WO2004035065A1 - Hypoglycemic substances containing zinc - Google Patents

Abstract

It is intended to develop drugs having hypoglycemic, α-glucosidase inhibitory, antiinflammatory, analgesic, anticancer, antiplatelet and antithrombotic effects as well as an effect of inhibiting blood coagulation causing heart attack and apoplexy by using zinc (II) complexes containing salicylic acid, aspirin, trigonelline and derivatives thereof together with a zinc source. As a result, medicinal preparations and preventives, which have hypoglycemic, α-glucosidase inhibitory, antiinflammatory, analgesic, anticancer, antiplatelet and antithrombotic effects as well as an effect of inhibiting blood coagulation causing heart attack and apoplexy and comprise organic zinc (II) complexes containing, as a ligand, a compound selected from among salicylic acid having a benzene ring, trigonelline having a pyridinium ring and derivatives thereof, are successfully developed.

Description

 Zinc-containing substance having hypoglycemic action

(Technical field)

 The present invention provides a hypoglycemic effect comprising a zinc (II) organic complex containing as a ligand a compound consisting of salicylic acid having a benzene ring, trigonelline having a pyridi-dimethyl ring, and a derivative thereof, and α-darkosida light. The present invention relates to a pharmaceutical preparation and a prophylactic agent having an inhibitory action, an anti-inflammatory action, an analgesic action, an anticancer action, an inhibitory action on platelet aggregation, an inhibitory action on blood coagulation, and an antithrombotic action. book

 (Background technology)

 This time, salicylic acid used as a ligand is contained in willow leaves and bark, and has been used as an analgesic and antipyretic since ancient times. Salicylic acid, which has been isolated and confirmed, has become widely used as an analgesic. However, the side effect of damaging the stomach and the severe bitter taste often caused the patient to stop taking it. Later, aspirin (acetylsalicylic acid), which is acetylated salicylic acid, was synthesized to overcome the disadvantages of salicylic acid. To date, aspirin has been shown to have anti-inflammatory, analgesic, anti-cancer, anti-platelet aggregation, anti-thrombotic effects, and anticoagulant effects that can cause heart attacks and strokes. Was. Aspirin, which has been taken for 100 years, is said to contribute to the recovery of patients' health and also to prevent diseases. On the other hand, some organic compounds and natural products have been developed as anti-diabetic drugs, anti-obesity drugs, anti-triglyceride drugs, and used as drugs. In addition, at present, there is no cure for type 1 (insulin-dependent) diabetes, which depends only on subcutaneous injection of insulin, and there is a need for the development of a therapeutic drug that can be orally administered instead of insulin. It also comes from stress, obesity, lack of exercise, aging, etc. 2

(Non-insulin dependent) Although some therapeutics have been developed and used clinically for type 2 diabetes, they are not universal and often have side-effect problems. One such treatment, vanadyl sulfate, is already being used in clinical trials in the United States and elsewhere. -On the other hand, zinc (II) ion, which is known to be less toxic than vanadium, It has been known from around the time that it has insulin-like activity (see Non-Patent Documents 1-3). Panadyl sulphate (zinc) ion (zinc sulphate and zinc salt) is an inorganic salt, so it is difficult for it to pass through biological membranes and is hardly taken into the living body. To overcome such problems, zinc (II) complexes with lower toxicity, better stability and better lipophilic insulin-like action than vanadium are more effective complexes than vanadyl complexes. Its development is desired. As zinc complexes, there have been patents (see Patent Documents 1-3) and papers (see Non-Patent Document 4) previously filed, and moreover, from a variety of low-toxic, highly active complexes, more useful blood glucose The development of a depressant is desired.

 Prior art document information relating to the invention of this application is as follows.

 [Non-Patent Document 1] L. Coulston and P. Dandona, Insulin-like effects of Zn on adipocytes, Diabetes, 29, 665-7 (1980)

 [Non-patent Literature Document 2] J.M.May and C.S.Contoreggi, The mechanism of the insulin-like effects of on oral ionic zinc, J. Biol.Chem., 257, 4362-8 (1982)

[Non-Patent Document 3] Shisheva, D. Gefel and Y. Shecliter, Insulinlike effects of zinc ion in vitro and in vivo (Zn ² + is the first agent other than vanadate that administration is able to restore tissue ability to metabolism glucose), .

Diabetes, 41, 982-8 (1992)

 [Non-Patent Document 4] E. Ueda, Y. Yoshikawa, Y. Isliino, H. Sakurai, and Y. Kojima, Potential insulino-mimetic agents of zinc (II) complexes with picolinamide.

derivatives: Preparations of complexes, in vitro and in vivo studies (Chem. Pharm. Bull., 50, 337-340 (2002)

 [Patent Document 1] Kojima, Sakurai, Zinc (II) Organic Complex Hypoglycemic Agent, PCT: WO 01/39769 A1

 [Patent Document 2] Kojima, Sakurai, Yoshikawa, hypoglycemic agent, PCT: WO 02/060432 A1

[Patent Document 3] Kojima, Sakurai, Yoshikawa, zinc-containing food, PCT: WO 02/089606 A1 Since zinc (II) ions (zinc sulfate, zinc chloride, etc.) are inorganic salts, they are difficult to pass through living organisms. It is difficult to be taken into the living body. In addition, dispirin salicylate, which has anti-inflammatory, analgesic, anti-cancer, anti-platelet aggregation, anti-thrombotic effects, and anticoagulant effect of causing heart attacks and strokes, etc. There are side effects. (Disclosure of the Invention)

 In order to overcome such a problem, the present invention provides a prophylactic / therapeutic treatment comprising a zinc (II) complex having a lower toxicity than zinc (II) ions, a moderate stability, and a moderate lipophilicity. The purpose is to provide drugs. Many zinc (II) complexes with hypoglycemic activity have been developed by the applicants, but complexes that can prevent and treat multiple health problems have not been developed so far.

 The present invention relates to a drug which can be safely prevented and treated as an anti-inflammatory action, an analgesic action, a pile cancer action, a platelet aggregation inhibitory action, a thrombotic preventive action, and a blood coagulation inhibitory action that causes heart attack and stroke. An object of the present invention is to provide a zinc (II) complex having aspirin and an analog thereof as a ligand. By coordinating aspirin to zinc, it became possible to enhance the absorption of aspirin and zinc, and at the same time, to exert a hypoglycemic effect having insulin-like activity. Diseases can cause health problems by combining various symptoms. Salicinoleic acid was converted to aspirin due to pain in the stomach and severe bitterness. However, to a lesser extent salicylic acid, aspirin also has stomach-damaging side effects. By converting aspirin into a zinc complex, absorption can be improved and side effects can be reduced.

 The problem to be solved by the present invention is a pharmaceutical composition containing a zinc (II) complex as an active ingredient, which causes diabetes, hypertension, inflammation, pain, carcinogenesis, and heart attack and stroke. The present invention relates to a pharmaceutical composition for treating blood coagulation and thrombus.

 The pharmaceutical composition of the present invention is preferably a pharmaceutical composition comprising a pharmaceutically acceptable simple substance and a mixture thereof in addition to the above-mentioned zinc (II) complex.

 The present invention relates to an agent comprising an organic compound capable of forming a complex with zinc and a zinc source. Preferred examples of the organic compound capable of forming a complex with zinc include, but are not limited to, organic compounds having a benzene ring and a pyridinium ring in addition to the general formulas (1) to (2). It is not done.

 As the zinc source used in the present invention, any zinc source suitable for administration to humans and / or other animals may be used.For example, a zinc mineral salt or a zinc organic complex is preferable. Are listed.

