Field of the invention

This invention relates to the prevention and treatment of thrombus formation using a histamine H1 receptor antagonist.

Background of the invention

Thrombus formation is the precipitating cause of diseases such as acute coronary syndromes, which comprises unstable angina, non-ST-segment-elevation myocardial infarction, or ST-segment-elevation myocardial infarction.

Current treatment of thrombus formation, e.g. in acute coronary syndrome, consist in administering aspirin (inhibition of platelet aggregation), clopidogrel (inhibition of platelet aggregation), heparin (inactivation of coagulation factors), glycoprotein llb/llla (GPIIbIIIa) antagonists such as abciximab or eptifibatide (inhibition of platelet aggregation), or related compounds. Combination of all these mentioned drugs is more effective than administering each of them separately. Typically such combination is given to a patient with acute coronary syndrome.

H1 receptor antagonists block the histamine type 1 receptor. They are currently used mainly for the symptomatic treatment of allergic diseases such as urticaria, allergic rhinitis, or anaphylactic reactions.

Mast cells, which produce and release histamine have been found both in coronary atherectomy specimens as well as in the adventitia of infarct-related arteries. Both the presence of mast cells [Forman, M. B., et al., N Engl J Med, 1985. 313(18):1138-41] and an elevation of the plasma histamine concentration [Sakata, Y., et al., Am J Cardiol, 1996. 77(12): 1121-6] has been described in patients with variant angina. However, these findings so far have not implied that diseases such as variant angina and acute coronary syndromes should be treated by H-i receptor antagonists because a link between thrombus formation and histamine has not been known.

Inflammation is involved in the pathogenesis of acute coronary syndromes as it can induce rupture of atherosclerotic plaques. According to the current recommendations by the Swiss Society of Cardiology, the role of inflammation in acute coronary syndromes is not clear enough to justify a general anti-inflammatory treatment. Singh, M., et al. [J Cardiovasc Pharmacol Then, 2003 June. 8(2): 135-48] propose to use histamine H1 receptor antagonists to impair the intense inflammatory reaction following reperfusion of ischemic myocardium. However, the authors did not recognize nor mention the usefulness of histamine H-i receptor antagonists for preventing and treating thrombus formation in acute coronary syndromes and variant angina.

The central phenomenon in acute coronary syndromes is thrombus formation leading to vascular occlusion. All the established treatments for acute coronary syndromes target thrombus formation. There is a need for additional effective treatment and prevention of thrombus formation which would complement and expand the existing methods of treatment and prevention.

Summary of the invention

The present invention relates to a method of treating and/or preventing thrombus related diseases, such as variant angina, acute coronary syndromes, transient ischemic attack, brain stroke, peripheral arterial occlusive disease, and the like, comprising administering a histamine Hi receptor specific antagonist, and the use of histamine H1 receptor specific antagonist in such a treatment and in the manufacture of medicaments for treating and/or preventing thrombus related diseases.

This novel method of treatment and prevention is based on the findings that tissue factor initiating thrombus formation is induced by histamine.

Brief description of the Figures

Fig. 1 : Histamine induces tissue factor (TF) expression in human aortic endothelial cells (HAEC) and human aortic vascular smooth muscle cells (HAVSMC). Bars represent at least 4 different experiments; error bars represent standard error of the mean. Representative blots are shown; all blots are equalized to acetylated tubulin expression (aT). Fig. 1A: Concentration dependency of histamine induced TF expression in HAEC. Maximal TF expression is about 18x the basal expression with 10"5 mol/l histamine. X axis: -log10 concentration of histamine. Y axis: Values are given as n-fold increase of unstimulated control. (*) p < 0.02 compared to unstimulated control. Fig. 1 B: Time dependency of TF expression in HAEC after stimulation with histamine. TF induction is maximal after 5 hours of stimulation (n=3). X axis: hours of stimulation. Y axis: Values are given as percentage of TF expression at 5 hours. Fig. 1 C: Histamine and TF expression in HAVSMC. Histamine increases basal TF expression in HAVSMC 3-fold. X axis: His: + or - indicates presence or absence of 10"5 mol/l histamine. Y axis: Values are given as n-fold increase of unstimulated control. (*) indicates significant increase in TF expression as compared to control (p < 0.05).

