FIBRES AFTER A KERATO-PLASTY OPERATION

Field of the invention The present invention relates to an ophthalmic composition comprising cobalamin, taurine and at least one tear substitute for use in the regeneration of corneal nerve fibres in subjects who have undergone a keratoplasty operation.

Background of the invention

The cornea is a transparent membrane that forms the anterior and outermost portion of the eyeball and represents the most powerful lens of the visual apparatus.

Because of its small size and the extensive branching of the peripheral axons of corneal neurons, the cornea is also the most densely innervated tissue in the body.

The corneal innervation is predominantly of the sensory type and is constituted by the terminations of the long ciliary nerves that derive from the nasociliary branch of the ophthalmic branch of the trigeminal nerve. The corneal nerves are narrow and elongated structures that extend in a layer of about 10 μηι and are very well detectable with the current instruments used during a routine ophthalmological visit.

Corneal nerves are arranged partly forming a deep plexus over the endothelium, partly crossing the corneal endothelium and projecting perpendicularly into the stroma, then forming a further plexus below the corneal epithelium, from which a small number of stromal nerves ending with free nerve endings projects towards the corneal epithelial layer.

Stromal nerve trunks penetrate radially into the peripheral corneal stroma before progressing anteriorly, proceeding in the cornea like a tree which divides into several smaller branches while projecting towards the superficial stroma. In other words, the nerve density increases and the diameter of the nerve tapers moving from the periphery to the center and anteriorly through the stroma. Corneal transplantation (keratoplasty) consists in the substitution of a part of the corneal tissue, usually the central portion, with a homologous graft from a non-living donor. This substitution involves all the corneal layers, including the endothelium in penetrating keratoplasty operations, and the corneal epithelial layers, Bowman's membrane and part of the stroma, in anterior lamellar keratoplasty. In both cases, cornea is cut for the entire circumference and the nerves, which from the periphery go into the corneal center, are completely cut off. This makes these two types of operation, albeit different by invasiveness, quite similar to each other as regards the consequences in terms of corneal denervation, which is complete in both cases. Consequence of this is a marked reduction in sensitivity (hypoaesthesia) of the transplanted cornea, which in many cases persists even for several years from the operation (Mathers 1988; Tugal Tutkun 1993). As a result of penetrating keratoplasty, for example, anomalies in the restoration of the nerve density of the subbasal nerve were noted even after 30 years from the operation and significant alterations of the subbasal nerve plexus remained evident even 40 years later (Al-Aqaba MA, et al. Organization of the regenerated nerves in human corneal grafts, Am J Ophthalmol. 2012; 153:29-37).

Moreover, the two operating techniques appear quite similar also as regards the onset and progress of the reinnervation process of the transplanted cornea, which includes a complex and wide range of phenomena that may limit, slow down or alter this reinnervation. When the reinnervation proceeds, most of the nerves regenerated in the tissue from the donor are a continuation of the subbasal nerves of the host, which cross the host/graft tissue junction to innervate the peripheral part of the graft cornea. However, the stromal nerve trunks from the host tissue may also stop at the edge of the graft and in some cases may be completely absent in the tissue from the donor. Even when the stromal nerves invade the tissue coming from the donor, these often innervate peripheral areas only after a long time and may also exhibit alterations, such as an increased tortuosity.

Finally, the stromal nerves can regenerate but do not contribute to the epithelial innervation: while in the normal cornea the epithelial nerves derive from the stromal nerves and there are numerous connections between them, as a result of keratoplasty these normal connections between the nerves can be almost completely absent in the donor tissue.

Related art

M.R. Romano et al., "Effects of vitamin B12 on the corneal nerve regeneration in rats", Experimental Eye Research 120 (2014) p. 109-1 17 investigates the effect of an ophthalmic solution comprising 0.05% by weight of vitamin B12 (cyanocobalamin), 0.5% by weight of taurine and 0.5% by weight of sodium hyaluronate, on the regeneration of nerve fibres in rats to which a superficial portion of corneal epithelium has been partially damaged with a corneal burr. Said study suggests that treatments with vitamin B12 accelerate the cicatrization and reinnervation processes of the epithelium after a wound injury thereof, similar to what happens with wounds, usually of an abrasive type, generated by trauma or in any case by unwanted injurious events suffered by the subject interested. To date, however, specific therapies that, in patients who have undergone corneal transplantation, have the purpose of regenerating the nerve fibres after their full surgical removal, are not known. To alleviate the discomfort due to the altered corneal sensitivity, patients who have undergone corneal transplantation can therefore only be suggested to use eye drops containing more or less viscous substances based on hyaluronic acid, cellulose derivatives, or other compounds on a daily basis in order to protect the surface of the eye.

