KR102644046B1 - Biomarker composition for drug addiction diagnosis, screening method of therapeutic agents for drug addiction, and pharmaceutical composition for preventing and treating drug addiction - Google Patents

Abstract

The present invention relates to a composition that can diagnose, prevent, or treat drug addiction based on SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein). Drug addiction can be diagnosed through the SYNCRIP protein or the gene encoding it, and the SYNCRIP By measuring expression, substances related to drug addiction treatment can be discovered, and substances that promote the expression or activity of SYNCRIP can be used to prevent or treat drug addiction.

Description

Biomarker composition for drug addiction diagnosis, screening method of therapeutic agents for drug addiction, and pharmaceutical composition for preventing and treating drug addiction }

The present invention relates to a biomarker composition for diagnosing drug addiction, a screening method for drug addiction treatment using the same, and a pharmaceutical composition for preventing or treating drug addiction.

Drug addiction is a chronic brain disease and mental illness in which the structure and function of the brain are changed, causing a person to become obsessed with drugs, compulsively crave them, and continue to use them despite knowing the negative and harmful consequences of drugs. Most drug addicts initially use drugs voluntarily out of curiosity or a sense of adventure, but as time goes by, they develop dependence on the drug, and eventually develop tolerance to the drug, which requires increasing the amount each time it is used. If you do not use it, you will fall into drug addiction, which causes withdrawal symptoms that cause unbearable mental and physical abnormalities.

This addiction, similar to other diseases such as heart disease, interferes with the normal and healthy function of tissues (organs) and causes very dangerous results. It becomes an addiction in which one cannot live a normal life without using addictive drugs, for example. For example, in addition to showing a tendency to become immersed in a specific area, symptoms such as paranoid schizophrenia, heart failure, increased blood pressure, vomiting, difficulty breathing, loss of sensory abilities, loss of consciousness, nightmares, and confusion, or after drug administration, may occur. -If left unattended for 12 hours, withdrawal symptoms such as anxiety, insomnia, hypersomnia, irritability, etc. may occur, and in severe cases, death may occur.

As the number of drug users is gradually increasing in Korea, there is a need for effective treatment methods for habitual drug users as well as prevention of drug addiction.

Because drug addiction lasts a lifetime if left untreated, various efforts to treat it have been carried out at a national level. Nevertheless, the clear mechanism of drug addiction has not been revealed, so treatments and treatments have not been developed.

Most drug addiction treatments developed to date are dopaminergic nervous system agonists or antagonists. The dopaminergic nervous system agonist can relieve narcolepsy or discomfort, but since it only acts on one conditional stimulus, there is a high possibility of recurrence and there is a risk of addiction to the treatment itself. Dopamine receptor antagonists have low responses to conditioned stimuli and low reward properties of the drug itself, but there is a risk of side effects related to the dopaminergic nervous system, such as dulled emotions, dysphoria, and tardive dyskinesia such as Parkinson's disease.

In order to solve the above-mentioned problems, various attempts have been made to develop substances that can treat drug addiction from natural products. For example, bergenin derivatives exhibit excellent inhibitory activity against drug addiction, have excellent safety, and do not show fatal side effects (Patent Document 1). The effect of such addiction treatment is extremely weak, so there is a limitation that it cannot be used in actual drug addiction treatment.

Although attempts have been made to develop various substances that can diagnose or treat drug addiction, there are still problems such as limitations and side effects, so there is an urgent need to develop safe and effective treatments related to drug addiction.

Patent Document 1. Korean Patent Publication No. 10-2012-0004965

The present invention was created to solve the above problems, and the purpose of the present invention is to provide a biomarker composition for diagnosing drug addiction and a kit for diagnosing drug addiction.

Another object of the present invention is to provide a method of providing information necessary for diagnosing drug addiction.

Another object of the present invention is to provide a method and a screening composition for screening candidate substances that can prevent or treat drug addiction.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating drug addiction.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing drug addiction containing SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) protein or the gene encoding it as an active ingredient.

In order to achieve the above other objects, the present invention provides a composition for diagnosing drug addiction, which includes as an active ingredient an agent capable of measuring the expression level of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein).

The agent for measuring the expression level of SYNCRIP includes a probe or primer that specifically binds to the SYNCRIP gene; And antibodies, peptides, aptamers or compounds that specifically bind to the SYNCRIP protein; It may be one or more selected from among.

The SYNCRIP gene may be composed of the base sequence represented by SEQ ID NO: 1.

In order to achieve the above other object, the present invention provides a kit for diagnosing drug addiction comprising the composition.

In order to achieve the above other object, the present invention provides a method of providing information necessary for diagnosing drug addiction, including the following steps.

(1) separating a biological sample from a subject and measuring the mRNA expression level of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) protein or SYNCRIP gene from the biological sample;

(2) comparing the expression level of the SYNCRIP protein or the mRNA expression level of the SYNCRIP gene with a normal control sample; and

(3) Step of determining drug addiction when the expression level of the SYNCRIP protein or the mRNA expression level of the SYNCRIP gene is lower than that of the normal control sample.

It may further include measuring the expression level of miR-137 from a biological sample of the subject.

In order to achieve the above object, the present invention includes the steps of: a) treating an animal with induced drug addiction or a sample isolated therefrom with a candidate substance; b) measuring the expression level of the SYNCRIP gene in the candidate treatment group; and c) selecting a drug addiction treatment material if the expression level of the SYNCRIP gene measured in the above step increases compared to the candidate substance untreated group (control group).

The SYNCRIP gene may be a base sequence represented by SEQ ID NO: 1.

The expression level can be measured using polymerase reaction (PCR), quantitative polymerase reaction (qPCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (competitive RT-PCR), and quantitative real-time polymerase reaction (qRT). It may be measured using any one method selected from the group consisting of - PCR), real time reverse transcription polymerase reaction (real time RT-PCR), RNase protection assay, northern blot analysis, and DNA chip analysis. there is.

It may further include measuring the expression level of miR-137 in the serum of the candidate treatment group.

When the expression level of the miR-137 increases compared to the candidate substance untreated group (control group), a step of determining the candidate substance as a drug addiction treatment substance may be further included.

In order to achieve the above other objects, the present invention provides a pharmaceutical composition for preventing or treating drug addiction containing a substance that promotes the expression or activity of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) as an active ingredient.

The substance that promotes the expression or activity of SYNCRIP may be a vector containing the SYNCRIP gene.

The vector may be any one selected from the group consisting of linear DNA plasmid DNA, recombinant non-viral vector, recombinant viral vector, and inducible gene expression vector system.

According to the present invention, the present invention relates to a biomarker composition for diagnosing drug addiction or detecting drug use, which contains the SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) protein or the gene encoding the same as an active ingredient, in the brain striatum of an animal model of drug addiction. We identified a decrease in the expression of SYNCRIP, and verified that when SYNCRIP expression was increased in the brain striatum of a drug addiction animal model, serum miR-137 expression, a drug addiction biomarker, was restored to the normal level, allowing for the diagnosis of drug addiction. It can be used as a biomarker for

In addition, it was experimentally confirmed that when the expression level of SYNCRIP increases, the decrease in serum miR-137 expression in drug addiction animal models is suppressed, and by measuring the expression of SYNCRIP, substances related to drug addiction treatment can be discovered. The above substances can be usefully used in related development fields such as medicine and food.

Figure 1 shows the results of comparing the expression level of miR137 in serum and striatum (brain) in control and drug addiction animal model (MA).
Figure 2 shows GL in HEK293TN cells (miR137-OE) transfected with SYNCRIP-3'UTR and a miR-137 overexpression vector and HEK293TN cells (control) transfected with SYNCRIP-3'UTR and a scrambled control vector. /This is a graph quantifying AP activity.
Figure 3 shows the results of Western blot analysis of the expression level of SYNCRIP in the brain striatum of a control group and the brain striatum of a drug addiction animal model.
Figure 4 is a graph comparing the expression level of SYNCRIP in the brain of a control and drug addiction animal model.
Figure 5 is a cleavage map of the SYNCRIP overexpression vector (Syncrip (NM_019666) Mouse Tagged ORF Clone vector (MR217725L4, OriGene Technologies, MD, USA)).
Figure 6 shows the results of comparing the expression level of miR-137 secreted in a SYNCRIP-overexpressing neuronal cell line (SYNCRIP-OE) and the control group.
Figure 7 shows the preparation of a transgenic animal model (DST ^SYNCRIP-OE -MA), a normal group (control), and a drug addiction animal model (MA) with increased SYNCRIP expression in the striatum of a mild drug addiction animal model, from which the drug This is the result of measuring the expression level of miR-137, an addiction biomarker, using qPCR.
Figure 8 shows the preparation of a transgenic animal model (Brain ^SYNCRIP-OE -MA), a normal group (control), and a drug addiction animal model (MA) with increased SYNCRIP expression in the striatum of a drug addiction animal model, from which drug addiction living organisms were prepared. This is the result of measuring the expression level of the indicator, miR-137, by qPCR.
Figure 9 schematically shows the experimental schedule design for evaluating egocentric spatial learning behavior using an underwater cross maze.
Figure 10 shows the structure of an underwater cross maze during the spatial learning period to evaluate behavior due to drug addiction.
Figure 11 shows the structure of an underwater cross maze to evaluate behavior due to drug addiction and to evaluate spatial learning after learning is completed.
Figure 12 is a graph showing the results of an underwater cross maze experiment for a drug addiction animal model (MA), a control group, and an animal model in which SYNCRIP overexpression was induced after drug addiction (MA-SYNCRIP; experimental group).
Figure 13 is a graph showing the degree of change in activity when methamphetamine is administered to a drug addiction animal model (MA) and an animal model in which SYNCRIP overexpression is induced after drug addiction (MA-SYNCRIP; experimental group).

