(12) Patent Application Publication (10) Pub. No.: US 2006/0120972 A1 Engels Et Al

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US 2006O120972A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0120972 A1 Engels et al. (43) Pub. Date: Jun. 8, 2006

(54) 9-(N-METHYL-PIPERIDYLIDEN-4)- Related U.S. Application Data THOXANTHENE FOR TREATMENT OF PULMONARY HYPERTENSION (60) Provisional application No. 60/626.249, filed on Nov. 9, 2004. (76) Inventors: Peter Engels, Gladbach (DE); Beate Publication Classification Schmitz, Koln (DE) (51) Int. Cl Correspondence Address: 3. if: 308: KNOBBE MARTENS OLSON & BEAR LLP A 6LX 9/70 (2006.015 2O4O MAN STREET A6IL 9/04 (2006.01) FOURTEENTH FLOOR (52) U.S. Cl...... 424/46; 514/320: 424/449 IRVINE, CA 92614 (US) 57 ABSTRACT (21) Appl. No.: 11/255,567 (57) Pimethixene is used as a medicament for the treatment of (22) Filed: Oct. 21, 2005 pulmonary hypertension. US 2006/0120972 A1 Jun. 8, 2006

9-(N-METHYL-PIPERIDYLIDEN-4)- Additionally, current evidence strongly Supports a therapeu THOXANTHENE FOR TREATMENT OF tic role for selective 5-HT, receptor antagonists that will PULMONARY HYPERTENSION offer distinct therapeutic advantages collectively in efficacy, rapidity of onset and absence of side effects. Such agents are CROSS-REFERENCE TO RELATED expected to be useful in the treatment of hypertension, APPLICATION disorders of the gastrointestinal track (e.g., irritable bowel 0001. This application claims priority under 35 U.S.C. S syndrome, hypertonic lower esophageal sphincter, and 119(e) to U.S. Provisional Application No. 60/626.249, filed motility disorders), restenosis, asthma and obstructive air Nov. 9, 2004, which is entirely incorporated herein by way disease, and prostatic hyperplasia (e.g., benign prostatic reference. hyperplasia). 0008 EP 1270.568 and WO 03/35646 describe selective BACKGROUND OF THE INVENTION 5HT2B receptor antagonists which are derivatives of 4-(thio or selenoxanthene-9-ylidene)-piperidine or acridine having 0002) 1. Field of the Invention in any case several side groups in the aromatic ring system. 0003. The present invention relates to the use of 9-(N- 0009. The involvement of 5-HT receptor activation in the methyl-piperidyliden-4)-thioxanthene as a medicament for development of pulmonary hypertension has been described the treatment of diseases aggravated by increased levels of by M. MacLean in TiPS, vol. 20, pg. 490-495 (1999). 5-HT and mediated by 5HT receptors. However, in this review article, the disorder state is referred 0004 2. Description of the Related Art to activation of 5HT and 5HT2A receptors. 0005 Serotonin, a neurotransmitter with mixed and com 0010 J.-M. Launay et al. describe in Nature Medicine plex pharmacological characteristics, was first discovered in Vol. 8, No. 10 (2002), p. 1129-1135 that serotonin as well as 1948, and subsequently has been the subject of substantial other agonists of the 5-HT2B receptor Support the develop ment of pulmonary hypertension. Further, the study showed research. Serotonin, also referred to as 5-hydroxytryptamine that 5HT2B receptor expression is upregulated in pulmonary (5-HT), acts both centrally and peripherally on discrete hypertension. 5-HT receptors. Currently, fourteen subtypes of serotonin receptor are recognized and delineated into seven families, 0011 Pimethixene is the common name of 9-(N-methyl 5-HT, to 5-HT7. Within the 5-HT, family, 5-HT, A, 5-HT piperidyliden-4)-thioxanthene. Pimethixene was developed and 5-HT. Subtypes are known to exist. These subtypes in the 1960s and has been used for the treatment of sleep share sequence homology and display similarities in their disorders, hyperactivity, anxiety, allergy, as bronchodilator, specificity for a wide range of ligands. Nomenclature and and for anesthesia. classification of 5-HT receptors have been reviewed (see Martin and Humphrey, Neuropharm. 1994, 33, 261-273 and SUMMARY OF THE INVENTION Hoyer et al., Pharm. Rev. 1994, 46, 157-203). 0012 Preferred embodiments provide for a method of treating a disease aggravated by increased levels of 5-HT 0006 The 5-HT, receptor, initially termed 5-HT, or comprising administering to a Subject a compound accord serotonin receptor like (SRL), was first characterized in rat ing to the formula: isolated stomach fundus (see Clineschmidt et al., J. Phar macol. Exp. Ther. 1985, 235, 696–708; Cohen and Wit tenauer, J. Cardiovasc. Pharmacol. 