JP2007315852A - Estimation method of lipid metabolism disorder - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply and certainly estimating or diagnosing whether a substance to be examined has the inducing possibility of a lipid metabolism disorder such as drug-induced phospholipidosis. <P>SOLUTION: The method for estimating or diagnosing the inducing possibility of the lipid metabolism disorder of the substance to be examined includes a process (1) for sampling a biospecimen from a mammal after the substance to be examined is administered to the mammal and a process (2) for measuring the specific in-vivo substance contained in the biospecimen to compare the obtained measuring result with the measuring result when the substance to be examined is not administered and judging that the substance to be examined has the inducing possibility of the lipid metabolism disorder when a significant difference is recognized in the measuring result. The specific in-vivo substance is at least one kind of a substance significantly varied by respectively administering amiodarone and a phenoxypropylamine compound. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for predicting or diagnosing whether or not a test substance may induce dyslipidemia. More specifically, the present invention relates to a method for easily and reliably predicting or diagnosing whether or not a test substance may induce lipid metabolism abnormality by measuring a change in a specific in-vivo substance.

  Drug administration may cause phospholipid accumulation in living tissues, and this condition is called drug-induced phospholipidosis. Drug-induced phospholipidosis is manifested by administration of various positive ion compounds having both hydrophilic and hydrophobic properties. (1) Digestion of drug-phospholipid conjugate with phospholipase Poor, (2) Inhibition of direct phospholipase activity by drug, (3) Decrease in phospholipase activity due to increase in lysosomal pH resulting from incorporation of drug into lysosome, (4) Increase in phospholipid synthesis by drug Yes. In addition, when the accumulated substances are neutral fat and sphingomyelin, they are called steatosis and sphingolipidosis, respectively, and these pathologies including phospholipidosis are dyslipidemia ( lipidosis). In order to create a drug candidate compound with low risk of dyslipidemia in humans, it is extremely easy to reliably and reliably predict the induction potential of dyslipidemia of a drug candidate compound at the stage of exploratory research for new drugs. is important. However, histopathological examination is a general method for diagnosing dyslipidemia in non-clinical safety evaluation during the development of new drugs. However, if dyslipidemia does not progress to some extent, it is observed as histopathological changes. It is difficult. In addition, in cases where dyslipidemia is observed when the drug candidate compound is administered to an experimental animal at a dose much higher than the clinically estimated use amount, or in cases where the observed degree of dyslipidemia is minor, Considering the risks and benefits when the drug candidate compound is administered to humans, it may proceed to the clinical trial stage, in which case lipid metabolism is measured using a biological sample collected by a method with less surgical invasion. It is necessary to proceed with clinical trials carefully while diagnosing abnormalities. For these reasons, there is a need for the development of a method for predicting dyslipidemia easily and reliably, or for reliably diagnosing dyslipidemia using biological samples collected by a method with little surgical invasion. Yes.

As such means, a means for predicting or diagnosing a substance that may induce dyslipidemia using a substance in a living body as a marker has been proposed. For example, phenacerucic acid (phenylacetylglycine, PAG; Magn. Reson. Chem., 39, pp. 559-565, 2001; Biomarkers, 5, pp. 410-423, 2000), lysobisphosphatidic acid (Biochim. Biophys. Acta, 1631, pp.136-146, 2003), lipid droplets in leukocytes (J. Appl. Toxicol., 26, pp.167-177, 2005), gene expression in leukocytes (Abstracts of American Society of Toxicology, pp.35) , 2006) is known to be usable as a biomarker for phospholipidosis, and can be expected to be used as a biomarker for evaluating the possibility of inducing a phospholipid metabolism disorder of a test substance. In addition, a method has been proposed for predicting the possibility of induction of abnormal lipid metabolism of a candidate compound in the development stage of a drug, using changes in the balance of the phenylalanine metabolic pathway as an index (International Publication WO2005 / 64344).
International Publication WO2005 / 64344 Magn. Reson. Chem., 39, pp.559-565, 2001 Biomarkers, 5, pp.410-423, 2000 Biochim. Biophys. Acta, 1631, pp.136-146, 2003 J. Appl. Toxicol., 26, pp.167-177, 2005 Abstracts of American Society of Toxicology, pp.35, 2006

An object of the present invention is to provide a method for easily and reliably predicting whether or not a test substance has a possibility of inducing lipid metabolism disorders such as drug-induced phospholipidosis. More specifically, it is an object of the present invention to provide a method for easily and reliably predicting the possibility of inducing dyslipidemia for a test substance by detecting a change in a specific in-vivo substance.
Another object of the present invention is to provide a method for easily and reliably diagnosing the presence or absence of onset of lipid metabolism disorders such as drug-induced phospholipidosis by detecting changes in specific in-vivo substances, and for the same It is to provide means.

