JPS60185715A - Bone formation accelerator - Google Patents

Abstract

PURPOSE:The titled accelerator that contains, as an active ingredient, 1alpha,25(R)- dihydroxyvitamin D326,23(S)-lactone, thus being used in treatment and prevention for osteoporosis, osteomalacia and so on, because of its powerful inhibition of boneresorption or reversely activation of osteoblasts. CONSTITUTION:The accelerator contains 1alpha,25(R)-dihydroxyvitamin D32,23(S)- lactone, as an active ingredient, which is represented by the formula and synthesized by, e.g., the hydroxylation of 25(R)-hydroxyvitamin D326,23(D)-lactone with 1alpha-hydroxylase, or the condensation of the A-ring skeleton with the C, D ring skeleton by the Wittlag reaction. The resulting drug is given orally or parenterally, such as intravenously or intraperitoneally. In case of oral administration, the preparation is made in a conventional manner by combining the ingredient with crystalline cellulose, polyvinyl pyrrollidone, lactose or starch.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an osteogenesis promoter. In more detail), the present invention relates to 1α, Z5 (R1-dihydroxyvitamin D, 26
, 23 (Relating to an osteogenesis promoter containing 81-lactone as an active ingredient.

Conventionally, as compounds useful for the treatment of osteoporosis, osteomalacia, etc., for example, 1α-hydroxycholecalciferol, 1
α,24-dihydroxycholecalciferol, 1α1
Active vitamin D such as 25-dihydroxycholecalciferol.

compounds are known.

On the other hand, recently, 1α-hydroxycholecalciferp
1α,25(R)-) A novel metabolite of human-xyconcalciferol
Dihydroxyvitamin D.

2s,2:t(81-lactone was isolated and identified (F
EBS LETTER8, 134, 207-211, 1
981; Arch, B% ochem, Biophys,
, 204, 3:19-391.

1980). ia, 2s(8)-dihydroxyvitamin D, 26, 23 (81-lactone's pharmacological actions include, for example, the action of lowering abnormally elevated calcium levels in serum (Japanese Patent Application Laid-open No. 5S-ITSS 16), tumor cell Growth inhibitory effect or cell killing effect (JP-A-58-21
0011) etc. have been reported.

The present inventors conducted a detailed study on the effect of 1α,25(8)-dihydroxyvitamin D, 26,2B (sl-lactone) on bone, and found that it has a strong bone resorption inhibitory effect or osteogenic cell activation. The present invention was achieved by discovering that such compounds are useful as bone formation promoters.

That is, the present invention is a bone formation promoting agent containing 1m, 25(2)-dihydroxyvitamin D, 26, 23(81-lactone) as an active ingredient.

The 1α,25(6)-dihydroxyvitamin D,26,2.3(Sl-lactone) used in the present invention is represented by the following. Method of causing 1α-hydroxylase to act on lactone (FEB8LETTER8, 13
4, 207-211.1981) or the corresponding A
A chemical synthesis method in which the i skeleton and the C, D ring skeleton are condensed by a WittLng reaction (J, Org, Chem, 48.
4433-4436.1983) etc.

This compound has the effect of suppressing the elution of calcium from bones (bone resorption), and the effect of activating bone-forming cells by increasing the alkaline phosphatase activity or fullergen synthesis ability of bone-forming cells. Therefore, the present compound is a useful compound as an osteogenesis promoter, and is effective in treating or preventing osteoporosis, osteomalacia, and the like.

1α,25(8)-dihydroxyvitamin D of the present invention,
26,25(S)-lactone is administered orally or parenterally, such as intravenously, intraperitoneally, intramuscularly, subcutaneously, rectally, or transdermally. Examples of dosage forms for oral administration include tablets, granules, powders, and capsules. The tablets are crystalline cellulose.

It is molded from polyvinylpyridone, lactose, starch, etc. using a conventional method. Granules and powders are also formed using the same base material by conventional methods. Capsules contain this compound in coconut oil.

It is formed by filling gelatin sotocapsules with a solution obtained by dissolving it in cottonseed oil, triglyceride ester of medium chain fatty acids, etc.

Examples of dosage forms for parenteral administration such as intravenous, intraperitoneal, intramuscular, and subcutaneous administration include injections obtained by dissolving the present compound in a solution such as alcohol and adding stabilizers, preservatives, and the like.

Dosage forms for transdermal administration include ointments, creams, etc., and dosage forms for intrarectal administration include soft capsules.

ta,zs(6)-dihydroxyvitamin D of the present invention,
The dosage of 2s, 2a(S)-lactone varies depending on the patient's age, sex, degree of disease, dosage form, etc., but is usually 0.01 to 100 μg per day for adults, preferably 0.1 to 30 μg. administered.

The present invention will be explained in more detail below with reference to Examples.

Example 1 Calcium elution test from bone in 1n vitro 1g, 25tBJ-dihydroxyvitamin D, 26°2
a Calcium leaching of (S)-lactone from bone (
The effect on bone resorption was investigated.

2-3 day old mice were given a
Cl, was administered intraperitoneally (o, s ~171 C1/hea
d) After 2-3 days, the parietal bone was removed. Approximately equal amounts of left and right parietal bones were removed, and one was used as a control group and the other was used as a treatment group. The first 24 hours were a pre-culture period, during which the cells were cultured in a modified BGJb medium containing 10 ml of fetal bovine serum. Thereafter, in the treatment group, 25(R)(OH)tDm was dissolved in modified BGJb culture medium containing 5 thioma serum.
For the control group, the same amount of ethanol was added and cultured for 96 hours. After completion of the culture, the parietal bones were washed with physiological saline and immersed in IN hydrochloric acid for about 24 hours to elute calcium, and the amount of radioactivity was measured using a liquid scintillation counter. A portion of the culture solution was also taken and the amount of radioactivity was measured in the same manner.

