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(12) Patent Application Publication (10) Pub. No.: US 2014/0010901 A1 Hibino Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2014/0010901 A1 Hibino Et Al US 20140010901A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0010901 A1 Hibino et al. (43) Pub. Date: Jan. 9, 2014 (54) BLEOMYCIN HYDROLASE PRODUCTION Publication Classification PROMOTOR (51) Int. Cl. A61E36/756 (2006.01) A63L/047 (2006.01) (75) Inventors: Toshihiko Hibino, Yokohama-shi (JP); A 6LX3/97 (2006.01) Shoko Yamada, Yokohama-shi (JP); A61E36/53 (2006.01) Hidekazu Fukushima, Yokohama-shi A61E36/23 (2006.01) (JP) (52) U.S. Cl. CPC ............... A61K 36/756 (2013.01); A61K 36/53 (2013.01); A61 K36/23 (2013.01); A61 K (73) Assignee: Shiseido Company, Ltd. 31/197 (2013.01); A61 K3I/047 (2013.01) USPC ........... 424/745; 424/769; 424/773; 424/777; (21) Appl. No.: 14/004,977 514/562; 514/738 (57) ABSTRACT (22) PCT Filed: Mar. 14, 2012 Provided is a novel bleomycin hydrolase production pro moter. (86). PCT No.: PCT/UP2012/056581 Provided is a bleomycin hydrolase production promoter, S371 (c)(1), natural moisturizing factor production promoter, and dry skin (2), (4) Date: Sep. 13, 2013 remedy, comprising as an active ingredient thereof one or a plurality of ingredients selected from the group consisting of (30) Foreign Application Priority Data chestnut rose extract, angelica root extract, cork tree bark extract, lamium album extract, rosemary extract, benzene Mar. 15, 2011 (JP) ................................. 2011-057126 sulfonyl GABA and erythritol. Patent Application Publication Jan. 9, 2014 Sheet 1 of 23 US 2014/0010901 A1 Fig.1 SPECIMEN 1 SPEC MEN 2 2 5 10 20 2 5 10 20 NUMBER OF TIMES OF TAPE STRIPPING Fig.2 SPECIMEN T N A M T, A: SUBJECTS NOT HAVING DRY SKIN N: SUBJECT HAVING SOMEWHAT DRY SKIN, MSUBJECT HAVING DRY SKN Patent Application Publication Jan. 9, 2014 Sheet 2 of 23 US 2014/0010901 A1 · | || ºsº&No.S,§? | --| 000|| 00‘G Patent Application Publication Jan. 9, 2014 Sheet 3 of 23 US 2014/0010901 A1 Fig.4 y - 0.2264x + 0.9652 R2 = 0.3294 Peason: r-0574 9 - manar- ---. 8 w-wawrwrmw 4-w 7 0 i 6 d 0. 5 t d st 4 - - w 2 - V 0. wrvmmam--------------------- 1 d 0. O ----------------------- O.OO 5.00 O.OO 5.00 2O,OO 25. OO BH amount Patent Application Publication Jan. 9, 2014 Sheet 4 of 23 US 2014/0010901 A1 - at x L H O l?) o * C. C. C. C dis C d do N. C. Patent Application Publication Jan. 9, 2014 Sheet 5 of 23 US 2014/0010901 A1 |ON Patent Application Publication Jan. 9, 2014 Sheet 6 of 23 US 2014/0010901 A1 Y E ; : - st 2as -a c- - S. 25 as SS i. Patent Application Publication Jan. 9, 2014 Sheet 7 of 23 US 2014/0010901 A1 **********ae, *********•••• Patent Application Publication Jan. 9, 2014 Sheet 8 of 23 US 2014/0010901 A1 O O Patent Application Publication Jan. 9, 2014 Sheet 9 of 23 US 2014/0010901 A1 Fig. 10 80% OO% 20% Ca Patent Application Publication Jan. 9, 2014 Sheet 10 of 23 US 2014/0010901 A1 Å | --HZW||— | Patent Application Publication Jan. 9, 2014 Sheet 11 of 23 US 2014/0010901 A1 Og0OO!!OGLOOZ0çZ00£ uone?uauð?IGE — olsegeIÐdEnl? Patent Application Publication Jan. 9, 2014 Sheet 12 of 23 US 2014/0010901 A1 ABSENCE OF UV IRRADAON RECOVERED 24 HOURS AFTER UW IRRADIATION AT 3On P7 RECOVERED 24 HOURS AFTER UV IRRADATION AT 60 RECOWERED 48 HOURS AFTER UN IRRADATION AT 30m RECOVEREDRRAD TATION 48 AT HOURS 60m AFTER LIV Patent Application Publication Jan. 9, 2014 Sheet 13 of 23 US 2014/0010901 A1 ---}N_LJO193JJE?) O| 0 (0%) uoissed dxa e Allee Patent Application Publication Jan. 9, 2014 Sheet 14 of 23 US 2014/0010901 A1 Fig.15 -earms...wor NAME SEQUENCE BH-1216 Koni (forward) 5 -CCGGGACCATCAGAGTTCCTTAGA A-3 BH-1016 Kon (forward) 5 -CCGGGTACCCAAGGTTTTTACAATCT-3 BH-86 K pn I (forward) 5 -CACGGTACCTGGGTAGTGTTCTTGAA - 3 BH-616 KonI (forward) 5 -CCAGGTACCTCCTTGTGACATATCGA - 3 BH-444 Kpni (forward) 5-AATGGTACCTTGGAGCGGGCCTGA-3 BH-216 Kipni (forward) 5 -AATGGTACCAGGGGGGAGTTTTGTCC - 3 BH-171 KpnI (forward) 5 - AATGAAGGT ACCCAGCCTCCCCGCCG - 3 BH-134 KpnI (forward) 5 - ACGGGTACCAGCCGGTTTCCTTTTTC - 3 BH-105 KpnI (forward) 5 -AATGGTACCGCGAGAGACAGGTCG - 3 BH+1 MUI (reverse) 5 -TAAA (ACGCGTTGGCGCCCACGCTGCCG - 3 -re re-em-ul Patent Application Publication Jan. 9, 2014 Sheet 15 of 23 US 2014/0010901 A1 Fig.16 PROBE SEQUENCE (sense) 5-TCTCCCAGCCTCAGTCTCCCAGCCTCAG-3 (anti-sense)5'- AGAGGGTCGGAGTCAGAGGGTCGGAGTC-3 www.wam. wrm-wiwww.wrwom (sense)5-CGCGAGGGGGGAGTTCGAGGGGGGAGTTT-3 (anti-sense) 5’-GCGCTCCCCCCTCA AGCTCCCCCCT CAAA-3 (sense) 5'-CCGGTTTCCTTTTTCGGGGTTTCCTTTTC-3 IRF-1/2 S (anti-sense) 5’-GGCCAAAGGAAAAAGCGCCAAAGGAAAAAG-3 (sense) 5’-GCAGCGCAATCCCGGCAGCGCAATCCCGGC-3' (anti-Sense) 5’-CGTCGCG ACCGCCGTCGCGTAGGGCCG-3 Man-ul- ------ -------all--------- Patent Application Publication Jan. 9, 2014 Sheet 16 of 23 US 2014/0010901 A1 Fig.17 GENE SEQUENCE ANNEALNG (forward) 5’-TGTGGTTTGGCTGTGATGTT-3 y BH (reverse) 5-GCACCATCCTGATCATCCTT-3 55 °C (forward) 5’-ACATGGAGGCCATCACTTTC-3 Calpan-I 55"C (reverse) 5’-GGTCCACGTGTTCCACTCT-3 (forward) 5’-AGCGACCAAGATCACTCCAT-3 (reverse) 5’-TGGGTGACT CAATTCTGCTG-3 58C (forward)5-TAGAGCCCTTGoTCACGTTT-3 M7F- 58C (reverse) 5’-GGGCATTGTCTAGGTGGAAA-3 IRF-1 k 58 "C (reverse) 5’-GCTCTTAGCATCTCGGCTGG A-3 (forward) 5’-TGGATGCATGCGGCTAGA-3 IRF-2-k 58 C (reverse) 5’-CATCTGAAATTCGCCTTCC-3 (forward) 5-ATTGTCAGTA AACGGGCAGG-3 GATA-1 (reverse) 5-TCTGAAT ACCATCCTTCCGC-3 Patent Application Publication Jan. 9, 2014 Sheet 17 of 23 US 2014/0010901 A1 Fig.18 A AF-2/Sp-1 Sp- IAP-2 -24 GATA-1" ' ".MZF-1 NF-Y. AF235; AP256 Sp-1.Sp-1/MZF-1 -56 IRF-1/2 - 20 B pG 3-1216 5assacagascar-Luc 3. G. 3-86 S3: is Lucas pG/3-616 al pGL3-444 liteLuc pG/3-216 unt Luc 1. N DIFFERENTIATED (2nM Ca2+) pG/3 -105 Luc D PROL. FERATING pG/36asic Luc O 100 20 O 3 OO 4OO 500 C -216 -17 5'-AGGGGGGAGTTTTGTCCCTCCGCGGACCCCGTTTCCCCAGCCTCAGCCT MZF- -134 Sp CCCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGAGCCGGTTCCTTTTTCC Sp-1/MZF-1 IRF-1/2 - 105 GGCGCTCCGGTGCGAGAGACAGGTCGGGCCCCCTAGGCAGCGAGCCGCAG CGCAATCCCGGCGCTCGCCCAAGGACCCTGGAAGCTACCGTTACCCCGCC GATA-1/2 Sp-1/AP-2 GGCAGCGTGGGCGCoA-3 Patent Application Publication Jan. 9, 2014 Sheet 18 of 23 US 2014/0010901 A1 V A - n A pCL3-616 del MZF-1 '''''' Luc ApGL3-616 del Sp-1r---LucC -616 de IRF-1/2" O 20 40 60 8O 1 OO RELATIVE LUCIFERASE ACTIVITY (8) B Sp-1 IRF-1/2 GAIA-1 - 72 - 12O -6 1 2 2 | HO PROBE 2. HOT PROBE+COLD PROBE Patent Application Publication Jan. 9, 2014 Sheet 19 of 23 US 2014/0010901 A1 Fig.20 A in- 12 186 g 14 ss. O.8 12 is . ;3 O6 O.8 re. 0.4 3. 0.2 a O2 Ot-to-risis. Ola Lyly", O IFN- Y (ng/ml) IL-17 A/F (ng/ml) sy B 5 is ; it 3 pol-216 H Luc AG3-216 . i UC OG/3-2I 6 8 2G13-2 6 L C O 20 40 60 80 100 120 RELATIVE LUCIFERASE ACTIVITY (%) (+IFN-r 100mg/ml O 20 AO 6O 8O OO 120 140 RELATIVE LUCIFERASE ACT WITY (96) C | 20 4 1 OO a 3.5 2 - 3 s 80 E 2.5 is 60 is a 2 as 3 40 E3uS 1.5 :: 20 1 -- () Cru O.5 CONTROL IRF- IRF-2 Opolf StRNA siRNA siRNA S RNA siRNA S RNA Patent Application Publication Jan. 9, 2014 Sheet 20 of 23 US 2014/0010901 A1 A 9 3 8 a 7 6 2 35 5 s:Fis 3 L1 2 W O Olivost CONFLUENCE B 2. soc.oo.3 80%. 100%l 20% C CONFLUENCE CONFLUENCE CONFLUENCE 3 2 1 80%. 100% 20% Ca2* CONFLUENCE CONFLUENCE Patent Application Publication Jan. 9, 2014 Sheet 21 of 23 US 2014/0010901 A1 Fig.22 A 14 O 120 E 100 3 e 2s 80 60 r- c1 40 20 in O - or is of the IFN- Y (ng/ml) IFN- r (ng/ml) B in 4 3 C. 'Égs 2 g; 2 - 2 g 1 - - E8 . 11. O 1 1 0 1 OO O 1 1 0 1 OO IL-4(ng/ml) IL-4(ng/ml) 12 12 G 10 10 - 0.8 0.8 0.6 O6 3 s: O4 0.4 It ai O. 2 O lists O - list, IL-4(ng/ml) IL-4(ng/ml) Patent Application Publication Jan. 9, 2014 Sheet 22 of 23 US 2014/0010901 A1 A SKIN OF HEALTHY VOLUNTEERS AD NON-LESONA SKN AD ESONAL SKN CONTROL NON- ESONAL (n-20). LESIONAL SKIN SKN (nat16) (n-6) Patent Application Publication Jan. 9, 2014 Sheet 23 of 23 US 2014/0010901 A1 Fig.24 EFFECTS ON EXPRESSION OF BH mRNA ?e ??S 92,2%@*$$````````(º:*QRRRR 49.3%??NSSSSSSSSSSSSS ?ºšº?????????????????????j 2,3%SSSSSSSSSSSS US 2014/0010901 A1 Jan. 9, 2014 BLEOMYCIN HYDROLASE PRODUCTION does not necessarily mean that the expression of filaggrin PROMOTOR decreases dramatically in the skin of atopic dermatitis patients. TECHNICAL FIELD PRIOR ART DOCUMENTS 0001. The present invention provides a bleomycin hydro lase production promoter, and a natural moisturizing factor Non-Patent Documents production promoter or a dry skin remedy comprising the SaC. 0007. Non-Patent Document 1: Blank, I. H., J. I. Derma tol., 18, 433 (1952) BACKGROUND ART 0008. Non-Patent Document 2: Blank, I. H., J. I. Derma tol., 21, 259 (1953) 0002 Keratin fibers of the epidermal granular layer aggre 0009. Non-Patent Document 3: Horii, I., et al., Br. J. Der gate by binding to a protein referred to as filaggrin during matol., 121, 587-592 (1989) keratinization, creating a specific form referred to as a “kera 0010. Non-Patent Document 4: Tanaka, M., et al., Br. J. tin pattern'.
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