JP2010163391A - Medicinal composition for treating diabetes or hyperlipidemia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicinal composition for the treatment and prevention of diabetes or hyperlipidemia, and a method for treating the same. <P>SOLUTION: The medicinal composition containing a Lypla 3 inhibitor as an active ingredient is useful for the prevention and treatment of the diabetes or hyperlipidemia. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a novel use of Lypla3 (Lpl2). More specifically, the present invention relates to a screening method for searching for a prophylactic / therapeutic agent for diabetes or hyperlipidemia using Lypla3, and a method for treating diabetes or hyperlipidemia.

Diabetes is a complex disease caused by hyperglycemia due to insufficient action of insulin. Diabetes often causes complications such as sensory paralysis, blindness, arteriosclerosis and the like, and is a disease that causes a great disability in daily life. In particular, non-insulin dependent diabetes mellitus (insulin-resistant diabetes, type 2 diabetes) is considered to be caused by environmental factors, and overeating and obesity are one of the major causes. For example, the amount of insulin secretion in the pancreas increases dramatically due to obesity, conversely, the pancreas is fatigued and the amount of insulin secretion decreases, eventually resulting in insufficient insulin action and hyperglycemia. Alternatively, insulin receptors decrease due to an increase in fat, resulting in insufficient insulin action and hyperglycemia. Conversely, excess glucose born from insufficient insulin action is stored as fat and obesity further progresses. Thus, obesity and diabetes are closely related in the onset mechanism.

Lypra3 (which may also be labeled as pla2g15 or Lpla2) was first cloned from THP-1 cells as a novel protein having lecithin: cholesterol acyltransferase (LCAT) -like activity (Patent Document 1). This enzyme has been reported to have lysophospholipase activity that hydrolyzes lysophosphatidylcholine to glycerophosphorylcholine (see Non-Patent Document 1). In addition, Lypla3 is presumed to be an enzyme that functions in the lysosome because it exhibits strong phospholipase A2 activity and 1-O-acetylamide synthase activity in an acidic environment (pH 4.5) (non-patent document). 2).

Moreover, as a result of creating and analyzing a Lypla3 (Lpl2) -deficient mouse, lyploidy accumulation in macrophages and spleen was observed in Lypla3-deficient mice, and phospholipid accumulation in pulmonary surfactant and spleen hypertrophy occurred in the first year of life. It became clear (see Non-Patent Document 3), and it was shown that administration of Lypla 3 to pulmonary surfactant is effective for alveolar proteinosis (see Patent Document 2). Furthermore, it is also reported that the symptoms of arteriosclerosis worsen when crossing a Lypla3 (LLPL) -deficient mouse with an arteriosclerosis model mouse, and that exposure of oxidized LDL to macrophages of a Lypla3 (LLPL) -deficient mouse promotes apoptosis. (See Non-Patent Document 4). In addition, there are also reports that it is effective for the treatment of arteriosclerosis by increasing the activity of Lpla2 in macrophages (patent document 3) and the prevention and treatment of diseases (cancer, neuropathy) caused by apoptosis (patent) Reference 4)

Patent Document 5 describes the relationship between LLPL (Lypla3) and arteriosclerosis. Here, it has been shown that a compound that increases the expression of LLPL (Lypla3) can be a therapeutic agent for arteriosclerosis.

International Publication No. 98/46767 Pamphlet International Publication No. 05/089386 Pamphlet International Publication No. 06/116746 Pamphlet International Publication No. 03/78624 Pamphlet International Publication No. 03/62416 Pamphlet

Biochem Biophys Res Commun. 1999; 257 (1): 50-6 J Biol Chem. 2002; 277 (12): 10090-9 Mol Cell Biol. 2006; 26 (16): 6139-48 Biochem Biophys Res Commun. 2005; 330 (1): 104-10

An object of the present invention is to provide a pharmaceutical composition for the treatment / prevention of diabetes or hyperlipidemia and a method for treating diabetes or hyperlipidemia.

As a result of intensive studies in order to solve the above-mentioned problems, the present inventors have found that the blood glucose level is lowered by suppressing the expression of Lypla3. Furthermore, it discovered that it leads to lipid metabolism improvement and weight loss effect. The present invention has been completed based on these findings.

That is, the present invention
(1) A pharmaceutical composition for preventing or treating diabetes or hyperlipidemia, characterized by comprising a Lypla3 inhibitor as an active ingredient,
(2) The composition according to (1), wherein the inhibitor is a functional nucleic acid that suppresses the expression of Lypla3,
(3) The composition according to (2) above, wherein the functional nucleic acid is siRNA,
(4) siRNA capable of suppressing Lypra3 gene expression by 80% or more,
(5) siRNA in which the double-stranded RNA portion has any one of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 in the sequence listing;
(6) A pharmaceutical composition comprising the siRNA according to (4) or (5) above,
(7) A method for treating diabetes or hyperlipidemia, comprising administering an effective amount of a Lypra3 inhibitor to a mammal,
(8) The method according to (7) above, wherein the inhibitor is a functional nucleic acid that suppresses the expression of Lypra3.
(9) The treatment method according to the above (8), wherein the functional nucleic acid is siRNA,
(10) Diabetes or hyperlipidemia screening method using inhibition of expression and / or action of Lypra3 as an index,
About.