Fig. 2: Histamine induced increase in TF expression and TF surface activity in HAEC is completely inhibited by the H1 receptor blocker mepyramine. Bars represent at least 4 different experiments; error bars represent standard error of the mean. Representative blots are shown; all blots are equalized to acetylated tubulin expression (aT). Fig 2A: Upper panel: Preincubation of HAEC with mepyramine (Me) completely prevents histamine (His) induced TF expression in a concentration dependent manner. X axis: His: + or - indicates presence or absence of 10'5 mol/l histamine. Me: -logio concentration of mepyramine. Y axis: Values are given as percentage of TF expression with 10"5 mol/l histamine only. (*) indicates significant decrease in TF expression as compared to stimulation with histamine (p < 0.0001). Middle and lower panel: Histamine induced TF expression in HAEC is suppressed in a similar manner after preincubation with the selective Hi receptor blockers chlorpheniramine (ChI, n=2) and diphenhydramine (Di, n=2). X axis: His: + or - indicates presence or absence of 10"5 mol/l histamine. ChI: -logio concentration of chlorpheniramine Di: -logio concentration of diphenhydramine Fig 2B: Pretreatment of HAEC with cimetidine (Cim) does not affect histamine induced expression of TF. X axis: His: + or - indicates presence or absence of 10"5 mol/l histamine. Cim: -logio concentration of cimetidine. Y axis: Values are given as percentage of TF expression with histamine only. Fig. 2C: Stimulation with histamine increases TF surface activity in HAEC. Increase in activity is completely suppressed by incubation with mepyramine (10"5 mol/l). X axis: His: + or - indicates presence or absence of 10"5 mol/l histamine. Me: + or - indicates presence or absence of 10"5 mol/l mepyramine Y axis: Values are given as n-fold increase of unstimulated control. (*) p < 0.01 as compared to control; (#) p < 0.01 as compared to stimulation with histamine. Fig. 2D: Pretreatment with mepyramine (10"5 mol/l), but not cimetidine (10~5 mol/l), significantly reduces histamine-induced increase in TF expression in HAVSMC. X axis: His: + or - indicates presence or absence of 10~5 mol/l histamine. Me: + or - indicates presence or absence of 10"5 mol/l mepyramine. Cim: + or - indicates presence or absence of 10"5 mol/l cimetidine. Y axis: Values are given as percentage of TF expression with histamine only. (*) p < 0.0001 as compared to control; (#) p < 0.0001 as compared to stimulation with histamine.

Detailed description of the invention

The present invention relates to a method of treating thrombus related diseases comprising administering a histamine H-i receptor specific antagonist, and the use of histamine H1 receptor specific antagonist in such a treatment and in the manufacture of medicaments for treating thrombus related diseases.

Likewise the present invention relates to a method of preventing thrombus related diseases, such as variant angina, acute coronary syndromes, transient ischemic attack, brain stroke, peripheral arterial occlusive disease, and the like, in particular variant angina and acute coronary syndromes, comprising administering a histamine H1 receptor specific antagonist, and the use of histamine H1 receptor specific antagonist in such a treatment and in the manufacture of medicaments for preventing thrombus related diseases.

Thrombus related diseases considered are, for example, variant angina, acute coronary syndromes, transient ischemic attack, brain stroke, peripheral arterial occlusive disease, and the like, in particular variant angina and acute coronary syndromes. Acute coronary syndromes comprise, for example, unstable angina, non-ST-segment-elevation myocardial infarction and ST-segment-elevation myocardial infarction. Hi receptor specific antagonists are widely known. An extensive report may be found e.g. in Wolff, M. E., Ed., Burger's Medicinal Chemistry and Drug Discovery, 5th edition, Vol. 5, John Wiley & Sons. H1 receptor specific antagonists are, for example, disclosed in patents EP-B-085 959, EP-B-133 534, EP-B-270 818, EP-B-271 192, US 2,712,020, US 4,254,129, US 4,727,076, US 4,837,325, and US 6,420,560. H1 receptor specific antagonists considered are, e.g., carbinoxamine, hydroxyzine, meclizine, promethazine, doxylamine, acrivastine, rupatadine, epinastine, fluticasone, mizolastine, ebastine, fexofenadine, cetirizine, levocetirizine, astemizole, loratidine, desloratidine, clemastine, dimetinden, chlorpheniramine, bropheniramine, terfenadine, oxatomide, azelastine, mepyramine (pyrilamine), or diphenhydramine. However, the invention is not restricted to the mentioned H1 receptor specific antagonists, but extends to all H1 receptor specific antagonists.

Preferred H1 receptor specific antagonists are the compound known as mepyramine, also named pyrilamine, N,N-dimethyl-N'-(4-methoxybenzyl)-N'-(2-pyridyl)ethylenediam ine, e.g. applied as the maleate salt; chlorpheniramine, 2-p-chloro-α-(2-dimethylaminoethyl) benzyl]pyridine; e.g. applied as the maleate salt; diphenhydramine, 2-diphenylmethoxy- N.N-dimethyl-ethylamine, preferably as the hydrochloride; cetirizine, [2-[4-[4- (chlorophenyOphenylmethyrj-i-piperazinylJethoxyjacetic acid; loratidine, ethyl 4-(8-chloro- 5,6-dihydro-11 H-benzo[5,6]cyclohepta[1 ,2-b] pyridin-11 -ylidene)-1 -piperidinecarboxylate; clemastine, (+)(2R)-2-[2-[(R)-p-chloro-α-methyl-α-phenylbenzyl-1 -oxy]ethyl]-1 - methylpyrrolidine, preferably as the fumarate salt; and dimetinden, N,N-dimethyl-3-[1-(2- pyridyl)ethyl]inden-2-ethylamine, e.g. applied as the maleate salt. Particularly preferred are chlorpheniramine, diphenhydramine, cetirizine, loratidine, clemastine, and dimetinden.

One aspect of the invention relates to a method of treating thrombus related diseases comprising administering a Hi receptor specific antagonist in a quantity effective against thrombus related diseases to a mammal in need thereof, for example to a human requiring such treatment. The treatment may be for prophylactic or therapeutic purposes. For the administration, the H1 receptor specific antagonist is preferably in the form of a pharmaceutical preparation comprising the H-i receptor specific antagonist in chemically pure form and optionally a pharmaceutically acceptable carrier and optionally adjuvants. The Hi receptor specific antagonist is used in an amount effective against thrombus related diseases. The dosage of the active ingredient depends upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, the mode of administration, and whether the administration is for prophylactic or therapeutic purposes. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 1 mg to approximately 500 mg, preferably from approximately 1 mg to approximately 100 mg, of a H1 receptor specific antagonist.

Pharmaceutical compositions for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as intravenous, intramuscular or subcutaneous administration, are especially preferred. The pharmaceutical compositions comprise from approximately 1% to approximately 95% active ingredient, preferably from approximately 20% to approximately 90% active ingredient.

For parenteral administration including intracoronary, intracerebrovascular, or peripheral vascular injection/infusion preference is given to the use of solutions of the H1 receptor specific antagonist, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example, can be made up shortly before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, viscosity-increasing agents, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes.

For oral pharmaceutical preparations suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, and also binders, such as starches, cellulose derivatives and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, flow conditioners and lubricants, for example stearic acid or salts thereof and/or polyethylene glycol. Tablet cores can be provided with suitable, optionally enteric, coatings. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient. Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The capsules may contain the active ingredient in the form of granules, or dissolved or suspended in suitable liquid excipients, such as in oils. Transdermal application is also considered, for example using a transdermal patch, which allows administration over an extended period of time, e.g. from one to twenty days.

Another aspect of the invention relates to the use of H1 receptor specific antagonists in the treatment of thrombus related diseases and in the manufacture of medicaments for treating thrombus related diseases. Such medicaments are manufactured by methods known in the art, especially conventional mixing, coating, granulating, dissolving or lyophilizing.

A H1 receptor specific antagonist can be administered alone or in combination with one or more other therapeutic agents, possible combination therapy taking the form of fixed combinations of a Hi receptor specific antagonist and one or more other therapeutic agents known in the treatment of thrombus related diseases, the administration being staggered or given independently of one another, or being in the form of a fixed combination.

Possible combination partners considered are aspirin, clopidogrel or other inhibitors of platelet aggregation, heparin or other inactivators of coagulation factors, GPIIbIIIa antagonists such as abciximab, eptifibatide or tirofiban, beta blockers, ACE inhibitors, HMG-CoA-reductase inhibitors, or related compounds.

Tissue Factor (TF), a 263 residue membrane-bound glycoprotein, is a key enzyme for initiation and propagation of thrombus formation; indeed, it is thought to play a pivotal role in acute coronary syndromes (ACS) [Moons, A.H., Levi, M. and Peters, R.J., Cardiovasc Res, 2002. 53(2):313-25]. The present invention is based on the observation that histamine induces TF expression and TF activity in cultured human aortic endothelial cells (HAEC) and human aortic vascular smooth muscle cells (HAVSMC).This effect is completely abolished by a H-i receptor antagonist, e.g. by mepyramine.

Augmented TF expression in HAEC and HAVSMC after stimulation with histamine is consistent with atherosclerosis as an inflammatory disease. Indeed, histamine induced TF expression may represent an important link between vascular inflammation and thrombus formation. Further, histamine evokes potent vascular contractions through activation of the Hi receptor [Okumura, K., et al., J Am Coll Cardiol, 1991. 17(2):338-45]. Hence, histamine induced vasospasm and initiation of thrombus formation are both mediated through the same receptor. The H-i receptor antagonist mepyramine completely inhibits the histamine induced increase in TF expression and surface activity. Taken together, these findings point to a previously unrecognized link between inflammation, vasospasm, and thrombus formation, and form the basis of the treatment and/or prevention of variant angina and acute coronary syndromes according to the invention.

Examples

HAEC and HA VSMC are cultured using the procedure described [Eto, M., et al., Circulation, 2002. 105(15): 1756-9]. Cells are grown to confluence in 6 cm culture dishes and rendered quiescent for 24 hours before stimulation with histamine. Mepyramine and cimetidine (both from Sigma, Germany) are added to the dishes 30 minutes prior to stimulation. TF protein expression is determined by Western Blot analysis as described by Eto et al. [loc. cit.]. An antibody to human TF (American Diagnostica) is used at 1 :1000 dilution. All blots are normalized to tubulin expression (anti-acetylated tubulin, 1 :5000 dilution, Sigma). TF surface activity is analyzed with a commercially available kit (American Diagnostica) according to the manufacturer's recommendations with some modifications as described [Cui, M. Z., et al., Arterioscler Thromb Vase Biol, 2003. 23(2):224-30]. A colorimetric assay for the detection of lactate dehydrogenase (LDH) is used according to the manufacturer's recommendations (Roche, Germany). Data are presented as mean +/- standard error of the mean. Unpaired t test is applied for comparison of two groups, ANOVA with Bonferroni correction for three or more groups. A p value < 0.05 is considered significant.

Stimulation of HAEC with histamine leads to a concentration- and time-dependent induction of TF expression up to 18-fold the basal level; maximal effects were observed after stimulation with 10"5 mol/l histamine (Fig. 1A) for 5 hours (Fig. 1 B). Further, the increase in total TF protein expression in response to stimulation with histamine is reflected in a 4-fold increase in TF surface activity after 5 hours of stimulation with 10~5 mol/l histamine (Fig. 2C). Similarly, TF expression is enhanced in HAVSMC after stimulation with histamine; maximal increase is about 3-fold as compared to control conditions (Fig. 1C). Histamine is known to exert most of its actions on cells through the Hi and / or H2 receptor. To determine the receptor involved in histamine-induced TF expression, the cells are pretreated with mepyramine or cimetidine to block the H-i and H2 receptor, respectively. Pretreatment with mepyramine completely abolishes histamine induced TF expression in HAEC (Fig. 2A) while it is not affected by cimetidine (Fig. 2B). Preincubation with chlorpheniramine and diphenhydramine, two other selective Hi receptor antagonists, equally prevents histamine induced TF expression in HAEC (Fig. 2A). Similarly, histamine induced increase in TF surface activity is completely inhibited by mepyramine (Fig. 2C). Pretreatment of histamine stimulated HAVSMC with mepyramine, but not cimetidine, significantly reduces TF expression (Fig. 2D). No cytotoxic effect of histamine, cimetidine, mepyramine, chlorpheniramine or diphenhydramine is observed at the concentrations used as measured by LDH-release into the cell supernatant (data not shown).

Biologically active TF is located at the cell surface. The increase in histamine induced expression of TF in HAEC is indeed paralleled by an increased TF surface activity. The relative increase of the latter is less pronounced as the increase in total protein expression. The distribution of TF in several cellular compartments as well as the expression of encrypted TF at the cell surface might account for this difference [Rao, L.V. and Pendurthi, U. R., Blood Coagul Fibrinolysis, 1998. 9 Suppl 1 :S27-35]. In contrast to unstimulated endothelial cells, vascular smooth muscle cells display a basal expression of TF. This may assure the rapid formation of a localized nidus of coagulation following endothelial denudation. Although clearly inducible, this basal expression might account for the observation that maximal TF expression after stimulation with histamine results in a 3- fold increase above basal expression in HAVSMC, while the increase is much more pronounced in HAEC.