JP2000281650A - 2-mercapto-pyridine-n-oxide derivative and oxovanadium (iv) complex - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an oxovanadium (IV) complex which has an insulin-like action and is useful as a medicine for treating diabetes, hypertension, and the like, by adding a mercapto-pyridine-oxide (derivative) as a ligand. SOLUTION: This oxovanadium (IV) complex containing a 2-mercapto- pyridine-N-oxide (derivative) as a ligand. The 2-mercapto-pyridine-N-oxide (derivative) is preferably a ligand of the formula [R1 to R4 are each hydrogen, a (substituted) hydrocarbon, a halogen, an alkoxy, a (substituted) amino or a (substituted) heterocyclic group]. A method for producing the oxovanadium (IV) complex includes a method comprising adding an oxovanadium sulfate solution to a solution of the target ligand and then isolating the formed oxovanadium (IV) complex. A medicinal composition contains the oxovanadium (IV) complex as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an oxovanadium (IV) complex containing 2-mercapto-pyridine-N-oxide or a derivative thereof as a ligand, and a pharmaceutical composition containing the same. About. The oxovanadium (IV) complex containing 2-mercapto-pyridine-N-oxide or a derivative thereof as a ligand according to the present invention acts like an insulin and is useful as a therapeutic agent for diabetes or hypertension.

[0002]

2. Description of the Related Art Despite dramatic advances in medical technology,
In Japan, various adult diseases such as Alzheimer's disease, ischemic heart disease, and diabetes are major issues. Among them, diabetes is a disease with many potential patients, and the number continues to increase every year. Currently, diabetes causes abnormalities in glucose, protein and lipid metabolism due to the absolute or relative deficiency of insulin, and also has the characteristics of chronic hyperglycemia, nephropathy, retinopathy and neurosis. It is defined as a disease that results in diabetic complications. In addition, the World Health Organization (WHO) states that diabetes is an insulin-dependent diabetes mellitus (Insulin indepenaent diabetic mellitu).
s, IDDM) and non-insulin dependent diabetes (Non-insu)
lin dependent diabetic mellitus (NIDDM). The pathology of IDDM is due to an absolute deficiency of insulin, in which B cells in the pancreas are destroyed by autoimmune insulitis and insulin is not synthesized or secreted at all. Therefore, at present, the only alternative is to rely on insulin injection, and therapeutic agents that can replace insulin are desired.

[0003] On the other hand, the pathological condition of NIDDM is caused by a relative shortage of insulin, and is caused by impaired insulin secretion and action. Obesity, stress, lack of exercise, and the like are known as factors relating to onset. 95-97% of diabetic patients in Japan are NIDDM patients,
DDM patients make up only 1-3%. In Europe and America I
DDM patients are said to account for 10-20% of the total. For the treatment of NIDDM, a number of synthetic drugs have already been developed, such as acarbose as an α-glucosidase inhibitor that promotes glucose digestion and absorption,
Sulfonylurea drugs such as tolbutamide, which promotes insulin secretion, and biguanide drugs such as buformin. However, these drugs have problems such as insufficient efficacy and side effects, and development of a drug having an insulin-like action that can be orally administered instead of insulin has been desired.

[0004] Among vanadium (V) known as one of the biological trace elements, it was accidentally discovered in 1977 that pentavalent V (vanadate) strongly inhibits Na ⁺ -K ⁺ ATPase like ouabain. Was. It is known that ouabain has an action similar to insulin involved in glucose transport and metabolism, and it was questioned what about pentavalent vanadium (V). Studies based on this question have shown that pentavalent vanadium (V) promotes the uptake of glucose into cells like insulin. In 1985, and subsequently in 1987, streptozotocin (STZ) -induced diabetic rats were reported to give pentavalent V orally normalization of blood glucose levels despite low blood insulin levels. Was. Furthermore, in rats, tetravalent V (vanadyl) is more than 10 times less toxic than pentavalent V. When pentavalent vanadium (V) is administered, most of it is present as tetravalent vanadium (V) in the body. It has been revealed to do.

[0005] In diabetes, impaired use of glucose in tissues causes energy production due to insufficient action of insulin.
In order to compensate for this, the breakdown of triglyceride in fat cells is promoted, and fatty acids (FFA) as an energy source
Release is promoted. Decomposition of neutral fat is promoted by activation of hormone-sensitive lipase, but catecholamine, glucagon, ACTH and the like activate this enzyme, and conversely inhibit insulin. In diabetes, in addition to insulin deficiency, increased secretion of glucagon occurs, which promotes the release of FFA from adipose tissue. In addition, previous studies have found that administration of a tetravalent vanadium (V) complex that normalizes blood glucose levels to STZ-induced diabetic rats does not restore blood insulin levels to normal levels. Vanadium (V) has been shown to act systemically, rather than directly acting on B cells of the pancreas to promote insulin synthesis and secretion.

