CN114533871A

CN114533871A - Application of targeted PLK3 in preventing and treating skin proliferative diseases

Info

Publication number: CN114533871A
Application number: CN202011301570.6A
Authority: CN
Inventors: 张亮; 华鹏
Original assignee: Shanghai Institute of Nutrition and Health of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2022-05-27

Abstract

The invention provides an application of targeted PLK3 in preventing and treating skin proliferative diseases. The invention provides application of a PLK3 inhibitor in preparing a preparation or a composition for preventing and/or treating skin proliferative diseases.

Description

Application of targeted PLK3 in preventing and treating skin proliferative diseases

Technical Field

The invention relates to the field of medicine, in particular to application of targeted PLK3 in preventing and treating skin proliferative diseases.

Background

The epidermis is the outermost barrier of the human body against the external environment, has a lifelong ability to regenerate, and is generally divided into the stratum corneum, the stratum granulosum, the stratum lucidum, the stratum spinosum and the stratum basale. Epidermal hyperplastic diseases (epidermias) are a group of noncancerous diseases with abnormally increased cell numbers in the epidermis due to overgrowth or abnormal differentiation of the epidermis, and mainly comprise psoriasis, chronic eczema, dermatitis, verrucous hyperplasia, papilloma hyperplasia and the like. In addition, ultraviolet radiation can also cause epidermal hyperplasia, resulting in symptoms such as erythema solare.

The current clinical treatment of psoriasis relies mainly on agents that suppress the immune response, such as the anti-inflammatory agent methotrexate and agents that suppress T cell activity such as cyclosporine. However, these immunosuppressive drugs have strong toxicity, such as methotrexate teratogenicity, and cyclosporin nephrotoxicity. In addition, drugs that induce epidermal apoptosis, such as anthralin or corticoids, can also be applied to psoriasis treatment by the topical route. However, anthralin is somewhat destructive to the epidermis, and is not suitable for use on the face and on the sides of bends, nor in areas at risk of ulceration. Its irritation to healthy skin also renders it unsuitable for prophylactic use.

Therefore, there is an urgent need in the art to develop a safe and effective active substance for preventing and treating skin proliferative diseases and a method for using the same.

Disclosure of Invention

The invention aims to provide a safe and effective active substance for preventing and treating skin proliferative diseases and an application method thereof.

In a first aspect of the invention there is provided the use of an inhibitor of PLK3 in the preparation of a formulation or composition for the prevention and/or treatment of a skin proliferative disease.

In another preferred embodiment, the skin proliferative disease is selected from the group consisting of: psoriasis, chronic eczema, dermatitis, wart hyperplasia, papilloma hyperplasia, pseudoepithelioma hyperplasia, or combinations thereof.

In another preferred embodiment, the skin is derived from a mammal.

In another preferred embodiment, the mammal includes both diseased and non-diseased mammals.

In another preferred embodiment, the mammal comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal comprises: rodents (e.g., rats, mice), primates (e.g., monkeys).

In another preferred embodiment, the composition is a pharmaceutical composition.

In another preferred embodiment, the PLK3 inhibitor is selected from the group consisting of: small molecule compounds, mirnas, antisense nucleic acids (e.g., antisense RNAs), mrnas, antibodies, gene editing agents, or combinations thereof.

In another preferred embodiment, the formulation or composition is used to inhibit epidermal cell proliferation.

In another preferred embodiment, the formulation or composition is used for:

(i) reducing the degree of erythema and scaling on the skin surface;

(ii) reducing the degree of epidermal thickening and hyperproliferation;

(iii) reducing the degree of skin immune cell infiltration;

(iv) any combination of (i) - (iii) above.

In another preferred embodiment, the composition comprises a pharmaceutical composition.

In another preferred embodiment, the PLK3 inhibitor comprises: GW (GW843682X), Wortmannin (Wortmannin), TAK-960, or a combination thereof.

