WO2011108534A1 - Method for diagnosis of amyotrophic lateral sclerosis (als) by analysis of prostaglandin d2 and metabolite thereof and creatinine, method for evaluation of effectiveness of medicinal agent in therapy, and system for estimation of tpgdm level in urine - Google Patents

Abstract

Disclosed are: a method for the diagnosis of amyotrophic lateral sclerosis (alleviated as "ALS", hereinafter) in a subject, including the determination of the degree of progression of symptoms and the monitoring of the progression in the patient; and a method for evaluating the effectiveness of a medicinal agent or the like in the treatment of ALS or the like. Specifically disclosed is a method for the diagnosis of ALS in a subject, including the determination of the degree of progression of symptoms and the monitoring of the progression in the patient, which comprises: (a) a step of collecting a sample from the subject; and (b) a step of analyzing prostaglandin D2 and a metabolite thereof in the sample by mass spectrometry or the like and a step of analyzing creatinine in the sample by mass spectrometry or the like. In some embodiments of the process, these substances contained in a sample such as urine and blood collected from an ALS patient or a subject who is suspected to be suffering from ALS are analyzed, whereby the diagnosis including the determination of the degree of progression of symptoms in the patient can be achieved and the progression of the disease can be monitored. Further, the effectiveness of a medicinal agent or the like or a therapy or the like can be evaluated. The urine component may be a substance capable of being measured, such as proteins, metabolites and hormones contained in urine.

Description

A method for diagnosing amyotrophic lateral sclerosis (ALS) by analyzing prostaglandin D2 and its metabolites and creatinine, a method for evaluating the effectiveness of drugs in treatment, and a system for estimating the concentration of tPGDM in urine

The present invention relates to the effectiveness of drugs in diagnostic methods and treatments, including determination of progress and monitoring of progression of amyotrophic lateral sclerosis (ALS) by analysis of prostaglandin D2, its metabolites and creatinine. The present invention relates to a method for evaluating and a system for estimating the concentration of tPGDM in urine.

Amyotrophic lateral sclerosis (hereinafter abbreviated as “ALS”), also known as Lou Gehrig's disease or motor neuron disease (MND), is one of several neurodegenerative diseases of the central nervous system. ALS affects 1 in 60,000 people, with an average onset age of 50 to 55 years, the most common adult-onset motor neuron disease, but with muscle atrophy and resembles muscular dystrophy It also has an aspect of muscular disease. Muscular dystrophy is defined as a hereditary disease that is primarily caused by skeletal muscle degeneration / necrosis and clinically shows progressive muscle weakness (MAAlderfer et al. J Pediatr Psychol. 33: 1046-1061 (2008), Z Wang, et al. ILAR J.50: 187-198 (2009)). However, ALS is characterized by rapid progressive degeneration of motor neurons in the brain, brainstem, and spinal cord (Cleveland et al., Nat. Rev. Neurosci., 2, 806-19 (2001)). Thus, it can be said that the pathology of ALS is characterized by a variety of patient symptoms such as having both sides of nerve and muscle, and also in the case of bulbar paralysis with articulation disorder. For this intractable disease that has not been well established as a diagnostic method, including progress determination and progress monitoring, the median survival time of the patient from the time of diagnosis is 5 years.

ALS has both sporadic with unknown genetic background and familial forms that have been elucidated. Familial ALS (FALS) is only 5-10% of all ALS cases. During the past decade, many basic and clinical studies have focused on elucidating the familial form of the disease, leading to the identification of eight genetic variants associated with FALS. Transgenic mice expressing a point mutant of the Cu / Zn superoxide dismutase-1 (SOD1) gene develop age-dependent progressive motor paralysis similar to human ALS by acquiring toxic function (Rosen 機能 et al. , Nature, 362: 59-62 (1993), Rosen et al. Hum Mol Genet, 3: 981-987 (1994) and Borchelt et al., Proc. Natl. Acad. Sci. USA, 91: 8292-8296 ( 1994)).

However, these gene mutations cannot explain sporadic ALS (SALS). The pathogenesis of SALS is multifactorial. On the other hand, many model systems have been utilized to elucidate the pathogenesis of ALS, including in vitro motor neuron primary cultures or spinal cord slice cultures, in vivo imaging studies and postmortem examination of tissue samples by SOD1 transgenic mice and rats described above (Subramaniam et al., Nat. Neurosci., 5: 301-307 (2002), Nagai et al., J. Neurosci., 21: 9246-9254 (2001), Menzies et al. Brain, 125: 1522-1533 (2002), Kim et al. J. Neuropathol. Exp. Neurol., 62: 88-103 (2003), and Ranganathan et al., Am. J. Pathol., 162: 823-835 (2003)). Although these studies have provided therapeutic targets and a number of clinical trials, there are no effective drugs that delay onset or prolong survival. Riluzole (Rilutek®, Aventis), a glutamate antagonist, is currently the only FDA (US Food Drug Administration) license available to treat ALS and insured in Japan Is a drug. Riluzole, however, can only extend life expectancy by a few months (Miller et al., Amyotrophic Lateral Sclerosis & Other Motor Neuron Disorders, 4: 191-206 (2003)). Creatine and α-tocophenol have a certain effect in alleviating the symptoms of ALS in SOD1 transgenic mice, but only a minor effect in human ALS patients (Groeneveld et al., Annals of Neurology, 53: 437-45 (2003), and Desnuelle et al., Amyotrophic Lateral Sclerosis & Other Motor Neuron Disorders, 2: 9-18 (2001)).

Furthermore, no bioactive substance has been identified as a biomarker for ALS.

Therefore, there is no specific diagnostic method including the determination of the degree of progression of ALS, and the final diagnosis is excluded from MRI, MRA, CT, cerebrovascular imaging, cerebrospinal fluid analysis, and the like. Diagnosis involves needle electromyography, which is also craftsmanship. Although creatine phosphokinase levels in blood tests are also noted as an indicator of muscle disorders, they do not necessarily correlate with onset or progression. The urinary creatinine level, which is increased due to kidney damage and widely used as an indicator thereof, has been reported to decrease in ALS, reflecting that this substance is synthesized in muscle (Narayanan et al., Clin. Chem., 26: 1119-1126 (1980), and Corbett et al., Neurology, 32: 550-552 (1982)).

However, it has not been established as a detailed analysis and diagnosis method. In recent years, a method has been developed for determining positive / negative by comparing protein analysis profiles of specimens at various parts of a patient using mass spectrometry (MS) by a proteomic technique. However, expensive equipment is required, and analysis does not target specific proteins, but rather determines complex peak differences in the entire MS spectrum (Pasinetti et.al. Neurology 66: 1218- 22 (2006)). The absence of such a positive diagnostic method delays the definitive diagnosis and, in combination with the age of onset, frequently causes extremely dangerous fall accidents, etc. due to doubts about the onset and its cause and behavior derived from life.

Cleveland et al., Nat. Rev. Neurosci., 2: 806-819 (2001) Rosen et al., Nature, 362: 59-62 (1993) Rosen et al. Hum Mol Genet, 3: 981-987 (1994) Borchelt et al., Proc. Natl. Acad. Sci. USA, 91: 8292-8296 (1994) Subramaniam et al., Nat. Neurosci., 5: 301-307 (2002) Nagai et al., J. Neurosci., 21: 9246-9254 (2001) Menzies et al. Brain, 125: 1522-1533 (2002) Kim et al. J. Neuropathol. Exp. Neurol., 62: 88-103 (2003) Ranganathan et al., Am. J. Pathol., 162: 823-835 (2003) Miller et al., Amyotrophic Lateral Sclerosis & Other Motor Neuron Disorders, 4: 191-206 (2003) Groeneveld et al., Annals of Neurology, 53: 437-445 (2003) Desnuelle et al., Amyotrophic Lateral Sclerosis & Other Motor Neuron Disorders, 2: 9-18 (2001) Pasinetti et.al. Neurology 66: 1218-1222 (2006)

There is an urgent need for improved methods for identifying therapeutic targets for ALS and improved methods for diagnosing the disease, including progress determination and progress monitoring.

An object of the present invention is to provide a method for evaluating the effect of drugs such as a diagnosis method and treatment including determination of the progress of ALS by analysis of prostaglandin D2, its metabolite and creatinine, and its monitoring.

Prostaglandin D2 promotes the activation of microglia and astrocytes, which are immunocompetent cells in the brain, and exacerbates the inflammatory response in chronic neurological diseases. In addition, the use of a prostaglandin D2 production inhibitor in Krabbe disease model mice reduces the symptoms (Mohri et al. Glia, lia42: 263-274 (2003), Mohri et al. J Neurosci 26: 4383-93). (2006) and Mohri et al. J Neuropathol Exp Neurol. 66: 469-80 (2007)).

In recent years, it is speculated that prostaglandin D2 is also produced in Duchenne muscular dystrophy. Increased prostaglandin D2 exacerbates myonecrosis in muscular dystrophy model mice. In addition, when a prostaglandin D2 production inhibitor is given to these mice, muscle necrosis is reduced (Taniike et al., Ministry of Health and Welfare, Mental and Neurological Research 8th-10th Research Report, Clinical Pathology and Treatment of Muscular Dystrophy and Related Diseases) Research on Page 30-32. (1999), Okinaga et al. Acta Neuropathol 104: 377-384. (2002) and Mohri I et.al.Am J.Path. 174: 1735-44 (2009)).

As a result of intensive studies to solve the above-mentioned problems, the present invention has been reached as follows.

The present invention provides a method for diagnosing ALS in a subject, including progress determination and progress monitoring. The method comprises (a) obtaining a sample from a subject, (b) analyzing the prostaglandin D2 and its metabolite concentration in the sample by mass spectrometry, etc., (c) analyzing the creatinine concentration, and (d) Comparing creatinine and prostaglandin D2 and its metabolite concentrations in the sample with positive or negative preparations, including progress and historical data.

The present invention also provides a method for evaluating the effectiveness of drugs in the treatment of ALS. The method comprises (a) obtaining a first sample from an ALS patient and a subject suspected of having amyotrophic lateral sclerosis (ALS), (b) prostaglandin D2 in the first sample by mass spectrometry or the like. And a step of analyzing the metabolite concentration, (c) a step of analyzing the creatinine concentration, (d) a step of administering a drug or the like to the subject, (e) a second sample from the subject after completion of step (d) (F) analyzing the prostaglandin D2 and its metabolite concentration in the second sample by mass spectrometry or the like, (g) analyzing creatinine, and (h) creatinine in the first sample Comparing the concentration and prostaglandin D2 and its metabolite concentration with the prostaglandin D2 and its metabolite concentration in the second sample.

The present invention is also a method for diagnosing amyotrophic lateral sclerosis in which the onset, progression, and therapeutic effect of amyotrophic lateral sclerosis are evaluated based on analysis of urine components of the patient. Here, the urine component refers to any measurable substance (protein, hormone, sugar, nucleic acid, lipid, and metabolite thereof) contained in urine. Furthermore, the present invention uses the relative relationship of substances contained in one or more measured urine as a basic concept of the diagnostic method.

According to the present invention, as a biomarker of ALS, a subject develops ALS by combining analysis of the amount or concentration of a prostaglandin D2 metabolite in urine of the subject and analysis of the amount or concentration of creatinine. It is possible to determine whether or not there is a high degree of accuracy, including the degree of progress and the progress. In addition, urinary prostaglandin D2 and metabolites of prostaglandin D2 can be used as biomarkers of ALS, and can be used, for example, in the development of treatment methods for ALS patients and the development of effective drugs for ALS patients. .

11,15-dioxo-9-hydroxy-2,3,4,5-tetraprostan-1,20-dioic acid (tetranor-PGDM, hereinafter abbreviated as “tPGDM”), a metabolite of prostaglandin D2, is liquid It is a figure shown by chromatograph mass spectrometry. It is a figure which shows the measurement result by liquid chromatography mass spectrometry of tPGDM which is a metabolite of the urinary prostaglandin D2 of the ALS patient S. It is a figure which shows the measurement result by the liquid chromatograph mass spectrometry of tPGDM which is a metabolite of prostaglandin D2 in a healthy person's urine. It is a figure which shows transition of the urinary tPGDM density | concentration in the day and night after 6 months of the ALS patient S shown to FIG. 1B. It is a figure which shows transition of the tPGDM density | concentration in urine of the ALS patient ("ALS" in a figure) including the patient S in the daytime and night. FIG. 3B is an enlarged view of a low concentration portion of the urine tPGDM concentration in an ALS patient during the day and night of FIG. 3A. It is a figure which shows transition of the tPGDM density | concentration in the healthy subject's urine in the daytime and night. FIG. 3C is an enlarged view of a low concentration portion of the urine tPGDM concentration in a healthy person day and night in FIG. 3C. It is the figure which compared the tPGDM average density | concentration in the urine of an ALS patient and a healthy person in 24 hours day and night shown in FIG.2, FIG.3A, FIG.3C. It is the figure which compared the tPGDM average density | concentration in the urine of an ALS patient and a healthy person in the daytime (from 6:00 am to 6:00 pm) shown in FIG.2, FIG.3A, FIG.3C. It is the figure which compared the urinary tPGDM average density | concentration of the ALS patient and healthy subject in the night (from 6:00 pm to 6:00 am) shown in FIG.2, FIG.3A, FIG.3C. It is the figure which compared the urinary tPGDM density | concentration in the day and night shown to FIG. 2, FIG. 3A, and FIG. 3C with the lowest value of each ALS patient, and the highest value of each healthy subject. It is the figure which showed the tPGDM density | concentration in the total urine collected about 24 hours (from morning to the next morning) of an ALS patient and a healthy subject. It is the figure which showed the total amount of tPGDM in the total urine collected about 24 hours (from morning to the next morning) of an ALS patient and a healthy person. Focusing on the patient's symptom walking function, he can walk at a degree of progression of 1, 2 for wheelchair life, 3 for bedridden life, 4 for ventilator wear, and the horizontal axis for the patient's urinary prosta It is the figure which showed the day / night average density | concentration of tPGDM which is a grandin D2 metabolite, and is the figure which showed the average of the average daytime night density | concentration of a healthy person with the horizontal broken line. For the ALS patient S shown in FIG. 1B, “Radicut” (manufactured by Mitsubishi Tanabe Seiyaku Co., Ltd., generic name “Edaravone”) is administered on the second day from 15 o'clock to 17 o'clock at 30 o'clock, including before and after It is the figure which showed transition of the tPGDM density | concentration in the total amount of urine of each time (all urinations are collected). It is the figure which showed the amount of tPGDM in the urine collection total amount shown to FIG. 7A. It is the figure which showed transition of the creatinine density | concentration over the day and night, Comprising: The urine sample is the figure which added the data over 2 days of the patient S to the data of five ALS patients using the same sample which performed the tPGDM analysis. It is the figure which showed transition of the creatinine density | concentration in urine of 5 healthy persons over the day and night. It is the figure which compared the urinary creatinine average density | concentration of the ALS patient of a day and night of FIG. 8A, FIG. 8B, and a healthy person. It is the figure which compared the urinary creatinine average density | concentration of the daytime ALS patient of FIG. 8A, FIG. 8B, and a healthy person. It is the figure which compared the urinary creatinine average density | concentration of the night ALS patient of FIG. 8A, FIG. 8B, and a healthy person. FIG. 9 is a comparison diagram between the maximum value of urinary creatinine concentration in ALS patients and the minimum value of healthy subjects in the day and night of FIGS. 8A and 8B. It is a figure which shows the creatinine density | concentration in the total urine collected about 24 hours (from morning to the next morning) of an ALS patient and a healthy person. It is a figure which shows the total amount of creatinine in the total urine collected about 24 hours (from morning to the next morning) of an ALS patient and a healthy person. It is the figure which investigated the relationship between the urinary creatinine day / night average density | concentration and the progress degree of a patient's symptom, Comprising: A broken line is a figure which shows the average of the healthy person's urine creatinine day / night average density | concentration. It is the figure which investigated the correlation of the tPGDM density | concentration and creatinine density | concentration in urine with the average density | concentration day and night about an ALS patient and a healthy subject. It is the figure which investigated the correlation of the tPGDM density | concentration in urine and the creatinine density | concentration with the density | concentration in the total urine collected for 24 hours about the ALS patient and the healthy subject. It is the figure which investigated the correlation of the amount of tPGDM in urine and the amount of creatinine by the total amount in the total urine collected for 24 hours about the ALS patient and the healthy subject. It is a figure which shows an example of the density | concentration transition model obtained by the system which estimates the highest density | concentration and the lowest density | concentration from the urine tPGDM density | concentration.

Amyotrophic lateral sclerosis (hereinafter abbreviated as “ALS”) is a disease in which muscle atrophy and muscle weakness in the whole body gradually progress to death. This disease is a kind of so-called intractable disease whose cause of development has not been fully elucidated and no treatment method has been established.

This embodiment provides a method for diagnosing ALS in a subject including progress determination and progress speed. The method includes (a) obtaining a sample from a subject, (b) analyzing the prostaglandin D2 and its metabolite concentration in the sample by mass spectrometry or the like, (c) analyzing the creatinine concentration, and (d ) Comparing the creatinine concentration in the sample and the prostaglandin D2 and its metabolite concentrations with a positive or negative sample, including the degree of progression and the person's past data.

As used herein, the term “sample” refers to a biomaterial isolated from a subject. The subject can be any suitable animal, but is preferably a mammal such as a mouse, rat, dog, monkey or human. It is contemplated that the methods of the foregoing invention can be used to diagnose ALS in animal models of disease when the subject is a non-human animal (eg, mouse, rat, monkey, dog, etc.).

The sample can be obtained by any suitable method known in the art, such as by biopsy, blood collection, urine collection, lumbar puncture (ie spinal puncture), ventricular puncture, and cisterna puncture. In a preferred embodiment of the invention, the sample is urine.

Many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and ALS are characterized by the accumulation or presence of protein abnormalities that contribute to the phenotype of the disease and are therefore known in the art as “Proteinopathies”. (Jellinger, Movement Disorders, 18, Suppl 6, S2-12 (2003) and Paulson, American Journal of Human Genetics, 64, 339-45 (1999)). Collectively analyzing all proteins and peptides present in a biological sample is often referred to in the art as a “proteome”.

On the other hand, at present, no specific substance or gene product has been identified as an ALS biomarker. The present invention obtains a sample by collecting urine with little burden on the patient, and measures prostaglandin D2 and its metabolite together with creatinine, thereby enabling early diagnosis including the degree of progression and the rate of progression.

The method of the present invention after sample preparation includes analysis of prostaglandin D2 and its metabolite concentration in the sample by mass spectrometry and the like, and analysis of creatinine concentration. Examples of suitable mass spectrometry for prostaglandin D2 and its metabolites include matrix-assisted laser desorption ionization mass spectrometry (MALDI Matrix Laser Desorption Ionization), matrix-assisted laser desorption ionization / time-of-flight (MALDI-TOF, (Proteinopathies Time-of-Flight)) Although there is mass spectrometry, since the substance to be measured is specified, detection and measurement methods using antibodies such as high performance liquid chromatography and ELISA are also possible. For example, liquid chromatography mass spectrometry (hereinafter “LC / MS”) in which a mass spectrometer is directly connected to high performance liquid chromatography (hereinafter abbreviated as “HPLC”) may be used.

In this embodiment, the prostaglandin D2 and its metabolite in the sample are preferably analyzed by “LC / MS” mass spectrometry.

This embodiment further provides a method for evaluating the effectiveness of drugs and the like in the treatment of ALS in a subject. The method comprises (a) obtaining a first sample from an ALS patient and a subject suspected of ALS, (b) analyzing prostaglandin D2 and its metabolite in the first sample by mass spectrometry or the like, (C) analyzing creatinine in the first sample, (d) administering a drug or the like to the subject, (e) obtaining a second sample from the subject after administering the drug or the like, (f) Analyzing the prostaglandin D2 and its metabolite in the second sample by mass spectrometry or the like, (g) analyzing the creatinine, (h) analyzing the prostaglandin D2 and its metabolite in the second sample. A step of analyzing, (i) a step of analyzing creatinine in the second sample, and (j) a ratio of creatinine concentration in the first sample and the second sample to prostaglandin D2 and its metabolite concentration. A process for by including a step of efficacy of such a drug is evaluated here.

In the present embodiment, since a specified biomarker called prostaglandin D2 is used as an index as a method for evaluating the effectiveness of drugs and the like in the treatment of ALS, any measurement method for prostaglandin D2 and its metabolites is used. Is also available.

The method in the present embodiment can be used to evaluate the effectiveness of any drug suitable for the treatment of ALS. Suitable drugs include those that are currently on the market, such as riluzole (Rilutek®, Aventis) and those that will be developed in the future.

Further, in the present embodiment, 11,15-dioxo-9-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic acid (tetranor-PGDM, hereinafter abbreviated as “tPGDM”) is the main prostaglandy. (Song, W., Wang, M., Ricciotti, E., et al. J.Biol.Chem 283, 1179-1188 (2008)). In (f), tPGDM, which is preferably a prostaglandin D2 metabolite, is detected by mass spectrometry. However, the detection substance and the detection method are not limited to this.

The diagnosis method for amyotrophic lateral sclerosis in this embodiment performs evaluation of the onset, progression, and therapeutic effect of amyotrophic lateral sclerosis based on analysis of urine components of patients. Here, the urine component refers to all measurable substances (for example, proteins, hormones, sugars, nucleic acids, lipids, and metabolites thereof) contained in urine. Furthermore, the basic concept of this diagnostic method is to make a diagnosis based on the relative relationship of substances contained in one or more measured urine.

The following examples serve to illustrate the present invention in more detail, but of course should not be construed as limiting to any form of its scope.

<Part 1>
A method for evaluating the effects of drugs, such as diagnosis and treatment, including the determination of the degree of progression of ALS by analysis of prostaglandin D2 and its metabolites:
Regarding extraction of prostaglandin D2 metabolite in urine in this example, tPGDM, which is a metabolite of prostaglandin D2, was extracted by the following procedure.
(I) Urine is basically stored frozen (−20 ° C. or lower).
(II) Thaw frozen urine with running water. You may leave it in the cold room from the previous day.
(III) After thawing, use a supernatant after light centrifugation. Note that the turbidity may not be removed.
(IV) Extraction and purification operations of Method 1 or Method 2 shown below are performed on 1 mL of the supernatant.
(V) A portion of urine (about 1.9 mL) is aliquoted into one 2 mL tube and stored frozen (−20 ° C. or lower). The remaining urine is stored for about 1 to 2 weeks until analysis results are obtained.
(VI) Creatinine is measured in a part of urine.

Method 1 [ethyl acetate extraction]:
(I) Collect 1 mL (50 to 1000 μL) of a urine sample or standard sample.
(Ii) Acidify with 20 μL of 10% acetic acid (2% final).
(Iii) Add internal standard (IS) (d6-PGDM). Use deuterium labeled tPGDM.
(Iv) Add 2 mL of ethyl acetate and stir (5 seconds or more).
(V) Centrifuge for 3 minutes or longer (at 4 ° C).
(Vi) Recover the ethyl acetate phase (care not to incorporate the aqueous phase).
(Vii) The above operations (iv) to (vi) are repeated 2 to 3 times.
(Viii) Concentrate the collected ethyl acetate phase (by SpeedVac etc.) to dryness.

Method 2 [Solid Phase Extraction / Sep-Pak C18 Plus]:
(I) Collect 1 mL (50 to 1000 μL) of a urine sample or standard sample.
(Ii) Acidify with 20 μL of 10% acetic acid (2% acetic acid).
(Iii) Add IS (internal standard) (d6-PGDM). Use deuterium labeled tPGDM.
(Iv) Conditioning of “Sep-Pak C18 Plus” (360 mg): wet with 2 mL of methanol and equilibrate with 2 mL of 0.5% acetic acid.
(V) Inject sample into Sep-Pak C18 Plus (Note: at a flow rate of 1 mL / min or less).
(Vi) Flow 0.5 mL of 0.5% acetic acid.
(Vii) Wash with 5 to 6 mL of hexane (there is no problem if it is too much. Water in “Sep-Pak C18 Plus” is sufficiently removed).
(Viii) Collect tPGDM with 2 mL of ethyl acetate.
(Ix) The collected ethyl acetate solution is concentrated (by SpeedVac or the like) to dryness.
(X) If water remains after drying, add ethyl acetate and perform ethyl acetate extraction.
(Xi) “Sep-Pak C18 Plus” after use is washed with 1 mL of isopropanol (IPA) and subsequently with about 2 mL of methanol (“Sep-Pak C18 Plus” can be used repeatedly several times).

[Example 1]
Detection of tPGDM in ALS patient S:
FIG. 1A shows the measurement results of a standard sample of tPGDM, which is a metabolite of prostaglandin D2, by liquid chromatography mass spectrometry (hereinafter “LC / MS”). Here, the measurement by LC / MS is basically the above-mentioned Song, W. et al. Conforms to other (2008). Detection was performed under the conditions of precursor ion; 327 amu, product ion; 155 amu. Specifically:

FIG. 1B shows ALS patient S (66 years old, male, body weight 58 kg, onset about 2 years, vocal phonation and gait disturbance, inability to stand up again on flat ground. SOD1 gene and monocyte / macrophage CD16 (FCγ receptor III The results of LC / MS measurement of tPGDM, which is a metabolite of prostaglandin D2 in urine, are shown. The measurement conditions by LC / MS are the same as in FIG. 1A.

FIG. 1C shows the LC / MS measurement results of tPGDM, which is a prostaglandin D2 metabolite in the urine of healthy subjects. The measurement conditions by LC / MS are the same as in FIG. 1A.

TPGDM, a metabolite of prostaglandin D2, is a highly water-soluble compound having a molecular weight of 328 and is excreted from urine in a short time. It extracted from urine using the ethyl acetate extraction method and the solid-phase extraction method, and this was analyzed by said LC / MS. In this analysis, negative ionization is performed, a precursor ion having a mass number of 327 amu is targeted, further fragmentation is performed, and a product ion of 155 amu is detected to detect a metabolite of prostaglandin D2 with high selectivity. be able to. FIG. 1A shows a chromatogram of a standard sample of tPGDM, which elutes at 18.2 minutes.

As shown in FIG. 1B, when urine of ALS patient S was analyzed, tPGDM was detected, and as a result of quantification from a calibration curve prepared with a standard sample of tPGDM, a concentration of 2.6 pg / μl (ng / ml) was obtained. Was found to be present in the urine.

As shown in FIG. 1C, when urine of a healthy person was analyzed, tPGDM was not detected.

It can be seen that tPGDM, which is a metabolite of prostaglandin D2, is specifically detected in the urine of patient S. Therefore, it can be seen that “tPGDM”, which is a metabolite of prostaglandin D2, becomes an “ALS biomarker”.

[Example 2]
The transition of the urinary tPGDM concentration in the day and night 6 months after the ALS patient S shown in Example 1 was verified. As shown in FIG. 2, tPGDM in patient S urine was detected at all times although the concentration varied with time.

[Example 3]
In addition to patient S, the transition of tPGDM concentration in urine during the day and night was newly verified in 5 ALS patients (male, 53-72 years old) and 6 healthy subjects.

FIG. 3A shows the transition of urinary tPGDM concentration in ALS patients during the day and night. FIG. 3B is an enlarged view of the low concentration region of FIG. 3A. FIG. 3C shows the transition of the urinary tPGDM concentration of a healthy person day and night. FIG. 3D is an enlarged view of the low concentration region of FIG. 3C.

Comparison of the average urinary tPGDM concentration of one ALS patient shown in FIG. 2 of Example 2 and 5 ALS patients shown in FIG. 3A of Example 3 and 6 healthy subjects shown in FIG. 3C. This is shown in FIG. FIG. 4A shows a comparison of average concentrations during the day and night, FIG. 4B compares the average concentrations during the day (from 6 am to 6 pm), and FIG. 4C illustrates the average concentrations during the night (from 6 pm to 6 am). Further, FIG. 4D shows a comparison between the lowest patient concentration and the highest healthy person concentration during the day and night.

）、８３．８２ｎｇ／ｍＬ（

）及び５６．２４ｎｇ／ｍＬ（

）であった。 A higher concentration of tPGDM was detected in the patient's urine, and the values of the patient and healthy subjects were analyzed by one-sided t-test. As a result, the significant differences in the average concentrations during the day and night, 24 hours, daytime and nighttime were 62.09 ng / mL (

), 83.82 ng / mL (

) And 56.24 ng / mL (

)Met.

）であった。 Furthermore, the significant difference between the lowest patient concentration in day and night and the highest healthy person concentration was 30.71 ng / mL (

)Met.

The calculation formula of t is as follows.

From the results of Examples 2 and 3, it was found that tPGDM was detected at a high concentration in the patient's urine at any time zone, significantly for healthy subjects.

So what time is the best time for urine collection? It can be seen from the data in FIG. 3 that the tPGDM concentration in the patient's urine shows the lowest value in the evening, then increases, and shows the highest value in the time zone from 3 am to 5 am. Therefore, it can be said that this time zone is optimal for urine collection.

[Example 4]
The total urine tPGDM concentration and total amount of 24-hour urine collection of 5 ALS patients and 6 healthy subjects were verified.

）、かつより多量に（有意差６７．７９μｇ（

））ｔＰＧＤＭが検出されることが明らかになった。この結果は実施例３の結果を追認している。 FIG. 5A shows the tPGDM concentration, and FIG. 5B shows the tPGDM amount. The patient's urine has a significantly higher concentration (significant difference 66.45 ng / mL (

) And in larger amounts (significant difference 67.79 μg (

)) It was revealed that tPGDM was detected. This result confirms the result of Example 3.

From the above results, it was found that tPGDM is meaningful and effective for use as a biomarker of ALS.

[Example 5]
The relationship between the progress of the patient's ALS symptoms and the tPGDM concentration was examined.