  As a result of intensive studies to solve the above problems, the present inventors have found that when a substance known to induce phospholipidosis, which is one of dyslipidemias, is administered to animals, 23 types of identification It has been found that fluctuations in in vivo substances occur. Based on this knowledge, predicting whether or not the test substance has the possibility of inducing lipid metabolism disorder by detecting a change in the biological substance after administering the test substance And by using this specific in vivo substance as a biomarker, the possibility of developing lipid metabolism disorders such as drug-induced phospholipidosis in mammals including humans was collected by a method with less surgical invasion. It has been found that simple and reliable diagnosis can be performed using a biological sample. The present invention has been completed based on these findings.

That is, according to the present invention, there is provided a method for predicting or diagnosing the possibility of inducing a lipid metabolism disorder of a test substance, comprising the following steps:
(1) A step of collecting a biological sample from a mammal after administering the test substance to a mammal other than a human;
(2) Measure the specific biological substance contained in the biological sample, and the measurement result obtained is a measurement result of the specific biological substance contained in the biological sample collected from a mammal not administered the test substance And a step of determining that the test substance may induce dyslipidemia when a significant difference is found in these measurement results, and the specific biological substance is amiodarone and Phenoxypropylamine compound (I) represented by the formula:
Methods are provided that are one or more substances that vary significantly with each administration of.

  According to a preferred embodiment of the above invention, the method of measuring a specific in vivo substance by metabolome analysis; the specific in vivo substance is L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4-hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2′-carboxyethyl-4,6- Dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-aspartic acid, trimethylamine N-oxide, hypotaurine, fructosyl lysine, betaine, L-asparagine, L-ornithine, L-lysine, The above method, which is one or more substances selected from the group consisting of L-methionine and L-histidine; Internal substances are L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4-hydroxy Selected from the group consisting of phenylacetylglycine, 3-indoxyl sulfate, 5-2'-carboxyethyl-4_6-dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-aspartic acid The above method wherein the specific biological substance is L-carnitine; the biological sample is urine, plasma, serum, or tissue homogenate; the tissue homogenate is There is provided the above method which is a lung homogenate or a liver homogenate; and the above method wherein the test substance is a drug candidate compound.

Further, according to the present invention, there is provided a screening method for a drug candidate compound, wherein a test substance determined to have a possibility of inducing a lipid metabolism disorder by the above prediction method is excluded from the test substance group, and the lipid metabolism disorder is detected. There are provided a method comprising the step of selecting a drug candidate compound that is not inducible, and a library comprising the drug candidate compound selected by the screening method.
Furthermore, the biomarker is used for predicting the possibility of the test substance to induce lipid metabolism disorders and the use of the specific in vivo substance as a biomarker to predict the possibility of the test substance to induce lipid metabolism disorders. Thus, the present invention provides a biomarker containing the above specific biological substance.

From another viewpoint, a method for diagnosing dyslipidemia, comprising the following steps:
(1) a step of collecting a biological sample from a mammal individual including a human;
(2) Measure a specific biological substance contained in the biological sample, compare the obtained measurement results with the measurement result of the specific biological substance contained in a biological sample collected from a healthy person, and measure these When a significant difference is found in the result, the individual includes a step of determining that the individual has a possibility of developing dyslipidemia, and the specific biological substance is amiodarone and phenoxypropyl represented by the above structural formula Methods are provided that are one or more substances that vary significantly with each administration of amine compound (I).

