WO2009007679A2 - Combination for use in the treatment of atherosclerosis comprising a mast cell inhibitor and a thromboxane a2 antagonist - Google Patents
Combination for use in the treatment of atherosclerosis comprising a mast cell inhibitor and a thromboxane a2 antagonist Download PDFInfo
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- WO2009007679A2 WO2009007679A2 PCT/GB2008/002247 GB2008002247W WO2009007679A2 WO 2009007679 A2 WO2009007679 A2 WO 2009007679A2 GB 2008002247 W GB2008002247 W GB 2008002247W WO 2009007679 A2 WO2009007679 A2 WO 2009007679A2
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- thromboxane
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- A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
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- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4174—Arylalkylimidazoles, e.g. oxymetazolin, naphazoline, miconazole
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
- A61K31/4535—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom, e.g. pizotifen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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- A61K31/60—Salicylic acid; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Definitions
- This invention relates to a novel pharmaceutical combination.
- Cardiovascular diseases such as coronary heart disease and stroke are major causes of death, disability, and healthcare expense, particularly in industrialised countries.
- Such diseases are often direct sequelae of atherosclerosis, a multifactorial condition that develops preferentially in subjects that smoke and/or present risk factors such as hypertension, diabetes mellitus, hypercholesterolemia, elevated plasma low density lipoprotein (LDL) and triglycerides.
- LDL low density lipoprotein
- Atherosclerotic lesions develop over many years. Pathological processes, such as cholesterol accumulation in the artery wall, foam cell formation, inflammation and cell proliferation are typically involved.
- HDLs high-density lipoproteins 1 LDLs, total cholesterol and triglycerides are all indicators in determining the risk of developing atherosclerosis and associated cardiovascular disorders, such as coronary artery diseases (e.g. angina pectoris, myocardial infarction, etc.), stroke (including cerebro-vascular accident and transient ischaemic attack) and peripheral arterial occlusive disease.
- coronary artery diseases e.g. angina pectoris, myocardial infarction, etc.
- stroke including cerebro-vascular accident and transient ischaemic attack
- peripheral arterial occlusive disease peripheral arterial occlusive disease.
- HMG-CoA reductase inhibitors hydroxymethylglutaryl-CoA reductase inhibitors
- statins cholesterol lowering drugs
- statins have significantly reduced mortality from coronary heart disease and stroke.
- these drugs suffer from the disadvantage that they are not equally effective in all patients and are known to have certain side effects (e.g. changes in liver function, myopathy and rhabdomyolysis), and atherosclerosis remains a major cause of death and disability.
- side effects e.g. changes in liver function, myopathy and rhabdomyolysis
- atherosclerosis remains a major cause of death and disability.
- a recent review article suggests that statins do not reduce serious cardiovascular events during the first four months of treatment in patients with acute coronary syndromes.
- Mast cells are cells that are present in most tissues in the vicinity of blood vessels, and are especially prominent near the boundaries between the extra-corporeal environment and the internal milieu, such as the skin, mucosal surfaces (such as the lungs, digestive tract, mouth, conjunctiva and nose). Mast cells play an important protective role, being involved in wound healing and defence against pathogens.
- mast cells In allergic reactions, mast cells remain inactive until an allergen binds to IgE already in association with the cell. Binding of two or more IgE molecules (crosslinking) leads to steric changes that cause disturbances to the cell membrane structure. This results in a complex sequence of reactions inside the cell that leads to activation and so-called "degranulation”.
- mast cells play a key role in the inflammatory process.
- Cross- linking of IgE receptors may stimulate mast cells to degranulate, and thereby release various mediators into the interstitium, including eicosanoids, such as prostaglandin D2 and leukotriene C4, cytokines, and preformed mediators, such as heparin, serine proteases and, particularly, histamine.
- Mast cells therefore play a central role in asthma, eczema, allergic rhinitis and allergic conjunctivitis. Mast cells are also implicated in the pathology associated with the autoimmune disorders such as rheumatoid arthritis and multiple sclerosis.
- Treatments of inflammatory conditions associated with mast cell degranulation include antihistamine drugs, which act by blocking the action of histamine on e.g. nerve endings and blood vessels.
- Chromone-based drugs (such as sodium cromoglycate and nedocromil) are thought to block ion channels essential for mast cell degranulation, stabilizing the cell and preventing release of histamine and related mediators.
- Other mast cell stabilisers include pemirolast, suplatast, repirinast, amlexanox, ketotifen, tazanolast and tranilast.
- pemirolast is an orally-active anti-allergic drug which is used in the treatment of conditions such as asthma, allergic rhinitis and conjunctivitis.
- the drug is presently marketed in e.g. Japan as the potassium salt.
- Suplatast is a Th2 cytokine inhibitor which inhibits the release of IL-4 and IL-5 from Th2 cells as well as the release of chemical mediators from mast cells. Suplatast is therefore indicated to be of use in the treatment of conditions such as asthma, allergic rhinitis, atopic dermatitis, interstitial cystitis and chronic nonbacterial prostatitis. See, for example, Tamoaki, Allergology International, 53, 55 (2004) and Suwaki et a/, International Immunopharmacology, 1, 2163 (2001 ). Suplatast has also been indicated in the possible treatment of acute eosinophilic myocarditis (see Umemoto et al, Heart Vessels, 18, 100 (2003)).
- Thromboxane A2 is an unstable cyclooxygenase product of arachidonic acid metabolism, which has been shown to be an important mediator of thrombosis, vasospasm and bronchoconstriction. As a potent bronchoconstrictor, it is involved in acute bronchoconstriction in asthmatic patients, as demonstrated by a markedly elevated level of its stable metabolite, thromboxane B2. Thromboxane A2 has an important role in bronchial hyperreactivity, a key feature of bronchial asthma. Moreover, there is accumulating evidence indicating that thromboxane A2 could be an immunomodulator.
- Thromboxane A2 may thus contribute to airflow limitation and bronchial hyperresponsiveness. Thromboxane A2 antagonists have been demonstrated to improve these symptoms. Such antagonists may act by way of a variety of mechanisms, for example by blocking the thromboxane TP receptor (e.g. terutroban and egualen), by inhibiting the enzyme thromboxane synthase (e.g. ozagrel and picotamide), or by selectively inhibiting platelet cyclooxygenase-1 , thereby inhibiting e.g. platelet aggregation (e.g. low-dose aspirin).
- thromboxane TP receptor e.g. terutroban and egualen
- thromboxane synthase e.g. ozagrel and picotamide
- platelet cyclooxygenase-1 e.g. platelet aggregation
- Thromboxane A2 antagonists have been well documented to be of potential use in various cardiovascular conditions (see, for example, Hiraku et a/, Jpn. J. Pharmacol., 41, 393 (1986), Vetrano et al, Giornale ltaliano di Cardioliga, 29, 524 (1999), Coto et al, J. Int. Med. Res., 26, 200 (1998), Osende et al, J. Thromb. Haemostasis, 2, 492 (2004), Sorbrera et al, Drugs of the Future, 31 , 867 (2006), Dogne et al, Expert Opin. Ther.
- US patent application US 2003/0109543 discloses combination products that are useful in the prevention or inhibition of platelet aggregation and thrombus formation comprising antiplatelet drugs, such as clopidrogrel and thromboxane A2 antagonists, more specifically ifetroban.
- antiplatelet drugs such as clopidrogrel and thromboxane A2 antagonists, more specifically ifetroban.
- US patent application US 2006/0024365 discloses dual retard pharmaceutical dosage forms comprising modified release high dose high solubility active ingredients in combination with immediate release low dose active ingredients.
- a wide variety of drugs, including some of those mentioned herein, are listed as potential candidates for the low dose active ingredient.
- US patent application US 2003/0104048 discloses novel pharmaceutical dosage forms comprising hydrophilic surfactant-containing fillers including pharmaceutically-active ingredients encapsulated by a shell.
- Various active compounds, including some of those mentioned herein, are listed amongst many possible drug candidates for use in such dosage forms.
- US patent application US 2007/0014733 discloses pharmaceutical compositions for the treatment of cardiovasular disorders comprising metabolites of nebivolol.
- Various active compounds, including some of those mentioned herein, are listed among the many active ingredients that may be combined with such metabolites in such compositions.
- US patent application US 2005/0181023 discloses pharmaceutical compositions comprising pemirolast for use in the inhibition or prevention of adhesions between tissue and/or organ surfaces.
- combination products comprising, mast cell inhibitors and thromboxane A2 antagonists is not specifically disclosed in any of the above- mentioned documents.
- the use of such combination products in the treatment of atherosclerosis and associated cardiovascular disorders, particularly in those patients with acute coronary syndromes, or abdominal aortic aneurysms is not disclosed in any of these documents.
- a combination product comprising: (a) one or more mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof; and (b) one or more thromboxane A2 antagonist, or a pharmaceutically-acceptabie salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast, which combination products are referred to hereinafter as "the combination products according to the invention".
- mast cell inhibitor includes any compound that is capable of inhibiting, to an experimentally-determinable degree, the degranulation of (and therefore release of e.g. histamine from) mast cells in in vitro and/or in vivo tests.
- Mast cell inhibitors include histamine H1 receptor antagonists that are known to inhibit mast cell activation to varying degrees, such as acrivastine, astemizole, azelastine, cetirizine, carebastine, desloratadine, ebastine, fexofenadine, ketotifen, levocabastine, levocetirizine, loratadine, mizolastine, norastemizole, olopatadine, oxatomide and terfenadine.
- Other mast cell inhibitors include those that are used for local inhibition of mast cell activation, such as cromoglycate (e.g.
- mast cell inhibitors include orally active non-antihistamine drugs that inhibit mast cell activation, such as pemirolast, repirinast, amlexanox, tazanolast, suplatast and tranilast.
- active metabolites of mast cell inhibitors such as acitazanolast (which is an active metabolite of tazanolast) and MY-1250 (5,6- dihydro-7,8-dimethyl-4,5-dioxo-4H-pyrano[3,2-c]quinoline-2-carboxylic acid, which is an active metabolite of repirinast).
- Preferred mast cell inhibitors include tranilast, more preferably ketotifen, particularly amlexanox, more particularly repirinast (or its active metabolite, MY- 1250) and tazanolast, and especially suplatast.
- Another preferred mast cell inhibitor is pemirolast.
- a combination product comprising:
- thromboxane A2 antagonist (b) one or more thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast.
- thromboxane A2 antagonist includes any compound that is capable of inhibiting, to an experimentally- determinable degree in in vitro and/or in vivo tests, the effects of thromboxane A2 by one or more of (i) blocking the thromboxane TP receptor, (ii) inhibiting the enzyme thromboxane synthase, or (iii) inhibiting (e.g. selectively) platelet cyclooxygenase-1 , thereby inhibiting e.g. platelet aggregation.
- Preferred thromboxane A2 antagonists include aspirin/acetylsalicylic acid, more preferably egualen, particularly ozagrel and, more particularly, picotamide and terutroban.
- salts that may be mentioned include acid addition salts and base addition salts.
- Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of an active ingredient with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze- drying or by filtration). Salts may also be prepared by exchanging a counter-ion of an active ingredient in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
- ketotifen examples include fumarate salts.
- Other salts of ketotifen, and salts of picotamide and suplatast, that may be mentioned include hydrochloride, bisulfate, maleate and tosylate salts.
- Preferred salts of suplatast include suplatast tosylate.
- Salts of tranilast, amlexanox, tazanolast, MY-1250, ozagrel, terutroban, egualen and aspirin include alkali metal salts, such as lithium, sodium and potassium salts.
- Preferred salts of ozagrel and egualen include sodium salts.
- Active ingredients that are employed in combination products according to the invention may be employed in diastereomerically-enriched and/or enantiomerically-enriched form.
- diastereomerically-enriched and enantiomerically-enriched we mean, respectively, any mixture of the diastereoisomers/enantiomers of an active ingredient, in which one isomer is present in a greater proportion than the other.
- enantiomers of e.g. ketotifen and terutroban
- optical purities enantiomeric excess; e.e.
- Preferred enantiomers of terutroban include S-18886, i.e. the R-enantiomer.
- Preferred combination products according to the invention include (in ascending order of preference) those in which:
- the mast cell inhibitor (a) in a composition of the invention is ketotifen or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) in a composition of the invention is aspirin or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is tranilast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is amlexanox or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is tazanolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable
- the mast cell inhibitor (a) is repirinast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is MY-1250 or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is ketotifen or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is ketotifen or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is ketotifen or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is tranilast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is tranilast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is tranilast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is tranilast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is amlexanox or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutical
- the mast cell inhibitor (a) is tazanolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is repirinast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is MY-1250 or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is amlexanox or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or
- the mast cell inhibitor (a) is amlexanox or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is tazanolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is tazanolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist
- the mast cell inhibitor (a) is tazanolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is repirinast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is repirinast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is repirinast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is MY-1250 or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is MY-1250 or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is MY-1250 or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is suplatast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is suplatast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is suplatast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is pemirolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is pemirolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is pemirolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof;
- the mast cell inhibitor (a) is pemirolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist
- the mast cell inhibitor (a) ⁇ is pemirolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof.
- Combination products according to the invention provide for the administration of mast cell inhibitor as hereinbefore defined in conjunction with a thromboxane A2 antagonist as hereinbefore defined, and may thus be presented either as separate formulations, wherein at least one of those formulations comprises a mast cell inhibitor, and at least one comprises a thromboxane A2 antagonist, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including mast cell inhibitor and a thromboxane A2 antagonist).
- a pharmaceutical formulation including one or more mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof; one or more thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast; and a pharmaceutically-acceptable adjuvant, diluent or carrier (which formulation is hereinafter referred to as a "combined preparation"); and
- A a pharmaceutical formulation including one or more mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier; and (B) a pharmaceutical formulation including one or more thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (A) and (B) are each provided in a form that is suitable for administration in conjunction with the other.
- a method of making a kit of parts as defined above comprises bringing component (A), as defined above, into association with a component (B), as defined above, thus rendering the two components suitable for administration in conjunction with each other.
- components (A) and (B) of the kit of parts may be: (i) provided as separate formulations (i.e. independently of one another), which are subsequently brought together for use in conjunction with each other in combination therapy; or
- kit of parts comprising:
- kits of parts described herein may comprise more than one formulation including an appropriate quantity/dose of mast cell inhibitor/salt/solvate, and/or more than one formulation including an appropriate quantity/dose of thromboxane A2 antagonist/salt/solvate, in order to provide for repeat dosing. If more than one formulation (comprising either active compound) is present, such formulations may be the same, or may be different in terms of the dose of either compound, chemical composition(s) and/or physical form(s).
- the combination products according to the invention find utility in the treatment of inflammatory conditions.
- Inflammatory conditions are typically characterized by activation of immune defence mechanisms, resulting in an effect that is more harmful than beneficial to the host. Such conditions are generally associated with varying degrees of tissue redness or hyperemia, swelling, hyperthermia, pain, itching, cell death and tissue destruction, cell proliferation, and/or loss of function.
- Inflammatory conditions include endometriosis and, more preferably, allergy (including allergic conjunctivitis and allergic rhinitis), ankylosing spondylitis, asthma, atopic dermatitis, chronic obstructive pulmonary disease, contact dermatitis, cystitis, gouty arthritis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), multiple sclerosis, osteoarthritis, pancreatitis, prostatitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, tendinitis, bursitis, Sjogren's syndrome, systemic lupus erythematosus, uveitis, urticaria, vasculitis, diabetic vascular complications, migraine, atherosclerosis and associated cardiovascular disorders.
- allergy including allergic conjunctivitis and allergic rhinitis
- ankylosing spondylitis asthma, atopic dermatitis, chronic obstruct
- Conditions that may be mentioned include endometriosis, migraine and, more preferably, asthma, chronic obstructive pulmonary disease, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis and, more particularly, atherosclerosis and associated cardiovascular disorders.
- Atherosclerosis will be understood by those skilled in the art to include any disease characterised by cholesterol accumulation in a blood vessel, especially an artery wall, foam cell formation, inflammation and cell proliferation.
- Cardiovascular disorders "associated with" atherosclerosis include aortic aneurysms (including abdominal and/or atherosclerotic aortic aneurysms) and, more preferably, arteriosclerosis, peripheral arterial occlusive disease, coronary artery diseases (e.g.
- angina pectoris including myocardial infarction, heart attack, etc
- coronary disease including cardiac disease and heart disease, such as ischaemic heart disease
- plaque or atheroma rupture and/or instability vascular or arterial disease
- ischaemic disease/ischaemia and stroke including cerebro-vascular accident and transient ischaemic attack.
- Patient groups that may be mentioned include those with acute coronary syndromes.
- acute coronary syndrome(s) will be understood by the skilled person to include any abnormal myocardial and ischaemic state, often but not exclusively associated with chest pain and/or an abnormal electrocardiogram (ECG). Such syndromes are the most common presentation of myocardial infarction (heart attack).
- ECG electrocardiogram
- the skilled person will appreciate that the term is largely synonymous with the term “unstable angina”, as opposed to “stable angina” (i.e. angina that develops during exertion and resolves at rest). Exertional angina that occurs at worsening rate (“crescendo angina”) will similarly be regarded by the skilled person as within the definition "unstable”.
- a method of treatment of an inflammatory disorder and in particular atherosclerosis and/or an associated cardiovascular disorder, which method comprises the administration of a combination product according to the invention to a patient in need of such treatment.
- a combination product according to the invention to a patient in need of such treatment.
- respective formulations comprising mast cell inhibitor (or salt/solvate thereof) and thromboxane A2 antagonist (or salt/solvate thereof) are administered, sequentially, separately and/or simultaneously, over the course of treatment of the relevant condition.
- the term "administration in conjunction with” includes that the two components of the combination product (mast cell inhibitor and thromboxane A2 antagonist) are administered (optionally repeatedly), either together, or sufficiently closely in time, to enable a beneficial effect for the patient, that is greater, over the course of the treatment of the relevant condition, than if either a formulation comprising the mast cell inhibitor, or a formulation comprising the thromboxane A2 antagonist, are administered (optionally repeatedly) alone, in the absence of the other component, over the same course of treatment. Determination of whether a combination provides a greater beneficial effect in respect of, and over the course of treatment of, a particular condition will depend upon the condition to be treated or prevented, but may be achieved routinely by the skilled person.
- the term "in conjunction with” includes that one or other of the two formulations may be administered (optionally repeatedly) prior to, after, and/or at the same time as, administration of the other component.
- the terms “administered simultaneously” and “administered at the same time as” include that individual doses of mast cell inhibitor and thromboxane A2 antagonist are administered within 48 hours (e.g. 24 hours) of each other.
- egualen in any form including as sole therapy
- a method of treatment of atherosclerosis and/or an associated cardiovascular disorder which method comprises the administration of egualen or a pharmaceutically-acceptable salt or solvate thereof to a patient in need of such treatment.
- treatment include the therapeutic, or palliative, treatment of patients in need of, as well as the prophylactic treatment and/or diagnosis of patients which are susceptible to, inflammatory disorders, such as atherosclerosis and associated cardiovascular disorders.
- Patients include mammalian (including human) patients.
- mast cell inhibitors and thromboxane A2 antagonists are preferably administered locally or systemically, for example orally, intravenously or intraarterially (including by intravascular stent and other perivascular devices/dosage forms), intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, or by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound(s) in pharmaceutically acceptable dosage form(s).
- Preferred modes of delivery include oral (particularly), intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal delivery.
- Mast cell inhibitors and thromboxane A2 antagonists will generally be administered together or separately in the form of one or more pharmaceutical formulations in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
- a pharmaceutically acceptable adjuvant, diluent or carrier which may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
- Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.
- Such pharmaceutically acceptable carriers may also impart an immediate, or a modified, release of either active ingredient, whether administered together in a combined preparation or in the form of a kit of parts.
- Suitable pharmaceutical formulations may be commercially available or otherwise are described in the literature, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995) and Martindale - The Complete Drug Reference (34 th Edition) and the documents referred to therein, the relevant disclosures in all of which documents are hereby incorporated by reference. Otherwise, the preparation of suitable formulations, and in particular combined preparations including both mast cell inhibitors and thromboxane A2 antagonists may be achieved non-inventively by the skilled person using routine techniques.
- the amount of active ingredients in the formulation(s) will depend on the severity of the condition, and on the patient, to be treated, as well as the compound(s) which is/are employed, but may be determined non-inventively by the skilled person.
- active ingredients may be administered at varying therapeutically effective doses to a patient in need thereof.
- the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe.
- the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease, as well as genetic differences between patients.
- Administration of active ingredients may be continuous or intermittent (e.g. by bolus injection).
- the dosage may also be determined by the timing and frequency of administration.
- Suitable doses of active ingredients include those referred to in the medical literature, such as Martindale - The Complete Drug Reference (34 th Edition) and the documents referred to therein, the relevant disclosures in all of which documents are hereby incorporated by reference. Suitable doses of active ingredients are therefore in the range of about 0.01 mg/kg of body weight to about 1 ,000 mg/kg of body weight. More preferred ranges are about 0.1 mg/kg to about 20 mg/kg on a daily basis, when given orally.
- peroral doses may be in the range of about 0.1 mg to about 1.2 g, such as about 0.5 mg to about 900 mg, per day, irrespective of whether the formulation employed is a combined preparation or a kit of parts as hereinbefore described.
- suitable peroral doses that may be mentioned are as follows: (1 ) for tazanolast, suitable lower limits of daily dose ranges are about 15 mg, such as about 25 mg, such as about 30 mg, such as about 50 mg, such as about 75 mg, such as about 100 mg, such as about 225 mg; and suitable upper limits are about 750 mg, such as about 500 mg, such as about 450 mg, such as about 350 mg, such as about 300 mg, such as about 225 mg, such as about 150 mg, such as about 80 mg, such as about 40 mg; (2) for repirinast, suitable lower limits of daily dose ranges are about 15 mg, such as about 25 mg, such as about 50 mg, such as about 75 mg, such as about 100 mg, such as about 150 mg; and suitable upper limits are about 1 g, such as about 900 mg, such as about 600 mg, such as about 400 mg, such as about 350 mg, such as about 300 mg, such as about 150 mg, such as about 100 mg, such as about 50 mg;
- suitable lower limits of daily dose ranges are about 5 mg, such as about 10 mg, such as about 20 mg, such as about 30 mg, such as about 35 mg, such as about 50 mg, such as about 75 mg; and suitable upper limits are about 500 mg, such as about 300 mg, such as about 275 mg, such as about 250 mg, such as about 200 mg, such as about 180 mg, such as about 150 mg, such as about 100 mg, such as about 60 mg, such as about 30 mg;
- suitable lower limits of daily dose ranges are about 25 mg, such as about 50 mg, such as about 75 mg, such as about 100 mg, such as about 150 mg, such as about 200 mg, such as about 300 mg; and suitable upper limits are to about 1 g, such as about 900 mg, such as about 800 mg, such as about 700 mg, such as about 600 mg, such as about 500 mg, such as about 250 mg, such as about 100 mg, such as about 50 mg;
- suitable lower limits of daily dose ranges are about 0.05 mg, such as about 0.1 mg, such as about 0.2 mg, such as about 0.3 mg, such as about 0.4 mg, such as about 0.5 mg; and suitable upper limits are about 5 mg, such as about 4 mg, such as about 3.5 mg, such as about 3 mg, such as about 2.5 mg, such as about 2.0 mg, such as about 1.0 mg, such as about 0.6 mg, such as about 0.3 mg; and
- suitable lower limits of daily dose ranges are about 0.5 mg, such as about 2 mg, such as about 20 mg, such as about 200 mg, such as about 300 mg; and suitable upper limits are about 1000 mg, such as about 800 mg, such as about 600 mg, such as about 450 mg, such as about 300 mg, such as about 200 mg, such as about 150 mg, such as about 100 mg, such as about 50 mg, per day, irrespective of whether the formulation employed is a combined preparation or a kit of parts as hereinbefore described.
- Suitable doses of pemirolast are known to those skilled in the art.
