WO2022002160A1 - Pcsk9抑制剂在制备治疗多种疾病产品中的应用 - Google Patents

Pcsk9抑制剂在制备治疗多种疾病产品中的应用 Download PDF

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WO2022002160A1
WO2022002160A1 PCT/CN2021/103749 CN2021103749W WO2022002160A1 WO 2022002160 A1 WO2022002160 A1 WO 2022002160A1 CN 2021103749 W CN2021103749 W CN 2021103749W WO 2022002160 A1 WO2022002160 A1 WO 2022002160A1
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pcsk9
group
inhibitor
skin
model
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French (fr)
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陈敏
王浥尘
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陈敏
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Priority claimed from CN202011228305.XA external-priority patent/CN114432447A/zh
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Priority to US18/024,010 priority Critical patent/US20230346773A1/en
Priority to EP21833682.4A priority patent/EP4268817A1/en
Publication of WO2022002160A1 publication Critical patent/WO2022002160A1/zh

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    • A61K31/5375—1,4-Oxazines, e.g. morpholine
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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Definitions

  • the invention belongs to the technical field of medicine, and specifically relates to the role of PCSK9 in the treatment of various diseases, and the application of PCSK9 inhibitors in the preparation of products for the treatment of various diseases.
  • Vitiligo is a common acquired localized or generalized skin depigmentation disease, and the prevalence of vitiligo in the world is 1% to 2%.
  • Depigmentation of the skin or mucous membranes caused by damage to melanocytes is mainly characterized by the appearance of discolored leukoplakia, which can occur in various parts of the body, and is common in the back of the fingers, wrist, forearm, face, neck and around the genitals. Most of the leukoplakia are distributed symmetrically, and in the initial stage, they are mostly nails to the size of a coin, and are approximately round, oval or irregular. At the onset of the disease, it is punctate depigmentation, the border is more obvious, and some edges are surrounded by pigment bands.
  • the cause is unknown. At present, it is mostly considered to be related to the following factors, including autoimmune factors, genetic factors, neuropsychiatric factors, etc. Vitiligo can be combined with autoimmune diseases. Specific antibodies of various organs can also be detected in serum, such as anti-thyroid antibodies, anti-parietal cell antibodies, anti-adrenal antibodies, anti-parathyroid antibodies, anti-smooth muscle antibodies, and anti-melanocyte antibodies. Wait. Antibodies and T lymphocytes can be produced in vitiligo patients, and the immune response may lead to the destruction of melanocytes. The toxic melanin precursors synthesized by the cells themselves and some chemicals that cause skin depigmentation may also selectively destroy melanocytes.
  • the disease occurs in the exposed and darkened parts, and the epidermal melanocytes are hyperactive, causing them to be depleted and declining, and may be caused by the excessive or accumulation of intermediates in the synthesis of melanin by melanocytes.
  • About 2/3 of the patients' onset is related to the development of skin lesions, mental trauma, overwork and anxiety, and some white scar lesions are symmetrical or distributed along the nerve segments.
  • Vitiligo is a refractory disease. Systemic treatment is mainly suitable for patients with generalized advanced vitiligo. Oral or intramuscular injection of hormones can stabilize advanced vitiligo as soon as possible.
  • Topical glucocorticoids are effective in the treatment of localized vitiligo, but long-term use of glucocorticoids causes many adverse reactions.
  • topical calcineurin inhibitors tacrolimus, pimecrolimus
  • Form vitamin D 3 derivative may be a combination therapy with NB-UVB, PUVA, etc., may be calcineurin inhibitor combination treatment with topical steroid and calcium.
  • Narrow-wave ultraviolet (NB-UVB) treatment of localized or generalized vitiligo has a certain effect. Current treatments are not yet able to meet clinical needs.
  • Acne (commonly known as youth bean) is a chronic inflammatory disease that occurs in hair follicles and sebaceous glands, with an incidence of about 9.4%, and has become the 8th most common disease in the world. It is more common in adolescence, and the occurrence of acne is closely related to the physiological and pathological changes of the skin during adolescence. The main clinical manifestations are acne, papules, pustules, nodules, cysts, scars, etc. The healing time is long, which has a serious impact on the appearance and psychology of patients. Acne is associated with multiple pathogenic mechanisms. Abnormal keratosis of the hair follicle or mouth is an important basis for the pathogenesis of this disease.
  • Inflammation and infection are the pathogenic factors of acne.
  • the sebaceous glands of acne patients are larger, the secretion of sebaceous glands increases, and the level of linoleic acid in sebum is relatively reduced, which affects the synthesis of fat and leads to the lack of fatty acids in the follicular epithelium, thereby inducing hyperkeratosis of the follicles, so that the epithelial cells cannot be shed normally, and the pilosebaceous glands of the hair follicles are in the mouth. If it becomes too small, the sebum cannot be discharged smoothly, resulting in acne.
  • the follicles and sebaceous glands are blocked to form an oxygen-deficient environment in the hair follicles and sebaceous glands, resulting in the proliferation of anaerobic Propionibacterium acnes, which decomposes sebum, produces chemokines, and leukocytes aggregate to form papules.
  • a large number of neutrophils in the hair follicles and sebaceous glands accumulate, and an inflammatory reaction occurs after swallowing Propionibacterium acnes, causing a large number of pus cells to accumulate to form pustules and cysts, which are easy to form pitted scars.
  • ichthyosis Diseases with abnormal keratinization mechanisms similar to acne include ichthyosis, keratosis pilaris (also known as lichen pilaris), keratosis pilaris, and porokeratosis. Corner plugs, ichthyosis manifests as a decrease in sweat glands and sebaceous glands, and keratinous plugs in hair follicles.
  • the above diseases are easy to recur and difficult to treat.
  • the drugs for treating abnormal keratinization, eliminating keratin plugs and acne are mainly retinoids.
  • Retinoic acid drugs can inhibit keratinization, reduce sebum secretion, promote normal keratinization of keratinocytes, and have immune-regulating and anti-inflammatory effects, thereby reducing the formation of acne, papules and pustules, and are widely used clinically to treat Dyskeratotic diseases such as acne, ichthyosis, keratosis pilaris, keratosis pilaris, and porokeratosis.
  • Dyskeratotic diseases such as acne, ichthyosis, keratosis pilaris, keratosis pilaris, and porokeratosis.
  • external use of retinoids can irritate the skin, easily lead to redness, swelling, tingling, and aggravate the original skin lesions. Long-term external use of retinoids can cause skin thinning, photosensitivity and skin barrier damage, etc. Oral retinoids There are adverse reactions such as liver damage and elevated blood
  • Alopecia areata is a non-scarring alopecia with generally normal local skin. Usually, it is a sudden patch of hair loss, which can affect the entire scalp in severe cases, which is called alopecia totalis (AT), and when it affects all the hairs of the body, including axillary hair and pubic hair, it is called alopecia universalis (alopecia).
  • AU alopecia universalis
  • autoimmune function has not been fully understood, but abnormal or unstable autoimmune function and neuropsychiatric factors are considered to be important related factors.
  • Many cases have neuropsychiatric trauma before the onset, such as long-term anxiety, anxiety, sadness, mental stress and emotional disturbance. Sometimes in the course of the disease, these mental factors can make the condition worsen rapidly.
  • the cure rate of alopecia areata is high, but the cure rate of alopecia areata caused by different causes is very different. Some patients with alopecia areata can recover spontaneously even without any treatment measures, and some patients with alopecia areata can only maintain their disease progression after several years of treatment.
  • Minoxidil is a common topical drug for the treatment of alopecia areata, which can promote the expansion of skin blood vessels, improve local blood circulation, and promote hair growth.
  • Glucocorticoids commonly used in severe alopecia areata mainly include prednisolone, compound betamethasone, etc., which can be administered orally, externally or intradermally.
  • immunosuppressive therapy can be used, and common drugs are cyclosporine and methotrexate. Glucocorticoids and immunosuppressants have many side effects.
  • Androgenetic alopecia is an androgen-dependent hereditary hair loss, which is a common and frequently-occurring disease. Usually men in their 20s to 30s have the disease. Hair loss is mainly at the top of the head, mostly starting from the hairline on both sides of the forehead, but also from the top. The alopecia area gradually expands upwards, and the hair also becomes thinner and thinner, and eventually most or all of the hair on the top of the head falls off, but the hair on the back of the occiput and the upper temporal side of both sides depends on it, showing a horseshoe-shaped appearance, and the hair in this band-shaped area remains normal.
  • the skin of the hair loss area is bright, the pores are reduced or a few fine vellus hairs remain.
  • the speed, extent and severity of hair loss is genetic and individual. Generally, it develops the fastest around the age of 30, and severe total baldness is rare. In women, diffuse hair loss occurs mostly on the crown of the head, with thinning of the hair on the crown of the head.
  • Epidemiological surveys in my country show that the prevalence of androgenetic alopecia is 21.3% in males and 6.0% in females. The etiology and pathogenesis of androgenetic alopecia are still unclear.
  • Androgenetic alopecia is a refractory type of alopecia, and animal models of the disease are often used as representative models of alopecia. Since androgens play a large role in the pathogenesis, new treatments in recent years have attempted to stop hair follicle shrinkage through anti-androgen effects.
  • Minoxidil is a non-specific drug for the treatment of hair loss. It is a first-line topical drug approved by the FDA for the treatment of hair loss. However, it may cause facial and limb hirsutism during use, and the therapeutic effect gradually disappears after discontinuation.
  • Finasteride is a type II 5a-reductase selective inhibitor, FDA-approved oral finasteride for the treatment of androgenetic alopecia, sustainable improvement of hair growth, but the existence of finasteride can cause sexual function Abnormal, transient reduction of sperm and gynecomastia and other adverse reactions, found to have teratogenic effects in animal experiments, it is not suitable for children and women of childbearing age. Cimetidine needs to be taken continuously for 5 months or more, and the side effects are gynecomastia, impotence, and decreased libido.
  • Oral contraceptives the main ones are sogonorrone, levonorgestrel (levonorgestrel), norgestrel, norethisterone, norgestimate (norethisteryl oxime), norethisterone diester Alcohol and norethisterone acetate, etc. It is often used to treat AGA in women, and hair will improve after 6 to 12 months of treatment.
  • Hair loss caused by anti-tumor drugs is the most common form of baldness in the growing period. While eliminating rapidly dividing cancer cells, anti-tumor drugs also attack rapidly dividing cells around the hair follicle to cause hair loss.
  • Minoxidil is a non-specific drug for the treatment of hair loss. It is a first-line topical drug approved by the FDA for the treatment of hair loss. However, it may cause hirsutism of the face and limbs during use, and the therapeutic effect gradually disappears after discontinuation.
  • Finasteride is a selective inhibitor of type II 5a-reductase, FDA-approved for the treatment of SA, and can improve hair growth continuously, but it can cause abnormal sexual function, transient sperm reduction and gynecomastia. and other adverse reactions. Therefore, there is a need to find more safe and effective drugs and products for treating hair loss.
  • Scar is the damage caused by physical, biological, chemical and other factors to the human skin and soft tissue, resulting in serious damage to the skin and soft tissue, which cannot be repaired normally by itself, and is replaced by fibrous tissue. Scars bring great physical and mental pain to patients, especially scars left after burns, scalds, and severe trauma. It is difficult to deal with scars. At present, only red and hard scars can be softened and shallower, wide scars can be narrowed, and thick scars can be thinned, but scars cannot be completely eliminated. It is therefore important to start interventions early in wound healing to reduce scarring, improve appearance, correct deformities, and restore function. At present, the commonly used methods to treat scars include: surgical treatment, laser treatment, cryotherapy and drug treatment.
  • glucocorticoids have anti-inflammatory, anti-viral, anti-shock and other functions, and have obvious anti-tissue fibrosis effects, but long-term use of glucocorticoids has many toxic and side effects.
  • Retinoic acid is an intermediate product of vitamin A metabolism in the body and is a vitamin A related drug. Retinoic acid can reduce local inflammation, promote epithelial cell growth, reduce collagen synthesis, reduce DNA synthesis in fibroblasts, and inhibit cell growth. The higher the concentration of retinoic acid, the more obvious the growth inhibition effect. However, the curative effect of retinoic acid is limited, and there are many toxic and side effects of systemic application. External application has obvious irritation to the skin, and it increases with the increase of concentration.
  • Pulmonary fibrosis is a diffuse lung disease with unknown etiology and complex pathogenesis. It is currently recognized as the result of excessive deposition of extracellular matrix due to excessive repair after lung injury.
  • the pathogenesis of pulmonary fibrosis is unknown, and it is currently recognized that repeated damage and excessive repair of alveolar epithelium is the key to the pathogenesis.
  • the pathological feature is that long-term chronic pulmonary inflammation and persistent alveolar damage lead to the accumulation of extracellular matrix metalloproteinase (MMP), especially the abnormal increase of MMP-2 and MMP-9, and the decrease of tissue inhibitor of metalloproteinase-1 (TIMP-1).
  • MMP extracellular matrix metalloproteinase
  • VEGF vascular endothelial growth factor
  • Glucocorticoids are commonly used as anti-inflammatory drugs to reduce the process of anti-fibrosis, but the efficacy is limited and there are many adverse reactions.
  • PCSK9 Proprotein convertase subtilisin/kexin type 9
  • the size of PCSK9 gene cDNA is 3617bp, encoding PCSK9 protein composed of 692 amino acids.
  • the PCSK9 precursor undergoes intramolecular autocatalytic separation of its N-terminal propeptide within the endoplasmic reticulum, and the separated N-terminal propeptide is linked to the catalytic domain, allowing the mature PCSK9 protein to leave the endoplasmic reticulum and enter the secretory pathway.
  • PCSK9 low-density lipoprotein
  • body decreased, that is, the level of PCSK9 was negatively correlated with the LDL receptor.
  • Several studies have shown that the loss of function of PCSK9 gene mutation can significantly reduce the LDL-C level and the incidence of coronary heart disease in different races. In view of the significant effect of inhibiting PCSK9 on reducing the incidence of LDL-C and coronary heart disease, a number of therapeutic regimens have been developed to block PCSK9 drugs to reduce the incidence of LDL-C and coronary heart disease.
  • PCSK9 inhibitors include two categories: 1. Prevent the binding of PCSK9 to LDL-R, such as monoclonal antibodies, peptidomimetics (polypeptide inhibitors), and antibody-mimicking protein drugs; 2. Inhibit the expression of PCSK9 molecules or interfere with PCSK9 secretion, such as small Molecular interfering RNA, antisense oligonucleotides, small molecule compound inhibitors, etc. Monoclonal antibodies have become a hot spot in new drug research due to their high blocking efficiency, accurate target sites and good stability. At present, the PCSK9-targeting monoclonal antibodies on the global market are all drugs that prevent the binding of PCSK9 to LDL-R.
  • evolocumab evolocumab
  • Repatha trade name Repatha
  • alizumab, alizumab trade name Praluent (Polida)
  • Eli lilly's LY3015014 Junshi Bio's recombinant humanized anti-PCSK9 monoclonal antibody (JS002)
  • Xinlitai Pharmaceutical industry's recombinant fully human anti-PCSK9 monoclonal antibody injection.
  • Inclisiran is an siRNA (Small Interfering RNA) drug, which is different from the monoclonal antibody that directly binds to PCSK9 molecules. It can inhibit the expression of the PCSK9 gene, so that the LDL receptor will not be degraded by PCSK9, thereby improving hepatocytes. Uptake of LDL particles, lowering LDL levels in the blood.
  • Alnylam's Inclisiran uses a proprietary technology that consists of lipid nanoparticles combined with GalNAc (N-acetylgalactosamine), which binds to the sialoglycoprotein receptors ASGR1 and ASGR2 expressed on the surface of liver cells.
  • the technology allows for subcutaneous administration and targeting the liver.
  • ALN-PCS and ALN-PCSsc of Affiris are also siRNA (small interfering RNA) drugs.
  • PCSK9 interfering RNAI inhibitor drugs also include Alnylam's Inclisran, Affiris' ALN-PCS and ALN-PCSsc.
  • PCSK9 mimetic peptides and PCSK9 mimetic antibody protein drugs include Pieris' DS9001 and Merck's 1G08.
  • PCSK9 antisense oligonucleotide drugs include SPC5001 of Santaris Pharma, etc.
  • PCSK9 inhibitors have not been used to treat vitiligo, alopecia, scarring, pulmonary fibrosis, and dyskeratotic diseases such as acne, ichthyosis, keratosis pilaris, keratosis pilaris, and porokeratosis related literature, patents and products.
  • the problem to be solved by the present invention is to clarify the role of PCSK9 gene in the pathogenesis of vitiligo, alopecia, dyskeratosis, scarring and pulmonary fibrosis, and the application of PCSK9 inhibitors in the preparation of products for the treatment of the above diseases.
  • the present invention discovers the key role of PCSK9 in the pathogenesis of vitiligo, alopecia, abnormal keratinization, scarring and pulmonary fibrosis through experimental research on animal models of related diseases, and the use of PCSK9 inhibitors in the preparation of products for the treatment of the above diseases Applications.
  • the present invention discovers the important role of PCSK9 gene in promoting skin pigmentation by establishing a representative depigmentation disease vitiligo animal model.
  • PCSK9 protein sequence is shown in SEQ ID NO: 1 in the Sequence Listing.
  • the present invention finds that the PCSK9 gene plays a key role in the pathogenesis of acne, and the application value of PCSK9 inhibitors in the preparation of products for treating acne.
  • the pathogenesis of abnormal keratinization diseases such as ichthyosis, keratosis pilaris, keratosis pilaris, and porokeratosis is characterized by the appearance of horn plugs and enlarged follicular openings similar to acne.
  • Androgenetic alopecia is a refractory type of alopecia, and animal models of this disease are often used as a representative model of alopecia.
  • the present invention finds that PCSK9 gene plays a key role in the pathogenesis of alopecia disease, and the application value of PCSK9 inhibitor in preparing medicine for treating alopecia disease.
  • the present invention finds that the PCSK9 gene plays a key role in the scar (scar) formation mechanism, and the application value of PCSK9 inhibitors in the preparation of products for preventing and treating scars (scar).
  • the C57BL/6 mouse is an animal model widely used at home and abroad to study the hair cycle: the hair cycle of each human hair follicle is not synchronized, and this mouse can show a unique hair cycle synchronization, so it is often used as a hair research model .
  • the melanocytes in the trunk skin of the mouse only exist in the hair follicles, and only synthesize melanin during the growth phase.
  • the appearance of the skin is black; during the degenerative phase, the production of melanin is reduced, and the skin is gray-black; in the telogen phase, the hair follicles stop producing melanin due to the shrinkage of the hair bulb, and the skin turns pink.
  • the invention adopts subcutaneous injection of testosterone propionate injection to create an experimental androgenetic alopecia model, and finds that PCSK9 gene knockout can obviously promote the hair growth of androgenetic alopecia model mice, and reduce the damage to subcutaneous hair follicles and sebaceous glands.
  • paraffin wax depilation is used to establish a non-specific hair loss model in mice, and it is confirmed that the PCSK9 inhibitor has a very obvious promoting effect on hair growth.
  • Rabbit ear is an animal model commonly used to measure the effect of acne-causing substances.
  • the size of the hair follicles and sebaceous glands varies greatly in rabbits like humans. With the increase of age, the ability of the animals to form acne also increases, so adult male rabbits are selected to replicate the acne model, so that the male hormones in the body have a certain stimulating effect on the skin.
  • Adapalene can remove keratin plugs by regulating the abnormal process of pilosebaceous epithelial keratinization, thereby preventing and eliminating acne lesions. Therefore, in this study, adapalene (trade name: Daphne) was selected as the positive control of the experiment. Taste.
  • PCSK9 monoclonal antibodies are respectively selected from the inhibitors that can obviously block PCSK9, and are treated by subcutaneous injection or skin external application.
  • Rabbit ear acne model compared with negative control group and positive drug (Duffvin). The results showed that the symptoms in the PCSK9 inhibitor group were significantly lighter than those in the model control group.
  • systemic or topical PCSK9 inhibitors can significantly inhibit the symptoms of rabbit ear acne model, reduce the formation of hair follicle corner plugs and blackheads, and are mild and non-irritating to the skin, showing that PCSK9 inhibitors have obvious therapeutic effects on acne.
  • PCSK9 inhibitors can significantly improve acne and inhibit the formation of inner corner plugs of hair follicles.
  • the pathogenesis of acne is complex, and the abnormal keratinization of the skin hair follicle orifice is an important basis for the pathogenesis of this disease.
  • the sebaceous glands of the hair follicles are blocked by keratinocytes, and the keratin and sebum are filled, forming acne, leading to clogged pores, and the growth of acne bacillus and staphylococcus aureus, which damages the skin tissue.
  • P. acnes induces and alters the pathway of complement activation, contributing to the inflammatory response.
  • dyskeratotic diseases such as ichthyosis and keratosis pilaris (also known as lichen pilaris)
  • the appearance of keratin plugs is similar to that of acne, and the hair follicle orifice is enlarged.
  • the invention applies 2% coal char solution on the inner side of the ears of New Zealand rabbits, once a day, for 14 days continuously, to establish an acne micro-comedone model, and visually observe the thickness, hardness, roughness of the ear and whether there is a black horn plug at the hair follicle orifice and other local skins. Changes in the microscopic experimental acne histological grading score. Through the above studies, it was found that PCSK9 knockout can significantly inhibit the symptoms of coal tar-induced rabbit acne model, reduce pore blockage, and significantly inhibit the formation of blackheads, confirming that PCSK9 plays a key role in the pathogenesis of acne.
  • representative PCSK9 monoclonal antibodies, PCSK9 polypeptide inhibitors, PCSK9 small molecule compound inhibitors and PCSK9 small interfering RNAs are respectively selected among the inhibitors that can obviously block PCSK9, and the rabbits are treated by subcutaneous injection or external application on the skin.
  • Ear acne model compared with negative control group and positive drug (Duffvin).
  • the results showed that the symptoms in the PCSK9 inhibitor group were significantly lighter than those in the model control group.
  • the activities and foraging of the rabbits were normal. Respiratory and central nervous system abnormalities.
  • PCSK9 inhibitors can significantly inhibit the symptoms of rabbit ear acne model, reduce the formation of hair follicle corner plugs and blackheads, and are mild and non-irritating to the skin, showing that PCSK9 inhibitors have obvious therapeutic effects on acne.
  • PSCK9 gene knockout can significantly promote the healing of skin wounds in rats, reduce scar (scar) formation, and can also significantly inhibit the proliferation of skin fibroblasts and promote fibroblast apoptosis. Inhibits the proliferation of skin fibroblasts and promotes their apoptosis to inhibit the formation of scars (scars).
  • PCSK9 inhibitors that can obviously block PCSK9
  • representative PCSK9 monoclonal antibodies PCSK9 polypeptide inhibitors
  • PCSK9 small molecule compound inhibitors PCSK9 small interfering RNAs are selected respectively.
  • Intravenous injection or skin topical intervention in the rat scar (scar) model it was found that the scar hyperplasia in the PCSK9 inhibitor group was significantly better than that in the model group.
  • various PCSK9 inhibitors can promote skin wound healing and reduce scar (scar) formation, showing that both systemic and external PCSK9 inhibitors can have obvious preventive and therapeutic effects on skin scars (scars), and no Obvious toxic side effects.
  • a pulmonary fibrosis model is established by injecting bleomycin into a rat's neck trachea.
  • PSCK9 gene knockout can significantly increase the levels of MMP-2, MMP-9 and VEGF in the lung tissue of a mouse model of pulmonary fibrosis, reduce the level of TIMP-1, and at the same time can increase the levels of SOD and CAT enzymes in peripheral blood, showing that the lung Fibrosis has an inhibitory effect.
