WO2019124267A1 - Test method for diagnosis of niemann-pick disease type c - Google Patents

Abstract

In order to realize a test method that has an improved accuracy of diagnosis of Niemann-Pick disease type C over that for the use of a single biomarker and that for conventional methods, the test method according to the present invention for the diagnosis of Niemann-Pick disease type C comprises: a step for measuring the amount of a first biomarker in a sample originating from blood collected from a living body and a step for measuring the amount of a second biomarker in a sample originating from urine collected from the living body.

Description

Test method for diagnosis of Niemann-Pick disease type C

The present invention relates to an inspection method and the like for diagnosis of Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a visceral neurological disease caused by mutations in the NPC1 (OMIM 607623) or NPC2 (OMIM 601015) gene (Non-patent Documents 1 and 2). Abnormal cholesterol efflux from the late endosomal / lysosomal compartment has been suggested as a mechanism associated with the pathogenesis of NPC. Early evidence has consistently shown that NPCs are associated with the accumulation of various lipids, including cholesterol, in humans (3). Such lipids include oxysterols such as cholestane-3β, 5α, 6β-triol and 7-ketocholesterol (Non-patent documents 4 to 9), bile acids (Non-patent documents 10 to 14), and glycosylated cholesterol ( Various cholesterol metabolites such as Non-Patent Document 15) can be mentioned. The NPC1 protein is a membrane protein within the lysosome and facilitates transport of cholesterol from the lysosome to the plasma membrane. NPC2 is a soluble protein in lysosomes and binds stoichiometrically with cholesterol. Based on these biochemical properties of NPC1 and NPC2, the mechanism of NPC is believed to be due, at least in part, to a defect in proper lipid migration in cells (Non-patent Document 2). This possibility has been demonstrated in several established murine NPC models as treatment with cyclodextrin (a cyclic oligosaccharide that promotes cholesterol transport across the plasma membrane) has shown positive therapeutic results. (Non-Patent Documents 16 to 18). In recent years, 380 or more pathogenic mutations have been registered in the database (Non-patent Document 19). Moreover, in recent years, the morbidity rate of classical NPC has been shown to be about 1 / 10,000, but the incidence rate of late-onset NPC is not sufficiently predicted (Non-Patent Document 19) ).

Since both NPC1 and NPC2 proteins have no enzymatic activity, no single clinically useful biomarker is known. Classically, staining with the antibiotic Filipin has been used, which is believed to correlate with the accumulation of cholesterol.

Both clinical and experimental evidence has shown that in NPC, sphingomyelin levels are increased in the liver and spleen (Non-patent documents 2 to 3). In mammals, biosynthesis of sphingomyelin originates from serine and palmitoyl CoA in the endoplasmic reticulum and is subjected to an enzymatic reaction by serine: palmitoyl CoA transferase (EC 2.3.1. 50) (Non-patent Document 20). Sphingomyelin is a major sphingolipid that exists outside the cell membrane. Because sphingomyelin is accumulated in NPC patients, the concentration of lysosphingomyelin (also known as sphingosyl phosphorylcholine (SPC)) in plasma has been proposed as a biomarker to screen NPCs (non- Patent Documents 5, 21 to 22).

Initial studies have shown that plasma SPC concentrations correlate with plasma concentrations of cholestane-3β, 5α, 6β-triol and 7-ketocholesterol, all of which are widely used as metrics for the diagnosis of NPC. It is accepted (nonpatent literature 5). In recent years, it has been reported that SPC is elevated in patients with acid sphingomyelinase deficiency as well as NPC patients, suggesting that plasma components may not affect the metabolism of SPC. (Non-Patent Document 21). Interestingly, in this study, selective accumulation of SPC in NPC can only be detected in plasma, not in dried filter paper samples, and SPC in red blood cells may not be affected by NPC-mediated metabolism. Have also been found (Non-Patent Document 21). Furthermore, lysosphingomyelin-509 is known as a novel biomarker whose chemical property accumulated in the plasma of NPC patients has not been determined (Non-patent documents 21 and 23 and Patent documents 1 and 2) .

Japanese Patent Publication "Tokukai 2014-533367 (Dec. 11, 2014)" International Publication Gazette "WO 2016-078762 (May 26, 2016)"

Staining with the antibiotic Filipin has the problem of poor reproducibility and quantification.

Cholesterol oxidation products such as cholestane-3β, 5α, 6β-triol and 7-ketocholesterol are to be used as target samples of the plasma to avoid the effects of artificial oxidation reactions that may occur before quantitative analysis After blood collection, the supernatant should be centrifuged immediately after freezing, and the supernatant should be cryopreserved under cryogenic temperature and transported to the laboratory while being kept frozen. Although preparation, storage and transport of such samples should be conducted according to a predetermined procedure, there is a problem in that cost and labor are generally required for arranging refrigerants such as dry ice and means for transporting in frozen state. There is. Furthermore, during transportation in summer, an accident may occur during transportation of the sample, for example, a frozen sample may be melted because all the dry ice is vaporized.

In addition, in order to use the above-mentioned two biomarkers contained in the sample for quantitative analysis, it is necessary to remove the solvent during the analysis operation and concentrate to dryness. There is a possibility that an artificial oxidation reaction may occur during this pretreatment, but even with this series of operations, even if a more detailed standard procedure is prepared, it is more difficult to carry out manual processing than the pretreatment operation at normal temperature and pressure. Oxidation reactions do not occur, or it is difficult to ensure that all samples are constant. In other words, cholestane-3β, 5α, 6β-triol and 7-ketocholesterol are oxidation products of cholesterol, and therefore, in addition to the endogenous accumulation to be quantified as a biomarker, artificially generated oxidation products during analytical procedures When the accumulation amount of the substance is significantly large, there is a problem that the normal control sample also exhibits a high level as recognized in the disease group.

The concentrations of compounds present in the plasma, such as cholestane-3β, 5α, 6β-triol and 7-ketocholesterol, and SPC also fluctuate to some extent under physiological conditions due to metabolic reduction and concentration by perspiration. However, in the previous researches, even in the case of a plurality of biomarkers, all of them have been measured to a single sample. Therefore, if the measured biomarkers rise or fall uniformly, there is no means to correct this.

One aspect of the present invention aims to realize a test method that improves diagnostic accuracy for Niemann-Pick disease type C in the case of using a biomarker alone and in comparison with the conventional method described above.