Below, we will look at various aspects and various implementations of the present invention in more detail.

As a result of diligent efforts to obtain a method for finding a substance capable of diagnosing drug addiction, the present inventors identified SYNCRIP that interacts with miR-137, a drug addiction biomarker, and completed the present invention based on this.

One aspect of the present invention relates to a biomarker composition for diagnosing drug addiction containing SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) protein or the gene encoding it as an active ingredient.

The biomarker composition may additionally include previously known drug addiction biomarkers, and previously known drug addiction biomarkers include, but are not limited to, miR-137.

In the present invention, 'SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein)' is a gene encoding a member of the cellular heterogeneous nuclear ribonucleoprotein (hnRNP) group. hnRNPs are RNA-binding proteins that form complexes with heterogeneous nuclear RNA (hnRNA) and regulate alternative splicing, polyadenylation, and other aspects of mRNA metabolism and transport. The encoded protein plays diverse roles in mRNA growth and is associated with multiprotein complexes, including the apoB RNA editing-complex and motor neuron (SMN) survival. To date, a number of variants related to the SYNCRIP gene have been observed, and its pseudogene is known to be located in the short sequence of chromosome 20.

In addition, the base sequence of the SYNCRIP gene can be indicated as SEQ ID NO: 1, which is registered in the National Center for Biotechnology Information (NCBI) as Gene ID: 10492. At this time, it may include a base sequence having sequence homology of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% with the base sequence represented by SEQ ID NO: 1. .

In the present invention, 'drug addiction' is a type of drug-induced disorder, which comprehensively refers to symptoms caused by administering or consuming a drug to which one is highly dependent. It is a type of drug dependence, and is characterized by physical, It can be defined as a state of long-term exposure to drugs, with a lack of self-control ability that causes one to continuously seek out or take drugs despite being in an emotional state such as mental damage.

Drug addiction is a disease that causes psychological and physical problems as well as interpersonal problems due to the use of illegal drugs such as heroin or legal drugs such as tranquilizers without medical supervision. In the present invention, drug addiction, drug abuse, drug dependence, and It may comprehensively include drug withdrawal.

In the present invention, the drug is not particularly limited as long as it is classified as a narcotic drug in the art, but is preferably any one or more selected from the group consisting of methamphetamine, amphetamine, cocaine, ecstasy, and methylphenitate, and is preferably Could be methamphetamine.

The above-mentioned ‘substance abuse’ may include cases where one or more of the four conditions below persist for more than one year and cause clinically significant disability or pain.

① Unable to perform important duties at work, school, or at home due to drug use (for example, frequent absences due to drug use, poor grades, disciplinary action such as suspension or expulsion, discord within the family, etc.). ② Use drugs even though you know it is physically dangerous (for example, driving after using drugs, riding a bicycle or motorcycle, operating machinery, etc.). ③ Cause legal problems due to drug use (such as being arrested for actions related to drug use). ④ Continuing to use drugs despite persistent or recurrent social or interpersonal problems that are caused or worsened by drug use (e.g., family discord or fighting).

In the present invention, 'drug dependence' comprehensively refers to addiction that occurs by administering or consuming a drug to which one is highly dependent, and is also referred to as drug addiction. More specifically, drug dependence can be defined as a state of long-term exposure to drugs, with a lack of self-control ability to continuously seek out or take drugs despite emotional states such as physical and mental damage. Specifically, drug dependence may include cases where three or more of the following seven conditions persist simultaneously for more than one year, resulting in clinically significant disability or suffering.

① The amount of drug used gradually increases, or the effect gradually decreases even when the same amount is used. ② If you stop using the drug or reduce the amount of use, withdrawal symptoms such as hand tremors, excessive sweating, rapid pulse, insomnia, nausea, vomiting, seeing or hearing empty things, anxiety, nervousness, and epilepsy may appear. To get rid of it, you need to use the same amount of medication as before. ③ Once you start using drugs, you end up using larger amounts and for a longer period of time than you thought. ④ I have tried to stop using drugs on my own but failed. ⑤ Because of drug use, people give up or become unable to engage in many important interpersonal relationships or occupational activities. ⑥ People continue to use drugs even after knowing that they have psychological or physical diseases caused by drug use, such as depression or liver disease.

In the present invention, 'substance withdrawal' refers to the repeated administration of a drug to which one is highly dependent, and the craving to take the drug during the withdrawal period during which the drug is not taken while the drug is highly dependent. ) is derived, and comprehensively refers to the symptoms of various mental disorders that occur including this.

In the present invention, 'diagnosis' refers to determining the susceptibility of an object to a specific disease or condition, determining whether an object currently has a specific disease or condition, and an object suffering from a specific disease or condition. Determining the prognosis or therametrics (e.g., monitoring the condition of a subject to provide information about the efficacy of treatment).

The relationship between SYNCRIP of the present invention and drug addiction has not yet been known, but through the present invention, it was confirmed that the expression of the SYNCRIP gene was significantly reduced in an animal model of drug addiction compared to the control group.

Furthermore, when SYNCRIP was overexpressed in a drug addiction animal model, the expression of the drug addiction-related biomarker, miR-137 gene, in serum was restored to normal levels. This means that SYNCRIP and drug addiction are interrelated, indicating that SYNCRIP can be used as a biomarker for diagnosing drug addiction according to the present invention. Drug addiction can be detected by measuring the expression level of the SYNCRIP protein of the present invention or the gene encoding it.

In the present invention, “expression” means the production of a protein or nucleic acid in a cell. 'Protein' is used interchangeably with 'polypeptide' or 'peptide' and refers to a polymer of amino acid residues, for example, as commonly found in proteins in their natural state. ‘Polynucleotide’ or ‘nucleic acid’ refers to deoxyribonucleotide (DNA) or ribonucleotide (RNA) in single- or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included. 'mRNA' refers to RNA that transfers genetic information (gene-specific base sequence) from a specific gene to a ribosome that specifies the amino acid sequence during the protein synthesis process.

Another aspect of the present invention relates to a composition for diagnosing drug addiction containing as an active ingredient an agent capable of measuring the expression level of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein).

The agent for measuring the expression level of SYNCRIP includes a probe or primer that specifically binds to the SYNCRIP gene; and an antibody, peptide, aptamer, or compound that specifically binds to the SYNCRIP protein.

In the present invention, a 'primer' is a nucleic acid sequence with a short free 3' hydroxyl group that can form base pairs with a complementary template and acts as a starting point for copying the template strand. It refers to the sequence. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.

In the present invention, 'probe' refers to a nucleic acid fragment such as RNA or DNA, ranging from a few bases to several hundred bases long, that can specifically bind to mRNA, and is labeled to determine the presence or absence of a specific mRNA and the expression level. can confirm. Probes may be manufactured in the form of oligonucleotide probes, single strand DNA probes, double strand DNA probes, RNA probes, etc. Selection of appropriate probes and hybridization conditions can be appropriately selected according to techniques known in the art.

In the present invention, the primer or probe can be chemically synthesized using phosphoramidite solid support synthesis or other well-known methods. Additionally, primers or probes can be modified in various ways according to methods known in the art to the extent that they do not interfere with hybridization with SYNCRIP mRNA. Examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologs, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) ) or charged linkers (e.g. phosphorothioate, phosphorodithioate, etc.), and binding of labeling material using fluorescence or enzymes.

Meanwhile, the agent for measuring the expression level of SYNCRIP may be an antibody, peptide, aptamer, or compound that specifically binds to the SYNCRIP protein.

In the present invention, 'antibody' is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to SYNCRIP of the present invention, and the antibody can be prepared according to a conventional method in the art. The types of antibodies include polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies are included. The antibody is meant to be intact, having two full-length light chains and two full-length heavy chains. Additionally, the above antibodies also include special antibodies such as humanized antibodies.

In the present invention, 'aptamer' refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure as a substance that can specifically bind to the analyte to be detected in the sample. Specifically, the presence of the target protein in the sample can be confirmed. The production of an aptamer involves determining and synthesizing the sequence of an oligonucleotide with selective and high binding affinity for the target protein to be identified (in the present invention, SYNCRIP protein) according to a general aptamer production method, and then 5' of the oligonucleotide. This may be achieved by modifying the terminal or 3' end with -SH, -COOH, -OH or NH ₂ so that it can be bound to the functional group of the aptamer chip, but is not limited thereto.

The diagnostic composition of the present invention containing the SYNCRIP protein-specific antibody may additionally contain an agent necessary for a method for detecting a known protein, and the method for detecting a known protein using the composition can be used without limitation. The expression level of SYNCRIP protein can be measured in the sample.

Since the nucleic acid sequences of the genes of the present invention are registered in gene banks, those skilled in the art can design antisense oligonucleotides, primer pairs, or probes that specifically amplify specific regions of these genes based on the sequences.

Antisense oligonucleotides, primers or probes of the present invention can be chemically synthesized using the phosphor amidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologs, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoro). amidate, carbamate, etc.) or charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).

In addition, another aspect of the present invention relates to a kit for diagnosing drug addiction, which includes an agent capable of measuring the expression level of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein).

The diagnostic kit of the present invention may include not only antibodies that recognize SYNCRIP protein as a marker or primers and probes that recognize SYNCRIP mRNA as a marker, but also one or more other component compositions, solutions, or devices suitable for the analysis method.

In a specific embodiment, the diagnostic kit may be a diagnostic kit characterized by including essential elements required to perform a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes each primer pair specific for the marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and is about 7bp to 50bp in length, more preferably about 10bp to 30bp in length. It may also include primers specific to the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, and the RNAse inhibitor DEPC. -Can include DEPC-water, sterilized water, etc.

