HK1156501B

HK1156501B - Respiratory disease treatment

Info

Publication number: HK1156501B
Application number: HK11110728.5A
Authority: HK
Inventors: Finch Harry; Fox Craig; Sajad Mohammed
Original assignee: Pulmagen Therapeutics (Inflammation) Limited
Priority date: 2008-08-07
Filing date: 2009-08-05
Publication date: 2015-10-09

Description

Treatment of respiratory diseases

The present invention relates to the use of the substantially pure 5R enantiomer of known glitazones (e.g. the known drugs pioglitazone and rosiglitazone) for the pulmonary administration by inhalation for the treatment of inflammatory respiratory diseases.

Background

A variety of respiratory diseases and conditions are well understood, many of which have cross-and inter-related etiologies. The most common of these diseasesTwo of the prevalent and prevalent types are Chronic Obstructive Pulmonary Disease (COPD) and asthma. Respiratory diseases have a significant inflammatory component. For example, current therapies for COPD and severe asthma focus primarily on the use of short-and long-acting bronchodilators, either as monotherapy or long-acting beta₂Combination therapy of the agonist bronchodilator with Inhaled Corticosteroids (ICS) to alleviate symptoms. For ICS alone or with beta₂The disappointing anti-inflammatory data of agonist combinations has led to an urgent need to find effective anti-inflammatory agents for COPD. It is clear that COPD is a chronic inflammatory disease involving complex interactions between the lungs and cells that may also be the systemic innate and adaptive immune response. One hypothesis that has been derived from intensive research is whether new corroborative anti-inflammatory agents can stop or slow COPD-specific hypofunction. Reducing the frequency and severity of exacerbations has become an increasingly important goal of COPD therapy because of the poor prognosis of patients after exacerbations. Anti-inflammatory therapies in COPD and asthma are expected to reduce the frequency and severity of exacerbations. Treatment is also expected to improve the decline in lung function and quality of life.

Therefore, new treatments for inflammatory respiratory diseases including the following are constantly being sought: asthma, COPD, allergic airway syndrome, bronchitis, cystic fibrosis, emphysema and pulmonary fibrosis (including idiopathic pulmonary fibrosis).

Peroxisome proliferator-activated receptor gamma receptor (PPAR γ) agonists are a class of drugs that increase the sensitivity of diabetic patients to glucose. It is believed that physiologically activated PPAR γ increases the sensitivity of peripheral tissues to insulin, thus facilitating glucose clearance from the blood and producing the desired anti-diabetic effect.

Many PPAR γ agonists are known from the patent and other literature, but currently there are only two approved clinical applications for diabetes, namely rosiglitazone and pioglitazone. See Campbell IW, Curr Mol Med.2005 May; 5(3): 349-63. Both of these compounds are thiazolidinediones ("TZDs" or "glitazones") which are administered in practice by the oral route for systemic delivery.

In addition to its effect on glucose metabolism, many reports have been published which indicate that rosiglitazone also exerts an anti-inflammatory effect. For example, (i) rosiglitazone has been reported to exert a consistent effect with anti-inflammatory effects in diabetic patients (Haffner et al, circulation.2002 Aug 6; 106 (6): 679-84; Marx et al, Arterioscler.Thromb.Vasc.biol.2003 Feb 1; 23 (2): 283-8); (ii) rosiglitazone has been reported to exert anti-inflammatory effects in a number of animal models of inflammation, including: carrageenan-induced paw swelling (Cuzzocrea et al, Eur. J. Pharmacol.2004Jan 1; 483 (1): 79-93), TNBS-induced colitis (Desreumanux et al, J. exp. Med.2001 Apr 2; 193 (7): 827-38; Sanchez-Hidalgo et al, biochem. Pharmacol.2005 Jun 15; 69 (12): 1733-44), experimental encephalomyelitis (Feinstein et al, Ann. Neurol.2002 Jun; 51 (6): 694-702), collagen-induced Arthritis (Cuzzocrea et al, Arthritis Rheum.2003 Decc; 48 (2004): 3544-56) and adjuvant-induced Arthritis (Eujirii et al, Eur. J. Pharmacol.2002; 448. Rheurael.19. Dec; 48 (2004): 3544-56), adjuvant-induced Arthritis (EB. J. Pharmacol. J. Res. No. 2; 19. 31; 19. J. No. 19, No. 23; Legione et al, 7; Legione; 7. 15; 7; Farlla. J. 15; Farlla. 7; Farlla. 7, 23; Farlla. J. 7; Falll.7; Falll.23; Falll.7, Falll.7; and 7; Falll.7; Falll. Inflammatory effects including iNOS expression in murine macrophages (Reddy et al, am. J. physiol. Lung cell. mol. physiol.2004 Mar; 286 (3): L613-9), TNF α -induced MMP-9 activity in human bronchial epithelial cells (Hetzel et al, Thorax.2003 Sep; 58 (9): 778-83), human airway smooth muscle cell proliferation (Ward et al, Br. J. Pharmacol. 2004Feb; 141 (3): 517-25) and MMP-9 release from neutrophils (WO 0062766-this is 2000?). PPAR γ agonists have also been shown to be effective in the following models: pulmonary fibrosis models (Milam et al, am.J.Physiol.Lung cell.mol.Physiol, 2008, 294 (5): L891-901) and pulmonary arterial hypertension models (Crossno et al, am.J.Physiol.Lung cell.mol.Physiol, 2007, 292 (4): L885-897).

Based on observations of anti-inflammatory activity in lung-associated cells, it has been shown that other PPAR γ agonists can be used for the treatment of inflammatory respiratory diseases, including asthma, COPD, cystic fibrosis and pulmonary fibrosis. See WO0053601, WO0213812 and WO 0062766. These studies suggest administration by both systemic oral and pulmonary inhalation routes.

Unfortunately, PPAR γ agonists also have deleterious cardiovascular effects, including haemodilution (haemodilution), peripheral and pulmonary oedema and Congestive Heart Failure (CHF). These effects are also thought to be caused by activation of PPAR γ. In particular, much effort has been devoted to the hypothesis that PPAR γ agonists prevent the normal maintenance of fluid balance by binding to the renal PPAR γ receptor. See Guan et al, nat. med.2005; 11(8): 861-6 and Zhang et al, Proc.Natl.Acad.Sci.USA.200528; 102(26): 9406-11. Treatment with PPAR γ agonists by the oral route of systemic delivery is also associated with an undue increase in body weight.

COPD patients are known to be at higher risk than other clinical populations of Congestive Heart Failure (CHF) (Curkendall et al, Ann epidemol, 2006; 16: 63-70; Padeletti M et al, Int J cardio.2008; 125 (2): 209-15), and it is therefore of paramount importance to keep systemic activation of PPAR γ receptors of these patients to a minimum to avoid the increased likelihood of CHF that will be observed. Administration of respiratory drugs by the inhalation route is one method of targeting anti-inflammatory drugs to the lung while keeping systemic exposure of the drug at a low point, thereby reducing the likelihood of systemic activity and observed side effects.

Pioglitazone has the following structural formula (I):

it may be named 5- {4- [2- (5-ethylpyridin-2-yl) ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione. The carbon atom at the 5-position of the thiazolidine-dione ring of pioglitazone (marked with an arrow in formula (I) above) is asymmetric and therefore pioglitazone has two enantiomers, the 5R and 5S enantiomers.

Rosiglitazone has the following structural formula (II) and can be designated 5- (4- {2- [ methyl (pyridin-2-yl) amino ] ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione the carbon atom at position 5 of the thiazolidine-dione ring of rosiglitazone (marked by the arrow in formula (II) below) is also asymmetric and therefore rosiglitazone also has two enantiomers, the 5R and 5S enantiomers.

The binding affinity of the 5S enantiomer of rosiglitazone to the PPAR γ receptor was higher than that of the 5R enantiomer (30nM to 2 μ M, Parks et al, 1998, bioorg. Med. chem. Lett.8 (24): 3657-8). As for the other member of the glitazone class, linaglitazone (Rivoglitazone), the 5S enantiomer also has a higher receptor binding affinity than the 5R enantiomer (see page 13 of WO 2007100027).

In practice, pioglitazone and rosiglitazone are administered by the oral route of systemic delivery as a mixture of the 5R and 5S enantiomers (1: 1 racemic mixture) to treat diabetes. It is known that the individual enantiomers of these compounds and members of the glitazone family generally rapidly reach equilibrium in vivo following oral administration (see, e.g., J.Clin.Pharmacol.2007, 47, 323-33; Rapid Commun.Mass Spectrum.2005, 19, 1125-9; J.Chromatograph, 835(2006), 40-46; Biopharmaceutics and Drug Disposition 1997, 18(4), 305-24; chem.Pharm.Bull 1984, 32, (11) 4460-65; T.J.Med.chem.1991, 34, 319-25), so there is virtually no difference between oral administration of substantially pure isomers and oral administration of racemic mixtures. In the case of pioglitazone in particular, no differences in activity have been noted in the submission to the U.S. Food and Drug Administration (FDA) after oral administration of the racemate or the individual enantiomers in a rodent diabetes model: (www.fda.gov/medwatch/SAFETY/2007/Sep PI/Actoplus Met PI.pdf)：

"(pioglitazone) contains one asymmetric carbon, and the compound is synthesized and used in a racemic mixture. The two enantiomers of pioglitazone interconvert in vivo. There is no difference in pharmacological activity between the two enantiomers ".

There appears to be no study of the effect of pulmonary inhalation of rosiglitazone or pioglitazone (or even any other glitazone) in either racemic or single enantiomeric form. Reports of possible equilibria of the 5R and 5S enantiomers of either compound or any other glitazone when in direct contact with lung tissue appear unpublished.

In general, the glitazones of PPAR γ agonists are characterized by the presence of a thiazolidine-2, 4-dione group (a) in the molecule, often as a constituent of a (thiazolidine-2, 4, dione-5-yl) methylphenyl group (B):

and the ring carbon atom indicated by the arrow is numbered at the 5-position of the thiazolidinone ring. The term "glitazone" as used herein refers to PPAR γ agonist compounds whose structure includes a thiazolidine-2, 4-dione group (a) or a (thiazolidine-2, 4, dione-5-yl) methylphenyl group (B).