In order to solve the above problems, a test method for diagnosis of Niemann-Pick disease type C according to one aspect of the present invention comprises an amount of a first biomarker in a blood-derived sample collected from a living body And measuring the amount of the second biomarker in a sample derived from urine collected from the living body.

In one embodiment of the present invention, there is also provided a method of measuring the amount of a first biomarker in a sample derived from blood collected from a living being, and a second biologic in a sample derived from urine collected from the living being And determining the amount of the marker, and providing a test method for evaluating the therapeutic effect of Niemann-Pick disease type C.

One embodiment of the present invention is also directed to an internal standard substance used for measuring the amount of the first biomarker in a blood-derived sample collected from a living body, and a sample derived from urine collected from the living body. A test kit for diagnosis of Niemann-Pick disease type C or evaluation of the therapeutic effect of Niemann-Pick disease type C comprising an internal standard substance to be used in measuring the amount of the biomarker of No. 2 is provided.

According to one aspect of the present invention, diagnostic accuracy may be improved for Niemann-Pick disease type C, as compared to the single use of a biomarker and the conventional method described above.

It is a figure which shows the chemical structure of the compound used by this research. A is D-erythro-sphingosyl phosphoryl choline (synthetic, SPC). B is sphingosyl phosphoryl choline (C17 base, IS _SPC ). C is 3β-sulfooxy-7β-N-acetylglucosaminyl-5-coren-24-noic acid (SNAG-Δ ⁵ -CA). D is 3β-sulfooxy-7β-hydroxy-23-nor-5- _{cholic acid} (IS _{bile acid} ). It is a figure which shows the calibration curve of IS _SPC and SPC which used LC-MS / MS. SPC and IS _SPC peak areas were quantified in MRM mode (SPC, 465.55>183.95; IS _SPC , 451.4> 183.90) using LC-MS / MS as described in the experimental procedures described below. Both SPC and IS _SPC were detected as [M + H] ⁺ ions using ESI positive mode. FIG. 7 shows selective accumulation of distinct SPCs in PBS (top) and plasma (bottom). Note that the amount of IS _SPC was nearly constant during the assay. FIG. 1 shows representative chromatograms for SPC and IS _SPC from NPC patients and controls (healthy people). SPC and IS _SPC concentrations were quantified in MRM mode (SPC, 465.55>183.95; IS _SPC , 451.4> 183.90) using LC-MS / MS as described in the experimental procedures described below. Both SPC and IS _SPC were detected as [M + H] ⁺ ions using ESI positive mode. ^* Represents a nonspecific peak. FIG. 6 shows the elevation of plasma SPC and lysosphingomyelin-509 and urinary SNAG-Δ ⁵ -CA in NPC patients. In FIG. 5A, plasma SPC concentrations in NPC patients (n = 5) and controls (n = 7) were determined using LC-MS / MS using sphingosyl phosphoryl choline (C17 base) as internal standard substance. It quantified. In FIG. 5B, plasma lysosphingomyelin-509 in NPC patients (n = 5) and controls (n = 7) was quantified. In FIG. 5C, the concentration of urinary SNAG-Δ ⁵ -CA in NPC patients (n = 5) and controls (n = 7) was quantified (p <0.05). FIG. 5 shows the correlation of plasma and urine biomarker concentrations in NPC patients. FIG. 6A shows the correlation between plasma SPC concentration and urinary bile acid metabolite SNAG-Δ ⁵ -CA concentration. FIG. 6B shows the correlation between plasma lysosphingomyelin-509 concentration and urine SNAG-Δ ⁵ -CA concentration. The concentration of SNAG-Δ ⁵ -CA was quantified using LC-MS / MS according to Non-Patent Document 11. 3β-sulfooxy-7β-hydroxy-23-nor-5-cholic acid was used as an internal standard.

[1. Test method for diagnosis of Niemann-Pick disease type C]
An inspection method for diagnosis of Niemann-Pick disease type C according to the present embodiment is
Measuring the amount of a first biomarker in a sample derived from blood collected from a living body (hereinafter referred to as "first step");
Measuring the amount of a second biomarker in a sample derived from urine collected from the living body (hereinafter referred to as “second step”);
including.

There is no restriction on the order of performing the first step and the second step, and the first step may be performed before the second step, or the second step may be performed before the first step. The first step and the second step may be performed in parallel.

The first and second biomarkers and other biomarkers whose amounts can be measured in this embodiment are collectively referred to hereinafter simply as "biomarkers".

In the present embodiment, examples of the living body (subject) include humans and animals other than humans (for example, mammals), and examples of mammals include mice, rats, rabbits, guinea pigs, primates excluding humans, and the like. Experimental animals; companion animals such as dogs and cats (pets); domestic animals such as cows, horses and pigs; humans. The living body (subject) may be a newborn. The inspection method according to the present embodiment may be performed as one of the items of the newborn screening test.

As used herein, "to measure the amount of biomarker" is intended to measure the amount or concentration of the biomarker in a biological sample or a sample obtained by purifying the same. The "amount of biomarker" may be an absolute amount (absolute mass or absolute concentration) or a relative amount (relative mass or relative concentration). In addition, for example, it may be a peak area in mass spectrometry, an emission intensity in luminescence measurement, or the like, or a value indicating how many times a predetermined standard is.

As used herein, "having Niemann-Pick disease type C" refers to having the etiology of Niemann-Pick disease type C, including when it has not yet developed typical clinical symptoms at this time It is a concept to gain. The pathogenesis of Niemann-Pick disease type C includes genetic factors, and more specifically, a gene encoding NPC1 protein or NPC2 protein with reduced function (ie mutation of NPC1 gene or mutation of NPC2 gene) Include, but are not limited to, other known and unknown etiologies may also be included.

(First step)
The "sample" used in the first step is a sample derived from blood collected from a living body (subject). Examples of blood-derived samples include whole blood, serum, plasma and the like. Among the samples derived from blood, serum and plasma are preferable, and plasma is more preferable. As a blood collection site, for example, a ovary fossa vein, a caudal cutaneous vein, and an ulnar cutaneous vein may be mentioned.

