CN101137384A - Combination of dehydroepiandrosterone or dehydroepiandrosterone-sulfate with a beta-agonist bronchodilator for treatment of asthma or chronic obstructive pulmonary disease - Google Patents
Combination of dehydroepiandrosterone or dehydroepiandrosterone-sulfate with a beta-agonist bronchodilator for treatment of asthma or chronic obstructive pulmonary disease Download PDFInfo
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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 beta2-agonist bronchodilator 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
The reference of related application
The application is the U. S. application series number of submitting on October 29th, 2,003 10/698,078 part continuation application, this application requires the priority of the U.S. Provisional Patent Application series number 60/492,232 of submission on July 31st, 2003, and this application is included in as a reference in full.
Background technology
Technical field
The present invention relates to contain the compositions of non-glucocorticoid steroid and β 2-agonist bronchodilatation medicine, described non-glucocorticoid steroid comprises dehydroepiandrosterone (DEHA), sulphuric acid DEHA or its salt.These compositionss can be used for treating asthma, chronic obstructive pulmonary disease (COPD) or other respiratory tract disease.
Background is described
Respiratory tract disease and various associated conditions are very common in the general population.Sometimes patient is with the inflammation that increases the weight of pulmonary's disease.Respiratory tract disease comprises asthma, chronic obstructive pulmonary disease (COPD) and lower respiratory illness, for example allergic rhinitis, adult respiratory distress syndrome and pulmonary fibrosis on other.
For example, asthma is one of modal disease in the industrialized country.In the U.S., it accounts for 1% of institute's unsoundness cost.Report that in the past decade popular the and mortality rate of asthma all increases frighteningly, and asthma is estimated to become main professional pneumonopathy in from now on 10 years.Asthma is the variable disease of a kind of feature, and airway obstruction is reversible in many cases.This process is relevant with pneumonia and the anaphylaxis with pulmonary sometimes.Many patients have the acute stage that is called " asthma attack ", and other people then suffer from chronic disease.It is believed that the asthma process is triggered by the antigen that irritated object sucks sometimes.This disease is commonly referred to " extrinsic asthma ".The morbidity of other asthmatic patient has immanent cause, therefore is called " intrinsic asthma ", comprises the disease of separate sources, replys the anaphylactic disease that mediated etc. as the disease of adenosine receptor mediation, immune IgE mediation.All asthmatic patients have the more distinctive symptoms of this disease: ictal bronchoconstriction, pneumonia and lung surface active substance reduce.Existing bronchodilatation medicine and anti-inflammatory drug be commercially available to be got and writes out a prescription and be used for the treatment of asthma.Modal anti-inflammatory drug glucocorticoid has many ill effects, but still is conventional prescription drugs.The more important thing is that great majority are used for the treatment of the medicine of asthma to many patient's nearly unavailables.
The feature of COPD is normally by chronic bronchitis, emphysema or airflow obstruction that the two causes simultaneously.Airway obstruction generally is not exclusively reversible, but the patient of 10-20% is having some improvement through treating really aspect the airway obstruction.In chronic bronchitis, airway obstruction is caused by chronic and unusual air flue mucus, inflammation, bronchospasm and infection excessive secretion.Except other reason of chronic cough, the feature of chronic bronchitis also has at least two years and recurred chronic cough, generation mucus (or the two all has) at least 3 months.The structural composition (elastin laminin) of emphysema destruction terminal bronchiole causes airway walls to be subsided and can not breathe out " outmoded " air.Emphysema cause the alveolar permanent destruction.Emophysematous feature is the unusual permanent expansion of the gas compartment of far-end terminal bronchiole, does not have tangible fibrosis with the destruction of airway walls.COPD also can cause the Secondary cases pulmonary hypertension.The Secondary cases pulmonary hypertension itself is the unusual disease that raises of a kind of pulmonary artery blood pressure.In cases with severe, right side heart must more firmly be worked than usual and be pumped blood with the antagonism high pressure.If this situation continues for a long time, right side heart increases, function reduces, and body fluid can accumulate in ankle (edema) and abdominal part.At last, the left side heart begins weak.The heart failure that is caused by pneumonopathy is called pulmonary heart disease.
The feature of COPD is to influence middle-aged and elderly people, is one of whole world morbidity and main causes of death.In the U.S., it influences about 1,000 4 million peoples and is the 4th dead reason, the 3rd reason that disables.Yet its M ﹠ M is also rising.Since nineteen eighty-two, estimate that this disease has risen 41% in that the U.S. is popular, the mortality rate that the age is adjusted between 1966 and 1985 has risen 71%.The mortality rate that the age of all reasons of this and the same period (having reduced by 22%) and cardiovascular disease (having reduced by 45%) is adjusted reduces opposite.In 1998, COPD caused the U.S.'s 112,584 people's death.
Yet COPD is preventible, and this is because it is believed that this sick main cause is to contact with flue gas.Long-term smoking is the modal reason of COPD.It accounts for the 80-90% of all cases.The probability that the smoker dies from COPD is higher 10 times than the non-smoker.This disease is rarely found in lifelong non-smoker, but they contact soluble at least some people's airway obstruction with the flue gas in the environment.The paathogenic factor of other proposition comprises air flue super-reaction or super quick, environmental air pollution and allergy.The airflow obstruction that COPD causes generally increases progressively in the people who continues smoking.This causes early stage deformity and shortens the life-span.Smoking cessation makes sickness rate reduce to the non-smoker, but the damage that smoking causes is irreversible.Other risk factor comprises: heredity, passive smoking, contact with contaminated air in work or the environment and the respiratory tract infection medical history of Childhood.The symptom of COPD comprises: chronic cough, uncomfortable in chest, have a rest and between moving period short of breath, dyspnea, mucus produce and increase and want clear clearing throat often.
Currently almost do not have symptom that method can alleviate COPD, prevent it to worsen, keep best pulmonary function and improve ADL and quality of life.Many patients will take medicine all the life, need to increase dosage and other medicines during worsening.The current COPD of being used for patient's prescription drugs comprises: the β 2-agonist of quick acting, anticholinergic bronchodilatation medicine, long-acting bronchodilatation medicine, antibiotic and expectorant.In current available COPD medicine, finding to use anticholinergic agents, β 2 adrenaline excitants and oral steroid has the short-term benefit but not have long term to the progress of this disease.Oral steroid is to recommend to use when acute exacerbation, and this medicine life-time service causes mortality rate and sickness rate to increase.
Fugitive and long-acting suction β 2 adrenaline excitants are realized the bronchorelaxing activity of short-term and are alleviated some symptoms of COPD patient, but the progress of disease is not had significant continuous action.Fugitive β 2 adrenaline excitants can improve COPD patient's symptom, for example improve mobility and produce to a certain degree bronchodilatation, in some serious cases even improved pulmonary function.The maximum effectiveness of finding newer long-acting imbedibility β 2 adrenaline excitants is suitable with fugitive β 2 adrenaline excitants.Though salmaterol only produces medium change to pulmonary function or do not change, find that it has improved symptom and quality of life.Use β2Ji Dongji can produce the cardiovascular effect, for example cause pulse frequency, blood pressure and Electrocardiographic change.Use β2Ji Dongji seldom to produce hypersensitivity reaction, for example gill fungus measles, angioedema, erythra and oropharynx edema.In these cases, should stop using β2Ji Dongji.Treat asthma continuously or COPD patient is not better than treating as required with bronchodilatation medicine ipratropium bromide or fenoterol, so this illustrates that they are not suitable for continued treatment.In addition, the modal direct ill effect of β 2 adrenaline excitants is to tremble, and can cause the reduction of blood plasma potassium content, heart rhythm disorders and arterial oxygen tension force to reduce during high dose.Compare with a kind of medicine with single, coupling β 2 adrenaline excitants and anticholinergic agents do not provide synergetic bronchorelaxing activity.Yet, to compare with a kind of medicine with single, about 90 days of imbedibility β 2 adrenaline excitants of ipratropium (ipratropium) adding standard dose have produced some improvement in stable COPD patient.In a word, the ill effect of use β 2 adrenaline excitants is for example trembled and heart rhythm disorders compares with the more frequent generation of anticholinergic agents.Therefore, anticholinergic agents or β 2 adrenaline excitants, perhaps two kinds of drug combinations can not work to all COPD patients.
Anticholinergic agents has been realized the bronchorelaxing activity of short-term and the improvement that produces some symptoms in COPD patient, but does not improve secular prognosis.Most of COPD patients have airway obstruction to a certain degree at least, and the available ipratropium bromide of this obstruction improves a little." lung health research " (The Lung HealthStudy) finds the vital capacity sign of the early stage COPD of men and women smoker and follows the tracks of and followed up a case by regular visits to them 5 years.Compared 3 kinds of Therapeutic Method during 5 years, the result shows that ipratropium bromide reduces significantly effect of nothing to patient's lung functions dischargeable capacity, and smoking cessation slows down the reduction of lung functions dischargeable capacity.Yet ipratropium bromide produces ill effect, for example heart sympton, hypertension, erythra and urinary retention.
Theophylline produces medium bronchorelaxing activity in COPD patient, but often has ill effect and therapeutic domain little.The serum-concentration of 15-20mg/l is the required and necessary careful monitoring serum levels of the best use of.Ill effect takes place when being included in the blood concentration of height change feels sick, diarrhoea, headache, excited, epilepsy and arrhythmia, even and this in many people, in the therapeutic dose scope, also can take place.The dosage of theophylline must come independent control according to individual's smoking habit, infection and other medicine, and this is pretty troublesome.Though it is said that theophylline has antiinflammatory action in asthma, particularly than low dosage the time, do not appear in the newspapers in COPD patient.The ill effect of theophylline and the frequent monitorings of needs (its serum levels) limit its application.
Oral glucocorticoid shows that in the acute exacerbation patient of COPD short-term improves effect, but long-term oral steroid is relevant with serious adverse, comprises osteoporosis and induces tangible diabetes.Find that the imbedibility corticosteroid does not have substantial short term efficacy to the histamine super-reaction of air flue.In the research of two 3 terms treatments of using the imbedibility fluticasones, medium and serious deterioration significantly reduces, and quality of life obtains the appropriateness improvement and do not influence pulmonary function.The COPD patient who suffers from more reversibility diseases looks to be benefited more more from the treatment of using the imbedibility fluticasone.
The mucolysis medicine has suitable beneficial effect to this sick frequency and persistent period of worsening, but pulmonary function is had harmful effect.Although evidence suggests the frequency that greatly reduces deterioration, yet, no matter be that other mucolysis medicine of N-acetylcystein does not all have remarkable effect to serious COPD (functional dischargeable capacity＜50%) patient.N-acetylcystein produces gastrointestinal side effect.Giving long oxygen therapy to the COPD of hypoxemia (hypoxaemic) and patients with congestive heart failure does not almost have effect to initial about 500 days mortality rate, but increases to some extent and kept stable in ensuing 5 years in male's survival rate after this.But in female patients, oxygen has all reduced mortality rate during whole research.Hypoxemic COPD patient is carried out continuous oxygen therapy reduced total mortality risk in 19.3.Yet, find up to now only to change living habit, smoking cessation and use oxygen therapy (in the hypoxemia patient) to change the long-term process of COPD for a long time.
Antibiotic prevents the further damage and the infection of ill pulmonary when the initial sign of respiratory tract infection also occurring through being everlasting.Expectorant helps lax mucus secretion and it is discharged air flue, and makes that breathing is more light.In addition, other prescription drugs can be used for treating the relevant disease of COPD.These medicines comprise: diuretic (giving as the treatment agent of avoiding the moisture relevant with right heart failure excessively to be detained), Folium Digitalis Purpureae (strengthening heart beating strength) and cough suppressant.The list of medications of back can help to alleviate the relevant symptom of COPD, but does not treat COPD.Therefore, currently do not have symptom that method can alleviate COPD, prevent it to worsen, keep best pulmonary function and improve ADL and quality of life.
Adult respiratory distress syndrome (ARDS) or hard lung, shock lung, pump lung and congestive atelectasis it is believed that it is to be gathered in lung by body fluid, and then cause lung stiff and cause.This disease can excite in 48 hours by the process of following various injury lungs, for example wound, head injury, shock, septicemia, repeatedly blood transfusion, Drug therapy, pulmonary embolisms, serious pneumonia, smoking, radiation, high latitude, almost be drowned etc.ARDS generally takes place as medical emergency case and can be caused by other disease that directly or indirectly causes intravascular fluid body " leakage " to advance lung.In ARDS, the diastole ability of lung is seriously undermined and alveolar and lung backing layer or endothelium is produced damage widely.The modal symptom of ARDS is to breathe effort, rapid, and flared nares is big, because histanoxia causes skin, lip and fingernail to be bluish violet, and anxiety and temporary transient respiratory arrest.The tentative diagnosis of ARDS can and be measured arterial blood gas and determine through the chest X-ray examination.In some cases, ARDS shows and other disease, acute myeloid leukaemia and using for example, and for example the acute tumor lysis syndrome (ATLS) that causes of cytarabin treatment back is relevant.Yet ARDS is relevant with following situation generally: wound, serious blood infection (for example septicemia) or other systemic disease, high dose radiation and chemotherapy and cause the multiple organ failure, MOF cause dead inflammatory response reaction in many cases.In precocious baby (" premature infant "), lung tissue or surfactant be zoon not all.Respiratory distress syndrome (RDS) takes place the premature infant is very serious problem.Now to the premature infant that suffers from RDS by ventilation with use oxygen and the surfactant preparation is treated.When the premature infant is survived from RDS, broncho-pulmonary dysplasia (BDP) takes place through regular meeting in them, and this is also referred to as premature infant's chronic lung disease, often is fatal.
The American of allergic rhinitis affects 1/5th estimates to account for 40-100 hundred million in the health care expenditure in every year, and betides all age brackets.Because many people think their symptom by mistake lasting flu or nasal sinuses problem, allergic rhinitis may not diagnosed out.Usually IgE combines with anaphylactogen in the nose and produces chemical mediator, the inducing cell process, and stimulating neuronal also causes potential inflammation.Its symptom comprises a nasal congestion, rhinorrhea, sneeze and titillation.As time passes, Allergic Rhinitis often produces sinusitis, otitis media companion's discharge and nasal polyp.About 60% Allergic Rhinitis also suffers from the asthma that asthma and allergic rhinitis increase the weight of.Thereby mast cell degranulation causes producing typical rhinitis symptom with the interactional preforming medium release of various cells, blood vessel and mucous gland.After contact allergy was former, most of early stage and late phase response took place in the nose.In chronic allergic rhinitis, observe late phase response and as the supersecretion and the hyperemia of main symptom.Contact antigen repeatedly and cause allergy one or more anaphylactogens.The patient also may be to nonspecific trigger material, for example cold air or the intensive abnormal smells from the patient overresponse that becomes.Non-allergic rhinitis can be by infection, and for example virus is induced, and is perhaps relevant with nasal polyp, as taking place in having the idiocratic patient of aspirin.
Medical condition, for example gestation or hypothyroidism can cause rhinitis with contacting with occupational factor or medicine.So-called NARES syndrome (nonallergic rhinitis with eosinophilia syndrome) is and the relevant nonallergic type rhinitis of oxyphil cell in the nasal discharge, often betides middle age and with certain anosmia.Treatment to anaphylaxis and non-allergic rhinitis is unsatisfactory.The saline that the oneself uses has improved nasal obstruction, sneeze and hyperemia, and generally has no side effect, and is pregnant patient's first-selected Therapeutic Method therefore.The brine spray agent is generally used for alleviating mucous membrane irritation or the drying relevant with various disease of the nose, the mucosa that reduces mucosal atrophy as far as possible and discharge crust or thicken.If use immediately before the intranasal administration of corticosteroid, the brine spray agent can help the inductive local excitation of prophylactic agent.Hydryllin, for example terfenadine and astemizole also are used for the treatment of allergic rhinitis; Yet use common and other medicines, for example ketoconazole is relevant with the ventricular arrhythmia that is known as Torsades de Points with the interactional hydryllin of erythromycin, or Secondary cases produces potential cardiac problems.Another kind of non-sedative antihistamine agent loratadine and cetirizine with to QT harmful effect at interval, or have nothing to do with serious cardiovascular detrimental effect.Yet it is drowsiness and be not widely used prescription drugs that cetirizine can produce the degree of depth.The hydryllin of non-sedating, for example loratadine can be alleviated sneeze, runny nose and nose, eye, palate titillation to a certain extent, but can test not be used for asthma or other more specific diseases.On the other hand, terfenadine, loratadine and astemizole show felicitous bronchorelaxing activity, reduce the bronchus hyperreactive of histamine and provide protection to resist the bronchospasm of motion or antigen induction.Yet the part in these curative effects need be with the dosage that is higher than current recommendation.The hydryllin of calm type helps to induce the sleep in night, if but take by day and can cause drowsiness and the infringement ability to act.Hydryllin in use usually with the Decongestant coupling to help the alleviation nasal congestion.Sympathomimetic drug can be used as vasoconstrictor and Decongestant.3 kinds of prescription drugs general Decongestants commonly used, pseudoephedrine, phenylpropanolamine and phenylephrine cause hypertension, cardiopalmus, tachycardia, agitation, insomnia and headache.The interaction of phenylpropanolamine and caffeine (dosage of the 2-3 cup coffee) blood pressure that can significantly raise.In addition, medicine such as pseudoephedrine can cause child's overreaction.Yet local Decongestant only can use in the limited duration, and is relevant with the expansible knock-on of nose because they excessively use.Anticholinergic agents can suffer from the patient of obvious rhinorrhea or be used for usually by taking in the specified disease that maror causes, and for example " sense of taste rhinitis ", and some curative effects can be arranged to common cold.For example,, can alleviate sneeze, rhinorrhea and nose and scratch where it itches, block the allergy in early stage and late period, but its produces sneeze, temporary headache even nose burn feeling if sodium cromoglicate is used as preventative nasal spray.Topical administration corticosteroid (for example, two beclometasones) is treated rhinitis effectively, particularly scratches where it itches, sneeze and runny symptom, but to the nasal obstruction weak effect.Yet the corticosteroid nasal spray can cause excitement, sensation of pricking, burn feeling or sneeze etc., and this depends on preparation.The perforation of local hemorrhage and barrier film also can take place sometimes, if particularly aerosol is not suitably on time.Topical steroids is more effective than sodium cromoglicate usually in treatment of allergic rhinitis.Though immunotherapy is expensive and inconvenient, often can provide curative effect, particularly the patient that other medicines are had side effects.The so-called blocking antibody and the medicine that can change the cell histamine release finally cause IgE to reduce and other many useful physiological changies.This acts in the disease (for example atopy patient's of middle ear infection recurrence allergy) of IgE mediation useful.
