Composition comprising beta-2-adrenergic receptor agonist for transdermal administration

Abstract

The present invention relates to a composition for transdermal administration, the composition comprising a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, a mixture thereof, or a salt thereof, and dimethyl sulfoxide. The invention further relates to a composition comprising a compound selected from the group comprising salbutamol, clenbuterol, or a salt thereof, and comprises at least one of a monoalcohol or water.

Description

[0001] COMPOSITION COMPRISING BETA-2-ADRENERGIC RECEPTOR AGONIST FOR TRANSDERMAL ADMINISTRATION

[0002] The present invention relates to a composition for transdermal administration.

[0003] Overweight and obesity are major risk factors for many diseases. One in two citizens of the European Union is overweight, more than one in three is pre-obese and one in six is considered obese with a Body Mass Index (BMI) of over 30. In the United States, the prevalence of obesity is even more pronounced. These groups of people are significantly more at risk of developing cardiovascular diseases and diabetes mellitus. The aesthetic problems of the overweight also lead to social isolation and can lead to mental illnesses such as depression. The clinical picture of lipedema in this regard is particularly distressing. Eipedema is a chronic and progressive disease of the subcutaneous fatty tissue of the upper and lower extremities, which almost exclusively affects women. The disease is poorly understood, but it appears to be due to a pathological increase in adipose tissue through hypertrophy and hyperplasia, accompanied by fluid retention. This leads to a marked, delineated increase in volume of the affected body parts, especially the extremities, compared to the rest of the body. Combating obesity therefore has a direct benefit for the physical and mental health of each individual and the population.

[0004] Drugs that increase fat loss belong for the most part to the class of anorectic drugs. These are largely derived from amphetamines and suppress the appetite. Another possibility to support weight reduction is the use of lipase inhibitors. However, these substances require oral or injectable administration. In the case of systemic administration, however, serious side effects must be expected, since these compounds largely interfere with the metabolism of neurotransmitters in the brain. A local administration of active substances is also known, whereby the active substance is applied directly into the site of action. One possibility to realise this is a direct injection into the fatty tissue. This is practised in some cases of small to medium local fat deposits by injecting a mixture of phosphatidylcholine and deoxycholate. However, this surface -active mixture leads to a localised lipolysis effect at the injection site and is typically associated with several injections and corresponding side effects such as pain, haematoma and the like.

[0005] A topical application essentially reduces systemic absorption and respective side effects. WO 2007 / 011743 Al discloses compositions for treating regional fat deposits comprising long -acting beta- 2 adrenergic receptor agonists salmeterol or formoterol and a compound that reduces desensitization of the target tissue to the long-acting P2 -adrenergic receptor agonist, for example, glucocorticosteroids and / or ketotifen. The composition, for example, may be administered by injection and / or transdermally. However, pharmaceutically active compounds often show no or only suboptimal clinical efficacy upon topical administration because of poor penetration into the skin.

[0006] EP 3 206 678 Bl discloses a pharmaceutical composition for topical administration comprising a prodrug for an agonist and / or an antagonist for an adrenergic receptor, wherein the prodrug is an ester, for or use in a therapeutic method of shaping a mammalian body by modulation of subcutaneous fat tissue. The numerous cited beta adrenergic receptor agonists (“beta-agonists”) include mirabegron, salbutamol, formoterol, salmeterol, fenoterol and clenbuterol. An absorption of the prodrug provides a transport route via the skin, but the availability of the active ingredient depends on the activity of enzymes which are capable of releasing the agonist from the prodrug, and whether a therapeutic effect can be achieved that way is unknown. However, the p2-adrenergic receptor agonists clenbuterol, formoterol, salmeterol, fenoterol and salbutamol, are not yet commercially available as a transdermal dosage form.

[0007] The object of the present invention is therefore to provide a composition allowing transdermal administration of P2 -adrenergic receptor agonists.

[0008] The object is achieved by the composition according to claim 1. The object further is achieved by the composition according to claim 5. The object further is achieved by a use according to claims 9 or 10. Advantageous embodiments are the subject of the dependent claims. The embodiments may be combined freely unless the context clearly indicates otherwise.

[0009] Accordingly, a composition for transdermal administration is provided, the composition comprising a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, a mixture thereof, or a salt thereof, and dimethyl sulfoxide.

[0010] It has surprisingly been found that dimethyl sulfoxide (DMSO) also allows for a transdermal administration of formoterol, salbutamol, salmeterol, or fenoterol. DMSO facilitated skin permeation and increased active delivery periods for formoterol, salbutamol, salmeterol, and fenoterol. It could further be shown that mixtures of dimethyl sulfoxide with alcohol, water, glycol ether or mixtures thereof could provide effective transdermal delivery of formoterol, salbutamol, salmeterol, and fenoterol.

[0011] As used herein, the term “formoterol” (INN), refers to a P2 -adrenergic receptor agonist (“beta2- agonist”). Formoterol also is denoted (±) N-[2-hydroxy-5-[l-hydroxy-2[[2-(p-methoxyphenyl)-2- propyl]amino]ethyl]phenyl]-formamide. Formoterol is available commercially as a racemic diastereomer, (R,R) plus (S,S) in a 1: 1 ratio. The formoterol salt that is commercially available is a racemic mixture of formoterol fumarate dihydrate depicted by the following formula (1):

[0012] As used herein, the term “salbutamol” (INN), refers to a P2 -adrenergic receptor agonist (“P2 -agonist”). Salbutamol also is denoted 4-[2-(tert-butylamino)-l-hydroxyethyl]-2-(hydroxymethyl)phenol and is known by CAS identifier 18559-94-9. Salbutamol is available commercially as a racemic diastereomer of R- and S-enantiomer in a 1 : 1 ratio. The salbutamol salt that is commercially available is a racemic mixture of salbutamol hemisulfate, depicted by the following formula (2):

[0013] As used herein, the term “salmeterol” (INN), refers to a long-acting P2 -adrenergic receptor agonist (“beta2 -agonist”). Salmeterol also is denoted 4-Hydroxy-al-[[[6-(4- phenylbutoxy)hexyl] amino] methyl] -1,3 -benzenedimethanol and is known by CAS identifier 89365- 50-4. Salmeterol is available commercially as a racemate (1: 1 mixture of its two enantiomers, two enantiomers, the (R)-form and the (S)-form) in the form of the salt of 1 -hydroxy-2 -naphthoic acid (= salmeterol xinafoate).

[0014] As used herein, the term “fenoterol” (INN), refers to a short-acting P2 -adrenergic receptor agonist (“beta2 -agonist”). Fenoterol also is denoted 5-[l-Hydroxy-2-[[2-(4-hydroxyphenyl)-l- methylethyl] amino] ethyl] - 1 ,3 -benzenediol or 1 -(3, 5 -Dihydroxyphenyl)- 1 -hydroxy-2- [ 1 -(4- hydroxyphenyl)isopropyl]aminoethane and is known by CAS identifier 13392-18-2. The molecule has two different stereogenic centers. Fenoterol is available commercially as a racemate of its (R,R)- and the (.S' .S) enantiomers.

[0015] As used herein, the term “clenbuterol” (INN), refers to a P2 -adrenergic receptor agonist (“P2 -agonist”) depicted by the following formula (3):

[0016] Clenbuterol also is denoted l-(4-amino-3,5-dichlorophenyl)-2-(tert-butylamino)ethanol and is known by CAS identifier 37148-27-9. Clenbuterol is available commercially as a racemic diastereomer of R- and S-enantiomer in a 1: 1 ratio. The clenbuterol salt that is commercially available for administration is a racemic mixture of clenbuterol monohydrochloride. As used herein, the term “topical” administration refers to that a substance, such as salbutamol, formoterol, salmeterol, fenoterol, or clenbuterol, is applied to a particular place on or in the body, in particular to body surfaces such as skin or mucous membranes. As used herein, the term “transdermal” administration refers to that a substance, such as salbutamol, formoterol, salmeterol, fenoterol, or clenbuterol, that upon administration permeates the upper skin layers and distributes in the deeper layers of the skin, such as dermis and subcutis as well as subcutaneous tissues.

[0017] The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. Preferred salts include ammonium, calcium, magnesium, potassium and sodium salts as well as hydrochloride, sulfate, hemisulfate, succinate, acetate, fumarate, tartrate, besilate and mesylate salts.

[0018] The term “solvent” as used herein refers to a substance, usually a liquid, which is capable of at least partially dissolving a compound, particularly one or several of an active compound selected from salbutamol, formoterol, salmeterol, fenoterol, and clenbuterol, thus providing a solution, a dispersion or a suspension. The amount of dissolved compound depends on the nature of the compound and the solvent, which may dissolve a compound in total or in parts. As used herein the term “solvent” also includes a dispersant. As used herein, the term solution also includes a dispersion or a suspension.

[0019] Regarding the solvents, given wt.-% or vol.% are calculated on the basis of a total weight or volume of the solvent(s) of 100 wt.-% or 100 vol.-%, respectively. Regarding the active compound, such as formoterol, salbutamol, salmeterol, fenoterol, or clenbuterol, given wt.-% refer to the total weight of the composition. For example, 100 g of a 5% formoterol solution in DMSO / ethanol (50 / 50, m / m) refers to 5 g formoterol and 47.5 g of each of DMSO and ethanol.

[0020] For effective transdermal delivery of a compound from a formulation solvent system, several requirements need to be met. First, an adequate solubility of the active compound in the formulation needs to be provided. Further, increased solubility of the active compound in skin (both epidermal and dermal) needs to be observed (Penetration Enhancement). Last, sufficient solvent retention (limitation of solvent evaporation) on the skin needs to be provided to ensure state of molecular dispersion of the payload. Solvents providing high solubility for a given compound do not automatically allow for a permeation in skin of that compound. For example, diethylene glycol monoethyl ether, which is commercially available under the tradename Transcutol®, can provide high solubility when used but insufficient permeation when used as only solvent.

[0021] For effective transdermal delivery of a compound selected of formoterol, salbutamol, salmeterol or fenoterol, dimethyl sulfoxide is used as solvent, where dimethyl sulfoxide may be used as only solvent or together with one or more further solvents in a mixture, preferably in a binary solvent mixture.

[0022] According to an embodiment is provided a composition for transdermal administration, the composition comprising a compound selected from the group of formoterol, salbutamol, salmeterol, fenoterol, or a salt thereof, and comprising:

[0023] - dimethyl sulfoxide in a range from > 5 wt.-% to < 100 wt.-%, and optionally

[0024] - water, a monoalcohol, glycerol, a glycol, a polyethylene glycol, a glycol ether, or mixtures thereof, in a range from > 5 wt.-% to < 95 wt.-%, wherein the wt.-% are based on a total weight of the solvent of 100 wt.-%.

[0025] In embodiments, a composition comprising formoterol, salmeterol, or fenoterol comprises 100 wt.-% DMSO, based on a total weight of the solvent of 100 wt.-%. Dimethyl sulfoxide provided very good permeation when used as only solvent with formoterol, salmeterol, and fenoterol.

[0026] Binary solvent mixtures comprising dimethyl sulfoxide and a monoalcohol, water or a glycol ether are preferred as solvent, particularly for composition comprising formoterol, salbutamol, salmeterol, or fenoterol as active ingredient.

