GB2140688A - Fatty acid containing compositions - Google Patents

Abstract

Compositions containing at least one fatty acid or salt thereof represented by the general formula: <IMAGE> wherein the carbon-carbon bond may be a single or a double bond (cis-form); m is zero or 6; and n is 7 or 8, and a carrier are useful for treating cerebral apoplexy and other symptoms and syndromes caused by hypertension.

Description

SPECIFICATION Pharmaceutical Composition for Reinforcing the Blood Vessels Field of the Invention The present invention relates to a pharmaceutical composition for reinforcing the blood vessels containing, as the active ingredient, at least one fatty acid or salt thereof represented by the general formula (1).

wherein the carbon-carbon bond represented by the formula

is a single bond or a double bond (cis-form); m is zero or 6; and n is 7 or 8, and pharmaceutically acceptable carriers.

The pharmaceutical composition of the present invention is useful as prophylactic and curative agent for treating hypertensive vascular injuries such as cerebral apoplexy, etc.

Description of the Prior Art Cerebral apoplexy is one of the serious hypertensive diseases and is one of the major death causes of Japanese people. Cerebral apoplexy, once it appears, gives thigh mortality rate and, even if it does not lead to be death, it is difficult to cure completely and often brings the patients to "a bedridden condition" needing the help of other persons. Thus, cerebral apoplexy is one of the incurable dieseases with which people are seized at the highest ratio. No sure method for curing this disease has yet been established other than taking an appropriate preventive treatment before its appearance.

Development of hypertension is caused by the interaction of genetic factors and environmental factors. Genetic factors connected with hypertension and cerebral apoplexy were investigated by the use of stroke-prone spontaneously hypertensive rats (hereinafter referred to as SHRSP) [100% of these rats spontaneously cause a serious hypertension and over 90% of them cause cerebral apoplexy and die: Japanese Circulation Journal, Vol. 41, pages 259-266, March (1977)]. As a result, it is believed that genetic factors connected with hypertension and those connected with cerebral apoplexy exist separately and these two kinds of genetic factors are closely related to each other. That is, when a person possesses both of these two genetic factors, the person is more liable to be seized with cerebral apoplexy.However, even if in the person the extent of contribution of genetic factors of hypertension is not so high (in other words, the person is not a serious hypertension patient), if the extent of contribution of genetic factors of cerebral apoplexy is high, the person is afflicted with cerebral apoplexy even if he or she is not severe hypertensive.

Drugs for treatment of hypertensive diseases currently in use, mainly are hypotensive drugs.

However, as can be inferred from the result of the above-mentioned experiments using SHRSP, in order to overcome hypertensive diseases, use of only hypotensive drugs is not sufficient and thus drugs with an entirely different action are further required. As to one of the latter drugs, there is considered a blood vessel reinforcing agent which is a drug for treatment of hypertensive diseases but reduces the incidence of brain vascular injuries.That is, in hypertensive state, a much higher load than in normal state is imposed on blood vessel system (particularly, an arterial system) and there appear functional dyscrasias of cell membranes such as hyperpermeability of the blood vessel wall cells, as a result, the blood vessel necrose resulting in breakage of blood vessels and bleeding (cerebral hemorrhage) and/or in formation of thrombi (cerebral infraction) at the necrosis sites of the blood vessels and other places.

In order to treat these symptoms and syndromes, use of a blood vessel reinforcing agent is considered.

As currently known blood vessel reinforcing agents, there are known compounds of vitamin P group such as rutin, hesperidin, eriocitrine and the like. Regrettably, because these blood vessel reinforcing agents are inferior in intestinal absorption and further, when administered by injection, are excereted quickly, their effect as the blood vessel reinforcing agent is regarded to be doubtful.

Moreover, these blood vessel reinforcing agents induce side-effects such -as anaphylaxis for blood vessel reinforcing agent, digestive disturbance and the like and thus are not satisfactory. Under such circumstances, the present inventors made extensive studies to develop a novel blood vessel reinfording agent and, as a result, have found out that compounds represented by the general formula (1) have a prophylactic activity for blood vessel injuries in hypertensive diseases and can be used as an effective blood vessel reinforcing agent. On the basis of the finding, the present invention has been completed.

Summary of the Invention An object of the present invention is to provide a pharmaceutical composition for reinforcing the blood vessels containing, as the active ingredient, at least one fatty acid or salt thereof represented by the general formula (1), and pharmaceutically acceptable carriers.

A further object of the present invention is to provide a pharmaceutical composition for treating hypertensive vascular injuries such as cerebral apoplexy.

These and other objects and features of the present invention will become more fully apparent from the following description and the accompanying drawings.

