JP4585186B2 - Novel preventive or therapeutic agent for obesity, diabetes and abnormal lipid metabolism - Google Patents

Description

  The present invention relates to an adenosine monophosphate activated protein kinase (AMP-activated protein kinase) activator (hereinafter abbreviated as “AMPK activator”) or a peroxisome proliferator activated receptor α. ] A novel prophylactic or therapeutic agent that enhances fatty acid oxidation of skeletal muscle and suppresses obesity, diabetes, and abnormal lipid metabolism by using a combination of an activator (hereinafter abbreviated as "PPARα activator") and leptin and its Regarding usage.

  It is known that fatty acid oxidation ability of skeletal muscle is reduced in type 2 diabetes with obesity and lipid metabolism abnormality. It is known that the pathological conditions of obesity, diabetes, and abnormal lipid metabolism are partially improved by exercise therapy, but the fatty acid oxidation ability of skeletal muscle is increased at that time. Acetyl-CoA carboxylase (ACC) is an enzyme that produces malonyl-CoA [Malonyl-CoA], a negative regulator of fatty acid oxidation. Type II ACC that is selectively expressed in skeletal muscle. It has been reported that in mice deficient, the onset of obesity and diabetes is suppressed without lowering the fatty acid oxidation ability of skeletal muscle when loaded with a high fat diet (Non-patent Document 1). There is also a report that mice that overexpressed uncoupling protein (UCP), which is involved in heat production, in skeletal muscle have increased oxygen consumption in skeletal muscle and reduced the development of obesity and diabetes. Patent Document 2). These findings suggest that if fatty acid oxidation in skeletal muscles can be enhanced by any method, diabetes and lipid metabolism abnormalities that develop on the basis of obesity can be suppressed.

  To date, the following substances are known as substances that enhance the fatty acid oxidation ability of skeletal muscle. 5-aminoimidazole-4-carboxamide 1-β-d-ribonulleoside (AICAR), an AMPK activator, suppresses insulin resistance when administered to obese rats (Non-patent Document 3). Metformin, known as an antidiabetic, enhances fatty acid oxidation of skeletal muscle through the activation of AMPK (Non-patent Document 4). It has been known that PPARα activators such as WY-14,643 and fibrate drugs enhance fatty acid oxidation of skeletal muscle through increased expression of fatty acid oxidation genes under the control of PPARα (Non-patent Document 5).

  On the other hand, it has been clarified that leptin enhances fatty acid oxidation of skeletal muscle by an in vitro experiment using isolated skeletal muscle and an experiment in which leptin is administered to rats (Non-patent Document 6). Although the involvement of AMPK activation in the action of leptin in fatty acid oxidation in skeletal muscle has been suggested (Non-Patent Document 7), there are still many unclear points regarding the details of the mechanism. However, based on these pharmacological findings, leptin has attracted attention as a new treatment strategy for obesity and diabetes. Leptin and its analogs and derivatives are disclosed in detail for use as regulators of body weight and obesity suppression in mammals including humans (Patent Document 1).

  Moreover, as a combination therapy for obesity and diabetes prevention, one using leptin and a β3 adrenergic receptor agonist (Patent Documents 2 and 3) and one using leptin and a nicotine analog (Patent Document 4) are disclosed. These combination therapies are mainly expected to promote energy metabolism and promote an appetite reducing effect.

  To date, no knowledge has been found regarding the combined effect with substances that enhance fatty acid oxidation ability by focusing on the action of leptin on fatty acid oxidation of skeletal muscle.

WO09 / 05309 Brochure WO97 / 16189 brochure WO98 / 18481 brochure WO98 / 03175 brochure Science 2001 Mar 30; 291 (5513): 2613-6. Nat. Med. 2000 Oct; 6 (10): 1115-20. Diabetes 2001 May; 50 (5): 1076-82. J. Clin. Invest. , 2001 Oct; 108 (8): 1167-74. J. Lipid Res. 1996 May; 37 (5): 907-25. J. Biol. Chem. 2002 Aug 16; 277 (33): 29424-30. Nature 2002 Jan 17; 415 (6869): 339-43.

  The present inventors paid attention to the action of leptin to enhance fatty acid oxidation in skeletal muscle, and in combination with a compound having fatty acid oxidation ability, additively synergistically increases fatty acid oxidation in skeletal muscle, resulting in obesity, diabetes and abnormal lipid metabolism. It is intended to develop a more effective preventive or therapeutic agent.

  While the present inventors examined the chronic effect of leptin on fatty acid oxidation of skeletal muscle, leptin increases fatty acid oxidation of skeletal muscle cells, and the effect is a substance that enhances fatty acid oxidation ability, that is, AMPK activator and PPARα. It has been found that it is further enhanced by the combined use with an agonist, and the present invention has been completed.