Examples of zinc mineral salts include zinc acetate, zinc chloride, zinc sulfate, and zinc nitrate. I can do it. When a mineral salt of zinc is used as the zinc source, the pH adjuster may be, for example, a basic aqueous solution such as potassium hydroxide, sodium hydroxide, lithium hydroxide, barium hydroxide, or the like. Alternatively, a buffer such as a citrate buffer or a phosphate buffer may be used in combination. Preferred examples of the zinc organic complex include a zinc organic complex having, as a ligand, a compound selected from the group consisting of salicylic acids, trigonelins, and derivatives thereof.

 The shape of the drug according to the present invention may be any of powder, granule, fatigue type, capsule, liquid, gel, and the like.

(Brief description of drawings)

 FIG. 1 shows a full chart of infrared absorption starch (IR) of a bis (salicylic acid) Z zinc (II) complex according to the present invention.

 Figure 2 shows the infrared absorption spectrum (IR) of bis (trigonelline) nozinc (II) complex.

FIG. 3 shows the change in blood glucose level when the bis (aspirin) Z zinc (Π) complex, [Zn (asp) ₂ (H ₂₀ ) ₂ ], was intraperitoneally administered once a day.

FIG. 4 shows changes in body weight when the bis (aspirin) / zinc ( _II ) complex, [Zn (asp) ₂ (H ₂₀ ) ₂ ], was intraperitoneally administered once a day.

Figure 5 represents 14 days, bis (aspirin) / zinc (II) complex, after administration _{[Zn (asp) 2 (H} 2 0) 2], and the transition of the blood glucose level when performing the glucose tolerance test ing. The complex non-administration group (violent) and the [Zn (asp) ₂ (H ₂ 0) ₂ ] complex administration group (〇) are plotted. In addition, * indicates the group of p 0.05 and the complex non-administered group.

Figure 6 shows the results of the sucrose tolerance test (OSTT) after administration of the bis (aspirin) / zinc ( _II ) complex, [Zn (asp) ₂ (H ₂ 0) ₂ ], aspirin and water. Changes in blood sugar levels.

The [Zn (asp) ₂ (H ₂ 0) ₂ ] complex administration group (〇), the aspirin administration group (Hata), and the complex non- (water) administration group (△) are plotted. In addition, * indicates p <0.05 vs aspirin administration group w.

FIG. 7 shows changes in the HbAlc value before and after administration of the bis (aspirin) / zinc (II) complex, [Zn (asp) ₂ (H ₂₀ ) ₂ ], and the administration. In addition, * indicates p <0.05 vs before KK_A ^y mouse FIG. 8 is a graph showing the relationship between the bis (salicylic acid) / zinc (II) complex concentration () and the α-dalcosidase inhibition rate (%) in Experimental Example 4.

FIG. 9 is a graph showing the relationship between the bis (aspirin) zinc (II) complex concentration (mM) and the _α- darcosidase inhibition rate (%) in Experimental Example 4.

(Best mode for carrying out the invention)

 The following production examples and examples are provided to illustrate the present invention, and the present invention is not limited to these examples and test examples.

 [Example 1]

The synthesis of bis (aspirin) Ζ zinc (II) complex, [Zn (asp) ₂ (Η ₂₀ ) ₂ ], was performed according to the literature (see Non-Patent Document 5). The molecular structure of the complex is shown in the following formula (3) (see Non-Patent Document 6).

[Non-Patent Document 5] K. Singla and H. Wadhwa, Int.J. Phamaseutics, 108, 173-185 (1994)

 [Non-Patent Document θ] U. Hartmann and Η. Vahrenkamp, Coordination Chem., 42, 161-7 (1994) [Example 2]

The synthesis of bis (salicylic acid) Z zinc (Π) complex, [Zn (sal) ₂ (0) ₂ ], is based on salicylic acid

Five

薷 ¾ ぇ for 弒 («26) (4 mmol) was dissolved in warm water, and an aqueous solution of barium hydroxide (2 mmol) was added to the solution, followed by stirring for 30 minutes. Further, an aqueous solution of zinc sulfate (2 mmol) is added, and the mixture is stirred with stirring. The resulting barium hydroxide was filtered off, the filtrate was concentrated, and the concentrated residue was recrystallized from water to obtain the desired product. Yield: 7 7% elemental analysis:. Found, C: 44.89, H: 3.67 , Calculated for _{Zn (C 7 H 5 0 3} ) 2 · 2H 2 0, C: 44.76, H:. 3.76 IR ( KB r): Fig. 1 shows a full chart.

[Embodiment 3]

The synthesis of the bis (trigonelline) Z zinc (II) complex, [Zn (tri) ₂ Cl ₂ ], is accomplished by dissolving trigoneline (2 mmol) and lithium hydroxide (2 mmol) in methanol, and after 30 minutes the solution Then, a methanol solution of zinc chloride (2 mmol) is added thereto, and the mixture is stirred overnight. The resulting precipitate was collected and washed with methanol to obtain the desired product.

Yield: 4 3% Elemental analysis:. Found, C: 40.47, H: 3.25 , N: 6.60, Calculated for _{Zn (C 7 H 7 N0 2} ) 2 Cl 2 · 0.4H 2 0, C: 40.25 , H: 3.57, N: 6.71.

 I R (KBr): The full chart is shown in Figure 2.

[Example 4]

Mono (aspirin) Z The zinc chloride (II) complex, Zn (asp) Cl, is synthesized from a solution of aspirin (10 mmol) and lithium hydroxide (10 mmol) in methanol, and a solution of zinc chloride (5 mmol) in methanol. And left stirring overnight. The resulting precipitate was filtered off, and the filtrate was concentrated to dryness. The residue was dissolved in water and allowed to stand overnight to obtain the desired product as a white precipitate. Yield: 8% Elementary analysis:. Found, C: 38.31, H: 2.81 , Zn (C 9 H 7 0 5) Calcd for Cl, C: 38.61, H: 2.52.

 (Pharmacological test example 1)

 The following experiment was performed according to the method described in Biol. Pharm. Bull., 18, 719-725 (1995).

Rat adipocytes were isolated by exsanguinating and killing male Wistar rats weighing 200 g under ether anesthesia using the rod-bell method. Biol. Chem., 239, 375 (1964)). Adipocytes were separated from the tissue. Cut the fat cells with scissors, Or 2 Omg bovine serum albumin (BSA) and KRB buffer (1 Omm Gunorekosu containing 2 mg Koranaze, 1 2 OmM N a C 1 , 1. 27 mM C a C 1 2, 1. 2mM Mg S 0 4, 4 . 75 mM KC 1, 1. 2mM K P0 4, and _{24 m MNaHC0 3; pH = 7.} 4) in, 3 7. For 1 hour. The fat cells were separated from the undigested tissue by filtration through a Nia Mesh (250 μπι), washed three times with the above buffer containing no collagenase, and adjusted to 2.5 × 10 ⁶ cells / ml. .

Effect on rat adipocytes of zinc (II) complexes, in siliconized vials, above isolated adipocytes (2. 5 X 1 0 ⁶ cells Zm 1), various concentrations (1 0 one ^4, 5 ^{X 1 0- 4, 1 0- 3} 3 7 ° C in VOS0 ₄ or zinc (II) in KRB buffer one lmL containing a 2 Omg B SA / m 1 complex), and 5 hours pre-incubator adipate 0.5 . One Ide, was added to the reaction mixture Epinefurin of 1 0- ⁵ M, 3:00 solution obtained in 3 7 ° C

 0

It was incubated for a while. The reaction was stopped by ice cooling, and the mixture was centrifuged at 3000 rpm for 10 minutes. For extracellular solution, free fatty acid (FFA) levels were determined using the NEFA kit, IC _5. Was measured (Table 1).

 【table 1】

 Table 1. Estimated IC50 values of zinc (II) complexes with several

 coordination mode.

 Complex Coordination mode

 VOSO4 ionic 1.00 (0.08)

Zn (tri) ₂ Cl ₂ 0 ₂ 0.78 (0.05) *

Zn (sal) ₂ 0 ₄ 0.90 (0.08) *

Zn (asp) 2 0 ₂ 0.57 (0.04) *

 Zn (asp) Cl O 1.03 (0.05)

 aspirin ― none

 ♦ Significance at p <0.05 vs. VOSO4

Table 1 to Table the IC ₅ o values for each zinc (II) complex relative to the VOS0 ₄ values (IC ₅ o = 1.00).

[Pharmacological test example 2]

For evaluation of the anti-diabetic effect, KK- ^Ay mice of type 2 diabetes model animals were used. In KK- ^Ay mice, once a day for 14 days, the blood glucose level in the abdominal cavity is 250 mg IdL or more. The complex was administered to give 1.5 mg Zn / kg body weight. At the time of complex administration, changes in body weight were also monitored. After the administration for 14 days, the animals were fasted for 16 hours, and glucose was administered to give 1 g glucose I kg body weight, and a glucose tolerance test was performed.

 In addition, HbAlc was measured before and after administration, and biochemical serum parameters were measured using blood obtained from orbital blood collection after administration.

In the complex administration group, changes in blood glucose levels and changes in body weight when the bis (aspirin) Z zinc (II) complex and [Zn (asp) ₂ (H ₂₀ ) ₂ ] were administered intraperitoneally were shown. Figures 3 and 4 show the results of the glucose tolerance test in Figure 5.

 [Pharmacological test example 3]

KK- ^Ay mice were used for the oral sucrose tolerance test (0STT). Water was orally administered to 9-week-old KK- ^Ay mice for 3 days. Thereafter, fasted about 1 6 hours, empty stomach in the early _{morning, Zn (asp) 2 (H} 2 0) 2 complex (5 nig Zn / kg body weight), aspirin _{(Zn (asp) 2 (H} 2 0) 2 complex throw The amount corresponding to aspirin at the time of administration) and pre-administered water. 60 minutes after the pre-administration, sucrose was administered to give a weight of 2 g / kg body weight, and a sucrose tolerance test was performed. Blood glucose was measured at 60 minutes before administration, at 0 minutes after administration of sucrose, at 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes after administration of sucrose. This was performed a total of seven times (Figure 6).

 [Pharmacological test example 4]

 The examination of mono-dalcosidase inhibitory activity was carried out by improving the Dehiqvist method described in JP-A-2002-316939.

0.1 M substrate (maltose and sucrose dissolved in 0.15 M HEPES buffer pH 6.8) or 4% starch solution (0.15 M HEPES buffer pH 6.8) 0 0.1 ml of the test substance solution and 0.1 ml of the enzyme solution were added to 1 ml, reacted at 37 ° C for 60 minutes, and then boiled to stop the reaction. The amount of generated glucose was measured by a glucose oxidase method (glucose CII test Co., Ltd.). As a blank test, add 0.15 M HEPES buffer ( _PH = 6.8) instead of the substrate solution, and take the absorbance of the same test as the plank value. The liquid As was determined. The enzyme solution used was a commercially available α-dalcosidase (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to 5 units / ml with a 0.015 M HEPES buffer solution (pH = 6.8). As a control, the absorbance Ac when a solvent was added instead of the test substance was measured, and α-gnorecosidase inhibitory activity was measured by the following equation (FIGS. 8 and 9). a-Darcosidase inhibitory activity (%) = [(Ac— As) / Ac] X 100

 As shown in Figure 3, the blood glucose level began to decrease after administration of the complex, and at the end of the 14-day administration, decreased by about 150 rag IdL as compared to before administration.

Body weight is one week after the start of the administration but decrease was not ¹ seen, then was steadily increased (Fig. 4). From these results, no major side effects such as weight loss due to administration of the bis (aspirin) Z zinc (II) complex were observed.

In the glucose tolerance test performed after the 14-day administration, as shown in Fig. 5, the bis (aspirin) Z zinc (Π) complex administration group did not receive the complex (feeding food and water freely for 14 days). Fasting blood glucose at O min during glucose loading was significantly lower than in the group. After the glucose load, the blood glucose level remained significantly lower than that of the group not administered the complex. These results suggested that administration of the bis (aspirin) / zinc (Π) complex improved glucose tolerance in KK- ^Ay mice (FIG. 5).

 In the sucrose tolerance test, it was observed that the zinc complex-administered group as a pretreatment tended to suppress an increase in blood glucose level after sucrose loading as compared to the groups administered aspirin or water. In addition, the blood glucose peak time was 30 minutes after sucrose loading in the zinc complex administration group, but peaked after 15 minutes in the other groups (Fig. 6). ).

 Figure 7 shows the change in HbAlc values before and after administration of the bis (aspirin) Z zinc (II) complex. The HbAlc value before administration was 7.2, 0.5. /. However, the value of HbAlc after administration decreased to 0.2% in 6.6 soils, which was significantly lower than the value before administration. These results indicated that the decrease in blood glucose of the bis (aspirin) Z zinc (II) complex was not temporary, but was persistent over a long period of time.

As shown in FIG. 8 and 9, bis (aspirin) Zinc (II) _{complex, [Zn (asp) 2 (} H 2 0) 2], and bis (salicylate) Z Zinc (II) complex, [Zn (sal) ₂ ], have been found to have the same activity of inhibiting the activity of dalcosidase. (Industrial applicability)

 According to the present invention, an agent comprising an organic compound capable of forming a complex with zinc and a zinc source is

It is less toxic than zinc (II) ion, has good stability, has good fat solubility, hypoglycemic action, α-darcosidase inhibitory action, anti-inflammatory action, analgesic action, anti-cancer action, It is expected to be a promising agent containing a zinc (II) complex that has platelet aggregation inhibitory action, thrombus preventive action, and blood coagulation inhibitory action that causes heart attack and stroke. In addition, improve the health status of diabetic patients and their spare groups, impaired glucose tolerance, diabetes (type 2 diabetes, etc.), insulin resistance syndrome (insulin receptor abnormalities, etc.), polycystic ovary syndrome, hyperlipidemia Is a drug that is effective in the prevention and treatment of cerebral disorders, atherosclerosis, cardiovascular disease, hyperglycemia, angina, hypertension, congestive heart failure, diabetic complications, or taste disorders. , Is what is expected.

 Further, the complex of the present invention is safe without substantial side effects even during long-term ingestion.

Claims

The scope of the claims 1. A blood bran lowering effect comprising an aspirin, a salicylic acid, a trigonelline, and an organic compound consisting of a derivative thereof and a zinc source capable of forming a complex with zinc, a blood bran lowering effect, an α_darcosidase inhibitory effect, A drug that can be safely prevented and treated as an inflammatory, analgesic, pile cancer, platelet aggregation inhibitory, blood coagulation inhibitory, and thrombus preventive. 2. The drug according to claim 1, wherein the zinc source is a mineral salt or an organic complex of zinc.  3. Salicylic acids are represented by the following general formula (1),

(In the formula, R represents a hydroxyl group, a lower alkoxy group, or an amino group which may be substituted with a lower alkyl group, and R ₂ are each independently hydrogen, a lower alkoxy group, a lower alkyl group, or a hydroxyl group. A derivative having a benzene ring represented by:  4. Trigonelins are represented by the following general formula (2),

(In the formula, R represents an amino group which may be substituted with a hydroxyl group, a lower alkoxy group, or a lower alkyl group, and each independently represents hydrogen, a lower alkoxy group, a lower alkyl group, or a hydroxyl group. R ₃ represents a hydrocarbon group.) A derivative having a pyridinium ring represented by the formula: 5. The drug according to claim 1, wherein the organic substance having a benzene ring and a pyridium ring comprises a compound represented by the general formulas (1) to (2) and a mixture thereof.