[0027] In embodiments, the composition comprises a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, or a salt thereof as active ingredient, and a solvent mixture comprising DMSO in a range from > 5 wt.-% to < 95 wt.-% and a monoalcohol in a range from > 5 wt.-% to < 95 wt.-%, based on a total weight of the solvent of 100 wt.-%. Preferred are short- chained monoalcohols comprising up to 6 aliphatic carbon atoms. In preferred embodiments, the monoalcohol is a C2-C5 alcohol selected from the group of ethanol, n-propanol, isopropanol, n- butanol, isobutanol, tert. -butanol, or mixtures thereof. In preferred embodiments, the monoalcohol is ethanol. In embodiments, the monoalcohol is ethanol, and the composition comprises dimethyl sulfoxide in a range of from > 5 wt.-% to < 55 wt.-%, or in a range of from > 10 wt.-% to < 50 wt.-%, and ethanol in a range of from > 45 wt.-% to < 95 wt.-%, or in a range of from > 50 wt.-% to < 90 wt.-%, based on a total weight of the solvents of 100 wt.-%. In such embodiments, the composition preferably comprises formoterol, salmeterol, fenoterol, or a salt thereof as active ingredient.

[0028] At least 10 wt.-% DMSO, based on 100 wt.-% of solvent, is preferred to provide sufficient permeation of formoterol, salbutamol, salmeterol, or fenoterol. In embodiments, the composition comprises a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol or a salt thereof, and as solvent dimethyl sulfoxide in a range from > 10 wt.-% to < 50 wt.-% and ethanol in a range from > 50 wt.-% to < 90 wt.-%, based on a total weight of the solvents of 100 wt.-%. In embodiments, the composition comprises formoterol, salmeterol, or a salt thereof, and as solvent a mixture of 50 wt.-% of each of dimethyl sulfoxide and ethanol, based on a total weight of the solvents of 100 wt.-%. In embodiments, the composition comprises fenoterol or a salt thereof, and as solvent dimethyl sulfoxide in a range from > 10 wt.-% to < 50 wt.-% and ethanol in a range from > 50 wt.-% to < 90 wt.-%, based on a total weight of the solvents of 100 wt.-%. These ranges provided good solubility and permeation of the compounds.

[0029] In embodiments, the composition comprises salbutamol or a salt thereof as active ingredient, and a solvent mixture comprising DMSO in a range from > 5 wt.-% to < 95 wt.-% and water in a range from > 5 wt.-% to < 95 wt.-%, based on a total weight of the solvent of 100 wt.-%. In embodiments, the composition comprises dimethyl sulfoxide in a range of from > 5 wt.-% to < 55 wt.-%, or in a range of from > 10 wt.-% to < 50 wt.-%, and water in a range of from > 45 wt.-% to < 95 wt.-%, or in a range of from > 50 wt.-% to < 90 wt.-%, based on a total weight of the solvents of 100 wt.-%.

[0030] For non-sterile medicinal products such as cutaneous preparations purified water is considered a suitable quality of water. The production and control of purified water is known to the skilled person and referred to in Ph. Eur. Monograph 0008. Purified water is a preferred type of water for use in the composition. Purified water is commercially available.

[0031] At least 10 wt.-% DMSO, based on 100 wt.-% of solvent, is preferred to provide sufficient permeation of salbutamol. In embodiments, the composition comprises salbutamol or a salt thereof, and as solvent dimethyl sulfoxide in a range from > 10 wt.-% to < 50 wt.-% and water in a range from > 50 wt.-% to < 90 wt.-%, based on a total weight of the solvents of 100 wt.-%. In embodiments, the composition comprises salbutamol or a salt thereof, and as solvent a mixture of 50 wt.-% of each of dimethyl sulfoxide and water, based on a total weight of the solvent of 100 wt.-%. These ranges provided good solubility and permeation of the compounds.

[0032] In other embodiments, the composition comprises salbutamol or a salt thereof, and as solvent a mixture of water in a range of from > 45 wt.-% to < 95 wt.-% and dimethyl sulfoxide in a range of from > 5 wt.-% to < 55 wt.-%, based on a total weight of the solvent of 100 wt.-%.

[0033] In other embodiments, a composition comprising a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, or a salt thereof, may comprise DMSO in a range from > 5 wt.-% to < 90 wt.-% and glycerol, a glycole such as propylene glycol (PG), a polyethylene glycol (PEG), dimethyl formamide (DMF), N-methyl-2 -pyrrolidone (NMP), a glycol ether, mixtures thereof, or mixtures with a monoalcohol in a range from > 10 wt.-% to < 95 wt.-%, based on a total weight of the solvent of 100 wt.-%. Gly coles and polyethylene glycols may be selected from the group of ethylene glycol, propylene glycol, and polyethylene glycol. A preferred glycol ether is diethylene glycol monoethyl ether. Diethylene glycol monoethyl ether (IUPAC name 2-(2-Ethoxyethoxy)ethanol) is commercially available, for example under the tradename Transcutol®.

[0034] In other embodiments, ternary solvent mixtures comprising dimethyl sulfoxide, a monoalcohol, and water are preferred for composition comprising formoterol, salbutamol, salmeterol, or fenoterol as active ingredient. In embodiments the composition comprises dimethyl sulfoxide, a monoalcohol and water, each in an amount in a range of from > 20 wt.-% to < 40 wt.-%, based on a total weight of the solvent of 100 wt.-%. The monoalcohol preferably is selected from ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert. -butanol, or mixtures thereof. In preferred embodiments, the monoalcohol is ethanol. In embodiments the composition comprises dimethyl sulfoxide in a range of from > 30 wt.-% to < 35 wt.-%, ethanol in a range of from > 30 wt.-% to < 35 wt.-%, and water in a range of from > 30 wt.-% to < 35 wt.-%, based on a total weight of the solvent of 100 wt.-%.

[0035] In embodiments, the composition comprises a compound selected from the group comprising formoterol, salmeterol, fenoterol, or a salt thereof in a range from > 0.01 wt.-% to < 25 wt.-%, preferably in a range from > 0.1 wt.-% to < 10 wt.-%, more preferably in a range from > 0.5 wt.-% to < 5 wt.-%, based on the total weight of the composition.

[0036] In embodiments, the composition comprises salbutamol or a salt thereof in a range from > 0.01 wt.-% to < 25 wt.-%, preferably in a range from > 1 wt.-% to < 10 wt.-%, more preferably in a range from > 2.5 wt.-% to < 5 wt.-%, based on the total weight of the composition.

[0037] In embodiments, the composition comprises a gel forming agent selected from the group comprising hydroxypropyl cellulose, preferably high-substituted hydroxypropyl cellulose (HPC-H), hydroxyethyl cellulose, hydroxypropyl methylcellulose (HPMC), and mixtures thereof in a range of from > 1 wt.-% to < 20 wt.-%, preferably in a range of from > 2 wt.-% to < 10 wt.-% or of from > 4 wt.-% to < 5 wt.- %, based on a total weight of the composition of 100 wt.-%. A gel composition preferably comprises DMSO as a solvent, DMSO in a range from > 5 wt.-% to < 95 wt.-%, preferably in a range of from > 5 wt.-% to < 55 wt.-%, or in a range of from > 10 wt.-% to < 50 wt.-%, based on a total weight of the solvent of 100 wt.-%. The further solvent preferably is selected from water or an monoalcohol such as ethanol. In preferred embodiments, a gel composition comprises hydroxypropyl cellulose, preferably high-substituted hydroxypropyl cellulose (HPC-H), in a range of from > 2.5 wt.-% to < 5 wt.-%, and formoterol, salbutamol, salmeterol or fenoterol in a range from > 2.5 wt.-% to < 10 wt.-%, based on the total weight of the composition, based on a total weight of the composition of 100 wt.-%, and a binary solvent mixture of DMSO in a range from > 10 wt.-% to < 50 wt.-% and ethanol or water in a range from > 50 wt.-% to < 90 wt.-% based on a total weight of the solvent of 100 wt.-%.

[0038] A further aspect refers to a composition for transdermal administration comprising formoterol, salbutamol, salmeterol, fenoterol, or a salt thereof, wherein the composition provides a transdermal flux of formoterol, salbutamol, salmeterol, fenoterol, of at least 5 pg / cm2over 24 hours as measured in a Franz Diffusion Cell with an orifice of 1 cm2at 32°C according to USP (725). In embodiments, the flux is at least 10 pg / cm2over 24 hours, preferably at least 20 pg / cm2over 24 hours, more preferably at least 30 pg / cm2over 24 hours, even more preferably at least 50 pg / cm2over 24 hours, more preferably 100 pg / cm2over 24 hours measured according to USP (725).

[0039] In embodiments, the transdermal flux is measured in an infinite dose experiment on human skin in a Franz Diffusion Cell with an orifice of 1 cm2at 32°C according to USP (725). The term infinite dose experiment as used herein refers to an experiment conducted with an amount of test preparation applied to the skin where a maximum absorption rate of the test substance is achieved and maintained as defined in OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 28, 2004 and described by Lau WM, Ng KW (2017) Finite and Infinite Dosing. In: Dragicevic N, Maibach HI (eds) Percutaneous Penetration Enhancers Drug Penetration Into / Through the Skin: Methodology and General Considerations. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 35-44.

[0040] A further aspect refers to compositions for transdermal administration comprising a compound selected from the group comprising salbutamol, clenbuterol, or a salt thereof, and at least one of a monoalcohol or water.

[0041] Preferred embodiments refer to compositions for transdermal administration comprising salbutamol or a salt thereof, and a monoalcohol, preferably ethanolic formulations of salbutamol.

[0042] According to an embodiment, a composition for transdermal administration is provided, the composition comprising salbutamol or a salt thereof, and comprising:

[0043] - a monoalcohol in a range from > 10 wt.-% to < 95 wt.-%, and

[0044] - water, dimethyl isosorbide, isopropylmyristate, glycerol, a glycol, a polyethylene glycol, a glycol ether, or mixtures thereof, in a range from > 5 wt.-% to < 90 wt.-%; wherein the wt.-% are based on a total weight of the solvent of 100 wt.-%.

[0045] It could be shown that ethanolic formulations comprising isopropylmyristate, water or glycol ether as further solvent could provide transdermal delivery of salbutamol.

[0046] Solvents selected from dimethyl isosorbide, isopropyl myristate, glycerol, a glycol, a polyethylene glycol, a glycol ether, or mixtures thereof can increase permeation. Glycoles and polyethylene glycols may be selected from the group of ethylene glycol, propylene glycol, and polyethylene glycol.

[0047] Preferably, the glycol ether is diethylene glycol monoethyl ether. Diethylene glycol monoethyl ether (IUPAC name 2-(2-Ethoxyethoxy)ethanol) is commercially available, for example under the tradename Transcutol®. Binary mixtures comprising a monoalcohol such as ethanol and water are usable as solvent for salbutamol. Binary mixtures simplify formulating and enhance compatibility with further excipients.