Fig. 6 focuses on the patient's symptom walking function, and can walk at a degree of progression of 1, 2 for wheelchair life, 3 for bedridden life, 4 for ventilator wear, and the horizontal axis and vertical axis for each patient It is the figure which showed the daytime night average density | concentration of FIG. 4A of tPGDM which is a urinary prostaglandin D2 metabolite. The average of the average daytime and nighttime concentration of healthy persons is indicated by a horizontal broken line. It can be seen that as the degree of progression proceeds from 1 to 3, the average concentration of tPGDM, which is a metabolite of prostaglandin D2 in the patient's urine, increases. The correlation coefficient for the degree of progress of this increase is 0.84. In addition, the correlation coefficient excluding data with a progression of 1 and skipping the value is 0.98.

The calculation formula of the correlation coefficient is as follows.

Therefore, it can be said that tPGDM is effective not only as a biomarker of ALS, but also in determining and determining the degree of progression.

[Example 6]
Changes in tPGDM during ALS treatment (monitoring of drug effect):
To the ALS patient S shown in FIG. 1B of Example 1, “Radicut” (manufactured by Mitsubishi Tanabe Pharma Corp., generic name “Edaravone”) was administered 16 months later (15 to 17 o'clock on the second day) 30 mg was mixed and administered to 500 mL of Ringer's solution Botacol R (Otsuka Pharmaceutical). Changes in the concentration and amount of tPGDM in the total amount of urine (collecting all urination) at each time from day 1 to day 5 including before and after were examined.

FIG. 7A shows the concentration of tPGDM before and after administration of “Radicut”, and FIG. 7B shows the total amount.

Since the amount of urine is unstable after infusion, more accurate analysis results can be obtained by using the total amount for the analysis of tPGDM. On the next day after the instillation (day 3 in FIGS. 7A and 7B), the amount of tPGDM in the urine slightly decreases, while on the second and third days after the infusion (day 4 and 5 in FIGS. 7A and 7B). A significant amount of tPGDM was detected in urine immediately after waking up.

[Summary of Examples]
In Example 1, excretion of tPGDM was observed in the urine of ALS patient S, which is not found in healthy subjects. This suggests that it can be a biomarker of ALS.

The result of FIG. 2 of Example 2 is the result after 6 months of the patient of FIG. 1B of Example 1. The urine tPGDM concentration rose from 2.6 ng / mL to a minimum of 5.0 ng / mL, a maximum of 24.3 ng / mL, and an average of 17.1 ng / mL over 6 months. During this time, the patient's symptoms were such that he was unable to raise his head when walking, unable to raise and lower his car by himself, and retreated his voice. Furthermore, the result of FIG. 7A of Example 6 is for the subsequent 10 months. Its concentration as a control prior to Radicut infusion rose to a minimum of 16.2 ng / mL, a maximum of 28.7 ng / mL, and an average of 22.7 ng / mL. The progression of symptoms during this period is difficulty in walking on its own. Thus, it was found that tPGDM in urine is a biomarker of ALS, and its concentration can be used for monitoring the progression rate of symptoms.

In Example 3, in addition to patient S, urine tPGDM concentration was analyzed for 5 ALS patients and 6 healthy subjects. Analysis of 6 patients versus 6 healthy subjects including patient S in Example 2 revealed that significantly high concentrations of tPGDM were excreted in the patient's urine. It was also revealed that the highest concentration of tPGDM was excreted in the patient urine collected around 3-5 am.

In Example 4, the tPGDM concentration and the total amount in the total urine volume obtained by collecting urine for 24 hours and pooling all were examined. As a result, the patient's urine showed significantly higher tPGDM concentration and total value.

From the above results, tPGDM is a biomarker suitable for the diagnosis of ALS.

From the data of patients S in Example 5 and Examples 1 and 2, it was revealed that there is a correlation between the urinary tPGDM concentration and the progress of ALS. This suggests that the method is also effective for monitoring ALS progression and recovery with treatment.

In Example 6, the concentration of urinary tPGDM decreased immediately after administration of radicut. This is considered to be a temporary inhibitory effect consistent with the rapid pharmacokinetics of Radicut (Toshiaki Watanabe et al. YAKUGAKU ZASSHI 124, 99-111 (2004) and Mitsubishi Tanabe Pharma Corporation Pharmaceutical Interview Form Japan Standard Product Classification Number: 87119). It is done. Further, after that (2 to 3 days after administration), a significant and large amount of excretion was confirmed. This seems to be a so-called rebound and is considered to reflect some effect of administration.

From this result alone, it is difficult to determine whether or not “Radicut” infusion is effective for treatment, but it is possible to analyze the drug efficacy using tPGDM as a biomarker of ALS. The effectiveness of treatment methods, such as combining methods, could be determined. Thus, the analysis of the present invention makes it possible to determine the effects of drugs and the like.

<Part 2>
Diagnosis of ALS by creatinine analysis:
Creatinine is an end product of creatine metabolism, and is an anhydride obtained by removing H ₂ O non-enzymatically from creatine. Creatine is synthesized in the liver and kidney from three amino acids, glycine, arginine, and methionine, and most of it is retained in skeletal muscle as creatine or creatine phosphate. In muscle cells, ATP is generated from creatine phosphate by a creatine kinase reaction and used for muscle contraction activity, and creatinine is generated from creatine generated as a metabolite thereof. Creatinine, one of the non-protein nitrogenous compounds in the blood, is filtered from the kidney glomeruli and excreted in the urine with little reabsorption.

Therefore, in renal diseases such as renal failure, the serum creatinine concentration increases and the urinary creatinine concentration decreases. Therefore, it is widely used as an assay method for renal function.

On the other hand, since this substance is synthesized in muscle, a decrease in creatinine concentration has been reported in both serum and urine in muscular diseases such as muscular dystrophy and polymyositis (Satoshi Yoshimura et al .: Japanese Clinical 53- -464-468 (1995), Osawa Susumu: Medical Technology 26,389-395 (1998)).

