JP2015020969A - Prevention/improvement agent for fatty liver - Google Patents

Abstract

[Problem] To provide a pharmaceutical, a non-pharmaceutical part or a food useful as an agent for preventing or improving fatty liver by suppressing the expression or action of fatty acid binding protein 1 (FABP1). As a compound that suppresses the expression or action of FABP1, a compound selected from the group consisting of (a) to (d), and a medicament useful as a prophylactic or ameliorating agent for fatty liver comprising the compound Or food. (A) siRNA targeting the FABP1 gene, (b) a nucleic acid construct for generating siRNA targeting the FABP1 gene in the cell, (c) an antisense nucleic acid targeting the transcription product of the FABP1 gene, (d) FABP1 Ribozymes targeting gene transcripts [selection] None

Description

  The present invention relates to prevention or improvement of fatty liver. Specifically, the present invention relates to a drug effective for the prevention or improvement of fatty liver and a screening method for an agent for preventing / ameliorating fatty liver.

  In recent years, along with an increase in metabolic syndrome, fatty liver in which neutral fat accumulates in the liver has also increased. Fatty liver progresses to cirrhosis and liver cancer as it progresses, and in recent years, it has become clear that it is involved in the exacerbation of metabolic syndrome and has become a pathological condition that should be the target of treatment. It has traditionally been thought that non-alcoholic fatty liver does not progress to a serious lesion. However, in recent years, it has become clear that non-alcoholic fatty liver progresses to cirrhosis and liver cancer through a pathological condition called NASH, and countermeasures are desired.

  Fatty liver is also found in about 30% of the patients in the nationwide medical docks. It has increased to nearly three times that of 20 years ago. In the future, with the establishment of diagnostic methods, the number of affected people is expected to increase further.

  Currently, there are no drugs that improve fatty liver itself, and dietary therapy and exercise therapy are the basic methods of improvement. In addition, antioxidants, antidiabetic agents, dyslipidemic agents and the like are used in combination depending on the medical condition.

  In addition, new drugs targeting fatty liver have been proposed or reported (for example, Patent Documents 1 and 2).

JP 2010-105948 A JP 2012-201620 A

  Current therapies for fatty livers depend on the patient's will for success or failure, treatment guidelines vary depending on hospital facilities, are difficult to enforce, take time to achieve therapeutic effects, There are many points to be solved, such as the fact that it may be difficult to carry out over a long period of time due to the environment, and the need for new treatment methods is high. Then, this invention makes it a main subject to provide a means effective in prevention or improvement of fatty liver.

  The present inventors have discovered that FABP3, which is a skeletal muscle type fatty acid binding protein (FABP), is expressed in the liver of the mouse in the course of studying in view of the above-mentioned problems, and FABP becomes fatty liver in the liver. I got the idea that it might play an important role. FABP is a small intracellular protein of 14 to 15 KDa and plays an important role in lipid transport. In humans, there are nine types of FABPs that are tissue-specific.

Based on the above idea, we decided to investigate in detail the involvement of FABP in fatty liver. First, when FABP1 showing liver-specific expression was forcibly expressed in the mouse liver, fatty liver was induced, and an increase in liver weight and neutral fat amount was observed. Based on this result, it was expected that fatty liver could be improved by limiting the expression or function of FABP1 in the liver. That is, the idea that suppression of FABP1 expression or function is effective for the prevention or improvement of fatty liver. In order to verify the effectiveness of the strategy of suppressing the expression or function of FABP1, an animal experiment using RNAi was conducted. As a result, the fatty liver was remarkably improved by suppressing the function of FABP1, and the effects exceeding the expectation that the liver weight and neutral fat were reduced were confirmed, and it was confirmed that the above strategy was effective. For prevention or improvement of fatty liver by suppressing the expression or function of FABP1, (i) the treatment method is not affected by the patient's intention and life as in the prior art, and (ii) it is more powerful than the conventional technique. Expected to be effective for treatment of fatty liver, (iii) Expected to be effective in a short period of time, while the conventional technology takes several months, (iv) FABP shows tissue-specific expression and is specific to FABP1 There is an advantage that a typical inhibitor is considered to have little influence on other organs, and (v) FABP is considered to have few side effects due to functional suppression because it is molecularly at the lowest layer.
The invention shown below is mainly based on the above results or knowledge.
[1] A prophylactic / ameliorating agent for fatty liver comprising as an active ingredient a compound that suppresses the expression or action of fatty acid binding protein 1 (FABP1).
[2] The preventive / ameliorating agent according to [1], wherein the compound is a compound selected from the group consisting of the following (a) to (d):
(a) siRNA targeting the FABP1 gene;
(b) a nucleic acid construct that generates siRNA targeting the FABP1 gene in a cell;
(c) an antisense nucleic acid that targets the transcript of the FABP1 gene;
(d) A ribozyme that targets the transcript of the FABP1 gene.
[3] The preventive / ameliorating agent according to [2], wherein the siRNA has the sequence of SEQ ID NO: 3 or 4.
[4] A medicine, quasi-drug or food comprising the preventive / ameliorating agent according to any one of [1] to [3].
[5] A screening method for a prophylactic / ameliorating agent for fatty liver, comprising examining whether or not a test substance exhibits an action of suppressing the expression or action of FABP1.
[6] The screening method according to [5], comprising the following steps (1) to (3):
(1) contacting FABP1 with a probe that binds to a fatty acid binding pocket of FABP1 in the presence of a test substance;
(2) a step of detecting the binding of the probe to FABP1; and (3) a step of determining the effectiveness of the test substance based on the detection result, wherein a decrease in the binding of FABP1 and the probe is an index of effectiveness. Step.
[7] Prepare a sample (control group) operated under the same conditions as in step (1) except in the absence of the test substance, and compare it with the detection results in the control group. The screening method according to [6], wherein sex is determined.
[8] The screening method according to [5], comprising the following steps (i) to (iii):
(I) contacting the test substance with a complex of FABP1 and a probe that binds to the fatty acid binding pocket of FABP1;
(Ii) detecting the binding of the probe to FABP1; and (iii) determining the effectiveness of the test substance based on the detection result, wherein a decrease in the binding of FABP1 and the probe is an index of effectiveness Step.
[9] Prepare a sample (control group) operated under the same conditions as in step (i) except in the absence of the test substance, and compare the detection results in the control group with the effectiveness in step (iii) The screening method according to [8], wherein sex is determined.
[10] The screening method according to [5], comprising the following steps (I) to (III):
(I) a step of bringing a test substance into contact with a cell into which a nucleic acid construct containing a promoter region of the FABP1 gene and a reporter gene under the control of the promoter region has been introduced;
(II) detecting the expression of the reporter gene; and (III) determining the effectiveness of the test substance based on the detection result, wherein the decrease in the expression of the reporter gene is an indicator of effectiveness.
[11] Prepare a sample (control group) operated under the same conditions as in step (I) except in the absence of the test substance, and compare the detection results in the control group with the effectiveness in step (III) The screening method according to [10], wherein sex is determined.
[12] The screening method according to [5], comprising the following steps (A) to (C):
(A) culturing cells expressing FABP1 in the presence of a test substance;
(B) detecting the expression level of FABP1 in the cell; and (C) determining the effectiveness of the test substance based on the detection result, wherein the decrease in the expression level of FABP1 is an index of effectiveness. .
[13] Prepare a sample (control group) operated under the same conditions as in step (A) except in the absence of the test substance, and compare the detection results in the control group with the effectiveness in step (C). The screening method according to [12], wherein sex is determined.

