WO2009122183A1 - Nouvelle combinaison destinée au traitement de troubles inflammatoires - Google Patents

Nouvelle combinaison destinée au traitement de troubles inflammatoires Download PDF

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Publication number
WO2009122183A1
WO2009122183A1 PCT/GB2009/000883 GB2009000883W WO2009122183A1 WO 2009122183 A1 WO2009122183 A1 WO 2009122183A1 GB 2009000883 W GB2009000883 W GB 2009000883W WO 2009122183 A1 WO2009122183 A1 WO 2009122183A1
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Prior art keywords
kit
parts
disease
ramatroban
suplatast
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PCT/GB2009/000883
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English (en)
Inventor
Johan Raud
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Cardoz Ab
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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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • 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
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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
  • LDLs low-density lipoproteins
  • 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.
  • 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.
  • 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 non- bacteria! prostatitis. See, for example, Tamoaki, Allergology International, 53, 55 (2004) and Suwaki et al, International Immunopharmacology, 1 , 2163 (2001 ).
  • Ramatroban is a thromboxane A2 antagonist and is known to have platelet aggregation inhibitory activity (see, for example, US patents Nos. 4,965,258, 6,362,214 and Expert Opin. Ther. Patents, 11, 1663 (2001 )).
  • the drug is known to be of potential utility in the treatment of allergic and inflammatory diseases, such as asthma, as well as the treatment of prevention of thrombosis and thromboembolism (see, for example, Ishizuka et al, Cardiovascular Drug Reviews, 22, 71 (2004)).
  • Suplatast and ramatroban are both presently marketed in e.g. Japan for the treatment of the treatment of allergic rhinitis (see, for example, Masuyama, Clin. Exp. All. Rev., 4, 27 (2004)).
  • US patent application US 2003/0104048 discloses novel pharmaceutical dosage forms comprising hydrophilic surfactant-containing fillers including pharmaceutically-active ingredients encapsulated by a shell. Suplatast and ramatroban are listed amongst many possible drug candidates for use in such dosage forms.
  • combination products comprising, specifically, suplatast and ramatroban is not 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.
  • a combination product comprising: (a) suplatast, or a pharmaceutically-acceptable salt or solvate thereof; and (b) ramatroban, or a pharmaceutically-acceptable salt or solvate thereof, which combination products are referred to hereinafter as "the combination products according to the invention".
  • 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 suplatast include supiatast tosylate.
  • Active ingredients that are employed in combination products according to the invention may be employed in diastereomerically- enriched and/or enantiomerically-enriched form.
  • diastereomerica ⁇ y-enriched and “enantiomericaiiy-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. suplatast
  • optical purities enantiomeric excess; e.e.
  • Combination products according to the invention provide for the administration of suplatast as hereinbefore defined in conjunction with ramatroban as hereinbefore defined, and may thus be presented either as separate formulations, wherein at least one of those formulations comprises suplatast, and at least one comprises ramatroban, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including suplatast and ramatroban).
  • 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.
  • component (A) and (B) of the kit of parts may be:
  • kit of parts comprising: (I) one of components (A) and (B) as defined herein; together with
  • kits of parts described herein may comprise more than one formulation including an appropriate quantity/dose of suplatast/salt/solvate, and/or more than one formulation including an appropriate quantity/dose of ramatroban/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 arteritis, diabetes mellitus, metabolic syndrome, endometriosis, acne, skin burns, rosacea, seborrheic dermatitis, skin ulcers, Marfan syndrome 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
  • Conditions that may be mentioned include endometriosis, atopic dermatitis, Marfan syndrome and, more preferably, migraine, 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), 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 ischaemic myocardial 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.
  • 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.
  • kits of parts as described herein by “administration in conjunction with”, we include that respective formulations comprising suplatast (or salt/solvate thereof) and ramatroban (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 (suplatast and ramatroban) are administered (optionaliy 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 suplatast, or a formulation comprising ramatroban, 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.
  • administered simultaneously and “administered at the same time as” include that individual doses of suplatast and ramatroban are administered within 48 hours (e.g. 24 hours) of each other.
  • Patients include mammalian (including human) patients.
  • suplatast and ramatroban 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. sub ⁇ ngually or buccally), rectally, transdermal ⁇ , 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.
  • Suplatast and ramatroban 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 (35 th Edition) and the documents referred to therein, the relevant disclosures in ail of which documents are hereby incorporated by reference. Otherwise, the preparation of suitable formulations, and in particular combined preparations including both suplatast and ramatroban 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 (35 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.
