to the present invention is reconstituted with said ophthalmic composition b) comprising sesamin, the reconstituted ophthalmic composition thus obtained advantageously shows a reduction in the oxidation rate of NGF during the storage period.

[0131] As discussed above and as demonstrated in the experimental section, the inventors have found that sesamin improves the tolerability of NGF when applied topically to the eye for the treatment of ocular conditions. In particular, in the presence of sesamin, ocular discomfort associated with NGF administration is greatly reduced.

[0132] Therefore, another object of the present invention is a combination, an ophthalmic composition or a kit as described above for use in the treatment of an ocular disorder.

[0133] Preferably, the ocular disorder is an ocular disorder known to be treatable by NGF.

[0134] Preferably, the use is by topical administration.

[0135] Preferably, the ophthalmic composition according to the second object of the invention or the ophthalmic composition of the kit according to the third object of the invention is used for treating an ocular disorder known to be treatable by NGF, wherein the composition is topically administered to the eye.

[0136] Preferably, the ocular disorder known to be treatable by NGF is a disorder of the cornea, conjunctiva, sclera, retina or optic nerve.

[0137] More preferably, the ocular condition known to be treatable by NGF is selected from: toxic and phototoxic keratopathy, iatrogenic, immune and neurotrophic corneal epithelial defects and ulcers, neurotrophic keratopathy, neuropathic corneal pain, keratoconjunctivitis sicca, dry eye associated with Sjögren's syndrome, retinitis pigmentosa, retinal detachment, post-ischemic retinal degeneration, phototoxic retinopathy, epiretinal membrane, macular hole, macular degeneration, optic neuropathy, glaucoma, scleritis, and episcleritis.

[0138] The number of daily applications of the ophthalmic composition of the present invention and the number of drops used in each application will vary with the NGF concentration and patient characteristics, such as age, type and severity of the condition, duration of treatment, presence of concurrent treatments, and similar factors within the knowledge and expertise of those skilled in the art. Typically, each application comprises instilling one or two drops of each of the ophthalmic composition according to the second purpose of the present invention or the composition of the kit according to the third purpose of the present invention in the form of a liquid ophthalmic preparation.

[0139] The ophthalmic compositions of the kit according to the third object of the present invention are respectively administered to patients.

[0140] In order to prevent ocular discomfort induced by topical application of NGF, the liquid ophthalmic composition a) and the liquid ophthalmic composition b) of the kit according to the third object of the present invention are preferably administered essentially simultaneously or sequentially in any order, with an interval of no more than 5 minutes, preferably no more than 2 minutes.

[0141] Experimental Section

[0142] Example 1

[0143] Autophagy is a key adaptive mechanism triggered by stress or injury. Autophagy occurs at low basal levels in all cells but can be activated by many stress stimuli under pathological conditions (e.g., starvation, inflammation) and by pharmacological agents.

[0144] Autophagy is a self-digestion process that participates in the degradation of proteins and organelles to improve the survival rate of cells in stress environments. There are three types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy (chaperonemediatedautophagy, CMA) (Glick et al, J.Pathol.2010, 221: 3–12). It has been shown that the change of macroautophagic activity in dorsal root ganglia (DRG) under injury conditions (Guo et al, Neurosci.Lett.2015, 599: 158–163). At the beginning of the macroautophagic process, Beclin-1 (upstream protein of the macroautophagic process) on the endoplasmic reticulum responds to the stress signal transduction pathway and initiates the formation of phagophores. In the next step, autophagy-related protein 5 (Atg5), microtubule-associated protein 1A / 1B-light chain 3 (LC3)-II and p62 participate in autophagosome formation (Glick et al, J. Pathol. 2010, 221: 3–12). Then, the autophagosome fuses with the lysosome, leading to the formation of autolysosomes and further degradation. When macroautophagy activity increases, the expression levels of LC3-II, Beclin-1 and Atg5 increase, but the level of p62 (delivering ubiquitinated cargo to the autophagosome) decreases.

[0145] The effect of NGF on autophagy in the presence and absence of sesamin was evaluated in dorsal root ganglion (DRG) neurons involved in the occurrence of NGF sensory side effects by analyzing the effects of different treatments on the levels of the above proteins involved in the autophagic pathway.

[0146] F11 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Cat# D6546; Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum and 2 mM glutamine (Sigma-Aldrich, USA). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.

[0147] Then, cells were plated at 7000 cells / cm 2 Seed and maintained in low serum medium for almost 2 weeks to induce differentiation into DRG. The complete composition of low serum medium is: DMEM supplemented with 2mM glutamine, 1% FBS (Sigma-Aldrich, St.Louis, MO, USA). The medium was replaced every 2 days. Immunofluorescence was used to determine cell differentiation (data not shown).

