Transdermal composition comprising a beta-2 adrenergic receptor agonist

Abstract

The present invention relates to a transdermal composition comprising a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, mixtures thereof or salts thereof, and dimethylsulfoxide. The present invention further relates to a composition comprising a compound selected from the group comprising salbutamol, clenbuterol or salts thereof, and comprising at least one of water or a monohydric alcohol.

Description

[0001] This invention relates to a transdermal application composition.

[0002] Overweight and obesity are major risk factors for many diseases. Half of the EU population is overweight, more than a third are pre-obese, and one in six are considered obese with a body mass index (BMI) over 30. The prevalence of obesity is even more pronounced in the United States. These populations have a significantly higher risk of cardiovascular disease and diabetes. The aesthetic problems associated with being overweight also lead to social isolation and may contribute to mental health issues such as depression. In this regard, lipedema is particularly distressing. Lipedema is a chronic, progressive disease of subcutaneous adipose tissue in the upper and lower extremities, affecting almost exclusively women. Little is known about this condition, but it appears to result from a pathological increase in adipose tissue through hypertrophy and hyperplasia, accompanied by fluid retention. This causes a noticeable, well-defined increase in volume in the affected limbs (especially the extremities) compared to other parts of the body. Therefore, combating obesity has direct benefits for the physical and mental health of every individual and population.

[0003] Most drugs that increase fat burning belong to the appetite suppressant class. These drugs are primarily derived from amphetamines and suppress appetite. Another possibility to support weight loss is the use of lipase inhibitors. However, these substances need to be administered orally or by injection. When administered systemically, serious side effects are to be expected, as these compounds significantly interfere with the metabolism of neurotransmitters in the brain.

[0004] Topical application of active substances, where the active substance is applied directly to the site of action, is also known. One possibility for achieving this is direct injection into adipose tissue. In some cases of small to medium-sized localized fat deposits, this has been practiced by injecting a mixture of phosphatidylcholine and deoxycholate. However, this surfactant mixture can cause localized lipolysis at the injection site and is often associated with multiple injections and corresponding side effects such as pain and hematoma.

[0005] Topical application significantly reduces systemic absorption and associated side effects. WO 2007 / 011743 A1 discloses a composition for treating localized fat deposits comprising a long-acting β2-adrenergic receptor agonist, salmeterol or formoterol, and a compound that reduces the desensitization of target tissues to the long-acting β2-adrenergic receptor agonist, such as a glucocorticoid and / or ketotifen. This composition can be administered, for example, by injection and / or transdermal application. However, due to the poor permeability of the active pharmaceutical compound to the skin, topical application typically yields no clinical efficacy or only suboptimal clinical efficacy.

[0006] EP 3 206 678 B1 discloses a pharmaceutical composition for topical application comprising a prodrug of an adrenergic receptor agonist and / or antagonist, wherein the prodrug is an ester, and the composition is used in therapeutic applications to shape the mammalian body by modulating subcutaneous adipose tissue. Numerous β-adrenergic receptor agonists (“β-agonists”) cited include mirabezon, salbutamol, formoterol, salmeterol, fenoterol, and clenbuterol. The absorption of the prodrug provides a transdermal transport pathway, but the utilization of the active ingredient depends on the activity of the enzyme capable of releasing the agonist from the prodrug, and whether a therapeutic effect can be achieved in this manner remains unclear. However, the β2-adrenergic receptor agonists clenbuterol, formoterol, salmeterol, fenoterol, and salbutamol are not yet commercially available in transdermal formulations.

[0007] Therefore, the object of the present invention is to provide a composition that allows transdermal administration of a β2-adrenergic receptor agonist.

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

[0009] Accordingly, a transdermal administration composition is provided, comprising a compound selected from the group consisting of formoterol, salbutamol, salmeterol, fenoterol, mixtures thereof, or salts thereof, and dimethyl sulfoxide.

[0010] Surprisingly, dimethyl sulfoxide (DMSO) has also been found to allow transdermal administration of formoterol, salbutamol, salmeterol, or fenoterol. DMSO promotes skin penetration of formoterol, salbutamol, salmeterol, and fenoterol and increases their active delivery cycle. Further evidence suggests that mixtures of DMSO with alcohols, water, glycol ethers, or mixtures thereof can provide efficient transdermal delivery of formoterol, salbutamol, salmeterol, and fenoterol.

[0011] As used herein, the term “formoterol” (INN) refers to a β2-adrenergic receptor agonist (“β2-agonist”). Formoterol is also represented as (±)N-[2-hydroxy-5-[1-hydroxy-2-[[2-(p-methoxyphenyl)-2-propyl]amino]ethyl]phenyl]formamide. Formoterol is commercially available as a 1:1 racemic diastereomeric mixture of (R,R) plus (S,S). Commercially available formoterol salts are racemic mixtures of formoterol fumarate dihydrate represented by the following formula (1):

[0012] (1).

[0013] As used herein, the term “salbutamol” (INN) refers to a β2-adrenergic receptor agonist (“β2-agonist”). Salbutamol is also represented as 4-[2-(tert-butylamino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol, with CAS identification number 18559-94-9. Salbutamol is commercially available as a racemic diastereomer in a 1:1 ratio of R-enantiomer and S-enantiomer. Commercially available salbutamol salts are racemic mixtures of salbutamol hemisulfates represented by the following formula (2):

[0014] (2).

[0015] As used herein, the term “salmeterol” (INN) refers to a long-acting β2-adrenergic receptor agonist (“β2-agonist”). Salmeterol is also represented as 4-hydroxy-α1-[[[6-(4-phenylbutoxy)hexyl]amino]methyl]-1,3-benzenedimethanol, with the CAS designation 89365-50-4. Salmeterol is commercially available as a racemic mixture (a 1:1 mixture of its two enantiomers, the (R)- and (S)- forms) in the form of 1-hydroxy-2-naphthoate (= salmeterol sineanoate).

[0016] As used herein, the term “fennotefuran” (INN) refers to a short-acting β2-adrenergic receptor agonist (“β2-agonist”). Fennotefuran is also represented as 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol or 1-(3,5-dihydroxyphenyl)-1-hydroxy-2-[1-(4-hydroxyphenyl)isopropyl]aminoethane, with the CAS designation 13392-18-2. The molecule has two distinct stereocenters. Fennotefuran is commercially available as its (R,R) enantiomer and (S,S) enantiomer racemic mixture.

[0017] As used herein, the term “clenbuterol” (INN) refers to a β2-adrenergic receptor agonist (“β2-agonist”), denoted by the following formula (3):

[0018] (3).

[0019] Clenbuterol is also known as 1-(4-amino-3,5-dichlorophenyl)-2-(tert-butylamino)ethanol, with CAS designation 37148-27-9. Clenbuterol is commercially available as a racemic diastereomer of the R-enantiomer and S-enantiomer in a 1:1 ratio. Commercially available clenbuterol salts for application are racemic mixtures of clenbuterol monohydrochloride.

[0020] As used herein, the term "topical" application refers to the application of a substance (e.g., salbutamol, formoterol, salmeterol, fenoterol, or clenbuterol) to a specific location on or within the body, particularly on the surface, such as the skin or mucous membranes. As used herein, the term "transdermal" application refers to the penetration of a substance (e.g., salbutamol, formoterol, salmeterol, fenoterol, or clenbuterol) into the upper layers of the skin and its distribution into deeper layers (e.g., the dermis and subcutaneous tissue) after application.

[0021] The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable, non-toxic alkali or acid. Preferred salts include ammonium salts, calcium salts, magnesium salts, potassium salts, and sodium salts, as well as hydrochlorides, sulfates, hemisulfates, succinates, acetates, fumarates, tartrates, benzenesulfonates, and methanesulfonates.

[0022] As used herein, the term "solvent" refers to a substance, typically a liquid, capable of at least partially dissolving a compound, particularly one or more active compounds selected from salbutamol, formoterol, salmeterol, fenoterol, and clenbuterol, thereby providing a solution, dispersion, or suspension. The amount of compound dissolved depends on the nature of the compound and the solvent; the solvent may dissolve the compound completely or partially. As used herein, the term "solvent" also includes dispersants. As used herein, the term "solution" also includes dispersions or suspensions.

[0023] Regarding solvents, the given wt.-% or vol.-% is calculated based on 100 wt.-% of the total solvent weight or 100 vol.-% of the total volume. Regarding active compounds, such as formoterol, salbutamol, salmeterol, fenoterol, or clenbuterol, the given wt.-% refers to the total weight of the composition. For example, 100 g of a 5% formoterol solution in DMSO / ethanol (50 / 50, m / m) means 5 g of formoterol and 47.5 g each of DMSO and ethanol.

[0024] For effective transdermal delivery of compounds from a formulation solvent system, several requirements must be met. First, sufficient solubility of the active compound in the formulation is necessary. Further, an increase in solubility of the active compound in the skin (epidermis and dermis) (enhanced penetration) needs to be observed. Finally, sufficient solvent retention on the skin (limiting solvent evaporation) is required to ensure the molecular dispersion of the payload. A solvent that provides high solubility for a particular compound does not automatically allow that compound to penetrate the skin. For example, diethylene glycol monoethyl ether, marketed under the trade name Transcutol®, provides high solubility during use but insufficient penetration when used as the sole solvent.

[0025] For effective transdermal delivery of compounds selected from formoterol, salbutamol, salmeterol, or fenoterol, dimethyl sulfoxide is used as a solvent, wherein dimethyl sulfoxide may be used as the sole solvent or in a mixture with one or more other solvents, preferably in the form of a binary solvent mixture.

[0026] According to one embodiment, a transdermal administration composition is provided, the composition comprising a compound selected from formoterol, salbutamol, salmeterol, fenoterol, or salts thereof, and comprising:

[0027] - Dimethyl sulfoxide ranging from ≥5 wt.% to ≤100 wt.% and optionally

[0028] - The range is from ≥5 wt.% to ≤95 wt.% of water, monohydric alcohol, glycerol, glycol, polyethylene glycol, ethylene glycol ether, or mixtures thereof.

[0029] Where wt.-% is based on a total solvent weight of 100wt.-%.

[0030] In this embodiment, the composition containing formoterol, salmeterol, or fenoterol contains 100 wt.-% DMSO based on a total solvent weight of 100 wt.-%. Dimethyl sulfoxide provides very good penetration when used as the sole solvent with formoterol, salmeterol, and fenoterol.

[0031] A binary solvent mixture containing dimethyl sulfoxide and a monohydric alcohol, water, or ethylene glycol ether is preferred as a solvent, especially for compositions containing formoterol, salbutamol, salmeterol, or fenoterol as active ingredients.

[0032] In an embodiment, the composition comprises: a compound selected from the group consisting of formoterol, salbutamol, salmeterol, fenoterol, or salts thereof as an active ingredient, and a solvent mixture comprising, based on a total solvent weight of 100 wt.%, ≥5 wt.% to ≤95 wt.% DMSO and ≥5 wt.% to ≤95 wt.% a monohydric alcohol. Preferably, it is a short-chain monohydric alcohol containing up to 6 aliphatic carbon atoms. In a preferred embodiment, the monohydric alcohol is a C2-C5 alcohol selected from ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, or mixtures thereof. In a preferred embodiment, the monohydric alcohol is ethanol.

