CN115120730A - Application of PCSK9 inhibitor in preparation of product for preventing and treating scars - Google Patents

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to an effect of proprotein convertase Bacillus subtilis convertase (PCSK9) in promoting skin wound healing and preventing and treating scar (scar) formation, and an application of a PCSK9 inhibitor in preparing a product for promoting skin wound healing and preventing and treating scar (scar). The PCSK9 inhibitor product for systemic or external use can be further developed, and can be used for preventing and treating scar (scar). The products have obvious curative effect and small adverse reaction.

Description

Application of PCSK9 inhibitor in preparation of product for preventing and treating scars

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an effect of PCSK9 in scar (scar) treatment and application of a PCSK9 inhibitor in preparation of scar (scar) treatment products.

Background

Scars (scars) are the symptoms that damage caused by physical, biological, chemical and other factors acts on skin soft tissues of a human body, the skin soft tissues are seriously damaged and cannot be completely and normally repaired, and fibrous tissues are replaced and repaired to leave the scars, so that the appearance and the function are influenced. The scars bring great physical and mental pains to patients, especially scars left after burns, scalds and severe trauma. The period of several years during the scar hyperplastic phase is almost overwhelming for the patient. The later atrophy period causes the patients to have complete facial disabilities and dysfunction, which causes great physical and mental disorders. Scars include a number of categories: mainly proliferative (hyperplastic) scars, keloids, superficial scars, atrophic scars, contracture scars, depressed scars and the like.

Hypertrophic scars are characterized by prominent surface, irregular appearance, uneven height, flushing, congestion, and tough texture. It has burning pain and pruritus. Hypertrophic scars are manifested by increased ambient temperature, agitation of mood, or increased symptoms when food is stimulated by spicy food. Hypertrophic scars often last months or years before progressive degenerative changes occur. The scar is characterized by: early local swelling becomes hard and congested with a tissue structure covered by a layer of atrophic epithelial cells in the surface layer, vasodilation in the middle layer with infiltration of inflammatory cells, and less collagen fibers and massive connective tissue proliferation in the bottom layer. The scar is raised above the skin surface, and early local thickening and hardening, and capillary congestion appears red or dark red. The scar base is generally non-adherent to deep tissue, can be pushed, has low contractility, mostly does not cause serious dysfunction, but mostly affects beauty on the face and exposed parts.

Keloid is essentially a connective tissue tumor on the skin, characterized by persistent strong hyperplastic force, often appearing as a crab-foot-like infiltrate to the surrounding healthy skin, with prominent lesions on the skin surface, uneven, irregular shape, hard and tough, and multiple sensations of itching. Keloid may be caused by dominant inheritance of autosome and mechanical stimulation of trauma, infection and other factors may make fiber cell produce great amount of abnormal collagen fiber. Scars are usually found in the sternum area, and are also commonly found in the shoulder, face, neck, ear, etc. The initial skin lesion is small and firm red papule which slowly increases and is round, oval or irregular, rises on the surface of the skin, often exceeds the original lesion part, is crab-foot-shaped and extends outwards, and the surface is smooth and bright. Scars on the face affect the beauty, secondary to burns, the wounded can have large-area skin damage, and serious patients can affect the function of the affected limbs.

Superficial scars are scars on the superficial layers of the skin, which are mostly caused by slight abrasion or superficial burn (superficial dermis), have the appearance slightly different from that of normal skin, have rough surface or changed pigment, generally have no dysfunction, and are gradually less obvious over time, so that the scars do not need to be treated generally.

Atrophic scar is the most unstable scar tissue, also called unstable scar, which is commonly seen in large area burns, especially deep and fatty layer wounds, and the wound is healed without skin grafting but only by the growth of peripheral epithelial cells. The scar is characterized by: the scar has a thin epidermis, a flat surface, poor local blood circulation, a bright white color and a hard substrate. Because the surface layer is only covered with a layer of atrophic epithelial cells, the skin is often damaged without external friction. Atrophic scars tend to form ulcers, are generally difficult to heal, and can lead to malignant changes over time. Because such scars often adhere to underlying muscles, nerves, and blood vessels, and are very constrictive, they can stretch normal tissue and cause or be more severe than proliferative scars. Since such scars are very destructive, care should be taken to prevent them and they should be treated early.

