Indian Patents. 268726:PHYSIOLOGICAL SUPPLEMENT OR MEDICAMENT FOR OPHTHALMIC USE AS EYE-DROPS

Title of Invention	PHYSIOLOGICAL SUPPLEMENT OR MEDICAMENT FOR OPHTHALMIC USE AS EYE-DROPS
Abstract	Use of L-carnitine and/or of one or more alkanoyl L-carnitines or one of their pharmaceutically acceptable salts for the preparation of an opthalmic physiological supplement or medicament in the form of eye-drops, for the treatment of corneal diseases.

Full Text	WO 2007/003481 PCT/EP2006/062919 The present invention relates to the use of L-carnitine and/or one or more alkanoyl L-carnitines for the preparation of a physiological supplement or medicament in the form of eye-drops useful for the treatment of corneal diseases. The cornea is the transparent, dome-shaped window covering the front of the eye. It is a powerful refracting surface, providing 2/3 of the eye's focusing power. Like the crystal on a watch, it gives us a clear window to look through. The cornea is extremely sensitive - there are more nerve endings in the cornea than anywhere else in the body. The adult cornea is only about 1/2 millimeter thick and is arranged in three main regions, or layers: - Epithelium: it functions primarily to block the passage of foreign material such as dust or water into the eye, and other layers of the cornea, and provide a smooth surface that absorbs oxygen and other needed cell nutrients that are contained in tears. This layer, which is about five cells deep, is filled with thousands of tiny nerve endings that make the cornea extremely sensitive to pain when rubbed or scratched. - Stroma: is located behind the epithelium, the stroma comprises about 90 percent of the cornea. It consists primarily of water and layered protein fibers that give the cornea its strength, elasticity, and form; and cells that nourish it. The unique shape, arrangement, and spacing of the protein fibers are essential in producing the cornea's light-conducting transparency. WO 2007/003481 PCT/EP2006/062919 2 Endothelium: this single layer of cells is located between the stroma and the aqueous humor. Because the stroma tends to absorb water, the endothelium's primary task is to pump excess water out of the stroma. Without this pumping action, the stroma would swell with water, become hazy, and ultimately opaque. Many diseases can damage this delicate structures. The main causes of impairment of the epithelial structure of the cornea are dry eye syndrome; corneal abrasions and injuries due to, for example, the application of contact lenses; and refractive laser surgery. Other diseases of the cornea are associated with impairment of the normal transparency of the corneal surface, caused, for example, by damage in the aftermath of keratitis, particularly bacterial, viral or fungal keratitis; by damage resulting from trauma and refractive laser surgery; as well as degenerative or hereditary diseases such as chronic and acute keratocono. The tear film, which coats the corneal epithelium and is essential for the homeostasis of the eye surface, performs an important optical function, acting as a lubricant between the eyelids and the eyeball and as a vehicle for oxygen, guaranteeing the metabolism of the cells of the corneal epithelium; it also performs a flushing function, ensuring the removal of external agents. Also important is its function as a carrier for growth factors, neuropeptides, and neuromodulators that regulate the activation, proliferation and differentiation of corneal and conjunctival epithelial WO 2007/003481 PCT/EP2006/062919 3 cells. It also transports immunoglobulins (IgA, IgAs, IgG, IgE, IgM), complement factors (C3, C4, C5), metalloproteases (MMP-2, 4, 9), enzymes (lysozyme, lactoferrin) and immune system cells (limphocyte), thus performing a fundamental defensive function against infections. Dry eye syndrome is characterised by a quantitative (hypolacrimation) and/or qualitative (dyslacrimation) impairment of the tear film of multifactorial origin which may or may not cause clinically significant damage to the eye surface. The prevalence of dry eye syndrome ranges from 10 to 40% in the adult population and there is a highly significant correlation with age. In the United States the prevalence of mild-to-moderate dry eye syndrome is up to approximately 10 million people (Am. J. Ophthalmol:, 1997; 124:723-728; Arch Ophthalmol, 2000; 118: 1264-1268). Various studies conducted in order to understand the mechanisms activated in this disease have shown that the tears of subjects affected by dry eye syndrome present: an increased evaporation rate, increased surface tension, reduced vitamin A concentration, increased osmolarity, reduced concentration of a number of proteins (lysozyme, lactoferrin), insufficient mucus production or qualitative changes in mucus production, with consequent inadequate reconstruction of the mucus layer, reductions in a number of growth factors (EGF, TGF-α, aFGF-bFGF, LG-F, HGF) (Contactologia, 1982; 4: 34-37), changes in concentration of inorganic elements, reduced androgens and dysregulation of T lymphocyte WO 2007/003481 PCT/EP2006/062919 4 activity (Cornea, 2005; 24: 1-7). Among the mechanisms activated in this disease the use of contact lenses has to be mentioned. The clinical signs regarded as being most closely related to this pathological condition are reduced break-up time (BUT test) and Schirmer test results (Pescosolido N.: Le alterazioni del film lacrimale. In Stendler P.: "il sistema lacrimale", Fabiano editore, Canelli (AT), 2000; pag. 237-330; hereinafter this reference will be referred to as Pescosolido 2000). The BUT test has to do with the mucin content of the tear film and, in the dry eye, yields only values below 5 seconds. The Schirmer test, on the other hand, has to do with the water content of the tear film and, in the dry eye, yields values below 5 millimetres in 5 minutes. The patient presents the following symptoms: foreign body sensation, burning, difficulty blinking, bruit on opening the eyelids, itching, eye fatigue, photophobia, blurred vision, and mucus extravasation at the inner canthi. The treatment of this syndrome is based on the use of the following: tear substitutes whose task is the regular moisturising of the cornea, but which do not exert any action on the basic causes of the disease and are endowed only with very short-lasting efficacy; inserts (plugs) in the lacrimal canaliculus; immunoregulators such as topical cyclosporin; topical steroids; anti-inflammatory agents (rumexilone and loteprednol); autologous serum (cytokine inhibitors); topical or systemic androgens; mucus (HETE eicosanoid) and aqueous (P2Y2 WO 2007/003481 PCT/EP2006/062919 5 agonists) secretogenic substances; acquaporins and agents such as antibiotics and detergents for the treatment of a disease often concomitant, blepharitis (Cornea, 2005; 24:1-7). Also used is treatment with iodide iontophoresis owing to its scavenger activity as a reducing agent and electron donor (Adv. Clin. Path., 2000; 4:11-17; Br. J. Ophthalmol, 2005; 89: 40-44). Even these latter treatments, despite exerting an action which may be regarded as more relevant to treating the causes of the disease, have failed to yield the anticipated results. The normal transparency of the corneal surface can be impaired by the aftermath of numerous diseases acquired, degenerative or congenital that damage the delicate structure of the various constituent components. The acquired disease conditions most commonly implicated are post-keratitis damage, particularly after herpetic keratitis, and damage occurring in the aftermath of trauma and laser refractive surgery. The minimum common denominator is the formation of corneal opacities (leucomas) that functionally jeopardise vision. The events involved in wound healing that occur in the corneal tissue after infection, caustic damages, injury and refractive ablative surgery are have a profound effect on the final morphological and refractive outcomes of the restitutio ad integrum process. The acute epithelial and stromal corneal lesions occurring immediately after injury and laser ablation are probably involved in the regulation of the subsequent corneal tissue repair events, and, among WO 2007/003481 PCT/EP2006/062919 6 the latter, keratocyte apoptosis probably plays a major role (Cornea, 2000;19:S7-12). This event is responsible for the corneal repair process since keratocyte apoptosis is the prime mover of the reproliferative stimulus. The stroma keratocytes underlying the initial acellular stroma therefore represent the cell source that mediates the subsequent healing of the surface stroma beneath the epithelium. As a result of the cellular repopulation, the activated keratocytes undergo myofibroblastic transformation (Invest. OphthalmoL Vis. Sci., 1998;39:487-501), thus proving responsible for the production of collagen fibres and of basic substances involved in the restituito ad integrum process. A recent scar is very similar to foetal tissue, it is rich in foetal and type III collagen with thick fibrils, wide interfibrillary spaces and a hydrophilic stroma. This explains the characteristic opacity. It also contains fibrinogen, fibronectin, laminin and weakly sulphurised keratan sulphate. The keratan sulphate/condroitin sulphate is reduced in the scar stroma. The myofibroblasts disappear in a few weeks to a few months. Gradually the scar changes and becomes less opaque. The resistance of the stromal scar tissue is 20% compared with normal tissue after three weeks, 60% after 1 year and 70% after 3-4 years. Corneal reinnervation is very slow. The cytokines play an important role in these events. IL-la, produced by the corneal epithelium, stimulates metalloprotease and collagenase synthesis through the keratocytes and the myofibroblasts. The TGF-β reduces the production of WO 2007/003481 PCT/EP2006/062919 7 collagenase and is involved in the formation of haze. IL-6 reduces the synthesis of metalloprotease thus increasing collagen synthesis. This process, however, is not self-controlled and, in many cases, abnormal, excessive healing occurs followed by a greater production of collagen type III, an increase of jaluronic acid, and an increase in lamellar disorganisation (Arch. Ophthalmol, 1990; 108: 665-675; Exp. Eye Res., 2004; 78 : 553-560; Ophthalmology, 2000; 107 : 1235-1245) or an accumulation keratocytes e dei myofibroblasts as shown by confocal microscopy (Progr. Retin. Eye Res., 1999; 18:311-356; J. Cataract Refract. Surg., 2000; 26 : 432-447; Exp. Eye Res., 2004; 78 : 553-560). These abnormalities are involved in the pathogenesis of the most feared complication of stromal regeneration after photorefractive keratectomy (PRK), namely, haze, with consequent impairment of the functional outcome. Haze is classified according to Heitzmann in 5 degrees on the basis of the visual impairment due to the reduced corneal transparency (Ophthalmology, 1998; 35 : 1411-1421); or according to the Fantes scale (Arch. Ophthalmol, 1990; 108 : 665-675) which distinguish 6 degrees of haze (0; 0.5; 1+; 2+; 3+; and 4+). Though the incidence of haze has been substantially reduced over recent years, as a result of the technological advances in the field of excimer lasers, it is still a fairly frequent complication even today and, in rare cases, would appear hard to reverse, even after months of cortisone therapy (0,5% of the eye with myopia not exceeding 6 diopters and 3-17% in the eye with myopia exceeding 10 diopters WO 2007/003481 PCT/EP2006/062919 8 (Ophthalmol. Clin. North. Am., 2001, 14 : 359-376). In cases of persistent haze (more than 15-18 months) which fails to respond to medical cortisonic therapy [an event that can occur with late-onset haze (4-12 months after the operation (Ophthalmology, 1997; 104 : 369-374; Cornea, 2004, 23 : 350-355), as the only feasible procedure is phototherapeutic keratectomy (PTK) with an excimer laser, a procedure used for the laser-assisted surgical removal of superficial stromal opacities combined with 0.02% mitomycin (J. Refract. Surg., 2003; 19:40-43; J. Refract Surg., 2003,19:449-454). The use of the activator of plasminogen and of TGF-al is still undergoing animal studies (J. Refract. Surg., 1997; 13:356-361; Invest. Ophthalmol. Vis. Sci., 2004;45:1329-1333), while INFp-2b, tested on man, has produced poor results (J. Refract. Surg., 1996;22:891-900). Indeed, while low degree haze is generally asymptomatic, or only causes a slight reduction in sensitivity to contrast, serious forms of haze can interfere with refraction and be accompanied by myopic regression due to exuberant scarring (J. Refract. Surg., 1995; 11.341- 347; Ophthalmology, 2000; 107:1235-1245). The corneal dystrophies (Bietti, 1971; Oftalmologia geriatrica) are rare disorders. They are defined as primitive corneal disorders not associated with trauma, earlier inflammation or systemic diseases. They affect both eyes, are hereditary and, for the most part, have an autosomal dominant trait. Corneal degeneration is far more common than dystrophies but the symptoms are generally less obvious. It is not hereditary, can be WO 2007/003481 PCT/EP2006/062919 9 unilateral or bilateral and the course is gradual or relatively stable. In each case, it is, by definition, permanent, or does not resolve itself spontaneously. Usually it involves more than one layer of the cornea. Distinctions can be drawn between primary or idiopathic and secondary forms. The first are often connected with ageing without being preceded by any specific pathological process, the second ones are always associated with eye diseases which precede them, whether acute, chronic, infectious or inflammatory in nature. Keratoconus is one of the most typical degenerative diseases. Keratoconus is a non-inflammatory ectasia of the paracentral portion of the cornea which evolves over time and takes a progressively conical form, with tapering of the tip. This corneal deformation results in severe astigmatism, often irregular, corrected wherever possible with hard gas-permeable corneal lenses. Only rarely are epikeratoplasty, photorefractive keratectomy or the use of intracorneal rings (INTACTS) considered (Cornea, 1987;6:131-139; Ophthalmology, 1992;99:1187- 1192; Surv. Ophthalmol., 1998;42:297-319;J. Cataract. Surg., 2000; 26 : 1117-1122; J. Refract.Surg., 2001;17:69-73) or also the use of hard gas-permeable lenses combined with INTACTS. All these techniques only correct the refractive defects but do not resolve the cause of the corneal ectasia and therefore do not stop the progress of keratoconus, which in its most severe form means a corneal transplant (Cornea, 1997;16:623-629; Cornea, 1997;16:414-419; Cornea, 1998; 17:468-470; Cornea, 2002;21:152-155 ). WO 2007/003481 PCT/EP2006/062919 10 Keratocomis starts at puberty and in 20% of cases perforating keratoplasty has to be performed (Ophthalmology, 1994; 101 : 439- 447). Past studies have not produced relevant results leading to an understanding of the physiopathology of the disease but only more recently, with the development of molecular techniques, have advances been made in understanding this abnormality. At present, however, we are not able to say that the presence of keratoconus is due to uniform anomalies which are specific to the extracellular matrix. There are areas where the elements of the basal membrane are absent indicating on-going proteolytic activity, and there are areas in which there are deposits of fibrotic substance which is also found in other pathologies. A cornea with keratoconus has low levels of inhibitory enzymes (TIMP) and greater activity of the enzymes which are capable of degrading the extracellular matrix. The TIMP play a predominant role in the thickness of the stroma and in the stability of Bowman's membrane, which is characteristically interrupted in keratoconus. Furthermore the decrease in one of these inhibitors, TIMP-1, could play an important role in apoptosis or in the anomalous behaviour of the cells found in keratoconus. Apoptosis is the programmed death of the cells. This death is necessary for reconstructing damaged cells and for the normal turn- over of many tissues. WO 2007/003481 PCT/EP2006/062919 11 In keratoconus, apoptosis is more frequently found in the stroma (Invest. Ophthalmol. Vis. Sci., 1998; 39 : 220-226) than in other corneal layers (Cornea, 2002; 21 : 206-209). This observation is important because a cornea with keratoconus is subject to chronic irritation caused by hard gas-permeable contact lenses, to greater friction and to moderate or severe atopy. Wilson suggests that mechanical trauma on the epithelium could cause apoptosis in the stromal cells of keratoconus (Exp. Eye Res., 1999; 69 : 225-266;Cornea, 2000; 19 : S7-S12). Furthermore, an increase in the levels of leukocytes commonly correlated to the LAR protein antigen present in keratoconus but not in normal corneas can be seen (Exp. Eye Res., 1999;68:283-293). LAR is a transmembrane phosphotyrosine transferase capable of stimulating apoptosis (Proc. Nati. Acad. Sci. USA, 1994;91:10868- 10872; J. Neurobiol., 2000;42:477-486). A third triggering mechanism of apoptosis is that it is inhibited by TIMP-1 (J. Clin. Invest., 1998;102:2002-2010; Blood, 1998;92:1342-1349) which in keratoconus proves to be reduced, as has been reported previously. In practice, given these statements, the phenomenon of apoptosis could play an important role in the pathogenesis of keratoconus. Sodium hyaluronate is a very well known compound used to protect corneal epithelial cells, especially in patients with dry eye syndrome or with Sjogren's syndrome. The action of sodium hyaluronate is due not only to its protective role of a mechanical type exerted on the epithelial cells as a result of its viscoelasticity, in WO 2007/003481 PCT/EP2006/062919 12 situations of reduced tear production, but also to the positive effects of its particular biological function on corneal epithelial cells by stimulating their migration (Exp. Eye Res., 1991 ;53:753-758). Taurine (or 2-aminoethanesulphonic acid) is considered an amino acid, even though it does not possess the characteristic carboxyl group (COOH) but the SO3H group. Taurine is only present in the animal realm, whereas vegetable foods do not possess this amino acid. Vitamin E is the main antioxidant of the cell membranes, and is found in the human body in 4 forms consisting of α-tocopherol, β- tocopherol, γ-tocopherol and δ-tocopherol. Of these the a form is the most frequent in the retina and in plasma and is the one with the greatest antioxidant and free radical scavenging activity. Vitamin A is essential for cell vitality, cytoskeletal organisation, it regulates the expression of proteins of the extracellular matrix, cell adhesion and wound repair (J. Cel.l Biochem., 2003; 89 : 837-847). Vitamin A is also involved in the production of angiogenic (inactivation) and anti-angiogenic (activation) factors in the endothelial cells (Exp. Eye Res., 2004; 78 : 945-955). Some of these, stromal derived factor 1 (SDF-1) are secreted under the effect of proinflammatory signals (IL- l,TNF-a, bacteria and viruses) and, in cooperation with other factors, are involved in the proliferation of the vascular endothelial cells. SDF-1 and its receptor CXCR4 have been found in human keratocytes (Mol. Vis. , 2003;9:96-102). Two other systems are also involved in corneal angiogenesis: vascular endothelial growth factor (VEGF) and fibroblast WO 2007/003481 PCT/EP2006/062919 13 growth factor b (b FGF). Anti-angiogenetic factors contrast these cytokines as the pigment epithelium derived factor (PEDF) (Mol. Vis., 2001, 7 : 154-163). IL-4 and TGF-β are also known to inhibit corneal angiogenesis in vivo (Acta Ophthalmol. Scand., 2002;80:238-247). Metalloproteases are able to intervene in the regulation of angiogenesis with inhibitory or potentiating effects. In the absence of vitamin A, the corneal epithelium becomes keratinised. Retinol and retinic acid (Vitamin A), which help to avoid the keratinisation of the corneal epithelium, are supplied by the tears. Nuclear receptors with retinoids are present in the epithelium and in the stroma. Cytidine-5'-diphosphate choline (CDP-choline), commonly known by the name of citicholine, is a precursor of phosphatidyl choline, the main phospholipid of the cell membranes. Due to the effect of the activation of certain lytic enzymes, the phospholipases, the catabolism of the membrane phospholipids is accelerated and, if the resynthesis mechanism is inadequate, toxic substances accumulate, such as the ceramides, which can activate the metabolic pathways which lead to cell apoptosis. A deterioration in the turn-over of the phospholipids adversely affects the validity of the membrane protection systems and puts cell function at risk. The use of inorganic elements is well known in the medical field, and a number of these are essential for the stability of the tear film (Pescosolido 2000). Previous uses of carnitine in the ophthalmological field are already known. WO 2007/003481 PCT/EP2006/062919 14 US Patent 5,037,851 describes the use of acetyl L-carnitine for the treatment of cataract. US 5,145,871 and 5,432,199 describe the use of acetyl D- carnitine for the treatment of glaucoma. US 5,883,127 describes the use of acetyl L-carnitine for the treatment of maculopathy and macular degeneration. Further uses of carnitine are also known. In Res 1992;18(8):355-365 the use of L-carnitine in the cardiological field is described. US 5,543,556 describes the use of acyl L-carnitine esters with gamma-hydroxybutyric acid for the inhibition of neuronal degeneration and in the treatment of coma. US 5811457 describes the use of propionyl L-carnitine for the treatment of chronic obliterating arteriopathy. None of the above-cited patents or publications describes or suggests the use of L-carnitine or of alkanoyl L-carnitine for the treatment of diseases of the cornea. In the medical field there is still a strongly perceived need for the availability of therapeutic agents or physiological supplement useful for the treatment of the above-mentioned corneal diseases. It has now been found that L-carnitine and/or one or more alkanoyl L-carnitines, or one of their pharmaceutically acceptable salts, are useful agents for the preparation of a physiological supplement or medicament in the form of eye-drops for the treatment of diseases of the cornea. WO 2007/003481 PCT/EP2006/062919 15 What is meant by pharmaceutically acceptable salt of L-carnitine is any salt of the latter with an acid that does not give rise to toxic or side effects. These acids are well known to pharmacologists and to experts in pharmacy. Non-limiting examples of such salts are: chloride, bromide, orotate, aspartate, acid aspartate, acid citrate, magnesium citrate, phosphate, acid phosphate, fumarate and acid fumarate, magnesium fumarate, lactate, maleate and acid maleate, oxalate, acid oxalate, pamoate, acid pamoate, sulphate, acid sulphate, glucose phosphate, tartrate and acid tartrate, glycerophosphate, mucate, magnesium tartrate, 2-amino-ethanesulphonate, magnesium 2-amino- ethanesulphonate, methanesulphonate, choline tartrate, trichloroacetate, and trifluoroacetate. What is meant by pharmaceutically acceptable salt of L-carnitine is also a salt approved by the FDA and listed in the publication Int. J. of Pharm. 