The elution rate of Ca was calculated using the following formula.

Table 1 shows the ratio of the Ca elution rate of the treated group to the control group in the parietal bones obtained from the same individual.

Table 1 1st trial 200 0.99±0.084 2000 0.9B±0.09 3 20000 0.87±0.06 6 2nd trial 200 0.96-1: 0.04 72000 0.
86±0.04 8 a) The treated group/control group shows the ratio of Ca elution rate, with a value of 1.00 or more indicating promotion of bone resorption, and a value of 1.00 or less indicating inhibition.

Example 2 La, 25-dihydroxyvitamin D, (1α.

1α, 25(6)-dihydroxyvitamin l), 26 for promotion of bone resorption by 25-(OH)*Da)
The effect of 2ρ-lactone was investigated.

The culture method was the same as that for the control group in the culture medium. The results are shown in Table 2.

Table 2 20 0 1.22±0.07 F3 20 20 1.16-1:0.06 820 200
1.02±0.07 720 2000 0.97±
0.09 8200 0 1.31=1=0.06 9
200 200 1.31f:0,07 11200
2000 1.71-1= 0.05 8200 0
1.47±0.12 8 200 2000 0.99±0.06 8 As is clear from Table 2, the promotion of bone resorption by 1α,25-(oH) was ) wDs 2
6 + 1α for osteogenic cells, 25 (published - (on) w
The effect of Ds-26s di-linked lactone was determined by alkaline phosphatase (ALP) activity and collagen synthesis ability (
The uptake of pH was measured by K.

+11 MC3T3-El cells obtained from the parietal bone of a newborn mouse using high ALP activity as an indicator were used. Cells are 5% Co
, α with 10% calf serum at 37°0 in 95% air
Cultured in MFM culture medium. Every 5th day, o, o o i
% pronase E, 0+02% EDTA, PBS (-) for 5 ubcultures.

Before experiments, 5 X 10' cells were incubated with 24 aMEM (10
% calf serum) and cultured in a 35 m plastic dish. After 5 days, the cells were transferred to aMEM (2% calf serum culture medium) supplemented with vitamin D and analogues, cultured, and ALP activity was measured 12 to 16 hours later.

ALP activity was measured using p-nitrophenyl phosphate as a substrate according to the method of Lowry et al. Add 10mM substrate, 0.7M2-7mi/-2-methyl-1-propatur, 1□MMgC1, and appropriate amount of sample to 0.1
M sodium carbonate buffer (pH 10) to a final volume of 11. This is 37°030 m 1ncu
-Bate the reaction with 2 ml of 0.1 N NaOH.
It was terminated by adding. The amount of p-nitrophenol produced was measured by absorbance at Δ10 nm. The results are shown in Table 3.

From the Curse Pack Table 3, 1α, 25 (loose-(OH)*Dszs, 2
It can be seen that a-lactone increases ALP activity.

(11) Collagen synthesis ability Cells were prepared using the same method as described in the ALP activity measurement section. Cells were transferred to LIE/ffMEM containing 50 μg of 7-scorbic acid, -17 minopyrropynitrile and 10 βCi of L-(3,4-”H':l proline) and incubated at 1 ncuba for 3 h.
te do it. After removing the culture medium, 0,2 MNaOH1
Add 50% TCAO02-containing 5-titanic acid to the homogenized culture solution and the white matter of the cells, respectively, and precipitate the mixture. 3 with 10% TCA containing 1qb tannic acid
Wash twice with ice-cold acetone. 0.05M NaOH
Add 1 ml and dissolve. Divide this into two, 100 m
0.01 containing Mcac4 and 2% MN z-thyl-maleimide.
2 Add MTris-MCI buffer (pH 7,5) and incubate for 37'03 hours in the presence of 25V collagenase.
Incubation. TCA was added to a final concentration of 10 cm to precipitate the false white matter, and the radioactivity of the supernatant was measured. This was defined as the amount of collagen solubilized with collagenase.

The results are shown in Table 4.

Table 4 1.25-(OH)tD.

0.5 (pgAt) 2. O1 2,0〃4.26 50, OII 3.88 'i''2% (Q)i), D, -26, m 720 (p
g/iAri 3.56 200 J/ 4.84 2000 /I 3.88 From Table 4, 1”+25CFt)-, (oi-t)t
D, 26, '23 (S) - It is found that lactone increases collagen synthesis ability.

Example 4 1α525 (pQ-dihydroxyvitamin Ds26,23 (Sl-lacto75) was added to fractionated coconut oil Ill.
Dissolve T, 9, and add 2 ” ” + ” 5 (
R)-dihydroxyvitamin D, 2 g, 2 a(
8) The following shell components were heated and dissolved to contain 0.5g of -lactone, and soft capsules were prepared at room temperature using a soft capsule making machine.

Pellet ingredient Ze La Chin 10 Heavy Group 2 Preservative o, 05s Titanium white o, 2 Water o, 2

Claims (1)

[Claims] 1α, 25 (■-dihydroxyvitamin D, 26°23
(An osteogenesis promoter containing Sl-lactone as an active ingredient.