Liver (right) in which skeletal muscle type FABP (FABP3) was forcibly expressed. The result of oil red O staining is shown. Left is control. The red part is lipid. Mouse liver (right) in which liver-type FABP (FABP1) was forcibly expressed. Two mice (mouse 1, mouse 2) were used in the experiment. Left is control. Liver weight (left) and triglyceride (right) of mice that forced FABP1 to be expressed. Design of FABP1 inhibitor. Two types of antisense nucleic acids (siFABP1-A: CUUGAGUUACCUCUGUGUUAG (SEQ ID NO: 3), siFABP1-B: CCUCUGUGUUAGUGGUUAUGG (SEQ ID NO: 4)) were designed. Inhibition of FABP1 by antisense nucleic acids. A significant decrease in fluorescence is observed in HeLa cells into which siFABP1-A has been introduced. Compared to siFABP1-A, there is little decrease in fluorescence when siFABP1-B is introduced. Improvement of fatty liver by administration of siFABP1-A. SiFABP1-A was administered to the liver of obese mice using adenovirus. One week later, the liver was removed and subjected to oil red O staining. Fat accumulation in the liver (right) of mice administered with siFABP1-A is significantly less than in the control (left). Liver weight (left) and triglyceride (right) of mice administered siFABP1-A.

  The first aspect of the present invention relates to a preventive / ameliorating agent for fatty liver. “Fatty liver” is a condition in which fat accumulates excessively in the liver. “Fatty liver” may be caused by or associated with alcohol consumption (alcoholic fatty liver) or not (nonalcoholic fatty liver). Prevention and improvement of fatty liver means prevention or improvement of fatty liver (including partial healing and symptom relief).

  The preventive / ameliorating agent of the present invention (hereinafter also referred to as “the drug of the present invention”) comprises a compound that suppresses the expression or action of fatty acid binding protein 1 (FABP1) as an active ingredient. It should be noted that the two terms “suppression” and “inhibition” have overlapping meanings and are often used interchangeably. Therefore, in this specification, the term “suppression” is used in a unified manner unless it is particularly necessary to distinguish between contexts.

  FABP1 is one of the FABP family molecules. As described above, FABP plays an important role in lipid transport, and in humans there are nine types of FABPs that are tissue-specific. FABP1 shows liver-specific expression. The amino acid sequence of FABP1 and the gene sequence encoding it are represented by SEQ ID NO: 1 (NCBI, ACCESSION: NP_001434, DEFINITION: fatty acid-binding protein, liver [Homo sapiens].) And SEQ ID NO: 2 (NCBI, ACCESSION: NM_001443, DEFINITION : Homo sapiens fatty acid binding protein 1, liver (FABP1), mRNA.)

