Pharmaceutical composition for inhibiting release of catecholamines from catecholaminergic neurons

Abstract

The present invention provides a means for effective treatment for dependence on a drug, or the like. More specifically, the present invention relates to a pharmaceutical composition used to inhibit the release of catecholamines from catecholaminergic neurons in a subject, said composition including a tyrosine hydroxylase (TH) inhibitor.

Description

Pharmaceutical composition for inhibiting catecholamine release from catecholaminergic neurons

[0001] The present invention relates to a pharmaceutical composition for inhibiting the release of catecholamines from catecholaminergic neurons, etc.

[0002] Some drugs, such as cannabis, heroin, cocaine, and amphetamines, are useful in moderate doses and may be used for medical purposes. However, because they produce a transient euphoria, they are easily used, and continued use in excessive doses can lead to drug abuse. Drug abuse is a major global social problem and a major medical challenge. Although drug sensitivity, reactivity, and dependency are thought to vary from person to person, the molecular basis for these differences remains unclear. In addition to the drug abuse mentioned above, addiction to alcohol, nicotine, sleeping pills, organic solvents, and other substances, as well as shopping, gaming, and gambling addictions, are also significant social issues.

[0003] In particular, it is estimated that there are more than 100,000 patients suffering from the above-mentioned addiction in Japan alone. It has been reported that dopaminergic neurons (dopamine neurons) in the mesolimbic system and the dopamine (DA) they produce are involved in this addiction (Non-Patent Document 1).

[0004] Therefore, methods for controlling dopamine have been investigated. Examples of methods for suppressing the effects of dopamine include blockers of dopamine receptors (e.g., D2 receptors). However, D2 blockers generally suppress neuronal activity excessively, resulting in side effects such as decreased motivation and emotional dullness, making it difficult to select the dosage and drug when using D2 blockers for addiction treatment. Furthermore, D2 blockers may have the adverse effect of activating the biosynthesis and release of DA via presynaptic autoreceptors (Non-Patent Document 2). Another method for further suppressing dopamine activity is to inhibit or attenuate dopamine biosynthesis. To date, the administration of α-methyltyrosine to inhibit dopamine biosynthesis has been reported as a therapeutic agent for chronic schizophrenia (Non-Patent Documents 3-5). Furthermore, the combination of drugs such as topiramate, ondansetron, and naltrexone has been found to be effective in treating alcoholism and drug addiction. However, these drugs are known to act on opioid receptors, and DA suppression using specific drugs is not available for the treatment of addiction. Currently, cognitive behavioral therapy is the main treatment for addiction in Japan (Non-Patent Document 6).

[0005] Special Publication No. 2010-537990

[0006] Wise RA and Robble MA, Annual Review of Psychology, 71, 79-106 (2020) Strange PG, Trends Pharmacol Sci, 29, 314-321 (2008) Carlsson, A. et al., Journal of Neural Transmission 34, 125-132 (1973) Engelman, K. et al., J Clin Invest. 1968;47(3):577-594 Carlsson, A. et al., Journal of Neural Transmission 34, 125-132 (1973) Matsumoto, T., Journal of Psychiatry and Neurology, 113, 999-1006 (2011)

[0007] To date, no effective treatment means or methods have been established for drug addiction or other disorders.

[0008] The present invention has been made in consideration of the above circumstances, and provides the following pharmaceutical compositions and the like used for inhibiting the release of catecholamines from catecholaminergic neurons.

[0009] [1] A pharmaceutical composition used to suppress the release of catecholamines from catecholaminergic neurons in a subject, the pharmaceutical composition comprising a tyrosine hydroxylase (TH) inhibitor. [2] The pharmaceutical composition according to [1], which is capable of suppressing an increase in the amount of catecholamines released from the catecholaminergic neurons. [3] The pharmaceutical composition according to [1], wherein the catecholamines comprise one or more selected from the group consisting of dopamine, noradrenaline, and adrenaline. [4] The pharmaceutical composition according to [2], wherein the catecholamine is dopamine.

[0010] [5] The pharmaceutical composition according to [1], wherein the tyrosine hydroxylase (TH) inhibitor is at least one selected from the group consisting of metyrosine, a coenzyme tetrahydrobiopterin (BH4) synthesis inhibitor, an inhibitory nucleotide for the nucleic acid sequence of TH, a TH expression inhibitor, a compound that suppresses TH phosphorylation, an inhibitor of an enzyme that phosphorylates TH, and an inhibitor of a signal that promotes TH phosphorylation. [6] The pharmaceutical composition according to [5], wherein the coenzyme BH4 synthesis inhibitor is selected from the group consisting of a sepiapterin reductase inhibitor, a GTP cyclohydrolase I inhibitor, a pyruvoyltetrahydropterin synthase inhibitor, and a quinonoid dihydropteridine reductase. [7] The pharmaceutical composition according to [2], wherein the increase in catecholamine release is induced by at least one selected from the group consisting of a sleeping pill, an anxiolytic, and an analgesic, as well as alcohol consumption, smoking, gambling, gaming, and a specific addiction. [8] The pharmaceutical composition according to [2], wherein the increase in the amount of catecholamine released is induced by the ingestion of at least one substance selected from the group consisting of amphetamine, methamphetamine, opioids, morphine, marijuana, heroin, caffeine, and nicotine.

[0011] [9] The pharmaceutical composition according to [1] above, which is used for the treatment of at least one condition selected from the group consisting of schizophrenia, Tourette's syndrome, bulimia, and addiction.

[10] The pharmaceutical composition according to [4] above, wherein the maximum amount of dopamine released by the increase is 200% or more of the subject's normal dopamine level.

[11] The pharmaceutical composition according to [4] above, wherein the pharmaceutical composition is administered to the subject at a dose that does not reduce the extracellular dopamine concentration related to the subject's dopaminergic neurons to 50% or less of the subject's normal extracellular dopamine concentration.

[12] The pharmaceutical composition according to [1] above, wherein the subject is a human.

[0012]

[13] A tyrosine hydroxylase inhibitor for use in suppressing the release of catecholamines from catecholaminergic neurons in a subject.

[14] A method for suppressing the release of catecholamines from catecholaminergic neurons in a subject, comprising administering to the subject a tyrosine hydroxylase inhibitor or the pharmaceutical composition described in [1] above.

[15] A method for treating schizophrenia, bulimia, or addiction, comprising administering to a subject a tyrosine hydroxylase inhibitor or the pharmaceutical composition described in [1] above.

[0013] According to the present invention, it is possible to provide a pharmaceutical composition or the like that can suppress the release of catecholamines (dopamine, etc.) from catecholaminergic neurons in a subject. Specifically, it is possible to provide a pharmaceutical composition or the like that suppresses the increase in the release amount (transient secretion amount) of catecholamines (dopamine, etc.) that occurs when taking certain drugs, etc., and that has few side effects. According to the present invention, it is possible to effectively treat schizophrenia, bulimia, addiction, etc., with few side effects.

[0014] Figure 1(1) shows the change in methamphetamine (METH)-induced locomotor activity in tyrosine hydroxylase (Th)-heterozygous mice. Figure 1(2) shows a graph comparing the cumulative locomotor activity after METH administration between wild-type and TH-heterozygous mice. Figure 2 shows the change in METH-induced locomotor activity in 6-week-old female Th-heterozygous mice. Figure 3 shows the time course of locomotor activity when wild-type mice were administered metyrosine (AMPT) followed by METH. Figure 4 shows a comparison of the AUC of locomotor activity when wild-type mice were administered AMPT followed by METH. Figure 5 shows the tissue dopamine levels in various brain regions 4 hours after AMPT administration in wild-type mice. Figure 6 shows a schematic diagram of the conditioned place preference test (CPP test). Figure 7 shows the results of the conditioned place preference test (CPP test) in AMPT-treated mice. Figure 8 shows a schematic diagram of the microdialysis method. FIG. 9 shows the changes in extracellular dopamine concentration caused by METH or cocaine after administration of AMPT using microdialysis.

[0015] The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be appropriately modified and implemented without departing from the spirit of the present invention. All publications cited in this specification, such as prior art documents, published patent applications, patent publications, and other patent documents, are incorporated herein by reference.

[0016] The present invention provides pharmaceutical compositions and the like used to inhibit the release of catecholamines from catecholaminergic neurons in a subject. The pharmaceutical compositions of the present invention contain, as an active ingredient, an inhibitor of tyrosine hydroxylase (TH), an enzyme involved in the biosynthesis of catecholamines. The present inventors have newly discovered that inhibiting the activity of tyrosine hydroxylase (TH), which is responsible for dopamine (DA) production, can not only inhibit DA production but also suppress the amount of DA release (exocytosis) from synaptic vesicles in DA neurons, thereby completing the present invention. Specifically, they have discovered that the above-mentioned novel effect can be achieved by inhibiting TH activity to an extent that does not significantly reduce the amount of DA in biological tissues, thereby completing the present invention. Before describing the pharmaceutical compositions and the like of the present invention, catecholamines and their biosynthesis will be described below.

[0017] 1. Catecholamines and Their Biosynthesis In this specification, catecholamines refer to physiologically active amines with a catechol as the basic structure. Examples of such physiologically active amines include dopamine (DA), noradrenaline (NOR), and adrenaline. Furthermore, examples of monoamines, which are neurotransmitters in the central nervous system of living organisms, include dopamine, noradrenaline, adrenaline, serotonin, and histamine. In the present invention, preferred catecholamines include dopamine and noradrenaline, and more preferably dopamine.

[0018] Dopamine (DA) is involved in physiological functions such as emotion, movement, learning (short-term memory, etc.), motivation, and drug addiction. Changes in DA metabolism may be involved in the pathophysiology of neuropsychiatric disorders such as Parkinson's disease. Noradrenaline (NOR) is involved in physiological functions such as maternal behavior, mood regulation, anxiety, stress response, and pain sensation.

[0019] Neurons in which catecholamines are biosynthesized, stored, transported, and released are called catecholaminergic neurons. In the present invention, catecholaminergic neurons preferably include dopaminergic neurons (also referred to as "dopamine neurons") and noradrenergic neurons (also referred to as "noradrenergic neurons"), more preferably dopaminergic neurons.

[0020] Specific locations of dopaminergic neurons in the brain include, for example, the midbrain reticular formation, substantia nigra, ventral tegmental area, and hypothalamus. More specifically, these include the nigrostriatal pathway, the mesencephalon-cortical pathway, the mesencephalon-limbic pathway, and the tuberoinfundibular tract. Of these, the nigrostriatal pathway is considered important for the motor function of the living body, and it is known that degeneration of the nigrostriatal pathway is observed in Parkinson's disease, for example.

[0021] Noradrenergic neurons are present in the brain and peripheral sympathetic nerves, and more specifically, examples of these include the locus coeruleus-cortical pathway, the reticular-hypothalamic pathway, the periventricular system, and the medulla-spinal pathway in the central nervous system.

[0022] Dopamine is synthesized in dopaminergic neurons through a two-step enzymatic reaction. Noradrenaline is synthesized in noradrenergic neurons through a three-step enzymatic reaction. The starting material for the biosynthesis of both dopamine and noradrenaline is tyrosine, and in the first step of biosynthesis, the aromatic ring is hydroxylated by tyrosine hydroxylase (TH) (EC 1.14.16.2) to produce L-3,4-dihydroxyphenylalanine (L-DOPA). L-DOPA is then decarboxylated by aromatic-L-amino acid decarboxylase (AADC) (EC 4.1.1.28) to produce dopamine. The produced dopamine is further converted to noradrenaline by dopamine-β-monooxygenase (EC 1.14.17.1). An outline of this process is shown below.

[0023]

[0024] (1) Tyrosine hydroxylase (TH) TH acts in the first step of dopamine biosynthesis and noradrenaline biosynthesis. The amino acid sequences of TH are known for humans under GenBank accession numbers KAI4069455.1 (528 aa), KAI4069454.1 (524 aa), KAI4069453.1 (497 aa), and KAI4069452.1 (403 aa), and for mice under GenBank accession numbers EDL18181.1 (498 aa), EDL18182.1 (478 aa), etc. In primates such as humans, various tyrosine hydroxylase mRNAs are generated from a single primary transcript by alternative mRNA splicing.

[0025] TH synthesizes L-DOPA in the cytoplasm using L-tyrosine as a specific substrate. This L-DOPA synthesis by TH is usually the rate-limiting step in catecholamine biosynthesis, and inhibition of this enzyme significantly affects catecholamine synthesis. Catecholamine biosynthesis by TH is subject to short-term regulation in the living body, including activation by phosphorylation of serine residues, followed by inhibition of enzyme activity by the final product (feedback inhibition). Long-term regulation includes regulation of TH protein amount by regulating the transcription and translation of the gene encoding TH.

[0026] (2) L-aromatic amino acid decarboxylase (AADC) AADC, which acts at the second step of dopamine and noradrenaline biosynthesis, catalyzes the conversion of L-DOPA to dopamine in the cytoplasm. However, all naturally occurring aromatic L-amino acids can be used as substrates for AADC.

[0027] 2. Vesicular transport, storage, and recycling of catecholamines. The catecholamine dopamine is produced in the cytoplasm of presynaptic neurons and then stored in intracellular vesicles by the vesicular monoamine transporter (vMAT). vMAT is a 12-transmembrane protein that is expressed in living organisms in two isoforms: vMAT1 and vMAT2. vMAT2 is primarily expressed in neurons, while vMAT1 is expressed in the intestine, lungs, pancreas, kidneys, etc.

[0028] It has been reported that vMAT2 interacts with TH and AADC via its cytoplasmic domain (Cartier, EA et al., J. Biological Chemistry Vol. 285, No. 3, pp. 1957-1966, 2010). This suggests that dopamine is synthesized in the synaptic vesicle membrane and is rapidly accumulated in synaptic vesicles after synthesis.

[0029] The vesicles that secrete neurotransmitters at the presynaptic terminals of nerve cells are also called synaptic vesicles. Dopamine, synthesized by TH or AADC and stored in synaptic vesicles, is released extracellularly (i.e., from dopaminergic neurons) by exocytosis. The process from synaptic vesicles to extracellular release can be divided into three steps: (1) docking, (2) priming, and (3) membrane fusion. In each of these steps, calcium ions (Ca 2+ ), synaptotagmin, synaptobrevin, SNARE proteins such as syntaxin, and SNAP-25 are thought to act.

[0030] Released neurotransmitters, such as dopamine, bind to receptors (e.g., dopamine receptors) on the postsynaptic surface adjacent to the presynaptic terminal, transmitting neuronal signals and then being metabolized. Meanwhile, presynaptic terminals that release neurotransmitters also have a recycling mechanism that takes up the released neurotransmitters and accumulates them intracellularly. This recycling mechanism includes the uptake process via transporters for each neurotransmitter, such as the dopamine transporter (DAT) on the plasma membrane, and the uptake process by endocytosis. Among these, DAT can be inhibited by cocaine, amphetamine, duloxetine hydrochloride, GBR 12783, GBR 12909, indatraline hydrochloride, rimcazole hydrochloride, etc., or can transport dopamine back from the intracellular to the extracellular space, resulting in increased extracellular dopamine concentrations.

[0031] 3. Pharmaceutical Composition As described above, the pharmaceutical composition of the present invention comprises an inhibitor of tyrosine hydroxylase (TH), an enzyme involved in the biosynthesis of catecholamines.

[0032] Examples of TH-specific inhibitors that can be used in the present invention include, but are not limited to, metyrosine ((2S)-2-amino-2-methyl-3-(4-hydroxyphenyl)propanoic acid), 3-chlorotyrosine, and 3-iodotyrosine. Metyrosine has the following structure and is commercially available as a medicine for pheochromocytoma, which exhibits excessive catecholamine secretion (e.g., "Demser Capsules" by Ono Pharmaceutical Co., Ltd.).

[0033]

[0034] As an alternative TH inhibitor, nucleotides capable of specifically inhibiting the expression of TH (antisense nucleotides, siRNA, etc.) can be used. Such nucleotides can be designed and prepared by means known in the art based on the known nucleotide sequence information of TH, and for example, known procedures described later in this specification can be referred to.

[0035] Furthermore, the tyrosine hydroxylation reaction by TH requires tetrahydrobiopterin (BH4) as a coenzyme (cofactor) (Urano et al., Vitamin, 2004, Vol. 78, No. 9, pp. 454-455 (DOI: https: / / doi.org / 10.20632 / vso.78.9_454_2)). Therefore, in the present invention, agents capable of inhibiting BH4 synthesis can also be used as TH inhibitors. GTP cyclohydrolase I, 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase are involved in the biosynthesis of BH4 (Ichinose (Washimi) et al., Japanese Pharmacology Journal, 2001, Vol. 118, No. 6, pp. 371-377, etc.). Therefore, for example, sepiapterin reductase inhibitors, GTP cyclohydrolase I inhibitors, and pyruvoyltetrahydropterin synthase inhibitors can be used, but are not limited to these. Alternatively, for example, the above-mentioned antisense nucleotides, siRNA, etc. capable of inhibiting the expression or translation of GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase, or sepiapterin reductase can be used to specifically inhibit these enzymes, thereby inhibiting TH and ultimately inhibiting dopamine synthesis.

