JP2009062322A - Hepatocyte regeneration promotor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hepatocyte regeneration promotor having a higher hepatocyte regeneration-promoting effect than those of conventionally used glycyrrhizin and its derivatives, and containing a glycyrrhetinic acid derivative as an active ingredient, and also the new glycyrrhetinic acid derivative capable of being used for the promotor. <P>SOLUTION: The hepatocyte regeneration promotor contains a compound or its pharmaceutically acceptable salt expressed by general formula (1) [wherein, X is H or OH], or a compound expressed by formula (12). As the pharmaceutically acceptable salt of the compound expressed by the general formula (1), an ammonium salt, an alkali metal salt, or a choline salt is suitable. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a hepatocyte regeneration promoter containing 11-deoxoglycyrrhetinic acid or a derivative thereof as an active ingredient, and an 11-deoxoglycyrrhetinic acid derivative.

Glycyrrhizin is isolated and purified from the roots of the leguminous plant (Glycyrrhiza glabra) and is known as one of the active ingredients of the herbal licorice. Glycyrrhizin and some of its derivatives are known to have many pharmacological actions, such as antispasmodic, analgesic, anti-inflammatory, anti-peptic ulcer effects and the like. In addition, glycyrrhetinic acid, which is an aglycone, is known to have various pharmacological actions such as anti-inflammatory, anti-allergic, and hyperlipidemia improving effects (see Non-Patent Document 1).
In Japan, glycyrrhizin preparations have been used clinically for 20 years as therapeutic agents (hepatic protection drugs) for chronic liver diseases such as hepatitis and hepatic cirrhosis. In Europe, the efficacy and safety of glycyrrhizin preparations for chronic hepatitis C patients are clinically evaluated, and the increase in serum alanine aminotransferase activity in chronic hepatitis C patients is reduced by intravenous administration. Has been reported (see Non-Patent Document 2).

The main pharmacological effect of glycyrrhizin preparations is thought to be its anti-inflammatory action, but there is an effective model experiment system for the presence or absence of a growth promoting action on hepatocytes, which are the main inflammatory sites such as hepatitis. There were no reports, and there were very few reports examined in detail.
Recently, the effect of glycyrrhizin and some of its derivatives on the proliferation of rat liver parenchymal cells has been studied using the rat primary hepatocyte culture system (in vitro experimental system). It has been found that glycyrrhizin and some derivatives have an EGF-like growth-promoting action that directly stimulates and promotes the growth of epithelial growth factor (EGF) receptors in hepatocytes. 3).
Inoue et al. 1996, Jpn. J. et al. Pharmacol. 71,281-289 van Rossum et al. 1999, J. MoI. Gastroenterol. Hepatol. 14,1093-1099 Kimura et al. , European Journal of Pharmacology 431 (2001) 151-161

However, with regard to derivatives of glycyrrhetinic acid, which is an aglycone of glycyrrhizin, the present situation is that sufficient studies as a hepatocyte regeneration promoter have not been made so far.
The present invention has been made in view of the above circumstances, and has a liver parenchymal cell regeneration promoting effect higher than that of conventional glycyrrhizin and its derivatives, and a liver parenchymal cell regeneration promoter containing a glycyrrhetinic acid derivative as an active ingredient, and the promotion thereof It is an object of the present invention to provide a novel glycyrrhetinic acid derivative that can be used as an agent.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that among glycyrrhetinic acid derivatives, particularly among 11-deoxo compounds, those having a higher hepatocyte regeneration promoting effect, The invention has been completed.

That is, to solve the above problem,
The invention according to claim 1 is a liver parenchymal cell regeneration promoter comprising a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

  (In the formula, X represents a hydrogen atom or a hydroxyl group.)

  Invention of Claim 2 is a compound represented by following formula (12).

  INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an agent for promoting the regeneration of liver parenchymal cells, which exhibits high effectiveness in promoting regeneration of liver damaged by liver disease, liver regeneration after living donor liver transplantation, and the like.

