JPH1087495A - Active vitamin d-containing preventive or therapeutic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicinal agent containing a specific vitamin D(s) as active ingredient, and useful for preventing/treating diseases due to RANTES production such as acidocytosis, bronchial asthma and nasal allergy. SOLUTION: This medicinal agent contains, as active ingredient, activae vitamin D(s) such as 1α-hydroxyvitamin D, 1α,24-dihydroxyvitamin D, 24,24- difluoro-1α,25-dihydroxyvitamin D, 26,26,26,27,27,27-hexafluoro-1α,25- dihydroxyvitamin D. This medicinal agent is administered in the form of a medicinal composition comprising this agent and a pharmaceutically permissible vehicle (e.g. vegetable oil).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an active vitamin D
It relates to a contained prophylactic or therapeutic agent. More specifically, the present invention comprises an inhibitor of RANTES production containing active vitamin D as an active ingredient, and the inhibitor and a pharmacologically acceptable excipient, resulting from the production of RANTES, or The present invention relates to a pharmaceutical composition for preventing and / or treating a disease in which the production of RANTES leads to an exacerbation of the disease.

[0002]

2. Description of the Related Art In recent years,
It has been found that secretory polypeptides having cell migration activity, collectively referred to as chemokines, are factors that induce cell infiltration in various inflammatory diseases. That is, allergic airway inflammation (Lukacs-NW et al., J-Leukoc-Bi
ol. 1996 Jan; 59 (1): 13-7), bronchial asthma (Konno-S
Et al., Int-Arch-Allergy-Immunol. 1996 Jan; 109 (1): 7
3-8), hypereosinophilia, allergic rhinitis (Bachert-C
Et al., Laryngorhinootologie. 1993 Dec; 72 (12): 585-
9), atopic dermatitis (Ying-S et al., J-Exp-Med. 1995 J
un 1; 181 (6): 2153-9), airway epithelial disorder (Wang-JH et al.,
Am-J-Respir-Cell-Mol-Biol. 1996 Jan; 14 (1): 27-3
5), rheumatoid arthritis (Robinson-E et al., Clin-Exp-Immu
nol. 1995 Sep; 101 (3): 398-407), inflammatory bowel disease, encephalomyelitis (Godiska-R et al., J-Neuroimmunol. 1995 May; 58).
(2): 167-76), conjunctivitis (Chihara-J et al., Int-Arch-Aller)
gy-Immunol. 1995 Apr; 106 (4): 428) and keratitis (Su-Y
H et al., J-Virol. 1996 Feb; 70 (2): 1277-81) and other ocular diseases, kidney diseases (Wenzel-UO et al., Am-J-Kidney-Dis. 1995 D).
It is known that chemokines are involved in pathogenesis such as ec; 26 (6): 982-94), and substances that suppress the production of chemokines are useful for their prevention and treatment.

[0003] RANTES (Regulated on Activation, Norma
lT Expressed, and presumably Secreted) is a recently discovered chemokine that has chemotactic activity on eosinophils, basophils, memory T cells, and monocytes. This suggests that RANs are responsible for T cell accumulation and activation in allergic inflammation settings.
It seems to indicate that TES is closely related. For example, RANTES is expressed when an allergen is administered subcutaneously to an allergic patient (Ying-S et al., J-Exp-Med. 1995
Jun 1; 181 (6): 2153-9), recombinant human RANT on dog skin
Inflammation is induced by administration of ES (Meur
er-R et al., J-Exp-Med. 1993 Dec 1; 178 (6): 1913-2.
1), in tears from conjunctivitis patients (Chihara-J et al., Int-Arch-All
ergy-Immunol. 1995 Apr; 106 (4): 428) and lymph nodes of patients infected with virus (Tedla-N et al., Am-J-Pathol. 1996).
May; 148 (5): 1367-73),
Synovial fibroblasts of rheumatoid arthritis produce RANTES (Rathanaswami-P et al., J-Biol-Chem. 1993 Mar 15;
268 (8): 5834-9), R in bronchoalveolar lavage fluid of asthmatics
That ANTES is detected (Alam-R et al., Am-J-Respir-Crit-
Care-Med. 1996 Apr; 153 (4 Pt 1): 1398-404), etc.
There are reports on experimental systems in vivo and in vitro and on patient cases. In addition, inflammatory bowel disease, kidney disease, rejection during kidney transplantation (Nadeau-KC et al., Proc-Natl
-Acad-Sci-USA. 1995 Sep 12; 92 (19): 8729-33).

