WO2002002553A1

WO2002002553A1 - Pharmaceutical compositions for treatment of diabetic nephropathy

Info

Publication number: WO2002002553A1
Application number: PCT/JP2001/005743
Authority: WO
Inventors: Atsuo Tahara; Takeshi Suzuki; Takeyuki Yatsu; Yuichi Tomura; Junko Tsukada
Original assignee: Yamanouchi Pharmaceutical Co., Ltd.
Priority date: 2000-07-04
Filing date: 2001-07-03
Publication date: 2002-01-10
Also published as: AU2001267911A1

Abstract

Pharmaceutical compositions for the treatment of overt and/or early diabetic nephropathy, containing as the active ingredient (Z)-4'-{4,4-difluoro-5-[2-oxo-2-(4-piperidino- piperidino)ethylidene]-2,3,4,5-tetrahydro-1H-1-benzazepine-1-carbonyl}-2-methyl-3-furanilide or pharmaceutically acceptable salts thereof.

Description

Description Pharmaceutical composition for the treatment of diabetic nephropathy Technical field

The present invention relates to a therapeutic agent for diabetic nephropathy, particularly a therapeutic agent for overt diabetic nephropathy and early diabetic nephropathy. Background art

Diabetic nephropathy is a type of microangiopathy caused by a chronic hyperglycemic state, which causes microalbuminuria to show signs of proteinuria, renal dysfunction, hypertension, edema, etc., leading to renal failure. The disease progresses through five stages: early nephropathy, early nephropathy, overt nephropathy, renal failure, and dialysis therapy. In general, diabetic nephropathy progresses relatively slowly in the early stages, and its lesions are also reversible.However, in the overt period in which proteinuria is positive, the lesions become irreversible and rapidly decrease renal function. End-stage renal failure (Di abetes, 46, S104-S111, 1997).

Characteristics of changes in renal function in the early stages of diabetes include microalbuminuria and glomerular hyperfiltration (Am. J. Med., 72, 375-380, 1982 / Br. Med. J., 4, 257-259, 1972). In early diabetic nephropathy, an increase in glomerular pressure is thought to be deeply involved in the progression of nephropathy, and it is well known that the glomerular hyperfiltration theory that diabetic nephropathy is caused by abnormal glomerular hemodynamics. ing. That is, glomerular hyperfiltration caused by a rise in glomerular pressure caused by changes in glomerular import arteriole and export arteriole resistance precedes renal tissue damage, which causes albuminuria and diabetic nephropathy. It is considered to trigger the onset of the disease (Am. J. Med., 80, 443-453, 1986b).

On the other hand, overt nephropathy is a stage in which proteinuria is clinically recognized, and in many cases, renal function (glomerular filtration value) has already started to decrease. The appearance of proteinuria in diabetic patients was considered to progress to renal failure over the course of a few years, making the disease difficult to treat, that is, difficult to treat. Treatment of diabetic nephropathy is based on three basic treatments: (1) strict blood sugar control, (2) blood pressure control, and (3) protein restricted diet. Angiotensin converting enzyme (AC) is used for blood pressure control. E) Inhibitors and calcium antagonists are used. However, with current therapies, it is difficult to stop the exacerbation if the nephropathy progresses to a certain extent and a large amount of proteinuria appears. In addition, progression to end stage renal failure dialysis has a worse prognosis than nondiabetic renal disease.

Bardou et al. Reported that administration of streptozotocin (STZ) to vasopressin-deficient rats to induce diabetes attenuated the development of nephropathy compared to normal STZ-induced diabetic rats. These results suggest that vasopressin functions as an exacerbating factor in diabetic nephropathy in vivo and may be a therapeutic target (Proc. Natl. Acad. Sci. USA, 96: 10397-10402, 1999).

Synthetic studies on vasopressin structural analogs have been actively conducted since the 1960s, and many peptide vasopressin receptor antagonists have been synthesized. However, among those skilled in the art, clinical expectations of vasopressin receptor antagonists have been diminished due to the following problems. The problem has a ®V _1A V ₂ antagonism, not found pure V _1A antagonist or V ₂ antagonists, it is impossible to orally administered for having ② peptide structure, (3) When administered chronically, the antagonistic effect disappears, leaving only the agonist effect. (4) The species difference is large and extremely effective in rats, but almost no antagonistic effect in dogs, monkeys and humans, etc. (Therapy, Vol.81, extra number, pp.309-314, 1999).

In fact, the STZ-induced diabetic nephropathy rats, Ru known as V _1A receptor antagonist 0PC- 21268 (chemical name: 1 one [1 one [4- [3- (Asechiruamino) Purobokishi] Benzoiru] piperidine - [4-yl] —1,2,3,4-tetrahydro-2-quinolinone) in the diet and a daily intake of 250 mg / kg reduced urinary protein excretion. (Journal of the American Society of Nephrology, Vol. 9, Abst. A3284, 1998). On the other hand, in human clinical studies, a significant decrease in the urinary excretion of trace albumin was observed 60 to 90 minutes after administration of 0PC-21268 lOOmg in patients with early-stage nephropathy of non-insulin-dependent diabetes mellitus. No significant decrease was reported for 0-120 minutes after administration in patients with renal phase nephropathy. (Journal of Diabetes and Its Complications, Vol. 9, No. 4, p326-329, 1995). There is a need to create therapeutic agents that can clearly reduce urinary protein and improve renal function in overt diabetic nephropathy, and that can be used in combination with antihypertensive treatment without affecting systemic hemodynamics. ing. In addition, even in early diabetic nephropathy, a therapeutic agent that reduces urinary albumin excretion and that can correct glomerular hyperfiltration by acting specifically on glomerular hemodynamics without affecting systemic hemodynamics In other words, there is a demand for a drug capable of suppressing the progress of nephropathy. Disclosure of the invention

The inventor considers a compound having extremely high affinity and selectivity for the human _V1A receptor to be at least as high as that of a rat without being affected by species differences, if it is overt diabetic nephropathy and Z or early We thought that it could be a therapeutic drug for diabetic nephropathy, and conducted intensive research with the aim of searching for a compound having such a profile.

As a result, the present inventor found that (Z) —4, — {4,4-difluoro-5— [2-oxo-2- (4-piperidinopiperidino) ethylidene] -1,2,3,4,5— Examples of tetrahydro- 1H-1-benzazepine-111-carbyl}-2-methyl-3-furanilide (hereinafter abbreviated as "Compound A") or a pharmaceutically acceptable salt thereof are described in Examples. As shown in FIG. 1, they were found to have extremely high affinity and selectivity for the human _V1A receptor.

Furthermore, as shown in Examples 2 and 3, this compound has an excellent ameliorating effect on evening proteinuria and renal glomerular lesions in rats with overt diabetic nephropathy without affecting blood pressure. Confirmed and completed the invention.

In addition, as shown in Examples 4 and 5, this compound reduces urinary albumin excretion and suppresses renal glomerular hyperfiltration in early-stage diabetic nephropathy rats without affecting systemic blood pressure The invention has been completed after confirming that the present invention has the effect.

Furthermore, the present inventors have completed the invention by finding that only 1Z2 fumarate stably forms a single crystal form in the process of studying the industrial production of compound A or a salt thereof as a pharmaceutical raw material. . That is, the present invention relates to a pharmaceutical composition for treating overt diabetic nephropathy comprising Compound A or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention relates to an agent for improving proteinuria or an agent for improving renal function in patients with overt diabetic nephropathy. Furthermore, the present invention relates to the use of Compound A or a pharmaceutically acceptable salt thereof for the manufacture of a therapeutic agent for overt diabetic nephropathy. Furthermore, the present invention relates to a method for treating overt glycemic nephropathy, which comprises administering to a patient a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof.

Further, the present invention relates to a pharmaceutical composition for treating early-stage diabetic nephropathy comprising Compound A or a pharmaceutically acceptable salt thereof as an active ingredient, and more particularly, an agent for improving albuminuria in patients with early-stage diabetic nephropathy Or an agent for improving renal glomerular hyperfiltration in patients with early-stage diabetic nephropathy. Furthermore, the present invention relates to the use of Compound A or a pharmaceutically acceptable salt thereof for the manufacture of a therapeutic agent for early-stage diabetic nephropathy. Furthermore, the present invention relates to a method for treating early-stage diabetic nephropathy, comprising administering to a patient a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention relates to a pharmaceutical composition for inhibiting the progress of diabetic nephropathy, comprising Compound A or a pharmaceutically acceptable salt thereof as an active ingredient. Furthermore, the present invention relates to the use of Compound A or a pharmaceutically acceptable salt thereof for the manufacture of an agent for suppressing the progress of diabetic nephropathy. Furthermore, the present invention relates to a method for suppressing the progress of diabetic nephropathy, comprising administering a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof to a patient. Furthermore, the present invention relates to a novel compound A · 1/2 fumarate.

'Compound A is a compound having a vasopressin receptor antagonistic activity, and a method for producing its hydrochloride is disclosed in Published Technical Report No. 97-9952.

In addition, International Publication No. WO 95/0655 discloses a _two- receptor binding test of rat and rabbits, and discloses a compound represented by the following general formula (I). V ₂ receptor has an antagonistic action, renal diseases based on these findings prevention (nephrosis, nephritis, diabetic nephropathy, chronic or acute renal failure) and many other diseases and Z or Osamu Although it is suggested that it may be useful for treatment, no specific experiment is described for each disease. Further, Compound A or a salt thereof is not specifically disclosed.

(Refer to the gazette for symbols in the formula)

The present inventor has, among the myriad of compounds included in the above formula (I), especially of compound A or salt thereof affinity-selectivity for V _{1 A} receptor of human is extremely excellent Was found to be an antagonist. In addition, Compound A or a salt thereof is effective in overt diabetic nephropathy at an extremely low dose of about 1% of the dose at which OPC-21268 reduced urinary protein excretion in STZ-induced diabetic nephropathy rats. It has been confirmed that proteinuria and renal glomerular lesions in rats are improved, and albuminuria and glomerular hyperfiltration in rats with early-stage diabetic nephropathy are improved, and the present invention has been completed based on the results. The medicament of the present invention is a remarkably excellent therapeutic agent for overt diabetic nephropathy, a therapeutic agent for early diabetic nephropathy, and an agent for inhibiting the progress of diabetic nephropathy.

In addition, since the 1Z2 fumarate of compound A does not form a polymorph in crystallization from a water-ethanol system of less than 10%, it is considered that there are few problems concerning stability and solubility. In general, when a compound having a crystalline polymorph (free form or salt) is used as a drug substance, if the drug substance contains more than one crystal form, the stability and solubility will vary, so the drug substance will not be dispersed. Standards cannot be guaranteed. It may also affect the pharmacokinetics of the drug and even its efficacy as a drug. Therefore, the 1/2 fumarate salt of compound A, which does not have a crystalline polymorph, is excellent as a pharmaceutical raw material in that a stable drug effect can be expected. Hereinafter, the present invention will be described in more detail, Compound A can form pharmaceutically acceptable acid and base addition salts with a wide variety of inorganic and organic acids or bases. Such salts also form part of the invention. For example, salts with inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, salts with organic acids such as fumaric acid, malic acid, citric acid, succinic acid, and mesylic acid, salts with alkali metals such as sodium and potassium, and calcium And salts with alkaline earth metals such as magnesium, and salts with organic bases such as diethanolamine. These salts can be produced by a conventional method. The compound A or a salt thereof, which is an active ingredient of the medicament of the present invention, includes a mixture of various isomers and all of its isolated, hydrated, and solvated forms. The compound A or a salt thereof, which is an active ingredient of the medicament of the present invention, includes a compound having a crystalline polymorph (free form or salt), and includes all such crystalline forms. Preferred as an active ingredient of the medicament of the present invention is a 1/2 fumarate of compound A.

Compound A or a salt thereof can be easily obtained by the production method described in the above-mentioned published technical report No. 97-9952, or by the production method of Example 1 described later, or according thereto.

The drug of the present invention is prepared as an oral solid preparation, an oral liquid preparation or an injection using an organic or inorganic carrier, excipient, and other additives suitable for oral or parenteral administration according to a conventional method. be able to. Most preferred are oral solid dosage forms that can be easily taken by patients and that are easy to store and carry.

As oral solid preparations, tablets, powders, fine granules, granules, capsules, pills, sustained-release preparations and the like are used. In such solid preparations, the one or more active substances comprise at least one inert diluent, such as lactose, mannitol, glucose, microcrystalline cellulose, starch, polyvinylpyrrolidone, aluminate metasilicate. Mixed with magnesium. The composition may be prepared in accordance with conventional practice with additives other than inert diluents, for example, binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose (HPMC), magnesium stearate, polyethylene glycol, starch, talc. Lubricants, disintegrants such as calcium cellulose glycolate, stabilizers such as lactose, solubilizing agents such as glutamic acid or aspartic acid, and coloring agents such as titanium oxide. . Tablets If necessary, the pills may be coated with a sugar coating such as sucrose, gelatin, agar, pectin, hydroxypropylcellulose, hydroxypropylmethylcellulose or the like, or a film of a gastric or enteric substance.

Oral liquid preparations include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc., and commonly used inert diluents such as purified water, ethanol, etc. Including The composition may contain, in addition to the inert diluent, auxiliaries such as wetting agents and suspending agents, sweetening agents, flavoring agents, fragrances, and preservatives.

Injections for intravenous injection, intramuscular injection, subcutaneous injection and the like include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Diluents for aqueous solutions and suspensions include, for example, distilled water for injection and physiological saline. Diluents for non-aqueous solutions and suspensions include, for example, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, alcohols such as ethanol, and polysorbate 80 Etc. Such compositions may also contain adjuvants such as preserving, wetting, emulsifying, dispersing, stabilizing (eg, lactose), and solubilizing agents (eg, glutamic acid, aspartic acid). . These are sterilized by, for example, filtration through a bacterial storage filter, blending of a bactericide or irradiation. They can also be used to produce a sterile solid composition which is dissolved in sterile water or a sterile solvent for injection before use. The dose of the compound A or a salt thereof, which is the active ingredient of the present invention, is appropriately determined depending on the individual case in consideration of the administration route, the symptoms of the disease, the age, sex, etc. of the administration subject. In the case of, the active ingredient per adult is about 1 to 50 Omg Z days, preferably 10 to 20 Omg / day, and this is orally administered once or in 2 to 4 times. .

The drug of the present invention is an insulin preparation, a sulfonylurea drug, a sulfonamide drug, a biguanide drug, an aldose reductase inhibitor, an α-dalcosidase inhibitor, an insulin sensitizer, a somatomedin C drug, and other hypoglycemic drugs. , Prostanoid-related drugs, AEC inhibitors, angiotensin Π receptor antagonists, tropoxane synthase inhibitors, vitamin Β preparations, Kampo preparations, calcium antagonists, diuretics, etc. Can be used together after a while You. Insulin preparations include insulin, neutral insulin, amorphous insulin zinc aqueous suspension, biphasic isophen insulin aqueous suspension, isophen insulin aqueous suspension, insulin zinc aqueous suspension, prominin insulin zinc aqueous suspension Aqueous suspension of crystalline zinc insulin; Toluptamide, glicloviramide, acetohexamide, tolazamide, chlorpropamide, dalipenclamide, daliclazide as sulfonylureas; dalibazole as sulfonamide; metformin hydrochloride as biguanide Buformin hydrochloride; epalrestat as an aldose reductase inhibitor; pogliose and acalcose as hypoglycosidase inhibitors; troglitazone and pioglitazone as insulin sensitizers; Medincerin as a medin C preparation; nateglinide as a hypoglycemic drug; beraprost, alprostadil alfadex, lipo-PGE limaprost alfadex as prostanoid-related drugs; enalapril maleate as an ACE inhibitor; Is oxcart kidney kimaru; and the like. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a multiplex recording diagram of a powder X-ray diffraction spectrum of a crystal of compound A · 1/2 fumarate under various crystallization conditions.

FIG. 2 shows a thermogravimetric analysis of TG-DSC of crystals of compound A · 1/2 fumarate. FIG. 3 shows the protocol of STZ-induced diabetic nephropathy rat test protocol showing overt proteinuria of Test Example 2.

