JP2000247891A - New calcium antagonist - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a strong and safe calcium antagonist useful as a therapeutic medicine for ischemic heart diseases, e.g. cardiotonic agents or cardiac infarction by including an uridine alkylphosphate compound as an active ingredient. SOLUTION: This calcium antagonist comprises an uridine 5'-alkyl phosphate compound of the formula (R is an alkyl), e.g. uridine 5'-hexadecyl phosphate an active ingredient. The compound of the formula is obtained by dissolving, e.g. uridine 5'-monophosphate and an alkyl alcohol in tert-butyl alcohol and reacting uridine 5'-monophosphate in the presence of dicyclohexylcarbodiimide usually at 60-100 deg.C for 2-20 hr. The compound of the formula can be administered by oral, parenteral, transrectal or percutaneous administration and the compound is preferably administered to each of adult patients at a daily total dose of 1-2,000 mg in the case of oral administration and 0.1-400 mg in the case of parenteral administration.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a new use of uridine 5'-alkylphosphate compounds, particularly as a calcium antagonist.

[0002]

2. Description of the Related Art Infectious diseases are being overcome by drugs such as antibiotics. On the other hand, circulatory diseases are caused by a rapid increase in fat intake due to the westernization of dietary habits and high chloride levels since ancient times in Japan. Along with the sodium diet, the number of cases is increasing, and it is feared that it will become a serious problem in the future of aging. In view of such a situation, various cardiovascular drugs have been developed. Among them, cardiovascular drugs having a calcium antagonism as a mechanism are expected to have great drug efficacy.

[0003] Calcium antagonists relax both the thick and thin parts of the coronary artery by inhibiting the influx of calcium ions into vascular smooth muscle cells, thereby increasing coronary blood flow and increasing oxygen to the myocardium. Increase supply. Conventional calcium antagonists are mainly nifedipine,
Verapamil and diltiazem are classified into three types. Of these, the nifedipine type (dihydropyridine type) calcium antagonist has an action of suppressing not only blood vessels but also the influx of calcium ions into cardiomyocytes, It is understood that compared with verapamil and diltiazem, it has a relatively weak cardiac inhibitory effect.

[0004]

SUMMARY OF THE INVENTION Calcium antagonists are
At present, attention has been paid to a therapeutic agent for ischemic heart disease such as angina pectoris and myocardial infarction, and there is a constant demand for providing a calcium antagonist having more potent and safe efficacy.

[0005]

In order to meet such a demand, the present inventors have conducted various studies on various nucleoside compounds, and found that uridine 5'-alkyl phosphate compounds have a remarkable calcium antagonism. Was confirmed, and the present invention was completed based on this finding.

Prior to the present invention, uridine 5'-monophosphate (UMP) was examined for the presence of a platelet agglutinating effect on uridine compounds, but it was reported that a negative result was obtained. (Pharmaceutical Magazine, 1
11 (9) 504-509 (1991)).

[0007] It has been reported that uridine 5'-alkyl phosphates inhibit sexual aggregation between α and α haploid (haploid) cells of yeast but do not affect cell growth. (FEMS Microbiol.
Lett. , 147, 17-22 (1997)). It has been shown that this aggregation inhibition is not due to the direct action of uridine 5'-alkyl phosphates on the yeast cell wall.

The gist of the present invention is that of formula I:

Embedded image (In the formula, R represents a linear or branched alkyl group.)
Calcium antagonists containing a uridine 5'-alkyl phosphate compound represented by the formula (1) as an active ingredient, in particular, a platelet aggregation inhibitor.

In the above formula I, the alkyl group represented by R may be linear or branched.
The carbon number does not usually exceed 30 and is preferably 4 to 24, and more preferably 16 to 20. Specific examples of such an alkyl group include butyl, pentyl,
Hexyl, heptyl, octyl, decyl, undecyl,
Examples thereof include linear alkyl such as dodecyl, tetradecyl, hexadecyl, heptadecyl, octadecyl, and eicosyl, and branched alkyl such as geranyl and farnesyl. Currently considered the most preferred is when R represents a 16 carbon n-hexadecyl group, ie uridine 5′-hexadecyl phosphate (UMPC16).

[0010] Uridine 5'-alkyl phosphate compound (I) as an active ingredient is not only in a free form, but also in a free form in a living body, such as hydrates, salts and esters, as long as it is pharmaceutically acceptable. It may be used in any form that can. Therefore, in the following description, the compound (I) is intended to include not only the free form but also any such pharmaceutically acceptable forms.

