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профилактика заболевания

Терапевтический эффект меланина (ENG №2)

2.Prohvlaxis
A second aspect of the present invention is that an active substance such as melanin can be used to prevent degenerative diseases of the nervous system which are caused by exposure of a mammal to toxic agents which cause such neurodegenerative diseases. Toxic agents which are known to cause neurodegenerative diseases includeN-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) andl-methyl-4-phenylpyridine (MPP+) and manganese dust for Parkinson's disease; quinolinic acid for
Huntington's chorea;ss-N-methylamino-L-alanine for amyotrophic lateral sclerosis, Parkinson's disease and
Alzheimer's disease; and aluminum has been implicated in
Alzheimer's disease. In addition to these agents, the toxic metabolite of MPTP,MPP+, has been field-tested as a herbicide under the name Cyperquat. The well-known herbicide Paraquat chemically resembles MPP+. Cyperquat and Paraquat are pyridine derivatives. Many analogs of
MPTP exist in the environment and could also be involved in idiopathic parkinsonism. One of the MPTP analogs, 4phenylpyridine, a constituent of peppermint and spearmint tea, was toxic to catecholamine neurons in vitro (Snyder et al., supra). Melanin can also be used to prevent the adverse effects caused by toxins which are absorbed, inhaled or ingested by a mammal. In addition to the toxins discussed above, other toxins include, but are not limited to, metals, metal-containing compounds, radioisotopes and radioactive compounds, including radioactively labelled therapeutics and diagnostics.

Metals include, but are not limited to, aluminum, lead and manganese. Melanin is especially useful as a chelating agent to lower or eliminate aluminum agents.

Melanin has been found to be able to bind MPTP as well as MPP+. Administration of melanin can thus effectively bind MPTP, MPP+ and other neurodegenerative disease-causing substances before the substances reach the tissue (especially brain tissue) they damage. The melanin can be administered by any means, but for present purposes it is preferred to administer it orally, by inhalation or suppositories. Suitable doses for this purpose are from about 0.5 to about 100 mg/kg, and preferably from about 1 to about 5 mg/kg of the active ingredient. This aspect of the invention is shown in Examples 1 and 2 below.

Alternatively, the administration of tyrosinase can increase the production of melanin in vivo, thereby causing the binding of neurodegenerative disease-causing substances before the substances reach the tissue (especially brain tissue) which they damage. The tyrosinase can be administered by any means, but for present purposes it is preferred to administer it orally, by inhalation or by suppositories. As in the case of treating melanin deficiency diseases, the amount of tyrosinase administered must be sufficient to catalyze the melanin producing reactions such that sufficient melanin is produced to alleviate the disease symptoms.

As is also the case with treatment of melanin deficiency diseases, another method by which the in vivo production of melanin may be enhanced us by the administration of the tyrosinase gene to the effected patient.

After administration, the tyrosinase gene transfects susceptible mammalian cells and tyrosinase is produced.

The tyrosinase, in turn, catalyzes the production of melanin from naturally occurring melanin precursors as explained above.

The most common method by which tyrosinase gene is introduced into the mammalian system is by its incorporation into a defective herpes simplex virus 1 (HSV-1) vector. Particularly, the defective HSV-1 vector, pHSVlac, developed by Geller et al., Science 241, 1667 (1988) is especially useful for this purpose as explained above. The amount of tyrosinase gene administered must be sufficient to transfect susceptible mammalian cells so that tyrosinase is produced therefrom.

Tyrosinase gene may also be introduced into the mammalian system using DNA contructs for retrovirus packaging cell lines as described in United States
Patent No. 4,861,719 incorporated herein by reference.

Briefly, a cell line containing a DNA construct such as pPAM3 (ATCC No. 40234) is used to transmit high titers of retroviral vectors which carry tyrosinase gene. An example of such a cell line is PA317 (ATCC No.

CLR 9078).

Useful DNA constructs such as pPAM3 are constructed by deleting all of the cis-acting elements except for the tRNA binding site from the genome of a replicationcompetent retrovirus. The particular cis-acting elements which are deleted are: the packaging signal; the site for initiation of second strand DNA synthesis; the site required for translation of reverse transcriptase during first strand DNA synthesis; and the provirus integration signal.