On the other hand, 1α-hydroxyvitamin D ₃ (1α
-OHD ₃ ), 1α, 24 (R) -dihydroxyvitamin D ₃ , 1α, 25-dihydroxyvitamin D _{3 and} other active forms of vitamin D have a calcium absorption promoting action in the small intestine,
Bone has effects such as regulating bone resorption and bone formation, and is well known as a therapeutic agent for various diseases caused by abnormal calcium metabolism. In addition, recently, in addition to the calcium metabolism regulating action and the bone metabolism regulating action described above, it has been shown that active vitamin D has an immunoregulatory action (for example, “Vitamin D—A New Flow”, Tatsuo Suda et al. Kodansha, 1984, 3rd edition, pp. 85-103). However, it was not clear how active vitamin D affects RANTES production.

[0005] The inventors of the present invention have conducted intensive studies on the effect of active vitamin D on RANTES production, and as a result, have found that active vitamin D has an inhibitory effect on RANTES production. It has been reached.

[0006]

That is, the present invention is an inhibitor of RANTES production containing active vitamin D as an active ingredient. Furthermore, the present invention comprises an inhibitor of RANTES production containing active vitamin D as an active ingredient and a pharmacologically acceptable excipient, resulting from the production of RANTES or the production of RANTES leading to an exacerbation of disease. It is a pharmaceutical composition for preventing and / or treating associated diseases.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Active vitamin D in the present invention
And refers to vitamin D which has a physiological action such as calcium bone metabolism regulating action to be distinguished from the absence of vitamin D of physiological effects itself, activated vitamin D _2, active vitamin D ₃ and their derivatives And specific examples thereof include, for example, 1α-hydroxyvitamin D, 1α, 24
-Dihydroxyvitamin D, 1α, 25-dihydroxyvitamin D, 1α, 24,25-trihydroxyvitamin D, 24,24 difluoro-1α, 25-dihydroxyvitamin D, 26,26,26,2
7,27,27-hexafluoro-1α, 25-dihydroxyvitamin D, 25-hydroxyvitamin D-26,23-lactone, 1α, 25
-Dihydroxyvitamin D-26,23-lactone. Among them, 1α-hydroxyvitamin D ₃ , 1α, 24
(R) -Dihydroxyvitamin D ₃ and 1α, 25-dihydroxyvitamin D ₃ are preferred, and especially 1α, 24 (R)-
Dihydroxyvitamin D ₃ is preferred.

[0008] These active ingredients are formulated alone or together with appropriate excipients as described below in a known manner to provide an inhibitor of RANTES production containing the active vitamin D of the present invention as an active ingredient. be able to. Specific examples of such preparations include oral preparations such as soft capsules, hard capsules, tablets and syrups, and injections of the above-mentioned active ingredient of the present invention by using a suitable excipient in a known manner. And external preparations.

Examples of such excipients include vegetable oils (eg, corn oil, cottonseed oil, coconut oil, almond oil, peanut oil, etc.), oily esters such as medium-chain fatty acid glycerides, mineral oils, petrolatum, animal oils and fats, and cellulose derivatives (crystalline cellulose). , Hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), polyvinylpyrrolidone, dextrin, lactose, mannitol, sorbitol, starch and the like.
If necessary, additives such as an antioxidant, a wetting agent, a viscosity stabilizer, and a coloring agent can be added. Specifically, oral preparations such as soft capsules, tablets and granules for dry syrup containing the active vitamin D of the present invention; external preparations such as ointments and creams are disclosed, for example, in JP-B-57-45415 and JP-B-63. No. 46728, JP-B-63-60007, and JP-A-61-87619.