Brief Description of the Drawings In the drawings: Fig. 1 shows curves indicating the relationship between the blood pressure and the age (days) of SHRSP fed on Diet-l and Diet-ll respectively; Fig. 2 shows curves indicating the relationship between the blood pressure and the age (days) of SHRSP fed on Diet-l to which methionine was administered chronically; Fig. 3 shows curves indicating the relationship between the rate of incidence of cerebral apoplexy and survival period of SHRSP fed on Diet-l, Diet-ll and Diet-lll respectively; Fig. 4(a) shows curves indicating the relationship between the blood pressure and the age (days) of SHRSP fed on Diet-l and Diet-IV respectively; Fig. 4(b) shows graphs indicating the relationship between the rate of incidence of cerebral apoplexy of SHRSP and the type of diets (Diet-l and Diet-lV);; Fig. 5 shows curves indicating the survival rate of SHRSP fed on a diet containing palmitoleic acid; Fig. 6 shows curves indicating the survival rate of SHRSP fed of a diet containing undecylenic acid or palmitic acid.

Detailed Description of Preferred Embodiments The present invention relates to a pharmaceutical composition for reinforcing the blood vessels containing, as the active ingredients, at least one fatty acid or salt thereof represented by the general formula (1),

wherein the carbon-carbon bond represented by the formula

is a single bond or a double bond (cis-form); m is zero or 6; and n is 7 or 8, and pharmaceutically acceptable carriers.

The fatty acid or salt thereof represented by the general formula (1) includes: cis-9 Hexadecenoic acid (palmitoleic acid, wherein the carbon-carbon bond represented by the formula

is a double bond; m is 6; and n is 7), n-Hexadecanoic acid (palmitic acid, wherein the carbon-carbon bond represented by the formula

is a single bond; m is 6; and n is 7), and 1 O-Undecenoic acid (undecylenic acid, wherein the carbon-carbon bond represented by the formula

is a double bond; m is zero; and n is 8).

The pharmaceutical composition for reinforcing the blood vessels according to the present invention has effects for recovering fragility and abnormal permeability of the blood vessels, effects for preventing aging of the blood vessels and can be able to introduce excellent results in prophylaxis and therapeutical effects for various blood vessel injuries of human beings and animals such as bleeding caused by fragility of the blood vessels.

Furthermore, the pharmaceutical composition for reinforcing the blood vessels according to the present invention is particularly effective for brain vascular injuries and suitable for prophylaxis and therapy of cerebral apoplexy.

The above-mentioned active ingredient, i.e., fatty acid or salt represented by the general formula (1) is suprior in intestinal absorptivity and stable in the blood, and therefore are excellent in duration of pharmaceutical effects and low in toxicity and can be used in administration for a long period of time.

Some of the fatty acid or salts represented by the general formula (1) are contained in various vegetable and animal oils and fats, typically in milk fats, fish oils, whale oil, apple and the like, in a fairly high content, which can be isolated from these raw materials according to conventional methods such as, for example, molecular distillation, counter current distribution, chromatography and the like, and are commercially available as standard reagents. From veiwpoint of practical purposes, the fatty acids or salts thereof as used as the active ingredient need not be isolated or purified, and can be used as in the form of crude products containing slight amounts of other fatty acids, etc.

The fatty acid or salt thereof represented by the general formula (1) may also be synthetic organic products prepared from appropriate starting materials.

In the present invention, pharmaceutically acceptable salts of the fatty acid represented by the general formula (1) can also be used as the active ingredient in the pharmaceutical composition for reinforcing the blood vessel of the present invention. As to typical examples of these pharmaceuticaliy acceptable salts, there can be mentioned alkali metal salts such as sodium salts, and potassium salts; alkaline earth metal salts such as calcium salts; other metal salts such as aluminum salts; ammonium salts; amine salts such as morpholine salts, piperadine salts, trimethylamine salts, diethylamine salts; and the like.

