KR101081358B1 - Method for diagnosis of Parkinson's disease by quantitative measuring of brain metabolites using magnetic resonance spectroscopy - Google Patents

Abstract

The present invention relates to a method for diagnosing Parkinson's disease by measuring quantitative changes in brain metabolites using Magnetic Resonance Spectroscopy (MRS). How to determine whether Parkinson's disease is induced in Parkinson's disease research animal model by analyzing the amount of brain metabolite glutamate complex, ie glutamate, glutamine and GABA complex / creatine, in the spectrum obtained by magnetic resonance spectroscopy , A method for diagnosing Parkinson's disease, a system for diagnosing Parkinson's disease, and a method for screening a Parkinson's disease therapeutic agent. In the method for diagnosing Parkinson's disease using magnetic resonance spectroscopy according to the present invention, the amount of glutamate complex (glutamate, glutamine and GABA complex) containing GABA present in a very small amount in a mammal can be analyzed by non-invasive method. It is more accurate than existing Parkinson's disease diagnosis methods, and has a quick and easy effect, which is useful for research and early diagnosis for Parkinson's disease.

Magnetic resonance spectroscopy, Parkinson's disease, brain tissue, glutamate complex, glutamate, glutamine, GABA, creatine

Description

Method for diagnosis of Parkinson's disease by quantitative measuring of brain metabolites using magnetic resonance spectroscopy}

The present invention relates to a method for diagnosing Parkinson's disease by measuring the amount of quantitative change in brain metabolites using magnetic resonance spectroscopy, and a method for screening a Parkinson's disease therapeutic agent.

Parkinson's Disease (Parkinson's Disease) is a chronic illness caused by lack of dopamine neurotransmitters in the brain. Dopamine is produced in nerve cells called the black matter of the brain, and the nerve cells of the black matter are connected to the part called the basal nucleus of the brain, and the basal nucleus is connected to the motor cortex and many other parts of the brain in a complex way to smooth and harmonize the movement of the human body. It is also a very important part to ensure that it is done correctly. However, Parkinson's disease is caused by a lack of dopamine, a substance secreted to control the function of the basal ganglia in black matter.

Parkinson's disease can be divided into primary and secondary symptoms. The primary symptoms are stiffness, tremor, slow or reduced movement of the body, imbalance, and impaired gait. Secondary symptoms refer to symptoms derived from primary symptoms or from involvement of the nervous system other than melanoma.

Therapies currently used for the treatment of Parkinson's disease include drug therapy, surgical therapy and physical therapy. In the case of drug therapy, dopamine, which is generally lacking in the brain, is supplemented, and neurotransmitter imbalance due to the lack of dopamine Drugs for the purpose of preventing or delaying the destruction of nerve cells and for controlling symptoms such as depression, such as amantadine, anticholinergic drugs, el-dopa, cinemet, madopa, dopamine Agonists, eldepril and antidepressants. However, since these drugs cannot revive dead neurons, they are not intended to cure, but to control symptoms.

In the case of surgical treatment, stereotactic brain surgery and transplantation are mainly used. Stereotactic brain surgery is performed to remove the symptoms of Parkinson's disease on the opposite side of the thalamus by artificially creating lesions in the sagittal. It aims to alleviate the symptoms or symptoms and has a weak therapeutic effect. Transplantation is a surgical technique developed due to the recent development of cell culture and surgical techniques, in which dopamine-producing cells are directly implanted into the brain. Especially, fetal nerve cells or the patient's own adrenal medulla cells are transplanted. Is developed. However, this method is ideal in that it fundamentally corrects the cause of Parkinson's disease, but the effect is still small, so it is too early to evaluate.

In addition, in the case of physiotherapy, most of the patient's symptoms are weak or not severe as a method of performing the exercise ability of the patient to help maximize, and prevents the joints to prevent.

     On the other hand, active research on the causes of Parkinson's disease is still ongoing, but the exact cause is still unknown. However, only the infection theory caused by viral encephalitis and the immune mechanism related to immunity, innately innate There are genetic theories, theories that free radicals destroy nerve cells, and that there is a problem with the production and metabolism of dopamine. However, there is not enough to explain Parkinson's disease as a whole. There is a need for more active research on this.

In addition, according to the recent statistics, the elderly population is rapidly increasing, and the incidence of Parkinson's disease is also increasing as the life expectancy is extended. This can be a very important problem.

Therefore, many researchers use Parkinson's disease animal model to identify the cause of Parkinson's disease and develop a therapeutic agent, but in many cases, Parkinson's disease is not properly induced or the desired animal model is not produced due to the physiological change of each animal. There are many problems with obtaining the results. In addition, there is no direct way to prove that Parkinson's disease animal models have Parkinson's disease, and we only have Parkinson's disease with these differences by observing differences in behavior between Parkinson's and normal animals. Indirectly confirmed, there is a disadvantage that the measurement results are inaccurate and time-consuming problem. In addition, accurate analysis required biopsy, an invasive method for the changes in cerebral metabolism in Parkinson's disease animal models.