The compound (I) of the present invention, that is, uridine 5'-alkyl phosphate, is generally
It can be synthesized from a monophosphate (a compound corresponding to R = H in the formula I) by a conventional method. For example, uridine 5'-monophosphate and various linear or branched alkyl alcohols are converted to t-
It can be produced by dissolving in butyl alcohol and reacting in the presence of dicyclohexylcarbodiimide. The reaction temperature varies depending on the type of the solvent, but is usually from 60 to 100 ° C, preferably from 70 to 90 ° C. The reaction time varies depending on the reaction temperature, and is usually from 2 to 20 hours, preferably from 6 to 8 hours. .

As described later, the fact that the compound (I) has an inhibitory effect was confirmed in an agglutination test using rabbit platelets. In addition, the fact that compound (I) has an inhibitory action on intracellular calcium ion inflow was also confirmed. Therefore, compound (I) is useful as a calcium antagonist, and is also useful as a platelet aggregation inhibitor.

The compounds (I) can be administered, for example, by oral, parenteral, buccal, rectal or transdermal administration, ie in a manner which is convenient for the application of the drug. For oral administration, they can be formulated as liquids or solids, for example, syrups, suspensions, emulsions, tablets, capsules or lozenges.

Liquid formulations generally comprise compound (I) together with optional additives such as suspending agents, preservatives, flavoring agents, coloring agents and the like, in a suitable liquid carrier (eg, ethanol, glycerin, polyethylene glycol, Oil, water)
It is prepared by suspending or dissolving in the medium.

The composition in tablet form can be prepared using any suitable pharmaceutical carrier customary for the manufacture of solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.

Compositions in capsule form can be prepared using conventional encapsulation procedures. For example, active ingredient-containing pellets can be prepared using a standard carrier and then filled into hard gelatin capsules. Alternatively,
A dispersion or suspension can be prepared using any suitable pharmaceutical carrier, for example, aqueous gums, celluloses, silicates or oils, and then the dispersion or suspension can be filled into soft gelatin capsules.

Compound (I) may also be administered parenterally by bolus injection or infusion. A typical parenteral composition consists of a solution or suspension of the compound in a sterile aqueous carrier or parenterally acceptable oil, such as polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil or sesame oil. Alternatively, the solution may be lyophilized and then reconstituted with a suitable solvent just prior to administration.

The pharmaceutical composition of the present invention is preferably in unit dosage form such as a tablet, capsule or ampoule.
Each dosage unit for oral administration contains 1 to 250 mg (preferably 0.1 to 60 mg for parenteral administration) of the compound (I).
(Converted as a free base).

In the case of adult patients, the daily dose may be, for example, 1 mg to 5 mg of compound (I) (in terms of free base).
00 mg, preferably 1 mg to 250 mg (for example, 5 mg
~ 200mg) oral dose, or 0.1mg ~ 10mg
0 mg, preferably 0.1 mg to 60 mg (for example, 1 mg
-40 mg) intravenous, subcutaneous or intramuscular dose;
It is administered 1 to 4 times a day. Compound (I)
May be administered by continuous intravenous infusion, preferably at a dose of up to 400 mg per day. Thus, the total daily dose by oral administration is in the range of 1 to 2000 mg and the total daily dose by parenteral administration is from 0.1 to 400 mg.
in the mg range. Suitably, the compounds will be administered for a period of continuous therapy, for example for a week or more.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The following description shows production examples, preparation examples and test examples of the compound (I) of the present invention.

Production Example 20 ml of uridine 5'-monophosphate (600 μmol) and hexadecyl alcohol (6 mmol)
After dissolving in t-butyl alcohol, 3 mmol of dicyclohexylcarbodiimide is added, and the mixture is heated at 80 ° C. for 20 hours. After completion of the reaction, the formed precipitate was filtered off, and t-
Butyl alcohol is removed under reduced pressure. The residue was washed several times with a mixture of hexane and acetone (1: 1), dried and then mixed with a mixture of chloroform and methanol (95: 1).
Dissolve in 5). This is loaded onto a silica gel column prepared with the same solution, and the column is sufficiently washed with the same solution to allow dicyclocarbodiimide to flow out. Thereafter, uridine 5′-hexadecyl phosphate is eluted by sequentially increasing the concentration of methanol. The solvent was removed from the eluate under reduced pressure to obtain a white precipitate. The yield per uridine 5'-monophosphate was 32.4.
%Met.