The fatty acid or salt represented by the general formula (1) of the present invention can be administered alone without mixed with any carrier or diluent, further it can be administered in the form of a pharmaceutical composition which is a mixture of the fatty acid or salt thereof represented by the general formula (1) as the active ingredient and pharmaceutically acceptable carriers. As to examples of these pharmaceutically acceptable carriers, there can be mentioned diluents and excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, surfactants, lubricants and the like, all of which are ordinary used in preparations of drugs of desired application forms. The administration unit forms of the pharmaceutical compositions of the present invention can be varied and selected from so as to meet the application purposes.Typical examples of the administration unit forms are tablets, pills, powders, granules, capsules, suppositories and other preparations in the form of solid and/or semi-solid state; liquors, suspensions, emulsions, injections (solutions, suspensions) and the like. Particularly, administration in the form of solid is preferable.In shaping into the form of tablets, there can be used as carriers, for example, excipients such as lactose, refined sugar, sodium chloride, a glucose solution, urea, starch, calcium carbonate, kaolin, crystalline cellulose, sillic acid, etc.; binding agents such as water, ethanol, propanol, simple syrup, a glucose solution, glycol, glycelin, a starch solution, a gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, calcium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents such as starch, sodium alginate, agar powder, laminalia powder, sodium bicarbonate, calcium carbonate, a fatty acid ester of polyoxyethylene sorbitan, sodium laurylsulfate, monoglyceride of stearic acid, lactose, etc.; disintegration inhibitors such as refined sugar, stearin, cacao butter, hydrogenated oils, etc.; absorption accelerators such as a quaternary ammonium base, sodium laurylsulfonate, etc.; wetting agents such as glycerin, starch, etc.; adsorbing agents such as starch, lactose, kaolin, bantonite, colloidal silicic acid, etc.; and lubricants such as purified talc, a stearic acid salt, boric acid powder, macrogol, a solid polyethylene glycol, etc. If necessary, the tablets can further be coated with usual coating materials to make them into coated tablets, for example, sugar-coated tablets, gelatin film-coated tablets, enteric layer-coated tablets, film-coated tablets, double layer tablets, multiple layer tablets, etc.In shaping into the form of pills, there can be used as carriers, for example, excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oils, kaolin, talc, etc.; binders such as powdered gum arabi, powdered tragacanth gum, gelatin, ethanol, etc.; and disintegrating agents such as laminaria, agar, etc. In shaping into the form of suppositories, there can be used as carriers, for example, a polyethylene glycol, cacao butter, a higher alcohol, an ester of a higher alcohol, gelatin, a semi-synthesized glyceride, etc. In preparation of injections, solutions and suspensions prepared are sterilized and preferably are adjusted so as to be isotonic to blood.In preparation of injections in the form of solutions, emulsions and suspensions, there can be used as diluents, for example, water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, a polyethylene sorbitan fatty acid esters, etc. At this time, sodium chloride, glucose or glycerin may be added to the injection solutions in an amount sufficient to make the injection solutions isotonic to blood. In shaping into the form of pastes, creams or gels, there can be used as diluents, for example, white petrolatum, paraffin, glycerin, a cellulose derivative, a polyethylene glycol, a silicone, bentonite, etc.If necessary, the pharmaceutical composition for reinforcing the blood vessel according to the present invention can further contain antioxidants for example, butylated hydroxytoluene, propyl gallate, quinone, cr-tocopherol, etc.; usual dissolving agents; buffer solutions; analgesic agents; preservatives; coating agents; perfumes; seasoning agents, sweetening agents and other medicines for example a platelet aggregation inhibitory agent; etc.

The amount of the active ingredient compound represented by the general formula (1) to be contained in the pharmaceutical composition of the present invention is not particularly restricted and can be selected from a wide range. Generally, 0.01% by weight or more of the active ingredient compound in terms of free fatty acid may be used. In the case of tablets, each tablets contains approximately 0.01-1.0 g of the active ingredient compound in terms of free fatty acid.

Administration method of the pharmaceutical composition for reinforcing the blood vessels of the present invention are not particularly restricted and can be administered by a suitable method for the respective types of administration unit forms. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally; injections are administered intraveneously singly or as a mixture with usual injectable transfusions such as a glucose solution, an amino acids solution, etc., if necessary, the injections are administered singly intramuscularly, intracutaneously, subcutaneously or intraperitoneally; and the suppositories are administered into rectum, and in the case of women, into vagina.

The dosage of the pharmaceutical composition of the present invention can suitably be selected from a wide range depending upon administration methods, conditions of the patient, etc. In general, the active ingredient compound in terms of free fatty acid can be administered in about 1-200 mg per kg of body weight per day, preferably in about 4150 mg per kg of body weight per day. Generally, the above-mentioned daily amount of the active ingredient compound is administered dividedly in 34 times a day.

The fatty acid or salt thereof represented by the general formula (1), as the active ingredient, contained in the pharmaceutical composition for reinforcing the blood vessels according to the present invention can be evaluated by comparing the results obtained from various pharmacological tests conducted by using the SHRSP in connection with the blood pressure, index of cerebral apoplexy and other items related to the activities with respect to several test diets each of which contains a different composition of the fatty acids or salts thereof as the active ingrediert.

Furthermore, the blood vessel reinforcing effect of fatty acid or salt thereof represented by the general formula (1), as the active ingredient, contained in the pharmaceutical composition according to the present invention can also be evaluated by determining the physical and morphological properties, for example the tensile strength of a piece of sample of the aorta enucleated from the SHRSP fed on the respective test diets.

The present invention will be explained in more detail by way of Examples for showing evaluation tests of the fatty acids or salts thereof represented by the general formula (1), as the active ingredient, contained in the pharmaceutical composition for reinforcing the blood vessels. However, the present invention is not restricted only to those Examples.

Example of Preparation of Tablets-i Tables containing the following compositions were prepared.

Palmitoleic acid 140 mg Starch 31.4mug Lactose 125 mg Polyvinyl pyrrolidone 1.8 mg Magnesium stearate 1.8 mg Total 300 mg Example of Preparation of Tablets-2 Tables were prepared in the same manner as in the above-mentioned example, except that palmitoleic acid was replaced by palmitic acid or undecylenic acid, respectively.

Example of Preparation of Film-Coated Tablets-i Film-coated tablets containing the following compositions were prepared.