  • representative PCSK9 monoclonal antibodies, PCSK9 polypeptide inhibitors, PCSK9 small molecule compound inhibitors and PCSK9 small interfering RNA inhibitors are respectively selected among the inhibitors that can obviously block PCSK9, and are treated by tail vein injection.
  • a rat model of pulmonary fibrosis Experiments have confirmed that PCSK9 inhibitors can significantly improve the laboratory indicators of pulmonary fibrosis, showing that PCSK9 inhibitors have obvious therapeutic effects on pulmonary fibrosis.
  • PCSK9 inhibitors have therapeutic effects on other similar diseases.
  • PCSK9 inhibitors can be used alone or in combination with other drugs or treatments, including traditional drugs and other targeted biologics.
  • the present invention relates to the application of PCSK9 inhibitors (blockers) in the preparation of products for the treatment of vitiligo, dyskeratotic diseases, alopecia diseases, scarring and pulmonary fibrosis.
  • the PCSK9 belongs to the proprotein convertase family (genebank serial number: 255738);
  • the dyskeratotic diseases include acne, ichthyosis, keratosis pilaris, keratosis pilaris and porokeratosis, etc.
  • the alopecia diseases include androgenetic alopecia or alopecia areata or alopecia caused by anti-tumor therapy.
  • the PCSK9 inhibitor of the present invention can be any conventional molecular biology or medicinal chemical means that can inhibit the expression or secretion of the PCSK9 gene, such as, but not limited to, knocking out the PCSK9 gene through existing molecular biology techniques. removal or silencing; in some embodiments, PCSK9 inhibitors can also be used or used, preferably, the above PCSK9 inhibitors (blockers) are PCSK9 small molecule compounds or PCSK9 interfering RNAs or PCSK9 monoclonal antibodies or PCSK9 mimetic peptides or PCSK9 mimetic antibody protein or PCSK9 antisense oligonucleotide or PCSK9 vaccine.
  • the PCSK9 small molecule compounds described in the present invention include but are not limited to R-IMPP, a product of Selleck Company, chemical formula: C 24 H 27 N 3 O 2 , molecular weight: 389.49, structural formula: Or Selleck company product PF-06446846, chemical formula: C 22 H 20 ClN 7 O, molecular weight: 433.04, structural formula: Or Selleck company product SBC-115076, chemical formula: C 31 H 33 N 3 O 5 , molecular weight: 527.61, structural formula: Or Selleck company product SBC-110736, chemical formula: C 26 H 27 N 3 O 2 , molecular weight: 413.51, structural formula:
  • the PCSK9 monoclonal antibody inhibitors of the present invention include, but are not limited to, ab84041 from Abcam, or evolocumab (evolocumab) jointly developed by Amgen and Astellas, Or alirocumab (alizumab, alizumab) jointly developed by Sanofi and Regeneron, or Junshi Bio’s recombinant humanized anti-PCSK9 monoclonal antibody (JS002), or Litai Pharmaceutical's recombinant fully human anti-PCSK9 monoclonal antibody injection, or Eli lilly's LY3015014, or Merck's 1G08.
  • the PCSK9 interfering RNAI inhibitors described in the present invention include, but are not limited to, Inclisran from Alnylam, ALN-PCS and ALN-PCSsc from Affiris.
  • PCSK9 mimetic peptide inhibitors and PCSK9 mimetic antibody protein inhibitors described in the present invention include, but are not limited to, DS9001 from Pieris and 1G08 from Merck.
  • PCSK9 antisense oligonucleotide inhibitors of the present invention include, but are not limited to, SPC5001 from Santaris Pharma.
  • the PCSK9 vaccine inhibitors of the present invention include, but are not limited to, AT04A and AT06A from Affiris.
  • PCSK9 inhibitors can be used alone or in combination with other drugs or treatments, including traditional drugs and other targeted biologics.
  • the present invention also provides a pharmaceutical composition, which is composed of the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient or main active ingredient, supplemented by a pharmaceutically acceptable carrier.
  • the compounds or compositions of the present invention can be prepared in any pharmaceutically acceptable dosage form, for example, those suitable for oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal , intranasal, inhalation, vaginal, intraocular, topical, subcutaneous, intrafat, intraarticular, intraperitoneal or intrathecal formulations for any mode of administration.
  • the dosage forms of the present invention are ointments, tablets, granules, oral liquids, capsules, drop pills, enemas, films or injections.
  • the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a new and better treatment method for the treatment of vitiligo, dyskeratotic diseases, alopecia diseases, scars and pulmonary fibrosis. It is revealed that systemic or external PCSK9 inhibitor (blocker) products can be further prepared, and new monomer drugs or compound preparations containing various PCSK9 inhibitors can be developed for the treatment of the above diseases.
  • Existing clinical trials have proved that such drugs containing PCSK9 inhibitors have significant curative effect in the treatment of the above diseases, less adverse reactions, and good patient tolerance. Especially, only external use can significantly improve symptoms, which is very suitable for actual clinical needs. It can provide the market with a series of new products with lower price, good curative effect and safety.
  • mice of different genotypes were divided into three groups: C57BL/6 mouse blank control group, C57BL/6 mouse model group and C57BL/6-PCSK9-/- mouse model group, with 6 mice in each group, 3 males and 3 females.
  • the black hair of 2cm ⁇ 2cm on the back was removed.
  • the other groups were smeared with 5% hydroquinone to decolorize the hair removal area, 0.5 mL each time, twice a day, and the blank control group animals were smeared with the same amount of normal saline every day. After 30 days of continuous smearing, obvious white spots appeared in the model group, indicating that the model was successful. After the modeling was completed, the pigment situation of the decolorized part where 5% hydroquinone was applied was observed.
  • mice were sacrificed, and the skin tissue on the back of the site where 5% hydroquinone was applied was cut out about 1 cm ⁇ 1 cm for histopathological examination.
  • the cut mouse skin tissue was fixed in 10% formalin for 1 hour, then rinsed with running water for 3-4 minutes, and placed in a phosphate buffer (pH 7.4) containing 0.1% dihydroxyphenylalanine at 37°C. Let stand for 1h. Then it was replaced with fresh Dopa reagent, and it was allowed to stand at 37°C for 12h. Rinse with running water and continue to fix with Bouin's solution for 24h. The specimens were taken out, rinsed with running water for 4 hours, dehydrated, transparent in xylene, embedded in paraffin, deparaffinized to water, and stained with hematoxylin-eosin for contrast. Dehydrated and routinely mounted.
  • brown-black staining was MC positive.
  • the number of MCs in each group in each high-power field of view was measured under a light microscope, 10 high-power fields were observed for each specimen, and the average number of MCs in every 100 epidermal basal cells was calculated.
  • the excised mouse skin tissue was fixed in 10% formalin, dehydrated, cleared with xylene, embedded in paraffin, sectioned, deparaffinized to water, washed with distilled water for several times, and then stained with Lillie with ferric chloride and iron.
  • the potassium cyanide mixture was soaked for 15-20 minutes, then differentiated with 1% acetic acid aqueous solution for several minutes, washed with distilled water for several times, and then rapidly dehydrated with 95% ethanol and absolute ethanol, transparent in xylene, and sealed with neutral gum. After Lillie staining, the melanin was dark green as positive.
  • the number of basal cells containing melanin granules in each group was determined under a light microscope. Each specimen was observed in 10 high-power fields, and the number of melanin granules in every 100 epidermal basal cells was calculated. the average number of basal cells.
  • the excised mouse skin tissue was fixed in 10% formalin. Then dehydrate, clear in xylene, embed in paraffin, make sections, deparaffinize to water, add 50 ⁇ L of peroxidase blocking agent (3% H 2 O 2 ) to each section for 10 min to block endogenous peroxides enzymatic activity.
  • the slices were immersed in 0.01mol ⁇ L-1PBS buffer and microwaved for 10min. Add mouse anti-human tyrosinase monoclonal antibody 50 ⁇ L, 37 °C, 60min. Add 50 ⁇ L of Elivision reagent, 37°C for 30min.
  • the sections were then placed in freshly prepared DAB-H 2 O 2 chromogenic solution for color development, rinsed with distilled water, stained with hematoxylin for 1 min, and sealed with neutral gum.
  • the positive product of tyrosinase is located in the cytoplasm of the cell, and yellow or brownish-yellow granules or clumps appear in the cells under microscope.
  • the two-level scoring method was used to determine the results.
  • the positive cell count was less than 5%, 0 points; 5%-25%, 1 point; 25%-50%, 2 points; 50%-75%, 3 points; >75%, 3 points 4 points. Classification by staining intensity: 1 point for light yellow; 2 points for yellow or dark yellow; 3 points for brown or brownish yellow. If the sum of the two is less than 2, it is negative (-); 2-3 is positive (+); 4-5 is moderately positive (2+); 6-7 is strongly positive (3+).
  • the experimental data were statistically processed by SPSS 16.0 system software. Statistical variables of experimental data were expressed as (x ⁇ s), using ⁇ 2 test and t test, and Wilcoxon rank sum test was used to compare the intensity of tyrosinase in animal skin tissue. The ⁇ value was taken on both sides, P>0.05 means no significant difference, P ⁇ 0.05 means significant difference, and P ⁇ 0.01 means very significant difference.
  • the tyrosinase intensity in the skin tissue of the C57BL/6 mouse model group was significantly lower than that of the blank control group (P ⁇ 0.01).
  • the C57BL/6-PCSK9-/- mouse model group could increase the intensity of tyrosinase in the skin tissue of the vitiligo mouse model (P ⁇ 0.01).
  • Knockout of PCSK9 gene can increase the number of epidermal MC and melanin granule-containing basal cells in the skin tissue of vitiligo model, and improve the tyrosinase intensity of skin tissue.
  • Equal amounts of siPcsk9-1 and 2 were mixed and then diluted to 20 ⁇ M with normal saline, and the diluted siRNA was mixed evenly with the lotion.
  • PCSK9 Interfering RNAi Inhibitor-2 RNA sequence identical to Alnylam's Inclisran; PCSK9 Interfering RNAi Inhibitor-3:
  • RNA sequence is the same as ALN-PCS from Affiris.
  • PCSK9 small molecule compound inhibitor 1 R-IMPP from Selleck Company, chemical formula: C 24 H 27 N 3 O 2 , molecular weight: 389.49, structural formula: PCSK9 small molecule compound inhibitor 2: Selleck company product PF-06446846, chemical formula:
  • PCSK9 monoclonal antibody 1 purchased from Abcam company (ab84041); PCSK9 monoclonal antibody 2: evolocumab (evolocumab); PCSK9 monoclonal antibody 3: alirocumab (alizumab).
  • PCSK9 polypeptide ab32727 of Abcam Company.
  • Positive therapeutic drug mometasone furoate cream (trade name: Ailosone, produced by Schering-Plough China Co., Ltd.)
  • the composition of excipient base includes methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), stearyl alcohol ( 5%), glycerin (20%), alkyl aryl polyglycol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethylparaben (0.1%) ), distilled water (about 31-55%), and mixed with an appropriate amount of PCSK9 inhibitor to form a mixed emulsion.
  • the cream base used in this example refers to the base component of the cream from which the active ingredient is removed.
  • the other groups were smeared with 5% hydroquinone to decolorize the hair removal area, 0.5 mL each time, twice a day, and the animals in the blank control group were smeared with an equal amount of normal saline every day. After 30 days of continuous smearing, the appearance of obvious white spots indicates that the model is successful. After modeling, the drug was administered in the above manner, once a day for 30 days.
  • the guinea pigs were sacrificed, and the skin tissue at the dosing site on the back of about 3 cm ⁇ 3 cm was cut for histopathological examination.
  • the cut guinea pig skin tissue was fixed in 10% formalin for 1 hour, then rinsed with running water for 3-4 minutes, placed in a phosphate buffer (pH 7.4) containing 0.1% dihydroxyphenylalanine, and kept at 37°C. Set 1h. Then it was replaced with fresh Dopa reagent, and it was allowed to stand at 37°C for 12h. Rinse with running water and continue to fix with Bouin's solution for 24h. The specimens were taken out, rinsed with running water for 4 hours, dehydrated, transparent in xylene, embedded in paraffin, deparaffinized to water, and stained with hematoxylin-eosin for contrast. Dehydrated and routinely mounted.
  • brown-black staining was MC positive.
  • the number of MCs in each group in each high-power field of view was measured under a light microscope, 10 high-power fields were observed for each specimen, and the average number of MCs in every 100 epidermal basal cells was calculated.
  • the cut guinea pig skin tissue was fixed in 10% formalin, dehydrated, cleared with xylene, embedded in paraffin, sectioned, deparaffinized to water, washed with distilled water for several times, and then stained with Lillie, with ferric chloride and ferricyanide.
  • the potassium chloride mixture was soaked for 15 to 20 minutes, then differentiated with 1% acetic acid aqueous solution for several minutes, washed with distilled water for several times, and then quickly dehydrated with 95% ethanol and absolute ethanol, transparent in xylene, and sealed with neutral gum. After Lillie staining, the melanin was dark green as positive.
  • the number of basal cells containing melanin granules in each group was determined under a light microscope. Each specimen was observed in 10 high-power fields, and the number of melanin granules in every 100 epidermal basal cells was calculated. the average number of basal cells.
  • the excised guinea pig skin tissue was fixed in 10% formalin. Then dehydrate, clear in xylene, embed in paraffin, make sections, deparaffinize to water, add 50 ⁇ L of peroxidase blocking agent (3% H 2 O 2 ) to each section for 10 min to block endogenous peroxides enzymatic activity.
  • the slices were immersed in 0.01mol ⁇ L-1PBS buffer and microwaved for 10min. Add mouse anti-human tyrosinase monoclonal antibody 50 ⁇ L, 37 °C, 60min. Add 50 ⁇ L of Elivision reagent, 37°C for 30min.
  • the sections were then placed in freshly prepared DAB-H 2 O 2 chromogenic solution for color development, rinsed with distilled water, stained with hematoxylin for 1 min, and sealed with neutral gum.
  • the positive product of tyrosinase is located in the cytoplasm of the cell, and yellow or brownish-yellow granules or clumps appear in the cells under microscope.
  • the two-level scoring method was used to determine the results.
  • the positive cell count was less than 5%, 0 points; 5%-25%, 1 point; 25%-50%, 2 points; 50%-75%, 3 points; >75%, 3 points 4 points. Classification by staining intensity: 1 point for light yellow; 2 points for yellow or dark yellow; 3 points for brown or brownish yellow. If the sum of the two is less than 2, it is negative (-); 2-3 is positive (+); 4-5 is moderately positive (2+); 6-7 is strongly positive (3+).
  • the experimental data were statistically processed by SPSS 16.0 system software. Statistical variables of experimental data were expressed as (x ⁇ s), using ⁇ 2 test and t test, and Wilcoxon rank sum test was used to compare the intensity of tyrosinase in animal skin tissue. The ⁇ value was taken on both sides, P>0.05 means no significant difference, P ⁇ 0.05 means significant difference, and P ⁇ 0.01 means very significant difference.
  • PCSK9 inhibitors can treat depigmentation diseases by increasing the number of epidermal MC and melanin granule-containing basal cells in the skin tissue of vitiligo model, and increasing the tyrosinase intensity in skin tissue.
  • mice SPF grade New Zealand rabbits, PCSK9-/-SPF grade New Zealand rabbits (using CRISPR gene editing technology to knock out Pcsk9 gene exon 2-3 to establish a Pcsk9 gene knockout rabbit model), 1.8-2.3 kg, male. Animals are derived from Southern Model Organisms.
  • the rabbits of different genotypes were divided into three groups: negative control group, model control group and PCSK9-/- model group, with 6 rabbits in each group.
  • the inner side of the right ear of the rabbit was taken as the observation area, the negative control group was coated with 95% alcohol, the model control group and the PCSK9-/- model group were coated with 2% coal tar solution (Alfa Aesar China Co., Ltd.), and 95% alcohol was used to prepare 2 % coal tar solution, apply a sterile cotton swab evenly to the opening of the ear canal on the inner side of the rabbit ear in a range of about 2cm ⁇ 2cm, once a day, 0.5mL each time, and wipe the previous application site with warm water for 14 days.
  • Acne model histological grading standard according to the histological grade is 3 grades.
  • Grade 0 means that there are only loose keratinized cells in the infundibulum, and no acne is generated "-";
  • Grade 1 means that the skin on the surface of the rabbit ear is red, or a small amount of dense keratinized material is seen in the infundibulum of the hair follicle, and the infundibulum does not expand "+”;
  • Grade 2 moderately dense keratinized material in the infundibulum of the hair follicle, extending to the sebaceous gland, with the proliferation of sebaceous gland ducts, the infundibulum expands "2+”;
  • Grade 3 extensive keratinized material in the hair follicle, caused by tight keratin embolism in the hair follicle The hair follicles were severely expanded, the epithelium of the sebaceous gland ducts proliferated significantly, the skin was raised
  • the rabbit ears of the negative control group were soft, thin and white, the capillaries were clear, and the pores in the opening of the rabbit ear tubes were uniform in size.
  • the ear thickness of rabbits increased and became hard, the hair follicles had black horn plugs, which were blackheads and comedones, and the hair follicles were raised in the form of papules.
  • the skin of the external auditory canal of the rabbits in the PCSK9-/- model group was slightly dry, and the pores were slightly enlarged. There was no black substance blocking the pores and no papules.
  • Knockout of PCSK9 can significantly inhibit the symptoms of coal tar-induced acne rabbit model, reduce pore clogging, and significantly reduce the formation of blackheads.
  • Equal amounts of siPcsk9-1 and 2 were mixed and then diluted to 20 ⁇ M with normal saline, and the diluted siRNA was mixed evenly with the lotion.
  • PCSK9 interfering RNAi inhibitor-2 The RNA sequence is the same as that of Alnylam's Inclisran;
  • PCSK9 interfering RNAi inhibitor-3 The RNA sequence is the same as that of Affiris' ALN-PCS.
  • PCSK9 small molecule compound inhibitor 1 R-IMPP, a product of Selleck, chemical formula: C 24 H 27 N 3 O 2 , molecular weight: 389.49, structural formula: PCSK9 small molecule compound inhibitor 2: Selleck company product PF-06446846, chemical formula:
  • PCSK9 monoclonal antibody 1 purchased from Abcam (ab84041); PCSK9 monoclonal antibody 2: evolocumab (evolocumab) jointly developed by Amgen and Astellas, trade name Repatha (Repatha); PCSK9 monoclonal antibody 3: alirocumab (alizumab, alizumab) jointly developed by Sanofi and Regeneron, trade name Praluent (Polida).
  • PCSK9 polypeptide-1 was purchased from Abcam Company (ab32727); PCSK9 polypeptide-2: DS9001 from Pieris Company.
  • Positive therapeutic drug 0.1% adapalene gel (trade name: Daphne, produced by Galderma, France)
  • excipient base composition includes methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), stearyl alcohol (5%) , glycerin (20%), alkyl aryl polyglycol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethylparaben (0.1%), distilled water (about 31-55%), respectively mixed with an appropriate amount or more of the PCSK9 inhibitor to form a mixed emulsion.
  • the cream base used in this example refers to the base component of the cream from which the active ingredient is removed.
  • compound group 1 (0.1% R-IMPP cream applied to the skin, twice a day)
  • compound group 2 (0.1% PF-06446846 cream applied to the skin, twice a day)
  • compound group 2 (0.1% PF-06446846 cream applied to the skin, twice a day)
  • Group 3 (0.1% SBC-115076 cream applied to the skin, twice a day)
  • compound group 4 (0.1% SBC-110736 cream applied to the skin, twice a day)
  • PCSK9 interfering RNA group-1 (0.1% PCSK9 small skin applied Interfering RNA-1 cream, 2 times a day
  • PCSK9 interfering RNA group-2 (0.1% Inclisran cream applied to the skin, 2 times a day
  • PCSK9 interfering RNA group-3 (0.1% ALN-PCS cream applied to the skin, daily 2 times
  • monoclonal antibody group 1 subcutaneous injection of PCSK9 monoclonal antibody ab84041, 1 mg/kg.d
  • monoclonal antibody group 2 sub
  • the inner side of the right ear of the rabbit was taken as the observation area, the left ear of all rabbits was used as a self-negative control, and 95% alcohol was applied.
  • the changes of local skin were observed with naked eyes, including ear thickness, hardness, roughness, and the presence or absence of black horn plugs at the mouth of the hair follicle. 18 hours after the last application, the samples were sacrificed, and the skin tissue was obtained by punching a hole in the application site with a 5mm punch, fixed in 10% formaldehyde, embedded in paraffin, and stained with HE for histopathological analysis.
  • Acne model histological grading standard according to the histological grade is 3 grades.
  • Grade 0 "—” means that there are only loose keratinized cells in the infundibulum, and no comedones are generated;
  • Grade 1 the skin on the surface of the rabbit ear is red, or a small amount of dense keratinized material is seen in the infundibulum of the hair follicle, and the infundibulum does not expand "+”;
  • Grade 2 moderately dense keratinized material in the infundibulum of the hair follicle, extending to the sebaceous gland, with the proliferation of sebaceous gland ducts, the infundibulum expands "2+”;
  • Grade 3 extensive keratinized material in the hair follicle, caused by tight keratin embolism in the hair follicle The hair follicles were severely expanded, the epithelium of the sebaceous gland ducts proliferated significantly, the skin was
  • the skin of the right ear of the rabbit was thinner than that of the left ear, most of the follicular papules subsided, the acne was reduced and flattened, the pores were reduced, and the skin was slightly dry.
  • the right ear of the monoclonal antibody treatment group, the polypeptide treatment group and the combined treatment group showed thin and soft skin, reduced acne, significantly reduced pores, no desquamation, and was basically close to the normal rabbit ear.
  • the right ear of the rabbits in the positive treatment group had mild redness, a little desquamation, a small amount of hair follicle horn plugs and acne, and no papules were found.
  • the left ear of the model group showed that the epidermis was thinner, hair follicles were visible, and the junction between the dermis and the epidermis was clear.
  • the epidermis was thickened, hyperkeratosis, the granular layer and the spinous layer were thickened, the hair follicles were enlarged, the horn plug blocked the opening of the hair follicle, and extended to the sebaceous gland.
  • Histological grading of experimental acne under microscope in each group (see Table 8): The right ear of the rabbit in the model group was compared with its left ear (blank control), and the difference was statistically significant (P ⁇ 0.05). The right ears of rabbits were compared, and the difference was statistically significant (P ⁇ 0.05). The histological grading of acne pimples in each group is shown in Table 8 below.
  • the effect of the treatment group was improved to varying degrees, and the effect of the monoclonal antibody group 1-3 was better than that of the other groups. Therefore, the effect is better.
  • the other groups are applied externally, and the effect will be reduced accordingly, but they are all better than the right ear of the model group.
  • the therapeutic effect has reached a level similar to that of the monoclonal antibody group with the best effect.
  • the combination therapy group adopts the method of external application, the administration method is easier than that of the monoclonal antibody group.
  • the thickness of the right ear epidermis, hair follicle volume and sebaceous gland diameter of the rabbits in the model group were compared with those of the left ear (blank control), and the differences were statistically significant (P ⁇ 0.05), indicating that the rabbit ear acne model was successfully replicated; the right ear epidermis of the rabbits in each treatment group was The thickness, hair follicle volume and sebaceous gland diameter were compared with those of the right ear of the model group, and the differences were statistically significant (P ⁇ 0.05) (see Table 9).
  • the thickness of ear epidermis, hair follicle area and sebaceous gland diameter in each group are shown in Table 9 below.