According to the present invention, a high therapeutic effect can be obtained by administering a Lypla3 inhibitor. Therefore, the pharmaceutical composition and the therapeutic method of the present invention are extremely useful for the treatment of diabetes or hyperlipidemia.

FIG. 1 shows the expression suppression effect in HEK293 cells of each shRNA designed against the Lypla3 gene. The vertical axis represents the expression level of the Lypla3 gene in each shRNA when the expression level of the Lypla3 gene in the negative control shRNA is 100%, and the horizontal axis represents the sequence number of each shRNA. FIG. 2 shows changes over time in body weight (g), blood glucose level (mg / dL), and food intake (g / day / g-mouse) in each mouse infected with an adenovirus expressing shRNA against Lypla3. . Marks on the graph are: x represents a mouse expressing shRNA with SEQ ID NO: 11 as a target sequence, Δ represents a mouse expressing shRNA with SEQ ID NO: 12 as a target sequence, and ○ represents a shRNA having SEQ ID NO: 13 as a target sequence Mice that express and □ indicate mice that express negative control shRNA, respectively. FIG. 3 shows the expression level of the Lypla3 gene in the liver of a mouse infected with an adenovirus expressing shRNA against Lypla3. The vertical axis shows the expression level of the Lypla3 gene in the mouse expressing each shRNA when the expression level of the Lypla3 gene in the liver of the mouse expressing the negative control shRNA is 100%. FIG. 4 shows genes whose expression changes due to suppression of Lypla3 expression in mouse liver, extracted as a result of microarray analysis. ShRNA targeting SEQ ID NO: 12 or SEQ ID NO: 13 when the expression level of each gene in mouse liver expressing negative control shRNA on day 3, 5 and 7 after adenovirus infection is 100% The relative expression level of each gene in a mouse expressing. FIG. 5 shows a list of consensus sequences in human Lypla3 mRNA and mouse Lypla3 mRNA (Part 1). FIG. 6 shows a list of consensus sequences in human Lypla3 mRNA and mouse Lypla3 mRNA (Part 2). FIG. 7 shows a list of consensus sequences in human Lypla3 mRNA and mouse Lypla3 mRNA (Part 3).

Hereinafter, embodiments of the present invention will be described in detail.
(A) Pharmaceutical composition and treatment method of the present invention

In one embodiment of the present invention, “a pharmaceutical composition for preventing or treating diabetes or hyperlipidemia characterized by containing a Lypla 3 inhibitor as an active ingredient” and “an effective amount of a Lypla 3 inhibitor” are administered. And a method for treating diabetes or hyperlipidemia, which is characterized by the above. Here, “Lypla3” is a known protein (GenBank: NM — 012320). The DNA sequence of Lypra3 is described in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence is described in SEQ ID NO: 2. “Lypla3” in the present invention is not limited to these sequences, and there are no limitations on the number of amino acid and DNA mutations and mutation sites as long as the function of the peptide represented by SEQ ID NO: 2 is retained. And

In the present invention, the “Lypla3 inhibitor” means a substance capable of inhibiting the activity of the Lypla3 gene (particularly the human Lypla3 gene) or its gene product. Examples of inhibitors include substances that affect gene transcription or translation and substances that affect the activity of gene products. The Lypla3 inhibitor also includes the Lypla3 promoter inhibitor. Examples of such inhibitors include nucleic acids such as antisense, siRNA or vectors containing them; polypeptides such as dominant negatives, antibodies (in polypeptides comprising a contiguous amino acid sequence shown in SEQ ID NO: 2) Such as an antibody that specifically binds to an epitope of the above), or an enzyme; a catalytic RNA, such as a ribozyme; an aptamer; a chemical molecule having a low molecular weight (for example, a molecular weight of less than 2000 Da). These inhibitors can be selected by, for example, the screening method disclosed in the present specification.

In the present invention, a preferred form of “Lypla3 inhibitor” is “functional nucleic acid”. The term “functional nucleic acid” as used herein refers to a function inherently acting in a series of processes for synthesizing proteins such as transcription and translation of DNA and mRNA in addition to suppressing the expression of endogenous genes such as intracellular DNA and mRNA. Means a nucleic acid capable of inhibiting For example, antisense, siRNA, miRNA, shRNA, ribozyme or an expression vector containing these. The functional nucleic acid of the present invention is a nucleic acid that can suppress the expression of Lypra3. Here, suppression of expression means suppressing 50% or more, preferably 80% or more, of the original expression level of mRNA or protein when compared with the control group.

