CN101119731A - Combination of dehydroepiandrosterone or dehydroepiandrosterone-sulfate with a leukotriene receptor antagonist for treatment of asthma or chronic obstructive pulmonary disease - Google Patents

Abstract

A pharmaceutical or veterinary composition, comprises a first active agent selected from a dehydroepiandrosterone and/or dehydroepiandrosterone-sulfate, or a salt thereof, and a second active agent comprising a leukotriene receptor antagonist for the treatment of asthma, chronic obstructive pulmonary disease, or any other respiratory disease. The composition is provided in various formulations and in the form of a kit. The products of this patent are applied to the prophylaxis and treatment of asthma, chronic obstructive pulmonary disease, or any other respiratory disease.

Description

Be used for the treatment of the dehydroepiandrosterone of asthma or chronic obstructive pulmonary disease or the compositions of dehydroepiandrosterone sulfate and LTRA

Background of invention

The application is the U. S. application series number of submitting on October 29th, 2,003 10/698,076 part continues, the latter requires the priority of the U.S. Provisional Patent Application series number 60/492,233 of submission on July 31st, 2003, and described application is included in this paper as a reference in full.

Invention field

The present invention relates to contain the compositions of non-glucocorticoid (comprising dehydroepiandrosterone (DHEA), sulphuric acid DHEA or its salt) and LTRA (LTRA).These compositionss can be used for treating asthma, chronic obstructive pulmonary disease (COPD) or other respiratory system disease.

Background is described

Respiratory disorder is relevant with multiple condition, and is very general in the general population.Under the certain situation, they are with inflammation, and inflammation can be aggravated lung symptoms.Respiratory disorder comprises the respiratory system disease of asthma, chronic obstructive pulmonary disease (COPD) and other upper and lower air flue, as allergic rhinitis, adult respiratory distress syndrome (ARDS) and pulmonary fibrosis.

For example, asthma is one of modal disease of industrialized country.It accounts for about 1% of all health care costs in the U.S. in estimation.It is reported that the spreading rate of asthma and mortality rate all increase with alarming speed over the past decade, and asthma is considered to next ten years professional pneumonopathy the most significant.Asthma be a kind of with variable, in most cases be that reversible respiratory tract obstruction is the disease of feature.This process is relevant with pulmonary infection, and the allergy with pulmonary also has relation in some cases.Acute attack can appear in many patients, be called as " asthma attack ", and other patient can suffer the torment of chronic disease.Asthma may suck antigen and shows effect in some cases because of irritated personnel.This disease is commonly referred to as " extrinsic asthma ".Therefore other asthma have intrinsic susceptibility to disease, are called as " intrinsic asthma ", comprise the asthma that different reasons cause, the allergy asthma that causes as the immunoreation of the asthma of adenosine receptor mediation, Ig-E mediation etc.All asthma all has following one group of symptom, and these symptoms are features of this disease: ictal bronchoconstriction, pulmonary infection and/or pulmonary surfactant reduce.Bronchodilator and anti-inflammatory drug are the treating asthma agent of being used always at present.Corticosteroid is modal anti-inflammatory drug, has very big ill effect, but still is widely used in clinical.The more important thing is that available most for the treatment of asthma agent now almost do not have effect to many patients.

The feature of COPD is an airflow obstruction, is generally caused jointly by chronic bronchitis, emphysema or the two.Airway obstruction is normally not exclusively reversible, but has the patient of 10-20% to show really that in treatment airway obstruction obtains some improvement.In chronic bronchitis, the unusual long-term excessive secretion of air flue mucus, inflammation, bronchospasm and infection cause airway obstruction.Chronic bronchitis also to have 3 months at least and occur chronic cough in successive every year in two years at least, to produce mucus or the two is a feature, is got rid of the reason that other cause chronic cough simultaneously.In emphysema, the structural detail of terminal bronchiole (elastin laminin) is destroyed, causes airway walls to be damaged and " waste and old " air of can't breathing out.Alveolar is by permanent damage in emphysema.Emophysematous feature is as far as the unusual irreversible expansion of the air cavity of terminal bronchiole, follows the air cavity wall to destroy simultaneously, but does not have tangible fibrosis.COPD also can cause condary pulmonary hypertension.Condary pulmonary hypertension itself is the unusual disease that raises of a kind of pulmonary artery blood pressure.Right side of heart must be worked than usual harder to resist this high pressure when serious.If this situation continues for some time, the right heart will enlarge and miopragia, and body fluid concentrates on ankle (edema) and abdominal part.The final left heart begins depletion.The heart failure that is caused by pulmonary disease is called as pulmonary heart disease.

The feature of COPD is invasion and attack middle age and old people, and it is one of cause of disease that M ﹠ M is the highest in the world wide.In the U.S., it attacks about 1,400 ten thousand people, and mortality rate occupies the 4th, and disability rate occupies the 3rd.But its M ﹠ M is still raising.According to estimates, since nineteen eighty-two, the prevalence rate of this disease has raise 41% in the U.S., and the mortality rate that ageadjustment is crossed has raise 71% between 1966-1985.The decline (having descended 45%) of the mortality rate of decline of the mortality rate of all causes of disease that this and same time ageadjustment are crossed (having descended 22%) and cardiovascular disease is opposite tendency.In 1998, COPD was total to dead 112,584 examples in the U.S..

But COPD can prevent, and is the contact smoke from cigarette because it is believed that its main cause.The long-term smoking is the modal cause of COPD.It accounts for the 80-90% of all cases.The probability that the people of a smoking dies from COPD is higher 10 times than the nonsmoker.This disease is rarely found on one's body lifelong nonsmoker, and the people of contact smoking environment has to small part trouble airway obstruction.Other causes of disease that propose comprise the pollution and the allergy of the super easy reactivity of trachea or hyperreaction, surrounding air.Airway obstruction among the COPD is normally progressive on one's body the chain smoker.This has caused early stage disable and shortening the life-span.Smoking cessation drops to sickness rate not smoke people's level value, but the injury that smoking causes is irreversible.Other dangerous factors comprise: hereditary, second-hand smoking, contact are during work and the air pollution in the environment, the respiratory tract infection history of Childhood.The symptom of COPD comprises: chronic cough, rapid breathing when uncomfortable in chest, static and firmly the time, breathe effort, the mucus that produces increases and frequent clear larynx.

Almost do not alleviate the COPD symptom at present, prevent to worsen, keep best pulmonary function and the method for raising daily routines vigor and quality of life.Many patients take Drug therapy in its remaining midium or long term in years, and the dosage of medicine constantly increases, and it is more to take medicine when sb.'s illness took a turn for the worse.The present prescription drugs of COPD patient comprises: quick-acting β 2-excitomotors, anticholinergic bronchodilators, long-acting bronchodilator, antibiotic and expectorant.In existing C OPD therapeutic agent, on the progress that stops disease, can reach short run effect rather than long-term effect by taking anticholinergic, beta 2-adrenergic excitomotor and oral steroid.Only recommend oral steroid when acute exacerbation, life-time service can make mortality rate and sickness rate rise.

Fugitive and long-acting suction beta 2-adrenergic agonist can be realized the short-term bronchiectasis and alleviate some symptoms of COPD patient, but the progress of disease is not shown the significant effect of keeping.Fugitive beta 2-adrenergic agonist can improve COPD patient's symptom, as improving motor capacity and cause to a certain degree bronchiectasis, even improves pulmonary function in some serious cases.Find that the maximum effect of new long-acting suction beta 2-adrenergic agonist can compare with fugitive beta 2-adrenergic agonist.Find that salmaterol can improve symptom and quality of life, although it is very little or do not have to the improvement of pulmonary function.Use β 2-agonist to impact, as changing pulse rate, blood pressure and electrocardiogram result to cardiovascular.In few case, use β 2-agonist can cause allergy, as urticaria, angioedema, erythra and oropharynx edema.These cases should be stopped using β 2-agonist.Treat asthma and COPD patient continuously not as treating when needed with bronchodilator ipratropium bromide or fenoterol, therefore illustrate that they are not suitable for use in keeping treatment.On the other hand, the modal ill effect immediately of beta 2-adrenergic agonist is to tremble, and this may cause the reduction of blood plasma potassium, dysrhythmia and arterial oxygen tension force to reduce when high dose.Unite and use the beta 2-adrenergic agonist to compare every kind of medicine of independent use with anticholinergic extra bronchiectatic activity is provided hardly.Yet, in the standard dose that sucks the beta 2-adrenergic agonist, add ipratropium and compared every kind of medicine of independent use in 90 days and can stablize COPD patient better.The occurrence frequency of beta 2-adrenergic agonist ill effect in a word, (as trembling and dysrhythmia) is higher than anticholinergic.Therefore, anticholinergic and beta 2-adrenergic agonist are ineffective to all COPD patients; The combination of these two kinds of medicaments also is like this.

Anticholinergic has been realized the short-term bronchiectasis and has been alleviated some symptoms, but can't improve long-term prognosis in COPD patient.Most of COPD patients have the airway obstruction to small part, can alleviate by ipratropium bromide." lung health research " is found: the vital capacity sign that early stage COPD is arranged in the masculinity and femininity smoker.Through the three kinds of treatments in 5 years relatively, find the decline not significantly effect of ipratropium bromide, the decline of the function available capacity of pulmonary is eased up and stop smoking for the function available capacity of patient pulmonary.But ipratropium bromide produces serious ill effect, and for example cardiac conditions, hypertension, erythra and bladder are detained.

Theophylline has slight bronchiectatic activity on one's body COPD patient, yet they have some common ill effects, and therapeutic domain is narrow.Need the serum-concentration of 15-20mg/l so that reach optimum efficiency, and must careful its serum levels of monitoring.Side effect comprises: feel sick, diarrhoea, headache, stimulate, irritated, epilepsy and cardiac arrhythmia, and they take place under the variable blood concentration of height, on many person, they in addition appear in the therapeutic domain.The dosage of theophylline must be adjusted one by one according to tobacco habit, infection and other treatment, and is very numerous and diverse.Though someone claims that theophylline has antiinflammation aspect asthma,, aspect COPD, still there is not report especially than under the low dosage.The side effect of theophylline limit their purposes with needing frequently to monitor.

Oral corticosteroid has shown the short term results that can improve the COPD acute exacerbation, but take oral steroid for a long time serious adverse is arranged, comprising osteoporosis with cause overt diabetes.Have now found that the corticosteroid of suction does not have actual shortterm effect at trachea aspect histamine super easy to be reactive.In two treatment in 3 years by a definite date researchs carrying out with the fluticasone that sucks, medium and severe exacerbation significantly reduces, and quality of life improves slightly but pulmonary function is not had influence simultaneously.Suffering from more with the fluticasone treatment that sucks, the COPD patient of reversibility disease seems more favourable.

Mucolytic agent has medium benefit for the lasting and frequency that worsens, but for pulmonary function side effect is arranged.Yet N-acetylcystein or other mucolytic agent all do not have significant effect for serious COPD patient (function available capacity＜50%), although once proof can greatly reduce the frequency that worsens.N-acetylcystein produces the intestines and stomach side effect.COPD and congestive heart failure patient to hypoxemia (hypoxaemic) use long term oxygen therapy, and at about 500 days, for almost not influence of mortality in said patients, still, male's survival rate had lifting subsequently, and remains unchanged during back 5 years.But in whole research process, oxygen has reduced the mortality rate of female patient.Hypoxemic COPD patient is continued to carry out oxygen therapy reach 19.3, can reduce total mortality risk.But, up to now, found that only life style changes, stopped smoking and using oxygen therapy (to hypoxemic patient) just can change the long-term process of COPD for a long time.

The also common first-selection of antibiotic is used for respiratory tract infection with other damage and the infection of prevention to sick lung.Expectorant helps lax air flue and remove mucus secretion from air flue, can help to make breathing smooth and easy.In addition, other medicines also can be used to treat the disease relevant with COPD.Comprising: diuretic (being detained when treating, for the excess water of being avoided relevant), Folium Digitalis Purpureae (cardiotonic is arranged) and cough medicine with right heart failure.The medicine of listing later can help to alleviate the symptom relevant with COPD, but can not treat COPD.Therefore, almost do not have to alleviate the COPD symptom now, prevent to worsen, keep best pulmonary function, improve the medicine of daily life vigor and quality of life simultaneously.

Poverty-stricken syndrome of acute respiratory (ARDS) or tetanic lung, shock lung, pump lung and congestive atelectasis are considered to owing to fluid accumulation in the lung and then cause pneumonocirrhosis to cause.This disease generally is to form in 48 hours because a variety of causes causes injury of lung, as wound, head injury, shock, septicemia, repeat blood transfusion, medicine, pulmonary infarction, severe pneumonia, smog suction, radiation, plateau, near drowning etc.In general, ARDS takes place as emergency case, may directly or indirectly be caused the disease of blood vessel seepage body fluid in lung and is caused by other.The expanding ability of lung sharply descends during ARDS, and air bag, substrate or the endothelium of lung caused major injury.The modal symptom of ARDS is that skin, lip and the breathing of fingernail cyanosis, anxiety and moment that breathing painstakingly fast, flaring of nares, histanoxia cause stagnated.By chest X-ray and measure arterial blood gas and can confirm the ARDS tentative diagnosis.ARDS is relevant with other diseases in some cases, treats the acute tumor lysis syndrome (ATLS) of back generation as acute myelogenous leukemia, with cytosine arabinoside.But, ARDS usually with radiotherapy, the chemotherapy of traumatic damage, serious symptom blood infection such as septicemia or other system disease, high dose, cause the multiple organ failure, MOF and cause the inflammatory reaction of death relevant in many cases.Premature infant's lung tissue or pulmonary surfactant ateliosis.The premature infant is exactly a very serious problem when the poverty-stricken syndrome of respiratory tract (RDS) takes place.The premature infant that RDS takes place is now generally with ventilating, cure by oxygen therapy and give the surfactant treatment.Usually also having bronchus dysplasia (BPD) even the premature infant of generation RDS is survived, be also referred to as premature infant's chronic lung disease, usually is fatal.

Have 1/5th American to suffer from allergic rhinitis, all age group all can take place, and annual medical expense is estimated to need 40 to 10,000,000,000 dollars.Because it is to continue flu or nasal sinuses problem that many people take for their symptom, allergic rhinitis are probably by mistaken diagnosis.Allergic rhinitis generally are to produce chemical mediator because IgE combines with the allergen of intranasal, and inducing cell reaction and nerve stimulation cause following inflammatory reaction.Symptom comprises eye and nasal congestion, watery nasal discharge, sneeze, rhinocnesmus, and eyes are itched.Sinusitis, otitis media companion's hydrops and nasal polyp usually can take place in allergic rhinitis patients after after a while.Have approximately 60% allergic rhinitis patients also suffer from asthma simultaneously allergic rhinitis increased the weight of asthma.Active medium that the mastocyte threshing discharges and various cell, blood vessel and mucous gland interact, thereby cause typical rhinitis symptom.After the most of early stage and late phase response of intranasal all occurs in allergen sensitization.But late phase response also sees chronic allergic rhinitis, follows Polyblennia and nasal cavity to stop up.Repeat to contact the allergy that can cause at one or more allergens.The patient also may become to nonspecific stimulation such as cold air and penetrating odor overresponse.The anallergic rhinitis also can infected institute be induced, as viral infection, or relevant with nasal polyp, as taking place among the aspirin idiosyncrasy patient.

Pregnancy, hypothyroidism and contact occupational factor or medicine also can cause rhinitis.So-called NARES syndrome (nonallergic rhinitis with eosinophilia syndrome) is a kind of anallergic rhinitis relevant with the eosinophilic granulocyte of intranasal secretions, and good sending out in middle age followed losing of olfactory sensation.Treatment to allergia and anallergic rhinitis is unsatisfactory.The oneself gives saline and has improved nasal obstruction, sneeze and hyperemia, and can not cause side effect usually, so first-selection is used for conceived patient.Usually alleviate mucous membrane irritation or the drying relevant, alleviate mucosal atrophy and remove mucosa that forms crust or the mucosa that thickens with brine spray with various nose conditions.If use immediately before intranasal drips corticosteroid, normal saline also helps to prevent drug induced local excitation.Antihistaminic (as Te Feinading and astemizole) also can be used for treating allergic rhinitis; But use antihistaminic once to cause ventricular arrhythmia (being called torsive ventricular tachycardia), usually with other drug such as ketoconazole and erythromycin interaction or cardiac trigger problem then.Loratadine (another kind of non-calm class antihistaminic) and cetirizine also do not find with to being separated with harmful effect or relevant between QT with the serious cardiovascular side effect.Yet cetirizine can cause drowsiness so not be widely used.Non-sedating class antihistaminic such as loratadine (Claritin) can suitably alleviate the Pruritus symptom of sneeze, watery nasal discharge and nose, eye, palate, but it is not also tried out in these indications or asthma.On the other hand, terfenadine, loratadine and terfenadine demonstrate extremely slight bronchiectasis effect, alleviate the bronchus allergy of histamine and prevent bronchospasm because of motion and antigen induction.Yet the ask for something in these benefits is higher than the dosage of present recommended dose.Calm class antihistaminic helps to cause the sleep at night, if but they cause drowsiness so by day use can weaken their effect.During use, antihistaminic is usually united use to help the alleviation nasal congestion with decongestant.Sympathomimetic drug can be used as vasoconstrictor and decongestant uses.Three kinds of systemic decongestant pseudoephedrine, phenylpropanolamine and phenylephrines that often use can cause that hypertension, cardiopalmus, cardiopalmus are hyperfunction, irritated, insomnia and headache.Phenylpropanolamine and caffeine (dosage of the 2-3 cup coffee) blood pressure that significantly to raise that interacts.In addition, treat with the medicine of pseudoephedrine and so on and may cause children ' s activity excessive.Yet local decongestant can only use in finite time, because excessively use can cause the expansion of bounce-back property nose.Anticholinergic agents can be used for treating the patient of obvious rhinorrhea or the case that some is special, and as " sense of taste rhinitis ", usually with to take in pungent food relevant, this medicine has been used to treat common cold to the latter.If will be for example sodium cromoglicate (Cromolyn) the preventative use of spraying can reduce sneeze, rhinorrhea and nose and scratches where it itches as nose, and early stage and allergy in late period capable of blocking, but can cause sneeze, temporarily have a headache, even nose calcination.Topical corticosteroid can effectively be treated rhinitis as beclomethasone (Vancenase), particularly treatment scratch where it itches, sneeze and watery nasal discharge symptom, but to the nasal obstruction poor effect.Yet according to the preparation difference, the spraying of corticosteroid nose can cause stimulation, twinge, calcination, even also can cause sneeze.Sometimes also local hemorrhage and perforation of nasal septum can take place, especially when aerocolloidal effect target is incorrect.The local steroid medicine that uses is generally than effective with sodium cromoglicate, when especially treating allergic rhinitis.Immunization therapy is normally useful, and especially for the patient that can have side effects with other medicines, but the immunization therapy expense is high and use inconvenient.The medicine of so-called blocking antibody and change cell histamine release causes IgE to reduce the most at last, can bring many other favourable physiological changies simultaneously.In the disease of this IgE-of acting on mediation is useful, for example suffers from the hypersensitivity among the atopy patient of recurrent otitis media.

Pulmonary fibrosis, interstitial lung disease (ILD) or interstitial pulmonary fibrosis comprise the chronic lung disease above 130 kinds; by the damage lung tissue, in alveolar wall generation inflammation, interstitial lung (or the tissue between the alveolar) cicatrization or fibrosis; make pneumosclerosis and damage lung, therefore gain the name.Respiratory arrest is one of first symptom of this class disease in the motion, also dry cough may occur.Usually symptom and x-ray fluoroscopy all can't accurately be distinguished dissimilar pulmonary fibrosiss.The patient of some pulmonary fibrosis knows pathogenic factor.Some pulmonary fibrosis patients' the cause of disease is unknown or special sending out.The process of this disease generally unpredictable and this disease is fatal inevitably.Its progress comprises that lung tissue thickens and sclerosis, inflammation and dyspnea.Most of patients needs oxygen uptake, and lung transplantation is unique cure method.

The modal in the world cancer of pulmonary carcinoma.2003, only the U.S. was with regard to New Development about 171900 routine pulmonary carcinoma (male's 91800 examples, women's 80100 examples), and Europe has 375000 examples approximately.Pulmonary carcinoma all is to cause the first cause of cancer mortality in masculinity and femininity.Estimate to have in 2003 157200 people to die from pulmonary carcinoma (wherein male 88400 people, women 68800 people), account for 28% of all cancer mortalities of single U.S..Compare the summation of colon cancer, breast carcinoma and carcinoma of prostate, more people die from pulmonary carcinoma (the ACS website, 2003, depth guide: pulmonary carcinoma: main statistics how?).Generally acknowledged smoking is the main cause that causes pulmonary carcinoma, has 90% case to be considered to relevant with Nicotiana tabacum L. approximately.The pulmonary carcinoma risk and every day amount of smoking, suction degree and between age of beginning smoking clear and definite dose-response relationship is arranged.Lifelong smoker's pulmonary carcinoma risk exceeds non smoker 20-30 doubly.Yet the pulmonary carcinoma risk reduces gradually along with the passage that stops the time after the smoking.Male ES's relative risk reduces rapidly with the length of smoking cessation time, but can not reach non smoker's level, and the reduction degree is not as good as women ES (Tyczynski etc., Lancet Oncol.4 (1): 45-55 (2003).

COPD and pulmonary carcinoma normally and deposit disease, potential COPD degree is carried out surgical operation to particular patient measurable whether the needs.For NSCLC (nonsmall-cell lung cancer), have only operation (associating or not combined radiotherapy or adjuvant chemotherapy) to cure.

1 annual survival rate of pulmonary carcinoma (diagnosing out the number of surviving at least 1 year after the cancer) was 42% in 1988, and this mainly is because the progress of surgical technic.

5 annual survival rates in all stages of nonsmall-cell lung cancer only are 15%.5 years relative survival rates of small cell lung cancer are about 6%.

For find NSCLC and the early treatment's that passed through to perform the operation patient before it is diffused into lymph node or other organ, average 5 annual survival rates are about 50%.Yet, only have 15% patients with lung cancer to be diagnosed out in this limitation stage early.

Obviously, in the space that grows a lot aspect the chemoprophylaxis of pulmonary carcinoma and the lung cancer therapy.

Dehydroepiandrosterone (DHEA) (3 beta-hydroxies androst-5-ene-17-ketone) is a kind of by the excretory natural steroid with tangible chemoproection characteristic of adrenal cortex.Epidemiological study shows that the low danger that can cause some kind tumor takes place of DHEA endogenous level increases, as the breast carcinoma of women before the menopause and men and women's bladder cancer.DHEA and DHEA analog such as DHEA-S (sulphuric acid DHEA) suppress tumorigenic ability and are considered to come from it to the active inhibition of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH).G6PDH is the rate-limiting enzyme of hexose monophosphate approach, and this approach is the main source of ribose in the born of the same parents-5-phosphoric acid and NADPH.Ribose-5-phosphoric acid is the essential substrate of synthetic kernel ribotide and deoxyribonucleotide.NADPH also is the synthetic and synthetic cofactor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) of biological nucleic acid.HMG CoA reductase is a kind of special enzyme, and producing 1 mole of mevalonic acid product needs 2 moles of NADPH.Therefore HMG CoA reductase exhausts it being very responsive to the NADPH of DHEA mediation, and the content of mevalonic acid reduces rapidly in the cell that DHEA handles.Mevalonic acid is that DNA is synthetic necessary, and DHEA can make people's cell be arrested in the G1 phase of cell cycle with the extremely similar mode of direct HMG CoA.Because G6PDH is essential for the synthetic mevalonic acid that generation is used for glairy isoprenylation of some cell processes and dolichol (the biosynthetic precursor of a kind of glycoprotein), thus isoprenylation that DHEA can be by exhausting mevalonic acid and then Profilin and glycoprotein synthesize the generation that suppresses tumor.Mevalonic acid is the synthetic center of a cholesterol precursor, also is non-sterol compounds such as the farnesylpyrophosphate and the synthetic center of the yak acyl pyrophosphoric acid precursor of the proteic post translational modification process of various participations; Also be the synthetic center of dolichol prerequisite, and that dolichol is a glycoprotein is synthetic necessary, glycoprotein participates in the communication and the cyto-architectural composition of cell pair cell.Know that already steroid hormone that the patient accepts the adrenal cortex source of suitable drug dose can increase the sickness rate of infectious disease.U.S. Patent No. 5527789 discloses a kind ofly treats method for cancer by giving patient DHEA and ubiquinone, and wherein said cancer is a kind of cancer to the DHEA sensitivity.