[0036] In one embodiment of the present invention, compounds that suppress TH phosphorylation (including small molecules, peptides, and antibodies), inhibitors of enzymes involved in intracellular TH phosphorylation, and inhibitors of signals that promote phosphorylation may also be used as TH inhibitors. For example, dominant-negative peptides based on regions containing the TH phosphorylation site may be used as TH inhibitors. The design and production of such peptides are known in the art, and reference can be made to the procedures described later in this specification.

[0037] Furthermore, antisense nucleotides against nucleotide regions involved in the transcription, splicing, and translation of TH can be used as inhibitors of TH expression. Such antisense nucleotides can be appropriately prepared by those skilled in the art using methods known in the art based on, for example, the promoter sequence of TH genome or the pre-mRNA sequence of TH.

[0038] In the pharmaceutical composition of the present invention, an inhibitor specific to aromatic-L-amino acid decarboxylase (AADC) can be used together with or instead of the TH inhibitor. Known AADC inhibitors include carbidopa, benserazide, brocresine, difluoromethyldopa, α-methyldopa, and monofluoromethyldopa. Some of these AADC inhibitors are commercially available as levodopa metabolic inhibitors for Parkinson's disease (e.g., "Neodopaston" by Daiichi Sankyo and "EC Dopar" by Kyowa Hakko Kirin).

[0039] As an AADC inhibitor, nucleotides capable of specifically inhibiting the expression of AADC (antisense nucleotides, siRNA, etc.) can also be used. Such nucleotides can be designed and produced by means known in the art based on the nucleotide sequence information of AADC.

[0040] AADC requires vitamin B6 (pyridoxal phosphate) as a cofactor. Therefore, dominant-negative peptides based on the region involved in the binding of AADC to vitamin B6 can be designed and used as AADC inhibitors. The design and production of such peptides are known in the art.

[0041] Furthermore, antisense nucleotides against nucleotide regions involved in the transcription, splicing, and translation of AADC can also be used as inhibitors of AADC expression (i.e., AADC inhibitors). Such antisense nucleotides can be appropriately prepared using methods known in the art based on, for example, the promoter sequence of AADC on the genome or the pre-mRNA sequence of AADC.

[0042] The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the active ingredient, or may be a pharmaceutical composition containing the active ingredient in combination with other ingredients such as pharmaceutically acceptable carriers or additives. The active ingredient may be a single ingredient or a combination of multiple ingredients. The active ingredient in the pharmaceutical composition of the present invention may be present in an amount of, for example, 0.1% by weight or more and 99.9% by weight or less, preferably 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less, 20% by weight or less, or 10% by weight or less.

[0043] The dosage form of the pharmaceutical composition of the present invention is not limited, and may be, for example, oral, parenteral (intravenous), intramuscular, oral mucosal, rectal, vaginal, transdermal, nasal, or inhalational, but is preferably a parenteral (intravenous) dosage form. The administration form of the pharmaceutical composition of the present invention is also not limited, and may be, for example, oral, parenteral (intravenous), intramuscular, oral mucosal, rectal, vaginal, transdermal, nasal, or inhalational, but is preferably a parenteral (intravenous) dosage form. The dosage of the pharmaceutical composition of the present invention is not limited, and may be, for example, 1 to 5,000 mg per day for an adult (e.g., weighing 60 kg), preferably 10 to 1,000 mg, 10 to 800 mg, 10 to 600 mg, 10 to 500 mg, 10 to 400 mg, 10 to 300 mg, 10 to 200 mg, 10 to 100 mg, 10 to 75 mg, or 10 to 50 mg. These daily doses may be administered in multiple doses, for example, two or more doses, three or more doses, etc.

[0044] When the pharmaceutical composition of the present invention contains a polynucleotide encoding a dominant-negative mutant, a polynucleotide that inhibits the expression or translation of TH or AADC, such a polynucleotide can be combined with an appropriate expression cassette including a promoter and designed to express the desired inhibitor after delivery into target cells. Such an expression cassette can be delivered to target cells, for example, using a suitable viral vector. In general, viral vectors are suitable for disease treatment because they can introduce genes into target cells with high efficiency. Viral vectors that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex viral vectors (Miller, AD et al. (1991) J. Virol. 65, 2220-2224; Miyake, S. et al. (1994) Proc. Natl. Acad. Sci. USA, 91, 8802-8806; Samulski, RJ et al. (1989) J. Virol. 63, 3822-3828; JP 2004-528836 A). When administering the pharmaceutical composition of the present invention containing the above-mentioned viral vector, the dosage is not limited, but can be, for example, 1 to 5,000 mg, preferably 10 to 1,000 mg, and more preferably 10 to 800 mg, 10 to 600 mg, 10 to 500 mg, 10 to 400 mg, 10 to 300 mg, 10 to 200 mg, 10 to 100 mg, 10 to 75 mg, 10 to 50 mg, etc. per day for an adult (e.g., body weight 60 kg). These daily dosages may be administered in multiple doses, for example, two or more, three or more, etc.

[0045] In the present invention, examples of pharmaceutically acceptable carriers or additives that can be used include excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, solubilizers, solubilizers, isotonicity agents, pH adjusters, stabilizers, etc.

[0046] Examples of formulations suitable for oral administration of the pharmaceutical compositions of the present invention include powders, tablets, capsules, fine granules, granules, liquids, and syrups. For oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, and glycine can be used, along with starch, preferably corn, potato, or tapioca starch, and various disintegrants such as alginic acid and certain double silicates, and granulation binders such as polyvinylpyrrolidone, sucrose, gelatin, and gum arabic. Lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often very effective for tableting. Similar solid compositions can also be filled into gelatin capsules. Suitable materials for this purpose include lactose or milk sugar, as well as high-molecular-weight polyethylene glycols. When aqueous suspensions and / or elixirs for oral administration are desired, the active ingredient may be used in combination with various sweeteners or flavorings, coloring agents or dyes, and, if necessary, emulsifying agents and / or suspending agents, and diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof may be used.

[0047] For the pharmaceutical compositions of the present invention, formulations suitable for parenteral administration include, for example, injections and suppositories. For parenteral administration, the active ingredient of the present invention can be dissolved in either sesame oil or peanut oil, or in aqueous propylene glycol. The aqueous solution should be appropriately buffered (preferably pH 8 or higher), if necessary, and the liquid diluent should first be rendered isotonic. Such a liquid diluent can be, for example, physiological saline. The resulting aqueous solution is suitable for intravenous injection, while the oily solution is suitable for intraarticular, intramuscular, and subcutaneous injection. The preparation of all these solutions under sterile conditions can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. Additionally, the active ingredient of the present invention can be administered topically, such as to the skin. In this case, topical administration is preferably carried out in the form of a cream, jelly, paste, or ointment, according to standard pharmaceutical practice.

[0048] 4. Effects of Pharmaceutical Composition and Evaluation Thereof The pharmaceutical composition of the present invention can suppress the release of catecholamines from catecholaminergic neurons induced, for example, by the ingestion of sleeping pills, anxiolytics, and analgesics, as well as by drinking alcohol, smoking, gambling, gaming, and specific addictions (various behaviors that increase catecholamine levels in the body, etc.), and preferably can suppress increases in the amount of catecholamine release.

[0049] Each of the above addictions is thought to involve the release of large amounts of neurotransmitters such as dopamine, norepinephrine, and serotonin. For example, in the case of nicotine addiction caused by smoking, nicotine taken into the body binds to nicotinic acetylcholine receptors in the brain, resulting in the release of large amounts of dopamine from the nucleus accumbens, which produces a strong sense of pleasure (from the Ministry of Health, Labor and Welfare's health information site for preventing lifestyle-related diseases, "Nicotine Addiction").

[0050] In one embodiment, the pharmaceutical composition of the present invention can be used for the treatment of, but is not limited to, schizophrenia, Tourette's syndrome, bulimia, or addictions (alcoholism, shopping addiction, drug addiction, gambling addiction, and gaming addiction). The dopamine hypothesis, which suggests that dopamine is involved in each of the above symptoms, has been proposed. Therefore, the pharmaceutical composition of the present invention can be used to treat diseases and symptoms associated with the transient release of large amounts of dopamine and the like.

[0051] Alternatively, the pharmaceutical composition of the present invention can suppress the release of catecholamines from catecholaminergic neurons, such as those induced by the ingestion of amphetamine, methamphetamine, opioids, morphine, marijuana, heroin, caffeine, or nicotine, or various low-molecular-weight compounds, and preferably can suppress the increase in the release of such catecholamines.

[0052] In the present invention, the inhibition of catecholamine (e.g., dopamine and / or noradrenaline) release from catecholaminergic neurons specifically means the inhibition of catecholamine release from neurons by exocytosis, which occurs when synaptic vesicles containing catecholamines fuse with the cell membrane of the neurons. Therefore, the inhibition of catecholamine release in the present invention does not mean the inhibition of catecholamine release from the neurons that may occur due to a decrease in the amount of catecholamine biosynthesis.

[0053] In the present invention, an increase in the release of catecholamines (e.g., dopamine and / or noradrenaline) from catecholaminergic neurons refers to the maximum release of catecholamines from synaptic vesicles of the neurons during excitation due to the aforementioned drug or addiction, which is, for example, 200% or more, 250% or more, 300% or more, 350% or more, 400% or more, 450% or more, or 500% or more of the normal catecholamine level in the subject. Quantitative measurement of catecholamine release can be performed using various methods known to those skilled in the art. For example, the method for measuring extracellular catecholamine levels used in the Examples below in this specification (described below) can be used. The pharmaceutical composition of the present invention inhibits the maximum transient increase in extracellular catecholamine levels in a subject to preferably 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, or 5% or less compared to the maximum level in a control individual, condition, etc. (control). The duration of this transient rise may range from a momentary rise to a sustained rise for several hours. Preferred examples of the duration of this transient rise are 10 seconds or more, 15 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 6 minutes or more, 7 minutes or more, 8 minutes or more, 9 minutes or more, 10 minutes or more, 15 minutes or more, and may be 30 minutes or less, 35 minutes or less, 40 minutes or less, 45 minutes or less, 50 minutes or less, 55 minutes or less, 1 hour or less, 2 hours or less, 3 hours or less, 4 hours or less, 5 hours or less, 6 hours or less, 8 hours or less, 10 hours or less, 12 hours or less, 18 hours or less, 24 hours or less, etc., but are not limited thereto.

[0054] The dosage of the pharmaceutical composition of the present invention to a subject is preferably a dose that does not reduce the extracellular catecholamine concentration in the subject's catecholaminergic neurons to 90% or less, 80% or less, 70% or less, 60% or less, or 50% or less of the subject's normal extracellular catecholamine concentration. In this specification, the term "extracellular catecholamine concentration in catecholaminergic neurons" refers to the extracellular (preferably, in the tissue of the brain region and outside the neuron) concentration measured in a brain region containing catecholaminergic neurons in a subject (e.g., midbrain, striatum, nucleus accumbens, substantia nigra, ventral tegmental area, hypothalamus, cerebral cortex, hippocampus, amygdala, etc.) according to the measurement procedures used in the Examples below. Measurement times can be measured in minutes, seconds, milliseconds, or microseconds.

[0055] In the present invention, the subject is not limited to, and includes various mammals, but is preferably a human.

[0056] Dopamine (DA) can be rapidly decomposed in body fluids such as blood, cerebrospinal fluid, and lymph, and therefore highly sensitive measurement using minute sample amounts may be required. In measuring the effects of the pharmaceutical composition of the present invention at a microscopic level, the amount, concentration, etc. of DA can be measured by techniques such as ELISA (e.g., Funakoshi's "Dopamine ELISA Kit"), HPLC, mass spectrometry, etc.

[0057] Preferably, the dopamine release level can be measured by a method known to those skilled in the art, such as the microdialysis method described in the Examples below. Microdialysis allows for continuous, time-dependent changes in tissue levels of biological substances while simultaneously observing the behavior of freely moving animals. This microdialysis method can be used to measure diurnal changes in monoamines (NE: norepinephrine, DA: dopamine, 5-HT: serotonin).

[0058] Furthermore, by administering a tracer such as DA to the body and using positron emission tomography (PET), it is possible to independently evaluate the functions of DA in the brain, from synthesis to reuptake and receptor activity. Examples of such tracers include known substances such as C-11 raclopride, C-11 NMSP, C-11 β-CFT, F-18 FP-DTPZ, and F-18 FDOPA (Annual Review Nerve 2013 J. Basic Neuroscience 52-62).

[0059] In one embodiment, the pharmaceutical composition of the present invention can be used for the treatment of, but is not limited to, schizophrenia, Tourette's syndrome, binge eating disorder, or addictions (alcoholism, shopping addiction, drug addiction, gambling addiction, gaming addiction).

[0060] Such an evaluation of addiction or the like can be performed using a method known to those skilled in the art, including, but not limited to, a conditioned place preference test (CPP method) using mice, as used in the Examples described later in this specification, and an intracerebral self-stimulation test (Gardner EL, Am J Addict, 2000).

[0061] 5. Other Inventions The present invention is not limited to the inventions relating to the pharmaceutical compositions described above, and may also provide inventions such as: a tyrosine hydroxylase inhibitor used to inhibit the release of catecholamines from catecholaminergic neurons in a subject; use of a tyrosine hydroxylase inhibitor for inhibiting the release of catecholamines from catecholaminergic neurons in a subject; use of a tyrosine hydroxylase inhibitor in the manufacture of a medicament for inhibiting the release of catecholamines from catecholaminergic neurons in a subject; a method for inhibiting the release of catecholamines from catecholaminergic neurons in a subject, comprising administering to the subject a tyrosine hydroxylase inhibitor or the pharmaceutical composition of the present invention; a method for treating schizophrenia, bulimia, or addiction, comprising administering to a subject a tyrosine hydroxylase inhibitor or the pharmaceutical composition of the present invention; a tyrosine hydroxylase inhibitor used to treat schizophrenia, bulimia, or addiction; and use of a tyrosine hydroxylase inhibitor in the manufacture of a medicament for treating schizophrenia, bulimia, or addiction. The explanations of the components and technical terms in these inventions may be appropriately applied to the explanations of the pharmaceutical composition of the present invention described above.

[0062] In this specification, terms not specifically explained are intended to refer to the scope of meaning that is commonly understood by those skilled in the art.

[0063] (1) Verification of changes in dopamine metabolites in the brain of Th heterozygous mice. Th heterozygous mice were used in this experiment. Th heterozygous mice lack one of the two Th genes in their genome. Western blotting of the striatum brain tissue confirmed that this deficiency reduced the amount of Th protein to approximately half that of the wild-type mice (data not shown).

[0064] To investigate the changes in dopamine metabolism in the brain of Th heterozygous mice, we measured the levels of dopamine and dopamine metabolites in the midbrain, striatum, and nucleus accumbens of Th heterozygous mice and wild-type mice using HPLC-electrochemical detection. No significant differences in dopamine levels were observed in each region (data not shown).

[0065] (2) Methamphetamine-induced changes in spontaneous locomotion in Th heterozygous mice Methamphetamine (METH) inhibits dopamine storage in the endoplasmic reticulum, increasing the dopamine concentration in the cytoplasm of presynaptic cells. At the same time, it reverses the dopamine transporter, releasing amines such as dopamine, noradrenaline, and serotonin from nerve terminals, ultimately resulting in neuroexcitation.

[0066] We examined METH-induced changes in spontaneous locomotor activity in Th heterozygous mice and wild-type mice (9-week-old male mice) when METH was administered at gradually increasing doses from 0 (saline only), 1.0, 2.0, and 3.0 mg / kg. Mice were individually placed in a cylindrical container with a diameter of 30 cm, and spontaneous locomotor activity was measured every 5 min using an infrared sensor (Supermex, Muromachi Kikai). To allow the mice to acclimate to the container, METH was administered intraperitoneally 120 min after the mice were placed in the container, and the METH-induced increase in spontaneous locomotor activity was measured (Fig. 1).

[0067] After administration of 1.0 mg / kg of METH, both mice showed only a similar response to saline alone, but administration of 2.0 mg / kg of METH significantly increased spontaneous motor activity. While wild-type mice continued to increase their motor activity from immediately after administration through 30 minutes after administration, Th heterozygous mice showed no further increase in motor activity after the initial administration and remained lower than wild-type mice. Administration of 3.0 mg / kg of METH further increased motor activity in both mice. The graph showing cumulative motor activity after METH administration is shown on the right side of Figure 1(1).

[0068] These results suggest that the locomotion following administration of 2.0 mg / kg of METH was significantly lower in Th heterozygous mice than in wild-type mice (Fig. 1(2)), indicating that METH responsiveness was reduced in Th heterozygous mice.

[0069] In a similar experiment conducted in 6-week-old female mice, a more significant difference in METH responsiveness was observed at 2.0 mg / kg METH (Fig. 2). This difference is thought to be due to the difference in dopamine metabolism between 6-week-old mice, which are still in the developmental stage before reaching adulthood, and adult mice.

[0070] (3) Effect of METH Administration under TH Inhibition in Th Heterozygous Mice Next, we examined changes in METH responsiveness when TH activity was suppressed by administering a TH inhibitor to wild-type mice. The TH inhibitor used was α-methyl DL-tyrosine (AMPT) (Merck, catalog number 120693-5g). Because AMPT is poorly soluble in saline, it was suspended in 0.5 w / v% methylcellulose 400 solution (Fujifilm Wako Pure Chemical Industries, Ltd., catalog number 133-17815) and administered intraperitoneally to mice 4 hours before METH administration. METH was dissolved in saline at the dose specified for each experiment and administered intraperitoneally. Control mice received only saline.

[0071] AMPT was administered intraperitoneally at 10 mg / kg or 20 mg / kg, or methylcellulose (control, CNTL) as a vehicle. Four hours after administration, 3.0 mg / kg METH was administered intraperitoneally to examine changes in METH-induced spontaneous locomotor activity. Control mice showed an increase in locomotor activity immediately after METH administration and continued to do so for 40 minutes after administration. However, the increase in METH-induced locomotor activity was significantly suppressed in AMPT-treated mice, with the 20 mg / kg group exhibiting a stronger suppression than the 10 mg / kg AMPT group (Fig. 3). Calculation of the area under the curve (AUC) for the locomotor activity up to 65 minutes after METH administration revealed a significant dose-dependent suppression of METH-induced spontaneous locomotor activity in AMPT-treated mice (Fig. 4).

[0072] (4) Changes in brain dopamine levels following AMPT administration. To examine the effects of AMPT on brain dopamine metabolism, wild-type mice (age-checked) were similarly administered AMPT and then dissected 4 hours later to examine dopamine and its metabolites in the midbrain, striatum, and nucleus accumbens. Dopamine levels decreased with increasing doses (10 mg / kg and 20 mg / kg), but only by approximately 10% to 20% at 10 mg / kg AMPT and approximately 30% to 50% at 20 mg / kg AMPT (Figure 5). Brain dopamine levels were measured by diluting 5 μL of mouse brain tissue supernatant 10-fold with 45 μL of 1x PBS, adding 50 μL of 0.2 M perchloric acid / 0.1 mM EDTA and 10 μL of 1 μM isoproterenol (ISO) as an internal standard, pipetting, and then allowing to stand on ice for 10 minutes. The mixture was then centrifuged at 20,000 x G for 15 minutes at 4°C. 10 μL of 1 M sodium acetate was added to the supernatant to neutralize the strong acid. The supernatant was then centrifuged again at 20,000 x G for 10 minutes at 4°C to prepare the dopamine measurement sample. The dopamine measurement sample was separated using a high-performance liquid chromatography (HPLC) reverse-phase column and detected using an electrochemical detector. Sample identification and concentration calculation were performed based on the retention time and peak area of ​​a standard substance with known concentration. A Welch's t-test was used to test for significance, with Bonferroni correction for three or more groups. A p<0.05 was considered significant.

[0073] (5) Effect of AMPT on METH-induced dependence Since AMPT suppressed METH-induced locomotor activity, we investigated whether AMPT administration had an inhibitory effect on METH-induced dependence by using the conditioned place preference test (CPP test). An outline of the experiment is shown in Figure 6.

[0074] The conditioned place preference test (CPP) was used to evaluate the dependence-forming potential of METH in AMPT-treated mice. A schematic diagram of the experiment is shown in Figure 6. The CPP score was calculated by subtracting the time spent in the METH-conditioned chamber during the pretest from the time spent in the METH-conditioned chamber during the posttest. Nine-week-old male wild-type mice were used in the experiment.

[0075] The CPP apparatus was divided into a black room with a metal grid floor and a white room with a wire mesh floor. During the habituation, pre-test, and post-test phases, mice were allowed to move between the two rooms via a T-shaped partition with two openings. On the other hand, during the conditioning phase, a rectangular partition without an opening prevented mice from moving between the rooms. The number of times the mice moved between the rooms and the time spent in each room were measured using an infrared detector (Neuroscience).

[0076] This test was divided into three phases: habituation / pretest, conditioning, and posttest. On days 1 and 2, mice were introduced into the apparatus from the white chamber to familiarize them with the apparatus. The number of transitions between the chambers and the time spent in each chamber were measured over a 900-second period. On day 3, as in days 1 and 2, mice were introduced into the apparatus for a 15-minute pretest, and the number of transitions between the chambers and the time spent in each chamber were measured. The mice were then divided into four groups for conditioning. Each day, mice were alternately placed in a black or white chamber, with no access to either chamber. Groups A and B were placed in the black chamber on days 4 and 6 and the white chamber on days 5 and 7. Groups C and D were placed in the white chamber on days 4 and 6 and the black chamber on days 5 and 7. Groups A and D were administered METH (3 mg / kg) while in the white chamber. Groups B and C were administered METH while in the black chamber and confined to their respective chambers for 45 minutes. On the eighth day, the mice are placed in the apparatus for 15 minutes under the same conditions as in the pre-test, and the number of times they move between the rooms and the time spent in each room are measured to evaluate which room they have come to prefer.

[0077] From the results obtained in this way, the dependence-forming potential of METH in mice was evaluated by calculating the CPP score (the time spent in the METH-conditioned chamber during the posttest minus the time spent in the METH-conditioned chamber during the pretest). The results showed that administration of 50 mg / kg of AMPT significantly reduced the dependence-forming potential of METH.

[0078] (6) Effects of AMPT administration on dopamine release in the brain. The amount of dopamine released from nerve terminals following AMPT administration was evaluated using microdialysis. Microdialysis is a technique for collecting extracellular substances at specific sites in living mice. A probe consisting of a semipermeable membrane is inserted into the head of the mouse, and Ringer's solution is perfused into the probe at a flow rate of 2.0 μl / min. Brain substances are then collected into the perfusion solution according to their concentration gradient (Figure 8). This technique allows for long-term, continuous monitoring of changes in extracellular substances in unanesthetized, unrestrained mice.

[0079] The amount of dopamine in the perfusate collected at regular intervals was measured using HPLC electrochemical detection. Specifically, samples collected from the living body were separated using a high-performance liquid chromatography (HPLC) reverse-phase column (150 × 4.6 mm) and detected using an electrochemical detector (first cell, +180 mV; second cell, -180 mV). The mobile phase consisted of 4.1 g / L sodium acetate adjusted to pH 4.1, 100 mg / L Na2EDTA, 100 mg / L octanesulfonic acid, and 20% methanol, perfused at 0.38 mL / min. Sample identification and concentration were calculated from the retention time and peak area of ​​known concentrations of standard compounds. After measurements, mice were euthanized, their brains were removed, fixed in formalin, and brain sections were prepared to confirm the intracerebral fixation of the microdialysis probe.

[0080] Figure 9 shows the changes in extracellular dopamine concentrations in the striatum measured by microdialysis. When 1.0 mg / kg METH was administered to mice administered 20 mg / kg or 50 mg / kg AMPT, the amount of extracellular dopamine released decreased dose-dependently, and METH-induced dopamine release was almost undetectable in mice administered 50 mg / kg AMPT. A similar experiment was performed using cocaine, which is known to inhibit the dopamine transporter, instead of METH. A significant increase in extracellular dopamine was also observed in mice administered 50 mg / kg AMPT.

[0081] These results indicate that AMPT administration inhibits the uptake of dopamine into vesicles via METH-mediated VMAT2 and suppresses dopamine release by reversing the rotation of the dopamine transporter. However, AMPT administration acts independently of METH-mediated suppression of dopamine release by cocaine, which acts on the dopamine transporter (inhibiting dopamine uptake).

[0082] (7) Discussion The above experiments yielded the following results. (i) Although the reduction in TH expression in heterozygous KO mice resulted in only a slight reduction in tissue DA levels, the reactivity and sensitivity to the addictive drug METH dramatically changed in response to the reduction in DA levels. (ii) The TH inhibitor AMPT dose-dependently suppressed the increase in extracellular DA caused by METH administration. (iii) Administration of AMPT suppressed the increase in extracellular DA after administration of METH, which is a VMAT2 agonist, but did not suppress the increase in DA caused by cocaine, a DAT inhibitor.

[0083] Comprehensive examination of these results revealed that the inhibitory effect of AMPT on METH sensitivity not only reduces intracellular DA, but also reveals a novel physiological effect in which TH activity regulates the amount of DA released from DA vesicles. This suggests that the administration of low doses of AMPT that do not significantly reduce intracellular DA can suppress the strong pleasurable stimulation caused by METH, providing a new drug for the treatment of addiction.

Claims

1. A pharmaceutical composition for use in inhibiting the release of catecholamines from catecholaminergic neurons in a subject, said pharmaceutical composition comprising a tyrosine hydroxylase (TH) inhibitor.

2. The pharmaceutical composition according to claim 1, which is capable of suppressing an increase in the amount of catecholamine released from the catecholaminergic neurons.

3. The pharmaceutical composition of claim 1, wherein the catecholamine comprises one or more selected from the group consisting of dopamine, noradrenaline, and adrenaline.

4. The pharmaceutical composition of claim 2, wherein the catecholamine is dopamine.

5. The pharmaceutical composition of claim 1, wherein the tyrosine hydroxylase (TH) inhibitor is at least one selected from the group consisting of metyrosine, inhibitors of the synthesis of the coenzyme tetrahydrobiopterin (BH4), inhibitory nucleotides against the nucleic acid sequence of TH, TH expression inhibitors, compounds that suppress the phosphorylation of TH, inhibitors of enzymes that phosphorylate TH, and inhibitors of signals that promote the phosphorylation of TH.

6. The pharmaceutical composition of claim 5, wherein the coenzyme BH4 synthesis inhibitor is selected from the group consisting of sepiapterin reductase inhibitors, GTP cyclohydrolase I inhibitors, pyruvoyltetrahydropterin synthase inhibitors, and quinonoid dihydropteridine reductase inhibitors.

7. The pharmaceutical composition according to claim 2, wherein the increase in the release of catecholamines is induced by the ingestion of sleeping pills, anti-anxiety drugs, and analgesics, as well as by at least one substance selected from the group consisting of alcohol consumption, smoking, gambling, gaming, and specific addictions.

8. The pharmaceutical composition according to claim 2, wherein the increase in catecholamine release is induced by the ingestion of at least one selected from the group consisting of amphetamine, methamphetamine, opioid, morphine, marijuana, heroin, caffeine, and nicotine.

9. The pharmaceutical composition according to claim 1, which is used for the treatment of at least one selected from the group consisting of schizophrenia, Tourette's syndrome, bulimia, and addiction.

10. The pharmaceutical composition of claim 4, wherein the maximum amount of dopamine released by said increase is 200% or more of the subject's baseline dopamine amount.

11. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is administered to the subject at a dose that does not reduce the extracellular dopamine concentration associated with dopaminergic neurons in the subject to 50% or less of the subject's baseline extracellular dopamine concentration.

12. The pharmaceutical composition of claim 1, wherein the subject is a human.

13. A tyrosine hydroxylase inhibitor used to inhibit the release of catecholamines from catecholaminergic neurons in a subject.

14. A method for inhibiting the release of catecholamines from catecholaminergic neurons in a subject, comprising administering to the subject a tyrosine hydroxylase inhibitor or the pharmaceutical composition of claim 1.

15. A method for treating schizophrenia, bulimia, or addiction, comprising administering to a subject a tyrosine hydroxylase inhibitor or the pharmaceutical composition of claim 1.

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