In another aspect, it may be a diagnostic kit that includes essential elements required to perform a DNA chip. A DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, agents, enzymes, etc. for producing a fluorescent probe. The substrate may also include cDNA or oligonucleotides corresponding to control genes or fragments thereof.

Samples used for analysis include blood, blood cells, brain tissue, nerve cells, cerebrospinal fluid, saliva, nasal fluid, sputum, joint sac fluid, amniotic fluid, ascites, cervical or vaginal secretions, and urine, which are intoxicated and can be distinguished from normal conditions. Includes biological samples from which specific proteins related to can be identified. Preferably, it can be measured from a biological sample, for example, any sample selected from the group consisting of blood, blood cells, brain tissue, nerve cells, and cerebrospinal fluid. The sample can be prepared to increase the detection sensitivity of the protein marker, for example, a serum sample obtained from a patient can be subjected to anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, It can be pretreated using methods such as sequential extraction or gel electrophoresis.

Another aspect of the present invention relates to a method of providing information necessary for diagnosing drug addiction, including the following steps.

(1) separating a biological sample from a subject and measuring the mRNA expression level of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) protein or SYNCRIP gene from the biological sample;

(2) comparing the expression level of the SYNCRIP protein or the mRNA expression level of the SYNCRIP gene with a control sample; and

(3) determining that the sample is in a state of drug addiction when the expression level of the SYNCRIP protein or the mRNA expression level of the SYNCRIP gene is lower than that of the control sample.

First, (1) a biological sample is separated from the subject, and the mRNA expression level of the SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) protein or SYNCRIP gene is measured from the biological sample.

In the present invention, 'subject' refers to any single object that is suspected of being drug addicted and for which a drug addiction diagnosis is required, including humans, cows, dogs, guinea pigs, rabbits, chickens, insects, mice, mice, etc. Additionally, any subject participating in a clinical research trial or a subject participating in an epidemiological study without any clinical signs of drug addiction disease is used as a control group. In one embodiment of the present invention, mice or mice were targeted.

In the present invention, a 'subject suspected of drug addiction' may be a subject who is expected to misuse addictive drugs, be addicted to drugs, or have withdrawal symptoms due to long-term drug abuse.

The 'biological sample' can be used without limitation as long as it is collected from a subject for whom drug addiction is to be diagnosed, for example, blood, blood cells, brain tissue, nerve cells, cerebrospinal fluid, saliva, nasal fluid, sputum, joint sac fluid, and amniotic fluid. , ascites, cervical or vaginal secretions, and urine, but is not particularly limited thereto, and is preferably any one selected from the group consisting of blood, blood cells, brain tissue, nerve cells, and cerebrospinal fluid. It may be one sample.

In the present invention, 'measurement' may preferably mean 'analysis', which means 'qualitative analysis' which means measuring and confirming the presence or absence of the target substance or the presence level (expression level) of the target substance. Alternatively, it may mean 'quantitative analysis' that measures and confirms changes in quantity. In the present invention, measurement can be performed including both qualitative and quantitative methods, and preferably, quantitative measurement can be performed.

The sample may be pretreated before use for detection or diagnosis. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc.

The measurement method is not particularly limited as long as the protein expression level is measured by a protein expression measurement method known in the art, for example, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, Ouchterlony immunodiffusion method. It may be any one or more selected from the group consisting of immunodiffusion, rocket immunoelectrophoresis, immunostaining, immunoprecipitation analysis, complement fixation analysis, FACS, and protein chip.

The gene expression level measurement is not particularly limited as long as it is a gene expression measurement method known in the art, but preferably polymerase reaction (PCR), quantitative polymerase reaction (qPCR), reverse transcription polymerase reaction (RT-PCR), competitive method. Competitive RT-PCR, quantitative real-time polymerase reaction (qRT-PCR), real time RT-PCR, RNase protection assay, northern blot analysis, and DNA chip. It may be any one method selected from the group consisting of analysis, and more preferably quantitative polymerase reaction (qPCR) or northern blot analysis.

Next, the expression level of SYNCRIP protein or mRNA expression of the SYNCRIP gene in the subject sample measured by the method in step (1) described above is compared with the SYNCRIP expression level of the normal control sample measured by the same method ((2) step). At this time, the sample is preferably of the same type or obtained by the same method between the subject and the normal control group.

Finally, if the expression level of the SYNCRIP protein or the mRNA expression level of the SYNCRIP gene is lower than that of the normal control sample, it is determined that the sample is in a state of drug addiction (step (3)). Specifically, if the SYNCRIP expression level of the subject sample measured by the above method is decreased compared to the sample of a healthy normal control group, it can be determined that the subject is in a state of drug addiction.

That is, the expression level of the biomarker (SYMCRIP) of the present invention is measured in a biological sample obtained from a subject suspected of drug addiction, and the biological sample of the present invention is measured from a biological sample of the same type or a normal control group obtained by the same method. After measuring the expression level of the marker (SYMCRIP), the two are compared. If the expression level of the subject is lower than that of the normal control group, the subject can be judged to be drug addicted.

Another aspect of the present invention relates to a screening method for a drug addiction treatment material comprising the following steps.

(a) treating a candidate substance in an animal in which drug addiction has been induced or a sample isolated therefrom;

(b) measuring the expression level of the SYNCRIP gene in the candidate treatment group; and

(c) If the expression level of the SYNCRIP gene measured in the above step increases compared to the candidate substance untreated group, selecting a drug addiction treatment substance.

In the present invention, 'animals with induced drug addiction' are animals whose drug addiction is induced due to long-term administration of drugs, and may be mammals other than humans, preferably mice, guinea pigs, pigs, birds, etc.

The ‘isolated sample’ may be tissue or cells derived from an animal that has been induced to be drug addicted. The sample includes tissue or cells capable of detecting the expression level of the SYNCRIP gene in an animal in which drug addiction is induced, and may preferably be a tissue or cell isolated from the brain of an animal in which drug addiction is induced. It is not limited.

In the present invention, the 'candidate material' refers to a substance to be tested for drug addiction treatment activity, and may include any molecule such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and a wide range of compounds. . Additionally, the 'candidate material' may be a cell transfected with a liposome or vector. The transfection may be performed using microinjection, calcium phosphate co-precipitation, electroporation, or liposome use, but is not limited thereto.

In the present invention, the 'untreated group' is also called the control group, which is an animal in which drug addiction was induced without treatment with the candidate material or a sample separated therefrom, and is a drug addiction that is in a parallel relationship with the group treated with the candidate material. This refers to the animal from which it was derived or a sample isolated from it.

In the present invention, the 'screening method' is a method of comparing the candidate substance with an untreated group (control group) by confirming the expression of SYNCRIP, which can upregulate miR-137, a biomarker of drug addiction, present in serum. It may be done by.

In the present invention, for drug addiction experiments, methamphetamine among addictive drugs was used to induce drug addiction in animals. Therefore, the drug addiction may be specifically caused by addictive drugs. The addictive drug is not particularly limited as long as it is classified as a narcotic or addictive drug in the art, but is preferably any one or more selected from the group consisting of methamphetamine, amphetamine, cocaine, ecstasy, and methylphenitate.

In an example of the present invention, it was confirmed that the expression of miR-137 was significantly higher in brain tissue in the drug addiction animal model compared to the normal animal model. On the other hand, it was confirmed that the expression of miR-137 was significantly lower in the serum of the drug addiction animal model compared to the normal animal model. This again confirms previous literature that the miR-137 gene acts as a biomarker for drug addiction.

As a result of the analysis, it was confirmed that overexpression of SYNCRIP normalized the expression level of miR-137 in the serum of a drug addiction model induced by methamphetamine administration compared to the control group. In other words, it was confirmed that the symptoms of drug addiction caused by drug administration and the decrease in serum miR-137 caused by this were significantly suppressed in the SYNCRIP overexpression animal model. SYNCRIP of the present invention prevents drug addiction caused by long-term drug administration. , it can be seen that it can be usefully applied as a target for preventing or treating withdrawal symptoms.

In addition, it was confirmed that SYNCRIP can induce the behavior (egocentric spatial learning evaluation) of an animal model addicted to a drug and showing withdrawal symptoms to a normal level, and the drug was administered again to an animal model that had a withdrawal period after being addicted to the drug. It was confirmed that it has the effect of suppressing the increase in activity that occurs when

In the present invention, 'prevention' refers to all actions that suppress or delay the onset and symptoms of drug addiction. In the present invention, 'treatment' refers to any action that improves or beneficially changes the symptoms of drug addiction or withdrawal symptoms due to drug addiction.

The present invention measures the expression level of the SYNCRIP gene in the group treated with the candidate substance b), and when the expression level of the SYNCRIP gene measured in step b) increases compared to the group not treated with the candidate substance (control group), it is used as a drug addiction treatment substance. You can select.

Specifically, the present invention measures the SYNCRIP gene expression level in animals with induced drug addiction or samples isolated from them in the absence of a candidate substance that can prevent or treat drug addiction (candidate substance untreated group), and the candidate substance is not treated. After measuring the SYNCRIP gene expression level in the presence of a substance and comparing the two, a substance that increases the SYNCRIP gene expression level in the presence of the candidate substance compared to the level in the absence of the candidate substance is predicted to be a substance for preventing or treating drug addiction. Or you can judge.

In the present invention, 'gene expression level measurement' is a process of confirming the expression level of the SYNCRIP gene in animals or samples isolated from drug addiction induced drug addiction in the absence and presence of candidate substances, and measures the amount of mRNA.