DHEA is a kind of 17-KS, the intravital a kind of main adrenocortical hormone of mammal of saying so on amount.Although DHEA is seemingly as the synthetic intermedium of gonad steroid, the major physiological function of DHEA is not also understood fully.Yet the level of known this hormone reduced (reach during old age initial level 5%) since 20 years old.Clinically, DHEA is by system and/or be used for the treatment of psoriasis, gout and hyperlipemia partly, and gives the patient after the coronary thrombosis.In mammal, DHEA has shown to have controlling body weight and antineoplastic action, and use is united with treatment menopausal symptom and manic depression, schizophrenia and Alzheimer's disease with estrogen clinically in Europe.40mg/kg/ days DHEA treatment terminal cancer and multiple sclerosis have been used clinically.The side effect of DHEA has androgen effect, hirsutism and the libido of moderate to strengthen.But these side effect can and/or utilize its analog to overcome by monitoring dosage.Known can be subcutaneous or orally give DHEA to strengthen the reaction of host to infecting, also can use transdermal to paste and send DHEA.Known DHEA also is the metabolic precursor thereof that strengthens the multiple medicine of mammalian immune reaction.DHEA works as prodrug one in other words: when changing into androstenediol, Androst-5-ene-3 beta, and 17-isoallopregnane-3 (β AED), androstene triol or Androst-5-ene-3 beta, 7 β can be used as immunomodulator during 17 beta-triols (β AET).Yet,, therefore be considered to its super immune-enhancing activity and come from it and can change into active higher metabolite because its on cell proliferation before changing into β AED and/or β AET has lymphocytotoxicity effect and depression effect.

Adenosine is the metabolic purine of a kind of participation intermedium, and can constitute the interior amboceptor of important lung of various diseases, and these diseases comprise bronchial asthma, COPD, CF, RDS, rhinitis, pulmonary fibrosis etc.Asthmatic patient has the reaction of remarkable bronchoconstriction to the adenosine of atomizing and normal individual does not have this reaction, and this discovery has illustrated the latent effect of its receptor.Asthma lagomorph model (the dust mite allergy rabbit model of human asthma) produces remarkable bronchoconstriction reaction to the adenosine of atomizing in a similar fashion, but not the demonstration of the rabbit of asthma is reactionless.The nearest work of carrying out with this animal model shows that inductive bronchoconstriction of adenosine and bronchial hyperreactivity can be mainly by stimulating adenosine receptor to mediate in the asthma.The object of not diagnosing out the high response airway disorders before the adenosine therapeutic is given when treating other disease and symptom demonstration can have side effects, comprising death.Adenosine is brought into play the modified unique effect of cellular metabolism in vivo.It can raise cell AMP, ADP and ATP level, this three is the energy intermediate product of cell.Adenosine can stimulate or reduce the active cAMP level of therefore regulating of adenyl cyclase.CAMP plays a role in release, cell division and the hormone of neurotransmitter discharge again.The main effect of adenosine is seemingly as protectiveness damage endocrine.As if adenosine play a role under any situation that ischemia, low oxygen tension or wound occur.The defective synthetic, that discharge, act on and/or degrade of having supposed adenosine can cause the brain exicitatory/amino acid neurotransmitter excessively active, and therefore causes various pathological states.Adenosine also is the symptom that determines bronchial asthma and other respiratory system disease, the main determining factor of inducing bronchoconstriction and air flue smooth muscle contraction.In addition, adenosine makes the asthmatic patient bronchoconstriction but do not have this effect in non-asthmatic patient.Other data show adenosine receptor also may participate in allergy and inflammatory reaction by the hyperkinesia that reduces central dopamine energy system.Supposed that the adjusting to the conduction of inflammatory cell surface signal can influence acute inflammation.It is said that adenosine can produce by stimulating neutrophilic leukocyte to suppress superoxides.Nearest evidence explanation, adenosine also has protective effect at apoplexy, CNS damage, epilepsy, ischemic heart desease, coronary artery bypass in roentgenization and the inflammation.In a word, adenosine seems by ATP adjusting cellular metabolism, as the methionine carrier, reduces the oxygen demand of cell and protect cell to avoid ischemic injuries.Adenosine is that a kind of cell of working as faces ischemia, anoxia, cellular stress and workload increase, or the tissue hormone or the intercellular courier that discharge when the ATP demand surpasses its supply.Adenosine is a kind of purine, and its formation is directly related with the catabolism of ATP.It seems scalable and comprises a series of physiological process of vasoconstriction, hormonal action, function of nervous system, platelet aggregation and lymphocyte differentiation.It also plays a role in DNA formation, ATP biosynthesis and general intermediate metabolism.This illustrates the formation of cAMP in its adjusting brain and the various peripheral organization.Adenosine passes through A ₁And A ₂Two kinds of receptors are regulated cAMP and are formed.Adenosine passes through A ₁Receptor reduces adenylate cyclase activity, and at A ₂The receptor for stimulating adenyl cyclase.Compare A ₂Receptor, adenosine A ₁Receptor is responsive more to adenosine.The CNS effect of adenosine is considered to usually by A ₁Receptor-mediated, and peripheral effect such as hypotension, bradycardia it is believed that it is by A ₂Receptor-mediated.

Now existing several different methods can be used for respiratory tract disease and treatment of conditions, though these methods all have limitation.Glucocorticoid, leukotriene inhibitors, anticholinergic, antihistaminic, oxygen therapy, theophylline and mucolytic are wherein arranged.Although the side effect of glucocorticoid has many reports, it is to use the most widely.Yet when treatment asthma, present available medicine is all only effective to the sub-fraction case, rather than all effectively.Also there is not a kind of Therapeutic Method can treat multiple other respiratory tract diseases now.Theophylline is a kind of important drugs of treatment asthma, is a kind of known adenosine receptor antagonists, it is reported that it can eliminate the bronchoconstriction of adenosine mediation in the asthma rabbit body.Also there is report to show that optionally adenosine A 1 receptor antagonists 8-cyclopenta-1,3-dipropyl xanthine (DPCPX) can suppress bronchoconstriction, inflammation and the bronchus allergy of adenosine mediation in the allergy rabbit body.Yet the therapeutic and the prophylactic use of existing adenosine A 1 receptor specific antagonists are limit by its toxicity.For example, the theophylline that once was widely used in treating asthma has the remarkable toxicity (the intestines and stomach, cardiovascular, nerve and biological disorderly) of frequent generation owing to its narrow therapeutic dose scope.Though carried out the many decades extensive studies, also do not existed the fact of adenosine receptor specific antagonists that can clinical use to confirm that these medicines are all toxic.

Can buy LTRA (LTRA) montelukast at present and prevent and the long-term treatment adult and the asthma of child patient more than 12 months, and alleviate and be grown up and pollinosis's symptom of child patient more than 2 years old.It is by Merck ﹠amp; Co., (Whitehouse StatioN NJ) sells with Singulair  (Menglusitena) Inc., and oral dose is a 4mg granule or 4,5 and the 10mg tablet.

Can buy the LTRA zafirlukast at present and come long-term treatment asthma.(Wilmington DE) sells with Accolate , and oral dose is 10mg and 20mg tablet by AstraZeneca Pharmaceuticals LP for it.

The pranlukast of SmithKline Beecham (Ultair) is a kind of LTRA of being held license and being gone through to sell in Japan by Ono Pharmaceutical.

U.S. Patent No. 5,660,835 (and corresponding PCT announces WO 96/25935) have been disclosed a kind of by giving the new method that object dehydroepiandrosterone (DHEA) or DHEA related compound come treatment target asthma or adenosine disappearance.This patent has also disclosed a kind of the suction maybe can breathe the new pharmaceutical composition that containing of dosage form can breathe granularity DHEA or DHEA related compound.

U.S. Patent No. 5,527,789 have disclosed and have a kind ofly come the treatment target method for cancer by giving object DHEA or DHEA related compound and ubiquinone (being used for the treatment of the heart failure that is caused by DHEA or DHEA related compound).

U.S. Patent No. 6,087,351 have disclosed and a kind ofly reduce or consume method in the body of adenosine in the subject by giving object DHEA or DHEA related compound.

The U.S. Patent Application Serial Number 10/454,061 that on June 3rd, 2003 submitted to has disclosed a kind of by giving the method that object DHEA or DHEA related compound come treatment target COPD.

The U.S. Patent Application Serial Number of submitting on June 17th, 2,003 10/462,901 has disclosed a kind of stable dry powder formulations that is sealed in the DHEA of the sucked form in the container.

The U.S. Patent Application Serial Number of submitting on June 17th, 2,003 10/462,927 has disclosed the stable dry powder formulations of the DHEA-S dihydrate crystal form of a kind of suitable treatment asthma and COPD.

Above-mentioned patent and patent application are included in this paper as a reference in full.

Obviously press for now the respiratory tract disease, pneumonopathy and the cancer that find a kind of new effective method to treat to treat at present or at least effectively and do not have a method of present side effects of pharmaceutical drugs.This sickness influence respiratory tract, particularly lung and pulmonary passageway comprise dyspnea, asthma, bronchoconstriction, pulmonary infection and allergy, surfactant exhaustion or low secretion etc.Also press in addition and find a kind of prevention and Therapeutic Method, this method only needs a spot of activating agent, thereby comparison is cheap and side effect is lighter.

In addition, also need the method for guaranteeing that the patient relatively cooperates and is convenient to take the required multiple chemical compound of prevention or treatment asthma, COPD or other respiratory system disease when taking medicine.

Summary of the invention

The invention provides a kind of compositions that contains at least two kinds of activating agents.First activating agent comprises non-glucocorticoid, as epiandrosterone (EA) or its salt.Second activating agent comprises LTRA (LTRA).Described compositions comprises the combination of first activating agent and second activating agent.The amount of the amount of first activating agent and second activating agent is in the said composition, is enough to the object that effectively prevention or treatment may be suffered from asthma, COPD or other respiratory system disease when giving the object said composition.Said composition also contains other bioactivator and formula components.Said composition is a kind of be fit to give object or patient, as the mankind or non-human animal's (as non-human mammal), medicinal or veterinary composition.

Described compositions can be used for treating asthma, COPD or has inflammation and other respiratory system disease of complication, comprises and bronchoconstriction, surfactant disappearance and/or the relevant symptom of allergy.

The present invention also provides the method for treatment asthma, COPD or other respiratory system disease, and this method comprises the described compositions of the object that needs this treatment.

The present invention also provides described first activating agent and second activating agent to be used for the application of the medicine of preventative or the above-mentioned asthma of therapeutic treatment, COPD or other respiratory system disease in manufacturing.

The present invention also provides and has comprised the medicine box that described compositions can be failed delivery device.Described defeated delivery device can arrive individual by delivering composition.Preferably, this defeated delivery device comprises that inhaler and aerosol or mist generating device are to carry granule.Preferably be transported to the air flue of object.More preferably be transported to the lung of object.Preferably be transported to desired area.

The major advantage of using described compositions is that the patient of this prevention of needs or treatment can partner treatment.Respiratory system disease such as asthma or COPD are multi-factor diseases, and each patient has different performances or symptom.Therefore Most patients is with the different aspect of multiple Drug therapy to palliate a disease.First activating agent (as DHEA or DHEA-S) can be treated specific patient colony convenient and effectively with the fixed combination of second activating agent (as montelukast, zafirlukast or pranlukast).Improve patient's compliance by the peculiar disease reason of simplifying therapy and paying close attention to each patient, therefore can more promptly treat their specific symptoms.In addition, another benefit of using first and second activating agents simultaneously is convenient or saves time.Uncomfortable especially true when regional when compositions being used for may occur on the subject's body, as said composition being used for the air flue of object.When giving object with compositions, also be like this by the invasive mode.

In addition, described first activating agent (as DHEA or DHEA-S) is a kind of anti-inflammatory agent, when with its conveying or deposit to alveolar membrane and the end of tiny air flue the most effective when peripheral airways rather than airtube slightly.Asthma and some COPD patient's airtube is narrow, and this has limited the conveying (because early stage deposition that low particle speed causes) of first activating agent (as DHEA) and to the effect of peripheral airways slightly of these ends.Use described compositions to improve lasting efficacy of drugs, thereby improved control disease.Anti-leukotriene medicine has alleviated the interstitial edema in the very little peripheral airways.This is also to increasing the peripheral airways diameter and being convenient to carry first activating agent effective.For antihistaminic also is so, this medicine also can alleviate the peripheral airways edema and be convenient to first active agent delivery to the end air flue slightly.

Accompanying drawing of the present invention can further be explained following some aspect of the present invention of being discussed as the part of description of the present invention.

The accompanying drawing summary

Fig. 1 has described the pure DHEA-S2H of micronization that single dose Acu-Breathe inhaler is carried ₂The function of O fines fraction and flow velocity.The result represents with DHEA-S.Also shown IDL data or almost anhydrous micronization DHEA-S among this figure, as seen from the figure, the result when flow velocity is 30L/min is set as zero point, does not therefore detect the quality that enters impacter this moment.

Fig. 2 has described the HPLC chromatogram after almost anhydrous DHEA-S mixes 1 week of storage 50 ℃ of independent storages and with lactose.The pure DHEA-S that contrast stores down for room temperature (RT).

Fig. 3 has described DHEA-S2H ₂O mixes the HPLC chromatogram after storing for 1 week 50 ℃ of independent storages and with lactose.The pure DHEA-S2H that contrast stores down for RT ₂O.

Fig. 4 has described the dissolubility of DHEA-S under two temperature and the function of NaCl concentration.

The function of the dissolubility of DHEA-S and sodium cation inverse concentration when Fig. 5 has described 24-25 ℃.

The function of the dissolubility of DHEA-S and sodium cation inverse concentration when Fig. 6 has described 7-8 ℃.

Fig. 7 has described that room temperature has and the dissolubility of DHEA-S and the function of NaCl concentration when not having buffer.

Fig. 8 has described 24-25 ℃ to be had and the dissolubility of DHEA-S and the function of sodium cation inverse concentration when not having buffer.

Fig. 9 has described DHEA-S solution concentration and time relation under two kinds of conditions of storage.

Figure 10 has described DHEA solution concentration and time relation under two kinds of conditions of storage.

Figure 11 has described the sketch map of atomizing experiment.

Figure 12 has described quality that is deposited on the DHEA-S in the bypass catcher and the functional relationship that places the original solution concentration in the aerosol apparatus.

Figure 13 has described the granular size that DHEA-S aerosol apparatus solution is impacted in cascade.Data are expressed as the meansigma methods of all 7 times spraying experiments.

Figure 14 has described DHEA to the HT-29SF cell inhibiting.

Figure 15 has described the influence of DHEA to HT-29SF cell period profile.

Figure 16 a and 16b have described the cytostatic reverse of the inductive HT-29 of DHEA.

Figure 17 has described the inductive HT-29SF cell of DHEA and has stayed in G ₁The reverse of phase.

Figure 18 has described the effect of DHEA-S to mast cell granuleization.

Figure 19 has described some suitable analog of DHEA.

Figure 20 has described some suitable analog of DHEA.

Figure 21 has described some suitable analog of DHEA.

Figure 22 has described the suitable modification to DHEA C-17 ketone.

DESCRIPTION OF THE PREFERRED

Definition

In the literary composition of the present invention, term " adenosine " and " surfactant " exhaust the level that comprises object and compare level before the object and reduce or exhaust, and it is basic identical with the level before this object, but owing to some other reason, by adjusting the level of these medicaments, the level before comparing will obtain some treatment benefits.

Part or all of respiratory system of the object that contacts with air represented here in term " air flue ".Described air flue comprises and is not limited to throat, trachea, nasal meatus, hole etc.Air flue also comprises trachea, bronchus, bronchioles, whole last ramuscule air flue, respiratory tract ramuscule air flue, alveolar duct and alveolar sac.

Disease or the situation relevant with inflammation on the object air flue represented here in term " airway inflammation ".Airway inflammation can be caused or is attended by following symptom by following symptom: allergy (one or more), asthma, disordered breathing, Cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), allergic rhinitis (AR), adult respiratory distress syndrome (ARDS), microorganism or viral infection, pulmonary hypertension, pneumonia, bronchitis, airway obstruction and bronchus constriction.

Biologically acceptable gaseous state, liquid state, solid-state carrier and composition thereof represented here in term " carrier ", the different drug delivery route that they are suitable for being scheduled to.Carrier is acceptable carrier pharmaceutically or veterinarily preferably.

" effective dose " represents to provide the amount of treatment or prevention benefit here.

" other therapeutic agent " is meant any therapeutic agent outside the described compositions first or second activating agent.

Term " prevention " here be illustrated in the people fall ill before or obtain the prophylactic treatment that carries out before the symptom of condition of illness condition, it just can make object avoid, prevent or reduce the symptom of disease or associated situation like this.Described object can be to suffer from this disease or individuality that the risk of the severity of symptoms of examining out before increases.

Term " respiratory system disease ", here expression disease or the situation relevant with respiratory system.Its example comprises and is not limited to: airway inflammation, allergy (one or more), disordered breathing, capsule fibroid become for example SARS of sick (CF), allergic rhinitis (AR), adult respiratory distress syndrome (ARDS), cancer, pulmonary hypertension, pneumonia, bronchitis, airway obstruction, bronchus constriction, infected by microbes and viral infection.

The treatment of the probability of the symptom that the patient be subjected to treatment like this shows disease or other situations represented to reduce here in term " treatment ".

The invention provides a kind of compositions that contains first activating agent and second activating agent, described first activating agent comprises non-glucocorticoid, and as epiandrosterone (EA), its analog or its salt, described second activating agent comprises LTRA (LTRA).Said composition also contains acceptable carrier, diluent, excipient, bioactivator or composition on medicine or the veterinary drug.Said composition can be used for treating asthma, COPD or other respiratory system disease.Other respiratory system disease that said composition can effectively be treated be pneumonopathy with respiratory system disease and with bronchoconstriction, pneumonia and/or allergy and the relevant symptom of pulmonary carcinoma.

Described first activating agent is epiandrosterone, its analog or acceptable salt pharmaceutically or on the veterinary drug.Described epiandrosterone, its analog or pharmaceutically or on the veterinary drug acceptable salt be selected from non-glucocorticoid with following chemical formula:

In the formula, dotted line is represented singly-bound or two key; R is a hydrogen or halogen; 5 H occurs with α or beta comfiguration, and perhaps the chemical compound of Formula I comprises the racemic mixture of two kinds of configurations; R ¹Be hydrogen or with covalently bound polyvalent mineral of this chemical compound or organic dicarboxylic acid;

Non-glucocorticoid with following chemical formula:

Or

Non-glucocorticoid with following chemical formula:

Or its combination,

In the formula, R1, R2, R3, R4, R5, R7, R8, R9, R10, R12, R13, R14 and R19 independently are H, OR, halogen, (C1-C10) alkyl or (C1-C10) alkoxyl, R5 and R11 independently are OH, SH, H, halogen, pharmaceutically acceptable ester, pharmaceutically acceptable thioesters, pharmaceutically acceptable ether, pharmaceutically acceptable thioether, pharmaceutically acceptable inorganic ester, pharmaceutically acceptable monosaccharide, disaccharide or oligosaccharide, the volution oxidative ethane, volution sulfur ethane (spirothirane),-OSO2R20,-OPOR20R21 or (C1-C10) alkyl, R5 and R6 are=O that R10 and R11 are=O together together; R15 be (1) when R16 be-C (O) is H during OR22, halogen, (C1-C10) alkyl or (C1-C10) alkoxyl, (2) when R16 be halogen, OH or (C1-C10) be H during alkyl, halogen, OH or (C1-C10) alkyl, (3) when being OH, R16 is H, halogen, (C1-C10) alkyl, (C1-C10) thiazolinyl, (C1-C10) alkynyl, formoxyl, (C1-C10) alkanoyl or epoxy, (4) when being H, R16 is OR, SH, H, halogen, pharmaceutically acceptable ester, pharmaceutically acceptable thioesters, pharmaceutically acceptable ether, pharmaceutically acceptable thioether, pharmaceutically acceptable inorganic ester, pharmaceutically acceptable monosaccharide, disaccharide or oligosaccharide, the volution oxidative ethane, volution sulfur ethane,-OSO2R20 or-OPOR20R21, or R15 and R16 are=O together; R17 and R18 independently for (1) when R6 be H OR, halogen, (C1-C10) alkyl or-C (O) is H during OR22,-OH, halogen, (C1-C10) alkyl or-(C1-C10) alkoxyl, (2) when R15 and R16 be H during together for=O, (C1-C10 alkyl) amino, the alkyl of ((C1-C10) alkyl) n amino-(C1-C10), (C1-C10) alkoxyl, the alkyl of hydroxyl-(C1-C10), (C1-C10) alkyl of alkoxyl-(C1-C10), (halogen) m (C1-C10) alkyl, (C1-C10) alkanoyl, formoxyl, (C1-C10) alkoxy carbonyl group or (C1-C10) alkanoyloxy, (3) R17 and R18 are=O together; (4) R17 or R18 form the 3-6 unit ring that contains 0 or 1 oxygen atom with the carbon that they connected; Or (5) R15 forms an epoxide ring with R17 with the carbon that they were connected; R20 and R21 independently are OH, pharmaceutically acceptable ester or pharmaceutically acceptable ether; R22 is H, (halogen) m (C1-C10) alkyl or (C1-C10) alkyl; N is 0,1 or 2; M is 1,2 or 3; Or its acceptable salt pharmaceutically or on the veterinary drug.

Preferably, in chemical formula (I), described multivalence organic dicarboxylic acid is SO ₂OM, phosphate or carbonate, wherein M comprises equilibrium ion, and wherein said equilibrium ion is H, sodium, potassium, magnesium, aluminum, zinc, calcium, lithium, ammonium, amine, arginine, lysine, histidine, triethylamine, ethanolamine, choline, triethanolamine, procaine, benzathine benzylpenicillin, Apiroserum Tham (tromethanine), pyrrolidine, piperazine, diethylamine, sulfatide

And phospholipid

R in the formula ²And R ³Can be identical or different, be straight or branched (C1-C14) alkyl or glucosiduronic acid

Hydrogen atom on 5 of Formula I can α or beta comfiguration perhaps this DHEA chemical compound mixture that is two kinds of configuration of compound appears.The representative compounds of above-mentioned Formula I includes, but not limited to DHEA, at this moment R and R ¹Be respectively hydrogen, contain a pair of key; 16-α bromine epiandrosterone, this moment, R was Br, R ¹Be H, contain a two key; 1-α-fluorine epiandrosterone, this moment, R was F, R ¹Be H, contain a two key; Etiocholanolone, R and R at this moment ¹Be respectively hydrogen, do not contain two keys; And dehydroepiandrosterone sulfate, this moment, R was H, R ¹Be SO ₂OM, M are sulfatide defined above (sulphatide) bases, unparalleled key.But also comprise other material.R is a halogen in the chemical compound of same preferred formula I, for example bromine, chlorine or fluorine, and R1 is a hydrogen, has two keys simultaneously.The chemical compound of most preferred formula I is 16-α-fluorine epiandrosterone.Other preferred chemical compound is DHEA and DHEA salt, as sulfate (DHEA-S).