The first biomarker is the amount of the above-mentioned blood-derived sample (such as whole blood, serum, or plasma) in individuals having Niemann-Pick disease type C and individuals not suffering from Niemann-Pick disease type C It is a biomarker that has a statistically significant difference. The first biomarker includes sphingophospholipids, cholestane-3β, 5α, 6β-triol, 7-ketocholesterol, bile acid A and the like. Examples of sphingophospholipids include sphingosyl phosphoryl choline (SPC), sphingosyl phosphoryl choline modified form, sphingomyelin, sphingomyelin modified form and the like.

In one embodiment, the first biomarker is lysosphingomyelin-509. The compound is compound 509 described in Patent Documents 1 and 2. Lyso sphingomyelin -509 (Compound 509) _{is, C 24 H 50 O 7 N} 2 P 509.3 having the empirical formula of the compound 509 (quasi-molecular M + H ions), more specifically 509.265 (m / z A substance having a pseudomolecular ion mass (as a monoisotopic pseudomolecular M + H ion). This is preferably according to the method of the present invention, more particularly according to the method of Example 1 and Example 2 as described in the specification of Patent Document 2, in a plasma sample derived from a subject from 509 m / z It may be a compound detected as MRM transition in ESI positive mode at 184 m / z (MRM transition).
The structural formula of compound 509 as [M + H] ⁺ is as follows, and preferably, the molecular weight of the compound of the following formula is 509.265 as monoisotopic pseudomolecular M + H ion (m / z). Lysosphingomyelin-509 (compound 509) is also referred to as ω-carboxy-sphingosyl phosphoryl choline.

In the present embodiment, as a method of measuring the amount of the biomarker (the first biomarker, the second biomarker, and optionally other biomarkers), the amount of the biomarker present can be quantitatively or semi-quantitatively The method is not particularly limited as long as it can be determined, for example, mass spectrometry, a method using an immunological method using an antibody that recognizes a biomarker to be measured, a chemiluminescence method, an electrochemical detection method, ultraviolet light A detection method, a differential refraction method, or the like can be used.

Mass spectrometry may be performed using a known mass spectrometer. Mass spectrometry using a mass spectrometer is excellent in sensitivity and accuracy, so accurate determination can be performed. Furthermore, it is also possible to measure, for example, the amount of two or more biomarkers at once by using a multichannel mass spectrometer capable of multicomponent simultaneous analysis. Furthermore, biomarkers for other diseases than Niemann-Pick disease type C can also be measured at the same time, and detection of various diseases can be attempted at one time. Moreover, in order to perform detection more accurately, it is preferable to use a tandem mass spectrometer (MS / MS). The mass spectrometer used in the detection method of the present invention is not particularly limited as long as it can be quantified. For example, it is possible to use conventionally known types of mass spectrometers such as quadrupole type and time of flight type. In one embodiment, liquid chromatography / mass spectrometry (LC-MS) is preferably used, and more preferably LC-MS / MS.

Examples of methods using antibodies include ELISA, quantitative western blotting, radioimmunoassay, immunochromatography, and immunoprecipitation. The type of ELISA method is not particularly limited, but is a so-called antigen measurement system (measurement of the amount of antigen contained in a biological sample), ELISA by direct adsorption method, ELISA by competition method, ELISA by sandwich method, and microflow An ELISA or the like specialized for measurement of a small amount of sample using a road type or microbeads can be mentioned.

The sample to be used for measurement of the amount of biomarker preferably contains an internal standard of known amount. The amount of biomarker can be calculated more accurately. In addition, variations between measurements can be suppressed, and diagnosis with high accuracy is possible. Furthermore, data comparison among multiple facilities becomes easy. In addition, by accumulating data at multiple facilities, the accuracy of the reference value can be further improved. As internal standard substances, analogues of biomarkers (for example, isomers, homologues, compounds having different numbers of atoms in the main chain, compounds having different functional groups, or compounds whose chemical properties are similar to those of the biomarker and stable Compounds that can be quantified), stable isotope labeled compounds of biomarkers, and the like.

The sample to be subjected to measurement of the amount of biomarker may be pretreated. Examples of pretreatment include solvent extraction, solid phase extraction, HPLC (such as online HPLC), and derivatization. By performing pretreatment using a solvent extraction method, there is an advantage that the extraction operation including the compound to be measured can be easily achieved. The pretreatment using the solid phase extraction method has an advantage that the extraction operation including the compound to be measured can be easily achieved with less organic solvent. Pretreatment using an HPLC (on-line HPLC or the like) method has an advantage that higher separation and purification with various kinds and precise fillers can be easily achieved as pretreatment. By performing pretreatment using derivatization, specific functional groups present in the compound are selectively chemically modified, and are pretreated by solvent extraction, solid phase extraction and HPLC (such as online HPLC). At the time of processing, there is an advantage that the degree of purification of the desired substance with respect to impurities is dramatically improved. In addition to this, there is also an advantage that high sensitivity analysis can be achieved by using a derivatization reagent that contributes to sensitivity improvement particularly in quantitative analysis.

In addition, as pretreatment, for example, (a) when a biological sample is mixed with an internal standard substance and diluted with dilution liquid, turbidity (fine particles) is generated, and / or (b) extract (purified product) The sample may be filtered or centrifuged, for example, when fine particles are generated in the For example, it may be obtained from a blood plasma sample which is collected several hours after a particularly high lipid diet, a subject with suspected or confirmed dyslipidemia (eg familial hypercholesterolemia etc.) Samples and samples with high salt concentration or high protein concentration for some reason, etc. have a high possibility of generation of microparticles in the above (a) and (b). Also, even when prepared at room temperature, it is transferred onto an autosampler maintained at a low temperature (eg, 4 ° C., 10 ° C. or its ambient temperature) from the point of sample stability, and cooled on the autosampler In some cases, precipitation may occur before the injection operation.

An example of a more specific method of the first step is described using sphingosyl phosphoryl choline (SPC) as an example (see also the examples described later). First, blood is collected from a subject by a known method, and plasma is prepared by a known method. Next, an organic solvent containing an internal standard of known amount is added to plasma to make a sample solution. Suitable internal standard substances for SPC include SPC-C17 (B in FIG. 1), SPC labeled with stable isotope, and the like. The sample solution is applied to a solid phase extraction column. Elute the SPC and internal standard and dry under a stream of nitrogen. It is then resuspended in a solvent suitable for mass spectrometry and mass spectrometry is performed. The concentration of SPC is calculated by comparing the peak area obtained with the peak area of the internal standard of known concentration.