Therefore, in order to obtain more accurate experimental results for the development of the cause and treatment of Parkinson's disease in animal models of many Parkinson's diseases reported so far, it is possible to confirm whether the animal model is properly induced by Parkinson's disease. The development of new technologies is required.

Therefore, the present inventors measured the amount of change in brain metabolites in an animal model of Parkinson's disease through a non-invasive method using magnetic resonance spectroscopy to determine whether the Parkinson's disease animal model is an animal with Parkinson's disease. The present invention has been completed by developing a technology that can be easily identified.

Accordingly, an object of the present invention is to provide a method for determining whether Parkinson's disease is developed in a Parkinson's disease animal model by measuring changes in brain metabolites in a Parkinson's disease animal model through a non-invasive method using magnetic resonance spectroscopy.

Another object of the present invention is to provide a method for diagnosing Parkinson's disease and a system for diagnosing Parkinson's disease by measuring the change in the level of glutamate complex (glutamate, glutamine and GABA) / creatine, which are brain metabolites, using magnetic resonance spectroscopy. To provide.

Another object of the present invention is to provide a method for screening a therapeutic agent for Parkinson's disease by measuring the change in the level of glutamate complex (glutamate, glutamine and GABA) / creatine, which is a brain metabolite using magnetic resonance spectroscopy. .

In order to achieve the object of the present invention as described above, the present invention comprises the steps of: (a) magnetic resonance spectroscopic measurement of the brain tissue region of the Parkinson's disease animal model and normal animal model in the magnetic resonance imaging device to obtain a spectrum; And (b) comparing the spectrum of the Parkinson's disease animal model obtained in step (a) with the spectrum of a normal animal model, and the level of glutamate complex (glutamate, glutamine and GABA complex) / creatine, a brain metabolite in the brain tissue. It provides a method for determining whether Parkinson's disease development of the Parkinson's disease animal model comprising the step of measuring and comparing.

In one embodiment of the present invention, the Parkinson's disease animal model is injected Parkinson's disease by injecting 20 ~ 30mg / kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneally Can be induced.

In one embodiment of the present invention, the magnetic resonance spectroscopy measurement may be performed by using a magnetic resonance imaging apparatus having a magnetic field strength of 9T or more.

In one embodiment of the present invention, when the ratio of glutamate complex (complex of glutamate, glutamine and GABA) / creatine is from normal animal model to Parkinson's disease animal model 1: 1.2 ~ 1: 2.0, the Parkinson's disease animal It can be determined that Parkinson's disease is developed in the model.

In one embodiment of the present invention, the brain tissue may be a striatum.

In addition, the present invention comprises the steps of (a) measuring the brain tissue of an individual judged or suspected of Parkinson's disease by magnetic resonance spectroscopy to obtain a spectrum; And (b) measuring and analyzing the level of glutamate complex (complex of glutamate, glutamine and GABA) / creatine in the spectrum obtained in step (a).

In one embodiment of the present invention, the analysis of step (b) is performed to determine the level of glutamate complex (complex of glutamate, glutamine and GABA) / creatine for brain subject of the subject of step (a). Glutamate complexes (glutamate, glutamine and GABA complexes) obtained by magnetic resonance spectroscopy / database of creatine levels, and glutamate complexes (glutamate, glutamine and GABA) obtained by magnetic resonance spectroscopy Complex) / creatinine levels to determine if Parkinson's disease has occurred.

In one embodiment of the present invention, it is determined that Parkinson's disease is caused when the glutamate complex / creatine level of the subject of step (a) is determined to be higher than the glutamate complex / creatine level of the normal subject. And if the glutamate complex / creatine level for the subject of step (a) is equal to or less than the glutamate complex / creatine level of the normal subject or less than that of the Parkinson's disease subject, Parkinson's disease is not induced. It can be judged that.

In one embodiment of the present invention, the glutamate complex / creatine level of the Parkinson's disease individual may be measured at a high rate of 1.2 to 2 times higher than the glutamate complex / creatine level of the normal individual.

In one embodiment of the present invention, the brain tissue may be a striatum.

In addition, the present invention comprises the steps of (a) measuring the brain tissue of the test subjects except for humans determined as Parkinson's disease by magnetic resonance spectroscopy to obtain a first spectrum; (b) treating Parkinson's disease therapeutic candidates to test subjects other than humans determined to be Parkinson's disease; (c) measuring the brain tissue of the test subject treated with the candidate Parkinson's disease therapeutic agent of step (b) by magnetic resonance spectroscopy to obtain a secondary spectrum; And (d) calculating and comparing the levels of glutamate complexes (complexes of glutamate, glutamine and GABA) / creatine in the primary and secondary spectra, to screen for Parkinson's disease therapeutics. .

