JPH069663A - Phospholipid derivative and cell damage protection agent - Google Patents

Abstract

PURPOSE:To provide a new compound having excellent bio-compatibility, tissue transferability, active oxygen elimination effect and cell damage protecting effect and useful for the treatment of ischemic cardiopathy, ischemic encephalopathy, circulatory disease, etc. CONSTITUTION:The compound of formula I (R<1> and R<2> are 16-22C unsaturated fatty acid acyl residue; X is H or salt-forming metal), e.g. the compound of formula II. The compound of formula I can be produced e.g. by reacting a phosphatidic acid chloride with ascorbic acid.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel phospholipid derivative, and a drug containing the same as an active ingredient for the prevention and treatment of diseases caused by cell damage caused by active oxygen.

[0002]

2. Description of the Related Art There are many active oxygen scavenging systems in the living body, which protect the living body from oxidative stress. It has been clarified that the active oxygen generated from the disturbance of the defense system is involved in the development of various diseases. Particularly in recent years, it has been noted that the damage of vascular endothelial cells due to active oxygen is deeply involved in the pathogenesis of arteriosclerosis.

Cellular disorders caused by active oxygen are considered to be a part of various diseases that occur in inflammation and the brain, heart, circulatory organ, digestive system, etc., and until now, investigation of drugs that eliminate these active oxygen in the living body Has been done. For example, an antioxidant compound having an action of scavenging active oxygen and decomposing lipid peroxide has been developed as an active oxygen scavenger by a multi-faceted approach (radical scavenger, Mebio,
5 , 90-94, 1988).

Further, a compound in which a fatty acid residue or an alkyl group is directly introduced in order to fat-solubilize vitamin C, which is a water-soluble antioxidant, has been studied (J. Med. Che.
m. , 31 , 793-798, 1988).

By the way, JP-A-3-291289, JP-A-4-99724 and JP-A-4-11.
7392 discloses a phospholipid derivative having an ascorbic acid skeleton. However, these publications only describe a phospholipid derivative in which two acyl groups are both palmitic acid residues and a phospholipid derivative derived from soybean lecithin or egg yolk lecithin, and the cytotoxic protective effects of these phospholipid derivatives are not shown. Is insufficient.

[0006] WO 90/12800 also discloses a phospholipid derivative having an ascorbic acid skeleton. However, this publication does not disclose a phospholipid derivative having an unsaturated fatty acid acyl group as a fatty acid residue. Thus, at present, no phospholipid derivative having an ascorbic acid skeleton in which acyl groups having a specific unsaturated bond are defined at both the 1- and 2-positions is known.

[0007]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel and useful phospholipid derivative and a cytotoxic agent having a higher cytotoxicity-protecting effect than conventional phospholipid derivatives having an ascorbic acid skeleton. Is.

[0008] Generally, as a substance which damages cells in vivo, for example, vascular endothelial cells, lipid peroxides and reactive oxygen species such as superoxide, hydroxy radical, singlet oxygen, hydrogen peroxide are known. . The present inventors, after culturing vascular endothelial cells, to create an experimental system to examine the endothelial cell damage by activated leukocytes by the release reaction of radioactive chromium, as a result of various studies of various compounds to suppress the vascular endothelial cell damage, The inventors have found that a specific phospholipid derivative has a strong inhibitory effect, and completed the present invention.

[0009]

That is, the present invention provides the following phospholipid derivative and a cytotoxic agent containing it as an active ingredient. (1) A phospholipid derivative represented by the following general formula [1].

[Chemical 2] (In the formula, R ¹ and R ² are the same or different from each other, unsaturated fatty acid acyl residues having 16 to 22 carbon atoms, and X represents a hydrogen atom or a salt-forming metal.) (2) The phosphorus described in (1) above. A cytotoxic agent comprising a lipid derivative as an active ingredient.

In the present invention, R ^{1 of the} general formula [1] is
Or as the unsaturated fatty acid acyl residue having 16 to 22 carbon atoms represented by R ² , for example, hexadecenoic acid, octadecenoic acid, eicosenoic acid, docosenoic acid or the like having 16 to 2 carbon atoms
The acyl residue of the unsaturated fatty acid of 2 etc. are mentioned. R ¹
And R ² may be the same or different. R ¹ and /
Alternatively, when R ² is an oleoyl group or a linoleoyl group, the cytotoxicity protecting effect is particularly high.

Examples of the salt-forming metal represented by X in the above general formula [1] include alkali metals such as Na and K; C
Examples thereof include pharmacologically acceptable salt-forming metals such as alkaline earth metals such as a and Mg.