[0048] Preferred are short-chained monoalcohols comprising up to 6 aliphatic carbon atoms. In embodiments, the monoalcohol is a C2-C5 alcohol selected from the group of ethanol, n-propanol, isopropanol, n- butanol, isobutanol, tert. -butanol, or mixtures thereof. In preferred embodiments, the monoalcohol is selected from ethanol, isopropanol, and n-butanol, isobutanol. Most preferred is ethanol.

[0049] In embodiments, the composition comprises salbutamol or a salt thereof, and as solvent water in a range of from > 45 vol.-% to < 55 vol.-% and ethanol in a range of from > 45 wt.-% to < 55 wt.-%, based on a total weight of the solvent of 100 wt.-%. In embodiments, the composition comprising salbutamol may comprise equal amounts of water and ethanol of 50 wt.-% each.

[0050] In embodiments, the composition comprises salbutamol or a salt thereof in a range from > 0.01 wt.-% to < 25 wt.-%, preferably in a range from > 1 wt.-% to < 10 wt.-%, more preferably in a range from > 2.5 wt.-% to < 5 wt.-%, based on the total weight of the composition.

[0051] A further aspect refers to a composition for transdermal administration comprising salbutamol or a salt thereof, wherein the composition provides a transdermal flux of salbutamol of at least 5 pg / cm2over 24 hours as measured in a Franz Diffusion Cell with an orifice of 1 cm2at 32°C according to USP (725). In embodiments, the flux is at least 10 pg / cm2over 24 hours, preferably at least 20 pg / cm2over 24 hours, more preferably at least 30 pg / cm2over 24 hours, even more preferably at least 50 pg / cm2over 24 hours, more preferably 100 pg / cm2over 24 hours measured according to USP (725).

[0052] In embodiments, the transdermal flux is measured in an infinite dose experiment on human skin in a Franz Diffusion Cell with an orifice of 1 cm2at 32°C according to USP (725). The term infinite dose experiment as used herein refers to an experiment conducted with an amount of test preparation applied to the skin where a maximum absorption rate of the test substance is achieved and maintained as defined in OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 28, 2004 and described by Lau WM, Ng KW (2017) Finite and Infinite Dosing. In: Dragicevic N, Maibach HI (eds) Percutaneous Penetration Enhancers Drug Penetration Into / Through the Skin: Methodology and General Considerations. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 35-44. Further embodiments refer to compositions for transdermal administration comprising clenbuterol or a salt thereof and comprising at least one of a monoalcohol or water.

[0053] According to an embodiment, a composition for transdermal administration is provided, the composition comprising clenbuterol or a salt thereof, and comprising:

[0054] - water in a range from > 45 vol.-% to < 95 vol.-%, and dimethyl isosorbide, isopropylmyristate, glycerol, a glycol, a polyethylene glycol, a glycol ether, or mixtures thereof, in a range from > 5 vol.- % to < 55 vol.-%; or

[0055] - a monoalcohol in a range from > 50 vol.-% to < 90 vol.-%, and water, isopropylmyristate, dimethyl isosorbide, glycerol, a glycol, a polyethylene glycol, a glycol ether, or mixtures thereof, in a range from > 10 vol.-% to < 50 vol.-%; wherein the vol.-% are based on a total volume of the solvent of 100 vol.-%.

[0056] It has been found that aqueous and alcoholic compositions of clenbuterol allowed for a transdermal administration. It could be shown that mixtures of water and at least one of ethanol, glycol ether or dimethyl isosorbide could provide effective transdermal delivery of clenbuterol, as well as mixtures of ethanol and isopropylmyristate.

[0057] Also, solvents selected from dimethyl sulfoxide, dimethyl isosorbide, glycerol, a glycol, a polyethylene glycol, a glycol ether, or mixtures thereof provided good permeation of clenbuterol. Glycoles and polyethylene glycols may be selected from the group of ethylene glycol, propylene glycol, and polyethylene glycol. Preferably, the glycol ether is diethylene glycol monoethyl ether. Diethylene glycol monoethyl ether (IUPAC name 2-(2-Ethoxyethoxy)ethanol) is commercially available, for example under the tradename Transcutol®.

[0058] For non-sterile medicinal products such as cutaneous preparations purified water is considered a suitable quality of water. The production and control of purified water is known to the skilled person and referred to in Ph. Eur. Monograph 0008. Purified water is a preferred type of water for use in the composition. Purified water is commercially available. Alcoholic solutions of clenbuterol are usable for transdermal application. Preferred are short-chained monoalcohols comprising up to 6 aliphatic carbon atoms. In embodiments, the monoalcohol is a C2-C5 alcohol selected from the group of ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert.- butanol, or mixtures thereof. In preferred embodiments, the monoalcohol is selected from ethanol, isopropanol, and n-butanol, isobutanol. Most preferred is ethanol.

[0059] In embodiments, the composition comprises clenbuterol or a salt thereof as active ingredient, and as solvent a monoalcohol selected from ethanol, isopropanol, isobutanol, or a mixture thereof, preferably ethanol, in a range from > 70 vol.-% to < 90 vol.-%, preferably in a range from > 70 vol.-% to < 80 vol.-%, and isopropylmyristate or water in a range from > 10 vol.-% to < 30 vol.-%, preferably in a range from > 20 vol.-% to < 30 vol.-%, based on a total volume of the solvents of 100 vol.-%. These ranges provided good solubility and permeation of clenbuterol. Particular for clenbuterol provided alcoholic solutions sufficient permeation after topic application to achieve a reduction in adipocyte size.

[0060] In other embodiments, the composition comprising a clenbuterol or a salt thereof, may comprise as solvent water in a range of from > 45 vol.-% to < 95 vol.-% and diethylene glycol monoethyl ether (Transcutol®) or dimethyl isosorbide in a range of from > 5 vol.-% to < 55 vol.-%, based on a total volume of the solvents of 100 vol.-%. For clenbuterol provided aqueous solutions comprising diethylene glycol monoethyl ether sufficient permeation after topic application to achieve a reduction in adipocyte size. In embodiments, the composition comprising clenbuterol may comprise water in a range from > 40 vol.-% to < 60 vol.-%, preferably in a range from > 45 vol.-% to < 55 vol.-%, and diethylene glycol monoethyl ether (Transcutol®) in a range from > 40 vol.-% to < 60 vol.-%, preferably in a range from > 45 vol.-% to < 55 vol.-%, based on a total volume of the solvent of 100 vol.-%. In embodiments, the composition comprising clenbuterol may comprise equal amounts of water and diethylene glycol monoethyl ether (Transcutol®) of 50 vol.-% each.

[0061] In embodiments, the composition comprising clenbuterol may comprise water in a range from > 80 vol.-% to < 90 vol.-% and dimethyl isosorbide in a range from > 10 vol.-% to < 20 vol.-%, based on a total volume of the solvent of 100 vol.-%. It was found that in vitro permeation of clenbuterol could be achieved from a formulation comprising only 12.5 vol.-% dimethyl isosorbide and as low as 0.1 wt.-% of clenbuterol. In embodiments, the composition comprises clenbuterol or a salt thereof in a range from > 0.01 wt.-% to < 25 wt.-%, preferably in a range from > 0.1 wt.-% to < 5 wt.-%, more preferably in a range from > 0. 1 wt.-% to < I wt.-%, based on the total weight of the composition.

[0062] A further aspect refers to a composition for transdermal administration comprising clenbuterol or a salt thereof, wherein the composition provides a transdermal flux of clenbuterol of at least 5 pg / cm2over 24 hours as measured in a Franz Diffusion Cell with an orifice of 1 cm2at 32°C according to USP (725). In embodiments, the flux is at least 10 pg / cm2over 24 hours, preferably at least 20 pg / cm2over 24 hours, more preferably at least 30 pg / cm2over 24 hours, even more preferably at least 50 pg / cm2over 24 hours, more preferably 100 pg / cm2over 24 hours measured according to USP (725).

[0063] In embodiments, the transdermal flux is measured in an infinite dose experiment on human skin in a Franz Diffusion Cell with an orifice of 1 cm2at 32°C according to USP (725). The term infinite dose experiment as used herein refers to an experiment conducted with an amount of test preparation applied to the skin where a maximum absorption rate of the test substance is achieved and maintained as defined in OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 28, 2004 and described by Lau WM, Ng KW (2017) Finite and Infinite Dosing. In: Dragicevic N, Maibach HI (eds) Percutaneous Penetration Enhancers Drug Penetration Into / Through the Skin: Methodology and General Considerations. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 35-44.

[0064] The compositions according to the invention are usable for transdermal administration. In embodiments, the compositions are in the form of atopical composition. Preferably, the topical composition is selected from the group consisting of a gel, an ointment, a cream, a foam, a lotion, a paste, a solution, a spray, or a plaster. Such formulations are usual topical compositions for application to a particular place on the body surface, such as the skin. Advantageously, atopical composition according to the invention provides for a transdermal administration of the compound. Preferably, the composition is in the form of a gel, an ointment, or a plaster, more preferred in the form of a gel or a spray. Dosage forms preferably are defined according to the definitions used by the skilled in the art, e.g. Pharm Eur, 10.0; 0132: Semi-solid preparations for cutaneous use. For the formulation of the respective formulations, the composition may comprise one or more pharmaceutically acceptable excipients in order to increase physical, chemical, and microbial stability as well as patient adherence of the formulation. The respective gel forming agents, surfactants, solubility enhancers, permeation enhancers, preservatives, antioxidants, complexing agents, cosolvents, emollients, fragrances, and colouring agents are known to the skilled person. Usable pharmaceutically acceptable carriers including hydrogels, cutaneous creams, cutaneous gels, cutaneous lotions, cutaneous ointments, cutaneous patches, cutaneous pastes, cutaneous poultices, cutaneous foams, cutaneous solutions, cutaneous sprays, and patches are known to the skilled person. Another preferred form of provision is a spray.

[0065] In embodiments, the composition comprises one or more pharmaceutical excipient(s). In embodiments, the composition comprises one or more pharmaceutical excipient(s) selected from the group comprising antioxidants, penetration enhancers, gel forming agent, ointment components, preservatives, colorants, odorants, stabilizers, triglycerides, and mixtures thereof, and preferably is a gel forming agent.

[0066] A preferred form of provision of the formulation is a gel. In embodiments, the composition comprises a gel forming agent selected from the group comprising hydroxypropyl cellulose, preferably high- substituted hydroxypropyl cellulose (HPC-H), hydroxyethyl cellulose, hydroxypropyl methylcellulose (HPMC), and mixtures thereof. The composition may comprise the gel forming agent, such as hydroxypropyl cellulose or hydroxyethyl cellulose, in a range of from > 1 wt.-% to < 20 wt.-%, preferably in a range of from > 2 wt.-% to < 10 wt.-%, based on a total weight of the composition of 100 wt.-%.

[0067] A gel composition preferably comprises DMSO as a solvent, DMSO in a range from > 5 wt.-% to < 95 wt.-%, preferably in a range of from > 5 wt.-% to < 55 wt.-%, or in a range of from > 10 wt.-% to < 50 wt.-%, based on a total weight of the solvent of 100 wt.-%. The further solvent preferably is selected from water or an monoalcohol such as ethanol.

