WO2002055081A2

WO2002055081A2 - Use of chelators in the treatment of macular degenerative disease

Info

Publication number: WO2002055081A2
Application number: PCT/IB2001/002265
Authority: WO
Inventors: Michel Xilinas
Original assignee: Michel Xilinas
Priority date: 2001-01-10
Filing date: 2001-11-28
Publication date: 2002-07-18
Also published as: WO2002055081A3; EP1408969A2; FR2819187A1

Abstract

A use of clioquinol and phanoquinone for the manufacture of pharmaceutical compositions for the treatment and prevention of pathological conditions related with macular eye disease influenced by the action of matrix matalloproteinases (MMPs) is disclosed. Also methods of treatments or prevention of such conditions are disclosed.

Description

USE OF CHELATORS IN THE TREATMENT OF MACULAR DEGENERATIVE DISEASE

TECHINCAL FIELD

The present invention relates to the use of a combination of two known compounds used together or each one of these two compounds used separately for the manufacture of a pharmaceutical composition for the prevention and treatment of macular degeneration. Further the invention relates a pharmaceutical composition for such prevention and treatment. The invention is also directed to methods of prevention or treatment of macular diseases related to impaired extracellular matrix degradation.

BACKGROUND OF THE INVENTION

Age-related macular degeneration (AMD) is a leading cause of visual loss. It is an eye disease that is present (at least to some degree) in millions of older persons. Early diagnosis of age-related macular degeneration is essential to successful treatment. Age-related macular degeneration affects the macula, a small portion of the retina. The retina is the light-sensing nerve tissue that lines the inside of the eye. All parts of the retina contribute to sight, but only the macula can provide the sharp, straight-ahead vision that is needed for driving and reading small print.

As a person ages, changes may occur in the macula. These changes can cause difficulties in reading and other tasks that require good central vision. It is not clear why these macular changes occur, but ageing evidently plays a major role in the process. Although AMD is a leading cause of visual loss, the majority of people with AMD continue to have almost normal vision throughout their lives. Even those who are severely affected, retain enough sight to move about independently and make use of helpful devices called low vision aids.

Usually, AMD does not develop until a person is 65 or older. A few people in their 40s and 50s, however, may be affected by AMD. Most patients with AMD have a form of the disease that develops very slowly. It is called the atrophic or "dry" form. In it, tiny yellowish deposits called drusen develop beneath the macula. Also, the layer of light-sensitive cells in the macula becomes thinner as some cells break down. These changes typically cause a dimming or distortion of vision that people find most noticeable when they try to read. Generally, if one eye has dry AMD, the other eye will also have some signs of the condition. Thus the person with dry AMD may eventually have visual problems in both eyes. However, the dry form of AMD rarely causes total loss of reading vision.

A much greater threat of visual loss arises when the dry form of AMD gives way to the exudative or "wet" or neovascular form of the disease. This condition arises in a small percentage of individuals with AMD. In the wet form, new blood vessels grow beneath the macula. These abnormal vessels leak fluid and blood, causing the light sensitive cells near them to sicken and die. This process generally produces marked disturbance of vision in the affected eye: straight lines look wavy, and later there may be blank spots in the field of vision. If the leakage and bleeding from new vessels continues, much of the nerve tissue in the macula may be killed or injured within a period of a few weeks or months. Such damage cannot be repaired, because the nerve cells of the macula do not grow back once they have been destroyed. Although only a small percentage of people with AMD develop the neovascular form, they make up the vast majority of those who experience serious visual loss from AMD. A clinical study (research effort) supported by the National Eye Institute of USA (NEI) found that there is a treatment that can help most people whose sight is threatened by the wet or neovascular form of AMD. This treatment is called laser photo-coagulation. In laser photo-coagulation, powerful light rays from a laser are directed into the eye and focused at a tiny spot on the macula. The aim of the laser treatment is to preserve vision by destroying abnormal blood vessels. In the NEI study, laser treatment reduced the risk of severe vision loss by more than half in people with neovascular AMD. However, this treatment is best applied soon after the new blood vessels develop, before they have reached and damaged the fovea (the central part of the macula).

According to NEI, laser photo-coagulation is of value only to the relatively few people who have the neovascular form of AMD, with new blood vessels actively growing in the macula and threatening to cause serious vision loss. There is no evidence, according to NEI, that laser treatment is of any value for people with the dry form of AMD. Because the laser cannot restore vision already lost from AMD, an eye whose macula has been badly damaged by this disease would not benefit from laser treatment, NEI reports. It important for AMD, and neovascular AMD in particular, should be detected as early as possible.

Drusen and the other macular changes typical of the atrophic type of AMD cannot be seen by the person who has them, but are visible to an eye care specialist examining the eye. Individuals who are middle-aged or older are advised to visit an eye care specialist regularly to be checked for early signs of

AMD, glaucoma, and other eye diseases that are linked to ageing.

AMD is a leading cause of visual loss in the USA. It is an eye disease that is present (at least to some degree) in millions of older Americans. Early diagnosis of age-related macular degeneration is essential to successful treatment. Age-related macular degeneration, affects the macula, a small portion of the retina. The retina is the light-sensing nerve tissue that lines the inside of the eye. All parts of the retina contribute to sight, but only the macula can provide the sharp, straight-ahead vision that is needed for driving and reading small print.

A person's chances of developing AMD are greater than average if he or she has a near relative with the disease. Medical science does not yet know what other factors may contribute to an individual's likelihood of experiencing

AMD.

Most patients with atrophic AMD have a very slow development in time. Pathologically, yellowish deposits called drusen develop beneath the macula. Also, the layer of light-sensitive cells in the macula becomes thinner as some cells break down. These changes typically cause a dimming or distortion of vision that people find most noticeable when they try to read. Generally, if one eye has dry AMD, the other eye will also have some signs of the condition. Thus the person with dry AMD may eventually have visual problems in both eyes. However, the dry form of AMD rarely causes total loss of reading vision. Generally, when dry AMD is found, the individual is encouraged to return for further check-ups. Also, he or she may be taught to perform a simple, at-home test for visual changes. The test involves looking at a piece of paper marked with a grid of straight lines.

If some of the lines begin to look curved or are not visible at all, this may be a valuable warning that new blood vessels are developing and laser treatment should be considered. Patients who use the grid are asked to look at it regularly and tell the doctor right away if they notice any changes in its appearance. If it is suspected that neovascular AMD is developing, a procedure called fluorescein angiography is generally advised. In this procedure, a dye called fluorescein is injected into the arm. Photographs are taken to show the movement of the dye as it reaches the eye and passes through the blood vessels of the retina. If there are new vessels leaking fluid or blood in the macula, the photographs will show their exact location, and serve as a guide for treatment. As with all tests and procedures, individuals are encouraged to ask their physicians about the benefits, risks and costs of various diagnostic and therapeutic alternatives.

