WO2008000142A1

WO2008000142A1 - Dopamine transporter agonist and its use

Info

Publication number: WO2008000142A1
Application number: PCT/CN2007/001839
Authority: WO
Inventors: Lihe Guo; Gang Zhao; Mingmin Xu; Xuesong Wang; Shaoyun Zang
Original assignee: Cell Star Bio-Technologies Co., Limited
Priority date: 2006-06-23
Filing date: 2007-06-11
Publication date: 2008-01-03
Also published as: CN101091706A; CN101091706B

Abstract

Use of flavonoids or their derivatives in the manufacture of medicaments for treatment of psychiatric and neurological disorders and composition containing these compounds. It is firstly proposed and proved that dopamine transporter can be agitated. A kind of dopamine transporter agonist is provided for treating psychiatric and neurological disorders, especially disorders related to dopaminergic nerve hyperactivity.

Description

Dopamine transporter agonist and use thereof

The present invention relates to the field of biomedicine, and more particularly, to a class of dopamine transporter agonists and their use. Background technique

Mental illnesses and neurological diseases severely affect the health of millions of people around the world. Some common mental illnesses and

/ or neurological diseases such as anxiety disorders, mood disorders, schizophrenia, personality disorders, psychosexual disorders > substance abuse And drug abuse and dependence affect people of all ages, and the duration of these diseases varies from weeks to decades.

With the development of neurobiology and molecular biology, people gradually understand the effects of some biochemical factors that cause mental and neurological diseases. Studies have found that important neurotransmitters such as dopamine (DA), norepinephrine, and serotonin play key physiological roles in psychiatric and neurological diseases. Dopamine is a major catecholamine neurotransmitter in the mammalian brain that controls many functions such as exercise, cognition, emotion, positive reinforcement, feeding, and endocrine regulation. Dopamine accounts for 80% of the brain's catecholamine neurotransmitters and is one of the most important neurotransmitters in the mammalian central nervous system. It plays a key regulatory role in the physiological and mental health of the human body.

Under normal physiological conditions, the process of nerve impulse transmission is as follows: When nerve cells transmit nerve impulses, neurotransmitters are released from neurons to synaptic spaces, and neurotransmitters bind to corresponding receptors on postsynaptic, resulting in physiological effects. At the same time, neurotransmitters are inactivated by different mechanisms. There are two main ways in which neurotransmitters are inactivated. One is the rapid metabolism of neurotransmitters to inactive substances; the other is the neurotransmitter that re-uptakes synaptic clefts through presynaptic membranes or glial cells. The re-uptake of neurotransmitters can be used repeatedly by neurons. The way of dopaminergic neurons belongs to the latter. The main mechanism of inactivation of dopamine is the reuptake of dopamine by the dopamine transporter (DAT) on the presynaptic membrane. DAT is a 12-transmembrane domain structural protein with sodium ion and chloride ion dependence in the presynaptic membrane, which is the basic substance for terminating dopamine and maintaining the balance of extracellular dopamine concentration. Therefore, the main function of dopamine transporters is to mediate dopamine reuptake, reduce dopaminergic concentrations in the synaptic cleft, regulate dopaminergic signaling in time course and intensity, and is the most important factor regulating and maintaining dopamine neurological homeostasis (Giros). Etc., Nature 1996, 379, 696). Dopaminergic hyperfunction (excessive dopamine secretion or high sensitivity of dopamine receptors in the brain) leads to disorder of dopaminergic neuroregulation, which is the main pathogenesis and important pathological links of many diseases, such as drug addiction and schizophrenia. , anxiety, emotional affliction, Drug abuse and dependence, Tourette's syndrome, muscle weakness, tardive dyskinesia, etc.

Although some recent advances have been made in the treatment of septic and neurological diseases, many patients cannot receive effective treatment, in part because the drugs are ineffective against them or the drugs have serious side effects such as anti-parasympathetic effects, myocardial toxicity, weight loss, etc. Editor-in-Chief Hao Wei. Psychiatry, Fourth Edition, Beijing: People's Medical Publishing House, 2001; Kapur, Shitij et al Current Opinion in Psychiatry. 17 (2) : 115-121, March 2004.

Therefore, there is an urgent need to further study the mechanism of regulating dopamine transport and to develop novel drugs for treating related psychiatric diseases or neurological diseases with remarkable effects and small side effects. Summary of the invention

The object of the present invention is to provide a class of dopamine transporter agonists which can regulate the transport of dopamine by promoting the transport function of dopamine transporters, maintain the balance of extracellular dopamine concentration, and can be used for the preparation of psychotic diseases and neurological diseases. medicine.

In a first aspect of the invention, there is provided a use of a flavonoid or a derivative thereof for the preparation of a pharmaceutical composition as a dopamine transporter agonist, or for the preparation of a medicament for treating a divine or neurological disease.

In another preferred embodiment of the invention, the agonist is an agonist of dopamine transporter reuptake.

In another preferred embodiment of the present invention, the mental disease or neurological disease is a mental disease or a neurological disease caused by hyperparaceptive hyperactivity.

In another preferred embodiment of the present invention, the flavonoid compound is selected from the group consisting of: apigenin, luteolin, quercetin, myricetin, poplarin, soybean aglycone, wogonin, baicalein, Genistein, morin, marigold, hesperetin, buckthorn gold, rhamnosin, or a combination thereof.

More preferably, the flavonoid is selected from the group consisting of: luteolin, apigenin, quercetin, soyin, or a combination thereof.

More preferably, the flavonoid is luteolin.

In another preferred embodiment of the present invention, the mental disease or neurological disease is a dopaminergic hyperfunction disorder.

In another preferred embodiment of the present invention, the mental or neurological disease is selected from the group consisting of: an addiction disorder, an anxiety disorder, Alzheimer's syndrome, anorexia nervosa, schizophrenia, Parkinson's syndrome, Insomnia, drug abuse and dependence, vomiting, irritable bowel syndrome, menopausal syndrome, Wilson's disease, chorea, demyelinating disease, mania, obsessive-compulsive disorder, or Tourette's syndrome.