Formulation Example 1 (intravenous injection) Compound (I) 1-40 mg Buffer pH up to about 7 Solvent up to 100 ml

Formulation Example 2 (bolus injection) Compound (I) 1-40 mg Buffer pH up to about 7 Cosolvent up to 5 ml

In the above Formulation Examples 1 and 2, specific examples of the buffer include citrate, phosphate and sodium hydroxide / hydrochloric acid, specific examples of the solvent include water, and specific examples of the co-solvent include propylene glycol. , Polyethylene glycol and alcohol.

Formulation Example 3 (Tablets) Compound (I) 1-40 mg Diluent / filler 50-250 mg Binder 5-25 mg Disintegrant 5-50 mg Lubricant 1-5 mg

In Formulation Example 3 above, specific examples of diluents / fillers include microcrystalline cellulose, lactose and starch, specific examples of binders include polyvinylpyrrolidone and hydroxypropylmethylcellulose, and specific examples of disintegrants Sodium starch glycolate and crospovidone, and specific examples of lubricants include magnesium stearate and sodium stearyl fumarate.

Formulation Example 4 (oral suspension) Compound (I) 1-40 mg Precipitating agent 0.1-10 mg Diluent 20-60 mg Preservative 0.01-1.0 mg Buffer pH up to about 5-8 Solvent 0-40mg Flavor 0.01-1.0mg Colorant 0.001-0.1mg

In Formulation Example 4, specific examples of the suspending agent include xanthin gum and microcrystalline cellulose.
As a specific example of the diluent, sorbitol solution, typically water, and as a specific example of the preservative, sodium benzoate,
Specific examples of the buffer include citrate, and specific examples of the cosolvent include alcohol, propylene glycol, polyethylene glycol, and cyclodextrin.

Test Example 1 Test material and test method 1-1. Test Materials: - uridine 5'-octyl phosphate as test compound _{(I: R = C 8 H} 17) (UMPC8), uridine 5 '
- dodecyl phosphate _{(I: R = C 12 H} 25) (UM
PC12), uridine 5'-hexadecyl phosphate _{(I: R = C 1 6} H 33) (UMPC16), uridine 5'-eicosyl phosphate (I: R = C ₂₀ H
₄₁ (UMPC20) and uridine 5'-tetracosyl phosphate _{(I: R = C 24 H} 49) (UMPC24)
Were synthesized according to the methods of Production Examples.

1-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) as a platelet aggregation inducer was obtained from Bachem Feinchemikalien AG (Bube).
ndorf, Switzerland).

The calcium-ion permeable carrier "A2318"
7 ", bovine plasma-derived thrombin, arachidonic acid, palmitic acid, adenosine 5'-diphosphate (ADP),
Ionomycin calcium salt and bovine serum albumin (BSA, fraction V, free of essential fatty acids)
Sigma Chemical Co. (St. Louis, MO, USA)
Was used. Hula 2 AM and Hula 2 are Dojin (Kumamoto,
Japan). Verapamil hydrochloride, 12-myristate 13-phorbol acetate (PMA) and Ficoll
-Paque is Wako Pure Chemical (Tokyo, Japan) and Resea, respectively.
rch Biochemicals International (Natick, MA,
USA) and Amersham Pharmacia Biotech (Tokyo, Japan). Solvents are of all grades.

The buffer composition is as follows:
(1) Basic Tyrode solution: 8.01 g of NaCl
/ L, KCl 0.195 g / L, MgCl ₂ 6H ₂ O 0.215
g / L, NaHCO ₃ 1.02 g / L, glucose 1.01 g / L,
pH 7.2; (2) Tyrode / Gelatin without Ca ²⁺ and EDTA: Same as buffer (1), but with gelatin (2.5 g / L) and Na ₂ EDTA (0.37).
(3 g / L), pH 6.5; (3) Tyrode / gelatin containing Ca ²⁺ : same as buffer (1), but with C
aCl containing _{2 2H 2 O (0.143g / L} ), pH7.2;
(4) Buffered saline containing HEPES-EDTA:
HEPES 1.00 g / L, NaCl 8.01 g / L, KCl 0.1.
195 g / L, dextrose 1.01 g / L, Na ₂ EDTA
0.373 g / L, pH 7.4.