(Second step)
The "sample" used in the second step is a sample derived from urine collected from a living body (subject). The living body (subject) in the first step and the living body (subject) in the second step are the same individual.

The second biomarker has a statistically significant difference in the amount of the above urine-derived sample between an individual suffering from Niemann-Pick disease type C and an individual not suffering from Niemann-Pick disease type C It is a biomarker. The second biomarker includes bile acid metabolites and the like. The bile acid metabolites, 3.beta .- Surufookishi -7β-N- acetylglucosaminyltransferase-5-cholenic-24-Noikku acid ^{(SNAG-Δ 5 -CA),} 3β- Surufookishi -7β-N- acetylglucosaminyl - 5-cholenic-24-Noikku acid glycine conjugates ^{(SNAG-Δ 5 -CG),} 3β- Surufookishi -7β-N- acetylglucosaminyltransferase-5-cholenic-24-Noikku acid taurine conjugate (SNAG-Δ ⁵ - CT) etc. The first biomarker and the second biomarker may be different from each other.

An example of a more specific method of the second step is described using SNAG-Δ ⁵ -CA as an example (see also the examples described later). First, urine is collected from a subject by a known method. Next, an aqueous solution containing a known amount of internal standard substance is added to the urine to make a sample solution. Preferred internal standards for SNAG-Δ ⁵ -CA include 3β-sulfooxy-7β-hydroxy-23-nor-5-cholic acid (D in FIG. 1) or stable isotope labeled SNAG-Δ ^5- CA etc. are mentioned. The sample solution is injected into a pre-equilibrated trapping column, washed with a solvent and eluted. Then, mass spectrometry is performed. The concentration of SNAG-Δ ⁵ -CA is calculated by comparing the peak area obtained with the peak area of the internal standard of known concentration.

(Determination of Niemann-Pick disease type C)
In this embodiment, the amount of the first biomarker in the sample from the subject measured in the first step, and the amount of the second biomarker in the sample from the subject measured in the second step Based on, Niemann-Pick disease type C is determined.

In one embodiment, the result of comparing the amount of the first biomarker in a sample derived from a subject with a reference value, and the result comparing the amount of a second biomarker in a sample derived from a subject with the reference value Based on the Niemann-Pick disease type C is determined. Therefore, the test method according to the present embodiment may include the step of comparing the amount of the first biomarker in the sample derived from the subject with the reference value of the first biomarker. The inspection method according to the present embodiment may include the step of comparing the amount of the second biomarker in the sample derived from the subject with the reference value of the second biomarker.

In this embodiment, data may be provided regarding the amount of the first biomarker in a sample derived from blood collected from a control subject. In this embodiment, data may be provided regarding the amount of the second biomarker in a sample derived from urine collected from a control subject. The data may be measured in advance, or may be measured during a series of operations of the inspection method according to the present embodiment. The control subject may be only an individual not suffering from Niemann-Pick disease type C, or only an individual suffering from Niemann-Pick disease type C, or both of them. Moreover, although a control subject may be one individual, it is preferable that there are two or more individuals, and the more the number, the more preferable because the accuracy is improved. Alternatively, a reference value derived from such data may be prepared. The reference value takes into account statistical variation in, for example, the lower limit value in a control subject consisting of an individual suffering from Niemann-Pick disease type C, and the average value of a control subject consisting of an individual not suffering from Niemann-Pick disease type C These values, values in the literature, or any of these may be considered in combination.

Whether the subject suffers from Niemann-Pick disease type C if at least one of the amount of the first biomarker and the amount of the second biomarker in the sample derived from the subject is greater than or equal to the reference value in the biomarker It can be determined that the patient is likely to suffer from Niemann-Pick disease type C. For example, if the amount of the first biomarker and the amount of the second biomarker are two-dimensionally plotted, and at least one of them is a reference value or more, you are suffering from Niemann-Pick disease type C, or Niemann-Pick disease type C It can be determined that there is a high possibility of suffering from Alternatively, the subject suffers from Niemann-Pick disease type C if both the amount of the first biomarker and the amount of the second biomarker in the sample from the subject are greater than or equal to the reference value in the biomarker. It can be determined that there is a high possibility of having or suffering from Niemann-Pick disease type C.

Alternatively, the amount of the biomarker in the sample derived from the subject is compared with a reference value, and the difference is determined by, for example, a statistical method such as t test, F test, chi-square test, or Mann-Whitney's U test. The probability of suffering from Niemann-Pick disease type C may be calculated according to

In another embodiment, the amount of the first biomarker and the amount of the second biomarker may be converted into one parameter (value) and used. In this case, for example, the amount of the first biomarker and the amount of the second biomarker are used to calculate a logistic regression score. The calculation method may be, for example, the method described in the literature: Pepe, MS, Cai, T., and Longton, G. (2006), Biometrics 62, 221-229. The reference value can also be calculated as one parameter (value) in the same manner. For this logistic regression score, if the subject is higher than the standard value, determine that the subject is likely to suffer from Niemann-Pick disease type C or is likely to suffer from Niemann-Pick disease type C. Can.

Alternatively, the logistic regression score in the sample derived from the subject is compared with the reference value, and the significant difference thereof is determined by a statistical method such as t test, F test, chi-square test, or Mann-Whitney's U test. The probability of suffering from Niemann-Pick disease type C may be calculated.

Since the test method according to the present embodiment utilizes two biomarkers respectively derived from different samples from the same subject, diagnostic accuracy is improved when using one biomarker alone and in comparison with the conventional method. Can improve.

One embodiment of the present invention is based on the amount of the first biomarker and the amount of the second biomarker in the sample from the subject measured in the above-mentioned test method, and the subject suffers from Niemann-Pick disease type C. The present invention provides a diagnostic method for Niemann-Pick disease type C, which includes the step of determining whether or not the patient is likely to suffer from Niemann-Pick disease type C. In one embodiment, when at least one of the amount of the first biomarker and the amount of the second biomarker in the sample derived from the subject measured in the above test method is equal to or greater than a reference value in the biomarker, It is determined that the subject has Niemann-Pick disease type C or is likely to suffer from Niemann-Pick disease type C. In one embodiment, when both the amount of the first biomarker and the amount of the second biomarker in the sample derived from the subject measured in the above-mentioned test method are equal to or higher than the reference value in the biomarker, Determining that the body suffers from Niemann-Pick disease type C or is likely to suffer from Niemann-Pick disease type C.

