CN107308145B - Application of phenformin in preparing anti-skin inflammation preparation - Google Patents

Abstract

The invention discloses an application of phenformin as a skin anti-inflammatory preparation. The invention firstly discovers that the phenformin can obviously inhibit the epidermal cells of the skin from secreting various inflammatory cytokines including interleukin 6/8(IL-6, IL-8) and tumor necrosis factor (TNF alpha), and also can obviously reduce the inflammatory reaction of the epidermal cells of the human skin induced by the commonly used 12-O-tetradecanoyl phorbol-13-acetate (TPA) and poly-inosinic-polycystic acid (poly I: C) (immunostimulant), thereby being used for preventing and treating diseases related to the skin inflammation. Therefore, the new application of the phenformin is developed, and a new prevention and treatment means is provided for the diseases related to the skin inflammation.

Description

Application of phenformin in preparing anti-skin inflammation preparation

Technical Field

The invention belongs to the field of pharmaceutical pharmacy, and particularly relates to an application of phenformin in preparation of an anti-skin inflammation preparation.

Background

Dermatitis (Dermatitis) is a generic term for inflammatory diseases of the skin caused by various internal and external infections or non-infectious factors, is not an independent disease, and has complex and various etiology and clinical manifestations, repeated onset and difficult clinical treatment. Modern medical research has suggested that the cause of dermatitis is very complex and may be related to the following factors: including internal factors such as chronic infectious disease (such as chronic cholecystitis, tonsillitis, intestinal parasitosis, etc.), endocrine and metabolic changes (such as menstrual disorder, pregnancy, etc.), blood circulation disorder (such as crus varicosis, etc.), neuropsychiatric factors, and genetic factors; external factors such as food (such as fish, shrimp, beef and mutton), inhalant (such as pollen, dust mite, etc.), living environment (such as cold, hot, dry, etc.), animal fur, and various physicochemical substances (such as cosmetics, soap, synthetic fiber, etc.). The current treatment policy aims at the treatment of etiology or various suspected pathogenic factors, infectious inflammation actively controls infection, and the general treatment principle of non-infectious inflammation is systemic drug treatment, anti-inflammation and itching relieving.

With the development of related fields such as molecular biology, it is found that dermatitis occurrence and development are closely related to interferon, inflammatory cytokines, tumor necrosis factor (TNF α), Fas-related death domain (FADD) protein, etc., such as guo, et al (old beauty, guo, CHENYan, etc.. new generation antihistamines for comparison study of the effects of 3 cytokines in mice with allergic contact dermatitis [ J ]. journal of clinical dermatology, 2006,35(1):6-8) study of the effects of mizolastine, loratadine and cetirizine on the production of Interferon (IFN), tumor necrosis factor (TNF α) and Interleukin (IL) -4 in mice with allergic contact dermatitis [ ACD ]; studies by Bonnet et al (Bonnet M C, Preukschat D, Welz P S, et al, the Adaptor Protein proteins peptide molecules from novel neuroptosis In Vivo and Prevents Skin Inflammation [ J ]. Immunity,2011,35(4): 572. Anoplom 582) indicate that mice with ablation of the Epidermal properties of FADD display spontaneous keratinocyte neoptosis and develop severe inflammatory Skin lesions within a few days of birth, and thus FADD is critical In Keratinocytes for maintaining a balance between Skin immune response and preventing Skin Inflammation. Therefore, research on the substances affecting the above-mentioned protein factors is an important direction for the development of preventive and therapeutic treatments of dermatitis-related diseases.

Phenformin (Phenformin) is a commonly used drug for treating adult non-insulin-dependent diabetes mellitus and partial insulin-dependent diabetes mellitus. The composition has effects in promoting glucose uptake and glycolysis of muscle cells, reducing glucose produced by liver, resisting hyperglycemia, and reducing insulin dosage. For obese diabetic patients, they can also reduce their body weight by suppressing appetite and absorbing glucose in the intestine. Because it has a high probability to induce diabetic complications, namely lactic acidosis, and 2016, 11 and 18 days, the State food and drug administration has issued the Notice on the stop of production and sale of phenformin.

Disclosure of Invention

In view of the above-mentioned deficiencies of the prior art, the inventors have conducted long-term technical and practical researches and unexpectedly found that phenformin can significantly inhibit the secretion of various inflammatory cytokines including interleukin 6/8(IL-6, IL-8) and tumor necrosis factor (TNF α) by epidermal cells of the skin, and also significantly reduce the inflammatory response of the epidermal cells of the skin induced by the commonly used 12-O-tetradecanoylphosphatide-13-acetate (TPA) and polyinosinic-polycytidylic acid (poly I: C) (immunostimulant), so that phenformin is found to have an anti-inflammatory effect on the skin, and can be developed and applied clinically for preventing and treating diseases related to the inflammation of the skin.

Specifically, the invention relates to the following technical scheme:

in one aspect of the present invention, the use of phenformin as an anti-inflammatory agent for the skin is disclosed, and therefore, is extremely effective for preventing and/or treating diseases associated with dermatitis (skin inflammation);

in another aspect of the invention, the use of phenformin as a skin anti-inflammatory agent for the preparation of a medicament for the prevention and/or treatment of a disease associated with skin inflammation is disclosed;

according to the present invention, the concept "prevention and/or treatment" means any measure suitable for treating a disease associated with inflammation of the skin, either for prophylactic treatment of such manifested disease or manifested symptoms, or to avoid recurrence of such disease, e.g. after the treatment period has ended or for treatment of symptoms of a disease that has already occurred.

In the sense of the present invention, a "skin anti-inflammatory agent" means a substance which blocks, delays or inhibits inflammatory cytokines, tumor necrosis factors and/or immunostimulants associated with skin inflammation;

wherein the inflammatory cytokines include, but are not limited to, interleukin 6/8(IL-6, IL-8);

such tumor necrosis factors include, but are not limited to, TNF α/TNF β;

the immunostimulant includes, but is not limited to, 12-O-tetradecanoyl phorbol-13-acetate (TPA)/polyinosinic-polycytidilic acid (poly I: C);

in the sense of the present invention, the concept of "inhibition" is to be understood as a slowing down (delaying) and/or reduction of the activity of inflammatory cytokines, tumor necrosis factors and/or immunostimulants, preferably by up to 10%, also preferably by up to 40%, more preferably by up to 60%, preferably by up to 70%, also preferably by up to 80%, more preferably by up to about 90%, also more preferably by up to 95%, yet more preferably by up to 98%, most preferably by 100% compared to inflammatory cytokines, tumor necrosis factors and/or immunostimulants which have not been administered phenformin.

In yet another aspect of the present invention, it is disclosed that diseases associated with skin inflammation include, but are not limited to, erythema of the skin, acantholysis of the skin, pemphigus, eczema, vitiligo, lichen planus, contact dermatitis, atopic dermatitis, burning, stinging and/or itching during inflammation, skin ulcers, acne;

for effective prevention and/or treatment of diseases associated with skin inflammation, it is possible in particular by administering phenformin pharmaceutical active ingredients which, taking into account the administration form, preferably have the following properties:

a) in the topical application of the skin anti-inflammatory formulations of the present invention: local applicability to topical administration over an arbitrary long period of time and high transdermal transport kinetics of the active ingredient (correspondingly applicable to parenteral administration);

b) the effective components are distributed uniformly in the range of action points to a great extent and the local concentration is avoided from being too high;

c) preferably enriched in the body part concerned, possibly in combination with a delay in delivery to the blood circulation (i.e. intravascular delivery);

d) inhibition, i.e., the inhibition or inhibition of the activity of inflammatory cytokines, tumor necrosis factor and/or immunostimulants associated with skin inflammation at the body site involved;

meanwhile, it is particularly noted that the use of phenformin as an active ingredient for the treatment of skin inflammation-related diseases has the following characteristics:

1) the possibility of achieving a concentration of phenformin in the epidermis or dermis of the skin sufficient to achieve a therapeutic effect by means of topical application of phenformin to the skin, without achieving a similarly high concentration of phenformin in other organs of the body as in systemic administration; without strong side effects on other organs or healthy skin tissue;

2) the aggregation state of phenformin upon topical administration may be liquid, gaseous or solid. Delivery to the skin may be achieved by direct contact of the phenformin or by a phenformin-containing formulation with the skin, or by diffusion through an intermediate layer such as air, a microporous membrane, or the like;

according to the present invention, not only is the effect of phenformin disclosed as an anti-inflammatory agent for the skin, but it is also disclosed that this effect can be enhanced when phenformin is administered in combination with at least one other pharmaceutically active ingredient. Phenformin may also be used in combination with other non-pharmaceutically active ingredients, as an alternative or in addition to other pharmaceutically active ingredients.

In view of the above, in another aspect of the present invention, a pharmaceutical composition comprising phenformin and at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient is disclosed.

Wherein the other pharmaceutically active ingredients are useful for the prevention and/or treatment of skin erythema, acantholysis, pemphigus, eczema, vitiligo, lichen planus, contact dermatitis, atopic dermatitis, burning, stinging and/or itching during inflammation, skin ulcers, acne;

other non-pharmaceutically active ingredients include pharmaceutically acceptable carriers, excipients and/or diluents. Such as pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, pharmacologically harmless salts such as sodium chloride, flavoring agents, vitamins such as vitamin a or vitamin E, tocopherols or similar vitamins or provitamins present in the human organism, antioxidants such as ascorbic acid, and stabilizers and/or preservatives for prolonging the use and shelf life of the pharmaceutically active ingredient or formulation, and other common non-pharmaceutically active ingredients or adjuvants and additives known in the art, and mixtures thereof. Other non-pharmaceutically active ingredients preferred according to the invention are in particular all substances which can form hydrogels, for example natural or synthetic water-soluble polymers which can form networks.

According to the use of the present invention, the administration of the pharmaceutical composition can be carried out topically, transdermally, nasally, enterally, parenterally, via infusion, via an endoscope, or as an aerosol or dry powder formulation. The specific dosage form of the pharmaceutical composition comprises oral preparation, injection, spray, cream, ointment, lotion, emulsion, gel or hydrogel.

Among these, the mode of administration is preferably topical and transdermal, carried out by applying the pharmaceutical composition on the skin, preferably a liquid, gas (aerosol or vapor), formulation containing phenformin, preferably as a cream, ointment, lotion, emulsion, or gel or hydrogel.

The preferred topical or transdermal administration of phenformin according to the present invention is highly advantageous for said skin inflammation related disorders. Preferred topical or transdermal administration according to the present invention allows for the topical application of high therapeutically effective concentrations of phenformin to the skin while reducing or completely avoiding systemic (blood circulation) loads and side effects on healthy skin tissue and tissue of other organs that do not require treatment.

At the same time, transport of phenformin from skin cells to the whole body can be inhibited or limited by means well known in the art. Examples of such means are mainly the targeted manipulation of the osmotic gradient through the skin (i.e. between the body part and the skin surface), the reduction of the water potential by using substances suitable for modifying the water potential of topical application of phenformin, which may be physiologically compatible salts such as sodium chloride, water-soluble polymers and other non-pharmaceutical substances.

The invention has the beneficial effects that: the invention firstly discovers that the phenformin can obviously inhibit the epidermal cells of the skin from secreting various inflammatory cytokines including interleukin 6/8(IL-6, IL-8) and tumor necrosis factor (TNF alpha), and also can obviously reduce the inflammatory reaction of the epidermal cells of the human skin induced by the commonly used 12-O-tetradecanoyl phorbol-13-acetate (TPA) and poly-inosinic-polycystic acid (poly I: C) (immunostimulant), thereby being used for preventing and treating diseases related to the skin inflammation. Therefore, the new application of the phenformin is developed, and a new prevention and treatment means is provided for the diseases related to the skin inflammation.

Drawings

FIG. 1 shows that different concentrations of phenformin inhibit various cytokines in epidermal cells in vitro cell culture (IL-6, IL-8,

TNF α) expression histogram;

FIG. 2 is a bar graph of inhibition of TPA-induced human skin cytokine expression by phenformin;

FIG. 3 is a bar graph of the expression of human skin cytokines induced by phenformin inhibitory immunostimulant Poly I: C;

FIG. 4 is a graph showing that phenformin treatment reduced TPA-induced inflammatory responses in the ears of mice; fig. 4a is a histopathological diagram of mouse ears under different treatment conditions, and fig. 4B is a histogram of analysis of mouse ear thickness under different treatment conditions.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

Interpretation of terms: in the sense of the present invention,

ointments are pharmaceutical preparations for external use, consisting of a matrix of a lubricating substance, such as vaseline, to which a truly medicinal, pharmaceutical and/or non-pharmaceutical active ingredient is added, for example by mixing;

a cream is an ointment according to the invention which may additionally comprise other constituent ingredients, for example cosmetically active ingredients such as fragrances, pigments and/or emulsifiers, for example lecithin. Creams should generally be distinguished from lotions, most of which depend on the viscosity grade being prepared. A cream is also understood to be an emulsion according to the invention.

Emulsions are macroemulsions or microemulsions based on water-in-oil or oil-in-water.

Gels are solutions of macromolecular substances, such as agarose, acrylic acid, alginic acid, silicone or acrylamide, in such high concentrations that the dissolved macromolecules combine into a spongy, three-dimensional structure with the presence of a fluid in their cavities under the appropriate combination and optional addition of other substances (e.g. salts, acids, fillers, buffer substances). The gel thus has a relatively fixed concentration. The viscosity is between liquid and solid.

Hydrogel means a gel which is characterized by a very high absorption capacity for water, preferably in the sense of the present invention 20 to 99%, more preferably 70 to 99%, and particularly preferably 80 to 99% of which consists of water, but which does not exhibit the rheological properties typical of fluids. In a particularly preferred embodiment, the hydrogel is transparent, permeable and at the same time easy to apply without affecting its morphology and integrity by applying the gel.

It is noted that the above-mentioned ointments, creams, lotions, milks or gels or hydrogels are carried out according to any standard method well known in the art. These methods and the concentrations to be selected for the components or substances to be used are known to the person skilled in the art.

As described in the background, phenformin has not been found to be useful for the prevention and treatment of diseases associated with skin inflammation;

in view of the above, in one embodiment of the present invention, the use of phenformin as an anti-inflammatory agent for the skin is disclosed, and thus, is extremely effective for preventing and/or treating diseases associated with dermatitis (skin inflammation);

in yet another embodiment of the present invention, the use of phenformin as a skin anti-inflammatory agent for the preparation of a medicament for the prevention and/or treatment of a disease associated with skin inflammation is disclosed;

wherein, the "skin anti-inflammatory agent" means a substance which blocks, delays or inhibits inflammatory cytokines, tumor necrosis factors and/or immunostimulants associated with skin inflammation;

wherein the inflammatory cytokines include, but are not limited to, interleukin-6/8 (IL-6, IL-8);

such tumor necrosis factors include, but are not limited to, TNF α/TNF β;

the immunostimulant includes, but is not limited to, 12-O-tetradecanoyl phorbol-13-acetate (TPA)/polyinosinic-polycytidilic acid (poly I: C);

in yet another embodiment of the present invention, it is disclosed that diseases associated with skin inflammation include, but are not limited to, erythema of the skin, acantholysis of the skin, pemphigus, eczema, vitiligo, lichen planus, contact dermatitis, atopic dermatitis, burning sensation during inflammation, stinging sensation and/or itching sensation, skin ulcers, acne;

in a further embodiment of the present invention, pharmaceutical compositions of phenformin with at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient for use according to the present invention are disclosed.

Wherein the other pharmaceutically active ingredients are useful for the prevention and/or treatment of skin erythema, acantholysis, pemphigus, eczema, vitiligo, lichen planus, contact dermatitis, atopic dermatitis, burning, stinging and/or itching during inflammation, skin ulcers, acne;

other non-pharmaceutically active ingredients include pharmaceutically acceptable carriers, excipients and/or diluents. Such as pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, pharmacologically harmless salts such as sodium chloride, flavoring agents, vitamins such as vitamin a or vitamin E, tocopherols or similar vitamins or provitamins present in the human organism, antioxidants such as ascorbic acid, and stabilizers and/or preservatives for prolonging the use and shelf life of the pharmaceutically active ingredient or formulation, and other common non-pharmaceutically active ingredients or adjuvants and additives known in the art, and mixtures thereof. Other non-pharmaceutically active ingredients preferred according to the invention are in particular all substances which can form hydrogels, for example natural or synthetic water-soluble polymers which can form networks.

In a further embodiment of the invention, the administration of the pharmaceutical composition according to the use of the invention can be carried out topically, transdermally, nasally, enterally, parenterally, via infusion, via endoscope, or as an aerosol or dry powder formulation. The specific dosage form of the pharmaceutical composition comprises oral preparation, injection, spray, cream, ointment, lotion, emulsion, gel or hydrogel.

Among these, the mode of administration is preferably topical and transdermal, carried out by applying the pharmaceutical composition on the skin, preferably a liquid, gas (aerosol or vapor), formulation containing phenformin, preferably as a cream, ointment, lotion, emulsion, or gel or hydrogel.

It should be noted that phenformin may induce lactic acidosis (incidence rate is about six parts per million (0.06%)) in the treatment of diabetic patients, and there has been no report of lactic acidosis caused by topical application. Therefore, the invention provides the local external use of the phenformin, which can effectively avoid the active ingredients of the medicine from acting on other parts of the body and is relatively safe. However, for safety reasons, it is not recommended to use diabetics with dermatitis-related disorders before extensive case trials have been carried out to verify their safety.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.

Examples

Materials and methods

1) Separating and culturing human original representative skin cells:

pretreating the tissue in 70% ethanol for 30 seconds, putting the pretreated tissue into PBS containing 2% streptomycin for sterilization, chopping the tissue into homogenate, transferring the homogenate into a 50ml centrifuge tube containing enzyme digestion solution, shaking the homogenate in 37 ℃ water bath for 1 hour, and then adding 0.25% trypsin for digestion for 30 minutes; after digestion, adding the same volume of DMED medium containing 10% fetal calf serum, repeatedly blowing, filtering, centrifuging at 1000Xg for 5 minutes, removing supernatant, repeating once, re-suspending cell sediment by using epidermal medium, and adding Rock inhibitor Y27632 for culture.

2) Phenformin-treated epidermal cells:

cultured epidermal cells (P3) were inoculated into a six-well plate using 2ml of medium per 30 ten thousand cells per well, the medium was changed after 24 hours of inoculation, and the epidermal cells were treated with different concentrations of phenformin (0,0.5mM/L, 1.0mM/L, 1.5mM/L) for 24 hours.

3) TPA/Poly (I: C) + phenformin treated epidermal cells:

cultured epidermal cells (P3) were inoculated into a six-well plate using 2ml of medium per 30 ten thousand cells, the medium was changed after 24 hours of inoculation, and then epidermal cells were treated with 5ug/ml TPA, 5ug/ml Poly (I: C), respectively, for 2 hours, and then epidermal cells were treated with phenformin (0,0.5mM/L, 1.0mM/L, 1.5mM/L) for 22 hours.

4) Cell collection RNA and protein extraction:

RNA was extracted using a TAKARA kit and sample proteins were extracted using the RIPA lysis method.

5) RT-PCR assay for inflammatory factor expression:

RNA samples are subjected to reverse transcription by using a TAKARA kit, and then the expression of various inflammatory factors including IL6, IL8 and TNF-alpha and ATF-3 is detected by adopting a real-time quantitative PCR technology.

6) ELISA assay for inflammatory factor levels:

taking cell culture solution supernatant: the sample is centrifuged at 1000Xg for 15 minutes at 4 ℃, the supernatant is taken as an Elisa experimental sample, and the R & D systemsValukine kit is adopted to detect the expression of IL-6, IL-8 and TNF-alpha.

7) Phenformin inhibits TPA-induced inflammatory responses in vivo:

the mouse inflammation model was established by inducing an inflammatory response in the right ear of C57 mice for 2 hours using TPA, followed by treatment with phenformin, plus Aceton as a control. After 24 hours, the ears were harvested and their thickness was measured, and the tissue structure of the ears and invasion of inflammatory cells were observed by HE staining.

Test results

1. As shown in figure 1, the phenformin inhibits the expression of skin epidermal inflammatory factors, has a dose response relationship, and has more obvious inhibition effect on IL-6, IL-8 and TNF-alpha when the concentration of the phenformin is higher within the concentration range of 0.5-1.5 mM/L.

2. In vitro, the phenformin can effectively inhibit inflammatory reaction of human skin epidermal cells caused by TPA to generate an anti-inflammatory effect, as shown in figure 2, the phenformin can effectively inhibit the expression of human skin cell inflammatory factors induced by TPA, and the inhibition effect is more obvious when the concentration of the phenformin is higher within the concentration range of 0.5-1.5 mM/L;

3. in vitro, phenformin can effectively inhibit inflammatory reaction of human skin epidermal cells caused by immunostimulant Poly I: C to generate anti-inflammatory effect, as shown in figure 3, phenformin can effectively inhibit TPA-induced human skin cell inflammatory factor expression, and the inhibition effect is more obvious when the concentration of phenformin is higher within the concentration range of 0.5-1.5 mM/L;

4. in animal (mouse) experiments (in vivo), phenformin can also eliminate skin inflammatory reaction induced by TPA in vivo, as shown in FIG. 4A, the addition of phenformin significantly reduces inflammatory reaction and inflammatory cell infiltration; FIG. 4B is a graph showing the quantitative analysis of the change in ear thickness in mice, representing significant differences (p <0.05) between the control (control) and the TPA + phenformin (TPA + phenformin) without phenformin.

As can be seen from the above tests, phenformin can effectively inhibit the expression of various inflammatory factors including IL-6, IL-8, TNF-alpha and the like in vivo or in vitro, and the higher the concentration of phenformin is, the stronger the anti-inflammatory effect is in a certain concentration range. The compound can be used as an anti-inflammatory drug for external application to the skin.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (1)

1. Use of phenformin for the preparation of an anti-skin inflammation formulation, characterized in that said anti-skin inflammation formulation is administered by infusion, topical or transdermal administration;

the skin inflammation is induced by 12-O-tetradecanoyl phorbol-13-acetic ester or poly-sarcosine.

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