Laser treatment is performed by a specially trained ophthalmologist in his office or in an eye clinic at a medical centre. A local anaesthetic may be used to prevent discomfort during the laser treatment session. The session generally takes only a few minutes. Soon afterwards, the individual is able to return home and continue his or her normal activities. The individuals usually will be asked to return to the doctor's office for follow-up appointments. If additional growth of new blood vessels is found, further laser treatment may be indicated. Between follow- up visits, the individuals can use the home test described above to detect any visual changes that might signal renewed blood vessel growth. At present, there is no proven method of preventing dry AMD or the onset of the neovascular form of the disease. There are many useful devices that can help a partially sighted person to make the most of his or her remaining vision. Called low vision aids, these devices have special lenses or electronic systems that produce enlarged images of nearby objects. If you need low vision aids, your eye care specialist can generally prescribe them. Often, he or she will be able to suggest further sources you might contact to get information about counselling, training and other special services for people with low vision. Generally, when dry AMD is found, the individual is encouraged to return for further check-ups. Also, he or she may seek to perform a simple, at-home test for visual changes. The test involves looking at a piece of paper marked with a grid of straight lines. At present, there is no proven method of preventing dry AMD or the onset of the neovascular form of the disease. There are many useful devices that can help a partially sighted person to make the most of his or her remaining vision. Called low vision aids, these devices have special lenses or electronic systems that produce enlarged images of nearby objects. If you need low vision aids, your eye care specialist can generally prescribe them. Often, he or she will be able to suggest further sources you might contact to get information about counselling, training and other special services for people with low vision.

PRIOR ART

The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that exist in both secreted and membrane bound forms. The enzymes are initially expressed as inactive pro-enzymes becoming activated by proteolytic cleavage of their amino termini. The MMPs play a key role in the normal physiology of connective tissue development, morphogenesis and wound healing but their unregulated activity is believed to be implicated in numerous disease processes including arthritis, tumour cell metastases and atherosclerotic disease.

Xilinas M. (PCT/IB01/00715 and PCT/IB01/00712) has presented an invention related to a new use of the known compounds phanquinone and clioquinol respectively, that pertains to their use for the manufacture of pharmaceutical compositions for the treatment or prevention of pathological conditions influenced by the action of MMPs.

In this invention a method is provided for the treatment of a subject having or suspected of having a pathological condition influenced by the action of MMPs in the development of macular degenerative disease including age-related macular degeneration. The activity of clioquinol and/or phanquinone is centred on the chelation of zinc in the eye that will restrict the different MMPs involved in the development of the macular diseases from their action on the extracellular matrix ECM. In this respect phanquinone and clioquinol have an inhibiting action on the enzymes.

It is aimed with the therapeutical or preventive treatment by clioquinol and/or phanquinone from one hand will inhibit or block or reduce or restrict the pathological effects of MMPs related to the development of the macular disease and from the other hand will re-establish the balance between the action of MMP and their corresponding TIMP thus remodulating their physiological balanced interactions.

The activity of MMPs is tightly regulated by the tissue inhibitors of metalloproteinases (TIMPs), a family of secreted proteins currently comprising four members (TIMP-1 , 2, 3, and -4). The balance between MMPs and TIMPs regulates the integrity of the proteinaceous extracellular matrix (ECM). And thus plays a key role in a wide range of physiological processes that include embryonic development, connective tissue remodelling, wound healing, glandular morphogenesis, and angiogenesis. An imbalance in MMP/TIMP expression has been implicated in various diseases such as erosive joint disease, cardiovascular disease and cancer.

The TIMPs form high affinity 1 :1 complexes with the active forms of most MMPs but show varies specificity for different pro-MMPs allowing TIMPs to control the activation of specific MMPs. Activities have also been ascribed to the

TIMPs that are independent of their ability to inhibit MMPs.

Isolated human Bruch's-choroid was examined as to the effect of ageing on the MMPs component of the extracellular matrix remodelling mechanisms (Hussain et al, Invest Opthalmol Vis Sci, 1999, 40:2676-82). It was shown that a matrix degrading mechanism in macular regions, increasing levels of inactive forms of metalloproteinase and scarcity of active forms of MMP-2 suggests possible involvement of impaired extracellular degradation in both ageing and macular degeneration. Further Kadonosono et al (Am J Ophthalmol, 1999, 128:382-4) evaluated the expression and localisation of MMP-7 in Batch's membrane choroidal neovascular membranes in age related macular degeneration. MMP-7 was expressed in all specimens and distinctly expressed in the thickened layer of the membrane and the basement membrane-like structure around the retinal pigment epithelial cells.

MMPs have been shown to be associated with diseases involving neovascularisation and abnormal cellular migration or proliferation. A number of diseases of this type affect the retina (Plantner et al, 1998, Exp Eye Res, 67: 637- 45). In the interphoto receptor matrix and the vitreous the most abundant metalloproteinase is the MMP-2. The level of MMP-2 is nearly double specifically in retinal pigment epithelium associated in the interphoto receptor matrix from eyes of patients with age related macular degeneration. It is concluded that the changes occurring in these patients and especially the neovascularisation which accompanies the exudative form is associated with the MMP-2 higher levels.

The MMPs play a key role in the normal physiology of connective (Brew et al, Biochem Biophys Acta, 2000, 1477:267-83). An important mechanism for the regulation of the activity of MMPs is via binding to a family of homologous proteins (TIMP-1 to TIMP-4). The two-domain TIMPs are of relatively small size, yet have been found to exhibit several biochemical and physiological/biological functions, including inhibition of active MMPs, proMMP activation, cell growth promotion, matrix binding, inhibition of angiogenesis and the induction of apoptosis. Mutations in TIMP-3 are the cause of Sorsby's fundus dystrophy in humans, a disease that results in early onset macular degeneration. Recently the high-resolution structures of TIMPs have been elucidated and their complexes with metalloproteinases, and the results of mutational and other studies of structure-function relationships that have enhanced our understanding of the mechanism and specificity of the inhibition of MMPs by TIMPs. Several intriguing questions, such as the basis of the multiple biological functions of TIMPs, the kinetics of TIMP-MMP interactions and the differences in binding in some TIMP- metalloproteinase pairs are discussed which, though not fully resolved, serve to illustrate the kind of issues that are important for a full understanding of the interactions between families of molecules.

Plantner et al (Exp Eye Res, 1998, 67:637-45) have shown that MMPs have increasingly been shown to be associated with diseases involving neovascularization and/or abnormal cellular migration or proliferation. A number of diseases of this type affect the retina. MMP-2, the most abundant MMP interphoto receptor matrix and vitreous, was measured with respect to age in normal human donor eyes and compared to donors with age-related macular degeneration. The level of MMP-2, was nearly doubled specifically in retinal pigment epithelium-associated interphoto receptor matrix from eyes with age- related macular degeneration, suggesting that MMP-2 may be associated with the changes that occur in age-related macular degeneration, especially the neovascularisation which accompanies the exudative form of the disease.