The test method according to the present embodiment may include the step of measuring the amount of additional biomarkers other than the first biomarker and the second biomarker.

Note that the test method according to the present embodiment includes “a step of measuring the amount of the first biomarker in the sample derived from blood and the amount of the second biomarker in the sample derived from urine. It can also be referred to as “test method for diagnosis of disease type C”. In addition, the test method according to the present embodiment includes “a step of testing using a first biomarker in a sample derived from blood and a second biomarker in a sample derived from urine. It can also be referred to as “test method for diagnosis of type”.

[2. Test method for evaluating the therapeutic effect of Niemann-Pick disease type C]
The present invention further provides a test method for evaluating the therapeutic effect of Niemann-Pick disease type C, which comprises the above-mentioned first step and the above-mentioned second step.

(First step and second step)
In one embodiment, the description of each process is [1. Test method for diagnosis of Niemann-Pick disease type C] is as described in the section.

(Evaluation of therapeutic effect of Niemann-Pick disease type C)
In this embodiment, the amount of the first biomarker in the sample from the subject measured in the first step, and the amount of the second biomarker in the sample from the subject measured in the second step To assess the therapeutic effect of Niemann-Pick disease type C.

In one embodiment, the result of comparing the amount of the first biomarker in the sample from the subject at the first time point with the amount of the first biomarker in the sample from the subject at the second time point; Based on the result of comparing the amount of the second biomarker in the sample from the subject at 1 time point with the amount of the second biomarker in the sample from the subject at the second time point, Evaluate the treatment effect. Therefore, the test method according to the present embodiment includes the amount of the first biomarker in the sample from the subject at the first time point and the amount of the first biomarker in the sample from the subject at the second time point A step of comparing may be included. In the test method according to the present embodiment, the amount of the second biomarker in the sample derived from the subject at the first time point and the amount of the second biomarker in the sample derived from the subject at the second time point A step of comparing may be included.

The first time point may be at some time before or after the start of treatment for Niemann-Pick disease type C (including after the end of treatment). The second time point may be later than the first time point and at a certain time point after the start of the treatment (including after the end of the treatment). In one aspect, if the amount of biomarker in the sample from the subject at the second time point is reduced compared to the amount of biomarker in the sample from the subject at the first time point, then there is a therapeutic effect It can be evaluated. In one aspect, if the amount of biomarker in the sample from the subject at the second time point is reduced compared to the amount of biomarker in the sample from the subject at the first time point, then there is a therapeutic effect It can be evaluated. If either the first biomarker or the second biomarker is decreased, it may be evaluated as having a therapeutic effect, or only if both have a therapeutic effect. May be Alternatively, the amount of the first biomarker and the amount of the second biomarker may be converted into one parameter (value) and compared as described above.

The test method according to the present embodiment may include the step of measuring the amount of additional biomarkers other than the first biomarker and the second biomarker.

Note that the test method according to the present embodiment includes “a step of measuring the amount of the first biomarker in the sample derived from blood and the amount of the second biomarker in the sample derived from urine. It can also be referred to as “test method for evaluating the treatment effect of disease type C”. In addition, the test method according to the present embodiment includes “a step of testing using a first biomarker in a sample derived from blood and a second biomarker in a sample derived from urine. It can also be referred to as “test method for evaluating the therapeutic effect of type”.

(Application to screening of therapeutic agents)
Test methods for evaluating the therapeutic effect of Niemann-Pick disease type C may be used for screening of therapeutic agents. That is, a candidate compound of a therapeutic agent can be administered to a subject, evaluation of the therapeutic effect of Niemann-Pick disease type C can be performed as described above, and it can be determined that a candidate compound having a therapeutic effect can be used as a therapeutic agent. .

[3. Test kit for diagnosis of Niemann-Pick disease type C or evaluation of treatment effect of Niemann-Pick disease type C]
The present invention further provides a test kit for diagnosing Niemann-Pick disease type C or for evaluating the therapeutic effect of Niemann-Pick disease type C. The said test kit can be suitably used for implementation of the test method for a diagnosis of Niemann-Pick disease type C mentioned above, or the test method for evaluation of the therapeutic effect of Niemann-Pick disease type C.

The test kit according to one embodiment includes an internal standard substance used when measuring the amount of the first biomarker in a sample derived from blood collected from a living body, and a sample derived from urine collected from the living body And an internal standard used to measure the amount of the second biomarker.

The description of the internal standard is described above [1. Test method for diagnosis of Niemann-Pick disease type C] is as described in the section. In the test kit, the internal standard may be included in the form of a solution, in the form of a solid, etc.

The test kit according to the present embodiment further includes a solid phase extraction tube, a solid phase extraction plate, a dilution liquid, a washing liquid, an elution liquid, and a container for the first biomarker. , Solid-phase extraction tube, solid-phase extraction plate, diluent, washing solution, eluate, container and the like, and at least one of instruction manual and the like. The test kit according to the present embodiment may include any combination of these. In one example, the test kit may include either a solid phase extraction tube or a solid phase extraction plate. In one example, the test kit may include a diluent, a washing solution and an eluent in combination. In one example, the test kit may be a kit suitable for pretreatment by solid phase extraction.

The solid phase extraction tube is a solid phase extraction tube for purifying a biomarker. As a solid phase extraction tube, a syringe type solid phase extraction tube is mentioned, for example. The solid phase extraction tube is more suitable when the number of samples to be processed at one time is relatively small (for example, but not limited to, about 1 to 20).

The solid phase extraction plate is a solid phase extraction plate for purifying a biomarker. As the solid phase extraction plate, for example, a solid phase extraction plate in 96 well format can be mentioned. The solid phase extraction plate is more suitable when the number of samples to be processed at one time is relatively large (but not limited to, for example, 20).