1987, 10, 176-181) and initially cloned from rat (see Foguet et al., EMBO 1992, 11, 3481-3487) followed by the cloning of the human 5-HT, receptor (see Schmucket al., FEBS Lett. 1994, 342, 85-90; Kursar et al., Mol. Pharmacol. 1994, 46, 227-234). The closely related 5-HT, receptor, widely distributed in the human brain, was first characterized as a 5-HT Subtype (see Pazos et al., Eur. J. Pharmacol. 1984, 106,539-546) and was Subsequently recognized as belonging to the 5-HT receptor family (see Pritchett et al., EMBO J. 1988, 7. CH3 4135-4140). 0007 Because of the similarities in the pharmacology of or a pharmaceutically acceptable salt thereof. ligand interactions at 5-HTP and 5-HT2 receptors, many of the therapeutic targets that have been proposed for 5-HT DETAILED DESCRIPTION OF THE receptor antagonists are also targets for 5-HT, receptor PREFERRED EMBODIMENT antagonists. Current evidence strongly supports a therapeu tic role for 5-HT2 receptor antagonists in treating anxi 0013 The preferred embodiments provide a new medi ety (e.g., generalized anxiety disorder, panic disorder and cament comprising a compound effective for treatment of obsessive compulsive disorder), alcoholism and addiction to disease aggravated by increased levels of 5-HT in blood or other drugs of abuse, depression, migraine, steep disorders, blood vessels, such as, but not limited to, pulmonary hyper feeding disorders (e.g., anorexia nervosa) and priapism. tension. US 2006/0120972 A1 Jun. 8, 2006

0014. The preferred embodiments provide the use of a right side of the heart, which pumps blood into the lungs, has compound according to formula: to pump against a higher resistance to blood flow. This makes it more difficult to pump the blood through the lungs, particularly when increased flow is needed, as when a Subject exercises. 0020. The compound of the preferred embodiments has been found to be a human 5-HT, receptor antagonist. Affin ity for the 5-HT, receptors was demonstrated using an in vitro binding assay utilizing cloned human 5-HT2B receptors radiolabelled with H-5HT, as well as luciferase reporter gene assays as shown in the examples. Antagonist properties for the human 5-HT, receptor were shown by counter N screening at human 5-HT, and 5-HT, receptors. Affinity CH3 for the human H receptor was determined using an in vitro binding assay utilizing cloned human H receptors radiola belled with H-mepyramine and antagonistic properties also called pimethixene, or a pharmaceutically acceptable were determined by a luciferase reporter gene assay, as salt thereof for the development of a medicament for the shown in the examples. treatment of disease aggravated by increased levels of 5-HT, 0021 Accordingly, the compound of the preferred Such as, but not limited to, pulmonary hypertension. embodiments is useful for treating diseases which can be 0015 The preferred embodiments further relate to a ameliorated by blockade of 5-HT, and in particular 5HT pharmaceutical composition comprising Such a compound receptors. Because of the similarities in the pharmacology of or a pharmaceutically acceptable salt thereof, in admixture ligand interactions at 5-HT2 and 5-HT2B receptors, many of with one or more pharmaceutically acceptable carriers. the therapeutic targets that have been proposed for 5-HT receptor antagonists are also targets for 5-HT2B receptor 0016 “Pharmaceutically acceptable salt” refers to those antagonists. salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise 0022 Experimental evidence supports a therapeutic role undesirable, formed with inorganic acids such as, but not for 5-HT, receptor antagonists in treating pulmonary limited to, hydrochloric acid, hydrobromic acid, sulfuric hypertension. In pulmonary hypertension, one of the most acid, nitric acid, phosphoric acid and the like, and organic profound increases in vascular responsiveness is observed acids such as, but not limited to, acetic acid, propionic acid, for serotonin. Two lines of evidence imply that this results glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic from a Switch in the receptor mediating vasoconstriction acid, Succinic acid, maleic acid, fumaric acid, tartaric acid, from predominantly 5-HT, to predominantly 5-HT. citric acid, benzoic