Summary of the invention

The Applicant has therefore addressed the problem of identifying a remedy for the regeneration of corneal nerve fibres in subjects who have undergone a keratoplasty operation, that is to say, an operation that involves the deliberate and preventive total cutting of the nerve fibres in the corneal inner layers.

With reference to the study of M.R. Romano et al. cited herein, the Applicant observes that said study investigates the accelerating effect of an ophthalmic composition comprising vitamin B12, taurine and sodium hyaluronate on the reconstruction process of damaged epithelial nerve fibres on rats which have suffered a lesion by means of partial mechanical removal of a superficial portion of the epithelium. The Applicant in this regard also notes that in the study of M.R. Romano et al. cited herein, referring to hypothesis of accidental lesions of superficial portions of corneal epithelium, and although it mentions a process semantically defined "reinnervation", the latter appears different in terms of extension, entity and mechanism with respect to the real reinnervation processes that can be established following a keratoplasty operation. The mechanical removal described in the study of M.R. Romano in fact caused a partial damage to the epithelium and only epithelial nerve fibres, without the least involvement or injury of the corneal innermost layers, and in particular the stroma and its nerves, in which the innervation remained still entirely present and functioning in its original natural form and indeed could thus form a substrate for the repair of the epithelial nerve fibres themselves.

Quite unexpectedly, the Applicant has instead found that an ophthalmic composition comprising cobalamin, taurine and a tear substitute allows obtaining, compared to what is evidenced by the study of M.R. Romano, a series of beneficial effects that are very different and more meaningful from a therapeutic point of view when used on subjects that have undergone a radically invasive trauma such as a keratoplasty operation, which involves the removal of a significant portion of the corneal tissue with the consequent total cutting of the nerve fibres even in the corneal inner layers, including the stromal nerve fibres, and the subsequent replacement of a part of the corneal tissue with a graft from a donor.

In particular, the Applicant has surprisingly and experimentally found that it is possible to achieve significant reinnervation with regeneration of corneal nerve fibres also at the level of stromal nerves following a keratoplasty operation, using an ophthalmic composition comprising cobalamin, taurine and at least one tear substitute.

More in particular, according to a first aspect, the present invention relates to an ophthalmic composition comprising cobalamin, taurine and at least one tear substitute, for use in the regeneration of corneal nerve fibres in subjects who have undergone a keratoplasty operation. Among the beneficial effects under the therapeutic profile that can be obtained by using the ophthalmic composition according to the present invention, the Applicant has in particular found the following:

- the regeneration of nerve fibres completely cut - and thus not only damaged - in the epithelium and also in a plurality of the corneal innermost layers of the epithelium itself and through a corneal tissue grafted from a donor: a deep and significant action in an extremely stressful clinical context for the patient;

- the absence of relevant symptomatology during the treatment phase with said composition, thus facilitating the post-operative course;

- an increased corneal sensitivity following treatment with said composition, which therefore reduces the need to daily use lubricating eye drops in order to protect the surface of the eye;

- the absence of significant anomalies in the re-epithelization of the transplanted cornea;

- the establishment of a synergy between cobalamin and taurine, in which the latter not only chemically stabilizes cobalamin in the ophthalmic composition, thus making its delivery on the cornea more effective, but also performs a combined and synergistic action on the reinnervation of the corneal nerve fibres. All this is therefore far beyond what can be foreseen on the basis of literature data suggesting, at most, an accelerating effect of vitamin B12 on the repair process of partially damaged epithelial nerve fibres following a partial mechanical lesion of the epithelial layer on rats. In the present description, a method for the regeneration of corneal nerve fibres, particularly at the level of stromal nerves, in subjects who underwent a keratoplasty operation is also described, which includes the use of an ophthalmic composition comprising cobalamin, taurine and at least one tear substitute.