The retrovirus vectors produced by PA317 cells are capable of infecting a variety of hosts including mouse, rat, cat, dog and human cells. Hemopoietic progenitor cells from human bone marrow and mouse embryo cells have been infected by retroviral vectors secreted from PA317 cells. -The vector titer from the PA317 cells is very high (up to 107 colony forming units/ml), and therefore, these cells are useful in mammalian gene therapy.

A further method of prophylaxis against the melanin deficiency diseases is to increase the concentration of naturally occurring melanin at the target cells in the central nervous system by the administration of melaninconcentrating hormone (MCH). Commonly, a combination of
MCH and tyrosinase or tyrosinase gene is administered as an effective combination for the treatment of melanin deficiency diseases. The tyrosinase or tyrosinase gene causes an increased melanin production, and the MCH induces the aggregation of melanin in the target cells and tissues.

3. NeuronRecoverv
Another aspect of the present invention is that an active substance such as melanin can be used to assist in the recovery of injured neurons. The neurons could be injured as a result of direct injury or disease. For example, it is known that MPTP destroys a substantial number of dopaminergic nerve terminals in the striatum of young mature mice, and that after five months there is a substantial, though incomplete, recovery of striated dopamine nerve terminal markers. Ricourte,
G.A. et al., Brain Res. 376, 117 (1986). It is also known that melanin is present in all neurons in the form of dark, irregularly shaped granules called Nissl bodies. Nissl bodies are scattered throughout the cytoplasm and occur in dendrites of the larger neurons.

They appear to be absent in the axon and axon-hillock.

In pathological conditions, there is a partial or complete reduction in the amount of Nissl bodies. For example, it is known that Nissl bodies disappear with nerve injury but reappear upon nerve recovery. Within certain regions of the brain, there are areas of neurons that have high concentrations of Nissl bodies, thereby rendering localized regions black. Examples of these areas include the substantia nigra and locus ceruleus.

It is known that Nissl bodies disappear with nerve injury but reappear upon nerve recovery. It has been found that the administration of melanin or a melanin derivative is able to aid in the recovery of neurons by accelerating the time frame for neuron recovery. This aspect of the invention is shown in Example 3 below.

It has also been found that the administration of tyrosinase, tyrosinase gene, MCH or combinations thereof aid in the recovery of neurons. The tyrosinase increases the production of melanin in vivo, and the melanin accelerates the time frame for neuron recovery. The administration of tyrosinase gene and/or MCH aids in neuron recovery by promoting the same reactions described above for treatment and prophylaxis of melanin deficiency diseases.

Melanin can also aid neuron recovery by acting as a carrier for nerve growth factor (NGF). NGF was originally derived from mouse sarcomas, moccasin snake venom and mouse salivary glands as a non-dialyzable, heat-labile protein molecule with a molecular weight of about 20,000 or about 44,000 (Levi-Montalcini, Science 237, 1154 (1987)). NGF is essential in the early differentiation stages of its target cells as evidenced by failure of chick embryo nerve cells to survive in vitro in the absence of the daily addition of nanogram quantities of NGF to the culture medium (Levi-Montalcini et al., Dev. Biol. 7, 653 (1963)).

NGF is a dimer of two identical subunits held together by noncovalent bonds. Although it is as yet unknown whether each NGF subunit is biologically active, it has been demonstrated that a covalently cross-linked form of the dimer maintains full activity (Stach et al.,
J. Biol. Chem. 249, 6668 (1974)).

NGF is taken up by nerves endings of the sympathetic or sensory fibers, and is retrogradely transported to the cell perikarya. Specifically, NGF is a trophic messenger conveyed through nerve fibers from peripheral cells to the invigorating neurons(Stöckel et al., Brain
Res. 76, 413 (1974); Hamburger et al., J. Neurosci. 1, 60 (1981)). NGF has the ability to direct growing or regenerating axons of sensory and sympathetic fibers along its concentration gradient (neurotropism) (Gundersen et al., Science 206, 1079 (1979)).