  Hereinafter, the present invention will be described in detail. In addition, the number notation of the carbon atom of the compound represented by the general formula (1) is as shown below, which is represented by the following formulas (2), (3), (11), and (12). The same applies to the compound to be prepared.

  (In the formula, X represents a hydrogen atom or a hydroxyl group.)

The hepatocyte regeneration promoter of the present invention comprises a compound represented by the general formula (1) (hereinafter abbreviated as compound (1)) or a pharmaceutically acceptable salt thereof as an active ingredient. .
In the formula, X represents a hydrogen atom or a hydroxyl group. That is, the compound (1) is more specifically a compound represented by the following formula (11) or (12) (hereinafter abbreviated as the compound (11) or the compound (12), respectively). Deoxoglycyrrhetinic acid or a derivative in which a hydrogen atom bonded to a carbon atom at the 24-position thereof is substituted with a hydroxyl group. Compound (12) is a novel compound.

In the present invention, examples of the pharmaceutically acceptable salt of compound (1) include ammonium salts such as compound (1) monoammonium salt; compound (1) monosodium salt, compound (1) monopotassium salt and the like. Examples include alkali metal salts; compound (1) choline salts and the like. In addition to these, calcium salts, magnesium salts, aluminum salts, various organic amine salts, and the like can also be used. Among these, ammonium salts are preferable.
These may be used alone or in combination of two or more.

  The liver parenchymal cell regeneration-promoting agent of the present invention is clinically used as an oral preparation such as tablets, powders, granules, capsules, fine granules, and liquid medicines; and parenteral preparations such as inhalants, suppositories, and injections by conventional methods. Can be used. The dose depends on the symptoms to be treated and the administration method, but usually 1 μg to 10 g per day for an adult can be administered in a single dose or divided into several times a day.

  Oral preparations include, for example, excipients, lubricants, plasticizers, surfactants, binders, disintegrating agents, wetting agents, stabilizers, corrigents, coloring agents, fragrances, buffers usually used in the production of these preparations. It can be produced according to a conventional method by blending an agent and the like.

Examples of the excipient include lactose, glucose, D-mannitol, fructose, dextrin, starch, sodium chloride, sodium hydrogen carbonate, calcium carbonate, sodium alginate, ethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, silicic anhydride and kaolin. Is mentioned.
Examples of the lubricant include magnesium stearate, calcium stearate, stearic acid, talc, corn starch, and macrogol.
Examples of the binder include gelatin, gum arabic, cellulose ester, and polyvinyl pyrrolidone.

  Examples of the plasticizer include polyethylene glycol, propylene glycol, glycerol, triacetin, medium chain fatty acid triglyceride, acetylglycerol fatty acid ester, and triethyl citrate.

Examples of the disintegrant include starch, agar, carmellose calcium, carmellose, and crystalline cellulose.
Examples of the wetting agent include gum arabic, polyvinyl pyrrolidone, methyl cellulose, carmellose sodium, hydroxypropyl cellulose and the like.

Examples of the flavoring agent include sucrose, honey, sodium saccharin, mint, eucalyptus oil, and cinnamon oil.
Examples of the colorant include iron oxide, β-carotene, chlorophyll, and a water-soluble edible tar dye.
Examples of the fragrance include lemon oil, orange oil, dl- or l-menthol.

In addition, when used as a parenteral preparation such as an inhalant or an injection, a pharmaceutical preparation such as a solution or suspension using distilled water for injection or a sterile non-aqueous solvent as a solvent can be exemplified. Examples of the base of the non-aqueous solvent or suspending agent include propylene glycol, polyethylene glycol, glycerin, olive oil, corn oil, ethyl oleate and the like.
Examples of the base when used as a suppository include cocoa butter and macrogol.
In addition to these, as long as the effects of the present invention are not hindered, parenteral preparations may be appropriately added with buffering agents, preservatives, antioxidants, and the like as necessary as pharmaceutically acceptable ingredients. it can.

