CN1739521A

CN1739521A - Application of pyrimidone compounds in preparing medicine

Info

Publication number: CN1739521A
Application number: CN 200510017135
Authority: CN
Inventors: 麻彤辉; 杨红
Original assignee: Northeast Normal University
Current assignee: Northeastern University China; Northeast Normal University
Priority date: 2005-09-12
Filing date: 2005-09-12
Publication date: 2006-03-01
Anticipated expiration: 2025-09-12
Also published as: CN100512816C

Abstract

The present invention relates to the application of pyrimidone compounds in preparing medicine for treating secreted diarrhea and autosomal dominant inheritance polycystic kidney disease. Cystic fibrosis transmembrane conductance regulator (CFTR) factor is one kind of chloridion channel protein activated with cyclic adenosine monophosphate, and during designing model, several CFTR activating ways are considered and applied in the screening process, so as to screen out pyrimidone compounds to prepare medicine for treating secreted diarrhea and autosomal dominant inheritance polycystic kidney disease. The present invention can prevent and treat swine diarrhea effectively and inhibit the liquid secretion of epidermis cell of autosomal dominant inheritance polycystic kidney cyst lining, without obvious toxicity.

Description

The application of pyrimidine (sulfur) ketone compounds in pharmacy

Technical field

The present invention relates to the application of pyrimidine (sulfur) ketone compounds in preparation treatment secreting type diarrhoea and autosomal dominant polycystic kidney disease medicine.The invention still further relates to a kind of method of setting up non-human mammal cystic fibrosis model.

Technical background

(cystic fibrosis transmembrane conductanceregulator CFTR) is a kind of activated Cl of adenosine monophosphate (adenosine 3 ' 5 '-cyclic monophosphate acidcAMP) that encircled to cystic fibrosis transmembrane conductance regulatory factor ^-Channel protein, this albumen is expressed in the epithelial cell that air flue, small intestinal, kidney, pancreas and the testis etc. of mammal are located.Hormone (as beta-adrenaline) or toxin (as cholera toxin) etc. activate and depend on the activated protein kinase of cAMP by improving intravital cAMP level, cause the phosphorylation of CFTR thus, thereby make CFTR Cl ^-Channel opener.CFTR is great expression on epithelial cell, and it is Cl ^-Cross over epithelial teleblem passage is provided, simultaneously it still is the check point of transhipment of the salt of transepithelial cell and water.CFTR Cl ^-The effect of passage and a variety of disease such as cystic fibrosis (cystic fibrosis, CF), male sterility, multicystic kidney disease and secreting type diarrhoea is relevant.

One, CFTR and secreting type diarrhoea

Diarrhoea can cause because of being exposed to various cause of diseases or virulence factor, and these factors comprise diarrhoea that cholera toxin, Escherichia coli endotoxin, AIDS cause, diarrhoea that ulcerative colitis causes, alimentary toxicosis or cause the other factors that intestinal secretion increases by CFTR.Secreting type diarrhoea is the main cause of developing country's death of child, approximately has 5,000,000 children to die from this kind disease (Gabriel et al., 1994 Science 166:107-109) every year).In developed country, diarrheal is pathogenic and lethal is also higher.In the U.S., there are every year 8 million peoples to suffer from acute diarrhea, the number of being admitted to hospital reaches 250000, and death toll surpasses 500 people.In addition, infectiousness secreting type diarrhoea also is one of principal disease of harm diversified economy animal.That uses mice studies show that CFTR Cl ^-Passage is that various agonist cause intestinal Cl ^-Excretory final common pathway (Snyderet al.1982 Bull.World Health Organ.60:605-613; Chao et al 1994 EMBO J.13:1065-1072; Kimberg et al.1971 J.Clin.Invest.50:1218-1230).

Liquid secretion plays crucial effects in gastrointestinal tract physiology.The normal condition lower intestinal tract mainly absorbs the liquid in the enteric cavity, electrolyte and nutrient substance, carries out a spot of basal secretion simultaneously and keeps intestinal mucosa humidifying and the food that helps to be mixed in the enteric cavity.The liquid secretion of intestinal is that the enteraden epithelial cell is from substrate direction finding enteric cavity side active transport Cl ^-The transepithelial liquid moving process that is driven (Thiagarajah JR., et al., 2003, Current Opinion in Pharmacology 3:594-599).Na on the enteraden epithelial cell basement membrane ⁺K ⁺-ATP enzyme and K ⁺The Na that channeling produces down ⁺And Cl ^-Concentration difference drives Cl ^-Enter in the cell by the NKCC device that cotransports from basement membrane one side; Cl ^-Enter the intestinal chamber by electrochemical action by the secretion of the CFTR chloride channel on the teleblem; Na ⁺With water along with Cl ^-Enter enteric cavity by the iuntercellular approach.Many physiology and pathological factor can activate Cl ^-Secretion with intestinal juice.For example the neuroregulation approach of intestinal secretion discharges pentahydroxy-color ammonia by the intestinal pheochromocyte, causes activation and the interior cAMP of glandular epithelium and the Ca of cholinergic and VIpergic nerve unit ⁺Increase, and then activate Cl ^-Passage and intestinal juice secretion process.Inflammatory mediator such as prostaglandin, histamine and interleukin etc. also can activate intestinal Cl by different signal pathways ^-Secretion.In addition, nucleotide and purine can also can pass through Ca by signal pathway ⁺⁺Stimulate intestinal Cl with the cAMP signal pathway ^-Secretion.

The CFTR chloride channel plays vital pivotal role in the activity of intestinal liquid secretion.The CFTR knock-out mice reduce owing to intestinal secretion and absorb increase cause intestinal obstruction (Grubb BR., et al., 1997, Am J Physiol, 273:258-266); Cholera toxin causes a large amount of secretions of small intestinal liquid at normal mouse, and does not have this effect at the CFTR knock-out mice.In addition, experiment in vitro also proves the intestinal mucosa that various agonist cause and the enterocyte Cl of cultivation ^-Secretion can be suppressed by anion channel blocker glibenclamide and 5-nitro-2-(3-phenylpropyl amine) benzoic acid [5-nitro2-(3-phenylpropylamino) benzoate, NPPB].