FIG. 4 shows the effect of Compound A on urinary albumin excretion.

FIG. 5 shows the effect of Compound A on urinary protein excretion.

FIG. 6 shows the effect of Compound A on systolic blood pressure.

FIG. 7 shows the effect of Compound A on renal glomerular lesions.

FIG. 8 shows the protocol of the diabetic overt nephropathy rat test protocol with hypertension in Test Example 3.

FIG. 9 shows the effect of compound A on urinary albumin excretion.

FIG. 10 shows the effect of Compound A on urinary protein excretion. FIG. 11 shows the effect of Compound A on systolic blood pressure.

FIG. 12 shows the effect of Compound A on renal glomerular lesions.

FIG. 13 shows the STZ-induced diabetic early nephropathy rat test protocol showing microalbuminuria of Test Example 4.

FIG. 14 shows the effect of Compound A on urinary albumin and type 4 collagen excretion.

FIG. 15 shows the effect of Compound A on systemic and renal hemodynamics.

FIG. 16 shows the effect of Compound A on glomerular hemodynamics.

FIG. 17 shows the STZ-induced diabetic early nephropathy rat test protocol showing microalbuminuria of Test Example 5.

FIG. 18 shows the effect of Compound A on urinary albumin and type 4 collagen excretion.

FIG. 19 shows the effect of Compound A on creatinine clearance and systolic blood pressure.

FIG. 20 shows the effect of Compound A on renal glomerular lesions.

FIG. 21 shows the effect of compound A on tubular lesions.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail based on examples and test examples, but the present invention is not limited to these examples and the like.

Example 1 '

Compound A (free form)

4 1 [(2-Methylfuran-3- 3-capillonamino)] Benzoic acid (104.30 g, 426 hire ol), thionyl chloride (37.3 ml, 511 ol) and dimethylformamide (0.70 ml) A mixed solution of tetrahydrofuran (500 ml) was stirred at room temperature for 7 hours. The reaction solution was concentrated under reduced pressure, acetonitrile (aoomi) was added, and the mixture was concentrated again. A solution of the residue in acetonitrile (300 ml) was added to (Z) —4 '-{4,4-difluoro-5- [2-oxo-2- (4-piperidinopiperidino) ethylidene] -1,2,3, 4, 5—tetra Hydro- 1 H-1-Benzazepine (138.48 g, 356 bandol, published technical report No. 97-9952) in acetonitrile (1,000 ml) solution was dripped slowly under ice-cooling, and after dripping for 1 hour at room temperature. Stir and leave overnight. The resulting crystals were collected by filtration to give Compound A hydrochloride (215.37 g) as a pale brown powder. To this hydrochloride (150.10 g) were added water (400 ml), a 1 M aqueous sodium hydroxide solution (1,875 ml) and chloroform (800 ml) under ice cooling, and the mixture was stirred at the same temperature for 15 minutes. After the organic layer was separated, the aqueous layer was extracted with black-mouthed form (300 ml × 2), and the organic layers were combined and dried over anhydrous sodium sulfate. Ethyl acetate (150 ml) and hexane (300 ml) were added to the residue obtained by concentrating the solvent under reduced pressure, and the mixture was stirred at room temperature for 19 hours. The precipitated solid was collected by filtration and dried under reduced pressure at 50 ° C. for 72 hours to obtain Compound A (127.35 g) as a pale yellow powder. Example 2

(1) Compound A · 1/2 fumarate (lot 2)

Compound A (0.50 g) was dissolved in ethanol (5 ml), a fumaric acid-ethanol solution (0.25 mol / l, 1.62 ml) was added, and the mixture was allowed to stand at room temperature for 116 hours. The precipitated solid was collected by filtration, washed with a mixed solution of ethanol and ethyl acetate (1: 2), and dried under reduced pressure at 60 ° C for 168 hours to give the title compound (0.48 g) as an orange-white crystalline substance. Obtained as a powder.

(2) Recrystallization of compound A · 12 fumarate (lot 6)

Compound A · 1/2 fumarate (2.08 g) was dissolved in a mixed solvent of ethanol and water (90:10, 40 ml) under reflux with heating, and then allowed to stand at room temperature for 159 hours. The precipitated solid was collected by filtration, washed with cold ethanol, and dried under reduced pressure at 50 ° C. for 336 hours to give the title compound (1.41 g) as a dark brownish white crystalline powder. '

Immediately 239-241 ° C

1H -Sat R (DMS0-d ₆ ) δ: 1.25-1.75 (1 OH, m), 1.75-1.95 (2H, i), .35-2.90 (7H, m), 2.65 (3H, s), 2.95- 3.25 (2H, m), 3.98 (1H, d, J-12.9Hz), 4.45 (1H, d, J = 12.9Hz),

6.53 (1H, s), 6.75-6.90 (2H, m), 7.00-7.23 (4H, m), 7.29 (1H, t, J = 7.5Hz), 7.47-

7.60 (4H, m), 9.78 (1H, s).

(3) Recrystallization of compound A · 1 Z2 fumarate (lot 9), Compound A · 1/2 fumarate (2.02 g) was dissolved in a mixed solvent of ethanol and water (90:10, 43 ml) with heating under reflux, followed by stirring at room temperature for 15 hours (using a stirrer). The precipitated solid was collected by filtration, washed with cold ethanol, and dried under reduced pressure at 50 for 336 hours to give the title compound (1.57 g) as a slightly brownish white crystalline powder.

(4) Recrystallization of compound A · 1/2 fumarate (lot 12)

Compound A · 1/2 fumarate (6.02 g) was dissolved in a mixed solvent of ethanol and water (90:10, 145 ml) with heating under reflux, followed by stirring at room temperature for 15 hours (using a stirring blade). The precipitated solid was collected by filtration, washed with cold ethanol, and dried under reduced pressure at 50 ° C for 431 hours to give the title compound (4.35 g) as a brownish white crystalline powder.

(5) Recrystallization of compound A · 1Z2 fumarate (lot 15)

Compound Α · Ethanol (151 ml) was added to 1Z2 fumarate (4.llg), and the mixture was stirred at 56 ° C for 30 minutes to obtain a homogeneous solution. At the same temperature, a fumaric acid-ethanol solution (0.5 M, 13.3 ml) was added, and the mixture was stirred for 10 minutes, and then stirred at room temperature for 15 hours (using a stirring blade under any stirring conditions). The precipitated solid was collected by filtration, washed with cold ethanol, and dried under reduced pressure at 50 ° C for 288 hours to give the title compound (4.31 g) as a slightly brownish white crystalline powder. Reference example 1

Compound A hydrochloride

Compound A (0.56 g) was dissolved in methanol (6 ml), concentrated by adding 4 N hydrochloric acid-ethyl acetate (4.5 ml), and the residue was dissolved by heating in water-ethanol (1:10, 3 ml), and the mixture was dissolved at room temperature. At rest overnight. The precipitated solid was collected by filtration, washed with a mixed solution of ethanol and ethyl acetate (1: 1), and dried under reduced pressure at 60 ° C. to give the title compound (0.31 g) as a pale yellow crystalline powder. I got it. mp 229-231 ° C

Reference example 2

Compound A

Compound A (0.50 g) was dissolved in ethanol (5 ml), a sulfuric acid-ethanol solution (lmol / 1, 0.81 ml) was added, and the mixture was allowed to stand at room temperature for 116 hours. The precipitated solid is collected by filtration, After washing with a mixed solution of ethyl ethyl acetate (1: 2), the mixture was dried under reduced pressure at 60 for 168 hours to obtain the title compound (0.31 g) as a pale yellow crystalline powder. mp 224-227T Reference example 3

Compound A1 / 2 succinate

Compound A (0.50 g) was dissolved in ethanol (5 ml), a succinic acid-ethanol solution (lmol / 1, 0.81 ml) was added, and the mixture was allowed to stand at room temperature for 116 hours. The precipitated solid was collected by filtration, washed with a mixed solution of ethyl acetate and ethyl acetate (1: 2), and dried under reduced pressure at 60 ° C for 168 hours to give the title compound (0.21 g) as a pale yellow solid. Obtained as orange crystalline powder. mp 187-190 ° C Reference example 4

Compound メタン · methanesulfonate

Compound A (0.50 g) was dissolved in ethanol (5 ml), methanesulfonic acid-ethanol solution (lmol / 1, 0.81 ml) was added, and the mixture was allowed to stand at room temperature for 116 hours. The precipitated solid was collected by filtration, washed with a mixed solution of ethyl acetate and ethyl acetate (1: 2), and dried under reduced pressure at 60 for 168 hours to give the title compound (0.45 g) as a pale orange crystalline powder. As obtained.

Immediately 256-258 ° C The X-ray powder diffraction pattern of the hydrochloride, sulfate, 1Z2 succinate and methanesulfonate of Compound A changed due to the difference in crystallization conditions, and polymorphism was observed. The powder X-ray diffraction pattern of 2 fumarate did not change due to the difference in crystallization conditions. In addition, 1Z2 fumarate showed the same TG-DSC curve under any crystallization conditions, and showed no peaks such as crystal transition and dehydration, and showed only one endothermic peak.

FIG. 1 shows a powder X-ray diffraction spectrum diagram of the compound A · 1Z2 fumarate obtained using the Cu—Ko; line of the crystal under the crystallization conditions shown in Table 1.

table 1

Recrystallization solvent Stirring

Example 2 (2) Lot 6 ethanol / water = 9: 1

Example 2 (3) Lot 9 ethanol Z water = 9: 1 stirrer

Example 2 (4) Lot 12 ethanol Z water = 9: 1 stirring blade

Example 2 (5) Lot 15 ethanol stirring blade The measurement conditions are as follows.

Equipment: Rigaku RIN IOGS type powder X-ray diffractometer

Sunset; Cu voltage; 40 kV current; 40 mA Scan speed 3.0 ° / min Figure 2 shows the results of TG-DSC thermogravimetric analysis of the crystal of compound A · 1/2 fumarate. The measurement conditions are as follows.

Apparatus; Rigaku TG8110 (TG-DSC) type thermal analyzer

Sample amount; about 10mg Sample cell; Aluminum open cell,

Nitrogen gas flow rate 50 ~ 100ml / min Heating rate; 10 ° C / min Example 3 Tablet

A capsule having the following formulation was produced.

Table 2

Example 4 Tablet

Compound Α · 100 g of 1Z2 fumarate, 2910300 g of HPMC, and 800 g of polysorbate were dissolved in 4550 g of a mixed solution of methanol and water (9: 1). Next, 442 g of lactose and 150 g of sodium bicarbonate were fluidized using a fluidized bed granulator (Niiglat, manufactured by Okawara Seisakusho), and the solution was sprayed to obtain a granulated powder. 834 g of the obtained granules, crystalline cellulose (trade name: Avicel PHI 02, manufactured by Asahi Kasei Corporation) 240 g, croscarmellose sodium (trade name: Ac—Di—So 1, manufactured by Asahi Kasei Corporation) 120 g, magnesium stearate 6 g The mixture is uniformly mixed, and the mixture is compressed using a rotary tableting machine (manufactured by Hata Seisakusho) with a tableting pressure of 700 kg / punch and 150 mg per tablet (Compound A

lOmg) Tablets with a diameter of 7.5 mm were produced. Example 5 Injection

Table 3

Test Example 1 Human Vasopressin V _1A Receptor Affinity and Selectivity Test

(1) Receptor binding test using cell membrane preparation expressing human vasopressin receptor

(Method)

① Preparation of membrane specimen

Human vasopressin (AVP) receptor subtype expression Chinese hamster ovary (CHO) cells and membrane preparations were prepared by reported methods (Br. J. Pharmacol., 125: 1463-1470, 1998). ) ₀ That is, the human expression vector pEF- BOS (Nucle Acids Res, 18 :... 5322, 1990) incorporate the human AVP receptor gene and dihydrofolate reductase gene, dihydrofolate reductase gene defect CH0 using LipofectAMINE reagent Transfection was performed on the cells. Thereafter, the CH0 cells were cultured for 2 weeks on MEM-alpha medium in the presence of 100 nM amethopterin and 10% FCS in the absence of nucleic acid, and the surviving colonies were removed.The concentration of amethopterin was further increased to 1 M. The cultivation was continued, and CH0 cells were selected. The cells grown to confluence were washed with PBS, and then collected in an ice-cooled hypotonic buffer (10 mM Tris-HC1, 5 mM EDTA, pH = 7.4) using a scraper. After homogenizing the collected cells and centrifuging (35, OOOXg 20 min, 4.C), the obtained membrane sample is suspended in a storage buffer (50 mM Tris-HCl, 10 mM MgCl ₂ , pH = 7.4). It became cloudy and stored at -80 ° C until use.

②Receptor binding experiment

In the saturation binding experiment, membrane samples were mixed with various concentrations of [¾] AVP (0.05-3. OnM) and incubated in a tube. In the displacement experiment, a constant concentration (0.5-1. OnM) [¾] AVP and membrane preparation were mixed with various concentrations of compound A fumarate and incubated. Atssay was performed in a total volume of 250 a 1, and Atsushi buffer was performed with 50 mM Tris-HCl, 10 mM MgCl ₂ , 0.1% BSA (pH = 7.4), and the incubation time was 1 hour at room temperature (25 ° C.). After incubation, add ice-cold Tris buffer (50 Tris-HCl, ΜιΜ MgCl ₂ , pH = 7.4) to stop the reaction, perform suction filtration, and perform membrane filtration on a 96-well GF / C UniFilter. Was recovered. After washing the filter, the radioactivity was measured. Non-specific binding was determined using the non-radioactive label AV 1 Μ). [¾] 50% inhibiting concentration of specific binding of AVP to each film sample of the compound Alpha (IC _5. Value) was determined from regression analysis of displacement curves of I spoon compound A. Inhibition constants (values) was determined from the _{= Ιϋ 50 / (1+ [ϋ} / ^) '([L] is the concentration of radioligand in the tube, (dissociation constant) was determined from the Scatchard plot analysis of saturation binding experiments value) .

(2) Receptor binding test using rat tissue membrane specimen

① Preparation of membrane specimen

After the rats were bled to death, the liver, kidneys and pituitary gland were removed. All operations were performed in a 4 ° C environment. Liver (J. Biol. Chem., 258: 9283—9289, 1983), kidney

(J. Biol. C em., 247: 6167-6175, 1972) and the anterior pituitary gland

(Biochem. Biophys. Res. Com Band., 115: 492-498, 1983) Membrane specimens were prepared according to a standard method.

②Receptor binding experiment

The test was conducted in the same manner as in the receptor binding test in (1).

(Result)

(1) is Hitopasopureshin receptor expressing cell membrane specimen receptor binding test compounds using Α showed high affinity for human V _1A receptor (= 0.30 ± 0.02 nM). Compound A was _{ν 1Β ¾ = 2¾500 ± 2500 nM} ), V 2 (381 ± 74 nM) at least 1000 times lower affinity than the V t _A receptor for the receptor.

(2) Receptor binding test using rat tissue membrane specimen

Compound A showed high affinity for rat liver _V1A receptor (= 0.50 ± 0.04 nM). Compound A pituitary V _1B ½ = 1510 ± 190 nM compared to V _1A receptors), renal It had low affinity for the gut V ₂ (= 72.2 ± 15.4 nM) receptor.

Affinity for human and V _{1 A} receptor in rats of Compound A, the selectivity shown in Table 4. Table 4

As shown in Table 4 Compound A showed a very strong affinity for rat Bok and V _{1 A} receptor in humans. Further, selectivity to V _{1 A} receptor The human showed about 9 times higher results in the case of the rat receptor. From the above results, Compound A was confirmed to be very potent and selective human V _{1 A} receptor拮钪agent.

On the other hand, 0PC-21268 did not exhibit little affinity for human V _{1 A} receptor, which is considered the reason was ineffective against overt stage diabetic nephropathy patients clinical. Test Example 2 STZ-induced diabetic nephropathy rat test with overt proteinuria

The improvement effect of Compound A in a model of severe diabetic overt nephropathy with proteinuria was investigated using enalapril maleate, a currently widely used therapeutic agent, as a control. '

(Method)

① Preparation of STZ-induced diabetic rats

Male Wistar rats (Nippon-charles River, 8 weeks old) were used for the experiment. Under anesthesia with pentobarbital (50 mg / kg), STZ (50 mg / kg) was dissolved in citrate buffer and administered via femoral vein to induce diabetes. In the normal group, the same volume of citrate buffer was administered intravenously instead of STZ.

② Examination of the effect of compound A on improving nephropathy in STZ-induced diabetic rats

The experimental protocol is shown in FIG. Sixteen weeks after the administration of STZ to induce diabetes, urinary albumin excretion, urinary protein excretion, blood pressure, and blood glucose levels were measured, and diabetic rats were treated to equalize the parameters. Divided into groups.

1) Normal group (n = 18) 2) Diabetes control group: Oral administration of 0.5% methylcellulose solution (n = 24)

3) Compound A administration group: Compound A · 1/2 fumarate (3mg / lig / day) orally (n = 24)

4) Enalapril administration group: Oral administration of enalapril maleate (1 Omg / kg / day) (n = 24) From the day of grouping, each drug was suspended in a 0.5% methylcellulose solution to give a solution of 5 ml / kg. The dose was administered once a day for eight consecutive weeks, and blood sampling and urine sampling (24-hour urine collection), body weight, urine volume, water consumption, and systolic blood pressure were measured every two weeks. The obtained blood was immediately subjected to slow protein, and the blood glucose level was measured based on a standard method. The urinary albumin concentration and urine protein concentration were measured from the obtained urine sample, and the amount of urinary albumin excretion and the amount of urinary protein excretion were calculated by multiplying the urine volume. In addition, the urinary type 4 collagen concentration was also measured, and the amount of urinary type 4 collagen excreted was calculated by correcting the urinary creatinine level at the same time. Urine glucose was measured based on a standard method.

After the urine collection experiment on the 8th week of continuous injection, whole blood was collected from the inferior aorta under ether anesthesia, plasma creatinine content and blood urea nitrogen were measured, and then perfusion was fixed. Was considered. Plasma creatinine was corrected for urine creatinine amount and body weight measured at the same time to calculate creatinine clearance. Systolic blood pressure was measured by a non-invasive non-invasive measurement method using cuff compression (PS-200A, RIKEN Development Co., Ltd.).

For renal tissue fixation and pathological examination, the kidney was perfused and fixed with saline and 10% formalin in 10% phosphate buffer under ether anesthesia. It was immersed and fixed in 10% buffered formalin solution. A paraffin section was prepared from this kidney fixing material, and subjected to H & E staining and PAS staining for microscopic examination. Glomerular lesions were classified into 100 glomeruli per rat according to the following grades. Normal No abnormality

Substrate increase in less than half of grade 1 mesangial region

Increased substrate in more than 1/2 of grade 2 mesangial region

Part of the glomerulus adheres to the Pauman's capsule and shows a hardened image

(Result)

① Effect of Compound A on body weight, blood sugar, urine sugar, urine output and water consumption In the normal group, weight gain was observed over time, but in the diabetic control group, no weight gain was observed, and the weight was significantly reduced as compared with the normal group. Blood glucose and urinary glucose levels were also significantly increased in the glycemic control group, and they continued to exhibit hyperglycemia and hyperuricemia throughout the test period. In the diabetic control group, a marked increase in urine volume and drinking water was observed throughout the test period. Compound A and enalapril had no effect on weight loss, hyperglycemia, urinary glucose or polydipsia in these diabetic controls.

② Effect of compound A on urinary albumin excretion, urinary protein excretion and urinary type 4 collagen excretion

The results of excretion of urinary albumin and protein in urine 8 weeks after continuous administration of drugs are shown in FIGS. Urinary albumin excretion and urinary protein excretion were significantly increased in the diabetic control group as compared with the normal group. Urinary albumin excretion and urinary protein excretion both showed an improvement effect in the compound A (3 mg / kg / day) administration group and in the enalapril (10 mg / kg / day) administration group. Urinary type 4 collagen excretion was also significantly increased in the diabetic control group as compared to the normal group, and a significant improvement effect was observed in the compound A administration group, but not in the enarabril administration group, although a significant improvement was observed. Admitted.

③ Effect of compound A on creatinine clearance, blood urea nitrogen and systolic blood pressure

A significant increase in creatinine clearance was observed in the diabetic control group compared to the normal group, and a significant decrease was observed in the compound A administration group. The enalapril treatment group only showed a decreasing trend. Regarding blood urea nitrogen, no remarkable difference was observed between the normal group, the diabetic control group, and all the drug administration groups.

FIG. 6 shows the results of the systolic blood pressure 8 weeks after continuous administration of the drug. Blood pressure was significantly increased in the diabetic control group compared to the normal group. The compound A administration group had no effect on blood pressure, but the enalapril administration group showed a significant hypotensive effect.

作用 Effect of compound A on renal glomerular lesions

Fig. 7 shows the incidence of glomerular lesions for each grade. Diabetes control compared to normal group In the group, the incidence of lesion and its grade increased significantly. Compound in drug administration group

In A, a significant improvement was observed in grade 1 lesions, and an improvement was also observed in grade 2 and 3 lesions, but enalapril did not show any improvement effect. Test Example 3 STZ-induced overt diabetic nephropathy rat test showing hypertension

To investigate the effect of compound A on improving nephropathy in diabetic nephropathy complicated with hypertension, diabetic rats with hypertension were prepared by performing STZ administration after uninephrectomy on spontaneously hypertensive rats. Was evaluated. Enalapril maleate was used as a control.

(Method)

(1) Preparation of diabetic nephropathy rats exhibiting hypertension

In the experiment, male spontaneously hypertensive rats (SHR, Japan Charles * River, 8 weeks old) were used. The right kidney was removed under anesthesia with pentobarbital (50 mg / kg, ip), and one week later, STZ (35 mg / kg) dissolved in citrate buffer was administered via femoral vein to induce diabetes. In the non-diabetic group, the right nephrectomy was not performed on the SHR, and the same volume of citrate buffer was administered intravenously instead of STZ.

(2) Examination of the effect of Compound A on improving nephropathy in diabetic nephropathy rats exhibiting hypertension FIG. 8 shows an experimental protocol. One week after STZ administration, urinary albumin excretion, protein excretion, systolic blood pressure, and blood glucose level were measured, and diabetic rats were divided into three groups so that each parameter became equal.

1) Non-diabetic group (n = 20)

2) Diabetes control group: Oral administration of 0.5% methylcellulose solution (n = 24)

3) Compound A administration group: Compound A · 1Z2 fumarate (3mg / kg / day) orally (n = 24)

4) Enalabril administration group: Oral administration of enalapril maleate (lOmg / kg / day) (n = 24) From the day of grouping, each drug was suspended in 0.5% methylcellulose at a volume of 5 ml / kg. Oral administration once a day for 8 weeks, blood collection, urine collection (measurement of urine volume and water consumption during 24-hour urine collection), body weight measurement and systolic blood pressure measurement were performed every two weeks. Blood glucose, urinary albumin excretion, urine protein excretion, urine type 4 collagen excretion, Urine glucose, creatinine clearance, blood urea nitrogen, and glomerular lesions were measured and calculated. .

(Result)

① Effect of Compound A on body weight, blood sugar, urine sugar, urine output and water consumption

The non-diabetic group gained weight over time, but the diabetic control group did not gain weight, and the weight of the diabetic control group was significantly lower than that of the non-diabetic group. Blood glucose and urine glucose levels were also significantly increased in the diabetic control group, and they continued to exhibit high blood glucose and high urine glucose throughout the test period. In the diabetic control group, a marked increase in urine volume and drinking water was always observed throughout the test period. Compound A and enalabril had no effect on weight loss, hyperglycemia / urinary glucose, or polydipsia in these diabetic controls.

The results of urinary albumin excretion and urinary protein excretion 8 weeks after continuous drug administration are shown in FIGS. 9 and 10. Urinary albumin excretion and urinary protein excretion were significantly increased in the diabetic control group compared to the non-diabetic group. Urinary albumin excretion The urinary protein excretion was improved in the compound A and enalapril treatment groups. Urinary type 4 collagen excretion was also significantly increased in the diabetic control group compared to the non-diabetic group, and a significant improvement was observed in the compound A and enalapril administration groups.

A significant increase in creatinine clearance was observed in the diabetic control group as compared to the non-diabetic group, and a significant lowering effect was observed in the compound A administration group. The group receiving enalapril only showed a decreasing trend. Blood urea nitrogen was significantly increased in the diabetic control group as compared to the non-diabetic group, but no improvement was observed by drug administration.

The results of systolic blood pressure 8 weeks after continuous drug administration are shown in FIG. In systolic blood pressure, The blood pressure increased with time in the non-diabetic group, but slightly decreased in the diabetic control group. The compound A administration group had no effect on blood pressure, but the enalapril administration group showed a significant hypotensive effect. Effect of Compound A on glomerular lesions of the kidney

Fig. 12 shows the glomerular lesion occurrence rates for each grade. The incidence and grade of lesions were significantly increased in the diabetic control group compared to the non-diabetic group. In the compound A and enalabril administration groups, lesion ameliorating effects were observed for each grade. The results of Test Examples 2 and 3 show that Compound A improves urinary albumin Z excretion and creatinine clearance at a lower dose than the control compound enalapril. At this time, the blood urea nitrogen level in the drug-administered group did not change from that in the normal group, suggesting that this improvement in creatinine clearance did not cause an excessive decrease in renal function.

Compound A exhibited a nephropathy improvement effect equal to or higher than that of enalapril despite having no antihypertensive effect, indicating that it has a kidney-specific mechanism of action independent of the antihypertensive effect It was suggested. On the other hand, in the case of renal glomerular lesions, in the diabetic control group, lesions that could be clearly differentiated from those in the non-diabetic group were observed in this study, and the significant improvement effect of Compound A was observed.

The effect of Compound A on the increase in urinary albumin / protein excretion is considered to be the result of lowering glomerular pressure by reducing elevated creatinine clearance and improving glomerular hyperfiltration.

Based on the above results, Compound A is lower than enalapril maleate, the most commonly used therapeutic agent, in diabetic nephropathy rats with overt proteinuria and in hyperglycemic diabetic nephropathy rats. It was confirmed that the dose can improve proteinuria, renal function and glomerular lesions. Furthermore, since it does not affect blood pressure, it was suggested that combination treatment with conventionally used antihypertensive drugs is also possible. Test Example 4 STZ-induced diabetic early nephropathy rat test showing microalbuminuria (4 Weekly oral administration)

The effect of improving the nephropathy and renal function in diabetic early nephropathy rats exhibiting microalbuminuria by continuous oral administration of Compound A for 4 weeks was examined using Enalabril, a currently widely used therapeutic agent, as a control.

(Method)

① Preparation of STZ-induced diabetic early nephropathy rats

Male Wistar rats (Nippon Charles' River, 10 weeks old) were used for the experiment. Figure 13 shows an outline of the protocol. Rats measured urinary albumin excretion before STZ administration and were equally divided into four groups.

1) Normal group: Oral administration of 0.5% methylcellulose solution (n = 9)

2) Diabetes control group: Oral administration of 0.5% methylcellulose solution (n = 9)

3) Compound A administration group: Compound A · 1/2 fumarate (3mg / kg / day) orally (n = 9)

4) Enalapril administration group: enalapril maleate (10 mg / kg / day) orally (n = 9)

Under anesthesia with pentobarbi (50 mg / kg), STZ (50 mg / kg) was dissolved in citrate buffer and administered via rat femoral vein to induce diabetes. The normal group received the same volume of saline intravenously instead of STZ. On the day after STZ administration, each drug was suspended in a 0.5% methylcellulose solution, and oral continuous administration was started once daily at a liquid volume of 5 ml / kg. Four weeks after drug administration, urine was collected in a metabolic cage for 24 hours while awake, and urinary albumin, type 4 collagen, and creatinine excretion were determined from the obtained urine samples.

② Effect of compound A on systemic hemodynamics and renal function

After the urine collection experiment, the rats continued to administer the drug once a day thereafter, and a renal clearance experiment was performed in the same week. On the day of measurement of systemic hemodynamics and renal function, two hours after administration of the drug, a polyethylene catheter for blood pressure measurement and blood collection was inserted into the right femoral artery of the rat under anesthesia with inactin (100 mg / kg). In addition, catheters for drug administration were inserted into the left and right femoral veins. For inulin clearance measurement, 1.5 ml / kg of 1 inulin solution dissolved in physiological saline from the force fenus of the right femoral vein After the administration, continuous injection was performed at a volume of 0.1 ml / min in the normal group and 0.2 ml / min in the diabetic group. After incising the left abdomen to expose the left kidney and exfoliate the left renal artery, a probe for measuring renal blood flow was attached and connected to an electromagnetic flowmeter. After exfoliating the left renal ureter, the distal side was ligated, a polyethylene catheter was inserted, and urine was collected over time. Blood pressure was continuously recorded via a polygraph system with a catheter inserted into the artery connected to a pressure transducer. Heart rate was measured by driving evening rice from the blood pressure pulse. Renal blood flow was also continuously recorded via a polygraph system, as was blood pressure. After a stable period of about 1 hour after the start of continuous inulin infusion, a 20-minute urine collection was started after confirming that hemodynamics and urine volume were stable. At an intermediate point 10 minutes after the start of urine collection, mean blood pressure, heart rate and renal blood flow were measured and used as basal values. At the same time, arterial blood was collected for immediate measurement of blood inulin concentration and hematocrit value, and immediately the same amount of saline was intravenously replaced.

③ Effect of compound A on glomerular hemodynamics

After collecting urine for 20 minutes, the left kidney was carefully detached from the surrounding tissues, and the capsule on the outer part of the kidney, where blood vessel control was small, was incised (diameter 1 awake, depth 0.3 mm). Then, the tip of the pencil-type CCD biological microscope mounted on the micromanipulator was guided obliquely to the kidney surface so as not to damage the tubule. By manipulating the micromanipulator, 2 to 6 glomeruli were observed per animal without obstruction of blood flow due to invasion of the experiment and showing glomerular import / export arterioles and glomerular circumference. The glomerular images were recorded continuously on tape by a video cassette recorder and used for measuring glomerular import, export arterioles and glomerular diameter. After the measurement of glomerular hemodynamics, blood for measuring blood creatinine concentration was collected from the abdominal aorta, and the left kidney was excised and the wet weight was measured.

④ Blood and urine parameters-measurement

In the same manner as in Test Example 1, the blood glucose level, urinary type 4 collagen excretion, urinary albumin excretion, urine protein excretion, and creatinine clearance were measured. The inulin concentration was determined by the anthrone method after removing the influence of glucose by the alkaline heating method. The inulin clearance was determined from the plasma and urinary inulin concentrations and defined as the glomerular filtration rate. (result)

① Effect of Compound A on body weight, blood glucose, urinary glucose and urine volume

Significant weight loss was observed in the diabetic control group as compared to the normal group. Blood glucose, urinary glucose and urine volume were also significantly increased in the diabetic control group compared to the normal group. Compound A and enalapril had no significant effect on weight loss, hyperglycemia, hyperuricemia and polyuria observed in these diabetic control groups.

②Effect of compound A on urinary albumin and type 4 collagen excretion

Fig. 14 shows urinary albumin and collagen type 4 excretion at 4 weeks after continuous drug administration in each group. Urinary albumin and type 4 collagen excretion were significantly increased in the diabetic control group compared to the normal group. On the other hand, a significant improvement effect was observed in both the compound A and enarabril administration groups.

(3) Effect of compound A on systemic and renal circulation

FIG. 15 shows the values of each parameter at 4 weeks after continuous administration of each group. Mean blood pressure did not change significantly between the normal group and the diabetic control group, and renal blood flow tended to increase but was not significant in the diabetic control group. On the other hand, creatinine clearance showed an increasing tendency in the diabetic control group as compared to the normal group, and a significant increase in glomerular filtration rate was observed. In the compound A administration group, mean blood pressure and renal blood flow did not show significant changes, but creatinine clearance and glomerular filtration rate tended to decrease. The mean blood pressure was significantly lower in the group receiving enalapril than in the diabetic control group. In addition, renal blood flow significantly increased, creatinine clearance tended to decrease, and glomerular filtration rate showed a slight increase.

作用 Effect of compound A on glomerular hemodynamics

FIG. 16 shows the values of each parameter overnight at 4 weeks of continuous drug administration in each group. Although the diameter of imported arterioles was significantly increased in the diabetic control group compared to the normal group, the diameter of imported arterioles was not significantly changed, and the ratio of imported Z exported arterioles was significantly increased. did. In addition, the glomerular diameter was significantly increased in the diabetic control group as compared to the normal group. In the compound A administration group, no significant change was observed in the diameter of the imported arteriole, but an increase in the diameter of the exported arteriole was observed. As a result, the increase in the ratio of the imported arteriole to the exported arteriole was significantly improved. There was also a tendency for the glomerular diameter to decrease. In the enalapril treatment group, no significant change was observed in the diameter of the imported arteriole, only a significant increase in the diameter of the exported arteriole was observed, and the increase in the ratio of the imported / exported arteriole was improved. There was also a tendency for the glomerular diameter to decrease.

In STZ-induced diabetic early nephropathy rats, oral administration of Compound A for 4 weeks showed an effect on urinary albumin and type 4 collagen excretion. In addition, by attenuating the increase in glomerular filtration rate without affecting macrohemodynamics such as systemic blood pressure and renal blood flow, it was suggested that glomerular hyperfiltration was directly suppressed. Compound A increased the diameter of exported arterioles and suppressed the increase in the ratio of exported arterioles of imported Z, suggesting that it has an improving effect on the increase in glomerular pressure. Thus, compound A diminished glomerular hyperfiltration and thus diabetic early nephropathy by expanding glomerular export arterioles without affecting systemic hemodynamics and acting specifically on glomerular hemodynamics it is conceivable that.

The results of this study suggest that enalapril, an ACE inhibitor, also dilates glomerular export arterioles, corrects glomerular hyperfiltration, and has an effect of improving diabetic early nephropathy. However, the glomerular filtration rate of the enalapril treatment group was increased to the same extent as that of the diabetic control group, suggesting that it has a mechanism of response due to macro-hemodynamic changes such as a strong hypotensive effect and an increase in renal blood flow. Was done. On the other hand, unlike enalapril, compound A suppresses glomerular hyperfiltration by a specific effect on glomerular hemodynamics independent of systemic hemodynamics, and is considered to have an effect of improving early diabetic nephropathy. Test Example 5 STZ-induced diabetic early nephropathy rat test with microalbuminuria (1

Oral continuous administration for 6 weeks)

The efficacy of enalapril, a currently widely used therapeutic agent, as a control was investigated for the effect of continuous oral administration of Compound A for 16 weeks on diabetic early nephropathy rats with microalbuminuria.

(Method) ① Preparation of STZ-induced diabetic early nephropathy rats

Male Wistar rats (Character's River, Japan, 13 weeks old) were used for the experiment. Figure 17 shows an outline of the protocol. Under anesthesia with pentobarbital (50 mg / kg), STZ (50 rag / kg) was dissolved in citrate buffer and administered from rat femoral vein to induce diabetes. The normal group received the same volume of saline intravenously instead of STZ.

② Examination of the effect of Compound A on improving nephropathy in STZ-induced diabetic early nephropathy rats

Four weeks after STZ administration, urinary albumin and type 4 collagen excretion, blood pressure, blood glucose level, and urinary glucose level were measured, and diabetic rats were divided into four groups so that each parameter became equal.

1) Normal group: Oral administration of 0.5 methylcellulose solution (n = 20)

2) Diabetes control group: 0.55¾ methylcellulose solution orally (n = 20)

3) Compound A administration group: Compound A · 1/2 fumarate (3mg / kg / day) orally (n = 20)

4) enalapril administration group: enalapril maleate (10mg / kg / day) orally (n = 20)

5) Compound A + enalapril administration group: Compound A · 1/2 fumarate (3 mg / kg / day) + enalapril maleate (1 Omg / kg / day) orally (n = 20)

From the day of grouping, each drug was suspended in 0.5% methylcellulose solution and administered orally once daily for 16 weeks at a volume of 5 ml / kg, and blood was collected and urine collected every 4 weeks (24-hour urine collection) , Body weight, urine output, water intake, and systolic blood pressure were measured. The obtained blood was immediately subjected to slow protein, and blood glucose was measured based on a standard method. The amount of albumin excreted in urine was calculated from the obtained urine sample. In addition, urinary type 4 collagen concentration was also measured, and the amount of urinary type 4 collagen excreted was calculated by correcting the urinary creatinine level at the same time. The urinary sugar level was measured based on a standard method. On the day after the urine collection experiment on the 16th week of continuous administration, whole blood was collected from the inferior aorta under ether anesthesia, the plasma creatinine concentration was measured, the right kidney was excised and weighed, and the perfusion was fixed and the left kidney was fixed. Extirpated and examined for tissue lesions.

Creatinine clearance, systolic blood pressure, blood glucose, urine in the same manner as in Test Example 1. The amount of medium 4 collagen excreted and the amount of urinary albumin excreted were measured, and renal tissue fixation and pathological examination were performed.

Glomerular lesions were classified into 100 glomeruli per rat according to the following score. Score 1 0: No abnormality / Score 1 1: Deposition of vitreous substance on a part of glomerular epithelial cells or snare wall / Score 1 2: Vitreous like epithelial cells of whole glomerulus or snare wall Deposition of substance is observed / score-3: In addition to the change of score 1-2, a part of glomerulus adheres to Poman's capsule and shows a hardened image

Tubular lesions were classified according to the following score 1 according to the appearance frequency of urinary casts in the tubules. Score — 0: No urinary cast Z score 1: 1: Small number of small casts in the cortex or extramedullary zone (less than 10 spots on section) Z score 1: 2: Small number of cortical and extramedullary zones Large urinary casts scattered in tubules (10 or more sections) / Scorer 3: Large urine casts scattered in cortical and extramedullary tubules (10 or more sections)

(Result)

Body weight gain was observed over time in the normal group, but no weight gain was observed in the diabetic control group, which was significantly reduced as compared to the normal group. Blood and urinary glucose levels were significantly increased in the diabetic control group from 4 weeks after the induction of diabetes, and they continued to exhibit hyperglycemia and hyperuricemia throughout the test period. In the diabetic control group, a marked increase in urine volume and water consumption was observed throughout the test period. Compound A and enalapril had no effect on weight loss, hyperglycemia / urinary glucose, or polydipsia in these diabetic controls.

②Effect of compound A on urinary albumin and type 4 collagen excretion

Fig. 18 shows the results of the excretion of urinary albumin and type 4 collagen 16 weeks after continuous administration of the drug. Urinary albumin excretion and urinary type 4 collagen excretion were significantly increased in the diabetic control group as compared with the normal group. The compound A administration group, the enalapril administration group and the compound A + enalapril administration group all showed a significant improvement effect on the increase in urinary albumin and type 4 collagen excretion. 3) Effect of compound A on creatinine clearance and systolic blood pressure Fig. 19 shows the results of creatinine clearance and systolic blood pressure 16 weeks after continuous drug administration. No significant change in creatinine clearance was observed in the diabetic control group as compared to the normal group, but a significant decrease was observed in the compound A administration group and the compound A + enalapril administration group, but not in the enalapril administration group. I was taken. Systolic blood pressure tended to decrease in the diabetic control group compared to the normal group. The compound A administration group had no effect on blood pressure, but the enalabril and compound A + enalapril administration groups showed a significant antihypertensive effect.

作用 Effect of compound A on renal glomerular and tubular lesions

The glomerular and tubular lesion occurrence rates are shown in FIG. 20 and FIG. In glomerular lesions, the incidence of lesions in the normal group was about 5% of the total, and most showed only mild lesions with a score of -1. On the other hand, the incidence and extent of lesions in the diabetic control group were significantly worse than in the normal group. In the drug-administered group, no clear difference was observed in any group from the diabetic control group.However, when focusing on scorer 3 showing a glomerular sclerosis image, compound A and compound A + enalapril were administered. There was an improvement trend in the group. The incidence and incidence of tubular lesions tended to be worse in the diabetic control group than in the normal group. There was a tendency to improve the frequency and degree of urinary cast in the compound A administration group, but no improvement was observed in the enalapril administration group and the compound A + enalapril group. Administration of Compound A had no effect on blood pressure, whereas administration of enalapril and Compound A + enalapril had a significant antihypertensive effect. It is well known that administration of an ACE inhibitor to a diabetic patient with hypertension to lower blood pressure decreases urinary albumin excretion, and in this study, a significant antihypertensive effect was also observed with enalabril administration. These results suggested that this antihypertensive effect is involved in part of the mechanism of action for improving nephropathy, including the reduction of urinary albumin excretion by enalapril. On the other hand, Compound A showed the same nephropathy-improving effect as enalapril despite having no antihypertensive effect. It was suggested to have a kidney-specific mechanism of action.

Improvement of glomerulosclerotic lesions and tubular lesions was observed with administration of Compound A, strongly suggesting that Compound A may have an effect of delaying the progression of renal lesions. Compound A has three problems with pasopressin receptor antagonists (① It has V _{1 A} V ₂ antagonistic activity, and no pure V _{1 A} antagonist or V ₂ antagonist has been found. Antagonism disappears when administered, only agonist action remains. (3) The species difference is large, and although extremely effective in rats, there is almost no antagonism in dogs, monkeys and humans.) It is a compound with an excellent profile and has therapeutic effects on rats with overt diabetic nephropathy (improvement of proteinuria, renal function and renal glomerular lesions) and therapeutic effects on rats with early diabetic nephropathy (albuminuria, kidney Improvement of glomerular hyperfiltration) is thought to be fully reflected in human clinical practice. Test Example 6 Clinical trial for patients with early-stage diabetic nephropathy

(Method)

The clinical test for patients with early-stage diabetic nephropathy was performed under the following conditions.

Target: 20 patients with early nephropathy due to type I diabetes (over 45 years)

Investigational product: Compound A-1/2 fumarate

Dosage form: 20 and 4 O mg tablets

Dosage: Brazepo, 20, 60 and 120 mg Z days (administered once or twice daily) Duration of administration: 6 weeks (28 days)

Dosage regimen: The study should be performed in a double-blind, multicenter, placebo-controlled, dose-escalating fashion. All patients receive the three doses of the test drug and placebo, and the placebo treatment period is randomly assigned during the test drug treatment period. The total treatment period for each patient is 24 weeks, with two weeks before and after the run-in period and a four-week period.

Evaluation index: (Primary index) Changes in albumin excretion from the start of administration to after treatment (24 weeks). (Secondary index) Albumin excretion from the last dose during the treatment period until the next treatment period change of. Changes in urine osmotic pressure, type 4 collagen, urinary N-acetyl / 3-D-glucose-aminidase and TGF-] 3 excretion, and intravenous arginine vasopressin.

Evaluation method:

Albumin excretion and urine osmolality should be assessed at baseline at the end of the run-in period using 24 hours x 3 urine collection samples. Albumin excretion and urine osmotic pressure should be reassessed at the end of each treatment period and at the beginning of the next treatment using 24 hours x 3 urine samples. Patients are hospitalized for at least 8 hours on the first day of each treatment period (Visits 4, 7, 10, and 13) and patients receive the first dose of each treatment period at the hospital. Patients are hospitalized for 24 hours on the last dosing day (Visit 4, 7, 10, and 13), and patients receive the last dose of each treatment period at the hospital. The amount of type 4 collagen, creatinine, N-acetyl-i3-D-darco-aminidase and TGF-j8 in urine was measured before randomization at Visit 1 and at 4, 7, 10, 13, and 14 before randomization. At this time, a small amount of urine is measured to evaluate the therapeutic effect of the biomarker. Intravenous arginine vasopressin is measured at visits 1, 4, 7, 10, 13, and 14. Industrial applicability

According to the present invention, proteinuria in patients with overt diabetic nephropathy, a therapeutic agent effective for improving reduced renal function and renal glomerular lesions, and Z or albuminuria in patients with early diabetic nephropathy The present invention can provide a therapeutic agent effective for improving renal glomerular hyperfiltration and suppressing the progress of disease.

Claims

The scope of the claims

1. (Z) 1 4'_ {4,4-Difluoro-5- [2-oxo-1 2- (4-piperidinopiperidino) ethylidene] 1,2,3,4,5-tetrahydro-1H-1-1 Benzozepine-1 —Chemical composition for the treatment of overt diabetic nephropathy comprising, as an active ingredient, 12-methyl-3-furanilide or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, which is an agent for improving proteinuria in a patient with overt diabetic nephropathy.

3. The pharmaceutical composition according to claim 1, which is an agent for improving renal function of a patient with overt diabetic nephropathy.

4. (Z) —4,1- {4,4 difluoro—5— [2-oxo—2 -— (4-piperidinopiperidino) ethylidene] for the manufacture of a therapeutic agent for overt diabetic nephropathy Use of 1,2,3,4,5-tetrahydro-lH-l-benzazepine-l-l-propanol} -12-methyl-3-furanilide or a pharmaceutically acceptable salt thereof.

5. (Z) —4,1- {4,4-difluoro-5— [2-oxo-1- (4-piperidinopiperidino) ethylidene] —2,3,4,5-tetrahydro-1H—1-benzobenzopine-1— A method for treating overt diabetic nephropathy, comprising administering to a patient a therapeutically effective amount of 12-methyl-3-furanilide or a pharmaceutically acceptable salt thereof.

6 · (Z) _4, ー {4,4-difluoro-5- [2-oxo-1- 2- (4-piperidinopiperidino) ethylidene] — 2,3,4,5-tetrahydro-1H-1 A pharmaceutical composition for treating early-stage diabetic nephropathy, comprising, as an active ingredient, 2-methyl-3-furanilide or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition according to claim 6, which is an agent for improving albuminuria in patients with early-stage diabetic nephropathy.

8. Claim 6 which is an agent for improving renal glomerular hyperfiltration in patients with early-stage diabetic nephropathy The pharmaceutical composition according to any one of the preceding claims.

9. (Z) —4,1- {4,4-difluoro-5- [2-oxo-1-2- (4-piperidinopiperidino) ethylidene] -2, for the manufacture of a therapeutic agent for early-stage diabetic nephropathy Use of 3,4,5-tetrahydro-1H_1-benzazepine-11-carbonyl} -12-methyl-3-furanilide or a pharmaceutically acceptable salt thereof.

1 0. (Z) 1,4,1 {4,4-difluoro-5 _ [2-oxo-1 2- (4-piridinopiperidino) ethylidene] — 2,3,4,5-tetrahydro-1H-1-benzazepine [0011] A method for treating early-stage diabetic nephropathy, which comprises administering to a patient a therapeutically effective amount of 12-methyl-3-furanilide or a pharmaceutically acceptable salt thereof.

1 1 · (Z) 1,4,1 {4,4 Difluoro-5- [2-oxo-2- (4-piridinobiperidino) ethylidene] 1,2,3,4,5-tetrahydro-1H— 1-benzozepine-1 Pharmaceutical composition for inhibiting progression of diabetic nephropathy, comprising, as an active ingredient, one-potile} -methyl-3-furanilide or a pharmaceutically acceptable salt thereof.

1 2. (Z) -4'- {4,4-difluor Π-5- [2-oxo-1- 2- (4-piperidinopiperidino) for the manufacture of an inhibitor of progression of diabetic nephropathy [Ethylidene] -2,3,4,5-tetrahydro-1H-1-benzazepine-11-potanol} -2-methyl-3-furanilide or a pharmaceutically acceptable salt thereof.

1 3. (Z) -4'- {4,4-difluoro-5- [2-oxo-1- 2- (4-piperidinopiperidino) ethylidene] — 2,3,4,5-tetrahydro-1H—1-benzazepine-1 A method for inhibiting the progression of glycemic nephropathy, comprising administering to a patient a therapeutically effective amount of 2-methyl-3-furanilide or a pharmaceutically acceptable salt thereof.

14. (Z) 1 4'— {4,4-Difluoro-5— [2-oxo_2— (4-pyridinopiperidino) ethylidene] 1,2,3,4,5-tetrahydro-1H—1-benzazepine-1 1-methyl-2- 3-methyl-3-furanilide 1/2 fumarate.