JPH06157338A - Production promoter for nerve growth factor - Google Patents

Abstract

PURPOSE:To provide a promoter of few side effects, useful for curing or preventing neurodegenerative diseases and for neurofunction recovery in peripheral nerve diseases. CONSTITUTION:The promoter containing, as active ingredient, a diacyl-type glyceroline lipid of the formula (R<1> and R<2> are each 8-30C acyl; X is H or lower alkyl, 3-8C cycloalkyl or 3 to 8-membered heterocycle; Y is H or counter cation, where, when X has a cation free from counter anion, OY is oxygen anion). The compound of the formula is e.g. phosphatidylethanolamine diacylated with a 8-30C fatty acid, phosphatidylserine, phosphatidylinositol, phosphatidylcholine, phosphatidic acid, phosphatidylglycerol, diphosphatidylglycerol (cardiolipin).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a diacylglycerophospholipid useful for medicine, particularly for treating or preventing neurodegenerative diseases such as senile dementia or Alzheimer's disease, and for recovering nerve function in peripheral nervous system diseases. Nerve growth factor [(Nerve growth fa
ctor), hereinafter abbreviated as NGF].

[0002]

2. Description of the Related Art NGF is one of the nutrients and growth factors necessary for the growth and function maintenance of nerve tissue, and in the peripheral nervous system,
In sensory and sympathetic nerves, and in the central nervous system, it is considered to be essential for maturation, differentiation and life support of large cell cholinergic neurons. Therefore, increasing NGF levels is useful for treating central dysfunctions such as Alzheimer's disease and vascular dementia, spinal cord injury, peripheral nerve injury, diabetic neuropathy and peripheral dysfunction such as amyotrophic lateral sclerosis. It is considered useful. However, NGF
Protein (molecular weight, monomer 13,000, dimer 26)
Thousand), and peripheral administration is ineffective because it cannot cross the blood-brain barrier. Therefore, rather than administering NGF, it is considered preferable to improve the central dysfunction and peripheral dysfunction by administering a substance that promotes NGF production in vivo to promote NGF biosynthesis. Based on this idea, attempts have been made to search for NGF production promoters, and catecholamines such as epinephrine, norepinephrine and dopamine have been found so far. However, since these compounds are hormonal substances, their administration in order to promote NGF synthesis destroys the quantitative balance of hormones in the body and is accompanied by side effects. Therefore, no satisfactory drug has been found yet.

[0003]

An object of the present invention is to provide an excellent NGF production promoter without the above-mentioned drawbacks.

[0004]

[Means for Solving the Problems] For the above-mentioned reasons, the inventors of the present invention have made extensive studies on a drug that promotes the production of NGF.
The present invention was completed by discovering that it has an effect of promoting the production of GF.

That is, the present invention has the general formula

[0006]

[Chemical 2]

(In the formula, R ¹ and R ^{2, which} may be the same or different, are an acyl group having 8 to 30 carbon atoms, X is a hydrogen atom, or a substituted or unsubstituted lower alkyl group, and 3 carbon atoms. ~ 8 cycloalkyl group or a 3-8 membered heterocycle, Y represents a hydrogen atom or a counter cation group, provided that when X has a cation group having no counter anion, OY is an oxygen anion. The present invention relates to a nerve growth factor production promoter containing a diacyl type glycerophospholipid represented by

R ¹ and R ² of the diacyl type glycerophospholipid represented by the above general formula may be the same or different and each is an acyl group having 8 to 30 carbon atoms, preferably 12 to 24.
Is an acyl group derived from the saturated or unsaturated fatty acid of. Examples of these acyl groups include octanoyl group, nonanoyl group, decanoyl group, tridecanoyl group,
Tetradecanoyl group, pentadecanoyl group, hexadecanoyl group, octadecanoyl group, eicosanoyl group, docosanoyl group, tetracosanoyl group, hexacosanoyl group, octacosanoyl group, and octenoyl group, decenoyl group, tridecenoyl group, tetradecenoyl group, pentadecenoyl group , Hexadecenoyl group, octadecenoyl group, eicosaenoyl group, docosaenoyl group, tetracosaenoyl group, tetradecadienoyl group, docosadienoyl group, hexadecatrienoyl group, eicosatetraenoyl group, eicosapentaenoyl group, docosahexae Examples thereof include a noyl group and the like.

In the above formula, X is an alkyl-substituted or unsubstituted amino group, an alkyl-substituted or unsubstituted ammonium group, a carboxyl group, an alkoxycarbonyl group,
A lower alkyl group substituted with one or more groups selected from an acyloxy group, an alkyl-substituted or unsubstituted carbamoyl group, an alkoxy group, an ester type or free phosphoric acid residue, and a hydroxy group, and a carbon number of 3 to 8 Is preferably a cycloalkyl group or a 3- to 8-membered heterocycle, and examples of the lower alkyl group include a methyl group, an ethyl group,
Examples of the cycloalkyl group having 3 to 8 carbon atoms such as propyl group and n-butyl group include cyclopropyl group, cyclopentyl group and cyclohexyl group, and examples of the 3 to 8 membered heterocycle include epoxy group, tetrahydrofuranyl group and tetrahydro group. Examples include pyranyl group, tetrahydrothienyl group, pyrrolidyl group, piperazyl group, furyl group, thienyl group, pyrrolyl group, pyridyl group, imidazolyl group and thiazolyl group.