More preferably, the psychotic or neurological disorder is selected from the group consisting of: an addiction disorder, schizophrenia, or drug abuse and dependence. In another preferred embodiment of the present invention, the addictive disease is an opioid addiction. More preferably, the opioid comprises, but is not limited to, opioid, morphine, heroin, cocaine, dulamine, methadone, amphetamine, wherein morphine, or cocaine is preferred.

In a second aspect of the invention, a method of determining a candidate drug useful for treating a psychiatric or neurological disorder, the method comprising the steps of:

(.1) In the test group, the amount of dopamine transporter to dopamine was measured in an in vitro system in the presence of a candidate substance; in the control group, in the absence of a candidate substance, in an in vitro system Determination of the amount of dopamine transporter to dopamine transport;

(2) Comparing the dopamine transport amount of the test group with the dopamine transport amount of the control group, if the test group has a higher dopamine transport amount (preferably significantly higher than, for example, 20% higher, more preferably 40% higher, more preferably high 60 The dopamine transport amount of the control group of % or higher indicates that the candidate substance is an agonist of the dopamine transporter, and thus can be used as a drug candidate for treating a mental disease or a neurological disease.

In another preferred embodiment of the present invention, the method further includes the following steps:

(3) administering a candidate substance of the agonist which has been shown to be a dopamine transporter obtained in the step (2) to a model animal of a mental disease or a neurological disease of a non-human mammal, and observing the behavior of the model animal To determine whether the symptoms of their treatment for mental illness or neurological disease have improved,

Among them, candidate substances which significantly improve the symptoms of treating mental illness or neurological diseases in model animals are candidates for the treatment of psychiatric diseases or neurological diseases.

In another preferred embodiment of the invention, the animal model is a mouse, a rat, or a monkey.

In another preferred embodiment of the invention, the following behavior or symptoms of the model animal are included in step (3) including, but not limited to, abnormal posture, irritation, gnaw, tearing, diarrhea, salivation or weight loss, or a combination thereof.

In a third aspect of the invention, a dopamine transporter agonist is provided, wherein the dopamine transporter agonist is capable of specifically promoting dopamine transporter uptake of dopamine and said dopamine transporter agonist is used as a therapeutic psychotropic A drug for a disease or a neurological disease.

In a fourth aspect of the invention, there is provided the use of a dopamine transporter agonist for the preparation of a pharmaceutical composition for the treatment of a psychiatric or neurological disorder. ,

In another preferred embodiment of the invention, the agonist is a flavonoid. More preferably, the agonist is selected from the group consisting of luteolin.

In a fifth aspect of the invention, a method of treating a psychiatric or neurological condition, the method comprising: administering an effective amount of a flavonoid or a derivative thereof to a subject in need of treatment. In another aspect, the present invention provides a pharmaceutical composition for treating a psychiatric disease or a neurological disease, the pharmaceutical composition comprising an effective amount of a flavonoid compound or a derivative thereof, and a pharmaceutically acceptable carrier .

In another preferred embodiment of the invention, the pharmaceutical composition comprises:

Flavonoids or derivatives thereof: 5-10 parts by weight;

A pharmaceutically acceptable carrier: 80-150 parts by weight;

Wherein, the total content of the flavonoid compound or its derivative is from 0.1 to 20% by weight based on the total weight of the pharmaceutical composition. .

In another preferred embodiment of the present invention, the pharmaceutical composition contains at least one flavonoid compound selected from the group consisting of apigenin, luteolin, quercetin, myricetin, poplarin, and soybean aglycone. , baicalein, baicalein, genistein, morin, marigold, lysin, buckthorn gold, or buckthorn.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS

Figure 1 shows the extraction and separation of sz ethanol extract into four parts, namely petroleum ether (szi), chloroform (εζ2), ethyl acetate (SZ3) and n-butanol (SZ4), which are at 10 μg/ml. The effect of concentration on the uptake activity of dopamine.

Figure 2 shows the effect of 'SZ3' divided into multiple sites on the polyacrylamide gel, which have an effect on the uptake of dopamine at a concentration of 10 g/ml. '

Figure 3 shows the uptake of dopamine by apigenin (SZ91) at concentrations of 1 μg/ml and 10 μg/ml.

Figure 4 shows the effect of different concentrations of luteolin (SZ92) on dopamine uptake.

Figure 5 shows the effect of purine acetylated SZ92' on dopamine uptake at a concentration of 10 μg/ml.

Figure 6 shows the activity of SZ92 on dopamine uptake.

Figure 1 shows the effect of SZ91 and SZ92 on the viability of glutamate transporters in COS-7 cells.

Figure 8 shows the effects of soybean aglycone, quercetin, genistein, and apigenin on the activity of glutamate transporters in COS-7 cells.

Figure 9 shows the score of Yanagida's score for withdrawal symptoms in the morphine addiction modeling of rats.

Figure 10 shows the scores of Yanagida Eiji who had withdrawal symptoms when the rats were treated with morphine addiction.

Figure 11 shows a comparison of the preference times of mice in each side of the white box.

Figure 12 shows a comparison of the pain sensation period (the hind limb time) of each group of mice.

Figure 13 shows a comparison of the total number of grids traversed within 2 hours for each test group of animals. detailed description

After long-term research and experiment, the present inventors first proposed the dopamine transporter agonism theory and proved that Dopa Amine transporters can be agonized, and this agonistic effect can serve as a new target for the treatment of psychiatric and neurological diseases. Moreover, the inventors obtained a class of dopamine transporter agonists which can significantly promote the transport of dopamine transporters or uptake of dopamine by drug screening. The present invention has been completed based on the above research results.