Detailed description of currently preferred embodiments of the invention The present invention can have, in one or more of the aspects thereof, one or more of the preferred features described hereinafter, which can be combined with one another as desired depending on the application requirements.

Within the scope of the present description and in the following claims, all numerical values indicating amounts, parameters, percentages and so on are to always be intended as preceded by the term "about", if not otherwise stated. Moreover, all numerical value ranges include all possible combinations of the maximum and minimum numerical values and all possible intermediate ranges, besides those specifically indicated below.

Within the scope of the present description and in the following claims, the term: tear substitute is meant to indicate a compound for topical application on the eye with viscosizing, mucomimetic and pseudoplastic properties.

Preferably, the subjects who underwent a keratoplasty operation are human subjects.

Preferably, the present invention relates to an ophthalmic composition comprising cobalamin, taurine and at least one tear substitute, for use in accelerating the regeneration of corneal nerve fibres in subjects who have undergone a keratoplasty operation. In a preferred embodiment, the regenerating action of corneal nerve fibres following a keratoplasty operation carried out by the composition comprising cobalamin, taurine and at least one tear substitute according to the present invention involves at least one corneal layer under the epithelium, such as Bowman's lamina and stroma.

Said keratoplasty operation can be either a penetrating or an anterior lamellar type. In a preferred embodiment, said operation is an anterior lamellar keratoplasty operation.

Preferably, said composition is administered to subjects who have undergone a keratoplasty operation for a period of at least 6 months, more preferably for a period of between 6 and 12 months, at a dose of 0.01 - 0.60 mg/day, more preferably 0.10 - 0.30 mg/day, of cobalamin, of 0.1 - 6 mg/day, more preferably of 1 - 3 mg/day, of taurine and a physiologically acceptable amount of said at least one tear substitute. Advantageously, the dosage regimens of the ophthalmic composition according to the present invention indicated above allow maximizing the therapeutic results in terms of regeneration of corneal nerve fibres, thereby reducing the time for recovery the sensitivity of the transplanted cornea.

In a preferred embodiment, said composition is administered to said subjects at a dose of 0.10 - 0.60 mg/day of cobalamin and 1 - 6 mg/day of taurine for the first 6 months and at a dose of 0.05 - 0.60 mg/day of cobalamin and 0.5 - 6 mg/day of taurine for the next 6 months.

The ophthalmic composition according to the present invention can be administered to said subjects starting from any moment following said keratoplasty operation, preferably starting from said keratoplasty operation. Advantageously, the dosage regimens of the ophthalmic composition according to the present invention described above allow the functional restoration of the corneal innervation both at the peripheral and central level even after penetrating keratoplasty, without encountering any side effects attributable to the therapy and, on the contrary, registering a correct corneal re-epithelialization process. Preferably, said at least one tear substitute is administered to said subjects at a dose of 0.1 to 6 mg/day, more preferably of 1 to 3 mg/day.

In a preferred embodiment, the ophthalmic composition according to the present invention exhibits a physiologically acceptable pH, more preferably between 6.5 and 7.5.

Preferably, the ophthalmic composition according to the present invention has a physiologically acceptable osmolarity, more preferably between 200 and 270 mOsm/kg, even more preferably between 220 and 260 mOsm/kg. Advantageously, osmolarity values in this range allow maintaining or possibly restoring the osmolarity of the tear film to physiologically acceptable values in the eye already after a few administrations.

Preferably, the ophthalmic composition according to the present invention has a Na7K ⁺ molar ratio of between 4.8 and 5.8, more preferably between 5.0 and 5.4. Advantageously, Na ⁺ /K ⁺ molar ratio values within this range contribute to maintaining or possibly restoring the Na ⁺ and K ⁺ levels of the tear film to physiologically acceptable levels in the eye already after a few administrations.

In a preferred embodiment, the ophthalmic composition according to the present invention comprises 0.03% - 0.07% by weight of cobalamin, more preferably 0.04% - 0.06% by weight of cobalamin. Preferably, cobalamin is cyanocobalamin (vitamin B12).