The compound that suppresses the expression or action of FABP1, which is an active ingredient of the present invention, is a compound that suppresses the FABP1 gene expression process (including transcription, post-transcriptional regulation, translation, and post-translational regulation). Examples of the compound are as follows. In the present invention, “suppression of expression” may be either transient suppression or permanent suppression, but transient suppression is preferable in view of the physiological function of FABP1.
(a) siRNA targeting the FABP1 gene
(b) Nucleic acid construct that generates siRNA targeting the FABP1 gene in cells
(c) Antisense nucleic acid targeting the transcript of the FABP1 gene
(d) Ribozymes targeting the transcripts of the FABP1 gene

  The above (a) and (b) are compounds used for expression suppression by so-called RNAi (RNA interference). In other words, according to the agent of the present invention containing the compound (a) or (b) as an active ingredient, the expression of FABP1 can be suppressed by RNAi. RNAi is a sequence-specific post-transcriptional gene repression process that can occur in eukaryotic cells. In RNAi for mammalian cells, a short double-stranded RNA (siRNA) having a sequence corresponding to the sequence of the target mRNA is used. Usually, siRNA is 21-23 base pairs. Mammalian cells are known to have two pathways (sequence specific pathway and sequence non-specific pathway) that are affected by double-stranded RNA (dsRNA). In sequence-specific pathways, relatively long dsRNAs are split into short interfering RNAs (ie siRNAs). On the other hand, a sequence non-specific pathway is considered to be caused by an arbitrary dsRNA regardless of the sequence as long as it has a predetermined length or more. In this pathway, dsRNA becomes an active form, namely PKR, which stops all protein synthesis by phosphorylating the translation initiation factor eIF2 and 2 ', 5' oligoadenyl, which is involved in the synthesis of RNAase L activation molecules. Acid synthase is activated. To minimize the progression of this non-specific pathway, it is preferable to use double stranded RNA (siRNA) shorter than about 30 base pairs (Hunter et al. (1975) J Biol Chem 250: 409-17 Manche et al. (1992) Mol Cell Biol 12: 5239-48; Minks et al. (1979) J Biol Chem 254: 10180-3; and Elbashir et al. (2001) Nature 411: 494-8. Wanna)

  In order to generate target-specific RNAi, a siRNA composed of a sense RNA homologous to a part of the mRNA sequence of the target gene and an antisense RNA complementary thereto is introduced into the cell or expressed in the cell. Just do it. The above (a) is a compound corresponding to the former method, and similarly the above (b) is a compound corresponding to the latter method.

  The siRNA targeting the target gene (FABP1 gene in the present invention) usually hybridizes with a sense RNA consisting of a sequence homologous to a continuous region in the mRNA sequence of the gene and an antisense RNA consisting of its complementary sequence. Double stranded RNA. The length of the “continuous region” here is usually 15 to 30 bases, preferably 18 to 23 bases, more preferably 19 to 21 bases.

  It is known that double-stranded RNA having an overhang of several bases at the end exhibits a high RNAi effect. Therefore, in the present invention, it is preferable to employ siRNA having such a structure. The length of the base forming the overhang is not particularly limited, but is preferably 2 bases (for example, TT, UU).

  You may decide to use siRNA which consists of modified RNA. Examples of modifications herein include phosphorothioation and the use of modified bases (eg, fluorescently labeled bases).

  siRNA can be designed and prepared by conventional methods. In designing siRNA, a sequence unique to a target sequence (continuous sequence) is usually used. Note that programs and algorithms for selecting an appropriate target sequence have been developed.

Examples of siRNA sequences targeting the FABP1 gene are listed below.
5'-CUUGAGUUACCUCUGUGUUAG-3 '(SEQ ID NO: 3. Sequence used in Examples described later)
5'-CCUCUGUGUUAGUGGUUAUGG-3 '(SEQ ID NO: 4. Sequence used in Examples described later)

  The “nucleic acid construct that generates siRNA in a cell” in (b) above refers to a nucleic acid molecule that, when introduced into a cell, produces a desired siRNA (siRNA that causes RNAi against a target gene) by a process in the cell. . One example of the nucleic acid construct is shRNA (short hairpin RNA). shRNA has a structure (hairpin structure) in which a sense RNA and an antisense RNA are linked via a loop structure part, and the loop structure part is cleaved in a cell to form a double-stranded siRNA, resulting in an RNAi effect. The length of the loop structure is not particularly limited, but is usually 3 to 23 bases.

  Another example of a nucleic acid construct is a vector that can express a desired siRNA. Such vectors include those that express shRNA that is converted to siRNA by a later process (inserted with a sequence encoding shRNA) (referred to as stem loop type or short hairpin type), sense RNA and antisense RNA. Are vectors that are expressed separately (referred to as tandem type). Those skilled in the art can prepare these vectors according to conventional methods (Brummelkamp TR et al. (2002) Science 296: 550-553; Lee NS et al. (2001) Nature Biotechnology 19: 500-505; Miyagishi M & Taira K (2002) Nature Biotechnology 19: 497-500; Paddison PJ et al. (2002) Proc. Natl. Acad. Sci. USA 99: 1443-1448; Paul CP et al. (2002) Nature Biotechnology 19: 505-508; Sui G et al. (2002) Proc Natl Acad Sci USA 99 (8): 5515-5520; Paddison PJ et al. (2002) Genes Dev. 16: 948-958 etc.). Currently, various RNAi vectors are available. You may decide to construct | assemble the vector of this invention using such a well-known vector. In this case, after preparing an insert DNA encoding a desired RNA (for example, shRNA), it is inserted into a cloning site of the vector to obtain an RNAi expression vector.

  The origin and structure of the vector are not limited as long as it has a function of generating siRNA that exerts RNAi action on the target gene in the cell. Accordingly, various viral vectors (adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, Sendai virus vectors, etc.), non-viral vectors (liposomes, positively charged liposomes, etc.) and the like can be used. it can. Examples of promoters that can be used in the vector are U6 promoter, H1 promoter, and tRNA promoter. These promoters are RNA polymerase III type promoters, and high expression efficiency can be expected.

