JP7244002B2 - Prophylactic and therapeutic drugs for alcoholism and food for prevention and treatment of alcoholism - Google Patents

Description

TECHNICAL FIELD The present invention relates to preventive and therapeutic drugs for alcoholism , and food products for prevention and treatment of alcoholism.

Alcohol dependence is said to be a disease that affects about 1 million people, which is equivalent to 1% of Japanese people. When alcohol is broken down in the liver, it produces acetaldehyde, which causes unpleasant symptoms. Accumulation of acetaldehyde in the body causes unpleasant symptoms. Therefore, drugs that inhibit aldehyde dehydrogenase, which converts acetaldehyde to acetic acid, are used as antialcoholic drugs. Discomfort caused by inhibition of aldehyde dehydrogenase prompts the patient to quit drinking, and continuous abstinence is expected. Disulfiram disclosed in Non-Patent Document 1, cyanamide disclosed in Non-Patent Document 2, and the like are known as anti-alcoholic drugs.

Acamprosate, which adjusts the balance between excitement and inhibition in the brain disturbed by alcohol, is sometimes used as a drug for alcoholism. Acamprosate suppresses alcohol-induced glutamate neuronal activity and suppresses the desire to drink.

Kitson T. M. , "Studies on the interaction between disulfiram and sheep liver cytoplasmic aldehyde dehydrogenase.", Biochem. J. , 1978, 175(1), 83-90 DeMaster EG, 3 others, "Metabolic activation of cyanamide by liver mitochondria, a requirement for the inhibition of aldehyde dehydrogenase enzymes.", Biochem. Biophys. Res. Commun. , 1982, 107(4), 1333-1339

The above-mentioned drugs used for alcoholism have large individual differences in efficacy and have many side effects. Therefore, there is a need for a drug that improves alcoholism by a new mechanism. Since it is still difficult to completely cure alcoholism, preventing the establishment of alcoholism is also an effective countermeasure against alcoholism.

The present invention has been made in view of the above circumstances, and in addition to the treatment of alcoholism, a prophylactic and therapeutic agent for alcoholism that can prevent the establishment of alcoholism, and a drug for treating alcoholism. The purpose is to provide prophylactic and therapeutic foods.

The present inventor has conducted extensive research on alcoholism, and has elucidated a new function of γ-oryzanol, a physiologically active substance contained in lipids such as rice bran. That is, it was found that γ-oryzanol acts on the intracerebral reward system to correct the abnormal activation state of dopamine, GABA and acetylcholinesterase (AChE), and that it is useful for the prevention and treatment of alcoholism. completed.

The prophylactic or therapeutic drug for alcoholism according to the first aspect of the present invention is
containing γ-oryzanol,
It is for suppressing alcohol preference of the administration subject.

The preventive or therapeutic food for alcoholism according to the second aspect of the present invention is
containing γ-oryzanol,
It is for suppressing the alcohol preference of the ingested subject.

According to the present invention, in addition to treating alcoholism, it is possible to prevent the establishment of alcoholism.

FIG. 4 is a diagram showing the inhibitory effect of γ-oryzanol on alcohol preference. FIG. 3 shows the preventive effect of γ-oryzanol on increased alcohol preference. FIG. 4 shows the expression level of tyrosine hydroxylase (TH) in the ventral tegmental area (VTA). FIG. 2 shows dopamine content in the nucleus accumbens (NAc). FIG. 2 shows the expression level of glutamic acid decarboxylase (GAD65) in NAc. FIG. 3 shows the expression level of GAD65 in the prefrontal cortex (mPFC). FIG. 4 is a diagram showing the expression level of AChE in VTA. FIG. 4 shows the expression level of AChE mRNA in neuroblastoma cell lines. FIG. 2 shows the amount of binding of transcription promoter Sp1 and transcription repressor Egr1 to the AChE promoter region in neuroblastoma cell lines.

(Embodiment)
An embodiment of the present invention will be described. The drug for treating alcoholism according to this embodiment contains γ-oryzanol. γ-Oryzanol is a physiologically active substance contained in brown rice. As shown in the examples below, γ-oryzanol can reduce alcohol preference and prevent excessive alcohol intake. γ-oryzanol can be extracted, for example, from rice bran oil or rice germ oil. Also, commercially available γ-oryzanol may be used.