Preparation of rabbit platelets: Plastic containers and siliconized glass containers were used for all platelet preparation and stimulation experiments. The washed rabbit platelets
Sugatani et al.'S method (J. Biol. Chem., 262, 5740).
1987). 4 each
5 mL of rabbit blood was added to 5 mL of 41 mM citric acid-85 m
M was added in trisodium citrate-2% glucose solution and centrifuged at 170 g for 10 minutes. Platelet-rich plasma, a platelet-rich plasma:
PRP) was layered with 10 mL Ficoll-Paque and 750
Centrifuged at g for 20 minutes. The platelet layer is 0.1 mM E
Mix gently with 40 mL of HEPES-buffered saline (pH 7.2) containing DTA and add 0.2 mL of Ficoll-Paq.
ue, and centrifuged at 750 g for 10 minutes. Again, the platelet layer was washed with 40 mL of H containing 0.1 mM EDTA.
Suspended in EPES-buffered saline (pH 7.2),
Centrifuged at 750 g for 10 minutes. Blood cells are 0.1 mM
Tyrode / gelatin containing EDTA (pH 6.5)
And suspended at 1.0 × 10 ⁹ cells / mL.

1-2. Test method:-(Platelet aggregation test) Platelets (1 × 10 ⁸ cells, 400
μL) are stimulated and activated in Tyrode / gelatin buffer containing 1 mM Ca ²⁺ . Aggregation activity is measured as a change in light transmission using an aggregometer (Nikko Hematracer, PAT-2A). Uridine 5'-alkyl phosphate dissolved in physiological saline containing 0.05% ethanol-0.005% dimethyl sulfoxide, and verapamil dissolved in physiological saline, unless otherwise indicated, stimulants such as PAF One minute before the addition of is added to the reaction. The ratio of platelet aggregation inhibition was [(control reaction−inhibition reaction) / control reaction] × 100.
Is calculated. The IC ₅₀ is obtained by regression analysis of the addition amount-reaction amount curve.

(Measurement of concentration of free calcium in cytosol) Rabbit platelets (1) suspended in HEPES-buffered saline (pH 7.4) containing 0.1 mM EDTA
(× 10 ⁹ cells / mL) was incubated with 1 × 10 ⁻⁶ M fura 2-acetoxymethyl ester at 37 ° C. for 45 minutes to be taken into cells, and washed once to remove extracellular dye. Tie road with 0.1mM EDTA /
The cells were resuspended in a gelatin buffer (pH 6.5) to a concentration of 1 × 10 ⁹ cells / mL. This platelet suspension (0.4
8 mL) was collected in a round-bottom test tube, and Ca ²⁺ ([C
a ²⁺ ] ₀ ) CaCl ₂ was added to a concentration of 1 mM.

Thereafter, these samples were transferred to a custom-made cuvette holder and placed in a JASCO (Tokyo, Japan) fluorescence spectrophotometer CAF-100 equipped with a recorder.
It was kept at 37 ° C. The magnet in the test tube was rotated to constantly agitate the cells. After holding at 37 ° C for at least 5 minutes,
Cells were activated by the addition of 0.01 mM of the above concentration of uridine 5'-alkyl phosphate for 1 minute, followed by the addition of 0.01 mL of the above concentration of aggregation-inducing substance. Hula 2 fluorescence is 340 nm (F340) and 380 nm
The measurement was continuously performed at an excitation wavelength of (F380) (each error ± 5 nm). [Ca ²⁺ ] i was measured by measuring the corresponding luminescence signal (500 ± 10 nm) in the same manner as the ratio signal (F340 / F380) represented as R340 / 380.

The intracellular Ca ²⁺ concentration [Ca ²⁺ ] i can be calculated from the following equation (G. Grynkiewicz et al., J. Biol.
Chem., 260, 3440, 1985). [Ca ²⁺ ] i = Kd × B × [(R−Rmin) / (Rmax−
R)] where Kd: Ca ^{2+ is} the dissociation constant of Hula 2 and in
In vivo, it can be calculated from the formula assuming 224 nM (G. Gry
nkiewicz et al., J. Biol. Chem., 260,
3440 1985); B: F in solution without Ca ²⁺ for F380 at inclusive solution in Ca ²⁺
R: Fluorescence ratio represented by F340 / F380; Rmax: the maximum luminescence signal value due to intracellular fura 2 when 0.01 mL of 20% Triton X-100 was added to lyse the cells; Rmin: Rmax After measurement, 150 mM Tris /
Luminescence signal value when 0.01 mL of 450 mM EGTA was added to remove Ca ²⁺ .

