GB2388540A

GB2388540A - New use of Ramatroban

Info

Publication number: GB2388540A
Application number: GB0211396A
Authority: GB
Inventors: Kevin Bacon; Yoshihisa Manabe; Akihiko Watanabe; Takashi Iino; Hiromi Sugimoto
Original assignee: Bayer AG
Current assignee: Bayer AG
Priority date: 2002-05-17
Filing date: 2002-05-17
Publication date: 2003-11-19
Also published as: GB0211396D0

Abstract

The invention provides the compound represented by the formula (I): <EMI ID=1.1 HE=46 WI=82 LX=627 LY=891 TI=CF> <PC>or acceptable salt thereof as a CRTH2 modulator. The compounds of the formula (I): (+)-(3R)-3-(4-fluorobenzenesulfonamido)-1,2,3 ,4-tetrahydrocarbazole-9-propionic acid or a pharmaceutically acceptable salt thereof has excellent CRTH2 antagonistic activity and is useful for the prophylaxis and treatment of diseases associated with the activity, in particular for the treatment of allergic diseases, such as asthma, allergic rhinitis, allergic conjuvatitis. In addition, eosinophil-related diseases, such as Churg-Strauss syndrome and sinusitis should be included as possible targets of a CRTH2 modulator. Moreover, basophil-related diseases, such as basophilic leukemia and basophilic leukocytosis should be also included.

Description

- New use of Ramatroban DETAILED DESCRIPTION OF INVENTION

5 TECHNICAL FIELD

The present invention relates to a CRTH2 modulator. More specifically, the present invention relates to a CRTH2 modulator, which comprises the compound represented by the formula (I): POOH N,S:3:

10 F (I)

(+)-(3R)-3-(4-fluorobenzenesulfonamido)-1,23,4-tetrahydrocarbazole-9propionic acid (Bay u 3405, ramatroban) or a pharmaceutically acceptable salt thereof, as an active ingredient. The compound is useful in therapy in particular in the treatment or prevention of allergic diseases, such as asthma, allergic rhinitis and allergic 15 conjunctivitis; eosinophilrelated diseases, such as Churg-Strauss syndrome and sinusitis; and basophil-related diseases, such as basophilic leukemia and basophilic leuPocytosis in human and other mammals.

BACKGROUND ART

Ramatroban is known as having platelet aggregation inhibitory activity and thromboxane A2 receptor antagonistic activity and useful in the treatment or pre-

vention of thrombosis, thromboembolism and ischaemia. It is also known to be useful as an antiasthrna agent, antiallergic agent and useful in allergic dermatitis 25 (Japanese patent Tokkyohei 4-50301, Japanese laidopen patent publication

- 2 Tokkaihei8-175991). However, it is not known that ramatroban has CRTH2 modulatory (antagonistic or agonistic) activity.

CRTH2 is a G-protein-coupled chemoattractant receptor, expressed on Th2 5 cells(Nagata et al. J. Inununol., 162, 1278-1286, 1999), eosinophils and basophils (Hirai et al., J. Exp. Med., 193, 255-261, 2001).

Th2-polarization has been seen in allergic diseases, such as asthma, allergic rhinitis, atopic dermatitis and allergic conjunctivitis (Romagnani S. Immunology Today, 18, 10 263-266, 1997; Hammad H. et al., Blood, 98, 1135-1141, 2001). Th2 cells regulate allergic diseases by producing Th2 cytokines, such as IL-4, IL-5 and IL-13 (Oriss et al., J. Irnmunol., 162, 1999-2007, 1999; Viola et al., Blood, 91, 2223-2230, 1998; Webb et al., J. Immunol., 165, 108-113, 2000; Dumont F.J., Exp. Opin. Ther. Pat., 12, 341-367, 2002). These Th2 cytokines directly or indirectly induce migration, 15 activation, priming and prolonged survival of effecter cells, such as eosinophils and basophils, in allergic diseases (Sanz et al., J. Immunol., 160, 5637-5645, 1998; Pope et al., J. Allergy Clin. Immunol., 108, 594-601, 2001; Teran L.M., Clin. Exp.

Allergy, 29, 287-290, 1999).