  According to a preferred embodiment of the above invention, the method of measuring a specific in vivo substance by metabolome analysis; the specific in vivo substance is L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4-hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2′-carboxyethyl-4,6- Dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-aspartic acid, trimethylamine N-oxide, hypotaurine, fructosyl lysine, betaine, L-asparagine, L-ornithine, L-lysine, The above method, which is one or more substances selected from the group consisting of L-methionine and L-histidine; Internal substances are L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4-hydroxy Selected from the group consisting of phenylacetylglycine, 3-indoxyl sulfate, 5-2'-carboxyethyl-4_6-dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-aspartic acid The above method wherein the specific biological substance is L-carnitine; the biological sample is urine, plasma, serum or tissue homogenate; the tissue homogenate is A method as described above, wherein the disorder is pulmonary homogenate or liver homogenate; and dyslipidemia is hereditary lipidosis, drug-induced lipidosis, or The above methods are provided wherein the fatty acid metabolism homeostasis is abnormal.

Also, the use of the above specific biological substance as a diagnostic marker for dyslipidemia and a diagnostic marker for dyslipidemia, comprising the above specific biological substance, are provided by the present invention. Is done.
Furthermore, a reagent for measurement capable of chemically or biologically detecting the above biomarker or the above diagnostic marker; a lipid metabolism abnormality comprising as an active ingredient a substance having an action of suppressing the fluctuation of the above specific biological substance A lipid metabolism disorder comprising as an active ingredient a drug for the prevention and / or treatment of infectious diseases; and a substance having an action of suppressing the expression of the specific biological substance or an action of enhancing the expression of the specific biological substance A medicament for the prevention and / or treatment of is also provided by the present invention.

  According to the prediction method of the present invention, it is possible to easily and surely determine whether or not a test substance has a possibility of inducing dyslipidemia without performing a long-term toxicity test using an experimental animal. New drug candidate compounds can be rapidly screened. In addition, the diagnostic method of the present invention is characterized in that a simple and highly accurate diagnosis can be performed without causing pain to the patient by using the diagnostic marker.

  The method of the present invention is a method for predicting the possibility of inducing dyslipidemia of a test substance, the following steps: (1) After administering the test substance to a mammal other than a human, a biological sample from the mammal And (2) measuring a specific biological substance contained in the biological sample, and specifying the measurement result obtained from the biological sample collected from a mammal not administered with the test substance. A step of determining that there is a possibility that the test substance induces dyslipidemia when there is a significant difference in the measurement results compared to the measurement results of the in vivo substance, and The method is characterized in that the in-vivo substance is one or more substances that vary significantly depending on the administration of amiodarone and the phenoxypropylamine compound (I) represented by the above structural formula.

  A method for predicting dyslipidemia caused by a drug candidate compound using a biosensor and a method for diagnosing dyslipidemia and related diseases are described in International Publication WO 2005/64344, and used for the prediction method. Type of biological sample (cell or tissue, etc.) to be collected, biological sample collection method, type of experimental animal, means for exposing the experimental animal to the test substance, and lipid metabolism disorder to which the diagnostic method is applied and its Related diseases and the like are described in detail and specifically. These explanations are useful for understanding the prediction method and diagnosis method of the present invention, and those skilled in the art can easily understand the present invention with reference to the above-mentioned publications. The entire disclosures of the above publications are incorporated herein by reference.

  The type of test substance applied in the prediction method of the present invention is not particularly limited. In addition to organic low molecular weight compounds, high molecular weight compounds, inorganic compounds, nucleic acids (including small molecule RNA and non-natural bases used for RNAi and the like) Any substance such as saccharides, lipids, or peptides (including oligopeptides or polypeptides) is included. In general, the prediction method of the present invention is preferably used as a screening means for drug candidate compounds, and the test substance is preferably a drug candidate compound.

  The kind of mammals other than humans used in the prediction method of the present invention is not particularly limited. For example, normal laboratory animals such as monkeys, rats, mice, guinea pigs, dogs, cats, and rabbits can be used. The method for administering the test substance to the mammal is not particularly limited, and an appropriate administration route can be selected according to the physicochemical properties of the test substance, the kind of animal, and the like. For example, an appropriate administration route such as oral administration, intravenous administration, or intraperitoneal administration can be selected.

  The type of biological sample is not particularly limited, and any biological sample such as urine, blood, saliva, lymph, tissue, or cell can be used, and an appropriate separation means is adopted depending on the type. be able to. For example, blood collected by blood collection can be used as the biological sample, and serum separated from the collected blood may be used as the biological sample. Further, a homogenate of a tissue collected from an organ or the like removed by means such as surgery can be used as a biological sample, and a homogenate supernatant may be used as a biological sample. For example, a tissue homogenate or a homogenate supernatant collected from an organ such as the lung or liver can be preferably used.

  The type of the specific biological substance contained in the biological sample is not particularly limited, but the specific biological substance includes two substances known to induce dyslipidemia: amiodarone and the above structural formula. It is necessary to use an in vivo substance that varies depending on each administration of the phenoxypropylamine compound (I) represented. About amiodarone, it is described in Toxicol. Appl. Pharmacol., 97, pp.124-133, 1987 and Hepatology, 8, pp.1063-1068, 1988 to develop dyslipidemia. The phenoxypropylamine compound (I) is a compound disclosed in WO 00/71517. Further, the lipid droplets in leukocytes observed by amiodarone administration are not observed by administration of phenoxypropylamine compound (I).

  The specific biological substance to be measured by the prediction method of the present invention may be a substance that is inherently present in the living body, preferably a substance that is inherently present in a certain concentration range, or lipid metabolism. It may be a substance that appears or disappears in an abnormality. The specific in-vivo substance may be any in-vivo substance or metabolite of the test substance. By measuring two or more kinds of specific in vivo substances, more accurate prediction may be possible. Specific biological substances include, for example, L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinoline Carboxylic acid, 4-hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2'-carboxyethyl-4_6-dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-asparagine One or more substances selected from the group consisting of acids, trimethylamine N-oxide, hypotaurine, fructosyl lysine, betaine, L-asparagine, L-ornithine, L-lysine, L-methionine, and L-histidine Preferably L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-hist , 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4-hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2'-carboxyethyl-4_6-dihydroxy Mention may be made of one or more substances selected from the group consisting of picolinate, guanidide acetate, 4-guanidinobutanoate and N-amidino-L-aspartic acid. Of these, L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4- Hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2′-carboxyethyl-4_6-dihydroxypicolinate are more preferred, and L-carnitine is most preferred. However, the specific in vivo substance is not limited to these.

  As used herein, the term “measurement” means to detect a specific in-vivo substance for the purpose of qualitative and / or quantitative, and includes concepts such as identification as well as concentration measurement. It should be interpreted in the broadest sense and the term should not be interpreted in any way restrictive. The term “measurement result” needs to be interpreted in the broadest sense including not only the measurement value of concentration but also an indicator such as the presence or absence of detection, and does not necessarily mean the obtained numerical value itself. For example, when the concentration of a specific in-vivo substance changes to a concentration below the detection limit, this result is also included in the concept of “measurement result”.

  The measuring means for the specific in vivo substance is not particularly limited, and any measuring means available to those skilled in the art can be used singly or in combination of two or more. For example, capillary electrophoresis and mass spectrometry are combined. It is preferable to perform measurement. See, for example, Anal. Chem., 77, pp. 78-84, 2005; Electrophoresis, 25, pp. 1964-1972, 2004; Journal of Proteome Research., 2, pp 488-494, 2003, etc. Can make this measurement easily. This method is characterized in that a specific in-vivo substance can be identified and quantified with high sensitivity and accuracy from the molecular weight and electrophoresis time. However, it is obvious to those skilled in the art that an appropriate measurement means can be adopted according to the type of the specific biological substance.

 Metabolome analysis means comprehensive measurement of the total amount of metabolites in the living body (metabolome). Metabolome measurement methods include capillary electrophoresis / mass spectrometry, gas chromatography / mass spectrometry, high performance liquid chromatography / mass spectrometry, Fourier transform ion cyclotron resonance mass spectrometry, nuclear magnetic resonance analysis (NMR), etc. Known (the forefront of metabolomic research, 2003, Springer Fairlark Tokyo Co., Ltd. (edited by Masaru Tomita, Takaaki Nishioka)).

  In addition, specific biological substances are untreated or appropriately derivatized, followed by separation techniques such as high-performance liquid chromatography and gas chromatography, UV, fluorescence, electrochemical detection methods, and free induction decay (FID: automatic). Induction decay), measurement methods that combine instrumental analysis methods such as mass spectrometry, enzyme immunoassay (ELISA), radioisotope immunoassay (RIA), chemiluminescent enzyme immunoassay (CLEIA), and other immune reactions The measurement can also be performed by a biochemical method that combines enzyme reaction and absorbance, fluorescence or luminescence detection.