- suitable lower limits of daily dose ranges are about 1 mg, such as about 2 mg, for example about 5 mg, such as about 10 mg, and more preferably about 20 mg
- suitable upper limits of daily dose ranges are about 200 mg, for example about 100 mg, such as about 80 mg, and more preferably about 60 mg.
- Daily peroral doses may thus be between about 2 mg and about 50 mg, such as about 5 mg and about 40 mg, and preferably about 10 mg and about 30 mg.
- Suitable individual doses may be about 40 mg, or about 20 mg (such as about 10 mg, more preferably about 6 mg (e.g. about 3 mg), per day.
- peroral doses may be in the range of about 1 mg to about 2 g, such as about 2 mg to about 1.75 g, per day, irrespective of whether the formulation employed is a combined preparation or a kit of parts as hereinbefore described.
- suitable peroral doses that may be mentioned are as follows: (I) for ozagrel, suitable lower limits of daily dose ranges are about 30, such as about 50 mg, such as about 100 mg, such as about 125 mg, such as about 175 mg, such as about 200 mg; and suitable upper limits are about 1 g, such as about 800 mg, such as about 750 mg, such as about 700 mg, such as about 600 mg, such as about 300 mg, such as about 100 mg, such as about 60 mg;
- suitable lower limits of daily dose ranges are about 100 mg, such as about 200 mg, such as about 300 mg, such as about 400 mg, such as about 500 mg, such as about 600 mg; and suitable upper limits are about 1.75 g, such as about 1.5 g, such as about 1.4 g, such as about 1.3 g, such as about 1.2 g, such as about 750 mg, such as about 500 mg, such as about 250 mg;
- suitable lower limits of daily dose ranges are about 1 mg, such as about 2 mg, such as about 3 mg, such as about 5 mg, such as about 8 mg, such as about 10 mg; and suitable upper limits are about 100 mg, such as about 75 mg, such as about 50 mg, such as about 40 mg, such as 30 mg, such as about 20 mg, such as about 12 mg, such as about 5 mg; and
- suitable lower limits of daily dose ranges are about 30 mg, such as about 50 mg, such as about 75 mg; and suitable upper limits are about 400 mg; such as about 325 mg, such as about 200 mg, such as about 120 mg, such as about 60 mg, per day, irrespective of whether the formulation employed is a combined preparation or a kit of parts as hereinbefore described.
- the dosage will comprise that which the medical practitioner, or other skilled person, will recognize as so-called "low dose” aspirin, i.e. not the higher doses that are normally prescribed to treat conditions such as pain.
- the medical practitioner or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient.
- the above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
- mast cell inhibitors as hereinbefore defined are combined, in accordance with the general and specific teaching described herein, with certain platelet aggregation inhibiting drugs, such as nitric oxide-donating derivatives of aspirin/acetylsalicylic acid (e.g. NCX-4016, NicOx S.A.) or, more preferably, anagrelide, argatroban, beraprost, cangrelor, cilostazol, dipyridamole, limaprost, parogrelil, procainamide, sarpogrelate (e.g.
- platelet aggregation inhibiting drugs such as nitric oxide-donating derivatives of aspirin/acetylsalicylic acid (e.g. NCX-4016, NicOx S.A.) or, more preferably, anagrelide, argatroban, beraprost, cangrelor, cilostazol, dipyridamole, limaprost, parogre
- the mast cell inhibitor (a) is acitazanolast (an active metabolite of tazanolast), or a pharmaceutically-acceptable salt or solvate thereof
- the thromboxane A2 antagonist (b) is selected from any one compound in the following list: anagrelide, argatroban, aspirin/acetylsalicylic acid, beraprost, cangrelor, cilostazol, DA-697b, DG-041 , dipyridamole, egualen, K-134, limaprost, ozagrel, parogrelil, picotamide, PL-2200, PRT-60128, procainamide, sarpogreiate (e.g.
- the mast cell inhibitor (a) is acrivastine, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A
- the mast cell inhibitor (a) is andolast, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
- mast cell inhibitor (a) is amlexanox, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
- mast cell inhibitor (a) is astemizole, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in
- mast cell inhibitor (a) is azelastine, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
- mast cell inhibitor (a) is cromoglycate, or a pharmaceutically-acceptable salt (e.g. disodium cromoglycate) or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
- mast cell inhibitor (a) is desloratadine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in
- the mast cell inhibitor (a) is levocabastine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
- the mast cell inhibitor (a) is levocetirizine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in
- the mast cell inhibitor (a) is loratadine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
- the mast cell inhibitor (a) is norastemizole, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
- mast cell inhibitor (a) is olopatadine, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in
- mast cell inhibitor (a) is oxatomide, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
- mast cell inhibitor (a) is suplatast, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
- mast cell inhibitor (a) is tazanolast, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
- mast cell inhibitor (a) is terfenadine, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in
- mast cell inhibitor (a) is tranilast, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A.
- the combination products/methods described herein may have the advantage that, in the treatment of the conditions mentioned hereinbefore, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have a broader range of activity than, be more potent than, produce fewer side effects than, or that it/they may have other useful pharmacological properties over, similar methods (treatments) known in the prior art for use in the treatment of inflammatory disorders (such as atherosclerosis and associated cardiovascular conditions) or otherwise.
- MonoMac-6 (MM6) cells (Ziegler-Heitbrock et al, Int. J. Cancer, 41 , 456 (1988)) are cultured (37°C/5% CO 2 ) in RPMI-1640 medium supplemented with 1 mM sodium pyruvate, ⁇ nonessential amino acids, 1-100 ⁇ g/mL insulin, 1 mM oxalacetic acid, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin and 10% (v/v) fetal bovine serum.
- TGF ⁇ (2 ng/ml) and 1 ,25(OH) 2 D3 (50 nM) are added, generally for about 2-4 days.
- differentiated or undifferentiated MM6 cells (at 1-15*10 6 /mL; 0.5-1 mL) are incubated for 5-30 minutes (at 37 0 C in PBS with calcium) with 25-50 ⁇ M arachidonic acid and 2-10 ⁇ M calcium ionophore A23187 (A23187 may also be used without arachidonic acid).
- the MM6 cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), and/or the thromboxane analogue U-46619, with or without A23187 and/or arachidonic acid as above.
- ADP adenosine diphosphate
- thromboxane analogue U-46619 adenosine diphosphate
- the MM6 incubations/stimulations above may also be performed in the presence of human platelets (from healthy donor blood) with an MM6:platelet ratio of 1 :10 to 1 :10000.
- the incubations/stimulations are stopped with two volumes of cold methanol and prostaglandin B 2 (PGB 2 ) added as internal standard.
- the samples are centrifuged and the supematants are diluted with water to reach a final methanol concentration of 30% and pH is adjusted to 3-4.
- Arachidonic acid metabolites in the supernatant are extracted on preconditioned (1 mL methanol followed by 1 mL H 2 O) C18 solid phase columns (Sorbent Technology, U.K.).
- Metabolites are eluted with methanol, whereafter one volume of water is added to the eluate.
- 76 ⁇ L of each sample is mixed with 39 ⁇ L H 2 O (other volume ratios may also be used).
- a Waters RCM 8*10 column is eluted with methanol/acetonitrile/H 2 O/acetic acid (30:35:35:0.01 v/v) at 1.2 mL/min.
- the absorbance of the eluate is monitored at 270 nm for detection and quantitation of PGB 2 and LTB 4 .
- Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring LTB 4 may also be used according to instructions from the kit manufacturers).
- EIA/ELISA kits enzyme immuno-assay kits
- PGE 2 prostaglandin E 2
- TXB 2 thromboxane B 2
- mast cell inhibitors tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast
- thromboxane A2 antagonists aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen
- test drug(s) mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone
- test drug(s) may also be added simultaneously with MM6 stimulation.
- the drugs are added to reach final concentrations of 1 nM to 100 ⁇ M (for comparison, some experiments are performed without the drugs).
- MM6 cells (at 1-10*10 6 /mL) are incubated (37°C/5% CO 2 ) for 4-24 hours (in RPMI-1640 with 1-10% fetal bovine serum, with or without supplements) with lipopolysaccharide (LPS, final concentration 1-100 ng/mL), phorbol-12-myristate- 13-acetate (PMA, final concentration 1-100 ng/mL) or an LPS/PMA mixture.
- LPS lipopolysaccharide
- PMA phorbol-12-myristate- 13-acetate
- the MM6 cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), arachidonic acid, calcium ionophore A23187 and/or the thromboxane analogue U-46619, with or without PMA and/or LPS as above.
- ADP adenosine diphosphate
- arachidonic acid adenosine diphosphate
- calcium ionophore A23187 calcium ionophore A23187
- the thromboxane analogue U-46619 with or without PMA and/or LPS as above.
- the MM6 cell incubations/stimulations may also be performed in the presence of human platelets (from healthy donor blood) with an MM6:platelet ratio of 1:10 to 1 :10000.
- test drug(s) mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone; as above regarding stock solutions and concentrations
- test drug(s) mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone; as above regarding stock solutions and concentrations
- PBMC peripheral blood mononuclear cells
- PMN polymorphonuclear cells
- PBMC or PMN are incubated for 5-30 minutes (at 37°C in PBS with calcium) with 25-50 ⁇ M arachidonic acid and 2-10 ⁇ M calcium ionophore A23187 (A23187 may also be used without arachidonic acid).
- the PBMC/PMN may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), and/or the thromboxane analogue U-46619, with or without A23187 and/or arachidonic acid as above.
- ADP adenosine diphosphate
- U-46619 adenosine diphosphate
- the PBMC/PMN incubations/stimulations above may also be performed in the presence of human platelets (from healthy donor blood) with a PBMC/PMN:platelet ratio of 1 :10 to 1 :10000.
- the incubations/stimulations are stopped with two volumes of cold methanol and prostaglandin B 2 added is as internal standard.
- the samples are centrifuged and the supernatants are diluted with water to reach a final methanol concentration of 30% and pH is adjusted to 3-4.
- Arachidonic acid metabolites in the supernatant are extracted on preconditioned (1 mL methanol followed by 1 mL H 2 O) C18 solid phase columns (Sorbent Technology, U.K.).
- Metabolites are eluted with methanol, after which one volume of water is added to the eluate.
- 76 ⁇ L of each sample is mixed with 39 ⁇ L H 2 O (other volume ratios may also be used).
- a Waters RCM 8x10 column is eluted with methanol/acetonitrile/H 2 O/acetic acid 30:35:35:0.01 v/v) at 1.2 mL/minute.
- the absorbance of the eluate is monitored at 270 nm for detection and quantitation of PGB 2 and LTB 4 .
- Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring LTB 4 may also be used according to instructions from the manufacturer(s).
- test drug(s) mass cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours prior to PBMC/PMN stimulation for inflammatory mediator release (see Example 1 above for details regarding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with PBMC/PMN stimulation). For comparison, some experiments are performed without the drugs.
- test drug(s) mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or
- cytokines and chemokines such as IL-1 ⁇ , IL- 6, TNF, IL-8, IL-10, IL-12p70, MCP-1 , PBMC/PMN (at 1-10*10 6 AnL) are incubated (37°C/5% CO 2 ) for 4-24 hours (in RPMI-1640 with 1-10% fetal bovine serum) with lipopolysaccharide (LPS, final concentration 1-100 ng/mL), phorbol- 12-myristate-13-acetate (PMA 1 final concentration 1-100 ng/mL) or an LPS/PMA mixture.
- LPS lipopolysaccharide
- PMA 1 phorbol- 12-myristate-13-acetate
- the PBMC/PMN cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), arachidonic acid, calcium ionophore A23187 and/or the thromboxane analogue U-46619, with or without PMA and/or LPS as above.
- ADP adenosine diphosphate
- arachidonic acid adenosine diphosphate
- calcium ionophore A23187 calcium ionophore A23187
- the thromboxane analogue U-46619 adenosine diphosphate
- the PBMC/PMN incubations/stimulations may also be performed in the presence of human platelets (from healthy donor blood) with a PBMC/PMN:platelet ratio of 1 :10 to 1 :10000.
- test drug(s) mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone, as above
- test drug(s) mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone, as above
- cytokine and chemokine concentrations in the supernatants are quantitated using a Cytometric Bead Array (BD Biosciences Pharmingen, San Diego, USA) according to the manufacturer's instructions.
- BD Biosciences Pharmingen San Diego, USA
- commercially available enzyme immuno-assay kits EIA/ELISA kits
- EIA/ELISA kits for measuring cytokines and chemokines may also be used according to instructions from the manufacturer(s).
- the cell pellets are stored frozen (-80 0 C) in RLT buffer (QIAGEN, Valencia, CA) until further processing for microarray experiments (see Example 12 below).
- Bone marrow-derived cultured mouse mast cells are obtained by culturing bone marrow cells from C57BL/6 mice.
- the bone marrow cells (from mouse femurs flushed with PBS) are cultured (37°C/5% CO 2 ) in 10% WEHI-3 or X-63 enriched conditioned RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum, 4 mM L-glutamine, 50 ⁇ M 2-mercaptoethanol, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM Hepes, and 100 ⁇ g/mL penicillin/streptomycin.
- Development of mast cells (which grow in suspension) is confirmed by expression of Kit (by flow-cytometry) on the cell surface and/or by toluidine blue staining (generally after at least 3-5 weeks of culture).
- Bone marrow-derived cultured mouse mast cells of connective tissue type are obtained by culturing bone marrow cells from C57BL/6 mice.
- the bone marrow cells are cultured (37°C/5% CO 2 ) in RPMI-1640 medium containing 10% filtered FCS, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/mL penicillin G, 100 ⁇ g/mL streptomycin, 0.1 mM MEM non-essential amino acids and 50 ⁇ M 2- ME, supplemented with 50 ng/mL recombinant murine stem cell factor and 1 ng/mL murine recombinant IL-4.
- Mast cell development is confirmed by expression of Kit (by flow-cytometry) on the cell surface and/or by toluidine blue staining (generally after at least 3-5 weeks of culture).
- Mouse mast cell lines MC/9 (obtained from ATCC, Product no CRL-8306) and C1.MC/C57.1 (Young et a/., Proc. Natl. Acad. ScL USA, 84, 9175 (1987)) may also be used.
- the MC/9 cells are cultured according to instructions from ATCC (https://www.atcc.org), and C1.MC/C57.1 cells are cultured as described in Rumsaeng et al ⁇ J. Immunol. 158, 1353 (1997)).
- the cultured mast cells are initially sensitized for 90 minutes at 37 C C (5% CO 2 ) with a monoclonal mouse anti-TNP IgE-antibody (lgEI-b4, ATCC, Rockville, MD, USA), used as a 15% hybridoma supernatant.
- a monoclonal mouse anti-TNP IgE-antibody (lgEI-b4, ATCC, Rockville, MD, USA), used as a 15% hybridoma supernatant.
- Cells to be used in the N-acetyl-beta-D- hexosaminidase (or histamine) or cytokine/chemokine release assays are then subjected to two washings with PBS and re-suspended in RPMI- 1640 medium supplemented with 0.2% bovine serum albumin (BSA) (Sigma) before the cells (at 0.5-10x10 6 /mL) are activated by addition of 100 ng/mL TNP- BSA (Biosearch Technologies, San Francisco, CA) with a coupling ratio of 9/1.
- BSA bovine serum albumin
- TNP-BSA The incubation (37°C/5% CO 2 ) with TNP-BSA is 30 minutes for the analysis of beta-hexosaminidase (or histamine) release and 4-24 hours for analysis of cytokine and chemokine release.
- Cells are incubated (37°C/5% CO 2 ) with test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours prior to addition of TNP-BSA (see Example 1 above for detail regarding drug stock solutions and concentration
- an enzymatic colourimetric assay is used for detection of IgE-dependent release of the granular mast cell enzyme beta- hexosaminidase.
- 60 ⁇ L from each well supernatant is transferred to a 96 well plate and mixed with an equal volume of substrate solution (7.5 mM p-nitrophenyl-N-acetyl-b-D-glucosaminide dissolved in 80 mM citric acid, pH 4.5). The mixture is incubated on a rocker platform for 2 hours at 37°C.
- beta-hexosaminidase is expressed as a percentage of total beta-hexosaminidase determined after cell lysis.
- EIA/ELISA kits enzyme immuno-assay kits for measuring histamine is used according to instructions from the manufacturer(s).
- cytokines and chemokines such as IL-6, IL-4, TNF, IL-1 ⁇ , KC 1 MCP-1 , IL-10, IL-12p70, IFN ⁇
- a Cytometric Bead Array (BD Biosciences Pharmingen, San Diego, USA) is used according to the manufacturer's instructions.
- Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring cytokines and chemokines may also be used according to instructions from the manufacturer(s).
- mast cell-inhibiting effects of the test drug(s) may also be studied using well established and documented experimental approaches and assays for analysing induced (with e.g. anti-lgE (with or without pretreatment of the cells with rat or mouse IgE), concanavalin A, protein L, compound 48/80, ionophore A23187, PMA) release of histamine, beta-hexosaminidase or tryptase from freshly isolated peritoneal rat or mouse mast cells.
- induced with e.g. anti-lgE (with or without pretreatment of the cells with rat or mouse IgE), concanavalin A, protein L, compound 48/80, ionophore A23187, PMA
- RAW 264.7 cells are cultured (37°C/5% CO 2 ) in DMEM, supplemented with 100 units /mL penicillin, and 100 ⁇ g/mL streptomycin and 10% fetal bovine serum.
- RAW 264.7 cells (at 1-10*10 6 /mL) are incubated (37°C/5% CO 2 ) for 4-24 hours (in DMEM with 1-10% fetal bovine serum, with or without supplements) with lipopolysaccharide (LPS 1 final concentration 1-100 ng/mL), phorbol-12-myristate-13-acetate (PMA, final concentration 1-100 ng/mL) or an LPS/PMA mixture.
- LPS 1 lipopolysaccharide
- PMA phorbol-12-myristate-13-acetate
- the RAW 264.7 cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), arachidonic acid, calcium ionophore A23187 and/or the thromboxane analogue U-46619, with or without PMA and/or LPS as above.
- ADP adenosine diphosphate
- arachidonic acid adenosine diphosphate
- calcium ionophore A23187 calcium ionophore A23187
- the thromboxane analogue U-46619 with or without PMA and/or LPS as above.
- the RAW 264.7 incubations/stimulations may also be performed in the presence of mouse or human (from healthy donor blood) platelets with a RAW 264.7:platelet ratio of 1 :10 to 1 :10000.
- test drug(s) mass cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours prior to RAW 264.7 stimulation for cytokine/chemokine release (see Example 1 above for details regarding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with RAW 264.7 stimulation).
- test drug(s) mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly,
- mice cytokine and chemokine concentrations in the supematants are quantitated using a Cytometric Bead Array (BD Biosciences Pharmingen, San Diego, USA) according to the manufacturer's instructions.
- BD Biosciences Pharmingen San Diego, USA
- Commercially available enzyme immuno-assay kits EIA/ELISA kits
- EIA/ELISA kits for measuring cytokines and chemokines may also be used according to instructions from the manufacturer(s).
- the cell pellets are stored frozen (-8O 0 C) in RLT buffer (QIAGEN, Valencia, CA) until further processing for microarray experiments (see Example 12 below).
- Test drug(s) mass cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to male Sprague- Dawley or Wistar rats weighing approximately 150-400 g (for comparison, some experiments are performed without the drugs).
- thromboxane A2 antagonist aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen
- stock solutions of drugs Prior to administration, stock solutions of drugs (see Example 1 above) are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used.
- a 0.5, 1.0 or 2.0% solution of carrageenan (Type IV Lambda, Sigma Chemical Co.) in 0.9% saline is injected into the subplantar region of one hind paw of anaesthetised rats. Before, and at indicated intervals 3-24 hours after carrageenan injection, the volume of the injected paw is measured with a displacement plethysmometer connected to a pressure transducer with a digital indicator.
- the degree of swelling indicates the degree of inflammatory edema.
- 3- 24 hours after carrageenan injection the rats are sacrificed and perfused with saline or PBS (other perfusion media may also be used).
- Plantar soft tissue biopsies from the inflamed paws are collected, weighed, stored frozen (samples for microarray analysis are frozen at -8O 0 C in TRlzol, Invitrogen, Carlsbad, CA), and, as described below (Example 10 and 12), subsequently analyzed with regard to 1 ) myeloperoxidase (MPO) accumulation, reflecting inflammatory neutrophil leukocyte accumulation; and/or 2) tissue gene expression using microarray technology.
- MPO myeloperoxidase
- Non-inflamed paw tissue from untreated rats provides base-line levels of MPO and gene expression.
- Tissue inflammation may also be studied using conventional histological and immunohistochemical techniques.
- Paw inflammation may also be induced by subplantar injection of compound 48/80 (48/80, 1-5 ⁇ g in 50-100 ⁇ l PBS or saline) (instead of carrageenan), followed by measurement of inflammatory paw swelling and collection of tissue biopsies for microarray and/or MPO analysis (as above) 30 min to 8 hours after 48/80 injection.
- Test drug(s) mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours (for comparison, some experiments are performed without the drugs).
- thromboxane A2 antagonist aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen
- stock solutions of drugs Prior to administration, stock solutions of drugs (see Example 1 above) are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used.
- 10-30 ⁇ l_ of a 2.0 or 4.0% solution of croton oil in acetone or ethanol is applied topically to one or both ears.
- punch biopsies of the ears are weighed to determine the inflammatory swelling of the ears (ear thickness may also be measured to determine the swelling).
- biopsies from the inflamed ears are collected, stored frozen (samples for microarray analysis are frozen at -80 0 C in TRIzol), and, as described below (Example 10 and 12), subsequently analyzed with regard to 1 ) myeloperoxidase (MPO) accumulation, reflecting inflammatory neutrophil leukocyte accumulation; and/or 2) tissue gene expression using microarray technology.
- MPO myeloperoxidase
- Non-inflamed ear biopsies from untreated mice provide base-line levels of swelling, MPO and gene expression. Tissue inflammation may also be studied using conventional histological and immunohistochemical techniques.
- Test drug(s) mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to male or female mice (for comparison, some experiments are performed without the drugs).
- thromboxane A2 antagonist aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen
- biopsies from the inflamed ears are collected, stored frozen (samples for microarray analysis are frozen at -80 0 C in TRIzol), and, as described below (Example 10 and 12), subsequently analyzed with regard to 1 ) myeloperoxidase (MPO) accumulation, reflecting inflammatory neutrophil leukocyte accumulation; and/or 2) tissue gene expression using microarray technology.
- MPO myeloperoxidase
- Non-inflamed ear biopsies from untreated mice provide base-line levels of swelling, MPO and gene expression. Tissue inflammation may also be studied using conventional histological and immunohistochemical techniques.
- mice Male CBA or NMRI mice weighing approximately 15-30 g, or male Wistar or Sprague-Dawley rats weighing approximately 150-450 g, are used (other strains of mice and rats may also be used). Acute tissue injury and acute inflammation is achieved in the distal part of the tail or one of the ears using a scalpel under aseptic conditions. One, two or three parallel, approximately 5-15 mm long, longitudinal cuts are made through all layers of the skin.
- Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneal ⁇ or orally every 2-24 hours, with the first dose given 1 minute to 24 hours before tissue injury (for comparison, some experiments are performed without the drugs).
- thromboxane A2 antagonist aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen
- Sprague-Dawley rats weighing 350-500 g are used (although other strains of rats may also be used). Animals are anesthetized with lsoflurane in oxygen and acute tissue injury and acute inflammation is achieved in the left common carotid artery as follows: After surgical exposure of the left common, external and internal carotid arteries and temporary cessation of local blood flow with temporary ligatures, a balloon catheter (2-French Fogarty) is passed through the external carotid into the aorta. Next, the balloon is inflated with sufficient water to distend the common carotid artery and then pulled back to the external carotid.
- a balloon catheter (2-French Fogarty) is passed through the external carotid into the aorta.
- the balloon is inflated with sufficient water to distend the common carotid artery and then pulled back to the external carotid.