The phospholipid derivative of the present invention can be chemically synthesized by reacting chloride of phosphatidic acid with ascorbic acid, or a known method of reacting phospholipid with ascorbic acid by using phospholipase D (see, for example, Japanese Patent Laid-Open No. Hei 10 (1999) -58242). 3-291289 gazette). Examples of the phospholipid include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin and the like. The obtained phospholipid derivative represented by the general formula [1] can be isolated and purified by a conventionally known method such as extraction, recrystallization, chromatography and the like.

The cytotoxic agent of the present invention contains the phospholipid derivative represented by the above general formula [1] as an active ingredient.

The agent for protecting cytotoxicity of the present invention protects against cell damage caused by active oxygen, and ischemic heart disease, ischemic brain disease, cardiovascular disease (eg arteriosclerosis), digestive organ disease (eg digestion). It is effective against diseases such as duct, liver, pancreas), skin diseases, cancer, lung diseases and inflammation.

When the phospholipid derivative of the present invention is used as a cytotoxic agent, the phospholipid derivative is mixed with a pharmacologically acceptable carrier, excipient, diluent or the like known per se, and according to a known method. It can be administered orally or parenterally as a pharmaceutical composition such as tablets, capsules, solutions, injections, suppositories and the like.

Further, since the phospholipid derivative of the present invention is an oil and fat component, the following dosage forms can be given for parenteral administration. For injection and infusion, there are aqueous suspensions, oil preparations such as liposome preparations and lipid microsphere preparations. Topically applied forms include intraocular eye drops and eye ointments, as well as rectal lipid surfactant-mixed micelle-type suppositories.

The dose varies depending on the administration subject, administration route, symptoms, etc., but when orally administered, the phospholipid derivative is usually used in a single dose of about 1 to 100 mg / kg body weight, preferably about 5 to 50 mg / kg. 1 to 1 kg body weight per day
Dosage about once.

When administered parenterally, for example, in the case of a suppository, about 5 to 20 mg / kg body weight as a phospholipid derivative is administered once or twice a day. For oil-based injections, about 0.1 to 20 mg / kg body weight as a phospholipid derivative
It is preferable to administer 2 times.

The phospholipid derivative of the present invention has both a glycerophospholipid skeleton and an ascorbic acid skeleton which are naturally present and is contained in foods, and therefore has low toxicity to living bodies.

The phospholipid derivative of the present invention can be used as a raw material for foods, pharmaceuticals and cosmetics, in addition to the above cytotoxic agent.

[0021]

The phospholipid derivative represented by the above general formula [1] of the present invention has excellent biocompatibility as compared with conventional phospholipid derivatives and is easily taken up by a biomembrane, so that it can be transferred to tissues. It becomes an excellent cytotoxic agent.

[0022]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a novel and useful phospholipid derivative and a cytotoxic agent having a high cytotoxic effect can be obtained. And this cytotoxic agent exhibits an excellent effect in the prevention and treatment of diseases caused by cell damage caused by active oxygen.

[0023]

EXAMPLES Next, examples of the present invention will be described. Unless otherwise specified,% in each example is% by weight. Example 1 1,2-Dioleoyl-sn-glycero-3-phosphocholine (hereinafter abbreviated as DOPC, manufactured by NOF CORPORATION) 1.
574 g was dissolved in 200 ml of diethyl ether. To this solution, 70.46 g of ascorbic acid was dissolved in 200 ml of H ₂ O, an aqueous solution adjusted to pH 4.5 with NaOH particles was added, and phospholipase D (Toyo Brewing Co., Ltd.) was added.
(Manufactured by K.K.) 902U. After stirring at 37 ° C. for 6 hours, the organic layer was separated, and the aqueous layer was further extracted twice with 100 ml of diethyl ether. The organic layers were combined, washed twice with water and then dried over MgSO ₄ . After the solvent was distilled off under reduced pressure, 1.558 g of a lard crude product was obtained.

The crude product thus obtained was subjected to silica gel chromatography (developing solvent; chloroform: methanol: water =
The product was purified by 13: 7: 1) to obtain 327 mg of the desired product (viscous oily substance). The mass analysis, NMR spectrum and infrared absorption spectrum of the obtained target substance were measured. The results are as follows.