33 (1986), 201-217, which is incorporated herein by way of a reference. One object of the present invention is the use of L-carnitine and/or of one or more alkanoyl L-carnitines selected from the group consisting of acetyl, propionyl, valeryl, isovaleryl, butyryl and isobutyryl L-carnitine, or one of their pharmaceutically acceptable salts, for the preparation of an ophthalmic physiological supplement or a medicament in the form of aeye-drops, for the treatment of corneal diseases in which said corneal disease is selected from the group comprising, de-epithelialising diseases, dry eye syndrome; infective WO 2007/003481 PCT/EP2006/062919 16 keratitis; acid or alkaline caustic damages; corneal abrasions and/or injuries due to mechanical action or contact lenses; degenerative disease of the corneal stroma such as acute or chronic keratocono, stromal damages caused by refractive laser surgery; and dystrophic diseases, impairment of the transparency of the surface of the cornea caused by various types of infective keratitis (viral, bacterial and fungal), or by injuries that damage the structure of the various components constituting the cornea, such as, for instance, injuries of a mechanical, post-surgical and post-laser-refractive surgery type (such as, for example, haze); hereditary or degenerative diseases such as chronic and acute keratoconus. A further object of the present invention is a physiological supplement or medicament for ophthalmic use, in the form of eye- drops, comprising as the active ingredient L-carnitine, or one of its pharmaceutically acceptable salts, in combination with humidifying agents such as sodium hyaluronate; antioxidants such as vitamin E; inorganic elements as components of enzymes present in the tear film such as manganese; inorganic and organic elements such as sodium, potassium and the amino acid taurine for the regulation of the cellular osmolarity, and optionally ophthalmologically acceptable excipients and/or diluents; in which: u L-carnitine is present preferably at a dose of 0.3-3%, and most preferably at a dose of 1%; WO 2007/003481 PCT/EP2006/062919 17 taurine is present preferably at a dose of 0.1-4%, and most preferably at a dose of 0.5%; sodium hyaluronate is present preferably at a dose of 0.05- 1.5%, and most preferably at a dose of 0.2%; vitamin E is present preferably at a dose of 0.05-1.0%, and most preferably at a dose of 0,2%; manganese is present preferably at a dose of 0.01-0.1 mg/L, and most preferably at a dose of 0.051 mg/L; zinc is present preferably at a dose of 0.5-1.5 mg/mL, and most preferably at a dose of 1.02 mg/mL; sodium is present preferably at dose of 5-5000 mg/L, and most preferably at a dose of 30 mg/L; potassium is present preferably at a dose of 1-1000 mg/L, and most preferably at a dose of 9 mg/L. The eye-drops according to the present invention may additionally contain further antioxidants such as, for example, vitamin C, Borage oil; epithelialising and anti-angiogenic agents; humidifying agents; inorganic elements; regulator of the cellular osmolarity; antibiotics; antiviral and antifungal agents. The following examples illustrate the invention. EXAMPLE 1 A clinical trial was conducted in which 43 patients suffering from dry eye syndrome were recruited. The patients recruited were all women aged from 35 to 77 years (mean age: 59.5 ± 10.4 years), 33 of whom were suffering from WO 2007/003481 PCT/EP2006/062919 18 Sjogren's syndrome, diagnosed on the basis of Fox et al.'s criteria (Arthritis Rheum, 1986; 29: 577-584; 1986). Patients were selected on the basis of the BUT test, the Schirmer test, the fiuorescein test and the rose bengal test (Pescosolido 2000; Arch. OphthalmoL, 1969;82: 10-14). The BUT test had to yield results ≤ 5 seconds, while the Schirmer test did not contraindicate inclusion in the trial. Damage to the surface of the eye was evaluated by means of the rose bengal staining test and the fiuorescein test. Damage in the rose Bengal staining test was determined by reference to the van Bijsterveld evaluation (Arch. OphthcdmoL, 1969;82: 10-14), dividing the exposed surface into 3 zones, with a score of 0 to 3 per zone. For abnormality of the fiuorescein test score both the affected surface (A) and the damage density (D) were evaluated, with a range from 0 to 3 (low and high) on the basis of severity Jap. Clin. Ophthalmol; 1994; 48: 183-188). On the basis of the test score results, patients were divided into 3 subgroups, namely, those with mild dry eye (A1D1, A1D2, A2D1), those with moderate dry eye (A1D3, A2D2, A3D1) and those with severe dry eye (A2D3, A3D2, A3D3). The right eye (R) of patients was treated with the eye-drops according to the invention, consisting of 1% L-carnitine, 0.5% taurine, 0.2% sodium hyaluronate, 0.2% water-soluble vitamin E, 1.02 mg/L zinc, 0.051 mg/L manganese, 30 mg/L sodium, and 9 mg/L potassium. WO 2007/003481 PCT/EP2006/062919 19 The left eye (L), which served as the control eye, the baseline values of which were no different from those of the right eye (R), was treated with 0.2% hyaluronic-acid-based eye-drops. Treatment efficacy was determined after 30 days, 4 hours after the last treatment. The results obtained are presented in Tables 1-3. WO 2007/003481 PCT/EP2006/062919 20 Each entry represents the mean ± standard deviation. Each entry represents the mean ± standard deviation. WO 2007/003481 PCT/EP2006/062919 21 EXAMPLE 2 In this clinical trial the patient sample recruited consisted of 16 patients, 8 men and 8 women, ranging in age from 21 to 32 years (mean age: 25 ± 4.2 years), who had been submitted to refractive laser surgery (PRK) in both eyes with myopia not exceeding 6 diopters. The right eye (R) was treated for 3 months with the eye-drops according to the present invention, while the left eye (L), which served as a control, was treated with 0.2% hyaluronic-acid-based eye-drop Both eyes were treated with antibiotic eye-drops for 5 days. A surface cortisonic was also administered in the left eye 5 days after the operation for 3 months. The efficacy of adequate re-epithelialisation after PRK was evaluated by means of biomicroscopy and the contrast sensitivity test. Biomicroscopy was performed with first vertical and then horizontal light target orientation, after 2, 3, 5 and 7 days of treatment, calculating the de-epithelialisation area. For the purposes of evaluating the optimal result after PRK, denoting normal re-epithelialisation and stromal repair, the contrast sensitivity test was performed (Pescosolido N., Guida automobilistica ed efficacia visiua; Canelli (AT), Fabva.no Ed., 2001; herinafter this reference will be referred to as Pescosolido 2001). Since the vision of an object or image cannot be limited to mimirnum separable perception (visual acuity), one important parameter evaluated was the contrast of the object. To study this parameter, the perception threshold was measured for a whole WO 2007/003481 PCT/EP2006/062919 22 range of objects of various sizes with increasingly reduced contrasts. The resulting assessment was the spatial contrast sensitivity function (spatial CSF) (Pescosolido 2001). For this function, test images were mainly used consisting of stripes with a sinusoidal luminance profile. These bars, alternating dark and light, were defined by their spatial frequency [cycles per degree (CPD) or number of pairs of stripes (black/white) per degree of visual angle] and by their contrast. The inverse of contrast (C) was contrast sensitivity (S) (S=1/C). Contrast is often expressed in terms of percentages, 98% being very high, and 3% very low {Pescosolido 2001). The contrast sensitivity test was performed using the Optec 6500 vision tester capable of receiving ETDRS and FACT test scores and software for the management and analysis of contrast sensitivity data. The system was capable of simulating the way in which the patient actually saw things. Moreover, it was capable of comparing patient simulations with standard representations. The examination was performed first after 10 days and then at 3 and 6 months postoperatively. Patients started treatment immediately after PRK. The right eye (R) was treated with the eye-drops according to the present invention mentioned in example 1 (2 drops, 4 times daily), while the left eye, which served as a control, was treated with 0.2% hyaluronic-acid-based eye-drops and surface cortisonic after postoperative day 5 (the latter only for the first 3 months, 3 times daily). WO 2007/003481 PCT/EP2006/062919 23 Single-dose antibiotic eye-drops was instilled in both eyes 4 times daily and a hydrogel contact lens was applied to both eyes after PRK for the first 5 days postoperatively. Two days after the operation (PRK) the patients treated with the eye-drops according to the present invention presented a de- epithelialisation area of 6.0 mm2 ±6.8 mm, whereas in the control eyes the de-epithelialisation area measured 8.4 mm2 ± 9.2 mm. After 3 days complete re-epithelialisation in the treated eyes was 77% as against 61% in the control eyes. After 5 days complete re- epithelialisation was 100% as against 90% in the control eyes. After 7 days re-epithelialisation was 100% in both eyes. Evaluation of the variation in contrast after 6 months' treatment in the eyes treated with the eye-drops according to the present invention (R) compared to the control eyes (L) showed a statistically significant difference. The results obtained are presented in Table 4. Each entry represents the mean ± standard deviation. L-carnitine and its alkanoyl derivatives are known compounds, the preparation process for which is described in US 4,254,053. WO 2007/003481 PCT/EP2006/062919 24 The physiological supplement or medicament according to the present invention may be bought with or without medical prescription. The physiological supplement or medicament according to the present invention are composed of active ingredients which are familiar to operators in the medical field and already in use in clinical practice, and their pharmacotoxicological profiles are known. Their procurement therefore is very easy, inasmuch as these are products which have been on the market now for a long time and are of a grade suitable for human or animal administration. In the following are reported non limiting examples of composition according to the present invention. Eye-drops - L-carnitine 1%; Taurine 0.5%; sodium hyaluronate 0.2%; vitamin E 0,2%; manganese 0.051 mg/L; zinc 1.02 mg/mL; sodium 30 mg/L; potassium 9 mg/L; Sodium mertiolate 0.02 mg/mL; Demineralized water; Volume 5 mL/vials. WO 2007/003481 PCT/EP2006/062919 25 CLAIMS 1. Use of L-carnitine and/or one or more alkanoyl L- carnitines or one of their pharmaceutically acceptable salts, for the preparation of a physiological supplement or medicament for the treatment of corneal diseases. 2. Use according to claim 1, in which the alkanoyl L- carnitine is selected from the group consisting of acetyl, propionyl, valeryl, isovaleryl, butyryl and isobutyryl L- carnitine. 3. Use according to claim 1, in which the pharmaceutically acceptable salt is selected from the group consisting of chloride, bromide, orotate, aspartate, acid aspartate, acid citrate, magnesium citrate, phosphate, acid phosphate, fumarate and acid fumarate, magnesium fumarate, lactate, maleate and acid maleate, oxalate, acid oxalate, pamoate, acid pamoate, sulphate, acid sulphate, glucose phosphate, tartrate and acid tartrate, glycerophosphate, mucate, magnesium tartrate, 2- amino-ethanesulphonate, magnesium 2-amino- ethanesulphonate, methanesulphonate, choline tartrate, trichloroacetate, and trifiuoroacetate. 4. Use according to claim 1 in which the physiological supplement or medicament for the treatment of corneal diseases is in the form of aeye-drops. WO 2007/003481 PCT/EP2006/062919 26 5. Use according to claim 4, in which the corneal disease is selected from the group comprising de-epithelialising diseases; dry eye syndrome; infective keratitis; acid or alkaline caustic damages; corneal abrasions and/or injuries; degenerative disease of the corneal stroma; stromal damages caused by refractive laser surgery; dystrophic diseases and impairment of the transparency of the surface of the cornea. 6. Use according to claim 5, in which the corneal abrasions and/or injuries are due to mechanical action or contact lenses. 7. Use according to claim 5, in which the degenerative disease of the corneal stroma is chronic and acute keratoconus. 8. Use according to claim 5, in which said impairment of the transparency of the surface of the cornea is due to infective keratitis, injuries and damages of the structure of the various components constituting the cornea; hereditary or degenerative diseases. 9. Use according to claim 8 in which keratitis is due to viral, bacterial or fungal agents; injuries and damages of the structure of the cornea are due to mechanical, post- surgical or post-laser-refractive surgery type; the hereditary or degenerative diseases is chronic and acute keratoconus. WO 2007/003481 PCT/EP2006/062919 27 10. Use according to claim 9 in which the post-surgical or post-laser-refractive surgery damages is haze. 11. A physiological supplement or medicament for ophthalmic use, in the form of eye-drops, comprising as the active ingredient L-carnitine, or one of its pharmaceutically acceptable salts, in combination with humidifying agents; antioxidants; inorganic and organic elements, and optionally ophthalmologically acceptable excipients and/or diluents. 12. The physiological supplement or medicament of claim 12, in which the humidifying agents is sodium hyaluronate; the antioxidant is vitamin E; the inorganic and organic elements are manganese, sodium, potassium and taurine. 13. Eye-drops having the following formulation: L-carnitine 1%; - Taurine 0.5%; - sodium hyaluronate 0.2%; - vitamin E 0,2%; - manganese 0.051 mg/L; - zinc 1.02 mg/mL; - sodium 30 mg/L; - potassium 9 mg/L. 14. The eye-drops of claim 14 further comprising antioxidants; vitamin C; Borage oil; epithelialising and anti-angiogenic WO 2007/003481 PCT/EP2006/062919 28 agents; humidifying agents; inorganic elements; regulator of the cellular osmolarity; antibiotics; antiviral and antifungal agents. Use of L-carnitine and/or of one or more alkanoyl L-carnitines or one of their pharmaceutically acceptable salts for the preparation of an opthalmic physiological supplement or medicament in the form of eye-drops, for the treatment of corneal diseases.