Preferably, the ophthalmic composition according to the present invention comprises 0.3% - 0.7% by weight of taurine, more preferably 0.4% - 0.6%.

Advantageously, the presence of taurine in this concentration range allows maximizing the stabilizing effect on cobalamin, promoting the onset of synergistic phenomena in the reinnervation of the transplanted cornea.

In a preferred embodiment, the ophthalmic composition according to the present invention comprises 0.3% - 0.7% by weight of said tear substitute.

Preferably, the tear substitute is selected from the group consisting of: at least one glycosaminoglycan, at least one cellulose derivative, at least one polyacrylic acid derivative, at least one water-soluble polymer and mixtures thereof.

Preferably, the glycosaminoglycans which can be used in the present invention are selected from the group consisting of: hyaluronic acid or a pharmaceutically acceptable salt thereof, more preferably sodium hyaluronate, chondroitin sulphate, and mixtures thereof.

In a preferred embodiment, said at least one glycosaminoglycan has a molecular weight ranging from 500 to 1200 kDa.

Advantageously, this molecular weight is more tolerable for the patient.

In a particularly preferred embodiment, the glycosaminoglycan is hyaluronic acid or a pharmaceutically acceptable salt thereof, more preferably sodium hyaluronate, commercially available and already known product in the field of ophthalmic compositions. Preferably, the cellulose derivatives which can be used in the present invention are selected from the group comprising: carboxymethylcellulose (CMC), hydroxypropylmethylcellulose (HPMC), methylcellulose (MC), hydroxyethylcellulose (HEC) and hydroxypropyl cellulose (HPC) and mixtures thereof.

Preferably, the polyacrylic acid derivatives which can be used in the present invention are selected from: carbopol, polycarbophil and the like, and mixtures thereof. Preferably, the water-soluble polymers which can be used in the present invention are selected from: polyvinyl alcohol (PVA), dextran, polyvinylpyrrolidone (PVP), polysorbate, and mixtures thereof.

In a particularly preferred embodiment, the composition according to the present invention comprises 0.03% - 0.07% by weight of cyanoobalamin, 0.3% - 0.7% by weight of taurine and 0.3% - 0.7% by weight of sodium hyaluronate. Advantageously, this specific combination of components and their presence in specific amounts allows obtaining optimal results in the regeneration of corneal nerve fibres. Commercial examples of products having these compositional features are, for example, the ophthalmic product laluvit, produced by the Applicant.

The ophthalmic composition according to the invention may contain one or more of the other possible ingredients known in the pharmaceutical technique for ophthalmic preparations.

In particular, the compositions may further contain at least one component selected from the group consisting of buffering agents, osmolarity adjusters, preservatives, antimicrobials and/or sequestering agents, and mixtures thereof.

Buffering agents or acidity regulators help maintain the pH of ophthalmic products as close as possible to the physiological one. This action is fundamental to allow good tolerability of the ophthalmic preparations and to preserve their efficacy.

Examples of buffering agents which may be used in the present invention are selected from the group comprising sorbitol, acetic acid, sodium phosphate, sodium hydroxide, sodium citrate, trisodium citrate, sodium carbonate, sodium borate, sodium bicarbonate, potassium phosphate, potassium citrate, potassium carbonate, boric acid, hydrochloric acid, acetic acid and magnesium chloride.

Examples of substances that regulate the osmolarity making isotonic ophthalmic solutions with tears are selected from the group comprising potassium chloride, propylene glycol, sodium chloride, glycerine, dextrose, dextran 40 and 70.

As usual, the compositions may contain other adjuvants, including preservatives, sequestering and antimicrobial agents, although these adjuvants tend to be avoided in eye drops by resorting to single-dose, preservative-free packages. The ophthalmic compositions according to the invention are conveniently prepared in the form of a solution or aqueous suspension in a pharmaceutically acceptable ophthalmic carrier. Preferably, the compositions according to the invention are prepared in the form of eye drops or gels. Such ophthalmic compositions can also be prepared in the form of ointment.

In order to assess the performance of the composition according to the invention, various experiments have been carried out, some of which are reported below, to be intended for illustrative and non-limiting purpose of the present invention.