Contracture scars are scars which are named after the features of the dysfunction they cause. After deep burns heal, scar contraction often causes appearance change and dysfunction, and long-term scar contracture can affect the development of bones, muscles, blood vessels, nerves and other tissues and should be treated as soon as possible. Clinically common deformities caused by scar contracture include blepharospexis, lipvalgus, mental-chest adhesion, hand scar contracture deformity, and contracture deformity on the flexed side or the extended side of each joint. The streak scar contracture on the flexion surface of the joint can be gradually stretched into web scar contracture after a long time, and is called web contracture scar. The larger webbed scars are usually found on the anterior cervical side, armpit, elbow, ankle joint, etc., while the smaller ones are found on the inner canthus, outer canthus, nasolabial sulcus, canthus, finge, etc., and appear in ring form on the open contracture scar of the body surface tunnel, which causes the narrow caliber of the body surface, affecting the normal function.

Depressed scars: when scar tissue causes a concave deformity on the body surface, it is called a depressed scar. It is commonly seen in scars left after smallpox, varicella or acne, and also in patients with trauma and skin infections. Simple depressed scars are only linear scars and depressions in their area, while extensive depressed scars may incorporate subcutaneous, muscular or skeletal tissue defects.

The treatment difficulty of scars is high, and at present, only reddish and hard scars can be softened and lightened, wide scars are narrowed, thick scars are thinned, and the scars cannot be completely and thoroughly eliminated. Therefore, it is very important to start intervention in the early stage of wound healing, which can effectively reduce scar formation, improve appearance, correct deformity, and restore function.

The commonly used methods for treating scars (scars) are: surgical treatment, laser treatment, cryotherapy, drug therapy, and the like. The commonly used drugs are mainly: glucocorticoids and tretinoin. The glucocorticoid has the functions of resisting inflammation, virus, shock and the like, and has obvious effect of resisting tissue fibrosis, the triamcinolone-A is the seed corticosteroid with the maximum anti-scar effect at present, and the glucocorticoid has many toxic and side effects after long-term use. The tretinoin is an intermediate product of vitamin A metabolism in vivo, is a vitamin A related drug, and can relieve local inflammation, promote epithelial cell growth, reduce collagen synthesis, reduce DNA synthesis of fibroblast, and inhibit cell growth. The higher the concentration of the retinoid drug is, the more obvious the growth inhibition effect is. However, the retinoic acid has limited curative effect, has few toxic and side effects in system application, has obvious skin irritation when externally applied and increases along with the increase of concentration. The existing method for treating scars (scars) can not meet the clinical requirement, and more new medicines with good curative effect, less side effect and low price are required to be searched to reduce the formation of scars (scars).

Proprotein convertase subtilisin (PCSK9) is a member of the proprotein convertase family, which is secreted in the liver as an inactive proenzyme. The size of the PCSK9 gene cDNA is 3617bp, and the PCSK9 protein consisting of 692 amino acids is encoded. The PCSK9 precursor undergoes intramolecular autocatalytic separation of its N-terminal propeptide within the endoplasmic reticulum, with the separated N-terminal propeptide attached to the catalytic region, allowing the mature PCSK9 protein to leave the endoplasmic reticulum and enter the secretory pathway. After PCSK9 is secreted extracellularly, it binds to low-density lipoprotein (LDL) receptors in the first epidermal growth factor-like region on the cell surface, and the PCSK9-LDL receptor complex can enter lysosome for degradation, leading to a decrease in the cell-surface LDL receptor, i.e., PCSK9 levels are inversely correlated with LDL receptors. In addition, multiple studies show that the loss of the function of the mutation of the PCSK9 gene can obviously reduce the LDL-C level and the incidence rate of coronary heart disease of different human species. In view of the significant effects of inhibiting PCSK9 on reducing the incidence of LDL-C and coronary heart disease, multiple treatment regimens have been developing drugs that block PCSK9 for reducing the incidence of LDL-C and coronary heart disease.