The diluent is, for example, a liquid for diluting a sample containing a biomarker. The diluent used to dilute the blood-derived sample may be appropriately selected by those skilled in the art depending on the type of biomarker, but it may be, for example, water, a solid phase containing an organic solvent to sufficiently dissolve the biomarker, and conditioned. Examples include an aqueous solution in which the biomarker is sufficiently retained on the solid phase when added to an extraction tube, and Solution 1 (75% water + 25% methanol + 0.1% H ₃ PO ₄ ) described later. The diluent used to dilute the urine-derived sample may be appropriately selected by those skilled in the art depending on the type of biomarker, but it may be, for example, water, a solid phase containing an organic solvent to sufficiently dissolve the biomarker and conditioned. An aqueous solution in which the biomarker is sufficiently retained on the solid phase when added to the extraction tube, mobile phase A described later (methanol / water = 5/95 containing 5 mM ammonium acetate) and the like can be mentioned.

The washing solution is, for example, a liquid for washing the solid phase extraction tube or the solid phase extraction plate in which the biomarker is held. Such a liquid can be appropriately selected by those skilled in the art depending on the type of biomarker, and examples include Solution 1 and Solution 2 described in the examples below. The washing solution may be contained in the kit may be one type or two or more types.

The eluate is, for example, a liquid for eluting the biomarker from the solid phase extraction tube or the solid phase extraction plate in which the biomarker is retained. Such a liquid can be appropriately selected by those skilled in the art depending on the type of biomarker, and examples thereof include Solution 3 described in the examples below.

The container is, for example, a container for mixing the sample containing the biomarker and the internal standard substance, or a container for collecting the eluate containing the biomarker and the internal standard substance. The container is, for example, a tube or a plate. The volume of the container may be, for example, about 1.5 mL to 2.0 mL. Examples of the material of the container include plastic, glass and the like.

The instruction manual is described above [1. Test method for diagnosis of Niemann-Pick disease type C] The contents of the test method for diagnosis of Niemann-Pick disease type C according to the present invention described in the section of the present invention, and / or the above [2. Test Method for Evaluating Therapeutic Effect of Niemann-Pick Disease Type C According to the Present Invention Described in the section of 2. The contents of the examination method for evaluating the treatment effect of Niemann-Pick disease type C are recorded.

In addition, the test kit according to the present embodiment is a control sample used in measurement, a first biomarker in a sample derived from blood collected from a control subject used when analyzing measurement results. Data on the amount, data on the amount of the second biomarker in the sample derived from urine collected from the control subject used when analyzing the measurement results, a reference value derived from at least one of these data (The reference value for the first biomarker, the reference value for the second biomarker, and / or the amount of the first biomarker and the amount of the second biomarker are converted into one parameter (value) A medium in which at least one of the data of the reference value of the case, etc.) is described or recorded may be included.

The test kit according to the present embodiment can be used, for example, in combination with any one or more of the following. These may be prepared by the user. Alternatively, in another embodiment, any one or more of these may be included as a component of the test kit.

Vials for autosamplers etc. (eg (a) vials made of glass or plastic compatible with LC-MS autosampler sample table used and vial caps with Teflon (R) seal, or (b) deep bottom 96 Well plate and organic solvent resistant dedicated seal for LC-MS analysis for the purpose of preventing solvent evaporation, dedicated mat for preventing solvent evaporation, or aluminum foil etc.),
Equipment for solvent removal (for example, (a) a dedicated instrument for solvent removal which has a nitrogen generator, nitrogen cylinder, etc., and a head compatible with 1.5 mL tubes and 96 wells, (b) centrifugal Concentrator, etc.),
Solid phase extraction apparatus (for example, pressure type of syringe type, pressure reduction type using vacuum pump or water flow pump, etc.),
Devices used for sample filtration or centrifugation (for both filtration and centrifugation, single (directly connected to a syringe)) and devices corresponding to 96 wells, etc. Centrifugal filter tubes (spins) The column may be packed with a solid phase extraction carrier, and may be in the form of solid phase extraction by centrifugation using a tube).
Analytical columns required for LC-MS, mobile phase solvents, etc. (water, organic solvents, and additives such as ionizing agents),
In addition, reagents (methanol for LC-MS, acetonitrile, MilliQ water, etc.) and equipment generally used for LC-MS as needed.

In addition, each operation described above may be appropriately implemented in the above-described inspection method.

[4. Inspection system for diagnosis of Niemann-Pick disease type C]
The invention further provides a test system for measuring the amount of the first biomarker and the amount of the second biomarker. The test system may include, as a component, software (a program) for measuring the amount of the first biomarker and / or the amount of the second biomarker. The program may be stored in a computer-readable recording medium, and as such a recording medium, "non-temporary tangible medium", for example, ROM (Read Only Memory), etc. Examples include tapes, disks, cards, semiconductor memories, and programmable logic circuits. Also, the program may be supplied to the computer via any transmission medium (communication network, broadcast wave, etc.) capable of transmitting the program.

[5. Other]
Above [1. Test Method for Diagnosis of Niemann-Pick Disease Type C] The result obtained by performing the test method described in the section can be used as one of diagnostic data when a doctor makes a diagnosis. Moreover, the above [1. Test method for diagnosis of Niemann-Pick disease type C]] By performing the test method described in the section, there is a need for a subject for which the result that there is a possibility of suffering from Niemann-Pick disease type C is obtained Depending on the result of the definite diagnosis by the doctor, the treatment can be performed. In particular, in the examination method according to the present embodiment, since Niemann-Pick disease type C can be detected with high accuracy, early diagnosis and early treatment can be realized.

More specifically, for example, based on the result obtained by performing the test method according to the present embodiment, when the doctor judges that "it may suffer from Niemann-Pick disease type C", As a result of a test method (such as bone marrow test, identification of mutation of NPC1 or NPC2 of the causative gene, or filipin staining), a definite diagnosis can be performed in consideration of family history and the like. Based on this, a treatment plan is made.

[6. Examples of specific embodiments according to the present invention]
In order to solve the above problems, a test method for diagnosis of Niemann-Pick disease type C according to one aspect of the present invention comprises an amount of a first biomarker in a blood-derived sample collected from a living body And measuring the amount of the second biomarker in a sample derived from urine collected from the living body.

In the test method according to one aspect of the present invention, the first biomarker is preferably a sphingophospholipid.

In the test method according to one aspect of the present invention, the sphingophospholipid is more preferably sphingosyl phosphoryl choline.

In the test method according to one aspect of the present invention, the sphingophospholipid is more preferably lysosphingomyelin-509.

In the test method according to one aspect of the present invention, the second biomarker is preferably a bile acid metabolite.