Compound (12) can be produced as follows.
A compound represented by the following formula (3) (hereinafter referred to as compound) by hydrolyzing the compound represented by the following formula (2) (hereinafter abbreviated as compound (2)) or a salt thereof to release sugar. (Abbreviated as (3)). The reaction is carried out, for example, by stirring the compound (2) or a salt thereof in ethanol together with an appropriate amount of sulfuric acid with heating, preferably heating and refluxing. After completion of the reaction, compound (3) is obtained by post-treatment and purification as necessary. For the post-treatment, for example, a known method such as extraction, dehydration, and vacuum concentration may be applied as necessary. For purification, a known method such as column chromatography or recrystallization may be appropriately selected.

Examples of the salt of compound (2) include ammonium salts such as compound (2) monoammonium salt; alkali metal salts such as compound (2) monosodium salt and compound (2) monopotassium salt; compound (2) choline salt Etc. In addition to these, calcium salts, magnesium salts, aluminum salts, various organic amine salts, and the like can also be used.
The salt of compound (2) can be obtained, for example, by allowing compound (2) to react with an inorganic base or organic base at a constant molar ratio, but a commercially available product may be used. Furthermore, since some commercially available glycyrrhizin salts such as glycyrrhizin monoammonium salt contain about several percent (for example, about 3 to 5%) of the salt of compound (2), the salt of compound (2) May be purified from this and taken out, or may be subjected to the above reaction as a mixture.

  Subsequently, the oxygen atom bonded to the carbon atom at the 11th position of the compound (3) is replaced with a hydrogen atom (that is, the carbonyl group containing the carbon atom at the 11th position is converted to a methylene group), and the compound is subjected to a reduction reaction (12) is obtained. For the reaction, for example, a method of reducing glycyrrhetinic acid described in JP-A-59-70638 can be applied. That is, compound (3) is reduced at room temperature with zinc and hydrochloric acid in a solvent, preferably dioxane. After completion of the reaction, insoluble matters are removed by filtration and the like, and then post-treatment and purification are performed as necessary to obtain the compound (12). For the post-treatment, for example, a known method such as extraction, dehydration, and vacuum concentration may be applied as necessary. For purification, a known method such as column chromatography or recrystallization may be appropriately selected.

  The pharmaceutically acceptable salt of compound (1) can be obtained, for example, by reacting compound (1) with an inorganic base or organic base at a certain molar ratio.

  Compound (1) or a pharmaceutically acceptable salt thereof has a liver parenchymal cell regeneration promoting effect superior to that of glycyrrhizin, a glycyrrhizin derivative, glycyrrhetinic acid, and pharmaceutically acceptable salts thereof. Here, the pharmaceutically acceptable salt of glycyrrhizin, a glycyrrhizin derivative, and glycyrrhetinic acid includes the same pharmaceutically acceptable salts of compound (1). Among these, the compound (11) or a pharmaceutically acceptable salt thereof has the highest regeneration promoting effect. Accordingly, the liver parenchymal cell regeneration promoter of the present invention exhibits an excellent effect on liver regeneration even when administered in a smaller amount than conventional promoters.

Hereinafter, the present invention will be described in more detail with specific examples. However, the present invention is not limited to the following examples.
[Example 1]
(Production of Compound (12))
<Production of Compound (3)>
250 ml of ethanol and 250 ml of 10% sulfuric acid were added to 28 g of glycyrrhizic acid monoammonium salt (purity 75%) containing compound (2) monoammonium salt, and the mixture was heated to reflux for 24 hours. Next, water was added to the reaction solution, extracted with chloroform, dried over sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (chloroform: methanol = 100: 1) to give 350 mg of compound (3). Obtained.
It was as follows when the physical property of the obtained compound (3) was confirmed.

