Combinations for use in ophthalmology

Abstract

The present invention relates to a combination of nerve growth factor and sesamin and its use in the treatment of ophthalmic disorders.

Description

[Technical Field]

[0001] The present invention relates to a combination of nerve growth factor and sesamin and its use in the treatment of ophthalmic disorders. [Background technology]

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

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

[0004] NGF, TrKA and p75 NTR is widely expressed in the visual system and plays an important role in trophism of both the anterior and posterior segments of the eye. For example, NGF regulates the development and differentiation of retinal ganglion cells, photoreceptors, optic nerve, and visual cortex, and promotes their survival and recovery after injury (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., Invest. Ophthalmol. Vis. Sci. 2002, 43(7):2334-2340; Garcia et al., Cytokine & Growth Factors 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).

[0005] In the anterior segment of the eye, TrKA and p75 NTR Nerve growth factor (NGF) is 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 demonstrated to maintain ocular surface homeostasis by acting in vitro, ex vivo, and in animal models on epithelial cell health, limbal stem cell differentiation, immunomodulation, and tear production (Lambiase et al., Curr Opin Ophthalmol. 2012, 23(4):296-302; Lambiase et al., Arch Ital Biol. 2011, 149(2):283-292).

[0006] The therapeutic efficacy of NGF in several disorders of the cornea, conjunctiva, and sclera has been demonstrated in animal models and clinical trials, particularly in phototoxic keratopathy (Rocco ML et al., Graefes Archive for Clinical and Experimental Ophthalmology 2018, 256:729-738), iatrogenic, immune, and neurotrophic corneal epithelial defects and ulcers, scleritis, and neurotrophic keratopathy (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., Arch Ophthalmol. 2000, 118(10):1446-1449; Bonini et al., Ophthalmology 2000, 107:1347-1351; Bonini et al., Ophthalmology 2018, 125(9):1332-1343), neuropathic corneal pain (Kenyon et al., Investigative Ophthalmology & Visual Science 2021, 62:842), keratoconjunctivitis sicca (Coassin et al., Graefes Arch Clin Exp Ophthalmol Albrecht Von Graefes Arch Klin Exp Ophthalmol 2005, 243(2):151-155; Sacchetti et al., Br. J. Ophthalmol 2020, 104:127-135), and Sjögren's syndrome-related dry eye (https: / / clinicaltrials.gov / ct2 / show / NCT05133180).

[0007] In the posterior segment of the eye, NGF is expressed in the retinal pigment epithelium, Müller cells, and retinal ganglion cells, and NGF receptors are found 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 demonstrated that NGF has a neuroprotective effect on optic nerve neurons under pathological conditions.

[0008] The therapeutic efficacy of NGF in retinal and optic neuropathy has been demonstrated in a number of clinical studies, including retinitis pigmentosa (Lenzi et al., Vision Res. 2005, 45(12):1491-1500; Sacchetti et al., 2017, Current Eye Research, 42:7, 1064-1068), retinal detachment (Sun et al., Ophthalmologica 2008, 222:58-61), diabetic retinopathy (Ali et al., Diabetes 2008, 57:889-898), retinal degeneration secondary to ischemia (Xien et al., Exp Eye Res 2014, 125:156-63), and phototoxic retinopathy (Rocco et al., Graefes Arch Clin Exp Ophthalmol 2018, 256:729-738; Garcia et al., J Neurochem 2014, 131(3):303-13), 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 Ist Super Sanita 2009, 45(4):439-442), optic neuropathy (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 (Lambiase et al., Proc Natl Acad Sci USA 2009, 106(32):13469-13474; Lambiase et al., Graefes Arch Clin Exp Ophthalmol Albrecht Von Graefes Arch Klin Exp Ophthalmol 1997, 235(12):780-785).

[0009] Pharmacokinetic studies have shown that after topical administration of NGF at a concentration of 200 μg / mL, high levels of the protein are 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 administration route is also suitable for treating retinal and optic nerve pathologies.

[0010] Topical administration of NGF to the ocular surface has been demonstrated to be safe in clinical trials. However, the main side effects reported by patients after topical ocular administration of NGF in clinical trials or after use for approved indications are ocular discomfort, including eye pain, eye or eyelid irritation, ocular itching, and ocular paresthesia. It has been suggested that this side effect is at least partially related to the therapeutic activity of NGF, as restoring ocular innervation and sensitivity may be associated with increased ocular surface symptoms. However, this has yet to be fully investigated.

[0011] Although the above-mentioned side effects are mild and transient, in a small number of cases, they cause the discontinuation of treatment and cause distress to patients.In addition, when patients experience eye pain, burning sensation and irritation as symptoms of pathology, the above-mentioned side effects may hinder the evaluation of the treatment of ocular disorders characterized by similar symptomatic symptoms, such as neuropathic corneal pain, dry eye and ocular autoimmune diseases.In these cases, the therapeutic effect of NGF may be masked or substantially reduced by the above-mentioned side effects.In addition, this side effect is particularly enhanced when topical application of NGF is used to treat pathologies at the back of the eye, and therefore high concentrations of NGF are used.