  The above (c) is a compound used for expression suppression by an antisense method. In other words, according to the agent of the present invention containing the compound (c) as an active ingredient, the expression of FABP1 can be suppressed by the antisense method. In the case of inhibiting expression by an antisense method, for example, an antisense construct that generates RNA complementary to a unique portion of mRNA encoding FABP1 when transcribed in a target cell is used. Such an antisense construct is introduced into a target cell, for example, in the form of an expression plasmid. On the other hand, as an antisense construct, an oligonucleotide probe that hybridizes with an mRNA / or genomic DNA sequence encoding FABP1 and inhibits its expression when introduced into a target cell can also be employed. As such an oligonucleotide probe, one that is resistant to endogenous nucleases such as exonuclease and / or endonuclease is preferably used.

  When a DNA molecule is used as the antisense nucleic acid, oligodeoxyribonucleotide derived from a region containing a translation initiation site (for example, a region of −10 to +10) of mRNA encoding FABP1 is preferable.

  The complementarity between the antisense nucleic acid and the target nucleic acid is preferably strict, but there may be some mismatch. The ability of an antisense nucleic acid to hybridize to a target nucleic acid generally depends on both the degree of complementarity and the length of both nucleic acids. Usually, the longer the antisense nucleic acid used, the more stable duplexes (or triplexes) can be formed with the target nucleic acid, even if the number of mismatches is large. One skilled in the art can ascertain the degree of acceptable mismatch using standard techniques.

  The antisense nucleic acid may be DNA, RNA, or a chimeric mixture thereof, or a derivative or modified form thereof. Moreover, it may be single-stranded or double-stranded. By modifying the base moiety, sugar moiety, or phosphate skeleton moiety, the stability, hybridization ability, etc. of the antisense nucleic acid can be improved. In addition, antisense nucleic acids can be used to promote cell membrane transport (eg, Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. W088 / 09810, published December 15, 1988) or substances that enhance affinity for specific cells may be added.

  The antisense nucleic acid can be synthesized by a conventional method, for example, using a commercially available automatic DNA synthesizer (for example, Applied Biosystems). For example, Stein et al. (1988), Nucl. Acids Res. 16: 3209 and Sarin et al., (1988), Proc. Natl. Acad. Sci. USA 85: 7448- 7451 etc. can be referred to.

  Strong promoters such as pol II and pol III can be used to enhance the action of antisense nucleic acids in target cells. That is, when a construct containing an antisense nucleic acid arranged under the control of such a promoter is introduced into a target cell, a sufficient amount of the antisense nucleic acid can be transcribed by the action of the promoter.

  Expression of the antisense nucleic acid can be performed by any promoter (inducible promoter or constitutive promoter) known to function in mammalian cells (preferably human cells). For example, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter from the 3 ′ end region of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), herpes zoster thymidine A promoter such as a kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445) can be used.

  In one embodiment of the present invention, expression suppression by a ribozyme is used (in the case of the compound (d) above). The target mRNA can be destroyed using a ribozyme that cleaves the mRNA with a site-specific recognition sequence, but preferably a hammerhead ribozyme is used. For the construction method of the hammerhead ribozyme, for example, Haseloff and Gerlach, 1988, Nature, 334: 585-591 can be referred to.

  As in the case of using the antisense method, for example, for the purpose of improving stability and target ability, a ribozyme may be constructed using a modified oligonucleotide. In order to generate an effective amount of a ribozyme in a target cell, for example, a nucleic acid construct in which DNA encoding the ribozyme is placed under the control of a strong promoter (for example, pol II or pol III) can be used. preferable.

In one embodiment of the present invention, a compound that suppresses the action of FABP1 is used as an active ingredient. Examples of such compounds are those that inhibit the binding of FABP1 to its ligand fatty acid. Specifically, antibodies that bind to or around the fatty acid binding pocket of FABP1, compounds that bind to FABP1 competitively with fatty acids, and the like can be employed as active ingredients. The type and form of the antibody here are not particularly limited. Any of a polyclonal antibody, an oligoclonal antibody and a monoclonal antibody may be used. Chimeric antibodies (eg, human and mouse chimeras), humanized antibodies, human antibodies, and the like can also be used. Furthermore, antibody fragments such as Fab, Fab ′, F (ab ′) ₂ , scFv and dsFv can also be used. Antibodies as active ingredients can be prepared by immunological techniques, phage display methods, ribosome display methods and the like. The antibody itself may not be used as an active ingredient, but a vector capable of expressing a desired antibody in cells may be used as an active ingredient. The same applies to compounds other than antibodies.

  The pharmaceutical preparation of the present invention can be formulated according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, diethylin sulfite, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

  The dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories. The drug of the present invention contains an active ingredient in an amount necessary for obtaining the expected effect (that is, a therapeutically effective amount). The amount of the active ingredient in the drug of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, within the range of about 0.1% by weight to about 95% by weight so as to achieve a desired dose.

  The drug of the present invention is applied to a subject by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) according to the dosage form. Is done. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed). When a nucleic acid construct is used as an active ingredient (for example, an embodiment using RNAi), not only in vivo administration but also ex vivo administration can be employed.

  The “subject” to which the agent of the present invention is administered is not particularly limited, and includes humans and non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows) Pigs, goats, sheep, dogs, cats, chickens, quails, etc.). In a preferred embodiment, the medicament of the present invention is applied to humans. Although the dosage varies depending on the patient's symptoms, age, sex, weight, etc., those skilled in the art can appropriately set an appropriate dosage. In setting the administration schedule, it is possible to consider patient symptoms and duration of drug effect.