In one embodiment of the present invention, the treatment of the candidate material in step (b) may be performed by oral administration or parenteral administration of the candidate material to the test subject.

In one embodiment of the present invention, the second spectrum of glutamate salt complex (glutamate, glutamine and GABA complex) / creatine level in the first spectrum glutamate complex (glutamate, glutamine and GABA complex) When compared to the level of / creatine, the candidate may be determined to be effective in treating Parkinson's disease.

Further, the present invention (a) means for obtaining a magnetic resonance signal from the brain tissue of the test subject using a magnetic field; (b) means for calculating a magnetic resonance signal obtained in step (a) as a biological function; (c) means for spectral imaging of the function calculated in step (b); (d) means for calculating glutamate complexes (complexes of glutamate, glutamine and GABA) / creatine levels in the spectrum of step (c); And (e) the glutamate complex (glutamate, glutamine and GABA complex) / creatine value of the test subject calculated in step (d) above with the glutamate complex (glutamate, glutamine and GABA complex) / creatine value of the normal subject. Provided is a non-invasive method for diagnosing Parkinson's disease, including means for quantitative comparison.

In the method for diagnosing Parkinson's disease using magnetic resonance spectroscopy according to the present invention, the amount of glutamate complex (glutamate, glutamine and GABA complex) containing GABA present in a very small amount in a mammal can be analyzed by non-invasive method. In addition, it is more accurate than the existing method for diagnosing Parkinson's disease, and has a quick and easy effect. Therefore, it is useful for research and early diagnosis for Parkinson's disease.

The present invention provides a method for confirming the development of Parkinson's disease in the animal model by measuring the biochemical change, ie, the brain metabolite changes in the brain tissue of the Parkinson's disease animal model using magnetic resonance spectroscopy. In more detail, (a) magnetic resonance spectroscopy measurements of brain tissue regions of Parkinson's disease animal models and normal animal models in magnetic resonance imaging apparatus to obtain spectra; And (b) comparing the spectrum of the Parkinson's disease animal model obtained in step (a) with the spectrum of a normal animal model, and the level of glutamate complex (glutamate, glutamine and GABA complex) / creatine, a brain metabolite in the brain tissue. It is characterized by providing a method for identifying the onset of Parkinson's disease in the animal model, including the step of measuring and comparing the.

In general, magnetic resonance spectroscopy is a technique for analyzing metabolites in the human body and has been used for the analysis of the composition of a substance and for the analysis of molecular structure.

In addition, magnetic resonance spectroscopy is a method of precisely observing the change in the magnetic resonance signal (frequency) with respect to the applied RF pulse when a test object is placed in the magnetic field, and quantitatively analyzing the structure, components, and states of the object. to be. Therefore, biochemical information according to the mechanism of metabolites in a given sample can be obtained in a method that is harmless to the subject, and this information is determined according to the type of molecular composition in the sample.

The magnetic resonance spectroscopy is known as an excellent technique among the analytical methods used in chemistry and biochemistry. Since 1990, it has been actively used in the clinical field. It is used in the field of brain neuroscience.

In addition, magnetic resonance spectroscopy is to use an infinite number of protons present in the human body each having a unique magnetic resonance frequency, analytical and chemically distinguish each element component and determine the molecular structure. Magnetic resonance spectroscopy data is expressed as a spectrum representing various signal intensities using a reference compound that provides the frequency of the reference point, and each peak position is expressed as a function of frequency (Hz). The protons that make up the different tissues have the same protons, but because of the surrounding environment, there is a slight difference in chemical and magnetic properties, that is, a local magnetic field. Because of this, the resonance frequency also shows a slight difference so that it can be easily distinguished by spectrum. This fine resonance frequency difference is called chemical shift and peaks at 4.7 ppm, for example for moisture.

Therefore, the present inventors measured whether or not Parkinson's disease was induced by measuring metabolites of brain tissue in an animal model of Parkinson's disease using such magnetic resonance spectroscopy.

Magnetic resonance spectroscopy according to the present invention may be carried out using magnetic resonance imaging equipment having a magnetic field strength of about 1.5T currently used in the clinic, preferably magnetic resonance imaging equipment having a magnetic field strength of 3T or more, more preferably Is effective using branch resonance imaging equipment with magnetic field strength of 9T or more. The use of high magnetic field equipment can increase the spin net magnetization compared with the use of the low magnetic field equipment, thereby shortening the shooting time and easily acquiring a small region of interest for acquiring a local region of the magnetic resonance image. In the case of magnetic resonance spectroscopy, the spectrum has an advantage that the resolution of the spectrum is improved because the peak of the component material is enlarged according to the magnetic field strength.

