Topical compositions containing tacrolimus

Abstract

The present invention relates to a composition for topical application comprising: a first discontinuous phase comprising a first oil and tacrolimus; a second discontinuous phase comprising a second oil; and a continuous aqueous phase; wherein the first oil is different from the second oil.

Description

[0001] This invention relates to a topical composition. Specifically, this invention relates to a topical composition comprising tacrolimus, which has improved skin permeability, stability, and patient compliance.

[0002] Atopic dermatitis is a common, chronic, relapsing inflammatory skin disease. The exact cause of the disease is controversial, but it is characterized by eczematous lesions, dry skin, and intense itching (pruritus). There is also strong evidence that the prevalence of atopic dermatitis has been increasing in recent years. Symptoms can range from mild to severe and can subsequently impair quality of life.

[0003] Current treatment options involve using a moisturizing cream, followed by other therapies in incremental increments. Topical application of mild corticosteroids such as hydrocortisone acetate is usually the next step, with increasingly potent corticosteroids used only as needed. However, there are several potential drawbacks associated with topical corticosteroids. These drawbacks (especially those of more potent corticosteroids) can include thinning of the skin, rapid tolerance, and rebound effects. Due to these and other potential side effects, the use of corticosteroids on the facial area is not recommended, even though atopic dermatitis that develops on the face can indeed have the most detrimental impact on a patient's quality of life.

[0004] Recently, topical macrolide immunosuppressants have been used to treat moderate to severe atopic dermatitis. Unlike corticosteroids, these compounds are generally well-tolerated and therefore can be used on the facial area.

[0005] Tacrolimus belongs to the macrolide immunosuppressant class of ascomycins. Mechanistically, tacrolimus does not directly inhibit cellular processes, but rather forms a complex with the specific cytoplasmic protein cis-trans-prolyl isomerase FKBP12 (also known as the FK506-binding protein, a member of the immunoaffinity protein family). It is this resulting complex that subsequently inhibits the inflammatory response.

[0006] In the presence of tacrolimus, the derived complex inhibits calcineurin, preventing the dephosphorylation of nuclear factor-AT (NF-AT) in activated T cells, thereby reducing the activity of genes encoding IL-2 and related cytokines in these cells.

[0007] While tacrolimus offers a clear advantage over corticosteroids in terms of tolerability, formulating it for topical application has proven challenging. The first difficulty is obtaining a chemically and physically stable product. The second difficulty is managing to deliver sufficient amounts of the active agent across the epidermal barrier to make it effective. Tacrolimus penetration is hampered by its molecular size (804 Da), which is significantly larger than the generally accepted easily penetrating size of 500 Da. Although disease conditions impair the skin barrier to some extent, strategies for improving tacrolimus penetration into the skin remain necessary.

[0008] Commercially, 0.03% and 0.1% anhydrous tacrolimus ointments (Protopic, supplied by Astellas) have been released. TM Both products contain a polar solvent (propylene carbonate) dispersed in a medium of liquid paraffin, white soft paraffin, hard paraffin, and beeswax. Propylene carbonate acts as a carrier and penetration enhancer for the drug. While the solvent is used to deliver tacrolimus to deeper layers of the skin, it further disrupts the already damaged skin barrier, potentially causing skin irritation. The lack of water limits the possibility of chemical degradation due to hydrolysis or pH incompatibility, and the occlusive nature of the bulk excipients creates a high degree of occlusion, facilitating the penetration of the active ingredient. However, as with most ointments, the lack of water and the presence of paraffin and wax components result in a very poor aesthetic profile (SEWolverton, Comprehensive Dermatologic Drug Therapy, 3rd ed., (2012), p13). This could severely limit patient compliance.

[0009] Several non-ointment tacrolimus formulations are known. For example, US 2005 / 0249757 discloses a pharmaceutical cream composition comprising a macrolide immunosuppressant, one or more cream-forming agents, and an effective amount of one or more skin penetration enhancers.

[0010] US 2012 / 0184511 discloses a liquid microemulsion of a macrolide immunosuppressant. The composition comprises a high level of surfactant and a polar, water-miscible liquid as a penetration enhancer.

[0011] EP 2596788 discloses an oil-in-water cream composition comprising tacrolimus, wherein diisopropyl sebacate may be used in the oil phase. The long-term stability of the composition at high temperatures (e.g., 40°C) is not disclosed.

[0012] Although known non-ointment formulations may have better effects than Protopic TMWhile these formulations offer superior aesthetic appeal, they often lack the chemical stability of the active ingredients. Furthermore, to achieve adequate penetration of the active ingredient, they typically require a high concentration of surfactants and / or polar, water-miscible liquids. This is exemplified by US 2005 / 0249757, US 2012 / 0184511, and EP 2596788. Due to the high content of these components, these non-ointment formulations often irritate the skin.

[0013] Therefore, one object of the present invention is to provide a formulation capable of delivering tacrolimus into the skin that has better aesthetic properties than prior art formulations, while being as gentle as possible on the skin barrier. In other words, one object of the present invention is to provide a tacrolimus formulation with better patient compliance than prior art formulations.

[0014] The alternative and / or additional purpose is to provide a tacrolimus formulation that has better skin penetration than existing formulations or at least provides a commercially useful alternative.

[0015] The alternative and / or additional purpose is to provide a formulation that exhibits improved chemical stability of tacrolimus compared to existing formulations.

[0016] According to a first aspect, the present invention provides a composition for topical application, comprising:

[0017] The first discontinuous phase contains the first oil and tacrolimus;

[0018] The second discontinuous phase containing the second oil; and

[0019] Continuous aqueous phase;

[0020] The first oil is different from the second oil.

[0021] The inventors have discovered that, unlike prior art non-ointment formulations, the compositions disclosed herein do not require high levels of polar, water-miscible liquids or high levels of surfactants to achieve good skin penetration. This presents several advantages. First, the composition is less irritating to the skin, providing improved patient compliance. This is particularly advantageous in cases where patients already have inflammatory skin conditions. Second, as tested in a porcine inflammation model, the composition exhibits excellent efficacy in vivo. Furthermore, surprisingly, tacrolimus possesses higher chemical stability than many existing formulations, especially non-ointment formulations. This will be explained in more detail below.

[0022] The invention will now be described further. In the following paragraphs, different aspects / implementations of the invention are defined in more detail. Unless expressly indicated to the contrary, each aspect / implementation so defined may be combined with any other aspect / implementation. In particular, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

[0023] This invention provides compositions for topical application. A composition for topical application is defined herein as a composition suitable for direct application to a portion of the human or animal body. Preferably, the composition is suitable for direct application to the skin, such as the face, scalp, feet, limbs, or torso.

[0024] The compositions of the present invention comprise a first discontinuous phase, a second discontinuous phase, and a continuous aqueous phase. In other words, the composition comprises a dispersion of the first and second discontinuous phases in a continuous aqueous phase. As used herein, the term "discontinuous phase" refers to a plurality of discrete regions forming oil droplets of that particular oil phase. It is not used to refer to a single oil droplet. These phases are physically and chemically distinct. It should be understood that the first discontinuous phase is not dispersed in the second discontinuous phase, and vice versa. In other words, the composition does not contain complex internal phases, such as those disclosed in WO 2005 / 082515. Figure 1 A simplified schematic diagram illustrating the structure of the composition according to the invention is provided. The inclusion of a continuous aqueous phase in the composition of the invention allows it to be provided as a lotion or cream, rather than an ointment. Therefore, compared to prior art ointments, the composition of the invention has improved aesthetic characteristics, thereby improving patient compliance. Preferably, the composition is in the form of a lotion or cream.