The MMP activity in Bruch's membrane and in the choroid are age dependent (Guo et al, Invest Ophthalmoi Vis Sci, 1999, 40:2676-82). A matrix- degrading mechanism essential for extracellular remodelling was shown to be present in Bruch's membrane. In macular regions, increasing levels of inactive forms of metalloproteinase and scarcity of active forms of MMP-2 suggests possible involvement of impaired extracellular degradation in both ageing and macular degeneration. Kodonosono et al (Am J Ophthalmoi, 1999, 128:382-4) showed that

MMPs are associated with neovascularisation and in particular MMP-7 was expressed in Bruch membrane of choroidal neovascular membranes in age- related macular degeneration. MMP-7 may be an important factor for the development of the sub-macular neovascular membrane in age-related macular degeneration.

Further Jomary et al (Neuroreport, 1997, 8:2169-72), found that the localisation of MMP-3 in neurodegenerative retinal disease is implicated in the regulation of remodelling of the ECM. The level of mRNA coding for TIMP-3 is increased in retinas affected by the photoreceptor degenerative disease, simplex retinitis pigmentosa, and mutations in TIMP-3 are associated with an inherited form of macular dystrophy. The comparison of TIMP-3 protein expression in normal retina and in those affected by RP and by age-related macular degeneration. Immunoreactive TIMP-3 is present in normal retinal pigment epithelium, and in degenerative retinas particularly at Bruch's membrane and additionally in photoreceptor-retaining regions in simplex RP. The pattern suggests a role for TIMP-3 in normal retinal homeostasis, and, in the disease state, in the modulation of extracellular matrix metabolism and neovascularisation.

Matrix metalloproteinases and metalloproteinase inhibitors are present in human interphoto receptor matrix and vitreous (Plantner et al, Curr Eye Res, 1998, 17:132-40) and it was established that MMPs and TIMPs were present in human interphoto receptor matrix, and vitreous. It was demonstrated that it is most likely that MMPs and TIMPs are involved in normal turnover within the ECM that surround the neural retina and play a role in a number of retinal diseases, particularly proliferative diabetic retinopathy and age-related macular degeneration. Nagase et al (Ann N Y Acad Sci, 1999, 878:1-11) found that differences in proteinase susceptibility between free TIMP-1 and the TIMP-1 -MMP-3 suggest that the residues around the disulfide bond between Cys1 and Cys70 in TIMP-1 may interact with MMPs. The crystal structure of the complex between TIMP-1 and the catalytic domain of MMP-3 has revealed that the alpha-amino group of

Cys1 bidentately chelates the catalytic zinc of MMP-3 and the Thr2 side chain occupies the S1¹ pocket. More particularly in Sorsby's fundus dystrophy (Felbor et al, J Med Genet,

1996, 33:233-6), showed that mutation occurrence of the Tyr168Cys mutation in an Sorsby's fundus dystrophy patient seem to affect the C-terminal region of the mature TIMP3 protein. In addition, all known mutations cause a change of an amino acid to a cysteine residue. This suggests a critical role for the additional C- terminal free thiol group in Sorsby's fundus dystrophy pathogenesis.

The evaluation of the gene encoding the tissue inhibitor of metalloproteinases-3 in various maculopathies (Felbor et al, Invest Ophthalmoi Vis Sci, 1997, 38:1054-9), showed that the mutations in the gene encoding the tissue inhibitor of TIMP3 cause Sorsby's fundus dystrophy, characterised by ECM irregularities in Bruch's membrane. In the 217 patients analysed Felbor et al, identified one sequence alteration (a G-to-C base change) in the 5'- untranslated region in a patient with age related macular degeneration. It is suggested that TIMP-3 is not a major factor in the cause of age related macular degeneration, adult vitelliform macular dystrophy, central areolar choroidal dystrophy, syndrome-associated macular dystrophies, cone-rod dystrophy, and in a group with unspecified macular degeneration and that only Sorsby's fundus dystrophy appears to be only associated with mutations in TIMP3.

Vettakkorumakankav and Ananthanarayanan (Biochim Biophys Acta, 1999, 1432:356-70VS) showed that TIMP binds only on zinc with definite stoichiometries. De La Paz et al, (Invest Ophthalmoi Vis Sci, 1998, 39:1256-60) showed that MMPS and their endogenous TIMPs are present in human vitreous and may be involved in the pathogenesis of vitreo-retinal diseases.

In general (Stohr et al, Genome Res, 1995, 5:483-7), the TIMPs play a crucial role in the physiological turnover of ECM by tightly regulating MMPs activities. Disturbances in the TIMP/MMP system have been implicated in many disease processes where loss of ECM integrity is a principal feature. More recently, it has been shown that mutations in TIMP3 cause the autosomal dominant disorder Sorsby's fundus dystrophy, in which condition characteristic ECM irregularities occur in Bruch's membrane.

Steen et al (Invest Ophthalmoi Vis Sci, 1998, 39:2194-200) have shown that MMPs play possible role in choroidal neovascularisation and support their role in the development of choroidal neovascularisation in macular degeneration. The localisation of MMP-2 and MMP-9 to the areas of new vessel formation and to the enveloping Bruch's-like membrane, respectively, suggests that MMP-2 and

MMP-9 may be co-operatively involved in the progressive growth of choroidal neovascular membranes in macular disease.

Changes of TIMP-3 in Bruch's membrane occur during ageing and in age-related macular degeneration (Kamei and Hollyfield Invest Ophthalmoi Vis Sci, 1999, 40:2367-75). TIMP-3 is present uniformly across Bruch's membrane in the normal samples. In samples from donors more than 50 years of age, immunostaining was intense and TIMP-3 increased with age. In macular degeneration eyes, TIMP-3 distribution in Bruch's membrane is abundant in areas of continuous soft drusen but absent in areas below the retinal pigment epithelium. During normal ageing, TIMP-3 content in Bruch's membrane of the macula shows a significant increase. TIMP-3 content in age related macular degeneration eyes Ms elevated relative to that of age-matched normal eyes. Higher levels of TIMP-3 may contribute to the thickening of Bruch's membrane observed in macular disease.

Brown PD (Expert Opin Investig Drugs 2000 Sep;9(9):2167-77) reported recently synthetic, potent, low molecular weight MMP inhibitors have been developed and, over the past five years and that these agents have begun clinical testing in patients with cancer, rheumatoid arthritis, osteoarthritis and acute macular degeneration

The basis of the invention was focused on the search for molecules that could:

Show a sufficient chelating activity towards zinc Have lipophilic chemical properties

Show a high penetration that would allow them to reach the retina of the eye

Show satisfactory pharmacokinetics that would allow them to be applied locally in the eye and also be absorbed orally in suitable formulations

Have a satisfactory toxicity profile based on experimental evidence in appropriate studies Have a satisfactory human safety profile based on clinical experience that would allow them to have a satisfactory risk benefit ration when treating macular disease

Have a weak chelating profile against other metals of the body and in particular iron, calcium and magnesium Show a satisfactory distribution in the eye

Show a satisfactory distribution within the cell parenchyma Show a satisfactory reduction of oxidative stress in the eye Our research has led us to the discovery and identification of clioquinol and phanquinone, two known chelators, that satisfy most of our research criteria as described above. Both molecules share a strong chelating effect on both zinc and copper while clioquinol has a stronger zinc chelating activity and phanquinone a stronger copper. However both molecules have separately, a sufficient chelating effect on both heavy metals and a very low or negligent chelating effect on iron, magnesium and calcium.

