CN116173063A - Application of MLKL inhibitor in preparation of preparation for promoting hair growth or improving androgenetic alopecia - Google Patents
Application of MLKL inhibitor in preparation of preparation for promoting hair growth or improving androgenetic alopecia Download PDFInfo
- Publication number
- CN116173063A CN116173063A CN202310384442.XA CN202310384442A CN116173063A CN 116173063 A CN116173063 A CN 116173063A CN 202310384442 A CN202310384442 A CN 202310384442A CN 116173063 A CN116173063 A CN 116173063A
- Authority
- CN
- China
- Prior art keywords
- mlkl
- hair growth
- nsa
- hair
- mice
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101001011663 Homo sapiens Mixed lineage kinase domain-like protein Proteins 0.000 title claims abstract description 67
- 102100030177 Mixed lineage kinase domain-like protein Human genes 0.000 title claims abstract description 67
- 230000003779 hair growth Effects 0.000 title claims abstract description 54
- 239000003112 inhibitor Substances 0.000 title claims abstract description 23
- 201000004384 Alopecia Diseases 0.000 title claims abstract description 22
- 230000001737 promoting effect Effects 0.000 title claims abstract description 22
- 206010068168 androgenetic alopecia Diseases 0.000 title claims abstract description 8
- 201000002996 androgenic alopecia Diseases 0.000 title claims abstract description 7
- 238000002360 preparation method Methods 0.000 title claims description 10
- 230000005764 inhibitory process Effects 0.000 claims abstract description 16
- 101100237799 Mus musculus Mlkl gene Proteins 0.000 claims 1
- 238000009472 formulation Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 abstract description 56
- 210000003780 hair follicle Anatomy 0.000 abstract description 27
- 241000699666 Mus <mouse, genus> Species 0.000 abstract description 24
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 abstract description 22
- 229960003473 androstanolone Drugs 0.000 abstract description 19
- 230000000694 effects Effects 0.000 abstract description 15
- 108090000623 proteins and genes Proteins 0.000 abstract description 14
- 102000004169 proteins and genes Human genes 0.000 abstract description 14
- 230000006907 apoptotic process Effects 0.000 abstract description 13
- 239000003814 drug Substances 0.000 abstract description 10
- 230000001338 necrotic effect Effects 0.000 abstract description 9
- 230000003698 anagen phase Effects 0.000 abstract description 7
- 230000003797 telogen phase Effects 0.000 abstract description 6
- 230000004913 activation Effects 0.000 abstract description 4
- 230000001225 therapeutic effect Effects 0.000 abstract description 4
- 238000010172 mouse model Methods 0.000 abstract description 3
- 150000003384 small molecules Chemical class 0.000 abstract description 3
- 108091000080 Phosphotransferase Proteins 0.000 abstract description 2
- 230000006872 improvement Effects 0.000 abstract description 2
- 102000020233 phosphotransferase Human genes 0.000 abstract description 2
- 230000031761 regulation of hair cycle Effects 0.000 abstract description 2
- 230000009466 transformation Effects 0.000 abstract description 2
- FNPPHVLYVGMZMZ-XBXARRHUSA-N necrosulfonamide Chemical compound COC1=NC=CN=C1NS(=O)(=O)C(C=C1)=CC=C1NC(=O)\C=C\C1=CC=C([N+]([O-])=O)S1 FNPPHVLYVGMZMZ-XBXARRHUSA-N 0.000 description 48
- 102000015735 Beta-catenin Human genes 0.000 description 22
- 108060000903 Beta-catenin Proteins 0.000 description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 21
- 210000003491 skin Anatomy 0.000 description 18
- ZFMITUMMTDLWHR-UHFFFAOYSA-N Minoxidil Chemical compound NC1=[N+]([O-])C(N)=CC(N2CCCCC2)=N1 ZFMITUMMTDLWHR-UHFFFAOYSA-N 0.000 description 15
- 210000004209 hair Anatomy 0.000 description 15
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 12
- 229960003632 minoxidil Drugs 0.000 description 11
- 108020004999 messenger RNA Proteins 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- 238000011529 RT qPCR Methods 0.000 description 9
- 231100000360 alopecia Toxicity 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 108020004459 Small interfering RNA Proteins 0.000 description 7
- 230000003660 hair regeneration Effects 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 239000013642 negative control Substances 0.000 description 7
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 7
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 6
- 230000003676 hair loss Effects 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000004055 small Interfering RNA Substances 0.000 description 6