In a second aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a1) PLK3 inhibitors;

(a2) optionally other medicaments for the prevention and/or treatment of psoriasis; and

(b) a pharmaceutically acceptable carrier.

In another preferred embodiment, said component (a1) is present in an amount of 0.1-99.9 wt%, preferably 10-99.9 wt%, more preferably 70-99.9 wt% based on the total weight of said pharmaceutical composition.

In another preferred embodiment, the PLK3 inhibitor is selected from the group consisting of: GW (GW843682X), Wortmannin (Wortmannin), TAK-960, or a combination thereof.

In another preferred embodiment, the PLK3 inhibitor is used to down-regulate (or reduce) the amount and/or activity of PLK 3.

In another preferred embodiment, the pharmaceutical composition is a liquid, solid, or semi-solid.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form, an injection, or an external pharmaceutical dosage form.

In another preferred embodiment, the dosage form of the pharmaceutical composition comprises tablets, granules, capsules, oral liquid or injection.

In another preferred embodiment, the composition is a liquid composition.

In another preferred embodiment, the composition is an oral formulation.

In another preferred embodiment, the carrier is selected from the group consisting of: an infusion solution carrier and/or an injection carrier, preferably, the carrier is one or more selected from the following group: normal saline, dextrose saline, or combinations thereof.

In another preferred embodiment, the composition or formulation may be used alone or in combination in the prevention and/or treatment of skin proliferative diseases.

In another preferred embodiment, the combination comprises: in combination with other agents for the prevention and/or treatment of skin proliferative diseases.

In another preferred embodiment, the other medicament for preventing and/or treating psoriasis is selected from methotrexate, cyclosporine, anthralin, corticoids, Tremfya (Tremfya), tofacitinib, E6201, Ruxolitinib (Ruxolitinib), or a combination thereof.

In a third aspect of the invention, there is provided a use of the pharmaceutical composition according to the second aspect of the invention for the preparation of a pharmaceutical product for the prevention and/or treatment of psoriasis.

In another preferred embodiment, the pharmaceutical composition is for:

(i) reducing the degree of erythema and scaling on the skin surface;

(ii) reducing the degree of epidermal thickening and hyperproliferation;

(iii) reducing the degree of skin immune cell infiltration;

(iv) any combination of (i) - (iii).

In a fourth aspect of the present invention, there is provided a method for slowing down epidermal cell proliferation, comprising the steps of: contacting said skin cells with a medically effective amount of a formulation or composition comprising an inhibitor of PLK 3.

In another preferred embodiment, the cell is derived from a mammal.

In another preferred embodiment, the mammal includes (but is not limited to) mouse, human.

In another preferred embodiment, the cells are cultured in vitro.

In another preferred embodiment, the cells are epithelial tissue cells.

In another preferred embodiment, the PLK3 inhibitor is selected from: GW (GW843682X), Wortmannin (Wortmannin), TAK-960, or a combination thereof.

In another preferred embodiment, the method is an in vitro method.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

In a fifth aspect of the invention, there is provided a method of treating a skin proliferative disorder, comprising the steps of: a safe and effective amount of a PLK3 inhibitor is administered to a subject in need thereof.

In another preferred embodiment, the skin proliferative disease is selected from the group consisting of: psoriasis, chronic eczema, dermatitis, wart hyperplasia, papilloma hyperplasia, or combinations thereof.

In another preferred embodiment, the subject is a human or a non-human mammal.

In another preferred embodiment, the subject is a psoriasis patient.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

FIG. 1A is a statistical graph of cell proliferation fold of primary mouse epidermal cells (MK) measured by CCK8 method for 3 days after DMSO (DM) or GW843682X (GW) treatment (vs. starting point on day 3). P <0.001(×), n ═ 3, two-tailed t-test.