In a word, non-glucocorticoid, suc as formula (I), (III) and those chemical compounds (IV), the dosage of the salt of their derivant and they is that per kilogram of body weight is about 0.05,0.1,1,5,20-100,500,1000,1500,1800,2500,3000,3600 milligrams.But other dosage of situation according to patient also are suitable.Formula (I), (III) and first activating agent (IV) can be changed a little according to known method or according to known method and be prepared, and this is well-known to those skilled in the art.Referring to U.S. Patent No. 4,956,355; British patent No.2,240,472; EPO patent application No.429; 187, PCT patent documentation No.WO 91/04030; U.S. Patent No. 5,859,000; Abou-Gharbia etc., J.Pharm.Sci.70:1154-1157 (1981); Merck Index MonographNo.7710 (the 11st edition, 1989) etc.

In some embodiments of the present invention, first activating agent can be an epiandrosterone analog or derivatives thereof.Simultaneously, the present invention also comprises the prodrug and the active metabolite of epiandrosterone.Be proficient in those skilled in the art and will know that chemical compound described here can have tautomerism, conformational isomerism, geometrical isomerism and/or enantiomerism.Be interpreted as the present invention includes any tautomerism, conformational isomerism, optical siomerism and/or the geometrical isomerism form of chemical compound with above-mentioned one or more use, and these multi-form mixture.

The metabolite of epiandrosterone, as below with reference to described in the document those, can be used as first activating agent: " with the capillary gas chromatography of the asthmatic children urine steroid of terbutaline treatment " (Capillary gas chromatographyof urinary steroids of terbutaline-treated asthmatic children, Chromatographia (1998), 48 (1/2), 163-165; " the androstenedione metabolism in people's lung fibroblast " (Androstenedione metabolism inhuman lung fibroblasts); Journal of Steroid Biochemistry (1986), 24 (4), 893-7; " the androsterone of human lung tissue in the culture and lung endotheliocyte and 5 α-androstane-3 α, the metabolism of 17-isoallopregnane-3 " (Metabolismof androsterone and 5 α-androstane-3 α, 17 β-diol in human lung tissue and in pulmonaryendothelial cells in culture), Journal of Clinical Endocrinology and Metabolism (1985), 60 (2), 244-50; " the testosterone metabolism of human lung tissue " (Testosterone metabolism by human lung tissue), Journal of Steroid Biochemistry (1978), 9 (1), 29-32; " the androsterone of human lung tissue in the culture and lung endotheliocyte and 5 α-androstane-3 α, the metabolism of 17-isoallopregnane-3 " (Metabolism of androsterone and5 α-androstane-3 α, 17 beta-diol in human lung tissue and in pulmonary endothelial cells inculture), Journal of clinical endocrinology and metabolism (in February, 1985), 60 (2), 44-50; " dehydroisoandrosterone of human lung tissue in the culture and lung endotheliocyte and the metabolism of androstenedione " (Metabolismof dehydroisoandrosterone and androstenedione in human pulmonary endothelial cells inculture), Journal of clinical endocrinology and metabolism (May nineteen eighty-three), 56 (5), 930-5; " the external metabolism of the dehydroisoandrosterone of people's lung and androstenedione " (Metabolism of dehydroisoandrosteroneand androstenedione by the human lung in vitro), Journal of Steroid biochemistry (1977Apr), 8 (4), 277-84; And " the external metabolism of the testosterone of Canis familiaris L. lung " (Testosterone metabolism in dog lungin vitro), Steroids and lipids research (1973), 4 (1), 17-23, these reference materials are included in this paper as a reference in full.

Other suitable analog of the DHEA that can be used as first activating agent has been described here.Figure 19 has described some suitable analog of DHEA, comprises the chemical compound of formula IA, IB, IC and ID.When narrating here the suitable R group, junction point CH ₂Group is represented or is represented with the atom of asterisk labelling.R1 and R3 can be the straight or branched alkyl, comprise benzyl and the optional alkyl that replaces, as aminoalkyl, hydroxy alkyl, ether and carboxylic acid, and optional aryl and the heteroaryl that replaces.For example, R1 and R3 can be

CF ₃CH ₃(CH ₂) _nWherein, the preferred 0-4 of n

Wherein, X=OH, O-alkyl, N

(by H, alkyl or acyl substituted)

M and p independently are 1-4

Wherein, Y=O, NH, N-alkyl or N-acyl group

Wherein, X=CO ₂H or amide

The example of the chemical compound of formula IA comprises

The substituent group that the R2 preferred diacids is deutero-or amino acid derived comprises chloracetyl derivant and acrylate derivative, or the optional aryl that replaces, as benzyl and assorted benzoyl.The example of the chemical compound of formula IB comprises

The example of the chemical compound of formula IC comprises

R4 can be the armaticity group, and the example of the suitable combination thing of formula ID comprises

Other suitable compound is included in the analog that the C-3 position keeps OH or replaces the modification of OH with NH.These analog usually can be from the hero-4-alkene 3 of C-17 acetal protection, and the 17-diketone begins preparation, as shown in figure 20.The chemical compound of formula IE can may as aroma substance, and can be alkynyl, thiazolinyl and alkyl from aryl-lithium reagent also from Grignard reagent.The example of R5 is

CH ₃(CH ₂) _nWherein, n=0-4

The example of the chemical compound of formula IE comprises

R6 can independently be different amine with R8, and can comprise the amine with the described degree of functionality of R1 group.The example of the chemical compound of suitable formula IF comprises

Suitable R 7 groups can also can have the described degree of functionality of R5 from Grignard reagent/organolithium reagent.The example of the chemical compound of formula IG comprises

The example of the chemical compound of formula IH comprises

Other suitable analog comprises that those are at the adorned chemical compound in the C-2 position of DHEA.Suitable modification is shown in Figure 21.R9 can be from alkylating agent, as alkyl, benzyl, assorted benzyl and other active halid derivant.The example of R9 comprises

The example of the chemical compound of formula IJ comprises

R10 can be an aromatic ester, but as contains the Arrcostab of aromatic ring or hetero-aromatic ring or enolization.

The example of the chemical compound of formula IK comprises

R11 can be a series of aromatic aldehydes or assorted aromatic aldehyde, as benzaldehyde and replace variant, pyridine carboxaldehyde or aldehyde that can not enolization, as (CH3) 3CCH=O.The example of the chemical compound of formula IL comprises

R12 can be the subclass of the amine of (for example) R6.The example of the chemical compound of formula IN comprises

Suitable modification to DHEA C-17 ketone is shown in Figure 22.Chemical compound shown in Figure 22 can be used as first activating agent.

Other suitable DHEA analog is described in United States Patent (USP) 6,635,629; European patent 934745; " dehydroepiandrosterone and analog suppress DNA combination and the air flue smooth muscle proliferation of AP-1 " (Dehydroepiandrosteroneand analogs inhibit DNA binding of AP-1 and airway smooth muscle proliferation), Journal of Pharmacology and Experimental Therapeutics (1998), 285 (2), 876-883; " dehydroepiandrosterone and related genera sterin are in the Repiration of vitro inhibition mitochondrial RNA (mt RNA) " (Dehydroepiandrosteroneand related steroids inhibit mitochondrial respiration in vitro), International Journal ofBiochemistry (1989), 21 (10), 1103-7, all these are included in this paper as a reference in full.

Second activating agent is the LTRA (LTRA) that can suppress bronchoconstriction.LTRA chemical compound of the present invention comprises 4,859,692; 5,294,636; 5,319,097; 5,482,963; 5,565,473; 5,583,152; The chemical compound of definition in 5,612,367 and 6,143,775 (its content is included into this paper as a reference).Preferred LTRA is montelukast, zafirlukast and pranlukast.

LTRA is by chemical formula V, (VI) and (VIII) definition:

LTRA is defined by chemical formula V:

In the formula: R1 be H, halogen ,-CF ₃,-CN ,-NO ₂Or N ₃R2 be low alkyl group, low-grade alkenyl, low-grade alkynyl ,-CF ₃,-CH ₂F ,-CHF ₂, CH ₂CF ₃, replacement or unsubstituted phenyl, replacement or unsubstituted benzyl, replacement or unsubstituted 2-phenethyl, two R2 groups that perhaps are connected on the identical carbon atoms can form a heteroatomic ring of 8 yuan at the most that is selected from O, S and N that contains 0-2; R3 is H or R2; CR3 R22 can be the standard amino acid base; R4 be halogen ,-NO ₂,-CN ,-OR3 ,-SR3, NR3 R3, NR3 C (O) R7 or R3; R5 be H, halogen ,-NO ₂,-N ₃,-CN ,-SR2 ,-NR3 R3 ,-OR3, low alkyl group or-C (O) R3; R6 is (CH ₂) s-C (R7R7)-(CH ₂) s-R8 or-CH ₂C (O) NR12 R12;

R7 is H or C _1-4Alkyl; R8 is A) contain 3-12 ring carbon atom and 1 or 2 monocycle or bicyclic heterocycles base that is selected from the ring hetero atom of N, S or O, and each ring in the heterocyclic radical forms by 5 or 6 atoms, or B) the W-R9 base; R9 contains nearly 20 carbon atoms, and is that (1) alkyl or (2) are acyclic or contain the alkyl-carbonyl of 1 heteroatomic monocycle organic carboxyl acid at most in ring; R10 is-SR11 ,-OR12 or-NR12 R12; R11 be low alkyl group ,-C (O) R14, unsubstituted phenyl or unsubstituted benzyl; R12 is H, R11 or is connected two R12 groups on the identical N and can forms one and contain 1-2 the heteroatomic 5 or 6 yuan of rings that are selected from O, S and N; R13 be low alkyl group, low-grade alkenyl, low-grade alkynyl ,-CF ₃, or replacement or unsubstituted phenyl, benzyl or 2-phenethyl; R14 is H or R13; R16 is H, C1-C4 alkyl or OH; R17 is low alkyl group, low-grade alkenyl, low-grade alkynyl or replacement or unsubstituted phenyl, benzyl or 2-phenethyl; R18 be low alkyl group, low-grade alkenyl, low-grade alkynyl ,-CF ₃Or replacement or unsubstituted phenyl, benzyl or 2-phenethyl; R19 be low alkyl group, low-grade alkenyl, low-grade alkynyl ,-CF ₃Or replacement or unsubstituted phenyl, benzyl or 2-phenethyl; R20 is H, C1-C4 alkyl, replacement or unsubstituted phenyl, benzyl, phenethyl or pyridine radicals, or is connected two R20 groups on the identical N and can forms one and contain 1-2 the heteroatomic saturated 5 or 6 yuan of rings that are selected from O, S and N; R21 is H or R17; R22 is R4, CHR7OR3 or CHR7SR2; M and m ' independence are 0-8; N and m ' independently are 0 or 1, and p and p ' independence are 0-8; When r be 1 and X2 be O, S, S (O) or S (O) ₂The time m+n+p be 1-10; When r be 1 and X2 when being CR3R16 m+n+p be 0-10; M+n+p is 0-10 when r is 0; M '+m '+p ' is 0-10; R and r ' independently are 0 or 1; S is 0-3; Q1 is-C (O) OR3,1H (or 2H)-tetrazolium-5-base ,-C (O) OR6 ,-C (O) NHS (O) ₂R13 ,-CN ,-C (O) NR12R12 ,-NR21S (O) ₂R13 ,-CN ,-NR12C (O) NR12R12 ,-NR21C (O) R18 ,-OC (O) NR12R12 ,-C (O) R19 ,-S (O) R18 ,-S (O) ₂R18 ,-S (O) ₂NR12R12 ,-NO ₂,-NR21C (O) OR17 ,-C (NR12R12)=NR12 ,-C (R13)=NOH; Perhaps if Q1 is-C (O) OH and R22 be-OH ,-SH ,-CHR7OH or-NHR3, then Q1 and R22 and the carbon atom that is connected them can form heterocycle by dehydration; Q2 is OH or NR20R20; W is O, S or NR3; X2 and X3 independently are O, S, S (O), S (O) ₂Or CR3R16; Y is-CR3=CR3-or-C ≡ C-; Z1 be Z2 independently for-HET (R3-R5)-; HET is the double-basis of benzene, pyridine, furan or thiophene; And pharmaceutically acceptable salt.

Definition

Below Suo Xie implication is:

The Et=ethyl

The Me=methyl

The Bz=benzyl

The Ph=phenyl

The t-Bu=tert-butyl

The i-Pr=isopropyl

The n-Pr=n-pro-pyl

The c-Hex=cyclohexyl

The c-Pr=cyclopropyl

1,1-c-Bu=1-pair-cyclobutyl

1,1-c-Pr=1,1-is two-cyclopropyl (HOCH for example ₂(1,1-c-Pr) CH ₂CO2 Me is 1-(methylol) cyclopropaneacetic acid methyl ester)

The c-=ring

The Ac=acetyl group

Tz=1H (or 2H)-tetrazolium-5-base

Th=2-or 3-thienyl

C ₃H ₅=pi-allyl

The c-Pen=cyclopenta

The c-Bu=cyclobutyl

Phe=benzene two bases (benzenediyl)

Pye=pyridine two bases

Fur=furan two bases

Thio=thiophene two bases

DEAD=azo-carboxylic acid diethylester

The DHP=dihydropyran

The DIAD=diisopropyl azo-2-carboxylic acid

The r.t.=room temperature

Alkyl, thiazolinyl and alkynyl comprise straight chain, side chain and circulus and their combination.

" alkyl " comprises " low alkyl group " and comprises and contain the nearly carbon plate section of 20 carbon atoms.The example of alkyl comprises octyl group, nonyl, norborny, undecyl, dodecyl, tridecyl, tridecyl, pentadecyl, eicosyl, 3,7-diethyl-2,2-dimethyl-4-propyl group nonyl, 2-(cyclo-dodecyl) ethyl, adamantyl etc.

" low alkyl group " is meant the alkyl that contains 1-7 carbon atom.The example of low alkyl group comprise methyl, ethyl, propyl group, isopropyl, butyl, the second month in a season-and tert-butyl, amyl group, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, suberyl, 2-methyl cyclopropyl, cyclopropyl methyl etc.

" low-grade alkenyl " is meant the thiazolinyl that contains 2-7 carbon atom.The example of low-grade alkenyl comprises vinyl, pi-allyl, isopropenyl, pentenyl, hexenyl, heptenyl, cyclopropanyl, cyclobutane base, cyclopentenyl, cyclohexenyl group, 1-acrylic, crotyl, 2-methyl-2-butene base etc.

" low-grade alkynyl " is meant the alkynyl that contains 2-carbon atom.The example of low-grade alkynyl comprises acetenyl, propargyl, 3-methyl-1-pentene alkynyl, 2-hexin base etc.

" alkyl-carbonyl " is meant the alkyl-carbonyl of the straight chain, side chain or the cyclic configuration that contain 1-20 carbon atom.The example of alkyl-carbonyl is 2-methylbutyryl base, octadecanoyl, 11-cyclohexyl undecanoyl etc.Therefore, 11-cyclohexyl undecanoyl is c-Hex-(CH ₂) ₁₀-C (O)-.

The phenyl, benzyl, 2-phenethyl and the pyridine radicals that replace are meant have 1 or 2 to be selected from low alkyl group, R10, NO on the aromatic ring ₂, SCF ₃, halogen ,-C (O) R7 ,-C (O) R10, CN, CF ₃And CN ₄The substituent structure of H.

Halogen is meant F, C1, Br and I.

The prodrug ester of Q1 (being Q1=-C (O) OR6) is meant the esters described in the following document, as Saari etc., J.Med.Chem., 21 (8): 746-753 (1978), Sakamoto etc., Chem.Pharm.Bull., 32 (6): 2241-2248 (1984) and Bundgaard etc., J.Med.Chem., 30 (3): 451-454 (1987).When definition R8, some representational monocycles or bicyclic heterocycles base are:

2,5-dioxo-1-pyrrolidinyl,

(3-pyridine radicals carbonyl) amino,

1,3-dihydro-1,3-dioxo-2H-iso-indoles-2-base,

1,3-dihydro-2H-iso-indoles-2-base,

2,4-imidazolinedione-1-base,

2,6-piperidine dione-1-base,

The 2-imidazole radicals,

2-oxo-1, the 3-Dioxol-4-yl (2-oxo-1,3-dioxolen-4-yl),

Piperidines-1-base,

Morpholine-1-base and

Piperazine-1-base.

When Q1 and R22 and the carbon atom that is connected them formed ring, so the ring that forms comprised lactone, lactams and thiolactone.

Definition and this molecule to any substituent group in the specific molecular (for example R1, R2, m, X etc.) are irrelevant in the definition in other place.Therefore ,-NR3R3 can represent-NHH ,-NHCH3 ,-NHC ₆H ₅Deng.

Two R3, R12 or R20 connect the heterocycle that forms by N and comprise cyclopyrrole alkane, piperidines, morpholine, thiomorpholine, piperazine and N methyl piperazine.

" standard amino acid " base can be CR3 R22, represent following aminoacid: alanine, agedoite, aspartic acid, arginine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine are (with F.H.C.Crick, experimental biology science meeting (Symposium of the Society ofExperimental Biology), 12,140 (1958)).

Following chemical compound described here contains one or more asymmetric centers, therefore can produce diastereomer and optical isomer.LTRA comprises that this possible diastereomer and their racemic mixture and optical activity split form.Available routine techniques splits optical activity (R) and (S) isomer.

Some chemical compounds described here contain the two keys of alkene, and except as otherwise noted, this comprises E and Z geometric isomer.

In the chemical compound of preferred chemical formula V:

R1 be H, halogen ,-CF ₃Or-CN;

R2 is C _1-4Alkyl ,-CF ₃,-CF2 ,-CH ₂F, or two R2 groups that are connected on the identical carbon atoms form a ring of 6 yuan at the most;

R3 is H or R2;

CR3R22 can be the standard amino acid base;

R4 is-OR3 ,-SR3, NR3R3, NHC (O) CH3 or R3;

R5 is H or halogen;

R6 is (CH ₂) s-C (R7R7)-(CH ₂) s-R8 or-CH ₂C (O) NR12R12;

R7 is H or C _1-4Alkyl;

R8 is A) contain 3-12 ring carbon atom and 1 or 2 monocycle or bicyclic heterocycles base that is selected from the ring hetero atom of N, S or O, and each ring in the heterocyclic radical forms by 5 or 6 atoms, or B) the W-R9 base;

R9 contains nearly 20 carbon atoms, and is (1) alkyl or (2) alkyl-carbonyl;

R10 is-SR11 ,-OR12 or-NR12R12;

R11 be low alkyl group ,-C (O) R14, unsubstituted phenyl or unsubstituted benzyl;

R12 is that M, R11 or two R12 groups being connected on the identical N can form the heteroatomic 5 or 6 yuan of rings that are selected from O, S and N that contain 1-2;

R13 be low alkyl group ,-CF ₃Or replacement or unsubstituted phenyl, benzyl or 2-phenethyl;

R14 is H or R13;

R16 is H, C _1-4Alkyl or OH;

R22 is R4, CH2OR3 or CH2SR2;

M and m ' independence are 0-4;

N and m ' independently are 0 or 1;

P and p ' independence are 0-4;

When r be 1 and X2 when being O or S m+n+p be 1-9;

When r be 1 and X2 when being CR3R16 m+n+p be 0-9;

M+n+p is 0-9 when r is 0;

M '+m '+p ' is 1-9;

R and r ' independently are 0 or 1;

S is 0-3;

Q1 is-C (O) OR3,1H (or 2H)-tetrazolium-5-base ,-C (O) OR6 ,-C (O) NHS (O) ₂R13 ,-C (O) NR12R12 ,-NHS (O) ₂R13; Perhaps if Q1 is-C (O) OH and R22 be-OH ,-SH ,-CH20H or-NHR3, then Q1 and R22 and the carbon atom that is connected them can form heterocycle by dehydration;

Q2 is OH;

W is O, S or NH;

X2 and X3 independently are O, S or CR3R16;

Y is (E)-CH=CH-;

Z1 be Z2 independently for-HET (R3-R5)-;

HET is the diradical of benzene, pyridine, furan or thiophene;

And pharmaceutically acceptable salt.

Organize in the preferred chemical compound at another, the R22 that is connected with Q1 α is low alkyl group, CF ₃Or replacement or unsubstituted phenyl.

The chemical compound of the chemical formula V that is more preferably can be used chemical formula (Va) expression:

In the formula: R1 is H, halogen, CF ₃Or CN;

R22 is R3 ,-CH ₂O ₃Or-CH ₂SR2;

Q1 is-C (O) OH, 1H (or 2H)-tetrazolium-5-base ,-C (O) NHS (O) ₂R13 ,-C (O) NR12R12 or-NHS (O) ₂R13;

M ' is 2 or 3;

P ' is 0 or 1;

M+p is 1-5;

Remaining definition as the definition in chemical formula V; With and pharmaceutically acceptable salt.

The chemical compound that another group is more preferably is shown in chemical formula (Va), wherein:

M ' is 0;

Remaining definition as the definition in chemical formula (Va).

The chemical compound of most preferred chemical formula (Va) has a low alkyl group on the α of Q1 base carbon.

Another chemical compound of organizing preferred chemical formula V can be used chemical formula (Vb) expression:

In the formula: R1 is H, halogen, CF ₃Or CN;

R22 be R3 ,-CH ₂O3 or-CH ₂SR2;

Q1 is-C (O) OH, 1H (or 2H)-tetrazolium-5-base ,-C (O) NHS (O) ₂R13 ,-C (O) NR12R12 or-NHS (O) ₂R13;

M is 0,2 or 3;

P is 0 or 1;

P ' is 1-4;

M+p is 0-4;

Remaining definition as the definition in chemical formula V; And pharmaceutically acceptable salt.

The chemical compound of representational chemical formula V is listed in U.S. Patent No. 5,565,473 Table I, and this patent is included into this paper as a reference.

The chemical compound of preferred chemical formula V is as follows:

Described compositions comprises the chemical compound of chemical formula V as second activating agent, or its pharmaceutically acceptable salt.Term " pharmaceutically acceptable salt " is meant from the salt of pharmaceutically acceptable nontoxic alkali (comprising inorganic base and organic base) preparation.Comprise aluminum, ammonium, calcium, copper, ferrum, ferrous, lithium, magnesium, manganese, bivalent manganese, potassium, sodium, zinc salt etc. derived from the salt of inorganic base.Preferred especially ammonium, calcium, magnesium, potassium and sodium salt.Salt derived from pharmaceutically acceptable organic nontoxic alkali comprises primary, the second month in a season and tertiary amine, the amine of replacement of amine that comprises the replacement of natural generation, cyclammonium and deacidite, as arginine, betanin, caffeine, choline, N, N '-dibenzyl-ethylenediamin, diethylamine, the 2-DEAE diethylaminoethanol, the 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glycosamine, aminoglucose, histidine, sea crust amine (hydrabamine), 2-aminopropane., lysine, methylglucosamine, morpholine, piperazine, piperidines, polyamino resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropyl amine (TPA), tromethane etc.