- -1 (3-methoxypyrazinyl) amino Chemical group 0.000 description 5
- 208000024963 hair loss Diseases 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 4
- 210000002615 epidermis Anatomy 0.000 description 4
- 238000003197 gene knockdown Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 239000012452 mother liquor Substances 0.000 description 4
- 239000010413 mother solution Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 230000031774 hair cycle Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000017074 necrotic cell death Effects 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 101710156256 Myosin phosphatase Rho-interacting protein Proteins 0.000 description 2
- 102100033729 Receptor-interacting serine/threonine-protein kinase 3 Human genes 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229960004039 finasteride Drugs 0.000 description 2
- DBEPLOCGEIEOCV-WSBQPABSSA-N finasteride Chemical compound N([C@@H]1CC2)C(=O)C=C[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H](C(=O)NC(C)(C)C)[C@@]2(C)CC1 DBEPLOCGEIEOCV-WSBQPABSSA-N 0.000 description 2
- 230000003325 follicular Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000000750 progressive effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 102000004082 Calreticulin Human genes 0.000 description 1
- 108090000549 Calreticulin Proteins 0.000 description 1
- 108010066551 Cholestenone 5 alpha-Reductase Proteins 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 102100030074 Dickkopf-related protein 1 Human genes 0.000 description 1
- 206010014418 Electrolyte imbalance Diseases 0.000 description 1
- 101000864646 Homo sapiens Dickkopf-related protein 1 Proteins 0.000 description 1
- 208000037093 Menstruation Disturbances Diseases 0.000 description 1
- 206010027339 Menstruation irregular Diseases 0.000 description 1
- 101150048823 Mlkl gene Proteins 0.000 description 1
- 101000783976 Mus musculus N(4)-(beta-N-acetylglucosaminyl)-L-asparaginase Proteins 0.000 description 1
- 241000234479 Narcissus Species 0.000 description 1
- 206010033557 Palpitations Diseases 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003098 androgen Substances 0.000 description 1
- 102000001307 androgen receptors Human genes 0.000 description 1
- 108010080146 androgen receptors Proteins 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000006909 anti-apoptosis Effects 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000000916 dilatatory effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001061 forehead Anatomy 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000000442 hair follicle cell Anatomy 0.000 description 1
- 230000003741 hair volume Effects 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000003658 preventing hair loss Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000009323 psychological health Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 210000004918 root sheath Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035946 sexual desire Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- LXMSZDCAJNLERA-ZHYRCANASA-N spironolactone Chemical compound C([C@@H]1[C@]2(C)CC[C@@H]3[C@@]4(C)CCC(=O)C=C4C[C@H]([C@@H]13)SC(=O)C)C[C@@]21CCC(=O)O1 LXMSZDCAJNLERA-ZHYRCANASA-N 0.000 description 1
- 229960002256 spironolactone Drugs 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 229940098465 tincture Drugs 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/4965—Non-condensed pyrazines
- A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Cosmetics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a novel therapeutic target and a small molecule inhibitor for promoting hair growth and improving androgenetic alopecia. We find that necrotic apoptosis key molecule-mixed lineage kinase domain like protein (MLKL) plays a key role in the regulation of hair cycle, and small molecule medicine NSA is utilized to target and inhibit the activation of MLKL, so that the transformation from telogen phase to growing phase of back hair follicle of mice can be accelerated, thereby promoting hair growth. Further, we utilized dihydrotestosterone DHT to induce an AGA mouse model, in which inhibition of MLKL was found to antagonize DHT inhibition of mouse hair growth, indicating that inhibition of MLKL may have some improvement effect on AGA. Therefore, MLKL can be used as a therapeutic target for promoting hair growth and improving AGA, and an inhibitor NSA of the MLKL can target and inhibit the activity of the MLKL to promote hair growth and treat AGA.