FIG. 1B is a Western blot of MK cells stably infected with LV-shRNAs (shScr, shPlk3-a, shPlk3-B, shPlk1-a, shPlk 1-B). ACTB is an internal reference protein. Numbers represent relative quantification of PLK1 or PLK3 target proteins. The ratio (target protein signal/reference protein signal) was calculated and compared to shScr lanes of each membrane. Quantification was performed using Image J. LV is a lentivirus. FIG. 1C is a statistical graph of day-3 cell proliferation fold of MK cells stably infected with LV-shRNAs (shScr, shPlk3-a, shPlk3-b, shPlk1-a, shPlk1-b) measured by CCK8 method. P <0.001 (. star.), P <0.01 (. star.), P <0.05 (. star.), n ═ 3, two-tailed t-test. LV is a lentivirus.

Fig. 2A is analysis of GW drug intervention effect in IMQ-induced mouse back skin psoriasis model. The experimental flow is schematically shown above. 7wo represents 7week old mice, IMQ/VAS means topical imiquimod or petrolatum control. DM/GW indicates DMSO solvent and GW84382X drug intervention. The sampling time point is the fifth day of the procedure. The lower left column is a photograph of the surface of the dorsal skin taken by a skin microscope. The scale bar is 0.5 cm. The middle and right columns are immunofluorescent staining (IF) photographs of dorsal skin sections. The scale bar is 100 um. Ki67 is a cell proliferation marker, CD104 is an epidermal basement membrane marker, and KRT14 is an epidermal basal cell marker. F4/80 is a dendritic cell marker. DAPI is nuclear fluorescent staining. The dotted line represents the upper surface of the skin. DM + VAS represents smearing DMSO solvent and vaseline; GW + VAS indicates the application of GW84382X medicine and vaseline; DM + IMQ for DMSO solvent and imiquimod cream; GW + IMQ indicates application of GW84382X drug and imiquimod cream.

Fig. 2B is a statistical characterization of the dorsal skin slice samples of the experimental sample points of fig. 2A. Where epidermal thickness refers to the distance of the basal membrane from the upper surface of the skin. P <0.01(×), n ═ 3, two-tailed t-test.

Figure 3A is an rig (rigosertib) drug intervention effect analysis of IMQ-induced mouse back skin psoriasis model. The experimental procedure, sampling time points, statistical analysis methods were the same as in 2A above. The left photograph is a photograph of the surface of the dorsal skin taken by a skin microscope. The scale bar is 0.5 cm. The right is the characteristic statistics of the dorsal cortex section sample. n.s. P >0.05, n ═ 3, two-tailed t-test. Fig. 3B is a statistical characterization of the dorsal cortex slice samples from the experimental sampling points in fig. 3A. Wherein DM + VAS represents that DMSO solvent and vaseline are smeared; RIG + VAS means small molecule inhibitor RIG and petrolatum applied with PLK 1; DM + IMQ for DMSO solvent and imiquimod cream; RIG + IMQ represent small molecule inhibitor RIG and imiquimod cream applied PLK 1.

Detailed Description

The inventor of the present invention has conducted extensive and intensive studies, and has found for the first time that it is unexpected that inhibition of PLK3 has the effect of slowing down epidermal cell proliferation, while inhibition of PLK1 has no similar effect. In particular, in a mouse psoriasis model, the inventors found that a small molecule inhibitor GW targeting both PLK3 and PLK1 had significant psoriasis antagonism with no visible side effects. While the small molecule inhibitor RIG specifically targeting PLK1 had no psoriasis antagonism. The inventors therefore propose: the technology for targeted inhibition of PLK3, including GW or other types of PLK3 small molecule inhibitors or other technologies for inhibiting expression and functions of PLK3, is a safe and effective new way for preventing and treating skin proliferative diseases. The present invention has been completed based on this finding.

PLK3

PLK3(Polo-like kinase 3), a serine/threonine kinase belonging to the Polo-like family (PLK). In mammals, PLK family members include PLK1, PLK2, PLK3, PLK4, and PLK 5. Among them, PLK1 is widely recognized as an important regulatory factor for promoting cell proliferation and a potential anticancer target, but the functions of other PLK proteins are mostly debated. Various in vitro cell culture studies have shown that PLK3 may have both cell proliferation promoting and cell proliferation inhibiting effects, and thus its function may be related to specific cell types and environmental conditions. However, neither PLK3 nor PLK1 have been reported to function in dermal skin cells.

