Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1999-10-25
2003-03-25
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S001100, C435S336000, C435S384000, C424S078040, C514S002600, C514S912000, C514S914000
Reexamination Certificate
active
06537808
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the use of nerve growth factor for the storage of corneas in culture, the in vitro production of corneal and conjunctival tissues and the treatment of corneal and conjunctival diseases. More particularly, the invention concerns the use of the neurotrophin named nerve growth factor (NGF)—known for its ability of influencing the development, the regeneration and the maintenance of the biological functions of neurons—for the treatment of corneal and conjunctival tissues, both for properly maintaining their functionality in vitro and for the therapy of pathologies of the ocular surface.
As it is known, corneal graft represents the only therapeutic approach to dystrophic, inflammatory or degenerative corneal diseases in the event that the diseases have irreversibly compromised the transparency or the normal structure of the cornea. Once isolated from cadaver, the corneas are “stored” up to the time when they are transplanted, by means of storage methods employing liquids derived from cell culture mediums. According to one of the most common methods, called “short/intermediate term storage”, the cornea is kept at 4° C. in culture mediums such as McCarey-Kaufman (MK) medium, or in different commercial liquids (among which Optisol-GS ), for a maximum period of 7 days. A longer term storage is possible with an equally common method, referred to as “organ culture”. This method is based on techniques similar to cell culture, and allows to store the explanted corneas, at temperatures comprised between 30 and 36° C., for more than one month.
None of the above methods affords an improvement of the biological features of the corneal tissues with the storage, the best result achievable being that of maintaining unchanged the quality of the tissues during the storage. This co-operates in making even more critical the already difficult situation in the field of corneal graft. Actually, more than one half of the corneas are discarded upon explantation, since the microscope examination shows them to be unsuitable for transplant.
Another major problem is represented by the need of having available single corneal cell populations, such as endothelial cells, epithelial cells and keratocytes, so as to be able to use them for transplantation. In particular situations involving the destruction of the ocular surface due to physical or chemical burns, or resulting from synechiating autoimmune diseases (such as cicatricial pemphigoid, Stevens-Johnson syndrome, etc.) or from post-infectious pathologies, it is of a critical importance to have available corneal epithelial cells (including stem cells) to be transplanted in place of the damaged ocular surface, and/or to have available conjunctival epithelial tissue for use in replacing the synechiated and retracted surface.
Considering the pathological situations that may affect the morphological and functional unit consisting of cornea (i.e., epithelium, stroma/keratocytes and endothelium) and conjunctiva, it is noted that there is a wide variety of disorders affecting this region which are, to date, of a difficult therapeutic approach, or are totally orphan of a valid treatment. Such pathologies or situations which may interfere with the normal surface integrity include all the impairments of the tear film functions, congenital or acquired corneal and/or conjunctival affections such as: neurotrophic and neuroparalytic keratitis and/or conjunctivitis; post-traumatic, post-infectious, post-surgical keratitis and/or conjunctivitis; keratitis and/or conjunctivitis due to laser treatment, to chemical, physical or metal burns; autoimmune, dystrophic, degenerative, post-inflammatory keratitis. These diseases appear with alterations of the corneal epithelium (spontaneously in the primary neuroparalytic forms or on a physical, infectious, immune or toxic basis in the secondary forms) which may end in an ulcer, leading in most cases to corneal perforation, either spontaneously or following microtraumas or superinfections. The clinical picture of this disease is characterized by a slow and difficult recovery, by superinfections and by the frequent failure of any therapy. The final outcome is often the opacification of the cornea, or the spontaneous perforation thereof.
The above diseases are generally treated by having recourse to bandage and to the use of lubricating substances and antibiotics, with a view to prevent the complications. The conjunctival covering is necessary when the perforation is imminent or has already occurred, and has the only purpose of preserving the anatomic integrity of the eyeball, while the visual function is sacrificed. The corneal graft, both by lamellar and by penetrating keratoplasty, is absolutely contra-indicated in view of the frequent occurrence of relapses even on the transplanted graft, and in view of the risk of superinfections. On the basis of in vitro studies the topical medical treatment with preparations containing fibronectin, plasmin, collagenase inhibitors, EGF (i.e., epidermal growth factor), autoserum has also been proposed. None of these drugs turned out to be resolutive, or able to block or to reduce the corneal injury, or to modify the final outcome of the pathology.
In particular, in the treatment of corneal burns quite good results have been obtained with the transplantation of marginal conjunctiva taken from the contra-lateral eye. When the affection is bilateral, recourse may be had to the donation of conjunctiva by close relatives or, when this is not possible, to the excision from cadaver. However, this method is not resolutive in the totality of the cases. In the case of autograft relapses of the disease may occur after years, while in the case of homograft rejection is frequent, and an immunosuppressive therapy is absolutely necessary, with the well-known consequences due to the side-effects.
Another kind of corneal-conjunctival diseases the treatment of which often gives unsatisfactory results are the herpetic infections. After the resolution of a first pathologic occurrence, herpetic infections often give rise to relapses, the prevention and the treatment of which are often unsuccessful.
Also the diseases affecting the corneal endothelium, such as, e.g., primary and secondary decompensation and endothelitis, characterized by a loss in the number and/or in the function of endothelial cells, represent a group of affections which presently lack any effective treatment.
Therefore, it is a primary object of the present invention to provide a solution to the problems referred to above—which problems are all related to the physiopathology of the corneal and conjunctival morphological and functional unit—by means of the use of a therapeutic agent allowing to maintain and restore the proper biological activity of the tissues.
The molecule known as nerve growth factor (NGF) is the first component of a complex family of neurotrophins, and is well-known for its trophic, tropic and differentiating action on cholinergic neurons of the central nervous system and on the peripheral sympathetic system. NGF is produced in several tissues in mammals, including man, and is released in the blood stream at higher levels during the growth and the differentiation of the nervous system. Biological, biochemical and molecular studies carried out in vitro on cell systems have evidenced a very high homology between murine NGF and human NGF. In man as in other animal species the NGF is normally present both in the cerebrospinal fluid and in the blood stream at levels of about 10-50 pg/ml. These concentrations increase in some inflammatory pathologies (autoimmune diseases, allergic diseases, etc.) and decrease in other pathologies (diabetes).
The NGF was discovered in 1951 by Prof. Rita Levi-Montalcini, of the Zoology Institute of the Washington University of St. Louis (see Levi-Montalcini R., Harvey Lect., 60:217, 1966) and represented a major advance in the study of the growth and differentiation mechanisms of the nerve cell, as it is capable of influencing the development and
Anabasis S.R.L.
Flood Michele C.
Tate Christopher R.
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