Description
Technical Field
The invention relates to the field of biotechnology, and relates to a novel therapeutic target for promoting hair growth and improving androgenetic alopecia (androgenetic alopecia, AGA). In particular to the application of an inhibitor of targeted inhibition of necrotic apoptosis key molecules-mixed lineage kinase domain like protein (MLKL) in preparing medicaments for promoting hair growth and improving AGA.
Background
In recent years, the incidence of hair loss has increased year by year. A resident health survey shows that the resident worry about the alopecia problem is arranged at the 7 th position of the health problem, and the 'hair volume anxiety' and the baldness become dull pain of a plurality of people. With the development of socioeconomic performance, the demand for hair loss treatment is increasing. AGA is the most common type of hair loss, mainly manifested by posterior forehead hairline and/or progressive reduction and thinning of head hair, also known as male pattern baldness. Alopecia seriously affects the appearance and psychological health of patients. The mainstream research considers that AGA is converted into Dihydrotestosterone (DHT) by 5 alpha-reductase under genetic background, and the DHT is combined with androgen receptor on hair follicle to lead to hair follicle micromation, so that the progressive reduction of hair follicle density is finally caused, the pathogenesis of AGA is not completely elucidated at present, and the targeting therapy is limited, so that the pathogenesis of AGA is explored, and further the guidance of searching a targeting therapy method is particularly important.
Although AGA is a very common disease, the approved treatments have limited options and are poorly effective. The drugs approved for treating male AGA at present mainly comprise finasteride and minoxidil, but the finasteride and minoxidil can take effect after long-term use. Adverse reactions such as decreased sexual desire and impotence may occur when the use of the narcissus. Propylene glycol in minoxidil solution may cause skin allergy; high sodium may lead to water sodium storage slip; minoxidil has the effect of dilating blood vessels, and some patients may have symptoms such as palpitation and dizziness when treated with minoxidil. Spironolactone for female AGA is at risk of causing irregular menstruation and electrolyte disturbance. The hair transplantation technique can redistribute hair and improve appearance, but belongs to invasive operation, is expensive, adopts autologous single hair follicle and follicular unit for transplantation in operation, and has limited quantity and unstable survival rate.
Through the above analysis, the problems and defects existing in the prior art are as follows:
(1) In the existing method for improving alopecia, the oral and external medicines have the problems of large side effect, slow onset of action, unstable curative effect, repeated illness state after stopping the medicines and the like.
(2) In the existing method for improving alopecia, the operation treatment cost is high, and the number is limited and the survival rate is unstable because the operation adopts autologous single hair follicle and follicular unit for transplantation.
Necrosulfonamide (NSA) N- [4- [ [ (3-methoxypyrazinyl) amino group]Sulfonyl group]Phenyl group]-3- (5-nitro-2-thienyl) -2-acrylamide (Necrosulfonamide), CAS: 1360614-48-7, formula C 18 H 15 N 5 O 6 S 2 。
Necrosulfonamide inhibits MLKL-mediated necrosis by preventing the function of the N-terminal CC region of MLKL. It inhibits downstream necrosis of RIP3 activation. Necrosulfonamide did not play any role in TNF- α and Smac-mimotic-induced apoptosis in Panc-1 cells that did not express RIP3, even at concentrations up to 5. Mu.M. Necrosulfonamide is effective in inhibiting necrosis in human cells.
After searching, the following reports are found: cn20221060780. X discloses the use of ultrasound in inducing hair follicle cells and hair growth, in particular: the secretion level of dihydrotestosterone induced alopecia inducing factors DKK1 and TGF-beta 1 is inhibited by 30kHz ultrasonic wave, and proliferation and anti-apoptosis effects of human hair papilla cells hDPC and external root sheath keratinocytes are induced to treat alopecia. CN202111668170.3 discloses the use of recombinant calreticulin for hair growth, protection of hair or hair loss prevention and related products. CN201410675650.6 discloses the use of microRNA-22 in hair development and androgen-induced hair loss. CN201810683534.7 discloses a novel stem cell hair tonic for promoting hair regeneration, which specifically comprises PDGF, VEGF, KGF, wherein the KGF concentration is 856.73 μg/ml, the PDGF concentration is 89.31 μg/ml, and the VEGF concentration is 118.65 μg/ml. No relationship between NSA and AGA was found to be reported.