[0012] Therefore, there is a need to develop an ophthalmic composition of NGF that does not cause ocular discomfort. 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. It has been reported to have several diverse physiological effects and is widely used as a dietary supplement, especially in Asia. For example, sesamin has been reported to exert hypoglycemic, reactive oxygen species (ROS) scavenging, antioxidant, anti-inflammatory, and anxiolytic effects, as well as to suppress diabetes-related cognitive decline (Farbood et al., Life Sciences 2019, 230:169-177; Guo et al., Nutritional Neuroscience 2016, 19(6):231-236). Summary of the Invention

[0013] The present inventors have now surprisingly found that when sesamin is administered in combination with nerve growth factor (NGF), it can reduce the side effects associated with the above-mentioned topical application to the ocular surface.

[0014] Accordingly, a first object of the present invention is a combination of nerve growth factor (NGF) and sesamin for use by topical administration in the treatment of ocular disorders. A second object of the present invention is an ophthalmic composition comprising nerve growth factor (NGF), sesamin and at least one ophthalmologically acceptable excipient.

[0015] 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. A fourth object of the present invention is a method for the treatment of ophthalmic disorders, comprising topically administering to a patient in need thereof the above combination or ophthalmic composition. [Brief explanation of the drawings]

[0016] [Figure 1]Figure 1 shows 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 in DRG after treatment with NGF (NGF) or NGF in combination with sesamin (NGF+DF24437), measured as described in Example 1 and expressed as relative optical units. [Figure 2] This figure shows the number of eye rubs after ocular administration of Vehicle 1 (Veh1), Vehicle 2 (Veh2), 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 = 4-8). ***p < 0.001 vs. Vehicle 1 and Vehicle 2 groups; ***p < 0.001 vs. rhNGF group. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is based on the observation that sesamin has the ability to reduce ocular discomfort induced by administration of nerve growth factor to the ocular surface. Accordingly, 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.

[0018] Preferably, the eye disorder is an eye disorder known to be treatable by NGF. Preferably, the combination is administered topically, more preferably topically to the eye.

[0019] According to one embodiment, the combination is contained in a single ophthalmic composition. Therefore, a second object of the present invention is an ophthalmic composition comprising nerve growth factor (NGF) and sesamin.

[0020] The ophthalmic compositions according to the present invention are conveniently administered topically to the eye. According to an alternative embodiment, the combination is a combination of two ophthalmic compositions, one containing nerve growth factor (NGF) and the other containing sesamin, administered simultaneously or sequentially.

[0021] Therefore, the third object of the present invention is to a) an ophthalmic composition comprising nerve growth factor (NGF); b) an ophthalmic composition containing sesamin; The kit includes:

[0022] Preferably, the nerve growth factor (NGF) is human nerve growth factor (hNGF). Preferably, the NGF has the amino acid sequence of SEQ ID NO: 1: SEQ ID NO:1: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR Alternatively, the NGF has the amino acid sequence of SEQ ID NO:2: SEQ ID NO:2: SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA Alternatively, the NGF is a mixture of NGFs having the sequences of SEQ ID NO:1 and SEQ ID NO:2.

[0023] Preferably, the NGF is recombinant hNGF (rhNGF) produced by recombinant DNA technology. Methods for producing rhNGF are known to those skilled in the art, for example, those described in WO0022119A1 and WO2013092776A1.

[0024] Preferably, the NGF has a purity of greater than 70%, more preferably greater than 80%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%. The purity of NGF can be determined by conventional means known to those skilled in the art, such as HPLC analysis.

[0025] Preferably, the sesamin is in the form of a purified compound, preferably with a purity of greater than 70%, more preferably greater than 80%, greater than 90%, or greater than 95%. In this application, when referring to the percentage purity of a protein or compound, it means the mass of the pure protein or compound divided by the total mass of a sample of that protein or compound multiplied by 100 percent.

[0026] 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 HPLC analysis.

[0027] According to an alternative embodiment, the sesamin is preferably in unrefined form as a constituent of sesame oil. 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.

[0028] Preferably, the ophthalmologically acceptable excipient is selected from ophthalmologically acceptable viscosity enhancing agents, penetration enhancers, buffering agents, osmolality adjusting agents, preservatives, antioxidants, surfactants, emulsifiers and polyethylene glycol.

[0029] The thickener is preferably selected from polyvinylpyrrolidone, cellulose ethers, preferably hydroxymethylcellulose, hydroxyethylcellulose (HEC), hydroxypropylmethylcellulose (HPMC) and methylcellulose, and gelling agents, preferably gellan gum, xanthan gum and carbopol-974. A preferred thickener according to the present invention is hydroxypropylmethylcellulose.

[0030] The penetration enhancer is preferably selected from cyclodextrins, chelating agents, crown ethers, bile acids and bile salts. Buffering agents are compounds capable of providing and maintaining a pH of the ophthalmic composition that is compatible with ophthalmic use, preferably a pH comprised between 6.5 and 8. A preferred buffer is phosphate buffer, although other buffers capable of maintaining a pH within the desired range suitable for ophthalmic use are also included.

