WO2009037503A2 - New combination - 012 for the treatment of respiratory diseases - Google Patents

New combination - 012 for the treatment of respiratory diseases Download PDF

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Publication number
WO2009037503A2
WO2009037503A2 PCT/GB2008/050831 GB2008050831W WO2009037503A2 WO 2009037503 A2 WO2009037503 A2 WO 2009037503A2 GB 2008050831 W GB2008050831 W GB 2008050831W WO 2009037503 A2 WO2009037503 A2 WO 2009037503A2
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Prior art keywords
antagonist
inhibitor
hydroxy
active ingredient
methyl
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PCT/GB2008/050831
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French (fr)
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WO2009037503A3 (en
Inventor
Lilian Alcaraz
Elaine Bridget Cadogan
Stephen Connolly
David John Nicholls
Alan Young
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Astrazeneca Ab
Astrazeneca Uk Limited
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Publication of WO2009037503A2 publication Critical patent/WO2009037503A2/en
Publication of WO2009037503A3 publication Critical patent/WO2009037503A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis

Definitions

  • the present invention relates to a combination of two or more pharmaceutically active substances for use in the treatment of respiratory diseases (for example chronic obstructive pulmonary disease (COPD) or asthma).
  • respiratory diseases for example chronic obstructive pulmonary disease (COPD) or asthma.
  • COPD chronic obstructive pulmonary disease
  • Respiratory diseases include Acute Lung Injury, Acute Respiratory Distress Syndrome (ARDS), occupational lung disease, lung cancer, tuberculosis, fibrosis, pneumoconiosis, pneumonia, emphysema, Chronic Obstructive Pulmonary Disease (COPD) and asthma.
  • ARDS Acute Respiratory Distress Syndrome
  • COPD Chronic Obstructive Pulmonary Disease
  • Asthma is generally defined as an inflammatory disorder of the airways with clinical symptoms arising from intermittent airflow obstruction. It is characterised clinically by paroxysms of wheezing, dyspnea and cough. It is a chronic disabling disorder that appears to be increasing in prevalence and severity. It is estimated that 15% of children and 5% of adults in the population of developed countries suffer from asthma. Therapy should therefore be aimed at controlling symptoms so that normal life is possible and at the same time provide basis for treating the underlying inflammation.
  • COPD is a term which refers to a large group of lung diseases which can interfere with normal breathing.
  • Current clinical guidelines define COPD as a disease state characterized by airflow limitation that is not fully reversible.
  • the airflow limitation is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases.
  • the most important contributory source of such particles and gases is tobacco smoke.
  • COPD patients have a variety of symptoms, including cough, shortness of breath, and excessive production of sputum; such symptoms arise from dysfunction of a number of cellular compartments, including neutrophils, macrophages, and epithelial cells.
  • the two most important conditions covered by COPD are chronic bronchitis and emphysema.
  • Chronic bronchitis is a long-standing inflammation of the bronchi which causes increased production of mucous and other changes. The patients' symptoms are cough and expectoration of sputum. Chronic bronchitis can lead to more frequent and severe respiratory infections, narrowing and plugging of the bronchi, difficult breathing and disability.
  • Emphysema is a chronic lung disease which affects the alveoli and/or the ends of the smallest bronchi.
  • the lung loses its elasticity and therefore these areas of the lungs become enlarged. These enlarged areas trap stale air and do not effectively exchange it with fresh air. This results in difficult breathing and may result in insufficient oxygen being delivered to the blood.
  • the predominant symptom in patients with emphysema is shortness of breath.
  • Corticosteroids also known as glucocorticosteroids or glucocorticoids
  • glucocorticosteroids are potent antiinflammatory agents. Whilst their exact mechanism of action is not clear, the end result of corticosteroid treatment is a decrease in the number, activity and movement of inflammatory cells into the bronchial submucosa, leading to decreased airway responsiveness. Corticosteroids may also cause reduced shedding of bronchial epithelial lining, vascular permeability, and mucus secretion. Whilst corticosteroid treatment can yield important benefits, the efficacy of these agents is often far from satisfactory, particularly in COPD.
  • steroids may lead to therapeutic effects
  • Recent studies have also highlighted the problem of the acquisition of steroid resistance amongst patients suffering from respiratory diseases. For example, cigarette smokers with asthma have been found to be insensitive to short term inhaled corticosteroid therapy, but the disparity of the response between smokers and non-smokers appears to be reduced with high dose inhaled corticosteroid (Tomlinson et al, Thorax 2005;60:282-287).
  • a further class of therapeutic agent used in the treatment of respiratory diseases are bronchodilators.
  • Bronchodilators may be used to alleviate symptoms of respiratory diseases by relaxing the bronchial smooth muscles, reducing airway obstruction, reducing lung hyperinflation and decreasing shortness of breath.
  • Types of bronchodilators in clinical use include ⁇ 2 adrenoceptor agonists, muscarinic receptor antagonists and methylxanthines. Bronchodilators are prescribed mainly for symptomatic relief and they are not considered to alter the natural history of respiratory diseases.
  • Combination products comprising a ⁇ 2 adrenoceptor agonist and a corticosteroid are available.
  • One such product is a combination of budesonide and formoterol fumarate (marketed by AstraZeneca under the tradename Symbicort ®), which has proven to be effective in controlling asthma and COPD, and improving quality of life in many patients.
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one or a salt thereof, and a second active ingredient selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; an antioxidant; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; a CRTh2 antagonist; a DPI antagonist; an Histone Deacetylase Inducer; an IKK2 inhibitor; a COX inhibitor; a lipoxygenase inhibitor; a leukotriene receptor antagonist; a MABA; an MPO inhibitor; a muscarinic antagonist; a p38 inhibitor; a PDE inhibitor; a PP
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2- (2- ⁇ 3-[(2-methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2- one or a salt thereof, and a second active ingredient selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; an antioxidant; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; a CRTh2 antagonist; a DPI antagonist; an Histone Deacetylase Inducer; an IKK2 inhibitor; a COX inhibitor; a lipoxygenase inhibitor; a leukotriene receptor antagonist; an MPO inhibitor; a muscarinic antagonist; a p38 inhibitor; a PDE inhibitor; a PPAR ⁇ agonist;
  • the first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2-methoxy- benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one or a salt thereof, may be in the form of a solvate (such as a hydrate).
  • the present invention provides a pharmaceutical product wherein the first and second active ingredients are in forms suitable for oral administration (for example for delivery to the lungs and/or airways).
  • the pharmaceutical product of the present invention comprises a first active ingredient and a second active ingredient, and it may comprise a third active ingredient.
  • the third active ingredient can be chosen from the list of second active ingredients but would normally have a different mechanism of action. So, for example, the second active ingredient might be a muscarinic antagonist and the third active ingredient might be: a non-steroidal glucocorticosteroid receptor agonist, corticosteroid, a CCRl antagonist or a PDE4 inhibitor.
  • a suitable salt of 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2-methoxy-benzylamino)-methyl]- phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one is, for example, a hydrochloride, hydrobromide (such as dihydrobromide), trifluoroacetate, sulphate, phosphate, acetate, fumarate, maleate, tartrate, lactate, citrate, pyruvate, succinate, oxalate, methanesulphonate, /?-toluenesulphonate, bisulphate, benzenesulphonate, ethanesulphonate, malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate, adipate, formate, glycolate, L-lactate, D-lactate, aspartate, malate, L-tartrate
  • the first and second active ingredients can be administered simultaneously (either in a single pharmaceutical preparation ⁇ that is, the active ingredients are in admixture ⁇ or via separate preparations), or sequentially or separately via separate pharmaceutical preparations.
  • a non-steroidal glucocorticoid receptor (GR) agonist is, for example, a compound disclosed in WO 2006/046916.
  • An antioxidant is, for example, Allopurinol, Erdosteine, Mannitol, N-acetyl cysteine choline ester, N-acetyl cysteine ethyl ester, N- Acetylcysteine, N-Acetylcysteine amide or Niacin.
  • a CCRl antagonist is, for example, a compound disclosed in WO2001/062728 or WO2001/098273, or a pharmaceutically acceptable salt thereof (such as a hydrochloride, trifluoroacetate, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt); BX471
  • a CCRl antagonist is, for example, a compound disclosed in WO2001/062728 or WO2001/098273 [N-(2 ⁇ (2S)-3[ ⁇ (3R)-l-[(4-chlorophenyl)methyl]-3-pyrrolidinyl ⁇ amino]- 2-hydroxypropoxy ⁇ -4-fluorophenyl)acetamide, N-(2 ⁇ (2S)-3 [ ⁇ (3 S)- 1 -[(4- chlorophenyl)methyl]-3-pyrrolidinyl ⁇ amino]-2-hydroxypropoxy ⁇ -4- fluorophenyl)acetamide, N-(2- ⁇ (2S)-3-[ 1 - ⁇ (4-chlorobenzoyl)-4-piperidinyl ⁇ amino]-2- hydroxypropoxy ⁇ -4-hydroxyphenyl)acetamide, (2- ⁇ [(2S)-3- ⁇ [(2R,5 S)- 1 -(4-chlorobenz
  • a CCRl antagonist is, for example, ⁇ /- ⁇ 2-[((25)-3- ⁇ [l-(4-chlorobenzyl)piperidin-4- yljamino ⁇ -2-hydroxy-2-methylpropyl)oxy]-4-hydroxyphenyl ⁇ acetamide (see WO 2003/051839), or, 2- ⁇ 2-Chloro-5- ⁇ [(2S)-3-(5-chloro-l ⁇ ,3H-spiro[l-benzofuran-2,4'- piperidin]- 1 '-yl)-2-hydroxypropyl]oxy ⁇ -4-[(methylamino)carbonyl]phenoxy ⁇ -2- methylpropanoic acid (see PCT publication no. WO 2008/010765), or a pharmaceutically acceptable salt thereof (for example a hydrochloride, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt).
  • a pharmaceutically acceptable salt thereof for example a hydrochloride
  • a chemokine antagonist (other than a CCRl antagonist), for example, 656933 (N-(2- bromophenyl)-N'-(4-cyano- 1 H- 1 ,2,3 -benzotriazol-7-yl)urea), 766994 (4-( ⁇ [( ⁇ [(2R)-4-(3 ,A- dichlorobenzyl)morpholin-2-yl]methyl ⁇ amino)carbonyl]-amino ⁇ methyl)benzamide), CCX-282, CCX-915, Cyanovirin N, E-921, INCB-003284, INCB-9471, Maraviroc, MLN- 3701, MLN-3897, T-487 (N- ⁇ l-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3- d]pyrimidin-2-yl]ethyl ⁇ -N-(pyridin-3-ylmethyl)-2-[
  • a corticosteroid is, for example, Alclometasone dipropionate, Amelometasone,
  • a CRTh2 antagonist is, for example, a compound from WO 2004/106302 or WO 2005/018529.
  • a DPI antagonist is, for example, L888839 or MK0525.
  • An histone deacetylase inducer is, for example, ADC4022, Aminophylline, a Methylxanthine or Theophylline.
  • IKK2 inhibitor is, for example, 2- ⁇ [2-(2-Methylamino-pyrimidin-4-yl)-lH-indole-5- carbonyl]-amino ⁇ -3-(phenyl-pyridin-2-yl-amino)-propionic acid.
  • a COX inhibitor is, for example, Celecoxib, Diclofenac sodium, Etodolac, Ibuprofen, Indomethacin, Meloxicam, Nimesulide, OC1768, OC2125, OC2184, OC499, OCD9101, Parecoxib sodium, Piceatannol, Piroxicam, Rofecoxib or Valdecoxib.
  • a lipoxygenase inhibitor is, for example, Ajulemic acid, Darbufelone, Darbufelone mesilate, Dexibuprofen lysine (monohydrate), Etalocib sodium, Licofelone, Linazolast, Lonapalene, Masoprocol, MN-OOl , Tepoxalin, UCB-35440, Veliflapon, ZD-2138, ZD- 4007 or Zileuton (( ⁇ )-l-(l-Benzo[b]thien-2-ylethyl)-l-hydroxyurea)
  • a leukotriene receptor antagonist is, for example, Ablukast, Iralukast (CGP 45715A), Montelukast, Montelukast sodium, Ontazolast, Pranlukast, Pranlukast hydrate (mono Na salt), Verlukast (MK-679) or Zafirlukast.
  • a MABA compound is a compound having dual activity as both a muscarinic antagonist and as a ⁇ 2 -adrenoceptor agonist, for example a MABA is a compound disclosed in: WO2004089892, WO2004106333, US20040167167, WO2005111004, WO2005051946, WO2006023457, WO2006023460, US20060223858, US20060223859, WO2007107828, WO2008000483, US7317102 or WO2008041095.
  • a MABA is: biphenyl-2- ylcarbamic acid 1 -[2-(4- ⁇ [(R)-2-(3-formylamino-4-hydroxypheny l)-2-hydroxyethylam- 2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl ester; or succinic acid salt and 1,2- ethanedisulfonic of biphenyl-2 -ylcarbamic acid l-[2-(2-chloro-4- ⁇ [(R)-2-hydroxy-2-(8- hydroxy-2-oxo- 1 ,2-dihydroquinolin-5-yl)ethylmino]methyl ⁇ -5- methoxyphenyIcarbamoyl)ethyl]piperidin-4-yl ester; or naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l-(9-[(R)
  • An MPO Inhibitor is, for example, a Hydroxamic acid derivative (N-(4-chloro-2-methyl- phenyl)-4-phenyl-4-[[(4-propan-2-ylphenyl)sulfonylamino]methyl]piperidine- 1 - carboxamide), Piceatannol or Resveratrol.
  • a muscarinic antagonist is, for example, Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine, Tiotropium bromide, Darotropium ((1R, 3R, 5S)-3-(2-cyano-2,2- diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3,2,l]octane bromide), 3(R)-(2-hydroxy-2,2- dithien-2-ylacetoxy)-l-(3-phenoxypropyl)-l-azoniabicyclo[2.2.2]octane bromide (see WO 01/04118), 3(R)-l-phenethyl-3-(9H-xanthene-9-carbonyloxy)-l- azoniabicyclo[2.2.2]
  • a muscarinic antagonist is Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine, Tiotropium bromide, 3(R)-(2-hydroxy-2,2-dithien-2- ylacetoxy)-l-(3-phenoxypropyl)-l-azoniabicyclo[2.2.2]octane bromide, or, 3(R)-I- phenethyl-3-(9H-xanthene-9-carbonyloxy)- 1 -azoniabicyclo[2.2.2]octane bromide, (3R)-3- [(2S)-2-cyclopentyl-2-hydroxy-2-thien-2-ylacetoxy]- 1 -(2-phenoxyethyl)- 1 - azoni
  • a muscarinic antagonist is, for example, Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine or Tiotropium bromide; or an oxazolyl quaternary ammonium salt (such as [2-((S)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]- dimethyl-(3 -phenoxy-propyl)-ammonium salt, [2-((R)-CyC lohexyl-hy droxy-phenyl- methyl)-oxazol-5-ylmethyl]-dimethyl-(3-phenoxy-propyl)-ammonium salt, [2-((R)- Cyc lohexyl-hy droxy-phenyl-methyl
  • a p38 Inhibitor is, for example, a compound from WO 2005/042502, 681323, 856553, AMG548 (2-[[(2S)-2-amino-3-phenylpropyl]amino]-3-methyl-5-(2-naphthalenyl)-6-(4- pyridinyl)-4(3H)-pyrimidinone), Array-797, AZD6703, Doramapimod, KC-706, PH 797804, R1503, SC-80036, SCIO469, 6-chloro-5-[[(2S,5i?)-4-[(4-fluorophenyl)methyl]- 2,5-domethyl-l-piperazinyl]carbonyl]-N,N,l-trimethyl- ⁇ -oxo-lH-indole-3-acetamide, VX702 or VX745 (5-(2,6-dichlorophenyl)-2-(phenylthio)-6H-pyrimi
  • a PDE Inhibitor such as a PDE4 inhibitor is, for example, 256066, Arofylline (3-(4- chlorophenyl)-3,7-dihydro-l -propyl- lH-Purine-2,6-dione), AWD 12-281 (N-(3,5-dichloro- 4-pyridinyl)-l-[(4-fluorophenyl)methyl]-5-hydroxy- ⁇ -oxo-lH-indole-3-acetamide), BAY 19-8004 (Bayer), CDC-801 (Calgene), Celgene compound (( ⁇ R)- ⁇ -(3,4- dimethoxyphenyl)-l,3-dihydro-l-oxo-2H-isoindole-2-propanamide), Cilomilast (cis-4- cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-cyclohexanecarboxylic acid),
  • a PDE5 Inhibitor is, for example, Gamma-glutamyl[s-(2-iodobenzyl)cysteinyl]glycine, Tadalafil, Vardenafil, sildenafil, 4-phenyl-methylamino-6-chloro-2-(l-imidazolyl)- quinazoline, 4-phenyl-methylamino-6-chloro-2-(3-pyridyl)-quinazoline, 1 ,3-dimethyl-6-(2- propoxy-5-methanesulphonylamidophenyl)-l,5-dihydropyrazolo[3,4-d]pyrimidin-4-one or l-cyclopentyl-3-ethyl-6-(3-ethoxy-4-pyridyl)-pyrazolo[3,4-d]pyrimidin-4-one.
  • a PPAR ⁇ agonist is, for example, Pioglitazone, Pioglitazone hydrochloride, Rosiglitazone Maleate, Rosiglitazone Maleate ((-)-enantiomer, free base), Rosiglitazone maleate/Metformin hydrochloride or Tesaglitizar.
  • a Protease Inhibitor is, for example, Alphal -antitrypsin proteinase Inhibitor, EPI-HNE4, UT-77, ZD-0892 or a compound from WO 2006/004532, WO 2005/026123, WO 2002/0744767 or WO 22002/074751; or a TACE Inhibitor (for example DPC-333, Sch- 709156 or Doxycycline).
  • a Statin is, for example, Atorvastatin, Lovastatin, Pravastatin, Rosuvastatin or Simvastatin.
  • a Thromboxane Antagonist is, for example, Ramatroban or Seratrodast.
  • a Vasodilator is, for example, A-306552, Ambrisentan, Avosentan, BMS-248360, BMS- 346567, BMS-465149, BMS-509701, Bosentan, BSF-302146 (Ambrisentan), Calcitonin Gene-related Peptide, Daglutril, Darusentan, Fandosentan potassium, Fasudil, Iloprost, KC-12615 (Daglutril) , KC-12792 2AB (Daglutril) , Liposomal treprostinil, PS-433540, Sitaxsentan sodium, Sodium Ferulate, TBC-11241 (Sitaxsentan), TBC-3214 (N-(2-acetyl- 4,6-dimethylphenyl)-3-[[(4-chloro-3-methyl-5-isoxazolyl)amino]sulfonyl]-2- thiophenecarboxamide), TBC-3711, Trapidil, Tre
  • Any second et seq active ingredient may be in the form of a solvate, for example, a hydrate.
  • the present invention provides a pharmaceutical product comprising the first and second active ingredients in admixture.
  • the pharmaceutical product may, for example, be a kit comprising a preparation of the first active ingredient and a preparation of the second active ingredient and, optionally, instructions for the simultaneous, sequential or separate administration of the preparations to a patient in need thereof.
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; an IKK2 inhibitor; a muscarinic antagonist; a p38 inhibitor; or, a PDE inhibitor.
  • GR Receptor non-steroidal Glucocorticoid Receptor
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient which is a non-steroidal Glucocorticoid Receptor (GR) Agonist for example, a compound disclosed in WO 2006/046916.
  • GR Glucocorticoid Receptor
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient which is a CCRl antagonist, for example, a compound disclosed in WO2001/062728 or WO2001/098273, or a pharmaceutically acceptable salt thereof (such as a hydrochloride, trifluoroacetate, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt); BX471 ((2R)-l-[[2-[(aminocarbonyl)amino]-4- chlorophenoxy]acetyl]-4-[(4-fluorophenyl)methyl]-2-methylpiperazine monohydrochloride) or CCX63
  • a CCRl antagonist is ⁇ /- ⁇ 2-[((25)-3- ⁇ [l-(4-chlorobenzyl)piperidin-4- yljamino ⁇ -2-hydroxy-2-methylpropyl)oxy]-4-hydroxyphenyl ⁇ acetamide, or, 2- ⁇ 2-Chloro- 5- ⁇ [(2S)-3-(5-chloro-l ⁇ ,3H-spiro[l-benzofuran-2,4'-piperidin]-r-yl)-2- hydroxypropyl] oxy ⁇ -4- [(methylamino)carbonyl]phenoxy ⁇ -2-methylpropanoic acid, or a pharmaceutically acceptable salt thereof (for example a hydrochloride, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt).
  • a pharmaceutically acceptable salt thereof for example a hydrochloride, sulphate, (hemi)fumarate, benzoate, furoate or succinate
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient which is a chemokine antagonist (not CCRl), for example, 656933 (N-(2-bromophenyl)-N'-(4-cyano-lH-l,2,3-benzotriazol-7-yl)urea), 766994 (4-( ⁇ [( ⁇ [(2R)-4-(3, 4-dichlorobenzyl)morpholin-2-yl]methyl ⁇ amino)carbonyl]- amino ⁇ methyl)benzamide), CCX-282, CCX-915, Cyanovirin N, E-921, INCB-003284, INCB-9471, Maraviroc
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a corticosteroid, for example, Alclometasone dipropionate, Amelometasone, Beclomethasone dipropionate, Budesonide, Butixocort propionate, Ciclesonide, Clobetasol propionate, Desisobutyrylciclesonide, Etiprednol dicloacetate, Fluocinolone acetonide, Fluticasone Furoate, Fluticasone propionate, Loteprednol etabonate (topical) or Mometasone furoate.
  • a corticosteroid for example, Alclo
  • the corticosteroid is selected from budesonide, fluticasone propionate, fluticasone fruoate mometasone furoate, beclomethasone dipropionate or butixocort propionate ester.