Therefore, in the art, by exploring influencing factors of hair cycle variation, finding signal pathways influencing hair growth, targeting key molecules to promote the transition from telogen to anagen phase, and thus finding new drugs for treating alopecia would be of great importance.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide a novel therapeutic target-necrotic apoptosis key molecule MLKL, and researches the relationship between Necrosulfonamide (NSA) and targeted inhibition of MLKL activity to promote the hair regeneration of mice and improve AGA.
The invention is realized by constructing Small-interfering RNA (siRNA) of targeted MLKL, detecting the knocking down condition of the siMLKL through RT-qPCR, injecting the siMLKL into the epidermis of a mouse in an intradermal injection mode, knocking down the expression of the MLKL in the epidermis, observing the hair growth condition, detecting the MLKL expression activation condition and the expression condition of a key molecule beta-Catenin in a Wnt signal of a hair growth related signal through RT-qPCR and Western Blot, and exploring the role of the MLKL in the hair growth of the mouse. Further, the MLKL expression activation condition and beta-Catenin expression condition are detected through RT-qPCR and Western Blot, and the targeted inhibition of the MLKL can promote the hair regeneration of mice, so that the key role of the MLKL in the hair regeneration is further verified.
The small molecular medicine for promoting the hair regeneration of the mice provided by the invention can be directly purchased, the working solution preparation method is simple, the operation is convenient, the remarkable effect is achieved, and the tendency of hair loss of the mice can be relieved and/or the hair state can be improved.
The technical scheme of the invention is as follows: the invention provides application of an MLKL inhibitor in preparation of a preparation for promoting hair growth or improving androgenetic alopecia.
Preferably, the MLKL inhibitor is simkl or NSA.
Preferably, the siMLKL is shown by sequences SEQ ID NO.1-SEQ ID NO. 6.
Preferably, the NSA concentration is 20uM-50uM.
Preferably, targeted inhibition of MLKL improves DHT inhibition of mouse hair growth.
Compared with the prior art, the invention has the advantages that:
the invention provides a new thought for applying an MLKL inhibitor NSA to hair research, and the NSA can accelerate the transition from the telogen phase of the back hair follicle of a mouse to the growing phase, thereby promoting the hair growth and up-regulating the expression of a hair growth key signal molecule beta-Catenin.
In addition, the medicine is used for an AGA mouse model, and the targeted inhibition MLKL is found to improve the inhibition effect of DHT on mouse hair growth, so that the targeted inhibition MLKL has a certain improvement effect on AGA, and a new direction is provided for preparing the medicine for promoting hair growth and improving AGA.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows RT-qPCR detection of knockdown of different siMLKL in HaCaT cells.
Fig. 2 is a graph of experimental results of the promotion of hair growth by knockdown of necrotic apoptosis key molecule MLKL in mice, wherein (a) groups of mice, after dehairing, injected intradermally with simkl, were photographed on days 0,8, 11, 13, 15 of intervention, 5 mice per group, for a total of 20 mice. External NSA promoted a shift from telogen to anagen in C57 mice compared to negative controls. (B) Hematoxylin and eosin (H & E) -stained tissue sections of transverse and longitudinal sections showed an increase in the number and density of hair follicles in NSA-treated mouse skin (scale: 1 mm). (C) cross-sectional hair follicle counts for different treatment groups. (D, E) mRNA expression of MLKL, beta-Catenin was changed in hair follicles of siMLKL, siNC treated mice compared to control. (F, G) protein expression changes of MLKL, p-MLKL, beta-Catenin, GAPDH were analyzed in the hair follicles of the siNC-treated mice compared to the control.
Fig. 3 is a molecular formula diagram of NSA.
FIG. 4 is a graph of experimental results of the effect of NSA on the promotion of hair growth in mice, wherein groups of mice after (A) dehairing were photographed on days 0,8, 11, 13, 15 of intervention, 5 mice per group, for a total of 20 mice. External NSA promoted a shift from telogen to anagen in C57 mice compared to negative controls. (B) Hematoxylin and eosin (H & E) -stained tissue sections of transverse and longitudinal sections showed an increase in the number and density of hair follicles in NSA-treated mouse skin (scale: 1 mm). (C) cross-sectional hair follicle counts for different treatment groups. (D, E) mRNA expression of MLKL, beta-Catenin in hair follicles of mice treated with 20uM-NSA, 50uM-NSA, as compared to control. (F, G) Western blot analysis of MLKL, p-MLKL, beta-Catenin, GAPDH was analyzed in mice hair follicles treated with 20uM-NSA, 50uM-NSA compared to control.
