HK1070587A - Combination of a selective pde4 inhibitor and an adrenergic beta-2 receptor agonist - Google Patents

Description

Combination of a selective PDE 4inhibitor and an adrenergic beta 2 receptor agonist

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The present invention relates to combinations of an inhalable selective PDE 4inhibitor and an adrenergic beta 2 receptor agonist, pharmaceutical compositions (including drug delivery devices) and the use of such a combination.

The combination of a selective PDE 4inhibitor and an adrenergic beta 2 receptor agonist is useful in the treatment of obstructive airways and other inflammatory diseases, in particular the obstructive airways diseases asthma, Chronic Obstructive Pulmonary Disease (COPD) and other obstructive airways diseases which are exacerbated by elevated bronchial reflexes, inflammation, bronchial hyperreactivity and bronchospasm. The combination is particularly useful for treating COPD.

Examples of specific diseases that can be treated with the present invention include the respiratory diseases asthma, acute respiratory distress syndrome, chronic pulmonary inflammatory disease, bronchitis, chronic obstructive pulmonary (airway) disease and pneumoconiosis and immune system diseases such as allergic rhinitis and chronic sinusitis.

3 ', 5' -cyclic nucleotide Phosphodiesterases (PDEs) comprise a large class of enzymes classified into at least eleven distinct families, which differ from each other structurally, biochemically and pharmacologically. Enzymes within each family are commonly referred to as isoenzymes or isoenzymes. A total of more than fifteen gene products are included in this class, with further diversity arising from differential splicing and post-translational processing of these gene products. The present invention is primarily concerned with four gene products of the fourth family of PDEs, namely PDE4A, PDE4B, PDE4C and PDE 4D. These enzymes are collectively referred to as isoforms or subtypes of the PDE4 isozyme family (PDE4 s).

PDE4s is characterized by selective, high affinity hydrolytic degradation of the second messenger cyclic nucleotide, adenosine 3 ', 5' -cyclic monophosphate (cAMP), and sensitivity to rolipram inhibition. Several selective PDE4s inhibitors have been discovered in recent years and beneficial pharmacological effects from this inhibition have been shown in various disease models: see, for example, Torphy et al, environ. health Persport.102 suppl.10, 79-84, 1994; duplantieret al, j.med.chem.39120-125, 1996; schneider et al, pharmacol, biochem, behav.50211-217, 1995; banner and Page, Br.J.Pharmacol.11493-98, 1995; barnette et al, j.pharmacol.exp.ther.273674-679, 1995; wright et al, "Differential in vivo and in vitro broncholorela xanthations activities of CP-80633, a selective phosphodiesterase 4 inhibitor", Can.J.Physiol.Pharmacol.751001-1008, 1997; manabe et al, "Anti-inflimatory and dbronchostatin promoters of KF19514, a phosphodiesterase 4 and1 inhibitor", Eur.J. Pharmacol.33297-107, 1997; ukita et al, "Novel, potential, and selective phosphorus-4 inhibitors as antisense agents: synthesis and biological activities of a series of 1-pyridines derivatives ", J.Med.chem.421088-1099, 1999.

Adrenergic beta receptors are present in the sympathetic nervous system. There are at least two types. Adrenergic beta 1 receptors are found in the heart and play an important role in regulating heart rate through the action of agonists epinephrine and norepinephrine. Adrenergic beta 2 receptors are present on several cell types in the lung (e.g., airway smooth muscle cells, epithelial cells, and various inflammatory cells), and adrenergic beta 2 receptor agonists are potent bronchodilators, leading to relaxation of airway smooth muscle. Sympathomimetic amines have a long history of use in the treatment of chronic airway diseases characterized by partially reversible airway narrowing, such as COPD and asthma, primarily as bronchodilators in the form of intravenous epinephrine. Subsequently, inhaled β -adrenergic drugs, such as isoproterenol, which are relatively non-selective for β 2 over β 1 receptors, were used, thus causing tachycardia at effective bronchodilator doses. More recently, inhalable β -adrenergic drugs, such as salbutamol, have been used which are more selective for the β 2 receptor, but are short acting. The inhalable beta-adrenergic drugs formoterol, N- [ 2-hydroxy-5- (1-hydroxy-2- ((2- (4-methoxyphenyl) -1-methylethyl) amino) ethyl) phenyl ] carboxamide, and salmeterol are both beta 2 receptor selective and long acting.

It has now surprisingly been found that the combination of a specific selective PDE 4inhibitor with an adrenergic beta 2 receptor agonist provides a significant benefit in the treatment of obstructive airways and other inflammatory diseases over both the individual drugs and other known combinations. The advantage of this combination is to provide optimal control of airway caliber and effective inhibition of inappropriate inflammation through the mechanism most appropriate for disease pathology, namely adrenergic beta 2 receptor agonism. In this way, the symptoms of the disease are controlled by correcting inappropriate airway neural reflexes, which cause coughing, mucus production, and dyspnea. By administering the adrenergic beta 2 receptor agonist in combination with a selective PDE 4inhibitor via the inhalation route, the benefits of each class are delivered without the undesirable peripheral effects. Furthermore, the specific combination of the present invention produces an unexpected synergy with efficacy greater than the maximum tolerated dose of the two drugs used alone.

The present invention therefore provides an inhalation combination of the following (a) and (b):

(a) selective PDE 4inhibitors of formula (I)

Or a pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₂-C₄) Alkenyl, phenyl, -N (CH)₃)₂、(C₃-C₆) Cycloalkyl group, (C)₃-C₆) Cycloalkyl (C)₁-C₃) Alkyl or (C)₁-C₆) Acyl, wherein the alkyl, phenyl or alkenyl radical may be interrupted by up to two-OH groups, (C)₁-C₃) Alkyl or-CF₃A group or up to three halogen substitutions;

R₂and R₃Each independently selected from the group consisting of H, (C)₁-C₁₄) Alkyl, (C)₁-C₇) Alkoxy (C)₁-C₇) Alkyl, (C)₂-C₁₄) Alkenyl, (C)₃-C₇) Cycloalkyl group, (C)₃-C₇) Cycloalkyl (C)₁-C₂) Alkyl, saturated or unsaturated (C)₄-C₇) Heterocycle (CH)₂)_nGroup consisting of, wherein n is 0, 1 or 2, containing one or two of oxygen, sulfur, sulfonyl, nitrogen and NR⁴As a hetero atom, wherein R⁴Is H or (C)₁-C₄) An alkyl group; or a group of formula (II):

wherein a is an integer from 1 to 5; b and c are 0 or 1; r⁵Is H, -OH, (C)₁-C₅) Alkyl, (C)₂-C₅) Alkenyl, (C)₁-C₅) Alkoxy group, (C)₃-C₆) Cycloalkoxy, halogen, -CF₃、-CO₂R⁶、-CONR⁶R⁷、-NR⁶R⁷、-NO₂or-SO₂NR⁶R⁷Wherein R is⁶And R⁷Each independently is H or (C)₁-C₄) An alkyl group; z is-O-, -S-, -SO₂-, -CO-or-N (R)⁸) -, wherein R⁸Is H or (C)₁-C₄) An alkyl group; y is (C)₁-C₅) Alkylene or (C)₂-C₆) Alkenylene optionally substituted by up to two (C)₁-C₇) Alkyl or (C)₃-C₇) Cycloalkyl substitution; wherein each alkyl, alkenyl, cycloalkyl, alkoxyalkyl or heterocyclic group may be substituted by 1 to 14, preferably 1 to 5 (C)₁-C₂) Alkyl, CF₃Or halo group substitution;

R⁹and R¹⁰Each independently selected from the group consisting of H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₆-C₁₀) Aryl and (C)₆-C₁₀) Aryloxy group;

and (b) an adrenergic beta 2 receptor agonist.

Furthermore, the present invention provides an inhaled combination of a selective PDE 4inhibitor of formula (I) as defined above and an adrenergic beta 2 receptor agonist for use as a medicament.

Furthermore, the present invention provides an inhaled combination of a selective PDE 4inhibitor of formula (I) as defined above and an adrenergic beta 2 receptor agonist for simultaneous, sequential or separate administration in the treatment of an obstructive airways or other inflammatory disease.

Further, the present invention provides a pharmaceutical composition comprising a selective PDE 4inhibitor of formula (I) as defined above, an adrenergic β 2 receptor agonist and a pharmaceutically acceptable excipient, diluent or carrier for administration by the inhaled route in the treatment of an obstructive airways or other inflammatory disease.

Furthermore, the present invention provides the use of a selective PDE 4inhibitor of formula (I) or an adrenergic β 2 receptor agonist as defined above in the manufacture of a medicament for simultaneous, sequential or separate administration of the two medicaments by the inhaled route in the treatment of an obstructive airways or other inflammatory disease.

Further, the present invention provides a method of treating an obstructive airways or other inflammatory disease which comprises simultaneously, sequentially or separately administering to a mammal in need of such treatment, by the inhaled route, an effective amount of a selective PDE 4inhibitor of formula (I) as defined above and an adrenergic β 2 receptor agonist.

Furthermore, the present invention provides an inhalation device for simultaneous, sequential or separate administration of a selective PDE 4inhibitor of formula (I) as defined above and an adrenergic beta 2 receptor agonist in the treatment of an obstructive airways or other inflammatory disease.

The selective PDE 4inhibitors have a greater affinity for the PDE4 isozyme than all other known PDE isozymes. Preferably, a selective PDE 4inhibitor according to the invention has an affinity for the PDE4 isozyme which is at least 100 times greater than its affinity for other PDE isozymes.

Preferred compounds of formula (I) include those wherein R is¹Is methyl, ethyl or isopropyl, and wherein R³Is (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₃-C₇) Cycloalkyl group, (C)₃-C₇) Cycloalkyl (C)₁-C₆) Alkyl or phenyl, optionally substituted with 1 or 2 groups selected from H, -OH, (C)₁-C₅) Alkyl, (C)₂-C₅) Alkenyl, (C)₁-C₅) Alkoxy, halogen, trifluoromethyl, -CO₂R⁶、-CONR⁶R⁷、-NR⁶R⁷、-NO₂or-SO₂NR⁶R⁷Wherein R is₆And R₇Each independently is H or (C)₁-C₄) Alkyl radical。

Preferred individual compounds of formula (I) include:

9-cyclopentyl-5, 6-dihydro-7-ethyl-3-phenyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (furan-2-yl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-pyridyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (4-pyridyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (3-thienyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

3-benzyl-9-cyclopentyl-5, 6-dihydro-7-ethyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3-propyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

3, 9-dicyclopentyl-5, 6-dihydro-7-ethyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (1-methylcyclohex-1-yl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

3- (tert-butyl) -9-cyclopentyl-5, 6-dihydro-7-ethyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-methylphenyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-methoxyphenyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (thiophen-2-yl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

3- (2-chlorophenyl) -9-cyclopentyl-5, 6-dihydro-7-ethyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-iodophenyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-trifluoromethylphenyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine; and

5, 6-dihydro-7-ethyl-9- (4-fluorophenyl) -3- (1-methylcyclohex-1-yl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

and pharmaceutically acceptable salts and solvates thereof.

Particularly preferred compounds of formula (I) include 9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-thienyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine and 9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (tert-butyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine and pharmaceutically acceptable salts and solvates thereof.

The synthesis of the compounds of formulA (I) is described in WO-A-96/39408.

Preferably, the adrenergic beta 2 receptor agonist used in the combination according to the invention is a selective adrenergic beta 2 receptor agonist, that is to say has a greater affinity for the adrenergic beta 2 receptor than all other known adrenergic beta receptors. Preferably, such a selective adrenergic beta 2 receptor agonist has an affinity for the adrenergic beta 2 receptor that is at least 100 times greater than its affinity for other adrenergic beta receptors.

Adrenergic beta 2 receptor agonists that are preferred for use in the present invention include salmeterol, formoterol, and pharmaceutically acceptable salts and solvates thereof.

Particularly preferred combinations include:

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-thienyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine or a pharmaceutically acceptable salt or solvate thereof and salmeterol or a pharmaceutically acceptable salt or solvate thereof;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-thienyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine or a pharmaceutically acceptable salt or solvate thereof and formoterol or a pharmaceutically acceptable salt or solvate thereof;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (tert-butyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine or a pharmaceutically acceptable salt or solvate thereof and salmeterol or a pharmaceutically acceptable salt or solvate thereof;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (tert-butyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine or a pharmaceutically acceptable salt or solvate thereof and formoterol or a pharmaceutically acceptable salt or solvate thereof.

The selective PDE 4inhibitor or adrenergic beta 2 receptor agonist used in accordance with the present invention may optionally be in the form of a pharmaceutically acceptable salt or solvate. Such a salt may be an acid addition salt or a base salt.

Suitable acid addition salts are formed from acids which form non-toxic salts, examples being hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogenphosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate salts.

Suitable base salts are derived from bases which form non-toxic salts, examples being the salts of sodium, potassium, aluminium, calcium, magnesium, zinc and diethanolamine.

For a review of suitable salts, see Berge et al, j.pharm.sci., 66, 1-19, 1977.

Pharmaceutically acceptable solvates of the selective PDE 4inhibitor and the adrenergic beta 2 receptor agonist or a salt thereof for use according to the invention include hydrates thereof.

The selective PDE 4inhibitor and the adrenergic beta 2 receptor agonist of the present invention may exist in one or more polymorphic forms.

The selective PDE 4inhibitors and adrenergic beta 2 receptor agonists of the invention (hereinafter referred to as "compounds of the invention") may contain one or more asymmetric carbon atoms and thus exist in two or more stereoisomeric forms (e.g., R' formoterol is a preferred embodiment). When such a compound contains an alkenyl or alkenylene group, cis/trans (or Z/E) isomerism may also be present. The invention includes these individual stereoisomers of the compounds of the invention, as appropriate and individual tautomeric forms thereof, and mixtures thereof.

Separation of the diastereomers or cis-trans isomers may be achieved by conventional techniques, such as fractional crystallization, chromatography or h.p.l.c. of a stereoisomeric mixture of the compounds of the invention or suitable salts or derivatives thereof. The individual enantiomers of the compounds of the invention can also be prepared from the corresponding optically pure intermediates, either by resolution, for example using a suitable chiral carrier for the h.p.l.c. of the corresponding racemate, or by fractional crystallization of the diastereomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as the case may be.

The invention also includes all suitable isotopic variations of the compounds of the invention or pharmaceutically acceptable salts thereof. Isotopic variations of the compounds of the present invention or pharmaceutically acceptable salts thereof are defined as wherein at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Can be incorporated into the compounds of the present invention and their pharmaceutical usesExamples of isotopes in the above-acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as²H、³H、¹³C、¹⁴C、¹⁵N、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F、³⁶And (4) Cl. Certain isotopic variations of the compounds of the present invention and pharmaceutically acceptable salts thereof, for example, wherein³H or¹⁴Those of radioactive isotopes, such as C, are useful for drug and/or substrate tissue distribution studies. Tritium, i.e.³H and carbon-14, i.e.¹⁴The C isotopes are particularly preferred because of their ease of preparation and detection. And, furthermore, by deuterium, i.e.²Substitution of isotopes such as H may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in some circumstances.

The types of diseases that can be treated with the combination of the present invention include, but are not limited to, asthma, chronic or acute bronchoconstriction, chronic bronchitis, small airway obstruction, emphysema, Chronic Obstructive Pulmonary Disease (COPD), COPD with chronic bronchitis, emphysema or dyspnea associated therewith, and COPD characterized by irreversible progressive airway obstruction.

Asthma and asthma

One of the most important respiratory disorders that can be treated with the therapeutic combination of the present invention is asthma, a chronic, increasingly widespread worldwide condition characterized by intermittent reversible airway obstruction, airway hyperreactivity, and inflammation. The cause of asthma remains to be determined, but the most common pathological manifestation of asthma is inflammation of the airways, which may be significant even in the airways of patients with mild asthma. This inflammation drives reflex airway events, leading to plasma protein extravasation, dyspnea, and bronchoconstriction. Based on bronchial biopsy and lavage studies, it has been clearly shown that asthma involves infiltration of mast cells, eosinophils and T lymphocytes into the airways of patients. Bronchial lavage (BAL) in atopic asthmatic patients shows activation of Interleukins (IL) -3, IL-4, IL-5 and granulocyte/macrophage-colony stimulating factor (GM-CSF), suggesting the presence of a T helper 2(Th-2) -like T cell population.

The therapeutic combinations of the present invention are useful in the treatment of atopic and non-atopic asthma. The term "atopy" denotes a genetic predisposition to the development of a type I (immediate) allergic response to a common environmental antigen. The most common clinical manifestations are allergic rhinitis, while bronchial asthma, atopic dermatitis and food allergies are less common. Thus, the expression "atopic asthma" as used herein is intended to be synonymous with "allergic asthma", i.e. the allergic manifestation of bronchial asthma in sensitive patients. The term "non-atopic asthma" as used herein is intended to mean all other asthma, especially essential or "real" asthma, which is provoked by various factors including vigorous exercise, irritant particles, psychological stress, etc.

Chronic Obstructive Pulmonary Disease (COPD)

The therapeutic combinations of the present invention are further useful in the treatment of COPD or COAD, including chronic bronchitis, emphysema or dyspnea associated therewith. COPD is characterized by progressive airway obstruction that is not fully reversible. Chronic bronchitis is associated with hyperplasia and hypertrophy of submucosal mucous secretory glands in the airways of large cartilage. Goblet cell proliferation, infiltration of mucosal and submucosal inflammatory cells, edema, fibrosis, mucus embolism and increased smooth muscle are all found in the terminal and respiratory bronchioles. Small airways are known to be the primary site of airway obstruction. Emphysema is characterized by destruction of the alveolar walls and loss of lung elasticity. A number of risk factors have also been identified in connection with the incidence of COPD. The link between smoking and COPD is well established. Other risk factors include exposure to coal ash and various genetic factors. See Sandford et al, "Genetic risk factor for respiratory inductive pulmonary disease", Eur.Respir.J.101380-1391, 1997. The incidence of COPD is increasing and has become a heavy economic burden for the population of industrialized countries. COPD itself also varies widely in clinical settings from simple chronic bronchitis without disability to patients with severe disability with chronic respiratory failure.

COPD is characterized by airway inflammation, which is also the case of asthma, but the inflammatory cells found in bronchoalveolar lavage and sputum of patients are neutrophils and macrophages, not eosinophils. Elevated levels of inflammatory mediators are also seen in COPD patients, including IL-8, LTB4 and TNF- α, and it has been found that the superficial epithelium and subepithelial membranes of the bronchi of such patients are infiltrated by T lymphocytes and macrophages. The use of beta agonists and anticholinergic bronchodilators may provide symptomatic relief in COPD patients, but the progression of the disease remains unchanged. Theophylline has been used to treat COPD, but has not met with much success, in part because of the tendency to have undesirable consequences. Steroids have also not been able to be satisfactory therapeutic agents for COPD because they are relatively ineffective as anti-inflammatory agents.

Thus, the use of the therapeutic combinations of the present invention to treat COPD and its associated and included obstructive airways diseases represents a significant advance in the art. With respect to the manner in which the desired therapeutic objectives are achieved using the therapeutic agent combinations of the present invention, the present invention is not limited to any particular mode of action or any hypothesis.

Bronchitis and bronchiectasis

In light of the specific and distinct inhibitory activities described above for the therapeutic combinations of the present invention, they can be used to treat bronchitis of any type, etiology or pathogenesis, including for example acute bronchitis, which has a short but severe course and is caused by cold, inhalation irritants or acute infections; catarrhal bronchitis, a form of acute bronchitis, with a large number of mucopurulent discharges; chronic bronchitis, a long-lasting form of bronchitis with a tendency to recur more or less after the quiescent phase, caused by recurrent episodes of acute bronchitis or chronic systemic disease, characterized by episodes of coughing, little or more sputum and secondary changes in the lung tissue; dry bronchitis, which is characterized by a small amount of mucus secreted; infectious asthmatic bronchitis, a syndrome known as secondary respiratory infection, which is caused by bronchospasm in asthmatic patients; proliferative bronchitis, which is bronchitis associated with productive cough.

The efficacy of the therapeutic combinations of the present invention in the treatment of atopic or non-atopic asthma, COPD or other chronic inflammatory airway diseases can be demonstrated using a number of different models known in the art, including the models described below.

Bronchodilatory ActivitycAMP is not only involved in smooth muscle relaxation, but also exerts an overall inhibitory effect on airway smooth muscle proliferation, both of which may arise from the PDE4 component of the invention elevating cAMP. Airway smooth muscle hypertrophy and hyperplasia can be modulated by cAMP, a condition that is a common morphological feature of chronic asthma.

Spasmolytic activity of in vitro bronchusThe ability of the therapeutic combination of the invention to cause relaxation of guinea pig tracheal smooth muscle was demonstrated in the following test procedure. Guinea pigs (350-500g) were sacrificed with thiopentasodium (100mg/kg i.p.). The trachea is cut open and sections of 2-3cm length are excised. Transecting trachea along the next cartilage plate cross section to obtain 3-5mm deep tissue ring. The proximal and distal rings are discarded. The rings were mounted vertically on a stainless steel support, one of which was fixed to the bottom of the organ bath and the other was connected to equidistant sensors. The rings were immersed in a Krebs solution bath (μ M composition: NaHCO) at 37 deg.C₃25；NaCl 113；KCl 4.7；MgSO₄·7H₂O 1.2；KH₂PO₄ 1.2；CaCl₂2.5; glucose 11.7), with O₂/CO₂(95: 5, v/v) aeration. The ring prepared in this way is stimulated with an electric field to contract. To confirm spasmolytic activity, the therapeutic combination tested according to the invention was dissolved in physiological saline and added to the organ bath at 5min intervals, increasing in number, to give a cumulative concentration-effect curve.

In the above test model, the therapeutic combination of the invention generally inhibited the contraction of the electric field-stimulated guinea pig tracheal ring preparation at a concentration ranging from 0.001 to 1.0 μ M.

Model of ozone-induced bronchial hyperreactivity-the ability of the therapeutic combination of the present invention to prevent an increase in the responsiveness of the airways to noxious stimuli, also known as bronchial hyperreactivity, was demonstrated by measuring the effect on guinea pig lung response activity. Adult guinea pigs (300-. The responsiveness of the airway to various stimuli is monitored in the basal state and after various interventions leading to changes in pulmonary mechanics. The test substance was administered i.t. or via aerosol at various times prior to challenge. Ozone pre-treatment of control animals resulted in a 3-100x increase in lung responsiveness, whereas the therapeutic combination of the present invention blocked this increase in a dose-dependent manner.

In the above test models, the therapeutic combinations of the present invention typically exhibit anti-inflammatory activity at a dosage range of 0.001 to 0.3mg/kgi.t.

Loose human bronchus-obtaining a human lung specimen dissected during cancer surgery within 3 days after removal. The small bronchi (ID. approx.2-5 mm) were excised, cut into small pieces, and placed in 2ml liquid nitrogen storage ampoules containing Fetal Calf Serum (FCS) containing 1.8M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectants. The ampoules were placed in polystyrene boxes (11X 22cm) and slowly frozen in a refrigerator kept at-70 ℃ with an average cooling rate of 0.6 ℃/min. After 3-15 hours, the ampoules were transferred to liquid nitrogen (-196 ℃) and stored until use. Before use, the tissue was exposed to-70 ℃ for 30-60min, and the ampoule was placed in a 37 ℃ water bath and thawed within 2.5 min. The bronchial fragments were then placed in Krebs-Henseleit solution (μ M: NaCl118, KCl 4.7, MgSO 2) containing 37 deg.C₄ 1.2，CaCl₂ 1.2，KH₂PO₄ 1.2，NaHCO₃25, glucose 11, EDTA 0.03), cut into rings, suspended in a 10ml organ bath and recorded under a preload of about 1 g. Application of an electric field stimulus induces a further increase in tension, which is known to induceActivation of the airway sample nerves produces tension via the release of acetylcholine and other natural mediators. The concentration-response curve is generated by cumulative additions, the latter concentration being added when the former concentration has produced the greatest effect. Papaverine (300 μ M) was added at the end of the concentration-response curve to induce complete relaxation of the bronchial ring. This effect is considered to be 100% relaxation.

In the above test model, the therapeutic combination of the invention typically produces a concentration-dependent relaxation of human bronchial ring preparations at a concentration range of 0.001 to 1.0 μ M, with preferred embodiments being active at a concentration range of 5.0nM to 500 nM.

Inhibition of capsaicin-induced bronchoconstrictionMale Dunkin-Hartley guinea pigs (400-]) And (6) anaesthetizing. According to a rectal thermometer, the animal was maintained at 37 ℃ using a heating pad, and a mixture of air and oxygen (45: 55 v/v) was passed through an endotracheal tube (about 8ml/kg, 1 Hz). Ventilation at the trachea is monitored using a pneumotachograph connected to a differential pressure sensor conforming to a respiratory pump. The pressure changes within the thoracic cavity are directly monitored via an intrathoracic cannula using a differential pressure sensor so that the pressure difference between the trachea and the thoracic cavity can be measured and displayed. Calculating airway resistance (R) per respiratory cycle from these measurements of airflow and transpulmonary pressure using a digital electronic breath analyzer₁ cmH₂O/l/s) and compliance (Cd)_dyn). Blood pressure and heart rate were recorded from the carotid artery using a pressure transducer.

