Here we present a US Government patent entitled “Melanin Therapy” or “Therapeutic uses of melanin.” The patent is a source for information about melanin, it’s function, specifically in mammals and the disease state resulting from effects of melanin loss.
| United States Patent | 5,703,051 |
| Berliner , et al. | December 30, 1997 |
Therapeutic uses of melanin
Abstract
The invention is directed to the treatment of degenerative diseases of tissues which have lost melanin and which share a common embryological basis as tissues of the nervous system by the administration of an active substance which causes an increased concentration of melanin in the tissue. Such active substances include melanin, melanin variants, melanin analogs, melanin derivatives, tyrosinase, tyrosinase gene, melanin-concentrating hormone and combinations thereof. Examples of such diseases include Parkinson’s disease, Alzheimer’s disease, retinitis pigmentosa, schizophrenia and dementia. The invention is also useful in assisting in the repair of neurons in a mammal having neuron damage by administering an effective amount of an active substance which causes an increased concentration of melanin in the neuron to aid in nerve repair. The invention is further useful in protecting a mammal from a toxin-induced disease or from the adverse effects of a toxin by administering an effective amount of the active substance described above.
| Inventors: | Berliner; David L (Atherton, CA), Erwin; Robert L. (Vacaville, CA), McGee; David R. (Vacaville, CA) |
|---|---|
| Assignee: | Biosource Technologies, Inc. (Vacaville, CA) |
| Family ID: | 24440247 |
| Appl. No.: | 07/988,739 |
| Filed: | December 10, 1992 |
Related U.S. Patent Documents
| Application Number | Filing Date | Patent Number | Issue Date | ||
|---|---|---|---|---|---|
| 609311 | Nov 5, 1990 | 5189024 | |||
| 331123 | Mar 31, 1989 | ||||
| 243736 | Sep 13, 1988 | ||||
| Current U.S. Class: | 514/8.4 ; 424/195.11; 424/94.4; 514/567; 514/63; 514/8.3 |
| Current CPC Class: | A61K 31/40 (20130101); A61K 31/69 (20130101); A61K 38/22 (20130101); A61K 47/48415 (20130101); A61K 47/4843 (20130101); A61K 48/00 (20130101); C12N 9/0059 (20130101); C12N 9/0071 (20130101); C12P 17/00 (20130101); C12Y 110/03001 (20130101); C12Y 114/18001 (20130101); A61K 38/22 (20130101); A61K 38/185 (20130101); A61K 33/00 (20130101); A61K 33/22 (20130101); A61K 38/185 (20130101); A61K 33/22 (20130101); A61K 31/40 (20130101); A61K 31/69 (20130101); A61K 38/00 (20130101); A61K 2300/00 (20130101); A61K 2300/00 (20130101); A61K 2300/00 (20130101); A61K 2300/00 (20130101); A61K 2300/00 (20130101) |
| Current International Class: | A61K 31/69 (20060101); A61K 38/22 (20060101); A61K 38/43 (20060101); A61K 47/48 (20060101); A61K 48/00 (20060101); A61K 31/40 (20060101); C12P 17/00 (20060101); C12N 9/02 (20060101); A61K 38/00 (20060101); A61K 031/40 (); A61K 047/06 (); A61K 047/46 () |
| Field of Search: | ;514/21,567 ;424/95,195.1 |
References Cited [Referenced By]
Foreign Patent Documents
| 363 792 | Oct 1989 | EP | |||
| WO 90/02551 | Mar 1990 | WO | |||
| WO 92/00373 | Jan 1992 | WO | |||
Other References
| Coderre, Jeffrey A. et al. “Selective Targeting of Boronophenylalanine to Melanoma in BALB/c Mice for Neutron Capture Therapy”. Cancer Research 47:6377-6383 (1987). . Chemical Abstracts 108:71398u (1988) Abstracting Cancer Res (1987) 47(23) 6377-83 reference. . della-Cioppa, et al., “Melanin Production in Escherichia Coli from a Cloned Tyrosinase Gene”, Biotechnology 8:634-638 (1990). . Coderre, et al., “Selective Delivery of Boron by the Melanin Precursor Analogue .sub.p Boronophenylalanine to Tumors Other Than Melanoma”, Cancer Research 50: 138-141 (1990). . Mishima, et al., “Treatment of Malignant Melanoma by Selective Thermal Neutron Capture Therapy Using Melanoma-Seeking Compound”, The Journal of Investigative Dermatology 92: 321S-325S (1989). . Derwent Publications Ltd., London GB AN 78-88551A(49) & JP-A-53 124 636 (Mori, K.) 31 Oct. 1978 Abstract. . Cotzias, et al., New England Journal of Medicine 276: 374-379 (1967). . Menn, et al., Mechanisms of Ageing & Development 21: 193-203 (1983). . Atschulte, et al., Clinical Pharm. & Therapeutics 19: 124-134 (1976). . Chemical Abstracts 112:91365t (1990).. |
Primary Examiner: Raymond; Richard L.
Attorney, Agent or Firm: Halluin; Albert P. Howrey & Simon
Parent Case Text
RELATED APPLICATIONS
This is a division of U.S. application Ser. No. 07/609,311, filed Nov. 5, 1990, now U.S. Pat. No. 5,210,076, which is a continuation-in-part of U.S. patent application Ser. No. 07/331,123, filed Mar. 31, 1989, now abandoned, which is a continuation-in-part of U.S. patent application Ser. No. 07/243,736, filed Sep. 13, 1988, now abandoned.
Claims
What is claimed is:
1. A method of administering a therapeutic agent to tissues within a mammal that share a common embryological basis as the nervous system using a carrier for the therapeutic agent wherein said carrier is a melanin wherein the therapeutic agent is used for a therapy with tissues in a mammal that share a common embryological basis with the nervous system.
2. The method of claim 1 wherein the therapeutic agent is administered to the brain of said mammal.
3. The method of claim 1 wherein the therapeutic agent is boron.
4. A method of administering nerve growth factor to tissues within a mammal that share a common embryological basis as the nervous system using a carrier for the nerve growth factor wherein said carrier is a melanin.
Description
BACKGROUND OF THE INVENTION
The present invention relates to the prophylaxis and treatment of degenerative diseases of the nervous system by the administration of an active substance which causes an increased concentration of melanin, melanin precursors, melanin derivatives, melanin variants and melanin analogs in the effected nervous system tissue. Such active substances include melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants, the enzyme tyrosinase, which catalyzes the reaction wherein naturally occurring melanin precursors are converted to melanin, tyrosinase gene, melanin-concentrating hormone and combinations thereof. Examples of such diseases include Parkinson’s disease, Alzheimer’s disease, retinitis pigmentosa and dementia. The present invention also relates to the treatment by the administration of melanin, melanin precursors or a melanin derivative of diseases of tissues which share a common embryological basis as tissues of the nervous system. The invention further relates to a method of preventing toxin-induced neurodegenerative diseases, toxin-induced diseases, or the adverse effects of toxins, and to a method for aiding the recovery of injured neurons, by the administration of the active substance which causes an increased concentration of melanin in the effected tissue.
A. Nervous System and Epidermis
The nervous system and epidermis have a common embryological basis and several common features.
1. Embryological Basis
During gastrulation the single layer of cells comprising the blastoderm migrate and fold to form the three germinal layers–ectoderm, endoderm and mesoderm. The germinal layers are the rudiments from which organs of the plant or animal develop. The ectoderm, for example, gives rise to the epidermis, central nervous system, i.e., the brain, spinal cord, spinal ganglia and nerves, various sensory organs and neural crest cell derivatives that includes cerebrospinal ganglia and melanocytes.