acid, cinnamic acid, mandelic acid, First, serotonin induced contractions of isolated blood ves methanesulfonic acid, ethanesulfonic acid, p-toluene sels from hypertensive animals become resistant to block by Sulfonic acid, salicylic acid, ascorbic acid and the like. selective 5-HT2A receptor antagonists, but remain sensitive to non-selective 5-HT2B receptor antagonists. Second, there 0017. The term “treatment” as used herein covers any is an increase in 5-HT, receptor mRNA in vessels from treatment of a disease in a mammal, particularly a human, hypertensive animals (see Watts et al., J. Pharmacol. Exp. and includes: Ther. 1996, 277, 1103-13 and Watts et al., Hypertension (i) preventing the disease from occurring in a Subject which 1995, 26, 1056-1059). This hypertension-induced shift in may be predisposed to the disease but has not yet been the population of receptor Subtype mediating constrictor diagnosed as having it; responses to 5-HT Suggests that selective block of vasocon strictor 5-HT, receptors may be of therapeutic benefit in the (ii) inhibiting the disease, i.e., arresting its development; or treatment of pulmonary hypertension. (iii) relieving the disease, i.e., causing regression of the disease. Pharmaceutical Compositions 0023. In applying the compound of the preferred embodi 0018. The term “disease state”, “disease', or “disease ments to treatment of the above conditions, administration aggravated by increased levels of 5-HT” as used herein is of the active compound and salts described herein can be via intended to cover all disease states which are mediated by any of the accepted modes of administration, including 5-HT, receptors due to increased serotonin level and/or inhalation, oral, parenteral and otherwise systemic route of plasma protein extravasation from the blood into the Sur administration. Any pharmaceutically acceptable mode of rounding tissue. Such disease states include, but are not administration can be used, including Solid, semi-solid or limited to, pulmonary hypertension. liquid dosage forms, such as, for example, tablets, Supposi 0.019 Pulmonary hypertension is an abnormal elevation tories, pills, capsules, powders, liquids, Suspensions, aero of the pressure in the blood vessels of the lungs, also called sols or the like, preferably in unit dosage forms suitable for “high blood pressure' of the lungs. In normal lungs, the single administration of precise dosages, or in Sustained or pressure in the blood vessels is about one-quarter of the controlled release dosage forms for the prolonged adminis pressure in the arteries of the body and can temporarily adapt tration of the compound at a predetermined rate. The com to increased pressures that occur during exercise. In pulmo positions will typically include a conventional pharmaceu nary hypertension, the Small arteries in the lungs are too tical carrier or excipient and at least the compound of the narrow, so the pressure rises in these vessels. As a result, the preferred embodiments or one of the pharmaceutically US 2006/0120972 A1 Jun. 8, 2006

acceptable salts thereof and, in addition, may include other 0029 DPIs typically administer a therapeutic agent in medicinal agents, pharmaceutical agents, carriers, adjuvants, the form of a free flowing powder that can be dispersed in etc. a patient’s air-stream during inspiration. In order to achieve 0024. The amount of the compound of the preferred a free flowing powder, the therapeutic agent can be formu embodiments administered can be dependent on the Subject lated with a Suitable excipient, such as lactose or starch. A being treated, the severity of the affliction, the manner of dry powder formulation can be made, for example, by administration and the judgment of the prescribing physi combining dry lactose having a particle size between about cian. However, an effective dose for inhalation, oral, 1 Lum and about 100 um with micronized particles of a parenteral and otherwise systemic routes of administration is pharmaceutical salt of the active agent and dry blending. in the range of about 0.01 to about 20 mg/kg/day, preferably Alternative, the agent can be formulated without excipients. about 0.1 to about 10 mg/kg/day. For an average 70 kg The formulation is loaded into a dry powder dispenser or human, the dose would be about 0.7 to about 1400 mg per into inhalation cartridges or capsules for use with a dry day, or preferably about 7 to about 700 mg/day. powder delivery device. 