WO2009152340A2

WO2009152340A2 - 2,3-substituted thiophenes in lung inflammation therapies

Info

Publication number: WO2009152340A2
Application number: PCT/US2009/047051
Authority: WO
Inventors: Andrew Garton; Neil Gibson; Graeme Griffin
Original assignee: Osi Pharmaceuticals, Inc.
Priority date: 2008-06-12
Filing date: 2009-06-11
Publication date: 2009-12-17
Also published as: WO2009152340A3

Abstract

Methods of treating inflammatory lung conditions comprising administering 2,3- substituted thiophenes of the formula (I).

Description

2.3-SUBSTITUTED THIOPHENES IN LUNG INFLAMMATION THERAPIES

BACKGROUND

The present invention relates to pharmaceuticals, kinase inhibitors, substituted thiophenes, c-kit inhibitors, disease treatment, modulation of airway resistance, and respiratory treatments, among other things.

It has been reported that increased stem cell factor and c-kit is asthmatic airways. YK Kim et al., J. Allergy Clin. Immunol. 100:389-399 (1997); S.Z. Al-Muhsen et al., Clin. Exp. Allergy 34, 911-916 (2004).

Asthma has been characterized as a Th2 type inflammatory disease that depends upon an inflammatory cascade involving numerous inflammatory cell types such as mast cells, eosinophils, and neutrophils, and a range of inflammatory-related chemokines and cytokines released by these cells.

Researchers have reported a chronic mouse model of allergic asthma using multiple cockroach allergen challenges. Using imatinib mesylate as an inhibitor of SCF/c-kit- associated receptor tyrosine kinase, an effect on chronic peribronchial allergen-induced fibrotic remodeling and airway responses was observed. A.A. Berlin et al., Lab. Investigation

86, 557-565 (2006); A.A. Berlin et al., Am. J. Respir. Crit. Care Med. 171 , 35-39 (2005); A.A.

Berlin et al., Clin. Exp. Immunol. 136, 15-20 (2004). Others have disclosed that inhibition of the SCF/c-kit pathway leads to significant decrease of histamine levels, mast cells and eosinophil infiltration, IL-4 production, and airway hyperresponsiveness. L. Reber et al., Eur.

J. Pharmac. 533, 327-340 (2006).

US2007/0054903 recites bis-aryl compounds and methods of treating numerous disorders, including asthma, therewith. US2007/0208053 recites heterobicyclic compounds and methods of treating numerous disorders, including asthma, therewith. US2005/0154014 and US6949563 disclose various 2,3-substituted thiophene compounds and the inhibition of c-kit and applications in cancer treatments therewith.

While not limiting of the present invention, there is a desire for treatments for inflammatory respiratory conditions such as asthma, COPD and others, including by the use of agents that may successfully inhibit inflammatory cell infiltration or recruitment to inflammation sites and may provide sustained effects on smooth muscle.

SUMMARY

The present invention provides methods treating and preventing inflammatory respiratory ailments, including asthma, comprising administering 2,3-substituted thiophene compounds as active agents. Such compounds can be used in the aspects described below.

i DETAILED DESCRIPTION

SUBSTITUTED THIOPHENES

Active agent(s) suitable in the present invention include one or more 2,3-substituted thiophenes or pharmaceutically acceptable salts or N-oxides thereof as described herein or in US 2005/0154014.

In some embodiments, an active agent used in the present invention is of the Formula

wherein:

R1 is selected from

R2 is selected from

; and wherein said quinolinyl group can be substituted by 1 to 2 groups independently selected from -OH, C_1-3alkoxy, or C_1-3alkoxy substituted by methoxy; and R3 is Co-₄alkyl.

In some embodiments, R2 is

that is optionally substituted by -OH, Ci- ₃alkoxy, or C_1-3alkoxy substituted by methoxy. In some embodiments, R3 is hydrogen.

In some embodiments, R1 is selected from:

R2 is selected from:

; and wherein said quinolinyl group can be substituted by methoxy.

In some embodiments, an active agent used in the present invention is selected from the compounds in Table 1 , below.

In some embodiments, an active agent used in the present invention is 3-{[(7- methoxyquinolin-4-yl)methyl]amino}-Λ/-[4-(trifluoromethyl)phenyl]thiophene-2-carboxamide (Compound 13 herein), or Λ/-(4-trifluoromethoxyphenyl) 3-[(quinolin-4-ylmethyl)amino] thiophene-2-carboxamide, or a pharmaceutically acceptable salt thereof, such as hydrochloride, di-hydrochloride, p-tosylate, or di-p-tosylate.

In some embodiments, an active agent used in the present invention can be a compound of Formula I having a biochemical IC50 against kit of about 0.1 μM or less, 0.05 μM or less, or 0.01 μM or less. Such compounds may also have a biochemical IC₅₀ against KDR of about 0.1 μM or less, 0.05 μM or less, or 0.01 μM or less. Suitable assays are described below and results shown in Table 1.

The compounds can be prepared by known methods, such as disclosed in US2005/0154014A1.