  • the epidermis thickness, hair follicle area and sebaceous gland diameter of the right ear of the model group increased significantly, while compound groups 1-4 and mAb groups 1-3 Compared with the left ear of the model group, the interfering RNA group 1-3, the polypeptide group and the combined treatment group also increased slightly, but the increase was not large.
  • the ears are basically the same. The main reason is that the administration method is injection, which belongs to the in vivo administration, so the effect is better. The other groups are applied externally, and the effect will be reduced accordingly, but they are also better than the right ear of the model group.
  • the therapeutic effect is also comparable to that of the positive treatment group, especially the combined treatment group, whose therapeutic effect has reached a level similar to that of the best monoclonal antibody treatment group 2-3.
  • the method of external application is easier to administer than the monoclonal antibody group.
  • the inventors believe that the combination therapy group also produces more excellent effects than the single use.
  • PCSK9 small molecule compound inhibitors can significantly inhibit the symptoms of coal tar-induced acne model in rabbit ears, reduce pore blockage and blackhead formation, and are mild and non-irritating to the skin. Has a therapeutic effect on acne.
  • mice SPF grade C57BL/6(B6) mice, C57BL/6-PCSK9-/- mice (using CRISPR gene editing technology to knock out the Pcsk9 gene exon 2-3 to establish a Pcsk9 gene knockout mouse model), Mice are derived from Southern Model Organisms.
  • mice with different genotypes were divided into three groups: C57BL/6 mouse negative control group, C57BL/6 mouse model group and C57BL/6-PCSK9-/- mouse model group, with 6 mice in each group, 3 males and 3 females.
  • the back of each mouse was depilated as an observation area.
  • mice in other groups were injected with testosterone propionate injection [8ml/(kg ⁇ d)] subcutaneously at the back of the neck, once a day for 60 days to establish SA model. After continuous subcutaneous injection of testosterone propionate for 30 days, the mice gradually appeared hair loss, which proved that the androgenetic alopecia model was successfully established. Observe hair growth.
  • the grading standards are as follows: normal skin dermal tissue cells, subcutaneous hair follicles, and sebaceous glands are marked as "-": no hyperplasia in the skin dermis, limited lesions of the hair follicles and sebaceous glands, and no subcutaneous inflammation, marked as " ⁇ ”: no skin and dermis tissue There is obvious hyperplasia and obvious cystic degeneration of hair follicles. No obvious hyperplasia of sebaceous glands, and no inflammation under the skin is marked as "+”: segmental hyperplasia of skin and dermis is not obvious, a small number of hair follicles have cystic degeneration, and sebaceous glands have Mild hyperplasia.
  • the hair length of the C57BL/6-PCSK9-/- mouse model group was longer than that of the C57BL/6(B6) mouse model group on the 10th, 20th, and 30th days of modeling, and the differences were statistically significant (P ⁇ 0.01).
  • the hair growth lengths of mice in each group are shown in Table 10 below.
  • Knockout of PCSK9 gene can significantly promote hair growth and reduce damage to subcutaneous hair follicles and sebaceous glands in mice with androgenetic alopecia.
  • Equal amounts of siPcsk9-1 and 2 were mixed and then diluted to 20 ⁇ M with normal saline, and the diluted siRNA was mixed evenly with the lotion.
  • PCSK9 interfering RNAi inhibitor-2 The RNA sequence is the same as that of Alnylam's Inclisran;
  • PCSK9 interfering RNAi inhibitor-3 The RNA sequence is the same as that of Affiris' ALN-PCS.
  • PCSK9 small molecule compound inhibitor 1 R-IMPP, a product of Selleck Company, chemical formula: C 24 H 27 N 3 O 2 , molecular weight: 389.49
  • structural formula: PCSK9 small molecule compound inhibitor 2 Selleck company product PF-06446846, chemical formula: C 22 H 20 ClN 7 O, molecular weight: 434.04, structural formula: PCSK9 small molecule compound inhibitor 3: SBC-115076, a product of Selleck, chemical formula: C 31 H 33 N 3 O 5 , molecular weight: 527.61
  • PCSK9 monoclonal antibody 1 purchased from Abcam (ab84041); PCSK9 monoclonal antibody 2: evolocumab (evolocumab) jointly developed by Amgen and Astellas, trade name Repatha (Repatha); PCSK9 monoclonal antibody 3: alirocumab (alizumab, alizumab) jointly developed by Sanofi and Regeneron, trade name Praluent (Polida).
  • PCSK9 polypeptide was purchased from Abcam Company (ab32727).
  • Preparation method of the treatment solution 60% ethanol is mixed with an appropriate amount of PCSK9 inhibitor to prepare solutions of different concentrations.
  • the rosin/paraffin mixture (1:1) was heated and melted, and then applied to the back, and then peeled off after solidification and hardening. . From the 2nd day after depilation, the corresponding drugs were applied to the depilation area, 2 times a day, 0.5mL per animal each time.
  • mice in each group were reserved for 40 days of naked eye observation, and the growth of back hair was recorded.
  • mice in each group were sacrificed by cervical dislocation.
  • the same parts of the back parallel to the spine were taken, fixed in 10% formaldehyde, dehydrated, embedded in paraffin, sectioned, stained with HE, sealed with neutral gum, and observed for hair follicle histology under light microscope.
  • Changes and morphological staging of hair follicles According to the international hair cycle scoring method, the hair follicles in each stage are scored as follows: 100 points for growth stage VI, 200 points for early regression, 300 points for middle regression, and 400 points for late regression.
  • the experimental data were statistically processed by SPSS 16.0 system software. Statistical variables of experimental data are expressed as (x ⁇ s), using ⁇ 2 test and t test, ⁇ value is two-sided, P>0.05 means no significant difference, P ⁇ 0.05 means significant difference, P ⁇ 0.01 means significant difference sex.
  • mice 2.1 Visually observe the changes in the hair plucking site of mice:
  • PCSK9 inhibitor can obviously prolong the growth period of hair follicles.
  • the skin color change time (days) of the dorsal hair-plucking area of the mice in each group is shown in Table 13 below.
  • mice 2.2 Hair growth in the back plucked area of mice:
  • the base of the hair follicle in the negative control group and the model control group became thinner and lighter in color
  • the lower part of the hair follicle degenerated
  • the dermal papilla became round and dense
  • the epithelial cord between the dermal papilla and the blastocyst was formed
  • the inner hair root sheath partially disappeared.
  • the hairs have rod-shaped ends.
  • the hair follicles in the PCSK9 inhibitor group and the positive control group were larger and longer, and most of the hair follicles were still in the VI stage of growth and in the early stage of regression.
  • the average hair cycle scores of the PCSK9 inhibitor group (mAb group, polypeptide group, compound group, siPcsk9 group) and positive control group were 158, 167, 165, 168, 172, and 185, respectively, indicating that Growth stage VI and early degenerative hair follicles.
  • the percentages of hair follicles in the growth stage VI of the inhibitor group (mAb group, polypeptide group, compound group, siPcsk9 group), positive control group, negative control group and model control group were 56%, 52%, 53%, 47%, and 47%, respectively. 42%, 13% and 11%, there were significant differences between the inhibitor group and the model control group in the percentage of growth stage VI hair follicles (P ⁇ 0.01); the percentage of growth stage VI hair follicles between the inhibitor group and the positive control group There was no significant difference (P>0.05). It is suggested that PCSK9 inhibitors can significantly prolong the growth period of hair follicles and promote hair growth.
  • siRNA sequences and modifications are shown in Table 15 below.
  • PCSK9 interfering RNAi inhibitor-2 the RNA sequence is the same as that of Alnylam's Inclisran
  • PCSK9 interfering RNAi inhibitor-3 the RNA sequence is the same as that of Affiris' ALN-PCS.
  • PCSK9 small molecule compound inhibitor 1 R-IMPP, a product of Selleck Company, chemical formula: C 24 H 27 N 3 O 2 , molecular weight: 389.49
  • structural formula: PCSK9 small molecule compound inhibitor 2 Selleck company product PF-06446846, chemical formula: C 22 H 20 ClN 7 O, molecular weight: 434.04, structural formula: PCSK9 small molecule compound inhibitor 3: SBC-115076, a product of Selleck, chemical formula: C 31 H 33 N 3 O 5 , molecular weight: 527.61
  • PCSK9 monoclonal antibody 1 purchased from Abcam (ab84041); PCSK9 monoclonal antibody 2: evolocumab (evolocumab) jointly developed by Amgen and Astellas, trade name Repatha (Repatha); PCSK9 monoclonal antibody 3: alirocumab (alizumab, alizumab) jointly developed by Sanofi and Regeneron, trade name Praluent (Polida).
  • PCSK9 polypeptide was purchased from Abcam Company (ab32727).
  • the preparation method of the treatment solution 75% ethanol is mixed with an appropriate amount of the above inhibitor to prepare solutions of different concentrations.
  • SPF grade Wistar rats were selected and randomly divided into compound group 1 (5% R-IMPP solution applied to the skin), compound group 2 (5% PF-06446846 solution applied to the skin), compound group 3 (5% R-IMPP solution applied to the skin) SBC-115076 solution), compound group 4 (skin application of 5% SBC-110736 solution), monoclonal antibody group 1 (subcutaneous injection of PCSK9 monoclonal antibody ab84041, 5mg/kg.d), monoclonal antibody group 2 (subcutaneous injection of evolocumab, 5mg /kg.d), monoclonal antibody group 3 (subcutaneous injection of alirocumab, 5mg/kg.d), PCSK9 interfering RNAi inhibitor group-1 (skin 5% PCSK9 small interfering RNA-1 solution), PCSK9 interfering RNAi inhibitor group -2 (5% PCSK9 small interfering RNA-2 solution applied to the skin), PCSK9 interfering RNAI inhibitor group-3 (5% PCSK9 small interfering
  • each rat selected an area of 4cm ⁇ 5cm on the back to take off the hair as the observation area. Except for the negative control group, the rats were subcutaneously injected with testosterone propionate injection [5ml/(kg ⁇ d)] at the back of the neck, once a day for 60 consecutive days to establish the SA model. After continuous subcutaneous injection of testosterone propionate for 4 weeks, the rats gradually appeared hair loss. The remaining hair became fine and brittle, which proved that the androgenetic alopecia model was successfully established.
  • the rats in the corresponding drug group were smeared or subcutaneously injected into the skin on the back of the rats in the drug group, smeared at 1 mL/(per time), twice a day, with an interval of 8 hours, and administered by subcutaneous injection, once a day.
  • the negative control group and the model control group were smeared with vehicle (75% ethanol solution), 1 mL/(only ⁇ time), twice a day, for 60 consecutive days.
  • the grading standards are as follows: normal skin dermal tissue cells, subcutaneous hair follicles, and sebaceous glands are marked as "-": no hyperplasia in the skin and dermis, limited lesions of the hair follicles and sebaceous glands, and no subcutaneous inflammation, marked as " ⁇ ”: no skin and dermis tissue There is obvious hyperplasia and obvious cystic degeneration of hair follicles. No obvious hyperplasia of sebaceous glands, and no inflammation under the skin is marked as "+”: segmental hyperplasia of skin and dermis is not obvious, a small number of hair follicles have cystic degeneration, and sebaceous glands have Mild hyperplasia.
  • the hair length of the rats in the PCSK9 inhibitor group was longer than that in the model control group on the 15th, 30th, 45th, and 60th days of administration, and the differences were statistically significant (P ⁇ 0.01).
  • the effect of each group on rat hair growth length is shown in Table 16 below.
  • the lesions of skin dermal tissue cells, subcutaneous hair follicles and sebaceous glands of the rats in the PCSK9 inhibitor group and the minoxidil tincture group were alleviated to different degrees.
  • the number of damaged hair follicles in the PCSK9 inhibitor group was significantly reduced, and the difference was statistically significant (P ⁇ 0.01).
  • the lesions of skin dermal tissue cells, subcutaneous hair follicles and sebaceous glands of the rats in the PCSK9 inhibitor group and the minoxidil tincture group were significantly reduced (P ⁇ 0.01).
  • the effects of each group on the rat skin hair follicles and sebaceous glands are shown in Table 17 below.
  • SPF grade rats SPF grade rats, PCSK9-/-SPF grade rats (using CRISPR gene editing technology to knock out Pcsk9 gene exon 2-3 to obtain Pcsk9 gene knockout rats), body weight (220 ⁇ 26) g, male. They were divided into model control group and PCSK9-/- group, with 6 animals in each group. Rats in each group were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (120 mg/kg), and then fixed on the operating table, and then a 4 ⁇ 5 cm piece of intact skin was selected on the left side of its back, depilated with 8% sodium sulfide, and used for tissue removal.
  • TUNEL is a method for detecting fragmentation of apoptotic DNA, widely used to identify and quantify apoptotic cells, or to detect excessive DNA fragmentation in a single cell, the assay relies on the use of terminal deoxynucleotidyl transferase (TdT) , TdT is an enzyme that catalyzes the attachment of deoxynucleotides labeled with fluorescent dyes or another label to the 3'-hydroxyl end of DNA double-strand breaks, and it can also label DNA with damaged cells.
  • concentration of the cell suspension used in the experiment was adjusted to 1 ⁇ 105/ml with MEM, and inoculated into 100ml culture flasks, 10ml per flask.
  • the cells were cultured under the conditions of 37° C., 5% CO 2 , and 95% humidity, and the cultured cells were pipetted into a cell solution after 24 h, 48 h and 72 h, and the cells were collected. Centrifuge at 2500 rpm for 5 min, remove the supernatant, and add 1 ml of 75% ethanol to each bottle for fixation. Staining was performed as required by the TUNEL Apoptosis Detection Kit, and the expression in the mean distribution of apoptotic fibroblast units in all fields was manually calculated with a manual counter for statistical comparison.
  • Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value can be measured by enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells.
  • the amount of MTT crystals formed in a certain range of cell number is proportional to the cell number.
  • Cells were seeded in a 48-well culture plate at a density of 1.0 ⁇ 10 5 /ml, and cultured at 100 ⁇ l per well in a 37° C., 5% CO 2 incubator until cells adhered. After culturing for 24h, 48h, and 72h in a 37°C, 5% CO 2 incubator, add 20 ⁇ l of MTT (5mg/ml) to each well for 4 hours, aspirate the medium, and add 100 ⁇ l of DMSO lysis buffer at 37° C., 5% CO 2 Incubate in the incubator for 20min, after the purple crystals are completely dissolved, measure the absorbance (optical density value) at 570nm on a microplate reader. The proliferation of fibroblasts in each group was compared.
  • SPSS 16.0 statistical software was used for data analysis. Measurement data were expressed as mean ⁇ standard deviation (x ⁇ s), one-way ANOVA was used for comparison, and t-test was used to compare pairwise data. P ⁇ 0.05 was considered statistically significant, and P ⁇ 0.01 was considered significant.
  • the wounds were routinely disinfected every day, and the wounds of rats were observed on the 1st, 3rd, 5th, 7th, 12th, and 20th. From the third day of the observation period, the wound recovery speed of the PCSK9-/- group was significantly faster than that of the model group, and the wound area became smaller. On the 12th day, the wounds in the PCSK9-/- group had basically recovered, leaving only a small amount of scabs, while the model group still had wounds of about 0.5 cm 2 in size. On the 20th day, the wounds in both groups had basically recovered, and the model control group had scars, while the PCSK9-/- group had less pigmentation and no obvious scars.
  • TUNEL is a method to detect apoptotic DNA fragmentation and is widely used to identify and quantify apoptotic cells.
  • the results showed that the apoptosis index of fibroblasts in PCSK9-/- group and model control group were 16.816 ⁇ 1.012 and 2.151 ⁇ 0.563, respectively, and the apoptosis of fibroblasts in PCSK9-/- group was significantly higher than that in model control group (P ⁇ 0.01).
  • Dimethyl sulfoxide can dissolve formazan in cells, and its light absorption value is measured at a wavelength of 490 nm with an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, MTT crystals form The amount is proportional to the number of cells.
  • the results showed that the average absorbance values of PCSK9-/- group and model control group were 0.31 and 0.52 after culturing for 24h, and the average absorbance values of PCSK9-/- group and model control group were 0.26 and 0.57 after culturing for 48h, respectively.
  • the mean absorbance values of PCSK9-/- group and model control group were 0.21 and 0.66, respectively.
  • Knockout of PSCK9 gene can significantly promote skin wound healing, while reducing fibroblast proliferation activity, promoting fibroblast apoptosis, and reducing scar (scar) formation, indicating that knockout of PSCK9 gene has preventive and therapeutic effects on scar (scar).
  • Equal amounts of siPcsk9-1 and 2 were mixed and then diluted to 20 ⁇ M with normal saline, and the diluted siRNA was mixed evenly with the lotion.
  • PCSK9 interfering RNAi inhibitor-2 The RNA sequence is the same as that of Alnylam's Inclisran;
  • PCSK9 interfering RNAi inhibitor-3 The RNA sequence is the same as that of Affiris' ALN-PCS.
  • PCSK9 small molecule compound inhibitor 1 R-IMPP, a product of Selleck Company, chemical formula: C 24 H 27 N 3 O 2 , molecular weight: 389.49
  • structural formula: PCSK9 small molecule compound inhibitor 2 Selleck company product PF-06446846, chemical formula: C 22 H 20 ClN 7 O, molecular weight: 434.04, structural formula: PCSK9 small molecule compound inhibitor 3: SBC-115076, a product of Selleck, chemical formula: C 31 H 33 N 3 O 5 , molecular weight: 527.61
  • PCSK9 monoclonal antibody 1 purchased from Abcam company (ab84041); PCSK9 monoclonal antibody 2: evolocumab (evolocumab); PCSK9 monoclonal antibody 3: alirocumab (alizumab).
  • PCSK9 polypeptide was purchased from Abcam Company (ab32727).
  • Positive therapeutic drug mometasone furoate cream (trade name: Ailosone, produced by Schering-Plough China Co., Ltd.)
  • the composition of excipient base includes methyl silicone oil (15%), stearic acid (6%), white petrolatum (5%), liquid paraffin (5%), stearyl alcohol ( 5%), glycerin (20%), alkyl aryl polyglycol ether (1%), fatty alcohol polyoxyethylene ether (1%), Tween-807 (1%), ethylparaben (0.1%) ), distilled water (about 31-55%), and mixed with an appropriate amount of PCSK9 inhibitor to form a mixed emulsion.
  • the cream base used in this example refers to the base component of the cream from which the active ingredient is removed.
  • Rats SPF grade male rats, weighing (210 ⁇ 28) g, were obtained from the Animal Center of Southern Medical University. Rats were numbered by body weight and divided into compound group 1 (0.5% R-IMPP cream applied to the skin), compound group 2 (0.5% PF-06446846 cream applied to the skin), and compound group 3 (0.5% PF-06446846 cream applied to the skin) by random arrangement.
  • %SBC-115076 cream compound group 4 (skin application of 0.5% SBC-110736 cream), monoclonal antibody group 1 (subcutaneous injection of PCSK9 monoclonal antibody ab84041, 3 mg/kg.d), monoclonal antibody group 2 (subcutaneous injection evolocumab, 3mg/kg.d), monoclonal antibody group 3 (subcutaneous injection of alirocumab, 3mg/kg.d), PCSK9 interfering RNA group-1 (skin application of 0.5% PCSK9 small interfering RNA-1 cream), PCSK9 interfering RNA group -2 (0.5% PCSK9 small interfering RNA-2 cream applied to the skin), PCSK9 interfering RNA group-3 (0.5% PCSK9 small interfering RNA-3 cream applied to the skin), peptide group (ab32727 of Abcam company subcutaneously injected, 3mg/kg d), positive treatment group (skin smeared with ilosone), blank control group (skin smeared
  • Rats in each group were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (120 mg/kg) and then fixed on the operating table, and then a 4 ⁇ 5 cm piece of intact skin was selected from the middle and left side of the back, and dehaired with 8% sodium sulfide. Tissue scissors were cut into a 2.4cm diameter round wound deep to the myofascia at the depilation site, and part of the muscle surface fascia was destroyed. Animals are kept in separate cages to prevent rats from biting and licking. The wounds were routinely disinfected with 2% iodine tincture daily, and the wound healing of the rats was observed.
  • the wounds were routinely disinfected every day, and the wounds of rats were observed on the 1st, 3rd, 5th, 7th, 12th, and 20th. From the 5th day, 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 was significantly faster than that of the model group, and the wound area gradually became smaller. The wound recovery speed of the PCSK9 polypeptide inhibitor treatment group was better than that of the model group and the blank control group from the 7th day.
  • the wounds in the small molecule compound treatment group, the PCSK9 small interfering RNA treatment group, and the PCSK9 monoclonal antibody had basically recovered, while the model group and the blank control group still had wounds of about 0.4 cm 2 and 0.36 cm 2 in size, respectively. There were still wounds of about 0.2cm 2 in the treatment group.
  • the wounds in each group had recovered, and the model control group and the blank control group left obvious scars, while the other groups only left varying amounts of pigmentation.
  • PCSK9 inhibitors can significantly promote skin wound healing and reduce scarring (scarring).
  • Reagents bleomycin (4mg/vial, Tianjin Taihe Pharmaceutical Co., Ltd.), mouse anti-mouse MMP monoclonal antibody (NEO Mark-ers company), mouse anti-mouse TIMP-1 polyclonal antibody (Wuhan Boster Company), enzyme-linked immunosorbent assay (ELISA) kit (R&D Company, USA), Quantscript RT Kit reverse transcription kit (TaKaRa Company, Dalian).
  • Rats were numbered by body weight and randomly divided into blank control group, model group, and PCSK9-/- group, with 6 rats in each group. Rats in each group were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (120 mg/kg), fixed on the operating table, and injected into the neck trachea. The blank control group was injected with normal saline (1.25ml/kg), and the model group and PCSK9-/- group were injected with 5U/mL bleomycin solution (5mg/kg), once a day for 14 consecutive days.
  • Peripheral venous blood was collected from the tail vein of animals in each group at 14 days after modeling, and the levels of superoxide dismutase (SOD) and catalase (CAT) in peripheral blood were detected.
  • SOD superoxide dismutase
  • CAT catalase
  • the rats in each group were sacrificed after blood collection, and the right lung tissue of the animals was collected and stored at -4°C for the detection of VEGF.
  • the left lung tissue was routinely embedded in paraffin and sectioned. Immunohistochemical staining was used to detect the expression of MMP isoforms and TIMP-1 in rat lung tissue.
  • the right lung tissue was taken out for grinding, tissue homogenization, centrifugation at 3000 r/min, and the supernatant was collected.
  • TIMP-1 and MMP isoforms were slightly expressed in the lung tissue of the blank control rats.
  • the expressions of MMP-2 and MMP-9 in the model group increased after modeling, while TIMP-1 decreased, and the difference was statistically significant compared with the blank control group (P ⁇ 0.05), indicating that the modeling was successful.
  • the expressions of MMP-2 and MMP-9 in the PCSK9-/- group were decreased, and the expression of TIMP-1 was up-regulated, and the difference was statistically significant compared with the model group (P ⁇ 0.05).
  • the comparison of the expression of TIMP-1 and MMP in the lung tissue of rats in each group is shown in Table 19 below.
  • VEGF protein and VEGF-mRNA in lung tissue of rats in each group are shown in Table 20.
  • the expression of VEGF protein and VEGF-mRNA in the model group was significantly decreased, and the difference was statistically significant compared with the blank control group (P ⁇ 0.05), indicating that the pulmonary fibrosis model was successfully established.
  • the expression of VEGF protein and VEGF-mRNA in PCSK9-/- group was significantly different (P ⁇ 0.05).
  • the comparison of VEGF protein and VEGF-mRNA expression levels in lung tissues of each group is shown in Table 20 below.