PCSK9 inhibitors include two broad classes: 1. prevent PCSK9 binding to LDL-R, e.g., monoclonal antibodies, peptidomimetics (polypeptide inhibitors), mimobody protein drugs; 2. inhibit the expression of the PCSK9 molecule or interfere the secretion of the PCSK9 molecule, such as small molecule interfering RNA, antisense oligonucleotide, small molecule compound inhibitor and the like. The monoclonal antibody is a hot spot for new drug research due to high blocking efficiency, accurate target position and good stability. At present, PCSK9 targeted monoclonal antibody drugs on the market all around the world are drugs preventing PCSK9 from binding with LDL-R. Including evocolumab (ilouzumab) developed by the combination of ann (Amgen) and antralae (Astellas), under the trade name retatha (rebetan); alirocumab (alisiuzumab, alilizumab) under the trade name pralutent (bolida) developed in combination with Sanofi (Sanofi) and regener (Regeneron), and LY3015014 from Eli lilly, a recombinant humanized anti-PCSK 9 monoclonal antibody (js002) from jun live, and a recombinant fully human anti-PCSK 9 monoclonal antibody injection from the believitai pharmaceutical industry. Clinical researches find that the drug has good tolerance to hypercholesterolemia, and the incidence of adverse reactions of a placebo group and an active treatment group is not obviously different. In addition, Inclisiran is a siRNA (small interfering RNA) drug, and unlike monoclonal antibodies which are directly combined with PCSK9 molecules, the Inclisiran can inhibit the expression of PCSK9 gene, so that LDL receptors are not degraded by PCSK9, and therefore, the Inclisiran improves the uptake of LDL particles by hepatocytes and reduces the LDL level in blood. Inclisiran from Alylam corporation uses proprietary technology to bind lipid nanoparticles to GalNAc (N-acetylgalactosamine), which binds to the sialoglycoprotein receptors ASGR1 and ASGR2 expressed on the surface of hepatocytes. This technique allows for subcutaneous administration and targeting to the liver. ALN-PCS and ALN-PCSSc from Affinis also belong to siRNA (small interfering RNA) drugs. PCSK9 interferes with RNAI inhibitor drugs and also Inclisran by Alnylam, ALN-PCS and ALN-PCSSc by Affinis. The relevant vaccine medicament designed by the pfeiri company and the AT04A and AT06A vaccines of the Affinis company, a patient only needs to receive the vaccine once a year to achieve the effect of reducing LDL for a long time, and the use frequency can be reduced. The development of PCSK9 mimic peptide and PCSK9 mimic antibody protein drug includes DS9001 of Pieris and 1G08 of Merck, etc. The PCSK9 antisense oligonucleotide drug includes SPC5001 from Santaris Pharma, and the like.

Until now, relevant documents, patents and products for treating scars (scars) by using the PCSK9 inhibitor are not found. We find for the first time that knocking out the PCSK9 gene can obviously inhibit the formation of scars (scars), and the PCSK9 inhibitor can obviously improve the scars (scars).

Disclosure of Invention

The problems to be solved by the invention are as follows: the function of the PCSK9 gene in a scar (scar) formation mechanism is clarified through an animal model, and the application of the PCSK9 inhibitor in the preparation of products for preventing and treating scars (scars) is also disclosed. According to the invention, a rat skin scar (scar) model is established, and the key role of the PCSK9 gene in a scar (scar) formation mechanism and the application value of the PCSK9 inhibitor in the preparation of products for preventing and treating scars (scars) are discovered.

Technical scheme of the invention

According to the invention, the research shows that the PSCK9 gene knock-out can obviously promote the healing of the skin wound of a rat and reduce the formation of scars. The proliferation of skin fibroblasts can be obviously inhibited by knocking out the PSCK9 gene, the apoptosis of the fibroblasts is promoted, and the PSCK9 is inhibited to inhibit the formation of scars (scars) by inhibiting the proliferation of the skin fibroblasts and promoting the apoptosis.

In the inhibitor (blocker) capable of obviously blocking PCSK9, representative PCSK9 monoclonal antibody, PCSK9 polypeptide inhibitor, PCSK9 small molecular compound inhibitor and PCSK9 small interfering RNA are respectively selected, a rat scar (scar) model is intervened through tail vein injection or external skin application, and compared with the model group, the result shows that the scar hyperplasia condition of the PCSK9 inhibitor group is obviously better than that of the model group, each PCSK9 inhibitor group has no systemic adverse reaction, the rat activity and foraging are normal, and the respiratory and central nervous system abnormal expression is not seen. Experimental research proves that various PCSK9 inhibitors can promote skin wound healing and reduce scar (scar) formation, and the PCSK9 inhibitors can be used for preventing and treating skin scars (scars) in a systemic or external mode and have no obvious toxic or side effect.