Methods for measuring gene expression levels include polymerase reaction (PCR), quantitative polymerase reaction (qPCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (competitive RT-PCR), and quantitative real-time polymerase reaction ( It may be any one method selected from the group consisting of qRT-PCR), real time reverse transcription polymerase reaction (real time RT-PCR), RNase protection assay, northern blot analysis, and DNA chip analysis, preferably It may be quantitative polymerase reaction (qPCR) or northern blot analysis.

The screening method for a drug addiction treatment substance of the present invention may further include measuring the expression level of miR-137 in the serum of the candidate treatment group. When the expression level of the miR-137 increases compared to the candidate substance untreated group (control group), a step of determining the candidate substance as a drug addiction treatment substance may be further included.

In the present invention, 'miR-137' is a substance that can diagnose drug addiction or whether withdrawal symptoms have occurred due to drug addiction. For the diagnosis of drug addiction or withdrawal symptoms, miR-137 is widely known as a biomarker. Specifically, when drug addiction or withdrawal symptoms occur, the expression level of miR-137 is relatively increased in brain tissue, and the expression level of miR-137 is relatively decreased in serum.

The relationship between the SYNCRIP gene of the present invention, drug addiction, and SYNCRIP and miR-137 was not yet known, but through the present invention, it was found that the expression of the SYNCRIP gene decreases during drug addiction. In addition, when SYNCRIP was overexpressed in animals, it was confirmed that the expression of the miR-137 gene, a drug addiction-related biomarker, significantly increased in serum. This means that the SYNCRIP gene can function as a screening marker for drug addiction treatment substances.

Candidate substances selected through the drug addiction prevention or treatment substance screening method can promote or increase the expression level of the SYNCRIP gene when compared to the candidate substance untreated group (control group) that did not administer the candidate substance.

Another aspect of the present invention relates to a composition or kit for screening drug addiction treatment substances that contains as an active ingredient an agent capable of measuring the expression level of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein).

The relationship between the SYNCRIP gene of the present invention, drug addiction, and SYNCRIP and miR-137 was not yet known, but through the present invention, it was found that the expression of the SYNCRIP gene decreases during drug addiction. In addition, when SYNCRIP was overexpressed in animals, it was confirmed that the expression of the miR-137 gene, a drug addiction-related biomarker, significantly increased in serum. This means that the SYNCRIP gene can function as a screening marker for drug addiction treatment substances. In other words, if the expression level of the SYNCRIP gene can be measured, substances for drug addiction prevention or treatment can be screened.

The agent capable of measuring the expression level may be any one selected from the group consisting of primers, probes, and antibodies that specifically bind to the SYNCRIP gene.

In the present invention, a 'primer' is a nucleic acid sequence with a short free 3' hydroxyl group that can form base pairs with a complementary template and acts as a starting point for copying the template strand. It refers to the sequence. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.

In the present invention, 'probe' refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to several hundred bases long, that can specifically bind to mRNA, and is labeled to determine the presence or absence of a specific mRNA and the expression level. can confirm. Probes may be manufactured in the form of oligonucleotide probes, single strand DNA probes, double strand DNA probes, RNA probes, etc. Selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art.

In the present invention, 'antibody' is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to SYNCRIP of the present invention, and the antibody can be prepared according to a conventional method in the art. The types of antibodies include polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies are included. The antibody is meant to be intact, having two full-length light chains and two full-length heavy chains. Additionally, the above antibodies also include special antibodies such as humanized antibodies.

Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating drug addiction containing a substance that promotes the expression or activity of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) as an active ingredient.

The relationship between the SYNCRIP gene of the present invention and drug addiction symptoms has not yet been known, but through the present invention, it has been found that the expression of the SYNCRIP gene decreases during drug addiction. In addition, it was confirmed that when SYNCRIP was overexpressed in animals, miR-137, a drug addiction marker, was restored to normal levels. In addition, when SYNCRIP was overexpressed in a drug addiction animal model, it was confirmed that spatial learning disability and drug-induced hyperactivity, which are withdrawal symptoms of drug addiction, were suppressed. This means that substances that promote the expression or activity of the SYNCRIP gene can function as substances for preventing or treating drug addiction.

The substance that promotes the expression or activity of SYNCRIP may include, but is not particularly limited to, a substance that promotes translation of SYNCRIP mRNA.

In one embodiment of the present invention, the substance that promotes the expression or activity of SYNCRIP may be a vector containing the SYNCRIP gene, or a drug addiction treatment substance screened according to the drug addiction treatment substance screening method described above.

The vector refers to a means for expressing a target gene in a host cell. The vector contains elements for expression of the target gene, and may include a replication origin, a promoter, an operator, a transcription termination sequence, etc., and may be inserted into the genome of the host cell. Appropriate enzymatic sites (e.g., restriction enzyme sites) for introduction and/or ribosome binding site (RBS) for translation into proteins and/or selectable markers to confirm successful introduction into host cells, IRES (Internal Ribosome Entry Site) may additionally be included. The vector may additionally include transcriptional control sequences (eg, enhancers, etc.) other than the promoter.

In addition, the vector may be plasmid DNA, recombinant vector, or other carrier known in the art, and specifically, linear DNA plasmid DNA, recombinant non-viral vector, recombinant viral vector, or inducible gene expression vector system. system), and the recombinant viral vector may include retrovirus, adenovirus, adeno associated virus, helper-dependent adenovirus, herpes simplex virus ( It may be a herpes simplex virus, lentivirus, or vaccinia virus vector, but is not limited thereto.

Additionally, the vector refers to an expression vector for gene therapy. In the present invention, 'gene therapy' refers to a method of inserting a normal gene into a cell with a genetic abnormality or providing a new function to normalize its function in order to treat various genetic diseases caused by genetic abnormalities. Therefore, in the present invention, the expression vector for gene therapy is an expression vector that normalizes the function by delivering a new function through cell fusion between cells with genetic abnormalities and normal cells by transferring and expressing the SYNCRIP gene to normal cells in the body. it means.

As for the SYNCRIP gene, as described above, a primer capable of specifically recognizing the gene is prepared from a known sequence, and the gene is amplified through a polymerase chain reaction using this primer, and the gene is amplified as described above. After introduction into the expression vector, it can be introduced into the cells according to the present invention as described below. Introduction methods are known and include, for example, liposome-mediated transfection, calcium phosphate method, DEAE-dextran-mediated transfection, positively charged lipid-mediated transfection, electroporation, transduction using a phage system, or infection using a virus. Methods, etc. are included, but are not limited thereto.

The composition according to an embodiment of the present invention may contain 0.00001 to 30% by weight of a substance that promotes the expression or activity of SYNCRIP based on the total weight of the composition.

By promoting the expression or activity of SYNCRIP according to the embodiments described herein, the expression of miR-137 related to drug addiction can be regulated to prevent or treat drug addiction or withdrawal symptoms resulting therefrom.

The pharmaceutical composition may be used to prevent or treat drug addiction. Additionally, the pharmaceutical composition may be provided in any formulation suitable for topical application. For example, it may be any route selected from the group consisting of oral administration, transdermal administration, intravenous administration, intramuscular administration, subcutaneous injection, and intrabrain administration.

The pharmaceutical composition according to the present invention can be formulated in a suitable form with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline solutions, carriers for parenteral administration such as aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the pharmaceutical composition according to the present invention may be used as a suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, and buffer, if necessary depending on the administration method or dosage form. , antioxidants, etc. may be appropriately included. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, current edition.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. Alternatively, it can be manufactured by placing it in a multi-capacity container.

Additionally, the pharmaceutical composition may be administered by any device capable of moving the active ingredient to target cells. The pharmaceutical composition of the present invention may be contained in a therapeutically effective amount for the treatment of diseases. In the present invention, the term 'treatment', unless otherwise stated, means reversing, alleviating, suppressing the progression of, or preventing one or more symptoms of the disease or disease to which this term applies. In addition, the term 'therapeutically effective amount' refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in tissue systems, animals, or humans as considered by researchers, veterinarians, doctors, or other clinicians, which means treatment. Includes an amount that induces relief of symptoms of the disease or disorder. It is obvious to those skilled in the art that the active ingredients included in the pharmaceutical composition of the present invention will vary depending on the effect.

Therefore, the optimal pharmaceutical composition content can be easily determined by a person skilled in the art, depending on the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of dosage form, and the patient's age, weight, general health, gender, and diet. , can be adjusted according to various factors, including administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs.

It is important to consider all of the above factors and include an amount that can achieve the maximum effect with the minimum amount without side effects. For example, the dosage of the composition of the present invention is 0.1 × 10 ⁵ to 1.0 × 10 ⁸ cells/kg (body weight), more preferably 0.5 × 10 ⁶ to 1.0 × 10 8 cells transformed with a vector containing the SYNCRIP gene, which is an active ingredient. It may be 1.0 × 10 ⁷ cells/kg (body weight). However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately. The number of administrations can be once or twice or more within the range of clinically acceptable side effects, and the administration site can be administered to one or two or more sites. For animals other than humans, the dosage per kg is the same as that for humans, or the above dosage is determined based on the volume ratio (e.g., average value) of the ischemic organ (e.g., heart, etc.) between the target animal and the human. The converted amount can be administered. Examples of animals subject to treatment according to the present invention include humans and other mammals, specifically humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, goats, horses, pigs, etc. This is included.

Hereinafter, the present invention will be described in more detail through examples, etc., but the scope and content of the present invention should not be construed as being reduced or limited by the examples below. In addition, based on the disclosure of the present invention, including the examples below, it is clear that a person skilled in the art can easily implement the present invention for which no specific experimental results are presented, and the patent to which such variations and modifications are attached It is natural that it falls within the scope of the claims.

In addition, the experimental results presented below describe only the representative experimental results of the examples and comparative examples, and the effects of each embodiment of the present invention that are not explicitly presented below will be described in detail in the corresponding section.