In the present invention, a preferred form of “functional nucleic acid” is siRNA. The siRNA of the present invention refers to siRNA that suppresses gene expression of Lypra3. Specifically, it refers to siRNA capable of suppressing Lypra3 gene expression by 50% or more, and preferably siRNA capable of suppressing 80% or more compared to the control group. siRNA is known to induce sequence-specific suppression of gene expression called RNA interference. siRNA is generally composed of a double-stranded RNA portion and an overhang portion at the 3 ′ end of the sense strand and the antisense strand. siRNAs can be designed by methods known to those skilled in the art. For example, a selected DNA sequence (preferably 19 to 21 bases) is directly converted into an RNA sequence (sense strand) and its antisense strand as a double-stranded RNA portion, and an overhang portion is added. The overhang portion is an arbitrary nucleic acid (ribonucleic acid or deoxynucleic acid) having 1 or 2 bases, and uridine (U) or thymidine (dT) is preferable. The siRNA of the present invention is not particularly limited as long as it is designed based on the DNA sequence of Lypla3, preferably human Lypla3 (SEQ ID NO: 1), and can suppress the expression of Lypla3. The suppression of expression is desirably specific for Lypla3. Whether it is specific or not can be confirmed by conducting a publicly available BLAST search. The overhang part is not essential.

The siRNA of the present invention also includes any molecule having the same effect as the siRNA of the present invention within the administration subject. An example of such a molecule is shRNA. shRNA is RNA having a short hairpin structure, and is an RNA molecule having a stem-loop structure so that a part of a single strand forms a complementary strand with another region. ShRNA in which the double-stranded RNA portion has the same structure as the siRNA of the present invention is also included in the siRNA of the present invention. In addition, a DNA that can express the siRNA of the present invention by being administered to an administration subject is also included in the siRNA of the present invention. Such a DNA is used by constructing a DNA encoding siRNA into an expression vector (for example, a vector such as adenovirus, adeno-associated virus, herpes virus, lentivirus).

The inventors of the present invention have confirmed that hypoglycaemia, and changes in genes related to insulin sensitivity and lipid metabolism are observed when siRNA capable of suppressing the expression of Lypla3, a Lypla3 inhibitor, is administered to mammals. Such findings suggest that an effective amount of a Lypla3 inhibitor is administered, and that a pharmaceutical composition containing the Lypla3 inhibitor as an active ingredient is effective for the treatment and prevention of diabetes and hyperlipidemia. is there.

When the active ingredient of the Lypra3 inhibitor is a functional nucleic acid, it is desirable to include a modification for improving the properties as a therapeutic agent or a transport carrier such as a liposome. To improve the therapeutic properties of the polynucleotide, nucleotide modifications or analogs can be introduced. For example, improved nuclease resistance and / or improved cell permeability. Nuclease resistance is brought about by any method known in the art that does not interfere with the biological activity of the antisense, siRNA, shRNA and / or ribozyme. An example of a modification that can be added to an oligonucleotide for the purpose of improving nuclease resistance is modification of a heteroatom's phosphorus or oxygen in the phosphate backbone. For example, methyl phosphate, phosphorothioate, phosphorodithioate, and morpholino oligomers. In addition, other modifications known in the art may greatly increase the stability against nucleases while maintaining physiological activity.

When the active ingredient of the Lypra3 inhibitor is a functional nucleic acid, an expression vector, particularly a mammalian expression vector, is used for in vivo expression, particularly for expression in a patient requiring the treatment of the present invention. Is desirable. Expression vectors are well known in the art and preferably include plasmids, cosmids, and viral expression systems. Examples of preferred virus expression systems are adenovirus, retrovirus, lentivirus and the like. In addition, methods for introducing vectors into cells and tissues are well known in the art. Preferred examples include transfection, lipofection, electroporation, and infection with recombinant viral vectors.

In the treatment of diabetes or hyperlipidemia, the above-mentioned Lypla3 inhibitor may be used as it is, but the inhibitor is usually used by using one or more pharmaceutical additives that are pharmaceutically acceptable. It is preferable to produce and administer a pharmaceutical composition containing as an active ingredient. When the Lypla3 inhibitor is used as a pharmaceutical composition, it can be used as a tablet, capsule, elixir, microcapsule or the like, or an injection such as a sterile solution or suspension, according to the usual method. .

The administration method of the Lypla inhibitor may be oral administration or parenteral administration, and the form of parenteral administration is not particularly limited, and intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, and the like can be performed.