Both small and large neuronal populations located in different brain areas have been shown to exhibit all of the properties and responses typical of sensory and sympathetic cells such as: 1) presence of specific receptors (Szutovitz et al., J. Biol. Chem. 251, 1516 (1976)); 2) retrograde transport of NGF (Seiler et al.,
Brain Res. 30, 23 (1984)); 3) increased neurotransmitter synthesis, particularly acetylcholine (Gnahn et al.,
Dev. Brain Res. 9, 45 (1983), Hefti et al., Brain Res.

293, 305 (1984), Mobley et al., Science 229, 284 (1985)); and 4) trophic response manifested as protection against exogenous NGF administration to selective noxious treatments or surgical transactions otherwise leading to cell death (Williams et al., Proc. Natl.

Acad. Sci U.S.A. 83, 9231 (1986), Kromer, Science 235, 214 (1987)).

NGF target cells include neural crest derivatives such as sympathoadrenal cells and sensory neurons.

Exemplary sympathoadrenal cells include long sympathetic neurons, short sympathetic neurons, paraganglia cells, small intensely fluorescent (SIF) cells, and normal and neoplastic chromaffin cells. Additional NGF target cells include those of the central nervous system such as cholinergic neurons and adrenergic, indoleaminergic and peptidergic neurons. Some cells of nonneuronal origin such as mast cells are also targets for NGF.


There has been found a high degree of homology in the cloned NGF gene of mice (Scott et al., Nature 302, 538 (1983)), humans (Ulbrich et al., Nature 303, 821 (1983)), bovine (Meier et al., EMBO J. 5, 1489 (1986) and chickens (Ebendal et al., EMBO J. 5, 1483 (1986)).

The human NGF gene is located on the proximal short arm of chromosome 1, and codes for a large polypeptide of 307 amino acids (Francke et al., Science 222, 1248 (1983)).

One method for producing NGF is taught by Rosenberg et al., Science242, 1575 (1988), and involves a retroviral vector constructed from Maloney murine leukemia virus (Wolf et al., Mol. Biol. Med. 5, 43 (1988), Varmus et al., RNA Tumor Viruses, R. Weiss, N. Teich, H.

Varmus, J. Coffin, Eds. (Cold Spring Harbor Press, Cold
Spring Harbor, NY, 1982) pp. 233-249). The vector contains the 777- bp HgA I-Pst I fragment of mouse NGF cDNA under control of the viral 5' long terminal repeat. The
NGF cDNA fragment was prepared in accordance with the techniques taught by Scott et al., Nature 302, 538 (1983) and Ulbrich et al., Nature 303, 821 (1983). The vector also contains a dominant selectable marker encoding the neomycin-resistance function of transposon Tn5 under control of an internal Rous sarcoma virus promoter.

Transmissible retrovirus is produced by transfecting vector DNA into PA137 amphotrophic producer cells (Miller et al., Mol. Cell Biol. 6, 2895 (1986)) by the calcium phosphate co-precipitation method (Graham et al., Virology 52, 456 (1973)) and by using medium from these cells to infectE 2 ecotropic producer cells (Mann et al., Cell 33, 153 (1983)) in the presence of
Polybrene (Sigma; 4pg/ml). Virus from theV 2 clone producing the highest titer is used to infect an established rat fibroblast cell line 208F (Quade,Virology 98, 461 (1979)) as described by Miyanohara et al., Proc.

Natl. Acad. Sci. U.S.A. 85, 6538 (1988).

Individual neomycin-resistant colonies, selected in medium containing the neomycin analog G418, are expanded and tested for NGF production and secretion by a twosite enzyme immunoassay, with commercially available reagents according to the manufacturer's protocols (Boehringer Mannheim). The NGF secreted by the clones is biologically active as determined by its ability to induce neurite outgrowth from PC12 rat pheochromocytoma cells (Greene et al., Proc. Natl. Acad. Sci. U.S.A. 73, 2424 (1976); Greene, Brain Res. 133, 350 (1977)).

Melanin readily binds nerve growth factor, and therefore can transport the nerve growth factor across the blood-brain barrier. The brain is an organ rich in nervous tissue, and therefore an organ where nerve growth factor is particularly useful. By transporting nerve growth factor across the blood-brain barrier and/or increasing the permeability of the blood-brain barrier, melanin is useful in allowing nerve growth factor to reach tissue where it is particularly useful and would not otherwise be capable of easily reaching.