Intravenous bolus administration of capsaicin induced acute episodes of bronchospasm when both basal tolerance and compliance were stable. Capsaicin was dissolved in 100% ethanol and diluted with phosphate buffered saline. The test therapeutic combinations of the present invention were administered when the response to capsaicin was stable, which was calculated after 2-3 such administrations, separated by 10 min. Reversal of bronchospasm was assessed over 1-8 hours following intratracheal or intraduodenal instillation or intravenous bolus injection. The spasmolytic activity of bronchus is expressed as capsaicinInitial, maximum tolerability after infusion (R)_D) Inhibition of (c). ED (electronic device)₅₀Values represent doses that resulted in a 50% decrease in capsaicin-induced tolerance. The duration of action is defined as the time, in minutes, at which bronchospasm is reduced by 50% or more. The effect on Blood Pressure (BP) and Heart Rate (HR) is ED₂₀Values are expressed, i.e. the dose that reduces BP or HR by 20% measured 5min after administration.

In the above test model, the therapeutic agent combination of the present invention generally exhibits bronchodilatory activity at a dosage range of 0.001 to 0.1mg/kgi.t. [ intratracheal ]. Furthermore, the combination administered i.t. exhibits an at least additive inhibitory effect on bronchospasm, each component alone being capable of inhibiting more than 50% of the control response observed.

LPS-induced pulmonary neutrophilia-recruitment and activation of neutrophils in the lung is considered an important pathological feature of COPD and severe asthma. Inhibition of one or both of these endpoints in animals therefore provides supportive evidence of the utility of the invention.

Male Wistar-Albino rats (150-. 1-24 hours after administration of the compound, the animals were challenged with an inhaled aerosol of bacterial Lipopolysaccharide (LPS) sufficient to induce significant pulmonary neutrophilia in the subsequent 1-24 hours. The neutrophilia is assessed by counting cells in bronchial washes or by measuring neutrophil production in lung washes or tissues. In such test systems, the therapeutic agents of the invention typically exhibit anti-inflammatory activity at a dosage range of 0.0001 to 0.1mg/kg i.t.. Surprisingly, the combination administered i.t. exerts an at least additive effect on inflammation, although neither component by itself exerts a significant anti-inflammatory effect. Furthermore, when used in combination as in the present invention, an equivalent anti-inflammatory effect of one of the high dose components can be observed at lower doses, thereby reducing the undesirable systemic effect.

Allergic guinea pig assayExperiments in which Dunkin-Hartley guinea pigs (400-600g body weight) were used to evaluate the therapeutic effect of the therapeutic combinations of the invention on dyspnea and bronchospasm symptoms (i.e.dyspnea or labored exertion, increased lung tolerance) and inflammatory symptoms (i.e.pulmonary neutrophilia and eosinophilia).

The crystallized and lyophilized class V ovalbumin (EA), aluminum hydroxide and mepyramine maleate used in this experiment are commercially available. The challenge and subsequent breath result readings were made in clear plastic boxes with internal dimensions of 10 x 6 x 4 inches. The box cover and the box body can be separated. The two are clamped together in use by a clamp and a soft rubber gasket is used to maintain an airtight seal between the box spaces. The atomizer is inserted through the center of the head end of the box space via a gas-tight seal, each end of the box also having an outlet. The pneumotachograph is inserted into one end of the cassette, connected to a volumetric pressure sensor, and then connected to the orbitometer (dynograph) by a suitable connector. While nebulizing the antigen, the outlet is open and the pneumotachograph is separated from the cassette space. The outlet was then closed and the pneumotachograph was connected to the cassette space during the recording of the breathing pattern. At challenge, 2ml of 3% antigen saline solution was placed in each nebulizer and aerosol was generated using air from a small diaphragm pump operating at 10psi and a flow rate of 8 l/min.

Guinea pigs were sensitized subcutaneously and i.p. by injection of 1ml saline suspension containing 1mg EA and 200mg aluminium hydroxide. They were used between 12 and 24 days after sensitization. To eliminate histamine factors in the response, guinea pigs were pretreated with 2mg/kg mepyramine i.p. 30min prior to aerosol challenge. Guinea pigs were then exposed to a saline aerosol of 3% EA for 1min accurately, and then the breathing curve was recorded for 30 min. Subsequently, lung inflammation after death was measured over 1-48 hours. The duration of continuous dyspnea is measured from the breath recordings.

The test therapeutic combinations of the present invention are typically administered i.t. 0.5-4 hours prior to challenge or via gasThe administration is carried out as an aerosol. The combination of compounds is dissolved in saline or a biocompatible solvent. The activity of the compounds was determined on the basis of their ability to reduce the magnitude and duration of dyspnea and bronchospasm symptoms and/or the magnitude of pulmonary inflammation compared to vehicle-treated controls. Tests to evaluate the therapeutic combination of the invention at a range of doses to derive ED₅₀It is defined as the dose (mg/kg) that inhibits the duration of symptoms by 50%.

Anti-inflammatory ActivityThe anti-inflammatory activity of the therapeutic agent combination of the invention is demonstrated by means of inhibition of eosinophil or neutrophil activation. In this assay, the eosinophil count was between 0.06 and 0.47X 10⁹L^-1In between non-atopic volunteers blood samples (50ml) were collected. Venous blood was collected into a centrifuge tube containing 5ml trisodium citrate (3.8%, pH 7.4).

Anticoagulated blood was diluted (1: 1, v: v) with phosphate buffered saline (PBS, containing neither calcium nor magnesium) in a 50ml centrifuge tube and layered on 15ml isotonic Percoll (density 1.082-1.085g/ml, pH 7.4). After centrifugation (30min, 1000 Xg, 20 ℃), the mononuclear cells at the plasma/Percoll interface were carefully aspirated and discarded.

The neutrophil/eosinophil/erythrocyte expulsed mass (volume about 5ml) was gently resuspended in 35ml of isotonic ammonium chloride solution (NH)₄Cl，155mM；KHC0₃10 mM; EDTA, 0.1 mM; 0 to 4 ℃ below zero. After 15min, cells were washed twice (10min, 400 Xg, 4 ℃) in PBS containing fetal calf serum (2%, FCS).

Eosinophils and neutrophils were separated using a magnetic cell separation system. Such a system is capable of separating cells in suspension on the basis of surface markers, comprising a permanent magnet in which pillars comprising a magnetizable steel matrix are placed. Before use, the column was equilibrated with PBS/FCS for 1 hour and then back-washed with ice-cold PBS/FCS via a 20ml syringe. A21G hypodermic needle was attached to the bottom of the column and 1-2ml of ice cold buffer was allowed to flow out of the needle.

After centrifugation of the granulocytes, the supernatant was aspirated and the cells were gently resuspended with 100. mu.l of magnetic particles (anti-CD 16 monoclonal antibody, conjugated with superparamagnetic particles). eosinophil/neutrophil/anti-CD 16 magnetic particle mixtures were incubated on ice for 40min and then diluted to 5ml with ice-cold PBS/FCS. The cell suspension was slowly introduced into the top of the column and the tap was opened to allow the cells to slowly move to the steel substrate. The column was then washed with PBS/FCS (35ml), which was carefully added to the top of the column to avoid disturbing magnetically labelled neutrophils that had been trapped within the steel matrix. Unlabeled eosinophils were collected in a 50ml centrifuge tube and washed (10min, 400 Xg, 4 ℃). The resulting discard block was resuspended in 5ml Hank's Balanced Salt Solution (HBSS) to enable assessment of cell number and purity prior to use. The column was removed from the magnet and the neutrophil fraction was eluted. The column was then washed with PBS (50ml) and ethanol (absolute) and stored at 4 ℃.

The total cells were counted using a micro-cytometer. A drop of lysis solution was added to the sample and recounted after 30 seconds to assess contamination of the red blood cells. Spin cell blood smears (100. mu.l sample, 3min, 500rpm) were prepared on a Shandon Cytospin 2 Cytospin machine. These preparations were stained, and at least 500 cells were examined by measuring the number of differential cells using an optical microscope. Cell viability was assessed by exclusion of trypan blue.

Eosinophils or neutrophils were diluted in HBSS and pipetted into a 96-well microtiter plate (MTP) at 1-10X 10 per well³And (4) cells. Each well contained 200 μ l of sample, which contained: 100 μ l of cell suspension; 50 μ l HBSS; 10. mu.l of bis-N-methylacridine nitrate; 20 μ l of activating stimulus; and 20. mu.l of test compound.

Samples were incubated with test compounds or vehicle for 10min, then a solution of activating stimuli fMLP (1-10 μ M) or C5a (1-100nM) in dimethylsulfoxide was added, then diluted in buffer so that the highest solvent concentration used was 1% (100 μ M test compound). The MTPs were stirred to facilitate mixing of the cells with the medium, and the MTPs were placed in a luminometer. Total chemiluminescence sum per well was measured simultaneously over 20minTransient curves, results are expressed in arbitrary units or as a percentage of fMLP induced chemiluminescence in the absence of test compound. Substituting the result into Hill equation, and automatically calculating IC₅₀The value is obtained.

In the above assay methods, the therapeutic combinations of the present invention are generally active at concentrations ranging from 0.0001. mu.M to 0.5. mu.M, with preferred embodiments being active at concentrations ranging from 0.1nM to 100 nM.

The anti-inflammatory activity of the therapeutic combination of the present invention was additionally demonstrated by virtue of its inhibitory effect on plasma extravasation into the airways of rats. In this assay, tracheal tissue is taken and the extent of plasma leakage is determined. This assay is also related to other chronic inflammatory diseases of the airways including, but not limited to, COPD, and therefore this section is not repeated.

Wistar albino rats (150-. Evans blue dye bound to plasma proteins was administered i.v. (30 mg/kg). After 10min, the test substance was administered i.t., and after 10min, capsaicin was administered i.v. (3 μ g/kg). After 30min, tracheal tissue was removed, extracted overnight with formamide, and absorbance was read at 620 nm. In some experiments, the order of administration was reversed, so that the compound was administered before the evans blue and inflammatory stimulant.

In the above test models, the therapeutic combinations of the present invention typically exhibit anti-inflammatory activity at a dosage range of 0.001 to 0.1mg/kgi.t.

As can be seen from the above, the therapeutic combinations of the present invention are useful in the treatment of inflammatory or obstructive airways diseases or other conditions involving obstruction of airways. In particular, they are useful in the treatment of bronchial asthma.

In view of their anti-inflammatory activity and their effect on airway hyperreactivity, the therapeutic combinations of the present invention are useful in the treatment of obstructive or inflammatory airway diseases, specifically prophylactic treatment. Thus, by regular administration over a prolonged period of time, the combination of compounds of the invention may be used to provide pre-protection against the onset of bronchoconstriction or other symptoms secondary to an obstructive or inflammatory airway disease. The compound combinations of the present invention may also be used to control, ameliorate or reverse the underlying state of such disorders.

Given their bronchodilator activity, the therapeutic combinations of the present invention are useful as bronchodilators, e.g. in the treatment of chronic or acute bronchoconstriction, and for symptomatic treatment of obstructive or inflammatory airway diseases.

Obstructive or inflammatory airways diseases to which the present invention is applicable include asthma; pneumoconiosis; chronic eosinophilic pneumonia; chronic obstructive airways or lung disease (COAD or COPD); and Adult Respiratory Distress Syndrome (ARDS), as well as airway hyperresponsiveness, is exacerbated by other drug therapies, such as aspirin or beta-agonist therapy.

The selective PDE 4inhibitor and the adrenergic beta 2 receptor agonist of the present invention may be administered alone or in combination, but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier.

The selective PDE 4inhibitor and the adrenergic beta 2 receptor agonist of the invention are preferably administered by inhalation, suitably in the form of a dry powder inhaler (alone or as a mixture, e.g. with lactose) or in the form of an aerosol, from a pressurised container, pump, nebuliser (atomiser) (preferably an atomiser which generates fine nebulisation using electro-hydraulics) or nebuliser (nebuliser), with or without the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a fluoroalkane, e.g. 1, 1, 1, 2-tetrafluoroethane (HFA 134A)^TM) Or 1, 1, 1, 2, 3, 3, 3-heptafluoropropane (HFA227 EA)^TM) Carbon dioxide, another perfluorinated hydrocarbon, e.g. Perflukron^TMOr other suitable gas. In the case of pressurized aerosols, the dosage units can be determined by means of a valve for metered release. The pressurised container, pump, spray, atomiser or nebuliser may contain a solution or suspension of the active compound,for example, using ethanol (optionally aqueous ethanol) or a mixture of an agent suitable for dispersion, solubilisation or delayed release and a propellant as the solvent, which may additionally contain a lubricant, for example sorbitan trioleate. Capsules, blisters and cartridges (made, for example, from gelatin or HPMC) for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch and a performance enhancing agent such as L-leucine, mannitol or magnesium stearate.

Prior to use in dry powder formulations or suspension formulations for inhalation, the compounds of the present invention will be micronized to a size suitable for inhalation release (generally considered to be less than 5 microns). Micronization can be achieved by a variety of methods, such as spiral jet milling, fluid bed jet milling, or crystallization using supercritical fluids.

Solution formulations suitable for use in atomiser which uses electro-hydraulics to generate fine aerosol may contain from 1 μ g to 10mg of a compound of the invention per starting volume which may vary from 1 to 100 μ l. A typical formulation may comprise a compound of the invention, propylene glycol, sterile water, ethanol and sodium chloride. Instead of propylene glycol, another solvent may be used, such as glycerol or polyethylene glycol.

Aerosol or dry powder formulations are preferably arranged to contain from 1 to 4000. mu.g of a compound of the invention per metered dose or "puff" for delivery to a patient. The total daily aerosol dose will be in the range 1 μ g to 20mg, which may be administered once daily, or more usually in divided doses.

Selective PDE 4inhibitor used: the preferred weight ratio (w/w) of adrenergic beta 2 receptor agonist will depend on the particular combination examined. This is due to the difference in potency of the individual compounds. In any event, the physician will determine the actual dosage of each compound which will be most suitable for any individual patient, which will vary with the age, weight and response of the particular patient.

It is understood that all references herein to treatment include curative, palliative and prophylactic treatment.

Experimental data-inhibition of Elastase Release from isolated human neutrophils

Venous blood (90ml) from male and female healthy volunteers was collected in 10ml of 3.8% (w/v) sodium citrate and 8ml aliquots were dispensed in 15ml polypropylene centrifuge tubes, each containing 4ml of Hanks Balanced Salt Solution (HBSS) with 6% dextran (average molecular weight 148,000). The tube was gently inverted to mix the dextran/blood and left at room temperature for 45 minutes to allow the red blood cells to settle. In a 50ml polypropylene centrifuge tube, a 16ml aliquot from the leukocyte-enriched supernatant was overlaid on a 10ml Ficoll-Hypaque pad and the tube was centrifuged at 400g for 35min at 21 ℃. The plasma, monocyte layer and Ficoll were removed, leaving a granulocyte-rich discard mass. The discard block was first resuspended in 10ml of ice-cold distilled water for 45 seconds to dissolve the contaminating red blood cells, followed by the addition of 10ml of ice-cold double concentrated Phosphate Buffered Saline (PBS) solution to each tube to restore osmolarity. The suspension was re-centrifuged at 200g for 10min at 4 ℃ to produce a neutrophil discard mass. The supernatant was removed and the expulsed mass was gently resuspended in a total volume of 10ml of ice-cold HBSS using a Pasteur pipette. The resulting neutrophil suspension was subjected to differential leukocyte counts using a Beckman Coulter ac.t5 hematology analyzer, and the cells were stored on ice until assayed. Shortly before the assay, an aliquot of the neutrophil suspension was removed and diluted to 4X 10 in ice-cold HBSS containing 2U/ml adenosine deaminase⁶Neutrophils/ml.

Inhibition of fMLP-induced elastase release was achieved in 96-well polystyrene microtiter plates using a 160 μ Ι assay volume. Elastase release was determined by measuring the rate of cleavage of the synthetic substrate MeOSuc-Ala-Ala-Pro-Val-p NA. For the measurement of elastase release, the assay wells contained 8 μ l of 100 μ g/ml cytochalasin B (in 10% DMSO/90% HBSS), 8 μ l of test compound (diluted in HBSS), 40 μ l of neutrophil suspension, and 96 μ l of 156 μ M MeOSuc-Ala-Ala-Pro-Val- ρ NA (in HBSS). Will measureThe plates were incubated at 37 ℃ for 10min, then 8 μ l of 2 μ M fMLP (in HBSS) was added and the rate of substrate cleavage was measured at 37 ℃ at λ 405nm for 3 min. Basal elastase release was determined by adding 8 μ l hbss instead of fMLP. The data in Table 1 below are IC₅₀The value, i.e., the active drug concentration (nM) required to achieve 50% inhibition of fMLP-induced elastase release. In the case of a combination experiment, 1000nM 9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-thienyl) -9H-pyrazolo [3, 4-c ] was added to the assay]-1, 2, 4-triazolo [4, 3-a]Pyridine (C), then a concentration response curve is generated from formoterol (F) or salmeterol (S).

TABLE 1 inhibition of Elastase Release (IC)₅₀Value, nM)

F	S	C	F+C	S+C
					＞1000	＞1000	＞1000	0.4	1.0

The use of a combination of a β 2 agonist (formoterol or salmeterol) and a PDE 4inhibitor (9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-thienyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine) has been shown to produce a synergistic inhibitory effect on proinflammatory neutrophil function. Individual treatments with these pharmacological agents achieved weak inhibition (μ M) of fMLP-induced elastase release from human neutrophils ex vivo, and their combined use dramatically enhanced this inhibition to very strong (nM).

Claims (17)

1. Inhalation combinations of the following (a) and (b):

(a) selective PDE 4inhibitors of formula (I)

Or a pharmaceutically acceptable salt or solvate thereof, wherein:

R¹is H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₂-C₄) Alkenyl, phenyl, -N (CH)₃)₂、(C₃-C₆) Cycloalkyl group, (C)₃-C₆) Cycloalkyl (C)₁-C₃) Alkyl or (C)₁-C₆) Acyl, wherein the alkyl, phenyl or alkenyl radical may be interrupted by up to two-OH groups, (C)₁-C₃) Alkyl or-CF₃A group or up to three halogen substitutions;

R²and R³Each independently selected from the group consisting of H, (C)₁-C₁₄) Alkyl, (C)₁-C₇) Alkoxy (C)₁-C₇) Alkyl, (C)₂-C₁₄) Alkenyl, (C)₃-C₇) Cycloalkyl group, (C)₃-C₇) Cycloalkyl (C)₁-C₂) Alkyl, saturated or unsaturated (C)₄-C₇) Heterocycle (CH)₂)_nGroup consisting of, wherein n is 0, 1 or 2, containing one or two of oxygen, sulfur, sulfonyl, nitrogen and NR⁴As a hetero atom, wherein R⁴Is H or (C)₁-C₄) An alkyl group; or a group of formula (II):

wherein a is an integer from 1 to 5; b and c are 0 or 1; r⁵Is H, -OH, (C)₁-C₅) Alkyl, (C)₂-C₅) Alkenyl, (C)₁-C₅) Alkoxy group, (C)₃-C₆) Cycloalkoxy, halogen, -CF₃、-CO₂R⁶、-CONR⁶R⁷、-NR⁶R⁷、-NO₂or-SO₂NR⁶R⁷Wherein R is⁶And R⁷Each independently is H or (C)₁-C₄) An alkyl group; z is-O-, -S-, -SO₂-, -CO-or-N (R)⁸) -, wherein R⁸Is H or (C)₁-C₄) An alkyl group; y is (C)₁-C₅) Alkylene or (C)₂-C₆) Alkenylene optionally substituted by up to two (C)₁-C₇) Alkyl or (C)₃-C₇) Cycloalkyl substitution; wherein each alkyl, alkenyl,The cycloalkyl, alkoxyalkyl or heterocyclic group may be substituted by 1 to 14, preferably 1 to 5 (C)₁-C₂) Alkyl, CF₃Or halo group substitution;

R⁹and R¹⁰Each independently selected from the group consisting of H, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy group, (C)₆-C₁₀) Aryl and (C)₆-C₁₀) Aryloxy group;

and (b) an adrenergic beta 2 receptor agonist.

2. A combination as claimed in claim 1, wherein R¹Is methyl, ethyl or isopropyl.

3. A combination as claimed in claim 1 or claim 2, wherein R is³Is (C)₁-C₆) Alkyl, (C)₂-C₆) Alkenyl, (C)₃-C₇) Cycloalkyl group, (C)₃-C₇) Cycloalkyl (C)₁-C₆) Alkyl or phenyl, optionally substituted with 1 or 2 groups selected from H, -OH, (C)₁-C₅) Alkyl, (C)₂-C₅) Alkenyl, (C)₁-C₅) Alkoxy, halogen, trifluoromethyl, -CO₂R⁶、-CONR⁶R⁷、-NR⁶R⁷、-NO₂or-SO₂NR⁶R⁷Wherein R is⁶And R⁷Each independently is H or (C)₁-C₄) An alkyl group.

4. A combination as claimed in any one of the preceding claims wherein the selective PDE 4inhibitor of formula (I) is selected from:

9-cyclopentyl-5, 6-dihydro-7-ethyl-3-phenyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (furan-2-yl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-pyridyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (4-pyridyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (3-thienyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

3-benzyl-9-cyclopentyl-5, 6-dihydro-7-ethyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3-propyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

3, 9-dicyclopentyl-5, 6-dihydro-7-ethyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (1-methylcyclohex-1-yl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

3- (tert-butyl) -9-cyclopentyl-5, 6-dihydro-7-ethyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-methylphenyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-methoxyphenyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (thiophen-2-yl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

3- (2-chlorophenyl) -9-cyclopentyl-5, 6-dihydro-7-ethyl-9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-iodophenyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-trifluoromethylphenyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine; and

5, 6-dihydro-7-ethyl-9- (4-fluorophenyl) -3- (1-methylcyclohex-1-yl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine;

and pharmaceutically acceptable salts and solvates thereof.

5. A combination as claimed in claim 4 wherein the selective PDE 4inhibitor of formula (I) is selected from 9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-thienyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine and 9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (tert-butyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine and pharmaceutically acceptable salts and solvates thereof.

6. A combination as claimed in any one of the preceding claims wherein the adrenergic beta 2 receptor agonist is selected from salmeterol, formoterol and pharmaceutically acceptable salts and solvates thereof.

7. A combination as claimed in claim 1, wherein:

the selective PDE 4inhibitor of formula (I) is 9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-thienyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine or a pharmaceutically acceptable salt or solvate thereof, and the adrenergic beta 2 receptor agonist is salmeterol or a pharmaceutically acceptable salt or solvate thereof;

the selective PDE 4inhibitor of formula (I) is 9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (2-thienyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine or a pharmaceutically acceptable salt or solvate thereof, and the adrenergic beta 2 receptor agonist is formoterol or a pharmaceutically acceptable salt or solvate thereof;

the selective PDE 4inhibitor of formula (I) is 9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (tert-butyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine or a pharmaceutically acceptable salt or solvate thereof, and the adrenergic beta 2 receptor agonist is salmeterol or a pharmaceutically acceptable salt or solvate thereof;

the selective PDE 4inhibitor of formula (I) is 9-cyclopentyl-5, 6-dihydro-7-ethyl-3- (tert-butyl) -9H-pyrazolo [3, 4-c ] -1, 2, 4-triazolo [4, 3-a ] pyridine or a pharmaceutically acceptable salt or solvate thereof and the adrenergic beta 2 receptor agonist is formoterol or a pharmaceutically acceptable salt or solvate thereof.

8. A combination as claimed in any preceding claim for use as a medicament.

9. A combination as claimed in any one of claims 1 to 7 for simultaneous, sequential or separate administration in the treatment of an obstructive airways or other inflammatory disease.

10. A pharmaceutical composition comprising a selective PDE 4inhibitor of formula (I) as defined in claim 1, an adrenergic β 2 receptor agonist and a pharmaceutically acceptable excipient, diluent or carrier for administration by the inhaled route in the treatment of an obstructive airways or other inflammatory disease.

11. A pharmaceutical composition as defined in claim 10 wherein the selective PDE 4inhibitor of formula (I) and the adrenergic β 2 receptor agonist are as defined in any one of claims 2 to 7.

12. Use of a selective PDE 4inhibitor of formula (I) or an adrenergic beta 2 receptor agonist as defined in claim 1 in the manufacture of a medicament for simultaneous, sequential or separate administration of the two drugs by the inhaled route in the treatment of an obstructive airways or other inflammatory disease.

13. Use according to claim 12, wherein the selective PDE 4inhibitor of formula (I) and the adrenergic β 2 receptor agonist are as defined in any one of claims 2 to 7.

14. A method of treatment of an obstructive airways or other inflammatory disease which comprises simultaneously, sequentially or separately administering to a mammal in need of such treatment, by the inhaled route, an effective amount of a selective PDE 4inhibitor of formula (I) as defined in claim 1 and an adrenergic β 2 receptor agonist.

15. A method as claimed in claim 14 wherein the selective PDE 4inhibitor of formula (I) and the adrenergic β 2 receptor agonist are as defined in any one of claims 2 to 7.

16. An inhalation device for the simultaneous, sequential or separate administration of a selective PDE 4inhibitor of formula (I) as defined in claim 1 and an adrenergic β 2 receptor agonist in the treatment of an obstructive airways or other inflammatory disease.

17. An inhalation device as claimed in claim 16, wherein the selective PDE 4inhibitor of formula (I) and the adrenergic β 2 receptor agonist are as defined in any one of claims 2 to 7.