In addition to the approved and marketed rosiglitazones and pioglitazones, there are a large number of glitazones known from the patent and scientific literature. Known examples include the following glitazones:

brief description of the invention

The present invention is based on the discovery that: that is, the 5R-enantiomer of glitazone is more effective than the 5S-enantiomer in the treatment of inflammatory respiratory diseases by inhalation. This principle is demonstrated from the treatment of inflammatory respiratory diseases in animal models by inhalation, where the 5R-enantiomer of pioglitazone and rosiglitazone has been shown to be active, while the 5S-enantiomer is essentially inactive. The results of this study lead to the conclusion that: inhaled pulmonary administration of the 5R-enantiomer of glitazone, in particular the 5R-enantiomer of pioglitazone or rosiglitazone, gives compounds with a more potent anti-inflammatory effect than that obtained with the same administration of the racemate, while having the benefit of lower systemic exposure and reduced side effects compared to oral administration.

Brief Description of Drawings

FIG. 1 is a bar graph showing the effect of intranasal administration of vehicle (0.2% Tween 80 in saline), 5S-pioglitazone (1. mu.g/kg or 3. mu.g/kg), 5R-pioglitazone (1. mu.g/kg or 3. mu.g/kg) or racemic pioglitazone (3. mu.g/kg) to the number of BAL cells induced by tobacco smoke 24 hours after final exposure.

FIG. 2 is a bar graph showing the effect of intranasal administration of vehicle (0.2% Tween 80 in saline), 5S-pioglitazone (1. mu.g/kg or 3. mu.g/kg), 5R-pioglitazone (1. mu.g/kg or 3. mu.g/kg) or racemic pioglitazone (3. mu.g/kg) to BAL neutrophil counts induced by tobacco smoke 24 hours after final exposure.

FIG. 3 is a bar graph showing the effect of intranasal administration of vehicle (0.2% Tween 80 in saline), 5S-rosiglitazone (3. mu.g/kg or 10. mu.g/kg), 5R-rosiglitazone (3. mu.g/kg or 10. mu.g/kg) or racemic rosiglitazone (10. mu.g/kg) to the number of tobacco smoke-induced BAL cells 24 hours after final exposure.

FIG. 4 is a bar graph showing the effect of intranasal administration of vehicle (0.2% Tween 80 salt solution), 5S-rosiglitazone (3. mu.g/kg or 10. mu.g/kg), 5R-rosiglitazone (3. mu.g/kg or 10. mu.g/kg) or racemic rosiglitazone (10. mu.g/kg) to the number of tobacco smoke-induced BAL neutrophils 24 hours after final exposure.

Detailed Description

The term "glitazone" as used herein has the above meaning, i.e. a PPAR γ agonist compound whose structure includes a thiazolidine-2, 4-dione group (a) or a (thiazolidine-2, 4, dione-5-yl) methylphenyl group (B).

The term "pioglitazone" or "pioglitazone component" as used herein refers to the compound 5- {4- [2- (5-ethylpyridin-2-yl) ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione of structural formula (I) above or a pharmaceutically acceptable salt thereof.

The term "rosiglitazone" or "rosiglitazone component" as used herein refers to the compound 5- (4- {2- [ methyl (pyridin-2-yl) amino ] ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione of structural formula (II) above or a pharmaceutically acceptable salt thereof.

The term "enantiomeric excess" or its abbreviation "e.e." as used herein is defined as the following percentage:

((R-S)/(R+S))x100％

wherein R and S are the respective weight fractions of the R enantiomer and the S enantiomer in the sample. Thus for the glitazone sample containing 95% by weight of the 5R-enantiomer and 5% of the 5S-enantiomer, the enantiomeric excess of the R enantiomer over the S enantiomer was ((95-5)/(95+5)) x100 ═ 90%.

In one aspect, the present invention provides a pharmaceutical composition suitable for pulmonary administration by inhalation, said composition comprising a glitazone, in particular pioglitazone or rosiglitazone, and one or more pharmaceutically acceptable carriers and/or excipients, wherein the glitazone content of the composition consists of at least 95% by weight of the 5R-enantiomer and less than 5% of the 5S-enantiomer.

In another aspect, the invention provides a glitazone (e.g. pioglitazone or rosiglitazone) for use in the treatment of inflammatory respiratory disease by pulmonary administration by inhalation, wherein the inhaled glitazone consists of at least 95% by weight of the 5R-enantiomer and less than 5% of the 5S-enantiomer.

In a further aspect, the invention provides the use of a glitazone (e.g. pioglitazone or rosiglitazone) in the manufacture of a medicament for pulmonary administration by inhalation for the treatment of inflammatory respiratory diseases wherein the glitazone content of the medicament consists of at least 95% by weight of the 5R-enantiomer and less than 5% of the 5S-enantiomer.

In yet another aspect, the invention provides a method of treating an inflammatory respiratory disease, the method comprising pulmonary administration by inhalation of a therapeutically effective amount of a glitazone (e.g., pioglitazone or rosiglitazone) to a patient suffering from such a disease, wherein the inhaled glitazone consists of at least 95% by weight of the 5R-enantiomer and less than 5% of the 5S-enantiomer.

In all aspects of the invention, the glitazone component (e.g. pioglitazone or rosiglitazone component) may be inhaled either nasally or orally. Preferably by oral inhalation.

In all aspects of the invention, the glitazone component (e.g. pioglitazone or rosiglitazone) should preferably contain as little 5S-enantiomer as possible. For example, the 5R-enantiomer may constitute at least 97%, or at least 98%, or at least 99% by weight of the glitazone component.

In all aspects of the invention, the glitazone component (e.g. pioglitazone or rosiglitazone component) may be used concomitantly with, or sequentially or simultaneously with, one or more other therapeutic agents for the prevention and treatment of respiratory diseases other than PPAR γ agonists.

In all aspects of the invention, the presently most preferred glitazone component is pioglitazone.

In all aspects of the invention, wherein the inflammatory respiratory disease may be selected from, for example, mild asthma, moderate asthma, severe asthma, steroid-resistant asthma, bronchitis, Chronic Obstructive Pulmonary Disease (COPD), cystic fibrosis, pulmonary edema, pulmonary embolism, pneumonia, pulmonary sarcoidosis, silicosis, pulmonary fibrosis, respiratory failure, acute respiratory distress syndrome, emphysema, chronic bronchitis, tuberculosis and lung cancer.

The glitazone component may be in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable inorganic and organic acids and bases.

Pharmaceutically acceptable inorganic bases include metal ions. More preferred metal ions include, but are not limited to, suitable alkali metal salts, alkaline earth metal salts, and other physiologically acceptable metal ions. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese, manganous, potassium, sodium, zinc and the like and salts thereof of conventional valency. Exemplary salts include aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc salts. Particularly preferred are ammonium, calcium, magnesium, potassium and sodium salts.

Salts derived from pharmaceutically acceptable organic non-toxic bases, including salts of the following amines: primary, secondary and tertiary amines, including in part trimethylamine, diethylamine, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; substituted amines, including naturally occurring substituted amines; a cyclic amine; and quaternary ammonium cations. Examples of such bases include arginine, betaine, caffeine, choline, N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine (hydrabamine), isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

Exemplary pharmaceutically acceptable acid addition salts of the compounds of the present invention may be prepared from acids including, without limitation, formic, acetic, propionic, benzoic, succinic, glycolic, gluconic, lactic, maleic, malic, tartaric, citric, nitric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, hydrochloric, hydrobromic, hydroiodic, isocitric, xinafoic (xinajoic) acid, tartaric, trifluoroacetic, pamoic, propionic, anthranilic, methanesulfonic (mesylic) acid, naphthalenedisulfonic (napadisylate) acid, oxalacetic, oleic, stearic, salicylic, p-hydroxybenzoic, nicotinic, phenylacetic, mandelic, enbo' nic (pamoic), methanesulfonic, phosphoric, phosphonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, benzoic, succinic, glycolic, malic, fumaric, maleic, fumaric, succinic, fumaric, butyric, toluenesulfonic, fumaric, and mixtures thereof, Sulfanilic acid, sulfuric acid, salicylic acid, cyclohexylsulfamic acid, alginic acid (algenic acid), beta-hydroxybutyric acid, galactaric acid, and galacturonic acid. Exemplary pharmaceutically acceptable salts include salts of hydrochloric acid and hydrobromic acid.

The compositions of the invention are useful for treating inflammatory respiratory diseases, such as asthma (mild, moderate or severe), e.g. bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma, post-exercise asthma, drug-induced asthma (including aspirin-and NSAID-induced asthma) and dust-induced asthma, steroid-resistant asthma; bronchitis, including infectious bronchitis and eosinophilic bronchitis; chronic Obstructive Pulmonary Disease (COPD); cystic fibrosis; pulmonary fibrosis, including cryptogenic fibrotic alveolitis, idiopathic pulmonary fibrosis, idiopathic interstitial pneumonia, pulmonary fibrosis complicated by antitumor therapy, and chronic infections, including tuberculosis and aspergillosis, and other fungal infections; pulmonary transplant complications; vascular inflammatory and thrombotic disorders of the pulmonary vasculature, and pulmonary hypertension (including pulmonary arterial hypertension); antitussive activity including chronic cough and iatrogenic cough associated with treatment of airway inflammation and secretory disorders; acute and chronic rhinitis including rhinitis medicamentosa and vasomotor rhinitis; perennial and seasonal allergic rhinitis including rhinitis nervosa (hay fever); nasal polyposis; acute viral infections including the common cold and acute viral infections caused by respiratory syncytial virus, influenza virus, coronavirus (including SARS) and adenovirus, pulmonary edema, pulmonary embolism, pneumonia, pulmonary sarcoidosis, silicosis, farmer's lung and related diseases; allergic pneumonia, respiratory failure, acute respiratory distress syndrome, emphysema, chronic bronchitis, tuberculosis, and lung cancer. In particular, the methods and compositions of the present invention include the prevention and treatment of the respiratory disease, COPD.

The term "chronic obstructive pulmonary disease" or "COPD" as used herein refers to a group of physiological symptoms including chronic bronchitis, chronic cough, expectoration, labored dyspnea and significant progressive airway constriction/airflow reduction that may or may not be partially reversible. Emphysema may also be present in the lungs. COPD is a disease characterized by: progressive airway airflow limitation due to abnormal inflammatory response to chronic inhaled particles.