According to the present inventors' study, the uptake and transport of dopamine by dopamine transporters can be stimulated, the uptake of synaptic interstitial dopamine can be enhanced, the extracellular dopamine concentration can be regulated, the dopamine homeostasis can be maintained in the synaptic cleft, and the postsynaptic dopa neurons can be reduced. Excitability. Stimulation of the transport of dopamine transporters can be achieved using dopamine transporter agonists.

As used herein, the "dopamine transporter agonist" includes all substances which enhance the uptake and transport function of dopamine transporter to dopamine, which are capable of fully or partially stimulating dopamine transporter transport or uptake of dopamine. More preferably, the "dopamine transporter agonist" is a flavonoid compound, a derivative thereof or the like.

As used herein, the term "flavonoid" refers to a generic term for a class of compounds in which two benzene rings are joined by a three carbon chain to form a C6-C3-C6 basic skeleton, and the structural formula is as in formula (I) or formula. (Π) shows:

In the formula, it is a hydrogen, a hydroxyl group, an alkoxy group or a halogen element.

More preferably, the flavonoids include, but are not limited to, apigenin (also referred to herein as SZ91), luteolin (also referred to herein as SZ92), quercetin. , myricetin, chrysin, dadzein, wogonin, baicalein, genistein, morin, marigold (quercetagetin), hesperetin, rhamnazin, rhamnetin.

More preferably, the flavonoid compound is selected from the group consisting of luteolin, apigenin, quercetin, or soybean aglycone; wherein the luteolin has a structure represented by formula (III); The substance has the structure shown in formula (IV):

Most preferably, the flavonoid compound is luteolin.

In the present invention, the dopamine transporter agonist may be present in the form of a pure flavonoid or a derivative thereof; or may be a mixture or extract containing a flavonoid compound or a derivative thereof (for example, extractable from a traditional Chinese medicine) The form exists; or it may be in the form of a Chinese medicinal material containing a flavonoid compound or a derivative thereof or an active site of a traditional Chinese medicine.

The inventors have found that the dopamine transporter agonist promotes the dopamine transporter to dopamine uptake, but has no effect or effect on other transporters (e.g., y-aminobutyrate transporter). This indicates that the dopamine transporter agonist is specific for promoting dopamine transporter uptake function. In the present invention, the mental diseases and neurological diseases include, but are not limited to, the following diseases: Addictive diseases

(addiction), anxiety, Alzheimer's disease, Nervous Anorexia, schizophrenia, Parkinson's disease, Parkinson's disease, Sleeping disorders, drug abuse and dependence, emesis, irritable bowel syndrome, menstrual dysphoria syndrome, Wilson's disease (Wilson) ' s disease ), Chorea, Demyelinating disorders > mania, obsessive - compulsive disorder or Tourette's syndrome; Addictive diseases, schizophrenia and drug abuse and dependence; most preferred are addictive diseases. .

Further, the addictive disease is an opioid drug addiction. Further, the opioids include, but are not limited to, opioids, morphine, heroin, cocaine, dulamine, methadone, amphetamine, with morphine, or cocaine being preferred. ·

In a preferred embodiment of the present invention, the dopamine protein agonist of the present invention enables the body to reduce the withdrawal symptoms caused by opioid addiction, thereby achieving a good effect of treating the addiction and abuse of opioids. Its mechanism of action is: The use of dopamine transporter agonists to promote dopamine transporter dopamine uptake in the synaptic cleft, thereby reducing the dopamine receptor dopamine receptor agonism and attenuating the dopamine neuron excitation. Therefore, it has a corrective effect on the abnormal hyperactivity of the dopaminergic nerve (reward effect system) caused by opioid addiction. In a preferred embodiment of the present invention, the dopamine protein agonist of the present invention enables the body to reduce symptoms of schizophrenia. Schizophrenia is associated with increased dopamine function in the brain. Dopamine transporter agonists promote the dopamine uptake of dopamine transporters in the synaptic cleft, thereby reducing the enhancement of dopamine function in the brain, thereby reducing the symptoms of schizophrenia.

The dopamine transporter agonists of the invention may also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base. 'These salts include (1⁄4 is not limited to) salts with the following inorganic acids: hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and salts with organic acids, while organic acids refer to acetic acid, oxalic acid, succinic acid, tartaric acid, Sulfonic acid and maleic acid. Its The salt thereof includes a salt formed with an alkali metal or an alkaline earth metal such as sodium, potassium, calcium or magnesium, in the form of an ester, a carbamate or other conventional "prodrug" (when administered in this form) At the time, it can be converted into an active part in the body).

When a dopamine transporter agonist is used to treat a psychiatric disorder and a neurological disorder, the amount of the drug depends on the nature and extent of the disease and the condition in which the patient has received treatment. The dosage is usually determined by the prescribing physician. The clinical dose is 0.5-300 mg of dopamine transporter agonist per kilogram of body weight per day.

The present invention also provides a composition comprising the dopamine transporter agonist, preferably, the composition is a pharmaceutical composition. 01至9%。 Preferably, a suitable amount of the dopamine transporter agonist of the total weight of the pharmaceutical composition of 0. 01-99%, preferably 0. 1-90%.