  The medicament of the present invention is provided in the form of, for example, a medicine, a quasi drug or a food. That is, this invention provides the pharmaceutical, quasi-drug, and foodstuff containing the chemical | medical agent of this invention as a preferable aspect. Examples of “food” in the present invention include general foods (cereals, vegetables, meat, various processed foods, confectionery, milk, soft drinks, alcoholic beverages, etc.), dietary supplements (supplements, nutritional drinks, etc.), food additives Products, foods for pet animals, and nutritional supplements for pet animals. In the case of a dietary supplement or food additive, it can be provided in the form of powder, granule powder, tablet, paste, liquid or the like. By providing it in the form of food, it becomes easy to ingest the active ingredient of the present invention on a daily basis or continuously.

  As is apparent from the above description, the present application is characterized by administering a therapeutically effective amount of the agent of the present invention to a patient with fatty liver or a potential patient (a person who may be affected in the future). Also provides treatment and prevention of fatty liver.

  The second aspect of the present invention relates to a screening method for a prophylactic / ameliorating agent for fatty liver. The screening method of the present invention is characterized by examining whether or not a test substance exhibits an action of suppressing the expression or action of FABP1, based on the knowledge that the inhibition of FABP1 is effective for the prevention and improvement of fatty liver. To do. Hereinafter, specific embodiments 1 to 4 of the screening method of the present invention will be described. The screening method of the present invention is performed in vitro as is apparent from the description.

1. Screening based on the ability of FABP1 to bind to fatty acids 1
In this embodiment, focusing on the binding between FABP1 and its ligand (fatty acid), a probe that binds to the fatty acid binding pocket of FABP1 (hereinafter also referred to as “binding probe”) is used. Specifically, the following steps (1) to (3) are performed.
(1) contacting FABP1 with a probe that binds to the fatty acid binding pocket of FABP1 in the presence of the test substance (2) detecting the binding of the probe to FABP1 (3) based on the detection result A step of determining effectiveness, wherein a decrease in the binding between FABP1 and the probe is an indicator of effectiveness

  In step (1), FABP1 is prepared, and a bound probe is brought into contact with the test substance. For example, a state in which FABP1, a test substance, and a binding probe coexist is formed in an appropriate container such as a culture dish or a microwell plate (for example, a 96-well plate or a 384-well plate). The order of addition is not particularly limited as long as the state in which the three elements coexist is formed. For example, a solution or suspension of FABP1 is prepared, a test substance is added, and then a binding probe is added. As the solvent, a buffer solution, physiological saline, a medium, or the like can be used. As the temperature condition, 4 ° C. to 37 ° C. can be adopted. The contact time is, for example, 3 minutes to 2 hours, preferably 5 minutes to 30 minutes. The optimum contact condition can be determined by a preliminary experiment.

  FABP1 may be naturally derived or recombinant. FABP1 can be prepared by an expression system using E. coli transformed with a vector capable of expressing the FABP1 gene. FABP1 is also commercially available, and commercially available FABP1 may be used.

  As a test substance, organic compounds or inorganic compounds having various molecular sizes can be used. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1 B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) Existing components or candidate components such as pharmaceuticals and nutritional foods are also preferable test substances. Extracts, cell extracts, culture supernatants, etc. may be used as test substances, etc. By adding two or more kinds of test substances at the same time, interactions between test substances, synergistic effects, etc. may be examined. The test substance may be derived from natural products or may be synthetic, in which case, for example, a combinatorial synthesis technique is used. It is possible to build an efficient screening system.

  As binding probes, use fluorescent probes such as 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), 12- (9-anthroyloxy) oleic acid (12-AO), fatty acid derivatives, cholesterol derivatives, etc. Can do.

In step (2), binding of the binding probe to FABP1 is detected. An appropriate detection method may be employed depending on the binding probe to be used. For example, when a fluorescent probe is used, the binding between FABP1 and the binding probe can be detected and evaluated by detecting fluorescence. In order to enable easy detection, when a fatty acid derivative, a cholesterol derivative or the like is used as a probe, it is preferable to use one labeled with a detectable labeling substance. Examples of the labeling substance include fluorescent dyes such as fluorescein, rhodamine, Texas red, oregon green, enzymes such as horseradish peroxidase, microperoxidase, alkaline phosphatase, β-D-galactosidase, chemical or bioluminescent compounds such as luminol, acridine dye, etc. , ³ H, ¹³ C, ³² P, ¹³¹ I, ¹²⁵ I, and the like, and biotin.

  In step (3), the effectiveness of the test substance is determined based on the detection result. In the present invention, a decrease in the binding between FABP1 and the binding probe is used as an index of effectiveness. That is, the test substance is determined to be effective when a decrease in binding between FABP1 and the binding probe is observed, and the test substance is determined to be ineffective when no decrease in binding between FABP1 and the binding probe is observed. When a plurality of test substances are used, the effectiveness of each test substance can be compared and evaluated based on the degree of decrease in binding.

  An effective test substance is selected based on the determination result in step (3). Usually, as a comparison target, prepared in the absence of the test substance (other conditions are the same as in step (1)) (hereinafter referred to as “control group”), which is also for FABP1 Detect binding of bound probe. Then, the effectiveness of the test substance is determined by comparing the detection result of the control group with the detection result of the test group. Thus, if the effectiveness of a test substance is determined by comparison with a control group, a more reliable determination result can be obtained.

  When the substance selected by the screening method of the present invention has a sufficient medicinal effect, the substance can be used as it is as an active ingredient of a preventive / ameliorating agent for fatty liver. On the other hand, when it does not have a sufficient medicinal effect, it can be used as an active ingredient of a fatty liver prevention / amelioration agent after improving its medicinal effect by altering such as chemical modification. Of course, even if it has a sufficient medicinal effect, the same modification may be applied for the purpose of further increasing the medicinal effect.