In the test method according to one aspect of the present invention, the bile acid metabolite is 3β-sulfoxy-7β-N-acetylglucosaminyl-5-colene-24-noic acid (SNAG-Δ ⁵ -CA) Is more preferred.

In the test method according to one aspect of the present invention, it is preferable to use an internal standard substance in measurement of the amount of the first biomarker.

In the test method according to one aspect of the present invention, it is preferable to use an internal standard substance in measurement of the amount of the second biomarker.

In the test method according to one aspect of the present invention, it is preferable to measure the amount of the first biomarker and the amount of the second biomarker by LC-MS.

In one embodiment of the present invention, there is also provided a method of measuring the amount of a first biomarker in a sample derived from blood collected from a living being, and a second biologic in a sample derived from urine collected from the living being And determining the amount of the marker, and providing a test method for evaluating the therapeutic effect of Niemann-Pick disease type C.

One embodiment of the present invention is also directed to an internal standard substance used for measuring the amount of the first biomarker in a blood-derived sample collected from a living body, and a sample derived from urine collected from the living body. A test kit for diagnosis of Niemann-Pick disease type C or evaluation of the therapeutic effect of Niemann-Pick disease type C comprising an internal standard substance to be used in measuring the amount of the biomarker of No. 2 is provided.

Examples will be shown below, and the embodiment of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it is needless to say that various aspects are possible as to details. Furthermore, the present invention is not limited to the embodiments described above, and various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining the disclosed technical means are also included. It is included in the technical scope of the invention. Also, all of the documents described in the present specification are incorporated by reference.

(Cross-reference to related applications)
This application claims the benefit of priority to Japanese Patent Application No. 2017-242131 filed on Dec. 18, 2017, the entire contents of which are hereby incorporated by reference. Included in this book. Also, the contents described in all the publications including the patent and patent application cited in the related application are all incorporated herein by reference.

[Experimental procedure]
(reagent)
D-erythro-sphingosyl phosphoryl choline (synthetic SPC) was purchased from Toronto Research Chemicals. Sphingosyl phosphoryl choline (C17 based, IS _SPC ) was purchased from Avanti Polar Lipids. Acetonitrile and methanol were purchased from Fischer Scientific. Deionized water was obtained from Milli-Q water system (Millipore). Ammonium acetate and formic acid were purchased from Kanto Chemical Co., Ltd. The Oasis HLB 96-well plate was purchased from Waters. 3β-sulfooxy-7β-hydroxy-23-nor-5-cholenic acid was synthesized as described in 11 and 24 and was used as an internal standard substance (IS _{bile acid} ) for SNAG-Δ ^5- CA Using. The other reagents used in this study were the highest grade commercially available. The chemical structures of the compounds used in this study are shown in FIG.

(Approved by the Ethics Review Board)
This study was approved by the Ethics Review Board of the National Center for Child Health Research.

(Patient sample)
All NPC patients were diagnosed by conventionally established methods (11 and 14).

(Solid phase extraction)
The preparation and analysis of plasma samples by a slightly modified LC-MS / MS assay is as described in [5]. Briefly, a portion of plasma (100 μL) was mixed with Solution 1 (900 μL, 75% water + 25% methanol + 0.1% H ₃ PO ₄ ) containing 2.169 pmol of IS _SPC . Then, the above suspension was previously washed with hexane (1 mL) and methanol (1 mL), followed by equilibration with Solution 1 (1 mL × 2) Oasis HLB 96-well plate (30 μm, 30 mg, manufactured by Waters) as described above. Put. The column was then filled with Solution 1 (1 mL) and Solution 2 (75% water + 25% methanol, 1
Washed with mL). After that, it was eluted with Solution 3 (3 times with 0.01% NH ₃ -containing methanol, 0.4 mL). Finally, after removing the solvent under N ₂ using a dry gas generator (Nippon Seiki Co., Ltd.), the residue is resuspended in Solution 4 (90% methanol + 10% water + 0.1% formic acid, 60 μL). It became cloudy.

(Quantification of plasma SPC and lyso sphingomyelin-509)
A portion (1 μL) was injected into a Nexara UPLC system connected to an LCMS 8030 plus mass spectrometer (Shimadzu Corporation). InertSustainSwift C18 column (SPC, lyso sphingomyelin-509 and IS _SPC) using mobile phase A (methanol / water = 5/95 containing 5 mM ammonium acetate) and mobile phase B (methanol containing 0.1% formic acid) 2.1 × 30 mm, 3 μm, separated by GL Science Inc. (3 minutes, flow rate 0.5 mL / minute). The temperature of the analysis column was kept at 50 ° C. using a column oven CTO-10 (Shimadzu Corporation). The column was maintained with 60% mobile phase B for the first 0.5 minutes, followed by a linear gradient with 60-100% mobile phase B for 0.5-1.5 minutes. The column was then washed with 100% mobile phase B for 1.5-2.0 minutes, followed by equilibration with 60% mobile phase B for 2.0-3.0 minutes. Data were acquired by multiple reaction monitoring (MRM) and electrospray (ESI) positive mode. Chromatographic data were collected using the software LabSolutions (Shimadzu Corporation). The details of the device are described in Tables 1 to 3.

(Determination of urinary bile acid metabolite SNAG-Δ ^5- CA)
The concentration of SNAG-Δ ⁵ -CA was quantified using an automated LC-MS / MS system with on-line sample purification as described in [11]. Briefly, a portion (50 μL) of a diluted urine sample (100 μL of urine + 100 μL of 2 μM IS _{bile acid} water) containing an internal standard, 20 mM aqueous ammonium acetate (pH 5.5) and methanol (9/1) In a trapping column (Shim-pack MAYI-C8, 5 μm, 4.6 × 10 mm, Shimadzu Corp.) at a flow rate of 1.0 mL / min using a two-component mobile phase containing Project. After 3 minutes, YAG-Pack Pro C18 (with a flow rate of 0.2 mL / min) using SNAG-Δ ^5- CA with 20 mM aqueous ammonium acetate (pH 5.5) and methanol (5/5, v / v) It was transferred to 5 μm, 2.0 × 150 mm, YMC). The temperature of the column was kept at 40 ° C.

An API 5000 mass spectrometer (AB Sciex) was used to detect SNAG-Δ ⁵ -CA. For the selected reaction monitoring mode, the combination of m / z 672.3 for Q1 and m / z 97.0 for Q3 was chosen. The residence time was set to 250 ms, and the collision energy was set to -70V. Data were analyzed using Analyst 1.4.1 software (AB Sciex). The details of the device are described in Tables 4 to 6.