In each experiment, three to four measurements were made for each inhibitor. The experiment was also set to be completed within one hour to minimize the effects of platelet reaction fluorescence, dye leakage and fluorescent labeling. Data presentation: The results shown in the tables and figures represent experiments performed at least twice, using adjusted platelets in isolation, providing averages (± range of averages) for multiple experiments, unless otherwise specified. ing.

2. Test results 2-1. UMPC for PAFs causing platelet aggregation
Effect of 16:-A dose-dependent effect of PAF and UMPC16 on platelet aggregation is shown in Figure 1. That is, FIG. 1 is a diagram showing the effect of UMPC16 on PAF which induces platelet aggregation.
B shows the effect of PC16 at different concentrations, B shows the dose-dependent effect of UMPC16 and palmitic acid on PAF inducing platelet aggregation, and C shows the effect of different concentrations of P
3 shows the inhibitory activity of UMPC16 on platelet aggregation by AF.

A to f in FIG. 1A indicate the following experimental conditions. That is, a tie road containing 1 mM CaCl ₂ /
Washed rabbit platelets (1 × 10 ⁸ cells, 0.38 mL) in gelatin buffer (pH 7.2) were dissolved in physiological saline containing 2.0% ethanol-2.0% dimethyl sulfoxide (UMPC16 (instruction)). Concentration) (10μ
L) and preincubation for 1 minute (a), followed by PAF dissolved in saline containing 0.1% BSA
(10 μL) (final concentration 8 × 10 ⁻¹¹ M) for 2 minutes (time at which a maximum response was obtained by PAF stimulation) (b). The inhibition rate of platelet aggregation was calculated by the above method, and the results were shown as a plurality of average values (range of the average value). c is a vehicle, d is 6 × 10 ^-6 M, and e is 8 × 10 ^-6
M and f show the case of 1 × 10 ⁻⁵ M.

As shown in FIG. 1A, the UMPC 16
It did not suppress platelet morphology and changes caused by 8 × 10 ⁻¹¹ M PAF, but inhibited platelet aggregation. UMPC16
Agglutination inhibition was dependent on the concentration of UMPC16 and PAF (see FIGS. 1B and C). On the other hand, palmitic acid (consisting of part of the alkyl of UMPC16) at a concentration of 1 to 10 μM did not inhibit platelet aggregation by PAF.

2-2. Thrombin, arachidonic acid, ADP, A23187 and PMA to induce platelet aggregation
The effect of UMPC16 on aggregation-inducing substances was examined in order to examine whether platelet aggregation inhibition by UMPC16 is only induced on PAF. That is, 2-1.
As in the case of FIG. 1, platelets (1 × 10 ⁸ cells,
0.38 mL) for 1 minute with UMPC16, followed by an aggregation-inducing substance (10 μL), ie, 0.3 μL.
PAF (final concentration 8 × 10 ⁻¹¹ M) dissolved in physiological saline containing 1% BSA, thrombin (final concentration 0.02 U / mL) dissolved in physiological saline, physiological saline containing 4% ethanol Arachidonic acid (final concentration 2 × 10
^-5 M), ADP dissolved in physiological saline (final concentration 2.5)
× 10 ⁻⁵ M) A23187 dissolved in physiological saline containing 4% dimethyl sulfoxide (final concentration 2 × 10 5
^-7 M) and PMA (final concentration 2 × 10 ^-8 M) dissolved in saline containing 4% ethanol, respectively.
Exposure for 4, 4, 2, 2, 4 and 30 minutes (time at which maximal response is obtained by stimulation with an agglutinogen in the presence of the vehicle). The inhibition rate of platelet aggregation was calculated by the above method, and the results were shown in FIG. 2 as a plurality of average values (± range of average value).

As is clear from FIG. 2, thrombin, arachidonic acid and ADP which induce platelet aggregation were clearly suppressed by UMPC16, similarly to PAF which induces platelet aggregation. The strength of the inhibition depends on the aggregation inducer and UMPC.
16 concentrations. PAF that induces aggregation (8 × 1
0 ⁻¹¹ M), thrombin (0.02 U / mL), arachidonic acid (2 × 10 ⁻⁵ M) and ADP (2.5 × 10 ⁻⁵ M)
The IC ₅₀ values for each 2.4 × 10 ^-6 M, 5.4
× 10 ^-6 M, 2.1 × 10 ^-6 M and 2.2 × 10 ^-6 M (data not shown). Conversely, platelet aggregation by A23187 at a concentration of 2 × 10 ⁻⁸ M to 2 × 10 ⁻⁷ M and platelet aggregation by PMA at a concentration of 2 × 10 ⁻⁹ M to 2 × 10 ⁻⁷ M were 1 × 10 8 Not inhibited by ^-5 M UMPC16. These results indicate that UMPC 16
Inhibition of F-stimulation as well as receptor-mediated stimulation, but no effect on extracellular calcium influx induced by A23187 or activation of the protein kinase C pathway.