Full Text

WO 2007/003481 PCT/EP2006/062919
The present invention relates to the use of L-carnitine and/or one
or more alkanoyl L-carnitines for the preparation of a physiological
supplement or medicament in the form of eye-drops useful for the
treatment of corneal diseases.
The cornea is the transparent, dome-shaped window covering the
front of the eye. It is a powerful refracting surface, providing 2/3 of the
eye's focusing power. Like the crystal on a watch, it gives us a clear
window to look through.
The cornea is extremely sensitive - there are more nerve endings
in the cornea than anywhere else in the body.
The adult cornea is only about 1/2 millimeter thick and is
arranged in three main regions, or layers:
- Epithelium: it functions primarily to block the passage of
foreign material such as dust or water into the eye, and other layers of
the cornea, and provide a smooth surface that absorbs oxygen and
other needed cell nutrients that are contained in tears. This layer,
which is about five cells deep, is filled with thousands of tiny nerve
endings that make the cornea extremely sensitive to pain when rubbed
or scratched.
- Stroma: is located behind the epithelium, the stroma comprises
about 90 percent of the cornea. It consists primarily of water and
layered protein fibers that give the cornea its strength, elasticity, and
form; and cells that nourish it. The unique shape, arrangement, and
spacing of the protein fibers are essential in producing the cornea's
light-conducting transparency.

