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WO2004112806A1 - Composition for treating or preventing neural disorders - Google Patents

Composition for treating or preventing neural disorders Download PDF

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Publication number
WO2004112806A1
WO2004112806A1 PCT/CA2004/000951 CA2004000951W WO2004112806A1 WO 2004112806 A1 WO2004112806 A1 WO 2004112806A1 CA 2004000951 W CA2004000951 W CA 2004000951W WO 2004112806 A1 WO2004112806 A1 WO 2004112806A1
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Prior art keywords
composition
cells
antler
extract
treatment
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PCT/CA2004/000951
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French (fr)
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WO2004112806A8 (en
Inventor
Binh Nguyen-Quoc
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Canada Changmin Nutraceutique Co. Ltd
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Publication of WO2004112806A1 publication Critical patent/WO2004112806A1/en
Publication of WO2004112806A8 publication Critical patent/WO2004112806A8/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • This invention concerns agents, and methods of their use, which protect a neuron and nervous system of animals or humans from injury or degeneration. b) Description of the prior art
  • Growth factors constitute a large family of proteins fundamentally devoted to reproduction, differentiation, maturation and survival of cells.
  • protein production through genetic engineering and biological techniques led to important discoveries on the physio-pathologic roles in mammalian bodies of several growth factors: as hyper- or hypo-production of growth factors by nearby or circulating cells has been linked to a large number of unrelated diseases, such as diabetes, angiogenesis, stroke, hypertensive arterial hypertrophy, atherosclerosis, restenosis, glomerular nephritis, cancer and so on.
  • neurotrophins As regards specifically cells of the nervous system, it has been shown in the art that the mammalian neuronal development is controlled by a family of growth factors, later called neurotrophins, whose most important member appears to be Nerve Growth
  • NGF Neuron Factor
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • stroke which are generally associated with neuron loss or death.
  • AD Alzheimer's disease
  • the incidence of many neurodegenerative diseases increases rapidly with aging. For example, the percent of the individuals below age sixty-five who have Alzheimer's disease is less than five percent, but this incidence increases almost exponentially over age sixty-five, and as many as forty-seven percent of individuals over eighty-five years of age may have some form of AD.
  • the brain of essentially all individuals studied over age eighty contain at least some age- and/or disease-related neuron loss.
  • aging itself is a significant factor for several types of neurodegenerative diseases and somatic degenerative, indicating that aging increases susceptibility to neuro loss. It is admitted that the causes of natural neuron loss and neurodegenerative diseases are still largely unknown. ' Neurons and entire regions of the nervous system can be severely damaged or killed during conditions or events such as strokes, drowning, carbon monoxide poisoning, cardiac arrest, or internal hemorrhaging due to rupture of an aneurysm. Various types of head injuries or other physical traumas, surgical damage, and certain types of poisons can also lead to permanent brain damage involving "excitotoxicity" (discussed below), which involves the excessive release or abnormal accumulation of excitatory neurotransmitters such as glutamic and aspartic acid.
  • Seizures or convulsions due to epilepsy, head trauma, or other causes also involve the excessive release of excitatory neurotransmitters; although relatively mild seizures are not presumed to cause neuronal death or permanent damage, some types of severe seizures which cannot be halted by normal drugs, such as temporal lobe epilepsy (status epilepticus), are believed to cause permanent brain damage and neuronal death due to excitotoxicity.
  • temporal lobe epilepsy status epilepticus
  • these so-called calcium channel blockers suppress activity at an entirely different type of calcium ion channel, known as the L-channel, which is not a pre-synaptic channel and which has no known direct effect on excitotoxic glutamate release at synapses.
  • One object of the present invention is to provide a composition for treating or preventing neuronal diseases comprising antler extract.
  • the neuronal disease can be an injury, an induced or natural degenerescence, acquired or physical or natural damage to nerve cells of an individual.
  • composition comprising the antler extract can be used as well as a neutraceutical as a cosmeceutical composition.
  • Another object of the present invention is the use of antler extract for the preparation of a composition for treating or preventing a neuronal disease.
  • a method for treating or preventing a neuronal disease comprising administrating antler extract to an individual, in a sufficient amount to partially or totally treat or prevent the occurrence or development of a neuronal disease in said individual. It will be understood that the composition is preferentially orally administered.
  • composition of the present invention can be administered by the sublingual route, or alternatively topically, intradermally, subcutaneously or by any other way allowing the delivery of the active compounds of the composition.
  • Another object of the present invention is to provide a method for regulating neuron development, maintenance and regeneration in the central and peripheral nervous systems of a mammal and to pharmaceutical compositions comprising antler powder useful for same.
  • the present invention is particularly useful in the treatment or prevention of developmental and cerebral anomalies and neuropathies in mammals and in particular humans.
  • compositions or agents comprised therein are able to either totally reverse, or at least partially reverse, the effect of biological, physical or chemical compounds involved in the injury or degeneration on that neuron.
  • the composition is able to reduce the effect of neuro-toxical compounds on the neuron, and thereby significantly increase the chances of that neuron surviving in the presence of such substance.
  • animal is intended to include any animal to which an agent of the invention can be administered for the indicated purpose, including both medicinal and veterinary purposes. Use in mammals and birds of all types is preferred, with use in humans being a primary utility.
  • one object of the subject invention is to provide that differentiation or growth promoting compounds found in antler extracts, and which have been extensively tested and proven to be safe as being capable of preventing or reducing neuronal injury or death due to ischemic/hypoxic events such as stroke or cardiac arrest, and to other events or conditions that cause excitotoxic damage to neurons, such as epileptic seizures and possibly some types of progressive neurodegenerative diseases.
  • Fig. 1 illustrates the releasing of LDH in the culture medium following treatment with different doses of antler extracts S or W;
  • Fig. 2 illustrates the activity of mitochondrial marker XTT in cells PC 12 after treatment with different antler extracts S or W;
  • Fig. 3 illustrates the release of LDH in the culture medium after chronic treatment with antler extracts S and W;
  • Fig. 4 illustrates the activity of mitochondrial marker XXT in cultured cells PC 12 following chronic treatment with antler extracts S and W;
  • Fig. 5 illustrates the effect of preventive treatment with antler extracts S and W on cultured cells PC 12;
  • Fig. 6 illustrates the effect of chronic preventive treatment on cultured PC 12 cells
  • Fig. 7 illustrates the effect of chronic preventive treatment with S and W antler extracts on cultured mammalian cells.
  • a composition comprising antler extract, which has been recently shown to have remedi activities on the nervous system and nerve cells in preventing or treating related diseases.
  • components of the antler extracts provide protection of neurons from injury caused by the effect of reducing or oxidizing agents.
  • these components can be used to protect neurons in different condition in vivo, and specifically those present in the central nervous system (CNS) of a human.
  • CNS central nervous system
  • One embodiment of the present invention is to provide a composition comprising antler extract in different concentrations, while the antler extract can be administered to an individual or a patient under pure form for the prevention or treatment of pathological conditions involving nerve cells and nervous tissue.
  • pathological conditions involving nerve cells and nervous tissue For example, natural, physical or toxicologically induced damages to the brain or other nervous system parts of the body, as well as several other types of progressive neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS) also involved in excessive neurotransmitter release or accumulation as a component of the disease process, can be modulated, treated or prevent with the composition of the present invention.
  • ALS amyotrophic lateral sclerosis
  • the antler extract composition of the present invention can also be useful in promoting protection against degenerative disorders and help in preserving the integrity of nerve cells and nervous system or brain for a longer period than what it occurs in nature.
  • the present invention also provides biologically active proteinaceous composition containing nerve growth factors in substantially homogeneous form by means of biochemical and physical preparation from antler tissue.
  • the invention encompasses these naturally occurring nerve growth factors in partially purified as well as substantially homogeneous form, as well as synthetically produced nerve growth factors, biologically active fragments thereof, and pharmaceutically acceptable salts and derivatives thereof.
  • biologically active polypeptide as used herein means naturally occurring polypeptide per se, as well as biologically active analogues thereof, including synthetically produced polypeptides and analogues thereof, as well as natural and pharmaceutically acceptable salts and pharmaceutically acceptable derivatives thereof.
  • biologically active polypeptide also encompasses biologically active fragments thereof, as well as biologically active sequence analogues thereof. Different forms may exist in nature.
  • the molecular weight of peptides or polypeptides found in the antler extract or fractions of the present invention can be between 250 to 500 000 Daltons (Da).
  • the antler extract can be fractionated into fractions containing polypeptides or peptides with different ranges of molecular weight.
  • a desired physiological effect can be obtained with a fraction, of the antler extract, comprising polypeptides having a molecular weight of between 1000 to 7000 DA, or between 20 000 to 30 000 DA, or again between 24 000 to 28 000 DA. This will allows to optimize and select a more selective effect of the fraction, or of the antler extract.
  • a nerve growth factor like effect can be found with polypeptides having a molecular weight between 20 000 to 30 000 Da.
  • a antler fraction according to another embodiment of the present invention, may comprise polypeptides having a molecular weight between 1000 and 7000, and have also a nerve protective or nerve stimulatory effects.
  • the invention features a method for protecting a neuron of an organism from neuronal injury. The method includes identifying an organism susceptible to neuronal injury, providing a physiologically acceptable composition including antler extract which causes protection of nerve cells, and administering the composition to the organism in an amount sufficient to protect the nerve cells.
  • Organisms e.g., human patients, susceptible to neuronal injury are identified by any of a number standard techniques.
  • Such patients will include those discussed above which are susceptible to, for example, or suffer from, strokes, anoxia and certain degenerative diseases. They will also include those patients which have no symptoms but are found to have abnormally high levels of metabolites or related compounds in the nervous system.
  • the antler extracts which are prepared under different forms, including for example but not limited to, pills, powder, cream or any other oral or topical acceptable forms, or by any other method, may be used for treatment of these patients.
  • Those skilled in the art will recognize how to determine, by routine experimentation, the amount of extract necessary to provide sufficient treatment or protection of nerve cells without causing significant deleterious or side effects, to the patient. Generally, this amount will be a balance between a level of extract where the potential of causing such deleterious effects is significant and a level where the extract provides complete protection against injury to the nerve cells or the nervous system.
  • the biologically active proteinaceous factors of the antler extracts of the present invention useful in the therapeutic and prophylactic method of the present invention may be also employed in such forms as liquid solutions, aqueous or not, suspensions, elixirs, or sterile liquid forms such as solutions or suspensions.
  • Suitable earners include diluents or fillers, sterile aqueous media and various non-toxic organic solvents.
  • the compositions may be formulated in the fo ⁇ n of powders as mentioned before, aqueous suspensions, or solutions, injectable solutions, elixirs, syrups and the like and may contain one or more stabilization agents such as human serum albumin, sugar or amino acid, antibacterial, and preserving agents in order to provide a pharmaceutically acceptable preparation.
  • Any inert carrier is preferably used, such as saline, or phosphate- buffered saline, or any such carrier in which the factors used in the method of the present invention have suitable solubility properties for use in the method of the present
  • the particular carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the antler extracts, the particular mode of administration and standard pharmaceutical practice.
  • solutions or suspensions of these factors in aqueous alcoholic media or in sesame or peanut oil or aqueous solutions of the soluble pharmaceutically acceptable salts can be employed.
  • the dosage regimen in carrying out the methods of this invention is that which insures maximum therapeutic response until improvement is obtained and thereafter the minimum effective level which gives relief.
  • Doses may vary, depending on the age, severity, body weight and other conditions of the patients With other forms of administration equivalent or adjusted doses will be administered depending on the route of administration.
  • one aspect of the present invention contemplates a method for regulating neuron development and/or maintenance and/or regeneration in a mammal comprising administering to said mammal an effective amount of antler extract for a time and under conditions sufficient to permit the differentiation and/or maintenance and/or regeneration of neural precursor cells into neurons.
  • Another aspect of the present invention relates to a method for enhancing and or stimulating and/or maintaining and/or regenerating the formation and/or survival of neurons in the nervous system of a mammal which comprises administering to said mammal an effective amount of antler extracts for a time and under conditions sufficient to effect an increase in and/or to maintain the number of neurons in the nervous system.
  • the antler extract enhances, stimulates, maintains (i.e. promotes survival) and/or regenerates immature neurons.
  • Yet another aspect of the present invention relates to a method for enhancing, stimulating and or maintaining the formation and/or survival of sensory neurons, for example sensory neurons, of the peripheral nervous system of a mammal which comprises administering to said mammal an effective amount of antler extract for a time and under conditions sufficient to effect an increase in and/or to maintain the number of neurons in the peripheral nervous system.
  • regulating neuron development, maintenance and regeneration is meant to include stimulating, enhancing and/or maintaining the formation and/or survival of neurons in the central and peripheral nervous systems of a mammal. It also includes the ability of said extract to assist the regeneration of properties associated with neuronal function following damage caused by disease or trauma. It is also includes stimulating, enhancing, maintaining and/or regenerating those properties associated with neurons such as, but not limited to, neurotransmitter type, receptor type and other features associated with this phenotype. In .particular, antler extracts or fractions can be used to induce, stimulate, enhance, maintain and/or regenerate the differentiation of immature nerve cells into fully mature neurons. This effect is titrable and occurs in the absence of proliferation of neuronal precursor cells.
  • antler extract also extends to the stimulation of the differentiation of precursor cells in embryonic dorsal root ganglia (DRG) into mature sensory neurons. Factors of these fractions might stimulate the differentiation of the sensory precursor cells. It was, therefore, surprisingly found in accordance with the present invention that antler extract stimulated the survival of nerve cells in the culture and that it acted as a survival factor for these neurons throughout lasting cultures under different conditions.
  • DRG embryonic dorsal root ganglia
  • Antler extracts can also potentiate the central nervous system.
  • the early steps in the development of the central nervous system from the embryonic precursor cells of the neural tube involves expansion of the precursor population and differentiation of these cells into mature neurons and glial cells. This phase is followed by a selective survival of neurons which have appropriately innervated the correct targets and is believed to be based on the limited availability of survival factors which are produced by the target cells.
  • Still yet another aspect of the invention contemplates a method of treatment of disease and injury in both the central and peripheral nervous systems in a mammal, said disease or injury including but not limited to one or more of Cerebral Palsy, trauma induced paralysis, vascular ischemia associated with stroke, neuronal tumors, motor- neuron disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis and peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and/or infectious diseases such as herpes, rubella, measles, chicken pox, HIV and/or HTLV-1 which comprises administering to said mammal an effective amount of antler extract for a time and under conditions sufficient to ameliorate the disease or injury.
  • the present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
  • Antlers from wapitis and red dear were taken at 60 days of growth, then frozen, despoiled, and crunched into rough particles (half of wapiti antler and half of red dear antler).
  • a first fraction (Fraction S) of the antler particles was kept at -80°C up until next processing step. Then it was placed at -20°C just before grinding into a fine powder.
  • the second fraction (Fraction W) was directly, without freezing, lyophilized and grinded into a fine powder.
  • the two fractions were diluted in bi-distilled water in proportion of 1:4 (w/v), agitated 2 minutes, and placed on ice for 5 minutes. The fractions were then centrifuged 5 minutes at 3000g.
  • the supematants about 5 ml, were first filtered through rock wool, then through a 0.22 ⁇ m filter, and filtrates were finally frozen at -20°C. Protein concentration of the two fractions were measured have been found to correspond at about 41 mg/ml (Fraction S) and 22 mg/ml (Fraction W). IGF-1 (the growth factor marker) was found at concentration of 5 and 10 ng/ml for each fraction respectively, and the means molecular weight was of about 60 000 Da for each fraction.
  • the toxicity assessment began with the study of the effects of extracts composed of aqueous fractions S and W following an acute treatment (on PC 12 cells in culture).
  • the cells survival was measured by LDH and XTT tests.
  • the LDH test determines the cellular toxicity on the basis of release in the medium of lactate dehydrogenase enzyme, and the XTT test, linked to the activity of mitochondrial succinate dehydrogenase, measures the percentage of living cells after a treatment. Once confluence is reached, PC 12 cells have been treated only once by extracts S or W at recommended concentrations. LDH and XTT tests were made 24 hours after.the addition of extracts S or W. For each dose and treatment, the experiments were executed three times with a minimum of three repetitions by experiment.
  • Deer velvet antler extracts S and W are not toxics on PC 12 cells, 24 hours after an acute treatment, even at concentrations of 50 ⁇ g/mL (Fig. 1). In presence of extract W,
  • XTT Test Deer velvet antler extracts S and W do not induce mortality of PC 12 cells compared to control, 24 hours after an acute treatment (Fig. 2). In fact, the activity of the mitochondrial marker is always at least equal or higher to the control.
  • the XTT test confirms the results obtained by the LDH method. It can be noted that at some concentrations, the XTT activity is significantly higher to the control, suggesting that deer velvet antler extract either lowers the mortality of PC12 cells, or stimulates their proliferation in the culture medium. From the results of these tests, the pursuing of the characterization of extracts with PC 12 cells might be considered, using concentrations in proteins between 1 and 50 ⁇ g/mL without leading to toxic effects.
  • Deer velvet antler extracts S and W are not toxics on PC 12 cells, one week after a chronic treatment (Fig. 3). However, an increase of LDH release in the culture medium treated with extract S at a concentration of 500 ⁇ g/mL can be observed. This cellular toxicity with a high concentration was not observed with the extract W prepared with a lyophilized deer velvet antler.
  • the XTT test shows on the opposite, that in presence of extract S at concentrations from 1 to 100 ⁇ g/mL, the number of surviving cells is higher compared to the control. This effect disappears while proceeding to the chronic treatment with extract S at a concentration of 500 ⁇ g/mL.
  • Treatments with extract W at concentrations from 1 to 100 ⁇ g/mL do not cause any change on cellular survival compared to the control.
  • the chronic treatment with 500 ⁇ g/mL of extract W leads to a significant increase of cellular survival compared to the control.
  • Fig. 6 The results of Fig. 6 show that in this test, a treatment with a concentration of hydrogen dioxide at 5.10 "5 M do not cause a significant mortality because the number of surviving cells after one week is the same in T and H 2 0 2 controls (Fig. 6). However, we note that even after a hydrogen dioxide treatment, the protecting effect of deer velvet antler as observed in conditions where only experimental stress intervenes (ex. medium change) is always significant. In this case, the treatment with hydrogen dioxide causes a very drastic effect on PC12 cells survival (Fig. 7).

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Abstract

The present invention relates to a method of use and composition containing antler extract as active component in the prevention or treatment of nerve cells or nervous system diseases. Particularly the composition of the invention is useful in treating or preventing nerve cells of or against degenerescence and oxidation.

Description

COMPOSITION FOR TREATING OR PREVENTING NEURAL
DISORDERS
BACKGROUND OF THE INVENTION a) Field of the invention
This invention concerns agents, and methods of their use, which protect a neuron and nervous system of animals or humans from injury or degeneration. b) Description of the prior art
It is known that unlike many other cell types, neurons cannot always be replaced in the adult, and this particularly in brain. Thus, neuron loss in the adult brain has crippling and generally irreversible consequences whether it is caused by age, disease, trauma, or combinations thereof.
Growth factors constitute a large family of proteins fundamentally devoted to reproduction, differentiation, maturation and survival of cells. In the last few years, the tremendous developments in. protein production through genetic engineering and biological techniques led to important discoveries on the physio-pathologic roles in mammalian bodies of several growth factors: as hyper- or hypo-production of growth factors by nearby or circulating cells has been linked to a large number of unrelated diseases, such as diabetes, angiogenesis, stroke, hypertensive arterial hypertrophy, atherosclerosis, restenosis, glomerular nephritis, cancer and so on.
As regards specifically cells of the nervous system, it has been shown in the art that the mammalian neuronal development is controlled by a family of growth factors, later called neurotrophins, whose most important member appears to be Nerve Growth
Factor (NGF), which exerts its effects on defined cell populations of neurons within the nervous system.
In other situations relating to the nervous system diseases, the cause of neuron loss during aging is unknown. It is now well recognized that almost everyone who lives long enough may develop nervous diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and stroke, which are generally associated with neuron loss or death. The incidence of many neurodegenerative diseases increases rapidly with aging. For example, the percent of the individuals below age sixty-five who have Alzheimer's disease is less than five percent, but this incidence increases almost exponentially over age sixty-five, and as many as forty-seven percent of individuals over eighty-five years of age may have some form of AD. The brain of essentially all individuals studied over age eighty contain at least some age- and/or disease-related neuron loss. Thus, aging itself is a significant factor for several types of neurodegenerative diseases and somatic degenerative, indicating that aging increases susceptibility to neuro loss. It is admitted that the causes of natural neuron loss and neurodegenerative diseases are still largely unknown. ' Neurons and entire regions of the nervous system can be severely damaged or killed during conditions or events such as strokes, drowning, carbon monoxide poisoning, cardiac arrest, or internal hemorrhaging due to rupture of an aneurysm. Various types of head injuries or other physical traumas, surgical damage, and certain types of poisons can also lead to permanent brain damage involving "excitotoxicity" (discussed below), which involves the excessive release or abnormal accumulation of excitatory neurotransmitters such as glutamic and aspartic acid. Seizures or convulsions due to epilepsy, head trauma, or other causes also involve the excessive release of excitatory neurotransmitters; although relatively mild seizures are not presumed to cause neuronal death or permanent damage, some types of severe seizures which cannot be halted by normal drugs, such as temporal lobe epilepsy (status epilepticus), are believed to cause permanent brain damage and neuronal death due to excitotoxicity.
These problems are severely aggravated by the inability of nerve cells to regenerate or repair themselves after injury; a child who suffers only a few minutes of perinatal asphyxia during birth may spend an entire lifetime horribly crippled by the injuries such damage can inflict, and many people who have suffered from strokes live the remainder of their lives partially paralyzed or unable to speak or remember different major events.
A substantial amount of research has been devoted to testing various drugs, or event different natural products or extracts to treat or prevent neuronal diseases or injuries. Among the drugs, for example, flunarizine, nicardipine, and nifedipine have been tested to determine whether they can reduce neuronal damage due to ischemia. Although these drugs are known as "calcium channel blockers," the results in tests to evaluate their protection against ischemic neuronal damage have been inconsistent. It is suspected that any beneficial effects they may have may be due to (1) vasodilatation (i.e., blood vessel dilating) effects, which in some animal models can provide more blood to a brain region that is being experimentally deprived of blood flow; and (2) reduction in the formation of highly reactive oxygen radicals. It should be emphasized that these so-called calcium channel blockers suppress activity at an entirely different type of calcium ion channel, known as the L-channel, which is not a pre-synaptic channel and which has no known direct effect on excitotoxic glutamate release at synapses.
Even though different approaches or drugs have been tested and used in the art to prevent or treat neuronal diseases or death or diseases of the nervous system, there is still large place to be provided with new compositions or products to reach this goal more physiologically.
SUMMARY OF THE INVENTION
One object of the present invention is to provide a composition for treating or preventing neuronal diseases comprising antler extract. The neuronal disease can be an injury, an induced or natural degenerescence, acquired or physical or natural damage to nerve cells of an individual.
The composition comprising the antler extract can be used as well as a neutraceutical as a cosmeceutical composition.
Another object of the present invention is the use of antler extract for the preparation of a composition for treating or preventing a neuronal disease.
In addition, in accordance with the present invention there is provided a method for treating or preventing a neuronal disease comprising administrating antler extract to an individual, in a sufficient amount to partially or totally treat or prevent the occurrence or development of a neuronal disease in said individual. It will be understood that the composition is preferentially orally administered.
It will be recognized by those skilled in the art that the composition of the present invention can be administered by the sublingual route, or alternatively topically, intradermally, subcutaneously or by any other way allowing the delivery of the active compounds of the composition.
Another object of the present invention is to provide a method for regulating neuron development, maintenance and regeneration in the central and peripheral nervous systems of a mammal and to pharmaceutical compositions comprising antler powder useful for same. The present invention is particularly useful in the treatment or prevention of developmental and cerebral anomalies and neuropathies in mammals and in particular humans.
By protection of a neuron from neuronal injury or degeneration is meant that the composition or agents comprised therein is able to either totally reverse, or at least partially reverse, the effect of biological, physical or chemical compounds involved in the injury or degeneration on that neuron. Alternatively, the composition is able to reduce the effect of neuro-toxical compounds on the neuron, and thereby significantly increase the chances of that neuron surviving in the presence of such substance.
The term "animal" is intended to include any animal to which an agent of the invention can be administered for the indicated purpose, including both medicinal and veterinary purposes. Use in mammals and birds of all types is preferred, with use in humans being a primary utility.
Accordingly, one object of the subject invention is to provide that differentiation or growth promoting compounds found in antler extracts, and which have been extensively tested and proven to be safe as being capable of preventing or reducing neuronal injury or death due to ischemic/hypoxic events such as stroke or cardiac arrest, and to other events or conditions that cause excitotoxic damage to neurons, such as epileptic seizures and possibly some types of progressive neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the releasing of LDH in the culture medium following treatment with different doses of antler extracts S or W;
Fig. 2 illustrates the activity of mitochondrial marker XTT in cells PC 12 after treatment with different antler extracts S or W;
Fig. 3 illustrates the release of LDH in the culture medium after chronic treatment with antler extracts S and W;
Fig. 4 illustrates the activity of mitochondrial marker XXT in cultured cells PC 12 following chronic treatment with antler extracts S and W; Fig. 5 illustrates the effect of preventive treatment with antler extracts S and W on cultured cells PC 12;
Fig. 6 illustrates the effect of chronic preventive treatment on cultured PC 12 cells; and
Fig. 7 illustrates the effect of chronic preventive treatment with S and W antler extracts on cultured mammalian cells.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention, may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
In accordance with the present invention, there is provided a composition comprising antler extract, which has been recently shown to have benefic activities on the nervous system and nerve cells in preventing or treating related diseases. Applicants have discovered that components of the antler extracts provide protection of neurons from injury caused by the effect of reducing or oxidizing agents. Thus, these components can be used to protect neurons in different condition in vivo, and specifically those present in the central nervous system (CNS) of a human. These compounds are particularly useful for treatment of humans suffering from strokes, anoxia or certain degenerative diseases.
One embodiment of the present invention is to provide a composition comprising antler extract in different concentrations, while the antler extract can be administered to an individual or a patient under pure form for the prevention or treatment of pathological conditions involving nerve cells and nervous tissue. For example, natural, physical or toxicologically induced damages to the brain or other nervous system parts of the body, as well as several other types of progressive neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS) also involved in excessive neurotransmitter release or accumulation as a component of the disease process, can be modulated, treated or prevent with the composition of the present invention.
The antler extract composition of the present invention can also be useful in promoting protection against degenerative disorders and help in preserving the integrity of nerve cells and nervous system or brain for a longer period than what it occurs in nature. The present invention also provides biologically active proteinaceous composition containing nerve growth factors in substantially homogeneous form by means of biochemical and physical preparation from antler tissue. The invention encompasses these naturally occurring nerve growth factors in partially purified as well as substantially homogeneous form, as well as synthetically produced nerve growth factors, biologically active fragments thereof, and pharmaceutically acceptable salts and derivatives thereof. It will be recognized to those skilled in the art that several other elements, such as but not limited to, calcium, sodium, potassium or any other salts or amino acids, lipids or compounds included in the antler extract composition synergistically act with the nerve growth factor where necessary on the nerve cells or the nervous system The term "biologically active polypeptide" as used herein means naturally occurring polypeptide per se, as well as biologically active analogues thereof, including synthetically produced polypeptides and analogues thereof, as well as natural and pharmaceutically acceptable salts and pharmaceutically acceptable derivatives thereof. The term biologically active polypeptide also encompasses biologically active fragments thereof, as well as biologically active sequence analogues thereof. Different forms may exist in nature. The molecular weight of peptides or polypeptides found in the antler extract or fractions of the present invention can be between 250 to 500 000 Daltons (Da). Preferbbly, the antler extract can be fractionated into fractions containing polypeptides or peptides with different ranges of molecular weight. For example, but not limited to, a desired physiological effect can be obtained with a fraction, of the antler extract, comprising polypeptides having a molecular weight of between 1000 to 7000 DA, or between 20 000 to 30 000 DA, or again between 24 000 to 28 000 DA. This will allows to optimize and select a more selective effect of the fraction, or of the antler extract. For example, a nerve growth factor like effect can be found with polypeptides having a molecular weight between 20 000 to 30 000 Da. Complementary to this, a antler fraction according to another embodiment of the present invention, may comprise polypeptides having a molecular weight between 1000 and 7000, and have also a nerve protective or nerve stimulatory effects. In another aspect, the invention features a method for protecting a neuron of an organism from neuronal injury. The method includes identifying an organism susceptible to neuronal injury, providing a physiologically acceptable composition including antler extract which causes protection of nerve cells, and administering the composition to the organism in an amount sufficient to protect the nerve cells. Organisms, e.g., human patients, susceptible to neuronal injury are identified by any of a number standard techniques. Such patients will include those discussed above which are susceptible to, for example, or suffer from, strokes, anoxia and certain degenerative diseases. They will also include those patients which have no symptoms but are found to have abnormally high levels of metabolites or related compounds in the nervous system. The antler extracts which are prepared under different forms, including for example but not limited to, pills, powder, cream or any other oral or topical acceptable forms, or by any other method, may be used for treatment of these patients. Those skilled in the art will recognize how to determine, by routine experimentation, the amount of extract necessary to provide sufficient treatment or protection of nerve cells without causing significant deleterious or side effects, to the patient. Generally, this amount will be a balance between a level of extract where the potential of causing such deleterious effects is significant and a level where the extract provides complete protection against injury to the nerve cells or the nervous system.
The biologically active proteinaceous factors of the antler extracts of the present invention useful in the therapeutic and prophylactic method of the present invention may be also employed in such forms as liquid solutions, aqueous or not, suspensions, elixirs, or sterile liquid forms such as solutions or suspensions. Suitable earners include diluents or fillers, sterile aqueous media and various non-toxic organic solvents. The compositions may be formulated in the foπn of powders as mentioned before, aqueous suspensions, or solutions, injectable solutions, elixirs, syrups and the like and may contain one or more stabilization agents such as human serum albumin, sugar or amino acid, antibacterial, and preserving agents in order to provide a pharmaceutically acceptable preparation. Any inert carrier is preferably used, such as saline, or phosphate- buffered saline, or any such carrier in which the factors used in the method of the present invention have suitable solubility properties for use in the method of the present invention.
The particular carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the antler extracts, the particular mode of administration and standard pharmaceutical practice. For parenteral administration, solutions or suspensions of these factors in aqueous alcoholic media or in sesame or peanut oil or aqueous solutions of the soluble pharmaceutically acceptable salts can be employed.
The dosage regimen in carrying out the methods of this invention is that which insures maximum therapeutic response until improvement is obtained and thereafter the minimum effective level which gives relief. Doses may vary, depending on the age, severity, body weight and other conditions of the patients With other forms of administration equivalent or adjusted doses will be administered depending on the route of administration.
Accordingly, one aspect of the present invention contemplates a method for regulating neuron development and/or maintenance and/or regeneration in a mammal comprising administering to said mammal an effective amount of antler extract for a time and under conditions sufficient to permit the differentiation and/or maintenance and/or regeneration of neural precursor cells into neurons.
Another aspect of the present invention relates to a method for enhancing and or stimulating and/or maintaining and/or regenerating the formation and/or survival of neurons in the nervous system of a mammal which comprises administering to said mammal an effective amount of antler extracts for a time and under conditions sufficient to effect an increase in and/or to maintain the number of neurons in the nervous system.
In one embodiment, the antler extract enhances, stimulates, maintains (i.e. promotes survival) and/or regenerates immature neurons. Yet another aspect of the present invention relates to a method for enhancing, stimulating and or maintaining the formation and/or survival of sensory neurons, for example sensory neurons, of the peripheral nervous system of a mammal which comprises administering to said mammal an effective amount of antler extract for a time and under conditions sufficient to effect an increase in and/or to maintain the number of neurons in the peripheral nervous system.
By "regulating neuron development, maintenance and regeneration" as used herein is meant to include stimulating, enhancing and/or maintaining the formation and/or survival of neurons in the central and peripheral nervous systems of a mammal. It also includes the ability of said extract to assist the regeneration of properties associated with neuronal function following damage caused by disease or trauma. It is also includes stimulating, enhancing, maintaining and/or regenerating those properties associated with neurons such as, but not limited to, neurotransmitter type, receptor type and other features associated with this phenotype. In .particular, antler extracts or fractions can be used to induce, stimulate, enhance, maintain and/or regenerate the differentiation of immature nerve cells into fully mature neurons. This effect is titrable and occurs in the absence of proliferation of neuronal precursor cells. The effect of antler extract also extends to the stimulation of the differentiation of precursor cells in embryonic dorsal root ganglia (DRG) into mature sensory neurons. Factors of these fractions might stimulate the differentiation of the sensory precursor cells. It was, therefore, surprisingly found in accordance with the present invention that antler extract stimulated the survival of nerve cells in the culture and that it acted as a survival factor for these neurons throughout lasting cultures under different conditions.
Antler extracts can also potentiate the central nervous system. The early steps in the development of the central nervous system from the embryonic precursor cells of the neural tube involves expansion of the precursor population and differentiation of these cells into mature neurons and glial cells. This phase is followed by a selective survival of neurons which have appropriately innervated the correct targets and is believed to be based on the limited availability of survival factors which are produced by the target cells. Still yet another aspect of the invention contemplates a method of treatment of disease and injury in both the central and peripheral nervous systems in a mammal, said disease or injury including but not limited to one or more of Cerebral Palsy, trauma induced paralysis, vascular ischemia associated with stroke, neuronal tumors, motor- neuron disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis and peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and/or infectious diseases such as herpes, rubella, measles, chicken pox, HIV and/or HTLV-1 which comprises administering to said mammal an effective amount of antler extract for a time and under conditions sufficient to ameliorate the disease or injury. The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope. EXAMPLE I Preparation of two model fractions of antler extracts
Antlers from wapitis and red dear were taken at 60 days of growth, then frozen, despoiled, and crunched into rough particles (half of wapiti antler and half of red dear antler). A first fraction (Fraction S) of the antler particles was kept at -80°C up until next processing step. Then it was placed at -20°C just before grinding into a fine powder. The second fraction (Fraction W) was directly, without freezing, lyophilized and grinded into a fine powder. The two fractions were diluted in bi-distilled water in proportion of 1:4 (w/v), agitated 2 minutes, and placed on ice for 5 minutes. The fractions were then centrifuged 5 minutes at 3000g. The supematants, about 5 ml, were first filtered through rock wool, then through a 0.22μm filter, and filtrates were finally frozen at -20°C. Protein concentration of the two fractions were measured have been found to correspond at about 41 mg/ml (Fraction S) and 22 mg/ml (Fraction W). IGF-1 (the growth factor marker) was found at concentration of 5 and 10 ng/ml for each fraction respectively, and the means molecular weight was of about 60 000 Da for each fraction.
EXAMPLE II Survival and toxicity tests following an acute treatment
Materials and Methods
The toxicity assessment began with the study of the effects of extracts composed of aqueous fractions S and W following an acute treatment (on PC 12 cells in culture). The cells survival was measured by LDH and XTT tests. The LDH test determines the cellular toxicity on the basis of release in the medium of lactate dehydrogenase enzyme, and the XTT test, linked to the activity of mitochondrial succinate dehydrogenase, measures the percentage of living cells after a treatment. Once confluence is reached, PC 12 cells have been treated only once by extracts S or W at recommended concentrations. LDH and XTT tests were made 24 hours after.the addition of extracts S or W. For each dose and treatment, the experiments were executed three times with a minimum of three repetitions by experiment.
LDH Test
Deer velvet antler extracts S and W are not toxics on PC 12 cells, 24 hours after an acute treatment, even at concentrations of 50 μg/mL (Fig. 1). In presence of extract W,
LDH values are lower than in the non-treated control, showing a lower mortality of cells in presence of the extract. These results show that extract W could protect PC 12 cells against cellular death induced by experimental stress such as culture medium change.
XTT Test , Deer velvet antler extracts S and W do not induce mortality of PC 12 cells compared to control, 24 hours after an acute treatment (Fig. 2). In fact, the activity of the mitochondrial marker is always at least equal or higher to the control. The XTT test confirms the results obtained by the LDH method. It can be noted that at some concentrations, the XTT activity is significantly higher to the control, suggesting that deer velvet antler extract either lowers the mortality of PC12 cells, or stimulates their proliferation in the culture medium. From the results of these tests, the pursuing of the characterization of extracts with PC 12 cells might be considered, using concentrations in proteins between 1 and 50 μg/mL without leading to toxic effects.
EXAMPLE III Survival and toxicity tests following a chronic treatment
The possible toxic effects of extracts S and W were studied after a chronic (long lasting repetitions) treatment on PC 12 cells. The cells were treated every two days during one week with different concentrations of S or W aqueous fractions. By the end of the week, the toxicity and survival of cells were evaluated by LDH and WTT tests. An additional treatment with concentrations of 500 μg/mL was added. LDH Test
Deer velvet antler extracts S and W are not toxics on PC 12 cells, one week after a chronic treatment (Fig. 3). However, an increase of LDH release in the culture medium treated with extract S at a concentration of 500 μg/mL can be observed. This cellular toxicity with a high concentration was not observed with the extract W prepared with a lyophilized deer velvet antler.
XTT Test
Deer velvet antler extracts S and W "do not induce mortality of PC 12 cells, one week after a chronic treatment (Fig. 4). The XTT test shows on the opposite, that in presence of extract S at concentrations from 1 to 100 μg/mL, the number of surviving cells is higher compared to the control. This effect disappears while proceeding to the chronic treatment with extract S at a concentration of 500 μg/mL. Treatments with extract W at concentrations from 1 to 100 μg/mL do not cause any change on cellular survival compared to the control. The chronic treatment with 500 μg/mL of extract W leads to a significant increase of cellular survival compared to the control.
EXAMPLE IV Preventive effect of deer velvet antler in presence of hydrogen dioxide
! After having showed that deer velvet antler extracts did not present any toxicity on PC 12 cells in culture, we studied the protecting effect of the extracts in preventive treatment. The hydrogen dioxide (H202) was chosen as a neurotoxic agent, because it is pro-oxidative and pro-apoptotic. For each dose and each S or W compound, experiments were executed three times with at least three repetitions. Once confluence was reached, PC 12 cells were treated only once with deer velvet antler extracts. A neurotoxic treatment with H202 at a final concentration of 5 10" 5M was realized 24 hours after the deer velvet antler treatment. A XTT test was made 24 hours after having added neurotoxin to evaluate the surviving cells rate. The results show that the treatment with 5 10"5M H202 leads to a decrease of 50% of PC12 living cells compared to the control which received no treatment (Fig. 5A). When the cells have received an acute treatment with extract S at concentrations of 5, 10 and 50 μg/ml, the number of surviving cells after a H202 treatment faces a slight decrease compared to the control which did not receive neurotoxin but stays higher to the control which has not been treated with deer velvet antler (Fig. 5A). Furthermore, after an acute treatment with extract S at concentrations of 1 and 100 μg/ml, the number of surviving cells is near twice superior to the control which received no treatment. These results suggest a preventive protective effect of extract S towards H202. Whether it is in presence of H202 or not (Figs. 2 and 5 A), an acute treatment with extract S, especially in concentrations of 1 and 100 μg/mL, leads to an increase of surviving PC 12 cells.
As demonstrated in figure 5B, an acute preventive treatment with extract W causes a protective effect similar to the one in extract S (Fig. 5A). We note that there is no apparent dose-dependent effect, but that concentrations of 1 and 100 μg/mL are the most effective. This phenomenon, often observed for raw extracts, may reflect complex interactions between a bioactive compound and endogenous inhibitors or the presence of several distinct compounds acting at different concentrations.
Chronic treatment
At this time, the preventive effect of deer velvet antler towards hydrogen dioxide has been studied with a repetitive treatments. Once confluence is reached, the PC 12 cells have been treated with extracts S or W at recommended concentrations every two days during 4 days, then at the 6th day, the cells have received at the same time an hydrogen dioxide treatment and the 4th treatment with deer velvet antler. The XTT test
- was made 24 hours after the last treatment.
The results of Fig. 6 show that in this test, a treatment with a concentration of hydrogen dioxide at 5.10"5M do not cause a significant mortality because the number of surviving cells after one week is the same in T and H202 controls (Fig. 6). However, we note that even after a hydrogen dioxide treatment, the protecting effect of deer velvet antler as observed in conditions where only experimental stress intervenes (ex. medium change) is always significant. In this case, the treatment with hydrogen dioxide causes a very drastic effect on PC12 cells survival (Fig. 7).
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WE CLAIM:
1. A composition for modulating neuronal diseases comprising antler extract and a physiologically acceptable diluent.
2. The composition of claim 1, wherein said antler extract is water soluble.
3. The composition of claim 1, wherein said modulation is treating or preventing said neuronal diseases.
4. The composition of claim 1, wherein said modulation of claim 1 is an anti- oxidative effect.
5. The composition of claim 1, wherein said neuronal disease is an injury, an induced or natural degenerescence, acquired or physical, chemical, or natural damage to nerve cells.
6. The composition of claim 1, wherein said administration is performed by oral, sublingual, or topical administration.
7. Use of antler extract for the preparation of a composition for modulating a neuronal disease or nerve cell physiology.
8. The use of claim 7, wherein said modulation is treating or preventing degenerescence or oxidation of nerve cells.
9. A method for modulating a neuronal disease comprising administrating antler extract to an individual in a sufficient amount to partially or totally treat or prevent the occurrence or development of a neuronal disease in said individual.
10. The method of claim 9, wherein said modulation is treating or preventing said neuronal disease.
11. The method of claim 9, wherein said neuronal disease is due to oxidation.
12. The method of claim 9, wherein said administration is performed by oral, sublingual, or topical administration.
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