2-3. Inhibitory effect of uridine 5'-alkyl phosphate compound (I) on PAF and thrombin which induces platelet aggregation:-Various uridine 5'-alkyl phosphate compounds (I), ie, UMPC8, UMPC12, UMPC
16, the platelet aggregation inhibitory effect of UMPC20 and UMPC24 was examined. That is, 2.0% ethanol-2.0
% Of UMPC8, UMPC12, UMPC16, UMP
MPC20 or UMPC24 (10 μL)
Tyrode / Gelatin buffer containing CaCl ₂ (pH 7.2)
Exposed to platelets (1 × 10 ⁸ cells, 0.38 mL) for 1 minute, and then PAF (final concentration 8 × 10 ⁻¹¹ M) (A) or physiological saline dissolved in saline containing 0.1% BSA. Thrombin dissolved in saline (final concentration 0.02U / mL)
Exposure to (B) for 2 or 4 minutes, respectively. The inhibition rate of platelet aggregation was calculated by the method described above, and the results are shown in FIG. 3 as a plurality of average values (range of average value).

As is apparent from FIGS. 3A and 3B, these uridine 5'-alkyl phosphate compounds (I)
Affects platelet aggregation by PAF and thrombin. Uridine 5'-alkyl phosphates containing C16 or C20 with a longer methylene chain of the pole head group have been reported to inhibit platelet aggregation-induced PAF from PAF stimulation (KD Newman et al., J. Clin. Invest.,
61, 395, 1978; TC Lee et al., A
rch. Biochem. Biophys., 223, 33, 1983.
Year; FH Valone et al., Thromb. Res., 40, 3
85, 1985), which is more potent than verapamil, a calcium channel inhibitor. That is, 1 ×
10 ⁻⁵ M UMPC16 and 1 × 10 ⁻⁵ M verapamil each showed 8 × 10 ⁻¹¹ M PAF against the control test.
Aggregation was suppressed by 0% and 19%. 8 × 10 ^-11 M
The IC ₅₀ value (2.4 × 10 ⁻⁶ M) of the aggregation inhibition of UMPC16 with respect to PAF is the IC ₅₀ value of UMPC20 (2.
2 × 10 ⁻⁶ M). Furthermore, UMPC1
6 and UMPC20 also inhibited aggregation of thrombin as well as PAF.

2-4. U to PAF causing aggregation
Characteristics of the inhibitory effect of MPC16: Next, it was examined whether the platelet aggregation inhibition induced by UMPC16 was reversed by supplemental calcium, similar to that seen with verapamil. That is, platelets (1 × 10 ⁸ cells, 0.3) in Tyrode / gelatin buffer (pH 7.2) containing 1 mM or 10 mM CaCl _2.
8 mL) was pre-incubated for 1 minute with UMPC16 (final concentration 1 × 10 ⁻⁵ M) (10 μL) dissolved in physiological saline containing 2.0% ethanol-2.0% dimethyl sulfoxide, and then 0.1%. The cells were exposed to PAF (final concentration 8 × 10 ⁻¹¹ M) dissolved in physiological saline containing% BSA for 2 minutes. The inhibition rate of platelet aggregation was calculated by the method described above, and the results are shown in FIG. 4 as a plurality of average values (±
Range of averages).

As can be understood from FIG. 4, when the calcium concentration was increased in the range of 1 mM to 10 mM in the platelet suspension, UMPC16 induced PAF aggregation.
Inhibition by was not reversed. Conversely, at a supplemental CaCl ₂ concentration of 10 mM, the extent of platelet aggregation induction by PAF was reduced to 72%.