The dose of Lypra3 inhibitor varies depending on the administration subject, administration method, etc. For example, in the case of oral administration, it is about 0.1 to 100 mg per day, preferably about 0.1 to 100 mg per day for patients with diabetes or hyperlipidemia (60 kg). About 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, for example, about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day for patients with diabetes or hyperlipidemia (60 kg). Can be injected intravenously.
(B) Screening method of the present invention

As another embodiment of the present invention, there is a screening method for a therapeutic agent for obesity or type II diabetes using inhibition of expression and / or action of Lypra3 as an index. As described above, the present inventors have found that suppression of the expression of the Lypra3 gene is deeply related to diabetes or hyperlipidemia and its progression. From this fact, Lypla3 is involved in the suppression of diabetes or hyperlipidemia or the progression thereof, a substance having the effect of suppressing the expression of the Lypla3 gene (mRNA expression) and reducing the production of Lypl3, or the function of Lypla3 It is considered that a substance having an action of lowering is useful as an effective component for preventing or treating diabetes or hyperlipidemia.

In the screening method of the present invention described below, among test substances, (B-1) a substance having an action of suppressing the expression of the Lypla3 gene (messenger RNA expression), and (B-2) a decrease in the production amount of Lypla3 Or (B-3) a prophylactic or therapeutic agent for diabetes or hyperlipidemia (hereinafter collectively referred to as “ It is also intended to obtain an active ingredient of “therapeutic agent for diabetes or hyperlipidemia”.

Examples of candidate substances that can be active ingredients for antidiabetic or hyperlipidemic agents include nucleic acids, peptides, proteins, organic compounds (including low molecular compounds and high molecular compounds), inorganic compounds, and the like. The screening method of the present invention can be carried out on samples containing these candidate substances (collectively referred to as “test substances”). Here, the sample containing the candidate substance includes a cell extract, an expression product of a gene library, a microorganism culture supernatant, a cell component, and the like.

(B-1) Screening method of substance having action of suppressing expression of Lypla3 gene In this screening, a substance having an action of suppressing the expression of Lypla3 gene is selected from test substances, and expression of Lypla3 mRNA is performed. This is a method of searching for the amount as an index and obtaining it as an active ingredient of a therapeutic agent for diabetes or hyperlipidemia.

Specifically, the method can be carried out by performing the following steps (1) to (3).
(1) contacting a test substance with a cell capable of expressing the Lypla3 gene;
(2) a step of measuring the expression level of the Lypla3 gene in a cell contacted with the test substance; and (3) a test substance whose measurement amount is smaller than the expression level of the Lypla3 gene in a control cell not contacted with the test substance. Process to select.

The cells used for such screening may be cells that can express the Lypla3 gene, whether natural or recombinant. The origin of the Lypla3 gene is not particularly limited, and may be derived from humans, or may be derived from mammals such as mice other than humans or other biological species, but is preferably human-derived Lypla3 gene. is there.

In addition, according to a standard method, a transformed cell prepared by introducing an expression vector having the cDNA of the Lypla3 gene and capable of expressing Lypl3 mRNA can also be used. The category of cells used for screening includes tissues that are aggregates of cells.

In the step (1) of the screening method (B-1) of the present invention, the condition for bringing the test substance into contact with the cell capable of expressing the Lypla3 gene is not particularly limited, but the cell does not die and the Lypla3 gene is expressed. It is preferable to select the culture conditions (temperature, pH, medium composition, etc.) to be obtained.

Selection of candidate substances can be performed, for example, by contacting a test substance with a cell capable of expressing the Lypla3 gene under the above conditions, and searching for a substance that suppresses the expression of the Lypla3 gene and decreases the expression level of the mRNA. . Specifically, the expression level of Lypra3 mRNA when cells capable of expressing the Lypla3 gene are cultured in the presence of the test substance is the same as that obtained when the corresponding cells capable of expressing the Lypla3 gene are cultured in the absence of the test substance. The test substance that has been brought into contact with the cell can be selected as a candidate substance using as an index the expression level lower than the expression level of mRNA (control expression level).

The measurement (detection and quantification) of the expression level of Lypla 3 mRNA is performed by Northern blotting using the expression level of Lypla 3 mRNA in a cell capable of expressing the Lypla 3 gene using an oligonucleotide having a sequence complementary to the base sequence of the Lypla 3 mRNA. It can be carried out by carrying out a known method such as RT-PCR method or real-time quantitative PCR method or a measuring method using a DNA array.

Screening methods The screening of substances having the effect of reducing the production of (B-2) Lypla3, from the test substance, a substance having an effect of lowering the production of Lypla3, searched as an index the production amount of Lypla3, It is a method of obtaining as an active ingredient of a therapeutic agent for diabetes or hyperlipidemia.