¹ H-NMR (400MHz, CDCl ₃ ) δppm: 5.69 (1H, s), 4.21 (1H, d, J = 11.0 Hz), 3.46 (1H, dd, J = 12.0, 4.4 Hz), 3.34 (1H, d , J = 11.0 Hz), 2.81 (1H, dt, J = 13.4, 6.7 Hz), 2.33 (1H, s), 2.20-2.17 (1H, m), 1.36 (3H, s), 1.25 (3H, s) , 1.21 (3H, s), 1.09 (3H, s), 1.08 (3H, s), 0.83 (3H, s). ¹³ C-NMR (100MHz, CDCl ₃ ) δppm: 200.0, 180.9, 169.5, 128.2, 80.5 , 64.2, 61.7, 55.5, 48.2, 45.4, 43.7, 43.1, 43.0, 40.9, 38.8, 37.7, 36.7, 32.9, 31.8, 30.9, 28.5, 28.4, 27.5, 26.5, 26.4, 23.4, 22.5, 18.5, 17.6, 16.9 .

<Production of Compound (12)>
50 mg of compound (3) was dissolved in 1 ml of dioxane, 54 mg of zinc powder and 0.2 mL of concentrated hydrochloric acid were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was then filtered, concentrated under reduced pressure, and recrystallized from hexane and ethanol to obtain 39 mg of compound (12) as white crystals (yield 80%).
It was as follows when the physical property of the obtained compound (12) was confirmed.

¹ H-NMR (400MHz, pyridine-d ₅ ) δppm: 5.48 (1H, br s), 4.51 (1H, d, J = 10.7 Hz), 3.71 (1H, d, J = 10.7 Hz), 3.64 (1H, dd, J = 11.5, 4.1 Hz), 2.45-2.42 (1H, m), 2.32-2.29 (2H, m), 1.57 (3H, s), 1.38 (3H, s), 1.27 (3H, s), 0.95 . (3H, s), 0.92 (3H, s), 0.89 (3H, s) 13 C-NMR (100MHz, pyridine-d 5) δppm: 179.6, 145.1, 122.8, 80.2, 64.7, 56.4, 48.8, 48.2, 44.5, 43.7, 43.3, 41.9, 40.2, 39.2, 39.0, 37.1, 33.5, 32.5, 32.0, 29.3, 28.7, 28.6, 27.4, 26.7, 26.3, 24.2, 23.7, 19.3, 17.1, 16.3.
LC / MS (m / z) ES + 473.2 [M + H] ⁺

[Example 2]
(Measurement of the number of nuclei and DNA synthesis ability of hepatocytes)
Using compound (11), the number of nuclei of liver parenchymal cells and the ability to synthesize DNA were measured by the following method.
<Isolation and culture of hepatocytes>
Wistar male rats (body weight 200-250 g) were used according to the method of Seglen et al. (Methods Cell Biol., 13, 29: 1975). Liver parenchymal cells isolated by in situ collagenase reflux method were added to Williams'E medium containing 0.1% bovine serum (containing 0.1 μg / mL aprotinin, 100 U / mL penicillin, 0.1 μg / mL streptomycin, 0.1 nM dexamethasone). The suspension was suspended, seeded on a collagen-coated culture dish (35-mmφ), and cultured at 37 ° C. in the presence of 5% CO ₂ for 3 hours. Three hours after the start of the culture, Compound (11) was added to the bovine neonatal serum-free Williams'E medium (serum-free medium), and the culture was further continued for 0-21 hours.

<Measurement of DNA synthesis ability>
According to the method of Morley and Kingdon (Anal. Biochem., 45, 298; 1972), the amount of ³ H-thymidine incorporated into the DNA fraction was measured to determine the DNA synthesis ability. The amount of specific ³ H-thymidine was determined by subtracting the amount counted in the presence of 10 μM aphidicolin. The ability to synthesize DNA was expressed in terms of unit time and ³ H-thymidine amount (dpm / mg protein / h) per unit protein.

<Measurement of the number of nuclei>
The method of Nakamura et al. (J. Biochem., 94, 1029; 1983) was partially modified. Liver parenchymal cells cultured for a certain period of time were washed with a phosphate buffer (PBS: pH 7.4) and treated with a 0.1 M citric acid solution containing 0.1% Triton X-100 to obtain naked nuclei. To this, the same volume of 0.3% trypan blue-PBS solution was added, and the measurement was performed with a hemocytometer.