Fig. 5 is a graph of experimental results of NSA improving DHT inhibition of mouse hair growth, wherein (a) the back skin photographs of mice from different treatment groups on days 0,8, 11, 13, 15, the external NSA promoted C57 mouse hair growth, while DHT inhibited hair growth, compared to the negative control. The nsa+dht group treated mice showed an increased trend in hair growth compared to DHT treated mice. (B) Cross and longitudinal sections hematoxylin and eosin (H & E) stained tissue sections showed the number and density of mouse hair follicles. (C) cross-sectional hair follicle counts for different treatment groups in Panel B. (D) mRNA expression levels of MLKL in hair follicles of mice in different treatment groups. (E, F, G, H) compared to the control, the WB assay results of MLKL, p-MLKL, β -Catenin, GAPDH were analyzed in mice hair follicles treated with 50uM NSA, DHT,50uM NSA+DHT, minoxidil.
FIG. 6 is a diagram of a specific experimental scheme.
Description of the embodiments
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Examples
1. Experimental protocol:
the application embodiment of the invention provides an application of a small molecule inhibitor for promoting the hair regeneration of mice, which comprises the following steps:
1. the configuration mode of the inhibitor is as follows: NSA is an inhibitor of necrotic apoptosis key molecule MLKL purchased from Selleck company, and has chemical formula of C18H15N5O6S2 shown in figure 3,
2. to investigate whether targeted inhibition of MLKL has a promoting effect on mouse hair growth, we constructed a 7 week old male C57 mouse back dehairing model, and after dehairing the back skin of the mice, respectively injecting simkl and a negative control siNC intradermally at a dose of 2nmol/20g body weight mice twice weekly with a frequency of 3% minoxidil topically as a positive control once daily. Taking pictures every other day to observe the hair growth condition of the mice, taking skin tissues of the mice on days 15-17, detecting mRNA expression levels of MLKL and a hair growth key signal molecule beta-Catenin by qPCR, detecting protein levels of MLKL, P-MLKL and beta-Catenin by WB, perfecting a cross section and a longitudinal section HE, and staining and observing the hair follicle number.
3. We used the drug to construct a 7 week old C57 mouse dehairing model by subcutaneous injection of 20uM,50uMNSA in saline (negative control) with 0.1% DMSO, 300uL each. The positive control group was administered 3% minoxidil tincture once daily. Taking pictures every other day to observe the hair growth condition of the mice, taking skin tissues of the mice on days 15-17, detecting mRNA expression levels of MLKL and beta-Catenin by qPCR, detecting protein levels of MLKL, P-MLKL and beta-Catenin by WB, perfecting cross section and longitudinal section HE, and observing the hair follicle number by dyeing.
4. Similarly, 7-week-old C57 mice were selected for back dehairing, and 50uMNSA,0.3ug/ml DHT,50uMNSA+0.3ug/ml DHT were subcutaneously injected with 0.1% DMSO, followed by re-examination of the index.
SiRNA sequence:
primer sequence:
the preparation method of the MLKL inhibitor for promoting the hair regeneration of the mice comprises the following steps:
NSA is an inhibitor of necrotic apoptosis key molecule MLKL purchased from Selleck company, and has a chemical formula of C18H15N5O6S2, a molecular formula shown in figure 1, and a molecular weight of 461.47.
Firstly, accurately weighing 4.6147mgNSA powder, placing the NSA powder in an EP tube, taking 1ml of sterile DMSO, adding the sterile DMSO into the EP tube, and shaking uniformly to obtain mother liquor, wherein the concentration of the mother liquor is 10mM, and when the sterile NSA powder is used, taking a certain amount of mother liquor, adding physiological saline according to the volume ratio to dilute the mother liquor, so that the concentration of a final working solution is 20uM and 50uM.