Experimental Example 1. Verification of drug addiction biomarkers

1) Drug addiction animal model (MA)

In this experiment, C57BL/6J mice over 7 weeks old were prepared to be used as experimental animals. During the experiment, they were allowed to consume standard rodent diet and water as desired, and the animals were provided by the Korea Institute of Science and Technology. Processed under strict approval by the Protection and Use Committee. The experimental animals were allowed to adapt for one week, and general behavioral abnormalities were observed. Animals found to have abnormalities were not used in this experiment. All experimental animals were allowed to freely consume water and feed under conditions of 12 hours of sunlight and 12 hours of light in a breeding facility with constant temperature and constant humidity during the experiment period.

In the present invention, a control group and a drug addiction animal model were prepared as follows, and 8 animals each were randomly assigned. First, a gradual ramp-up protocol was used to develop methamphetamine addiction. Specifically, for the first three days (days 1 to 3) of the first week, 0.5 to 1.5 mg/kg of methamphetamine is injected once a day, and for the next three days (days 4 to 6), 1.5 to 2.5 mg/kg of methamphetamine is injected. After injecting twice a day and taking a day off, 2.5-4.0 mg/kg of methamphetamine was injected three times a day for six days (days 8 to 13) of the second week. Afterwards, injections were stopped for 1 week to prepare an animal model of drug addiction.

The control group for this was prepared by repeatedly injecting sterile saline, not methamphetamine, at 10 mL/kg intraperitoneally at the same time for 2 weeks, and then stopping the injection for 1 week.

2) Analysis of drug addiction-related miR-137 biomarker expression

To prepare cDNA through reverse transcription, a control group and a drug addicted animal model were anesthetized, their heads were cut off to collect brain striatum and serum, and a total of 50 ng of RNA was isolated from them. For miRNAs, cDNA was amplified using TaqMan Universal Master Mix II (Thermo Fisher Scientific) without uracil-DNA Glycosylase (UNG) according to the manufacturer's protocol. Next, the expression of mature miRNAs was quantified using TaqMan MicroRNA Assays (Thermo Fisher Scientific) and quantitative real-time PCR (qPCR). The qPCR reaction was performed in CFX connect (Bio-Rad). All reactions were measured in triplicate.

The relative abundance of miRNA and mRNA measured from the above results was confirmed using the 2 ^-ddCt method. snoRNA202 was used as a normalization control for quantifying miRNAs present in the mouse brain. The standard miRNA, miR-16, was used as a standardization control for quantifying miRNAs present in serum.

Figure 1 shows the results of comparing the expression level of miR-137 in serum and striatum (brain) in control and drug addiction animal model (MA). According to this, in the drug addiction animal model (MA) addicted to methamphetamine, the expression level of miR-137 in the intracerebral striatum was significantly increased compared to the control.

In addition, in the drug addiction animal model (MA) addicted to methamphetamine, it was confirmed through qPCR that the expression level of miR-137 in serum was significantly reduced compared to the control.

This may show the same tendency as that miR-137 acts as a biomarker that can diagnose drug addiction and withdrawal symptoms due to drug addiction. Therefore, it was confirmed once again that miR-137 acts as a biomarker that can confirm the presence or absence of drug addiction and withdrawal symptoms due to drug addiction.

Experimental Example 2. Confirmation of mutual influence of miR-137 and SYNCRIP

miRNA 3'UTR target expression clone for mouse Syncrip NM_019666.2 (SYNCRIP-3'UTR) (MmiT031985-MT05, GeneCopoeia), Human pre-microRNA Expression Construct Lenti-miR-137 (PMIRH137PA-1, System Biosciences), and Lucifer. A control vector was prepared for lase analysis.

For the luciferase assay, it was performed according to the manufacturer's protocol. Briefly, HEK293TN cells were first cultured in 12-well cell culture plates using EndoFectin Lenti transfection reagent (GeneCopoeia), which contained SYNCRIP-3'UTR and miR. -137 overexpression vector (or scrambled control vector) was processed and co-transfected. After 16 hours, the medium was changed, and the medium was recovered after 32 hours.

For each cell, the Gaussia luciferase (GL) activity was normalized to alkaline phosphatase (AP) activity using the Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia) according to the manufacturer's protocol. The activity of each enzyme was quantified by reading the absorbance with a Synergy HTX Multi-Mode Microplate Reader (BioTek, VT, USA).

Figure 2 shows GL in HEK293TN cells (miR137-OE) transfected with SYNCRIP-3'UTR and a miR-137 overexpression vector and HEK293TN cells (control) transfected with SYNCRIP-3'UTR and a scrambled control vector. /This is a graph quantifying AP activity.

As shown in Figure 2, it was confirmed that GL/AP was greatly reduced in cells overexpressing miR-137 (miR137-OE), indicating that miR-137 binds to SYNCRIP mRNA and suppresses the expression of the SYNCRIP gene. .

Experimental Example 3. Confirmation of the effect of SYNCRIP on drug addiction

1) Preparation of drug addiction animal model

The experimental animals used were C57BL/6J mice over 7 weeks of age purchased from the Research Animal Resource Center of the Korea Institute of Science and Technology, and treated in accordance with strict approval from the Animal Care and Use Committee. The experimental animals were allowed to adapt for at least one week before the experiment, and general behavioral abnormalities were observed. Animals that were observed to have abnormalities were not used in this experiment. All experimental animals were allowed to freely consume water and feed under conditions of 12 hours of sunlight and 12 hours of light in a breeding facility with constant temperature and constant humidity during the experiment period.

A gradual ramp-up protocol was used to induce methamphetamine addiction. Specifically, for the first three days (days 1 to 3) of the first week, 0.5 to 1.5 mg/kg of methamphetamine is injected once a day, and for the next three days (days 4 to 6), 1.5 to 2.5 mg/kg of methamphetamine is injected. After injecting twice a day and taking a day off, 2.5-4.0 mg/kg of methamphetamine was injected three times a day for six days (days 8 to 13) in the second week. Afterwards, injections were stopped for 1 week to prepare an animal model of drug addiction.

The control group for this was prepared by repeatedly injecting sterile saline, not methamphetamine, at 10 mL/kg intraperitoneally at the same time for 2 weeks, and then stopping the injection for 1 week.

2) SYNCRIP expression analysis (Western blot)

The control and drug addiction animal models were anesthetized, their heads were cut off, and the striatum of the brain was collected. Proteins were extracted from the cerebrostriatum (hereinafter also referred to as brain) using RIPA buffer (Thermo Fisher Scientific). At this time, the extraction process was carried out according to the manufacturer's protocol. In the extracted protein samples, the protein concentration was quantified using Protein Assay Dye Reagent Concentrate (Bio-Rad) and a spectrophotometer.

First, the protein (50 μg/lane) sample was loaded on SDS-PAGE and transferred to a PVDF membrane. The PVDF membrane was treated with 5% skim milk for 20 minutes and then washed. The primary antibody was treated at 4°C, reacted overnight, and then incubated at room temperature for 2 hours. The primary antibodies used at this time were as follows: rabbit anti-hnRNP Q (1:10,000) (ab184946, Abcam, Cambridge, UK) and mouse anti-β-actin (1:500) (sc-47778, Santa Cruz Biotechnology, CA) , USA)

Then, the primary antibody-bound membrane was washed, and donkey anti-rabbit IgG-HRP (1:2,000) (sc-2317, Santa Cruz Biotechnology) or donkey anti-mouse IgG-HRP (1:2,000) (sc-2318, Santa Cruz Biotechnology) was incubated at room temperature for 2 hours. HRP signals were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and Image Quant LAS4000 (GE Healthcare Bio-Sciences, Uppsala, Sweden).

For quantitative densitometric comparison, Image J was used. In this experiment, samples were processed correspondingly from blots for the same sample, and a loading control (β-actin) was performed on the same blot as a target (SYNCRIP).

3) Expression level of SYNCRIP (qPCR)

To prepare cDNA through reverse transcription, a control group and a drug addicted animal model (MA) were anesthetized, their heads were cut off to collect brain striatum and serum, and a total of 50 ng of RNA was extracted from this. separated. For miRNAs, cDNA was amplified using TaqMan Universal Master Mix II (Thermo Fisher Scientific) without UNG according to the manufacturer's protocol. Next, the expression of mature miRNAs was quantified using TaqMan MicroRNA Assays (Thermo Fisher Scientific) and quantitative real-time PCR (qPCR). The qPCR reaction was performed in CFX connect (Bio-Rad). All reactions were measured in triplicate.

The relative abundance of miRNA and mRNA measured from the above results was confirmed using the 2 ^-ddCt method. snoRNA202 was used as a normalization control for quantifying miRNAs present in the mouse brain. The standard miRNA, miR-16, was used as a standardization control for quantifying miRNAs present in serum.

Figure 3 shows the results of Western blot analysis of the expression level of SYNCRIP in the brain striatum of a control group and the brain striatum of a drug addiction animal model. According to this, it was confirmed that SYNCRIP expression was decreased in the cerebrostriatum in an animal model (MA) addicted to methamphetamine by abusing it. This is a significantly reduced value compared to the control group. In other words, it can be seen that SYNCRIP protein is significantly reduced in a state of drug addiction.

Figure 4 is a graph comparing the expression level of SYNCRIP in the brain of a control and drug addiction animal model. According to this, it was confirmed that SYNCRIP expression was decreased in the cerebrostriatum in a drug addiction animal model (MA) addicted to methamphetamine. It can be seen that this is a significantly increased number compared to the control group. This means that the SYNCRIP gene of the present invention can be used as a biomarker for drug addiction.

Experimental Example 4. Confirmation of the effect of SYNCRIP on the expression level of miR-137

The following experiment was conducted to confirm that drug addiction markers increase when the expression of SYNCRIP is promoted in nerve cells.