Method for confirming the development of Parkinson's disease in the animal model using the magnetic resonance spectroscopy according to the present invention, (a) magnetic resonance spectroscopy of the brain tissue region of the Parkinson's disease animal model and normal animal model in the magnetic resonance imaging equipment Obtaining; And (b) comparing the spectra of the Parkinson's disease animal model obtained in step (a) with the spectrum of a normal animal model, and the levels of glutamate complex (glutamate, glutamine and GABA complex) / creatine, which are brain metabolites in the brain tissue. Measuring the change.

Herein, the Parkinson's disease animal model can be used for all Parkinson's disease laboratory animals (except humans) that are manufactured and sold in the market to study Parkinson's disease, and in one embodiment of the present invention, the neurons are damaged to cause Parkinson's disease. Parkinson's disease-induced mice were used by injecting mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), known as neurotoxins.

In addition, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) may be injected into the mouse through a parenteral administration method, preferably 20 ~ 30mg / kg of MPTP can be injected into the abdominal cavity of mice to cause Parkinson's disease.

In addition, the brain tissue of the normal animal and the Parkinson's disease-induced animal brains, the non-invasive method of the brain portion of the animal model, preferably the striatum region through the magnetic resonance spectroscopy method for the brain tissue, that is, the spectrum of the striatum I can learn it.

On the other hand, the present inventors have measured whether the Parkinson's disease animal model really caused Parkinson's disease by measuring the amount of change in brain metabolites by magnetic resonance spectroscopy measurement of brain tissue in the subjects of normal animals and Parkinson's disease animal model through the above method In this case, as the brain metabolite, the complexes of glutamate, glutamine and GABA, creatine and N-acetyl aspartic acid were measured.

In general, in the case of brain disease, a method of measuring metabolic changes in specific areas of the brain is used to study pathophysiological mechanisms.In the case of metabolic diseases, when a disease occurs, a change in a specific metabolite is known. By measuring the amount of metabolite change, the disease is diagnosed or predicted.

In metabolites associated with metabolic diseases, particularly known brain metabolites, N-acetylaspaetate (NAA), creatine (Cr), choline (Cho), glutamate and glutamine (glutamine), lactate (liptate), and lipid (lipid), among which NAA is not present in the supporting cells and is distributed evenly throughout the brain where the nerve cells are located, indicating damage or dysfunction of nerve cells in the living body. In particular, the ratio of NAA / Cr based on creatine is used as an index indicating neuronal integrity. In addition, choline (Cho) is a biosynthetic precursor of cell membrane lipids released during the degradation of myelin, and synthesized with acetyl CoA to produce acetylcholine, a neurotransmitter, and cell membrane It is known to increase in cell membrane destruction, tumor and gliosis in relation to the synthesis of. Creatine (Cr) is a stable metabolite that exists uniformly throughout the brain and is used as a reference in measuring changes in other metabolites.

In addition, gammaaminobutyrate (GABA), a specific amino acid that exists only in the mammalian brain and is known as an inhibitory neurotransmitter in the central nervous system of higher animals, is a glutamate decarboxylase (glutamate) present at the nerve endings of the brain. It is synthesized from glutamic acid by decarboxylase, and is used in about one third of the whole brain cell's connection part. It counteracts excitatory toxins, reducing the production of free radicals by toxins and protecting brain cells from free radicals. Function In particular, in brain disease patients such as dementia patients, the excretion of excitatory neurotransmitters such as glutamic acid is high while the concentration of inhibitory neurotransmitters such as GABA is low, thereby accelerating brain cell death.

Lipids in the brain are about 86% more likely to be present in the brain when tuberculosis is induced, but 20 to 21% are known in nonspecific inflammatory granulomas or cystic dystrophy. It is used to distinguish between tuberculoma and other nonspecific inflammatory granulomas or cerebral cystitis. In addition, lactate is rarely found in normal areas and is produced as a result of anaerobic glycolysis, which is caused by poorly supplied oxygen, indicating that lactate content is increased, indicating a lack of tissue oxygen deficiency and ischemia. Reflect.

As such, measuring the amount of change in brain metabolites can be said to be one of the important ways to determine whether there is a brain disorder.

On the other hand, in the diagnosis of Parkinson's disease, the above-mentioned measurement of the amount of change in brain metabolites is used as an important method, but still has a relatively low reliability in the accuracy. Therefore, in order to more accurately diagnose the onset of Parkinson's disease, a method of measuring the amount of change in more types of brain metabolites is needed. On the other hand, GABA is present in a very small amount in mammals compared to other types of brain metabolites and is a modified metabolite of glutamate, which is difficult to identify in glutamine complexes by spectroscopy by chemical transfer. There was a difficulty in using it as a data for research.

