JPH0782148A - Immunosuppressive agent - Google Patents

Abstract

PURPOSE:To obtain an immunosuppressive agent having excellent actions, comprising a specific 5-fluorouracil derivative as an active ingredient. CONSTITUTION:This immunosuppressive agent comprises a compound of the formula (R is 1-6C alkoxymethyl) such as 3-[3-(6-benzoyloxy-3-cyano-2- pyridyloxycarbonyl)benzoyl]-1-(ethoxymethyl)-5-fluorouracil as an active ingredient. The compound has excellent characteristics of (i) good adsorption, (ii) duration, (iii) good stability, (iv) extremely low toxicity of alimentary canal such as diarrhea, vomit or hemorrhage of alimentary canal and (v) big difference between a dose to exhibit immunosuppressive action of its own and a dose to cause toxicity, excellent therapeutic index and high safety.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunosuppressive agent containing a 5-fluorouracil derivative.

[0002]

BACKGROUND OF THE INVENTION The present inventors have conducted extensive research on compounds having various pharmacological actions, but
In the process, a specific 5-fluorouracil derivative having a carcinostatic action was found (JP-A-63-201127).

On the other hand, 5-fluorouracil (5-FU)
And 5'-deoxy-5-fluorouridine (5'-DF
It has already been reported that UR) has an immunosuppressive action (Ohta, Y., et al., Gann, 71 , 190-196, (1980)).
However, according to the report, the immunosuppressive action of 5'-DFUR is weaker than that of 5-FU, and no relationship can be found between these pharmacological actions.

However, as a result of further studies, the present inventors have found a new fact that the above-mentioned specific 5-fluorouracil derivative has a particularly excellent immunosuppressive action, and completed the present invention here. Came to do.

[0005]

That is, the present invention has the general formula

[0006]

[Chemical 2]

[In the formula, R represents a C ₁ -C ₆ alkoxymethyl group. ] The present invention relates to an immunosuppressive agent containing a 5-fluorouracil derivative as an active ingredient.

The 5-fluorouracil derivative of the above-mentioned general formula (1), which is an active ingredient in the present invention, has an extremely excellent immunosuppressive action and low toxicity, and is used as an active ingredient compound for human and animal immunosuppressive agents. Especially useful. In particular, the above compounds have (1) good absorbability, (2) good persistence, (3) good stability, (4) diarrhea, mango, and gastrointestinal toxicity such as gastrointestinal bleeding, etc. are extremely low, (5) It has excellent features such as a large difference between the dose that exerts its original immunosuppressive action and the dose that induces toxicity, an excellent therapeutic index, and high safety.

Accordingly, the present invention provides an immunosuppressive agent containing an effective amount of the 5-fluorouracil derivative of the general formula (1) together with a pharmacologically acceptable carrier.

In the present specification, the C ₁ -C ₆ alkoxymethyl group represented by the above general formula (1) includes methoxymethyl, ethoxymethyl, 1-propoxymethyl, isopropoxymethyl, 1-butoxymethyl, 2- Examples thereof include butoxymethyl, tert-butoxymethyl, 1-pentyloxymethyl and 1-hexyloxymethyl groups.

Among the compounds of the above general formula (1), the compound in which R is an ethoxymethyl group or a methoxymethyl group is preferable. In particular, 3- [3- (6-benzoyloxy-
3-Cyano-2-pyridyloxycarbonyl) benzoyl] -1- (ethoxymethyl) -5-fluorouracil is the most preferred compound.

The compound represented by the general formula (1) is
It is publicly known and can be produced by a publicly known method (see the above-mentioned JP-A-63-201127).

The above-mentioned compounds are usually administered to mammals including human in the form of common pharmaceutical preparations. The preparation is prepared by using a diluent or an excipient such as a filler, a filler, a binder, a disintegrant, a surface active agent and a lubricant which are usually used. As this pharmaceutical preparation, various forms can be selected according to the therapeutic purpose, and typical examples thereof include tablets, pills, powders, liquids,
Suspensions, emulsions, granules, capsules, suppositories, injections (solutions, suspensions, etc.), ointments and the like can be mentioned.

In the case of molding in the form of tablets, as a carrier, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid or other excipients, simple syrup, glucose solution, Binders such as starch solution, gelatin solution, carboxymethylcellulose, cerac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters , Disintegrants such as sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, etc., disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, etc., quaternary ammonium base, absorption promotion of sodium lauryl sulfate, etc. , Glycerin, humectants, such as starch, starch, lactose, kaolin, bentonite, adsorbent such as colloidal silicic acid, purified talc, stearates, boric acid powder, lubricant and the like such as polyethylene glycol can be exemplified. Further, the tablet may be a tablet coated with a usual coating, if necessary, such as a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet or a multi-layer tablet.

In the case of molding in the form of pills, as a carrier, for example, an excipient such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, etc., a binder such as gum arabic powder, tragacanth powder, gelatin, etc., Laminaran,
A disintegrating agent such as agar can be exemplified.

