阅读说明：本技术 Pk11195的新用途 (New application of PK11195 ) 是由 吕金鹏 姜松周 杨莹 安晓虹 高榕荫 曹妍 宋国强 恽常军 于 2019-11-01 设计创作，主要内容包括：本发明属于医药技术领域,涉及PK11195的新用途,具体涉及PK11195或其药学上可接受的盐在制备治疗和/或预防色素增多性疾病的药物/化妆品的用途,特别是在制备治疗和/或预防雀斑、妊娠斑、黄褐斑、咖啡斑、色痣等各种遗传性、紫外线、炎症因素诱导的色素沉着的药物/化妆品的用途。(The invention belongs to the technical field of medicines, relates to a new application of PK11195, and particularly relates to an application of PK11195 or a pharmaceutically acceptable salt thereof in preparing medicines/cosmetics for treating and/or preventing hyperpigmentation diseases, in particular to an application in preparing medicines/cosmetics for treating and/or preventing pigmentation induced by various hereditary, ultraviolet rays and inflammatory factors such as freckles, cyasma, chloasma, coffee spots, pigmented nevi and the like.)

Use of PK11195 for the preparation of medicaments/cosmetics for the treatment and/or prevention of hyperchromic disorders.

2. Use according to claim 1, characterized in that: the hyperchromic disease is freckle, cyasma, chloasma, coffee stain, pigmented nevus, ultraviolet induced pigmentation, and hereditary black skin.

Use of a pharmaceutically acceptable salt of PK11195 in the preparation of a medicament/cosmetic for the treatment and/or prevention of a hyperchromic disorder.

4. Use according to claim 3, characterized in that: the pigmentation diseases include freckle, cyasma, chloasma, coffee stain, pigmented nevus, ultraviolet induced pigmentation, and hereditary black skin.

5. Use of a pharmaceutical composition for the preparation of a medicament/cosmetic for the treatment and/or prevention of a hyperchromic disorder, characterized in that: the pharmaceutical composition contains a therapeutically effective amount of PK11195 or a pharmaceutically acceptable salt thereof.

6. Use according to claim 5, characterized in that: the hyperchromic disease is freckle, cyasma, chloasma, coffee stain, pigmented nevus, ultraviolet induced pigmentation, and hereditary black skin.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a new application of PK 11195.

Background

The skin melanin plays an important role in the aspects of people's normal social interaction, protection of people from ultraviolet radiation and the like. Melanin synthesis can be induced by various factors in vivo and in vitro, such as alpha-melanocyte stimulating hormone (alpha-MSH), adrenocorticotropic hormone (ACTH), and ultraviolet rays. However, if a large amount of melanin accumulates in the skin, hyperpigmentation disorders such as freckles, cyasma, chloasma, coffee spots, moles, uv-induced pigmentation, hereditary black skin can result. The hyperchromic disease not only seriously affects the appearance of the patient, but also exerts a great mental stress thereon, and may even induce skin cancer such as melanoma and the like. Currently, people pay more attention to the prevention and treatment of the diseases along with the improvement of living standard.

At present, various compounds for inhibiting pigment synthesis are applied to the prevention and treatment of pigment-increasing diseases, such as hydroquinone and arbutin (hydroquinone derivatives) which are used as whitening agents are widely applied to medicines/cosmetics. In addition, hydroquinone, kojic acid and the like are widely used whitening agents at present. However, the whitening agents have certain toxic and side effects in practical application, and kojic acid is very easy to oxidize in the air, so that the whitening agents are very unstable in properties. Hydroquinone is highly toxic to melanocytes and thus easily causes skin toxicity, and in addition, it causes chloasma and mental problems if it is used for a long period of time, so that it has been gradually prohibited from clinical use. Arbutin, as a hydroquinone derivative, also causes skin toxicity. Therefore, there is an increasing need to develop drugs/cosmetics that inhibit the synthesis of pigments with novel action patterns and definite therapeutic effects.

PK11195 (CAS: 85532-75-8), chemical name 1- (2-chlorophenyl) -N-methyl-N- (1-methylpropyl) -3-isoquinoline carboxamide. The molecule is C₂₁H₂₁ClN₂O, molecular weight 352.86. As a white solid; no smell, slightly bitter taste. Dissolving in ethanol, methanol, dichloromethane, and DMSO. The melting point is 136-138 ℃. The chemical structural formula is as follows:

PK11195 is an isoquinoline carboxamide, which selectively binds to the Peripheral Benzodiazepine Receptor (PBR). PK11195 has nM concentration level affinity with PBR, and is one of the most widely used PBR ligands at present. The expression of PBR is obviously up-regulated in the process of nerve damage, so PK11195 is widely applied to early diagnosis of central nervous system diseases. In addition, PK11195 is also reported to have anti-inflammatory effect, but the pigment synthesis inhibition effect is not reported at home and abroad at present.

Disclosure of Invention

In view of the above, the present invention provides a new use of PK11195, and in particular relates to a use of PK11195 in preparing a medicament/cosmetic for treating and/or preventing hyperpigmentation diseases, especially in preparing a medicament/cosmetic for treating and/or preventing freckles, cyasma, chloasma, coffee spot, moles, uv-induced pigmentation, and hereditary black skin.

In one aspect, the invention provides the use of PK11195 for the preparation of a medicament/cosmetic for the treatment and/or prevention of hyperchromic disorders. Further, the hyperchromic disease is freckle, cyasma, chloasma, coffee spot, pigmented nevus, ultraviolet-induced pigmentation, and hereditary black skin.

In another aspect, the invention provides the use of a pharmaceutically acceptable salt of PK11195 for the manufacture of a medicament/cosmetic for the treatment and/or prevention of hyperchromic disorders. Further, the hyperchromic disease is freckle, cyasma, chloasma, coffee spot, pigmented nevus, ultraviolet-induced pigmentation, and hereditary black skin.

In another aspect, the present invention provides a use of a pharmaceutical composition for the preparation of a medicament/cosmetic for the treatment and/or prevention of a hyperchromic disease, said medicament/cosmetic composition comprising a therapeutically effective amount of PK11195 or a pharmaceutically acceptable salt thereof. Further, the hyperchromic disease is freckle, cyasma, chloasma, coffee spot, pigmented nevus, ultraviolet-induced pigmentation, and hereditary black skin.

The invention proves that PK11195 can inhibit the synthesis of melanin in a normal state, can inhibit the synthesis of melanin induced by endogenous factors (alpha-MSH and ACTH) and environmental factors (ultraviolet rays) through a large amount of modern pharmacological scientific researches and in-vitro and in-vivo pharmacodynamic experiments, has the efficacy of treating diseases with hyperpigmentation, and particularly treats freckles, cyasma, chloasma, coffee spots, naevus, ultraviolet ray-induced pigmentation and hereditary black skin.

Firstly, the method comprises the following steps: effect of PK11195 on melanin synthesis in Normal human Primary melanocytes

Experiments show that: PK11195 had no significant effect on human primary melanocyte proliferation and showed lower toxicity.

Experiments show that: PK11195 can obviously inhibit the synthesis of human primary melanocyte melanin.

Experiments show that: PK11195 can inhibit melanin synthesis by inhibiting the expression of key proteins (MITF, TYR) for melanin synthesis in human primary melanocytes.

II, secondly: effect of PK11195 on the Synthesis of Zebra Fish melanin

Experiments show that: PK11195 can remarkably inhibit the synthesis of melanin of zebra fish

Thirdly, the method comprises the following steps: effect of PK11195 on UV-induced dorsal skin pigmentation in Guinea pigs

Experiments show that: PK11195 can remarkably reduce pigmentation of back skin of guinea pigs caused by ultraviolet rays.

Has the advantages that: the invention provides an application of PK11195 or a pharmaceutically acceptable salt thereof in preparing a medicament/cosmetic for treating and/or preventing hyperpigmentation diseases, in particular an application in preparing a medicament/cosmetic for treating and/or preventing freckles, cyasma, chloasma, coffee spots, pigmented nevi, ultraviolet-induced pigmentation and hereditary black skin. Experiments prove that PK11195 can inhibit the synthesis of melanin by inhibiting the expression of important proteins (MITF and TYR) for the synthesis of melanin in melanocytes; PK11195 can inhibit melanin synthesis of skin of zebra fish and guinea pig. Provides a new effective treatment option for hyperchromia diseases.

Drawings

Figure 1 is the effect of PK11195 on the growth of human primary melanocytes.

FIG. 2 is the effect of PK11195 on alpha-MSH-induced melanin synthesis in human primary melanocytes.

Fig. 3 is the effect of PK11195 on ACTH-induced melanin synthesis in human primary melanocytes.

FIG. 4 is a graph showing the effect of PK11195 on UV-induced melanin synthesis in human primary melanocytes.

FIG. 5 shows the effect of PK11195 on the expression of MITF and TYR, key proteins in melanin synthesis in human primary melanocytes.

FIG. 6 is a graph of the effect of PK11195 on the synthesis of melanin in zebrafish.

Fig. 7 is a photograph of the effect of PK11195 on melanin synthesis in guinea pig back skin (back skin photograph).

FIG. 8 is a graph of the effect of PK11195 on the synthesis of melanin in the skin of guinea pigs on the back (ammoniacal silver staining of melanin in the back skin).

Fig. 9 is a chart of the color difference of PK11195 versus control over time on the skin of guinea pig backs.

Detailed Description

To further illustrate the present invention, a series of examples are given below, which are purely illustrative and are intended to be a detailed description of the invention only and should not be understood as limiting the invention.

The following are some of the pharmacodynamic tests and results of the present invention:

a first part: effect of PK11195 on melanin synthesis in human Primary melanocytes

First, PK11195 influences the proliferation of human primary melanocytes

Taking normal primary melanocyte in exponential growth phase, digesting, counting, inoculating the cells in 96-well culture plate, inoculating density of 5 × 10⁴One seed/ml, the inoculation amount is 180 mu l/hole, the mixture is placed at 37 ℃ and 5 percent CO₂Culturing in an incubator for 24 hours; adding PK11195 with different concentrations, and culturing for 48 hours; adding 20 mul MTT into each hole, and reacting for 4 hours in an incubator at 37 ℃; the supernatant was aspirated off, 200. mu.l DMSO was added to each well and shaken on a plate shaker for 10 min; measuring the absorbance of each well at 570nm wavelength with a microplate reader,and (5) calculating the cell proliferation rate. The results are shown in FIG. 1.

The experimental results are as follows: compared with the blank control group, the PK11195 administration group has no significant influence on the growth of the primary melanocyte of the normal human (P > 0.05).

And (4) experimental conclusion: PK11195 had no significant effect on human primary melanocyte proliferation and showed lower toxicity.

Second, the influence of PK11195 on the synthesis of melanin in human primary melanocytes

Collecting human primary melanocyte in exponential growth phase, digesting, counting, inoculating to 6-well plate with concentration of about 1 × 10⁵The inoculation amount is 2 ml/ml, the mixture is placed at 37 ℃ and 5 percent CO₂Culturing in an incubator for 24 hours; treating melanocyte with alpha-MSH, ACTH and ultraviolet ray, adding PK11195 with different concentrations, and culturing for 48 hr; collecting the above cells after administration, adding 100 μ l non-denaturing lysis solution (containing 1nM PMSF), and lysing at 4 deg.C for 20 min; centrifuging at 4 deg.C and 12000r/min for 10min, collecting supernatant for protein quantification (BCA method), and calculating total protein content; adding 100 μ l NaOH (containing 10% DMSO) into the melanin precipitate at the lower layer, and placing in a water bath at 80 deg.C for lysis for 2 hr; the completely dissolved melanin was added to a 96-well plate at 100. mu.l/well, and the absorbance at 405nm was measured, as shown in FIG. 2, FIG. 3, and FIG. 4. In figure 2 compared to the blank control group,^*P﹤0.05，^***p < 0.001; in contrast to the alpha-MSH treated group,^###p < 0.001. In fig. 3 and 4, the data of the ACTH treatment group and the uv treatment group,^*P﹤0.05，^**P﹤0.01，^***P﹤0.001。

the experimental results are as follows: compared with a blank control group, PK11195 can obviously inhibit the melanin synthesis of human primary melanocytes; compared with the alpha-MSH treatment group, the ACTH treatment group and the ultraviolet treatment group, PK11195 can obviously inhibit melanin synthesis induced by the factors and has dose dependence.

And (4) experimental conclusion: PK11195 can obviously inhibit the synthesis of human primary melanocyte melanin.

Influence of PK11195 on expression of important proteins MITF and TYR for melanin synthesis in human primary melanocytes

Taking human primary melanocytes in an exponential growth phase, digesting, counting, inoculating the cells into a 6-pore plate, treating the melanocytes by using alpha-MSH, adding PK11195 with different concentrations, collecting the cells after 48 hours, adding cell lysate, centrifuging, taking supernatant BCA (burst cutting) method to determine the protein concentration, adjusting the protein lysate to the same concentration, uniformly mixing the protein lysate with 2 times of loading buffer solution with the same volume, boiling for 5 minutes, and performing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); after electrophoresis, transferring the protein to an NC membrane by 100V voltage for 1 hour, and sealing by TBST (0.05% Tween-20) containing 5% skimmed milk for 1 hour; respectively incubating the membrane with MITF, TYR and beta-actin primary antibody (1: 200) dissolved in a confining liquid at 4 ℃ overnight, washing with TBST for 5 minutes and 3 times; then HRP labeled antibody was added to bind the primary antibody and the membrane was incubated at room temperature for 1 hour. The membrane was removed and washed 4 times with TBST for 5 minutes each. And (3) performing chemiluminescence detection, namely dripping the premixed luminescent liquid on the membrane, and photographing and imaging by using a gel imaging system. And the results were analyzed using quality one software from Bio-Rad. The results are shown in FIG. 5.

The experimental results are as follows: compared with a blank control group, PK11195 can obviously inhibit the protein expression of MITF and TYR in human primary melanocytes, and the difference is significant (P < 0.05); compared with the alpha-MSH treatment group, PK11195 can obviously inhibit the protein expression of MITF and TYR in human primary melanocytes, and the difference is significant (^###P﹤0.001)

And (4) experimental conclusion: PK11195 inhibits melanin synthesis by inhibiting the expression of melanin synthesis-important proteins (MITF, TYR) in human primary melanocytes.

A second part: effect of PK11195 on the Synthesis of Zebra Fish melanin

Zebra fish breeding method is performed according to The Zebraphis Book. The water temperature of the circulation system is kept at 28.5 ℃, the fixed illumination time is 14 hours every day, the dark time is 10 hours, and the feeding is carried out once every morning and evening. One female fish and one male fish were placed in the spawning tank the afternoon one day before embryo collection and separated by a partition. The partition plate is pulled away after the light is turned on the next day, and embryos can be collected after 20 minutes. Embryos were washed and cultured in egg water and placed in a light incubator at 28.5 ℃. Zebrafish embryos at the same time period were placed in 6-well plates and embryos developed to 35 hours, washed and added with different concentrations of PK 11195. After further culturing for 25 hours, the change in melanin synthesis in zebrafish was observed by taking pictures with a stereoscope. The results are shown in FIG. 6.

The experimental results are as follows: compared with a blank control group, the PK11195 administration group remarkably inhibits the synthesis of the melanin of the zebra fish.

And (4) experimental conclusion: PK11195 obviously inhibits the synthesis of the melanin of the zebra fish.

And a third part: effect of PK11195 on UV-induced Back pigmentation in Guinea pigs

After the guinea pig was acclimatized (10 animals, male and female halves), long hair on the back of the guinea pig was cut off with scissors, short hair was shaved with an electric shaver, and the skin on the back of the guinea pig was irradiated with SH4B ultraviolet light, and the total area was 6 x 6cm². The lamp tube is 15cm away from the back of the guinea pig, and the disposable irradiation dose is 500mJ/cm²Continuously for one week. One week after back skin of guinea pig is irradiated, stable pigmentation is formed locally, the irradiation region is divided into two medicine areas, one is coated with PK11195 (1%), the other is coated with blank matrix, the medicine area of each medicine area is a circle with a diameter of 1cm, the medicine areas of the two medicine areas are separated by 0.5cm, and the medicine is coated twice per day. For three weeks, the hairs were shaved to a minimum with an electric razor before application. Color changes of the skin of guinea pig dorsal skin were measured with a CR-400 colorimeter before, 1 week after, 2 weeks, and 3 weeks after the application of ultraviolet rays, respectively, and the colorimetric values were each measured value-the measurement value before the irradiation of ultraviolet rays, and photographs were taken for objective comparison. After four weeks, skin tissues were taken, fixed in 4% paraformaldehyde solution and sectioned in normal paraffin. Changes in melanin in the skin of the guinea pig dorsal skin were determined by Masson-Fontana amine silver stain. The results are shown in FIG. 7, FIG. 8, FIG. 9. In comparison with the control group in figure 9,^*P＜0.05。

the experimental results are as follows: PK11195 inhibits uv-induced melanin synthesis in the skin of guinea pig dorsal region compared to the blank control group.

And (4) experimental conclusion: PK11195 reduces uv-induced melanin pigmentation of the skin of guinea pig back.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.