JP2543627B2 - Active vitamin D-containing therapeutic agent - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to relates to a novel preventive and therapeutic agent for diseases caused by preparative Ronbokisan A ₂ production.

[0002]

2. Description of the Related Art In recent years,
It has become clear that various free radicals including oxygen radicals are one of the factors that induce various diseases, and the increase in lipid peroxides associated therewith is closely related to the pathogenesis of various organs. Is known to exist. That is, autoimmune disease, emphysema, adult respiratory distress syndrome, shock, disseminated intravascular coagulation syndrome, gastric mucosal disorder, acute pancreatitis, Crohn's disease, ulcerative colitis, ischemic enteritis, ischemic heart disease, cerebrovascular disorder, It is known that free radicals are involved in the etiology of Parkinson's disease, and substances that suppress the production of lipid peroxides are useful for their prevention and treatment.

On the other hand, thromboxane A ₂ is a substance in which arachidonic acid released from a biological membrane by phospholipase A ₂ is converted by cyclooxygenase and thromboxane A ₂ synthase, and has a strong platelet aggregation action and vasoconstriction action. It is known that it has physiological effects such as, and is closely related to various pathological conditions. That is, a substance that suppresses the production of thromboxane A ₂ is useful for the prevention and treatment of ischemic heart disease, cerebrovascular disorder, bronchial asthma, adult respiratory distress syndrome, shock, disseminated intravascular coagulation syndrome, renal disease and the like. is there.

On the other hand, 1α-hydroxyvitamin D, 1
α, 24 (R) -dihydroxyvitamin D, 1α, 25
-Active vitamin Ds such as dihydroxyvitamin D are
It has a calcium absorption promoting action in the small intestine and a bone resorption and bone formation regulating action in bone, and is well known as a therapeutic drug for various diseases caused by abnormal calcium metabolism. In addition, recently, the aforementioned calcium metabolism regulating action,
It has been shown that active vitamin D has an immunoregulatory action in addition to the bone metabolism regulating action. However, active vitamin D produces lipid peroxide and thromboxane A ₂
It is not known what effect it will have on production.

Therefore, the present inventors have found that this active vitamin D produces lipid peroxide and thromboxane A _{2 in} a
As a result of diligent studies on what kind of effect it has, the inventors have found that active vitamin D has an action of suppressing lipid peroxide production and thromboxane A ₂ production, and arrived at the present invention.

[0006]

Means for Solving the Problems That is, the present invention is a preventive or therapeutic agent for diseases caused by the activated vitamin D to the active ingredient and to belt Ronbokisan A ₂ production.

The active vitamin D in the present invention includes active vitamin D ₂ , active vitamin D ₃ and derivatives thereof, and specific examples thereof include 1
α-hydroxyvitamin D, 1α, 24-dihydroxyvitamin D, 1α, 25-dihydroxyvitamin D, 1
α, 24,25-trihydroxyvitamin D, 24,2
4-difluoro-1α, 25-dihydroxyvitamin D, 26,26,26,27,27,27-hexafluoro-1α, 25-dihydroxyvitamin D, 25-hydroxyvitamin D ₃ -26,23-lactone, 1α,
Examples include 25-dihydroxyvitamin D ₃ -26,23-lactone. Among them, 1α-hydroxyvitamin D ₃ , 1α, 24 (R) -dihydroxyvitamin D ₃ ,
1α, 25-dihydroxyvitamin D ₃ is preferred.

[0008] These active ingredients are prepared by a known method using suitable excipients, etc. to prepare soft capsules, hard capsules, tablets,
It can be used as an oral preparation such as a syrup, an injection, or an external preparation.

Examples of such excipients are vegetable oils (eg corn oil, cottonseed oil, coconut oil, almond oil, peanut oil, etc.), oily esters of medium-chain fatty acid glycerides, mineral oils, petrolatum, animal oils and fats, cellulose derivatives (crystalline cellulose). , Hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), polyvinylpyrrolidone, dextrin, lactose, mannitol, sorbitol, starch and the like.

The dose of the active ingredient is usually 0.01 to 10
μg / day / person, preferably 0.25 to 4.0 μg
/ Day / person, the number of administrations is usually 1 to 3 times / day,
It is preferable to prepare the preparation so as to satisfy such conditions.

The preventive / therapeutic agent of the present invention can be used in combination with an existing drug therapy / therapeutic agent.

Next, the present invention will be described in detail with reference to examples.

[0013]

Example 1 Male ICR mice (7 to 8 weeks old) were fed with E.
coli derived lipopolysaccharide (LPS) 20 mg /
kg was intraperitoneally administered to induce endotoxin shock.

The amount of lipid peroxide is determined by the method of Ohkawa et al. (Anal. Biochem. 95, 351-358 (197).
According to 9)), the amount of hepatic malondialdehyde (MDA) was measured. That is, 8 hours after LPS administration, the liver was removed, perfused through the portal vein with ice-cold physiological saline and washed, then 10% homogenate was prepared with physiological saline, and the thiobarbituric acid (TBA) method was used. The amount of liver MDA was measured. The amount of protein was measured by the Biuret method.

As the amount of thromboxane A _{2, the} amount of stable metabolite thromboxane B ₂ (TXB ₂ ) was measured. That is, 3.8% EDTA-N 8 hours after LPS administration
0.8 ml of blood was collected from the right ventricle with a syringe filled with 0.1 ml of a ₂ , 10 ^-4 M indomethacin, and plasma was separated.
The immunoreactive TXB ₂ (iTXB ₂ ) value in this plasma was measured by radioimmunoassay. In addition, the survival rate after 24 or 48 hours was observed. The results are summarized in Tables 1 to 4 below.

Incidentally, 1α-hydroxyvitamin D ₃ , 1
α, 25-dihydroxyvitamin D ₃ , 1α, 24
(R) -Dihydroxyvitamin D ₃ (0.02 μg / k
g) was dissolved in 1% ethanol-0.5% Triton X physiological saline and orally administered simultaneously with LPS administration.

Table 1 shows the measurement results of liver MDA.

[0018]

[Table 1] Mice receiving LPS had an average MDA value of 4.59 n
mol / mg protein and normal mice (average 1.57 nmol
/ Mg protein) was clearly increased. On the other hand, 1α-hydroxyvitamin D ₃ , 1α, 25-dihydroxyvitamin D ₃ and 1α, 24 (R) -dihydroxyvitamin D ₃ 0.02 μg / kg increased the liver MDA value by 34.0, respectively. Suppressed by 24.8 and 35.3%.

Table 2 shows the measurement results of plasma iTXB ₂ .

[0020]

[Table 2] Plasma iTXB ₂ of LPS-administered mice was clearly higher than that of normal mice. On the other hand, 1α-
Hydroxyvitamin D ₃ , 1α, 25-dihydroxyvitamin D ₃ and 1α, 24 (R) -dihydroxyvitamin D ₃ 0.02 μg / kg increased plasma iTXB ₂ by 14.0, 24.5 and 28. respectively. Suppressed by 7%.

Tables 3 and 4 show the results of survival rate.

[0022]

[Table 3]

[0023]

[Table 4] As is clear from Table 3, LPS administration reduced the survival rate, and the survival rate at 24 hours after administration was 40.0, 30.0%, but 1α-hydroxyvitamin D ₃ 0.02 μg
/ Kg administration improved the survival rate to 70.0, 90.0% (Experiments 1 and 2).

As is clear from Table 4, the survival rate was similarly improved by administration of 1α, 25-dihydroxyvitamin D ₃ and 1α, 24 (R) -dihydroxyvitamin D ₃ 0.02 μg / kg.

[0025]

Example 2 Male BALB / c mice (6 weeks old) were injected with 0.3 mg of Propionibacterium acnes heat killed from the tail vein, and after 7 days, the method of Munthe-Kaas et al. (J. Ex.
p. ac. 141, 1-10 (1975)).
nes-activated liver adherent cells) were collected.

That is, the liver was removed and 10 ml of Hank's solution (HBSS: pH 7.4) at 4 ° C. was perfused from the portal vein for 1 minute, followed by HBSS containing 0.1% collagenase and 0.1%.
HBSS containing% pronase E was perfused. After that, the liver was cut into a paste and washed twice with HBSS, and then the liver was digested by shaking with HBSS containing 0.05% collagenase and 0.1% pronase E at 37 ° C for 20 minutes. did. Undigested substances were removed by filtration with stainless steel gauze, and washed with HBSS four times to obtain liver non-parenchymal cells.