It is well known to those skilled in the art that, based on the co-pathogenesis of the disease and the results shown in the above experiments, it is presumed that PCSK9 inhibitors (blockers) have preventive and therapeutic effects on various types of scars (scars). PCSK9 inhibitors (blockers) can be used alone or in combination with other drugs or therapies, including traditional drugs and other targeted biologies.

Based on the research, the invention relates to application of a PCSK9 inhibitor (blocking agent) in preparation of products for preventing and treating scars (scars), wherein the PCSK9 is proprotein convertase Bacillus subtilis convertase/kexin 9 type (genebank serial number: 255738) and belongs to the proprotein convertase family.

The PCSK9 inhibitor of the present invention may be any conventional product or method capable of inhibiting the expression or secretion of the PCSK9 gene by molecular biological or medicinal chemical means, such as but not limited to, the knock-out or silencing of the PCSK9 gene by existing molecular biological techniques; in some embodiments, a PCSK9 inhibitor may also be or be used, preferably, the PCSK9 inhibitor (blocker) is a PCSK9 small molecule compound or a PCSK9 interfering RNAi inhibitor or a PCSK9 monoclonal antibody or a PCSK9 mimetic peptide or a PCSK9 mimetic antibody protein or a PCSK9 antisense oligonucleotide or a PCSK9 vaccine.

Preferably, the PCSK9 inhibitor (blocker) is a PCSK9 small molecule compound or a PCSK9 interfering RNA or a PCSK9 monoclonal antibody or a PCSK9 mimetic peptide or a PCSK9 mimetic antibody protein or a PCSK9 antisense oligonucleotide or a PCSK9 vaccine.

In some examples, the PCSK9 small molecule compounds of the present invention include, but are not limited to, the seleck product R-IMPP, formula: c ₂₄ H ₂₇ N ₃ O ₂ Molecular weight: 389.49, structural formula:

or product PF-06446846 from the company Selleck, of formula: c ₂₂ H ₂₀ ClN ₇ O, molecular weight: 433.04, structural formula:

or Selleck company SBC-115076, formula: c ₃₁ H ₃₃ N ₃ O ₅ Molecular weight: 527.61, structural formula:

or Selleck corporation SBC-110736, formula: c ₂₆ H ₂₇ N ₃ O ₂ Molecular weight: 413.51, structural formula:

in some examples, the PCSK9 monoclonal antibody inhibitor described in the present invention includes, but is not limited to, ab81041 from Abcam, or evolocumab developed by a combination of Amgen and Astellas (Astellas), or alirocumab developed by a combination of Sanofi and regeners (Regeneron), or recombinant humanized anti-PCSK 9 monoclonal antibody (js002) from juniper organisms, or recombinant fully human anti-PCSK 9 monoclonal antibody injection from tei lilly, or LY3015014 from Eli lilly, or 1G08 from Merck.

In some examples, PCSK9 interference RNAI inhibitors described herein include, but are not limited to, inlisran by alantam, ALN-PCS and ALN-PCSsc by Affiris.

In some examples, the PCSK9 peptidomimetic inhibitors and PCSK9 mimetibody protein inhibitors described herein include, but are not limited to, DS9001 by Pieris, 1G08 by Merck, and the like.

In some examples, PCSK9 antisense oligonucleotide inhibitors described herein include, but are not limited to, SPC5001 from Santaris Pharma.

In some examples, the PCSK9 vaccine inhibitors described herein include, but are not limited to, AT04A and AT06A of Affiris, inc.

Based on the above mechanism of action of PCSK9, it is well known to those skilled in the art that PCSK9 inhibitors (blockers) have therapeutic effects on scar diseases caused by various causes.

The PCSK9 inhibitors (blockers) may be used alone or in combination with other drugs or treatments, including traditional drugs and other targeted biologies.

The invention also provides a pharmaceutical composition, which is prepared by taking the compound or the pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient and assisting with a pharmaceutically acceptable carrier.