4. Melanin As A Carrier For Other
Therapeutic Aaents
An additional aspect of melanin therapy is the use of melanin as a carrier for other therapeutic agents.

Two therapeutic agents with which melanin is especially useful as a carrier are boron and nerve growth factor.

However, melanin is also useful as a carrier with any other agent which binds to melanin.

Boron is particularly useful in the treatment of cancerous tumors with neutron capture therapy (thermal neutron activated radiotherapy). As explained above,boron-l0 isotopes which have been irradiated by slow neutrons, release significant amounts of radiation in their immediate vicinity. By targeting boron to cancerous and/or tumorous areas, the radiation emitted from theboron-l0 isotopes is selectively lethal to the cancerous cells in the immediate vicinity of the boron.

Melanin has been found to be a very useful carrier for boron. The boron is strongly bound by melanin, and the boron/melanin complex thus created can be attached to an antibody so that the complex is delivered to a specific cancerous site. As noted in United States
Patent No. 4,824,659, an antibody conjugate must have a sufficiently large number of boron atoms in order to function as an efficient therapeutic agent. The strong binding of boron to melanin allows the described antibody conjugates to have sufficient boron atoms to be efficient therapeutic agents.

Melanin is particularly useful as a boron carrier when the cancerous cells to be treated are located in the brain. The binding of boron to melanin permits relatively easy transport of boron across the bloodbrain barrier. Melanin facilitates the boron transport cross the blood brain barrier in two ways: 1) melanin itself easily crosses the blood-brain barrier; and 2) melanin analog causes an increase in the permeability of the blood-brain barrier.

The antibodies conjugated to the melanin/boron complex may be any type of immunoglobulin molecule having a region which specifically binds to an antigen of therapeutic interest. These immunoglobulin molecules include whole immunoglobulins such as IgA, IgD, IgE,
IgG, IgM and the like, or immunoglobulin fragments such as Fab, Fab', F(ab)1, F(ab')2 and the like. Useful antibodies also include hybrid antibodies or hybrid antibody fragments.

In addition to being a useful carrier for boron, melanin is also a useful carrier for nerve growth factor. Like boron, nerve growth factor binds strongly to melanin.

As discussed in detail above, nerve growth factor is a non-dialyzable, heat-labile protein molecule with a molecular weight of about 20,000 or about 44,000 (Levi-Montalcini, Science 237, 1154 (1987)). The NGF molecule is a dimer of two identical subunits held together by noncovalent bonds. NGF target cells include neural crest derivatives which include sympathoadrenal cells and sensory neurons. NGF has the ability to direct growing or regenerating axons of sensory and sympathetic fibers along its concentration gradient (Gundersen et al., Science 206, 1079 (1979)).

When bound to melanin, NGF can relatively easily cross the blood-brain barrier, because of melanin's ability to cross the blood-brain barrier and melanin analog's ability to increase the permeability of the blood-brain barrier. The brain is an area of particular usefulness for NGF due to the large amount of nerve tissue present in this organ. The use of melanin as a carrier for NGF aids the transport of this therapeutic agent to an area where it is particularly useful, and would not easily reach otherwise.

E. PharmaceuticalComnositions andDeliverv
Pharmaceutical compositions containing the active substance of the present invention (i.e. melanin, melanin derivatives, tyrosinase, tyrosinase gene, MCH and combinations thereof) in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, topical, aerosol, suppository, parenteral or spinal injection.In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugarcoated or enteric-coated by standard techniques.For parenterals, the carrier will usually comprise sterile water, though other ingredients, for example, to aid solubility or for preservative purposes, may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, pH adjusting agents, isotonicity adjusting agents and the like may be employed. For topical administration, the carrier may take a wide variety of forms depending on the form of preparation, such as creams, dressings, gels, lotions, ointments or liquids.

Aerosols are prepared by dissolving or suspending the active ingredient in a propellant such as ethyl alcohol or in propellant and solvent phase. Suppositories are prepared by mixing the active ingredient with a lipid vehicle such as theobroma oil, cacao butter, glycerin, gelatin, or polyoxyethylene glycols. The pharmaceutical compositions for topical or aerosol form will generally contain from about 1% by weight to about 40% by weight, depending on the particular form employed.