Therefore, the present inventors were able to confirm whether Parkinson's disease was developed in an experimental animal model by measuring changes in brain metabolites including GABA using magnetic resonance spectroscopy, and the glutamate complexes, namely glutamate and Levels of the complex of glutamine and GABA were measured in normal animal models and Parkinson's disease animal models, respectively, based on creatine levels, and then compared to confirm the development of Parkinson's disease in animal models of Parkinson's disease.

According to an embodiment of the present invention, the glutamate complex (glutamate), which is a brain metabolite present in the brain tissue, is measured by magnetic resonance spectroscopy in a normal mouse group and an experimental group of mice induced with Parkinson's disease with MPTP. N-acetyl aspartic acid / creatine and choline / creatine, together with the level of the complex (glutamin and GABA) / creatine, respectively, and the results of N-acetyl aspartic acid / creatine, The choline / creatine levels were measured to be very similar as they showed a difference of about 0.01, whereas the glutamate complex (complex of glutamate, glutamine and GABA) / creatine was normal. The experimental group measured 4.90 ± 1.19 and the experimental group measured 6.24 ± 1.22. Was born indicate that there is a difference in degree, the experimental group showed about 1.3 times higher than the normal control group (see Table 1 and Fig. 2). Therefore, in the present invention, the glutamate complex (complex of glutamate, glutamine and GABA) / creatinine is a Parkinson's disease animal model when the ratio of normal animal model to Parkinson's disease animal model is 1: 1.2 to 1: 2.0. It can be judged that the disease is developed, the higher the glutamate complex / creatine level in the Parkinson's disease animal model, the more profound the symptoms of Parkinson's disease. It can be judged to be insignificant.

Therefore, as described above, the Parkinson's disease animal model, which is conventionally sold for Parkinson's disease research, has a problem that Parkinson's disease is not properly induced so that accurate results are not obtained. As a result of observing the difference between the normal animal model and the behavior is used in the experiment, this method not only takes a lot of time, but also has an objective and inaccurate problem to objectively determine the development of Parkinson's disease.

However, the method according to the present invention obtains the spectrum by measuring the magnetic resonance spectroscopy in the laboratory animals for the study of Parkinson's disease and by measuring the level of glutamate complex (complex of glutamate, glutamine and GABA) / creatine, It can provide a simple and accurate way to identify whether Parkinson's disease is the animal.

In addition, the present invention comprises the steps of (a) measuring the brain tissue of an individual judged or suspected of Parkinson's disease by magnetic resonance spectroscopy to obtain a spectrum; And (b) measuring and analyzing the level of glutamate complex (complex of glutamate, glutamine, and GABA) / creatine in the spectrum obtained in step (a), to provide a non-invasive method for diagnosing Parkinson's disease. have.

In addition, the analysis of step (b) is obtained by measuring the brain tissue of Parkinson's disease subjects by magnetic resonance spectroscopy of glutamate complex (complex of glutamate, glutamine and GABA) / creatine for the subject of step (a) Database for glutamate complexes (complexes of glutamate, glutamine and GABA) / creatinine and glutamate complexes (complexes of glutamate, glutamine and GABA) / creatine levels obtained by magnetic resonance spectroscopy of normal tissue brain tissue Determining whether Parkinson's disease has occurred compared to the database, in particular, the glutamate complex / creatine level for the subject of step (a) is higher than the glutamate complex / creatin level of the normal subject If measured, it is determined that Parkinson's disease is caused, and the dog of step (a) If the glutamate complex / creatine level for the sieve is determined to be equal to or lower than the glutamate complex / creatine level of the normal individual, it can be determined that Parkinson's disease is not induced.

In addition, in the present invention, glutamate complex / creatine level of the Parkinson's disease individual is measured 1.2 to 2 times higher than that of normal glutamate complex / creatine level, so glutamate of the individual suspected or suspected of Parkinson's disease Parkinson's disease can be determined to occur when the complex / creatinine level is 1.2 to 2 times higher than that of the normal individual glutamate complex / creatinine level.

Further, the present invention, (a) measuring the brain tissue of the test subjects except for humans determined as Parkinson's disease by magnetic resonance spectroscopy to obtain a first spectrum; (b) treating Parkinson's disease therapeutic candidates to test subjects other than humans determined to be Parkinson's disease; (c) measuring the brain tissue of the test subject treated with the candidate Parkinson's disease therapeutic agent of step (b) by magnetic resonance spectroscopy to obtain a secondary spectrum; And (d) calculating and comparing the levels of glutamate complex (complex of glutamate, glutamine and GABA) / creatine in the primary and secondary spectra to provide a method for screening a therapeutic agent for Parkinson's disease. Can be.

As described above, Parkinson's disease can be diagnosed as having Parkinson's disease when the level of glutamate complex / creatine is higher than that of the normal group by magnetic resonance spectroscopy.

Thus, if a candidate has an activity as a Parkinson's disease therapeutic agent, the administration of Parkinson's disease-induced subjects will result in reduced levels of glutamate complex / creatine measured prior to administration of the candidate.

Therefore, in the analysis of step (d), the ratio of the glutamate complex (glutamate, glutamine and GABA) / creatine level in the secondary spectrum is equal to the glutamate complex (complex of glutamate, glutamine and GABA) / creatine in the primary spectrum. If lower than the ratio, the candidate may be determined to have therapeutic efficacy for Parkinson's disease.

In addition, the method of treating the candidate substance of the Parkinson's disease therapeutic agent in step (b) may be treated by the parent oral administration such as oral administration or subcutaneous, subcutaneous, intravenous and intramuscular injection to the test subject.

Furthermore, the present invention can provide a system capable of diagnosing Parkinson's disease in a non-invasive way by measuring the amount of metabolic changes in brain tissue using magnetic resonance spectroscopy, more specifically, the system, (a) a magnetic field Means for obtaining a magnetic resonance signal from the brain tissue of the test subject using; (b) means for calculating a magnetic resonance signal obtained in step (a) as a biological function; (c) means for spectral imaging of the function calculated in step (b); (d) means for calculating glutamate complexes (complexes of glutamate, glutamine and GABA) / creatinine in the spectrum of step (c); And (e) the glutamate complex (glutamate, glutamine and GABA complex) / creatine value of the test subject calculated in step (d) above with the glutamate complex (glutamate, glutamine and GABA complex) / creatine value of the normal subject. Means for quantitative comparisons.

In order to acquire magnetic resonance signals in magnetic resonance spectroscopy using magnetic resonance imaging equipment, first, an experimental object to be photographed is moved to a space inside a magnet system, that is, a position where a static magnetic field is generated. A gradient magnetic field and a high frequency magnetic field are then applied to generate a magnetic resonance signal from the spin in the object. Thereafter, magnetic resonance signals can be obtained, and each signal can be calculated by a biological function, that is, a frequency function, and then imaged as a spectrum. The spectrum is subjected to the same process as that of the method for a normal object. Compared with the spectrum obtained through the diagnosis of the presence of Parkinson's disease can be diagnosed.

At this time, the diagnosis can be determined by measuring and comparing the glutamate complex (complex of glutamate, glutamine and GABA) / creatine levels in the spectra obtained from the test subjects and the normal subjects. If it is higher than the level, Parkinson's disease can be diagnosed. If the test subject's level is less than or equal to that of normal, Parkinson's disease can be diagnosed as not. In addition, the test subject may include all animals including humans.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Determination of Parkinson's Disease-Related Biochemical Changes in Brain Tissue in Parkinson's Disease Animal Model

In the present invention, the change of biochemicals generated in the brain tissue of the animal model was measured in the Parkinson's disease animal model using magnetic resonance spectroscopy to verify whether the animal model is a Parkinson's disease animal model. To this end, first, Parkinson's animal model was produced. Parkinson's animal model was used to purchase C57BL / 6 mice, which are widely used for experiments, and 1-methyl-4-phenyl-1, Parkinson's disease was induced in the mice by injecting 25 mg / kg of 2,3,6-tetrahydropyridine (MPTP) for 5 days. In this case, the control group was injected with saline (0.1ml) in the abdominal cavity of C57BL / 6 mice for 5 days, and the process of preparing Parkinson's disease mouse through intraperitoneal injection is shown in the photograph of FIG. 1. Afterwards, the magnetic resonance spectroscopy method was performed to compare changes or levels of brain metabolites, ie, glutamate complex (complex of glutamate + glutamine + GABA) / creatine (Cr), with those of the control group. In other words, ¹ H magnetic resonance spectroscopy was performed, and the magnetic resonance apparatus was used with a Bruker 9.4 T / 20 cm magnet BioSpec 94/20 instrument (bruker biospin, Germany). The implementation conditions were 5000 ms of repetition time, 2.2 ms of echo time, 20 ms of mixing time, 2048 of number of excitation, and the size of the voxel was 2.5x1. The voxel was located in the striatum, a typical site associated with the development of Parkinson's disease. At this time, all conditions including the voxel positions of the control group and the experimental group were performed for the same experimental conditions. In addition, single voxel spectroscopy (SVS) using a single voxel was performed to compare the same sites, and the quantitative comparison of measured metabolites was carried out using Java-based magnetic resonance user interface (jMRUI, version: 3.0, The analysis was performed using the EU project TMR, FMRX-CT97-0160, and statistical analysis was performed using the SPSS software package (SPSS version 12.01, Chicago, USA). Numerical results of the analyzed brain metabolites are shown in Table 1 and FIG. 2.