[0033] In this embodiment, the monohydric alcohol is ethanol, and based on a total solvent weight of 100 wt.%, the composition comprises ≥5 wt.% to ≤55 wt.%, or ≥10 wt.% to ≤50 wt.%, of dimethyl sulfoxide, and ≥45 wt.% to ≤95 wt.%, or ≥50 wt.% to ≤90 wt.%, of ethanol. In such embodiments, the composition preferably comprises formoterol, salmeterol, fenoterol, or a salt thereof as the active ingredient.

[0034] Based on 100 wt.-% solvent, at least 10 wt.-% DMSO is preferred to provide sufficient penetration of formoterol, salbutamol, salmeterol, or fenoterol. In embodiments, the composition comprises a compound selected from the group comprising formoterol, salbutamol, salmeterol, fenoterol, or salts thereof, and as a solvent, a range of ≥10 wt.-% to ≤50 wt.-% dimethyl sulfoxide and a range of ≥50 wt.-% to ≤90 wt.-% ethanol based on 100 wt.-% of the total solvent weight. In embodiments, the composition comprises formoterol, salmeterol, or salts thereof, and as a solvent, a mixture of 50 wt.-% each of dimethyl sulfoxide and ethanol based on 100 wt.-% of the total solvent weight. In an embodiment, the composition comprises fenotero or a salt thereof, and as a solvent, a range of ≥10 wt.-% to ≤50 wt.-% of dimethyl sulfoxide and a range of ≥50 wt.-% to ≤90 wt.-% of ethanol based on 100 wt.-% of the total solvent weight. These ranges provide good solubility and permeability of the compound.

[0035] In one embodiment, the composition comprises salbutamol or a salt thereof as the active ingredient, and a solvent mixture comprising, based on a total solvent weight of 100 wt.%, ≥5 wt.% to ≤95 wt.% DMSO and ≥5 wt.% to ≤95 wt.% water. In another embodiment, based on a total solvent weight of 100 wt.%, the composition comprises, based on a total solvent weight of ≥5 wt.% to ≤55 wt.%, or ≥10 wt.% to ≤50 wt.%, dimethyl sulfoxide and, based on a total solvent weight of ≥45 wt.% to ≤95 wt.%, or ≥50 wt.% to ≤90 wt.% water.

[0036] For non-sterile pharmaceutical preparations such as dermatological formulations, purified water is considered to be of suitable quality. The production and control of purified water are known to those skilled in the art and are mentioned in European Pharmacopoeia Monograph 0008. Purified water is the preferred type of water used in compositions. Purified water is commercially available.

[0037] Based on 100 wt.-% solvent, at least 10 wt.-% DMSO is preferred to provide sufficient penetration of salbutamol. In one embodiment, the composition comprises salbutamol or a salt thereof, and as a solvent, a mixture of ≥10 wt.-% to ≤50 wt.-% dimethyl sulfoxide and ≥50 wt.-% to ≤90 wt.-% water, based on 100 wt.-% of the total solvent weight. In another embodiment, the composition comprises salbutamol or a salt thereof, and as a solvent, a mixture of 50 wt.-% each of dimethyl sulfoxide and water, based on 100 wt.-% of the total solvent weight. These ranges provide good solubility and penetration of the compound.

[0038] In other embodiments, the composition comprises salbutamol or a salt thereof, and a mixture of water (ranging from ≥45wt.-% to ≤95wt.-% based on 100wt.-% of the total solvent weight) and dimethyl sulfoxide (ranging from ≥5wt.-% to ≤55wt.-%) as a solvent.

[0039] In other embodiments, compositions comprising compounds selected from the group consisting of formoterol, salbutamol, salmeterol, fenoterol, or salts thereof may comprise 100 wt.-% of DMSO in the range of ≥5 wt.-% to ≤90 wt.-% based on the total weight of the solvent, and ≥10 wt.-% to ≤95 wt.-% of glycerol, glycols such as propylene glycol (PG), polyethylene glycol (PEG), dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), ethylene glycol ethers, mixtures thereof, or mixtures thereof, in the range of PG to ≤95 wt.-%. The glycols and polyethylene glycols may be selected from the group consisting of ethylene glycol, propylene glycol, and polyethylene glycol. A preferred ethylene glycol ether is diethylene glycol monoethyl ether. Diethylene glycol monoethyl ether (IUPAC name 2-(2-ethoxyethoxy)ethanol) is commercially available, for example under the trade name Transcutol. ® Commercially available.

[0040] In other embodiments, for compositions containing formoterol, salbutamol, salmeterol, or fenoterol as active ingredients, a ternary solvent mixture comprising dimethyl sulfoxide, a monohydric alcohol, and water is preferred. In embodiments, the composition comprises dimethyl sulfoxide, a monohydric alcohol, and water, each comprising ≥20 wt.% to ≤40 wt.% based on a total solvent weight of 100 wt.%. The monohydric alcohol is preferably selected from ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, or mixtures thereof. In a preferred embodiment, the monohydric alcohol is ethanol. In embodiments, based on a total solvent weight of 100 wt.%, the composition comprises ≥30 wt.% to ≤35 wt.% dimethyl sulfoxide, ≥30 wt.% to ≤35 wt.% ethanol, and ≥30 wt.% to ≤35 wt.% water.

[0041] In an embodiment, based on the total weight of the composition, the composition comprises a compound selected from the group consisting of formoterol, salmeterol, fenoterol, or salts thereof, in a range of ≥0.01 wt.-% to ≤25 wt.-%, preferably in a range of ≥0.1 wt.-% to ≤10 wt.-%, and more preferably in a range of ≥0.5 wt.-% to ≤5 wt.-%.

[0042] In an embodiment, based on the total weight of the composition, the composition contains salbutamol or a salt thereof, ranging from ≥0.01 wt.-% to ≤25 wt.-%, preferably from ≥1 wt.-% to ≤10 wt.-%, and more preferably from ≥2.5 wt.-% to ≤5 wt.-%.

[0043] In this embodiment, the composition comprises a gelling agent selected from the group consisting of hydroxypropyl cellulose (preferably highly substituted hydroxypropyl cellulose (HPC-H)), hydroxyethyl cellulose, hydroxypropyl methyl cellulose (HPMC), and mixtures thereof, wherein, based on 100 wt.-% of the total weight of the composition, the gelling agent ranges from ≥1 wt.-% to ≤20 wt.-%, preferably from ≥2 wt.-% to ≤10 wt.-%, or from ≥4 wt.-% to ≤5 wt.-%. The gel composition preferably comprises DMSO as a solvent, wherein, based on 100 wt.-% of the total weight of the solvent, the DMSO ranges from ≥5 wt.-% to ≤95 wt.-%, preferably from ≥5 wt.-% to ≤55 wt.-%, or from ≥10 wt.-% to ≤50 wt.-%. Other solvents are preferably selected from water or monohydric alcohols such as ethanol. In a preferred embodiment, the gel composition comprises: 100 wt.-% of hydroxypropyl cellulose (preferably highly substituted hydroxypropyl cellulose (HPC-H)) in the range of ≥2.5 wt.-% to ≤5 wt.-% based on the total weight of the composition, and ≥2.5 wt.-% to ≤10 wt.-% of formoterol, salbutamol, salmeterol, or fenoterol, in the range of ≥2.5 wt.-% to ≤10 wt.-% based on the total weight of the composition; and a binary solvent mixture of DMSO in the range of ≥10 wt.-% to ≤50 wt.-% and ≥50 wt.-% to ≤90 wt.-% based on the total weight of the solvent, and ethanol or water in the range of ≥50 wt.-% to ≤90 wt.-%.

[0044] On the other hand, it relates to a transdermal administration composition comprising formoterol, salbutamol, salmeterol, fenoterol, or a salt thereof, wherein the composition provides a transdermal flux of formoterol, salbutamol, salmeterol, and fenoterol, according to USP (725), in a 1 cm² area. 2 The concentration measured in the Franz diffusion cell at 32°C was at least 5 µg / cm³ over 24 hours. 2In this implementation, the flux, as measured by USP(725), is at least 10 µg / cm³ over 24 hours. 2 Preferably, the concentration is at least 20 µg / cm³ within 24 hours. 2 More preferably, at least 30 µg / cm³ within 24 hours. 2 Even more preferably, at least 50 µg / cm³ within 24 hours. 2 More preferably, 100 µg / cm within 24 hours 2 .

[0045] In this implementation, the transdermal flux is based on USP (725) at a depth of 1 cm. 2 Measurements were taken on human skin in an unlimited dose assay at 32°C in a Franz diffusion cell at the orifice. As used herein, the term unlimited dose assay refers to an assay performed with a measured amount of the test preparation applied to the skin at which the absorption of the test substance reaches its maximum and is maintained, as defined in OECD Publications Series on Environmental Health and Safety, Test and Evaluation, 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.

[0046] On the other hand, it relates to transdermal application compositions comprising a compound selected from the group consisting of salbutamol, clenbuterol, or salts thereof, and at least one of water or a monohydric alcohol.

[0047] Preferred embodiments involve transdermal administration compositions comprising salbutamol or a salt thereof and a monohydric alcohol (preferably an ethanolic formulation of salbutamol).

[0048] According to one embodiment, a transdermal administration composition is provided, the composition comprising salbutamol or a salt thereof, and comprising:

[0049] - Monohydric alcohols ranging from ≥10 wt.% to ≤95 wt.% and

[0050] - The range is ≥5 wt.% to ≤90 wt.% of water, isosorbide dimethyl ether, isopropyl myristate, glycerin, glycol, polyethylene glycol, ethylene glycol ether or mixtures thereof;

[0051] Where wt.-% is based on a total solvent weight of 100wt.-%.

[0052] It has been shown that ethanol formulations containing isopropyl myristate, water, or glycol ether as other solvents can provide transdermal delivery of salbutamol.

[0053] Solvents selected from isosorbide dimethyl ether, isopropyl myristate, glycerol, glycol, polyethylene glycol, ethylene glycol ether, or mixtures thereof can increase penetration. The glycol and polyethylene glycol may be selected from the group consisting of ethylene glycol, propylene glycol, and polyethylene glycol. Preferably, the ethylene glycol ether is diethylene glycol monoethyl ether. Diethylene glycol monoethyl ether (IUPAC name 2-(2-ethoxyethoxy)ethanol) is commercially available, for example under the trade name Transcutol. ® Commercially available.

[0054] Binary mixtures containing monohydric alcohols such as ethanol and water can be used as solvents for salbutamol. Binary mixtures simplify formulation and enhance compatibility with other excipients.

[0055] Preferably, the monohydric alcohol is a short-chain monohydric alcohol containing up to six aliphatic carbon atoms. In embodiments, the monohydric alcohol is a C2-C5 alcohol selected from ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, or mixtures thereof. In a preferred embodiment, the monohydric alcohol is selected from ethanol, isopropanol, n-butanol, and isobutanol. Ethanol is most preferred.