[0025] The first and second discontinuous phases comprise a first oil and a second oil, respectively. The first oil differs from the second oil, i.e., it is chemically different from the second oil. Preferably, the first oil and / or the second oil are pharmaceutically acceptable oils. Examples of oils that can be used in this invention include: coconut oil, squalane, isopropyl myristate, isopropyl isostearate, isopropyl palmitate, modified triglycerides, caprylic / capric triglycerides, fractionated triglycerides, tridecanoic acid esters, trihexanoic acid esters, tricaprylic acid esters, tricaprylic acid / capric acid esters, tricaprylic acid / capric acid esters, tricaprylic acid / capric acid / laurate, tricaprylic acid / capric acid / linoleic acid esters, tricaprylic acid / capric acid / stearate, trilaurylic acid esters, trilinoleic acid esters, trilinolenic acid esters, trilinolenic acid trilinolenic acid esters, trioleic acid tri(undecanoic acid) esters, linoleic acid esters, saturated polyethylene glycol-modified glycerides, and oils primarily containing C8-C. 12The composition comprises the synthesis of fatty acid chains including medium-chain triglycerides, long-chain triglycerides, modified triglycerides, fractionated triglycerides, isostearyl isostearate, diisopropyl adipate, mineral oil, polydimethylsiloxane, cyclodimethylsiloxane, hydrogenated polyisobutylene, heptamethylnonane, and mixtures thereof. Preferably, the composition does not contain a wax component that is solid at 25°C.

[0026] It should be understood that the first oil and the second oil form two distinct discontinuous phases. In some exemplary embodiments, the first oil and the second oil are substantially immiscible. However, it is also possible to form two distinct discontinuous phases from two miscible or at least partially miscible oils. This can be achieved by dispersing the first oil and the second oil separately in a continuous phase without pre-mixing the oils. Preferably, the first discontinuous phase does not contain the second oil, and the second discontinuous phase does not contain the first oil.

[0027] The first discontinuous phase comprises tacrolimus. It should be understood that the term "tacrolimus" includes anhydrous tacrolimus and tacrolimus hydrate. Tacrolimus is a macrolide, a natural product belonging to the class of polyketide compounds. It is an immunosuppressant and is known to treat skin conditions. The source of tacrolimus used in this invention is preferably anhydrous tacrolimus, although it will be appreciated that other sources of tacrolimus, such as tacrolimus hydrate, can be used. The amount of tacrolimus introduced into the compositions based on this document is in the anhydrous form of tacrolimus. Adjusting the amounts used in the preparation of the compositions according to the source used to provide the desired amount in the final composition will be within the capabilities of those skilled in the art.

[0028] Preferably, the first oil comprises diisopropyl adipate. As shown in the examples, diisopropyl adipate, diethyl sebacate, and dibutyl adipate all exhibit similar solubility profiles for tacrolimus. However, the inclusion of diisopropyl adipate as part of the first oil surprisingly results in significantly higher tacrolimus chemical stability than when diethyl sebacate or dibutyl adipate is included. Not wishing to be theoretically limited, it is thought that the two ester bonds present in all three oils are more protected in diisopropyl adipate due to steric hindrance caused by the proximity / conformation of the methyl group near the bond. It is believed that degradation of this bond could subsequently affect the stability of the active agent, thus explaining the increased stability when diisopropyl adipate is included. The first discontinuous phase advantageously comprises an oil with high solubility for tacrolimus because this allows for limiting the amount of the first oil for a given overall tacrolimus concentration. Because the compositions disclosed herein contain two discontinuous phases, using an oil with poor solubility for tacrolimus in the first discontinuous phase will result in a high total oil content in the formulation, leading to an unpleasant, greasy appearance. The amount of the first oil has a significant impact on the total oil level of the formulation in which the first and second oils are used in a preferred ratio. Preferably, the composition contains less than 1% by weight of diethyl sebacate and / or less than 1% by weight of dibutyl adipate. Preferably, the composition does not contain diethyl sebacate and / or does not contain dibutyl adipate.

[0029] Another oil with good solubility for tacrolimus is ethylene glycol salicylate. This oil is frequently used in existing technology systems. However, the inventors have found that the physical stability of the compositions of the present invention is significantly higher than that of ethylene glycol salicylate when diisopropyl adipate is used. Not wishing to be limited by theory, it is considered that diisopropyl adipate has a higher logP and therefore less "polarity" than ethylene glycol salicylate, resulting in a lower tendency to partition into the aqueous phase. Preferably, the composition comprises less than 1% by weight of ethylene glycol salicylate. More preferably, the composition does not contain ethylene glycol salicylate.

[0030] Preferably, in addition to diisopropyl adipate, the first oil comprises caprylic / capric triglyceride in combination with isopropyl myristate and / or isopropyl palmitate. Alternatively, in addition to diisopropyl adipate, the first oil may comprise castor oil and / or squalane. These oils increase the viscosity of diisopropyl adipate, thereby improving its processability. They can also improve the physical stability of the composition, especially when using low amounts of surfactant. Preferably, the first oil comprises diisopropyl adipate and / or isopropyl myristate and / or caprylic / capric triglyceride, and / or consists of diisopropyl adipate and / or isopropyl myristate and / or caprylic / capric triglyceride.

[0031] The inventors have also discovered that caprylic / capric triglyceride and / or isopropyl myristate have reasonable solubility characteristics for tacrolimus and provide emollient properties. Therefore, the first oil is particularly preferred to comprise, or consist of, a combination of diisopropyl adipate and caprylic / capric triglyceride and / or isopropyl myristate.

[0032] Preferably, based on the weight of the first oil, the first oil comprises 10% to 40% by weight of diisopropyl adipate and / or 10% to 40% by weight of isopropyl myristate and / or 2% to 80% by weight of caprylic / capric triglyceride. More preferably, based on the weight of the first oil, the first oil comprises 20% to 30% by weight of diisopropyl adipate and / or 20% to 30% by weight of isopropyl myristate and / or 40% to 60% by weight of caprylic / capric triglyceride. Preferably, the first oil consists of diisopropyl adipate and / or isopropyl myristate and / or caprylic / capric triglyceride, preferably in the amounts described above.

[0033] Preferably, the second oil comprises or is composed of an emollient oil; more preferably, it comprises or is composed of an oil selected from or composed of oils including mineral oil, polydimethylsiloxane, cyclodimethylsiloxane, hydrogenated polyisobutane, heptamethylnonane, and mixtures of two or more thereof. Most preferably, the second oil comprises or is composed of mineral oil. The second oil contributes to the emollient properties of the formulation and provides a blocking layer to promote the diffusion of tacrolimus. Therefore, the diffusion properties of the composition are enhanced. Preferably, tacrolimus is substantially insoluble in the second oil. In other words, the second oil is preferably used as a non-solvent oil phase.

[0034] Preferably, the first discontinuous phase comprises a first oil in an amount of at least 90% by weight, more preferably at least 95% by weight, even more preferably at least 97.5% by weight, and most preferably at least 99% by weight. Preferably, the first discontinuous phase comprises a first oil in an amount of up to 99.75% by weight. Preferably, the first discontinuous phase comprises tacrolimus in an amount of 0.025% to 10% by weight, more preferably 0.05% to 5% by weight, even more preferably 0.25% to 2.5% by weight, and most preferably about 0.5% by weight. Preferably, the first discontinuous phase consists of a first oil and tacrolimus, more preferably in the amounts described above.

[0035] Preferably, the second discontinuous phase contains at least 90% by weight of the second continuous phase, more preferably at least 95% by weight, even more preferably at least 99% by weight, and most preferably the second discontinuous phase is composed of the second oil.

[0036] Preferably, tacrolimus is predominantly in the first discontinuous phase. Predominantly in the first discontinuous phase means at least 90% by weight of tacrolimus in said discontinuous phase, preferably at least 95% by weight, more preferably at least 99% by weight. This property of the composition results from the use of one or more oils with good solubility characteristics for tacrolimus in the first discontinuous phase, while a non-solvent oil is used in the second discontinuous phase. Preferably, tacrolimus is substantially soluble in the first oil and / or substantially insoluble in the second oil. "Substantially soluble" means a solubility of at least 0.1% by weight, preferably at least 0.2% by weight, at 25°C. "Substantially insoluble" means a solubility of less than 0.1% by weight, preferably less than 0.05% by weight, at 25°C.

[0037] As a result of the selection of the oil phase, the continuous aqueous phase of the present invention does not need to contain high amounts of polar water-miscible liquids, such as alcohols and glycols, to achieve effective skin penetration or solubilization of tacrolimus. These liquids are typically required as penetration enhancers in conventional tacrolimus formulations. Preferably, the compositions of the present invention contain less than 10% by weight of C1-C4 alcohols, polyethylene glycol, ethylene glycol, propylene glycol, butylene glycol, pentanediol, glycerin, diethylene glycol monoethyl ether, propylene carbonate, or mixtures thereof. In other words, these components are not required to be present in the composition, but when one or more of these components are present, the total amount of these components is less than 10% by weight of the composition. More preferably, the composition contains less than 5% by weight of C1-C4 alcohols, polyethylene glycol, ethylene glycol, propylene glycol, butylene glycol, glycerin, diethylene glycol monoethyl ether, propylene carbonate, or mixtures thereof, even more preferably less than 2% by weight, more preferably less than 1% by weight, more preferably less than 0.5% by weight, more preferably less than 0.2% by weight, and most preferably less than 0.1% by weight. Preferably, these compounds are not present in the composition.

[0038] By using the first and second discontinuous phases described herein, it is facilitated to use only low levels of polar water-miscible liquids (or none at all) and to minimize skin irritation caused by the composition. This is particularly advantageous in cases where the patient already has inflamed skin. The fact that such low levels of polar water-miscible liquids can be used is surprising, as tacrolimus formulations, especially non-ointment formulations, typically require high concentrations of these solvents as penetration enhancers.

[0039] Preferably, the compositions of the present invention comprise a surfactant. The surfactant can be incorporated into one or more discontinuous phases and / or a continuous aqueous phase. Suitable surfactants include alkyl polyethylene glycol ethers, alkyl polyethylene glycol esters, ethoxylated alcohols, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, ionic or nonionic surfactants, hydrogenated castor oil / polyoxyethylene glycol adducts containing 25 to 60 ethoxy groups, castor oil / polyoxyethylene glycol adducts containing 25 to 45 ethoxy groups, sorbitan fatty acid esters (e.g., Span 20 or Span 80), block copolymers of ethylene oxide and propylene oxide (e.g., Pluronic L121 or Pluronic F68), or mixtures thereof. Alternatively, polymeric surfactants based on modified crosslinked copolymers of acrylic acid, such as Pemulen Tr-1 and Pemulen Tr-2 (Lubrizol Corporation), can be used. It should be understood that other suitable surfactants can be used.

[0040] Preferably, the composition comprises two or more surfactants, such as a first surfactant incorporated into the first and / or second discontinuous phase, and a different second surfactant incorporated into the continuous aqueous phase. The first and second surfactants are preferably selected from the list above. The first surfactant is readily soluble in or dispersed in the first and / or second oil, and is preferably selected from Laureth-4 (polyoxyethylene (4) monododecyl ether), polysorbate 80, Span 80, and mixtures of two or more thereof. The second surfactant is readily soluble in or dispersed in the continuous aqueous phase, and is preferably selected from polysorbate 20, Pluronic L121, Pluronic F68, PEG-40 hydrogenated castor oil, Span 20, and mixtures of two or more thereof. Most preferably, the first surfactant is Laureth-4 (polyoxyethylene (4) monododecyl ether), and the second surfactant is polysorbate 20.

[0041] Alternatively, the composition may contain only one surfactant. In this embodiment, the surfactant is preferably a polymeric surfactant based on a modified crosslinked copolymer of acrylic acid. Suitable surfactants are Pemulen Tr-1 and Pemulen Tr-2 (Lubrizol Corporation). Although these surfactants dissolve in the first and / or second discontinuous phases during composition preparation, the surfactants are partitioned between the discontinuous and continuous aqueous phases in the resulting composition.

[0042] Preferably, the total surfactant content of the compositions disclosed herein is less than 5% by weight of the composition, more preferably less than 2.5% by weight, even more preferably less than 2% by weight, even more preferably less than 1% by weight, and most preferably less than 0.5% by weight. Preferably, the total surfactant content is at least 0.1% by weight. As used herein, the term "total surfactant content" refers to the sum of the weight percentages of all surfactants present in the composition. As described above, surfactants can be incorporated into one or more discontinuous phases and / or continuous aqueous phases.

[0043] The use of the first and second discontinuous phases described herein facilitates the use of low levels of surfactant and minimizes skin irritation caused by the composition. This is particularly advantageous in cases where the patient already has inflamed skin. The fact that such low levels of surfactant can be used is surprising, as tacrolimus cream (non-ointment) formulations typically require very high levels of surfactant to act as penetration enhancers or allow for the formation of physically stable products.

[0044] Another advantage of using preferred low levels of polar water-miscible liquids and / or surfactants is that tacrolimus surprisingly exhibits higher chemical stability than in known formulations. Not wishing to be limited by theory, the inventors believe that polar water-miscible liquids and surfactants influence the partitioning of tacrolimus between the oil and aqueous phases. Tacrolimus is oil-soluble. However, the presence of polar water-miscible liquids and surfactants increases the compatibility of tacrolimus with the aqueous phase. The inventors have found that this can lead to significantly greater water exposure of tacrolimus, resulting in its decomposition. Conversely, in the compositions of the present invention, preferred low levels of polar water-miscible liquids and / or surfactants mean less aqueous exposure of tacrolimus, thus resulting in higher chemical stability.

[0045] Preferably, tacrolimus is chemically stable for at least 12 months at 5°C ± 3°C, as measured at 60% RH ± 5%. This stability was measured after storage in a sealed amber glass jar, which was then exposed to air. "Chemically stable" means 100% ± 5% as determined by HPLC relative to the measurement at t = 0.

[0046] Preferably, tacrolimus is chemically stable for at least 12 months at 25°C ± 2°C, as measured at 60% RH ± 5%. This stability was measured after storage in a sealed amber glass jar. The jar was sealed to air. Again, "chemically stable" means 100% ± 5% as determined by HPLC relative to the measurement at t = 0.

[0047] Preferably, tacrolimus is chemically stable for at least 6 months at 40°C ± 3°C. This stability was measured after storage in a sealed amber glass jar, which was then exposed to air. "Chemically stable" has the same meaning as described above.

[0048] Preferably, the composition comprises 0.005% to 2% tacrolimus by weight of the composition, more preferably 0.01% to 1% tacrolimus, even more preferably 0.03% to 0.5% tacrolimus, and most preferably about 0.1% tacrolimus. Advantageously, the present invention allows for tacrolimus loadings comparable to those of known ointment formulations while achieving improved aesthetics and patient compliance.

[0049] Preferably, the composition comprises 5 to 40% by weight of a first discontinuous phase, more preferably 10% to 30% by weight, more preferably 10% to 25% by weight, even more preferably 15% to 25% by weight, and most preferably about 20% by weight.

[0050] Preferably, the composition comprises 5% to 40% by weight of a second discontinuous phase, more preferably 10% to 30% by weight, more preferably 10% to 25% by weight, even more preferably 15% to 25% by weight, and most preferably about 20% by weight. Surprisingly, the permeability of the formulation is maximized when the amount of the second discontinuous phase is relatively low. This is demonstrated in the in vitro model in the examples.

[0051] Preferably, the first oil is present in an amount of 5% to 40% by weight of the composition, more preferably 10% to 30% by weight, even more preferably 10% to 25% by weight, even more preferably 15% to 25% by weight, and most preferably about 20% by weight. Preferably, the second oil is present in an amount of 5% to 40% by weight of the composition, more preferably 10% to 30% by weight, even more preferably 10% to 25% by weight, even more preferably 15% to 25% by weight, and most preferably about 20% by weight.

[0052] Preferably, the weight ratio of the second oil to the first oil in the composition is 1:3 to 3:1, more preferably 1:2 to 2:1, even more preferably 2:3 to 3:2, and most preferably about 1:1. As demonstrated in the examples, the inventors have found that the ratio of the second oil to the first oil should not be too high in order to maximize the penetration of the formulation. This is demonstrated in the in vitro models in the examples.

[0053] Preferably, the composition contains at least 10% by weight of water, more preferably at least 20% by weight, more preferably at least 30% by weight, even more preferably at least 40% by weight, and most preferably at least 50% by weight. Preferably, the composition contains at most 70% by weight of water. The excellent solubility of tacrolimus in the first discontinuous phase means that a relatively high water content can be used without causing excessive aqueous exposure of the active agent. The relatively high level of water makes the skin feel less oily, further improving patient compliance.

[0054] Preferably, the composition comprises at most two discontinuous phases. Preferably, the composition does not contain any oil other than the first and second oils described herein.

[0055] Preferably, the composition further comprises at least one pharmaceutically acceptable excipient.

[0056] Preferably, the compositions of the present invention have an average droplet diameter of 1 to 30 μm, more preferably 1 to 20 μm (i.e., the diameter of the discontinuous phase droplets). This droplet diameter contrasts with microemulsions, which typically have an average droplet diameter of 1 to 100 nm. In the context of the present invention, the droplet diameter is measured using a Malvern Mastersizer 2000 laser diffraction particle size analyzer.

[0057] The compositions of the present invention can be in the form of macroemulsions. Macroemulsions are known in the art and differ from microemulsions in that they have a larger average droplet diameter. Furthermore, unlike microemulsions, macroemulsions are thermodynamically unstable. Due to this relative thermodynamic instability, macroemulsions are typically prepared in a different manner than microemulsions. For example, macroemulsion formation usually requires stirring / shearing, while microemulsions typically form effectively and spontaneously.

[0058] Alternatively, the compositions of the present invention may be in the form of a multi-microbubble dispersion. Multi-microbubble dispersions are known in the art and are disclosed in the following references of Sebba: "Biliquid Foams", J. Colloid and Interface Science, 40 (1972) 468-474 and "The Behaviour of Minute Oil Droplets Encapsulated in a Water Film", Colloid Polymer Sciences, 252 (1979) 392-396; Hicks "Investigating the Generation, Characterisation, and Structure of Biliquid Foams", PhD Thesis, University of Bristol, 2005; Crutchley "The Encapsulation of Oils and Oil Soluble Substances Within Polymer Films", PhD Thesis, The University of Leeds, 2006 and Lye and Stuckey, Colloid and Surfaces, 131 (1998) 119-136. Microbubbles are also disclosed in US-A-4,486,333 and WO 97 / 32559. A suitable method for preparing multi-microbubble dispersions is described in WO 03 / 064024.

[0059] Preferably, the composition is in the form of a lotion or cream. Lotions and creams in the context of this invention differ from, for example, shaving foam, which has a much higher surfactant content to produce the desired foam during use. The inventors have discovered that the use of two discontinuous phases as described herein can provide physically and chemically stable tacrolimus lotion or cream compositions with good patient tolerability and perceived aesthetics, good efficacy, and low irritation.

[0060] Preferably, the compositions of the present invention are dispersible in water. Preferably, the compositions of the present invention are dilute in water. This increases the flexibility of use of the invention, for example, by wetting the hair to improve the application of the composition through the hair to the scalp, or by rinsing the preparation from any local surface when desired or needed, or by easily removing the product by rinsing it from accidentally soiled clothing. These advantages improve the user experience and improve patient compliance.

[0061] Preferably, the pH of the composition is 3.5 to 6, more preferably 4 to 5.5, even more preferably 4.5 to 5.25, and most preferably 4.5 to 5. The inventors have found that controlling the pH of the composition within these limits improves the stability of tacrolimus and the composition as a whole. This is surprising because lower pH values ​​are routinely used in the prior art (see, for example, US 2011 / 0201639 and US 8574562). It should be understood that the pH can be adjusted to an appropriate value or range using any suitable acid or base. Advantageously and preferably, the pH of the composition can be stabilized by incorporating a suitable buffer solution into the continuous aqueous phase. Suitable buffer systems with pH within the specified range are familiar to those skilled in the art.

[0062] Preferably, as measured at 60% RH ± 5%, the composition is physically stable for at least 12 months at 5°C ± 3°C. Stability is measured after storage in a sealed amber glass jar. The jar is sealed to air. “Physically stable” means that the composition presents as a uniform cream with no significant rheological or appearance changes from t=0.

[0063] Preferably, as measured at 60% RH ± 5%, the composition is physically stable for at least 12 months at 25°C ± 2°C. Stability is measured after storage in a sealed amber glass jar. The jar is sealed to air. Similarly, "physically stable" means that the composition presents as a uniform cream with no significant rheological or appearance changes from t=0.

[0064] Preferably, as measured at 60% RH ± 5%, the composition is physically stable for at least 6 months at 40°C ± 3°C. Stability is measured after storage in a sealed amber glass jar. The jar is sealed to air. "Physically stable" has the same meaning as described above.

[0065] Preferably, the compositions of the present invention further comprise a gelling agent and / or a rheology modifier, such as a viscosity modifier. The gelling agent may be, for example, selected from alginate gum or salts thereof, guar gum, locust bean gum, xanthan gum, gum arabic, gelatin, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose or salts thereof, bentonite, magnesium aluminum silicate, "carbomer" (a salt of a cross-linked polymer of acrylic acid), or polyglycerol methacrylate or dispersions thereof in glycols. It should be understood that other suitable gelling agents may be used. Additionally, some gelling agents (e.g., carbomer) have been found to also act as chemical buffers, thereby preventing undesirable changes in the pH of the composition during storage and use. In the case of using a viscosity modifier, it is preferably a polymeric cellulose thickener. The inclusion of a gelling agent and / or a rheology modifier provides additional anti-emulsion stratification stability and ensures that the concentration of the active agent is uniform throughout the composition. The use of these components is described in WO97 / 32559. The choice of gelling / thickening agent also allows for control of formulation viscosity from easily pourable thin lotions to thick creams with significant flow resistance.

[0066] Preferably, the composition of the present invention comprises 0.05% to 5.0% by weight of a gelling agent, more preferably 0.1% to 2.0% by weight, and more preferably 0.2% to 1.0% by weight. In one embodiment of the present invention, the composition has a gel consistency.

[0067] The compositions of the present invention may also contain other additives, such as preservatives (e.g., to prevent microbial spoilage), buffers (to control pH and avoid instability and damage to the acidic shell of the skin), and antioxidants. When preservatives are used, their presence is preferably 0.5% to 1% by weight of the composition, more preferably 0.6% to 0.8% by weight. Preservatives are preferably selected from the group consisting of benzyl alcohol, phenoxyethanol, sodium benzoate, and combinations of two or more thereof. More preferably, the preservative is phenoxyethanol. These additives may be included in the continuous or discontinuous phase of the multi-microbubble dispersion. It should be understood that the included additives will be in the same amount and type as the materials found to be effective and useful. Care should be taken in the selection and amount of these additives to prevent impairment of other performance advantages of the present invention.

[0068] In a particularly preferred embodiment, the composition of the present invention comprises:

[0069] The first discontinuous phase contains diisopropyl adipic acid and tacrolimus;

[0070] A second discontinuous phase containing mineral oil; and

[0071] Continuous aqueous phase;

[0072] Preferably, the tacrolimus is substantially present in the first discontinuous phase;

[0073] The composition comprises less than 1% by weight of a surfactant;

[0074] The composition comprises less than 5% by weight of C1-C4 alcohols, polyethylene glycol, ethylene glycol, propylene glycol, butylene glycol, pentanediol, glycerol, diethylene glycol monoethyl ether, propylene carbonate, and mixtures of two or more thereof; and

[0075] The composition is in the form of a lotion or cream.

[0076] In a further particularly preferred embodiment, the composition of the present invention comprises:

[0077] The first discontinuous phase contains diisopropyl adipic acid and tacrolimus;

[0078] A second discontinuous phase containing mineral oil; and

[0079] Continuous aqueous phase;

[0080] Preferably, the tacrolimus is substantially present in the first discontinuous phase;

[0081] The composition comprises less than 1% by weight of a surfactant;

[0082] The composition comprises less than 1% by weight of C1-C4 alcohols, polyethylene glycol, ethylene glycol, propylene glycol, butylene glycol, pentanediol, glycerol, diethylene glycol monoethyl ether, propylene carbonate, and mixtures of two or more thereof; and

[0083] The composition is in the form of a lotion or cream.

[0084] According to the second aspect, compositions as described herein are provided for the treatment of psoriasis or atopic dermatitis.

[0085] According to a third aspect, a composition as described herein is provided for the manufacture of a medicament for the treatment of psoriasis or atopic dermatitis.

[0086] According to the fourth aspect, a composition as described herein is provided for use in treating a human or animal body via therapy.

[0087] According to the fifth aspect, a method for treating psoriasis or atopic dermatitis in human or animal patients is provided, the method comprising administering an effective amount of the composition as described herein to the patient in need.

[0088] The compositions described herein can be applied to the scalp or other skin surfaces via hair. Preferably, in this embodiment, the hair is moistened (e.g., by using water with or without shampoo, and then towel-dried). The product can then be applied to the scalp in an appropriate amount and massaged into the scalp through the hair. The hair can then be allowed to air dry or dried using a hair dryer. Advantageously, this aqueous dispersible form of the formulation allows for uniform distribution of the active agent on the skin using this method. Alternatively or additionally, the composition can be massaged into the scalp through dry hair and allowed for an appropriate time (which can be 8 to 12 hours), after which excess or residue can be rinsed off with water with or without shampoo. Preferably, the composition is applied to humans or animals in unit dosage forms.

[0089] According to a sixth aspect, the present invention provides packaging comprising the compositions disclosed herein. Preferably, the packaging is a tube or a pumpless packaging. For example, a squeezeable tube for topical application of the composition.

[0090] According to a seventh aspect, the present invention provides a method for manufacturing a composition for topical application, the method comprising:

[0091] (i) Provide a first oil containing tacrolimus;

[0092] (ii) Provide a second oil;

[0093] (iii) Provide aqueous components; and

[0094] (iv) Dispersing the first oil and the second oil in the aqueous component to form a composition for topical application, the composition comprising: a first discontinuous phase comprising the first oil and tacrolimus; a second discontinuous phase comprising the second oil; and a continuous aqueous phase;

[0095] The first oil is different from the second oil.

[0096] Preferably, the composition prepared by this method is the composition described above. Methods for preparing such oil-in-water dispersions are known in the art, for example, G. Godwin, Harry's Cosmeticology, 7th edition, 1982. Those skilled in the art will understand that other manufacturing methods may be used as appropriate.

[0097] As described above, the composition can be a multi-microbubble dispersion. A suitable method for preparing a multi-microbubble dispersion is described in US-A-44863333. Those skilled in the art will understand that other manufacturing methods may be used as appropriate.

[0098] Preferably, the method further includes packaging the composition.

[0099] The foregoing aspects can be freely combined with any of the foregoing aspects disclosed herein.

[0100] The invention will now be described in conjunction with the following non-limiting drawings:

[0101] Figure 1 This is a simplified schematic diagram illustrating the structure of the composition according to the present invention. The lighter circles represent the first discontinuous phase (containing the first oil and tacrolimus), the darker circles represent the second discontinuous phase (containing the second oil), and the background represents the continuous aqueous phase.

[0102] Figure 2 This is the HPLC chromatogram of the tacrolimus standard at t=0 used in Example 1. Peaks were observed for tacrolimus (19.863 min), tacrolimus 19-epimer (16.803 min), and ascomycin (18.976 min). HPLC conditions were: column: Luna C18(2), 3 μm particle size, 4.6 x 150 mm column (Waters), flow rate: 1.5 mL / min, column temperature: 25 °C.

[0103] Figure 3 This is the HPLC chromatogram of Sample 1 from Example 1 at t=0. The HPLC conditions were: column: Luna C18(2), 3 μm particle size, 4.6 x 150 mm column (Waters), flow rate: 1.5 mL / min. Column temperature: 25 °C.

[0104] Figure 4 This is the HPLC chromatogram of Sample 1 from Example 1 at 40°C for t = 6 months. The HPLC conditions were: column: Luna C18(2), 3 μm particle size, 4.6 x 150 mm column (Waters), flow rate: 1.5 mL / min, column temperature: 25°C.

[0105] Figure 5 This is the HPLC chromatogram of sample 9 from Example 1 at t=0. The HPLC conditions were: column: Luna C18(2), 3 μm particle size, 4.6 x 150 mm column (Waters), flow rate: 1.5 mL / min. Column temperature: 25 °C.

[0106] Figure 6 This is the HPLC chromatogram of sample 9 from Example 1 at 40°C for t = 6 months. The HPLC conditions were: column: Luna C18(2), 3 μm particle size, 4.6 x 150 mm column (Waters), flow rate: 1.5 mL / min, column temperature: 25°C.

[0107] Figure 7 This is a bar chart showing the effect of the compositions of samples 1, 2, 3, and 6 on erythema scores 48 hours after initial application of the composition. Error bars represent the standard error of the mean.

[0108] Figure 8This is a bar chart showing the in vitro diffusion of samples 1, 13, 14, and 15, in μg / cm³. 2 The y-axis indicates the average cumulative tacrolimus diffusion, in μg / cm³. 2 The error bars represent the standard deviation.

[0109] Figure 9 This is a dose-response graph comparing the corresponding variations of tacrolimus ointment with samples 1, 22, 23, and 24 (tacrolimus concentrations of 0.1%, 0.03%, 0.01%, and 0.003%, respectively) in a Landrace Swine model of allergic contact dermatitis. Black circles: variations of tacrolimus ointment; gray circles: variations of tacrolimus in sample 1; dashed line: sample 21 (control carrier). The x-axis represents tacrolimus concentration, and the y-axis represents the erythema score on day 10 (24 hours after challenge). Error bars represent standard deviation.

[0110] The invention will now be described in conjunction with the following non-limiting examples.

[0111] Example 1

[0112] The following compositions were prepared, and the stability of tacrolimus was measured in initial assays and after 6 months at 40°C:

[0113]

[0114]

[0115] Key components:

[0116] 1) Dispersed aqueous phase

[0117] 2) API non-solvent oil phase

[0118] 3) API solvent oil phase

[0119] 4) Gel / Thickener

[0120] 5) pH / water adjustment

[0121] Each component was prepared separately and then added sequentially while being properly stirred. Components 1-3 in the combination each constitute 50% by weight of the final formulation in each case, component 4 constitutes 48% by weight, and component 5 constitutes 2% by weight.

[0122] Based on the relative size of the phases, the substance is distributed proportionally between the two phases. For example, if the two oil phases have the same mass, an equal mass of surfactant is added to each oil phase. If the mass of the first oil phase is twice that of the second oil phase, the surfactant is distributed between the two phases at a mass ratio of 2:1.

[0123] Each sample was stored in a sealed, hermetically sealed glass container, with the headspace comprising no more than 5% of the container's total usable volume. Each container containing the sample was stored at a constant temperature of 40°C in a standard laboratory incubator (e.g., a Memmert IF260PLUS incubator). Each sample was stored for six months, after which its chemical stability was measured.

[0124] The stability assay at 100±5% indicates that tacrolimus is chemically stable for 6 months at 40°C. The stability assay at 100±2% indicates that tacrolimus has particularly high chemical stability.

[0125] The chemical stability of tacrolimus in each sample after storage was determined by HPLC. The HPLC method is as follows:

[0126]

[0127]

[0128] Mobile phase preparation:

[0129] Solution A: A 6 mM orthophosphate solution in water is prepared by adding 0.4 mL to 1000 mL of purified water (typically 3 L is prepared).

[0130] Solution B: A mixture of acetonitrile and tert-butyl methyl ether is prepared in a ratio of 81:19% (typically 3L is prepared).

[0131] The mobile phases A and B are prepared into a mixture of solutions A and B in the proportions detailed in the table above.

[0132] Sample preparation

[0133] Acetonitrile was used as the sample diluent.

[0134] Prepare the solution in the amber glass container.

[0135] step:

[0136] 1. Accurately weigh approximately 1.0g of sample into a 10ml volumetric flask, minimizing the amount of sample remaining on the neck of the flask.

[0137] 2. Add approximately 5 mL of sample diluent to the volumetric flask and vortex mix for 2 minutes.

[0138] 3. Allow it to equilibrate to room temperature, then dilute the volumetric flask to the required volume using the sample diluent. Mix thoroughly by stirring for 1 hour.

[0139] 4. Transfer the solution of the aliquots to 2 ml centrifuge tubes and centrifuge at 13,000 rpm for 10 minutes.

[0140] 5. Filtered through a 0.45μm PTFE syringe filter.

[0141] Preparation of standard products:

[0142] A solution of tacrolimus in acetonitrile with a concentration of 100 μg / mL was prepared.

[0143] The HPLC chromatogram of the tacrolimus standard using this method is shown in... Figure 2 The HPLC chromatogram of sample 1 is shown in the figure. Figure 3 (t=0) and Figure 4 (t = 6 months, 40℃). The HPLC chromatogram of sample 9 is shown in... Figure 5 (t=0) and Figure 6 (t = 6 months, 40℃)

[0144] Approximate retention time:

[0145]

[0146] Discussion of stability results

[0147] The stability data show that, in terms of the stability of tacrolimus, Sample 1 (which uses diisopropyl adipate, isopropyl myristate, and caprylic / capric triglyceride for the first discontinuous phase and does not contain polar water-miscible liquids) is the most stable formulation.

[0148] Tacrolimus was less stable in samples 2, 3, 4, 5, and 9. These formulations contained 12%–30% by weight of various polar, water-miscible liquids (propylene glycol, Transcutol P...). TM (Hexanediol or pentanediol). This lower stability is believed to be attributed to the influence of polar non-aqueous solvents on the partitioning of tacrolimus between the oil and aqueous phases. Specifically, the greater aqueous exposure of tacrolimus in samples 2, 3, 4, 5, and 9 compared to Example 1 is believed to lead to increased degradation of tacrolimus. The enhanced chemical stability of sample 1 relative to sample 9 can be seen in… Figure 3-6 This can be seen from the text.

[0149] Sample 10 was identical to Sample 1, except that it had an increased level of surfactant Laureth 4 (9.4 wt%). The measured tacrolimus stability was significantly lower than that of Sample 1. Again, this is believed to be attributed to the surfactant affecting the partitioning of tacrolimus between the oil and aqueous phases. In particular, the greater aqueous exposure of tacrolimus in Sample 10 is thought to lead to increased degradation of tacrolimus compared to Sample 1.

[0150] A comparison of Samples 1, 7, and 8 shows that the use of diisopropyl adipate (Example 1) in the first discontinuous phase produces greater tacrolimus stability than the use of diethyl sebacate or dibutyl adipate (Samples 7 and 8, respectively) in the first discontinuous phase. This is surprising because all three oils have similar solubility profiles for tacrolimus (see discussion in Example 2).

[0151] All samples were observed to be physically stable after 6 months at 40°C, meaning each sample showed a uniform cream with no significant rheological or appearance changes from t=0.

[0152] Example 2

[0153] In this embodiment, the solubility of tacrolimus in various oil solvents was measured by HPLC.

[0154] Weigh approximately 40 mg of tacrolimus, add a known amount (1-2 g) of oil, and stir at room temperature for 5 hours. If, by visual inspection, the tacrolimus fails to dissolve, add up to 10 g of oil. If the tacrolimus appears to dissolve completely with 10 g of oil, incubate the sample at 4°C overnight. If, upon cooling, the tacrolimus precipitates, add additional oil and repeat the above steps until dissolved or add another 10 g of oil. Then centrifuge all samples to ensure the removal of any undissolved tacrolimus and analyze the supernatant by HPLC as described in Example 1.

[0155] If the final formulation is subjected to refrigeration or exposure to low temperatures, freezing of the oil solvent provides confidence that tacrolimus will remain in solution. It is not unreasonable for commercial products to undergo such conditions. Importantly, tacrolimus will not precipitate from the solution. Such precipitation could lead to physical or chemical instability of the product or result in uneven distribution of tacrolimus.

[0156] result:

[0157] Based on the above method, the solubility data of tacrolimus in oil solvents are as follows:

[0158] solvent Solubility (%wt) Diethyl sebacate 3.26* Dibutyl adipic acid 3.24* diisopropyl adipic acid 3.08* castor oil 1.44 Caprylic / Capric Triglyceride 0.44 Isopropyl palmitate 0.26 Isopropyl myristate 0.25 Isopropyl isostearate 0.19 heptylmethylnonane 0.006 Cyclodimethylsiloxane 0.004 polydimethylsiloxane 0.004 mineral oil 0.003

[0159] *HPLC determinations are within 95% of the theoretical experimental maximum value. The maximum possible solubility is greater than stated.

[0160] Based on the above data, oils can be divided into three categories: good solvents (>1% solubility), medium solvents (0.15-1% solubility), and effective non-solvents (<0.1%).

[0161] Although tacrolimus has similar solubility in diethyl sebacate, diisopropyl adipate, and dibutyl adipate, it is surprising that diisopropyl adipate is significantly more effective than diethyl sebacate or dibutyl adipate in stabilizing tacrolimus (see Example 1).

[0162] Without being bound by theory, it is believed that the two ester bonds present in all three oils are more protected in diisopropyl adipate by steric hindrance caused by the proximity / conformation of the methyl group near the bond. It is thought that degradation of this bond may subsequently affect the stability of the surfactant, thus explaining the increased stability in Example 1.

[0163] Example 3

[0164] In vivo experiments using an allergic contact dermatitis model in Landrace pigs (five pigs) compared Protopic... TM (0.1% tacrolimus), petrolatum control, and samples 1-3 and 6.

[0165] This method involves applying dinitrofluorobenzene (DNFB) and dinitrochlorobenzene (DNCB) to skin patches to induce contact hypersensitivity, followed by Protopic TM (0.1% tacrolimus), petrolatum control and one of samples 1-3 and 6 were used for treatment.

[0166] This method is based on the protocol described by Mollison et al., J Invest Dermatol. 1999 May; 112(5):729-38: "amacrolactam inhibitor of T helper type 1 and T helper type 2 cytokine biosynthesis for topical treatment of inflammatory skin diseases".

[0167] method:

[0168] Day 0: Apply 100 μl of 10% dinitrobenzene (DNFB) in acetone / DMSO / olive oil (45 / 5 / 50 v / v / v) to the outer sides of both ears and bilateral lower abdomen, covering an area of ​​approximately 20 cm. 2 Each of them.

[0169] Day 3:Apply 100 μl of 5% dinitrobenzene (DNFB) in acetone / DMSO / olive oil (45 / 5 / 50 v / v / v) to the inner ear patches of both ears and to bilateral areas of the lower thoracic cavity, approximately 20 cm in length. 2 .

[0170] Day 9: 61 μl of 0.6% dinitrochlorobenzene (DNCB) in acetone / olive oil (95 / 5v / v) was applied to 18 rectangular test areas – 9 on each side of the pig (3.5 x 3.5 cm, 12.25 cm). 2 ).

[0171] Day 9 + 30 minutes: Apply 61 μl of the test reagent to the test area (3.5 x 3.5 cm, 12.25 cm). 2 ).

[0172] Scoring / evaluation was performed 24 and 30 hours after the initial application of the test formulation, followed by reapplication of the test formulation. Further scoring was conducted 48 hours later.

[0173] The block's score is as follows:

[0174] 0 – No erythema

[0175] 0.5 – Suspicious erythema

[0176] 1 – Faint or scattered erythema

[0177] 2 – Moderate erythema, without induration

[0178] 3 – Intense erythema, with focal areas of edema or induration.

[0179] 4 – Extreme erythema with uniform induration or edema

[0180] result:

[0181]

[0182] Pigs rated 3 or higher on the 24-hour sensitization score for the Vaseline control will be used for comparative purposes. Pigs that do not respond strongly to sensitization will not be allowed to make adequate distinctions in determining the efficacy of the active formulation.

[0183] The results after 48 hours also showed Figure 7 (The error bars represent the standard error of the mean). For example, from... Figure 7 As is evident in the table above, the erythema scores of samples 1-3 and 6 at 48 hours were similar. These erythema scores were significantly lower than those of the petrolatum control carrier and Protopic. TM Therefore, the formulations of the present invention are superior to those such as Protopic.TM Existing ointment formulations have higher efficacy in vivo.

[0184] Example 4

[0185] Other compositions were prepared using different surfactant systems. For comparison, the composition of Sample 1 is included in the table.

[0186]

[0187] The component numbers and proportions (1-5) are the same as in Example 1.

[0188] Based on the relative size of the phases, the substance is distributed proportionally between the two phases. For example, if the two oil phases have the same mass, an equal mass of surfactant is added to each oil phase. If the mass of the first oil phase is twice that of the second oil phase, the surfactant is distributed between the two phases at a mass ratio of 2:1.

[0189] For Examples 11 and 12, the components were heated to 70°C before assembly. In the case of Example 12, a high-shear rotator (Silverson) was used at 7000 rpm.

[0190] Example 5

[0191] The composition was prepared in which the second oil and its amount were varied. For comparison, the composition of Sample 1 is included in the table.

[0192]

[0193] Sample 13 Mineral oil replaced with polydimethylsiloxane

[0194] Sample 14 Additional mineral oil

[0195] Sample 15 There is no second discontinuous phase (comparative).

[0196] Based on the relative size of the phases, the substance is distributed proportionally between the two phases. For example, if the two oil phases have the same mass, an equal mass of surfactant is added to each oil phase. If the mass of the first oil phase is twice that of the second oil phase, the surfactant is distributed between the two phases at a mass ratio of 2:1.

[0197] The formulation was tested in an in vitro diffusion assay using an artificial membrane in a Franz chamber. The membrane used was the Strat-M membrane from Merck Millipore and was described as a synthetic, non-animal model for transdermal diffusion assays that predict diffusion in human skin. A phosphate-buffered saline solution containing 5% bovine serum albumin and 5% isopropanol was used as the acceptor phase. A surface area of ​​0.64 cm² was used. 2 cells and about 2cm 3 Receptor volume was determined. Approximately 40 mg of the formulation was administered at 0, 24, and 48 hours, cells were agitated, and incubated at 37°C. The receptor phase was sampled and replaced at 24, 48, and 72 hours. Tacrolimus concentration was determined by HPLC according to the above method, and individual chamber receptor volumes were adjusted. The average cumulative tacrolimus per square centimeter of membrane was then plotted for each sample (see [link to relevant documentation]). Figure 8 ).

[0198] The data clearly show that Samples 1 and 13 exhibit excellent in vitro diffusion properties. These are formulations with the same ratio of second to first discontinuous phase (but containing either mineral oil or polydimethylsiloxane). Sample 15 (without a second discontinuous phase) is significantly inferior to Samples 1 and 13, indicating that the presence of the second oil phase significantly improves permeation. However, Sample 14 shows that an excess of the second discontinuous phase may have adverse effects on in vitro diffusion. Therefore, there exists an optimal level of second discontinuous phase that maximizes the in vitro diffusion of the formulation.

[0199] Furthermore, it is noteworthy that there was no statistically significant difference between the results of samples 1 and 13. Polydimethylsiloxane is far less likely to mix with the API oil phase during application because it is known to have lower miscibility with other oils compared to mineral oil. Therefore, the lack of a statistically significant difference between samples 1 and 13 suggests that the improved permeability relative to the single API oil phase system (sample 15) is not primarily due to mixing of the oils during application (which leads to changes in the saturation of tacrolimus in the resulting mixture).

[0200] Example 6

[0201] Using water-miscible liquids (Transcutol P) at different levels in the aqueous phase TM To prepare various simplified formulations.

[0202] Element %wt / wt oil phase Tacrolimus 0.13 Caprylic / Capric Triglyceride 12.73 diisopropyl adipic acid 6.36 Isopropyl myristate 6.36 Butylated hydroxytoluene 0.13 Aqueous phase Phenoxyethanol 0.89 Sodium citrate 0.209 Citric acid 0.121 <![CDATA[Transcutol P TM ]]> x water Appropriate amount (up to 100)

[0203] (Transcutol P TM =Diethylene glycol monoethyl ether)

[0204] No surfactants are added. This ensures that the oil and aqueous phases can mix vigorously but separate easily when left to stand overnight, making sampling and analysis more direct. Furthermore, the absence of mineral oil or gelling agents again makes sampling and analysis more direct.

[0205] The formulation was prepared by manually and vigorously shaking the two phases together. They were then allowed to stand overnight, and the oil phase was removed. The two phases were centrifuged and tacrolimus was analyzed using the HPLC method described herein.

[0206] The prepared samples and their associated tacrolimus (API) recovery levels are shown in the table below:

[0207]

[0208] As can be seen from the table, the higher the level of polar non-aqueous solvent incorporated into the aqueous phase, the greater the degree to which tacrolimus partitions into the aqueous phase. This increased aqueous exposure is believed to partially explain the chemical stability trend observed in Example 1.

[0209] Example 7

[0210] Another composition was prepared by using sodium benzoate as a preservative instead of phenoxyethanol.

[0211]

[0212] Key components:

[0213] 1) Dispersed aqueous phase

[0214] 2) API non-solvent oil phase

[0215] 3) API solvent oil phase

[0216] 4) Gel / Thickener

[0217] 5) Preservatives

[0218] 6) pH / water adjustment

[0219] Each component was prepared separately, and then added sequentially while being properly stirred. In each case, components 1-3 were combined to form 50% by weight of the final formulation, component 4 to form 43% by weight, component 5 to form 5% by weight, and component 6 to form 2% by weight.

[0220] Based on the relative size of the phases, the substance is distributed proportionally between the two phases. For example, if the two oil phases have the same mass, an equal mass of surfactant is added to each oil phase. If the mass of the first oil phase is twice that of the second oil phase, the surfactant is distributed between the two phases at a mass ratio of 2:1.

[0221] Example 8

[0222] Local tolerance studies were conducted in miniature pigs to evaluate the local tolerance of the two topical tacrolimus creams and commercially available ointments according to the invention and to determine the amount of tacrolimus in the skin after twice-daily application to miniature pigs for 4 weeks by skin application.

[0223] method:

[0224] Four (4) males were used in the study. Miniature pigs. Eight (8) application sites, each measuring 2.5 × 2.5 cm, were tattooed on the back of each animal, and each formulation, namely Sample 1 carrier, Sample 1 with 0.03% tacrolimus, Sample 1 with 0.1% tacrolimus, and Sample 2 with 0.1% tacrolimus, were applied. 0.1% tacrolimus ointment was applied to two different application sites on each animal.

[0225] Administer medication to animals according to the following schedule.

[0226]

[0227] Assess the following: mortality rate, clinical signs, weight, and autopsy; gross examination of treated and untreated skin and histopathology. Skin biopsies for bioanalysis should be collected on day 28, 4 hours after the first dose, and at autopsy. Additionally, examine the application site for response to treatment and score erythema, edema, and other skin reactions.

[0228] Biopsies were performed on all application sites for bioanalysis 4 hours + / - 10 minutes after the first dose on the last day of administration (day 28) and at necropsy. Two biopsies (one for backup) were taken from each application site at each sampling time. A total of 16 biopsies were performed per animal at each sampling time. Approximately 5 mm of biopsy tissue was taken from the edge of the application site. Prior to collecting skin samples, the stratum corneum was separated from the skin at the sampling biopsy site using commercial adhesive tape, peeling 40 times every two biopsies.

[0229] Biopsies were collected using a 5 mm diameter punch. From the biopsy, the epidermis was separated from the dermis using a scalpel in the most efficient manner possible. Subcutaneous tissue was then removed from the dermis using a scalpel and discarded. The biopsies were weighed and immediately rapidly frozen in liquid nitrogen and stored below -80°C until analysis. Tacrolimus levels in the tissue samples were analyzed using a validated bioassay.

[0230] result:

[0231] Miniature pigs were treated with topical tacrolimus cream twice daily for four weeks using three different formulations: Sample 21 (carrier), Sample 22 (0.03% tacrolimus), and Sample 1 (0.1% tacrolimus). The 0.1% ointment was well tolerated, and no test events associated with skin reactions or histopathological changes were observed.

[0232] Bioanalytical analysis showed that the tested substances were mainly located in the epidermis, with only a small amount in the dermis. Treatment with sample 0.1% tacrolimus cream resulted in approximately three times higher tacrolimus levels compared to treatment with sample 1 (0.03% tacrolimus cream), indicating a dose-response relationship. Treatment with 0.1% tacrolimus ointment resulted in an exposure level approximately 30% lower than that of the sample treated with 10.1% tacrolimus cream. Therefore, it can be concluded that, at a given tacrolimus loading, the compositions of the present invention exhibit superior in vivo performance compared to existing ointment formulations (e.g., Protopic). TM Improved skin permeability. Data is summarized in the table below.

[0233] surface:

[0234] Tacrolimus levels were measurable in skin biopsies of miniature pigs after 28 days of application of the test product twice daily.

[0235]

[0236] Example 9

[0237] In vivo experiments were conducted in Landrace pigs (8 females) using an allergic contact dermatitis model to compare the dose response of commercially available tacrolimus ointment with the compositions of the present invention containing tacrolimus of varying strengths. The following test formulations were compared: Protopic TM (0.1% tacrolimus ointment), Protopic TM (0.03% tacrolimus ointment), 0.01% tacrolimus ointment, 0.003% tacrolimus ointment, Sample 1 (containing 0.1% tacrolimus), Samples 21 and 22 (same as Sample 1 but without tacrolimus and containing 0.03% tacrolimus, respectively), Sample 23 (same as Sample 1 but containing 0.01% tacrolimus), and Sample 24 (same as Sample 1 but containing 0.003% tacrolimus). 0.01% and 0.003% tacrolimus ointments were prepared according to Example 1 of EP1093371B1 (using the method disclosed in Example 1 of EP-A-0474126), wherein the amount of tacrolimus was appropriately reduced.

[0238] The method involves applying dinitrofluorobenzene (DNFB) and dinitrochlorobenzene (DNCB) to skin patches to induce contact hypersensitivity reactions, followed by treatment with one of the test formulations.

[0239] This method is based on the protocol described by Mollison et al., J Invest Dermatol. May 1999; 112(5):729-38: "amacrolactam inhibitor of T helper type 1 and T helper type 2 cytokine biosynthesis for topical treatment of inflammatory skin diseases".

[0240] method:

[0241] Day 0: Apply 100 μl of 10% dinitrobenzene (DNFB) in acetone / DMSO / olive oil (45 / 5 / 50 v / v / v) to the outer sides of both ears and bilateral lower abdomen, covering an area of ​​approximately 20 cm. 2 Each of them.

[0242] Day 3: Apply 100 μl of 5% dinitrobenzene (DNFB) in acetone / DMSO / olive oil (45 / 5 / 50 v / v / v) to the inner ear of both ear regions and to the bilateral areas of the lower thoracic cavity, approximately 20 cm in length. 2 .

[0243] Day 9: 60 μl of 0.6% dinitrochlorobenzene (DNCB) in acetone / olive oil (95 / 5v / v) was applied to 18 rectangular test areas – 9 areas on each side of the pig (3.5 x 3.5 cm, 12.25 cm). 2 ).

[0244] Day 9 + 30 minutes and 6 hours: Apply 60 μl of the test reagent to the test area (3.5 x 3.5 cm, 12.25 cm). 2 ).

[0245] Scoring was performed 24 and 30 hours after the initial application of the test formulation, followed by reapplication of the test formulation. Erythema scoring was performed 24 and 48 hours later.

[0246] The erythema caused by allergic contact dermatitis was scored in the test area as follows:

[0247] 0 – No erythema

[0248] 0.5 – Suspicious erythema

[0249] 1 – Faint or scattered erythema

[0250] 2 – Moderate erythema, without induration

[0251] 3 – Intense erythema, with focal areas of edema or induration.

[0252] 4 – Extreme erythema with uniform induration or edema

[0253] result:

[0254]

[0255] The results after 24 hours also showed Figure 9 (The error bars represent the standard deviation). The following conclusions were drawn from the study of Sample 1 and tacrolimus ointment:

[0256] 1. Clearly, the lowest concentrations of tacrolimus (0.003% and 0.01%) were ineffective in both the cream and ointment variants.

[0257] 2. The baseline of the cream sample according to the present invention is lower than that of the ointment, indicating that the cream carrier may have an independent beneficial effect on inflammation compared with the ointment.

[0258] 3. A dose-response trend of the anti-inflammatory effect of tacrolimus was observed in the claimed compositions of the present invention, and it is comparable to that of tacrolimus in ointment variants.

[0259] The foregoing detailed description has been provided by way of explanation and illustration, and is not intended to limit the scope of the appended claims. Many variations of the presently preferred embodiments shown herein will be apparent to those skilled in the art, and remain within the scope of the appended claims and their equivalents.

Claims

1. A composition for topical application, comprising: A first discontinuous phase comprising a first oil and tacrolimus, wherein the first oil comprises diisopropyl adipate; The second discontinuous phase containing the second oil; and Continuous aqueous phase; The first oil is different from the second oil, and the first discontinuous phase and the second discontinuous phase are physically and chemically different from each other. in, Tacrolimus is substantially soluble in the first oil and substantially insoluble in the second oil.

2. The composition of claim 1, wherein, The first oil also contains isopropyl myristate and / or caprylic / capric triglyceride.

3. The composition according to claim 1 or claim 2, wherein, The second oil comprises an oil selected from the group consisting of: mineral oil, polydimethylsiloxane, cyclodimethylsiloxane, hydrogenated polyisobutane, heptamethylnonane, and mixtures of two or more thereof.

4. The composition of claim 1 or 2, wherein the total content of surfactant is less than 5% by weight of the composition.

5. The composition of claim 1 or 2, wherein the total content of surfactant is less than 1% by weight of the composition.

6. The composition of claim 1 or 2, wherein the total content of surfactant is less than 0.5% by weight of the composition.

7. The composition of claim 1 or 2, comprising less than 5% by weight of C1-C4 alcohols, polyethylene glycol, ethylene glycol, propylene glycol, butanediol, pentanediol, glycerol, diethylene glycol monoethyl ether, propylene carbonate, and mixtures thereof.

8. The composition of claim 1 or 2, comprising less than 1% by weight of C1-C4 alcohols, polyethylene glycol, ethylene glycol, propylene glycol, butanediol, pentanediol, glycerol, diethylene glycol monoethyl ether, propylene carbonate, and mixtures thereof.

9. The composition of claim 1 or 2, comprising less than 0.5% by weight of C1-C4 alcohols, polyethylene glycol, ethylene glycol, propylene glycol, butanediol, pentanediol, glycerol, diethylene glycol monoethyl ether, propylene carbonate, and mixtures thereof.

10. The composition according to claim 1 or 2, wherein, The composition has a weight ratio of the second oil to the first oil of 1:3 to 3:

1.

11. The composition according to claim 1 or 2, wherein, The composition has a weight ratio of the second oil to the first oil of 2:3 to 3:

2.

12. The composition according to claim 1 or 2, wherein, The composition has a weight ratio of the second oil to the first oil of 1:

1.

13. The composition according to claim 1 or 2, wherein, The composition comprises 10 to 30% by weight of a second discontinuous phase.

14. The composition according to claim 1 or 2, wherein, The composition comprises 15 to 25% by weight of a second discontinuous phase.

15. The composition according to claim 1 or 2, wherein, The composition contains at least 10% by weight of water.

16. The composition of claim 1 or 2, wherein, The composition contains at least 50% by weight of water.

17. The composition according to claim 1 or 2, wherein, The composition comprises up to two discontinuous phases.

18. The composition of claim 1 or 2, wherein, The average droplet diameter of the composition is 1 to 30 μm.

19. The composition according to claim 1 or 2, wherein, The average droplet diameter of the composition is 1 to 20 μm.

20. The composition according to claim 1 or 2, wherein, The pH of the composition is 3.5 to 6.

21. The composition according to claim 1 or 2, wherein, The pH of the composition is 4 to 5.

5.

22. The composition according to claim 1 or 2, wherein, The pH of the composition is 4.5 to 5.

23. The composition of claim 1 or 2, wherein, as measured at 60% RH ± 5%, the composition is stable at 25°C ± 2°C for at least 12 months; and / or wherein, as measured at 60% RH ± 5%, the composition is stable at 40°C ± 3°C for at least 6 months.

24. The composition of claim 1 or 2, comprising: The first discontinuous phase contains diisopropyl adipic acid and tacrolimus; A second discontinuous phase containing mineral oil; and Continuous aqueous phase; The composition contains less than 1% by weight of a surfactant. The composition comprises less than 5% by weight of C1-C4 alcohols, polyethylene glycol, ethylene glycol, propylene glycol, butylene glycol, pentanediol, glycerol, diethylene glycol monoethyl ether, propylene carbonate, and mixtures of two or more thereof; and The composition is in the form of a lotion or cream.

25. The composition of claim 24, wherein at least 90% by weight of the tacrolimus is present in the first discontinuous phase.

26. The composition of claim 1 or 2, for treating human and / or animal skin by topical application.

27. A method of manufacturing a composition for topical application, the method comprising: (i) Provide a first oil containing tacrolimus, the first oil further containing diisopropyl adipate; (ii) Provide a second oil; (iii) Provide aqueous components; and (iv) Disperse the first oil and the second oil in the aqueous component to form a composition for topical application; The first oil is different from the second oil, and the first discontinuous phase and the second discontinuous phase are physically and chemically different from each other. Tacrolimus is substantially soluble in the first oil and substantially insoluble in the second oil.

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