First, SH-SY5Y neural cells were purchased from the Korea Cell Line Bank, distributed on a 100 ㆈ dish, and then cultured in a 37°C CO ² incubator for cell culture. Afterwards, Syncrip (NM_019666) Mouse Tagged ORF Clone vector (MR217725L4, OriGene Technologies, MD, USA) was used to promote (over expression) SYNCRIP in the nerve cells (Figure 5).

In this experiment, pLenti-C-mGFP-P2A-Puro vector (OriGene Technologies) was used as a control.

To prepare cDNA through reverse transcription, a total of 50 ng of RNA was isolated from the sample. For miRNAs, cDNA was amplified using TaqMan Universal Master Mix II (Thermo Fisher Scientific) without UNG according to the manufacturer's protocol. Next, the expression of mature miRNAs was quantified using TaqMan MicroRNA Assays (Thermo Fisher Scientific) and quantitative real-time PCR (qPCR). The qPCR reaction was performed in CFX connect (Bio-Rad). All reactions were measured in triplicate.

The relative abundance of miRNA and mRNA measured from the above results was confirmed using the 2 ^-ddCt method. snoRNA202 was used as a normalization control for quantifying miRNAs present in the mouse brain. The standard miRNA, miR-16, was used as a standardization control for quantifying miRNAs present in serum.

Figure 6 shows the results of comparing the expression level of miR137 secreted in a SYNCRIP-overexpressing neuronal cell line (SYNCRIP-OE) and the control group. According to this, it was confirmed through qPCR that when the SYNCRIP gene was overexpressed in a cultured cell line, the expression level of the drug addiction marker gene miR-137 secreted by the cells was also significantly increased. In other words, since overexpression of SYNCRIP in neurons can increase the secretion amount of the miR-137 marker due to drug addiction, it can be seen that the secretion amount of miR-137, a drug addiction biomarker, can be upregulated through the SYNCRIP gene. It is possible to screen therapeutic substances applicable to the treatment and prevention of drug addiction through the expression level of the SYNCRIP gene, and it suggests that agents that promote the SYNCRIP gene can be applied as therapeutic substances.

Experimental Example 5. Verification of SYNCRIP-1

1) Drug addiction animal model (MA) and drug addiction transgenic animal model (DST) ^SYNCRIP-OE +MA) Preparation

The experimental animals used were C57BL/6J male mice over 7 weeks of age purchased from the Research Animal Resource Center of the Korea Institute of Science and Technology, and treated in accordance with the strict approval of the Animal Care and Use Committee. The experimental animals were allowed to adapt for at least one week before the experiment, and general behavioral abnormalities were observed. Animals that were observed to have abnormalities were not used in this experiment. All experimental animals were allowed to freely consume water and feed under conditions of 12 hours of sunlight and 12 hours of light in a breeding facility with constant temperature and constant humidity during the experiment period.

For animal experiments, a Lentivirus system was constructed to ensure smooth synthesis of SYNCRIP transcript and for long term expression. Specifically, Syncrip (NM_019666) Mouse Tagged ORF Clone vector (MR217725L4, OriGene Technologies, MD, USA) and its control pLenti-C-mGFP-P2A-Puro vector (OriGene Technologies) were packaged into Lentivirus and used (Figure 5). .

After anesthetizing the experimental animal with ketamine-xylazine mixture (ketamine 120 mg/kg, xylazine 8 mg/kg), the scalp and skull were opened and a hole was drilled in the skull with Bregma AP +1.1, ML ±1.65 mm as the coordinates. , 0.2, 0.7, and 0.7 ul of Lentivirus were injected at the coordinates of DV -3.0, -2.6, and -2.2 mm, respectively. The injected virus specifically targeted only cholinergic interneurons in the brain striatum by Cre-lox recombination. After lentivirus injection, it was sutured with silk suture. After a 4-week resting period and virus infection period after surgery, a transgenic animal model (DST ^SYNCRIP-OE ) overexpressing SYNCRIP was prepared.

The transgenic animal model (DST ^SYNCRIP-OE ) was brought into a state of mild drug addiction (maintained in a hyperexcitable state without any significant symptoms) by injecting (abuse) small amounts of methamphetamine over a long period of time. Specifically, a transgenic animal model of mild drug addiction (DST ^SYNCRIP-OE +MA) was prepared by repeatedly injecting 1.0 mg/kg of methamphetamine intraperitoneally every two days for two weeks.

The control for this was intraperitoneally administered 10 mL/kg of sterile saline, not methamphetamine, to a control animal model (treated with the same amount of pLenti-C-mGFP-P2A-Puro lentivirus, which does not express SYNCRIP), rather than a transgenic animal model. It was prepared by repeated injections once every two days at the same time for 2 weeks.

As a comparison group, a drug addiction animal model (MA) prepared in the same manner as the above addiction process was used, except that non-transgenic normal animals were used.

2) Analysis of drug addiction-related miR-137 biomarker expression

To prepare cDNA through reverse transcription, the control group, drug addiction animal model (MA), and drug addiction transgenic animal model (DST ^SYNCRIP-OE +MA) were anesthetized, serum was collected, and a total of 50 ng of RNA was isolated. For miRNAs, cDNA was amplified using TaqMan Universal Master Mix II (Thermo Fisher Scientific) without UNG according to the manufacturer's protocol. Next, the expression of mature miRNAs was quantified using TaqMan MicroRNA Assays (Thermo Fisher Scientific) and quantitative real-time PCR (qPCR). The qPCR reaction was performed in CFX connect (Bio-Rad). All reactions were measured in triplicate.

The relative abundance of miRNA and mRNA measured from the above results was confirmed using the 2 ^-ddCt method. snoRNA202 was used as a normalization control for quantifying miRNAs present in the mouse brain. The standard miRNA, miR-16, was used as a standardization control for quantifying miRNAs present in serum.

Figure 7 shows the preparation of a transgenic animal model (DST ^SYNCRIP-OE -MA), a normal group (control), and a drug addiction animal model (MA) with increased SYNCRIP expression in the striatum of a mild drug addiction animal model, from which the drug This is the result of measuring the expression level of miR-137, an addiction biomarker, using qPCR.

According to Figure 7, it was confirmed that the expression level of serum miR-137 was decreased even in an animal model of mild drug addiction (MA) in which low concentrations of methamphetamine were repeatedly administered to show only a mild drug addiction state. Specifically, in the mild drug addiction animal model (MA), it can be confirmed through qPCR that the expression level of miR-137 is significantly reduced compared to the control group.

It can be seen that through the expression level of miR-137 in the blood, it is possible to detect both drug addiction with clear withdrawal symptoms and drug addiction with mild withdrawal symptoms. As previously known, miR-137 is a major biomarker for diagnosing drug addiction.

When SYNCRIP expression was increased in the striatum of a drug addiction animal model (DST ^SYNCRIP-OE -MA), the expression level of blood miR-137, a drug addiction marker, increased compared to the MA group, showing no significant difference from the normal group (control). Recovery to this level was confirmed through qPCR. If SYNCRIP is overexpressed in nerve cells, even mild drug addiction without withdrawal symptoms can be recovered to a level similar to that of a normal group. Through this, the SYNCRIP gene can not only diagnose mild drug addiction without withdrawal symptoms to severe drug addiction with withdrawal symptoms, but can also be used as a screening biomarker for therapeutic substances, and substances that express SYNCRIP or promote its activity. In the case of , it can be seen that it can be used as a pharmaceutical composition that can treat drug addiction.

Experimental Example 6. Verification of SYNCRIP-2

1) Drug addicted transgenic animal model (Brain ^SYNCRIP-OE +MA) Preparation

The experimental animals used were C57BL/6J male mice over 7 weeks of age purchased from the Research Animal Resource Center of the Korea Institute of Science and Technology, and treated in accordance with the strict approval of the Animal Care and Use Committee. The experimental animals were allowed to adapt for at least one week before the experiment, and general behavioral abnormalities were observed. Animals that were observed to have abnormalities were not used in this experiment. All experimental animals were allowed to freely consume water and feed under conditions of 12 hours of sunlight and 12 hours of light in a breeding facility with constant temperature and constant humidity during the experiment period.

For animal experiments, a Lentivirus system was constructed to ensure smooth synthesis of SYNCRIP transcript and for long term expression. Specifically, Syncrip (NM_019666) Mouse Tagged ORF Clone vector (MR217725L4, OriGene Technologies, MD, USA) (Figure 5) and its control pLenti-C-mGFP-P2A-Puro vector (OriGene Technologies) were packaged into Lentivirus and used. .

After anesthetizing the experimental animal with ketamine-xylazine mixture (ketamine 120mg/kg, xylazine 8mg/kg), open the scalp and skull, drill a hole in the skull with Bregma AP +1.1, ML ±1.65 mm as the coordinates, and DV Lentivirus of 0.2, 0.7, and 0.7 ul was injected at coordinates of -3.0, -2.6, and -2.2 mm, respectively. The injected virus specifically targeted only cholinergic interneurons in the brain striatum by Cre-lox recombination. After lentivirus injection, it was sutured with silk suture. After a 4-week resting period and virus infection period after surgery, a transgenic animal model (DST ^SYNCRIP-OE ) overexpressing SYNCRIP was prepared.

A gradual ramp-up protocol was used to make the transgenic animal model (DST ^SYNCRIP-OE ) addicted to methamphetamine. Specifically, for the first three days (days 1 to 3) of the first week, 0.5 to 1.5 mg/kg of methamphetamine is injected once a day, and for the next three days (days 4 to 6), 1.5 to 2.5 mg/kg of methamphetamine is injected. After injecting twice a day and taking a day off, 2.5-4.0 mg/kg of methamphetamine was injected three times a day for six days (days 8 to 13) in the second week. Afterwards, injections were stopped for 1 week to prepare a transgenic animal model (DST ^SYNCRIP-OE +MA).