[0031] The osmolality adjusting agent is a salt that can make the ophthalmic composition isotonic with ocular fluids. A preferred salt is sodium chloride (NaCl), but other ophthalmologically acceptable salts, such as potassium chloride (KCl), calcium chloride (CaCl), and magnesium chloride (MgCl), and mixtures thereof, can also be used.

[0032] The preservative is preferably selected from quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride. The surfactant and emulsifier is preferably selected from linoleic acid sodium salt, Kolliphor HS15, polysorbates, preferably Tween 80, and poloxamers, preferably Kolliphor P188.

[0033] 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:

[0034] [Table A]

[0035] As used herein, "Kolliphor HS15," also known as macrogol (15)-hydroxystearate, refers to a pH-independent nonionic surfactant having the following structure: polyethylene glycol (15)-hydroxystearate.

[0036] 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 comprises one or more excipients selected from trehalose, mannitol, cellulose ethers, buffers, surfactants, emulsifiers, polyethylene glycol and antioxidant compounds.

[0037] 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, a cellulose ether, a buffer and polyethylene glycol as excipients.

[0038] According to another preferred embodiment, the sesamin-containing ophthalmic composition of the kit according to the third object of the present invention contains a buffer and polyethylene glycol as excipients. The combination according to the first object of the present invention, 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 a form suitable for topical administration to the ocular surface.

[0039] Thus, the combination according to the first object of the present invention, 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 conveniently administered topically to the eye. 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, preferably a lyophilized powder, which is dissolved in a suitable vehicle at the time of use to form a liquid ophthalmic preparation. 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.

[0040] According to an alternative preferred embodiment, said ophthalmic composition according to the second object of the invention is in the form of a powder formulation, preferably a lyophilized powder formulation. According to a preferred embodiment, in the kit of the present invention, at least one of the ophthalmic compositions a) and b) is in the form of a liquid ophthalmic preparation. Preferably, in the kit of the present invention, the ophthalmic composition containing nerve growth factor (NGF) and the ophthalmic composition containing sesamin are both in the form of a liquid ophthalmic preparation.

[0041] Preferably, in the kit according to the present invention, one of the ophthalmic composition containing nerve growth factor (NGF) and the ophthalmic composition containing sesamin is in the form of a liquid ophthalmic formulation, and the other is in the form of a powder formulation, preferably a freeze-dried powder formulation.

[0042] More preferably, the ophthalmic composition a) containing nerve growth factor (NGF) is in the form of a powder formulation (preferably a freeze-dried powder formulation), and the ophthalmic composition b) containing sesamin is in the form of a liquid ophthalmic formulation.

[0043] Even more preferably, the liquid ophthalmic composition b) comprising sesamin is a sesamin-containing reconstitution solvent for the powder (eg, lyophilized powder) ophthalmic composition a) comprising nerve growth factor (NGF).

[0044] According to an alternative preferred embodiment, in the kit according to the present invention, the ophthalmic composition containing nerve growth factor (NGF) and the ophthalmic composition containing sesamin are both in the form of a powder formulation.

[0045] By "liquid ophthalmic preparation" according to the present invention is meant a preparation suitable for use in application to the surface of the eye and for dispensing in small volume units, for example drops, from an eye dropper container. Preferably, the liquid ophthalmic formulation comprises NGF and / or sesamin dissolved or suspended in a liquid vehicle, preferably an aqueous liquid vehicle, and at least one of the ophthalmologically acceptable excipients as described above.

[0046] Preferably, the liquid ophthalmic preparation has an osmolality of 200 to 380 mOsmol / kg, more preferably 250 to 330 mOsmol / kg, and even more preferably 280 to 320 mOsm / Kg.

[0047] Preferably, the liquid ophthalmic preparation has a pH of 6.5 to 8, more preferably 6.8 to 7.5, and even more preferably 7 to 7.4. When the liquid ophthalmic formulation contains NGF as described above, it is preferably present in the liquid ophthalmic formulation at a concentration of between 5 μg / ml and 300 μg / ml.

[0048] When the ophthalmic formulation is for use in the treatment of ocular disorders affecting the anterior portion of the eye, preferably the cornea, conjunctiva and sclera, the NGF is present in the liquid ophthalmic formulation at a concentration of 5 μg / ml to 50 μg / ml, more preferably 10 μg / ml to 50 μg / ml, and even more preferably 10, 20, 30 or 40 μg / ml.

[0049] Alternatively, when the ophthalmic formulation is for use in the treatment of an ocular disorder affecting the posterior portion of the eye, preferably the retina or optic nerve, the NGF is preferably present in the liquid ophthalmic formulation at a concentration of 50 μg / ml to 300 μg / ml, more preferably 100 μg / ml to 250 μg / ml, and even more preferably 150 or 200 μg / ml.