Because diarrhoea finally all can cause dehydration, therefore, one of target of treatment is a correct dehydration, adopts methods such as fluid infusion and feeding at present usually.The CFTR inhibitor of high-affinity will have important effect aspect the treatment secreting type diarrhoea.Though benzidine-2-carboxylic acid (diphenylamine-2-carboxylate, DPC), NPPB and glibenclamide also can suppress the activity of CFTR under the condition of high concentration, but their above-mentioned activity is nonspecific (Cabantchik et al., 1992, Am.J.Physiol.262:C803-C827; McDonough et al., 1994, Neuron 13:623-634; Schultzet al., 1999, Physiol.Rev.79:S109-S144.Edwards et al., 1993, Br.J.Pharmacol.110:1280-1281; Rabe et al., 1995, Pflugers Arch.429:659-662; Yamazaki et al., 1997, Circ.Res.81:101-109).Up to the present, also do not suppress CFTR Cl by specificity ^-Channel function is treated secreting type diarrheal active drug.

Two, CFTR and autosomal dominant polycystic kidney disease

Multicystic kidney disease (Polycystic kidney disease, PKD) be a kind of common hereditary, the cyst that forms the increase of a plurality of carrying out property with renal tubules is a feature, is the one of the main reasons (Wilson PD.2004, NEngl J Med.350:151-164) of end-stage renal failure.Multicystic kidney disease can be divided into autosomal dominant polycystic kidney disease (autosomal dominant polycystic kidney disease by mode of inheritance, ADPKD) and recessive hereditary multicystic kidney disease (autosomal recessive polycystic kidney disease, ARPKD) two kinds.The ARPKD sickness rate is low, is about 1/20000, is more common in infant, and how dead in early days infant is.Crowd's sickness rate of ADPKD is higher, is about 1/400～1/1000.About 50% ADPKD patient advanced into end-stage renal failure at 60 years old, accept among the patient of renal transplantation and dialysis 8～10% clinically, and the end-stage renal failure patient of ADPKD (Hateboer N., et al., 1999, Lancet, 353:103-107).ADPKD is except the kidney performance, and pathological changes also relates to other a plurality of organs, for example the cyst formation of liver, pancreas, spleen and intracranial aneurysm, mitral valve prolapse etc.Have been found that the Disease-causing gene of three ADPKD at present: be positioned at the PKD1 of 16p13.3, the PKD2 that is positioned at 4q21-23 and PKD3 (Ariza M., et al., 1997, J Med Genet, 34:587-589).The all bigger and mutational spectrum more complicated of ADPKD gene does not have tangible mutantional hotspot, and various sudden changes are distributed in whole gene.The pathogeny of ADPKD is also not fully aware of on cell and molecular level, causes ADPKD still to lack effective treatment means so far.

The ADPKD pathology change comprise tubular ectasia and the extracellular matrix that the liquid abnormal secretion causes in renal cells paraplasm and apoptosis, the renal tubules reinvent unusually (GRANTHAM, J.J.1993, J.Am.Soc.Nephrol, 3:1841-1857).Studies have shown that the cAMP level that increases unusually in the liquid secretion of cyst lining epithelial cells and the polycystic kidney tissue is relevant, plays a crucial role in the formation and development of ADPKD cyst.More and more evidences shows the Cl that is subjected to the activated CFTR chloride channel mediation of cAMP ^-Secretion in the ADPKD cyst, play an important role in the fluid accumulation process (Perso A., etal., 2000, J Am Soc Nephrol.11:2285-2296; Murcia NS., et al., 1999, Kidney Int.55:1187-1197; Hanaoka K., et al., 1998, J Am Soc Nephrol.9:903-916), prompting is by suppressing CFTR Cl ^-The formation and development of gathering the prevention cyst of liquid may become the New Policy that ADPKD treats in the ADPKD cyst thereby transport activity is blocked.

Three, CFTR and cystic fibrosis

(cystic fibrosis CF) is a kind of mortality genetic diseases that is caused by the CFTR recessive mutation to cystic fibrosis.By cystic fibrosis patient and CF mouse models result of study being shown CFTR in the fluid transport of small intestinal and pancreas and male fertility, play an important role (Grubb et al., 1999.Physiol.Rev.79:S193-S214; Wong, P.Y., 1997, Mol.Hum.Repord.4:107-110).Although carrying out property pulmonary function pathological changes is to cause CF patient's morbidity and dead common cause, yet the CFTR sudden change causes still unclear at present (the Pilewski et al. of mechanism of cystic fibrosis patient air flue pathological changes, 1999, Physiol.Rev.79:S215-S255).Utilizing tissue or animal to set up the cystic fibrosis model is extremely important to pulmonary lesion and the mechanism of causing a disease of illustrating cystic fibrosis and causing.Studies show that more and more the reason that pulmonary's cystic fibrosis produces is because air flue submucosal gland liquid secretion deficiency and macromole excessive secretion have caused lung fine hair to remove the infection of afunction and antibacterial.Yet owing to from the air flue material of lung transplant serious pathological changes is arranged all, research is greatly limited.Utilizing the CFTR inhibitor to set up large animal cystic fibrosis model will be extremely important to research submucosal gland CFTR role in water, salt and macromole secretion process.The discovery of the CFTR inhibitor of high-affinity will play a significant role aspect the CF large animal model setting up, and it is to research CF pathogeny and to seek effective treatment approach also significant equally.

Summary of the invention

The purpose of this invention is to provide the application of pyrimidine (sulfur) ketone compounds in preparation treatment secreting type diarrhoea and autosomal dominant polycystic kidney disease medicine.Another object of the present invention provides a kind of method of setting up non-human mammal cystic fibrosis model.

The invention provides high-affinity pyrimidine (sulfur) ketone CFTR inhibitor and handle relevant symptom or the situation method of curee's cystic fibrosis transmembrance regulator (CFTR) ion transport dysfunction with it.Pyrimidine (sulfur) ketone compounds is by finding on the basis that has utilized at the model discrimination of screening and the direct bonded inhibitor of CFTR a large amount of chemical compounds.Owing in the Model Design process, consider the multiple activated pathway of CFTR, and with multiple activated pathway use in conjunction in screening process, thereby the pyrimidine among the present invention (sulfur) ketone compounds thinks to be combined in Cl ^-Transhipment passage place.By to contain chemical compound in the various combinatorial chemical library of 100,000 kinds of structures screen we obtained several belong to pyrimidine (sulfur) ketone compounds can suppress CFTR Cl effectively ^-The chemical compound of transport function.These chemical compounds are structurally all different with known CFTR inhibitor or activator.The strongest wherein active CFTR inhibitor can effectively suppress people's air flue cell Cl in concentration during for 10M ^-Electric current.When being 100 μ g/kg, concentration can suppress fluid accumulation in the mouse small intestine that cholera toxin causes effectively.Inject once a day, can effectively prevent the generation of piglet diarrhea in continuous 7 days by 0.25mg/kg when the diarrheal piglet is taken place, and do not show tangible toxicity in vivo effect.When being 500 μ g/kg, concentration can suppress autosomal dominant inheritance, AD polycystic kidney cyst lining epithelial cells liquid secretion effectively.The CFTR inhibitor that we found in addition is rapid, reversible and be that CFTR is special to the inhibitory action of CFTR.

The structure of the pyrimidine among the present invention (sulfur) ketone compounds comprises a heterocycle of being made up of six atoms, be connected with a carbonyl or ghiourea group on the heterocycle, being pyrimidinones when being connected with a carbonyl on the heterocycle, is the pyrimidine thioketone compounds when being connected with a ghiourea group on the heterocycle.Particularly among the present invention in pyrimidine (sulfur) the ketone compounds molecular formula, wherein X is respectively hydrogen, organic group, halogen atom, nitro, azo group, hydroxyl or sulfydryl arbitrarily arbitrarily; Y ₁, Y ₂Be respectively hydrogen, any organic group, any halogen atom, nitro, azo group, hydroxyl or sulfydryl; A ₁Be oxygen atom or sulphur atom; A ₂Be respectively hydrogen, any organic group, any halogen atom, nitro, azo group, hydroxyl or sulfydryl.

Pyrimidine under some concrete condition (sulfur) ketone compounds has following molecular formula:

X is methyl, ethyl, propyl group, isopropyl or pi-allyl, Y ₁Be alkyl or the hydrogen of C1-C6, Y ₂Be alkyl or the hydrogen of C1-C6, Y ₁With Y ₂Identical or different, A ₁Be oxygen atom or sulphur atom, A ₂Be alkyl or the hydrogen of C1-C6.

Some concrete forms of chemical compound among the present invention are:

(1) N-(2, the 3-3,5-dimethylphenyl)-6-methyl-2-sulfur-4-(4-aminomethyl phenyl)-1,2,3,4-tetrahydropyrimidine-5-amide

(2) N-(2, the 4-3,5-dimethylphenyl)-6-methyl-2-sulfur-4-(4-aminomethyl phenyl)-1,2,3,4-tetrahydropyrimidine-5-amide

(3) N-(2, the 3-3,5-dimethylphenyl)-4-(4-ethylphenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine-5-amide

(4) N-(2, the 4-3,5-dimethylphenyl)-4-(4-ethylphenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine-5-amide

(5) N-(2, the 3-3,5-dimethylphenyl)-4-(4-isopropyl phenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine-5-amide

(6) N-(2, the 4-3,5-dimethylphenyl)-4-(4-isopropyl phenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine-5-amide

(7) 4-(4-allyl phenyl)-N-(2,3-diethyl phenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine-5-amide

(8) 4-(4-allyl phenyl)-N-(2,4-diethyl phenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine-5-amide

(9) N-(2, the 6-3,5-dimethylphenyl)-6-methyl-2-sulfur-4-(4-aminomethyl phenyl)-1,2,3,4-tetrahydropyrimidine-5-amide

(10) N-(2, the 6-3,5-dimethylphenyl)-4-(4-ethylphenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine-5-amide

(11) N-(2, the 6-3,5-dimethylphenyl)-4-(4-isopropyl phenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine-5-amide

(12) 4-(4-allyl phenyl)-N-(2,6-diethyl phenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine-5-amide

The invention still further relates to the dosage form of above-mentioned pyrimidine (sulfur) ketone compounds preparation treatment secreting type diarrhoea and autosomal dominant polycystic kidney disease medicine.Chemical compound among the present invention can be made the form of solid, semisolid, liquid or gas with suitable, pharmaceutical carrier, diluent, excipient or adjuvant etc.For example tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant and aerosol.Chemical compound among the present invention can pass through the number of ways administration.That these approach comprise is oral, under the oral cavity, rectum, vein, peritoneum, subcutaneous, air flue etc.

Chemical compound in dosage form among the present invention can be its pharmaceutical salts form, and they also can separately or be united or use jointly with other pharmaceutical active compounds.

When making oral medicine, the chemical compound among the present invention can be made sheet shape, powder, granule or capsule separately or with proper additive.Additive that can be conventional for example with lactose, mannitol, corn starch, potato starch etc., or the bonding agent of cellulose, cellulose derivative, acacin, corn starch or gelatin, lubricant such as Pulvis Talci, magnesium stearate, or be mixed and made into oral medicine with diluent, buffer agent, wetting agent, antistaling agent and flavoring agent etc. as required.

The injection type of the chemical compound among the present invention can for the compound dissolution among the present invention, suspend, be emulsified in water or the nonaqueous solvent (as vegetable oil, synthetic fatty glyceride, esters, higher fatty acids or propylene glycol).Can add conventional cosolvent, isotonic agent, suspending agent, emulsifying agent, stabilizing agent and antistaling agent when needing.

Chemical compound among the present invention can be made the form of aerosol and pass through inhalation.Chemical compound among the present invention can be made aerosol with compressions such as dichlorodifluoromethane, propane, nitrogen together.

Chemical compound among the present invention can also be mixed and made into suppository as different substrate such as emulsifying base, water-soluble base.The suppository that chemical compound is made among the present invention can pass through rectally.The carrier of suppository can be that cocoa butter, carbowax and polyvinyl alcohol etc. at room temperature are solid, and the carrier that under body temperature, melts.

When being form of medication with oral or rectum, the chemical compound among the present invention can be made the measurement unit form with syrup, ingredients and suspending agent etc.Measurement unit can be teaspoon, soupspoon, sheet or bolt.Contain a certain amount of one or more inhibitor in each measurement unit.During with intramuscular injection or intravenous injection can be with the compound dissolution among the present invention in sterilized water, generic physiological saline or other allow medicinal carrier.

Along with the object of being treated or administering mode or route of administration difference, dosage is also different.Because the needed dosage of different mammals differs greatly, for example the per weight servant so the dosage range of the chemical compound among the present invention is very wide, for example can be 0.1-10mg/kg/ individuality/sky than low 20,30 even 40 times of the needed dosage of rat.Also there is influence in same different administering mode to dosage.For example, the chemical compound among lumbar injection 10-20mg the present invention once can effectively be blocked by the inductive diarrhea of mouse of cholera toxin, but dosage will improve tens of times and just can reach effect same when oral.

Representative dosage forms can be the solution that is suitable for drug administration by injection; Take 2-6 time tablet every day; Take once slow releasing capsule or tablet or the like every day.Slow release effect can be by utilizing dissolved gum capsule material or the capsule that slowly discharges or other known controllable releasable material realization under condition of different pH under different osmotic.

Chemical compound can comprise that the CFTR inhibitor together uses with other preparation with pharmaceutically active among the present invention.

Medicinal excipient such as diluent, carrier, adjuvant are that routine is easy to get, and other allows that medicinal appurtenance such as pH regulator agent, buffer agent, osmotic pressure regulator, stabilizing agent, wetting agent etc. are easy to get for routine.

Using dosage can change the tolerance degree of side reaction to some extent owing to specific compound, the order of severity of disease symptoms, treatment target among the present invention.For a certain definite chemical compound among the present invention, determine using dosage by distinct methods.

CFTR inhibitor among the present invention can be by the dosage of 50-500 μ g/kg body weight, makes non-human cystic fibrosis animal model for 1-3 time by intraperitoneal, subcutaneous or other route of administration administration every day.

CFTR inhibitor among the present invention can be treated secreting type diarrhoea and autosomal dominant polycystic kidney disease effectively, acts on rapid, reversible and does not show tangible toxicity in vivo effect.CFTR inhibitor among the present invention can make non-human mammals such as pig, cattle, sheep occur and the similar phenotypes of cystic fibrosis when carrying out long-term, a large amount of administration.

CFTR inhibitor among the present invention is any situation, disorder, disease, symptom that is associated with CFTR activity (as the ion transport activity of CFTR) in relevant symptom or the situation of treatment CFTR.The situation that these CFTR are relevant, disorder, disease, symptom can be treated by the activity such as the CFTR ion transport activity that suppress CFTR.

CFTR inhibitor of the present invention can be used for treating relevant symptom or the situation of undesired increase with the especially acute small intestinal liquid of the excretory undesired increase of small intestinal liquid.Proved already that the CFTR activity caused that under the effect of the various agonist that comprise cholera toxin intestinal secretion increases, the CFTR inhibitor uses under the dosage that can effectively suppress the CFTR ion transport can reduce the intestinal liquid secretion.Experiment shows to the mouse small intestine liquid secretion, can suppress the excessive secretion of the small intestinal liquid that causes by cholera toxin effectively for through the CFTR of lumbar injection non-toxic dosage inhibitor mice, therefore the CFTR inhibitor can be used for the treatment of enteritis sexual disorder and diarrhoea, especially secreting type diarrhoea.

The CFTR inhibitor also might be used for the treatment of the diarrhoea that AIDs causes (the relevant diarrhoea of AIDs) and struvite gastrointestinal disturbance such as ulcerative colitis, inflammatory bowel disease (IBD), Crohn (Crohn ' s) disease and similar conditions.

CFTR inhibitor among the present invention also can be used for treating the disease such as polycystic kidney one class, and can further be developed as male contraceptive pill by the activity that suppresses CFTR in the testis.

Especially meaningfully, the CFTR inhibitor among the present invention can be used to induce the cystic fibrosis pathological changes the non-human animal.For example will effectively suppress the CFTR inhibitor of CFTR channel activity dosage and on lung, simulate the CFTR functional defect of finding in the cystic fibrosis effectively.The route of administration of CFTR inhibitor is given and detailed argumentation hereinbefore.

Be applicable to that the animal of using the inhibitor induced animal model among the present invention comprises especially mammal of humans and animals, for example non-human primates (monkey, orangutan, gorilla, chimpanzee etc.), Rodents (rat, mice, gerbil jird, hamster, rabbit, ferret etc.), rabbit, pig, horse, Canis familiaris L., cat etc.Wherein most interested is the large animal model.

The CFTR inhibitor also can contact with the tissue that exsomatizes and make the vitro disease model.These tissues can contact with the CFTR inhibitor of CFTR active dose in these tissues of effective reduction and produce the vitro human disease model in 15 minutes to 2 hours.Interested tissue includes but not limited to lung (comprising bronchus and air flue), liver, pancreas, testis etc.The tissue that application CFTR inhibitor was handled carries out physiology, biochemistry, genomics or other research and can identify the novel therapeutic target molecule that plays an important role in the disease Pathophysiology.For example never the in vitro tissue separated of the mankind of cystic fibrosis can be exposed to the inhibitor that is enough to induce the cystic fibrosis disease, and this class research can be identified the novel therapeutic type target molecule that plays an important role in cystic fibrosis pathology physiology.

Description of drawings

Fig. 1 is the principle schematic of CFTR inhibitor screening technology of the present invention.Utilize multiple activator to activate and stablize cotransfection pig CFTR and a kind of Cl ^-/ I ^-The CFTR that expresses on the epithelial cell of responsive yellow fluorescence protein (YFP).After adding test-compound, in the cell culture hole, add and contain I ^-Solvent is measured I ^-Internal flow velocity.

The representative change in fluorescence curve of Fig. 2 for being measured in the single cell culture hole that screening obtained.The matched group that has shown no activator and test-compound among the figure, non-activity test-compound group suppresses the change in fluorescence curve that the CFTR chloride channel is opened active test-compound group with having.

Fig. 3 a is the chemical constitution of the resulting pyrimidine thioketone class CFTR chloride channel inhibitor of screening.

Fig. 3 b is the complete chemical constitution of CFTR chloride channel inhibitor, and relative activity is respectively 0.9 (CFTRinh-1), 0.9 (CFTRinh-2), 0.9 (CFTRinh-3), 0.9 (CFTRinh-4), 1 (CFTRinh-5), 0.8 (CFTRinh-6), 0.9 (CFTRinh-7), 0.1 (CFTRinh-8), 0.6 (CFTRinh-9), 0.9 (CFTRinh-10), 0.8 (CFTRinh-11), 0.2 (CFTRinh-12).

The specific embodiment

Embodiment 1

Present embodiment is described the foundation of CFTR micromolecular inhibitor high-flux cell screening model

Research cell membrane CFTR Cl ^-Ion channel opening status and the basis of screening its inhibitor are to set up highly sensitive, high specificity, reliable and stable and be suitable for carrying out the cell model of high flux screening.At first we use pig CFTR expression plasmid (SV40 promoter, zeocin is a selected marker) and to fluorescence green protein mutant YFP-H148Q expression plasmid (the CMV promoter of halogen sensitivity, G418 is a selected marker) stablize cotransfection Fischer rat thyroid (fischer rat thyroid, FRT) epithelial cell line.Select fluorescence intensity height, rapid, the basic chloride ion permeability of growth and on cytoplasma membrane, stablize the clone FRT/pCFTR/YFP-H148Q/I152L of high expressed.When carrying out high flux screening, with the FTR cell suspension in F-12Coon ' s culture fluid (containing 10% hyclone, 2mM L-Gln, 500u/mL mycillin, 500ug/ml Zeocin), above-mentioned cell suspension is joined in 96 orifice plates (Corning-Costar 3904) of black wall clear bottom, cell quantity is 20,000 in every hole.Cultivated 16-24 hour, and made cell grow up to monolayer, on FluoStar Galaxy luminoscope, measure the chloride channel function.When carrying out the short circuit current mensuration of chloride channel mediation, on Snapwell supporting carrier (Corning-Costar), every hole number is about 500,000 with cell culture.Grew 8-10 days, and treated that iuntercellular closely connected (striding the cell electrical impedance) formation back and carries out chloride ion transmembrane current mensuration with Ussing Chamber.

Embodiment 2

Present embodiment is described CFTR micromolecular inhibitor high-flux cell screening technique and result

1. chemical compound

Various class medicine micromolecular compound (molecular weight ranges is between the 350-550 dalton) storehouse of 100,000 kinds of structures is available from ChemBridge company (ChemBridge Co.San Diego).Compound concentrations is the DMSO solution of 10mM, is sub-packed on 96 orifice plates.During primary dcreening operation, above-mentioned micromolecular compound is screened.The positive compound that screens is carried out postsearch screening and dose-effect analysis.Definite high-affinity positive compound is carried out structural analysis, and buy or synthetic its analog further detects.

2.CFTR the high throughput screening system of inhibitor

Primary dcreening operation is measured on the screening system at the Beckman high-flux cell and is carried out.This system mainly by automatic mechanical hand, 3 meters long slideways, CO2 gas incubator, multi-functional object stages, know ink recorder, wash the plate machine, Tissue Culture Plate cap removing apparatus, agitator, shrouding machine, liquid transfer device and fluorescence detector etc. form.Fluorescence detector is FLUOstar (Galaxy, BMGLab Technologies), and this detector contains two syringe pumps, and excitation wavelength is that (500 ± 10nm), emission wavelength is HQ535/30M (535 ± 15nm) to HQ500/20X.This system controls by central host, and control software is provided by Beckman company.

3.CFTR micromolecular inhibitor high-flux cell screening technique

The density of FTR/pCFTR/YFP-H148Q/I152L cell by every hole 20,000 is laid in 96 orifice plates, and (37 ℃, humidity is 90%, CO in the carbon dioxide incubator ₂Concentration is 5%, and air concentration is 95%) be cultured to the formation monolayer.Below all screening sequences are automation processes by robotic arm manipulation: Tissue Culture Plate is taken out from the carbon dioxide incubator, remove lid, with phosphate buffer (PBS) washed cell three times, each 300 μ l, the last 50 μ l PBS that keep deliver on the liquid-transfering device, add the mixture that 10 μ l contain a kind of activator and (contain 5 μ M FSK in every hole, 100 μ M IBMX and 25 μ M APG), adding tested micromolecular compound to the final concentration of individualized compound after 5 minutes is 10 μ M.Deliver to fluor tester after 15 minutes, measure the fluorescent quenching kinetics of iodide ion by YFP in the stream pair cell in the chloride channel.The method of fluorescence analysis is: with the speed METHOD FOR CONTINUOUS DETERMINATION of 5 point/seconds 14 seconds, wherein preceding 2 seconds was baseline.In the cell culture hole, inject 160 μ l after 2 seconds fast and contain the 137mM NaI PBS of (replacing NaCl), continue to measure 12 seconds.Utilize self-defining cubic equation to calculate the initial velocity of change in fluorescence.

4. elementary The selection result

The purpose of this research is to find the inhibitor that can directly block wild type CFTR chloride channel, therefore at first we make the CFTR chloride channel be in the continuous openness state with a kind of mixture (containing 5 μ M FSK, 100 μ M IBMX and 25 μ M APG) of activator.The screening concentration of chemical compound is 10 μ M.Chemical compound adds behind the 15min of back fast injection I in system ^-, and measure fluorescence kinetics variation (being the internal flow velocity of I-) situation (Fig. 1) in the cell.

As negative control, more for a short time being illustrated in of the slope of curve measured compound activity big more (Fig. 2) under the concentration with the PBS saline solution of the mixture that contains activator for we.In 100,000 kinds of chemical compounds that screened, 99,995 chemical compounds when being 10 μ M, concentration are arranged to I ^-Not obviously influence of internal flow velocity (slope of curve reduces degree＜10%), have 5 chemical compounds to make I ^-Internal flow velocity reduced 10-50%.The positive compound that is screened all has pyrimidine (sulfur) ketone compounds core texture, and (Fig. 3 a).

We so that 196 chemical compounds with pyrimidine (sulfur) ketone compounds core texture have been carried out the fluorescence activity analysis is wherein active the highest shown in Fig. 3 b.

Embodiment 3

Present embodiment is described CFTR micromolecular inhibitor property testing method and result

1. chemical compound

FSK, IBMX, APG, purchase that (Sigma Chemical Co.St.Louis, Mo.) CFTRinh-1, CFTRinh-5, CFTRinh-7 are synthetic voluntarily in Sigma company

2.CFTR inhibitor suppresses the active dynamic analysis of CFTR ion transport

The FRT/pCFTR/EYFP-H148Q cell is pressed every hole 30, the density of 000 cell is laid on 96 orifice plates (Corning-Costar 3904) of black wall clear bottom, cultivating composition is F-12Coon ' s culture fluid (containing 10% hyclone, 2mM glutamine, 500u/mL mycillin), in the carbon dioxide incubator (37 ℃, humidity is 90%, CO ₂Concentration is 5%, and air concentration is 95%) cultivate form after 24 hours behind the monolayer standby.

Tissue Culture Plate is taken out from the carbon dioxide incubator, with phosphate buffer (PBS) washed cell three times, each 300 μ l, the last 40 μ l PBS that keep, in every hole, add mixture (the 5 μ M forskolin that 10 μ l contain the CFTR activator, 100 μ M IBMX, 50 μ M genistein) PBS solution, after 5 minutes, add 10 μ l CFTR inhibitor to be measured again, respectively at the 2nd, 4,6,8,10,15,20,30,60 minute that adds the CFTR inhibitor, carry out fluorescence analysis.The method of fluorescence analysis is: with the speed METHOD FOR CONTINUOUS DETERMINATION of 5 point/seconds 14 seconds, wherein preceding 2 seconds was baseline.Inject 160 μ l after 2 seconds fast and contain the 137mM NaI PBS of (replacing NaCl) in the cell culture hole, continue to measure 12 seconds, the functional experiment of cell chloride ion transhipment is directly with fluorescent value and time changing curve reflection.Calculate the initial velocity of change in fluorescence.

The result shows when CFTRinh-1 concentration is 0.4-0.7 μ M wild type CFTR is had significant inhibitory effect.This activity just can reach maximum (t within 10min _1/2=5min).When CFTRinh-5 concentration is 0.3-0.6 μ M, wild type CFTR there is significant inhibitory effect.This activity just can reach maximum (t within 10min _1/2=4min).When CFTRinh-7 concentration is 0.3-0.6 μ M, wild type CFTR there is significant inhibitory effect.This activity just can reach maximum (t within 10min _1/2=5min).

3.CFTR inhibitor suppresses the active reversibility Analysis of CFTR ion transport

The FRT/CFTR/EYFP-H148Q cell is pressed every hole 30, the density of 000 cell is laid on 96 orifice plates (Corning-Costar 3904) of black wall clear bottom, cultivating composition is F-12Coon ' s culture fluid (containing 10% hyclone, 2mM glutamine, 500u/mL mycillin), in the carbon dioxide incubator (37 ℃, humidity is 90%, CO ₂Concentration is 5%, and air concentration is 95%) cultivate form after 24 hours behind the monolayer standby.

Tissue Culture Plate is taken out from the carbon dioxide incubator, with phosphate buffer (PBS) washed cell three times, each 300 μ l, the last 40 μ l PBS that keep, in every hole, add mixture (the 5 μ M forskolin that 40 μ l contain the CFTR activator, 100 μ M IBMX, 50 μ M genistein) PBS solution, after 5 minutes, add 10 μ l CFTR inhibitor to be measured again, act on after 15 minutes, with phosphate buffer (PBS) washed cell three times, each 300 μ l keep 50 μ l PBS for the last time, add mixture (the 5 μ M forskolin that 10 μ l contain the CFTR activator in every hole, 100 μ M IBMX, 50 μ Mgenistein) PBS solution is respectively at behind the mixture that adds activator the 5th, 10,15,20,30,60 minutes, carry out fluorescence analysis.The method of fluorescence analysis is: with the speed METHOD FOR CONTINUOUS DETERMINATION of 5 point/seconds 14 seconds, wherein preceding 2 seconds was baseline.Inject 160 μ l after 2 seconds fast and contain the 137mM NaI PBS of (replacing NaCl) in the cell culture hole, continue to measure 12 seconds, the functional experiment of cell chloride ion transhipment is directly with fluorescent value and time changing curve reflection.Calculate the initial velocity of change in fluorescence.

The result shows, the activation recovering of removing CFTR behind the CFTRinh-1 5min in the system is more than 1/2; The activation recovering of removing CFTR behind the CFTRinh-5 5min in the system is more than 1/2; The activation recovering of removing CFTR behind the CFTRinh-7 7min in the system is more than 1/2

4.CFTR inhibitor is by directly acting on its function of performance with CFTR

CFTRinh-1, three kinds of inhibitor of CFTRinh-5 and CFTRinh-7 all can suppress the activation to CFTR by FSK, IBMX, APG, can also suppress to comprise the activity of GEN, CPT-cAMP, CPX, 8-MPO, UCCF-029 and UCCF-853.Because above-mentioned these CFTR activator structurally almost do not have dependency, thereby we think CFTRinh-1, and CFTRinh-5 and CFTRinh-7 are by bringing into play its function with the CFTR direct interaction.

5.CFTR inhibitor suppresses CFTR ion transport reactive electro physiological measurement result

On 6 hole Snapwell supporting carriers (Corning-Costar), every hole number is 500,000 with cell culture.Grew 8-10 days, after treating that iuntercellular closely connects (striding the cell electrical impedance) formation, with Ussing Chamber carry out the chloride ion transmembrane current measure the Snapwell of the monolayer FRT cell will be in the polar growth state put into Ussing chamber (Physiologic Instruments, Inc) in.Then basement membrane one side is added and contain 130mM NaCl, 2.7mMKCl, 1.5mM KH ₂PO ₄, 1mM CaCl ₂, 0.5mM MgCl ₂, the 10mM glucose, 10mM Na-Hepes (pH7.3) solution, and the adding of teleblem side contains the 65mM gluconic acid sodium salt, 65mM NaCl, 2.7mM KCl, 1.5mM KH ₂PO ₄, 2mMCaCl ₂, 0.5mM MgCl ₂, 10mM glucose, the solution of 10mM Na-Hepes (pH7.3).System in 37 ℃ of continuous bubbling airs, is carried out permeability with 250 μ g/ml amphotericin Bs to basement membrane then and handles.During mensuration the Ussing groove is linked to each other with the DVC-1000 voltage clamp (World Precision Instruments), measure the short circuit current between Ag/AgCl electrode and the 1M KCl agarose bridge.

We utilize electrophysiological method to study CFTRinh-1, and CFTRinh-5 and CFTRinh-7 suppress the active specificity of CFTR.We add CFTRinh-1 (or CFTRinh-5, or CFTRinh-7) in epithelial membrane one side of the FRT cell of expressing CFTR, measure its influence to short circuit current.Discover CFTRinh-1, CFTRinh-5 and CFTRinh-7 all can fast and effeciently suppress the short circuit current of FRT cell, and this inhibition is active and its concentration is proportionate.

6.CFTR inhibitor oxicity analysis

This experiment is to CFTRinh-1, and the toxicity of CFTRinh-5 and CFTRinh-7 has been carried out external respectively and the body inner analysis.Utilize dihydro rhodamine method to CFTRinh-1, the cytotoxicity of CFTRinh-5 and CFTRinh-7 is measured.The result shows that common insulation is 24 hours when concentration is 100 μ M, the CFTRinh-1 that we found, and CFTRinh-5 and CFTRinh-7 all do not have tangible toxicity to the FRT cell.

Give mice through 1 milligram/kg body weight of lumbar injection above-claimed cpd, injection every day once, continuous injection 7 days or disposable high dose (10mg/kg) lumbar injection, mice diet and amount of drinking water are normal, serum electrolyte, concentration of glucose, liver function index, serum creatine, amylase, hematocrit etc. do not have significant difference with the normal control Mus.

7.CFTR inhibitor multiple drug resistance activity (MDR-1) analysis result

Since CFTR structurally with ABC (ATP-binding cassette, ABC) transport protein MDR-1 has homology, thereby we have studied CFTRinh-1, CFTRinh-5 and CFTRinh-7 are to the inhibitory action of MDR-1.We utilize mensuration ³The accumulation degree of H-vincristine in human bronchial cell 9HTEo-/Dx measured multiple drug resistance albumen (multidrug resistance protein-1, activity MDR-1).Concrete grammar is for to induce 9HTEo-/Dx cellular expression MDR-1 (Rasola et al.1994 J.Biol.Chem.269:1432-1436) with amycin, then cell is pressed 200, the amount in 000/ hole is laid in 24 orifice plates, after 48 hours above-mentioned cell (is contained 130mM NaCl with cleaning mixture, 2mM KCl, 1mM KH ₂PO ₄, 2mM CaCl ₂, 2mM MgCl ₂, 10mM Na-HEPES pH7.3) and washing and contain with 200 μ l ³H-vincristine (0.7 μ M; 1 μ Ci/ml) and the solution of testing sample 37 ℃ the insulation 1 hour.At last with above-mentioned cell with the cleaning mixture of prior pre-cooling washing 3 times, cell is measured radiological dose after with 0.25M NaOH cracking.Work as CFTRinh-1, its transport activity to MDR-1 transhipment vincristine did not all have inhibitory action when CFTRinh-5 and CFTRinh-7 were 5 μ M.

Embodiment 4

Present embodiment is described CFTR inhibitor micromolecular inhibitor and is suppressed inductive mouse small intestine secretion experimental technique of cholera toxin and result.

Laboratory animal fasting (can't help water) was anaesthetized after 12 hours, perform the operation in the knot section of three 2-10 centimeter length of the other ligation of ileal segment.During the enteral administration, injection 0.2-2 milliliter phosphate buffer includes following composition: first section (not having other composition) in every section enteric cavity; Second section (containing 10-100 microgram CFTRinh-1 or CFTRinh-5 or CFTRinh-7); The 3rd section (containing 1-10 microgram cholera toxin and 10-100 microgram CFTRinh-1 or CFTRinh-5 or CFTRinh-7); The 4th section (containing 1-10 microgram cholera toxin and 10-100 microgram CFTRinh-1 or CFTRinh-5 or CFTRinh-7 non-activity analog); The 5th section (containing 1-10 microgram cholera toxin).Postoperative is closed the abdominal cavity, wakens animal, makes it free movable.Put to death animal in 6 hours, take out each section small intestinal, weigh and measure length, calculated weight/length ratio.Found that liquid gathers in a large number after 6 hours in the small intestinal of having injected cholera toxin, cause the intestinal segment extreme expansion, injected cholera toxin and CFTRinh-1 simultaneously, CFTRinh-5 and CFTRinh-7 experimental group liquid reduce more than 80% enteral gathering.

During intravenously administrable, laboratory animal is divided into two groups, all animals are in the knot section of two 2-10 centimeter length of the other ligation of ileal segment.Injection 0.2-2 milliliter phosphate buffer in every section enteric cavity includes 1-10 microgram cholera toxin respectively, and postoperative is closed the abdominal cavity, keeps the narcotism of animal.Give the first treated animal intravenous drip CFTRinh-1 or CFTRinh-5 or CFTRinh-7 normal saline solution in postoperative 2 hours by 0.25 milligram/kg body weight, the second treated animal intravenous drip does not have active CFTRinh-1 or CFTRinh-5 or CFTRinh-7 analog normal saline solution to drip off in 4 hours.Take out each section small intestinal, weigh and measure length, calculated weight/length ratio.Found that, intravenous injection CFTRinh-1 or CFTRinh-5 or CFTRinh-7 group, liquid reduces more than 60% enteral gathering.

Embodiment 5

Present embodiment is described CFTR inhibitor micromolecular inhibitor and is suppressed pig transmissible diarrheal therapeutical effect experimental technique and result

This problem has been carried out a diarrhea research on the spot on kind pig farm, the west of a city, Changchun.Serious yellow scour of piglet to have occurred popular on this kind pig farm at that time, mainly betides 3-10 days the newborn piglet in birth back, presents whole nest morbidity more.Sick pig shows as watery diarrhea, becomes thin and serious dehydration morbidity back death in 3-7 days rapidly.Each nest mortality rate is up to 50-100%.Studies show that, the newborn piglet that uses synthetic CFTRinh-1 or CFTRinh-5 or CFTRinh-7 (0.5 milligram/kilogram) that 10 hairs are given birth to infectious diarrhea and serious dehydration carries out intramuscular injection separately, 8 sick pigs tide over a critical period and rehabilitation as a result, and curative effect is very remarkable.And have only 2 survivals in 10 piglets of DMSO injection.

In addition, the piglet infectious diarrhea high-incidence season to 3 nests selected at random totally 33 piglets by 0.25mg/kg carry out once a day, continuous 7 days preventive usage, none is suffered from diarrhoea in the piglet 30 days as a result.The blood biochemical analysis shows the equal no significant differences of index such as liver function, renal function, electrolyte, blood glucose and blood plasma total protein of medication group and DMSO matched group, shows no tangible toxicity in vivo effect.

Embodiment 6

Present embodiment is described CFTR inhibitor micromolecular inhibitor and is induced pig cystic fibrosis model experiment method and result

Press 0.25mg/kg body weight dosage intramuscular injection CFTRinh-1 or CFTRinh-5 or CFTRinh-7 to newborn piglet, twice of every day (every 12 hours), drug administration by injection continuously.Successive administration 90 days.The pathological change similar to the cystic fibrosis symptom appears in body of gland under pulmonary and the bronchial mucosa.

Embodiment 7

Present embodiment is described experimental technique and the result of CFTR inhibitor micromolecular inhibitor inhibition to the influence of the former foster ADPKD cyst lining epithelial cells liquid secretion speed of being commissioned to train of cAMP stimulation.

It is foster that ADPKD cyst lining epithelial cells former is commissioned to train: will separate the ADPKD mice (Pkd2 that obtains ^WS25/-Type) and people's cyst of kidney tissue (polycystic kidney tissue and isolating single cyst are from accepting renal transplantation or because complication and the part or all of sick kidney of excision is obtained the patient in advance and agreed clinically.Can obtain 1-2 time specimen in general every month) wash blister cavities 3 times with PBS, spend the night at 4 ℃ of enzymolysis with XIV type protease, with serum-free DME-F12 culture fluid flushing blister cavities, collect cyst lining epithelial cells then.Above-mentioned epithelial cell is continued to cultivate, increase at serum-free DME-F12 culture fluid (containing 2% hyclone, insulin, transferrins, ethanolamine, phosphoethanolamine, hydrocortisone, 3, tretinoin, Niu Chuiti extract).

The Cl of ADPKD cyst lining epithelial cells CFTR mediation ^-Amperometric determination: through trypsinization, high density is layered on permeable charge material, and (Transwell-COL Costar) goes up cultivation 24 hours with above-mentioned cell, change the bottom culture fluid into the DME-F12 mixed-culture medium then and (contain 2% hyclone, insulin, transferrins, ethanolamine, phosphoethanolamine, hydrocortisone, 3, tretinoin, cattle hypophysis extract), top surface can obtain to have polar simple columnar epithelium cellular layer in about about 10 days to air continuation cultivation.Measure the Cl of CFTR mediation with Ussing chamber ^-Electric current, assay method is: the epithelial Snapwell of monolayer ADPKD that will be in the polar growth state put into Ussing chamber (Physiologic Instruments, Inc) in.Then basement membrane one side is added and contain 130mM NaCl, 2.7mMKCl, 1.5mM KH ₂PO4,1mM CaCl ₂, 0.5mM MgCl ₂, the 10mM glucose, the CFTRinh-1 of 10mM Na-Hepes (pH7.3) and variable concentrations or CFTRinh-5 or CFTRinh-7 solution, and the adding of epithelial membrane one side contains the 65mM gluconic acid sodium salt, 65mM NaCl, 2.7mM KCl, 1.5mM KH ₂PO ₄, 2mMCaCl ₂, 0.5mM MgCl ₂, 10mM glucose, the solution of 10mM Na-Hepes (pH7.3).System in 37 ℃ of continuous bubbling airs, is carried out permeability with 250 μ g/ml amphotericin Bs to basement membrane then and handles.During mensuration the Ussing groove linked to each other with the DVC-1000 voltage clamp (WorldPrecision Instruments), measure the short circuit current between Ag/AgCl electrode and the 1M KCl agarose bridge, discover CFTRinh-1, CFTRinh-5 and CFTRinh-7 all can fast and effeciently suppress the short circuit current of FRT cell, and this inhibition is active and its concentration is proportionate.

ADPKD cyst lining epithelial cells liquid secretion is measured: will add the CFTR specific inhibitor CaCl of cAMP and variable concentrations in the culture fluid of the polar simple columnar epithelium cellular layer of having of above-mentioned acquisition bottom respectively when measuring the transepithelial liquid secretion with filter paper method ₂, 0.5mM MgCl ₂, 10mM glucose, the CFTRinh-1 of 10mM Na-Hepes (pH7.3) and variable concentrations or CFTRinh-5 or CFTRinh-7.Discover CFTRinh-1, CFTRinh-5 and CFTRinh-7 all can fast and effeciently suppress ADPKD cyst lining epithelial cells liquid secretion.

Embodiment 8

Present embodiment is described CFTR inhibitor micromolecular inhibitor and is suppressed experimental technique and the result that isolated culture ADPKD patient cyst of kidney is organized the influence of liquid secretion speed.

Fresh ADPKD kidney is kept in the DME-F12 tissue culture medium of pre-cooling, separating the volume weight that is in the kidney surface is the cyst tissue of 5-50g.Asepsis injector is with the thorough sucking-off of cyst chamber liquid, and liquid volume in the accurate recording cyst is divided into A, B, C group with cyst.Again inject the mixing cyst chamber liquid of 1/3 volume in the A group cyst; The mixing cyst chamber liquid that contains 10mol/L CFTRinh-1 or CFTRinh-5 or CFTRinh-7 of injection 1/3 volume in the B group cyst; The 10mol/L CFTRact-09 (activator that a kind of CFTR is special) that contains of injection 1/3 volume mixes cyst chamber liquid in the C group cyst.Above-mentioned A, B, three groups of cysts of C are placed the DME-F12 tissue culture medium (containing 5% hyclone, insulin, transferrins, selenium, hydrocortisone, 3, penicillin and streptomycin) of containing 10mol/L forskolin respectively, in the carbon dioxide incubator (37 ℃, humidity is 90%, CO ₂Concentration is 5%, and air concentration is 95%) cultivate and measure the variation of respectively organizing cyst weight after 24 hours, obtain the speed of cyst per surface area liquid secretion divided by the surface area of cyst with the variation of cyst weight.With the A group is contrast, found that CFTRinh-1, and CFTRinh-5 and CFTRinh-7 all can significantly suppress cyst liquid secretion speed.

Claims

1, the application of pyrimidine (sulfur) ketone compounds in pharmacy is characterized in that the application of pyrimidine (sulfur) ketone compounds in preparation treatment secreting type diarrhoea and autosomal dominant polycystic kidney disease medicine.

2, the dosage form of pyrimidine (sulfur) ketone compounds preparation treatment secreting type diarrhoea and autosomal dominant polycystic kidney disease medicine is characterized in that pyrimidine (sulfur) ketone compounds can make the form of solid, semisolid, liquid or gas with suitable, pharmaceutical carrier, diluent, excipient or adjuvant: i.e. tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant and aerosol; Pyrimidine (sulfur) ketone compounds can be by the number of ways administration: comprise under oral, oral cavity, rectum, vein, the peritoneum, subcutaneous, air flue; Pyrimidine (sulfur) ketone compounds can also be its pharmaceutical salts form, can separately or unite or use jointly with other pharmaceutical active compounds.

3, the method for building up of a boar cystic fibrosis model, it is characterized in that to newborn piglet by 0.25mg/kg body weight dosage intramuscular injection CFTRinh-1 or CFTRinh-5 or CFTRinh-7, twice of every day, 12 hours at interval, continuously drug administration by injection is 90 days, and the pathological change similar to the cystic fibrosis symptom appears in body of gland under pulmonary and the bronchial mucosa.