• In addition to the dopamine transporter agonist as an active ingredient, the composition may also contain a pharmaceutically acceptable carrier including, but not limited to, fillers, disintegrants, lubricants, glidants, effervescent agents, Flavoring agents, coating materials, dietary products, or slow/controlled release agents. The composition may be prepared in the form of a solid or gel, such as a pill, a tablet, a capsule or the like, depending on the mode of administration; or in a liquid form such as an injection or suspension. They are suitable for oral administration, rectal administration, topical administration or parenteral administration, or intravenous administration. In the present invention, a "pharmaceutically acceptable" ingredient is a substance which is suitable for use in humans and/or animals without excessive adverse side effects (e.g., toxicity, irritability, and allergy), i.e., has a reasonable benefit/risk ratio. A "pharmaceutically acceptable carrier" is a pharmaceutically or food acceptable solvent, suspending agent or excipient for delivering the dopamine transporter agonist to an animal or human. The carrier can be a liquid or a solid. ·

The solid composition for oral administration of the present invention can be used in the form of tablets, pills, capsules, powders, granules, drops, and the like. These solid compositions are mixed with one or more active substances and at least one inert diluent, for example, lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, and polyethylene. Pyrrolidone, Qiongyi, table glue, magnesium aluminosilicate, magnesium aluminate. The composition may further contain an additive other than an inert diluent according to a usual method, for example, a lubricant such as magnesium stearate, a disintegrant such as calcium cellulose glycolate, a stabilizer such as lactose, glutamic acid or Tianyi. A co-solvent such as a cis. If it is a tablet or a preparation, it may be coated with a sugar coating such as sucrose, gelatin, hydroxymethylcellulose, hydroxypropylmethylcellulose or phthalate or a gastric-soluble or enteric film as needed. .

The liquid composition for oral administration includes a pharmaceutically acceptable emulsion, a solution, a suspending agent, a syrup, an elixir, etc., and the commonly used inert diluent includes purified water, and drunk. The composition may contain, in addition to the inert diluent, an auxiliary agent such as a wetting agent, a suspending agent, a sweetener, a flavoring agent, a fragrance, and a preservative. ''Injectables for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and opacifiers. The aqueous solution and the suspension include distilled water for injection and physiological saline. The water-insoluble solution and suspending agent include diethylene glycol, polyethylene glycol, cocoa butter, olive oil, castor oil and the like, alcohol such as ethanol, gum arabic, Tween 80 and the like. These compositions may also contain isotonic agents, preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizers (for example, lactose), and solubilizing agents (for example, Glutamate, aspartic acid). Sterile purposes can be achieved by sterilizing the above composition by filtration and using a sterilizing agent. Then, a sterile solid composition is prepared by using the above composition, and it can be utilized by dissolving it in a solvent with water or sterilizing before use.

Examples of specific pharmaceutically acceptable carriers and excipients that can be used in formulating the oral dosage forms of the present invention are described in U.S. Patent No. 3,903,297, issued toS. Techniques and compositions for making useful dosage forms of the invention are described in the following documents: 7 Modern Pharmaceutics, Chapters 9 and 10 (Editor Banker & Rhodes, 1979): Lieberman et al., Pharmaceutical Formulations: Tablets (Pharmaceutical Dosage Forms-. Tablets) (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Edition (1976).

The composition of the dopamine transporter agonist or dopamine transporter agonist of the present invention can also be used in combination with other drugs for treating psychotic diseases and neurological diseases, depending on various needs. Further, the dopamine transporter agonist of the present invention can also be used in combination with an analgesic or analgesic drug having an addiction, thereby achieving an analgesic effect while preventing animal addiction.

The invention also encompasses a method of treating a dopaminergic hyperreactive disorder, the method comprising: administering to a subject in need thereof an effective amount of a dopamine transporter agonist. Preferably, the dopamine transporter agonist is a xanthone compound or a derivative thereof.

Furthermore, the present invention also encompasses a method of screening for a medicament for treating a mental disorder or a neurological disorder such as a dopaminergic hyperactive disorder. The method comprises screening for a substance that promotes dopamine transporter transport or uptake of dopamine. Such screening can be accomplished by establishing a cellular or animal model that can be used to observe the transport or uptake of dopamine transporters.

The main advantages of the invention are:

(1) The dopamine transporter agonism theory was first proposed, and it was demonstrated that the dopamine transporter can be excited, and this agonistic effect can be used as a new target for treating hypertonic diseases of dopamine neurons.

(2) The first screening of a class of dopamine transporter agonists provides a new drug for the clinical treatment of psychotic or neurological diseases, especially for the treatment of dopaminergic hyperactivity.

(3) The dopamine transporter agonist does not affect the analgesic effect of opioids, which will have broad application prospects in clinical practice. '

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Percentage and number of copies, unless otherwise stated Weight calculation.

Unless otherwise defined, all professional and scientific terms used herein are the same as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the present invention.

The sources of the drugs used in the following examples are as follows:

Luteolin, apigenin, soybean aglycone, quercetin and genistein were purchased from China National Drug Control Institute;

1640 medium culture, calf serum was purchased from GIBC0 company;

3⁄4-DA, 3⁄4-GABA, 3⁄4-Glutamic Acid was purchased from Amersham Pharmacia Biotech; morphine hydrochloride (batch number 20030124) was purchased from Shenyang No. 1 Pharmaceutical Factory; naloxone hydrochloride (lot number) 20040211) purchased from Beijing Sihuan Pharmaceutical Factory; Chinese hamster ovary cells (CH0) and COS-7 cells were purchased from ATCC, USA;

Balb/c mice and SD rats were purchased from the Chinese Academy of Sciences Animal Center, Sprague-Dawley rats were purchased from Shanghai Slack Laboratory Animal Center; C57BL/6 mice were purchased from the Chinese Academy of Sciences Animal Center.

Other chemical reagents were purchased from Sigma. Example 1 Screening and Detection of MT Agonists

1. Establishment of an in vitro screening model for DAT agonists

An active screening cell line targeting dopamine transporter (DAT) and y-aminobutyrate transporter (GAT-1) was established.大鼠 The DAT and GAT-1 full-length cDNA coding sequences of DAT (GenBank accession number GI310097; GAT-1 GenBank accession number GI204221) were cloned into pCDNA3 vector (American Irwitrogen) by conventional molecular biology methods. In the multiple cloning site, the Chinese hamster ovary cells (CH0) were transfected by electroporation, and cultured for 48 hours with 1640 medium containing G418. After 10 days, all the cells in the control group died, and the experimental group formed many cell clones. After picking up the clone for one week, the cells were covered with the bottom of the well, and the medium was aspirated, and digested with trypsin. The cells in each well were seeded in respective wells of two 96-well plates. After the cells have filled the bottom of the well, one of the plates is used for isotope flow measurement. The cells in the well-transported wells of the corresponding plate were expanded stepwise, and the isotope flow rate was measured at each stage, and the cell line with the highest transport activity was selected. Finally, the cell clones with the highest transport activity were selected (named as D8, G1 cells) Seed preservation, establishment of a CH0 cell line that permanently expresses two neurotransmitters (DAT, GAT-1) of dopamine and Y-aminobutyric acid.