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a corticosteroid, for example, Budesonide, Fluticasone Furoate or Fluticasone propionate.
  • the corticosteroid is budesonide.
  • Budesonide and its preparation is described, for example, in Arzneistoff-Forschung (1979), 29 (11), 1687-1690, DE 2,323,215 and US 3,929,768.
  • Presently available formulations of budesonide are marketed under the tradename 'Entocort ®'.
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is an IKK2 inhibitor, for example, 2- ⁇ [2-(2- Methylamino-pyrimidin-4-yl)- 1 H-indole-5 -carbonyl]-amino ⁇ -3 -(phenyl-pyridin-2-yl- amino)-propionic acid.
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is an MPO Inhibitor, for example, a Hydroxamic acid derivative (N-(4-chloro-2-methyl-phenyl)-4-phenyl-4-[[(4-propan-2- ylphenyl)sulfonylamino]methyl]piperidine-l-carboxamide), Piceatannol or Resveratrol.
  • a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothi
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a MABA compound, for example a compound disclosed in: WO2004089892, WO2004106333, US20040167167, WO2005111004, WO2005051946, WO2006023457, WO2006023460, US20060223858, US20060223859, WO2007107828, WO2008000483, US7317102 or WO2008041095.
  • a MABA is: biphenyl-2-ylcarbamic acid l-[2-(4- ⁇ [(R)-2-(3-formylamino-4- hydroxyphenyl)-2-hydroxyethylam-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl ester; or succinic acid salt and 1 ,2-ethanedisulfonic of biphenyl-2-ylcarbamic acid l-[2-(2- chloro-4- ⁇ [(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydroquinolin-5- yl)ethylmino]methyl ⁇ -5-methoxyphenyIcarbamoyl)ethyl]piperidin-4-yl ester; or naphthalene- 1, 5 -disulfonic acid salt of biphenyl-2-ylcarbamic acid l-(9-[(R)-2-hydroxy
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a muscarinic antagonist, for example, Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine, Tiotropium bromide, Darotropium ((1R, 3R, 5S)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8- azoniabicyclo[3,2,l]octane bromide), 3(R)--(
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a muscarinic antagonist, for example
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a muscarinic antagonist, for example, Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine or Tiotropium bromide; or an oxazolyl quaternary ammonium salt (such as [2-((S)-Cyclohexyl-hydroxy-phenyl-methyl)- oxazol-5-ylmethyl]-dimethyl-(3-phenoxy-propy
  • the muscarinic receptor antagonist is a long acting muscarinic receptor antagonist, that is a muscarinic receptor antagonist with activity that persists for more than 12 hours.
  • long acting muscarinic receptor antagonists include tiotropium bromide.
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is Tiotropium bromide.
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is Oxitropium bromide or Tiotropium bromide; or an oxazolyl quaternary ammonium salt (such as [2-((S)-Cyclohexyl-hydroxy-phenyl- methyl)-oxazol-5 -ylmethyl] -dimethyl-(3-phenoxy-propyl)-ammonium salt, [2-((R)- Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]-dimethyl-(3-phenoxy-propyl)- ammonium salt, [2-((R)- Cyclohe
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a p38 inhibitor, for example, a compound from WO 2005/042502, 681323, 856553, AMG548 (2-[[(2S)-2-amino-3-phenylpropyl]amino]- 3-methyl-5-(2-naphthalenyl)-6-(4-pyridinyl)-4(3H)-pyrimidinone), Array-797, AZD6703, Doramapimod, KC-706, PH 797804, R1503, SC-80036, SCIO469, 6-chloro-5-[[(25,5i?)-4- [(4-fluorine
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a PDE Inhibitor: such as a PDE4 inhibitor ⁇ for example, 256066, Arofylline (3-(4-chlorophenyl)-3,7-dihydro-l-propyl-lH-Purine-2,6- dione), AWD 12-281 (N-(3,5-dichloro-4-pyridinyl)-l-[(4-fluorophenyl)methyl]-5- hydroxy- ⁇ -oxo-lH-indole-3-acetamide), BAY19-8004 (Bayer), CDC-801 (Calgene),
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a PDE4 inhibitor, for example, 256066, Arofylline (3-(4-chlorophenyl)-3,7-dihydro-l-propyl-lH-Purine-2,6-dione), AWD 12-281 (N-(3,5-dichloro-4-pyridinyl)-l-[(4-fluorophenyl)methyl]-5-hydroxy- ⁇ -oxo-lH-indole-3- acetamide), BAY 19-8004 (Bayer), CDC-801 (Calgene), Celgene compound (( ⁇ R)- ⁇
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a PDE4 inhibitor, for example AWD 12-281 (N-(3,5-dichloro-4-pyridinyl)-l-[(4-fluorophenyl)methyl]-5-hydroxy- ⁇ -oxo-lH-indole-3- acetamide) or roflumilast.
  • a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malon
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- ⁇ 3-[(2- methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is roflumilast.
  • the first active ingredient and the second active ingredient of the pharmaceutical product of the present invention may be administered simultaneously, sequentially or separately to treat respiratory diseases.
  • simultaneously is meant that the active ingredients are in admixture, or they could be in separate chambers of the same inhaler.
  • sequential it is meant that the active ingredients are administered, in any order, one immediately after the other. They still have the desired effect if they are administered separately, but when administered in this manner they are generally administered less than 4 hours apart, conveniently less than two hours apart, more conveniently less than 30 minutes apart and most conveniently less than 10 minutes apart, for example less than 10 minutes but not one immediately after the other.
  • the active ingredients of the present invention may be administered by oral or parenteral (e.g. intravenous, subcutaneous, intramuscular or intraarticular) administration using conventional systemic dosage forms, such as tablets, capsules, pills, powders, aqueous or oily solutions or suspensions, emulsions and sterile injectable aqueous or oily solutions or suspensions.
  • the active ingredients may be delivered to the lung and/or airways via oral administration in the form of a solution, suspension, aerosol or dry powder formulation.
  • These dosage forms will usually include one or more pharmaceutically acceptable ingredients which may be selected, for example, from an adjuvant, carrier, binder, lubricant, diluent, stabilising agent, buffering agent, emulsifying agent, viscosity- regulating agent, surfactant, preservative, flavouring or colorant.
  • pharmaceutically acceptable ingredients may be selected, for example, from an adjuvant, carrier, binder, lubricant, diluent, stabilising agent, buffering agent, emulsifying agent, viscosity- regulating agent, surfactant, preservative, flavouring or colorant.
  • the first and second active ingredients are administered via a single pharmaceutical composition (that is, the first and second active ingredients are in admixture). Therefore, the present invention further provides a pharmaceutical composition comprising, in admixture, a first active ingredient which is 4-hydroxy-7-[li?- hydroxy-2-(2- ⁇ 3-[(2-methoxy-benzylamino)-methyl]-phenyl ⁇ -ethylamino)-ethyl]-3H- benzothiazol-2-one bis-malonate, and a second active ingredient as defined above.
  • compositions of the present invention can be prepared by mixing the first active ingredient with the second active ingredient and a pharmaceutically acceptable adjuvant, diluent or carrier. Therefore, in a further aspect of the present invention there is provided a process for the preparation of a pharmaceutical composition, which comprises mixing the first and second active ingredients and a pharmaceutically acceptable adjuvant, diluent or carrier.
  • each active ingredient administered in accordance with the present invention will vary depending upon the particular active ingredient employed, the mode by which the active ingredient is to be administered, and the condition or disorder to be treated.
  • the first active ingredient is administered via inhalation.
  • the dose of the first active ingredient (that is 4-hydroxy-7-[ li?-hydroxy-2-(2- ⁇ 3-[(2-methoxy-benzylamino)-methyl]-phenyl ⁇ - ethylamino)-ethyl]-3H-benzothiazol-2-one bis-malonate in: salt form, solvate form, or, solvate of salt form) will generally be in the range of from 0.1 microgram ( ⁇ g) to 5000 ⁇ g, 0.1 to 1000 ⁇ g, 0.1 to 500 ⁇ g, 0.1 to 100 ⁇ g, 0.1 to 50 ⁇ g, 0.1 to 5 ⁇ g, 5 to 5000 ⁇ g, 5 to 1000 ⁇ g, 5 to 500 ⁇ g, 5 to 100 ⁇ g, 5 to 50 ⁇ g, 5 to 10 ⁇ g, 10 to 5000 ⁇ g, 10 to 1000 ⁇ g, 10 to 500 ⁇ g, 10
  • the second active ingredient is administered by inhalation.
  • the dose of the second active ingredient will generally be in the range of from 0.1 microgram ( ⁇ g) to 5000 ⁇ g, 0.1 to 1000 ⁇ g, 0.1 to 500 ⁇ g, 0.1 to 100 ⁇ g, 0.1 to 50 ⁇ g, 0.1 to 5 ⁇ g, 5 to 5000 ⁇ g, 5 to 1000 ⁇ g, 5 to 500 ⁇ g, 5 to 100 ⁇ g, 5 to 50 ⁇ g, 5 to 10 ⁇ g, 10 to 5000 ⁇ g, 10 to 1000 ⁇ g, 10 to 500 ⁇ g, 10 to 100 ⁇ g, 10 to 50 ⁇ g, 20 to 5000 ⁇ g, 20 to 1000 ⁇ g, 20 to 500 ⁇ g, 20 to 100 ⁇ g, 20 to 50 ⁇ g, 50 to 5000 ⁇ g, 50 to 1000 ⁇ g, 50 to 500 ⁇ g, 50 to 100 ⁇ g, 100 to 5000 ⁇ g, 100 to 1000 ⁇ g or 100 to
  • the present invention provides a pharmaceutical product wherein the molar ratio of first active ingredient to second active ingredient is from 1:1000 to 1000:1, such as from 1:100 to 100:1, for example from 1:50 to 50:1, for example 1:20 to 20: 1.
  • the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient as defined above, and a second active ingredient as defined above, wherein each active ingredient is formulated for inhaled administration.
  • the pharmaceutical product is in the form of a pharmaceutical composition comprising the first and second active ingredients in admixture, and which composition is formulated for inhaled administration.
  • the active ingredients of the present invention are conveniently delivered via oral administration by inhalation to the lung and/or airways in the form of a solution, suspension, aerosol or dry powder (such as an agglomerated or ordered mixture) formulation.
  • a metered dose inhaler device may be used to administer the active ingredients, dispersed in a suitable propellant and with or without an additional excipient such as ethanol, a surfactant, lubricant or stabilising agent.
  • a suitable propellant includes a hydrocarbon, chlorofiuorocarbon or a hydrofluoroalkane (e.g. heptafluoroalkane) propellant, or a mixture of any such propellants, for example in a pressurised metered dose inhaler (pMDI).
  • Preferred propellants are P 134a and P227, each of which may be used alone or in combination with other another propellant and/or surfactant and/or other excipient.
  • a nebulised aqueous suspension or, preferably, solution may also be employed, with or without a suitable pH and/or tonicity adjustment, either as a unit-dose or multi-dose formulation.
  • a suitable device for delivering a dry powder is Turbuhaler®.
  • the pharmaceutical product of the present invention can, for example, be administered: via an inhaler having the first and second active ingredients in separate chambers of the inhaler such that on administration the active ingredients mix in either the mouthpiece of the inhaler or the mouth of a patient or both (for simultaneous use); or, where the first and second active ingredients are in separate inhalers, via separate inhalers (for separate or sequential use); or the first and second active ingredients are in admixture in an inhaler when the inhaler is supplied to a patient (for simultaneous use).
  • a dry powder inhaler may be used to administer the active ingredients, alone or in combination with a pharmaceutically acceptable carrier (such as lactose), in the later case either as a finely divided powder or as an ordered mixture.
  • a pharmaceutically acceptable carrier such as lactose
  • the dry powder inhaler may be single dose or multi-dose and may utilise a dry powder or a powder-containing capsule.
  • Metered dose inhaler, nebuliser and dry powder inhaler devices are well known and a variety of such devices is available.
  • the combination of the present invention may be used to treat diseases of the respiratory tract such as obstructive diseases of the airways including: asthma, including bronchial, allergic, intrinsic, extrinsic, exercise-induced, drug-induced (including aspirin and NSAID- induced) and dust-induced asthma, both intermittent and persistent and of all severities, and other causes of airway hyper-responsiveness; chronic obstructive pulmonary disease (COPD); bronchitis, including infectious and eosinophilic bronchitis; emphysema; bronchiectasis; cystic fibrosis; sarcoidosis; farmer's lung and related diseases; hypersensitivity pneumonitis; lung fibrosis, including cryptogenic fibrosing alveolitis, idiopathic interstitial pneumonias, fibrosis complicating anti-neoplastic therapy and chronic infection, including tuberculosis and aspergillosis and other fungal infections; complications of lung transplantation; vascul
  • the present invention further provides a pharmaceutical product according to the invention for simultaneous, sequential or separate use in therapy.
  • the present invention further provides the use of a pharmaceutical product according to the invention in the manufacture of a medicament for the treatment of a respiratory disease, in particular chronic obstructive pulmonary disease, asthma, rhinitis, emphysema or bronchitis (such as chronic obstructive pulmonary disease or asthma; for example chronic obstructive pulmonary disease).
  • a respiratory disease in particular chronic obstructive pulmonary disease, asthma, rhinitis, emphysema or bronchitis (such as chronic obstructive pulmonary disease or asthma; for example chronic obstructive pulmonary disease).
  • the present invention still further provides a method of treating a respiratory disease which comprises simultaneously, sequentially or separately administering:
  • the present invention provides the use of a pharmaceutical product, kit or composition as hereinbefore described for the treatment of a respiratory disease, in particular chronic obstructive pulmonary disease, asthma, rhinitis, emphysema or bronchitis (such as chronic obstructive pulmonary disease or asthma; for example chronic obstructive pulmonary disease).
  • a respiratory disease in particular chronic obstructive pulmonary disease, asthma, rhinitis, emphysema or bronchitis (such as chronic obstructive pulmonary disease or asthma; for example chronic obstructive pulmonary disease).
  • the term “therapy” also includes “prophylaxis” unless there are specific indications to the contrary.
  • the terms “therapeutic” and “therapeutically” should be construed accordingly. Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of, the condition or disorder in question. Persons at risk of developing a particular condition or disorder generally include those having a family history of the condition or disorder, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition or disorder.
  • SCX Solid phase extraction with a sulfonic acid sorbent
  • the reaction was poured onto a mixture of ethyl acetate (3OmL), saturated aqueous sodium thiosulfate solution (3OmL) and saturated aqueous sodium bicarbonate solution (3OmL), and was stirred vigorously for 10 minutes.
  • the aqueous phase was separated and extracted with ethyl actetate (3OmL).
  • the combined organics were washed with water (5OmL), dried (magnesium sulfate) and evaporated.
  • the aqueous phase was extracted with ethyl acetate (3x250mL) and the combined organics were washed with water (25OmL), brine (25OmL), dried over sodium sulfate, filtered and evaporated.
  • the crude material was purified in two portions by column chromatography eluting with dichloromethane: ethyl acetate (95:5 to 80:20 gradient) to give a yellow oil (total of 17.2g).
  • H292 cells were grown in 225cm2 flasks incubator at 37°C, 5% CO 2 in RPMI medium containing, 10% (v/v) FBS (foetal bovine serum) and 2 mM L-glutamine.
  • Adherent H292 cells were removed from tissue culture flasks by treatment with AccutaseTM cell detachment solution for 15 minutes. Flasks were incubated for 15 minutes in a humidified incubator at 37°C, 5% CO 2 . Detached cells were re-suspended in RPMI media (containing 10% (v/v) FBS and 2 mM L-glutamine) at 0.05 x 10 6 cells per mL. 5000 cells in 100 ⁇ L were added to each well of a tissue-culture -treated 96-well plate and the cells incubated overnight in a humidified incubator at 37°C, 5% CO 2 .
  • the culture media was removed and cells were washed twice with 100 ⁇ L assay buffer and replaced with 50 ⁇ L assay buffer (HBSS solution containing 1OmM HEPES pH7.4 and 5 mM glucose). Cells were rested at room temperature for 20 minutes after which time 25 ⁇ L of rolipram (1.2 mM made up in assay buffer containing 2.4% (v/v) dimethylsulphoxide) was added. Cells were incubated with rolipram for 10 minutes after which time Compound Z was added and the cells were incubated for 60 minutes at room temperature. The final rolipram concentration in the assay was 300 ⁇ M and final vehicle concentration was 1.6% (v/v) dimethylsulphoxide. The reaction was stopped by removing supernatants, washing once with 100 ⁇ L assay buffer and replacing with 50 ⁇ L lysis buffer. The cell monolayer was frozen at -80 0 C for 30 minutes (or overnight).
  • the concentration of cAMP (cyclic adenosine monophosphate) in the cell lysate was determined using AlphaScreenTM methodology. The frozen cell plate was thawed for 20 minutes on a plate shaker then 10 ⁇ L of the cell lysate was transferred to a 96-well white plate. 40 ⁇ L of mixed AlphaScreenTM detection beads pre-incubated with biotinylated cAMP, was added to each well and the plate incubated at room temperature for 10 hours in the dark. The AlphaScreenTM signal was measured using an EnVision spectrophotometer (Perkin-Elmer Inc.) with the recommended manufacturer's settings. cAMP concentrations were determined by reference to a calibration curve determined in the same experiment using standard cAMP concentrations.
  • a concentration response curve for Compound Z was constructed and data was fitted to a four parameter logistic equation to determine both the PEC50 and Intrinsic Activity. Intrinsic Activity was expressed as a fraction relative to the maximum activity determined for formoterol in each experiment. A result for Compound Z is in Table 1.
  • Membranes were prepared from human embryonic kidney 293 (HEK293) cells expressing recombinant human ⁇ lo receptor. These were diluted in Assay Buffer (5OmM HEPES, ImM EDTA, 0.1% gelatin, pH 7.4) to provide a final concentration of membranes that gave a clear window between maximum and minimum specific binding.
  • Assay Buffer 5OmM HEPES, ImM EDTA, 0.1% gelatin, pH 7.4
  • Membranes containing recombinant human adrenergic beta 1 receptors were obtained from Euroscreen. These were diluted in Assay Buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, 0.1% gelatin, pH 7.4) to provide a final concentration of membranes that gave a clear window between maximum and minimum specific binding.
  • Assay Buffer 5OmM HEPES, ImM EDTA, 12OmM NaCl, 0.1% gelatin, pH 7.4
  • Iodocyanopindolol (0.036 nM final concentration) and 10 ⁇ L of Compound Z (10x final concentration) were added to each test well.
  • 10 ⁇ L vehicle (10% (v/v) DMSO in Assay Buffer; defining maximum binding) or 10 ⁇ L Propranolol (10 ⁇ M final concentration; defining non-specific binding (NSB)).
  • NBS non-specific binding
  • the plates were incubated for 2 hours at room temperature and then filtered onto PEI coated GF/B filter plates, pre-soaked for 1 hour in Assay Buffer, using a 96-well plate Tomtec cell harvester. Five washes with 250 ⁇ L wash buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, pH 7.4) were performed at 4°C to remove unbound radioactivity. The plates were dried then sealed from underneath using Packard plate sealers and MicroScint-0 (50 ⁇ L) was added to each well. The plates were sealed (TopSeal A) and filter-bound radioactivity was measured with a scintillation counter (TopCount, Packard BioScience) using a 3-minute counting protocol.
  • a scintillation counter TopCount, Packard BioScience
  • Membranes containing recombinant human Dopamine Subtype D2s receptors were obtained from Perkin Elmer. These were diluted in Assay Buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, 0.1% gelatin, pH 7.4) to provide a final concentration of membranes that gave a clear window between maximum and minimum specific binding.
  • Assay Buffer 5OmM HEPES, ImM EDTA, 12OmM NaCl, 0.1% gelatin, pH 7.4
  • Assays were performed in U-bottomed 96-well polypropylene plates. 30 ⁇ L [ 3 H]- spiperone (0.16 nM final concentration) and 30 ⁇ L of Compound Z (10x final concentration) were added to each test well. For each assay plate 8 replicates were obtained for [ 3 H] -spiperone binding in the presence of 30 ⁇ L vehicle (10% (v/v) DMSO in Assay Buffer; defining maximum binding) or 30 ⁇ L Haloperidol (10 ⁇ M final concentration; defining non-specific binding (NSB)). Membranes were then added to achieve a final volume of 300 ⁇ L.
  • vehicle % (v/v) DMSO in Assay Buffer; defining maximum binding
  • 30 ⁇ L Haloperidol (10 ⁇ M final concentration; defining non-specific binding (NSB)
  • the plates were incubated for 2 hours at room temperature and then filtered onto PEI coated GF/B filter plates, pre-soaked for 1 hour in Assay Buffer, using a 96-well plate Tomtec cell harvester. Five washes with 250 ⁇ L wash buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, pH 7.4) were performed at 4°C to remove unbound radioactivity. The plates were dried then sealed from underneath using Packard plate sealers and MicroScint-0 (50 ⁇ L) was added to each well. The plates were sealed (TopSeal A) and filter-bound radioactivity was measured with a scintillation counter (TopCount, Packard BioScience) using a 3-minute counting protocol.
  • a scintillation counter TopCount, Packard BioScience
  • Rats LPS challenge in CRL:CD rats causes an influx of inflammatory cells into the lungs. Rats are challenged either with an aerosol of 0.9% w/v saline or O.lmg/mL LPS in 0.9% saline for 30 min or an intratracheal dose of 0.1-10 ⁇ g/kg. This is repeated up to 8 times according to the experimental protocol. Rats are dosed with vehicle, standard compound or test compound by the appropriate route and frequency at various time points before and after challenge depending upon the experimental protocol. Test compound groups could either be the same compound at different doses or single doses of different compounds or a combination of the two. Test compounds are given by intraperitoneal, intravenous or subcutaneous injection or by inhalation or intratracheal administration.
  • the rats are euthanized at various time points after challenge depending upon the nature of the study, but typically 4hr after LPS challenge with ImL pentobarbitone sodium.
  • a tracheotomy is performed and a cannula inserted.