[0056] In one embodiment, the composition comprises salbutamol or a salt thereof, and water in the range of ≥45 vol.-% to ≤55 vol.-% based on 100 wt.-% of the total solvent weight, and ethanol in the range of ≥45 wt.-% to ≤55 wt.-% as a solvent. In another embodiment, the composition comprising salbutamol may comprise equal amounts of water and ethanol, each 50 wt.-%

[0057] In an embodiment, the composition comprises salbutamol or a salt thereof, ranging from ≥0.01 wt.-% to ≤25 wt.-% based on the total weight of the composition, preferably from ≥1 wt.-% to ≤10 wt.-%, and more preferably from ≥2.5 wt.-% to ≤5 wt.-%.

[0058] On the other hand, it relates to a transdermal administration composition comprising salbutamol or a salt thereof, wherein the transdermal flux of salbutamol provided by the composition, according to USP (725), is within a 1 cm² area. 2 The concentration measured in the Franz diffusion cell at 32°C was at least 5 µg / cm³ over 24 hours. 2 In this implementation, the flux, as measured by USP(725), is at least 10 µg / cm³ over 24 hours. 2Preferably, the concentration is at least 20 µg / cm³ within 24 hours. 2 More preferably, at least 30 µg / cm³ within 24 hours. 2 Even more preferably, at least 50 µg / cm³ within 24 hours. 2 More preferably, 100 µg / cm within 24 hours 2 .

[0059] In this implementation, the transdermal flux is based on USP (725) at a depth of 1 cm. 2 Measurements were taken on human skin in an unlimited dose assay at 32°C in a Franz diffusion cell at the orifice. As used herein, the term unlimited dose assay refers to an assay performed with a measured amount of the test preparation applied to the skin at which the absorption of the test substance reaches its maximum and is maintained, as defined in OECD Publications Series on Environmental Health and Safety, Test and Evaluation, 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.

[0060] Further embodiments involve transdermal application compositions comprising clenbuterol or a salt thereof, and at least one of water or a monohydric alcohol.

[0061] According to one embodiment, a transdermal administration composition is provided, the composition comprising clenbuterol or a salt thereof, and comprising:

[0062] - Water ranging from ≥45 vol.% to ≤95 vol.% and isosorbide dimethyl ether, isopropyl myristate, glycerin, glycol, polyethylene glycol, ethylene glycol ether, or mixtures thereof ranging from ≥5 vol.% to ≤55 vol.%; or

[0063] - Monohydric alcohols ranging from ≥50 vol.-% to ≤90 vol.-% and water, isopropyl myristate, dimethyl isosorbide, glycerol, glycol, polyethylene glycol, ethylene glycol ethers or mixtures thereof ranging from ≥10 vol.-% to ≤50 vol.-%;

[0064] Where vol.-% is based on the total solvent volume of 100 vol.-%.

[0065] Aqueous and alcoholic compositions of clenbuterol have been found to allow for transdermal administration. It can be demonstrated that mixtures of water and at least one of ethanol, glycol ether or isosorbide dimethyl ether, as well as mixtures of ethanol and isopropyl myristate, can provide efficient transdermal delivery of clenbuterol.

[0066] Furthermore, solvents selected from dimethyl sulfoxide, isosorbide dimethyl ether, glycerol, glycol, polyethylene glycol, ethylene glycol ethers, or mixtures thereof also provide good penetration of clenbuterol. The glycol and polyethylene glycol may be selected from the group consisting of ethylene glycol, propylene glycol, and polyethylene glycol. Preferably, the ethylene glycol ether is diethylene glycol monoethyl ether. Diethylene glycol monoethyl ether (IUPAC name 2-(2-ethoxyethoxy)ethanol) is commercially available, for example under the trade name Transcutol. ® Commercially available.

[0067] For non-sterile pharmaceutical preparations such as dermatological formulations, purified water is considered to be of suitable quality. The production and control of purified water are known to those skilled in the art and are mentioned in European Pharmacopoeia Monograph 0008. Purified water is the preferred type of water used in compositions. Purified water is commercially available.

[0068] An alcoholic solution of clenbuterol can be used for transdermal applications. Preferably, it is a short-chain monohydric alcohol containing up to six aliphatic carbon atoms. In embodiments, the monohydric alcohol is a C2-C5 alcohol selected from ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, or mixtures thereof. In a preferred embodiment, the monohydric alcohol is selected from ethanol, isopropanol, n-butanol, and isobutanol. Ethanol is most preferred.

[0069] In this embodiment, the composition comprises clenbuterol or a salt thereof as the active ingredient, and a monohydric alcohol selected from ethanol, isopropanol, isobutanol, or mixtures thereof (preferably ethanol) as a solvent, ranging from ≥70 vol.-% to ≤90 vol.-%, preferably from ≥70 vol.-% to ≤80 vol.-%, based on a total solvent volume of 100 vol.-%, and isopropyl myristate or water, ranging from ≥10 vol.-% to ≤30 vol.-%, preferably from ≥20 vol.-% to ≤30 vol.-%. These ranges provide good solubility and permeability of clenbuterol. Particularly for clenbuterol, the alcoholic solution provides sufficient penetration after topical application to achieve a reduction in adipocyte size.

[0070] In other embodiments, compositions containing clenbuterol or its salts may contain water as a solvent, ranging from ≥45 vol.-% to ≤95 vol.-% based on a total solvent volume of 100 vol.-%, and diethylene glycol monoethyl ether (Transcutol®) or isosorbide dimethyl ether, ranging from ≥5 vol.-% to ≤55 vol.-% based on a total solvent volume of 100 vol.-%. For clenbuterol, aqueous solutions containing diethylene glycol monoethyl ether provide sufficient penetration after topical application to achieve a reduction in adipocyte size. In embodiments, compositions containing clenbuterol may contain water ranging from ≥40 vol.-% to ≤60 vol.-% (preferably from ≥45 vol.-% to ≤55 vol.-%) based on a total solvent volume of 100 vol.-%, and diethylene glycol monoethyl ether (Transcutol®) ranging from ≥40 vol.-% to ≤60 vol.-% (preferably from ≥45 vol.-% to ≤55 vol.-%) based on a total solvent volume of 100 vol.-%. ® In embodiments, compositions containing clenbuterol may comprise equal amounts of water and diethylene glycol monoethyl ether (Transcutol), each 50 vol.-% in volume. ® ).

[0071] In embodiments, compositions containing clenbuterol, based on a total solvent volume of 100 vol.-%, may contain ≥80 vol.-% to ≤90 vol.-% water and ≥10 vol.-% to ≤20 vol.-% isosorbide dimethyl ether. It has been found that in vitro permeation of clenbuterol can be achieved by formulations containing only 12.5 vol.-% isosorbide dimethyl ether and as low as 0.1 wt.-% clenbuterol.

[0072] In an embodiment, the composition contains clenbuterol or a salt thereof, in an amount ranging from ≥0.01 wt.-% to ≤25 wt.-%, preferably from ≥0.1 wt.-% to ≤5 wt.-%, and more preferably from ≥0.1 wt.-% to ≤1 wt.-%, based on the total weight of the composition.

[0073] On the other hand, it relates to a transdermal administration composition comprising clenbuterol or a salt thereof, wherein the composition provides a transdermal flux of clenbuterol according to USP (725) at a depth of 1 cm. 2 The concentration measured in the Franz diffusion cell at 32°C was at least 5 µg / cm³ over 24 hours. 2 In this implementation, the flux, as measured by USP(725), is at least 10 µg / cm³ over 24 hours. 2 Preferably, the concentration is at least 20 µg / cm³ within 24 hours. 2 More preferably, at least 30 µg / cm³ within 24 hours. 2Even more preferably, at least 50 µg / cm³ within 24 hours. 2 More preferably, 100 µg / cm within 24 hours 2 .

[0074] In this implementation, the transdermal flux is based on USP (725) at a depth of 1 cm. 2 Measurements were taken on human skin in an unlimited dose assay at 32°C in a Franz diffusion cell at the orifice. As used herein, the term unlimited dose assay refers to an assay performed with a measured amount of the test preparation applied to the skin at which the absorption of the test substance reaches its maximum and is maintained, as defined in OECD Publications Series on Environmental Health and Safety, Test and Evaluation, 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.

[0075] The compositions according to the invention can be used for transdermal application. In embodiments, the compositions are in the form of topical compositions. Preferably, the topical compositions are selected from gels, ointments, creams, foams, lotions, pastes, solutions, sprays, or plasters. Such formulations are conventional topical compositions for application to specific locations on the body surface (e.g., skin). Advantageously, the topical compositions according to the invention provide for transdermal application of the compound. Preferably, the compositions are in the form of gels, ointments, or plasters, more preferably in the form of gels or sprays. The dosage form is preferably according to the definition used by those skilled in the art, for example, European Pharmacopoeia, 10.0; 0132: Semi-solid preparations for skin.

[0076] For the formulation of the corresponding preparation, the composition may contain one or more pharmaceutically acceptable excipients to increase the physical, chemical, and microbiological stability of the preparation and patient compliance. Relevant gelling agents, surfactants, solubility enhancers, penetration enhancers, preservatives, antioxidants, complexing agents, cosolvents, emollients, fragrances, and colorants are known to those skilled in the art. Available pharmaceutically acceptable carriers, including hydrogels, skin creams, skin gels, skin lotions, skin ointments, skin patches, skin pastes, skin mud dressings, skin foams, skin solutions, skin sprays, and patches, are known to those skilled in the art. Another preferred form of delivery is a spray.

[0077] In one embodiment, the composition comprises one or more pharmaceutical excipients. In another embodiment, the composition comprises one or more pharmaceutical excipients selected from the group consisting of antioxidants, penetration enhancers, gelling agents, ointment components, preservatives, colorants, odorants, stabilizers, triglycerides, and mixtures thereof, and preferably gelling agents.

[0078] A preferred form of the formulation is a gel. In embodiments, the composition comprises a gel-forming agent selected from the group consisting of hydroxypropyl cellulose (preferably highly substituted hydroxypropyl cellulose (HPC-H)), hydroxyethyl cellulose, hydroxypropyl methyl cellulose (HPMC), and mixtures thereof. The composition may contain a gel-forming agent, such as hydroxypropyl cellulose or hydroxyethyl cellulose, ranging from ≥1 wt.-% to ≤20 wt.-% based on 100 wt.-% of the total weight of the composition, preferably ranging from ≥2 wt.-% to ≤10 wt.-%.

[0079] The gel composition preferably contains DMSO as a solvent, with DMSO ranging from ≥5 wt.% to ≤95 wt.% based on 100 wt.% of the total solvent weight, preferably from ≥5 wt.% to ≤55 wt.% or from ≥10 wt.% to ≤50 wt.% of the total solvent weight. Other solvents are preferably selected from water or monohydric alcohols such as ethanol.

[0080] In this embodiment, the composition comprises a gelling agent selected from the group consisting of hydroxypropyl cellulose (preferably highly substituted hydroxypropyl cellulose (HPC-H)), hydroxyethyl cellulose, hydroxypropyl methyl cellulose (HPMC), and mixtures thereof, in the range of ≥1 wt.-% to ≤20 wt.-%, preferably ≥2 wt.-% to ≤10 wt.-%, or ≥4 wt.-% to ≤5 wt.-%, based on 100 wt.-% of the total weight of the composition. The gel composition preferably contains DMSO as a solvent, in the range of ≥5 wt.-% to ≤95 wt.-%, preferably ≥5 wt.-% to ≤55 wt.-%, or ≥10 wt.-% to ≤50 wt.-%, based on 100 wt.-% of the total weight of the solvent. Other solvents are preferably selected from water or monohydric alcohols such as ethanol. In a preferred embodiment, the gel composition comprises 100 wt.% of hydroxypropyl cellulose (preferably highly substituted hydroxypropyl cellulose (HPC-H)) ranging from ≥2.5 wt.% to ≤5 wt.% based on the total weight of the composition, and ≥0.1 wt.% to ≤10 wt.% of formoterol, salbutamol, salmeterol, fenoterol, or clenbuterol, and a binary solvent mixture of 100 wt.% to ≤50 wt.% of DMSO and ≥50 wt.% to ≤90 wt.% of ethanol or water based on the total weight of the solvent.

[0081] Suitable excipients selected from ointment bases, gelling agents, antioxidants, pH adjusters, preservatives, colorants, and stabilizers are preferably selected from the group consisting of: α-tocopherol, acetic acid, ammonia solution, anhydrous citric acid, ascorbic acid, ascorbyl palmitate, bentonite, benzalkonium chloride, benzyl alcohol, butylparaben, calcium acetate, carbomer, sodium carboxymethyl cellulose, carnauba wax, carrageenan, castor oil, cetearyl alcohol polyether-30, veteth-10, cetearyl alcohol, cetyl alcohol, cholesterol, coconut oil, colloidal silica, corn oil, cropovidone, diethylene glycol monoethyl ether, polydimethylsiloxane 100, docosanol, sodium docusate, disodium edetate. Disodium, ethyl cellulose, ethylparaben, red iron oxide, formic acid, gelatin, glyceryl monostearate, glyceryl oleate, sodium hyaluronate, hydrochloric acid, hydrogenated castor oil, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, isopropyl myristate, isopropyl palmitate, lanolin, lavender oil, lecithin, linoleic acid, magnesium silicate, magnesium stearate, medium-chain triglycerides, menthol, methyl cellulose, methylparaben, mineral oil, myristic acid, myristol, oleic acid, olive oil, paraffin wax, peanut oil, PEG 6-32 stearate / ethylene stearate, PEG / PPG-18 / 18 polydimethylsiloxane, PEG-100 stearate, PEG-5 oleate, PEG-60 hydrogenated castor oil, PEG-75 lanolin, poloxamer, polyacrylic acid, polyethylene, polyethylene glycol, polysorbate, povidone, propylene glycol, quaternium-15, shea butter, simethicone, sodium bisulfite, sodium chloride, sodium hydroxide, sodium lauryl ether sulfate-2, sodium lauryl sulfate, sodium metabisulfite, sodium silicate, sorbic acid, stearic acid, thyme oil, triethanolamine, urea, vitamin E polyethylene glycol succinate, wax, xanthan gum, zinc acetate, zinc oxide, and zinc stearate.

[0082] In embodiments, the composition may comprise monoacylglycerol, diacylglycerol, or triacylglycerol. Preferably, the triacylglycerol is selected from the group consisting of glyceryl monostearate and glyceryl oleate. In embodiments, the composition comprises triacylglycerol, preferably selected from the group consisting of glyceryl monostearate and glyceryl oleate, and the triacylglycerol ranges from ≥1 wt.% to ≤20 wt.% based on the total weight of the composition.

[0083] In embodiments, the composition may comprise cyclodextrin, ranging from ≥1 wt.% to ≤20 wt.% based on 100 wt.% of the total solvent weight. Adding cyclodextrin can increase the solubility of the active compound. Preferred cyclodextrins are pharmaceutically acceptable cyclodextrins, such as β-cyclodextrin and γ-cyclodextrin, as well as β-cyclodextrin derivatives used as pharmaceutical excipients, such as sulfobutyl ether of β-cyclodextrin (SBE-β-CD), hydroxypropyl derivative of β-cyclodextrin (HP-β-CD), and randomly methylated β-cyclodextrin (RM-β-CD).

[0084] The compositions described herein can be used for transdermal administration of an active compound selected from the group consisting of formoterol, salbutamol, salmeterol, fenoterol, clenbuterol, mixtures thereof, or salts thereof to subcutaneous adipose tissue. Subcutaneous adipose tissue refers to the tissue layer directly beneath the skin of mammals, containing adipocytes. Transdermal administration of the compositions results in an increased local concentration of the compound in adipocytes, while significantly reducing systemic drug concentrations.

[0085] This composition provides efficient transdermal delivery of the active compound. This results in high uptake in subcutaneous adipose tissue, which responds by reducing the amount of subcutaneous adipose tissue and / or decreasing the size of individual fat cells. Reducing the amount of subcutaneous adipose tissue will reduce overweight caused by fat cells and reduce obesity and lipedema.

[0086] Furthermore, transdermal application of formoterol, clenbuterol, and salbutamol to mice resulted in a significant increase in the expression of browning markers in the inguinal white adipose tissue.

[0087] Therefore, another aspect relates to the use of the composition described above in the manufacture of a medicament for treating obesity or lipedema or for providing lipolysis. A related aspect relates to the composition described above for treating obesity or lipedema, or for providing lipolysis. Thus, the composition may be a pharmaceutical composition. In another aspect, a method for treating obesity or lipedema or for providing lipolysis is provided, wherein the method comprises the step of administering a therapeutically effective amount of the composition described herein to a subject in need (e.g., a mammal such as a human).

[0088] As used in this article, the term "lipolysis" refers to the process of applying active ingredients to achieve a localized reduction in the amount of subcutaneous fat tissue. The term "lipolysis" can also refer to a treatment or cosmetic procedure.

[0089] In other embodiments, the composition may be a cosmetic composition for cosmetic purposes. In another aspect, the present invention relates to the use of the composition described above in a cosmetic method for reducing the amount of subcutaneous adipose tissue in a subject by topical application. In other words, in embodiments, the composition described above is used in a cosmetic method for reducing the amount of subcutaneous adipose tissue in a subject by topical application. In another aspect, a cosmetic method for reducing the amount of subcutaneous adipose tissue in a subject (e.g., a mammal such as a human) is provided, wherein the method comprises applying the composition described above. A cosmetic method without therapeutic effect can be produced by applying a compound selected from the group consisting of formoterol, salbutamol, salmeterol, fenotrol, or clenbuterol to a local site of the body to reduce the volume of subcutaneous adipose tissue.

[0090] On the other hand, the use of compounds selected from the group consisting of formoterol, salbutamol, salmeterol, fenoterol, clenbuterol, mixtures thereof, or salts thereof in the manufacture of transdermal pharmaceutical products, preferably for the treatment of obesity or lipedema, or for the provision of lipolysis. In other words, the use of compounds selected from the group consisting of formoterol, salbutamol, salmeterol, fenoterol, clenbuterol, mixtures thereof, or salts thereof as transdermal pharmaceutical products, preferably for the treatment of obesity or lipedema, or for the provision of lipolysis, is also permitted. Compounds selected from the group consisting of formoterol, salbutamol, salmeterol, fenoterol, clenbuterol, mixtures thereof, or salts thereof can be administered transdermally using compositions as described above. This allows for the transdermal application of the compound. For a description of the compounds, their salts, compositions, and uses, refer to the description above.

[0091] On the other hand, it relates to compositions as described herein, used for transdermal therapy, preferably for treating obesity or lipedema, or for providing lipolysis. For a description of the compounds and their salts, compositions, and uses, refer to the above description.

[0092] 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 pertains.

[0093] The following examples are intended to illustrate the invention in more detail, but do not constitute a limitation thereof.

[0094] The attached diagram shows:

[0095] Figure 1 Permeation time curves of formoterol fumarate dihydrate from a 6.17% (w / w) solution in a binary mixture (50 / 50, %w / w) of pure dimethyl sulfoxide and ethanol across 400 µm thick human abdominal skin. Data are expressed as mean ± SD, n=6.

[0096] Figure 2Mice were treated for 10 days with a solution containing 6.17% (w / w) formoterol fumarate dihydrate (FF) in a 50 / 50 (%w / w) mixture of dimethyl sulfoxide and ethanol. The gene expression of the thermogenic markers UCP1, CIDEA, DIO2, PGC1α, and TNFα was significantly reduced. Figure 2 Data are presented as mean ± SD; n = 8–14. Significance analysis was performed using Student's t-test for PGC1α and Welch's t-test for UCP1, CIDEA, DIO2, and TNFa, to account for different SDs. *p < 0.05.

[0097] Figure 3 Clenbuterol, derived from a binary mixture of four ethanols (EtOH), water (W), Transcutol® (TC), and isosorbide dimethyl ether (DMI), was permeation-time profiled through 400 µm thick human abdominal skin.

[0098] Figure 4 Histological sections of mouse inguinal white adipose tissue after treatment with a gel containing 0.4% (w / v) clenbuterol in a 50 / 50 (%v / v) mixture of water and Transcutol® for 10 days: stained with hematoxylin and eosin. Figure 4 B vs. placebo control ( Figure 4 A) Comparison, and after UCP1 staining Figure 4 D compared to placebo control ( Figure 4 C) Comparison.

[0099] Figure 5 Mice were treated for 10 days with a gel containing 0.4% (w / v) clenbuterol in a 50 / 50 (%v / v) mixture of water and Transcutol®. Gene expression of the thermogenic markers UCP1, CIDEA, DIO2, PGC1α, and TNFα was significantly reduced. Figure 5 Data are presented as mean ± SD; n = 3–8. Significance analysis was performed using Student's t-test for CIDEA and Welch's t-test for PGC1α to account for different SDs. *p < 0.05.

[0100] Figure 6 Mice were treated for 10 days with a gel containing 1% (w / v) clenbuterol in a 25 / 75 (%v / v) mixture of water and ethanol. The gene expression of the thermogenic markers UCP1, CIDEA, DIO2, PGC1α, and TNFα was significantly reduced. Figure 6Data are presented as mean ± SD; n = 8–14. Significance analysis was performed using Welch's t-test for CIDEA and PGC1α to account for different SDs. *p < 0.05.

[0101] Figure 7 Permeation time curves of salbutamol hemisulfate through rat skin from a 5% (w / w) binary mixture (50 / 50 and 90 / 10 (w / w)) of water and dimethyl sulfoxide (DMSO). Data are presented as mean ± SD, n=5 (50 / 50 (w / w)) and n=4 (90 / 10 (w / w)).

[0102] Figure 8 Histological sections of white adipose tissue in the groin of mice after treatment with a gel containing 10% (w / w) salbutamol hemisulfate (by solution weight) for 10 days with either placebo or a 50 / 50 (% w / w) mixture of water and ethanol: stained with hematoxylin and eosin. Figure 8 B vs. placebo control ( Figure 8 A) Comparison, and after UCP1 staining Figure 8 D compared to placebo control ( Figure 8 C) Comparison.

[0103] Figure 9 Mice were treated for 10 days with a gel containing 10% (w / w) salbutamol hemisulfate (by solution weight) in a 50 / 50 (% w / w) mixture of water and ethanol. The gene expression of the thermogenic markers UCP1, CIDEA, DIO2, PGC1α, and TNFα was significantly reduced. Figure 9 Data are presented in categories A, B, C, D, and E. Data are expressed as mean ± SD; n = 8. Significance analysis was performed using the Student's t-test.

[0104] Figure 10 Permeation of salbutamol hemisulfate from a 2.5% (w / w) gel (by solution weight) of a 50 / 50 (%w / w) mixture of water and dimethyl sulfoxide through 400 µm thick ablation of human abdominal skin. Data are presented as mean ± SD, n=6.

[0105] Figure 11 : Permeation time curves of salmeterol sineproterenol from a 5% (w / w) binary mixture (50 / 50 (%w / w)) of ethanol (EtOH) and dimethyl sulfoxide (DMSO) via mouse skin. Data are expressed as mean ± SD, n=6.

[0106] Figure 12Penetration-time curves of fennoteulobromate from 5% (w / w) binary mixtures (50 / 50 and 90 / 10 (w / w)) of ethanol (EtOH) and dimethyl sulfoxide (DMSO) via mouse skin. Data are presented as mean ± SD, n=5.

[0107] Figure 13 Permeation-time curves of salbutamol hemisulfate, salmeterol sine benzoate, formoterol fumarate dihydrate, and fenotrol hydrobromide in a 72 µM solution of a ternary mixture of DMSO, ethanol, and water (30 / 35 / 35, % (w / w / w)) through 600 µm thick rat skin.

[0108] Figure 14 Permeation-time curves (unlimited dose) of salbutamol hemisulfate, salmeterol sinecurate, formoterol fumarate dihydrate, and fenotrol hydrobromide in full-thickness rat skin were compared from a 72 μM solution of a mixture of dimethyl sulfoxide (DMSO), ethanol (EtOH), and water (30 / 35 / 35 w / w / w). Data are presented as mean ± SD, n=6.

[0109] Material

[0110] Formoterol fumarate dihydrate (FF), clenbuterol (CLB), salbutamol hemisulfate (SS), salmeterol sine benzoate, and fenotrol hydrobromide were purchased from Swapnroop Drugs & Pharmaceuticals (Aurangabad, India) at analytical grade or higher. Brij® 98, dimethyl isosorbide (DMI), and triethylamine (99.5%) were products of Acros Organics (Geel, Belgium). Cocoyl caprylate, poloxamer 407, and Soluplus® were gifts from BASF SE (Ludwigshafen, Germany). Hydroxyethyl cellulose 250, isopropyl myristate, Miglyol® 812 (MCT812), polyethylene glycol 300, propylene glycol, and sodium lauryl sulfate were purchased from Caesar & Loretz GmbH (Hilden, Germany). Sodium fluoride and sodium orthovanadate were purchased from Carl Roth (Karlsruhe, Germany). Methanol and tetrahydrofuran were purchased from Fisher Scientific (Schwerte, Germany). Dow Corning TM BIO-PSA TM7-4202 was a gift from DDP Specialty Electronic Materials (Midland, MI, USA). Transcutol® P was a gift from Gattefossé SAS (Saint-Priest Cedex, France). DuroTAK® 387-2054 and DuroTAK® 387-2510 were gifts from Henkel (Düsseldorf-Holthausen, Germany). Pharmaceutical grade dimethyl sulfoxide was purchased from ITW Reagents (Monza, Italy). Parteck® MXP was a gift from Merck KGaA (Darmstadt, Germany). Hydroxypropyl cellulose H and hydroxypropyl cellulose SL were gifts from Nippon Soda Co., Ltd. (Tokyo, Japan). Dulbecco modified Eagle medium (DMEM), anhydrous ethanol, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), Nonidet® P 40 substitute (NP-40), and sodium deoxycholate were purchased from Sigma-Aldrich (Taufkirchen, Germany). 85% orthophosphate 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 TM and SYBR TM Green Master Mix was purchased from ThermoFisher, Waltham, MA, USA. The ProtoScript® II First Strand cDNA Synthesis Kit was purchased from New England Biolabs, MA, USA.

[0111] method

[0112] Solubility screening in excipients and solvents:

[0113] To assess the solubility of compounds in solvents of different polarities, 100 mg of the compound was added to 4 mL of solvent in a sealed beaker and stirred in the dark for 24 hours. The resulting suspension was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was then filtered through a 0.22 µm PES or PTFE syringe filter. The solution was analyzed by HPLC-UV or using a fluorescence detector, and diluted appropriately with 0.1 M HCl, water, or MeOH as needed. Because some of the compounds used were expensive and most solubility assessments were screening in nature, experiments were performed once or in triplicate. For some solvents, if the solubility was higher than 25 mg / mL, an increasing amount of the compound was added until a clear solution could not be obtained, or until 100 mg / mL was reached, and the final concentration at which a clear solution was produced was recorded.

[0114] Gel preparation:

[0115] A gel is prepared by dissolving the corresponding compound in its solvent mixture and then continuously adding a gelling agent under stirring until it is completely dissolved.

[0116] High-performance liquid chromatography (HPLC):

[0117] For each compound, analysis was performed on an LC-2040C 3D Plus system equipped with a C18 reversed-phase column (Inertsil ODS-3, 150 mm in length, 2.1 mm in inner diameter, and 3 µm in particle size) and an integrated diode array and a coupled RF-20A fluorescence detector (Shimadzu, Kyoto, Japan).

[0118] For clenbuterol, the flow rate was set to 0.3 mL / min, and the mobile phase 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 maintained at 25 °C. The injection volume was 2 µL, and the detector was set to 242 nm for UV detection. The limit of quantitation was 100 ng / mL.

[0119] For formoterol fumarate dihydrate, the flow rate was set to 0.3 mL / min, using a mixture of 35 / 65 (% v / v) MeOH and 0.2% TEA solution adjusted to pH 3 with orthophosphoric acid as the mobile phase. The column temperature was maintained at 40 °C, and the autosampler at 25 °C. The injection volume was 2 µL, and the detector was set to 210 nm for UV detection, or fluorescence detection at an excitation wavelength of 228 nm and an emission wavelength of 300 nm. The limit of quantitation was 100 ng / mL.

[0120] For salbutamol hemisulfate, the flow rate was set to 0.3 mL / min, and the mobile phase 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 maintained at 25 °C. The injection volume was 2 µL, and fluorescence detection was performed at an excitation wavelength of 220 nm and an emission wavelength of 310 nm. The limit of quantitation was less than 100 ng / mL.

[0121] For salmeterol benzoate, the flow rate was set at 0.3 mL / min, and the mobile phase consisted of 55 / 45 (% v / v) acetonitrile and 0.2% TEA solution adjusted to pH 3 with orthophosphoric acid. The column temperature was maintained at 40 °C, and the autosampler was maintained at 25 °C. The injection volume was 2 µL, and fluorescence detection was performed at an excitation wavelength of 340 nm and an emission wavelength of 415 nm. The limit of quantitation was less than 100 ng / mL.

[0122] For fennotefurantoin 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 was maintained at 25 °C. The injection volume was 2 µL, and fluorescence detection was performed at an excitation wavelength of 285 nm and an emission wavelength of 345 nm. The limit of quantitation was less than 100 ng / mL.

[0123] Preparation of human skin membrane:

[0124] Abdominal skin from the patient consenting to the plastic surgery was obtained and collected immediately postoperatively at Dreifaltigkeits-Krankenhaus, Weserling, Germany 50389. Ethical approval was granted by the Ethics Committee of the University of Bonn under approval reference number 082 / 20. Subcutaneous adipose tissue was carefully removed with a scalpel, and the resulting full-thickness skin was frozen at -35°C until use. For the permeability test, the skin was thawed at room temperature and then cut to a thickness of 400µm using a dermatograph (Aesculap® Acculan GA643, Tuttlingen, Germany).

[0125] Franz diffusion cell (FDC) experiment:

[0126] Using a receiving cell with an 8mL volume and 1cm 2Permeation experiments were conducted using a vertical Franz diffusion cell (SESAnalysensysteme, Bechenheim, Germany) with an effective diffusion area. Human abdominal skin, cut to a thickness of 400 µm according to OECD Guideline 408, was placed in the cell with the stratum corneum facing the air. The skin surface temperature was maintained at 32 °C. The receiving solution consisted of phosphate-buffered saline (pH 7.4) and 6% (w / w) Brij® 98. A 200 µL volume of the formulation was applied to the skin in the donor chamber, and 0.5 mL samples were taken at specified time points, immediately replaced with fresh receiving solution.

[0127] Processing of ex vivo human abdominal tissue:

[0128] Post-operatively, without pre-freezing, abdominal skin containing subcutaneous adipose tissue was cut into appropriately sized rectangular slices and placed in a culture dish containing 20 mL of DMEM (containing 10% fetal bovine serum and 1% penicillin / streptomycin). 0.2 mL of the preparation was applied to the skin using a syringe and spread on a 6 cm² surface. 2 On a rectangular area, the culture dish was sealed and incubated in a drying oven at 32°C for 3 days to simulate the temperature of the outer skin layer. After incubation, the skin was removed from the culture dish, and adipose tissue samples adjacent to the dermis were taken for further gene expression analysis by real-time PCR.

[0129] In vivo gel treatment in mice:

[0130] Wild-type (WT) C57BL / 6 mice were randomly divided into groups of eight, treated with either the test drug (Verum) or a placebo. Twenty-four hours before application, the fur on the flanks of the mice was carefully shaved with an electric razor. 50 mg of gel was applied to each flank once daily for 10 days. Twenty-four hours after the last gel application, the animals were sacrificed, and white adipose tissue (WATi) from the groin was harvested. Browning marker expression was analyzed by qPCR and Western blotting, followed by histological analysis after staining with hematoxylin and eosin, and UCP1 antibody.

[0131] Preparation of histological sections:

[0132] Tissue samples were fixed in 4% paraformaldehyde solution, dehydrated with ethanol (EtOH), embedded in paraffin, and sectioned using a microtome. The sections were stained with hematoxylin and eosin (H&E) and UCP1-specific antibody, respectively.

[0133] RNA isolation and real-time polymerase chain reaction (qPCR):

[0134] mRNA was extracted from tissue homogenized with Trizol and treated with chloroform, followed by precipitation with propan-2-ol and washing with 75% EtOH. The concentration of isolated RNA was analyzed spectrophotometrically using a Nanodrop 200 (ThermoFisher Scientific, Waltham, MA, USA).

[0135] cDNA was synthesized using the ProtoScript II First Strand cDNA Synthesis Kit according to the manufacturer's instructions. Amplification was analyzed by staining with SYBR Green Master Mix and measuring on a QuantStudio 5 qPCR system (ThermoFisher, Waltham, MA, USA). For mouse samples, 2... -ΔCT The method calculates the relative expression of the gene mRNA relative to the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT) normalized. For human samples, 2 -ΔCT The method was used to calculate the relative expression of gene mRNA relative to the housekeeping gene ribosomal protein L13a (RPL13A). Primer sequences are listed in Table 1.

[0136] Table 1: Primer sequences used for mouse gene qPCR.

[0137]

[0138] Protein blot:

[0139] To separate proteins, lysis buffer was determined using radioimmunoprecipitation with 50 mM Tris pH 7.5, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, and a mixture of protease inhibitors (Roche, Basel, Switzerland). Protein concentration was quantified using Bradford assay. Proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. For Western blotting, primary antibodies against UCP1 (Sigma-Aldrich, Taufkirchen, Germany) and Calnexin, as well as anti-rabbit secondary antibodies, were used. Proteins were visualized using Amersham ECL Western Blotting reagent and detected using an ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL, USA). Quantification was performed using ImageJ software.

[0140] Statistical analysis:

[0141] Statistical significance tests were performed using GraphPad Prism 8 software. Different statistical significance tests were conducted depending on the sample size of each group, whether multiple comparisons were considered, and whether the groups had the same standard deviation; these are appropriately noted in the relevant sections of the results.

[0142] Example 1

[0143] Solubility determination of formoterol in several solvents

[0144] As described above, the solubility of formoterol fumarate dihydrate in water, tetrahydrofuran (THF), diisosorbide dimethyl ether (DMI), propylene carbonate (PC), Transcutol® (TC), Miglyol® 812 (MCT812), cocoyl capryloyl decanoate (CC), ethanol (EtOH), polyethylene glycol 300 (PEG300), propylene glycol (PG), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and methanol (MeOH) was tested. The results are listed in Table 2 below.

[0145] Table 2: Solubility of formoterol fumarate dihydrate in water, tetrahydrofuran (THF), diisosorbide dimethyl ether (DMI), propylene carbonate (PC), Transcutol® (TC), Miglyol® 812 (MCT812), cocoyl capryloyl decanoate (CC), ethanol (EtOH), polyethylene glycol 300 (PEG300), propylene glycol (PG), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and methanol (MeOH).

[0146]

[0147] As shown in Table 2, formoterol fumarate dihydrate is insoluble in the nonpolar excipients MCT812 and cocoyl octanoyl decanoate because no amount of dissolved formoterol fumarate dihydrate (FF) can be detected, which is certainly due to its salt form (Table 5). Furthermore, formoterol fumarate dihydrate (FF) also exhibits low solubility in highly polar PCs. Solubility in water, THF, and DMI is less than 1 mg / mL, while in polar protic solvents containing alcohol groups (such as EtOH or TC), solubility is approximately an order of magnitude higher: the highest solubility is found in aprotic polar solvents (such as DMSO, DMF, and NMP).

[0148] Example 2

[0149] In vitro permeability determination of formoterol fumarate dihydrate

[0150] In the Franz diffusion cell, the in vitro permeability of formoterol from DMSO or a mixture of DMSO and ethanol over 24 hours via detached human abdominal skin was determined in an unlimited-dose experiment. The Franz diffusion cell allows for the measurement of the amount of the compound diffused through a membrane (such as human abdominal skin or mouse skin) into the receiving medium. As used herein, the term unlimited-dose experiment refers to an experiment conducted using a quantity of the test preparation applied to the skin at which the absorption of the test substance reaches its maximum and is maintained, as defined in OECD Publications Series on Environmental Health and Safety, Test and Evaluation, No. 28 (2004), and described by LauWM, 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.

[0151] The Franz diffusion cell has a donor chamber and a acceptor chamber, connected together by a clamp connecting lug. A membrane is installed between the donor and acceptor chambers. The acceptor chamber has a sampling arm. The test formulation is applied to the donor chamber. The acceptor chamber contains the receiving solution. The orifice of the Franz diffusion cell is 1 cm. 2 The experiment was conducted at 32°C according to USP(725).

[0152] Human abdominal skin membranes were prepared by removing subcutaneous fat tissue with a scalpel and then normalizing it to a thickness of 400 µm using a dermabrasion tool (Aesculap GA 643).

[0153] A 6.17 wt.-% formoterol fumarate dihydrate solution (w / w, equivalent to 5% free formoterol base) was prepared by dissolving formoterol fumarate dihydrate in DMSO. A 6.17 wt.-% formoterol fumarate dihydrate solution in a DMSO and ethanol mixture (50:50, w / w) was prepared by dissolving formoterol fumarate dihydrate in DMSO and adding ethanol. 200 mg of the formulation containing formoterol was placed in the donor chamber.

[0154] Samples were taken at 2, 3, 4, 5, 6, 8 and 24 hours and analyzed by HPLC-UV. Figure 1 It shows the number of centimeters per 24 hours. 2 The amount of formoterol permeated through the skin of mice. From Figure 1 It can be seen that formoterol applied in DMSO or DMSO / ethanol permeated human skin extensively within 24 hours. A 6.17% (w / w) pure DMSO solution (equivalent to 5% free formoterol base) produced 8.2 ± 2.4 µg / (cm³) of formoterol permeation through the skin of the human abdomen. 2 The flux is 0.8 ± 0.6 hours.

[0155] Example 3

[0156] In vivo assay of the lipolysis effect of formoterol formulation

[0157] As previously described, the effects of a solution containing 6.17% (w / w) formoterol fumarate dihydrate (FF) in a 50 / 50 (% w / w) mixture of dimethyl sulfoxide and ethanol were tested in C57BL / 6 mice. Browning marker expression in inguinal white adipose tissue (WATi) was analyzed by qPCR. The browning markers tested were uncoupling protein-1 (UCP1), cell death-inducing DFFA-like effector A (CIDEA), and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α). Another marker evaluated was type II iodothyronine deiodinase (DIO2).

[0158] Figure 2 The effect of treating mice with 6.17% (w / w) formoterol fumarate dihydrate solution for 10 days on the expression of browning markers was shown. Figure 2 It can be seen that, compared with the placebo control, the treatment significantly increased the mRNA expression of browning markers UCP1, CIDEA and PGC1α in white adipose tissue, while DIO2 and TNFα showed no significant changes.

[0159] Example 4

[0160] Determination of the solubility of clenbuterol in several solvents

[0161] As described above, the solubility of clenbuterol in water, isopropyl myristate, Miglyol® 812, propylene carbonate, isosorbide dimethyl ether, polyethylene glycol 300 (PEG300), propylene glycol, Transcutol®, and ethanol was tested. The results are listed in Table 3 below.

[0162] Table 3: Solubility of clenbuterol in water, isopropyl myristate (IPM), Miglyol® 812, propylene carbonate, isosorbide dimethyl ether, polyethylene glycol 300 (PEG300), propylene glycol, Transcutol®, and ethanol. Data are expressed as mean ± SD; n=3.

[0163]

[0164] As shown in Table 3, the solubility of isosorbide dimethyl ether (DMI), polyethylene glycol 300 (PEG300), propylene glycol (PG), Transcutol® (TC), and ethanol (EtOH) is greater than 25 mg / mL.

[0165] The solubility of clenbuterol in a binary mixture of water and ethanol, Transcutol® (TC), and isosorbide dimethyl ether (DMI) was further tested. The results are shown in Table 4 below. As can be seen from Table 4, the solubility increases as the amount of water decreases.

[0166] Table 4: Solubility of clenbuterol in water and binary mixtures of increased amounts of ethanol (EtOH), isosorbide dimethyl ether (DMI), and Transcutol® (TC). Data are expressed as mean ± SD; n = 3.

[0167]

[0168] Example 5

[0169] In vitro permeability of clenbuterol was determined in a Franz diffusion cell.

[0170] As described in Example 2, the in vitro permeability of clenbuterol from a binary solvent mixture of water and ethanol, Transcutol® and isosorbide dimethyl ether, and a solution based on ethanol and isosorbide dimethyl ether, was determined in a Franz diffusion cell over 24 hours through detached human abdominal skin.

[0171] For the mixture in EtOH, the loading of clenbuterol was set at 1%, while the loading of solutions containing Transcutol® and isosorbide dimethyl ether was adjusted to 0.1% and 0.4%, respectively, due to reduced solubility.

[0172] Samples were taken hourly at 1–8 hour and 22–24 hour intervals and analyzed by HPLC-UV. Table 5 below summarizes the compositions and results.

[0173] Table 5: Composition, flux, and extrapolated lag time of clenbuterol (CLB) formulations containing water, ethanol (EtOH), Transcutol® (TC), isopropyl myristate (IPM), and dimethyl isosorbide (DMI) tested in FDC permeation experiments. Data are presented as mean ± SD; n = 3–6.

[0174]

[0175] Figure 3 The permeation time curves for clenbuterol in the tested binary mixtures of ethanol / isopropyl myristate (EtOH_IPM), ethanol / water (EtOH_W), Transcutol® / water (TC_W), and isosorbide dimethyl ether / water (DMI_W) are shown in Table 5 and Figure 3 As can be seen, the formulation containing EtOH showed the highest permeation rate after 24 hours, despite a high hysteresis time. For the formulation containing isosorbide dimethyl ether, clenbuterol was only detected in the receiving solution after 22 hours, resulting in the lowest permeation rate and the longest hysteresis time among the tested formulations. The formulation containing Transcutol® showed the lowest hysteresis time of 1.4 hours and 1.8 µg / (cm²). 2 The flux was 1000 × h and was selected for further in vivo mouse experiments.

[0176] Example 6

[0177] In vivo assay of the lipolysis effect of aqueous clenbuterol formulation

[0178] The effect of 0.4 wt.% clenbuterol in the Transcutol® / water (50:50 v / v) aqueous formulation on adipocyte size after transdermal administration was tested in mice. A 0.4% clenbuterol (w / v) solution was prepared by dissolving clenbuterol in diethylene glycol monoethyl ether (Transcutol®) and then adding water. 2.5% (w / w) hydroxypropyl cellulose H was added to the solution with stirring, and the resulting suspension was tightly sealed until complete dissolution of the gelling agent was observed. Mice were treated with the gelling agent, and the expression of browning markers in inguinal white adipose tissue (WATi) was analyzed by qPCR as described above, followed by histological analysis after hematoxylin and eosin staining and UCP1 antibody staining.

[0179] Figure 4 Histological sections of white adipose tissue in the groin, stained with hematoxylin and eosin. From Figure 4 B shows that, compared to the placebo... Figure 4 Compared to A, mice treated with the test drug showed increased lipid droplets and decreased adipocyte size. In antibody-stained sections, the placebo ( Figure 4C) and the test drug treatment group ( Figure 4 No difference in UCP1 expression was observed between sections D). However, changes in cell morphology were also observed in these sections. These morphological changes, from unilocular to multilocular adipocytes, are typical of brown and beige adipocytes.

[0180] Figure 5 The results show the effects of treatment with aqueous clenbuterol gel on the expression of browning markers UCP1, CIDEA, DIO2, PGC1α, and TNFα in mice, respectively. Figure 5 From A, B, C, D, and E. Figure 5 It can be seen that, compared with the placebo control, the treatment significantly increased the mRNA expression of browning markers CIDEA and PGC1α in white adipose tissue, while UCP1, DIO2 and TNFα showed no significant changes.

[0181] Example 7

[0182] In vivo assay of the lipolysis effect of ethanol / water-based clenbuterol formulation

[0183] The effect of 1 wt.% clenbuterol in an ethanol / water (75:25, v / v) aqueous formulation on adipocyte size after transdermal administration was tested in mice. A 1% clenbuterol (w / v) solution was prepared by dissolving clenbuterol in ethanol and then adding water. 2.5% (w / w) hydroxypropyl cellulose H was added to produce a gelling formulation. Mice were treated with the gelling formulation, and the expression of browning markers in inguinal white adipose tissue (WATi) was analyzed by qPCR as described above, followed by histological analysis after hematoxylin and eosin staining and UCP1 antibody staining.

[0184] Figure 6 The results show the effects of treatment of mice with ethanol / aqueous clenbuterol gel on the expression of browning markers UCP1, CIDEA, DIO2, PGC1α, and TNFα, respectively. Figure 6 From A, B, C, D, and E. Figure 6 It can be seen that, compared with the placebo control, the treatment significantly increased the mRNA expression of browning markers CIDEA and PGC1α in inguinal white adipose tissue, while UCP1, DIO2 and TNFα showed no significant changes.

[0185] Example 8

[0186] Determination of the solubility of salbutamol hemisulfate in several solvents

[0187] As described above, the solubility of salbutamol hemisulfate in Transcutol® (TC), ethanol (EtOH), dimethyl sulfoxide (DMSO), methanol (MeOH), propylene glycol (PG), and water was tested. The results are listed in Table 6 below.

[0188] Table 6: Solubility of salbutamol hemisulfate in Transcutol®, ethanol, dimethyl sulfoxide, methanol, and propylene glycol. Data are presented as n=1.

[0189]

[0190] As can be seen from Table 6, the solubility in water is higher than 100 mg / mL.

[0191] Example 9

[0192] In vitro permeability of salbutamol hemisulfate was determined in a Franz diffusion cell.

[0193] As described in Example 2, the in vitro permeability of salbutamol hemisulfate from a binary solvent mixture of water and dimethyl sulfoxide (DMSO) through mouse skin over 24 hours was determined in a Franz diffusion cell.

[0194] Samples were taken at 1, 2, 4, 6, 8, 9, 10, 12, and 24 hours and analyzed by HPLC using a fluorescence detector. Table 7 below summarizes the compositions and results.

[0195] Table 7: Composition, flux, and extrapolated lag time of salbutamol hemisulfate from a binary mixture of water and dimethyl sulfoxide (DMSO) tested in permeation experiments. Data are expressed as mean ± SD, n=5 (50 / 50(w / w)) and n=4 (90 / 10(w / w)).

[0196]

[0197] Figure 7 The permeation time curves for salbutamol hemisulfate from a binary mixture of water and dimethyl sulfoxide (DMSO) are shown. (From Table 7 and...) Figure 7 It can be seen that the formulation exhibits good penetration and flux after 24 hours.

[0198] Example 10

[0199] In vivo determination of the effect of ethanol / aqueous salbutamol hemisulfate preparation on lipolysis

[0200] The effect of a gel containing 10% (w / w) salbutamol hemisulfate (by solution weight) in a 50 / 50 (% w / w) mixture of water and ethanol on adipocyte size after transdermal application was tested in mice. A 10% (w / w) salbutamol hemisulfate solution was prepared by dissolving salbutamol hemisulfate in water and then adding ethanol. The solution was gelled by adding 5% (w / w) hydroxypropyl cellulose H. Mice were treated with the gelling agent for 10 days, and the expression of browning markers in inguinal white adipose tissue (WATi) was analyzed by qPCR as described above. Histological analysis was performed after hematoxylin and eosin staining and UCP1 antibody staining.

[0201] Figure 8 Histological sections of inguinal white adipose tissue stained with hematoxylin and eosin, and stained with UCP1 antibody, are shown. Figure 8 B shows that, with Figure 8 Compared to the placebo group (shown in Figure A), mice treated with the test drug showed increased lipid droplets and decreased adipocyte size. In antibody-stained sections, the placebo group (e.g., ...) Figure 8 (as shown in C) and the test drug treatment group (e.g., Figure 8 No difference in UCP1 expression was observed between (as shown in D).

[0202] Figure 9 The effects of water-based salbutamol hemisulfate gel treatment on the expression of browning markers UCP1, CIDEA, DIO2, PGC1α, and TNFα in mice were shown, respectively. Figure 9 From A, B, C, D, and E. Figure 9 It can be seen that the treatment did not significantly increase the mRNA expression of browning markers in inguinal white adipose tissue; however, all browning marker genes in the experimental drug treatment group showed an increasing trend. TNFα was not increased compared to placebo. It is speculated that the rapid evaporation of ethanol on the skin in mice may lead to insufficient time for osmotic enhancement, resulting in insufficient salbutamol hemisulfate levels in white adipose tissue, and that less volatile osmotic enhancers improve in vivo delivery.

[0203] Example 11

[0204] In vitro permeability assay of salbutamol hemisulfate from aqueous DMSO formulation

[0205] The permeability of salbutamol hemisulfate from a 2.5% (w / w) gel (by solution weight) based on a 50 / 50 (% w / w) mixture of water and dimethyl sulfoxide was evaluated over 24 hours through a 400 µm thick layer of exfoliated human abdominal skin. The gel of 2.5% (w / w) salbutamol hemisulfate with a 50 / 50 (% w / w) mixture of water and DMSO as the solvent matrix was prepared by gelation with an additional 4% (w / w) HEC 250 (by total solution weight). Samples were taken at 5 hours and 24 hours.

[0206] Figure 10 Displayed per cm after 5 hours and 24 hours 2 The amount of salbutamol hemisulfate permeating through the skin of the human abdomen. From Figure 10 It can be seen that the concentration reached 212.2±84.4µg / cm after 24 hours. 2 The high permeability of salbutamol hemisulfate.

[0207] Compared to an ethanol-based aqueous formulation, this aqueous DMSO solvent system exhibited nearly double the permeability when the salbutamol hemisulfate concentration was normalized. Therefore, it is hypothesized that formulations based on a binary mixture of water and DMSO can provide increased permeability.

[0208] Example 12

[0209] Determination of the solubility of salmeterol sineproterone acetate in several solvents

[0210] As described above, the solubility of salmeterol sinemetidine in water, ethanol, propylene glycol, dimethyl sulfoxide, methanol, and Transcutol® was tested. The results are listed in Table 8 below.

[0211] Table 8: Solubility of salmeterol sinemetidine in water, ethanol, propylene glycol, dimethyl sulfoxide, methanol, and Transcutol®. Data are presented as n=1.

[0212]

[0213] As can be seen from Table 8, the solubility is greater than 50 mg / mL for dimethyl sulfoxide, methanol and Transcutol®.

[0214] Example 13

[0215] In vitro permeability of salmeterol oxadixate determined in a Franz diffusion cell

[0216] As described in Example 2, the in vitro permeability of salmeterol sineproterone acetate from a 5% (w / w) binary mixture (50 / 50 (%w / w)) of ethanol (EtOH) and dimethyl sulfoxide (DMSO) across mouse skin over 24 hours was determined in a Franz diffusion cell. Samples were taken at 1, 2, 4, 6, 8, 10, 12, and 24 hours and analyzed by HPLC using a fluorescence detector. The results are summarized in Table 9 below.

[0217] Table 9: Compositions of salmeterol sinemetidine derived from a binary mixture of ethanol and dimethyl sulfoxide (DMSO), the resulting fluxes, and the extrapolated hysteresis times tested in permeation experiments. Data are expressed as mean ± SD, n = 6.

[0218]

[0219] Figure 11 The permeation time curves of salmeterol sinemetidine from the binary mixture of ethanol and dimethyl sulfoxide (DMSO) tested are shown. (From Table 9 and...) Figure 11 It can be seen that the formulation exhibits good penetration and flux after 24 hours.

[0220] Example 14

[0221] Determination of the solubility of fennotefuran hydrobromide in several solvents

[0222] As described above, the solubility of fenoterol hydrobromide in water, ethanol, propylene glycol, dimethyl sulfoxide, methanol, and Transcutol® was tested. The results are listed in Table 10 below.

[0223] Table 10: Solubility of fenoterol hydrobromide in dimethyl sulfoxide, ethanol, methanol, propylene glycol, Transcutol®, and water. Data are presented as n=1.

[0224]

[0225] As can be seen from Table 10, the solubility is greater than 50 mg / mL for dimethyl sulfoxide, ethanol, methanol, propylene glycol, Transcutol® and water.

[0226] Example 15

[0227] In vitro permeability of fennotefurano hydrobromide was determined in a Franz diffusion cell.

[0228] As described in Example 2, the in vitro permeability of fennotefuranohydrobromide from 5% (w / w) binary mixtures (50 / 50 and 90 / 10 (w / w)) of ethanol and dimethyl sulfoxide (DMSO) across mouse skin over 24 hours was determined in a Franz diffusion cell. Samples were taken at 1, 2, 4, 6, 8, 10, 12, and 24 hours and analyzed by HPLC using a fluorescence detector. The results are summarized in Table 11 below.

[0229] Table 11: Composition, flux, and extrapolated lag time of fennoteulobromate from a binary mixture of ethanol and dimethyl sulfoxide (DMSO) tested in permeation experiments. Data are expressed as mean ± SD, n=5.

[0230]

[0231] Figure 12 The permeation time curves of salmeterol sinemetidine from the binary mixture of ethanol and dimethyl sulfoxide (DMSO) tested are shown. (From Table 11 and...) Figure 12 It can be seen that both formulations showed good penetration and flux after 24 hours.

[0232] Example 16

[0233] In vitro permeability of compounds derived from ternary mixtures was determined in a Franz diffusion cell.

[0234] As described in Example 2, the in vitro permeability of a ternary mixture (30 / 35 / 35, %w / w / w) of salbutamol hemisulfate, salmeterol sinetetroxide, formoterol fumarate dihydrate, and fenotrol hydrobromide, based on 3% (w / w) salbutamol, through approximately 600 µm of mouse skin over 24 hours was determined in a Franz diffusion cell. The skin surface temperature was maintained at 37 °C. Otherwise, the experiment was performed as described above. Samples were taken at 0, 1, 2, 4, 6, 8, 12, and 24 hours and analyzed by HPLC using a fluorescence detector. The results are summarized in Table 12 below.

[0235] Table 12: Composition, flux, and extrapolated lag time of a ternary mixture (30 / 35 / 35, %w / w / w) of dimethyl sulfoxide (DMSO), ethanol, and water tested in the permeation experiment.

[0236]

[0237] Figure 13 The permeation time curves of salmeterol sineproterone acetate from the tested ternary mixture of dimethyl sulfoxide (DMSO), ethanol, and water are shown. (From Table 12 and...) Figure 13It can be seen that the ternary formulation provides good penetration and flux after 24 hours.

[0238] Example 17

[0239] In vitro permeability of formoterol through mouse skin was determined in a Franz diffusion cell.

[0240] As described in Example 2, the in vitro permeability of formoterol from a mixture of DMSO and ethanol through the lateral ventral skin of mice over 24 hours was determined in a Franz diffusion cell using an unlimited dose experiment (as defined in OECD Environmental Health and Safety Publications Series Testing and Evaluation, 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).

[0241] Mouse lateral ventral skin membranes were prepared by removing skin from mice and trimming the fur to 0.5 mm. Full-thickness lateral ventral skin membranes were used, with a thickness of approximately 600 µm.

[0242] Solutions of 5 wt.-% formoterol fumarate dihydrate (w / w, equivalent to 4.1% free formoterol base) or 1 wt.-% formoterol fumarate dihydrate (w / w, equivalent to 0.82% free formoterol base) in a DMSO and ethanol mixture (50:50 or 10:90, w / w) were prepared by dissolving formoterol fumarate dihydrate in DMSO and adding ethanol. 200 µl of the formulation containing formoterol was placed in the donor chamber. Samples were taken after 24 hours and analyzed by HPLC using a fluorescence detector. The results are summarized in Table 13 below.

[0243] Table 13: Composition, flux, and extrapolated lag time of formoterol fumarate dihydrate from a binary mixture of dimethyl sulfoxide (DMSO) and ethanol (EtOH) tested in an unlimited-dose permeation experiment. Data are expressed as mean ± SD, n=6.

[0244]

[0245] As can be seen from Table 13, the formulation showed good penetration and flux after 24 hours.

[0246] Example 18

[0247] In vitro permeability of salbutamol through mouse skin determined in a Franz diffusion cell.

[0248] As described in Example 17, the in vitro permeability of salbutamol hemisulfate from a mixture of binary and ternary solvents through mouse skin over 24 hours was determined in a Franz diffusion cell. Samples were taken after 24 hours and analyzed by HPLC using a fluorescence detector. Table 14 below summarizes the composition and results.

[0249] Table 14: Composition, flux, and extrapolated lag time of salbutamol hemisulfate from a mixture of dimethyl sulfoxide (DMSO), ethanol (EtOH), glycerol, water, and urea solution (20% (w / w) aqueous solution) tested in an unlimited dose permeation experiment. Data are expressed as mean ± SD, n=6.

[0250]

[0251] As can be seen from Table 14, the formulation showed good penetration and flux after 24 hours.

[0252] Example 19

[0253] In vitro permeability assay of salbutamol from gelled DMSO formulation

[0254] As described in Example 17, the permeability of salbutamol hemisulfate through full-thickness rat lateral ventral skin over 24 hours in 2% or 5% (w / w) gels (by solution weight) of a 30 / 35 / 35% (w / w / w) ternary mixture matrix based on dimethyl sulfoxide, ethanol, and water was evaluated in a Franz diffusion cell at an unlimited dose. The gels were prepared by gelling the 30 / 35 / 35% (w / w / w) ternary mixture of dimethyl sulfoxide, ethanol, and water with an additional 2% or 5% (w / w) of highly substituted hydroxypropyl cellulose (HPC-H) (by total solution weight). The samples used for comparison were “gelling agent-free” 30 / 35 / 35% (w / w / w) ternary mixtures of dimethyl sulfoxide, ethanol, and water. Samples were taken after 24 hours and analyzed by HPLC using a fluorescence detector. Table 15 below summarizes the composition and results.

[0255] Table 15: Composition, resulting flux, and extrapolated lag time of 2% (w / w) salbutamol hemisulfate from a 30 / 35 / 35% (w / w / w) mixture of dimethyl sulfoxide (DMSO), ethanol (EtOH), and water with and without 2% and 5% (w / w) hydroxypropyl cellulose H grade (HPC H) as a gelling agent at unlimited dose. Data are expressed as mean ± SD, n=6.

[0256]

[0257] As shown in Table 15, the formulation exhibited good penetration and flux after 24 hours. There was no significant difference in penetration and flux over 24 hours with varying amounts of gelling agent.

[0258] Example 20

[0259] In vitro permeability of salbutamol through rat and human skin was determined in a Franz diffusion cell.

[0260] As described in Example 17, the in vitro permeability of salbutamol hemisulfate from a 30 / 35 / 35% (w / w / w) ternary mixture of dimethyl sulfoxide, ethanol, and water through human and mouse skin was determined in a Franz diffusion cell over 24 hours. Human abdominal skin membranes were prepared by removing subcutaneous adipose tissue with a scalpel and then normalizing to a thickness of 400 µm using a dermabrasion scalpel (Aesculap GA 643). Samples were taken after 24 hours and analyzed by HPLC using a fluorescence detector. Table 16 below summarizes the composition and results.

[0261] Table 16: Composition, flux, and extrapolated lag time of a 30 / 35 / 35% (w / w / w) mixture of 5% (w / w) salbutamol hemisulfate (API) in dimethyl sulfoxide (DMSO), ethanol (EtOH), and water at unlimited dose. Data are expressed as mean ± SD, n=6.

[0262]

[0263] As can be seen from Table 16, the formulation exhibits good penetration and flux over 24 hours.

[0264] Example 21

[0265] In vitro permeability of compounds derived from ternary mixtures through mouse skin was determined in a Franz diffusion cell.

[0266] As described in Example 16, the in vitro permeability of a 72 µM solution of salbutamol hemisulfate, salmeterol sinecurate, formoterol fumarate dihydrate, and fenotrol hydrobromide from a ternary mixture (30 / 35 / 35, %w / w / w) of DMSO, ethanol, and water through mouse skin over 24 hours was determined in a Franz diffusion cell at unlimited doses. Samples were taken at 0, 1, 2, 4, 6, 8, 12, and 24 hours and analyzed by HPLC using a fluorescence detector.

[0267] Figure 14 The permeation time curves of salbutamol hemisulfate, salmeterol sine, formoterol fumarate dihydrate, and fenotrol hydrobromide in a 72 μM solution of a mixture of dimethyl sulfoxide (DMSO), ethanol (EtOH), and water (30 / 35 / 35 w / w / w) are shown. Figure 14 It can be seen that the ternary formulation provides good penetration after 24 hours.

[0268] from Figure 14 It can be seen that the formulation exhibits good penetration and flux after 24 hours.

[0269] Example 22

[0270] The in vitro permeability of salbutamol, salmeterol, formoterol, and fenoterol through mouse and human skin was determined in a Franz diffusion cell.

[0271] As described in Example 17, the in vitro permeability of a 72 μM solution of salbutamol hemisulfate, salmeterol sine benzoate, formoterol fumarate dihydrate, and fenotrol hydrobromide in a 30 / 35 / 35% (w / w / w) ternary mixture of dimethyl sulfoxide, ethanol, and water was determined over 24 hours in a Franz diffusion cell. Samples were taken after 24 hours and analyzed by HPLC using a fluorescence detector. Table 17 below summarizes the compositions and results.

[0272] Table 17: Composition, flux, and extrapolated lag time of 72 μM salbutamol hemisulfate, salmeterol sine, formoterol fumarate dihydrate, and fenotrol hydrobromide (API) solutions in a mixture of dimethyl sulfoxide (DMSO), ethanol (EtOH), and water (30 / 35 / 35 w / w / w) via rat skin at unlimited doses. Data are expressed as mean ± SD, n=6.

[0273]

[0274] As can be seen from Table 17, the formulation showed good penetration and flux after 24 hours.

[0275] In summary, the results indicate that dimethyl sulfoxide allows for transdermal administration of formoterol, salbutamol, salmeterol, and fenoterosterol. Further results demonstrate that efficient transdermal delivery of salbutamol and clenbuterol can be achieved through the application of aqueous and ethanol formulations.

Claims

1. A transdermal administration composition comprising a compound selected from the group consisting of formoterol, salbutamol, salmeterol, fenoterol, mixtures thereof, or salts thereof, and dimethyl sulfoxide.

2. The transdermal application composition according to claim 1, wherein, The composition comprises a compound selected from formoterol, salbutamol, salmeterol, fenoterol, or a salt thereof, and includes: - Dimethyl sulfoxide ranging from ≥5 wt.% to ≤100 wt.% and optionally - The range is from ≥5 wt.% to ≤95 wt.% of water, monohydric alcohol, glycerol, glycol, polyethylene glycol, ethylene glycol ether, or mixtures thereof. Where wt.-% is based on a total solvent weight of 100wt.-%.

3. The composition according to claim 2, wherein, Based on a total solvent weight of 100 wt.%, the composition comprises ≥5 wt.% to ≤55 wt.% of dimethyl sulfoxide and ≥45 wt.% to ≤95 wt.% of ethanol.

4. The composition according to claim 2, wherein, Based on a total solvent weight of 100 wt.%, the composition comprises ≥5 wt.% to ≤55 wt.% of dimethyl sulfoxide and ≥45 wt.% to ≤95 wt.% of water.

5. A transdermal application composition, wherein, The composition comprises a compound selected from the group consisting of salbutamol, clenbuterol, or salts thereof, and contains at least one of water or a monohydric alcohol.

6. The composition according to claim 5, wherein, The composition comprises salbutamol or a salt thereof, and includes: - Monohydric alcohols ranging from ≥10 wt.% to ≤95 wt.% and - The range is ≥5 wt.% to ≤90 wt.% of water, isosorbide dimethyl ether, isopropyl myristate, glycerin, glycol, polyethylene glycol, ethylene glycol ether or mixtures thereof; Where wt.-% is based on a total solvent weight of 100wt.-%.

7. The composition according to claim 5, wherein, The composition comprises clenbuterol or a salt thereof, and includes: - Water ranging from ≥45 vol.% to ≤95 vol.% and isosorbide dimethyl ether, isopropyl myristate, glycerin, glycol, polyethylene glycol, ethylene glycol ether, or mixtures thereof ranging from ≥5 vol.% to ≤55 vol.%; or - Monohydric alcohols ranging from ≥50 vol.-% to ≤90 vol.-% and water, isopropyl myristate, dimethyl isosorbide, glycerol, glycol, polyethylene glycol, ethylene glycol ethers or mixtures thereof ranging from ≥10 vol.-% to ≤50 vol.-%; Where vol.-% is based on the total solvent volume 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 gels, ointments, creams, foams, lotions, pastes, solutions, sprays or plasters, and is preferably in the form of gels or sprays.

9. Use of the composition according to any one of claims 1 to 8 in the manufacture of a medicament for treating obesity or lipedema or for providing fat breakdown.

10. Use of the composition according to any one of claims 1 to 8 in a cosmetic method of reducing the amount of subcutaneous adipose tissue in a subject by topical application of the composition.

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