The compounds or compositions of the invention may be prepared in any pharmaceutically acceptable dosage form, for example, in a formulation suitable for any mode of administration, oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, inhalation, vaginal, intraocular, topical, subcutaneous, intraadipose, intraarticular, intraperitoneal or intrathecal.

In a preferred embodiment, the dosage form of the present invention is a paste, a tablet, a granule, an oral liquid, a capsule, a drop pill, an enema, a film or an injection.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a new and better treatment method for preventing and treating scars (scars), and PCSK9 inhibitor (blocker) products for preventing and treating scars (scars) can be further prepared through the disclosure of the invention, so that new monomer medicines or compound preparations containing various PCSK9 inhibitors are developed and used for preventing and treating scars (scars). The existing research results prove that the product containing the PCSK9 inhibitor has the advantages of remarkable curative effect, small side effect and good tolerance, and can provide a series of new products with good curative effect, safety and economy for the market.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any number between the two endpoints are optional unless otherwise specified in the invention. 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 invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention, in addition to those described in the art and in accordance with the teachings of the present invention.

Example 1 Effect of PSCK9 knock-out on scar (scar) model in rats

1. Experimental methods

1.1 grouping and modeling of experimental animals: SPF-grade rats, PCSK 9-/-SPF-grade rats (Pcsk9 gene exon 2-3 was knocked out by using CRISPR gene editing technology to obtain Pcsk9 gene knocked-out rats), body weight (220 + -26) g, male. Divided into model control group and PCSK 9-/-group, each group having 6 animals. Each group of rats was anesthetized with 2% sodium pentobarbital (120mg/kg) by intraperitoneal injection and then fixed on an operating table, and then a 4X 5cm piece of intact skin was selected on the left side of the back, 8% sodium sulfide was used for depilation, and a 2.4cm diameter circular wound reaching the myofascial depth was cut at the depilation site with tissue scissors, and a part of the myofascial surface was destroyed. In order to prevent rats from biting and licking, the animals are raised in cages. The wound surface is coated with 2% iodine tincture for routine disinfection every day, and the wound surface healing condition of rats is observed. Collecting materials at 20d, anesthetizing rat with 2% sodium pentobarbital, and collecting wound skin edge tissue about 0.5cm ² The method is used for culturing the fibroblast and carrying out subsequent experiments.

1.2 fibroblast culture and characterization

Preparing wound tissue into 1mm × 1mm, inoculating into culture dish, and adding 0.5ml DMEM culture solution.

1.3 TUNEL kit for detecting in situ apoptosis

TUNEL is a method of detecting apoptotic DNA fragmentation widely used to identify and quantify apoptotic cells, or to detect excessive DNA fragmentation in individual cells, and relies on the use of terminal deoxynucleotidyl transferase (TdT), an enzyme that catalyzes the attachment of deoxynucleotides labeled with a fluorescent dye or another marker to the 3' -hydroxyl end of a DNA double strand break, which can also label cells with DNA damage by other means besides the apoptotic process. The cell suspension for the experiment was adjusted to 1X 105/ml with MEM and inoculated into 100ml culture flasks at l0ml per flask. Culturing at 37 deg.C under 5% CO2 and 95% humidity, respectively blowing and beating the cultured cells into cell solution after 24h, 48h and 72h, and collecting cells. Centrifuging at 2500rpm for 5min, removing supernatant, and adding 75% ethanol lml into each bottle for fixation. Staining was performed as required for the TUNEL apoptosis test kit, expression in the average distribution of apoptotic fibroblast units in all fields was calculated manually with a manual counter and statistically compared.

1.4 detection of cell proliferation by MTT method

Chemical name of MTT: 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide, trade name: thiazole blue. The detection principle is as follows: succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to the water-insoluble bluish-violet crystalline Formazan (Formazan) and deposit in the cells, whereas dead cells do not. Dimethyl sulfoxide (DMSO) can solubilize the formazan in cells, which is measured by an enzyme-linked immunosorbent assay for its light absorbance values, which indirectly reflects the viable cell number. The amount of MTT crystals formed is proportional to the number of cells over a range of cell numbers. Cells were seeded at a density of 1.0X 105/ml in 48-well plates and cultured in a 5% CO2 incubator at 100. mu.1/well 37 ℃ until the cells were adherent. Culturing in a 5% CO2 incubator at 37 ℃ for 24h, 48h and 72h, adding 20 mu 1 MTT (5mg/ml) into each hole, continuing culturing for 4h, removing the culture medium, adding 100 mu 1 DMSO lysate, incubating in a 5% CO2 incubator at 37 ℃ for 20min, and testing the absorbance (optical density value) at 570nm on a microplate reader after the purple crystals are completely dissolved. The growth of the fibers in each group was compared.

1.4 statistical methods

Data analysis was performed using SPSS 16.0 statistical software. The measured data are expressed by means of standard deviation plus or minus (x plus or minus s), single-factor analysis of variance is adopted for comparison, two-by-two data comparison is carried out by adopting t test, the difference is more than 0.05, the difference is statistically significant, and the difference is more than 0.01.

2. Results of the experiment

2.1 rat wound Observation results

The wound surface was routinely sterilized every day, and rat wound surfaces were observed at 1d, 3d, 5d, 7d, 12d, and 20 d. The PCSK 9-/-group had significantly faster wound recovery and smaller wound area than the model group starting on day 3 of the observation period. By day 12, PCS wasThe K9-/-group wound surface had substantially recovered, leaving only a small amount of scabs, while the model group still had 0.5cm ² The wound surface of the left and right size. By day 20, both groups had substantial recovery of the wound surface, with the model control group leaving scars, while the PCSK 9-/-group left little pigmentation with no visible scars.

2.2 TUNEL method for detecting apoptosis of fibroblast

TUNEL is a method of detecting apoptotic DNA fragmentation widely used to identify and quantify apoptotic cells. The results showed that the fibroblast apoptosis index in PCSK 9-/-group and model control group were 16.816 ± 1.012 and 2.151 ± 0.563, respectively, and that fibroblast apoptosis in PCSK 9-/-group was significantly more than in model control group (P < 0.01).

2.3 detection of fibroblast proliferation by MTT method

Dimethyl sulfoxide (DMSO) solubilizes formazan in cells, and measuring its light absorption at 490nm using an enzyme linked immunosorbent assay, indirectly reflects the number of viable cells, over a range of cell numbers, in which the amount of MTT crystal formation is directly proportional to the number of cells. The results show that: the average absorbance values of the PCSK 9-/-group and the model control group after 24h of culture were 0.31 and 0.52, respectively, the average absorbance values of the PCSK 9-/-group and the model control group after 48h of culture were 0.26 and 0.57, respectively, and the average absorbance values of the PCSK 9-/-group and the model control group after 72h of culture were 0.21 and 0.66, respectively. The absorbance values of the PCSK 9-/-group and the model control group are obviously reduced after being cultured for 24 hours, the absorbance values of the PCSK 9-/-group are more obviously reduced after being cultured for 72 hours, and the difference between the absorbance values of the PCSK 9-/-group and the model control group is more obvious. The experimental result shows that PCSK9 knock-out can obviously reduce the proliferation activity of fibroblasts.

3. Conclusion of the experiment

The knockout of the PSCK9 gene can obviously promote the healing of skin wounds, simultaneously reduce the proliferation activity of fibroblasts, promote the apoptosis of the fibroblasts and reduce the formation of scars (scars), and shows that the knockout of the PSCK9 gene has the effects of preventing and treating the scars (scars).

Example 2 Effect of various PSCK9 inhibitors on rat scar model

1. Experimental methods

1.1 materials:

(1) PCSK9 interfering RNA-1 sequence and modification

Gene	5'-3'Sense	5'-3'Antisense
			siPCSK9-1	GccuGGAGuuuAuucGGAAdT*dT	UUCCgAAuAAACUCcAGGCdT*dT
siPCSK9-2	AGGuGuAucuccuAGAcAcdT*dT	GUGUCuAGGAGAuAcACCUdT*dT

Mixing siPcsk9-1 and 2 with equal amount, diluting to 20 μ M with normal saline, and mixing diluted siRNA and skin lotion.

PCSK9 interferes with RNAi inhibitor-2: the RNA sequence was identical to Inclisran by Alnylam; PCSK9 interferes with RNAi inhibitor-3: the RNA sequence was the same as ALN-PCS of Affinis.

(2) PCSK9 small molecule compound inhibitor 1: product R-IMPP from the company Selleck, of formula: c ₂₄ H ₂₇ N ₃ O ₂ Molecular weight: 389.49, structural formula:

PCSK9 small molecule compound inhibitor 2: product PF-06446846 from Selleck, chemical formula: c ₂₂ H ₂₀ ClN ₇ O, molecular weight: 434.04, structural formula:

PCSK9 small molecule compound inhibitor 3: selleck company SBC-115076, formula: c ₃₁ H ₃₃ N ₃ O ₅ Molecular weight: 527.61, structural formula:

PCSK9 small molecule compound inhibitor 4: selleck corporation SBC-110736, formula: c ₂₆ H ₂₇ N ₃ O ₂ Molecular weight: 413.51, structural formula:

(3) PCSK9 monoclonal antibody 1: purchased from Abcam corporation (ab 84041); PCSK9 monoclonal antibody 2: evolocumab (elouitumumab); PCSK9 monoclonal antibody 3: alirocumab (alicetuximab).

(4) A PCSK9 polypeptide: ab32727 by Abcam.

(5) Positive therapeutic agents: mometasone furoate cream (trade name: Allosone, manufactured by Xianlingbao Chinese Co., Ltd.) (6) experimental animals: SPF-grade black guinea pigs, body mass (252. + -. 18) g, half male and half female.

(7) The preparation method of the treatment cream comprises the following steps: the excipient matrix comprises 15 percent of methyl silicone oil, 6 percent of stearic acid, 5 percent of white vaseline, 5 percent of liquid paraffin, 5 percent of octadecanol, 20 percent of glycerol, 1 percent of alkylaryl polyglycol ether, 1 percent of fatty alcohol polyoxyethylene ether, 1 percent of tween-807, 0.1 percent of ethylparaben and 31 to 55 percent of distilled water, which are respectively mixed with a proper amount of PCSK9 inhibitor to form a mixed emulsion. As used in this example, the cream base refers to the base component of the cream from which the active ingredient is removed.

1.2 grouping and modeling of experimental animals: male SPF grade rats, weighing 210 ± 28 g, were obtained from southern medical centers. Rats were numbered according to body weight and classified by the random arrangement method into compound group 1 (skin-applied 0.5% R-IMPP cream), compound group 2 (skin-applied 0.5% PF-06446846 cream), compound group 3 (skin-applied 0.5% SBC-115076 cream), compound group 4 (skin-applied 0.5% SBC-110736 cream), mab group 1 (subcutaneous injection of PCSK9 monoclonal antibody ab81041, 3mg/kg. d), mab group 2 (subcutaneous injection of evolocumab, 3mg/kg. d), mab group 3 (subcutaneous injection of alirocumab, 3mg/kg. d), PCSK9 interfering RNA group-1 (skin-applied 0.5% PCSK9 small interfering RNA-1 cream), cream-injected PCSK9 interfering RNA group-2 (skin-applied 0.5% PCSK9 small interfering RNA-2), PCSK9 interfering RNA group-3 (skin-applied 0.5% PCSK9 small interfering RNA-3), and polypeptide group 727 (subcutaneous Abcam), 3mg/kg. d), positive treatment group (applying Alloloson to skin), blank control group (applying cream base to skin), model group (injecting normal saline into tail vein, applying cream base to skin), and 6 pieces of each group. Each group of rats was anesthetized with 2% sodium pentobarbital (120mg/kg) by intraperitoneal injection and then fixed on an operating table, and then a 4X 5cm piece of intact skin was selected on the left side of the back, 8% sodium sulfide was used for depilation, and a 2.4cm diameter circular wound reaching the muscular fascia was cut at the depilation site with tissue scissors, and a part of the muscular surface fascia was destroyed. The animals are raised in cages to prevent rats from biting and licking. The wound surface is coated with 2% iodine tincture for routine disinfection every day, and the wound surface healing condition of rats is observed.

2. Results of the experiment

2.1 rat wound observation results

The wound surface was routinely sterilized every day, and rat wound surfaces were observed at 1d, 3d, 5d, 7d, 12d, and 20 d. The wound recovery speed of the small molecule compound treatment group, the PCSK9 small interfering RNA treatment group, the PCSK9 monoclonal antibody treatment group and the positive treatment group is obviously higher than that of the model group from the 5 th day, and the wound area gradually becomes smaller. The speed of wound recovery from day 7 of the PCSK9 polypeptide inhibitor-treated group was better than that of the model group and the blank control group. On day 12, the small molecule compound treatment group, PCSK9 small interfering RNA treatment group, and PCSK9 mab wound surface had substantially recovered, while the model group and blank control group, respectively, still had about 0.4cm ² And 0.36cm ² The size of wound surface is 0.2cm in the polypeptide inhibitor treatment group ² Left and right large wound surfaces. By day 20, the wound surface had recovered in each group, and the model control group and the blank control group left significant scars, while the other groups left only unequal amounts of pigmentation.

3. Conclusion of the experiment

The PCSK9 inhibitor can obviously promote the healing of skin wound and reduce the formation of scar.

Sequence listing

<110> Chenmin

Application of PCSK9 inhibitor in preparation of product for preventing and treating scars

<150> 2021103213902

<151> 2021-03-25

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 692

<212> PRT

<213> person (Homo sapiens)

<400> 1

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln

690

Claims (11)

1. Use of a PCSK9 inhibitor for the manufacture of a product for the prevention and treatment of scarring, characterised in that PCSK9 is the proprotein convertase subtilisin/kexin 9 type, belonging to the proprotein convertase family.
2. 2. The use according to claim 1, characterized in that said scars include but are not limited to proliferative scars, keloids, superficial scars, atrophic scars, contracture scars and depressed scars.
3. 3. The use of claims 1 and 2 wherein the PCSK9 inhibitor comprises but is not limited to a PCSK9 small molecule compound inhibitor, a PCSK9 interfering RNAi inhibitor, a PCSK9 monoclonal antibody inhibitor, a PCSK9 peptidomimetic inhibitor, a PCSK9 mimobody protein inhibitor, a PCSK9 antisense oligonucleotide inhibitor or a PCSK9 vaccine inhibitor.
4. 4. The use of claims 1 and 2 wherein the PCSK9 inhibitor is a PCSK9 small molecule compound inhibitor, a PCSK9 interfering RNAi inhibitor, a PCSK9 monoclonal antibody inhibitor.
5. 5. The use of claims 1 and 2 wherein the PCSK9 small molecule compound inhibitor comprises but is not limited to a compound of formula I, formula II, formula III or formula IV,

   

   

   

   。
6. 6. formula I, formula II, formula III, formula IV

   The use of claims 1 and 2 wherein the PCSK9 monoclonal antibody inhibitor includes, but is not limited to, antibody ab84041 from Abcam, or eloitumumab, or alisitumumab, or recombinant humanized anti-PCSK 9 monoclonal antibody JS002, or recombinant fully human anti-PCSK 9 monoclonal antibody, or PCSK9 monoclonal antibody LY 3015014.
7. 7. The use of claims 1 and 2, wherein the PCSK9 interfering RNAi inhibitor comprises but is not limited to an Inclisran injection, an ALN-PCS injection or an ALN-PCSsc injection.
8. 8. The use of claims 1 and 2 wherein the PCSK9 peptidomimetic inhibitor and PCSK9 mimetibody protein inhibitor include, but are not limited to, the polypeptide ab32727 from Abcam, or the mimetibody protein DS9001, or the PCSK9 human antibody-antigen binding fragment 1G 08.
9. 9. The use according to claims 1 and 2, wherein the PCSK9 antisense oligonucleotide inhibitor comprises but is not limited to SPC5001 from Santaris Pharma; the PCSK9 vaccine inhibitor includes but is not limited to PCSK9 vaccine AT04A or PCSK9 vaccine AT 06A.
10. 10. Use according to claims 1 and 2, characterized in that the PCSK9 inhibitor can be used alone or in combination with other therapeutic methods or drugs for the prophylaxis and treatment of scar disorders.
11. 11. The product of claim 10 wherein the PCSK9 inhibitor is selected from the group consisting of a PCSK9 small molecule compound inhibitor, a PCSK9 interfering RNAi inhibitor, a PCSK9 monoclonal antibody inhibitor, a PCSK9 peptidomimetic inhibitor, a PCSK9 mimobody protein inhibitor, a PCSK9 antisense oligonucleotide inhibitor, and a PCSK9 vaccine inhibitor.