2. Screening based on the ability to inhibit the binding of FABP1 to fatty acids 2
In this embodiment, the effect of the test substance on the complex of FABP1 and the binding probe is examined to determine the effectiveness of the test substance. Specifically, the following steps are performed. In addition, since it is the same as that of the said aspect about the matter which is not mentioned especially, the overlapping description is abbreviate | omitted and corresponding description is used.
(I) a step of bringing a test substance into contact with a complex of FABP1 and a probe that binds to the fatty acid binding pocket of FABP1 (ii) a step of detecting binding of the probe to FABP1 (iii) a test substance based on the detection result A step of determining the effectiveness of the step, wherein a decrease in the binding between FABP1 and the probe is an indicator of effectiveness

  In the contact step (i) of this embodiment, a complex of FABP1 and a binding probe (FABP1 to which a binding probe is bound) is prepared, and a test substance is brought into contact with the complex. The conditions in this case (container, solvent, temperature, time, etc.) are in accordance with the above embodiment. The operation of step (ii) is the same as step (2) of the above aspect.

  Also in this embodiment, the effectiveness of the test substance is determined using “decrease in binding between FABP1 and the binding probe” as an index (step (iii)). A test substance that causes a decrease in the binding between FABP1 and the binding probe, that is, a substance that inhibits binding and promotes dissociation of the binding probe from FABP1, is selected as an effective compound. Also in this aspect, it is preferable to determine the effectiveness of the test substance based on the comparison with the detection result of the control group.

3. Screening using a reporter assay In this embodiment, a so-called reporter assay is used. Specifically, the following steps are performed. In addition, since it is the same as that of the said aspect about the matter which is not mentioned especially, the overlapping description is abbreviate | omitted and corresponding description is used.
(I) a step of bringing a test substance into contact with a cell into which a nucleic acid construct containing a promoter region of the FABP1 gene and a reporter gene under the control of the promoter region has been introduced;
(II) a step of detecting the expression of the reporter gene (III) a step of determining the effectiveness of the test substance based on the detection result, wherein the decrease in the expression of the reporter gene is an indicator of the effectiveness

  In step (I), a cell into which a nucleic acid construct containing a promoter region of the FABP1 gene and a reporter gene under the control of the promoter region is prepared is prepared. “Promoter” refers to a functional region that regulates the initiation of transcription of a gene under its control. “Under control” is synonymous with “under control” and means that the promoter and the reporter gene are functionally linked. By placing the reporter gene under the control of the promoter, transcription of the reporter gene is controlled (regulated) by the promoter. Typically, the reporter gene is directly linked to the promoter, but other sequences may be interposed between the promoter and the reporter gene as long as the reporter gene is transcriptionally controlled by the promoter. An example of the promoter region of the FABP1 gene is shown in SEQ ID NO: 7. As long as the promoter activity is shown, a part of the promoter region shown here may be used. As the reporter gene, β-galactosidase gene (lacZ), chloramphenicol acetyltransferase gene (CAT), firefly-derived luciferase gene (Luc) and the like can be used.

  By transfecting an appropriate cell with the nucleic acid construct, cells necessary for step (I) (cells for screening) can be obtained. Various animal cells (human cells, non-human mammalian cells, etc.) can be used as the cells into which the nucleic acid construct is introduced.

  A test substance is brought into contact with the prepared screening cells. For example, a test substance is added to a culture solution for screening cells. Alternatively, the screening cell culture medium is replaced with one containing a test substance. The culture method may be in accordance with a conventional method, and can be determined according to the type of cell for screening. The contact time with the test substance may be set through a preliminary experiment, and is, for example, 30 minutes to 48 hours.

  In step (II) following step (I), the expression of the reporter gene is detected. The detection may be performed according to a conventional method. For general-purpose reporter genes that can be used in the present invention, basic detection methods have been established, and appropriate methods and conditions can be easily selected or set with reference to books and past papers. Is possible.

  In step (III), the effectiveness of the test substance is determined based on the detection result. In the present invention, decrease in reporter gene expression is used as an index of effectiveness. That is, it is determined that the test substance is effective when a decrease in the expression of the reporter gene is observed, and it is determined that the test substance is not effective when the decrease in the expression of the reporter gene is not recognized. When a plurality of test substances are used, the effectiveness of each test substance can be compared and evaluated based on the degree of decrease in binding. In this embodiment as well, it is preferable to determine the effectiveness of the test substance based on the comparison with the detection result of the control group.

4). Screening using cells expressing FABP1 In this embodiment, screening is performed using cells expressing FABP1. Specifically, the following steps are performed. In addition, since it is the same as that of the said aspect about the matter which is not mentioned especially, the overlapping description is abbreviate | omitted and corresponding description is used.
(A) A step of culturing cells expressing FABP1 in the presence of a test substance (B) A step of detecting the expression level of FABP1 in the cells (C) A step of determining the effectiveness of the test substance based on the detection result A step in which a decrease in the expression level of FABP1 is an indicator of effectiveness

  In step (A), cells in which FABP1 is expressed (hereinafter referred to as “FABP1-expressing cells”) are prepared and cultured in the presence of a test substance. As the cells, liver cells, HepG2 cell lines, HepaRG cell lines, human iPSC-derived hepatocytes and the like can be used. A cell newly prepared from a living body or a passage cell thereof may be used, or a commercially available cell line may be used. Cells (genetically modified cells) that have been engineered to forcefully express FABP1 can also be used. The cell species is not particularly limited. Examples of species include humans, mice, rats, hamsters and monkeys.

  In order to culture FABP1-expressing cells in the presence of a test substance, for example, after seeding FABP1-expressing cells in an appropriate container (such as a culture dish or a microwell plate), a predetermined time (for example, 30 minutes to 1 week) has elapsed. The test substance may be added to the culture solution or replaced with a culture solution to which the test substance has been added. Immediately after seeding, the test substance may be added or replaced with a culture solution containing the test substance. In addition, a culture solution to which a test substance is added in advance may be used, and a “state in which the test substance is present in the culture solution” may be formed simultaneously with sowing.

  The culture time in the presence of the test substance is not particularly limited, but for example, 30 minutes to 48 hours, preferably 30 minutes to 16 hours. The optimal culture time can be determined by preliminary experiments.

  Matters that are not mentioned in the present specification (medium, culture temperature, etc.) may be in accordance with general culture conditions for culturing the cells to be used. The culture conditions may be determined with reference to past reports and books or through preliminary experiments. The culture temperature is usually 37 ° C.

  In step (B), the FABP1 expression level of the FABP1-expressing cell that has undergone step (A), that is, the FABP1-expressing cell cultured in the presence of the test substance for a predetermined time is detected. The method for detecting the FABP1 expression level is not particularly limited. For example, it can be detected by quantifying the amount of mRNA of the FABP1 gene by RT-PCR or by measuring the amount of FABP1 protein immunologically (for example, by ELISA or Western blotting).

  In step (C), the effectiveness of the test substance is determined based on the detection result in step (B). In the present invention, a decrease in the expression level of FABP1 is used as an index of effectiveness. That is, the test substance is determined to be effective when a decrease in the expression level of FABP1 is observed, and is determined to be ineffective when the decrease in the expression level of FABP1 is not observed. When a plurality of test substances are used, the effectiveness of each test substance can be compared and evaluated based on the degree of decrease in the expression level. In this embodiment as well, it is preferable to determine the effectiveness of the test substance based on the comparison with the detection result of the control group.

  The discovery that FABP3, a skeletal muscle type FABP, is expressed in the liver of a mouse becomes fatty liver (Figure 1), and the idea that FABP may play an important role in liver fat accumulation. Obtained. Based on this idea, we decided to investigate in detail the involvement of FABP in fatty liver.

1. FABP1 Forced Expression in Liver (1) Method FABP1 showing liver-specific expression was forcedly expressed in mouse liver by the following method. The cDNA encoding the human FABP1 gene was cloned into the pENTR-1A vector (life technologies), which is an adenovirus shuttle vector. The prepared pENTR-FABP1 and the adenovirus vector pAd / CMV / V5-DEST vector (life technologies) were homologously recombined using LR clonase to prepare pAd-FABP1. pAd-FABP1 was digested with the restriction enzyme PacI and linearized, and then transfected into 293A cells using Lipofectamine 2000 to produce FABP1-expressing adenovirus. The resulting virus particles were amplified by reinfection several more times with 293A cells, and then the virus particles were purified by cesium chloride density gradient ultracentrifugation. When adenoviral vectors are injected from the tail vein, almost all are trapped in the liver, so that genes can be efficiently expressed in the liver. After anesthetizing the mouse, the prepared FABP1-expressing adenovirus was injected into the tail vein to express FABP1 in the liver. Mice were dissected one week after virus administration.

(2) Results As shown in FIG. 2, in the liver (right) in which FABP1 was expressed as compared to the control (left), fat accumulation (red portion in the original figure is fat) increased and fatty liver was induced. That is, it was shown that fatty liver is induced not only by FABP3 but also by FABP1. When the liver was removed from the mouse after the test and the liver weight and the amount of triglyceride were examined, the liver weight and triglyceride amount were increased by the expression of FABP1 (FIG. 3). In other words, FABP1 expression promoted liver hypertrophy (fat accumulation and increased liver burden). That is, it was shown that when the expression of FABP in the liver was increased, fat in the liver decreased, resulting in fatty liver. These results suggest that FABP1 has a function to control liver fat accumulation. From this, conversely, decreasing FABP1 expression in the liver was expected to reduce fat in the liver and improve fatty liver.

2. Effect of suppressing FABP1
We decided to investigate whether inhibition of FABP1 is effective in improving fatty liver. Specifically, the effect and influence when FABP1 was suppressed with an antisense drug were investigated.
(1) Method
Two types of antisense nucleic acids (siFABP1-A: CUUGAGUUACCUCUGUGUUAUA (SEQ ID NO: 3), siFABP1-B: CCUCUGUGUUAGUGGUUAUGG (SEQ ID NO: 4)) were designed (FIG. 4). ShDNAs (SEQ ID NOs: 5 and 6) for expressing these siRNAs were prepared. The prepared shDNA was ligated to pENTR-U6 vector (life technologies), which is an adenovirus shuttle vector, to prepare pENTR-siFABP-A, B. In order to examine the FABP1 effect, this vector was co-transfected into HeLa cells with pIRES2-FABP-GFP expressing GFP together with FABP1. The pENTR-siFABP-A, which showed a high FABP1 expression inhibitory effect, and the adenovirus vector pAd / BLOCK-iT / DEST vector were homologously recombined using LR clonase to prepare pAd-siFABP1. This was digested with the restriction enzyme PacI and linealized, and then transfected into 293A cells using Lipofectamine 2000 to produce FABP1-suppressed adenovirus. The resulting virus particles were amplified by reinfection several more times with 293A cells, and then the virus particles were purified by cesium chloride density gradient ultracentrifugation. After anesthesia of the mouse, FABP1-suppressing adenovirus was injected into the tail vein to express siFABP1 in the liver. Mice were dissected one week after adenovirus administration.

(2) Results As shown in FIG. 5, the use of siFABP1-A resulted in a significant decrease in fluorescence (high FABP1 inhibitory effect). Therefore, siFABP1-A was administered to the liver of obese mice (C57 black / 6J) who became obese by eating a high fat diet using adenovirus. The results one week after administration are shown in FIG. The control (left) exhibited fatty liver, whereas the siFABP1-A-treated mouse (right) dramatically improved fatty liver. When the liver was removed from the mouse after the test and the liver weight and triglyceride were examined, the siFABP1-A-treated mice showed a decrease in liver weight (improvement of liver enlargement) and a decrease in triglyceride in the liver. It was seen (Figure 7). That is, it was demonstrated that the fatty liver is improved by a nucleic acid (siFABP1-A) that suppresses the expression of FABP1.

3. Summary As described above, it was found that the strategy of suppressing FABP1 is effective in improving fatty liver. This strategy includes (i) that the treatment method is not affected by the patient's will and life as in the prior art, (ii) that it can be expected to have a powerful fatty liver treatment effect compared to the prior art, and (iii) the prior art In contrast, it takes several months, but a short-term effect is expected. (Iv) FABP shows tissue-specific expression, so an inhibitor specific to FABP1 has little effect on other organs. (V) Since FABP is molecularly at the lowest layer, it has advantages such as being considered to have few side effects due to function suppression, and clinical application is greatly expected.

  According to the present invention, fatty liver can be prevented or improved by limiting the expression or function of a fatty acid binding protein responsible for transporting a fat-soluble substance in the liver. That is, a new treatment method targeting fatty liver is provided.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Sequence number 3: Description of artificial sequence: siFABP1-A
SEQ ID NO: 4: Description of artificial sequence: siFABP1-B
SEQ ID NO: 5: Description of artificial sequence: FABP1-shDNA-A
SEQ ID NO: 6: Description of artificial sequence: FABP1-shDNA-B

Claims (13)

  An agent for preventing / ameliorating fatty liver comprising as an active ingredient a compound that suppresses the expression or action of fatty acid binding protein 1 (FABP1). The preventive / ameliorating agent according to claim 1, wherein the compound is a compound selected from the group consisting of the following (a) to (d):
(a) siRNA targeting the FABP1 gene;
(b) a nucleic acid construct that generates siRNA targeting the FABP1 gene in a cell;
(c) an antisense nucleic acid that targets the transcript of the FABP1 gene;
(d) A ribozyme that targets the transcript of the FABP1 gene.   The preventive / ameliorating agent according to claim 2, wherein the siRNA has the sequence of SEQ ID NO: 3 or 4.   A medicine, quasi-drug or food comprising the preventive / ameliorating agent according to any one of claims 1 to 3.   A screening method for a prophylactic / ameliorating agent for fatty liver, comprising examining whether or not a test substance exhibits an action of suppressing the expression or action of FABP1. The screening method according to claim 5, comprising the following steps (1) to (3):
(1) contacting FABP1 with a probe that binds to a fatty acid binding pocket of FABP1 in the presence of a test substance;
(2) a step of detecting the binding of the probe to FABP1; and (3) a step of determining the effectiveness of the test substance based on the detection result, wherein a decrease in the binding of FABP1 and the probe is an index of effectiveness. Step.   Prepare a sample (control group) operated under the same conditions as in step (1) except that the test substance is not present, and compare the detection results in the control group to determine the efficacy in step (3) The screening method according to claim 6, wherein: The screening method according to claim 5, comprising the following steps (i) to (iii):
(I) contacting the test substance with a complex of FABP1 and a probe that binds to the fatty acid binding pocket of FABP1;
(Ii) detecting the binding of the probe to FABP1; and (iii) determining the effectiveness of the test substance based on the detection result, wherein a decrease in the binding of FABP1 and the probe is an index of effectiveness Step.   Prepare a sample (control group) operated under the same conditions as in step (i) except that the test substance is not present, and compare the detection results in the control group to determine the effectiveness in step (iii) The screening method according to claim 8, wherein: The screening method according to claim 5, comprising the following steps (I) to (III):
(I) a step of bringing a test substance into contact with a cell into which a nucleic acid construct containing a promoter region of the FABP1 gene and a reporter gene under the control of the promoter region has been introduced;
(II) detecting the expression of the reporter gene; and (III) determining the effectiveness of the test substance based on the detection result, wherein the decrease in the expression of the reporter gene is an indicator of effectiveness.   Prepare a sample (control group) operated under the same conditions as in step (I) except that the test substance is not present, and compare the detection results with the control group to determine the effectiveness in step (III) The screening method according to claim 10, wherein: The screening method according to claim 5, comprising the following steps (A) to (C):
(A) culturing cells expressing FABP1 in the presence of a test substance;
(B) detecting the expression level of FABP1 in the cell; and (C) determining the effectiveness of the test substance based on the detection result, wherein the decrease in the expression level of FABP1 is an index of effectiveness. .   Prepare a sample (control group) that was operated under the same conditions as in step (A) except in the absence of the test substance, and determine the effectiveness in step (C) compared to the detection results in the control group The screening method according to claim 12, wherein:

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