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Endothelium: this single layer of cells is located between the
stroma and the aqueous humor. Because the stroma tends to absorb
water, the endothelium's primary task is to pump excess water out of
the stroma. Without this pumping action, the stroma would swell with
water, become hazy, and ultimately opaque.
Many diseases can damage this delicate structures.
The main causes of impairment of the epithelial structure of the
cornea are dry eye syndrome; corneal abrasions and injuries due to,
for example, the application of contact lenses; and refractive laser
surgery.
Other diseases of the cornea are associated with impairment of
the normal transparency of the corneal surface, caused, for example,
by damage in the aftermath of keratitis, particularly bacterial, viral or
fungal keratitis; by damage resulting from trauma and refractive laser
surgery; as well as degenerative or hereditary diseases such as
chronic and acute keratocono.
The tear film, which coats the corneal epithelium and is essential
for the homeostasis of the eye surface, performs an important optical
function, acting as a lubricant between the eyelids and the eyeball and
as a vehicle for oxygen, guaranteeing the metabolism of the cells of the
corneal epithelium; it also performs a flushing function, ensuring the
removal of external agents.
Also important is its function as a carrier for growth factors,
neuropeptides, and neuromodulators that regulate the activation,
proliferation and differentiation of corneal and conjunctival epithelial

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3
cells. It also transports immunoglobulins (IgA, IgAs, IgG, IgE, IgM),
complement factors (C3, C4, C5), metalloproteases (MMP-2, 4, 9),
enzymes (lysozyme, lactoferrin) and immune system cells (limphocyte),
thus performing a fundamental defensive function against infections.
Dry eye syndrome is characterised by a quantitative
(hypolacrimation) and/or qualitative (dyslacrimation) impairment of
the tear film of multifactorial origin which may or may not cause
clinically significant damage to the eye surface. The prevalence of dry
eye syndrome ranges from 10 to 40% in the adult population and there
is a highly significant correlation with age.
In the United States the prevalence of mild-to-moderate dry eye
syndrome is up to approximately 10 million people (Am. J. Ophthalmol:,
1997; 124:723-728; Arch Ophthalmol, 2000; 118: 1264-1268).
Various studies conducted in order to understand the
mechanisms activated in this disease have shown that the tears of
subjects affected by dry eye syndrome present: an increased
evaporation rate, increased surface tension, reduced vitamin A
concentration, increased osmolarity, reduced concentration of a
number of proteins (lysozyme, lactoferrin), insufficient mucus
production or qualitative changes in mucus production, with
consequent inadequate reconstruction of the mucus layer, reductions
in a number of growth factors (EGF, TGF-α, aFGF-bFGF, LG-F, HGF)
(Contactologia, 1982; 4: 34-37), changes in concentration of inorganic
elements, reduced androgens and dysregulation of T lymphocyte

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activity (Cornea, 2005; 24: 1-7). Among the mechanisms activated in
this disease the use of contact lenses has to be mentioned.
The clinical signs regarded as being most closely related to this
pathological condition are reduced break-up time (BUT test) and
Schirmer test results (Pescosolido N.: Le alterazioni del film lacrimale. In
Stendler P.: "il sistema lacrimale", Fabiano editore, Canelli (AT), 2000;
pag. 237-330; hereinafter this reference will be referred to as
Pescosolido 2000).
The BUT test has to do with the mucin content of the tear film
and, in the dry eye, yields only values below 5 seconds. The Schirmer
test, on the other hand, has to do with the water content of the tear
film and, in the dry eye, yields values below 5 millimetres in 5
minutes.
The patient presents the following symptoms: foreign body
sensation, burning, difficulty blinking, bruit on opening the eyelids,
itching, eye fatigue, photophobia, blurred vision, and mucus
extravasation at the inner canthi.
The treatment of this syndrome is based on the use of the
following: tear substitutes whose task is the regular moisturising of the
cornea, but which do not exert any action on the basic causes of the
disease and are endowed only with very short-lasting efficacy; inserts
(plugs) in the lacrimal canaliculus; immunoregulators such as topical
cyclosporin; topical steroids; anti-inflammatory agents (rumexilone
and loteprednol); autologous serum (cytokine inhibitors); topical or
systemic androgens; mucus (HETE eicosanoid) and aqueous (P2Y2

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agonists) secretogenic substances; acquaporins and agents such as
antibiotics and detergents for the treatment of a disease often
concomitant, blepharitis (Cornea, 2005; 24:1-7).
Also used is treatment with iodide iontophoresis owing to its
scavenger activity as a reducing agent and electron donor (Adv. Clin.
Path., 2000; 4:11-17; Br. J. Ophthalmol, 2005; 89: 40-44).
Even these latter treatments, despite exerting an action which
may be regarded as more relevant to treating the causes of the disease,
have failed to yield the anticipated results.
The normal transparency of the corneal surface can be impaired
by the aftermath of numerous diseases acquired, degenerative or
congenital that damage the delicate structure of the various
constituent components. The acquired disease conditions most
commonly implicated are post-keratitis damage, particularly after
herpetic keratitis, and damage occurring in the aftermath of trauma
and laser refractive surgery. The minimum common denominator is
the formation of corneal opacities (leucomas) that functionally
jeopardise vision. The events involved in wound healing that occur in
the corneal tissue after infection, caustic damages, injury and
refractive ablative surgery are have a profound effect on the final
morphological and refractive outcomes of the restitutio ad integrum
process.
The acute epithelial and stromal corneal lesions occurring
immediately after injury and laser ablation are probably involved in the
regulation of the subsequent corneal tissue repair events, and, among

WO 2007/003481 PCT/EP2006/062919
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the latter, keratocyte apoptosis probably plays a major role (Cornea,
2000;19:S7-12). This event is responsible for the corneal repair process
since keratocyte apoptosis is the prime mover of the reproliferative
stimulus. The stroma keratocytes underlying the initial acellular
stroma therefore represent the cell source that mediates the
subsequent healing of the surface stroma beneath the epithelium. As a
result of the cellular repopulation, the activated keratocytes undergo
myofibroblastic transformation (Invest. OphthalmoL Vis. Sci.,
1998;39:487-501), thus proving responsible for the production of
collagen fibres and of basic substances involved in the restituito ad
integrum process.
A recent scar is very similar to foetal tissue, it is rich in foetal
and type III collagen with thick fibrils, wide interfibrillary spaces and a
hydrophilic stroma. This explains the characteristic opacity. It also
contains fibrinogen, fibronectin, laminin and weakly sulphurised
keratan sulphate. The keratan sulphate/condroitin sulphate is
reduced in the scar stroma.
The myofibroblasts disappear in a few weeks to a few months.
Gradually the scar changes and becomes less opaque. The resistance
of the stromal scar tissue is 20% compared with normal tissue after
three weeks, 60% after 1 year and 70% after 3-4 years. Corneal
reinnervation is very slow. The cytokines play an important role in
these events. IL-la, produced by the corneal epithelium, stimulates
metalloprotease and collagenase synthesis through the keratocytes
and the myofibroblasts. The TGF-β reduces the production of

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collagenase and is involved in the formation of haze. IL-6 reduces the
synthesis of metalloprotease thus increasing collagen synthesis.
This process, however, is not self-controlled and, in many cases,
abnormal, excessive healing occurs followed by a greater production of
collagen type III, an increase of jaluronic acid, and an increase in
lamellar disorganisation (Arch. Ophthalmol, 1990; 108: 665-675; Exp.
Eye Res., 2004; 78 : 553-560; Ophthalmology, 2000; 107 : 1235-1245)
or an accumulation keratocytes e dei myofibroblasts as shown by
confocal microscopy (Progr. Retin. Eye Res., 1999; 18:311-356; J.
Cataract Refract. Surg., 2000; 26 : 432-447; Exp. Eye Res., 2004; 78 :
553-560).
These abnormalities are involved in the pathogenesis of the most
feared complication of stromal regeneration after photorefractive
keratectomy (PRK), namely, haze, with consequent impairment of the
functional outcome. Haze is classified according to Heitzmann in 5
degrees on the basis of the visual impairment due to the reduced
corneal transparency (Ophthalmology, 1998; 35 : 1411-1421); or
according to the Fantes scale (Arch. Ophthalmol, 1990; 108 : 665-675)
which distinguish 6 degrees of haze (0; 0.5; 1+; 2+; 3+; and 4+).
Though the incidence of haze has been substantially reduced over
recent years, as a result of the technological advances in the field of
excimer lasers, it is still a fairly frequent complication even today and,
in rare cases, would appear hard to reverse, even after months of
cortisone therapy (0,5% of the eye with myopia not exceeding 6
diopters and 3-17% in the eye with myopia exceeding 10 diopters