The small molecular medicine is dissolved in pure DMSO to prepare mother solution, the concentration of the mother solution is 10mM, when the solution is used, a certain amount of mother solution is taken, physiological saline is added according to the volume ratio to dilute the mother solution, the concentration of the final working solution is 20uM and 50uM, and the final working solution is injected into the back skin of a mouse dehairing model intradermally once every other day.
2. Experimental results:
evidence of the effect of the examples. The embodiment of the invention has a plurality of positive effects in the research and development or use process, and has a great advantage compared with the prior art, and the following is described with reference to data, charts and the like in the test process.
1. Screening of MLKL-targeting siRNA in HaCaT cells
To explore whether the necrotic apoptosis key molecule MLKL is involved in the regulation of hair cycle and its role in hair growth. We constructed 3 MLKL-targeted siRNA (Small-interfering RNA, siRNA) to knock down the MLKL gene in HaCaT cells and examined the mRNA and protein expression levels of MLKL in HaCaT cells by RT-qPCR to evaluate their transfection efficiency. The results showed that, compared to the control group, the necrotic apoptosis key molecule MLKL was significantly knocked down successfully in HaCaT cells, with siRNA-RIPK3-03 being most efficient in silencing expression of MLKL (P < 0.05) (see fig. 1). Therefore, we selected siRNA-MLKL-03 for subsequent experiments.
2. Effect of knockdown of MLKL on hair growth in mouse back skin
To investigate whether transfection of siMLK into C57 mice skin has a promoting effect on hair growth, we constructed a 7 week old male C57 mice back dehairing model, and after dehairing the back skin of the mice, siMLKL and negative control siNC were injected intradermally, at a dose of 2nmol/20g body weight mice, twice weekly, with topical 3% minoxidil as positive control, once daily. The back hair growth of the mice was observed by photographing every other day. Compared with the control group, the back skin of mice in the siMLK group and the minoxidil group is blackened earlier, and the skin hair growth of the siMLK group is faster than that of the siNC group. On day 15 of local intradermal injection of simkl, most of the hair of the NSA group mice was observed to be in the anagen phase earlier than the control group. Thus, intradermal injection of simkl had a promoting effect on hair growth in C57 mice, and further hair follicle counting of sections of tissue sections stained with hematoxylin and eosin (H & E) from different treatment groups found a superfluous control of hair follicle density in simkl groups, the differences were statistically significant (P < 0.05) (see fig. 2a, b, C).
Further we extracted mouse epidermal RNAs from each group, and detected mRNA expression of MLKL and β -Catenin, a key signal molecule for hair growth, in mouse epidermis after simkl transfection using RT-qPCR technique, the results showed that mRNA expression levels of MLKL in mouse epidermis were down-regulated (see fig. 2D) and β -Catenin were up-regulated (fig. 2E), the difference was statistically significant (P < 0.05) compared to control group. The protein expression levels of MLKL, P-MLKL and beta-catenin are detected by using the WB technology, and the protein expression levels of MLKL and pMLKL in the back skin of mice are found to be down-regulated, and the expression level of the key protein beta-catenin of a Wnt/beta-catenin signal pathway is up-regulated (P < 0.05) (FIG. 2F, G and H).
MLKL inhibitor NSA promotes mouse hair growth
The previous study found that intradermal injection of simkl had a promoting effect on hair growth in C57 mice. Further, in order to find a simple preparation substance to replace simkl, we first applied a necrotic apoptosis key molecule MLKL inhibitor NSA to hair research to investigate whether NSA has a promoting effect on hair growth in vivo, we constructed a 7-week-old male C57 mouse back dehairing model, dehaired mice were injected subcutaneously with 0.1% DMSO in normal saline, 20uM NSA, and 50uM NSA every other day, topically applied 3% minoxidil as a positive control, and photographed every other day to observe hair growth. Compared with the control group, the skin of the back of the mice in NSA group and minoxidil group was blackened earlier, and the skin hair growth in 50uMNSA group was faster than that in 20uM group. On day 15 of local intradermal injection of NSA, most of the hair of the NSA group of mice was observed to grow earlier than the control group (fig. 4A). Thus, NSA significantly promoted hair growth in C57 mice.