Attributable to the production of the RANTES of the present invention, or
Prevention of diseases in which RANTES production leads to exacerbation of the disease and / or
Alternatively, a therapeutic pharmaceutical composition comprises the above-mentioned inhibitor of RANTES production containing the active vitamin D of the present invention as an active ingredient, and a pharmacologically acceptable excipient. Such diseases include hypereosinophilia, bronchial asthma, airway epithelial disorders, allergic airway inflammation, atopic dermatitis, allergic / nonallergic rhinitis, nasal allergy, rheumatoid arthritis, inflammatory bowel disease, brain Examples include eye diseases such as myelitis, conjunctivitis and keratitis, and renal diseases.

When the pharmaceutical composition of the present invention is used as a prophylactic or therapeutic agent, its active ingredient, active vitamin D
The dose of is a necessary amount effective for such prevention or treatment, and is generally decided according to the patient's age, body weight, type of combination therapy, frequency of treatment, type of administration of desired effect, etc. Not always, but when used as a therapeutic agent
00 μg to 0.01 μg / day, more preferably 20 μg to
0.1 μg / day, and the administration frequency is usually 1 to 3 times / day. In the case of an external preparation, more preferably 1 μg to 20 μg
g / day. Usually 5 μg when used as a prophylactic agent
-0.05 μg / day, more preferably 0.5 μg-0.1
μg / day, and the number of administrations is usually 1 to 3 times / day.
It is preferable to prepare the preparation so as to satisfy such conditions.

[0012]

Next, the present invention will be described in detail with reference to examples. In the following,% indicates% by weight.

Example 1 Human neonatal skin fibroblasts (cryopreserved product, product name: cryoNHDF-Neo, manufacturer: Cl)
Thawed after inoculation, subcultured three times, subcultured, and counted the number of cells using a hemocytometer.
Well culture plate (Falcon No.3078, area 0.8cm ² / hole)
At ⁴ × 10 ⁴ cells / cm ² and a liquid volume of 400 L. The next day, the medium was replaced with a new culture solution, and the cells were further cultured for one day. FB in culture
Using a mixed solution of S (fibroblast basal medium, Clonetics) and FGM-2 (additional factor set, Clonetics), the cells were cultured in an incubator at a temperature of 37 ° C and a gas phase of 5CO2. According to the composition of the experimental group, 0, 10 ^-11 , 10 ^-10 , 10 ^-9 , 1
Add 1/8 volume (50 L) of a culture solution containing 1α, 24 (R) -dihydroxyvitamin D and 1 ethanol at each concentration of 0 ^-8 , 10 ^-7 , and 10 ^-6 M, and gently pipette. Mixed.
Next, TNF-α (product name: recombinant Human TNF-
α, manufacturer: R & D, 100 ng / mL and IFN-γ (product name:
recombinant Human IFN-γ, manufacturer: R & D) 100ng / mL
50 L of a culture solution containing or not containing was added and mixed.

After 24 hours, the culture supernatant was collected and stored at −20 ° C. until it was used for measurement. Thaw it at room temperature and use Amersha
The RANTES concentration in the solution was quantified by an immobilized sandwich ELISA method (enzyme immunoassay) using a cytokine measurement kit No. RPN2147 from m. Absorbance measurement was performed using a plate reader (Molecular Devices, Vmax kinetic micropla
Using a te reader), control of the reader and acquisition and storage of data were performed with a Macintosh computer connected to the reader and software SOFTmax881 / Ver.2.2. Statistical significance was tested by Dunnett's two-sided multiple comparison test. The results are shown in Table 1.

[0015]

[Table 1]

From Table 1, it can be seen that human dermal fibroblasts are TNF-α + I
The production of RANTES by FN-γ stimulation, whereas the addition of 10 ^-7 M 1α, 24 (R) -dihydroxyvitamin D ₃ inhibits the production of RANTES by fibroblasts. You can see that it was done. On the other hand, 1α,
24 (R) -dihydroxyvitamin D ₃ alone 10 ^-10 to 1
0 ^-7 did not induce the production of RANTES in the range of M.