[0148] F11 cells maintained in 10% FBS medium were used as controls (control, undifferentiated). After two weeks of treatment with low serum, morphological and functional analyses were performed when cells were fully differentiated. Undifferentiated control cells underwent the same analysis at 7 days to avoid significant cell death due to confluence.

[0149] Fully differentiated cells were treated with 1 μM rhNGF alone or in combination with 30 nM sesamin for 24 hours.

[0150] Control and treated cells were collected and lysed in ice-cold RIPA buffer (Thermo Scientific, USA) with freshly added protease and phosphatase inhibitors. Protein lysates (30 μg) were separated on 4% to 12% precast gels using MOPS or MES buffer (Thermo Scientific, USA), depending on the size of the target protein. The proteins were then electroblotted on polyvinyldifluoride membranes (PVDF; Sigma, StLoius, USA), and nonspecific binding sites were blocked for 15 minutes at room temperature in blocking buffer (Thermo Scientific).

[0151] Afterwards, the membrane was incubated overnight at 4°C with the following primary antibodies diluted in blocking buffer: rabbit anti-Atg5 1:1000 (Abcam, ab228668, UK), rabbit anti-LC3 1:2000 (Abcam, ab192890, UK), rabbit anti-Beclin 1:2000 (Abcam, ab207612, UK), rabbit anti-P62 1:500 (Abcam, ab91526, UK), rabbit anti-vinculin 1:1000 (Abcam, ab219649, UK) and HRP-conjugated actin 1:1000 (Cell Signaling, #5125, USA). Peroxidase-conjugated anti-rabbit IgG 1:10000 (Abcam, UK) was used as a secondary antibody. Immunoreactive bands were visualized by ECL (Thermo Scientific, USA) according to the manufacturer's instructions. Using ImageJ software, the relative optical density of the immunoreactive bands was determined and normalized to that of anti-vinculin or anti-actin (depending on the molecular weight of the protein analyzed and the gel / buffer used). The phosphorylated form was normalized to the total form.

[0152] Figure 1The results of the analysis are shown, wherein the amounts of Atg5, Beclin-1, LC3-I and LC3-II detected after each treatment are expressed as relative optical density units (RU). As can be seen from the figure, treatment with NGF alone significantly increases the levels of Atg5 and Beclin necessary for performing autophagy, while reducing the level of p62 (autophagy-specific substrate). In addition, the combination of increased levels of microtubule-associated proteins LC3-I and LC3-II and the reduction of autophagy substrate p62 are consistent with active autophagy-mediated protein degradation. When sesamin is combined with NGF, autophagy is significantly reduced. The results obtained show that sesamin can inhibit the stress response induced by treating cells with NGF.

[0153] Example 2

[0154] An eye wiping test was performed to evaluate ocular discomfort induced by ocular administration of rhNGF alone or in combination with sesamin.

[0155] Experiments were performed on male Sprague-Dawley rats (300 g to 320 g) housed in the animal care facility of the Department of Pharmacy, University of Naples (Charles River, Italy). Food and water were available ad libitum.

[0156] The rats were divided into 4 experimental groups:

[0157] Group 1 - Vehicle 1 control group.

[0158] Animals were treated with Vehicle 1 in water for injection (WFI) having the following composition:

[0159] Element concentration Trehalose dihydrate 50.40mg / ml Mannitol 13.1mg / ml Anhydrous Na2HPO4 3.06mg / mL Na2H2PO4 dihydrate 1.16mg / mL HPMC 1.07mg / ml PEG 6000 10.7mg / ml

[0160] Group 2 - Vehicle 2 control group.

[0161] Animals were treated with Vehicle 2 in water for injection (WFI) having the following composition:

[0162] Element concentration Trehalose dihydrate 50.40mg / ml Mannitol 13.1mg / ml Anhydrous Na2HPO4 3.06mg / mL Na2H2PO4 dihydrate 1.16mg / mL HPMC 1.07mg / ml PEG 6000 10.7 mmg / ml DMSO 0.6％

[0163] Group 3 - rhNGF treated group:

[0164] Animals were treated with 20 μg / ml rhNGF in Vehicle 1 (n=8);

[0165] Group 4-rhNGF+sesamin treatment group:

[0166] Animals were treated with 20 μg / ml rhNGF and 3.5 μg / ml sesamin in Vehicle 2 (n=8).

[0167] All tested compositions were prepared immediately prior to use from lyophilized placebo or rhNGF reconstituted in a 1 mL volume of vehicle.