0025. One of ordinary skill in the art of treating such 0030) Examples of DPI delivery devices provided com diseases will be able, without undue experimentation and in mercially include Diskhaler (GlaxoSmithKline, Research reliance upon personal knowledge and the disclosure herein, Triangle Park, N.C.) (see, e.g., U.S. Pat. No. 5,035,237): to ascertain a therapeutically effective amount of the com Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 6,378, pound for a given disease. 519: Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769); and Rotahaler (GlaxoSmithKline) 0026. For solid compositions, conventional non-toxic (see, e.g., U.S. Pat. No. 4,353.365). Further examples of Solid carriers include, for example, pharmaceutical grades of suitable DPI devices are described in U.S. Pat. Nos. 5,415, mannitol, lactose, cellulose, cellulose derivatives, sodium 162, 5,239,993, and 5,715,810 and references therein. crosscarmeillose, starch, magnesium Stearate, sodium sac charin, talcum, glucose, Sucrose, magnesium carbonate, and 0031 MDI’s typically discharge a measured amount of the like may be used. The active compound as defined above therapeutic agent using compressed propellant gas. Formu may be formulated as Suppositories using, for example, lations for MDI administration include a solution or suspen polyalkylene glycols, e.g. PEG (polyethyleneglycol) or PEG sion of active ingredient in a liquefied propellant. While derivatives, acetylated triglycerides and the like, as the chlorofluorocarbons, such as CC1F, conventionally have carrier. Liquid pharmaceutically administrable compositions been used as propellants, due to concerns regarding adverse can, for example, be prepared by dissolving, dispersing, etc. affects of such agents on the ozone layer, formulations using an active compound as defined above and optional pharma hydrofluoroalkanes (HFA), such as 1,1,1,2-tetrafluoroethane ceutical adjuvants in a carrier, Such as, for example, water, (HFA 134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA saline, aqueous dextrose, glycerol, ethanol, and the like, to 227) have been developed. Additional components of HFA thereby form a solution or suspension. If desired, the phar formulations for MDI administration include co-solvents, maceutical composition to be administered may also contain Such as ethanol or pentane, and Surfactants, such as Sorbitan minor amounts of nontoxic auxiliary Substances such as trioleate, oleic acid, lecithin, and glycerin. (See, for wetting or emulsifying agents, pH buffering agents and the example, U.S. Pat. No. 5.225,183, EP 0717987 A2, and WO like, for example, Sodium acetate, Sorbitan monolaurate, 92/22286.) triethanolamine sodium acetate, Sorbitan monolaurate, tri 0032. Thus, a suitable formulation for MDI administra ethanolamine oleate, etc. The composition or formulation to tion can include from about 0.01% to about 5% by weight be administered will, in any event, contain a quantity of the of a pharmaceutical salt of active ingredient, from about 0% active compound(s) in an amount effective to alleviate the to about 20% by weight ethanol, and from about 0% to about symptoms of the Subject being treated. 5% by weight surfactant, with the remainder being the HFA 0027 Pimethixene or a pharmaceutically acceptable salt propellant. In one approach, to prepare the formulation, thereof may be formulated in a suitable form for adminis chilled or pressurized hydrofluoroalkane is added to a vial tration by inhalation (e.g. via an aerosol) or insufflation containing the pharmaceutical salt of active compound, ethanol (if present) and the Surfactant (if present). To prepare (either through the mouth or nose). a Suspension, the pharmaceutical salt can be provided as 0028. One manner for administering a compound of the micronized particles. The formulation is loaded into an preferred embodiments is inhalation. Inhalation is an effec aerosol canister, which forms a portion of an MDI device. tive means for delivering an agent directly to the respiratory Examples of MDI devices developed specifically for use tract. There are three general types of pharmaceutical inha with HFA propellants are provided in U.S. Pat. Nos. 6,006, lation devices: nebulizer inhalers, dry powder inhalers 745 and 6,143,277. (DPI), and metered-dose inhalers (MDI). Nebulizer devices produce a stream of high Velocity air that causes a thera 0033. In an alternative preparation, a suspension formu peutic agent to spray as a mist which is carried into the lation is prepared by spray drying a coating of Surfactant on patient's respiratory tract. The therapeutic agent is formu micronized particles of a pharmaceutical Salt of active lated in a liquid form Such as a solution or a suspension of compound. (See, for example, WO 99/53901 and WO micronized particles of respirable size, where micronized is 00/61108.) For additional examples of processes of prepar typically defined as having about 90% or more of the ing respirable particles, and formulations and devices Suit particles with a diameter of less than about 10 Lum. A typical able for inhalation dosing see U.S. Pat. Nos. 6,268,533, formulation for use in a conventional nebulizer device is an 5,983,956, 5,874,063, and 6,221,398, and WO99/55319 and isotonic aqueous solution of a pharmaceutical Salt of the WO OO/30614. active agent at a concentration of the active agent of between 0034. With regard to construction of the delivery device, about 0.05 g/mL and about 10 mg/mL. any form of aerosolization known in the art, including but US 2006/0120972 A1 Jun. 8, 2006

not limited to nebulization, atomization or pump aerosoliza Preferably, the composition comprises about 0.2 to about 2% tion of a liquid formulation, and aerosolization of a dry by weight of pimethixene or a pharmaceutically acceptable powder formulation, can be used in the practice of the salt thereof in solution. preferred embodiments. A delivery device that is uniquely designed for administration of Solid formulations is envi 0041 Transdermal or “pulsed transdermal administra sioned. Often, the aerosolization of a liquid or a dry powder tion may be supported by cremes, gels, dispersions and the formulation will require a propellent. The propellent can be like. any propellent generally used in the art. Examples of useful 0042. The composition of the preferred embodiments propellents include chlorofluorocarbons, hydrofluorocar may also be formulated for administration in any convenient bons, hydrochlorofluorocarbons, and hydrocarbons, includ way by analogy with other topical compositions adapted for ing trifluoromethane, dichlorodifluoroethane, dichlorotet use in mammals. These compositions may be presented for rafluoroethanol, and 1,1,1,2-tetrafluoroethane, and use in any conventional manner with the aid of any of a wide combinations thereof. variety of pharmaceutical carriers or vehicles. For such topical administration, a pharmaceutically acceptable non 0035) Systems of aerosol delivery, such as the pressur toxic formulation can take the form of semisolid, liquid, or ized metered dose inhaler and the dry powder inhaler are Solid, Such as, for example, gels, creams, lotions, Solutions, disclosed in Newman, Aerosols and the Lung, Clarke, S. W. Suspensions, ointments, powders, or the like. As an example, and Davia, D. editors, pp 197-22 and may be used in the active component may be formulated into a syrup or gel connection with the preferred embodiments. using ethanol, propylene glycol, propylene carbonate, poly 0036) Dosage forms or compositions containing pime ethylene glycols, diisopropyl adipate, glycerol, water, etc., thixene compound or a pharmaceutically acceptable salt with appropriate gelling agents, such as Carbomers, Klucels, thereof in the range of about 0.25 to about 95% by weight etc. If desired, the formulation may also contain minor with the balance made up from non-toxic carrier may be amounts of non-toxic auxiliary Substances such as preser prepared. Vatives, antioxidants, pH buffering agents, surface active agents, and the like. Actual methods of preparing Such 0037 For oral administration, a pharmaceutically accept dosage forms are known, or will be apparent, to those skilled able non-toxic composition is formed by the incorporation in the art; for example, see Remington's Pharmaceutical of any of the normally employed excipients, such as, for Sciences, Mack Publishing Company, Easton, Pa., 19th example pharmaceutical grades of mannitol, lactose, cellu Edition, 1995. lose, cellulose