COMPOSITIONS The active compounds and compound combinations of the invention can be administered unformulated or formulated in unit dosage form with or without one or more carriers in any desired manner for any desired administration mode such as oral, inhalation, and parenteral (including subcutaneous, intramuscular, and intravenous). Such formulations may provide for immediate, delayed, or sustained release of active agents.

TARGETS AND INDICATIONS

The present invention includes treating and preventing inflammatory respiratory ailments mediated by c-kit/SCF alone or in combination with other mechanisms.

The present invention includes treating respiratory ailments, including ailments mediated by one or more of c-kit/SCF, PDGFR, CSF1 R, or KDR. The present invention includes treating inflammatory respiratory ailments by inhibiting inflammatory cell infiltration and/or reducing inflammatory cytokine levels, including Th2 cytokines.

The present invention includes modulating airway resistance to treat inflammatory respiratory ailments and treating conditions for which such modulation is beneficial. Indications include inflammatory respiratory ailments, including allergy-related conditions, asthma, hypereosinophilic disease, COPD and related conditions, rhinitis and related conditions, and other inflammatory or allergic lung diseases. In some embodiments, the condition is asthma.

USE

The present invention provides methods of treating and preventing inflammatory respiratory ailments, including allergy-related conditions, asthma, hypereosinophilic disease,

COPD and related conditions, rhinitis and related conditions, and other inflammatory or allergic lung diseases, as discussed above. The present invention includes carrying out the above methods and treating such ailments by administration of one or more 2,3-substituted thiophenes, as discussed above.

The present invention includes carrying out the above methods and treating such ailments by administering compounds 2,3-substituted thiophenes such as those described herein, in

US2005/0154014, and the like. The present invention includes treating and preventing inflammatory respiratory ailments in a human or other mammal with a therapeutically effective amounts of a compound(s) or composition of the invention as described.

Dosing route, strength, and frequency will depend upon the particular properties of the active compound(s) being utilized, the condition(s) being treated or prevented, and should be separately determined for each situation.

In some embodiments, a compound or composition of the present invention is orally or inhalation administered to a subject in a therapeutically effective amount.

In some embodiments, a compound or composition of the present invention is administered orally or by inhalation to a subject in a therapeutically effective amount at a strength of about 1-5, 5-15, 15-50, or 50-100 mg/kg per day. In some embodiments thereof, administration frequency is QD. In other embodiments, the dosing schedule can be QD, BID,

TID, or QID. Treatment can commence when indicated and continue as long as indicated.

The compound(s) may be administered in combination with or sequentially with additional agent(s) that are useful against a respiratory condition being treated, such as additional agent(s) for treating asthma, COPD, or other lung inflammation condition treatments.

PROTEIN KINASE ASSAYS

Table 1 shows IC₅₀ ranges for 2,3-substituted thiophenes. The actual mean value obtained for each compound was lower by less than one order of magnitude than the value shown. NT indicates that the compound was not tested. Testing methods are described below.

TABLE 1

Summary of Protein Kinase Assays

Protein kinase assays were done by ELISA-based assay methods. ELISA assays used poly(Glu:Tyr) (Sigma, St. Louis, MO) as the substrate bound to the surface of 96-well assay plates; phosphorylation was then detected using an antiphosphotyrosine antibody conjugated to HRP. The bound antibody was then quantitated using ABTS as the peroxidase substrate by measuring the absorbance at 405/490 nm. All assays used purified recombinant kinase catalytic domains that were either expressed in insect cells or in bacteria. Recombinant Kit protein was expressed as an NH2-terminal glutathione S-transferase fusion protein in insect cells and was initially purified as a nonphosphorylated (nonactivated) enzyme with a relatively high Km for ATP (400 μmol/L). In some assays, an activated (tyrosine phosphorylated) form of the enzyme was prepared by incubation with 1 mmol/L ATP for 1 hour at 30 ⁰C. The phosphorylated protein was then passed through a desalting column to remove the majority of the ATP and stored at -80 ⁰C in buffer containing 50% glycerol. The resultant preparation had a considerably higher specific activity and a lower Km for ATP (25 μmol/L) than the initial nonphosphorylated preparation. The inhibition of Kit autophosphorylation was assayed by incubation of the nonphosphorylated enzyme at 30 ⁰C in the presence of 200 μmol/L ATP and various concentrations of test compound. The reaction was stopped by removal of aliquots into SDS-PAGE sample buffer followed by heating to 100 ⁰C for 5 minutes. The degree of phosphorylation of Kit was then determined by immunoblotting for both total Kit and phosphorylated Kit.

