HK1060311B - Preventives and remedies for complications of diabetic - Google Patents

Description

Preventive and therapeutic agent for diabetic complications

Technical Field

The present invention relates to a preventive and therapeutic agent for diabetic complications, which contains a compound having an inhibitory activity against 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase as an active ingredient. In particular, it relates to a pharmaceutical agent for preventing and treating the onset and progression of diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and diabetic vascular disorder.

Background

It is known that diabetes is complicated with complications such as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic vascular disorder, and the like, and strict blood sugar control is required for the prevention and treatment thereof. In these complications, tissue fibrosis or calcification is found. In a hyperglycemic state, glycated proteins are produced as a cell function regulator, or intracellular polyol channels are activated to accumulate sorbitol, intracellular Proteases (PKC) are activated, which causes dysfunction of glomerular cells, nerve cells or vascular endothelial cells, and induces accumulation or calcification of extracellular matrix.

Extracellular matrix known as hyperfunction expression in diabetes is type IV collagen or fibronectin (Cagliero E.et al.: J.Clin.invest., 82, 735-containing 738 (1998); Haneda M.et al.: Diabetologia, 34, 198-containing 200 (1991); DoiT.Et. et al.: Proc.Natl.Acad.Sci.USA, 89, 2873-containing 2877(1992)), and recently, it has been newly reported that osteopontin expression in kidney and blood vessel is significantly promoted in diabetic state, which is associated with diabetic nephropathy or diabetic vascular disorder (Takekmoto M.et al.: Arterio scaler. Thromb.Vasc. biol., 20, 624-containing 628 (2000); Takemomoto M.et al.: Ann.NYAcad.Sci., 357, 2000)). From these facts, it is expected that inhibition of expression of extracellular matrix fibronectin which is accelerated in the kidney or blood vessel in the diabetic state will have a preventive effect on onset or development of diabetic nephropathy or diabetic vascular disorder.

At present, as a medicament for preventing and treating diabetic complications such as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and diabetic vascular disorder, a medicament for regulating the nature of tissue disorders such as expression and production of extracellular matrix including osteopontin has not been found, and a medicament having an excellent therapeutic effect on diabetic complications is expected.

However, compounds having an inhibitory activity against HMG-CoA reductase which have been known so far have, in addition to a cholesterol synthesis inhibitory activity, cell proliferation inhibitory activity, cell binding inhibitory activity, intimal thickening inhibitory activity, and prevention and treatment of osteoporosis. In addition, inhibition of endothelial injury in carotid arteries induced accumulation of fibronectin at intimal hypertrophy sites of neointima (Kitahara M.et al: Jpn.J. Pharmacol., 77, 117-. However, the effect on osteopontin expression is not known.

The purpose of the present invention is to provide a drug which can prevent and treat diabetic complications such as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and diabetic vascular disorder by inhibiting the expression of osteopontin in diabetic nephropathy or blood vessels.

Disclosure of Invention

In view of the above-described situation, the present inventors administered a compound HMG-CoA reductase inhibitor represented by formula (1) represented by (+) -bis { (3R, 5S, 6E) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolyl ] -3, 5-dihydroxy-6-heptenoic acid } -calcium (hereinafter, referred to as pitavastatin calcium) to Streptozotocin (STZ) -induced diabetic rats, and studied the influence on the expression level of osteopontin mRNA in the kidney and blood vessels in detail. As a result, it was found that the compound represented by formula (1) or its lactone exhibits a significant osteopontin mRNA expression inhibitory effect and is therefore useful as a preventive and therapeutic agent for diabetic complications, leading to the completion of the present invention.

That is, the present invention provides a preventive and therapeutic agent for diabetic complications, which comprises a compound represented by the formula (1) or a lactone thereof as an active ingredient.

(wherein R represents an organic group and X represents-CH)₂CH₂-or-CH ═ CH-, M represents a hydrogen atom, C_1-10Alkyl groups or physiologically tolerated cationic groups. )

The invention also provides the application of (+) -bis { (3R, 5S, 6E) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolyl ] -3, 5-dihydroxy-6-heptenoic acid } -calcium in preparing medicaments for treating diabetic nephropathy, diabetic retinopathy and diabetic vascular disorder.