  • VEGF protein (pg/ml) VEGF-mRNA model control group 37.23 ⁇ 4.16 0.52 ⁇ 0.19 Blank control group 54.26 ⁇ 3.95* 0.83 ⁇ 0.13* PCSK9-/-group 56.12 ⁇ 3.67* 0.85 ⁇ 0.15*
  • the levels of SOD and CAT enzymes in peripheral blood of rats in each group are shown in Table 21.
  • the peripheral blood SOD and CAT enzyme activities of the rats in the model group were decreased, and the difference was statistically significant compared with the blank control group (P ⁇ 0.05). The differences were statistically significant (P ⁇ 0.05).
  • PSCK9 gene knockout can significantly reduce the levels of MMP-2 and MMP-9 in the lung tissue of the pulmonary fibrosis mouse model, increase the levels of TIMP-1 and VEGF, and increase the levels of SOD and CAT enzymes in peripheral blood. Has inhibitory effect.
  • Reagents bleomycin (4mg/vial, Tianjin Taihe Pharmaceutical Co., Ltd.), mouse anti-mouse MMP monoclonal antibody (NEO Mark-ers company), mouse anti-mouse TIMP-1 polyclonal antibody (Wuhan Boster Company), enzyme-linked immunosorbent assay (ELISA) kit (R&D Company, USA), Quantscript RT Kit reverse transcription kit (TaKaRa Company, Dalian).
  • PCSK9 small molecule compound inhibitor (Selleck company product R-IMPP), chemical formula: C 24 H 27 N 3 O 2 , molecular weight: 389.49
  • PCSK9 monoclonal antibody was purchased from Abcam (ab84041)
  • PCSK9 polypeptide was purchased from Abcam (ab32727)
  • Rats were numbered according to their body weight and divided into groups according to their body weight by random arrangement table method, and divided into small molecule compound treatment group (tail vein injection of PCSK9 small molecule compound inhibitor, 3 mg/kg.d), siPcsk9 treatment group (Tail vein injection of PCSK9 small interfering RNA, 3mg/kg.d), monoclonal antibody treatment group (tail vein injection of PCSK9 monoclonal antibody, 3mg/kg.d), peptide treatment group (tail vein injection of PCSK9 polypeptide inhibitor, 3mg/kg/d) kg.d), model group (injection of physiological saline into tail vein, 3 mg/kg.d), blank control group (injection of physiological saline into tail vein, 3 mg/kg.d), 12 animals in each group, half male and half male.
  • Rats in each group were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (120 mg/kg), fixed on the operating table, and injected into the neck trachea.
  • the control group was injected with normal saline (1.25ml/kg), the model group and each treatment group were injected with 5U/mL bleomycin solution (5mg/kg), starting one week after modeling, each treatment group was injected with corresponding PCSK9 via tail vein respectively.
  • the blank control group and the model group were injected with the same volume of normal saline through the tail vein, once a day for 14 consecutive days.
  • Peripheral venous blood was collected from the tail vein of animals in each group after modeling and 14 days after treatment, and the levels of superoxide dismutase (SOD) and catalase (CAT) in peripheral blood were detected.
  • Rats in each group were sacrificed twice after blood collection (after modeling and 14 days after treatment, 6 rats in each group), and the right lung tissue of the animals was collected and stored in a -4°C refrigerator for the detection of VEGF.
  • the left lung tissue was routinely embedded in paraffin, sectioned, and immunohistochemical staining was used to observe the expression of MMP isoforms and TIMP-1 in rat lung tissue.
  • the right lung tissue was taken out, ground, homogenized, centrifuged at 3000 r/min at high speed, and the supernatant was collected to detect VEGF protein in lung tissue by ELISA, and the expression of VEGF-mRNA by reverse transcription polymerase chain method.
  • TIMP-1 and MMP subtypes were expressed in small amounts in the lung tissue of the rats in the blank control group, and there was little change after modeling and 14 days of treatment.
  • the expressions of MMP-2 and MMP-9 in the model group increased after modeling and 14 days after treatment, while TIMP-1 decreased, and the difference was statistically significant compared with the blank control group (P ⁇ 0.05).
  • the expressions of MMP-2 and MMP-9 in each PCSK9 inhibitor group were decreased, and the expression of TIMP-1 was up-regulated, and the difference was statistically significant compared with the model group (P ⁇ 0.05).
  • the comparison of the expression of TIMP-1 and MMP in the lung tissue of rats in each group is shown in Table 23 below.
  • VEGF protein and VEGF-mRNA in lung tissue of rats in each group are shown in Table 24.
  • the expression of VEGF protein and VEGF-mRNA in the lung tissue of the rats in the blank control group did not change significantly (P>0.05); Academic significance (P ⁇ 0.05).
  • the expressions of VEGF protein and VEGF-mRNA in each PCSK9 inhibitor group were enhanced compared with those after modeling, and the differences were statistically significant compared with the model group (P ⁇ 0.05).
  • the comparison of VEGF protein and VEGF-mRNA expression levels in lung tissue of each group is shown in Table 24 below.
  • the levels of SOD and CAT enzymes in peripheral blood of rats in each group are shown in Table 25.
  • the activities of SOD and CAT enzymes in the peripheral blood of the rats in the blank control group had no significant changes (P>0.05).
  • the peripheral blood SOD and CAT enzyme activities of the rats in the model group decreased, and the difference was statistically significant compared with the blank control group (P ⁇ 0.05).
  • the peripheral blood SOD and CAT enzyme activities of the PCSK9 inhibitor groups were enhanced, Compared with the model group, the difference was statistically significant (P ⁇ 0.05).
  • the comparison of SOD and CAT enzyme levels in peripheral blood of each group is shown in Table 25 below.
  • Each PCSK9 inhibitor can significantly reduce the levels of MMP-2 and MMP-9 in the lung tissue of the pulmonary fibrosis mouse model, and increase the levels of TIMP-1 and VEGF; at the same time, all of them can increase the levels of SOD and CAT enzymes in peripheral blood, showing that they have a significant effect on the lung tissue. Fibrosis has therapeutic effects.

Abstract

本发明提供了前蛋白转化酶枯草杆菌转化酶（PCSK9）抑制剂在治疗多种疾病的产品中的应用，所述PCSK9抑制剂为PCSK9小分子化合物或PCSK9干扰RNA或PCSK9单克隆抗体或PCSK9模拟肽或PCSK9模拟抗体蛋白或PCSK9反义寡核苷酸或PCSK9疫苗。

Description

PCSK9抑制剂在制备治疗多种疾病产品中的应用 技术领域
本发明属于医药技术领域，具体涉及PCSK9在治疗多种疾病中的作用，以及PCSK9抑制剂在制备治疗多种疾病的产品中的应用。
背景技术
白癜风是一种常见的后天性限局性或泛发性皮肤色素脱失病，全世界白癜风患病率为1％～2％。由黑素细胞受损而引起皮肤或者黏膜的色素脱失，以出现褪色的白斑为主要特征，全身各部位可发生，常见于指背、腕、前臂、颜面、颈项及生殖器周围等。白斑多数对称分布，初期多为指甲至钱币大，近似圆形、椭圆形或不规则形。也有起病时，即为点状减色斑，境界多明显，有的边缘绕以色素带。病因尚不清楚。目前多考虑与以下因素有关，包括自身免疫因素、遗传因素、神经精神因素等。白癜风可以合并自身免疫病，血清中还可以检出多种器官的特异性抗体，如抗甲状腺抗体、抗胃壁细胞抗体、抗肾上腺抗体、抗甲状旁腺抗体、抗平滑肌抗体、抗黑素细胞抗体等。白癜风患者体内可以产生抗体和T淋巴细胞，免疫反应可能导致黑素细胞被破坏。而细胞本身合成的毒性黑素前身物及某些导致皮肤脱色的化学物质对黑素细胞也可能有选择性的破坏作用。本病好出现在暴露及色素加深的部位，表皮黑色素细胞功能亢进，使其耗损而衰退，并可能是因为黑色素细胞合成黑色素的中间产物过量或积聚引起。约2/3的患者起病与皮损发展、精神创伤、过度劳累以及焦虑有关，有些白瘢风损害对称或沿神经节段分布。白癜风是难治性疾病，系统治疗主要适用于泛发型进展期白癜风患者，口服或肌内注射激素可以使进展期白癜风尽快趋于稳定。外用糖皮质激素对于局限型白癜风的治疗有效，但长期使用糖皮质激素引起的不良反应多。对于不适于使用激素的部位，或为避免长期应用激素产生不良反应，外用钙调神经磷酸抑制药(他克莫司、吡美莫司)具有一定的效果。维生素D 3衍生物可与NB-UVB、PUVA等联合治疗，也可以与外用激素和钙调神经磷酸酶抑制药联合治疗。窄波紫外线(NB-UVB)治疗局限型或泛发性白癜风有一定效果。目前的治疗方法还不能满足临床需求。
痤疮(俗称青春豆)是好发于毛囊皮脂腺的慢性炎症性疾病，发病率约9.4％，已成为世界第8大流行性疾病。于青春期多见，痤疮的产生与青春期皮肤的生理病理变化密切相关。临床表现主要有粉刺、丘疹、脓疱、结节、囊肿、疤痕等，愈合时间长，给患者的容貌和心理带来严重的影响。痤疮与多个发病机制相关，毛囊口角化异常是本病发病的重要基础，炎症和感染是痤疮的发病因素。痤疮患者的皮脂腺较大，皮脂腺分泌增加，皮脂中亚油酸水平相对减少，影响脂肪的合成，导致滤泡上皮缺乏脂肪酸，从而诱导滤泡过度角化，使上皮细胞不能正常脱落，毛囊皮脂腺口过度变小，皮脂不能顺畅排出，形成粉刺。毛囊皮脂腺口被堵塞而形成毛囊皮脂腺内缺氧的环境，造成厌氧性的痤疮丙酸杆菌大量繁殖，分解皮脂，产生化学趋化因子，白细胞聚集形成丘疹。毛囊皮脂腺内的大量中性粒细胞聚集，吞咽痤疮丙酸杆菌后发生炎症反应，使得大量脓细胞堆积而形成脓疱、囊肿，愈后易形成凹陷性疤痕。当面部、额部、颊部、下颌部、胸部、背部及肩部出现大小不等的结节、囊肿，常继发化脓感染，破溃后常流出带血的胶冻状脓液，以后形成窦道。雄激素水平升高是加速痤疮产生的关键环节，使皮肤角化异常堵塞毛囊皮脂腺导管，导致细菌滞留、繁殖，产生炎症。
与痤疮具有类似角化异常机制的疾病包括鱼鳞病、毛周角化病(又名毛发苔藓)、毛囊角化病和汗孔角化症等，毛周角化病见毛囊口扩大，内有角栓，鱼鳞病表现为汗腺和皮脂腺减少，毛囊内出现角质性栓塞。以上疾病容易反复出现，治疗困难。治疗角化异常，消除角栓和粉刺的药物主要是维甲酸类药物。维甲酸类药物可抑制角化，减少皮脂分泌，促进角质形成细胞的正常角化，并具有调节免疫和抗炎作用，从而减少粉刺、丘疹和脓疱的形成，在临床上被广泛应用来治疗痤疮、鱼鳞病、毛周角化病、毛囊角化病和汗孔角化症等角化异常性疾病。但维甲酸类药物外用对皮肤有刺激，容易导致红肿、刺痛，加重原有皮损，长期外用维甲酸类药物可导致皮肤变薄，光敏和皮肤屏障受损等，而口服维甲酸类药物有肝损和血脂升高等不良反应。因此，临床上需要更多治疗此类疾病的药物。
另一方面，面部痤疮粉刺在人群中非常普遍，使皮肤看起来暗黄粗糙、毛孔粗大，影响美观，市场上 真正能够解决此问题的产品非常稀缺，因此，需要寻找安全、有效的控制粉刺的产品来满足广大爱美群体的需求。
斑秃(alopecia areata，AA)是一种非瘢痕性脱发，局部皮肤大体正常。通常情况下为突发的脱发斑，严重时可累及整个头皮，此时称为全秃(alopecia totalis，AT)，当累及包括腋毛、阴毛在内的全身所有毛发时，称为普秃(alopecia universalis，AU)，容易给患者的容貌和心理带来严重的影响。斑秃的病程较长，但预后较好，越早介入治疗，治愈几率越高。目前病因尚未完全明了，自身免疫功能异常或不稳定、神经精神因素被认为是重要相关因素。不少病例发病前有神经精神创伤，如长期焦急、忧虑、悲伤、精神紧张和情绪不安等现象。有时病人在病程中，这些精神因素可使病情迅速加重。斑秃的治愈机率较高，但不同病因引起的斑秃，治愈概率有很大区别。部分斑秃患者，即便没有采取任何治疗措施，也可以自然痊愈，还有部分斑秃患者持续数年的治疗，也仅能维持病情不再发展。米诺地尔是一种治疗斑秃常见的外用药物，可促进皮肤血管扩张、改善局部血液循环、促进毛发生长。严重斑秃常用的糖皮质激素，主要包括泼尼松龙、复方倍他米松等等，可以口服、外用或皮内注射。对于不适用糖皮质激素类药物的患者，可采用免疫抑制剂治疗，常见的药物有环孢素、甲氨蝶呤。糖皮质激素和免疫抑制剂副作用多。
雄激素源性秃发(AGA)是一种雄激素依赖性的遗传性毛发脱落，是常见病、多发病。多为20～30岁左右的男性发病。脱发主要在头顶部，多先从前额两侧发际开始，也有自顶部开始者。脱发区逐渐向上扩延，头发也渐变得稀少纤细，终而头顶部头发大部或全部脱落，但枕后及双侧颞上方头发依存，呈马蹄形外观，此带形区内头发保持正常。脱发处皮肤光亮，毛孔缩小或残留少许细软毳毛。脱发的速度、范围和严重程度，受遗传和个体影响。一般30岁左右发展最快，严重全秃者少见。女性多为发生于头顶的弥漫性脱发，头顶头发变稀疏。我国流行病学调查显示男性雄激素性脱发患病率为21.3％，女性为6.0％。雄激素性脱发的病因及发病机制尚不明确，一般认为雄激素及其受体在本病的发生中起关键作用，Ⅱ型5a-还原酶是其发病的重要因素。正常生理状态下，雄激素在体内对毛发的生长发育起一定的刺激促进作用，但在某些特定部位能诱导毛发脱失；睾酮是体内主要的雄激素，它经5a-还原酶转化为二氢睾酮，后者可引起终毛向毳毛转变，最终导致脱发。目前尚无理想治疗方法，全身及局部治疗可参照其他脱发性疾病处理。雄激素源性秃发是脱发性疾病难治的类型，该病的动物模型通常作为脱发性疾病的代表模型。由于雄激素在发病中起很大作用，因而近年来的新治疗方法企图通过抗雄激素的效应来终止毛囊的变小。米诺地尔是一种非特异性治疗脱发的药物，是FDA批准用于治疗脱发的一线外用药物，但使用过程中可能引起面部和四肢多毛症，停用后治疗效果逐渐消失。非那雄胺是一种Ⅱ型5a-还原酶选择性抑制剂，FDA批准口服非那雄胺用于治疗雄激素性脱发，可持续改善头发的生长情况，但非那雄胺存在引起性功能异常，精子一过性减少和男性乳房发育异常等不良反应，在动物实验中发现有致畸作用，故不宜用于小儿和育龄妇女。西咪替丁需连服5个月或更久，副作用为男性乳房发育、阳痿、性欲降低等。口服避孕药：主要有的索高诺酮、左炔诺孕酮(左旋甲基炔诺孕酮)、炔诺孕酮、炔诺酮、诺孕酯(肟炔诺酯)、双酯炔诺醇和醋炔诺酮等。常用来治疗女性AGA，治疗6～12个月后头发会有所改善。
抗肿瘤药物引起的脱发是最常见的生长期秃发，抗肿瘤药物在消除迅速分裂的癌细胞的同时，也攻击毛囊四周迅速分裂的细胞引起脱发。
脱发治疗较困难，容易反复。米诺地尔是一种非特异性的治疗脱发药物，是FDA批准用于治疗脱发的一线外用药物，但使用过程中可能引起面部和四肢多毛症，停用后治疗效果逐渐消失。非那雄胺是一种Ⅱ型5a-还原酶选择性抑制剂，FDA批准用于治疗SA，可持续改善头发的生长情况，但存在引起性功能异常，精子一过性减少和男性乳房发育异常等不良反应。因此，需要寻找更多安全、有效的治疗脱发的药物和产品。
疤痕是物理、生物、化学等因素的损害作用于人体皮肤软组织，导致皮肤软组织的严重损伤而不能完全自行正常修复，转由纤维组织替代修复留下的即影响外观又影响功能的症状。疤痕给患者带来的是巨大的肉体痛苦和精神痛苦，尤其是烧伤、烫伤、严重外伤后遗留的疤痕。对于瘢痕的处理难度较大，目前只能做到使发红、发硬的疤痕变软、变浅，宽的疤痕变窄，粗的疤痕变细，还不能完全彻底消除疤痕。因此在伤口愈合早期即开始进行干预非常重要，可以有效减少疤痕的形成，改善外观，纠正畸形，恢复功能。目前常用来治疗疤痕方法有：手术治疗、激光治疗、冷冻治疗和药物治疗等。常用的药物主要有：糖皮质激素和维甲酸。糖皮质激素有抗炎、抗病毒、抗休克等功能，并且有明显的抗组织纤维化的效应，但糖皮质激素长期使用毒副作用多。维甲酸是维生素A在体内代谢的中间产物，是维生素A类相关药物，维甲酸能减轻局部炎症，促进上皮细胞生长，减少胶原合成，使成纤维细胞的DNA合成减少，抑制细胞生长。 维甲酸类药物浓度越大，抑制生长作用越明显。但维甲酸疗效有限，系统应用毒副作用也不少，外用对皮肤刺激明显，并随着浓度增加而增加。
肺纤维化(pulmonary fibrosis)是一种病因未明、发病机制复杂的弥漫性肺部疾病，目前公认是肺损伤后过度修复导致细胞外基质过度沉积的结果。肺纤维化的发病机制不明，目前公认为肺泡上皮反复损伤与过度修复是发病的关键。病理特点是长期慢性肺部炎症及肺泡持续性损伤导致细胞外基质金属蛋白酶(MMP)聚集，特别是MMP-2、MMP-9异常增多，金属蛋白酶组织抑制剂-1(TIMP-1)降低，使其平衡关系破坏，导致肺大量细胞外基质聚集，组织细胞重构和胶原过度沉积。同时可能抑制组织中血管内皮生长因子(VEGF)表达，降低肺微小静脉血管的通透性，抑制血管内皮细胞分裂增殖及血管再生，加重肺组织损伤，并最终导致弥漫性肺间质疾病一肺纤维化。目前无有效的抗纤维化药物，常用糖皮质激素抗炎以降低抗纤维化进程，但疗效有限，不良反应多。
对于上述疾病，目前的治疗方法远不能满足临床需求，需要寻找更多疗效好、副作用少、价格便宜的新药来控制疾病进展，减少复发和并发症，降低死亡率。
前蛋白转化酶枯草杆菌转化酶(proprotein convertase subtilisin/kexin type 9，PCSK9)是前蛋白转换酶家族中的一员，前蛋白转换酶在肝脏内作为一种不活跃酶原分泌。PCSK9基因cDNA的大小为3617bp，编码692个氨基酸组成的PCSK9蛋白。PCSK9前体在内质网内经过分子内自动催化分离其N-末端前肽，分离后的N-末端前区与催化区相连，允许成熟的PCSK9蛋白离开内质网并进入分泌途径。PCSK9分泌至细胞外后，在细胞表面的第一表皮生长因子样区域与低密度脂蛋白(LDL)受体结合，PCSK9-LDL受体复合体可进入溶酶体降解，从而导致细胞表面LDL受体下降，即PCSK9水平与LDL受体成负相关关系。而多项研究显示，PCSK9基因的突变功能丧失可使不同人种的LDL-C水平及冠心病发生率明显下降。鉴于抑制PCSK9对降低LDL-C及冠心病发生率的显著作用，已有多项治疗方案正在研发阻断PCSK9的药物，用于降低LDL-C及冠心病的发生率。
PCSK9抑制剂包括两大类：1、阻止PCSK9与LDL-R的结合，如单抗、模拟肽(多肽抑制剂)、模拟抗体蛋白药；2、抑制PCSK9分子的表达或干扰PCSK9分泌，如小分子干扰RNA、反义寡核苷酸、小分子化合物抑制剂等。单克隆抗体由于阻断效率高、靶位准确和稳定性好而成为新药研究的热点。目前，全球上市的PCSK9靶向单抗药物，均为阻止PCSK9与LDL-R结合的药物。包括安进(Amgen)和安斯泰来(Astellas)联合开发的的evolocumab(依洛尤单抗)，商品名Repatha(瑞百安)；赛诺菲(Sanofi)和再生元(Regeneron)联合开发的alirocumab(阿利西尤单抗，阿利珠单抗)，商品名Praluent(波立达)，以及Eli lilly公司的LY3015014，君实生物的重组人源化抗PCSK9单克隆抗体(JS002)，和信立泰药业的重组全人源抗PCSK9单克隆抗体注射液。临床研究发现，以上药物治疗高胆固醇血症耐受性良好，安慰剂组与积极治疗组不良反应的发生率无明显区别。此外，Inclisiran是一款siRNA(小干扰RNA)药物，不同于直接与PCSK9分子结合的单抗，它可以抑制PCSK9基因的表达，使LDL受体不会因PCSK9而被降解，从而改善肝细胞对LDL颗粒的摄取，降低血液中的LDL水平。Alnylam公司的Inclisiran采用专有技术，由脂质纳米颗粒与GalNAc(N-乙酰半乳糖胺)结合而成，而GalNAc可与肝细胞表面表达的唾液酸糖蛋白受体ASGR1和ASGR2结合。该技术允许皮下给药并靶向肝脏。Affiris公司的ALN-PCS和ALN-PCSsc也属于siRNA(小干扰RNA)药物。PCSK9干扰RNAI抑制剂药物还有Alnylam公司的Inclisran，Affiris公司的ALN-PCS和ALN-PCSsc。辉瑞公司设计的一种相关的疫苗药物，以及Affiris公司的AT04A和AT06A疫苗，患者只需每年接受一次疫苗即可达到长期降低LDL的效果，可以减少使用频率。PCSK9模拟肽和PCSK9模拟抗体蛋白药药物研发包括Pieris公司的DS9001和Merck公司的1G08等。PCSK9反义寡核苷酸药物包括Santaris Pharma公司的SPC5001等。
到目前为止，尚未见到应用PCSK9抑制剂治疗白癜风、脱发、疤痕、肺纤维化，以及痤疮、鱼鳞病、毛周角化病、毛囊角化病和汗孔角化症等角化异常性疾病的相关文献、专利和产品。
发明内容
本发明需要解决的问题是：明确PCSK9基因在白癜风、脱发性疾病、角化异常性疾病、疤痕以及肺纤维化发病机制中的作用，以及PCSK9抑制剂在制备治疗以上疾病的产品中的应用。本发明通过相关疾病的动物模型实验研究，发现了PCSK9在白癜风、脱发性疾病、角化异常性疾病、疤痕以及肺纤维化发病机制中的关键作用，以及PCSK9抑制剂在制备治疗以上疾病产品中的应用。
本发明通过建立代表性的色素脱失性疾病白癜风动物模型，发现了PCSK9基因在促进皮肤色素生成 中的重要作用。
PCSK9蛋白序列如序列表中序列1所示。
本发明通过建立兔耳痤疮模型，发现了PCSK9基因在痤疮发病机制中起关键作用，以及PCSK9抑制剂在制备治疗痤疮的产品中的应用价值。鱼鳞病、毛周角化病、毛囊角化病和汗孔角化症等角化异常性疾病的发病机制中表现为与痤疮类似出现角栓，毛囊口扩大。由于目前尚没有成熟的鱼鳞病、毛周角化病、毛囊角化病和汗孔角化症等角化异常性疾病的动物研究模型，因此本研究选取兔耳痤疮粉刺模型作为代表来研究PCSK9在以上角化异常性疾病的发病机制中的作用，以及PCSK9抑制剂在改善皮肤角化异常，抑制角栓形成中的作用。
雄激素性脱发是脱发性疾病中难治的类型，该病的动物模型通常作为脱发性疾病的代表模型。本发明通过建立雄激素性脱发动物模型，发现了PCSK9基因在脱发性疾病的发病机制中起关键作用，以及PCSK9抑制剂在制备治疗脱发性疾病的药物中的应用价值。
本发明通过建立大鼠皮肤疤痕(瘢痕)模型，发现了PCSK9基因在疤痕(瘢痕)形成机制中起关键作用，以及PCSK9抑制剂在制备预防和治疗疤痕(瘢痕)的产品中的应用价值。
通过建立大鼠肺纤维化模型明确PCSK9基因在肺纤维化疾病机制中的作用，以及PCSK9抑制剂在制备治疗肺纤维化疾病的产品中的应用。
本发明的技术方案：
C57BL/6小鼠是目前国内外广泛应用的研究毛发周期的动物模型：人类各个毛囊的毛发周期不同步，而该小鼠可表现出独特的毛发周期同步性，故它常用来作为毛发研究模型。该小鼠躯干皮肤的黑素细胞只存在于毛囊中，而且仅在生长期时合成黑色素；在毛发的生长期，由于毛球部黑素细胞不断产生黑素，并传递给毛囊角质形成细胞，使皮肤外观呈现黑色；退行期时，黑素生成减少，皮肤呈灰黑色；在休止期由于毛球部微缩消失，毛囊停止生成黑素，皮肤变为粉红色。休止期的毛发经拔除后局部可被诱导出高度同步的新毛发周期，组织学上同该鼠自然周期变化一致。故可从皮肤颜色的变化来推断毛发周期的改变。本发明采用了皮下注射丙酸睾酮注射液造成实验性雄激素性脱发模型，发现PCSK9基因敲除能明显促进雄激素性脱发模型小鼠毛发生长，减轻对皮下毛囊和皮脂腺的损伤。本发明采用石蜡脱毛建立小鼠非特异性脱发模型，证实PCSK9抑制剂对毛发生长具有非常明显的促进作用。本发明通过雄激素性脱发模型实验，发现PCSK9抑制剂组小鼠毛发生长速度明显快于模型对照组，证实PCSK9抑制剂在治疗脱发性疾病中的作用和机制。本发明在能够明显阻断PCSK9的制剂中，分别选取了具有代表性的单克隆抗体、多肽抑制剂、小分子化合物抑制剂和PCSK9小干扰RNA，通过皮下注射或者皮肤外用治疗小鼠脱发模型。结果发现，PCSK9抑制剂组疗效均明显优于模型对照组，各组均未出现明显不良反应。实验证实，系统或外用PCSK9抑制剂对毛发生长具有明显的促进作用。
兔耳是一种常用来衡量引起粉刺物质作用强度的动物模型，兔和人一样毛囊皮脂腺大小变化很大。动物随着年龄的增加，形成粉刺的能力也增加，因此选择成年雄性家兔来复制痤疮模型，使其体内的雄性激素对皮肤具有一定的刺激作用。阿达帕林可通过调节毛囊皮脂腺上皮角化异常过程来去除角质栓，从而起到防止及消除粉刺皮损的作用，故本研究选择阿达帕林(商品名：达芙文)作为实验的阳性对照品。本发明又在能够明显阻断PCSK9的抑制剂中，分别选取了具有代表性的PCSK9单克隆抗体、PCSK9多肽抑制剂、PCSK9小分子化合物抑制剂和PCSK9小干扰RNA，通过皮下注射或者皮肤外用治疗兔耳痤疮模型，与阴性对照组和阳性药物(达芙文)比较。结果发现，PCSK9抑制剂组的症状均明显轻于模型对照组。实验证实，系统或外用PCSK9抑制剂均能明显抑制兔耳痤疮模型症状，减少毛囊角栓和黑头粉刺形成，并对皮肤温和无刺激，显示PCSK9抑制剂对痤疮具有明显的治疗作用。本研究人员首次发现敲除PCSK9基因能够明显抑制痤疮皮损的形成，PCSK9抑制剂能够明显改善粉刺，抑制毛囊内角栓的形成。痤疮的发病机制复杂，其中皮肤毛囊口的异常角化是本病发病的重要基础。毛囊皮脂腺口被角质细胞堵塞，角化物和皮脂充塞其中，形成粉刺，导致毛孔堵塞，痤疮杆菌、金黄葡萄球菌生长，侵害皮肤组织。丙酸痤疮杆菌可诱导和改变补体的激活路径，有助于炎症反应。鱼鳞病、毛周角化病(又名毛发苔藓)等角化异常性疾病的发病机制中表现为与痤疮类似出现角栓，见毛囊口扩大。
本发明于新西兰家兔耳内侧面涂2％煤焦溶液，每天1次，连续涂14d，建立痤疮微粉刺模型，肉眼观察耳厚薄、硬度、粗糙程度和毛囊口有无黑色角栓等局部皮肤的变化，进行镜下实验性粉刺组织学分级评分。通过以上研究发现，PCSK9基因敲除能明显抑制煤焦油诱导的兔痤疮模型症状，减少毛孔堵塞，明显抑制黑头粉刺形成，证实PCSK9在痤疮的发病机制中起着关键作用。
本发明在能够明显阻断PCSK9的抑制剂中，分别选取了具有代表性的PCSK9单克隆抗体、PCSK9多肽抑制剂、PCSK9小分子化合物抑制剂和PCSK9小干扰RNA，通过皮下注射或者皮肤外用治疗兔耳痤疮模型，与阴性对照组和阳性药物(达芙文)比较。结果发现，PCSK9抑制剂组的症状均明显轻于模型对照组，各PCSK9抑制剂组均未出现局部和全身不良反应，如红斑、水肿、脱屑等，家兔活动和觅食正常，未见呼吸和中枢神经系统异常表现。实验证实，系统或外用PCSK9抑制剂均能明显抑制兔耳痤疮模型症状，减少毛囊角栓和黑头粉刺形成，并对皮肤温和无刺激，显示PCSK9抑制剂对痤疮具有明显的治疗作用。
本发明通过研究发现，PSCK9基因敲除能明显促进大鼠皮肤创伤的愈合，减少疤痕(瘢痕)形成，也能明显抑制皮肤成纤维细胞的增殖，促进成纤维细胞凋亡，显示抑制PSCK9能通过抑制皮肤成纤维细胞增殖，促进其凋亡，来抑制疤痕(瘢痕)的形成。本发明又在能够明显阻断PCSK9的抑制剂(阻断剂)中，分别选取了具有代表性的PCSK9单克隆抗体、PCSK9多肽抑制剂、PCSK9小分子化合物抑制剂和PCSK9小干扰RNA，通过尾静脉注射或者皮肤外用干预大鼠疤痕(瘢痕)模型，结果发现，PCSK9抑制剂组的疤痕增生情况均明显好于模型组。研究证实，各种PCSK9抑制剂均能促进皮肤创伤愈合，减少疤痕(瘢痕)形成，显示无论系统还是外用PCSK9抑制剂均能对皮肤疤痕(瘢痕)具有明显的预防和治疗作用，且均未见明显毒副作用。
本发明于大鼠颈部气管切开注入博莱霉素建立肺纤维化模型。研究发现PSCK9基因敲除能明显增加肺纤维化小鼠模型肺组织中MMP-2、MMP-9和VEGF水平，降低TIMP-1水平，同时可提高外周血中SOD、CAT酶水平，显示对肺纤维化具有抑制作用。本发明又在能够明显阻断PCSK9的抑制剂中，分别选取了具有代表性的PCSK9单克隆抗体、PCSK9多肽抑制剂、PCSK9小分子化合物抑制剂和PCSK9小干扰RNA抑制剂，通过尾静脉注射治疗肺纤维化大鼠模型。实验证实，PCSK9抑制剂均能明显改善肺纤维化实验室指标，显示PCSK9抑制剂对肺纤维化具有明显的治疗作用。
以上动物实验中，各PCSK9抑制剂组均未出现全身不良反应，动物活动和觅食正常，未见呼吸和中枢神经系统异常表现。
本领域技术人员公知，基于以上PCSK9的作用机制，PCSK9抑制剂对其他类似疾病均具有治疗作用。PCSK9抑制剂可以单独使用，也可以联合其他药物或者治疗方法一起使用，包括传统药物和其他靶向生物制剂。
基于上述研究，本发明涉及了PCSK9抑制剂(阻断剂)在制备治疗白癜风、角化异常性疾病、脱发性疾病、疤痕以及肺纤维化的产品中的应用。其中所述PCSK9属前蛋白转换酶家族(genebank序列号：255738)；所述角化异常性疾病包括痤疮、鱼鳞病、毛周角化症、毛囊角化病和汗孔角化症等；所述脱发性疾病包括雄激素性脱发或斑秃或抗肿瘤治疗引起的脱发等。
本发明所述的PCSK9抑制剂可以为常规的任何的分子生物学或者药物化学手段能够抑制PCSK9基因表达或者分泌作用的产品或方法，例如但不限于通过现有分子生物学技术对PCSK9基因进行敲除或者沉默；在一些实施例中，还可以为或者采用PCSK9抑制剂，优选的，上述PCSK9抑制剂(阻断剂)为PCSK9小分子化合物或PCSK9干扰RNA或PCSK9单克隆抗体或PCSK9模拟肽或PCSK9模拟抗体蛋白或PCSK9反义寡核苷酸或PCSK9疫苗。
在一些实例中，本发明所述的PCSK9小分子化合物包括但不限于Selleck公司产品R-IMPP，化学式：C 24H 27N 3O 2，分子量：389.49，结构式：
Figure PCTCN2021103749-appb-000001
或Selleck公司产品PF-06446846，化学式：C 22H 20ClN 7O，分子量：433.04，结构式：
Figure PCTCN2021103749-appb-000002
或Selleck公司产品SBC-115076，化学式：C 31H 33N 3O 5， 分子量：527.61，结构式：
Figure PCTCN2021103749-appb-000003
或Selleck公司产品SBC-110736，化学式：C 26H 27N 3O 2，分子量：413.51，结构式：
Figure PCTCN2021103749-appb-000004
在一些实例中，本发明所述的PCSK9单克隆抗体抑制剂包括但不限于Abcam公司ab84041，或安进(Amgen)和安斯泰来(Astellas)联合开发的evolocumab(依洛尤单抗)，或赛诺菲(Sanofi)和再生元(Regeneron)联合开发的alirocumab(阿利西尤单抗，阿利珠单抗)，或君实生物的重组人源化抗PCSK9单克隆抗体(JS002)，或信立泰药业的重组全人源抗PCSK9单克隆抗体注射液，或Eli lilly公司的LY3015014，或Merck公司的1G08。
在一些实例中，本发明所述的PCSK9干扰RNAI抑制剂包括但不限于Alnylam公司的Inclisran，Affiris公司的ALN-PCS和ALN-PCSsc。
在一些实例中，本发明所述的PCSK9模拟肽抑制剂和PCSK9模拟抗体蛋白抑制剂包括但不限于Pieris公司的DS9001和Merck公司的1G08等。
在一些实例中，本发明所述的PCSK9反义寡核苷酸抑制剂包括但不限于Santaris Pharma公司的SPC5001。
在一些实例中，本发明所述的PCSK9疫苗抑制剂包括但不限于Affiris公司的AT04A和AT06A。
PCSK9抑制剂(阻断剂)可以单独使用，也可以联合其他药物或者治疗方法一起使用，包括传统药物和其他靶向生物制剂。
本发明还提供一种药物组合物，以本发明所述化合物或其药学上可接受的盐为活性成分或主要活性成分，辅以药学上可接受的载体组成。
本发明所述的化合物或组合物可制备为药学上允许的任何剂型，例如为适于口服、肠胃外、腹膜内、静脉内、动脉内、透皮、舌下、肌内、直肠、透颊、鼻内、吸入、阴道、眼内、局部、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。
在一种优选的实施方式中，本发明所述的剂型为膏剂、片剂、冲剂、口服液剂、胶囊剂、滴丸剂、灌肠剂、膜剂或注射剂。
本发明与现有技术相比其有益效果是：本发明为白癜风、角化异常性疾病、脱发性疾病、疤痕以及肺纤维化的治疗提供了新的、更好的治疗方法，通过本发明的揭示可进一步制备系统或外用的PCSK9抑制剂(阻断剂)产品，进而开发出包含各类PCSK9抑制剂的单体新药或复方制剂，用于治疗以上疾病。已经有的临床实验证明此类包含PCSK9抑制剂的药物治疗以上疾病疗效显著、不良反应小、患者耐受性好，尤其是仅仅外用也能明显改善症状，非常适宜于实际临床需求。可为市场提供一系列价格较低，疗效好，安全的新产品。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通 常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明，均属于本发明的保护范围。
实施例1：
PCSK9基因敲除对小鼠色素生长的影响：
1.实验方法：
1.1实验动物：
SPF级C57BL/6(B6)小鼠，C57BL/6-PCSK9-/-小鼠(利用CRISPR基因编辑技术，敲除Pcsk9基因exon 2-3，建立Pcsk9基因敲除小鼠模型)，体质量(20±4.6)g，来源于南模生物。
1.2动物分组与造模：
将不同基因型小鼠分三组：C57BL/6小鼠空白对照组、C57BL/6小鼠模型组和C57BL/6-PCSK9-/-小鼠模型组，每组6只，雌雄各3只。脱去背部2cm×2cm面积黑色毛发，除空白对照组外，其余各组均在脱毛区涂抹5％氢醌脱色，每次0.5mL，每天2次，空白对照组动物每天涂抹等量生理盐水。连续涂抹30d，模型组出现明显白斑表明模型成功。造模结束后，观察涂抹5％氢醌脱色部位色素情况。
1.3观察指标及测试方法：
1.3.1组织学观察实验：
观察结束后，处死小鼠后，剪取背部涂抹5％氢醌部位的皮肤组织约1cm×1cm，做组织病理学检查。
1.3.2白癜风动物模型表皮黑素细胞(MC)计数：
将剪取的小鼠皮肤组织置10％福尔马林中固定1h，后流水冲洗3～4min，放入含0.1％二羟基苯丙氨酸的磷酸盐缓冲液(pH7.4)中，于37℃静置1h。然后换入新鲜的Dopa试剂中，37℃静置12h。流水冲洗，用Bouin氏液继续固定24h。将标本取出后流水冲洗4h后脱水，二甲苯透明，石蜡包埋，脱蜡至水，用苏木素-伊红对比染色。脱水，常规封固。Dopa-氧化酶染色后，棕黑色染色为MC阳性。分别于光镜下测定每个高倍镜视野内各组MC数量，每个标本观察10个高倍镜视野，计算每100个表皮基底细胞中MC的平均数量。
1.3.3白癜风小鼠模型表皮含黑素颗粒基底细胞计数：
将剪取的小鼠皮肤组织置10％福尔马林中固定，脱水，用二甲苯透明，石蜡包埋，制切片，脱蜡至水，蒸馏水洗涤数次后进行Lillie染色，用氯化铁和铁氰化钾混合液浸染15～20min，再以1％醋酸水溶液分化数分钟，蒸馏水洗涤数次，然后用95％乙醇及无水乙醇迅速脱水，二甲苯透明，中性树胶封固。Lillie染色后，黑色素呈暗绿色为阳性，分别于光镜下测定各组含黑素颗粒的基底细胞数量，每个标本观察10个高倍镜视野，计算每100个表皮基底细胞中含黑素颗粒的基底细胞的平均数量。
1.3.4白癜风小鼠模型皮肤组织酪氨酸酶活性的检查：
将剪取的小鼠皮肤组织置10％福尔马林中固定。然后脱水，二甲苯透明，石蜡包埋，制切片，脱蜡至水后，每张切片加过氧化酶阻断剂(3％H 2O 2)50μL，10min，阻断内源性过氧化物酶活性。切片浸入0.01mol·L-1PBS缓冲液中，微波处理10min。加鼠抗人酪氨酸酶单克隆抗体50μL，37℃、60min。加Elivision试剂50μL，37℃30min。然后将切片置新鲜配制的DAB-H 2O 2显色液中显色，蒸馏水冲洗，苏木素对比染色1min，中性树胶固封。酪氨酸酶阳性产物定位于细胞的细胞质内，显镜下细胞内出现黄色或棕黄色颗粒、或团块状为阳性表达。用二级计分法判定结果，阳性细胞计数<5％为0分；5％～25％为1分；25％～50％为2分；50％～75％为3分；>75％为4分。按染色强度分类:淡黄色1分；黄或深黄2分；褐或棕黄色3分。两者相加小于2分为阴性(—)；2～3分为阳性(+)；4～5分为中等阳性(2+)；6～7分为强阳性(3+)。
1.4统计学方法：
实验数据采用SPSS 16.0系统软件进行统计学处理。实验数据统计变量以(x±s)表示，采用χ2检验和t检验，动物皮肤组织酪氨酸酶强度比较均用Wilcoxon秩和检验。α值取双侧，P>0.05表示差异无显著性，P<0.05表示差异有显著性，P<0.01为差异有极显著性。
2.实验结果：
2.1 PCSK9基因敲除对氢醌脱色所致白癜风小鼠模型表皮MC、含黑素颗粒基底细胞计数的影响：
经氢醌脱色后，与C57BL/6-PCSK9-/-小鼠模型组比较，C57BL/6小鼠模型组皮肤组织中表皮MC含量降低(P<0.01)，含黑素颗粒基底细胞计数降低(P<0.01)。各组小鼠表皮MC、含黑素颗粒基底细胞计数情况 (n＝6，x±s)见下表1。
表1各组小鼠表皮MC、含黑素颗粒基底细胞计数情况(n＝6，x±s)
组别 表皮黑素细胞计数 含黑素颗粒基底细胞计数
空白对照组 35.6±2.2* 38.7±2.1*
C57BL/6-PCSK9-/-小鼠模型组 28.5±2.8* 33.5±3.2*
C57BL/6小鼠模型组 12.7±1.3 17.3±2.6
注：*与C57BL/6小鼠模型组比较，P＜0.01。
2.2 PCSK9基因敲除对氢醌脱色所致白癜风鼠模型皮肤组织酪氨酸酶强度的影响：
经氢醌脱色后，C57BL/6小鼠模型组皮肤组织中酪氨酸酶强度与空白对照组比较明显降低(P<0.01)。与C57BL/6小鼠模型组比较，C57BL/6-PCSK9-/-小鼠模型组可提高白癜风鼠模型皮肤组织中酪氨酸酶的强度(P<0.01)。各组氢醌脱色所致小鼠皮肤组织酪氨酸酶的强度(n＝6)见下表2。
表2各组氢醌脱色所致小鼠皮肤组织酪氨酸酶的强度(n＝6)
组别 阳性细胞数(个) 颜色(个) 总数(个) 强度
空白对照组 50％(3) 棕黄色(2) 5 +++
C57BL/6-PCSK9-/-小鼠模型组 33.3％(2) 棕黄色(2) 4 +++*
C57BL/6小鼠模型组 16.7％(1) 黄色(1) 2 +
注：*与C57BL/6小鼠模型组比较，P＜0.01。
3.实验结论：
PCSK9基因敲除可增加白癜风模型皮肤组织中表皮MC和含黑素颗粒基底细胞数量，提高皮肤组织酪氨酸酶强度。
实施例2：
PCSK9抑制剂对小鼠色素生长的影响：
1.实验方法：
1.1材料：
(1)PCSK9干扰RNA-1序列及修饰如下表3所示。
表3
基因 5'-3'Sense 5'-3'Antisense
siPCSK9-1 GccuGGAGuuuAuucGGAAdT*dT UUCCgAAuAAACUCcAGGCdT*dT
siPCSK9-2 AGGuGuAucuccuAGAcAcdT*dT GUGUCuAGGAGAuAcACCUdT*dT
将siPcsk9-1和2等量混合后用生理盐水稀释至20μM，取稀释后siRNA与润肤液均匀混合。
PCSK9干扰RNAi抑制剂-2：RNA序列与Alnylam公司的Inclisran相同；PCSK9干扰RNAi抑制剂-3：
RNA序列与Affiris公司的ALN-PCS相同。
(2)PCSK9小分子化合物抑制剂1：Selleck公司产品R-IMPP，化学式：C 24H 27N 3O 2，分子量：389.49，结构式：
Figure PCTCN2021103749-appb-000005
PCSK9小分子化合物抑制剂2：Selleck公司产品PF-06446846，化学式：
C 22H 20ClN 7O，分子量：434.04，结构式：
Figure PCTCN2021103749-appb-000006
PCSK9小分子化合物抑制剂3：Selleck公司产品 SBC-115076，化学式：C 31H 33N 3O 5，分子量：527.61，结构式：
Figure PCTCN2021103749-appb-000007
PCSK9小分子化合物抑制剂4：Selleck公司产品SBC-110736，化学式：C 26H 27N 3O 2，分子量：413.51，结构式：
Figure PCTCN2021103749-appb-000008
(3)PCSK9单克隆抗体1：购于Abcam公司(ab84041)；PCSK9单克隆抗体2：evolocumab(依洛尤单抗)；PCSK9单克隆抗体3：alirocumab(阿利西尤单抗)。
(4)PCSK9多肽：Abcam公司的ab32727。
(5)阳性治疗药：糠酸莫米松乳膏(商品名：艾洛松，先灵葆雅中国有限公司生产)
(6)实验动物：SPF级黑色豚鼠，体质量(252±18)g，雌雄各半。
(7)治疗乳膏制备方法：赋形剂基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，分别与适量以上PCSK9抑制剂混匀形成混合乳剂。本实施例所用的乳膏基质是指乳膏除去活性成分的基质成分。
1.2动物分组与造模：
选取SPF级黑色豚鼠，体质量(252±18)g，按体质量编号，采用随机排列表法分为化合物组1(皮肤涂抹0.1％R-IMPP乳膏)、化合物组2(皮肤涂抹0.1％PF-06446846乳膏)、化合物组3(皮肤涂抹0.1％SBC-115076乳膏)、化合物组4(皮肤涂抹0.1％SBC-110736乳膏)、单抗1组(皮下注射PCSK9单克隆抗体ab84041，1mg/kg.d)、单抗2组(皮下注射evolocumab，1mg/kg.d)、单抗3组(皮下注射alirocumab，1mg/kg.d)、PCSK9干扰RNA组-1(皮肤涂抹0.1％PCSK9小干扰RNA-1乳膏)、PCSK9干扰RNA组-2(皮肤涂抹0.1％PCSK9小干扰RNA-2乳膏)、PCSK9干扰RNA组-3(皮肤涂抹0.1％PCSK9小干扰RNA-3乳膏)、多肽组(皮下注射Abcam公司ab32727，0.5mg/kg.d)、阳性治疗组(皮肤涂抹艾洛松)、空白对照组(涂抹凡士林)、模型对照组(涂抹凡士林)，每组10只，雌雄各半。除空白对照组外，其余各组均在脱毛区涂抹5％氢醌脱色，每次0.5mL，每天2次，空白对照组动物每天涂抹等量生理盐水。连续涂抹30d，出现明显白斑表明模型成功。造模结束后，按照以上方式进行给药，每天一次，连续30d。
1.3观察指标及测试方法：
1.3.1组织学观察实验：
给药结束后，处死豚鼠后，剪取背部给药部位的皮肤组织约3cm×3cm，用于做组织病理学检查。
1.3.2白癜风动物模型表皮黑素细胞(MC)计数：
将剪取的豚鼠皮肤组织置10％福尔马林中固定1h，后流水冲洗3～4min，放入含0.1％二羟基苯丙氨酸的磷酸盐缓冲液(pH7.4)中，于37℃静置1h。然后换入新鲜的Dopa试剂中，37℃静置12h。流水冲洗，用Bouin氏液继续固定24h。将标本取出后流水冲洗4h后脱水，二甲苯透明，石蜡包埋，脱蜡至水，用苏木素-伊红对比染色。脱水，常规封固。Dopa-氧化酶染色后，棕黑色染色为MC阳性。分别于光镜下测定每个高倍镜视野内各组MC数量，每个标本观察10个高倍镜视野，计算每100个表皮基底细胞中MC的平均数量。
1.3.3白癜风豚鼠模型表皮含黑素颗粒基底细胞计数：
将剪取的豚鼠皮肤组织置10％福尔马林中固定，脱水，用二甲苯透明，石蜡包埋，制切片，脱蜡至水，蒸馏水洗涤数次后进行Lillie染色，用氯化铁和铁氰化钾混合液浸染15～20min，再以1％醋酸水溶液分化数分钟，蒸馏水洗涤数次，然后用95％乙醇及无水乙醇迅速脱水，二甲苯透明，中性树胶封固。Lillie染色后，黑色素呈暗绿色为阳性，分别于光镜下测定各组含黑素颗粒的基底细胞数量，每个标本观察10个高倍镜视野，计算每100个表皮基底细胞中含黑素颗粒的基底细胞的平均数量。
1.3.4白癜风豚鼠模型皮肤组织酪氨酸酶活性的检查：
将剪取的豚鼠皮肤组织置10％福尔马林中固定。然后脱水，二甲苯透明，石蜡包埋，制切片，脱蜡至水后，每张切片加过氧化酶阻断剂(3％H 2O 2)50μL，10min，阻断内源性过氧化物酶活性。切片浸入0.01mol·L-1PBS缓冲液中，微波处理10min。加鼠抗人酪氨酸酶单克隆抗体50μL，37℃、60min。加Elivision试剂50μL，37℃30min。然后将切片置新鲜配制的DAB-H 2O 2显色液中显色，蒸馏水冲洗，苏木素对比染色1min，中性树胶固封。酪氨酸酶阳性产物定位于细胞的细胞质内，显镜下细胞内出现黄色或棕黄色颗粒、或团块状为阳性表达。用二级计分法判定结果，阳性细胞计数<5％为0分；5％～25％为1分；25％～50％为2分；50％～75％为3分；>75％为4分。按染色强度分类:淡黄色1分；黄或深黄2分；褐或棕黄色3分。两者相加小于2分为阴性(—)；2～3分为阳性(+)；4～5分为中等阳性(2+)；6～7分为强阳性(3+)。
1.4统计学方法：
实验数据采用SPSS 16.0系统软件进行统计学处理。实验数据统计变量以(x±s)表示，采用χ2检验和t检验，动物皮肤组织酪氨酸酶强度比较均用Wilcoxon秩和检验。α值取双侧，P>0.05表示差异无显著性，P<0.05表示差异有显著性，P<0.01为差异有极显著性。
2.实验结果：
2.1各种PCSK9抑制剂对氢醌脱色所致白癜风豚鼠模型表皮MC、含黑素颗粒基底细胞计数的影响：
经氢醌脱色后，与空白对照组比较，模型组豚鼠皮肤组织中表皮MC含量降低(P<0.01)，含黑素颗粒基底细胞计数降低(P<0.01)。各组PCSK9抑制剂分别作用于氢醌脱色所致的白癜风豚鼠模型后，PCSK9抑制剂各给药组均可提高白癜风模型动物皮肤组织中表皮MC计数(P<0.01)；各PCSK9抑制剂组均可提高白癜风豚鼠模型动物皮肤组织中含黑素颗粒基底细胞计数(P<0.01)。各组对氢醌脱色所致豚鼠表皮MC、含黑素颗粒基底细胞计数的影响(n＝10，x±s)见下表4。
表4各组对氢醌脱色所致豚鼠表皮MC、含黑素颗粒基底细胞计数的影响(n＝10，x±s)
组别 表皮黑素细胞计数 含黑素颗粒基底细胞计数
模型对照组 15.2±2.1 21.6±1.8
空白对照组 43.5±2.3 42.8±3.2
阳性治疗组 42.6±2.1* 41.2±2.9*
单抗组1 35.6±3.3* 38.5±4.3*
单抗组2 46.2±3.8* 41.2±3.9*
单抗组3 43.5±3.6* 39.6±3.7*
多肽组 29.7±3.5* 32.4±3.6*
化合物组1 33.6±2.9* 36.5±3.8*
化合物组2 39.3±3.2* 38.8±3.9*
化合物组3 36.1±2.8* 36.9±3.6*
化合物组4 34.6±2.6* 36.8±3.7*
干扰RNA组1 31.8±2.6* 33.2±2.9*
干扰RNA组2 32.6±2.8* 36.1±3.2*
干扰RNA组3 33.8±2.7* 35.3±3.1*
注：*与模型对照组比较，P＜0.01。
2.2各种PCSK9抑制剂对氢醌脱色所致白癜风豚鼠模型皮肤组织酪氨酸酶强度的影响：
经氢醌脱色后，模型组豚鼠皮肤组织中酪氨酸酶强度与空白对照组比较明显降低(P<0.01)。将各种PCSK9抑制剂分别作用于氢醌脱色所致的白癜风豚鼠模型后，各种PCSK9抑制剂组均可提高白癜风豚鼠模型动物皮肤组织中酪氨酸酶的强度(P<0.01)。各组对氢醌脱色所致豚鼠皮肤组织酪氨酸酶强度的影响(n＝10)见下表5。
表5各组对氢醌脱色所致豚鼠皮肤组织酪氨酸酶强度的影响(n＝10)
组别 阳性细胞数(个) 颜色(个) 总数(个) 强度
模型对照组 10％(1) 黄色(1) 2 +
空白对照组 40％(4) 棕黄色(3) 7 +++
阳性治疗组 30％(3) 棕黄色(3) 6 +++*
单抗组1 30％(3) 棕黄色(3) 6 +++*
单抗组2 40％(4) 棕黄色(4) 7 +++*
单抗组3 30％(3) 棕黄色(4) 7 +++*
多肽组 20％(2) 黄色(2) 4 ++*
化合物组1 30％(3) 棕黄色(2) 5 +++*
化合物组2 40％(4) 棕黄色(3) 7 +++*
化合物组3 30％(3) 棕黄色(3) 6 +++*
化合物组4 30％(3) 棕黄色(3) 6 +++*
干扰RNA组1 20％(2) 黄色(2) 4 ++*
干扰RNA组2 30％(3) 黄色(2) 5 +++*
干扰RNA组3 30％(3) 黄色(2) 5 +++*
注：*与模型对照组比较，P＜0.01。
3.实验结论：
各种PCSK9抑制剂可通过增加白癜风模型皮肤组织中表皮MC和含黑素颗粒基底细胞数量，提高皮肤组织酪氨酸酶强度来治疗色素脱失性疾病。
实施例3：
PCSK9基因敲除对兔耳痤疮模型的影响：
1.实验方法：
1.1动物分组与造模：
实验动物：SPF级新西兰家兔，PCSK9-/-SPF级新西兰家兔(利用CRISPR基因编辑技术，敲除Pcsk9基因exon 2-3，建立Pcsk9基因敲除兔模型)，1.8～2.3kg，雄性。动物来源于南模生物。
分组与造模：将不同基因型兔分三组：阴性对照组、模型对照组和PCSK9-/-模型组，每组6只。取兔右耳内侧脱毛处理作为观察区，阴性对照组涂95％酒精，模型对照组和PCSK9-/-模型组均涂2％煤焦油溶液(Alfa Aesar中国公司)，用95％酒精配制成2％的煤焦油溶液，用无菌棉签均匀涂于家兔耳内侧面耳导管开口处约2cm×2cm范围，每天1次，每次0.5mL，并且用温水擦拭前次涂药部位，连续涂14d，建立痤疮微粉刺模型。肉眼观察局部皮肤的变化，耳厚薄、硬度、粗糙程度和毛囊口有无黑色角栓等。末次涂18h后处死取材，使用5mm打孔器在涂药部位打孔取皮肤组织，10％甲醛固定，石蜡包埋切片，HE染色后，进行病理组织学观察分析。
1.2观察指标：
痤疮模型组织学判定分级标准：按组织学级别为3级。0级为漏斗部仅有松散的角化的细胞，无粉刺生成“—”；1级为兔耳表面皮肤发红，或毛囊漏斗部见少量致密角化物质，漏斗部不扩张“+”；2级为毛囊漏斗部见中等致密角化物质，并向皮脂腺延伸，伴随皮脂腺导管的增生，漏斗扩张“2+”；3级为毛囊内有广泛的角化物质，毛囊中紧密的角质栓塞引起毛囊重度扩张，皮脂腺导管上皮明显增生，皮肤凸起、瘢痕，部分皮脂腺发生退行变“3+”。
2.实验结果：
涂煤焦油14d后，阴性对照组兔耳柔软、薄白，毛细血管清晰，兔耳管开口处可见毛孔大小均匀。模型对照组兔耳厚度增加、变硬，毛囊口有黑色角栓，呈黑头粉刺状，毛囊口隆起呈丘疹状，表面粗燥，触之较硬，部分融合成片。PCSK9-/-模型组兔的外耳道皮肤轻微干燥，毛孔稍有增大，肉眼观察未见有黑色物质堵塞毛孔，未见丘疹。
组织切片观察：与阴性对照组比较，模型对照组兔耳见不同程度的颗粒层、棘层肥厚，毛囊口角质化，皮肤组织结构被破坏，PCSK9-/-模型组仅见皮肤棘层轻微增厚，毛囊面积轻度增大。各组镜下实验性粉刺组织学分级见下表6。
表6各组粉刺的组织学分级
从表6可以看出，敲除Pcsk9基因的模型组，确实能够有效的降低痤疮微粉刺的发病率，也由此可知，
组别 n _ 1+ 2+ 3+
阴性对照组 10 10 0 0 0
模型对照组 10 0 1 4 5
PCSK9-/-模型组 10 8 2 0 0
抑制Pcsk9基因也确实能够达到类似的效果。
3.实验结论：
PCSK9基因敲除能明显抑制煤焦油诱导的兔痤疮模型症状，减少毛孔堵塞，明显减少黑头粉刺形成。
实施例4：
PCSK9抑制剂对兔耳痤疮模型的影响：
1.实验方法：
1.1材料：
(1)PCSK9干扰RNA-1序列及修饰，如下表7所示。
表7
基因 5'-3'Sense 5'-3'Antisense
siPCSK9-1 GccuGGAGuuuAuucGGAAdT*dT UUCCgAAuAAACUCcAGGCdT*dT
siPCSK9-2 AGGuGuAucuccuAGAcAcdT*dT GUGUCuAGGAGAuAcACCUdT*dT
将siPcsk9-1和2等量混合后用生理盐水稀释至20μM，取稀释后siRNA与润肤液均匀混合。
PCSK9干扰RNAi抑制剂-2：RNA序列与Alnylam公司的Inclisran同；PCSK9干扰RNAi抑制剂-3：RNA序列与Affiris公司的ALN-PCS同。
PCSK9小分子化合物抑制剂1：Selleck公司产品R-IMPP，化学式：C 24H 27N 3O 2，分子量：389.49，结构式：
Figure PCTCN2021103749-appb-000009
PCSK9小分子化合物抑制剂2：Selleck公司产品PF-06446846，化学式：
C 22H 20ClN 7O ，分子量：434.04，结构式：
Figure PCTCN2021103749-appb-000010
PCSK9小分子化合物抑制剂3：Selleck公司产品 SBC-115076，化学式：C 31H 33N 3O 5，分子量：527.61，结构式：
Figure PCTCN2021103749-appb-000011
PCSK9小分子化合物抑制剂4：Selleck公司产品SBC-110736，化学式：C 26H 27N 3O 2，分子量：413.51，结构式：
Figure PCTCN2021103749-appb-000012
(3)PCSK9单克隆抗体1：购于Abcam公司(ab84041)；PCSK9单克隆抗体2：安进(Amgen)和安斯泰来(Astellas)联合开发的evolocumab(依洛尤单抗)，商品名Repatha(瑞百安)；PCSK9单克隆抗体3：赛诺菲(Sanofi)和再生元(Regeneron)联合开发的alirocumab(阿利西尤单抗，阿利珠单抗)，商品名Praluent(波立达)。
(4)PCSK9多肽-1购于Abcam公司(ab32727)；PCSK9多肽-2：Pieris公司的DS9001。
(5)阳性治疗药：0.1％阿达帕林凝胶(商品名：达芙文，法国高德美制药公司生产)
(6)实验动物：普通级新西兰家兔，1.9～2.4kg，雄性，来源于上海斯莱克实验动物有限责任公司。
治疗乳膏制备方法：赋形剂基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，分别与适量以上PCSK9抑制剂混匀形成混合乳剂。
本实施例所用的乳膏基质是指乳膏除去活性成分的基质成分。
1.2动物分组与造模：
按体重编号，采用随机排列表法分为化合物组1(皮肤涂抹0.1％R-IMPP乳膏，每天2次)、化合物组2(皮肤涂抹0.1％PF-06446846乳膏，每天2次)、化合物组3(皮肤涂抹0.1％SBC-115076乳膏，每天2次)、化合物组4(皮肤涂抹0.1％SBC-110736乳膏，每天2次)、PCSK9干扰RNA组-1(皮肤涂抹0.1％PCSK9小干扰RNA-1乳膏，每天2次)、PCSK9干扰RNA组-2(皮肤涂抹0.1％Inclisran乳膏，每天2次)、PCSK9干扰RNA组-3(皮肤涂抹0.1％ALN-PCS乳膏，每天2次)、单抗1组(皮下注射PCSK9单克隆抗体ab84041，1mg/kg.d)、单抗2组(皮下注射evolocumab，1mg/kg.d)、单抗3组(皮下注射alirocumab，1mg/kg.d)、多肽组(皮下注射Abcam公司ab32727，0.5mg/kg.d)、联合治疗组1(皮肤涂抹0.1％PF-06446846乳膏和0.1％阿达帕林凝胶，每天分别2次)、联合治疗组2(皮肤涂抹0.1％PCSK9小干扰RNA-1乳膏和0.1％阿达帕林凝胶，每天分别2次)、阳性治疗组(涂抹0.1％阿达帕林凝胶)、模型对照组(涂抹乳膏基质)，每天2次，每组10只。取兔右耳内侧脱毛处理作为观察区，所有家兔左耳作为自身阴性对照，涂抹95％酒精，模型组和治疗组右耳内侧均涂2％煤焦油(Alfa Aesar中国公司，用95％酒精配制成2％的煤焦油溶液)，用无菌棉签均匀涂于家兔耳内侧面耳导管开口处约2cm×2cm范围，每天1次，每次0.5mL，并且用温水擦拭前次涂药部位，连续涂14d，建立痤疮微粉刺模型。肉眼观察局部皮肤的变化，包括耳厚薄、硬度、粗糙程度和毛囊口有无黑色角栓等。末次涂18h后处死取材，使用5mm打孔器在涂药部位打孔取皮肤组织，10％甲醛固定，石蜡包埋切片，HE染色后，进行病理组织学观察分析。
1.3观察指标：
痤疮模型组织学判定分级标准：按组织学级别为3级。0级“—”为漏斗部仅有松散的角化的细胞，无粉刺生成；1级为兔耳表面皮肤发红，或毛囊漏斗部见少量致密角化物质，漏斗部不扩张“+”；2级为毛囊漏斗部见中等致密角化物质，并向皮脂腺延伸，伴随皮脂腺导管的增生，漏斗扩张“2+”；3级为毛囊内有广泛的角化物质，毛囊中紧密的角质栓塞引起毛囊重度扩张，皮脂腺导管上皮明显增生，皮肤凸起、瘢痕，部分皮脂腺发生退行变“3+”。
在显微镜下观察其组织病理改变情况，并用Biomias99图像分析系统测量一张切片上5处不同表皮的厚度，计算平均值；检测4张切片中位置相同而结构最完整的2个毛囊的面积和4个皮脂腺的直径，计算各自的平均值，然后将各组兔左、右外耳道数据相减，即得各兔的左、右耳表皮厚度差、毛囊面积差和皮脂 腺直径差。
1.4统计学处理：
用SPSS16软件进行统计分析。自身左右对照采用配对t检验，各组间比较用t检验，P<0.05为差异有统计学意义。
2.实验结果：
肉眼观察：涂煤焦油14d后，所有组兔左耳皮肤柔软，其外耳道毛囊口排列整齐，未见粉刺、丘疹及脓疱等。模型对照组兔右耳涂煤焦油后耳厚度增加、变硬，表面粗燥，毛囊口有黑色角栓，形成黑头粉刺，毛囊口隆起呈丘疹状，触之较硬，部分融合成片。siPcsk9治疗组右耳表现为耳部皮肤粗燥肥厚，丘疹较前变平，仍可见毛囊角栓，毛孔稍由缩小。小分子化合物治疗组表现为兔右耳与左耳相比，皮肤变薄，大部分毛囊性丘疹消退，粉刺减少变平，毛孔缩小，皮肤稍干燥。单抗治疗组、多肽治疗组和联合治疗组右耳表现为皮肤变薄柔软，粉刺减少，毛孔明显缩小，无脱屑，基本接近正常兔耳。阳性治疗组兔右耳与左耳相比，皮肤轻度发红，有少许脱屑，少量毛囊角栓和粉刺，未见丘疹。
组织切片观察：模型组左耳显示表皮较薄，可见毛囊，真皮与表皮交界清楚。模型组右耳造模后见表皮增厚，角化过度，颗粒层和棘层增厚，毛囊扩大，角栓堵塞毛囊口，并向皮脂腺延伸，毛囊漏斗部充满角化物质并扩大呈壶状；真皮上层毛细血管扩张，毛囊周围散在炎症细胞浸润，少量中性粒细胞浸润；皮脂腺数量增多，皮脂腺体积增大。
各组镜下实验性粉刺组织学分级(见表8)：模型组兔右与其左耳(空白对照)进行比较，差异有统计学意义(P<0.05)，各治疗组兔右耳与模型组兔右耳进行比较，差异均有统计学意义(P均<0.05)。各组痤疮粉刺的组织学分级如下表8所示。
表8各组痤疮粉刺的组织学分级
从表8可以看出，具有抑制PCSK9功效的化合物组1-4、单抗组1-3、干扰RNA组1-3、多肽组以及联合
组别/组织学分级 n _ 1+ 2+ 3+
模型组左耳 10 10 0 0 0
模型组右耳 10 0 1 5 4
化合物组1 10 6 3 1 0
化合物组2 10 7 2 1 0
化合物组3 10 5 3 2 0
化合物组4 10 5 2 3 0
单抗组1 10 8 2 0 0
单抗组2 10 9 1 0 0
单抗组3 10 9 1 0 0
干扰RNA组1 10 4 5 1 0
干扰RNA组2 10 6 3 1 0
干扰RNA组2 10 5 4 1 0
多肽组 10 5 4 1 0
联合治疗组1 10 8 2 0 0
联合治疗组2 10 8 1 1 0
阳性治疗组 10 6 2 2 0
治疗组与模型组右耳相比，均具有不同程度的效果提升，其中单抗组1-3的效果要优于其它组别，考虑其主要原因在于给药方式为注射，属于体内给药，故而效果更优，其它组别均为外用涂抹，效果相应会有所降低，但也均优于模型组右耳，同时，也与阳性治疗组治疗效果相当，尤其是其中的联合治疗组，其治疗效果已经达到了与效果最好的单抗组相近似的程度，考虑到联合治疗组采用的是外用涂抹的方式，给药方式要比单抗组跟容易，发明人认为，联合治疗组必然产生了比单独使用更加优异的效果。
模型组兔右耳表皮厚度、毛囊画积和皮脂腺直径与其左耳(空白对照)进行比较，差异有统计学意义(P<0.05)，提示兔耳痤疮模型复制成功；各治疗组兔右耳表皮厚度、毛囊画积和皮脂腺直径与模型组兔右耳进行比较，差异均有统计学意义(P<0.05)(见表9)。各组耳表皮厚度、毛囊面积和皮脂腺直径如下表9所示。
表9各组耳表皮厚度、毛囊面积和皮脂腺直径
*为与模型组右耳比较P<0.05。
组别 n 表皮厚度(mm) 毛囊面积(mm 2) 皮脂腺直径(mm)
模型组左耳 10 0.1238±0.0082* 0.1052±0.0978* 0.0426±0.0695*
模型组右耳 10 0.2913±0.0242 0.4821±0.1743 0.4127±0.1436
化合物组1 10 0.2433±0.0196* 0.2726±0.0842* 0.0512±0.0327*
化合物组2 10 0.1968±0.0121* 0.2353±0.0812* 0.0467±0.0313*
化合物组3 10 0.2245±0.0185* 0.2836±0.0851* 0.0542±0.0346*
化合物组4 10 0.2276±0.0193* 0.2853±0.0879* 0.0561±0.0362*
单抗治疗组1 10 0.2325±0.0189* 0.2218±0.0826* 0.0465±0.0312*
单抗治疗组2 10 0.1532±0.0156* 0.1533±0.0562* 0.0432±0.0267*
单抗治疗组3 10 0.1547±0.0149* 0.1526±0.0723* 0.0435±0.0285*
干扰RNA组1 10 0.2546±0.0211* 0.2954±0.1065* 0.0781±0.0518*
干扰RNA组2 10 0.1856±0.0179* 0.1836±0.0856* 0.0561±0.0423*
干扰RNA组3 10 0.1987±0.0185* 0.1921±0.0933* 0.0615±0.0476*
多肽组 10 0.2362±0.0192* 0.2462±0.1142* 0.0532±0.0415*
联合治疗组1 10 0.1723±0.0115* 0.2046±0.0782* 0.0448±0.0296*
联合治疗组2 10 0.1851±0.0118* 0.2136±0.0789* 0.0463±0.0302*
阳性治疗组 10 0.2461±0.0205* 0.2915±0.0867* 0.0569±0.0836*
从表9可以看出，模型组右耳与模型组左耳和阳性治疗组相比，其表皮厚度、毛囊面积及皮脂腺直径均有大幅增长，而化合物组1-4、单抗组1-3、干扰RNA组1-3、多肽组以及联合治疗组与模型组左耳相比，虽也略有增长，但增长幅度不大，其中，单抗治疗组2-3的数据值与模型组左耳基本相当，考虑其主要原因在于给药方式为注射，属于体内给药，故而效果更优，其它组别均为外用涂抹，效果相应会有所降低，但也均优于模型组右耳，同时，也与阳性治疗组治疗效果相当，尤其是其中的联合治疗组，其治疗效果已经达到了与效果最好的单抗治疗组2-3相近似的程度，考虑到联合治疗组采用的是外用涂抹的方式，给药方式要比单抗组跟容易，发明人认为，联合治疗组同样产生了比单独使用更加优异的效果。
3.实验结论：
PCSK9小分子化合物抑制剂、PCSK9干扰RNA、PCSK9单克隆抗体、PCSK9多肽抑制剂均能明显抑制煤焦油诱导的兔耳痤疮模型症状，减少毛孔堵塞和黑头粉刺形成，并对皮肤温和无刺激，显示对痤疮具有治疗作用。
实施例5：
PCSK9基因敲除对雄性激素性脱发(SA)的影响：
1.实验方法：
1.1动物分组与造模：
实验动物：SPF级C57BL/6(B6)小鼠，C57BL/6-PCSK9-/-小鼠(利用CRISPR基因编辑技术，敲除Pcsk9基因exon 2-3，建立Pcsk9基因敲除小鼠模型)，小鼠来源于南模生物。
将不同基因型小鼠分三组：C57BL/6小鼠阴性对照组、C57BL/6小鼠模型组和C57BL/6-PCSK9-/-小鼠模型组，每组6只，雌雄各3只。每只小鼠背部进行脱毛处理，作为观察区。除阴性对照组外，其他组小鼠颈后皮下注射丙酸睾酮注射液[8ml/(kg·d)]，每天1次，连续60d，建立SA模型。连续皮下注射丙酸睾丸 酮30天后小鼠逐渐出现毛发脱落，证明雄性激素性脱发模型成功建立。观察毛发生长情况。
1.2观察指标及测试方法：
每10天于每只小鼠背部观察区拔取10根毛发，用游标卡尺测量毛发长度。实验结束后，取实验观察区皮肤，进行常规组织脱水、石蜡包埋、HE染色，光镜镜检，观察小鼠皮肤毛囊和皮脂腺组织病理学改变。对各组病变进行半定量分析。分级标准如下：皮肤真皮组织细胞和皮下毛囊、皮脂腺结构正常记为“—”：皮肤真皮未见有增生，毛囊、皮脂腺病变局限，皮下未见有炎症记为“±”：皮肤真皮组织未见有明显增生，毛囊明显囊性变.皮脂腺未见有明显增生，皮下未见有炎症记为“+”：皮肤真皮组织有节段性增生，不明显，少部分毛囊有囊性变，皮脂腺有轻度增生肥大.皮下未见有明显炎症记为“++”：皮肤真皮组织细胞有不同程度节段性增生，部分毛囊囊性变，表现毛囊大小不均匀，周边部无细胞。皮脂腺有增生，增生腺体内细胞核较少，个别小鼠皮下有轻度炎性增生记为“+++”。
2.实验结果：
2.1对小鼠毛发生长的影响：
C57BL/6-PCSK9-/-小鼠模型组在建模第10、20、30天的毛发长度均长于C57BL/6(B6)小鼠模型组，差异均有统计学意义(P<0.01)。各组小鼠毛发生长长度如下表10所示。
表10各组小鼠毛发生长长度
组别 10天毛发长度(mm) 20天毛发长度(mm) 30天毛发长度(mm)
阴性对照组 3.42±0.41 5.32±0.52 6.83±0.59*
C57BL/6小鼠模型组 2.61±0.36 3.06±0.41 3.92±0.45
C57BL/6-PCSK9-/-小鼠模型组 3.03±0.38 4.96±0.43* 6.26±0.51*
*为与C57BL/6(B6)小鼠组模型组比较P<0.01。
2.2对小鼠观察区皮肤组织真皮浅层毛囊形态的影响：
C57BL/6(B6)小鼠模型组部分皮肤真皮组织细胞有不同程度节段性增厚，皮下有轻度淋巴细胞增生；部分小鼠皮下毛囊有明显囊性变，毛囊大小不等，周边有轻度纤维化，毛囊周边细胞消失或细胞层次明显减少，皮脂腺数目增多，部分腺体有肥大，肥大腺体细胞核明显减少，正常毛囊数减少。C57BL/6-PCSK9-/-小鼠模型组皮肤真皮组织细胞及皮下毛囊、皮脂腺病变与C57BL/6小鼠模型组比较有不同程度减轻，皮肤损伤毛囊数明显减少，差异有统计学意义(P<0.01)。各组对小鼠皮肤毛囊和皮脂腺的影响(只)如下表11所示。
表11各组对小鼠皮肤毛囊和皮脂腺的影响(只)
组别 — ± + ++ +++ P值
阴性对照组 6 0 0 0 0 <0.01
C57BL/6小鼠模型组 0 0 1 1 4 ——
C57BL/6-PCSK9-/-小鼠模型组 0 3 2 1 0 <0.01
*为与C57BL/6(B6)小鼠组模型组比较P<0.01。
3.实验结论：
PCSK9基因敲除能明显促进雄性激素性脱发模型小鼠毛发生长，减轻对皮下毛囊和皮脂腺的损伤。
实施例6：
PCSK9抑制剂对小鼠毛发生长的影响：
1.实验方法：
1.1材料：
(1)PCSK9干扰RNA-1序列及修饰，如下表12所示。
表12
基因 5'-3'Sense 5'-3'Antisense
siPCSK9-1 GccuGGAGuuuAuucGGAAdT*dT UUCCgAAuAAACUCcAGGCdT*dT
siPCSK9-2 AGGuGuAucuccuAGAcAcdT*dT GUGUCuAGGAGAuAcACCUdT*dT
将siPcsk9-1和2等量混合后用生理盐水稀释至20μM，取稀释后siRNA与润肤液均匀混合。
PCSK9干扰RNAi抑制剂-2：RNA序列与Alnylam公司的Inclisran同；PCSK9干扰RNAi抑制剂-3：RNA序列与Affiris公司的ALN-PCS同。
(2)PCSK9小分子化合物抑制剂1：Selleck公司产品R-IMPP，化学式：C 24H 27N 3O 2，分子量：389.49，结构式：
Figure PCTCN2021103749-appb-000013
PCSK9小分子化合物抑制剂2：Selleck公司产品PF-06446846，化学式：C 22H 20ClN 7O，分子量：434.04，结构式：
Figure PCTCN2021103749-appb-000014
PCSK9小分子化合物抑制剂3：Selleck公司产品SBC-115076，化学式：C 31H 33N 3O 5，分子量：527.61，结构式：
Figure PCTCN2021103749-appb-000015
PCSK9小分子化合物抑制剂4：Selleck公司产品SBC-110736，化学式：C 26H 27N 3O 2，分子量：413.51，结构式：
Figure PCTCN2021103749-appb-000016
(3)PCSK9单克隆抗体1：购于Abcam公司(ab84041)；PCSK9单克隆抗体2：安进(Amgen)和安斯泰来(Astellas)联合开发的evolocumab(依洛尤单抗)，商品名Repatha(瑞百安)；PCSK9单克隆抗体3：赛诺菲(Sanofi)和再生元(Regeneron)联合开发的alirocumab(阿利西尤单抗，阿利珠单抗)，商品名Praluent(波立达)。
(4)PCSK9多肽购于Abcam公司(ab32727)。
治疗溶液制备方法：60％乙醇分别与适量以上PCSK9抑制剂混匀配制成不同浓度溶液。
1.2动物分组与造模：
选取SPF级C57BL/6小鼠，按体重编号，采用随机排列表法分为化合物组1(皮肤涂抹2％R-IMPP溶液)、化合物组2(皮肤涂抹2％PF-06446846溶液)、化合物组3(皮肤涂抹2％SBC-115076溶液)、化合物组4(皮肤涂抹2％SBC-110736溶液)、单抗1组(皮下注射PCSK9单克隆抗体ab84041，5mg/kg.d)、单抗2组(皮下注射evolocumab，5mg/kg.d)、单抗3组(皮下注射alirocumab，5mg/kg.d)、PCSK9干扰RNA组-1(皮肤涂抹2％PCSK9小干扰RNA-1溶液)、PCSK9干扰RNA组-2(皮肤涂抹2％PCSK9小干扰RNA-2溶液)、PCSK9干扰RNA组-3(皮肤涂抹2％PCSK9小干扰RNA-3溶液)、多肽组(皮下注射Abcam公司ab32727，3mg/kg.d)、阳性对照组(皮肤涂抹2％米诺地尔溶液)、阴性对照组(皮肤涂抹60％乙醇)、模型对照组(皮肤涂抹60％乙醇)，每组10只，雌雄各半。小鼠经乙醚麻醉后用松香/石蜡混合物(1:1)热融化后涂于背部，待凝固变硬后揭去，以小鼠背部光滑，无伤无毛根为净，脱毛面积约3cm×4cm。脱毛后第2日起分别于脱毛区涂抹相应药物，每日2次，每次0.5mL/只。
1.3观察指标及测试方法：
1.3.1肉眼观察：
涂药后第2天起，观察小鼠背部拔毛部位皮肤每天颜色的变化确定毛囊的生长状态以及肉眼直观观察脱毛区毛发生长情况，每日对每鼠脱毛区新毛生长状况评分1次。各组预留5只小鼠肉眼观察40天，并记录背部毛发生长情况。
1.3.2组织学观察实验：
第17天各组分别颈椎脱臼处死5只小鼠，背部平行脊柱相同部位取材，10％甲醛固定，脱水，石蜡包埋，切片，HE染色，中性树胶封片，光镜下观察毛囊组织学变化并对毛囊进行形态学分期。依据国际毛发周期评分法，对各期毛囊作以下评分：生长Ⅵ期为100分，退行早期为200分，退行中期为300分，退行 晚期为400分。每只小鼠随机选择50个毛囊，确定各组毛囊所处的周期，计算平均毛发周期评分及生长期VI期、退行早期、退行中期、退行晚期毛囊所占百分比。
1.4统计学方法：
实验数据采用SPSS 16.0系统软件进行统计学处理。实验数据统计变量以(x±s)表示，采用χ2检验和t检验，α值取双侧，P>0.05表示差异无显著性，P<0.05表示差异有显著性，P<0.01为差异有显著性。
2.实验结果：
2.1肉眼观察小鼠拔毛部位的变化：
C57BL/6小鼠在拔毛后，造模局部可被诱导出高度同步的新生毛发周期，即在拔毛后第1～5天虽然拔毛局部仍然为粉红色，但组织学上毛囊已呈现出生长Ⅰ～Ⅲ期的表现；第7天皮肤颜色变为黑色，组织学上毛囊已经进入生长Ⅳ期；第9～10天拔毛区组织学上毛囊为生长Ⅵ期，至拔毛后第18天局部皮肤变为灰黑色，组织学毛囊已呈退行期改变，到拔毛后第20天以后拔毛局部又变为粉红色，组织学毛囊则进入休止期。
在本实验中，阴性对照组与模型对照组小鼠于拔毛后第7天背部皮肤由粉红色变为黑色，拔毛后第17天左右背部皮肤颜色又由黑色变为灰黑色。而抑制剂组与阳性对照组小鼠于拔毛后第6天左右背部皮肤由粉红色变为黑色，拔毛后第20天左右背部皮肤颜色又由黑色变为灰黑色。具体变化时间见如表13，可见，各抑制剂组小鼠拔毛区皮肤颜色变黑、变灰黑时间及黑色持续时间与阳性对照组相比，无显著性差异(P均＞0.05)，与阴性对照组、模型对照组相比均有极显著性差异(P均＜0.01)。提示PCSK9抑制剂有明显的延长毛囊生长期的作用。各组小鼠背部拔毛区皮肤颜色变化时间(天)如下表13所示。
表13各组小鼠背部拔毛区皮肤颜色变化时间(天)
组别 皮肤颜色变黑时间 皮肤颜色变灰黑时间 皮肤黑色持续时间
模型对照组 7.26 17.27 10.01
阴性对照组 7.09 17.56 10.47
阳性对照组 6.29 19.09 12.80
化合物组1 5.96* 20.25* 14.29*
化合物组2 5.45* 17.32* 10.86*
化合物组3 5.63* 18.12* 11.65*
化合物组4 5.78* 18.46* 12.32*
单抗组1 6.20* 19.26* 13.06*
单抗组2 5.23* 16.83* 10.21*
单抗组3 5.35* 16.65* 13.43*
多肽组 6.13* 19.38* 13.25*
干扰RNA组1 6.23* 19.21* 12.98*
干扰RNA组2 5.66* 18.23* 11.87*
干扰RNA组3 6.08* 18.52* 12.16*
注：*与阴性对照组、模型对照组比较，P＜0.01。
2.2小鼠背部拔毛区毛发的生长情况：
阴性对照组与模型对照组小鼠拔毛后第11天左右背部拔毛区出现新生毛发，第37天左右拔毛区毛发与非实验区毛发长度一致。抑制剂组与阳性对照组小鼠拔毛后第7天左右背部拔毛区出现新生毛发，第19天左右拔毛区毛发与非实验区毛发长度一致。具体毛发生长时间见表14，可见，抑制剂组小鼠新毛发生长速度与阳性对照组相比，无显著性差异(P均＞0.05)，抑制剂组与阴性对照组、模型对照组相比均有显著性差异(P均＜0.01)。提示抑制剂有明显的促毛发生长的作用。各组小鼠背部拔毛区毛发生长时间(天)如下表14所示。
表14各组小鼠背部拔毛区毛发生长时间(天)
Figure PCTCN2021103749-appb-000017
Figure PCTCN2021103749-appb-000018
注：*与阴性对照组、模型对照组比较均有显著性差异(P均＜0.01)。
2.3毛囊组织学观察：
拔毛后第17天可见阴性对照组与模型对照组毛囊基底变细，颜色变淡，毛囊下段退化，毛乳头变圆、致密，毛乳头和囊胚间上皮索形成，内毛根鞘部分消失，毛发呈杆状末端。而PCSK9抑制剂组与阳性对照组毛囊较大，较长，多数仍为生长VI期和退行早期毛囊。拔毛后第17天，PCSK9抑制剂组(单抗组、多肽组、化合物组、siPcsk9组)与阳性对照组平均毛发周期评分分别为158、167、165、168、172分、185分，提示生长VI期和退行早期毛囊。而阴性对照组与模型对照组平均毛发周期评分分别为306分、345分，对应于毛囊处于退行中晚期。抑制剂组(单抗组、多肽组、化合物组、siPcsk9组)、阳性对照组、阴性对照组和模型对照组生长Ⅵ期毛囊所占百分比分别为56％、52％、53％、47％、42％、13％和11％，抑制剂组与模型对照组生长Ⅵ期毛囊所占百分比比较均有显著性差异(P＜0.01)；抑制剂组与与阳性对照组生长Ⅵ期毛囊所占百分比均无显著性差异(P＞0.05)。提示PCSK9抑制剂有明显延长毛囊生长期、促进毛发生长的作用。
实施例7：
各种PCSK9抑制剂对雄性激素性脱发(SA)大鼠模型的影响：
1.实验方法：
1.1材料：
(1)siRNA序列及修饰，如下表15所示。
表15
基因 5'-3'Sense 5'-3'Antisense
siPCSK9-1 GccuGGAGuuuAuucGGAAdT*dT UUCCgAAuAAACUCcAGGCdT*dT
siPCSK9-2 AGGuGuAucuccuAGAcAcdT*dT GUGUCuAGGAGAuAcACCUdT*dT
PCSK9干扰RNAi抑制剂-2：RNA序列与Alnylam公司的Inclisran同；PCSK9干扰RNAi抑制剂-3：RNA序列与Affiris公司的ALN-PCS同。
(2)PCSK9小分子化合物抑制剂1：Selleck公司产品R-IMPP，化学式：C 24H 27N 3O 2，分子量：389.49，结 构式：
Figure PCTCN2021103749-appb-000019
PCSK9小分子化合物抑制剂2：Selleck公司产品PF-06446846，化学式：C 22H 20ClN 7O，分子量：434.04，结构式：
Figure PCTCN2021103749-appb-000020
PCSK9小分子化合物抑制剂3：Selleck公司产品SBC-115076，化学式：C 31H 33N 3O 5，分子量：527.61，结构式：
Figure PCTCN2021103749-appb-000021
PCSK9小分子化合物抑制剂4：Selleck公司产品SBC-110736，化学式：C 26H 27N 3O 2，分子量：413.51，结构式：
Figure PCTCN2021103749-appb-000022
(3)PCSK9单克隆抗体1：购于Abcam公司(ab84041)；PCSK9单克隆抗体2：安进(Amgen)和安斯泰来(Astellas)联合开发的evolocumab(依洛尤单抗)，商品名Repatha(瑞百安)；PCSK9单克隆抗体3：赛诺菲(Sanofi)和再生元(Regeneron)联合开发的alirocumab(阿利西尤单抗，阿利珠单抗)，商品名Praluent(波立达)。
(4)PCSK9多肽购于Abcam公司(ab32727)。
治疗溶液制备方法：75％乙醇分别与适量以上抑制剂混匀配制成不同浓度溶液。
1.2动物分组与造模：
选取SPF级Wistar大鼠，采用随机排列表法分为化合物组1(皮肤涂抹5％R-IMPP溶液)、化合物组2(皮肤涂抹5％PF-06446846溶液)、化合物组3(皮肤涂抹5％SBC-115076溶液)、化合物组4(皮肤涂抹5％SBC-110736溶液)、单抗1组(皮下注射PCSK9单克隆抗体ab84041，5mg/kg.d)、单抗2组(皮下注射evolocumab，5mg/kg.d)、单抗3组(皮下注射alirocumab，5mg/kg.d)、PCSK9干扰RNAi抑制剂组-1(皮肤涂抹5％PCSK9小干扰RNA-1溶液)、PCSK9干扰RNAi抑制剂组-2(皮肤涂抹5％PCSK9小干扰RNA-2溶液)、PCSK9干扰RNAI抑制剂组-3(皮肤涂抹5％PCSK9小干扰RNA-3溶液)、多肽组(皮下注射Abcam公司ab32727，3mg/kg.d)、阳性对照组(皮肤涂抹5％米诺地尔酊)、阴性对照组(涂抹75％乙醇)、模型对照组(涂抹75％乙醇)，每组10只，雌雄各半。
实验前每只大鼠选取背部4cmx5cm面积的区域将毛脱去作为观察区。除阴性对照组外，大鼠颈后皮下注射丙酸睾酮注射液[5ml/(kg·d)]，每天1次，连续60d，建立SA模型。连续皮下注射丙酸睾丸酮4周后大鼠逐渐出现毛发脱落.残存毛发变得纤细、质脆，证明雄性激素性脱发模型成功建立。造模同时于对应药物组大鼠背部观察区皮肤涂抹或皮下注射给药，涂抹1mL/(只·次)，每日2次，给药间隔8h，皮下注射给药，每天1次。阴性对照组和模型对照组涂抹赋型剂(75％乙醇溶液)，1mL/(只·次)，每日2次，连续60d。
1.3观察指标及测试方法：
给药每15天于每只大鼠背部观察区拔取10根毛发，用游标卡尺测量毛发长度。给药60d后，取实验观察区皮肤，进行常规组织脱水、石蜡包埋、HE染色，光镜镜检，观察大鼠皮肤毛囊和皮脂腺组织病理学改变。对各组病变进行半定量分析。分级标准如下：皮肤真皮组织细胞和皮下毛囊、皮脂腺结构正常记为“—”：皮肤真皮未见有增生，毛囊、皮脂腺病变局限，皮下未见有炎症记为“±”：皮肤真皮组织未见有明显增生，毛囊明显囊性变.皮脂腺未见有明显增生，皮下未见有炎症记为“+”：皮肤真皮组织有节段性增生，不明显，少部分毛囊有囊性变，皮脂腺有轻度增生肥大.皮下未见有明显炎症记为“++”：皮肤真 皮组织细胞有不同程度节段性增生，部分毛囊囊性变，表现毛囊大小不均匀，周边部无细胞。皮脂腺有增生，增生腺体内细胞核较少，个别大鼠皮下有轻度炎性增生记为“+++”。
2.实验结果：
2.1对大鼠毛发生长的影响：
PCSK9抑制剂组大鼠在给药第15、30、45、60天的毛发长度均长于模型对照组，差异均有统计学意义(P<0.01)。各组对大鼠毛发生长长度的影响如下表16所示。
表16各组对大鼠毛发生长长度的影响
Figure PCTCN2021103749-appb-000023
*与模型对照组比较差异均有统计学意义(P均<0.01)。
2.2对大鼠观察区皮肤组织真皮浅层毛囊形态的影响：
模型组大鼠部分皮肤真皮组织细胞有不同程度节段性增厚，大鼠皮下有轻度淋巴细胞增生；部分大鼠皮下毛囊有明显囊性变，毛囊大小不等，增大毛囊腔内有脱落角化物.周边有轻度纤维化，毛囊周边细胞消失或细胞层次明显减少，腔内似有钙化物染成蓝色，皮脂腺数目增多，部分腺体有肥大，肥大腺体细胞核明显减少，正常毛囊数减少。PCSK9抑制剂组和米诺地尔酊组大鼠皮肤真皮组织细胞及皮下毛囊、皮脂腺病变与模型组比较有不同程度减轻。PCSK9抑制剂组大鼠皮肤损伤毛囊数与模型对照组比较明显减少，差异有统计学意义(P<0.01)。与模型对照组比较，PCSK9抑制剂组和米诺地尔酊组大鼠皮肤真皮组织细胞及皮下毛囊、皮脂腺病变明显减轻，差异有统计学意义(P<0.01)。各组对大鼠皮肤毛囊和皮脂腺的影响(只)如下表17所示。
表17各组对大鼠皮肤毛囊和皮脂腺的影响(只)
Figure PCTCN2021103749-appb-000024
Figure PCTCN2021103749-appb-000025
注：*为与模型对照组比较差异有统计学意义(P<0.01)。
实施例8：
PSCK9基因敲除对大鼠疤痕(瘢痕)模型的影响：
1.实验方法：
1.1实验动物分组与造模：
SPF级大鼠，PCSK9-/-SPF级大鼠(利用CRISPR基因编辑技术，敲除Pcsk9基因exon 2-3，获得Pcsk9基因敲除大鼠)，体重(220±26)g，雄性。分为模型对照组和PCSK9-/-组，每组各6只。各组大鼠用2％戊巴比妥钠(120mg/kg)腹腔注射麻醉后固定于手术台上，然后在其背部左侧选择一块4×5cm的完整皮肤，8％硫化钠脱毛，用组织剪在脱毛处各剪成一直径为2.4cm圆形深达肌筋膜的伤口，破坏部分肌肉表面筋膜。为防止大鼠撕咬、舔蹭，动物分笼饲养。创面每日涂2％碘酊常规消毒，观察大鼠创面愈合情况。于第20d取材，用2％戊巴比妥钠麻醉大鼠，取创面皮缘组织约0.5cm 2，用于成纤维细胞的培养和后续实验。
1.2成纤维细胞培养和鉴定：
取创面组织制成1mm*1mm大小，接种于培养皿，加入0.5mlDMEM培养液。
1.3 TUNEL试剂盒检测原位细胞凋亡：
TUNEL是一种检测凋亡DNA片段化的方法，广泛用于鉴定和定量凋亡细胞，或检测单个细胞中过多的DNA断裂，该测定依赖于末端脱氧核苷酸转移酶(TdT)的使用，TdT是催化用荧光染料或另一种标记物标记的脱氧核苷酸与DNA双链断裂的3'－羟基末端连接的酶，它还可以通过除凋亡过程之外的其他方式标记具有DNA损伤的细胞。将实验用的细胞悬液用MEM调节浓度至1×105/ml，接种于100ml培养瓶中，每瓶l0ml。于37℃、5％CO 2、95％湿度条件下培养，分别于24h、48h、72h后将培养细胞吹打成细胞溶液，收集细胞。于2500rpm离心5min，去上清，每瓶加入75％乙醇lml固定。按照TUNEL细胞凋亡检测盒要求进行染色，用手动计数器手动计算在所有视野中凋亡的成纤维细胞单元的平均分布中的表达，进行统计学比较。
1.4 MTT法检测细胞增殖：
MTT化学名：3－(4，5－二甲基噻唑－2)－2，5－二苯基四氮唑溴盐，商品名：噻唑蓝。检测原理：活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶的蓝紫色结晶甲臢(Formazan)，并沉积在细胞中，而死亡细胞无此功能。二甲基亚砜(DMSO)能溶解细胞中的甲臢，用酶联免疫检测仪测定其光吸收值，可间接反映活细胞数量。在一定细胞数范围内MTT结晶形成的量与细胞数成正比。将细胞以1.0×10 5/ml密度接种于用48孔培养板中，每孔100μ1、37℃、5％CO 2孵箱中培养至细胞贴壁。于37℃、5％CO 2孵箱中培养24h、48h、72h后每孔加入MTT(5mg/ml)20μ1继续培养4h，吸弃培养基，加入DMSO裂解液100μ1，37℃，5％CO 2孵箱中孵育20min，紫色结晶完全溶解后在酶标仪上在570nm下测试吸光度(光密度值)。对各组的成纤维的增殖情况进行比较。
1.4统计学方法：
采用SPSS 16.0统计软件进行数据分析。计量资料以均数±标准差(x±s)表示，采用单因素方差分析比较，两两数据比较采用t检验，P<0.05为差异有统计学意义，P<0.01为差异显著。
2.实验结果：
2.1大鼠创面观测结果：
创面每天常规消毒，第1d、3d、5d、7d、12d、20d观察大鼠创面。在观察期第3天开始PCSK9-/-组伤口恢复速度明显比模型组快，创面面积变小。到第12天时，PCSK9-/-组创面已经基本恢复，只留下少量结痂，而模型组仍有0.5cm 2左右大小的创面。到第20天时，两组创面均已经基本恢复，模型对照组留下疤痕，而PCSK9-/-组只留下较少色素沉着，未见明显疤痕。
2.2 TUNEL法检测成纤维细胞凋亡：
TUNEL是一种检测凋亡DNA片段化的方法，广泛用于鉴定和定量凋亡细胞。结果显示PCSK9-/-组和模型对照组中的成纤维细胞凋亡指数分别为16.816±1.012和2.151±0.563，PCSK9-/-组中的成纤维细胞凋亡显著多于模型对照组(P<0.01)。
2.3 MTT法检测成纤维细胞增殖：
二甲基亚砜(DMSO)能溶解细胞中的甲臢，用酶联免疫检测仪在490nm波长处测定其光吸收值，可间接反映活细胞数量，在一定细胞数范围内，MTT结晶形成的量与细胞数成正比。结果显示：培养24h后PCSK9-/-组和模型对照组的平均吸光度值分别为0.31和0.52，培养48h后PCSK9-/-组和模型对照组的平均吸光度值分别为0.26和0.57，培养72h后PCSK9-/-组和模型对照组的平均吸光度值分别为0.21和0.66。PCSK9-/-组与模型对照组比较吸光度值在培养24h后开始明显降低，培养72h后PCSK9-/-组吸光度值降低更为明显，与模型对照组比较差距更加明显。实验结果表明PCSK9敲除能明显降低成纤维细胞的增殖活性。
3.实验结论：
PSCK9基因敲除能明显促进皮肤伤口愈合，同时降低成纤维细胞增殖活性，促进成纤维细胞凋亡，减少疤痕(瘢痕)形成，显示敲除PSCK9基因对疤痕(瘢痕)具有预防和治疗作用。
实施例9：
各种PSCK9抑制剂对大鼠疤痕模型的影响：
1.实验方法：
1.1材料：
(1)PCSK9干扰RNA-1序列及修饰，如下表18所示。
表18
基因 5'-3'Sense 5'-3'Antisense
siPCSK9-1 GccuGGAGuuuAuucGGAAdT*dT UUCCgAAuAAACUCcAGGCdT*dT
siPCSK9-2 AGGuGuAucuccuAGAcAcdT*dT GUGUCuAGGAGAuAcACCUdT*dT
将siPcsk9-1和2等量混合后用生理盐水稀释至20μM，取稀释后siRNA与润肤液均匀混合。
PCSK9干扰RNAi抑制剂-2：RNA序列与Alnylam公司的Inclisran同；PCSK9干扰RNAi抑制剂-3：RNA序列与Affiris公司的ALN-PCS同。
(2)PCSK9小分子化合物抑制剂1：Selleck公司产品R-IMPP，化学式：C 24H 27N 3O 2，分子量：389.49，结构式：
Figure PCTCN2021103749-appb-000026
PCSK9小分子化合物抑制剂2：Selleck公司产品PF-06446846，化学式：C 22H 20ClN 7O，分子量：434.04，结构式：
Figure PCTCN2021103749-appb-000027
PCSK9小分子化合物抑制剂3：Selleck公司产品SBC-115076，化学式：C 31H 33N 3O 5，分子量：527.61，结构式：
Figure PCTCN2021103749-appb-000028
PCSK9小分子化合物抑制剂4：Selleck公司产品SBC-110736，化学式：C 26H 27N 3O 2，分子量：413.51，结构式：
Figure PCTCN2021103749-appb-000029
(3)PCSK9单克隆抗体1：购于Abcam公司(ab84041)；PCSK9单克隆抗体2：evolocumab(依洛尤单抗)；PCSK9单克隆抗体3：alirocumab(阿利西尤单抗)。
(4)PCSK9多肽购于Abcam公司(ab32727)。
(5)阳性治疗药：糠酸莫米松乳膏(商品名：艾洛松，先灵葆雅中国有限公司生产)
(6)实验动物：SPF级黑色豚鼠，体质量(252±18)g，雌雄各半。
(7)治疗乳膏制备方法：赋形剂基质组成成分包括甲基硅油(15％)、硬脂酸(6％)、白凡士林(5％)、液体石蜡(5％)、十八醇(5％)、甘油(20％)、烷基芳基聚乙醇醚(1％)、脂肪醇聚氧乙烯醚(1％)、吐温-807(1％)、尼泊金乙酯(0.1％)、蒸馏水(约31-55％)，分别与适量以上PCSK9抑制剂混匀形成混合乳剂。本实施例所用的乳膏基质是指乳膏除去活性成分的基质成分。
1.2实验动物分组与造模：
SPF级雄性大鼠，体重(210±28)g，来源于南医大动物中心。大鼠按体重编号，采用随机排列表法分为化合物组1(皮肤涂抹0.5％R-IMPP乳膏)、化合物组2(皮肤涂抹0.5％PF-06446846乳膏)、化合物组3(皮肤涂抹0.5％SBC-115076乳膏)、化合物组4(皮肤涂抹0.5％SBC-110736乳膏)、单抗1组(皮下注射PCSK9单克隆抗体ab84041，3mg/kg.d)、单抗2组(皮下注射evolocumab，3mg/kg.d)、单抗3组(皮下注射alirocumab，3mg/kg.d)、PCSK9干扰RNA组-1(皮肤涂抹0.5％PCSK9小干扰RNA-1乳膏)、PCSK9干扰RNA组-2(皮肤涂抹0.5％PCSK9小干扰RNA-2乳膏)、PCSK9干扰RNA组-3(皮肤涂抹0.5％PCSK9小干扰RNA-3乳膏)、多肽组(皮下注射Abcam公司ab32727，3mg/kg.d)、阳性治疗组(皮肤涂抹艾洛松)、空白对照组(皮肤涂抹凡士林)、模型组(尾静脉注射生理盐水)，每组各6只，各组给药每天2次。各组大鼠用2％戊巴比妥钠(120mg/kg)腹腔注射麻醉后固定于手术台上，然后在其背部中左侧选择一块4×5cm的完整皮肤，8％硫化钠脱毛，用组织剪在脱毛处各剪成一直径为2.4cm圆形深达肌筋膜的伤口，破坏部分肌肉表面筋膜。动物分笼饲养防止大鼠撕咬、舔蹭。创面每日涂2％碘酊常规消毒，观察大鼠创面愈合情况。
2.实验结果：
2.1大鼠创面观测结果：
创面每天常规消毒，第1d、3d、5d、7d、12d、20d观察大鼠创面。自第5天开始小分子化合物治疗组、PCSK9小干扰RNA治疗组、PCSK9单抗治疗组和阳性治疗组伤口恢复速度明显比模型组快，创面面积逐渐变小。PCSK9多肽抑制剂治疗组自第7天开始伤口恢复速度好于模型组和空白对照组。第12天，小分子化合物治疗组、PCSK9小干扰RNA治疗组、PCSK9单抗创面已经基本恢复，而模型组和空白对照组分别仍有约0.4cm 2和0.36cm 2大小的创面，多肽抑制剂治疗组仍有0.2cm 2左右大小创面。到第20天时，各组创面均已经恢复，模型对照组和空白对照组留下明显疤痕，而其他组只留下数量不等的色素沉着。
3.实验结论：
各PCSK9抑制剂均能明显促进皮肤创面愈合，减少疤痕(瘢痕)形成。
实施例10：
PSCK9基因敲除对大鼠肺纤维化模型的影响：
1.实验方法：
1.1材料：
(1)试剂：博来霉素(4mg/支，天津太和制药有限公司)，鼠抗鼠MMP单克隆抗体(NEO Mark-ers公司)，鼠抗鼠TIMP-1多克隆抗体(武汉博士德公司)，酶联免疫吸附试验(ELISA)试剂盒(美国R&D公司)，Quantscript RT Kit逆转录试剂盒(大连TaKaRa公司)。
(2)实验动物：SPF级Wistar大鼠，PCSK9-/-SPF级Wistar大鼠(利用CRISPR基因编辑技术，敲除Pcsk9基因exon 2-3，获得Pcsk9基因敲除大鼠)，体重(181±22)g，雄性。动物来源于南模生物。
1.2动物分组与造模：
大鼠按体重编号，采用随机排列表法分为空白对照组、模型组、PCSK9-/-组，每组各6只。各组大鼠用2％戊巴比妥钠(120mg/kg)腹腔注射麻醉后固定于手术台上，颈部气管切开注药。空白对照组注入生理盐水(1.25ml/kg)，模型组及PCSK9-/-组分别注入5U/mL博莱霉素溶液(5mg/kg)，每天1次，连续14天。
1.3观察指标及测试方法：
各组动物分别于造模后14d时经尾静脉取外周静脉血，检测外周血超氧化物歧化酶(SOD)、过氧化氢 酶(CAT)水平。各组取血后处死大鼠，取动物右肺组织于-4℃冰箱储存用于检测VEGF。取左侧肺组织常规石蜡包埋、切片，用免疫组织化学染色法检测大鼠肺组织中MMP亚型及TIMP-1表达。检测VEGF时，取出右肺组织进行研磨、组织匀浆，以3000r/min高速离心，取上清液，采用ELISA法检测肺组织VEGF蛋白，应用反转录聚合酶链法测定VEGF-mRNA表达。
1.4统计学方法
数据分析采用SPSS 16.0统计软件。计量资料以均数±标准差(x±s)表示，比较采用单因素方差分析，两两比较采用t检验，P<0.05为差异有统计学意义。
2.实验结果
2.1PSCK9基因敲除对大鼠肺组织MMP的影响
空白对照组大鼠肺组织TIMP-1和MMP亚型均有少量表达。模型组大鼠MMP-2、MMP-9表达在造模后升高，TIMP-1降低，与空白对照组比较差异有统计学意义(P<0.05)，说明造模成功。PCSK9-/-组的MMP-2、MMP-9表达降低，TIMP-1表达上调，与模型组比较差异有统计学意义(P<0.05)。各组大鼠肺组织TIMP-1和MMP表达的比较(n＝6，x±s)如下表19所示。
表19各组大鼠肺组织TIMP-1和MMP表达的比较(n＝6，x±s)
组别 TIMP-1 MMP-2 MMP-9
模型对照组 5.71±0.63 3.86±0.29 5.17±0.39
空白对照组 8.95±0.56* 2.41±0.18* 3.29±0.22*
PCSK9-/-组 8.87±0.58* 2.43±0.21* 3.32±0.23*
注：*与模型对照组比较，P<0.05。
2.2 PSCK9基因敲除对肺组织中VEGF的影响：
各组大鼠肺组织VEGF蛋白及VEGF-mRNA表达见表20。模型组VEGF蛋白、VEGF-mRNA表达明显降低，与空白对照组比较差异有统计学意义(P<0.05)，说明肺纤维化模型造模成功。PCSK9-/-组VEGF蛋白及VEGF-mRNA表达与模型组比较差异均有统计学意义(P<0.05)。各组肺组织中VEGF蛋白及VEGF-mRNA表达水平的比较(n＝6，x±s)如下表20所示。
表20各组肺组织中VEGF蛋白及VEGF-mRNA表达水平的比较(n＝6，x±s)
组别 VEGF蛋白(pg/ml) VEGF-mRNA
模型对照组 37.23±4.16 0.52±0.19
空白对照组 54.26±3.95* 0.83±0.13*
PCSK9-/-组 56.12±3.67* 0.85±0.15*
注：*与模型对照组比较，P<0.05。
2.3 PSCK9基因敲除对外周血中SOD、CAT酶活性的影响：
各组大鼠外周血SOD、CAT酶水平见表21。模型组大鼠外周血SOD、CAT酶活性降低，与对空白对照组比较差异有统计学意义(P<0.05)；PCSK9-/-组大鼠外周血SOD、CAT酶活性均增强，与模型组比较差异均有统计学意义(P<0.05)。各组外周血中SOD、CAT酶水平的比较(n＝6，x±s)如下表21所示。
表21各组外周血中SOD、CAT酶水平的比较(n＝6，x±s)
组别 SOD(U/ml) CAT(kU/g)
模型对照组 143.36±15.23 8.87±1.21
空白对照组 223.65±13.28* 15.16±1.29*
PCSK9-/-组 221.76±13.57* 14.98±1.32*
注：*与模型对照组比较，P<0.05。
3.实验结论：
PSCK9基因敲除能明显降低肺纤维化小鼠模型肺组织中MMP-2、MMP-9水平，增加TIMP-1和VEGF水平，同时可提高外周血中SOD、CAT酶水平，显示对肺纤维化具有抑制作用。
实施例11：
PSCK9抑制剂对肺纤维化大鼠模型的影响：
1.实验方法：
1.1材料：
(1)试剂：博来霉素(4mg/支，天津太和制药有限公司)，鼠抗鼠MMP单克隆抗体(NEO Mark-ers公司)，鼠抗鼠TIMP-1多克隆抗体(武汉博士德公司)，酶联免疫吸附试验(ELISA)试剂盒(美国R&D公司)，Quantscript RT Kit逆转录试剂盒(大连TaKaRa公司)。
(2)siRNA序列及修饰，如下表22所示。
表22
基因 5'-3'Sense 5'-3'Antisense
siPCSK9-1 GccuGGAGuuuAuucGGAAdT*dT UUCCgAAuAAACUCcAGGCdT*dT
siPCSK9-2 AGGuGuAucuccuAGAcAcdT*dT GUGUCuAGGAGAuAcACCUdT*dT
(3)PCSK9小分子化合物抑制剂(Selleck公司产品R-IMPP)，化学式：C 24H 27N 3O 2，分子量：389.49
(4)PCSK9单克隆抗体购于Abcam公司(ab84041)
(5)PCSK9多肽购于Abcam公司(ab32727)
(6)实验动物：SPF级Wistar大鼠，雌雄各半，鼠龄51～55d，体重(180±21)g，来源于南京医科大学动物中心。
1.2动物分组与造模：
大鼠按体重编号，采用随机排列表法分为按体重编号，采用随机排列表法分为小分子化合物治疗组(尾静脉注射PCSK9小分子化合物抑制剂，3mg/kg.d)、siPcsk9治疗组(尾静脉注射PCSK9小干扰RNA，3mg/kg.d)、单抗治疗组(尾静脉注射PCSK9单克隆抗体，3mg/kg.d)、多肽治疗组(尾静脉注射PCSK9多肽抑制剂，3mg/kg.d)、模型组(尾静脉注射生理盐水，3mg/kg.d)、空白对照组(尾静脉注射生理盐水，3mg/kg.d)，每组各12只，雌雄各半。各组大鼠用2％戊巴比妥钠(120mg/kg)腹腔注射麻醉后固定于手术台上，颈部气管切开注药。对照组注入生理盐水(1.25ml/kg)，模型组及各治疗组注入5U/mL博莱霉素溶液(5mg/kg)，于造模后一周开始，各治疗组分别尾静脉注射相应PCSK9抑制剂溶液，空白对照组及模型组尾静脉注射等量生理盐水，每天1次，连续14天。
1.3观察指标及测试方法：
各组动物分别于造模后和治疗14d时经尾静脉取外周静脉血，检测外周血超氧化物歧化酶(SOD)、过氧化氢酶(CAT)水平。各组取血后分2次处死大鼠(造模后和治疗14d，每组6只)，取动物右肺组织于-4℃冰箱储存用于检测VEGF。取左侧肺组织常规石蜡包埋、切片，用免疫组织化学染色法观察大鼠肺组织中MMP亚型及TIMP-1表达。检测VEGF时，取出右肺组织进行研磨、组织匀浆，以3000r/min高速离心，取上清液，采用ELISA法检测肺组织VEGF蛋白，应用反转录聚合酶链法测定VEGF-mRNA表达。
1.4统计学方法：
数据分析采用SPSS 16.0统计软件。计量资料以均数±标准差(x±s)表示，比较采用单因素方差分析，两两比较采用t检验，P<0.05为差异有统计学意义。
2.实验结果：
2.1各PCSK9抑制剂对大鼠肺组织MMP的影响：
空白对照组大鼠肺组织TIMP-1和MMP亚型均有少量表达，在造模及治疗14d后变化不大。模型组大鼠MMP-2、MMP-9表达在造模及治疗14d后均升高，TIMP-1均降低，与空白对照组比较差异有统计学意义(P<0.05)。如表23所示，治疗14d后，各PCSK9抑制剂组的MMP-2、MMP-9表达均降低，TIMP-1表达均上调，与模型组比较差异有统计学意义(P<0.05)。各组大鼠肺组织中TIMP-1和MMP表达的比较(n＝6，x±s)如下表23所示。
表23各组大鼠肺组织中TIMP-1和MMP表达的比较(n＝6，x±s)
组别 TIMP-1 MMP-2 MMP-9
模型组 5.53±0.72 3.91±0.36 5.21±0.32
空白对照组 8.96±0.48* 2.42±0.21* 3.29±0.21*
化合物组 8.56±0.58* 2.67±0.32* 3.43±0.34*
siPcsk9治疗组 8.24±0.45* 2.98±0.26* 3.56±0.25*
单抗治疗组 7.87±0.51* 3.24±0.28* 3.64±0.27*
多肽治疗组 7.53±0.54* 3.05±0.31* 3.71±0.23*
注：*与模型对照组比较，P<0.05。
2.2各PCSK9抑制剂对肺组织中VEGF的影响：
各组大鼠肺组织VEGF蛋白及VEGF-mRNA表达见表24。造模及治疗14d后，空白对照组大鼠肺组织VEGF蛋白及VEGF-mRNA表达无明显变化(P>0.05)；模型组VEGF蛋白、VEGF-mRNA表达明显降低，与空白对照组比较差异有统计学意义(P<0.05)。治疗14d后，各PCSK9抑制剂组VEGF蛋白及VEGF-mRNA表达与造模后比较均增强，与模型组比较差异均有统计学意义(P<0.05)。各组肺组织中VEGF蛋白及VEGF-mRNA表达水平比较(n＝6，x±s)如下表24所示。
表24各组肺组织中VEGF蛋白及VEGF-mRNA表达水平比较(n＝6，x±s)
Figure PCTCN2021103749-appb-000030
注：*与模型对照组比较，P<0.05。
2.3各PCSK9抑制剂对外周血中SOD、CAT酶活性的影响：
各组大鼠外周血SOD、CAT酶水平见表25。造模及治疗14d后，空白对照组大鼠外周血SOD、CAT酶活性均无明显变化(P>0.05)。模型组大鼠外周血SOD、CAT酶活性降低，与空白对照组比较差异有统计学意义(P<0.05)；治疗14d后，各PCSK9抑制剂组大鼠外周血SOD、CAT酶活性均增强，与模型组比较差异均有统计学意义(P<0.05)。各组外周血中SOD、CAT酶水平的比较(n＝6，x±s)如下表25所示。
表25各组外周血中SOD、CAT酶水平的比较(n＝6，x±s)
Figure PCTCN2021103749-appb-000031
Figure PCTCN2021103749-appb-000032
注：*与模型对照组比较，P<0.05。
3.实验结论：
各PCSK9抑制剂均能明显降低肺纤维化小鼠模型肺组织中MMP-2、MMP-9水平，增加TIMP-1和VEGF水平；同时均可提高外周血中SOD、CAT酶水平，显示对肺纤维化具有治疗作用。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (11)

  1. PCSK9抑制剂在制备治疗多种疾病产品中的应用，其特征在于，所述PCSK9是前蛋白转化酶枯草杆菌转化酶/kexin9型，属前蛋白转换酶家族。
  2. 根据权利要求1所述的应用，其特征在于，所述疾病为白癜风、脱发、疤痕、肺纤维化或角化异常性疾病；所述角化异常性疾病包括但不限于痤疮、鱼鳞病、毛周角化病、毛囊角化病或汗孔角化症。
  3. 根据权利要求1和2所述的应用，其特征在于，所述PCSK9抑制剂包括但不限于PCSK9小分子化合物抑制剂、PCSK9干扰RNAi抑制剂、PCSK9单克隆抗体抑制剂、PCSK9模拟肽抑制剂、PCSK9模拟抗体蛋白抑制剂、PCSK9反义寡核苷酸抑制剂或PCSK9疫苗抑制剂。
  4. 根据权利要求3所述的应用，其特征在于，所述PCSK9抑制剂为PCSK9小分子化合物抑制剂、PCSK9干扰RNAi抑制剂或PCSK9单克隆抗体抑制剂。
  5. 根据权利要求3所述的应用，其特征在于，所述PCSK9小分子化合物抑制剂包括但不限于如式Ⅰ、式Ⅱ、式Ⅲ或式Ⅳ所示的化合物，
    Figure PCTCN2021103749-appb-100001
  6. 根据权利要求3所述的应用，其特征在于，所述PCSK9单克隆抗体抑制剂包括但不限于Abcam公司的抗体ab84041，或依洛尤单抗，或阿利西尤单抗，或重组人源化抗PCSK9单克隆抗体JS002，或重组全人源抗PCSK9单克隆抗体，或PCSK9单克隆抗体LY3015014。
  7. 根据权利要求3所述的应用，其特征在于，所述的PCSK9干扰RNAi抑制剂包括但不限于Inclisran注射剂、ALN-PCS注射剂或ALN-PCSsc注射剂。
  8. 根据权利要求3所述的应用，其特征在于，所述的PCSK9模拟肽抑制剂和PCSK9模拟抗体蛋白抑制剂包括但不限于Abcam公司的多肽ab32727，或模拟抗体蛋白DS9001，或PCSK9人抗体-抗原结合片段1G08。
  9. 根据权利要求3所述的应用，其特征在于，所述PCSK9反义寡核苷酸抑制剂包括但不限于Santaris Pharma公司的SPC5001；所述PCSK9疫苗抑制剂包括但不限于PCSK9疫苗AT04A或PCSK9疫苗AT06A。
  10. 根据权利要求1所述的应用，其特征在于，所述PCSK9抑制剂单独使用或联合其他治疗药物用于制备治疗白癜风、脱发、疤痕、肺纤维化或角化异常性疾病的产品。
  11. 根据权利要求10所述的应用，其特征在于，所述PCSK9抑制剂选自PCSK9小分子化合物抑制剂、PCSK9干扰RNA、PCSK9单克隆抗体、PCSK9模拟肽、PCSK9模拟抗体蛋白、PCSK9反义寡核苷酸或PCSK9疫苗。
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