DISEASES, AND SEPSIS

The present invention provides methods for preventing and/or treating diseases, namely pulmonary diseases, vascular diseases, and sepsis in mammals. Moreover, the present invention relates to N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) -ethyl]- 2,2-diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof, as well as to pharmaceutical compositions containing them, for use in preventing and/or treating these diseases.

Background of the Invention

Pulmonary diseases, vascular diseases, and sepsis are diseases that are worldwide affecting a high number of patients and that even may become life-threatening. In particular, pulmonary diseases (lung diseases) are some of the most common medical conditions in the world. Besides infection, e.g. due to bacteria or viruses, lung diseases may be caused also by inflammation without infection. Several tens of millions of people suffer from lung diseases having symptoms like cough and shortness of breath.

Vascular diseases, in particular vasculitis, belong to a class of diseases of the blood vessels, including arteries and veins of the circulatory system of the human body. It is defined as a subgroup of cardiovascular diseases. Vasculitis is an inflammation of blood vessels, either venous or arterial and of all sizes. It causes changes in the blood vessel walls, including thickening, weakening, narrowing or scarring. These changes can restrict blood flow, resulting in organ and tissue damage. Vasculitis might affect just one organ, or several. The condition can be short term (acute) or long lasting (chronic).

Smoking, infections, high cholesterol values and high blood pressure as well as genetics and lack of physical activity are considered as the main reasons both for pulmonary diseases and vascular diseases. Also air pollution plays an important role in particular in cities with high traffic and industrial emissions. Sepsis is a life-threatening condition that arises when the body's response to infection causes injury to its own tissues and organs. The most common primary sources of infection resulting in sepsis are the lungs, the abdomen, and the urinary tract, while typically about 50% of all sepsis cases start as an infection in the lungs. Infections leading to sepsis mainly are bacterial, but may be fimgal or viral. In severe sepsis and septic shock, usually broad-spectrum antibiotics are recommended as well as hemodynamic optimization and even the use of glucocorticosteroids as anti-inflammatory measure.

Even if there exists a number of actives that are suitable in treating the above mentioned diseases, most of them show significant deficiencies in efficacy, side effect profile and/or safety.

EP 0 569 802 claims N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) - ethyl]-2,2-diphenylacetamide among other aryl acetamides as compounds with high affinity towards kappa-opioid receptors (KOR) for us as analgesics and neuroprotectives.

EP 0 752 246 claims the use of N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine- l-yl)-ethyl]-2,2-diphenylacetamide for the preparation of a medicament for the treatment of inflammatory diseases of the intestine.

EP 2 561 870 claims the use of KOR agonists, especially N-methyl-N-[(lS)-l-phenyl-2- ((3S)-3-hydroxypyrrolidine-l-yl)-ethyl]-2,2-diphenylacetamid e, for the treatment of diarrhea-predominant and alternating irritable bowel syndrome (IBS).

In view thereof a strong need still exists for finding new drugs that are able to provide for an improved overall performance including efficacy, side effect profile and safety in the treatment and prevention of pulmonary diseases, vascular diseases, and sepsis. In particular, new therapeutic agents are necessary, which are both highly effective in the treatment and/or prevention of pulmonary diseases, vascular diseases, and sepsis and which do not show severe side effects, such as on the central nervous system (CNS) or other side effects.

Therefore, it is an object of the present invention to provide an improved effective and safe way of preventing and treating pulmonary diseases, vascular diseases, and sepsis free of side effects.

The above-mentioned object has been solved by the inventors of the present invention who surprisingly found out that N-methyl-N-[( 1 S)- 1 -phenyl-2-((3 S)-3-hydroxypyrrolidine- 1 -yl)- ethyl]-2,2-diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof effectively and safely treats and prevents pulmonary diseases, vascular diseases, and sepsis while at the same time does not cause side effects, such as CNS side effects.

Summary of the Invention

Therefore, the subject-matter of the present invention is N-methyl-N-[(lS)-l-phenyl-2- ((3S)-3-hydroxypyrrolidine-l-yl)-ethyl]-2,2-diphenylacetamid e or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof, as well as a pharmaceutical composition containing them, for use in preventing and/or treating pulmonary diseases, vascular diseases, and sepsis. In other words, the present invention relates to the use of N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) -ethyl]- 2,2-diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof in a method of treating and/or preventing pulmonary diseases, vascular diseases, and sepsis. The present invention also relates to the use of these compounds in the preparation of pharmaceutical compositions (also referred to as medicaments) for treating and/or preventing these diseases as well as the pharmaceutical compositions per se. Detailed Description of the Invention

N-Methyl-N-[(l S)- 1 -phenyl-2-((3S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2- diphenylacetamide (asimadoline; EMD 61753) is a known compound (e.g. from EP 0 569 802 A) which acts as a peripherally selective k-opioid receptor (KOR) agonist. Because of its poor ability to cross the blood brain barrier, N-methyl-N-[(lS)-l-phenyl-2-((3S)-3- hydroxypyrrolidine-l-yl)-ethyl]-2,2-diphenylacetamide lacks the psychotomimetic effects of centrally acting KOR agonists. Based thereon, it was found to be effective in the treatment of gastrointestinal diseases, inflammatory and non-inflammatory diseases of the gastrointestinal tract, of the urinary system and eating disorders as well as of pain and neuropathy (EP 1 572 640 A).

Now it was surprisingly found that N-methyl-N-[(lS)-l-phenyl-2-((3S)-3- hydroxypyrrolidine-l-yl)-ethyl]-2,2-diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof is effective in treating and/or preventing pulmonary diseases, vascular diseases, and sepsis.

According to the invention N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l- yl)-ethyl]-2,2-diphenylacetamide or a derivative or prodrug thereof can be present in the form of their acids or their bases or in the form of their salts, in particular the pharmaceutically (physiologically) acceptable salts, or in the form of their solvates, in particular their hydrates.

The“pharmaceutically acceptable salts” can be base addition salts or acid addition salts.

Base addition salts include salts of the compounds according to the invention with inorganic bases, such as alkali metal hydroxides, alkaline earth metal hydroxides, or with organic bases, such as mono-, di- or triethanolamine, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N ^' - dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, and tromethamine.

Acid addition salts include salts of the compounds according to the invention with inorganic acids, such as hydrochloric acid, sulfuric acid or phosphoric acid, or with suitable organic carboxylic or sulfonic acids or with amino acids. These are chosen, for example, from the group comprising chlorides, bromides, iodides, hydrochlorides, hydrobromides, sulfonates, methanesulfonates, sulfates, hydrogen sulfates, sulfites, hydrogen sulfites, phosphates, nitrates, methanoates, acetates, propionates, lactates, citrates, glutarates, maleates, malonates, malates, succinates, tartrates, oxalates, fumarates, benzoates, p-toluenesulfonates and/or salts of amino acids, preferably the proteinogenic amino acids. The hydrochloride is particularly preferred.

According to the invention N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l- yl)-ethyl]-2,2-diphenylacetamide can be present in the form of its derivatives and prodrugs.

“Derivative” in the present invention means any compound that is derivable fromN-methyl- N-[(l S)- 1 -phenyl-2-((3S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2-diphenylacetamide by a small number of chemical reactions, preferably by one or two reaction steps only (e.g. by acid addition, esterification or etherification at the 3-hydroxypyrrolidine group). Particularly preferred are the derivatives comprising an acid covalently bonded at the 3- hydroxypyrrolidine group. The acid preferably is selected from monobasic carboxylic acids, dibasic carboxylic acids and hydroxymonobasic carboxylic acids (such as disclosed in EP 1 572 640 B).

The term“prodrug” means any precursor compound that is metabolized into N-methyl-N- [( 1 S)- 1 -phenyl-2-((3 S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2-diphenylacetamide. Preferred prodrugs comprise an ester group or an ether group at the 3-hydroxypyrrolidine group.

As mentioned above, one advantage of N-methyl-N-[(lS)-l-phenyl-2-((3S)-3- hydroxypyrrolidine-l-yl)-ethyl]-2,2-diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof is that these compounds pass the blood-brain barrier to only a small extent. This makes it possible for the compounds to be used in particular as peripherally acting agents in treating and/or preventing pulmonary diseases, vascular diseases, and sepsis with no or only mild centrally mediated side effects, such as dysphoria, sedation, polyuria or insomnia. It is of particular advantage that these compounds show no dysphoric or sedative action. This makes it possible to administer the compounds over a relatively long period of time. For example, a long-term administration, in particular a daily administration, is made possible.

According to the invention N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l- yl)-ethyl]-2,2-diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof can be used in particular for therapeutic and/or prophylactic treatment, diagnosis and/or therapy of diseases chosen from the group of pulmonary diseases, vascular diseases, and sepsis.

The invention also provides the use of N-methyl-N-[(lS)-l-phenyl-2-((3S)-3- hydroxypyrrolidine-l-yl)-ethyl]-2,2-diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof for the preparation of a medicament for therapeutic and/or prophylactic treatment of diseases chosen from the group of pulmonary diseases, vascular diseases, and sepsis.

Pulmonary diseases are preferably selected from the group of inflammatory pulmonary diseases, e.g. asthma, chronic obstructive pulmonary disease (COPD), infectious pulmonary diseases, e.g. pneumonia, mucoviscidosis, as well as granulomatous or fibrotic pulmonary diseases, either as a consequence of chronic pulmonary inflammation or independent thereof, e.g. Wegener’s granulomatosis, pulmonary fibrosis, and interstitial lung disease (ILD).

Vascular diseases are preferably selected from small vessel vasculitis, e.g. leucocytoclastic vasculitis, Beliefs syndrome, eosinophilic granulomatosis with polyangiitis, Henoch- Schonlein purpura, microscopic polyangiitis, granulomatosis with polyangiitis, medium vessel vasculitis, e.g. Buerger's disease, cutaneous vasculitis, Kawasaki disease, polyarteritis nodosa and large vessel vasculitis, e.g. polymyalgia rheumatica, Takayasu's arteritis, temporal arteritis and disease accompanied by vasculitis, e.g. rheumatic diseases or collagen diseases.

Sepsis preferably is defined as systemic inflammatory response syndrome, severe sepsis or septic shock.

In the context of the present invention, the term“prophylactic treatment” is understood as meaning in particular that the compounds according to the invention can be administered before symptoms of a disease occur or the risk of a disease exists.

N-methyl-N-[( 1 S)- 1 -phenyl-2-((3 S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2- diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof according to the invention can be used by itself or in combination with known substances for treatment of diseases chosen from the group of pulmonary diseases, vascular diseases, and sepsis.

The pharmaceutical compositions according to the invention may contain, next to N-methyl- N-[(l S)- 1 -phenyl-2-((3S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2-diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof, other substances known to be effective in the treatment of diseases chosen from the group of pulmonary diseases, vascular diseases, and sepsis such as immunosuppressants, e.g. glucocorticosteroids, or biologies (biopharmaceuticals). By using these combinations of actives a synergistic effect in efficacy against pulmonary diseases, vascular diseases, and sepsis could be observed and might yield a reduction of the latter ones (“sparing effect”).

It is further preferred to incorporate into the pharmaceutical compositions at least one opioid receptor antagonist, preferably chosen from the group comprising naloxone, naltrexone, cyprodime, naltrindole, norbinaltorphimine, nalmefene, nalorphine, nalbuphine, naloxonazine, methylnaltrexone and/or ketylcyclazocine, and/or a steroidal anti inflammatory drug, preferably chosen from the group of hydrocortisone, hydrocortisone acetate, prednisolone, methylprednisolone, prednisone, betamethasone, hydrocortisone- 17- valerate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone- 17-butyrate, flunisolide, fluticasone propionate, triamcinolone acetonide, beclomethasone dipropionate, budesonide and/or hydrocortisone- 17-butyrate and/or a nonsteroidal anti inflammatory drug (NSAID), preferably chosen from the group of aspirin, ibuprofen, diclofenac and/or naproxen, and/or an opioid receptor agonist, preferably chosen from the group comprising tramadol, pethidin, codein, piritramid, morphin, levomethadon, fentanyl, alfentanil, remifentanil and/or sufentanil, and/or an antibiotic.

N-Methyl-N-[(l S)- 1 -phenyl-2-((3S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2- diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof according to the invention can be administered by conventional methods, for example orally, dermally, intranasally, transmucosally, pulmonally, enterally, buccally, rectally, intraurethral, aural, by inhalation, by means of injection, for example intravenously, parenterally, intrap eritoneally, intradermally, subcutaneously and/or intramuscularly and/or locally, for example on painful areas of the body. Oral administration is particularly preferred.

N-Methyl-N-[(l S)- 1 -phenyl-2-((3S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2- diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof according to the invention can be used in particular for the preparation of medicaments by being brought into a suitable dosage form together with at least one carrier substance or auxiliary substance (excipient), for example in the form of injection solutions, drops, juices, syrups, sprays, suspensions, tablets, patches, capsules, plasters, suppositories, ointments, creams, lotions, gels, emulsions, aerosols or in multiparticulate form, for example in the form of pellets or granules.

Pharmaceutical dosage forms with delayed release (sustained release formulation) are furthermore preferred for oral administration of the compounds according to the invention. Examples of formulations with delayed release are sustained release matrix tablets, multilayered tablets, the coating of which can be, for example, constructed to be resistant to gastric juice, such as coatings based on shellac, sustained release capsules or formulations using biodegradable polymers, for example poly(lactic acid) polymers.

In particular, the excipients used according to the invention are known to the skilled person. These include for example carrier materials, diluents, wetting agents, emulsifiers, dyestuffs, preservatives, disintegrating agents, lubricants, salts for influencing the osmotic pressure, buffer substances, aromas, binders, solvents such as organic solvents, gelling agents, detergents, oils, alcohols, solubilizers, humectants, fillers, bioadhesives, bactericides, surfactants, colorants thickeners, softening agents, moisturizing agents, oils, fats, waxes, water, alcohols, polyols, polymers or other suitable components of a pharmaceutical preparation.

Examples of suitable excipients are water, plant oils, benzyl alcohols, polyethylene alcohols/glycols, gelatine, soya, carbohydrates (such as lactose, tragacanth or starch), lecithin, glycerol triacetate and other fatty acid glycerides, fatty acid salts, talc and cellulose. Examples of gelling agents as suitable excipients are natural gelling agents, such as pectin, agarose, gelatine and casein, or modified natural gelling agents, such as methyl cellulose, hydroxymethyl cellulose, hydroxymethylpropyl cellulose and carboxymethyl cellulose or full synthetic gelling agents, such as polyvinylalcohols, poly(meth)acrylacids, polyacrylamide, polyvinylpyrrolidone polypropylene glycol and polyethylene glycol. The pH value of the formulation can be stabilized using buffer systems consisting of polyacids and their salts. Examples for such polyacids are citric acid, tartaric acid, phosphoric acid and malic acid.

The compounds according to the invention are commercially available e.g. from Sigma Aldrich or BOC Sciences.

As used herein, an effective amount of N-methyl-N-[(lS)-l-phenyl-2-((3S)-3- hydroxypyrrolidine-l-yl)-ethyl]-2,2-diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof essentially means an amount that is effective to exhibit biological activity, preferably wherein the biological activity is defined as an effective prevention/treatment of diseases chosen from the group of pulmonary diseases, vascular diseases, and sepsis, at the site(s) of activity in a mammalian subject. This is intended to exclude undue adverse side effects (in particular centrally mediated side effects, such as dysphoria, sedation, polyuria or insomnia).

The effective amount of N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) - ethyl]-2,2-diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof, based on the weight relative to N-methyl-N-[(lS)- 1 -phenyl-2-((3S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2-diphenylacetamide, is typically from about 0.1 mg to 15 mg, more preferably from 0.5 mg to 12 mg, even more preferably 1.0 to 10 mg, most preferably from 2.5 to 8 mg per day. Particularly preferred is a range between 0.5 and 15 mg orally administered per day, even more preferred between 1 and 12 mg orally administered per day and most preferred between 2.5 and 10 mg (for example 3, 4, 5, 6, or 7 mg) orally administered per day. Above the maximum values the risk of centrally mediated side effects (CNS effects), such as dysphoria, sedation, polyuria or insomnia, significantly increases. Above 15 mg per day significant CNS effects are observed.

A preferred dosage regime for administration is once per week to three times daily, more preferred every second day to twice daily, most preferred once daily. The composition preferably contains from about 0.001% to about 10.0% (w/w), more preferably from about 0.01% to about 5.0% (w/w), most preferably from about 0.05 to about 1.0% (w/w) of N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) -ethyl]-2,2- diphenylacetamide or a derivative, solvate, hydrate, or prodrug thereof or a pharmaceutically acceptable salt thereof, based on the weight relative to N-methyl-N-[(lS)-l-phenyl-2-((3S)- 3-hydroxypyrrolidine-l-yl)-ethyl]-2,2-diphenylacetamide, based on the weight of the total composition. Particularly suitable values out of these ranges are amounts of 0.1 , 0.3, 0.5, and 1.0%. If the amount is below the minimum values of the indicated ranges, the treatment and/or prevention is ineffective. On the other hand, if the amount is above the maximum values mentioned, CNS side effects are observed.

The pharmaceutical compositions according to the invention are prepared in a suitable way known to the skilled person. When prepared as an inhalable dry powder formulation being one of the preferred compositions of the invention the process for preparation may include the following process steps.

N-Methyl-N-[(l S)- 1 -phenyl-2-((3S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2- diphenylacetamide hydrochloride is micronized in a way that the mass median aerodynamic diameter (MMAD) of the resulting particles is preferably less than 10 pm. Suitable micronizing equipment is well known in the art and includes a variety of grinding and milling machinery, e.g. compressive-type mills such as mechanofusion mills, impact mills such as ball mills, homogenizers, micro fluidizers, jet mills, low shear mixers such as Turbular ^® power blender and high-shear mixers such as MiPro ^® power blender.

In the second step of the process for preparing an inhalable dry powder formulation, carrier particles are admixed with the micronized crystalline drug substance to give the desired inhalable dry powder formulation. The carrier particles make the micronized drug substance less cohesive and improve its flowability. This makes the powder easier to handle downstream, for example when filling the dry powder formulation into capsule. The micronized drug substance particles tend to adhere to the surface of the carrier particles whilst stored in a dry powder inhaler device but are dispersed from the surfaces of the carrier particles on inhalation into the respiratory tract to give a fine suspension. The larger carrier particles are mostly deposited in the oropharyngeal cavity.

The carrier particles may be composed of any pharmacological inert material or combination of materials which is acceptable for inhalation. They are suitably composed of one or more crystalline sugars including monosaccharides, disaccharides, polysaccharides and sugar alcohols such as arabinose, glucose, fructose, ribose, mannose, sucrose, trehalose, lactose, lactose, maltose, starches, dextran, mannitol or sorbitol. An especially preferred carrier is lactose, for example lactose monohydrate or anhydrous lactose.

Preferably substantially all (by weight) of the carrier particles have a diameter of 20 to 1000 pm, more preferably 50 to 500 pm, but especially 20 to 250 pm. The diameter of substantially all (by weight) of the carrier particles is suitably less than 355 pm. this provides good flow and entrainment characteristics and improves release of the active particles in the airways to increase deposition of the active particles in the lower lung. It will be understood that, throughout, the diameter of the particles referred to is the aerodynamic diameter of the particles.

When desirable, one or more force control agents such as magnesium stearate are included in dry powder formulations for inhalation. The force control agent leads to a general improvement in the inhalable fine particle fraction in dry powder N-methyl-N-[(lS)-l- phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl)-ethyl]-2,2-dipheny lacetamide formulations. It stabilizes the carrier materials and the drug substance by suppressing or slowing down undesirable morphological phase transitions. It also enhances the dosing efficiency of inhalable dry powder N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) - ethyl]-2,2-diphenylacetamide formulations by improving powder flowability.

Other suitable force control agents include amino acids such as leucine, phospholipids such as lecithin or fatty acid derivatives such as calcium stearate. However, magnesium stearate is especially preferred. It is preferably added in particularly small amounts, for example 0.1 to 0.5% by weight, more preferably 0.1 to 2% by weight, but especially about 0.25 to 1% by weight, based on the total formulation, of magnesium stearate.

The force control agent is preferably in particulate form but it may be added in liquid or solid form and for some materials, especially where it may not be easy to form particles of the material and/or where those particles should be especially small, it may be preferred to add the material in a liquid, for example as a suspension or a solution.

The dry powder may be contained as unit doses in capsules of, for example, gelatine or plastic, or in blisters (e.g. of aluminium or plastic), for use in a dry powder inhalation device, which may be a single dose or multiple dose device. Preferably the total weight of powder per capsule is from 5 mg to 50 mg. Alternatively, the dry powder may be contained in a reservoir in a multi-dose dry powder inhalation (MDDPI) device adapted to deliver, for example, 3-25 mg of dry powder per actuation. A suitable device for delivery of dry powder in encapsulated form is described in US 3,991,761 or WO 05/113042, while suitable MDDPI devices include those described in WO 97/20589 and WO 97/30743.

The following non-limiting examples describe the present invention in more detail.

Brief description of the drawings

Figure 1 is a diagram that shows cytosolic Ca ²⁺ concentration in single endothelial cells from mouse pulmonary venules in situ before and during application of 5 x 10 ⁵ CFU/ml D39 wild type S. pneumoniae (right). Control group without S. pneumoniae (left).

Figure 2 is a diagram that shows that the Ca ²⁺ signals observed in endothelial cells from mouse pulmonary venules are mediated by pneumolysin (maximal cytosolic concentrations of Ca ²⁺ signals in endothelial cells of mouse pulmonary venules in situ at baseline and during application of wild type S. pneumoniae (left), pneumolys in-deficient S. pneumoniae (middle), and pneumolysin (right)).

Figure 3 shows Ca ²⁺ signals in human endothelial cells (HPMEC) following treatment with S. pneumoniae or pneumolysin (maximal cytosolic concentrations of Ca ²⁺ signals in pulmonary micro vascular endothelial cells in vitro at baseline and during application of wild type S. pneumoniae (left), and pneumolysin (right).

Figure 4 shows [Ca ²⁺ ] _cyt in HPMEC under various conditions ([Ca ²⁺ ] _cyt in HPMEC (mean ± SEM) during the incubation with HEPES buffer (grey bar: basal conditions) or during application of 5 x 10 ⁵ CFU/mL S. pneumoniea D39 variants and pneumolysin (5 ng/mL), resp. (black bars). WT : wild type D39, ACPS: capsule-deficient D39, APLY : pneumolysin- deficient D39, PLY: isolated pneumolysin from wild type D39.

* p<0.05 vs. basal condition, * p<0.05 vs. control).

Figure 5 shows KOR expression in HPMEC (H) and T-Jurkat lymphocytes (TL) (Western Blot).

Figure 6 shows (A) KOR mRNA expression following 6 h stimulation of HPMEC with vehicle, LPS (1 or 2 pg/mL), D39 wild type (SP257), pneumolysin deficient or capsule deficient D39; (B) KOR protein in HPMEC following 6 h stimulation with vehicle, D39 wild type or pneumolysin deficient D39 (PN196), mean ± SEM, * p<0.05 vs. vehicle condition, ^# p<0.05 vs. D39.

Figure 7 shows [Ca ²⁺ ] _cyt response (Fura-2 ratio) in HPMEC (mean ± SEM) pre-incubated with D39 (45 min, 5 x 10 ⁵ CFU/mL) following application of asimadoline.

Figure 8 shows grouped [Ca ²⁺ ] _cyt response in HUVEC following pre-incubation with WT D39 and treatment with asimadoline (Group data analysis of [Ca ²⁺ ] _cyt response (mean ± SEM) in HUVECS following aryl 241 (100 mM) or asimadoline (100 mM) and 30 minute pretreatment with 5 x 10 ⁵ CFU/mL S. pneumoniea D39. * p<0.05 vs. WT.)

Figure 9 shows Ca ²⁺ signals in single endothelial cells from mouse pulmonary venules in situ before and during application of WT D39 followed by application of asimadoline in KOR WT and KOR KO mice (Group data analysis of [Ca ²⁺ ] _cyt response (mean ± SEM) in endothelial cells of intact pulmonary arterioles following asimadoline (100 mM) and 30 minute pretreatment with 5 x 10 ⁵ CFU/mL S. pneumoniea D39 in wild type (WT) or KOR mice. * p<0.05 vs. WT).

Figure 10 is a graph showing the fraction of rolling platelets in pulmonary arterioles or venules of WT or KOR mice following asimadoline (100 mM) and 60 minutes

pretreatment with 5 x 10 ⁵ CFU/mL S. pneumoniea D39 (group data analysis of fraction of rolling platelets (PLT) (mean ± SEM) in pulmonary arterioles or venules of WT or KOR mice following asimadoline (100 mM) and 60 minute pretreatment with 5 x 10 ⁵ CFU/mL S. pneumoniea D39.

^# p<0.05 vs. WT, n=4-5).

Figure 11 shows the fraction of sticking platelets in pulmonary arterioles or venules of WT or KOR mice following asidomaline (100 mM) and 60 minutes pretreatment with 5 x 10 ⁵ CFU/mL S. pneumoniea D39 (group data analysis of fraction of sticking platelets (PLT) (mean ± SEM) in pulmonary arterioles or venules of WT or KOR mice following asimadoline (100 mM) and 60 minute pretreatment with 5 x 10 ⁵ CFU/mL S. pneumoniea D39.

^# p<0.05 vs. WT, n=4-5).

EXAMPLES

A. Active sensing of S. pneumoniae by endothelial cells of pulmonary microvessels in situ

C57BL/6 mice were anaesthetized with i.p. administered ketamine and xylazine. Subsequently the rats were trachetomized, intubated and ventilated. Following sternotomy heparin was applied via cardial puncture and depending on the size of the animal 10 to 15 mL whole blood was aspirated. The arterial cannula for perfusion of the lung was introduced transmurally into the right ventricle and fixated. For drainage of the effluent a venous cannula was placed in the left atrium. The lung was eviscerated en block together with the heart. During the experiment a continuous positive respiration pressure of 5 cm H ₂ 0 was kept and the lunge was perfused by means of a roller pump with autologous whole blood plus HEPES buffer (150 nM Na ⁺ , 5 mM K ⁺ , 1 mM Ca ²⁺ , 1 mM Mg ²⁺ , 0.16 mM dextrane and 10 mM glucose; pH 7.4, 295 mOsm) at 37°C. The pulmonary arterial pressure and left atrial pressure were 7 cm H ₂ 0 and 2 cm H ₂ 0, resp. The lung surface was kept wet using saline heated to 37°C. Endothelial cells of distinct capillary region were loaded with Fura-2 using a micro catheter. The endothelial cytosolic concentration of Ca ²⁺ ([Ca ²⁺ ] _cyt ) was measured by epifluorescence microscopy and Fura-2 real-time imaging in situ applying the method from the group of Bhattacharya et al. (see e.g. Kiefmann R. Blood 2008; 111 :5205- 5214).

S. pneumoniae (5 x 10 ⁵ CFU/mL, wild type (WT), resuspended in HEPES buffer) were applied to the isolated perfused rat lung model. The bacteria were able to induce a Ca ²⁺ signal in endothelial cells of intact venules. The Ca ²⁺ signals were induced in form of spikes (Figure 1). Of note, the cytosolic Ca ²⁺ levels are returning to the baseline following each spike. This points towards an active generation of the Ca ²⁺ signal. Since the venules are exclusively perfused with HEPES buffer lacking blood components such as leukocytes or erythrocytes, we assume that endothelial cells can directly sense contact to pneumococci.

B. Signal mediation via pneumolysin

Point mutated S. pneumoniae lacking pneumolysin (5 x 10 ⁵ CFU/mL) and isolated pneumolysin (5 ng/mL), respectively, were tested in the model described under A. It was shown that the lack of the bacterial envelope did not have an impact on Ca ²⁺ signaling. Pneumolysin alone was also able to induce Ca ²⁺ signals. Of note, the applied concentration of pneumolysin did not lead to cell lysis. Pneumolysin-deficient S. pneumoniae, however, were no longer able to induce Ca ²⁺ signals in endothelial cells of intact venules (Figure 2). In summary, the Ca ²⁺ signal in endothelial cells of pulmonary venules is mediated by the virulence factor pneumolysin.

C. S. pneumoniae- induced Ca ²⁺ spikes in human pulmonary microvascular endothelial cells in vitro

Human primary pulmonary microvascular endothelial cells (HPMEC, PromoCell, Heidelberg, Germany) were cultured in a specific medium for endothelial cells containing penicillin and streptomycin at 37°C and 5% C0 ₂ . For measurement of cytosolic Ca ²⁺ concentrations HPMEC were seeded on a special microscope slide and incubated until confluence of the endothelial cells (2-3 days) at 37°C and 5% C0 ₂ . Following exchange of the medium with HEPES buffer (150 nM Na ⁺ , 5 mM K ⁺ , 1 mM Ca ²⁺ , 1 mM Mg ²⁺ , 0.16 mM dextrane and 10 mM glucose; pH 7.4, 295 mOsm) und staining with Fura-2-AM the cytosolic Ca ²⁺ concentration was measured via fluorescence microscopy.

HPMEC were treated with S. pneumoniae (wild type and pneumolysin-deficient, resp., 5 x 10 ⁵ CFU/mL) or wild type pneumolysin (5 ng/mL) for 45 min. It was shown that human primary endothelial cells react on S. pneumoniae treatment with a Ca ²⁺ signal, too (Figure 3). As can be seen from the figure the Ca ²⁺ signal is observed after latency. A rapid increase of [Ca ²⁺ ] _cyt was seen followed by a short plateau phase and a decrease of [Ca ²⁺ ] _cyt to basal levels. Signaling is analog to the signal observed in Example A. Moreover, the signal is mediated via wild type pneumolysin. It was shown that application of pneumolysin lead to Ca ²⁺ signals whereas no signals were observed following application of pneumolysin- deficient S. pneumoniae (Figure 4).

D. Influence of S. pneumoniae (D39) on KOR expression

The expression of KOR on HPMEC was determined on mRNA level using PCR technique. Treatment with D39 resulted in a 22fold increased KOR expression on mRNA level (Figure 5 A). The endotoxin from E. coli, lipopolysaccharid (LPS, 2 ng/mL), which is commonly used for experimental sepsis studies, was only able to slightly up-regulate KOR expression.

In a subsequent experiment KOR expression on protein level was determined. Applying FACS technique it was shown that KOR expression in HPMEC was more than doubled 6 h following treatment with WT D39 whereas pneumolysin-deficient D39 did not affect KOR expression (Figure 5 B).

The expression of KOR on HPMEC and T-Jurkat lymphocytes was determined on protein level using Western Blot technology in addition (Figure 6).

E Effects of asimadoline on S. pneumoniae- induced Ca ²⁺ signaling in human pulmonary microvascular endothelial cells or human umbilical vein endothelial cells in vitro

HPMEC were cultured as described in Example C. The cells were pretreated with S. pneumoniae (wild type, 5 x 10 ⁵ CFU/mL) for 45 min. Treatment with asimadoline (100 mM induced a strong [Ca ²⁺ ] _cyt response (measured as Fura-2 ratio) (Figure 7). Subsequently the experiment was repeated with human umbilical vein endothelial cells (HUVEC). Following pretreatment with S. pneumoniae (wild type, 5 x 10 ⁵ CFU/mL) for 30 min vehicle, asimadoline (100 mM) or a aryl 241 (100 mM; a structural analog of a KOR agonist devoid of functional activity at KOR) were added. Treatment with asimadoline resulted in a strong [Ca ²⁺ ] _cyt response. No such effect was observed in cells treated with vehicle or the structural analog without KOR activity (Figure 8).

F. Effects on lungs of KOR wild type and knockout mice in situ

Effects of KOR agonists on shedding of adhesion molecules and recruitment of neutrophilic granulocytes to the endothelium were determined in the model of isolated perfused mouse lung. KOR WT mice (KOR ^+/+ , C57/BF6) or KOR knockout mice (KOR , B6.129S2- OprkltmlKff/J) were treated with WT D39 alone or in combination with a KOR agonist (asimadoline). WT D39 were administered i.v. 2 h before lungs were removed.

Fungs were re-perfused with HEPES buffer and calcein/Fluo-4 for 20 min. Subsequently, lungs were perfused with a KOR agonist (asimadoline) for 30 min and Ca ²⁺ signals were measured using real-time imaging. Treatment with the KOR agonist caused a Ca ²⁺ signal in WT lungs similar to the ones obtained in vitro. In contrast no Ca ²⁺ signal was observed in lungs from KOR knockout mice (Figure 9).

For determination of leukocyte recruitment to the endothelium blood of mice was obtained from pre-treated mice via heart puncture followed by isolation of neutropilic granulocytes. These neutrophilic granulocytes were fluorescence marked and re-infused in the isolated perfused lungs. Neutrophil rolling and sticking was monitored by video recordings. The proportion of platelets which were rolling or sticking was higher both in arteries and veins of KOR knockout mice compared to their WT littermates. Results are shown in Table 1. Table 1: Influence of k-opioid receptors on platelet endothelial cell interaction in mouse lungs in situ. Shown are data for rolling and sticking of platelets in lungs obtained from KOR WT and KOR KO mice. Data were obtained for arteries and veins.

Subsequently, perfusion experiments were performed with asimadoline. Briefly, isolated perfused lungs from KOR WT or KOR KO mice pretreated with 5 x 10 ⁵ CFU/mL S. pneumoniea D39 were administered with asimadoline (100 mM) and platelet rolling and sticking was monitored by video recordings. The proportion of platelets which were rolling or sticking was higher both in arteries and veins of KOR knockout mice compared to their WT littermates (Figures 10 and 11.). Moreover, rolling and sticking were both reduced following treatment with asimadoline in WT animals compared to untreated control (Table 1)·

Taken together the examples shown above clearly show that KOR agonists can influence the signaling of endothelial cells of the lung induced by S. pneumoniae. Moreover, treatment with KOR agonist asimadoline also lead to down-regulation of adhesion molecules E- selectin and ICAM-l on endothelial cells of the lung counteracting the up-regulation induced by S. pneumoniae or TNFa. In addition KOR agonist treatment resulted in the shedding of E-selectin induced by S. pneumoniae. KOR agonists reduce the activation of leukocyte- endothelial cell interactions resulting in a reduction of the inflammatory infiltrate in the lung.

G. Vasculitis model in mice

C57BL/6 mice receive an intradermal injection of LPS. On the following day vasculitis is induced by intradermal injection of TNF-a. In addition Evan’s blue is injected. 24 hours following the injection of TNF-a mice are scarified. Ear thickness is measured and the degree of vasculitis is assessed by counting petechiae. The content of Evan’s blue in the ear tissue is a marker for vascular permeability. Ears are analyzed by histology, FACS and RT-qPCR.

Treatment with example N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) - ethyl]-2, 2-diphenyl acetamide hydrochloride (s.c.) resulted in a reduction of ear thickness and a reduced number of petechiae. In histology a reduced inflammatory infiltrate was seen.

Examples of Pharmaceutical Compositions

Composition Example injection glasses: 100 g N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) -ethyl]-2,2- diphenylacetamide hydrochloride and 5 g disodium hydrogen phosphate are dissolved in 3 L bidest. water. The pH is adjusted to 5.8 with 2 N aqueous hydrochloric acid. Following sterile filtration the solution is filled into injection glasses, lyophilized under sterile conditions and sterilely sealed. Each injection glass contains 5 mg N-methyl-N-[(lS)-l- phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl)-ethyl]-2,2-dipheny lacetamide.

Composition Example suppositories:

20 g N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) -ethyl]-2,2- diphenylacetamide hydrochloride are molten together with 100 g soya lecithin and 1400 g cocoa butter. The molten mixture is poured into molds. Each suppository contains 15 mg N- methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl)-e thyl]-2,2- diphenylacetamide.

Composition Example solution:

A solution is prepared consisting of 1 g N-methyl-N-[(lS)-l-phenyl-2-((3S)-3- hydroxypyrrolidine-l-yl)-ethyl]-2,2-diphenylacetamide hydrochloride, 9.38 g sodium dihydrogen phosphate dihydrate, 28.48 g disodium hydrogen phosphate dodecahydrate and 0.1 g benzalkonium chloride in 940 mL bidest. water. The pH is adjusted to 5.8. Bidest. water is added until a total volume of 1 L is reached. The resulting solution is sterilized by irradiation.

Composition Example tablets:

A mixture of 1 kg N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) -ethyl]- 2,2-diphenylacetamide hydrochloride, 4 kg lactose, 1.2 kg potato starch, 0.2 kg talcum and 0.1 kg magnesium stearate are pressed to tablets applying standard methods in a way that each tablet contains 10 mgN-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-y l)- ethyl]-2,2-diphenylacetamide.

Composition Example lozenge:

Tablets prepared as described above are coated with a coating containing sucrose, potato starch, talcum, tragacanth and a colouring agent.

Composition Example ampulla:

A solution of 1 kg N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) -ethyl]- 2,2-diphenylacetamide hydrochloride in 60 L bidest. water is sterile filtered, lyophilized under sterile conditions and sterilely sealed. Each ampulla contains 10 mg N-methyl-N- [( 1 S)- 1 -phenyl-2-((3 S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2-diphenylacetamide.

Composition Example capsule:

As a specific embodiment of an oral composition of a compound of the present invention, 10 mg of N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) -ethyl]-2,2- diphenylacetamide hydrochloride is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatine capsule.

Composition Example inhalable formulation:

31.82 kg of lactose monohydrate for inhalation (200M) are used as the coarser excipient component. 1.68 kg of lactose monohydrate (5 pm) are used as the finer excipient component. In the resulting 33.5 kg of excipient mixture the proportion of the finer excipient component is 5%.

About 0.8 to 1.2 kg of lactose monohydrate for inhalation (200M) are added to a suitable mixing container through a suitable granulating sieve with a mesh size of 0.5 mm. Then alternate layers of lactose monohydrate (5 pm) in batches of about 0.05 to 0.07 kg and lactose monohydrate for inhalation (200M) in batches of 0.8 to 1.2 kg are sieved in. Lactose monohydrate for inhalation (200M) and lactose monohydrate (5 pm) are added in 31 and 30 layers, respectively (tolerance: ±6 layers). The ingredients sieved in are then mixed together (mixing at 900 rpm).

To prepare the final mixture, 32.87 kg of the excipient mixture (1.1) and 0.13 kg of micronized N-methyl-N-[(lS)-l-phenyl-2-((3S)-3-hydroxypyrrolidine-l-yl) -ethyl]-2,2- diphenylacetamide hydrochloride (such as the one described in the general description above) are used. The content of active substance in the resulting 33.0 kg of inhalable powder is 0.4%.

About 1.1 to 1.7 kg of excipient mixture are added to a suitable mixing container through a suitable granulating sieve with a mesh size of 0.5 mm. Then alternate layers of N-methyl-N- [( 1 S)- 1 -phenyl-2-((3 S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2-diphenylacetamide hydrochloride in batches of about 0.003 kg and excipient mixture in batches of 0.6 to 0.8 kg are sieved in. The excipient mixture and the active substance are added in 46 or 45 layers, respectively (tolerance: ±9 layers). The ingredients sieved in are then mixed together (mixing at 900 rpm). The final mixture is passed through a granulating sieve twice more and then mixed (mixing at 900 rpm).

Inhalation capsules (inhalettes) having the following composition were produced using the mixture obtained accordingly.

N-methyl-N-[( 1 S)- 1 -phenyl-2-((3 S)-3-hydroxypyrrolidine- 1 -yl)-ethyl]-2,2- diphenylacetamide hydrochloride: 0.0225 mg lactose monohydrate (200 M): 5.2025 mg lactose monohydrate (5 mhi): 0.2750 mg hard gelatine capsule: 49.0 mg

Total: 54.5 mg