<Quantification of protein>
According to a modified method of Lowry et al. (Anal. Biochem., 47, 184; 1972), bovine serum albumin was measured as a standard substance.

<Statistical processing>
Statistical significance in the measurement results was evaluated by one-way analysis of variance followed by paired comparison test of each group with respect to the control group by Dunnett method. A risk rate of 0.05% or less was considered significant (n = 3).

[Example 3]
The number of liver parenchymal cells and the ability to synthesize DNA were measured in the same manner as in Example 2 except that the compound (12) obtained in Example 1 was used instead of the compound (11). .

[Comparative Example 1]
The number of hepatocytes and the ability to synthesize DNA were measured by the same method as in Example 2 except that glycyrrhetinic acid was used instead of compound (11).

<Measurement results and discussion>
The measurement results are shown in FIGS. 1 and 2, “GA” represents “glycyrrhetinic acid”, “(11)” represents “compound (11)”, and “(12)” represents “compound (12)”. “GA analogs” indicates “glycyrrhetinic acid derivative”, and “M” indicates “mol / L”.
FIG. 1 is a graph showing the dose-dependent effects of glycyrrhetinic acid, compound (11) and compound (12) on DNA synthesis and proliferation of primary cultured hepatocytes in 4 hours of culture, (a) is DNA synthesis ability, (B) shows the dose-dependent effect on the number of nuclei.
FIG. 2 is a similar graph for 21 hours of culture.
The experimental conditions were as follows.
Seeding density: 3.3 × 10 ⁴ cells / cm ² (seeding 3 hours before drug addition).
Culture time: 4 hours (FIG. 1) and 21 hours (FIG. 2) after replacement with serum-free medium.
The symbols “★” and “★★” in FIGS. 1 and 2 indicate a significant difference from the control (drug-untreated group). ★ P <0.05, ★★ P <0.01 (mean ± SEM, n = 3).

All of glycyrrhetinic acid, compound (11) and compound (12) showed a promoting effect on the proliferation of primary cultured hepatocytes. That is, as shown in FIG. 1, the stimulating effect was shown in a dose-dependent manner as early as 4 hours under low density culture conditions. The potency of these compounds is stronger in the order of Compound (11); 4 × 10 ⁻¹¹ M << Compound (12); 4 × 10 ⁻⁹ M <Glycyrrhetinic acid; 5 × 10 ⁻⁹ M from the EC ₅₀ value. As is clear from 1 and 2, the EC ₅₀ values of DNA synthesis and the number of nuclei almost coincided.
From the above results, when the oxygen atom bonded to the 11th carbon atom of glycyrrhetinic acid is removed and the carbonyl group is converted to a methylene group, the effect of promoting the growth of primary cultured hepatocytes can be enhanced. It was suggested. In particular, the growth promoting effect of compound (11) was expressed about 100 times stronger than glycyrrhetinic acid at EC ₅₀ value.

  INDUSTRIAL APPLICABILITY The present invention can be used for therapeutic agents such as promoting regeneration of damaged liver and liver regeneration after living-donor liver transplantation.

It is a graph which shows the dose-dependent effect of glycyrrhetic acid, a compound (11), and a compound (12) in DNA synthesis and proliferation of primary culture hepatocytes in culture | cultivation of Example 2 and 3 and the comparative example 1 for 4 hours, (a ) Shows the ability to synthesize DNA, and (b) shows the dose-dependent effect on the number of nuclei. It is a graph which shows the dose dependence effect of glycyrrhetinic acid, a compound (11), and a compound (12) in DNA synthesis and proliferation of primary culture hepatocytes in 21 hours of culture | cultivation of Example 2 and 3 and the comparative example 1, (a ) Shows the ability to synthesize DNA, and (b) shows the dose-dependent effect on the number of nuclei.

Claims (2)

A liver parenchymal cell regeneration promoter comprising a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

(In the formula, X represents a hydrogen atom or a hydroxyl group.) The compound represented by following formula (12).

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