Melanin, whether used as a therapeutic agent or as the carrier for another therapeutic agent is soluble in aqueous solution. Particularly, melanin is soluble in aqueous solutions of pH 5 or higher, and preferably, aqueous solutions of pH 8 or higher.

There are unique considerations in the treatment of central nervous system dysfunction. Unlike other tissues, brain tissue is not redundant. It is highly differentiated, compartmentalized, and cannot be replaced. Thus, neuropharmaceutics must be found nontoxic to normal tissue. The real problem, however, has been to find the most efficacious route of circumventing the blood-brain barrier. Although melanin has been found to be capable of crossing the blood-brain barrier, other devices or methods for transport across the bloodbrain barrier can even further enhance melanin's ability to cross the blood-brain barrier.

One way to bypass the barrier is by intracerebrospinal fluid administration by lumbar puncture or by the intraventricular route. Catheterization using the Ommaya reservoir is used, but logistics dictate that to be a last-resort method.

Because the barrier is selective, some drugs can be administered orally. Certain lipophilic chemicals or agents that mimic the neural amino acids can bypass the barrier by mere diffusion or by transport via the energy dependent membrane-bound carrier, respectively. Melanin derivatives can be prepared to add lipid and/or carbohydrate groups to the melanin to make it move lipophilic and hence enhance its ability to cross the blood-brain barrier.

An example of a drug administered intrathecally is methotrexate, an antineoplastic agent, in the treatment of meningeal leukemia. The sodium salt of methotrexate is administered in solution in doses of 12 mg per square meter of body surface or in an empirical dose of 15 mg.

The drug is given every two to five days until the cell count of the cerebrospinal fluid returns to normal.

L-dopa can be used to compensate for the depletion of dopamine that occurs in parkinsonism because it also passes freely through the blood-brain barrier.

Transient reversible modification of the bloodbrain barrier is accomplished in either of two ways -osmotic opening or metrazol opening. The first method is based on increasing capillary permeability by osmotically-induced shrinkage of the endothelial cells which caused widening of the intercellular tight junctions. The osmotic load is generally a hyperosmotic water-soluble agent such as mannitol or arabinose.

Briefly, under general anesthesia, a transfemoral catheter is introduced into the internal carotid or vertebral artery and 150-300 ml infusion of 25% mannitol is administered at 6-10 mg/sec for 30 seconds. The intravenous infusion of melanin or tyrosinase is begun approximately five to seven minutes before the mannitol infusion and is continued for 15 minutes. The transfemoral catheter is removed and the patient observed for 24-48 hours.

Alternatively, the active agent (melanin or tyrosinase) may be linked to the osmotic agent (mannitol, arabinose, glucose or other sugar moiety), and a single infusion may be used. Conventional techniques may be used to link the active agent and the osmotic agent. The linked agent itself will then cause the osmotically-induced shrinkage of the endothelial cells in order to widen the tight intercellular junctions.

The linked agent may be designed such that the active agent (melanin or tyrosinase) is cleaved from the linked agent after the blood brain barrier has been crossed.

In the second method, capillary permeability is increased by eliciting seizure activity using a central nervous stimulant such as pentylenetetrazol. The technique is similar to that of osmotic opening with the replacement of mannitol infusion by parental delivery of the stimulant.

A drug also can be disguised so that is able to cross the blood-brain barrier. One method of accomplishing the disguise is to prepare a redox system as described by Bodor, sura. In this system a derivative of the drug is prepared which is capable of crossing the blood-brain carrier and which is converted in tissue to the drug or to a substance having the activity of the drug. In the case of melanin or tyrosinase, a derivative is prepared by attaching melanin or tyrosinase to a dihydrotrigonelline carrier such as described in
Bodor, sura.

A similar method of disguising a drug so that it will cross the blood brain barrier is to create a redox system in which the drug is coupled to a pyridinium carrier as described by Bodor, N. et al., Pharmac. Ther.

19, 337 (1986). Commonly used pyridinium carriers include substituted nicotinic acid and nicotinamide.

After coupling, the drug-carrier complex is reduced, yielding a dihydropyridine. The reduced complex is then administered systemically. The reduced complex will cross the blood brain barrier due to its enhanced membrane permeability, and it will also be distributed elsewhere in the body.