Palmitoleic acid 150 g Avicel 40 g (A trademark for microcrystalline cellulose manufactured by Asahi Chemical Industry Co., Ltd.) Corn starch 30 g Magnesium stearate 29 Hydroxypropylmethyl cellulose 10 9 Polyethylene glycol 6000 3 9 Castor oil 40 g Methanol 40g Palmitoleic acid, Avicel, corn starch and magnesium stearate were mixed together and ground.

Then, the mixture was shaped into tablets by using a pounder of 10 mm diameter. The tablets thus prepared were coated with a film coating agent consisting of hydroxylprppylmethyl cellulose, polyethylene glycol 6000, caster oil and methanol to obtain film-coated tablets.

Example of Preparation of Film-Coated Tablets-2 Film-coated tablets containing the following compositions were prepared.

Undecylenic acid 1 50 g Citric acid lOg Lactose 33.5 9 Calcium secondary phosphate 70.0 g Pluronic F-68 30.0 g Sodium laurylsulfate 15.0 g Polyvinyl pyrrolidone 15.0 g Polyethylene glycol (Carbowax 1500) 4.5 g Polyethylene glycol (Carbowax 6000) 45.0 g Corn starch 30.0 g Dry sodium laurylsulfate 3.0 g Dry magnesium stearate 3.0 g Ethanol q.s.

Undecylenic acid, citric acid, lactose, calcium secondary phosphate, Pluronic F-68 and sodium laurylsulfate were mixed together. The mixture was sieved through a screen (No. 60), then was granulated by using an ethanol solution containing polyvinyl pyrrolidone, Carbowax 1500 and Carbowax 6000. The admixed powder was made into pasty granuless by adding ethanol, if necessary.

Corn starch was added to the mixtuere and mixing operation was continued until homogeneous granules were formed. Then, the granules were sieved through a screen (No. 10), and were placed in a tray and dried for 12 to 14 hours in an oven at 100 C. The dried granules were sieved through a screen (No. 1 6) and then admixed with dry sodium laurylsulfate and dry magnesium stearate. The thus obtained mixture was pressed into a desired shape by using a tablet machine. The core portion of these tablets thus prepared were treated with a varnish and then talc was applied thereupon to prevent adsorption of moisture. The surface of thus treated core portions were covered with an undercoat layer.

For an oral administration use, the varnish coating was conducted in sufficient times. In order to make the surface of the tablets completely round and smooth, the under coating layer and the smooth coating layer were applied further. These coated tablets were colored until the desired color of the coated tablets was obtained. After drying, the coated tablets were polished to obtain homogeneous and glossy surface thereof.

EXAMPLE 1 Prophylactic activity for the blood vessel injuries in hypertensive diseases performed by palmitoleic acid, which is one of the fatty acids represented by the general formula (1), as the active ingredient, contained in the pharmaceutical composition according to the present invention can clearly be shown in the following test results.

All tests were conducted by using male SHRSP at the age of two months. In the first test, SHRSP were fed on two kinds of diets, i.e., Diet-l and Diet-ll, as shown in Table 1 , for about 10 months. Nine (9) SHRSP were used for Diet-l, and eight (8) SHRSP were used for Diet-Il. The blood pressures and incidences of cerebral apoplexy of these SHRSP were observed.

TABLE 1 Component Diet-l Diet-Il Water, % by weight 9.3 9.0 Proteins, % by weight 20.5 25.3 Oils and fats, % by weight 4.5 Fibers, % by weight 3.3 3.5 Ashes, % by weight 4.85 6.46 Phosphorus, % by weight 0.63 0.74 Soluble nitrogen-free substances, 59.8 49.6 % by weight Sodium chloride, % by weight 1.05 0.98 Thiamine, mug/100 g 1.4 1.5 Riboflavin, mug/100 g 1.0 0.83 Niacin, mg/1 00 g 11 11 Vitamin A, IU/100 g 770 1,200 Carotene, mcg/100 g 50 430 Calcium, % by weight 0.683 0.825 Cobalt, ppm 2.9 0.9 Copper, ppm 21.6 7.7 Iron, ppm 214 387 Magnesium, ppm 224 775 Manganese, ppm 1 ni 4 89.9 Potassium, % by weight 0.650 0.754 Selenium, ppm 0.53 0.33 Sodium, % by weight 0.284 0.261 TABLE 1 (contd.) Component Diet-l Diet-ll Zinc, ppm 52.2 57.5 Nitrate nitrogen (nitrate, 3 1 4 nitric acid), ppm Amino acids composition of proteins Alanine, % by weight 0.99 1.50 Valine, % by weight 1.03 1.27 Glycine, % by weight 0.95 1.33 Isoleucine, % by weight 0.88 1.06 Leucine, % by weight 1.63 2.22 Proline, % by weight 1.63 1.77 Threonine, % by weight 0.77 1.02 Serine, % by weight 1.01 1.26 Methionine, % by weight 0.38 0.51 Hydroxyproline, % by weight 0.06 0.17 Phenylalanine, % by weight 1.00 1.23 Aspartic acid, % by weight 1.65 2.29 Glutamic acid, % by weight 4.37 4.46 Tyrosine, % by weight 0.67 0.86 Lysine, % by weight 0.96 1.34 Histidine, % by weight 0.67 0.39 Arginine, % by weight 0.52 0.73 Cystine, % by weight 0.22 0.26 The accompanied Fig. 1 shows the effects of Diet-l and Diet-il on the blood pressure, in which the relationship between the age (days) and blood pressure of the SHRSP are given. As can be seen from Fig. 1 that the rise in blood pressure was slightly suppressed at the initial stage in the case of Diet-ll.