Although by definition the ectoderm is the outer-most of the germinal layers, it is not long during gastrulation that by migration and invagination cells once on the surface are displaced into the interior of the developing embryo. Because nervous tissue and epidermis have a shared origin, it is not uncommon for embryologic diseases to affect seemingly unrelated organs such as brain and skin.
Another example of cell migration and invagination during development is the adrenal gland. The medulla of the gland is a highly specialized adjunct to the sympathetic nervous system and derived from the ectoderm. The cortex, on the other hand, is derived from endoderm and mesoderm. The adrenal medulla secretes the catecholamines adrenaline (epinephrine) and noradrenaline (norepinephrine).
2. Cell Structure and Coloration
Early in development, the neural crest cells lie dorsal to the neural tube. Soon they migrate laterally and ventrally, basically associating with ectodermally-derived structures as in the areas of the epidermis-dermis junction.
Melanocytes are the cells found in the skin and are epidermal derivatives that are responsible for coloration. Those cells have polygonal cell bodies and long dendritic processes that ramify between epithelial cells throughout the lower strata of the epidermis. Pigmented cells are not restricted to cutaneous structures but can be found associated with various internal structures of ectodermal origin, as in the brain, spinal cord or adrenal medulla.
3. Nervous System
Neurons have a polygonal cell body and two types of arborizing processes, the axon and one or more dendrites. One region of the brain is called the substantia nigra (for black substance) because of its highly pigmented character. Many of the neurons of the substantia nigra contain significant quantities of melanin, and it is the melanin that confers on those cells the dark coloration. It has been seen that cell death in the normal Substantia nigra appears to be related to the content of neuromelanin per cell. Mann, D. M. et al., Brain 97, 489 (1974).
The substantia nigra is one region of the brain that is involved in the coordination (planning and programming) of neural signals for gross and slow, steady movements (ramp movements) and posture. The substantia nigra is part of that portion of the brain known as the basal ganglia which is itself part of the midbrain.
Two other highly pigmented areas of the brain are the locus ceruleus and the pituitary gland. The locus ceruleus is an eminence in the superior angle of the floor of the fourth ventricle. The hypophysis (pituitary gland), like the adrenal gland, arises from two embryological sources. The anterior pituitary arises as an epithelial outgrowth from the roof of the mouth. One of the hormones that it secretes is melanocyte stimulating hormone. The posterior pituitary is derived from a downgrowth of hypothalamic nerve tracts.
B. Degenerative Diseases of the Nervous System
The term “degenerative” as applied to diseases of the nervous system is used to designate a group of disorders in which there is gradual, generally symmetric, relentlessly progressive wasting away of neurons, for reasons still unknown. Many of the conditions so designated depend on genetic factors and thus appear in more than one member of the same family. This general group of diseases is therefore frequently referred to as heredodegenerative. A number of other conditions, not apparently differing in any fundamental way from the hereditary disorders, occur only sporadically, i.e., as isolated instances in a given family. For all diseases of this class William Gowers in 1902 suggested the now-familiar term “abiotrophy,” by which he meant “defective vital endurance” of the structures affected, leading to their premature death. This term, of course, tells nothing of the true nature of the defects. It is to be assumed that their basis must be some disorder of the metabolism of the parts involved.
Within relatively recent times there has been some elucidation of the nature of a number of metabolic nervous disorders which, in their symmetric distribution and gradually progressive course, resemble the degenerative diseases under discussion. It is to be expected that with advances in knowledge others of the latter group will eventually find their place in the metabolic category. The degenerative diseases of the nervous system manifest themselves by a number of common syndromes easily distinguished by their clinical attributes, the recognition of which can assist the clinician in arriving at the diagnosis of a disorder of this class.
1. General Considerations
It is a characteristic of the degenerative diseases that they begin insidiously and run a gradually progressive course which may extend over many years. The earliest changes may be so slight that it is frequently impossible to assign any precise time of onset. However, as with other gradually developing conditions, the patient or his family may give a history implying an abrupt appearance of disability. This is particularly likely to occur if there has been an injury, or if some other dramatic event has taken place in the patient’s life, to which illness might conceivably be related. In such a case, skillful taking of the history may bring out that the patient or family has suddenly become aware of a condition which had, in fact, already been present for some time but had passed unnoticed. Whether trauma or other stress may bring on or aggravate one of the degenerative diseases is still a question that cannot be answered with certainty. From all that is known it would seem highly improbable that this could happen. In any event, it must be kept in mind that the disease processes under discussion by their very nature develop spontaneously without relation to external factors.
Family history of degenerative nervous diseases is a significant feature of this class of diseases. Another significant feature is that in general their ceaselessly progressive course is uninfluenced by all medical or surgical measures. Dealing with a patient with this type of illness is often, therefore, an anguishing experience for all concerned. Yet symptoms can often be alleviated by wise and skillful management, and the physician’s kindly interest may be of great help even when curative measures cannot be offered.
The bilaterally symmetric distribution of the changes brought about by these diseases has already been mentioned. This feature alone may serve to distinguish conditions in this group from many other diseases of the nervous system. At the same time, it should be pointed out that, in the earliest stages, greater involvement on one side or in one limb is not uncommon. Sooner or later, however, despite the asymmetric beginning, the inherently generalized nature of the process asserts itself.
A striking feature of a number of disorders of this class is the almost selective involvement of anatomically or physiologically related systems of neurons. This is clearly exemplified in amyotrophic lateral sclerosis, in which the process is almost entirely limited to cortical and spinal motor neurons, and in certain types of progressive ataxia, in which the Purkinje cells of the cerebellum are alone affected. Many other examples could be cited (e.g., Friedreich’s ataxia) in which certain neuronal systems disintegrate, leaving others perfectly intact. An important group of the degenerative diseases has therefore been called “system diseases” (“progressive cerebrospinal system atrophies”), and many of these are strongly hereditary. It must be realized, however, that selective involvement of neuronal systems is not exclusively a property of the degenerative group, since several disease processes of known cause have similarly circumscribed effects on the nervous system. Diphtheria toxin, for instance, selectively attacks the myelin of the peripheral nerves, and triorthocresyl phosphate affects particularly the corticospinal tracts in the spinal cord as well as the peripheral nerves. Another example is the special vulnerability of the Purkinje cells of the cerebellum to hyperthermia. On the other hand, several of the conditions included among the degenerative diseases are characterized by pathologic changes that are diffuse and unselective. These exceptions nevertheless do not detract from the importance of affection of particular neuronal systems as a distinguishing feature of many of the diseases under discussion.
Since etiologic classification is impossible, subdivision of the degenerative diseases into individual syndromes rests on descriptive criteria, based largely on pathologic anatomy but to some extent on clinical aspects as well. In the terms used to designate many of these syndromes, the names of a number of distinguished neurologists and neuropathologists are commemorated. A useful way of keeping in mind the various disease states is to group them according to the outstanding clinical features that may be found in an actual case. The classification outlined in Table 1 is based on such a plan.