Pulmonary fibrosis, interstitial lung disease (ILD) or interstitial pulmonary fibrosis comprise the chronic lung disease more than 130 kinds, and these pneumonopathy are by destroying lung tissue, produce inflammation in alveolar wall, scabbing in stroma (or the tissue between alveolar) or fibrosis and make the stiff pulmonary that influences of pulmonary.Asthma may be one of initial symptoms of these diseases in the time of between moving period, and dry cough may occur.Only be not enough to distinguish the dissimilar of pulmonary fibrosis usually by symptom or X-ray examination.Some pulmonary fibrosis patients' reason is known, and some patients' reason is unknown or idiopathic.Common this disease of unpredictable baby of the cause of disease of this kind disease unavoidably is fatal.Its development comprises thickening and stiff, inflammation and dyspnea of lung tissue.Most of people need give oxygen therapy, and unique Therapeutic Method is lung transplantation.
Pulmonary carcinoma is modal in the world cancer.In the period of 2003, only the U.S. just will increase about 171,900 routine pulmonary carcinoma (male's 91,800 examples, and women's 80,100 examples) newly, and Europe has 375,000 examples approximately.Pulmonary carcinoma all is first causes of cancer mortality in the men and women.Estimation 2003 is only in the U.S. just 157,200 routine patients with lung cancer death (male's 88,400 examples, women's 68,800 examples), accounts for 28% of total cancer mortality number.The people who dies from pulmonary carcinoma is than dying from also many (American Cancer Society website (the AmericanCancer Society Web site) that colon cancer, breast carcinoma and carcinoma of prostate are added up, 2003, " depth guide: pulmonary carcinoma: what crucial statistics is? " (Detailed Guide:Lung Cancer:What are the Key Statistics? )).Smoking is known to be the main cause of pulmonary carcinoma, and it is relevant with Nicotiana tabacum L. that about 90% case is thought.Between the age of the radical of risk for lung cancer and smoking every day, the degree of suction and beginning smoking tangible dosage-response relation is arranged.Lifelong smoker suffers from risk for lung cancer than the high 20-30 of non-smoker doubly.Yet risk for lung cancer reduced with the time that begins to give up smoking.Male ES's relative risk sharply reduced with the time that begins to give up smoking, but did not reach non-smoker's low-risk, and also fell manyly (Tyczynski etc., Lancet Oncol.4 (1): 45-55 (2003)) not as the women ES.
The disease that COPD and pulmonary carcinoma are fallen ill often simultaneously, and potential COPD degree can indicate certain patient whether to need surgical operation.With regard to NSCLC (nonsmall-cell lung cancer), only surgical operation (in conjunction with or not combination with radiotherapeutic or adjuvanticity chemotherapy) be curing property.
1 annual survival rate of patients with lung cancer in 1998 (living through the number in 1 year after diagnosing out cancer at least) is 42%, and this is because the raising of surgical technic to a great extent.
It only is 15% that the nonsmall-cell lung cancer patient's of all each phases 5 annual survival rates add together.With regard to small cell lung cancer, the relative survival rate in 5 years about 6%.
Just diagnose out NSCLC and carried out in early days before diffusing to lymph node or other organ with regard to the people of surgical intervention, its average survival rate in 5 years is 50% approximately.Yet, only have the people of 15% trouble pulmonary carcinoma to obtain diagnosis in this early stage, localized stage.
The Therapeutic Method of lung cancer drugs prevention and pulmonary carcinoma obviously also has a lot of leeway of improving.
Dehydroepiandrosterone (DEHA) (3 beta-hydroxies androst-5-ene-17-ketone) is the excretory natural steroid with obvious chemoproection performance of adrenal cortex.Epidemiological study show the endogenous level of DEHA low with the cancer of suffering from some form, for example the risk of the bladder cancer that all has in breast carcinoma and the two kinds of sexes before women's menopause increases relevant.It is believed that DEHA and DEHA analog, for example the ability of DEHA-S (sulphuric acid DEHA) inhibition cancer formation is because it suppresses the active noncompetitive of glucose-6-phosphate dehydrogenase (G6PD) (G6PDH).G6PDH is the rate-limiting enzyme of hexose monophosphoric acid 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 necessary substrate of synthetic rna or DNA (deoxyribonucleic acid).NADPH participates in the synthetic cofactor with 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) of biological nucleic acid.HMG CoA reductase is a kind of enzyme of uniqueness, and every generation 1 product of moles one mevalonate needs 2 moles NADPH.Therefore, HMG CoA reductase looks that the NADPH shortage that DHEA is mediated should have hypersensitivity, and should demonstrate mevalonate storehouse in the shortage born of the same parents fast through the cell of DHEA processing.DNA is synthetic need mevalonate, DHEA to be similar to directly with the utmost point mode of (direct) HMG CoA makes people's cell stop at the G1 phase of cell cycle.Because G6PDH is that to produce the mevalonic acid be used for cell processing (dolichol of for example proteinic isoprenylation and the biosynthetic a kind of precursor of glycoprotein is synthetic) required, DHEA is by exhausting mevalonic acid and Profilin matter isoprenylation and synthesizing of glycoprotein suppress cancer formation by this.Mevalonate is synthetic cholesterol and the synthetic various non-sterol compound (for example, farnesyl pyrophosphate and pyrophosphoric acid ridge cattle ester) that relates to protein post-translational modification and relate to cell-cell and get in touch center precursor with the synthetic required dolichol of cyto-architectural glycoprotein.The patient who accepts adrenal cortex source steroid hormone with suitable pharmaceutical doses shows that the sickness rate increase of infectious disease knows already.U.S. Patent number 5,527,789 have disclosed a kind ofly by using DHEA to the patient and ubiquinone is resisted method for cancer, and wherein said cancer is a kind of cancer to the DHEA sensitivity.
DHEA is a kind of 17-KS, and it quantitatively is a kind of main adrenocortical steroid hormone of finding in mammal.Though as intermediate, do not understand fully by the major physiological function of DHEA in the gonad steroid hormone is synthetic for DHEA.Yet, the level of known this hormone begin in 10 years to reduce at second of life (old age reach initial level 5%).DHEA is used for treating the patient who suffers from psoriasis, gout, hyperlipemia by general or local making clinically, and is applied to coronary artery (operation) back patient.In mammal, DHEA shows the effect with optimization body weight (weight optimizing) and anticancer formation, and reverse symptoms of menopause with the estrogen coupling as medicine clinically in Europe, also be used for the treatment of manic depression, schizophrenia and presenile dementia.The dosage that DHEA is used for the treatment of terminal cancer and multiple heavy sclerosis clinically is 40mg/kg/ days.Gentle androgenic effect, hirsutism and hypersexuality are observed side effect.These side effect can and/or use analog to overcome by monitoring dosage.Known subcutaneous or Orally administered DHEA improves the host to replying of infecting, and sends DHEA as using paster.DHEA is also known to be the precursor that finally improves in the metabolic pathway of the stronger medicine that mammalian immune replys.That is, DHEA is as former medicine: when it is converted into androstenediol or Androst-5-ene-3 beta, 17-isoallopregnane-3 (β AED), or androstene triol (androstenetriol) or Androst-5-ene-3 beta during 17 beta-triols (β AET), can play the effect of immunomodulator.Yet DHEA had certain lymph toxicity and inhibitory action in external on cell proliferation before being converted into β AED and β AET.Therefore, it is believed that to improve performance be because it is converted into has more due to the active metabolite by using more excellent immunity that DHEA obtains.
Adenosine is a kind of purine that relates to intermediate supersession, can constitute important medium in various pneumonopathy, comprises bronchial asthma, COPD, CF, RDS, rhinitis, pulmonary fibrosis etc.Asthmatic patient has the reaction of significant bronchoconstriction to the aerosolization adenosine and the normal person does not have this phenomenon and shows that its receptor has potential effect.The rabbit animal model (the dust mite allergy Rabbit Model of people's asthma) of suffering from asthma has the obvious bronchoconstriction reaction of similar fashion to the aerosolization adenosine, and it is reactionless not have the Rabbit Model of asthma.Recently the inductive bronchoconstriction of the adenosine that studies show that asthmatic patient of this animal model and bronchus overreaction may be mainly by stimulating the adenosine receptor mediation.When the adenosine therapeutic administration when previously not diagnosing out the object of other disease of trouble with overreaction air flue, show that also adenosine can cause ill effect, comprises death.As the regulator of cellular metabolism, adenosine plays a part unique in vivo.It can promote the cellular level as the AMP of cellular energy intermediate, ADP and ATP.Thereby adenosine can stimulate or reduce the level that the activity of adenyl cyclase is regulated cAMP.CAMP and then in neurotransmitter release, cell division and hormone discharge, work.It seems that the main effect of adenosine be to play protectiveness to damage endocrine effect.When local ischemia, oxygen tension reduced or damage generation, adenosine worked.Thereby the defective of synthesizing, discharge, working and/or degrading that it is believed that adenosine causes the active excessive various pathologic state that cause of cerebral irritation acidic amino acid neurotransmitter.The symptom that adenosine also is considered to bronchial asthma and other respiratory tract disease takes place, and induces the main determining factor of bronchoconstriction and air flue smooth muscle contraction.In addition, adenosine causes bronchoconstriction but do not have this effect in no asthmatic patient in asthmatic patient.Other data show that adenosine receptor also may participate in allergy and inflammatory response by the overacfivity that reduces central dopamine energy system.It is believed that the signal transduction of regulating the inflammatory cell surface has influenced acute inflammation.It is said that adenosine is by activating the generation that neutrophilic leukocyte suppresses superoxides.Recent evidence shows that adenosine also shields in apoplexy, CNS wound, epilepsy, ischemic heart desease, coronary artery bypass, contact radiation and inflammation.In a word, adenosine shows can regulate cellular metabolism by ATP, as the carrier of methionine, can reduce the oxygen demand and the protection cell of cell and avoid ischemia injury.Adenosine is courier in a kind of tissue hormone that cell suffers ischemia, anoxia, cell is emergent and workload increases and/or discharge when the demand to ATP surpasses its supply or born of the same parents.Adenosine is that a kind of purine and its generation directly relate to the ATP metabolism.It shows a series of physiological process of scalable, comprises vascular tone, hormonal action, function of nervous system, platelet aggregation and lymphocyte differentiation.It also can form at DNA, work in ATP biosynthesis and the whole intermediate supersession.It is said that the cAMP that it can be regulated in brain and the various peripheral tissues forms.Adenosine is by two kinds of receptor A
1And A
2Regulating cAMP forms.Adenosine passes through A
1Receptor reduces the activity of adenyl cyclase, and passes through A
2The receptor activation adenyl cyclase.Adenosine A
1Receptor compares A
2Receptor is more responsive to adenosine.The CNS effect that it is believed that adenosine is by A
1Receptor-mediated, and peripheral action, for example hypotension, bradycardia be it is said by A
2Receptor-mediated.
Many medicines have been used for the treatment of respiratory tract disease, but they all have limitation generally.These medicines have glucocorticoid, leukotriene inhibitors, anticholinergic agents, hydryllin, oxygen therapy, theophylline and mucolytic.Although glucocorticoid has proved retroaction, it is still one of the most widely used medicine.Yet most of available medicines are only effective in the minority case, and useless at all when treatment asthma.Current do not have available Therapeutic Method to many other respiratory tract diseases.Theophylline is known as a kind of important drugs for the treatment of asthma to be the antagonist of adenosine receptor, it is reported that it has eliminated the bronchoconstriction of adenosine mediation in the rabbit that suffers from asthma.Also report a kind of selectivity A
1Receptor antagonist, 8-cyclopenta-1,3-dipropyl xanthine (DPCPX) can suppress the bronchoconstriction and the bronchus overreaction of the adenosine mediation of irritated rabbit.Yet, current available adenosine A
1The therapeutic of receptor-specific antagonist and prophylactic use are subject to its toxicity.For example, theophylline has been widely used in treatment asthma, but because its narrow therapeutic dosage range often causes frequently, significant toxicity (gastrointestinal, cardiovascular, nerve and biological disorderly).DPCPX toxicity is bigger can not be used for clinical.Although through the broad research of many decades, there is not specific adenosine receptor antagonists to can be used for the clinical general toxicity that fact proved these medicines.
Now, β 2-agonist bronchodilatation medicament of salmeterol commercially availablely can be used to treat that asthma, COPD (comprising chronic bronchitis, emphysema) and other pneumonopathy causes pants, short of breath and dyspnea.Salmaterol also is used to prevent the dyspnea (bronchospasm) between moving period.Salmaterol uses with the aerosol of oral suction.In Britain, it is with Servent
TMAccuhaler
TM, Serevent
TMDiskhaler
TMAnd Servent
TMInhaler is sold (Glaxo Wellcome, Britain).
Now, the commercially available bronchoconstriction that can be used to treat COPD (comprising chronic bronchitis, emphysema) patient of β 2-agonist bronchodilatation medicament formoterol.It with the capsule form that contains dry powder formulations (
) sell, said preparation contains the formoterol fumarate of 12 μ g and the lactose of 25mg, and only uses
Inhaler (Schering Corp., Kenilworth, NJ) oral suction.
U.S. Patent number 5,660,835 (with the open WO96/25935 of the PCT of correspondence) disclosed a kind ofly treats the asthma of study subject or the new method that adenosine exhausts by use dehydroepiandrosterone (DHEA) or DHEA related compound to study subject.This patent has also disclosed a kind of relating to and can suck the new pharmaceutical composition that maybe can breathe preparation, and said preparation contains DHEA or the DHEA related compound that can breathe size particles.
U.S. Patent number 5,527,789 have disclosed and have a kind ofly resisted the cancer of study subject by use DHEA or DHEA related compound to study subject, and use ubiquinone and resist the method for DHEA or the inductive heart failure of DHEA related compound.
U.S. Patent number 6,087,351 have disclosed and a kind ofly reduce or exhaust method in the body of adenosine in the study subject tissue by use DHEA or DHEA related compound to study subject.
The U.S. Patent Application Serial 10/454,061 that on June 3rd, 2003 submitted to has disclosed a kind of by use the method that DHEA or DHEA related compound are treated the COPD of study subject to study subject.
The U.S. Patent Application Serial 10/462,901 that on June 17th, 2003 submitted to has disclosed a kind of stable DHEA dry powder formulations that is sealed in the Sprayable in the container.
The U.S. Patent Application Serial of submitting on June 17th, 2,003 10/462,927 has disclosed a kind of stable dry powder formulations that is suitable for treating the DHEA-S two crystalline hydrate forms of asthma and COPD.
Above patent and patent application are included in as a reference in full.
Be badly in need of novel and effectively be used for the treatment of the medicine of respiratory tract disease, pneumonopathy and cancer, described disease can not be treated now or existing at least medicine is invalid and can not avoid obvious harmful side effect.Influence respiratory tract, say that more specifically the disease that influences lung and lung airway comprises exhausting of dyspnea, asthma, bronchoconstriction, pneumonia and allergy, surfactant or hyposecretion etc.In addition, clearly need to have the Therapeutic Method that the low dosage activating agent was used and needed to preventative and therapeutic, this can make not only cheapness but also be difficult for causing deleterious side effect of these Therapeutic Method.
In addition, need guarantee that the patient is ready to take medicine, and need and help take treatment or the required multiple chemical compound of prevention of asthma, COPD or other respiratory tract disease.
Summary of the invention
The invention provides the compositions that comprises two kinds of activating agents at least.First activating agent contains non-glucocorticoid, for example epiandrosterone (EA) or its salt.Second activating agent contains β 2-agonist bronchodilatation medicine.Said composition comprises the combination of first activating agent and second activating agent.When said composition was applied to study subject, wherein the amount of first activating agent and second activating agent was enough to effectively prevent or treat the study subject that is in trouble asthma, COPD or other respiratory tract disease danger.Said composition also can comprise other bioactivator and preparation composition.Said composition is to be suitable for being applied to study subject or patient, for example people or non-human animal's (for example non-human mammal) pharmaceutical composition or veterinary composition.
Said composition can be used for treatment because of inflammation and the caused asthma of sequela, COPD or other respiratory tract disease, comprises with bronchoconstriction, surfactant exhausting and/or irritated relevant disease.
The present invention also provides the method for treatment asthma, COPD or other respiratory tract disease, and described method comprises the study subject applying said compositions to this treatment of needs.
The present invention also provides first activating agent and the application of second activating agent in the medicine of producing above-mentioned prevention or treatment asthma, COPD or other respiratory tract disease.
The present invention also provides a kind of test kit that described compositions and delivery apparatus are housed.This delivery apparatus can be delivered to study subject with described compositions.The inhaler of the delivery of particles that provides with aerosol or spraying generator is be provided delivery apparatus.Preferably be delivered to the air flue of study subject.More preferably be delivered to the pulmonary of study subject.Preferably directly be delivered to desired area.
The major advantage of use said composition is the patient's of this prevention of needs or treatment compliance.For every patient, respiratory tract disease, for example asthma or COPD are multifactorial and show different Signs and symptom.Therefore, Most patients is alleviated the different aspect of disease with multiple Drug therapy.First activating agent, for example DHEA or DHEA-S and second activating agent, for example the fixed combination of salmaterol or formoterol provides targeted therapy method more easily for certain patient subgroups of determining.Should be able to improve patient's compliance by unique disease attribute of simplifying medicine and being primarily focused on every patient, thereby treat its specific symptom with effective and efficient manner.In addition, the additional advantage of using first and second activating agents in single administration simultaneously is convenient or saves time.When said composition is applied to certain zone that may have uncomfortable study subject health, when for example said composition being applied to the air flue of study subject, this is particularly desirable.It is also particularly desirable when the method that said composition is applied to study subject is invasive.
In addition, when first activating agent, for example DHEA or DHEA-S send or are deposited on the far-end peripheral airways of alveolar membrane and tiny air flue but not conduction during air flue, and it is the most effective anti-inflammatory drug.Asthma and number of C OPD patient's conduction air flue shrinks, and this has limited first activating agent that acts on these far-end peripheral airways, and for example DHEA's sends (because lower particle speed causes deposition more early).Therefore, coupling bronchodilatation medicine (β 2-agonist, the anti-gill fungus drug toxicity (antimuscarinic) that makes tonicity raise and reverse) helps anti-inflammatory drug is delivered to the far-end peripheral airways.Use this combination to improve persistent pharmacological action, thereby change improvement into disease control.Anti-leukotriene medicine reduces the interstitial edema in the minimum peripheral airways.This also has increases peripheral airways diameter and the effect that promotes to send first activating agent.Hydryllin also reduces the peripheral airways edema and helps the terminal air flue of first activating agent to send, and also is ideal.
Follow the accompanying drawing of this patent to form the part of content of the present invention, and further illustrate some aspects of addressing below the present invention.
The accompanying drawing summary
Fig. 1 has described the pure micronization DHEA-S-2H that sends as the single dose Acu-Breathe inhaler of flow velocity function
2The fine fraction of O.The result represents with DHEA-S.The IDL data of anhydrous in fact micronization DHEA-S also are shown in this figure, and wherein because no detectable microgranule enters ram, 30L/ minute result is set to zero point.