derivatives, sodium crosscarmellose, starch, magnesium Stearate, Sodium saccharin, talcum, glucose, 0043 Preferably the pharmaceutical composition is Sucrose, magnesium, carbonate, and the like. Such compo administered in a single unit dosage form for continuous sitions take the form of Solutions, Suspensions, tablets, pills, treatment or in a single unit dosage form ad libitum when capsules, powders, Sustained release formulations and the relief of symptoms is specifically required. like. Such compositions may contain about 1 to about 95% by weight of a compound of the preferred embodiments, EXAMPLES more preferably about 2 to about 50% by weight, most 0044) Example 1 preferably about 5 to about 8% by weight. 0038 Parenteral administration is generally character Cloned Human 5-HT Receptor Binding Assay ized by injection, either Subcutaneously, intramuscularly or 0045. The following describes an in vitro binding assay intravenously. Injectables can be prepared in conventional utilizing cloned 5-HT, receptors radiolabelled with H forms, either as liquid solutions or Suspensions, Solid forms 5HT. Suitable for Solution or Suspension in liquid prior to injec tion, or as emulsions. Suitable excipients are, for example, Receptor Binding Assay water, Saline, dextrose, glycerol, ethanol or the like. In 0046) HEK 293 cells transiently transfected with an addition, if desired, the pharmaceutical composition to be expression plasmid pXMD1-hu2B encoding the human administered may also contain minor amounts of non-toxic 5-HT, receptor (see Schmucket al., FEBS Left., 1994, 342, auxiliary Substances Such as wetting or emulsifying agents, 85-90) were used as described previously (Schmuck et al., pH buffering agents and the like, Such as for example, Eur. J. Pharmacol., 1996, 8, 959-967). Two days after Sodium acetate, Sorbitan monolaurate, triethanolamine ole transfection cells were harvested, pelleted at 500 g for 5 min ate, triethanolamine Sodium acetate, etc. at 4° C., gently resuspended in ice-cold buffer1 (50 mM 0039. A more recently devised approach for parenteral TRIS pH 7.7, 4 mM CaCl) and homogenized using a administration employs the implantation of a slow-release or Polytron PT 1200 tissue homogenizer (position 6 for 30s). Cells were pelleted at 50,000 g, 4° C. for 10 min, washed Sustained-release system, Such that a constant level of dos with buffer and pelleted again. The final pellet was resus age is maintained (see, e.g., U.S. Pat. No. 3,710,795). pended in incubation buffer (50 mM TRIS pH 7.7, 4 mM 0040. The percentage of active compound contained in CaCl, 10 uM pargyline and 0.1% by weight ascorbic acid). Such parenteral compositions is highly dependent on the The binding assay consisted of 300 ul of membrane suspen specific nature thereof, as well as the activity of the com sion (protein concentration=0.3 to 0.5 mg/ml), 150 ul of pound and the needs of the Subject. However, percentages of competing drug and 50 ul of H5-HT at a final concentra pimethixene or a pharmaceutically acceptable salt thereof of tion of 4 to 5 nM. The mixture was incubated at 37° C. for about 0.1 to about 10% by weight in solution are employ 30 min and the assay terminated by rapid filtration and two able, and will be higher if the composition is a solid which washing steps with 5 ml of cold 20 mM Iris-NCI pH=7.5, will be subsequently diluted to the above percentages. and 154 mM NaCl over Whatman GFB filters. Filters were US 2006/0120972 A1 Jun. 8, 2006

counted by liquid Scintilation. Non-specific binding was neohP4 encoding the human Histamine H. H. H. or H determined in the presence of an excess of 5-HT (100 uM). receptor, respectively. Recombinant cell lines were har Bound radioligand represented less than 1% of free radio vested after confluent growth, homogenized in ice-cold 50 ligand. In competition experiments, specific binding repre mM Na/potassium phosphate buffer (pH 7.4) and used for sented about 60% of total binding. Results expressed as pK, radioligand binding studies. Cell homogenates (40-50 jug of values are shown in Table 1 protein) were incubated for 30 min at 25° C. in 50 mM Example 2 Na/potassium phosphate buffer (pH 7.4) in 400 ul with the 5-HT, and 5-HT. Receptor Binding Methods various concentrations of either H-mepyramine, H 0047 The following describes receptor binding methods thiotidine, H-R-a-Methylhistamine, and H-pyramil in which the ligand with high affinity for 5-HT, receptors amine for cells expressing recombinant human H. H. H. were counter screened at 5-HT, and 5-HT, receptors to and H receptors, respectively. The nonspecific binding was demonstrate selectivity. defined in the presence of 1 LM mianserin. In displacement studies, cell homogenated were incubated either with 1 nM 0048 5-HT, receptors were labelled with Hket H-mepyramine, 15 nM H-thiotidine, 0.5 nM H-R- anserin in human cortex, in HEK293 cells expressing a C.