Specific examples of such X include nitrogen-containing groups such as 2-aminoethyl group, 3-aminopropyl group and 2-trimethylammoniumethyl group, 2-amino-2-carboxyethyl group (serine residue). Amino acid residues, such as
A group containing a phosphoric acid residue such as a phosphonooxymethyl group,
Examples thereof include sugar residues such as glucose and inositol.

Examples of the counter cation group include sodium ion, potassium ion, lithium ion and ammonium ion. If a cationic group is present in X, the cationic group can form an ion pair with OY.

Specific examples of the diacyl-type glycerophospholipid according to the present invention include the following compounds diacylated with the same or different fatty acids having 8 to 30 carbon atoms and pharmaceutically acceptable salts thereof. .

[0013]

[Chemical 3]

[0014]

[Chemical 4]

(In the formula, R ¹ , R ² and Y are the same as above, R ³ and R ⁴ may be the same or different, and are an acyl group having 8 to 30 carbon atoms).

These diacyl glycerophospholipids are
For example, Shinsei Chemistry Laboratory 4 "Lipid II (phospholipid)"
It is publicly known in the literature such as Tokyo Kagaku Dojin (1991) edited by The Biochemical Society of Japan, and is also partially commercially available. In addition, various derivatives of these diacyl type glycerophospholipids,
It can be synthesized by a known method described in the above documents and the like.

The diacyl type glycerophospholipid of the present invention is
Either oral or parenteral dosage forms are possible. In the case of oral administration, it can also be administered by a usual method such as capsules, tablets and powders. Further, in the case of parenteral administration, it is administered in the form of injections, infusions and the like. Further, a sustained release agent is also effective. $ To formulate the active ingredient of the present invention, a surfactant, an excipient, a coloring agent, a preservative, a coating aid and the like are appropriately used. It can also be used in combination with other drugs.

[0018]

[Examples] Examples showing the NGF production promoting action of the NGF production promoting agent containing a diacyl type glycerophospholipid according to the present invention will be shown below, but the present invention is not limited to these examples. .

Example 1 Fibroblast established LM cells derived from mouse connective tissue were
Contains 0.5% peptone (manufactured by Difco Laboratories) 19
9 cells (made by Flow Laboratories) containing 2 × 10 ⁴ cells
Resuspend the cells in a number of holes / hole, place them in a flat-bottom 96-well microplate (Nunc), and put them in a CO ₂ incubator (3
The cells were cultured at 7 ° C. in an atmosphere of 5% CO ₂ -95% air) for 3 days. The above medium was treated with phosphatidylethanolamine (fatty acid composition 14: 0 2%, 16: 0 17%, 16: 1 51
%, 18: 1 Baxenoic acid 30%) 0.5% bovine serum albumin (Ar
(Mour Pharmaceutical Co., Ltd.)-containing 199 medium was dissolved at each concentration or replaced with a test compound-free medium, and the cells were cultured in a CO ₂ incubator. After 24 hours of culture, the amount of NGF contained in the culture supernatant was measured by enzyme immunoassay (Korsching and Thoenen, Proc. Natl. Acad. Sci., U.
SA, 80. 3513-3516, 1983.). The results are shown in Table 1.
Shown in.

[0020]

[Table 1]

Method for measuring NGF An anti-mouse βNGF antibody (prepared using βNGF prepared from mouse submandibular gland as an antigen) solution (pH 8.3) on a polystyrene 96-well microplate (MS-3496F manufactured by Sumitomo Bakelite Co., Ltd.) ) Is dispensed into each hole by 50 μl,
It was left at 37 ° C. for 4 hours. After removing the antibody not adsorbed on the microplate, each well was washed three times with a washing solution.
Standard βNGF (Toyobo) solution or sample solution 4
After 0 μl was dispensed into each well and left at 4 ° C. for 18 hours, the standard βNGF or the sample solution was removed, and each well was washed 3 times. β-galactosidase-labeled anti-βNGF monoclonal antibody (Boehringer Mammheim) solution (40 mU / m
50 μl of each solution was added to each well and left at 37 ° C. for 4 hours, then the enzyme-labeled antibody was removed and the plate was washed 3 times. Four
-Methylumbelliferyl-β-D-galactoside (Si
(manufactured by gma) (20 μg / ml, pH 7.6) was dispensed into each well by 100 μl, reacted at room temperature for 1.5 hours, and then 0.2 M glycine-
100 μl of sodium hydroxide buffer solution (pH 10.3) was dispensed into each well to stop the enzyme reaction, and the fluorescence intensity of the produced 4-methylumbelliferone was measured with a plate reader, and the amount of NGF was determined from the standard curve. Calculations were made and the results are shown in Table 1. The NGF production promoting activity of the test compound was represented by the relative value of the NGF amount produced by the test compound-treated cells with respect to the NGF amount produced by the untreated cells to which the test compound was not added.

Example 2 As a test compound, a bacterium (Alteromonas putrifaciens SCRC-2871; Micro Works Kenjo No. 1624)
No.)-Derived phosphatidylethanolamine (fatty acid composition 14: 0 2%, 15: 0 21%, 16: 1 23%, 17: 1 9%, 18: 0 1%, 18: 1
NGF production promoting activity was examined in the same manner as in Example 1 except that oleic acid 2%, 18: 1 vaccenic acid 7%, 20: 5 11%) was used.
The results are shown in Table 2.

[0023]

[Table 2]

Example 3 Phosphatidyl ethano-derived from pig liver as a test compound
Luamine (fatty acid composition 15: 0 7%, 18: 0 26%, 18: 1 oleic acid 6
%, 18: 2 9%, 20: 4 35%) (Serdary Research Laboratories
NGF production promoting activity was examined in the same manner as in Example 1 except that the product manufactured by Catalog No. A-33) was used. The results are shown in Table 3.

[0025]

[Table 3]

Example 4 Dioleoylphosphatidylethano-as a test compound
Example 1 except that Luamine (SIGMA, P-0510) was used
The NGF production promoting activity was examined in the same manner as in. The results are shown in Table 4.

[0027]

[Table 4]

Example 5 As a test compound, dilauroylphosphatidyl ethano-
NGF production promoting activity was examined in the same manner as in Example 1 except that ruamine (SIGMA, P-627) was used. The results are shown in Table 5.

[0029]

[Table 5]

Example 6 As a test compound, bovine brain-derived phosphatidylserine (Serd
ary Research Laboratories, Catalog No. A-37) was used to examine NGF production promoting activity in the same manner as in Example 1. The results are shown in Table 6.

[0031]

[Table 6]

Example 7 Pig liver-derived phosphatidylinositol as a test compound
(Serdary Research Laboratories, Catalog No. A-
NGF production promoting activity was examined in the same manner as in Example 1 except that 38) was used. The results are shown in Table 7.

[0033]

[Table 7]

Comparative Example 1 LM cells were cultured in the same manner as in Example 1 except that epinephrine (050-04081 manufactured by Wako Pure Chemical Industries, Ltd.) known as an NGF production promoter was used as a test compound. ,
The NGF production promoting activity of epinephrine was examined. The results are shown in Table 8.

[0035]

[Table 8]

When epinephrine was added at 0.025 mg / ml or more, the NGF production promoting effect was observed for the first time, and the maximum activity value (about 500%) was obtained at the addition of 0.1 mg / ml. On the other hand, diacyl glycerophospholipids generally showed NGF production promoting activity at a lower concentration, and in particular, phosphatidylethanolamine had remarkably high NGF production promoting activity.

[0037]

EFFECTS OF THE INVENTION NG with strong diacyl glycerophospholipid
Since it exhibits F production promoting activity, the nerve growth factor production promoting agent of the present invention is a preventive and therapeutic drug for central dysfunction, particularly Alzheimer's dementia and cerebral ischemic pathology, and also peripheral nerve function recovery and nerve regeneration promotion. Used as an agent.

Claims (3)

[Claims] 1. A general formula: (In the formula, R ¹ and R ^{2, which} may be the same or different, are an acyl group having 8 to 30 carbon atoms, X is a hydrogen atom, or a substituted or unsubstituted lower alkyl group, and 3 to 8 carbon atoms. Represents a cycloalkyl group or a 3- to 8-membered heterocycle,
Y represents a hydrogen atom or a counter cation group. However, X
When a cation group having no counter anion is contained therein, OY represents an oxygen anion), and a nerve growth factor production promoter containing a diacyl type glycerophospholipid as an active ingredient. 2. X is an alkyl-substituted or unsubstituted amino group, an alkyl-substituted or unsubstituted ammonium group, a carboxyl group, an alkoxycarbonyl group, an acyloxy group, an alkyl-substituted or unsubstituted carbamoyl group, an alkoxy group, an ester type. Or a lower alkyl group substituted with one or more groups selected from a free phosphoric acid residue and a hydroxy group, a cycloalkyl group having 3 to 8 carbon atoms, or a 3 to 8 membered heterocycle. The production promoter for nerve growth factor according to 1. 3. A diacyl-type glycerophospholipid, which is diacylated with a fatty acid having 8 to 30 carbon atoms, such as phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylcholine, phosphatidic acid, phosphatidylglycerol, or diphosphatidylglycerol (cardiolipin), or The nerve growth factor production promoter according to claim 1, which is a pharmaceutically acceptable salt thereof.