In patients with this disease, poor gas exchange in the lungs results in decreased oxygen levels, increased carbon dioxide levels and shortness of breath in the blood. Chronic airway obstruction in COPD is complicated by the loss of lung elasticity caused by enzyme destruction of the lung parenchyma. Rather than being a single pathological condition, COPD is a comprehensive term that includes chronic obstructive bronchitis and emphysema.

Compositions suitable for oral or nasal administration by inhalation are known and may include carriers and/or diluents known for use in such compositions. The composition may contain from 0.01 to 99% by weight of a pioglitazone or rosiglitazone component. Preferably, the unit dose contains from 1 μ g to 50mg of the pioglitazone or rosiglitazone component.

The most suitable dosage level may be determined by any suitable method known to those skilled in the art. It will be understood, however, that the specific amount employed in any particular patient will depend upon a variety of factors including the activity of the specific compound being employed, the age, body weight, diet, general health and sex of the patient, the time of administration, the route of administration, the rate of excretion, the use of any other drug, and the severity of the disease undergoing therapy. The optimum dosage can be determined by clinical trials necessary in the art.

The compositions of the invention may be used in combination with other therapeutic agents which may be useful in the treatment/prevention/inhibition or amelioration of the diseases or conditions for which the compounds of the invention are useful. Thus, such other therapeutic agents may be administered, by a route and in an amount conventionally employed, either simultaneously or sequentially with the glitazone component, particularly the pioglitazone or rosiglitazone component. If the compounds of the present invention are administered concurrently with one or more other therapeutic agents, a pharmaceutical composition containing such other therapeutic agents in addition to the pioglitazone component is preferred. Thus, the pharmaceutical compositions of the present invention include, in addition to the glitazone component, in particular the pioglitazone or rosiglitazone component, pharmaceutical compositions which additionally contain one or more other active ingredients.

Suitable therapeutic agents for use in combination therapy with the glitazone component/composition of the invention, in particular pioglitazone or rosiglitazone component/composition, include one or more other therapeutic agents selected from the group consisting of: anti-inflammatory agents, bronchodilators, expectorants, antitussives, leukotriene inhibitors and antibiotics.

Suitable therapeutic agents for use in combination therapy with the glitazone component/composition of the invention, in particular pioglitazone or rosiglitazone component/composition, include: (1) steroid drugs, such as corticosteroids, e.g. beclomethasone (e.g. as the monopropionate or dipropionate ester), flunisolide, fluticasone (e.g. as the propionate or furoate ester), ciclesonide, mometasone (e.g. as the furoate ester), mometasone desonide, rofleponide, hydrocortisone, prednisone, prednisolone, methylprednisolone, naftifine, deflazacort, haloprednisolone acetate, fluocinolone acetate, clocortolone, tipredane (tipre)dane), prednisone ester, alclometasone dipropionate, halometasone, rimexolone, delosolone propionate, triamcinolone, betamethasone, fludrocortisone, deoxycorticosterone, etoposide (etiprendnol dicloacetate), and the like. Steroid drugs may also include steroids developed clinically or preclinically for respiratory diseases, such as GW-685698, GW-799943, GSK870086, QAE397, NCX-1010, NCX-1020, NO-dexamethasone, PL-2146, NS-126 (formerly ST-126), and the compounds mentioned in the following international patent applications: WO0212265, WO0212266, WO02100879, WO03062259, WO03048181 and WO 03042229. Steroid drugs may additionally include the next generation of molecules under development with reduced side-effect profiles, such as selective glucocorticoid receptor agonists (SEGRA), including the compounds mentioned in ZK-216348 and International patent applications WO-00032585, WO-000210143, WO-2005034939, WO-2005003098, WO-2005035518 and WO-2005035502, as well as functional equivalents and functional derivatives thereof; (2) beta 2-adrenoceptor agonists, such as salbutamol, bambuterol, terbutaline, fenoterol, formoterol fumarate, salmeterol xinafoate, arformoterol tartrate (arfomoterol tartrate), indacaterol ((indacaterol) QAB-149), carmoterol (carmoterol), pirbuterol, BI 1744CL, GSK159797, GSK59790, GSK159802, GSK 246444, GSK678007, GSK96108, clenbuterol, procaterol, bitolterol and bromsalterol, TA-2005 and the compounds of the following patents or patent applications: EP1440966, JP05025045, WO/18007, WO/, US2002/, US2005/, WO/42193, WO/, WO 76933, WO/, WO 42160, WO/42164, WO/, WO 99764, WO/16578, WO/, WO 22547, WO/32921, WO/, WO 37773, WO/37807, WO 043979, WO/45618, WO/, EP1460064, WO/, EP01477167, US2004/, WO 108675, WO/108676, WO/, WO 071388, WO/, WO, WO05/065650, WO05/066140, WO05/070908, WO05/092840, WO05/092841, WO05/092860, WO05/092887, WO05/092861, WO05/090288, WO05/092087, WO05/080324, WO05/080313, US20050182091, US20050171147, WO05/092870, WO05/077361, DE10258695, WO05/111002, WO05/111005, WO05/110990, US2005/0272769, WO05/110359, WO05/121065, US2006/0019991, WO06/016245, WO06/014704, WO06/031556, WO06/032627, US2006/0106075, US2006/0106213, WO 0106213/363672, WO 0106213/0106213, WO 363672/363672; (3) leukotriene modulators, such as montelukast or pranlukast; (4) anticholinergics, e.g. selective muscarinic-3 (M3) receptor antagonists, e.g. ipratropium bromide, tiotropium (tiotropium), tiotropium bromide (Spiriva)) Glycopyrrolate, NVA237, LAS34273, GSK656398, GSK233705, GSK573719, LAS35201, QAT370 and oxytropium bromide; (5) phosphodiesterase-IV (PDE-IV) inhibitors, such as roflumilast or cilomilast; (6) antitussives such as codeine or dexramorphan; (7) non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen or ketoprofen; (8) expectorants, such as N-acetylcysteine or fudosteine (fudostein); (9) expectorant/mucokinetic (mucokinetic) modulators, such as ambroxol, hypertonic solutions (e.g. saline or mannitol) or surfactants; (10) peptide expectorants, such as recombinant human deoxyribonuclease I (alpha-strand enzyme and rhDNase) or spirodiclodin; (11) antibiotics such as azithromycin, tobramycin and aztreonam; and (12) p38MAP kinase inhibitors, such as GSK 856553 and GSK 681323.

In one aspect, the present invention provides a combination of a glitazone component of the present invention, in particular pioglitazone or rosiglitazone component, in combination with other anti-inflammatory and bronchodilatory drugs (i.e. a triple combination), for inhaled administration, including but not limited to salmeterol xinafoate/fluticasone propionate (Advair/Seretide)) Formoterol fumarate/budesonide (Symbicort)) Formoterol fumarate/mometasone furoate, formoterol fumarate/beclometasone dipropionate (Foster)) Formoterol fumarate/fluticasone propionate (FlutiForm)) Indacaterol/mometasone furoate, indacaterol/QAE-397, GSK159797/GSK 685698, GSK159802/GSK 685698, GSK642444/GSK 685698, formoterol fumarate/ciclesonide, and arformoterol tartrate/ciclesonide.

In a further aspect, the invention provides combinations of the glitazone component of the invention, in particular pioglitazone or rosiglitazone component, with other bronchodilator drug combinations, in particular B2 agonist/M3 antagonist combinations (i.e. triple combinations) including, but not limited to, salmeterol xinafoate/tiotropium bromide, formoterol fumarate/tiotropium bromide, BI 1744 CL/tiotropium bromide, indacaterol/NVA 237, indacaterol (indacterol)/QAT-370, formoterol/LAS 34273, GSK159797/GSK 573719, GSK159802/GSK573, GSK642444/GSK 573719, GSK159797/GSK 233705, GSK159802/GSK 233705, GSK642444/GSK 233705 and compounds of the same molecule which have both β 2 agonist and M3 antagonist activity (binary functionality), for example GSK 1081.

Thus in a further aspect the present invention provides a kit for use in the treatment of a respiratory disease in a patient, said kit comprising a first dosage form comprising a composition suitable for pulmonary administration by inhalation, said composition comprising glitazone, in particular pioglitazone or rosiglitazone, and one or more pharmaceutically acceptable carriers and/or excipients, wherein the glitazone content of the composition consists of at least 95% by weight of the 5R-enantiomer and less than 5% of the 5S-enantiomer; the kit further comprises a second dosage form of, for example, the other therapeutic agents described above, selected from the group consisting of anti-inflammatory agents, bronchodilators, expectorants, antitussives, leukotriene inhibitors and antibiotics.

For delivery by inhalation, the active compound is preferably in the form of microgranules. Microgranules can be prepared by a variety of techniques including spray drying, freeze drying, and micronization. After the pulverization to produce microparticles, the Particle Size Distribution (PSD) of the compound is examined and is often described in the art by determining the values of d10, d50, and d 90. The average particle diameter, i.e., the average equivalent diameter, is defined as 50% of the powder (particles) being larger than the equivalent diameter, and the other 50% being smaller than the equivalent diameter. Therefore, the average particle diameter is expressed as equivalent d 50. For inhalation applications, a d50 of less than 10 microns, preferably less than 5 microns, is desired.

For example, the compositions of the present invention may be formulated as a suspension for delivery by a nebulizer or as an aerosol in a liquid propellant, for example for a Pressurized Metered Dose Inhaler (PMDI). Suitable propellants for use in PMDI are known to those skilled in the art and include CFC-12, HFA-134a, HFA-227, HCFC-22 (CCl)₂F₂) And HFA-152 (CH)₄F₂And isobutane).

In a preferred embodiment of the invention, the composition of the invention is in the form of a dry powder for delivery using a Dry Powder Inhaler (DPI). Many DPI types are known.

Microparticles for delivery by inhalation may be formulated with excipients to aid delivery and release. For example, in a dry powder formulation, the microgranules may be formulated with large carrier particles that facilitate injection into the lung by DPI. Suitable carrier particles are known and include lactose particles; their mass median aerodynamic diameter may be greater than 90 μm.

The aerosol formation can be effected using, for example, a pressure-driven jet nebulizer or an ultrasonic nebulizer, preferably a propellant-driven metered aerosol, or by administering the propellant-free micronized active compound, for example from an inhalation capsule or other "dry powder" delivery system.

As mentioned above, the compositions may be metered in depending on the inhaler system used. In addition to the active compounds, the administration forms may additionally contain excipients, for example propellants (e.g. Frigen in the case of metered aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, fillers (e.g. lactose in the case of powder inhalers) or other active compounds (where appropriate).