As a potential anticancer target, a number of small molecule inhibitors of PLK1 have been developed. For example, rig (rigosetib) is a small molecule drug that specifically inhibits PLK1 without affecting PLK 3. On the other hand, specific inhibitors against PLK3 have not been reported so far, but small molecule drug GW (GW843682X, CAS: 660868-91-7) has been found to selectively inhibit PLK1 and PLK3 without affecting the effects of other similar kinases.

It is understood that "inhibitor targeting PLK 3" as used herein comprises a small molecule compound, miRNA, antisense nucleic acid (e.g. antisense RNA), mRNA, antibody or gene editing agent that specifically inhibits PLK3 or selectively inhibits PLK 3. As used herein, an "inhibitor targeting PLK 3" may down-regulate the amount or activity of PLK3, e.g., act on PLK3 gene and its expression products, down-regulate the amount or activity of PLK3 gene, mRNA, cDNA, or PLK3 protein.

Psoriasis disease

Psoriasis, commonly known as "psoriasis", is a common epidermal proliferative disease caused by the interaction of various factors such as environmental factors, genetic factors, immune factors, etc., and is mainly manifested as squamous erythema or plaque of the skin, which can be localized in one place or widely distributed throughout the body. Psoriasis affects not only the epidermis but also joint swelling and pain, abnormal nails, metabolic syndrome, cardiovascular diseases, etc. by causing systemic immune system disorders. Immune-related cytokines, mainly including TNF, IL-23, IL-17, etc., are thought to contribute to the pathogenesis of psoriasis. The mouse skin is treated by Imiquimod (IMQ) medicine, so that psoriasis can be quickly induced by activating IL-23/IL-17 signal paths, and the model is a widely applied psoriasis research model. The pathological and histological features of the mouse model are highly similar to those of human psoriasis, including erythema on the skin, thickening of the epidermis, change of differentiation of keratinocytes, angiogenesis, infiltration of skin immune cells and the like.

Active ingredient

As used herein, "active ingredient of the present invention" refers to inhibitors targeting PLK3, which can be used to reduce or down-regulate the activity or amount of PLK 3. It is understood that the term includes a single compound, a combination of two or more compounds, or an extract. In addition, the term also includes any agent that can down-regulate the activity or amount of PLK3, including gene editing agents or RNA interference-type agents. For example, Cas 9-based gene editing reagents can be used to inactivate the PLK3 gene.

Pharmaceutical composition

The invention provides a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and effective amounts of the following active ingredients: an inhibitor targeting PLK 3.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical composition of the invention can be prepared into injections, oral preparations (tablets, capsules, oral liquids), transdermal agents and sustained-release agents. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the active ingredient of the invention is administered at a daily dose of about 0.00001mg to 50mg per kg of animal body weight, preferably 0.0001mg to 10mg per kg of animal body weight. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

The main advantages of the invention include:

(1) the invention discovers for the first time that the technology for inhibiting the expression and the function of PLK3 is a safe and effective new way for preventing and treating skin proliferative diseases, and does not have similar effect in the aspect of inhibiting PLK 1.

(2) The compositions of the present invention prepared with the PLK3 inhibitor have no significant side effects on the skin.

(3) The PLK3 inhibitor directly inhibits epithelial cell proliferation, but not widely inhibits the immune response of the organism, so the PLK3 inhibitor has no toxic effect of drug immunosuppressive drugs.

(4) The invention discovers for the first time that the inhibition of PLK3 has an important role in preventing and treating psoriasis.

The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and alterations to the present invention, which fall within the scope of the invention.