Creatine works by affecting the energy production mechanism of cells. When creatine was given by diet to mice that had been genetically engineered to develop a mutation that caused ALS, the average life span was increased by 26 days compared to mice that did not receive creatine. The effect of oral administration of creatine is dose-dependent, and the addition of 1% and 2% to drinking water has been reported to significantly improve the degree of life extension (Peter Kliveny et al. Nature Medicine 5, 347-350 ( 1999)). This effect of creatine was also demonstrated by Adhihetty and Beal (Neuromolecular Med 10, 275-90 (2008)).

Urinary creatinine concentration decreases in both kidney disease and muscle disease, and is effective in diagnosing these diseases (Gaku Yoshimura et al .: Japanese Clinical 53-Increase-464-468 (1995) and Susumu Osawa: Medical Technology 26 , 389-395 (1998)).

In this example, urine prepared in the first part tPGDM analysis is used for the analysis of creatinine in urine, and the analysis method is based on an existing method used in a renal disease test. Specifically, a creatinine measurement kit manufactured by Wako Pure Chemical Industries, Ltd. is used.

The urinary creatinine was analyzed in 6 ALS patients and 2 healthy subjects who analyzed tPGDM in Examples 2 and 3, and 3 healthy subjects.

[Example 7]
Transition of urinary creatinine concentration between ALS patients and healthy subjects 24 hours a day:
Using the same urine sample as used in Examples 2 and 3, and urine samples of 3 healthy subjects, the transition of urinary creatinine concentration in ALS patients during the day and night for 24 hours was verified. FIG. 8A shows the creatinine concentration in the urine of an ALS patient, and FIG.

）である。また、全員の測定回数を取り入れた場合の有意差は、下記の計算から－１０４０．５３μｇ／ｍＬ（

）である。 [Example 8]
Comparison of average creatinine concentrations in ALS patients and healthy individuals:
FIG. 9A is a graph comparing the average concentrations of creatinine in urine of ALS patients and healthy subjects day and night. Significant difference is −1135.52 μg / mL except for one patient with high concentration (

). In addition, the significant difference when taking the number of measurements of all members is -1040.553 μg / mL (

).

）である。また、全員の測定回数を取り入れた場合の有意差は、下記の計算から－１０１３．４１μｇ／ｍＬ（

）である。 FIG. 9B is a graph comparing the average concentrations of urinary creatinine in ALS patients and healthy individuals in the daytime. Significant difference is −1149.12 μg / mL except for one patient with high concentration (

). In addition, the significant difference when taking the number of measurements of all members is -1013.41 μg / mL (

).

）である。また、全員の測定回数を取り入れた場合の有意差は、下記の計算から－１０６９．１１μｇ／ｍＬ（

）である。 FIG. 9C is a diagram comparing the average concentrations of urinary creatinine in ALS patients and healthy individuals at night. Significant difference is −1128.77 μg / mL except for one patient with high concentration (

). In addition, the significant difference when taking the number of measurements of all members is −1069.11 μg / mL (

).

FIG. 9D is a diagram comparing the patient maximum concentration and the normal normal concentration of urinary creatinine in ALS patients and healthy subjects during the day and night. There is no significant difference in patients at low concentrations.

[Example 9]
The total urinary creatinine concentration and total amount of 24-hour urine collection of 5 ALS patients and 5 healthy subjects were verified.

）であり、総量では－１６９５．５２ｍｇ（

）である。 FIG. 10A shows the creatinine concentration, and FIG. 10B shows the creatinine amount. In terms of concentration, the significant difference in t-test was −1318.6 μg / mL (

), And the total amount is −1695.52 mg (

).

）、総量では－１７５７．５１ｍｇ（

）である。したがって、患者の尿には、健常者に対して有意に、低濃度、少量のクレアチニンが検出されることが明らかになった。この結果は上記の実施例８の結果を追認している。 In addition, except for one patient with high concentration, the significant difference is -1491 μg / mL in concentration (

), The total amount is -1757.51 mg (

). Therefore, it was revealed that a low concentration and a small amount of creatinine were detected in the urine of the patient, which was significantly lower than that of a healthy person. This result confirms the result of Example 8 above.

It is known that the amount of creatinine in urine is greatly affected by kidney diseases. In this example, it was revealed that the urinary creatinine concentration of ALS patients was significantly lower than that of healthy subjects.

In the first to sixth embodiments, tPGDM was analyzed. The analysis of creatinine uses the same sample as that used for the analysis of tPGDM in the first part.

In the creatinine analysis of the present example, it was clearly possible to distinguish from healthy individuals in ALS patients that could not be distinguished from healthy individuals by tPGDM analysis.

A highly accurate diagnosis method is possible by combining tPGDM analysis and creatinine analysis.

[Example 10]
Relationship between ALS progression and creatinine concentration:
Using the same sample as the ALS patient whose tPGDM concentration was analyzed in Example 6, the creatinine concentration was analyzed.

As described in Example 5, the patient's ALS progression standard was divided into a wheelchair life, a wheelchair life, a bedridden life, and a ventilator wearing.

FIG. 11 shows the relationship between the patient's symptom progression degree and the average concentration of creatinine in FIG. 9A day and night. The broken line in the figure shows the average of the average concentration of creatinine day and night for healthy subjects.

Except for one ALS patient with a progression of 1, the creatinine concentration of all patients was significantly lower than the average creatinine concentration of healthy subjects indicated by the broken line, but no correlation with the progression was observed.

[Summary of Examples]
The urinary creatinine concentration and total amount are significantly lower in ALS patients than in healthy individuals. Its concentration does not correlate with the progression of symptoms, but the analysis can be applied to diagnose ALS. The optimum time for collecting urine is around 7pm. Furthermore, the creatinine concentration and total amount are low even in patients with low tPGDM concentration and total amount. Therefore, a patient overlooked by tPGDM analysis can also be detected, and ALS diagnosis with higher accuracy can be performed by complementing tPGDM analysis and combining both.

<Part 3>
Diagnosis method of ALS by combination of tPGDM analysis and creatinine analysis:
The muscle contains a nitrogen compound that stores energy called creatine phosphate. When this is broken down into creatine by the action of an enzyme, it releases energy, and the muscle moves using that energy. Creatine is turned into creatinine when it finishes its role.