In the case of molding in the form of suppositories, polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like can be used as carriers.

Capsules are usually prepared by mixing the active ingredient compound with the various carriers exemplified above and filling hard gelatin capsules, soft capsules and the like.

When prepared as an injection, the solution, emulsion and suspension are preferably sterilized and isotonic with blood. When molding into these forms, a diluent such as water or ethyl alcohol is used. , Macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used. In this case, a sufficient amount of salt, glucose or glycerin for preparing an isotonic solution may be contained in the pharmaceutical preparation, and a usual solubilizing agent, buffer, soothing agent, etc. may be added. May be.

If necessary, coloring agents, preservatives, fragrances,
Flavors, sweeteners, and other pharmaceuticals may be included in the pharmaceutical preparation.

In forming the paste, cream and gel, white petrolatum, paraffin, glycerin, cellulose derivatives, polyethylene glycol, silicone, bentonite and the like can be used as a diluent.

The amount of the active ingredient compound represented by the general formula (1) to be contained in the immunosuppressive agent of the present invention is not particularly limited and may be appropriately selected within a wide range. It is preferably 70% by weight.

The administration method of the above-mentioned pharmaceutical preparation is not particularly limited, and it is determined according to various preparation forms, patient's age, sex and other conditions, patient's symptom level and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. The injections are administered intravenously alone or in admixture with usual replenishers such as glucose and amino acids, and if necessary, they are administered intramuscularly, intracutaneously, subcutaneously or intraperitoneally alone. Suppositories are administered rectally.

The dose of the above-mentioned pharmaceutical preparation is appropriately selected according to the usage, the age of the patient, the sex and other conditions, the degree of disease and the like, but the amount of the compound of the present invention which is an active ingredient is usually about 1 per 1 kg of body weight per day. The dose is preferably about 200 mg, and the preparation can be administered in 1 to 4 divided doses per day.

[0024]

[Examples] Examples of pharmacological tests conducted on the active ingredient compounds of the present invention will be given below, and then preparation examples of the immunosuppressive agents of the present invention will be given as formulation examples.

[0025]

[Pharmacological Test Example 1] As the active ingredient compound of the present invention, 3- [3] described in Example 1 in JP-A-63-201127 was used.
-(6-benzoyloxy-3-cyano-2-pyridyloxycarbonyl) benzoyl] -1- (ethoxymethyl) -5-fluorouracil was used. 1% of the compound
Put in an aqueous solution of hydroxypropylmethylcellulose (HPMC), stir for about 30 minutes, and then sonicate for about 10 minutes in ice water to obtain a uniform suspension form.
When diluted with an aqueous solution of C, the compound concentrations are 5, 10,
20 and 40 mg / 10 ml suspensions were prepared.

1. Effect test on humoral immunity Sheep-preserved blood (Cosmobio, SRBC) was washed with physiological saline, and 2.1 × 10 was used using an automatic blood cell counter (Sysmex CC-130 manufactured by Toa Medical Electronics Co., Ltd.). After adjusting the physiological saline to ⁹ cells / ml, 0.25 ml of the saline was administered through the tail vein of the mouse for sensitization. From the same day of sensitization (the drug administration on the sensitization day was 1 hour after SRBC sensitization), the test reagents were administered by oral gavage once a day for 4 days (experimental group). In addition, a control group was prepared without addition of the reagent (1% HPMC 10 ml / kg administration).

On the 7th day of drug administration, blood was collected from the posterior vena cava of each mouse under ether anesthesia, serum was collected, and the antibody titer (hemolysin titer) by the immunohemolytic reaction was determined by the method of Matsuhashi et al. Nao, Biochemistry Experimental Method 15, "Introduction to Immunological Experiments", pp. 111-115, Academic Publishing Center (198
3 years)).

That is, the hemolytic titer was measured by inactivating each mouse antiserum at 56 ° C. for 30 minutes and then preparing a double dilution series starting from a 10-fold dilution with gelatin-barbital buffer (GVB). 50 μl of the solution was placed in a 96-well microplate, 50 μl of complement serum (saturated by cold) from which the heterologous antibody obtained from guinea pig was removed as a complement was added and mixed to a concentration of 5.2 × 10 ⁸ cells / ml. 50 μl of the adjusted SRBC suspension was added, mixed well, and kept at 37 ° C. for 24 hours. GVB-complement-red blood cells and normal serum-complement-red blood cell combinations were made as controls.

The hemolytic titer was determined by preparing a reference hemolytic sample and comparing the size of the circle of red blood cells that sank on the bottom of the microplate with that of each sample. Hemolyzed sample is 5.2 x 10 ⁸ SRBC suspension 1m / ml
Centrifuge l, add 0.48 ml of distilled water to the precipitate, completely lyse the erythrocytes, and then add 0.5 ml of 1.7% saline to the SRBC suspension and GVB in the following proportions. Made by mixing. Place 150 μl of each mixed sample in a microplate and perform 2
It was kept warm for 4 hours and used as a criterion for judgment.

