JPS6259092B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an antiplatelet agent containing 24,25-dihydroxycholecalciferol as an active ingredient. In recent years in Japan, factors such as the increase in the elderly population and the Westernization of dietary habits and social environments have become
There is a tendency for diseases associated with platelet hyperfunction to increase, and among antiplatelet therapies, antithrombotic therapy is being applied. These diseases include cerebrovascular disease (cerebral infarction, transient ischemic attack), ischemic heart disease (myocardial infarction), thrombosis associated with artificial materials (heart-lung machine, artificial kidney, A-V shunt, cardiac artificial valves, catheters,
Artificial blood vessels, etc.), small vessel disorders (thrombotic thrombocytopenic purpura, small angiopathic hemolytic anemia, etc.), thrombocytosis, and other diseases in which platelet hyperfunction has some involvement in these conditions include diabetes. ,
These include chronic nephritis, kidney transplantation, nephrotic syndrome, hypertension, multiple sclerosis, preeclampsia, cancer, encephalemia, paroxysmal nocturnal hemoglobinuria, Kawasaki disease, Raynaud's disease, and vibration-related disorders. Currently, drugs such as deipyridamole, trapidil, and ticlopidine are used as antiplatelet therapy for these diseases, but they have problems in terms of side effects. As a result of intensive research on substances that are endogenous to healthy humans and have proven safety, the present inventors discovered 24,25-dihydroxycholecalciferol (hereinafter referred to as this substance or 24,25-dihydroxycholecalciferol). 25−
(OH) ₂ -D ₃ ) has been found to have numerous physiologically active effects, and has already been shown to have anti-hypercalcemic and anti-hypercalcemic effects.
It has been found to have anti-ulcer effects, prevent immune function decline, regulate magnesium metabolism, anti-hyperphosphatemia, regulate blood sugar, and anti-tumor effects. Subsequently, as a result of repeated research, it was discovered that this substance has an antiplatelet effect with few side effects, and the present invention was achieved. All of these substances are known substances and have the following structures. For example, Anthony W.Norman, Vitamin
D; MOLECULAR BIOLOGY AND CLINICAL
NUTRITION, MARCEL DEKKER, INC., p.1
~92 (1980). The present inventors used animal platelets such as rats and human platelets to study the property of inhibiting platelet aggregation, and found that 24·25-(OH) ₂ -D ₃ has an inhibitory effect on platelet aggregation. I found out. This substance is 24R・25−(OH) ₂ −D ₃ , 24S・25−
Although it may be (OH) ₂ -D ₃ or a mixture thereof, 24R·25-(OH) ₂ -D ₃ is particularly preferred. The antiplatelet agent of the present invention contains the above-mentioned substance as an active ingredient, and can be used in various formulations as shown below. The antiplatelet agent of the present invention can be administered orally, parenterally or rectally, with oral administration being preferred. Furthermore, the antiplatelet agent of the present invention can also be used as an antithrombotic agent as a subconcept thereof. Preparations containing this substance as an active ingredient include tablets, powders,
Granules, suppositories, capsules, alcohol solutions,
It is used in dosage forms such as oily solutions and aqueous suspensions. As the oily solvent, triglyceride esters of intermediate fatty acids, corn oil, cottonseed oil, peanut oil, fish liver oil, oily esters, etc. are used.
Also preferred are cacao oil and glycerin. Other ingredients include lactose, starch, talc, magnesium stearate, sorbic acid, sorbic acid salts,
Sugar or its derivative alcohol, physiological saline, surfactants, antioxidants, or other pharmaceutical agents may be used in combination with this substance. The substance may be contained in a unit dosage form from 2x10 ^-5 to 4% by weight, preferably from 2x10 ^-4 to 1% by weight. In addition, this substance is 0.1 μg per day for adults.
1×10 ⁵ μg, preferably 0.5 to 1×10 ⁴ μg is administered. Next, we will describe the results of investigating the acute toxicity of this substance. Acute toxicity: This substance was dissolved in ethanol and triglyceride ester of intermediate fatty acids to give an ethanol concentration of 2%, and administered orally (po) to 10 male ICR mice (body weight 25±3 g). . The dose is 100mg/Kg. The animals were observed for symptoms of toxicity for two weeks after administration, but all 10 animals survived without any abnormalities. After slaughter, blood, biochemical tests, autopsy findings, and histopathological examinations were performed, but there was no difference in any way from the control group to which only triglyceride ester of intermediate fatty acid containing 2% ethanol was administered.
Therefore, the LD ₅₀ value for oral administration of this substance is 100mg/
1α-(OH)-D ₃ (orally administered), which is said to be an active vitamin D ₃ analogue.
(LD ₅₀ is less than 1 mg/Kg), this substance can be said to be extremely safe. The pharmacological effects of the present invention will be specifically explained below with reference to Examples. The substance used in the examples is 24R・25-(OH) ₂ -D ₃ , and the structure of the optical isomer at position 24 is confirmed in Tetrahedron Letters No.
26, p. 2203-2206, 1975. Example 1 Platelet rich plasma (hereinafter abbreviated as PRP) was obtained from a 20-week-old normal Wistar male rat. Using this, Adenosine in vitro.
The effect of 24R·25-(OH) ₂ -D ₃ on diphosphate (hereinafter abbreviated as ADP)-induced platelet aggregation was investigated. Chitrate blood was collected from 20-week-old normal Wistar male rats (titrate was 1/9 volume of whole blood). This was centrifuged at 1500 rpm for 6 minutes, and the supernatant was collected and used as PRP. For platelet aggregometry, Payton
Lumiaggregation Module Model 1000 was used. Take 1.5 μ of a 2 mg/ml ethanol solution of 24R・25−(OH) ₂ −D ₃ and add it to 250 ml of PRP for 2 minutes.
After incubation at 37°C, 30 μM of ADP was added and subjected to measurement. At this time, 24R.25-(OH) ₂ -D ₃ has a final concentration of 12 μg/ml (0.6% ethanol PRP). As a control, 1.5μ of ethanol was added. The results, expressed as maximum platelet aggregation rate, were
44.5%, and 37.0% when 24R·25-(OH) ₂ -D ₃ was added. This is compared to the control 24R・25−(OH) ₂ −D ₃
The platelet aggregation inhibition rate was approximately 17.0%. This platelet aggregation inhibition rate was determined by the following formula.

[Table] Control maximum platelet aggregation rate Example 2 Using PRP obtained from fresh venous blood of normal humans, the effect of 24R・25-(OH) ₂ -D ₃ on blood platelet aggregation was evaluated as in Example 1. The same consideration was given. The final concentration of 24R・25−(OH) ₂ −D ₃ is 1 μg/ml.
The flocculant ADP was 5 μM. As a result, 24R・25−(OH) ₂ −D ₃ relative to the control
The platelet aggregation inhibition rate was 37.6%. Example 3 Using PRP obtained from streptozotocin (hereinafter abbreviated as STZ)-induced diabetic rats and their control group, 24R and 25-
The effect of (OH) ₂ −D ₃ was investigated. STZ 65mg/ to 6 week old Wistar Kondo male rats
Kg was administered intraperitoneally after a 48-hour fast. One week after administration, blood was collected from the tail vein and the blood sugar level was 500-600.
mg/dl and whose urinary glucose excretion was +++++ (2%) using Hemacombistats (manufactured by Miles Sankyo Co., Ltd.) test strips were subjected to titrate blood collection. Normal Wistar Kondo male rats of the same age were used as controls. Preparation of PRP and measurement of platelet aggregation were performed in Example 1.
I did it in the same way. PRP is also PPP
(Platelet Poor Plasme; the remainder after removing PRP
Obtained by centrifugation at 3000 rpm for 10 minutes. ) and used with a platelet count of 300,000/ ^mm3 . flocculant
ADP was 30 μM. The results are shown in Table 1 below. In this example, 24R.25-(OH) ₂ -D ₃ was prepared as an ethanol solution with a final concentration as shown in the table below. The control was added with ethanol.

[Table] In addition, as above for normal rats, the platelet count was set to 30.
For PRP diluted to 10,000/ ^mm3 , ADP30μ
For M, the maximum platelet aggregation rate was 15.0 to 17.0% regardless of the concentration of 24R.25-(OH) ₂ -D ₃ . As shown in Table 1 above, the effect on ADP-induced platelet aggregation using PRP in STZ-induced diabetic rats.
The effect of 24R.25-(OH) ₂ -D ₃ was dependent on its concentration, and at a low concentration of 1×10 ⁻² ng/ml, it showed a remarkable platelet aggregation inhibiting effect of 25.1%. Example 4 The effect of 24R.25-(OH) ₂ -D ₃ on ADP-induced platelet aggregation was investigated using PRP in a hereditary hypertension model SHR (spontaneous hypertensive rats). Titrate blood was collected from the abdominal aorta of 7- to 8-week-old SHR rats, and PRP was prepared as in Example 1.
Platelet aggregation was measured. The final concentration of 24R・25−(OH) ₂ −D ₃ is 1 ng/ml.
and ethanol was used as a control. ADP is 30
It was set as μM. The results showed that the platelet aggregation inhibition rate was 18.3 compared to the control.
%. Example 5 Japanese white male rabbits were given a solid feed containing 1% cholesterol orally ad libitum, and after about 3 months, an increase in serum lipid components was confirmed. Using 24R・
The effect of 25-(OH) ₂ -D ₃ on ADP-induced platelet aggregation was investigated. Preparation of PRP and measurement of platelet aggregation were performed in Example 1.
I followed. The final concentration of 24R・25−(OH) ₂ −D ₃ is
The concentration was 1 ng/ml, and the ADP was 30 μM. Ethanol was used as a control. The result was a platelet aggregation inhibition rate of 15.8% compared to the control. Example 6 A 6-week-old male Wistar rat was subjected to bilateral nephrectomy, and a marked increase in serum BUN (serum urea nitrogen) was confirmed on the 3rd postoperative day. Prepare PRP and its ADP
The effect of 24R·25-(OH) ₂ -D ₃ on induced platelet aggregation was investigated. Preparation of PRP and measurement of platelet aggregation were performed according to Example 1. The final concentration of 24R·25-(OH) ₂ -D ₃ was 1 ng/ml, and ADP was 30 μM. Ethanol was used as a control. As a result, a platelet aggregation inhibition effect of 17.3% was obtained compared to the control. Example 7 10-week-old Wistar male rats were given 24R and 25-
(OH) ₂ −D ₃ at 100 μg/Kg and MCT (C ₈ ~ _{C 10}
triglyceride ester of carboxylic acid) was administered orally by gavage. ADP was injected intravenously 6 hours after administration. As a control, all rats in the MCT solvent-only administration group died, but the mortality rate in the substance administration group was 63%. That is, this result indicates the antithrombotic effect of this substance. Example 8 Formulation Example 5 mg of 24R・25-(OH) ₂ −D ₃ was added to 1 kg of MCT, which was irradiated with a 400 W high-pressure mercury lamp for 72 hours while bubbling argon to eliminate peroxide.
Dissolve 24R・25−(OH) ₂ −D ₃ in one capsule.
The following shell components were dissolved by heating to contain 0.5 μg of the following, and soft capsules were prepared by a conventional method using a soft capsule making machine. Shell formulation example Gelatin 10 parts by weight Glycerin 2 parts by weight Preservative (ethylparaben) 0.05 parts by weight Titanium white 0.2 parts by weight Water 0.2 parts by weight (parts by weight in final form) Similarly, 1 μg, 2 μg, 5
Products containing μg or 10 μg were prepared, respectively.

Claims (1)

[Claims] 1. An antiplatelet agent containing 24,25-dihydroxycholecalciferol as an active ingredient. 2. The antiplatelet agent according to claim 1, wherein the 24,25-dihydroxycholecalciferol is 24R,25-dihydroxycholecalciferol. 3. The antiplatelet agent according to claim 1 or 2, which is an antithrombotic disease agent.