The drug for treating alcoholism according to this embodiment is produced by a known method. The drug for treating alcoholism contains 0.1-99% by weight, 1-50% by weight, preferably 1-20% by weight of γ-oryzanol as an active ingredient. The drug for treating alcoholism may be, for example, a combination drug blended with a pharmacologically acceptable carrier. Pharmaceutically acceptable carriers are various organic or inorganic carrier substances used as pharmaceutical ingredients. Pharmaceutically acceptable carriers are, for example, excipients, lubricants, binders and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, tonicity agents and buffers in liquid formulations. It is also used as an analgesic, etc. in therapeutic drugs for alcoholism. Additives such as preservatives, antioxidants, coloring agents and sweetening agents may also be added as necessary.

The administration route of the therapeutic drug for alcoholism according to the present embodiment is not particularly limited. The drug for treating alcoholism is preferably orally administered. The dosage of the therapeutic drug for alcoholism is appropriately determined according to the subject's sex, age, body weight, symptoms, and the like. The drug for treating alcoholism is administered in an effective amount of γ-oryzanol. An effective amount is that amount of γ-oryzanol required to inhibit, ameliorate or treat alcoholism and to slow, inhibit, prevent, reverse or cure the condition being treated or treated. quantity.

The drug for treating alcoholism according to the present embodiment contains γ-oryzanol as an active ingredient of 0.01 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg per day, based on the body weight of the administration subject, for example. kg to 200 mg/kg, more preferably 0.2 mg/kg to 20 mg/kg. The drug for treating alcoholism may be administered once a day or in more divided doses. When the drug for treating alcoholism is administered in divided doses, the drug for treating alcoholism is preferably administered from 1 to 4 times per day. Also, the drug for treating alcoholism may be administered at different dosing frequencies, such as daily, every other day, once a week, every other week, once a month, and the like. The timing of administration of the therapeutic drug for alcoholism may be determined as appropriate according to the expected therapeutic effect. For example, a therapeutic agent for alcoholism may be pre-administered post-alcoholism or prophylactically. Amounts outside the above ranges can also be used, if desired.

As shown in the examples below, γ-oryzanol has the effect of preventing an increase in alcohol preference. Therefore, the therapeutic drug for alcoholism according to the present embodiment may be used as a preventive drug for alcoholism.

The drug for treating alcoholism according to the present embodiment contains γ-oryzanol as an active ingredient. γ-Oryzanol corrects the abnormal activation state of dopamine, GABA and AChE, and induces aversion to alcohol. Therefore, the drug for treating alcoholism can suppress alcohol intake and treat alcoholism. In addition, the preventive drug for alcoholism can prevent the establishment of alcoholism.

γ-oryzanol acts on both the hypothalamus and intracerebral reward system to alleviate dependence and preference for animal fat (Chisayo Kozuka, 7 others, "Impact of brown rice-specific γ-oryzanol on epigenetic modulation of dopamine D2 receptors in brain striatum in high-fat-diet-induced obesity in mice”, Diabetologia, 2017, Vol.60, 8, p.1502-1511). Specifically, habitual overdose of animal fat promotes DNA methylation in the promoter region of the dopamine receptor gene in the intracerebral reward system such as the striatum, and decreases dopamine receptor expression. In contrast, γ-oryzanol inhibits DNA methyltransferase, reduces DNA methylation, and normalizes dopamine receptor expression in the brain reward system.

The examples below show that γ-oryzanol tends to increase the content of the neurotransmitter dopamine in the nucleus accumbens. The increase in brain reward system dopamine content in the suppression of alcoholism is distinct from the normalization of dopamine receptor expression as in excess animal fat intake. γ-oryzanol ameliorates or prevents alcohol dependence by mechanisms of not only increasing dopamine content in the brain reward system, but also correcting abnormal activity of GABA and AChE.

In addition, γ-oryzanol may be encapsulated in biocompatible particles in order to improve the pharmacokinetics of γ-oryzanol. Biocompatible particles can be made, for example, from polymers. As the polymer used in the present embodiment, a biocompatible polymer having low irritation and toxicity to living bodies and having biocompatibility that is decomposed and metabolized after administration is preferable.