The said method can be specifically implemented by performing the following process (1 ')-(3').
(1 ′) contacting a test substance with a cell capable of producing Lypla3 or a cell fraction prepared from this cell;
(2 ′) a step of measuring the production amount of Lypla3 in the cell or cell fraction contacted with the test substance, and (3 ′) a control cell in which the test substance is not contacted with the test substance (cells capable of producing Lypla3) Or a step of selecting a test substance smaller than the production amount of Lypla in a cell fraction (prepared from a cell capable of producing Lypla3) that is not brought into contact with the test substance.

The cells (including control cells) used for such screening are not limited to natural or recombinant cells, but may be any cells that can express Lypla gene and produce Lypl3. The origin of the Lypla3 gene is not particularly limited, and may be derived from humans, or may be derived from mammals such as mice other than humans or other biological species, but is preferably human-derived Lypla3 gene. is there.

In addition, according to a standard method, a transformed cell prepared by introducing an expression vector having the cDNA of the Lypla3 gene and capable of producing Lypla3 can also be used. The category of cells used for screening includes tissues that are aggregates of cells.

In the step (1 ′) of the screening method (B-2) of the present invention, the condition for bringing the test substance into contact with a cell capable of producing Lypla3 is not particularly limited, but the cell does not die and the Lypla3 gene is expressed. In addition, it is preferable to select culture conditions (temperature, pH, medium composition, etc.) capable of producing Lypla3.

Selection of candidate substances can be performed, for example, by contacting a test substance with a cell capable of producing Lypla3 or a cell fraction thereof under the above conditions to search for a substance that decreases the production amount of Lypla3. Specifically, the amount of Lypla3 produced when culturing a cell capable of producing Lypla3 or a cell fraction thereof in the presence of a test substance is cultivated in the absence of the test substance, the cell capable of producing Lypla3 corresponding to the above or a cell fraction thereof. The test substance brought into contact with the cell or cell fraction can be selected as a candidate substance, using as an index the production amount (control production amount) of Lypla obtained in this case.

The measurement (detection and quantification) of the production amount of Lypra3 is carried out by measuring the amount of Lypla3 obtained from the cells capable of producing Lypla3 or its cell fraction using Western blotting, immunoprecipitation, ELISA using an antibody against the Lypla3 (anti-Lypla3 antibody). It can implement by performing well-known methods, such as.

(B-3) Screening method for substance having action of reducing action of Lypla The screening is carried out by searching for a substance having an action of lowering the action of Lypla from the test substances using the action of Lypla3 as an index. Or it is the method of acquiring as an active ingredient of a therapeutic drug for hyperlipidemia.

Specifically, the method can be carried out by performing the following steps (1 ″) to (3 ″).
(1 ″) a step of contacting a test substance with a cell capable of producing Lypra3 or a cell fraction prepared from the cell,
(2 ″) a step of detecting the action of Lypla3 in the cell or cell fraction contacted with the test substance, and (3 ″) a control cell (cell capable of producing Lypla3) in which the detected action does not contact the test substance ) Or cell test (prepared from cells capable of producing Lypla3), a step of selecting a test substance having a lower action than Lypla3.

As the cells used in the screening and the method and conditions for contacting with the test substance in the step (2 ″), those described in the screening method of (B-2) described above can be used similarly.

Selection of a candidate substance can be performed by contacting a test substance with a cell capable of producing Lypla3 or a cell fraction thereof under the above conditions and searching for a substance that reduces the action of Lypla3. Specifically, the action of Lypla3 produced when cultivating a cell capable of producing Lypla3 or a cell fraction thereof in the presence of a test substance cultivates a cell or cell fraction corresponding to the above in the absence of the test substance. In this case, the test substance can be selected as a candidate substance using as an index the lower than the action (control action) of Lypra3 obtained.

Here, examples of the action of Lypla include esterase activity. This measurement itself can be performed according to a known method (for example, J. Biol. Chem, 264, 14723-14728, 1989). Other examples include lysophospholipase activity (see Biochem Biophys Res Commun. 1999, Vol. 257 (1), pages 50-56). In addition, what is known to those skilled in the art as an action of Lypla3 is also included.

Substances selected by the screening methods (B-1) to (B-3) described above have the effect of suppressing the expression of the Lypla3 gene in the cells to reduce the production of Lypla3 or the action of the Lypla3. It can be used as an active ingredient of a composition for preventing diabetes or hyperlipidemia and its progression or a composition for improving diabetes or hyperlipidemia. Candidate substances selected by the above screening method can be further screened using a non-human animal with a disease state having diabetes or hyperlipidemia. The candidate substance thus selected is in a medicinal effect test, safety test using a non-human animal having diabetes or hyperlipidemia, and a patient (human) having diabetes or hyperlipidemia or in a previous state thereof. It may be used for clinical trials on patients (humans), and by conducting these tests, it is possible to select and acquire active ingredients of a more practical composition for preventing or treating diabetes or hyperlipidemia .