[0050] When the liquid ophthalmic preparation contains sesamin, it is preferably present in the liquid ophthalmic preparation at a concentration of 0.5 to 20 μg / ml. 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.

[0051] According to another preferred embodiment, the sesamin is present in the liquid ophthalmic formulation at 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, and conveniently 1.5 to 2 μg / ml.

[0052] Thus, when sesamin is present in the formulation in an unrefined form in sesame oil, the amount of sesame oil present in the liquid ophthalmic formulation can be an amount that results in the above concentrations of sesamin. Preferably, when the liquid ophthalmic formulation contains sesamin, the vehicle is an aqueous vehicle further containing an ophthalmologically acceptable organic solvent, preferably dimethyl sulfoxide (DMSO).

[0053] Preferably, the DMSO is present in the liquid ophthalmic formulation at a concentration of 0.1 to 1.2% v / v, more preferably 0.2 to 0.9% v / v, even more preferably 0.2% v / v or 0.6% v / v.

[0054] Preferably, the liquid ophthalmic formulations of the present invention comprise one or more excipients selected from trehalose, mannitol, cellulose ethers, buffers, polyethylene glycol, surfactants, emulsifiers, and optionally antioxidant compounds.

[0055] According to one embodiment, the liquid ophthalmic formulation of the present invention comprises trehalose, mannitol, a cellulose ether, a buffering agent, polyethylene glycol, and optionally an antioxidant compound.

[0056] According to another embodiment, the liquid ophthalmic formulation 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.

[0057] According to a further embodiment, the liquid ophthalmic formulation of the present invention comprises trehalose, mannitol, a buffering agent, one or more surfactants, and optionally an antioxidant compound. Preferably, the polyethylene glycol has a molecular weight of 4000 to 8000 g / mol, more preferably 5000 to 7000 g / mol, more preferably 5500 to 6500 g / mol, and even more preferably 6000 g / mol. Preferably, when the polyethylene glycol has a molecular weight of 6000 g / mol (PEG 6000), it is present in the liquid ophthalmic formulation at a concentration comprised between 5 and 15 mg / ml, preferably between 8 and 12 mg / ml.

[0058] Preferably, the antioxidant compound is selected from L-methionine and cysteine. Preferably, the antioxidant compound L-methionine is present in the liquid ophthalmic formulation at a concentration comprised between 0.005 and 0.02 mg / ml, more preferably at a concentration of 0.01 mg / ml.

[0059] Preferably, trehalose is present in said liquid ophthalmic formulation at a concentration comprised between 30 and 60 mg / ml, more preferably between 40 and 55 mg / ml, more preferably between 45 and 53 mg / ml, and even more preferably at a concentration of 45 and 50 mg / ml.

[0060] Preferably, mannitol is present in said liquid ophthalmic preparation in a concentration comprised between 5 and 20 mg / ml, more preferably between 10 and 15 mg / ml. Preferably, the concentration of cellulose ether in the liquid ophthalmic formulation is such that it results in a kinematic viscosity of about 1 to 25 cSt as determined at 25° C. using a capillary viscometer.

[0061] Preferably, said cellulose ether is hydroxypropyl methylcellulose, more preferably hydroxypropyl methylcellulose at a concentration comprised between 0.5 and 2 mg / ml, preferably between 0.8 and 1.2 mg / ml.

[0062] Preferably, the buffer in the liquid ophthalmic preparation has a composition and concentration that results in a pH of the liquid ophthalmic preparation of 6.5 to 8, more preferably 6.8 to 7.5, and even more preferably 7 to 7.4. Preferably, the buffer is a phosphate buffer and / or sodium hydroxide.

[0063] Preferably, said one or more surfactants are Kolliphor P188 (Poloxamer 188) and / or Kolliphor HS15 as defined above, and most preferably each of said one or more surfactants is present in said liquid ophthalmic formulation in a concentration comprised between 1 and 10 mg / ml, more preferably between 1.5 and 6 mg / ml, more preferably between 1.5 and 5 mg / ml.

[0064] 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 the form of a powder, it can be obtained by lyophilization of the liquid ophthalmic formulation described above, and more preferably, it is obtained by lyophilization of the liquid ophthalmic formulation described above.

[0065] According to one embodiment, in the kit of the present invention, the ophthalmic composition a) containing nerve growth factor (NGF) is in the form of a powder formulation, preferably a freeze-dried powder formulation, and the ophthalmic composition b) containing sesamin is in the form of a liquid ophthalmic formulation.

[0066] According to this particular embodiment, the ophthalmic composition a) comprises NGF and at least one or more excipients selected from trehalose, mannitol, cellulose ethers, buffers, polyethylene glycols, surfactants, emulsifiers, and optionally antioxidant compounds.

[0067] Preferably, said ophthalmic composition a) comprises NGF, as well as trehalose, mannitol, a buffer, one or more surfactants, and optionally an antioxidant compound, according to the concentrations defined above.

[0068] More preferably, said ophthalmic composition a) comprises NGF, as well as trehalose, mannitol, a buffering agent, Kolliphor P188, and optionally an antioxidant compound, according to the concentrations defined above.