WO 2007/003481 PCT/EP2006/062919
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(Ophthalmol. Clin. North. Am., 2001, 14 : 359-376). In cases of
persistent haze (more than 15-18 months) which fails to respond to
medical cortisonic therapy [an event that can occur with late-onset
haze (4-12 months after the operation (Ophthalmology, 1997; 104 :
369-374; Cornea, 2004, 23 : 350-355), as the only feasible procedure is
phototherapeutic keratectomy (PTK) with an excimer laser, a procedure
used for the laser-assisted surgical removal of superficial stromal
opacities combined with 0.02% mitomycin (J. Refract. Surg.,
2003; 19:40-43; J. Refract Surg., 2003,19:449-454). The use of the
activator of plasminogen and of TGF-al is still undergoing animal
studies (J. Refract. Surg., 1997; 13:356-361; Invest. Ophthalmol. Vis.
Sci., 2004;45:1329-1333), while INFp-2b, tested on man, has produced
poor results (J. Refract. Surg., 1996;22:891-900).
Indeed, while low degree haze is generally asymptomatic, or only
causes a slight reduction in sensitivity to contrast, serious forms of
haze can interfere with refraction and be accompanied by myopic
regression due to exuberant scarring (J. Refract. Surg., 1995; 11.341-
347; Ophthalmology, 2000; 107:1235-1245).
The corneal dystrophies (Bietti, 1971; Oftalmologia geriatrica) are
rare disorders. They are defined as primitive corneal disorders not
associated with trauma, earlier inflammation or systemic diseases.
They affect both eyes, are hereditary and, for the most part, have an
autosomal dominant trait.
Corneal degeneration is far more common than dystrophies but
the symptoms are generally less obvious. It is not hereditary, can be

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9
unilateral or bilateral and the course is gradual or relatively stable. In
each case, it is, by definition, permanent, or does not resolve itself
spontaneously.
Usually it involves more than one layer of the cornea.
Distinctions can be drawn between primary or idiopathic and
secondary forms. The first are often connected with ageing without
being preceded by any specific pathological process, the second ones
are always associated with eye diseases which precede them, whether
acute, chronic, infectious or inflammatory in nature. Keratoconus is
one of the most typical degenerative diseases.
Keratoconus is a non-inflammatory ectasia of the paracentral
portion of the cornea which evolves over time and takes a progressively
conical form, with tapering of the tip. This corneal deformation results
in severe astigmatism, often irregular, corrected wherever possible with
hard gas-permeable corneal lenses. Only rarely are epikeratoplasty,
photorefractive keratectomy or the use of intracorneal rings (INTACTS)
considered (Cornea, 1987;6:131-139; Ophthalmology, 1992;99:1187-
1192; Surv. Ophthalmol., 1998;42:297-319;J. Cataract. Surg., 2000;
26 : 1117-1122; J. Refract.Surg., 2001;17:69-73) or also the use of
hard gas-permeable lenses combined with INTACTS. All these
techniques only correct the refractive defects but do not resolve the
cause of the corneal ectasia and therefore do not stop the progress of
keratoconus, which in its most severe form means a corneal transplant
(Cornea, 1997;16:623-629; Cornea, 1997;16:414-419; Cornea,
1998; 17:468-470; Cornea, 2002;21:152-155 ).

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Keratocomis starts at puberty and in 20% of cases perforating
keratoplasty has to be performed (Ophthalmology, 1994; 101 : 439-
447).
Past studies have not produced relevant results leading to an
understanding of the physiopathology of the disease but only more
recently, with the development of molecular techniques, have advances
been made in understanding this abnormality.
At present, however, we are not able to say that the presence of
keratoconus is due to uniform anomalies which are specific to the
extracellular matrix. There are areas where the elements of the basal
membrane are absent indicating on-going proteolytic activity, and
there are areas in which there are deposits of fibrotic substance which
is also found in other pathologies. A cornea with keratoconus has low
levels of inhibitory enzymes (TIMP) and greater activity of the enzymes
which are capable of degrading the extracellular matrix. The TIMP play
a predominant role in the thickness of the stroma and in the stability
of Bowman's membrane, which is characteristically interrupted in
keratoconus. Furthermore the decrease in one of these inhibitors,
TIMP-1, could play an important role in apoptosis or in the anomalous
behaviour of the cells found in keratoconus.
Apoptosis is the programmed death of the cells. This death is
necessary for reconstructing damaged cells and for the normal turn-
over of many tissues.

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In keratoconus, apoptosis is more frequently found in the stroma
(Invest. Ophthalmol. Vis. Sci., 1998; 39 : 220-226) than in other
corneal layers (Cornea, 2002; 21 : 206-209).
This observation is important because a cornea with keratoconus
is subject to chronic irritation caused by hard gas-permeable contact
lenses, to greater friction and to moderate or severe atopy. Wilson
suggests that mechanical trauma on the epithelium could cause
apoptosis in the stromal cells of keratoconus (Exp. Eye Res., 1999; 69 :
225-266;Cornea, 2000; 19 : S7-S12). Furthermore, an increase in the
levels of leukocytes commonly correlated to the LAR protein antigen
present in keratoconus but not in normal corneas can be seen (Exp.
Eye Res., 1999;68:283-293).
LAR is a transmembrane phosphotyrosine transferase capable of
stimulating apoptosis (Proc. Nati. Acad. Sci. USA, 1994;91:10868-
10872; J. Neurobiol., 2000;42:477-486). A third triggering mechanism
of apoptosis is that it is inhibited by TIMP-1 (J. Clin. Invest.,
1998;102:2002-2010; Blood, 1998;92:1342-1349) which in
keratoconus proves to be reduced, as has been reported previously.
In practice, given these statements, the phenomenon of apoptosis
could play an important role in the pathogenesis of keratoconus.
Sodium hyaluronate is a very well known compound used to
protect corneal epithelial cells, especially in patients with dry eye
syndrome or with Sjogren's syndrome. The action of sodium
hyaluronate is due not only to its protective role of a mechanical type
exerted on the epithelial cells as a result of its viscoelasticity, in

WO 2007/003481 PCT/EP2006/062919
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situations of reduced tear production, but also to the positive effects of
its particular biological function on corneal epithelial cells by
stimulating their migration (Exp. Eye Res., 1991 ;53:753-758).
Taurine (or 2-aminoethanesulphonic acid) is considered an
amino acid, even though it does not possess the characteristic
carboxyl group (COOH) but the SO3H group. Taurine is only present in
the animal realm, whereas vegetable foods do not possess this amino
acid.
Vitamin E is the main antioxidant of the cell membranes, and is
found in the human body in 4 forms consisting of α-tocopherol, β-
tocopherol, γ-tocopherol and δ-tocopherol. Of these the a form is the
most frequent in the retina and in plasma and is the one with the
greatest antioxidant and free radical scavenging activity.
Vitamin A is essential for cell vitality, cytoskeletal organisation, it
regulates the expression of proteins of the extracellular matrix, cell
adhesion and wound repair (J. Cel.l Biochem., 2003; 89 : 837-847).
Vitamin A is also involved in the production of angiogenic (inactivation)
and anti-angiogenic (activation) factors in the endothelial cells (Exp.
Eye Res., 2004; 78 : 945-955). Some of these, stromal derived factor 1
(SDF-1) are secreted under the effect of proinflammatory signals (IL-
l,TNF-a, bacteria and viruses) and, in cooperation with other factors,
are involved in the proliferation of the vascular endothelial cells. SDF-1
and its receptor CXCR4 have been found in human keratocytes (Mol.
Vis. , 2003;9:96-102). Two other systems are also involved in corneal
angiogenesis: vascular endothelial growth factor (VEGF) and fibroblast

WO 2007/003481 PCT/EP2006/062919
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growth factor b (b FGF). Anti-angiogenetic factors contrast these
cytokines as the pigment epithelium derived factor (PEDF) (Mol. Vis.,
2001, 7 : 154-163). IL-4 and TGF-β are also known to inhibit corneal
angiogenesis in vivo (Acta Ophthalmol. Scand., 2002;80:238-247).
Metalloproteases are able to intervene in the regulation of angiogenesis
with inhibitory or potentiating effects. In the absence of vitamin A, the
corneal epithelium becomes keratinised. Retinol and retinic acid
(Vitamin A), which help to avoid the keratinisation of the corneal
epithelium, are supplied by the tears. Nuclear receptors with retinoids
are present in the epithelium and in the stroma.
Cytidine-5'-diphosphate choline (CDP-choline), commonly known
by the name of citicholine, is a precursor of phosphatidyl choline, the
main phospholipid of the cell membranes. Due to the effect of the
activation of certain lytic enzymes, the phospholipases, the catabolism
of the membrane phospholipids is accelerated and, if the resynthesis
mechanism is inadequate, toxic substances accumulate, such as the
ceramides, which can activate the metabolic pathways which lead to
cell apoptosis. A deterioration in the turn-over of the phospholipids
adversely affects the validity of the membrane protection systems and
puts cell function at risk.
The use of inorganic elements is well known in the medical field,
and a number of these are essential for the stability of the tear film
(Pescosolido 2000).
Previous uses of carnitine in the ophthalmological field are
already known.

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US Patent 5,037,851 describes the use of acetyl L-carnitine for
the treatment of cataract.
US 5,145,871 and 5,432,199 describe the use of acetyl D-
carnitine for the treatment of glaucoma.
US 5,883,127 describes the use of acetyl L-carnitine for the
treatment of maculopathy and macular degeneration.
Further uses of carnitine are also known.
In Res 1992;18(8):355-365 the use of L-carnitine in the
cardiological field is described.
US 5,543,556 describes the use of acyl L-carnitine esters with
gamma-hydroxybutyric acid for the inhibition of neuronal degeneration
and in the treatment of coma.
US 5811457 describes the use of propionyl L-carnitine for the
treatment of chronic obliterating arteriopathy.
None of the above-cited patents or publications describes or
suggests the use of L-carnitine or of alkanoyl L-carnitine for the
treatment of diseases of the cornea.
In the medical field there is still a strongly perceived need for the
availability of therapeutic agents or physiological supplement useful
for the treatment of the above-mentioned corneal diseases.
It has now been found that L-carnitine and/or one or more
alkanoyl L-carnitines, or one of their pharmaceutically acceptable
salts, are useful agents for the preparation of a physiological
supplement or medicament in the form of eye-drops for the treatment
of diseases of the cornea.

WO 2007/003481 PCT/EP2006/062919
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What is meant by pharmaceutically acceptable salt of L-carnitine
is any salt of the latter with an acid that does not give rise to toxic or
side effects.
These acids are well known to pharmacologists and to experts in
pharmacy. Non-limiting examples of such salts are: chloride, bromide,
orotate, aspartate, acid aspartate, acid citrate, magnesium citrate,
phosphate, acid phosphate, fumarate and acid fumarate, magnesium
fumarate, lactate, maleate and acid maleate, oxalate, acid oxalate,
pamoate, acid pamoate, sulphate, acid sulphate, glucose phosphate,
tartrate and acid tartrate, glycerophosphate, mucate, magnesium
tartrate, 2-amino-ethanesulphonate, magnesium 2-amino-
ethanesulphonate, methanesulphonate, choline tartrate,
trichloroacetate, and trifluoroacetate.
What is meant by pharmaceutically acceptable salt of L-carnitine
is also a salt approved by the FDA and listed in the publication Int. J.
of Pharm. 33 (1986), 201-217, which is incorporated herein by way of a
reference.
One object of the present invention is the use of L-carnitine
and/or of one or more alkanoyl L-carnitines selected from the group
consisting of acetyl, propionyl, valeryl, isovaleryl, butyryl and
isobutyryl L-carnitine, or one of their pharmaceutically acceptable
salts, for the preparation of an ophthalmic physiological supplement or
a medicament in the form of aeye-drops, for the treatment of corneal
diseases in which said corneal disease is selected from the group
comprising, de-epithelialising diseases, dry eye syndrome; infective

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keratitis; acid or alkaline caustic damages; corneal abrasions and/or
injuries due to mechanical action or contact lenses; degenerative
disease of the corneal stroma such as acute or chronic keratocono,
stromal damages caused by refractive laser surgery; and dystrophic
diseases, impairment of the transparency of the surface of the cornea
caused by various types of infective keratitis (viral, bacterial and
fungal), or by injuries that damage the structure of the various
components constituting the cornea, such as, for instance, injuries of
a mechanical, post-surgical and post-laser-refractive surgery type
(such as, for example, haze); hereditary or degenerative diseases such
as chronic and acute keratoconus.
A further object of the present invention is a physiological
supplement or medicament for ophthalmic use, in the form of eye-
drops, comprising as the active ingredient L-carnitine, or one of its
pharmaceutically acceptable salts, in combination with humidifying
agents such as sodium hyaluronate; antioxidants such as vitamin E;
inorganic elements as components of enzymes present in the tear film
such as manganese; inorganic and organic elements such as sodium,
potassium and the amino acid taurine for the regulation of the cellular
osmolarity, and optionally ophthalmologically acceptable excipients
and/or diluents;
in which: u
L-carnitine is present preferably at a dose of 0.3-3%, and most
preferably at a dose of 1%;

WO 2007/003481 PCT/EP2006/062919
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taurine is present preferably at a dose of 0.1-4%, and most
preferably at a dose of 0.5%;
sodium hyaluronate is present preferably at a dose of 0.05-
1.5%, and most preferably at a dose of 0.2%;
vitamin E is present preferably at a dose of 0.05-1.0%, and
most preferably at a dose of 0,2%;
manganese is present preferably at a dose of 0.01-0.1
mg/L, and most preferably at a dose of 0.051 mg/L;
zinc is present preferably at a dose of 0.5-1.5 mg/mL, and
most preferably at a dose of 1.02 mg/mL;
sodium is present preferably at dose of 5-5000 mg/L, and
most preferably at a dose of 30 mg/L;
potassium is present preferably at a dose of 1-1000 mg/L,
and most preferably at a dose of 9 mg/L.
The eye-drops according to the present invention may
additionally contain further antioxidants such as, for example,
vitamin C, Borage oil; epithelialising and anti-angiogenic agents;
humidifying agents; inorganic elements; regulator of the cellular
osmolarity; antibiotics; antiviral and antifungal agents.
The following examples illustrate the invention.
EXAMPLE 1
A clinical trial was conducted in which 43 patients suffering from
dry eye syndrome were recruited.
The patients recruited were all women aged from 35 to 77 years
(mean age: 59.5 ± 10.4 years), 33 of whom were suffering from

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Sjogren's syndrome, diagnosed on the basis of Fox et al.'s criteria
(Arthritis Rheum, 1986; 29: 577-584; 1986).
Patients were selected on the basis of the BUT test, the Schirmer
test, the fiuorescein test and the rose bengal test (Pescosolido 2000;
Arch. OphthalmoL, 1969;82: 10-14).
The BUT test had to yield results ≤ 5 seconds, while the Schirmer
test did not contraindicate inclusion in the trial.
Damage to the surface of the eye was evaluated by means of the
rose bengal staining test and the fiuorescein test. Damage in the rose
Bengal staining test was determined by reference to the van Bijsterveld
evaluation (Arch. OphthcdmoL, 1969;82: 10-14), dividing the exposed
surface into 3 zones, with a score of 0 to 3 per zone.
For abnormality of the fiuorescein test score both the affected
surface (A) and the damage density (D) were evaluated, with a range
from 0 to 3 (low and high) on the basis of severity Jap. Clin.
Ophthalmol; 1994; 48: 183-188).
On the basis of the test score results, patients were divided into 3
subgroups, namely, those with mild dry eye (A1D1, A1D2, A2D1),
those with moderate dry eye (A1D3, A2D2, A3D1) and those with
severe dry eye (A2D3, A3D2, A3D3).
The right eye (R) of patients was treated with the eye-drops
according to the invention, consisting of 1% L-carnitine, 0.5% taurine,
0.2% sodium hyaluronate, 0.2% water-soluble vitamin E, 1.02 mg/L
zinc, 0.051 mg/L manganese, 30 mg/L sodium, and 9 mg/L
potassium.

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The left eye (L), which served as the control eye, the baseline
values of which were no different from those of the right eye (R), was
treated with 0.2% hyaluronic-acid-based eye-drops.
Treatment efficacy was determined after 30 days, 4 hours after
the last treatment.
The results obtained are presented in Tables 1-3.

WO 2007/003481 PCT/EP2006/062919
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Each entry represents the mean ± standard deviation.

Each entry represents the mean ± standard deviation.

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EXAMPLE 2
In this clinical trial the patient sample recruited consisted of 16
patients, 8 men and 8 women, ranging in age from 21 to 32 years
(mean age: 25 ± 4.2 years), who had been submitted to refractive laser
surgery (PRK) in both eyes with myopia not exceeding 6 diopters.
The right eye (R) was treated for 3 months with the eye-drops
according to the present invention, while the left eye (L), which served
as a control, was treated with 0.2% hyaluronic-acid-based eye-drop
Both eyes were treated with antibiotic eye-drops for 5 days. A surface
cortisonic was also administered in the left eye 5 days after the
operation for 3 months.
The efficacy of adequate re-epithelialisation after PRK was
evaluated by means of biomicroscopy and the contrast sensitivity test.
Biomicroscopy was performed with first vertical and then
horizontal light target orientation, after 2, 3, 5 and 7 days of
treatment, calculating the de-epithelialisation area.
For the purposes of evaluating the optimal result after PRK,
denoting normal re-epithelialisation and stromal repair, the
contrast sensitivity test was performed (Pescosolido N., Guida
automobilistica ed efficacia visiua; Canelli (AT), Fabva.no Ed., 2001;
herinafter this reference will be referred to as Pescosolido 2001).
Since the vision of an object or image cannot be limited to
mimirnum separable perception (visual acuity), one important
parameter evaluated was the contrast of the object. To study this
parameter, the perception threshold was measured for a whole

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range of objects of various sizes with increasingly reduced
contrasts. The resulting assessment was the spatial contrast
sensitivity function (spatial CSF) (Pescosolido 2001). For this
function, test images were mainly used consisting of stripes with a
sinusoidal luminance profile. These bars, alternating dark and
light, were defined by their spatial frequency [cycles per degree
(CPD) or number of pairs of stripes (black/white) per degree of
visual angle] and by their contrast. The inverse of contrast (C) was
contrast sensitivity (S) (S=1/C). Contrast is often expressed in
terms of percentages, 98% being very high, and 3% very low
{Pescosolido 2001).
The contrast sensitivity test was performed using the Optec
6500 vision tester capable of receiving ETDRS and FACT test scores
and software for the management and analysis of contrast
sensitivity data. The system was capable of simulating the way in
which the patient actually saw things. Moreover, it was capable of
comparing patient simulations with standard representations. The
examination was performed first after 10 days and then at 3 and 6
months postoperatively. Patients started treatment immediately
after PRK. The right eye (R) was treated with the eye-drops
according to the present invention mentioned in example 1 (2
drops, 4 times daily), while the left eye, which served as a control,
was treated with 0.2% hyaluronic-acid-based eye-drops and surface
cortisonic after postoperative day 5 (the latter only for the first 3
months, 3 times daily).

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Single-dose antibiotic eye-drops was instilled in both eyes 4
times daily and a hydrogel contact lens was applied to both eyes
after PRK for the first 5 days postoperatively.
Two days after the operation (PRK) the patients treated with
the eye-drops according to the present invention presented a de-
epithelialisation area of 6.0 mm2 ±6.8 mm, whereas in the control
eyes the de-epithelialisation area measured 8.4 mm2 ± 9.2 mm.
After 3 days complete re-epithelialisation in the treated eyes was
77% as against 61% in the control eyes. After 5 days complete re-
epithelialisation was 100% as against 90% in the control eyes. After
7 days re-epithelialisation was 100% in both eyes.
Evaluation of the variation in contrast after 6 months'
treatment in the eyes treated with the eye-drops according to the
present invention (R) compared to the control eyes (L) showed a
statistically significant difference.
The results obtained are presented in Table 4.

Each entry represents the mean ± standard deviation.
L-carnitine and its alkanoyl derivatives are known compounds,
the preparation process for which is described in US 4,254,053.

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The physiological supplement or medicament according to the
present invention may be bought with or without medical prescription.
The physiological supplement or medicament according to the
present invention are composed of active ingredients which are familiar
to operators in the medical field and already in use in clinical practice,
and their pharmacotoxicological profiles are known.
Their procurement therefore is very easy, inasmuch as these are
products which have been on the market now for a long time and are
of a grade suitable for human or animal administration.
In the following are reported non limiting examples of
composition according to the present invention.
Eye-drops
- L-carnitine 1%;
Taurine 0.5%;
sodium hyaluronate 0.2%;
vitamin E 0,2%;
manganese 0.051 mg/L;
zinc 1.02 mg/mL;
sodium 30 mg/L;
potassium 9 mg/L;
Sodium mertiolate 0.02 mg/mL;
Demineralized water;
Volume 5 mL/vials.

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CLAIMS
1. Use of L-carnitine and/or one or more alkanoyl L-
carnitines or one of their pharmaceutically acceptable
salts, for the preparation of a physiological supplement
or medicament for the treatment of corneal diseases.
2. Use according to claim 1, in which the alkanoyl L-
carnitine is selected from the group consisting of acetyl,
propionyl, valeryl, isovaleryl, butyryl and isobutyryl L-
carnitine.
3. Use according to claim 1, in which the pharmaceutically
acceptable salt is selected from the group consisting of
chloride, bromide, orotate, aspartate, acid aspartate,
acid citrate, magnesium citrate, phosphate, acid
phosphate, fumarate and acid fumarate, magnesium
fumarate, lactate, maleate and acid maleate, oxalate,
acid oxalate, pamoate, acid pamoate, sulphate, acid
sulphate, glucose phosphate, tartrate and acid tartrate,
glycerophosphate, mucate, magnesium tartrate, 2-
amino-ethanesulphonate, magnesium 2-amino-
ethanesulphonate, methanesulphonate, choline tartrate,
trichloroacetate, and trifiuoroacetate.
4. Use according to claim 1 in which the physiological
supplement or medicament for the treatment of corneal
diseases is in the form of aeye-drops.

WO 2007/003481 PCT/EP2006/062919
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5. Use according to claim 4, in which the corneal disease is
selected from the group comprising de-epithelialising
diseases; dry eye syndrome; infective keratitis; acid or
alkaline caustic damages; corneal abrasions and/or
injuries; degenerative disease of the corneal stroma;
stromal damages caused by refractive laser surgery;
dystrophic diseases and impairment of the transparency
of the surface of the cornea.
6. Use according to claim 5, in which the corneal abrasions
and/or injuries are due to mechanical action or contact
lenses.
7. Use according to claim 5, in which the degenerative
disease of the corneal stroma is chronic and acute
keratoconus.
8. Use according to claim 5, in which said impairment of
the transparency of the surface of the cornea is due to
infective keratitis, injuries and damages of the structure
of the various components constituting the cornea;
hereditary or degenerative diseases.
9. Use according to claim 8 in which keratitis is due to
viral, bacterial or fungal agents; injuries and damages of
the structure of the cornea are due to mechanical, post-
surgical or post-laser-refractive surgery type; the
hereditary or degenerative diseases is chronic and acute
keratoconus.

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10. Use according to claim 9 in which the post-surgical or
post-laser-refractive surgery damages is haze.
11. A physiological supplement or medicament for
ophthalmic use, in the form of eye-drops, comprising as
the active ingredient L-carnitine, or one of its
pharmaceutically acceptable salts, in combination with
humidifying agents; antioxidants; inorganic and organic
elements, and optionally ophthalmologically acceptable
excipients and/or diluents.
12. The physiological supplement or medicament of claim
12, in which the humidifying agents is sodium
hyaluronate; the antioxidant is vitamin E; the inorganic
and organic elements are manganese, sodium,
potassium and taurine.
13. Eye-drops having the following formulation:
L-carnitine 1%;

- Taurine 0.5%;
- sodium hyaluronate 0.2%;
- vitamin E 0,2%;
- manganese 0.051 mg/L;
- zinc 1.02 mg/mL;
- sodium 30 mg/L;
- potassium 9 mg/L.
14. The eye-drops of claim 14 further comprising antioxidants;
vitamin C; Borage oil; epithelialising and anti-angiogenic

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agents; humidifying agents; inorganic elements; regulator
of the cellular osmolarity; antibiotics; antiviral and
antifungal agents.

Use of L-carnitine and/or of one or more alkanoyl L-carnitines or one of their pharmaceutically acceptable salts
for the preparation of an opthalmic physiological supplement or medicament in the form of eye-drops, for the treatment of corneal
diseases.

Documents:

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Patent Number

268726

Indian Patent Application Number

4879/KOLNP/2007

PG Journal Number

38/2015

Publication Date

18-Sep-2015

Grant Date

14-Sep-2015

Date of Filing

14-Dec-2007

Name of Patentee

SIGMA-TAU INDUSTRIE FARMACEUTICHE RIUNITE S.P.A.

Applicant Address

VIALE SHAKESPEARE, 47, I-00144 ROME

Inventors:

#	Inventor's Name	Inventor's Address
1	PESCOSOLIDO NICOLA	VIA PAOLO ALBERA, 33, I-00181 ROMA
2	KOVERECH ALEARDO	VIA AURELIA ANTICA, 200, I-00165 ROME (RM)

PCT International Classification Number

A61K 31/221

PCT International Application Number

PCT/EP2006/062919

PCT International Filing date

2006-06-06

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	05014337.9	2005-07-01	EUROPEAN UNION