Mixed sample composition (μl): 100% hemolysis 75% hemolysis 50% hemolysis insoluble hemolysis hemolysis 100 75 50 0 SRBC suspension 0 25 50 100 GVB 200 200 200 200 200 In this experiment, hemolysis of 75% or more If it is recognized as positive, the highest dilution is the titer (hemolysin titer)
And In addition, in order to make the red blood cell circle formed on the bottom of the plate as clear as possible, the determination was carried out 24 hours after the completion of heat retention.

The results of the above tests are shown in Table 1. In addition, each result is represented by the average value ± standard deviation, the significant difference between the average values,
Nonparametric Dunet test
nnett's test), p <0.05 is marked with *, and p <0.01 is marked with **.

[0032]

[Table 1]

From Table 1 above, the SRBC of control mice
On the 7th day of sensitization, the average hemolysin titer was 686, whereas in the group administered with the active ingredient compound of the present invention (experimental group), it was 4
It is clear that continuous administration for a day causes a dose-dependent decrease.

2. Effect test on cell-mediated immunity The abdomen of each mouse that had been acclimated and raised for 1 week after arrival was shaved, and an intact mouse was selected the next day, and sensitized antigen (7% picryl chloride (p
icryl chloride, Tokyo Kasei reagent special grade) ethanol solution)
100 μl was applied to the abdomen. From the next day, the reagent to be used is 1. Sensitization (experimental group) was carried out by oral gavage once a day for 6 days at the same dose as in the effect test on humoral immunity. The same was applied to the control group.

On the 7th day of sensitization, the thickness of both ears of the mouse was measured using a dial thickness gauge (manufactured by Ozaki Seisakusho) and used as the previous value. Then 10 on each ear of the mouse
Each μl (5 μl each on the front and back of the ear) was applied with a provoking antigen (1% picryl chloride olive oil solution), and after 24 hours, the thickness of both ears was measured again and set as the post-value.

In the determination, the difference between the posterior value and the anterior value of the left and right ears was obtained, and the average thereof was taken as the increase amount of the ear thickness. In addition, the inhibition rate (%) was calculated from the obtained increase amount according to the following formula.

Inhibition rate (%) = (1−T / C) × 100 T = increase in ear thickness in the test substance administration group (experimental group) C = increase in ear thickness in the control group

The results of the above tests are shown in Table 2 as in Table 1.

[0039]

[Table 2]

From Table 2, the active ingredient compound of the present invention was tested for the delayed hypersensitivity reaction after 24 hours with picryl chloride.
Inhibition tendency at high dose (46 at 20 mg / kg)
%, 31% at 40 mg / kg administration).

3. Effect test on immune organs The test substance was added to the mouse as described above in 1.
And 2. In the same dose as in the above test, the mice were orally administered once daily for 7 days, and the general condition of the mice was observed every day (before and after administration of the test substance) during the administration period.

The body weights of the mice in each group were measured daily from the day of grouping to the day of autopsy (the day after the final administration) before the administration of the test substance on that day.

On the day of autopsy, blood was collected from the posterior vena cava under anesthesia with ether, and the number of peripheral leukocytes was determined using an automatic hemocytometer (Sysmex F-800, manufactured by Toa Medical Electronics Co., Ltd.).

Further, on the day of autopsy, blood was collected and exsanguinated to death, and then the weights of thymus and spleen as immune organs were measured.

As a result, the general condition of each group did not change during the administration period.

The results of peripheral leukocyte count, thymus weight and spleen weight are shown in Table 3 as in Table 1.

[0047]

[Table 3]

From Table 3, the active ingredient compound of the present invention was administered to the peripheral leukocyte count at 20.5 at a dose of 40 mg / kg.
%, And 20 mg /% of thymus weight
69.1% for both kg and 40 mg / kg
And 74.5%, and the spleen weight showed a decreasing tendency of 16.9% at 40 mg / kg administration.

Hereinafter, the immunosuppression of the present invention using 3- [3- (6-benzoyloxy-3-cyano-2-pyridyloxycarbonyl) benzoyl] -1- (ethoxymethyl) -5-fluorouracil as an active ingredient compound. Formulation examples of agents are given below.

Formulation Example 1 Tablets of the above composition were prepared in one tablet.

Formulation Example 2 Tablets of the above composition were prepared in one tablet.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Takeshi Imaoka 2-7-303 Misora-cho, Otsu-shi, Shiga (72) Inventor Akiaki Motoyama 8-20-402 Fujimidai, Otsu-shi, Shiga (72) Invention Utsunomiya Faithful 72, 2112 Mikami, Yasu-machi, Yasu-gun, Shiga Prefecture

Claims (1)

[Claims] 1. A general formula: [Wherein R represents a C ₁ -C ₆ alkoxymethyl group. ] The immunosuppressive agent which uses the 5-fluorouracil derivative represented by these as an active ingredient.