-Methylhistamine, or 15 nM H-pyramilamine and cloned human 5-HT, receptor and in HEK293 cells expressing the rat 5-HT2A receptor. For competition binding increasing concentrations of competing ligands. The incu studies the ligand concentration was approximately 0.1 nM. bations were stopped by rapid dilution with 3 ml of ice-cold For Saturation binding studies concentrations of radioligand 50 mM Na/potassium phosphate buffer (pH 7.4). The ranged from 0.01 nM to 2.0 nM. Assays were conducted in bound radioactivity was separated by filatration through 0.5 ml of assay buffer (50 mM Tris-HCl, 4 mM calcium Whatman GF/C filters that had been treated with 0.3% chloride, 0.1% by weight ascorbic acid) (pH 7.4 at 4° C.). polyethyleneimine. Filters were washed twice in 3 ml of Non-specific binding was defined with 10 mM unlabelled buffer and radioactivity retained on the filters was measured ketanserin. After 60 min incubation at 32° C., membranes by liquid Scintillation counting. were harvested onto filters treated with 0.1% by weight of 0055. The concentration of Pimethixene producing 50% polyethylenimine and the bound radioactivity was deter inhibition of binding (ICs) was determined using iterative mined. curve fitting techniques. 0049 Human 5-HT, receptors were labelled in HEK293 cells as described above except that the radioligand was 0056 Proceeding as in the example above, the compound H-5HT and that the assay buffer contained pargyline in a of the preferred embodiments was found to have no affinity concentration of 10 mM and 0.1% by weight of ascorbic for the human H. and H4 receptor while it showed high acid. For competition binding studies the radioligand con affinity to the H1 receptor. centration was approximately 0.4 nM while for saturation binding studies the concentration of H-5HT ranged from TABLE 1. 0.05 to 8 nM. Non-specific binding was defined with 10 mM 5-HT. Incubations were for 120 min at 4° C. RECEPTORAFFINITY: PKVALUES OF THE PIMETHIXENE 0050 5-HT, receptors were labelled in Cos-7 cells Pimethixene Methysergide Pizotifen expressing the human 5-HT, receptor and in NIH-3T3 Receptor pKi values expressing the rat 5-HT, receptor. 5HT2A 10.2 8.54 5HT2B 10.6 8.85 8.54 0051) Assays were conducted as described for the 5-HT, 5HT2 8.6 receptor except that the radioligand was Himesulergine. H 11.1 The radioligand concentration for competition studies was H2 low activity H low activity approximately 0.2 nM while for saturation binding studies H4 low activity the concentration ranged from 0.1 to 18 nM. Non-specific M 8.6 binding was defined with 10 uM unlabelled mesulergine. D2 8.2 0.052 Competition radioligand binding data were ana Alpha 1A 7.6 lyzed using a four parameter logistic equation and iterative curve-fitting techniques to obtain estimates of the ICso and 0057 Summary of pKi values of Pimethixene for the Hill slope. Kd values, determined from saturation binding respective receptor Subtypes. pKi values were calculated studies were then used to calculate inhibition dissociation using the equation of Cheng and Prusoff pKi-log (ICso/ constants (Ki). 1+used concentration of Agonist/ECs). ECso values were 0053 Proceeding as in the example above the compound calculated from corresponding dose-response experiment of the preferred embodiments was found to have selective using 5-HT, histamine, epinephrine (alpha 1A adrenergic affinity for the 5-HT, receptor versus 5HT. receptor. receptor), dopamine (D2 dopaminergic receptor), and ace Results are shown in Table 1 tylcholine (M1 muscarinic receptor) as agonists. Example 3 0.058. The results of Table 1 show that the compound of Cloned Human H. H. and H. Receptor Binding the preferred embodiments has a selective affinity for the Assay 5HT and H receptor, particularly have a much higher 0054 CHO cells were stably transfected with an expres affinity2AB for the 5HT receptor than for the 5HT receptor sion plasmid pCineohH1, pCineohH2, pCineohH3 and pCi or other monoaminergic receptors. US 2006/0120972 A1 Jun. 8, 2006