The control for this was intraperitoneally administered 10 mL/kg of sterile saline, not methamphetamine, to a control animal model (treated with the same amount of pLenti-C-mGFP-P2A-Puro lentivirus, which does not express SYNCRIP), rather than a transgenic animal model. It was prepared by repeated injections at the same time for 4 weeks. As a comparison group, a drug addiction animal model (MA) prepared in the same manner as the above addiction process was used, except that non-transgenic normal animals were used.

2) Analysis of drug addiction-related miR-137 biomarker expression

To prepare cDNA through reverse transcription, a control group and a drug-addicted transgenic animal model (experimental group) were anesthetized, serum was collected, and a total of 50 ng of RNA was isolated from them. For miRNAs, cDNA was amplified using TaqMan Universal Master Mix II (Thermo Fisher Scientific) without UNG according to the manufacturer's protocol. Next, the expression of mature miRNAs was quantified using TaqMan MicroRNA Assays (Thermo Fisher Scientific) and quantitative real-time PCR (qPCR). The qPCR reaction was performed in CFX connect (Bio-Rad). All reactions were measured in triplicate.

The relative abundance of miRNA and mRNA measured from the above results was confirmed using the 2 ^-ddCt method. snoRNA202 was used as a normalization control for quantifying miRNAs present in the mouse brain. The standard miRNA, miR-16, was used as a standardization control for quantifying miRNAs present in serum.

Figure 8 shows the preparation of a transgenic animal model (Brain ^SYNCRIP-OE -MA), a normal group (control), and a drug addiction animal model (MA) with increased SYNCRIP expression in the striatum of a drug addiction animal model, from which drug addiction living organisms were prepared. This is the result of measuring the expression level of the indicator, miR-137, by qPCR.

As shown in Figure 8, it was confirmed that the expression level of miR-137 in the serum of the comparison group (MA) addicted to simple methamphetamine was significantly decreased compared to the control group. In other words, it was reaffirmed that drug addiction such as methamphetamine can be diagnosed through a decrease in serum miR-137.

Additionally, when SYNCRIP was overexpressed (Brain ^SYNCRIP-OE +MA), the expression level of miR-137, a drug addiction marker gene, in serum was restored to normal level. It can be seen that SYNCRIP of the present invention can prevent or treat drug addiction.

Experimental Example 7. SYNCRIP treatment efficacy on methamphetamine withdrawal-induced animal model

1) Preparation of drug addiction animal model

The experimental animals used were C57BL/6J mice over 7 weeks of age purchased from the Research Animal Resource Center of the Korea Institute of Science and Technology, and treated in accordance with strict approval from the Animal Care and Use Committee. The experimental animals were allowed to adapt for at least one week before the experiment, and general behavioral abnormalities were observed. Animals that were observed to have abnormalities were not used in this experiment. All experimental animals were allowed to freely consume water and feed under conditions of 12 hours of sunlight and 12 hours of light in a breeding facility with constant temperature and constant humidity during the experiment period.

A gradual ramp-up protocol was used to induce methamphetamine addiction. Specifically, for the first three days (days 1 to 3) of the first week, 0.5 to 1.5 mg/kg of methamphetamine is injected once a day, and for the next three days (days 4 to 6), 1.5 to 2.5 mg/kg of methamphetamine is injected. After injecting twice a day and taking a day off, 2.5-4.0 mg/kg of methamphetamine was injected three times a day for six days (days 8 to 13) in the second week. Afterwards, injections were stopped for 1 week to prepare an animal model of drug addiction.

The control group for this was prepared by repeatedly injecting sterile saline, not methamphetamine, at 10 mL/kg intraperitoneally at the same time for 2 weeks, and then stopping the injection for 1 week.

2) Induction of SYNCRIP overexpression in drug addiction animal model (MA)

We sought to determine whether SYNCRIP would have a therapeutic effect when treated with an animal model (MA) in which withdrawal due to drug addiction was induced. For this purpose, a lentivirus capable of overexpressing Syncrip (NM_019666) Mouse Tagged ORF Clone vector (MR217725L4, OriGene Technologies, MD, USA) was injected into the brain striatum of the drug addiction animal model (MA) prepared in step 1) above (Figure 5). Specifically, after anesthetizing a drug addicted animal model (MA) with ketamine-xylazine mixture (ketamine 120mg/kg, xylazine 8mg/kg), the scalp and skull were opened and the skull was placed with Bregma AP +1.1, ML ±1.65 mm as the coordinates. After drilling a hole, 0.2, 0.7, and 0.7 ul of lentivirus was injected at the coordinates of DV -3.0, -2.6, and -2.2 mm, respectively. After lentivirus injection, it was sutured with silk suture. After 4 weeks of post-surgery stabilization and virus infection period, an animal model (MA-SYNCRIP; experimental group) in which SYNCRIP overexpression was induced after drug intoxication was prepared. All experimental animals were allowed to freely consume drinking water and feed under conditions of 12 hours of sunlight and 12 hours of light in a breeding facility with constant temperature and constant humidity during the experiment period.

3) Evaluation of egocentric spatial learning in drug addiction animal model (MA), control group, and animal model that induced SYNCRIP overexpression after drug addiction (MA-SYNCRIP; experimental group)

To evaluate cognitive dysfunction, which is one of the representative symptoms of drug addiction, an animal model of drug addiction (MA), a control group, and an animal model in which SYNCRIP overexpression was induced after drug addiction (MA-SYNCRIP; experimental group) were used. The experiment was performed with a cross maze.

The water cross maze is a cross-shaped maze consisting of four open branches (30 × 6 cm) filled with water from the bottom to a height of 10 cm . When viewed from the starting point of the underwater cross maze, a hidden platform was placed on the right branch. After placing the experimental animals at the starting point, they were allowed to freely find the hidden platform for 5 minutes. If the experimental animal could not find the platform within 5 minutes, it was guided directly to the platform and learned the location of the platform. The learning process was conducted 6 times a day for a total of 3 days, and the intervals between each learning process within a day were 10 minutes, 10 minutes, 3 hours, 10 minutes, and 10 minutes (Figures 9 and 10).

After the experimental animals were trained for 3 days, a probe test was performed the next day (FIG. 11). In the test, the experimental animals were placed on a branch opposite the starting point used during the learning period and were asked to find the platform according to visual clues. Where the platform is ( If you go to the right side (the branch marked with a star) that you learned during the learning period, you are evaluated as having performed allocentric spatial learning. If you go to the right side (the branch marked with a star) that you learned during the learning period, you are evaluated as having performed egocentric spatial learning. The number of mice evaluated as having performed spatial learning was measured as a percentage and shown on the graph.

Figure 12 is a graph showing the results of an underwater cross maze experiment for a drug addiction animal model (MA), a control group, and an animal model in which SYNCRIP overexpression was induced after drug addiction (MA-SYNCRIP; experimental group). According to this, drug addicted animals The model (MA) confirmed that egocentric spatial learning was abnormally strengthened compared to the control group.

On the other hand, in an animal model (MA-SYNCRIP) in which SYNCRIP overexpression was induced after drug addiction, it was confirmed that spatial learning ability was restored to the control level.

Experimental Example 8. Activity evaluation on methamphetamine withdrawal induction animal model according to SYNCRIP treatment

A drug addiction animal model (MA), a control group, and an animal model in which SYNCRIP overexpression was induced after drug addiction (MA-SYNCRIP; experimental group) were prepared according to Experimental Example 7. In all animal models, an open field experiment was performed for 20 minutes before drug (methamphetamine) injection to measure activity, and this was used as a baseline.

As comparison group 1, 4.0 mg/kg of methamphetamine was administered to the control group, and an open field experiment was performed again for 20 minutes to measure activity.

As comparison group 2, 4.0 mg/kg of methamphetamine was administered to a drug addicted animal model (MA), and an open field experiment was performed again for 20 minutes to measure activity.

As an experimental group, an animal model (MA-SYNCRIP) in which SYNCRIP overexpression was induced after drug addiction was prepared, and after administering 4.0 mg/kg of methamphetamine, an open field experiment was performed again for 20 minutes to measure activity.

Finally, the activity of each group compared to the baseline was compared, the percentage increase was calculated, and the degree of hyperactivity due to methamphetamine is shown in Figure 12.