  The present invention enhances skeletal muscle fatty acid oxidation by using a combination of a leptin and a PPARα activator such as metformin or AICAR that activates AMPK and a fibrate, and obesity, diabetes, and abnormal lipid metabolism. Have been found and a method for using the same.

That is, the present invention
1) A prophylactic or therapeutic agent for obesity, diabetes and lipid metabolism disorders, characterized by combining leptin and a substance that enhances fatty acid oxidation ability,
2) Obesity, diabetes, and abnormal lipid metabolism characterized by combining a first drug formulated with leptin as an active ingredient and a second drug formulated with a substance that enhances fatty acid oxidation ability as an active ingredient Preventive or therapeutic agent,
3) A prophylactic or therapeutic agent for obesity, diabetes and abnormal lipid metabolism, characterized by containing as an essential component a substance that enhances leptin and fatty acid oxidation ability,
4) The preventive or therapeutic agent according to any one of 1) to 3), wherein the substance that enhances the ability to oxidize fatty acids is an AMPK activator or a PPARα activator,
5) The preventive or therapeutic agent according to 4), wherein the AMPK activator is metformin or 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR),
6) The preventive or therapeutic agent according to 4), wherein the PPARα activator is fibrate or WY-14,643,
7) Use for the prevention or treatment of obesity, diabetes and dyslipidemia, characterized by using a combination of leptin and a substance that enhances fatty acid oxidation ability;
8) Use for prevention or treatment according to claim 7, wherein leptin and a substance that enhances fatty acid oxidation ability are used simultaneously or continuously.
9) Use for prevention or treatment according to 7) or 8), wherein the substance that enhances the ability to oxidize fatty acids is an AMPK activator or a PPARα activator,
10) Use for prevention or treatment according to 9), wherein the AMPK activator is metformin or 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR),
The present invention relates to a novel preventive and therapeutic agent that suppresses obesity, diabetes, and abnormal lipid metabolism, and uses thereof for prevention or treatment.

  The present invention can enhance fatty acid oxidation of skeletal muscle by using leptin in combination with a drug such as metformin or AICAR that activates AMPK and a PPARα activator such as a fibrate, obesity, diabetes, and lipids. A novel preventive and therapeutic agent, and a preventive and therapeutic method thereof are provided for patients suffering from metabolic disorders.

  The present invention relates to a preventive or therapeutic agent for obesity, diabetes, and abnormal lipid metabolism, which is a combination of leptin and a substance that enhances fatty acid oxidation ability, and a method for using the same.

  The leptin used in the present invention is a human or mammalian natural leptin, recombinant leptin, artificial leptin or leptin analog and leptin derivative, and can be purchased and used as a reagent. It is desirable to use human leptin when it relates to a preventive or therapeutic agent for human obesity, diabetes and abnormal lipid metabolism, and a method for using the agent.

  The substance that enhances the ability to oxidize fatty acids is an AMPK activator or a PPARα activator. Preferred examples of the AMPK activator include metformin and 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Examples of PPARα activators include fibrate drugs and WY-14,643. Among them, fenofibrate and WY-14,643 are preferable. If necessary, a mixture of these AMPK activators and PPARα activators or a combination thereof may be used.

  A prophylactic or therapeutic agent for obesity, diabetes, and lipid metabolism abnormality comprising a combination of leptin and a substance that enhances the ability to oxidize fatty acids is a physiologically acceptable carrier or excipient for these active ingredients separately or simultaneously. , Mixed with binders, diluents, etc., orally, or parenterally, spray inhalation or rectal as granules, powders, tablets, capsules, syrups, suppositories, suspensions, solutions, etc. Can be administered. When the active ingredients are formulated separately, they can be mixed and administered at the time of use, or each can be administered simultaneously or continuously to the same patient with a time difference.

  The preventive or therapeutic agent for obesity, diabetes and dyslipidemia of the present invention can be formulated by conventional methods.

  The dosage of each active ingredient used in the combination can vary depending on the active ingredient contained, the method of administration and the condition of the subject to be treated. Usually leptin or an analog thereof is administered in a daily dose of about 0.01 mg / kg to about 100 mg / kg, preferably in a single dose, in two or three divided doses per day or in a sustained release form. The combined AMPK activator or PPARα activator is preferably administered in a single dose, in two or three divided doses per day or in a sustained release form. Preferably, metformin is combined with a daily dose of about 250 mg / body to about 750 mg / body as the AMPK activator, and about 100 mg / body to about 300 mg / body of fenofibrate as the PPARα activator.

  Next, the present invention will be described with reference to specific examples, but the present invention is not limited to these examples.

