Title:  Use of nerve growth factor for the storage, culture or treatment of cornea

United States Patent:  6,537,808

Issued: March 25, 2003

Inventors:  Lambiase; Alessandro (Rome, IT)

Assignee:  Anabasis S.R.L. (Rome, IT)

Appl. No.:  403568

Filed:  October 25, 1999

PCT Filed:  November 21, 1997

PCT NO:  PCT/IT97/00292

PCT PUB.NO.:  WO98/48002

PCT PUB. Date: October 29, 1998

The nerve growth factor (NGF) is used for the storage of corneas in culture, for the production and the storage in vitro of single cell populations of the corneal morphological and functional unit (i.e., epithelium, stroma/keratocytes and endothelium) and of the conjunctival epithelium, and for the production and the storage of corneal and conjunctival tissues, in particular for transplantation purposes. The NGF is also proposed for use in the therapy and/or the prophylaxis of diseases of the corneal surface, wherein a lack of integrity of the corneal and conjunctival morphological and functional unit occurs, in particular for pathologies having a dystrophic or neurodystrophic basis, both congenital and acquired.

DESCRIPTION OF THE INVENTION

The studies that led to the present invention started from the concept of cornea as a morphological and functional unit, wherein the single layers (i.e. epithelium, stroma and endothelium) all carry out a function of an equal importance in the preservation of the tissues integrity and in the recovery mechanisms. In this perspective, a key role is played by the trophic support offered by the corneal sensory innervation, whose damage brings about a damage to the whole morphological and functional unit.

In view of the fact that no evidence existed in the prior art of an effect of the NGF (i.e., one of the many mediators released by the sensory endings in other regions of the body) on the ocular surface, the question has been initially posed whether such neurotrophin might play a trophic-reparatory action on the corneal tissue of neuroectodermal embryonal derivation, since several targets of the neurotrophin show a similar embryogenetic derivation. Thus, cornea and conjunctiva have been analyzed for the presence of the NGF and of the NGF high affinity receptor (TrkA, tyrosine kinase A), using a monoclonal antibody (TrkA-antibody; Santa Cruz, USA) with immunohisto-chemistry techniques (Bhattacharyya A. et al., J. Neurosci. 17:7007, 1997). In fact, the presence of the specific receptor is a key requirement for the activity of the molecule under examination.

It has thus been found that all of the corneal cells (i.e., epithelial cells, endothelial cells and keratocytes) express TrkA and that, at the same time, the corneal somatosensory innervation is able to release NGF. This discovery allows to hypothesize, on one hand, that the NGF, probably released by the nerve endings, would play a physiopathologic role in all corneal reparative mechanisms (both at the surface and at a deeper level) and, on the other hand, that several superficial corneal diseases accompanied by primary damage to the innervation (such as neurotrophic or dystrophic keratitis and recurrences of herpetic infections) or with secondary damage to the innervation (such as chemical or physical burns and post-infectious, autoimmune, post-surgical lesions, or lesions resulting from laser treatment) would admit as a fundamental etiologic step the lack of NGF release.

The same finding also allows to hypothesize that the NGF would be essential to the maintenance of the whole corneal tissue in culture, i.e. in all such conditions wherein the cornea as a whole, intended as a morphological and functional unit comprising epithelium, stroma and endothelium, or some cells thereof, are deprived of the trophic support provided by the release of the NGF by the corneal nerve endings.

In addition, since the effects that have been observed after administration of exogenous NGF occur at concentrations close to the physiological level it can be hypothesized that a possible physiopathogenic mechanism in the corneal affections considered herein consists in a reduction of the local levels of the NGF below the threshold capable of assuring the corneal and/or conjunctival integrity.

Accordingly, the present invention specifically provides, according to a first aspect thereof, the use of the nerve growth factor for the storage of corneas in culture and for the storage and the production in vitro of corneal and conjunctival tissues, and of single corneal or conjunctival cell populations.

Preferably, the NGF is added to a culture medium of the kind suitable for the storage of corneas, or for the in vitro culture of corneal or conjunctival cells or tissues, in amounts comprised between 100 pg/ml and 200 ng/ml, optionally together with other nutrients and other biologically active agents. The NGF to be used to that aim can be of a murine or human origin, including recombinant NGF, and it may be used in lyophilized form, dissolved in solution, in a culture medium or in any other available solvent, so as to obtain a final concentration comprised in the range specified above.

Suitable methods of extracting and purifying the NGF are reported in the literature mentioned above. For the purpose of experimenting the present invention, the Bocchini and Angeletti technique (cited above) has been adopted, the latter being synthetically set forth below. The submaxillary glands of adult male mice are explanted under sterile conditions and the tissues are homogenized, centrifuged and dialyzed; then the suspension is passed through subsequent cellulose columns, thereby separating the NGF by adsorption. The NGF is then eluted from the column by means of a buffer containing 0.4 M sodium chloride. The samples so obtained are analyzed by spectrophotometer at a wavelength of 280 nm to identify the NGF-containing fractions. The latter are dialyzed, and the NGF thus obtained is lyophilized under sterile conditions and kept in refrigerator at -20^oC.