At all locations in the body (in the brain as well as elsewhere in the body) the reduced drug-carrier complex will be subject to oxidation. However, the rate of oxidation can be controlled to some extent by selected substitution of the pyridine ring. Following oxidation, the charged drug-carrier complex is rapidly eliminated from the peripheral blood system by renal and/or biliary processes. However, the compound will be retained in the brain due to its size and charge. The cleavage of the drug from the oxidized carrier will also occur in both the brain and the periphery, and if this cleavage occurs at a more rapid rate than the efflux of complex from the brain, a sustained release of the drug in the brain will be achieved.In the case of melanin or tyrosinase, a drug-carrier complex is prepared by coupling the melanin or tyrosinase to nicotinyl chloride as described by Bodor, N. et al., supra.

A further alternative method for delivering melanin or tyrosinase to target areas of the brain is to transport the tyrosinase gene into the brain by means of a defective Herpes simplex virus-1(HSV-1) vector using a method described by Geller, A.I. et al., Science 241, 1667 (1988). Particularly, the defective HSV-1 vector described by Geller, A.I. et al., sura, is pHSVlac, which contains the Escherichia coli lacZ gene under the control of the HSV-1 immediate early 4/5 promoter.

In order to use this HSV-1 vector in the present invention, the tyrosinase gene (as isolated and identified by Huber, M. et al., Biochemistry 24, 6038 (1985)) is inserted into the defective HSV-1 vector in place of the E. coli lacZ gene using conventional techniques. This new vector containing the tyrosinase gene can then enter the brain where the tyrosinase gene will be replicated and transcribed to produce tyrosinase which in turn will catalyze melanin production in the immediate vicinity of the target cells.

The tyrosinase gene may also be introduced into the mammalian system using DNA constructs for retrovirus packaging cell lines as described in United States
Patent No. 4,861,719, the disclosure of which is incorporated herein by reference. In this procedure, a cell line containing a DNA construct such as pPAM3 (ATCC No.

40234) is used to transmit high titers of retroviral vectors which carry tyrosinase gene. An example of a useful cell line for this purpose is PA317 (ATCC No. CLR 9078).

Useful DNA constructs such as pPAM3 are constructed by deleting all of the cis-acting elements except for the tRNA binding site from the genome of a replication competent retrovirus. The particular cis-acting elements which are deleted are: the packaging signal; the site for initiation of second strand DNA synthesis; the site required for translation of reverse transcriptase during first strand DNA synthesis; and the provirus integration signal.

The retrovirus vectors produced byP A3 17 cells are capable of infecting a variety of hosts including mouse, rat, cat, dog and human cells. Hemopoietic progenitor cells from human bone marrow and mouse embryo cells have been infected by retroviral vectors secreted from PA317 cells. The vector titer from the PA317 cells is very high (up to 107 colony forming units/ml), and therefore, these cells are useful in mammalian gene therapy.

As with most neurologic drugs, there is no established dosage of melanin or tyrosinase. The regimen is determined empirically for each patient. The optimal dose is that which produces maximal improvement with tolerated side effects. For example, an initial dose of 0.5-1.0 gm/day with the total daily dosage increasing in increments not more than 0.75 gm every three to seven days as tolerated, is a recommended regimen. Although the optimal therapeutic dosage should not exceed 8 gm per day, patients may be given more as required. It is worth emphasizing that in both of the above cases, optimal dosage is determined empirically and balances the benefits and adverse side effects.

F. Examples
The invention is further illustrated by the following non-limiting examples. Example 1 demonstrates melanin's capability of chelating toxins such as MPTP.

Example 2 shows that toxin-induced Parkinson's disease can be prevented if the toxin cannot bind to melanin in the brain. Since administered melanin can chelate toxins, it prevents the toxins from binding to melanin in the brain and causing neurodegenerative diseases.

Example 7 demonstrates that melanin can be used to aid neuron recovery. Example 8 shows the use of melanin for the treatment of Parkinson's disease.

EXAMPLES 1-4
Extractions of Melanin
Melanin produced in the following examples was extracted from the growth medium by the following procedure.

Cultures were filtered through glasswool to remove mycelium. Alternatively, particulate matter and cells were removed from the growth medium by centrifugation at 5,000 X gravity. The pH of the melanin containing medium was then reduced to about 3.0 withHCl. The precipitated melanin was removed by centrifugation at 6,800 X gravity. The precipitate was then removed and resolubilized at pH 8.0. The resolubilized melanin was washed by doubling the value of the liquid with sterile distilled H2O. The process of precipitation, removal, resolubilization and washing is repeated 4 times in order to substantially remove any non-precipitable impurities. The product may be dried to completion in an oven at200 C for 48 hours, if desired.

EXAMPLE 1
Conventional Melanin Production
This Example sets forth a conventional method for the production of melanin as taught by Hopwood, D.A. et al., "Genetic Manipulation of Streptomyces: A Laboratory
Manual" The John Innes Foundation (1985).

Melanin production byStretomvces lividans TK64 (pIJ702).

Preparation of Growth Medium
MMT MEDIUM was prepared from the following ingredients as described below.

MM MEDIUM:
L-asparagine 0.5 g
K2HPO4 0.5 g
MgSO4.7H2O 0.2 g
FeSO4.7H2O 0.01 g H2O- 1000 ml
The ingredients were dissolved in water, adjusted to pH 7.0-7.2 with NaOH, 100 ml placed into 500 ml flasks, and autoclaved for 20 minutes.

The following sterile stocks were prepared:
*Difco Casaminoacids (30%)(50x Stock)
*Glucose (50%) (50x Stock)
*CuSO4.5 H2O (0.50%) (1000x Stock) *Tyrosinase Inducer:
L-methionine (1%)
L-tyrosine (3%) (33.3x Stock)
L-leucine (5%) *Tiger Milk:
L-arginine (0.75%)
L-cystine (0.75%)
L-histidine(1.0%) (133.3x Stock)
DL-homoserine (0.75%)
L-phenylalanine (0.75%) Does not dissolve
L-proline (0.75%) completely forms a
adenine (0.15%) white, milk-like
uracil (0.15%) solution
nicotinamide(0.018)
thiamine (0.01%) * All of these stocks were autoclaved prior to making the medium.

The following ingredients were combined to prepare MMT medium:
100 ml MM MEDIUM
2 ml Casaminoacids
2 ml Glucose
750 ul Tiger Milk
For tyrosine and melanin production, the following ingredients were also included:
100 ulCuSO.5 H2O
3 ml Tyrosinase Inducer
Inoculation and Growth of TK64(pIJ702)
A small amount of the bacteria were scraped from the top of the plate and transferred into 10 ml of sterile water which was mixed and pipetted into six-500 ml flasks containing 100 ml of MMT. Cultures were grown at30*C, and 120 RPM for 3 days.

Results
Melanin was purified as described above. The yield of melanin was about 100 mg/l, dry weight.

EXAMPLE 2
Production of Melanin In A Bioreactor
Preparation of Growth Medium
The growth medium was prepared as in Example 4.

The medium contains 1.5 grams per liter of tyrosine.

This medium contains no glucose or other carbon source except amino acids.

Inoculation and Growth of TK64(pIJ702}
Spore stock of S. lividans TK64 (pIJ702) was diluted 1:10 in water. A starter culture was produced by adding 501 of dilute spore stock to 250 ml of culture medium in a 1 liter flask. The starter culture was incubated at 30'C with shaking until it reached midlog phase.

Starter culture was then transferred to a 30 liter fermentor containing 20 liters of growth medium. Incubation was at30'C with constant mixing at 225 RPM until the optical density reached a constant. Aeration during fermentation was by constant air flow at 1 liter of air per minute for 40 hours, and by 2.5 liters per minute for 40-60 hours, then by 3.0 liters per minute for the remaining 60-120 hours.

Results
Melanin was purified as described above. The yield of melanin was about 1.7 grams per liter dry weight.

EXAMPLE 3
Production of Melanin In A Bioreactor
Preparation of Growth Medium
The growth medium was prepared as in Example 4.

The medium contains 1.5 grams per liter of tyrosine.

This medium contains no glucose or other carbon source except amino acids.

Inoculation and Growth of TK64(pIJ702)
Spore stock of S. lividans TK64 (pIJ702) was diluted 1:10 in water. A starter culture was produced by adding 501 of dilute spore stock to 250 ml of culture medium in a 1 liter flask. The starter culture was incubated at30'C with shaking until it reached midlog phase. Starter culture was then transferred to a 42 liter fermentor containing 35 liters of growth medium.

Incubation was at30'C with constant mixing at 225 RPM until the optical density reached a constant. Aeration was by constant airflow at 1.5 liters of air per minute for 36 hours, 4.0 liters per minutes for 36-48 hours, and 5.0 liters per minute for the final 48-120 hours.

Antifoam was added daily after 48 hours.

Results
Melanin was purified as described above. The yield of melanin was about 2.0 grams per liter.

Melanin was also produced in E. coli K38 (pGP1-2) containing different tyrosinase expression vectors such as pBS620.3 orpBS636 as described in U.S. Serial No.

filed filed November 2, 1990 entitled "Melanin
Production by Transformed Microorganisms" (Attorney
Docket No. 18604.92847) incorporated herein by reference. It is preferred to produce melanin in E.

coli.

EXAMPLE 4
Production of Melanin Analoas
A. Preparation of the Growth Medium
MMT MEDIUM was prepared from the following ingredients as described below.

MM MEDIUM:
L-asparagine 0.5 g
K2HPO4 0.5 g
MgSO4.7H20 0.005 g
FeSO4.7H2O 0.01 g
H20 1000 ml
These ingredients were dissolved in water, adjusted to pH 7.0-7.2 with NaOH, 100 ml placed into 500 ml flasks, and autoclaved for 20 minutes.

The following sterile stocks were prepared:
*Casein-Peptone Hydrolyzate (30%) (50x Stock)
*Glucose (50%) (50x Stock)
*CuSO4g5 HzO (0.50%) (1000x Stock)
*Tyrosinase Inducer:
L-methionine (1%)
L-tyrosine (3%) (33.3x Stock)
L-leucine (5%)
*Tiger Milk:
L-arginine (0.75%)
L-cystine (0.75%)
L-histidine (1.0%) (133.3x Stock)
DL-homoserine (0.75%)
L-phenylalanine (0.75%) Does not
L-proline (0.75%) dissolve
adenine (0.15%) completely forms
uracil (0.15%) a white, milk
nicotinamide (0.01%) like solution
thiamine(0.01%)
*All of these stocks were autoclaved prior to making the medium.

The following ingredients were combined to prepare
MMT medium:
100 ml MM MEDIUM
2 ml Casein-Peptone Hydrolyzate
2 ml Glucose
750 ul Tiger Milk 100 ul CuSO45 H2O
3 ml Tyrosinase Inducer
B. Inoculation and Growth of the Streptomyces
lividens plasmidpIJ702
TheStrentomvces lividens containing plasmid pIJ702 was inoculated into the MMT growth medium at a rate of 2 x 105 spores/ml. TheStretomvces lividens was then allowed to grow for 24 hours at30'C and pH 6.8 with 85% dissolved oxygen provided to the media.

C. Additions to the Growth Medium
After theStretomvces lividens had been growing for 24 hours, tyrosine and metal ions were added to the growth medium. The tyrosine was added to a final concentration of 1.6 grams/liter,CuSO4v5H2O was added to a final concentration of 0.2 grams/liter and FeCl3 was added to a final concentration of 4 mM.

D. Purification of A Melanin Analog
After 72 hours of fermentation following the addition of the tyrosine and metal ions, the melanin analog was extracted from the growth medium by the following procedure
Cultures were filtered through glasswool to remove mycelium. Alternatively, particulate matter and cells were removed from the growth medium by centrifugation at 5,000 x gravity. The pH of the melanin containing medium was then reduced to about 3.0 withHC1. The precipitated melanin was removed by centrifugation at 6,800 x gravity. The precipitate was then removed and resolubilized at pH 8.0. The resolubilized melanin was washed by doubling the amount of the liquid with sterile distilled H2O. The process of precipitation, removal, resolubilization and washing was repeated 4 times in order to substantially remove any non-precipitable impurities.The product may be dried to completion in an oven at200 C for 48 hours, if desired.