This was dispersed in a plastic dish at a density of 2 × 10 ⁶ / ml in RPMI 1640 medium containing 20% fetal calf serum (FCS), and cultured at 37 ° C. for 18 hours. After culturing, non-adherent cells were removed, and a calcium-free phosphate buffer (PBS) containing 10% FCS and 0.02% disodium ethylenediaminetetraacetate (PBS) was added at 1 ° C.
Liver adherent cells were obtained by incubating for 5 minutes. After washing the cells 3 times with HBSS, RPM containing 10% FCS
The density was adjusted to 1 × 10 ⁶ / ml with I 1640 medium.
E. 3 by adding 1 μg / ml of LPS derived from E. coli
ITXB _{2 in} the supernatant after culturing at 7 ° C. for 24 hours was measured by radioimmunoassay.

Note that 1α, 25-dihydroxyvitamin D ₃ (0.1 to 10 nM) was dissolved in ethanol and added simultaneously with LPS so that the final ethanol concentration would be 0.2%. The results are shown in Table 5.

[0029]

[Table 5] The average value of iTXB ₂ in the culture supernatant was ₂ by the addition of LPS.
It was 3.1 ng / ml, which was a clear increase compared to the non-stimulated (average 4.7 ng / ml). On the other hand, 1α, 25-dihydroxyvitamin D ₃ (0.1 to 10 nM) had iTXB ₂ values in the culture supernatant of 53.2 and 45.7, respectively.
And suppressed by 63.3%.

[0030]

[Example 3] 5 BALB / c male mice (7 weeks old)
% Oyster glycogen was administered intraperitoneally, and 4 days later, HB
The peritoneal cavity was washed with SS to obtain peritoneal macrophages (OG-activated peritoneal macrophages). This cell is HBSS
After washing twice at 10%, RPMI 164 containing 10% FCS
The mixture was dispersed in a 0 medium at a density of 1 × 10 ⁶ / ml in a plastic dish and cultured at 37 ° C. for 2 hours. After the culture, non-adherent cells were removed to obtain cells attached to the dish.

After washing this with HBSS twice, 10% F
The density was adjusted to 1 × 10 ⁶ / ml with RPMI 1640 medium containing CS. E. 1 μg of LPS derived from E. coli
/ T in the supernatant after 24 hours incubation at 37 ° C
XB ₂ was measured by radioimmunoassay.

It should be noted that 1α-hydroxyvitamin D ₃ , 1
α, 25-dihydroxyvitamin D ₃ and 1α, 24
(R) -Dihydroxyvitamin D ₃ (10 nM each)
Is dissolved in ethanol and the final ethanol concentration is 0.2%
Was added at the same time as LPS. The results are shown in Table 6.

[0033]

[Table 6] The average value of iTXB ₂ in the culture supernatant was 5 when LPS was added.
It was 2.0 ng / ml, which was a clear increase compared to the case without stimulation (mean 4.9 ng / ml). In contrast, 1α-hydroxyvitamin D ₃ , 1α, 25-dihydroxyvitamin D
₃ and 1α, 24 (R) -dihydroxyvitamin D ₃
(10 nM) suppressed the iTXB ₂ value in the culture supernatant by 11.9, 35.6 and 30.4%, respectively.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location A61K 31/59 ACJ A61K 31/59 ACJ ACV ACV

Claims (2)

(57) [Claims] 1. A preventive or therapeutic agent for diseases caused by the active vitamin D to the active ingredient and to belt B <br/> Nbokisan A ₂ production. 2. Active vitamin D is 1α-hydroxyvitamin D, 1α, 24-dihydroxyvitamin D, 1
α, 25-dihydroxyvitamin D, 1α, 24,25
-Trihydroxyvitamin D, 24,24-difluoro-1α, 25-dihydroxyvitamin D, 26,26,
26,27,27,27-hexafluoro-1α, 25
The preventive / therapeutic agent according to claim 1, which is selected from the group consisting of -dihydroxyvitamin D, 25-hydroxyvitamin D ₃ -26,23-lactone, 1α, 25-dihydroxyvitamin D ₃ -26,23-lactone. .