[0168] Animal care complied with Italian legislation on the protection of animals used for experimental and other scientific purposes, as well as with EEC regulations (OJ of ECL 135358 / 1 12 / 18 / 1986).

[0169] The above treatment was applied as eye drops once a day in a volume of 10 μL. After application, an eye rub test was performed.

[0170] The animals were placed on a 50 x 50 cm table for a 10 minute acclimation period. 10 μL of the solution was applied to the left eye of the animal and the number of eye rubs was counted over a 30 minute period.

[0171] The test results are in Figure 2 Shown in.

[0172] As can be seen from the results obtained, rats treated with rhNGF alone showed a significant increase in the number of eye rubbing compared to rats treated with Vehicle 1 or Vehicle 2 (respectively, *** p<0.001), while mice receiving the combination of rhNGF and sesamin produced modestly increased or no significant increase in the number of eye rubbings relative to either Vehicle 1 or Vehicle 2. In addition, significant differences were observed between the rhNGF and sesamin+rhNGF groups ( ○○○ p<0.001).

[0173] Therefore, sesamin can alleviate the ocular discomfort induced by rhNGF.

[0174] All data are expressed as mean ± SEM. Data analysis was performed using GraphPad Prism 8. Statistical analysis was performed by two-way ANOVA followed by Tukey's test for multiple comparisons, as appropriate. Statistical significance was set at * p<0.05.

[0175] Example 3

[0176] Preparation of the formulation according to the invention

[0177] Formulation 1 – Liquid ophthalmic formulation containing NGF and sesamin

[0178] The following excipients were weighed and dissolved in an appropriate volume of WFI: trehalose dihydrate (5.04% w / v), mannitol (1.31% w / v), anhydrous Na2HPO4 (0.3065% w / v), NaH2PO4*H2O (0.116% w / v), hydroxypropyl methylcellulose (0.107% w / v), PEG 6000 (1.07% w / v).

[0179] Wait for the excipient to dissolve completely.

[0180] Separately, about 2 to 4 mg of sesamin powder was dissolved in 1, 2 or 3 mL of DMSO, and the solution was shaken to obtain complete dissolution of the powder. The dissolved sesamin was added to solution A and stirred for 5 minutes. WFI was added until the defined volume (500 mL) and filtered with a 0.22 μm filter.

[0181] Defined concentrations of rhNGF were added.

[0182] Formulation 2: Liquid ophthalmic formulation containing NGF

[0183] Weigh the following excipients and dissolve them in an appropriate volume of WFI: Trehalose dihydrate (5.04% w / v), mannitol (1.31% w / v), anhydrous Na2HPO4 (0.3065% w / v), NaH2PO4*H2O (0.116% w / v), hydroxypropyl methylcellulose (0.107% w / v), PEG 6000 (1.07% w / v) in WFI. Wait for the excipients to dissolve completely. Check the pH (about 7.3). Add WFI until the defined volume (e.g., liters (Lt)). Filter the preparation using a 0.22 μm filter. Add defined concentration of rhNGF.

[0184] Preparation 3: Liquid ophthalmic preparation containing sesamin

[0185] Solution A : Weigh the following excipients and dissolve them in an appropriate volume of WFI: Trehalose dihydrate (2.52% w / v), mannitol (0.65% w / v), hydroxypropyl methylcellulose (0.0535% w / v), PEG 6000 (0.535% w / v) in WFI. Wait for the excipients to dissolve completely. Add WFI until the defined volume (e.g. 1 liter).

[0186] Solution B : About 9 mg of sesamin powder was dissolved in 10 mL of DMSO, and the solution was shaken to obtain complete dissolution of the powder.

[0187] Add 300 μL of solution B to 50 mL of solution A. Stir for 5 minutes. Aliquot the final formulation into glass vials for the lyophilization process.

[0188] After the lyophilization process, the cake was reconstituted with WFI and filtered through 0.22 μm.

[0189] Preparation 4: Liquid ophthalmic preparation containing sesamin

[0190] 50 to 100 μL of sesame oil was added to 5 mL of WFI and homogenized by a homogenizer Ultraturraxt25 basic (IKA) at 15000 rpm for 5 minutes using an 8G probe.

[0191] PEG 6000 (final concentration 0.25%) was added to the first emulsion and homogenized at 15000 rpm for another 5 minutes.

[0192] Preparation 5: Liquid ophthalmic preparation containing sesamin

[0193] About 45 mg of micronized sesamin powder (sesamin + PEG6000, or sesamin + trehalose, or sesamin + mannitol) was weighed and dissolved in 500 mL of WFI (or WFI + 0.6% DMSO). The preparation was stirred for 10 minutes and filtered with a 0.22 μm filter.