Results: A cell line with high expression of DAT and GAT-1 transporter was obtained by measuring the isotope flow rate. A cell screening platform for DAT agonists was established.

COS-7 cell culture: 1640 (GIBCO) medium, 10% FBS (PAA), penicillin and streptomycin were 100 IU/nd, respectively, cultured at 37 °C. After the cells are full, 0.25% trypsin (Shanghai Shenggong Bioengineering Co., Ltd.) digests and inoculates 2, polyamine transporter agonist in vitro screening and selection specificity determination

D8 cells were cultured in a 48-well plate (Costar) to cover the plate (approximately 60,000 cells per well). Discard the liquid. Wash once with PBS, aspirate the PBS solution, add 90 ul of HBS dOraM Hepes, lOOmM NaCl, PH8. 0) per well, incubate at 25 °C for 10 minutes, and add lOul of HBS reaction solution to each well. The experimental group and the positive control were added with 80 ul of HBS, 10 ul of different concentrations of the drug, and lOul 3⁄4-DA (Amersham Pharmacia Biotech), 100 mM vitamin C and ΙΟΟμΜpajilin. Incubate for 20 minutes at 25 ° C, wash three times with PBS solution in ice bath, lyse for 60 minutes with lysate, and aspirate the lysis of each well. Add the solution to 1.2 ml of scintillation fluid and put it into the liquid scintillation counter (Beckman) The isotope content (DMP value) is measured in LS 5000TA), which is used to measure the effect of the drug on the transport activity of dopamine transporter. For the GABA transporter assay, the method was similar to the dopamine transporter, replacing the 3⁄4-DA in the reaction system with G1 cells for D8 cells, 50 nM 3⁄4-GABA (Amersham Pharmacia Biotech), and the other methods were unchanged. 'The response to glutamate transporter uptake activity using cos-7 cells as a screening model, 3⁄4-glutamate and 3⁄4-fucaffeine' for the isotopes in the above reactions, with 1 mM glutamic acid and aspartame The acid was a negative control and the other methods were unchanged. Result

1. In vitro screening of dopamine transporter agonists

The ethanol extract of Suzi (SZ, purchased from Xuhui Chinese Herbal Pieces Factory) was extracted and separated into four parts, namely petroleum ether '(S.Z1), chloroform (SZ2), ethyl acetate (SZ3) and n-nonanol (SZ4). ), activity tracking showed that SZ3 had the strongest dopamine uptake at 10 μg/ml, which was 8 times higher than that of the control group, as shown in Figure 1. ' ' SZ3 was separated by polyacrylamide gel and divided into multiple parts (SZ2, SZ3, SZ4, SZ5, SZ6, SZ8, SZ9). Activity tracking showed that at the concentration of 10 μg/ml, SZ9 The most vigorous, about 8 times the control group, see Figure 2.

Further purification and separation of SZ9, obtained pure SZ91 (identified as apigenin) and SZ92 (identified as luteolin), and acetylated SZ92 after acetylation of SZ92, the three ingested at a concentration of 10 ug / ml Dopamine activity was 2 times, 5 times and 2 times in the control group, respectively, see Figure 3, Figure 4, Figure 5. According to Fig. 4, different concentrations of SZ92 (0. 03~l(H3⁄4/ml)' have an agonistic effect on DAT, and exhibit a dose dependency in the concentration range of 0.03 g/ml~l. OO g/ml. The concentration of DAT uptake by the drug was increased to 150% of the negative control group (D8 cells without drug), that is, the concentration at which DAT activity increased by 50% was called EC ₅ o, to evaluate the biological activity of the DAT agonist. The EC50 activity of SZ92 reached 1.36.25ηΜ, as shown in Figure 6. 2. Study on the specificity of dopamine transporter agonist selection

To verify the selectivity specificity of the dopamine transporter agonist, luteolin (SZ92), acetylated SZ92, soy aglycone, quercetin, genistein, apigenin at 10 g/ml were determined in a similar manner as described above. At the concentration, the DAT uptake activity of D8 cells and the transaminase transport activity of G-1 fine sputum were compared with the HBS group. Furthermore, using the analysis software Origin 6. 0, the DMP values of the D8 cells and the G-1 cell blank group (ie, the HBS group) were denominators and normalized.

The results are shown in Table 1: Lutein (SZ92), acetylated SZ92, soybean aglycone, quercetin, genistein, apigenin at a concentration of 10 g/inl, D8 cells increased DAT uptake activity, while on G-1 The aminobutyric acid transporter of the cell has no agonistic effect. Among them, luteolin (SZ92) had the strongest DAT (0. 01) and more than 4 times that of the control group (HBS group).

Table 1

Compound 1 ^3⁄4 -D uptakes i ³ H- ABJ uptake

CHO DS CHO G-1

HBS 0.08 1.00 0.01 100 Celery ^ 3⁄4 0.02 1.80 0.03 0Ρ8 Surgery 3⁄4 cable 0.01 4.30 0.02 1.04

ZM SZ92 0.04 1.97 0.04 1 D0 Soybean 3⁄4ζ 0.Q2 2.S9 0.03 1.02 槲 0.03 1.45 0.04 1 J01 Dyeing 0.05 1.65 0.05 1.02

Moreover, the inventors also examined the effects of different concentrations of each candidate drug on the glutamate transporter of COS-7 cells. It was found that luteolin (SZ92), soybean aglycone, quercetin, genistein, and apigenin had no agonistic effects on glutamate transporters of COS-7 cells at different concentrations, as shown in Figure 7. 8.

Therefore, it can be seen from the present examples that among the various candidates, the flavonoids luteolin (SZ92), soybean aglycone, quercetin, genistein, and apigenin have obvious specific excitability to dopamine transfer protein. The role, especially SZ92, is the most stimulating. Each candidate drug had no agonistic effect on glutamate transport of COS-7 cells and Y-aminobutyrate transport of G-1 cells. Example 2. Isolation and Composition Preparation of Luteolin (SZ92)

1. Isolation and purification of active ingredients from traditional Chinese medicine decoction pieces

A. Preparation method of water extract Selected for the treatment of mental and neurological diseases, some Chinese herbal medicines Su Zi (SZ) 50-100g, decoction to boiling, 40 minutes later, the decoction is filtered, by freeze dryer (LyoPro3000, Denmark Jouari, company) Dry powder, sample retention for in vitro experiments. B. Separation, purification and activity tracking of SZ active ingredients from traditional Chinese medicine decoction pieces

Take Chinese Herbal Medicine SZ 4 kg, add 5L 95°/. Industrial alcohol is leached 4 times at room temperature for 3 days. The filtrate was combined and concentrated under reduced pressure. The obtained extract was 213 g, which was dissolved in 500 ml of water. It was extracted three times with petroleum ether three times each time, and the petroleum ether layer was separated, combined, concentrated under reduced pressure, and dried to give 115 g of petroleum ether. The remaining aqueous layer was extracted three times with chloroform, and each portion of chloroform was partitioned from chloroform, and then concentrated, concentrated under reduced pressure, and dried to give 9 g of chloroform layer. The remaining aqueous layer was extracted three times with ethyl acetate, 450 ml each time, and the ethyl acetate layer was separated, combined, concentrated under reduced pressure, and dried to give ethyl acetate. Combined with chloroform, the ethyl acetate part was obtained as a total of 8g of extract. Activity tracking showed that SZ3 had the strongest activity; SZ3 was divided into 9 parts by polyacrylamide gel column, and activity tracking showed that SZ9 had the strongest activity; continue to separate SZ-9 part: 10ml acetone dissolved sample after 3g 200- 300 mesh silica gel sample; 50g 200-300 mesh silica gel column; stripping agent: chloroform: methanol = 30: 1; developing agent: chloroform: methanol = 10: 1; combining the same two components, get SZ91 200mg; SZ92 1. lg; SZ91 is dissolved in chloroform and then added with a little methanol. After standing overnight, the precipitate is precipitated. The precipitate is washed several times with acetone. After dissolving, the sample plate is prepared. Developer: Chloroform: methanol = 10: 1, SZ91 pure product. Further storage of SZ92 parts: Take sample weight l. lg, 100ml acetone dissolved sample, 2. 5g 200-300 mesh silica gel sample, 30g 200-300 mesh silica gel column; eluent: chloroform: methanol = 50: 1, Developing agent: chloroform: methanol = 10: 1 ; Take SZ92, dissolve it with '20 ml of methanol, divide it into LH-20 gel several times, and obtain SZ92 pure product, developing solvent: chloroform: methanol = 10:1. 10 mg of each of SZ91 and SZ92 was weighed, dissolved in 5 ml of DMS0, and detected by hydrogen spectroscopy at 400 Hz. 'The separation of the above parts and the D8, G-Nl, S6 and other cell activity tracking (as in Example 1), finally determined that the pure products SZ91 (apigenin) and SZ92 (luteolin) are dopamine transporter agonists.

2. Preparation of luteolin injection

An aqueous solution was prepared by dissolving 5000 lignin in lOQOrag water and dissolved by heating. The mixture is mixed, and the injection liquid which is packed into a concentration of 10 mg / 2 ml / is filled in a vial and sealed to be sterilized.

3. Preparation of luteolin tablets

The luteolin tablet is prepared according to a method known to a person skilled in the art, wherein the tablet contains 5-10% luteolin (mass percentage) as needed, and the luteolin content can be increased or decreased. . Taking 100 g of luteolin, 560 g of microcrystalline cellulose, 380 g of anhydrous lactose, 200 g of magnesium stearate, 30 g of silica, tablets are prepared according to well-known tableting techniques and equipment, and stearic acid is removed from the above formula. Mix all ingredients except magnesium for 25-30 minutes, then sift the stearic acid 'magnesium and continue mixing. Then punched into pieces.

4. Acetylation Preparation of SZ92

Weigh S2502 250mg into a desiccator to dry, to ensure that the reaction is carried out in an anhydrous environment; dissolve the sample with 5ml of anhydrous pyridine, add 5ml of acetic anhydride and mix, stir at room temperature for 45 minutes, and then complete the reaction by thin layer chromatography. Adding 0.7 ml of physiological saline 100 ml and a little chloroform three times, 250 ml each time, the chloroform extract is dried over anhydrous sodium sulfate, and concentrated under reduced pressure; dissolved in 100 ml of ethanol, and the white insoluble matter is filtered off, and the solution is filtered off; The insoluble matter was dissolved by adding chloroform, a small amount of ethanol was added, and the crystals were precipitated overnight. , 5. Preparation of luteolin liposome '

1. Phosphatidylcholine (PC) 400mg, cholesterol (Choi) 40mg, vitamin E1⁄2g, luteolin 2mg, dissolved in '20ml absolute ethanol, sonicated all luteolin, formulated into 1mg/ml drug weak.

2. Preparation of luteolin liposomes by thin layer evaporation method. The ratio was the same as 1, without the addition of luteolin, to prepare 3⁄4 white liposome. 'Lignin's liposome encapsulation rate test: 10ug/ral of luteolin ethanol solution, 200-500nra UV-visible scan, luteolin has a strong absorption peak at 353nm, and the blank liposomes were quantified by this wavelength. And the drug-containing liposome 5ral, add NaC1015M solution ^ il, mix, ? After centrifugation at 00 brpm for 20 min, the supernatant was removed, and the precipitate was dissolved in absolute ethanol and reconstituted in a 100 ml volumetric flask. The absorbance of the drug-containing liposome was determined at 353 nm using a precipitate of the blank liposome as a reference. . 5 ml of blank liposome and drug-containing liposome were separately taken up in a 100 ml volumetric flask with absolute ethanol, and the absorbance value of the drug-containing liposome was measured at 353 nm = A hit/A. _S X 100%. In this experiment, the luteolin liposome encapsulation rate was 98 ° /. . · Example 4. Luteolin treatment of morphine addiction in rats.

I. Drug treatment in the modeling of addiction

Thirty Sprague-Dawley rats of clean grade were weighed 200 ± 10 g, half male and half female. Raised at room temperature 22- 24 V, humidity 50-70%, atmospheric environment, from 'Right Nights and Nights', given basic rat Chow feed, free water (sterilized pure water).

One week later, the large 'mouse was randomly divided into 3' groups: saline group, empty liposome group, and luteolin (SZ92) liposome-embedded group. Intraperitoneal injection of morphine hydrochloride, 6 times on the first day (8: 00, 9: 30 12: 00, 15: 00, 19: 00, 22: 00), the morphine dose was 2, 4, ' 6, 8, 8, 8 mg ' kg- ¹ , 2 times the next day (8:00, 11: 00), morphine dose 8mg · kg" ¹ , intraperitoneal injection (ip) naloxone hydrochloride (4mg · kg" ¹ after the last injection of morphine hydrochloride for 3h ), observe all withdrawal symptoms within 30rain and score. The empty liposomes and luteolin (SZ92) liposomes were intraperitoneally injected at a time of half an hour before morphine injection, at the same dose as each morphine injection. Rats with naloxone urged withdrawal to change the various withdrawal symptoms The score of the Yanagida Keji score (Table 2) was scored.

** Score every 15 minutes, record accumulated points within 1 hour

*Only one rating (one hour observation period)

(Highly irritating, abnormal posture such as writhing reaction, standing, stretching, licking hair, washing face, etc., individual rats are extremely violent, constantly striking iron and Chu, strong desire to come out, especially male rats react more strongly than female rats Results: After naloxone addiction, the morphine withdrawal symptoms of each group of rats were scored by Yantian Zhisi: the saline group (control 1) was 19.1, and the empty lipid group (group) was 15..25'. 5。 The liposome-embedded SZ92 group was 11.4. Compared with the control group 1, the morphine withdrawal syndrome of the liposome-encapsulated SZ92 group was significantly lower (0.01), with a reduction of 59% in the control group (Fig. 9).

The above results indicate that SZ92 can significantly reduce morphine withdrawal symptoms. Second, addiction modeling after drug treatment

Referring to the method of the first, after the morphine dose escalation method establishes the addiction model, before the naloxone addiction 3 (ki _n with different doses) The animals were treated with liposomes embedded in SZ92, and naloxone-induced morphine withdrawal symptoms in each group were scored according to the improved Yanagida score.

RESULTS: After naloxone addiction, liposomal-embedded SZ92 had a significant inhibitory effect on morphine addiction at doses of 0.53 mg/kg and 1.67 mg/kg (compared with the control group, 0.05 um and ^ ). 01), 1. The 67mg/Kg group withdrew Yanta Zhiji scored the lowest score (Figure 10). This suggests that SZ92 can also significantly reduce morphine withdrawal symptoms after morphine addiction. 'Examples · 5. Luteolin treatment of cocaine addiction in mice

(1) Preparation of the shuttle box: A 32cmX 16 _C mX 30cm rectangular box with an open top is made of colored plexiglass plates, and the middle is partitioned into two equal parts by a baffle, and a 10 cm×10 cm square channel is provided in the middle of the baffle plate. Animals pass freely. The sides of the box are painted white and black, and covered with clear glass. Place the shuttle box in a large isolation box with a 5W incandescent lamp and an image surveillance system on top.

(2) Preconditioning stage: experimental animals Bl/c57 mice, male, 20-25 g, 8 weeks old, 70 rats. The first half of the day. Shuttle in the box for 10 minutes, the second half for 20 minutes, the third half for 20 minutes, and check the preferred side stay time, screening 50 mice with similar preference scores for modeling. · , .

(3) Modeling stage: The mice were randomly divided into normal group, model group and SZ92 treatment group (dose were

1. 67mg/Kg, 3. 33mg/Kg 6. 67mg/Kg), 10 in each group. On the 1st, 2nd, 3rd, and 4th morning, the treatment group injected the drug SZ92, the normal group and the model group were injected with the same volume of solvent; 30 minutes later, the model group and the treatment group were injected intraperitoneally with cocaine (purchased from China Drug Testing Center) 20 ing /Kg, the normal group was injected with the same volume of normal saline, and the animals were placed on the non-preferred side (white); each group of animals was injected with normal saline for 30 minutes after the first, second, third, and fourth afternoons, and the animals were placed in preference. side. .

(4) + Detection: After 12 hours of modeling on the fourth day, the Pico2000 video system recorded the residence time of the animals in a white box for 20 minutes.

After 20 minutes of observation, the results showed that: the model group had a preference time of 654. 9 seconds in the white side box, and the normal control group had a preference time of 469 seconds in the white side box, which was significantly lower than the model group (0.001), cocaine dependence model. success. The preference time of liposome-embedded SZ92 treatment group (1.7 mg/Kg, 3. 3 mg/Kgs 6. 7 mg/Kg) was 535.1 seconds, 516. 9 3⁄4 616. 3 seconds, respectively, which were significantly lower than the model. Group (0. 05 and / 0. 001) (Figure 11).

The above results indicate that liposome-encapsulated SZ92 1⁄2 cocaine addiction has a significant inhibitory effect, and the dependence of cocaine on animals can be completely blocked at the dose of 3.3 mg/Kg'. 'Example 6 _: Effect of luteolin on morphine analgesic effect' Take Kunming mice, female, 20-25 g 30. They were randomly divided into 3 groups: saline group, empty liposome group and luteolin (SZ92) liposome-embedded group. Subcutaneous injection of luteolin (SZ92) liposome (5mg SZ92 / kg body weight / day) or the same volume ' 30 minutes after the solvent (liposome), morphine (5 mg / kg body weight / day) was injected subcutaneously. The control group was injected subcutaneously with the same volume of liposomes and physiological salts. Twenty minutes after morphine injection, the animals were placed on a 55-inch hot plate. The time range of the hind paws was 10-30 s, and the test was repeated at intervals of 15 min. The average of the two test results was the final result. The standard of the test is the time of the animal's hind paw (latency of pain).

RESULTS: The pain sensation period (the hindlimb time) of each group was: saline group 13. 381 seconds, empty liposome group

47. 244 seconds, luteolin liposomal embedding group (SZ92) 46. 181 seconds. The hindlimb time of the empty liposome group was significantly greater than that of the saline group (0.01). There was no significant difference in pain latency between the empty liposome group and the SZ92 group (P>0.05), indicating that morphine had significant analgesic effect (Fig. 12). ). '

The above results indicate that SZ92 does not affect the analgesic effect of morphine while preventing animals from becoming addicted to morphine. Example 7. Luteolin treatment of mouse schizophrenia model 'Experimental animals and groupings: References (An animal model of schizophrenia established by Wu Jinhua et al. using different experimental mouse strains. Acta Physiologica Sinica, 2003, 55: 381-387 ). Inbred C57BL/6 small, rat, male, body weight (20 ± 2 g). It is divided into normal group, model group and treatment group.

The treatment group (SZ) was intraperitoneally injected with liposomes embedded in SZ92. The normal group and the model group were replaced with the same volume of liposomes, and the mice were placed in a 48 0mX 24 cmX 20 cm rectangular experiment box with an open top. The box is covered with glass and pre-adapted for 30 minutes. '

After the pre-conditioning, the model group and the different concentration treatment groups were respectively treated with 0.6 mg/kg dizocilpine maleate (referred to as MK801,

Sigma) was injected intraperitoneally into the experimental mice, and the normal group was replaced with the same volume of physiological saline instead of MK801.

Detection: Animal activity was recorded after 10 minutes. The bottom of the experiment box is virtually divided into 8 areas of the same size. The activity of the mice is recorded by the Pico2000 video system. The technicians record the number of times the mice enter each area per unit time (15 min), and record them separately. The total number of grids traversed by the movement behavior during the entire detection period (2h). The result of the total number of cells in the model group is about 3,600, and the total number of cells in the liposome-embedded SZ92 is lower than that in the model group, at 0. 17 mg / Kg to 16. 7rag / Kg In the dose range, the total number of grids crossed by animals within 2 hours was decreased in a concentration-dependent manner. 16. The total number of grids in the 7rag'/Kg group was the lowest, about 50% of the model group.

13).

The above results indicate that the liposome package: ^ SZ92 has anti-schizophrenia effect. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the present invention.

Claims

Rights request

Use of a flavonoid or a derivative thereof for the preparation of a pharmaceutical composition as a dopamine transporter agonist, or for the preparation of a medicament for treating a psychotic or neurological disease.

The use according to claim 1, wherein the flavonoid is selected from the group consisting of: apigenin, luteolin, quercetin, myricetin, poplarin, soybean aglycone, and wogonin , baicalein, genistein, morin, marigold, hesperetin, buckthorn gold, rhamnosin, or a combination thereof.

The use according to claim 2, wherein the flavonoid compound is selected from the group consisting of: luteolin, apigenin, quercetin, soybean aglycone, or a combination thereof.

The use according to claim 1, wherein the mental disease or neurological disease is a dopaminergic hyperfunction.

The use according to claim 4, wherein the mental or neurological disease is selected from the group consisting of: an addiction disorder, an anxiety disorder, Alzheimer's syndrome, anorexia nervosa, schizophrenia, Parkinson's syndrome, insomnia, substance abuse and dependence, vomiting, irritable bowel syndrome, menopausal syndrome, Wilson's disease, chorea, demyelinating disease, mania, obsessive-compulsive disorder, or Tourette's synthesis Sign.

6. A method of determining a drug candidate useful for treating a psychiatric disease or a neurological disease, the method comprising the steps of:

(1) In the test group, the amount of dopamine transporter to dopamine was measured in an in vitro system in the presence of a candidate substance; in the control group, in the absence of a candidate substance, in an in vitro system Determination of the amount of dopamine transporter to dopamine transport;

(2) The amount of dopamine transported in the assay group and the dopamine transporter in the control group. If the dopamine transporter in the test group is significantly higher than the dopamine transporter in the control group, the candidate substance is an agonist of the dopamine transporter. As a drug candidate for the treatment of mental or neurological diseases.

7. The method according to claim 6, wherein the method further comprises the following steps: '·

A dopamine transporter agonist, wherein the dopamine transporter agonist is capable of specifically promoting dopamine transporter uptake of dopamine, and the dopamine transporter agonist is used for the preparation of a therapeutic psychiatric or neurological disorder Drug.

9. Use of a dopamine transporter agonist, characterized in that it is used for the preparation of a pharmaceutical composition for treating a psychiatric or neurological disease.

A method for treating a psychiatric disease or a neurological disease, characterized in that the method comprises: administering an effective amount of a flavonoid compound or a derivative thereof to a subject in need of treatment.