JP2002128664A - Diabetes treatment / prevention agent consisting of probucol - Google Patents

Abstract

(57) Abstract: The present invention provides a novel diabetic, preferably an oral diabetic. More specifically, the present invention provides a novel antidiabetic drug based on an antioxidant action in pancreatic β cells and an action of reducing the amount of fat accumulated in β cells. The present invention relates to an agent for treating or preventing diabetes, comprising probucol or a salt or solvate thereof. The present invention also relates to a pancreatic β-cell protective agent comprising probucol or a salt thereof or a solvate thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a prophylactic or therapeutic drug for diabetes comprising probucol or a salt or solvate thereof. Further, the present invention relates to a β-cell protective agent comprising probucol or a salt thereof or a solvate thereof, which has a function of reducing the amount of fat accumulated in β-cells and a function of suppressing oxidative stress of β-cells.

[0002]

2. Description of the Related Art Diabetes has been known for a long time, and a description of diabetes was made around 1500 BC. In the East, the symptoms and treatments have been described in the late Han era under the name of "depletion". Diabetes had a short life expectancy after onset, and most of the patients died from diabetic coma. Diabetes has been classified into type 1 insulin-dependent diabetes mellitus (IDDM), type 2 non-insulin-dependent diabetes mellitus (NIDDM), and other diabetes. Insulin-dependent diabetes mellitus (IDDM) is due to absolute insulin deficiency, such as by destruction of pancreatic β-cells, but only accounts for about 5% of Japanese diabetic patients. Non-insulin-dependent diabetes mellitus (NIDDM), a type 2 diabetes mellitus, develops due to decreased insulin secretion and insulin resistance due to a decrease in the number and function of pancreatic β cells, and most Japanese diabetic patients have this disease. It is said to be a type of diabetes.

[0003] Insulin is synthesized and secreted by β cells of the islets of Langerhans of the pancreas. β cells produce insulin in response to glucose concentration, but there is no specific receptor for glucose in the cell membrane of β cells, and glucose is converted to β
It produces insulin by metabolizing it in cells. Thus, glucose has physiological effects that promote insulin secretion and biosynthesis, but rather strongly suppresses pancreatic β cells when they are continuously exposed to hyperglycemia. Pancreatic β-cell damage due to such chronic hyperglycemia is called β-cell glucose toxicity. In recent years, Robertson et al. Reported that β-cell glucose toxicity was determined to be “glucose desensitizatio” in terms of its location and degree, clinical findings, and mechanism of development.
n) ”and“ strictly defined (pancreatic β-cell) glucose toxicity (glucose t
oxicity). The former is caused by hyperglycemia for a relatively short period (within 24 hours), and the glucose responsiveness of insulin secretion is characteristically impaired.
On the other hand, the latter are caused by moderate to long-term chronic hyperglycemia, resulting in extensive, partly irreversible β-cell dysfunction. In many cases of type 2 diabetes with chronic hyperglycemia, it is thought that both types coexist to modify the pathology of diabetes.
It is considered to be a useful classification for correctly analyzing and understanding the molecular biological background of cellular glucose toxicity. The involvement of oxidative stress and decreased transcription factor function has been suggested for the latter,
That is, it is “glucose toxicity in a narrow sense”.

[0004] While "glucose refractory" mainly exhibits impairment of the glucose-responsive mechanism of insulin secretion, "glucose toxicity in a narrow sense" is characterized by a decrease in insulin biosynthesis and a progressive, partially irreversible effect. Insulin secretion disorder. In the background, insulin biosynthesis disorder and pancreatic β
There is a decrease in cell number to fibrosis of the islets. In recent years, in type 2 diabetes model animals capable of observing pancreatic β-cells over time, decreases in insulin mRNA and insulin content in β-cells that are consistent with the onset of diabetes have been observed. This is thought to be the same in humans, and in cases where sulfonylurea (SU) drugs have become secondary ineffective, such as decreased pancreatic β-cell endocrine granules, decreased pancreatic β-cell count, and fibrosis of pancreatic islet tissue. Found in autopsy cases. "Glucose toxicity in a narrow sense" is considered to greatly contribute to the onset of type 2 diabetes by progressing and accelerating such a decrease in insulin biosynthesis and a decrease in the number of pancreatic β cells.

[0005] A series of studies by the inventors so far have highlighted oxidative stress as a cause of glucose toxicity (Kaneto H., et al., Biochem J.,
320,85-863 (1996); Matsuoka T., et al., J. Clin.
Invest., 99, 144-150 (1997); Kaneto H., et al.,
Diabetes, 48, A240 (1999): Kajimoto et al., Diabetes Front
ier, 10 (4), 531-536 (1999)). In the first place, the mechanism of cell damage caused by hyperglycemia includes various mechanisms such as an increase in osmotic pressure, activation of a sorbitol pathway, and enhancement of a glycation reaction. In such a situation, we focused on the enhanced glycation reaction and the resulting increased oxidative stress for the following two reasons. One is that in diabetic individuals, chronic hyperglycemia results in the induction of glycation in many tissues and organs throughout the body (including crystallin in the lens) and the activity produced during the Maillard reaction. This is the fact that oxygen and the like cause tissue damage and contribute to the development of diabetic complications. In fact, recently, it has been shown that glycation and oxidative stress are induced in pancreatic islets and pancreatic β cells exposed to hyperglycemia in diabetic animals. The second reason we focused on oxidative stress is the fact that the expression of antioxidant enzymes is extremely weak in pancreatic islets (0-1 of the liver).
About 5%). Therefore, it was speculated that β cells would be very fragile once oxidative stress was induced in β cells.

The present inventors have proposed a type 2 diabetes model, C
57BL / KsJ-db / db mice were fed a diet containing 2.5% N-acetylcysteine, which is an antioxidant, 0.5% vitamin E, and 0.5% vitamin C, from 6 weeks of age (anti- Oxidant-treated mice) showed a significant improvement in glucose tolerance function with an increase in glucose-responsive insulin secretion. At that time, an increase in the amount of insulin mRNA and insulin in the β-cells was also observed, indicating that insulin biosynthesis, which had been reduced due to glucose toxicity, was restored. Furthermore, β
A remarkable increase in the number of cells was observed, and the background was suppression of apoptosis by antioxidants. In addition, it was found that the expression of PDX-1 in β cells, which is also reduced in expression along with oxidative stress induction and diabetes, was significantly restored by antioxidant treatment. None of the above effects of antioxidants on β-cell count, insulin biosynthesis ability and transcription factor activity were observed in non-diabetic model animals, but were caused by increased oxidative stress associated with chronic hyperglycemia in diabetic mice. It was considered that the antioxidant had a remarkable effect on the resulting cytotoxicity (Kajimoto et al., Diabetes Frontier, 10 (4), 531-536 (1999)). On the other hand, probucol has the chemical formula

[0007]

Embedded image

4,4'-isopropylidenedithio-bis (2,6-di-tert-butylphenol), which is used as a therapeutic agent for hyperlipidemia, such as familial high cholesterol, xanthomas, etc. It is a drug that has already been used in the clinic as a therapeutic and prophylactic agent for blood bleeding and arteriosclerosis. Also, it has been reported that probucol strongly suppresses LDL oxidation and is also effective as a preventive agent for restenosis after percutaneous coronary angioplasty (PTCA). But,
There is no known effect on diabetes or beta cells.

[0009]

SUMMARY OF THE INVENTION The present invention provides a novel diabetic, preferably an oral diabetic.
More specifically, the present invention provides a novel antidiabetic drug based on an antioxidant action in pancreatic β cells and an action of reducing the amount of fat accumulated in β cells.

[0010]

Means for Solving the Problems The present inventors have studied the effects of antioxidants on diabetes, and found that probucol, which is known as an antihyperlipidemic agent having an antioxidant effect on LDL, He found that probucol has an antioxidant effect on glycation and oxidative stress in pancreatic β-cells and a reducing effect on the amount of fat accumulated in pancreatic β-cells, and that probucol is effective as a therapeutic and preventive drug for diabetes, especially type 2 diabetes I found it.

That is, the present invention relates to an agent for treating and preventing diabetes, comprising probucol or a salt or solvate thereof. Further, the present invention relates to a β-cell protective agent comprising probucol or a salt or solvate thereof.

[0012] The present inventors have compared C57BL / KsJ-db / db mice, a type 2 diabetes model animal, with 6
From the age of the week, a diet containing 1% probucol was given freely without restriction (probucol group). The control group was also given normal food ad libitum without restriction. At 10 and 16 weeks, glucose tolerance (intraperitoneal glucose tolerance test) was evaluated and morphological and functional analysis of pancreatic tissue was performed.

That is, 10 weeks (n = 20) and 16 weeks (n
= 8), the intraperitoneal glucose tolerance test of both groups
0 g / kg body weight glucose is intraperitoneally administered and loaded,
Immediately after administration, 30 minutes and 120 minutes of plasma insulin concentration (FIGS. 1 and 2), and immediately after administration, 30 minutes, 60 minutes, 90 minutes and 120 minutes of blood glucose levels (FIGS. 3 and 4). Examined. Fig. 1 (10 weeks)
And FIG. 2 (16 weeks). The horizontal axis is the time after load (minutes)
And the vertical axis indicates the amount of insulin secretion (pmol / l). In each figure, a white square mark (□) indicates a group treated with probucol, and a black circle mark (●) indicates a control (control group). In addition, the results of the blood glucose level are shown in FIGS. 3 (10 weeks) and 4 (16 weeks).
Shown in The horizontal axis shows the time (minutes) after loading, and the vertical axis shows the blood glucose level (mg / dl). In each figure, a white square mark (□) indicates a group treated with probucol, and a black circle mark (●) indicates a control (control group). In the intraperitoneal glucose tolerance test, significant (p <0.
05) Improved blood glucose levels and significant (p <0.05) retention of plasma insulin levels.

At 16 weeks (n = 8), lipid metabolism was examined. The results for free fatty acids (FFA) are shown in FIG. Neutral fat (TG) and total cholesterol in plasma (TC
hol. 6) is shown in FIG. The vertical axis in FIG. 5 shows the equivalent (mEq / l) of free fatty acids (FFA), and the vertical axis in FIG. 6 shows the respective amounts (mg / dl). In addition, neutral fat (T
The result (n = 4) of the content (μg) of G) is shown in FIG.
The vertical axis in FIG. 7 indicates the amount of TG (μg / ng-DNA).
The gray part in FIGS. 5, 6 and 7 is the control (control group).
, And the black part indicates the probucol administration group. Based on these results, serum triglyceride (Tr
igriceride (TG)), Free Fatty Acid
(FFA)) and total cholesterol (Total Choles)
terol (T. Chol.)) significant (p <0.05)
A decrease was observed. At the same time, TG in the pancreatic islets of Langerhans
A decrease in content (16 weeks) was also observed. This indicated that the reduction of lipid toxicity (Lipotoxicity) was also at least partially involved in β-cell protection.

Further, for histological examination, 1
Pancreatic islets of Langerhans at weeks 0 and 16 were compared by insulin staining. The results are shown in FIG. 8 (10 weeks) and FIG.
(6 weeks) is shown by a photograph instead of a drawing. The upper side of each figure (the right side when the binding side is up) is for the probucol administration group, and the lower side (the left side when the binding side is up) is for the control group. As a result, retention of the insulin content in the pancreatic islets of Langerhans was observed. Also, 10
Weekly and 16-week pancreatic islets of Langerhans were compared by staining with 4-hydroxynonenal (4-HNE). The results are shown in FIGS. 10 (10 weeks) and 11 (1
(6 weeks) is shown by a photograph instead of a drawing. The upper side of each figure (the right side when the binding side is up) is for the probucol administration group, and the lower side (the left side when the binding side is up) is for the control group. 4-Hydroxynonenal is a kind of lipid peroxide, which reflects the peroxidation state of lipids, and is a kind of oxidative stress marker. Therefore, as a result, reduction of oxidative stress in pancreatic islets of Langerhans could be observed. Further, 16-week-old pancreatic islets of Langerhans were stained with heme oxygenase expressed by oxidative stress and compared. This enzyme is expressed when subjected to oxidative stress, and the expressed enzyme is stained brown. The results are shown in FIG. 12 (10 weeks) and FIG.
3 (16 weeks) is shown with a photograph instead of a drawing. The upper side of each figure (the right side when the binding side is up) is for the probucol administration group, and the lower side (the left side when the binding side is up) is the control group. As a result, in the probucol administration group, reduction of oxidative stress in the pancreatic islets of Langerhans was observed.

FIGS. 14 and 15 show the results of weight change and feed intake of both groups during the test period, respectively. The horizontal axis of FIG. 14 and FIG. 15 shows the breeding period (week),
The vertical axis in FIG. 14 indicates body weight (g), and the vertical axis in FIG. 15 indicates feed intake (g / day). In FIGS. 14 and 15, the gray areas indicate the control (control group), and the black areas indicate the probucol-administered group. As a result, between the two groups, there was no significance in the amount of food consumed and body weight throughout the entire process.

From the above results, it was shown that probucol protects pancreatic β cells and reduces glucose tolerance by reducing oxidative stress and lipid toxicity (Lipotoxicity) of β cells. These results once again show the involvement of oxidative stress in β-cell damage in type 2 diabetes and show the usefulness of probucol administration in the treatment of type 2 diabetes.

[0018]

As described above, the present invention provides a therapeutic or preventive agent for diabetes comprising probucol or a salt or solvate thereof. The present invention also provides a β-cell protective agent comprising probucol or a salt or solvate thereof. The β-cell protective action of the present invention includes an action by reducing the amount of fat accumulated in β-cells. In addition, the β cell protective action of the present invention includes an action of suppressing oxidative stress of β cells. Therefore, the present invention also provides an agent for decreasing the amount of fat accumulated in β-cells and an agent for suppressing oxidative stress of β-cells, comprising probucol or a salt thereof or a solvate thereof.

Probucol, the active ingredient of the present invention, comprises:
It is a known substance already used clinically (for example, manufactured and sold under a name such as “Shinrestal” (registered trademark)), and obtained by a known method,
Can be manufactured. Probucol, which is an active ingredient of the present invention, can be used as it is, but may be used as a salt such as a sodium salt thereof.
It may be used in the form of a solvate such as a hydrate.

The active ingredient of the present invention can be used for treating and preventing diabetes, and the target diabetes is preferably type 2 diabetes. It may be due to any factor such as a genetic factor or an extrinsic factor, and is useful for the treatment and prevention of diabetes due to a decrease in the number and function of β cells.

The medicament of the present invention may be administered orally or parenterally, but oral administration is preferred. As the dose, in the mouse experiment described above,
Since the administration was about 50 mg / day for a body weight of 20 to 40 g, this was converted to a human dose of about 750 mg / day. Therefore, although it depends on the actual condition and condition of the patient, it is usually 0.
About 1 to 2000 mg / day, about 0.1 to 1000 mg / day, or about 1 to 1000 mg / day is preferable. In addition, in the above-described mouse experiments, even when the active ingredient of the present invention was administered, no significant change in body weight or feed intake was observed. No toxicity is found in the active ingredient of the present invention in the range.

The active ingredient of the present invention can be suitably formulated according to the administration method. It can be formulated by ordinary methods such as tablets, solutions, emulsions and the like. If necessary, the preparation can be made into a sustained-release preparation by an ordinary method.

[0023]

Next, the present invention will be described in more detail with reference to specific examples, but the present invention is not limited to these specific examples and test examples.

Example 1 (breeding of mice) C57BL / KsJ-d which is a type 2 diabetes model animal
b / db mouse (Clear Japan
n (Tokyo))) were divided into 20 animals per group, and from 6 weeks of age, each group was given a diet containing 1% probucol freely without restriction (probucol group). The control group was also given normal food ad libitum without restriction. At 10 and 16 weeks, glucose tolerance (intraperitoneal glucose tolerance test) was evaluated and morphological and functional analysis of pancreatic tissue was performed.

Example 2 (Intraperitoneal glucose tolerance test) After fasting overnight, 1 g of glucose per 1 kg of body weight was intraperitoneally administered. After a certain period of time, blood was collected, and the blood glucose level was measured using a glucose analyzer (Termo, Tokyo). The results are shown in FIGS. In addition, the insulin concentration in plasma was measured from the collected blood using a Labis radioimmunoassay kit (Shibagoat (Gunma)). 1 without result
And FIG.

Example 3 (Measurement of Accumulated Fat in Pancreatic Islet) After fasting for 12 hours, the abdomen was opened under anesthesia, the duodenal orifice of the common bile duct was ligated, and collagenase was injected to isolate the islet. Thereafter, a fraction containing neutral fat was extracted by treating with hypertonic saline, and the neutral fat content was measured using a TG analysis kit (Kainos (Tokyo)). FIG. 7 shows the results.

Example 4 (Histological analysis) After fasting for 12 hours, the abdomen was opened under anesthesia to remove the pancreas,
After formalin fixation, paraffin embedding was performed to prepare sections. Immunohistological staining was performed using an anti-insulin antibody, an anti-4-hydroxynonenal antibody, or an anti-heme oxygenase antibody. The results are shown in FIGS.

[0028]

EFFECT OF THE INVENTION The present invention relates to β-
It is intended to provide a novel drug capable of protecting β cells and improving β cell functions by suppressing oxidative stress of cells and / or reducing the amount of accumulated fat. The medicament of the present invention is effective in treating and preventing diabetes, particularly type 2 diabetes. Accordingly, the present invention provides a therapeutic / prophylactic agent for diabetes comprising probucol or a salt or solvate thereof, a method for treating diabetes using the same, and a use thereof for producing a therapeutic / prophylactic agent for diabetes. .

[Brief description of the drawings]

FIG. 1 shows the results of plasma insulin concentration at 10 weeks. The horizontal axis indicates the time (minutes) after loading, and the vertical axis indicates the amount of insulin secretion (pmol / l). In each figure, a white square (□) indicates a probucol administration group, and a black circle (●)
Indicates a control (control group).

FIG. 2 shows the results of plasma insulin concentration at 16 weeks. The horizontal axis indicates the time (minutes) after loading, and the vertical axis indicates the amount of insulin secretion (pmol / l). In each figure, a white square (□) indicates a probucol administration group, and a black circle (●)
Indicates a control (control group).

FIG. 3 shows the results of blood glucose levels at 10 weeks.
The horizontal axis indicates the time (minutes) after loading, and the vertical axis indicates the amount of insulin secretion (pmol / l). In each figure, a white square mark (□) indicates a group treated with probucol, and a black circle mark (●) indicates a control (control group).

FIG. 4 shows blood glucose results at 16 weeks.
The horizontal axis indicates the time (minutes) after loading, and the vertical axis indicates the amount of insulin secretion (pmol / l). In each figure, a white square mark (□) indicates a group treated with probucol, and a black circle mark (●) indicates a control (control group).

FIG. 5 shows the results of free fatty acids (FFA) at 16 weeks (n = 8). The vertical axis is free fatty acids (FFA)
, The gray part indicates the control (control group), and the black part indicates the probucol-administered group.

FIG. 6 shows plasma neutral fat (TG) and total cholesterol (T. Cho) at 16 weeks (n = 8).
l. ) Shows the results. The vertical axis indicates the respective amount (mg / d
l), the gray part indicates a control (control group),
The black part indicates the probucol administration group.

FIG. 7 shows DNA1n in Langerhans island.
The result (n = 4) of content (μg) of neutral fat (TG) per g is shown. The vertical axis indicates the amount of TG (μg / ng-DN)
A), the gray part indicates a control (control group),
The black part indicates the probucol administration group.

FIG. 8 is a photograph instead of a drawing, showing the results of a comparison between pancreatic islets of Langerhans at 10 weeks of age and insulin staining. The lower side is that of the control group, and the upper side is that of the probucol administration group.

FIG. 9 is a photograph instead of a drawing, showing the results of insulin staining of pancreatic islets of Langerhans of 16-week mice, which were compared with each other. The lower side is that of the control group, and the upper side is that of the probucol administration group.

FIG. 10 is a photograph instead of a drawing, showing the results of 4-HNE staining of pancreatic islets of Langerhans of mice at 10 weeks of age. The lower side is that of the control group, and the upper side is that of the probucol administration group.

FIG. 11 is a photograph, instead of a drawing, showing the results of 4-HNE staining of pancreatic islets of Langerhans from 16-week mice. The lower side is that of the control group, and the upper side is that of the probucol administration group.

FIG. 12 is a photograph instead of a drawing, showing the results of staining and comparing heme oxygenase (Heme Oxygenase) expressed by oxidative stress in the pancreatic islet of Langerhans of a 10-week mouse. The lower side is that of the control group, and the upper side is that of the probucol administration group.

FIG. 13 is a photograph instead of a drawing, showing the results of staining and comparing heme oxygenase (Heme Oxygenase) expressed by oxidative stress in pancreatic islets of Langerhans at 16 weeks of mice. The lower side is that of the control group, and the upper side is that of the probucol administration group.

FIG. 14 shows the results of weight change of both groups during the test period. The horizontal axis shows the breeding period (week), the vertical axis shows the body weight (g), the gray part shows the control (control group), and the black part shows the probucol administration group.

FIG. 15 shows the results of feed intake of both groups during the test period. The horizontal axis indicates the breeding period (weeks), the vertical axis indicates the feed intake (g / day), the gray part indicates the control (control group), and the black part indicates the probucol administration group.

Claims (5)

[Claims] 1. An agent for treating and preventing diabetes comprising probucol or a salt or solvate thereof. 2. The method according to claim 1, wherein the diabetes is type 2 diabetes. 3. A β-cell protective agent comprising probucol or a salt or solvate thereof. 4. The β-cell protective agent according to claim 3, wherein the β-cell protective action is an action of reducing the amount of fat accumulated in β-cells. 5. The β-cell protecting agent according to claim 3, wherein the β-cell protecting effect is a β-cell oxidative stress suppressing effect.