Activated c-kit Kinase Bench Assay cDNA encoding the kit tyrosine kinase domain was isolated from K562 cells and cloned into a baculovirus expression vector for protein expression as a fusion protein with GST (Glutathione S-Transferase) in insect cells. Following purification, the enzyme was incubated with ATP to generate a tyrosine phosphorylated, activated form of the enzyme, which was used in kinase assays to determine the ability of compounds to inhibit phosphorylation of an exogenous substrate by the kit tyrosine kinase domain. Phosphorylation of c-kit protein

Column Buffer: 5OmM HEPES pH 7.4125mM NaCI; 10% Glycerol; 1 mg/mL BSA; 2mM DTT; 200 μM NaVO₃

Phosphorylation Buffer: 5OmM HEPES pH 7.4; 125mM NaCI; 24mM MgCI₂ 1 mM MnCI₂; 1 % Glycerol; 200 μM NaVO₃; 2mM DTT; 2mM ATP. 75μl_ purified GST-kit tyrosine kinase protein (approximately 150μg) is incubated with

225μl_ phosphorylation buffer for 1 h at 3O⁰C. In a cold room, a desalting column (e.g. Pharmacia PD-10 column) is equilibrated using 25ml_ of column buffer. Phosphorylated protein is applied to the column followed by sufficient column buffer to equal 2.5ml_ total (in this case 2.2ml_). The phosphorylated kit protein is then eluted with 3.5ml_ column buffer, and collected into a tube containing 3.5ml_ glycerol (final concentration of 50% glycerol). After mixing, aliquots are stored at -2O⁰C or -7O⁰C.

Kinase activity is determined in an ELISA-based assay that measures the ability of kit to phosphorylate an exogenous substrate (poly Glu:Tyr) on tyrosine residues in the presence of ATP. Substrate phosphorylation is monitored by quantitation of the degree of binding of an antibody that recognizes only the phosphorylated tyrosine residues within the substrate following incubation with kit. The antibody used has a reporter enzyme (e.g. horseradish peroxidase, HRP) covalently attached, such that binding of antibody to the phosphorylated substrate can be determined quantitatively by incubation with an appropriate HRP substrate (e.g. ABTS). The stock reagents used are as follows:

13.3 μg/mL PGT stock solution: Add 66.7μl_ 10mg/ml_ PGT to 5OmL PBS. 1X wash buffer: Dilute 2OX wash buffer (KPL #50-63-00) to 1 X with H₂O. Assay Buffer: 5OmM Hepes, pH 7.4; 125mM NaCI; 24mM MgCI_2, 1 mM MnCI₂; 1% Glycerol; 200μM Vanadate -add immediately prior to use; 2mM DTT - add immediately prior to use.

Assay buffer + ATP: Add 5.8μL of 75mM ATP to 12mL of assay buffer. Activated GST-c-kit(TK): Dilute 1 :500 in assay buffer. Block Buffer: PBS containing 0.5% Tween-20, 3% BSA

200μM Vanadate - add immediately prior to use. pY20-HRP: Add 6.2μL of a 100μg/mL stock of pY20-HRP to 1 OmL of block buffer. ABTS substrate: KPL 3 50-66-06, use as provided. Assay protocol

Each well of a 94-well immulon-4 microtitre plate is coated with 75μL of 13.3μg/mL PGT stock solution, incubated overnight at 37⁰C and washed once with 250μL 1X wash buffer.

To the negative control wells, 50μL of assay buffer (without ATP) are added, all other wells contain 50μL assay buffer +ATP. To positive and negative control wells, 10μL 5% DMSO is added, other wells contain 10μL of test compounds (at concentrations between 1 OnM and 100μM) dissolved in 5% DMSO. 30μl_ of activated GST-c-kit are added to initiate the assay, which is incubated at RT for 30min, and then stopped by the addition of 50μl_/well of 0.5M EDTA. The plate is washed 3X with 1X wash buffer, and then 75μl_ of a phospho-tyrosine-specific antibody-HRP conjugate (e.g. pY20-HRP, Calbiochem) in block buffer are added. The plate is incubated at RT for 2h, and then washed 3X with 1X wash buffer. 100μL of ABTS substrate are then added, the plate is incubated at RT for 30min, and the reaction stopped by the addition of 100μl_ of 1% SDS. The reaction is quantitated by measuring the OD at 405/49OnM on a microtitre plate reader.

Comparison of the assay signals obtained in the presence of compound with those of controls (in the presence and absence of ATP, with no compound added), allows the degree of inhibition of kinase activity to be determined over a range of compound concentrations. These inhibition values are fitted to a sigmoidal dose-response inhibition curve to determine the IC₅₀ values (Ae. the concentration of compound that reduces the kinase activity to 50% of the control activity).

Mechanistic assays of protein kinase inhibition in intact cells. Cells were seeded the day before use into 96-well plates for quantitative 96-well

ELISA-based assays of the cellular effects of compound or into 10-cm dishes for analysis by immunoblotting. The cells were treated with various concentrations of compound for 3 hours before lysis (the final concentration of DMSO in the assay was 0.1 %), and as required, the appropriate ligand was added for the final 15 minutes of the compound treatment period [100 ng/mL stem cell factor (R&D Systems), 50 ng/mL VEGF165 (R&D Systems), and 25 ng/mL PDGF-BB (R&D Systems)]. Lysates were then prepared in buffer containing 50 mmol/L Tris- HCI (pH 7.4), 150 mmol/L NaCI, 10% glycerol, 1% Triton X-100, 0.5 mmol/L EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and 1 mmol/L sodium orthovanadate. ELISA-based assays of target protein phosphorylation were done by transferring lysates into a second 96-well plate that was precoated with the appropriate capture antibody. The captured target proteins were then probed with an antiphosphotyrosine antibody-HRP conjugate using a chemiluminescent HRP substrate (Pierce) for detection by luminometry. In experiments done to evaluate the effect of plasma protein binding of compound on its ability to affect cellular processes, purified human plasma proteins albumin (Sigma) and α1-acid glycoprotein (Sigma) were incorporated into the quantitative 96-well assays at concentrations approximating those found in vivo (3% and 0.05%, respectively). In these experiments, plasma proteins were added to the cell culture medium before compound addition and the DMSO stock solution of compound was also initially diluted into cell culture medium containing plasma proteins to ensure preequilibration of compound binding to plasma protein. For immunoblotting analysis, lysates were cleared of insoluble material by centrifugation at 15,000 x g for 5 minutes at 4 ⁰C and the resultant supernatant was subjected to immunoprecipitation with the appropriate antibody coupled to Protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ), followed by SDS-PAGE and immunoblotting with the same antiphosphotyrosine antibody-HRP conjugate and chemiluminescent detection. Alternatively, for highly abundant protein targets (S6, Erk, and p70S6K), lysates were analyzed directly by SDS-PAGE and immunoblotting. Compound 13 was evaluated in two mouse models of inflammatory lung ailments. In response to challenge, the data demonstrate efficacy of Compound 13, inhibition of inflammatory cell infiltration (including eosinophils, granulocytes, and lymphocytes (T-cells and B-cells)) into the lungs, reduction of airway hyperresponsiveness, and reduction of inflammatory cytokine levels.

FIRST MOUSE MODEL

Compound 13 was evaluated in a murine model of cockroach allergen-induced hyperreactive airway inflammation. See A.A. Berlin et al., Lab. Investigation 86, 557-565 (2006), which is incorporated herein in its entirety for all purposes including the descriptions of the murine model.

Studies utilized Balb/c/J mice sensitized with soluble cockroach antigens in incomplete Freund's adjuvant intraperitoneally. After 14 days the mice were again sensitized with soluble cockroach antigen by an intranasal administration. Then 3 additional intranasal challenges every 4 days were given. Subsequently, 4 days after the 4th intranasal we mice were instilled with an intratracheal injection of cockroach antigen followed by a second intratracheal administration of allergen 4 days later. Prior to the intratracheal challenges the allergic mice received Compound 13 or a control treatment. The delivery route of Compound 13 was oral gavage using a dose response of compound, 25, 2.5, 0.25 mg/kg, 30 minutes before the allergen and each day between the final 2 allergen challenges. Labrafil was the vehicle for Compound 13. As a positive control for treatment one group of animals was treated with montelucast, which is presently approved for clinical use. After 24 hours post-final allergen challenge the mice were examined for airway hyperreactivity, inflammation, and peribronchial eosinophil and neutrophil accumulation. This response is dependent upon Th2 type immune responses (anti-IL-4 or anti-IL-13 attenuate the response). Blood samples were collected for PK analysis. The studies used 10 animals total in each group, including a group of naϊve controls. Histological analysis consisted of peribronchial eosinophil enumeration and assessment of the overall inflammatory picture, including staining for goblet cell development (Ae., mucus production), and staining for mast cells using a T-blue stain. Tissues were saved for further processing if desired for mRNA and protein (cytokine) analyses. Measurement of airway hyperreactivity: Airway hyperreactivity was assessed using a

Buxco mouse plethysmograph specifically designed for the low tidal volumes (Buxco, Troy, NY) using a direct ventilation method for assessment of airway resistance (this is not PENH). The mice were anesthetized with sodium pentobarbital and intubated via cannulation of the trachea with an 18 gauge metal tube. The mouse was subsequently ventilated with a mouse ventilator (tidal volume = 0.25 ml, frequency = 120 breaths/min, positive end-expiratory pressure -1.0 cm H₂O). The plethysmograph were sealed and readings monitored by computer. Since the box is a closed system, a change in lung volume is represented by a change in box pressure (Pbox) which is measured by a differential transducer. The system is calibrated with a syringe that delivered a known volume of 2 ml. A second transducer was used to measure the pressure swings at the opening of the trachea tube (Paw), referenced to the body box (i.e., pleural pressure), and to provide a measure of transpulmonary pressure (Ptp=Paw-Pbox). The trachea transducer was calibrated at a constant pressure of 20 CmH₂O. Resistance was calculated by the Buxco software by dividing the change in pressure (Ptp) by the change in flow (F) (_Ptp/_F; units= cmH₂O/ml_/sec) at two time points from the volume curve based upon a percentage of the inspiratory volume. Once the mouse was hooked up to the box it was ventilated for 3 minutes prior to acquiring readings. Once baseline levels stabilized and initial readings taken, a methacholine challenge was given. After determining a dose response curve, an optimal dose was chosen. This dose was used throughout the rest of the experiments. After the methacholine challenge, the response was monitored and the peak airway resistance recorded as a measure of airway hyperreactivity.

Alteration of airway physiology by c-kit blockade: Compound 13 was administered by oral gavage at three different concentrations as indicated above. The data in Figure 1 illustrates that findings from these studies related to the induction of AHR using sensitive instrumentation made specifically for low tidal volumes of the mouse. Data represent the mean +/- SE of 6-8 mice/group. These measurements utilize anesthetized and ventilated animals monitored by computer. The AHR data demonstrate that Compound 13 causes a dose dependent decrease in the airway hyperreactivity induced by an allergen stimulus. Alteration of eosinophil accumulation by c-kit blockade Eosinophils are most often associated with development of asthma and can determine the severity and intensity of the disease. One way to assess the intensity of eosinophil accumulation is to use histologic enumeration of the peribronchial eosinophils. The parameter has been used in these studies to determine whether increasing doses of Compound 13 would alter the eosinophil accumulation response. Initial experiments found an inconsistent decrease in the number of eosinophils, likely due to the low total numbers of eosinophils in the control group, and any effects observed were not dose-dependent. Figure 2 shows enumeration of peribronchial eosinophils were not significantly inhibited in animals treated with Compound 13. Data represents mean ± SE from 4-5 mice/gp.

Cytokine levels after blockade of c-kit: In order to further examine one potential mechanism of the altered physiologic response, whole lung tissue from challenged mice was isolated and subjected to analysis of Th1 and Th2 cytokine expression levels. Samples from control mice with no allergen had no detectable levels of the asthma-related cytokines. Figures 3 and 4 depict observed cytokine levels in two experiments. Based on the average of the two experiments, compound 13 reduced the levels of all three cytokines assessed (IL-4, IL-5 and IL-13) in the lungs of the sensitized mice.

SECOND MOUSE MODEL

Compound 13 was evaluated in an ovalbumin-induced murine asthma model.

Study Outline: The potential anti-asthmatic activity of three test items at three doses was assessed in the ovalbumin-induced experimental murine asthma model. Asthma was induced by immunization with OVA/Alum on days 0 and 14 and by intranasal challenge with OVA on days 14, 25 and 26. The lung response to NECA challenge was assessed 4h after OVA on day 26. On day 27 the mice were culled and cell influx, pulmonary cytokines and systemic anti-OVA antibodies. Mice were treated with test or control items once daily on days 24, 25 and 26.

Inflammatory cell types present within the lung airways and tissue: Following asthma induction 1.4x10⁶ cells were observed within the BALF of vehicle treated mice, this was significantly reduced to 5.5 x10⁶ by treatment with 25mg/kg Compound 13 (group 3F).

Of these cells 70%, -9.7x10⁵ were granulocytes of which 80% -7.7x10⁵ were eosinophils, 20% -1.7x10⁵ and were neutrophils. There were also -2.3 x 10⁵ lymphocytes (17% of cells). Of these lymphocytes -1.5 x105 were MHCII- T cells (70%) and -6.9x10⁴ were B cells (29%, MHCII+). Finally 6%, ~9.4x10⁴ auto-fluorescent macrophages.

Dexamethasone treatment significantly reduced the number of granulocytes and eosinophils, and also increased the number of macrophages. Treatment with 25mg/kg Compound 13 (group 3F) significantly reduced the numbers of granulocytes and eosinophils.

BALF cytokine levels: Following asthma induction ~220pg/ml IL-4, ~25pg/ml IL-5 and ~9pg/ml IL-5 were found in the Bronchoalveolar Lavage Fluid (BALF), these were all reduced (but not significantly) by dexamethasone treatment, treatment with Compound 13 at 25mg/kg (group 3F) reduced IL-5 levels to a similar extent as dexamethasone.

Serum antibody levels: Levels of neither anti-OVA IgE, IgGI nor lgG2a isotypes were significantly affected by any of the treatments when administered on days 24-16. Penh percentage increase above baseline: The percentage increase above baseline for vehicle treated mice (1 F) rose to 600% after challenge with 400μg/ml NECA. At the same NECA challenge there was only a 200% increase above baseline Penh in dexamethasone treated mice (2F, significantly lower than 1 F). Test Materials: Compound 13 reconstituted in Labrafil.

Positive Control: Dexamethasone (water soluble) Sigma D12915 White powder 2-8°C. A stock solution of the dexamethasone vehicle, 0.5% CMC/0.25% Tween 80, was prepared in dH₂0 and dissolved overnight on a magnetic stirrer. Test System: Species/Strain: Mouse / BALB/c. Gender: Female. Total No. of Animals: n=40. Age:

Young adults, 6-7 weeks of age at study initiation. Body Weight: Weight variation of animals at the time of treatment initiation did not exceed ± 20% of the mean weight. Animals Health: The health status of the animals used in this study was examined on arrival. Only animals in good health were acclimatized to laboratory conditions and were used in the study. Acclimation: At least 7 days. Housing: During acclimation and following dosing, animals were housed within a limited access rodent facility and kept in groups of maximum 10 mice, in polypropylene cages, fitted with solid bottoms and filled with wood shavings as bedding material. Food and Water: Animals were provided ad libitum a commercial rodent diet and free access to drinking water, supplied to each cage via polyethylene bottles with stainless steel sipper tubes. Environment: Automatically controlled environmental conditions were set to maintain temperature at 20 - 24°C with a relative humidity (RH) of 30 - 70%, a 12:12 hour light: dark cycle and 10-30 air changes/hr in the study room. Temperature and RH were monitored daily by both manual measurements and the control computer. The light cycle was monitored by the control computer. Identification: Animals were given a unique animal identification number. This number also appears on a cage card, visible on the front of each cage. The cage card also contains the study number. Randomization: Animals were randomly assigned to experimental groups. Termination: At the end of the study surviving animals were euthanized by cervical dislocation. Justification: The mouse was selected since it represents the species of choice for this experimental animal model. The BALB/c strain of mouse is highly susceptible to Ovalbumin-induced asthma. Test Groups:

1 F: Vehicle. 2F: Dexamethasone. 3F: Compound 13 25 mg/kg. 4F: Compound 13 2.5 mg/kg. 5F: Compound 13 0.25 mg/kg. For each group: size (n) =10; dose vol = 10 mL/kg per oral (PO) once daily on days 24-26 2h prior to OVA challenge. Test Procedures: Days 0 & 14 Sensitization: i.p. injection of OVA/Alum; Days 14, 25&26 OVA challenge: i.n. administration of OVA; Day 26 NECA challenge: AHR-Penh; Test and Control Items: x1 daily days 24-26. Day 27 Analyze response: BALF cell types, BALF cytokines, Serum IgG/lgE. Observations and Examinations:

Penh was investigated by whole body plethysmography 4h after OVA challenge on day 26 in 8/10 mice. The animals were challenged with PBS or increasing concentrations of NECA for 2 min then Penh was assessed for 5min. The remaining 2 mice per group were also exposed to 400μg/ml NECA for 2 min to compensate for the exposure of the other animals in that group to the NECA.

Sample Collection: On day 27 all mice were culled and a blood sample taken from each mouse and serum prepared for lgG1/lgG2a/lgE measurement. Bronchoalveolar lavage was carried out and part of the bronchoalveolar lavage fluid (BALF) was used to analyze the cell influx via FACS analysis, part was used to study cytokine levels. The cellular markers used for cell differentiation were: CCR3 (eosinophil), CD3 (T cells), MHC Il (T/B cells) and B220 (B cells). Macrophages and neutrophils are identified by their lack of these other markers and their granularity/size.

Parameters measured: A - Cell differentials within bronchoalveolar lavage fluid (BALF) using FACS (eosinophils, neutrophils, macrophages, T cells, B cells). B - Cytokine levels within BALF (IL-4, IL-5, IL-13). C - Serum OVA-specific IgG/lgE. D - Determine penh as an assessment of airways hyper responsiveness in response to NECA challenge. E - Determine mast cell protease levels in serum.

Clinical Signs: Careful clinical examinations were carried out daily. Observations included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions (e.g. diarrhea) and autonomic activity (e.g. lacrimation, salivation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of bizarre behavior, tremors, convulsions, sleep and coma were also examined.

No deviations to the protocol occurred during the course of this study. Data Evaluation:

Evaluation was primarily based on the Penh response to NECA, FACS analysis of the cell types within the BAL and lung tissue, serum antibody titers and cytokine levels within the BALF. Where appropriate, analysis of the data by statistical methods (ANOVA with Tukey post hoc analysis, WinStat 2005.1 for FACS data and BALF cytokine levels and the nonparametric Mann-Whitney U test WinStat 2005.1 (with Bonferroni correction) for penh and serum antibody levels) has been applied to determine significance of treatment effects. Results: Clinical Signs: No adverse clinical signs related to test item were observed during the course of this study.

Cell numbers within the BALF (Figure 5 shows the total number of cells within the airways/mouse. Graph shows mean+SEM; ^*, represents significantly different to 1 M (p<0.05 by ANOVA with tukey post-hoc analysis)): On day 27 the number of cells obtained following bronchoalveolar lavage were counted using a haemocytometer. Following asthma induction in vehicle treated mice 1.4x10⁶ leucocytes were observed within the BALF, this was significantly reduced to 5.5 x10⁵ by treatment with 25mg/kg Compound 13 (group 3F).

Counts of inflammatory cell types present within the lung airways: On day 27 the types of inflammatory cell present within the lung airways of mice were assessed via FACS analysis. Total Granulocytes (Figure 6 shows the number of leukocytes within the airways (BAL) that are granulocytes.): Following asthma induction 9.7x10⁵ granulocytes were found in vehicle treated mice (group 1 F, vehicle). This level was reduced significantly by treatment with dexamethasone (2F) (1.2x10⁵ granulocytes), 25mg/kg Compound 13 (3F) (3x10⁵granulocytes). Eosinophils (Figure 7 shows the number of granulocytes within the airways (BAL) that are eosinophils. Graph shows mean+SEM; ^*, represents significantly different to 1 F (p<0.05 by ANOVA with tukey post-hoc analysis).): Airway granulocytes were further classified as either eosinophils or neutrophils by their expression of the marker CCR3. Of the airway granulocytes in vehicle treated mice (1 F), 7.7x10⁵ were CCR3+ eosinophils. Treatment with dexamethasone significantly reduced the number of eosinophils to 2.1x10⁴. Treatment with 25mg/kg Compound 13 (3F) significantly reduced the number of eosinophils to 2.25x10⁴. Treatment with 2.5mg/kg Compound 13 (4F) reduced eosinophil influx slightly but not significantly. Neutrophils (Figure 8 shows the number of granulocytes within the airways (BAL) that are neutrophils. Graph shows mean+SEM.): Of the granulocytes found in BALF from vehicle treated mice 1.7x10⁵ were neutrophils (CCR3-) were found in BALF from vehicle treated mice. Treatment with dexamethasone or any of the test items did not significantly affect the number of neutrophils. Total Lymphocytes (Figure 9 shows the number of leukocytes within the airways (BAL) that are lymphocytes. Graph shows mean+SEM.): After asthma induction in vehicle treated mice 2.3 x 105 lymphocytes constituted were found in the lung airways. This was not significantly affected by any of the treatment groups. Treatment with 25mg/kg Compound 13 (3F), 2.5mg/kg Compound 13 (4F), 25mg/kg C37590 (9F) or 2.5mg/kg Compound 13 (4F) reduced lymphocyte numbers but not significantly. T cells (Figure 10 shows the number of lymphocytes within the airways (BAL) that are T cells. Graph shows ean+SEM. ^*, represents significantly different to 1 F (p<0.05 by ANOVA with tukey post- hoc analysis).): Airway lymphocytes were further classified as either T cells or B cells by their expression of the marker MHCII. 1.5 x10⁵ MHCII- T cells were found in vehicle treated mice, this was reduced but not significantly by dexamethasone (2M), 25mg/kg Compound 13 (3F), or 2.5mg/kg Compound 13 (4F) treatment. B cells (Figure 1 1 shows the number of lymphocytes within the airways (BAL) that are B cells. Graph shows mean+SEM.): 6.9x10⁴ B cells (MHCII+) were found in the vehicle treated mice; this was not significantly affected by dexamethasone treatment or by any of the test items. Macrophages (Figure 12 shows the number of leukocytes within the airways (BAL) and tissue that are macrophages. Graph shows mean+SEM. ^*, represents significantly different to 1 F (p<0.05 by ANOVA with tukey post-hoc analysis).): Following asthma induction 9.4x10⁴ auto-fluorescent macrophages were found in BALF from vehicle treated mice. Treatment with dexamethasone significantly increased the number of macrophages within the airways to 2.4 x105. Treatment with test items did not significantly affect macrophage levels.

BALF cytokine levels: On day 27 bronchoalveolar lavage was carried out and the levels of the cytokines IL-4, IL-5 and IL-13 within the BALF were assessed by Luminex analysis. IL-4 (Figure 13 shows the level of IL-4 within the BALF. Graph shows mean+SEM.): Following asthma induction ~220pg/ml IL-4 was found in the BALF, this was reduced but not significantly to ~22pg/ml by dexamethasone treatment. Treatment with test items did not affect the BALF IL-4 level. IL-5 (Figure 14 shows the level of IL-5 within the BALF. Graph shows mean+SEM.): Following asthma induction ~25pg/ml IL-5 was found in the BALF, this was reduced to 3pg/ml by dexamethasone treatment. Treatment with Compound 13 at 25mg/kg (group 3F) reduced IL-5 levels to a similar extent to dexamethasone. IL-13 (Figure 15 shows the level of IL-13 within the BALF. Graph shows mean+SEM.): Following asthma induction ~9pg/ml IL-13 was found in the BALF of vehicle treated mice. This level was basically baseline and there was high variation within groups with respect to IL-13 production.

Serum antibody levels: On day 27 a blood sample was taken from each mouse and the levels of OVA specific IgE, IgGI and lgG2a were analyzed in the serum by ELISA. For IgGI and lgG2a the levels were measured by titrating out the sample and determining the dilution of the serum that gives a baseline OD measurement (endpoint titration). For IgE a control antibody was used as a standard. IgE (Figure 16 shows the Serum OVA specific IgE level. Graph shows mean+SEM.): Following asthma induction ~10,000pg/ml OVA specific IgE was found in the serum, treatment with dexamethasone on days 24-26 did not significantly affect this, nor did treatment with any of the test items. IgGI (Figure 17 shows the Serum OVA specific IgGI endpoint titre. Graph shows mean+SEM.): Following asthma induction OVA specific IgGI was found in the serum at a titer of -1.3x10⁸, this was not significantly affected by dexamethasone treatment. The greatest reduction in anti-OVA IgGI was seen upon treatment with Compound 13 at 25mg/kg (3F, titer of 6.2 x107) although this did not reach statistical significance. lgG2a (Figure 18 shows the Serum OVA specific lgG2a endpoint titre. Graph shows mean+SEM.): In keeping with asthma being a Th2 mediated disease, lower levels of anti-OVA lgG2a were found than for the IgGI isotype. In vehicle treated animals a titer of 6.Ox 10² was found. This level was not significantly affected by any of the treatments.