When chemical compound of the present invention is alkalescence, can be from pharmaceutically acceptable non-toxic acid (comprising mineral acid and organic acid) preparation salt.This acid comprises acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethyl sulfonic acid, Fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroxyethylsulfonic acid., lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, glactaric acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulphuric acid, tartaric acid, p-methyl benzenesulfonic acid etc.Special optimization citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, phosphoric acid and tartaric acid.

Be interpreted as, when following Therapeutic Method is discussed, when mentioning the chemical compound of chemical formula V, also comprise pharmaceutically acceptable salt.

The ability of the chemical compound antagonism leukotriene effect of chemistry formula V makes them can be used for preventing in human subjects or reversing by the inductive symptom of leukotriene.This antagonism to the leukotriene effect illustrates that this chemical compound or its pharmaceutical composition can be used to treat in the mammal especially mankind, prevention or alleviation: 1) comprise asthma, chronic bronchitis and relevant obstructive airway diseases are at interior pulmonary disease, 2) allergy and anaphylaxis, as allergic rhinitis, contact dermatitis, allergic conjunctivitis etc., 3) inflammation, as arthritis or inflammatory bowel, 4) pain, 5) dermatosis, as psoriasis, atopic eczema etc., 6) cardiovascular disease, as angina pectoris, myocardial ischemia, hypertension, platelet aggregation etc., 7) renal insufficiency that causes ischemia to cause owing to immunity or chemical substance (cyclosporin), 8) migraine or cluster headache, 9) ocular disease, as uveitis, 10) chemical, immunity or infectiousness stimulate the hepatitis that causes, 11) wound or shock state, as burn, endotoxemia etc., 12) allograft rejection, 13) prevention and the relevant side effect of administer cytokines (as interleukin II and tumor necrosis factor) treatment, 14) chronic lung disease becomes sick as the capsule fibroid, bronchitis and other little air flue or big airways disease, and 15) cholecystitis.

The chemical compound prevention of chemistry formula V or the big young pathbreaker of therapeutic dose change with the order of severity of symptom to be treated and with the chemical compound and the route of administration thereof of specific chemical formula V.It also will become with age, body weight and the reaction of individual patient.Usually, when being used for asthma, antiallergic action and anti-inflammatory purpose, the daily dose scope is every kilogram of about 0.001-100 of weight of mammal, preferred every kilogram of 0.01-10mg, and every kilogram of 0.1-1mg most preferably is with single dose or broken dose administration.On the other hand, in some cases, also can use dosage outside these scopes.

When intravenous was used compositions, the suitable dose scope of chemical compound that is used for the chemical formula V of asthma, anti-inflammatory or antiallergic action was the about 0.001-25mg of every kg body weight every day (preferred 0.01-1mg).

When using Orally administered composition, the suitable dose scope of chemical compound that is used for the chemical formula V of asthma, anti-inflammatory or antiallergic action is the about 0.01-100mg of every kg body weight every day, preferred every kilogram of about 0.1-10mg.

Except above-mentioned common formulations, the chemical compound of chemical formula V also can pass through United States Patent(USP) Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200 and 4,008,719 described controlled release methods and/or defeated delivery device give, and these patents are included into this paper as a reference.

Contain in the compound compositions of chemical formula V and also can contain the biosynthetic inhibitor of leukotriene, as EP138,481, described in EP 115,394, EP 136,893 and the EP 140,709 those, these patents are included into this paper as a reference.Contain in the compound compositions of chemical formula V and also can contain: the biosynthetic inhibitor of (1) leukotriene, (2) prostaglandin antagonists; (3) histidine decarboxylase inhibitor; (4) leukotriene antagonist; (5) H1 or H2-receptor antagonist; (6) K+/H+ATP enzyme inhibitor; (7) mast cell stabilizers; (8) serotonin antagonist, and/or (9) anticholinergic is (as United States Patent(USP) Nos. 4,208,423; 5,603,918; 5,955,058; 6,299,861; 6,455,524 is described).

A kind of preferred second activating agent is Menglusitena (Montelukast Sodium), and it is a kind of selectivity and Orally active LTRA that suppresses cysteinyl leukotriene CysLT1 receptor.The chemistry of Menglusitena is called [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolyl) vinyl] phenyl]-3-[2-(1-hydroxyl-1-Methylethyl) phenyl] propyl group] sulfo-] methyl] cyclopropaneacetic acid list sodium salt.

Menglusitena is a kind of hygroscopicity optical activity white or off-white powder.It is soluble in ethanol, first alcohol and water, and is almost insoluble in acetonitrile.It can obtain by commercial.Every 10mg film coating Singulair  sheet contains the 10.4mg Menglusitena, and its mole equals the 10.0mg free acid, and various inert fraction.But every 5mg Singulair  chewable tablet contains the 5.2mg Menglusitena, and its mole equals the 5.0mg free acid, and various inert fraction.

The also available chemical formula of LTRA (VI) expression:

Wherein, group＞X-Y-Z-is selected from down group:

(a)＞CRc-CRaRb-NRd-

(b)＞C＝N-Za-

(c)＞C＝CRa-Zb-

(d)＞N-CRa＝N-

(e)＞N-CRbRe-CRcRf-Zb-

(f)＞N-N＝N-

(g)＞N-NRg-CO-

(h)＞N-N＝C.ORd-

Wherein "＞" represents two keys that separate,

Ra is hydrogen or (1-4C) alkyl;

Rb and Rc are respectively hydrogen, perhaps form unsaturated bond with existing carbon-carbon bond;

Rd is hydrogen or optional 1 or 2 two key or triple-linked (1-10C) alkyl of containing, and carbon atom wherein can be chosen wantonly by oxygen or sulfur and substitute, described (1-10C) alkyl also can be chosen wantonly and have the substituent group that is selected from down group: (1-4C) alkoxyl, cyano group, carboxyl, 1H-tetrazolium-5-base, carbamyl, N-(1-4C) carbamyl, N, N-two [(1-4C) alkyl] carbamyl and (1-4C) alkoxy carbonyl, perhaps Rd is (3-8C) cycloalkyl, (3-8C) alkyl of cycloalkyl-(1-4C), (2-6C) alkyl of alkanoyl or phenyl-(1-4C), its phenyl moiety can be chosen wantonly and have the substituent group that is selected from down group: cyano group, halogen, (1-4C) alkyl, (1-4C) alkoxyl and trifluoromethyl;

Re and Rf independently are hydrogen or (1-4C) alkyl;

Rg is (1-4C) alkyl;

Za be oxygen, sulfur or formula-N (Rd)-the imino group of replacement, wherein Rd has any implication defined above;

Zb is oxygen or sulfur;

Radicals R 1.L-represents the acylamino-of following formula: R1.W.CO.NH-or R1.W.CS.NH-, and wherein R1 is optional (2-10C) alkyl that contains one or more fluoro substituents; Perhaps R1 is the alkyl of phenyl-(1-6C), (1-6C) moieties wherein can be chosen wantonly and have fluorine or (1-4C) alkoxy substituent, and phenyl moiety wherein can be chosen wantonly and has the substituent group that is selected from down group: halogen, (1-4C) alkyl, (1-4C) alkoxyl and trifluoromethyl; Or R1 is (3-8C) cycloalkyl or (3-8C) alkyl of cycloalkyl-(1-6C), and any one annulus can be chosen wantonly to contain a unsaturated bond and can choose wantonly and have 1 or 2 (1-4C) alkyl substituent;

W is oxygen, sulfur, imino group or the key that directly connects R1;

R2 is hydrogen, halogen, (1-4C) alkyl or (1-4C) alkoxyl;

Q is optional one or more substituent phenylenes that independently are selected from down group that have: halogen, hydroxyl, (1-4C) alkyl, (1-4C) alkoxyl and trifluoromethyl;

A1 is (1-2C) alkylidene or ethenylidene;

A2 is methylene, ethenylidene or the key that directly connects M;

M is the acidic-group that is selected from down group: carboxyl, the acyl group sulfonamide residue of formula--CO.NH.SOm R3 and 1H-tetrazolium-5-base, wherein m is the integer of integer 1 or 2, R3 is (1-6C) alkyl, (3-8C)-cycloalkyl, (6-12C) aryl, (wherein at least one is a carbon atom to contain 5-12 atom, at least one is selected from oxygen, sulfur and nitrogen) heteroaryl, (6-12C) alkyl of aryl-(1-4C), wherein any one aromatic portion or assorted virtue part can have 1 or 2 and be selected from down the substituent group of organizing: halogen, (1-4C) alkyl, (1-4C) alkoxyl, trifluoromethyl, nitro and amino;

Or its its pharmaceutically acceptable salt.

The chemical compound of some chemical formula (VI), for example R1 wherein comprises the carbon atom of asymmetric replacement, can optically-active exists with the racemic mixture form and separates.In addition, the chemical compound of some formula I, for example wherein Rd or-A1.Q.A2-contains the chemical compound of ethenylidene, can exist with isolating stereoisomeric forms in any ratio (' E ' and ' Z ') and separate according to this group.Some chemical compounds can more than one tautomeric forms exist.Some chemical compounds can have polymorphism.Be interpreted as, the present invention includes any raceme, optical activity, tautomerism, polymorphic or stereoisomeric forms in any ratio with leukotriene antagonist characteristic, or its mixture, preparation optical activity form (for example by resolving racemic mixtures form or synthetic) from the optical activity initial substance and prepare ' E ' and ' Z ' stereoisomer (for example by its mixture of chromatography) independently and how the method for the code test mensuration leukotriene antagonist characteristic by hereinafter described be well known in the art.

In this description, expression one class group such as Ra, Rb, Rc and do not have other implication.Be interpreted as, " (1-6C) alkyl " this class term comprises the straight or branched alkyl, but when mentioning that a kind of alkyl for example only represents straight chain (" just ") group when " propyl group ", branched chain isomer will be mentioned especially as " isopropyl ".Similarly regulation is used to other class group, for example " alkylidene " and " alkenylene " etc.

The occurrence of groups such as Ra, Rb, Rc is as follows:

Its occurrence is when Ra, Re, Rf, Rg or R2 are (1-4C) alkyl, for example methyl, ethyl or propyl group.

Its occurrence is when R2 is (1-4C) alkoxyl, for example methoxy or ethoxy; And occurrence is when it is halogen, for example fluorine, chlorine or bromine.

Its occurrence is when Rd is (1-10C) alkyl, for example methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, the second month in a season-butyl, 3-methyl butyl, amyl group or hexyl; When its occurrence when containing 1 or 2 two key or triple-linked alkyl is, for example vinyl, pi-allyl, 1-acrylic, 2-methacrylic, 3-methyl but-2-ene base, 1,3-pentadiene base, 2-propynyl or 3-butynyl; And when its be 1 or 2 carbon atom during by the alkyl of oxygen or sulfur its occurrence be for example 2-methoxy ethyl or 2-methylmercaptoethyl.

For example, Rd takes up an official post and selects substituent occurrence to be: for (1-4C) alkoxyl is methoxy or ethoxy; For N-(1-4C) alkylcarbamoyl group is N-methyl-or N-ethyl carbamyl; For N, N-two (1-4C) alkylcarbamoyl group is N, N-dimethylamino formoxyl; For (1-4C) alkoxy carbonyl is methoxycarbonyl, ethoxy carbonyl or uncle-butoxy carbonyl.

Its occurrence is when Rd is (3-8C) cycloalkyl, for example cyclopropyl, cyclopenta or cyclohexyl; Occurrence is when it is the alkyl of (3-8C) cycloalkyl-(1-4C), for example cyclopropyl methyl, cyclopentyl-methyl or cyclohexyl methyl; Occurrence is when it is (2-6C) alkanoyl, for example acetyl group or propiono; Occurrence is when it is the alkyl of phenyl-(1-4C), for example benzyl, 1-phenylethyl or 2-phenylethyl.

Its occurrence is when R1 is (2-10C) alkyl, for example ethyl, propyl group, isopropyl, butyl, isobutyl group, the second month in a season-butyl, tert-butyl, amyl group, 1-ethyl propyl, hexyl, heptyl, 1-ethyl pentyl group or nonyl; Occurrence is when it contains one or more fluoro substituents, for example 2,2, and 2-trifluoroethyl or seven fluoropropyls.

Its occurrence comprises when R1 is the alkyl of phenyl-(1-6C), for example benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenyl propyl, 2-phenyl propyl, 3-phenyl propyl, 1-methyl isophthalic acid-phenylethyl, 1-phenyl butyl and 1-phenylpentyl; (1-6C) occurrence of optional (1-4C) alkoxy substituent is on the moieties, for example methoxy or ethoxy.

Definition as mentioned, some that can occur on the phenyl moiety of R1 or Rd or its part chosen substituent occurrence wantonly and comprised, for example: can be selected from fluorine, chlorine and bromine for halogen; Can be selected from methyl and ethyl for (1-4C) alkyl; 1 can be selected from methoxyl group and ethyoxyl for (1-4C) alkoxyl.

Its occurrence is when R1 is (3-8C) cycloalkyl, for example cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl or suberyl; Occurrence is when it is the alkyl of (3-8C) cycloalkyl-(1-6C), for example cyclobutylmethyl, cyclopentyl-methyl, cyclohexyl methyl, 1-cyclopenta ethyl, 2-cyclopenta ethyl, 1-cyclopenta propyl group, 1-cyclohexyl propyl group, 1-cyclopenta butyl, 1-cyclohexyl butyl; The occurrence that contains the group of unsaturated bond in cycloalkyl ring is, for example the alkyl (as cyclohexenyl group methyl or 1-(cyclohexenyl group) butyl) of cyclohexenyl group or cyclohexenyl group-(1-6C); The occurrence of optional (1-4C) alkyl substituent on this group annulus is, for example methyl, ethyl or isopropyl.

The occurrence of Q be between-phenylene or right-phenylene, preferably have fluorine, chlorine, (1-4C) alkyl, (1-4C) alkoxyl or trifluoromethyl substituent.

Its occurrence is when A1 is (1-2C) alkylidene, for example methylene, vinyl or ethylidene.

Its occurrence is when R3 is (1-6C) alkyl, for example methyl, ethyl, propyl group, isopropyl or butyl; Occurrence is when it is (3-8C) cycloalkyl, for example cyclopenta or cyclohexyl; Occurrence is when it is (6-12C) aryl, for example phenyl, 1-naphthyl or 2-naphthyl; Occurrence is when it is heteroaryl, for example furyl, thienyl or pyridine radicals; Occurrence is when it is the alkyl of (6-12C) aryl-(1-4C), for example benzyl, 1-naphthyl methyl or 2-naphthyl methyl; Or pyridylmethyl.

The optional substituent occurrence that can occur on the aromatic portion of R3 or assorted virtue part or its part comprises above at described in the phenyl moiety of R1 those.

For example, above-mentioned group be worth more specifically comprise following these:

For R1: ethyl, propyl group, isopropyl, butyl, isobutyl group, the second month in a season-butyl, tert-butyl, amyl group, the 1-ethyl propyl, hexyl, heptyl, the 1-ethyl pentyl group, nonyl, seven fluoropropyls, benzyl, 4-benzyl chloride base, the 4-trifluoromethyl benzyl, the 4-methyl-benzyl, the 1-phenylethyl, the 2-phenylethyl, 1-methyl isophthalic acid-phenylethyl, the 1-phenyl propyl, the 1-phenylpentyl, α-luorobenzyl, α-methoxy-benzyl, cyclobutyl, cyclopenta, cyclohexyl, cyclopentyl-methyl, cyclohexyl methyl, 2-cyclopenta ethyl, 1-cyclopenta butyl, 1-cyclohexyl propyl group, 1-cyclohexyl butyl, 5-methyl-2-(1-Methylethyl) cyclohexyl and 1-cyclohexene-4-base;

For R2: hydrogen, fluorine, chlorine, bromine, methyl and methoxyl group;

For R3: methyl, isopropyl, butyl, cyclopenta, phenyl, 4-chlorphenyl, 4-aminomethyl phenyl, 2-aminomethyl phenyl, naphthyl, thienyl and 6-chloropyridine 3-base;

For Ra: hydrogen and methyl;

For Rb and Rc: hydrogen, Rb and Rc and existing carbon-carbon bond form unsaturated bond together;

For Rd: hydrogen, methyl, ethyl, propyl group, butyl, amyl group, hexyl, pi-allyl, propargyl, 3-methyl butyl, 3-methyl but-2-ene base, 2-carbamyl ethyl, carboxyl methyl, carboxy ethyl, N-ethyl carbamyl methyl, N, N-dimethylamino formoxyl methyl, 2-carboxy vinyl, 2-(methoxycarbonyl) vinyl, 2-methoxy ethyl, 3-methoxy-propyl, cyclopenta, cyclopropyl methyl, acetyl, benzyl, 3-cyano group benzyl and 4-benzyl chloride base;

For Re and Rf: hydrogen, methyl and ethyl;

For Rg: methyl, ethyl and propyl group;

For A1: methylene and vinyl;

For A2: key and methylene;

For Q :-phenylene and right-phenylene (optional fluorine, chlorine, hydroxyl, methyl, methoxyl group or the trifluoromethyl substituent of having); And

For W: oxygen, imino group, sulfo-and key.

The example of interested especially special groups comprise following these:

For R1: butyl, amyl group, 1-ethyl pentyl group, 1-phenyl propyl, α-luorobenzyl, α-methoxy-benzyl, cyclopenta and cyclopentyl-methyl;

For R2: hydrogen;

For R3: phenyl and 2-aminomethyl phenyl;

For Ra: hydrogen;

For Rb and Rc: hydrogen, Rb and Rc and existing carbon-carbon bond form unsaturated bond together;

For Rd: hydrogen, methyl, ethyl, propyl group, hexyl, pi-allyl, propargyl, 3-methyl butyl, 3-methyl but-2-ene base, carboxyl methyl, carboxy ethyl, N-ethyl carbamyl methyl, N, N-dimethylamino formoxyl methyl, 2-methoxy ethyl, cyclopenta, cyclopropyl methyl, acetyl, benzyl and 3-cyano group benzyl;

For Re or Rf: hydrogen;

For Rg: propyl group;

For A1: methylene;

For A2: key;

For Q :-phenylene and right-phenylene (optional hydroxyl or the methoxyl group substituent group of having); And

For W: oxygen, imino group and key.

In the chemical compound of formula (VI), top definition comprises many subclass of chemical compound, for example:

(i) indole and the indoline of chemical formula (VIa)

(ii) benzisoxa  azoles, benzisothiazole and the indazole of chemical formula (VIb);

(iii) benzo [b] furan of chemical formula (VIc) and benzo [b] thiophene;

The (iv) benzimidazole of chemical formula (VId):

(v) 1 of chemical formula (VIe), 4-benzoxazine and 1,4-benzothiazine;

(the vi) benzotriazole of chemical formula (VIf);

(the vii) indazolone of chemical formula (VIg); With

(the viii) indazole of chemical formula (VIh);

Wherein, in each subclass, m, R1-R3, Ra-Rg, Za, Zb, A1, A2, Q, W and M have any implication defined above; With and pharmaceutically acceptable salt.

In above-mentioned subclass, other subclass of The compounds of this invention comprise following these:

(ix) those chemical compounds of chemical formula (VIa), wherein Rb and Rc form unsaturated bond with existing carbon-carbon bond;

(x) those chemical compounds of chemical formula (VIe), wherein Zb is oxygen or sulfur, Rb and Rc are hydrogen;

Wherein, in (ix) and each subclass (x), the group of all the other types has any implication defined above; With and pharmaceutically acceptable salt.

In above-mentioned subclass, the preferred value of A1 is, for example methylene; A2 preferred value be for example directly to connect the key of M; The preferred value of Q is, for example right-phenylene (optionally replaced by methoxyl group, especially relative with A1 be adjacent methoxyl group); The preferred value of M is the group of carboxyl, 1H-tetrazolium-5-base or formula-CO.NH.SO2R4, and wherein R4 is a phenyl, optional by as mentioned to the definition of R3 for example the 2-aminomethyl phenyl replace.In a word, preferred group R1.L-is with the benzene part of alternate with the X base but not adjacent with Z base mode convolution I.The preferred value of R1.L-is, for example R1.W.CO.NH-; The preferred value of W is, for example oxygen, imino group or key; The preferred value of R1 is when W is oxygen or imino group, for example cyclopenta; The preferred value of R1 is when W is key, for example cyclopentyl-methyl.

One group of preferred chemical compound of the present invention comprises the indole derivatives with following chemical formula (VIIa),

Indazole derivative with following chemical formula (VIIb),

Benzo [b] thiophene derivant with following chemical formula (VIIc),

Benzimidizole derivatives with following chemical formula (VIId),

Have 2 of following chemical formula (VIIe), 3-dihydrobenzo-1,4- oxazine derivatives,

Benzotriazole derivatives with following chemical formula (VIIf),

And have following chemical formula (VIIg) indazole derivative,

Wherein, R1, R2, Ra, Rd, Re, Rf, W, Q, A2 and M have any implication defined above; With and pharmaceutically acceptable salt.For chemical formula (VIIa) and derivant (VIIb), when M was carboxyl, the special preferred value of Rd comprised methyl, propyl group, 2-methoxy ethyl, N-ethyl carbamyl methyl and cyclopenta.For chemical formula (VIIa) and derivant (VIIb), when M is formula-CO.NH SO ₂During the group of R4 (wherein R4 is a phenyl), the special preferred value of Rd comprises hydrogen, methyl, 2-methoxy ethyl and N-ethyl carbamyl methyl.For chemical formula (VIIa) and derivant (VIIb), when M is formula-CO.NH.SO ₂During the group of R4 (wherein R4 is the 2-aminomethyl phenyl), the special preferred value of Rd comprises methyl and N, N-dimethylamino formoxyl methyl.For derivant (VIIg), when R1.L-be Rl.W.CO.NH-(wherein R1.W-is a cyclopentyloxy) and M be carboxyl or-CO.NH.SO ₂During R4 (wherein R4 is a phenyl), the special preferred value of Rd is a methyl.For derivant (VIIg), when R1.L-be R1.W.CO.NH-(wherein R1 is a cyclopentyl-methyl), W be key and M be carboxyl or-CO.NH.SO ₂During R4 (wherein R4 is the 2-aminomethyl phenyl), the special preferred value of Rd is a N-ethyl carbamyl methyl.