(Combination effect with PPARα activator)
1. Materials and Methods Mouse C2C12 myoblasts (ATCC CRL-1772) were purchased from Dainippon Pharmaceutical and cultured in Dulbecco's modified high glucose medium (DMEMH) containing 10% calf serum at 37 ° C. and 5% CO ₂ . Cells were grown to 70-80% confluence in a 25 cm ² flask and then cultured in DMEMH containing 2% horse serum for 5-7 days to induce differentiation from myoblasts to myotubes. After 24 hours of culture in serum-free DMEMH, leptin and WY-14,643 were treated for 22 hours. Leptin (R & D Systems) was dissolved in a phosphate buffer (PBS) and added to the cells. WY-14,643 (Calbiochem) was dissolved in dimethyl sulfoxide and added to the cells. The fatty acid oxidation reaction was measured using [ ¹⁴ C] carbon dioxide production from [ ¹⁴ C] palmitic acid as an index. [ ¹⁴ C] Palmitic acid (0.1 μCi / ml), 0.25 mM palmitic acid, and 0.5% (W / V) bovine serum albumin were used for fatty acid oxidation reaction. A 20 ml glass vial for a liquid scintillation counter containing a 25 mm glass filter pre-moistened with Solen 350 was connected to the flask with candy rubber and sealed with 95% O ₂ and 5% CO ₂ gas. The fatty acid oxidation reaction was performed at 37 ° C. for 2 hours. The reaction was stopped with 8% perchloric acid, and the amount of [ ¹⁴ C] carbon dioxide collected on the glass filter was measured with a liquid scintillation counter. The results are shown in FIG. That is, the results shown in FIG. 1 show that differentiated C2C12 myoblasts were treated with leptin 10 and 30 nM for 24 hours in the presence or absence of WY-14,643 (10 ⁻⁴ M), and 0.25 mM [ ¹⁴ C] palmitin. It is the graph which measured the amount of [ ^14C ] carbon dioxide production for 2 hours after adding an acid. The value indicates an average value ± standard deviation when three independent experiments are performed.

(Combination effect of metformin)
According to Example 1, instead of WY-14,643, metformin (manufactured by Sigma) was dissolved in dimethyl sulfoxide and tested in the same manner. The result is shown in FIG. That is, the results shown in FIG. 2 are obtained by treating leptin 10 and 30 nM in the presence or absence of metformin (2 mM) in differentiated C2C12 myoblasts for 24 hours and adding 0.25 mM [ ¹⁴ C] palmitic acid. It is the graph which measured the amount of [ ¹⁴ C] carbon dioxide production for 2 hours. The value indicates an average value ± standard deviation when three independent experiments are performed.

(Combination effect of AICAR)
In accordance with Example 1, AICAR (manufactured by Toronto Research Chemicals) was dissolved in purified water instead of WY-14,643 and tested in the same manner. The result is shown in FIG. That is, the results shown in FIG. 3 are obtained by treating leptin 10 and 30 nM in the presence or absence of AICAR (2 mM) in differentiated C2C12 myoblasts for 24 hours and adding 0.25 mM [ ¹⁴ C] palmitic acid. It is the graph which measured the amount of [ ¹⁴ C] carbon dioxide production for 2 hours. The value indicates an average value ± standard deviation when three independent experiments are performed.

  From the above results, it is clear that metformin, AICAR and WY-14,643 increased fatty acid oxidation ability in skeletal muscle cells, but leptin synergistically increases their action, as is apparent from FIGS. It became.

  Combined and combined with leptin and PPARα activator WY-14,643 or AMPK activator metformin or AICAR, fatty acid oxidation of skeletal muscles is further enhanced, leading to excellent prevention of obesity, diabetes and lipid metabolism abnormalities. It was suggested that it could be a treatment method.

  As described above, AMPK activator metformin, AICAR or PPARα activator WY-14,643 increases fatty acid oxidation ability in skeletal muscle cells, but when used in combination with leptin, the fatty acid oxidation action is synergistic. It became clear that it increased.

  Combining leptin with drugs such as metformin and AICAR that activate AMPK and PPARα activators such as fibrates, fatty acid oxidation in skeletal muscle cells is further enhanced and promoted, leading to superior obesity, diabetes, and lipid metabolism The present invention provides an agent for preventing or treating abnormalities, or a method for using the same.

The measurement result which shows the combined use effect with the PPAR (alpha) activator with respect to the fatty acid oxidation enhancement by the leptin in C2C12 skeletal muscle cell. The measurement result which shows the combined use effect with metformin with respect to the fatty acid oxidation enhancement by the leptin in a C2C12 skeletal muscle cell. The measurement result which shows the combined use effect with AICAR with respect to the fatty acid oxidation enhancement by the leptin in a C2C12 skeletal muscle cell.

Claims (2)

A prophylactic or therapeutic agent for obesity, diabetes or abnormal lipid metabolism characterized by combining 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR) and leptin and allowing them to act for 24 hours or more . A combination of a first drug formulated with leptin as an active ingredient and a second drug formulated with 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR) as an active ingredient The preventive or therapeutic agent for obesity, diabetes or lipid metabolism abnormality according to claim 1.

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