In this invention a new therapy is proposed based on the ability of the two known substances, clioquinol and phanquinone to chelate zinc and copper. Using mass spectometry, Kidany et al (1974, Jap Analyst 23:1375-1378), analysed quantitatively the clioquinol chelates with Fe, Co, Ni, Cu and Zn, and obtained the following results: in the case of cobalt, nickel, copper and zinc chelates, the molecular ion peaks, corresponding to 2:1 ratio metal chelates were obtained in every spectrum, whereas in the case of Fe (III) the ratio was 3:1. In monkey micro autoradiography, it was demonstrated histochemically that zinc- positive granules in the hippocampus of animals, and the neuropathology of Ammon's horn in man that the high radioactivity in Sommer's sector of Ammon's horn in the 20-minute autoradiography was due to Zn (II) chelates of clioquinol mainly in the terminal axodendritic boutons of the mossy fibers. There is an extremely high penetration of intravenously administered clioquinol into the nervous system meaning that the there is no blood brain barrier to the clioquinol penetration (Shiraki, 1979, Handbook of Clinical Neuroly, 37:115- 139).

Both molecules are lipophilic and are well absorbed by oral administration. Satisfactory plasma concentrations are achieved by oral administration, 250 mg of clioquinol give plasma concentrations varying between 1 and 5 microg/ml. Phanquinone also has been given at dosages of 50 mg three times daily in the past achieving satisfactory clinical response. Clioquinol more specifically in man and when given at dosages of 7.4 to 9.3 mg/kg/day achieved concentrations in the range of 2J to 5.8 microg/ml in plasma (Shiraki, 1979).

Due to the lipophilicity of both molecules they easily cross the blood brain barrier and can be found in the cerebro-spinal fluid and in the brain tissue. In electron microscope autoradiographs of mice 18 hours after intraperitoneal administration of Zn⁶⁵CI it was observed that large numbers of grains due to Zn⁶⁵ in the mossy fibre endings on granule cells and their processes, as well as on the pyramidal cells of the hippocampus were present. The Zn⁶⁵ grains were found mostly over synaptic vesicles and less frequently over the mitochondria in presynapses of the mossy endings, but not over the synapses of granule and pyramidal cells or their processes in the hippocampus.

As examples of the brain and nervous system in general activity of both clioquinol and phanquinone and in relation to the A beta amyloid the following experiments have been described earlier (WO 99/09981 ) on the effect of phanquinone in A beta (25-35) dose response and induced toxicity in PC12 cells. In the presence of phanquinone the toxic effect of A beta amyloid (25-35) was virtually abolished and even at concentrations of A beta as high as 1 microM, the presence of phanquinone completely counteracted the toxic effect of A beta. On the other hand it was shown that in the presence of increasing concentrations of phanquinone of over 0.1 microg/ml resulted in drastic reduction of the toxic effect of A beta (25-35) which were completely abolished at a concentration of 1 microg/ml of phanquinone.

The data described above (WO 99/09981 published under the PCT Treaty) show that both clioquinol and phanquinone reduced the neurotoxicity in the brain by sequestrating zinc and copper. The chelation of both or either of these metals is the basis of the treatment described here to treat or prevent age related macular disease.

DISCLOSURE OF THE INVENTION

According to the present invention the new use of clioquinol and/or phanquinone for the manufacture of a pharmaceutical composition for the treatment or prevention of pathological conditions influenced by the action of MMPs in eye macular disease is provided. Using biochemical assays we have shown that clioquinol inhibits the metalloproteinases-1 (MMP-1 ) at concentrations of 10 microM. The inhibitory effect of clioquinol is increasing by increasing the concentration of the drug from 0.1 to 1 and further to lOmicroM. In a continuation of this experiment whan the clioquinol concentration was increase to 100 microM the inhibition of the MMP-1 was practically total. An overwhelming increase of the clioquinol concentration to

1 mM and 10 mM confirmed the specific inhibitory effects of clioquinol.

In a similar manner clioquinol was found to inhibit biochemical activity of MMP-2, MMP-3, MMP-7 and MMP-9 in an increasing manner proportionally to the clioquinol concentration (0.1 , 1 , 10 and lOOmicroM). Phanquinone shares with clioquinol a similar chelating activity towards zinc. Both molecules are also chelating copper and other metals. The inhibitory activity of phanquinone as to the MMPs is postulated to be similar to that shown already experimentally for clioquinol.

Ointment and eye drop preparations of clioquinol and phanquinone in suitable vehicles can achieve local concentrations in the eye of 100 microM that will be sufficient to inhibit in the macular tissues the metalloproteinases activity and override their deleterious degenerative effects. Various diseases are influenced by MMPs. Examples of such diseases are age related macular degeneration as well as tumour metastasis and angiogenesis, rheumatoid arthritis, corneal ulceration, osteoporosis and osteoarthritis, multiple sclerosis, diabetic complications, periodontal disease and atherosclerosis. The common feature for the pathological conditions which may be influenced by MMPs is that such conditions involve tissue breakdown. In general, the cause of the diseases influenced by MMPs is due to an over expression and over-activity of the MMPs leading to increased degradation of tissue. Clioquinol and phanquinone separately or in combination are two molecules intended to be used for the prevention or treatment of macular degenerative disease. Achieving concentrations by local eye administration as an ointment or as eye drops preferably, at concentrations preferably of 1% of either clioquinol and/or phanquinone will be sufficient based on our MMP studies of clioquinol, to modulate the effect of the MMPs by decreasing them and reestablish a balance between the MMP and corresponding TIMP activity, Using such formulation concentrations inhibition of MMPs can be achieved as necessary ( of the order of 100 microM ).

The effects of clioquinol and/or phanquinone can by maintained as long as required therapeutically for periods ranging from three weeks up to several years or decades. Such effects will act both in a therapeutic as well as prophylactic manner as clinically required.

The two molecules have been already used for other indications and are relatively safe for use. Clioquinol was related to the SMON epidemic only in Japan where the drug was used in excessive and off-label doses. Several millions of patients have used both clioquinol and/or phanquinone in the past with relatively no serious adverse events. Both molecules are known chelators of zinc and copper: clioquinol relatively less than phanquinone. Both these molecules penetrate well the blood brain barrier. Both these molecules have been proposed for the treatment of

Alzheimer's disease due to their ability to chelate zinc and copper and to reduce oxidative reactions in the brain. Amyloid in Alzheimer's disease is aggregated in the presence of mainly zinc but also copper and clioquinol and phanquinone are used to chelate the two metals in the brain and block the aggregation of amyloid and the formation of plaques.

Clioquinol or phanquinone or their combination are therefore indicated for the treatment of macular diseases, including age related macular degeneration.