The material selected in this way is subjected to structural analysis as necessary, and then, depending on the type of the material, chemical synthesis, biological synthesis (including fermentation), or genetic engineering, And can be used to create a composition for preventing or treating diabetes or hyperlipidemia.

EXAMPLES The present invention will be described below with reference to examples. However, the examples are provided for better understanding of the present invention, and the scope of the present invention is intended to be limited to these examples. It is not a thing.

Screening of short hairpin RNA (shRNA) sequence against mouse Lypla3 gene
[Purpose] To screen for shRNA sequences with high inhibitory effect used for functional analysis of Lypla3.
[Method] (1) Design of Lypla3 shRNA Sequence and Plasmid Construction Oligo for shRNA preparation for 20 target sequences (19-21 bp) for mouse Lypla3 gene (Genbank Accession No. NM_133792) was designed. The oligo for shRNA preparation consists of sense strand + loop sequence (TTCAAGAGA) + antisense strand + terminator sequence (TTTTTT). Using BLOCK-iT U6 RNAi Entry Vector Kit (Invitrogen), oligos for shRNA preparation were introduced into the pENTR / U6 vector according to the attached manual to construct pENTR / U6-Lypla3 shRNA. On the other hand, as a Lypla3 expression vector, pCI-Lypla3 was constructed by introducing the mouse Lypla3 ORF into the XhoI / NotI site of the pCI-neo vector (Promega).
(2) In vitro screening of Lypla3 shRNA
HEK293 cells (5 x 10 ⁵ cells / 2ml / well) were seeded in a 6-well plate using 10% serum-containing D-MEM (High Glucose) medium, and cultivation was started in a 37 ° C, 5% CO ₂ environment . On the next day, using FuGENE6 transfection reagent (Roche), pENTR / U6-Lypla3 shRNA and pCI-Lypla3 were introduced into the cells so that the molecular weight ratio was 1: 1. In order to correct the transfection efficiency, a luciferase expression plasmid was simultaneously introduced into the cells. 72 hours after introduction of the plasmid, a solubilizer was added, and total RNA was column-extracted using RNeasy plus kit (Qiagen) according to the manual. Next, a reverse transcription reaction was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using 2 μg of total RNA. Using the synthesized cDNA and Lypla3 primer set (SEQ ID NOs: 3 and 4), the amount of Lypla3 mRNA was measured by quantitative real-time PCR using SYBR Premix Ex Taq II (Takara Bio Inc.). At this time, it was corrected by the amount of luciferase mRNA measured simultaneously (the luciferase primer set (SEQ ID NOs: 5 and 6)) to obtain relative values.
[Results] 20 types of shRNA against the Lypla3 gene were designed, and the expression of Lypla3 mRNA in HEK293 cells co-expressed with the Lypla3 gene and each shRNA was confirmed by quantitative real-time PCR. As a result, negative control was performed as shown in FIG. In contrast, 90% or more of shRNAs that suppress Lypla3 expression could be obtained in multiple sequences.
[Interpretation] The shRNA sequence for Lypla3 gene was obtained by in vitro screening. By using RNAi (including shRNA and siRNA) having a target sequence that has a high inhibitory effect in this screening, a high Lypla3 expression inhibitory effect can be expected.