[0069] Advantageously, the ophthalmic composition a) comprising nerve growth factor (NGF) in the form of a powder formulation, preferably a lyophilized powder formulation, is useful as a component of a kit according to the present invention, providing a convenient and economical means of providing stable lyophilized NGF in a form that can be rapidly and easily reconstituted in an appropriate sesamin-containing reconstitution vehicle for administering NGF and sesamin to a patient in need of treatment.

[0070] Advantageously, the ophthalmic composition b) of the kit according to the present invention, which comprises sesamin in the form of a liquid ophthalmic formulation, may also be a sesamin-containing reconstitution solvent for the powder (e.g., freeze-dried powder) ophthalmic composition a) of the kit according to the present invention, which comprises nerve growth factor (NGF).

[0071] Thus, the ophthalmic composition b) comprises sesamin, an ophthalmologically acceptable organic solvent, and one or more ophthalmologically acceptable excipients as described above. Preferably, the ophthalmic composition b) comprises sesamin, an ophthalmologically acceptable organic solvent, an osmolality adjuster, and optionally a surfactant.

[0072] Preferably, said sesamin is present in said ophthalmic composition b) in a concentration comprised between 0.25 and 2.5 μg / ml, more preferably between 0.5 and 2 μg / ml. Preferably, the ophthalmologically acceptable organic solvent is dimethyl sulfoxide (DMSO), which is present in said liquid ophthalmic composition b) at a concentration comprised between 0.1 and 1.2% v / v, more preferably between 0.2 and 0.9% v / v, and even more preferably at a concentration of 0.2% v / v or 0.6% v / v.

[0073] Preferably, the osmolality adjusting agent is sodium chloride (NaCl), which is present in the ophthalmic composition b) at a concentration comprised between 0.5 and 3 mg / ml, more preferably between 1 and 2 mg / ml, and even more preferably at a concentration of 1.5 mg / ml.

[0074] Preferably, said surfactant is Kolliphor HS15, which is present in said ophthalmic composition b) at a concentration comprised between 0.10% w / v and 0.20% w / v, more preferably at a concentration of 0.15% w / v.

[0075] Preferably, said ophthalmic composition b) according to the invention is an aqueous composition. Surprisingly, when an ophthalmic composition a) comprising nerve growth factor (NGF) in the form of a powder formulation, preferably a freeze-dried powder formulation, is reconstituted with said ophthalmic composition b) comprising sesamin according to the present invention, the reconstituted ophthalmic composition so obtained advantageously exhibits a reduced rate of oxidation of NGF during storage, as shown in the examples below.

[0076] As discussed above and demonstrated in the Examples 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, the ocular discomfort associated with the administration of NGF is significantly reduced in the presence of sesamin.

[0077] Therefore, a further object of the present invention is a combination, an ophthalmic composition or a kit as described above for use in the treatment of an eye disorder. Preferably, the eye disorder is an eye disorder known to be treatable by NGF.

[0078] Preferably, said use is by topical administration. 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, is for use in the treatment of an ophthalmic disorder known to be treatable by NGF, wherein the composition is administered topically to the eye.

[0079] Preferably, the eye disorder known to be treatable by NGF is a corneal disorder, a conjunctival disorder, a scleral disorder, a retinal disorder or an optic nerve disorder. More preferably, the ocular disorders known to be treatable with NGF are selected from toxic and phototoxic keratopathy, iatrogenic, immune and neurotrophic corneal epithelial defects and ulcers, neurotrophic keratopathy, neuropathic corneal pain, keratoconjunctivitis sicca, Sjogren's syndrome-associated dry eye, retinitis pigmentosa, retinal detachment, retinal degeneration secondary to ischemia, phototoxic retinopathy, epiretinal membrane, macular hole, macular degeneration, optic neuropathy, glaucoma, scleritis and episcleritis.

[0080] The number of daily doses of the ophthalmic composition of the present invention and the number of drops used in each dose can vary depending on the concentration of NGF and the patient's characteristics, such as age, type and severity of the condition, duration of treatment, presence of concurrent therapy, and similar factors within the knowledge and skill of those skilled in the art. Typically, each dose consists of one or two drops of the ophthalmic composition according to the second object of the present invention or the composition of the kit according to the third object of the present invention in the form of a liquid ophthalmic preparation.

[0081] The ophthalmic compositions of the kit according to the third object of the invention are administered separately to the patient. To prevent ocular discomfort induced by topical administration 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 substantially simultaneously or sequentially in any order, with an interval of not more than 5 minutes, preferably not more than 2 minutes, between each other.

[0082] Experimental section [Example]

[0083] Autophagy is an important adaptive mechanism triggered by stress or injury. It occurs at a low basal level in all cells but can be activated by numerous stressful stimuli, such as starvation, inflammation, and in pathological conditions, as well as by pharmacological agents.

[0084] Autophagy is a self-digestion process involved in protein and organelle degradation that improves cell survival in stressful environments. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) (Glick et al., J. Pathol. 2010, 221:3-12). Alterations in macroautophagy activity in dorsal root ganglia (DRG) have been demonstrated in injury conditions (Guo et al., Neurosci. Lett. 2015, 599:158-163). At the initiation of the macroautophagy process, beclin-1 (an upstream protein in the macroautophagy process) in the endoplasmic reticulum responds to stress signaling pathways and initiates isolation membrane formation. 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). The autophagosome then fuses with lysosomes, leading to autolysosome formation and further degradation. When macroautophagy activity increases, the expression levels of LC3-II, Beclin-1, and Atg5 increase, whereas the level of p62 (which delivers ubiquitinated cargo to autophagosomes) decreases.

[0085] The effect of NGF on autophagy in the presence and absence of sesamin was evaluated by analyzing the influence of different treatments on the levels of the above proteins involved in the autophagy pathway in dorsal root ganglion (DRG) neurons, which are involved in the generation of NGF-induced sensory side effects.

[0086] F11 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, catalog number D6546; Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum and 2 mM glutamine (Sigma-Aldrich, USA). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.

[0087] The cells were then cultured at 7000 cells / cm 2 The cells were seeded in 100% PBS and maintained in low-serum medium for approximately 2 weeks to induce differentiation into DRGs. The complete composition of the low-serum medium was DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2 mM glutamine and 1% FBS. The medium was changed every 2 days. Cell differentiation was confirmed using immunofluorescence (data not shown).

[0088] F11 cells maintained in 10% FBS medium were used as a control (control, undifferentiated). Morphological and functional analyses were performed on fully differentiated cells after 2 weeks of treatment with low serum. Undifferentiated control cells underwent the same analyses at 7 days to avoid dramatic cell death due to confluence. Fully differentiated cells were treated with 1 μM rhNGF alone or in combination with 30 nM sesamin for 24 hours.

[0089] Control and treated cells were harvested and lysed in ice-cold RIPA buffer (Thermo Scientific, USA) containing freshly added protease and phosphatase inhibitors. Protein lysates (30 μg) were separated on 4%–12% precast gels using MOPS or MES buffer (Thermo Scientific, USA) depending on the size of the protein of interest. Proteins were then electroblotted onto polyvinyldifluoride (PVDF) membranes (Sigma, St. Louis, MO), and nonspecific binding sites were blocked in blocking buffer (Thermo Scientific) for 15 min at RT.

[0090] The membranes were then 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 1:10,000 (Abcam, UK) was used as a secondary antibody. Immunoreactive bands were visualized by ECL (Thermo Scientific, USA) according to the manufacturer's instructions. The relative optical density of the immunoreactive bands was determined using ImageJ software and normalized to the relative optical density of anti-vinculin or anti-actin (depending on the molecular weight of the protein analyzed and the gel / buffer used). The phosphorylated forms were normalized to the total forms.

[0091] Figure 1 shows the results of the analysis, where the amounts of Atg5, Beclin-1, and LC3-I and LC3-II detected after each treatment are expressed as relative optical density units (RU). As can be seen, treatment with NGF alone significantly increased the levels of Atg5 and Beclin, which are essential for autophagy, while decreasing the level of p62 (an autophagy-specific substrate). Furthermore, the combination of increased levels of the microtubule-associated proteins LC3-I and LC3-II and decreased levels of the autophagy substrate p62 is consistent with active autophagy-mediated protein degradation. When sesamin was combined with NGF, autophagy was significantly reduced. The results demonstrate that sesamin can suppress the stress response induced by treatment of cells with NGF. [Example]

[0092] An eye rub test was performed to evaluate the ocular discomfort induced by ocular administration of rhNGF alone or in combination with sesamin. Experiments were performed on male Sprague-Dawley rats (300-320 g) housed in the animal facility of the Department of Pharmacy, University of Naples, Charles River, Italy. Food and water were available ad libitum.

[0093] The rats were divided into four experimental groups: Group 1 - Vehicle 1 control group Animals were treated with Vehicle 1 having the following composition in water for injection (WFI):

[0094] [Table B]

[0095] Group 2 - Vehicle 2 control group Animals were treated with Vehicle 2 having the following composition in water for injection (WFI):

[0096] [Table C]

[0097] Group 3 - rhNGF treatment group: Animals were treated with 20 μg / ml rhNGF in vehicle 1 ( n = 8); Group 4 - rhNGF + Sesamin treatment group: Animals were treated with 20 μg / ml rhNGF and 3.5 μg / ml sesamin in vehicle 2 (n=8).

[0098] All compositions tested were prepared immediately before use from lyophilized placebo or rhNGF reconstituted in a volume of 1 mL of vehicle. Animal care complied with Italian and European Economic Community regulations concerning the protection of animals used for experimental and other scientific purposes (Official Journal of the European Communities L135 358 / 1 12 / 18 / 1986).

[0099] The treatments described above were administered as eye drops in a volume of 10 μL once daily, followed by eye rub testing. The animals were placed on a 50x50cm table for a 10 minute habituation period. 10 μL of the solution was applied to the left eye of the animals and the number of eye rubs was counted for 30 minutes.

[0100] The test results are shown in Figure 2. As can be seen from the results obtained, rats treated with rhNGF alone showed a significant increase in the number of eye rubs compared to rats treated with Vehicle 1 or Vehicle 2 (***p<0.001, respectively), whereas mice administered rhNGF in combination with sesamin showed a moderate or no significant increase in the number of eye rubs compared to Vehicle 1 or Vehicle 2. Furthermore, a significant difference was observed between the rhNGF group and the Sesamin + rhNGF group (***p<0.001).

[0101] Therefore, sesamin can alleviate rhNGF-induced eye discomfort. All data are expressed as mean ± SEM. Data analysis was performed using GraphPad Prism 8. Statistical analysis was performed by two-way analysis of variance followed by Tukey's test for multiple comparisons where appropriate. Statistical significance was set at *p<0.05. [Example]

[0102] Preparation of formulations according to the invention Formulation 1 - Liquid ophthalmic formulation containing NGF and sesamin Weigh out the following excipients and solubilize them in the appropriate volume of WFI: trehalose dihydrate (5.04% w / v), mannitol (1.31%, w / v), anhydrous NaHPO (0.3065% w / v), NaHPO·HO (0.116% w / v), hydroxypropyl methylcellulose (0.107%, w / v), PEG 6000 (1.07%, w / v). Wait for complete solubilization of the excipients. Separately, approximately 2-4 mg of sesamin powder was solubilized in 1, 2, or 3 mL of DMSO and the solution was shaken to ensure complete solubilization of the powder. The solubilized sesamin was added to Solution A and stirred for 5 minutes. WFI was added to the defined volume (500 mL) and filtered through a 0.22 μm filter. A defined concentration of rhNGF is added.

[0103] Formulation 2: Liquid ophthalmic formulation containing NGF Weigh out the following excipients and solubilize them in an appropriate volume of WFI: trehalose dihydrate (5.04% w / v), mannitol (1.31%, w / v), anhydrous NaHPO (0.3065% w / v), NaHPO·HO (0.116% w / v), (hydroxypropyl methylcellulose (0.107%, w / %)), PEG 6000 (1.0% w / v) in WFI. Wait for complete solubilization of the excipients. Check the pH (approximately 7.3). Add WFI to a defined volume (e.g., Lt). Filter the formulation through a 0.22 µm filter. Add a defined concentration of rhNGF.

[0104] Formulation 3: Liquid ophthalmic formulation containing sesamin Solution A : Weigh out the following excipients and solubilize them in the appropriate volume of WFI: Trehalose dihydrate (2.52% w / v), Mannitol (0.65% w / v), Hydroxypropylmethylcellulose (0.0535% w / v), PEG 6000 (0.535% w / v) in WFI. Wait for complete solubilization of the excipients. Add WFI to the defined volume (e.g., 1 L). Solution BApproximately 9 mg of sesamin powder is solubilized in 10 mL of DMSO, and the solution is shaken to achieve complete solubilization of the powder. Add 300 μL of Solution B to 50 mL of Solution A. Stir for 5 minutes. Aliquot the final formulation into glass vials and undergo the lyophilization process. After the freeze-drying process, the cake is reconstituted with WFI and filtered through 0.22 μm.

[0105] Formulation 4: Liquid ophthalmic formulation containing sesamin Add 50-100 μL of sesame oil to 5 mL of WFI and homogenize for 5 min at 15,000 rpm with a homogenizer Ultraturrax t25 basic (IKA) using an 8G probe. PEG6000 (final concentration 0.25%) is added to the first emulsion and homogenized at 15000 rpm for an additional 5 minutes.

[0106] Formulation 5: Liquid ophthalmic formulation containing sesamin Approximately 45 mg of micronized sesamin powder (sesamin + PEG6000 or sesamin + trehalose or sesamin + mannitol) was weighed and solubilized in 500 mL of WFI (or WFI + 0.6% DMSO). The formulation was stirred for 10 minutes and filtered through a 0.22 μm filter.

[0107] Formulation 6: Lyophilized NGF ophthalmic formulation and sesamin-containing reconstitution solvent Weigh out the following excipients and solubilize them in the appropriate volume of WFI: trehalose dihydrate (5.04% w / v), mannitol (1.31%, w / v), anhydrous NaHPO (0.3065% w / v), NaHPO·HO (0.116% w / v), Kolliphor P188 (0.4% w / v), L-methionine (0.001% w / v), and 1.0 N sodium hydroxide (enough to achieve a pH of 7.20 ± 0.20). Wait for complete solubilization of the excipients. The formulation is filtered through a 0.22 μm filter. rhNGF is added at a concentration of 0.020 mg / mL. After adding rhNGF, the formulation so obtained is dispensed into 2R glass vials and then freeze-dried using a VirTis Advantage Pro freeze dryer (SP Scientific), each vial containing a nominal volume of 0.5 mL. Separately, sesamin-containing reconstitution solvents A and B are prepared as follows. Solvent A: DMSO (0.5% w / v), sesamin (1.5 μg / mL), NaCl (1.5 mg / mL), and WFI (qb); sesamin was previously solubilized in 100% DMSO. 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-solubilized in 100% DMSO. The solvent was filtered through a 0.22 μm filter. The lyophilized NGF ophthalmic formulation was reconstituted with 1 mL or 2 mL of reconstitution solvent A or B to give a final concentration of 10 μg / mL or 5 μg / mL of rhNGF, respectively.

[0108] Formulation 7: Lyophilized ophthalmic formulation containing NGF and sesamin Weigh out the following excipients and solubilize them in 100 ml of WFI: trehalose dihydrate (5.04% w / v), mannitol (1.3% w / v), anhydrous NaHPO (0.3065% w / v), NaHPO·H0 (0.116% w / v), Kolliphor P188 (0.4% w / v), L-methionine (0.001% w / v), Kolliphor HS15 (0.3% w / v), and 1.0 N sodium hydroxide (enough to achieve a pH of 7.20 ± 0.20). Wait for complete solubilization of the excipients. Separately, 0.008 mg of sesamin (powder) was pre-solubilized in 5.5 mg of 100% DMSO, sonicated for 2 minutes, and then added to the above excipient solution. The formulation was then filtered through a 0.22 μm filter. Add 0.020 mg of rhNGF. The following formulation is obtained:

[0109] [Table D]

[0110] The formulation so obtained is dispensed into 2R glass vials and then freeze-dried using a VirTis Advantage Pro freeze dryer (SP Scientific), each vial containing a nominal volume of 0.5 mL.

[0111] Reconstitute the freeze-dried formulation with 1 mL or 2 mL of the appropriate solvent (WFI or saline solution 0.9% NaCl) to obtain a final concentration of rhNGF of 10 μg / mL or 5 μg / mL, respectively. [Example]

[0112] In-use stability of Formulation 6 drug product An in-use stability study of the rhNGF lyophilized formulation (according to Formulation 6) after reconstitution with sesamin-containing reconstitution solvent B or WFI was performed as follows. Studies were conducted on the lyophilized formulations reconstituted in solvent B or WFI and stored at different temperatures (+2 / +8°C, or 25°C-60% relative humidity) for more than 28 days to examine possible impurity formation in the rhNGF reconstituted liquid formulation in glass vials. Impurities were determined as the percentage (w / w) of oxidized NGF relative to the total NGF contained in the reconstituted liquid formulation. The results are reported in Table 1 (+2 / +8°C) and Table 2 (25°C-60% relative humidity).

[0113] [Table 1]

[0114] [Table 2]

[0115] As shown above, samples reconstituted with solvent B surprisingly exhibited a reduced rate of oxidation during storage compared to samples reconstituted with WFI alone (Table 1). A significant decrease in oxidation rate between rhNGF formulations reconstituted with WFI and reconstitution solvent B was observed in samples stored at higher temperatures (Table 2).

Claims

1. A combination of nerve growth factor (NGF) and sesamin for topical administration in the treatment of eye 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) An ophthalmic composition containing nerve growth factor (NGF), b) An ophthalmic composition containing sesamin and A kit that includes this.

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

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

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

9. The ophthalmic composition according to claim 2, or the kit according to claim 3, wherein the composition comprises one or more excipients selected from trehalose, mannitol, cellulose ether, buffer, polyethylene glycol, surfactant, emulsifier, and antioxidant compound.

10. The ophthalmic composition according to claim 2, in the form of a liquid ophthalmic formulation.

11. The kit according to claim 3, wherein at least one of the ophthalmic compositions a) and b) is in the form of a liquid ophthalmic formulation.

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

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

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

16. The ophthalmic composition according to claim 2, in the form of a powder formulation.

17. The kit according to claim 3, 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. The ophthalmic composition according to claim 2, for use by topical administration in the treatment of eye disorders known to be treatable by NGF.

19. The kit according to claim 3 for use by topical administration in the treatment of an eye disorder known to be treatable by NGF.

20. The combination for use according to claim 1, the ophthalmic composition for use according to claim 18, or the kit for use according to claim 19, wherein the ophthalmic disorder is selected from corneal disorder, conjunctival disorder, scleral disorder, retinal disorder, or optic nerve disorder.

21. A combination, ophthalmic composition, or kit for use according to claim 20, wherein the ocular disorder is selected from toxic and phototoxic keratopathy, iatrogenic, immunogenic and neurotrophic corneal epithelial defects and ulcers, neurotrophic keratopathy, neuropathic corneal pain, keratoconjunctivitis sicca, Sjögren's syndrome-related dry eye, retinitis pigmentosa, retinal detachment, retinal degeneration secondary to ischemia, phototoxic retinopathy, epiretinal membrane, macular hole, macular degeneration, optic neuropathy, glaucoma, scleritis, and episcleritis.

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