Further, in order to examine whether UMPC16 is effective even after the addition of PAF, the effect of the time of UMPC16 addition on platelet aggregation was examined. That is,
UMPC16 or the solvent for the control test was added to 8 × 10 ⁻⁸ M
At a specified time before and after PAF addition to platelets,
Calculate the platelet aggregation inhibition rate by the above method and plot the results
FIG. 5 shows a plurality of average values (± range of average value).
1 × 10 1 min before adding 8 × 10 ⁻¹¹ M PAF
^In addition to incubating with ^-5 M UMPC16,
8 × 10 ^-11 M PAF and 1 × 10 ^-5 M UMP
Even when C16 was added, aggregation by PAF was 100%.
Was inhibited. On the other hand, UMPC16 was added 10 minutes after addition of PAF.
Seconds, 30 seconds and 1 minute after addition to platelets,
Platelet aggregation was 29% and 42%, respectively, for the control test.
% And about 75% (FIG. 5). These findings are
UMPC16 causes disaggregation like verapamil,
It shows that there was an inhibitory effect on PAF stimulation even when added after PAF.

2-5. PAF elicits [Ca ²⁺ ] i response
Effect of UMPC16 on and ionomycin:-To investigate the effect of UMPC16 on PAF and ionomycin that induces [Ca ²⁺ ] i reaction, UMPC16 and a solvent were preincubated directly with platelets in warm water at 37 ° C for 1 minute. The maximum reaction, in the presence of a solvent,
It was measured by PAF stimulation. Solvent (a), PAF (b, 8 × 10 ⁻¹¹ M), UMPC1
^{6 (c, 1 × 10- 5} M), ionomycin (d, 4 ×
1 ^-8 M) was added. The results are shown in FIG. 6, which is a representative example of an experiment performed at least four times.

PA that changes calcium concentration in platelets
The effects of verapamil and UMPCs on F and ionomycin were compared. That is, platelets (10 ⁹ cells / mL)
Is combined with a solvent, verapamil (final concentration 1 × 10 ⁻¹ M) or UMPCs (final concentration 1 × 10 ⁻⁵ M), and the above 2.4.
Incubate for 1 minute as described in FIG.
After treatment, they were exposed to PAF (final concentration 4 × 10 ⁻¹¹ M) and ionomycin (final concentration 4 × 10 ⁻⁸ M). maximum
[Ca ²⁺ ] i levels were measured by Hula-2 fluorescence as described above. The results are the average of the results of 4-6 measurements ±
It is shown in Table 1 as SD.

[0051]

TABLE 1 Elevated [Ca ²⁺ ] i agonist Saline Verapamil UMPC16 nM PAF 474.7 ± 74.2 199.8 ± 67.3 167.4 ± 35.8 Ionomycin 204.9 ± 28.8 ND 230.1 ± 19.0 ND: not performed

From this result, UMPC 16 determines that [Ca²⁺]
attenuates the effects of PAF that induce i²⁺] i
Do not attenuate the action of ionomycin that induces
Understandable (FIG. 6, Table 1). 1 mM extracellular calcium
In the presence, the mean of unstimulated platelets [Ca²⁺] i is 5
2.0 ± 18.4 nM (n = 15), and UMPC16
This value was not changed. 8 × 10^-11M PAF
When added, platelets [Ca ²⁺] i is 474.7 ± 74.2
nM, but UMPC 16
[Ca²⁺] I attenuated the rise. By UMPC16
[Ca²⁺] The degree of inhibition of the i reaction is due to verapamil
Although higher (Table 1), the mode of platelet aggregation inhibition
It was very similar.

3. Analysis of test results The above test results show that UMPC16
Thrombin, arachidonic acid and A to induce platelet aggregation
DP also inhibited directly in a dose-dependent manner, indicating that the inhibition was not restricted to PAFs that elicit platelet activation (FIGS. 1 and 2). Of the UMPC compounds, U
MPC16 has an inhibitory effect on platelet activation of UMP
It is stronger than C8 or UMPC12 (FIG. 3). Arachidonic acid is metabolized by cyclooxygenase and thromboxane A ₂ synthetase via endoperoxide and thromboxane A _2, which is thought to induce the activity of phospholipase C pathway. Stimulation PAF, thrombin, by thromboxane A ₂ and ADP is transmitted through the receptor to elicit a protein phosphorylation 40-kDa, induces the activation of phospholipase C, which are associated with protein kinase C system . UMPC16 is responsible for platelet aggregation and [Ca ²⁺ ] by stimulation through these receptors.
inhibited the action of PAF to induce changes in i (FIGS. 2 and 6).
And Table 1).

Increasing the calcium concentration in the platelet suspension did not restore the inhibition of UMPC16 to induce aggregation on PAF (FIG. 4), which differs from the mode of activity of verapamil. Other findings indicate that the inhibitory effect of verapamil on neutrophils is not only to inhibit calcium influx, but also to have a direct inhibitory activity on the protein kinase C enzyme activated by verapamil. ing. On the other hand, UMPC16 is a calcium ion permeable carrier, and A2318 that induces platelet aggregation.
7 and did not inhibit the action of ionomycin to alter [Ca ²⁺ ] i (FIGS. 2 and 6). Furthermore, UMPC16 did not inhibit PMA, which causes platelet aggregation (FIG. 2).
PMA directly activates protein kinase C, which phosphorylates the 40 kDa protein in platelets, and does not require extracellular Ca ²⁺ .

As shown in FIG. 5, when added to platelets previously aggregated with PAF, UMPC16 at a concentration of 1 × 10 ⁻⁵ M can inhibit aggregation. That is, deaggregation is caused. Verapamil is available for 40-kDa and 20-
By reversing the phosphorylation of the kDa protein,
It has been reported that platelets aggregated with PAF cause disaggregation (G. Bonadonna et al., Thromb. Heamos., 5).
6, 308, 1986). In a study of the kinetics of PAF binding to rabbit platelets, platelets fully aggregated by PAF show disaggregation while reducing the amount of PAF to the receptor, ie, maintain platelet aggregation Therefore, it has been elucidated that this indicates that PAF binding is required for the receptor.

As described above, the mode of activation of UMPC16 inhibits calcium influx through an aggregation-inducing substance that specifically binds to a receptor, and inhibits binding of an aggregation-inducing substance, like verapamil, a calcium channel inhibitor. It can be thought that it is due to things. In addition, inhibition of a reaction dependent on intracellular calcium may affect the inhibitory activity of UMPC16 on platelet response. In conclusion, it can be said that UMPC16 more effectively inhibits receptors through the platelet response than verapamil.

[0057]

Industrial Applicability The nucleoside 5'-alkyl phosphate compound (I) of the present invention has a calcium antagonism, and particularly has a calcium antagonism based on the inhibition of extracellular fluid calcium from flowing into cells. Since it is effective in preventing arrhythmia and has an excellent inhibitory action on platelet aggregation, it is useful as a preventive or therapeutic agent for thrombosis, embolism or arteriosclerosis.

[Brief description of the drawings]

FIG. 1. UMP for PAF that induces platelet aggregation
It is a figure showing the effect of C16. A is PA that induces aggregation
F shows effects at different concentrations of UMPC16 on F, B shows UMP on PAF to induce platelet aggregation
Shows dose-dependent effects of C16 and palmitic acid, where C is UMPC on platelet aggregation by different concentrations of PAF.
16 shows the inhibitory activity of the present invention.

FIG. 2 is a graph showing the inhibitory effect of UMPC16 on an aggregation-inducing substance that induces platelet aggregation.

FIG. 3 is a graph showing the inhibitory effect of uridine 5′-alkyl phosphates on a platelet aggregation inducer. A shows the case where the inducer is PAF, and B shows the case where the inducer is thrombin.

FIG. 4. UMPC16 of PAF that induces platelet aggregation
FIG. 4 is a graph showing the effect of extracellular calcium on the inhibitory activity of lipase.

FIG. 5 shows the effect of adding UMPC16 at various times to PAF that induces platelet aggregation.

FIG. 6: PAF that changes platelet calcium concentration
FIG. 3 is a view showing the effect of UMPC16 on ionomycin and ionomycin.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Akira Matsukawa 3-407-3, Showa-cho, Hamadera, Sakai-shi, Osaka F-term (reference) 4C057 BB02 DD01 LL09 LL10 LL23 4C086 AA01 AA02 EA17 MA01 MA04 NA05 NA14 ZA40 ZA45 ZA54 ZC50

Claims (5)

[Claims] ## STR1 ## Formula I: ## STR1 ## (In the formula, R represents a linear or branched alkyl group.)
A calcium antagonist comprising as an active ingredient a uridine 5′-alkyl phosphate compound represented by the formula: 2. The calcium antagonist according to claim 1, wherein R is an alkyl group having no more than 30 carbon atoms. 3. The calcium antagonist according to claim 2, wherein R is an alkyl group having 4 to 24 carbon atoms. 4. The calcium antagonist according to claim 1, wherein the uridine 5′-alkyl phosphate compound is used in a free form or any pharmaceutically acceptable form capable of giving a free form in vivo. . 5. The calcium antagonist according to claim 1, which is used as a platelet aggregation inhibitor.