[0194] Formulation 6: Lyophilized NGF ophthalmic formulation and reconstitution solvent containing sesamin

[0195] The following excipients were weighed and dissolved in an appropriate volume of WFI: trehalose dihydrate (5.04% w / v), mannitol (1.31% w / v), anhydrous Na2HPO4 (0.3065% w / v), NaH2PO4*H2O (0.116% w / v), Kolliphor P188 (0.4% w / v), L-methionine (0.001% w / v), 1.0 N sodium hydroxide quantified to pH 7.20±0.20.

[0196] Wait for the excipient to dissolve completely.

[0197] The preparation was filtered through a 0.22 μm filter.

[0198] rhNGF was added at a concentration of 0.020 mg / mL.

[0199] After loading with rhNGF, the formulation thus obtained was dispensed into 2R glass vials and subsequently freeze-dried using a VirTisAdVantage Pro freeze dryer (SP Scientific). Each vial contained a nominal volume of 0.5 mL.

[0200] Separately, reconstitution solvents A and B containing sesamin were prepared as follows.

[0201] Solvent A: DMSO (0.5% w / v), sesamin (1.5 μg / mL), NaCl (1.5 mg / mL) and WFI (qb); sesamin was pre-dissolved in DMSO 100%.

[0202] Solvent B: DMSO (0.5% w / v), Kolliphor HS15 (0.15% w / v), sesamin (1.5 μg / mL), NaCl (1.5 mg / mL) and WFI (qb); sesamin was pre-dissolved in DMSO 100%.

[0203] The solvent was filtered through a 0.22 μm filter.

[0204] The lyophilized NGF ophthalmic formulations were reconstituted with 1 mL or 2 mL of reconstitution solvent A or B to obtain a final concentration of 10 μg / mL or 5 μg / mL of rhNGF, respectively.

[0205] Preparation 7: Freeze-dried ophthalmic preparation containing NGF and sesamin

[0206] The following excipients were weighed and dissolved in 100 ml WFI: Trehalose dihydrate (5.04% w / v), mannitol (1.31% w / v), anhydrous Na2HPO4 (0.3065% w / v), NaH2PO4*H2O (0.116% w / v), Kolliphor P188 (0.4% w / v), L-methionine (0.001% w / v), Kolliphor HS15 (0.3% w / v), 1.0 N sodium hydroxide quantified to pH 7.20 ± 0.20.

[0207] Wait for the excipient to dissolve completely.

[0208] Separately, 0.008 mg of sesamin (powder) was preliminarily dissolved in 5.5 mg of DMSO 100%, sonicated for 2 minutes, and then added to the above excipient solution.

[0209] The preparation was then filtered through a 0.22 μm filter.

[0210] Add 0.020 mg rhNGF.

[0211] The following preparations were obtained:

[0212]

[0213] The formulation thus obtained was dispensed into 2R glass vials and subsequently freeze-dried using a VirTis AdVantage Pro freeze dryer (SP Scientific). Each vial contained a nominal volume of 0.5 mL.

[0214] The lyophilized formulations were reconstituted with 1 mL or 2 mL of the appropriate solvent (WFI or 0.9% NaCl saline solution) to obtain a final concentration of 10 μg / mL or 5 μg / mL of rhNGF, respectively.

[0215] Example 4

[0216] Drug Product In-Use Stability of Formulation 6

[0217] The in-use stability study of the rhNGF lyophilized formulation (according to Formulation 6) after reconstitution with the reconstitution solvent B containing sesamin or with WFI was conducted as follows.

[0218] The lyophilized formulations reconstituted in solvent B or WFI stored at different temperatures (+2°C / +8°C, or 25°C-60% relative humidity) for more than 28 days were tested to verify possible impurity formation in the reconstituted liquid formulation of rhNGF in glass vials.

[0219] Impurities were determined as the percentage of oxidized NGF relative to the total NGF contained in the reconstituted liquid formulation (w / w).

[0220] The results are reported in Table 1 (+2°C / +8°C) and Table 2 (25°C-60% relative humidity).

[0221] Table 1: Impurities of rhNGF after reconstitution with reconstitution solvent B or WFI and storage at +2°C / +8°C.

[0222]

[0223] Table 2: Impurities of rhNGF after reconstitution with reconstitution solvent B or WFI and storage at +25°C -60% RU

[0224]

[0225] As shown above, samples reconstituted with solvent B unexpectedly showed a reduction in oxidation rate during storage period compared to samples reconstituted with WFI alone (Table 1).

[0226] In samples stored at higher temperatures, a significant decrease in oxidation rate was observed between rhNGF formulations reconstituted with WFI or reconstitution solvent B (Table 2).

Claims

1. A combination of nerve growth factor (NGF) and sesamin for use by topical administration in the treatment of ocular disorders known to be treatable by NGF.

2. An ophthalmic composition comprising nerve growth factor (NGF), sesamin and at least one ophthalmologically acceptable excipient.

3. A medicine kit comprising: a) an ophthalmic composition comprising nerve growth factor (NGF), and b) An ophthalmic composition comprising sesamin.

4. The combination for use according to claim 1, the ophthalmic composition according to claim 2 or the drug kit according to claim 3, wherein the NGF has the amino acid sequence of SEQ.ID NO.1 or SEQ ID NO.

2.

5. The combination for use according to claim 1 or 4, the ophthalmic composition according to claim 2 or 4, or the medicine kit according to claim 3 or 4, wherein the NGF is recombinant human NGF.

6. A combination for use according to claim 1, 4 or 5, an ophthalmic composition according to claim 2, 4 or 5, or a kit according to claim 3, 4 or 5, wherein the purity of the NGF is higher than 70%, more preferably higher than 80% w / w, higher than 90% w / w, higher than 95% w / w, higher than 98% w / w or higher than 99% w / w.

7. The combination for use according to claim 1, 4, 5 or 6, the ophthalmic composition according to claim 2, 4, 5 or 6, or the kit according to claim 3, 4, 5 or 6, wherein the sesamin is a purified compound.

8. The combination for use, the ophthalmic composition or the kit according to claim 7, wherein the purity of sesamin is higher than 70% w / w, more preferably higher than 80% w / w, higher than 90% w / w or higher than 95% w / w.

9. An ophthalmic composition according to claim 2, 4, 5, 6, 7 or 8, or a kit according to claim 3, 4, 5, 6, 7 or 8, wherein the composition comprises one or more excipients selected from the group consisting of trehalose, mannitol, cellulose ethers, buffers, polyethylene glycols, surfactants, emulsifiers and antioxidant compounds.

10. The ophthalmic composition according to claim 2, 4, 5, 6, 7, 8 or 9, which is in the form of a liquid ophthalmic preparation.

11. The kit of claim 3, 4, 5, 6, 7, 8 or 9, wherein at least one of the ophthalmic compositions a) and b) is in the form of a liquid ophthalmic formulation.

12. The ophthalmic composition of claim 10, wherein the liquid ophthalmic formulation comprises NGF at a concentration of 5 μg / ml to 300 μg / ml.

13. The kit according to claim 11, wherein the ophthalmic composition a) is in the form of a liquid ophthalmic preparation and comprises NGF at a concentration of 5 μg / ml to 300 μg / ml.

14. The ophthalmic composition according to claim 10 or 12, wherein the liquid ophthalmic preparation comprises sesamin at a concentration of 0.5 to 20 μg / ml.

15. The kit according to claim 11 or 13, wherein the ophthalmic composition b) is in the form of a liquid ophthalmic preparation and comprises sesamin at a concentration of 0.5 to 20 μg / ml.

16. The ophthalmic composition according to claim 2, 4, 5, 6, 7, 8 or 9, which is in the form of a powder preparation.

17. The kit of claim 3, 4, 5, 6, 7, 8, 9, 11 or 15, wherein the ophthalmic composition a) is in the form of a powder formulation and the ophthalmic composition b) is in the form of a liquid ophthalmic formulation.

18. An ophthalmic composition according to claim 2, 4, 5, 6, 7, 8, 9, 10, 12, 14 or 16, or a kit according to claim 3, 4, 5, 6, 7, 8, 9, 11, 13, 15 or 17, for use in treating an ocular condition known to be treatable by NGF by topical administration.

19. The combination for use according to claim 1, the ophthalmic composition or the kit for use according to claim 18, wherein the ocular disorder is selected from the group consisting of a disorder of the cornea, conjunctiva, sclera, retina or optic nerve.

20. A combination, ophthalmic composition or kit for use according to claim 19, wherein the ocular condition is selected from the group consisting of toxic and phototoxic keratopathy, iatrogenic, immune and neurotrophic corneal epithelial defects and ulcers, neurotrophic keratopathy, neuropathic corneal pain, keratoconjunctivitis sicca, dry eye associated with Sjögren's syndrome, retinitis pigmentosa, retinal detachment, post-ischemic retinal degeneration, phototoxic retinopathy, epiretinal membrane, macular hole, macular degeneration, optic neuropathy, glaucoma, scleritis, and episcleritis.

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