Suppression of Lypla3 gene expression in mouse liver using adenovirus
[Objective] An adenovirus expressing shRNA that suppresses Lypla3 expression obtained in Example 1 is prepared, and functional analysis of Lypla3 in mouse liver is performed.
[Method] (1) Construction of adenovirus plasmid Among the shRNAs obtained in Example 1, cggaggaatg ctatttctca a (SEQ ID NO: 11), cacgccaaac tctttctact a (SEQ ID NO: 12), and cagctggcta ctgccataca a (SEQ ID NO: 12) in the DNA sequence of mouse Lypla3 Three types of shRNA targeting SEQ ID NO: 13) were introduced into the pAdx-RGD-DEST plasmid (a plasmid in which a Gataway cassette (Invitrogen) was introduced into pAdx (Clontech)) by LR clonase (Invitrogen) reaction. PAdx-RGD-Lypla3 shRNA virus plasmid was constructed. These are expressed as siRNA in which a double-stranded RNA portion has a sequence of cggaggaaug cuauuucuca a (SEQ ID NO: 14) cacgccaaac ucuuucuacu a (SEQ ID NO: 15) cagcuggcua cugccauaca a (SEQ ID NO: 16) in vivo.
(2) Preparation and concentration of adenovirus
20 μg of pAdx-RGD-Lypla3 shRNA plasmid was treated with restriction enzyme PacI (NEB) overnight, treated with phenol / chloroform, precipitated with ethanol, and redissolved in 20 μl of TE buffer. PacI-treated plasmid was introduced into HEK293 cells (70% confluent, 10cm-dish, 10% serum-containing D-MEM (High Glucose) medium) with Lipofectamine 2000 transfection reagent (Invitrogen), and at 37 ° C, 5% CO ₂ environment Was cultured. After transfection, change the medium every 4 to 5 days until CPE (cytopathic effect) is seen. When about 50% of the cells are removed by CPE, collect the cells and medium, and centrifuge at 1000 rpm for 5 minutes. Cells were pelleted and resuspended in 2 ml of medium. The cell pellet was frozen 5 times with Freeze & Thaw (completely frozen in dry ice / methanol and thawed in a 37 ° C. warm bath), and the cells were disrupted and centrifuged at 3000 rpm for 15 minutes to collect the virus solution (supernatant). This virus solution was infected with HEK293 cells (70% confluent, 15 cm-dish × 2), cultured in a 37 ° C., 5% CO ₂ environment, and the virus solution was recovered in the same manner as described above. Further, HEK293 cells (70% confluent, 15 cm-dish × 20 cells) were reinfected, and finally 10 ml of virus solution was collected. 37 ° C. The virus solution with Benzonase (Novagen, Inc.), was treated for 30 minutes, the supernatant ^{CsCl / PBS (1.50g / cm 3} ) 0.5mL, CsCl / PBS (1.35g / cm3) 2.5mL, CsCl / PBS ( 1.25 g / cm 3) 4 mL was added to the stacked tubes in this order. The white virus fraction was collected by ultracentrifugation at 35000 rpm, 16 ° C. for 1 hour using a SW41Ti rotor (Beckman). This virus fraction was put into a dialysis cassette Slide-A-Lyzer10000MWCO (PIERCE) and dialyzed against 3 liters of PBS + 10% Glycerol. The collected virus was immediately divided into tubes and stored at −80 ° C. Adenovirus infection titer was measured according to the manual using Adeno-X rapid titer kit (Clontech).
(3) Adenovirus infection to C57BL / 6J mice
C57BL / 6J mice (8 weeks old mouse, Charles River Japan) were infected with 3 types of Lypla3 shRNA-expressing adenovirus (3 × 10 ⁹ ifu / mouse) by tail vein injection. The mice are individually caged and are freely fed under the breeding conditions of breeding temperature 24 ± 1 ° C, humidity 55 ± 5%, lighting time 12 hours (from 8:00 to 20:00) and food CRF-1 (Oriental Yeast Co., Ltd.). Feeded and raised. After virus infection, the blood glucose level was measured at any time using the body weight and glucocardidia α (Arkray) at a rate of once every 3 to 4 days. Dissection was performed 14 days after virus infection, and the liver was collected.
(4) Analysis of Lypla3 expression level in liver Total RNA was extracted from the collected liver using QIAZOL Lysis Reagent (Qiagen) according to the manual. Furthermore, total RNA was column purified using RNeasy mini kit (Qiagen) according to the manual. Next, a reverse transcription reaction was performed using High Capacity cDNA Reverse Transcription Kit using 2 μg of total RNA. The amount of mRNA of Lypla3 was measured by quantitative real-time PCR using SYBR Premix Ex Taq II (Takara Bio Inc.) using the synthesized cDNA and Lypla3 primer set (SEQ ID NOs: 3 and 4). At this time, it was corrected by the amount of mRNA of the β-actin primer set (SEQ ID NOs: 7 and 8) measured simultaneously to obtain a relative value.
[Results] As a result of measuring changes in body weight and blood glucose level of C57BL / 6J mice infected with each Lypla3 shRNA-expressing adenovirus, as shown in Fig. 2, the body weight was not significantly different from the control group. Values were significantly reduced in all shRNA expressing mice groups. In addition, as shown in FIG. 3, the expression level of Lypla3 in mouse liver 14 days after virus infection was about 50% for shRNA with SEQ ID NO: 11 as the target sequence, and about 10% with shRNA with SEQ ID NO: 12 as the target sequence. In addition, about 60% of the expression was suppressed by shRNA using SEQ ID NO: 13 as a target sequence.
[Interpretation] By suppressing the expression of the Lypla3 gene in the liver, a significant decrease in blood glucose level was observed as needed. This effect was also seen with all three separately designed shRNAs. From this, it became clear that the blood glucose level could be lowered by suppressing Lypla3 in the liver.