In an evaluation of the effects of an addition of NGF to various culture mediums for explanted corneas (both at 4^oC. and at 30-36^o C.), a general improvement of the biological features of the corneal tissue has been obtained with amounts of NGF comprised between 100 pg/ml and 200 ng/ml. The optimal response has been obtained with a concentration of about 100 ng/ml. Specifically, after 7 days of culture the improvements obtained were an increase of the endothelial cells density (increase from 10 to 25%), a reduction of the endothelial cells mortality (absence of trypan blue positive endothelial cells), a better endothelial morphology (i.e., quantitatively, a 3/3 trophism vs. a 2/3 trophism in the controls without addition of NGF), a higher viability of the keratocytes and a remarkably better appearance of the epithelium. In addition, some corneas that before being placed in culture were considered not suitable for transplantation turned out to be suitable after being cultured for 7 days in presence of NGF.

A similar experimental evaluation of the effects of the NGF as additive in the culture medium of corneal cells (i.e., epithelial cells, endothelial cells and keratocytes) showed that the addition of murine NGF in amounts comprised between 100 pg/ml and 200 ng/ml (with an optimal response at the concentration of about 100 ng/ml) induces the proliferation and differentiation of the various cell populations, and, moreover, it assists the rooting of the cell populations on a number of supporting tissues (such as lamellae of corneal stroma, amniotic membrane, etc.). Further, such addition favors the interaction between the different cell types in cell co-cultures. Thus, the invention may be also employed for culturing mixed corneal cell lines in order to obtain an artificial cornea useful for transplantation.

Also the conjunctival epithelial cell cultures have shown a proliferation and differentiation increase, as well as an increase in the number of goblet cells, when cultured in presence of murine NGF at the same concentrations specified above. The graft of epithelial conjunctival tissue thus obtained is extremely advantageous for replacing the conjunctiva of patients suffering from kerato-conjunctivitis sicca (dry eye syndrome), both primary and secondary to other pathologies.

According to another aspect of the invention, there is provided, therefore, a culture medium for the storage of corneas or for the storage and the production in vitro of corneal and/or conjunctival tissues, or of single corneal or conjunctival cell populations, characterized in that it contains an effective amount of NGF, preferably comprised between 100 pg/ml and 200 ng/ml, most preferably about 100 ng/ml.

As set forth before, according to a further aspect of the invention there is provided the use of the nerve growth factor in the manufacture of a medicament for the therapy and/or the prophylaxis of corneal and/or conjunctival diseases.

Specifically, the invention is useful for the production of medicinal products suitable for the treatment and/or the prophylaxis of congenital and/or acquired corneal and/or conjunctival diseases chosen from the group consisting of: neurotrophic and neuroparalytic keratitis and/or conjunctivitis; herpetic keratitis and/or conjunctivitis; post-traumatic, post-infectious, post-surgical keratitis and/or conjunctivitis; keratitis and/or conjunctivitis due to impairment of the tear film functions, to laser treatment, to chemical, physical or metal bums; autoimmune, dystrophic, degenerative and post-inflammatory keratitis.

Preferably, a medicinal product according to the invention suitable for topical administration contains, alone or in combination with one or more other active ingredients, from 10 to 500 .mu.g of NGF per ml, an optimal concentration being about 250 .mu.g of NGF per ml. Such product may be in the form of ophthalmic solution for eye-drops, or in the form of a gel, an ointment, a cream or a powder, or it can be added to a local bandage or to a medicinal contact lens.

According to another embodiment of the invention, the pharmaceutical product for the manufacture of which the NGF is proposed is a medicament indicated for the therapy and/or the prophylaxis of primary and secondary endothelial ophthalmic pathologies. For this use, the preferred formulations contain (also in this case optionally in combination with other active ingredients) from 1 to 250 .mu.g of NGF per ml, and the administration is carried out by introduction in the anterior chamber of the eye.

In vivo studies on animals concerning the treatment of disorders of the corneal endothelium have been carried out by administration in the anterior chamber of the eye of aqueous solutions containing NGF at concentrations comprised between 1 and 250 .mu.g/ml. In particular, in rabbits wherein an endothelial damage had been introduced by means of a cryoprobe a complete restoration of the endothelial density has been obtained after 15 days of treatment with NGF. The administration of NGF in endothelial pathologies, both of a dystrophic nature and acquired, both with loss of the number of endothelial cells and with loss of the functionality thereof, has been shown to restore a proper endothelial function.

In a series of studies on man for ascertaining the efficiency of the treatment with NGF in the topical treatment of corneal and/or conjunctival affections, a number of pathologies have been selected on the basis of a primary or secondary involvement of the somato-sensorial corneal nerve plexus. Murine NGF (2.5S), obtained with the purification method summarized above, has been employed in such studies. The NGF has been topically administered at a concentration of about 250 mg/ml, diluted in balanced salt solution.

The table shown further on summarizes the results obtained from a study on 5 patients affected by torpid corneal ulcer due to neurodistrophic keratitis (2 eyes), or after keratoplasty (2 eyes) or due to bum by alkali (2 eyes). The therapeutic schedule consisted in the instillation of one-two drops of the preparation of the invention with a daily frequency divided as follows: every 2 hours for the first 2 days, 6 times a day up to the 2nd day after the complete re-epithelization of the cornea and twice a day for the following 15 days.

The topical treatment with the NGF was started after 15 days of treatment with autoserum, in the absence of signs of improvement of the clinical picture. All of the patients treated showed clear signs of recovery within 2 weeks from the start of the treatment with NGF, and none of them evidenced any occurrence of local or systemic side-effects during the treatment or in the following period. Once suspended, the therapy should be immediately restored in the event that the first signs or symptoms of any relapse of the epithelial pathology appear.

The concerned 5 cases are discussed in detail below, and the main data are summarized in the following table.

1st Case

A 9-year-old female, affected by a congenital anophthalmos, showed in the other eye a corneal ulcer since more than 20 days. The ulcer, topically treated with antibiotics and steroids, did not show any tendency to healing. The ulcer was about 7 mm in diameter and more than 2/3 of the corneal stroma in depth. The clinical examination showed corneal anaesthesia, on the basis of which a neurotrophic keratitis was diagnosed. After 15 days of treatment with autoserum, having ascertained a progressive worsening of the clinical picture, the topical treatment with NGF was started.

After 4 days the corneal ulcer was reduced to about a 3.5 mm in diameter, and after 12 days the cornea was completely healed and the treatment with the NGF was discontinued. The patient showed a central leukoma with some neo-vessels and, although reduced, a corneal sensitivity was present, while it was completely absent before the treatment. After a 8 months follow-up the patient showed 5/10 of visual acuity and the corneal sensitivity was still present.

2nd Case

A 26-year-old woman, affected by syndactilia and deficit of the VIII pair of cranial nerves, showed since about 2 months a corneal ulcer which progressively deepened until it reached an extent of 7 mm and a depth up to the Descemet membrane. The clinical examination showed a complete corneal anaesthesia.

After 2 weeks of therapy with the NGF apparent signs of recovery were present, with a reduction in the ulcer depth. After 6 weeks of therapy the cornea was completely re-epithelized, while a central leukoma with some neo-vessels was left. In addition, some corneal sensitivity was recovered, although reduced.

3rd Case

A man aged 25, who had undergone enucleation of the left eye following the onset of a corneal ulcer after a penetrating keratoplasty, showed since about 1 month a corneal ulcer in the right eye, into a corneal graft. The corneal ulcer was about 5 mm in diameter and its depth was 1/2 of the corneal stroma. Corneal anaesthesia was also present.

After 2 weeks of treatment with NGF a corneal healing process was evident, with reduction of the extent and the depth of the ulcer. After 4 weeks of treatment with NGF the ulcer was completely healed and a central leukoma with some neo-vessels was left. A marked corneal hypoesthesia was still present.

4th Case

A 56-year-old man showed a bilateral corneal ulcer due to a burn by alkali. The right eye, after having undergone surgery by penetrating keratoplasty, had been enucleated after the onset of an ulcer. The left eye, after undergoing lamellar keratoplasty, developed a torpid ulcer of about 7 mm in diameter, which did not show any sign of healing.

After 2 weeks of treatment with NGF the onset of a recovery process was evident, and the process was complete after 5 weeks.

5th Case

A 56-year-old man showed a bilateral corneal ulcer due to a burn by hydrochloric acid. In the right eye an ulcer of about 4 mm in diameter was present, while in the left eye the ulcer was bigger (more than 8 mm in diameter) and deeper. The patient showed a marked corneal hypohestesia.

After 2 weeks of treatment with NGF the right eye was completely healed, with a residual central leukoma, while the ulcer in the left eye was reduced both in extent and in depth, although a neo-vessel pannus was present. After 3 weeks of treatment both corneas were fully re-epithelized, a central leukoma was left and corneal sensitivity was present, although reduced. After a 2 months follow-up both corneas remained re-epithelized, and the visual acuity of the right eye was 3/10, while that of the left eye was 1/10. In addition, a corneal sensitivity was still present in both eyes.

 

The foregoing data clearly show the effectiveness of the use of the NGF not only for storing and producing in vitro corneal and/or conjunctival tissue, as a whole or partially, or for storing and producing in vitro the single cell types of which the tissues consist, but also for treating and preventing human or animal diseases affecting the corneal morphological and functional unit or the conjunctiva, which diseases have not found, so far, an effective therapy.

The present invention has been disclosed with particular reference to some specific embodiments thereof, but it should be understood that modifications and changes may be made by the persons skilled in the art without departing from the scope of the invention as defined in the appended claims.

Claim 1 of 19 Claims

What is claimed is:

1. A method for treating or reducing the risk of a corneal or conjunctival disease, comprising:

topically administering an effective amount of nerve growth factor on an ocular surface of an eye of a subject in need thereof to treat or reduce the risk of the corneal or conjunctive disease,

wherein the disease is at least one member selected from the group consisting of keratitis due to impairment of tear film function, conjunctivitis due to impairment of tear film function, autoimmune keratitis, and autoimmune conjunctivitis.
 

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