TABLE 1 ______________________________________ Clinical Classification of the Degenerative Diseases of the Nervous System ______________________________________ I. Syndrome in which progressive dementia is an outstanding feature in the absence of other prominent neurologic signs A. Diffuse cerebral atrophy 1. Senile dementia 2. Alzheimer’s disease B. Circumscribed cerebral atrophy (Pick’s disease) II. Syndrome in which progressive dementia is combined with other neurologic signs A. Principally in adults 1. Huntington’s chorea 2. Cerebrocerebellar degeneration B. In children and adults 1. Amaurotic family idiocy (neuronal lipidoses) 2. Leukodystrophy 3. Familial myoclonus epilepsy 4. Hallervorden-Spatz disease 5. Wilson’s disease (hepatolenticular degeneration, Westphal-Strumpell pseudosclerosis) III. Syndrome chiefly manifested by gradual development of abnormalities of posture or involuntary movements A. Paralysis agitans (Parkinson’s disease) B. Dystonia musculorum deformans (torsion dystonia) C. Hallervorden-Spatz disease and other restricted dyskinesias D. Familial tremor E. Spasmodic torticollis IV. Syndrome chiefly manifested by slowly developing ataxia A. Cerebellar degenerations B. Spinocerebellar degenerations (Friedrich’s ataxia, Marie’s hereditary ataxia) V. Syndrome with slowly developing muscular weakness and wasting A. Without sensory changes; motor system disease 1. In adults a. Amyotrophic lateral sclerosis b. Progressive muscular atrophy c. Progressive bulbar palsy d. Primary lateral sclerosis 2. In children or young adults a. Infantile muscular atrophy (Werdnig-Hoffmann disease) b. Other forms of familial progressive muscular atrophy (including Wohlfart-Kugelberg-Welander syndrome) c. Hereditary spastic paraplegia B. With sensory changes 1. Progressive neural muscular atrophy a. Peroneal muscular atrophy (Charcot-Marie-Tooth) b. Hypertrophic interstitial neuropathy (Dejerine-Sottas) 2. Miscellaneous forms of chronic progressive neuropathy VI. Syndrome chiefly manifested by progressive visual loss A. Hereditary optic atrophy (Leber’s disease) B. Pigmentary degeneration of the retina (retinitis pigmentosa) ______________________________________
2. Parkinson’s Disease
Perhaps the disorder the general public is most familiar with is Parkinson’s disease, or paralysis agitans. In early stages of the disease, there may be slight disturbances of posture, locomotion, facial expression or speech. The manifestations may be asymmetric, e.g, a slight tremor of the fingers on one hand at rest. The symptoms then become bilateral and the patient tends to assume a stooped posture. Gait disturbances increase and there is a moderate generalized disability. After a number of years the disability, bradykinesia, weakness and rigidity progress to the point of complete invalidism.
Because of the prevalence of Parkinson’s disease, it has been the focus of much neurological research. As early as 1953 it was recognized that it was common for there to be a depletion of dopaminergic transmission and a loss of the melanin-containing cells of the substantia nigra. It is not fully clear whether the changes are the result of “demelanination” by cells or actual cell death.
Current therapy for Parkinsonism is the oral administration of levodopa (L-dopa), which is 3-(3,4-dihydroxyphenyl)-L-alanine. Because L-dopa is a precursor of epinephrine and melanin there are certain contraindications. Apparently levodopa can exacerbate malignant melanomas or other skin lesions and can have untoward effects in patients with cardiovascular or pulmonary disease, asthma, or renal, hepatic or endocrine disease.
The deficiency of dopamine synthesis that characterizes Parkinsonism prompted the notion of transplanting dopamine neurons, particularly those of the adrenal medulla, into the brain as replacement therapy. Following successful transplants and alleviation of symptoms in the rotational rat model and in primates with induced lesions, the first transplants of fetal adrenal medulla were made to the striatum in two patients with severe Parkinsonism. Some rewarding effects were registered. Additional successful cases have been reported in the literature. Nevertheless, it is a complicated procedure which requires fetal donor tissue, and there have been a few unexplained deaths in those same studies.
3. Alzheimer’s Disease
Alzheimer’s Disease (AD) generally presents a clinical picture of gradual loss of intellectual capabilities. The incidence of AD in a number of surveys averages between four and five percent of the U.S. population. This translates to approximately 1.3 million cases of severe AD and an additional 2.8 million patients with mild to moderate impairment. The diagnosis of AD is complicated by the lack of a specific clinical marker. Currently a physician must depend on longitudinal observation for the gradual manifestation of the typical neuropathological features, and the support of a diffusely slow electroencephalogram, reduced cerebral blood flow and particular patterns on positron emission tomographic scanning.
Post-mortem examination of the brain shows a generalized atrophy. There are extensive histologic changes in AD dominated by the presence of intracellular amyloid plaques and neurofibrillary tangles. Plaques and tangles are rare, however, in the basal ganglia and substantia nigra. Many specimens from AD patients demonstrate a loss of pigmentation in the area of the locus ceruleus, which is a major source of noradrenergic synthesis in the brain.
Proposed treatments for Alzheimer’s disease include the administration of memory-enhancing compounds such as those described in U.S. Pat. No. 4,752,610, as well as the administration of substances such as gangliosides and peptide growth factors which aid the regeneration of injured nerve cells (Terry, R. D. et al., Ann. Neurol. 14, 497 (1983)).
4. Schizophrenia and Other Diseases
Dopaminogenic neuronal activity may be abnormal in cases of schizophrenia. There is a reduction in fresh volume of substantia nigra in brains of schizophrenics with the majority of that due to a reduction of cell body volume in the medial portions of that region. Nevertheless, the reduction by cells is not as contributory to the fresh volume loss as is reduction of the neuropil. It is unknown whether those observations have a bearing on the hypothesis that dopamine neurons are overactive in schizophrenia.
Human diseases of the basal ganglia result in hyperkinetic or hypokinetic activity. For example, progressive familial myoclonic epilepsy (Unver-Richt-Lundberg-Lafora disease) is characterized by first generalized convulsive seizures followed by myoclonic jerks of increasing frequency and severity, and progressive dementia. Pathologic investigation reveals atypical cellular architecture in the substantia nigra. In Hallervorden-Spatz disease the patient presents a variable clinical picture that includes abnormalities of posture and muscle tone, involuntary movements and progressive dementia.
5. Retinitis Pigmentosa (RP)
Because the eye is an ectodermal derivative, that organ, like the brain, contains pigmented cells. Melanocytes are contained in the choroid, which is the structure that supports the multilayered, photosensitive retina. The outermost layer is comprised of pigmented epithelial cells. Those layers of pigmented cells absorb light that passes through the retina and minimizes interference due to reflection.
RP is an ophthalmologic disease characterized by progressive visual field loss and night blindness. The primary defect is at the level of the photoreceptor and pigmented cells of the retina. Currently, there is no known therapy for RP except for cases of vitamin A deficiency and removal of cataracts. Numerous low vision aids such as various magnifiers, telescopes and image intensifiers are available as supportive therapy.
C. Xeroderma Pigmentosum (XP)
XP is characterized by extreme cutaneous photosensitivity at wavelengths of 280 to 310 nm. Although dermatology textbooks often refer to the occurrence of XP in all races, there are few reports of XP in blacks. Patients sustain severe sunburns, hyperpigmented macules are prevalent and the skin becomes thickened and hyperkeratotic. Because the defect is manifest embryologically, other ectodermal derivatives are often affected. Thus, ophthalmic changes include photophobia and increased lacrimation, and neurologic abnormalities include microcephaly, retardation, deafness and ataxia. Cutaneous malignancies develop in virtually all patients with XP. Psoralens have been administered to promote a natural tan in fair-skinned patients in hopes of providing some photoprotection.
D. Melanin
For the purposes of the present description, melanins are defined below and are further described and 5 classified as in the book entitled “Melanins, ” by R. A. Nicolaus, published in 1968 by Hermann, 115, Boulevard Saint-Germain, Paris, France, which work in its entirety is incorporated herein by reference. As defined by Nicolaus, melanins constitute a class of pigments which are widespread in the animal and vegetable kingdoms While the name “melanin” in Greek means black, not all melanins as pigments are black but may vary from brown to yellow.
Mammalian colors are determined chiefly by two types, eumelanins and phaeomelanins. Eumelanins are derived from the precursor tyrosine and are generally insoluble and black or brown in color. Phaeomelanins have as their precursors tyrosine and cysteine and are generally alkali-soluble and lighter in color. Allomelanins (“allo” meaning other) are formed from nitrogen-free precursors, primarily catechol and 1,8-dihydroxynaphthalene (see The Merck Index, Tenth Edition, page 827, item 5629, Melanins). Quinones are the usual intermediates in allomelanin synthesis. The synthesis of melanins occurs in nature as well as being produced synthetically. A further group of low molecular weight yellow, red and violet pigments is known as trichochromes. The trichochromes are usually classified with the melanins, since they serve as pigments and are derived from the oxidation of tyrosine.
The biosynthetic pathway by which melanin is produced is shown below as reported by Hearing, V. J. et. al., Int. J. Biochem. 19, 1141 (1987).
E. Tyrosinase
The enzyme, tyrosinase, plays a key role in the synthesis of melanin and its derivatives. In mammals, tyrosinase is a glycosylated enzyme found in melanocytes.
It has been theorized that tyrosinase functions by means of separate catalytic sites; one site for tyrosinase hydroxylase activity, another site for dopa oxidase activity, and a third independent site for dopa as a cofactor. Hearing, V. J. et al., Biochem. J., 157 549 (1976). Tyrosinase may also play a role in catalyzing the oxidation of 5,6-dihydroxyindole to indole-5,6-quinone. Korner, A. M. et al., Science 217, 1163 (1982). In vivo, mammalia tyrosinase undergoes extensive modification. When initially synthesized, tyrosinase has an apparent molecular weight of about 55,000. Glycosylation of the enzyme occurs as it is transferred through the Golgi complex and delivered to the melanocytes. Imokawa, G. et al., J. Invest. Derm., 85, 165 (1985). During this modification of tyrosinase, sialic acid and 4 mol of asparagine-linked carbohydrate chains (containing mannose, glucosamine, galactose and fucose) are added to each mole of tyrosinase. Ferrini, V. et al., Int. J. Biochem. 19, 229 (1987). The glycosylated tyrosinase has an apparent molecular weight of about 70,000. Laskin, J. D. et al., J. Biol. Chem. 261, 16626 (1986) .
The glycosylated tyrosinase is delivered to the melanocytes by coated vesicles. In the melanocytes, the tyrosinase is membrane bound and aggregates into a high molecular weight form. In vivo, tyrosinase is under active metabolic control involving an active degradation system which results in a biological half-like of about ten hours. Jimenez, M. et al., Fed. Proc. Fodn. Am. Socs. Exp. Biol. 45, 1714 (1986).
F. Tyrosinase Gene
The gene for human tyrosinase has been isolated, sequenced and cloned (PCT application WO 88/02372, published Apr. 7, 1988). The cloned gene encodes a polypeptide of 548 amino acids with a molecular weight of 62,160, excluding a hydrophobic signal peptide.
The gene for Streptomyces glaucescens tyrosinase has also been isolated and sequenced (Huber, M. et al., Biochemistry 24, 6038 (1985)). Nearly all of the codons used end in either G or C, and the overall G+C content of the gene is 71.4%. Id.
In order to isolate the S. glaucescens tyrosinase gene, the KpnI fragment of plasmid pMEA4 containing the S. glaucescens gene (Hintermann, G. et al., Mol. Gen. Genet. 200, 422 (1985)) is cloned into the PvuII site of pBR322 with KpnI linkers (P-L Biochemicals). Two resulting plasmids (pMEA6 and pMEA7) contain the tyrosinase gene in opposite directions. (Huber, M. et al., supra). Plasmid DNA is then isolated by conventional techniques such as those described by Maniatis, T. et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor (1982).
Restriction endonucleases are then used according to the suppliers’ instructions (Boehringer, Amsterdam) to perform digestions, and the fragments are recovered by low-melting agarose gels as described by Weislander, L., Anal. Biochem. 98, 305 (1979). The nucleotide sequences are then determined using the methods of Maxam, A. M. et al., Methods. Enzymol. 65, 499 (1980).
G. Melanin Concentrating Hormone
Melanin concentrating hormone (MCH) is a peptide which has been isolated from fish pituitary gland, characterized and synthesized (Kawauchi, H. et al., Nature 305, 321 (1983)). MCH has also been localized by immunohistochemistry in the brain and pituitary gland of salmons, frogs and rats (Baker, B. J. et al., Gen. Comp. Endocrinol. 50, 1423 (1983), Naito, N. et al., Neurosci. Lett. 70, 81 (1986), Skotfitsch, G. et al., Proc. Natl. Acad. Sci. USA 83, 1528 (1986) and Zamir, N. et al., Brain Research 373,240 (1986)).
A mammalian MCH-like substance has been recognized by salmon MCH-directed antiserum in radioimmunoassay and immunohistochemistry (Zamir, N. et al., Proc. Natl. Acad. Sci. USA, supra). This mammalian MCH has been diluted in parallel with synthetic MCH, but exhibits distinct chromatographic properties on both RP-HPLC and gel chromatography. Id. The persistence of this mammalian MCH in the mammalian hypothalamo-neurohypophyseal system suggests a role in posterior pituitary function, such as the regulation of food and water intake. Id.
Other functions of this mammalian MCH peptide have also been suggested. Due to the identification of MCH fibres in the human median eminence and pituitary stalk, it has been suggested that the peptide causes the aggregation or concentration of melanin in cells of the central nervous system and may be involved in the regulation of anterior pituitary function (Pelletier, G. et al., Brain Research 423, 247 (1987)). Furthermore, Sekiya, K. et al. in Neuroscience 25, 925 (1988) suggest that MCH may act as a neurotransmitter and/or neuromodulator in the central nervous system or may regulate pituitary portal-blood system and/or the neurosecretory system in mammals.
SUMMARY OF THE INVENTION
The present invention is directed to therapeutic uses of melanin, melanin precursors, melanin derivatives, melanin analogs and related substances. One particular aspect of the invention relates to the treatment of certain diseases by the administration of active substances which cause an increased melanin concentration in the patient’s central nervous system (CNS). Such substances include melanin, melanin precursors, melanin derivatives, melanin analogs, melanin variants melanin-concentrating hormone (MCH), tyrosinase, tyrosinase gene and combinations thereof. These diseases include those of tissues which have lost melanin and which share a common embryological basis as the nervous system.
More specifically, the present invention is directed to the administration of melanin, a melanin precursor, a melanin variant, a melanin analog or a melanin derivative to replace lost melanin in the treatment of diseases which exhibit a decrease in the production of melanin, one or more melanin precursors or one or more derivatives or analogs of melanin and/or exhibit an increase in the catabolism or excretion of melanin, one or more melanin precursors or one or more derivative of analogs of melanin. Alternatively, the administration of MCH causes the concentration of available melanin and/or one or more melanin precursors in particular areas of the CNS, and the administration of tyrosinase or tyrosinase gene allows the patient’s body to produce more melanin by increasing the conversion of assuming no precursor deficiencies melanin precursors to melanin. The present invention is especially useful for treating diseases which exhibit a neurological dysfunction or disorder. Such diseases include Parkinson’s disease, Alzheimer’s disease, retinitis pigmentosa, depression, schizophrenia and other diseases such as those listed in Table 1 above. Tissues which share a common embryological basis as the nervous system include epithelium and the adrenal medulla. An example of a disease of the epithelium is xeroderma pigmentosum.
The present invention is also useful for assisting the recovery of neurons in a mammal having neuron injury by administering an effective amount of an active substance which causes an increased concentration of melanin in the neuron to aid in nerve recovery. Melanin, a melanin precursor, a melanin analog, a melanin variant or a melanin derivative can be administered to accomplish this result. Alternatively, the melanin necessary to aid nerve recovery may be concentrated in the CNS by administration of MCH, or may be produced in the patient’s body by administering tyrosinase which catalyzes naturally occurring melanin precursors to melanin. Furthermore, the administration of tyrosinase gene causes the production of tyrosinase in the patient’s body, thereby catalyzing the conversion of the naturally occurring melanin precursors to melanin. The present invention is further useful in protecting a mammal from a disease, such as a neurode-generative disease, or the adverse effects of toxins upon exposure to toxins such as neurodegenerative disease-causing substances, by administering an effective amount of melanin, melanin precursor, melanin derivative, melanin analog, melanin variant, MCH, tyrosinase, tyrosinase gene or a combination thereof.
A further aspect of the present invention relates to the use of melanin, a melanin precursor, a melanin derivative, a melanin analog and/or a melanin variant as a carrier for other therapeutic agents. Melanin is particularly useful as a carrier for therapeutic agents which do not easily cross the blood-brain barrier. One of the unique properties of melanins is their ability to cross the blood-brain barrier.
DETAILED DESCRIPTION OF THE INVENTION
One aspect of the present invention relates to the treatment of a mammal having a disease of a tissue which exhibits a melanin deficiency by the administration to the mammal of an effective amount of an active substance which causes an increased concentration of melanin in the effected tissue. Such active substances include but are not limited to melanin, melanin precursorsz melanin derivatives, melanin analogs, melanin variants tyrosinase, tyrosinase gene, melanin-concentrating hormone and combinations thereof. The tissue includes those which share a common embryological basis with the nervous system. The present invention is especially useful for treating such diseases which exhibit a neurological dysfunction or disorder.
The present invention is also useful for assisting the recovery of neurons in a mammal having neuron injury by administering an effective amount of the same active substance described above. The increased concentration of melanin in the affected neuron, caused by administration of the active substance aids in nerve recovery.
The present invention is further useful in protecting a patient from a disease, such as a neurodegenerative disease, or the adverse affects of toxins upon exposure to toxins, such as neurodegenerative disease-causing substances, by administering an effective amount of the active substance. The increased melanin concentration caused by administration of the active substance causes chelation or scavenging of the toxin.
The present invention still further relates to the use of melanin as a carrier for other therapeutic agents. Due to specific properties of melanin, it is particularly useful as a carrier for other therapeutic agents.
A. Definitions
In order to provide a clear and consistent understanding of the specification and claims, including the scope given to such terms, the following definitions are provided:
Administration: The application or delivery of a drug to a mammal in need of the drug. This term is intended to include any means of administration which accomplishes the application or delivery of the drug (i.e., topical, oral, aerosol, suppository, intravenous, intramuscular, injection, e.g., into the brain or the cerebrospinal fluid or other parts of the nervous system, peritoneally and the like). The term is also intended to include any means necessary to accomplish such administration, such as a sugar loading procedure to enable a drug to cross the blood-brain barrier. The term is further intended to include the in vivo production of a drug or aggregation of a drug moderated by another substance such as an enzyme (tyrosinase) or enzyme gene (tyrosinase gene) to moderate production of a drug (melanin) or its precursors, or a concentrating hormone (MCH) to moderate drug (melanin) concentration.
Blood-Brain Barrier: The blood-brain barrier is made up of brain microvessel endothelial cells characterized by tight intercellular junctions, minimal pinocytic activity, and the absence of fenestra. These characteristics endow these cells with the ability to restrict passage of most small polar blood-borne molecules (e.g., neurotransmitter catecholamines, small peptides) and macromolecules (e.g., proteins) from the cerebrovascular circulation to the brain. The blood-brain barrier contains highly active enzyme systems as well, which further enhance the already very effective protective function. It is recognized that transport of molecules to the brain is not determined solely by molecular size but by the permeabilities governed by specific chemical characteristics of the permeating substance. Thus, besides molecular size and lipophilicity, the affinity of the substances to various blood proteins, specific enzymes in the blood, or the blood-brain barrier will considerably influence the amount of the drug reaching the brain.
Common Embryological Basis: This term is intended to include all tissues which are derived from the same germinal layer, specifically the ectoderm layer, which forms during the gastrulation stage of embryogenesis. Such tissues include, but are not limited to, brain, epithelium, adrenal medulla, spinal chord, retina, ganglia and the like.
Degenerative Diseases of the Nervous System: This term is intended to include any of the diseases referred to in Table 1 as well as other brain disturbances including, but not limited to, depression, dementia and schizophrenia. This term is used interchangeably with the terms “diseases with a neurological dysfunction or disorder” or “neurodegenerative diseases,” which are intended to have the same meaning.
Melanin: Melanins are polymers produced by polymerization of reactive intermediates. The polymerization mechanisms include but are not limited to autoxidation, enzyme catalyzed polymerization and free radial initiated polymerization. The reactive intermediates are produced chemically or enzymatically from precursors. Suitable enzymes include,. but are not limited to peroxidases and catalases, polyphenol oxidases, tyrosinases, tyrosine hydroxylases or laccases. The precursors which are connected to the reactive intermediates are hydroxylated aromatic compounds. Suitable hydroxylated aromatic compounds include, but are not limited to 1) phenols, polyphenols, aminophenols and thiophenols of aromatic or polycyclic aromatic hydrocarbons, including but not limited to phenol, tyrosine, pyrogallol, 3-aminotyrosine, thiophenol and .alpha.-naphthol; 2) phenols, polyphenols, aminophenols, and thiophenols of aromatic heterocyclic or heteropolycyclic hydrocarbons such as but not limited to 2-hydroxypyrrole, 4-hydroxy-1,2-pyrazole, 4-hydroxypyridine, 8-hydroxyquinoline, and 4,5-dihydroxybenzothiazole. The term melanin includes naturally occurring melanins which are usually high molecular weight polymers (generally, molecular weights in the millions) and low molecules weight polymers as well as melanin analogs as defined below. Naturally occurring melanins include eumelanins, phaeomelanins, neuromelanins and allomelanins. The term melanin is also intended to include trichochromes when used hereafter. The term “melanin” is further intended to include both melanin, melanin precursors, melanin analogs, melanin variants and melanin derivatives unless the context dictates otherwise.
Melanin Analog: Melanin in which a structural feature that occurs in naturally occuring or enzymatically produced melanins is replaced by an unusual substituent divergent from substituents traditionally present in melanin. An example of an unusual substituent is selinium in place of sulfur, such as selinocysteine.
Melanin Deficiency: This term is intended to refer to a condition in diseased tissue in which melanin is absent, present in a lower amount when compared to normal tissue, or functionally non-active. The deficiency may be caused by a decrease in the synthesis of melanin and/or an increase in the catabolism or excretion of melanin. The melanin may be functionally non-active as the result of a substance binding to it which destroys the melanin’s activity.
Melanin Derivative: This term is intended to include any derivative of melanin which is capable of being converted in tissue to either melanin or a substance having melanin activity. An example of a melanin derivative is melanin attached to a dihydrotrigonelline carrier such as described in Bodor, N., Ann. N.Y. Acad. Sci. 507, 289 (1987) to enable the melanin to cross the blood-brain barrier. The term melanin derivatives is also intended to include chemical derivatives of melanin, such as an esterified melanin.
Melanin Variant: Melanin variants are defined to include various subsets of melanin substances that occur as families of related materials. Included in these subsets, but not limited thereto, are:
(1) Naturally occurring melanins produced by whole cells that vary in their chemical and physical characteristics;
(2) Enzymatically produced melanins prepared from a variety of precursor substrates under diverse reaction conditions;
(3) Melanin analogs in which a structural feature that occurs in (1) or (2) above is replaced by an unusual substituent divergent from the traditional; and
(4) Melanin derivatives in which a substituent in a melanin produced in (1), (2) or (3) above is further altered by chemical or enzymatic means.
Neurodegenerative Disease-Causing Substance: Any substance which can cause a neurodegenerative disease in a mammal. Examples of such substances include N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-henylpyridine (MPP.sup.+) and manganese dust for Parkinson’s disease; guinolinic acid for Huntington’s chorea; and .beta.-N-methylamino-L-alanine for amyotrophic lateral sclerosis, Parkinson’s disease and Alzheimer’s disease.
Tyrosinase: An enzyme which, in mammals, catalyzes: (a) the hydroxylation of tyrosine to dopa (3,4-dihydroxyphenylalanine); (b) the oxidation of dopa to dopaquinone; and (c) may catalyze the oxidation of 5,6-dihydroxyindole to indole-5,6-quinone. All of these reactions which are catalyzed by tyrosinase take place in the biosynthetic pathway which produces melanin. Tyrosinase is most commonly found in a glycosylated form in vivo.
B. Melanin
Naturally occurring melanins include such melanins as eumelanins, phaeomelanins, neuromelanins and allomelanins. Trichochromes which are low molecular weight polymers derived from the oxidation of tyrosine are also considered melanins for the purpose of this invention. Melanins and melanin variants are as defined above. Melanin variants are considered melanins for the purpose of this invention unless the context indicates otherwise.
Naturally occurring melanin is formed through natural biochemical pathways which involve the hydroxylation and decarboxylation of the amino acids phenylalanine and tyrosine. In one possible anabolic pathway, tyrosine is hydroxylated to form the catecholamine dopa, which is 3,4-dihydroxyphenylalanine, then the diol is oxidized to form the diketone 3,4-dioxyphenylalanine (also known as dopaquinone). The dopaquinone is cyclized to form 5,6-indolequinones, and it is the polymerization of those indolequinones that produces melanin. There are alternative pathways for melanin production. However, in each of those alternatives an understanding of the mechanisms in the final steps remains elusive.
Another pathway for natural melanin production involves the use of the neurotransmitters epinephrine (adrenaline) and norepinephrine (noradrenaline). Epinephrine is oxidized to form adrenochrome, then adrenolutin is produced and finally melanin. But melanin production is more intimately involved with the neural system because tyrosine and phenylalanine are also the precursors for the neurotransmitters epinephrine, norepinephrine and dopamine.
It is not uncommon for metabolic pathways such as these to be intimately involved, for it is a hallmark of “biological economy” that characterizes life processes. Thus, one amino acid building block such as phenylalanine can be used in a number of ways. Similarly, any one of the intermediates in a pathway such as dopamine can serve as starting material for an end product. Catabolism of the end product or intermediates ultimately produces the same building blocks for reconstruction at a later time, or produces unusable catabolites or detoxifies harmful intermediates for removal. Because those pathways are fully integrated, it is common for the end products such as melanin or epinephrine to serve as regulators for the pathway. That phenomenon is known as feedback inhibition. Thus, melanin could inhibit one of the enzymes early in the melanin biosynthetic pathway such as tyrosinase. In that way, when melanin concentration is low, tyrosinase activity is high and a large amount of tyrosine is converted into dopa for eventual production of melanin. When there is sufficient melanin, tyrosine hydroxylase activity is low and less melanin is produced. That scheme of regulatory economy is typical of metabolism, as is most noted in the endocrine system, of which the neurotransmitters are a part.
The metabolic pathway machinery for the production of products such as melanin and epinephrine from the amino acid building blocks, although likely to be present in all cells, finds maximal presence in those cells that have a high demand for those products, as in the brain. Brain cells have high levels of tyrosinase because there is high demand for dopamine, for example. The substantia nigra, that region of the brain where cells are highly pigmented because of the concentration of melanin, is noted for cells with high levels of tyrosinase. In fact, if one performs immunohistochemical analyses of brain sections using an antityrosinase antibody, the substantia nigra would be a region of the brain heavily labelled. Because of the intimate relationship between melanin and dopamine, it is not unexpected that the substantia nigra and its pigmented cells have high levels of tyrosinase.
Naturally occurring melanin can be prepared synthetically or isolated from natural sources. Natural sources include beef eyes, squid, hair, bacteria such as Streptococcus antibioticus, and brain, among others. Melanins can be prepared synthetically, as described by Froncisz, W. et al., Arch. Biochem. Biophys. 202, 289 (1980) and Lyden, A. et al., Arch. Int. Pharmacodyn. 259, 230 (1982), among others.
Since melanins are polymers of indole quinones, they are polar molecules with exposed amino, keto and carboxyl functionalities. The presence of these charged groups allows melanin to act as an effective ionic sponge or chelator. A variety of drugs such as chloroquine and chlorpromazine have a high affinity for melanin (Larson, B. et al., Biochem. Pharmac. 28, 1181 (1979)). Further, there is a high uptake by melanin of serotonin, and moderate uptake of dopamine, noradrenaline and adrenaline, while L-dopa and L-tyrosine have no affinity for melanin (Lindquist, N. G., Acta Radiol. Suppl. 325, 67 (1973)). As mentioned earlier, melanin also has a high affinity for the neurotoxic parkinsonism drug MPTP. High concentrations of MPTP can be found in the substantia nigra and locus cereuleus of animals and patients that have been exposed to the neurotoxin (Snyder, S. H. et al., Neurology 36, 250 (1986)). Melanin also readily crosses the blood-brain barrier, and is therefore useful as a carrier for other therapeutic agents which must reach brain tissue to produce their therapeutic responses.
Melanin has also been used as a chelator for uranium (Takashi, S. et al., J.Chem.Technol.Biotechnol. 40, 133 (1987)) and as a sorbent for clarifying and stabilizing wine (USSR 924,098). Melanin has additional anti-toxin characteristics as a free radical scavenger or oxygen scavenger, and as such can serve as a terminator of free radical chain reactions. As a free radical scavenger, melanin may play an important role in preserving cells from the toxic effects of O.sub.2. Geremia, C. et al., Comp. Biochem. Physiol. 79B, 67 (1984).
Melanin has many other interesting properties such as ultraviolet absorption, which has been utilized to prepare optical lenses (U.S. Pat. No. 4,698,374) as well as cosmetic creams (Jap. 49-071149). Melanin has both semiconductor (Culp, C. J. et al., J.Appl.Phys. 46, 3658 (1975)) and superconductor (Cope, F. W., Physiol. Chem. Phys. 10, 233 (1978)) properties.
Melanin variants have all of the above-described properties of naturally occurring melanins, but in addition, melanin analogs cause an increase in the permeability of the blood-brain barrier. The mechanism for this increased permeability of the blood-brain barrier is not known.
Due to their ability to increase the permeability of the blood-brain barrier, the melanin variants are also useful as carriers for other therapeutic agents. Two examples of such therapeutic agents which will cross the blood-brain barrier when linked to melanin are boron and nerve growth factor.
Irradiated boron has a very high energy for a very short distance, and is therefore selectively lethal to cells in its immediate vicinity. For this reason, boron has been used in neutron capture therapy of cancerous cells.
Neutron capture therapy entails the delivery of the boron-10 isotope to cancerous areas of the body, and subsequent irradiation of the boron-10 isotope. Boron-10 readily absorbs thermal neutrons (slow neutrons) and the activated nucleus rapidly decays to lithium 7 and an alpha particle. The alpha particles are cytotoxic, so they kill the cancerous cells surrounding the boron and thus, cause tumor reduction. The boron which is used for neutron capture therapy should have at least a 20% natural abundance of the boron-10 isotope.
The boron may be carried to the cancerous site by a variety of vehicles. Conventional vehicles include steroids, antibodies, nucleosides, chlorpromazine, thiouracil, amino acids, porphyrins and liposomes, Fairfield et al., Int. J. Radiation Oncology Biol. Phys. 11, 831 (1985). It has now been found that melanin (including melanin variants) is also an effective vehicle for the transport of boron to cancerous sites in the body.
Melanin binds boron very strongly, and is therefore an excellent vehicle for the boron when combined with an antibody specific to the cancerous cells. As noted in U.S. Pat. No. 4,824,659, there has been a need for a more efficient method to conjugate boron-10 and antibody, and melanin provides that efficient method. The antibody directs the boron/melanin complex to the specific cancerous site where the boron-10 is irradiated with slow neutrons.
A preferred source of slow neutrons for irradiation of the boron-10 isotope is a 2 keV beam. The amount of boron atoms necessary in the cancerous tissue for effective neutron capture therapy is generally quantified as a molar concetration which is about 10.sup.4 to 10.sup.6 higher than that needed for diagnostic techniques (Fairchild et al., supra).
The therapeutic efficacy of neutron capture therapy is generally dependent on the ratio of tumor dose of boron to maximum normal tissue dose of boron. This ratio is termed the “advantage factor” and should be greater than 1. Id. The higher the advantage factor is above 1, the more efficatious is the neutron capture therapy. It is generally difficult to achieve an advantage factor significantly greater than 1 because the treatment volumes must be extended beyond the bulk tumor in order to include unseen microscopic extensions of the tumor growth. The presence of normal tissues within this extended treatment volume then limits the tumor dose to the normal-tissue tolerance dose.
An additional advantage of using melanin as the vehicle to transport boron to a tumor site is the ability of melanin to cross the blood-brain barrier, and melanin analog’s activity which causes an increase in the permeability of the blood-brain barrier. These properties of melanin permit easier treatment of brain tumors than is possible with the conventional boron vehicles which do not easily cross the blood-brain barrier and do not increase the permeability of the blood-brain barrier.
In addition to being an excellent carrier or vehicle for boron, melanin has also been found to be a very useful carrier for nerve growth factor. Like boron, nerve growth factor binds strongly to melanin. The major advantage of using melanin as a carrier for nerve growth factor in the ability to get nerve growth factor across the blood-brain barrier.
As discussed above, not only does melanin easily cross the blood-brain barrier, but the melanin variants also increase the permeability of the blood-brain barrier. Due to the substantial amount of nerve tissue in the brain, the ability to easily transport nerve growth factor across the blood-brain barrier is a major improvement over conventional nerve growth factor therapy. This major improvement over conventional the nerve growth factor therapy is attributable to the use of melanin as a carrier for the therapeutic agent.
C. Neurodegenerative Disease Effects on Melanin-Containing Cells
In order to develop therapy programs for any disease, it is useful to identify (a) potential causes of the disease, in an attempt to avoid them; (b) potential manifestations of the disease, in an attempt to identify aspects of the disease which may be treated, and (c) drugs which are similar to known therapeutic drugs. Little is known as to the cause-and-effect relationship in the neurodegenerative diseases. One problem in these diseases is that few animal models exist which can be utilized to gain the necessary understanding of each disease and its treatment.
Post-mortem examination of the brain shows a generalized atrophy. There are extensive histologic changes in Alzheimer’s disease (AD) dominated by the presence of intracellular amyloid plaques and neurofibrillary tangles. Plaques and tangles are rare, however, in the basal ganglia and substantia nigra. Many specimens from AD patients demonstrate a loss of pigmentation in the area of the locus ceruleus, which is a major source of noradrenergic synthesis in the brain.
Dopaminogenic neuronal activity may be abnormal in cases of schizophrenia. There is a reduction in fresh volume of substantia nigra in brains of schizophrenics with the majority of that due to a reduction of cell body volume in the medial portions of that region. Nevertheless, the reduction by cells is not as contributory to the fresh volume loss as is reduction of the neuropil. It is unknown whether those observations have a bearing on the hypothesis that dopamine neurons are overactive in schizophrenia.
Human diseases of the basal ganglia result in hyperkinetic or hypokinetic activity. For example, progressive familial myoclonic epilepsy (Unver-Richt-Lundberg-Lafora disease) is characterized by first generalized convulsive seizures followed by myoclonic jerks of increasing frequency and severity, and progressive dementia. Pathologic investigation reveals atypical cellular architecture in the substantia nigra. In Hallervorden-Spatz disease the patient presents a variable clinical picture that includes abnormalities of posture and muscle tone, involuntary movements and progressive dementia.
Retinitis pigmentosa is an ophthalmologic disease characterized by progressive visual field loss and night blindness. The primary defect is at the level of the photoreceptor and pigmented cells of the retina. Currently, there is no known therapy for retinitis pigmentosa except for cases of vitamin A deficiency and removal of cataracts. Numerous low-vision aids such as various magnifiers, telescopes and image intensifiers are available as supportive therapy.
Probably the most studied disease in terms of brain pathology has been Parkinson’s disease. It is well known that substantial changes occur within the substantia nigra of patients suffering from Parkinsonism. As previously discussed, the substantia nigra is one of the most heavily pigmented areas of the brain and consequently contains significant amounts of melanin. It has been demonstrated that cell death in the substantia nigra in Parkinson’s disease is related to a loss of melanin in the neurons of the substantia nigra (Mann et al., supra; Hirsch, E. et al., Nature 334, 345 (1988)). Furthermore, it has been established that MPTP, which can cause Parkinson’s disease, binds to neuromelanin (D’Amato et al. (1986), supra) and is concentrated in the substantia nigra and locus cereuleus (Snyder et al., supra).
The common factor in each of these diseases is that a tissue which is highly pigmented, i.e., one which contains melanin, is involved in the disease. In almost every instance, there is a decreased melanin content, i.e., a loss of pigment, which may lead to cell death. As described further below, applicant has discovered that treatment of neurodegenerative diseases with melanin can ameliorate the primary neurological symptoms of the disease.
D. Aspects of the Invention
In its broadest aspects, therapeutic uses of melanin include: 1) melanin as a drug, 2) melanin as a drug delivery agent and 3) melanin as a target for various types of radiation. In instances in which selected delivery or target cell sequestration is not required native melanin as well as melanin variants, melanin analog and melanin derivatives can be used. Melanins, melanin variants, melanin analogues, and melanin derivatives can be produced with predictable molecular weights, particle sizes, and compositions. Consequently, melanins can now be attached to antibodies and thus targeted for specific cell (e.g. liver cells).
Melanin has a number of properties which can be exploited to both alter cellular metabolism and/or remove intra- and intercellular toxins. Such properties include, oxygen and free radical scavenging, metal binding, binding of organic cationic species (MPP.sup.+ is one example), catalysis of coupled redox reactions. These properties are not interdependent, and, melanin can be selectively altered and optimized.
Drugs can be covalently bound to melanin or just adsorbed on its surface. They can be attached in such a manner that induced cellular metabolism at the target cell would cause release of the therapeutic agent. The melanin derivatives are preferred for this type of application.
Melanin absorbs various types of radiation as well as being capable of binding boron. Melanin can be used to absorb ultraviolet rays in skin creams as well as to translate irradiation to cover tissue. Each of these broad aspects is further described below.
1. Therapy
One aspect of the present invention is that an active substance such as melanin can be used to treat neurodegenerative diseases or diseases of tissues which share a common embryological basis with the nervous system. As discussed above, the loss of melanin can be seen in many neurodegenerative diseases. For example, the retina suffers a loss of pigmented cells in retinitis pigmentosa. In Alzheimer’s disease there is a generalized atrophy and a loss of pigment, i.e., melanin, in the area of the locus ceruleus, which is a major source of noradrenergic synthesis in the brain. A reduction in fresh volume of the substantia nigra, especially of the neurophil, has been seen in schizophrenics. A typical cellular architecture also exists in Unver-Richt-Lundberg-Lafora disease.
Probably the most studied disease in terms of brain pathology has been Parkinson’s disease. It is well known that substantial changes occur within the substantia nigra of patients suffering from Parkinsonism. As previously discussed, the substantia nigra is one of the most heavily pigmented areas of the brain and consequently contains significant amounts of melanin. It has been demonstrated that cell death in the substantia nigra in Parkinson’s disease is related to a loss of melanin in the neurons of the substantia nigra (Mann et al., supra; Hirsch, E. et al., Nature 334, 345 (1988)). Furthermore, it has been established that MPTP, which can cause Parkinson’s disease, binds to neuromelanin (D’Amato et al. (1986), supra) and is concentrated in the substantia nigra and locus cereuleus (Snyder et al., supra).
It has now been found that the administration of melanin to a mammal having a disease of tissue which exhibits a melanin deficiency, such as the neurodegenerative diseases discussed above, is capable of ameliorating the primary neurological symptoms of the neurodegenerative disease which is treated. Similar improvement in overall functional ability is also improved. Furthermore, secondary motor manifestations of the neurodegenerative diseases are also proportionately improved upon administration of melanin. The melanin can be administered by any means which will insure that it reaches the desired tissue. In many instances, the administration may require mechanisms for crossing the blood-brain barrier. Several mechanisms are described below and others are known in the art.
Since the treatment of the disease will require many separate doses of melanin, some mechanisms will be more preferred than others. Suitable doses for this purpose are from about 0.5 to about 150 mg/kg/day and preferably from about 1 to about 50 mg/kg/day of the active ingredient. Proper doses are determined as described below.
Melanin can also be used for ameliorating Alzheimer’s disease since it is capable of aiding the recovery of injured neurons (discussed in further detail below). Suitable doses for this purpose are as described above, and the optimal dose is determined as described below.
An alternative method for treating these nervous system diseases with melanin is to enhance the in vivo production of melanin by administering tyrosinase to the effected patient. Tyrosinase catalyzes at least two, and possibly three, of the reactions in the biosynthetic pathway which produces melanin.
Naturally, occurring tyrosine in the human body is hydroxylated to 3,4-dihydroxyphenylalanine (dopa), and the hydroxylation is catalyzed by tyrosinase. Tyrosinase also catalyzes the subsequent oxidation of dopa to dopaquinone. The dopaquinone is a precursor for two separate biosynthetic pathways for the production of melanin. Therefore, both tyrosinase-catalyzed reactions which lead to the production of dopaquinone (the hydroxylation of tyrosine to dopa and the oxidation of dopa to dopaquinone) are important reactions in the human body’s production of melanin.
One pathway from dopaquinone to melanin involves a ring closure and hydrogenation of dopaquinone to produce leucodopachrome. This is followed by partial oxidation of leucodopachrome to dopachrome, and decarboxylation and hydroxylation of the dopachrome to 5,6-dihydroxyindole. The 5,6-dihydroxyindole is then oxidized to indole-5,6-quinone, and it is at this step that tyrosinase is again believed to serve as a catalyst. Korner, A. M. et al., Science 217, 1163 (1982). Tyrosinase is believed to catalyze this oxidation reaction. The indole-5,6-quinone is then converted to melanin or eumelanin.
The other pathway from dopaquinone to melanin involves the addition of cysteine to dopaquinone to produce 5-S-cysteinyldopa, followed by the oxidation of 5-S-cysteinyldopa to 5-S-cysteinyldopaquinone. A ring closure of the 5-S-cysteinyldopaquinone then yields 7-alanyl-5-hydroxy-3-carboxy-2H-1,4-benzothiazine which is subsequently decarboxylated to yield 7-alanyl-5-hydroxy-2H-1,4-benzothiazine. At this point, the 7-alanyl-5-hydroxy-2H-1,4-benzothiazine is converted to melanin and pheomelanin. Tyrosinase does not play any additional role in this melanin production pathway.
It has now been found that the administration of tyrosinase to a mammal having a disease of a tissue which exhibits a melanin deficiency, such as the neurodegenerative diseases discussed above, is capable of ameliorating the primary neurological symptoms of the neurodegenerative disease which is treated. Similar improvement in overall functional ability is also improved. Furthermore, secondary motor manifestations of the neurodegenerative diseases are also proportionately improved upon administration of tyrosinase. These improvements are believed to be due to the increased in vivo production of melanin brought about by the increased tyrosinase-mediated catalysis of reactions along the biosynthetic pathway responsible for the production of melanin.
The tyrosinase can be administered by any means which will insure that it reaches the desired tissue. In many instances, the administration will require mechanisms for crossing the blood-brain barrier. Several mechanisms are described below and others are known in the art. Since the treatment of the disease will require many separate doses of tyrosinase, some mechanisms will be more preferred than others. 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. Proper doses are determined as described below.
Tyrosinase can also be used for ameliorating the symptoms of Alzheimer’s disease since it increases the production of melanin in vivo, and melanin is capable of aiding the recovery of injured neurons (discussed in further detail below). Suitable doses for this purpose are as described above, and the optimal dose is determined as described below.
Another method by which the in vivo production of melanin may be enhanced is 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. This vector is useful for transneuronally transporting genes from peripheral neurons to the primary target cells in the brain (Ugolini et al., Science 243, 89 (1989)). The amount of tyrosinase gene administered must be sufficient to transfect susceptible mammalian cells so that tyrosinase is produced therefrom.
A further method of treating 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 melanin-concentrating 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.
2. Prophylaxis
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 include N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP.sup.+) and manganese dust for Parkinson’s disease; quinolinic acid for Huntington’s chorea; .beta.-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, MP.sup.+, has been field-tested as a herbicide under the name Cyperquat. The well-known herbicide Paraquat chemically resembles MPP.sup.+. 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, 4-phenylpyridine, 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
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