Gene variation analysis of Lypla3 expression-suppressed mouse liver using DNA microarray
[Purpose] The downstream factor is searched for by the variable gene analysis using a DNA microarray about the blood glucose level lowering effect by Lypla3 suppression in the liver shown in Example 2.
[Method] (1) Infection of C57BL / 6J mice with adenovirus Adenovirus (3x10 ⁹ ifu / mouse) expressing two types of shRNA targeting SEQ ID NOS: 12 and 13 used in Example 2 was used as C57BL / 6J mice (8 week old mice, Charles River Japan) were infected by tail vein injection. The mice are individually caged and are freely fed under the breeding conditions of breeding temperature 24 ± 1 ° C, humidity 55 ± 5%, lighting time 12 hours (from 8:00 to 20:00) and food CRF-1 (Oriental Yeast Co., Ltd.). Feeded and raised. Dissection was performed 3 days, 5 days, and 7 days after virus infection, and the liver was collected.
(2) Implementation of DNA microarray Total RNA was extracted from the collected liver using QIAZOL Lysis Reagent (Qiagen). Furthermore, total RNA was column purified using RNeasy mini kit (Qiagen). The grade of purified total RNA was confirmed with RNA1000Chip / BioAnalyzer (Agilent). 500 ng of total RNA was converted to cDNA, in vitro transcribed, and Cy3 labeled according to the manual using Agilent Low RNA Input Linear Amplification Kit PLUS, One-Color (Agilent), and finally 1.65 μg of Cy3 cRNA Was used as a probe. The probe was hybridized (65 ° C., 17 hours) with Whole Mouse Genome Oligo Microarray (Agilent) using a hybrid oven. After washing, reading was performed with an Agilent Microarray Scanner (Agilent), and data analysis was performed using analysis computer software GeneSpring (Agilent).
[Results] As a result of microarray analysis, representative genes among the genes whose expression is fluctuated due to suppression of Lypla3 expression are summarized in FIG. Transcription factors ChREBP (Biochimie.2005; 87 (1): 81-6, J Lipid Res.2006; 47 (9): 2028-41, J Biol Chem) involved in lipid acid synthesis such as Fasn, Accα, Elovl6, SPOT14 .2005; 280 (12): 12019-27), the expression of genes that are regulated is suppressed, and conversely, the gene involved in triglyceride metabolism, Lpl (J Lipid Res. 2002; 43 (12): 1997 -2006) and the gene Irs1 (J Clin Invest. 2006; 116 (1): 101-14) involved in insulin sensitivity were found to be up-regulated.
[Interpretation] As a result of microarray analysis, the expression of genes regulated by the transcription factor ChREBP is suppressed, suggesting that suppression of Lypla3 expression in the liver results in suppression of the transcription factor ChREBP activity. Is done. It has been reported that suppression of ChREBP activity specifically in the liver improves lipid metabolism throughout the body and decreases blood glucose and body weight (Diabetes 2006; 55: 2159-2170). By suppressing the expression, it is considered that the blood sugar level is lowered, the lipid metabolism is improved in the whole body in the long term, and the weight is reduced accordingly. In addition, since the gene involved in triglyceride metabolism is up-regulated, the effect of improving lipid metabolism is expected, and since the gene involved in insulin sensitivity is up-expressed, the effect of improving insulin resistance is expected. . This suggests that Lypla3 is useful not only for diabetes drugs but also for the treatment and prevention of hyperlipidemia, obesity, arteriosclerosis, and metabolic syndrome.

Method for confirming the inhibitory effect of Lypla3 siRNA <br/> In the mRNA sequences predicted from the DNA sequences of human Lypla3 (NM_012320) and mouse Lypla3 (NM_133792) (human: SEQ ID NO: 9, mouse: SEQ ID NO: 10), A sequence having 19 consecutive base matches was selected (FIGS. 5 to 6). Lypla3 expression suppression effect of siRNA containing these sequences is calculated by introducing siRNA into a human or mouse liver-derived cell line using a commercially available transfection reagent, quantifying the Lypla3 mRNA sequence by quantitative real-time PCR, and calculating the suppression rate Can be confirmed.

Claims (10)

A pharmaceutical composition for preventing or treating diabetes or hyperlipidemia, which comprises a Lypla3 inhibitor as an active ingredient. The composition of claim 1, wherein the inhibitor is a functional nucleic acid that suppresses the expression of Lypla3. The composition according to claim 2, wherein the functional nucleic acid is siRNA. An siRNA that can suppress the expression of Lypla3 gene by 80% or more. An siRNA in which the double-stranded RNA portion has any one of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. A pharmaceutical composition comprising the siRNA according to claim 4 or 5. A method for treating diabetes or hyperlipidemia, comprising administering an effective amount of a Lypra3 inhibitor to a mammal. The method according to claim 7, wherein the inhibitor is a functional nucleic acid that suppresses the expression of Lypla3. The method according to claim 8, wherein the functional nucleic acid is siRNA. A screening method for a therapeutic agent for diabetes or hyperlipidemia, wherein inhibition of expression of Lypra3 is used as an index.

Images