Penh assessment: The enhanced pause (Penh) can be represented in a variety of ways. Here we have shown the percentage increase above baseline which is most commonly used as well as the mean data. Percentage increase above baseline (Figure 19 shows the percentage increase in Penh with increasing concentrations of the mast cell activator NECA- groups 1 F-5F. Graph shows mean+SEM. ^*, P<0.05 lower for group 2F than for 1 F.): The percentage increase above baseline for vehicle treated mice (1 F) rose to 600% after challenge with 400μg/ml NECA. At the same NECA challenge there was only a 200% increase above baseline Penh in dexamethasone treated mice (2F). Treatment with Compound 13 did not affect the percentage increase above baseline. Mean Penh (Figure 20 shows the Mean serum mast cell protease levels. Graph shows mean+SEM. ^*, represents significantly different to 1 F (p<0.05 by ANOVA with tukey post-hoc analysis).): The mean Penh for vehicle treated mice was about 1.3 before challenge (control) and following challenge with PBS. This rose to peak at 8.6 following challenge with 400μg/ml NECA. Treatment with dexamethasone reduced the control Penh to 0.6 and peak following 400μg/ml NECA to 2 (significantly lower than for vehicle treated mice). It does not appear that treatment with any of the test items significantly affected the peak Penh following 400μg/ml NECA.

Conclusion: In view of the findings obtained under the conditions of this study, treatment with Compound 13 significantly inhibits leukocyte recruitment to the lungs and exhibits statistically significantly lower eosinophil and granulocyte recruitment as well as reduced IL-5 expression.

The present invention and the descriptions thereof are not bound or limited by any scientific explanation or theory. Any section headings or subheadings herein are for the reader's convenience and are non-limiting.

Unless otherwise indicated, language and terms refer to their broadest reasonable interpretation as understood by the skilled artisan.

In that a salt, solvate, or hydrate of a compound includes the compound itself, a recitation of a compound is intended to embrace such forms.

The term "active agent" of the invention refers to a compound of the invention in any salt, polymorph, crystal, solvate, or hydrated form.

Claims

1. A method of treating or preventing an inflammatory respiratory condition comprising administering to a subject in need thereof a therapeutically effective amount of an active agent according to Formula I:

wherein:

R1 is selected from

R2 is selected from

, , , or ; and wherein said quinolinyl group can be substituted by 1 to 2 groups independently selected from -OH, Ci_-3alkoxy, or Ci_-3alkoxy substituted by methoxy; and

R3 is C₀-₄alkyl.

2. The method of Claim 1 , wherein the condition is asthma.

3. The method of Claim 1 , wherein the subject is a patient diagnosed with asthma.

4. The method of any one of Claims 1-3, wherein, R2 is

that is optionally substituted by -OH, C_1-3alkoxy, or C_1-3alkoxy substituted by methoxy.

5. The method of any one of Claims 1-4, wherein R3 is hydrogen.

6. The method of any one of Claims 1-5, wherein R1 is selected from:

R2 is selected from:

, , or ; and wherein said quinolinyl group can be substituted by methoxy.

7. The method of any one of Claims 1-3, wherein the active agent comprises 3-{[(7- methoxyquinolin-4-yl)methyl]amino}-Λ/-[4-(trifluoromethyl)phenyl]thiophene-2-carboxamide or a pharmaceutically acceptable salt or N-oxide thereof.

8. The method of any one of Claims 1-6, wherein the active agent has a biochemical IC₅₀ against kit of about 0.1 μM or less.

9. The method of any one of Claims 1-6, wherein the active agent has a biochemical IC50 against kit of about 0.01 μM or less.

10. The method of any one of Claims 1-6, wherein the active agent has a biochemical IC₅₀ against kit of about 0.01 μM or less and a biochemical IC50 against KDR of about 0.01 μM or less.

11. The method of any one of Claims 1-10, wherein the active agent is administered in an amount of about 1-5 mg/kg per day.

12. The method of any one of Claims 1-10, wherein the active agent is administered in an amount of about 15-50 mg/kg per day.

13. The method of any one of Claims 1-10, wherein the active agent is administered in an amount of about 50-100 mg/kg per day.

14. The method of any one of Claims 1-13, wherein the active agent is administered orally.

15. The method of any one of Claims 1-13, wherein the active agent is administered by inhalation.

16. The method of any one of Claims 1 or 4-15, wherein the condition is an allergic lung disease.

17. The method of any one of Claims 1 or 4-15, wherein the condition is selected from asthma, hypereosinophilic disease, COPD, and rhinitis.

18. The method of any one of Claims 1 or 4-15, wherein the condition is hypereosinophilic disease.

19. The method of any one of Claims 1 or 4-15, wherein the condition is COPD.

20. The method of any one of Claims 1-19, wherein the condition is mediated by at least c- kit/SCF.

21. The method of any one of Claims 1-19, wherein the condition is treated by inhibiting inflammatory cell infiltration and/or reducing inflammatory cytokine levels.

22. The method of any one of Claims 1-19, wherein the condition is treated by modulating airway resistance.