Experimental procedures in the following examples, in which specific conditions are not specified, can be carried out by methods conventional in the art, for example, with reference to the molecular cloning, A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989, or according to the conditions recommended by the supplier. Methods for sequencing DNA are conventional in the art and tests are available from commercial companies.

The materials and methods applied in this example are listed below:

materials and methods

1) An animal model.

1.1) BALB/c mice.

The mouse is purchased from Shanghai Ling biological science and technology limited company, and is a wild type mouse model widely used for immunology and physiological research.

2) Model of psoriasis induced by imiquimod cream

BALB/c female mice of 7wo (7weekolds) were selected, shaved on the first day and depilated locally with vetting depilatory cream, 2 spots on top and bottom of the dorsal skin, 4 spots in total were tested, and on the next day 5% imiquimod cream and petrolatum control induction were performed on 4 depilated sites, one daily application and three days post application. Imiquimod cream was purchased from Aldara and petrolatum was purchased from bio-engineering (shanghai) gmbh (a 510146-0500).

3) Intraperitoneal injection of PLK3 inhibitor GW and PLK1 inhibitor RIG

Each time 6 female mice were entered into the experiment, randomly grouped, 3 injected with DMSO for the control group, 3 injected with PLK3 inhibitor GW or PLK1 inhibitor RIG for the experimental group, respectively, shaved hair on the first day, and injected with DM (DMSO) and GW, respectively, on the third day IMQ (imiquimod cream) and VAS (petrolatum) for the control, imiquimod and petrolatum daily, DM and GW and RIG injected every 2 days, and samples were collected for analysis on day 6. Injecting small molecule inhibitor GW or small molecule inhibitor RIG into abdominal cavity. The injection amount was 100ul of GW or RIG at a concentration of 2.5mg/ml per mouse. Injections were given every 2 days. GW is available from MCE (HY-11003) and RIG is available from Selleck (ON-01910).

4) Mouse dorsal skin sample collection and analysis

The data is collected after the mice are coated with IMQ for 3 days, the skin surface morphology of the dorsal skin is recorded by using a skin microscope, then the mice are killed by using a carbon dioxide lethal device, shaved, respectively treated by imiquimod and treated by vaseline, cleaned by PBS after the adipose tissues on the dermis surface are removed by using an operating knife, and placed in an OCT embedding medium to prepare an embedding block. The embedded blocks were sectioned using a Leica cryomicrotome and stained with Ki67, F4/80. After staining, pictures were taken using a Zeiss fluorescence microscope followed by semi-quantitative fluorescence analysis using Image J and the relative Ki67, F4/80 expression levels were counted for skin length per unit of skin. OCT Tissue Tek was purchased from Sakura (4583). Ki67 was purchased from ABCAM (ab8191) and F4/80 was purchased from BioLegend (123101).

5) Immunofluorescence

Sections were taken out of a-80 ℃ freezer, dried at room temperature for 10 minutes, fixed with 4% paraformaldehyde for 10 minutes, washed with PBS for 3 times, 5 minutes each, and the fixed sections were blocked with blocking solution ((2.5% conventional donkey serum, 2.5% conventional sheep serum, 1% BSA, 0.3% Triton X-100) for 1 hour, then incubated overnight at four degrees for one time, washed with PBS secondary antibody for 1 hour at room temperature, washed away excess secondary antibody with PBS followed by mounting buffer blocking, and observed for photography Ki67(Rabbit,1:500), F4/80(Rat,1:200), 4% paraformaldehyde from Bioworker (WH1013), PBS from HYCLONE (SH30256.01) conventional donkey serum, conventional sheep serum from Jackson, BSA from Saint 36101ES 25).

6) MK cell isolation and culture

Newborn mice were sacrificed within 3 days, their dorsal skin was taken, digested at 4 ℃ overnight in 2.5mg/ml dispase II (Roche), the dermis and epidermis were separated, the epidermis was digested in Trypsin-Versene (Lonza) for 15 minutes into single cells, passed through a 100um cell sieve (Bd Falcon), washed with PBS and cultured with CNT-PR (cellntec) medium.