Further, the incidence of cerebral apoplexy up to 220 days after the birth was 0% in the case of Diet-ll as compared with 60% in the case of Diet-i. Also, in observation of natural death, the incidence of cerebral apoplexy was 94% in Diet-l, while 13% in Diet-ll, thus there was observed significant difference between the two diets in stroke incidence.

It was firstly presumed that the slight suppression effect of Diet-ll on the blood pressure was due to the difference in protein composition between Diet-l and Diet-ll. Accordingly, the following test was conducted, thus eight (8) SHRSP were fed on Diet-l containing 1.5% by weight of methionine, and the relationship between the age (days) and the blood pressure of these SHRSP being administered methionine chronically was investigated. The results are shown in Fig. 2. In this test, other group of ten (10) SHRSP were used as control, being fed on Diet-l without containing methionine. The results are also shown in Fig. 2. As can clearly be seen form Fig. 2, that the sulfur amino acid (e.g., methionine) as a protein-constituting amino acid suppresses the rise of the blood pressure significantly, and reduces the incidence of the cerebral apoplexy to about 1/3. It should be noted the fact that, the amount of methionine added to Diet-l was 1.5% by weight, and said amount was far larger than the difference of the methonine content between Diet-l and Diet-ll [0.51-0.38=0.13 (g per 100 g ofdiet)]. It become clear from this fact that, the cerebral apoplexy-reducing effect of Diet-ll is not owing to its protein composition only.

Then, a compositional analysis was conducted for fats in Diet-l and Diet-ll, and the results are shown in Table 2.

TABLE 2 Composition of fatty acids Content (% by weight) contained in fats in diets Diet-l Diet-ll Lauricacid 0.3 Myristic acid 0.4 0.2 Palmitic acid 13.7 14.5 Palmitoleic acid 0.3 3.3 Stearic acid 4.5 3.4 Oleic acid 20.7 22.0 Linoelicacid 52.1 41.5 Arachidic acid 0.1 0.2 Gadoleic acid 5.2 4.8 Behenic acid 0.3 0.4 Erucic acid 0.5 0.7 In the above Table 2, the content of palmitoleic acid (POA) differs largely between the two diets, thus Diet-ll (containing 6.2% by weight of fat) contains about 1 5 times higher quantity of POA as compared to Diet-l (containing 4.5% by weight of fat).

Next, in order to confirm whether palmitoleic acid has an effect for suppressing the incidence of cerebral apoplexy or not, Diet-lll having the composition similar to that of Diet-l was prepared. In Diet III, the amino acids content was adjusted to about the same level as In Diet-l, and the fat content was also adjusted to about the same level as in Diet-l. On the other hand, the content of palmitoleic acid was lowered to 0.7% by weight which is about 1/5 contents as compared with 3.3% by weight contained in Diet-Il. The result of compositional analysis of Diet-lll is shown in Table 3. Further, the results of compositional analysis of fatty acids in the fats contained in Diet-lll is shown in Table 4.

TABLE 3 Component Diet-lll Water, % by weight 9.6 Proteins, % by weight 19.0 Oils and fats, % by weight 4.1 Fibers, % by weight 3.3 Ashes, % by weight 6.30 Phosphorus, % by weight 0.68 Soluble nitrogen free substances, 57.7 % by weight Sodium chloride, % by weight 1.18 Thiamine, mug/100 g 2.0 Riboflavin, mug/100 g 1.2 Niacin, mug/100 g 13 TABLE 3 (contd.) Component Diet-lll Vitamin A, IU/100 g 1,000 Carotene, mcg/100 9 140 Calcium % by weight 0.869 Cobalt, ppm 2.7 Copper, ppm 10.9 Iron, ppm 350 Magnesium, ppm 0.111 Manganese, ppm 114 Potassium, % by weight 0.826 Selenium, ppm 0.35 Sodium, % by weight 0.403 Zinc, ppm 58.0 Nitrate nitrogen (nitrate, nitric 4 acid), ppm Amino acids composition of proteins Alanine, % by weight 1.02 Valise, % by weight 0.83 Glycine, % by weight 0.89 Isoleucine, % by weight 0.68 Leucine, % by weight 1.53 Proline, % by weight 1.38 Threonine, % by weight 0.76 Serine, % by weight 1.05 Methionine, % by weight 0.39 Hydroxyproline, % by weight 0.06 Phenylalanine, % by weight 0.90 Aspartic acid, % by weight 1.70 Glutamic acid, % by weight 3.68 Tyrosine, % by weight 0.65 Lysine, % by weight 0.88 Histidine, % by weight 0.47 Arginine, % by weight 0.62 Cystine, % by weight 0.30 TABLE 4 Composition of fatty acids Content (% by weight) contained in fats in diet Diet-lll Lauricacid 0.1 Myristic acid 0.9 Palmitic acid 13.7 Palmitoleic acid 0.7 Stearic acid 3.2 Oleic acid 23.7 Linoleic acid 48.6 Arachidic acid 0.2 Gadoleic acid 5.0 Behenic acid 0.4 Erucic acid 0.4 In this test, 1%sodium chloride aqueous solution was used as a drinking water for the purpose of shortening the test period. SHRSP (10 rats for each groups) were fed on the respective diets, i.e., Diet-l, Diet-ll and Diet-lll, with 1%sodium chloride aqueous solution as a drinking water, and observed that their blood pressures were increased sharply and the incidence of cerebral apoplexy was increased as shown in the following Table 5.

TABLE 5 Incidence of cerebral apoplexy Test period Diet-l Diet-Il Diet-Ill After 6 weeks 63% 0% 39% After 10 weeks 79% 61% 77% The incidence of cerebral apoplexy at 6 weeks after the administration of the diet was in the order of Diet-l (highest: 63%), Diet-lll (middle: 39%) and Diet-ll (lowest: 0%) respectively. After 10 weeks, the incidence of cerebral apoplexy increased to 79% in Diet-l, 77% in Diet-lll and 61% in Diet-Il.These results are shown in Fig. 3 as the relationships between the incidence of cerebral apoplexy and survival period (days). Thus, the peak of incidence of cerebral apoplexy appeared first in Diet-l, second in Diet-Ill and lastly in Diet-ll. This indicates that palmitoleic acid (POA) suppresses the brain vascular injuries without having an effect for reducing the blood pressure.

In order to confirm the above indication, Diet-lV containing 20% by weight of total fats, so as to contain about 44-fold higher quantity of POA relative to Diet-l was prepared by adding a milk fat which contains about 3% by weight of POA to Diet-l, and it was investigated whether or not Diet-IV is effective for preventing the brain vascular injuries. In conducting this test, usual tap water was used as a drinking water. One test group consisting of 14 SHRSP was fed on Diet-l, and the other test group consisting of 20 SHRSP was fed on Diet-lV. The results are shown in Fig. 4(a) and 4(b). There was no difference in rise of the blood pressure between Diet-l and Diet-lV. The incidence of cerebral apoplexy was about 83% in Diet-l in observation of the total natural death, while it was only about 5% in Diet-lV.

Further, survival period of SHRSP was apparently longerin Diet-lV with average for 325 days as compared with average 306 days in Diet-l.

From the above test results, it was confirmed that palmitoleic acid (POA) has prophylactic activity for hypertensive blood vessel injuries, said activity of POA has not been known before.

EXAMPLE 2 (1) Each of 3-month old SHRSP were divided into 5 groups as shown in Table 6. Each test groups was fed on diet FR-2 (manufactured by Funahashi Nojo K.K.) with 1%sodium chloride aqueous solution as a drinking water. The test animals were observed with elapse of time and their survival rate was determined. The all test groups showed no difference in dietary intake and drinking water intake.

TABLE 6 Amount of fatty Number (n) acid added in the Test group of SHRSP Fatty acid diet (% by weight) Experiment-1 Control n=1 1 Not added Test group A n=9 Palmitoleic 1 acid Experiment-2 Control n=10 Not added Test group B n=10 Undecylenic 1 acid Test group C n=10 Palmitic 1 acid Test results of Experiment-l and -2 are shown in Fig. 5 and Fig. 6 respectively. It was confirmed that palmitoleic acid, undecylenic acid and palmitic acid, all of which are the active ingredient compounds of the fatty acid represented by the general formula (1) of the present invention, exhibit very excellent prophylactic activities for cerebral apoplexy.

(2) In the above test (1), the changes of systolic blood pressures of the SHRSP used in both control group and test group A with time (days) were determined. The results are shown in Table 7 as follows.

TABLE 7 Test group 0 Days 10 Days 23 Days 38 Days Control 198.9+4.7 n=1 1 201.1+5.5 n=9 187.5+6.6 n=3 (244.0+36.4) n=3 Test group A 199.3+6.6 n=9 201.3+8.5 n=9 1 89.0+4.0 n=7 196.7+6.6 n=6 (Mean+S.E.) There was no difference in the blood pressure of the SHRSP used in both control group and test group A. Accordingly, it does not follow that palmitoleic acid reduces the blood pressure and, as its result, the cerebral apoplexy is suppressed.

(3) Spontaneously hypertensive rats (SHR) and Wister Kyoto rats (WKY) which are the maternal ancestor of the SHR were fed separately on a diet (MF: manufactured by Oriental Yeast Co., Ltd.) containing 1% by weight of palmitoleic acid for 4 weeks. Then, the contents of palmitoleic acid in the plasma and the thoracic aorta as well as platelet aggregation activity were measured.

Fatty Acid Analysis Each of the thoracic aorta was taken out from the SHR and WKY tested, and placed in a glassmade homogenizer together with 19-fold quantity of 1.15%-KCI aqueous solution. The thoracic aorta was homogenized to obtain a homogenate having the concentration of 5%.

0.1 Milliliter of plasma or the tissue homogenate was placed in a centrifugal tube witti a stopper, and 20 micrograms of pentadecanoic acid was added in the tube as the internal standard. Further, 3 ml of a chloroform-methanol mixture (2:1) was added, subsequently, deproteinization and extraction of the total lipids were conducted. After 10 minutes centrifugation at 3,000 r.p.m., the supernatant was transferred to a test tube with a screw cap. The solution was concentrated to dryness at 400C or lower temperature under a nitrogen gas stream. To the residue thus obtained was added 0.2-0.25 mi of 5%-HCl/methanol solution (manufactured by Tokyo Kasei Kogyo Co., Ltd.). After capped the test tube containing the mixture was subjected to transmethylation for 2 hours on an aluminum brock of 100 C.

Then after cooling the mixture to a room temperature, 1.0 ml of n-hexane was added; the mixture was thoroughly stirred to extract methyl esters of the fatty acids into the hexane layer; then 1 ml of water was added and stirred smoothly, and the mixture was subjected to centrifugal separation for 5 minutes at 3,000 r.p.m. The hexane layer of the upper portion was taken to another test tube and the layer was dried under a nitrogen gas stream.Prior to the analysis, the resulting residue was re-dissolved in 0.1 ml of hexane and a part of the resulting solution (1-3 microliters) was used as the sample for gas chromatography analysis with the following conditions: Gas Chromatography: Apparatus: Shimadsu GC-4M (FID) Column: 3%-Silar 1 OC on Chromosorb W-HP 80-100 mesh, 3 mmx2 mm glass column Temperature: Inlet and detector: 2800C Column: 1 60-2200C at 5 C/min.

Carrier gas: Nitrogen gas 50 ml/min Detection: FID Integrator: YHP 3390A The results are shown in Table 8 as follows.

TABLE 8

Plasma Aorta Item SHR WKY SHR WKY Control POA adm. Control POA adm. Control POA adm. Control POA adm.

n=5 n=4 n=5 n=4 n=5 n=4 n=5 n=4 POA concentration 1.70 5.73 2.03 4.90 2.08 4.68 2.00 3.83 (mg/dl plasma) (0.22) (0.61) (0.27) (0.53) (0.30) (0.61) (0.60) (0.41) Total fatty acid 1.22 1.27 1.72 1.46 47.35 65.81 53.41 59.31 concentration (mg/ml) (0.07) (0.07) (0.08) (0.12) (5.13) (7.40) (8.31) (6.62) Composition of total fatty acids (%) Palmitic acid 22.59 22.15 21.37 20.26 27.75 26.10 25.36 22.93 (0.45) (0.71) (0.33) (0.49) (0.34) (0.44) (0.63) (0.53) Palmitoleic acid 1.39 4.52 1.17 3.35 4.35 7.09 3.48 6.47 (0.13) (0.40) (0.12) (0.19) (0.37) (0.33) (0.52) (0.18) Stearic acid 7.31 6.68 7.36 7.97 6.99 6.69 8.02 7.39 (0.18) (0.19) (0.26) (0.42) (0.24) (0.19) (0.58) (0.23) Oleic acid 13.15 14.01 11.47 11.60 28.40 28.39 28.28 27.77 (0.49) (0.36) (0.47) (0.38) (0.84) (0.75) (1.21) (0.68) Linoleic acid 30.31 31.05 28.93 28.22 22.40 22.62 25.33 26.18 (0.74) (1.42) (0.56) (0.85) (0.89) (1.01) (1.17) (1.11) Arachldonic acid 14.56 12.70 18.52 19.34 2.69 2.04 2.37 2.20 (0.96) (0.21) (1.21) (1.03) (0.12) (0.19) (0.28) (0.14) Docosahexaenoic acid 4.59 4.32 4.30 3.95 1.25 1.12 1.06 0.98 (0.19) (0.28) (0.09) (0.51) (0.94) (0.12) (0.14) (0.16) The ratio of concentration of palmitoleic acid (POA) to concentration of total fatty acid in plasma was significantly higher in the POA-administered groups than that in the control groups. This indicates that POA was absorbed well.The ratio of concentration of POA to concentration of total fatty acids in thoracic aorta was also significantly higher in the POA-administered groups than that in the control groups, and the POA concentration in thoracic aorta was higher than that in plasma. This indicates that POA has a higher affinity with aorta and its prophylactic effect for cerebral apoplexy is based on the reinforcement activity of the blood vessels by POA.

Platelet Aggregation Activity Platelet aggregation activity was measured in accordance with Born's turbidity test method [Nature: Vol. 1 94, 927, (1 962)] which is widely used for evaluation of platelet aggregation inhibitors.

Thus, the blood sample was drawn from a vein by mixing at a proportion of blood (9) to 3.8%trisodium citrate solution (1). The mixture of blood sample was centrifuged at 200 g for 1 5 minutes to obtain the supernatant liquid fraction as a platelet rich plasma (PRP). The remaining sediment fraction was further centrifuged at 800 g for 10 minutes to obtain the supernatant liquid as a platelet poor plasma (PPP). The number of platelet contained in the PRP was adjusted by diluting it with PPP so as to prepare PRP-sample containing 400,000 platelets perl.

Platelet aggregation was measured with an aggregometer (manufactured by Nikoh Bio-Science K.K.). Thus, 200 ,ul of the PRP-sample was placed in a cuvette cell and kept for 1 minute at 370C in the aggregometer. Then, ADP, collagen or Ca-inophore A231 87 (manufactured by Sigma Co.), as a platelet aggregation inducer, was added to the PRP-sample respectively. The aggregation was measured as transmittance and the maximum aggregation rate (%) was calculated from the following formula: A,-A, Aggregation rate (%)= x 100 A2-A1 wherein A,: transmittance of PRP A2: transmittance of PPP A3: transmittance of PRP added with an aggregation inducer The results are shown in Table 9 as follows.

TABLE 9

Maximum aggregation rate (%) ADP Collagen A23187 1.25 M 2.5 M 5.0 M 2.5 g/ml 5.0 g/ml 2x10-5M Control 31.2#4.2 53.2#9.3 64.2#8.4 62.4#35.5 81.0#4.4 63.8#35.8 SHR POA administered 41.5#7.3 54.3#6.8 67.5#6.2 71.2#16.0 78.0#5.2 67.8#38.0 Control 45.8#3.1 65.6#1.9 72.6#6.1 80.0#0.0 81.4#3.1 80.0#1.6 WKY POA administered 50.3#3.9 63.6#7.2 74.4#7.8 73.5#5.1 76.8#4.7 80.5#5.3 As can be seen from the results shown in Table 9, there is no difference between POA administered test groups and control groups with respect to the platelet aggregation induced by ADP, collagen and Ca-ionophore A23 1 87. This indicates that the prophylactic effect of palmitoleic acid for cerebral apoplexy is not based on the suppression of platelet aggregation.

Acute Toxicity Each of fatty acids or salts thereof represented by the general formula (1) used as the active ingredient in pharmaceutical composition according to the present invention has low toxicity. Examples are shown as follows.

Undecylenic acid (in rats, p.o.): 2.5 g/kg Palmitic acid (in mice, i.v.): 57+3.4 mg/kg (4) The blood vessel reinforcing effects of fatty acid or salt thereof represented by the general formula (1) can also be evaluated from other pharmacological test results, for example by determining the change of physical and morphological properties of aorta. [Japanese Heart Journal, Vol. 1 6, pages 296-298 (1975)].

Thus, SHRSP of after weaning or three months old were fed on a diet added with 1% by weight of palmitoleic acid as the test group. While, the same age of SHRSP were fed on the same diet without added with palmitoleic acid as the control group.

SHRSP of both the test group and the control group were decapitated at the same time before SHRSP of the control group were died due to the cerebral apoplexy. Pieces of aorta were enucleated respectively from SHRSP of both the test group and the control group. Each pieces of aorta was kept in a cold physiological saline (at 40C) for 24 hours, then was trimmed into an aortic strip of 3x 10 mm in size having T-shaped ends for connecting to the chacks. After the thickness of each strip was measured by a micrometer, the load-elongation curve of the strip was measured by Instron (manufactured by Shimadzu Corp.).

The piece of aorta obtained from SHRSP of test group fed on a diet containing palmitoleic acid showed a higher tensile strength over that of shown by the piece of aorta obtained from SHRSP of the control group. This indicates that palmitoleic acid has reinforcing effects for the blood vessel.

Claims (9)

1. A pharmaceutical composition containing, as the active ingredient, at least one fatty acid or salt thereof represented by the general formula

wherein the carbon-carbon bond represented by the formula

is a single bond or a double bond (cis-form); m is zero or 6; and n is 7 or 8, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to Claim 1, wherein said composition is a solid form pharmaceutical composition.

3. The pharmaceutical composition according to Claim 1 or 2, wherein said fatty acid is cis-9hexadecenoic acid (palmitoleic acid).

4. The pharmaceutical corriposition according to Claim 1 or 2, wherein said fatty acid is 10undecenoic acid (undecylenic acid).

5. The pharmaceutical composition according to Claim 1 or 2, wherein said fatty acid is n-hexadecanoic acid (palmitic acid).

6. A fatty acid or salt as defined in Claim 1 for use as a prophylactic and/or curative agent for treating inter alia hypertensive vascular injuries.

7. A fatty acid or salt according to Claim 6, wherein said use is as a prophylactic and/or curative agent for treating cerebral apoplexy.

8. A fatty acid or salt according to Claim 6, wherein said fatty acid is palmitoleicacid.

9. A fatty acid or salt according to Claim 7, wherein said fatty acid is palmitoleic acid.