  • the airway is then lavaged using 3 mL sterile PBS at room temperature.
  • the PBS is left in the airway for 10 seconds before being removed.
  • the PBS containing cells is placed into a 15 mL centrifuge tube on ice. This process is repeated three times. An aliquot of BAL fluid is removed and counted on Sysmex (Sysmex UK, Milton Keynes).
  • Cytospin slides are prepared by adding a 100 ⁇ l aliquot of BAL fluid into cytospin funnels in a Shandon Cytospin3 operated at 700 rpm for 5 min. Slides are stained on the Hema-Tek-2000 automatic slide stainer, using Wright-Giemsa stain and typically, 200 cells are counted under a microscope. Cells are classified as eosinophils, neutrophils and mononuclear cells (mononuclear cells included monocytes, macrophages and lymphocytes) and are expressed as a percentage of the total count.
  • Example 2 Evaluation of compound activity on intra- alveolar neutrophil migration after aerosol challenge with lippopolysaccharride (LPS) in the guinea-pig.
  • LPS lippopolysaccharride
  • mice Male Dunkin-Hartley guinea-pigs (300-60Og) are placed into open fronted guinea-pig holding cones attached at random around a cylindrical aerosol chamber. Guinea-pigs are held in the challenge cones and exposed to an aerosol of vehicle, or LPS at concentrations of 0.1-30 ⁇ g/ml in 0.9%saline per group Aerosols are generated using 2 jet nebulisers per column with a flow rate of 12 L/m. 10ml of the challenge agent is placed into each nebuliser. Alternatively animals receive an intratracheal dose of 0.1-10 ⁇ g/kg. This is repeated up to 8 times according to the experimental protocol.
  • Guinea-pigs are dosed with vehicle, standard compound or test compound by the appropriate route and frequency at various time points before and after challenge depending upon the experimental protocol.
  • Test compound groups could either be the same compound at different doses or single doses of different compounds or a combination of the two.
  • Test compounds are given by intraperitoneal, intravenous or subcutaneous injection or by inhalation or intratracheal administration.
  • Challenged guinea-pigs are killed by anaesthesia overdose (0.5ml Euthetal i.p.) at 4h-24h post challenge. The lungs are then lavaged.
  • HBSS Hanks Buffered Salt Solution
  • EDTA EDTA -free
  • the lavaging is performed with gentle massaging of the chest to ensure appropriate agitation of the fluid in the lungs.
  • the washes are harvested into a 15ml conical, polypropylene centrifuge tube, an aliquot of BAL fluid is removed and counted on Sysmex (Sysmex UK, Milton Keynes).
  • Cytospin slides are prepared by adding a 100 ⁇ l aliquot of BAL fluid into cytospin funnels in a Shandon Cytospin3 operated at 700 rpm for 5 min. Slides are stained on the Hema-Tek-2000 automatic slide stainer, using Wright-Giemsa stain and typically, 200 cells are counted under a microscope. Cells are classified as eosinophils, neutrophils and mononuclear cells (mononuclear cells included monocytes, macrophages and lymphocytes) and are expressed as a percentage of the total count.
  • mice Male C57BL/6/J or BALB/C mice (20-35g) are placed in Perspex exposure boxes in groups of up to 20 and exposed to an aerosol of either 0.3 mg/ml LPS or 0.9% w/v saline.
  • the LPS Sigma, E.Coli, Ref L-3755, Serotype 026:B6, Lot no. 11 lk4078
  • An aerosol is generated using two jet nebulisers operated at a flow rate of 12 L/min (6L/min for each nebuliser) for 15 min.
  • animals receive an intratracheal dose of 0.1-10 ⁇ g/kg. This may be repeated up to 8 times.
  • mice are dosed with vehicle, standard compound or test compound by the appropriate route and frequency at various time points before and after challenge depending upon the experimental protocol.
  • Test compound groups could either be the same compound at different doses or single doses of different compounds or a combination of the two.
  • Test compounds are given by intraperitoneal, intravenous or subcutaneous injection or by inhalation or intratracheal administration.
  • mice are killed with an overdose of Euthatal i.p 30 minutes, l-24hr after LPS challenge.
  • the trachea is cannulated (Portex intravenous cannula) and the airways lavaged with 3 x 0.3ml of Isoton II (Beckman Coulter Ref. 8448011 Lot no.25775).
  • Isoton II Beckman Coulter Ref. 8448011 Lot no.25775
  • lOO ⁇ l of the BALF is added to a cytospin funnel and spun, using a ThermoShandon Cytospin model 3 or 4, at 700 rpm for 5 min.
  • Cells on the slide are stained on the Hema-Tek-2000 automatic slide stainer, using Wright-Giemsa stain and differential cell counts carried out to differentiate eosinophils, neutrophils and lymphomononuclear cells (including monocytes, macrophages and lymphocytes). Typically, 200 cells are counted per slide and each cell type expressed as a percentage of the total count. BALF total white cell count is measured using a Sysmex (Sysmex UK, Milton Keynes).
  • mice Male Dunkin-Hartley guinea-pigs (300-60Og) are weighed and dosed with either vehicle or compound in an appropriate vehicle according to the experimental protocol via the intratracheal route under recoverable gaseous anaesthesia (5% halothane in oxygen). Following dosing, the animals are administered supplemental oxygen and monitored until full recovery. Typically a dose volume of 0.5 mL/kg is used for the intratracheal route. In a dose response study, animals are dosed with compound or vehicle two hours prior to the administration of histamine. Test compound groups could either be the same compound at different doses or single doses of different compounds or a combination of the two.
  • the guinea-pigs are anaesthetised with pentobarbitone (1 mL/kg of 60 mg/mL solution intraperitoneally) approximately 30 minutes prior to the first bronchoconstrictor administration.
  • the trachea is cannulated (Portex intravenous cannula, 200/300/070 (orange) or 200/300/060 (yellow)) and the animal ventilated using a constant volume respiratory pump (Harvard Rodent Ventilator model 683) at a rate of 60 breath/min and a tidal volume of 5 ml/kg.
  • a jugular vein is cannulated (Portex intravenous catheter 200/300/010 (green)) for the administration of histamine or maintenance anaesthetic (0.1 mL of pentobarbitone solution, 60 mg/mL, as required).
  • the animals are then transferred to a Flexivent System (SCIREQ, Montreal, Canada) in order to measure airway resistance.
  • the animals are ventilated (quasi-sinusoidal ventilation pattern) at 60 breaths/min at a tidal volume of 5 mL/kg.
  • a positive end expiratory pressure of 2-3 CmH 2 O is applied.
  • Respiratory resistance is measured using the Flexivent "snapshot" facility (1 second duration, 1 Hz frequency).
  • the animals are given histamine dihydrochloride or methacholine in ascending doses (Histamine; 0.5, 1, 2, 3 and 5 ⁇ g/kg, Lv., methacholine; 3, 10 and 30 ⁇ g/kg, i.v.) at approximately 4-minute intervals via the jugular catheter. After each administration of histamine the peak resistance value is recoreded. Guinea pigs are euthanised with approximately 1.OmL pentobarbitone sodium (Euthatal) intravenously after the completion of the lung function measurements.
  • Percentage bronchoprotection produced by a compound is calculated at each dose of histamine as follows:
  • % change R veh is the mean of the maximum percentage change in airway resistance in the vehicle treated group.
  • Rats are dosed via the appropriate route with vehicle, standard compound or test compound at various time points before and after challenge depending upon the experimental protocol. Rats are euthanised with 0.5 mL pentobarbitone sodium (Euthatal) intraperitoneally at various times after challenge. A tracheotomy is performed and the trachea cannulated. The airway is then lavaged using 3 mL sterile PBS at room temperature. The PBS is left in the airway for 10 seconds before being removed. The PBS containing cells is placed into a 15 mL centrifuge tube on ice. This process is repeated three times. The final volume recovered is recorded. An aliquot of BAL fluid is removed and counted using a Sysmex (Sysmex UK, Milton Keynes).
  • Cytospin slides are prepared by adding a 100 ⁇ l aliquot of BAL fluid into cytospin funnels in a Shandon Cytospin 3 operated at 700 rpm for 5 min. Slides are stained on the Hema- Tek-2000 automatic slide stainer, using Wright-Giemsa stain and typically, 200 cells are counted under a microscope. Cells are classified as eosinophils, neutrophils and mononuclear cells. Mononuclear cells included monocytes, macrophages and lymphocytes.
  • mice 20-25g male BALB/c mice are sensitized to ovalbumin by i.p administration of 100 ⁇ g of grade V ovalbumin (Sigma) adsorbed onto lmg of aluminium hydroxide gel mixture (Fisher Scientific UK) in 0.3 ml saline. Groups of mice are pre-dosed with compound if required, a minimum of two weeks after sensitization. They are then dosed daily for 1-8 days as study protocol specified, with test compound or 0.25 ml vehicle.
  • mice are placed in perspex chambers (20x11x1 lcm, 10 mice max./chamber) and administered an aerosol challenge of 20mg ml "1 ovalbumin for 36 min (8 ml for 18 min followed by another 8 ml for 18 min). Aerosol delivery is achieved using a DeVilbiss jet nebulizer with a flow rate of 61 min "1 . 24h after the last dose the mice are killed with euthatal 0.2 ml i.p.
  • trachea is cannulated using a pink luer mount Portex cannula cut to lcm and the lungs are lavaged using 3 washes of ImI of
  • Isoton IL Isoton IL.
  • cytospins lOO ⁇ l of the BALF is added to a cytospin funnel and spun, using a ThermoShandon Cytospin model 3 or 4, at 700 rpm for 5 min.
  • Cells on the slide are stained on the Hema-Tek-2000 automatic slide stainer, using Wright-Giemsa stain and differential cell counts carried out to differentiate eosinophils, neutrophils and lymphomononuclear cells (including monocytes, macrophages and lymphocytes).
  • BALF total white cell count is measured using a Sysmex (Sysmex UK, Milton Keynes).
  • mice undergo whole body exposure to main stream smoke (50 min/12 cigarettes) and fresh air once or twice a day for 1-9 days.
  • Mice are dosed via the appropriate route with vehicle, standard compound or test compound at various time points before and after challenge depending upon the experimental protocol.
  • mice are either killed with euthatal 0.2 ml i.p. and broncho-aveolar lavage fluid obtained for analysis of white blood cell infiltration (as described above) or lung function is assessed using a Flexivent System (SCIREQ, Montreal, Canada).
  • SCIREQ Flexivent System
  • EMMS forced manoeuvres system
  • Mice are anaesthetised with pentobarbitone (1/lOdilution at a dose volume of 1 mL/kg intraperitoneally).
  • the trachea is cannulated and the animal transferred to the Flexivent System where they are ventilated (quasi-sinusoidal ventilation pattern) at a rate of 150 breath/min and a tidal volume of 10 ml/kg in order to measure airways resistance. Respiratory resistance is measured using the Flexivent "snapshot" facility (1 second duration, 1 Hz frequency).
  • Mice are euthanised with approximately 0.5mL pentobarbitone sodium (Euthatal) intravenously after the completion of the lung function measurements.
  • Example 8 Evaluation of bronchodilator activity in the guinea-pig isolated tracheal ring preparation.
  • Guinea-pigs 300-60Og are killed by cervical dislocation and the trachea removed. After clearing the adherent connective tissue, the trachea is cut into four ring segments (2-3 cartilage rings in width) and suspended in 10ml organ baths containing modified Krebs' solution (gassed with 5% CO 2 , 95% O 2 at 37°C). The tracheal rings are attached to an isometric force transducer for the measurement of isometric tension. The tissues are washed and a force of Ig was applied to each tissue. The rings are precontracted with methacholine (1 ⁇ M) and a cumulative (10 "9 M - 10 "5 M) isoprenaline concentration effect curve is constructed.
  • Responses are expressed as a percentage relaxation of the methacholine induced contraction.
  • the rings are washed and a second concentration of methacholine (l ⁇ M) is added. Once the contraction has reached a plateau isoprenaline or the compound under investigation is added until a maximum effective dose is reached.
  • Data are collected using the ADInstruments chart4forwindows software, which measures the maximum tension generated at each concentration of agonist and the response expressed as percentage relaxation.

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Abstract

The invention provides a pharmaceutical product, kit or composition comprising a first active ingredient which is 4-hydroxy-7-[1R-hydroxy-2-(2-{3-[(2-methoxy-benzylamino)- methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one or a salt thereof, and a second active ingredient selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; an antioxidant; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; a CRTh2 antagonist; a DPI antagonist; an Histone Deacetylase Inducer; an IKK2 inhibitor; a COX inhibitor; a lipoxygenase inhibitor; a leukotriene receptor antagonist; a MABA,; an MPO inhibitor; a muscarinic antagonist; a p38 inhibitor; a PDE inhibitor; a PPARγ agonist; a protease inhibitor; a Statin; a thromboxane antagonist; a vasodilator; or, an ENAC blocker (Epithelial Sodium-channel blocker); and its use in the treatment of respiratory disease.

Description

NEW COMBINATION - 012
The present invention relates to a combination of two or more pharmaceutically active substances for use in the treatment of respiratory diseases (for example chronic obstructive pulmonary disease (COPD) or asthma).
The essential function of the lungs requires a fragile structure with enormous exposure to the environment, including pollutants, microbes, allergens, and carcinogens. Host factors, resulting from interactions of lifestyle choices and genetic composition, influence the response to this exposure. Damage or infection to the lungs can give rise to a wide range of diseases of the respiratory system (or respiratory diseases). A number of these diseases are of great public health importance. Respiratory diseases include Acute Lung Injury, Acute Respiratory Distress Syndrome (ARDS), occupational lung disease, lung cancer, tuberculosis, fibrosis, pneumoconiosis, pneumonia, emphysema, Chronic Obstructive Pulmonary Disease (COPD) and asthma.
Among the most common of the respiratory diseases is asthma. Asthma is generally defined as an inflammatory disorder of the airways with clinical symptoms arising from intermittent airflow obstruction. It is characterised clinically by paroxysms of wheezing, dyspnea and cough. It is a chronic disabling disorder that appears to be increasing in prevalence and severity. It is estimated that 15% of children and 5% of adults in the population of developed countries suffer from asthma. Therapy should therefore be aimed at controlling symptoms so that normal life is possible and at the same time provide basis for treating the underlying inflammation.
COPD is a term which refers to a large group of lung diseases which can interfere with normal breathing. Current clinical guidelines define COPD as a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases. The most important contributory source of such particles and gases, at least in the western world, is tobacco smoke. COPD patients have a variety of symptoms, including cough, shortness of breath, and excessive production of sputum; such symptoms arise from dysfunction of a number of cellular compartments, including neutrophils, macrophages, and epithelial cells. The two most important conditions covered by COPD are chronic bronchitis and emphysema.
Chronic bronchitis is a long-standing inflammation of the bronchi which causes increased production of mucous and other changes. The patients' symptoms are cough and expectoration of sputum. Chronic bronchitis can lead to more frequent and severe respiratory infections, narrowing and plugging of the bronchi, difficult breathing and disability.
Emphysema is a chronic lung disease which affects the alveoli and/or the ends of the smallest bronchi. The lung loses its elasticity and therefore these areas of the lungs become enlarged. These enlarged areas trap stale air and do not effectively exchange it with fresh air. This results in difficult breathing and may result in insufficient oxygen being delivered to the blood. The predominant symptom in patients with emphysema is shortness of breath.
Therapeutic agents used in the treatment of respiratory diseases include corticosteroids. Corticosteroids (also known as glucocorticosteroids or glucocorticoids) are potent antiinflammatory agents. Whilst their exact mechanism of action is not clear, the end result of corticosteroid treatment is a decrease in the number, activity and movement of inflammatory cells into the bronchial submucosa, leading to decreased airway responsiveness. Corticosteroids may also cause reduced shedding of bronchial epithelial lining, vascular permeability, and mucus secretion. Whilst corticosteroid treatment can yield important benefits, the efficacy of these agents is often far from satisfactory, particularly in COPD. Moreover, whilst the use of steroids may lead to therapeutic effects, it is desirable to be able to use steroids in low doses to minimise the occurrence and severity of undesirable side effects that may be associated with regular administration. Recent studies have also highlighted the problem of the acquisition of steroid resistance amongst patients suffering from respiratory diseases. For example, cigarette smokers with asthma have been found to be insensitive to short term inhaled corticosteroid therapy, but the disparity of the response between smokers and non-smokers appears to be reduced with high dose inhaled corticosteroid (Tomlinson et al, Thorax 2005;60:282-287). A further class of therapeutic agent used in the treatment of respiratory diseases are bronchodilators. Bronchodilators may be used to alleviate symptoms of respiratory diseases by relaxing the bronchial smooth muscles, reducing airway obstruction, reducing lung hyperinflation and decreasing shortness of breath. Types of bronchodilators in clinical use include β2 adrenoceptor agonists, muscarinic receptor antagonists and methylxanthines. Bronchodilators are prescribed mainly for symptomatic relief and they are not considered to alter the natural history of respiratory diseases.
4-Hydroxy-7-[ li?-hydroxy-2-(2- {3-[(2-methoxy-benzylamino)-methyl]-phenyl} - ethylamino)-ethyl]-3H-benzothiazol-2-one and its bis-malonate, dihydrochloride, bis- trifluoroacetate and tartrate salts are β2 adrenoceptor agonists and are disclosed in PCT/SE2007/000233 (published as WO2007/106016). The compound and its salts show at least a 5-fold selectivity of β2 adrenoceptor agonism over adrenergic αlD, adrenergic βl and dopamine D2 activities.
Combination products comprising a β2 adrenoceptor agonist and a corticosteroid are available. One such product is a combination of budesonide and formoterol fumarate (marketed by AstraZeneca under the tradename Symbicort ®), which has proven to be effective in controlling asthma and COPD, and improving quality of life in many patients.
In view of the complexity of respiratory diseases such as asthma and COPD, it is unlikely that any one mediator can satisfactorily treat a respiratory disease alone. Moreover, whilst combination treatments using a β2 adrenoceptor agonist and a corticosteroid deliver significant patient benefits, there remains a medical need for new therapies against respiratory diseases such as asthma and COPD, in particular for therapies with disease modifying potential.
Accordingly, the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl} -ethylamino)-ethyl]-3H-benzothiazol-2-one or a salt thereof, and a second active ingredient selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; an antioxidant; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; a CRTh2 antagonist; a DPI antagonist; an Histone Deacetylase Inducer; an IKK2 inhibitor; a COX inhibitor; a lipoxygenase inhibitor; a leukotriene receptor antagonist; a MABA; an MPO inhibitor; a muscarinic antagonist; a p38 inhibitor; a PDE inhibitor; a PPARγ agonist; a protease inhibitor; a Statin; a thromboxane antagonist; a vasodilator; or, an ENAC blocker (Epithelial Sodium-channel blocker).
In one particular aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2- (2-{3-[(2-methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2- one or a salt thereof, and a second active ingredient selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; an antioxidant; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; a CRTh2 antagonist; a DPI antagonist; an Histone Deacetylase Inducer; an IKK2 inhibitor; a COX inhibitor; a lipoxygenase inhibitor; a leukotriene receptor antagonist; an MPO inhibitor; a muscarinic antagonist; a p38 inhibitor; a PDE inhibitor; a PPARγ agonist; a protease inhibitor; a Statin; a thromboxane antagonist; a vasodilator; or, an ENAC blocker (Epithelial Sodium-channel blocker).
The first active ingredient, which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2-methoxy- benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one or a salt thereof, may be in the form of a solvate (such as a hydrate).
In one particular aspect the present invention provides a pharmaceutical product wherein the first and second active ingredients are in forms suitable for oral administration (for example for delivery to the lungs and/or airways).
The pharmaceutical product of the present invention comprises a first active ingredient and a second active ingredient, and it may comprise a third active ingredient. The third active ingredient can be chosen from the list of second active ingredients but would normally have a different mechanism of action. So, for example, the second active ingredient might be a muscarinic antagonist and the third active ingredient might be: a non-steroidal glucocorticosteroid receptor agonist, corticosteroid, a CCRl antagonist or a PDE4 inhibitor.
A suitable salt of 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2-methoxy-benzylamino)-methyl]- phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one is, for example, a hydrochloride, hydrobromide (such as dihydrobromide), trifluoroacetate, sulphate, phosphate, acetate, fumarate, maleate, tartrate, lactate, citrate, pyruvate, succinate, oxalate, methanesulphonate, /?-toluenesulphonate, bisulphate, benzenesulphonate, ethanesulphonate, malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate, adipate, formate, glycolate, L-lactate, D-lactate, aspartate, malate, L-tartrate, D-tartrate, stearate, 2- furoate, 3-furoate, napadisylate (naphthalene- 1, 5 -disulfonate or naphthalene- 1 -(sulfonic acid)-5 -sulfonate), edisylate (ethane- 1 ,2-disulfonate or ethane- 1 -(sulfonic acid)-2- sulfonate), isethionate (2-hydroxyethylsulfonate), 2-mesitylenesulphonate, 2- naphthalenesulphonate, 2,5-dichlorobenzenesulphonate, D-mandelate, L-mandelate, cinnamate, benzoate, adipate, esylate, malonate, mesitylate (2-mesitylenesulphonate), napsylate (2-naphthalenesulfonate), camsylate (camphor- 10-sulphonate), formate, glutamate, glutarate, glycolate, hippurate (2-(benzoylamino)acetate), orotate, xylate (p- xylene-2-sulphonate), pamoic (2,2'-dihydroxy- 1 , 1 '-dinaphthylmethane-3 ,3'-dicarboxylate), palmitate or furoate.
The first and second active ingredients can be administered simultaneously (either in a single pharmaceutical preparation {that is, the active ingredients are in admixture} or via separate preparations), or sequentially or separately via separate pharmaceutical preparations.
A non-steroidal glucocorticoid receptor (GR) agonist is, for example, a compound disclosed in WO 2006/046916.
An antioxidant is, for example, Allopurinol, Erdosteine, Mannitol, N-acetyl cysteine choline ester, N-acetyl cysteine ethyl ester, N- Acetylcysteine, N-Acetylcysteine amide or Niacin.
A CCRl antagonist is, for example, a compound disclosed in WO2001/062728 or WO2001/098273, or a pharmaceutically acceptable salt thereof (such as a hydrochloride, trifluoroacetate, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt); BX471
((2R)-l-[[2-[(aminocarbonyl)amino]-4-chlorophenoxy]acetyl]-4-[(4-fluorophenyl)methyl]- 2-methylpiperazine monohydrochloride) or CCX634.
A CCRl antagonist is, for example, a compound disclosed in WO2001/062728 or WO2001/098273 [N-(2{(2S)-3[{(3R)-l-[(4-chlorophenyl)methyl]-3-pyrrolidinyl}amino]- 2-hydroxypropoxy } -4-fluorophenyl)acetamide, N-(2 {(2S)-3 [ {(3 S)- 1 -[(4- chlorophenyl)methyl]-3-pyrrolidinyl}amino]-2-hydroxypropoxy}-4- fluorophenyl)acetamide, N-(2- {(2S)-3-[ 1 - {(4-chlorobenzoyl)-4-piperidinyl} amino]-2- hydroxypropoxy } -4-hydroxyphenyl)acetamide, (2- { [(2S)-3- { [(2R,5 S)- 1 -(4-chlorobenzyl)- 2,5 -dimethylpiperidin-4-yl] amino } -2-hydroxy-2-methylpropyl] oxy} -4-fluorophenyl)acetic acid, (2-{[(2S)-3-{[(3S,4R)-l-(4-chlorobenzyl)-3-methylpiperidin-4-yl]amino}-2-hydroxy- 2-methylpropyl] oxy }-4-fluorophenyl)acetic acid, (2-{[(2S)-3-{[(3R,4R)-l-(4- chlorobenzyl)-3 -methylpiperidin-4-yl] amino } -2-hydroxy-2-methylpropyl] oxy } -4- fluorophenyl)acetic acid, (2-{[(2S)-3-{[(2R,4S,5S)-l-(4-chlorobenzyl)-2,5- dimethylpiperidin-4-yl] amino } -2-hydroxy-2-methylpropyl] oxy} -4-fluorophenyl)acetic acid, (2-{[(2S)-3-{[(2R,4R,5S)-l-(4-chlorobenzyl)-2,5-dimethylpiperidin-4-yl]amino}-2- hydroxy-2-methylpropyl]oxy} -4-fluorophenyl)acetic acid, (2- { [(2S)-3- {[(2S,4R,5R)- 1 -(4- chlorobenzyl)-2,5-dimethylpiperidin-4-yl]amino}-2-hydroxy-2-methylpropyl]oxy}-4- fluorophenyl)acetic acid, (2-{[(2S)-3-{[(2S,4S,5R)-l-(4-chlorobenzyl)-2,5- dimethylpiperidin-4-yl] amino } -2-hydroxy-2-methylpropyl] oxy} -4-fluorophenyl)acetic acid, Methyl (2- { [(2S)-3- {[ 1 -(4-chlorobenzyl)piperidin-4-yl] amino} -2- hydroxypropyl]oxy}-4-fluorophenyl)propanoate, N-[2-({2S}-3-[(l-[4-chlorobenzyl]-4- piperidinyl)amino]-2-hydroxypropoxy)-4-chlorophenyl acetamide, N-[2-({2S}-3-[(l-[4- chlorobenzyl]-4-piperidinyl)amino]-2-hydroxy-2-methylpropoxy)-4-hydroxyphenyl] acetamide, N-[2-({2S} -3-[(l-[4-chlorobenzyl]-4-piperidinyl)amino]-2-hydroxy-2- methylpropoxy)-4-fluorophenyl] acetamide, N- [5 -chloro- [2-( {2S}-3-[(l -[4-chlorobenzyl] - 4-piperidinyl)amino]-2-hydroxy-2-methylpropoxy)-4-hydroxyphenyl] acetamide, N-[5- chloro-[2-({2S}-3-[(l-[4-chlorobenzyl]-4-piperidinyl)amino]-2-hydroxy-2- methylpropoxy)-4-hydroxyphenyl] propaneamide, (2- { [(25)-3 - { [ 1 -(4- chlorobenzyl)piperidin-4-yl]amino}-2-hydroxy-2-methylpropyl]oxy}-4- fluorophenyl)methanesulfonic acid, N-5-chloro-(2-{(2S)-3-[l-{(4-chlorobenzyl)-4- piperidinyl}amino]-2-hydroxypropoxy}-4-hydroxyphenyl)-N'-cyclopropyl-urea, N-(2- {(2S)-3-[ 1 - {(4-chlorobenzyl)-4-piperidinyl} amino] -2-hydroxypropoxy} -phenyl)-N' -ethyl- urea, (2S)-l-(2-ethylphenoxy)-3[(l-[4-chlorobenzyl]4-piperidinyl)amino]propan-2-ol, (2S)- 1 -[2-(-hydroxyethyl)phenoxy]-2-methyl-3 [(I -[4-chlorobenzyl]-4- piperidinyl)amino]propan-2-ol, 2-({2S}-3-[(l-[4-chlorobenzyl]-4-piperidinyl)amino]-2- hydroxy-2-methylpropoxy)benzaldehyde, 2-({2S}-3-[(l-[4-chlorobenzyl]-4- piperidinyl)amino]-2-hydroxypropoxy)-N-cyclopropylbenzamide, Methyl 2-({2S}-3-[(l- [4-chlorobenzyl]-4-piperidinyl)amino]-2-hydroxypropoxy)-4-fluorobenzoate, N-(2-{[(2S)- 3-(5-chloro-l Η,3H-spiro[ 1 -benzofuran-2,4'-piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} -4- hydroxyphenyl)acetamide, N-(2-{[(2S)-3-(5-chloro-l'H-spiro[l,3-benzodioxole-2,4'- piperidin]-r-yl)-2-hydroxypropyl]oxy}-4-hydroxyphenyl)acetamide, 2-{[(2S)-3-(5-chloro- 1 Η,3H-spiro[ 1 -benzofuran-2,4'-piperidin]-r-yl)-2-hydroxypropyl]oxy} -4-hydroxy-N- methylbenzamide, 2- {[(2S)-3-(5-chloro- 1 Η,3H-spiro[l -benzofuran-2,4'-piperidin]- 1 '-yl)- 2-hydroxypropyl]oxy}-4-hydroxybenzoic acid, N-(2-{[(2S)-3-(5-chloro-lΗ,3H-spiro[2- benzofuran- 1 ,4'-piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} -4-hydroxyphenyl)acetamide; 2-{[(2S)-3-(5-chloro-lΗ,3H-spiro[2-benzofuran-l,4'-piperidin]-r-yl)-2- hydroxypropyl]oxy}-4-hydroxy-N-methylbenzamide, N-(2-{[(2S)-3-(5-fluoro-l'H,3H- spiro[ 1 -benzofuran-2,4'-piperidin]-r-yl)-2-hydroxypropyl]oxy} -4- hydroxyphenyl)acetamide, 2-{[(2S)-3-(5-fluoro-lΗ,3H-spiro[l-benzofuran-2,4'- piperidin]-r-yl)-2-hydroxypropyl]oxy}-4-hydroxy-N-methylbenzamide, N-[2-({(2S)-3- [(2R)-5-chloro- 1 Η,3H-spiro[ 1 -benzofuran-2,3'-pyrrolidin]-r-yl]-2-hydroxypropyl} oxy)-4- hydroxyphenyljacetamide, N-(2-{[(2S)-3-(5-chloro-lΗ,3H-spiro[l-benzofuran-2,4'- piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} -4-hydroxyphenyl)urea, 4-fluoro-2- {[(2S)-3-(5- fluoro- 1 Η,3H-spiro[l -benzofuran-2,4'-piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} benzoic acid, N-(2-{[(2S)-3-(5-chloro-lΗ,3H-spiro[l-benzofuran-2,4'-piperidin]-r-yl)-2- hydroxypropyl]oxy}-4-fluorophenyl)urea, N-(2-{[(2S)-2-amino-3-(5-fluoro-lΗ,3H- spiro[ 1 -benzofuran-2,4'-piperidin]- 1 '-yl)propyl]oxy} -4-hydroxyphenyl)acetamide, 2-[(2S)- 3-(5-chlorospiro[benzofuran-2(3H),4'-piperidin]-r-yl)-2-hydroxypropoxy]-benzaldehyde, (αS)-5-chloro-α-[[2-(2-hydroxyethyl)phenoxy]methyl]-Spiro[benzofuran-2(3H),4'- piperidine]- 1 '-ethanol, (αS)-5-chloro-α-[[2-(hydroxymethyl)phenoxy]methyl]- Spiro[benzofuran-2(3H),4'-piperidine]-r-ethanol, N-(2-{[(2S)-3-(5-chloro-lΗ,3H-spiro[l- benzofuran-2,4'-piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} -5-chloro-4- hydroxyphenyl)acetamide, 2-Chloro-5-{[(25)-3-(5-chloro-rH,3H-spiro[l-benzofuran-2,4'- piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} -(4- {acetylamino}phenoxy)acetic acid, 5-{[(2S)-3- (5-Chloro- 1 'H,3H-spiro[l -benzofuran-2,4'-piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} -(4- {acetylamino}phenoxy)acetic acid, {2-Chloro-5-{[(25)-3-(5-chloro-rH,3H-spiro[l- benzofuran-2,4'-piperidin]-r-yl)-2-hydroxypropyl]oxy}-4-
[(methylamino)carbonyl]phenoxy} acetic acid, 2- {2-Chloro-5- {[(25)-3-(5-chloro- 1 'H,3H- spiro[ 1 -benzofuran-2,4'-piperidin]-r-yl)-2-hydroxypropyl]oxy} -4- [(methylamino)carbonyl]phenoxy}-2-methylpropanoic acid, (2-Chloro-5-{[(25)-3-(5- chloro- 1 'H,3H-spiro[l -benzofuran-2,4'-piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} -4- {[(35)-3- hydroxypyrrolidin-l-yl]carbonyl}phenoxy)acetic acid, 5-Chloro-2-{[(25)-3-(5-chloro- 1 'H,3H-spiro[l -benzofuran-2,4'-piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} -4- (cyanomethoxy)benzoic acid, 2-{[(2S)-3-(5-chloro-lΗ,3H-spiro[l-benzofuran-2,4'- piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} -5-chloro-4-(2,2-difluoroethoxy)benzoic, 5-Chloro- 2-{[(25)-3-(5-chloro-rH,3H-spiro[l-benzofuran-2,4'-piperidin]-r-yl)-2- hydroxypropyl]oxy}-4-(3,3,3-trifluoropropoxy)benzoic acid, N-(2-{3-[5-chloro-l'H,3H- spiro[l-benzofuran-2,4'-piperidin]-r-yl]propoxy}phenyl)acetamide, Methyl 3-(2-{[(2S)-3- (5-chloro-rH3H-spiro[l-benzofuran-2,4'-piperidin]-l '-yl)-2-hydroxypropyl]oxy}-4- fluorophenyl)propanoic acid, N-(2-{[(2S)-3-({spiro[indole-2-4'-piperidin]-3(lH)-one}-r- yl)-2-hydroxypropyl]oxy}-4-hydroxyphenyl)acetamide, or (2-{[(25)-3-(5-Chloro-l'H,3H- spiro[ 1 -benzofuran-2,4'-piperidin]-r-yl)-2-hydroxypropyl]oxy} -A- fluorophenyl)methanesulfonic acid, or a pharmaceutically acceptable salt thereof (for example as described above; (such as a hydrochloride, trifluoroacetate, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt))]; BX471 ((2R)-l-[[2- [(aminocarbonyl)amino]-4-chlorophenoxy]acetyl]-4-[(4-fluorophenyl)methyl]-2- methylpiperazine monohydrochloride); or CCX634.
Also, a CCRl antagonist is, for example, Λ/-{2-[((25)-3-{[l-(4-chlorobenzyl)piperidin-4- yljamino} -2-hydroxy-2-methylpropyl)oxy]-4-hydroxyphenyl} acetamide (see WO 2003/051839), or, 2-{2-Chloro-5-{[(2S)-3-(5-chloro-lΗ,3H-spiro[l-benzofuran-2,4'- piperidin]- 1 '-yl)-2-hydroxypropyl]oxy} -4-[(methylamino)carbonyl]phenoxy} -2- methylpropanoic acid (see PCT publication no. WO 2008/010765), or a pharmaceutically acceptable salt thereof (for example a hydrochloride, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt).
A chemokine antagonist (other than a CCRl antagonist), for example, 656933 (N-(2- bromophenyl)-N'-(4-cyano- 1 H- 1 ,2,3 -benzotriazol-7-yl)urea), 766994 (4-( {[({ [(2R)-4-(3 ,A- dichlorobenzyl)morpholin-2-yl]methyl}amino)carbonyl]-amino}methyl)benzamide), CCX-282, CCX-915, Cyanovirin N, E-921, INCB-003284, INCB-9471, Maraviroc, MLN- 3701, MLN-3897, T-487 (N-{l-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3- d]pyrimidin-2-yl]ethyl}-N-(pyridin-3-ylmethyl)-2-[4-(trifluoromethoxy)phenyl]acetamide) or Vicriviroc.
A corticosteroid is, for example, Alclometasone dipropionate, Amelometasone,
Beclomethasone dipropionate, Budesonide, Butixocort propionate, Ciclesonide, Clobetasol propionate, Desisobutyrylciclesonide, Etiprednol dicloacetate, Fluocinolone acetonide, Fluticasone Furoate, Fluticasone propionate, Loteprednol etabonate (topical) or Mometasone furoate.
A CRTh2 antagonist is, for example, a compound from WO 2004/106302 or WO 2005/018529.
A DPI antagonist is, for example, L888839 or MK0525.
An histone deacetylase inducer is, for example, ADC4022, Aminophylline, a Methylxanthine or Theophylline.
An IKK2 inhibitor is, for example, 2-{[2-(2-Methylamino-pyrimidin-4-yl)-lH-indole-5- carbonyl]-amino} -3-(phenyl-pyridin-2-yl-amino)-propionic acid.
A COX inhibitor is, for example, Celecoxib, Diclofenac sodium, Etodolac, Ibuprofen, Indomethacin, Meloxicam, Nimesulide, OC1768, OC2125, OC2184, OC499, OCD9101, Parecoxib sodium, Piceatannol, Piroxicam, Rofecoxib or Valdecoxib.
A lipoxygenase inhibitor is, for example, Ajulemic acid, Darbufelone, Darbufelone mesilate, Dexibuprofen lysine (monohydrate), Etalocib sodium, Licofelone, Linazolast, Lonapalene, Masoprocol, MN-OOl , Tepoxalin, UCB-35440, Veliflapon, ZD-2138, ZD- 4007 or Zileuton ((±)-l-(l-Benzo[b]thien-2-ylethyl)-l-hydroxyurea)
A leukotriene receptor antagonist is, for example, Ablukast, Iralukast (CGP 45715A), Montelukast, Montelukast sodium, Ontazolast, Pranlukast, Pranlukast hydrate (mono Na salt), Verlukast (MK-679) or Zafirlukast.
A MABA compound is a compound having dual activity as both a muscarinic antagonist and as a β2-adrenoceptor agonist, for example a MABA is a compound disclosed in: WO2004089892, WO2004106333, US20040167167, WO2005111004, WO2005051946, WO2006023457, WO2006023460, US20060223858, US20060223859, WO2007107828, WO2008000483, US7317102 or WO2008041095. For example a MABA is: biphenyl-2- ylcarbamic acid 1 -[2-(4- {[(R)-2-(3-formylamino-4-hydroxypheny l)-2-hydroxyethylam- 2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl ester; or succinic acid salt and 1,2- ethanedisulfonic of biphenyl-2 -ylcarbamic acid l-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8- hydroxy-2-oxo- 1 ,2-dihydroquinolin-5-yl)ethylmino]methyl} -5- methoxyphenyIcarbamoyl)ethyl]piperidin-4-yl ester; or naphthalene-l,5-disulfonic acid salt of biphenyl-2-ylcarbamic acid l-(9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydro- quinolin-5-yl)ethylamino]nonyl}piperidin-4-yl ester.
An MPO Inhibitor is, for example, a Hydroxamic acid derivative (N-(4-chloro-2-methyl- phenyl)-4-phenyl-4-[[(4-propan-2-ylphenyl)sulfonylamino]methyl]piperidine- 1 - carboxamide), Piceatannol or Resveratrol.
A muscarinic antagonist is, for example, Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine, Tiotropium bromide, Darotropium ((1R, 3R, 5S)-3-(2-cyano-2,2- diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3,2,l]octane bromide), 3(R)-(2-hydroxy-2,2- dithien-2-ylacetoxy)-l-(3-phenoxypropyl)-l-azoniabicyclo[2.2.2]octane bromide (see WO 01/04118), 3(R)-l-phenethyl-3-(9H-xanthene-9-carbonyloxy)-l- azoniabicyclo[2.2.2]octane bromide or (3R)-3-[(2S)-2-cyclopentyl-2-hydroxy-2-thien-2- ylacetoxy]- 1 -(2 -phenoxyethyl)-l-azoniabicyclo[2.2.2]actane bromide (see WO 01/04118); or a quaternary ammonium salt (such as [2-((S)-Cyclohexyl-hydroxy-phenyl-methyl)- oxazol-5-ylmethyl]-dimethyl-(3-phenoxy-propyl)-ammonium salt, [2-((R)-Cyclohexyl- hydroxy-phenyl-methyl)-oxazol-5 -ylmethyl] -dimethyl-(3 -phenoxy-propyl)-ammonium salt, [2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]- dimethyl-(2- phenethyloxy-ethyl)-ammonium salt, [2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol- 5-ylmethyl]- [3-(3,4-dichloro-phenoxy)-propyl] dimethyl-ammonium salt, [2-((R)- Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]-[2-(3,4-dichloro-benzyloxy)- ethyl]- dimethyl-ammonium salt, [2-(4-Chloro-benzyloxy)-ethyl]-[2-((R)-Cyclohexyl- hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]- dimethyl-ammonium salt, or (i?)-l-[2-(4- Fluoro-phenyl)-ethyl]-3-((5)-2-phenyl-2-piperidin- 1 -yl-propionyloxy)- 1 -azonia- bicyclo[2.2.2]octane; wherein the counter-ion is, for example, chloride, bromide, sulfate, methanesulfonate, benzenesulfonate (besylate), toluenesulfonate (tosylate), napthalene- bissulfonate (napadisylate), phosphate, acetate, citrate, lactate, tartrate, mesylate, maleate, fumarate or succinate.
In one aspect of the invention a muscarinic antagonist is Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine, Tiotropium bromide, 3(R)-(2-hydroxy-2,2-dithien-2- ylacetoxy)-l-(3-phenoxypropyl)-l-azoniabicyclo[2.2.2]octane bromide, or, 3(R)-I- phenethyl-3-(9H-xanthene-9-carbonyloxy)- 1 -azoniabicyclo[2.2.2]octane bromide, (3R)-3- [(2S)-2-cyclopentyl-2-hydroxy-2-thien-2-ylacetoxy]- 1 -(2-phenoxyethyl)- 1 - azoniabicyclo[2.2.2]actane bromide or Darotropium ((1R, 3r, 5S)-3-(2-cyano-2,2- diphenylethy^-δ^-dimethyl-δ-azoniabicyclofS^JJoctane bromide).
In another aspect a muscarinic antagonist is, for example, Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine or Tiotropium bromide; or an oxazolyl quaternary ammonium salt (such as [2-((S)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]- dimethyl-(3 -phenoxy-propyl)-ammonium salt, [2-((R)-CyC lohexyl-hy droxy-phenyl- methyl)-oxazol-5-ylmethyl]-dimethyl-(3-phenoxy-propyl)-ammonium salt, [2-((R)- Cyc lohexyl-hy droxy-phenyl-methyl)-oxazol-5 -ylmethyl] - dimethyl-(2-phenethyloxy- ethyl)-ammonium salt, [2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5 -ylmethyl] - [3-(3,4-dichloro-phenoxy)-propyl] dimethyl-ammonium salt, [2-((R)-Cyclohexyl-hydroxy- phenyl-methyl)-oxazol-5-ylmethyl]-[2-(3,4-dichloro-benzyloxy)-ethyl]- dimethyl- ammonium salt, or [2-(4-Chloro-benzyloxy)-ethyl]-[2-((R)-Cyclohexyl-hydroxy-phenyl- methyl)-oxazol-5 -ylmethyl]- dimethyl-ammonium salt; wherein the counter-ion is, for example, chloride, bromide, sulfate, methanesulfonate, benzenesulfonate (besylate), toluenesulfonate (tosylate), napthalenebissulfonate (napadisylate), phosphate, acetate, citrate, lactate, tartrate, mesylate, maleate, fumarate or succinate).
A p38 Inhibitor is, for example, a compound from WO 2005/042502, 681323, 856553, AMG548 (2-[[(2S)-2-amino-3-phenylpropyl]amino]-3-methyl-5-(2-naphthalenyl)-6-(4- pyridinyl)-4(3H)-pyrimidinone), Array-797, AZD6703, Doramapimod, KC-706, PH 797804, R1503, SC-80036, SCIO469, 6-chloro-5-[[(2S,5i?)-4-[(4-fluorophenyl)methyl]- 2,5-domethyl-l-piperazinyl]carbonyl]-N,N,l-trimethyl-α-oxo-lH-indole-3-acetamide, VX702 or VX745 (5-(2,6-dichlorophenyl)-2-(phenylthio)-6H-pyrimido[l,6-b]pyridazin-6- one).
A PDE Inhibitor: such as a PDE4 inhibitor is, for example, 256066, Arofylline (3-(4- chlorophenyl)-3,7-dihydro-l -propyl- lH-Purine-2,6-dione), AWD 12-281 (N-(3,5-dichloro- 4-pyridinyl)-l-[(4-fluorophenyl)methyl]-5-hydroxy-α-oxo-lH-indole-3-acetamide), BAY 19-8004 (Bayer), CDC-801 (Calgene), Celgene compound ((βR)-β-(3,4- dimethoxyphenyl)-l,3-dihydro-l-oxo-2H-isoindole-2-propanamide), Cilomilast (cis-4- cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-cyclohexanecarboxylic acid), a compound in WO2006098353 from Kyowa Hakko Kogyo Co. Ltd. Japan, 2-(3,5-dichloro-4- pyridinyl)- l-(7-methoxyspiro[ 1 ,3-benzodioxole-2, 1 '-cyclopentan]-4-yl)ethanone (CAS number 185406-34-2)), Compound from Pfizer (2-(3,4-difluorophenoxy)-5-fluoro-N-[cis- 4-[(2-hydroxy-5-methylbenzoyl)amino]cyclohexyl]-)-3-pyridinecarboxamide), Compound from Pfizer (2-(3,4-difluorophenoxy)-5-fluoro-N-[cis-4-[[2-hydroxy-5- (hydroxymethyl)benzoyl]amino]cyclohexyl]-3-pyridinecarboxamide,), CT2820, GPD- 1116, Ibudilast, IC 485, KF 31334, KW-4490 (Kyowa Hakko Kogyo), Lirimilast ([2-(2,4- dichlorobenzoyl)-6-[(methylsulfonyl)oxy]-3-benzofuranyl])-urea), Merck Compound (N- cyclopropyl-1 ,4-dihydro-4-oxo- 1 -[3-(3-pyridinylethynyl)phenyl]-)- 1 ,8-naphthyridine-3- carboxamide), Oglemilast (N-(3,5-dichloro-4-pyridinyl)-4-(difluoromethoxy)-8- [(methylsulfonyl)amino])-l-dibenzofurancarboxamide), ONO6126, ORG 20241 (4-(3,4- dimethoxyphenyl)-N-hydroxy-)-2-thiazolecarboximidamide), PD189659/PD168787 (Parke-Davis), Pentoxifylline (3 ,7-dihydro-3 ,7-dimethyl- 1 -(5 -oxohexyl)-)- 1 H-purine-2,6- dione), Pfizer compound (5-fiuoro-N-[4-[(2-hydroxy-4-methyl- benzoyl)amino]cyclohexyl]-2-(thian-4-yloxy)pyridine-3-carboxamide), Pfizer UK 500,001, Piclamilast (3-(cyclopentyloxy)-N-(3,5-dichloro-4-pyridinyl)-4-methoxy- benzamide), PLX-369 (WO 2006026754), Rofαimilast (3-(cyclopropylmethoxy)-N-(3,5- dichloro-4-pyridinyl)-4-(difluoromethoxy)benzamide), SCH 351591 (N-(3,5-dichloro-l- oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinolinecarboxamide), SeICID(TM) CC- 10004 (Calgene), T-440 (Tanabe), Tetomilast (6-[2-(3,4-diethoxyphenyl)-4-thiazolyl]- 2-pyridinecarboxylic acid), Tofimilast (9-cyclopentyl-7-ethyl-6,9-dihydro-3-(2-thienyl)- 5H-pyrazolo[3,4-c]-l,2,4-triazolo[4,3-a]pyridine), TPI 1100, UCB 101333-3 (N,2- dicyclopropyl-ό^hexahydro- 1 H-azepin- 1 -yl)-5 -methyl-4-pyrimidinamine), V-I l 294 A (Napp), VM554/VM565 (Vernalis), or Zardaverine (6-[4-(difiuoromethoxy)-3- methoxypheny 1] -3 (2H)-pyridazinone) .
A PDE5 Inhibitor is, for example, Gamma-glutamyl[s-(2-iodobenzyl)cysteinyl]glycine, Tadalafil, Vardenafil, sildenafil, 4-phenyl-methylamino-6-chloro-2-(l-imidazolyl)- quinazoline, 4-phenyl-methylamino-6-chloro-2-(3-pyridyl)-quinazoline, 1 ,3-dimethyl-6-(2- propoxy-5-methanesulphonylamidophenyl)-l,5-dihydropyrazolo[3,4-d]pyrimidin-4-one or l-cyclopentyl-3-ethyl-6-(3-ethoxy-4-pyridyl)-pyrazolo[3,4-d]pyrimidin-4-one.
A PPARγ agonist is, for example, Pioglitazone, Pioglitazone hydrochloride, Rosiglitazone Maleate, Rosiglitazone Maleate ((-)-enantiomer, free base), Rosiglitazone maleate/Metformin hydrochloride or Tesaglitizar.
A Protease Inhibitor is, for example, Alphal -antitrypsin proteinase Inhibitor, EPI-HNE4, UT-77, ZD-0892 or a compound from WO 2006/004532, WO 2005/026123, WO 2002/0744767 or WO 22002/074751; or a TACE Inhibitor (for example DPC-333, Sch- 709156 or Doxycycline).
A Statin is, for example, Atorvastatin, Lovastatin, Pravastatin, Rosuvastatin or Simvastatin.
A Thromboxane Antagonist is, for example, Ramatroban or Seratrodast.
A Vasodilator is, for example, A-306552, Ambrisentan, Avosentan, BMS-248360, BMS- 346567, BMS-465149, BMS-509701, Bosentan, BSF-302146 (Ambrisentan), Calcitonin Gene-related Peptide, Daglutril, Darusentan, Fandosentan potassium, Fasudil, Iloprost, KC-12615 (Daglutril) , KC-12792 2AB (Daglutril) , Liposomal treprostinil, PS-433540, Sitaxsentan sodium, Sodium Ferulate, TBC-11241 (Sitaxsentan), TBC-3214 (N-(2-acetyl- 4,6-dimethylphenyl)-3-[[(4-chloro-3-methyl-5-isoxazolyl)amino]sulfonyl]-2- thiophenecarboxamide), TBC-3711, Trapidil, Treprostinil diethanolamine or Treprostinil sodium. An ENAC (Epithelial Sodium-channel blocker) is, for example, Amiloride, Benzamil, Triamterene, 552-02, PSA14984, PSA25569, PSA23682 or AER002.
Any second et seq active ingredient may be in the form of a solvate, for example, a hydrate.
In one particular aspect the present invention provides a pharmaceutical product comprising the first and second active ingredients in admixture. Alternatively, the pharmaceutical product may, for example, be a kit comprising a preparation of the first active ingredient and a preparation of the second active ingredient and, optionally, instructions for the simultaneous, sequential or separate administration of the preparations to a patient in need thereof.
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; an IKK2 inhibitor; a muscarinic antagonist; a p38 inhibitor; or, a PDE inhibitor.
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient which is a non-steroidal Glucocorticoid Receptor (GR) Agonist for example, a compound disclosed in WO 2006/046916. In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient which is a CCRl antagonist, for example, a compound disclosed in WO2001/062728 or WO2001/098273, or a pharmaceutically acceptable salt thereof (such as a hydrochloride, trifluoroacetate, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt); BX471 ((2R)-l-[[2-[(aminocarbonyl)amino]-4- chlorophenoxy]acetyl]-4-[(4-fluorophenyl)methyl]-2-methylpiperazine monohydrochloride) or CCX634.
In another aspect a CCRl antagonist is Λ/-{2-[((25)-3-{[l-(4-chlorobenzyl)piperidin-4- yljamino} -2-hydroxy-2-methylpropyl)oxy]-4-hydroxyphenyl} acetamide, or, 2- {2-Chloro- 5-{[(2S)-3-(5-chloro-lΗ,3H-spiro[l-benzofuran-2,4'-piperidin]-r-yl)-2- hydroxypropyl] oxy } -4- [(methylamino)carbonyl]phenoxy } -2-methylpropanoic acid, or a pharmaceutically acceptable salt thereof (for example a hydrochloride, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt). For example N-{2-[((2S)-3-{[l-(4- chlorobenzyl)piperidin-4-yl]amino}-2-hydroxy-2-methylpropyl)oxy]-4- hydroxyphenyl} acetamide as a benzoate salt, or, 2-{2-Chloro-5-{[(2S)-3-(5-chloro- 1 Η,3H-spiro[ 1 -benzofuran-2,4'-piperidin]- l'-yl)-2-hydroxypropyl]oxy} -4- [(methylamino)carbonyl]phenoxy} -2-methylpropanoic acid as the free acid.
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient which is a chemokine antagonist (not CCRl), for example, 656933 (N-(2-bromophenyl)-N'-(4-cyano-lH-l,2,3-benzotriazol-7-yl)urea), 766994 (4-({[({ [(2R)-4-(3, 4-dichlorobenzyl)morpholin-2-yl]methyl}amino)carbonyl]- amino}methyl)benzamide), CCX-282, CCX-915, Cyanovirin N, E-921, INCB-003284, INCB-9471, Maraviroc, MLN-3701, MLN-3897, T-487 (N-{l-[3-(4-ethoxyphenyl)-4-oxo- 3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-N-(pyridin-3-ylmethyl)-2-[4- (trifluoromethoxy)phenyl]acetamide) or Vicriviroc. In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a corticosteroid, for example, Alclometasone dipropionate, Amelometasone, Beclomethasone dipropionate, Budesonide, Butixocort propionate, Ciclesonide, Clobetasol propionate, Desisobutyrylciclesonide, Etiprednol dicloacetate, Fluocinolone acetonide, Fluticasone Furoate, Fluticasone propionate, Loteprednol etabonate (topical) or Mometasone furoate.
In one embodiment of the present invention the corticosteroid is selected from budesonide, fluticasone propionate, fluticasone fruoate mometasone furoate, beclomethasone dipropionate or butixocort propionate ester.
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a corticosteroid, for example, Budesonide, Fluticasone Furoate or Fluticasone propionate.
In one embodiment of the present invention the corticosteroid is budesonide. Budesonide and its preparation is described, for example, in Arzneimittel-Forschung (1979), 29 (11), 1687-1690, DE 2,323,215 and US 3,929,768. Presently available formulations of budesonide are marketed under the tradename 'Entocort ®'.
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is an IKK2 inhibitor, for example, 2-{[2-(2- Methylamino-pyrimidin-4-yl)- 1 H-indole-5 -carbonyl]-amino } -3 -(phenyl-pyridin-2-yl- amino)-propionic acid. In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is an MPO Inhibitor, for example, a Hydroxamic acid derivative (N-(4-chloro-2-methyl-phenyl)-4-phenyl-4-[[(4-propan-2- ylphenyl)sulfonylamino]methyl]piperidine-l-carboxamide), Piceatannol or Resveratrol.
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl} -ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a MABA compound, for example a compound disclosed in: WO2004089892, WO2004106333, US20040167167, WO2005111004, WO2005051946, WO2006023457, WO2006023460, US20060223858, US20060223859, WO2007107828, WO2008000483, US7317102 or WO2008041095. For example a MABA is: biphenyl-2-ylcarbamic acid l-[2-(4-{[(R)-2-(3-formylamino-4- hydroxyphenyl)-2-hydroxyethylam-2,5-dimethylphenylcarbamoyl)ethyl]piperidin-4-yl ester; or succinic acid salt and 1 ,2-ethanedisulfonic of biphenyl-2-ylcarbamic acid l-[2-(2- chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-l,2-dihydroquinolin-5- yl)ethylmino]methyl} -5-methoxyphenyIcarbamoyl)ethyl]piperidin-4-yl ester; or naphthalene- 1, 5 -disulfonic acid salt of biphenyl-2-ylcarbamic acid l-(9-[(R)-2-hydroxy-2- (8-hydroxy-2-oxo- 1 ,2-dihydro-quinolin-5-yl)ethylamino]nonyl}piperidin-4-yl ester.
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a muscarinic antagonist, for example, Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine, Tiotropium bromide, Darotropium ((1R, 3R, 5S)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8- azoniabicyclo[3,2,l]octane bromide), 3(R)-(2-hydroxy-2,2-dithien-2-ylacetoxy)-l-(3- phenoxypropyl)-l-azoniabicyclo[2.2.2]octane bromide (see WO 01/04118), 3(R)-I- phenethyl-3-(9H-xanthene-9-carbonyloxy)-l-azoniabicyclo[2.2.2]octane bromide or (3R)- 3-[(2S)-2-cyclopentyl-2-hydroxy-2-thien-2-ylacetoxy]- 1 -(2-phenoxyethyl)- 1 - azoniabicyclo[2.2.2]actane bromide (see WO 01/04118); or a quaternary ammonium salt (such as [2-((S)-CyC lohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]-dimethyl-(3- phenoxy-propyl)-ammonium salt, [2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5- ylmethyl]-dimethyl-(3-phenoxy-propyl)-ammonium salt, [2-((R)-Cyclohexyl-hydroxy- phenyl-methyl)-oxazol-5-ylmethyl]- dimethyl-(2-phenethyloxy-ethyl)-ammonium salt, [2- ((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]- [3-(3,4-dichloro-phenoxy)- propyl] dimethyl-ammonium salt, [2-((R)-Cy clohexyl-hydroxy-phenyl-methyl)-oxazol-5- ylmethyl]-[2-(3,4-dichloro-benzyloxy)-ethyl]- dimethyl-ammonium salt, [2-(4-Chloro- benzyloxy)-ethyl]-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]- dimethyl-ammonium salt, or (i?)-l-[2-(4-Fluoro-phenyl)-ethyl]-3-((5)-2-phenyl-2- piperidin-l-yl-propionyloxy)-l-azonia-bicyclo[2.2.2]octane; wherein the counter-ion is, for example, chloride, bromide, sulfate, methanesulfonate, benzenesulfonate (besylate), toluenesulfonate (tosylate), napthalenebissulfonate (napadisylate), phosphate, acetate, citrate, lactate, tartrate, mesylate, maleate, fumarate or succinate.
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a muscarinic antagonist, for example
Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine, Tiotropium bromide, 3(R)-(2- hydroxy-2,2-dithien-2-ylacetoxy)- 1 -(3-phenoxypropyl)- 1 -azoniabicyclo[2.2.2]octane bromide, or, 3(R)-l-phenethyl-3-(9H-xanthene-9-carbonyloxy)-l- azoniabicyclo[2.2.2]octane bromide, (3R)-3-[(2S)-2-cyclopentyl-2-hydroxy-2-thien-2- ylacetoxy]-l -(2-phenoxyethyl)- l-azoniabicyclo[2.2.2]actane bromide or Darotropium ((1R, 3r, 5S)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3,2,l]octane bromide).
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a muscarinic antagonist, for example, Aclidinium bromide, Glycopyrrolate (such as R,R-, R,S-, S,R-, or S,S-glycopyrronium bromide), Oxitropium bromide, Pirenzepine, telenzepine or Tiotropium bromide; or an oxazolyl quaternary ammonium salt (such as [2-((S)-Cyclohexyl-hydroxy-phenyl-methyl)- oxazol-5-ylmethyl]-dimethyl-(3-phenoxy-propyl)-ammonium salt, [2-((R)-Cyclohexyl- hydroxy-phenyl-methyl)-oxazol-5 -ylmethyl] -dimethyl-(3 -phenoxy-propyl)-ammonium salt, [2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]- dimethyl-(2- phenethyloxy-ethyl)-ammonium salt, [2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol- 5-ylmethyl]- [3-(3,4-dichloro-phenoxy)-propyl] dimethyl-ammonium salt, [2-((R)- Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]-[2-(3,4-dichloro-benzyloxy)- ethyl]- dimethyl-ammonium salt, or [2-(4-Chloro-benzyloxy)-ethyl]-[2-((R)-Cyclohexyl- hydroxy-phenyl-methyl)-oxazol-5 -ylmethyl]- dimethyl-ammonium salt; wherein the counter-ion is, for example, chloride, bromide, sulfate, methanesulfonate, benzenesulfonate (besylate), toluenesulfonate (tosylate), napthalenebissulfonate (napadisylate), phosphate, acetate, citrate, lactate, tartrate, mesylate, maleate, fumarate or succinate).
In one aspect of the invention the muscarinic receptor antagonist is a long acting muscarinic receptor antagonist, that is a muscarinic receptor antagonist with activity that persists for more than 12 hours. Examples of long acting muscarinic receptor antagonists include tiotropium bromide.
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is Tiotropium bromide.
In a further aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is Oxitropium bromide or Tiotropium bromide; or an oxazolyl quaternary ammonium salt (such as [2-((S)-Cyclohexyl-hydroxy-phenyl- methyl)-oxazol-5 -ylmethyl] -dimethyl-(3-phenoxy-propyl)-ammonium salt, [2-((R)- Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]-dimethyl-(3-phenoxy-propyl)- ammonium salt, [2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]- dimethyl-(2-phenethyloxy-ethyl)-ammonium salt, [2-((R)-Cyclohexyl-hydroxy-phenyl- methyl)-oxazol-5-ylmethyl]- [3-(3,4-dichloro-phenoxy)-propyl] dimethyl-ammonium salt, [2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-ylmethyl]-[2-(3,4-dichloro- benzyloxy)-ethyl]- dimethyl-ammonium salt, or [2-(4-Chloro-benzyloxy)-ethyl]-[2-((R)- Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5 -ylmethyl] - dimethyl-ammonium salt; wherein the counter-ion is, for example, chloride, bromide, sulfate, methanesulfonate, benzenesulfonate (besylate), toluenesulfonate (tosylate), napthalenebissulfonate (napadisylate), phosphate, acetate, citrate, lactate, tartrate, mesylate, maleate, fumarate or succinate).
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a p38 inhibitor, for example, a compound from WO 2005/042502, 681323, 856553, AMG548 (2-[[(2S)-2-amino-3-phenylpropyl]amino]- 3-methyl-5-(2-naphthalenyl)-6-(4-pyridinyl)-4(3H)-pyrimidinone), Array-797, AZD6703, Doramapimod, KC-706, PH 797804, R1503, SC-80036, SCIO469, 6-chloro-5-[[(25,5i?)-4- [(4-fluorophenyl)methyl]-2,5-domethyl- 1 -piperazinyl]carbonyl]-7V,7V, 1 -trimethyl-α-oxo- lH-indole-3-acetamide, VX702 or VX745 (5-(2,6-dichlorophenyl)-2-(phenylthio)-6H- pyrimido[ 1 ,6-b]pyridazin-6-one).