20 PGD2, a ligand for CRTH2, is produced from mast cells, another important effecter cells in allergic diseases (Nagata et al., FEBS Lett. 459, 195-199, 1999; Hirai et al., J. Exp. Med., 193, 255-261, 2001). PGD2 induces migration and activation of Th2 cells, eosinophils, and basophils, via CRTH2 (Hirai et al., J. Exp. Med., 193, 255 261, 2001; Gervais et al. , J. Allergy Clin. Immunol., 108, 982-9887 2001).

Therefore, antagonists which inhibit the binding of CRTH2 and PGD2 should be useful for the treatment of allergic diseases, such as asthma, allergic rhinitis, atopic dermatitis and allergic conjunctivitis. In addition, several experimental evidence has demonstrated the contribution of eosinophils in sinusitis (Hamilos et al., Arn. J. 30 Respir. Cell and Mol. Bioh, 15, 443-450, 1996; Fan et al., J. Allergy Clin. Immunol., 106, 551-558, 2000), and Churg-Strauss syndrome (Coffin et al., J. Allergy Clin.

- 3 Immunol., 101, 116-123, 1998; Kurosawa et al., Allergy, 55, 785-787, 2000). In the tissues of these patients, mast cells can be observed to be colocalized with eosinophils (Khan et al., J. Allergy Clin. Irnmunol., 106, 1096-1101, 2000). It is suggested that PGD2 production from mast cells induces the recruitment of 5 eosinophils. Therefore, CRTH2 modulators are useful not only for the treatment of allergic diseases, but also for the treatment of other eosinophil-related diseases.

CRTH2 also may be involved in the recruitment of basophils in some basophil related diseases, because of high expression of CRTH2 on basophils.

10 SUMMARY OF THE INVENTION

This invention is to provide a CRTH2 modulator, which comprises the compound represented by the formula (I): POOH ó,>Os H H 3 F (I) 15 (+)(3R)-3-(4-fluorobenzenesulfonamido)- l,2,3,4-tetrahydrocarbazole-9propionic acid or a pharmaceutical acceptable salt thereof as an effective agent.

The compound represented by the formula (1) shows excellent CRTH2 antagonistic activity. They are, therefore, suitable especially for the prophylaxis and treatment of 20 diseases associated with CRTH2 activity, in particular for the treatment or prevention of allergic diseases, such as asthma, allergic rhinitis, atopic dermatitis and allergic conjunctivitis; eosinophil-related diseases, such as Churg-Strauss syndrome and sinusitis; and basophil-related diseases, such as basophilic leukemia and basophilic leukocytosis.

- 4 EMBODIMENT OF THE INVENTION

It is a discovery of the present invention that ramatroban or a pharmaceutically acceptable salt thereof has CRTH2 antagonistic activity and can be regulated to 5 control diseases that are caused by aberrant activity of this receptor and diseases whose symptoms can be ameliorated by stimulating or inhibiting the activity of CRTH2.

More specifically, ramatroban and the salt thereof inhibit the binding of [3H]PGD2 to 10 CRTH2 transfectants in a dose-dependent manner. Ramatroban and its salts inhibit PGD2-induced Ca2+ flux of CRTH2 transfectants in a dose-dependent manner.

Ramatroban and its salts inhibit PGD2-induced chemotaxis of eosinophils and CD4+ T cells in a dose-dependent manner. These results demonstrate that ramatroban and its analogues can inhibit migration of CRTH2expressing cells, such as eosinophils, 15 basophils and Th2 cells, by the inhibition of the binding of [3H]PGD2 to CRTH2.

Therefore, ramatroban and its analogues should be very useful for the treatment of allergic diseases, such as asthma, allergic rhinitis, atopic dermatitis and allergic con junctivitis, eosinophil-related diseases, such as Churg-Strauss syndrome and 20 sinusitis, and basophil-related diseases, such as basophilic leukemia and basophilic leukocytosis. Ramatroban has been well known and can be prepared by the methods described in Japanese patent Tokkyohei 4-50301.

Typical salts of the compound shown by the formula (I) include salts prepared by reaction of the compounds of the present invention with a mineral or organic acid, or an organic or inorganic base. Such salts are known as acid addition and base addition salts, respectively.

- 5 Acids to form acid addition salts include inorganic acids such as, without limitation, sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, hydriodic acid and the like, and organic acids, such as, without limitation, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, 5 succinic acid, citric acid, benzoic acid, acetic acid, and the like.