Further staining of the tissues by hematoxylin and eosin (H & E) showed that both skin hair follicle density and number of the NSA treated mice increased and that the promotion was more pronounced in the 50um msa treated mice (fig. 4A, B and C). In animal models, RT-qPCR results suggested no significant change in mRNA levels of MLKL in control and NSA groups (fig. 4D), NSA upregulated the mcan expression level of β -Catenin (fig. 4E). The protein expression levels of MLKL, P-MLKL and beta-catenin are detected by using the WB technology, no obvious change is found in the protein level of MLKL in the back skin of mice, the protein expression level of pMLKL is down-regulated, and the expression level of the key protein beta-catenin of a Wnt/beta-catenin signal channel is up-regulated (P < 0.05) (FIG. 4F, G, H).
4. MLKL inhibitor NSA improves the inhibition of DHT on mouse hair growth
The 7 week old male C57 mouse dehairing model was randomly divided into four groups of five. The mice were given their own injections of 0.1% DMSO in physiological saline, 0.3ug/ml DHT,50uMNSA and 50uM NSA+DHT subcutaneously once every other day, 3% minoxidil topically as a positive control, and were photographed for back hair growth on days 0,8, 11, 13, 15. The results showed that the skin darkening was most pronounced on day 15 in the NSA group, hair growth was more accelerated, and the minoxidil group was higher than the negative control group, while the skin remained mostly pink in the DHT group, with the nsa+dht group hair being significantly faster than the DHT group (fig. 5A). Thus, NSA can significantly accelerate the transition of hair follicles from telogen to anagen, promoting hair growth in C57 mice. Hematoxylin and eosin (H & E) stained tissues showed an increase in the density and number of NSA-treated mice and minoxidil group hair follicles compared to the control group, and an increase in the density and number of nsa+dht-treated mice hair follicles compared to DHT-treated mice (fig. 5b, c). There was no change in the mRNA level of MLKL (fig. 5D). The protein expression level of MLKL was not significantly changed in the treatment group, the protein expression level of p-MLKL was down-regulated in the NSA and NSA+DHT groups, and the protein expression level of β -catenin was up-regulated (FIGS. 5E,5F,5G, 5H).
3. Conclusion:
1. the invention discovers that the target apoptosis inhibiting key molecule MLKL can regulate and control the hair cycle for the first time, promote the transformation from the resting period to the growing period of mice, promote the hair growth and be a possible new target for treating alopecia diseases. DHT can be used to construct a mouse AGA model by causing apoptosis of hair papilla cells, resulting in abnormal hair cycle. To investigate whether MLKL inhibitors have an effect on AGA, we induced an AGA mouse model with DHT, and found that the use of the MLKL inhibitor NSA antagonizes the inhibitory effect of DHT on mouse hair growth.
2. The invention provides a new thought for using an MLKL inhibitor for promoting hair growth for the first time, and an inhibitor NSA of a necrotic apoptosis key molecule MLKL is used for researching hair, so that the MLKL inhibitor can promote hair growth and improve AGA.
The foregoing is a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to apply equivalents and modifications to the technical solution and the concept thereof within the scope of the present invention as defined in the appended claims.
Claims (5)
- Use of an mlkl inhibitor for the preparation of a formulation for promoting hair growth or improving androgenetic alopecia.
- 2. Use according to claim 1, characterized in that the MLKL inhibitor is simkl or NSA.
- 3. Use according to claim 1, characterized in that the siMLKL is represented by the sequence SEQ ID No.1-SEQ ID No. 6.
- 4. Use according to claim 1, characterized in that the NSA concentration is 20uM-50uM.