  • Suitable doses of suplatast are known to those skilled in the art.
  • Suitable lower limits of peroral daily dose ranges for suplatast are thus about 0.5 mg, such as about 2 mg, such as about 20 mg, such as about 50 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 about 1000 mg, such as about 800 mg, such as about 600 mg, such as about 450 mg.
  • suitable doses of ramatroban are known to those skilled in the art.
  • suitable lower limits of peroral daily dose ranges for ramatroban are, thus about 0.5 mg, such as about 2 mg, such as about 20 mg, such as about 30 mg, such as about 60 mg, such as about 80 mg, such as about 100 mg; and suitable upper limits are about 400 mg, such as about 200 mg, such as about 150 mg.
  • 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.
  • 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 ⁇ t 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 ⁇ M) 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°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:piatelet 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 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, 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 8x10 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 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 manufacturer(s).
  • EIA/ELISA kits enzyme immunoassay kits
  • PGE 2 prostaglandin E 2
  • TXB 2 thromboxane B 2
  • Stock solutions of suplatast and ramatroban are prepared in ethanol, DMSO, N- methyl-2-pyrrolidone, PEG 400, propylene glycol and/or deionized water or physiological saiine solution, with sonication, warming and adjustment of pH as needed (other vehicles may also be used).
  • test drug(s) salivary-derived neuropeptides
  • test drug(s) salivary-derived neuropeptides
  • the drugs are added to reach final concentrations of 1 nM to 100 ⁇ M (for comparison, some experiments are performed without the drugs).
  • differentiated or undifferentiated MM6 cells 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 1 final concentration 1-100 ng/mL) or an LPS/PMA mixture.
  • LPS lipopolysaccharide
  • PMA 1 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) suplatast in combination with ramatroban, supiatast alone and ramatroban alone; as above regarding stock solutions and concentrations
  • test drug(s) for 1 minute to 24 hours prior tc- MM6 stimulation (for comparison, some experiments are performed without the drugs; test drug(s) may also be added simultaneously with MM6 stimulation).
  • PBMC peripheral blood mononuclear cells
  • PMN polymorphonuclear cells
  • PBMC or PMN 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 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 PGB2 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).
  • supematants from the PBMC/PMN 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 ).
  • PGE 2 prostaglandin E 2
  • TXB 2 thromboxane B 2
  • test drug(s) salivary derived from fetal bovine serum
  • test drug(s) salivary derived from a source of fetal bovine serum
  • ramatroban fetal bovine serum
  • suplatast alone a cell line
  • ramatroban a cell line
  • ramatroban a cell line
  • ramatroban a cell line
  • test drug(s) may also be added simultaneously with PBMC/PMN stimulation.
  • some experiments are performed without the drugs.
  • 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 /mL) 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, final concentration 1-100 ng/mL) or an LPS/PMA mixture.
  • LPS lipopolysaccharide
  • PMA 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:plateiet ratio of 1 :10 to 1 :10000.
  • test drug(s) serum-derived neurotrophic factor(s)
  • test drug(s) serum-derived neurotrophic factor(s)
  • suplatast in combination with ramatroban, suplatast alone and ramatroban alone, as above test drug(s)
  • test drug(s) may also be added simultaneously with PBMC/PMN stimulation.
  • 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/EUSA kits
  • EIA/EUSA 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).
  • 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 fiow-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 al., Proc. Natl. Acad. ScL USA, 84, 9175 (1987)) may also be used.
  • the MC/9 cells are cultured according to instructions from ATCC (http://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 0 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) (suplatast in combination with ramatroban, suplatast alone and ramatroban 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.
  • 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 (- 8O 0 C) in RLT buffer (QIAGEN, Valencia, CA) until further processing for microarray experiments (see Example 12 below).
  • 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, 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 DMEIvI, supplemented with 100 units /imL 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, final concentration 1 -100 ng/mL), phorboi-12-myristate-13-acetate (PMA, final concentration 1-100 ng/mL) or an LPS/PMA mixture.
  • LPS lipopolysaccharide
  • PMA phorboi-12-myristate-13-acetate
  • the RAW 264.7 cells may also be stimulated with documented biologicaily 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) salivary-derived neuropeptides
  • test drug(s) salivary-derived neuropeptides
  • ramatroban fetal bovine serum
  • suplatast alone fetal bovine serum
  • ramatroban fetal bovine serum
  • ramatroban fetal bovine serum
  • test drug(s) may also be added simultaneously with RAW 264.7 stimulation.
  • some experiments are performed without the drugs.
  • mouse 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.
  • Cytometric Bead Array BD Biosciences Pharmingen, San Diego, USA
  • E1A/ELISA kits enzyme immuno-assay kits
  • 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) (suplatast in combination with ramatroban, suplatast alone and ramatroban alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally or orally every 2-24 hours to male Sprague-Dawiey or Wistar rats weighing approximately 150- 400 g (for comparison, some experiments are performed without the drugs).
  • stock solutions of drugs are diluted as needed in e.g.
  • 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.
  • 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.
  • mice 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 -80°C in TRIzol, 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.
  • 48/80 48/80, 1-5 ⁇ g in 50-100 ⁇ l PBS or saline
  • MPO analysis as above
  • Test drug(s) (suplatast in combination with ramatroban, suplatast alone and ramatroban 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).
  • 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.
  • biopsies from the inflamed ears are collected, stored frozen (samples for microarray analysis are frozen at -8O 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) are administered subcutaneously, intravenously, intraperitoneal ⁇ or orally every 2-24 hours to male or female mice (for comparison, some experiments are performed without the drugs).
  • stock solutions of drugs are diluted as needed in e.g. 0.5% or 1% methylcellulose in water (for oral treatment) or saline (for parenteral administration).
  • phorbol 12- myristate 13-acetate PMA
  • TPA tetradecanoyl phorbol acetate
  • arachidonic acid 1-5 mg arachidonic acid in 10-30 ⁇ l acetone or ethanol
  • biopsies from the inflamed ears are collected, stored frozen (samples for microarray analysis are frozen at -8O 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) (suplatast in combination with ramatroban, suplatast alone and ramatroban alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally 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).
  • 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% methyicellulose in water (for oral treatment) or saline (for parenteral administration). Other vehicles may also be used.
  • Example 9 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 -8O 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/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.
  • 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) (suplatast in combination with ramatroban, suplatast alone and ramatroban alone) at doses of 0.03 to 50 mg/kg are administered subcutaneously, intravenously, intraperitoneally 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).
  • 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.
  • Example 10 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 -8O 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. 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 is abundant in neutrophil leukocytes and is often used as a marker for the detection of neutrophil accumulation in inflamed tissue.
  • MPO myeloperoxidase
  • the tissues are homogenised in 0.5% hexadecyltrimethyl-arnmonium 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 ⁇ 2 -tetramethylbenzidine catalysed by MPO. Values are expressed as MPO units/mg tissue.
  • Rat aortic smooth muscle cells are isolated as previously described (Hedin et a/, Arterioscler. Thromb. Vase. Biol., 17, 1977 (1997)). Cells are cultured (37°C/5% CO 2 ) in Ham's medium F-12 supplemented with 10% fetal bovine serum, 50 ⁇ g/mL L-ascorbic acid, 50 ⁇ g/mL streptomycin, 50 lU/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 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).
  • the ceils 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
  • RASMCs starved RASMCs are stimulated with either 10 ng/ml lfiF-1 or 10% fetal bovine serum for 12-48 hours (other well established mitogens such as PDGF may also be used).
  • Test drug(s) (suplatast in combination with ramatroban, suplatast alone and ramatroban alone) are added 1 minute to 24 hours prior to stimulation (see Example 1 above for details regarding 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 ⁇ Ci [3H]-thymidine for 8 hours before the end of the stimulation period.
  • the plates are then washed with ice-cold PBS 1 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.
  • HBSMCs Human bronchial smooth muscle cells
  • DMEM fetal bovine serum
  • 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).
  • 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 manufacturer(s)), or conventional Western blot and/or immunohistochemicai 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 1 instructions. Other conventional tests of cell proliferation may also be used.
  • Test drug(s) (supiatast in combination with ramatroban, supiatast alone and ramatroban 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 Test drug(s) (supiatast in combination with ramatroban, supiatast alone and ramatroban 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
  • This assay is essentially according to Rao et a/ (J. Pharmacol. Exp. Ther. 269, 917 (1994)) (other strains of mice may also be used).
  • Test drug(s) (suplatast in combination with ramatroban, suplatast alone and ramatroban alone) at doses of 0.03 to 50 mg/kg are administered subcutaneousiy, intravenously, intraperitoneal ⁇ or orally every 2-24 hours to the animals (for comparison, some experiments are performed without the drugs).
  • stock solutions of drugs are diluted as needed in e.g. 0,5% or 1% methylcellulose in water (for oral treatment) or saline (for parenteral administration).
  • 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, peritonea! inflammation may also be induced by intraperitoneal injection of pro-inflammatory concentrations of other well established proinflammatory 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 ⁇ .
  • proinflammatory 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, T
  • 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).
  • 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 0 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-I p 1 KC, MCP-1 , IL-10, IL-12p70, lFN ⁇ ) content as described in Example 1 and 4 above.
  • inflammatory mediators LTB 4 , PGE 2 , TXB 2 and/or mouse cytokines/chemokines (e.g. IL-4, IL-6, TNF, IL-I p 1 KC, MCP-1 , IL-10, IL-12p70, lFN ⁇ ) content as described in Example 1 and 4 above.
  • cytokines/chemokines e.g. IL-4, IL
  • 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 manufacturer(s). 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 a/ (J. Pharmacol. Exp. Ther.
  • tissue biopsies from the inflamed peritonea! cavity are collected, weighed, stored frozen (samples for microarray analysis are frozen at -8O 0 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.
  • Test drug(s) (supiatast in combination with ramatroban, suplatast alone and ramatroban 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).
  • 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.
  • 1-100 mg zymosan A (Sigma, cat. no.
  • peritoneal inflammation may also be induced by intraperitoneal injection of pro-inflammatory concentrations of other well established pro-inflammatory stimuli such as anti-rat-lgE (with or without intraperitoneal pretreatment with rat IgE for 1-3 days), concanavaiin A, protein L 1 compound 48/80, carrageenan, proteose peptone, LPS, PMA, thioglycolate, arachidonic acid, fMLP, TNF, IL-1 ⁇ .
  • pro-inflammatory stimuli such as anti-rat-lgE (with or without intraperitoneal pretreatment with rat IgE for 1-3 days), concanavaiin A, protein L 1 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 proinflammatory 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°C, 3-10 mih), and cell-free lavage fluid supernatant is stored frozen (-2O 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. lL-4, IL-6, TNF, IL-I p, KC, MCP-1 , IL-10, IL-12p70, IFN ⁇ ) essentially as described in Example 1 and 4 above.
  • rat cytokines/chemokines e.g. lL-4, IL-6, TNF, IL-I p, KC, MCP-1 , IL-10, IL-12p70, IFN ⁇
  • 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 manufacturer(s). 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 (-2O 0 C to - 80°) until analysis of myeloperoxidase (MPO) content basically as described by Rao et a/ (J. Pharmacol. Exp.
  • tissue biopsies from the inflamed peritoneal cavity are collected, weighed, stored frozen (samples for microarray analysis are frozen at -80 0 C in TRIzoi, 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 a/, 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 at, 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 0 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) 1 fMLP, and/or the thromboxane analogue U- 46619, with or without A23187 and/or arachidonic acid as above.
  • ADP adenosine diphosphate
  • 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:p late let 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/ ⁇ 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 manufacturer(s).
  • 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) salivary-derived neuropeptides
  • test drug(s) salivary-derived neuropeptides
  • NB4 or HL-60 stimulation for inflammatory mediator release
  • chemokines and mediators such as IL-Ip 1 IL-6, TNF, IL-8, IL-10, IL-12p70, MCP-1 , PAF, C5a, differentiated or undifferentiated NB4 or HL-60 cells (at 1-10*10 6 /mL) are incubated (37 0 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) 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).
  • cytokine/chemokine and mediator 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
  • 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 trisodium citrate (Becton Dickinson, Meylan, France).
  • Whole blood platelet P-selectin expression reflecting platelet activity
  • leukocyte CD11 b 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).
  • Hepes buffered saline 150 mM NaCI, 5 mM KCI, 1 mM MgSO4, 10 mM Hepes, pH 7.4
  • 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, 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 leukocyte activating stimuli such as N-formyl-methionyl-leucyl- phenylalan
  • 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) (suplatast in combination with ramatroban, suplatast alone and ramatroban 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).
  • RPE R-phycoerythrin
  • MAb monoclonal antibody
  • 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 (GPlX) 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 CD11b expression is reported as mean fluorescence intensity (MFI) of the total leukocyte population and of leukocyte subpopulations.
  • MFI mean fluorescence intensity
  • PKAs Platelet-leukocyte aggregates
  • 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) (suplatast in combination with ramatroban, suplatast alone and ramatroban 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).
  • Effects of the drugs on the eiastase- or CaCI 2 - induced aneurysmal increases in aortic diameter are typically measured immediately before and one, two, three and/or four weeks after challenge with elastase or CaCI 2 (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 ef a/ ⁇ 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 IL-1 ⁇ , IL-4, IL-6, TNF, IL-8, IMO 1 !M2p70, MCP-1 , IFN ⁇ ) and release and/or activation of matrix metalloproteases (MMPs), diced tissues are incubated (37°C/5% CO 2 ) for IL-1 ⁇ , IL-4, IL-6, TNF, IL-8, IMO 1 !M2p70, MCP-1 , IFN ⁇ ) and release and/or activation of matrix metalloproteases (MMPs), diced tissues are incubated (37°C/5% CO 2 ) for IL-1 ⁇ , IL-4, IL-6, TNF, IL-8, IMO 1 !M2p70, MCP-1 , IFN ⁇ ) and release and/or activation of matrix metalloproteases (MMPs)
  • MMPs matrix metalloproteases
  • the diced tissues can also be incubated (37°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) Prior to exposing the tissues to the inflammatory stimuli above, the tissues are incubated with test drug(s) (suplatast in combination with ramatroban, suplatast alone and ramatroban alone) for 1 minute to 24 hours (test drug(s) may also be added simultaneously with the stimuli above).
  • 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.
  • MM6 Human macrophage cell-line MonoMac-6 (MM6) (Ziegler-Heitbrock et a/, 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,
  • MM6 cells were seeded in 96-well plates at a density of 1 x10 5 cells/mL (100 ⁇ l_ 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.

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Abstract

L'invention porte sur des produits de combinaison comprenant: (a) du suplatast, ou un sel ou solvate pharmaceutiquement acceptable de ce dernier; et (b) du ramatroban, ou un sel ou solvate pharmaceutiquement acceptable de ce dernier. Les produits de combinaison selon l'invention trouvent une utilité particulière dans le traitement de l'athérosclérose et des états associés.
PCT/GB2009/000883 2008-04-04 2009-04-03 Nouvelle combinaison destinée au traitement de troubles inflammatoires WO2009122183A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005063253A1 (fr) * 2003-12-26 2005-07-14 Taiho Pharmaceutical Co., Ltd. Composition medicinale pour le traitement de symptomes allergiques
WO2009007674A2 (fr) * 2007-07-11 2009-01-15 Cardoz Ab Nouvelle association pour une utilisation dans le traitement de troubles inflammatoires

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005063253A1 (fr) * 2003-12-26 2005-07-14 Taiho Pharmaceutical Co., Ltd. Composition medicinale pour le traitement de symptomes allergiques
WO2009007674A2 (fr) * 2007-07-11 2009-01-15 Cardoz Ab Nouvelle association pour une utilisation dans le traitement de troubles inflammatoires

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ISHIZUKA T ET AL: "RAMATROBAN (BAY U 3405): A NOVEL DUAL ANTAGONIST OF TXA2 RECEPTOR AND CRTH2, A NEWLY IDENTIFIED PROSTAGLANDIN D2 RECEPTOR", CARDIOVASCULAR DRUG REVIEWS, NEVA PRESS, BRANFORD, CT, US, vol. 22, no. 2, 1 June 2004 (2004-06-01), pages 71 - 90, XP008078031, ISSN: 0897-5957 *
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, 1 May 2006 (2006-05-01), pages 143 - 148, XP008078040, ISSN: 1567-2700 *
SHIOKOSHI TAKAHIRO ET AL: "Downregulation of nitric oxide accumulation by cyclooxygenase-2 induction and thromboxane A2 production in interleukin-1beta-stimula ted rat aortic smooth muscle cells.", JOURNAL OF HYPERTENSION MAR 2002, vol. 20, no. 3, March 2002 (2002-03-01), pages 455 - 461, XP009120606, ISSN: 0263-6352 *
TAJIMA K ET AL: "GENERAL PHARMACOLOGY OF SUPLATAST TOSILATE (IPD-1151T), A NEW ANTI-ALLERGIC DRUG: EFFECTS ON RESPIRATORY AND CARDIOVASCULAR SYSTEMS, GASTROINTESTINAL TRACT AND OTHER TISSUES", OYO YAKURI - PHARMACOMETRICS,, vol. 43, no. 6, 1 January 1992 (1992-01-01), pages 531 - 548, XP008042748, ISSN: 0300-8533 *

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