Brief description of the drawings

FIG. 1(a) is a bar graph showing the effect of pitavastatin calcium affecting protein secretion of osteopontin in the culture supernatant of rat aortic smooth muscle cells cultured at normal glucose concentration. FIG. 1(b) is a bar graph showing the effect of atorvastatin affecting protein secretion of osteopontin in the culture supernatant of rat aortic smooth muscle cells cultured at normal glucose concentration.

FIG. 2(a) is a bar graph showing the effect of mevalonate addition on the inhibition of intracellular osteopontin mRNA expression in rat aortic smooth muscle cells cultured at normal glucose concentrations by pitavastatin calcium. FIG. 2(b) is a bar graph showing the effect of mevalonate addition on inhibition of protein secretion by osteopontin in the culture supernatant of rat aortic smooth muscle cells cultured at normal glucose concentration.

Detailed Description

The present invention will be described in detail below.

The compounds represented by the formula (1) or their lactones are known as HMG-CoA reductase inhibitors, but it is not completely clear whether these compounds are useful agents for inhibiting osteopontin expression and treating diabetic complications.

The compounds represented by the formula (1) or their lactones are described in, for example, U.S. Pat. No. 4739073 and european patent No. 114027; european patent application publication No. 367895; U.S. patent nos. 5001255, 4613610, 4851427, 4755606, 4808607, 4751235, 4939159, 4822799, 4804679, 4876280, 4829081, 4927851, 4588715; and f.g. katawala, Medical Research Reviews, 11, 121-; european patent application publication No. 324347; european patent application publication No. 300278; united states patent nos. 5013749, 5872130 and 58563236, 4231938, 4444784, 4346227, 5354772, 4346227, 5354772, 5273995, 5177080, 3983140, 2648897, 5260440 or Bioorganic & Medicinal Chemistry, 5, pp437 (1997), 2569746, 304063 or 585656563236.

Particularly lovastatin described in U.S. Pat. No. 4231938, simvastatin described in U.S. Pat. No. 4444784, pravastatin described in U.S. Pat. No. 4346227, fluvastatin described in U.S. Pat. No. 5354772, atorvastatin described in U.S. Pat. No. 5273995, cerivastatin described in U.S. Pat. No. 5177080, mevastatin described in U.S. Pat. No. 3983140, and rosuvastatin (rosuvastatin) described in Japanese patent No. 2648897, U.S. Pat. No. 5260440, or Bioorganic & Medicinal Chemistry, 5, pp437, (1997), namely, monocalcium di (+) -7- [4- (4-fluorophenyl) -6-isopropyl-2- (N-methyl-N-methanesulfonamidopyrimidin-5-yl ] - (3R, 5S) -dihydroxy- (E) -6-heptenoate. Similarly, pitavastatin calcium is described in japanese patent No. 2569746, european patent No. 304063, or us patent No. 5856336.

In the above formula (1), the organic group represented by R is preferably a group having a ring structure selected from an indolyl group, an indenyl group, a pyridyl group, a pyrrolopyridyl group, a pyrazolopyridyl group, a thienopyridyl group, a pyrimidinyl group, a pyrazolyl group, a pyrrolyl group, an imidazolyl group, an indolizinyl group, a quinolyl group, a naphthyl group, a hexahydronaphthyl group, a cyclohexyl group, a phenylsilylphenyl group, a phenylthienyl group and a phenylfuryl group. Among these cyclic organic groups, hexahydronaphthyl, indolyl, pyridyl, pyrimidyl, pyrrolyl and quinolyl groups are particularly preferable. These cyclic structures may have a hydroxyl group or C_1-10Alkyl (including linear, branched, cyclic), alkoxyalkyl, alkylcarbonyloxy, substituted amino, substituted sulfamoyl, halophenyl, phenyl, and like substituents, and particularly preferred is a cyclic organic group having an isopropyl group, a cyclopropyl group, and a p-fluorophenyl group. The physiologically acceptable salts of the compounds represented by the formula (1) are selected from alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and sodium salts, organic ammonium salts and ammonium salts such as phenethylamine salts, and sodium salts and calcium salts are more preferable.

Further, among the above compounds, compounds having an effect of HMG-CoA reductase inhibitors represented by lovastatin, pravastatin, simvastatin, fluvastatin, cerivastatin (serivastatin), atorvastatin, rosuvastatin and pitavastatin calcium are selected. Among them, pitavastatin calcium is particularly preferable.

As shown in examples described later, the compounds represented by the formula (1) significantly inhibit the STZ-induced osteopontin gene expression in the kidney and blood vessels of diabetic rats and the osteopontin gene expression in cultured vascular smooth muscle cells of rats. Therefore, the compounds represented by the formula (1) or their lactones have an effect of preventing and treating diabetic complications such as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and diabetic vascular disorder by inhibiting the expression of osteopontin. By using the compound of the present invention, it is possible to prevent and treat complications caused by excessive osteopontin expression due to diabetes, and to develop a novel experimental system related to osteopontin gene expression, screen a novel medicine, and the like.

Examples of administration forms when the compound of the present invention is used as a medicine include oral administration such as tablets, capsules, granules, powders, and syrups, and non-oral administration such as intravenous injection, intramuscular injection, transdermal absorption agents, suppositories, inhalants, eye drops, and nasal drops. In the preparation of pharmaceutical preparations of various dosage forms, the active ingredient is used alone or in combination with other pharmaceutically acceptable excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, corrigents, perfumes, coating agents, carriers, diluents, and the like, as appropriate.

Among these administration methods, oral administration is preferred, and for oral preparations, for example, the method described in Japanese patent laid-open No. 2-6406, patent No. 2774037, and WO97/23200, in which pH is adjusted to adjust the pH, is preferred in view of the stability of the active ingredient.

The dose for medical use of the present invention varies depending on the body weight, age, sex, symptoms and the like of a patient, and in the case of an adult, the compound represented by formula (1) is usually administered in an amount of 0.01 to 100mg per day, and particularly preferably 0.1 to 10mg 1 or 2 times per day.

Examples

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1: streptozotocin (STZ) -induced inhibition of renal and vascular osteopontin mRNA expression in diabetic rats

The effect of pitavastatin calcium on Streptozotocin (STZ) -induced renal and vascular osteopontin mRNA expression in diabetic rats was determined by the following method.

That is, Wistar rats (body weight: about 300g) were injected with 35mg of STZ dissolved in physiological saline at a concentration of 50mg/mL through the tail vein per 1kg of body weight, and after about 1 hour, a 0.5% carboxymethylcellulose solution (3mg/kg) of the drug to be tested (pitavastatin calcium) was forcibly orally administered at a weight of 1mL (3mg/kg) per 1kg of body weight using a gastric probe. Then, the same amount of the drug was forcibly orally administered 1 time a day. In the control group, only the same amount of 0.5% carboxymethyl cellulose solution was forcibly administered orally. On the 2 nd day of the test, it was confirmed that the blood glucose level obtained by collecting blood through the tail vein was 200mg/dL or more.

After 24 hours of 7-day administration, blood was collected under ether anesthesia, and the kidney and the thoracic aorta were removed. A predetermined amount of the tissue pieces were disrupted by a polytron homogenizer from ISOGEN (Wako pure chemical industries, Ltd.) to extract total RNA. The resulting total RNA was precipitated with isopropanol. The precipitate was washed with ice-cold 70% ethanol and stored at-80 ℃ with 70% ethanol.

Using the total RNA obtained, osteopontin mRNA was detected by the RNA transfer blotting technique (Northern blotting method) according to the assay method. That is, the total RNA precipitated at 70% was centrifuged at 15000 rpm, the supernatant was removed, dried at room temperature, and dissolved in a small amount of TE buffer (10mM Tris-HCl buffer-1 mM EDTA solution). Mu.l of the resulting solution was diluted with 990. mu.l of TE buffer, and the amount of RNA was calculated by measuring the absorbance of the solution at 260nm ultraviolet light. To a certain amount (10 or 20. mu.g) of total RNA (final volume: 6. mu.l) was added a 40% aqueous solution (3.5. mu.l) of deionized glyoxal) 0.1M NaHPO₄The buffer (2.4. mu.l) and dimethyl sulfoxide (11.8. mu.l) were heated at 50 ℃ for 1 hour to denature the RNA. After cooling at room temperature, 6.3. mu.l of a 10mM sodium phosphate buffer (pH: 6.8) containing 50% glycerol and 0.4% bromophenol blue was added to the solution, and the mixture was subjected to electrophoresis using 1.5% agarose gel. According to the protocol, RNA was blotted from an agarose gel onto a nylon membrane using 20 × standard saline-citric acid buffer (SSC). After washing the nylon membrane with 2 XSSC, the RNA was immobilized on the nylon membrane by heating to 80 ℃ under vacuum. The osteopontin partial DNA fragment was excised from pCRIIrOP vector using Eco R-I restriction enzyme, and the resulting fragment was digested with ProbeQuant^TMG-50Micro Columns (Amersham pharmacia Biotech Co., Ltd.) was purified. The obtained osteopontin partial DNA fragment was used by rediprime^TM(Amersham Pharmacia Biotech Co., Ltd.)³²P-labeled probe, hybridized with nylon membrane overnight at 65 ℃. Radioactivity of the probe bound to the nylon membrane was detected by an X-ray membrane, and the bond strength was calculated using NIH image. As an internal standard control RNA, 18 strrna (18S) was used, and the expression level was calculated from the bond strength ratio. Osteopontin mRNA was determined in the same manner in normal rat experiments.

Table 1 shows the results of example 1.

In table 1, opnmna/18S represents the ratio of the bond strength of osteopontin mRNA to the bond strength of 18StRNA calculated from NIH image, and the% inhibition rate represents the inhibition rate for each control group. Values for OPNmRNA/18S represent the mean. + -. standard deviation of 3 animals.

TABLE 1

	Kidney (A)	Aorta with superior arterial resistance
			OPNmRNA/18S% inhibition	OPNmRNA/18S% inhibition
	Normal control group normal administration group	1.343±0.4621.667±0.321 -24.1			2.400±1.3452.433±0.929 -1.4
Diabetes administration group for diabetes control group			3.233±0.1151.933±0.874* 40.2	3.200±0.3611.300±0.781** 59.4

*: significantly worse risk than control; p is 0.016

**: significantly worse risk than control; p is 0.036

OPN: osteopontin

The ratios of osteopontin mRNA to 18S in kidney and aorta rose from 1.343 to 3.233 and from 2.400 to 3.200, respectively, in streptozotocin-induced diabetic rats. Pitavastatin calcium had no effect on osteopontin expression in the kidney and aorta of normal rats (1.667 and 2.433, respectively), but the STZ-induced osteopontin mRNA expression in the kidney and aorta of diabetic rats was significantly reduced to 1.933 (inhibition rate: 40.2%) and 1.300 (inhibition rate: 59.4%), respectively.

Example 2: inhibition of protein secretion of osteopontin in rat aortic smooth muscle cells

The protein secretion effect of pitavastatin calcium and atorvastatin on osteopontin in the culture supernatant of rat aortic smooth muscle cells cultured at normal glucose concentration was measured by the following method.

First, rat aortic smooth muscle cells were cultured (parentage)Passage 5-10) were plated onto 6-well culture dishes and plated with a plate containing 10% fetal bovine serum (FBS: BioWhittaker Co.) Low glucose (1000mg/L) Dulbecco's Modified Eagle's Medium (DMEM) in 5% CO₂And confluent culture at 37 deg.C. Then, the medium was replaced with the same medium to which the test drugs (pitavastatin calcium and atorvastatin) were added, and the medium was incubated for 48 hours. Each 1 well was replaced with 1.5mL of the same medium containing no FBS, and the culture was further continued for 48 hours, and the supernatant was recovered. To a fixed amount (0.5 to 1mL) of this solution, 0.14M common salt and 50mM Tris hydrochloric acid buffer (pH7.4) were added in equal amounts, and then 50. mu.L of anion-exchanged DEAE cellulose suspended at a concentration of 50% and swollen and equilibrated with the buffer and DE52 (manufactured by Whatman) were added, and the mixture was gently stirred at 4 ℃ for 1 hour to adsorb osteopontin to DE 52.

After centrifugation, the settled DE52 gel was washed several times with this buffer, and then 60. mu.l of 0.2M Tris hydrochloric acid buffer (pH6.8) containing 5% 2-mercaptoethanol, 4% SDS, 5mM EDTA, 20% glycerol, and 0.01% bromophenol blue was added thereto, followed by heat treatment at 95 ℃ for 5 minutes. After cooling at room temperature, centrifugation was carried out, and a certain amount (30. mu.L) of the supernatant was subjected to 10% SDS polyacrylamide gel electrophoresis. After the migration, the migration protein was transferred onto a nitrocellulose membrane according to the protocol, and western blotting (western blotting) was performed according to the protocol. Specifically, the nitrocellulose membrane was shaken in TBS-T containing 3% bovine serum albumin (Tris-buffered saline containing 0.2% Tween-20) for 1 hour or more, and then shaken in an anti-osteopontin antibody (MPIIIB1O 1; manufactured by American Research Products) solution diluted with 1/1000 in the same solution for 1 hour. Further, after shaking for 1 hour in a solution in which an anti-mouse IgG antibody was bound with horseradish peroxidase (Horseradish peroxidase) diluted to 1/5000 with TBS-T containing 3% bovine serum albumin, the solution was washed with TBS-T several times. Using ECL^TMChemiluminescence was detected by an X-ray membrane (Amersham pharmacia Biotech). The bond strength was calculated using NIH image.

The above measurements were performed at concentrations of 0 (control), 0.03 and 0.3 μ M for pitavastatin calcium and at concentrations of 0 (control), 0.3 and 3 μ M for atorvastatin, respectively.

Fig. 1 shows the results of example 2.

FIG. 1(a) shows the bond strength of osteopontin calculated with NIH image at various concentrations of pitavastatin calcium, and FIG. 1(b) shows the bond strength of osteopontin calculated with NIH image at various concentrations of atorvastatin.

As can be seen from fig. 1, both pitavastatin calcium and atorvastatin significantly reduced the protein secretion amount of osteopontin in the culture supernatant of rat cultured aortic smooth muscle cells.

Example 3: effect of mevalonic acid on osteopontin mRNA expression and osteopontin protein secretion inhibition of rat aortic smooth muscle cells

The effect of mevalonate on inhibition of osteopontin mRNA expression in rat aortic smooth muscle cells cultured at normal glucose concentration and protein secretion of osteopontin protein in culture supernatant by pitavastatin calcium was determined by the following method.

Rat aortic smooth muscle cells (passage cultured for 5-10 passages) were plated on 6-well culture dishes in 10% FBS-containing low glucose (1000mg/L) DMEM at 5% CO₂And confluent culture at 37 deg.C. Then, the medium was replaced with the same medium supplemented with pitavastatin calcium (8. mu.M) and/or mevalonate (100. mu.M), and cultured for 48 hours. 1.5mL of the same medium without FBS was replaced per 1 well and incubated for another 48 hours.

After culturing, the cells attached to the culture dish were homogenized with ISOGEN, RNA was extracted as in example 1, and the amount of osteopontin mRNA was determined by northern blotting.

The culture supernatant was collected, and osteopontin was adsorbed on DE52, followed by electrophoresis in the same manner as in example 2 to measure the protein secretion amount of osteopontin by Western blotting.

The above measurements were performed without addition of pitavastatin calcium and mevalonate, with addition of only pitavastatin calcium, and with addition of pitavastatin calcium and mevalonate.

Fig. 2 shows the results of example 3.

FIG. 2(a) shows the ratio of the bond strength of osteopontin mRNA to the bond strength of 18StRNA calculated based on NIH image in the above 3 cases. FIG. 2(b) shows the calculated protein bond strengths of osteopontin based on NIH image in the above 3 cases.

FIG. 2 shows that although pitavastatin calcium inhibited osteopontin mRNA expression and protein secretion of osteopontin from rat cultured smooth muscle cells, the inhibitory effect of these pitavastatin calcium was abolished by addition of mevalonate. It is thus concluded that mRNA expression and protein secretion of osteopontin in aortic smooth muscle cells are inhibited by the addition of pitavastatin calcium to inhibit the production of mevalonate.

Industrial applicability

The compound represented by the formula (1) of the present invention shows a specific and effective inhibitory action on osteopontin expression which is accelerated in the kidney and aorta in a diabetic state without affecting osteopontin expression in a normal state, and significantly inhibits osteopontin production in these organs in a diabetic state.

Therefore, the compound represented by the formula (1) is useful as a prophylactic and therapeutic agent for diabetic complications associated with increased osteopontin expression, such as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and diabetic vascular disorder.

Claims (3)

1. Use of (+) -bis { (3R, 5S, 6E) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolinyl ] -3, 5-dihydroxy-6-heptenoic acid } -calcium for the manufacture of a medicament for the treatment of diabetic nephropathy.
2. Use of (+) -bis { (3R, 5S, 6E) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolinyl ] -3, 5-dihydroxy-6-heptenoic acid } -calcium for the manufacture of a medicament for the treatment of diabetic retinopathy.
3. Use of (+) -bis { (3R, 5S, 6E) -7- [ 2-cyclopropyl-4- (4-fluorophenyl) -3-quinolinyl ] -3, 5-dihydroxy-6-heptenoic acid } -calcium for the manufacture of a medicament for the treatment of diabetic vascular disorder.