[0006] Vanadium (IV) oxide sulfate is known as a drug having an insulin-like action, and is already used in clinical tests in the United States and other countries. However, since vanadium (IV) oxide sulfate is an inorganic salt, it is difficult to permeate a biological membrane and is hardly taken into a living body. It is also known that some compounds of a tetravalent vanadium complex are effective for treating insulin-dependent diabetes. For example, vanadium (IV) having cysteine methyl ester as a ligand (H. Sakurai, et al., J. Clin. Biochem. Nu.
tr., 8, 193 (1990)), vanadium (IV) having a dihydroxydicarboxylate or a hydroxypyranone derivative as a ligand (JH McNeill, et al., J. Med. Che.
m., 35, 1489 (1992)), vanadium (IV) having an N-substituted dithiocarbamine salt as a ligand (H. Watanab)
e, et al., J. Med. Chem., 37, 876 (1994)). However, these vanadium (I
V) The compound has not reached a practical stage in terms of action and side effects.

The toxicity caused by the administration of vanadium (V) drugs is mainly nephrotoxicity, but at present the problem of toxicity has not been solved. Thus, the present inventors
By increasing the fat solubility of the complex, it is possible to increase the absorption rate of vanadium into the body, and by reducing the dose of the complex, it is possible to reduce the toxicity. 2-mercaptopyridine-N- having a pyridine skeleton as a ligand is considered. A complex was synthesized using oxide and its tautomer, 1-hydroxy-2-pyridinethione, and the effect of normalizing blood glucose was examined.

It is known that 2-mercaptopyridine-N-oxide and 1-hydroxy-2-pyridinethione used as ligands form complexes with various metals. Among them, zinc complexes are known to exhibit an antibacterial effect, and are added to shampoos and the like. To date, no toxicity has been reported for either ligand. From these facts, if this vanadium complex can exhibit an effect in a living body, it can be expected to be a therapeutic agent for diabetes with few side effects. Therefore, 2-mercaptopyridine-N-oxide, 1-hydroxy-2
-Focused on vanadium (IV) complexes having pyridinethione as a ligand.

[0009]

DISCLOSURE OF THE INVENTION The present inventors aimed to develop an oral therapeutic agent for IDDM which can replace insulin, and clarify the mechanism of action.
Using oxovanadium (IV) complex having 1-hydroxy-2-pyridinethione or a derivative thereof as ligand as a mercaptopyridine-N-oxide or a tautomer thereof, in vitro and in vivo.
The study was carried out. In vivo, blood glucose and FF
It is known that there is a positive correlation between A and FFA
Could be able to lower the blood sugar level if the release of
The effect of inhibiting the release of lipase was examined. In addition, in vivo, in addition to blood glucose concentration and FFA, urea nitrogen (BUN), which is an indicator of nephrotoxicity, and triglyceride to investigate its effect on lipid metabolism in order to clarify the mechanism of action, Changes in serum parameters such as (TG) and total cholesterol (TCHO) were measured and examined.

At present, there is no cure for insulin-dependent diabetes mellitus only by subcutaneous injection of insulin, and the present invention aims to develop a therapeutic drug for oral diabetes for IDDM, and has a novel insulin-like action. It is intended to provide an oxovanadium (IV) complex. The present invention also provides a pharmaceutical composition containing the oxovanadium (IV) complex as an active ingredient.

[0011]

The present inventors have conducted intensive studies and as a result, have found that an oxovanadium (IV) complex containing a 2-mercapto-pyridine-N-oxide derivative as a ligand has an insulin-like action. They have found that they can be used as therapeutic agents for diabetes, and have completed the present invention. The oxovanadium (IV) complex of the present invention is a novel compound.

The present invention relates to 2-mercapto-pyridine-
The present invention relates to an oxovanadium (IV) complex containing N-oxide or a derivative thereof as a ligand. More specifically, the 2-mercapto-pyridine-N-oxide or a derivative thereof of the present invention is 2-mercapto-pyridine-N-oxide which may have a substituent, and more specifically, the following formula (I) ,

[0013]

Embedded image

(Wherein R ¹ , R ² , R ³ and R ⁴ are each independently a hydrogen atom, a hydrocarbon group which may have a substituent,
It represents a halogen atom, an alkoxy group, an amino group which may be substituted, and a heterocyclic group which may have a substituent. ).

[0015] The present invention also relates to a pharmaceutical composition containing the above oxovanadium (IV) complex. The above-mentioned oxovanadium (IV) complex of the present invention has an insulin-like action, and the present invention relates to a pharmaceutical composition having an insulin-like action or a pharmaceutical composition for treating diabetes or hypertension. The pharmaceutical composition of the present invention is preferably a pharmaceutical composition further comprising a pharmaceutically acceptable carrier in addition to the oxovanadium (IV) complex described above.

[0016]

DETAILED DESCRIPTION OF THE INVENTION 2-Mercapto-pyridine-N-oxide or a derivative thereof according to the present invention is vanadium (I).
As long as it can be a ligand of V) and does not inhibit the insulin-like action of the present invention, it may have a substituent on the pyridine ring. As the substituent, various organic residues can be used, but a hydrocarbon group which may have a substituent, a halogen atom, an alkoxy group, an amino group which may be substituted, And the like may be a heterocyclic group.

Examples of the hydrocarbon group include a lower alkyl group, a lower alkenyl group, a lower alkynyl group, a cycloalkyl group, a cycloalkenyl group and an aryl group. The “lower alkyl group” in the ligand represented by the general formula (I) of the present invention is a straight-chain or branched alkyl having 1 to 15, preferably 1 to 10, and more preferably 1 to 6 carbon atoms. And more specifically, for example,
Examples include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a secondary butyl group, a tertiary butyl group, a pentyl group, and a hexyl group. Examples of the “lower alkenyl group” include a linear or branched alkenyl group having 2 to 15, preferably 2 to 10, and more preferably 2 to 6 carbon atoms. More specifically, for example, a vinyl group , Allyl group, butenyl group, pentenyl group and the like. Examples of the “lower alkynyl group” include a linear or branched alkynyl group having 2 to 15, preferably 2 to 10, and more preferably 2 to 6 carbon atoms, and more specifically, for example, an ethynyl group , Propynyl group, butynyl group, pentynyl group and the like.

The "cycloalkyl group" includes 5 to 30 carbon atoms, preferably 5 to 20 carbon atoms, and more preferably 6 carbon atoms.
And a monocyclic, polycyclic or condensed cyclic alkyl group of from 10 to 10. As the "cycloalkenyl group", those having an unsaturated group such as one or more double bonds in the above cycloalkyl group are mentioned. No.

The above “hydrocarbon group” may further have a substituent. Examples of these substituents include a lower alkoxy group derived from the aforementioned lower alkyl group, a hydroxyl group, an amino group, a halogen atom, a heterocyclic group, a nitro group, and the like. The “halogen atom” includes chlorine, bromine, iodine and the like. As the “alkoxy group”, a lower alkoxy group derived from the lower alkyl group described above is preferable. Examples of the substituent of the “optionally substituted amino group” include the aforementioned hydrocarbon groups and acyl groups derived from these hydrocarbon groups.

The "heterocyclic group" is a group having at least one nitrogen atom, oxygen atom or sulfur atom in the ring,
Each ring has 5 to 20 members, preferably 5 to 10 members, more preferably 5 to 7 members, and may be condensed with a carbocyclic group such as a cycloalkyl group, a cycloalkenyl group or an aryl group. Examples include well-saturated or unsaturated monocyclic, polycyclic or condensed cyclic ones. The above-mentioned heterocyclic group may further have a substituent, and as these substituents, a lower alkoxy group derived from the above-mentioned lower alkyl group,
Examples include a hydroxyl group, an amino group, a halogen atom, a heterocyclic group, and a nitro group.

As a method for producing the oxovanadium (IV) complex of the present invention, a oxovanadium (IV) complex is formed by adding a vanadium oxide solution to a solution of a target ligand, and the oxovanadium (IV) complex is isolated. Can be manufactured. More specifically, reference can be made to the embodiments described below.

The oxovanadium (IV) complex of the present invention has an insulin-like action as apparent from the test examples described later, and can be used as a therapeutic agent for diabetes or hypertension. Accordingly, the present invention provides a pharmaceutical composition comprising the oxovanadium (IV) complex of the present invention and a pharmaceutically acceptable carrier.

For the treatment, the oxovanadium (IV) complex of the present invention is used as an active ingredient, and an organic or inorganic solid or liquid excipient suitable for oral administration, parenteral administration or external (topical) administration. Can be used in the form of a pharmaceutical preparation containing it together with a pharmaceutically acceptable carrier. The pharmaceutical preparations include capsules, tablets, dragees, granules, inhalants, suppositories, solutions, lotions, suspensions, emulsions, ointments,
It may be a gel or the like. If necessary,
Auxiliaries, stabilizers, wetting or emulsifying agents, buffers and other commonly used additives may be included.

The therapeutically effective dose of the oxovanadium (IV) complex of the present invention varies depending on the age and symptoms of the patient, but the average dose of the oxovanadium (IV) complex of the present invention is about 0.1 mg / person. A dose of from about 1 to about 1000 mg / person may be administered once or several times a day.

The present inventors have proposed 2-mercaptopyridine-
An oxovanadium complex of the present invention was produced using N-oxide and 1-hydroxy-2-pyridinethione, respectively (Examples 1 and 2 described later). An oxovanadium complex (hereinafter referred to as VO-MPNO) produced using 2-mercaptopyridine-N-oxide;
When various physical properties such as IR, UV, and ESR with an oxovanadium complex (hereinafter referred to as VO-HPT) produced using hydroxy-2-pyridinethione were examined, electrons of both complexes were delocalized. And they could not be clearly distinguished. Infrared absorption spectrum (I
R) are shown in FIG. 2 (VO-MPNO) and FIG.
O-HPT).

Next, Table 1 shows the test results of the bis (1-oxy-2-pyridinethiolate) oxovanadium (IV) complex on rat adipocytes.

[0027]

[Table 1]

Free "blank" of FFA in Table 1
Indicates a value due to spontaneous release of cells, and “control” indicates a value due to epinephrine stimulation. “VOSO ₄ ” in Table 1 indicates oxovanadium sulfate. The VO-HPT of the present invention exhibited a fatty acid release inhibitory effect that was equal to or higher than that of vanadium sulfate at the same concentration, which is representative of tetravalent vanadium. And
The effect was concentration dependent. The IC ₅₀ indicating the test drug concentration that inhibits the release of fatty acids by 50% is 1.9.
× 10 ⁻⁴ M. Further, the VO-MPNO of the present invention
Also exhibited the same or higher fatty acid releasing effect as vanadium sulfate at the same concentration, which is representative of tetravalent vanadium. And the effect was concentration dependent. IC ₅₀ indicating test drug concentration that inhibits fatty acid release by 50%
Was 1.8 * 10 <^-4> M.

Next, using the STZ-induced diabetic rat, the complex of the present invention was tested for its effect on normalizing blood glucose in vivo. Complexes in STZ-induced diabetic rats were 5 mg / kg as vanadium and 2.5 mg / kg as vanadium
Administered intraperitoneally. The dose was administered for the first two weeks, half the dose for the next week, and terminated three weeks later. FIG. 4 shows the change in body weight of the 5 mg / kg intraperitoneal administration group, FIG. 5 shows the change in blood glucose level, FIG. 6 shows the change in body weight of the 2.5 mg / kg intraperitoneal administration group, and FIG. Show. In each case, the data in the figures are shown as mean ± standard deviation.

In each of the administration groups, the blood glucose level gradually decreased after the administration, and from about the third day after administration, the normal blood glucose level of 1
It showed a value in the range of 00 to 200 mg / dl. However, if the dose is switched to half after 15 days,
In the group of rats to which the dose was low, a tendency of increasing the blood sugar level was observed. On the other hand, in a group of rats with a large dose,
Normal blood glucose levels continued to be maintained after administration was stopped. In addition, the change in body weight of the rats in the small dose group showed a tendency to increase for a while after the administration, but then to a declining trend. In the high-dose group of rats, the body weight was reduced simultaneously with the administration.

STZ-induced diabetic rats were orally administered 10 mg / kg of the complex as vandium for 21 days. As a result, there were rats in which the blood glucose level was normalized and those in which hyperglycemia was maintained. Therefore, the former group was divided into two groups, a sensitive group and a latter group as a non-sensitive group. FIG. 8 shows changes in body weight of the susceptible group after oral administration, and FIG. 9 shows changes in blood glucose level. FIG. 10 shows a change in body weight of the insensitive group, and FIG. 11 shows a change in blood glucose level. In each case, the data in the figures are shown as mean ± standard deviation. In the susceptible group, the blood glucose level gradually decreased after administration, and then slightly increased, but remained almost normal during the administration period. However, after the administration was discontinued, it showed a slightly increasing tendency.
The change in body weight slightly decreased until the third day after the administration, but increased thereafter during the administration period. On the other hand, in the non-sensitive group, the blood glucose level was lower than before administration, but did not reach the normal range. From day 22 to 28
By the day, the dose was doubled to 20 mg / kg, but the blood glucose level was not normalized. Changes in body weight did not differ significantly from the susceptible group.

A complex containing 5 mg / kg of vanadium as a complex was added to STZ-induced diabetic rats having a blood sugar level of 600 mg / dl or more.
Was administered intraperitoneally for 14 days, no administration was performed for the next 7 days, and the same amount was administered for the next 7 days. Figure 12 shows the weight change.
FIG. 13 shows changes in the blood glucose level. In each case, the data in the figures are shown as mean ± standard deviation. As a result, the blood sugar level gradually decreased and showed a normal value. However, when administration was stopped, it began to rise again. After 7 days of withdrawal, 5 mg / kg complex was administered again as vanadium, but no further follow-up was possible due to the reduced number of rats.

In the above experiment, the insulin concentration (μU
/ Ml), FFA (mEq / L), BUN (mg / d
l), TG (mg / dl) and TCHO (mg / dl)
Were measured for serum parameters. Table 2 shows the results.

[0034]

[Table 2]

"Sensitivity" and "insensitivity" in the table mean the susceptibility group and the insensitivity group in the oral administration group described above, and "ip" indicates intraperitoneal administration and "po. "
Indicates oral administration, intraperitoneal administration group consisted of 5 mice, and oral administration consisted of 10 mice as a group, 6 sensitive groups and 4 non-sensitive groups. All data in the table are shown as mean ± standard deviation.

Insulin concentration did not increase significantly during the administration period. Therefore, it is thought that the mechanism of action of the complex is not improving insulin secretion but acting systemically. The change in FFA showed a positive correlation with the blood glucose level, and decreased as the blood glucose level decreased.
BUN, which is an indicator of nephrotoxicity, increased during the intraperitoneal administration during the administration period. In particular, 5 mg / Vanadium
The increase was significant when the kg complex was administered.
However, oral administration did not show any significant variation during the administration period. Therefore, it is considered that toxicity is considerably reduced by oral administration. Regarding TG and TCHO, the relationship between the blood glucose level and TG is not clear from this data, but there was an increasing tendency during the administration period. However, for other complexes, TG and TCHO have been reported to decrease with decreasing blood glucose levels, and this mechanism will be further investigated.

From the above results, it can be seen that the VO-HPT of the present invention
The (VO-MPNO) complex has been found to be useful as an oral treatment for IDDM. However, its mechanism of action has not been completely elucidated, and further elucidation of the mechanism of action is required in the future.

[0038]

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

Example 1 Preparation of bis (1-oxy-2-pyridinethiolate) oxovanadium (IV) complex (VO-MPNO) 2-mercaptopyridine-N-oxide (10 mM) was added to 10 ml of an aqueous sodium hydroxide solution (10 mM). ) Was dissolved and adjusted to pH 5-6 with hydrochloric acid. An aqueous solution of vanadium oxide (5 mM) was added dropwise to the solution while stirring. The precipitate formed after stirring for 30 minutes was filtered through a glass filter, washed sufficiently with water, and dried to obtain a purple target product, 1.286.
g (80.6% yield). Molecular weight 317. IR 960 cm ^-1 (a full chart is shown in FIG. 2) UV λ ₁ 512 nm (ε = 74.5); λ ₂ 600 nm (ε = 24.0) Magnetic susceptibility χ _g 3.546 × 10 ⁻⁶ cgs unit μ _eff 1.74 ESR g value _{(g 0 1.990, g η 1.955} , g ⊥ 2.008) A value _{(A 0 83.4, A η 142.5} , A ⊥ 53.85) analysis calculated Value C: 37.60 H: 2.51 N: 8.78 found value C: 37.61 H: 2.45 N: 8.87

Example 2 Preparation of bis (1-oxy-2-pyridinethiolate) oxovanadium (IV) complex (VO-HPT) 1-hydroxy-2-pyridinethione (10 mM) was added to 10 ml of an aqueous sodium hydroxide solution (10 mM). ) Was dissolved. An aqueous solution of vanadium oxide (5 mM) was added dropwise to the solution while stirring. After stirring for 30 minutes, the resulting precipitate was filtered with a glass filter, washed sufficiently with water and dried to obtain 1.256 g (yield: 78.8%) of a purple target product. Molecular weight 317. IR 960 cm ^-1 (the full chart is shown in FIG. 2) UV λ ₁ 512 nm (ε = 42.3); λ ₂ 600 nm (ε = 13.0) Magnetic susceptibility _{ｇ g} 3.728 × 10 ^-6 cgs unit μ _eff 1.79 ESR g value _{(g 0 1.990, g η 1.954} , g ⊥ 2.008) A value _{(A 0 85.1, A η 162.0} , A ⊥ 46.65) analysis calculated Value C: 37.60 H: 2.51 N: 8.78 found C: 37.41 H: 2.53 N: 8.87

Next, the oxovanadium complex of the present invention (I
5 shows pharmacological data of V). Pharmacological Test Example 1 Biol. Pharm, Bull. , 18 (5), 71
The following tests were performed according to the method described in 9-725 (1995).

Isolation of Rat Adipocytes Wistar male rats were killed by blood loss and killed, followed by the method of rod bell (J. Biol. Chem., 239, 375 (196).
According to 4)), adipocytes were separated from adipose tissue around epididymis. Cut adipose tissue with scissors, 20mg per ml
KRB buffer containing bovine serum albumin (BSA) and 2 mg collagenase (120 mM NaCl, 1.2
7 mM CaCl ₂ , 1.2 mM MgSO ₄ ,
4.75 mM KCl, 1.2 mM KH ₂ PO ₄ ,
And 24 mM NaHCO ₃ ; pH = 7.4)
Digested for 1 hour at 7 ° C. Adipocytes were separated from undigested tissue by filtration through a nylon mesh (250 μm), washed three times with the above-mentioned buffer not containing collagenase, and adjusted to 2.5 × 10 ⁶ cells / ml.

Bis (1-oxy-2-pyridinethiolee)
G) Oxovanadium (IV) complex in rat adipocytes
Effect on grease separated above in siliconized vial
Fat cells (2.5 × 10 ⁶Cells / ml) at various concentrations (1
0^-4, 5 × 10^-4, 10³) VOSO₄, VO-
1 ml of KR containing 20 mg BSA / ml of MPNO
Pre-incubation for 0.5 hour at 37 ° C in B buffer
I did it. Then 10^-5 Reaction mixture with M epinephrine
To the mixture and incubate the resulting solution at 37 ° C for 3 hours.
I got it. The reaction was stopped by ice cooling and the mixture was
 Centrifuged at rpm for 10 minutes. About extracellular solution
Free fatty acid (FFA) levels using a NEFA kit
And measured.

The free fatty acids (FFA) in the sample were converted to acyl-CoA, AMP and AMP by the action of acyl-CoA synthetase (ACS) in the presence of Conzyme A (CoA) and adenosine-5-trisodium triphosphate (ATP). Produces pyrophosphate (Ppi). Acyl-C formed
oA is oxidized by the action of acyl-CoA oxidase (ACOD) to produce 2,3-trans-enoyl-CoA and hydrogen peroxide. The generated hydrogen peroxide is converted into ME by the action of peroxidase (POD).
HA and 4-aminoantipyrine are quantitatively oxidatively condensed to form a blue-violet dye. By measuring the blue-violet absorbance, the FFA concentration in the sample is determined.

The composition of the buffer used here was: 1) KRB + 2% BSA 2) NaCl 120 mM 3) CaCl ₂ 1.27 mM 4) MgSO ₄ 1.2 mM 5) KCl 4.75 m 6) KH ₂ PO ₄ 1.2 mM 7) NaHCO ₃ 24 mM. The results are shown in Table 1 above. Table 3 shows IC ₅₀ (mM) values obtained from the results.

[0046]

[Table 3]

[0047] As shown in Table 1 and 3, complexes of the present invention, it is possible to significantly suppress the free fatty acids from rat adipose cells compared to VOSO _4, is excellent as a therapeutic agent for diabetes It became clear.

Pharmacological Test Example 2 Effect of oxovanadium (IV) complex on (STZ) -induced diabetic rats (1) Preparation of STZ-induced diabetic rats 0.1 M Citrate buffer (pH 5.
0) 30 mg of STZ was dissolved in 5 ml. This solution was intravenously injected at a rate of 1 ml for a rat body weight of 200 g. The following experiment was performed using a rat whose blood glucose level was increased and became constant. (2) VO-HP was injected into a diabetic rat having an increased blood glucose level (STZ) by intravenously injecting streptozone (STZ).
The T complex was orally administered as vanadium so as to be 10 mg / kg body weight. Blood glucose was measured over time using serum collected from rats. The results of each experiment are shown in FIGS.

The administration method was as follows. (A) Intraperitoneal administration (3 weeks) 31 mg (5 mg as V) and 16 mg (2.5 mg as V) of the complex were suspended in 5 ml of a 5% acacia solution. 1 ml of this solution was intraperitoneally administered to 200 g of each rat for 14 days. From day 15, the solution was reduced to half (2.5 mg and 1.25 mg as V).
The administration was similarly performed intraperitoneally until the first day. (B) Intraperitoneal administration (2 weeks + 1 week) 31 mg (5 mg as V) of the complex was suspended in 5 ml of a 5% acacia solution. 600 mg of this solution
1ml per 1 / 200g / l rat
It was intraperitoneally administered for 4 days, the drug was withdrawn from day 15 to day 20, and again intraperitoneally from day 21 to day 27. (C) Oral administration (3 weeks) 63 mg (10 mg as V) of the complex was suspended in 5 ml of a 5% acacia solution. This solution was used to weigh 2 rats.
Rats that were orally administered 1 ml per 00 g for 21 days and whose blood sugar level did not decrease were orally administered (20 mg as V) by doubling the complex for 7 days from day 22.

(3) Measurement of Blood Sugar Level When a coloring agent is allowed to act on the reagent, glucose in the sample is rapidly converted from α-type to β-type by the action of mulotase contained in the coloring solution. β-D-glucose is oxidized under the action of glucose oxidase (GOD), and at the same time produces hydrogen peroxide. The produced hydrogen peroxide quantitatively oxidizes and condenses phenol and 4-aminoantipyrine in the color reagent solution by the action of coexisting peroxidase to produce a red dye. The glucose concentration in the sample is determined by measuring the red absorbance.

(4) Measurement of Blood Parameters Blood serum levels, insulin levels, free fatty acid (FFA) levels, and triglyceride (T) levels were measured using serum collected from the rat subclavian vena cava during and after the administration of the complex.
G) The concentration, total cholesterol (TCHO) concentration, and urinary cord nitrogen (BUN) concentration were measured. Blood collection was performed on the day indicated below, with the start of administration as day 0. (A) Intraperitoneal administration (3 weeks); Day 0, 14, 21, 28 (b) Intraperitoneal administration (2 weeks + 1 week); Day 14 (c) Oral administration (3 weeks); 0, 14, Days 21 and 28

(5) Measurement of Insulin Concentration When the antibody in the sample is reacted with an antibody bead obtained by binding an anti-insulin antibody to glass beads and an anti-insulin antibody labeled with an enzyme (peroxidase), the reaction is carried out using the following method. A) -insulin-enzyme-labeled anti-insulin antibody "sandwich is formed,
Since the amount of enzyme bound to the antibody beads is proportional to the amount of insulin in the sample, the enzyme activity is measured using a coloring reagent (o-phenylenediamine and water peroxide) to obtain a standard solution with a known content in advance. The insulin content in the sample can be determined from the calibration curve prepared using the method.

(6) Measurement of TG concentration TG in the sample is hydrolyzed by the action of lipoprotein lipase (LPL) to produce glycerol. Glycerol is converted to glycerol-3-phosphate in the presence of glycerol kinase (GK), and is further converted to dihydroxyacetone-3-phosphate by glycerol-3-phosphate dehydrogenase (G-3-PDH). In the process, reduced coenzyme (NADH) is generated from coenzyme oxidized nicotinamide dinucleotide (NAD +), and the generated NAD
H reduces nitrotetrazolium blue (NTB) by the action of diaphorase to form a formazan dye. The TG concentration in the sample is determined by measuring the absorbance of the dye.

(7) Measurement of TCHO concentration Free and ester cholesterol in the sample is dissociated into lipid (cholesterol component) and protein by the action of lipoprotein lipase (LPL) and a surfactant, and then cholesterol esterase Starts the hydrolysis of cholesterol. The generated cholesterol and endogenous cholesterol then generate hydrogen peroxide by the action of cholesterol oxidase. This hydrogen peroxide oxidizes the leuco dye by peroxidase (POD), and develops a blue color. The TCHO concentration in the sample is determined by measuring the absorbance of the blue dye.

(8) Measurement of BUN Concentration Urea in a sample is decomposed into ammonia and carbon dioxide by the action of urease. At the same time, ammonia gas is generated by keeping the pH of the reaction system alkaline. This ammonia gas changes bromcresol green from yellow to green. The BUN concentration in the sample is determined by measuring the absorbance of the green dye.

Table 2 summarizes the above experimental results.
From the results of the above experiments, it was revealed that the complex of the present invention was able to lower the blood glucose level of STZ-induced diabetic rats, and was excellent as a therapeutic agent for diabetes because it had few side effects.

[0057]

Industrial Applicability The oxovanadium (IV) complex of the present invention is highly stable and has a fat-soluble insulin-like action and a hypotensive action. Therefore, the oxovanadium (IV) complex of the present invention can be used for glucose intolerance, diabetes (such as type II diabetes), insulin resistance syndrome (such as insulin receptor abnormality), polycystic ovary syndrome, hyperlipidemia, and atheroma. Atherosclerosis, cardiovascular disease (angina, heart failure, etc.), hyperglycemia, or hypertension, or angina, hypertension, pulmonary hypertension, congestive heart failure, diabetic complications (eg, diabetic necrosis, diabetic It is useful as a medicament for use as a prophylactic / therapeutic agent for arthropathy, diabetic glomerulosclerosis, diabetic skin disorder, branuripathic neuropathy, diabetic self-disorder, diabetic retinopathy, etc.

[Brief description of the drawings]

FIG. 1 shows the situation of delocalization of the complex of the present invention.

FIG. 2 shows an IR chart of the complex VO-MPNO of the present invention.

FIG. 3 shows an IR chart of the complex VO-HPT of the present invention.

FIG. 4 shows that the complex VO-HPT of the present invention was 5 mg / vol.
1 shows changes in body weight when intraperitoneally administered in kg.

FIG. 5 shows that the complex VO-HPT of the present invention was 5 mg / vol.
1 shows changes in blood glucose level when intraperitoneal administration of kg is performed.

FIG. 6 shows that the complex VO-HPT of the present invention has a diameter of 2.5 m.
2 shows changes in body weight when g / kg was intraperitoneally administered.

FIG. 7 shows that the complex VO-HPT of the present invention had a length of 2.5 m.
FIG. 3 shows changes in blood glucose level when g / kg was intraperitoneally administered.

FIG. 8 shows 10 mg of the complex VO-HPT of the present invention.
1 shows changes in body weight when orally administered for 3 weeks.

FIG. 9 shows 10 mg of the complex VO-HPT of the present invention.
5 shows changes in blood glucose level when orally administered for 3 weeks.

FIG. 10 shows that the complex VO-HPT of the present invention was
mg / kg, orally for 3 weeks, and 20 mg / k
g shows changes in body weight when orally administered for one week.

FIG. 11 shows that the complex VO-HPT of the present invention was
mg / kg, orally for 3 weeks, and 20 mg / k
g shows the change in blood glucose level after oral administration for one week.

FIG. 12 shows that the complex VO-HPT of the present invention was administered to STZ-induced diabetic rats having a blood glucose level of 600 mg / dl for 5 m.
2 shows changes in body weight when g / kg was intraperitoneally administered.

FIG. 13 shows that the complex VO-HPT of the present invention was administered to STZ-induced diabetic rats having a blood glucose level of 600 mg / dl for 5 m.
FIG. 3 shows changes in blood glucose level when g / kg was intraperitoneally administered.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61K 31/00 609 A61K 31/00 609H 609E 31/555 31/555

Claims (6)

[Claims] 1. An oxovanadium (IV) complex containing 2-mercapto-pyridine-N-oxide or a derivative thereof as a ligand. 2. The 2-mercapto-pyridine-N-oxide or a derivative thereof is represented by the following formula (1): (Wherein, R ¹ , R ² , R ³ , and R ⁴ each independently represent a hydrogen atom, a hydrocarbon group which may have a substituent, a halogen atom, an alkoxy group, an amino group which may be substituted. Oxovanadium (IV) complex according to claim 1, wherein the ligand is a heterocyclic group which may have a substituent. 3. The oxovanadium (IV) complex according to claim 2, wherein the hydrocarbon group is a lower alkyl group. 4. The oxovanadium (IV) complex according to claim ² , wherein R ¹ , R ² , R ³ , and R ⁴ are hydrogen atoms. 5. A pharmaceutical composition comprising the oxovanadium (IV) complex according to claim 1. 6. The pharmaceutical composition according to claim 5, which is a therapeutic agent for diabetes.