Incidental embodiment has described particular compound of the present invention.Yet; in these chemical compounds; particularly preferably be N-[4-[5-(cyclopentyloxy carbonyl) amino-1-methylindole-3-ylmethyl]-the 3-anisoyl] benzsulfamide; N-[4-[5-(cyclopentyloxy carbonyl) amino-1-(N-ethyl carbamyl methyl) indol-3-yl methyl]-the 3-anisoyl] benzsulfamide; N-[4-[5-(cyclopentyloxy carbonyl) amino-1-methylindazole-3-ylmethyl]-the 3-anisoyl] benzsulfamide; N-[4-[5-(cyclopentyloxy carbonyl) amino-1-methylindole-3-ylmethyl]-the 3-anisoyl]-the 2-methyl benzenesulfonamide; N-[4-[5-(2-cyclopenta acetylamino)-1-(N; N-dimethylamino formoxyl methyl) indol-3-yl methyl]-the 3-anisoyl]-the 2-methyl benzenesulfonamide; N-[4-[6-(cyclopentyloxy carbonyl) amino-2; 3-dihydrobenzo-1; 4- piperazine-4-ylmethyl]-the 3-anisoyl] benzsulfamide; N-[4-[6-(2-cyclopenta acetylamino)-2; 3-dihydrobenzo-1; 4- piperazine-4-ylmethyl]-the 3-anisoyl] benzsulfamide; N-[4-[5-(cyclopentyloxy carbonyl) amino benzo [b] thiene-3-yl-methyl]-the 3-anisoyl] benzsulfamide; N-[4-[6-(2-cyclopenta acetylamino) benzimidazole-1-ylmethyl]-the 3-anisoyl]-benzsulfamide; N-[4-[6-(2-cyclopenta acetylamino)-2] 3-dihydrobenzo-1; 4- piperazine-4-ylmethyl]-the 3-anisoyl]-the 2-methyl benzenesulfonamide; N-[4-[6-(cyclopentyloxy carbonyl) amino-3-methoxyl group indazole-1-ylmethyl]-the 3-anisoyl] benzsulfamide; N-[4-[5-(N '-the cyclopenta urea groups)-1-methylindole-3-ylmethyl]-the 3-anisoyl]-the 2-methyl benzenesulfonamide; N-[4-[6-(2-cyclopenta acetylamino) benzotriazole-1-ylmethyl]-the 3-anisoyl] benzsulfamide; N-[4-[5-(cyclopentyloxy carbonyl) amino indole-3-ylmethyl]-the 3-anisoyl] benzsulfamide; N-[4-[5-(cyclopentyloxy carbonyl) amino-1-(2-methoxy ethyl) indol-3-yl methyl]-the 3-anisoyl]-benzsulfamide; N-[4-[5-(2-cyclopenta acetylamino)-1-methylindole-3-ylmethyl]-the 3-anisoyl] benzsulfamide; N-[4-[6-(2-cyclopenta acetylamino)-3-(N-ethyl carbamyl methoxyl group) indazole-1-ylmethyl]-the 3-anisoyl]-2-methyl benzenesulfonamide and the amino benzimidazole of N-[4-[6-(cyclopentyloxy carbonyl)-1-ylmethyl]-the 3-anisoyl] benzsulfamide, they can free acid forms or use with its corresponding pharmaceutically acceptable salt.

The example of suitable pharmaceutically acceptable salt is by forming the salt that physiologically acceptable cationic alkali forms, as alkali metal (especially sodium and potassium), alkaline-earth metal (especially calcium and magnesium), aluminum and ammonium salt, and the salt made from suitable organic base such as triethylamine, morpholine, piperidines and triethanolamine.For those chemical compounds that is the formula I of alkalescence basically, the example of suitable pharmaceutically acceptable salt comprises acid-addition salts, for example those salt made from strong acid example hydrochloric acid, sulphuric acid or phosphoric acid.

The chemical compound of a kind of chemical formula that is more preferably (VI) is a zafirlukast, and it has following chemical formula:

Zafirlukast is a kind of LTRA, its chemistry 4 (5-cyclopentyloxy-carbonylamino-1-methyl-indol-3-yl methyl)-3 methoxyl groups-N-neighbour-tosyl yl-benzamides by name.It is extremely faint yellow amorphous fine powder of a kind of white, and is water insoluble basically, is slightly soluble in methanol, is dissolved in oxolane, dimethyl sulfoxide and acetone fully.Zafirlukast can obtain with Accolate  by commercial, and the oral tablet of this a kind of 20mg (AstraZeneca PharmaceuticalsLP, Wilmington, DE).

The also available chemical formula of LTRA (VIII) expression:

In the formula,

A represents singly-bound or chooses wantonly by 1,2 or 3 to contain the straight or branched alkyl of 1-10 carbon atom and/or methylene, ethylidene, trimethylene, tetramethylene, ethenylidene, allylidene, butenylidene, Aden's dialkylene (butadienylene) or the ethynylene that phenyl replaces;

B represents

(i) 4-8 unit carbocyclic ring, not replaced or have 1,2 or 3 optional carbon atom to be substituted (this ring can be chosen wantonly by oxo, sulfo-and/or hydroxyl and replace) by oxygen, nitrogen and/or sulphur atom, or

The (ii) divalent group represented of following formula:

Or

T represents oxygen atom or sulphur atom;

R1 represents the group that following general formula is represented:

(iv) straight or branched alkyl, the alkenyl or alkynyl of 20 carbon atoms at the most; Wherein R5 and R6 represent hydrogen atom or halogen atom or not replaced or 1,2,3,4 or 5 optional carbon atom is arranged by the carbocyclic ring of oxygen atom, sulphur atom, halogen atom, nitrogen-atoms, phenyl ring, thiphene ring, naphthalene nucleus, a 4-7 carbon atom, carbonyl, carbonyl oxygen base, hydroxyl, carboxyl, azido and/or the alternate straight or branched alkyl of nitro, alkenyl or alkynyl respectively;

R2 represents the straight or branched alkyl of hydrogen atom or 1-6 carbon atom;

R3 represents that (wherein R7 represents the straight or branched alkyl of hydrogen atom or 1-6 carbon atom for the group of hydrogen atom, halogen atom, hydroxyl, nitro, general formula-COOR7.) or straight or branched alkyl, alkoxyl or the alkylthio group (alkylthio) of 1-6 carbon atom;

R4 represents

(i) when representing closed loop, B is the group that following general formula is represented:

-U-(CH ₂) _n-COOR ⁸

-(CH ₂) _p-COOR ⁸or

(wherein, U represents oxygen atom or sulphur atom; R8 represents the straight or branched alkyl of hydrogen atom or 1-6 atom, and n and m represent the integer of 1-10 respectively, p and q represent respectively 0 or the integer of 1-10), or

(ii) when B be not the group that following general formula is represented during representative ring:

(wherein R8, p and q definition as mentioned, condition are that p is not 0 when B represents the group of following formula:

);

And its nontoxic salts, its preparation method and contain their medicaments as active component.

The chemical compound of general formula (IB) also is a noval chemical compound, except leukotriene has antagonistic activity, finds that they have the activity of inhibition to 5, lipoxidase and aldose reductase for the first time.

In general formula (VIII), the example of the group of R5 and R6 representative is as follows:

Hydrogen atom, halogen atom

The alkyl of 1-20 carbon atom

The alkenyl or alkynyl of 2-20 carbon atom

The alkoxyl of 1-19 carbon atom or alkylthio group

The alkene oxygen base of 3-19 carbon atom, alkenylthio group (alkenylthio), alkynyloxy group or alkynes sulfenyl (alkynylthio)

By the alkyl of 1-19 carbon atom of halogen atom and/or hydroxyl replacement

By the alkenyl or alkynyl of 2-19 carbon atom of halogen atom and/or hydroxyl replacement

By the alkoxyl or the alkylthio group of 1-18 carbon atom of halogen atom and/or hydroxyl replacement

By alkene oxygen base, alkenylthio group, alkynes sulfenyl or the alkynyloxy group of 3-18 carbon atom of halogen atom and/or hydroxyl replacement

The alkoxyalkyl of 19 carbon atoms, alkene oxygen base alkyl or alkoxyl thiazolinyl at the most

The cycloalkyl of 4-7 carbon atom, cycloalkyloxy or cycloalkylthio

Phenyl, phenoxy group or thiophenyl

The alkyl of 1-19 carbon atom, therebetween or terminal carbocyclic ring, phenyl ring, naphthalene nucleus or the thiphene ring that contains 4-7 carbon atom arranged

The alkoxyl of 18 carbon atoms, alkylthio group, alkene oxygen base, alkenylthio group, alkynyloxy group or alkynes sulfenyl at the most, therebetween or terminal carbocyclic ring, phenyl ring, naphthalene nucleus or the thiphene ring that contains 4-7 carbon atom arranged

Thiophenyl alkoxyl or phenoxy alkoxy, moieties wherein are the groups that contains 1-17 carbon atom

The carboxyl alkoxyl of 19 carbon atoms or alkoxy carbonyl alkoxyl at the most

The alkoxyl carbonyl oxygen base alkoxyl of 3-19 carbon atom

The thiazolinyl carbonyl oxygen base of 3-20 carbon atom

The alkyl-carbonyl of 2-20 carbon atom

The azido alkyl of 19 carbon atoms, 4-nitro alkyl, aminoalkyl, alkyl amino alkyl, dialkyl aminoalkyl at the most

The azido alkoxyl of 18 carbon atoms, nitro alkoxyl, aminoalkoxy, alkyl amino alkoxyl, dialkyl amido alkoxyl at the most

The alkenyl carbonyl amino of 3-19 carbon atom

1-19 carbon atom alkyl amino

Further by the above-mentioned group of halogen atom, hydroxyl, azido, nitro and/or carboxyl substituted

In the above-mentioned group, the preferred group of R5 and R6 is as follows:

Hydrogen atom

Halogen atom

The straight or branched alkyl of 1-20 carbon atom

The straight or branched alkoxyl of 1-19 carbon atom

The straight or branched alkene oxygen base of 3-19 carbon atom

The straight or branched alkynyloxy group of 3-19 carbon atom

The straight or branched alkylthio group of 1-19 carbon atom

The straight or branched alkyl of 1-18 the carbon atom that is replaced by halogen atom

The straight or branched alkoxyalkyl of 2-19 carbon atom

Optional cycloalkyl, cycloalkyl-alkyl (moieties wherein contains 1-8 carbon atom) or the cycloalkyl alkoxy (moieties wherein contains 1-8 carbon atom) that is replaced by the straight or branched alkyl of 1-8 carbon atom, hydroxyl, halogen atom and/or nitro

Optional phenyl, phenylalkyl (moieties wherein is the group that contains 1-8 carbon atom), phenyl alkoxyl (moieties wherein is the group that contains 1-8 carbon atom) or the phenyl alkene oxygen base (alkenyl part wherein is the group that contains 2-8 carbon atom) that is replaced by the straight or branched alkyl of 1-8 carbon atom, hydroxyl, halogen atom and/or nitro

Optional naphthyl, naphthyl alkyl (moieties wherein is the group that contains 1-8 carbon atom), naphthyl alkoxyl (moieties wherein is the group that contains 1-8 carbon atom) or the naphthyl alkene oxygen base (alkenyl part wherein is the group that contains 2-8 carbon atom) that is replaced by straight or branched alkyl, hydroxyl, halogen atom and/or nitro

By straight or branched alkoxyl, alkene oxygen base or the alkoxyl alkoxyl of 18 carbon atoms at the most of carbonyl, carbonyl oxygen base and/or hydroxyl replacement

By the straight or branched alkoxyl of 1-17 carbon atom of phenoxy group or thiophenyl replacement

By the straight or branched alkoxyl of the cyclosubstituted 1-18 of a thiophene carbon atom

By straight or branched alkyl, thiazolinyl, alkoxyl or the alkene oxygen base of azido or nitro or amino 18 carbon atoms at the most that replace, described amino is optional to be replaced (comprising dialkyl amido) by the alkyl of 1-6 carbon atom

By straight or branched alkyl, thiazolinyl, alkoxyl or the alkene oxygen base of carboxyl and alternate 18 carbon atoms at the most of amino this group of two types

The alkyl of 1-20 carbon atom is meant methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, tridecyl, myristyl, pentadecyl, cetyl, heptadecyl, octadecyl, nonadecyl, eicosyl and their isomerism group in the present invention.

Thiazolinyl and alkynyl with the corresponding 2-20 of an above-mentioned group carbon atom.

The alkyl of 1-6 carbon atom is meant methyl, ethyl, propyl group, butyl, amyl group or hexyl in the present invention, perhaps its isomerism base.

The cycloalkyl of 4-7 carbon atom is meant cyclobutyl, cyclopenta, cyclohexyl or suberyl in the present invention.

Halogen atom is meant chlorine, bromine, iodine or fluorine atom in the present invention.

For the chemical compound of chemical formula (VIII), when certain carbon atom was substituted by another atom, ring or group, any carbon atom can be replaced, as long as this substituting itself is to be acceptable chemically or physically.For example, " centre or end at isobutyl group substitute with phenyl ring " is meant isopropyl phenyl, dimethyl benzene ylmethyl or 2-phenyl propyl.When a carbon atom is replaced, can suitably add or remove hydrogen atom.For example, " substitute with nitrogen-atoms " and be meant N-propyl group amino methyl at 2 of amyl group.

Simultaneously, for example, 2-(phenoxy group) ethyoxyl and 5-(2-chloro-4-nitrophenylsulfenyl)-5-methylpent-2 alkene oxygen base has replaced amyl group and 6 respectively, 1,2,3,4 or 5 optional carbon atom of 8-dimethyl ninth of the ten Heavenly Stems-3-thiazolinyl, so they are also included within the present invention.

The isocyclic example of representing with B in general formula (VIII) of 4-8 unit is as follows, and this carbocyclic ring is not replaced or have 1,2 or 3 optional carbon atom to be substituted (this ring can be chosen wantonly by oxo, sulfo-and/or hydroxyl and replace) by oxygen, nitrogen and/or sulphur atom:

(above-mentioned ring can be chosen wantonly by hydroxyl and replace)

Above-mentioned carbocyclic ring can be saturated rings or unsaturated ring, or aromatic ring or non-aromatic ring.

Above-mentioned any ring all is preferred.And when this ring and phenyl ring condensed, preferred especially following fused benzene rings promptly had the ring of following general formula

As follows:

B wherein is that the chemical compound of open group shown in the following formula also is preferred:

Or

The chemical compound of a kind of chemical formula that is more preferably (VIII) is a pranlukast, and its chemical formula is:

Pranlukast is a kind of chemistry 4-oxo-8-[4-(4-phenyl butoxy) benzoyl-amido by name]-LTRA of 2-(tetrazolium-5-yl)-4H-1-.alpha.-5:6-benzopyran semihydrate.Pranlukast can be by commercial obtain (OnoPharmaceutical Co, Ltd., Osaka, Japan) in Japan.

First and second activating agents are used to treat respiratory tract and pulmonary disease, as mentioned above, also can give following any additional agents itself or its pharmaceutically acceptable salt, and all are called as " reactive compound or activating agent ".First and second activating agents also can be co-administered, with unpack format or be combined into pharmaceutically or acceptable preparation on the veterinary drug.As described below, but described reactive compound or their salt systemic administration or local application.

The present invention also provides the method for treatment asthma, COPD or other respiratory system disease, and described method comprises the described compositions of the object that needs this treatment.This method is used to prevention or therapeutic purposes.This method comprises method in a kind of body.Described method can effectively be treated multiple disease, regardless of its cause of disease, and comprising taking steroid, adenosine or adenosine receptor metabolism or resulting anomaly, or other reason.Described method comprises treatment respiratory tract disease and pneumonopathy, no matter be by reducing adenosine or adenosine receptor level, reduce hypersensitivity or other mechanism, especially in lung, liver, heart and brain, or the organ of this treatment of any needs to adenosine.Here other respiratory system disease of mentioning comprises that the capsule fibroid becomes sick (CF), dyspnea, emphysema, stridulate pulmonary hypertension, pulmonary fibrosis, pulmonary carcinoma, airway hyperreactivity, adenosine or adenosine receptor level raise, and be especially relevant with infectious disease, the pulmonary branches trachea shrinks, pneumonia, pulmonary allergy, surfactant disappearance, chronic bronchitis, bronchoconstriction, dyspnea, lung airway obstacle or block, the test of cardiac function adenosine, the lung vasoconstriction, respiratory disorder, adult respiratory distress syndrome (ARDS), use some drugs, as the other medicines that raise the medicine of adenosine or adenosine level or for example treat supraventricular tachycardia (SVT), and implement adenosine stress test, infant respiratory distress syndrome (baby RDS), pain, allergic rhinitis, pulmonary surfactant reduces, Serious Atypica Respiratory Syndrome (SARS) etc.

In one embodiment, the invention provides the method for prevention or treatment asthma, comprise needing the group of objects of this treatment compound, the amount of compositions to be enough to prevention or treatment asthma in this object.

In one embodiment, the invention provides the method for prevention or treatment COPD, comprise needing the group of objects of this treatment compound, the amount of compositions to be enough to prevention or treatment COPD in this object.

In one embodiment, the invention provides the method for prevention or treatment bronchoconstriction, pneumonia or pulmonary allergy, comprise and need the group of objects of this treatment compound, the amount of compositions to be enough to prevention or treatment bronchoconstriction, pneumonia or pulmonary allergy in this object.

In one embodiment, the invention provides the method that reduces or eliminate the adenosine in the object tissue, comprise needing the group of objects of this treatment compound, the amount of compositions to be enough to reduce or eliminating the interior adenosine of described object tissue.

The present invention also provides described first activating agent and second activating agent to be used for the treatment of application in the medicine of asthma, COPD or other respiratory system disease (comprising pulmonary carcinoma) in manufacturing.Described medicine comprises compositions as herein described.

Giving first activating agent of object and the daily dose of second activating agent will change with total treatment plan, first activating agent that will use and second activating agent, preparation type, route of administration and patient's state.Embodiment 11-18 has described the atomization preparation prepared according to the present invention, can spray in the respiratory tract or intranasal or pass through inhalation by device.For feeding drug into pulmones, preferred lipid formulations.For the other biological activating agent, FDA has stipulated the recommended amounts as the bioactive additive of the daily absorption of people, as vitamin and mineral.But for the special disease of treatment or improve and to use the amount that exceeds hundreds of even several thousand times experimenter's the immunoreation.In most cases the dosage range recommended of pharmacopeia all is very wide, and the doctor can therefrom obtain instructing.The amount of typical medicaments described in the invention is positioned at the daily dosage range of being recommended of taking at present, also can be below or above this level.Treatment generally cooperates non-glucocorticoid or other suitable bioactivator from the bronchodilator of low dosage, increases each patient's dosage then gradually.But higher and lower dosage comprises that predose all should be within the defined scope of the present invention.

First and second activating agents that the present invention is used or the preferable range of other treatment agent will be with the different of route of administration and used dosage form change to some extent, and those skilled in the art can select and prepares according to known method and component.Reactive compound can single dose (once a day) or the mode administration of multidose (every day for several times).The compositions and the method for prevention and treatment respiratory tract disease, heart disease and cardiovascular disease can be used for treating adult, child and baby, also can be used for treating and suffer from the non-human animal who states disease.Though what the present invention mainly paid close attention to is treatment people's disease, also can be used as veterinary medicine and treats other mammals, as Canis familiaris L., cat and heavy livestock and wild animal." adenosine " of term " height " and " low " level comprises following two kinds of situations with " adenosine receptor " and " adenosine disappearance ": compare same object adenosine level before, the adenosine of this moment higher or lower (even disappearance); And the adenosine level of this moment is in normal range but because some other symptoms in this patient or change by reducing or the level or the hypersensitivity of rising adenosine or adenosine receptor can obtain to treat benefit.Therefore, this treatment helps the experimenter to form good life style.Though use that first activating agent can reduce or even eliminate the normal or higher intravital adenosine level of object of adenosine level before the treatment, further use second activating agent and will help short-term to improve patient's breathing.Further add other therapeutic agent and will help the unfavorable low adenosine level that raises, can be observed this rising by using treatment of the present invention, especially when optimal dose.

Other therapeutic agent that can mix the present composition is the various human and animals' of being used for one or more of therapeutic agent.

Except that first and second activating agents, described compositions also can comprise ubiquinone and/or folinic acid.Ubiquinone is a kind of chemical compound of representing with following formula:

Or its pharmaceutically acceptable salt.

Preferably, ubiquinone is the chemical compound as above-mentioned chemical formulation, wherein n=1-10 (ubiquinone _1-10), more preferably n=6-10 (ubiquinone _6-10), n=10 (ubiquinone most preferably ₁₀).Ubiquinone is used with the disease of treatment targeting or the therapeutic dose of symptom, and its dosage will depend on the type and the route of administration of the symptom of object, other medicament of using and used preparation simultaneously.The preferable amount of ubiquinone is that the total amount of every day is that every kg body weight is about 0.1, about 1, about 3, about 5, about 10, about 15, about 30-50, about 100, about 150, about 300, about 600, about 900, about 1200mg.The total amount of preferred every day is about 1-150mg/kg, and about 30-100mg/kg most preferably is about 5-50mg/kg.Ubiquinone is a kind of material of natural generation, and can obtain by commercial.

Activating agent of the present invention contained dosage range in compositions is very wide.For example, the amount of the activating agent that is comprised in the compositions can be to account for 0.001%, 1%, 2%, 5%, 10%, 20%, 40%, 90%, 98%, 99.999% of compositions.Above-mentionedly can suitably adjust every kind of content of medicines when eclipsed other drug being arranged when adding in addition in the compositions with activity activating agent.Yet the dosage of reactive compound will change to some extent according to the different of subject age, body weight and the state of an illness.Treatment can promptly be lower than the amount of the present invention's first activating agent optimal dose from low dose.Second activating agent also can this be handled, up to reaching ideal level.Vice versa, and for example under the situation of using multivitamin and/or mineral, the experimenter at first takes these medicines and is stabilized on the level of expection, and then uses first reactive compound.Dosage can increase up to reaching expection and/or ideal effect.In general, the concentration that activating agent uses preferably can produce effective result and can not cause any excessively deleterious or deleterious side effect, can single unit dose administration, also subunit dosage multiple dosing in the suitable time of every day easily if desired.Second treatment or diagnostic medicine can the dosed administrations that can achieve the goal effectively known in the art.If it is overlapping that the activity of second medicine and principal agent has, the dosage of wherein a kind of medicine or two kinds of medicines should be adjusted to obtain expected effect can not occur over-drastic side effect because exceeding dosage range.Therefore, when adding other analgesic and anti-inflammatory agent in the compositions, used dosage should be known in the artly to reach the amount of its intended purposes or lower dosage during than its independent use.

Pharmaceutically acceptable salt should be to can be used for pharmacological, materia medica or veterinary salt, can be prepared into the form of alkali metal or alkali salt, as sodium salt, potassium salt or calcium salt.Organic salt and ester also are applicable to the present invention.Reactive compound comprises whole body drug-delivery preparation and local application's preparation preferably with the form administration of medicinal or veterinary composition.Wherein preferred preparation should be suitable for sucking or be suitable for administration in administration in respiratory tract administration, oral administration, oral, rectally, vagina administration, intranasal administration, feeding drug into pulmones, eye drops, ophthalmic administration, intracavitary administration, the trachea, the organ, local application's (comprising oral cavity, Sublingual, corium and ophthalmic), parenteral (comprise under subcutaneous, the corium, intramuscular, intravenous and intra-articular injection) and stride the corium administration.

The present invention also provides the medicine box that contains described compositions and defeated delivery device.Compositions is suitable for being prepared into the form of single or multiple unit dose, and makes bulk, the known any method preparation of available pharmaceutical field.Compositions in the medicine box can be made the form of preparation, and perhaps first and second activating agents also can provide respectively with other components, also comprises the description of dosage form and using method in the medicine box.Medicine box can also comprise other drug, and those medicines of describing as this patent when by the parenteral administration, can install to independently in the container with carrier, and carrier should be aseptic.Compositions of the present invention can also provide with freeze dried form, is divided in the different sterile chambers so that add lipid carrier before injection.See U.S. Patent No. 4,956,355; British patent No.2,240,472; EPO patent application No.429,187; PCT patent WO 91/04030; Mortensen, S.A. etc., Int.J.Tiss.Reac.XII (3): 155-162 (1990); Greenberg, S. etc., J.Clin.Pharm.30:596-608 (1990); Folkers, K. etc., P.N.A.S. (USA) 87:8931-8934 (1990), relevant preparation method and chemical compound ratio are included in by above-mentioned list of references.

Compositions of the present invention provides with various whole body preparations and topical formulations.That whole body of the present invention and topical formulations are selected from is oral, in the cheek, in the lung, in the rectum, intrauterine, Intradermal, part, skin, parenteral, tumor, in the intracranial, lung, in buccal, Sublingual, nose, subcutaneous, the blood vessel, in the sheath, can suck, can breathing, in intraarticular, intracavity, implantable, percutaneous, iontophoresis, ophthalmic, eyes, vagina, optical, intravenous, intramuscular, the gland, in the trachea, intralymphatic, slow release and casing preparation.The actual fabrication of these different preparations and mixing are known in the art, here do not need to describe in detail.But described compositions is used one or many every day.

Be suitable in the respiratory tract, intranasal, lung and the preparation of inhalation is preferred, local administration preparation, oral formulations and parenteral preparation also are preferred.All preparation methoies all comprise the step that reactive compound is added relative carrier, and carrier comprises one or more helper components.In general, the preparation method of preparation is that relative equably liquid-carrier of reactive compound or the solid carrier accurately cut apart are mixed, and again the product plastotype is become required preparation if desired.

Be suitable for liquid preparations for oral administration and can be included in the one unit, as hard capsule, cachet, rhombus or tablet, every kind of dosage form all contains the reactive compound of predetermined close; Powder or granule; Solution that water or on-aqueous liquid are made or suspension; Oil-in-water or water in oil emulsion.

Be suitable for comprising by the compositions of parenteral administration the sterilized water injection or the non-water injection of reactive compound, preparation preferably oozes with receptor blood etc.These preparations can contain antioxidant, buffer, antibacterial and make compositions and the isoosmotic solute of receptor blood.Water or non-water sterile suspension contain the medicine and the thickening agent of suspension.Compositions can place the container of single dose or multiple dose, and in the ampoule and vial that for example seals, form that also can lyophilized powder stores, and adds sterile liquid carrier before use, as saline or water for injection.

Intranasal spraying or the preparation that drips are made up of the purification of aqueous solutions of reactive compound, wherein also contain antiseptic and isotonic agent.This preparation is preferably adjusted to the degree identical with nasal mucosa with pH with osmotic pressure.

The preparation that is suitable for rectum or intravaginal administration can adopt the form of suppository, adds suitable carriers such as cocoa butter or hydrogenated fat or hydrogenated fatty acid.

Ophthalmic preparation can just will be adjusted to the degree that is complementary with eyes with its pH and osmotic pressure by preparing with intranasal spray agent similar methods.Generally with adhesive carrier preparation, as wet goods, this is known in the art to ophthalmic preparation, can drip to ophthalmic at an easy rate like this and can not flow out.

The compositions that is suitable for the local skin use preferably adopts the form of ointment, cream, lotion, paste, gel, spray, aerosol or oil preparation.Available carrier comprises the mixture of vaseline, lanoline, polyethylene glycerol, ethanol, dermal penetration enhancer and above-mentioned two or more carriers.Being suitable for the compositions that corium penetrates administration can adopt the form that can paste with the single skin that the application site epidermis closely contacts to prolong the time of contact.

Here first and second activating agents that disclosed can suck by the mode of any appropriate, instil (entering in the lung) in the respiratory system of receptor in breathing, intranasal administration or the lung, preferably contain Powdered by preparation or liquid intranasal, lung in, can breathe and maybe can suck particulate aerosol or spray and come administration.The sucked granule that contains reactive compound can be sucked by the experimenter, promptly by sucking or intranasal spraying or give the experimenter by being instilled in respiratory tract or the lung.According to the present invention, preparation can contain can suck liquid or solid active chemical compound granule, is small enough to comprising size and can passes the sucked granule that oral cavity and throat enter bronchus and alveolar.In general, particulate diameter is about 0.05,0.1,0.5,1,2-4,6,8,10 microns.More preferably, about 0.5 micron to can breathing or suck less than about 5 microns granule.The solids precipitation that can not suck in aerosol or the spray is swallowed down in throat.Therefore the granule that can not suck in the aerosol is few more good more.For instiling in intranasal spraying or the lung, preferred particle size is about 8,10,20,25-35,50,100,150,250,500 microns, so that be trapped in nasal cavity or instil and directly be deposited in the lung.Liquid preparation can be sprayed onto in respiratory tract (nose) and the lung, during especially for neonate and baby.

The composition of liquid medicine of the used reactive compound of preparation aerosol can prepare by reactive compound and stable carrier are mixed mutually, as pyrogen-free sterilized water.The preparation that the micron order that contains reactive compound can suck particulate solid particle composition is with mortar and pestle exsiccant reactive compound to be ground, and then ground compositions is crossed 400 purpose sieves to smash or to isolate bigger agglomerate.The solid particle composition of being made up of reactive compound can also contain dispersant to promote forming of aerosol.Suitable dispersant is a lactose, lactose can with reactive compound with suitable mixed, mix as weight ratio by 1: 1.U.S. Patent Application Serial Number 10/462,901 and 10/462,927 discloses the stable dry powder formulations of DHEA of Sprayable and the stable dry powder formulations (these patent applications are included in this paper as a reference in full) of DHEA-S dihydrochloride dihydrate crystal form respectively.

The drop aerosol that contains reactive compound can prepare by any suitable mode, as uses aerosol apparatus, sees U.S. Patent No. 4,501,729 (this patent is originally included this paper in as a reference).Aerosol apparatus has commercial device, and this device is to make compressed gas, is generally air or oxygen and quickens by a narrow mouth or by ultrasonic vibrations the solution or the suspension of active component to be become curative aerosol.Being used in suitable compositions in the aerosol apparatus comprises with liquid-carrier and dilutes dissolved activating agent, activating agent accounts for more than the 40%w/w of compositions, but below the preferred 20%w/w of the content of carrier, carrier is generally the ethanol water of water or dilution, preferably by adding sodium chloride itself and body fluid etc. is oozed.If compositions is not aseptic, can add anti-corrosion composition, as methyl hydroxy benzoate, antioxidant, flavoring agent, volatile oil, buffer agent and surfactant.The solid particle aerosol that contains reactive compound equally also can prepare with any solid particle medicinal aerosol generator.Aerosol generator can become the solid particle medication preparation granule medicament that can suck, and produces the aerosol that contains predetermined dose of medication of certain volume with the speed that is suitable for the human body administration.The example of this aerosol generator comprises metered-dose inhaler and insufflator.

Can use and to produce that the aerocolloidal any device of liquid or solid granule such as aerosol or spray generator are defeated passs compositions of the present invention.These devices produce respirable granules (as mentioned above), and produce the aerosol or the spray foam volume of the medicament that contains predetermined dose dosage with the speed that is suitable for giving the human or animal.An illustrative type of solid particle aerosol or spray generator is an insufflator, and it is applicable to the powder that gives fine pulverizing.In insufflator, the kit that powder if can effectively be carried out the dosing of treatment as herein described is contained in capsule or the medicated bag.These capsules or medicated bag are made by gelatin, paper tinsel or plastics usually, can be pierced through or open by original position, and powder was inhaled this device by suction effect or manually-operated pump afterwards.The compositions of using in the insufflator can be made up of first and second medicaments separately, also can be made up of the mixture of powders that contains first and second medicaments, and medicament accounts for the 0.01-100%w/w of said preparation usually.Compositions contains the 0.01%w/w that has an appointment, about 1%w/w, about 5%w/w first and second medicaments to about 20%w/w, about 40%w/w, about 99.99%w/w usually.But the medicament of other compositions and other amounts also is suitable within the scope of the present invention.

In one embodiment, described compositions is passed by aerosol apparatus is defeated.This method is particularly useful for patient or the object that oneself could suck or breathe compositions.When being in a bad way, patient or object earn a bare living by artificial ventilator.This aerosol apparatus can use acceptable carrier on pharmaceutically any or the veterinary drug, as light salt brine solution.Aerosol apparatus is a kind of device that powder pharmaceutical compositions is transported to the target spot in patient or the object air flue.

Also can provide and be fit to different dosing method and defeated multi-form compositions of passing approach.In one embodiment, described compositions comprises respirable preparation, as aerosol or spray foam.Compositions of the present invention in batch, become unit form and become the form of implant, capsule, vesicatory or medicated bag to provide, these forms all can be opened or be pierced through as known in the art.Medicine box also is provided, and it comprises defeated delivery device and the compositions of the present invention that is contained in the isolating container, randomly also comprises other excipient and therapeutic agent, also comprises and instructs the description of using this kit components.

In one embodiment, with defeated this medicament of passing of suspension dosing absorption (MDI) preparation.Can use the defeated delivery device that is equipped with propellant such as hydrofluoroalkane (HFA) to fail and pass this MDI preparation.Preferably, the HFA propellant contains 100 parts/1,000,000 (PPM) or water still less.

In one embodiment, defeated delivery device comprises the defeated Diskus (DPI) of passing single agent or the described preparation of multi-agent.Single agent inhaler can the disposable drug form provide, and this disposable medicine box has sterilely added enough expendable preparation in advance.The inhaler mode of can also pressurizeing provides inhaler, preparation be provided at can pierce through or openable capsule or medicated bag in.Medicine box also can randomly comprise the medicament that loads in the independent container, as other treatment chemical compound, excipient, surfactant (being intended as therapeutic agent and preparation composition), antioxidant, flavoring agent and coloring agent, filler, ethereal oil, buffer agent, dispersant, surfactant, antioxidant.Flavoring agent, extender, propellant and antiseptic and other are used for the suitable additives of different preparations.

Now the present invention has been done general description, by the present invention may be better understood with reference to some special embodiment, therefore the embodiment that this paper comprised is used to explain the present invention, and unless otherwise indicated, these embodiment also do not mean that the present invention or its embodiment are made qualification.

Embodiment

Embodiment 1 and 2: folinic acid and DHEA are to the vivo effect of adenosine level

Young male Fischer 344 rats (120 gram) are fed the dehydroepiandrosterone (DHEA) that is dissolved in the carmellose (300mg/kg) or methyltestosterone (40mg/kg) by gavage, once a day, continue 14 days.Folinic acid (50mg/kg) once a day, continues 14 days by intraperitoneal injection.In the time of the 15th day, put to death animal by intracranial microwave pulse (1.33kw, 2450MHZ, 6.5 seconds), this processing can stop the further metabolism of adenosine immediately with all cerebral tissue degeneration.Take out heart, quick freezing is 10 seconds in liquid nitrogen.Liver and lung are whole to be taken out, and quick freezing is 30 seconds in liquid nitrogen.Then with brain tissue slice.Extract in-house adenosine, make it to be derivatized to 1, N6-ethenylidene adenosine, and utilize spectrofluorometry to analyze (J.of Neuroscience Methods 25:243 (1988)) with high performance liquid chromatography (HPLC) according to the method for Clark and Dar.The result of these tests is summarised in the following table 1.The result represents with meansigma methods ± SEM, p＜0.05 when the κ representative is compared with matched group, and the ψ representative is compared p＜0.05 with DHEA or methyltestosterone processed group.

Table 1:DHEA, δ-1-methyltestosterone and folinic acid are to the vivo effect of adenosine level in the various tissues of rat

Processing method	Adenosine (nmols) in the born of the same parents/mg protein
					The heart	Liver	Lung	Brain
	Contrast DHEA (300mg/kg) methyltestosterone (40mg/kg)	10.6±0.6 (n＝12)κ 6.7±0.5 (n＝12)κ 8.3±1.0 (n＝6)κ	14.5±1.0 (n＝12)κ 16.4±1.4 (n＝12)κ 16.5±0.9 (n＝6)κ	3.1±0.2 (n＝6)κ 2.3±0.3 (n＝6)κ N.D.					0.5±0.04 (n＝12)κ 0.19±0.01 (n＝12)κ 0.42±0.06 (n＝6)κ

Methyltestosterone (120mg/kg) folinic acid (50mg/kg) DHEA (300mg/kg)+folinic acid (50mg/kg) methyltestosterone (120mg/kg)+folinic acid (50mg/kg)	6.0±0.4 (n＝6)κ 12.4±2.1 (n＝5)κ 11.1±0.6 (n＝5)ψ 9.1±0.4 (n＝6)ψ	5.1±0.5 (n＝6)κ 16.4±2.4 (n＝5)κ 18.8±1.5 (n＝5)ψ N.D.	N.D. N.D. N.D. N.D.	0.32±0.03 (n＝6)κ 0.72±0.09 (n＝5)κ 0.55±0.09 (n＝5)ψ 0.60±0.06 (n＝6)ψ
					N.D.=does not determine

The presentation of results rat of these tests gives DHEA or methyltestosterone once a day, gives two Zhou Houke and makes a plurality of intraorganic adenosine depletions.Exhaust that the most serious is in brain (DHEA handles and exhausts 60%, and the methyltestosterone of high dose is handled and exhausted 34%) and heart (DHEA handles and exhausts 37%, and the methyltestosterone of high dose is handled exhaustion 22%).Add folinic acid again and can eliminate the adenosine depletion of steroid mediation fully.Give the interior adenosine level rising of organ that folinic acid can make all tests separately.

Embodiment 3: determining of dosage ground and can breathe in the aerojet of anhydrous DHEA-S

Mode with treating asthma is assessed DHEA-S.Solid-state stability (Nakagawa, H., Yoshiteru, T. and Fujimoto, Y., 1981, Chem.Pharm.Bull., 29 (5), the 1466-1469 of the dehydroepiandrosterone sodium sulfate (NaDHEA-S) of caked and grinding have been studied; Nakagawa, H., Yoshiteru, T. and Sugimoto, I., 1982, Chem.Pharm.Bull., 30 (1), 242-248).DHEA-S is the most stable, and crystallization is a dihydrate form.The DHEA-S anhydrous form has low degree of crystallinity, and is unusual moisture absorption.As long as DHEA-S is not suction when storing, its anhydrous form is stable.The crystalline material of retaining part does not have moisture needs specific production and packing technique.For durable product, it is essential in its development process its sensitivity to moisture being reduced to minimum.

(1) micronization of DHEA-S

Use jet mill (Jet-O-Mizer#00 series 100-120PSI nitrogen) the anhydrous DHEA-S of micronization.The sample that makes about 1g by jet mill once, with the sample of about 2g by jet mill 2 times.The particle suspending that will obtain from each grinder is in hexane, and DHEA-S is insoluble in hexane, adds the Spa85 surfactant, to stop caking.Supersound process gained solution 3 minutes, granule disperses fully.On Malvern MastersizerX, test dispersive solution with small size sampler (SVS) adnexa.Test a dispersive sample 5 times.The particle mean size of abrasive material or D (v, 0.5) are not 52.56 microns, and %RSD (relative standard deviation) is 7.61 (5 values).D (v, 0.5) once is 3.90 microns by jet mill, and %RSD is 1.27, and the D (v, 0.5) by jet mill 2 times is 3.25 microns, and %RSD is 3.10.This proof can obtain to be applicable to the DHEA-S of the granularity of suction by jet grinding.

(2) HPLC analyzes

Use two bottles (A: by once, 150mg; B, by twice, 600mg) micronized medicine is measured the degraded of jet grinding micronization process Chinese medicine.The DHEA-S of the equal portions (weight) that will obtain from A bottle and B bottle compares with the standard solution of the acetonitrile-aqueous solution (1: 1) that does not grind DHEA-S (10mg/ml).The chromatographic peak area that does not grind the HPLC analysis of pharmaceutical standards solution is 23.427, preparation A bottle equal portions (weight) sample and B bottle equal portions (weight) sample in acetonitrile-aqueous solution (1: 1).The chromatographic peak area of A bottle and B bottle is respectively 11.979 and 11.677.Clearly, do not take place and detectedly to degrade at spraying abrasive micropowder process Chinese medicine.

(3) ejection dose study

In the Nephele pipe, adopt HPLC to analyze the DHEA-S powder collection.To the test three Diskuses in each with each air velocity carry out three times the test (the DPI device of Rotahaler, Diskhaler and IDL).One end of Nephele pipe is loaded onto glass filter (Gelman Science, A/E type, 25 μ m), it is connected with air flow, with the dosage of collection from the medicine of each Diskus ejection of test.Fix one at the other end of Nephele pipe and have opening to accept the respectively silicone adapter of the spout of the Diskus of test.Make required air-flow, promptly 30,60 or 90L/min manage by Nephele.Then each Diskus spout is inserted in silicone rubber adapter, continue to be blown into air-flow about 4 seconds, remove this pipe then, cover this opening of each pipe with lid.Remove the lid that this pipe does not comprise filter, the water-acetonitrile solution (1: 1) of 10 milliliters of HPLC levels is added in this pipe.Again cover lid shook pipe 1-2 minute.Remove lid from pipe then, solution is transferred in 10 milliliters of plastic injectors that filter (Cameo13N Syringe Filter, Nylon, 0.22 μ m) is housed.The solution Direct Filtration of equivalent in the HPLC bottle, is used for HPLC pharmaceutical analysis after a while.The micronization DHEA-S (about 12.5 or 25 milligrams) that use is contained in gelatine capsule (Rotahaler) or the Ventodisk vesicatory (Diskhaler and single agent DPI (IDL)) carries out above-mentioned ejection dosetest.When the micronization DHEA-S that weighs (only using the B bottle), when being added to it in gelatine capsule or the vesicatory (blister), a little gathering appears in micronised powder.With 30,60 and the result of the ejection dosage test carried out of the flow velocity of 87.8L/min be presented in the table 2.Table 2 is results of 3 different in flow rate in the Rotahaler test, the result of 3 different in flow rate in the result of 3 different in flow rate and the multi-agent test in the Diskhaler test.

Table 2: the comparison of three kinds of different powder inhaler ejection dosage

Suction apparatus	Airflow rate (L/min)	Ejection dosage (%)
			Rotahaler	87.8	73.2，67.1，68.7
On average		69.7
			Rotahaler (research for the second time)	87.8	16.0，24.5，53.9

On average		31.5
Diskhaler	87.8	65.7，41.6，46.5
			On average		51.3
Diskhaler (research for the second time)	87.8	57.9，59.9，59.5
			On average		59.1
			The IDL multi-agent	87.8	71.3，79.0，67.4
On average		72.6
			IDL multi-agent (research for the second time)	87.8	85.7，84.6，84.0
On average		84.8
Rotahaler	60	58.1，68.2，45.7
			On average		57.3
Diskhaler	60	63.4，38.9，58.0
			On average		68.2
			The IDL multi-agent	60	78.8，83.7，89.6
On average		84.0
Rotahaler	30	34.5，21.2，48.5
			On average		34.7
Diskhaler	30	53.8，53.4，68.7
					58.6
The IDL multi-agent	30	78.9，88.2，89.2
			On average		85.4

(4) can breathe Research on dose

The cascade impactor of use standard (Andersen) carries out breathing dosage (but respirable fraction) research, this sampler is made up of the reserve filter in inlet cone (using sampler preseparator to replace at this), 9 workbench, 8 collecting boaries and 8 the aluminum workbench, these 8 aluminum workbench are being supported together by 3 alligator clamps and O-ring liner, and wherein each sampler workbench comprises a plurality of accurate borings.When air communication was crossed sampler, a plurality of aerojet streams in each workbench carried the surface that granule blows to this workbench collecting board with gas.The size of the gaseous blast of each workbench is constant, but the air-flow size of each workbench diminishes successively.Whether granule strikes the gaseous blast speed that will depend on each workbench on each workbench and the cutoff value of work at present platform.All granules of being collected by first workbench are not all along with the air-flow around this panel edges enters next workbench, at that granule bump onboard or by this plate and enter next workbench, and the like, enough be used for bump up to the speed of gaseous blast.Granule is upspring when preventing to carry out the cascade impactor test, and each sampler plate all wraps and scribbles hexane-oils and fats (fine vacuum) solution (100: 1).As mentioned above, the granule cutoff value on the sampler plate changes according to different air velocitys.For example, workbench 2 when 60L/min corresponding to cutoff value greater than 6.2 micron particle, when 30L/min corresponding to cutoff value greater than 5.8 micron particle; The granularity cutoff value of workbench 3 when 90L/min is greater than 5.6 microns.Thereby, when comparable airflow rate, preferably use similar granule cutoff value, that is, and in the scope of 5.6-6.2 micron.The device of the test Diskus that U.S. Phamacopeia proposes is made up of the spout adapter that is connected in glass trunnion (replacing with 50 milliliters round-bottomed flasks) (being silicone in this embodiment) and the glass end pharyngeal canal (leader) and the Andersen sampler of guiding preseparator.This preseparator sample comprises the washings that obtains from spout adapter, glass trunnion, terminal pharyngeal canal and preseparator.Before carrying out the cascade impactor test, earlier with 5 milliliters of acetonitriles: water (1: 1) solvent is put in this preseparator.To 3 kinds of different Diskuses, respectively with 30,60 and the airflow rate of 90L/min carry out twice this cascade impactor test.Adopt HPLC to analyze the medicine of on the cascade impactor plate, collecting, to each Diskhaler and multi-agent cascade impactor test carrying out medicine mass balance (massbalance), comprise the amount that remains in the medicine in the vesicatory of measuring, remain in the amount of the medicine in the device (only Diskhaler), remain in silicone rubber spout adapter, the glass trunnion, the amount of the medicine that can not suck on glass end pharyngeal canal and the preseparator, they are all blended together a duplicate samples, measure respirable dosage then, promptly in the workbench 2 30 and the air draught of 60L/min under by the dosage of filter impaction sampler plate and in workbench 1 in the 90L/min test dosage by filter impaction sampler plate.

Table 3: cascade impactor test (90L/min)

Suction apparatus	Preseparator (%)	Vesicatory (%)	Can breathe dosage (%)	Device (%)	Mass balance (%)
						Diskhaler	72.7	6.6	2.9	22.1	104.3
Diskhaler	60.2	10.1	2.4	13.3	86.0
Multi-agent	65.8	3.9	3.8	26.5 *a	100.0
						Multi-agent	73.3	3.8	3.6	19.3 *a	100.0
Multi-agent *b	78.7	2.8	4.6	13.9 *a	100.0
						Multi-agent *c	55.9	5.0	1.2	37.9 *a	100.0

^*A: do not wash the multi-agent device, because solvent can be attacked the SLA component.Obtain the multi-agent device and be detained the percentage ratio difference.

^*B: toasted exsiccant medicine 80 minutes

^*C: toasted exsiccant medicine 20 hours

Following conclusion derives from ejection dosage and cascade impactor test.But obtain low inhalant value in the cascade impactor test, this is the cause of assembling because of drug particles, even the drug particles of assembly can not be separated under the highest airflow rate.Our suggestion is, the assembly of medicine is the static of setting up in the mechanical lapping processing that is used for reducing granularity and the result who causes, and this state is further absorbed compound by ensuing pellet moisture.The method of micronization that produces the DHEA-S crystal (being dihydrate form) of the complete hydration of less static or less moisture absorption should be able to provide and reduce the more free-pouring powder of assembling probability.

Embodiment 4: the mensuration that the spray of anhydrous DHEA sulfate is done and can be breathed dosage

(1) micronization of medicine

The anhydrous DHEA sulfate of 1.5g is dissolved in 100 milliliter of 50% ethanol: in the water, obtain 1.5% solution.(Switzerland) this solution is done in spray for Buchi, Flawil, and inlet temperature is that 55 ℃, outlet temperature are that 40 ℃, 100% inhaler, 10% pump, stream of nitrogen gas are that 40mbar, spraying flow velocity are 600 units to spray dried device with B-191Mini.To spray dryed product and be suspended in the hexane, and add the Span85 surfactant and assemble to reduce.The supersound process dispersion liquid cools off 3-5 minute to disperse this dispersion liquid of test on the Malvem Mastersizer X that is equipped with small size sampler (SVS) adnexa fully.The particle mean size of finding two batches of spray dries is respectively 5.07 ± 0.70 microns and 6.66 ± 0.91 microns.Adopt optical microscope to disperse the macroscopy of thing to confirm to each batch, little breathed granularity has been given birth in spray dry labor.The particle mean size of each batch is respectively 2.4 microns and 2.0 microns.This proof can be sprayed DHEA-S the dried granularity that becomes to be suitable for sucking.

(2) can breathe Research on dose

As described in embodiment 3, carry out the cascade impactor test.Carry out four cascade impactor tests, wherein three are used IDL multi-agent device, and one is used Diskhaler, and the air velocity of all tests all is 90L/min.The results are shown in the table 4 of all cascade impactor tests.Compare the increase that the anhydrous substances that spray is done in these tests has produced twice with the dosage breathed of the anhydrous DHEA-S of micronization.Really obtained the dosage breathed that increases though compare the spray drying method with jet grinding, the % that can breathe dosage remains low.This is because may be the result that anhydrous form absorbs water and assembles and cause.

Table 4: the cascade impactor result of the test that the drug products that uses spray to do carries out

Device	Diskhaler	The multi-agent device	The multi-agent device	The multi-agent device
					Vesicatory quantity
	3	3	4	4
					The medicine of every vesicatory (mg)	38.2	36.7	49.4	50.7
Preseparator (%)	56.8	71.9	78.3	85.8
					Device (%)	11.2	7.9	8.9	7.6
Vesicatory (%)	29.0	6.4	8.2	4.8
					Can breathe dosage (%)	5.6	7.8	5.3	2.6
The mass balance response rate (%)	102.7	94.0	103.3	98.1

Embodiment 5:DHEA-S dihydrate (DHEA-S2H ₂O) mensuration that aerojet is ground and can be breathed dosage

(1) recrystallization of DHEA-S dihydrate

Anhydrous DHEA-S is dissolved in the boiling mixture of 90% ethanol/water.In dry ice/ethanol bath, cool off this solution fast, make the DHEA-S recrystallization.Leach crystal,, be placed in the vacuum desiccator under the room temperature 36 hours altogether then with cold washing with alcohol twice.In the dry run, regularly stir this material, to destroy big aggregation block with scraper.After the drying, make this material by 500 tm screen.

(2) micronization and physical chemistry test

In nitrogen in jet mill micronization DHEA-S, Venturi tube pressure is that 40PSI, grinder pressure are that 80PSI, feed arrangement are 25, the product feed rate is about 120-175g/ hour.Use 5 BET assay determination surface areas, use Micromeritics TriStar surface area analyser to carry out this BET as adsorbed gas (P/Po=0.05 to 0.30) and analyze with nitrogen.Use Micromeritics Satum Digisizer by the laser diffraction analysis particle size distribution, wherein particle suspending uses Sodium docusate salt as dispersant in mineral oil.Adopt KarlFischer titration (Schott Titroline KF) to measure the water content of drug substance.As standard, all relative standard deviations of three tests are less than 1% with pure water.Directly powder is added in the titration culture medium.Before the micronization and the physicochemical characteristics of the DHEA-S dihydrate behind the micronization be listed in the table 5.

Table 5: before the micronization and the physicochemical characteristics of the DHEA-S dihydrate behind the micronization

Characteristic	Caking	Micronization
			Granularity (D 50％)	31 microns	3.7 micron
Surface area (m 2/g)	Do not measure	4.9
			Water (%w/w)	8.5	8.4
Impurity	There is not tangible peak	There is not tangible peak

Measured significant change only is the variation of granularity.The loss of water is also not obvious, and the increase of impurity is also not obvious.The surface area of micronization material and mean size are that the particulate surface area of irregular shape of 3-4 micron is consistent.The micronization success reduces to the scope that is suitable for sucking through granularity, and does not produce measurable variation in solid state chemistry.

(3) the DHEA-S dihydrate is aerosolized

With single agent Acu-Breathe device assessment DHEA-S dihydrate.About 10 milligrams of pure DHEA-S dihydrates are filled in the paper tinsel vesicatory, and sealing.Impel these vesicatories to enter in 8 workbench stepwise impaction samplers of Andersen that flow velocity is 30-75L/min, this sampler has a glass binary collision device trunnion.Clean the 1-5 workbench of Andersen impaction sampler together, obtain the fractional estimated value of fine powder.To merge into a test sample from the medicine that a plurality of workbench collections obtain and make the better sensitivity of the method.The result of this campaign is presented among Fig. 1.Under all flow velocitys, the fine particle fraction that dihydrate produces is higher than the in fact mark of anhydrous substances.Owing to use single agent inhaler that the dihydrate powder is aerosolized, therefore can rationally draw the aerosol feature significantly better than anhydrous substances conclusion in fact.Higher degree of crystallinity and stable moisture are to cause dihydrate to have the most possible factor of the aerosol feature of excellence like this.This specific characteristic of DHEA-S dihydrate is not also reported in the prior art.Though the improvement of the DHEA-S aerosol performance of dihydrate form clearly, pure drug substance may not be best preparation.Use the bigger carrier of granularity can improve the aerosol performance of micronized medicine material usually.

Embodiment 6: have or the situation of free from lactose under the stability of anhydrous DHEA-S and DHEA-S dihydrate

Adopt high pressure liquid chromatography (HPLC) to measure the initial purity (time=0) of anhydrous DHEA and DHEA-S dihydrate.With pure form of powder or with 50: 50 ratio DHEA and DHEA-S dihydrate are mixed with lactose respectively then, are placed in the open glass bottle, then with temperature maintenance 50 ℃ of 4 week.Utilize these conditions to make these preparations be subjected to stress, to predict their long-time stability result.Sealing only contains the control bottle of DHEA-S (anhydrous or dihydrate), and its temperature is remained on 25 ℃ to 4 weeks.Obtain sample in the 0th, 1,2 and 4 weeks, and carry out HPLC and analyze, measure the amount of degraded, promptly measure the amount of degraded by the formation of measuring DHEA.After 1 week, the in fact anhydrous DHEA-S (50%, nominal) that is mixed with lactose in 50 ℃ of sealed glass jars of storing down produces the tone of brown, and is darker than milk-sugar mixture.This change color is accompanied by stratographic significant change (as shown in Figure 1).Main degradation product is dehydroepiandrosterone or DHEA.From the quantitative data of Fig. 2, the amount of DHEA is higher than the amount in other two duplicate samples in the mixture.For the DHEA% in the quantitative assay sample, with the area at DHEA peak the gross area (the results are shown in Table 6) divided by DHEA-S and DHEA peak.The higher degraded ratio of mixture shows between lactose and the in fact anhydrous DHEA-S special reaction has taken place.Increasing corresponding with DHEA is that As time goes on the brown of quickening the powder of storage (accelerated storage) also increases.Taking by weighing sample is used for caking phenomenon that the chemical analysis process takes place and has confirmed also that the material that quickens to store demonstrated with the time and concern more closely.Based on these results, can not prepare in fact anhydrous DHEA-S with lactose.This is very disadvantageous, because the lactose the most frequently used suction excipient that is dry powder formulations.Continuation then means with in fact anhydrous form will be restricted to preparation pure powder, perhaps will use new excipient need carry out safety research widely.

Show 6:50 ℃ of percentage ratio from the DHEA of anhydrous DHEA-S formation

Preparation	Time (week)	1	2	4
					Contrast	2.774	2.694	2.370	2.666
Independent DHEA-S		9.817	14.954	20.171
					DHEA-S+ lactose (50: 50)		24.085	30.026	38.201

Different with Fig. 2, in fact there is not DHEA to produce (see figure 3) after 1 week of storage down at 50 ℃.In addition, the color of material does not change.In fact the moisture of DHEA-S dihydrate did not change after 50 ℃ of 1 weeks of storage.Compare with initial 8.8%, quickening to store the back moisture is 8.66%.The DHEA% that records in this stable program is presented in the table 7.

The DHEA percentage ratio that forms by the DHEA-S dihydrate under showing 7:50 ℃

Preparation	Time (week)	1	3	4
					Contrast	0.213			0.218
Independent DHEA-S		0.216	0.317	0.374
					DHEA-S: lactose (50: 50)		0.191	0.222	0.323

By comparison diagram 1,2 and table 6,7, the dihydrate form of DHEA-S is more stable form as can be seen, is applicable to further research.Do not report in existing patent and the research document that DHEA-S dihydrate and milk-sugar mixture are with respect to the excellent compatibility of anhydrous substances in fact yet.Next part will be described the dissolubility of this material, as a part of aerosol apparatus solution R﹠D work.

Embodiment 7:DHEA-S dihydrate/milk-sugar mixture, can breathe dosage and stability mensuration

(1) DHEA-S dihydrate/milk-sugar mixture

The lactose (Foremost Aero Flo 95) of the DHEA-S dihydrate of weight such as hand mix and suction grade makes mixture pass through 500 μ m sieve then, makes premix.Then this premix and remaining lactose are put in the BelArt Micro grinder, obtain the DHEA-S mixture of 10%w/w.Blender is connected with the variable voltage power supply, to regulate the speed of agitator.The voltage of blender is respectively with 30%, 40%, 45% and 30% total head circulation 1,3,1.5 and 1.5 minute.Content uniformity (contentuniformity) by HPLC assay determination mixture.Table 8 shows the result of this mixture content homogeneity sample.Desired value is the DHEA-S of 10%w/w.Mixture content is gratifying, near desired value and content difference.

The content difference of the mixture of table 8:DHEA-S dihydrate and lactose

Sample	％DHEA-S，w/w
		1	10.2
2	9.7
		3	9.9
4	9.3

5	9.4
		Meansigma methods	9.7
RSD	3.6％

(2) DHEA-S dihydrate/milk-sugar mixture is aerosolized

This about 25 milligrams powder is filled in the paper tinsel vesicatory, and sealing, use single agent device aerosolized then with 60L/min.Two doses of vesicatories are used in each test respectively, and the result of fine particle fraction (material among the workbench 1-5) is presented in the table 9.The aerosol result of the mixture of powders of this preliminary study is gratifying for the respiratory medications delivery system.Optimizing mixture of powders and vesicatory/unit configuration is to obtain higher fine particle fraction.The whole particle size distribution of test 2 is presented in the table 10.The aerocolloidal average diameter of this DHEA-S is about 2.5 microns.This diameter is less than the average diameter that adopts the measured micronized DHEA-S dihydrate of laser diffraction.The granule of irregular shape can be used as smaller particles and plays a role on aerodynamic, this is because their the longest yardstick tends to arrange with airflow field.Therefore, can see usually between two kinds of methods and there are differences.Diffractometry is the quality control test that is used to import material, and cascade impactor then is the quality control test that is used for final products.

Table 9: the fine particle fraction of milk-sugar mixture in two different tests

Test	Total powder weight (mg) in two vesicatories	The DHEA-S that collects among the workbench 1-5 (mg)	The fine powder mark, %
				1	52.78	1.60	31
2	57.09	1.62	29

Table 10: the particle size distribution of aerosolized DHEA-S dihydrate/milk-sugar mixture

Size (μ m)	6.18	9.98	3.23	2.27	1.44	0.76	0.48	0.27
									Under granule %	100	87.55	67.79	29.87	10.70	2.57	1.82	0.90

(3) stability of DHEA-S dihydrate/milk-sugar mixture

Also this lactose preparation is placed 50 ℃ accelerated stability program.Result about DHEA-S content is presented in the table 11.Contrast is the mixture at room temperature storage.DHEA-S content does not all have as time passes and the trend that changes under any condition, within the scope of the sample that all results gather in the uniform content property testing (referring to table 11).In addition, change color not taking place, does not observe irregular place in the chromatograph yet.Mixture shows chemical stability.

The stress stability that table 11:DHEA-S dihydrate/milk-sugar mixture is 50 ℃

Time (week)	%DHEA-S w/w, collating condition	%DHEA-S w/w, stress condition
			0	9.7	9.7
1	9.6	9.6
			1.86	9.5	9.7
3	10	9.9

The spray agent of embodiment 8:DHEA-S

The dissolubility of DHEA-S.

To be added to according to the excessive DHEA-S dihydrate that " recrystallization of DHEA-S dihydrate (embodiment 5) " make in the solvent mediation, make its balance at least 14 hours, regularly shake simultaneously.Then this suspension is filtered 0.2 micron syringe filter, dilution is immediately then analyzed to carry out HPLC.For preparing refrigerative sample, before using syringe and filter be kept in the electric refrigerator at least 1 hour.Sucking pure water can cause cough to stimulate.Therefore, importantly halide ion will be added in the spray agent, sodium chloride is to make salt commonly used.Because DHEA-S is a sodium salt, therefore owing to the cause of common ion effect, sodium chloride can reduce dissolubility.Fig. 4 has shown as the DHEA-S of the function of sodium chloride concentration at room temperature (24-26 ℃) and the dissolubility when cold preservation (7-8 ℃).The dissolubility of DHEA-S descends along with sodium chloride concentration.Under all sodium chloride concentrations, reduce storage temperature and all can reduce dissolubility.When high sodium chloride concentration, temperature effect a little less than.In three parallel tests, the dissolubility of about 25 ℃ and 0% sodium chloride is 16.5-17.4mg/mL, and relative standard deviation is 2.7%.In 0.9% sodium chloride cold preservation situation, the solubility range of three tests is 1.1-1.3mg/mL, and relative standard deviation is 8.3%.

Balance between the solid-state and liquid DHEA-S is as follows:

NaDHEA-S _Solid-state DHEA-S ^-+ Na ⁺

K=[DHEA-S ^-] [Na ⁺]/[NaDHEA-S] _Solid-state

Because the solid-state concentration of DHEA-S is constant (that is, the dihydrate that physical state is stable), this balanced type can be simplified shown as:

Ksp＝[DHEA-S ^-][Na ⁺]

Based on this hypothesis, the DHEA-S dissolubility and the inverse of total sodium cation concentration to be mapped, the collinear inclination of gained equals Ksp.This shows in Fig. 5 and Fig. 6 respectively, is respectively the balance under room temperature and the cold preservation situation.Based on correlation coefficient, the data under this model and room temperature and the cold preservation situation can both rationally be coincide, and under these two kinds of situations, equilibrium constant is respectively 2236 and 665mM ²In order to make the dissolubility maximum, the level of sodium chloride should be low as much as possible.Spraying should be 20mM or 0.12% sodium chloride with the minimum halide ion content of solution.

In order to assess the DHEA-S concentration of solution, 10 ℃ (promptly 15 ℃) in use descend the temperature in the aerosol apparatus.Interpolation between equilibrium constant and absolute temperature inverse, promptly the Ksp 15 ℃ the time is about 1316mM ²The DHEA-S of each mole provides 1 mole sodium cation to solution, therefore:

Ksp＝[DHEA-S][Na ⁺]＝[DHEA-S ^-][Na ⁺+DHEA-S ^-]

＝[DHEA-S ^-] ²+[Na ⁺][DHEA-S ^-]

It calculates [DHEA-S-] with quadratic equation.Ksp is 1316mM ²20mM Na+ solution be DHEA-S-or the 10.7mg/mL of 27.5mM.Therefore, 0.12% sodium chloride solution of selecting 10mg/mL DHEA-S for use is used for further test for candidate's preparation well.The estimated value of this formula will not take in owing to water evaporates the concentration affects that causes from aerosol apparatus.The pH that contains 0.12% sodium chloride solution of 10mg/mL DHEA-S is 4.7-5.6.Though this is an acceptable value for sucking preparation, assessed the effect of using the 20mM phosphate buffer.Buffer and non-buffer solubility results at room temperature are presented among Fig. 7.The existence of buffer has suppressed dissolubility in the preparation, when especially sodium chloride levels is low.As shown in Figure 8, the dissolubility data of buffer solution drops on the straight line identical with the balance straight line of non-buffer solution.It is caused by extra sodium cation content that the dissolubility that uses buffer to cause descends.Making the dissolubility maximum is an important target, and has reduced dissolubility with the buffer formulated.The stability of NaDHEA-S was not significantly increased when in addition, the research of Ishihora and Sugimoto (1979, Drug.Dev.Indust.Pharm., 5 (3) 263-275) was presented at neutral pH.

Stability study.

In 0.12% sodium chloride, prepare the DHEA-S preparation of 10mg/mL, be used for the solution-stabilized Journal of Sex Research of short-term.The sample aliquot of this solution is added in the transparent vial, respectively in room temperature (24-26 ℃) and 40 ℃ of preservations.DHEA-S content, DHEA content and outward appearance in the every day test sample.On each time point, from each bottle, obtain double sample and dilution.Fig. 9 and 10 has shown the relation of DHEA-S content and this search time length.Under acceleration environment, solution shows degraded faster, and is preserving appearance muddiness two days later.At room temperature the solution of Bao Cuning was more stable, a little precipitation occurred at the 3rd day.Stopped this research at the 3rd day.The decomposition of DHEA-S is accompanied by the increase of DHEA content, as shown in figure 10.Because DHEA is insoluble in water, it only needs can produce on a small quantity muddy solution (quickening to preserve) or crystalline deposit (room temperature preservation) in preparation.This has just explained the visual assessment substantially understate early that why the DHEA-S dissolubility the carried out dissolubility of this chemical compound: a spot of DHEA will cause the experimenter to draw the conclusion of the solubility limit that exceeds DHEA-S.Though this is not a preparation that commercial viability is arranged, in clinical trial, should be stable at the solution of preparing again that day.The aerosol properties of said preparation is described with the lower part.

Spraying research.

Use Pari ProNeb Ultra compressor and LC Plus aerosol apparatus to make the DHEA-S atomizing.Figure 11 has shown the sketch map of this assay device.5 ml solns are added in the aerosol apparatus, continue to atomize, up to output material become visually not obvious till (4.5 to 5 minutes).Use has the Califonia InstrumentsAS-6 type 6 workbench impaction samplers test atomized soln of USP trunnion.After atomizing one minute, operate 8 seconds of this impaction sampler with 30L/min, collect sample.In the every other time of this test, with about 33L/min aerosol is introduced in the bypass catcher, clean gathering-device, aerosol apparatus and impaction sampler with mobile phase, carry out HPLC and analyze.In aerosol apparatus, use 0.12% sodium chloride solution of 5 milliliters of DHEA-S.This volume is chosen as the actual upper limit of using in the clinical research.The results are shown in the following table of first 5 tests.

Table 12: the result who uses DHEA-S to spray and study

Solution-aerosol apparatus #	Remain in the amount in the aerosol apparatus, mg	Be deposited on the amount in the catcher, mg	Be deposited on the amount in the sampler, mg	Amount to mg
					10mg/mL-1	17.9 *	16.3	0.38	34.6
10mg/mL-2	31.2	17.2	0.48	49.0
					7.5mg/mL-1	19.3	16.3	0.35	36.0
7.5mg/mL-1	21.7	15.4	0.30	37.4
					5.0mg/mL-1	14.4	10.6	0.21	25.2

^*Only assess the liquid of from aerosol apparatus, pouring out, do not weigh aerosolized before and afterwards weight or clean whole unit

Made aerosol apparatus #1 dry about 5 minutes, and make aerosol apparatus #2 drying be less than about 4.5 minutes slightly.In all cases, the liquid volume that remains in the aerosol apparatus approximately is 2 milliliters.After taking out from aerosol apparatus, this liquid is muddy at first, and it is clear to become in 3-5 minute then.Should be after the time even crossed, the solution of 10mg/mL also shows a spot of coarse deposit.Look like in this liquid thin air bubble and caused initial muddiness.DHEA-S has demonstrated surface activity (that is, promoting foam to form), and this makes the bubble stabilizes in the liquid.Deposit among the 10mg/mL shows that the dissolubility of medicine surpasses the dissolubility in other environment in the aerosol apparatus environment.Therefore, the extra spray testing shown under lower concentration, carry out table 13.Table 13 shows " dosage " linear excessive data to solution concentration.

Table 13: the result of the extra spray testing that use DHEA-S carries out

Solution-aerosol apparatus #	Remain in the amount in the aerosol apparatus, mg	Be deposited on the amount in the catcher, mg	Be deposited on the amount in the sampler, mg	Amount to mg
					6.25mg/mL-2	17.8	12.1	0.24	30.1
7.5mg/mL-3	21.2	13.8	0.33	35.3

Dry aerosol apparatus #3 has used and has been less than about 4.5 minutes slightly and reaches dry.With quality in the bypass catcher and initial solution concentration mapping, as shown in figure 12.Sxemiquantitative ground demonstrates favorable linearity from 0 to 7.5mg/mL, and collected afterwards amount begins to occur departing from.Though the dissolubility that cooling reduces is also included within the calculating of 10mg/mL solution, any concentration affects of medicine and sodium chloride content all is left in the basket.Therefore, to form precipitate be possible to the supersaturation by spraying liquid.Data shown in Figure 12 and after spraying in the 10mg/mL solution more observed granules show that as the notional evidence of clinical preparation, the highest solution concentration is approximately 7.5mg/mL.One aerosol sample is introduced in the cascade impactor, carried out grain size analysis.The tendency that does not have the detectable particle size distribution relevant with solution concentration or aerosol apparatus quantity.The average particle size distribution that is obtained in all spray testings is presented among Figure 13.The aerosol particle measured value discloses/advertisement result consistent (that is, median diameter is about 2 microns) with this aerosol apparatus.Though in vitro tests proof nebulizer formulation can be failed and pass respirable DHEA-S aerosol, said preparation is unsettled, and needs 4-5 minute continuous atomizing.Therefore, stable DPI preparation has significant advantage.The DHEA-S dihydrate is accredited as the most stable solid-state state that is used for the DPI preparation.If contact with water before atomizing and keep its stability by eliminating this anhydrous form, anhydrous form also is fit to give with aerosol apparatus.For the clinical trial of DHEA-S, best spray agent is 0.12% sodium chloride solution of the DHEA-S of 7.5mg/mL.The pH that does not need the buffer system preparation also is an acceptable.By sodium cation concentration being reduced to the minimum water solubility maximization that makes DHEA-S.The minimum sodium chloride levels of no buffer can realize this target.This is that it will can not produce precipitation in atomization process with the highest drug level of the Cl-acquisition of 20mM.This preparation at room temperature at least can stable existence 1 day.

Embodiment 9: the preparation of test model

Obtain cell culture HT-29SF cell, it is that (subbreed Md.) can be in the PC-1 culture medium of the serum-free of definition (Ventrex, Portland, Me.) interior growth fully for ATCC, Rockville for the HY-29 cell.Cultured cell is cultivated in this culture medium, and cultivation temperature is 37 ℃ and (contains 5%CO ₂Wet air in).(Gibco, Grand Island N.Y.) went down to posterity after the digestion, change a subculture in per 24 hours with pancreatin/EDTA when cell covered with.Under this condition of culture, the time that the HT-27SF cell doubles when being in exponential phase is 24 hours.

Flow cytometry

Cell is inoculated into the 60mm culture dish in duplicate, each culture dish inoculation 10 ⁵Individual cell.In cell culture, add the distribution of 0,25,50 or 200 μ M DHEA with the analysis of cells cycle.Add 0 or 25 μ M DHEA in the cell culture and be used to analyze the counter-rotating of the influence of DHEA cell cycle, add MVA, CH, RN, MVA in the culture medium and add CH or MVA and add that CH adds RN or what does not add.Culture after 0,24,48,74 hour with trypsinization, fixing and with Bauer etc., Cancer Res., 46, the method dyeing that 3173-3178 (1986) describes.In brief, centrifugal collecting cell is resuspended in the saline of cold phosphate-buffered.Cell is fixed with 70% ethanol, and washing is then in being resuspended to the saline of cold phosphate-buffered.Add the hypotonic dyeing liquor of 1ml (50 μ g/ml propidium iodides (Sigma Chemical Co.) then, 20 μ g/ml RNA enzyme A (BoehringerMannheim, Indianapolis, Ind.), 30 μ g/ml Polyethylene Glycol, 0.1%Triton X-100, be dissolved in the 5mM citrate buffer), left standstill under the room temperature 10 minutes, add 1ml etc. then and ooze dyeing liquor (propidium iodide, Polyethylene Glycol, Triton X-100 is dissolved in the 0.4M sodium chloride solution), with flow cytometer (the Becton Dickinson Immunocytometry Systems that is equipped with pulse amplitude/pulse area dual resolution design, San Jose CA) analyzes.With analyzing the minimum analysis 2 * 10 of each sample after the fluorescence beam correction ⁴Individual cell, the interior fluorescence intensity enhanced cell sum of each passage is represented with s in 1024 passages, and resulting rectangular histogram is analyzed with Cellfit analysis programme (Becton Dickinson).

The influence of DHEA cell growth

Cell inoculation in the 30mm culture dish, 4 culture dishs of every kind of cell inoculation, each culture dish is inoculated 25000 cells, adds 0,12.5,25,50 or 200 μ M DHEA after 2 days.1, uses Coulter calculating instrument (Z type after 24,48 and 72 hours; Coulter Electronics, Inc.Hialeah FL) carries out cell counting.DHEA (AKZO, Switzerland, Basel) is dissolved in the dimethyl sulfoxide, filtration sterilization, be stored in-20 ℃ standby.

What Figure 14 showed is the inhibition of DHEA to the growth of HT-29 cell.Point is represented cell number, and horizontal stripe is represented SEM.Each data point is represented 4 multiple holes, test triplicate.Unconspicuous local explanation SEM is also littler than symbol for the SEM horizontal stripe.Compare with matched group, add behind the 12.5 μ M DHEA behind 72 hours, 25 or 50 μ M DHEA behind 48 hours, 200 μ M DHEA and cell number to be reduced in 24 hours, illustrate DHEA can time dependent mode and dose-dependent mode suppress the growth of cell.

The influence of DHEA cell cycle

For the influence of checking the DHEA cell cycle to distribute, inoculation HT-29SF cell (10 ⁵Cell/60mm culture dish), handle with 0,25,50 or 200 μ M DHEA after 48 hours.What Figure 15 showed is the influence of DHEA to the HT-29SF cell cycle distribution.24, harvesting after 48 and 72 hours is fixed with ethanol, then with propidium iodide dyeing, by flow cytometry analysis dna content/cell.Be in G with the calculating of Cellfit cell cycle analysis program ₁, S and G ₂The percentage of cells of M phase.Use tetragonal labelling in order to distinguish the S phase.The data that twice mensuration obtains show with typical rectangular histogram.The test triplicate.

Initial 24 hours inner cell cells in culture period profile after 25 or 50 μ M DHEA handle do not change.But along with the prolongation in processing time, the cell proportion that is in the S phase reduces gradually, is in G ₁, S and G ₂The percentage of cells of M phase is calculated with Cellfit cell cycle analysis program.Use tetragonal labelling in order to distinguish the S phase.The data that twice mensuration obtains show with typical rectangular histogram.The test triplicate.

Initial 24 hours inner cell cells in culture period profile after 25 or 50 μ M DHEA handle do not change, and still along with the prolongation in processing time, the cell proportion that is in the S phase reduces gradually, is in G ₁The percentage of cells of phase increased in processing in back 72 hours.Be in G ₂The cell of M phase had an instantaneous obvious increase in back 48 hours in processing.It is similar to handle resulting result with 200 μ M DHEA, begins to reduce at the back cell proportion that was in the S phase in 24 hours of processing, and lasts till off-test always.This explanation can time dependent mode and dose-dependent mode with the HT-29SF blocking-up at G ₁Phase.

The cell growth of embodiment 10:DHEA mediation and the counter-rotating of cell cycle effect

The growth inhibiting counter-rotating of DHEA mediation.

Cell is inoculated as stated above, with the culture medium culturing that contains 0 or 25 μ M DHEA, adds mevalonic acid (" MVA " in the culture medium after 2 days; MM), zamene (SQ; 80 μ M), cholesterol (CH; 15 μ g/ml), MVA+CH, ribonucleotide (RN; Uridine, cytidine, adenosine and guanosine, final concentration are 30 μ M), dezyribonucleoside (DN; Thymidine, deoxycytidine, deoxyadenosine and deoxyguanosine, final concentration are 20 μ M), RN+DN, MVA+CH+RN or the culture medium of not adding above-mentioned substance.All chemical compounds all available from Sigma Chemical Co. (St.Louis, Mo.).Cholesterol dissolves immediately with ethanol before use.The result shows, even RN and DN reach the also not influence of growth of Cmax pair cell when not containing DHEA.

What Figure 16 showed is the cytostatic counter-rotating of the inductive HT-29SF of DHEA.In A, added 2 μ M MVA, 80 μ M SQ, 15 μ g/ml CH or MVA+CH in the culture medium or do not added above-mentioned substance (CON).In B, culture medium has been added the mixture of RN, wherein contains uridine, cytidine, adenosine and the guanosine of 30 μ M; The perhaps mixture of DN wherein contains thymidine, deoxycytidine, deoxyadenosine and the deoxyguanosine of 20 μ M; RN adds DN (RN+DN); Or MVA adds CH and adds RN (MVA+CH+RN).Respectively at before handling or handle and carried out cell counting in back 48 hours, the value added of inner cell number during the growth of cell is calculated as and handled in 48 hours.Cylindricality is represented the cell growth percent of untreated control; Horizontal stripe is represented SEM.The cell number value added of untreated control group is 173,370 " 6518.Each data point has represented in four independent trialss four to repeat the resulting data meansigma methodss of culture dish.Statistical analysis adopts Si Shi t check, compares κ with matched group and represents p＜0.01; ψ represents p＜0.001.The not influence of growth that under the condition of no DHEA, adds the above-mentioned substance pair cell.

Under these conditions, the inductive growth inhibited of DHEA can partly be overcome by MVA or MVA+CH.Add SQ or CH separately and do not have this effect.The cell inhibitory effect active part of this explanation DHEA is that the disappearance by the endogenous mevalonic acid causes, and the disappearance of endogenous mevalonic acid can cause early stage instant biosynthetic inhibition in the cholesterol path, and this is that the cell growth is necessary.And, can make its growth obtain part behind adding RN or the RN+DN and recover, but adding DN does not have this effect, illustrate that the exhaustion in mevalonic acid and nucleotide pond participates in the inductive growth inhibited effect of DHEA.But add these materials, comprise uniting adding the growth inhibitory effect that MVA, CH and RN can not overcome DHEA fully, illustrate that the biochemical pathway that DHEA has cellulotoxic effect or has other participates.

The counter-rotating of DHEA cell cycle effect

The HT-29SF cell is handled ability with the cell cycle specific effect that detects its blocking-up DHEA mediation with 25 μ M DHEA and other chemical compounds such as MVA, CH or RN.After processing 48 and 72 hours by the flow cytometry cell cycle distribution.

The counter-rotating situation that makes the inductive HT-29SF cell cycle arrest of DHEA that Figure 17 shows.Cell inoculation (10 ⁵Cell/60mm culture dish) handled with 0 or 25 μ M DHEA in back 48 hours.Culture medium is added 2 μ M MVA; 15 μ g/mlCH; The RN mixture that contains 30 μ M uridines, cytidine, adenosine and urine purine nucleoside; MVA adds CH (MVA+CH); Or MVA adds CH and adds RN (MVA+CH+RN) or do not add any material.Harvesting after 48 or 72 hours is fixed with ethanol, propidium iodide dyeing, each intracellular dna content of flow cytometry analysis.Be in G with the calculating of Cellfit cell cycle analysis program ₁, S and G ₂The percent of M phase cell.Use tetragonal labelling in order to distinguish the S phase.The data that twice mensuration obtains show with typical rectangular histogram.Test repeats 2 times.The not influence of distribution that under the condition of no DHEA, adds the above-mentioned substance cell cycle.

Along with the prolongation in processing time, DHEA can reduce the ratio of the cell that is in the S phase gradually.Add in 48 hours behind the MVA and can partly stop this effect, but this effect disappears after 72 hours, adds the minimizing that MVA+CH also can partly stop S phase cell in 72 hours, illustrate that MVA and CH are that cell is grown needed when extending contact time.Add MVA, CH and RN the cell growth is recovered, but still can't return to the S phase cell proportion of untreated matched group.Add separately behind CH or the RN and all not have what effect in 48 or 72 hours.From morphology, cell becomes circle behind the adding DHEA, and this variation only needs to add MVA and just can stop in culture medium.Shown DHEA handles some back 72 hours DNA rectangular histogram the cell subsets that exists dna content obviously to reduce also is described among Fig. 4.Because HT-29 cell line is the known chromosomal cell mass (68-72 of various numbers that contains; ATCC), thus it represented one to contain and reduce chromosomal subgroup.

Conclusion

The embodiment 9-10 proof that provides above can make the growth of cell be suppressed with the known DHEA concentration handler colon carcinoma cell line HT-29SF of endogenous mevalonic acid that exhausts, and cause G1 to stagnate, add MVA and in culture medium, can partly stop this effect.It is similar with specificity 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor such as lovastatin and the viewed effect of anti-handkerchief fourth in many aspects to the effect of protein prenylation that DHEA produced.But, DHEA is not the biosynthesis that directly suppresses mevalonic acid, but bring into play the influence of its cell cycle and cell growth in polyphenic mode, this mode relates to the biosynthesis of ribonucleotide or deoxyribonucleotide, can also have other factors to participate in.

Embodiment 11: metered-dose inhaler

Activating agent	The amount that at every turn excites
		Zafirlukast DHEA stabilizing agent Arcton 11 dichlorodifluoromethane	25.0μg 400mg 5.0μg 23.70mg 61.25mg

Embodiment 12: metered-dose inhaler

Activating agent	The amount that at every turn excites
		Zafirlukast DHEA stabilizing agent Arcton 11 dichlorodifluoromethane	25.0μg 400mg 7.5μg 23.67mg 61.25mg

Embodiment 13: metered-dose inhaler

Activating agent	The amount that at every turn excites
		Zafirlukast DHEA stabilizing agent Arcton 11 dichlorodifluoromethane	25.0μg 400.0mg 15.0μg 23.56mg 61.25mg

Embodiment 14: metered-dose inhaler

Activating agent	The amount that at every turn excites
		Zafirlukast DHEA stabilizing agent Arcton 11 dichlorodifluoromethane	25.0μg 400.0mg 15.0μg 23.56mg 61.25mg

Among the embodiment 15-18 below, first and second activating agents are by micronization, and with lactose in the top ratio moistening in bulk of giving.Mixture is filled in hard gelatin capsule or the cartridge case or the special two paper tinsel blister packages of packing into (Rotadisks blister package, use inhaler such as Rotahaler inhaler (Glaxo ^) use Glaxo ^Inhaler, or fail with the Diskhaler inhaler and to pass blister package (Glaxo ^).

Embodiment 15: the quantitative powder preparation

Activating agent	The content of each cartridge case or each bubble
		Zafirlukast DHEA lactose Ph.Eur.	72.5 μ g 1.00mg to 12.5 or 25.0mg

Embodiment 16: the quantitative powder preparation

Activating agent	The content of each cartridge case or each bubble
		Zafirlukast DHEA lactose Ph.Eur.	72.5 μ g 1mg to 12.5 or 25.0mg

Embodiment 17: the quantitative powder preparation

Activating agent	The content of each cartridge case or each bubble
		Zafirlukast DHEA lactose Ph.Eur.	72.5 μ g 1mg to 12.5 or 25.0mg

Embodiment 18: the quantitative powder preparation

Activating agent	The content of each cartridge case or each bubble
		Zafirlukast DHEA lactose Ph.Eur.	72.5 μ g 1mg to 12.5 or 25.0mg

Embodiment 19: the combination of sulphuric acid DHEA and leukotriene antagonist

Leukotriene is synthetic (Gilchrist in cell (being not only mastocyte), M., McCauley, S.D. and Befus, A.D. (2004) " expression, location and adjusting of NOS in the human mast cell system: " (Expression to the effect of leukotriene generation, localization, and regulation ofNOS in human mast cell lines:effectson leukotriene production) Blood 104,462-469).We have measured the threshing that whether can reduce mastocyte in the rat peritoneum mastocyte with sulphuric acid DHEA after the compound 48/80 stimulation.Can quantize the threshing of mastocyte by measuring histamine (in the mastocyte secretory granule contained another kind of material).The thing of surrogate markers easily that the histamine that discharges by simple measurement discharges as leukotriene.

The rat peritoneum mastocyte

Rat peritoneum mastocyte (2 * 10 with fresh separated ⁵Cell) in the balanced salt solution that contains 150mM NaCl (pH7.4), 2.7mM KCL, 0.9mM CaCl2,4mM Na2HPO4,2.7mM KH2PO4,1.75mg/ml BSA and 0.1 μ g/ml compound 48/80, cultivated 5 minutes in advance in 37 ℃.Add DHEAS or water (contrast) afterwards and with mixture 37 ℃ of incubations 2 minutes.

After the incubation mixture is cooled to 4 ℃, 4500rpm is centrifugal 5 minutes then.Collect supernatant and mix 30 minutes so that protein precipitation with 5%TCA (trichloroacetic acid) at 4 ℃.Contracting agent supernatant and mix after centrifugal 15 minutes of the 9500xg with 0.25M HCl.Then sample and 2M NaOH and 0.2%OPT (phthalic aldehyde (ortho-phtalaldehyde)) are mixed and be incorporated in 4 ℃ of dark culturing 30 minutes, add 0.5M H afterwards ₂SO ₄Cessation reaction.

Measure the amount that fluorescence intensity quantizes excretory histamine with spectrofluorimeter (GeminiXS, Molecular Devices) at λ ex=360nm and λ em=450nm.The result is expressed as the excretory inhibition percentage ratio of contrast histamine.

Figure 18 shows, cultivates sulphuric acid DHEA and causes the concentration dependent to the rat hypertrophy cell threshing to suppress, and the maximum effectiveness during 0.1uM is 69.9%.EC50 is 170nM.It should be noted that and adopt present experimental condition, the dissolubility of DHEAS is height-limited during 10-4M, and this pseudo-biological effect (spurious biological effect) significant when this concentration has been described.

LTRA is very effective for the treatment respiratory tract disease, their LT capable of blocking after mastocyte discharges to the effect of target tissue.By above discovery as seen, the combination of sulphuric acid DHEA and LTRA will play a role with property taken advantage of or cooperative mode: DHEAS will reduce the mastocyte threshing and reduce histamine and leukotriene release simultaneously, and LTRA will block the pharmacological action of the leukotriene of any release then.Therefore, the patient will be benefited from the combination of these two kinds of medicines.

Remove sulphuric acid DHEA, other suitable non-glucocorticoid also can be used as first activating agent, includes but not limited to isoandrosterone and derivant thereof, analog and pharmaceutically acceptable salt.The chemical compound described of the I of formula, III and IV here for example.

Though described the present invention in conjunction with aforementioned preferred embodiments, it should be understood that, under the situation that does not depart from Confucian classics of the present invention, can make various changes.

Content on all publications, patent and patent application and the webpage is included in this paper as a reference in full at this, and the degree of including in reaches as these independent publications, patent or patent application and shown situation about will include in full as a reference separately or respectively.

Claims (19)

1. pharmaceutical composition, it comprises pharmaceutically or acceptable carrier on the veterinary drug, first activating agent and and second activating agent, can effectively treat asthma, chronic obstructive pulmonary disease or respiratory tract or pulmonary disease,

(a) described first activating agent is at least a non-glucocorticoid with the non-glucocorticoid shown in non-glucocorticoid shown in the following chemical formula (III) and the chemical formula (IV) that is selected from

In the formula, R1, R2, R3, R4, R5, R7, R8, R9, R10, R12, R13, R14 and R19 independently are H, OR, halogen, (C1-C10) alkyl or (C1-C10) alkoxyl, R5 and R11 independently are OH, SH, H, halogen, pharmaceutically acceptable ester, pharmaceutically acceptable thioesters, pharmaceutically acceptable ether, pharmaceutically acceptable thioether, pharmaceutically acceptable inorganic ester, pharmaceutically acceptable monosaccharide, disaccharide or oligosaccharide, the volution oxidative ethane, volution sulfur ethane,-OSO2R20,-OPOR20R21 or (C1-C10) alkyl, R5 and R6 are=O that R10 and R11 are=O together together; R15 be (1) when R16 be-C (O) is H during OR22, halogen, (C1-C10) alkyl or (C1-C10) alkoxyl, (2) when R16 be halogen, OH or (C1-C10) be H during alkyl, halogen, OH or (C1-C10) alkyl, (3) when being OH, R16 is H, halogen, (C1-C10) alkyl, (C1-C10) thiazolinyl, (C1-C10) alkynyl, formoxyl, (C1-C10) alkanoyl or epoxy, (4) when being H, R16 is OR, SH, H, halogen, pharmaceutically acceptable ester, pharmaceutically acceptable thioesters, pharmaceutically acceptable ether, pharmaceutically acceptable thioether, pharmaceutically acceptable inorganic ester, pharmaceutically acceptable monosaccharide, disaccharide or oligosaccharide, the volution oxidative ethane, volution sulfur ethane,-OSO2R20 or-OPOR20R21, or R15 and R16 are=O together; R17 and R18 independently for (1) when R6 be H OR, halogen, (C1-C10) alkyl or-C (O) is H during OR22,-OH, halogen, (C1-C10) alkyl or-(C1-C10) alkoxyl, (2) when R15 and R16 be H during together for=O, (C1-C10 alkyl) amino, the alkyl of ((C1-C10) alkyl) n amino-(C1-C10), (C1-C10) alkoxyl, the alkyl of hydroxyl-(C1-C10), (C1-C10) alkyl of alkoxyl-(C1-C10), (halogen) m (C1-C10) alkyl, (C1-C10) alkanoyl, formoxyl, (C1-C10) alkoxy carbonyl group or (C1-C10) alkanoyloxy, (3) R17 and R18 are=O together; (4) R17 or R18 form the 3-6 unit ring that contains 0 or 1 oxygen atom with the carbon that they connected; Or (5) R15 forms an epoxide ring with R17 with the carbon that they were connected; R20 and R21 independently are OH, pharmaceutically acceptable ester or pharmaceutically acceptable ether; R22 is H, (halogen) m (C1-C10) alkyl or (C1-C10) alkyl; N is 0,1 or 2; M is 1,2 or 3; Or its acceptable salt pharmaceutically or on the veterinary drug; With

(b) described second activating agent is a LTRA.

2. pharmaceutical composition as claimed in claim 1 is characterized in that, described first activating agent is the non-glucocorticoid with following chemical formula

In the formula, dotted line is represented singly-bound or two key; R is a hydrogen or halogen; 5 H occurs with α or beta comfiguration, and perhaps the chemical compound of Formula I comprises the racemic mixture of two kinds of configurations; R ¹Be hydrogen or with covalently bound polyvalent mineral of this chemical compound or organic dicarboxylic acid.

3. pharmaceutical composition as claimed in claim 1 is characterized in that, described first activating agent is the non-glucocorticoid with chemical formula (I), and wherein, described multivalence organic dicarboxylic acid is SO ₂OM, phosphate or carbonate, wherein M comprises equilibrium ion, and wherein said equilibrium ion is H, sodium, potassium, magnesium, aluminum, zinc, calcium, lithium, ammonium, amine, arginine, lysine, histidine, triethylamine, ethanolamine, choline, triethanolamine, procaine, benzathine benzylpenicillin, Apiroserum Tham, pyrrolidine, piperazine, diethylamine, sulfatide

And phospholipid

R in the formula ²And R ³Can be identical or different, be straight or branched (C ₁-C ₁₄) alkyl or glucosiduronic acid

4. pharmaceutical composition as claimed in claim 3 is characterized in that, described first activating agent is a dehydroepiandrosterone.

5. pharmaceutical composition as claimed in claim 3 is characterized in that, described first activating agent is a dehydroepiandrosterone sulfate.

6. pharmaceutical composition as claimed in claim 1 is characterized in that, described LTRA is montelukast, zafirlukast or pranlukast.

7. pharmaceutical composition as claimed in claim 1 also comprises ubiquinone or its acceptable salt pharmaceutically or on the veterinary drug, and wherein said ubiquinone has following chemical formula

In the formula, n is 1-12.

8. pharmaceutical composition as claimed in claim 1 is characterized in that, described pharmaceutical composition comprises can suck the granule that maybe can breathe size.

9. pharmaceutical composition as claimed in claim 8 is characterized in that, described granular size is about 0.01-10 μ m.

10. pharmaceutical composition as claimed in claim 8 is characterized in that, described granular size is about 10-100 μ m.

11. medicine box that comprises defeated delivery device and the described pharmaceutical composition of claim 1.

12. medicine box as claimed in claim 11 is characterized in that, described defeated delivery device is aerosol generator or spray generator.

13. medicine box as claimed in claim 12 is characterized in that, described aerosol generator comprises inhaler.

14. medicine box as claimed in claim 13 is characterized in that, described inhaler device is carried the various predetermined closes of preparation.

15. medicine box as claimed in claim 13 is characterized in that, described inhaler comprises aerosol apparatus or insufflator.

16. a method that reduces asthma probability or treatment asthma in object, described method comprise object prevention that needs this treatment or the described pharmaceutical composition of claim 1 for the treatment of effective dose.

17. a method that reduces chronic obstructive pulmonary disease probability or treatment chronic obstructive pulmonary disease in object, described method comprise object prevention that needs this treatment or the described pharmaceutical composition of claim 1 for the treatment of effective dose.

18. in object, treat respiratory tract, lung or malignant disease or symptom for one kind, or reduce adenosine or adenosine receptor level or to the method for the sensitivity of adenosine or adenosine receptor, described method comprises object prevention that needs this treatment or the described pharmaceutical composition of claim 1 for the treatment of effective dose.

19. method as claimed in claim 18, it is characterized in that described disease or symptom comprise asthma, chronic obstructive pulmonary disease (COPD), the capsule fibroid becomes sick (CF), dyspnea, emphysema, stridulate, pulmonary hypertension, pulmonary fibrosis, airway hyperreactivity, adenosine or adenosine receptor level raise, the adenosine hypersensitivity, infectious disease, the pulmonary branches trachea shrinks, respiratory inflammation or allergy, pulmonary surfactant or ubiquinone disappearance, chronic bronchitis, bronchoconstriction, dyspnea, lung airway obstacle or block, the test of cardiac function adenosine, the lung vasoconstriction, respiratory disorder, adult respiratory distress syndrome (ARDS), use the medicine of rising adenosine or adenosine level, infant respiratory distress syndrome (baby RDS), pain, allergic rhinitis, cancer or chronic bronchitis.

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