7) Infection with LV-shRNAs, DM, GW stimulation

Primary cells were cultured for 16h after synchronization in CNT-basal (cellntec) culture and were subsequently cultured in CNT-PR and infected with LV-shRNAs (shScr, shPlk3-a, shPlk3-b, shPlk1-a, shPlk1-b) or DM or GW treatment to maintain a final concentration of 500 nM. shScr is a control group, shPlk3-a and shPlk3-b is an experimental group. DM is in contrast to GW. LV is a lentivirus.

8) CCK8 detection of cell growth fold

MK species were cultured in 96-well plates at 100ul medium per well, and the proliferation assay of CCK8 was performed according to the CCK8 kit (Biyun day), with 16 hours of cell seeding defined as day 0, and the assay was performed daily, with the day three being the end point, and the assay results on day three/day 0 being the fold proliferation.

9)Western Blots

Cell lysis is carried out by using cell lysis solution, and column type animal tissue/cell total protein extraction kit is usedExtracting protein with the (yase), and running the protein pre-made gel (yase) to a proper position with a bromophenol blue indicator by electrophoresis. The membrane is transferred, the sealing is carried out, the primary antibody and the secondary antibody are carried out, and the color development is carried out according to the method provided by the merchant. The primary antibodies used were PLK3 (rabbit, CST,1:1000), PLK1 (rabbit, CST,1:1000), ACTB (mouse, Genscript,1:1000), respectively. WB primary anti-diluent, WB secondary anti-diluent, protein-free rapid blocking solution (5 x), TBS/Tween buffer solution (10 x), trichrome pre-dyed protein Marker 10 kDa-250 kDa, PVDF membrane (0.45 μm), HRP-labeled secondary antibody, goat anti-mouse IgG, HRP-labeled secondary antibody, goat anti-rabbit IgG, Omni-ECL^TMUltrasensitive chemiluminescent detection kits were all purchased from yase. The Bio-rad development system was used for visualization, followed by Image J for quantification of the protein of interest.

10) Plasmid construction

shScr: pLKO.1-puro (Addgene) was selected. The lentiviral plasmid is used as a vector, puromycin (puro) is used as a marker for screening a stable cell line, enzyme cutting sites Age I and EcoR I are respectively positioned at the side of 2 sides of a nonsense sequence scramble sequence, and sequences required for constructing the plasmid are scramble-AgeI (SEQ ID NO:1) and scramble-EcoRI (SEQ ID NO: 2).

shPlk 3-a: pLKO.1-puro was selected. The lentiviral plasmid is used as a vector, puromycin is used as a marker for screening a stable cell line, the restriction enzyme sites Age I and EcoR I are respectively positioned at the 2 sides of the shRNA sequence of Plk3, and the sequences required for constructing the plasmid are shPlk3a-AgeI (SEQ ID NO:3) and shPlk3a-EcoRI (SEQ ID NO: 4).

shPlk 3-b: pLKO.1-puro was selected. The lentiviral plasmid is used as a vector, puromycin is used as a marker for screening a stable cell line, the restriction enzyme sites Age I and EcoR I are respectively positioned at the 2 sides of the shRNA sequence of Plk3, and the sequences required for constructing the plasmid are shPlk3b-AgeI (SEQ ID NO:5) and shPlk3b-EcoRI (SEQ ID NO: 6).

shPlk 1-a: pLKO.1-puro was selected. The lentiviral plasmid is used as a vector, puromycin is used as a marker for screening a stable cell line, and the sequences required for constructing plasmids, in which the restriction enzyme sites Age I and EcoR I are respectively positioned at the 2 sides of the shRNA sequence of Plk1, are shPlk1a-AgeI (SEQ ID NO:7) and shPlk1a-EcoRI (SEQ ID NO: 8).

shPlk 1-b: pLKO.1-puro was selected. The lentiviral plasmid is used as a vector, puromycin is used as a marker for screening a stable cell line, and the sequences required for constructing plasmids, in which the restriction enzyme sites Age I and EcoR I are respectively positioned at the 2 sides of the shRNA sequence of Plk1, are shPlk1b-AgeI (SEQ ID NO:9) and shPlk1b-EcoRI (SEQ ID NO: 10).

11) Image analysis and statistical method

CCK8 was measured for cell proliferation fold at day 3, the seeded cells 16h were recorded as day 0 and the data were obtained from day 3 CCK 8/day 0 values and plotted using Graphpad with n being 3. WB quantification was performed by first calculating the ratio (target protein signal/reference protein signal) and then comparing it with shScr lanes of each membrane. Quantification was performed using Image J. Ki67 was counted as the number of Ki67 positive cells in 100um long skin, F4/80 was counted as the expression level of F4/80 in 100um long skin, and the area occupied by the positive cells was analyzed using Image J, where n was 3. The inventor considers that P <0.05 is statistically significant, and has 1 star mark, P <0.01, 2 star marks, P <0.001, 3 star marks, and P >0.05, and has no statistical significance.

Sequence of

Table 1. sequences required for plasmid construction:

example 1 Effect of inhibition of PLK3 in inhibition of epidermal cell proliferation

In vitro cell culture, the fold proliferation of primary mouse epidermal cells (MK) was measured by the CCK8 method for 3 days after dmso (dm) or GW843682X (GW) treatment.

Experimental results show that GW drug treatment can significantly slow down the proliferation rate of primary mouse epidermal cells (MK) (fig. 1A).

Since GW has both effects of inhibiting PLK1 and PLK3, the present inventors further performed shRNA knockdown on PLK1 and PLK3, respectively, to confirm their functions in epidermal cell proliferation. Lentiviral shRNA expression vectors for PLK1 and PLK3 were first constructed, respectively, and their ability to knock down target proteins in MK cells was confirmed by Western Blots analysis (FIG. 1B). Subsequently, MK cells stably infected with LV-shRNAs (shScr, shPlk3-a, shPlk3-b, shPlk1-a, shPlk1-b) were further analyzed for their effect on MK cell proliferation by measuring the 3-day cell proliferation fold by the CCK8 method.

As a result of the experiment, it was found that knocking down PLK3 could significantly inhibit the proliferation of MK cells, while knocking down PLK1 had no similar effect (fig. 1C). This indicates that PLK3 is a potent target for inhibiting epidermal cell proliferation, and PLK1 is not.

Example 2 effectiveness of Targeted inhibition of PLK3 in inhibiting skin proliferative diseases

Psoriasis is a chronic inflammatory disease closely associated with inflammation, and its typical symptoms are manifested as erythema, roughness, abnormal thickening, hyperproliferation of the epidermis, and in addition, a large amount of infiltration of immune cells, represented by dendritic cells, in the skin. Psoriasis models were prepared on the dorsal skin of 7week old BALB/c mice by standard IMQ stimulation and were also treated with GW or solvent DM controls to evaluate their effect.

Each time 6 female mice were entered into the experiment and randomly grouped, 3 were DM solvent treated control groups and 3 GW treated experimental groups.

The experimental results show that under IMQ stimulation, the GW-treated group (GW + IMQ) showed significantly reduced psoriasis symptoms relative to the DM solvent-treated control group (DM + IMQ) (fig. 2A), mainly comprising:

1) the skin mirror photo shows that the degree of erythema and scale on the surface of the skin is obviously reduced;

2) immunofluorescent staining of sections of the classical cell proliferation marker Ki67 and the epidermal basement membrane marker CD104 showed a significant reduction in the degree of epidermal thickening and hyperproliferation phenomena;

3) immunofluorescent staining of sections of dendritic cell marker F4/80 showed a significant reduction in the degree of skin immune cell infiltration.