EXAMPLES

EXAMPLE 1

12 patients visited at the eye clinic of Fondazione Banca degli Occhi del Veneto after anterior lamellar corneal transplantation for keratoconus have undergone to a therapeutic treatment in order to assess the performance of a composition according to the present invention with respect to eye drops containing 0.4% sodium hyaluronate and salts such as potassium and magnesium for moistening and lubricating the ocular surface.

The 12 patients underwent a 12-month treatment, during which, at 3, 6 and 12 months from the beginning of the treatment, the presence of corneal innervation was examined (in terms of presence of central corneal nerves and progression of reinnervation), ocular symptomatology, the need to resort to lubricating eye drops or other additional eye drops, and corneal sensitivity.

During experimentation, patients have also simultaneously undergone to the standard post- transplant therapeutic treatment, which therefore also included the administration of an eye drops antibiotic (used for 2 months in decreasing doses) and of cortisone eye drops (used for 3 months in decreasing doses) during the day and a mydriatic medication and an ophthalmic gel with antibiotic and cortisone in the evening for a week.

For testing, the 12 patients were divided into the following two groups: - Group A: 7 patients, who were topically administered with an ophthalmic aqueous solution containing 0.5% by weight of sodium hyaluronate, 0.5% by weight of taurine and 0.05% by weight of vitamin B12, having pH 7, osmolarity 240 mOsm/kg and molar ratio Na7K ⁺ 5.2 at a dose of 0.4 ml/day for the first 6 months of treatment and 0.3 ml/day for the following 6 months, corresponding to the administration of 2 mg/day of sodium hyaluronate, 2 mg/day of taurine and 0.2 mg/day of vitamin B12 during the first 6 months of treatment and 1 .5 mg/day of sodium hyaluronate, 1 .5 mg/day of taurine and 0.15 mg/day of vitamin B12 in the following 6 months of treatment;

- Group B: 5 patients, who were topically administered, as a comparative composition, with Oftaial (produced by the Applicant, sterile ophthalmic solution containing 0.4% by weight of sodium hyaluronate having pH 7, osmolarity 240 mOsm/kg and molar ratio Na7K ⁺ 5.2) at a dose of 0.4 ml/day for the first six months of treatment and 0.3 ml/day for the following 6 months, corresponding to the administration of 1 .6 mg/ day of sodium hyaluronate in the first 6 months of treatment and 1 .2 mg/day of sodium hyaluronate in the following 6 months of treatment. The overall results of the trial at the end of the 12 months of treatment are shown in the following Table 1 .

Table 1 - Trial results

Performance evaluation Corneal reinnervation

Corneal reinnervation was evaluated under a confocal microscope. The presence of central corneal nerves in at least one of the three annual checks at 3, 6 and 12 months was interpreted as evidence of corneal reinnervation. In the trial, 57% of patients in Group A showed signs of corneal reinnervation, while only 20% of patients in Group B showed such signs. It was also found that reinnervation of patients in Group A was not associated with anomalies in the re-epithelialization of the transplanted cornea.

Effects on ocular symptomatology

Evaluation with the OSDI questionnaire revealed no symptoms in all patients during all evaluations, excluding a female patient, in Group B, for which instead a moderate dry eye condition was evident during all assessments.

Use of additional eye drops

Three patients in Group A had to add to the standard treatment the administration of Netildex (ophthalmic gel containing 2 mg/ml Netilmicin / 1 mg/ml Dexamethasone) for some periods during post-transplantation, due to the occurrence of stromal rejection phenomena. One patient in Group B was treated with Luxazone (eye drops containing 2mg/ml of dexamethasone) 6 months after transplantation, while 2 additional patients in Group B used supplemental lubricants.

Corneal sensitivity Corneal sensitivity was assessed with the retractable nylon wire Cochet-Bonnet extensometer. All patients in Group A showed a sensitivity in at least one of the three evaluations at 3, 6 and 12 months from the start of treatment, while 3 of 5 patients in group B reported complete absence of corneal sensitivity (corneal anesthesia in all the evaluations available). As can be seen from the checks carried out, the composition according to the present invention has shown to have beneficial effects on the regeneration of corneal nerve fibres on patients who have undergone keratoplasty, in the absence of treatment-related symptoms and leading to increased corneal sensitivity and less need for lubricating eye drops in the post-operative course.