阅读说明：本技术 一类化合物促皮肤损伤修复的用途 (Application of compound in promoting skin injury repair ) 是由 李英杰 骆衍新 白亮亮 蔡都 王小琳 禹汇川 唐冠楠 黄美近 汪建平 于 2020-06-19 设计创作，主要内容包括：本发明涉及聚醚化合物在在制备促皮肤修复和/或再生药物或药物组合物中的用途。本发明证实,包括莫能霉素(Monensin A)、南昌霉素(Nanchangmycin)、拉沙里菌素(Lasalocid A)、尼日利亚菌素(Negericin)、沙利霉素(Salinomycin)、马杜拉霉素(Maduramycin)、甲基盐霉素(Narasin)在内的聚醚化合物体外能显著促进人表皮角质细胞和人胚肾上皮细胞的增殖。其中,莫能霉素(Monensin A)还能明显促进小鼠表皮成纤维细胞的增殖,并在BALB/C小鼠以及糖尿病和肥胖模型小鼠皮肤损伤模型中显著促进损伤愈合,疗效优于相同浓度的人重组EGF。(The invention relates to an application of polyether compound in preparing a medicine or a pharmaceutical composition for promoting skin repair and/or regeneration. The invention proves that polyether compounds including Monensin (Monensin A), nanchanmycin (Nanchangmycin), Lasalocid (Lasalocid A), nigericin (Negericin), Salinomycin (Salinomycin), Maduramycin (Maduramycin) and Narasin (Narasin) can remarkably promote the proliferation of human epidermal keratinocytes and human embryonic kidney epithelial cells in addition. Wherein, the Monensin A can also obviously promote the proliferation of epidermal fibroblasts of mice, and obviously promote the injury healing in BALB/C mice and mice skin injury models of diabetes and obesity models, and the curative effect is better than that of human recombinant EGF with the same concentration.)

1. Use of a polyether compound or a derivative thereof, a chelated form, a hydrated form or a pharmaceutically acceptable salt or ester thereof, for the preparation of a medicament or pharmaceutical composition for promoting epithelial tissue repair and/or regeneration.

2. Use according to claim 1, wherein the polyether compound comprises 2 to 12 ether oxygen atoms and 1 monobasic organic acid group;

preferably, 2 to 10 ether oxygen atoms are contained;

preferably, 2 to 8 ether oxygen atoms are contained;

preferably, 2 to 7 ether oxygen atoms are contained;

preferably, 2 to 6 ether oxygen atoms are contained;

preferably, 2 to 5 ether oxygen atoms are contained;

preferably, 2 ether oxygen atoms are contained.

3. Use according to claim 1, wherein the polyether compound comprises a cyclic ether structure;

preferably, at least 1 tetrahydrofuran and/or 1 tetrahydropyranyl group is included;

preferably, at least 1 tetrahydrofuran group and 1 tetrahydropyran group are included;

preferably, the tetrahydrofuran group and the tetrahydropyran group are directly connected by a carbon-carbon single bond;

preferably, either one of the tetrahydrofuran group and the tetrahydropyran group is attached to the other group at the carbon in the 2-position;

preferably, the tetrahydrofuran group and the tetrahydropyran group are both attached to each other at the carbon in position 2.

4. The use of claim 1, wherein the polyether compound comprises at least one of the structures of formula I, formula II, formula III, formula IV, formula V, formula VI, or formula VII,

wherein R is₁₀₁To R₁₃₉、R₂₀₁To R₂₅₁、R₃₀₁To R₃₃₃、R₄₀₁To R₄₄₅、R₅₀₁To R₅₄₃、R₆₀₁To R₆₄₃、R₇₀₁To R₇₅₃Each is independently selected from one or more of hydrogen, methyl, ethyl, propyl, hydroxyl, methoxy, hydroxymethyl, hydroxyl or halogen;

preferably, X is selected from one or more of hydrogen, methyl, ethyl, propyl.

5. The use according to claim 1, wherein the polyether compound is selected from at least one of Monensin (Monensin a), nanchanmycin (Nanchangmycin), Lasalocid (Lasalocid a), nigericin (negicin), Salinomycin (Salinomycin), Narasin (Narasin), maduramicin (maduramicin);

preferably, the Monensin (Monensin a), nanchanmycin (Nanchangmycin), Lasalocid (Lasalocid a), nigericin (negicin), Salinomycin (Salinomycin), naramycin (Narasin), Maduramycin (Maduramycin) have the following structural formulas, respectively:

6. use according to claim 1, wherein the polyether compound is synthetic or isolated from a microbial fermentation broth.

7. Use according to claim 1, wherein the polyether compound is used at a concentration of between 1 and 1000 nM.

8. The use of claim 1, wherein said promotion of epithelial tissue repair and/or regeneration comprises promotion of repair and/or regeneration of a skin wound or scar;

preferably, the skin wound is selected from the group consisting of wounds, burns, ulcers;

preferably, the wound is selected from an excision wound, a cut, a puncture wound or a puncture wound.

9. The use of claim 1, wherein the medicament or pharmaceutical composition is formulated for topical or transdermal administration.

10. The use of claim 1, wherein the medicament or pharmaceutical composition is formulated as a gel, spray, paste, ointment, cream, lotion, ointment, oil, aqueous solution, suspension, dispersion, patch, adhesive, bandage, dressing, depot or reservoir.

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of a polyether compound in promoting repair and/or regeneration of epithelial tissues.

Background

The human skin has large area and is directly contacted with the outside, and the human skin is the organ which is most easily damaged by the outside. The normal healing of skin wound is vital to maintain the steady state of the body, if abnormal healing occurs, such as large-area wound or burn, difficult healing of chronic wound surface caused by chronic diseases, slow healing of drug-induced wound surface, blocked healing of wound surface caused by infection and the like, not only the loss of skin structure and function can be caused, but also the pain and aesthetic damage caused by the loss of skin structure and function often cause heavy psychological burden to the patient, and the physical and psychological health of the patient is seriously influenced. Promoting healing of acute and chronic skin wounds to the maximum recovery of tissue function is one of the major concerns of current research.

Recombinant cytokines and polypeptides are common biological agents for treating skin lesions. Clinically, the recombinant basic fibroblast growth factor (bFGF) which is a new Chinese medicine class I is clinically applied to the treatment of wounds, burns and scalds; another clinical medicine for treating wound, KANGFUXIN, contains active polypeptide of Periplaneta americana as main ingredient. In addition, some of the short-segment skin repair peptides which have been patented, such as the lizard-derived polypeptide Tylotoin, the repair polypeptides AH90 and CW49 isolated from rana grahami, and the small-molecule remodeling peptide targeting fibroblast growth factor receptor 2(FGFR2), have the curative effect of repairing skin injury. However, the treatment of skin lesions with polypeptides and recombinant proteins presents challenges: 1) the protein or the polypeptide is prepared by a genetic engineering or industrial synthesis method, so the preparation cost is high; 2) the protein and the polypeptide have large molecular weight, are unstable and need harsher transportation conditions; 3) is easy to be hydrolyzed by hydrolase in vivo, and has unstable curative effect. In contrast, small molecule compounds have natural advantages: 1) the molecular weight is small, and the degradation is not easy; 2) the preparation cost is low; 3) can be continuously used in vivo, etc. To date, there are no small molecule compounds available for clinical treatment of skin wounds. The search for lead compounds that enhance the endogenous regenerative capacity of the skin has become a great need in society.

The polyether compound is a polyether polyketone compound containing a carboxylic acid group and a plurality of five-membered or six-membered ether rings. Monensin (Monensin a), nanchanmycin (Nanchangmycin), Lasalocid (Lasalocid a), nigericin (negicin), Salinomycin (Salinomycin), Narasin (Narasin), and Maduramycin (Maduramycin) are representatives of this class of compounds, which have been used successively as anticoccidial and antibacterial agents for feed additives and can promote the growth and development of animals. Such compounds can bind to metal cations and can carry the metal cation compound across the cell membrane into the cell. The mechanism of action of the composition for killing coccidiosis is mainly high-affinity Na⁺Ions, make a large amount of Na⁺Enter cells to interfere with coccidian cells Na⁺Normal penetration of (2). The compound is mainly prepared by a biological fermentation method and synthesized by utilizing the secondary metabolism of production bacteria, and has mature extraction and separation processes and low preparation cost.

It has been shown that intracellular Na⁺The enhancement of ions plays an important role in tissue repair and regeneration. Tseng AS et al observed Xenopus laevis (Xenopus laevis) tail injury site Na⁺The accumulation of ions, monensin, can increase intracellular Na⁺Ion, remarkably promotes the repair and regeneration of the damaged tail of Xenopus laevis [4]. However, no report on the application of the compound in the repair treatment of mammal tissue damage exists at present, and no related patent application exists. The antibiotics have definite action mechanism, low toxic and side effect and mature preparation process, but are only limited to be applied to feed additives in animal husbandry at present. Further research and development of application of the compounds in tissue repair and regenerationThe application range of the method is expanded, and the method has important significance. In view of this, the invention is particularly proposed.

Disclosure of Invention

At present, the application of polyether compounds, especially polyether antibiotics, is mainly reported as follows: 1) mainly used for resisting coccidiosis in animal husbandry; 2) the additive is added to livestock feed, so that the growth can be obviously promoted, the utilization rate of the feed can be improved, and diseases can be controlled; 3) inhibiting breast cancer cell activity; 4) has killing effect on plasmodium.

In one aspect, the present invention provides the use of a polyether compound or a derivative thereof, a chelated form, a hydrated form or a pharmaceutically acceptable salt or ester thereof, for the manufacture of a medicament or pharmaceutical composition for promoting epithelial tissue repair and/or regeneration.

In some embodiments, the polyether compound contains 2 to 12 ether oxygen atoms and 1 monobasic organic acid group.

In some embodiments, from 2 to 10 ether oxygen atoms are included.

In some embodiments, from 2 to 8 ether oxygen atoms are included.

In some embodiments, from 2 to 7 ether oxygen atoms are included.

In some embodiments, from 2 to 6 ether oxygen atoms are included.

In some embodiments, from 2 to 5 ether oxygen atoms are included.

In some embodiments, 2 ether oxygen atoms are included.

In some embodiments, the polyether compound comprises a cyclic ether structure.

In some embodiments, at least 1 tetrahydrofuran and/or 1 tetrahydropyranyl group is included.

In some embodiments, at least 1 tetrahydrofuran group and 1 tetrahydropyran group are included.

In some embodiments, the tetrahydrofuran group and the tetrahydropyran group are directly connected by a carbon-carbon single bond.

In some embodiments, either one of the tetrahydrofuran group and the tetrahydropyran group is attached to the other group at the carbon in the 2-position.

In some embodiments, the tetrahydrofuran group and the tetrahydropyran group are both attached to each other at the carbon in the 2-position.

In some embodiments, the polyether compound is at least one of the structures of formula I, formula II, formula III, formula IV, formula V, formula VI, or formula VII,

wherein R is₁₀₁To R₁₃₉、R₂₀₁To R₂₅₁、R₃₀₁To R₃₃₃、R₄₀₁To R₄₄₅、R₅₀₁To R₅₄₃、R₆₀₁To R₆₄₃、R₇₀₁To R₇₅₃Each independently selected from one or more of hydrogen, methyl, ethyl, propyl, hydroxy, methoxy, hydroxymethyl, hydroxy or halogen.

In some embodiments, X is selected from one or more of hydrogen, methyl, ethyl, propyl.

In some embodiments, the polyether compound is selected from at least one of Monensin (Monensin a), nanchanmycin (Nanchangmycin), Lasalocid (Lasalocid a), nigericin (negicin), Salinomycin (Salinomycin), naramycin (Narasin) and Maduramycin (Maduramycin).

In some embodiments, the Monensin (Monensin a), nanchanmycin (Nanchangmycin), Lasalocid (Lasalocid a), nigericin (negicin), Salinomycin (Salinomycin), naramycin (Narasin), Maduramycin (Maduramycin) have the following structural formulae, respectively:

the epidermis of mammalian skin is a continuous, multilayered epithelium that is highly regenerative. Under physiological conditions, the epidermis is constantly aging and exfoliating, while basal cell division, proliferation and differentiation are constantly replacing [1 ]. Thus, skin possesses a delicate, complex, ordered set of mechanisms to protect itself and restore tissue integrity when damaged, a process that is coordinated by multiple cells and cytokines [2 ]. The process of skin injury repair can be divided into four stages: hemostasis → inflammation → epidermal tissue regeneration → epidermal remodeling. Among them, epidermal tissue regeneration is the most important stage of the entire set of repair mechanisms. At this stage, granulation tissue consisting of fibroblasts at the sprouting sites of new capillaries grows from the wound edges and bottoms and spreads towards the center and surface of the defect, filling the depressed wound and becoming a new substrate for later keratinocyte (keratinocytes) migration. Subsequently, the basal keratinocytes begin to proliferate, migrate under the clot from the wound edge to the wound center, form a monolayer of epithelial cells on the granulation tissue, and eventually proliferate, differentiate into squamous epithelium. During the period, fibroblasts from the periphery of the wound and bone marrow are activated by macrophages, secrete collagen and form scars; wherein a part of the fibroblasts differentiate into myofibroblasts to promote the condensation of the wound margin. In fact, the ability of the epidermis to self-renew, repair is maintained primarily by basal stem cells. The epidermal stem cells have strong self-renewal capacity, complete self-renewal through asymmetric division, and simultaneously form a new daughter cell, namely a transient expansion cell. The transiently expanded cells continue to divide, expand, move up to form additional layers of cells, such as layers of spiked cells, granular cells, etc. [1 ]. During wound repair, cells involved in repair secrete a variety of cytokines, including EGF, TGF- β, FGF and VEGF, among others, which in turn regulate cell proliferation, migration and differentiation [3 ]. However, skin wounds often have difficulty healing due to severe tissue defects or inadequate repair mechanisms. Aiming at the problem, an effective clinical treatment means is not available at present except for preventing wound infection. The key target of skin injury repair is found, the repair mechanism is clarified, and a safe and effective skin injury treatment scheme is favorably found.

In one embodiment of the present invention, the polyether compounds Monensin (Monensin a), nanchanmycin (Nanchangmycin), Lasalocid (Lasalocid a), nigericin (negicin), Salinomycin (Salinomycin), Narasin (Narasin), maduramicin (maduramicin) were demonstrated to significantly promote the proliferation of human epidermal keratinocytes (HACAT) and human embryonic kidney epithelial cells (HEK293) cells in a dose-dependent relationship.

In one embodiment of the present invention, it was demonstrated that Monensin (Monensin a) has a significant pro-proliferative effect on mouse epidermal fibroblasts (MDF) in a dose-dependent relationship.

In some embodiments, the polyether compound is synthetic or isolated from a microbial fermentation broth.

In some embodiments, the polyether compound is used at a concentration of 1-1000 nM.

In some embodiments, the promotion of epithelial tissue repair and/or regeneration comprises promotion of repair and/or regeneration of a skin wound or scar.

In some embodiments, the skin wound is selected from the group consisting of a wound, a burn, a scald, and an ulcer.

In some embodiments, the wound is selected from an excision wound, a cut, a puncture wound, or a puncture wound.

In some embodiments, the medicament or pharmaceutical composition is formulated for topical or transdermal administration.

In some embodiments, the medicament or pharmaceutical composition is formulated as a gel, spray, paste, ointment, cream, lotion, ointment, oil, aqueous solution, suspension, dispersion, patch, adhesive, bandage, dressing, depot or reservoir.

Drawings

Fig. 1 is a structural formula of some polyether compounds, in some embodiments, wherein: FIG. 1A is Monensin (Monensin A), FIG. 1B is Nanchangmycin (Nanchangmycin), FIG. 1C is lasalocin (Lasalocid A), FIG. 1D is nigericin (Negericin), FIG. 1E is Salinomycin (Salinomycin), FIG. 1F is Narasin (Narasin), and FIG. 1G is Maduramycin (Maduramycin);

FIGS. 2A and 2B show that Monensin (Monensin A), nanchanmycin (Nanchangmycin), Lasalocid (Lasalacicin A), nigericin (Negericin), Salinomycin (Salinomycin), Maduramycin (Maduramycin) and Narasin (Narasin) significantly promote proliferation of human epidermal keratinocytes (HACAT) and human embryonic kidney epithelial cells (HEK293), respectively;

FIG. 3 shows that Monensin (Monensin A) significantly promoted proliferation of mouse epidermal fibroblasts (MDF);

FIG. 4A shows representative pictures of skin injury healing effects of 0-14 days Monensin (Monensin A) and EGF in BALB/C mice, and FIG. 4B shows the quantitative results of the wound areas of the mice in each group;

FIG. 5A shows the growth of Monensin A and EGF BSK-Lepr on days 0, 16, and 24^em2Cd479Representative pictures of skin injury healing effect of/Gpt mice, and fig. 5B shows the quantitative results of the wound area of each group of mice.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

The invention is tested on in vitro cell and mouse animal models, and the test scheme is a conventional method.

Example 1 evaluation of in vitro cell proliferation promoting Activity of 7 polyether Compounds on human epidermal keratinocytes (HACAT) and human embryonic Kidney epithelial cells (HEK293)

HACAT and HEK293 cells in logarithmic growth were seeded in 96-well plates at 5000 cells per well, and after the cells were fully attached to the wall, polyether compounds were added at the concentrations of Table 1 and cultured for 3 days. To the cell culture plate, 20. mu.L/well of MTS assay was added and incubated at 37 ℃ for 4 hours. The absorbance (OD value) was measured at a wavelength of 490nm using a microplate reader. Relative cell viability (OD)_{Medicine adding device}-OD_{Blank group})/(OD_{Control group}-OD_{Blank group})*100％。

TABLE 1 concentration of polyether compound added to different cells (unit: nM)

As shown in FIG. 2, the proliferation-promoting effects of Monensin (Monensin A), nanchanmycin (Nanchangmycin), Lasalocid (Lasalocid A), nigericin (Negericin), Salinomycin (Salinomycin), Maduramycin (Maduramycin), Narasin (Narasin) on HACAT and HEK293 cells were dose-dependent and ranged between 1-1000 nM.

Example 2 Proliferative Effect of Monensin (Monensin A) on mouse epidermal fibroblasts (MDF)

Inoculating mouse epidermal fibroblast (MDF) in logarithmic growth into a 96-well plate, wherein 5000 cells are in each well, and adding Monensin (Monensin A) with different concentrations to culture for 3 days after the cells are completely attached to the wall. The cell viability was measured by the MTS method, and the relative cell viability (relative cell viability) was calculated.

As shown in FIG. 3, Monensin A significantly promoted MDF cell proliferation in a dose-dependent manner. The optimum concentration of Monensin (Monensin A) for promoting MDF is between 1-2 nM.

Example 3 repair of skin injury model in BALB/C mice by Monensin A

BALB/C mice (female, 6-8 weeks old, weight 20-25g) were anesthetized, shaved on their backs, and then depilated clean with depilatory cream. A biopsy punch with a 6mm caliber was used to punch holes in the back of the mice, and a digital camera recorded the wounds with a 10mm aperture silicone sheet as a reference. Mice were randomized into 4 groups, physiological saline (Vehicle), Monensin a high dose (Monensin a250nM), Monensin a low dose (Monensin a125nM), and human recombinant EGF (EGF) Growth factor (EGF250 nM). The preparation is administered once every two days, and is administered for 7 times, 20 μ L each time, and wound is recorded by using digital camera with 10mm aperture silica gel sheet as reference. After administration, the wound was adhered to the entire back using a 3M medical transparent patch to prevent skin shrinkage and drug spillage. Wound and silica gel pore areas were measured using Image J and wound relative area and wound area relative values were calculated. Wound relative area-wound area/silica gel pore area 100%; relative wound area of wound (relative area of wound) is 100% of the relative wound area on day N/day 0.

The results are shown in fig. 4, and the Monensin (Monensin a) can remarkably promote the healing of skin wounds and is dose-dependent. Monensin (Monensin a) showed significant effect starting on day 6 of wound healing and the therapeutic effect was superior to that of EGF at the same concentration on days 8-14.

Example 4 Moonemycin (Monensin A) promotes BSK-Lepr^em2Cd479Gpt mouse skin injury repair

BSK-Lepr^em2Cd479A/Gpt mouse (female, 6-8 weeks old, weight 40-55g) was anesthetized, shaved on its back, and depilated cleanly with depilatory cream. The back of the mouse was punched with a 6mm bore biopsy punch and the wound was recorded using a digital camera with a 10mm bore silicone sheet as a reference. Mice were randomized into 4 groups, physiological saline (Vehicle), Monensin a high dose (Monensin a250nM), Monensin a low dose (Monensin a125nM), and EGF (EGF250nM), respectively. The drug is administered once every 4 days, 7 times continuously, 20 μ L each time, and the wound surface is recorded by photographing. After administration, the wound was adhered to the entire back using a 3M medical transparent patch to prevent skin shrinkage and drug spillage. The wound and silica gel pore areas were measured using Image J,relative wound area and relative wound area values were calculated. Wound relative area-wound area/silica gel pore area 100%; relative wound area of wound (relative area of wound) is 100% of the relative wound area on day N/day 0.

As shown in FIG. 5, from day 16, Monensin A significantly promoted BSK-Lepr^em2Cd479the/Gpt mice had skin wounds that healed in a dose-dependent manner. EGF showed significant effects on days 16-28 of wound healing. The curative effect of the Monensin (Monensin A) is equivalent to that of EGF at the same concentration.

Reference to the literature

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2.Geoffrey C.Gurtner,Sabine Werner,Yann Barrandon,et al.Wound repair and regeneration[J].Nature,2008,453(7193):314-321.

3.Sun B K,Siprashvili Z,Khavari P A.Advances in skin grafting and treatment of cutaneous wounds[J].Science,2014,346(6212):941-945.

4.A.-S,Tseng,W,et al.Induction of Vertebrate Regeneration by a Transient Sodium Current[J].Journal of Neuroscience,2010,30(39):13192-13200.