Example 4 Functional Test of Pimethixene in the mCPP Model in Guinea Pigs 0059. In accordance to the protocol published by Markowitz, et. al. (1987) and Olesen et. al. (2000), guinea pigs were used to determine the amount of plasma protein released at the blood vessels within the Dura mater after mCPP application. 0060. After deep anesthesia the test compounds pime thixene, methysergide or pizotifen were injected intravenous N in the Vena jugularis at different concentrations. 2 minutes CH3 later 1 g/kg body weight mGPP were injected and followed by the i.v. application of EvansBlue as marker for the protein plasma extravasation. 15 minutes later animals were tran or a pharmaceutically acceptable salt thereof. Scranial perfused and decapitated. A defined area of the dura 2. The method according to claim 1, wherein the admin mater was isolated and transferred into an eppendorf tube istration is performed by a mode selected from the group containing 200 ul formamid. Because EvansBlue binds to consisting of inhalation, oral, parenteral, Systemic, transder plasma protein, the amount of bound marker dye is a mal, and topical. measure of extravasation of plasma protein. Formamid dis 3. The method according to claim 2, wherein the admin solves EvansBlue from the protein which can be calorimet ric detected and quantified at 600 nm. istration is by inhalation. 4. The method according to claim 3, wherein the inhala TABLE 2 tion is performed with a nebulizer inhaler, a dry powder inhaler, or a metered dose inhaler. DETERMINATION OF PLASMA PROTEIN EXTRAVASATION 5. The method according to claim 2, wherein the admin AS EVANSBLUE EXTINCTION AT 600 NM. istration is by oral administration. Dose in 6. The method according to claim 5, wherein the oral |lgkg Pimethixene in Pizotifen in Methysergide n administration is performed with a form selected from the group consisting of Solution, Suspension, tablet, pill, cap 100 not determined - 0.230 - 0.018 7 0.189 - 0.026 3 10 not determined - 0.144 - 0.035 8 0.228 0.064 5 Sule, powder, and Sustained release formulations. 1 not determined - 0.404 - 0.025 6 0.250 + 0.027 5 7. The method according to claim 6, wherein the form O.1 0.126 - 0.028 5 not determined - 0.265 + 0.034 5 comprises about 1 to about 95% by weight of the compound. 0.01 0.157+ 0.040 4 not determined — not determined — 0.001 0.269 + 0.022 5 not determined — not determined — 8. The method according to claim 7, wherein the form comprises about 2 to about 50% by weight of the compound. 9. The method according to claim 2, wherein the admin 0061 An extinction of 0.300+0.038 was determined for istration is by parenteral administration. the positive control (mCPP agonist), an extinction of 10. The method according to claim 9, wherein parenteral 0.130+0.024 was measured for the negative control. administration is performed with a solution of about 0.1 to 0062. As shown in Table 2, pimethixene inhibited the about 10% by weight of the compound. plasma protein extravasation at concentrations 100 to 1000 fold lower than the reference compounds methysergide or 11. The method according to claim 2, wherein the admin pizotifen. istration is topical or transdermal. 12. The method according to claim 11, wherein adminis 0063. The references cited herein are entirely incorpo tration is performed with a form selected from the group rated herein by reference. consisting of gel, cream, lotion, Solution, Suspension, oint 0064. Many modifications and variations of the embodi ment, powder, and dispersion. ments described herein may be made without departing from 13. The method according to claim 1, wherein the com the scope, as is apparent to those skilled in the art. The pound is administered at a dose of about 0.01 to about 20 specific embodiments described herein are offered by way of mg/kg/day. example only. 14. The method according to claim 13, wherein the What is claimed is: compound is administered at a dose of about 0.1 to about 10 1. A method of treating pulmonary hypertension compris mg/kg/day. ing administering to a subject a compound according to the formula: