Leoligin and derivatives thereof as calcium channel blockers for the prevention and / or treatment of related diseases

Abstract

The present invention provides compounds of formula (I) which act as a calcium channel blocker, for use in the prevention and / or treatment of diseases associated with dysfunction of the calcium channels and / or imbalance in intracellular calcium homeostasis, such as hypertension, Raynaud's disease, migraine, migraine prophylaxis, viral infections, angina, arrhythmias, subarachnoid hemorrhage, hypertrophic cardiomyopathy, esophageal spasm, and pulmonary hypertension, as well as a composition and a pharmaceutical composition comprising said compounds.

Description

LEOLIGIN AND DERIVATIVES THEREOF AS CALCIUM CHANNEL BLOCKERS FOR THE PREVENTION AND / ORTREATMENT OF RELATED DISEASESField of the Invention

[0001] The present invention relates to the use of leoligin and derivatives thereof as calcium channel blockers. The invention specifically relates to the prevention, reduction and / or treatment of pathological disorders caused by dysfunction of calcium channels and / or imbalance in intracellular calcium homeostasis.Background Art

[0002] Calcium ions (Ca2+) are the major signaling ions in cells. The entry of calcium ions into cells and into different cellular compartments is regulated by specific calcium channels and transporters.

[0003] Dysfunction of calcium channels causes imbalance in intracellular calcium homeostasis, which can lead to severe pathological disorders including cardiovascular disease, cardiac arrhythmias, cancer, muscle disease, neurological diseases, allergies, and inflammation.

[0004] Calcium channel blockers (CCBs) are defined as a class of clinically used molecules, peptides or proteins that selectively block the entry of Ca2+into cells through inhibition of calcium-permeable ion channels, resulting in decrease of intracellular Ca2+concentration.

[0005] Currently known calcium channel blockers include phenylalkylamines such as verapamil, or dihydropyridines (DHP) such as amlodipine, felodipine, isradipine, lacidipine, nicardipine, nifedipine, niguldipine, niludipine, nimodipine, nisoldipine, nitrendipine, nivaldipine, and the like.

[0006] In general, calcium channel blockers are used alone or in combination with other drugs such as angiotensin receptor blockers (ARB) and / or diuretics, for the treatment of hypertension, coronary heart disease (CAD) , angina pectoris, heart failure such as congestive heart failure, left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiac myopathy, supraventricular and ventricular arrhythmias, atrial fibrillation, atrial flutter, detrimental vascular remodeling, myocardial infarction, atherosclerosis, renal insufficiency, diabetes, primary and secondary pulmonary hypertension, renal failure, diabetic nephropathy, glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria of primary renal disease, and also renal vascular hypertension, diabetic retinopathy, the management of other vascular disorders, such as migraine, peripheralvascular disease, Raynaud's disease, luminal hyperplasia, cognitive dysfunction (such as Alzheimer's diseases), glaucoma and stroke.

[0007] One example of a commonly used non-dihydropyridines is the phenylalkylamine verapamil, which is used for the treatment of stroke, coronary artery disease, angina pectoris or heart arrythmias e.g., EP0229644B1).

[0008] Calcium channel blockers may cause side effects such as for example, dizziness, fatigues, flushing, or swellings in belly, ankles or feet. Non- dihydropyridines may cause constipation and intestinal complications, or slow heartbeat and a reduction of the heart’s pumping ability. In rare cases, commonly used CCBs caused gastrointestinal bleeding and mortality. Mechanisms behind side effects remain mostly elusive, and patients must adapt their treatment using alternatives to CCBs. Furthermore, animal experiments showed placental transduction and potential embryotoxicity of some CCBs, which limits use for pregnant women.

[0009] Leoligin, 5-methoxyleoligin (5-ML) and their derivatives are naturally occurring lignans from Edelweiss (Leontopodium niva / e ssp. a / pinurri) that exhibit potent pro-arteriogenic and pro-angiogenic activity (WC2022043481A). Leoligin displays anti-inflammatory activity with low or no toxicity, making it a candidate for the treatment of vein graft disease. Furthermore, leoligin is a cholesterol efflux inducer for macrophages, making it a potential candidate for the treatment of atherosclerosis. US2013053438A discloses a pharmaceutical composition comprising leoligin or 5-ML for stimulating angiogenesis and / or prevention and / or treatment of an angiogenic disorder.

[0010] Taken together, there is still need for novel calcium channel blockers with higher sensitivity and selectivity, and lower toxic potential than current drugs available.Summary of invention

[0011] It is the objective of the present invention to provide compounds and compositions which act as calcium channel blockers to provide means for the medical interference in diseases and disorders associated with imbalance in intracellular calcium homeostasis.

[0012] The objective is solved by the subject matter of the present invention.

[0013] According to the invention, there is provided a compound of general formula (I) or a composition comprising a compound of general formula (I)whereinR1is selected from the group consisting of H, C^alkyl, halogen, -CF3, -CN, -0Ra, -C(O)Ra, -C(O)ORa, and -NRaRa, or two R1together form a 6-membered cyclic group;R2is selected from the group consisting of H, C^alkyl, C2-6alkenyl, C3-6cycloalkyl, C3-6cycloalkenyl, phenyl, -NRaRa; and -C(O)Ra; each Raindependently of one another denotes H C^alkyl, C2-6alkenyl; C3-6cycloalkyl, C3-6cycloalkenyl, or phenyl; n denotes 0, 1, 2, or 3; for use as a calcium channel blocker in the prevention and / or treatment of diseases associated with dysfunction of the calcium channels and / or imbalance in intracellular calcium homeostasis such as hypertension, Raynaud’s disease, migraine, migraine prophylaxis, viral infections, angina, arrhythmias, subarachnoid hemorrhage, hypertrophic cardiomyopathy, esophageal spasm, and pulmonary hypertension.

[0014] According to a further embodiment, R1is selected from the group consisting of H, C / ^alkyl, halogen, -CF3, — CN,-ORa, -C(O)Ra, and -C(O)ORa.

[0015] R2may denote H, C2-6alkenyl, or -C(O) Ra.

[0016] Ramay denote hydrogen, methyl, ethyl, propyl, propenyl, or methylpropenyl.

[0017] According to one embodiment of the invention, the compound is administered systemically to the subject in need thereof.

[0018] The compound may be administered orally, sublingually, or intravenously.

[0019] The compound may be administered through infusion, via inhalation, or dermal application.

[0020] According to a further embodiment, the compound is selected from the group consisting of:

[0021] According to one embodiment of the invention, the compound is selected from the group consisting of:

[0022] One embodiment of the invention relates to a composition comprising a compound for use according as described herein.

[0023] One embodiment of the invention relates to a pharmaceutical composition comprising a compound for use as described herein.Brief description of drawings

[0024] Fig. 1 depicts the electrical current changes caused by calcium channel inhibition through 10 pM 5-ML (n=7) or 10 pM Verapamil (n=6), each added 180s after the start of the experiment, compared to continuously applied 10 mM extracellular Ca2+(n=7). Electrical current is shown as absolute values (A) and normalized to the time point of drug application (B). Lanthanum (La3+), was introduced 270s after the start of the experiment, causing a block of electric currents. Experiments were performed using the patch clamp technique on single HEK cells overexpressing CAV1.2.

[0025] Fig. 2 shows the in si / ico determined binding affinity of Verapamil, Leoligin, 5-ML, and leoligin derivatives compound 14, compound 16 and compound 20 to calcium channel Cavl.2.Description of Embodiments

[0026] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person.

[0027] As used herein, the term "halogen" denotes fluorine, chlorine, bromine, or iodine, particularly fluorine or chlorine.

[0028] Unless specified otherwise, the term “alkyl”, when used alone or in combination with other groups or atoms, refers to a saturated straight or branched chain consisting solely of 1 to 6 hydrogen-substituted carbon atoms, and includes methyl, ethyl, propyl, isopropyl, n-butyl, 1-methylpropyl, isobutyl, t- butyl,2,2-dimethylbutyl, 2,2-dimethylpropyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl and the like.

[0029] Unless specified otherwise, the term “alkenyl” refers to a partially unsaturated straight or branched chain consisting solely of 2 to 6 hydrogensubstituted carbon atoms that contains at least one double bond, and includes vinyl, allyl, 2-methylprop-l-enyl, but-l-enyl, but-2-enyl, but-3-enyl, buta-1,3- dienyl, penta-1, 3-dienyl, penta-2, 4-dienyl, 2-methylbut-l-enyl, 2-methylpent-l- enyl, 4-methylpent-l-enyl, 4-methylpent-2-enyl, 2-methylpent-2-enyl, 4- methylpenta-1, 3-dienyl, hexen-l-yl and the like.

[0030] Unless specified otherwise, the term “cycloalkyl”, when used alone or in combination with other groups or atoms, refers to monocyclic hydrocarbon rings, bicyclic hydrocarbon rings or spirohydrocarbon rings, which each may be either saturated or unsaturated (cycloalkenyl). The term unsaturated means that in the ring system in question there is at least one double bond, but no aromatic system is formed. Cycloalkyl itself may be linked to the molecule as substituent via any suitable position of the ring system.

[0031] Examples of saturated hydrocarbon rings are cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0032] Examples of saturated hydrocarbon rings are cycloprop-l-enyl, cycloprop- 2-enyl, cyclobut-l-enyl, cyclobut-2-enyl, cyclopent-l-enyl, cyclopent-2-enyl, cyclopent-3-enyl, cyclohex-l-enyl, cyclohex-2-enyl, and cyclohex-3-enyl.

[0033] In certain embodiments, two R1substituents in the formula can be linked together to form a cyclic ring. Unless specified otherwise, the term “cyclic ring” refers to an unsaturated ring such as, for example, a cyclic alkyl ring, or saturated ring , for example, an aryl ring.

[0034] The term “calcium channels” refers to calcium ion (Ca2+) channels.Calcium ions are important cellular signaling factors which can regulate molecular processes such as gene expression and regulation, protein phosphorylation and modification, cellular activities such as energy metabolism, cell division, proliferation, differentiation, and apoptosis, and tissue-level functions such as embryo formation and development, cardiac and muscle contraction, neuronal communication, memory, and learning (2 - 4). Intracellular calcium homeostasis is maintained by calcium channels, which are proteins that transport calcium ions across the plasma membrane and the membrane of the organelles. Thus, calcium channels remove intracellular calcium overload or allow influx when needed.

[0035] Calcium channels are classified into several types based on their properties and functions. The main types of calcium channels include Voltage- Gated Calcium Channels (VGCCs), Voltage-Independent Calcium Channels, Second Messenger-Operated Calcium Channels, Transient Receptor Potential (TRP) Channels, Calcium release-activated calcium channel, and Mitochondrial Calcium Channels. One example of a voltage-dependent calcium channel is the long-lasting (L)-type calcium channel, which is, to name a few examples, responsible for the excitation-contraction coupling of skeletal, smooth, cardiac muscle. One isoform of the L-type calcium channel is, e.g., Cavl.2.

[0036] Calcium channel blockers refer to a class of clinically used molecules, peptides or proteins that selectively block the entry of Ca2+into cells through inhibition of calcium-permeable ion channels, resulting in decrease of intracellular Ca2+concentration.

[0037] Leoligin (A) is a lignan, which was isolated from the roots of Edelweiss (Leontopodium aipinum Cass.). The IUPAC name for leoligin is: [(2S,3 / ?,4A)-4- (3, 4-di methoxy benzyl) -2- (3, 4-di methoxy phenyl) tetra hydrofuran -3-yl] methyl (2Z)- 2- methyl but-2-enoat.

[0038] Derivatives of leoligin include but are not limited to natural derivatives of leoligin, such as 5-methoxyleoligin (=[(2S,3 / ?,4A)-4-(3,4-dimethoxybenzyl)-2- (3, 4, 5 -tri methoxy phenyl) tetra hydro-furan -3-yl] methyl -(22) -2- methyl but-2-en- oate). The chemical formula of 5-methoxyleoligin is C28H36O8. Optically pure 5- methoxyleoligin is a furan lignin, which may for example be isolated from the roots of Leontopodium species or synthesized via various synthetic pathways.

[0039] The present invention solves the above identified objective since it was surprisingly found that compounds of formula (I) may act as calcium channel blockers, thus providing means for the medical interference in diseases and disorders associated with imbalance in intracellular calcium homeostasis.

[0040] Specifically, the compound to be used in accordance with the present invention or the compound as comprised in the pharmaceutical composition or formulation of the present invention may be obtained from plants belonging to the genus Leontopodium, optionally followed by standard derivatization reactions. It is particularly preferred that the compounds provided herein may be obtained from Leontopodium a / pinum, in particular Leontopodium aipinum Cass., which is commonly known under the trivial name "edelweiss". According to another nomenclature "edelweiss" is also known under the scientific term "Leontopodiumniva / e subsp. a / pinum (Cass.) Greater". However, the terms " Leontopodium alpinum Cass" and "Leontopodium niva / e subsp. aipinum (Cass.) Greater" refers to the same plant species and merely reflects a regrouping of the species in botanical nomenclature. Accordingly, these terms can be used interchangeably in context of the present invention and any definitions and explanations given herein in respect of Leontopodium aipinum Cass, also applies to Leontopodium nivaie subsp. aipinum (Cass.) Greater, mutatis mutandis, and vice versa.

[0041] Of course, it is envisaged herein that the compounds to be used according to the present invention may be obtained from other Leontopodium species, including but not limited to commercial cultivars, such as Leontopodium hybrids.

[0042] Of course, the compounds of formula (I) as provided herein may be obtained from corresponding cell culture, cell suspension culture or a comparable in vitro cultivation technique, such as callus culture and the like. A person skilled in the art will be aware of corresponding means and methods for establishing and maintaining corresponding cultures. In a preferred embodiment of the invention, the cell culture is derived from roots of Leontopodium species described herein above, in particular Leontopodium aipinum (edelweiss). Most preferably, the cell culture is derived from hairy roots.

[0043] Based on his general knowledge and the teaching provided herein a skilled person is readily in the position to obtain the compounds to be used herein, in particular leoligin, from Leontopodium species. Generally, the person skilled in the art is capable of preparing an extract from plants belonging to the genus Leontopodium by standard techniques. A preferred method for extracting these compounds from the roots of Leontopodium aipinum is for example provided in US2014 / 0371306A1. An artisan will be aware how to adapt this protocol for extracting the compounds from further Leontopodium species and in particular from roots of these plants. A skilled person will also be aware of alternative protocols to be used in this context.

[0044] The term extract is well known in the art and used accordingly herein. For example, this term may refer to preparations of fluid consistence (fluid extracts and tinctures), semisolid consistence (viscous extracts, syrup concentrate) or solid consistence (dried extracts), which are usually prepared using fresh or dried plant material.

[0045] The extract obtained from Leontopodium species is an extract that is received by the use of an organic or nonorganic solvent. Suitable solvents arehexane, heptane, petroleum benzene, acetone, chloroform, dichloromethane, ethyl acetate, diethylether, liquid carbon dioxide, ethanol, tertiary butyl methyl ether (tBMe) and mixtures of water and alcohol. The extract may be obtained by extracting the plant material, in particular roots, with any of the solvents separately. It is further possible to subsequently extract the obtained extract with a second solvent or mixtures of different solvents. An exemplary, non-limiting solvent to be used in a first extraction step is hexane. However, any of the above solvents can be used in such a first extraction step. This first extraction step may be followed by (a) subsequent second (or further) extraction step with at least one of the above exemplary solvents, e.g. dichloromethane, chloroform or tertiary butyl methyl ether (tBMe). Extraction of the compounds disclosed herein, in particular leoligin and / or its (di)methoxy-derivative(s)) is also illustrated in US2014 / 0371306A1.

[0046] Preferably, dichloromethane and methanol are used as extraction solvents. In subsequent extraction, it is preferred that the compounds are first extracted with n-hexane, followed by a subsequent extraction with dichloromethane, chloroform or tBMe. The lignan content can be increased by a second or further extracting steps. Also, the use of chromatographic methods, such as Sephadex-l_H20-column chromatography and in particular silica gel column chromatograph is advantageous in this context.

[0047] Further chromatographic methods to increase the content of leoligin and / or its (di)methoxy-derivative(s)) can be used in addition or in the alternative to the above described methods. Exemplary, non-limiting chromatographic methods to be used in this context are reversed phase column chromatography or (semi)-preparative HPLC using water / acetonitrile mixtures or comparable solvent mixtures known in the art. Alternatively, techniques of liquid-liquid extractions (discontinuous or continuous methods) can be used to increase the content of leoligin and its derivatives. An exemplary liquid-liquid extraction is high speed counter current chromatography using a solvent system of two not mixable solvents.

[0048] The preparation of the basic extract of Leontopodium species, in particular Leontopodium a / pinum, may comprise mechanical pulping, sonication, use of mortars and pestles, freeze-thawing cycles, use of blenders (like Waring Blenders, Polytron), liquid homogenization and maceration (see also appended examples), or e.g. Dounce homogenization, Potter-Elvehjem, French Press etc.However, the extracts may also be obtained by disrupting the cells and cells from the Leontopodium species by any mechanical / physical or chemical means, like by use of detergents.

[0049] The cells and plants to be employed in order to obtain the basic extract may be cells of natural origin as well as cultured cells or plants. It is preferred herein that the cells or plants and in particular roots of the plants are dried before mechanical disruption / maceration as described herein above. The cells or plants may be air dried, lyophilized (freeze-dried) or, though less preferred, dried in an oven. It is preferred herein that the "cell(s)" and "plant(s)" to be used as a basic material are fresh, i.e. harvested shortly before the extract is prepared. Nonetheless, it is possible to store the basic material before its use in the preparation of the extract. For example, the basic material may be lyophilized (freeze-dried) or simply frozen and stored at low temperatures, e.g. at about -20 to -30° C, or as low as -80° C.

[0050] Preferably, the extract is an enriched extract, i.e. contains leoligin and / or its derivatives in a high amount. Such an enriched extract can, for example, be obtained by taking advantage of silica gel chromatography. Silica gel column chromatography is well known in the art and described in detail in standard textbooks, such as "Preparative Chromatography Techniques" by Hostettmann, K. Marston, Andrew Hostettmann, Maryse, Springer-Verlag GmbH, 2007, 260 p.

[0051] In an alternative embodiment, the compounds of formula (I) may be synthesized. An exemplary synthetic pathway of leoligin and derivatives thereof, such as 5-methoxyleoligin, is shown in e.g., US2014 / 0371306A1 and US2017 / 0157301A1. The shown synthetic pathway might be adapted by a change of the corresponding educts to obtain other compounds of the present invention. A skilled person will be aware of methods of synthesizing the compounds of the present invention, in particular 5-methoxyleoligin, or may deduce corresponding methods e.g. from Li Hong Hu, J. Nat. Prod. 68, 342-8. (2005) or Linder et al. Chem. Sci. 10(22) :5815-5820 (2019).

[0052] According to a specific embodiment, key steps of the synthetic production of leoligin and derivatives thereof, as described in Linder et al., involve (i) establishing an enantiomerically pure status upon lipase mediated kinetic resolution; (ii) formation of a tetrahydrofuran ring system upon diastereoselective radical cyclization; (iii) regio- and stereo-selective arylation via hydroboration and subsequent Suzuki-Miyaura cross-coupling in a single-operation cascade and,finally, (iv) esterification employing a modified Mitsunobu protocol (Linder et al. Chem. Sci. 10(22) :5815-5820 (2019)).

[0053] As used herein, the term “patient” or “subject” refers to both, humans and other animals, particularly warm-blooded animals and / or mammals. Thus, the methods and compositions described herein are applicable to both human therapy and veterinary applications. Preferably, the patient is a mammal. A "subject" mammal can include, but is not limited to, a human or non-human mammal, such as a primate, bovine, equine, canine, ovine, feline, or rodent. Preferably, the patient or subject is a human.

[0054] The term "treatment" relates to any treatment which improves the health status, reduces or inhibits unwanted tissue damage and / or prolongs and / or increases the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and / or decrease the recurrence in an individual who currently has or who previously has had a disease.

[0055] The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably.

[0056] As used herein, "preventing” or “prevention" of a disease, disorder or condition refers to the reduction of the occurrence of the disorder or condition in a treated subject relative to an untreated control subject or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control subject.

[0057] The terms "protect", "prevent", "prophylactic", "preventive", or "protective" relate to the prevention and / or treatment of diseases associated with dysfunction of the calcium channels and / or imbalance in intracellular calcium homeostasis.

[0058] The term "effective amount" as used herein refers to an amount of the compound of the present invention effective for "treating" or "preventing" a disease or disorder in a subject. The effective amount may cause any changes observable or measurable in a subject as described in the definition of "treating", "treatment", "preventing", or "prevention" above. The "effective amount" may vary depending, for example, the compound, the disease and / or symptoms of thedisease, severity of the disease and / or symptoms of the disease or disorder, the age, weight, and / or health of the subject to be treated, and the judgment of the prescribing physician. An appropriate amount in any given instance may be ascertained by those skilled in the art or capable of determination by routine experimentation.

[0059] The pharmaceutical compositions or pharmaceutically acceptable formulations described herein comprise a compound of formula (I) as active ingredient in an effective amount. The compound of formula (I) to be used in accordance with the present invention may be obtained from Leontopodium plants as described herein above and / or chemically synthesized.

[0060] The pharmaceutical composition described herein is formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The "effective amount" of the pharmaceutical composition for purposes herein is thus determined by such considerations.

[0061] The skilled person knows that the effective amount of pharmaceutical composition administered to an individual will, inter alia, depend on the nature of the compound. For example, if said compound is leoligin the total pharmaceutically effective amount of composition administered systemically as described herein will be in the range of about 0.01 mg / kg / day [minimum dose] to 10 mg / kg / day [maximum dose] of patient body weight, although, as noted above, this will be subject to therapeutic discretion.

[0062] More preferably, this dose is at least 0.25 mg / kg / day, and most preferably for humans between about 0.05 and 5 mg / kg / day.

[0063] The length of treatment needed to observe changes and the interval following treatment for responses to occur may vary depending on the desired effect. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art.

[0064] Pharmaceutical compositions of the invention preferably comprise one or more pharmaceutically acceptable carriers. As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Thecarrier is selected for administration by the selected route of administration. The use of such media and agents for pharmaceutically active substances is well known in the art (Rowe R.C. et al, Handbook of Pharmaceutical Excipients, 2012, 7th edition, Pharmaceutical Press, London UK). Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated.

[0065] Non-limiting pharmaceutically suitable materials that may be incorporated in pharmaceutical preparations of the present invention include solubilizing / diluting agents, antioxidants, enteric coatings, absorption enhancers, pH adjusting agents and buffers, dispersing agents, coatings, antibacterial and antifungal agents, absorption delaying agents, osmolarity adjusters, isotonic agents, preservative agents, stabilizers, surfactants, thickening agents, solvents, co-solvents, emollients, coloring agents, wetting agents and ligands / pilot / targeting molecules. Methods for preparing appropriate formulations are well known in the art.

[0066] As used herein, the term “systemic administration” refers to a route of administration of a product, specifically the compositions and formulations described herein, into the circulatory system of a subject or patient. Specifically, the term “systemic administration” as used herein refers to administration via the respiratory tract, buccal administration, sublingual administration or parenteral administration.

[0067] As used herein, the term “systemic administration” preferably excludes enteral administration, i.e. absorption of the drug through the gastrointestinal tract.

[0068] The term "sublingual administration", synonymous with “sublingual delivery”, as used herein refers to the pharmacological route of administration by which substances diffuse into the blood through tissues under the tongue.

[0069] The term "buccal administration", synonymous with “buccal delivery”, as used herein refers to a topical route of administration by which drugs held or applied in the buccal area (in the cheek) diffuse through the oral mucosa and enter directly into the bloodstream.

[0070] Specifically, formulations for sublingual administration comprise tablets, capsules, bars, lozenges or granules having a size so small (generally not exceeding 9-10 mm diameter) that they can be placed in the sublingual cavity of the mouth, whereas formulations for buccal, peribuccal or orobuccaladministration comprise tablets, capsules, bars, lozenges or granules having a greater size, which are dissolved or chewed in the oral cavity.

[0071] In both cases, sublingual or buccal administration, the delivery system consists in placing a given product (tablet, lozenge etc.) inside the oral cavity and waiting until it is dissolved after a short time, generally a few minutes. It is a form of very fast intake, wherein the product comes into contact with the mucous membrane, and spreads, dissolving, evenly in the epithelium under the tongue, an area full of blood vessels, entering into the circulatory system, i.e. into the blood, much faster than oral administration (where the tablet is swallowed). Such an effect is explained in view of the fact that the oral hematic system discharges the active ingredient in the superior vena cava thus avoiding the portal venous system responsible of the known effect of first pass splanchnic metabolism, thereby improving systemic bioavailability.

[0072] In a specific embodiment, leoligin or the derivative thereof, is provided as orosoluble and / or effervescent formulation for sublingual, or buccal or orobuccal or peribuccal use, containing leoligin or a derivative thereof, specifically 5-ML, in combination with physiologically acceptable carriers and / or excipients.

[0073] The orosoluble and / or effervescent compositions of the present invention are preferably characterized by the fact of having a high palatability, which guarantees easy administration in the oral cavity. Said high palatability (meant as very pleasant taste) also provides for a lower impulse to swallow, which helps to improve the absorption rate and bioavailability of the active ingredient due to a longer period of permanence of the composition in the oral cavity.

[0074] The orosoluble and / or effervescent compositions of the present invention can therefore be formulated in tablets, lozenges, powders, capsules and / or granules, preferably in the form of an orosoluble and / or effervescent tablet.

[0075] According to the present invention the term "orosoluble" refers to a composition capable of melting immediately, releasing the active ingredient contained therein when in contact with the oral mucosa.

[0076] In this way, the active ingredient can be absorbed directly into the oral mucosa, thus bypassing the hepatic system. According to the invention, the term orosoluble is therefore used to indicate compositions to be introduced in the oral cavity.

[0077] The compositions of the present invention can be formulated with further excipients, such as sweeteners or flavorings (added to improve the organolepticcharacteristics of the products); antioxidants-antimicrobials (used to prolong the shelf life of the product); gliding agents (used to improve the flow properties of the granules or powder and thus to facilitate the filling of the matrix in a homogeneous manner, allowing to achieve uniform tablets by weight); solubilizing agents (for favoring the solubilization of the composition); and / or pH regulating agents.

[0078] The excipients of the conventional type most commonly used are: lactose, glucose, saccharose, mannite (or mannitol), kaolin, talc, bentonite, titanium dioxide, xylitol, maltitol, sorbitol, sucralose, acesulfame K, aspartame, neohesperidin, fructose, dextrose, maltose, "spray-dried" malt, aspartate sodium, maltodextrin, sodium chloride, hydroxypropylmethylcellulose, erythritol, citrus extract, silica gel, vegetable fibers (as for example, the pea fiber), flavoring agents and aromas, such as mint flavor (peppermint, spearmint, fresh mint), anethole of star anise, vanilla, sage, liver, chicken, grapefruit, peach, orange, lemon or lime, sodium glutamate and fish flour.

[0079] To obtain the orosoluble for, the compositions of the invention are for example formulated using the excipients described above.

[0080] More preferably, the orosoluble compositions according to the invention are formulated with magnesium oxide, magnesium hydroxide, alginic acid, stearic acid, hydrogenated vegetable oils (palm, oleic, behenic), cocoa butter, cocoa mass, xylitol, maltitol, sorbitol, mannitol, sucralose, acesulfame K, cyclamate, aspartame, sucrose, neohesperidin, fructose, dextrose, maltose, spray dried malt, aspartate sodium, maltodextrin, inositol, inulin, chitosan, beer yeast or a mixture of two or more of the foregoing excipients. In particular, in order to obtain the orosoluble form, said excipients are present in the compositions of the invention in an amount from 20% to 95% by weight, relative to the total weight of the formulation.

[0081] Further provided herein is a pharmaceutical liquid composition for buccal or sublingual administration comprising leoligin or a derivative thereof, an alcoholic solvent, and, optionally, one or more pharmaceutically acceptable carriers and / or excipients.

[0082] The liquid pharmaceutical composition according to the present invention is in the form of solution, suspension, nano-suspension, emulsion, microemulsion, multiple emulsion and the like meant for administration through oral mucosa preferably, in the form of solution.

[0083] Another embodiment of the present invention relates to pharmaceutical liquid spray composition for buccal or sublingual administration consisting essentially of leoligin or a derivative thereof, and an alcoholic solvent.

[0084] In another preferred embodiment, the present invention relates to pharmaceutical liquid spray compositions for buccal or sublingual administration consisting of leoligin or a derivative thereof, an alcoholic solvent and water.

[0085] In a specific embodiment, systemic administration of the leoligin or derivative thereof may be via parenteral administration, e.g. by intramuscular selfinjection.

[0086] In cases where parenteral administration is elected as the route of administration, pharmaceutical compositions of the present invention may be provided to patients in combination with additional pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions or emulsions. Formulations to be used for in vivo administration are preferably sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.

[0087] Pharmaceutically acceptable carriers for parenteral formulations include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Aqueous solvents include water, alcohol, specifically ethanol, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils.

[0088] Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose, and the like.

[0089] Formulations suitable for oral administration, specifically buccal or sublingual administration, can consist of liquid solutions, such as an effective amount of active agent(s) suspended in diluents / solubilizers, such as water, vegetable or animal oils, saline or PEG 400; capsules such as soft shell capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; suspensions in an appropriate liquid; and suitable emulsions.

[0090] Aqueous solutions suitable for oral use, specifically buccal or sublingual delivery, are prepared by dissolving the active compound(s) in water and addingsuitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well- known suspending agents. Examples of non-aqueous solvents are alcohol, benzyl benzoate, butyl alcohol, polyethylene glycol, propylene glycol, N,N- dimethylacetamide, ethyl oleate, oleyl oleate, glyceryl trioleate, glyceryl dioleate, glyceryl monooleate, cetyl alcohol, stearyl alcohol, capric acid, undecenoic acid, undecanoic acid, lauric acid, oleic acid, synthetic glycerides of saturated fatty acids with 8 to 12 carbon atoms, polyoxyethylene derivatives of glycerol, bees' wax, glycerin, mineral oil, vegetable oil such as but not limited to corn oil, cottonseed oil, peanut oil, canola oil, sesame oil, safflower oil, soybean oil, arachis oil, castor oil, linseed oil, soya bean oil, sunflower seed oil, olive oil, fish liver oil, and any combination thereof.

[0091] Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

[0092] Cellulose acetate phthalate concentrations generally used are 0.5-9.0% of the core weight. The addition of plasticizers improves the water resistance of this coating material, and formulations using such plasticizers are more effective than when cellulose acetate phthalate is used alone. Cellulose acetate phthalate is compatible with many plasticizers, including acetylated monoglyceride; butyl phthalyl butyl glycolate; dibutyl tartrate; diethyl phthalate; dimethyl phthalate; ethyl phthalyl ethyl glycolate; glycerin; propylene glycol; triacetin; triacetin citrate; and tripropionin. It is also used in combination with other coating agents such as ethyl cellulose, in drug controlled-release or time-release preparations.

[0093] Generally, the formulations described herein are prepared by contacting the components of the pharmaceutical composition uniformly and intimately withliquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. The carrier may be a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles, such as fixed oils and ethyl oleate, are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, e.g., polyarginine or tripeptides proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or nonionic surfactants such as polysorbates, poloxamers, or PEG.

[0094] According to a preferred embodiment, leoligin or its derivatives are administered as inhalable powder. In spray and aerosol compositions, leoligin is delivered to the mouth in a dosed amount through a nozzle or orifice. The composition may be delivered through a pump spray, a pressurized spray or as an aerosol. Moreover, it is easy to apply and it is possible to ensure the delivery of the composition to the oral mucosa and / or airways.

[0095] For example, compounds of formula (I) can be formulated for delivery into the upper respiratory system. Exemplary formulations include nasal, bronchial, oral, and pulmonary formulations. In a specific embodiment, a composition comprising leoligin as described herein is formulated as an aerosol. The aerosol can be a liquid or powdered aerosol. In some embodiments, the composition contains one or more pharmaceutically acceptable excipients such as glycerin. The composition can contain 0.01%-20% w / v of the esterified green tea polyphenol and 10% to 20% glycerol.

[0096] The composition, shape, and type of dosage forms of leoligin as described herein will typically vary depending on their use. These and other ways in which specific dosage forms encompassed by this disclosure will vary from one anotherwill be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990).

[0097] In a preferred embodiment, a compound of formula (I) is delivered to oral, nasal, or bronchial tissue in a suitable topical dosage form.

[0098] Nasal spray drug products contain therapeutically active ingredients dissolved or suspended in solutions or mixtures of excipients in non-pressurized dispensers that deliver a spray containing a metered dose of the active ingredient. The dose can be metered by the spray pump or could have been premetered during manufacture. A nasal spray unit can be designed for unit dosing or can discharge up to several hundred metered sprays of formulation containing the drug substance. Nasal sprays are applied to the nasal cavity for local and / or systemic effects.

[0099] Inhalation solution and suspension drug products are typically aqueousbased formulations that contain therapeutically active ingredients and can also contain additional excipients. Aqueous-based oral inhalation solutions and suspension must be sterile. Inhalation solutions and suspensions are intended for delivery to the lungs by oral inhalation for local and / or systemic effects and are to be used with a specified nebulizer.

[0100] An inhalation spray drug product consists of the formulation and the container closure system. The formulations are typically aqueous based and must be sterile. Inhalation sprays are intended for delivery to the lungs by oral inhalation for local and / or systemic effects. Inhalation spray drug products containing the disclosed compositions can also contain additional excipients.

[0101] Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile e.g, a gaseous propellant, such as freon), or in a squeeze bottle. Examples of sprayable aerosol preparations include but are not limited to metered dose inhalers, dry powder inhalers, and nebulizers. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton, Pa. (1990).

[0102] In some embodiments, the effect of a compound of formula (I) and compositions thereof on a subject is compared to a control. For example, the effect of the composition on a particular symptom, pharmacologic, or physiologicindicator can be compared to an untreated subject or the condition of the subject prior to treatment. In some embodiments, the symptom, pharmacologic, or physiologic indicator is measured in a subject prior to treatment, and again one or more times after treatment is initiated. In some embodiments, the control is a reference level, or an average determined from measuring the symptom, pharmacologic, or physiologic indicator in one or more subjects that do not have the disease or condition to be treated (for example, healthy subjects). In some embodiments, the effect of the treatment is compared to a conventional treatment that is known in the art.

[0103] According to a specific aspect, the present invention relates to the use of crystalline leoligin or crystalline compounds of formula (I) as a medicament in the light of the pharmaceutical efficacy of the anhydrous form according to the invention. To prepare a medicament which can be inhaled, particularly an inhalable powder, which contains the anhydrous, crystalline leoligin described by the present invention, methods known from the prior art may be used. In this respect, reference is made, for example, to the teaching of DE-A-179 22 07. Accordingly, a further aspect of the present invention relates to inhalable powders characterized in that they contain anhydrous, crystalline leoligin or its derivatives.

[0104] Preferably, the following physiologically acceptable excipients are used in an inhalable powder formulation: monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, sucrose, maltose), oligo- and polysaccharides (e.g. dextran), polyalcohols (e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these excipients with one another. Preferably, mono- or disaccharides are used, while the use of lactose or glucose is preferred, particularly, but not exclusively, in the form of their hydrates.

[0105] According to the invention, any pharmaceutically acceptable salt of a compound of formula (I) may be used for the formulations described herein.When the term leoligin salt is used within the scope of the present invention, this is to be taken as a reference to leoligin. A reference to leoligin corresponds to the free ammonium cation. The leoligin salt accordingly contains an anion as the counter-ion. Leoligin salts which may be used within the scope of the present invention are preferably compounds which contain, in addition to leoligin as counter-ion (anion), chloride, bromide, iodide, methanesulfonate, p- toluenesulfonate, and / or methylsulfate.

[0106] The concentration of a salt of a compound of formula (I) based on the proportion of the compound of formula (I) in the finished pharmaceutical preparation depends on the therapeutic effect sought.

[0107] Other pharmacologically acceptable adjuvants may be added to the formulation according to the invention. By adjuvants and additives are meant, in this context, any pharmacologically acceptable and therapeutically useful substance which is not an active substance but can be formulated together with the active substance in the pharmacologically suitable solvent, in order to improve the qualities of the active substance formulation. Preferably, these substances have no pharmacological effects or no appreciable or at least no undesirable pharmacological effects in the context of the desired therapy. The adjuvants and additives include, for example, other stabilizers, complexing agents, antioxidants, and / or preservatives which prolong the shelf life of the finished pharmaceutical formulation, flavorings, vitamins, and / or other additives known in the art. The additives also include pharmacologically acceptable salts such as sodium chloride, for example.

[0108] The preferred excipients include antioxidants such as ascorbic acid, for example, provided that it has not already been used to adjust the pH, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins occurring in the human body.

[0109] Preservatives can be added to protect the formulation from contamination with pathogenic bacteria. Suitable preservatives are those known from the prior art, particularly benzalkonium chloride or benzoic acid or benzoates such as sodium benzoate in the concentration known from the prior art.Examples

[0110] The Examples which follow are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to limit the scope of the invention in any way. The Examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art.Example 1 - Testing calcium channel blocking abilities of 5-ML in vitro

[0111] To test if 5-ML acts as a calcium channel blocker, the ability of 5-ML to inhibit or block calcium flow into the cell was tested in vitro (Fig. 1). Fig.l shows the mean electrical current values (Fig. 1A) and mean electrical current normalized to the time point of drug application (Fig. IB) of continuously applied10 mM extracellular Ca2+(n=7), and 10 pM calcium channel blocker Verapamil (n=6) or 10 pM 5-ML (n=7) applied after 180 seconds (s) during patch clamp experiments to determine electrical current changes due to calcium flux. Experiments were performed on single cells, using HEK cells overexpressing CAV1.2 L-type voltage gated calcium channels.

[0112] Over the measuring period of 350 s, cells were perfused extracel I u la rly with Ca2+, causing a detectable electrical current. After 270s, Lanthanum (La3+), an ion that blocks calcium flux by binding to the channel irreversibly, was introduced, causing a block of electric currents. As a positive control, Verapamil, a well-known calcium channel blocker with high affinity to L-type voltage gated channels, was used. Verapamil was applied at approximately 180 s and caused a reduction in recorded currents. Similar perfusion with 5-ML at approximately 180 s caused a reduction in recorded currents, to a comparable degree as Verapamil. As a result, it was shown that 5-ML acts as a calcium channel blocker.Example 2 - Testing calcium channel binding affinity in si / ico

[0113] In si / ico molecular docking studies were performed to test the binding affinity between ligands and different calcium channels (Fig. 3). Fig. 3 shows an overview of the binding affinities of Leoligin, 5-ML and derivatives to different calcium channels. The binding affinity is shown per amount of ligand in the pM and nM range. Experiments were conducted using LigandScout version 4.4.8 software to determine druggable binding pockets in calcium channels, and AutoDock Vina 1.1 for binding affinity scores.

[0114] As a next step, the binding affinity was tested in siiico for human L-type voltage gated calcium channels Cavl.2 (Fig. 2). Fig. 2 shows the in siiico determined binding affinity of Leoligin, Verapamil, 5-ML, and its derivatives compound 14, compound 16 and compound 20 (referred to as SOGE-37, SOGE-39 and SOGE-44, respectively) to calcium channel Cavl.2. Binding affinity was highest for Verapamil (18.67%). 5-ML as well as compound 20 (referred to as SOGE-44) showed binding affinities between 10.46% and 12.83%, respectively.Leoligin showed the lowest measured binding affinity at 4.15%. As a result, the in siiico determined binding affinity of 5-ML to human CAV1.2 is comparable to well-known calcium channel blocker Verapamil.References[1] Shah, K., Seeley, S. L., Schulz, C., Fisher, J. L., & Rao, S. G. (2022). Calcium channels in the heart: Disease states and drugs. Cells, 11 (6), 943.[5] Dylan Cooper; Manjari Dimri. (2023). Biochemistry, Calcium Channels. Treasure Island (FL): StatPearls Publishing.

[0014] Godfraind, T. (2006). Calcium-channel modulators for cardiovascular disease. Expert Opinion on Emerging Drugs, 11 (1), 49-73.

[0015] Kistamas, K., Veress, R., Horvath, B., Banyasz, T., Nanasi, P. P., & Eisner, D. (2020). Calcium handling defects and cardiac arrhythmia syndromes. Frontiers in Pharmacology, 11.

[0016] Monteith, G. R., Davis, F. M., & Roberts-Thomson, S. J. (2012). Calcium channels and pumps in Cancer: Changes and consequences. Journal of Biological Chemistry, 287(38), 31666-31673.

[0017] Gommans, 1., Vlak, M. H., De Haan, A., & Van Engelen, B. (2002). Calciumregulation and muscle disease. Journal of Muscle Research and Cell Motility, 23(1), 59-63.

[0018] Nam, J. H., & Kirn, W. K. (2020). The Role ofTRP Channels in Allergie Inflammation and its Clinical Relevance. Current Medicinal Chemistry, 27(9), 1446-1468.

[0019] Linder, T., Geyrhofer, S., Papaplioura, E., Wang, L., Atanasov, A. G., Stuppner, H., Dirsch, V. M., Schnurch, M., & Mihovilovic, M. D. (2020). Design and synthesis of a compound library exploiting 5-Methoxyleoligin as potential cholesterol efflux promoter. Molecules, 25(3), 662.

[0020] Wang, L., Ladurner, A., Latkolik, S., Schwaiger, S., Linder, T., Hosek, J., Palme, V., Schilcher, N., Polansky, O., Heiss, E.H., Stangl, H., Mihovilovic, M. D., Stuppner, H., Dirsch, V. M., & Atanasov, A. G. (2016). Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages. Journal of Natural Products, 79(6), 1651-1657.

[0021] Reisinger, U., Schwaiger, S., Zeller, 1., Me ner, B., Stigler, R., Wiedemann, D., Mayr, T., Seger, C., Schachner, T., Dirsch, V. M., Vollmar, A. M., Bonatti, J., Stuppner, H., Laufer, G., & Bernhard, D. (2009). Leoligin, the major lignan from Edelweiss, inhibits intimal hyperplasia of venous bypass grafts. Cardiovascular Research, 82(3), 542-549.

Claims

Claims1. A compound of formula (I)whereinR1is selected from the group consisting of H, C^alkyl, halogen, -CF3, -CN, - 0Ra, -C(O) Ra, -C(O)ORa, and -NRaRa, or two R1together form a 6-membered cyclic ring;R2is selected from the group consisting of H, C^alkyl, C2-6alkenyl, C3_ 6cycloalkyl, C3-6cycloalkenyl, phenyl, -NRaRa; and -C(O) Ra; each Raindependently of one another denotes H C^alkyl, C2-6alkenyl;C3-6cycloalkyl, C3-6cycloalkenyl, or phenyl; n denotes 0, 1, 2, or 3; for use as a calcium channel blocker in the prevention and / or treatment of diseases associated with dysfunction of the calcium channels and / or imbalance in intracellular calcium homeostasis, such as hypertension, Raynaud’s disease, migraine, migraine prophylaxis, viral infections, angina, arrhythmias, subarachnoid hemorrhage, hypertrophic cardiomyopathy, esophageal spasm, and pulmonary hypertension.

2. The compound for use according to claim 1, whereinR1is selected from the group consisting of H, C / ^alkyl, halogen, -CF3, — CN,- ORa, -C(O)Ra, and -C(O)ORa.

3. The compound for use according to claim 1 or 2, whereinR2denotes H, C2-6alkenyl, or -C(O)Ra.

4. The compound for use according to any one of claims 1 to 3, whereinRadenotes hydrogen, methyl, ethyl, propyl, propenyl, or methyl-propenyl.

5. The compound for use according to any one of claims 1 to 4, wherein the compound is administered systemically to the subject in need thereof.

6. The compound for use according to any one of claims 1 to 5, wherein the compound is administered orally, sublingually, or intravenously.

7. The compound for use according to claim 6 wherein the compound is administered through infusion, via inhalation, or dermal application.

8. The compound for use according to any one of claims 1 to 7, wherein the compound selected from the group consisting of:

9. The compound for use according to any one of claims 1 to 8, wherein the compound selected from the group consisting of:

10. A composition comprising a compound for use according to any one of claims 1 to 9.

11. A pharmaceutical composition comprising a compound for use according to any one of claims 1 to 9.

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