Example 2 After receiving keratinocytes (product name: NHEK (F), manufacturer: Cascade) derived from human newborn skin, the flask was allowed to stand at 37 ° C. for 24 hours while keeping the flask sealed. Subculture three times in subconfluents, count the number of cells using a hemocytometer, and use a 48-well culture plate (Falc
on No. 3078, area 0.8 cm ² / hole) at 1 × 10 ⁵ cells / cm ² and a liquid volume of 500 L. The next day, the culture was removed and replaced with 400 L of a new culture. A mixture of HuMedia-KG medium and a growth additive set (human EGF, insulin, bovine pituitary extract, antibiotics) (Kurabo Co., Ltd.) was used as the culture medium. The temperature was 37 ° C and the gas phase was 5CO2 in an incubator. Cultured under the conditions.
According to the composition of the experimental group, 0, 10 ^-11 , 10 ^-10 , 10
Add 1/8 volume (50 L) of a culture solution containing 1α, 24-dihydroxyvitamin D ₃ and 1 ethanol at each concentration of ^-9 , 10 ^-8 , 10 ^-7 , and 10 ^-6 M, and gently pipette. Was mixed.
Next, TNF-α (product name: recombinant Human TNF-
α, manufacturer: R & D, 50 ng / mL and IFN-γ (product name: r
(ecombinant Human IFN-γ, manufacturer: R & D) 50 L of a culture solution containing or not containing 10 ng / mL was added and mixed.

After 24 hours, the culture supernatant was collected and stored at −20 ° C. until it was used for measurement. Thaw it at room temperature and use Amersha
By immobilized sandwich ELISA (enzyme-linked immunosorbent assay) using m-company's cytokine measurement kit NO.RPN2147,
The RANTES concentration in the solution was quantified. The absorbance measurement was performed using a plate reader (Molecular Devices, Vmax kinetic microp
Using a late reader), control of the reader and acquisition and storage of data were performed with a Macintosh computer connected to the reader and software SOFTmax881 / Ver.2.2. Statistical significance was tested by Dunnett's two-sided multiple comparison test. The results are shown in Table 2.

[0019]

[Table 2]

As shown in Table 2, human skin keratinocytes produced RANTES upon stimulation of TNF-α + IFN-γ. On the other hand, when 10 ^-9 or 10 ^-8 M of 1α, 24 (R) -di-hydroxyvitamin D ₃ was added, the production of RANTES by keratinocytes was suppressed. On the other hand, in the unstimulated state, 1α, 24 (R) -di-hydroxyvitamin D ₃ alone had no effect on the production of RANTES in the range of 10 ⁻¹² to 10 ⁻⁷ M.

Example 3 Normal human bronchial epithelial cells (cryopreserved product, product name: cryoNHBE, manufacturer: Clonetics)
Thaw and inoculate a collagen-coated culture flask (Falcon
No. 40520, 75 cm ² in area), subculture twice, subculture and count the number of cells using a hemocytometer.
Collagen-coated culture plate with holes (Falcon No.4040
8, 2 cm ² / hole) at 3 × 10 ⁴ cells / cm ^{2 at} a volume of 1000 L. Use BEGM (basal medium, supplementary factor set, Clonetics) as the culture solution, and incubate at 37 ° C.
Cultivation was carried out under the conditions of ° C and 5CO2 in the gas phase. The culture medium was replaced with a new culture medium every other day and cultured until confluence was reached. When it becomes confluent, replace it with a new culture medium 930L,
Immediately after that, according to the composition of the experimental group, 0, 2 × 10 ^-10 , 2 ×
10 ^-9 , 2 × 10 ^-8 , 2 × 10 ^-7 , 2 × 10 ^-6 M (final concentration: 0, 10
^-11 , 10 ^-10 , 10 ^-9 , 10 ^-8 , 10 ^-7 M)
A culture solution containing (R) -dihydroxyvitamin D ₃ and 2 ethanol (final concentration: 0.1) was added to 1/20 volume (50 L) and mixed gently. 0, 2 × 10 ^-8 ,
2 × 10 ⁻⁷ , 2 × 10 ⁻⁶ M (final concentration: 0, 10 ⁻⁹ , 10 ⁻⁸ , 10
^-7 M) of each concentration of Dexamethasone. next
TNF-α (product name: Recombinant Human TNF-α, manufacturer: BECTON DICKINSON) 5 g / mL (final concentration: 50 ng / m)
L) and IFN-γ (product name: Recombinant Human IFN-
γ, manufacturer: BECTON DICKINSON) 5 g / mL (final concentration: 5)
(0 ng / mL) or 10 L of a culture solution each containing or not containing the mixture was gently mixed.

After 24 hours, the suspended components are centrifuged (10,000 rpm, 2
min., 4 ° C) The removed culture supernatant was collected and stored at -80 ° C until used for measurement. Thaw this at room temperature, and use Amersham
The RANTES concentration in the solution was quantified by a solid phase sandwich ELISA (enzyme-linked immunosorbent assay) using the cytokine measurement kit NO. Absorbance measurement was performed using a plate reader (Molecular Devices, THERMO max microplate r).
The reader control and data acquisition and storage were performed using a Macintosh computer connected to the reader and software SOFTmax881 / Ver.2.2. Statistical significance was tested by Dunnett's two-sided multiple comparison test. The results are shown in Table 3.

[0023]

[Table 3]

As shown in Table 3, normal human bronchial epithelial cells produced RANTES upon stimulation of TNF-α + IFN-γ. In contrast, 1α, 24 (R) in the range of 10 ^-11 to 10 ^-7 M
When -dihydroxyvitamin D ₃ was added, the production of RANTES by bronchial epithelial cells was suppressed. On the other hand, Dexame
thasone also suppressed RANTES production in the range of 10 ^-9 to 10 ^-7 M.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI A61K 31/59 ABY A61K 31/59 ABY ACD ACD ACJ ACJ ACV ACV ADA ADA // C07C 401/00 C07C 401/00 (72) Invention Person Hiroaki Mitsuhashi 4-3-2 Asahigaoka, Hino-shi, Tokyo Inside the Tokyo Research Center, Teijin Limited (72) Inventor Tomohiro Ota 4-2-2, Asahigaoka, Hino-shi, Tokyo Inside the Tokyo Research Center Teijin Limited

Claims (5)

[Claims] 1. RANT containing active vitamin D as an active ingredient
An inhibitor of ES production. 2. The active vitamin D is 1α-hydroxyvitamin D, 1α, 24-dihydroxyvitamin D, 1α, 25-dihydroxyvitamin D, 1α, 24,25-trihydroxyvitamin D, 24,24 difluoro-1α, 25-dihydroxyvitamin D, 26,26,26,27,27,27-hexafluoro-1α, 25-dihydroxyvitamin D, 25-hydroxyvitamin D-26,23-lactone, 1α, 25-dihydroxyvitamin D-26 The inhibitor according to claim 1, which is one or more active vitamin D selected from the group consisting of, 23-lactone. 3. The active vitamin D is 1α-hydroxyvitamin D ₃ , 1α, 24 (R) -dihydroxyvitamin D ₃ ,
Or 1α, 25-dihydroxyvitamin D _3.
The described inhibitors. 4. A method comprising the inhibitor according to any one of claims 1 to 3 and a pharmacologically acceptable excipient, caused by the production of RANTES, or the production of RANTES leads to an exacerbation of disease. Pharmaceutical composition for prevention and / or treatment of associated diseases. 5. The method according to claim 1, wherein the disease is hypereosinophilia, bronchial asthma,
Eye diseases such as respiratory epithelial disorder, allergic airway inflammation, atopic dermatitis, allergic / non-allergic rhinitis, nasal allergy, rheumatoid arthritis, inflammatory bowel disease, encephalomyelitis, conjunctivitis or keratitis, or kidney disease The pharmaceutical composition according to claim 4, which is