Statistical analysis by the inventors further confirmed the significance of these differences (fig. 2B). Meanwhile, in the vaseline control stimulation condition (DM + VAS), the appearance of epidermis, thickness of epidermis, degree of proliferation of epidermis, and degree of infiltration of immune cells of GW-treated group (GW + VAS) were not significantly different from those of solvent-treated control group (fig. 2A, 2B). Indicating that GW had no significant side effects on the skin.

Comparative example 1

The inventors tested RIG drugs in the same manner. RIG was found to have no psoriasis antagonism similar to GW (fig. 3A, 3B).

Comparative example 1 demonstrates that targeted inhibition of PLK3, but not PLK1, is an effective approach in preventing and treating skin proliferative diseases.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Nutrition and health institute of Chinese academy of sciences

<120> application of targeted PLK3 in preventing and treating skin proliferative diseases

<130> P2020-2167

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 57

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

ccggcaacaa gatgaagagc accaactcga gttggtgctc ttcatcttgt tgttttt 57

<210> 2

<211> 57

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

aattaaaaac aacaagatga agagcaccaa ctcgagttgg tgctcttcat cttgttg 57

<210> 3

<211> 58

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

ccggcagcgc gagaagatcc taaatctcga gatttaggat cttctcgcgc tgtttttg 58

<210> 4

<211> 58

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

aattcaaaaa cagcgcgaga agatcctaaa tctcgagatt taggatcttc tcgcgctg 58

<210> 5

<211> 58

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

ccggtcagcg cgagaagatc ctaaactcga gtttaggatc ttctcgcgct gatttttg 58

<210> 6

<211> 58

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

aattcaaaaa tcagcgcgag aagatcctaa actcgagttt aggatcttct cgcgctga 58

<210> 7

<211> 58

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ccggcctctc aaagtcctca ataaactcga gtttattgag gactttgaga ggtttttg 58

<210> 8

<211> 58

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

aattcaaaaa cctctcaaag tcctcaataa actcgagttt attgaggact ttgagagg 58

<210> 9

<211> 58

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

ccggcctcaa ctatttccgc aattactcga gtaattgcgg aaatagttga ggtttttg 58

<210> 10

<211> 58

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

aattcaaaaa cctcaactat ttccgcaatt actcgagtaa ttgcggaaat agttgagg 58

Claims

1. Use of a PLK3 inhibitor for the preparation of a formulation or composition for the prevention and/or treatment of a skin proliferative disorder.

2. The use according to claim 1, wherein the skin proliferative disorder is selected from the group consisting of: psoriasis, chronic eczema, dermatitis, wart hyperplasia, papilloma hyperplasia, pseudoepithelioma hyperplasia, or combinations thereof.

3. The use of claim 1, wherein the formulation or composition is for inhibiting epidermal cell proliferation.

4. The use of claim 1, wherein the formulation or composition is for:

(i) reducing the degree of erythema and scaling on the skin surface;

(ii) reducing the degree of epidermal thickening and hyperproliferation;

(iii) reducing the degree of skin immune cell infiltration;

(iv) any combination of (i) - (iii) above.

5. The use of claim 1, wherein said PLK3 inhibitor comprises: GW (GW843682X), Wortmannin (Wortmannin), TAK-960, or a combination thereof.

6. A pharmaceutical composition, comprising:

(a1) PLK3 inhibitors;

(b) a pharmaceutically acceptable carrier.

7. The composition of claim 6, wherein the other psoriasis-preventing and/or-treating agent is selected from the group consisting of methotrexate, cyclosporine, anthralin, corticoids, Tremfya, tofacitinib, E6201, ruxotinib, and combinations thereof.

8. Use of a pharmaceutical composition according to claim 6 for the preparation of a pharmaceutical product for the prevention and/or treatment of psoriasis.

9. A method of slowing epidermal cell proliferation, comprising the steps of: contacting said skin cells with a medically effective amount of a formulation or composition comprising an inhibitor of PLK 3.

10. The method of reducing epidermal cell proliferation of claim 9, wherein said cell is derived from a mammal.