  Measuring a specific biological substance contained in a biological sample, and comparing the obtained measurement result with a measurement result of the specific biological substance contained in a biological sample collected from a mammal not administered with the test substance; When a significant difference is recognized in these measurement results, it can be determined that the test substance may induce dyslipidemia. In the present specification, “significant difference” generally means that a significant difference is obtained between two measurement results by a well-known and conventional statistical analysis method, but not necessarily by a strict statistical method. If an apparent difference is recognized, it may be determined that there is a significant difference.

  When the test substance shows an inducibility of dyslipidemia, the concentration of the specific biological substance, tissue distribution, etc. vary. In this specification, the term “variation” refers to one or two types, for example, an increase or decrease in the concentration of a specific biological substance, disappearance, appearance, change in distribution within a tissue or cell or in a living body, or increased expression. It is a concept that includes the above, and should not be construed as limiting in any way. This fluctuation is caused by the measurement result of the specific biological substance contained in the biological sample collected from the animal administered with the test substance and the specific biological substance contained in the biological sample collected from the mammal not administered with the test substance. This can be easily confirmed by comparing with the measurement results. In general, fluctuations can be easily confirmed by setting the non-administration group of the test substance as the control group, but the measurement results obtained in advance from healthy mammals are prepared as standard results. You may determine the presence or absence of a significant difference compared with a result. It goes without saying that such an embodiment is also included in the method of the present invention.

  Using the above prediction method, a safe drug candidate compound that does not induce dyslipidemia can be screened. In this screening method, a test substance that may induce dyslipidemia is selected from the test substance group to be screened using the prediction method described above, and the test substance is excluded from the test substance group. And a step of obtaining the remaining test substance group as a screening result. Substances contained in the screening results thus obtained are safe substances that have no possibility of inducing dyslipidemia or extremely low possibility, and can be suitably used as pharmaceuticals. It is. In addition, a library composed of safe substance groups obtained in this way is useful as a substance source group for the development of new medicines.

  The method of the present invention provided from another viewpoint is a method for diagnosing dyslipidemia, comprising the following steps: (1) collecting a biological sample from an individual mammal including human; (2) the living body The specific biological substance contained in the sample is measured, and the obtained measurement result is compared with the measurement result of the specific biological substance contained in the biological sample collected from a healthy person, and there is a significant difference between these measurement results. And the phenoxypropylamine compound (I) wherein the individual includes a step of determining that the individual has the possibility of developing dyslipidemia, and the specific in vivo substance is amiodarone and the structural formula above. It is characterized in that it is one or more substances that vary significantly depending on the administration of each of these. Examples of mammals include, but are not limited to, humans, pets such as dogs and cats, and livestock such as cows and pigs. Preferably, diagnosis can be performed on human subjects.

  In the above diagnostic method, examples of dyslipidemia include hereditary lipidosis (eg, Gaucher disease, Niemann-Pick disease (types A to C), Fabry disease, Wolman disease, cholesterol ester storage disease, cerebral tendon xanthoma disease, Phytosterolemia, refsum disease, Tay-Sachs disease, systemic (GM1) gangliosidosis, sulfatid lipidosis (black-stained leukotrophy), galactosylceramide lipidosis, etc., drug-induced lipidosis (eg, PLsis, steatosis, Sphingomyelin accumulation disease, etc.), fatty acid metabolism homeostasis abnormality (for example, fatty acid β oxidation abnormality, etc.) can be mentioned, but are not limited thereto. In the present specification, the term “dyslipidemia” includes diseases associated with abnormal lipid metabolism, diseases caused by abnormal lipid metabolism, and the like. Such diseases are known as diseases related to dyslipidemia, such as hypertension, atherosclerosis, arteriosclerosis, myocardial infarction, fatty liver, hepatitis, cirrhosis, diabetes, dementia, Alzheimer's disease, heart In addition to diseases and chronic fatigue syndrome, pulmonary fibrosis, blindness, and encephalopathy are examples of drug-induced lipidosis-related diseases. However, the dyslipidemia which is the target of the diagnostic method of the present invention is not limited to these.

  The above diagnostic method can be performed basically in the same manner as the above prediction method. Preferably, urine or blood can be used as a biological sample separated from an individual. As a measurement result obtained from a healthy person, a measurement result obtained from a healthy person is prepared in advance as a standard result, and the result can be used. If there is a significant difference between the measured value obtained from the test individual and the measurement result obtained from the healthy subject, the test individual has already developed dyslipidemia or has dyslipidemia. Risk of developing. In the present specification, the term “having the possibility of developing dyslipidemia” means that the individual can clearly confirm that the individual has already developed dyslipidemia by histopathological means, Pathological evidence that the individual is suffering from dyslipidemia but other clinical data strongly suspected of developing dyslipidemia, or dyslipidemia in the future It should be interpreted in the broadest sense, including when there is a risk of developing it.

  The specific in-vivo substance is useful as a diagnostic marker for dyslipidemia. As a diagnostic marker, one kind of specific in vivo substance may be used, or two or more kinds of specific in vivo substances may be used in combination. For the convenience of the above diagnostic method, it is also desirable to provide a measuring reagent capable of specifically detecting the diagnostic marker by chemical or biological means, and each of the two or more types of diagnostic markers is specific. It is also desirable to provide a kit by combining the detection reagents that can be detected. Examples of the reagent that can specifically detect a specific in-vivo substance include labeled antibodies that specifically react with the specific in-vivo substance, but are not limited to this specific reagent.

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
Example 1
With amiodarone and phenoxypropylamine compound (I) as test substances, the presence or absence of phospholipidosis was examined by histopathological examination and phosphatidylcholine determination in lung tissue. In addition, intermediate and final metabolites in urine and serum were comprehensively analyzed using CE-TOFMS.

  Amiodarone was purchased from SIGMA, and phenoxypropylamine compound (I) was synthesized by Mitsubishi Pharma Corporation and used in the experiment. Six-week-old Crl: CD (SD) rats were purchased from Japanese Charles River and used for experiments after habituation for 5 days. The animals were randomly divided into 3 groups of 4 animals. The solvent control group was treated with 0.5% hydroxypropylmethylcellulose solution (HPMC), the amiodarone-administered group and the phenoxypropylamine compound (I) -administered group with HPMC, respectively. The suspension was orally administered repeatedly for 14 days at doses of 150 mg / kg and 100 mg / kg. Immediately after the last dose, all animals were transferred to metabolic cages and urine collected for 24 hours. At 24 hours after the final administration, the animals were anesthetized with diethyl ether and blood was collected. After exsanguination, the lungs were collected. After separating serum from blood by a conventional method, an extract with methanol / chloroform was added to CE-TOFMS. Urine was added to CE-TOFMS after removing impurities by centrifugation. The analysis by CE-TOFMS was performed according to the method described in Japanese Patent Application No. 2005-258684. The lung was divided into two parts, and a part of the tissue piece was prepared according to a conventional method to observe a histopathological change. The other tissue piece was frozen in liquid nitrogen immediately after collection, and phosphatidylcholine was quantified according to a conventional method.

  Histopathological examination revealed foamy alveolar macrophage aggregation, a characteristic of phospholipidosis, in all patients in the amiodarone-administered group. On the other hand, in the phenoxypropylamine compound (I) -administered group and the solvent control group, there was no observation suspicious of phospholipidosis in any individual. The results of phospholipid quantification in the lung are shown in FIG. The amount of phosphatidylcholine in the amiodarone-administered group and the phenoxypropylamine compound (I) -administered group was significantly increased as compared with the solvent control group. The increase rate of the phenoxypropylamine compound (I) administration group was about 1.5 times, while the amiodarone administration group showed an increase rate of about 3.5 times. Under these experimental conditions, the amiodarone administration group was more phenoxypropylamine compound (I ) From the administration group, the degree of phospholipidosis was considered to be severe.

After simultaneous analysis of in vivo substances by CE-TOFMS, comparison of each in vivo substance amount by metabolomic differential display method is performed by Williams' multiple comparison test and the amount of phosphatidylcholine in lung tissue for each individual The correlation coefficient of Pearson and Spearman was obtained between the two, and peaks with a correlation coefficient of 0.5 or more or -0.5 or less were extracted. For each peak, candidate substances were narrowed down based on accurate mass and electric mobility, and the structure of the substance was estimated by a mass spectrometer (MS / MS). Substances for which standard substances were available were compared and identified. As a result, as shown in Table 1, as metabolites that fluctuate in both the amiodarone-administered group and the phenoxypropylamine compound (I) -administered group, trimethylamine N-oxide, hypotaurine, guanidide acetate, 4-guanidinobuta are derived from urine. Noate, 4-beta-acetylaminoethylimidazole, L-carnitine, N-amidino-L-aspartic acid, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, fructosyl lysine, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, phenylacetylglycine, 4-hydroxyphenylacetylglycine, 3-indoxyl sulfate, and 5-2′-carboxyethyl-4_6-dihydroxypicolinate were found and Trimethylamine N-oxide, betaine, L-asparagine, L-ornithine, L- Jin, L- methionine and L- histidine were found. Substances identified or estimated were in vivo metabolites derived from amino acid metabolic pathways such as histidine and tryptophan, β-oxidation pathway of fatty acids and urea cycle. Among them, phenylacetylglycine has been reported to be useful as a biomarker for drug-induced phospholipidosis (Biomarkers, 5, pp. 410-423, 2000; Magn. Reson. Chem., 39, pp. 559). -565, 2000), suggesting that the prediction / diagnosis method of the present invention has high accuracy.

It is the figure which showed the amount of phosphatidylcholine in a lung tissue. In the figure, compound (I) shows the result of phenoxypropylamine compound (I). Data show mean values + standard deviation, and ^** and p <0.01 (Dunnett's multiple comparison test).

Claims (12)

A method for predicting the possibility of inducing dyslipidemia of a test substance, comprising the following steps:
(1) collecting a biological sample from a mammal after administering the test substance to a mammal other than a human; and
(2) Measure the specific biological substance contained in the biological sample, and the measurement result obtained is a measurement result of the specific biological substance contained in the biological sample collected from a mammal not administered the test substance And a step of determining that the test substance may induce dyslipidemia when a significant difference is found in these measurement results, and the specific biological substance is amiodarone and Phenoxypropylamine compound (I) represented by the formula:
A method that is one or more substances that vary significantly with each administration of. The method according to claim 1, wherein the measurement of the specific in vivo substance is performed by metabolomic analysis. Specific biological substances are L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4 -Hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2'-carboxyethyl-4,6-dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-aspartic acid, Claims that are one or more substances selected from the group consisting of trimethylamine N-oxide, hypotaurine, fructosyl lysine, betaine, L-asparagine, L-ornithine, L-lysine, L-methionine, and L-histidine Item 3. The method according to Item 1 or 2. Specific biological substances are L-carnitine, 4-beta-acetylaminoethylimidazole, N-acetyl-L-histidine, 1-ribosylimidazole-4-acetate, L-glutamic acid, 4-hydroxy-2-quinolinecarboxylic acid, 4 -Hydroxyphenylacetylglycine, 3-indoxyl sulfate, 5-2'-carboxyethyl-4_6-dihydroxypicolinate, guanidide acetate, 4-guanidinobutanoate, N-amidino-L-aspartic acid 3. The method according to claim 1 or 2, which is one or more substances selected from the group consisting of: The method according to claim 1 or 2, wherein the specific in-vivo substance is L-carnitine. The method according to any one of claims 1 to 6, wherein the biological sample is urine, plasma, serum, or tissue homogenate. The method of claim 6, wherein the tissue homogenate is a lung homogenate or a liver homogenate. A screening method for a drug candidate compound, wherein a test substance determined to have a possibility of inducing dyslipidemia by the prediction method according to any one of claims 1 to 7 is excluded from a test substance group. And selecting a drug candidate compound that is not likely to induce dyslipidemia. A biomarker for predicting the possibility of inducing dyslipidemia of a test substance, comprising the specific in vivo substance according to claim 1. A method for diagnosing dyslipidemia, comprising the following steps:
(1) a step of collecting a biological sample from a mammal individual including a human;
(2) Measure a specific biological substance contained in the biological sample, compare the obtained measurement results with the measurement result of the specific biological substance contained in a biological sample collected from a healthy person, and measure these The method includes a step of determining that the individual has a possibility of developing dyslipidemia when a significant difference is found in the results, and the specific in vivo substance is amiodarone and the phenoxypropylamine compound according to claim 1 The method which is 1 type, or 2 or more types of substance which changes significantly by each administration of (I). The method according to claim 10, wherein the dyslipidemia is inherited lipidosis, drug-induced lipidosis, or fatty acid metabolism homeostasis abnormality. A diagnostic marker for dyslipidemia, comprising the specific in-vivo substance according to claim 10.