For inhalation, a variety of systems are available which can utilize inhalation techniques to generate and administer aerosols of optimal particle size for the patient. Except for using connectors (mist containers, expanders) and pear-shaped containers (e.g. Nebulilator)Volumatic) And automatic means for emitting the spray (Autohaler)) In addition, many technical solutions are available for metered aerosols, especially in the case of powder inhalers (e.g. Diskhaler)RotadiskTurbohalerOr an inhaler, such as described in EP-A-0505321).

Process for the preparation of glitazone enantiomers

Chiral HPLC can be used to separate glitazones (see, e.g., methods 1-3 and 5-7 below). Chiral columns include CHIRALPAK AD, AD-H, AS-V, 50801, IA, IC, OD, OF, OG, OJ, OK and OZ. Preferred HPLC chiral columns are CHIRALPAK AD-H and CHIRALPAK IA eluted with ethanol and varying TFA, preferably 0.05-0.2% TFA.

An alternative method of resolution is shown in scheme 2 (see also example 10), using pioglitazone as an example.

Scheme 2

Recent review articles of optical resolution methods: e.fogassy et al, Tetrahedron: asymmetry, 2008, 19, 519 and 536; wilen, Topics in stereoschemistry, Wiley-Interscience: NY, 1972, 6, 107, main eds e.l.eliel, n.l.allinger; newman, Optical Resolution Procedures of Chinese Compounds 1-3, Resolution Information Center (Resolution Information Center), NY, 1978-; j.jaques, s.h.wilen, a.collett, enertomers racemes and solutions, Wiley-Interscience: NY, 1991; scheldon, Chirotechnology, marcel dekker, NY, 1993; optical resolution via diastereometric salt format, CRC Press, 2002, eds David Kozma.

Common chiral acid resolving agents include, without limitation, dibenzoyl tartaric acid, dimethoxybenzoyl tartaric acid, ditoluoyltartaric acid, tartaric acid, phencyphos, chlorocyphos, anilyphos, 1 '-binaphthyl-2, 2' -diester hydrogen phosphate, camphorsulfonic acid, bromocamphorsulfonic acid, camphoric acid, phenethylsulfonic acid, malic acid, mandelic acid, 4-bromomandelic acid, 4-methylmandelic acid, lactic acid, and chalcone sulfonic acid.

More preferred chiral acid resolving agents include (-) -O, O '-dibenzoyl-L-tartaric acid (anhydrous), (-) -O, O' -dibenzoyl-L-tartaric acid monohydrate, (-) -di-O-p-toluyl-L-tartaric acid, L- (+) -tartaric acid, and (-) -di-p-methoxyphenyl (p-anisyl) -L-tartaric acid.

The most preferred chiral acid resolving agent is (-) -O, O' -dibenzoyl-L-tartaric acid (anhydrous).

The resolution can be carried out using chiral acid resolving agents in different stoichiometric relationships. The resolving agent described above may be used in a ratio of 1 part (I) to 10 parts of chiral acid (II), more preferably in a ratio of 1 part (I) to 5 parts of chiral acid (II), most preferably in a ratio of 1 part (I) to 2 parts of chiral acid (II). Even more preferably a ratio of 1 part (I) to 1 part of chiral acid (II).

A variety of solvents can be used to form diastereomeric salts (III) with chiral acids (II). Preferred solvents may include ethyl acetate, dichloromethane, tetrahydrofuran, 1, 4-dioxane, acetone, acetonitrile, MeOH, EtOH, IPA, 1-propanol, 1, 2-dimethoxyethane, diethyl ether, dichloroethane, t-butyl methyl ether, 1-butanol, 2-butanol, t-butanol, 2-butanone, toluene, cyclohexane, heptane, hexane, H₂O, DMF, Petroleum Ether and CHCl₃。

The most preferred solvent is acetonitrile.

The solvents may be used in combination with each other, with a preferred combination comprising 10% HCl/H₂O、10％H₂O/DMF、10％H₂O/EtOH、10％H₂O/IPA, different parts of methanol and water and HCl, and different parts of CHCl₃And ethyl acetate, different parts of EtOH and water, different parts of IPA and water, different parts of 1-propanol and water, and different parts of CHCl₃And dioxane.

More preferred solvent combinations include different parts of CHCl₃And dioxane, varying parts of CHCl₃And ethyl acetate, different parts of methanol and water and different parts of HCl and methanol and water.

Common chiral amine resolving agents include, but are not limited to, 1-phenylethylamine, cinchonidine, cinchonine, quinidine, codeine, morphine, strychnine, brucine, quinine, ephedrine, aminobutanol, dehydroabietylamine (dehydroabietylamine), 2-phenylglycinol, 1, 2-cyclohexanediamine, 1-naphthylethylamine, 2-amino-1-phenylpropane-1, 3-diol, 4-chloro-1-aniline, N- (4-methoxybenzyl) -1-phenylethylamine, fenchyamine, N-benzyl-1-phenylethylamine, N- (4-dimethylaminobenzyl) -1-phenylethylamine, 3-amino-2, 2-dimethyl-3-phenylpropan-1-ol, sparteine, proline, serine, phenylalanine, lysine, threonine, valine, histidine, alanine, glutamic acid and glutamine, arginine, homoarginine, N-alpha-acetyl lysine, N-acetyl lysine and ornithine.

The most preferred chiral amine resolving agent is L-lysine.

The resolution can be carried out using chiral amine resolving agents in different stoichiometric relationships. The resolving agent described above may be used in a ratio of 1 part (I) to 10 parts chiral amine (IV), more preferably in a ratio of 1 part (I) to 5 parts chiral amine (IV), most preferably in a ratio of 1 part (I) to 1 part chiral amine (IV).

A variety of solvents can be used to form diastereomeric salts (V) with chiral amines (IV). Preferred solvents may include ethyl acetate, dichloromethane, tetrahydrofuran, 1, 4-dioxane, acetone, acetonitrile, MeOH, EtOH, IPA, 1-propanol, 1, 2-dimethoxyethane, diethyl ether, dichloroethane, t-butyl methyl ether, 1-butanol, 2-butanol, t-butanol, 2-butanone, toluene, cyclohexane, heptane, hexane, H₂O, DMF, Petroleum Ether and CHCl₃。

References k.akiba et al, bull.chem.soc.jpn., 1974, 47(4), 935-937; r.a. bartsch et al, JACS, 2001, 123(31), 7479, disclose the decomposition of heterocyclic nitrosamines and are applicable to the synthesis and resolution of glitazones. Thus, using pioglitazone as an example, an alternative method of synthesis/resolution can be seen in scheme 3:

scheme 3

Of course, the final product can be recrystallized to improve enantiomeric purity.

References t.sohda et al, chem.pharm.bull, 1984, 32(11), 4460-5 disclose chiral syntheses of related compounds and are applicable to the synthesis of other glitazones. Thus, using pioglitazone as an example, an alternative method of synthesis can be seen in scheme 4:

scheme 4

References j.r.falck et al, bioorg.med.chem.lett., 2008, 18, 1768-. Thus, using pioglitazone as an example, an alternative method of synthesis can be seen in scheme 5 (see also example 13).

Scheme 5

In the case of rosiglitazone, the reference J.chem.Soc.Perkin Trans.I, 1994, 3319-3324 indicates that 5- [1- {4- [3- (methyl-pyridin-2-yl-amino) -propoxy ] -phenyl } -meth- (E) -ylidene ] -thiazolidine-2, 4-dione can be reduced using biocatalysis. The yeast strains suggested for biocatalysis are Rhodotorula rubra (or Rhodotorula mucilaginosa) and Rhodotorula glutinis (Rhodotorula glutinis). Thus, for example with pioglitazone, an alternative preparation of glitazones can be seen in scheme 6:

scheme 6

Alternatively, asymmetric hydrogenolysis of 5- [1- {4- [2- (5-ethyl-pyridin-2-yl) -ethoxy ] -phenyl } -meth- (E) -ylidene ] -thiazolidine-2, 4-dione in the presence of a chiral ligand using rhodium and an iridium catalyst is a method known to those skilled in the art (see also example 12 below).

The following examples illustrate the preparation of pioglitazone and rosiglitazone enantiomers, and the biological results on which the present invention is based:

chemical examples

General experimental details

Abbreviations used in the experimental section:

c is concentration; h is h; h₂O is distilled water; HPLC ═ high performance liquid chromatography; LCMS ═ liquid chromatography-mass spectrometry; MeOH ═ methanol; TFA ═ trifluoroacetic acid; DMSO ═ dimethyl sulfoxide; HCl ═ hydrochloric acid; EtOH ═ ethanol; IPA ═ isopropyl alcohol; EtOAc ═ ethyl acetate; THF ═ tetrahydrofuran; NH (NH)₄Cl ═ ammonium chloride; LDA ═ lithium diisopropylamide; min is minutes; RT ═ room temperature; rt ═ retention time; e.e. ═ enantiomeric excess; MP-Carbonate ═ macroporous methyl polystyrene triethyl ammonium Carbonate (0.5% inorganic antistatic agent). d.e. ═ diastereomeric excess; SL-W003-2 ═ S- [ (S) -2- (2 i-diphenylphosphinophenyl) ferrocenyl]Ethyldicyclohexylphosphine; rh (COD)₂BF₄Rhodium (1, 5-cyclooctadiene) tetrafluoroborate I); pioglitazone ═ 5- {4- [2- (5-ethylpyridin-2-yl) ethoxy]Benzyl } -1, 3-thiazolidine-2, 4-dione; L-DBTA ═ L-dibenzoyltartaric acid

The nomenclature of the structures is given using ACD laboratory version 10.

NMR spectra were obtained from the following mass spectrometer: a Varian Unity Inova 400 mass spectrometer with a 5mm reverse phase detection triple resonance probe operating at 400MHz, or a Bruker AvanceDRX 400 mass spectrometer with a 5mm reverse phase detection triple resonance TXI probe operating at 400MHz, or a Bruker Avance DPX 300 mass spectrometer with a standard 5mm dual frequency probe operating at 300 MHz. Shifts are given in ppm relative to tetramethylsilane. Optical rotation was measured using an AA-10R automated polarimeter with a 5x25mm jacketed sample tube. The asymmetric hydrogenolysis experiments were performed using a Biotage engage hydrogenation apparatus.

All solvents and commercial reagents were used as received.

Using liquid chromatography-mass spectrometry (LC/MS) and liquid chromatography systems:

method 1

CHIRALPAK AD-H (250X30mm, 5 μm), eluted with EtOH + 0.05% TFA-flow 30 ml/min. detection-Embedded UV detection set to 250nM wavelength

Method 2

CHIRALPAK 1A (250X4.6mm, 5. mu.M) eluted with EtOH + 0.05% TFA-flow rate of 0.7 ml/min. detection-Embedded DAD set to 280nM wavelength

Method 3

CHIRALCEL OD-RH (150X4.6mm), with 90% MeOH + 10% H₂O elution-flow rate 0.5 ml/min. detection-Embedded UV detection set to 254nM wavelength

Method 4

Waters Micromass ZQ2000 with C18-reverse phase column (100X 3.0mm Higgins Clipeus, particle size 5 μm) with A: water + 0.1% formic acid; b: acetonitrile + 0.1% formic acid. Gradient:

detection-MS, ELS, UV (100. mu.l split to MS with embedded UV detector). MS ionization method-electrospray (positive ion)

Method 5

CHIRALPAK IA (250X21mm, 5 μm), eluting with ethanol + 0.2% TFA-flow 13 ml/min. detection-Embedded UV detection set to 220nM wavelength

Method 6

Chiral-AGP (150X4.0mm, 5. mu.M), with A: 86% 10mM potassium dihydrogen phosphate buffer (pH 7.0); b: 14% acetonitrile + 0.1% formic acid elution-flow rate 0.8 ml/min. detection-Embedded DAD set to 254nM wavelength

Method 7

CHIRALPAK 1A (250X4.6mm, 5. mu.M) with A, 0.05% TFA/EtOH; b, heptane; d, IPA (A: B: D: 40: 30) elution-flow rate 0.7 ml/min. detection-Embedded DAD set to 225nM wavelength

detection-MS, ELS, UV PDA. MS ionization method-electrospray (positive/negative ion)

Method 8

Waters Micromass ZQ2000 with Acquity BEH or Acquity BEH Shield RP 181.7. mu.M 100X2.1mm C18-reverse phase column, using A: water + 0.1% formic acid; b: acetonitrile + 0.1% formic acid. Gradient:

Method 9

Agilent 1100 series with CHIRALPAK IA (150X4.6mm, 5 μm) was prepared using A: heptane, B: ethanol + 0.05% TFA elution-flow rate 0.5 ml/min. Detection-embedded polarimeter and UV detection set at 270nM wavelength. Gradient:

method 10

Phenomenex Gemini C18-reversed phase column (250X 21.20mm, 5 μm particle size) with A: water + 0.1% formic acid; b: methanol + 0.1% formic acid. Gradient-50% A/50% B-5% A/95% B in 15 min-flow rate 18 mL/min. Detection-an embedded UV detector set at 254nM wavelength.

Method 11

Phenomenex Luna 3 micron C18(2)30X4.6mm, prepared from A: water + 0.1% formic acid; b: acetonitrile + 0.1% formic acid. Gradient:

detection-MS, ELS, UV (200. mu.l/min split to MS with embedded HP1100DAD detection). MS ionization method-electrospray (positive and negative ion)

All reactions were carried out under nitrogen atmosphere unless otherwise indicated. Racemic rosiglitazone was used as the free base, whereas racemic pioglitazone was used as the free base or as the noted HCl salt.

Example 1

(5R) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione trifluoroacetate salt

The title compound (480mg) was isolated using method 1. LCMS (method 4): rt 6.00 min, M/z 357[ M-CF₃CO₂H⁺]。[α]_D ²⁵+104 ° (c 1.0, MeOH). e.e. (method 2) not less than 98%, Rt 4.69 minutes.¹H NMR(400MHz，DMSO-d₆)：12.02-11.88(1H，bs)，8.68-8.60(1H，d，J 1.7)，8.32-8.23(1H，d，J 7.7)，7.90-7.82(1H，d，J 8.4)，7.14-7.06(2H，d，J 8.7)，6.85-6.78(2H，d，J 8.7)，4.85-4.78(1H，dd，J 4.4，8.9)，4.35-4.27(2H，t，J 6.2)，3.40-3.34(2H，t，J 6.1)，3.28-3.21(1H，dd，J 4.3，14.3)，3.05-2.97(1H，dd，J 9.0，14.3)，2.77-2.67(2H，q，J 7.6)，1.22-1.14(3H，q，J 7.5)。

Example 2

(5S) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione trifluoroacetate salt

The title compound (674mg) was isolated using method 1. LCMS (method 4): rt 6.01 min, M/z 357[ M-CF₃CO₂H⁺]。[α]_D ²⁵76 ° (c 1.0, MeOH). e.e. (method 2) not less than 98%, Rt 7.00 minutes.¹H NMR(400MHz，DMSO-d₆)：12.02-11.88(1H，bs)，8.68-8.60(1H，d，J 1.7)，8.32-8.23(1H，d，J 7.7)，7.90-7.82(1H，d，J 8.4)，7.14-7.06(2H，d，J 8.7)，6.85-6.78(2H，d，J 8.7)，4.85-4.78(1H，dd，J 4.4，8.9)，4.35-4.27(2H，t，J 6.2)，3.40-3.34(2H，t，J 6.1)，3.28-3.21(1H，dd，J 4.3，14.3)，3.05-2.97(1H，dd，J 9.0，14.3)，2.77-2.67(2H，q，J 7.6)，1.22-1.14(3H，q，J 7.5)。

Example 3

(5R) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione

MP-Carbonate (389mg, 1.06mmol) was added to (5R) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy]Benzyl } -1, 3-thiazolidine-2, 4-dione trifluoroacetate salt (100mg, 0.21mmol) in MeOH (100mL) was stirred at room temperature for 2h, and after filtration the resin was washed with MeOH (3X10 mL). The filtrate was concentrated in vacuo to give the title compound (35mg, 47%). e. (method 2) 92.90%, Rt 6.27 min.¹H NMR(400MHz，DMSO-d₆)：12.44-11.11(1H，bs)，8.34-8.29(1H，d，J 1.9)，7.55-7.49(1H，dd，J 2.2，7.9)，7.24-7.20(1H，d，J 7.8)，7.12-7.05(2H，d，J 8.6)，6.84-6.77(2H，d，J 8.6)，4.78-4.71(1H，dd，J 4.3，9.1)，4.30-4.19(1H，d，J 4.3)，3.24-3.18(2H，d)，3.11-3.03(2H，t，J 6.6)，3.00-2.92(1H，dd，J9.2，14.2)，2.59-2.50(2H，q，J 7.6)，1.17-1.09(3H，t，J 7.7)。

Example 4

(5R) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione hydrochloride

Add approximately 1.25M HCl/MeOH (0.33mL, 0.33mmol) to (5R) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy]To a suspension of benzyl } -1, 3-thiazolidine-2, 4-dione (from example 3) (30mg, 0.084mmol) and MeOH (5mL) was stirred at room temperature for 1 h. The solvent was removed in vacuo to give the title compound (32.4mg, 100%). LCMS (method 4): rt 5.95 min, M/z 357[ M-HCl ]⁺]. e (method 3) 93.2%, Rt 12.10 min. According to single crystal X-ray diffraction analysis, the stereochemistry of C-5 is (R) configuration. [ alpha ] to]_D ²⁴+108°(c 1.0，MeOH)。¹H NMR(400MHz，DMSO-d₆)：12.03-11.88(1H，bs)，8.68-8.62(1H，d，J 1.7)，8.34-8.25(1H，d，J 7.9)，7.91-7.83(1H，d，J8.3)，7.14-7.05(2H，d，J 8.7) 6.86-6.77(2H, d, J8.7), 4.85-4.77(1H, dd, J4.3, 8.9), 4.38-4.28(2H, t, J6.0), 3.42-3.36(2H, t, J6.2), 3.28-3.20(1H, dd, J9.0, 14.2), 3.06-2.96(1H, dd, J9.0, 14.2), 2.77-2.67(2H, q, J7.7), 1.23-1.15(3H, t, J7.7). Followed by MeOH-EtOAc or MeOH-Et₂Recrystallization of O afforded the title compound, which was e.e. > 97%.

Example 5

(5S) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione

An analogous method to that described in example 3 was used to select from (5S) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy]The title compound was prepared starting from benzyl } -1, 3-thiazolidine-2, 4-dione trifluoroacetate (28mg, 37%). e. (method 2) 90.9%, Rt 9.21 min.¹H NMR(400MHz，DMSO-d₆)：12.44-11.11(1H，bs)，8.34-8.29(1H，d，J 1.9)，7.55-7.49(1H，dd，J 2.2，7.9)，7.24-7.20(1H，d，J 7.8)，7.12-7.05(2H，d，J 8.6)，6.84-6.77(2H，d，J8.6)，4.78-4.71(1H，dd，J 4.3，9.1)，4.30-4.19(1H，d，J 4.3)，3.24-3.18(2H，d)，3.11-3.03(2H，t，J 6.6)，3.00-2.92(1H，dd，J9.2，14.2)，2.59-2.50(2H，q，J 7.6)，1.17-1.09(3H，t，J 7.7)。

Example 6

(5S) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione hydrochloride

Analogously to the process described in example 4, starting from (5S) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy]The title compound (25.7mg, 100%) was prepared starting from benzyl } -1, 3-thiazolidine-2, 4-dione (from example 2). LCMS (method 4): rt 5.94 min, M/z 357[ M-HCl ]⁺]. e. (method 3) 92.7%, Rt 13.25 min. According to single crystal X-ray diffraction analysis, the stereochemistry of C-5 is (S) configuration. [ alpha ] to]_D ²³-104°(c 1.0，MeOH)。¹H NMR(400MHz，DMSO-d₆): 12.03-11.88(1H, bs), 8.68-8.62(1H, d, J1.7), 8.34-8.25(1H, d, J7.9), 7.91-7.83(1H, d, J8.3), 7.14-7.05(2H, d, J8.7), 6.86-6.77(2H, d, J8.7), 4.85-4.77(1H, dd, J4.3, 8.9), 4.38-4.28(2H, t, J6.0), 3.42-3.36(2H, t, J6.2), 3.28-dd 3.20(1H, dd, J9.0, 14.2), 3.06-2.96(1H, dd, J9.0, 14.2), 2.77-2.67(2H, 7.7, 7.7.7, 7.7.7.7, 7, 7.7.7, 7.7.. Followed by MeOH-EtOAc or MeOH-Et₂Recrystallization of O afforded the title compound, which was e.e. > 97%.

Example 7

(5R) -5- (4- {2- [ methyl (pyridin-2-yl) amino ] ethoxy } benzyl } -1, 3-thiazolidine-2, 4-dione trifluoroacetate salt

The title compound (149mg) was isolated using method 5. Rt 7.14 min.¹H NMR(400MHz，DMSO-d₆)：12.04-11.86(1H，s)，7.99-7.94(1H，dd，J 1.1，6.2)，7.92-7.83(1H，t，J 6.6)，7.27-7.15(1H，d，J 8.7)，7.13-7.05(2H，d，J 8.6)，6.88-6.82(1H，t，J 6.6)，6.81-6.76(2H，d，J 8.7)，4.83-4.78(1H，dd，J 4.4，8.8)，4.18-4.12(2H，t，J 5.3)，3.99-3.94(2H，t，J 5.3)，3.27-3.20(1H，dd，J 4.2，14.4)，3.18(3H，s)，3.05-2.97(1H，dd，J 8.9，14.6)。

Example 8

(5R) -5- (4- {2- [ methyl (pyridin-2-yl) amino ] ethoxy } benzyl) -1, 3-thiazolidine-2, 4-dione hydrochloride

Analogously to the methods described in examples 3 and 4, starting from (5R) -5- {4- [ 2-methyl-2-pyridinylamino) ethoxy]The title compound was prepared starting from benzyl } -1, 3-thiazolidine-2, 4-dione trifluoroacetate (38mg, 64%). [ alpha ] to]_D ²⁶+100 ° (c 1.0, MeOH). LCMS (method 4): rt 5.41 min, M/z 358[ M-HCl ]⁺]. e. (method 6) 85.7%, Rt 8.03 min.¹H NMR(400MHz，DMSO-d₆)：12.09-11.81(1H，s)，7.98-7.94(1H，dd，J 1.1，6.3)，7.93-7.82(1H，m)，7.31-7.14(1H，bs)，7.13-7.05(2H，d，J 8.5)，6.90-6.82(1H，t，J 6.7)，6.80-6.76(2H，d，J 8.5)，4.84-4.78(1H，dd，J 4.4，8.9)，4.19-4.12(2H，t，J 5.2)，4.02-3.94(2H，t，J 5.2)，3.27-3.21(1H，dd，J 4.4，10.1)，3.20(3H，s)，3.05-2.97(1H，dd，J 9.0，14.2)。

R-enantiomers having greater than 90% e.e. can be obtained using literature methods: J.chem.Soc.Perkin Trans.1.1994, 3319-3324.

Example 9

(5S) -5- (4- {2- [ methyl (pyridin-2-yl) amino ] ethoxy } benzyl) -1, 3-thiazolidine-2, 4-dione hydrochloride monohydrate

The title compound (123mg) was obtained using the following literature method: J.chem.Soc.PerkinTrans.1.1994, 3319-3324. [ alpha ] to]_D ²³100 ° (c 1.0, MeOH). LCMS (method 4): rt 5.44 min, M/z 358[ M-HCl ]⁺]. e. (method 6) 92.7%, Rt 8.99 min. According to single crystal X-ray diffraction analysis, the stereochemistry of C-5 is (S) configuration.¹H NMR(400MHz，DMSO-d₆)：12.01-11.88(1H，s)，7.98-7.94(1H，dd，J 1.4，6.1)，7.93-7.86(1H，t，J 7.7)，7.31-7.18(1H，m)，7.12-7.05(2H，d，J 8.7)，6.90-6.83(1H，t，J 6.3)，6.81-6.75(2H，d，J 8.7)，4.83-4.78(1H，dd，J 4.5，8.8)，4.19-4.13(2H，t，J 5.1)，4.02-3.96(2H，t，J 5.1)，3.26-3.22(1H，m)，3.21(3H，s)，3.05-2.97(1H，dd，J 8.8，14.0)。

Example 10

(5R) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy ] benzyl } -1, 3-thiazolidine-2, 4-dione (-) -O, O' -dibenzoyl-L-tartrate

To (5R) -5- {4- [2- (5-ethylpyridin-2-yl) ethoxy]To a mixture of benzyl } -1, 3-thiazolidine-2, 4-dione hydrochloride (50mg) (example 4) and (-) -dibenzoyl-L-tartaric acid (50mg) was added MeOH (1.5 mL). In the dropwise addition of H₂The clear solution was stirred rapidly while O until cloudy. The reaction was allowed to stand at ambient temperature for more than 48 hours and the solid was collected by filtration to give the title compound (43 mg). (method 7) 99.01% Rt 10.83 minutes, 0.98% Rt 15.83 minutes; d.e.98.03%.

Reaction of (-) -dibenzoyl-L-tartaric acid (1.0g, 2.79mmol) with H₂A slurry of O (20mL) was stirred at ambient temperature and 5- {4- [2- (5-ethylpyridin-2-yl) ethoxy ] was added over 5 minutes]M of benzyl } -1, 3-thiazolidine-2, 4-dione hydrochloride (1.01g, 2.57mmol)eOH (20 mL). When the addition was complete, the product of example 10a (5mg) was added and the reaction stirred for 93 hours. The reaction was filtered and the solid was dried to give the title compound (0.863 g). (method 7) 79.72%, Rt 10.82 min; 20.27%, Rt 15.14 min; d.e.59.45%.

Dissolve the product of example 10b (0.863g) in MeOH (8.5mL) containing 1M HCl (1.21mL) and add H dropwise₂O (5 mL). The product of example 10a (1mg) was added followed by dropwise addition of H₂O (2.3 mL). The reaction was stirred for 22 hours, filtered and the solid was washed with H₂After washing with O-MeOH (2: 1, 3mL), it was dried under high vacuum at 40 ℃ to give the title compound (0.582 g). (method 7) 93.2%, Rt 10.82 min; 6.8 percent, Rt 15.14 minutes; d.e.86.4%.

Dissolve the product of example 10c (0.582g) in MeOH (5.5mL) containing 1M HCl (0.795mL) and add H dropwise₂O (2 mL). The product of example 10a (1mg) was added followed by dropwise addition of H₂O (3.5 mL). The reaction was stirred for 22 hours, filtered and the solid was washed with H₂O-MeOH (2: 1, 3mL) and dried at 40 deg.C under high vacuum to give the title compound (0.453 g). (method 7) 97.3%, Rt 10.65 minutes; 2.7%, Rt 14.83 min; d.e.94.6%.¹H NMR(400MHz，DMSO-d₆)：14.25-13.60(bs，1H，D₂O is exchangeable), 12.05-12.00(bs, 1H, D)₂O interchangeable), 8.37(d, J ═ 2.0Hz, 1H), 8.02(d, J ═ 7.6Hz, 4H), 7.73(t, J ═ 7.6Hz, 2H), 7.60(t, J ═ 7.6Hz, 5H), 7.29(d, J ═ 8.0Hz, 1H), 7.13(d, J ═ 8.8Hz, 2H), 6.86(d, J ═ 8.8Hz, 2H), 5.88(s, 2H), 4.86(q, J ═ 4.4Hz, 1H), 4.30(t, J ═ 6.8Hz, 2H), 3.30(dd, J ═ 4.0)&10.0Hz，1H)，3.13(t，J＝6.8Hz，2H)，3.04(dd，J＝5.2 & 9.2Hz，1H)，2.60(q，J＝7.6Hz，2H)，1.17(t，J＝7.6Hz，3H)。

Example 11

11a. dissolve the product of example 10d in MeOH (2.25mL) containing 37% HCl (0.134mL) at 35 ℃. After the solution was filtered, EtOAc (9mL) was poured into the stirred solution and the mixture was stirred for 20 minutes. The white solid was collected by filtration, washed with EtOAc and dried at 30 ℃ under high vacuum to give the title compound (0.181 g). (method 7) 98.3%, Rt 10.65 minutes; 1.7%, Rt 14.83 min, e.e.96.6%. LCMS (method 8): rt 2.90 min 99.39%, m/z 357[ MH ]⁺-HCl]. LCMS (method 11) Rt 2.91 min, m/z 357[ MH⁺-HCl]。¹H NMR(400MHz，DMSO-d₆)：12.0(1H，s)，8.70(1H，d，J 1.7Hz)，8.36(1H，bd，J 8.3Hz)，7.93(1H，d，J8.2Hz)，7.15，6.87(4H，A2B2q，J 8.7Hz)，4.86(1H，dd，J 4.4，8.9Hz)，4.38(2H，t，J 6.3Hz)，3.44(2H，t，J 6.2Hz)，3.29(1H，dd，J 4.3，14.2Hz)，3.06(1H，dd，J 9.0，14.3Hz)，2.78(2H，q，J 7.6Hz)，1.23(3H，t，J 7.6Hz)。

The product of example 10c (1g) was reacted as described for example 11a to give the title compound (473 mg). (method 7) 95.6%, Rt 10.65 minutes; 4.3%, Rt 14.83 min, e.e.91.3%. All other characteristic data were the same as those of example 11a.

Example 12

Preparation of Rh (COD) in a glove box₂BF₄(10.6mg, 26.1. mu. mol) with DStock solution of CM (9.5 mL). Ligand SL-W003-2(8.4mg, 12.5. mu. mol) was weighed into a vial, and 500. mu.L of Rh (COD) was added₂BF₄The solution was stored. Reacting 5- [1- {4- [2- (5-ethyl-pyridin-2-yl) -ethoxy]-phenyl } -meth- (E) -ylidene]A solution of thiazolidine-2, 4-dione (78.2 mg of 4.5mL stock solution in 35mL MeOH) was added to the reaction vial containing the catalyst solution. For vials N₂(5x) and H₂(5x) purging, and finally charging 25bar of H₂. After 18 hours, H is released₂The reaction vial is filled with N₂Purge (3 ×). A sample of the reaction vial was removed and diluted with an equal volume of EtOH containing 1% formic acid. e. (method 9) 75%, Rt 36.11 min.

Example 13

Methanesulfonic acid 2- (5-ethyl-pyridin-2-yl) -ethyl ester

Methanesulfonyl chloride (15.56mL) was added to a solution of 2- (5-ethyl-pyridin-2-yl) -ethanol (25.34g) and triethylamine (46.7mL) in DCM (130mL) at 0 deg.C under a nitrogen atmosphere, and the reaction mixture was warmed to room temperature and stirred overnight. An additional portion of methanesulfonyl chloride (3.88mL) was added and the reaction stirred for 30 minutes. The reaction was diluted with DCM and then H₂O and brine wash. The organic layer was dried (MgSO)₄) After that, the solution was concentrated to give the title compound (38.24g, as red oil).¹H NMR(400MHz，CDCl₃)：8.39(d，J＝2.2Hz，1H)，7.47(dd，J＝7.7，2.3Hz，1H)，7.14(d，J＝7.7Hz，1H)，4.64(t，J＝6.5Hz，2H)，3.19(t，J＝6.5Hz，2H)，2.90(s，3H)，2.64(q，J＝7.6Hz，2H)，1.25(t，J＝7.6Hz，3H)。

13b.3- {4- [2- (5-Ethyl-pyridin-2-yl) -ethoxy ] -phenyl } -propionic acid methyl ester

A solution of the product of example 13a (12.72g) in toluene (50mL) was added dropwise to a stirred mixture of methyl 3- (4-hydroxyphenyl) propionate (10g) and potassium carbonate (23.01g) in toluene (180 mL). The reaction was heated to reflux for 23 hours and then left at room temperature for 90 hours. Reaction mixture with H₂O treatment and extraction with three portions of ether. The combined extracts are washed with H₂O twice, once with brine, dried and evaporated. The residue was purified by chromatography (eluting with 20-40% EtOAc/petroleum ether (bp ═ 40-60 ℃)) to give the title product (10.73g as a yellow solid).¹HNMR(400MHz，CDCl₃)：8.39(d，J＝2.1Hz，1H)，7.44(dd，J＝7.8，2.4Hz，1H)，7.18(d，J＝7.8Hz，1H)，7.08，6.83(A₂B₂q，J＝8.6Hz，4H)，4.31(t，J＝6.7Hz，2H)，3.66(s，3H)，3.22(t，J＝6.7Hz，2H)，2.87(t，J＝7.6Hz，2H)，2.66-2.55(m，4H)，1.24(t，J＝7.6Hz，3H)。

13c.3- {4- [2- (5-Ethyl-pyridin-2-yl) -ethoxy ] -phenyl } -propionic acid

The product of example 13b (10.73g) was dissolved in 1, 4-dioxane/water (350/100mL), lithium hydroxide monohydrate (4.3g) was added and the reaction stirred overnight. Removing dioxane by evaporation under reduced pressure, and mixing the suspension with H₂After dilution with O, it was treated with 2.0M HCl until pH 6-7. The solid was filtered and dried to give the title compound (9.57 g).¹H NMR(400MHz，CDCl₃)：8.41(d，J＝2.3Hz，1H)，7.52(dd，J＝7.9，2.3Hz，1H)，7.25(d，J＝7.9Hz，1H)，7.11，6.81(A₂B₂q, J ═ 8.6Hz, 4H), 4.27(t, J ═ 6.6Hz, 2H), 4.0(bs, 2H, COOH + water) 3.24(t, J ═ 6.6Hz, 2H), 2.90(t, J ═ 7.6Hz, 2H), 2.68-2.59(m, 4H), 1.24(t, J ═ 7.6Hz, 3H).

(R) -4-benzyl-3- (3- {4- [2- (5-ethyl-pyridin-2-yl) -ethoxy ] -phenyl } -propionyl) -oxazolidin-2-one

A suspension of the product of example 13c (9.0g) and (R) - (+) -4-benzyl oxazolidinone (2.67g) with triethylamine (8.38mL) and toluene (90mL) was heated to 80 ℃. Pivaloyl chloride (3.71mL) was added dropwise to maintain the temperature between 80 ℃ and 85 ℃. The reaction was then heated to reflux for 22.5 hours. The reaction was cooled to room temperature and the reaction was allowed to stand at H₂Partition between O-EtOAc, extract the aqueous layer twice with EtOAc and dry the combined organics (MgSO)₄) And concentrated. The crude product was purified by silica gel chromatography (eluting with 0-40% EtOAc-cyclohexane) to give the title product (2.06g as a white solid).¹H NMR(300MHz，CDCl₃)：8.39(d，J＝2.2Hz，1H)，7.45(dd，J＝7.9，2.2Hz，1H)，7.36-7.24(m，3H)，7.21-7.12(m，5H)，6.84(d，J＝8.6Hz，2H)，4.65(m，1H)，4.32(t，J＝6.6Hz，2H)，4.16(m，2H)，3.33-3.12(m，5H)，3.04-2.87(m，2H)，2.74(dd，J＝9.5，13.4Hz，1H)，2.63(q，J＝7.6Hz，2H)，1.24(t，J＝7.6Hz，3H)。

(R) -4-benzyl-3- ((R) -3- {4- [2- (5-ethyl-pyridin-2-yl) -ethoxy ] -phenyl } -2-thiocyanato-propionyl) -oxazolidin-2-one

At-78 deg.C in an argon atmosphereNext, a solution of the product of example 13d (0.20g) in THF (4mL) was added dropwise to a solution of LDA (0.528mmol) in THF/hexane (3/0.3 mL). After 30 minutes, a solution of N-thiocyanic acid succinimide (JACS, 2004, 126, 10216-7) (0.137g) in THF (2mL) was added dropwise. After a further 110 minutes at-78 ℃ saturated NH was added₄The reaction was quenched with aqueous Cl (5mL) and warmed to room temperature. The mixture was extracted with three portions of 15mL EtOAc and the combined organic layers were dried (Na)₂SO₄) Then evaporating at least the volume under reduced pressure. Toluene (20mL) was added and the mixture was evaporated under reduced pressure. The residue was dissolved in DCM (3mL) and stored at-20 ℃ for 16 h. The soluble material was purified by silica gel chromatography (eluting with 20-50% EtOAc/cyclohexane) and the impurity-containing fractions were repurified in the same manner to give the title product and succinimide (0.121g, colorless semi-solid) in a 1: 2.4 molar ratio.¹H NMR(300MHz，CDCl₃): 8.60(bs, 1H succinimide) 8.40(d, J ═ 2.3Hz, 1H), 7.47(dd, J ═ 7.8, 2.3Hz, 1H), 7.39-7.12(m, 8H), 6.85(d, J ═ 8.7Hz, 2H), 5.07(dd, J ═ 8.1, 7.2Hz, 1H), 4.63(m, 1H), 4.32(t, J ═ 6.7Hz, 2H), 4.24-4.11(m, 2H), 3.47(dd, J ═ 14.0, 8.0Hz, dd1H), 3.33(dd, J ═ 13.4, 3.2Hz, 1H), 3.28-3.16(m, 3H), 2.84(dd, J ═ 9, 3.4, 13.4, 3.2Hz, 1H), 3.28-3.16(m, 3H), 2.84(dd, J ═ 9, 3, 13.4, 6H, 6.6H), 7H, 6.6 (J ═ 6H), 7H, 6H).

Thiocarbamic acid S- ((R) -2- ((R) -4-benzyl-2-oxo-oxazolidin-3-yl) -1- {4- [2- (5-ethyl-pyridin-2-yl) -ethoxy ] -benzyl } -2-oxo-ethyl) ester

The product of example 13e (51mg) was dissolved in THF/water (2/1ml) under argon and treated with tris (dimethylphosphineoxide) platinum (tet.lett., 2002, 43, 8121) (4 mg). The reaction was heated to 40 ℃ for 2 hours and then cooled to room temperature. EtOAc (10mL) was added and the organic phase was dried (Na)₂SO₄) Concentrated and chromatographed on silica gel (2g) (eluting with 40-70% EtOAc/cyclohexane) to give 1: 3.3 molesThe title product and succinimide (0.029g, as a colorless semisolid) in molar ratio.¹H NMR(400MHz，CDCl₃): 8.66(bs, 1H succinimide) 8.39(d, J ═ 2.0Hz, 1H), 7.47(dd, J ═ 7.9, 2.2Hz, 1H), 7.37-7.17(m, 6H), 7.17, 6.81(a, 6H)₂B₂q J ═ 8.5Hz, 4H), 5.74(t, J ═ 7.9Hz, 1H), 5.63(bs, 2H), 4.53(m, 1H), 4.30(t, J ═ 6.7Hz, 2H), 4.08(dd, J ═ 8.9, 2.5Hz, 1H), 3.95(dd, J ═ 8.9, 7.8Hz, 1H), 3.31(dd, J ═ 13.5, 3.1Hz, 1H), 3.26-3.18(m, 3H), 2.96(dd, J ═ 13.5, 8.1Hz, 1H), 2.74(s, 4H succinimides), 2.72(dd, J ═ 13.5, 9.8Hz, 1H), 2.63(q, 7.6, J ═ 6, 2.24H), t, 6.6H, 3.6 Hz, 3H).

Dissolve the product of example 13f in MeOH (10mL) and add H₂O (10ml), giving a turbid solution. It was left at room temperature for 2.5 hours, then 1M HCl (0.15mL) was added. After the solution was evaporated to dryness, the residue was dissolved in MeOH (0.1mL) containing concentrated HCl (0.0015 mL). EtOAc (5mL) was added and the solution was slightly concentrated under reduced pressure to precipitate a white solid. The solution was removed and the solid (7mg) was washed with EtOAc. The product was purified by preparative HPLC (method 10), and fractions containing the first eluting component were combined, treated with 1M HCl (1mL) and evaporated to dryness to give the title product and succinimide (0.002g, colorless gum) in a 1: 0: 0.19 molar ratio. (method 7) 99.175%, Rt 10.65 min; 0.825%, Rt 14.83 min; e.e.98.35%. LCMS (method 11) Rt 2.9 min, m/z 357[ MH⁺]。¹H NMR(300MHz，d₄-MeOH)：8.62(bd，1H)，8.42(dd，J＝8.2，2.0Hz，1H)，7.98(d，J＝8.2Hz，1H)，7.16，6.85(A₂B₂q, J ═ 8.7Hz, 4H), 4.68dd, J ═ 8.7, 4.2Hz, 1H), 4.38(t, J ═ 5.8Hz, 2H), 3.34(m masked, 1H), 3.11(dd,j ═ 14.3, 8.8Hz, 1H), 2.87(q, J ═ 7.6Hz, 2H), 2.68(s, 4H succinimide), 1.33(t, J ═ 7.6Hz, 3H).

Biological results:

example 14

Preclinical mouse model of COPD inflammation-tobacco smoke induced pneumonia.

Previous studies have demonstrated that the number of inflammatory cells recovered in bronchoalveolar lavage fluid (BAL) is significantly elevated 24 hours after a final Tobacco Smoke (TS) exposure of 4 or 11 consecutive days of daily Tobacco Smoke (TS) exposure, the 24 hour post-exposure time point being used in the studies reported herein.

The preparation of TS exposure protocol, bronchoalveolar lavage (BAL) obtained, cytospin slide (cytospin slide) for differential cell count in mice is summarized below.

Mice were exposed to TS daily for 4 or 11 consecutive days

In this exposure protocol, mice were exposed to 5 of each group in a separate clear polycarbonate chamber (27cmx16cmx12 cm). The TS of the cigarette was allowed to enter the exposure chamber at a flow rate of 100 ml/min. To minimize any potential problems caused by repeated exposure to high levels of TS (6 cigarettes), the amount of cigarettes exposed to TS was increased stepwise over the exposure period up to a maximum of 6 cigarettes. The exposure protocol used for 4 days was as follows:

day 1: 4 cigarettes (Exposure about 32 minutes)

Day 2: 4 cigarettes (Exposure about 32 minutes)

Day 3: 6 cigarettes (Exposure about 48 minutes)

Day 4: 6 cigarettes (Exposure about 48 minutes)

The exposure protocol used for 11 day exposure was as follows:

day 1: 2 cigarettes (Exposure about 16 minutes)

Day 2: 3 cigarettes (Exposure for about 24 minutes)

Day 3: 4 cigarettes (Exposure about 32 minutes)

Day 4: 5 cigarettes (Exposure about 40 minutes)

Day 5-11: 6 cigarettes (Exposure about 48 minutes)

Another group of mice was exposed to air daily for the same duration as a control (no TS exposure).

Bronchoalveolar lavage fluid (BAL) analysis

Bronchoalveolar lavage was performed as follows: a Portex nylon intravenous cannula (ping luer fitting) cut to about 8mm was inserted into the trachea. Phosphate Buffered Saline (PBS) was used as lavage fluid. A volume of 0.4ml was gently instilled, withdrawn 3 times using a 1ml syringe, and then added to the microcentrifuge tube, which was kept on an ice bath prior to subsequent measurements.

Cell counting:

lavage fluid was separated from the cells by centrifugation, and the supernatant was decanted and frozen for subsequent analysis. The cell pellet was resuspended in a known volume of PBS and the total number of cells of the stained (Turks dye) aliquot of suspension was counted under the microscope using a hemocytometer.

Differential cell counts were performed as follows:

diluting the remaining cell pellet to about 10⁵Individual cells/ml. A volume of 500. mu.l was added to the cytospin funnel and centrifuged at 800rpm for 8 minutes. Slides were allowed to air dry and stained with a 'Kwik-Diff' solution (Shandon) according to the patent specification. When air dried, cover the lid and perform a differential cell count using an optical microscope. A fair operator uses light microscopy to count up to 400 cells. Cells were resolved using standard morphometric techniques.

Medical treatment

Rodents (e.g., mice and rats) are obligate nasal breathers (e.g., mice) and thus oral delivery of inhalation test materials (e.g., therapeutics) does not result in good lung exposure. Thus, in rodents, delivery of therapeutic agents to the lungs is typically achieved intranasally, intratracheally, or by inhalation of a systemic aerosol exposure in an exposure chamber.

The exposure room method utilizes a large number of test materials, generally reserved for inhalation toxicology studies rather than pharmacological efficacy studies. Intratracheal administration is a very effective delivery method, as almost all test materials are delivered to the lungs, but this is a completely invasive technique. Especially for the study of mice, it is technically quite laborious, since the trachea diameter is quite small. The intranasal route is less invasive than the intratracheal route and is therefore particularly suitable for repeated administration studies, for example in the 4-11 day mouse model described below. After intranasal administration, approximately 50% of the administered dose will be delivered to the lungs (Eyles JE, Williamson ED and Alpar HO.1999, Int J Pharm, 189 (1): 75-9).

As an alternative to oral inhalation, mice were given intranasally vehicle (0.2% Tween 80 in saline), 5S-pioglitazone (prepared as in example 6) (3. mu.g/kg), 5S-pioglitazone (prepared as in example 6) (1. mu.g/kg), 5R-pioglitazone (prepared as in example 4) (3. mu.g/kg), 5R-pioglitazone (prepared as in example 4) (1. mu.g/kg) or racemic pioglitazone (3. mu.g/kg). The mouse control group received vehicle 1 hour daily before exposure to air (up to 50 min/day). BAL was performed 24 hours after the final TS exposure. All compounds were administered as HCl salts in doses converted to bases.

In a second experiment, mice were dosed intranasally with vehicle (0.2% Tween 80 salt solution), 5S-rosiglitazone (prepared as in example 9) (3. mu.g/kg), 5S-rosiglitazone (prepared as in example 9) (10. mu.g/kg), 5R-rosiglitazone (prepared as in example 8) (3. mu.g/kg), 5R-rosiglitazone (prepared as in example 8) (10. mu.g/kg) or racemic rosiglitazone (10. mu.g/kg). The mouse control group received vehicle 1 hour daily before exposure to air (up to 50 min/day). BAL was performed 24 hours after the final TS exposure. All compounds were administered as HCl salts in doses converted to the free base.

Data management and statistical analysis

All results are presented as individual data points for each animal and the mean of the groups is calculated. Since the test for normal was positive, the data were subjected to one-way analysis of variance test (ANOVA), followed by Bonferroni correction for multiple comparisons to test for significance between treatment groups. A "p" value < 0.05 was considered statistically significant. Percent inhibition of cell data was automatically calculated using an Excel spreadsheet using the following formula:

the inhibition data for the other parameters were calculated manually using the above formula.

As shown in figure 1, there was a clear difference between the activity of both enantiomers of pioglitazone on the total number of BAL cells after exposure to TS. Pioglitazone 5R-enantiomer (e.e.97.8%) significantly inhibited BAL cell influx induced by TS when administered by intranasal routes of 1. mu.g/kg and 3. mu.g/kg. In contrast, 5S-enantiomer (e.e.97.5%) failed to inhibit BAL cell inflammation at any of the doses determined.

After observation of BAL cell spin smears, BAL neutrophil counts were determined. Consistent with activity on BAL total cells, both doses of pioglitazone 5R-enantiomer significantly inhibited BAL neutrophil counts induced by TS exposure, while pioglitazone 5S-enantiomer did not function (fig. 2).

Racemic pioglitazone (which contains 50% pioglitazone 5R-enantiomer) at a dose of 3 μ g/kg also significantly inhibited the total BAL cell count and BAL neutrophils induced by TS.

As shown in figure 3, the activity of both enantiomers of rosiglitazone on the total number of BAL cells was significantly different after exposure to TS. Rosiglitazone 5R-enantiomer (e.e.85.7%) significantly inhibited TS-induced influx of BAL cells when administered by intranasal routes of 3 and 10 μ g/kg. In contrast, 5S-enantiomer (e.e.92.7%) failed to inhibit BAL cell inflammation at any of the doses determined.

After observation of BAL cell spin smears, BAL neutrophil counts were determined. Consistent with activity on BAL total cells, both doses of rosiglitazone 5R-enantiomer significantly inhibited BAL neutrophil counts induced by TS exposure, while rosiglitazone 5S-enantiomer did not (fig. 2).

Racemic rosiglitazone (which contains 50% of the 5R-enantiomer of rosiglitazone) at a dose of 10 μ g/kg also significantly inhibited the total BAL cell count and BAL neutrophils induced by TS.

In summary, the results of both studies identified the 5R-enantiomer of both pioglitazone and rosiglitazone as having the anti-inflammatory activity required to inhibit BAL cell influx, whereas the 5S-enantiomer did not.

Although the enantiomeric excess of the rosiglitazone 5R-enantiomeric preparation was less than optimal (i.e. 85.7% rather than > 90%), a difference in activity of the two enantiomers was observed. This indicates that the optimal preparation of the 5R-enantiomer of rosiglitazone may have similar or even higher activity than the data provided herein. Thus, the data provided herein represent the effective effect obtainable for a preparation containing at least 95% by weight of the 5R-enantiomer of rosiglitazone.

Claims

1. Use of a glitazone in the manufacture of a medicament for pulmonary administration by inhalation for the treatment of an inflammatory respiratory disease wherein the glitazone content of the medicament consists of at least 95% by weight of the 5R enantiomer and less than 5% by weight of the 5S enantiomer, and wherein the glitazone is pioglitazone or rosiglitazone.

2. The use as claimed in claim 1, wherein the inflammatory respiratory disease is selected from mild asthma, moderate asthma, severe asthma, steroid-resistant asthma, bronchitis, chronic obstructive pulmonary disease, cystic fibrosis, pulmonary edema, pulmonary embolism, pneumonia, pulmonary sarcoidosis, silicosis, pulmonary fibrosis, respiratory failure, acute respiratory distress syndrome, emphysema, tuberculosis and lung cancer.

3. The use as claimed in claim 2, wherein the bronchitis is chronic bronchitis.

4. The use as claimed in claim 1 wherein the inflammatory respiratory disease is chronic obstructive pulmonary disease.

5. A pharmaceutical composition suitable for pulmonary administration by inhalation, the composition comprising glitazone and one or more pharmaceutically acceptable carriers and/or excipients, and wherein the glitazone content of the composition consists of at least 95% by weight of the 5R enantiomer and less than 5% by weight of the 5S enantiomer, and wherein the glitazone is pioglitazone or rosiglitazone.

6. A pharmaceutical composition as claimed in claim 5 additionally comprising one or more other therapeutic agents selected from the group consisting of: anti-inflammatory agents, bronchodilators, expectorants, antitussives, leukotriene inhibitors and antibiotics.

7. A kit for treating a respiratory disease in a patient, the kit comprising a first dosage form comprising a composition as claimed in claim 5 and a second dosage form comprising an additional therapeutic agent selected from the group consisting of: anti-inflammatory agents, bronchodilators, expectorants, antitussives, leukotriene inhibitors and antibiotics.