Fig. 2 is purified anhydrous in fact DHEA-S in bulk and is stored in 50 ℃ of 1 HPLC chromatogram after week with the mixture of lactose.Contrast is the pure DHEA-S that is stored in room temperature (RT).
Fig. 3 is purified DHEA-S-2H in bulk
2O and be stored in 50 ℃ of 1 HPLC chromatogram after week with the mixture of lactose.Contrast is the pure DHEA-S-2H that is stored in RT
2O.
Fig. 4 has described in two kinds of temperature sodium chloride concentrations and the deliquescent functional relationship of DHEA-S.
Fig. 5 has described in the inverse of 24-25 ℃ of Na ion concentration and the deliquescent functional relationship of DHEA-S.
Fig. 6 has described in the inverse of 7-8 ℃ of Na ion concentration and the deliquescent functional relationship of DHEA-S.
Fig. 7 is sodium chloride concentration and the deliquescent functional relationship of DHEA-S when room temperature has or do not have buffer.
Fig. 8 has described at 24-25 ℃, has or the inverse and the deliquescent functional relationship of DHEA-S of Na ion concentration when not having buffer.
Fig. 9 has described under two kinds of preservation conditions, the solution concentration of DHEA-S and time relation.
Figure 10 has described under two kinds of preservation conditions, the solution concentration of DHEA and time relation.
Figure 11 is the sketch map of spraying experiment.
Figure 12 has described DHEA-S amount that is deposited in the bypass catcher and the functional relationship that is placed in the initial soln concentration in the aerosol apparatus.
Figure 13 has described the particle diameter that DHEA-S spray solution cascade bump obtains.The data that provide are meansigma methodss of all 7 spraying experiments.
Figure 14 has described DHEA to the effect of HT-29SF cell inhibiting.
Figure 15 has described the effect of DHEA to the cell cycle distribution of HT-29SF cell.
Figure 16 a and 16b have described the inductive growth inhibiting reverse to DHEA in the HT-29 cell.
Figure 17 has described the inductive G of DHEA in the HT-29SF cell
1The reverse of stagnating.
Figure 18 EGF, IBMX have been described and DHEA right
3The H-thymus pyrimidine mixes the effect in human smooth muscle's culture.
Figure 19 has described single ability that suppresses the propagation of EGF stimulation with DHEA and coupling IBMX.
Figure 20 has described some suitable DHEA analog.
Figure 21 has described some suitable DHEA analog.
Figure 22 has described some suitable DHEA analog.
Figure 23 has described the suitable modification to the C-17 ketone of DHEA.
Description of Preferred Embodiments
Definition
The term of this paper " adenosine " comprises with the previous level of study subject with exhausting of " surfactant " to be compared, the reduction of this levels of substance or exhaust in the study subject, and with identical with the previous level of this study subject basically level, but owing to some other reasons, the previous level that change these materials could reach the therapeutic benefit in the patient.
The part or the whole respiratory system of the study subject of term used herein " air flue " expression ingress of air.Air flue includes, but is not limited to throat, tracheobronchial tree, nasal passage, nasal sinuses etc.Air flue also comprises trachea, bronchus, bronchioles, terminal bronchiole, respiratory tract bronchioles, alveolar duct and alveolar sac.
The disease that term used herein " airway inflammation " expression is relevant with the airway inflammation of study subject.Airway inflammation can be caused or accompanied with it by following disease: allergy, asthma, respiratory disorder, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), allergic rhinitis (AR), adult respiratory distress syndrome (ARDS), microorganism or viral infection, pulmonary hypertension, pneumonia, bronchitis, cancer, airway obstruction and bronchoconstriction.
Term used herein " carrier " representation is gas, liquid, solid carrier and composition thereof, is suitable for required different administration approach acceptable carrier biology.This carrier preferably pharmacy or veterinary is acceptable.
" effective dose " used herein expression provides the amount of therapeutic or preventative benefit.
Any curative drug first or second activating agent of " other curative drug " expression in described compositions.
Term used herein " prevention " is illustrated in the prophylactic treatment that study subject is attacked by a disease before or the condition worse of previously diagnosing out was carried out in the past, thereby makes study subject can avoid, prevent or reduce the symptom of the disease that takes a disease or the probability of relevant disease.The object that this study subject takes a disease disease or the risk of the condition worse previously diagnosed out increases.
Disease or disease that term used herein " respiratory tract disease " expression is relevant with respiratory system.Example includes, but is not limited to airway inflammation, allergy, respiratory disorder, cystic fibrosis (CF), allergic rhinitis (AR), adult respiratory distress syndrome (ARDS), cancer, pulmonary hypertension, pneumonia, bronchitis, airway obstruction, bronchoconstriction, microorganism and viral infection, for example SARS.
The Therapeutic Method that the symptom of disease or other disease of showing the study subject that term used herein " treatment ", " treatment " or " therapeutic " expression is given this Therapeutic Method may reduce.
The invention provides the compositions that includes first activating agent and second activating agent, wherein first activating agent contains the non-glucocorticoid steroid, for example epiandrosterone (EA), its analog or its salt, and second activating agent contains β 2-agonist bronchodilatation medicine.Said composition also can contain pharmaceutically or veterinarily acceptable carrier, diluent, excipient, bioactivator or composition.Said composition can be used for treating asthma, COPD or other respiratory tract disease.Other respiratory tract disease that said composition also can be used for treating is pneumonopathy and the respiratory tract disease relevant with pulmonary carcinoma with bronchoconstriction, pneumonia and/or allergy.
Described first activating agent is epiandrosterone, its analog or pharmaceutically or veterinarily acceptable salt.Epiandrosterone, its analog or pharmaceutically or veterinarily acceptable salt be selected from non-glucocorticoid steroid with following chemical formula
Wherein dotted line is represented singly-bound or two key; R is a hydrogen or halogen; 5 H is α or beta comfiguration, and perhaps the chemical compound shown in the Formula I comprises the racemic mixture of these two kinds of configurations; R
1Be hydrogen or the polyvalent mineral that is covalently attached to this chemical compound or organic dicarboxylic acid;
Non-glucocorticoid steroid shown in the following chemical formula
Non-glucocorticoid steroid shown in the following chemical formula
Their combination,
Respectively do for oneself H, OR, halogen, (C1-C10) alkyl or (C1-C10) alkoxyl of R1, R2, R3, R4, R5, R7, R8, R9, R10, R12, R13, R14 and R19 wherein; R5 and R11 respectively do for oneself 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, volution oxidative ethane (spirooxirane), volution sulfur ethane (spirothirane) ,-OSO2R20 ,-OPOR20R21 or (C1-C10) alkyl; R5 and R6 combine and are=O; R10 and R11 combine and are=O; R15 be (1) when R16 be-during C (O) OR22, be H, halogen, (C1-C10) alkyl or (C1-C10) alkoxyl, (2) when R16 be halogen, OH or (C1-C10) during alkyl, be H, halogen, OH or (C1-C10) alkyl, (3) when R16 is OH, be H, halogen, (C1-C10) alkyl, (C1-C10) alkenyl, (C1-C10) alkynyl, formoxyl, (C1-C10) alkanoyl or epoxy radicals (4) are OR when R16 is H, 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 combine and are=O; R17 and R18 respectively do for oneself (1) when R6 be H, OR, halogen, (C1-C10) alkyl or-during C (O) OR22, be H,-OH, halogen, (C1-C10) alkyl or-(C1-C10) alkoxyl, (2) when R15 and R16 combine be=during O, be H, (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) carbalkoxy or (C1-C10) alkanoyloxy, (3) R17 and R18 combine and are=O; (4) R17 or R18 combine with its carbon that is connected and form the 3-6 unit ring that contains 0 or 1 oxygen atom; Or (5) R15 combines formation epoxide ring with the carbon that R17 is connected with them; R20 and R21 respectively do for oneself 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 their pharmacy or veterinarily acceptable salt.
With regard to chemical formula (I), the multivalence organic dicarboxylic acid is SO preferably
2OM, phosphate or carbonate, wherein M comprises equilibrium ion.The example of equilibrium ion is H, sodium, potassium, magnesium, aluminum, zinc, calcium, lithium, ammonium, amine, arginine, lysine, histidine, triethylamine, ethanolamine, choline, triethanolamine (triethanoamine), procaine, benzathine penicillin G, Apiroserum Tham (tromethanine), pyrrolidine, piperazine, diethylamine, sulfatide
And phospholipid
R that can be identical or different wherein
2And R
3Be straight or branched (C1-C14) alkyl or glucosiduronic acid
But the mixture that 5 H α of Formula I or beta comfiguration or DHEA can these two kinds of configuration of compound provides.The exemplary compounds of above Formula I includes, but is not limited to contain the DHEA of two keys, wherein R and R
1Be respectively hydrogen; Contain 16-α bromine (generation) epiandrosterone of two keys, wherein R is Br, R
1Be H; Contain 16-α-fluorine (generation) epiandrosterone of two keys, wherein R is F, R
1Be H; The etiocholanolone that lacks two keys, wherein R and R
1Be respectively hydrogen; Lack the dehydroepiandrosterone sulfate of two keys, wherein R is H, R
1Be SO
2OM, and M is above-mentioned sulfatide (sulphatide) group.Yet also can comprise other chemical compound.Chemical compound shown in the formula I also preferably contains two keys, and R is a halogen, for example bromine, chlorine or fluorine, and R1 is the chemical compound of hydrogen.Chemical compound shown in the formula I is 16-α-fluorine (generation) epiandrosterone most preferably.Other preferred chemical compound is DHEA and DHEA salt, for example sulfate (DHEA-S).
The non-glucocorticoid sterone, for example formula (I), (III) and (IV) shown in chemical compound, their derivant and the application dosage of salt thereof generally be about 0.05,0.1,1,5,20-100,500,1000,1500,1800,2500,3000,3600mg/kg body weight.Yet other dosage also is suitable also being considered by this patent.Formula (I), (III) and (IV) shown in first activating agent can be in accordance with known methods, the perhaps version manufacturing of these methods of understanding of those skilled in the art.For example, referring to U.S. Patent number 4,956,355; British Patent No. 2,240,472; EPO number of patent application 429,187; PCT patent publication No. WO91/04030; U.S. Patent number 5,859,000; Abou-Gharbia etc., J.Pharm.Sci.70:1154-1157 (1981); Merck catalogue monograph (Merck Index Monograph) No. 7710 (the 11st edition, 1989) etc.
In some embodiments of the present invention, described first activating agent can be an epiandrosterone analog or derivatives thereof.The present invention also comprises the former medicine and the active metabolite thereof of epiandrosterone.Those skilled in the art will appreciate that chemical compound as herein described can show tautomerism, configuration isomery, geometrical isomerism and/or enantiomerism.It should be understood that any tautomerism, configuration isomery, optical siomerism and/or the geometrical isomerism form that the present invention includes chemical compound with one or more purposes described herein, and these various multi-form mixture.
The metabolite of epiandrosterone, for example be described in the capillary gas chromatography (Capillary gaschromatography of urinary steroids of terbutaline-treated asthmatic children) of urine steroid of asthmatic children that can be used as the terbutaline treatment of first activating agent below with reference to document, Chromatographia (1998), 48 (1/2), 163-165; Androstenedione metabolism in people's lung fibroblast (Androstenedione metabolism in human lung fibroblasts), Journal of SteroidBiochemistry (1986), 24 (4), 893-7; Androsterone and 5 α-androstane-3 α, human lung tissue and (the Metabolism of androsterone and5 α-androstane-3 α of the metabolism in the lung endotheliocyte that 17-isoallopregnane-3 is being cultivated, 17 β-diol in human lung tissue and in pulmonary endothelial cellsin 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; Androsterone and 5 α-androstane-3 α, human lung tissue and (the Metabolism of androsterone and5 alpha-androstane-3 alpha of the metabolism in the lung endotheliocyte that 17-isoallopregnane-3 is being cultivated, 17 beta-diol in human lung tissue and in pulmonaryendothelial cells in culture), Journal of clinical endocrinology and metabolism (in February, 1985), 60 (2), 44-50; The metabolism (Metabolism of dehydroisoandrosterone and androstenedione in humanpulmonary endothelial cells in culture) in people's lung endotheliocyte of cultivating of dehydroepiandrosterone and androstenedione, Journal of clinical endocrinology andmetabolism (May nineteen eighty-three), 56 (5), 930-5; The vitro human lung is to the metabolism (Metabolism of dehydroisoandrosterone and androstenedione by the humanlung in vitro) of dehydroepiandrosterone and androstenedione, Journal of steroid biochemistry (in April, 1977), 8 (4), 277-84; Testosterone metabolism in the external Canis familiaris L. lung (Testosterone metabolism in dog lung in vitro), Steroids andlipids research (1973), 4 (1), 17-23, these documents all include in as a reference in full.
Other suitable DHEA analog that can be used as first activating agent is described in the literary composition.Figure 20 has described some suitable DHEA analog, comprises the chemical compound shown in formula IA, IB, IC and the ID.In the description of this paper to the suitable R group, junction point is with the CH2 group or have the atom of asterisk labelling to represent.R1 and R3 are the alkyl of straight or branched, comprise benzyl and the optional alkyl that replaces, for example aminoalkyl, hydroxy alkyl, ether and carboxylic acid, and aryl and the heteroaryl that can choose replacement wantonly.R1 and R3 for example,
The example of the chemical compound shown in the formula IA is
R2 preferably diacid derives or amino acid derived substituent group, may comprise chloracetyl derivant and acrylate derivatives, the perhaps optional aryl that replaces, for example benzyl and assorted benzoyl.The example of the chemical compound shown in the formula IB is
The example of chemical compound shown in the formula IC comprises,
R4 is aromatic radical in itself, and the example of suitable compound shown in the formula ID comprises,
Other suitable analog comprises by keeping OH in the C-3 position or replacing the analog that OH modifies with NH.As shown in figure 21, from the hero-4-alkene 3 of C-17 acetal protection, the 17-diketone begins to make these analog usually.Chemical compound shown in the formula IE can for example be aromatic chemical compound derived from Grignard reagent and possible aryl-lithium reagent in essence, and also alkynyl, alkenyl and alkyl.The example of R5 is CH2 (CH2)
n, n=0-4 wherein
The example of chemical compound shown in the formula IE comprises
Respectively do for oneself various amine and can comprise amine of R6 and R8 with the described degree of functionality of R1 group.The example of suitable compound shown in the formula IF comprises,
Suitable R 7 groups can derive from Grignard reagent/organolithium reagent, therefore can have the described degree of functionality of R5.The example of chemical compound shown in the formula IG comprises,
The example of chemical compound shown in the formula IH comprises
Other suitable analog comprises the wherein adorned chemical compound in C-2 position of DHEA.Suitable trim is described in Figure 22.R9 can derive from alkylating reagent, for example alkyl, benzyl, assorted benzyl and other active halid derivant.The example of R9 comprises,
The example of chemical compound shown in the formula IJ comprises,
But R10 aromatic ester, but the Arrcostab that for example has aryl or heteroaryl ring or enolization.The example of chemical compound shown in the formula IK comprises,
R11 is a series of aromatic series and assorted aromatic aldehyde, and the variant of benzene carboxylic aldehyde and replacement thereof for example adjoins pyridine carboxylic aldehyde or aldehyde that can not enolization, for example (CH3) 3CCH=O.The example of chemical compound shown in the formula IL comprises,
R12 is an amine, for example R6 subgroup.Comprising of chemical compound shown in the formula IN,
The suitable modification thing of the C-17 ketone of DHEA is described in Figure 23.The chemical compound that is described in Figure 23 also can be used as first activating agent.
Other suitable DHEA analog is described in U.S. Patent number 6,635,629; European patent 934745; Dehydroepiandrosterone and analog suppress the combination and airway smooth muscle propagation (Dehydroepiandrosterone and analogs inhibit DNA binding of AP-1 and airwaysmooth muscle proliferation) of DNA and AP-1, Journal of Pharmacology and ExperimentalTherapeutics (1998), 285 (2), 876-883: dehydroepiandrosterone and related steroid are at the effect of vitro inhibition mitochondrial respiratory (Dehydroepiandrosterone and related steroids inhibit mitochondrialrespiration in vitro), International Journal of Biochemistry (1989), 21 (10), 1103-7, all these documents all include in as a reference in full.
Second activating agent is the bronchodilatation medicine that contains long-acting beta 2-agonist.The bronchodilatation medicine loosens tightly around the muscle band of air flue, makes more air turnover pulmonary and improve breathing by this.The bronchodilatation medicine also helps to remove mucus from pulmonary, and promptly when airway open, mucus can more freely move and more easily by expectoration or removing.The bronchodilatation medicine has fugitive and long-acting form: fugitive form is alleviated or is stopped symptoms of asthma, and long-acting form control symptoms of asthma and prevention of asthma outbreak.
Salmaterol contain chemical formula V and (VI) shown in chemical compound.Chemical compound (Britain patent description book number 1200886) shown in the chemistry formula V is:
Wherein X1 is a hydroxy alkyl, and R1 and R are respectively hydrogen atoms, and R3 is straight or branched C
1-6Alkyl, aralkyl or aryloxy alkyl.A chemical compound in this group developed be used for clinical.Now, Aerolin [(α 1-tert-butyl amino methyl)-4-hydroxyl-m-dimethylbenzene-α 1, α 3-glycol; Above-mentioned X1=CH
2OH, R1=--H; R2=--H; The R3=tert-butyl group] be to be widely used in the prescription drugs of treatment as diseases such as bronchial asthma and chronic bronchitiss.The success of Aerolin is its binding mode, and particularly it is renderd a service with selective stimulating effect to beta-2-adrenoreceptor and interrelates.
The β 2-stimulant of all present clinical uses has short shortcoming action time when suction is used.Therefore, action time, long β 2-stimulant obviously had advantage when treatment bronchial asthma and relevant disease.
Long-acting beta 2-stimulant also comprises the chemical compound shown in the chemical formula (VI):
Wherein m is the integer of 2-8, and n is the integer of 1-7, and prerequisite is that the m+n sum is 4-12; Ar represents can choose wantonly by one or two and is selected from halogen atom, C
1-3Alkyl or C
1-3The phenyl that alkylenedioxy group (alkylenedioxy) shown in the substituent group of alkoxyl or formula--O (CH2) pO--(wherein p is 1 or 2) replaces; R1 and R2 represent hydrogen atom or C1-3 alkyl respectively, and prerequisite is that the carbon atom sum among R1 and the R2 is no more than 4; And their physiologically acceptable salt or solvate (for example, hydrate).
Chemical compound has one or two asymmetric carbon atom shown in the general formula (VI), promptly
Carbon atom in the group, and when R1 is different group with R2, the carbon atom that these groups connected.
Therefore, chemical compound of the present invention comprises all enantiomers, diastereomer and their mixture, comprises racemic modification.Preferably
Carbon atom in the group is the chemical compound of R configuration.
In the general-purpose type (VI), chain--(CH2)
m--, for example-(CH2)
3--,--(CH
2)
4--,-(CH
2)
5--,--(CH
2)
6-or--(CH
2)
7--; Chain--(CH
2)
n--, for example--(CH
2)
2--, (CH
2)
3--,--(CH
2)
4--,--(CH
2)
5--or (CH
2) 6---.
Chain--(CH2)
m--and--(CH2)
nIn carbon number sum 6-12 (comprising 6 and 12) preferably, for example 7,8,9 or 10.The summation of m+n is that 7,8 or 9 chemical compound is particularly preferred.
Preferably wherein m is 3 and n is 6, or m is 4 and n is 3,4 or 5, and perhaps m is 5 and n is 2,3,4 or 5, and perhaps m is 6 and n is a chemical compound shown in 2 or 3 the general formula (VI).
For example R1 and R2 can be respectively methyl, ethyl, propyl group or isopropyl, if perhaps among R1 and the R2 is propyl group or isopropyl, another is hydrogen atom or methyl.Therefore, but for example R1 hydrogen atom or methyl, ethyl or propyl group.But for example R3 hydrogen atom or methyl.
R1 and R2 difference be hydrogen atom or methyl preferably.
Preferred one group of chemical compound is that wherein R1 and R2 all are hydrogen atoms.Organize in the preferred chemical compound at another, R1 is a hydrogen atom, and R2 is C
1-3Alkyl, particularly methyl.Also having another to organize in the preferred chemical compound, R1 and R2 all are methyl.
Chain in the general formula (VI)
, for example--(CH
2)
4O (CH
2)
4--, (CH
2)
5O (CH
2)
2--(CH
2)
5O (CH
2)
3,-(CH
2)
5O (CH
2)
4--,
The optional substituent example that is illustrated on the phenyl with Ar comprises bromine, iodine or particularly chlorine or fluorine atom, or methyl, ethyl, methoxy or ethoxy.Generally, the preferred unsubstituted phenyl of Ar.Ar is by a substituent group, particularly fluorine or chlorine atom or methoxyl group or methyl substituted phenyl in another preferred example.
The physiologically acceptable acceptable acid addition salts of chemical compound comprises derived from inorganic and organic acid acid-addition salts, for example hydrochlorate shown in the logical formula V, hydrobromate, sulfate, phosphate, maleate, tartrate, citrate, benzoate, 4-methoxybenzoic acid salt, 2-or 4-hydroxy benzoate, the 4-chloro-benzoate, right-toluene fulfonate, metilsulfate (methanesulphonate), Ascorbate, Salicylate, acetate, fumarate, succinate, lactate, glutarate, gluconate, tricarballylic acid salt, hydroxyl naphthalene-carboxylic acid salt (as the 1-hydroxyl-or 3-hydroxyl-2-naphthalene-carboxylic acid salt) or oleate.These chemical compounds also can form salt with suitable alkali.The example of this salt is alkali metal (for example sodium and potassium) and alkaline-earth metal (for example calcium and magnesium) salt.
The chemical compound of second activating agent has the selective stimulating effect to beta-2-adrenoreceptor, and this has the binding mode that has superiority especially.Stimulation is confirmed in Cavia porcellus, and wherein compound exhibits causes the isolating trachea diastole that PEG2 α shrinks.In another test, when chemical compound of the present invention through sucking or oral route when being applied to clear-headed Cavia porcellus, its demonstration can provide the protective effect of resisting the inductive bronchoconstriction of histamine.In two tests, compound exhibits of the present invention long especially acting duration.These chemical compounds (effect) are confirmed in rat or Cavia porcellus.These chemical compounds have weak to isolating rat or Guinea Pig Left tremulous pulse (β1-Shen Shangxiansushouti tissue) or do not have the isolating trachea diastole that the concentration of doing the time spent causes PGF2 α-contraction.Chemical compound of the present invention also shows and suppresses spasm former (spasmagen) and inflammation former (inflammagen) the people's tissue from sensitization, for example anaphylaxis release (U.S. Patent number 4,992,474 in the lung fragment; 5,126,375; With 5,225,445; Include in as a reference in full).
Chemical compound of the present invention can be used for treatment and reversibility airway obstruction diseases associated, for example asthma, COPD and chronic bronchitis.
Chemical compound of the present invention also can be used for treating premature infant, depression and congestive heart failure, also can be used for treating inflammatory or allergic skin diseases, psoriasis, proliferative dermatosis, glaucoma, and be used for the treatment of and reduce that gastric acid can benefit, the disease of gastric ulcer and peptic gastric ulcer particularly.
One group of chemical compound with particular importance of good long-acting time (is included U.S. Patent number 4,992,474 as a reference in full; 5,126,375 and 5,225,445) shown in chemical formula (VII), and physiologically acceptable salt and solvate thereof:
Wherein R1 and R2 such as general formula (VI) definition; M is the integer of 3-6, and n is the integer of 2-6, and Ar is a phenyl or by methoxyl group or methyl or be more preferably the phenyl that the fluorine or chlorine atom replaces, in each example, and chain--(CH
2)
m-and-(CH
2)
n-middle carbon number sum is the integer (comprising 7 and 10) that is selected from 7-10, for example 7,8 or 9.
Wherein R1 and R2 be respectively hydrogen atom be chemical compound shown in preferred one group of chemical formula (VII).
Organize in the chemical compound shown in the preferred chemical formula (VII) at another, R1 is hydrogen or methyl and R2 is a methyl.
In also having the chemical compound shown in one group of chemical formula (VII), R1 and R2 are respectively hydrogen atoms, and Ar is a phenyl or or by methoxyl group or methyl or be more preferably the phenyl that the fluorine or chlorine atom replaces.
One group of chemical compound shown in the preferred especially chemical formula (VII) and physiologically acceptable salt and solvate, wherein R1 and R2 are respectively hydrogen atom or methyl, m is 4 or 5, and n is 2,3 or 4, and Ar is a phenyl or by chlorine or fluorine atom or methoxyl group or methyl substituted phenyl.
The chemical compound of particular importance is: 4-hydroxyl-α 1[[[6-(4-phenyl butoxy) hexyl] amino] methyl]-1,3-benzene dimethanol (benzenedimethanol) and physiologically acceptable salt thereof; 4-hydroxyl-α 1[[[6-(3-phenyl propoxyl group) hexyl] amino] methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; 4-hydroxyl-α 1-[[[6-(2-phenyl ethoxy) hexyl] amino] methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; 4-hydroxyl-1-[[[5-(4-phenyl butoxy) amyl group] amino] methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; 4-hydroxyl-1-[[[1-methyl-6-(2-phenyl ethoxy) hexyl] amino] methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; 4-hydroxyl-1-[[[1-methyl-5-(3-phenyl propoxyl group) amyl group] amino] methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; 4-hydroxyl-α 1-[[[1-methyl-5-(4-phenyl butoxy) amyl group] amino] methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; 4-hydroxyl-α 1-[[[1-ethyl-6-(2-phenyl ethoxy) hexyl] amino] methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; α 1-[[[1,1-dimethyl-6-(2-phenyl ethoxy) hexyl] amino] methyl-4-hydroxyl-1,3-benzene dimethanol and physiologically acceptable salt thereof; 1-[[[6-[2-(4-fluoro phenyl) ethyoxyl]-1-methyl hexyl] amino] methyl]-4-hydroxyl-1,3-benzene dimethanol and physiologically acceptable salt thereof; 4-hydroxyl-α 1-[[[6-[3-(4-methoxyphenyl) propoxyl group]-1-methyl hexyl] amino] methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; 4-hydroxyl-α 1-[[[1-methyl-6-(4-phenyl butoxy) hexyl] amino] methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; 4-hydroxyl-α 1-[[[6-[2-(4-aminomethyl phenyl) ethyoxyl] hexyl] amino] methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; α 1-[[[6-[2-(3-chlorophenyl) ethyoxyl] hexyl] amino] methyl]-4-hydroxyl-1,3-benzene dimethanol and physiologically acceptable salt thereof; 4-hydroxyl-α 1-[[[6-[2-(4-methoxyphenyl) ethyoxyl] hexyl]-amino]-methyl]-1,3-benzene dimethanol and physiologically acceptable salt thereof; α 1-[[[6-[3-(4-fluoro phenyl) propoxyl group] hexyl] amino] methyl]-4-hydroxyl-1,3-benzene dimethanol and physiologically acceptable salt thereof.
Therefore, the present invention also be provided for treating or the formula V of the reversibility airway obstruction relevant disease of prevention and human or animal's study subject shown in chemical compound and physiologically acceptable salt and solvate.Compositions and the operation instructions of using it for the reversibility airway obstruction relevant disease of treatment or prevention and human or animal's study subject that the present invention also provides chemical compound shown in the formula V and physiologically acceptable salt and solvate and contains them.
Chemical compound of the present invention can be formulated as is convenient to form of administration.Therefore, scope of the present invention comprises that preparation is used for the pharmaceutical composition of chemical compound shown at least a chemical formula V of containing of people or veterinary drug or its physiologically acceptable salt or solvate.This compositions can be used with physiologically acceptable carrier or excipient, and optional medical preparation with interpolation uses.
Recommendation dosage every day of second activating agent is 0.0005mg-100mg, this can potion or two doses use easily.Employed exact dose will depend on patient's age and situation and route of administration certainly.Therefore, sucking the suitable dose of using is 0.0005mg-10mg, and Orally administered is 0.02mg-100mg, and it is 0.001mg-2mg that parenteral is used.
The salmaterol chemical compound is according to U.S. Patent number 4,992,474; 5,126,375 and 5,225,445 described methods prepare and separate, and these documents are included in as a reference in full.
Formoterol contains the alpha-amido xylyl alcohol derivant shown in the chemical formula (VIII):
Wherein among A and the B represents hydrogen atom or hydroxyl, and another is
(R1 that wherein is different from R2 represents hydrogen atom or C to group
1-7Alkyl, and R2 represent hydrogen atom or--the CO--R4 group, wherein R4 represents hydrogen atom, C
1-7Hydroxy alkyl or C
2-10And R3 represents C the alkanoylamino alkyl),
3-7Alkyl, C
6-12Cycloalkyl-alkyl or
(wherein Alk represents straight or branched C to group
1-7Alkylidene, and X, Y are identical with Z or have nothing in common with each other, each represents hydrogen atom, hydroxyl, C
1-7Alkanoylamino, C
1-7Alkyl or C1-7 alkoxyl).
Therefore formoterol has high activity and is suitable as the bronchodilatation medicine breathing smooth muscle as β-adrenal gland's energy stimulant.
Known so far all cpds, isonorin and should to breathe heavily peaceful (Trimetoquinol) be the chemical compound with bronchorelaxing activity particularly, these chemical compounds be the bronchodilatation medicine known and since its have strong effect and extensively sold.The bronchodilatation medicine also should not have ill effect to heart, and promptly it should have high selectivity, and Aerolin can satisfy this requirement and also extensively be sold.
In addition, be similar to the known 3-of the having amino-4-hydroxy-α of chemical compound-isopropyl amino methyl benzylalcohol (HOII P numbers 85197: " chemical abstracts " (ChemicalAbstract) of The compounds of this invention on the structure, 5211121d (1958)), 3-ethoxy carbonyl amino-4-hydroxy-α-isopropyl amino methyl benzylalcohol (belgian patent numbers 765,986), α-(isopropyl amino methyl)-4-hydroxyl-3-urea groups benzylalcohol (Japanese Unexamined Patent Publication No 2676/1971).These patents and patent application are included this paper in as a reference in full.
In representing the chemical formula of The compounds of this invention (VIII), the example of R1 is hydrogen atom, alkyl, for example methyl, ethyl, propyl group, isopropyl, normal-butyl, the tert-butyl group, 1,3-dimethylbutyl, 1,3-dimethyl amyl group, 2,3-dimethylbutyl, 2,3,3-tributyl etc.; The example of R4 is hydrogen atom, hydroxy alkyl; for example hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl etc.; alkanoylamino alkyl, for example Methanamide methyl (formamidemethyl), acetyl-amino methyl, acetyl-amino ethyl, acetyl-amino propyl group, bytyry amino-ethyl etc.The example of the alkyl of R3 have propyl group, isopropyl, just-butyl, tert-butyl, 1,3-dimethylbutyl, 1,3-dimethyl amyl group, 2,3-dimethylbutyl, 2,3,3-trimethyl butyl etc.; The example of the cycloalkyl of R3 has cyclopentyl-methyl, 2-ring ethyl, cyclohexyl methyl, 2-cyclohexyl ethyl, 3-cyclohexyl-1-methyl-propyl etc.The example of Alk has alkylidene, for example methylene, ethylidene, propylidene, butylidene, 1-methyl ethylidene, 1-ethyl ethylidene, 1-methyl propylidene, 1-ethyl propylidene, 2-methyl propylidene, 3-methyl butylidene, 2-ethyl butylidene etc.The example of X, Y and Z is hydrogen atom, hydroxyl, alkanoylamino; for example carbonylamino group, acetyl-amino, propiono amino, bytyry amino etc.; alkyl; for example methyl, ethyl, propyl group, isopropyl, isobutyl group, tert-butyl etc.; or alkoxyl, for example methoxyl group, ethyoxyl, propoxyl group, isopropoxy, butoxy etc.
Useful especially chemical compound of the present invention is 3-formamido-4-hydroxyl-α-[N-(1-methyl-2-right-hydroxy phenyl ethyl) amino-methyl] benzylalcohol, 3-formamido-4-hydroxyl-α-[N-(1-methyl-2-right-methoxyphenyl ethyl) amino methyl] benzylalcohol, 4-hydroxy-3-methyl amino-α-(N-tert-butyl amino methyl) benzylalcohol etc.
When the A of the chemical formula of representing The compounds of this invention (VIII) is a hydroxyl, and B is
During group, chemical compound of the present invention is shown in chemical formula (VIII):
Wherein R1, R2 have and the identical meaning of formula (VII) with R3, and say that more specifically formula (VIII) comprises following three kinds of structural formulas:
In the above structural formula, R3 and R4 have the meaning of the formula of being same as (VII).
Preferred formoterol is N-[2-hydroxyl-5-(1-hydroxyl-2-((2-(4-methoxyphenyl)-1-Methylethyl) amino)-ethyl) phenyl] Methanamide, or the solvate of its pharmaceutically acceptable salt or formoterol or described salt.Preferably it is its Fumaric acid salt form.Formoterol is the bronchodilatation medicine that is used for the treatment of inflammatory or obstructive airway diseases.
Particularly preferred Fu Yingteluo is the Fu Yingteluo fumarate shown in the following chemical formula:
The pharmaceutically acceptable salt of formoterol comprises, mineral acid for example, example hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid, and organic acid, as Fumaric acid, maleic acid, acetic acid, lactic acid, citric acid, tartaric acid, ascorbic acid, succinic acid, 1,3-propanedicarboxylic acid, gluconic acid, tricarballylic acid, oleic acid, benzoic acid, p-Methoxybenzoic acid, salicylic acid, neighbour. and right. hydroxy benzoic acid, right. the salt of chlorobenzene formic acid, methanesulfonic acid, right-toluenesulfonic acid and 3-hydroxyl-2-naphthalene-carboxylic acid.
Formoterol is the mixture of any isomeric forms or isomeric forms, for example pure enantiomer, the mixture of enantiomer, racemic modification or its mixture.It is solvate forms, and for example U.S. Patent number 3,994,974 or 5,684,199 described its hydrates, or certain crystal form, and for example WO95/05805 is described.These patents and patent application are included this paper in as a reference in full.Preferred formoterol is the fumarate of formoterol, particularly dihydrate form.
Suitable dosage every day of formoterol or its salt or solvate, particularly formoterol fumarate dihydrate is: but 1-72 μ g during suction, 1-60 μ g for example, 3-50 μ g normally, preferred 6-48 μ g is as 6-24 μ g.Used exact dose depends on the efficient of disease to be treated, patient and suction apparatus.The unit dose of formoterol and frequency of administration thereof can be selected like this.Formoterol, but the appropriate units dosage 1-72 μ g of the fumarate of formoterol and dihydrate particularly, 1-60 μ g for example, 3-48 μ g normally, preferred 6-36 μ g, particularly 12-24 μ g.These unit dose can be suitably according to above-mentioned suitable every day dosage once a day or administered twice.With regard to the consumption of needs, the formoterol unit dose of preferred 6 μ g-12 μ g.
The formoterol chemical compound is by U.S. Patent number 3,994,974 and 5,684,199 described method preparations and separate (these full patent texts are included this paper in as a reference).The fumarate of formoterol is commercially available to be got.It with the capsule form that contains dry powder formulations (
) sell, said preparation contains the formoterol fumarate of 12 μ g and the lactose of 25mg, only uses
Inhaler (Schering Corp., Kenilworth, NJ) oral suction.
Be used for the treatment of first and second activating agents of respiratory tract and pulmonary disease and following other arbitrary medicine can itself or use with above-mentioned pharmaceutically acceptable salt form, all be called " reactive compound or medicine ".The form that first and second activating agents also can make up mutually, separate or unite in pharmacy or veterinarily acceptable dosage form and use.These activating agents or its salt can general as described below or local applications.
The present invention also provides the method for treatment asthma, COPD or other respiratory tract disease, and described method comprises the study subject applying said compositions to this treatment of needs.This method is preventative or the therapeutic purpose.This method comprises method in the body.This method can be treated multiple disease effectively, and its cause of disease no matter, comprises that steroid is used, due to adenosine or adenosine receptor metabolism or synthetic unusual or other cause of disease.This method comprises that by reducing particularly adenosine in the organ of lung, liver, heart and brain or this treatment of any other needs or adenosine receptor level, reduction are treated respiratory tract disease and pneumonopathy to over anaphylaxis or other mechanism of adenosine.Other alleged respiratory tract disease of this paper comprises cystic fibrosis (CF), out of breath, emphysema, stridulate, pulmonary hypertension, pulmonary fibrosis, pulmonary carcinoma, the air flue hyperreactive, adenosine or adenosine receptor level raise, particularly relevant disease with various infectious disease, the pulmonary branches trachea shrinks, pneumonia, lung hypersensitivity disease, surfactant exhausts, chronic bronchitis, bronchoconstriction, dyspnea, lung airway obstacle or obstruction, the adenosine test of cardiac function is caused, the lung vasoconstriction, respiratory disorder, adult respiratory distress syndrome (ARDS), using of some drugs is caused, the medicine and the other medicines of adenosine and adenosine level for example can raise, as the medicine for the treatment of supraventricular tachycardia (SVT), carry out the caused of the emergent test of adenosine, infant respiratory distress syndrome (baby RDS), pain, allergic rhinitis, pulmonary surfactant reduces, Serious Atypica Respiratory Syndrome (SARS) etc.
In one embodiment, the present invention is the method for a kind of prevention or treatment asthma, and this method comprises to the study subject of this treatment of needs uses the compositions that is enough to prevent or treat the amount of study subject asthma.
In one embodiment, the present invention is the method for a kind of prevention or treatment COPD, and this method comprises to the study subject of this treatment of needs uses the compositions that is enough to prevent or treat the amount of study subject COPD.
In one embodiment, the present invention is the method for a kind of prevention or treatment bronchoconstriction, pneumonia or lung hypersensitivity disease, and this method comprises the compositions of amount from treatment study subject to the study subject of this treatment of needs that use bronchoconstriction, pneumonia or the lung hypersensitivity disease of enough preventions or.
In one embodiment, the present invention is a kind of method that reduces or exhaust the adenosine in the study subject tissue, and this method comprises to the study subject of this treatment of needs uses the compositions that is enough to reduce or exhaust the amount of adenosine in this study subject tissue.
The present invention also provides first activating agent and second activating agent to be used for the treatment of asthma, COPD or other respiratory tract disease in production, comprises the application in the lung cancer drugs.This medicine comprises the described compositions of this description.
Being applied to first activating agent of study subject and dosage every day of second activating agent becomes according to designed total therapeutic scheme, employed first activating agent and second activating agent, dosage form, route of administration and patient's situation.Embodiment 11-18 has shown and has been used for using or using through suction with respiratory tract or nose sending aerosolization preparation of the present invention.With regard to using in the lung, the preferred liquid preparation.With regard to other bioactivator, FDA has recommended to be used for replenishing the amount that people take in extra bioactivator every day, for example vitamin and mineral.Yet when being used for the treatment of particular disorder or improve the immunne response of study subject, the used dosage of these medicines can high hundreds and thousands of times.The often non-constant width of dosage range that pharmacopeia is recommended, the medical worker can therefrom draw instruction.The amount of the described exemplary medicine of this patent can be in the consumption per day scope of present recommendation, also can be below or above those levels.Treatment generally can start from coupling low dosage bronchodilatation medicine and non-glucocorticoid steroid or other suitable bioactivator, every the patient's that raises then dosage titre.Yet higher Yu lower amount comprises that primary quantity also can use within the scope of the invention.
The preferable range of first activating agent and second activating agent or any other curative drug becomes according to route of administration and used dosage form, and this is that (medicine) worker can understand and can be according to known method and component production.Reactive compound can single dose (once a day) or multidose (every day for several times) use.The compositions and the method for prevention and treatment respiratory tract, heart and cardiovascular disease can be used for treating adult and child and the non-human animal who suffers from described disease.Though the present invention relates generally to the treatment of people's study subject, also can be used for veterinary's purpose and treat the non-human mammal study subject, for example dog and cat and large-scale domestic and wild animal." height " of term " adenosine " and " adenosine receptor " is used to represent when compare the disease of adenosine level higher or lower (even exhausting) with the former adenosine level of same study subject with " low " level and " adenosine exhausts "; With adenosine level in normal range, but because some other disease or changes of patient, the disease that level that can be by reducing or increase adenosine or adenosine receptor or over anaphylaxis obtain the therapeutic benefit.Therefore, the mode that this Therapeutic Method can client's particular design assists to regulate (titration) patient.Although use that first activating agent can reduce or even exhaust the adenosine level of the study subject that before treatment, has normal or higher level, further use the breathing situation that second activating agent can improve study subject in a short time.Add other curative drug again and can help titration adenosine bad low-level (this can observe) after using medicine of the present invention, particularly until the best titre that obtains suitable dosage.
Can comprising into, other curative drug of the present composition is one or more various curative drugs that can be applied to humans and animals.
Except that first activating agent and second activating agent, said composition also can contain ubiquinone and/or folinic acid.Ubiquinone is the chemical compound shown in the following formula or its pharmaceutically acceptable salt:
Chemical compound shown in the preferably above chemical formula of ubiquinone, wherein n=1-10 (ubiquinone
1-10), more preferably n=6-10 (ubiquinone
6-10), n=10 (ubiquinone most preferably
10).Ubiquinone uses therapeutic goal disease or disease with therapeutic dose, and this dosage is according to the type of the disease of study subject, the other medicines of being used, used preparation and route of administration and become.The total amount that ubiquinone is used every day is preferably about 0.1,1,3,5,10,15,30-50,100,150,300,600,900,1200mg/kg body weight.Total amount every day more preferably from about 1-150mg/kg, about 30-100mg/kg, most preferably from about 5-50mg/kg.Ubiquinone is natural materials and commercially available getting.
Activating agent of the present invention accounts for the scope of the amount of said composition can be very wide.For example, activating agent can account for said composition about 0.001%, 1%, 2%, 5%, 10%, 20%, 40%, 90%, 98%, 99.999%.If the other medicines that comprise have the described overlapping activity of this patent, the amount of every kind of activating agent will be adjusted.Yet the dosage of reactive compound can become according to age, body weight and the disease of study subject.Treatment can begin than the present invention's first activating agent of low dosage, for example is lower than optimal dose.But second activating agent is similar operations also, until obtaining desired level.Or vice versa, for example with regard to multivitamin and/or mineral, can stablize the desired level that gives these products of study subject, uses first reactive compound again.Dosage can increase until the required and/or the best use of that obtains under this condition.In a word, activating agent does not preferably cause unsuitable concentration harmful or toxic side effect to be used so that useful effect to be provided, and can single dose unit use or if desired easily subunit use in the suitable time of every day.Second therapeutic or diagnostic medicine are used with the amount that is effective to required application known in the art.When second medicine has with main medicine eclipsedly when active, be no more than dosage range thereby the dosage that can adjust another kind or two kinds of medicines is realized required effect and avoid adverse side effect.Therefore, for example when other analgesic or anti-inflammatory agent are added said composition, these medicines can required application quantity known in the art or the dosage used separately a little less than itself add.
Pharmaceutically acceptable salt should be pharmacology and pharmacy or veterinarily acceptable, can be prepared into alkali metal or alkali salt, for example sodium salt, potassium salt or calcium salt.Organic salt and ester be also suitable to be used for the present invention.Activating agent comprises that preferably with pharmacy or veterinary's compositions whole body and topical formulations are applied to study subject.In these compositionss, preferred preparation be suitable for sucking or can be in respiratory tract, mouthful cheek, oral cavity, rectum, vagina, nose, lung, in the eye, eye, intracavity, trachea in (intratraccheal), the organ, the preparation used such as part (comprising a mouthful cheek, Sublingual, corium and ophthalmic), parenteral (comprising subcutaneous, intradermal, intramuscular, intravenous and intraarticular) and transdermal.
The present invention also provides dress to include the test kit of said composition and delivery apparatus.Compositions can be easily with single or multiple unit dosage form and in bulk providing, and any method preparation that can know by pharmaceutical field.Having the compositions that has been formulated into together or first and second activating agents to separate with other composition in the test kit provides, and the operation instructions of its prescription of explanation and application program.This test kit also can be equipped with other medicines, and for example this patent is described, and when parenteral was used, these medicines can be contained in the container separately and provide with the carrier of can sterilizing.The present composition also can be positioned in the autonomous container of sterilization and provide with lyophilized form, adds the liquid carrier before using.Referring to, for example U.S. Patent number 4,956, and 355; British Patent No. 2,240,472; EPO patent application series number 429,187; PCT patent disclosure WO91/04030; Mortensen, SA etc., Int.J.Tiss.Reac.XII (3): 155-162 (1990); Greenberg, S etc., JClin.Pharm.30:596-608 (1990); Folkers, K. etc., Proc.Natl.Acad.Sci U.S. 87:8931-8934 (1990), the related preparation of these documents and chemical compound partly draw and are reference.
Compositions of the present invention provides with various generals and locality preparation.General of the present invention or topical formulations be selected from oral cavity, mouthful cheek, in the lung, in the rectum, intrauterine, intradermal, part, skin, parenteral, tumor, in the intracranial, lung, in mouthful cheek, Sublingual, nose, subcutaneous, the blood vessel, in the sheath, can suck, through respiratory tract, intraarticular, intracavity, implantable, transdermal, through iontophoresis, ophthalmic, eye, transvaginal, in eye, intravenous, intramuscular, body of gland, in the organ, in the lymph, slow release and enteric coating preparation.The actual fabrication mixed method of these different preparations is known in the art, need not to describe in detail.But said composition once-a-day administration or several.
Preferred be fit in breathing, nose, lung and suck the preparation of using, for example local, oral and parenteral formulation.All preparation methoies include reactive compound and the blended step of delivery bulk phase that constitutes one or more compositions.In a word, can prepare this preparation, product can be processed into the shape of required preparation then if desired by reactive compound evenly and is closely mixed mutually with liquid carrier, fine graded solid carrier or with the two.
Being suitable for Orally administered compositions can provide by discontinuous unit, for example contains capsule, cachet, lozenge or the tablet of scheduled volume reactive compound separately; Powder or granule; The solution of aqueous or non-aqueous liquid or suspension; Or oil-in-water or water in oil emulsion.
Be suitable for sterile aqueous or non-aqueous injection solution that compositions that parenteral uses comprises reactive compound, these preparations preferably ooze with blood of receiver etc.These preparations can contain antioxidant, buffer, press down antibacterial and make compositions and receiver's the isoosmotic solute of blood.Aqueous or non-aqueous sterile suspensions can contain suspending agent and thickening agent.Compositions can be contained in single dose or the multi-dose container and provide, for example Mi Feng ampoule and bottle, and can lyophilized form or the lyophilization condition under store, only need add sterile liquid carrier, for example saline or water for injection before use.
The nasal preparation and the preparation that can instil contain the aqueous solution of the purification of reactive compound and antiseptic and isotonic agent.This preparation preferably oozes status adjustment for compatible with nasal mucosa with pH with waiting.
The preparation of rectum or vaginal application can be with suitable carrier, and for example cupu oil or hydrogenant fat or hydrogenant aliphatic carboxylic acid are made suppository provides.
Ophthalmic preparation can be similar to the method preparation of nasal spray, except pH and wait ooze the factor preferably to be adjusted to be complementary with eye.Ear (otical) preparation is generally used viscous carrier body known in the art, for example wet goods preparation, thus can easily use into ear and do not have leakage.
The compositions that is suitable for being applied topically to skin preferably adopts ointment, ointment, lotion, paste, spray, aerosol or oil preparation form.Spendable carrier comprises vaseline, lanoline, Polyethylene Glycol, ethanol, dermal penetration enhancer and one or more combination thereof.The compositions that is suitable for transdermal administration can be suitable for keeping the long-time discontinuous paster that closely contacts with receiver's epidermis to be provided.
First and second activating agents that this paper discloses can by any suitable method in suction, breathing, nasal administration or lung, instils (entering pulmonary) use the into respiratory system of study subject, preferably by generation by the aerosol or the spray application that maybe can suck granulometric composition in the nose of powder or liquid, the lung, through breathing.Contain maybe can the sucking granule and sucked of reactive compound, promptly through suction or nasal administration or through being instilled into respiratory tract or pulmonary itself by study subject through breathing.Preparation can contain the liquid or solid granule that can breathe the reactive compound that maybe can suck, thus described granule of the present invention comprise volume enough little can be after suction through port and larynx and continuing enter bronchus and alveolar maybe can suck granule through breathing.Particulate diameter is 005,0.1,0.5,1 substantially approximately, 2-4,6,8,10 microns.Particularly the about 0.5 μ m of diameter is to being can be through breathing the granule maybe can suck less than 5 μ m.The granule that can not suck size in aerosol or the spraying easily is deposited on the throat place and is swallowed.Therefore, can not breathe particulate quantity in the aerosol should be minimum.With regard to instiling in nasal administration or the lung, preferred particle size range is 8,10,20 approximately, 25-35,50,100,150,250,500 μ m (diameter) to be to guarantee staying nasal cavity or to instil and directly be deposited into pulmonary.But liquid preparation spirt respiratory tract (nose) and pulmonary particularly are applied to neonate and baby.
The composition of liquid medicine that is used to produce aerocolloidal reactive compound can be by with reactive compound and stable carrier, and for example apyrogenic sterilized water combination prepares.Containing the dried particulate solid particle composition of breathing of micronization reactive compound can make micronized compositions separate by 400 eye mesh screens or tell big aggregation and prepare by the reactive compound of doing with mortar and pestle porphyrize again.The solid particle composition that contains reactive compound can be chosen wantonly to contain and can promote to form aerocolloidal dispersant.Suitable dispersant is can any proper proportion, for example blended lactose of 1: 1 weight ratio and reactive compound.The DHEA-S that the DHEA that U.S. Patent Application Serial 10/462,901 and 10/462,927 has disclosed aerosolizable form respectively stablizes dry powder formulations and two crystalline hydrate forms stablizes dry powder formulations (these patent applications are included in as a reference in full).
The aerosol that contains the liquid particles of reactive compound can be by any suitable method production, for example aerosol apparatus.Referring to, for example U.S. Patent number 4,501,729 (these contents draw be with reference to).Aerosol apparatus is by Compressed Gas (generally being air or oxygen) being quickened through narrow Venturi tube nozzle or making the solution of active component or the commercialization device that suspension is converted into the therapeutic aerosol mist by ultrasonic stirring.The compositions that is suitable for aerosol apparatus is made up of the active component of liquid carrier preparation, this active component accounts for the 40%w/w of compositions, but preferably contain the carrier (generally being the alcohol water blend of water or dilution) that is less than 20%w/w, preferably by adding, for example sodium chloride makes it to ooze with body fluid etc.Optional additive comprises antiseptic (if compositions is not a sterile preparation), for example methyl hydroxybenzoate, antioxidant, flavoring agent, ethereal oil, buffer reagent and surfactant.The aerosol that contains the solid particle of reactive compound can be produced with any commercially available granule medicament aerosol generator similarly.As explained above, solid particle medicament administration to the aerosol generator of study subject is produced the aerosol that can breathe granule and contain pre-metered doses of medication in a large number with the speed that is suitable for human body and uses.The ion of this aerosol generator comprises inhaler and the insufflator that definite dose drug is housed.
Described compositions can produce the aerocolloidal delivery apparatus of liquid or solid granule with any, and for example aerosol or spraying generator are sent.As explained above, these devices are to be suitable for aerosol or the spraying that speed that the human or animal uses produces respirable granule and contains pre-metered doses of medication in a large number.A kind of illustrative type of solid particle aerosol or spraying generator is the insufflator that is suitable for using the thin powder of lappingout.In insufflator, powder for example can effectively carry out the compositions of definite dosage of treatment described herein, is contained in capsule or the cartridge case.These capsules or cartridge case are generally made with gelatin, tinsel or plastics, and can bore a hole in position or open, and send and powder flows through the air or the manually operated pump of device when air-breathing.The compositions that insufflator uses can be only be made of first and second activating agents or the mixture of powders (generally accounting for the 0.01-100%w/w of compositions) that contains first and second medicines.The amount of first and second activating agents that said composition is contained generally is 0.01%w/w, 1%w/w/, 5%-20%w/w, 40%w/W, 99.99%w/W approximately.Yet the amount of other composition and other medicines also is fit to be contained in the scope of the present invention.
In one embodiment, described compositions is sent by aerosol apparatus.This device is for relying on patient or the study subject that self-ability can not composition for inhalation to be particularly useful.In cases with severe, patient or study subject rely on artificial respirator to earn a bare living.Aerosol apparatus can use the acceptable carrier of any pharmacy or veterinary, for example low saline solution.Aerosol apparatus is the device that powder pharmaceutical compositions is delivered to patient or the target position of study subject in air flue.
Said composition is also customizable, and multi-form different administration method and the route of administration of providing is provided.In one embodiment, said composition contains respirable preparation, for example aerosol or spray.Compositions of the present invention can in bulkly provide with form and the form of opening implant, capsule, blister package or the cartridge case that maybe can bore a hole known in the art unit dose.The present invention also provides test kit, and this test kit is equipped with delivery apparatus, compositions of the present invention and optional other excipient and curative drug are housed in autonomous container, and the operation instructions that use the test kit composition.
In one embodiment, use the suspension of determining dosage to suck (MDI) formulation delivered said composition.This MDI preparation can make delivery apparatus send, and described delivery apparatus uses propellant, for example hydrofluoroalkane (HFA).Each 1,000,000 parts (ppm) preferably contains 100 parts or water still less in the HFA propellant.
In one embodiment, this delivery apparatus comprises the Diskus (DPI) that can send the single or multiple dosage composition.Monodose inhaler can be used as disposable test kit and provides, the aseptic in advance preparation of enough once using that is equipped with of this test kit.Inhaler can be used as the pressurization inhaler and provides, and preparation be contained in can perforation or openable capsule or cartridge case in.Test kit also can be chosen the reagent that is equipped with as other appropriate addn of different preparations wantonly in container independently, for example other therapeutic compound, excipient, surfactant (as curative drug and preparation composition), antioxidant, seasoning and coloring agent, filler, ethereal oil, buffer agent, dispersant, surfactant, antioxidant, flavoring agent, extender, propellant and antiseptic.
The present invention has been carried out behind the describe, in general terms, can understand the present invention better with reference to specific embodiment, unless spell out, the embodiment of this paper does not really want to limit the present invention or its any embodiment for purposes of illustration.
Embodiment
Young bull Fischer344 rat (120 gram) with gavage use every day dehydroepiandrosterone (DHEA) that carboxymethyl cellulose prepares (300mg/kg) or methyltestosterone (40mg/kg) once, totally 14 days.Every day, intraperitoneal was used folinic acid (50mg/kg) once, totally 14 days.At the 15th day, put to death rat by the cranium brain being carried out microwave pulse (1.33 kilowatts, 2450 megahertzes, 6.5 seconds (s)), this microwave pulse makes all brain albuminous degenerations immediately and prevents that adenosine is by further metabolism.Take out rat heart quick-freezing in liquid nitrogen caused death in 10 seconds.Liver and lung take out quick-freezing together and caused death in 30 seconds.Dissect cerebral tissue then.Adenosine is organized in extraction, and deriving is 1, and N6-ethenylidene adenosine also detects with fluorescence spectrum by high performance liquid chromatography (HPLC) according to the method for Clark and Dar (J.of Neuroscience Methods 25:243 (1988)) and to analyze.These result of experiment are summarized in the following table 1.The result represents with meansigma methods ± SEM, compares κ p＜0.05 with matched group; Compare ψ p＜0.05 with DHEA or methyltestosterone processed group.
Act in table 1:DHEA, δ-1-methyltestosterone and the body of folinic acid adenosine level in the various rat tissues
| Treatment | Adenosine (nmol) in the born of the same parents/mg albumen | |||
| Heart | Liver | Lung | Brain | |
| Contrast DHEA (300mg/kg) methyltestosterone (40mg/kg) methyltestosterone (120mg/kg) folinic acid (50mg/kg) DHEA (300mg/kg)+folinic acid (50mg/kg) methyltestosterone (120mg/kg)+folinic acid (50mg/kg) | 10.6±0.6 (n＝12)K 6.7±0.5 (n＝12)K 8.3±1.0 (n＝6)K 6.0±0.4 (n＝6)K 12.4±2.1 (n＝5)K 11.1±0.6 (n＝5)ψ 9.1±0.4 (n＝6)ψ | 14.5±1.0 (n＝12)K 16.4±1.4 (n＝12)K 16.5±0.9 (n＝6)K 5.1±0.5 (n＝6)K 16.4±2.4 (n＝5)K 18.8±1.5 (n＝5)ψ N.D. | 3.1±0.2 (n＝6)K 2.3±0.3 (n＝6)K N.D. N.D. N.D. N.D. N.D. | 0.5±0.04 (n＝12)K 0.19±0.01 (n＝12)K 0.42±0.06 (n＝6)K 0.32±0.03 (n＝6)K 0.72±0.09 (n＝5)K 0.55±0.09 (n＝5)ψ 0.60±0.06 (n＝6)ψ |
| The N.D=undetermined | ||||
These result of experiment show uses many organs consumption that DHEA or the rat in two weeks of methyltestosterone demonstrate adenosine every day.Adenosine consumes in brain (use DHEA and consumed 60%, and the methyltestosterone of administered with high dose has consumed 34%) and heart (use DHEA and consumed 37%, and the methyltestosterone of administered with high dose has consumed 22%) obviously high.Use folinic acid simultaneously and eliminated the adenosine consumption of steroid mediation fully.List causes adenosine level to increase in the organ of all researchs with folinic acid.
Embodiment 3: but the mensuration of anhydrous DHEA-S of airbrasive and inhalation dose
DHEA-S treats agent as asthma and assesses.Studied the two solid-state stability (Nakagawa, H., Yoshiteru, T. and Fujimoto, Y. (1981) Chem.Pharm.Bull.29 (5) 1466-1469 of in bulk and abrasive material of dehydroepiandrosterone sulfate sodium (NaDFEA-S); Nakagawa, H., Yoshiteru, T. and Sugimoto, I. (1982) Chem.Pharm.Bull.30 (1) 242-248).Dihydrate form is the most stable crystal form of DHEA-S.DHEA-S anhydrous form degree of crystallinity is low and be highly susceptible to moisture absorption.As long as do not absorb water when storage, the anhydrous form of DHEA-S can be kept stable.Make the partially crystallizable material need the production and the packing technique of specialization away from moisture.With regard to the robot product, must reduce sensitivity in process of production as far as possible to moisture.
(1) micronization of DHEA-S
Use airbrasive machine (Jet-O-Mizer Series#00,100-120 PSI nitrogen) to make anhydrous DHEA-S micronization.About 1g sample passes through the airbrasive machine once, and about 2g sample passes through the airbrasive machine twice.Each particle suspending that grinds is in the hexane that does not dissolve DHEA-S and add the Spa85 surfactant to prevent gathering.The solution supersound process that obtains 3 minutes is shown as fully and scatter.Dispersive solution is tested on Malvern Mastersizer X with sampler (SVS) adnexa of small size.The sample test of a dispersed substance 5 times.The particle diameter of abrasive material or D (v, 0.5) intermediate value is not 52.56 μ m and RSD (relative standard deviation) % of 5 test values is 7.61.By airbrasive machine D (v, 0.5) once is 3.90 μ m and RSD% is 1.27, and the D (v, 0.5) by twice in airbrasive machine is 3.25 μ m and RSD% is 3.10.This confirms that DHEA-S can be the big or small granule that is suitable for sucking through airbrasive.
(2) HPLC analyzes
Use the micronized medicine (A of two bottles; By once; 150mg) with (B; By twice; 600mg) be determined at the degraded of airbrasive micronization process Chinese medicine.The DHEA-S standard solution (10mg/ml) that waits weight DHEA-S and not grinding of the bottle A of the solution of acetonitrile-water (1: 1) preparation and B compares.The chromatographic peak area that does not grind the HPLC analysis of pharmaceutical standards solution (10mg/ml) is 23,427.Bottle A and B etc. heavy micronization DHEA-S with preparation (5mg/ml) in the acetonitrile-aqueous solution (1: 1).The chromatographic peak area of bottle A and B is respectively 11,979 and 11,677.Obvious no detectable drug degradation in the micronized process of airbrasive.
(3) hair spray quantity research
The DHEA-S powder collection is analyzed in the Nephele test tube and through HPLC.Each of 3 kinds of Diskuses being tested (Rotahaler, the DPI device of Diskhaler and IDL) repeats 3 experiments with every kind of air velocity.One end of Nephele test tube is loaded onto glass filter (Gelman Sciences, A/E type, 25 μ m), is connected with airflow line then to collect the medication amount of every kind of Diskus eruption being tested.Fixedly have the silicone adapter of opening at the other end of Nephele test tube, this opening links to each other with the interface of every kind of Diskus being tested.Obtain required 30,60 or 90L/ minute air-flow by the Nephele test tube.The interface of Diskus is inserted the silicone rubber adapter, ventilated continuously about 4 seconds, remove test tube then and at the end of each test tube end cap (end-cap) of screwing on.Take off the end cap of the test tube of not being with filter and in test tube, add the water-acetonitrile solution (1: 1) of 10ml HPLC level, covered end cap and tube shaken 1-2 minute.Take off end cap and solution is transferred to 10ml from test tube then and be equipped with in the plastic injector of filter (Cameo 13NSyringe Filter, Nylon, 0.22 μ m).The solution Direct Filtration of equivalent is advanced the HPLC bottle be used for after a while HPLC pharmaceutical analysis.The micronization DHEA-S (about 12.5 or 25 milligrams) that use is contained in gelatine capsule (Rotahaler) or the Ventodisk bubble-cap (Diskhaler and single agent DPI (IDL)) carries out above-mentioned eruption dosetest.In the time of in taking by weighing micronization DHEA-S (only using the B bottle) adding gelatine capsule or bubble-cap, micronised powder shows minor agglomeration may.With 30,60 and the hair spray that carries out of the air velocity of 87.8L/min measure the table 2 that the results are shown in of examination.Table 2 has been summed up the result of the Rotahaler test of carrying out with 3 kinds of different in flow rate, the result of the Diskhaler test of carrying out with 3 kinds of different in flow rate and carry out the multiple dose result of experiment with 3 kinds of different in flow rate.
Table 2: the comparison of three kinds of different powder inhaler eruption dosage
| Suction apparatus | Airflow rate (L/min) | Eruption dosage (%) |
| Rotahaler | 87.8 | 73.2，67.1，68.7 |
| Meansigma methods | 69.7 | |
| Rotahaler (research for the second time) | 87.8 | 16.0，24.5，53.9 |
| Meansigma methods | 31.5 | |
| Diskhaler | 87.8 | 65.7，41.6，46.5 |
| Meansigma methods | 51.3 | |
| Diskhaler (research for the second time) | 87.8 | 57.9，59.9，59.5 |
| Meansigma methods | 59.1 | |
| The IDL multi-agent | 87.8 | 71.3，79.0，67.4 |
| Meansigma methods | 72.6 | |
| IDL multi-agent (research for the second time) | 87.8 | 85.7，84.6，84.0 |
| Meansigma methods | 84.8 | |
| |
60 | 58.1，68.2，45.7 |
| Meansigma methods | 57.3 | |
| |
60 | 63.4，38.9，58.0 |
| Meansigma methods | 68.2 | |
| The IDL multi-agent | 60 | 78.8，83.7，89.6 |
| Meansigma methods | 84.0 | |
| |
30 | 34.5，21.2，48.5 |
| Meansigma methods | 34.7 | |
| |
30 | 53.8，53.4，68.7 |
| 58.6 | ||
| The IDL multi-agent | 30 | 78.9，88.2，89.2 |
| Meansigma methods | 85.4 |
(4) can breathe Research on dose
The sampling cascade ram (Andersen) of use standard carries out breathing dosage (can breathe component) research, this sampler is made up of inlet cone (the ram preseparator is replaced at this), 9 platforms, 8 collecting boaries and reserve filter with 8 aluminum platforms, these aluminum platforms are linked together by 3 alligator clamps and O-type ring seal washer, and wherein each ram platform has a plurality of fine drilling hole nozzles.When air communication was crossed sampler, the multi beam air-flow in each platform was given birth to the collecting board surface that granule blows to this platform with gas.The air-flow constant magnitude of each platform, but the air-flow of every next platform is less than last one.Whether granule strikes on the given arbitrarily platform depends on its aerodynamic diameter.The scope that is collected in the granular size of each platform depends on the air velocity of platform and holding back a little of a last platform.Any granule of being collected by first platform is not all along with the air-flow around this panel edges enters next platform, can clash into or by entering next platform at this platform place granule, and the like, be enough to produce bump until the speed of air-flow.In order to prevent that granule is upspring in cascade ram test process, each ram plate all scribbles hexane-oils and fats (fine vacuum) solution (100: 1).As mentioned above, the granular size on the ram plate is held back a little and is become according to different air velocitys.For example, the cutoff value of platform 2 correspondence in the time of 60L/ minute is greater than 6.2 micron particle, and the cutoff value of correspondence is greater than 5.8 micron particle in the time of 30L/ minute; The particle diameter cutoff value of platform 3 in the time of 90L/ minute is greater than 5.6 microns.Therefore, when the airflow rate that equates, preferably use similar trapped particles value, that is, and the 5.6-6.2 micron.The device of the test Diskus of American Pharmacopeia suggestion is made up of the spout adapter that is connected in glass trunnion (improved 50 milliliters of round-bottomed flasks) (this example is silicone) and the far-end glass pharyngeal canal (guiding port) and the Andersen sampler of last logical preseparator.The sample of this preseparator comprises the washings that obtains from spout adapter, glass trunnion, far-end pharyngeal canal and preseparator.Before carrying out cascade ram experiment, with 5 milliliters of acetonitriles: water (1: 1) solvent places this preseparator, and this experiment is carried out twice with 3 kinds of different Diskuses with 90L/ minute airflow rate with 30,60.Analyze the medicine of on cascade ram plate, collecting with HPLC, each Diskhaler and the experiment of multiple dose cascade ram are carried out drug quality balance (mass balance), comprise and measure the medication amount that remains in the bubble-cap, residue in the medication amount in the device (only being Diskhaler), remain in silicone rubber spout adapter, the glass trunnion, the medication amount that can not suck on far-end glass pharyngeal canal and the preseparator, all medicines are merged into a duplicate samples measure and can breathe dosage, promptly air velocity is 30 and the dosage that passes through filter ram plate of platform 1 during the experiment of the dosage that passes through filter ram plate of platform 2 and 90L/ minute 60L/ minute the time.
Table 3: cascade ram experiment (90L/ minute)
| Suction apparatus | Preseparator (%) | Bubble-cap (%) | 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 |
| Multiple dose | 65.8 | 3.9 | 3.8 | 26.5 *a | 100.0 |
| Multiple dose | 73.3 | 3.8 | 3.6 | 19.3 *a | 100.0 |
| Multiple dose *b | 78.7 | 2.8 | 4.6 | 13.9 *a | 100.0 |
| Multiple dose *c | 55.9 | 5.0 | 1.2 | 37.9 *a | 100.0 |
*A: do not wash the multiple dose device, because solvent can be attacked the SLA component.Obtain multiple dose device percent retention difference.
*B: oven drying medicine 80 minutes
*C: oven drying medicine 20 hours
Based on eruption dosage and cascade ram result of experiment, because drug particles is assembled, the dose value breathed that obtains in the experiment of cascade ram is low, even accumulative drug particles can not separate in high gas flow rate test.The gathering of drug particles is due to the static that produces in the mechanical lapping process that is used for reducing granular size, and ensuing pellet moisture absorbs and makes this situation more complicated.The method of micronization that produces complete hydrate crystal (the being dihydrate form) form of less static or hygroscopicity DHEA-S should be able to provide assembles the powder more freely that flows that probability reduces.
Embodiment 4: the spray drying of anhydrous DHEA-S and the mensuration that can breathe dosage
(1) micronization of medicine
1.5g anhydrous DHEA-S is dissolved in 100ml50% ethanol: obtain 1.5% solution in the water.(Switzerland) spray drying, inlet temperature are that 55 ℃, outlet temperature are that 40 ℃, air exhauster are that 100%, 10% pump, stream of nitrogen gas are that 40mbar, spraying flow velocity are 600 units for Buchi, Flawil with B-191 Mini spray dryer for this solution.Spray-dired product is suspended in the hexane, adds the Span85 surfactant and assemble to reduce.Supersound process is also cooled off and dispersion liquid was disperseed fully in 3-5 minute, and dispersive solution is tested on the Malvern Mastersizer X that small size sampler (SVS) adnexa is housed.The mean diameter of finding two batches of spray-dried material is respectively 5.07 ± 0.70 μ m and 6.66 ± 0.91 μ m.Confirm that with each batch of optics microscopic visual measurement dispersion liquid spray drying has produced the granule of little breathed size.The mean diameter of each batch is respectively 2.4 μ m and 2.0 μ m.The granule that the sprayable drying of this proof DHEA-S is suitable for sucking for size.
(2) can breathe Research on dose
As described in embodiment 3, carry out the experiment of cascade ram.Carry out four cascade ram experiments, wherein use IDL multiple dose device for three times, once use Diskhaler, the air velocity of all experiments all is 90L/ minute.Cascade ram result of experiment is listed in the following table 4.Anhydrous DHEA-S compares with micronization, and the dosage breathed of the spray drying anhydrous substances that produces in these experiments has increased twice.Compare with airbrasive, spray drying looks and can obtain the higher dosage breathed, and remains low yet can breathe dosage %.This may be to cause because anhydrous form absorbs moisture.
Table 4: the cascade ram experimental result of using spray-dired drug products to carry out
| Device | Diskhaler | The multiple dose device | The multiple dose device | The multiple dose device |
| The bubble- |
3 | 3 | 4 | 4 |
| The medicine of every piece of bubble-cap (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 |
| Bubble-cap (%) | 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 grinds and can breathe dosage
(1) recrystallization of DHEA-S dihydrate
Anhydrous DHEA-S is dissolved in the boiling mixed liquor of 90% ethanol/water.This solution of cooling makes the DHEA-S recrystallization fast in dry ice/methanol bath.Leach crystal, with cold washing with alcohol twice, then in room temperature in vacuum desiccator dry 36 hours.In the dry run, regularly stir to destroy big aggregation with scraper.After the drying, this material is by 500 μ m sieves.
(2) micronization and physical and chemical testing
With nitrogen micronization DHEA-S dihydrate in the airbrasive machine, Venturi tube pressure is that 40PSI, grinding pressure are that 80PSI, charging are set to 25, the product feed rate is about 120-175g/ hour.Use Micromeritics TriStar surface area analyser to carry out 5 BET assay determination surface areas as adsorbed gas (P/Po=0.05 to 0.30) with nitrogen.Use Micromeritics Satum Digisizer by the laser diffraction analysis particle size distribution, wherein particle suspending uses sodium dioctyl sulfosuccinate as dispersant in mineral oil.Measure the moisture of medicine by Karl Fischer titration (Schott Titroline KF).As standard substance, all relative standard deviations of three tests are less than 1% with pure water.Powder directly adds in the titration medium.The physicochemical property of DHEA-S dihydrate is summarized in table 5 before and after the micronization.
Table 5: the physicochemical property of DHEA-S dihydrate before and after the micronization
| Characteristic | The loose powder caking | Micronization |
| Granular size (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 particle diameter.The increase of moisture loss and impurity is all not obvious.The surface area of micronization material and size intermediate value are that the irregularly shaped particles of 3-4 micron is consistent.Micronization successfully is reduced to particle diameter the scope that is suitable for sucking, and does not have measurable variation in its solid state chemistry character.
(3) the DHEA-S dihydrate is aerosolized
With single dose Acu-Breathe device assessment DHEA-S dihydrate.The DHEA-S dihydrate that about 10mg is pure is filled and is sealed in the paper tinsel bubble-cap.Making these bubble-caps enter flow velocity is 30-75L/ minute, is equipped with in the 8 platform cascade rams of Andersen of the two ram trunnions of a glass.The 1-5 platform of drip washing Andersen ram obtains the estimated value of fines fraction together.Collection is merged into a test sample from the medicine of a plurality of platforms makes the method more sensitive.This campaign the results are shown in Fig. 1.Under all flow velocitys, the fines fraction that dihydrate produces is higher than that anhydrous substances in fact produces.Owing to use the single dose inhaler that the dihydrate powder is aerosolized, therefore can rationally draw the aerosol performance significantly better than the conclusion of anhydrous substances in fact.Higher degree of crystallinity and stable moisture are most possibly to cause dihydrate to have the factor of the aerosol properties of excellence like this.This peculiar property of DHEA-S dihydrate yet there are no report in any existing document.Though the DHEA-S aerosol performance improvement of dihydrate form clearly, pure medicine may not be an optimal formulation.Use the bigger carrier of particle diameter can improve the aerosol character of micronized medicine usually.
Embodiment 6: add and the stability of anhydrous DHEA-S and DHEA-S dihydrate when not adding lactose
Adopt high pressure liquid chromatography (HPLC) to measure the initial purity (time=0) of anhydrous DHEA and DHEA-S dihydrate.Then will with 50: 50 ratio and lactose be blended or the DHEA-S of pure powder type is placed in the opening vial, kept for 4 weeks for 50 ℃.Predict their long-time stability result for the preparation stress application with these conditions.Sealing only contains the contrast bottle of DHEA-S (anhydrous or dihydrate) and kept for 4 weeks in 25 ℃.As measure the formation of DHEA, in the sampling of the 0th, 1,2 and 4 weeks and carry out HPLC and analyze the amount of measuring degraded.After 1 week, be stored in 50 ℃ of sealed glass jars with the blended in fact anhydrous DHEA-S of lactose (50%, nominally) and produce than dark filbert of milk-sugar mixture.This change color is with stratographic significant change as shown in Figure 1.Main degradation product is DHEA.From the quantitative situation of Fig. 2, the amount of DHEA is higher than other two duplicate samples in the mixture.For the DHEA% in the quantitative sample estimates, with the area at DHEA peak the gross area (seeing Table 6) divided by DHEA-S and DHEA peak.The higher degradation rate of mixture shows between lactose and the in fact anhydrous DHEA-S special interaction.When DHEA increases, the brown of quickening to store (test) powder also in time passing and deepen.The caking phenomenon that takes place in the process that takes by weighing of sample for chemical analysis confirms that also the material that quickens to store passes in time and become more tacky.Based on these results, in fact anhydrous DHEA-S is impossible with the lactose preparation.Because lactose is the most frequently used suction excipient of dry powder formulations, this is very disadvantageous.Continue to mean that with in fact anhydrous form preparation will be limited to pure powder, perhaps will carry out more fully safety research and come with new excipient.
Show the 6:50 ℃ of formed DHEA% of anhydrous DHEA-S
| Preparation | Time (week) | 1 | 2 | 4 |
| Contrast | 2.774 | 2.694 | 2.370 | 2.666 |
| DHEA-S (separately) | 9.817 | 14.954 | 20.171 | |
| DHEA-S+ lactose (50: 50) | 24.085 | 30.026 | 38.201 |
Opposite with Fig. 2 is not have DHEA in fact after 50 ℃ of 1 weeks of storage and produce (referring to Fig. 3).In addition, the color no change of this material.50 ℃ of moistures of storing 1 all backs DHEA-S dihydrate do not have substantial change.Quickening to store the back moisture is 8.66%, and initial value is 8.8%.The DHEA% that records in this stable program is shown in table 7.
Show the 7:50 ℃ of formed DHEA percentage ratio of DHEA-S dihydrate
| Preparation | Time (week) | 1 | 3 | 4 |
| Contrast | 0.213 | 0.218 | ||
| DHEA-S (separately) | 0.216 | 0.317 | 0.374 | |
| DHEA-S: lactose (50: 50) | 0.191 | 0.222 | 0.323 |
Comparison Fig. 1 and 2 and table 6 and 7 dihydrate form of DHEA-S as can be seen are the more stable forms of further studying.Compare with anhydrous substances in fact, the superior compatibility of DHEA-S dihydrate and lactose is not appeared in the newspapers in patent and research document.Next part will be reported the dissolubility as this material of an aerosol apparatus solution development part.
Embodiment 7:DHEA-S dihydrate/milk-sugar mixture can be breathed dosage and stable mensuration
(1) DHEA-S dihydrate/milk-sugar mixture
The DHEA-S dihydrate of weight such as manual mixing and a suction level lactose (Foremost Aero Flo 95) make mixture cross 500 μ m sieves with the preparation premix then.This premix and remaining lactose place BelArt Micro grinder to obtain the DHEA-S mixture of 10%w/w then.But blender is connected with variable-voltage power supply regulates agitator speed.The voltage of blender circulated 1,3,1.5 and 1.5 minute by 30%, 40%, 45% and 30% of total head respectively.Analyze the content uniformity of measuring mixture by HPLC.Table 8 has shown the measurement result of this mixture content homogeneity sample.Desired value is the DHEA-S of 10%w/w.Mixture content is near desired value and content uniformity, and is satisfactory.
The content uniformity of the mixture of table 8:DHEA-S dihydrate and lactose
| Sample | ％DHEA-S，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 powder of about 25mg is filled and is sealed in the bubble-cap, carries out aerosolized with single dose device with 60L/ minute then.Two pieces of bubble-caps are used in each test respectively, and the measurement result of fines fraction (material on the platform 1-5) is shown in table 9.The aerosol measurement result of this raw powder mixture is satisfied for the respiratory medications delivery system.Optimize mixture of powders and bubble-cap/unit configuration and might obtain higher fines fraction.Total particle size distribution of test 2 is shown in table 10.The median diameter of DHEA-S is about 2.5 μ m in this aerosol.This diameter is less than the median diameter with the measured micronization DHEA-S dihydrate of laser diffraction.Because particulate longest dimension in irregular shape may be in line with airflow field, their performances on aerodynamic are as than granule.Therefore, it is normal there are differences between two kinds of methods.Diffractometry is the Quality Control test of input material, and the cascade bump is the Quality Control test of final products.
Table 9: the fines fraction of milk-sugar mixture in two kinds of different experiments
| Test | Total powder weight (mg) of two pieces of bubble-caps | The DHEA-S that collects among the platform 1-5 (mg) | Fines fraction, % |
| 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 |
100 | 87.55 | 67.79 | 29.87 | 10.70 | 2.57 | 1.82 | 0.90 |
(3) stability of DHEA-S dihydrate/milk-sugar mixture
This lactose preparation also places 50 ℃ accelerated stability program.DHEA-S content the results are shown in table 11.Contrast is the mixture that is stored in room temperature.DHEA-S content is not all passed under arbitrary condition and the trend that changes in time, and all results are in the collected sample scope of uniform content property testing (referring to table 11).In addition, color does not change, and does not perhaps observe irregular in chromatograph yet.It seems that mixture be chemically stable.
Table 11:DHEA-S dihydrate/milk-sugar mixture is in 50 ℃ stress stability
| Time (week) | DHEA-S% w/w under the collating condition | DHEA-S% w/w under the |
| 0 | 9.7 | 9.7 |
| 1 | 9.6 | 9.6 |
| 1.86 | 9.5 | 9.7 |
| 3 | 10 | 9.9 |
The nebulizer formulation of embodiment 8:DHEA-S
The dissolubility of DHEA-S.
Excessive DHEA-S dihydrate according to " DHEA-S dihydrate recrystallization method (embodiment 5) " preparation adds solvent medium, and balance at least 14 hours is regularly shaken simultaneously.This suspension filters through 0.2 micron syringe filter then, dilutes immediately to carry out HPLC and analyzes.Be preparation cooling sample, syringe and filter are with before being kept in the electric refrigerator at least 1 hour.Suck pure water and can produce the cough stimulation.Therefore, it is important halogen ion (the most frequently used salt is sodium chloride) being added in the nebulizer formulation.Because DHEA-S is a sodium salt, so sodium chloride can reduce dissolubility because of the common ion effect.The functional relationship of sodium chloride concentration and the DHEA-S dissolubility when room temperature (24-26 ℃) and cooling (7-8 ℃) is shown in Fig. 4.The dissolubility of DHEA-S reduces along with sodium chloride concentration.For all sodium chloride concentrations, reduce storage temperature and all can reduce dissolubility.During high sodium chloride concentration a little less than the effect of temperature.In three repeated experiments, the dissolubility when about 25 ℃ and 0% sodium chloride is 16.5-17.4mg/mL, and relative standard deviation is 2.7%.When cooling, 0.9% sodium chloride, the scope of three repeated trials is 1.1-1.3mg/mL, and relative standard deviation is 8.3%.
Equilibrium relation between solid and the solution state DHEA-S is as follows:
K=[DHEA-S
-] [Na
+]/[NaDHEA-S]
Solid
Because the constant concentration (that is, the dihydrate of physically stable) of solid DHEA-S, this flat relation weighing apparatus can be reduced to:
Ksp＝[DHEA-S
-][Na
+]
Based on this hypothesis, mapping is linear and slope equals Ksp to the DHEA-S dissolubility to the inverse of total sodium cation concentration.Equilibrium relation under room temperature and the cooling situation is shown in Fig. 5 and Fig. 6 respectively.Based on correlation coefficient, the data when this model and room temperature and chilling temperature can both rationally be coincide, and equilibrium constant is respectively 2236 and 665mM
2In order to make the dissolubility maximum, the level of sodium chloride should be low as far as possible.The minimum halogen ion concentration of aerosol apparatus solution should be 20mM or 0.12% sodium chloride.
Be the DHEA-S concentration of assessment solution, take to make during use 10 ℃ (promptly 15 ℃) of temperature decline of aerosol apparatus.Insertion value between equilibrium constant and the absolute temperature inverse, the Ksp in the time of 15 ℃ is about 1316mM
2Every mole DHEA-s provides 1 mole sodium cation to this solution, therefore:
Ksp＝[DHEA-S][Na
+][DHEA-S
-][Na
++DHEA-S
-]
＝[DHEA-S
-]
2+[Na
+][DHEA-s
-]
Calculate [DHEA-S with quadratic equation
-].Ksp is 1316mM
220mM Na
+Solution is the DHEA-S of 27.5mM
-Or 10.7mg/mL.Therefore, selecting 0.12% sodium chloride solution of 10mg/mL DHEA-S for use is that good candidate's preparation is with further test.The estimated value of this formula is also undeclared 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 value is to suck the acceptable pH level of preparation, assessed the effect of using the 20mM phosphate buffer.Buffer and non-buffer are shown in Fig. 7 in the solubility results of room temperature.Exist buffer to suppress dissolubility in the said preparation, when especially sodium chloride levels is low.As shown in Figure 8, the dissolubility data of buffer solution drops on the same balanced line of non-buffer solution.It is that sodium cation content by extra adding causes that the dissolubility that uses buffer to cause descends.Increasing dissolubility as far as possible is important target, but said preparation uses buffer to reduce dissolubility.In addition, Ishihora and Sugimoto ((1979), Drug.Dev.Indust.Pharm., 5 (3) 263-275) do not confirm that the stability of NaDHEA-S is significantly increased when neutral pH.
Stability study.
The DHEA-S preparation of preparation 10mg/mL is used for the stability of solution program research of short-term in 0.12% sodium chloride.This solution of equal portions added the vial settled accounts and respectively at room temperature (24-26 ℃) and 40 ℃ of preservations.Every day sample for reference DHEA-S content, DHEA content and outward appearance.On each time point, from each bottle, take out two duplicate samples and dilution.The content of DHEA-S is shown in Fig. 9 and 10 during this research.Under acceleration environment, solution demonstrate faster degradation rate and preserve occur two days later muddy.The solution of preserving under the room temperature was more stable, observed a little precipitation at the 3rd day.This research stopped at the 3rd day.As shown in figure 10, the decomposition of DHEA-S increases with DHEA content.Because DHEA is insoluble in water, only need just can produce on a small quantity muddy solution (quickening to preserve) or crystalline deposit (room temperature preservation) in this preparation.This just may be interpreted as what previous visually rank DHEA-S dissolubility substantially understate the dissolubility of this chemical compound: a small amount of DHEA will cause the experimenter to draw the conclusion that DHEA-S has surpassed solubility limit.It is stable that this solution should be easy to keep when clinical trial is prepared again.The aerosol properties of said preparation is described with the lower part.
Aerosol apparatus research.
Use Pari ProNeb Ultra compressor and LC Plus aerosol apparatus to make the DHEA-S atomizing.Illustrating of this assay device in Figure 11.5 milliliters of this solution aerosol apparatus of packing into continues spraying until ejecta fog (41/2 to 5 minute).Use has the solution of the Califonia Instruments AS-6 type 6 platform rams test atomizing of USP trunnion.Spray after one minute, collect sample 8 seconds to operate this ram in 30L/ minute.This experiment all At All Other Times in, with about 33L/ minute aerosol is introduced the bypass catcher.Clean gathering-device, aerosol apparatus and ram and analyze with mobile phase with HPLC.In aerosol apparatus, use 0.12% sodium chloride solution of 5mlDHEA-S.This volume is elected the used actual upper bound of clinical research as.Preceding 5 result of the tests are listed in the table below.
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 ram, mg | Total amount, 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 test the liquid of from aerosol apparatus, pouring out, the weight of the aerosolized front and back of not weighing or cleaning whole unit
Operation aerosol apparatus # 1 made it dry in about 5 minutes, and aerosol apparatus # 2 operation was slightly less than about 4.5 minutes.In each case, the liquid volume that residues in aerosol apparatus approximately is 2ml.After taking out from aerosol apparatus, this liquid is muddy at first, becomes clarification then in 3-5 minute.Should also demonstrate a small amount of rough deposit in the solution of 10mg/mL after the time even crossed.Seem it to be that bubble tiny in this liquid causes initial muddiness.DHEA-S shows surface activity (that is, can promote foaming), and this has stablized the bubble in this liquid.Deposit in the 10mg/mL solution shows at the dissolubility of aerosol apparatus environment Chinese medicine higher.Therefore, the spraying of other shown in the table 13 experiment is carried out under low concentration.Table 13 has provided " dosage " linear other data to solution concentration.
Table 13: other spraying experimental result of using DHEA-S to carry 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 ram, mg | Total amount, 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 |
Aerosol apparatus #3 drying was slightly less than 4.5 minutes.Quality in the bypass catcher is to the Figure 12 that is illustrated in of initial solution concentration work.Have favorable linearity from 0 to 7.5mg/mL, the amount of Shou Jiing begins to flatten afterwards.To the influence that dissolubility reduces, ignored the influence of concentration though also considered cooling in the calculating of 10mg/mL solution to medicine and sodium chloride content.Therefore, may be spray liquid supersaturation and formed precipitate.Data shown in Figure 12 and spraying back more observed granules in 10mg/mL solution show that the highest solution concentration as concept nature clinical trial preparation evidence is 7.5mg/mL approximately.Aerosol sample introduced carry out granularmetric analysis in the cascade ram.No detectable trend between particle size distribution and solution concentration or the aerosol apparatus number.Average particle size distribution in all spraying experiments is shown in Figure 13.Aerosol particle diameter measured value and this aerosol apparatus deliver/advertisement result consistent (that is the about 2 μ m of median diameter).Though experiment in vitro proof nebulizer formulation can be sent respirable DHEA-S aerosol, said preparation is unstable and need 4-5 minute continuous atomizing.Therefore, stable DPI preparation has significant advantage.The DHEA-S dihydrate becomes through evaluation and is used for the most stable solid-state of DPI preparation.With regard to 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 of the said preparation of no buffer system also can accept.Farthest increase the water solubility of DHEA-S by reducing sodium cation concentration as far as possible.The minimum sodium chloride levels of no buffer can realize this target.This is not produce the sedimentary 20mM of using C1 in atomization process
-The highest drug level that obtains.Said preparation can be kept at least in room temperature and stablize 1 day.
Embodiment 9: the preparation of experimental model
Obtain cell culture, HT-29 SF cell, this is that (subbreed Md.) can adapt in complete synthetic serum-free PC-1 culture medium (Ventrex, Portland, Me.) interior growth the HY-29 cell for ATCC, Rockville.Original seed is cultivated and is maintained at this culture medium (containing in the wet air of 5%CO2) at 37 ℃.When cell is paved with, culture with pancreatin/EDTA (Gibco, Grand Island, N.Y.) dissociate after inoculation once more, added a subculture in per 24 hours.Under this condition, the HT-27 SF cell of exponential phase times the time be 24 hours.
Flow cytometry
With cell with 10
5/ 60-mm is seeded in the culture dish, and is duplicate.In cell culture, add 0,25,50 or 200 μ M DHEA with the analysis of cells period profile.Culture contacts with 0 or 25 μ M DHEA analyzes the influence that the DHEA cell cycle reverses, and is added with MVA, CH, RN, MVA in the culture medium and adds CH or MVA and add CH and add RN or do not add.Culture after 0,24,48,74 hour with trypsinization, with Bauer etc., Cancer Res., 46,3173-3178 (1986) is described to improve one's methods fixing and dyeing.In brief, centrifugal collecting cell is resuspended in cold phosphate buffered saline (PBS).Cell is fixed with 70% ethanol, and washing is resuspended in phosphate buffered saline (PBS).Add the hypotonic dyeing liquor of 1ml then and (contain 50 μ g/ml propidium iodides (Sigma Chemical Co.), 20 μ g/ml RNA enzyme A (BoehringerMannheim, Indianapolis, Ind.), 30 μ g/ml Polyethylene Glycol, the 5mM citrate buffer of 0.1%Triton X-100), place under the room temperature and add 1ml etc. after 10 minutes and ooze dyeing liquor and (contain propidium iodide, Polyethylene Glycol, the 0.4M sodium chloride solution of Triton X-100), with being furnished with two flow cytometer (the Becton Dickinson Immunocytometry Systems that differentiate of pulse width/pulse area, San Jose CA) analyzes.Fluorescence beam is proofreaied and correct the minimum analysis 2 * 10 in back
4The sample of individual cell/sample, data are expressed as 1024 fluorescence intensities and strengthen the total cellular score in each passage in the passage, and the rectangular histogram of gained is analyzed with Cellfit analysis programme (Becton Dickinson).
The influence of DHEA cell growth
Cell is seeded in the culture dish with 25000 cell/30-mm, and is quadruplicate, 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) measures cell number.DHEA (AKZO, Basel, Switzerland) is dissolved in the dimethyl sulfoxide, filtration sterilization, be stored in-20 ℃ standby.
Figure 14 has shown the inhibition of DHEA to the growth of HT-29 cell.Each point is represented cell number, and the bar thick stick is represented SEM.Each data point repeats 4 times, the test triplicate.The unconspicuous local explanation SEM in-less-than symbol of SEM bar thick stick.Compare with matched group, contact 12.5 μ M DHEA72 hour, contact 25 or 50 μ M DHEA48 hour, 200 μ M cell number reduction after DHEA24 hour, illustrate that DHEA has produced the inhibition of time and dose dependent to growth.
The effect of DHEA cell cycle
For the influence of checking the DHEA cell cycle to distribute, inoculation HT-29 SF cell (10
5Individual cell/60mm culture dish), handle with 0,25,50 or 200 μ M DHEA after 48 hours.Figure 15 shows the influence of DHEA to the cell cycle distribution of HT-29 SF cell.24, harvesting after 48 and 72 hours, ethanol is fixed, and propidium iodide dyeing is measured dna content/cell with flow cytometry analysis.Calculate G with Cellfit cell cycle analysis program
1, S and G
2The percent of M phase cell.The S phase with tetragonal labelling to show difference.The representative rectangular histogram that has shown duplicate mensuration gained.This tests triplicate.
Do not change after initial 24 hours with the cell cycle distribution in the cell culture of 25 or 50 μ M DHEA processing.Yet along with the contact DHEA time increases, S phase cell proportion reduces gradually, has calculated with Cellfit cell cycle analysis program to be in G
1, S and G
2The percentage of cells of M phase.The S phase with tetragonal labelling to show difference.The representative rectangular histogram that has shown duplicate mensuration gained.This tests triplicate.
Do not change after initial 24 hours with the cell cycle distribution in the cell culture of 25 or 50 μ M DHEA processing.Yet along with the contact DHEA time increases, S phase cell proportion reduces gradually, and G
1The phase percentage of cells increased after 72 hours.G
2M phase cell obviously had of short duration increase after 48 hours.The result who contacts gained with 200 μ M DHEA is similar, but G after 24 hours
1Phase cell percentage ratio increases fast, and S phase cell proportion reduces, and this situation continues to betide during the entire process.This shows that DHEA produces G with time and dosage relying party formula in the HT-29-SF cell
1The phase blocking-up.
The cell growth of embodiment 10:DHEA mediation and the reverse of cell cycle effect
The growth inhibiting reverse of DHEA mediation.
Inoculating cell adds the culture medium that contains 0 or 25 μ MDHEA after 2 days as stated above, adds mevalonic acid (" MVA " in this culture medium; MM), zamene (SQ; 80 μ M), cholesterol (CH; 15 μ g/ml), MVA+CH, ribonucleotide (RN; Final concentration respectively is uridnine, cytidine, adenosine and the guanosine of 30 μ M), dezyribonucleoside (DN; Final concentration respectively is thymidine, deoxycytidine, deoxyadenosine and the deoxyguanosine of 20 μ M), RN+DN, MVA+CH+RN or the culture medium of adding ingredient not.All chemical compounds all derive from Sigma Chemical Co (St.Louis, Mo.).Cholesterol is with the preceding ethanol that is dissolved in immediately.RN and DN are the not influences of growth of their pair cells when showing no DHEA with the Cmax use.
Figure 16 shows the cytostatic reverse of the inductive HT-29 SF of DHEA.In A, added 2 μ M MVA, 80 μ MSQ, 15 μ g/ml CH or MVA+CH in the culture medium or do not made to add (CON).In B, culture medium has been added the mixture (uridnine, cytidine, adenosine and the guanosine that contain 30 μ M) of RN; The perhaps mixture of DN (thymidine, deoxycytidine, deoxyadenosine and the deoxyguanosine that contain 20 μ M); RN adds DN (RN+DN); Or MVA adds CH and adds RN (MVA+CH+RN).Assessed before and after handling 48 hours cell number, the value added of cell number during the growth of cell is calculated as and handles 48 hours.The bar cylindricality is represented the cell growth percent of untreated control; The bar thick stick is represented SEM.Cell number in the untreated control has increased by 173,370 " 6518.Each data point has represented four of four independent trialss to repeat culture dish.Use Si Shi t test to carry out statistical analysis, than matched group, κ p＜0.01; ψ p＜0.001.When noting no DHEA, the growth of substance pair cell does not almost have influence.
Under these conditions, the inductive growth inhibited of DHEA can partly overcome by adding MVA or MVA+CH.Add SQ or CH separately and do not have this effect.This shows that the cell inhibitory activity of DHEA partly is that consumption by the endogenous mevalonic acid and the biosynthesis that suppresses early stage intermediate product in the cholesterol approach mediate.In addition, find that growth obtains part and recovers after adding RN and adding RN+DN, but do not have this effect after adding DN, show that the consumption in mevalonic acid and nucleotide storehouse relates to the growth inhibited effect of DHEA.But do not have which kind of recovery condition can overcome (comprise uniting and add MVA, CH and RN) the growth inhibited effect of DHEA fully, show that DHEA has cytotoxic effect or may relate to other biochemical route.
The reverse of DHEA cell cycle effect
With 25FM DHEA and many other chemical compounds, handle HT-29 SF cell as MVA, CH or RN and can prevent the cell cycle specific effect of DHEA to test them.After handling 48 and 72 hours, pass through the flow cytometry cell cycle distribution.
Figure 17 shows the reverse result that the inductive HT-29 SF of DHEA cell is stagnated.Inoculating cell (10
5Individual cell/60mm culture dish), handle with 0 or 25 μ M DHEA after 48 hours.Culture medium is added with 2FMMVA; 15Fg/ml CH; The RN mixture that contains uridnine, cytidine, adenosine and uridnine that final concentration is 30 μ M; MVA adds CH (MVA+CH); Or MVA adds CH and adds RN (MVA+CH+RN) or do not add any material.Collecting cell after 48 or 72 hours, ethanol is fixed, propidium iodide dyeing, the dna content of each cell of cells were tested by flow cytometry.Calculate G with Cellfit cell cycle analysis program
1, S and G
2The percent of M phase cell.The S phase with tetragonal labelling to show difference.The typical rectangular histogram that has shown duplicate mensuration gained.This experiment repeats 2 times.Notice that the substance cell cycle is made progress during no DHEA does not almost have influence.
Along with increase time of contact, DHEA reduces the ratio of S phase cell gradually.Can partly stop this effect in initial 48 hours behind the adding MVA, but can not stop this effect after 72 hours, also can partly stop the S phase to exhaust in 72 hours that add MVA+CH, illustrate that at the period of contact MVA and the CH that increase all be that cell cycle progression is required.Add MVA, CH and RN obviously to recovering the most effective, still still can't return to the S phase cell percentage ratio of untreated control culture.Add separately CH or RN and a little effect is arranged and no effect in 72 hours 48.From morphology, cell is to become circle to the reaction of DHEA, and this variation only needs to add MVA and just can stop in culture medium.Some DNA rectangular histogram after contact DHEA72 shown in Figure 4 hour also shows the cell subsets that has the obvious minimizing of dna content.Because known HT-29 cell line is to contain the chromosomal cell mass (68-72 of different numbers; ATCC), therefore above-mentioned cell has represented one to contain the separative less chromosomal cell subsets that carries.
Conclusion
The embodiment 9-10 proof that provides above is external, and HT-29 SF human colon adenocarcinoma cell contacts with the known DHEA that consumes the various concentration of endogenous mevalonic acid can cause growth inhibited and G
1Stagnate, can partly stop these effects and in culture medium, add MVA.DHEA produced to the effect of protein prenylation in many aspects with specificity 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor, similar as lovastatin and the viewed effect in his spit of fland of health piperazine.Yet different with the biosynthetic direct inhibitor of mevalonic acid, DHEA has mediated the effect that its cell cycle is made progress and cell is grown in the pleiotropy mode, relates to the biosynthesis of ribonucleotide or deoxyribonucleotide, also may relate to other factors.
Embodiment 11: quantitative inhaler
| Active component | The target that at every turn excites |
| Salmaterol DHEA stabilizing agent Arcton 11 dichlorodifluoromethane | 25.0μg400mg5.0μg23.70mg61.25mg |
Embodiment 12: quantitative inhaler
| Active component | The target that at every turn excites |
| Salmaterol (as Hydroxynaphthoate) DHEA-S stabilizing agent Arcton 11 dichlorodifluoromethane | 25.0μg400mg7.5μg23.67mg61.25mg |
Embodiment 13: quantitative inhaler
| Active component | The target that at every turn excites |
| Formoterol fumarate DHEA stabilizing agent Arcton 11 dichlorodifluoromethane | 12.0μg400.0mg15.0μg23.56mg61.25mg |
Embodiment 14: quantitative inhaler
| Active component | The target that at every turn excites |
| Formoterol fumarate DHEA-S stabilizing agent Arcton 11 dichlorodifluoromethane | 12.0gg400.0mg15.0gg23.56mg61.25mg |
Among the embodiment 15-18 below, first and second activating agents are by micronization, and mix with lactose is in bulk according to the above ratio.This mixture be filled in hard gelatin capsule or the cartridge case or the special two paper tinsel blister packages of packing in (by inhaler, as the Rotahaler inhaler (
) use the Rotadisks blister package,
, or with the Diskhaler inhaler (
) use blister package).
Embodiment 15: quantitative dry powder formulations
| Active component | / cartridge case or bubble-cap |
| Salmaterol (hydroxynaphthoic acid salt) DHEA lactose Ph.Eur. | 72.5 μ g1.00mg to 12.5 or 25.0 mg |
Embodiment 16: quantitative dry powder formulations
| Active component | / cartridge case or bubble-cap |
| Salmaterol (hydroxynaphthoic acid salt) DHEA-S lactose Ph.Eur. | 12.5 μ g1mg to 12.5 or 25.0 mg |
Embodiment 17: quantitative dry powder formulations
| Active component | / cartridge case or bubble-cap |
| Salmaterol (hydroxynaphthoic acid salt) DHEA lactose Ph.Eur. | 72.5pg1mg to 12.5 or 25.0mg |
Embodiment 18: quantitative dry powder formulations
| Active component | / cartridge case or bubble-cap |
| Salmaterol (hydroxynaphthoic acid salt) DHEA-S lactose Ph.Eur. | 72.5 μ g1mg to 12.5 or 25.0mg |
Embodiment 19:cAMP is to the effect of Human airway smooth muscle's cell function of asthmatic patient
In following research, assessed DHEA-S and how to have improved the effect that medicine suppresses the growth of people ASM cell by cAMP.This studies selected mitogenesis former (EGF) at asthmatic patient or suffer to have increased in the bronchoalveolar lavage fluid (BAL) of asthmatic patient of anaphylactogen invasion and attack.Concentration in the BAL liquid that employed concentration and these research obtain is approaching.
People ASM cell derives from the far-end trachea in accordance with the graft donor of the examination board of directors of association (University ofPennsylvania ' s Institutional Review Board) of the University of Pennsylvania.Dissecting the far-end trachea removes connective tissue and produces about 1 * 10 with enzymic digestion
4Individual ASM cell.These cells are grown to converge in 10% hyclone and be incorporated in the serum-free medium that contains 1% bovine serum albumin static 24 hours.Subsequently, cell and diluent or EGF (1ng/ml) were hatched 24 hours.After tentatively hatching, make cell with [
3H]-thymus pyrimidine contact and the DNA that uses the thymus pyrimidine method of mixing to measure in 24 hours are synthetic.Scrape cell and place on the filter membrane and use the β rolling counters forward.All experiment personnel selection ASM cell lines are triplicate at least.The all conditions of each experiment repeats six times.Data are expressed as mean+/-standard error, carry out statistical analysis with Bonferroni-Dunn verification and ANOVA, and P＜0.05 shows that difference is meaningful.
In some experiments, cell and specific somatomedin and 10 μ M DHEA-S are hatched.It is synthetic to measure DNA with the thymus pyrimidine method of mixing as mentioned above.The synthetic concentration of DNA of calculating inhibition 50% growth factor-induced is as IC
50
Also detected DHEA-S and regulated cAMP concentration the mitotic effect of ASM.Known cAMP is the important medium of smooth muscle proliferation second signal pathway.The normally used anti-asthmatic medicament that can change cAMP concentration and/or adenosine concentration has three classes.The medicine of these types is methylxanthine, beta-2-agonists and phosphodiesterase inhibitor.With regard to every kind of these drug type, isobutyl methylxanthine (IBMX) suppresses smooth muscle proliferation as the tool compound.In these experiments, IC
50The DHEA-S of concentration adds in the cell of handling with IBMX simultaneously and detects the synthetic effect of its DNA to growth factor-induced.
Figure 18 EGF, IBMX have been described and DHEA-S right
3The H-thymus pyrimidine mixes the effect in human smooth muscle's culture.These data show are single all can not to suppress smooth muscle proliferation with IBMX or DHEA-S or coupling.And the EGF of 1ng/ml concentration stimulates
3Mixing of H thymus pyrimidine increases about 10 times.
Whether this serial experiment has been studied singly can suppress the propagation that EGF stimulates with DHEA-S with the IBMX coupling.As can be seen from Figure 19, the DHEA-S of 10 μ M concentration reduces ASM and breeds about 11% (relatively second and the 3rd bar post).IBMX (0.1-100 μ M) mixes thymus pyrimidine has not same-action (bar post 8-11).Yet, when 10 μ M DHEA-S, repeating the research, concentration dependent form (the bar post 4-7) smooth muscle proliferation that IBMX causes IC50 to be about 10 μ M suppresses.
These data show that coupling DHEA-S and IBMX have stronger inhibition smooth muscle proliferation effect than single with arbitrary medicine.IBMX assesses the effect of cAMP as the tool compound.Therefore, these experiments show the medicine that can regulate cAMP, and for example beta-agonists can be treated respiratory tract disease with the DHEA-S coupling.Do not want to be subject to any mechanism of action, expectation to the inhibitory action of smooth muscle proliferation can prevent in wait until the Airway Remodeling remodeling that arrives seen in the serious asthmatic patient.These results show coupling DHEA-S and cAMP regulator, and for example beta-agonists can reduce airway smooth muscle and reinvent remodeling.
Embodiment 20:DHEA-S and Aerolin are to the effect of histamine release
Model 1: rat peritoneum mastocyte
The rat peritoneum mastocyte (2 * 10 of fresh separated
5Individual cell) preincubate 5 minutes in 37 ℃ of balanced salt solutions that containing 150mMNaCl (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.Then, add DHEA-S, DHEA-S and Aerolin or water (contrast) and mixture hatched 2 minutes in 37 ℃.
After hatching mixture is chilled to 4 ℃, 4500rpm is centrifugal 5 minutes then.Collect supernatant and mix 30 minutes precipitating proteins with 5%TCA (trichloroacetic acid) in 4 ℃.9500 * g collects supernatant and mixes with 0.25M HCl after centrifugal 15 minutes.With sample and 2M NaOH and mixed 4 ℃ of wall light cultivations 30 minutes that are incorporated in of 0.2%OPT (phthalic aldehyde (ortho-phtalaldehyde)), add 0.5M H then
2SO
4With cessation reaction.
Come the excretory histamine amount of quantitative assay with fluorophotometer (GeminiXS, Molecular Devices) at λ ex=360nm and λ em=450nm place mensuration fluorescence intensity.The result represents to suppress the excretory percentage rate of contrast histamine.
Having studied concentration is the Aerolin of 1,10 and 100 μ M, and this scope is known have been comprised and the bonded EC of beta 2 adrenoreceptor
50These concentration are not to compound 48/80-inductive histamine release or faint inhibition (seeing table) arranged.The DHEA-S of 30 μ M has suppressed 46% mastocyte release.When 30 μ MDHEA-S and 10 or during the coupling of 100pM Aerolin, observed than single coordinate repression with any medicine high 93%.
Because beta-2-agonists confirmed in the treatment respiratory tract disease effectively, based on above discovery as can be known coupling DHEA-S with beta-2-agonists can double or cooperative mode reduction autopath in the harm of histamine release.Therefore, the patient will benefit from the coupling of these two kinds of medicines.
Except that sulphuric acid DHEA, other suitable non-glucocorticoid steroid also can be used as first activating agent, includes, but is not limited to epiandrosterone and derivant thereof, analog and pharmaceutically acceptable salt.For example, the described chemical compound of the formula I of this paper, III and IV.
Though described the present invention in conjunction with the preferred embodiment that is provided, it should be understood that under the situation that does not break away from the present invention's design and can make various improvement.
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Claims (19)
1. pharmaceutical composition, it comprises pharmacy or veterinarily acceptable carrier, first activating agent that can effectively treat asthma, chronic obstructive pulmonary disease or respiratory tract disease or pneumonopathy and second activating agent,
(a) described first activating agent is to be selected from the non-glucocorticoid steroid with following chemical formula:
At least a with in the non-glucocorticoid steroid of following chemical formula;
Or its pharmacy or veterinarily acceptable salt;
In the formula, R1, R2, R3, R4, R5, R7, R8, R9, R10, R12, R13, R14 and R19 respectively do for oneself H, OR, halogen, (C1-C10) alkyl or (C1-C10) alkoxyl; R5 and R11 respectively do for oneself 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, volution oxidative ethane, volution sulfur ethane ,-OSO2R20 ,-OPOR20R21 or (C1-C10) alkyl, R5 and R6 combine and are=O; R10 and R11 combine and are=O; 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) alkenyl, (C1-C10) alkynyl, formoxyl, (C1-C10) alkanoyl or epoxy radicals, (4) are OR when R16 is H, 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 combine and are=O; R17 and R18 respectively do for oneself (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 combine to=O, be H, (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) carbalkoxy or (C1-C10) alkanoyloxy, (3) R17 and R18 combine and are=O; (4) carbon that connected of R17 or R18 and they combines and forms the 3-6 unit ring that contains 0 or 1 oxygen atom; Or (5) R15 forms an epoxide ring with R17 with the carbon that they were connected; R20 and R21 respectively do for oneself OH, pharmaceutically acceptable ester or pharmaceutically acceptable ether; R22 is H, (halogen) m (C 1-C10) alkyl or (C1-C10) alkyl; N is 0,1 or 2; M is 1,2 or 3; With
(b) described second activating agent is a β 2-agonist bronchodilatation medicine.
2. pharmaceutical composition as claimed in claim 1 is characterized in that, described first activating agent is the non-glucocorticoid steroid with following chemical formula
In the formula, dotted line is represented singly-bound or two key; R is a hydrogen or halogen; 5 H exists with α or beta comfiguration, and perhaps chemical compound shown in the Formula I comprises the racemic mixture of these 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 steroid with chemical formula (I), and wherein, described multivalence organic dicarboxylic acid is SO
2OM, phosphate or carbonate, wherein M comprises equilibrium ion, and described 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
Or phospholipid
R that can be identical or different wherein
2And R
3Be straight or branched (C
1-C
14) alkyl or glucosiduronic acid
4. pharmaceutical composition as claimed in claim 2 is characterized in that, described first activating agent is a dehydroepiandrosterone.
5. pharmaceutical composition as claimed in claim 2 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 β 2-agonist bronchodilatation medicine is salmaterol or formoterol.
7. pharmaceutical composition as claimed in claim 1 also includes ubiquinone or its pharmacy or veterinarily acceptable salt, and wherein said ubiquinone has following chemical formula
Wherein, n is 1-12.
8. pharmaceutical composition as claimed in claim 1 is characterized in that, described pharmaceutical composition includes big I and sucks or respirable granule.
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. one kind is equipped with the delivery apparatus and the test kit of pharmaceutical composition according to claim 1.
12. test kit as claimed in claim 11 is characterized in that, described delivery apparatus is aerosol generator or spray generator.
13. test kit as claimed in claim 11 is characterized in that, described aerosol generator comprises inhaler.
14. test kit as claimed in claim 13 is characterized in that, described inhaler can be sent the preparation of single predetermined close.
15. test kit as claimed in claim 13 is characterized in that, described inhaler comprises aerosol apparatus or insufflator.
16. one kind is reduced the method that study subject is suffered from asthma probability or treatment asthma, described method comprises pharmaceutical composition as claimed in claim 1 from treatment effective dose to the study subject of this treatment of needs that use prevention or.
17. one kind is reduced the method that study subject is suffered from chronic obstructive pulmonary disease probability or treatment chronic obstructive pulmonary disease, described method comprises pharmaceutical composition as claimed in claim 1 from treatment effective dose to the study subject of this treatment of needs that use prevention or.
18. respiratory tract, lung or malignant disease or disease for the treatment of study subject, or reduce adenosine or adenosine receptor level or to the method for adenosine or adenosine receptor sensitivity, described method comprises pharmaceutical composition as claimed in claim 1 from treatment effective dose to the study subject of this treatment of needs that uses prevention or.
19. method as claimed in claim 18, it is characterized in that described disease or disease comprise asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), out of breath, emphysema, stridulate, pulmonary hypertension, pulmonary fibrosis, the air flue hyperreactive, adenosine or adenosine receptor level raise, the adenosine over anaphylaxis, infectious disease, the pulmonary branches trachea shrinks, respiratory inflammation or allergy, pulmonary surfactant or ubiquinone exhaust, chronic bronchitis, bronchoconstriction, dyspnea, lung airway obstacle or obstruction, the adenosine test of cardiac function is caused, the lung vasoconstriction, respiratory disorder, adult respiratory distress syndrome (ARDS), adenosine or adenosine level promote using of medicine, infant respiratory distress syndrome (baby RDS), pain, allergic rhinitis, cancer or chronic bronchitis.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US49223203P | 2003-07-31 | 2003-07-31 | |
| US60/492,232 | 2003-07-31 | ||
| US10/698,078 | 2003-10-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101137384A true CN101137384A (en) | 2008-03-05 |
Family
ID=39161042
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2004800262371A Pending CN101137384A (en) | 2003-07-31 | 2004-07-30 | Combination of dehydroepiandrosterone or dehydroepiandrosterone-sulfate with a beta-agonist bronchodilator for treatment of asthma or chronic obstructive pulmonary disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101137384A (en) |
-
2004
- 2004-07-30 CN CNA2004800262371A patent/CN101137384A/en active Pending
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