- 5. The use according to any one of claims 1-4, wherein targeted inhibition of MLKL improves DHT inhibition of mouse hair growth.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310384442.XA CN116173063A (en) | 2023-04-12 | 2023-04-12 | Application of MLKL inhibitor in preparation of preparation for promoting hair growth or improving androgenetic alopecia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310384442.XA CN116173063A (en) | 2023-04-12 | 2023-04-12 | Application of MLKL inhibitor in preparation of preparation for promoting hair growth or improving androgenetic alopecia |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116173063A true CN116173063A (en) | 2023-05-30 |
Family
ID=86442601
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310384442.XA Pending CN116173063A (en) | 2023-04-12 | 2023-04-12 | Application of MLKL inhibitor in preparation of preparation for promoting hair growth or improving androgenetic alopecia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116173063A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115154575A (en) * | 2022-07-15 | 2022-10-11 | 中南大学湘雅三医院 | Application of small molecule drug in promoting mouse hair regeneration |
| CN115721716A (en) * | 2022-07-13 | 2023-03-03 | 苏州翊鹏医药科技有限公司 | Use of MDH2 inhibitors in the treatment of androgenetic alopecia |
-
2023
- 2023-04-12 CN CN202310384442.XA patent/CN116173063A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115721716A (en) * | 2022-07-13 | 2023-03-03 | 苏州翊鹏医药科技有限公司 | Use of MDH2 inhibitors in the treatment of androgenetic alopecia |
| CN115154575A (en) * | 2022-07-15 | 2022-10-11 | 中南大学湘雅三医院 | Application of small molecule drug in promoting mouse hair regeneration |
Non-Patent Citations (1)
| Title |
|---|
| SONGFENG CHEN ET AL: ""RIPK1/RIPK3/MLKL-mediated necroptosis contributes tocompression-induced rat nucleus pulposus cells death"", 《APOPTOSIS》, vol. 22, no. 5, 31 May 2017 (2017-05-31), pages 626 - 638, XP036203861, DOI: 10.1007/s10495-017-1358-2 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2690845C2 (en) | Method for stimulating hair growth and preventing hair loss of patient, substance therefor and method for producing substance | |
| RU2470677C1 (en) | Method for stimulation of repair and trophic skin processes | |
| CN111329832B (en) | Nanometer lipid carrier microneedle for treating alopecia and application thereof | |
| CN110548031B (en) | Composition for enhancing hair growth induction ability of adipose-derived stem cells comprising udenafil as active ingredient | |
| CN109432128A (en) | Application of the dental pulp mescenchymal stem cell in curing psoriasis | |
| CN116173063A (en) | Application of MLKL inhibitor in preparation of preparation for promoting hair growth or improving androgenetic alopecia | |
| US20230144955A1 (en) | Peptide for preventing or treating hair loss, and use thereof | |
| Ozturk et al. | The effect of stromal vascular fraction for patients with androgenetic alopecia | |
| CN113876955B (en) | Use of PCSK9 inhibitors for the preparation of hair growth promoting products | |
| RU2405572C1 (en) | Method of face and neck skin rejuvenation in women | |
| RU2322250C1 (en) | Method for treating alopecia | |
| RU2612936C1 (en) | Method for treating trigeminal neuralgia | |
| CN109453294B (en) | Application of Xinmaian in promoting hair growth | |
| CN107625781B (en) | Application of miRNA inhibitor in preparation of medicine for preventing and treating myocardial infarction | |
| CN114432332A (en) | Application of circUTRN in preparation of medicine for treating heart failure, recombinant vector and medicine for treating heart failure | |
| CN103356617A (en) | Application of deacetylase inhibitors in preparation of medicaments for promoting insulin secretion | |
| Tan et al. | Concentrated growth factor from autologous platelet promotes hair growth in androgenetic alopecia | |
| KR102260252B1 (en) | Method of analyzing circadian rhythm through skin cells, method of normalizing circadian rhythm disorder, and information providing system using circadian rhythm information | |
| Wu et al. | Triamcinolone acetonide suppressed scar formation in mice and human hypertrophic scar fibroblasts in a dose-dependent manner | |
| CN115779028B (en) | Traditional Chinese medicine composition and preparation method and application thereof | |
| WO2021147740A1 (en) | Use of mapk/erk pathway inhibitor in antagonizing skin aging and radiation-induced premature aging | |
| CN108685896B (en) | Application of oroxylin A in preparation of medicine for treating and/or preventing chronic peripheral vascular occlusive diseases | |
| CN106334188A (en) | Medicine action target for treating skin melanin related diseases and whitening skins | |
| CN116421625A (en) | Biological product for promoting hair regeneration and preparation method and application thereof | |
| Marques et al. | The efficacy of using platelet-rich plasma (PRP) in the treatment of androgenic alopecia: a literature review |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |