CN101002940A - Medicine composition for treating polycystic renal disease - Google Patents

Abstract

The invention relates to a pharmaceutical composition for treating polycystic kidney disease. The active ingredients of the pharmaceutical composition are composed of cyclooxygenase 2 (COX-2) specific inhibitors and thiazolidinedione drugs in effective doses. The preferred combination of the present invention is: the cyclooxygenase 2 specific inhibitor is selected from celecoxib, and the thiazolidinediones are selected from rosiglitazone. The in vivo experiments also confirmed that the combined treatment of rosiglitazone and celecoxib improved the disease course and histological lesions of polycystic kidney disease Han:SPRD rats better than a single drug. The pharmaceutical composition for treating polycystic kidney disease of the invention not only has low toxicity, but also has better effect than a single drug, and is a promising polycystic kidney disease therapeutic drug.

Description

Pharmaceutical composition for treating polycystic kidney disease and its application

technical field

The invention relates to the field of medicines, in particular to a pharmaceutical composition for treating polycystic kidney disease, an application thereof and a pharmaceutical preparation containing the same.

Background technique

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, with a worldwide incidence of about 1/400-1/1000. Its main pathological feature is the formation of multiple fluid cysts in the cortex and medulla of the bilateral kidneys and progressive growth, which eventually destroys the structure and function of the kidneys, leading to renal failure. ADPKD is also a systemic disease. In addition to involving the kidneys, it can also cause organ lesions such as liver and pancreatic cysts, heart valve disease, and cerebral aneurysms (Extrarenal manifestations of ADPKD, Perrone RD, Kidney Int.1997; 51(6): 2022 -36). At present, there are about 1.5 million ADPKD patients in my country, 50% of whom enter end-stage renal failure after the age of 60, accounting for 5-10% of the causes of end-stage renal failure. It can be seen that ADPKD is a kind of genetic disease that seriously endangers human health.

The two pathogenic genes PKD1 and PKD2 that cause ADPKD are located on chromosomes 16 and 4, respectively, and were cloned in 1994 and 1996. Studies have found that 85% of patients are due to PKD1 gene mutations, and 10-15% of patients are caused by PKD2 gene mutations. In some patients, no two types of gene mutations were found, suggesting that there may be other pathogenic genes (such as PKD3), but they have not been located yet. The pathogenesis of ADPKD is relatively complex, and its molecular pathogenesis and pathophysiological changes can be summarized as follows: ADPKD patients have heterozygous mutations in a pair of alleles (PKD1 or PKD2) in the embryonic stage through parental inheritance or spontaneous mutation, and the individual is born with a heterozygous mutation. Under the action of environmental factors such as toxins and infections, homozygous mutations (two hits) occur in some segmental somatic cells of the kidney, causing abnormal functions of the encoded proteins polycystin-1 and polycystin-2, causing cell cycle regulation and intracellular Metabolic abnormalities lead to cyst formation (The molecular basis of focal cystformation in human autosomal dominant polycystic kidney disease type I.; Qian F, Watnick TJ, Onuchic LF, Germino GG.; Cell. 1996; 87(6): 979-87). The pathophysiological changes of ADPKD have the following characteristics: (1) Abnormal proliferation and apoptosis of epithelial cells: polycystic kidney disease is a tumor-like disease, the proliferation and apoptosis indexes of cyst epithelial cells are significantly increased, the expression of various growth factor receptors is up-regulated, cystic kidney disease The mitogenic factors contained in the fluid bind to the dislocated receptors on the lumen of the renal tubule, forming an autocrine and paracrine loop, which stimulates the cyst to continue to grow; (2) Abnormal fluid secretion and accumulation: cyst lining epithelial cell lumen membrane There is a Cl ^- secretion transporter, called cystic fibrosis transmembrane regulator (cystic fibrosis transmembrane regulator, CFTR), under the stimulation of cyclic adenosine monophosphate (cAMP), CFTR secretes Cl ^- increase, through the effect of charge, Na ⁺ Enter the cyst cavity through the tight junction between cells. Under the action of osmotic pressure, water enters the cyst cavity from the epithelial cells, causing fluid to accumulate in the cyst cavity, and the cyst grows progressively; (3) Extracellular matrix remodeling and interstitial fibrosis: Immunohistochemical studies found that renal cyst tissue Increases in fibrin, type I, type IV collagen and laminin, and lack of heparan sulfate glycosides lead to remodeling of the extracellular matrix and reduced compliance of the renal tubular basement membrane, which is conducive to the progressive growth of cysts. Interstitial fibrosis; (4) cell polarity change with poor differentiation: cell polarity change causes Na ⁺ -K ⁺ -ATPase to be located on the membrane surface of the tubule cell lumen, continuously secrete fluid into the cyst cavity, and a variety of differentiation and maturation indicators Lost (Recent advances in understanding thepathogenesis of polycystic kidney disease: therapeutic implications., Cowley BD Jr., Drugs 2004; 64: 1285-1294).

Due to the great harm and complex pathogenesis of ADPKD, its early treatment is quite difficult, and there are no effective intervention measures and therapeutic drugs in clinical practice. Once it develops to end-stage renal failure, it can only rely on renal replacement therapy (dialysis, kidney transplantation). Therefore, how to delay the progression of polycystic kidney disease through early drug intervention is an urgent problem to be solved (Therapies to slow polycystic kidney disease, Torres VE., Nephron Exp Nephrol. 2004; 98 (1): e1-7).

Cyclooxygenase (COX), also known as PGH2 synthase and prostaglandin synthase, is the key rate-limiting enzyme in the initial step of prostaglandin (PGs) synthesis. The kidney, especially the renal medulla, is one of the most active tissues for the synthesis of PGs. The main sites of medullary PGs synthesis are medullary collecting duct epithelial cells and renal interstitial cells, which are mainly involved in water and salt metabolism. Prostaglandins bind to receptors and exert different biological effects by regulating intracellular cAMP and Ca ²⁺ levels. COX products, such as PGE1, PGE2, and PGI2, promote renin release in whole animals, isolated perfused kidneys, and isolated glomeruli. PGs are also involved in the proliferation of mesangial cells, the synthesis of extracellular matrix and the secretion of other vasoactive substances. According to the difference in structure and function, COX can be divided into two isozymes, called structural COX-1 and inducible COX-2. The former is widely distributed, while the latter is only locally distributed. Under the action of inflammatory stimuli, such as tumor-promoting agents and various cell growth factors, the expression increases sharply by 10-80 times. At present, it is believed that the above-mentioned stimulating signals are activated through two pathways of tyrosine kinase receptor and serpentine receptor, and then activate protein kinase A, protein kinase C, JAK-STAT and other signal transmission pathways, causing cell proliferation and extracellular matrix production increase. Another author believes that in glomerulonephritis, COX-2 induces inflammation and interstitial fibrosis by increasing the production of PGE2, activating EP3 receptors, and up-regulating the expression of pathogenic α-integrins. A number of studies have shown that specific COX-2 inhibitors can inhibit mesangial cell proliferation and extracellular matrix synthesis, improve interstitial inflammation and fibrosis, and in vitro experiments have also confirmed that it can inhibit colon cancer, lung cancer and other tumor cell proliferation , to promote apoptosis.

In US patent application US2004024042, its inventors concluded that COX-2 specific inhibitors may have a therapeutic effect on polycystic kidney disease.

Since it has been reported in recent years that the COX-2 specific inhibitors that have been marketed have cardiotoxicity, the safety of large-scale clinical use of COX-2 specific inhibitors is controversial. The patent does not specify the specific dosage of COX-2 specific inhibitors for the treatment and prevention of polycystic kidney disease.