Mass Spectrometry Fab (Neg) Matrix: triethanolamine [M−H] ⁻ : 857 [M-ascorbic acid] ⁻ : 699

¹ H-NMR (270 MHz, TMS standard, CDCl ₃ : CD ₃ OD = 2:
1) δppm 0.89 (t, 6H, J = 6.6Hz), 1.30 (m, 40H), 1.62 (m, 4H),
2.03 (m, 8H), 2.30-2.37 (m, 4H), 4.01 (m, 5H), 4.16-
4.23 (m, 1H), 4.42 (m, 1H), 4.84 (s, 1H), 5.24 (m, 1
H), 5.35 (m, 4H)

¹³ C-NMR (67.8 MHz, CDCl ₃ : CD ₃ OD = 2: 1) δppm 174.44, 174.09, 172.43, 153.47, 130.35, 130.10, 11
9.17, 76.15, 70.92, 69.47, 66.13, 64.02, 63.39, 3
4.61-22.82, 14.23

IR (neat) cm ^-1 3300 (br, OH stretch), 2920 (CH stretch), 2850 (CH stretch), 1740
(C = O expansion / contraction), 1460 (CH ₃ , CH ₂ deflection), 1060 (PO expansion / contraction)

From these results, it can be seen that the viscous oil obtained in Example 1 above is a phospholipid derivative having a structure represented by the following formula.

[Chemical 3]

Example 2 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (hereinafter abbreviated as DLoPC, manufactured by NOF CORPORATION)
391 mg was dissolved in 50 ml of diethyl ether. To this solution, 17.61 g of ascorbic acid was added to 50 ml of H 2.
Phospholipase D (manufactured by Toyo Brewery Co., Ltd.) dissolved in ₂ O and added with an aqueous solution adjusted to pH 4.5 with NaOH particles
The reaction was carried out in the presence of 225 U. After stirring at 37 ° C for 6 hours, the organic layer was separated, and the aqueous layer was further diluted with diethyl ether 2
Extracted twice with 5 ml. Combine the organic layers, wash twice with water,
It was dried over MgSO ₄ . After the solvent was distilled off under reduced pressure, 256 mg of a lard-like crude product was obtained.

The crude product thus obtained was subjected to silica gel chromatography (developing solvent; chloroform: methanol: water =
The product was purified by 13: 7: 1) to obtain 94 mg of the desired product (viscous oily substance). The mass analysis, NMR spectrum and infrared absorption spectrum of the obtained target substance were measured. The results are as follows.

Mass Spectrometry Fab (Neg) Matrix: Triethanolamine [M−H] ⁻ : 853 [M-Ascorbic Acid] ⁻ : 695

¹ H-NMR (270 MHz, TMS standard, CDCl ₃ : CD ₃ OD = 2:
1) δppm 0.90 (t, 6H, J = 6.6Hz), 1.22-1.45 (m, 36H), 1.61 (m, 4
H), 2.02-2.07 (m, 8H), 2.30-2.38 (m, 4H), 2.78 (t, 4
H, J = 5.6Hz), 3.94-4.40 (m, 7H), 4.83 (s, 1H), 5.21-
5.45 (m, 4H)

¹³ C-NMR (67.8 MHz, CDCl ₃ : CD ₃ OD = 2: 1) δppm 174.40, 174.01, 172.32, 153.28, 130.49, 130.31, 12
8.41, 128.26, 119.15, 76.10, 70.77, 69.41, 66.18,
64.10, 62.91, 34.56-22.89, 14.20

IR (neat) cm ^-1 3450 (br, OH stretch), 2925 (CH stretch), 2855 (CH stretch), 1740
(C = O expansion / contraction), 1460 (CH ₃ , CH ₂ deflection), 1060 (PO expansion / contraction)

From these results, it can be seen that the viscous oil obtained in Example 2 above is a phospholipid derivative having a structure represented by the following formula.

[Chemical 4]

Reference Examples 1 and 2 The phospholipid derivative used in Comparative Examples was produced by the following method.
The DOPC of Example 1 was replaced with 1,2-dipalmitoyl-sn-
The procedure was carried out in the same manner as in Example 1 except that glycero-3-phosphocholine (Reference Example 1) or soybean lecithin (Reference Example 2) was used, and the target phospholipid derivative (hereinafter, DPPA-ASA, SOYPA-, respectively). Abbreviated as ASA). The mass spectrometry, NMR spectrum, and infrared absorption spectrum of the obtained DPPA-ASA are shown.

Mass Spectrometry Fab (Neg) Matrix: triethanolamine [MH] ^- : 805 [M-ascorbic acid] ^- : 647

¹ H-NMR (270 MHz, TMS standard, CDCl ₃ : CD ₃ OD = 2:
1) δppm 0.89 (t, 6H, J = 6.8Hz), 1.27 (m, 48H), 1.62 (m, 4H),
2.30-2.37 (m, 4H), 4.03 (m, 5H), 4.15-4.22 (m, 1H),
4.42 (m, 1H), 4.81 (s, 1H), 5.24 (m, 1H)

¹³ C-NMR (67.8 MHz, CDCl ₃ : CD ₃ OD = 2: 1) δppm 174.37, 174.03, 172.50, 153.19, 118.96, 76.21, 70.
71, 69.14, 66.35, 64.11, 62.84, 34.54-22.95, 14.20