Since nitrogen in the body is excreted only from the kidneys, all creatinine is excreted from the kidneys into the urine via the blood. For this reason, as renal disease progresses, serum creatinine concentration begins to rise when kidney function falls below half of normal. Moreover, the concentration in urine is lowered by kidney disease.

On the other hand, the ALS biomarker tPGDM developed by this method is elevated in urine in ALS disease.

¡ALS can be diagnosed with higher accuracy by combining tPGDM analysis and creatinine analysis.

[Example 11]
Correlation between tPGDM concentration and creatinine concentration:
The data in the following figure is used from the first and second parts respectively. FIG. 12A shows a correlation between both average concentrations of day and night. The horizontal axis represents the creatinine average concentration, and the vertical axis represents the tPGDM average concentration.

FIG. 12B shows the concentrations of tPGDM and creatinine in the total urine obtained by collecting urine for 24 hours day and night, and FIG. 12C shows the total amounts of tPGDM and creatinine in the total urine volume.

Using two parameters, tPGDM concentration and creatinine concentration, or total amount, it became clear that 6 ALS patients and 2 healthy subjects can be completely separated.

[Creating a system (program) that uses circadian rhythm data to estimate the maximum and minimum concentrations of patients at any concentration at any time of urine collection]
A circadian rhythm (circadian rhythm) was confirmed in the time course change of the urine tPGDM concentration (FIG. 3). tPGDM is a metabolite of prostaglandin D2 known as a sleep substance. (In short, Urade Y, Hayaishi O., “Prostaglandin D2 and sleep regulation.” Biochim Biophys Acta. 1999 Jan 4; 1436 (3): 606-15. And Hayaishi O, Urade Y., “Prostaglandin D2 in sleep -wake regulation: recent progress and perspectives. ”Neuroscientist. 2002 Feb; 8 (1): 12-5.)

The basis of the circadian rhythm of tPGDM concentration in urine is thought to be derived from the above. However, it is difficult to specify a large number of new patients and to collect urine multiple times. Therefore, it was possible to create a system (program) for estimating the maximum and minimum concentrations of a patient at an arbitrary concentration at an arbitrary urine collection time by using the circadian rhythm data. FIG. 13 shows the prototype model. When the patient's urine collection time and tPGDM concentration are entered and the calculation is clicked, the maximum concentration and the minimum concentration of the patient are estimated from a reference curve prepared in advance from the data of changes in urine tPGDM concentration over time (shown in FIG. 13). Displayed. In addition, an estimation graph of the concentration change for 24 hours is displayed.

With this system, it is possible to grasp the patient's condition and to compare patients with each other by estimating the time-dependent change of each patient's concentration and the highest and lowest concentrations, thereby enabling more accurate diagnosis.

In this system, as shown in FIG. 13, when the urine collection time and tPGDM concentration on the screen are entered and the calculation is clicked, the maximum concentration and the minimum concentration are displayed on the screen and a graph showing the concentration at each time is displayed. As for creatinine, the circadian rhythm is confirmed in FIGS. 8A to 11, and a similar system can be created.

[Preferred embodiment]
Further, the present invention can predict a time-dependent change in sleep substances, human growth hormone, hormones linked to the sexual cycle, and blood glucose level after meals, so that an estimation system based on the above known data is possible.

Examples of utilization of the present invention include the development and establishment of treatment methods for ALS patients and the development of effective drugs for ALS patients.

Claims (11)

A method of using prostaglandin D2 and its metabolite as a biomarker for amyotrophic lateral sclerosis (ALS). A method of combining prostaglandin D2 and its metabolite analysis and creatinine analysis as a diagnostic method for amyotrophic lateral sclerosis (ALS). A method for diagnosing amyotrophic lateral sclerosis (ALS) in a subject comprising:
(A) obtaining a sample from a subject;
(B) measuring prostaglandin D2 and its metabolite in the sample;
(C) measuring creatinine in a sample; and (d) comparing prostaglandin D2 and its metabolite concentration and creatinine concentration of a positive or negative sample of the sample, wherein the subject is diagnosed as ALS Comprising the steps of: The method according to claim 3, wherein the standard is a positive standard. The method according to claim 3, wherein the preparation comprises a sample obtained from a patient with amyotrophic lateral sclerosis or a subject suspected of having amyotrophic lateral sclerosis. A method for evaluating the effectiveness of a drug in the treatment of amyotrophic lateral sclerosis comprising:
(A) obtaining a first sample from a subject with amyotrophic lateral sclerosis and suspected of amyotrophic lateral sclerosis;
(B) analyzing prostaglandin D2 and its metabolite in the first sample by mass spectrometry or the like;
(C) analyzing creatinine;
(D) a step of administering a drug or the like to the subject,
(E) obtaining the second sample from the subject after completion of step (d);
(F) analyzing prostaglandin D2 and its metabolite in the second sample by mass spectrometry or the like;
(G) the step of analyzing creatinine, and (h) the prostaglandin D2 and its metabolite concentration and creatinine concentration of the first sample, and the prostaglandin D2 and its metabolite concentration and creatinine concentration of the second sample. A method comprising a step of comparing, wherein the effectiveness of a drug or the like is evaluated here. The effectiveness of a drug or the like is measured as a change in prostaglandin D2 and its metabolite concentration and creatinine concentration in the second sample compared to the prostaglandin D2 and its metabolite concentration and creatinine concentration in the first sample. The method according to claim 6. 4. The method according to claim 3, wherein the subject is a human. The method according to claim 6, wherein the patient with amyotrophic lateral sclerosis and the subject suspected of having amyotrophic lateral sclerosis are ALS model animals and humans selected from the group consisting of mice, rats and dogs. . Based on the analysis of the patient's urine component, the onset, progression, and therapeutic effect of amyotrophic lateral sclerosis is evaluated.
The urine component is a measurable substance selected from the group comprising proteins, hormones, sugars, nucleic acids, lipids, and metabolites thereof contained in urine,
The method according to any one of claims 3 to 5, wherein the diagnosis is performed based on a relative relationship between one or more measured urine components. A system that estimates the maximum and minimum concentrations of a patient at any tPGDM concentration at any urine collection time using circadian rhythm (circadian rhythm) data over time of tPGDM concentration in urine.

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