TABLE 1

Content of Brain Metabolites as Measured by Magnetic Resonance Spectroscopy

Average value ± ST Average value ± ST F value V value P value Control mouse Parkinson's disease mouse Glx / Cr 4.90 ± 1.19 6.24 ± 1.22 5.5415 -2.614 0.001 NAA / Cr 1.35 ± 0.49 1.17 ± 0.27 24.218 -2.797 0.024 Cho / Cr 1.01 ± 0.29 1.02 ± 0.25 16.467 -0.035 0.121

In the table, Glx is a complex of glutamate + glutamine + GABA, NAA is N-acetylaspartate, Cho is choline, and Cr is creatine.

As shown in Table 1, in the saline-injected control group and MPTP-injected Parkinson's disease mice, the ratios of NAA / Cr and Cho / Cr among brain metabolites showed similar levels. The ratio of Glx / Cr, a complex of glutamate, glutamine and GABA, was significantly higher in Parkinson's disease mice (6.24 ± 1.22) compared to the control (4.90 ± 1.19). In addition, as shown in FIG. 2, it was found that a peak of Glx / Cr, which is hardly identified in a Parkinson's disease-induced mouse, is generally detected.

Therefore, the present inventors can know that the Parkinson's disease-induced mice can be easily measured by comparing the spectrum of brain metabolite changes in mice obtained by magnetic resonance spectroscopy. there was.

<Predictive Example 1>

Parkinson's disease animal model Detecting the degree of Parkinson's disease in brain tissue

Using the magnetic resonance spectroscopy according to the present invention can determine the degree of Parkinson's disease pathogenesis in the brain tissue of Parkinson's disease animal model. That is, the experimental animals are the same as in Example 1, and the induction of Parkinson's disease causes Parkinson's disease in the mouse by injecting MPTP as in Example 1. In order to observe changes in brain metabolism in Parkinson's disease animal models and control models (eg, normal models), magnetic resonance spectroscopy was performed on normal mice before Parkinson's disease was induced, Magnetic resonance spectroscopy is performed to obtain spectra for brain metabolites. At this time, the conditions for carrying out the magnetic resonance spectroscopy are performed in the same manner as described in Example 1.

Then, comparing the spectra of the brain metabolites of normal mice and Parkinson's disease-induced mice obtained by the above method, the glutamate complex (Glx) / creatine (Cr) ratio in normal mice with Parkinson's disease-induced mice It will be higher, and the increased degree will be proportional to the degree of Parkinson's disease. Therefore, the degree of Parkinson's disease can be determined accordingly. For example, when the level of glutamate complex (Glx) / creatin (Cr) detected in Parkinson's disease mice compared to normal mice is 1.2 to 2.0 times or more, Parkinson's disease may be induced.

<Predictive Example 2>

Clinical Trials of Parkinson's Disease Therapeutics Using Magnetic Resonance Spectroscopy

Magnetic resonance spectroscopy according to the present invention can determine the effect of Parkinson's disease therapeutic agent in Parkinson's disease animal model. The experimental animals were subjected to the same conditions as in Example 1, and the induction of Parkinson's disease was also induced in Parkinson's disease in mice using MPTP as in Example 1. Thereafter, the change of the brain metabolites in the Parkinson's disease animal model is compared with the case of the normal mouse group through magnetic resonance spectroscopy to determine whether Parkinson's disease is induced. Thereafter, a potential Parkinson's disease treatment is administered to the mice induced with Parkinson's disease, followed by magnetic resonance spectroscopy. As a result, if potential Parkinson's disease treatments are effective, the glutamic acid complex / creatine ratio will be lower in animals treated with the potential Parkinson's disease compared to mice receiving Parkinson's disease, and the level of the lowered level of Parkinson's disease treatment It will be proportional to the effect, so you can see how effective the Parkinson's disease drug is.

1 is a photograph showing a process of injecting the neurotoxin MPTP intraperitoneally of the mouse to induce Parkinson's disease.

Figure 2 shows the spectrum of the brain tissue, that is, striatum, measured by magnetic resonance spectroscopy in the normal group and Parkinson's disease-induced experimental group, the amount of the brain metabolite glutamate complex, creatine and NAA .

Figure 3 is a graph showing the levels of the brain metabolite glutamate complex / creatine in the striatum of the normal group and Parkinson's disease-induced experimental group, the thick line in the square box shows the median of each group.