- Test drug(s) mass cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneal ⁇ or orally every 2-24 hours, with the first dose given 1 minute to 24 hours before tissue injury (for comparison, some experiments are performed without the drugs).
- thromboxane A2 antagonist aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen
- stock solutions of drugs Prior to administration, stock solutions of drugs (see Example 1 above) are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used. 2-48 hours after injury, the animals are anesthetized with lsoflurane in oxygen and their left carotid arteries exposed.
- Clamps are put on the very proximal part of the common and internal carotid arteries, respectively, and then the vessel between the clamps is gently flushed with sterile saline and/or TRIzol, removed, weighed and stored frozen (samples for microarray analysis are frozen at -80 0 C in TRIzol), and, as described below (Example 10 and 12), subsequently analyzed with regard to 1 ) myeloperoxidase (MPO) accumulation, reflecting inflammatory neutrophil leukocyte accumulation; and/or 2) tissue gene expression using microarray technology.
- MPO myeloperoxidase
- Corresponding non-injured/inflamed vessels from untreated rats provide base-line levels of MPO and gene expression. Tissue reactions and inflammation in response to injury may also be studied using conventional histological and immunohistochemical techniques.
- MPO myeloperoxidase
- the enzyme myeloperoxidase (MPO) is abundant in neutrophil leukocytes and is often used as a marker for the detection of neutrophil accumulation in inflamed tissue.
- the tissues are homogenised in 0.5% hexadecyltrimethyl-ammonium bromide, and freeze- thawed.
- the MPO activity of the supernatant is determined spectrophotometrically as the change in absorbance at 650 nm (25 0 C) occurring in the redox reaction of H 2 O 2 -tetramethylbenzidine catalysed by MPO. Values are expressed as MPO units/mg tissue.
- Rat aortic smooth muscle cells are isolated as previously described
- RASMCs are seeded in 24-well plates at a density of approximately 4x10 4 cells per well in F- 12/10% fetal bovine serum (plates with larger numbers of wells per plate and appropriate lower numbers of cells per well may also be used). After 24 hours, the cells are synchronized in G0/G1 phase by starvation in Ham's medium F-12 supplemented with 0.1 % bovine serum albumin (BSA), 50 ⁇ g/mL L-ascorbic acid, 50 ⁇ g/mL streptomycin and 50 IU/mL penicillin (F-12/0.1% BSA) for 24-48 hours.
- BSA bovine serum albumin
- test drug(s) mass cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) are added 1 minute to 24 hours prior to stimulation (see Example 1 above for details re'garding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with stimulation).
- thromboxane A2 antagonist aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen
- the cells are labelled with 1 ⁇ Ci [3H]-thymidine for 8 hours before the end of the stimulation period.
- the plates are then washed with ice-cold PBS, incubated overnight with ice-cold 10% (w/v) trichloroacetic acid, lysed in 0.2 M sodium hydroxide, and radioactivity is measured in a liquid scintillation counter.
- the stimulated RASMC proliferation may also be analyzed using commercially available bromodeoxyuridine (BrdU) cell proliferation assays (for example Cell Proliferation ELISA, BrdU, from Roche Applied Science), the cell proliferation reagent WST-1 (Roche Diagnostics Scandinavia AB, Bromma, Sweden) (both according to the manufacturer's instructions), or by cell counting.
- BrdU bromodeoxyuridine
- HBSMCs Human bronchial smooth muscle cells
- DMEM fetal bovine serum
- FBS 1 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 0.12 IU/mL insulin, and with or without 2 ⁇ g/mL amphotericin B.
- FBS 1 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 0.12 IU/mL insulin, and with or without 2 ⁇ g/mL amphotericin B.
- FBS 1 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 0.12 IU/mL insulin, and with or without 2 ⁇ g/mL amphotericin B.
- FBS 1 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 0.12 IU/mL insulin, and with or without 2 ⁇ g/mL amphotericin B.
- FBS 1 100 units/mL penicillin, 100 ⁇ g/mL strepto
- HBSMCs (at 80% confluence, corresponding to approximately 8*10 5 /25 cm 2 flask) are incubated (37°C/5% CO 2 ) for 24-48 hours (in DMEM with 1-10% fetal bovine serum, with or without supplements) with different combinations of IL-1 ⁇ and TNF- ⁇ (both at 1-50 ng/mL).
- test drug(s) mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone, as above
- test drug(s) mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone, as above
- cytokine and chemokine concentrations in the supernatants are quantitated using commercially available enzyme immuno-assay kits (EIA/ELISA kits) according to instructions from the manufacturers).
- EIA/ELISA kits enzyme immuno-assay kits
- the cells are then collected and stored frozen (-80 0 C) in RLT buffer (QIAGEN 1 Valencia, CA) until further processing for microarray experiments (see Example 12 below).
- RNA from mouse, rat and human tissues is isolated using TRIzol (Invitrogen, Carlsbad, CA) followed by RNeasy cleanup (QIAGEN, Valencia, CA) according to manufacturers' protocols.
- Total RNA from the cell incubations/stimulations described in the examples above and below is isolated using RNeasy Mini Kit (QIAGEN), with or without RNase-Free DNase set (QIAGEN), according to the manufacturer's protocol(s).
- microarray analysis is performed using GeneChip® Human Genome U133 Plus 2.0 Array, GeneChip® Mouse Genome 430 2.0 Array or GeneChip® Rat Genome 230 2.0 Array, or corresponding newer version of these chips (all arrays from Affymetrix, Santa Clara, CA) according to the manufacturer's protocols.
- the microarray expression data is analyzed using e.g. GeneChip Operating Software (Affymetrix) and Bioconductor/R (www.bioconductor.org). Other relevant software may also be used.
- Gene expression from the different species may also be analyzed using Human Genome Survey Microarray V2.0, Mouse Genome Survey Microarray V2.0 or Rat
- Genome Survey Microarray (or corresponding newer version of these arrays) according to protocols from the manufacturer Applied Biosystems (Foster City,
- microarray expression data are analyzed using e.g. 1700
- Chemiluminescent Microarray Analyzer (Applied Biosystems, Foster City, CA) supplied with an Oracle® database of annotations, GeneSpring 7.2 (Agilent
- Gene expression may also be analyzed using quantitative or semi-quantitative PCR. Analysis of gene expression at the protein level may be analyzed using commercially available enzyme immuno-assay kits (EIA/ELISA kits) (according to instructions from the manufacturers)), or conventional Western blot and/or immunohistochemical approaches.
- EIA/ELISA kits enzyme immuno-assay kits
- Proliferation of stimulated and unstimulated mouse mast cells, MonoMac-6 cells, RAW 264.7 cells, NB4 cells, HL-60 cells and HBSMC described in the examples above and below is measured using the cell proliferation reagent WST-1 (Roche Diagnostics Scandinavia AB, Bromma, Sweden) or commercially available bromodeoxyuridine (BrdU) cell proliferation assays (for example Cell Proliferation ELISA, BrdU, from Roche Applied Science) according to the manufacturers' instructions. Other conventional tests of cell proliferation may also be used.
- Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) are added 1-120 minutes prior to induction of platelet aggregation (see Example 1 above for details regarding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with induction of platelet aggregation). For comparison, some experiments are performed without the drugs.
- Test drug(s) mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to the animals (for comparison, some experiments are performed without the drugs).
- thromboxane A2 antagonist aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen
- peritoneal inflammation may also be induced by intraperitoneal injection of pro-inflammatory concentrations of other well established pro-inflammatory stimuli such as anti-mouse-lgE (with or without intraperitoneal pretreatment with mouse IgE for 1-3 days), concanavalin A, carrageenan, proteose peptone, LPS, PMA, thioglycolate, arachidonic acid, fMLP, TNF, IL-1 ⁇ .
- Test drug(s) may also be administered simultaneously with intraperitoneal injection of zymosan or the other pro-inflammatory stimuli).
- zymosan or one or more of the other pro-inflammatory stimuli
- the animals are sacrificed.
- the peritoneal cavity is then flushed with 1-3 mL of a lavage buffer (ice-cold PBS with or without 3-5 mM EDTA or 5-10 units/mL heparin).
- a lavage buffer ice-cold PBS with or without 3-5 mM EDTA or 5-10 units/mL heparin.
- Total and differential leukocyte counts in the lavage fluid are done with a hemocytometer following staining with Turk's solution and/or in cytospin preparations stained with May-Grunwald Giemsa or a modified Wright's (Diff-Quik) stain, respectively, by light microscopy using standard morphological criteria. Other established methods for determining total and differential leukocyte counts may also be used.
- the remaining lavage fluid is centrifuged (300-3000 x g, 4°C, 3-10 min), and cell-free lavage fluid supernatant is stored frozen (-20 0 C to -80°) until analyzed for content of inflammatory mediators LTB 4 , PGE 2 , TXB 2 and/or mouse cytokines/chemokines (e.g. IL-4, IL-6, TNF, IL-1 ⁇ , KC, MCP-1 , IL-10, IL-12p70, IFN ⁇ ) content as described in Example 1 and 4 above.
- the histamine content in the lavage fluid supernatant is determined by using commercially available histamine enzyme immuno-assay kits (EIA/ELISA kits) according to instructions from the manufacturers).
- Inflammatory peritoneal cell activation may also be studied by measuring beta-hexosaminidase activity in the lavage fluid using the beta-hexosaminidase assay described in Example 3.
- the cell pellets of the lavage fluid are resuspended in 0.1-1.0 mL 0.05 M KHPO 4 pH 6.0 with 0.5% HTAB and stored frozen (-20 0 C to -80°) until analysis of myeloperoxidase (MPO) content as described by Rao et al (J. Pharmacol. Exp. Ther. 269, 917-25 (1994)).
- tissue biopsies from the inflamed peritoneal cavity are collected, weighed, stored frozen (samples for microarray analysis are frozen at -80 0 C in TRIzol, Invitrogen, Carlsbad, CA) 1 and, as described in Example 12, subsequently analyzed with regard to tissue gene expression using microarray technology.
- Non-inflamed peritoneal cavities from untreated animals provide base-line levels of MPO, inflammatory mediators, cytokines/chemokines and gene expression. Tissue inflammation may also be studied using conventional histological and immunohistochemical techniques.
- Test drug(s) mass cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to the animals (for comparison, some experiments are performed without the drugs).
- thromboxane A2 antagonist aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen
- peritoneal inflammation may also be induced by intraperitoneal injection of pro-inflammatory concentrations of other well established proinflammatory stimuli such as anti-rat-lgE (with or without intraperitoneal pretreatment with rat IgE for 1-3 days), concanavalin A, protein L, compound 48/80, carrageenan, proteose peptone, LPS, PMA, thioglycolate, arachidonic acid, fMLP, TNF, IL-1 ⁇ .
- proinflammatory stimuli such as anti-rat-lgE (with or without intraperitoneal pretreatment with rat IgE for 1-3 days), concanavalin A, protein L, compound 48/80, carrageenan, proteose peptone, LPS, PMA, thioglycolate, arachidonic acid, fMLP, TNF, IL-1 ⁇ .
- Test drug(s) may also be administered simultaneously with intraperitoneal injection of zymosan or the other pro-inflammatory stimuli). 2- 24 hours after injection of zymosan (or one or more of the other stimuli), the animals are sacrificed. The peritoneal cavity is then flushed with 10-20 ml of a lavage buffer (e.g. ice-cold PBS with or without 3-5 mM EDTA or 5-10 units/mL heparin).
- a lavage buffer e.g. ice-cold PBS with or without 3-5 mM EDTA or 5-10 units/mL heparin.
- Total and differential leukocyte counts in the lavage fluid are done with a hemocytometer following staining with Turk's solution and/or in cytospin preparations stained with May-Grunwald Giemsa or a modified Wright's (Diff- Quik) stain, respectively, by light microscopy using standard morphological criteria. Other established methods for determining total and differential leukocyte counts may also be used.
- the remaining lavage fluid is centrifuged (300-3000 x g, 4 0 C, 3-10 min), and cell-free lavage fluid supernatant is stored frozen (-20 0 C to -80°) until analyzed for content of the inflammatory mediators LTB 4 , PGE 2 , TXB 2 and/or rat cytokines/chemokines (e.g. IL-4, IL-6, TNF, IL-1 ⁇ , KC 1 MCP-1 , IL-10, IL-12p70, IFN ⁇ ) essentially as described in Example 1 and 4 above.
- IL-4, IL-6, TNF, IL-1 ⁇ , KC 1 MCP-1 , IL-10, IL-12p70, IFN ⁇ essentially as described in Example 1 and 4 above.
- the histamine content in the lavage fluid supernatant is determined by using commercially available histamine enzyme immuno-assay kits (EIA/ELISA kits) according to instructions from the manufacturers). Inflammatory peritoneal cell activation may also be studied by measuring beta-hexosaminidase activity in the lavage fluid using the beta-hexosaminidase assay described in Example 3.
- the cell pellets of the lavage fluid are resuspended in 0.1-1.0 ml_ 0.05 M KHPO 4 pH 6.0 with 0.5% HTAB and stored frozen (-20 0 C to -80°) until analysis of myeloperoxidase (MPO) content basically as described by Rao et al (J. Pharmacol. Exp.
- tissue biopsies from the inflamed peritoneal cavity are collected, weighed, stored frozen (samples for microarray analysis are frozen at -80°C in TRIzol, Invitrogen, Carlsbad, CA), and, as described in Example 12, subsequently analyzed with regard to tissue gene expression using microarray technology.
- Non-inflamed peritoneal cavities from untreated animals provide base-line levels of MPO, inflammatory mediators, cytokines/chemokines and gene expression. Tissue inflammation may also be studied using conventional histological and immunohistochemical techniques.
- Human NB4 cells (Lanotte et al, Blood, 77, 1080 (1991 )) are cultured (37°C/5% CO2) in RPMI-1640 medium supplemented with 100 units/mL penicillin, 100 ⁇ g/mL streptomycin and 10% (v/v) fetal bovine serum. For differentiation, 1-5 ⁇ M all-frans-retinoic acid (ATRA) is added, generally every third day.
- ATRA all-frans-retinoic acid
- Human HL-60 cells (Steinhilber et al, Biochim. Biophys. Acta 1178, 1 (1993)) are cultured (37°C/5% CO2) in RPMI-1640 medium supplemented with 100 units/mL penicillin, 100 ⁇ g/mL streptomycin and 10-20% (v/v) fetal bovine serum.
- ATRA (1-5 ⁇ M
- DMSO 1-2%)
- PMA 100-500 ng/mL
- vitamin D3 (1-15 ⁇ M
- differentiated or undifferentiated NB4 or HL-60 cells are incubated for 5-30 minutes (at 37°C in PBS with calcium) with 10-40 ⁇ M arachidonic acid and/or 2-10 ⁇ M calcium ionophore A23187.
- the NB4 and HL-60 cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), fMLP, and/or the thromboxane analogue U- 46619, with or without A23187 and/or arachidonic acid as above.
- ADP adenosine diphosphate
- fMLP adenosine diphosphate
- thromboxane analogue U- 46619 with or without A23187 and/or arachidonic acid as above.
- the NB4 and HL-60 incubations/stimulations above may also be performed in the presence of human platelets (from healthy donor blood) with an NB4/HL-60:platelet ratio of 1 :10 to 1 :10000.
- the incubations/stimulations are stopped with 1 mL cold methanol and prostaglandin B 2 (PGB 2 ) added as internal standard.
- the samples are centrifuged and the supematants are diluted with water to reach a final methanol concentration of 30% and pH is adjusted to 3-4.
- Arachidonic acid metabolites in the supernatant are extracted on preconditioned (1 mL methanol followed by 1 mL H 2 O) C18 solid phase columns (Sorbent Technology, U.K.).
- Metabolites are eluted with methanol, whereafter one volume of water is added to the eluate.
- 76 ⁇ L of each sample is mixed with 39 ⁇ L H 2 O (other volume ratios may also be used).
- a Waters RCM 8*10 column is eluted with methanol/acetonitrile/H 2 0/acetic acid (30:35:35:0.01 v/v) at 1.2 mL/min. The absorbance of the eiuate is monitored at 270 nm for detection and quantitation of PGB 2 and LTB 4 .
- Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring LTB 4 may also be used according to instructions from the kit manufacturers).
- the supernatants from the NB4/HL-60 incubations/stimulations above may also be analysed with regard to content of the inflammatory mediators prostaglandin E 2 (PGE 2 ) and/or thromboxane B 2 (TXB 2 ).
- EIA/ELISA kits enzyme immuno-assay kits
- test drug(s) mass cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours prior to NB4 or HL-60 stimulation for inflammatory mediator release (see Example 1 above for details regarding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with NB4/HL- 60 stimulation). For comparison, some experiments are performed without the drugs.
- test drug(s) mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250)
- chemokines and mediators such as IL-1 ⁇ , IL-6, TNF 1 IL-8, IL-10, IL-12p70, MCP-1 , PAF 1 C5a, differentiated or undifferentiated NB4 or HL-60 cells (at 1-10> ⁇ 10 6 /mL) are incubated (37°C, 5% CO2) for 4-24 hours (in RPMI-1640 with 1-10% fetal bovine serum, with or without supplements) with lipopolysaccharide (LPS 1-100 ng/mL), phorbol-12-myristate-13-acetate (PMA 1-100 ng/mL) or calcium ionophore A23187 (1-10 ⁇ M), or combinations of these stimuli.
- LPS 1-100 ng/mL lipopolysaccharide
- PMA 1-100 ng/mL phorbol-12-myristate-13-acetate
- calcium ionophore A23187 1-10 ⁇ M
- the NB4 and HL-60 cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP) and/or the thromboxane analogue U-46619, with or without LPS, PMA and/or A23187 as above.
- ADP adenosine diphosphate
- the NB4 and HL-60 incubations/stimulations may also be performed in the presence of human platelets (from healthy donor blood) with an NB4/HL-60:platelet ratio of 1 :10 to 1 :10000.
- test drug(s) mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone, as above
- test drug(s) mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone, as above
- cytokine/chemokine and mediator concentrations in the supernatants are q ⁇ antitated using a Cytometric Bead Array (BD Biosciences Pharmingen, San Diego, USA) according to the manufacturer's instructions.
- BD Biosciences Pharmingen San Diego, USA
- enzyme immuno-assay kits EIA/ELISA kits
- the cell pellets are stored frozen (-80 0 C) in RLT buffer (QIAGEN, Valencia, CA) until further processing for microarray experiments (see Example 12 above).
- effects of the drugs on spontaneous or stimulated adhesion and/or migration of these cells may also be analyzed (freshly isolated human blood polymorphonuclear cells (PMN) isolated according to standard protocols may also be used).
- PMN human blood polymorphonuclear cells isolated according to standard protocols may also be used.
- Spontaneous or stimulated (with fMLP, IL-8, PAF, LTB 4 or other relevant PMN activating factors) adhesion of the PMN or neutrophil-like cells to e.g. cultured endothelial cells or protein-coated artificial surfaces are studied using well established and documented experimental approaches and assays.
- Venous blood is collected by venepuncture without stasis, using siliconized vacutainer tubes containing 1/10 volume of 129 mM tris ⁇ dium citrate (Becton
- leukocyte CD11b expression reflecting leukocyte activity
- single platelet and platelet-platelet microaggregate counting and platelet-leukocyte aggregates (PLAs) are measured using flow cytometric assays, essentially as described previously (see e.g. Li et al. Circulation 100, 1374 (1999) for reference).
- ⁇ l_ aliquots of whole blood are added to 45 ⁇ l_ Hepes buffered saline (150 mM NaCI, 5 mM KCI, 1 mM MgSO4, 10 mM Hepes, pH 7.4) containing appropriately diluted antibodies (see below) in the absence or presence of platelet activating stimuli such as adenosine diphosphate (ADP), U- 46619, U-44069, platelet activating factor (PAF), arachidonic acid, collagen or thrombin, and/or leukocyte activating stimuli such as N-formyl-methionyl-leucyl- phenylalanine (fMLP), arachidonic acid, PAF, LPS 1 A23187 or LTB 4 .
- platelet activating stimuli such as adenosine diphosphate (ADP), U- 46619, U-44069, platelet activating factor (PAF), arachidonic acid, collagen or thrombin, and/or
- test drug(s) Prior to exposing the blood to the platelet and/or leukocyte activating stimuli + the antibodies, blood samples (0.1-1 ml) are incubated with test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1-60 minutes (test drug(s) may also be added simultaneously with the stimuli above).
- test drug(s) mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or
- Platelet P- selectin expression is determined by R-phycoerythrin (RPE)-CD62P monoclonal antibody (MAb) AC1.2 (Becton Dickinson, San Jose, CA, USA).
- Leukocyte CD11 b expression is determined by fluorescein isothiocyanate (FITC)-conjugated MAb BEAR 1 (Immunotech, Marseille, France). FITC and RPE conjugated isotypic MAbs are used as negative controls.
- Fluorescent beads (SPHEROTM Rainbow particles, 1.8-2.2 ⁇ m) used for platelet counting are from PharMingen (San Diego, CA, USA).
- Platelets are identified with FITC conjugated anti-CD42a (GPIX) MAb Beb 1 (Becton Dickinson), and leukocytes are identified with RPE conjugated anti-CD45 MAb J33 (Immunotech).
- Samples drug-treated or untreated blood + antibodies with or without the stimuli, as above are incubated at room temperature in the dark for 20 min. Afterwards, the samples are diluted and mildly fixed with 0.5% (v/v) formaldehyde saline, and analysed for the various platelet and leukocyte parameters with a Beckman-Coulter EPICS XL-MCL flow cytometer (Beckman-Coulter Corp., Hialeah, FL).
- Platelet P-selectin expression data are reported as the percentages of P-selectin positive cells in the platelet population and as absolute counts of P-selectin positive platelets.
- Leukocyte CD11 b expression is reported as mean fluorescence intensity (MFI) of the total leukocyte population and of leukocyte subpopulations.
- Platelet-leukocyte aggregates (PLAs) are presented as both absolute counts and percentages of platelet-conjugated leukocytes in the total leukocyte population and among lymphocytes, monocytes, and neutrophils. Other relevant reagents, experimental conditions/approaches, equipment and modes of analysis to measure corresponding platelet and leukocyte activation in human whole blood may also be used.
- Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.3 to 200 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to the animals (drugs, up to 400 mg/kg/day, may also be continuously administered subcutaneously or intraperitoneally using e.g.
- tissue leukocyte accumulation gene expression and protease activity is measured in specimens of the aortic aneurysmal tissue using established/conventional biochemical, histological, immunohistochemical, immunological, micoroarray and zymographical techniques. Examples of how to handle collected tissues and measure tissue gene expression is described in Examples 5 and 12 above. Effects of the drugs may also be studied in corresponding rat models of elastase- induced aortic aneurysms, for example essentially according to Holmes et al (J. Surg. Res., 63, 305 (1996)), or CaCI2-induced aortic aneurysms, for example essentially according to lsenburg et al (Circulation, 115, 1729 (2007)).
- Samples of human atherosclerotic carotid or femoral arteries or abdominal aortic aneurysms obtained during routine surgery are used to study drug effects on spontaneous or induced inflammation, gene expression and protease activity in the diseased arterial tissues. Before the incubations/treatments below, the tissues are kept on ice in PBS without Ca and Mg (other established tissue media may also be used).
- diced tissues are incubated (37°C/5% CO 2 ) for 1-24 hours (in RPM 1-1640 with 1-10% fetal bovine serum; other established tissue media may also be used) with or without lipopolysaccharide (LPS, final concentration 1-100 ng/mL), phorbol-12-myristate-13-acetate (PMA, final concentration 1-100 ng/mL) or an LPS/PMA mixture.
- LPS lipopolysaccharide
- PMA phorbol-12-myristate-13-acetate
- the diced tissues can also be incubated (37 0 C with or without 5% CO 2 ) for 5 min to 24 hours (in RPMI-1640 with 1-10% fetal bovine serum; other established media may also be used) with or without anti-lgE, concanavalin A, protein L or compound 48/80. Stimulation of IgE-dependent release of inflammatory mediators may also be performed using pre-incubation with anti-TNP IgE followed by TNP-BSA challenge essentially as described in Example 3.
- test drug(s) mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours (test drug(s) may also be added simultaneously with the stimuli above).
- the test drugs remain present during the incubations with the proinflammatory stimuli.
- inflammatory mediators e.g. histamine, tryptase, cytokines, chemokines
- gene expression and protease e.g. MMPs such as MMP2, MMP3, MMP9
- MMPs such as MMP2, MMP3, MMP9
- Egualen e.g. egualen sodium
- egualen at doses of 1 to 100 mg/kg is administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to the animals (egualen, up to 400 mg/kg/day, may also be continuously administered subcutaneously or intraperitoneally using e.g. Alzet® Osmotic Pumps according to instructions by the manufacturer).
- Alzet® Osmotic Pumps e.g. Alzet® Osmotic Pumps according to instructions by the manufacturer.
- stock solutions of egualen are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration).
- Effects of egualen on the elastase- or CaCI2-induced aneurysmal increases in aortic diameter are typically measured immediately before and one, two, three and/or four weeks after challenge with elastase or CaCI2 (other measurement intervals may also be used).
- Effects of egualen on inflammation e.g. tissue leukocyte accumulation
- gene expression and protease activity is measured in specimens of the aortic aneurysmal tissue using established/conventional biochemical, histological, immunohistochemical, immunological, micoroarray and zymographical techniques.
- MM6 Human macrophage cell-line MonoMac-6 (MM6) (Ziegler-Heitbrock et al, Int. J. Cancer, 41, 456 (1988)) were cultured (37°C/5% CO 2 ) in RPMI-1640 medium supplemented with 1 mM sodium pyruvate, ⁇ nonessential amino acids, 10 ⁇ g/mL insulin, 1 mM oxalacetic acid, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin and 10% (v/v) fetal bovine serum.
- MM6 cells were seeded in 96-well plates at a density of 1x10 5 cells/mL (100 pi ⁇ per well).
- the Proliferation of the MM6 cells was measured using the Cell Proliferation Reagent WST-1 (Roche Diagnostics Scandinavia AB, Bromma, Sweden) or by cell counting using a microscope.
- the WST-1 reagent is designed to be used for spectrophotometric quantification of e.g. cell growth in proliferation assays and was used according to the manufacturers' instructions.
- the wavelength for measuring absorbance was 450 nm.
- MY-1250 and/or picotamide were added at the start of the experiments and 46 hours later the effects of the drugs on MM6 cell proliferation were studied using the WST-1 reagent described above.
- Example 22 The procedure described in Example 22 was repeated for tazanolast (synthesised according to a procedure described in European patent EP 481 118 B1 ).
- Tazanolast was dissolved in DMSO and then diluted in sterile saline leading to final DMSO concentrations in the MM6 incubations of 0.03%, 0.1% and 0.3% for the final tazanolast concentrations 3 ⁇ M, 10 ⁇ M and 30 ⁇ M, respectively.
- the effects of treatment with tazanolast (alone and in combination) on MM6 proliferation were compared with control treatments containing DMSO at concentrations corresponding to the DMSO concentrations used in the respective tazanolast treatments.
- Example 22 The procedure described in Example 22 was repeated for pemirolast (potassium salt; purchased from American Custom Chemicals Corporation, San Diego, USA; stock solutions were made in sterile saline).
- mast cell inhibitors e.g. tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast, more particularly, suplatast or preferably pemirolast
- thromboxane A2 antagonists e.g. aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban and/or egualen.
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Abstract
There is provided combination products comprising (a) a mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof; and (b) a thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof. Such combination products find particular utility in atherosclerosis and related conditions.
Description
NEW COMBINATION FOR USE IN THE TREATMENT OF INFLAMMATORY DISORDERS
Field of the Invention
This invention relates to a novel pharmaceutical combination.
Background and Prior Art
Cardiovascular diseases, such as coronary heart disease and stroke are major causes of death, disability, and healthcare expense, particularly in industrialised countries. Such diseases are often direct sequelae of atherosclerosis, a multifactorial condition that develops preferentially in subjects that smoke and/or present risk factors such as hypertension, diabetes mellitus, hypercholesterolemia, elevated plasma low density lipoprotein (LDL) and triglycerides.
Atherosclerotic lesions (or plaques) develop over many years. Pathological processes, such as cholesterol accumulation in the artery wall, foam cell formation, inflammation and cell proliferation are typically involved.
Levels of high-density lipoproteins (HDLs)1 LDLs, total cholesterol and triglycerides are all indicators in determining the risk of developing atherosclerosis and associated cardiovascular disorders, such as coronary artery diseases (e.g. angina pectoris, myocardial infarction, etc.), stroke (including cerebro-vascular accident and transient ischaemic attack) and peripheral arterial occlusive disease.
Patients with high overall cholesterol and/or triglycerides levels are at a significant risk, irrespective of whether or not they also have a favourable HDL level. Patients with normal overall cholesterol levels but low HDL levels are also at increased risk. Recently, it has also been noted that the level of risk of cardiovascular disease associated with high levels of apolipoprotein B (ApoB; which carries lipids in very low-density lipoproteins (VLDLs) and LDLs), and/or
low levels of apolipoprotein A-I (ApoA-l; which carries lipids in HDLs), is extremely high.
Drugs that reduce LDL levels in serum can reduce the build-up of atherosclerotic plaques, and can reduce (long term) the risk of plaque rupture and associated thrombo-embolic complications. There are several types of drugs that can help reduce blood cholesterol levels. The most commonly prescribed are the hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors (hereinafter defined together, irrespective of their generic name, as "statins"), including simvastatin and atorvastatin. These drugs prevent directly the formation of cholesterol in the liver and thus reduce the risk of cardiovascular disease.
Other prescribed drug categories include resins (such as cholestyramine and colestipol), which act by binding bile acids, so causing the liver to produce more of the latter, and using up cholesterol in the process. Further, the B vitamin niacin has been reported at high doses to lower triglycerides and LDL levels in addition to increasing HDL levels. Fibrates (such as gemfibrozil and fenofibrate) are known to lower triglycerides and can increase HDL levels.
The introduction of cholesterol lowering drugs such as statins has significantly reduced mortality from coronary heart disease and stroke. However, these drugs suffer from the disadvantage that they are not equally effective in all patients and are known to have certain side effects (e.g. changes in liver function, myopathy and rhabdomyolysis), and atherosclerosis remains a major cause of death and disability. Indeed, a recent review article (Briel et al, JAMA, 295, 2046 (2006)) suggests that statins do not reduce serious cardiovascular events during the first four months of treatment in patients with acute coronary syndromes.
There is thus a real clinical need for safer and/or more effective treatments of atherosclerosis and associated cardiovascular disorders, particularly in those patients with acute coronary syndromes. Furthermore, there is no established drug treatment for the arterial disease abdominal aortic aneurysm, a potentially fatal disease associated with atherosclerosis.
Mast cells (mastocytes) are cells that are present in most tissues in the vicinity of blood vessels, and are especially prominent near the boundaries between the extra-corporeal environment and the internal milieu, such as the skin, mucosal surfaces (such as the lungs, digestive tract, mouth, conjunctiva and nose). Mast cells play an important protective role, being involved in wound healing and defence against pathogens.
In allergic reactions, mast cells remain inactive until an allergen binds to IgE already in association with the cell. Binding of two or more IgE molecules (crosslinking) leads to steric changes that cause disturbances to the cell membrane structure. This results in a complex sequence of reactions inside the cell that leads to activation and so-called "degranulation".
In this respect, mast cells play a key role in the inflammatory process. Cross- linking of IgE receptors (as well as direct (e.g. physical or chemical) injury) may stimulate mast cells to degranulate, and thereby release various mediators into the interstitium, including eicosanoids, such as prostaglandin D2 and leukotriene C4, cytokines, and preformed mediators, such as heparin, serine proteases and, particularly, histamine.
Histamine dilates post capillary venules, activates the endothelium, and increases blood vessel permeability. This leads to local oedema (swelling), warmth, redness, and the attraction of other inflammatory cells to the site of release. It also irritates nerve endings (leading to itching or pain).
Mast cells therefore play a central role in asthma, eczema, allergic rhinitis and allergic conjunctivitis. Mast cells are also implicated in the pathology associated with the autoimmune disorders such as rheumatoid arthritis and multiple sclerosis.
Treatments of inflammatory conditions associated with mast cell degranulation include antihistamine drugs, which act by blocking the action of histamine on e.g. nerve endings and blood vessels. Chromone-based drugs (such as sodium cromoglycate and nedocromil) are thought to block ion channels essential for mast cell degranulation, stabilizing the cell and preventing release of histamine
and related mediators. Other mast cell stabilisers include pemirolast, suplatast, repirinast, amlexanox, ketotifen, tazanolast and tranilast.
In particular, pemirolast is an orally-active anti-allergic drug which is used in the treatment of conditions such as asthma, allergic rhinitis and conjunctivitis. The drug is presently marketed in e.g. Japan as the potassium salt.
Suplatast is a Th2 cytokine inhibitor which inhibits the release of IL-4 and IL-5 from Th2 cells as well as the release of chemical mediators from mast cells. Suplatast is therefore indicated to be of use in the treatment of conditions such as asthma, allergic rhinitis, atopic dermatitis, interstitial cystitis and chronic nonbacterial prostatitis. See, for example, Tamoaki, Allergology International, 53, 55 (2004) and Suwaki et a/, International Immunopharmacology, 1, 2163 (2001 ). Suplatast has also been indicated in the possible treatment of acute eosinophilic myocarditis (see Umemoto et al, Heart Vessels, 18, 100 (2003)).
Certain studies have been reported that relate to the potential use of mast cell inhibitors in the prevention of restenosis (see Miyazawa et al, J. Cardiovasc. Pharmacol., 30, 157 (1997), Ohsawa et al, Am. J. Heart, 136, 1081 (1998), European patent application EP 766 963 (which discloses that pemirolast exhibits an inhibitory effect on the proliferation of vascular smooth muscle cells), Singh et al, J. Cardiovasc. Pharmacol. Ther., 8, 135 (2003), Tamai et al, Am. J. Heart, 138, 968 (1999), Holmes et al, Circulation, 106, 1243 (2002), Matsumura et al, ibid., 99, 919 (1999), Siaura et al, Eur. J. Pharmcol., 433, 163 (2001 ), Philippe et al, Annales de Cardiologie et d-Angeiologie, 54, 201 (2005) and Schainfeld, Cathet. Cardiovasc. Intervent, 56, 421 (2002)). See also Zhang and Lin, Chin. J. New Drugs and Clin. Rem., 23, 795 (2004) and US Patent No. 6,585,995.
International patent application WO 2004/071531 mentions certain mast cell inhibitors for the treatment of cerebral diseases involving mast cell activation caused by ischemia.
Thromboxane A2 is an unstable cyclooxygenase product of arachidonic acid metabolism, which has been shown to be an important mediator of thrombosis, vasospasm and bronchoconstriction. As a potent bronchoconstrictor, it is
involved in acute bronchoconstriction in asthmatic patients, as demonstrated by a markedly elevated level of its stable metabolite, thromboxane B2. Thromboxane A2 has an important role in bronchial hyperreactivity, a key feature of bronchial asthma. Moreover, there is accumulating evidence indicating that thromboxane A2 could be an immunomodulator.
Thromboxane A2 may thus contribute to airflow limitation and bronchial hyperresponsiveness. Thromboxane A2 antagonists have been demonstrated to improve these symptoms. Such antagonists may act by way of a variety of mechanisms, for example by blocking the thromboxane TP receptor (e.g. terutroban and egualen), by inhibiting the enzyme thromboxane synthase (e.g. ozagrel and picotamide), or by selectively inhibiting platelet cyclooxygenase-1 , thereby inhibiting e.g. platelet aggregation (e.g. low-dose aspirin). Thromboxane A2 antagonists have been well documented to be of potential use in various cardiovascular conditions (see, for example, Hiraku et a/, Jpn. J. Pharmacol., 41, 393 (1986), Vetrano et al, Giornale ltaliano di Cardioliga, 29, 524 (1999), Coto et al, J. Int. Med. Res., 26, 200 (1998), Osende et al, J. Thromb. Haemostasis, 2, 492 (2004), Sorbrera et al, Drugs of the Future, 31 , 867 (2006), Dogne et al, Expert Opin. Ther. Patents, 11, 1663 (2001 ) and Ishizuka et al, Cardiovascular Drug Reviews, 22, 71 (2004), J. Cardiovasc. Pharmacol., 41 , 571 (2003) and Vascular Disease Prevention, 3, 143 (2006)). Further egualen is known to be of use in the treatment of gastritis (see, for example, Akagi et al, Pharmacology, 63, 203 (2001 )).
US patent application US 2003/0109543 discloses combination products that are useful in the prevention or inhibition of platelet aggregation and thrombus formation comprising antiplatelet drugs, such as clopidrogrel and thromboxane A2 antagonists, more specifically ifetroban.
US patent application US 2006/0024365 discloses dual retard pharmaceutical dosage forms comprising modified release high dose high solubility active ingredients in combination with immediate release low dose active ingredients. A wide variety of drugs, including some of those mentioned herein, are listed as potential candidates for the low dose active ingredient.
US patent application US 2003/0104048 discloses novel pharmaceutical dosage forms comprising hydrophilic surfactant-containing fillers including pharmaceutically-active ingredients encapsulated by a shell. Various active compounds, including some of those mentioned herein, are listed amongst many possible drug candidates for use in such dosage forms.
US patent application US 2007/0014733 discloses pharmaceutical compositions for the treatment of cardiovasular disorders comprising metabolites of nebivolol. Various active compounds, including some of those mentioned herein, are listed among the many active ingredients that may be combined with such metabolites in such compositions.
US patent applications US 2006/0135577 and US 2006/0148830 disclose novel antagonists of the LPA receptor (and especially the EDG-2 receptor) for use in inter alia disorders of the urinary system, inflammation etc. Certain active compounds, including some of those mentioned herein, are mentioned separately among the many different active ingredients that may be combined with such novel compounds.
Sugimoto et al (Arzneimittel-Forschung., 47, 837 (1997)) studied the effect of the PAF receptor antagonist, apafant, on bronchial hyperresponsiveness and down- regulation of beta-adrenoceptors induced by endotoxin in guinea pigs. It was also reported in that study that ketotifen and ozagrel (individually) prevented bronchial hyperresponsiveness and endotoxin-induced deterioration of the beta- adrenergic system. Takehana et al (Nippon Yakurigaku Zasshi - Folia Pharmacologica Japonica., 95, 121 (1990)) reported that oral administration of ozagrel and tranilast suppressed antigen-induced inflammatory edema in the skin. Behrendt et al (Hautarzt, 43, 139 (1992)) administered acetylsalicylic acid and ketotifen for pain relief in patients with angiolipomas. Delaney (Clinical Allergy, 13, 247 (1983)) studied the effect of ketotifen on aspirin-induced asthma attacks.
Finally, US patent application US 2005/0181023 discloses pharmaceutical compositions comprising pemirolast for use in the inhibition or prevention of adhesions between tissue and/or organ surfaces.
The use of combination products comprising, mast cell inhibitors and thromboxane A2 antagonists is not specifically disclosed in any of the above- mentioned documents. Further, the use of such combination products in the treatment of atherosclerosis and associated cardiovascular disorders, particularly in those patients with acute coronary syndromes, or abdominal aortic aneurysms, is not disclosed in any of these documents.
Disclosure of the Invention
According to the invention, there is provided a combination product comprising: (a) one or more mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof; and (b) one or more thromboxane A2 antagonist, or a pharmaceutically-acceptabie salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast, which combination products are referred to hereinafter as "the combination products according to the invention".
In the context of the present application, the term "mast cell inhibitor" includes any compound that is capable of inhibiting, to an experimentally-determinable degree, the degranulation of (and therefore release of e.g. histamine from) mast cells in in vitro and/or in vivo tests. Mast cell inhibitors that may be mentioned include histamine H1 receptor antagonists that are known to inhibit mast cell activation to varying degrees, such as acrivastine, astemizole, azelastine, cetirizine, carebastine, desloratadine, ebastine, fexofenadine, ketotifen, levocabastine, levocetirizine, loratadine, mizolastine, norastemizole, olopatadine, oxatomide and terfenadine. Other mast cell inhibitors that may be mentioned include those that are used for local inhibition of mast cell activation, such as cromoglycate (e.g. disodium cromoglycate), nedocromil (such as nedocromil sodium) and andolast. However, preferred mast cell inhibitors include orally active non-antihistamine drugs that inhibit mast cell activation, such as pemirolast, repirinast, amlexanox, tazanolast, suplatast and tranilast Also included within this definition are active metabolites of mast cell inhibitors, such
as acitazanolast (which is an active metabolite of tazanolast) and MY-1250 (5,6- dihydro-7,8-dimethyl-4,5-dioxo-4H-pyrano[3,2-c]quinoline-2-carboxylic acid, which is an active metabolite of repirinast).
Preferred mast cell inhibitors include tranilast, more preferably ketotifen, particularly amlexanox, more particularly repirinast (or its active metabolite, MY- 1250) and tazanolast, and especially suplatast.
Another preferred mast cell inhibitor is pemirolast.
According to a further aspect of the invention, there is provided a combination product comprising:
(a) pemirolast, or a pharmaceutically-acceptable salt or solvate thereof; and
(b) one or more thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast.
For the avoidance of doubt, preferred features of combination products according to the invention mentioned hereinbefore or hereinafter may be imported into/applied to this aspect of the invention.
According to a still further aspect of the invention, there is provided a combination product comprising:
(a) one or more mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof, provided that the mast cell inhibitor is not pemirolast; and (b) one or more thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast.
For the avoidance of doubt, preferred features of combination products according to the invention mentioned hereinbefore or hereinafter may be imported into/applied to this aspect of the invention.
In the context of the present application, the term thromboxane A2 antagonist includes any compound that is capable of inhibiting, to an experimentally- determinable degree in in vitro and/or in vivo tests, the effects of thromboxane A2 by one or more of (i) blocking the thromboxane TP receptor, (ii) inhibiting the
enzyme thromboxane synthase, or (iii) inhibiting (e.g. selectively) platelet cyclooxygenase-1 , thereby inhibiting e.g. platelet aggregation.
Preferred thromboxane A2 antagonists include aspirin/acetylsalicylic acid, more preferably egualen, particularly ozagrel and, more particularly, picotamide and terutroban.
Pharmaceutically-acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of an active ingredient with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze- drying or by filtration). Salts may also be prepared by exchanging a counter-ion of an active ingredient in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
Preferred salts of ketotifen include fumarate salts. Other salts of ketotifen, and salts of picotamide and suplatast, that may be mentioned include hydrochloride, bisulfate, maleate and tosylate salts. Preferred salts of suplatast include suplatast tosylate. Salts of tranilast, amlexanox, tazanolast, MY-1250, ozagrel, terutroban, egualen and aspirin that may be mentioned include alkali metal salts, such as lithium, sodium and potassium salts. Preferred salts of ozagrel and egualen include sodium salts.
Active ingredients that are employed in combination products according to the invention (and in particular ketotifen and terutroban) may be employed in diastereomerically-enriched and/or enantiomerically-enriched form. By "diastereomerically-enriched" and "enantiomerically-enriched" we mean, respectively, any mixture of the diastereoisomers/enantiomers of an active ingredient, in which one isomer is present in a greater proportion than the other. For example, enantiomers (of e.g. ketotifen and terutroban) with optical purities (enantiomeric excess; e.e.) of greater than 90% may be employed. Preferred enantiomers of terutroban include S-18886, i.e. the R-enantiomer.
Preferred combination products according to the invention include (in ascending order of preference) those in which:
(i) the mast cell inhibitor (a) in a composition of the invention is ketotifen or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) in a composition of the invention is aspirin or a pharmaceutically-acceptable salt or solvate thereof; (ii) the mast cell inhibitor (a) is tranilast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof; (iii) the mast cell inhibitor (a) is amlexanox or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof; (iv) the mast cell inhibitor (a) is tazanolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof;
(v) the mast cell inhibitor (a) is repirinast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof; (vi) the mast cell inhibitor (a) is MY-1250 or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof; (vii) the mast cell inhibitor (a) is ketotifen or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof; (viii) the mast cell inhibitor (a) is ketotifen or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof; (ix) the mast cell inhibitor (a) is ketotifen or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof;
(x) the mast cell inhibitor (a) is ketotifen or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
(xi) the mast cell inhibitor (a) is tranilast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof;
(xii) the mast cell inhibitor (a) is tranilast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof; (xiii) the mast cell inhibitor (a) is tranilast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof; (xiv) the mast cell inhibitor (a) is tranilast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof; (xv) the mast cell inhibitor (a) is amlexanox or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof;
(xvi) the mast cell inhibitor (a) is tazanolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof; (xvii) the mast cell inhibitor (a) is repirinast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof; (xviii) the mast cell inhibitor (a) is MY-1250 or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof; (xix) the mast cell inhibitor (a) is amlexanox or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof; (xx) the mast cell inhibitor (a) is amlexanox or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof;
(xxi) the mast cell inhibitor (a) is amlexanox or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
(xxii) the mast cell inhibitor (a) is tazanolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof; (xxiii) the mast cell inhibitor (a) is tazanolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist
(b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof;
(xxiv) the mast cell inhibitor (a) is tazanolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
(xxv) the mast cell inhibitor (a) is repirinast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof; (xxvi) the mast cell inhibitor (a) is repirinast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof; (xxvii) the mast cell inhibitor (a) is repirinast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof;
(xxviii) the mast cell inhibitor (a) is MY-1250 or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof;
(xxix) the mast cell inhibitor (a) is MY-1250 or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof; (xxx) the mast cell inhibitor (a) is MY-1250 or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof; (xxxi) the mast cell inhibitor (a) is suplatast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof; (xxxii) the mast cell inhibitor (a) is suplatast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof;
(xxxiii) the mast cell inhibitor (a) is suplatast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof;
(xxxiv) the mast cell inhibitor (a) is suplatast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof;
(xxxv) the mast cell inhibitor (a) is suplatast or a pharmaceutically-acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof; (xxxvi) the mast cell inhibitor (a) is pemirolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is aspirin or a pharmaceutically-acceptable salt or solvate thereof;
(xxxvii)the mast cell inhibitor (a) is pemirolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is egualen or a pharmaceutically-acceptable salt or solvate thereof;
(xxxviii) the mast cell inhibitor (a) is pemirolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is ozagrel or a pharmaceutically-acceptable salt or solvate thereof;
(xxxix) the mast cell inhibitor (a) is pemirolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist
(b) is picotamide or a pharmaceutically-acceptable salt or solvate thereof; and
(xl) the mast cell inhibitor (a) ■ is pemirolast or a pharmaceutically- acceptable salt or solvate thereof; and the thromboxane A2 antagonist (b) is terutroban or a pharmaceutically-acceptable salt or solvate thereof.
For the avoidance of doubt, preferred features of combination products according to the invention mentioned hereinbefore or hereinafter may be imported into/applied to any one of the above-mentioned specific combinations (i) to (xl) above.
Combination products according to the invention provide for the administration of mast cell inhibitor as hereinbefore defined in conjunction with a thromboxane A2 antagonist as hereinbefore defined, and may thus be presented either as
separate formulations, wherein at least one of those formulations comprises a mast cell inhibitor, and at least one comprises a thromboxane A2 antagonist, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including mast cell inhibitor and a thromboxane A2 antagonist).
Thus, there is further provided:
(1 ) a pharmaceutical formulation including one or more mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof; one or more thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast; and a pharmaceutically-acceptable adjuvant, diluent or carrier (which formulation is hereinafter referred to as a "combined preparation"); and
(2) a kit of parts comprising components:
(A) a pharmaceutical formulation including one or more mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier; and (B) a pharmaceutical formulation including one or more thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (A) and (B) are each provided in a form that is suitable for administration in conjunction with the other.
According to a further aspect of the invention, there is provided a method of making a kit of parts as defined above, which method comprises bringing component (A), as defined above, into association with a component (B), as defined above, thus rendering the two components suitable for administration in conjunction with each other.
By bringing the two components "into association with" each other, we include that components (A) and (B) of the kit of parts may be:
(i) provided as separate formulations (i.e. independently of one another), which are subsequently brought together for use in conjunction with each other in combination therapy; or
(ii) packaged and presented together as separate components of a "combination pack" for use in conjunction with each other in combination therapy.
Thus, there is further provided a kit of parts comprising:
(I) one of components (A) and (B) as defined herein; together with
(II) instructions to use that component in conjunction with the other of the two components.
The kits of parts described herein may comprise more than one formulation including an appropriate quantity/dose of mast cell inhibitor/salt/solvate, and/or more than one formulation including an appropriate quantity/dose of thromboxane A2 antagonist/salt/solvate, in order to provide for repeat dosing. If more than one formulation (comprising either active compound) is present, such formulations may be the same, or may be different in terms of the dose of either compound, chemical composition(s) and/or physical form(s).
The combination products according to the invention find utility in the treatment of inflammatory conditions.
Inflammatory conditions are typically characterized by activation of immune defence mechanisms, resulting in an effect that is more harmful than beneficial to the host. Such conditions are generally associated with varying degrees of tissue redness or hyperemia, swelling, hyperthermia, pain, itching, cell death and tissue destruction, cell proliferation, and/or loss of function. Inflammatory conditions that may be mentioned include endometriosis and, more preferably, allergy (including allergic conjunctivitis and allergic rhinitis), ankylosing spondylitis, asthma, atopic dermatitis, chronic obstructive pulmonary disease, contact dermatitis, cystitis, gouty arthritis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), multiple sclerosis, osteoarthritis, pancreatitis, prostatitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, tendinitis, bursitis, Sjogren's syndrome, systemic lupus erythematosus, uveitis, urticaria, vasculitis, diabetic vascular complications, migraine, atherosclerosis and associated cardiovascular
disorders. Conditions that may be mentioned include endometriosis, migraine and, more preferably, asthma, chronic obstructive pulmonary disease, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis and, more particularly, atherosclerosis and associated cardiovascular disorders.
The term "atherosclerosis" will be understood by those skilled in the art to include any disease characterised by cholesterol accumulation in a blood vessel, especially an artery wall, foam cell formation, inflammation and cell proliferation. Cardiovascular disorders "associated with" atherosclerosis include aortic aneurysms (including abdominal and/or atherosclerotic aortic aneurysms) and, more preferably, arteriosclerosis, peripheral arterial occlusive disease, coronary artery diseases (e.g. angina pectoris, myocardial infarction, heart attack, etc), coronary disease (including cardiac disease and heart disease, such as ischaemic heart disease), and may also include plaque or atheroma rupture and/or instability, vascular or arterial disease, ischaemic disease/ischaemia and stroke (including cerebro-vascular accident and transient ischaemic attack).
Patient groups that may be mentioned include those with acute coronary syndromes. The term "acute coronary syndrome(s)" will be understood by the skilled person to include any abnormal myocardial and ischaemic state, often but not exclusively associated with chest pain and/or an abnormal electrocardiogram (ECG). Such syndromes are the most common presentation of myocardial infarction (heart attack). The skilled person will appreciate that the term is largely synonymous with the term "unstable angina", as opposed to "stable angina" (i.e. angina that develops during exertion and resolves at rest). Exertional angina that occurs at worsening rate ("crescendo angina") will similarly be regarded by the skilled person as within the definition "unstable".
According to a further aspect of the invention there is provided a method of treatment of an inflammatory disorder, and in particular atherosclerosis and/or an associated cardiovascular disorder, which method comprises the administration of a combination product according to the invention to a patient in need of such treatment.
With respect to the kits of parts as described herein, by "administration in conjunction with", we include that respective formulations comprising mast cell inhibitor (or salt/solvate thereof) and thromboxane A2 antagonist (or salt/solvate thereof) are administered, sequentially, separately and/or simultaneously, over the course of treatment of the relevant condition.
Thus, in respect of the combination product according to the invention, the term "administration in conjunction with" includes that the two components of the combination product (mast cell inhibitor and thromboxane A2 antagonist) are administered (optionally repeatedly), either together, or sufficiently closely in time, to enable a beneficial effect for the patient, that is greater, over the course of the treatment of the relevant condition, than if either a formulation comprising the mast cell inhibitor, or a formulation comprising the thromboxane A2 antagonist, are administered (optionally repeatedly) alone, in the absence of the other component, over the same course of treatment. Determination of whether a combination provides a greater beneficial effect in respect of, and over the course of treatment of, a particular condition will depend upon the condition to be treated or prevented, but may be achieved routinely by the skilled person.
Further, in the context of a kit of parts according to the invention, the term "in conjunction with" includes that one or other of the two formulations may be administered (optionally repeatedly) prior to, after, and/or at the same time as, administration of the other component. When used in this context, the terms "administered simultaneously" and "administered at the same time as" include that individual doses of mast cell inhibitor and thromboxane A2 antagonist are administered within 48 hours (e.g. 24 hours) of each other.
The use of egualen (in any form including as sole therapy) in the treatment of atherosclerosis and/or associated cardiovascular disorders as defined herein is not known. According to a still further aspect of the invention, there is provided a method of treatment of atherosclerosis and/or an associated cardiovascular disorder, which method comprises the administration of egualen or a pharmaceutically-acceptable salt or solvate thereof to a patient in need of such treatment. For the avoidance of doubt, preferred features of combination
products according to the invention mentioned hereinbefore or hereinafter may be imported into/applied to this aspect of the invention.
For the avoidance of doubt, in the context of the present invention, the terms "treatment", "therapy" and "therapy method" include the therapeutic, or palliative, treatment of patients in need of, as well as the prophylactic treatment and/or diagnosis of patients which are susceptible to, inflammatory disorders, such as atherosclerosis and associated cardiovascular disorders.
"Patients" include mammalian (including human) patients.
In accordance with the invention, mast cell inhibitors and thromboxane A2 antagonists are preferably administered locally or systemically, for example orally, intravenously or intraarterially (including by intravascular stent and other perivascular devices/dosage forms), intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, or by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound(s) in pharmaceutically acceptable dosage form(s). Preferred modes of delivery include oral (particularly), intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal delivery.
Mast cell inhibitors and thromboxane A2 antagonists will generally be administered together or separately in the form of one or more pharmaceutical formulations in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Such pharmaceutically acceptable carriers may also impart an immediate, or a modified, release of either active ingredient, whether administered together in a combined preparation or in the form of a kit of parts.
Suitable pharmaceutical formulations may be commercially available or otherwise are described in the literature, for example, Remington The Science and Practice
of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995) and Martindale - The Complete Drug Reference (34th Edition) and the documents referred to therein, the relevant disclosures in all of which documents are hereby incorporated by reference. Otherwise, the preparation of suitable formulations, and in particular combined preparations including both mast cell inhibitors and thromboxane A2 antagonists may be achieved non-inventively by the skilled person using routine techniques.
The amount of active ingredients in the formulation(s) will depend on the severity of the condition, and on the patient, to be treated, as well as the compound(s) which is/are employed, but may be determined non-inventively by the skilled person.
Depending on the disorder, and the patient, to be treated, as well as the route of administration, active ingredients may be administered at varying therapeutically effective doses to a patient in need thereof.
However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease, as well as genetic differences between patients.
Administration of active ingredients may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration.
Suitable doses of active ingredients include those referred to in the medical literature, such as Martindale - The Complete Drug Reference (34th Edition) and the documents referred to therein, the relevant disclosures in all of which
documents are hereby incorporated by reference. Suitable doses of active ingredients are therefore in the range of about 0.01 mg/kg of body weight to about 1 ,000 mg/kg of body weight. More preferred ranges are about 0.1 mg/kg to about 20 mg/kg on a daily basis, when given orally.
However, suitable doses of mast cell inhibitors are known to those skilled in the art. For example, peroral doses may be in the range of about 0.1 mg to about 1.2 g, such as about 0.5 mg to about 900 mg, per day, irrespective of whether the formulation employed is a combined preparation or a kit of parts as hereinbefore described.
In this respect, suitable peroral doses that may be mentioned are as follows: (1 ) for tazanolast, suitable lower limits of daily dose ranges are about 15 mg, such as about 25 mg, such as about 30 mg, such as about 50 mg, such as about 75 mg, such as about 100 mg, such as about 225 mg; and suitable upper limits are about 750 mg, such as about 500 mg, such as about 450 mg, such as about 350 mg, such as about 300 mg, such as about 225 mg, such as about 150 mg, such as about 80 mg, such as about 40 mg; (2) for repirinast, suitable lower limits of daily dose ranges are about 15 mg, such as about 25 mg, such as about 50 mg, such as about 75 mg, such as about 100 mg, such as about 150 mg; and suitable upper limits are about 1 g, such as about 900 mg, such as about 600 mg, such as about 400 mg, such as about 350 mg, such as about 300 mg, such as about 150 mg, such as about 100 mg, such as about 50 mg;
(3) for amlexanox, suitable lower limits of daily dose ranges are about 5 mg, such as about 10 mg, such as about 20 mg, such as about 30 mg, such as about 35 mg, such as about 50 mg, such as about 75 mg; and suitable upper limits are about 500 mg, such as about 300 mg, such as about 275 mg, such as about 250 mg, such as about 200 mg, such as about 180 mg, such as about 150 mg, such as about 100 mg, such as about 60 mg, such as about 30 mg;
(4) for traήilast, suitable lower limits of daily dose ranges are about 25 mg, such as about 50 mg, such as about 75 mg, such as about 100 mg, such as about 150 mg, such as about 200 mg, such as about 300 mg; and
suitable upper limits are to about 1 g, such as about 900 mg, such as about 800 mg, such as about 700 mg, such as about 600 mg, such as about 500 mg, such as about 250 mg, such as about 100 mg, such as about 50 mg; (5) for ketotifen, suitable lower limits of daily dose ranges are about 0.05 mg, such as about 0.1 mg, such as about 0.2 mg, such as about 0.3 mg, such as about 0.4 mg, such as about 0.5 mg; and suitable upper limits are about 5 mg, such as about 4 mg, such as about 3.5 mg, such as about 3 mg, such as about 2.5 mg, such as about 2.0 mg, such as about 1.0 mg, such as about 0.6 mg, such as about 0.3 mg; and
(6) for suplatast, suitable lower limits of daily dose ranges are about 0.5 mg, such as about 2 mg, such as about 20 mg, such as about 200 mg, such as about 300 mg; and suitable upper limits are about 1000 mg, such as about 800 mg, such as about 600 mg, such as about 450 mg, such as about 300 mg, such as about 200 mg, such as about 150 mg, such as about 100 mg, such as about 50 mg, per day, irrespective of whether the formulation employed is a combined preparation or a kit of parts as hereinbefore described.
Suitable doses of pemirolast are known to those skilled in the art. For example suitable lower limits of daily dose ranges are about 1 mg, such as about 2 mg, for example about 5 mg, such as about 10 mg, and more preferably about 20 mg; and suitable upper limits of daily dose ranges are about 200 mg, for example about 100 mg, such as about 80 mg, and more preferably about 60 mg. Daily peroral doses may thus be between about 2 mg and about 50 mg, such as about 5 mg and about 40 mg, and preferably about 10 mg and about 30 mg. Suitable individual doses may be about 40 mg, or about 20 mg (such as about 10 mg, more preferably about 6 mg (e.g. about 3 mg), per day. These doses/dose ranges are all irrespective of whether the formulation employed is a combined preparation or a kit of parts as hereinbefore described.
Similarly, suitable doses of thromboxane A2 antagonists are known to those skilled in the art. For example, peroral doses may be in the range of about 1 mg to about 2 g, such as about 2 mg to about 1.75 g, per day, irrespective of whether
the formulation employed is a combined preparation or a kit of parts as hereinbefore described.
In this respect, suitable peroral doses that may be mentioned are as follows: (I) for ozagrel, suitable lower limits of daily dose ranges are about 30, such as about 50 mg, such as about 100 mg, such as about 125 mg, such as about 175 mg, such as about 200 mg; and suitable upper limits are about 1 g, such as about 800 mg, such as about 750 mg, such as about 700 mg, such as about 600 mg, such as about 300 mg, such as about 100 mg, such as about 60 mg;
(II) for picotamide, suitable lower limits of daily dose ranges are about 100 mg, such as about 200 mg, such as about 300 mg, such as about 400 mg, such as about 500 mg, such as about 600 mg; and suitable upper limits are about 1.75 g, such as about 1.5 g, such as about 1.4 g, such as about 1.3 g, such as about 1.2 g, such as about 750 mg, such as about 500 mg, such as about 250 mg;
(III) for terutroban and egualen, suitable lower limits of daily dose ranges are about 1 mg, such as about 2 mg, such as about 3 mg, such as about 5 mg, such as about 8 mg, such as about 10 mg; and suitable upper limits are about 100 mg, such as about 75 mg, such as about 50 mg, such as about 40 mg, such as 30 mg, such as about 20 mg, such as about 12 mg, such as about 5 mg; and
(IV) for aspirin/acetylsalicylic acid, suitable lower limits of daily dose ranges are about 30 mg, such as about 50 mg, such as about 75 mg; and suitable upper limits are about 400 mg; such as about 325 mg, such as about 200 mg, such as about 120 mg, such as about 60 mg, per day, irrespective of whether the formulation employed is a combined preparation or a kit of parts as hereinbefore described. In the case of aspirin/acetylsalicylic acid, the dosage will comprise that which the medical practitioner, or other skilled person, will recognize as so-called "low dose" aspirin, i.e. not the higher doses that are normally prescribed to treat conditions such as pain.
In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an
individual patient. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Wherever the word "about" is employed herein, for example in the context of doses of active ingredients, it will be appreciated that such variables are approximate and as such may vary by ± 10%, for example ± 5% and preferably ± 2% (e.g. ± 1 %) from the numbers specified herein.
Further combination products that may be mentioned include those in which mast cell inhibitors as hereinbefore defined are combined, in accordance with the general and specific teaching described herein, with certain platelet aggregation inhibiting drugs, such as nitric oxide-donating derivatives of aspirin/acetylsalicylic acid (e.g. NCX-4016, NicOx S.A.) or, more preferably, anagrelide, argatroban, beraprost, cangrelor, cilostazol, dipyridamole, limaprost, parogrelil, procainamide, sarpogrelate (e.g. sarpogrelate hydrochloride), ticlopidine, tirofiban and triflusal, as well as those defined in the literature by way of following developmental drug codes: DA-697b (see international patent application WO 2007/032498; Daiichi Seiyaku Co Ltd), DG-041 (deCODE Genetics Inc), K-134 (CAS RN 189362-06-9), PL-2200 (CAS RN 50-78-2), PRT-60128 (Portola Pharmaceuticals Inc), SH-529 (an iloprost/beta-cyclodextrin clathrate; Bayer Schering Pharma AG) and YY-280 (a combination therapy of ticlopidine and EGb-761 (tanamin; a Ginkgo biloba extract; Yuyu Inc.)). For the avoidance of doubt, preferred features of combination products according to the invention mentioned hereinbefore or hereinafter may be imported into/applied to any one of the above-mentioned mast cell inhibitor/platelet aggregation inhibiting drug combinations.
For the avoidance of doubt, the following specific combinations are disclosed herein: (1 ) those in which the mast cell inhibitor (a) is acitazanolast (an active metabolite of tazanolast), or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in the following list: anagrelide, argatroban, aspirin/acetylsalicylic acid, beraprost, cangrelor, cilostazol, DA-697b, DG-041 , dipyridamole, egualen, K-134, limaprost, ozagrel, parogrelil,
picotamide, PL-2200, PRT-60128, procainamide, sarpogreiate (e.g. sarpogrelate hydrochloride), SH-529, terutroban, ticlopidine, tirofiban, YY-280, a nitric oxide-donating derivative of aspirin/acetylsalicylic acid (e.g. NCX-4016), or a pharmaceutically-acceptable salt or solvate of any of these compounds (which list is referred to hereinafter as List A);
(2) those in which the mast cell inhibitor (a) is acrivastine, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A; (3) those in which the mast cell inhibitor (a) is andolast, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(4) those in which the mast cell inhibitor (a) is amlexanox, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(5) those in which the mast cell inhibitor (a) is astemizole, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in
List A;
(6) those in which the mast cell inhibitor (a) is azelastine, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(7) those in which the mast cell inhibitor (a) is carebastine, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A; (8) those in which the mast cell inhibitor (a) is cetirizine, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(9) those in which the mast cell inhibitor (a) is cromoglycate, or a pharmaceutically-acceptable salt (e.g. disodium cromoglycate) or
solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(10) those in which the mast cell inhibitor (a) is desloratadine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in
List A;
(1 1 ) those in which the mast cell inhibitor (a) is ebastine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(12) those in which the mast cell inhibitor (a) is fexofenadine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A; (13) those in which the mast cell inhibitor (a) is ketotifen, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(14) those in which the mast cell inhibitor (a) is levocabastine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(15) those in which the mast cell inhibitor (a) is levocetirizine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in
List A;
(16) those in which the mast cell inhibitor (a) is loratadine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(17) those in which the mast cell inhibitor (a) is mizolastine, or a pharmaceuticaliy-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(18) those in which the mast cell inhibitor (a) is MY-1250, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A; (19) those in which the mast cell inhibitor (a) is nedocromil, or a pharmaceutically-acceptable salt (e.g. nedocromil sodium) or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(20) those in which the mast cell inhibitor (a) is norastemizole, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(21 ) those in which the mast cell inhibitor (a) is olopatadine, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in
List A;
(22) those in which the mast cell inhibitor (a) is oxatomide, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(23) those in which the mast cell inhibitor (a) is permirolast, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A; (24) those in which the mast cell inhibitor (a) is repirinast, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(25) those in which the mast cell inhibitor (a) is suplatast, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A;
(26) those in which the mast cell inhibitor (a) is tazanolast, or a pharmaceutically-acceptable salt or solvate thereof, and the
thromboxane A2 antagonist (b) is selected from any one compound in List A;
(27) those in which the mast cell inhibitor (a) is terfenadine, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in
List A; and
(28) those in which the mast cell inhibitor (a) is tranilast, or a pharmaceutically-acceptable salt or solvate thereof, and the thromboxane A2 antagonist (b) is selected from any one compound in List A.
For the avoidance of doubt, preferred features of combination products according to the invention mentioned hereinbefore or hereinafter may be imported into/applied to any one of the above-mentioned mast cell inhibitor/platelet aggregation inhibiting drug combinations (1 ) to (28).
The combination products/methods described herein may have the advantage that, in the treatment of the conditions mentioned hereinbefore, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have a broader range of activity than, be more potent than, produce fewer side effects than, or that it/they may have other useful pharmacological properties over, similar methods (treatments) known in the prior art for use in the treatment of inflammatory disorders (such as atherosclerosis and associated cardiovascular conditions) or otherwise.
The invention is illustrated by the following examples.
Examples
Example 1 MonoMac-6 Cell Inflammatory Mediator Release Assays
MonoMac-6 (MM6) cells (Ziegler-Heitbrock et al, Int. J. Cancer, 41 , 456 (1988)) are cultured (37°C/5% CO2) in RPMI-1640 medium supplemented with 1 mM sodium pyruvate, ^nonessential amino acids, 1-100 μg/mL insulin, 1 mM oxalacetic acid, 100 units/mL penicillin, 100 μg/mL streptomycin and 10% (v/v)
fetal bovine serum. For differentiation, TGFβ (2 ng/ml) and 1 ,25(OH)2D3 (50 nM) are added, generally for about 2-4 days.
To stimulate release of the inflammatory mediator leukotriene B4 (LTB4), differentiated or undifferentiated MM6 cells (at 1-15*106/mL; 0.5-1 mL) are incubated for 5-30 minutes (at 370C in PBS with calcium) with 25-50 μM arachidonic acid and 2-10 μM calcium ionophore A23187 (A23187 may also be used without arachidonic acid). The MM6 cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), and/or the thromboxane analogue U-46619, with or without A23187 and/or arachidonic acid as above. The MM6 incubations/stimulations above may also be performed in the presence of human platelets (from healthy donor blood) with an MM6:platelet ratio of 1 :10 to 1 :10000. The incubations/stimulations are stopped with two volumes of cold methanol and prostaglandin B2 (PGB2) added as internal standard. The samples are centrifuged and the supematants are diluted with water to reach a final methanol concentration of 30% and pH is adjusted to 3-4. Arachidonic acid metabolites in the supernatant are extracted on preconditioned (1 mL methanol followed by 1 mL H2O) C18 solid phase columns (Sorbent Technology, U.K.). Metabolites are eluted with methanol, whereafter one volume of water is added to the eluate. For reverse phase HPLC, 76 μL of each sample is mixed with 39 μL H2O (other volume ratios may also be used). A Waters RCM 8*10 column is eluted with methanol/acetonitrile/H2O/acetic acid (30:35:35:0.01 v/v) at 1.2 mL/min. The absorbance of the eluate is monitored at 270 nm for detection and quantitation of PGB2 and LTB4. Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring LTB4 may also be used according to instructions from the kit manufacturers). Using commercially available enzyme immuno-assay kits (EIA/ELISA kits) according to instructions from the manufacturer(s), supematants from the MM6 incubations/stimulations above may also be analysed with regard to content of the inflammatory mediators prostaglandin E2 (PGE2) and/or thromboxane B2 (TXB2).
Stock solutions of mast cell inhibitors (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) and thromboxane A2 antagonists (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen) are
prepared in ethanol, DMSO, N-methyl-2-pyrrolidone, PEG 400, propylene glycol and/or deionized water or physiological saline solution, with sonication, warming and adjustment of pH as needed (other vehicles may also be used). Cells are incubated (at 37°C/5% CO2 in PBS without calcium or in RPMI-1640 with 1-10% fetal bovine serum, with or without supplements) with test drug(s) (mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours prior to MM6 stimulation for inflammatory mediator release (test drug(s) may also be added simultaneously with MM6 stimulation). The drugs are added to reach final concentrations of 1 nM to 100 μM (for comparison, some experiments are performed without the drugs).
To stimulate release of inflammatory cytokines and chemokines such as IL-1 β, IL- 6, TNF, IL-8, IL-10, IL-12p70, MCP-1, differentiated or undifferentiated MM6 cells (at 1-10*106/mL) are incubated (37°C/5% CO2) for 4-24 hours (in RPMI-1640 with 1-10% fetal bovine serum, with or without supplements) with lipopolysaccharide (LPS, final concentration 1-100 ng/mL), phorbol-12-myristate- 13-acetate (PMA, final concentration 1-100 ng/mL) or an LPS/PMA mixture. The MM6 cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), arachidonic acid, calcium ionophore A23187 and/or the thromboxane analogue U-46619, with or without PMA and/or LPS as above. The MM6 cell incubations/stimulations may also be performed in the presence of human platelets (from healthy donor blood) with an MM6:platelet ratio of 1:10 to 1 :10000. Cells are incubated (at 37°C/5% CO2 in RPMI-1640 with 1-10% fetal bovine serum, with or without supplements) with test drug(s) (mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone; as above regarding stock solutions and concentrations) for 1 minute to 24 hours prior to MM6 stimulation (for comparison, some experiments are performed without the drugs; test drug(s) may also be added simultaneously with MM6 stimulation). After spinning down the cells after the incubations/stimulations, human cytokine and chemokine concentrations in the supernatants are quantitated using a Cytometric Bead Array (BD Biosciences Pharmingen, San Diego, USA) according to the manufacturer's instructions. Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring cytokines and chemokines may also be used
according to instructions from the manufacturers). The cell pellets are stored frozen (-800C) in RLT buffer (QIAGEN1 Valencia, CA) until further processing for microarray experiments (see Example 12 below).
Example 2
Human Peripheral Blood Cell Inflammatory Mediator Release Assays
Human peripheral blood mononuclear cells (PBMC) or polymorphonuclear cells (PMN) are isolated by Lymphoprep or Ficoll-Paque separation (with or without Polymorphoprep separation and/or Dextran sedimentation) from healthy donor blood using established protocols.
To stimulate release of the inflammatory mediator leukotriene B4 (LTB4), PBMC or PMN (at 1-15χ106/mL; 0.5-1 mL) are incubated for 5-30 minutes (at 37°C in PBS with calcium) with 25-50 μM arachidonic acid and 2-10 μM calcium ionophore A23187 (A23187 may also be used without arachidonic acid). The PBMC/PMN may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), and/or the thromboxane analogue U-46619, with or without A23187 and/or arachidonic acid as above. The PBMC/PMN incubations/stimulations above may also be performed in the presence of human platelets (from healthy donor blood) with a PBMC/PMN:platelet ratio of 1 :10 to 1 :10000. The incubations/stimulations are stopped with two volumes of cold methanol and prostaglandin B2 added is as internal standard. The samples are centrifuged and the supernatants are diluted with water to reach a final methanol concentration of 30% and pH is adjusted to 3-4. Arachidonic acid metabolites in the supernatant are extracted on preconditioned (1 mL methanol followed by 1 mL H2O) C18 solid phase columns (Sorbent Technology, U.K.). Metabolites are eluted with methanol, after which one volume of water is added to the eluate. For reverse phase HPLC, 76 μL of each sample is mixed with 39 μL H2O (other volume ratios may also be used). A Waters RCM 8x10 column is eluted with methanol/acetonitrile/H2O/acetic acid 30:35:35:0.01 v/v) at 1.2 mL/minute. The absorbance of the eluate is monitored at 270 nm for detection and quantitation of PGB2 and LTB4. Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring LTB4 may also be used according to instructions from the manufacturer(s). Using commercially available enzyme immuno-assay kits
(EIA/ELISA kits) according to instructions from the manufacturers), supernatants from the PBMC/PMN incubations/stimulations above may also be analysed with regard to content of the inflammatory mediators prostaglandin E2 (PGE2) and/or thromboxane B2 (TXB2). Cells are incubated (at 370C in PBS without calcium or in RPMI-1640 with 0-10% fetal bovine serum) with test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours prior to PBMC/PMN stimulation for inflammatory mediator release (see Example 1 above for details regarding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with PBMC/PMN stimulation). For comparison, some experiments are performed without the drugs.
To stimulate release of inflammatory cytokines and chemokines such as IL-1 β, IL- 6, TNF, IL-8, IL-10, IL-12p70, MCP-1 , PBMC/PMN (at 1-10*106AnL) are incubated (37°C/5% CO2) for 4-24 hours (in RPMI-1640 with 1-10% fetal bovine serum) with lipopolysaccharide (LPS, final concentration 1-100 ng/mL), phorbol- 12-myristate-13-acetate (PMA1 final concentration 1-100 ng/mL) or an LPS/PMA mixture. The PBMC/PMN cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), arachidonic acid, calcium ionophore A23187 and/or the thromboxane analogue U-46619, with or without PMA and/or LPS as above. The PBMC/PMN incubations/stimulations may also be performed in the presence of human platelets (from healthy donor blood) with a PBMC/PMN:platelet ratio of 1 :10 to 1 :10000. Cells are incubated (at 37°C/5% CO2 in RPMI-1640 with 1-10% fetal bovine serum) with test drug(s) (mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone, as above) for 1 minute to 24 hours prior to PBMC/PMN stimulation for cytokine/chemokine release (for comparison, some experiments are performed without the drugs; test drug(s) may also be added simultaneously with PBMC/PMN stimulation). After spinning down the cells after the incubations/stimulations, human cytokine and chemokine concentrations in the supernatants are quantitated using a Cytometric Bead Array (BD Biosciences Pharmingen, San Diego, USA) according to the manufacturer's
instructions. Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring cytokines and chemokines may also be used according to instructions from the manufacturer(s). The cell pellets are stored frozen (-800C) in RLT buffer (QIAGEN, Valencia, CA) until further processing for microarray experiments (see Example 12 below).
Example 3
Mouse Mast Cell Inflammatory Mediator Release Assays
Bone marrow-derived cultured mouse mast cells (mMCs) are obtained by culturing bone marrow cells from C57BL/6 mice. The bone marrow cells (from mouse femurs flushed with PBS) are cultured (37°C/5% CO2) in 10% WEHI-3 or X-63 enriched conditioned RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum, 4 mM L-glutamine, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM Hepes, and 100 μg/mL penicillin/streptomycin. Development of mast cells (which grow in suspension) is confirmed by expression of Kit (by flow-cytometry) on the cell surface and/or by toluidine blue staining (generally after at least 3-5 weeks of culture).
Bone marrow-derived cultured mouse mast cells of connective tissue type (CT- type) are obtained by culturing bone marrow cells from C57BL/6 mice. The bone marrow cells are cultured (37°C/5% CO2) in RPMI-1640 medium containing 10% filtered FCS, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/mL penicillin G, 100 μg/mL streptomycin, 0.1 mM MEM non-essential amino acids and 50 μM 2- ME, supplemented with 50 ng/mL recombinant murine stem cell factor and 1 ng/mL murine recombinant IL-4. Mast cell development is confirmed by expression of Kit (by flow-cytometry) on the cell surface and/or by toluidine blue staining (generally after at least 3-5 weeks of culture).
Mouse mast cell lines MC/9 (obtained from ATCC, Product no CRL-8306) and C1.MC/C57.1 (Young et a/., Proc. Natl. Acad. ScL USA, 84, 9175 (1987)) may also be used. The MC/9 cells are cultured according to instructions from ATCC (https://www.atcc.org), and C1.MC/C57.1 cells are cultured as described in Rumsaeng et al {J. Immunol. 158, 1353 (1997)).
For activation/stimulation through cross-linking of the IgE-receptor, the cultured mast cells are initially sensitized for 90 minutes at 37CC (5% CO2) with a monoclonal mouse anti-TNP IgE-antibody (lgEI-b4, ATCC, Rockville, MD, USA), used as a 15% hybridoma supernatant. Cells to be used in the N-acetyl-beta-D- hexosaminidase (or histamine) or cytokine/chemokine release assays (see below) are then subjected to two washings with PBS and re-suspended in RPMI- 1640 medium supplemented with 0.2% bovine serum albumin (BSA) (Sigma) before the cells (at 0.5-10x106/mL) are activated by addition of 100 ng/mL TNP- BSA (Biosearch Technologies, San Francisco, CA) with a coupling ratio of 9/1. The incubation (37°C/5% CO2) with TNP-BSA is 30 minutes for the analysis of beta-hexosaminidase (or histamine) release and 4-24 hours for analysis of cytokine and chemokine release. Cells are incubated (37°C/5% CO2) with test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours prior to addition of TNP-BSA (see Example 1 above for detail regarding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with TNP-BSA stimulation). For comparison, some experiments are performed without the drugs. After the incubations/stimulations, the samples are centrifuged and the supematants analysed with regard to content of beta- hexosaminidase (or histamine) and/or cytokines/chemokines as described below. The cell pellets are stored frozen (-8O0C) in RLT buffer (QIAGEN, Valencia, CA) until further processing for microarray experiments (see Example 12 below).
For detection of IgE-dependent release of the granular mast cell enzyme beta- hexosaminidase, an enzymatic colourimetric assay is used. 60 μL from each well supernatant is transferred to a 96 well plate and mixed with an equal volume of substrate solution (7.5 mM p-nitrophenyl-N-acetyl-b-D-glucosaminide dissolved in 80 mM citric acid, pH 4.5). The mixture is incubated on a rocker platform for 2 hours at 37°C. After incubation, 120 μL of glycine (0.2 M, pH 10.7) is added to each well and the absorbance at 405 and 490 nm is measured using an Emax Precision Microplate Reader (Molecular Devices, Sunnyvale, CA). Release of
beta-hexosaminidase is expressed as a percentage of total beta-hexosaminidase determined after cell lysis. For detection of IgE-dependent release of granular mast cell histamine, commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring histamine is used according to instructions from the manufacturer(s).
For detection of IgE-dependent release of mouse mast cell cytokines and chemokines such as IL-6, IL-4, TNF, IL-1 β, KC1 MCP-1 , IL-10, IL-12p70, IFNγ, a Cytometric Bead Array (BD Biosciences Pharmingen, San Diego, USA) is used according to the manufacturer's instructions. Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring cytokines and chemokines may also be used according to instructions from the manufacturer(s).
In addition to the mast cell experiments above, mast cell-inhibiting effects of the test drug(s) (as above) may also be studied using well established and documented experimental approaches and assays for analysing induced (with e.g. anti-lgE (with or without pretreatment of the cells with rat or mouse IgE), concanavalin A, protein L, compound 48/80, ionophore A23187, PMA) release of histamine, beta-hexosaminidase or tryptase from freshly isolated peritoneal rat or mouse mast cells.
Example 4
RAW 264.7 Cell Inflammatory Mediator Release Assays
RAW 264.7 cells are cultured (37°C/5% CO2) in DMEM, supplemented with 100 units /mL penicillin, and 100 μg/mL streptomycin and 10% fetal bovine serum.
To stimulate release of inflammatory cytokines and chemokines such as IL-6, TNF, IL-1 β, KC, MCP-1 , IL-10, IL-12p70, IFNγ, RAW 264.7 cells (at 1-10*106/mL) are incubated (37°C/5% CO2) for 4-24 hours (in DMEM with 1-10% fetal bovine serum, with or without supplements) with lipopolysaccharide (LPS1 final concentration 1-100 ng/mL), phorbol-12-myristate-13-acetate (PMA, final concentration 1-100 ng/mL) or an LPS/PMA mixture. The RAW 264.7 cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), arachidonic acid, calcium ionophore A23187
and/or the thromboxane analogue U-46619, with or without PMA and/or LPS as above. The RAW 264.7 incubations/stimulations may also be performed in the presence of mouse or human (from healthy donor blood) platelets with a RAW 264.7:platelet ratio of 1 :10 to 1 :10000. Cells are incubated (at 37°C/5% CO2 in DMEM with 1-10% fetal bovine serum, with or without supplements) with test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours prior to RAW 264.7 stimulation for cytokine/chemokine release (see Example 1 above for details regarding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with RAW 264.7 stimulation). For comparison, some experiments are performed without the drugs. After spinning down the cells after the incubations/stimulations, mouse cytokine and chemokine concentrations in the supematants are quantitated using a Cytometric Bead Array (BD Biosciences Pharmingen, San Diego, USA) according to the manufacturer's instructions. Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring cytokines and chemokines may also be used according to instructions from the manufacturer(s). The cell pellets are stored frozen (-8O0C) in RLT buffer (QIAGEN, Valencia, CA) until further processing for microarray experiments (see Example 12 below).
Example 5 Rat Paw Inflammation Induced by Carrageenan
This assay is essentially according to that described by Winter et al (Proc. Soc. Exp. Biol. Med., 111, 544 (1962)). Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to male Sprague- Dawley or Wistar rats weighing approximately 150-400 g (for comparison, some
experiments are performed without the drugs). Prior to administration, stock solutions of drugs (see Example 1 above) are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used. 1 minute to 24 hours after the first drug dose, a 0.5, 1.0 or 2.0% solution of carrageenan (Type IV Lambda, Sigma Chemical Co.) in 0.9% saline is injected into the subplantar region of one hind paw of anaesthetised rats. Before, and at indicated intervals 3-24 hours after carrageenan injection, the volume of the injected paw is measured with a displacement plethysmometer connected to a pressure transducer with a digital indicator. The degree of swelling indicates the degree of inflammatory edema. 3- 24 hours after carrageenan injection, the rats are sacrificed and perfused with saline or PBS (other perfusion media may also be used). Plantar soft tissue biopsies from the inflamed paws are collected, weighed, stored frozen (samples for microarray analysis are frozen at -8O0C in TRlzol, Invitrogen, Carlsbad, CA), and, as described below (Example 10 and 12), subsequently analyzed with regard to 1 ) myeloperoxidase (MPO) accumulation, reflecting inflammatory neutrophil leukocyte accumulation; and/or 2) tissue gene expression using microarray technology. Non-inflamed paw tissue from untreated rats provides base-line levels of MPO and gene expression. Tissue inflammation may also be studied using conventional histological and immunohistochemical techniques. Paw inflammation may also be induced by subplantar injection of compound 48/80 (48/80, 1-5 μg in 50-100 μl PBS or saline) (instead of carrageenan), followed by measurement of inflammatory paw swelling and collection of tissue biopsies for microarray and/or MPO analysis (as above) 30 min to 8 hours after 48/80 injection.
Example 6
Mouse Ear Inflammation Induced by Croton Oil
This assay is essentially according to that described by Tonelli et al (Endocrinology 77, 625 (1965)) (other strains of mice may also be used). Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell
inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours (for comparison, some experiments are performed without the drugs). Prior to administration, stock solutions of drugs (see Example 1 above) are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used. 1 minute to 24 hours after the first drug dose, 10-30 μl_ of a 2.0 or 4.0% solution of croton oil in acetone or ethanol is applied topically to one or both ears. At indicated intervals 4-12 hours after croton oil application, the animals are sacrificed, and punch biopsies of the ears are weighed to determine the inflammatory swelling of the ears (ear thickness may also be measured to determine the swelling). The biopsies from the inflamed ears are collected, stored frozen (samples for microarray analysis are frozen at -800C in TRIzol), and, as described below (Example 10 and 12), subsequently analyzed with regard to 1 ) myeloperoxidase (MPO) accumulation, reflecting inflammatory neutrophil leukocyte accumulation; and/or 2) tissue gene expression using microarray technology. Non-inflamed ear biopsies from untreated mice provide base-line levels of swelling, MPO and gene expression. Tissue inflammation may also be studied using conventional histological and immunohistochemical techniques.
Example 7
Mouse Ear Inflammation Induced by Phorbol Ester or Arachidonic Acid
These assays are essentially according to those described by Chang et al (Eur. J. Pharmacol. 142, 197 (1987)) (although other strains of mice may also be used). Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to male or female mice (for comparison, some experiments are performed without the drugs). Prior to administration, stock solutions of drugs (see Example 1 above) are diluted as
needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used. 1 minute to 24 hours after the first drug dose, 1-10 μg of phorbol 12-myristate 13-acetate (PMA), tetradecanoyl phorbol acetate (TPA), or 1-5 mg arachidonic acid in 10-30 μl acetone or ethanol is applied topically to one or both ears. 4-12 hours after PMA or TPA application, and 30 min to 6 hours after arachidonic acid application, the animals are sacrificed, and punch biopsies of the ears are weighed to determine the inflammatory swelling of the ears (ear thickness may also be measured to determine the swelling). The biopsies from the inflamed ears are collected, stored frozen (samples for microarray analysis are frozen at -800C in TRIzol), and, as described below (Example 10 and 12), subsequently analyzed with regard to 1 ) myeloperoxidase (MPO) accumulation, reflecting inflammatory neutrophil leukocyte accumulation; and/or 2) tissue gene expression using microarray technology. Non-inflamed ear biopsies from untreated mice provide base-line levels of swelling, MPO and gene expression. Tissue inflammation may also be studied using conventional histological and immunohistochemical techniques.
Example 8 Acute Tissue Reaction and Inflammation in Response to Injury in Mouse and Rat
Male CBA or NMRI mice weighing approximately 15-30 g, or male Wistar or Sprague-Dawley rats weighing approximately 150-450 g, are used (other strains of mice and rats may also be used). Acute tissue injury and acute inflammation is achieved in the distal part of the tail or one of the ears using a scalpel under aseptic conditions. One, two or three parallel, approximately 5-15 mm long, longitudinal cuts are made through all layers of the skin. Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneal^ or orally every 2-24 hours, with the first dose given 1 minute to 24 hours before tissue injury (for comparison,
some experiments are performed without the drugs). Prior to administration, stock solutions of drugs (see Example 1 above) are diluted as needed in e.g. 0.5% or 1% methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used. 2-48 hours after injury, the animals are killed and the injured segments of the tissues are removed, weighed and stored frozen (samples for microarray analysis are frozen at -80°C in TRIzol), and, as described below (Example 10 and 12), subsequently analyzed with regard to 1 ) myeloperoxidase (MPO) accumulation, reflecting inflammatory neutrophil leukocyte accumulation; and/or 2) tissue gene expression using microarray technology. Corresponding non-injured/non-inflamed tissues from untreated animals provide base-line levels of MPO and gene expression. Tissue reactions and inflammation in response to injury may also be studied using conventional histological and immunohistochemical techniques.
Example 9
Acute Tissue Reaction and Inflammation in Response to Injury in Rat
Male Sprague-Dawley rats weighing 350-500 g are used (although other strains of rats may also be used). Animals are anesthetized with lsoflurane in oxygen and acute tissue injury and acute inflammation is achieved in the left common carotid artery as follows: After surgical exposure of the left common, external and internal carotid arteries and temporary cessation of local blood flow with temporary ligatures, a balloon catheter (2-French Fogarty) is passed through the external carotid into the aorta. Next, the balloon is inflated with sufficient water to distend the common carotid artery and then pulled back to the external carotid. This procedure is repeated three times, and then the catheter is removed, the external carotid ligated and the wound closed. Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneal^ or orally every 2-24 hours, with the first dose given 1 minute to 24 hours before tissue injury (for comparison, some experiments are performed without the drugs). Prior to administration, stock
solutions of drugs (see Example 1 above) are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used. 2-48 hours after injury, the animals are anesthetized with lsoflurane in oxygen and their left carotid arteries exposed. Clamps are put on the very proximal part of the common and internal carotid arteries, respectively, and then the vessel between the clamps is gently flushed with sterile saline and/or TRIzol, removed, weighed and stored frozen (samples for microarray analysis are frozen at -800C in TRIzol), and, as described below (Example 10 and 12), subsequently analyzed with regard to 1 ) myeloperoxidase (MPO) accumulation, reflecting inflammatory neutrophil leukocyte accumulation; and/or 2) tissue gene expression using microarray technology. Corresponding non-injured/inflamed vessels from untreated rats provide base-line levels of MPO and gene expression. Tissue reactions and inflammation in response to injury may also be studied using conventional histological and immunohistochemical techniques.
Example 10
Inflammatory Accumulation of Tissue Myeloperoxidase
The enzyme myeloperoxidase (MPO) is abundant in neutrophil leukocytes and is often used as a marker for the detection of neutrophil accumulation in inflamed tissue. To determine inflammatory myeloperoxidase accumulation in inflamed mouse and rat tissues (as described in Example 5-9 above), the tissues are homogenised in 0.5% hexadecyltrimethyl-ammonium bromide, and freeze- thawed. The MPO activity of the supernatant is determined spectrophotometrically as the change in absorbance at 650 nm (250C) occurring in the redox reaction of H2O2-tetramethylbenzidine catalysed by MPO. Values are expressed as MPO units/mg tissue.
Example 11
Smooth Muscle Cell Assays
Rat aortic smooth muscle cells (RASMCs) are isolated as previously described
(Hedin et al, Arterioscler. Thromb. Vase. Biol., 17, 1977 (1997)). Cells are cultured (37°C/5% CO2) in Ham's medium F-12 supplemented with 10% fetal
bovine serum, 50 μg/mL L-ascorbic acid, 50 μg/mL streptomycin, 50 ILJ/mL penicillin (F-12/10% fetal bovine serum), grown to confluence, serially passaged by trypsinization, and used in experiments after 2-6 passages. RASMCs are seeded in 24-well plates at a density of approximately 4x104 cells per well in F- 12/10% fetal bovine serum (plates with larger numbers of wells per plate and appropriate lower numbers of cells per well may also be used). After 24 hours, the cells are synchronized in G0/G1 phase by starvation in Ham's medium F-12 supplemented with 0.1 % bovine serum albumin (BSA), 50 μg/mL L-ascorbic acid, 50 μg/mL streptomycin and 50 IU/mL penicillin (F-12/0.1% BSA) for 24-48 hours. To estimate DNA synthesis, starved RASMCs are stimulated with either 10 ng/ml IGF-1 or 10% fetal bovine serum for 12-48 hours (other well established mitogens such as PDGF may also be used). Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) are added 1 minute to 24 hours prior to stimulation (see Example 1 above for details re'garding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with stimulation). For comparison, some experiments are performed without the drugs. The cells are labelled with 1 μCi [3H]-thymidine for 8 hours before the end of the stimulation period. The plates are then washed with ice-cold PBS, incubated overnight with ice-cold 10% (w/v) trichloroacetic acid, lysed in 0.2 M sodium hydroxide, and radioactivity is measured in a liquid scintillation counter. The stimulated RASMC proliferation may also be analyzed using commercially available bromodeoxyuridine (BrdU) cell proliferation assays (for example Cell Proliferation ELISA, BrdU, from Roche Applied Science), the cell proliferation reagent WST-1 (Roche Diagnostics Scandinavia AB, Bromma, Sweden) (both according to the manufacturer's instructions), or by cell counting. In separate experiments (to study gene expression), larger numbers of starved RASMCs (1-5 x 106 cells per well) are stimulated with 10 ng/ml IGF-1 or 10% fetal bovine serum (or PDGF) as above, or with LPS (1-100 ng/mL), with 1-10% fetal bovine serum for 4-48 hours (all stimuli with and without test drug(s) as above). The cells are then collected and stored frozen (-800C) in RLT buffer (QIAGEN1 Valencia, CA) until further processing for microarray experiments (see Example 12 below).
Human bronchial smooth muscle cells (HBSMCs, Promocell, Heidelberg, Germany) are cultured in DMEM supplemented with 10% FBS1 100 units/mL penicillin, 100 μg/mL streptomycin, 0.12 IU/mL insulin, and with or without 2 μg/mL amphotericin B. Prior to the experiments, cells may be growth arrested for 24 hours in low-FBS (0.3-5%), insulin-free medium. To stimulate formation and release of inflammatory cytokines and chemokines such as IL-8 and eotaxin, HBSMCs (at 80% confluence, corresponding to approximately 8*105/25 cm2 flask) are incubated (37°C/5% CO2) for 24-48 hours (in DMEM with 1-10% fetal bovine serum, with or without supplements) with different combinations of IL-1β and TNF-α (both at 1-50 ng/mL). Cells are incubated (at 37°C/5% CO2 in DMEM with 0.3-10% fetal bovine serum, with or without supplements) with test drug(s) (mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone, as above) for 1 minute to 24 hours prior to HBSMC stimulation (see Example 1 above for details regarding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with HBSMC stimulation). For comparison, some experiments are performed without the drugs. After the incubations/stimulations, human cytokine and chemokine concentrations in the supernatants are quantitated using commercially available enzyme immuno-assay kits (EIA/ELISA kits) according to instructions from the manufacturers). The cells are then collected and stored frozen (-800C) in RLT buffer (QIAGEN1 Valencia, CA) until further processing for microarray experiments (see Example 12 below).
Example 12
Analysis of Gene Expression
Total RNA from mouse, rat and human tissues (see Example 5 to 9, 15, 16, 19 and 20) is isolated using TRIzol (Invitrogen, Carlsbad, CA) followed by RNeasy cleanup (QIAGEN, Valencia, CA) according to manufacturers' protocols. Total RNA from the cell incubations/stimulations described in the examples above and below (mouse mast cells, MonoMac-6, PBMC, PMN, RAW 264.7, RASMC, HBSMC, NB4, HL-60) is isolated using RNeasy Mini Kit (QIAGEN), with or without RNase-Free DNase set (QIAGEN), according to the manufacturer's protocol(s). Depending on the species from which the different tissues and cells
originate, microarray analysis is performed using GeneChip® Human Genome U133 Plus 2.0 Array, GeneChip® Mouse Genome 430 2.0 Array or GeneChip® Rat Genome 230 2.0 Array, or corresponding newer version of these chips (all arrays from Affymetrix, Santa Clara, CA) according to the manufacturer's protocols. The microarray expression data is analyzed using e.g. GeneChip Operating Software (Affymetrix) and Bioconductor/R (www.bioconductor.org). Other relevant software may also be used.
Gene expression from the different species may also be analyzed using Human Genome Survey Microarray V2.0, Mouse Genome Survey Microarray V2.0 or Rat
Genome Survey Microarray (or corresponding newer version of these arrays) according to protocols from the manufacturer Applied Biosystems (Foster City,
CA). These microarray expression data are analyzed using e.g. 1700
Chemiluminescent Microarray Analyzer (Applied Biosystems, Foster City, CA) supplied with an Oracle® database of annotations, GeneSpring 7.2 (Agilent
Technologies, Inc., Palo Alto, CA) and Microarray Suite version 5.0 software
(MAS 5.0, Affymetrix). Other relevant software may also be used.
Gene expression (mRNA levels) may also be analyzed using quantitative or semi-quantitative PCR. Analysis of gene expression at the protein level may be analyzed using commercially available enzyme immuno-assay kits (EIA/ELISA kits) (according to instructions from the manufacturers)), or conventional Western blot and/or immunohistochemical approaches.
Example 13
Cell Proliferation Assays
Proliferation of stimulated and unstimulated mouse mast cells, MonoMac-6 cells, RAW 264.7 cells, NB4 cells, HL-60 cells and HBSMC described in the examples above and below (with or without growth arrest for 24-48 hours in 0.1-5% fetal bovine serum prior to the addition of the respective test drugs and/or stimuli described in the Examples above and below for 24-72 hours) is measured using the cell proliferation reagent WST-1 (Roche Diagnostics Scandinavia AB, Bromma, Sweden) or commercially available bromodeoxyuridine (BrdU) cell proliferation assays (for example Cell Proliferation ELISA, BrdU, from Roche
Applied Science) according to the manufacturers' instructions. Other conventional tests of cell proliferation may also be used.
Example 14 Platelet Aggregation Tests
Aggregation of rabbit or human platelets (in platelet rich plasma or whole blood) induced by adenosine diphosphate (ADP), arachidonic acid, collagen or the thromboxane analogue U-46619 is analyzed using aggregometry, for example as described by Bertele et al {Eur. J. Pharmacol. 85, 331 (1982)). Induced (as above) platelet aggregation may also be analyzed using washed human or rabbit platelets and/or with other established aggregometry or other corresponding methods for measuring platelet aggregation. Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) are added 1-120 minutes prior to induction of platelet aggregation (see Example 1 above for details regarding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with induction of platelet aggregation). For comparison, some experiments are performed without the drugs.
Example 15 Mouse Peritoneal Inflammation-Induced by Zymosan and Other Stimuli
This assay is essentially according to Rao et al (J. Pharmacol. Exp. Ther. 269, 917 (1994)) (other strains of mice may also be used). Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to the animals (for comparison, some experiments are performed without the drugs).
Prior to administration, stock solutions of drugs (see Example 1 above) are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used. 1 minute to 24 hours after the first drug dose, 0.5-2 mg zymosan A (Sigma, cat. no. Z4250) in 0.5-1 mL sterile PBS (sonicated and well mixed) is injected intraperitoneally (instead of using zymosan A, peritoneal inflammation may also be induced by intraperitoneal injection of pro-inflammatory concentrations of other well established pro-inflammatory stimuli such as anti-mouse-lgE (with or without intraperitoneal pretreatment with mouse IgE for 1-3 days), concanavalin A, carrageenan, proteose peptone, LPS, PMA, thioglycolate, arachidonic acid, fMLP, TNF, IL-1 β. Test drug(s) may also be administered simultaneously with intraperitoneal injection of zymosan or the other pro-inflammatory stimuli). 2-24 hours after injection of zymosan (or one or more of the other pro-inflammatory stimuli), the animals are sacrificed. The peritoneal cavity is then flushed with 1-3 mL of a lavage buffer (ice-cold PBS with or without 3-5 mM EDTA or 5-10 units/mL heparin). Total and differential leukocyte counts in the lavage fluid are done with a hemocytometer following staining with Turk's solution and/or in cytospin preparations stained with May-Grunwald Giemsa or a modified Wright's (Diff-Quik) stain, respectively, by light microscopy using standard morphological criteria. Other established methods for determining total and differential leukocyte counts may also be used. The remaining lavage fluid is centrifuged (300-3000 x g, 4°C, 3-10 min), and cell-free lavage fluid supernatant is stored frozen (-200C to -80°) until analyzed for content of inflammatory mediators LTB4, PGE2, TXB2 and/or mouse cytokines/chemokines (e.g. IL-4, IL-6, TNF, IL-1 β, KC, MCP-1 , IL-10, IL-12p70, IFNγ) content as described in Example 1 and 4 above. The histamine content in the lavage fluid supernatant is determined by using commercially available histamine enzyme immuno-assay kits (EIA/ELISA kits) according to instructions from the manufacturers). Inflammatory peritoneal cell activation may also be studied by measuring beta-hexosaminidase activity in the lavage fluid using the beta-hexosaminidase assay described in Example 3. The cell pellets of the lavage fluid are resuspended in 0.1-1.0 mL 0.05 M KHPO4 pH 6.0 with 0.5% HTAB and stored frozen (-200C to -80°) until analysis of myeloperoxidase (MPO) content as described by Rao et al (J. Pharmacol. Exp. Ther. 269, 917-25 (1994)). Identical cell pellets from separate animals are stored frozen (-8O0C) in RLT buffer (QIAGEN, Valencia, CA) until further
processing for microarray experiments (see Example 12). At the time of flushing the peritoneal cavity with lavage buffer, tissue (peritoneal wall, intestines and/or other intra- or retroperitoneal organs/tissues) biopsies from the inflamed peritoneal cavity are collected, weighed, stored frozen (samples for microarray analysis are frozen at -800C in TRIzol, Invitrogen, Carlsbad, CA)1 and, as described in Example 12, subsequently analyzed with regard to tissue gene expression using microarray technology. Non-inflamed peritoneal cavities from untreated animals provide base-line levels of MPO, inflammatory mediators, cytokines/chemokines and gene expression. Tissue inflammation may also be studied using conventional histological and immunohistochemical techniques.
Example 16
Rat Peritoneal Inflammation-Induced by Zymosan and Other Stimuli
Male Wistar or Sprague Dawley rats weighing approximately 150-450 g are used. Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to the animals (for comparison, some experiments are performed without the drugs). Prior to administration, stock solutions of drugs (see Example 1 above) are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used. 1 minute to 24 hours after the first drug dose, 1-100 mg zymosan A (Sigma, cat. no. Z4250) in 1-10 mL sterile PBS (sonicated and well mixed) is injected intraperitoneally (instead of using zymosan A, peritoneal inflammation may also be induced by intraperitoneal injection of pro-inflammatory concentrations of other well established proinflammatory stimuli such as anti-rat-lgE (with or without intraperitoneal pretreatment with rat IgE for 1-3 days), concanavalin A, protein L, compound 48/80, carrageenan, proteose peptone, LPS, PMA, thioglycolate, arachidonic acid, fMLP, TNF, IL-1 β. Test drug(s) may also be administered simultaneously with intraperitoneal injection of zymosan or the other pro-inflammatory stimuli). 2-
24 hours after injection of zymosan (or one or more of the other stimuli), the animals are sacrificed. The peritoneal cavity is then flushed with 10-20 ml of a lavage buffer (e.g. ice-cold PBS with or without 3-5 mM EDTA or 5-10 units/mL heparin). Total and differential leukocyte counts in the lavage fluid are done with a hemocytometer following staining with Turk's solution and/or in cytospin preparations stained with May-Grunwald Giemsa or a modified Wright's (Diff- Quik) stain, respectively, by light microscopy using standard morphological criteria. Other established methods for determining total and differential leukocyte counts may also be used. The remaining lavage fluid is centrifuged (300-3000 x g, 40C, 3-10 min), and cell-free lavage fluid supernatant is stored frozen (-200C to -80°) until analyzed for content of the inflammatory mediators LTB4, PGE2, TXB2 and/or rat cytokines/chemokines (e.g. IL-4, IL-6, TNF, IL-1 β, KC1 MCP-1 , IL-10, IL-12p70, IFNγ) essentially as described in Example 1 and 4 above. The histamine content in the lavage fluid supernatant is determined by using commercially available histamine enzyme immuno-assay kits (EIA/ELISA kits) according to instructions from the manufacturers). Inflammatory peritoneal cell activation may also be studied by measuring beta-hexosaminidase activity in the lavage fluid using the beta-hexosaminidase assay described in Example 3. The cell pellets of the lavage fluid are resuspended in 0.1-1.0 ml_ 0.05 M KHPO4 pH 6.0 with 0.5% HTAB and stored frozen (-200C to -80°) until analysis of myeloperoxidase (MPO) content basically as described by Rao et al (J. Pharmacol. Exp. Ther., 269, 917-25 (1994)). Identical cell pellets from separate animals are stored frozen (-800C) in RLT buffer (QIAGEN, Valencia, CA) until further processing for microarray experiments (see Example 12). At the time of flushing the peritoneal cavity with lavage buffer, tissue (peritoneal wall, intestines and/or other intra- or retroperitoneal organs/tissues) biopsies from the inflamed peritoneal cavity are collected, weighed, stored frozen (samples for microarray analysis are frozen at -80°C in TRIzol, Invitrogen, Carlsbad, CA), and, as described in Example 12, subsequently analyzed with regard to tissue gene expression using microarray technology. Non-inflamed peritoneal cavities from untreated animals provide base-line levels of MPO, inflammatory mediators, cytokines/chemokines and gene expression. Tissue inflammation may also be studied using conventional histological and immunohistochemical techniques.
Example 17
NB4 and HL-60 Cell Inflammatory Mediator Release Assays
Human NB4 cells (Lanotte et al, Blood, 77, 1080 (1991 )) are cultured (37°C/5% CO2) in RPMI-1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin and 10% (v/v) fetal bovine serum. For differentiation, 1-5 μM all-frans-retinoic acid (ATRA) is added, generally every third day.
Human HL-60 cells (Steinhilber et al, Biochim. Biophys. Acta 1178, 1 (1993)) are cultured (37°C/5% CO2) in RPMI-1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin and 10-20% (v/v) fetal bovine serum. For differentiation ATRA (1-5 μM), DMSO (1-2%), PMA (100-500 ng/mL) or vitamin D3 (1-15 μM) is added for 5 days.
To stimulate formation and release of the inflammatory mediator leukotriene B4 (LTB4), differentiated or undifferentiated NB4 or HL-60 cells (at 1-15* 106AnL) are incubated for 5-30 minutes (at 37°C in PBS with calcium) with 10-40 μM arachidonic acid and/or 2-10 μM calcium ionophore A23187. The NB4 and HL-60 cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP), fMLP, and/or the thromboxane analogue U- 46619, with or without A23187 and/or arachidonic acid as above. The NB4 and HL-60 incubations/stimulations above may also be performed in the presence of human platelets (from healthy donor blood) with an NB4/HL-60:platelet ratio of 1 :10 to 1 :10000. The incubations/stimulations are stopped with 1 mL cold methanol and prostaglandin B2 (PGB2) added as internal standard. The samples are centrifuged and the supematants are diluted with water to reach a final methanol concentration of 30% and pH is adjusted to 3-4. Arachidonic acid metabolites in the supernatant are extracted on preconditioned (1 mL methanol followed by 1 mL H2O) C18 solid phase columns (Sorbent Technology, U.K.). Metabolites are eluted with methanol, whereafter one volume of water is added to the eluate. For reverse phase HPLC, 76 μL of each sample is mixed with 39 μL H2O (other volume ratios may also be used). A Waters RCM 8*10 column is eluted with methanol/acetonitrile/H20/acetic acid (30:35:35:0.01 v/v) at 1.2 mL/min. The absorbance of the eiuate is monitored at 270 nm for detection and quantitation of PGB2 and LTB4. Commercially available enzyme immuno-assay
kits (EIA/ELISA kits) for measuring LTB4 may also be used according to instructions from the kit manufacturers). Using commercially available enzyme immuno-assay kits (EIA/ELISA kits) according to instructions from the manufacturers), the supernatants from the NB4/HL-60 incubations/stimulations above may also be analysed with regard to content of the inflammatory mediators prostaglandin E2 (PGE2) and/or thromboxane B2 (TXB2). Cells are incubated (at 37°C in PBS without calcium or in RPMI-1640 with 1-20% fetal bovine serum, with or without supplements) with test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours prior to NB4 or HL-60 stimulation for inflammatory mediator release (see Example 1 above for details regarding drug stock solutions and concentrations; test drug(s) may also be added simultaneously with NB4/HL- 60 stimulation). For comparison, some experiments are performed without the drugs.
To stimulate formation and release of inflammatory cytokines, chemokines and mediators such as IL-1 β, IL-6, TNF1 IL-8, IL-10, IL-12p70, MCP-1 , PAF1 C5a, differentiated or undifferentiated NB4 or HL-60 cells (at 1-10><106/mL) are incubated (37°C, 5% CO2) for 4-24 hours (in RPMI-1640 with 1-10% fetal bovine serum, with or without supplements) with lipopolysaccharide (LPS 1-100 ng/mL), phorbol-12-myristate-13-acetate (PMA 1-100 ng/mL) or calcium ionophore A23187 (1-10 μM), or combinations of these stimuli. The NB4 and HL-60 cells may also be stimulated with documented biologically active concentrations of adenosine diphosphate (ADP) and/or the thromboxane analogue U-46619, with or without LPS, PMA and/or A23187 as above. The NB4 and HL-60 incubations/stimulations may also be performed in the presence of human platelets (from healthy donor blood) with an NB4/HL-60:platelet ratio of 1 :10 to 1 :10000. Cells are incubated (at 37°C, 5% CO2 in RPMI-1640 with 1-10% foetal bovine serum, with or without supplements) with test drug(s) (mast cell inhibitor in combination with thromboxane A2 antagonist, mast cell inhibitor alone and thromboxane A2 antagonist alone, as above) for 1 minute to 24 hours prior to NB4 or HL-60 stimulation for cytokine/chemokine/mediator release (for
comparison, some experiments are performed without the drugs; test drug(s) may also be added simultaneously with NB4/HL-60 stimulation). After spinning down cells, human cytokine/chemokine and mediator concentrations in the supernatants are qυantitated using a Cytometric Bead Array (BD Biosciences Pharmingen, San Diego, USA) according to the manufacturer's instructions. Commercially available enzyme immuno-assay kits (EIA/ELISA kits) for measuring the cytokines/chemokines and mediators may also be used according to instructions by the manufacturers). The cell pellets are stored frozen (-800C) in RLT buffer (QIAGEN, Valencia, CA) until further processing for microarray experiments (see Example 12 above).
In addition to studying the effects of the drugs above on release of mediators and chemokines/cytokines from the neutrophil-like NB4 and HL-60 cells, effects of the drugs on spontaneous or stimulated adhesion and/or migration of these cells may also be analyzed (freshly isolated human blood polymorphonuclear cells (PMN) isolated according to standard protocols may also be used). Spontaneous or stimulated (with fMLP, IL-8, PAF, LTB4 or other relevant PMN activating factors) adhesion of the PMN or neutrophil-like cells to e.g. cultured endothelial cells or protein-coated artificial surfaces are studied using well established and documented experimental approaches and assays. Migration (stimulated with fMLP, IL-8, PAF, LTB4 or other relevant PMN chemotactic factors) of the PMN or neutrophil-like cells are studied using well established and documented experimental approaches and assays, e.g. migration through commercially available protein-coated membranes designed for such migration studies.
Example 18
Platelet and Leukocyte Activation in Human Whole Blood
Venous blood is collected by venepuncture without stasis, using siliconized vacutainer tubes containing 1/10 volume of 129 mM trisόdium citrate (Becton
Dickinson, Meylan, France). Whole blood platelet P-selectin expression
(reflecting platelet activity), leukocyte CD11b expression (reflecting leukocyte activity), single platelet and platelet-platelet microaggregate counting, and platelet-leukocyte aggregates (PLAs) are measured using flow cytometric assays, essentially as described previously (see e.g. Li et al. Circulation 100, 1374 (1999)
for reference). Briefly, 5 μl_ aliquots of whole blood are added to 45 μl_ Hepes buffered saline (150 mM NaCI, 5 mM KCI, 1 mM MgSO4, 10 mM Hepes, pH 7.4) containing appropriately diluted antibodies (see below) in the absence or presence of platelet activating stimuli such as adenosine diphosphate (ADP), U- 46619, U-44069, platelet activating factor (PAF), arachidonic acid, collagen or thrombin, and/or leukocyte activating stimuli such as N-formyl-methionyl-leucyl- phenylalanine (fMLP), arachidonic acid, PAF, LPS1 A23187 or LTB4. Prior to exposing the blood to the platelet and/or leukocyte activating stimuli + the antibodies, blood samples (0.1-1 ml) are incubated with test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1-60 minutes (test drug(s) may also be added simultaneously with the stimuli above). For comparison, some blood samples are stimulated as above without exposure to the drug(s). Platelet P- selectin expression is determined by R-phycoerythrin (RPE)-CD62P monoclonal antibody (MAb) AC1.2 (Becton Dickinson, San Jose, CA, USA). Leukocyte CD11 b expression is determined by fluorescein isothiocyanate (FITC)-conjugated MAb BEAR 1 (Immunotech, Marseille, France). FITC and RPE conjugated isotypic MAbs are used as negative controls. Fluorescent beads (SPHERO™ Rainbow particles, 1.8-2.2 μm) used for platelet counting are from PharMingen (San Diego, CA, USA). Platelets are identified with FITC conjugated anti-CD42a (GPIX) MAb Beb 1 (Becton Dickinson), and leukocytes are identified with RPE conjugated anti-CD45 MAb J33 (Immunotech). Samples (drug-treated or untreated blood + antibodies with or without the stimuli, as above) are incubated at room temperature in the dark for 20 min. Afterwards, the samples are diluted and mildly fixed with 0.5% (v/v) formaldehyde saline, and analysed for the various platelet and leukocyte parameters with a Beckman-Coulter EPICS XL-MCL flow cytometer (Beckman-Coulter Corp., Hialeah, FL). Platelet P-selectin expression data are reported as the percentages of P-selectin positive cells in the platelet population and as absolute counts of P-selectin positive platelets. Leukocyte CD11 b expression is reported as mean fluorescence intensity (MFI) of the total leukocyte population and of leukocyte subpopulations. Platelet-leukocyte aggregates (PLAs) are presented as both absolute counts and percentages of
platelet-conjugated leukocytes in the total leukocyte population and among lymphocytes, monocytes, and neutrophils. Other relevant reagents, experimental conditions/approaches, equipment and modes of analysis to measure corresponding platelet and leukocyte activation in human whole blood may also be used.
Example 19
Experimental Abdominal Aortic Aneurysms
Test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) at doses of 0.3 to 200 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to the animals (drugs, up to 400 mg/kg/day, may also be continuously administered subcutaneously or intraperitoneally using e.g. Alzet® Osmotic Pumps according to instructions by the manufacturer). (For comparison, some experiments are performed without the drugs.) Prior to administration, stock solutions of drugs (see Example 1 above) are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used. Immediately after or up to 7 days after the first drug dose, production of experimental abdominal aortic aneurysms (and measurements of aortic diameters) is performed using periaortic CaCI2 application essentially according to Longo ef al {J. Clin. Invest, 110, 625, 2002) or using aortic elastase perfusion essentially according to Pyo et al (J. Clin. Invest, 105, 1641 (2000)). (Other strains of mice than those used in Longo ef al and Pyo et al may also be used.) Effects of the drugs on the elastase- or CaCI2-induced aneurysmal increases in aortic diameter are typically measured immediately before and one, two, three and/or four weeks after challenge with elastase or CaCI2 (other measurement intervals may also be used). Effects of the drugs on inflammation (e.g. tissue leukocyte accumulation), gene expression and protease activity is measured in specimens of the aortic aneurysmal tissue using established/conventional biochemical, histological, immunohistochemical, immunological, micoroarray and
zymographical techniques. Examples of how to handle collected tissues and measure tissue gene expression is described in Examples 5 and 12 above. Effects of the drugs may also be studied in corresponding rat models of elastase- induced aortic aneurysms, for example essentially according to Holmes et al (J. Surg. Res., 63, 305 (1996)), or CaCI2-induced aortic aneurysms, for example essentially according to lsenburg et al (Circulation, 115, 1729 (2007)).
Example 20
Human Arterial Tissue ex vivo
Samples of human atherosclerotic carotid or femoral arteries or abdominal aortic aneurysms obtained during routine surgery are used to study drug effects on spontaneous or induced inflammation, gene expression and protease activity in the diseased arterial tissues. Before the incubations/treatments below, the tissues are kept on ice in PBS without Ca and Mg (other established tissue media may also be used).
To stimulate release of inflammatory cytokines and chemokines (e.g. IL-1 β, IL-4, IL-6, TNF, IL-8, IL-10, IL-12p70, MCP-1 , IFNγ) and release and/or activation of matrix metalloproteases (MMPs), diced tissues are incubated (37°C/5% CO2) for 1-24 hours (in RPM 1-1640 with 1-10% fetal bovine serum; other established tissue media may also be used) with or without lipopolysaccharide (LPS, final concentration 1-100 ng/mL), phorbol-12-myristate-13-acetate (PMA, final concentration 1-100 ng/mL) or an LPS/PMA mixture.
To stimulate release of inflammatory histamine, tryptase, cytokines and chemokines (see above), and to activate matrix metalloproteases, the diced tissues can also be incubated (370C with or without 5% CO2) for 5 min to 24 hours (in RPMI-1640 with 1-10% fetal bovine serum; other established media may also be used) with or without anti-lgE, concanavalin A, protein L or compound 48/80. Stimulation of IgE-dependent release of inflammatory mediators may also be performed using pre-incubation with anti-TNP IgE followed by TNP-BSA challenge essentially as described in Example 3.
Prior to exposing the tissues to the inflammatory stimuli above, the tissues are incubated with test drug(s) (mast cell inhibitor (tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast or, more particularly, suplatast or preferably pemirolast) in combination with thromboxane A2 antagonist (aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban or egualen), mast cell inhibitor alone and thromboxane A2 antagonist alone) for 1 minute to 24 hours (test drug(s) may also be added simultaneously with the stimuli above). The test drugs remain present during the incubations with the proinflammatory stimuli.
Effects of the drugs on the release of inflammatory mediators (e.g. histamine, tryptase, cytokines, chemokines), gene expression and protease (e.g. MMPs such as MMP2, MMP3, MMP9) activity in the vasular tissues are examined using established/conventional biochemical, histological, immunohistochemical, immunological, microarray and zymographical techniques. Examples of how to handle tissues and measure tissue gene expression are described in Examples 5 and 12 above, and examples of how to measure the inflammatory mediators are described in Examples 1 and 3 above.
Example 21
Effect of Egualen on Experimental Abdominal Aortic Aneurysms
Egualen (e.g. egualen sodium) at doses of 1 to 100 mg/kg is administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to the animals (egualen, up to 400 mg/kg/day, may also be continuously administered subcutaneously or intraperitoneally using e.g. Alzet® Osmotic Pumps according to instructions by the manufacturer). (For comparison, some experiments are performed without egualen.) Prior to administration, stock solutions of egualen (made in sterile saline or other established and appropriate vehicles/solvents) are diluted as needed in e.g. 0.5% or 1 % methylcellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used. Immediately after or up to 7 days after the first egualen dose, production of experimental abdominal aortic aneurysms (and measurements of aortic diameters) is performed using periaortic CaCI2 application essentially according to Longo et al (J. Clin. Invest, 110, 625, 2002) or using aortic elastase perfusion
essentially according to Pyo et al (J. Clin. Invest, 105, 1641 (2000)). (Other strains of mice than those used in Longo et al and Pyo et al may also be used.) Effects of egualen on the elastase- or CaCI2-induced aneurysmal increases in aortic diameter are typically measured immediately before and one, two, three and/or four weeks after challenge with elastase or CaCI2 (other measurement intervals may also be used). Effects of egualen on inflammation (e.g. tissue leukocyte accumulation), gene expression and protease activity is measured in specimens of the aortic aneurysmal tissue using established/conventional biochemical, histological, immunohistochemical, immunological, micoroarray and zymographical techniques. Examples of how to handle collected tissues and measure tissue gene expression is described in Examples 5 and 12 above. Effects of egualen may also be studied in corresponding rat models of elastase- induced aortic aneurysms, for example essentially according to Holmes et al (J. Surg. Res., 63, 305 (1996)), or CaCI2-induced aortic aneurysms, for example essentially according to lsenburg et al (Circulation, 115, 1729 (2007)).
Example 22
Inhibition of Macrophage Proliferation by an Active Metabolite of
Repririnast and Picotamide
Cells from the human macrophage cell-line MonoMac-6 (MM6) (Ziegler-Heitbrock et al, Int. J. Cancer, 41, 456 (1988)) were cultured (37°C/5% CO2) in RPMI-1640 medium supplemented with 1 mM sodium pyruvate, ^nonessential amino acids, 10 μg/mL insulin, 1 mM oxalacetic acid, 100 units/mL penicillin, 100 μg/mL streptomycin and 10% (v/v) fetal bovine serum. At the start of the experiment, MM6 cells were seeded in 96-well plates at a density of 1x105 cells/mL (100 pi¬ per well).
Proliferation of the MM6 cells was measured using the Cell Proliferation Reagent WST-1 (Roche Diagnostics Scandinavia AB, Bromma, Sweden) or by cell counting using a microscope. The WST-1 reagent is designed to be used for spectrophotometric quantification of e.g. cell growth in proliferation assays and was used according to the manufacturers' instructions. The wavelength for measuring absorbance was 450 nm.
Stock solutions of the active metabolite of repirinast MY-1250 (5,6-dihydro-7,8- dimethyl-4,5-dioxo-4H-pyrano[3,2-c]quinoline-2-carboxylic acid, CAS RN 63768- 47-8) (sodium salt; purchased from Anthem Biosciences Pvt. Ltd., Bangalore, India as the free acid and converted to the sodium salt by adding one equivalent of aqueous NaOH) and picotamide (HCI salt; synthesised according to a procedure described in UK patent application GB 2080288 and converted to the HCI salt by adding 2.8 equivalents of aqueous HCI) were made in sterile saline.
Untreated MM6 cells increased from 1x105 cells/mL at the start of the experiment to 1.4x105 ± 0.06x105 cells/mL and 2.3x105 ± 0.10x105 cells/mL after 24 and 48 hours, respectively (mean values ± sem, n=4 for each of the three time-points).
MY-1250 and/or picotamide were added at the start of the experiments and 46 hours later the effects of the drugs on MM6 cell proliferation were studied using the WST-1 reagent described above. The WST-1 reagent was added at 44 hours after the start of the experiments and the average absorbance of untreated control cells exposed to the WST-1 reagent was 1.35 (n=5).
After treatment with 0.3 μM, 1 μM and 10 μM picotamide the MM6 proliferation was 114%, 96% and 89%, respectively, of the proliferation of untreated control cells (mean values, n=5 for each concentration).
After treatment with 1 μM, 3 μM and 10 μM MY-1250 the MM6 proliferation was 89%, 96% and 94%, respectively, of the proliferation of untreated control cells (mean values, n=5 for each concentration).
After combination treatment with 0.3 μM picotamide + 1 μM MY-1250, 1 μM picotamide + 3 μM MY-1250 and 10 μM picotamide + 10 μM MY-1250 the MM6 proliferation was 70%, 77% and 69%, respectively, of the proliferation of untreated control cells (mean values, n=5 for each combination).
Example 23
Inhibition of Macrophage Proliferation by Tazanolast and Picotamide
The procedure described in Example 22 was repeated for tazanolast (synthesised according to a procedure described in European patent EP 481 118
B1 ). Tazanolast was dissolved in DMSO and then diluted in sterile saline leading to final DMSO concentrations in the MM6 incubations of 0.03%, 0.1% and 0.3% for the final tazanolast concentrations 3 μM, 10 μM and 30 μM, respectively. The effects of treatment with tazanolast (alone and in combination) on MM6 proliferation were compared with control treatments containing DMSO at concentrations corresponding to the DMSO concentrations used in the respective tazanolast treatments.
The average absorbance of untreated control cells exposed to the WST-1 reagent was 0.89 (n=5).
After treatment with 0.3 μM, 3 μM and 10 μM picotamide the MM6 proliferation was 106%, 117% and 110%, respectively, of the proliferation of untreated control cells (mean values, n=5 for each concentration).
After treatment with 3 μM, 10 μM and 30 μM tazanolast the MM6 proliferation was 98%, 93% and 89%, respectively, of the proliferation of untreated control cells (with DMSO as above) (mean values, n=5 for each concentration).
After combination treatment with 0.3 μM picotamide + 3 μM tazanolast, 3 μM picotamide + 10 μM tazanolast and 10 μM picotamide + 30 μM tazanolast the MM6 proliferation was 62%, 77% and 75%, respectively, of the proliferation of untreated control cells (with DMSO as above) (mean values, n=5 for each combination).
Example 24
Inhibition of Macrophage Proliferation by Pemirolast and Picotamide
The procedure described in Example 22 was repeated for pemirolast (potassium salt; purchased from American Custom Chemicals Corporation, San Diego, USA; stock solutions were made in sterile saline).
The average absorbance of untreated control cells exposed to the WST-1 reagent was 0.89 (n=5).
After treatment with 0.3 μM, 1 μM and 10 μM picotamide the MM6 proliferation was 114%, 96% and 89%, respectively, of the proliferation of untreated control cells (mean values, n=5 for each concentration).
After treatment with 10 μM, 30 μM and 100 μM pemirolast the MM6 proliferation was 98%, 86% and 94%, respectively, of the proliferation of untreated control cells (mean values, n=5 for each concentration).
After combination treatment with 0.3 μM picotamide + 10 μM pemirolast, 1 μM picotamide + 30 μM pemirolast and 10 μM picotamide + 100 μM pemirolast the MM6 proliferation was 80%, 80% and 78%, respectively, of the proliferation of untreated control cells (mean values, n=5 for each combination).
One or more of the above-described examples demonstrate a clear synergistic effect for the combination of mast cell inhibitors (e.g. tranilast, ketotifen, amlexanox, particularly repirinast (or its active metabolite, MY-1250) or tazanolast, more particularly, suplatast or preferably pemirolast) and thromboxane A2 antagonists (e.g. aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban and/or egualen).
Claims
1. A combination product comprising:
(a) one or more mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof; and
(b) one or more thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast.
2. A combination product as claimed in Claim 1 , provided that the mast cell inhibitor is not pemirolast.
3. A combination product as claimed in Claim 1 , wherein the mast cell inhibitor is selected from tranilast, ketotifen, repirinast, MY-1250, amlexanox, tazanolast, suplatast and pemirolast.
4. A combination product as claimed in Claim 3, wherein the mast cell inhibitor is selected from tranilast, ketotifen, repirinast, MY-1250, amlexanox, tazanolast and suplatast.
5. A combination product as claimed in Claim 4, wherein the mast cell inhibitor is selected from repirinast, MY-1250, amlexanox, tazanolast and suplatast.
6. A combination product as claimed in Claim 5, wherein the mast cell inhibitor is suplatast.
7. A combination product as claimed in Claim 3, wherein the mast cell inhibitor is pemirolast.
8. A combination product as claimed in any one of the preceding claims, wherein the thromboxane A2 antagonist is selected from aspirin/acetylsalicylic acid, ozagrel, picotamide, terutroban and egualen.
9. A combination product as claimed in Claim 8, wherein the thromboxane A2 antagonist is selected from picotamide, terutroban and ozagrel.
10. A combination product as claimed in any one of the preceding claims which comprises a pharmaceutical formulation including one or more mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof; one or more thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast; and a pharmaceutically-acceptable adjuvant, diluent or carrier.
1 1. A combination product as claimed in any one of Claims 1 to 9, which comprises a kit of parts comprising components:
(A) a pharmaceutical formulation including one or more mast cell inhibitor, or a pharmaceutically-acceptable salt or solvate thereof, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier; and
(B) a pharmaceutical formulation including one or more thromboxane A2 antagonist, or a pharmaceutically-acceptable salt or solvate thereof, provided that the thromboxane A2 antagonist is not ramatroban or seratrodast, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (A) and (B) are each provided in a form that is suitable for administration in conjunction with the other.
12. A method of making a kit of parts as defined in Claim 11 , which method comprises bringing component (A) into association with a component (B), thus rendering the two components suitable for administration in conjunction with each other.
13. A kit of parts comprising:
(I) one of components (A) and (B) as defined in Claim 11 ; together with
(II) instructions to use that component in conjunction with the other of the two components.
14. A kit of parts as claimed in Claim 11 or Claim 13, wherein components (A) and (B) are suitable for sequential, separate and/or simultaneous use in the treatment of an inflammatory disorder.
15. The use of a combination product as defined in any one of Claims 1 to 1 1 , 13 or 14, for the manufacture of a medicament for the treatment of an inflammatory disorder.
16. A method of treatment of an inflammatory disorder, which method comprises the administration of a combination product as defined in any one of Claims 1 to 11 , 13 or 14, to a patient in need of such treatment.
17. A kit of parts as claimed in Claim 14, a use as claimed in Claim 15, or a method as claimed in Claim 16, wherein the disorder is selected from asthma, chronic obstructive pulmonary disease, endometriosis, migraine, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus or ulcerative colitis.
18. A kit of parts as claimed in Claim 14, a use as claimed in Claim 15, or a method as claimed in Claim 16, wherein the disorder is atherosclerosis or an associated cardiovascular disorder.
19. The use of egualen, or a pharmaceutically-acceptable salt or solvate thereof, for the manufacture of a medicament for the treatment of atherosclerosis or an associated cardiovascular disorder.
20. A method of treatment of atherosclerosis or an associated cardiovascular disorder, which method comprises the administration of egualen, or a pharmaceutically-acceptable salt or solvate thereof, to a patient in need of such treatment.
21. A kit of parts, use or method as claimed in Claim 18, a use as claimed in Claim 19, or a method as claimed in Claim 20, wherein the disorder is atherosclerosis.
22. A kit of parts, use or method as claimed in Claim 18, a use as claimed in Claim 19, or a method as claimed in Claim 20, wherein the cardiovascular disorder associated with atherosclerosis is selected from an aortic aneurysm, arteriosclerosis, peripheral arterial occlusive disease, a coronary artery disease, a coronary disease, plaque rupture and/or instability, atheroma rupture and/or instability, a vascular disease, an arterial disease, an ischaemic disease, ischaemia and stroke.
23. A kit of parts, use or method as claimed in Claim 22, wherein the coronary artery disease is selected from angina pectoris, myocardial infarction and heart attack.
24. A kit of parts, use or method as claimed in Claim 22, wherein the coronary disease is selected from a cardiac disease and a heart disease.
25. A kit of parts, use or method as claimed in Claim 22, wherein the stroke is selected from cerebro-vascular accident and transient ischaemic attack.
26. A kit of parts, use or method as claimed in Claim 22, wherein the disorder is plaque rupture and/or instability, or atheroma rupture and/or instability.
27. A kit of parts, use or method as claimed in Claim 22, wherein the disorder is an aortic aneurysm.
28. A kit of parts, use or method as claimed in any one of Claims 14 to 27, wherein the patient has an acute coronary syndrome.
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011058331A1 (en) * | 2009-11-13 | 2011-05-19 | Cardoz Ab | Pemirolast for the treatment of systemic low grade inflammation |
CN103450087A (en) * | 2013-09-13 | 2013-12-18 | 上海海虹实业(集团)巢湖今辰药业有限公司 | Preparation method of paeonol and ozagrel conjugate |
JP5997416B1 (en) * | 2015-03-23 | 2016-09-28 | キッセイ薬品工業株式会社 | A treatment for dry eye associated with Sjogren's syndrome |
CN110637788A (en) * | 2019-11-05 | 2020-01-03 | 云南洛宇生物科技有限公司 | Method for establishing rabbit atherosclerosis animal model |
CN112654357A (en) * | 2018-07-06 | 2021-04-13 | 阿尔杰农制药股份有限公司 | Compositions and methods for treating non-alcoholic steatohepatitis |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4086349A (en) * | 1975-07-30 | 1978-04-25 | Mitsubishi Yuka Pharmaceutical Co., Ltd. | Quinolopyran-4-one-2-carboxylic acid derivatives and salts thereof as novel compounds and as medicines for treatment of allergic asthma |
US4122274A (en) * | 1977-05-25 | 1978-10-24 | Bristol-Myers Company | 3-Tetrazolo-5,6,7,8-substituted-pyrido[1,2-a]pyrimidin-4-ones |
EP0316174A1 (en) * | 1987-11-10 | 1989-05-17 | Tokyo Tanabe Company Limited | Aqueous preparation of 9-methyl-3-(1H-tetrazol-5-yl)-4H-Pyrido[1,2-a]pyrimidin-4-one potassium salt |
EP0766963A1 (en) * | 1994-05-31 | 1997-04-09 | Tokyo Tanabe Company Limited | Arteriosclerosis depressant |
US20020103219A1 (en) * | 2000-10-05 | 2002-08-01 | Jeremy Jacob | Stable viscous liquid formulations of amlexanox for the prevention and treatment of mucosal diseases and disorders |
EP1285921A1 (en) * | 2001-08-10 | 2003-02-26 | Dinamite Dipharma S.p.A. | A process for the preparation of high purity pemirolast |
US6585995B1 (en) * | 1999-09-21 | 2003-07-01 | Hanson Stephen R | Methods and compositions for treating platelet-related disorders |
-
2008
- 2008-06-25 WO PCT/GB2008/002247 patent/WO2009007679A2/en active Application Filing
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4086349A (en) * | 1975-07-30 | 1978-04-25 | Mitsubishi Yuka Pharmaceutical Co., Ltd. | Quinolopyran-4-one-2-carboxylic acid derivatives and salts thereof as novel compounds and as medicines for treatment of allergic asthma |
US4122274A (en) * | 1977-05-25 | 1978-10-24 | Bristol-Myers Company | 3-Tetrazolo-5,6,7,8-substituted-pyrido[1,2-a]pyrimidin-4-ones |
EP0316174A1 (en) * | 1987-11-10 | 1989-05-17 | Tokyo Tanabe Company Limited | Aqueous preparation of 9-methyl-3-(1H-tetrazol-5-yl)-4H-Pyrido[1,2-a]pyrimidin-4-one potassium salt |
EP0766963A1 (en) * | 1994-05-31 | 1997-04-09 | Tokyo Tanabe Company Limited | Arteriosclerosis depressant |
US6585995B1 (en) * | 1999-09-21 | 2003-07-01 | Hanson Stephen R | Methods and compositions for treating platelet-related disorders |
US20020103219A1 (en) * | 2000-10-05 | 2002-08-01 | Jeremy Jacob | Stable viscous liquid formulations of amlexanox for the prevention and treatment of mucosal diseases and disorders |
EP1285921A1 (en) * | 2001-08-10 | 2003-02-26 | Dinamite Dipharma S.p.A. | A process for the preparation of high purity pemirolast |
Non-Patent Citations (23)
Title |
---|
"PEMIROLAST POTASSIUM" DRUGS OF TODAY / MEDICAMENTOS DE ACTUALIDAD, J.R. PROUS SS.A. INTERNATIONAL PUBLISHERS, ES, vol. 28, no. 1, 1 January 1992 (1992-01-01), pages 29-31, XP008066535 ISSN: 0025-7656 * |
AKAGI MASAAKI ET AL: "Inhibitory effect of egualen sodium: A new stable derivative of azulene on histamine release from mast cell-like cells in the stomach" PHARMACOLOGY (BASEL), vol. 63, no. 4, November 2001 (2001-11), pages 203-209, XP008086936 ISSN: 0031-7012 * |
COLLINS C E ET AL: "Anti-platelet therapy in active Crohn's disease: An open trial using picotamide, a thromboxane antagonist" GASTROENTEROLOGY, vol. 108, no. 4 SUPPL., 1995, page A799, XP008086946 & 95TH ANNUAL MEETING OF THE AMERICAN GASTROENTEROLOGICAL ASSOCIATION AND DIGESTIVE DISEASE WEEK; SAN DIEGO, CALIFORNIA, USA; MAY 14-17, 1995 ISSN: 0016-5085 * |
COLLINS C E ET AL: "PICOTAMIDE INHIBITION OF EXCESS IN VITRO THROMBOXANE B2 RELEASE BY COLORECTAL MUCOSA IN INFLAMMATORY BOWEL DISEASE" ALIMENTARY PHARMACOLOGY & THERAPEUTICS, BLACKWELL SCIENTIFIC PUBLICATIONS LTD., CAMBRIDGE, GB, vol. 10, no. 3, 1996, pages 315-320, XP009040236 ISSN: 0269-2813 * |
DOGNE J-M ET AL: "Recent developments of thromboxane modulators" EXPERT OPINION ON THERAPEUTIC PATENTS, ASHLEY PUBLICATIONS, GB, vol. 11, no. 11, 2001, pages 1663-1675, XP002445841 ISSN: 1354-3776 * |
ISAJI M ET AL: "TRANILAST INHIBITS THE PROLIFERATION, CHEMOTAXIS AND TUBE FORMATIONOF HUMAN MICROVASCULAR ENDOTHELIAL CELLS IN VITRO AND ANGIOGENESIS IN VIVO" BRITISH JOURNAL OF PHARMACOLOGY, BASINGSTOKE, HANTS, GB, vol. 122, no. 6, November 1997 (1997-11), pages 1061-1066, XP000997892 ISSN: 0007-1188 * |
ISHIZUKA T: "THROMBOXANE A2 RECEPTOR BLOCKADE PREVENTS ATHEROSCLEROTIC PROCESS BY ITS ANTI-INFLAMMATORY EFFECT" VASCULAR DISEASE PREVENTION, BENTHAM SCIENCE PUBLISHERS, GB, vol. 3, no. 2, May 2006 (2006-05), pages 143-148, XP008078040 ISSN: 1567-2700 * |
JONES N L ET AL: "Ketotifen therapy for acute ulcerative colitis in children: A pilot study" DIGESTIVE DISEASES AND SCIENCES, PLENUM PUBLISHING CO, US, vol. 43, no. 3, 1 March 1998 (1998-03-01), pages 609-615, XP008086243 ISSN: 0163-2116 * |
MARSHALL J K ET AL: "Ketotifen treatment of active colitis in patients with 5-aminosalicylate intolerance" CANADIAN JOURNAL OF GASTROENTEROLOGY, MEDICARE PUBL., OAKVILLE, CA, vol. 12, no. 4, 1998, pages 273-275, XP008086245 ISSN: 0835-7900 * |
MATSUMURA ET AL: "Suppression of atherosclerotic development in watanabe heritable hyperlipidemic rabbits treated with an oral antiallergic drug, tranilast" MEDLINE, 23 February 1999 (1999-02-23), XP002953929 * |
MINOCHA A ET AL: "Role of ketotifen in pathogenesis of chronic ulcerative colitis" GASTROENTEROLOGY, SAUNDERS, PHILADELPHIA, PA,, US, vol. 104, no. 4 SUPPL, 1993, page A746, XP008086242 ISSN: 0016-5085 * |
MYOU SHIGEHARU ET AL: "Effect of ozagrel hydrochloride, a thromboxane synthetase inhibitor, on alcoholic beverage-induced bronchoconstriction in asthmatic patients" PROSTAGLANDINS LEUKOTRIENES AND ESSENTIAL FATTY ACIDS, vol. 66, no. 4, April 2002 (2002-04), pages 397-401, XP008086945 ISSN: 0952-3278 * |
NOBUHIDE O ET AL: "Long-term prospective pilot study with tranilast for the prevention of stricture progression in patients with Crohn's disease" GUT, BRITISH MEDICAL ASSOCIATION, LONDON,, GB, vol. 56, no. 4, April 2007 (2007-04), pages 599-600, XP008086231 ISSN: 0017-5749 * |
SAIURA A ET AL: "Tranilast inhibits transplant-associated coronary arteriosclerosis in a murine model of cardiac transplantation" EUROPEAN JOURNAL OF PHARMACOLOGY, AMSTERDAM, NL, vol. 433, no. 2-3, 21 December 2001 (2001-12-21), pages 163-168, XP008086230 ISSN: 0014-2999 * |
SHIMIZU T ET AL: "INHIBITORY EFFECTS OF AZELASTINE AND TRANILAST ON LEUKOTRIENE B4 AND LEUKOTRIENE C4, GENERATION BY RAT COLONIC MUCOSA" PROSTAGLANDINS LEUKOTRIENES AND ESSENTIAL FATTY ACIDS, CHURCHILL LIVINGSTONE MEDICAL JOURNALS,, GB, vol. 53, no. 5, 1995, pages 355-358, XP001042184 ISSN: 0952-3278 * |
SORBERA L A ET AL: "Terutroban sodium. Prostanoid TP receptor antagonist, antithrombotic agent, antiatherosclerotic agent" DRUGS OF THE FUTURE, BARCELONA, ES, vol. 31, no. 10, October 2006 (2006-10), pages 867-873, XP008086411 ISSN: 0377-8282 * |
SUWAKI T ET AL: "Modification of eosinophil function by suplatast tosilate (IPD), a novel anti-allergic drug" INTERNATIONAL IMMUNOPHARMACOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 1, no. 12, November 2001 (2001-11), pages 2163-2171, XP002443957 ISSN: 1567-5769 * |
TAKAHASHI N ET AL: "Beneficial effect of combination therapy with ozagrel and pranlukast in exercise-induced asthma demonstrated by krypton-81m ventilation scintigraphy: A case report" ANNALS ACADEMY OF MEDICINE SINGAPORE, vol. 29, no. 6, November 2000 (2000-11), pages 766-769, XP008086937 ISSN: 0304-4602 * |
TAMAOKI JUN: "Role of a Th2 cytokine inhibitor in asthma treatment" ALLERGOLOGY INTERNATIONAL, BLACKWELL SCIENCE, CARLTON,, AU, vol. 53, no. 2, June 2004 (2004-06), pages 55-60, XP002443956 ISSN: 1323-8930 * |
TEH L G ET AL: "Does the addition of ketotifen to non-steroidal anti-inflammatory drugs confer any additional benefit in rheumatoid arthritis?" BRITISH JOURNAL OF CLINICAL PHARMACOLOGY, BLACKWELL SCIENTIFIC PUBL, GB, vol. 17, no. 2, 1984, pages 157-159, XP008086082 ISSN: 0306-5251 * |
YAMADA N ET AL: "MY-1250, a major metabolite of the anti-allergic drug repirinast, induces phosphorylation of a 78-kDa protein in rat mast cells" BIOCHEMICAL PHARMACOLOGY, PERGAMON, OXFORD, GB, vol. 44, no. 6, 25 September 1992 (1992-09-25), pages 1211-1213, XP023732179 ISSN: 0006-2952 [retrieved on 1992-09-25] * |
YANAGIHARA Y ET AL: "Immunopharmacological studies on TBX, a new antiallergic drug. (4) Effects on type II to IV allergic reactions and immunological functions in animal models" JAPANESE JOURNAL OF PHARMACOLOGY, JPTHE JAPANESE PHARMACOLOGICAL SOCIETY, KYOTO, vol. 51, no. 1, 1 January 1989 (1989-01-01), pages 93-100, XP008066536 ISSN: 0021-5198 * |
ZHANG W ET AL: "Development of new therapeutic effect and mechanism of tranilast" ZHONGGUO XIN YAO YU LINCHUANG ZAZHI - CHINESE JOURNAL OF NEW DRUGS AND CLINICAL REMEDIES, SHANGHAI SHI YIYAO GUANLIJU KE-JI QINGBAO YANJIUSUO ZHONGGUO, CN, vol. 23, no. 11, November 2004 (2004-11), pages 795-798, XP008086232 ISSN: 1007-7669 * |
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