Figure 13 is a graph showing the degree of change in activity when methamphetamine is administered to a drug addiction animal model (MA), an animal model in which SYNCRIP overexpression is induced after drug addiction (MA-SYNCRIP; experimental group). According to this, the control group ( control) and drug addiction animal model (MA) showed a significant increase in activity due to methamphetamine injection. On the other hand, in an animal model (MA-SYNCRIP) in which SYNCRIP overexpression was induced after drug addiction, it was confirmed that the increase in activity was significantly suppressed even when methamphetamine was administered. In other words, it was confirmed that SYNCRIP has the effect of suppressing the dependent effects of drugs even after drug addiction or withdrawal due to drug addiction occurs.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Biomarker composition for drug addiction diagnosis, screening method of therapeutic agents for drug addiction, and pharmaceutical composition for preventing and treating drugs Addiction <130>HPC-9500 <150> KR 2020/0077727 <151> 2020-06-25 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 2754 <212> DNA <213> Artificial Sequence <220> <223> SYNCRIP <400> 1 actcgcgccg gacacaggga gcagcgagca cgcgtttccc gcaacccgat accatcggac 60 aggatttctc cgcctcagcc caacggggag atctctggaa acatggctac agaacatgtt 120 aatggaaatg gtactgaaga gcccatggat actacttctg cagttatcca ttcagaaaat 180 tttcagacat tgcttgatgc tggtttacca cagaaagttg ctgaaaaact agatgaaatt 240 tacgttgcag ggctagttgc acatagtgat ttagatgaaa gagctattga agctttaaaa 300 gaattcaatg aagacggtgc attggcagtt cttcaacagt ttaaagacag tgatctctct 360 catgttcaga acaaaagtgc ctttttatgt ggagtcatga agacttacag gcagagagaa 420 aaaacaaggga ccaaagtagc agattctagt aaaggaccag atgaggcaaa aattaaggca 480 ctcttggaaa gacaggcta cacacttgat gtgaccactg gacagaggaa gtatggagga 540 ccacctccag attccgttta ttcaggtcag cagccttctg ttggcactga gatatttgtg 600 ggaaagatcc caagagatct atttgaggat gaacttgttc cattatttga gaaagctgga 660 cctatatggg atcttcgtct aatgatggat ccactcactg gtctcaatag aggttatgcg 720 tttgtcactt tttgtacaaa agaagcagct caggaggctg ttaaactgta taataatcat 780 gaaattcgtt ctggaaaaca tattggtgtc tgcatctcag ttgccaaacaa taggcttttt 840 gtgggctcta ttcctaagag taaaaccaag gaacagattc ttgaagaatt tagcaaagta 900 acagagggtc ttacagacgt cattttatac caccaaccgg atgacaagaa aaaaaacaga 960 ggcttttgct ttcttgaata tgaagatcac aaaacagctg cccaggcaag gcgtaggtta 1020 atgagtggta aagtcaaggt ctgggggaat gttggaactg ttgaatgggc tgatcctata 1080 gaagatcctg atcctgaggt tatggcaaag gtaaaagtgc tgtttgtacg caaccttgcc 1140 aatactgtaa cagaagagat tttagaaaag gcatttagtc agtttgggaa actggaacga 1200 gtgaagaagt taaaagatta tgcgttcatt cattttgatg agcgagatgg tgctgtcaag 1260 gctatggaag aaatgaatgg caaagacttg gagggagaaa atattgaaat tgtttttgcc 1320 aagccaccag atcagaaaag gaaagaaaga aaagctcaga ggcaagcagc aaaaaatcaa 1380 atgtatgacg attactacta ttatggtcca cctcatatgc cccctccaac aagaggtcga 1440 gggcgtggag gtagaggtgg ttatggatat cctccagatt attatggata tgaagattat 1500 tatgattatt atggttatga ttaccataac tatcgtggtg gatatgaaga tccatactat 1560 ggttatgaag attttcaagt tggagctaga ggaaggggtg gtagaggagc aaggggtgct 1620 gctccatcca gaggtcgtgg ggctgctcct ccccgcggta gagccggtta ttcacagaga 1680 ggaggtcctg gatcagcaag aggcgttcga ggtgcgagag gaggtgccca acaacaaaga 1740 ggccgcgggg tacgtggtgc gaggggtggc cgcggtggaa atgtaggagg aaagcgcaaa 1800 gctgatgggt acaaccagcc agattccaag cggcgccaga ccaataatca gaactggggc 1860 tcccaaccca ttgctcagca accgctccaa ggtggtgatc attctggtaa ctatggttac 1920 aaatctgaaa accaggagtt ttatcaggat acttttgggc aacagtggaa gtagaaacag 1980 tagggcctct gtaaaattgg agactgatag gttgatcaga aactcaccct aaatctgaac 2040 gggtgccgct ataatttgtg acatctggca agatttccct ttatgtatat attttaacaa 2100 tccgcttgga cacgaacaaa gccacacttc taactgcttc tggcgaactg attttattt 2160 taattttttt caataaagat attcttagat actgaaagaa atagttaatg agtttgcatt 2220 tgtgcttgag aaaatttggc tcaagtccat ttggctgtag tgtcaacgat gtttccagta 2280 gtgtttagat ttggtgtctt caaaggtagt tgattaaaac caagtgtgtc tttaatatct 2340 tgtatcagaa taactttgta tgttaccaac ttaaattgct agaataaggt aaattgatac 2400 acaactgcta tttttaattt agaactttga cctaatttgg gttttcaaaa ccattttggc 2460 tacttgtatt ctttatgctg ttgtttattt caataaaaaaa ttcacaccta aatgtatact 2520 tactaaaatt gtgtttacaa ttcgtttttc acaaaatttc ctgcaaattt ggttcaaatt 2580 gtatagcatg tcaaggccaa ttaaagggtt ttgtgccttg ttaattcttg tgtggaatat 2640 gtctgcacat tacacaacac tgatttattg cagttttctg cttctggttt aaagtgctat 2700 tttacaacag acttcatgtt cccatcaaaa ataaaaagat aatacatgta gtaa 2754

Claims (17)

delete Contains as an active ingredient an agent that can measure the expression level of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) protein,
A composition for diagnosing drug addiction, wherein the gene encoding the SYNCRIP protein consists of a base sequence represented by SEQ ID NO: 1. According to paragraph 2,
The agent for measuring the expression level of SYNCRIP includes a probe or primer that specifically binds to the SYNCRIP gene; And antibodies, peptides, aptamers or compounds that specifically bind to the SYNCRIP protein; A composition characterized in that it is one or more selected from among. delete A kit for diagnosing drug addiction comprising the composition according to claim 2. (1) measuring the protein expression level of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) from a biological sample isolated from a subject;
(2) comparing the expression level of the SYNCRIP protein with that of a normal control sample; and
(3) determining that the SYNCRIP protein expression level is lower than that of the normal control group as a result of the comparison in step (2), and determining that the SYNCRIP protein is in a state of drug addiction,
A method of providing information necessary for diagnosing drug addiction, characterized in that the gene encoding the SYNCRIP protein is represented by SEQ ID NO: 1. According to clause 6,
A method of providing information necessary for diagnosing drug addiction, further comprising measuring the expression level of miR-137 from a biological sample of the subject. (a) preparing a candidate substance treatment group by treating a mammal other than a human with induced drug addiction or a sample isolated therefrom with the candidate substance;
(b) measuring and comparing SYNCRIP protein expression levels in the candidate material treatment group and the candidate material non-treatment group; and
(c) selecting the candidate material as a drug addiction treatment material if the SYNCRIP protein expression level of the candidate material treated group is higher than the candidate material untreated group (control group) in the results obtained from step (b);
The drug is methamphetamine,
A method for screening a substance for treating drug addiction caused by methamphetamine, characterized in that the gene encoding the SYNCRIP protein is represented by SEQ ID NO: 1. delete According to clause 8,
The expression level can be measured using Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay, complement fixation assay, A screening method for a drug addiction treatment material, characterized in that measurement is performed using any one method selected from the group consisting of FACS and protein chip. According to clause 8,
A screening method for a drug addiction treatment material, further comprising measuring the expression level of miR-137 in the serum of the candidate material treated group and the candidate material untreated group. According to clause 11,
If the expression level of miR-137 in the group treated with the candidate material is not significantly different from the expression level of miR-137 in the group not treated with the candidate material (control group), it further includes the step of determining the candidate material as a treatment material for drug addiction. A screening method for drug addiction treatment substances, characterized in that: The substance used to treat drug addiction caused by methamphetamine selected by the screening method from Paragraph 8 is used as the active ingredient,
The active substance is characterized by promoting or activating protein expression of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein),
A pharmaceutical composition for preventing or treating drug addiction caused by methamphetamine, characterized in that the gene encoding the SYNCRIP protein is represented by SEQ ID NO: 1. According to clause 13,
A pharmaceutical composition for preventing or treating drug addiction, wherein the drug addiction treatment material that promotes protein expression of SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) is a vector containing a gene encoding SYNCRIP. According to clause 14,
The vector is for preventing or treating drug addiction, characterized in that it is any one selected from the group consisting of linear DNA plasmid DNA, recombinant non-viral vector, recombinant viral vector, and inducible gene expression vector system. Pharmaceutical composition. a) treating a candidate substance in a mammal other than a human whose drug addiction has been induced by methamphetamine;
b) Mammals other than humans that overexpress SYNCRIP (synaptotagmin binding cytoplasmic RNA interacting protein) protein and are addicted to drugs; and measuring the expression level of the SYNCRIP gene in a sample isolated from a mammal other than a human in which drug addiction was induced by treatment with the candidate substance in step a);
c) mammals other than humans that overexpress the SYNCRIP gene in step b) above and are addicted to drugs; And measuring the level of expression of the gene for miR-137 in a sample isolated from a mammal other than a human in which drug addiction was induced by treatment with the candidate substance in step a); and
d) The expression level of the SYNCRIP gene in a sample isolated from a mammal other than a human in which drug addiction was induced by treating the candidate substance in step a) measured in step b) above was determined in a sample that overexpressed the SYNCRIP gene and was addicted to the drug. If there is no significant difference from the expression level of the SYNCRIP gene in samples isolated from mammals other than humans, and
The expression level of the miR-137 gene in the serum sample isolated from a mammal other than a drug-addicted human overexpressing the SYNCRIP gene in step b) measured in step c) is the result of treatment with the candidate substance in step a). If there is no significant difference from the expression level of the miR-137 gene in samples isolated from mammals other than humans with induced drug addiction,
A step of selecting the candidate substance as a substance for treating drug addiction when all of the following are met,
A method for screening a substance for treating drug addiction caused by methamphetamine, characterized in that the SYNCRIP gene consists of a base sequence represented by SEQ ID NO: 1.
According to clause 16,
The sample in step b) is a brain sample,
A screening method for a drug addiction treatment substance, characterized in that the sample in step c) is a serum sample.