(Statistical analysis)
Data were expressed as mean ± standard deviation. The mean values of the two groups were compared using Student's t-test. Differences were considered statistically significant with p <0.05.

〔result〕
(Validity of the assay)
Alternative biopsies of lysosphingomyelin-509 using reverse-phase chromatography with MS / MS-based detection to quantify plasma SPC and lysosphingomyelin-509 concentrations with the highest possible sensitivity and accuracy SPC was adopted as a marker. Under the assay conditions of this study, both SPC and IS _SPC showed linear responses in the range of 1 to 200 fmol (FIG. 2). Under these analysis conditions, the intra-day error CV (%) was 7% at 216 nM, 6% at 108 nM, and 6% at 21.6 nM (n = 5). In order to assess the recovery of SPC in biological samples, we first examined the recovery of SPC in PBS as a blank matrix. When a known amount of SPC (11 nM) was added, it was detected in PBS with 119% recovery (Figure 3 and Table 7). Similarly, recovery when known amounts of SPC (11 nM) were added to the plasma was 120% under this assay condition. The daily error CV (%) when adding known amounts of SPC to plasma was 19% at 108 nM SPC, 6% at 11 nM SPC, and 11% at 1 nM SPC (Table 8).

(Plasma SPC concentration and lysosphingomyelin-509 concentration in NPC patients)
Based on the results of this validation study, plasma SPC concentrations in NPC patients were first assessed. As shown in FIG. 4, the SPC peak (eluted at 1.5 minutes) was higher in all NPC patients compared to the control (healthy) (upper part in FIG. 4) but the amount of IS _SPC Did not change (the lower part of FIG. 4). In this study, the mean plasma SPC concentration in NPC patients is 8.2 ± 2.8 nM (median 7.0 nM; maximum 11.7 nM; minimum 5.1 nM; n = 5), and plasma SPC in controls The mean concentration was 3.1 ± 1.4 nM (median 2.9 nM; maximum 4.8 nM; minimum 1.5 nM; n = 7) (FIG. 5A). Thus, the mean rise in plasma SPC concentration in NPC patients was 2.6 times that of controls (Table 9).

Similarly, the mean of multiple medians of plasma lysosphingomyelin-509 in NPC patients was 65.2 times (maximum 73.2; min, 26.7; n = 5) (FIG. 5B) . To further confirm that the accumulation of these two biomarkers is consistent with the already characterized biomarkers of NPC, SNAG-Δ ⁵ -CA, an established biomarker for NPC in urine Tried to determine if it was rising in NPC patients. Therefore, the urinary SNAG-Δ ^5- CA concentration in samples collected from the same individual was quantified using 3β-sulfooxy-7β-hydroxy-23-nor-5- _{cholic acid} as IS _{bile acid} . According to LC-MS / MS assay, urinary SNAG-Δ ⁵ -CA concentration in samples of NPC patients is 2477 ± 2968 ng / mL (median 1332 ng / mL; maximum 7731 ng / mL; minimum 449 ng / mL; n = 5 And 39 ± 73 ng / mL (median 18 ng / mL; maximum 204 ng / mL; minimum 0 ng / mL; n = 7) for the control (FIG. 5C).

(Correlation between plasma SPC, plasma lyso sphingomyelin-509 and urinary bile acid metabolite SNAG-Δ ^5- CA)
FIG. 6A shows a summary of the correlation between plasma SPC and urinary bile acid metabolite SNAG-Δ ⁵ -CA. As shown in FIG. 6A, all NPC patients evaluated in this study showed higher concentrations in either SPC or SNAG-Δ ⁵ -CA. These results indicate that combined quantification of plasma SPC and urinary bile acid metabolite SNAG-Δ ⁵ -CA using LC-MS / MS provides a more reliable diagnostic criterion for NPC than a single biomarker Prove it. Similarly, a correlation between plasma lysosphingomyelin-509 and urinary bile acid metabolite SNAG-Δ ^5- CA concentration was clearly shown (FIG. 6B). In this experiment, the sensitivity of NPC patients is 100 by the combination of plasma biomarkers and urine metabolites, ie the combination of SPC concentration and SNAG-Δ ⁵ -CA concentration or lyso sphingomyelin-509 concentration and SNAG-Δ ⁵ -CA concentration. % And the specificity was 100%.

The present invention can be used for a clinical test kit for diagnosis of Niemann-Pick disease type C, an analysis method package including software, and the like.

Claims (11)

1. Measuring the amount of the first biomarker in a blood-derived sample collected from a living body;
   Measuring the amount of the second biomarker in a sample derived from urine collected from the living body, a test method for diagnosing Niemann-Pick disease type C.
2. The test method according to claim 1, wherein the first biomarker is a sphingophospholipid.
3. The test method according to claim 2, wherein the sphingophospholipid is sphingosyl phosphoryl choline.
4. The test method according to claim 2, wherein the sphingophospholipid is lyso sphingomyelin-509.
5. The test method according to any one of claims 1 to 4, wherein the second biomarker is a bile acid metabolite.
6. The test method according to claim 5, wherein the bile acid metabolite is 3β-sulfooxy-7β-N-acetylglucosaminyl-5-coren-24-noic acid (SNAG-Δ ^5- CA).
7. The test method according to any one of claims 1 to 6, wherein an internal standard substance is used in the measurement of the amount of the first biomarker.
8. The test method according to any one of claims 1 to 7, wherein an internal standard substance is used in the measurement of the amount of the second biomarker.
9. The test method according to any one of claims 1 to 8, wherein the amount of the first biomarker and the amount of the second biomarker are measured by LC-MS.
10. Measuring the amount of the first biomarker in a blood-derived sample collected from a living body;
    Measuring the amount of the second biomarker in a sample derived from urine collected from the living body, and a test method for evaluating the therapeutic effect of Niemann-Pick disease type C.
11. An internal standard substance used to measure the amount of the first biomarker in a blood-derived sample collected from a living body;
    Diagnosis of Niemann-Pick disease type C or evaluation of the therapeutic effect of Niemann-Pick disease type C, including an internal standard substance used to measure the amount of the second biomarker in a sample derived from urine collected from the living body Test kit for

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