Preferably, polylactic acid, polyglycolic acid, lactic acid/glycolic acid copolymer, or lactic acid/aspartic acid copolymer is used as the biocompatible polymer. Suitably, the biocompatible particles comprise polylactide glycolide copolymer (PLGA) or polyethylene glycol/chitosan modified-PLGA (PEG/CS-PLGA) as the biocompatible polymer.

In another embodiment, there is provided a preventive or therapeutic food for alcoholism containing γ-oryzanol as an active ingredient. The food is manufactured by a known method. The food is ingested (eating and drinking) as, for example, functional food, food with function claims, food for specified health uses, food with nutrient function, and the like.

The content of γ-oryzanol in the food for prevention or treatment of alcoholism is not particularly limited. For example, the food contains 90 wt%, 80 wt%, 70 wt%, 60 wt%, 50 wt%, 40 wt%, 30 wt%, 20 wt%, 10 wt%, 5 wt%, 3 wt%, 2 wt%, 1 wt% or 0.1 wt% γ-oryzanol.

Foods for prevention or treatment of alcoholism contain, for example, γ-oryzanol as an active ingredient of 0.01 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg, per day, based on the body weight of the subject to be administered. ~200 mg/kg, more preferably between 0.2 mg/kg and 20 mg/kg.

Preferably, the packaging, package insert, sales pamphlet, or the like of the food for prevention or treatment of alcoholism states that the food is taken for the purpose of prevention or treatment of alcoholism.

The forms of food for prevention or treatment of alcoholism are, for example, sweets such as candy, cookies, tablets, chewing gum and jelly, processed grain products such as noodles, bread, rice and biscuits, sausage, ham and fish paste. and other paste products, dairy products such as butter and yogurt, sprinkles and seasonings. Foods for the prevention or treatment of alcoholism include beverages, which may be, for example, nutritional drinks, soft drinks, black and green teas, and the like. The food for prevention or treatment of alcoholism may contain additives such as sweeteners, flavoring agents and coloring agents.

Yet another embodiment provides a preventive or therapeutic supplement for alcoholism comprising γ-oryzanol. Alcoholism prophylactic or therapeutic supplements may be provided in liquid, powder, tablet or capsule form, or the like. In another embodiment, there is also provided an additive for the prevention or treatment of alcoholism comprising γ-oryzanol.

In another embodiment, a method of treating alcoholism is provided by administering γ-oryzanol to an alcoholic patient. Also provided is a method of preventing alcoholism in a subject by administering γ-oryzanol to the subject. In another embodiment, an antialcoholic, antialcoholic or abstinence aid comprising γ-oryzanol is provided.

Also, according to the examples below, γ-oryzanol directly suppresses the expression of AChE. Therefore, γ-oryzanol can be used as an AChE expression inhibitor.

Hereinafter, the present invention will be described in more detail with test examples, but the present invention is not limited by the test examples.

(Test Example 1 Evaluation of ethanol preference test with γ-oryzanol using alcoholism model mice)
A diet containing γ-oryzanol was fed to alcoholism model mice, and changes in ethanol preference were examined.

In breeding C57BL/6J mice (11 weeks old, male), a mouse strain reported to have a high preference for alcohol, the ethanol concentration in the drinking water bottle was increased from 5% to 20%, and the ethanol intake was increased. and established mice with steadily increased ethanol preference for 2 months (EtOH group). Ethanol preference was evaluated by placing water and 20% ethanol jelly (each containing 3% sucrose) diagonally in an open field (50 x 50 x 50 cm plastic box) and placing one mouse in each group. , by measuring the weight of the jelly food consumed in 30 minutes. Ethanol preference was defined as the ratio of the weight of the ethanol jelly eaten to the weight of the initial ethanol jelly.

In addition to the EtOH group, a group ingested 1% γ-oryzanol-containing feed after an increase in ethanol preference was observed (EtOH + Orz group), a group ingested 1% γ-oryzanol-containing feed at the same time as ethanol administration (EtOH + int Orz group), and a control group to which sterilized water was administered as a control, were evaluated for ethanol preference.

(result)
Fig. 1 shows the preference for ethanol 2 months and 3.5 months after the start of ethanol administration. Compared to the control group, the EtOH group and the EtOH+Orz group showed a significant increase in their preference for ethanol two months after the start of ethanol administration. From the 2nd month onwards, in the EtOH+Orz group fed with the γ-oryzanol-containing feed, the preference for ethanol was significantly reduced after 1.5 months of feed change. As shown in FIG. 2, EtOH+int. In the Orz group, ethanol preference did not increase after 2 months when alcohol preference is normally established. These results indicated that the feeding of 1% γ-oryzanol-containing feed improved the preference for ethanol and had a preventive effect.

(Test Example 2 Evaluation of dopamine signals in the ventral tegmental area by γ-oryzanol using alcoholism model mice)
Changes in the expression level of TH, a rate-limiting enzyme for dopamine synthesis, in the VTA of the brains of mice fed a γ-oryzanol-containing diet and having a reduced preference for ethanol were examined. Also, the content of dopamine in the NAc, to which VTA dopamine neurons project, was measured using an HPLC-electrochemical detector.

Control group, EtOH group, EtOH+Orz group and EtOH+int. VTA was collected from each mouse in the Orz group. Proteins were extracted from VTA and Western blotting was used to compare TH protein expression. As an endogenous control in the nucleus, the expression level of β-actin protein was determined, and the value obtained by dividing the expression level of TH protein by the expression level of β-actin protein was used for comparison of the expression levels.

Equal volumes of tissue pieces were collected from the NAc of each mouse in the control group, EtOH group and EtOH+Orz group, and dopamine was extracted with a perchlorate buffer. Dopamine was quantified using an HPLC system (manufactured by Acom) equipped with a reversed-phase column and an electrochemical detector.

(result)
The EtOH group, the EtOH+Orz group, and the EtOH+int. FIG. 3 shows relative values of TH protein expression levels in the Orz group. Compared to the control group, the EtOH group showed a decrease in the TH protein expression level, and the EtOH+Orz group showed a tendency to recover the TH protein expression level. Compared to the TH protein expression level in the EtOH group, the EtOH + int. The Orz group showed significantly enhanced TH protein expression. FIG. 4 shows dopamine content in NAc. Compared with the EtOH group, the EtOH+Orz group tended to increase the dopamine content.

From these results, in the VTA, which is a reward system control region, the expression level of TH protein, which was decreased by ethanol intake, was increased by feeding γ-oryzanol-containing feed, thereby increasing the dopamine content in NAc and ethanol preference. It was shown that sexuality improvement and preventive effect can be obtained.

(Test Example 3 Evaluation of GAD65 expression levels in the nucleus accumbens and prefrontal cortex by γ-oryzanol using alcoholism model mice)
Changes in the expression level of GAD65 in the reward control region NAc and the aversive control region mPFC of mice that were fed a γ-oryzanol-containing diet and had decreased preference for ethanol were examined. GAD65 degrades glutamic acid to produce GABA.

Control group, EtOH group, EtOH+Orz group and EtOH+int. NAc and mPFC were collected from each Orz group of mice. Proteins were extracted from NAc and mPFC and Western blotting was used to compare GAD65 protein expression. The expression level of β-actin protein in the neuronal nucleus was determined, and the value obtained by dividing the expression level of GAD65 protein by the expression level of β-actin protein was used for comparison of the expression levels.

(result)
The EtOH group, the EtOH+Orz group, and the EtOH+int. FIG. 5 shows the relative expression levels of GAD65 protein in the Orz group. The expression level of GAD65 protein in the NAc, to which dopaminergic nerves are projected from the VTA, was higher in the EtOH+Orz group and the EtOH+int. It decreased significantly in the Orz group. FIG. 6 shows the relative expression levels of GAD65 protein in mPFC. In the mPFC, the expression level of GAD65 protein was significantly increased in the EtOH+Orz group compared to the EtOH group.

Based on these results, it is considered that the expression of GAD65, which was increased by ethanol intake, was significantly reduced by feeding γ-oryzanol-containing feed in the NAc, which is a reward system control region, and thus the preference for ethanol was suppressed. On the other hand, in the aversive control region mPFC, the expression of GAD65 was increased, and it is thought that the aversive behavior towards ethanol increased.

(Test Example 4 Evaluation of AChE expression level in ventral tegmental area by γ-oryzanol using alcoholism model mice)
Changes in the expression level of AChE in the VTA of mice fed a γ-oryzanol-containing diet and having a reduced preference for ethanol were examined.