Base addition salts include those derived from inorganic bases, such as, without limitation, ammonium hydroxide, alkaline metal hydroxide, alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, and organic bases, such as, 10 without limitation, ethanolamine, triethylamine, tris(hydroxyrnethyl)arninomethane, and the like. Examples of inorganic bases include, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

15 The compound of the present invention or a salts thereof, depending on its substituents, may be modified to form lower alkylesters or known other esters; and/or hydrates or other solvates. Those esters, hydrates, and solvates are included in the scope of the present invention.

20 The compound of the present invention may be administered in oral forms, such as, without limitation normal and enteric coated tablets, capsules, pills, powders, granules, elixirs, tinctures, solution, suspensions, syrups, solid and liquid aerosols and emulsions. They may also be administered in parenteral forms, such as, without limitation, intravenous, intraperitoneal, subcutaneous, intramuscular, and the like 25 forms, well-known to those of ordinary skill in the pharmaceutical arts. The compounds of the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using transdermal delivery systems well-known to those of ordinary skilled in the art.

30 The dosage regimen with the use of the compounds of the present invention is selected by one of ordinary skill in the arts, in view of a variety of factors, including,

- 6 without limitation, age, weight, sex, and medical condition of the recipient, the severity of the condition to be treated, the route of administration, the level of metabolic and excretory function of the recipient, the dosage form employed, the particular compound and salt thereof employed.

The compounds of the present invention are preferably formulated prior to administration together with one or more pharmaceutically-acceptable excipients.

Excipients are inert substances such as, without limitation carriers, diluents, flavoring agents, sweeteners, lubricants, solubilizers, suspending agents, binders, tablet 10 disintegrating agents and encapsulating material.

Yet another embodiment of the present invention is pharmaceutical formulation comprising a compound of the invention and one or more pharrnaceutically acceptable excipients that are compatible with the other ingredients of the 15 formulation and not deleterious to the recipient thereof. Phammaceutical formulations of the invention are prepared by combining a therapeutically effective amount of the compounds of the invention together with one or more pharmaceutically-acceptable excipients therefore. In making the compositions of the present invention, the active ingredient may be mixed with a diluent, or enclosed within a carrier, which may be in 20 the form of a capsule, sachet, paper, or other container. The carrier may serve as a diluent, which may be solid, semi-solid, or liquid material which acts as a vehicle, or can be in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, 25 sterile injectable solutions and sterile packaged powders.

For oral administration, the active ingredient may be combined with an oral, and non-toxic, phamlaceutically-acceptable carrier, such as, without limitation, lactose, starch, sucrose, glucose, sodium carbonate, mannitol, sorbitol, calcium carbonate, 30 calcium phosphate, calcium sulfate, methyl cellulose, and the like; together with, optionally, disintegrating agents, such as, without limitation, maize, starch, methyl

- 7 cellulose, agar bentonite, xanthan gum, alginic acid, and the like; and optionally, binding agents, for example, without limitation, gelatin, natural sugars, beta-lactose, corn sweeteners, natural and synthetic gums, acacia, tragacanth, sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the lice; and, optionally, 5 lubricating agents, for example, without limitation, magnesium stearate, sodium stearate, stearic acid, sodium oleate, sodium benzoate, sodium acetate, sodium chloride, talc, and the like.

In powder forms, the carrier may be a finely divided solid which is in admixture with 10 the finely divided active ingredient. The active ingredient may be mixed with a carrier having binding properties in suitable proportions and compacted in the shape and size desired to produce tablets. The powders and tablets preferably contain from about I to about 99 weight percent of the active ingredient which is the novel composition of the present invention. Suitable solid carriers are magnesium carboxy 15 methyl cellulose, low melting waxes, and cocoa butter.

Sterile liquid formulations include suspensions, emulsions, syrups and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile organic solvent, or a mixture of both sterile water 20 and sterile organic solvent.

The active ingredient can also be dissolved in a suitable organic solvent, for example, aqueous propylene glycol. Other compositions can be made by dispersing the finely divided active ingredient in aqueous starch or sodium carboxymethyl cellulose 25 solution or in a suitable oil.

The formulation may be in unit dosage form, which is a physically discrete unit containing a unit dose, suitable for administration in human or other mammals. A unit dosage form can be a capsule or tablets, or a number of capsules or tablets. A 30 "unit dose" is a predetermined quantity of the active compound of the present invention, calculated to produce the desired therapeutic effect, in association with

( - 8 one or more excipients. The quantity of active ingredient in a unit dose may be varied or adjusted from about 0.1 to about 1000 milligrams or more according to the particular treatment involved.

5 Typical oral dosages of the compound of the present invention, when used for the indicated effects, will range from about 1 mg Ikg/day to about 10 mg/kglday. The compounds of the present invention may be administered in a single daily dose, or the total daily dose may be administered in divided doses, two, three, or more times per day. Where delivery is via transderrnal forms, of course, administration is 1 0 continuous.

EXAMPLES

The present invention will be described as a form of examples. In the example 15 below, all quantitative data, if not stated otherwise, relate to percentages by weight.

EXAMPLE I

Human CRTH2 was amplified from human eosinophil cDNA using the primers 20 described in Nagata et al. J. Immunol., 162, 1278-1286, 1999 and cloned into pEAK vector (Edge Bio Systems). The cloned CRTH2 gene (40 fig) , inserted into pEAK vector, was transfected into host cells, such as L1. 2 cells, K562 cells, and HEK293 cells, at a cell density of 1x107 cells/50 pi, by using electroporation apparatus (Gene Pulser 11; BioRad) at 250V/1,000 pE. One day after the transfection, puromycin 25 (1 g/ml, Sigma) was added into the cell culture plates. Two weeks aRer the transfection, grown cells were picked up for further growth.

EXAMPLE 2

30 CRTH2 transfectants were washed once with PBS and resuspended in binding buffer (25 mM HEPES pH 7.6, 5 mM MgCl2, I mM CaCI2, 0.5% BSA, 0. 1% NaN3). 100 pi

- 9 - of cell suspension (2 x 105 cells), [3H]-labeled PGD2, and various concentrations of the ramatroban were then mixed in a 96-well U-bottom polypropylene plate and incubated for 60 min at room temperature to allow binding to occur. ARer incubation, the cell suspension was transferred to a filtration plate (#MAFB, 5 Millipore) and washed 3 times with binding buffer containing 0.5 M NaCI.

Scintillant was added to the filtration plate, and radioactivity remaining on the filter was measured by TopCount (Packard), a scintillation counter. Non-specific binding was determined by incubating the cell suspension and [3H]-labeled PGD2 in the presence of 500 nM of unlabeled PGD2. Puromycin-resistant Ll.2 transfectants 10 bound to [3H]- labeled PGD2 with high affinity (KD = 6.3 nM). Ramatroban inhibited the binding of [3H]-labeled PGD2 to CRTH2 transfectants in a dose-dependent manner, with ICso= 45 rim.

EXAMPLE 3

Ca2+ loading buffer was prepared by mixing 5 pi of Fluo-3AM (2 mM in DMSO, final I '1M, Molecular Probes) and 10,1 of pluronic F-127 (Molecular Probes) and diluting the resulting mixture in 10 ml of Ca2+ assay buffer (20 mM HEPES PH 7.6, 0.1% BSA, I mM probenecid, Hanks' solution). The CRTH2 transfectanteds cells 20 which were prepared in Example l were washed with PBS, resuspended in Ca2+ loading buffer at l x 107 cells/ml, and incubated for 60 min at room temperature.

After incubation, cells were washed and resuspended in Ca2+ assay buffer, then dispensed into transparent-bottom 96-well plates (#3631, Costar) at 2 x I Os cells/well. Cells were incubated with various concentrations of ramatroban for 25 5 minutes at room temperature. The emitted 480 nm fluorescence was measured on FDSS6000, a Ca2+ -measurement apparatus (Hamamatsu Photonics, Hamamatsu, Japan). The transfectant showed PGD2induced Ca2+ nuX in a concentration dependent manner. Ramatroban inhibited 10 nM PGD2-induced Ca2+ flux in a dose dependent manner, with IC50= 30 nM.

( - 10 EXAMPLE 4

Animal model: Eosinophilia is induced in the airway of cynomolgus monkeys (Macaca fascicularis, weighing 4.0-9.0 kg) by PGD2 inhalation (10-100 ng/ml, 5 10 min.) with the use of an appropriate nebulizer, such as an ultrasonic nebulizer (TUR-3200, Nihon KohUen). Rarnatroban is given 1 hr before the inhalation. A pediatric fiberoptic bronchoscope (Olympus, model BF3C30) is guided past the carina into the distal lung until the tip of the bronchoscope is wedged in the bronchoalveolar region. A 15 ml aliquot of saline is instilled slowly down one 10 channel of the bronchoscope followed by 2-3 ml of air to completely empty the bronchoscope channel. The fluid is aspirated back into a syringe. Typical recovery volumes are greater than 60%. The volume is recorded. Cell number is counted and cell differentiation is determined by using stained cytospin specimens.

15 EXAMPLE 5

Human polymorphonuclear cells were isolated from heparinized venous blood of healthy donors by laying the blood on Mono-Poly Resolving Medium (ICN Biomedicals, Co.Ltd) and centrifuging it at 400 x g for 30 min. at room temperature.

20 After centrifugation, eosinophils were purified from the the lower layer of polymorphonuclear cells by CD16-negative selection using antiCD16-conjugated magnetic beads (Miltenyi Biotech GmbH).

Human eosinophils were washed with PBS and resuspended in chemotaxis buffer (20 25 mM HEPES pH 7.6, 0.1% BSA, Hanks' solution) at 6 x]o6 cellslml. Fifty p1 of the cel1 suspension (3 x 105 cells/well) was then dispensed into the upper chamber and 30 p1 of ligand solution (PGD2, 10 nM, final concentration) was added to the lower chamber of a 96-well chemotaxis chamber (Diameter = 5 mm, #] 06-5, Neuro Probe).

Cells were preincubated with various concentrations of ramatroban at 37 C for 30 10 minutes. Chemotaxis is then allowed to occur in a humidified incubator at 37 C, 5% CO2 for 2 hours. The number of cells migrating into the lower chamber was

( counted by FACS can (B ecton-D i ckinson) Ramatroban inhibited PGD2 induced chemotaxis of human eosinophils in a dose-dependent manner with IC50=50 nM.

EXAMPLE 6

Human mononuclear cells were isolated from heparinized venous blood of healthy donors by laying the blood on Mono-Poly Resolving Medium (ICN Biomedicals, Co.Ltd) and centrifuging it at 400 x g for 30 min. at loom temperature. After centrifugation, CD4+ T lymphocytes were purified from mononuclear cells by using 10 CD4+ T cell isolation kit (Miltenyi Biotec GmbH).

Human CD4+ T lymphocytes were washed with PBS and resuspended in chemotaxis buffer (20 mM HEPES pH 7.6, 0.1% BSA, Hanks' solution) at 6 x 106 cells/ml.

Fifty al of the cell suspension (3 x 105 cells/well) was then dispensed into the upper 15 chamber and 30 pi of ligand solution (PGD2, 10 nM, final concentration) was added to the lower chamber of a 96-well chemotaxis chamber (Diameter = 3 mm, #106-3, Neuro Probe). Cells were preincubated with various concentrations of compound at 37 C for 10 minutes. Chemotaxis is then allowed to occur in a humidified incubator at 37 C, 5% CO2 for 4 hours. The number of cells migrating into the lower chamber 20 was counted by FACScan (Becton-Dickinson). Ramatroban inhibited PGD2-induced chemotaxis of CD4+ T lymphocytes in a dose- dependent manner with IC50= 60 nM.

Claims

f - 12 CLAIMS

(1) A CRTH2 modulator comprising the compound represented by the formula (I) POOH 0N,,0 H H:3:

5 (1)

(+)-3R)-3-(4-fluorobenzenesulfonamido)- 1,2,3,4-tetrahydrocarbazole-9 propionic acid or a pharmaceutical acceptable salt thereof as an effective agent. 10 (2) A CRTH2 modulator as claimed in claim 1, wherein said CRTH2 modulator is effective for treating or preventing eosinophil- related diseases or basophil related diseases.

(3) A CRTH2 modulator as claimed in claim 2, wherein said eosinophilrelated; 15 diseases are one selected from the group consisting of ChurgStrauss syn drome and sinusitis. I (4) A CRTH2 modulator as claimed in claim 2, wherein said basophil-related diseases are one selected from the group consist of basophilic leukemia and 20 basophilic leukocytosis.

(5) Use of the compound represented by the formula (I) as claimed in claim 1, for manufacturing a medicament for the treatment and/or prophylaxis of eosinophil-related diseases or basophil-related diseases.

( - 13 (6) Process for controlling eosinophil-related diseases or basophil-related di seases in humans and animals by administration of an effective amount of the compound represented by the formula (I).