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  Pharmaceutical Patents  

 

Title:  Therapy of diseases of the eye, the inner ear and the central nervous system
United States Patent: 
7,488,749
Issued: 
February 10, 2009

Inventors:
 Schraermeyer; Ulrich (Tubingen, DE)
Assignee:
  Cevec Pharmaceuticals GmbH (Cologne, DE)
Appl. No.: 
10/505,030
Filed:
 February 12, 2003
PCT Filed:
 February 12, 2003
PCT No.:
 PCT/DE03/00415
371(c)(1),(2),(4) Date:
 February 25, 2005
PCT Pub. No.:
 WO03/070269
PCT Pub. Date:
 August 28, 2003


 

Pharm Bus Intell & Healthcare Studies


Abstract

The present invention relates to 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and/or 5-S-cysteinyldopa (CD) as medicaments, as well as their use, and the use of tyrosinase for the preparation of a medicament for prophylaxis or therapy of diseases induced by oxidative stress. Furthermore, the present invention relates to the use of gene therapy vectors comprising a tyrosinase gene. Further, the present invention relates to cells modified by a tyrosinase gene.

Description of the Invention

This application claims priority to PCT/DE 03/00415, filed on Feb. 12, 2003, the entire contents of which are hereby incorporated by reference.

The present invention relates to 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and/or 5-S-cysteinyldopa (CD) as medicaments, as well as their use, and the use of tyrosinase for the preparation of a medicament for prophylaxis or therapy of diseases induced by oxidative stress. Furthermore, the present invention relates to the use of gene therapy vectors comprising a tyrosinase gene. Further, the present invention relates to cells modified by a tyrosinase gene.

Tyrosinase is the most important enzyme in the synthesis of melanin, a pigment present in cells of the skin, the eye and the central nervous system.

Tyrosinase is an integral membranaceous glycoprotein consisting of 529 amino acids. The enzyme catalyses the hydroxylation of tyrosine to dihydroxyphenylalaline (DOPA) and subsequently its transformation into dopaquinone. In addition, melanin is produced by different intermediate steps (Stage-III-melanosome) which may proceed mostly spontaneously. 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and 5-S-cysteinyldopa (CD) belong to these intermediate steps.

According to today's standard of knowledge the formation of melanin in mammalian retinal pigment epithelium, including the human eye, takes place only prenatally.sup.2;5. In humans the tyrosinase is active in retinal pigment epithelium only until about 6 weeks prior to the end of pregnancy, and is then not produced any more.sup.4. Melanin and tyrosinase, respectively, can be found in the eye in embryonically distinct tissues: in the uvea consisting of iris and chorioidea, as well as in the pigment epithelium of the retina (RPE), iris (IPE) and ciliary body. The only pigmented tissue which is claimed that no more melanin synthesis takes places there postnatally, is the RPE. This is based on work by Miyamoto and Fitzpatrick (1957).sup.4 in which it could be shown that no tyrosinase activity is to be found in postnatal RPE. According to the present state or the art, the enzyme itself has not been detected on protein level in RPE cells of adult mammals or humans, and no continuative studies have been performed either.

Whereas melanin predominantly represents a physical light shield in the skin, its functions in the eye are more complex.sup.6. A physical light shield is merely exhibited by the melanin of the iris (function as an aperture). Anatomically seen from the outside, the melanin in the retinal pigment epithelium (RPE) is located behind the sensitive photoreceptors of the retina (NH) which are to be protected. Here the pigment can reduce the scattered radiation by absorption, or protect tissues, such as endothelial cells of the choroid. Melanin has also a chemical protection effect. As a scavenger, melanin reduces the formation of lipid peroxidate ions which are toxic to tissues and which are formed in the RPE by means of a high concentration of oxygen, incidence of light from the outside and a pronounced activity in phagocytosis. The high concentration of oxygen results from an especially good blood circulation of the chorioidea which lies beneath it. Phagocytosis of secreted external segments of photoreceptors of the retina is one of the major tasks of the RPE and causes additional oxidative stress.sup.1. During phagocytosis superoxide is released extracellularly and intracellularly and which secondarily induces the formation of hydroxyl radicals and hydrogen peroxide.sup.1.

Here, melanin is protective by reducing free radicals to molecular oxygen and hydrogen peroxide. Therefore, melanin has also protective effects in case of inflammation of the eye. Furthermore, melanin represent an important storage for zinc since it is able to bind heavy metals, such as zinc, medicaments and other cytotoxic substances and to store them. The RPE contains a high concentration of zinc which is for the most part bound to melanin.sup.7. Since zinc is a necessary co-factor for about 300 enzymes, such as the superoxide dismutase, the carbonic anhydrase, the retinal dehydrogenase, the collagenase and the catalase.sup.8, a loss of melanin, e.g. in the case of AMD, may be associated with a loss of function of these enzymes. The enzymes mentioned before excerpt important functions especially in the eye. The activity of catalase is reduced in the eye of elderly people and especially in the case of AMD patients.sup.9. Thus, the cells are i.e. less protected against oxidative damages. Zinc which is reduced locally in the RPE and in the melanocytes of the choroid by the loss of melanin as well as systemically in the serum in the case of AMD, can also affect certain gene expressions. Zinc is required for various, i.e. anti-oxidatively operating, enzymes as a co-factor. A deficiency and complete absence, respectively, of melanin leads e.g. to the following diseases of the eye: Albinism, Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome (CHS), age-related macula degeneration (AMD). In the CNS, the Parkinson's disease correlates with a loss of melanin containing dopaminergic neurons of the substantia nigra. The content of melanin of the inner ear correlates with the sensitivity against oxidative stress caused by noise.sup.10.

A causal therapy for Albinism, Hermansky-Pudlak syndrome and Chediak-Higashi syndrome is not known by now. In the case of albinos, the extreme photophobia is countered by the use of sun glasses.

A causal therapy of AMD is not known at present. Treatments with coagulation by argon laser can only be applied in the case of non-subfoveal CNV and they are just as the photodynamic therapy not able to prevent relapses.

The currently used standard therapies in the case of Parkinson's disease with L-DOPA show side effects and often lose their effectiveness after several years. Also dopamine agonists, MAO-inhibitors, amantadine and anticholinergics, show side effects or affect only single impacts of the disease.

The problem of the present invention is therefore to provide means for the prevention or therapy of diseases which are induced by oxidative stress in cells. A further problem is to provide means for the prevention or therapy of diseases of the eye, the inner ear and the central nervous system (CNS) which are correlated with a deficiency of the pigment melanin.

The problem is solved by the subject matter defined in the patent claims.

The herein used term "tyrosinase" relates to a protein or polypeptide having the amino acid sequence which is specific for an enzyme with the biological activity of the tyrosinase as previously described. One example for a tyrosinase is the human melanogenic tyrosinase (EC 1.14.18.1). This tyrosinase is an integral membranaceous glycoprotein consisting of 529 amino acids (GenBank no. M27160). The tyrosinase belongs together with the human tyrosinase-related proteins I+II (TRP I+II) (GenBank no. for TRP I is AL138753 and for TRP II D17547), the Lysosome Associated Membrane Protein (Lamp; GenBank no. P11279) and gp100 (pmel 17; GenBank no. P40967) to one protein family. Therefore, the term tyrosinase includes proteins belonging to that protein family as well as potential modifications of the tyrosinase such as posttranslational modifications.

Further, the term tyrosinase comprises a nucleic acid having the sequence encoding one of the previously illustrated polypeptides. Thereby, not only the hitherto known sequences but also the sequences of derivatives, i.e. substitutions, additions, deletions, insertions, inversions, and also the sequences exhibiting fragments and modifications are encompassed by said term. The nucleic acid comprises not only the respective cDNA but may furthermore contain the genomic locus including the introns, regulational elements, etc. A nucleic acid is comprised by the term "tyrosinase" if the protein or polypeptide encoded by it exhibits the biological activity of the tyrosinase.

The herein used term "vector" or "gene transfer vector" relates to natural occurring or artificially generated constructs and organisms for the uptake, propagation, expression or transfer of nucleic acids in cells. Examples for gene transfer vectors are viruses, such as adenoviruses, adeno-associated viruses, lenti viruses, retro viruses, pox viruses, alpha viruses, baculoviruses, rabies viruses or herpes viruses. The gene transfer vectors are capable of autonomous replication inside a cell or of integration into the genome of the cells. The gene transfer vector is constructed such that it contains at least one desired therapeutic polynucleotide to be replicated and/or expressed. Furthermore, the gene transfer vectors may contain further polynucleotides such as a selection marker.

Oxidative stress is e.g. triggered by phagocytosis. Surprisingly, the inventor found that the tyrosinase and its metabolites such as 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and/or 5-S-cysteinyldopa (CD) may offer a protective function at time points of increased exposure to reactive oxygen species and may protect cells and tissues against oxidative stress.

Oxidative stress means the formation of reactive oxygen species in cells or tissues. These oxygen species are superoxide anion, hydroxyl radical and H.sub.2O.sub.2. These species are formed e.g. during the reactions of the respiratory chain in the mitochondria of each cell. The risk of a cellular damage increases if the tissue exhibits a high metabolic activity (e.g. brain) or is intensely exposed to light (eye, skin). The oxygen species (superoxide anion, H.sub.2O.sub.2) are eliminated by anti-oxidative enzymes (superoxide dismutase, catalase, glutathion peroxidase and others). However, if this elimination is not completely accomplished hydroxyl radical is formed which causes severe cell damages and cell death: Cellular ageing and ageing of whole organisms are ascribed to the damages caused by reactive oxygen species. A well known cellular damage triggered by oxidative stress is the lipid peroxidation. Oxidative stress causes neurological degenerative diseases (e.g. Parkinson's disease, age-related macula degeneration, Alzheimer's disease, degenerative diseases of the inner ear) and cardiovascular diseases (e.g. heart attack, atherosclerosis) but also the formation of tumours (e.g. melanoma).

Lipofuscin is a brownish-yellowish, electron-dense, auto-fluorescent pigment which is accumulated in the lysosomes of postmitotic cells such as nerve cells, myocardial cells, pigment epithelium cells of the eyes and the skin cells, especially if exposed to the oxidative stress. Oxidative stress causes lipid peroxidation. Said peroxidative lipids cannot be completely degraded and are in part transformed into lipofuscin. Lipofuscin has a considerable pathological impact especially in the retinal pigment epithelium and is presumable caused by the incomplete degradation of outer segments of visual cells. These are especially easily transformed into lipofuscin if reactive oxygen species are present, because of their high content of unsaturated fatty acids. By means of the content of lipofuscin the age of a cell can be concluded. The spots of lipofuscin in the skin of ageing humans are referred to as ageing pigment. The exact chemical composition of lipofuscin is not known. One component consists of ethanolamine.

Drusen are deposits of residual cellular material of unknown origin under the pigment layer between or within the Bruch's layer and the RPE. It is possible that they are formed as a consequence of a dysfunction of phagocytosis and degradation of retinal pigment cells; and it is assured that in the case of complex diseases of the retinal, pigment epithelium, choroid complex, including AMD in humans, drusen play a role. Funduscopically so called soft drusen having a diffuse boundary or hard drusen having sharp boarders are discriminated. If drusen are formed in the region of the macula, this is associated with a pronounced loss of sight.

Therefore, one aspect of the present invention is to provide 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and/or 5-S-cysteinyldopa (CD) as medicaments. A further aspect of the present invention is the use of tyrosinase, 5,6-dihydroxyindole (DHI), 5,6-dihydroxylindole-2-carboxylic acid (DHICA) and/or 5-S-cysteinyldopa (CD) for the preparation of a medicament for the treatment or prophylaxis of diseases induced by oxidative stress. In particular, the medicaments are used for the treatment of drusen or of an increased content of lipofuscin or for prophylaxis of the formation of drusen or of the formation of lipofuscin. Preferably, the diseases comprise diseases of the eye, in particular age-related macula degeneration (AMD), albinism, Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome (CHS), choroidal and retinal neovascularisation, diabetic retinopathy and retinopathy of the prematury, degenerative diseases of the inner ear (presbycusis) and diseases of the CNS, in particular Parkinson's disease.

However, the therapeutic may not only be administered in terms of a chemical substance (5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5-S-cysteinyldopa (CD)) or in form of a protein or a polypeptide (tyrosinase) but also in terms of a nucleic acid molecule encoding that polypeptide. The nucleic acid molecule may be introduced into the target cells by means of gene transfer vectors.

The expression of the tyrosinase gene in the gene transfer vectors is controlled by promoters. Viral or non-viral promoters which are constitutively, tissue specifically or controllably active may be used for the expression. For example the SV40 or cytomegalovirus promoter can be used for the constitutive expression of a gene. The use of the tissue specific promoter allows the tissue specific expression, e.g. in melanocytes, IPE or RPE cells. An example for such a tissue specific promoter is the transthyretin promoter that has a good activity in RPE and IPE cells. The gene expression may be controlled quantitatively and qualitatively by the use of a controllable expression system. One example for a controllable gene expression system is the tetracycline dependent gene expression system or the RU 486 system.

Both viral and non-viral vectors may be used for the gene transfer into the target cells. Examples for viral vectors are adenoviral vectors, AAV vectors, retroviral vectors and lentiviral vectors. Examples for non-viral vectors are liposomes and cationic lipids. Furthermore, any kind of vector or carrier can be used which is able to introduce the nucleic acid into the target cell.

The vectors may be injected e.g. subretinally or into the choroid for the treatment of diseases of the eye. For the treatment of diseases of the CNS, vectors may be injected e.g. in the substantia nigra, the putamen or striatum. Preferred target cells are stromal bone marrow cells, fibrocytes, melanocytes, retinal pigment epithelium cells, pigment epithelium cells of the iris and of the ciliary body, pericytes, endothelial cells, dopaminergic neurons, neurons, Schwann cells, astrocytes, microglia, Muller cells or neuronal gliocytes.

In a preferred embodiment of the present invention the formation of tyrosinase is induced by genetic modifications of RPE cells with adenoviral vectors thereby protecting the retinal tissue against light induced oxidative stress and, furthermore, reducing the formation of lipofuscin and a neovascularisation. For example, in the case of patients with AMD up to 60% of the cellular volume of RPE cells are filled with lipofuscin which interferes with the function of the cells. Moreover, the tyrosinase can be used for the elimination of reactive oxygen species such as H.sub.2O.sub.2, superoxide anion and hydroxyl radicals in lysosomes.

In a further preferred embodiment of the present invention the formation of tyrosinase is induces by genetic modifications of cells of the CNS with adenoviral vectors which leads to the increase of synthesis of dopamine and to the binding of iron and elimination of reactive oxygen species by the metabolites of the tyrosinase. Furthermore, thereby the lysosomal activity in neurons is activated.

The use of the tyrosinase is preferred in case of pigment deficiency diseases and hypopigmention of all kinds, such as vitiligo, allopecia, graying of hair, achromatosis, piebaldism, albinism, retinopathy of the prematury (ROP), retinitis pigmentosa, diabetic retinopathy, age-related macula degeneration, Parkinson's disease, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome.

Furthermore, one aspect of the present invention resides in the genetic modification of cells which are then administered to the patient. Thereby, the cells may be transduced by the gene transfer vectors previously described. Furthermore, the cells may be transfected by the nucleic acid molecule comprising the tyrosinase gene.

In the case of diseases of the eye, for example cells originating from the adrenal cortex, pigment epithelial cells of the iris, retinal pigment epithelium cells, stem cells from the bone marrow, sertoli cells from the gonads, glomus cells from the carotids, fibroblasts or astrocytes, neuronal stem cells or other cells of the body may protect against oxidative damages in the choroid (e.g. at endothelial cells) or the retina by genetic alteration with the tyrosinase gene. The herein mentioned cells may be obtained both from foetuses and autologously. At the same time, the lysosomal activity of the cells may be increased thereby reducing the formation of lipofuscin. The formation of nitrogen monoxide (NO) may be activated by the formation of DOPA which exhibits a positive effect on the flow of the blood in the choroid. These mechanisms prevent neovascularisation in the case of diseases of the eyes, such as age-related macula degeneration, diabetic retinopathy or retinopathy of the prematury.

For instance, foetal cells obtained from brains of human foetuses, e.g. from the ventral mid-brain, or dopaminergic neurons may be used. Furthermore, various different cell types, among them also non-neuronal cells e.g. cells from the adrenal cortex, pigment epithelium cells of the iris, stem cells from the bone marrow, sertoli cells from the gonads, glomus cells from the carotids, fibroblasts or astrocytes or neuronal stem cells e.g. from brains of adult vertebrates, may be used.

In the case of Parkinson's disease, transplanted cells which are modified with the tyrosinase gene, may develop neural protective effects by the antioxidative effect of their melanin granula.

In a further preferred embodiment of the invention, dopaminergic neurons or other neuronal cells of the CNS are to be transduced with the tyrosinase gene in the case of patients with Parkinson's disease in order to at the one hand activate the formation of melanin and/or to on the other hand enhance the production of dopamine. Preferably, the used cells are autologous cells.

A further aspect of the present invention is to provide 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and/or 5-S-cysteinyldopa (CD) as medicaments. The substances may not only be administered as chemical substances but are also produced during the administration of tyrosinase polypeptides or during the gene-therapeutic modification of cells with the tyrosinase gene. Due to the expression of tyrosinase the substances according to the invention are formed as intermediate steps of the melanin synthesis and may diffuse e.g. from the modified cells of the RPE into the choroid or retina, or from the modified CNS cells into the CNS and actively exhibit the described protective functions.

A further aspect of the present invention is to provide a combinatory composition consisting on the one hand of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), 5-S-cysteinyldopa (CD) and/or tyrosinase and on the other hand of zinc as medicaments. In particular, the invention relates to the provision of the combinatory composition for the treatment of age-related macula degeneration, diabetic retinopathy, Parkinson's disease, for the reduction of the neoplasm of blood vessels, of lipofuscin and of drusen and of degenerative diseases of the inner ear (presbycusis).

Thereby zinc may be administered simultaneously or after the other substances used in the combinatory composition. Preferably, the concentration of melanin is first increased in the cells affected by the disease, e.g. the choroid, the substantia nigra or in RPE by administration of the gene transfer vectors previously described. Subsequently, the concentration of zinc is increased in the artificially pigmented tissues by the substitution with zinc, thereby improving the anti-oxidative protective effects of the melanin and its metabolites.sup.11. The administration of zinc may occur orally. The blood vessels of the choroid and neuronal cells in the CNS cells may be protected by reducing the oxidative stress. The neoplasm of blood vessels, the formation of lipofuscin and drusen may be reduced by the combination therapy according to the present invention.
 

Claim 1 of 1 Claim

1. A pharmaceutical formulation comprising 5,6-dihydroxyindole (DHI) and zinc combined with a pharmaceutically acceptable buffer, diluent or excipient.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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