Control group, EtOH group, EtOH+Orz group and EtOH+int. Proteins were extracted from the VTA of mice in each Orz group, and the expression of AChE protein was compared using Western blotting. The expression level of β-actin protein in the neuronal nucleus was determined, and the value obtained by dividing the expression level of AChE protein by the expression level of β-actin protein was used for comparison of the expression levels.

(result)
The EtOH group, the EtOH+Orz group, and the EtOH+int. FIG. 7 shows the relative expression levels of AChE protein in the Orz group. Compared to the control group, the expression level of AChE protein in the EtOH group was significantly increased, and the EtOH+Orz group and the EtOH+int. In the Orz group, the expression level of AChE protein was significantly decreased compared to the EtOH group.

From this result, in the VTA, which is the reward system control region, the expression of AChE protein, which was increased by ethanol intake, was significantly reduced by feeding γ-oryzanol-containing feed, and the acetylcholine concentration in the VTA was increased, leading to an increase in ethanol preference. It was suggested that improvement and preventive effects could be obtained.

(Test Example 5 Evaluation of AChE expression level in neuroblastoma cell line by addition of γ-oryzanol and study of expression control mechanism)
Using a neuroblastoma cell line (SH-SY5Y) in which AChE expression has been confirmed, changes in the expression level of AChE due to the addition of γ-oryzanol were examined. We also investigated the AChE mRNA transcriptional control mechanism.

γ-oryzanol at various concentrations from 0 to 60 μM was added and administered for 2 days to SH-SY5Y that had been differentiated with retinoic acid in advance. Two hours after the addition of carbachol, an ACh receptor agonist, mRNA was extracted and purified from SH-SY5Y, and the expression level of AChE mRNA was compared using the reverse transcription quantitative polymerase chain reaction (RT-QPCR) method. The nucleotide sequence of the forward primer and the nucleotide sequence of the reverse primer used for RT-QPCR are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Furthermore, in order to elucidate the binding of transcription factors to the AChE promoter region and the transcription control mechanism, the chromatin sedimentation method was used to measure and compare the DNA binding activities of transcription factors to the promoter region. One hour after the addition of 40 μM γ-oryzanol, the cells were fixed, the protein-DNA complex was extracted so as not to be destroyed, and the transcription factor-DNA fragment complex bound to the promoter region was recovered using a transcription factor-specific antibody. Binding domain-specific primers were used to compare the proportion of transcription factors bound to the AChE promoter region.

It has been reported that in the AChE promoter region, the transcription promoter Sp1 and the transcription repressor Egr1 are responsible for transcription regulation through competitive binding to the same DNA sequence (Getman DK et al., J. Biol. Chem., 1995, 270: 23511-23519 and Mutero A et al., J. Biol. Chem., 1995, 270:1866-1872).

(result)
FIG. 8 shows the relative values of the AChE mRNA expression level in cells to which each concentration of γ-oryzanol was added, when the expression level of AChE mRNA in cells to which γ-oryzanol was not added (0 μM) was set to 1. Addition of 40 μM and 60 μM γ-oryzanol significantly decreased the AChE expression level compared to 0 μM. The expression level of AChE mRNA decreased in a concentration-dependent manner of added γ-oryzanol. FIG. 9 shows the DNA binding amounts of the transcription promoter Sp1 and the transcription repressor Egr1 in the AChE promoter region. Addition of 40 μM γ-oryzanol significantly increased the DNA-binding activity of Egr1.

These results indicate that γ-oryzanol suppresses AChE mRNA expression by increasing the binding activity of the transcription repressor Egr1 to the AChE promoter region.

The present invention is capable of various embodiments and modifications without departing from the broader spirit and scope of the invention. Moreover, the embodiment described above is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of equivalent inventions are considered to be within the scope of the present invention.

INDUSTRIAL APPLICABILITY The present invention is suitable for pharmaceuticals such as antialcoholic drugs, functional foods, and the like.

Claims (2)

containing γ-oryzanol,
For suppressing alcohol preference of the administration subject,
A prophylactic or therapeutic drug for alcoholism. containing γ-oryzanol,
for suppressing alcohol preference of the ingested subject,
A preventive or therapeutic food for alcoholism.

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