IR (neat) cm ^-1 3300 (br, OH stretch), 2920 (CH stretch), 2850 (CH stretch), 1740
(C = O expansion / contraction), 1465 (CH ₃ , CH ₂ bending), 1060 (PO expansion / contraction)

Example 3 Pharmacological tests of the phospholipid derivatives obtained in Examples 1 and 2 were conducted as follows. 1) Culture of bovine vascular endothelial cells After removing 5 to 10 cm of bovine carotid artery blood vessels, P added with antibiotics (penicillin, streptomycin, etc.)
Lightly wash with BS (phosphate buffered saline) and use the same antibiotic-containing MEM (Eagle medium, minimum essen)
Tial medium), cooled with ice and brought back to the culture room.

The blood vessel was further washed several times with MEM medium containing antibiotics. Then, the fat adhering to the blood vessel was removed cleanly, the bifurcation was cut with scissors, and the blood vessel was longitudinally cut so as to pass through the bifurcation. The blood vessel was pinned in tension on a flat fixation surface with the intimal side facing up. Using a # 11 scalpel, the endothelial cells were peeled off by lightly touching the intimal surface. At that time, the female was preliminarily treated with 20% FBS (fetal bovine serum).
The MEM medium (containing antibiotics) was moistened to smoothen the movement of the female and prevent smooth muscle cell contamination.

Endothelial cells attached to the scalpel were dispersed in 10 ml of the above MEM medium and centrifuged at 800 rpm for 5 minutes. After that, the above MEM was added to the precipitate, and the mixture was dispersed with a pipette until a dozen of endothelial cells gathered to form a rice ear shape, which was then seeded and cultured in a plastic petri dish.

2) Reactive oxygen protection test using vascular endothelial cells Bovine carotid artery endothelial cells isolated and cultured by the method of 1) above were sown on a 96-well microplate at 2 × 10 ⁴ cells per well and confluent. I chose The test drug obtained in Examples 1 and 2 was added thereto to each concentration to give 24
The cells were cultured for a period of time and incorporated into endothelial cells. Thereafter, ⁵¹ Cr-sodium chromate was added to each well in an amount of 0.5 μCi, and the mixture was further cultured for 18 hours to incorporate ⁵¹ Cr-sodium chromate into the cells.

After that, it was washed 3 times with Hanks' solution and 4 × 1.
0 ⁵ cell / well in white blood cells (human peripheral blood from Ficoll (trade name, separated neutrophils Pharmacia)) was added, 12-O-tetradecanoyl - stimulated phorbol-13-acetate at 10 ng / ml did. This substance acts on the leukocyte membrane, stimulates the NADPH-dependent pentasaccharide phosphate cycle, promotes the production of active oxygen, and damages endothelial cells. At this time, radioactivity is released from cells damaged by active oxygen.

The radioactivity released into the culture medium after 5 hours was measured with a γ-scintillation counter and used as the amount released in the state in which the test drug was taken up. The total amount of ⁵¹ Cr taken up in the endothelial cells was 0.1% Triton X-100, and the cell membrane was dissolved to measure the radioactivity released in the culture solution. It was defined as the amount released. In addition, white blood cells and 12-O-tetradecanoyl-
The amount of radioactivity without addition of phorbol-13-acetate was defined as the amount released without stimulation.

The injury rate of endothelial cells is calculated by the following calculation formula [1], the release rate of ⁵¹ Cr (specific rele).
as of ⁵¹ Cr: SL).

[Equation 1]

Next, the inhibition rate of endothelial cell damage was calculated by the following calculation formula [2]. The results are shown in Table 1.

[Equation 2]

Comparative Examples 1 to 5 The inhibitory rate of endothelial cell damage was determined in the same manner as in Example 3 except that the phospholipid derivative obtained in Reference Examples 1 or 2 or the compound shown in Table 1 was used as the test drug. The results are shown in Table 1.

[0051]

[Table 1]

As is clear from the above test results, the compounds obtained in Examples 1 and 2 had a statistically significant inhibitory effect on endothelial cell damage due to active oxygen in a concentration-dependent manner. The same effect was not observed in SOYPA-ASA, other phospholipids, and ascorbic acid.

Example 4 A tablet was produced by a conventional method using the following ingredients.
The composition per tablet is as follows. Adults are given 2 to 6 tablets per adult after each meal.

Claims (2)

[Claims] 1. A phospholipid derivative represented by the following general formula [1]. [Chemical 1] (In the formula, R ¹ and R ² are the same or different from each other and have an unsaturated fatty acid acyl residue having 16 to 22 carbon atoms, and X represents a hydrogen atom or a salt-forming metal.) 2. A cytotoxic agent comprising the phospholipid derivative according to claim 1 as an active ingredient.