Claims (14)

(a) An animal model of Parkinson's disease that induced Parkinson's disease by injecting 20-30 mg / kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into the abdominal cavity in a magnetic resonance imaging apparatus; Obtaining spectra by measuring magnetic resonance spectra of brain tissues of the normal animal model; And (b) comparing the spectrum of the Parkinson's disease animal model obtained in step (a) with the spectrum of the normal animal model to determine the levels of glutamate complexes (glutamate, glutamine and GABA complexes) / creatine, which are brain metabolites, in the brain tissue. A method of confirming whether Parkinson's disease develops in a Parkinson's animal model, comprising the step of measuring and comparing. delete The method of claim 1, The magnetic resonance spectroscopy is a method of identifying whether Parkinson's disease occurs in a Parkinson's animal model, characterized in that by using a magnetic resonance imaging device having a magnetic field strength of 9T or more. The method of claim 1, If the ratio of glutamate complex (complex of glutamate, glutamine and GABA) / creatinine is 1: 1.2 to 1: 2.0 in normal animal model and Parkinson's disease animal model, it is determined that Parkinson's disease is developed in the Parkinson's disease animal model. Parkinson's animal model characterized in that the development of Parkinson's disease. The method of claim 1, The brain tissue is a striatum (striatum), Parkinson's animal model characterized in that the development of Parkinson's disease. (a) measuring the brain tissues of the subjects except humans judged or suspected of Parkinson's disease by magnetic resonance spectroscopy to obtain a spectrum; And (b) measuring and analyzing the level of glutamate complex (complex of glutamate, glutamine and GABA) / creatine in the spectrum obtained in step (a); In the analysis of step (b), the levels of glutamate complex (complex of glutamate, glutamine and GABA) / creatine for the individual of step (a) were measured by magnetic resonance spectroscopy of brain tissues of Parkinson's disease individuals except humans. Database of obtained glutamate complexes (complexes of glutamate, glutamine and GABA) / creatinine and glutamate complexes (complexes of glutamate, glutamine and GABA) / creatine levels obtained by magnetic resonance spectroscopy Determining whether Parkinson's disease has occurred compared to the database for the subject, wherein the glutamate complex / creatine level for the subject of step (a) is determined to be higher than the glutamate complex / creatine level of the normal subject. If it is determined that Parkinson's disease is caused, the subject of step (a) If the glutamate complex / creatine level for the same as the normal or glutamate complex / creatine level of the normal individual or less than the level of Parkinson's disease, except the human, it is determined that Parkinson's disease is not caused , Non-invasive method for diagnosing Parkinson's disease. delete delete The method of claim 6, Non-invasive Parkinson's disease diagnostic method, characterized in that the glutamate complex / creatine level of the individual except Parkinson's disease is measured at a ratio of 1.2 to 2.0 times higher than the normal glutamate complex / creatine level. The method of claim 6, Non-invasive Parkinson's disease diagnosis method characterized in that the brain tissue is striatum. (a) measuring brain tissues of experimental subjects except humans determined to be Parkinson's disease by magnetic resonance spectroscopy to obtain a first spectrum; (b) treating Parkinson's disease therapeutic candidates to test subjects other than humans determined to be Parkinson's disease; (c) measuring the brain tissue of the test subject treated with the candidate Parkinson's disease therapeutic agent of step (b) by magnetic resonance spectroscopy to obtain a secondary spectrum; And (d) calculating and comparing the levels of glutamate complex (complex of glutamate, glutamine and GABA) / creatine in the primary and secondary spectra, the method of screening a Parkinson's disease therapeutic agent. The method of claim 11, Candidate treatment of step (b) is a method for screening a Parkinson's disease therapeutic agent, characterized in that the treatment of the candidate substance to the test subject by oral or parenteral administration. The method of claim 11, In step (d), the second spectrum of glutamate complex (complex of glutamate, glutamine and GABA) / creatine level is higher than that of glutamate complex (complex of glutamate, glutamine and GABA) / creatine of primary spectrum When reduced, the candidate is determined to have a therapeutic effect on Parkinson's disease. (a) means for obtaining magnetic resonance signals from brain tissues of experimental subjects except humans using magnetic fields; (b) means for calculating a magnetic resonance signal obtained in step (a) as a biological function; (c) means for spectral imaging of the function calculated in step (b); (d) means for calculating glutamate complexes (complexes of glutamate, glutamine and GABA) / creatine levels in the spectrum of step (c); And (e) Glutamate complexes (complexes of glutamate, glutamine and GABA) / creatine levels of the test subjects other than humans calculated in step (d) above; A Parkinson's disease diagnostic system in a non-invasive method comprising means for quantitatively comparing numerical values.

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