The treatment is directed whenever there is an over expression or over-activity of one or more of the MMPs that results in pathological conditions and vitro-retinal disease. The treatment is indicated for neuro-degenerative retinal diseases, including exudative and atrophic age related macular degeneration as well as for photoreceptor degenerative disease, simplex retinitis, proliferative diabetic retinopathy, Sorsby's fundus dystrophy, macular hole, presumed ocular histoplasmosis syndrome, proliferative diabetic retinopathy, epiretinal membrane, vitreo-macular traction syndrome, macro-aneurysm with subretinal haemorrhage, central retinal vein occlusion with vitreous haemorrhage and proliferative vitreoretinopathy or in any other vitro-retinal condition clioquinol and/or phanquinone will modulate and re-establish the equilibrium between the enzymatic activity of the respective MMPs and their TIMPs inhibitors.

Both clioquinol and phanquinone possess significant anti-oxidative effects due to their capacity to chelate zinc in neural tissue. This effect is more evident with phanquinone than clioquinol with due to the higher chelating affinity of the later towards copper. Clioquinol (iodo- chlorohydroxyquinol, iodo- chloro hydroxyquinoline, chloro- iodoquine, 7-iodo -5-chloro-8 -hydroxyquinoline, 5-Chloro- 7-iodo -8- hydroxy quinoline, 5-Chloro-7-iodo-8-quinolinol, 5-Chloro- 8-hydroxy- 7- iodoquinoline) is a known antiamoebic and antibacterial drug that has been used extensively as such in the past and sold by CIBA-GEIGY as

ENTEROVIOFORME as well as by several other companies. Recently clioquinol has been shown to have effects on the amyloid in Alzheimer's disease (Dement Geriatr Cogn Disord 2001 Nov-Dec;12(6):408-14) and we have shown in our research in 20 patients with Alzheimer's disease that clioquinol crosses the blood-brain barrier and has greater affinity for zinc and copper ions than for calcium and magnesium ions. We treated for 21 days at doses of 20 mg/day to 10 patients and 80 mg/day to another 10 patients. Cerebrospinal fluid (CSF) investigations revealed a significant increase in Tau protein and growth- associated protein (GAP43). Clinical ratings showed slight improvement after 3 weeks treatment.

Phanquinone, (4J-phenanthroline-5,6-dione) has hitherto been used for the treatment of parasites and in particular giardia and amoebas. Phanquinone has been sold by CIBA-GEIGY under the trademark ENTOBEX. Phanquinone has received renewed interest in recent years and we have suggested it for the treatment of Alzheimer's disease (WO 99/09981). Phanquinone has a large number of clinical investigations completed in the past where it has shown a good safety profile when given orally in the treatment of parasitic disease and has shown no toxic effects when used at dosages up to 150mg daily for periods ranging from three weeks to six months.

Clioquinol is a chelating agent with proven activity as to the sequestration by chelation of metals including zinc, iron, manganese, copper, mercury, nickel, copper and cobalt. Injectable preparations of clioquinol have been reported as well penetrating in the brain and crossing the blood brain barrier and achieving concentrations in the brain of the order of 20microg/ml. The concentration of clioquinol was also found to be high in areas of the brain like the hippocampus that are directly involved in Alzheimer's disease.

It has been also used in combination with clioquinol in different concentrations for the same indications as clioquinol and marketed in combination by CIBA-GEIGY as MEXAFORME and MEXASE.

Recently extensive research has been undertaken for using clioquinol and phanquinone in the treatment of Alzheimer's disease. From experimental data it has been shown that clioquinol has the ability to chelate several heavy metals in vivo and in the brain and in particular zinc and copper. Phanquinone has the ability to chelate copper as well as zinc in the brain. The ability of copper chelation is three times more pronounced with phanquinone compared to clioquinol. On the contrary clioquinol has a two fold at least more pronounced ability to chelate zinc in comparison to phanquinone. It has also been shown that phanquinone possesses the same beneficial properties as clioquinol as to the abolishment of the amyloid formation and the resolubilisation of amyloid in the brain. Clioquinol has been given in the past at dosages of 250mg three times daily for periods of up to three months for the treatment of intestinal amoebiasis. It has been also used extensively in the treatment of intestinal infectious diarrhoea. The only reported toxicity with clioquinol was the one reported in Japan when excessive dosages were given and for long periods of time reaching up to two years. Then a neurotoxicity called sub-acute myelo optic neurotoxicity (SMON) was developed. In recent research it has been shown that this toxicity is related to the vitamin B₁₂ metabolism. Based on the studies showing that both clioquinol and phanquinone are potent chelators of zinc and copper and recent data showing that amyloid in Alzheimer's disease is precipitated only in the presence of zinc and copper extensive research was undertaken to evaluate the beneficial effects of both molecules in the treatment of Alzheimer's disease. These effects were studied in relation to the ability of both molecules to abolish precipitation of amyloid in vitro. Further it has been shown that both molecules can resolobilise precipitated amyloid taken from brains of Alzheimer's patients. The physicochemical properties of clioquinol have confirmed its chelating activity (Kidani et al, 1974, Jap Analyst 23:1375-78, Tateishi et al, 1973, Psychiat Neurol. Jap. 75: 187-196).

Based on experimental data intravenous clioquinol achieves high concentrations in the brain of the order of 20microg/ml when administered at dosages of 10-20mg/kg (Tateishi et al 1975 and Tamura, 1975, Sci Biol suppl,

28:69-77).

Clioquinol after many decades of use all over the world showed toxicity only in Japan and under special conditions. All cases of neurotoxicity that were documented with pathology showed toxicity at dosages from 23 to 50mg/kg/daily and for treatment duration from 13 to 313 days.

Recently in our research we have linked the toxicity of clioquinol described in Japan with a B₁₂ explanation (J Neurol Sci 2000 Feb 1 ;173(1 ):40-4) and concluded that chronic treatment with clioquinol may alter the tissue homeostasis of vitamin B(12) in the brain. This fact made us propose in our clinical studies the use of clioquinol in combination with B₁₂ co-administration

The ability of clioquinol to abolish Aβ amyloid aggregation in the presence of zinc has been confirmed experimentally. When zinc (30μM) is added it reduces the solubility of Aβ (2.5μM in TBS at a pH of 7.4) from 95% to 43%. W en clioquinol is added (120μM) to the zinc and Aβ it abolishes the zinc-induced aggregation from 43% to 70%.

Therefore the administration of clioquinol will chelate zinc and other heavy metals in the brain and in specific brain and due to its lipophilicity is well absorbed per os achieving high cerebrospinal fluid and brain concentrations.

Considerable evidence now indicates that the accumulation of Aβ amyloid (Aβ) in the brain cortex is very closely related to the cause of AD. Aβ is a normal component of biological fluids whose function is unknown. Aβ accumulates in a number of morphologies varying from highly insoluble amyloid to deposits that can be extracted from post-mortem tissue in aqueous buffer. The factors behind the accumulation are unknown, but we have systematically appraised the solubility of synthetic Aβ peptide in order to get some clues as to what kind of pathological environment could induce the peptide to precipitate. Aβ has three principal vulnerabilities- zinc, copper and low pH (Bush,

1994). The precipitation of Aβ by copper is dramatically exaggerated under mildly acidic conditions (e.g. pH 6.9), suggesting that the cerebral lactic acidosis that complicates AD could contribute to the precipitation of Aβ and this event could be mediated by copper. A consideration of the involvement of zinc and copper in plaque pathology is contemptible since the regulation of these metals in the brain has been shown to be abnormal in AD.