Therefore, it is necessary to further study and explore the effective therapeutic dose of COX-2 specific inhibitors and evaluate their safety, so as to find more effective and safer drugs for the treatment of polycystic kidney disease.

Contents of the invention

The inventor selected celecoxib (celecoxib, trade name: Celebrex), which has relatively small toxic and side effects of COX-2 (cyclooxygenase 2) inhibitors, and carried out research on the treatment of polycystic kidney disease. Through cell experiments, it was found that: COX -2-specific inhibitors can inhibit the proliferation of cyst lining epithelial cells and promote apoptosis, and the inhibitory effect is dose-dependent; and through animal experiments, it shows that celecoxib (10mg/kg·d) in the small and medium dose group can improve ADPKD animals Model Han: SPRD rats progress in disease course and protect renal function. The main mechanism is that celecoxib inhibits the high expression of cyclooxygenase 2, reduces the release of PGE2, down-regulates the expression of inflammatory mediators, and reduces inflammation and interstitial fibrosis. This study is the first at home and abroad to prove that cyclooxygenase 2 inhibitors can effectively treat polycystic kidney disease with small and medium doses through in vivo and in vitro experiments.

Due to the safety problems of specific cyclooxygenase 2 inhibitors, it is necessary to further study and find other more safe and effective drugs for the treatment and prevention of polycystic kidney disease.

The researchers of the present invention unexpectedly found through animal experiments and clinical studies that thiazolidinediones include troglitazone, rosiglitazone, pioglitazone, and ciglitazone. ) and englitazone (englitazone), etc., can dose-dependently inhibit cyst lining epithelial cell proliferation and promote apoptosis. In vivo experiments show that it can improve the course of disease and histological changes of ADPKD animal Han:SPRD rats, which can be used as Drugs to prevent and treat polycystic kidney disease. The use of thiazolidinediones for the prevention and treatment of polycystic kidney disease is new and has not been reported in domestic and foreign literature.

What is even more surprising is that the inventors of the present invention used the primary cultured cyst lining epithelial cells of ADPKD patients and polycystic kidney disease Han:SPRD rats as the main research platform, and confirmed that the cyst lining epithelial cells of the 3rd to 5th generation and Han :SPRD rats can well reflect the pathophysiological characteristics of ADPKD patients, and can be used as a reliable platform for evaluating drug efficacy; Unexpected synergistic effect, their combined use can dose-dependently inhibit the proliferation of cyst lining epithelial cells, and has the effect of promoting apoptosis. The present invention compares the curative effect of combined treatment with different compatibility methods at the same time, and finds that the combined use of cyclooxygenase 2-specific inhibitors and thiazolidinediones is higher than the sum of the effects of the single use of the two drugs on the proliferation inhibition rate and apoptosis rate , low-dose combined drug is better than high-dose single drug, which provides a reference for in vivo experiments and clinical applications. The present invention further proves in vivo experiments that the combined use of cyclooxygenase 2-specific inhibitors and thiazolidinediones at low doses can significantly improve the course and histological lesions of polycystic kidney disease, and has a good therapeutic synergistic effect.

Therefore, based on the above findings, the present invention provides a pharmaceutical composition for the prevention and treatment of polycystic kidney disease, which comprises: a) a cyclooxygenase 2 specific inhibitor, and b) thiazolidinediones.

In the pharmaceutical composition of the present invention, cyclooxygenase 2-specific inhibitors include, but are not limited to: celecoxib, rofecoxib, paracoxib, valdecoxib, etoricoxib, meloxicam, nimesulide, radicicol and their pharmaceutically acceptable salts. Preference is given to celecoxib, meloxicam, radicicol and their pharmaceutically acceptable salts. Particular preference is given to celecoxib.

In the pharmaceutical composition of the present invention, the thiazolidinediones are selected from rosiglitazone, troglitazone, pioglitazone, ciglitazone, englitazone (englitazone) and their pharmaceutically acceptable salts. Rosiglitazone, troglitazone, pioglitazone and their pharmaceutically acceptable salts are preferred. Particular preference is given to rosiglitazone, pioglitazone and their pharmaceutically acceptable salts.

The pharmaceutically acceptable salts described in the present invention include the salts formed by cyclooxygenase 2 specific inhibitors and thiazolidinediones with inorganic acids or organic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, Nitric acid, phosphoric acid, acetic acid, maleic acid, fumaric acid, citric acid, citric acid, oxalic acid, succinic acid, tartaric acid, malic acid, mandelic acid, trifluoroacetic acid, pantothenic acid, methanesulfonic acid and p-toluenesulfonic acid Salt.

In a preferred example of the present invention, particularly preferred salts of cyclooxygenase 2 specific inhibitors and thiazolidinediones are maleate, fumarate, celecoxib and rosiglitazone. Citrate, citrate, succinate, tartrate, pantothenate, or malate.

In the pharmaceutical composition of the present invention, the molar ratio of cyclooxygenase 2-specific inhibitors to thiazolidinedione drugs is 1:0.1-40, preferably 1:0.25-20, more preferably 1:0.5-10, More preferably 1:1-5, especially preferably 1:1-2.5.

In the pharmaceutical composition of the present invention, a particularly preferred example is a pharmaceutical composition formed by combining celecoxib and pioglitazone or a pharmaceutically acceptable salt thereof, wherein the molar ratio of celecoxib to pioglitazone is 1: 0.125-40 , preferably 1:0.25-20, more preferably 1:0.5-10, further preferably 1:1-5, particularly preferably 1:1-2.5.

In the pharmaceutical composition of the present invention, another particularly preferred pharmaceutical composition is a pharmaceutical composition formed by combining celecoxib and rosiglitazone or a pharmaceutically acceptable salt thereof, wherein celecoxib and rosiglitazone The molar ratio is 1: 0.125-40 (weight ratio is 1: 0.155-49.6), preferably 1: 0.25-20 (weight ratio is 1: 0.31-24.8), more preferably 1: 0.5-10 (weight ratio is 1:0.62-12.4), more preferably 1:1-5 (1:1.24-6.2 by weight ratio), especially preferably 1:1-2.5 (1:1.24-3.1 by weight ratio).

The pharmaceutical composition of the present invention can be alone or preferably mixed with a pharmaceutically acceptable carrier, excipient or diluent, and administered to mammals, including humans, according to standard pharmaceutical practices.

Therefore, another aspect of the present invention provides a pharmaceutical preparation, which contains the above-mentioned cyclooxygenase 2 specific inhibitor and the above-mentioned thiazolidinedione drug or a pharmaceutically acceptable salt thereof as active ingredients and a or multiple pharmaceutically acceptable carriers. Wherein the molar ratio of cyclooxygenase 2-specific inhibition to thiazolidinedione drugs or salts thereof is 1:0.1-40, preferably 1:0.25-20, more preferably 1:0.5-10, further preferably 1 : 1-5, particularly preferably 1: 1-2.5.

In the pharmaceutical preparation of the present invention, a particularly preferred example is a pharmaceutical preparation containing celecoxib and pioglitazone or a pharmaceutically acceptable salt thereof as active ingredients, wherein the molar ratio of celecoxib to pioglitazone is 1:0.125-40 , preferably 1:0.25-20, more preferably 1:0.5-10, further preferably 1:1-5, particularly preferably 1:1-2.5.

In the pharmaceutical preparation of the present invention, another particularly preferred example is a pharmaceutical composition formed by combining celecoxib and rosiglitazone or a pharmaceutically acceptable salt thereof, wherein the molar ratio of celecoxib to rosiglitazone is 1:0.125-40 (weight ratio is 1:0.155-49.6), preferably 1:0.25-20 (weight ratio is 1:0.31-24.8), more preferably 1:0.5-10 (weight ratio is 1:0.62 -12.4), more preferably 1:1-5 (weight ratio is 1:1.24-6.2), especially preferably 1:1-2.5 (weight ratio is 1:1.24-3.1).

The pharmaceutical preparations of the present invention can be administered orally or parenterally. Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.

The pharmaceutical preparations of the present invention may be in a form suitable for oral use, such as tablets, sustained-release tablets, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs agent.

Formulations of the present invention for oral use may be prepared according to any method known in the art for the preparation of oral pharmaceutical compositions, and such compositions may contain one or more substances selected from the group consisting of sweeteners, flavoring agents, opaque, coloring and preservative agents to provide pharmaceutically aesthetic and palatable preparations.

Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients can be: inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as microcrystalline cellulose, sodium carboxymethylcellulose, corn starch or alginic acid ; binders such as starch, gelatin, polyvinylpyrrolidone or gum arabic; and lubricants such as magnesium stearate, stearic acid or talc.

Tablets may be uncoated or they may be coated by techniques well known in the art to mask the unpleasant taste of the drug or to delay its disintegration and absorption in the gastrointestinal tract, and thus over a longer period of time. Sustained effect within. For example, water soluble taste masking materials such as hydroxypropylmethylcellulose or hydroxypropylcellulose or time delay materials such as ethylcellulose, cellulose acetate butyrate may be used.

Oral formulations of the invention may also be presented as hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate and kaolin, or as soft gelatin capsules, wherein the active ingredient is mixed with a water-soluble carrier such as polyethylene glycol alcohol or oily medium such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials in admixture with excipients or dispersants suitable for the manufacture of aqueous suspensions. Said excipients include: suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic. The dispersant can be a natural phospholipid such as lecithin, or a condensation product of alkylene oxide and fatty acid, such as polyoxyethylene stearate, or a condensation product of alkylene oxide and long-chain aliphatic alcohol, such as heptadecene Oxycetyl alcohols, or condensation products of alkylene oxides with partial esters derived from fats and hexitols, such as polyoxyethylene sorbitan monooleate.

The aqueous suspensions of the present invention may also contain one or more preservatives such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents , and one or more sweetening agents such as sucrose, saccharin or aspartame.

Oily suspensions of the present invention can be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those described above and flavoring agents may also be added to provide a palatable oral preparation. These pharmaceutical composition formulations can be preserved by adding antioxidants such as butylated hydroxyanisole or alpha-tocopherol.

The dispersible powders and granules of the present invention are suitable for use to prepare aqueous suspensions by adding water. It provides the active ingredient in admixture with a dispersing agent, suspending agent and one or more preservatives. Examples of suitable dispersing and suspending agents are those mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions can be preserved by the addition of antioxidants such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as liquid paraffin, or a mixture of these oils. Suitable emulsifiers are natural phospholipids, such as soybean lecithin, and esters or partial esters derived from fatty acids with hexitol anhydrides, such as sorbitan monooleate, and condensation products of said partial esters with alkylene oxides , such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring, preservative and antioxidant agents.

Syrups and elixirs of the invention may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain buffers, preservatives, flavoring and coloring agents and antioxidants.

The pharmaceutical preparations of the present invention may be in the form of sterile injectable aqueous solutions. Vehicles or solvents that may be employed include water, Ringer's solution, and isotonic sodium chloride solution.

The sterile injectable preparation of the present invention may also be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oily phase. For example, the active ingredient may first be dissolved in a mixture of soybean oil and lecithin. The oil solution can then be introduced into a mixture of water and glycerin and processed to form a microemulsion.

The injection solution or microemulsion of the present invention can be introduced into the patient's bloodstream by local bolus instillation. Alternatively, it may be advantageous to administer the solution or microemulsion in a manner that maintains a constant circulating concentration of the composition of the invention. To maintain such a constant concentration, a continuous intravenous delivery device can be used.

The pharmaceutical preparations of the present invention may be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. This suspension may be formulated according to methods known in the art using suitable dispersing and suspending agents which have been mentioned above. Sterile injectable preparations can also be sterile injectable solutions or suspensions dissolved in non-toxic parenterally acceptable diluents or solvents. For example a solution in 1,3-butanediol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The pharmaceutical formulation of the invention may also be in the form of a suppository for rectal administration of the drug. It can be prepared by mixing the pharmaceutical composition of the invention with suitable non-irritating excipients. Such excipients are solid at ordinary temperatures. But it is liquid at rectal temperature, so it can melt in the rectum and release the active drug. The non-irritating excipients include coconut oil, glycerinated gelatin, hydrogenated vegetable oil, a mixture of polyethylene glycols of different molecular weights and fatty acid esters of polyethylene glycol.

For topical use, creams, ointments, gels, solutions or suspensions, etc., containing the pharmaceutical compositions of this invention may be employed. Topical applications also include aqueous mouthwashes and mouthwashes.

The pharmaceutical compositions of this invention may be administered in intranasal form through the use of suitable intranasal vehicles and delivery devices, or may be administered transdermally using nasal patches well known to those skilled in the art. To be administered in a transdermal delivery system, the dosage should, of course, be continuous rather than intermittent throughout the dosage regimen.

When the pharmaceutical preparation of the present invention is administered to humans, the daily dosage is usually determined by the prescribing physician, and the dosage generally varies with the age, weight, sex and response of the individual patient and the severity of the patient's symptoms. Usually, the dosage for adult is 0.1-70 mg active ingredient/kg body weight per day, preferably 0.2-10 mg active ingredient/kg body weight per day.

Another aspect of the present invention provides a method for preparing a pharmaceutical preparation, comprising mixing the above-mentioned pharmaceutical composition of the present invention with one or more of the above-mentioned pharmaceutically acceptable carriers or excipients.

Another aspect of the present invention is to use the pharmaceutical composition of the present invention to prepare medicines for preventing and treating polycystic kidney disease.

The present invention also provides a method for screening effective drugs for preventing and treating polycystic kidney disease, which comprises the following steps:

(1) provide a kind of ADPKD patient cyst lining epithelial cell and described polycystic kidney disease Han:SPRD rat with primary culture;

(2) contacting the drug to be screened with primary cultured ADPKD patient cyst lining epithelial cells, or/and administering the drug to be screened to the polycystic kidney disease Han:SPRD rats;

(3) Observe any of the following phenomena: high expression of cyclooxygenase 2 is inhibited, PGE2 release is reduced, the expression of inflammatory mediators is down-regulated, inflammation and interstitial fibrosis are alleviated, and the proliferation or apoptosis of cyst lining epithelial cells is inhibited;

(4) If any of the phenomena listed in step (3) occurs, it is determined that the screened drug is an effective drug for preventing and treating polycystic kidney disease.

Another object of the present invention is to provide a method for preventing and treating polycystic kidney disease in mammals, especially humans, which comprises administering to said mammals a specific inhibitor of cyclooxygenase 2 and a thiazolidinedione drug.

The "combined use" mentioned in the present invention includes administering the cyclooxygenase 2-specific inhibitor and thiazolidinedione drugs separately or simultaneously to mammals, or administering the cyclooxygenase 2-specific Sex inhibitors and thiazolidinediones are combined to form a pharmaceutical composition or pharmaceutical preparation for administration to mammals.

In the method of the present invention, the molar ratio of the cyclooxygenase 2-specific inhibitor and thiazolidinediones used in combination is 1:0.1-40, preferably 1:0.25-20, more preferably 1:0.5- 10, more preferably 1:1-5, especially preferably 1:1-2.5.

In the method of the present invention, the cyclooxygenase 2-specific inhibitors used in combination include, but are not limited to: celecoxib, rofecoxib, paracoxib, valdecoxib, etoricoxib, meloxicam , nimesulide, radicicol and its pharmaceutically acceptable salts. Celecoxib, meloxicam and radicicol are preferred. Particular preference is given to celecoxib.

In the method of the present invention, the thiazolidinedione drugs used in combination include, but are not limited to: rosiglitazone, troglitazone, pioglitazone, ciglitazone, emglitazone and their pharmaceutically acceptable Salt. Rosiglitazone, troglitazone, pioglitazone and their pharmaceutically acceptable salts are preferred. Particular preference is given to rosiglitazone, pioglitazone and their pharmaceutically acceptable salts.

The pharmaceutically acceptable salts described in the method of the present invention include cyclooxygenase 2-specific inhibitors and thiazolidinediones and salts formed with inorganic acids or organic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, Sulfuric acid, nitric acid, phosphoric acid, acetic acid, maleic acid, fumaric acid, citric acid, citric acid, oxalic acid, succinic acid, tartaric acid, malic acid, mandelic acid, trifluoroacetic acid, pantothenic acid, methanesulfonic acid and p-toluenesulfonic acid salt formed. Particularly preferred salts of cyclooxygenase 2 specific inhibitors and thiazolidinediones are maleate, fumarate, citrate, citrate, celecoxib and rosiglitazone. Succinate, Tartrate, Pantothenate or Malate.

The molar ratio of celecoxib and pioglitazone used in combination in the method of the present invention is 1: 0.125-40, preferably 1: 0.25-20, more preferably 1: 0.5-10, further preferably 1: 1-5, especially Preferably it is 1:1-2.5.

Description of drawings

Figure 1 Primary culture and identification of human cyst lining epithelial cells

A. Observation of primary cultured human cyst lining epithelial cells under an inverted phase-contrast microscope

B. Adhesion spots can be seen under the transmission electron microscope

C. Microvilli can be seen under the transmission electron microscope

D, anti-cytokeratin positive

E. Alkaline phosphatase staining was gray-black positive reaction

F. Positive for vimentin monoclonal antibody

Figure 2 BrdU method to detect the average increase in the proliferation inhibition rate of cyst lining epithelial cells when 25uM rosiglitazone was combined with different concentrations of celecoxib

Figure 3 BrdU method to detect the average increase in the proliferation inhibition rate of cyst lining epithelial cells when 10uM celecoxib was combined with different concentrations of rosiglitazone

Figure 4 BrdU method to detect the proliferation inhibition rate of cyst lining epithelial cells after rosiglitazone 50uM alone, celecoxib 10uM, rosiglitazone 50uM and celecoxib 10uM for 48 hours

Figure 5 BrdU method to detect the proliferation inhibition rate of cyst lining epithelial cells in the single administration of rosiglitazone 25uM, celecoxib 20uM, combined administration of rosiglitazone 25uM and celecoxib 20uM for 48 hours

Figure 6 BrdU method to detect the proliferation inhibition rate of cyst lining epithelial cells after rosiglita 50uM alone, celecoxib 20uM, rosiglitazone 50uM and celecoxib 20uM for 48 hours

Figure 7 Flow cytometry detection of the apoptosis rate of cyst lining epithelial cells after rosiglitazone 50uM, 100uM alone, celecoxib 20uM, 50uM, rosiglitazone 50uM and celecoxib 20uM for 48 hours

Figure 8 Transmission electron microscope observation of the ultrastructure of cyst lining epithelial cells in combination with rosiglitazone 50uM and celecoxib 20uM for 48 hours

A. Chromatin edge assembly B. Formation of a large number of apoptotic bodies

Figure 9 Comparison of serum urea nitrogen levels in each group in animal experiments

Figure 10 Comparison of kidney weight/body weight in each group in animal experiments

The following examples are only used to further explain the present invention, but not to limit the scope of the present invention.

Example

Example 1 Experimental study on the combined application of a certain concentration of rosiglitazone and celecoxib to inhibit the proliferation of cyst lining epithelial cells

1. Experimental method

1. Primary cultured human cyst lining epithelial cells, identified by immunohistochemistry and electron microscopy.

The specimens were obtained from nephrectomy polycystic kidney tissue. Cut the superficial and transparent vesicles, peel off the superficial fibrous membrane, and cut the vesicle wall into debris. After being digested with 0.1% collagenase for 1 hour, the cells were inoculated into culture flasks coated with rat tail collagen, and the culture medium was D-Valine modified MEM containing 20% fetal bovine serum. When cells are close to confluence, digest and passage. Inverted phase-contrast microscope observation. Immunohistochemistry and cell enzyme chemistry were used for cell identification, and electron microscopy was used to observe adhesion plaques and microvilli. Carry out the next cell experiment with the 3rd-5th passage primary culture.

2. BrdU method was used to detect the inhibitory effect of rosiglitazone 25uM combined with different concentrations of celecoxib on the proliferation of cyst lining epithelial cells.

Cell proliferation was measured by BrdU method. Cell subculture ADPKD cyst lining epithelial cells in logarithmic growth phase were seeded in 96-well plates (1×10 ⁴ /well), and incubated with DMEM+F12 medium containing 10% fetal bovine serum for 24 hours (37°C, 5% CO2) , Change the serum-free DMEM+F12 medium to synchronize for 24h. Discard the supernatant, add rosiglitazone 25uM and serum-free DMEM+F12 culture solution containing different concentrations of celecoxib (10, 20, 50, 100, 200, 400uM) for 24, 48, 72 hours respectively, and set The dosing group was the control group. Each group has 6 replicate wells. BrdU detection was carried out according to the instructions of Roche, and the brief description is as follows: add 10ul BrdU labeling solution to each well, incubate at 37°C for 6 hours, add 200ul pre-cooled fixative solution to each well to fix for 30 minutes, add 100ul ribonuclease to each well and incubate at 37°C For 30 minutes, add 100ulanti-BrdU-POD to each well and incubate at 37°C for 30 minutes (wash 3 times with 0.01mol/L PBS buffer containing 250ul 10% fetal bovine serum between each step), add 100ul peroxide to each well Enzyme substrate solution + substrate enhancer, incubate at room temperature for 20 minutes to develop color, measure the absorbance with a microplate reader, the measurement wavelength is 405nm, and the reference wavelength is 490nm. After trypan blue staining, the living cells reached more than 95%, indicating that the drug does not produce obvious cytotoxicity at this experimental concentration. The formula for calculating the inhibition rate of cell proliferation is: (absorbance value of the control group-absorbance value of the drug-dosed group)/absorbance value of the control group×100%, to calculate the increment of the inhibition rate of cell proliferation.

3. BrdU method was used to detect the inhibitory effect of the combination of celecoxib 10uM and different concentrations of rosiglitazone on the proliferation of cyst lining epithelial cells.

The method was the same as above, adding celecoxib 10uM and serum-free DMEM+F12 culture solution containing different concentrations of rosiglitazone (10, 20, 50, 100, 200, 400uM) for 24, 48, 72 hours respectively. Six replicate wells were set up for each group, and the increment of cell proliferation inhibition rate was calculated.

2. Experimental results

1. Primary culture and identification of human cyst lining epithelial cells.

Primary cultured human cyst lining epithelial cells were positive for anti-cytokeratin and vimentin monoclonal antibodies, but negative for anti-factor VIII, cytodesmin, actin, and fibroblast-specific protein monoclonal antibodies. Alkaline phosphatase staining showed gray-black positive reaction, and adhesion plaques and microvilli were seen under the electron microscope, which proved that it was derived from epithelial cells without fibroblast contamination, as shown in Figure 1. Cyst lining epithelial cells proliferate significantly faster than normal tubular epithelial cells, which can well reflect the pathophysiological characteristics of ADPKD patients and can be used as a reliable platform for evaluating drug efficacy.

2. BrdU method detection showed that: the combination of rosiglitazone 25uM (11.84mg/L) and different concentrations of celecoxib can dose-dependently increase the inhibitory effect on the proliferation of cyst lining epithelial cells (see Figure 2), Compared with the group without rosiglitazone, there is a statistical difference (P<0.05); but when celecoxib is 100-400uM (38.14-152.55mg/L), especially when it is above 200uM, the toxicity to cells obviously increase.

3. BrdU assay showed that the combination of celecoxib 10uM (3.81mg/L) and different concentrations of rosiglitazone could dose-dependently increase the inhibitory effect on the proliferation of cyst lining epithelial cells (see Figure 3), Compared with the group without celecoxib, there was a statistical difference (P<0.05); but when rosiglitazone was above 400uM (189.38mg/L), the toxicity to cells increased.

Example 2 Comparison of curative effects of combination therapy of rosiglitazone and celecoxib in different concentrations.

1. Experimental method

1. The BrdU method was used to detect the inhibitory effect of the combination of rosiglitazone and celecoxib in different concentrations on the proliferation of cyst lining epithelial cells.

The method is the same as above, and the medicines are added according to the following schemes alone or in combination, respectively acting on the cyst lining epithelial cells for 48 hours. Six replicate wells were set up in each group, and the cell proliferation inhibition rate was calculated.

Single use group: rosiglitazone 25uM (11.84mg/L), rosiglitazone 50uM (23.67mg/L), rosiglitazone 100uM (47.35mg/L); celecoxib 10uM (3.81mg/L ), Celecoxib 20uM (7.62mg/L), Celecoxib 50uM (19.05mg/L).

Compatibility combination group:

Rosiglitazone 25uM/celecoxib 10uM (molar ratio 5:2, weight ratio 3.1:1), rosiglitazone 25uM/celecoxib 20uM (molar ratio 5:4, weight ratio 1.6:1), Rosiglitazone 25uM/celecoxib 50uM (molar ratio 1:2, weight ratio 1:1.6); rosiglitazone 50uM/celecoxib 10uM (molar ratio 5:1, weight ratio 6.2:1), Rosiglitazone 50uM/celecoxib 20uM (molar ratio 5:2, weight ratio 3.1:1), rosiglitazone 50uM/celecoxib 50UM (molar ratio 1:1, weight ratio 1.2:1); Rosiglitazone 100uM/celecoxib 10uM (molar ratio 10:1, weight ratio 12.4:1), rosiglitazone 100uM/celecoxib 20uM (molar ratio 5:1, weight ratio 6.2:1), Rosiglitazone 100uM/celecoxib 50uM (molar ratio 2:1, weight ratio 2.5:1).

2. Using flow cytometry and transmission electron microscopy to detect the effects of single and combined use of thiazolidinedione drugs and COX-2 specific inhibitors on the apoptosis of cyst lining epithelial cells at the best compatible concentration

Primary cultured ADPKD cyst lining epithelial cells in the logarithmic growth phase were inoculated in 6-well plates (5×10 ⁵ /well), and the best compatible concentrations of rosiglitazone alone, celecoxib alone, rosiglitazone + celecoxib used in combination for cells, each group set up 3 duplicate wells, after 48 hours of drug action, digest with trypsin, wash 2 times with pre-cooled PBS, resuspend cells in binding buffer, add AnnexinV/FITC and propidium iodide Color development was performed in the dark, detected by flow cytometry, and the apoptosis index was analyzed by MCYCLE software. Treat the cells in the same way, after digestion and centrifugation, fix with 2.5% glutaraldehyde at 4°C for 1 hour, then wash with sodium cacodylate buffer for 3 times, fix with 1% starve acid for 1 hour, wash with PBS for 3 times, and routinely embed , LKB ultrathin microtome section, lead staining, observation of cell ultrastructure under transmission electron microscope.

2. Experimental results

1. The BrdU method was used to detect the inhibitory rate of proliferation of cyst lining epithelial cells in different combinations of rosiglitazone and celecoxib. The results of some combinations and single use are summarized as follows:

(1) Rosiglitazone 50uM, celecoxib 10uM alone, and rosiglitazone 50uM/celecoxib 10uM in combination (molar ratio 5:1, weight ratio 6.2:1), their proliferation inhibition rates were 24.96% ± 6.25%, 12.80% ± 2.79%, 46.88% ± 3.55%, the proliferation inhibition rate of the combination of the two drugs is higher than the sum of the proliferation inhibition rate achieved by the two drugs alone, the results prove that the combination of the two drugs has a significant effect synergistic effect (see Figure 4).

(2) Rosiglitazone 25uM, celecoxib 20uM alone, and rosiglitazone 25uM/celecoxib 20uM in combination (molar ratio 5:4, weight ratio 1.6:1), their proliferation inhibition rates were 11.32% ± 4.74%, 25.80% ± 5.97%, 46.07% ± 4.62%, the proliferation inhibition rate of the combination drug is also higher than the sum of the proliferation inhibition rate achieved by the two drugs alone, and the combination of the two drugs has obvious synergistic effect (See Figure 5).

(3) Rosiglitazone 50uM, celecoxib 20uM alone, and rosiglitazone 50uM/celecoxib 20uM in combination (molar ratio 5:2, weight ratio 3.1:1), their proliferation inhibition rates were 24.96%±6.25%, 25.80%±5.97% and 60.50%±3.27%, the combined drug proliferation inhibition rate is also higher than the sum of the proliferation inhibition rate achieved by the two drugs alone, the combination of the two drugs has obvious synergistic effect (See Figure 6).

2. Cyst lining epithelial cells detected by flow cytometry Rosiglitazone 50uM, rosiglitazone 100uM, celecoxib 20uM, celecoxib 50uM; Rosiglitazone 50uM/celecoxib 20uM (molar ratio 5: 2, weight ratio 3.1: 1), their apoptosis rates were 12.3% ± 2.5%, 18.5% ± 3.8%, 10.9% ± 1.7%, 17.9% ± 2.6%, 30.5% ± 3.9% % (see Figure 7). In addition, the transmission electron microscope found that the apoptotic cells in the combination group increased significantly, and the chromatin margins and apoptotic bodies increased significantly (see Figure 8).

The above test results clearly show that the different concentrations of the thiazolidinedione drug rosiglitazone and the COX-2 specific inhibitor celecoxib can significantly increase the number of cyst lining epithelial cells compared with the two drugs used alone. Proliferation inhibition rate, and can more significantly promote cyst lining epithelial cell apoptosis, with a significant synergistic effect.

Example 3 Comparison of curative effects of other thiazolidinediones and COX-2 specific inhibitors in combination with different concentrations.

According to the method of Example 2, the combination of thiazolidinedione drugs troglitazone and pioglitazone and COX-2 specific inhibitors meloxicam and radicicol alone and in different concentrations were determined. The inhibitory rate of proliferation of cyst lining epithelial cells and the apoptosis rate of cyst lining epithelial cells. The results show that the combination of thiazolidinediones and COX-2 specific inhibitors at different concentrations can also significantly increase the inhibition rate of cyst lining epithelial cell proliferation compared with the two drugs alone, and can significantly promote cyst formation. Apoptosis of lining epithelial cells, with a marked synergistic effect.

Example 4. Rosiglitazone and Celecoxib Combined Treatment of Polycystic Kidney Disease Animal Model Han: Comparison of Curative Effects in SPRD Rats

1. Experimental method

1. Animal experiment protocol

Male Han: SPRD heterozygous (Cy/+) rats were randomly divided into groups: unintervented control group, TZDs treatment group (rosiglitazone 10 mg/kg·d), Cox-2 inhibitor treatment group (celecoxib 10 mg/kg d). kg·d) and combined therapy group (rosiglitazone 4 mg/kg·d+celecoxib 3 mg/kg·d), 10 rats in each group. From the 3rd week after weaning to the 11th week, the treatment group was given the pure drug (suspended in 1% CMC-Na) for oral administration, and the control group was given 1% CMC-Na for oral administration, and measured every week Record your weight.

2. Monitoring of parameters and biochemical indicators

At the 11th week, the rats were weighed, and 2ml of blood was collected retroorbitally, and the serum was collected for liver and kidney function, blood sugar, blood lipid and other related analysis, and whole blood was collected for blood routine analysis. Urine was collected in the metabolic cage (after 8 hours of water deprivation, enter the metabolic cage, fasted for water, and the urine collector collected urine for 12 hours) for urine osmotic pressure and urine protein quantitative analysis. Soften-II rat tail volume marker sphygmomanometer was used to measure blood pressure, and the blood pressure was measured in a quiet state, and the average value was taken 3 times for each mouse.

3. Histological Analysis

After the male heterozygous rats were sacrificed, the kidneys and livers were weighed and fixed for histological analysis. After the contralateral kidney was perfused with 4% paraformaldehyde, part of the renal tissue (including the cortex, medulla and papilla) was fixed in 10% neutral formalin, embedded in paraffin, made into 3um sections for HE and PAS And Masson staining, IDA-2000 pathological graphic analysis system to quantify the histological indicators of cystic diseases: cyst index (cyst index), renal fibrosis score (fibrosis score) and interstitial inflammatory cell infiltration. Cyst index: Randomly select 20 fields of view under 100 times magnification and analyze the average area percentage of cyst small lumen in the image by using graphic processing software. According to foreign literature reports, the area exceeds the average area of glomeruli ( Small lumens of 9,000um ² ) were counted as cysts. Judgment index of fibrosis score: Randomly select 20 fields of view under 100 times magnification and analyze the average value of the area percentage of dot matrix in the image occupied by Masson's staining of renal interstitial fibrosis staining by graphic processing software. Evaluation index of interstitial inflammatory cell infiltration: 0 no infiltration; 1 focal mild; 2 focal moderate; 3 diffuse mild; 4 diffuse moderately severe.

4. Statistical analysis

All data ends with Said, SPSS 11.0 was used for statistics, and t-test or analysis of variance was used for comparison between groups.

2. Experimental results

After 8 weeks of treatment, there was no difference in body weight and liver function in the clinical indicators between each treatment group and the control group, but arterial systolic blood pressure, 24h urine protein decreased, serum urea nitrogen (P<0.05), and urine osmotic pressure increased (P<0.05). 0.01), kidney weight/body weight (P<0.05), cyst index, fibrosis index (P<0.01), interstitial inflammatory cell infiltration (P<0.05) in histological indicators were all significantly improved (see Table 1), low There were statistically significant differences in urine osmolality, serum urea nitrogen (see Figure 9), kidney weight/body weight (see Figure 10), cyst index, and fibrosis index between the dose combination therapy group and the larger dose of single drug therapy group (P< 0.05), while the arterial systolic blood pressure, 24h urine protein quantification, and interstitial inflammatory cell infiltration did not reach statistical differences (P<0.05).

The above results show that the combination of thiazolidinedione drug rosiglitazone and COX-2 specific inhibitor celecoxib is better than either drug alone in improving the course and histological progression of polycystic kidney disease. curative effect.

Table 1: Parameters and histological indicators of male Han:SPRD heterozygous (cy/+) rats in each group

index Control group 0 (N=10) Rosiglitazone group 10mg/kg·d (N=10) Celecoxib group 10mg/kg·d (N=10) Low-dose combination therapy group Rosiglitazone 4mg/kg·d+celecoxib

3mg/kg·d(N＝10) Mean arterial pressure (mmHg) urine osmolality (mosm/H ₂ O) 24h urine protein quantification (mg) alanine aminotransferase (U) serum urea nitrogen (umoI/L) body weight (g) kidney weight/body weight (%) cyst index (%) Fibrosis index (%) Inflammatory cell infiltration 119±9.4876±10386±16.846.5±7.815.6±2.8368±24.51.63±0.1251.9±6.216.7±4.32.7±0.63 96.3±7.3 ^** 1624±178 ^** 54±12.1 ^** 39.8±6.312.2±1.7 ^* 365±23.71.38±0.11 ^* 30.7±6.7 ^** 8.9±3.7 ^** 1.68±0.52 ^* 102±12.1 ^* 1520±148 ^** 49±13.1 ^** 42.2±7.511.8±2.1 ^* 359±30.71.36±0.14 ^** 33.6±7.8 ^** 9.0±3.2 ^** 1.56±0.49 ^* 87.8±8.7 ^**▲ 1930±123 ^**▲ 48±14.7 ^** 45.5±6.99.7±1.48 ^*▲ 362±29.61.16±0.12 ** ^▲ 22.9±5.6 ^**▲ 4.9±2.4 ^**▲ 1.50± 0.56 ^*

Compared with the control group ^* P<0.05 ^** P<0.01, compared with the single drug group ^▲ P<0.05

Example 5. Comparison of Curative Effects of Other Thiazolidinedione Drugs and COX-2 Specific Inhibitors in Combined Treatment of Polycystic Kidney Disease Animal Model Han:SPRD Rats

According to the same method of Example 4, the combination of thiazolidinedione drugs troglitazone and pioglitazone respectively combined with COX-2 specific inhibitors meloxicam and radicicol was determined to treat animals with polycystic kidney disease Efficacy of the model Han: SPRD rats. The results showed that: the combination of thiazolidinediones and COX-2 specific inhibitors was compared with the two drugs alone, and the combination of thiazolidinediones troglitazone and pioglitazone and COX-2 specific inhibitors Met The combination of loxicam and radicicol is more effective in improving the course and histological progression of polycystic kidney disease than any single drug.

Example 6

The compound tablet of pioglitazone and celecoxib is made according to the following formula: (all used contents are weight percentages)

Pioglitazone 11.2%

Celecoxib 9%

Microcrystalline Cellulose 75.8%

PVP-k30 2%

Magnesium Stearate 2%

Mix pioglitazone, celecoxib, microcrystalline cellulose, and PVP-k30, use ethanol as a soft material, granulate through a 12-mesh sieve, ventilate and dry below 70°C, granulate, add magnesium stearate, mix well and press Slices, ready to serve. Wherein the weight ratio of celecoxib to pioglitazone is 1:1.24.

Example 7

The compound tablet of rosiglitazone and celecoxib is made according to the following formula: (all used contents are weight percentages)

Rosiglitazone Maleate 14%

Celecoxib 4.5%

Microcrystalline Cellulose 77.5%

PVP-k30 2%

Magnesium Stearate 2%

Mix rosiglitazone maleate, celecoxib, microcrystalline cellulose, and PVP-k30, use ethanol as a soft material, granulate through a 12-mesh sieve, ventilate and dry below 70°C, granulate, add stearin Magnesium acid and then mixed and pressed into tablets. Wherein the weight ratio of celecoxib to rosiglitazone maleate is 1:3.1.

Example 8

According to the same method of embodiment 7, the compound tablet of rosiglitazone and celecoxib has been obtained according to the following formula: (all contents used are weight percentages)

Rosiglitazone Maleate 10%

Celecoxib 8%

Microcrystalline Cellulose 50%

Calcium Carbonate 20%

Calcium bicarbonate 3%

PVP-k30 4%

Magnesium Stearate 6%

Wherein the weight ratio of celecoxib to rosiglitazone maleate is 1:1.24.

Example 9

According to the same method of embodiment 7, the compound tablet of rosiglitazone and celecoxib has been obtained according to the following formula: (all contents used are weight percentages)

Rosiglitazone Maleate 2.1%

Celecoxib 13%

Microcrystalline Cellulose 50%

Calcium carbonate 21.9%

Calcium bicarbonate 3%

PVP-k30 4%

Magnesium Stearate 6%

Wherein the weight ratio of celecoxib to rosiglitazone maleate is 1:0.155.

Example 10

According to the same method of embodiment 7, the compound tablet of rosiglitazone and celecoxib has been obtained according to the following formula: (all contents used are weight percentages)

Rosiglitazone Maleate 24.8%

Celecoxib 2%

Microcrystalline Cellulose 43.2%

Calcium carbonate 20%

Calcium bicarbonate 3%

PVP-k30 4%

Magnesium Stearate 3%.

Wherein the weight ratio of celecoxib to rosiglitazone maleate is 1:12.4.

Example 11

Rosiglitazone-celecoxib compound tablet is made according to the following formula: (all contents used are weight percentages)

Rosiglitazone Maleate 15.5%

Celecoxib 2.5%

Microcrystalline Cellulose 45%

Calcium bicarbonate 30%

PVP-k30 3%

Magnesium stearate 4%

The preparation method is the same as in Example 7, wherein the weight ratio of celecoxib to rosiglitazone maleate is 1:6.2.

Example 12

Rosiglitazone-celecoxib compound capsules were prepared according to the following formula: (all contents used are weight percentages)

Rosiglitazone Maleate 4%

Celecoxib 6.5%

Calcium Carbonate 45%

Starch 44.5%

Mix calcium carbonate and starch, sieve, then mix evenly with appropriate amount of ethanol-dispersed rosiglitazone maleate and celecoxib powder, pass through a 120-mesh sieve, dry at 60-70°C for 2 hours, and mix well The medicinal powder is filled into empty capsules. Wherein the weight ratio of celecoxib to rosiglitazone maleate is 1:0.62.

Example 13

The pioglitazone celecoxib compound capsules were prepared according to the following formula: (all contents used are weight percentages)

Pioglitazone 2%

Celecoxib 6.5%

Calcium Carbonate 45.5%

Starch 46%

Mix calcium carbonate and starch, sieve, then mix evenly with appropriate amount of ethanol-dispersed rosiglitazone maleate and celecoxib powder, pass through a 120-mesh sieve, dry at 60-70°C for 2 hours, and mix well The medicinal powder is filled into empty capsules. Wherein the weight ratio of celecoxib to pioglitazone is 1:0.31.

Example 14

Made rosiglitazone and celecoxib hair compound oral liquid by following formula: (contents used are all percentages by weight)

Rosiglitazone Maleate 14%

Celecoxib 6%

D, L-malic acid Appropriate amount

Methylparaben 0.5%

Propylparaben 0.5%

Sucrose 69%

Sucrose monostearate 10%

Edible flavor Appropriate amount

Add distilled water to 1000ml

Dissolve sucrose in an appropriate amount of distilled water to dissolve, and prepare 10% of rosiglitazone maleate, celecoxib, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, and sucrose monostearate Alcohol solution, add to the above solution, stir evenly while adding, then add distilled aqueous solution of D, L-malic acid, adjust the pH value to about 5, add distilled water to 1000ml, mix well, take a sample to measure the content and pH value, pass After that, it can be sterilized by canning. Wherein the weight ratio of celecoxib to rosiglitazone maleate is 1:2.3.

Example 15

The drop pill preparation of pioglitazone and celecoxib composition is made according to the following formula: (all used contents are weight percentages)

Pioglitazone 35%

Celecoxib 5%

Macrogol 600 60%

Heat polyethylene glycol 600 on a water bath to melt it, add pioglitazone and celecoxib, mix well, keep warm at 90°C for later use, preheat the dripping device, and pre-cool the coolant liquid paraffin to 10-15°C, Drop at a speed of 50-70 drops. Collect the dripping pills in the cooling liquid, filter off the cooling liquid, absorb the paraffin with filter paper, and dry it in the air. Wherein the weight ratio of celecoxib to rosiglitazone maleate is 1:7.

Example 16: Injection vials

Celecoxib and pioglitazone are dissolved in 5 L of double distilled water with 100 g of active ingredients and 5 g of disodium hydrogen phosphate in a weight ratio of 1:3, and the resulting solution is adjusted to pH 6.5 with 2N hydrochloric acid, and sterile filtered , transferred into injection vials, lyophilized under sterile conditions, and sealed under sterile conditions. Each injection vial contains 20 mg of active ingredient.

Example 17: Injection Ampoule

Celecoxib and rosiglitazone maleate are dissolved in 10L of double-distilled water with 100 grams of active ingredients in a weight ratio of 1: 3.1, and the solution formed is sterile filtered and poured into ampoules. Freeze-dried under conditions and sealed under sterile conditions. Each ampule contains 10 mg of active ingredient.

Embodiment 18: suppositories

The mixture of celecoxib and rosiglitazone maleate in a weight ratio of 1:2 consisting of 30 grams of active ingredients, 100 grams of soybean lecithin and 1400 grams of cocoa butter was melted, poured into molds, and cooled. Each suppository contains 20 mg of active ingredient.

Example 19: Ointment

500 mg of the active ingredient of the composition composed of celecoxib and rosiglitazone maleate at a weight ratio of 1:4 was mixed with 99.5 g of petrolatum under aseptic conditions.

Embodiment 20: sugar-coated tablet

The pharmaceutical composition containing celecoxib and rosiglitazone maleate was prepared according to the method of Example 7, and compressed into tablets, followed by coating with sucrose, potato starch, talc, tragacanth gum and dyes by conventional methods.

Claims (41)

1, a kind of pharmaceutical composition is characterized in that said composition comprises: thiazolidinediones medicine a) epoxidase 2 specific inhibitors, and b), wherein a) and b) mol ratio be 1: 0.1-40.

2, the pharmaceutical composition of claim 1 is characterized in that wherein said epoxidase 2 specific inhibitors are selected from celecoxib, rofecoxib, para and examine former times, valdecoxib, Ai Tuokao former times, meloxicam, nimesulide and radicicol; Described thiazolidinediones medicine is selected from rosiglitazone, troglitazone, pioglitazone, ciglitazone, englitazone and their pharmaceutically useful salt.

3. the pharmaceutical composition of claim 2, wherein said salt are the salt that thiazolidinediones medicine and mineral acid or organic acid form.

4. the pharmaceutical composition of claim 3, wherein said salt comprise the salt that thiazolidinediones medicine and hydrochloric acid, bromine hydracid, iodine hydracid, sulphuric acid, nitric acid, phosphoric acid, acetic acid, maleic acid, fumaric acid, citric acid, citric acid, oxalic acid, succinic acid, tartaric acid, malic acid, mandelic acid, trifluoroacetic acid, pantothenic acid, methanesulfonic acid and p-methyl benzenesulfonic acid form.

5. the pharmaceutical composition of claim 2 is characterized in that wherein said epoxidase 2 specific inhibitors are selected from celecoxib, meloxicam and radicicol; Described thiazolidinediones medicine is selected from rosiglitazone, troglitazone, pioglitazone and their pharmaceutically useful salt.

6. the pharmaceutical composition of claim 5, it is characterized in that wherein said epoxidase 2 specific inhibitors are celecoxibs, described thiazolidinediones medicine is rosiglitazone or pioglitazone or its pharmaceutically useful salt, and wherein the mol ratio of celecoxib and pioglitazone or rosiglitazone or its pharmaceutically useful salt is 1: 0.125-40.

7. the pharmaceutical composition of claim 6 is characterized in that wherein said salt is maleate, fumarate, citrate, citrate, succinate, tartrate, pantothenate or malate.

8, each described pharmaceutical composition of claim 1-7, the mol ratio that it is characterized in that wherein said epoxidase 2 specific inhibitors and described thiazolidinediones medicine is 1: 0.25-20.

9, each described pharmaceutical composition of claim 1-7, the mol ratio that it is characterized in that wherein said epoxidase 2 specific inhibitors and described thiazolidinediones medicine is 1: 0.5-10.

10. each described pharmaceutical composition of claim 1-7, the mol ratio that it is characterized in that wherein said epoxidase 2 specific inhibitors and described thiazolidinediones medicine is 1: 1-5.

11. each described pharmaceutical composition of claim 1-7, the mol ratio that it is characterized in that wherein said epoxidase 2 specific inhibitors and described thiazolidinediones medicine is 1: 1-2.5.

12. a pharmaceutical preparation is characterized in that pharmaceutical composition that it contains among the claim 1-11 each as active component and one or more pharmaceutically useful carriers, wherein the mol ratio of active component is 1: 0.1-40.

13. the pharmaceutical preparation of claim 12 is characterized in that wherein said active component is celecoxib and pioglitazone or its pharmaceutically useful salt, wherein the mol ratio of celecoxib and pioglitazone is 1: 0.125-40.

14. the pharmaceutical preparation of claim 12 is characterized in that wherein said active component is celecoxib and rosiglitazone or its pharmaceutically useful salt, wherein the mol ratio of celecoxib and rosiglitazone is 1: 0.125-40.

15. each pharmaceutical preparation of claim 12-14, the mol ratio that it is characterized in that wherein said active component is 1: 0.25-20.

16. each pharmaceutical preparation of claim 12-14, the mol ratio that it is characterized in that wherein said active component is 1: 0.5-10.

17. each pharmaceutical preparation of claim 12-14, the mol ratio that it is characterized in that wherein said active component is 1: 1-5.

18. each pharmaceutical preparation of claim 12-14, the mol ratio that it is characterized in that wherein said active component is 1: 1-2.5.

19. each pharmaceutical preparation of claim 12-14 is characterized in that wherein said pharmaceutical preparation is oral administration form, drug administration by injection form or topical form.

20. the pharmaceutical preparation of claim 19 is characterized in that wherein said oral administration form comprises tablet, slow releasing tablet, lozenge, aqueous solution or oil suspension, dispersion powder or granule, emulsion, hard or soft capsule, syrup or elixir.

21. the pharmaceutical preparation of claim 19 is characterized in that wherein said drug administration by injection form is the aqueous solution of aseptic injection or the oil-in-water microemulsion of aseptic injection.

22. the pharmaceutical preparation of claim 19 is characterized in that wherein said topical form is suppository, cream, unguentum, gel, solution or suspension.

23. one kind prepares each the method for pharmaceutical preparation of claim 12-22, it is characterized in that the pharmaceutically useful carrier of wherein said active component and one or more is mixed.

24. the application of each pharmaceutical composition in the medicine of preparation treatment multicystic kidney disease among the claim 1-10.

25, the described application of claim 24 is characterized in that described medicine is with oral administration form, drug administration by injection form or topical form administration.

26, a kind of screening prevents and treats the method for multicystic kidney disease active drug, it is characterized in that said method comprising the steps of:

(1) provides a kind of with former ADPKD patient's cyst lining epithelial cells of being commissioned to train foster and described multicystic kidney disease Han:SPRD rat;

(2) with the medicine of desire screening with contact with former ADPKD patient's cyst lining epithelial cells of being commissioned to train foster or/and the medicine that this multicystic kidney disease Han:SPRD rat is screened with desire;

(3) observe following any phenomenon: epoxidase 2 high expresseds are suppressed, PGE2 discharge reduce, the expression of inflammatory mediator is reduced, inflammation and interstitial fibrosis are alleviated, cyst lining epithelial cells propagation is suppressed or apoptosis;

(4), judge that then this screened medicine is to preventing and treating the active drug of multicystic kidney disease if listed arbitrary phenomenon takes place in (3) step.

27. a method of preventing and treating the mammal multicystic kidney disease is characterized in that uniting a kind of epoxidase 2 specific inhibitors of use and a kind of thiazolidinediones medicine to described mammal.

28. the method for claim 27 is characterized in that describedly uniting that to use be with described epoxidase 2 specific inhibitors and thiazolidinediones medicine respectively individually successively or be applied to described mammal simultaneously.

29. the method for claim 27 is characterized in that describedly uniting that to use be that medication preparation with described epoxidase 2 specific inhibitors and thiazolidinediones medicine becomes pharmaceutical composition or pharmaceutical preparation to be applied to mammal.

30. each method among the claim 27-29 is characterized in that described mammal is human.

31. each method among the claim 27-29 is characterized in that the mol ratio of described epoxidase 2 specific inhibitors and thiazolidinediones medicine is 1: 0.1-40.

32, the method for claim 27 is characterized in that wherein said epoxidase 2 specific inhibitors are selected from celecoxib, rofecoxib, para and examine former times, valdecoxib, Ai Tuokao former times, meloxicam, nimesulide and radicicol; Described thiazolidinediones medicine is selected from rosiglitazone, troglitazone, pioglitazone, ciglitazone, englitazone and their pharmaceutically useful salt.

33. the method for claim 32 is characterized in that wherein said salt is the salt of thiazolidinediones medicine and mineral acid or organic acid formation.

34. the method for claim 33 is characterized in that wherein said salt comprises the salt that thiazolidinediones medicine and hydrochloric acid, bromine hydracid, iodine hydracid, sulphuric acid, nitric acid, phosphoric acid, acetic acid, maleic acid, fumaric acid, citric acid, citric acid, oxalic acid, succinic acid, tartaric acid, malic acid, mandelic acid, trifluoroacetic acid, pantothenic acid, methanesulfonic acid and p-methyl benzenesulfonic acid form.

35. the method for claim 32 is characterized in that wherein said epoxidase 2 specific inhibitors are selected from celecoxib, meloxicam and radicicol; Described thiazolidinediones medicine is selected from rosiglitazone, troglitazone, pioglitazone and their pharmaceutically useful salt.

36. the method for claim 35, it is characterized in that wherein said epoxidase 2 specific inhibitors are celecoxibs, described thiazolidinediones medicine is rosiglitazone or pioglitazone or its pharmaceutically useful salt, and wherein the mol ratio of celecoxib and pioglitazone or rosiglitazone or its pharmaceutically useful salt is 1: 0.125-40.

37. the pharmaceutical composition of claim 36 is characterized in that wherein said salt is maleate, fumarate, citrate, citrate, succinate, tartrate, pantothenate or malate.

38, each described method of claim 32-37, the mol ratio that it is characterized in that wherein said epoxidase 2 specific inhibitors and described thiazolidinediones medicine is 1: 0.25-20.

39, each described method of claim 32-37, the mol ratio that it is characterized in that wherein said epoxidase 2 specific inhibitors and described thiazolidinediones medicine is 1: 0.5-10.

40. each described method of claim 32-37, the mol ratio that it is characterized in that wherein said epoxidase 2 specific inhibitors and described thiazolidinediones medicine is 1: 1-5.

41. each described method of claim 32-37, the mol ratio that it is characterized in that wherein said epoxidase 2 specific inhibitors and described thiazolidinediones medicine is 1: 1-2.5.

Images