[0068] In embodiments, the composition comprises a gel forming agent selected from the group comprising hydroxypropyl cellulose, preferably high-substituted hydroxypropyl cellulose (HPC-H), hydroxyethyl cellulose, hydroxypropyl methylcellulose (HPMC), and mixtures thereof in a range of from > 1 wt.-% to < 20 wt.-%, preferably in a range of from > 2 wt.-% to < 10 wt.-% or of from > 4 wt.-% to < 5 wt.- %, based on a total weight of the composition of 100 wt.-%. A gel composition preferably comprises DMSO as a solvent, DMSO in a range from > 5 wt.-% to < 95 wt.-%, preferably in a range of from > 5 wt.-% to < 55 wt.-%, or in a range of from > 10 wt.-% to < 50 wt.-%, based on a total weight of the solvent of 100 wt.-%. The further solvent preferably is selected from water or an monoalcohol such as ethanol. In preferred embodiments, a gel composition comprises hydroxypropyl cellulose, preferably high-substituted hydroxypropyl cellulose (HPC-H), in a range of from > 2.5 wt.-% to < 5 wt.-%, and formoterol, salbutamol, salmeterol, fenoterol or clembuterol in a range from > 0.1 wt.-% to < 10 wt.- %, based on a total weight of the composition of 100 wt.-%, and a binary solvent mixture of DMSO in a range from > 10 wt.-% to < 50 wt.-% and ethanol or water in a range from > 50 wt.-% to < 90 wt.-% based on a total weight of the solvent of 100 wt.-%.

[0069] Suitable excipients selected from ointment bases, gel formers, antioxidants, pH modulators, preservatives, colorants, and stabilizers preferably are selected from the group comprising alpha tocopherol, acetic acid, ammonia solution, anhydrous citric acid, ascorbic acid, ascorbyl palmitate, bentonite, benzalkonium chloride, benzyl alcohol, butylparaben, calcium acetate, carbomers, carocymethyl cellulose sodium, carnauba wax, carrageenan, castor oil, ceteareth-30, veteth-10, cetostearyl alcohol, cetyl alcohol, cholesterol, coconut oil, colloidal silicon dioxide, com oil, crospovidone, diethylene glycol monoethyl ether, dimethicone 100, docosanol, docusate sodium, etate disodium, ethylcellulose, ethylparaben, ferric oxide red, formic acid, gelatin, gylceryl monostearate, gylceryl oleate, hyaluronate sodium, hydrochloric acid, hydrogenated castor oil, hydroxyethyl cellulose, hydroxyproyl cellulose, hypromellose, isopropyl myristate, isopropyl palmitate, lanolin, lavender oil, lecithin, linoleic acid, magnesium silicate, magnesium stearate, medium -chain triglycerides, menthol, methylcellulose, methylparaben, mineral oil, myristic acid, myristyl alcohol, oleic acid, olive oil, paraffin, peanut oil, PEG 6-32 stearate / glycol stearate, PEG / PPG-18 / 18 dimethicone, PEG-100 stearate, PEG-5 oleate, PEG-60 hydrogenated castor oil, PEG-75 lanolin, poloxamers, polyacrylic acid, polyethylene, polyethylene glycols, polysorbates, povidone, propylene glycol, quatemium-15, shea butter, simethicone, sodium bisulfite, sodium chloride, sodium hydroxide, sodium laureth-2 sulfate, sodium lauryl sulfate, sodium metabisulfite, sodium silicate, sorbic acid, stearic acid, thyme oil, triethanolamine, urea, vitamin E polyethylene succinate, wax, xanthan gum, zinc acetate, zinc oxide, and zinc stearate. In embodiments, compositions may comprise a mono-, di- or triglyceride. Preferred are triglycerides are selected from the group comprising glyceryl monostearate and glyceryl oleate. In embodiments, the composition comprises a triglyceride, preferably selected from the group comprising glyceryl monostearate and glyceryl oleate, in a range from > I wt.-% to < 20 wt.-%, based on a total weight of the composition.

[0070] In embodiments, the composition may comprise a cyclodextrin in a range of from > I wt.-% to < 20 wt.-%, based on a total weight of the solvent of 100 wt.-%. The addition of cyclodextrins may increase the solubility of the active compound. Preferred cyclodextrins are pharmaceutically acceptable cyclodextrins such as P-cyclodextrin and gamma-cyclodextrin as well as -cyclodextrin derivatives used as excipients in medicines such as the sulfobutylether of P-cyclodextrin (SBE-P-CD), the hydroxypropyl derivative of P-cyclodextrin (HP-P-CD), and randomly methylated P-cyclodextrin (RM-P-CD).

[0071] The compositions as described herein are usable for transdermal administration of the active compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, clenbuterol, a mixture thereof, or a salt thereof to the subcutaneous fat tissue. Subcutaneous fat tissue refers to a tissue layer just beneath the skin of a mammal, comprising adipocytes. A transdermal administration of the composition results in a locally increased concentration of the compound in the adipocytes with significantly reduced systemic drug concentration.

[0072] The composition provides effective transdermal delivery of the active compound. This results in a high uptake in subcutaneous fat tissue, to which fat tissue will respond by decreasing the quantity of subcutaneous fatty tissue and / or decrease the size of the single fat cells. Decreasing the quantity of subcutaneous fatty tissue will reduce overweight caused by fat cells and reduce obesity and lipedema.

[0073] Further, it was shown for formoterol, clenbuterol and salbutamol that upon transdermal application on mice in-vivo the expression of browning markers was significantly increased in inguinal white adipose tissue.

[0074] Thus, another aspect refers to a use of the compositions as described above is in the manufacture of a medicament for the treatment of obesity or lipedema or for providing lipolysis. A related aspect relates to the compositions as described above for use in the treatment of obesity or lipedema, or for providing lipolysis. The composition hence may be a pharmaceutical composition. In another aspect, a method for the treatment of obesity or lipedema, or for providing lipolysis is provided, wherein the method comprises the step of administering to a subject in need thereof, for example a mammal such as a human, a therapeutically effective amount of a composition as described herein.

[0075] The term “lipolysis” as used herein refers to a procedure in which an active ingredient is applied to achieve local decrease of the quantity of subcutaneous fat tissue. The term “lipolysis” may refer to a treatment or to a cosmetic procedure.

[0076] The composition in other embodiments may be a cosmetic composition for cosmetic use. In another aspect, the invention refers to a use of the composition as described above in a cosmetic method for decreasing the quantity of subcutaneous fat tissue of a subject by topical application. In other words, in embodiments, the composition as described above is for use in in a cosmetic method for decreasing the quantity of subcutaneous fat tissue of a subject by topical application. In another aspect, a cosmetic method for decreasing the quantity of subcutaneous fat tissue of a subject, for example a mammal such as a human, is provided, wherein the method comprises the administration of the composition as described above. A decrease of the volume of subcutaneous fat tissue at a local site of the body by application of a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, or clenbuterol may result in a cosmetic method without therapeutic benefit.

[0077] A further aspect refers to a use of a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, clenbuterol, a mixture thereof, or a salt thereof for the manufacture of a transdermal medicament, preferably for use in the treatment of obesity or lipedema, or for providing lipolysis. In other words, a further aspect refers to a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, clenbuterol, a mixture thereof, or a salt thereof for use as a transdermal medicament, preferably for use in the treatment of obesity or lipedema, or for providing lipolysis. The compounds selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, clenbuterol, a mixture thereof, or a salt thereof can be administered transdermally using a composition as described above. This allows for a transdermal application of the compounds. For the description of the compounds and salts thereof, the composition and the uses, reference is made to the description above. A further aspect refers to a composition as described herein for use in a transdermal treatment, preferably for use in the treatment of obesity or lipedema, or for providing lipolysis. For the description of the compounds and salts thereof, the composition and the uses, reference is made to the description above.

[0078] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0079] The Examples, which follow serve to illustrate the invention in more detail but do not constitute a limitation thereof.

[0080] The figures show:

[0081] Figure 1 Permeation time profile of formoterol fumarate dihydrate over 400 pm thick human abdominal skin from a 6.17% (w / w) solution in pure dimethyl sulfoxide and a binary mixture with ethanol (50 / 50, %w / w). Data is presented as mean ± SD, n = 6.

[0082] Figure 2 Genexpression of thermogenesis markers UCP1, CIDEA, DIO2, PGCla, and TNFa after treatment of mice with 6.17% (w / w) formoterol fumarate dihydrate (FF) containing solution based on a 50 / 50 (%w / w) mixture of dimethyl sulfoxide and ethanol for 10 days in Figures 2 A, B, C, D and E, respectively. Data is presented as mean ± SD; n = 8-14. Analysis of significance was conducted by Student’s t-test for PGCla and by Welch’s t-test for UCP1, CIDEA, DIO2 and TNFa to account for differing SD. *p < 0.05.

[0083] Figure 3 Permeation time profiles of clenbuterol over 400 pm thick human abdominal skin from four binary mixtures of ethanol (EtOH), water (W), Transcutol® (TC), and dimethyl isosorbide (DMI).

[0084] Figure 4 Histologic sections of inguinal white adipose tissue of mice after treatment with 0.4% (w / v) clenbuterol containing gel based on a 50 / 50 (%v / v) mixture of water and Transcutol® for 10 d after haematoxylin and eosin staining in Figure 4B compared to placebo control (Figure 4A) and after UCP1 staining in Figure 4D compared to placebo control in Figure 4C. Figure 5 Genexpression of thermogenesis markers UCP1, CIDEA, DIO2, PGCla, and TNFa after treatment of mice with 0.4% (w / v) clenbuterol containing gel based on a 50 / 50 (%v / v) mixture of water and Transcutol® for 10 days in Figures 5 A, B, C, D and E, respectively. Data is presented as mean ± SD; n = 3-8. Analysis of significance was conducted by Student’s t-test for CIDEA and by Welch’s t-test for PGCla to account for differing SD. *p < 0.05.

[0085] Figure 6 Genexpression of thermogenesis markers UCP1, CIDEA, DIO2, PGCla, and TNFa after treatment of mice with 1% (w / v) clenbuterol containing gel based on a 25 / 75 (%v / v) mixture of water and ethanol for 10 days in Figures 6A, B, C, D and E, respectively. Data is presented as mean ± SD; n = 8-14. Analysis of significance was conducted by Welch’s t-test for CIDEA and PGCla to account for differing SD. *p < 0.05.

[0086] Figure 7 Permeation time profile of salbutamol hemisulfate over murine skin from 5% (w / w) binary mixtures of water and dimethyl sulfoxide (DMSO) (50 / 50 and 90 / 10 (w / w)). Data is presented as mean ± SD, n = 5 (50 / 50 (w / w)) and n = 4 (90 / 10 (w / w)).

[0087] Figure 8 Histologic sections of inguinal white adipose tissue of mice after treatment with placebo or 10% (w / w) salbutamol hemisulfate containing gel, referring to the weight of solution, on base of a 50 / 50 (%w / w) mixture of water and ethanol for 10 days after haematoxylin and eosin staining in Figure 8B compared to placebo control (Figure 8A) and after UCP1 staining in Figure 8D compared to placebo control in Figure 8C.

[0088] Figure 9 Genexpression of thermogenesis markers UCP1, CIDEA, DIO2, PGCla, and TNFa after treatment of mice with 10% (w / w) salbutamol hemisulfate containing gel, referring to the weight of solution, on base of a 50 / 50 (%w / w) mixture of water and ethanol for 10 days in Figures 9A, B, C, D and E, respectively. Data is presented as mean ± SD; n = 8. Analysis of significance was conducted by Student’s t-test.

[0089] Figure 10 Permeation of salbutamol hemisulfate from a 2.5% (w / w) gel, referring to the weight of solution, on base of a 50 / 50 (%w / w) mixture of water and dimethyl sulfoxide over 400 pm thick dermatomed human abdominal skin. Data is presented as mean ± SD, n = 6.

[0090] Figure 11 Permeation time profile of salmeterol xinafoate over murine skin from a 5% (w / w) binary mixture of ethanol (EtOH) and dimethyl sulfoxide (DMSO) (50 / 50 (%w / w)). Data is presented as mean ± SD, n = 6. Figure 12 Permeation time profile of fenoterol hydrobromide over murine skin from 5% (w / w) binary mixtures of ethanol (EtOH) and dimethyl sulfoxide (DMSO) (50 / 50 and 90 / 10 (w / w)). Data is presented as mean ± SD, n = 5.

[0091] Figure 13 Permeation time profile of salbutamol hemisulfate, salmeterol xinafoate, formoterol fumarate dihydrate, and fenoterol hydrobromide over 600 pm thick murine skin from a 72 pM solution in a ternary mixture of DMSO, ethanol and water (30 / 35 / 35, %(w / w / w)).

[0092] Figure 14 Comparison of permeation time profiles of salbutamol hemisulfate, salmeterol xinafoate, formoterol fumarate dihydrate and fenoterol hydrobromide over full thickness murine skin from 72 pM solutions in mixtures of dimethyl sulfoxide (DMSO), ethanol (EtOH) and water (30 / 35 / 35 w / w / w) in infinite dose. Data is presented as mean ± SD, n=6.

[0093] Materials

[0094] Formoterol fumarate dihydrate (FF), clenbuterol (CLB), salbutamol hemisulfate (SS), salmeterol xinafoate, and fenoterol hydrobromide were of analytical grade or higher and procured from Swapnroop Drugs & Pharmaceuticals (Aurangabad, India). Brij® 98, dimethyl isosorbide (DMI), and triethylamine (99.5%) were products from Acros Organics (Geel, Belgium). Cocoyl caprylocaprate, Poloxamer 407, and Soluplus® were kind gifts from BASF SE (Ludwigshafen, Germany). Hydroxyethyl cellulose 250, isopropyl myristate, Miglyol® 812 (MCT812), polyethylene glycol 300, propylene glycol, and sodium dodecyl sulfate were purchased from Caesar & Loretz GmbH (Hilden, Germany). Sodium fluoride and sodium orthovanadate were bought from Carl Roth (Karlsruhe, Germany), methanol and tetrahydrofuran were purchased from Fisher Scientific (Schwerte, Germany). DowComing™ BIO-PSA™ 7-4202 was a generous gift from DDP Specialty Electronic Materials (Midland, MI, USA). Transcutol® P was kindly donated by Gattefosse SAS (Saint-Priest Cedex, France). DuroTAK® 387-2054 and DuroTAK® 387-2510 were gifts from Henkel (Diisseldorf- Holthausen, Germany). Pharma grade dimethyl sulfoxide was obtained from ITW Reagents (Monza, Italy). Parteck® MXP was a donation by Merck KGaA (Darmstadt, Germany). Hydroxypropyl Cellulose H and Hydroxypropyl Cellulose SL were kindly donated by Nippon Soda Co., Ltd. (Tokyo, Japan). Dulbecco’s modified eagle medium (DMEM), ethanol abs., ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), Nonidet® P 40 substitute (NP-40), and sodium deoxy cholate were purchased from Sigma- Aldrich (Taufkirchen, Germany). Orthophosphoric acid 85% was a product of Th. Geyer GmbH & Co. KG (Renningen, Germany). Phosphate buffered saline powder and propan-2 -ol were purchased from VWR International GmbH (Darmstadt, Germany). Trizol™ and SYBR™ Green Master Mix were procured from ThermoFisher, Waltham, MA, USA. ProtoScript® II First Strand cDNA Synthesis Kit was purchased from New England Biolabs, MA, USA.

[0095] Methods

[0096] Solubility Screening in Excipients and Solvents:

[0097] For assessment of solubilities of compounds in solvents of various polarities an amount of 100 mg compound was added to 4 mL of solvent in a sealable beaker and stirred for 24 h in the dark. The resulting suspensions were centrifuged at 15,000 rpm for 10 min and the supernatant was then fdtered over PES or PTFE syringe fdters with a 0.22 pm pore diameter. The solutions were analyzed by HPLC-UV or by using a fluorescence detector and appropriately diluted with 0. 1 M HC1, water or MeOH if needed. Due to the expense of some of the used compounds and the screening character of most solubility assessments the experiments were carried out once or in triplicate. For some solvents, if solubilities were higher than 25 mg / mL, increasing amounts of compound were added until no clear solution could be obtained, or 100 mg / mL were reached and the last concentration yielding a clear solution was noted.

[0098] Preparation of Gels:

[0099] Gels were prepared by dissolving the respective compound in its solvent mixture and subsequent successive addition of gelling agent under stirring until the agent was completely dissolved.

[0100] High Performance Liquid Chromatography (HPLC):

[0101] For every compound analysis was conducted on an LC-2040C 3D Plus system with an integrated diode array and a coupled RF-20A fluorescence detector (Shimadzu, Kyoto, Japan), equipped with a C18 reverse phase column (Inertsil ODS-3, 150 mm length, 2.1 mm inner diameter, 3 pm particle size).

[0102] For clenbuterol the flow rate was set to 0.3 mL / min and eluent consisted of 25 / 75 (%v / v) methanol (MeOH) and 0.2% (v / v) triethylamine (TEA) solution adjusted to pH 3 with orthophosphoric acid. The column temperature was maintained at 40°C and the autosampler temperature was kept at 25 °C. The injection volume was 2 pL and the detector was set to 242 nm for UV detection. The limit of quantification was lOO ng / mL.

[0103] For formoterol fumarate dihydrate the flow was set to 0.3 mL / min and a solvent mixture of 35 / 65 (%v / v) MeOH and 0.2% TEA solution adjusted to pH 3 with orthophosphoric acid was used as mobile phase. The column temperature was maintained at 40°C and the autosampler kept at 25°C. The injection volume was 2 pL and the detector was set to 210 nm for UV detection or fluorometric detection was conducted at an excitation wavelength of 228 nm and an emission wavelength of 300 nm. The limit of quantification was 100 ng / mL.

[0104] For salbutamol hemisulfate the flow rate was set to 0.3 mL / min and the eluent consisted of 6 / 94 (%v / v) MeOH and 0.2% (v / v) TEA solution adjusted to pH 3 with orthophosphoric acid. The column temperature was maintained at 40°C and the autosampler was kept at 25°C. The injection volume was 2 pL and the fluorometric detection was conducted at an excitation wavelength of 220 nm and an emission wavelength of 310 nm. The limit of quantification was below 100 ng / mL.

[0105] For salmeterol xinafoate the flow rate was set to 0.3 mL / min and the mobile phase consisted of 55 / 45 (%v / v) ACN and 0.2% TEA solution adjusted to pH 3 with orthophosphoric acid. The column temperature was maintained at 40°C and the autosampler kept at 25°C. The injection volume was 2 pL and the fluorometric detection was conducted at an excitation wavelength of 340 nm and an emission wavelength of 415 nm. The limit of quantification was below 100 ng / mL.

[0106] For fenoterol hydrobromide the flow rate was set to 0.3 mL / min and the mobile phase consisted of 25 / 75 (%v / v) MeOH and 0,01 M KH2PO4 buffer adjusted to pH 6.5 with 0,1 M NaOH. The column temperature was maintained at 40°C and the autosampler kept at 25°C. The injection volume was 2 pL and the fluorometric detection was conducted at an excitation wavelength of 285 nm and an emission wavelength of 345 nm. The limit of quantification was below 100 ng / mL.

[0107] Preparation of human skin membranes:

[0108] Human abdominal skin from plastic surgery was obtained from consenting patients and collected immediately after surgery that was performed at Dreifaltigkeits-Krankenhaus, 50389 Wesseling, Germany. Ethical approval was given by the ethics council of the University of Bonn under approval reference 082 / 20. The subcutaneous adipose tissue was carefully removed with a scalpel and the resulting full thickness skin was frozen at -35°C until use. For permeation experiments skin was thawed at room temperature and subsequently cut to a thickness of 400 pm with a dermatome (Aesculap® Acculan GA643, Tuttlingen, Germany). Franz Diffusion Cell (FDC) Experiments:

[0109] Vertical Franz diffusion cells with a receptor volume of 8 mL and effective diffusion area of 1 cm2were used for permeation experiments (SES Analysensysteme, Bechenheim, Germany). Human abdominal skin cut to a thickness of 400 pm as in accordance with OECD guideline 408 was placed upon the cells with the stratum comeum facing the air. The skin surface temperature was maintained at 32°C. The receiver consisted of phosphate buffered saline pH 7.4 with 6% (w / w) Brij®98. A volume of 200 pL of formulation was applied to the skin in the donor compartment and samples of 0.5 mL were taken at defined timepoints and immediately replaced with fresh receptor solution.

[0110] Treatment of live human abdominal tissue ex-vivo:

[0111] Human abdominal skin with underlying subcutaneous fatty tissue was cut into appropriately sized rectangular pieces after surgery without prior freezing and placed into petri dishes that were filled with 20 mL of DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin. A volume of 0.2 mL formulation was applied to the skin with a syringe and spread across a rectangular area of 6 cm2. Dishes were closed and incubated in a drying cabinet for 3 d at 32°C to simulate the outer skin temperature. After incubation, skin was taken from the dishes and fatty tissue directly adjacent to the dermis was sampled for further analysis of gene expression by real-time PCR.

[0112] Treatment of mice with gels in-vivo'.

[0113] Wild type (WT) C57BL / 6 mice were split into groups of eight and treated with verum and a placebo, respectively. Mice were carefully shaved on the flanks with an electric razor 24 h before application. 50 mg of gel were applied once a day to each flank for 10 d. 24 h after the last application of gel the animals were sacrificed and inguinal white adipose tissue (WATi) was removed and analyzed for browning marker expression by qPCR, western blot and histologically after hematoxylin and eosin, as well as UCP1 antibody staining.

[0114] Preparation of histologic sections:

[0115] Tissue samples were fixed in 4% paraformaldehyde solution, dehydrated with ethanol (EtOH), embedded in paraffin and cut with a microtome. Sections were stained with hematoxylin and eosin (H&E) and with UCP1 specific antibodies, respectively. Isolation of RNA and Real-time Polymerase Chain Reaction (qPCR):

[0116] Tissues were homogenized with Trizol for mRNA extraction and treated with chloroform with subsequent precipitation by addition of propan-2 -ol and washing with 75% EtOH. The concentration of isolated RNA was spectrophotometrically analyzed with a Nanodrop 200 (ThermoFisher Scientific, Waltham, MA, USA).

[0117] Synthesis of cDNA was conducted with a ProtoScript II First Strand cDNA Synthesis Kit according to the manufacturer's instructions. Analysis of amplification was performed by staining with SYBR Green Master Mix and measurement on a QuantStudio 5 qPCR system (ThermoFisher, Waltham, MA, USA). The 2ACTmethod was used to calculate the relative mRNA expression of genes normalized to the housekeeping genes Hypoxanthine Phosphoribosyltransferase 1 (HPRT) for murine- and Ribosomal Protein L13a (RPL13A) for human samples. Primer sequences are listed in Table 1.

[0118] Table 1: Primer sequences used for qPCR of murine genes.

[0119] Western Blot:

[0120] To isolate proteins a radioimmunoprecipitation assay lysis buffer containing 50 mM Tris pH 7.5, 150 mM sodium chloride, 1% NP-4O, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.1 mM EDTA, 0. 1 mM EGTA, 1 mM NasVCK 10 mM NaF, containing a protease inhibitor cocktail (Roche, Basel, Switzerland), was used. The protein content was quantified by Bradford assay. Proteins were separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. For immunoblot primary antibodies of UCP1 (Sigma- Aldrich, Taufkirchen, Germany) and Calnexin and secondary anti -rabbit antibodies were used. Amersham ECL Western Blotting reagent was used to visualize proteins and detection was performed with an ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL, USA). Quantification was conducted using Image J software.

[0121] Statistical Evaluation:

[0122] Tests for statistical significance were conducted using the software GraphPad Prism 8. Depending on the number of samples in each group, whether multiple comparisons needed to be accounted for and whether groups were of the same standard deviation, different tests for statistical significance were performed, that are properly denoted in the respective sections of the results.

[0123] Example 1

[0124] Determination of solubility of formoterol in several solvents

[0125] Solubility of formoterol fumarate dihydrate was tested in water, tetrahydrofiiran (THF), dimethyl isosorbide (DMI), propylene carbonate (PC), Transcutol® (TC), Miglyol® 812 (MCT812), cocoyl caprylocaprate (CC), ethanol (EtOH), polyethylene glycol 300 (PEG300), propylene glycol (PG), dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), N-Methyl-2 -pyrrolidone (NMP) and methanol (MeOH) as described above. The results are listet in Table 2 below.

[0126] Table 2: Solubility of formoterol fumarate dihydrate in water, tetrahydrofiiran (THF), dimethyl isosorbide (DMI), propylene carbonate (PC), Transcutol® (TC), Miglyol® 812 (MCT812), cocoyl caprylocaprate (CC), ethanol (EtOH), polyethylene glycol 300 (PEG300), propylene glycol (PG), dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), N-Methyl-2 -pyrrolidone (NMP) and methanol (MeOH).

[0127] As can be taken from Table 2, formoterol fumarate dihydrate was insoluble in the apolar excipients MCT812 and cocoyl caprylocaprate as no amount of dissolved formoterol fumarate dihydrate (FF) could be detected, which is surely due to its salt form (Tab. 5). Then again, the solubility of formoterol fumarate dihydrate (FF) was also low in highly polar PC. In water, THF, and DMI the solubility was below 1 mg / mL and about a magnitude higher in polar protic solvents containing alcoholic groups, such as EtOH or TC: The highest solubility was found in polar aprotic solvents, such as DMSO, DMF, and NMP.

[0128] Example 2

[0129] Determination of in vitro permeation of formoterol fumarate dihydrate

[0130] The in vitro permeation of formoterol from DMSO or a mixture of DMSO and ethanol through excised human abdominal skin was determined over a time period of 24 h in Franz Diffusion Cell as infinite dose experiment. The Franz Diffusion Cell allows the measurement of the amount of a compound diffused across a membrane, such as abdominal human skin or mouse skin, in an acceptor medium. The term infinite dose experiment as used herein refers to an experiment conducted with an amount of test preparation applied to the skin where a maximum absorption rate of the test substance is achieved and maintained as defined in OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 28, 2004 and described by Lau WM, Ng KW (2017) Finite and Infinite Dosing. In: Dragicevic N, Maibach HI (eds) Percutaneous Penetration Enhancers Drug Penetration Into / Through the Skin: Methodology and General Considerations. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 35-44.

[0131] The Franz Diffusion Cell had a donor compartment and a receptor compartment attached to each other using clamp attachment lug. Between donor compartment and receptor compartment was a membrane mounted. The receptor compartment had a sampling side arm. The formulation to be determined was applied to the donor compartment. A receiver fluid was contained in the receptor compartment. The Franz Diffusion Cell had an orifice of 1 cm2. Experiments were conducted at 32°C according to USP (725). Human abdominal skin membranes were prepared by removing subcutaneous fatty tissue with a scalpel and then normalising to 400 pm thickness with a dermatome (Aesculap GA 643).

[0132] A solution of 6.17 wt.-% formoterol fumarate dihydrate (w / w, equivalent to 5% formoterol free base) was prepared by dissolving formoterol fumarate dihydrate in DMSO. A solution of 6.17 wt.-% formoterol fumarate dihydrate in a mixture of DMSO and ethanol (50:50, w / w) was prepared by dissolving formoterol fumarate dihydrate in DMSO and adding ethanol thereto. 200 mg of formulations comprising formoterol, were provided in the donor compartment.

[0133] Samples were taken after 2, 3, 4, 5, 6, 8 and 24 hours and analysed via HPLC-UV. Figure 1 shows the amount of formoterol permeated per cm2of murine skin over 24 h. As can be taken from Figure 1, formoterol applied in DMSO or DMSO / ethanol permeated human skin within 24 h in substantial amounts. The permeation from a 6.17% (w / w) (equivalent to 5% formoterol free base) solution in pure DMSO across human abdominal skin resulted in a flux of 8.2 ± 2.4 pg / (cm2xh) with a lag time of 0.8 ± 0.6 h.

[0134] Example 3

[0135] Determination of the effect of a formoterol formulation on lipolysis in vivo

[0136] The effect of 6.17% (w / w) formoterol fumarate dihydrate (FF) containing solution based on a 50 / 50 (%w / w) mixture of dimethyl sulfoxide and ethanol was tested in vivo in C57BL / 6 mice as described above. Inguinal white adipose tissue (WATi) was analyzed for browning marker expression by qPCR. Browning markers tested for were Uncoupling -protein- 1 (UCP1), cell death-inducing DFFA-like effector A (CIDEA), and peroxisome proliferator-activated receptor gamma coactivator 1 -alpha (PGCla). Another marker that was evaluated was Type II iodothyronine deiodinase (DIO2).

[0137] Figure 2 shows the results of treatment of mice with 6.17% (w / w) formoterol fumarate dihydrate solution for 10 days on expression of browning markers. As can be taken from Figure 2, treatment significantly increased mRNA expression of the browning markers UCP1, CIDEA and PGCla, while DIO2 and TNFa were not significantly changed in white adipose tissue compared to placebo controls. Example 4

[0138] Determination of solubility of clenbuterol in several solvents

[0139] Solubility of clenbuterol was tested in water, isopropyl myristate, Miglyol® 812, propylene carbonate, dimethyl isosorbide, polyethylene glycol 300 (PEG300), propylene glycol, Transcutol®, and ethanol as described above. The results are listed in Table 3 below.

[0140] Table 3: Solubility of clenbuterol in water, isopropyl myristate (IPM), Miglyol® 812, and propylene carbonate, dimethyl isosorbide, polyethylene glycol 300 (PEG300), propylene glycol, Transcutol®, and ethanol. Data is presented as mean ± SD; n = 3.

[0141] As can be taken from Table 3, for dimethyl isosorbide (DMI), polyethylene glycol 300 (PEG300), propylene glycol (PG), Transcutol® (TC), and ethanol (EtOH), the solubility was above 25 mg / mL.

[0142] Solubility of clenbuterol was further tested in binary mixtures of water with ethanol, Transcutol® (TC), and dimethyl isosorbide (DMI). The results are listed in Table 4 below. As can be taken from Table 4, solubility increased with decreasing amount of water.

[0143] Table 4: Solubility of clenbuterol in binary mixtures of water with increasing amounts of ethanol

[0144] (EtOH), dimethyl isosorbide (DMI), and Transcutol® (TC). Data is presented as mean ± SD; n = 3. Example 5

[0145] Determination of in vitro permeation of clenbuterol in Franz Diffusion Cell

[0146] The in vitro permeation of clenbuterol from binary solvent mixtures of water with ethanol, Transcutol®, and dimethyl isosorbide, as well as a solution based on ethanol and dimethyl isosorbide through excised human abdominal skin was determined over a time period of 24 h in Franz Diffusion Cell as described in example 2.

[0147] For mixtures in EtOH the drug load of clenbuterol was set to 1%, while Transcutol®, and dimethyl isosorbide containing solutions were adjusted to 0.1 and 0.4% respectively, due to reduced solubility.

[0148] Samples were taken hourly after 1 - 8 and 22-24 hours and analysed via HPLC-UV. Table 5 below summarises the compositions and results.

[0149] Table 5: Compositions, resulting flux and extrapolated lag times of the clenbuterol (CLB) formulations containing water, ethanol (EtOH), Transcutol® (TC), isopropyl myristate (IPM), and dimethyl isosorbide (DMI), tested in FDC permeation experiments. Data is presented as mean ± SD; n

[0150] Figure 3 illustrates the permeation time profiles of clenbuterol from the tested binary mixtures of ethanol / isopropyl myristate (EtOH IPM), ethanol / water (EtOH W), Transcutol® / water (TC_W), and dimethyl isosorbide / water (DMI W). As can be taken from Table 5 and Figure 3, the EtOH containing formulations showed the highest permeated amounts after 24 h, albeit connected to high lag times. For the dimethyl isosorbide containing formulation, clenbuterol was only detectable in the receiver after 22 h, resulting in the lowest permeated amount and the longest lag time of the tested formulations. The Transcutol® containing formulation showed the lowest lag time of 1.4 h and a flux of 1.8 pg / (cm2xh) and was chosen for further in-vivo experiments in mice. Example 6

[0151] Determination of the effect of an aqueous clenbuterol formulation on lipolysis in vivo

[0152] The effect of 0.4 wt.-% clenbuterol in an aqueous formulation of Transcutol® / water (50:50 v / v) on adipocyte size after transdermal administration was tested in vivo in mice. A solution of 0.4% clenbuterol (w / v) was prepared by dissolving clenbuterol in diethylene glycol monoethyl ether (Transcutol®) before the addition of water. 2.5 % (w / w) hydroxypropyl cellulose H was added to the solution under stirring and the resulting suspension was left tightly sealed until complete dissolution of the gelling agent was observed. Mice were treated with the gelled formulation and inguinal white adipose tissue (WATi) was analyzed for browning marker expression by qPCR and histologically after hematoxylin and eosin, as well as UCP1 antibody staining as described above.

[0153] Figure 4 shows histological sections of inguinal white adipose tissue after haematoxylin and eosin staining. As can be seen in Figure 4B an increase in lipid droplets and reduction in adipocyte size in verum treated mice compared to placebo Figure 4A was seen. No differences in UCP1 expression from antibody-stained sections could be observed between placebo (Figure 4C) and verum treated groups (Figure 4D) was seen. However, a change in cell morphology was visible in these sections as well. These morphologic changes from uni- to multilocular adipocytes are typical for brown and beige adipocytes.

[0154] Figure 5 shows the results of treatment of mice with aqueous clenbuterol gel on expression of browning markers UCP1, CIDEA, DIO2, PGCla, and TNFa in Figures Figure 5 A, B, C, D and E, respectively. As can be taken from Figure 5, treatment significantly increased mRNA expression of the browning markers CIDEA and PGCla, while UCP1, DIO2 and TNFa were not significantly changed in white adipose tissue compared to placebo control.

[0155] Example 7

[0156] Determination of the effect of an ethanolic / aqueous clenbuterol formulation on lipolysis in vivo

[0157] The effect of 1 wt.-% clenbuterol in an ethanolic aqueous formulation of ethanol / water (75:25, v / v) on adipocyte size after transdermal administration was tested in vivo in mice. A solution of 1% clenbuterol (w / v) was prepared by dissolving clenbuterol in ethanol before the addition of water. 2.5 % (w / w) hydroxypropyl cellulose H was added to result in a gelled formulation. Mice were treated with the gelled formulation and inguinal white adipose tissue (WATi) was analyzed for browning marker expression by qPCR and histologically after hematoxylin and eosin, as well as UCP1 antibody staining as described above.

[0158] Figure 6 shows the results of treatment of mice with ethanolic / aqueous clenbuterol gel on expression of browning markers UCP1, CIDEA, DIO2, PGCla, and TNFa in Figures Figure 6A, B, C, D and E, respectively. As can be taken from Figure 6, treatment significantly increased mRNA expression of the browning markers CIDEA and PGCla, while UCP1, DIO2 and TNFa were not significantly changed in inguinal white adipose tissue compared to placebo control.

[0159] Example 8

[0160] Determination of solubility of salbutamol hemisulfate in several solvents

[0161] Solubility of salbutamol hemisulfate was tested in Transcutol® (TC), ethanol (EtOH), dimethyl sulfoxide (DMSO), methanol (MeOH) and propylene glycol (PG) and water as described above. The results are listet in Table 6 below.

[0162] Table 6: Solubility of salbutamol hemisulfate in Transcutol®, ethanol, dimethyl sulfoxide, methanol and propylene glycol. Data is presented as n = 1.

[0163] As can be taken from Table 6, for water the solubility was above 100 mg / mL.

[0164] Example 9

[0165] Determination of in vitro permeation of salbutamol hemisulfate in Franz Diffusion Cell The in vitro permeation of salbutamol hemisulfate from binary solvent mixtures of water with dimethyl sulfoxide (DMSO) through murine skin was determined over a time period of 24 h in Franz Diffusion Cell as described in example 2.

[0166] Samples were taken after 1, 2, 4, 6, 8, 9, 10, 12 and 24 hours and analysed via HPLC using a fluorescence detector. Table 7 below summarises the compositions and results.

[0167] Table 7 : Compositions, resulting flux and extrapolated lag times of salbutamol hemisulfate from binary mixtures of water and dimethyl sulfoxide (DMSO), tested in permeation experiments. Data is presented as mean ± SD; n = 5 (50 / 50 (w / w)) and n = 4 (90 / 10 (w / w)).

[0168] Figure 7 illustrates the permeation time profiles of salbutamol hemisulfate from the tested binary mixtures of water and dimethyl sulfoxide (DMSO). As can be taken from Table 7 and Figure 7, the formulations showed good permeated amounts after 24 h and flux.

[0169] Example 10

[0170] Determination of the effect of an ethanolic / aqueous salbutamol hemisulfate formulation on lipolysis in vivo

[0171] The effect of 10% (w / w) salbutamol hemisulfate containing gel, referring to the weight of solution, on base of a 50 / 50 (%w / w) mixture of water and ethanol on adipocyte size after transdermal administration was tested in vivo in mice. A solution of 10% (w / w) salbutamol hemisulfate was prepared by dissolving salbutamol hemisulfate in water before the addition of ethanol. 5 % (w / w) hydroxypropyl cellulose H was added to the solution for gellation. Mice were treated with the gelled formulation for 10 days and inguinal white adipose tissue (WATi) was analyzed for browning marker expression by qPCR and histologically after hematoxylin and eosin, as well as UCP1 antibody staining as described above. Figure 8 shows histological sections of inguinal white adipose tissue after haematoxylin and eosin staining and UCP1 antibody staining. As can be seen in Figure 8B an increase in lipid droplets and reduction in adipocyte size in verum treated mice compared to placebo as depicted in Figure 8A was seen. No differences in UCP1 expression from antibody-stained sections could be observed between placebo, depicted in Figure 8C, and verum treated groups, depicted in Figure 8D, was seen.

[0172] Figure 9 shows the results of treatment of mice with aqueous salbutamol hemisulfate gel on expression of browning markers UCP1, CIDEA, DIO2, PGCla, and TNFa in Figures Figure 9A, B, C, D and E, respectively. As can be taken from Figure 9, treatment did not significantly increase mRNA expression of browning markers in inguinal white adipose tissue, although verum treated groups showed an increased trend for all browning marker genes. TNFa was not increased compared to placebo. It is assumed that a rapid evaporation of ethanol on the murine skin in-vivo could have resulted in inadequate time for permeation enhancement and thus insufficient amounts of salbutamol hemisulfate in white adipose tissue, and that a less volatile permeation enhancer would improve delivery in-vivo.

[0173] Example 11

[0174] Determination of in-vitro permeation of salbutamol hemisulfate from aqueous DMSO formulation

[0175] The permeation of salbutamol hemisulfate from a 2.5% (w / w) gel, referring to the weight of solution, on base of a 50 / 50 (%w / w) mixture of water and dimethyl sulfoxide over 400 pm thick dermatomed human abdominal skin was evaluated over a time period of 24 h. A gel of 2.5% (w / w) salbutamol hemisulfate on solvent base of a 50 / 50 (%w / w) mixture of water and DMSO was prepared by gelling with an additional 4% (w / w) HEC 250, referring to the total weight of solution. Samples were taken after 5 h and 24 h.

[0176] Figure 10 shows the amount of salbutamol hemisulfate permeated per cm2of human abdominal skin after 5 h and 24 h. As can be taken from Figure 10, high permeated amounts of 212.2 ± 84.4 pg / cm2salbutamol hemisulfate after 24 h were achieved.

[0177] Comparing this solvent system of aqueous DMSO formulation to the ethanolic aqueous formulation, the permeated amount was almost doubled, when normalized to the concentration of salbutamol hemisulfate. Accordingly, it is assumed that formulations based on binary mixtures of water and DMSO could provide increased permeation.

[0178] Example 12

[0179] Determination of solubility of salmeterol xinafoate in several solvents

[0180] Solubility of salmeterol xinafoate was tested in water, ethanol, propylene glycol, dimethyl sulfoxide, methanol and Transcutol® as described above. The results are listed in Table 8 below.

[0181] Table 8: Solubility of salmeterol xinafoate in water, ethanol, propylene glycol, dimethyl sulfoxide, methanol and Transcutol®. Data is presented as n = 1.

[0182] As can be taken from Table 8, for dimethyl sulfoxide, methanol and Transcutol® the solubility was above 50 mg / mL.

[0183] Example 13

[0184] Determination of in vitro permeation of salmeterol xinafoate in Franz Diffusion Cell

[0185] The in vitro permeation of salmeterol xinafoate from a 5% (w / w) binary mixture of ethanol (EtOH) and dimethyl sulfoxide (DMSO) (50 / 50 (%w / w)) through murine skin was determined over a time period of 24 h in a Franz Diffusion Cell as described in example 2. Samples were taken after 1, 2, 4, 6, 8, 10, 12 and 24 hours and analysed via HPLC using a fluorescence detector. Table 9 below summarises the results.

[0186] Table 9: Composition, resulting flux and extrapolated lag time of salmeterol xinafoate from binary mixtures of ethanol and dimethyl sulfoxide (DMSO), tested in permeation experiments. Data is presented as mean ± SD; n = 6.

[0187] Figure 11 illustrates the permeation time profdes of salmeterol xinafoate from the tested binary mixture of ethanol and dimethyl sulfoxide (DMSO). As can be taken from Table 9 and Figure 11, the formulation showed a good permeated amount after 24 h and flux.

[0188] Example 14

[0189] Determination of solubility of fenoterol hydrobromide in several solvents

[0190] Solubility of fenoterol hydrobromide was tested in water, ethanol, propylene glycol, dimethyl sulfoxide, methanol and Transcutol® as described above. The results are listed in Table 10 below.

[0191] Table 10: Solubility of fenoterol hydrobromide in dimethyl sulfoxide, ethanol, methanol, propylene glycol, Transcutol® and water. Data is presented as n = 1.

[0192] As can be taken from Table 10, for dimethyl sulfoxide, ethanol, methanol, propylene glycol, Transcutol® and water the solubility was above 50 mg / mL.

[0193] Example 15

[0194] Determination of in vitro permeation of fenoterol hydrobromide in Franz Diffusion Cell

[0195] The in vitro permeation of fenoterol hydrobromide from a 5% (w / w) binary mixture of ethanol and dimethyl sulfoxide (DMSO) (50 / 50 and 90 / 10 (w / w)) through murine skin was determined over a time period of 24 h in a Franz Diffusion Cell as described in example 2. Samples were taken after 1, 2, 4, 6, 8, 10, 12 and 24 hours and analysed via HPLC using a fluorescence detector. Table 11 below summarises the results. Table 11 : Compositions, resulting flux and extrapolated lag times of fenoterol hydrobromide from binary mixtures of ethanol and dimethyl sulfoxide (DMSO), tested in permeation experiments. Data is presented as mean ± SD; n = 5.

[0196] Figure 12 illustrates the permeation time profiles of salmeterol xinafoate from the tested binary mixtures of ethanol and dimethyl sulfoxide (DMSO). As can be taken from Table 11 and Figure 12, both formulations showed a good permeated amount after 24 h and flux.

[0197] Example 16

[0198] Determination of in vitro permeation of compounds from a ternary mixture in Franz Diffusion Cell

[0199] The in vitro permeation of salbutamol hemisulfate, salmeterol xinafoate, formoterol fumarate dihydrate, and fenoterol hydrobromide of equimolar solutions based on 3% (w / w) salbutamol from a ternary mixture of DMSO, ethanol and water (30 / 35 / 35, %w / w / w) through murine skin of about 600 pm was determined over a time period of 24 h in a Franz Diffusion Cell. The skin surface temperature was maintained at 37°C. Otherwise experiments were performed as described above. Samples were taken after 0, 1, 2, 4, 6, 8, 12 and 24 hours and analysed via HPLC using a fluorescence detector. Table 12 below summarises the results.

[0200] Table 12: Compositions, resulting flux and extrapolated lag times from ternary mixtures of dimethyl sulfoxide (DMSO), ethanol and water (30 / 35 / 35, %w / w / w) tested in permeation experiments.

[0201] Figure 13 illustrates the permeation time profiles of salmeterol xinafoate from the tested ternary mixtures of dimethyl sulfoxide (DMSO), ethanol and water. As can be taken from Table 12 and Figure 13, the ternary formulations provided a good permeated amount after 24 h and flux. Example 17

[0202] Determination of in vitro permeation of formoterol through murine skin in a Franz Diffusion Cell

[0203] The in vitro permeation of formoterol from a mixture of DMSO and ethanol through mouse flank skin was determined over a time period of 24 h in Franz Diffusion Cell as described in Example 2 as infinite dose experiment as defined in OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 28, 2004 and described by Lau WM, Ng KW (2017) Finite and Infinite Dosing. In: Dragicevic N, Maibach HI (eds) Percutaneous Penetration Enhancers Drug Penetration Into / Through the Skin: Methodology and General Considerations. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 35-44.

[0204] Mouse flank skin membranes were prepared by removing the skin from the mice and trimming the fur to 0.5 mm. The flank skin membranes were used in full thickness, which is about 600 pm.

[0205] A solution of 5 wt.-% formoterol fumarate dihydrate (w / w, equivalent to 4.1% formoterol free base) or 1 wt.-% formoterol fumarate dihydrate (w / w, equivalent to 0.82 % formoterol free base) in a mixture of DMSO and ethanol (50:50 or 10:90, w / w) were prepared by dissolving formoterol fumarat dihydrate in DMSO and adding ethanol thereto. 200 pl of formulations comprising formoterol were provided in the donor compartment. Samples were taken after 24 hours and analysed via HPLC using a fluorescence detector. Table 13 below summarises the results.

[0206] Table 13: Compositions, resulting flux and extrapolated lag time of formoterol fumarate dihydrate from binary mixtures of dimethyl sulfoxide (DMSO) and ethanol (EtOH), tested in permeation experiments in infinite dose. Data is presented as mean ± SD, n=6.

[0207] As can be taken from Table 13, the formulations showed a good permeated amount after 24 h and flux. Example 18

[0208] Determination of in vitro permeation of salbutamol through murine skin in a Franz Diffusion Cell

[0209] The in vitro permeation of salbutamol hemisulfate from binary and ternary solvent mixtures through murine skin was determined over a time period of 24 h in Franz Diffusion Cell as described in example 17. Samples were taken after 24 hours and analysed via HPLC using a fluorescence detector. Table 14 below summarises the compositions and results.

[0210] Table 14: Compositions, resulting flux and extrapolated lag time of salbutamol hemisulfate from mixtures of dimethyl sulfoxide (DMSO), ethanol (EtOH), glycerol, water and a urea solution (20 % (w / w) in water), tested in permeation experiments in infinite dose. Data is presented as mean ± SD,

[0211] As can be taken from Table 14, the formulations showed a good permeated amount after 24 h and flux.

[0212] Example 19

[0213] Determination of in-vitro permeation of salbutamol from gelled DMSO formulations

[0214] The permeation of salbutamol hemisulfate from a 2% or 5% (w / w) gels, referring to the weight of solution, on base of a 30 / 35 / 35% (w / w / w) ternary mixture of dimethyl sulfoxide, ethanol and water over full thickness murine flank skin was evaluated over a time period of 24 h in a Franz Diffusion Cell as described in example 17 in infinite dose. Gels were prepared by gelling a 30 / 35 / 35% (w / w / w) mixture of dimethyl sulfoxide, ethanol and water with an additional 2% or 5% (w / w) of high- substituted hydroxypropyl cellulose (HPC-H), referring to the total weight of solutions. Sample for comparison was a 30 / 35 / 35% (w / w / w) ternary mixture of dimethyl sulfoxide, ethanol and water denoted “without gelling agent“. Samples were taken after 24 h and analysed via HPLC using a fluorescence detector. Table 15 below summarises the compositions and results.

[0215] Table 15: Compositions, resulting flux and extrapolated lag time of 2 % (w / w) salbutamol hemisulfate from mixtures of dimethyl sulfoxide (DMSO), ethanol (EtOH) and water 30 / 35 / 35 % (w / w / w) with and without 2 % and 5 % (w / w) Hydroxypropyl cellulose grade H (HPC H) as gelling agent in infinite dose. Data is presented as mean ± SD, n=6.

[0216] As can be taken from Table 15, the formulations showed a good permeated amount after 24 h and flux. There was no significant difference in permation over 24 h and flux depending on the amount of gelling agent.

[0217] Example 20

[0218] Determination of in-vitro permeation of salbutamol through murine and human skin in a Franz

[0219] Diffusion Cell

[0220] The in vitro permeation of salbutamol hemisulfate from a 30 / 35 / 35% (w / w / w) ternary mixture of dimethyl sulfoxide, ethanol and water through human and murine skin was determined over a time period of 24 h in a Franz Diffusion Cell as described in example 17. Human abdominal skin membranes were prepared by removing subcutaneous fatty tissue with a scalpel and then normalising to 400 pm thickness with a dermatome (Aesculap GA 643). Samples were taken after 24 hours and analysed via HPLC using a fluorescence detector. Table 16 below summarises the compositions and results. Table 16: Compositions, resulting flux and extrapolated lag time of salbutamol hemisulfate (API) from 5 % (w / w) solutions in mixtures of dimethyl sulfoxide (DMSO), ethanol (EtOH) and water 30 / 35 / 35 % (w / w / w) in infinite dose. Data is presented as mean ± SD, n=6.

[0221] As can be taken from Table 16, the formulations showed a good permeated amount after 24 h and flux.

[0222] Example 21

[0223] Determination of in-vitro permeation of compounds from a ternary mixture through murine skin in a Franz Diffusion Cell

[0224] The in vitro permeation of salbutamol hemisulfate, salmeterol xinafoate, formoterol fumarate dihydrate, and fenoterol hydrobromide of 72 pM solutions from a ternary mixture of DMSO, ethanol and water (30 / 35 / 35, %w / w / w) through murine skin was determined over a time period of 24 h in a Franz Diffusion Cell in infinite dose as described in example 16. Samples were taken after 0, 1, 2, 4, 6, 8, 12 and 24 hours and analysed via HPLC using a fluorescence detector.

[0225] Figure 14 illustrates the permeation time profiles of 72 pM solutions of salbutamol hemisulfate, salmeterol xinafoate, formoterol fumarate dihydrate and fenoterol hydrobromide in mixtures of dimethyl sulfoxide (DMSO), ethanol (EtOH) and water (30 / 35 / 35 w / w / w) over murine skin. As can be taken from Figure 14, the ternary formulations provided a good permeated amount after 24 h.

[0226] As can be taken from Figure 14, the formulations showed a good permeated amount after 24 h and flux.

[0227] Example 22

[0228] Determination of in-vitro permeation of salbutamol, salmeterol, formoterol and fenoterol through murine and human skin in a Franz Diffusion Cell The in vitro permeation of 72 pM solutions of salbutamol hemisulfate, salmeterol xinafoate, formoterol fumarate dihydrate and fenoterol hydrobromide in a 30 / 35 / 35% (w / w / w) ternary mixture of dimethyl sulfoxide, ethanol and water through murine skin was determined over a time period of 24 h in a Franz Diffusion Cell as described in example 17. Samples were taken after 24 hours and analysed via HPLC using a fluorescence detector. Table 17 below summarises the compositions and results.

[0229] Table 17: Compositions, resulting flux and extrapolated lag time of salbutamol hemisulfate, salmeterol xinafoate, formoterol fumarate dihydrate and fenoterol hydrobromide (API) over murine skin from 72 pM solutions in mixtures of dimethyl sulfoxide (DMSO), ethanol (EtOH) and water (30 / 35 / 35 w / w / w) in infinite dose. Data is presented as mean ± SD, n=6.

[0230] As can be taken from Table 17, the formulations showed a good permeated amount after 24 h and flux.

[0231] In summary, the results show that dimethyl sulfoxide allows for a transdermal administration of formoterol, salbutamol, salmeterol, and fenoterol. The results show further that effective transdermal delivery of salbutamol and clenbuterol can be achieved via application of aqueous and ethanolic formulations.

Claims

C l a i m s1. A composition for transdermal administration, the composition comprising a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, a mixture thereof, or a salt thereof, and dimethyl sulfoxide.

2. The composition for transdermal administration according to claim 1, wherein the composition comprises a compound selected from the group of formoterol, salbutamol, salmeterol, fenoterol, or a salt thereof, and comprises:- dimethyl sulfoxide in a range from > 5 wt.-% to < 100 wt.-%, and optionally- water, a monoalcohol, glycerol, a glycol, a polyethylene glycol, a glycol ether, or mixtures thereof, in a range from > 5 wt.-% to < 95 wt.-%, wherein the wt.-% are based on a total weight of the solvent of 100 wt.-%.

3. The composition according to claim 2, wherein the composition comprises dimethyl sulfoxide in a range of from > 5 wt.-% to < 55 wt.-% and ethanol in a range of from > 45 wt.-% to < 95 wt.-%, based on a total weight of the solvent of 100 wt.-%.

4. The composition according to claim 2, wherein the composition comprises dimethyl sulfoxide in a range of from > 5 wt.-% to < 55 wt.-% and water in a range of from > 45 wt.-% to < 95 wt.-%, based on a total weight of the solvent of 100 wt.-%.

5. A composition for transdermal administration, wherein the composition comprises a compound selected from the group comprising salbutamol, clenbuterol, or a salt thereof, and comprises at least one of a monoalcohol or water.

6. The composition according to claim 5, wherein the composition comprises salbutamol or a salt thereof, and comprises:- a monoalcohol in a range from > 10 wt.-% to < 95 wt.-%, and- water, dimethyl isosorbide, isopropylmyristate, glycerol, a glycol, a polyethylene glycol, a glycol ether, or mixtures thereof, in a range from > 5 wt.-% to < 90 wt.-%; wherein the wt.-% are based on a total weight of the solvent of 100 wt.-%.

7. The composition according to claim 5, wherein the composition comprises clenbuterol or a salt thereof, and comprises:- water in a range from > 45 vol.-% to < 95 vol.-%, and dimethyl isosorbide, isopropylmyristate, glycerol, a glycol, a polyethylene glycol, a glycol ether, or mixtures thereof, in a range from > 5 vol.-% to < 55 vol.-%; or- a monoalcohol in a range from > 50 vol.-% to < 90 vol.-%, and water, isopropylmyristate, dimethyl isosorbide, glycerol, a glycol, a polyethylene glycol, a glycol ether, or mixtures thereof, in a range from > 10 vol.-% to < 50 vol.-%; wherein the vol.-% are based on a total volume of the solvent of 100 vol.-%.

8. The composition according to any one of the preceding claims, wherein the composition is in the form of a topical composition, preferably selected from the group consisting of a gel, an ointment, a cream, a foam, a lotion, a paste, a solution, a spray, or a plaster, and preferably is in the form of a gel or spray.

9. Use of the composition according to any one of claims 1 to 8 for the manufacture of a medicament for the treatment of obesity or lipedema, or for providing lipolysis.

10. Use of the composition according to any one of claims 1 to 8 in a cosmetic method for decreasing the quantity of subcutaneous fat tissue of a subject by topical application of the composition.

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