In our research in this field we have shown recently (Neuron 2001 that Jun;30(3):665-76) that the treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. The overall conclusions from these experiments can be resumed as follows: Divalent metals (most probably zinc) maintain Aβ in an insoluble state in both healthy and disease states suggesting a role in Aβ homeostasis. Other metals, possibly calcium and/or magnesium may operate to maintain Abeta in the soluble state. Amyloid deposits in AD tissue also respond to chelation treatment at the appropriate concentration. This probably reflects the removal of zinc bound to less soluble diffuse amyloid and the outer layers of plaques. A mild treatment with chelating agents allows rapid resolubilisation of the aggregated Aβ. Abundant ephemeral deposits of this type may be present at all times in both affected the unaffected individuals but may only be detectable as background staining by immunohistology. The results suggest that metal chelators will have a therapeutic use in AD and serve a prophylactic function in reducing amyloid burden in the clinical state. Clioquinol is the only chelator that enriched in the brain due to its hydrophobicity, making it an excellent candidate for development towards clinical trials.

Recently direct evidence has been obtained that these metals are integral components of the Aβ deposits in the brain in AD. It was found that zinc- and copper- specific chelators can dramatically redisolve a significant proportion of Aβ extracted from post-mortem AD affected brain tissue, compared to the amount extracted from the tissue by buffer in the absence of chelators.

In the past phanquinone has also been shown in experimental models to possess central nervous system effects that were translated with behavior changes.

Phanquinone has been used extensively in the past for the treatment of parasitic disease and specifically in the treatment of giardiasis. It has been also used in combination with clioquinol in different concentrations for the same indications as clioquinol. Recently we have undertaken extensive research for using phanquinone in the treatment of Alzheimer's disease. From experimental data it has been shown that phanquinone has the ability to chelate copper as well as zinc in the brain. The ability of copper chelation is three times more pronounced with phanquinone compared to clioquinol. It has also been shown that phanquinone possesses the same beneficial properties as clioquinol as to the abolishment of the amyloid formation and the resolubilisation of amyloid in the brain.

DETAILED DESCRIPTION OF THE INVENTION

In the following the invention will be explained in further detail. The proposed mechanism of action of the invention is not to limit the invention to the said mechanism. At present the applicant has shown experimentally that clioquinol in biochemical assays inhibits directly the MMP-1 , MMP-2, MMP-3, MMP-7 and MMP-9. He also believes that clioquinol and/or phanquinone by competition chelate zinc from a common pool. Both clioquinol and/or phanquinone have the physico-chemical ability to penetrate to tissues including the retina, biological fluids, cells and pathological formations including drusen, atheromas, metastatic cells, degenerative cells, neo angiogenesis cells and inflammatory tissues. When clioquinol and/or phanquinone enter the biological area involved in the pathological condition, the zinc (II) ion is captured from the free pool existing due to the equilibrium between zinc dependent MMPs and TIMPs. Clioquinol and/or phanquinone having chelated a zinc (II) ion then moves away from the area involved in the pathological condition and into the interstitial fluid, the lymph, the blood, the urine or the bile and is cleared from the body. The deprivation of zinc from the direct environment of the zinc requiring and zinc dependent MMPs inhibits their action.

MMPs are directly involved in macular degenerative disease in man.

From several studies it has been established that the highest concentration of zinc in the body is in the choroid of the eye in a wide range of species including man. It has been clearly shown in monkeys that clioquinol achieves high concentration in the choroid in the same anatomic where the concentration of zinc is high and the MMPs and TIMPs are located.

Further clioquinol and phanquinone due to their capacity to chelate copper share a scavenger anti-oxidative effect.

The pharmaceutical composition manufactured using clioquinol and/or phanquinone preferably comprises one or more pharmaceutical acceptable carriers and, optionally, one or more further active constituent(s). The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. In a preferred embodiment, the clioquinol and/or phanquinone and optionally, further active constituents in the pharmaceutical composition are purified.

It will be appreciated that the amount of clioquinol and/or phanquinone and, optionally, further active constituents required for said treatment or prevention will vary according to the route of administration, the disorder to be treated, the condition, age, the file history of the subject, and the galenic formulation of the pharmaceutical composition etc. When treating a patient diagnosed as having an eye disease, the amount of phanquinone and of clioquinol is preferably effective to provide for at least a partially inhibition of at least one of the enzymes belonging to the group of MMPs.

In general, a suitable therapeutically effective amount of phanquinone and/or of clioquinol in the oral pharmaceutical composition is, for example 1 mg to 1 g, preferably 10 mg to 50 mg of phanquinone and/or 1 mg to 1 g of clioquinol, preferably 20 mg to 250 mg. If vitamin B₁₂ is selected as further active ingredients of the pharmaceutical composition, the amount given is preferably the amount necessary to inhibit any detrimental effects of clioquinol administration and 5 micro g to 2 mg. In case of local applied ointment or eye drop pharmaceutical composition the effective concentration is, for example 0.1 to 3 % of phanquinone and/or of clioquinol.

The actually administered amounts of phanquinone and/or clioquinol, and optionally further active constituents, such as vitamin B₁₂, may be decided by a supervising physician. If the pharmaceutical composition in addition to clioquinol and/or phanquinone comprises further active constituents they may be in the same composition for administering in combination concurrently, or in different compositions for administering substantially simultaneously but separately, or sequentially, the further active ingredients may be administered prior or subsequently to the administering of phanquinone and/or clioquinol.

Pharmaceutical formulations include those suitable for parenteral

(including intramuscular and intravenous), oral, rectal, local eye, including ointments and drops, or intradermal administration, although oral and local eye administrations are the preferred routes. Thus, the pharmaceutical composition may be formulated as tablets, pills, syrups, capsules, suppositories, formulations for transdermal application, powders, especially lyophilised powders for reconstitution with a carrier for intravenous administration, eye ointments, eye drops, eye solutions for eye injection. The pharmaceutical preparation may be prepared using conventional carriers. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapy is administered. The carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polivinylpyrrolidone (polyvidone or povidone), polysorbate 80, polyethylene glycol (Macrogol), gum, tragacanth, gelatine, starch, lactose, or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium, lauryl sulfate; a glidant, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; and/or a flavouring agent, such as peppermint, methyl salicylate, orange flavouring.

Pharmaceutical formulations suitable for oral administration, e. g. tablets and pills, may be obtained by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by mixing the constituent(s), and compressing the mixture obtained in a suitable apparatus into tablets having suitable size. Prior to the mixing, the clioquinol and/or phanquinone may be mixed with a binder, a lubricant, an inert diluent and/or a disintegrating agent and the further optionally present constituents may be mixed with a diluent, a lubricant and/or a surfactant. In a preferred embodiment, free-flowing clioquinol and/or phanquinone powder is mixed in a binder, such as microcrystalline cellulose, and a surfactant, such as lauryl sulphate, until a homogeneous mixture is obtained. Subsequently, another binder, such as polyvidone, is transferred to the mixture under stirring. When a uniform distribution is obtained as aqueous solution of vitamin B₁₂ is added under constant stirring. Said mixture is passed through granulating sieves and dried by desiccation before being compressed into tablets in a standard compressing apparatus.

In a second preferred embodiment, free-flowing clioquinol and/or phanquinone powder is mixed with surfactants and/or emulsifying agents, such as Sapamine (N-(4'-stearoyl amino phenyl)-trimethylammonium methyl sulphuric acid) and lactose monohydrate until a uniform distribution of the constituents is obtained. A second preparation containing a disintegrating agent, such as maize starch, is added to the clioquinol and/or phanquinone mixture while being continuously stirred. Such a second preparation may be prepared by adding excess boiling water to a maize starch suspended in cold water. The final mixture is granulated and dried as above and mixed with maize starch and magnesium stearate and finally compressed into tablets in a standard apparatus.

A tablet may be coated or uncoated. An uncoated tablet may be scored. A coated tablet may be coated with sugar, shellac, film or other enteric coating agents.

Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of the above constituents. An aqueous or oily carrier may be used. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Formulations for parenteral administration also include a lyophilised powder comprising clioquinol and/or phanquinone and, optionally an aqueous solution of carboxymethylcellulose and lauryl sulphate.

When the pharmaceutical formulation is a capsule, it may contain a liquid carrier, such as fatty oil, e.g. cacao butter.

Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatine, mal, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol water, ethanol and the like. The compositions may be solutions, including eye drops, suspensions, ointments, creams, drops, emulsion, tablets, pills, capsules, powders, sustained release formulations and the like. The composition may be formulated as a suppository, with traditional binders and carriers such as triglycerides. In one embodiment of the pharmaceutical composition, clioquinol and/or phanquinone and the optionally, further active constituents, are comprised as separate pharmaceutical entities. By one example, one entity may comprise vitamin B₁₂. The two entities, may administered simultaneously or sequentially. For example, the entity comprising clioquinol and/or phanquinone can be administered, followed by vitamin B₁₂ administered within a day, week, or month of clioquinol and/or phanquinone administration. If two entities are administered sequentially, the entity comprising phanquinone and/or clioquinol, is preferably administered for one to three weeks followed by a wash out period of one to four weeks, during which the entity comprising vitamin B₁₂ is administered but not the entity comprising phanquinone and/or clioquinol. After the wash out period, the treatment may be repeated.

The pharmaceutical composition may be provided as a pack or kit comprising one or more entities containing one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally, associated with such entities may be a notice in the form described by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of the manufacture, use or sale for human administration. Other features and advantages of the invention will be apparent from the following examples illustrating by way of example the principles of the invention.

Example 1

Preparation of a pharmaceutical composition comprising phanquinone, clioquinol and vitamin B₁₂.

250 g phanquinone and 250 g of clioquinol was mixed with 200g of N'-(4'- steatoryl aminophenyl) - trimethylamonium methyl sulphuric acid) and 1025 g lactose mono-hydrate for a period of 5 minutes. 300 g of boiling water was added in one go to a mixture of 100 g maize starch in 100 g cold water. The maize suspension, cooled to 40° C was added to the phanquinone and clioquinol containing powder mixture under continuous stirring. Subsequently, an aqueous solution of 5 g vitamin B₁₂was added. The mixture was granulated using a 2.5 mm sieve and desiccated for 18 hours at 40° C. The dry granules were mixed with

400 g maize starch and 20 g magnesium stearate. The final mixture was formulated into tablets having a diameter of 8.0 mm and a weight of 500 mg.

Example 2

250 g phanquinone and 250 g of clioquinol were mixed with 200 g Sapamine (N- (4'- steatoryl aminophenyl) - trimetthylammonium methyl sulphuric acid) and 1025 g lactose mono-hydrate for a period of 5 minutes. 300 g of boiling water was added in one go to a mixture of 100 g maize starch in 100 g cold water. The maize suspension, cooled to 40°C was added to the phanquinone and clioquinol containing powder mixture under continuous stirring. Subsequently, an aqueous solution of 5 g vitamin B₁₂ was added. The mixture was granulated using a 2.5 mm sieve and desiccated for 18 hours at 40°C. The dry granules were mixed with 400 g maize starch and 20 g magnesium stearate. The final mixture was formulated into tablets having a diameter of 8.0 mm and weight of 200 mg. Example 3

Preparation of a pharmaceutical composition comprising phanquinone or clioquinol

An ointment for use according to the present invention may be prepared by addition of 1 % clioquinol to a common ointment base comprising Vaseline and white wax. Alternative may an ointment be prepared by addition of 0.5 % phanoquinone to an ointment base comprising Vaseline and white wax..

Example 4

Inhibition study of MMPs by clioquinol

An enzyme assay was conducted with five of the enzymes belonging to the

MMP group. Specifically, the assay was conducted for MMP-1 , MMP-2, MMP-3, MMP- 7, and MMP-9 at various concentrations. The MMP-1 , MMP-3, and MMP-7 were initially pre-incubated in 60 min at 37EC and MMP-2 and MMP-9 were pre-incubated in 60 min at 25EC in an aqueous vehicle of 50 mM MOPS, 10mM CaCI₂.2H₂0, 10 μM ZnCI₂, 0,05% Brij 35, pH 7.2 and a concentration of clioquinol of 100 μM. A test substrate of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ was subsequently added to obtain a concentration of 25 μM. MMP-1 was incubated for 2 hours at 37EC, MMP-2 was incubated for 3 hours at 25EC, MMP-3 was incubated for 90 min at 37EC. MMP-7 was incubated for 90 min at 37EC, and MMP-9 was incubated for 2 hours at 25EC. The activity of the enzymes was measured by fluorometric quantitation of Mca-Pro-Leu-Gly- OH. The results are indicated in Table I below.

Table I

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variation are not to be regarded a departure from the spirit and scope of the invention, and all such modifications, as would be obvious to a person skilled in the art, are intended to be included in the scope of the following claims.

Claims

C L A I M S

1. A use of one or more chelators for the manufacture of a pharmaceutical composition for treatment or prevention of age related macular degenerative disease, wherein the chelators:

Show a chelating activity towards zinc, Have lipophilic chemical properties,

Show a high penetration that allow them to reach the retina of the eye, and Have satisfactory toxicity profile.

2. The use according to claim 1, wherein the one or more chelators are selected from clioquinol, phanquinone and mixtures thereof.

3. The use according to claim 2, wherein the disease influenced by impairment of the action of matrix metalloproteinases includes the MMP-1 , MMP-2, MMP-3, MMP-4, MMP-7, MMP-9, MMP-12 and MMP-14 among other possible MMPs.

4. The use according to claim 2 or 3, wherein the diseases are influenced by impairment or imballance in the action of MMPs or of their respective tissue inhibitor(s) metalloproteinases (TIMPs) or by an anti-oxidant effect.

5. The use according to claims 2 to 4, wherein the diseases are neurodegenerative retinal diseases, including exudative and atrophic age related macular degeneration as well as photoreceptor degenerative disease, simplex retinitis, proliferative diabetic retinopathy,

Sorsby's fundus dystrophy, macular hole, presumed ocular histoplasmosis syndrome, proliferative diabetic retinopathy, epiretinal membrane, vitreomacular traction syndrome, macroaneurysm with subretinal haemorrhage, central retinal vein occlusion with vitreous haemorrhage and proliferative vitreoretinopathy or in any other vitro-retinal condition.

6. The use according to claims 2 to 5, wherein clioquinol modulates the zinc and copper concentrations in the eye and reduces, down grades or inhibits the enzymatic activity of MMPs or re-establishes the activity balance between MMPs and their corresponding TIMP inhibitors.

7. The use according to claims 2 to 6, wherein clioquinol is administered in an amount of 1 mg to 1 g, or preferably 20 mg to 250 mg.

8. The use according to claim 2 or 6, wherein phanquinone in an amount of 1 mg to 1 g or preferably 10 mg to 50 mg is used in combination with clioquinol. in an amount of 1 mg to 1 g, or preferably 20 mg to 250 mg.

9. The use according to claims 2 to 6, wherein phanquinone in an amount 1 mg to 1 g or preferably 10 mg to 50 mg is administered without clioquinol.

10. The use according to claim 2 or 9, wherein clioquinol and/or phanquinone is administered in one pharmaceutical preparation.

11. The use according to claims 2 to 10 where either clioquinol or phanquinone or their combination are administered one to four times daily.

12. The use according to any of the preceding claims, wherein a metal salt or prosthetic group is administered prior to, concurrent with, or subsequent to the administration of clioquinol, phanquinone or their combination.

13. The use according to claim 12, wherein, the prosthetic group is vitamin B₁₂.

14. The use according to claim 13, wherein the amount of vitamin B₁₂ is effective to inhibit a detrimental side effect of clioquinol administration.

15. The use according to claim 13, wherein the amount of vitamin B₁₂ is 5 microg to 2 mg.

16. The use according to claim 13, wherein the amount of vitamin B₁₂ is 0.5 mg to 1 mg.

17. The use according to any of the preceding claims, wherein the pharmaceutical composition is formulated for ocular, oral, parenteral or intradermal administration.

18. A method of treating a subject having or suspected of having a pathological condition influenced by the chelating action on zinc on MMPs and/or their respective TIMPs, comprising administering an amount of clioquinol, or of phanquinone or of their combination to treat or prevent the pathological condition related to macular degenerative disease or vitro-retinal eye diseases.

19. A method of treating a subject having or suspected of having a pathological condition influenced by oxidative stress in addition to deregulation of MMP/TIMP balanced interaction, comprising administering to the subject an amount of clioquinol, or of phanquinone or of the combination of clioquinol and phanquinone effective to treat or prevent to macular degenerative disease or vitro-retinal eye diseases.

20. A method of treating a subject having or suspected of having a pathological condition influenced by MMPs and/or of their respective TIMP deregulation in the eyes, comprising administering to the said subject of:

(a) an amount of clioquinol or of phanquinone or of a combination of clioquinol and phanquinone to treat or prevent the disease influenced by the pathological imbalance between MMPs and/or of their respective TIMPs or of oxidative stress, (b) an amount of a compound or of a mixture of compounds selected from the group comprising metal salts or prosthetic groups different from clioquinol or of phanquinone having another activity, to treat or prevent the disease influenced by the pathological imbalance between MMPs and/or of their respective TIMPs or of oxidative stress.

21. The method according to claim 20, wherein the total amount of the compound(s) in (b) is sufficient for increasing the effect of the prevention or treatment of a pathological condition influenced by the action of MMPs or for impeding any detrimental side effect.

22. A method according to claim 19, 20 or 21 , wherein the daily dose administered amount of clioquinol is 1 mg to 1 g.

23. A method according to claims 19, 20 or 21, wherein an amount of clioquinol 1 mg to 250 mg is administered one to four times daily.

24. A method according to claim 19, 20 or 21 , wherein the daily dose administered amount of phanquinone is 1 mg to 1 g.

25. A method according to claims 19, 20 or 21 , wherein an amount of phanquinone 1 mg to 250 mg is administered one to four times daily.

26. The method according to claim 20, wherein the amount of the compound(s) in (b) is 5 microg to 250 mg.

27. The method according to claim 20, wherein the composition different from clioquinol and of phanquinone having another activity is vitamin B₁₂.

28. The method according to claim 20, wherein the amount of vitamin B₁₂ is 5 microg to 2 mg.

29. The method according to claim 20, wherein the amount of vitamin B₁₂ is 0.5 mg to 1 mg.

30. A method of treating a subject having or suspected of having pathological conditions or diseases influenced by MMPs comprising administering:

(a) an amount of clioquinol effective to treat or prevent the imbalance between the action of MMPs and/or their respective TIMPs or the oxidative stress on the eye diseases, and

(b) a mixture of phanquinone and vitamin B₁₂, the amount of phanquinone being influenced by the chelating action on the zinc dependent MMPs causing the pathological condition and the disease and the amount of vitamin B₁₂ being effective to inhibit detrimental side effects of clioquinol administration.

31. The method according to claims 20 or 30, wherein (a) clioquinol and (b) the compound(s) are comprised in a single pharmaceutical composition.

32. The method according to claims 21 or 30, wherein (a) clioquinol and (b) the compound(s) are administered substantially simultaneously.

33. The method according to claims 21 or 30, wherein (a) clioquinol and (b) the compound(s) are administered sequentially.

34. The method according to claims 21 or 30, wherein clioquinol and vitamin B ₂ are administered sequentially.

35. The method according to claims 21 or 30, wherein phanquinone and vitamin B₁₂ are administered sequentially.

36. The method according to claim 21 and 30, wherein phanquinone is administered in a first period followed by a second period, wherein clioquinol is administered.

37. The method according to claim 21 and 30, wherein clioquinol is administered in a first period followed by a second period, wherein phanquinone is administered.

38. The method according to claim 36-37, wherein the first period is one to three weeks and the second one to four weeks.

39. The use according to claim 1-17, wherein clioquinol and/or phanquinone is formulated for oral administration.

40. The use according to claim 1-17, wherein clioquinol and/or phanquinone is formulated for local ocular administration.

41. The use according to claim 1-17, wherein clioquinol and/or phanquinone is formulated for intra-ocular administration.

42. The use according to claim 1-17, wherein clioquinol and/or phanquinone is formulated for parenteral administration.

43. The use according to claim 1-17, wherein clioquinol and/or phanquinone is formulated for transdermal administration.

44. The use according to claim 1-17, wherein clioquinol and/or phanquinone is used for the treatment or prevention of age related macular degenerative disease for periods lasting several months to twenty years.

45. The use according to claim 40 and 41 , where the concentration of the formulation of clioquinol is 0.1 to 5 %.

46. The use according to claim 40 and 41, where the concentration of phanquinone is 0.1 to 5%.