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a PDE Inhibitor: such as a PDE4 inhibitor {for example, 256066, Arofylline (3-(4-chlorophenyl)-3,7-dihydro-l-propyl-lH-Purine-2,6- dione), AWD 12-281 (N-(3,5-dichloro-4-pyridinyl)-l-[(4-fluorophenyl)methyl]-5- hydroxy-α-oxo-lH-indole-3-acetamide), BAY19-8004 (Bayer), CDC-801 (Calgene), Celgene compound ((βR)-β-(3,4-dimethoxyphenyl)-l,3-dihydro-l-oxo-2H-isoindole-2- propanamide), Cilomilast (cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]- cyclohexanecarboxylic acid), a compound in WO2006098353 from Kyowa Hakko Kogyo Co. Ltd. Japan, 2-(3,5-dichloro-4-pyridinyl)-l-(7-methoxyspiro[l,3-benzodioxole-2,r- cyclopentan]-4-yl)ethanone (CAS number 185406-34-2)), Compound from Pfizer (2-(3,4- difluorophenoxy)-5-fluoro-N-[cis-4-[(2-hydroxy-5-methylbenzoyl)amino]cyclohexyl]-)-3- pyridinecarboxamide), Compound from Pfizer (2-(3,4-difluorophenoxy)-5-fluoro-N-[cis-4- [[2-hydroxy-5-(hydroxymethyl)benzoyl]amino]cyclohexyl]-3-pyridinecarboxamide,), CT2820, GPD-1116, Ibudilast, IC 485, KF 31334, KW-4490 (Kyowa Hakko Kogyo), Lirimilast ([2-(2,4-dichlorobenzoyl)-6-[(methylsulfonyl)oxy]-3-benzofuranyl])-urea), Merck Compound (N-cyclopropyl-l,4-dihydro-4-oxo-l-[3-(3-pyridinylethynyl)phenyl]-)- 1 ,8-naphthyridine-3-carboxamide), Oglemilast (N-(3,5-dichloro-4-pyridinyl)-4- (difluoromethoxy)-8-[(methylsulfonyl)amino])-l-dibenzofurancarboxamide), ONO6126, ORG 20241 (4-(3 ,4-dimethoxyphenyl)-N-hydroxy-)-2-thiazolecarboximidamide), PD 189659/PD 168787 (Parke-Davis), Pentoxifylline (3,7-dihydro-3,7-dimethyl-l-(5- oxohexyl)-)-lH-purine-2,6-dione), Pfizer compound (5-fluoro-N-[4-[(2-hydroxy-4-methyl- benzoyl)amino]cyclohexyl]-2-(thian-4-yloxy)pyridine-3-carboxamide), Pfizer UK 500,001, Piclamilast (3-(cyclopentyloxy)-N-(3,5-dichloro-4-pyridinyl)-4-methoxy- benzamide), PLX-369 (WO 2006026754), Roflumilast (3-(cyclopropylmethoxy)-N-(3,5- dichloro-4-pyridinyl)-4-(difluoromethoxy)benzamide), SCH 351591 (N-(3,5-dichloro-l- oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinolinecarboxamide), SeICID(TM) CC- 10004 (Calgene), T-440 (Tanabe), Tetomilast (6-[2-(3,4-diethoxyphenyl)-4-thiazolyl]- 2-pyridinecarboxylic acid), Tofimilast (9-cyclopentyl-7-ethyl-6,9-dihydro-3-(2-thienyl)- 5H-pyrazolo[3,4-c]-l,2,4-triazolo[4,3-a]pyridine), TPI 1100, UCB 101333-3 (N,2- dicyclopropyl-6-(hexahydro- 1 H-azepin- 1 -yl)-5 -methyl-4-pyrimidinamine), V-I l 294 A (Napp), VM554/VM565 (Vernalis), or Zardaverine (6-[4-(difluoromethoxy)-3- methoxyphenyl]-3(2H)-pyridazinone); or a PDE5 Inhibitor, for example, Gamma- glutamyl[s-(2-iodobenzyl)cysteinyl]glycine, Tadalafil, Vardenafil, sildenafil, 4-phenyl- methylamino-6-chloro-2-( 1 -imidazolyl)-quinazoline, 4-phenyl-methylamino-6-chloro-2- (3-pyridyl)-quinazoline, l,3-dimethyl-6-(2-propoxy-5-methanesulphonylamidophenyl)- l,5-dihydropyrazolo[3,4-d]pyrimidin-4-one or l-cyclopentyl-3-ethyl-6-(3-ethoxy-4- pyridyl)-pyrazolo[3,4-d]pyrimidin-4-one}. In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a PDE4 inhibitor, for example, 256066, Arofylline (3-(4-chlorophenyl)-3,7-dihydro-l-propyl-lH-Purine-2,6-dione), AWD 12-281 (N-(3,5-dichloro-4-pyridinyl)-l-[(4-fluorophenyl)methyl]-5-hydroxy-α-oxo-lH-indole-3- acetamide), BAY 19-8004 (Bayer), CDC-801 (Calgene), Celgene compound ((βR)-β-(3,4- dimethoxyphenyl)-l,3-dihydro-l-oxo-2H-isoindole-2-propanamide), Cilomilast (cis-4- cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-cyclohexanecarboxylic acid), a compound in WO2006098353 from Kyowa Hakko Kogyo Co. Ltd. Japan, 2-(3,5-dichloro-4- pyridinyl)- l-(7-methoxyspiro[ 1 ,3-benzodioxole-2, 1 '-cyclopentan]-4-yl)ethanone (CAS number 185406-34-2)), Compound from Pfizer (2-(3,4-difluorophenoxy)-5-fluoro-N-[cis- 4-[(2-hydroxy-5-methylbenzoyl)amino]cyclohexyl]-)-3-pyridinecarboxamide), Compound from Pfizer (2-(3,4-difluorophenoxy)-5-fluoro-N-[cis-4-[[2-hydroxy-5- (hydroxymethyl)benzoyl]amino]cyclohexyl]-3-pyridinecarboxamide,), CT2820, GPD- 1116, Ibudilast, IC 485, KF 31334, KW-4490 (Kyowa Hakko Kogyo), Lirimilast ([2-(2,4- dichlorobenzoyl)-6-[(methylsulfonyl)oxy]-3-benzofuranyl])-urea), Merck Compound (N- cyclopropyl-1 ,4-dihydro-4-oxo- 1 -[3-(3-pyridinylethynyl)phenyl]-)- 1 ,8-naphthyridine-3- carboxamide), Oglemilast (N-(3,5-dichloro-4-pyridinyl)-4-(difluoromethoxy)-8- [(methylsulfonyl)amino])-l-dibenzofurancarboxamide), ONO6126, ORG 20241 (4-(3,4- dimethoxyphenyl)-N-hydroxy-)-2-thiazolecarboximidamide), PD189659/PD168787 (Parke-Davis), Pentoxifylline (3 ,7-dihydro-3 ,7-dimethyl- 1 -(5 -oxohexyl)-)- 1 H-purine-2,6- dione), Pfizer compound (5-fluoro-N-[4-[(2-hydroxy-4-methyl- benzoyl)amino]cyclohexyl]-2-(thian-4-yloxy)pyridine-3-carboxamide), Pfizer UK 500,001, Piclamilast (3-(cyclopentyloxy)-N-(3,5-dichloro-4-pyridinyl)-4-methoxy- benzamide), PLX-369 (WO 2006026754), Roflumilast (3-(cyclopropylmethoxy)-N-(3,5- dichloro-4-pyridinyl)-4-(difluoromethoxy)benzamide), SCH 351591 (N-(3,5-dichloro-l- oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinolinecarboxamide), SeICID(TM) CC- 10004 (Calgene), T-440 (Tanabe), Tetomilast (6-[2-(3,4-diethoxyphenyl)-4-thiazolyl]- 2-pyridinecarboxylic acid), Tofimilast (9-cyclopentyl-7-ethyl-6,9-dihydro-3-(2-thienyl)- 5H-pyrazolo[3,4-c]-l,2,4-triazolo[4,3-a]pyridine), TPI 1100, UCB 101333-3 (N,2- dicyclopropyl-6-(hexahydro- 1 H-azepin- 1 -yl)-5 -methyl-4-pyrimidinamine), V-I l 294 A (Napp), VM554/VM565 (Vernalis), or Zardaverine (6-[4-(difiuoromethoxy)-3- methoxypheny 1] -3 (2H)-pyridazinone) .
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is a PDE4 inhibitor, for example AWD 12-281 (N-(3,5-dichloro-4-pyridinyl)-l-[(4-fluorophenyl)methyl]-5-hydroxy-α-oxo-lH-indole-3- acetamide) or roflumilast.
In another aspect the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2- methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- malonate, and a second active ingredient is roflumilast.
The first active ingredient and the second active ingredient of the pharmaceutical product of the present invention may be administered simultaneously, sequentially or separately to treat respiratory diseases. By simultaneously is meant that the active ingredients are in admixture, or they could be in separate chambers of the same inhaler. By sequential it is meant that the active ingredients are administered, in any order, one immediately after the other. They still have the desired effect if they are administered separately, but when administered in this manner they are generally administered less than 4 hours apart, conveniently less than two hours apart, more conveniently less than 30 minutes apart and most conveniently less than 10 minutes apart, for example less than 10 minutes but not one immediately after the other.
The active ingredients of the present invention may be administered by oral or parenteral (e.g. intravenous, subcutaneous, intramuscular or intraarticular) administration using conventional systemic dosage forms, such as tablets, capsules, pills, powders, aqueous or oily solutions or suspensions, emulsions and sterile injectable aqueous or oily solutions or suspensions. The active ingredients may be delivered to the lung and/or airways via oral administration in the form of a solution, suspension, aerosol or dry powder formulation. These dosage forms will usually include one or more pharmaceutically acceptable ingredients which may be selected, for example, from an adjuvant, carrier, binder, lubricant, diluent, stabilising agent, buffering agent, emulsifying agent, viscosity- regulating agent, surfactant, preservative, flavouring or colorant. As will be understood by those skilled in the art, the most appropriate method of administering the active ingredients is dependent on a number of factors.
In another embodiment the first and second active ingredients are administered via a single pharmaceutical composition (that is, the first and second active ingredients are in admixture). Therefore, the present invention further provides a pharmaceutical composition comprising, in admixture, a first active ingredient which is 4-hydroxy-7-[li?- hydroxy-2-(2-{3-[(2-methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H- benzothiazol-2-one bis-malonate, and a second active ingredient as defined above.
The pharmaceutical compositions of the present invention can be prepared by mixing the first active ingredient with the second active ingredient and a pharmaceutically acceptable adjuvant, diluent or carrier. Therefore, in a further aspect of the present invention there is provided a process for the preparation of a pharmaceutical composition, which comprises mixing the first and second active ingredients and a pharmaceutically acceptable adjuvant, diluent or carrier.
It will be understood that the therapeutic dose of each active ingredient administered in accordance with the present invention will vary depending upon the particular active ingredient employed, the mode by which the active ingredient is to be administered, and the condition or disorder to be treated.
In one embodiment of the present invention, the first active ingredient is administered via inhalation. When administered via inhalation the dose of the first active ingredient (that is 4-hydroxy-7-[ li?-hydroxy-2-(2- {3-[(2-methoxy-benzylamino)-methyl]-phenyl} - ethylamino)-ethyl]-3H-benzothiazol-2-one bis-malonate in: salt form, solvate form, or, solvate of salt form) will generally be in the range of from 0.1 microgram (μg) to 5000 μg, 0.1 to 1000 μg, 0.1 to 500 μg, 0.1 to 100 μg, 0.1 to 50 μg, 0.1 to 5 μg, 5 to 5000 μg, 5 to 1000 μg, 5 to 500 μg, 5 to 100 μg, 5 to 50 μg, 5 to 10 μg, 10 to 5000 μg, 10 to 1000 μg, 10 to 500 μg, 10 to 100 μg, 10 to 50 μg, 20 to 5000 μg, 20 to 1000 μg, 20 to 500 μg, 20 to 100 μg, 20 to 50 μg, 50 to 5000 μg, 50 to 1000 μg, 50 to 500 μg, 50 to 100 μg, 100 to 5000 μg, 100 to 1000 μg or 100 to 500 μg. The dose will generally be administered from 1 to 4 times a day, conveniently once or twice a day, and most conveniently once a day.
In one embodiment of the present invention the second active ingredient is administered by inhalation. When administered via inhalation the dose of the second active ingredient will generally be in the range of from 0.1 microgram (μg) to 5000 μg, 0.1 to 1000 μg, 0.1 to 500 μg, 0.1 to 100 μg, 0.1 to 50 μg, 0.1 to 5 μg, 5 to 5000 μg, 5 to 1000 μg, 5 to 500 μg, 5 to 100 μg, 5 to 50 μg, 5 to 10 μg, 10 to 5000 μg, 10 to 1000 μg, 10 to 500 μg, 10 to 100 μg, 10 to 50 μg, 20 to 5000 μg, 20 to 1000 μg, 20 to 500 μg, 20 to 100 μg, 20 to 50 μg, 50 to 5000 μg, 50 to 1000 μg, 50 to 500 μg, 50 to 100 μg, 100 to 5000 μg, 100 to 1000 μg or 100 to 500 μg. The dose will generally be administered from 1 to 4 times a day, conveniently once or twice a day, and most conveniently once a day.
In another embodiment the present invention provides a pharmaceutical product wherein the molar ratio of first active ingredient to second active ingredient is from 1:1000 to 1000:1, such as from 1:100 to 100:1, for example from 1:50 to 50:1, for example 1:20 to 20: 1.
In one embodiment, the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient as defined above, and a second active ingredient as defined above, wherein each active ingredient is formulated for inhaled administration. In a further aspect of this embodiment, the pharmaceutical product is in the form of a pharmaceutical composition comprising the first and second active ingredients in admixture, and which composition is formulated for inhaled administration.
The active ingredients of the present invention are conveniently delivered via oral administration by inhalation to the lung and/or airways in the form of a solution, suspension, aerosol or dry powder (such as an agglomerated or ordered mixture) formulation. For example a metered dose inhaler device may be used to administer the active ingredients, dispersed in a suitable propellant and with or without an additional excipient such as ethanol, a surfactant, lubricant or stabilising agent. A suitable propellant includes a hydrocarbon, chlorofiuorocarbon or a hydrofluoroalkane (e.g. heptafluoroalkane) propellant, or a mixture of any such propellants, for example in a pressurised metered dose inhaler (pMDI). Preferred propellants are P 134a and P227, each of which may be used alone or in combination with other another propellant and/or surfactant and/or other excipient. A nebulised aqueous suspension or, preferably, solution may also be employed, with or without a suitable pH and/or tonicity adjustment, either as a unit-dose or multi-dose formulation. A suitable device for delivering a dry powder is Turbuhaler®.
The pharmaceutical product of the present invention can, for example, be administered: via an inhaler having the first and second active ingredients in separate chambers of the inhaler such that on administration the active ingredients mix in either the mouthpiece of the inhaler or the mouth of a patient or both (for simultaneous use); or, where the first and second active ingredients are in separate inhalers, via separate inhalers (for separate or sequential use); or the first and second active ingredients are in admixture in an inhaler when the inhaler is supplied to a patient (for simultaneous use).
A dry powder inhaler may be used to administer the active ingredients, alone or in combination with a pharmaceutically acceptable carrier (such as lactose), in the later case either as a finely divided powder or as an ordered mixture. The dry powder inhaler may be single dose or multi-dose and may utilise a dry powder or a powder-containing capsule.
Metered dose inhaler, nebuliser and dry powder inhaler devices are well known and a variety of such devices is available.
The combination of the present invention may be used to treat diseases of the respiratory tract such as obstructive diseases of the airways including: asthma, including bronchial, allergic, intrinsic, extrinsic, exercise-induced, drug-induced (including aspirin and NSAID- induced) and dust-induced asthma, both intermittent and persistent and of all severities, and other causes of airway hyper-responsiveness; chronic obstructive pulmonary disease (COPD); bronchitis, including infectious and eosinophilic bronchitis; emphysema; bronchiectasis; cystic fibrosis; sarcoidosis; farmer's lung and related diseases; hypersensitivity pneumonitis; lung fibrosis, including cryptogenic fibrosing alveolitis, idiopathic interstitial pneumonias, fibrosis complicating anti-neoplastic therapy and chronic infection, including tuberculosis and aspergillosis and other fungal infections; complications of lung transplantation; vasculitic and thrombotic disorders of the lung vasculature, and pulmonary hypertension; antitussive activity including treatment of chronic cough associated with inflammatory and secretory conditions of the airways, and iatrogenic cough; acute and chronic rhinitis including rhinitis medicamentosa, and vasomotor rhinitis; perennial and seasonal allergic rhinitis including rhinitis nervosa (hay fever); nasal polyposis; acute viral infection including the common cold, and infection due to respiratory syncytial virus, influenza, coronavirus (including SARS) and adenovirus.
Accordingly, the present invention further provides a pharmaceutical product according to the invention for simultaneous, sequential or separate use in therapy.
The present invention further provides the use of a pharmaceutical product according to the invention in the manufacture of a medicament for the treatment of a respiratory disease, in particular chronic obstructive pulmonary disease, asthma, rhinitis, emphysema or bronchitis (such as chronic obstructive pulmonary disease or asthma; for example chronic obstructive pulmonary disease).
The present invention still further provides a method of treating a respiratory disease which comprises simultaneously, sequentially or separately administering:
(a) a therapeutically effective dose of a first active ingredient as defined above; and,
(b) a therapeutically effective dose of a second active ingredient as defined above; to a patient in need thereof.
In a further aspect the present invention provides the use of a pharmaceutical product, kit or composition as hereinbefore described for the treatment of a respiratory disease, in particular chronic obstructive pulmonary disease, asthma, rhinitis, emphysema or bronchitis (such as chronic obstructive pulmonary disease or asthma; for example chronic obstructive pulmonary disease).
In the context of the present specification, the term "therapy" also includes "prophylaxis" unless there are specific indications to the contrary. The terms "therapeutic" and "therapeutically" should be construed accordingly. Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of, the condition or disorder in question. Persons at risk of developing a particular condition or disorder generally include those having a family history of the condition or disorder, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition or disorder.
General Preparative Methods
There follow methods for the bis-malonate and bis-trifluoroacetate salts of 4-hydroxy-7- [li?-hydroxy-2-(2-{3-[(2-methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H- benzothiazol-2-one, and also assays by which the activity of these compounds may be assessed. Data showing the activity of 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2-methoxy- benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- trifluoroacetate (called Compound Z in the assays and Table 1 below) is also presented.
H NMR spectra were recorded on a Varian Inova 400 MHz or a Varian Mercury-VX 300 MHz instrument. The central peaks of chloroform-J (5H 7.27 ppm), dimethylsulfoxide-J,? (δH 2.50 ppm), acetonitrile-Jj (δH 1.95 ppm) or methanol-^ (δH 3.31 ppm) were used as internal references. Column chromatography was carried out using silica gel (0.040-0.063 mm, Merck). Unless stated otherwise, starting materials were commercially available. All solvents and commercial reagents were of laboratory grade and were used as received.
The following method was used for LC/MS analysis: Instrument Agilent 1100; Column Waters Symmetry 2.1 x 30 mm; Mass APCI; Flow rate 0.7 ml/min; Wavelength 254 nm; Solvent A: water + 0.1% TFA; Solvent B: acetonitrile + 0.1% TFA; Gradient 15-95%/B 8 min, 95% B 1 min. Analytical chromatography was run on a Symmetry Cis-column, 2.1 x 30 mm with 3.5 μm particle size, with acetonitrile/water/0.1% trifluoroacetic acid as mobile phase in a gradient from 5% to 95% acetonitrile over 8 minutes at a flow of 0.7 ml/min.
The abbreviations or terms used in the examples have the following meanings: SCX: Solid phase extraction with a sulfonic acid sorbent
HPLC: High performance liquid chromatography DMF: 7V,7V-Dimethylformamide
Preparation 1
7- [2-(2- {3- [(2-Chloro-benzylamino)-methyl] -phenyl}-ethylamino)- lR-hydroxy-ethyl] - 4-hydroxy-3H-benzothiazol-2-one bis-trifluoroacetate salt
Dess-Martin periodinane (450mg) was added to a solution of the (2-chlorobenzyl)-[3-(2- hydroxyethyl)-benzyl]-carbamic acid tert-butyl ester (330mg) in dry dichloromethane
(3OmL) and stirred, under nitrogen, for 1 hour. The reaction was poured onto a mixture of ethyl acetate (3OmL), saturated aqueous sodium thiosulfate solution (3OmL) and saturated aqueous sodium bicarbonate solution (3OmL), and was stirred vigorously for 10 minutes. The aqueous phase was separated and extracted with ethyl actetate (3OmL). The combined organics were washed with water (5OmL), dried (magnesium sulfate) and evaporated. A portion of the crude aldehyde (220mg) was dissolved in methanol (1OmL) and 7-(2-amino- li?-hydroxy-ethyl)-4-hydroxy-3H-benzothiazol-2-one hydrochloride (lOOmg) was added followed by acetic acid (22μL) and stirred under nitrogen. After 1 hour, sodium cyanoborohydride (36mg) was added and the reaction stirred for 18 hours. The reaction was quenched with a few drops of 0.880 ammonia solution and concentrated. The residue was dissolved in acetonitrile/propan-2-ol [1:1] and loaded onto conditioned Tosic-65A resin (2g, Argonaut). The resin was washed with acetonitrile/propan-2-ol [1:1] (5OmL) and eluted with propan-2-ol / 0.880 ammonia solution [4: l](30mL). The elution fraction was concentrated. The residue was dissolved in dichloromethane (5mL) and trifluoroacetic acid (2mL) was added, stirred for 20 minutes and concentrated. Purification by HPLC, method A, and subsequent trituration with diethyl ether afforded the title compound (98mg). m/z 484 (M+H)+ (Agilent multimode)
1H NMR (500MHz, DMSO) δ 11.67 (s, IH), 10.28 (s, IH), 9.49 (s, 2H), 9.00 (s, IH), 8.84 (s, IH), 7.62 (d, IH), 7.55 (d, IH), 7.50 - 7.28 (m, 6H), 6.93 (d, IH), 6.78 (d, IH), 6.51 (s, IH), 4.91 (d, IH), 4.33 - 4.18 (m, 4H), 3.24 - 2.91 (m, 6H).
Preparation 2
4-Hydroxy-7-[lR-hydroxy-2-(2-{3-[(2-methoxybenzylamino)methyl]phenyl}- ethylamino)ethyl] -3H-benzothiazol-2-one bis-trifluoroacetate salt
Prepared from [3-(2-hydroxyethyl)benzyl]-(2-methoxybenzyl)carbamic acid tert-butyl ester (252mg) and 7-(2-amino-li?-hydroxy-ethyl)-4-hydroxy-3H-benzothiazol-2-one hydrochloride (O.lg) using the method of Preparation 1. The crude product was purified by HPLC method B. The purified material was dissolved in acetonitrile (2mL), evaporated and the residue was triturated with diethyl ether and filtered to give the title compound as a white solid (81mg). m/z 480 (M+H)+ (Agilent multimode)
1U NMR (400 MHz, DMSO, 900C) δ 7.39 - 7.30 (m, 6H), 7.06 (d, IH), 6.99 - 6.92 (m, 2H), 6.77 (d, IH), 4.93 - 4.90 (m, IH), 4.14 (s, 2H), 4.09 (s, 2H), 3.81 (s, 3H), 3.25 - 3.00 (m, 6H). 7H exchangeable not seen at elevated temperature.
Preparation 3 (3-Formyl-phenyl)-acetic acid ethyl ester
/V-bromosuccinimide (1.78g) and 2,2'-azobis(2-methylpropionitrile) (16mg) were added to a solution of ethyl-3-methylphenylacetate (1.76mL) in chloroform (18mL) and heated to reflux for 3 hours. On cooling the mixture was diluted with chloroform (4OmL), washed sequentially with saturated aqueous sodium bicarbonate solution (2x50mL), brine (5OmL), dried (sodium sulfate) and concentrated. The residue was dissolved in nitrogen degassed dimethylsulfoxide (5OmL) and sodium bicarbonate (13.5g) added. The mixture was heated at 1000C, under nitrogen for 30min. The reaction was cooled in an ice-bath and poured into brine (30OmL). The aqueous phase was extracted with diethyl ether (3x300mL). The combined organics were dried (sodium sulfate) and concentrated. Purification on silica, eluting with 10% diethyl ether in z'sø-hexane, to afford the subtitle compound as a yellow oil (1.23g). 1R NMR (400 MHz, CDCl3) δ 10.04 (s, IH), 7.82 - 7.78 (m, 2H), 7.59 - 7.48 (m, 2H), 4.17 (q, 2H), 3.70 (s, 2H), 1.26 (t, 3H).
Preparation 4 (2-Chlorobenzyl)-[3-(2-hydroxy-ethyl)-benzyl]-carbamic acid tert-bntyl ester
2-Chlorobenzylamine (0.28g) was added to a solution of (3-formyl-phenyl)-acetic acid ethyl ester (Preparation 3; 0.38g,) and acetic acid (114μL) in ethanol (1OmL). After 1 hour, sodium triacetoxyborohydride (1.27g) was added and the reaction mixture stirred for 18 hours. A few drops of 0.880 ammonia solution were added and the reaction concentrated. The residue was dissolved in ethanol (5mL) and loaded onto a conditioned SCX cartridge (1Og Varian). The cartridge was washed with ethanol (3x20mL) and eluted with ethanol/0.880 ammonia solution [4:l](2xl5mL). The combined elution fractions were evaporated. The residue was dissolved in dimethylformamide (5mL) and a solution of di-tert-butyl dicarbonate (0.48g) in dimethylformamide (5mL) added. The resulting mixture was stirred for 18 hours, then evaporated. The residue was dissolved in ethanol (1OmL) and calcium chloride (0.44g) added, followed by sodium borohydride (0.30g). The reaction mixture was stirred for 3 hours at room temperature. 2M Aqueous potassium carbonate solution (2OmL) was added and the reaction concentrated to ~20mL. The aqueous suspension was extracted with ethyl acetate (3x10OmL). The combined organics were washed with brine (10OmL), dried (magnesium sulfate) and concentrated.
Purification on silica, eluting with z'so-hexane/ethyl acetate [4:1], to afford the subtitle compound as a pale yellow oil (330mg). m/z 376 (M+H)+ (APCI)
1U NMR (300MHz, DMSO, 900C) δ 7.40 - 7.36 (m, IH), 7.33 - 7.17 (m, 4H), 7.12 - 7.00 (m, 3H), 4.46 (s, 2H), 4.37 (s, 2H), 4.25 (t, IH), 3.61 (td, 2H), 2.70 (t, 2H), 1.39 (s, 9H).
Preparation 5
7- [2-(2- {3- [(2-Chloro-benzylamino)-methyl] -phenyl}-ethylamino)- 1/f-hydroxy-ethyl] - 4-hydroxy-3H-benzothiazol-2-one bis-trifluoroacetate salt Dess-Martin periodinane (450mg) was added to a solution of the (2-chlorobenzyl)-[3-(2- hydroxyethyl)-benzyl]-carbamic acid tert-butyl ester (330mg) in dry dichloromethane (3OmL) and stirred, under nitrogen, for 1 hour. The reaction was poured onto a mixture of ethyl acetate (3OmL), saturated aqueous sodium thiosulfate solution (3OmL) and saturated aqueous sodium bicarbonate solution (3OmL), and was stirred vigorously for 10 minutes. The aqueous phase was separated and extracted with ethyl actetate (3OmL). The combined organics were washed with water (5OmL), dried (magnesium sulfate) and evaporated. A portion of the crude aldehyde (220mg) was dissolved in methanol (1OmL) and 7-(2-amino- li?-hydroxy-ethyl)-4-hydroxy-3H-benzothiazol-2-one hydrochloride (lOOmg) was added followed by acetic acid (22μL) and stirred under nitrogen. After 1 hour, sodium cyanoborohydride (36mg) was added and the reaction stirred for 18 hours. The reaction was quenched with a few drops of 0.880 ammonia solution and concentrated. The residue was dissolved in acetonitrile/propan-2-ol [1:1] and loaded onto conditioned Tosic-65A resin (2g, Argonaut). The resin was washed with acetonitrile/propan-2-ol [1:1] (5OmL) and eluted with propan-2-ol / 0.880 ammonia solution [4: l](30mL). The elution fraction was concentrated. The residue was dissolved in dichloromethane (5mL) and trifluoroacetic acid (2mL) was added, stirred for 20 minutes and concentrated. Purification by HPLC, method A, and subsequent trituration with diethyl ether afforded the title compound (98mg). m/z 484 (M+H)+ (Agilent multimode)
1H NMR (500MHz, DMSO) δ 11.67 (s, IH), 10.28 (s, IH), 9.49 (s, 2H), 9.00 (s, IH), 8.84 (s, IH), 7.62 (d, IH), 7.55 (d, IH), 7.50 - 7.28 (m, 6H), 6.93 (d, IH), 6.78 (d, IH), 6.51 (s, IH), 4.91 (d, IH), 4.33 - 4.18 (m, 4H), 3.24 - 2.91 (m, 6H).
Preparation 6
4-Hydroxy-7- [ l/?-hydroxy-2-(2- {3- [(2-methoxy-benzylamino)-methyl] -phenyl}- ethylamino)-ethyl]-3H-benzothiazol-2-one bis-malonate salt
Figure imgf000034_0001
a) 3- [2-(Tetr ahydro-pyran-2-yloxy)-ethyl] -benzaldehyde
To a solution of 2-[2-(3-bromo-phenyl)-ethoxy]-tetrahydro-pyran (2Og) in anhydrous tetrahydrofuran (30OmL) at -780C was added n-butyl lithium (2.5M in hexanes, 33.6ml) dropwise over 5 minutes. The dark solution was kept at -780C for 30 minutes then N, N- dimethylformamide (16.35ml) was added. The mixture was stirred -780C for half hour then quenched with aqueous ammonium acetate, and allowed to warm to room temperature. The resulting mixture was extracted with ethyl acetate, and the organics washed with brine, dried over sodium sulfate and concentrated in vacuo to give a yellow/orange oil (16.Og). The material was used in the next step without further purification. 1R NMR (400 MHz, DMSO) δ 10.00 (s, IH), 7.80 (s, IH), 7.76 (d, IH), 7.61 (d, IH), 7.53 (t,lH), 4.59 - 4.56 (m, IH), 3.84 (dt, IH), 3.66 - 3.53 (m, 2H), 3.40 - 3.33 (m, IH), 2.94 (t, 2H), 1.74 - 1.54 (m, 2H), 1.51 - 1.32 (m, 4H).
b) (2-Methoxy-benzyl)- {3-[2-(tetrahydro-pyran-2-yloxy)-ethyl]-benzyl}-amine
To a solution of 3-[2-(tetrahydro-pyran-2-yloxy)-ethyl]-benzaldehyde (1Og) and 2- methoxybenzylamine (6g) in toluene (15OmL) was added TsOH (lOOmg). The mixture was heated at reflux in a Dean-Stark apparatus for a couple of hours. About ImL water was displaced. The reaction was checked by TLC, z'sø-hexane/EtOAc (4/1, Rf -0.5). The mixture was cooled and washed with aqueous bicarbornate, then concentrated in vacuo. The residue was azeotroped twice with toluene and was taken up in ethanol (150ml). The solution was cooled to 00C and sodium borohydride (1.65g) was added slowly. The reaction was left to warm to room temperature and was stirred overnight. Water was added (15OmL) and ethanol removed under vacuum. The residue was partitioned between aqueous bicarbonate and ethyl acetate. The organic layers were dried over sodium sulfate and concentrated in vacuo to give a yellow oil (6.5 g). The material was used in the next step without further purification. 1R NMR (300 MHz, DMSO) δ 7.33 (dd, IH), 7.27 - 7.13 (m, 4H), 7.09 (d, IH), 6.94 (d, IH), 6.89 (d, IH), 4.56 (t, IH), 3.85 - 3.72 (m, IH), 3.76 (s, 3H), 3.76 (s, 2H), 3.64 (s, 2H), 3.70 - 3.49 (m, 4H), 3.41 - 3.28 (m, IH), 2.81 (t, IH), 2.50 (quintet, IH), 1.75 - 1.29 (m, 4H).
c) (l-Methoxy-benzyO-IS-β-^etrahydro-pyran-l-yloxyJ-ethyll-benzylJ-carbamic acid tert-buty\ ester
To a solution of (2-methoxybenzyl)-{3-[2-(tetrahydro-pyran-2-yloxy)-ethyl]-benzyl}- amine (15g) in dichloromethane (20OmL) at O0C was added 7V,7V-diisopropylamine (3.7ml) and di-tert-butyl dicarbonate (4.2g). The reaction was stirred for 4 hour and allowed to warm to room temperature. The reaction was partitioned between dichloromethane and aqueous bicarbonate. The organic layer was washed with brine, dried over sodium sulfate then concetrated in vacuo to give a yellow oil (18.Ig). The material was used in the next step without further purification. [M+H-BOC] 356
d) (2-Methoxy-benzyl)-{3-[2-(hydroxy)-ethyl]-benzyl}-carbamic acid tert-bntyl ester
(2-Methoxy-benzyl)- {3-[2-(tetrahydro-pyran-2-yloxy)-ethyl]-benzyl} -carbamic acid tert- butyl ester (51 g) was dissolved in a mixture of THF (25OmL), AcOH (225mL) and water (15OmL) and the resulting mixture refluxed for a total of 10 hours. The majority of the tetrahydrofuran and acetic acid was evaporated and the pH of was adjusted to 8 using saturated aqueous sodium bicarbonate solution. The aqueous phase was extracted with ethyl acetate (3x250mL) and the combined organics were washed with water (25OmL), brine (25OmL), dried over sodium sulfate, filtered and evaporated. The crude material was purified in two portions by column chromatography eluting with dichloromethane: ethyl acetate (95:5 to 80:20 gradient) to give a yellow oil (total of 17.2g). 1R NMR (300 MHz, DMSO) δ 7.30 - 7.18 (m, 2H), 7.11 (d, 2H), 7.07 - 6.88 (m, 4H), 4.63 (t, IH), 4.40 - 4.23 (m, 4H), 3.76 (s, 3H), 3.58 (td, 2H), 2.70 (t, 2H), 1.39 (s, 9H).
e) 4-Hydroxy-7- [ li?-hydroxy-2-(2- {3- [(2-methoxy-benzylamino)-methyl] -phenyl}- ethylamino)-ethyl]-3H-benzothiazol-2-one
A vigorously stirred solution of 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2-methoxy- benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one bis- trifluoroacetate prepared according to procedure from (2-methoxy-benzyl)-{3-[2-
(hydroxy)-ethyl]-benzyl} -carbamic acid tert-butyl ester (as decribed in Preparation 2, 56mg) in water (3mL) was treated dropwise with 1 drop of concentrated aqueous ammonia. After stirring for 2 minutes, the resultant precipitate was filtered off and washed with water before being dried under vacuum at room temperature. Yield 26mg m/z 480 (M+H)+ (Agilent multimode)
1R NMR (400MHz, DMSO) δ 7.23 (d, IH), 7.24-7.14 (m, 4H), 7.04 (d, IH), 6.96-6.89 (m, 2H), 6.85 (d, IH), 6.68 (d, IH), 4.61-4.58 (m, IH), 3.76 (s, 3H), 3.67 (s, 2H), 3.65 (s, 2H), 2.80-2.74 (m, 2H), 2.72-2.63 (m, 4H). 5 exchangeable protons not seen at elevated temperature.
f) 4-Hydroxy-7- [ IR -hydroxy-2-(2- {3- [(2-methoxy-benzylamino)-methyl] -phenyl}- ethylamino)-ethyl]-3H-benzothiazol-2-one bis-malonate salt
To a solution 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2-methoxy-benzylamino)-methyl]- phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one (lOOmg) in a mixture of ethanol (2mL) and acetonitrile (1OmL) at reflux (~90°C) was added dropwise a solution of malonic acid (65mg) in ethanol (0.5mL). Precipitate formed during addition but redissolved on complete addition of acid. Acetonitrile was added until slight cloudiness persisted then the mixture refiuxed until the solution cleared. The solution was cooled to ~70-80°C and seed crystals added (~lmg). The mixture was allowed to cool to room temperature and stirred slowly under nitrogen overnight. The solution was further cooled in an ice bath for 2 hours. The solid was collected by filtration, washed with acetonitrile, then with ether (twice) and dried at 500C under high vacuum to give yield 93mg of the title compound. [M+H]+ 480
1R NMR (400 MHz, DMSO) δ 7.41 - 7.28 (m, 4H), 7.31 (s, IH), 7.24 (d, IH), 7.05 (d, IH), 6.97 (t, IH), 6.92 (d, IH), 6.76 (d, IH), 4.89 - 4.82 (m, IH), 4.01 (s, 2H), 3.94 (s, 2H), 3.79 (s, 3H), 3.14 (t, 2H), 3.09 - 2.86 (m, 4H), 2.80 (s, 4H). 7 exchangable protons not observed.
Preparation 7
4-Hydroxy-7- [ lR-hydroxy-2-(2- {3- [(2-methoxy-benzylamino)-methyl] -phenyl}- ethylamino)-ethyl] -3H-benzothiazol-2-one bis-trifluoroacetate salt
Figure imgf000037_0001
a) [3-(2-Hydroxyethyl)benzyl]-(2-methoxybenzyl)carbamic acid tert-butyl ester
Prepared from 2-methoxybenzylamine (0.41 Ig) and (3-formyl-phenyl)-acetic acid ethyl ester (Preparation 3; 356mg) using the method of Preparation 4. Purification on silica, eluting with 60% diethyl ether / z'sø-hexane gave the subtitle compound as a pale yellow oil
(504mg).
1U NMR (300 MHz, CDCl3) δ 7.28 - 7.05 (m, 7H), 6.93 (t, IH), 6.84 (d, IH), 4.49 - 4.38
(m, 4H), 3.84 (q, 2H), 3.78 (s, 3H), 2.84 (t, 2H), 1.47 - 1.47 (m, 9H).
b) 4-Hydroxy-7-[lR-hydroxy-2-(2-{3-[(2- methoxybenzylamino)methyl] phenyl} ethylamino)ethyl] -3H-benzothiazol-2-one bis- trifluoroacetate salt
Prepared from [3-(2-hydroxyethyl)benzyl]-(2-methoxybenzyl)carbamic acid tert-butyl ester (252mg) and 7-(2-amino-li?-hydroxy-ethyl)-4-hydroxy-3H-benzothiazol-2-one hydrochloride (O.lg) using the method of Preparation 5. The crude product was purified by HPLC method B. The purified material was dissolved in acetonitrile (2mL), evaporated and the residue was triturated with diethyl ether and filtered to give the title compound as a white solid (81mg). m/z 480 (M+H)+ (Agilent multimode)
1U NMR (400 MHz, DMSO, 900C) δ 7.39 - 7.30 (m, 6H), 7.06 (d, IH), 6.99 - 6.92 (m, 2H), 6.77 (d, IH), 4.93 - 4.90 (m, IH), 4.14 (s, 2H), 4.09 (s, 2H), 3.81 (s, 3H), 3.25 - 3.00 (m, 6H). 7H exchangeable not seen at elevated temperature.
Adrenergic β2 mediated cAMP production
Cell preparation
H292 cells were grown in 225cm2 flasks incubator at 37°C, 5% CO2 in RPMI medium containing, 10% (v/v) FBS (foetal bovine serum) and 2 mM L-glutamine.
Experimental Method
Adherent H292 cells were removed from tissue culture flasks by treatment with Accutase™ cell detachment solution for 15 minutes. Flasks were incubated for 15 minutes in a humidified incubator at 37°C, 5% CO2. Detached cells were re-suspended in RPMI media (containing 10% (v/v) FBS and 2 mM L-glutamine) at 0.05 x 106 cells per mL. 5000 cells in 100 μL were added to each well of a tissue-culture -treated 96-well plate and the cells incubated overnight in a humidified incubator at 37°C, 5% CO2. The culture media was removed and cells were washed twice with 100 μL assay buffer and replaced with 50 μL assay buffer (HBSS solution containing 1OmM HEPES pH7.4 and 5 mM glucose). Cells were rested at room temperature for 20 minutes after which time 25 μL of rolipram (1.2 mM made up in assay buffer containing 2.4% (v/v) dimethylsulphoxide) was added. Cells were incubated with rolipram for 10 minutes after which time Compound Z was added and the cells were incubated for 60 minutes at room temperature. The final rolipram concentration in the assay was 300 μM and final vehicle concentration was 1.6% (v/v) dimethylsulphoxide. The reaction was stopped by removing supernatants, washing once with 100 μL assay buffer and replacing with 50 μL lysis buffer. The cell monolayer was frozen at -800C for 30 minutes (or overnight).
AlphaS creen™ cAMP detection
The concentration of cAMP (cyclic adenosine monophosphate) in the cell lysate was determined using AlphaScreen™ methodology. The frozen cell plate was thawed for 20 minutes on a plate shaker then 10 μL of the cell lysate was transferred to a 96-well white plate. 40 μL of mixed AlphaScreen™ detection beads pre-incubated with biotinylated cAMP, was added to each well and the plate incubated at room temperature for 10 hours in the dark. The AlphaScreen™ signal was measured using an EnVision spectrophotometer (Perkin-Elmer Inc.) with the recommended manufacturer's settings. cAMP concentrations were determined by reference to a calibration curve determined in the same experiment using standard cAMP concentrations. A concentration response curve for Compound Z was constructed and data was fitted to a four parameter logistic equation to determine both the PEC50 and Intrinsic Activity. Intrinsic Activity was expressed as a fraction relative to the maximum activity determined for formoterol in each experiment. A result for Compound Z is in Table 1.
Selectivity Assays
Adrenergic αlD Membrane Preparation
Membranes were prepared from human embryonic kidney 293 (HEK293) cells expressing recombinant human αlo receptor. These were diluted in Assay Buffer (5OmM HEPES, ImM EDTA, 0.1% gelatin, pH 7.4) to provide a final concentration of membranes that gave a clear window between maximum and minimum specific binding.
Experimental Method
Assays were performed in U-bottomed 96-well polypropylene plates. 10 μL [3H]-prazosin
(0.3 nM final concentration) and 10 μL of Compound Z (1Ox final concentration) were added to each test well. For each assay plate 8 replicates were obtained for [3H]-prazosin binding in the presence of 10 μL vehicle (10% (v/v) DMSO in Assay Buffer; defining maximum binding) or lOμL BMY7378 (10 μM final concentration; defining non-specific binding (NSB)). Membranes were then added to achieve a final volume of 100 μL. The plates were incubated for 2 hours at room temperature and then filtered onto PEI coated GF/B filter plates, pre-soaked for 1 hour in Assay Buffer, using a 96-well plate Tomtec cell harvester. Five washes with 250 μL wash buffer (5OmM HEPES, ImM EDTA, pH 7.4) were performed at 4°C to remove unbound radioactivity. The plates were dried then sealed from underneath using Packard plate sealers and MicroScint-0 (50 μL) was added to each well. The plates were sealed (TopSeal A) and filter-bound radioactivity was measured with a scintillation counter (TopCount, Packard BioScience) using a 3-minute counting protocol.
Total specific binding (Bo) was determined by subtracting the mean NSB from the mean maximum binding. NSB values were also subtracted from values from all other wells. These data were expressed as percent of Bo. Compound concentration-effect curves
(inhibition of [3H] -prazosin binding) were determined using serial dilutions typically in the range 0.1 nM to 10 μM. Data was fitted to a four parameter logistic equation to determine the compound potency, which was expressed as pIC50 (negative log molar concentration inducing 50% inhibition of [3H]-prazosin binding). Result is shown in Table 1 below. Adrenergic βl
Membrane Preparation
Membranes containing recombinant human adrenergic beta 1 receptors were obtained from Euroscreen. These were diluted in Assay Buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, 0.1% gelatin, pH 7.4) to provide a final concentration of membranes that gave a clear window between maximum and minimum specific binding.
Experimental Method Assays were performed in U-bottomed 96-well polypropylene plates. 10 μL [125I]-
Iodocyanopindolol (0.036 nM final concentration) and 10 μL of Compound Z (10x final concentration) were added to each test well. For each assay plate 8 replicates were obtained for [125I]-Iodocyanopindolol binding in the presence of 10 μL vehicle (10% (v/v) DMSO in Assay Buffer; defining maximum binding) or 10 μL Propranolol (10 μM final concentration; defining non-specific binding (NSB)). Membranes were then added to achieve a final volume of 100 μL. The plates were incubated for 2 hours at room temperature and then filtered onto PEI coated GF/B filter plates, pre-soaked for 1 hour in Assay Buffer, using a 96-well plate Tomtec cell harvester. Five washes with 250 μL wash buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, pH 7.4) were performed at 4°C to remove unbound radioactivity. The plates were dried then sealed from underneath using Packard plate sealers and MicroScint-0 (50 μL) was added to each well. The plates were sealed (TopSeal A) and filter-bound radioactivity was measured with a scintillation counter (TopCount, Packard BioScience) using a 3-minute counting protocol.
Total specific binding (Bo) was determined by subtracting the mean NSB from the mean maximum binding. NSB values were also subtracted from values from all other wells. These data were expressed as percent of Bo. Compound concentration-effect curves (inhibition of [125I]-Iodocyanopindolol binding) were determined using serial dilutions typically in the range 0.1 nM to 10 μM. Data was fitted to a four parameter logistic equation to determine the compound potency, which was expressed as pICso (negative log molar concentration inducing 50% inhibition of [125I]-Iodocyanopindolol binding). A result is shown in Table 1 below.
Dopamine D2
Membrane Preparation
Membranes containing recombinant human Dopamine Subtype D2s receptors were obtained from Perkin Elmer. These were diluted in Assay Buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, 0.1% gelatin, pH 7.4) to provide a final concentration of membranes that gave a clear window between maximum and minimum specific binding.
Experimental Method
Assays were performed in U-bottomed 96-well polypropylene plates. 30 μL [3H]- spiperone (0.16 nM final concentration) and 30 μL of Compound Z (10x final concentration) were added to each test well. For each assay plate 8 replicates were obtained for [3H] -spiperone binding in the presence of 30 μL vehicle (10% (v/v) DMSO in Assay Buffer; defining maximum binding) or 30 μL Haloperidol (10 μM final concentration; defining non-specific binding (NSB)). Membranes were then added to achieve a final volume of 300 μL. The plates were incubated for 2 hours at room temperature and then filtered onto PEI coated GF/B filter plates, pre-soaked for 1 hour in Assay Buffer, using a 96-well plate Tomtec cell harvester. Five washes with 250 μL wash buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, pH 7.4) were performed at 4°C to remove unbound radioactivity. The plates were dried then sealed from underneath using Packard plate sealers and MicroScint-0 (50 μL) was added to each well. The plates were sealed (TopSeal A) and filter-bound radioactivity was measured with a scintillation counter (TopCount, Packard BioScience) using a 3-minute counting protocol.
Total specific binding (Bo) was determined by subtracting the mean NSB from the mean maximum binding. NSB values were also subtracted from values from all other wells. These data were expressed as percent of Bo. Compound concentration-effect curves
(inhibition of [3H] -spiperone binding) were determined using serial dilutions typically in the range 0.1 nM to 10 μM. Data was fitted to a four parameter logistic equation to determine the compound potency, which was expressed as pICso (negative log molar concentration inducing 50% inhibition of [3H] -spiperone binding). A result for Compound Z is shown in Table 1.
Table 1
Figure imgf000043_0001
The present invention will now be further explained by reference to the following illustrative Examples.
Example 1
Evaluation of compound activity on intra-alveolar neutrophil migration after aerosol challenge with lippopolysaccharride (LPS) in the CRL:CD rat.
LPS challenge in CRL:CD rats causes an influx of inflammatory cells into the lungs. Rats are challenged either with an aerosol of 0.9% w/v saline or O.lmg/mL LPS in 0.9% saline for 30 min or an intratracheal dose of 0.1-10μg/kg. This is repeated up to 8 times according to the experimental protocol. Rats are dosed with vehicle, standard compound or test compound by the appropriate route and frequency at various time points before and after challenge depending upon the experimental protocol. Test compound groups could either be the same compound at different doses or single doses of different compounds or a combination of the two. Test compounds are given by intraperitoneal, intravenous or subcutaneous injection or by inhalation or intratracheal administration.
The rats are euthanized at various time points after challenge depending upon the nature of the study, but typically 4hr after LPS challenge with ImL pentobarbitone sodium. A tracheotomy is performed and a cannula inserted. The airway is then lavaged using 3 mL sterile PBS at room temperature. The PBS is left in the airway for 10 seconds before being removed. The PBS containing cells is placed into a 15 mL centrifuge tube on ice. This process is repeated three times. An aliquot of BAL fluid is removed and counted on Sysmex (Sysmex UK, Milton Keynes). Cytospin slides are prepared by adding a 100 μl aliquot of BAL fluid into cytospin funnels in a Shandon Cytospin3 operated at 700 rpm for 5 min. Slides are stained on the Hema-Tek-2000 automatic slide stainer, using Wright-Giemsa stain and typically, 200 cells are counted under a microscope. Cells are classified as eosinophils, neutrophils and mononuclear cells (mononuclear cells included monocytes, macrophages and lymphocytes) and are expressed as a percentage of the total count.
Example 2 Evaluation of compound activity on intra- alveolar neutrophil migration after aerosol challenge with lippopolysaccharride (LPS) in the guinea-pig.
Male Dunkin-Hartley guinea-pigs (300-60Og) are placed into open fronted guinea-pig holding cones attached at random around a cylindrical aerosol chamber. Guinea-pigs are held in the challenge cones and exposed to an aerosol of vehicle, or LPS at concentrations of 0.1-30μg/ml in 0.9%saline per group Aerosols are generated using 2 jet nebulisers per column with a flow rate of 12 L/m. 10ml of the challenge agent is placed into each nebuliser. Alternatively animals receive an intratracheal dose of 0.1-10μg/kg. This is repeated up to 8 times according to the experimental protocol.
Guinea-pigs are dosed with vehicle, standard compound or test compound by the appropriate route and frequency at various time points before and after challenge depending upon the experimental protocol. Test compound groups could either be the same compound at different doses or single doses of different compounds or a combination of the two. Test compounds are given by intraperitoneal, intravenous or subcutaneous injection or by inhalation or intratracheal administration. Challenged guinea-pigs are killed by anaesthesia overdose (0.5ml Euthetal i.p.) at 4h-24h post challenge. The lungs are then lavaged. After the trachea is exposed and cannulated using a luer fitting cannula (orange =size 8FG), the lungs are lavaged with 3 x 5ml aliquots of Hanks Buffered Salt Solution (HBSS, EDTA -free). The lavaging is performed with gentle massaging of the chest to ensure appropriate agitation of the fluid in the lungs. The washes are harvested into a 15ml conical, polypropylene centrifuge tube, an aliquot of BAL fluid is removed and counted on Sysmex (Sysmex UK, Milton Keynes). Cytospin slides are prepared by adding a 100 μl aliquot of BAL fluid into cytospin funnels in a Shandon Cytospin3 operated at 700 rpm for 5 min. Slides are stained on the Hema-Tek-2000 automatic slide stainer, using Wright-Giemsa stain and typically, 200 cells are counted under a microscope. Cells are classified as eosinophils, neutrophils and mononuclear cells (mononuclear cells included monocytes, macrophages and lymphocytes) and are expressed as a percentage of the total count.
Example 3
Evaluation of compound activity on intra-alveolar neutrophil migration after aerosol challenge with lippopolysaccharride (LPS) in the mouse.
Male C57BL/6/J or BALB/C mice (20-35g) are placed in Perspex exposure boxes in groups of up to 20 and exposed to an aerosol of either 0.3 mg/ml LPS or 0.9% w/v saline. The LPS (Sigma, E.Coli, Ref L-3755, Serotype 026:B6, Lot no. 11 lk4078) is made up in 0.9% w/v saline. An aerosol is generated using two jet nebulisers operated at a flow rate of 12 L/min (6L/min for each nebuliser) for 15 min. Alternatively animals receive an intratracheal dose of 0.1-10μg/kg. This may be repeated up to 8 times.
Mice are dosed with vehicle, standard compound or test compound by the appropriate route and frequency at various time points before and after challenge depending upon the experimental protocol. Test compound groups could either be the same compound at different doses or single doses of different compounds or a combination of the two. Test compounds are given by intraperitoneal, intravenous or subcutaneous injection or by inhalation or intratracheal administration.
Mice are killed with an overdose of Euthatal i.p 30 minutes, l-24hr after LPS challenge. When circulation has ceased, the trachea is cannulated (Portex intravenous cannula) and the airways lavaged with 3 x 0.3ml of Isoton II (Beckman Coulter Ref. 8448011 Lot no.25775). For cytospins, lOOμl of the BALF is added to a cytospin funnel and spun, using a ThermoShandon Cytospin model 3 or 4, at 700 rpm for 5 min. Cells on the slide are stained on the Hema-Tek-2000 automatic slide stainer, using Wright-Giemsa stain and differential cell counts carried out to differentiate eosinophils, neutrophils and lymphomononuclear cells (including monocytes, macrophages and lymphocytes). Typically, 200 cells are counted per slide and each cell type expressed as a percentage of the total count. BALF total white cell count is measured using a Sysmex (Sysmex UK, Milton Keynes).
Example 4
Evaluation of lung function in anaesthetised guinea-pigs.
Male Dunkin-Hartley guinea-pigs (300-60Og) are weighed and dosed with either vehicle or compound in an appropriate vehicle according to the experimental protocol via the intratracheal route under recoverable gaseous anaesthesia (5% halothane in oxygen). Following dosing, the animals are administered supplemental oxygen and monitored until full recovery. Typically a dose volume of 0.5 mL/kg is used for the intratracheal route. In a dose response study, animals are dosed with compound or vehicle two hours prior to the administration of histamine. Test compound groups could either be the same compound at different doses or single doses of different compounds or a combination of the two.
The guinea-pigs are anaesthetised with pentobarbitone (1 mL/kg of 60 mg/mL solution intraperitoneally) approximately 30 minutes prior to the first bronchoconstrictor administration. The trachea is cannulated (Portex intravenous cannula, 200/300/070 (orange) or 200/300/060 (yellow)) and the animal ventilated using a constant volume respiratory pump (Harvard Rodent Ventilator model 683) at a rate of 60 breath/min and a tidal volume of 5 ml/kg. A jugular vein is cannulated (Portex intravenous catheter 200/300/010 (green)) for the administration of histamine or maintenance anaesthetic (0.1 mL of pentobarbitone solution, 60 mg/mL, as required).
The animals are then transferred to a Flexivent System (SCIREQ, Montreal, Canada) in order to measure airway resistance. The animals are ventilated (quasi-sinusoidal ventilation pattern) at 60 breaths/min at a tidal volume of 5 mL/kg. A positive end expiratory pressure of 2-3 CmH2O is applied. Respiratory resistance is measured using the Flexivent "snapshot" facility (1 second duration, 1 Hz frequency). Once stable baseline resistance value has been obtained the animals are given histamine dihydrochloride or methacholine in ascending doses (Histamine; 0.5, 1, 2, 3 and 5μg/kg, Lv., methacholine; 3, 10 and 30 μg/kg, i.v.) at approximately 4-minute intervals via the jugular catheter. After each administration of histamine the peak resistance value is recoreded. Guinea pigs are euthanised with approximately 1.OmL pentobarbitone sodium (Euthatal) intravenously after the completion of the lung function measurements.
Percentage bronchoprotection produced by a compound is calculated at each dose of histamine as follows:
%changeRveh - % changeRcmpd
% bronchoprotection = ■
% changeRv ?eh
Where % change Rveh is the mean of the maximum percentage change in airway resistance in the vehicle treated group.
Example 5
Evaluation of Compounds on Antigen induced Eosinophilia in Ovalbumin Sensitised Brown Norway Rats.
On day 0 of the study Brown Norway rats are given a subcutaneous injection of 500 μg ovalbumin adsorbed onto 100 mg aluminium hydroxide in 0.4 mL saline in two distinct sites, approximately 0.2 mL per site. Day 14 and 15 following sensitisation the rats are challenged with aerosolised ovalbumin for 15 minutes. The rats are placed in groups of 10 in an acrylic box (internal dimensions 320mm wide x 320mm deep x 195 mm high, 2OL volume). 8mL of 10 mg/mL ovalbumin in 0.9% saline, or 0.9% saline alone, is placed in each of two jet nebulizers (Sidestream®, Profile Respiratory Systems Ltd.). Compressed air at 6 L/min is passed through each nebulizer and the output of the nebulizers is passed into the box containing the rats.
Rats are dosed via the appropriate route with vehicle, standard compound or test compound at various time points before and after challenge depending upon the experimental protocol. Rats are euthanised with 0.5 mL pentobarbitone sodium (Euthatal) intraperitoneally at various times after challenge. A tracheotomy is performed and the trachea cannulated. The airway is then lavaged using 3 mL sterile PBS at room temperature. The PBS is left in the airway for 10 seconds before being removed. The PBS containing cells is placed into a 15 mL centrifuge tube on ice. This process is repeated three times. The final volume recovered is recorded. An aliquot of BAL fluid is removed and counted using a Sysmex (Sysmex UK, Milton Keynes).
Cytospin slides are prepared by adding a 100 μl aliquot of BAL fluid into cytospin funnels in a Shandon Cytospin 3 operated at 700 rpm for 5 min. Slides are stained on the Hema- Tek-2000 automatic slide stainer, using Wright-Giemsa stain and typically, 200 cells are counted under a microscope. Cells are classified as eosinophils, neutrophils and mononuclear cells. Mononuclear cells included monocytes, macrophages and lymphocytes.
Example 6
Evaluation of Compounds on Antigen induced eosinophilia in ovalbumin sensitised mice.
20-25g male BALB/c mice are sensitized to ovalbumin by i.p administration of 100 μg of grade V ovalbumin (Sigma) adsorbed onto lmg of aluminium hydroxide gel mixture (Fisher Scientific UK) in 0.3 ml saline. Groups of mice are pre-dosed with compound if required, a minimum of two weeks after sensitization. They are then dosed daily for 1-8 days as study protocol specified, with test compound or 0.25 ml vehicle.
Each day of the 1-8 days, 1 hour after dosing, the mice are placed in perspex chambers (20x11x1 lcm, 10 mice max./chamber) and administered an aerosol challenge of 20mg ml"1 ovalbumin for 36 min (8 ml for 18 min followed by another 8 ml for 18 min). Aerosol delivery is achieved using a DeVilbiss jet nebulizer with a flow rate of 61 min"1. 24h after the last dose the mice are killed with euthatal 0.2 ml i.p. and blood samples are taken (in EDTA tubes) for differential cell count analysis, the trachea is cannulated using a pink luer mount Portex cannula cut to lcm and the lungs are lavaged using 3 washes of ImI of
Isoton IL. For cytospins, lOOμl of the BALF is added to a cytospin funnel and spun, using a ThermoShandon Cytospin model 3 or 4, at 700 rpm for 5 min. Cells on the slide are stained on the Hema-Tek-2000 automatic slide stainer, using Wright-Giemsa stain and differential cell counts carried out to differentiate eosinophils, neutrophils and lymphomononuclear cells (including monocytes, macrophages and lymphocytes).
Typically, 200 cells are counted per slide and each cell type expressed as a percentage of the total count. BALF total white cell count is measured using a Sysmex (Sysmex UK, Milton Keynes).
Example 7 Evaluation on the effect of compound on lung function and BAL-neutrophilia following acute smoke exposure in the mouse
BALB/c or C57BL6/J mice undergo whole body exposure to main stream smoke (50 min/12 cigarettes) and fresh air once or twice a day for 1-9 days. Mice are dosed via the appropriate route with vehicle, standard compound or test compound at various time points before and after challenge depending upon the experimental protocol. On the final day of the experiment, mice are either killed with euthatal 0.2 ml i.p. and broncho-aveolar lavage fluid obtained for analysis of white blood cell infiltration (as described above) or lung function is assessed using a Flexivent System (SCIREQ, Montreal, Canada). Alternatively lung mechanics are measured using a forced manoeuvres system (EMMS).
Mice are anaesthetised with pentobarbitone (1/lOdilution at a dose volume of 1 mL/kg intraperitoneally). The trachea is cannulated and the animal transferred to the Flexivent System where they are ventilated (quasi-sinusoidal ventilation pattern) at a rate of 150 breath/min and a tidal volume of 10 ml/kg in order to measure airways resistance. Respiratory resistance is measured using the Flexivent "snapshot" facility (1 second duration, 1 Hz frequency). Mice are euthanised with approximately 0.5mL pentobarbitone sodium (Euthatal) intravenously after the completion of the lung function measurements.
Example 8 Evaluation of bronchodilator activity in the guinea-pig isolated tracheal ring preparation.
Guinea-pigs (300-60Og) are killed by cervical dislocation and the trachea removed. After clearing the adherent connective tissue, the trachea is cut into four ring segments (2-3 cartilage rings in width) and suspended in 10ml organ baths containing modified Krebs' solution (gassed with 5% CO2, 95% O2 at 37°C). The tracheal rings are attached to an isometric force transducer for the measurement of isometric tension. The tissues are washed and a force of Ig was applied to each tissue. The rings are precontracted with methacholine (1 μM) and a cumulative (10"9M - 10"5M) isoprenaline concentration effect curve is constructed. Responses are expressed as a percentage relaxation of the methacholine induced contraction. The rings are washed and a second concentration of methacholine (lμM) is added. Once the contraction has reached a plateau isoprenaline or the compound under investigation is added until a maximum effective dose is reached. Data are collected using the ADInstruments chart4forwindows software, which measures the maximum tension generated at each concentration of agonist and the response expressed as percentage relaxation.

Claims

C L A I M S
1. A pharmaceutical product comprising, in combination, a first active ingredient which is 4-hydroxy-7-[ li?-hydroxy-2-(2- {3-[(2-methoxy-benzylamino)-methyl]-phenyl} - ethylamino)-ethyl]-3H-benzothiazol-2-one or a salt thereof, and a second active ingredient selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; an antioxidant; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; a CRTh2 antagonist; a DPI antagonist; an Histone Deacetylase Inducer; an IKK2 inhibitor; a COX inhibitor; a lipoxygenase inhibitor; a leukotriene receptor antagonist; a MABA; an MPO inhibitor; a muscarinic antagonist; a p38 inhibitor; a PDE inhibitor; a PPARγ agonist; a protease inhibitor; a Statin; a thromboxane antagonist; a vasodilator; or, an ENAC blocker (Epithelial Sodium-channel blocker).
2. A kit comprising: a preparation of a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2- {3-[(2-methoxy-benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H- benzothiazol-2-one or a salt thereof, a preparation of a second active ingredient which is selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; an antioxidant; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; a CRTh2 antagonist; a DPI antagonist; an Histone Deacetylase Inducer; an IKK2 inhibitor; a COX inhibitor; a lipoxygenase inhibitor; a leukotriene receptor antagonist; a MABA; an MPO inhibitor; a muscarinic antagonist; a PDE inhibitor; a PPARγ agonist; a protease inhibitor; a Statin; a thromboxane antagonist; a vasodilator; or, an ENAC blocker (Epithelial Sodium-channel blocker), and optionally, instructions for the simultaneous, sequential or separate administration of the preparations to a patient.
3. A pharmaceutical composition comprising, in admixture: a first active ingredient which is 4-hydroxy-7-[li?-hydroxy-2-(2-{3-[(2-methoxy- benzylamino)-methyl]-phenyl}-ethylamino)-ethyl]-3H-benzothiazol-2-one or a salt thereof; a second active ingredient which is selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; an antioxidant; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; a CRTh2 antagonist; a DPI antagonist; an Histone Deacetylase Inducer; an IKK2 inhibitor; a COX inhibitor; a lipoxygenase inhibitor; a leukotriene receptor antagonist; a MABA; an MPO inhibitor; a muscarinic antagonist; a p38 inhibitor; a PDE inhibitor; a PPARγ agonist; a protease inhibitor; a Statin; a thromboxane antagonist; a vasodilator; or, an ENAC blocker (Epithelial Sodium-channel blocker); and, a pharmaceutically acceptable adjuvant, diluent or carrier.
4. A method of treating a respiratory disease, which method comprises simultaneously, sequentially or separately administering: (a) a therapeutically effective dose of a first active ingredient which is 4-hydroxy-7- [li?-hydroxy-2-(2-{3-[(2-methoxy-benzylamino)-methyl]-phenyl}-ethylamino)- ethyl]-3H-benzothiazol-2-one or a salt thereof; and,
(b) a therapeutically effective dose of a second active ingredient which is selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; an antioxidant; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; a CRTh2 antagonist; a DPI antagonist; an Histone Deacetylase Inducer; an IKK2 inhibitor; a COX inhibitor; a lipoxygenase inhibitor; a leukotriene receptor antagonist; a MABA; an MPO inhibitor; a muscarinic antagonist; a p38 inhibitor; a PDE inhibitor; a PPARγ agonist; a protease inhibitor; a Statin; a thromboxane antagonist; a vasodilator; or, an ENAC blocker (Epithelial Sodium-channel blocker); to a patient in need thereof.
5. A pharmaceutical product, kit or composition or method as claimed in claim 1, 2, 3 or 4 wherein the second active ingredient selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; a CCRl antagonist; a chemokine antagonist (not CCRl); a corticosteroid; an IKK2 inhibitor; a muscarinic antagonist; a p38 inhibitor; or, a PDE inhibitor.
6. A pharmaceutical product as claimed in claim 1 wherein the first active ingredient is in the form of a salt which is a hydrochloride, hydrobromide (such as dihydrobromide), trifluoroacetate, sulphate, phosphate, acetate, fumarate, maleate, tartrate, lactate, citrate, pyruvate, succinate, oxalate, methanesulphonate, p- toluenesulphonate, bisulphate, benzenesulphonate, ethanesulphonate, malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate, adipate, formate, glycolate, L- lactate, D-lactate, aspartate, malate, L-tartrate, D-tartrate, stearate, 2-furoate, 3- furoate, napadisylate (naphthalene- 1, 5 -disulfonate or naphthalene- 1 -(sulfonic acid)- 5-sulfonate), edisylate (ethane- 1 ,2-disulfonate or ethane- 1 -(sulfonic acid)-2- sulfonate), isethionate (2-hydroxyethylsulfonate), 2-mesitylenesulphonate, 2- naphthalenesulphonate, 2,5-dichlorobenzenesulphonate, D-mandelate, L-mandelate, cinnamate, benzoate, adipate, esylate, malonate, mesitylate (2- mesitylenesulphonate), napsylate (2-naphthalenesulfonate), camsylate (camphor- 10- sulphonate), formate, glutamate, glutarate, glycolate, hippurate (2- (benzoylamino)acetate), orotate, xylate (p-xylene-2-sulphonate), pamoic (2,2'- dihydroxy-l,r-dinaphthylmethane-3,3'-dicarboxylate), palmitate or furoate.
7. A pharmaceutical product, kit or composition or method as claimed in any one of the preceding claims wherein the first active ingredient is in the form of a salt which is a bis-malonate.
8. A pharmaceutical product, kit or composition or method as claimed in any one of the preceding claims wherein the the second active ingredient is a muscarinic antagonist which is a muscarinic antagonist which is Aclidinium bromide, Glycopyrrolate, Oxitropium bromide, Pirenzepine, telenzepine, Tiotropium bromide, 3(R)-(2-hydroxy-2,2-dithien-2-ylacetoxy)-l-(3-phenoxypropyl)-l- azoniabicyclo[2.2.2]octane bromide, 3(R)-l-phenethyl-3-(9H-xanthene-9- carbonyloxy)-l-azoniabicyclo[2.2.2]octane bromide or (3R)-3-[(2S)-2-cyclopentyl- 2-hydroxy-2-thien-2-ylacetoxy]-l-(2-phenoxyethyl)-l-azoniabicyclo[2.2.2]actane bromide.
9. A pharmaceutical product, kit or composition or method as claimed in any one of the preceding claims wherein the second active ingredient is Tiotropium bromide.
10. A pharmaceutical product, kit or composition or method as claimed in any one of claims 1 to 7 wherein the second active ingredient is a CCRl antagonist.
11. A pharmaceutical product, kit or composition or method as claimed in any one of claims 1 to 7 wherein the second active ingredient is a corticosteroid.
12. A pharmaceutical product, kit or composition or method as claimed in any one of claims 1 to 7 wherein the second active ingredient is a PDE4 inhibitor.
13. Use of a pharmaceutical product, kit or composition as claimed in claim 1-3 or 5-12 in therapy.
14. Use of a pharmaceutical product, kit or composition as claimed in claim 1-3 or 5-12 in the manufacture of a medicament for the treatment of a respiratory disease.
15. Use of a pharmaceutical product, kit or composition as claimed in claim 1-3 or 5-12 for the treatment of a respiratory disease.
16. Use according to claim 14 or 15, wherein the respiratory disease is chronic obstructive pulmonary disease, asthma, rhinitis, emphysema or bronchitis.
17. Use according to claim 14 or 15, wherein the respiratory disease is chronic obstructive pulmonary disease.
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