USOO9084734B2
(12) United States Patent (10) Patent No.: US 9,084,734 B2 Collier et al. (45) Date of Patent: Jul. 21, 2015
(54) PEPTIDE PERSONAL CARE COMPOSITIONS (52) U.S. Cl. AND METHODS FOR THEIR USE CPC. A61 K8/64 (2013.01); A61K 8/66 (2013.01); A61O 1/02 (2013.01); A61O I/04 (2013.01); (75) Inventors: Katherine D. Collier, Los Altos, CA (US); Anthony Day, San Francisco, CA (Continued) (US); Hans de Nobel, Heemstede (NL); (58) Field of Classification Search David A. Estell, San Francisco, CA None (US); Grant C. Ganshaw, Tracy, CA See application file for complete search history. (US); Marc Kolkman, Oegsteest (NL); (56) References Cited Raj Lad, San Mateo, CA (US); Jeffrey V. Miller, Menlo Park, CA (US); U.S. PATENT DOCUMENTS Christopher J. Murray, Soquel, CA 3,755,560 A 8/1973 Yancey et al. (US); Scott D. Power, San Bruno, CA 12/1975 Laughlin et al. (US); Brian Schmidt, Half Moon Bay, 3,929,678 A CA (US); Anita van Kimmenade, San (Continued) Bruno, CA (US); Gudrun Vogtentanz, Sunnyvale, CA (US) FOREIGN PATENT DOCUMENTS Assignee: EP O O65, 193 5, 1982 (73) Danisco US Inc., Palo Alto, CA (US) EP 776 657 11, 1996 (*) Notice: Subject to any disclaimer, the term of this (Continued) patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 2284 days. Altschul, et al., J. Mol. Biol. 215:403-410 1990 “Basic Local Align (21) Appl. No.: 11/919,717 ment Search Tool'. (22) PCT Fled: Apr. 25, 2006 (Continued) (86) PCT NO.: PCT/US2006/O15711 Primary Examiner — Carlos Azpuru S371 (c)(1), (74) Attorney, Agent, or Firm — Danisco US Inc. (2), (4) Date: Aug. 25, 2011 (57) ABSTRACT The present invention provides peptides and Supported pep (87) PCT Pub. No.: WO2O06/121610 tides for treating various diseases and conditions. In particu PCT Pub. Date: Nov. 16, 2006 larly preferred embodiments, the present invention provides compositions and methods for personal care. In some (65) Prior Publication Data embodiments the present invention provides compositions US 2012/OO 14885 A1 Jan. 19, 2012 for use in skin and/or hair care, as well as cosmetic compo sitions. IN alternative particularly preferred embodiments, Related U.S. Application Data the present invention provides peptides and Supported pep (60) Provisional application No. 60/678,601, filed on May tides for treating diseases of the skin, such as rosacea. In some 5, 2005. particularly preferred embodiments, the Supported peptides of the present invention are anti-VEGF peptides. In alterna (51) Int. C. tive particularly preferred embodiments, the anti-VEGF pep A6 IK 8/02 (2006.01) tides are expressed on a scaffold protein. In some most pre A6 IK 8/64 (2006.01) ferred embodiments, the scaffold protein comprises BBI. (Continued) 31 Claims, 27 Drawing Sheets
(E3:28 YNLYgWT- (SEC) NO:1) CK3282 TLWPTFW (SEQ NO:2) CK3283 -NripHW (SEC) NO:3) E328i SLWAFW (SEQ NO:4) C328 -APWNSI (SEQ iD NO:5) CK328i -APWNTHT (SEQED NO:6) CK3289 -TLWPSYW (SEQED NO:7) Cossess W. W.
YNLYGWT(5) CK3728i (SEQED NO:8) APWNSHI (2) CK37286 (SEQ D NO9) Ariasi (2 cK37287 (SEQ NC:0
TLWPTFW cK37282 (SEQ ID NO:2) rwessw (5) cK37289 (SECR D NO:11) Ngweir CR37283 (SEC) NO:3) SEWPAFW (2 CK37284 (SEQ NO:2) US 9,084,734 B2 Page 2
(51) Int. Cl. Derian et al., Cell Growth Different, 8:743-749 1997). A6 IK 8/66 (2006.01) Devereux et al., Nuc. Acids Res., 12:387 (1984). “A Comprehensive A61O I/02 (2006.01) set of sequence analysis programs for the VAX'. A61O I/04 (2006.01) Ferrariet al., J. Bact. 170:289-295 (1988 “Transcription of Bacillus A61O I/10 (2006.01) subtilis Subtilisin and Expression of Subtilisin in Sporulation A61O 702 (2006.01) Mutants. A61O 9/00 (2006.01) Hahn et al., Mol. Microbiol., 21:763-775 (1996 “Regulatory inputs A61O 15/00 (2006.01) for the synthesis of ComK, the competence transcription factor of A61O 1704 (2006.01) Bacillus subtilis'. A61O 19/02 (2006.01) Hengen, TIBS 20:285-286 (1995 “Methods and reagants'. A61O 19/04 (2006.01) Henikoffet al., Proc. Natl. Acad Sci. USA 89:10915 1989. A61O 19/10 (2006.01) Henner et al., J. Bact. 170: 296-300 (1988 "Location of the Targets C07K 7/06 (2006.01) of the hpr–97, sacU32(hy), and sacQ36(Hy) Mutations in Upstream Regions of the Subtilisin Promoter'. (52) U.S. Cl. Higgins and Sharp, Gene 73:237-2441988)"Clustal a package for CPC. A61O 1/10 (2013.01); A61O 702 (2013.01); performing multiple sequence alignment On a microcomputer'. A61O 9/00 (2013.01); A61O 15/00 (2013.01); Kajino at al., Appl. Env, Microbiol. 66:638-642 2000 “A Protein A61O 1704 (2013.01); A61O 19/02 (2013.01); Disulfide Isomerase Gene Fusion Expression System That Increases A61O 19/04 (2013.01); A61O 19/10 (2013.01); the Extracellular Productivity of Bacillus brevis". C07K 7/06 (2013.01) Karlin et al., Proc. Natl Acad. Sci USA90:5873 |1993]: “Applica tions and statistics for multiple high-scroinf segments in molecular (56) References Cited sequences.'. Kennedy, Am. J. Clin. Neutr., 68: 1406S-14 12S 1998 “The Bow U.S. PATENT DOCUMENTS man-Birk inhibitor from soybeans as an anticarcinogenic agent'. Landon, Meth. Enzymol. 47: 145-149 (1977 "Cleavage at Aspartyl 4,152,416 A 5/1979 Spitzer et al. 4.421,769 A 12/1983 Dixon et al. Prolyl Bonds”. 4,683, 195 A 7, 1987 Mullis et al. Laskowski and Kato, Ann. Rev. Biochem., 49:593-626 1980) “Pro 4,683.202 A 7, 1987 Mullis et al. tein inhibitors of proteinase'. 4,937,370 A 6, 1990 Sabatelli Linet al., Eur, J. Biochem., 212:549-555 (1993. “The 0.25-mm X-Ray 4,965,188 A 10, 1990 Mullis et al. structure of the Bowman birk-Tipe inhibitor from mung bean in 4,999,186 A 3, 1991 Sabatelli et al. ternary complex with porcine trypsin'. 5,011,681 A 4, 1991 Ciotti et al. Livingstone and Barton, Comput. Appl. Biosci., 9:745-756 (1993 5,073,371 A 12, 1991 Turner et al. "Protein sequence alignments. a strategy for the hierarchical analy 5,073.372 A 12/1991 Turner et al. sis of residue conservation". 5,087,372 A 2/1992 Toyomoto et al. Matthes et al., EMBO J., 3:801-805 (1984 “Simultaneous rapid 5,411,873. A 5, 1995 Adams et al. chemical synthesis of over one hundred oligonucleotides On a 5,429,950 A 7, 1995 Power et al. microscale'. 5,679,543 A 10, 1997 Lawlis 5,827,508 A 10, 1998 Tanner et al. Meima et al., J. Biol. Chem., 277:6994-7001, 2002 “The bdbDC 5,935,556 A 8, 1999 Tanner et al. Operon of Bacillus subtilis Encodes Thiol-disulfide Oxidoreductases 5,968,485 A 10, 1999 Robinson Required for Competence Development'. 5,972,316 A 10, 1999 Robinson Morinaga et al., Biotechnol. 2:646-649 (1984 “Improvement of 6,063,611 A 5/2000 VanSolingen et al. Oligonucleotide-Directed Site-Specific Mutagensis. Using Double 6,537,968 B1 3/2003 Ledey et al. Stranded Plasmid DNA. Neidhardt et al., J. Bacteriol., 119: 736-747 1974). FOREIGN PATENT DOCUMENTS Nelson and Long, Anal. Biochem., 180: 147-151 (1989) “A General Method of Site-Specific Mutagensis. Using a Modification of the WO WO96,03964 2, 1996 Thermus Aquatics Polymerase Chain Reaction'. WO WO 98.22085 5, 1998 Odani and Ikenaka, J. Biochem., 71: 839-848 1972 "Studies on WO WO98,22085 5, 1998 Soybean Tryspin Inhibitors'. WO WOOO,06110 2, 2000 Paineet al., J. Invest. Dermatol., 116:587-595 2001“An Alternative WO WOOO,24372 4/2000 Approach to Depigmentation by Soybean Extracts via Inhibition of WO WOO 1/74317 10, 2001 the PAR-2 Pathway'. WO WOO1,794.79 10, 2001 WO WOO2/O83O88 10, 2002 Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444-2448 1988 WO WOO3,O72049 9, 2003 “Improved tools for biological sequence comparison'. WO 2005046709 * 5/2005 Sagarin, Cosmetics, Science and Technology, 2nd Edition, vol. 1, pp. WO WO2005/046709 5, 2005 32-43 (1972 “Emollient Creams and Lotions'. WO WO2005/0473O2 5, 2005 Sahu et al., J. Immunol. 157: 884-891, 1996 “Inhibition of Human Complement by a C3-Binding Peptide Isolated from a Phage-Dis OTHER PUBLICATIONS played Random Peptide Library'. Sarkar and Sommer, Biotechn., 8:404–407 1990. Beucage et al., Tetrahedr. Lett., 22:1859-1869 1981 Sayre et al., J. Soc. Cosmet. Chem., 41: 103-109 1990 “Physical "Deoxynucleoside Phosphoramidites—A New Class of Key Interme sunscreens'. diates for Deoxypolnucleotide Synthesis'. Shaw et al., J. Mol. Biol. 320:303-309|2002 “Novel Combination of Billings et al., Pro. Natl. Acad. Sci., 89:3120-3124 (1992). Two Classic Catalyic Schemes". Birk, Int. J. Pept. Protein Res., 25:113-131 (1985. “The Bowman Song et al., J. Mol. Biol., 275:347-63 (1998 “Kunitz-type Soybean Birk inhibitor. Trypsin Inhibitor Revisited Refined Structure of its Complex with Bode and Huber, Eur, J. Biochem. 204:433-451 1992). “Natural Porcine Trypsin Reveals an Insight Into the Interaction Between a protein proteinase inhibitors and their interaction with proteinases". Homologous Inhibitor From Erythrina Cafra and Tissue-type Chen et al., J. Biol. Chem., 267: 1990-1994 1992. Plasminogen Activator'. Chou et al., Proc. Natl. Acad. Sci. USA 71:1748-1752 (1974): “Non Taylor, J. Theor. Biol., 119:205-218 1986) “The Classification of Selective Inhibition of Transformed Cell Browth by a Protease Inhibi Amino Acid Conservation'. for ss. Ullrich & Schlessinger, Cell 61:203-212 1990). US 9,084,734 B2 Page 3
(56) References Cited display of conformationally constrained peptides.” Protein Engi neering 12(9): 797-806, 1999. OTHER PUBLICATIONS Fernandez-Carneado, J., et al., "Surface Grafting onto Template Assembled Synthetic Protein Scaffolds in Molecular Recognition.” van Tilbeurgh et al., Meth. Enzymol. 160:45-59 1988 “Flurogenic Biopolymers 55(6): 451-458, 2000. and Chromogenic Glycosidases as Substrates and Ligands of Car Murphy, W.L., et al., “Sustained release of vascular endothelial bohydrates'. growth factor from mineralized poly(lactide-co-glycolide) scaffolds for tissue engineering.” Biomaterials 21:2521-2527, 2000. Voss et al., Eur, J. Biochem. 242: 122-131 1996 "Crystal structure Tsunogae, Y., et al., “Crystallization of Bowman-BirkType Protease of the bifunctional soybean Bowman-Birk inhibitor at 0.28-nm reso Inhibitor (Peanut) and Its Complex with Trypsin.” J. Biochem. 100: lution '. 243-246, 1986. Werner & Wemmer, Biochem., 31:999-1010 1992 “Three-Dimen International Search Report and the Written Opinion of the Interna sional Structure of Soybean Trypsin/Chymotrypsin Bowman-Birk tional Searching Authority for International Application No. PCT/ Inhibitor in Solution'. US2006/015711 mailed Dec. 5, 2006. Yavelow et al., Cancer Res. (Suppl.) 43:24.54s-2459s 1983 “Bow International Preliminary Report on Patentability for International man-Birk Soybean Protease Inhibitor as an Anticarcinogen'. Application No. PCT/US2006/015711 mailed Nov. 6, 2007. Yavellow et al., Proc. Natl. Acad. Sci. USA 82:5395-5399 (1985 Siemeister, et al., “The pivotal role of VEGF in tumor angiogenesis: “Nanomolar Concentrations of Bowman-Birk soybean protease Molecular facts and therapeutic opportunities'. Cancer and inhibitor suppress x-ray induced transformation in vitro". Metastasis Reviews, 17:241-8. Christmann, A., et al., “The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface * cited by examiner
U.S. Patent US 9,084,734 B2
US 9,084,734 B2
3
Resp. Diff. U.S. Patent Jul. 21, 2015 Sheet 4 of 27 US 9,084,734 B2
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Resp. Diff. U.S. Patent Jul. 21, 2015 Sheet 5 Of 27 US 9,084,734 B2
irker d His Tag (409) ...ifkar OOC loop E loop D is pCB04WT phagemid plasmid ria Ciarase 5070 bp Loop AS ra (332SN CA Spe (3228}
Signal
Peptide
linker N is ag-N linker -
Cated Beta lacta?iase Gere I pME22-N terminal Stuffer
Ebs (328O - CA Stifer Sequence Bbs (32271 sts?
Truncate? Sequence U.S. Patent Jul. 21, 2015 Sheet 6 of 27 US 9,084,734 B2
Amber TAG Stop-N s His tag irker
CA CK3728-ys
Signé.
Sequence U.S. Patent Jul. 21, 2015 Sheet 7 Of 27 US 9,084,734 B2
9"501-1
U.S. Patent Jul. 21, 2015 Sheet 9 Of 27 US 9,084,734 B2
& WFG-3A O pCMO4-library / / - 150 A BA f : -100 s 3 or S O
OOC 0.01 io i
im 1000
8)
8 w
ACO
1000 10000 ligand, nM
U.S. Patent Jul. 21, 2015 Sheet 10 of 27 US 9,084,734 B2
U.S. Patent Jul. 21, 2015 Sheet 11 of 27 US 9,084,734 B2
for SS 23
O 200 400 6. 800 { ine, Sec FIG 11
Peptide inhibition (48,80) 100 |------CK37283 - - - CK3728i |-e-CK37282 H-a-Ab U.S. Patent Jul. 21, 2015 Sheet 12 of 27 US 9,084,734 B2
U.S. Patent Jul. 21, 2015 Sheet 13 Of 27 US 9,084,734 B2
s s F. a or r= r - a a e
s
Fi. iii. apr E proilater region.
cort - a -a -s are are: aaa M
Aircrccan is is CAAAATA ACAATCG (ACCAA api E promoter region
ACGAGCC AAAAAAGCC CGCCCCIG CAAATCGGA, GCCGCA aprE promoter region
AAAAICCCG AAGEA AACACCCCC CAATGccGee cecaTCTGAT api E prototer region
apri promoter region Trcaron arccorrector ArcaGa Anacrifar api E promoter region 2. carcargor TGAAAAAATArcaccataan arcCarter circaceae aprE promoter region cacaccago Toronic Gartric concert ToccGGGC aprE promoter region 35 caccaccar TAAccTAAAAAAccAreacArcaccata arcacar api E prototer region 4. ) AccArcror ArcGrc incretar GAAAAAGT ArcGaGrc apri promoter region
45 TCACGGAAA, AGAGAA CAAFACT AAAAAGA AAAACARE
U.S. Patent Jul. 21, 2015 Sheet 14 of 27 US 9,084,734 B2
ap:E promoter region CAAAAAAAG GCACAA AAAAC CACEAA AAAAACA apri profiloter region
s 5 CAGAAAGC AAGAA GCACCG AAA AAAGGAGAGG Apri: signal peptide agrS promoter region
R S K K S , , F is 6 GAAAGAGE AAACAAAA AAGGA, AGC (CGEAA Ari signal peptide
o F A. S A A D D 85 CGIAAFC ACGAGCCG CA AEA, CGCGCA (CCGAGA BCE 3
Y S W W E. E. H. G S S N G E TO ACAG. AGAGGAACA. GGCAACTA AGATAGA ACGGAAT BCE 3 Noor V N E R G E W Q , K. G. M. S so T AGAAGAA CGAGGCGAA AAGCAGE AAAAGGGAE. ACCCCAG BCE. S --~ • I. C. W. Y G E. W. N. Y E S K 8 GAAG GACGGAA GTAAACT AGAAAGA GAAAGGCA
3CEO3
R. D. O. it R. A. A Y S S 3. AAGAGA (GGGGAATAAC GATSCCA (CAGCAA AACE CC
BCE 3
a S J K E. E. W. K 901 AGAGGATA: ATTGACGATC CATAGTAAA (GAAAAAGTA AAAGAGACG
BCE 3
* E . Aa. D G. : y : 9 S. (AGGCEC GATAGACC GGEAAA (ACARA GGAAC
U.S. Patent Jul. 21, 2015 Sheet 15 Of 27 US 9,084,734 B2
BCE (3
S. D. N. N. Y K E. E. A. K. F Oi TCACACA ACACCCGAA 'AATAAAA (AACAAGCCA ACACT
ECE 3
a E i? S E Y Y I Y e (5 GAAAAC, CACACIC ACCACACA CCGAACTG AIATAC(AAA BCE. 3
o A. N. E. S. G. S W N N O I K 10 GCAAAGA ACCGAAG AGAGA CGGGGACAA CAAATAAAA
A. E. E P R N N CGAGCA AAGAAGIGA, CCGGA, CGGAEAAG ACCCAAAA BCE 3
s I W i. W G G S. V A. A. 8 3. A-GA TAS GTACAGC ACAGGAG CAGGACC CACAGAS
N Q A. D. P N W . Y A F H. F 2S. CCCAAACA GCCAGA CEAAC(TCA CGAGAI CATIA
A G H G. C. N. R. D. V Y A 30 GCAGGAAAC AEGGACAA AA TACGAGA CCAAGTAGAT ATGATTAGA
* G. A. A F W S. E . G S. A. A G 3 (CAAiGGAGCA. GCGAAG AGGAAG GGGGACAAG GCAGCACAG
BCE (3
8 G G D E A | A : (CACCC CTTFA (ACAACCAC AACCCAT CACTTAC
EiE 3
E R N S A S T : R. D. E . 5 (GAGAAAAA AAA.C. G.C.A.A.G. COAACC AAAGASA. ECE 3
St.
S S A. A. G. A. S. G W T E 5. CACGCA CGAAC CAGGCCAAA CAAG GGGGACAG F.G. 14C U.S. Patent Jul. 21, 2015 Sheet 16 of 27 US 9,084,734 B2
BCE O 3
e A E s Sc: S G r F R. E. R. R E 55 A.G.GIGAACT ACCCAC GTACAG GAGGGAAAA AAAAACAA BCEOs
MP are are Mr -r- - - -ar -r- X- are 1- or St. CB iiike: s a s r. pps perspor. 6 AGCCA, CEGEAAG ACCAAA CGCCASG ACAGGAEA
EB fusior. site 1st CBD linker an Sac pDP ess C C D C A C : 61 ACGGAECCA GACGATGAGA GT CAAA.C. CGGGA CAAIGCECAT
B ------it. {{GAAA AAACCA, AGTGG GCSAA. GCGIGAA
BB --- S C ; A. S A. Y A 751. AGGCATA GTCATGAA. AAGCGA. CGCCCGA, GAC CASC
O Cw F C v. D if Tr F C. W. E. (C. K. 3 CAAG GCGCACA CAGGAE CGASAS CATAAA
RB e S E R. E. E H H H H Stop (SEQ D NO:36) (CAAGCGAGGA, CGAAAAGA AACACAC ACCACACCA AAAAGTA
9 (1 U.S. Patent Jul. 21, 2015 Sheet 17 Of 27 US 9,084,734 B2
EcoRi () ar: Promoter Region apri Signal Peptide
- 3 3 pJM103BB his Psi (1511) A - 1st CBD linker Fisic Site Y Barnhi (1655) S\\\\\ Saci (1674) WW s \\\BB Bla? N. e1 \\ Sphi (1768) wsstvessessesses Sai (1815) W 6x-S Hindi (1945) FG 15 A eitiator
2}{ w 188 - Active BB Einactive BB 16 - 1S. 40 E. : 80 an 80 40 20 y Sir (1X) BE *Rati i BCE. S. FG U.S. Patent Jul. 21, 2015 Sheet 18 of 27 US 9,084,734 B2
3E3BEck.8
Bai HE S&CE
D E S S K C C E C. A. C. Y {{ACCAA GATAGAGC (CTAAAEC (GATCAA TGCCAG CCAGGCG CACTCCGA GACGGAC AA CGCTAG ACGCGACAA
233 8.
N Y G W T C. R. C. S. M. R. N S AAAiGA CGCTGAC (CCSCCA CAiiATGCC. TCCAAICC AAAACA ACCCACCGA ACAGOGACGT CGCTAACGC AGACAAGG
233 (3.
C E S A C. R. S. C A. C Y N Y G W e GECAAGG CCCCAAAAG CCCCAT, AIAACCG ACGGGRGAC AAGACA. GGAGC (ACCGAA AAGAA GCCACCG 2BSck.8
s C E C D F : D. F. C. Y E. P. K R S s ECC EACACA CSACE CAEAGCA CAAACAA ACAAAAACG CAGCGAG GCC GAAGAC GAACCGG ACAGG
12BBCK8. E E D R E N + (SEQ D NO:38) 201 GCGAGGACGA TAAAGAGAAC TAA (SEQ NC:37) CCCCCGC ATTCCTTG AT
FG U.S. Patent Jul. 21, 2015 Sheet 19 Of 27 US 9,084,734 B2
1 4. E. Purified Fusion Protei
8.
BCE BCE 88 8Bi-- 3iv 3r? VE E
--2 BBicket - - -Peptide -a-BB
O 5 20 25 Binder uM
U.S. Patent Jul. 21, 2015 Sheet 20 Of 27 US 9,084,734 B2
F. A a tra a crr a rr ra r
F. : is as as a us as a al
Fi, C.
Apri signal cleavage site
ESSEE Neil Sir GT
S A Q. A S D W W I. K K F AGCGCGCAGG CTACGA TRACAACTG AAAAAAGAA CGACGA TCCCCCICC CACCCTACA ACACTCAC CiC AAACCCC
F K N W .E. A. E. E. E. A P J C CECACAAA ACAAATGACC III CERC (AAEC (CCCGGE GAAGAG. GTACGG AACAAGAACG ACTAAAAAG (CGCGSCACCA
• G. H. C. K A A E E E . A CCCCTCACG CAAA CTC CCCCGAG ACCACCAAGC (CAACTACA CSCCAGTGA GTCGAGAA GAGACA GCCCG ACGGAS
K E R. N K A K W C E. E. S. CGAAAGAAA AGAAAAA ACGCEAAA GTAGACGCA CAGAAGAGAC ATC CCAir TGAACATT CAC.A.G. G.C.C.C.C. PT
Q Q if G W. E. G y p if K W E a GATEGO CAAAAAIG GIGACGG ACCAA AAA AAAAAC GTAC CACAATCC (ACGA (AACAAA F.G. 20 U.S. Patent Jul. 21, 2015 Sheet 21 of 27 US 9,084,734 B2
R G N S P Y K G. C. R. K. A. A 2. CCGGGCC (ACAAOCA TCOCEACA AAGCCAACG EAAA GCGC AGCCACCGA, AGCCA AACAATG TCCAGGC ATCGACCA
A S Y M K Q S A W S E W S. GCAACAC CATACAIGA CAAACAATC CGCCGCTG ACPGAAG GAGGAA (ATGACA GEGAGA (GAGGACGAC AAGACECA
I K N E. E. F K. K. A A W W AEAAAAGAC, AACCGAAG AAAAAAA AGCGACAAA. GCGCG AGTCG GGAACT AAA CGACGT (GAAAAAC
A Y W A S R. A. S. S. E. W. F' T : & GCIAGE AGAIGCTC GAAAACCA CACCCAAG CACCAA AACAATAA CACAAA CTCCA (ACGCCA AAAGGA
W. A. E. K. R ) N E. G. S. S. S C A {{CCAAA AACGCGCCA (AACACCCA TCCCiCTA GOC (AGC CAACCAC GACGCGC ACACGG AAGCCGAGA (AACTAG
* A A E. A. E. G. W. K. A. P A W Y K C. GCACTCGC (GAAGCCAGG (GCGTAAAGC ACCCA. GICACA ACGGACCA CGACCC CGEA ACG GACAAA CAAGAAAG
o E K A W E S E. R. F E. W. E. S. AAGAGA GAAGGAAA GCGGC CGAAAAA CGAAGAGAG CCAAACE ATCCAT, CGCCAAAAGA. GACCAA (CCACC
A E. R. A K G A C E 6 G. GCAAGAAA AA CGCTAA AACAGGGC ACCCACA GGCGAAA EGAGC AACGGA TCAGA TGAGGGAA AACCGCA
o G E. Y S Y i S A A. Y. S. CGEACCTGAA ACACTCG AACAP AGCTGGCAC CCCGGCA GCECAC AAGAA AAG ACAS CGACCAS (AACCGA
FI U.S. Patent Jul. 21, 2015 Sheet 22 of 27 US 9,084,734 B2
o F. A E A E E R K E S. K (1 ACATICGC (GAAACAGCT GAAGAGCGTA AAGAACT CAG CGACAAACT (AAAAGC AGCCA 'CCA, CCAGC (C. CAA
K A E. A. R. G. W. N. F. G. i ST. AAACAA.G. CGAAGCCA AGGGC AAAC (GAAGA GGASC GACGAG GCACCGAA AAGAAA CAGAAAC iP)
O A K A F. G. A. H. A. G. N. K R. 3{1 CGCTAAAGCA IGGGCIC ACGGGAAA. (CTGAACTG AAAACTGACA GGACG AAACAGAG (GCACCTT GACAGAC TTGACT
A F A C E W A K N Q K E 85. AAECCCCGC IECGCAAC CAAGAAGG CEAAAAACCA. AAAAICCC TAAGGGACG AAAGCGTAG CTCCAAC GATTGG TAAGGGA
F D E E. E. F. E. A K. A. S. GACAAG AAAAAGAAA ACGAA, GCACAAAS. EACGG2 AAACAT TICTA AGAAAAC CCACC AAGAAC
F W A. G. R. E. E. S K S E S. CGA GCGGAAAA GAACCAAG CACAAACA GAACAAC CAAAACAA CGACCAT AGC GGC GTAG TAG CEG TAGO
E. R. C. E y R v W A. K. N Y N CGAAAAACA AGAAGGCC (AEGAG ACCAA AAACAEAA GACT G TCTCCACCA CAATCACAFC AACACGA (ACTA
E W K. D. W. E. E. Y A P GAAAICCITC GGACGATAC AAAGAGA AAGAA ACCCCC CASCAAG ACCGCAG ACTAA AAAACA AAA CGAG
H C K A A. P. R Y E E G A (, TGGGCCGT CACTCCAAAG CCGCCC EAAAACGAA GAA CGGG AACCACCCA GTGACGC GAGAACSAGG AAGC (CGAACCAC
U.S. Patent Jul. 21, 2015 Sheet 23 Of 27 US 9,084,734 B2
Y A R. S. E E. K. R W W I A K W . . . CICGAGC AAAAAGCCAG CAAA (ACC GIGIGAAI CCAAAGE (AGACAAG GCC AACTGG CACAACAA ACGAAA
A A N D E f : G F p if GAGAAAG CEAACGAG (CAAGAA ACAAGGA, ECACA CTAC(TC GATTCACA AGECAC AACTTCCTA AGGGAEGAA
* R Y A. G. A. K G p W Y S G. S. (CAAACAAC CCACCCTC CAAAACGTCA ACCACT ACICICI TGATAG GCGACCAC GT:CAG GAiAAGA AGASA CAA
o R W E S K E A E R. Y S CACGEACGE GAAGEC C. ACAAACA GOAAAA CGAAAAE CTCCiCACA AITCGAA TAGAAC, AAC(ACT COATAC
E. A. A S E ) A. E. E S S A. T. E. F. S 5. AAAGCGCAA CCAGAAGA GCGAAGAG ACAGICAG CAAEGAAAC TCACGT AGAGCC AGA CTCTC CACAAGC (TAC C.
E A K. S. E. E. A. A. K. E A 4. AACTACAGAA. ACGCTACAA AGTCAGAAGA AGCCAAAA GAAACGCAA ICACC GACCAC, CACTCTC CAC T CET(AC Enteropeptidase cleavage linker hi N-teri 33
it E E. E. G. S G. S. G. O K (AGAAACGA CGAACTGA, CGGCCG (GAGAGARA GACAA.A.G.A. (TCIGIGC (CTGAACC AGACCAAGCC CTCTAC(CT ACTGT CTG
N. t. E.
} E S S {} GATGAGAGCT. CT (SEQD NO:39) CTACECrce: A GA (SEQ D NO:40)
U.S. Patent Jul. 21, 2015 Sheet 24 of 27 US 9,084,734 B2
Fig. if ae ee ea ee
fig. 3
Fig. 2 (C
we w w area w x we ww.
apri proRoter
GAATCCA TCC TACAAAA AACAGACC GATTCA CAAGAGG AAAAGAAA GAATIA GTCGAGE AAAAAGG agrE proEacter
5 AAGAGCT CAAAAAACC CGCCCC (GAAACGGA (CCG.C.A. C{AAA. S. ACSAA. ACE AGACA agEE protecte:
{}. AAAATCCC (ATAICCTT. AAACACCGC (CAATCCCCC, CCCCACCA AAAGGG CAAACAA. GCGCCG CGACCGC GGCGAGACT
U.S. Patent Jul. 21, 2015 Sheet 25 Of 27 US 9,084,734 B2
aprE profiloter
GiCTTGCT GGCGAACT CACITAT CCCCCC CCAAAA; ACAGAAAGA ACCGCACA AGAAAAA AGAAGGAGGG AGAGAA arE projecter 2: trip Ticar C TACC0 if CTGAAAGT iAir TA AAACTA AAAAAGAAG ATAGGAAAA (SACACAA AAAAAACC EAAAAA arE promoter 35 ICACACT (AAAAAA, ATCACGAAA ACCAGE SACCCAA AGAGACGA AACT Trrif A "fag"GCTAir AAGGAAA AGAGGCC aprE profiloter
GCACACGCAG GTCAT GAA CGAAirfi! (CGACAGGAA GCCGGGAC CCGC CAGAAACT CAAAAAA (CECA. AACGCC apri promoter
CAGACCA AACEAAA AAAEATGAC ACACA AAAACA AGCCA AAGGAT TCACG AAAGCGEA ACIGAA arE proELoter
a TAC CAGTC TApriprict CT. CTGA f(AAAAAG AC(AG AGAGACAG AAAAAGCAA (SAAAAACA ACEACA AAAAGCCA agrE promoter
AS CAGAA AAGCASAG AAAACC AAAAAGA AAAACAC GAGAGCCTT TACGCCTC ACTAACG ATACT: AAGTAG aprE tomote CAAAAAAAI GGGCACA. AAAAA ECACAA CAAAAAC Agitprit pia CCCACAGAT TAiAATAA (CAGAAA Air AAC apri profiloter
AAAAAG CAAGA IGCTACA GAAAAAI (AGAGAGA CAAAAAAA CCC aprE promoter Cutinase signal peptide ApriE signal peptide
W R S K R. . . . S E A SO GAAAAG GIGAAGAAA AAAGA CACCG (GCCA CCACECA C CCG (AACACC AGGAACAA CAAACGAA
21 U.S. Patent Jul. 21, 2015 Sheet 26 of 27 US 9,084,734 B2
Catinase
A. A S C S W C A A A. A a SS ACGCGGCGG CCCIGCC GICCGCGI, GCACGTCG CGGCGGCC GCACCGCC (GAGAACEA CAGGCAGACA, CGCACAGC GCCGCCSAGG Cultinase
T. P G A p F P A W A N E E R o 7. CGCCGGA ACACGGAG (GCAC GCGCGC AAGAC GCACCGCCA GTCCCCC (CGCTAAA CGACAGCC AAACCC Cutinase
is S. Y S S Q S E S C R S. G.C.A.G.GGCCC CACACCACC AGCAGCCA GA. GGGGGGC: GAGCGCGC GCACCGG, AGG CCGC CCCCCCGC (CGACACC Cutinase
Y R. P. R D G G. G. W. R. H. P W I 38 ACTACGGC CCCGCGACC GGGCAGGGG (GGCGGCGC ACCGGGA ACAAGCC GGCCGSA (CCAGCCC (CGEACCAG ACECACA Cutinase
* G N. : T. G. A G S A G is . " 85. CCGGGGC AAGGCACCG GTGCCGGGCC GCCACCAE GCCGGCTGO AGACACCCCG ACCCGC CACGG CCCG CAGGAA CGCCGAACC Cutinase
S H A S G F W W A A. A. E. T. S. S. ACGCACG GGCAAGECAC GGCGGG GGCGGCGGC (GGAAACCC AASCCA CGTCCC, CCAAASCAC ACCCCCC. CCCA Cutinase
N A C. R. E. A C T D Y I, W R a 35. AAEGCGGA, CCGGGCGGGA AAGCCGCC GCCGGAC ACGGACG ACGCCA GCCCCCC EACAGGG ACCGACCGA, AGACCAGC Cutinase
a E N T p y : if Y S K N (; R o (O GAGAACGAC ACCCCCTACG GCACC TAC CGGCAAGCC AATACCGGGC ACCGCG. GGGGGAGC (CGGGATAAG GCCGCGAG. A. GGCCCG Cutinase
W S. G. S O G. G ( S A GAGCGGCAC CGGGCAF CCCAGGGG (GGGCGGCC GACAGGCC AGCGG AAGACCCGTA AGGGCECAC CACCGCCGAG (AGACCGG F.G. 21C U.S. Patent Jul. 21, 2015 Sheet 27 Of 27 US 9,084,734 B2
Cultinase
G Q R V R A p : p i is G. GGGCAGGATA CGAGGGGCG ACCACGGG CGACCAGC (CTACACCC CCCGCCTA, CCTCCCACCC AIGCTCCCC (CACTCC AEGA Cutinase S R R G CGGCCTGGGG CACGACAGCG CCTCGCAGCG GCGGCAGCAG. GGGCCGATG (CCGACCCC (CCCC (GAGCCAR CCCCC CCGCACA Cultinase
e S { { { ) A F i. N. A 2 G CCTGACC CC GCGCCGT CACACCA.C.G. CCCCCTA, CCTCAACGC ACCACACAC, SCCACCCCCA CGGGTAC CAAAGGA (ACCCA Cutinase
C E V Y R. R. A. N W F G E. R. R. 2 CAGCCGGTC. ACGGCGGC CAAGIGCG GEGECGGG GCGAACGGCG CGECASA (CCCCACC, CACACGE CACAAGACC CCCCCCGC Cutinase
a Y V S W C. S. ( . A y R SO. ACGAG ACCGAGC (GGCGGAG GGGGGGCC ATCGGGC AAG ACTCC GAA GCC (CCAGCCAC CCCACCCCC ATACGCC Cltinase S T A W E R F C I. is D B C D A R 3, CGAGCACGGC AGGCCGC CCAGCTGA GGAGACCA AGACGCCCGC (CCGCCG TACAAGS, AACGCGAC, ACCACC TGGGC Cultinase AV744:
A Y { A S C E S S 4 (1 CFACE TCT AC(CCCCCA GTCAGTTC (CACTEC (CGGC CGAGCAAGA TGCCGCGC CACGCACAE ACCAAAG ACAEACCAC Eiriker 2 Cutinase
Sai
E. R. R ( , ) N N ) p p &5. GTGAACGE AGAGGG ACAACAAGA, CCACCG (GATCC (S EQ is NO.41) ACAATCG TCCCAAA GTTACT AGAAAGCC CTAG (S EQ D NO:42)
FI . 21 US 9,084,734 B2 1. 2 PEPTIDE PERSONAL CARE COMPOSITIONS al., 1993: Asano et al., 1998: Mesiano et al., 1998: Luo et AND METHODS FOR THEIR USE al., 1998a and 1998b; and Borgstrom et al., 1996 and 1998). CROSS-REFERENCE TO RELATED RTKs comprise a large family of transmembrane receptors APPLICATIONS for polypeptide growth factors with diverse biological activi ties. The intrinsic function of RTKs is activated upon ligand The present application claims priority to International binding, which results in phosphorylation of the receptor and Application No. PCT/US06/015711, filed Apr. 25, 2006 and multiple cellular Substrates, and Subsequently in a variety of U.S. Provisional Patent Application Ser. No. 60/678,601 filed cellular responses. (See, Ullrich & Schlessinger, Cell 61:203 May 5, 2005, the contents of which are fully incorporated 10 212 1990). herein by reference. Angiogenesis, involving VEGF and RTKs is not only involved in cancer development, as many other diseases or SEQUENCE LISTING conditions affecting different physiological systems are angiogenesis-dependent, such as arthritis and atherosclerotic The sequence listing Submitted via EFS, in compliance 15 plaques (bone and ligaments), diabetic retinopathy, neovas with 37 C.F.R.S 1.52(e), is incorporated herein by reference. cular glaucoma, macular degeneration, ocular herpes, tra The sequence listing text file submitted via EFS contains the choma and corneal graft neovascularization (eye), psoriasis, file “GC874-US-SEQ-LIST.txt created on Aug. 24, 2011, Scleroderma, rosacea, hemangioma and hypertrophic scar which is 78,808 bytes in size. ring (skin), vascular adhesions and angiofibroma (blood sys tem). FIELD OF THE INVENTION VEGF is an angiogenesis factor of major importance for skin vascularization (Detmar 2000). VEGF expression is The present invention provides peptides and Supported upregulated in the hyperplastic epidermis of psoriasis (Det peptides for treating various diseases and conditions. In par 25 marandYeo et al. 1995), in healing wounds and in other skin ticularly preferred embodiments, the present invention pro diseases characterized by enhanced angiogenesis (Detmar vides compositions and methods for personal care. In some 2000, supra). Targeted overexpression of VEGF in the epi embodiments, the present invention provides compositions dermis of transgenic mice was reported to result in enhanced for use in skin and/or hair care, as well as cosmetic compo skin vascularization with equal numbers of tortuous and leaky sitions. In alternative particularly preferred embodiments, the 30 blood vessels (See e.g., Brown et al. 1998). Also, chronic present invention provides peptides and Supported peptides synthesis of VEGF in mouse skin leads to the first histologi for treating diseases of the skin, such as rosacea. In some cally equivalent murine model of human psoriasis (Xia et al. particularly preferred embodiments, the Supported peptides 2003) that is reversible by binding agents specific for VEGF. of the present invention are anti-VEGF peptides. In alterna Proteases are involved in a wide variety of biological pro tive particularly preferred embodiments, the anti-VEGF pep 35 cesses. Disruption of the balance between proteases and pro tides are expressed on a scaffold protein. In some most pre tease inhibitors is often associated with pathologic tissue ferred embodiments, the scaffold protein comprises BBI. destruction. Indeed, various studies have focused on the role of proteases in tissue injury, and it is thought that the balance BACKGROUND OF THE INVENTION between proteases and protease inhibitors is a major determi 40 nant in maintaining tissue integrity. Serine proteases from Angiogenesis is the development of a blood Supply to a inflammatory cells, including neutrophils, are implicated in given area of tissue. Angiogenesis is part of normal embry various inflammatory disorders, such as pulmonary emphy onic development and revascularization of wound beds, as sema, arthritis, atopic dermatitis and psoriasis. well as due to the stimulation of vessel growth by inflamma Proteases also appear to function in the spread of certain tory or malignant cells. Angiogenesis is also the process 45 cancers. Normal cells exist in contact with a complex protein through which tumors or inflammatory conditions derive a network, called the extracellular matrix (ECM). The ECM is blood Supply through the generation of microVessels. a barrier to cell movement and cancer cells must devise ways Angiogenesis is regulated in normal and malignant cancer to break their attachments, degrade, and move through the tissues by the balance of angiogenic stimuli and angiogenic ECM in order to metastasize. Proteases are enzymes that inhibitors that are produced in the target tissue and at distant 50 degrade other proteins and have long been thought to aid in sites (See, Fidleret al., 1998; and McNamara et al., 1998). freeing the tumor cells from their original location by chew Vascular endothelial growth factor-A (VEGF, also known as ing up the ECM. Recent studies have Suggested that they may vascular permeability factor, “VPF) is a primary stimulant of promote cell shape changes and motility through the activa angiogenesis. VEGF is a multifunctional cytokine that is tion of a protein in the tumor cell membrane called Protease induced by hypoxia and oncogenic mutations and can be 55 Activated Receptor-2 (PAR2). This leads to a cascade of produced by a wide variety of tissues (See, Kerbel et al., intracellular reactions that activates the motility apparatus of 1998; and Mazure et al., 1996). the cell. Thus, it is hypothesized that one of the first steps in The recognition of VEGF as a primary stimulus of angio tumor metastasis is a reorganization of the cell shape. Such genesis in pathological conditions has led to various attempts that it forms a distinct protrusion at one edge facing the to block VEGF activity. Inhibitory anti-VEGF receptor anti 60 direction of migration. The cell then migrates through a blood bodies, soluble receptor constructs, antisense strategies, RNA vessel wall and travels to distal locations, eventually reattach aptamers against VEGF and low molecular weight VEGF ing and forming a metastatic tumor. For example, human receptor tyrosine kinase (RTK) inhibitors have all been pro prostatic epithelial cells constitutively secrete prostate-spe posed for use in interfering with VEGF signaling (See, cific antigen (PSA), a kallikrein-like serine protease, which is Siemeister et al., 1998). In fact, monoclonal antibodies 65 a normal component of the seminal plasma. The protease acts against VEGF have been shown to inhibit human tumor to degrade the extracellular matrix and facilitate invasion of Xenograft growth and ascites formation in mice (See, Kim et cancerous cells. US 9,084,734 B2 3 4 Synthetic and natural protease inhibitors have been shown Small (60-90 amino acid residues in length) and contain no to inhibit tumor promotion in vivo and in vitro. Previous disulfide bonds. Eglin C, however, is highly resistant to dena investigations have indicated that certain protease inhibitors turation by acidification or heat regardless of the lack of belonging to a family of structurally-related proteins classi disulfide bonds to help stabilize its tertiary structure. The fied as serine protease inhibitors or SERPINS, are known to 5 protein occurs naturally in the leech Hirudo medicinalis. inhibit several proteases including trypsin, cathepsin G, As noted above, protease inhibitors interfere with the thrombin, and tissue kallikrein, as well as neutrophil elastase. action of proteases. Naturally occurring protease inhibitors The SERPINS are extremely effective at preventing/sup can be found in a variety of foods such as cereal grains (oats, pressing carcinogen-induced transformation in vitro and car barley, and maize), Brussels sprouts, onion, beetroot, wheat, cinogenesis in animal model systems. Systemic delivery of 10 finger millet, and peanuts. One source of interest is the Soy purified protease inhibitors apparently reduces joint inflam bean. The average level of protease inhibitors present in soy mation and cartilage and bone destruction as well. beans is around 1.4 percent and 0.6 percent for Kunitz and Topical administration of protease inhibitors finds use in Bowman-Birk respectively, two of the most important pro Such conditions as atopic dermatitis, a common form of tease inhibitors. Notably, these low levels make it impractical inflammation of the skin, which may be localized to a few 15 to isolate the natural protease inhibitor for clinical and other patches or involve large portions of the body. The depigment applications. Indeed, despite much research in the personal ing activity of protease inhibitors and their capability to pre care arena, there remains a need in the art for personal care vent ultraviolet-induced pigmentation have been demon compositions that have desired characteristics without unde strated both invitro and in vivo (See e.g., Paine et al., J. Invest. sirable chemical modification of the proteins. There also Dermatol., 116:587-595 (2001). Protease inhibitors have remains a need in the art for a method of delivering a protein also been reported to facilitate wound healing. For example, into a personal care composition so as to effectively deliver secretory leukocyte protease inhibitor was demonstrated to the protein in a useable form. reverse the tissue destruction and speed the wound healing process when topically applied. In addition, serine protease SUMMARY OF THE INVENTION inhibitors can also help to reduce pain in lupus erythematosus 25 patients (See e.g., U.S. Pat. No. 6,537,968). The present invention provides peptides and Supported The Bowman-Birk protease inhibitor (BBI) is a designa peptides for treating various diseases and conditions. In par tion of a family of stable, low molecular weight trypsin and ticularly preferred embodiments, the present invention pro chymotrypsin enzyme inhibitors found in Soybeans and vari vides compositions and methods for personal care. In some ous other seeds, mainly leguminous seeds and vegetable 30 embodiments, the present invention provides compositions materials. BBI comprises a family of disulfide bonded pro for use in skin and/or hair care, as well as cosmetic compo teins with a molecular weight of about 8kD (See e.g., Chouet sitions. In alternative particularly preferred embodiments, the al., Proc. Natl. Acad. Sci. USA 71:1748-1752 (1974: present invention provides peptides and Supported peptides Yavellow et al., Proc. Natl. Acad. Sci. USA 82:5395-5399 for treating diseases of the skin, Such as rosacea. In some 1985; and Yavellow et al., Cancer Res. (Suppl.) 43:24.54 35 particularly preferred embodiments, the Supported peptides S-2459 s 1983). BBI has a pseudo-symmetrical structure of of the present invention are anti-VEGF peptides. In alterna two tricyclic domains each containing an independent native tive particularly preferred embodiments, the anti-VEGF pep binding loop, the native loops containing binding sites for tides are expressed on a scaffold protein. In some most pre both trypsin and chymotrypsin (See, Liener, in Summerfield ferred embodiments, the scaffold protein comprises BBI. and Bunting (eds), Advances in Legume Science, Royal Bot. 40 In some preferred embodiments, the present invention pro Gardens, Kew, England). These binding sites each have a vides cosmetic and/or pharmaceutical compounds Suitable canonical loop structure, which is a motif found in a variety of for improving the appearance of skin. The present invention serine proteinase inhibitors (Bode and Huber, Eur. J. Bio further provides peptides that block binding of a protein. In chem. 204:433-451 1992). Commonly, as in one of the some preferred embodiments, the protein is VEGF. In some soybean inhibitors, one of the native loops inhibits trypsin and 45 particularly preferred embodiments, the peptide is expressed the other inhibits chymotrypsin (See, Chen et al., J. Biol. in a protease-resistant scaffold. In some especially preferred Chem., 267: 1990-1994 1992; Werner & Wemmer, Bio embodiments, the scaffold is a protease inhibitor (e.g., BBI, chem., 31:999-1010 1992; Lin et al., Eur. J. Biochem. STI, or Eglin chymotrypsin inhibitor). In some most pre 212:549-555 1993; and Voss et al., Eur. J. Biochem., 242: ferred embodiments, the protease inhibitor is BBI. 122-131 1996) though in other organisms (e.g., Arabidop 50 In some embodiments, the present invention provides per sis), both loops are specific for trypsin. Sonal care compositions comprising a scaffold, wherein the STI inhibits the proteolytic activity of trypsin by the for scaffold comprises at least one protease inhibitor and at least mation of a stable stoichiometric complex (See e.g., Liu, one peptide selected from the group consisting of SEQ ID Chemistry and Nutritional Value of Soybean Components. In: NOS:1-17. In alternative embodiments, any of the additional Soybeans, Chemistry, Technology and Utilization, pp. 32-35. 55 peptide sequences provided herein find use in the present Aspen Publishers, Inc., Gaithersburg, Md., 1999). STI con invention. In further embodiments, additional sequences find sists of 181 amino acid residues with two disulfide bridges use in the present invention, including but not limited to SEQ and is roughly spherically shaped (See e.g., Song et al., J. ID NOS:20-25, 31,32-34, 43, and 238. In yet furtherembodi Mol. Biol., 275:347-63 (1998). The trypsin inhibitory loop ments, the at least one peptide sequence is selected from the lies within the first disulfide bridge. The Kunitz-type soybean 60 group consisting of YNLYGWT (SEQ ID NO:1), KYY trypsin inhibitor (STI) has played a key role in the early study LYWW (SEQID NO:239), WYTLYKW (SEQID NO:240), of proteinases, having been used as the main Substrate in the TYRLYWW (SEQ ID NO:241), RYSLYYW (SEQ ID biochemical and kinetic work that led to the definition of the NO:242), YYLYYWK (SEQ ID NO:243), NYQLYGW standard mechanism of action of proteinase inhibitors. (SEQ ID NO:244), TLWKSYW (SEQ ID NO:245), Eglin C is a small monomeric protein that belongs to the 65 TKWPSYW (SEQ ID NO:246), PLWPSYW (SEQ ID potato chymotrypsin inhibitor family of serine protease NO:247), RLWPSYW (SEQIDNO:248), TLWPKYW (SEQ inhibitors. The proteins that belong to this family are usually ID NO:249), KYDLYWW (SEQ ID NO:33), RYDLYWW US 9,084,734 B2 5 6 (SEQ ID NO:250), DYRLYWW (SEQ ID NO:251), DYK selected from non-water-resistant Sunscreens, very water-re LYWW (SEQID NO:34), EYKLYWW (SEQ ID NO:252), sistant Sunscreens, and water-in-silicone Sunscreens. In some and RYPLYWW (SEQ ID NO:253). In some particularly embodiments, the hair care composition is radioprotective. preferred embodiments, the peptide sequence comprises the The present invention further provides personal care com motifset forth in SEQID NO:31. In yet further embodiments, positions that are oral care compositions. In some preferred the scaffold comprises an amino acid sequence selected from embodiments, the oral care compositions are selected from the group consisting of SEQID NOS:22-25. the group consisting of toothpastes, tooth gels, mouth rinses, In yet additional embodiments, the protease inhibitor is mouthwashes, anti-caries compositions, tooth whitening selected from the group consisting of Bowman-Birk inhibi compositions, chewing gums, denture adhesives, and breath tor, soybean trypsin inhibitor, and Elgin chymotrypsin inhibi 10 fresheners. tor. In some particularly preferred embodiments, the Bow The present invention also provides personal care compo man-Birk inhibitor is a modified Bowman-Birk inhibitor. In sitions that are cosmetic compositions. In some preferred further embodiments, the scaffold comprises from about embodiments, the cosmetic compositions are selected from 0.001 weight percent to about 5 weight percent of the per eye gels, eye shadows, high-melting point lipsticks, lipsticks, Sonal care composition, while in alternative embodiments, 15 lip glosses, lip balms, mascaras, eyeliners, pressed powder the scaffold comprises from about 0.01 weight percent to formulations, and foundations. In some preferred embodi about 2.0 weight percent of the personal care composition, ments, the makeup compositions comprise at least one pig and in yet additional embodiments, the scaffold comprises ment. from about 0.01 weight percent to about 1 weight percent of In some preferred embodiments, the makeup composition the personal care composition. comprising at least one pigment is a mascara selected from The present invention also provides personal care compo non-waterproof mascaras, waterproof mascaras, Volumizing sitions comprising skin care compositions. In some preferred mascaras, lengthening mascaras, curling mascaras, anhy embodiments, the skin care compositions are selected from drous waterproof mascaras, water-based mascaras, and eye the group consisting of skin creams, lotions, sprays, emul lash or eyebrow treatments. sions, colloidal Suspensions, foams, aerosols, liquids, gels, 25 In yet additional embodiments, the makeup compositions sera, and Solids. In additional embodiments, the skin care are pressed powder formulations selected from loose pow compositions are moisturizing body washes, body washes, ders, blushes, eye shadows, and bronzing powders. In still antimicrobial cleansers, skin protective creams, body lotions, further embodiments, the makeup compositions are founda facial creams, moisturizing creams, facial cleansing emul tions selected from water-in-oil foundations, water-in-sili sions, facial gels, facial Sera, Surfactant-based facial cleans 30 cone foundations, oil-in-water foundations, anhydrous ers, facial exfoliating gels, anti-acne treatments, facial toners, makeup Sticks, and cream-to-powder foundations. exfoliating creams, facial masks, after shave balms, pre-shave The present invention further provides personal care com balms, tanning compositions, skin lightening compositions, positions having a scaffold, wherein the scaffold comprises skin redness reduction compositions, Sunscreens, depilato the amino acid sequence set forth in SEQ ID NO:19. In ries, hair growth inhibitors, and radioprotectives. In addi 35 additional embodiments, the scaffold further comprises the tional embodiments, the skin care compositions comprise amino acid sequence(s) set forth in SEQ ID NOS:20 and/or topically applied over-the-counter compositions, anti-fungal 21. In alternative embodiments, the amino acid sequence(s) treatments, anti-acne treatments, skin protectants, Sun set forth in SEQID NOS: 20 and/or 21 is/are replaced by at screens, deodorants, and antiperspirants. least one peptide having an amino acid sequence selected In some preferred embodiments, the skin care composi 40 from the group consisting of SEQID NOS:1-17. In alterna tions are capable of lightening the skin tone, while in alter tive embodiments, any of the peptide sequences provided native embodiments the skin care compositions are capable of herein find use as a replacement of SEQID NOS:20 and/or reducing redness in skintone. In yet further embodiments, the 21. In alternative embodiments, any of the additional peptide skin care compositions are capable of preventing skin tone sequences provided herein find use in the present invention. darkening, while in additional embodiments, the skin care 45 In further embodiments, additional sequences find use in the compositions are capable of preventing skin color develop present invention, including but not limited to SEQID NOS: ment. In some preferred embodiments, the skin care compo 20-25, 31.32-34,43, and 238. In yet further embodiments, the sitions are radioprotective. In alternative embodiments, the at least one peptide sequence is selected from the group skin care compositions comprise at least one radioprotec consisting of YNLYGWT (SEQ ID NO:1), KYYLYWW tives. In some particularly preferred embodiments, the radio 50 (SEQ ID NO:239), WYTLYKW (SEQ ID NO:240), TYR protectives are selected from the group consisting of Sun LYWW (SEQ ID NO:241), RYSLYYW (SEQ ID NO:242), screens. In some most preferred embodiments, the Sunscreens YYLYYWK (SEQ ID NO:243), NYQLYGW (SEQ ID are selected from non-water-resistant Sunscreens, very water NO:244), TLWKSYW (SEQ ID NO:245), TKWPSYW resistant Sunscreens, and water-in-silicone Sunscreens. (SEQ ID NO:246), PLWPSYW (SEQ ID NO:247), The present invention also provides personal care compo 55 RLWPSYW (SEQ ID NO:248), TLWPKYW (SEQ ID sitions that are capable of preventing hair growth. In some NO:249), KYDLYWW (SEQIDNO:33), RYDLYWW (SEQ embodiments, the hair is selected from the group consisting ID NO:250), DYRLYWW (SEQ ID NO:251), DYKLYWW of facial hair, leg hair, arm hair, and torso hair. (SEQ ID NO:34), EYKLYWW (SEQ ID NO:252), and The present invention also provides personal care compo RYPLYWW (SEQ ID NO:253). In some particularly pre sitions that are hair care compositions. In some embodiments, 60 ferred embodiments, the peptide sequence comprises the the hair care composition is selected from the group consist motifset forth in SEQID NO:31. In yet further embodiments, ing of shampoos, conditioners, hair styling compositions, the scaffold comprises an amino acid sequence selected from hair colorants, permanent wave formulations, creams, gels, the group consisting of SEQID NOS:22-25. mousses, sprays, emulsions, colloidal Suspensions, liquids, The present invention also provides methods for making foams, and solids. In further embodiments, the hair care com 65 the personal care compositions of the present invention, com positions comprise at least one radioprotectant. In some pre prising combining an effective amount of the scaffold and at ferred embodiments, the radioprotectant is a Sunscreen least one physiologically acceptable carrier or excipient. US 9,084,734 B2 7 8 The present invention further provides methods for modi NO:239), WYTLYKW (SEQ ID NO:240), TYRLYWW fying the skin tone of a subject, comprising the steps of (SEQ ID NO:241), RYSLYYW (SEQ ID NO:242), providing at least one composition comprising a personal YYLYYWK (SEQ ID NO:243), NYQLYGW (SEQ ID care composition of the present invention; ii) providing a NO:244), TLWKSYW (SEQ ID NO:245), TKWPSYW Subject to be treated; and applying the composition to the (SEQ ID NO:246), PLWPSYW (SEQ ID NO:247), Subject in an area in which modifications to the Subject's skin RLWPSYW (SEQ ID NO:248), TLWPKYW (SEQ ID tone is desired. In some embodiments, the modification of NO:249), KYDLYWW (SEQIDNO:33), RYDLYWW (SEQ skin tone comprises lightening the Subject’s skin tone. In ID NO:250), DYRLYWW (SEQ ID NO:251), DYKLYWW Some alternative embodiments, the modification of skin tone (SEQ ID NO:34), EYKLYWW (SEQ ID NO:252), and comprises reducing redness in the Subject's skin tone. In yet 10 RYPLYWW (SEQ ID NO:253). In some particularly pre additional embodiments, the methods comprise a personal ferred embodiments, the peptide sequence comprises the care composition comprising the amino acid sequence set motifset forth in SEQID NO:31. In yet further embodiments, forth in SEQ ID NO:19. In alternative embodiments of the the scaffold comprises an amino acid sequence selected from methods, the personal care composition comprises at least the group consisting of SEQID NOS:22-25. one of the amino acid sequences set forth in SEQID NOS:20 15 In some embodiments, the present invention provides cos and 21. In yet further embodiments of the methods, at least metic and/or pharmaceutical compounds for improving the one of the amino acid sequences set forth in SEQID NOS:20 appearance of skin comprising at least one polypeptide or a and 21 is replaced by at least one peptide having an amino peptide. In some preferred embodiments, the polypeptide or acid sequence selected from the group consisting of SEQID peptide binds to VEGF. In alternative embodiments, the bind NOS:1-17. In alternative embodiments, any of the additional ing of the polypeptide or peptide to VEGF blocks the down peptide sequences provided herein find use as a replacement stream activity of VEGF. In some embodiments, the com of SEQID NOS:20 and/or 21. In further embodiments, addi pounds comprise at least one peptide, while in other tional sequences find use in the present invention, including embodiments, the compounds comprise at least one polypep but not limited to SEQID NOS:20-25,31,32-34, 43, and 238. tide. In some preferred embodiments, the peptide has an In yet further embodiments, the at least one peptide sequence 25 amino acid sequence selected from the group consisting of is selected from the group consisting ofYNLYGWT (SEQID SEQID NOS:1-17. In still further embodiments, the peptide NO:1), KYYLYWW (SEQID NO:239), WYTLYKW (SEQ has an amino acid sequence selected from the group consist ID NO:240), TYRLYWW (SEQ ID NO:241), RYSLYYW ing of KYDLYWW (SEQID NO:33) and DYKLYWW (SEQ (SEQ ID NO:242), YYLYYWK (SEQ ID NO:243), NYQ ID NO:34). In additional preferred embodiments, the peptide LYGW (SEQ ID NO:244), TLWKSYW (SEQ ID NO:245), 30 has a conserved binding sequence, the sequence being XXL TKWPSYW (SEQ ID NO:246), PLWPSYW (SEQ ID WPXWC (SEQID NO:15). In some preferred embodiments, NO:247), RLWPSYW (SEQIDNO:248), TLWPKYW (SEQ the sequence comprises SEQID NO:15. In further embodi ID NO:249), KYDLYWW (SEQ ID NO:33), RYDLYWW ments, the sequence comprises an amino acid sequence (SEQ ID NO:250), DYRLYWW (SEQ ID NO:251), DYK selected from the group consisting of SEQID NOS:1-17. In LYWW (SEQID NO:34), EYKLYWW (SEQ ID NO:252), 35 further embodiments, the sequence comprises an amino acid and RYPLYWW (SEQ ID NO:253). In some particularly sequence selected from the group consisting of SEQID NOS: preferred embodiments, the peptide sequence comprises the 22-25. In alternative preferred embodiments, the compounds motifset forth in SEQID NO:31. In yet further embodiments, have a sequence, the sequence being at least 70%, preferably the scaffold comprises an amino acid sequence selected from 80%, more preferably 90%, and most preferably 95% the group consisting of SEQID NOS:22-25. 40 homologous to the sequences set forth herein. In some pre The present invention also provides methods for modifying ferred embodiments, the polypeptide has a molecular weight the hair growth of a Subject, comprising the steps of provid that is preferably between 500 Daltons and 30,000 Daltons, ing the personal care composition of the present invention; more preferably between 1000 Daltons and 10,000 Daltons, providing a subject to be treated; and applying the composi and most preferably from 1500 Daltons to 8,000 Daltons. tion to the Subject in an area in which modifications to the 45 In some preferred embodiments, the compounds find use in Subjects hair growth is desired. In some embodiments, the the improvement of skin in an organism (i.e., Subject) having modification of hair modification of hair growth comprises a skin disorder. In some preferred embodiments, the skin inhibiting the growth of the subjects hair, wherein the hair to disorder is an angiogenic skin disorder. In additional pre be inhibited is selected from the group consisting of facial air, ferred embodiments, the skin disorder is at least one selected underarm hair, leg hair, torso hair, and arm hair, and head hair. 50 from the group consisting of psoriasis, venous ulcers, acne, In yet additional embodiments, the methods comprise a per rosacea, warts, eczema, hemangiomas and lymphangiogen Sonal care composition comprising the amino acid sequence esis, etc. In some particularly preferred embodiments, the set forth in SEQID NO:19. In alternative embodiments of the skin disorder is rosacea. methods, the personal care composition comprises at least In other preferred embodiments, the present invention pro one of the amino acid sequences set forth in SEQID NOS:20 55 vides cosmetic and/or pharmaceutical compounds for and 21. In yet further embodiments of the methods, at least improving the appearance of skin. In these preferred embodi one of the amino acid sequences set forth in SEQID NOS:20 ments, the compounds comprise at least one peptide or and 21 is replaced by at least one peptide having an amino polypeptide and at least one scaffold, the peptide or polypep acid sequence selected from the group consisting of SEQID tide being expressed in the scaffold. In some particularly NOS:1-17. In some alternative embodiments, any of the addi 60 preferred embodiments, the at least one peptide or polypep tional peptide sequences provided herein find use as a tide is a loop. In other particularly preferred embodiments, replacement of SEQID NOS:20 and/or 21. In further embodi the loop is closed by a disulfide bond. In some preferred ments, additional sequences find use in the present invention, embodiments, the polypeptide or peptide binds to VEGF. In including but not limited to SEQID NOS:20-25, 31, 32-34, alternative embodiments, the binding of the polypeptide or 43, and 238. In yet further embodiments, the at least one 65 peptide to VEGF blocks the downstream activity of VEGF. In peptide sequence is selected from the group consisting of Some particularly preferred embodiments, the peptide is YNLYGWT (SEQ ID NO:1), KYYLYWW (SEQ ID expressed in a protease-resistant scaffold. In some especially US 9,084,734 B2 10 preferred embodiments, the scaffold is a protease inhibitor desired. In some embodiments, the peptide binds to a vascular (e.g., BBI, STI, or Eglin chymotrypsin inhibitor). In some endothelial growth factor (VEGF). In some preferred most preferred embodiments, the protease inhibitor is BBI. embodiments, the vascular endothelial growth factor (VEGF) In some preferred embodiments, the compounds further is VEGF-A. In further preferred embodiments, the scaffold is comprise at least one peptide. Preferably, the peptide has an 5 selected from the group consisting of Bowman-Birk inhibi amino acid sequence selected from the group consisting of tor, soybean trypsin inhibitor, and Eglinchymotrypsin inhibi SEQ ID NOS: 1-17. Most preferably, the compounds com tor. In some particularly preferred embodiments, the scaffold prise an amino acid sequence selected from the group con is Bowman-Birk inhibitor. In some further embodiments, the sisting of SEQ ID NOS:22-25. In some preferred embodi scaffold comprises the amino acid sequence set forth in SEQ ments, the peptide has a conserved binding sequence, the 10 ID NO:19. In still further embodiments, the scaffold com sequence being XXLWPXWC (SEQ ID NO:15). In some prises at least one of the amino acid sequences set forth in preferred embodiments, the compounds have a sequence, the SEQ ID NOS:20 and 21. In some particularly preferred sequence being at least 70%, preferably 80%, more prefer embodiments, at least one of the amino acid sequences set ably 90%, and most preferably 95% identical to the sequences forth in SEQ ID NOS:20 and 21 is replaced by at least one set forth herein. The peptide molecular weight is preferably 15 peptide having an amino acid sequence selected from the between 500 Daltons and 45,000 Daltons, more preferably group consisting of SEQ ID NOS:1-17. In still further par between 1000 Daltons and 12,000 Daltons, and most prefer ticularly preferred embodiments, the scaffold and the peptide ably from 1500 Daltons to 10,000 Daltons. In some preferred are encoded by an amino acid sequence selected from the embodiments, the compounds comprise at least one polypep group consisting of SEQID NOS:22-25. tide. The present invention also provides methods for decreas The present invention provides compositions comprising ing the activity of a vascular endothelial growth factor com at least one peptide selected from the group consisting of SEQ prising the steps of: i) providing a Subject; and ii) adminis ID NOS:1-17, wherein the peptide binds to a vascular endot tering the composition comprising at least one peptide that helial growth factor. In some preferred embodiments, the binds to the vascular endothelial growth factor to the subject, peptide is expressed in a protease resistant scaffold. In alter 25 under conditions such that the activity of the vascular endot native preferred embodiments, the scaffold comprises a pro helial growth factor is decreased. In some embodiments, the tease inhibitor. In some more preferred embodiments, the vascular endothelial growth factor (VEGF) is VEGF-A. In protease inhibitor is selected from the group consisting of Some particularly preferred embodiments, the composition Bowman-Birk Inhibitor, soybean trypsin inhibitor, and Eglin comprises an amino acid sequence selected from the group chymotrypsin inhibitor. In some most preferred embodi 30 consisting of SEQID NOS:22-25. ments, the scaffold is Bowman-Birk inhibitor. In still further In some additional preferred embodiments, the compounds embodiments, the protease resistant scaffold and the peptide are used for the improvement of skin in an organism (i.e., a comprise a fusion protein. IN some particularly preferred Subject) having a skin disorder. In additional preferred embodiments, the composition comprises an amino acid embodiments, the skin disorder is at least one selected from sequence selected from the group consisting of SEQID NOS: 35 the group consisting of psoriasis, venous ulcers, acne, rosa 22-25. In additional embodiments, the scaffold comprises the cea, warts, eczema, hemangiomas and lymphangiogenesis, amino acid sequence set forth in SEQ ID NO:19. In still etc. In some particularly preferred embodiments, the skin further embodiments, the scaffold comprises at least one of disorder is rosacea. the amino acid sequences set forth in SEQID NOS:20 and 21. In yet further embodiments, the present invention provides In yet additional embodiments, at least one of the amino acid 40 cosmetic and/or pharmaceutical compositions comprising at sequences set forth in SEQID NOS:20 and 21 is replaced by least one polypeptide or peptide, as set forth herein, and a at least one peptide having an amino acid sequence selected physiologically acceptable carrier or excipient. Preferably, from the group consisting of SEQID NOS:1-17. the compound is present in an amount of about 0.0001% to The present invention also provides cosmetic and/or phar about 5% by weight based on the total weight of the compo maceutical compositions comprising the at least one peptide 45 sition. Also preferably, the compound is present in an amount that binds to a vascular endothelial growth factor. In some of about 0.001% to about 0.5% by weight based on the total embodiments, the composition is capable of modulating weight of the composition. The composition may be in the angiogenesis. In additional embodiments, the composition form of an emulsified vehicle, such as a nutrient cream or further comprises a scaffold comprising a protease inhibitor. lotion, a stabilized gel or dispersion system, a treatment In some preferred embodiments, the protease inhibitor is 50 serum, a liposomal delivery system, a topical pack or mask, a selected from the group consisting of Bowman-Birk Inhibi Surfactant-based cleansing system such as a shampoo or body tor, soybean trypsin inhibitor, and Eglinchymotrypsin inhibi wash, an aerosolized or sprayed dispersion or emulsion, a hair tor. In some preferred embodiments, the scaffold is Bowman or skin conditioner, styling aid, or a pigmented product Such Birk inhibitor. In some particularly preferred embodiments, as makeup, as well as other Suitable make-up and cosmetic the scaffold comprises the amino acid sequence set forth in 55 preparations. In some embodiments, the carrier is preferably SEQID NO:19. In some alternative embodiments, the scaf at least one selected from the group consisting of water, fold comprises at least one of the amino acid sequences set propylene glycol, ethanol, propanol, glycerol, butylene gly forth in SEQID NOS:20 and 21. In further preferred embodi col and polyethylene glycol. ments, at least one of the amino acid sequences set forth in In yet further embodiments, the present invention provides SEQ ID NOS:20 and 21 is replaced by at least one peptide 60 means for decreasing VEGF activity and/or levels. In some having an amino acid sequence selected from the group con preferred embodiments, the VEGF activity and/or levels are sisting of SEQID NOS:1-17. decreased in the epidermis. In some embodiments, the The present invention also provides methods for modulat method comprising applying an effective amount of at least ing angiogenesis comprising: i) providing a composition one of the compounds described herein to an organism in comprising a peptide contained within a scaffold; ii) provid 65 need thereof. ing a Subject to be treated; and iii) applying the composition In additional embodiments, the present invention provides to the Subject in an area in which angiogenesis modulation is applications for hair and/or skin treatment, as well as appli US 9,084,734 B2 11 12 cations wound healing, treatment of proliferative diseases, FIG. 9 provides the BBI gene and amino acid sequences etc. Thus, the present invention provides compositions and (SEQID NOS:18 and 19, respectively) designed for efficient methods Suitable for application in?on humans and otherani cloning. This sequence comprises the expression cassette mals. used in E. coli. It codes for the pro-BBI protein with a C-ter minal His tag and has extra sequences at the 5' and 3' ends for BRIEF DESCRIPTION OF THE DRAWINGS cloning into the E. coli expression vector. The protein signal sequence is italicized while the trypsin loop (CTKSNPPQC; FIG. 1 provides a sequence summary of VEGF binding SEQ ID NO:20) and chymotrypsin loop (CALSYPAQC; phage clones (SEQID NOS:1-12). Twenty-four phage clones SEQID NO:21) are highlighted in bold and boxed. were sequenced after 3 rounds of panning. The sequence 10 FIG. 10 provides an SDS PAGE gel showing the results of alignment tree indicates a highly conserved sequence motif refolding anti-VEGF BBI. Anti-VEGF BBI was refolded in ACXLWPXXWC (SEQ ID NO:14). The number in paren the presence or absence of subtilisin BPN'Y217L. The lanes theses represents the frequency of that sequence within the 24 are as follows: Lane 1: Hampton Foldit 11, refolding buffer, clones sequenced after the third round of panning. -subtilisin; Lane 2: Hampton Foldit 11 refolding buffer, 15 +subtilisin; Lane 3: Hampton Foldit 13 refolding buffer, FIG. 2 provides results of a phage ELISA to demonstrate -subtilisin; Lane 4, Hampton Foldit 13 refolding buffer, the binding of unique clones to VEGF and not to BSA. +Subtilisin; and Lane 5, molecular weight markers. Equivalent amounts of phage were evaluated to determine FIG. 11 provides a graph showing that BBI-VEGF1 (SEQ their relative binding affinity to hVEGFs. The clone number ID NO:22) binds specifically to VEGF. and randomized sequence (SEQ ID NOS:1-7, and 13) are FIG. 12 provides a graph showing HUVEC results for indicated below each symbol. Target-bound phage were designated peptides. detected with anti-M13-HRP. The HRP was monitored with FIG. 13 provides sequences of three BBI-VEGF fusions, ABTS Substrate at an Aaos, after 30 minutes (n-3). BBI-VEGF1 (SEQ ID NO:22), BBI-VEGF2 (SEQ ID FIG.3 provides results of a BIACORE binding analysis of NO:23) and BBI-VEGF12 (SEQ ID NO:24). Fusions BBI VEGF binding peptides. Binding curves were obtained as 25 VEGF1 and BBI-VEGF2 have only one of the binding loops described in the Examples. Data were fit to a two state reac replaced; fusion BBI-VEGF12 has both of the binding loops tion model with conformation change: Analyte (A) binds to replaced. ligand (B) to form complex AB. Complex AB changes to AB FIG.14 provides the DNA and amino acid sequences of the which cannot dissociate directly to A+B. Panel A provides aprE-BCE 103-BBI-Histag expression cassette (EcoRI-Hin results for biotinylated peptide CK37281. kal=2.84 e M' 30 dIII) cloned into the plM103 integration vector (SEQ ID s', ka 1=0.0122 s', ka2=1.5 e-3 s' kd2=3.36 e NOS:35 and 36). s'K–1.92 e M. Panel B provides results for CK37283 FIG. 15 provides a schematic map of the plM103BBIhis (6000 RU VEGF, 3500 RUTNFono buffer only subtraction); expression vector. ka1 =1.24 e M' s', ka 1–0.318 s', ka2=6.34 e-3 s', FIG. 16 provides the DNA and amino acid sequences of kd2=1.23 es' K-4.90 e M. Panel C provides results for 35 12BBIck81 from the BCE 103 fusion site (at the BamHI) to v114 control peptide (1000 RU VEGF, 850 RU TNFC. Data the end of the gene (SEQID NOS:37 and 38). The CK37281 were fit to a 1:1 Langmuir binding ka1 =7.51 e M' s' peptide sequences (ACYNLYGWTC (SEQ ID NO:43)) are kd1=0.167 s'K–2.23 e7 M. inserted into both the trypsin and chymotrypsin inhibitory FIG. 4 provides plasmid maps used in the Examples. Panel loops. A provides the map for pCB04WT expression phagemid for 40 FIG. 17 provides a graph showing titers of active versus expression of C-terminal His6x tagged beta-lactamase. Panel inactive 2BBIck81 (by trypsin inhibition) and the ratio of the B provides the map for pME22 N-terminal stuffer phagemid activities of BCE 103 cellulase to 2BBck81 with various thiol for cloning using Bbs1 restriction sites. Panel C provides the reducing agents added during the growth of the culture. In this map for pCM01 N-terminal avEGF-BLA fusion expression Figure, BME-2-mercaptoethanol, Cyt=cysteine, phagemid. 45 Glut reduced glutathione, DTT-dithiothreitol). FIG. 5 provides a summary of N-terminal fusion cloning FIG. 18 provides a graph showing activation of BCE-link2 strategy using Bbs 1 cloning sites (SEQID NOS:227-237). 2BBIck81 with 2-mercaptoethanol (bME) after partial puri FIG. 6 provides an SDS-PAGE gel of His-tag purified fication by ion exchange chromatography. beta-lactamase fusions with peptides. IMAC purified BLA FIG. 19 provides a graph showing results from a competi versions and different peptides were concentrated and loaded 50 tion analysis of 2BBlck81 versus anti-VegE antibody binding onto an SDS PAGE gel (4-12%). Lanes 1 & 10: MW markers. to VegF. Lane 2: pCB04 (WT with 6xhis tag); Lanes 3,4,5,6: pCM01 FIG. 20 provides the sequence of the synthetic DNA frag aVEGF-BLA N-terminal fusion protein scaffold; and Lanes ment carrying the H. insolens PDI (hiPDI) that was inserted 7,8: pCM02 achymotrypsin-BLA N-terminal fusion protein. into the B. subtilis BBI expression vector, as well as the amino FIG.7 provides a graph showing that a VEGF peptide-BLA 55 acid sequence (SEQ ID NOS:39 and 40) fusion binds specifically to VEGF. Increasing concentrations FIG.21 provides the DNA and amino acid sequences of the of pCM01 (aVEGF peptide-BLA fusion) and pCB04 (WT) aprE-cutinase expression cassette that was ligated into the were added to VEGF coated wells of a microtiter plate. EcoRI-BamHI sites of p2JM103-link2-2BBIck81 (SEQ ID Residual bound nitrocefin activity was measured after wash NOS:41 and 42). ing 5x with nitrocefin assay buffer (0.125% n-octyl-beta-D- 60 glucopyranoside in PBS). DESCRIPTION OF THE INVENTION FIG. 8 provides a graph showing inhibition of VEGF induced HUVEC proliferation by anti-VEGF peptide (filled The present invention provides peptides and Supported circles). Proliferation was monitored by radioactive incorpo peptides for treating various diseases and conditions. In par ration of H thymidine (n=3). Anti-VEGF antibody (open 65 ticularly preferred embodiments, the present invention pro circles) was used as a positive control, as described in the vides compositions and methods for personal care. In some Examples. embodiments, the present invention provides compositions US 9,084,734 B2 13 14 for use in skin and/or hair care, as well as cosmetic compo are written left to right in 5' to 3' orientation; amino acid sitions. In alternative particularly preferred embodiments, the sequences are written left to right in amino to carboxy orien present invention provides peptides and Supported peptides tation, respectively. It is to be understood that this invention is for treating diseases of the skin, Such as rosacea. In some not limited to the particular methodology, protocols, and particularly preferred embodiments, the Supported peptides reagents described, as these may vary, depending upon the of the present invention are anti-VEGF peptides. In alterna context they are used by those of skill in the art. tive particularly preferred embodiments, the anti-VEGF pep Furthermore, the headings provided herein are not limita tides are expressed on a scaffold protein. In some most pre tions of the various aspects or embodiments of the invention ferred embodiments, the scaffold protein comprises BBI. which can be had by reference to the specification as a whole. As described in greater detail herein, the present invention 10 Accordingly, the terms defined immediately below are more provides compositions for use in numerous aspects of per fully defined by reference to the specification as a whole. Sonal care, including but not limited to hair and skin care, as Nonetheless, in order to facilitate understanding of the inven well as cosmetics (e.g., make-up). For example, the present tion, a number of terms are defined below. invention provides compositions that find use in daily per Definitions Sonal care, skin care, Sun care (e.g., Sunscreens, as well as 15 As used herein, the term “scaffold’ refers to a protease tanners), hair care (e.g., shampoos, leave-on and/or rinse off inhibitor having a heterologous and/or modified peptide conditioners, hair tonics, hair sprays, gels, foams, mousses, sequence incorporated therein. In preferred embodiments, the setting products, hair colorants, permanent formulations, term “scaffold’ refers to a wild-type protein sequence into other styling and cleaning products, etc.), after-Sun care for which a variant sequence is introduced. In some embodi skin, hair and lips, oral care (e.g., toothpastes and gels, ments, the scaffold has portions (e.g., parts or all of one or mouthwashes, rinses, etc.), bathing (e.g., washes, shower both loops), that are replaced with heterologous sequence(s). Soaps, bath Soaps, salts, pearls, etc.), skin lighteners, cleans For example, the BBI sequences having anti-VEGF (AV) ing treatments for skin conditions (e.g., pimples, acne, skin sequences incorporated as provided herein, find use as scaf toners, etc.), depilatories, wet wipes, deodorants, anti-perspi folds. Indeed, the present invention encompasses BBI-based rants, facial masks, shaving (e.g., shaving creams, gels, etc.), 25 sequences, but which have structural and functional differ after-shave, skin peeling (e.g., exfoliants), intimate care prod ences from wild-type BBI. ucts (e.g., feminine hygiene products), personal fresheners, As used herein, the term “vascular endothelial growth fac and foot care. The present invention also provides composi tor” (VEGF) refers to proteins with the ability to stimulate tions that find use in cosmetics (e.g., foundations, mascara, vascular growth, including those designated “VEGF-A’ eye shadows, eye liners, lipsticks, lip glosses, blushers, etc.). 30 known to those of skill in the art. It is contemplated that the compositions of the present inven As used herein, the term “anti-VEGF (“aVEGF and tion will find use various forms, including but not limited to 'AV') refers to peptides and other compositions that recog Solids, liquids, colloidal Suspensions, emulsions, oils, gels, nize (i.e., bind) to VEGF. In preferred embodiments, these aerosols, foams, powders, pump sprays, etc., as well as being peptides/compositions modulate VEGF activity. used in conjunction with items such as wet wipes, etc. Indeed, 35 The term “angiogenesis' refers to the biological processes it is contemplated that the present invention will find use in which result in the development of blood vessels and/or any suitable form for the intended use(s). increase in the vascularity of tissue in an organism. In par Unless otherwise indicated, the practice of the present ticular embodiments herein, the term refers to the process invention involves conventional techniques commonly used through which tumors or other rapidly proliferating tissue in molecular biology, microbiology, and recombinant DNA, 40 derive a blood Supply through the generation of microVessels. which are within the skill of the art. Such techniques are The terms “angiogenic disease.” “angiogenic disorder.” known to those of skill in the art and are described in numer and “angiogenic skin disorder are used in reference to a ous texts and reference works (See e.g., Sambrook et al., disorder, generally a skin disorder or related disorder which “Molecular Cloning: A Laboratory Manual’. Second Edition occurs as a consequence of or which results in increased (Cold Spring Harbor), 1989); and Ausubel et al., “Current 45 vascularization in tissue. Oftentimes, the etiology of the Protocols in Molecular Biology' 1987). All patents, patent angiogenic disease is unknown. However, whetherangiogen applications, articles and publications mentioned herein, both esis is an actual cause of a disease state or is simply a condi Supra and infra, are hereby expressly incorporated herein by tion of the disease state is unimportant, but the inhibition of reference. angiogenesis in treating or reversing the disease state or con Unless defined otherwise herein, all technical and scien 50 dition is an important aspect of the present invention. Thus, it tific terms used herein have the same meaning as commonly is not intended that the present invention be limited to any understood by one of ordinary skill in the art to which this particular mechanisms of action. Examples of angiogenic invention pertains. For example, Singleton and Sainsbury, skin disorders which are suitable for treatment utilizing com Dictionary of Microbiology and Molecular Biology, 2d Ed., pounds of the present invention include, but are not limited to John Wiley and Sons, NY (1994); and Hale and Marham, The 55 psoriasis, acne, rosacea, warts, eczema, hemangiomas and HarperCollins Dictionary of Biology, Harper Perennial, N.Y. lymphangiogenesis, Sturge-Weber syndrome, neurofibroma (1991) provide those of skill in the art with a general dictio tosis, tuberous sclerosis, chronic inflammatory disease, and naries of many of the terms used in the invention. Although arthritis. Any skin disorder which has as a primary or second any methods and materials similar or equivalent to those ary characterization, increased vascularization, is considered described herein find use in the practice of the present inven 60 an angiogenic skin disorder herein. Thus, the compounds tion, the preferred methods and materials are described provided by the present invention find use in treatment of a herein. Accordingly, the terms defined immediately below are wide variety of diseases and/or conditions. more fully described by reference to the Specification as a The term "rosacea' is used to describe acne, rosacea, or whole. Also, as used herein, the singular “a,” “an and “the erythematosa characterized by vascular and follicular dila includes the plural reference unless the context clearly indi 65 tion typically involving the nose and contiguous portions of cates otherwise. Numeric ranges are inclusive of the numbers the cheeks. Rosacea may vary from very mild but persistent defining the range. Unless otherwise indicated, nucleic acids erythema to extensive hyperplasia of the sebaceous glands US 9,084,734 B2 15 16 with deep-seated papules and pustules and be accompanied according to the present invention is provided. For treatment by telangiectasia at the affected erythematous sites. This con of those infections, conditions or disease states which are dition is also referred to as “hypertrophic rosacea' or "rhino specific for a specific animal Such as a human patient, the term phyma.” depending upon the severity of the condition. It is organism refers to that specific animal. intended that the term encompass all of the various forms of 5 The “host cells' used in the present invention generally are the condition. prokaryotic or eukaryotic hosts which contain an expression The term “wart' is used to describe a small, usually hard vector and/or gene of interest. Host cells are transformed or growth on the skin. Also known as a “verruca warts are transfected with vectors constructed using recombinant DNA flesh-colored growths of the skin which are characterized by circumscribed hypertrophy of the papillae of the corium, with 10 techniques. Such transformed host cells are capable of either thickening of the malpighian, granulation and keratin layers replicating vectors encoding the protein variants or express of the epidermis. Verucca Vulgaris, a subset of warts or ver ing the desired protein variant. In the case of vectors which ruca, is characterized by infection of the keratinocytes with encode the pre- or prepro-form of the protein variant, Such human papillomavirus. variants, when expressed, are typically secreted from the host The term “psoriasis” is used to describe a skin condition 15 cell into the host cell medium. which is characterized by the eruption of circumscribed, dis The term “effective amount” is used throughout the speci crete and confluent, reddish, silvery-scaled maculopapules. fication to describe concentrations or amounts of compounds Although it is not intended that the present invention be according to the present invention which may be used to limited to any particular body area, psoriatic lesions typically produce a favorable change in the disease or condition occur on the elbows, knees, Scalp and trunk. Microscopically, 20 treated, whether that change is hair growth or prevention of these lesions demonstrate characteristic parakeratosis and hair growth. elongation of rete ridges. As used herein, “active' (and “actives') refers to a compo The term "acne is used to describe a condition of the skin sition that imparts a benefit to a subject being treated. For characterized by inflammatory follicular, papular and pustu example, in preferred embodiments, the present invention lar eruptions involving the sebaceous apparatus. Although 25 provides personal care compositions comprising BBI-AV, a there are numerous forms of acne, the most common form is “primary active' which functions to provide benefit to the known as acne simplex or acne Vulgaris which is character area to which it is applied. Thus, in Some embodiments, ized by eruptions of the face, upper back and chest and is BBI-AV is present in skin care formulations and serves to primarily comprised of comedones, cysts, papules and pus modify the skin tone of subjects to which is applied. It is not tules on an inflammatory base. The condition occurs prima- 30 intended that the term be limited to BBI-AV, as there are rily during puberty and adolescence due to an overactive additional constituents present in the personal care composi sebaceous apparatus which is believed to be affected by hor tions of the present invention which impart benefits. In some monal activity. preferred embodiments, these additional constituents are The term “eczema' is a generic term used to describe acute encompassed by the designation “secondary actives. Pri or chronic inflammatory conditions of the skin, typically 35 mary and secondary actives are collectively referred to as erythematous, edematous, papular, Vesicular and/or crusting. “actives' herein. These conditions are often followed by lichenification, scal As used herein, 'vitamin B compound means a com ing and occasionally, by duskiness of the erythema and, infre pound having the formula: quently, hyperpigmentation. Eczema is often accompanied by the sensation of itching and burning. Eczema vesicles form 40 due to intraepidermal spongiosis. Eczema is sometimes referred to colloquially as “tetter,” “dry tetter, and “scaly tetter.” There are numerous subcategories of eczema, all of O which are treated by one or more of the compounds according N to the present invention. 45 As used herein, “CK' followed by an integer refers to a wherein R is —CONH2. (i.e., niacinamide), —COOH (i.e., specific peptide. Various peptide sequences that find use with nicotinic acid) or —CH-OH (i.e., nicotinyl alcohol); deriva the present invention are provided herein (See e.g., FIG. 1). tives thereof, and salts of any of the foregoing. As an example, CK37281 refers to the peptide sequence As used herein, “non-vasodilating” means that an ester YNLYGWT (SEQ ID NO:1) which is also is included in 50 does not commonly yield a visible flushing response after various othersequences (e.g., “ACYNLYGWTCGGG” (SEQ application to the skin in the Subject compositions. It is con ID NO:238). templated that the majority of the general population would As used herein, in some embodiments, the “compound' not experience a visible flushing response, although Such comprises the "complete' protein, (i.e., in its entire length as compounds may cause vasodilation not visible to the naked it occurs in nature (or as mutated)), while in other embodi- 55 eye. ments it comprises a truncated form of a protein. Thus, in As used herein, “retinoid' includes all natural and/or syn Some embodiments, the compounds of the present invention thetic analogs of Vitamin A and/or retinol-like compounds are either truncated or be “full-length. In addition, in some which possess the biological activity of Vitamin A in?on the embodiments, the truncation is located at the N-terminal end, skin, as well as the geometric isomers and stereoisomers of while in other embodiments the truncation is located at the 60 these compounds. However, it is not intended that the term be C-terminal end of the protein. In further embodiments, the limited to these compounds, as the term encompasses vitamin compound lacks one or more portions (e.g., Sub-sequences, A alcohol (retinol) and its derivatives such as vitamin A signal sequences, domains or moieties), whether active or aldehyde (retinal), Vitamin Aacid (retinoic acid) and vitamin not. A esters (e.g., retinyl acetate, retinyl propionate and retinyl The term “organism' is used throughout the specification 65 palmitate), etc. It is further intended that the term encompass to describe an animal, preferably a human, to whom treat all-trans-retinoic acids and 13-cis-retinoic acids. It is also ment, including prophylactic treatment, with the compounds intended that the term encompass compositions that are US 9,084,734 B2 17 18 encapsulated, as well as provided for use in various forms. As used herein, the term “plasmid' refers to a circular The terms “retinol and “retinal preferably comprise the double-stranded (ds) DNA construct used as a cloning vector, all-trans compounds. The retinoid preferably used for the and which forms an extrachromosomal self-replicating formulation of the present invention is all-trans-retinol, gen genetic element in some eukaryotes or integrates into the host erally referred to as “retinol herein. chromosomes. As used herein, “carotenoid' is used in reference to B-caro As used herein, the term “expression” refers to the process tene, lycopene, lutein, astaxanthin, Zeaxanthin, cryptoxan by which a polypeptide is produced based on the nucleic acid thin, citranaxanthin, canthaxanthin, bixin, B-apo-4-carotenal, sequence of the gene or the chemical synthetic peptide. The B-apo-8-carotenal, B-apo-8-carotenoic esters, alone, as well process includes both transcription and translation of the gene as in combination. Carotenoids which are preferably used are 10 to produce polypeptide?protein. B-carotene, lycopene, lutein, astaxanthin, Zeaxanthin, cit As used herein, the term "gene' means the segment of ranaxanthin and canthaxanthin. In some embodiments, caro DNA involved in producing a polypeptide chain that may or tenoids are utilized in crystalline form, as well as in formu may not include regions preceding or following the coding lations, including but not limited to dry powders (See e.g., dry region. powders, as described in EP 0065 193; hereby incorporated 15 As used herein, the terms “nucleic acid molecule' and by reference). “nucleic acid sequence' include sequences of any form of In some embodiments, the preferred use in the case of nucleic acid, including, but not limited to RNA, DNA and lycopene, astaxanthin and canthaxanthin is of lycopene-, cDNA molecules. It will be understood that, as a result of the astaxanthin- and canthaxanthin-containing dry powders, for degeneracy of the genetic code, a multitude of nucleotide example LYCOVITR), LUCANTINR Pink and LUCAN sequences encoding a given protein may be produced, in TINR Red (10% dry powders respectively of lycopene, astax addition to mutant proteins. anthin and canthaxanthin, commercially available from As used herein, "codon” refers to a sequence of three BASF AG, Ludwigshafen, Germany. nucleotides in a DNA or mRNA molecule that represents the As used herein, the term “bioactivity” refers to a cause and instruction for incorporation of a specific amino acid into a effect relationship between a composition and a biological 25 polypeptide chain. system. Thus, the term is used as by those skilled in the art of As used herein, the term “disulfide bridge' or “disulfide biotechnology and biological sciences as the phrase that bond refers to the bond formed between the sulfur atoms of describes a cause and effect relationship between a molecular cysteine residues in a polypeptide or a protein. In this inven composition and living biological matter (e.g., tissue, cells, tion, a disulfide bridge or disulfide bond may be non-naturally etc.). 30 occurring and introduced by way of point mutation. As used herein as a noun, the term “bioactive' refers a As used herein, the term "salt bridge” refers to the bond composition that exhibits bioactivity upon administration to formed between oppositely charged residues, amino acids in living biological matter (e.g., tissue, cells, etc.). The term is a polypeptide or protein. In this invention, a salt bridge may used synonymously with “bioactive compound.” be non-naturally occurring and introduced by way of point As used herein, “silicone gum' means high molecular 35 mutation. weight silicones having an average molecular weight in As used herein, an “enzyme refers to a protein or polypep excess of about 200,000 and preferably from about 200,000 to tide that catalyzes at least one chemical reaction. about 4,000,000. It is intended that the definition encompass As used herein, the term “activity” refers to any activity non-volatile polyalkyl and polyaryl siloxane gums. associated with a particular protein, such as enzymatic activ As used herein, the term “polypeptide' refers to a com 40 ity associated with a protease. In some embodiments, the pound made up of a single chain of amino acid residues linked activity is biological activity. In further embodiments, activ by peptide bonds. The term “protein herein may be synony ity encompasses binding of proteins to receptors which mous with the term “polypeptide' or may refer, in addition, to results in measurable downstream effects (e.g., VEGF bind a complex of two or more polypeptides. The exact meaning is ing to its cognate receptor). "Biological activity” refers to any that known to those in the art. 45 activity that would normally be attributed to that protein by As used herein, the terms "expression cassette' and one skilled in the art. “expression vector” refer to nucleic acid constructs generated As used herein, the term “protease' refers to an enzyme recombinantly or synthetically, with a series of specified that degrades peptide bonds. nucleic acid elements that permit transcription of a particular As used herein, "peptide bond refers to the chemical bond nucleic acid in a target cell. The recombinant expression 50 between the carbonyl group of one amino acid and the amino cassette can be incorporated into a plasmid, chromosome, group of another amino acid. mitochondrial DNA, plastid DNA, virus, or nucleic acid frag As used herein, “wild-type' refers to a sequence or a pro ment. Typically, the recombinant expression cassette portion tein that is native or naturally occurring. of an expression vector includes, among other sequences, a As used herein, "point mutations' refers to a change in a nucleic acid sequence to be transcribed and a promoter. The 55 single nucleotide of DNA, especially where that change term "expression cassette' may be used interchangeably results in a sequence change in a protein. herein with “DNA construct” and its grammatical equiva As used herein, "mutant refers to a version of an organism lents. or protein where the version is other than wild-type. The As used herein, the terms “vector” and “cloning vector” change may be effected by methods well known to one skilled refer to nucleic acid constructs designed to transfer nucleic 60 in the art, for example, by point mutation in which the result acid sequences into cells. ing protein may be referred to as a mutant. As used herein, the term “expression vector” refers to a As used herein, "mutagenesis' refers to the process of vector that has the ability to incorporate and express heter changing a composition (e.g., protein) from a wild-type com ologous DNA fragments in a foreign cell. Many prokaryotic position (e.g., protein) into a mutant or variant composition and eukaryotic expression vectors are commercially avail 65 (e.g., protein). able. Selection of appropriate expression vectors is within the As used herein, “substituted” and “substitutions' refer to knowledge of those of skill in the art. replacement(s) of an amino acid residue or nucleic acid base US 9,084,734 B2 19 20 in a parent sequence. In some embodiments, the Substitution a point of initiation of synthesis when placed under condi involves the replacement of a naturally occurring residue or tions in which synthesis of a primer extension product which base. is complementary to a nucleic acid strand is induced, (i.e., in As used herein, “modification' and “modify” refer to any the presence of nucleotides and an inducing agent such as change(s) in an amino acid or nucleic acid sequence, includ DNA polymerase and at a suitable temperature and pH). The ing, but not limited to deletions, insertions, interruptions, and primer is preferably single stranded for maximum efficiency Substitutions. In some embodiments, the modification in amplification, but may alternatively be double stranded. If involves the replacement of a naturally occurring residue or double stranded, the primer is first treated to separate its base. strands before being used to prepare extension products. Pref As used herein, “functional portion of a secreted polypep 10 erably, the primer is an oligodeoxyribonucleotide. The primer tide' and its grammatical equivalents refers to a truncated must be sufficiently long to prime the synthesis of extension secreted polypeptide that retains its ability to fold into a products in the presence of the inducing agent. The exact normal, albeit truncated, configuration. In some embodi lengths of the primers will depend on many factors, including ments, it is contemplated that sufficient residues of a domain temperature, source of primer and the use of the method. of the naturally secreted polypeptide must be present to allow 15 As used herein, the term “probe' refers to an oligonucle it to fold in its normal configuration independently of the otide (i.e., a sequence of nucleotides), whether occurring desired polypeptide to which it is attached. However, in most naturally as in a purified restriction digest or produced syn cases, the portion of the secreted polypeptide are both cor thetically, recombinantly or by PCR amplification, which is rectly folded and result in increased secretion as compared to capable of hybridizing to another oligonucleotide of interest. its absence. Similarly, in most cases, the truncation of the A probe may be single-stranded or double-stranded. Probes secreted polypeptide means that the functional portion retains are useful in the detection, identification and isolation of a biological function. In a preferred embodiment, the cata particular gene sequences. It is contemplated that any probe lytic domain of a secreted polypeptide is used, although other used in the present invention will be labeled with any functional domains may be used, for example, the Substrate “reporter molecule, so that is detectable in any detection binding domains. Additionally preferred embodiments utilize 25 system, including, but not limited to enzyme (e.g., ELISA, as the catalytic domain and all or part of the linker region. well as enzyme-based histochemical assays), fluorescent, As used herein, "loop” refers to a sequence of amino acids, radioactive, and luminescent systems. It is not intended that for example 3-20 amino acids, more preferably 5-15 amino the present invention be limited to any particular detection acids, even more preferably 5-10amino acids, and most pref system or label. erably 7-9 amino acids, which connects structural elements of 30 As used herein, the term “target, when used in reference to a protein. Such elements include, but are not limited to beta the polymerase chain reaction, refers to the region of nucleic sheets and helical elements and the connecting loop of a acid bounded by the primers used for polymerase chain reac beta-hairpin. In some embodiments, the loop is further stabi tion. Thus, the “target' is sought to be sorted out from other lized through the use of covalent linkages. In some preferred nucleic acid sequences. A “segment is defined as a region of embodiments, the covalent linkages comprise disulfide 35 nucleic acid within the target sequence. bonds, especially as provided herein. In alternative embodi As used herein, the term “polymerase chain reaction' ments, the loops are stabilized by the use of other means, (“PCR) refers to the method well-known in the art (See e.g., including but not limited to amides, hydrogen bonds, and/or U.S. Pat. Nos. 4,683, 1954,683.202, and 4,965,188, hereby salt bridges. In most embodiments, the loops are located on incorporated by reference), for increasing the concentration the Surface of proteins and may be altered, as provided herein, 40 of a segment of a target sequence in a mixture of genomic to confer additional (e.g., desirable) properties to the requisite DNA without cloning or purification. This process for ampli proteins. fying the target sequence consists of introducing a large As used herein, "oligonucleotide' refers to a short nucle excess of two oligonucleotide primers to the DNA mixture otide sequence which may be used, for example, as a primer containing the desired target sequence, followed by a precise in a reaction used to create mutant proteins. 45 sequence of thermal cycling in the presence of a DNA poly As used herein, the terms “an oligonucleotide having a merase. The two primers are complementary to their respec nucleotide sequence encoding a gene' and “polynucleotide tive strands of the double stranded target sequence. To effect having a nucleotide sequence encoding a gene.” means a amplification, the mixture is denatured and the primers then nucleic acid sequence comprising the coding region of a gene annealed to their complementary sequences within the target or in other words the nucleic acid sequence which encodes a 50 molecule. Following annealing, the primers are extended gene product. The coding region may be present in either a with a polymerase so as to form a new pair of complementary cDNA, genomic DNA or RNA form. When present in a DNA Strands. The steps of denaturation, primer annealing and form, the oligonucleotide or polynucleotide may be single polymerase extension can be repeated many times (i.e., dena stranded (i.e., the sense strand) or double-stranded. Suitable turation, annealing and extension constitute one “cycle'; control elements such as enhancers/promoters, splice junc 55 there can be numerous "cycles') to obtain a high concentra tions, polyadenylation signals, etc. may be placed in close tion of an amplified segment of the desired target sequence. proximity to the coding region of the gene if needed to permit The length of the amplified segment of the desired target proper initiation of transcription and/or correct processing of sequence is determined by the relative positions of the prim the primary RNA transcript. Alternatively, the coding region ers with respect to each other, and therefore, this length is a utilized in the expression vectors of the present invention may 60 controllable parameter. By virtue of the repeating aspect of contain endogenous enhancers/promoters, splice junctions, the process, the method is referred to as the “polymerase intervening sequences, polyadenylation signals, etc. or a chain reaction' (hereinafter "PCR). Because the desired combination of both endogenous and exogenous control ele amplified segments of the target sequence become the pre mentS. dominant sequences (in terms of concentration) in the mix As used herein, the term “primer' refers to an oligonucle 65 ture, they are said to be “PCR amplified.” otide, whether occurring naturally as in a purified restriction As used herein, the terms “PCR product,”“PCR fragment.” digest or produced synthetically, which is capable of acting as and “amplification product” refer to the resultant mixture of US 9,084,734 B2 21 22 compounds after two or more cycles of the PCR steps of mined using methods known to those skilled in the art of denaturation, annealing and extension are complete. These crystallography and protein characterization/analysis. terms encompass the case where there has been amplification In some embodiments, modification is preferably made to of one or more segments of one or more target sequences. the “precursor DNA sequence' which encodes the amino acid As used herein, the term “amplification reagents’ refers to sequence of the precursor enzyme, but can be by the manipu those reagents (deoxyribonucleotide triphosphates, buffer, lation of the precursor protein. In the case of residues which etc.), needed for amplification except for primers, nucleic are not conserved, the replacement of one or more amino acidtemplate and the amplification enzyme. Typically, ampli acids is limited to Substitutions which produce a variant fication reagents along with other reaction components are which has an amino acid sequence that does not correspond to placed and contained in a reaction vessel (test tube, microw 10 ell, etc.). one found in nature. In the case of conserved residues, such As used herein, the term “RT-PCR refers to the replication replacements should not result in a naturally-occurring and amplification of RNA sequences. In this method, reverse sequence. Derivatives provided by the present invention fur transcription is coupled to PCR, most often using a one ther include chemical modification(s) that change the char enzyme procedure in which a thermostable polymerase is 15 acteristics of the protein. employed, as described in U.S. Pat. No. 5,322,770, herein In some preferred embodiments, the protein gene is ligated incorporated by reference. In RT-PCR, the RNA template is into an appropriate expression plasmid. The cloned protein converted to cDNA due to the reverse transcriptase activity of gene is then used to transform or transfect a host cell in order the polymerase, and then amplified using the polymerizing to express the protein gene. In some embodiments, this plas activity of the polymerase (i.e., as in other PCR methods). mid replicates in the hosts, in the sense that it contains the As used herein, the term “hybridization” refers to the pro well-known elements necessary for plasmid replication or the cess by which a strand of nucleic acid joins with a comple plasmid may be designed to integrate into the host chromo mentary strand through base pairing, as known in the art. some. The necessary elements are provided for efficient gene As used herein, “maximum stringency” refers to the level expression (e.g., a promoter operably linked to the gene of of hybridization that typically occurs at about Tm-5°C. (5° 25 interest). In some embodiments, these necessary elements are C. below the Tm of the probe): “high stringency” at about 5° Supplied as the gene's own homologous promoter if it is C. to 10°C. below Tm; “intermediate stringency” at about 10° recognized, (i.e., transcribed, by the host), a transcription C. to 20° C. below Tm; and “low stringency” at about 20° C. terminator (a polyadenylation region for eukaryotic host to 25°C. below Tm. As will be understood by those of skill in cells) which is exogenous or is Supplied by the endogenous the art, a maximum stringency hybridization can be used to 30 terminator region of the protein gene. In some embodiments, identify or detect identical polynucleotide sequences whilean a selection gene Such as an antibiotic resistance gene that intermediate or low stringency hybridization can be used to enables continuous cultural maintenance of plasmid-infected identify or detect polynucleotide sequence homologs. host cells by growth in antimicrobial-containing media is also The phrases “substantially similar and “substantially iden included. tical in the context of two nucleic acids or polypeptides 35 As used herein, the terms “restriction endonucleases” and typically means that a polynucleotide or polypeptide com “restriction enzymes' refer to bacterial enzymes, each of prises a sequence that has at least 75% sequence identity, which cut double-stranded DNA at or near a specific nucle preferably at least 80%, more preferably at least 90%, still otide sequence. more preferably 95%, most preferably 97%, sometimes as As used herein, the term “recombinant DNA molecule' as much as 98% and 99% sequence identity, compared to the 40 used herein refers to a DNA molecule which is comprised of reference (i.e., wild-type) sequence. Sequence identity may segments of DNA joined together by means of molecular be determined using known programs such as BLAST, biological techniques. ALIGN, and CLUSTAL using standard parameters. (See e.g., The term “recombinant protein’ or “recombinant polypep Altschul, et al., J. Mol. Biol. 215:403-410 1990: Henikoffet tide' as used herein refers to a protein molecule which is al., Proc. Natl. Acad. Sci. USA 89:10915 1989: Karinet al., 45 expressed from a recombinant DNA molecule. Proc. Natl Acad. Sci. USA90:5873 |1993); and Higgins et al., The term “native protein’ as used herein to indicate that a Gene 73:237-244 1988). Software for performing BLAST protein does not contain amino acid residues encoded by analyses is publicly available through the National Center for vector sequences; that is the native protein contains only Biotechnology Information. Also, databases may be searched those amino acids found in the protein as it occurs in nature. using FASTA (Pearson et al., Proc. Natl. Acad. Sci. USA 50 A native protein may be produced by recombinant means or 85:2444-2448 (1988). may be isolated from a naturally occurring source. As used herein, “equivalent residues' refers to proteins that As used herein the term “portion' when in reference to a share particular amino acid residues. For example, equivalent protein (as in “a portion of a given protein') refers to frag resides may be identified by determining homology at the ments of that protein. The fragments may range in size from level of tertiary structure for a protein (e.g., VEGF) whose 55 four amino acid residues to the entire amino acid sequence tertiary structure has been determined by X-ray crystallogra minus one amino acid. phy. Equivalent residues are defined as those for which the As used herein, the term “fusion protein’ refers to a chi atomic coordinates of two or more of the main chain atoms of meric protein containing the protein of interest (i.e., VEGF a particular amino acid residue of the protein having putative and fragments thereof) joined to an exogenous protein frag equivalent residues and the protein of interest (Non N, CA on 60 ment (the fusion partner which consists of a non-VEGF pro CA, C on C and O on O) are within 0.13 nm and preferably 0.1 tein). In some embodiments, the fusion partner enhances nm after alignment. Alignment is achieved after the best solubility of the VEGF proteinas expressed in a host cell, may model has been oriented and positioned to give the maximum provide an affinity tag to allow purification of the recombi overlap of atomic coordinates of non-hydrogen protein atoms nant fusion protein from the host cell or culture Supernatant, of the proteins analyzed. The preferred model is the crystal 65 or both. If desired, the fusion protein may be removed from lographic model giving the lowest R factor for experimental the protein of interest (i.e., VEGF and/or fragments thereof) diffraction data at the highest resolution available, deter by a variety of enzymatic or chemical means known to the art. US 9,084,734 B2 23 24 The terms “in operable combination.” “in operable order.” the oligonucleotide or polynucleotide will contain at a mini and “operably linked as used herein refer to the linkage of mum the sense or coding strand (i.e., the oligonucleotide or nucleic acid sequences in Such a manner that a nucleic acid polynucleotide may single-stranded), but may contain both molecule capable of directing the transcription of a given the sense and anti-sense Strands (i.e., the oligonucleotide or gene and/or the synthesis of a desired protein molecule is polynucleotide may be double-stranded). produced. The term also refers to the linkage of amino acid As used herein, a "composition comprising a given poly sequences in Such a manner so that a functional protein is nucleotide sequence” refers broadly to any composition con produced. taining the given polynucleotide sequence. The composition As used herein the term "coding region' when used in may be in any form, particularly a form that is suitable for reference to structural gene refers to the nucleotide sequences 10 administration. which encode the amino acids found in the nascent polypep tide as a result of translation of a mRNA molecule. The As used herein, a compound is said to be “ina form Suitable coding region is bounded, in eukaryotes, on the 5' side by the for administration' when the compound may be administered nucleotide triplet ATG’ which encodes the initiator to a human or other animal by any desired route (e.g., topical, methionine and on the 3' side by one of the three triplets which 15 oral, etc.). specify stop codons (i.e., TAA, TAG, TGA). As used herein, 'safe and effective amount” refers to a As used herein, the term "structural gene' refers to a DNA Sufficient amount of a material that significantly induces a sequence coding for RNA or a protein. In contrast, “regula positive modification to the area upon which the material is tory genes are structural genes which encode products which applied and also does not result in the production of serious control the expression of other genes (e.g., transcription fac side effects (at a reasonable risk/benefit ratio). The safe and tors). effective amount of the material may vary with the particular As used herein, the term “purified’ or “to purify” refers to skin or other body part being treated, the age of the Subject the removal of contaminants from a sample. For example, being treated, the severity of the condition being treated, the recombinant VEGF oraVEGF polypeptides are expressed in duration of treatment, the nature of concurrent therapy, the host cells and the polypeptides are purified by the removal of 25 specific material used, the particular carrier utilized, etc. host cell proteins; the percent of recombinant VEGF or Those of skill in the art are capable of adjusting the concen aVEGF polypeptides is thereby increased in the sample. tration of the personal care compositions provided herein for As used herein, the term “substantially pure' when applied the desired application of the compositions. to the proteins or fragments thereof of the present invention As used herein, the term "personal care composition' means that the proteins are essentially free of other sub 30 stances to an extent practical and appropriate for their refers to a product for application to the skin, hair, nails, oral intended use. In particular, the proteins are sufficiently pure cavity and related membranes for the purposes of improving, and are sufficiently free from other biological constituents of cleaning, beautifying, treating, and/or caring for these Sur the host cells so as to be useful in, for example, protein faces and membranes. sequencing, and/or producing pharmaceutical preparations. 35 In some embodiments, the personal care composition is in As used herein, the term “target protein’ refers to protein the form of an emulsified vehicle. Such as a nutrient cream or (e.g., enzyme, hormone, etc.), whose action would be blocked lotion, a stabilized gel or dispersioning system, Such as skin by the binding of the variant inhibitors provided for herein. softener, a nutrient emulsion, a nutrient cream, a massage As used herein, the terms “variant sequence” and “variant cream, a treatment serum, a liposomal delivery system, a sequences’ refer to the short polypeptide sequence(s) that 40 topical facial pack or mask, a Surfactant-based cleansing sys replace the binding loops of the wild-type protease inhibitor. tem. Such as a shampoo or body wash, an aerosolized or The variant sequence does not need to be of the same length sprayed dispersion or emulsion, a hair or skin conditioner, as the binding loop sequence it is replacing in the wild-type styling aid, or a pigmented product such as makeup in liquid, protease inhibitor. cream, Solid, anhydrous or pencil form. However, it is not The term "isolated when used in relation to a nucleic acid, 45 intended that the present invention be limited to any particular as in “an isolated oligonucleotide' or “isolated polynucle form, as various forms find use in the present invention. otide' refers to a nucleic acid sequence that is identified and Personal care products can be classified/described as cos separated from at least one contaminant nucleic acid with metic, over-the-counter ("OTC) compounds that find use in which it is ordinarily associated in its natural Source. Isolated personal care applications (e.g., cosmetics, skin care, oral nucleic acid is such presentina form or setting that is different 50 care, hair care, nail care). In some embodiments, the repeat from that in which it is found in nature. In contrast, non sequence protein polymer is added to a personal care compo isolated nucleic acids as nucleic acids such as DNA and RNA sition Such as a hair care composition, a skin care composi found in the state they exist in nature. For example, a given tion, a nail care composition, a cosmetic composition, or any DNA sequence (e.g., a gene) is found on the host cell chro combinations thereof. mosome in proximity to neighboring genes; RNA sequences, 55 As used herein, "cosmetic composition” refers to compo Such as a specific mRNA sequence encoding a specific pro sitions that find use in the cosmetics. The Food Drug and tein, are found in the cell as a mixture with numerous other Cosmetic Act (FD&C Act) definition is used herein. Thus, mRNAs which encode a multitude of proteins. However, cosmetics are defined by their intended use, as articles isolated nucleic acid encoding a VEGF protein includes, by intended to be rubbed, poured, sprinkled, or sprayed on, intro way of example, such nucleic acid in cells ordinarily express 60 duced into, or otherwise applied to the human body for ing a VEGF protein where the nucleic acid is in a chromo cleansing, beautifying, promoting attractiveness, or altering somal location different from that of natural cells, or is oth appearance. These compositions provide non-therapeutic erwise flanked by a different nucleic acid sequence than that benefits and are not regulated as pharmaceuticals. However, found in nature. The isolated nucleic acid, oligonucleotide, or in Some situations, cosmetic compositions are incorporated polynucleotide may be present in single-stranded or double 65 into pharmaceutical compositions to provide cosmetic ben Stranded form. When an isolated nucleic acid, oligonucle efits (e.g., products that treat skin or hair diseases, but also otide or polynucleotide is to be utilized to express a protein, contain cosmetic compositions for their coloring or other US 9,084,734 B2 25 26 benefits). Also, it is intended that the present invention and wrinkles, improving appearance and skin tone, increas encompass the use of cosmetics on animals other than ing skin firmness and/or Suppleness, decreasing sagging of humans. skin, increasing skin glow and clarity, increasing the skin As used herein, the terms “pharmaceutical compositions' renewal process, and/or removing Vellus hair. Improving the and “therapeutic compositions' refer to compositions such as visual appearance of skin also encompasses regulating drugs that provide medical benefits, rather than solely cos wrinkles, atrophy, skin lightening, skin darkening, skin metic benefits. In the United States, pharmaceutical and Smoothness, and/or reducing the visual appearance of pores. therapeutic compositions are approved by the Food and Drug As used herein, "modifying skintone' refers to a change in Administration for treatment and/or prevention of particular skin color, either darkening or lightening the color of the skin. conditions. 10 However, in other contexts, the term is also used in reference As used herein, the term “drug is defined as it is in the to modifications in the muscular and connective tissue health FD&C Act definition. Thus, drugs are defined as articles of the skin (i.e., it is not related to the color of the skin, but the intended for use in the diagnosis, cure, mitigation, treatment firmness of the skin). or prevention of disease, and articles (other than food) As used herein, “lightening skin tone' and “lightening intended to affect the structure or any function of the body of 15 skin’ refer to a decrease in skin darkness visualized by eye man or other animals. and/or mechanical means. It is intended that the term encom As used herein, “leave-on” refers to a composition that is pass any range of observable lightening (i.e., “whitening) of applied to a subject and not removed (e.g., cleansed by wash the skin tone. In some embodiments, the term encompasses ing, rinsing, etc.) for a period of typically at least several decreasing the concentration of melanin present in the skin, hours (e.g., 4-12 hours) before the area exposed to the com including skin areas with hyperpigmentation due to the pres position is cleansed. ence of age spots, melasma, chloasma, freckles, post-inflam As used herein, a "rinse-off composition is a composition matory hyperpigmentation, and/or skin-induced hyperpig that is applied and cleansed (e.g., by washing, rinsing, etc.) mentation. Soon after its application (generally within about 30 minutes As used herein, "hyperpigmented region” refers to a local of application). In some preferred embodiments, rinse-off 25 ize region of darker skin relative to the base skin tone of a compositions are formulated so as to deposit an effective particular individual. For example, in preferred embodi amount of active(s) on the area treated. ments, hyperpigmented regions are areas with localized As used herein, the term "cosmetic benefit” refers to a increases in melanin. desired cosmetic change that results from the administration As used herein, preventing skin darkening.” “preventing of a personal care composition. Cosmetic benefits include but 30 darkening of skin tone.” “preventing skin tone darkening.” are not limited to improvements in the condition of skin, hair, “preventing skin color development, and other equivalent nails, and the oral cavity. In preferred embodiments, at least phrases refer to skin tone that is observed as less skin pig one cosmetic benefit is provided by the skin care, hair care, mentation or darkness compared to untreated skin after UV nail care, and makeup compositions of the present invention. radiation and/or Sun exposure. Thus, in some particularly As used herein, "cosmetically acceptable” refers to mate 35 preferred embodiments, the compositions of the present rials that are Suitable for use in contact with tissues of humans invention provide a benefit by preventing the skin darkening and/or other animals, without undue toxicity, incompatibility, effects associated with exposure to sun and/or UV radiation. instability, irritation, allergic responses, etc., commensurate It is intended that the term encompass any range of observable with a reasonable benefit/risk ratio. difference between sun-exposed skin that is protected from As used herein, “skin care composition” refers to compo 40 darkening and Sun-exposed skin that is not protected. sitions that are applied to skin in order to provide beneficial As used herein, “evening skintone' refers to the evening of properties, including but not limited to wrinkle minimizing, skin color in an application area. In preferred embodiments, wrinkle removal, decoloring, coloring, skin softening, skin the phrase refers to the use of compositions of the present Smoothing, depilation, cleansing, etc. In some particularly invention to make the skin color the same or provide less of a preferred embodiments, the present invention provides skin 45 contrast in the skin color in one or more skin region. care compositions that improve skin tone. In these embodi As used herein, “reducing redness in skin tone' refers to a ments, the improvement comprises lessening of wrinkles, lessening of red color in skin, as observed visually or by other Smoothing skin texture, modifying skin coloration, and other CaS. desired cosmetic benefits. In further embodiments, the skin As used herein, “inhibiting hair growth' and “inhibition of care composition is in a form selected from the group con 50 hair growth” refer to an observed lessening of hair length sisting of body washes, moisturizing body washes, deodorant and/or thickness. Thus, in some preferred embodiments, body washes, antimicrobial cleansers, skin protecting treat application of a personal care composition of the present ments, body lotions, facial creams, moisturizing creams, invention provides a benefit in lessening hair length and/or facial cleansing emulsions, Surfactant-based facial cleansers, thickness as compared to an area in which a personal care facial exfoliating gels, facial toners, exfoliating creams, facial 55 composition of the present invention has not been applied. In masks, after shave lotions, balms, and/or radioprotective some embodiments, the observed reduction of hair growth compositions (e.g., Sunscreens). and/or thickness is a range from less than 1% to more than As used herein, “improving the visual appearance of skin' 99%, as compared to untreated areas, while in other embodi refers to any benefit achieved through use of the personal care ments, the observed reduction is from about 100% to about compositions of the present invention. Examples of benefits 60 90%, from about 90% to about 80%, from about 80% to about include but are not limited to reducing the visual appearance 70%, from about 70% to about 60%, from about 60% to about of pores (e.g., by reducing pore size), reducing imperfections 50%, from about 50% to about 40%, from about 40% to about and/or blemishes in skin color, including lightening hyper 30%, from about 30% to about 20%, from about 20% to about pigmented regions of skin and/or evening skin pigmentation, 10%, from about 10% to about 1%. Indeed, it is not intended relieving dryness, eliminating rough, dry spots, improving 65 that the term be limited to any particular percentage reduc the skin's ability to retain moisture and/or protect itself from tion, as long as the reduction is observable by visual (i.e., by environmental stresses, reducing the appearance offine lines eye) or other means. It is also intended that the term encom US 9,084,734 B2 27 28 pass preventing hair growth' to any degree, as described toothpastes, tooth gels, mouth rinses, breath fresheners, whit above. It is not intended that the term be limited to the com ening treatments, and inert carrier Substrates. plete prevention of hair growth (i.e., there is no observed As used herein, the term “dispersed phase' is used as by growth of hair). those of skill in the art of emulsion technology as the phase As used herein, "hair care composition” refers to compo that exists as Small particles or droplets Suspended in and sitions that are applied to hair to provide beneficial properties Surrounded by a continuous phase. The dispersed phase is Such as thickening, thinning, coloring, decoloring, cleansing, also known as the “internal' or “discontinuous’ phase. conditioning, softening, shaping, etc. In some embodiments, As used herein, “penetration enhancers' refer to composi the hair care composition is in a form selected from the group tions that facilitate penetration through the upper stratum consisting of shampoos, conditioners, anti-dandruff treat 10 corneum barrier to the deeper skin layers. Examples of pen ments, styling aids, styling conditioners, hair repair or treat etration enhancers include, but are not limited to, propylene ment Sera, lotions, creams, pomades, and chemical treat glycol, aZone, ethoxydiglycol, dimethyl isosorbide, urea, ments. In other embodiments, the styling aids are selected ethanol, dimethyl Sulfoxide, micoroemulsions, liposomes, from the group consisting of sprays, mousses, rinses, gels, and nanoemulsions. foams, and combinations thereof. In further embodiments, 15 As used herein, the terms "emulsifier” and 'surfactant” the chemical treatments are selected from the group consist refer to compounds that disperse and Suspend the dispersed ing of permanent waves, relaxers, and permanents, semi phase within the continuous phase of a material. Surfactants permanents, temporary color treatments and combinations find particular use in products intended for skin and/or hair thereof. cleansing. In particular embodiments, the term "surfactant As used herein, "makeup compositions' refer to cosmetic (s) is used in reference to Surface-active agents, whether preparations that are used to beautify, caring for, maintaining, used as emulsifiers or for other surfactant purposes such as or augment the appearance of a human or other animal. skin cleansing. "Makeup compositions include, but are not limited to color In various embodiments, the present invention also cosmetics, such as mascaras, lipsticks, lip liners, eye shad includes “protectants' such as UV absorbers (e.g., octyl ows, eye-liners, rouges, face powders, foundations, blushes, 25 methoxycinnamate, benzophenone-3, titanium dioxide, and and nail polish. In some embodiments, the cosmetic compo octyl salicylate); film-forming agents (e.g., VP/Eicosene sition of the present invention is in a form selected from the copolymer); cosmeceutical agents (e.g., peptides and pro group consisting of eye gels, eye shadows, high-melting point teins, alpha hydroxy acids, and retinol and retinoic acid lipsticks, lipsticks, lip glosses, lip balms, mascara, brow lin derivatives); antioxidants (e.g., tocopherol and derivatives ers, eyeliners, pressed powder formulations, foundations, 30 thereof and ascorbic acid and derivatives thereof); Vitamins protein coated pigments, and combinations thereof. In further (e.g., B, D, K and their derivatives); antiperspirant actives embodiments, the cosmetic compositions comprise makeup (e.g., aluminum hydroxide and zirconium hydroxide); depil compositions. In yet another embodiment, the nail care com ating agents (e.g., thioglycolate salts); anti-acne agents (e.g., position is in a form selected from the group consisting of nail salicylic acid and benzoyl peroxide); abrasives and exfoliants enamel, cuticle treatment, nail polish, nail treatment, and 35 (e.g., silicates, pumice, and polyethylene); and extracts of polish remover. plant, fruit, vegetable and/or marine sources. As used herein, "oral care compositions' refer to personal Thus, in Some embodiments, the present invention pro care compositions Suitable for use in the mouth, including but vides compositions comprising any organic UV-A and UV-B not limited to forms selected from the group consisting of filter, for example but not limited to the following:
CAS-Nr. Nr. Compound (=Acid) 4-Aminobenzoicacid 150-13-0 3-(4'-Trimethylammonium)-benzylidenbornan-2-on-methylsulfate 52793-97-2 3,3,5-Trimethyl-cyclohexyl-Salicylate (Homosalatum) 118-56-9 2-Hydroxy-4-methoxy-benzophenon (Oxybenzonum) 131-57-7 2-Phenylbenzimidazol-5-sulfonic acid and their Calcium-, Sodium 27SO3-81-7 and Triethanolaminosalts 6 3,3'-(1,4-Phenylendimethin)-bis(7,7-dimethyl 90457-82-2 2-oxobicyclo2.2.1]heptan-1-methansolfonicacid) and salts thereof 4-Bis(polyethoxy)amino-benzoesäurepolyethoxy-ethylester 11301O-52-9 4-Dimethylamino-benzoicacid-2-ethylhexylester 21245-02-3 Salicylicacid-2-ethylhexylester 118-60-5 4-Methoxy-cinnamicacid-2-isoamylester 71617-10-2 11 4-Methoxy-cinnamicacid-2-ethylhexylester S466-77-3 12 2-Hydroxy-4-methoxy-benzophenon-5-sulfonicacid 4O65-45-6 (Sulisobenzonum) and the sodiumsalt 13 3-(4'-Sulfobenzyliden)-bornan-2-on and salts thereof S8O3O-58-6 14 3-Benzylidenbornan-2-on 16087-24-8 15 1-(4'-Isopropylphenyl)-3-phenylpropan-1,3-dion 63260-25-9 16 4-Isopropylbenzylsalicylat 94134-93-7 17 3-Imidazol-4-yl-acrylicacid und ihr Ethylester 104-98-3 18 2-Cyano-3,3-diphenylacrylicacidethylester S232-99-5 19 2-Cyano-3,3-diphenylacrylicacid-2'-ethylhexylester 61.97-30-4 2O Menthyl-o-aminobenzoat oder: 134-09-8 5-Methyl-2-(1-methylethyl)-2-aminobenzoat 21 Glyceryl p-aminobenzoat oder: 136-44-7 4-Aminobenzoicacid-1-glyceryl-ester 22 2,2'-Dihydroxy-4-methoxybenzophenon (Dioxybenzone) 131-53-3 23 2-Hydroxy-4-methoxy-4-methylbenzophenon (Mexenon) 1641-17-4 US 9,084,734 B2 29 30 -continued
CAS-Nr. Nr. Compound (=Acid) 24 Triethanolamin Salicylat 2174-16-5 25 Dimethoxyphenylglyoxalsäure oder: 4732-70-1 3,4-dimethoxy-phenyl-glyoxal-Saures Natrium 26 3-(4'Sulfobenzyliden)-bornan-2-on und seine Salze S6039-58-8 27 4-tert-Butyl-4'-methoxy-dibenzoylmethan 70356-09-1 28 2,2'44'-Tetrahydroxybenzophenon 131-55-S 29 2,2'-Methylen-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3- 103597-45-1 tetramethylbuty)phenol 30 2,2'-(1,4-Phenylen)-bis-1H-benzimidazol-4,6- 18O898-37-7 disulfonicacid, Sodiumsalt 31 24-bis-(4-(2-Ethylhexyloxy)-2-hydroxyphenyl- 187393-00-6 6-(4-methoxyphenyl)-(1,3,5)-triazin 32 3-(4-Methylbenzyliden)-campher 36861-47-9 33 4-Bis(polyethoxy)paraaminobenzoicacidpolyethoxyethylester 11301O-52-9 34 2,4-Dihydroxybenzophenon 131-56-6 35 2,2'-Dihydroxy-4,4'-dimethoxybenzophenon-5,5'- 3121-60-6 disodiumsulfonat 36 Benzoicacid.2-4-(diethylamino)-2-hydroxybenzoyl-hexylester 3O2776-68-7 37 2-(2H-Benzotriazol-2-yl)-4-methyl-6-2-methyl-3-1,3,3,3-tetramethyl- 155633-54-8 1-(trimethylsily)oxydisiloxanylpropylphenol 38 1,1-(2,2'-Dimethylpropoxy)carbonyl-4,4-diphenyl-1,3-butadien 3636O2-15-7
In some embodiments, the present invention provides com invention encompasses any compound that provides a color to positions comprising pigments, including, but not limited to 25 the composition and/or imparts a color to the Surface (e.g., inorganic pigments based on metaloxides and/or other in skin and/or hair) to which the composition is applied. In some water slightly soluble or insoluble metal compounds such as embodiments, the dyes and pigments are chosen from the list Zinc oxides (ZnO), titanium (TiO), iron (e.g., FeO), Zirco nium (ZrO2), silica (SiO2), manganese (e.g., MnO), alu of cosmetic colorants provided by the Cosmetics Directive or minium (Al2O), cer (e.g., CeO), and mixed compositions 30 the EC. In most cases, these dyes and pigments are identical of these oxides, as well as blends thereof. In some preferred to the dyes approved for foods. Preferred pigments/dyes embodiments, the metaloxides are microfine, while in alter include for example, titanium dioxide, mica, iron oxides (e.g., native preferred embodiments, the metaloxides are pigment FeO, FeO, Fe0(OH)) and/or tin oxide. Advantageous grade. In yet additional embodiments, the pigments are pigments/dyes include for example, carmine, Berlin blue, “coated such that they are surface treated. In some preferred 35 chrome oxide green, ultramarine blue and/or manganese vio embodiments, the coating comprises a thin, hydrophobic film let. In some preferred embodiments, the pigments/dyes layer, while in other embodiments, the coating comprises a include, but are not limited to those in the following table. The thin, hydrophilic film layer. Colour Index Numbers (CIN) those known in the art (See, As used herein, the terms “pigment.” “color pigment, and Society of Dyers and Colourists, Rowe Colour Index, 3rd “dye' used in reference to the compositions of the present Edition, Bradford, England, 1971).
CHEMICAL OR OTHERNAME CIN COLOR Pigment Green O006 green Acid Green 1 0020 green 2,4-Dinitrohydroxynaphthalene-7-sulfonic acid O316 yellow Pigment Yellow 1 1680 yellow Pigment Yellow 3 1710 yellow Pigment Orange 1 1725 orange 2,4-Dihydroxyazobenzene 1920 orange Solvent Red 3 2010 re -(2'-Chloro-4'-nitro-1'-phenylazo)-2-hydroxynaphthalene 2O85 re Pigment Red 3 2120 re Ceres red; Sudan red; Fat Red G 2150 re Pigment Red 112 2370 re Pigment Red 7 242O re Pigment Brown 1 2480 brown 4-(2-Methoxy-5'-sulfodiethylamido-1'-phenylazo)-3-hydroxy-5'- 2490 re chloro-2',4'-dimethoxy-2-naphthanilide Disperse Yellow 16 2700 yellow -(4-Sulfo-1-phenylazo)-4-aminobenzene-5-sulfonic acid 3015 yellow 2,4-Dihydroxyazobenzene-4'-sulfonic acid 4270 orange 2-(2,4-Dimethylphenylazo-5-sulfo)-1-hydroxynaphthalene-4-sulfonic 14700 re acid 2-(4-Sulfo-1-naphthylazo)-1-naphthol-4-sulfonic acid 472O re 2-(6-Sulfo-2,4-xylylazo)-1-naphthol-5-sulfonic acid 4815 re 1-(4'-Sulfophenylazo)-2-hydroxynaphthalene 5510 orange 1-(2-Sulfo-4-chloro-5-carboxy-1-phenylazo)-2-hydroxynaphthalene SS25 re 1-(3-Methylphenylazo-4-sulfo)-2-hydroxynaphthalene SS80 re 1-(4'.(8)-Sulfonaphthylazo)-2-hydroxynaphthalene 562O re 2-Hydroxy-1,2-azonaphthalene-1'-sulfonic acid 5630 re
US 9,084,734 B2 33 34 -continued
CHEMICAL OR OTHERNAME CIN COLOR 1-Hydroxy-4-(4-methylphenylamino)anthraquinone 6O725 violet Acid Violet 23 6O730 violet 1,4-Di(4'-methylphenylamino)anthraquinone 61S6S green 1,4-Bis(O-sulfo-p-toluidino)anthraquinone 61570 green Acid Blue 80 61585 blue Acid Blue 62 62O45 blue N,N'-Dihydro-1,2,1',2'-anthraquinone azine 698OO blue Vat Blue 6: Pigment Blue 64 6982S blue Vat Orange 7 71105 Orange indigo 73OOO blue indigo-disulfonic acid 73O15 blue 4,4'-Dimethyl-6,6'-dichlorothioindigo 73360 red 5,5'-Dichloro-7,7-dimethylthioindigo 73385 violet Quinacridone Violet 19 73900 violet Pigment Red 122 73915 red Pigment Blue 16 74100 blue Phthalocyanine 74160 blue Direct Blue 86 7418O blue Chlorinated Phthalocyanines 74260 green Natural Yellow 6.19; Natural Red 1 75100 yellow Bixin, Nor-Bixin 75120 Orange Lycopene 75.125 yellow trans-alpha-, beta- and gamma-caroteine 751.30 Orange Keto- and/or hydroxyl derivates of carotene 75135 yellow Guanine or pearlizing agent 75170 white ,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione 753OO yellow Complex Salt (Na, Al, Ca) of carminic acid 75470 red Chlorophylla and b; copper compounds of chlorophylls and 75810 green Chlorophyllins Aluminum 77OOO white Hydrated alumina 770O2 white Hydrous aluminum silicates 77004 white Ultramarine 77.007 blue Pigment Red 101 and 102 77015 red Barium sulfate 7712O white Bismuth oxychloride and its mixtures with mica 77163 white Calcium carbonate 77220 white Calcium sulfate 77231 white Carbon 77266 black Pigment Black 9 77267 black Carbo medicinalis vegetabilis 77268 black Chromium oxide 77288 green Chromium oxide, hydrous 77289 green Pigment Blue 28, Pigment Green 14 77346 green Pigment Metal 2 77400 brown Gold 7748O brown ron oxides and hydroxides 77489 Orange ron oxide 77491 red ron oxide, hydrated 77492 yellow ron oxide 77499 black Mixtures of iron (II) and iron(III) hexacyano-ferrate 77510 blue Pigment White 18 77713 white Manganese ammonium diphosphate 77742 violet Manganese phosphate: Mn(PO), 7 H2O 77745 red Silver 7782O white Titanium dioxide and its mixtures with mica 77891 white Zinc oxide 77947 white 6,7-Dimethyl-9-(1'-D-ribityl)-isoalloxazine, lactoflavine yellow Sugar coloring brown Capsanthin, capsorubin Orange Betanin red Benzopyrylium salts, Anthocyans red Aluminum, zinc, magnesium and calcium Stearate white Bromothymol blue blue Bromocresol green green Acid Red 195 red
In yet further embodiments, compositions of the present 60 naphthylazo)-2-hydroxynaphthalene-3-carboxylic acid, invention further comprise one or more Substances from the aluminum salt of 1-(4-Sulfo-1-phenylazo)-2-naphthol-6-sul following group: 2,4-dihydroxyaZobenzene, 1-(2-chloro-4'- fonic acid, aluminum salt of 1-(4-sulfo-1-naphthylazo)-2- nitro-1'-phenylazo)-2-hydroxynaphthalene, Ceres Red, 2-(4- naphthol-3,6-disulfonic acid, 1-(4-sulfo-1-naphthylazo)-2- Sulfo-1-naphthylazo)-1-naphthol-4-Sulfonic acid, calcium naphthol-6,8-disulfonic acid, aluminum salt of 4-(4-sulfo-1- salt of 2-hydroxy-1,2-azonaphthalene-1'-sulfonic acid, cal 65 phenylazo)-1-(4-Sulfophenyl)-5-hydroxypyrazolone-3- cium and barium salts of 1-(2-sulfo-4-methyl-1-phenylazo)- carboxylic acid, aluminum and Zirconium salts of 4.5- 2-naphthylcarboxylic acid, calcium salt of 1-(2-sulfo-1- dibromofluorescein, aluminum and Zirconium salts of 2.4.5, US 9,084,734 B2 35 36 7-tetrabromofluorescein, 3',4',5',6'-tetrachloro-2,4,5,7- These are available under the name Reflecks. In addition to tetrabromofluorescein and its aluminum salt, aluminum salt the color, as a result of their particle size of from 40 nm to 180 of 2,4,5,7-tetraiodofluorescein, aluminum salt of quinophtha mm, they have a glitter effect. lone disulfonic acid, aluminum salt of indigo disulfonic acid, In yet further embodiments, effect pigments which are red and black iron oxide (CIN: 77 491 (red) and 77 499 available under the trade name Metasomes Standard/Glitter (black)), iron oxide hydrate (CIN: 77 492), manganese in various colors (yellow, red, green, blue) from Flora Tech ammonium diphosphate and titanium dioxide. find use in the compositions of the present invention. The In yet further embodiments, oil-soluble natural dyes, such glitter particles are present here in the mixtures with various as, for example, paprika extracts, 3-carotene or cochenille auxiliaries and dyes (such as, for example, the dyes with the 10 Colour Index (CI) Numbers 19140, 77007, 77289, 77491). find use in the present invention. In some embodiments, the dyes and pigments are present In yet additional embodiments, gel cream compositions of either individually or in a mixture. In alternative embodi the present invention comprise pearlescent pigments. In some ments, they are mutually coated with one another, different preferred embodiments, various pearlescent pigments find coating thicknesses generally giving rise to different color use in the present invention, including but not limited to 15 effects. In some embodiments, the total amount of dyes and “natural pearlescent pigments” (e.g., “pearl essence' gua color-imparting pigments is chosen from a range of concen nine/hypoxanthine mixed crystals from fish scales. “mother trations (e.g., from about 0.1% by weight to about 30% by of pearl ground mussel shells), and "monocrystalline weight; preferably from about 0.5 to about 15% by weight; pearlescent pigments” (e.g., bismuth oxychloride BiOCl); and most preferably from about 1.0 to about 10% by weight, and “layer Substrate pigments” (e.g. mica/metal oxide). in each case based on the total weight of the preparations). Bases for pearlescent pigments include, but are not limited The yet further embodiments, the present invention pro to pulverulent pigments, castor oil dispersions of bismuth vides methods for the preparation of the compositions of the oxychloride and/or titanium dioxide, bismuth oxychloride present invention. In some embodiments, these methods and/or titanium dioxide on mica. The luster pigment listed include combining and heating the constituents of the oil under CIN 77163, for example, is particularly advantageous. 25 phase and/or the water phase separately, and then combining An additional group of pearlescent pigments based on them together with stirring. In some preferred embodiments, mica/metal oxide find particular use in the present invention the phases are homogenized. In some particularly preferred is provided below. embodiments, the compositions are stirred with moderate to high input of energy, advantageously using a gear rim dis 30 persing machine at a rotary number up to at most 10000 rpm COATING LAYER (preferably in the range from about 2500 to about 7700 rpm). GROUP THICKNESS COLOR Cosmetic Formulations Comprising the Present Invention Silver-white pearlescent pigments TiO2:40-60 nm silver It is contemplated that the present invention will find use in Interference pigments TiO2: 60-80 nm. yellow numerous personal care compositions. It is not intended that TIO:80-100 nm red 35 the present invention be limited to any particular format or TiO2: 100-140 nm blue type of composition. The following description provides TiO2: 120-160 nm green Color luster pigments Fe2O3 bronze exemplary, not limiting compositions comprising the follow Fe2O. copper ing invention. Fe2O3 red Emulsions comprises one group of customary, commonly Fe2O3 red-violet 40 Fe2O3 red-green used cosmetics. The term "emulsion' is generally used in Fe2O. black reference to a heterogeneous system of two liquids which are Combination pigments TiO2/Fe2O. gold shades immiscible or miscible only to a limited extent with one TiO2/Cr2O. green another, which are usually referred to as “phases.” One phase TiO2/Berlin blue deep blue is typically in the form of droplets (i.e., the “dispersed.” TiO2 carmine red 45 “discontinuous” or “internal' phase), while the other liquid forms a continuous (i.e., “coherent or “external) phase. In some preferred embodiments, the pearlescent pigments Less common forms of application include multiple emul available from Merck under the trade names Timiron, Colo sions (i.e. those in which the droplets of the dispersed or rona or Dichrona find use in the present invention. However, discontinuous phase, comprise for their part droplets of a it is not intended that the present invention be limited to the 50 further dispersed phase, such as water/oil/water W/O/W specific pigments listed herein. Indeed, pearlescent pigments emulsions and oil/water/oil IO/W/O emulsions). that find use in the present invention are obtainable from If the oil phase is finely distributed in the water phase, then numerous sources. For example, other Substrates apart from this is an oil-in-water emulsion (O/W emulsion; e.g. milk). mica can be coated with further metal oxides, such as, for The basic character of an O/W emulsion is determined by the example, silica and the like. SiO particles coated with, for 55 water. These emulsions are generally less greasy on the skin, example, TiO, and FeO (“ronaspheres'), which are sold by are rather matting, and absorb more rapidly into the skin than Merck and are particularly suitable for the optical reduction W/O (water-in-oil) emulsions. of fine lines find use in the present invention. Those of skill in the art are familiar with a large number of In alternative embodiments, the Substrate (e.g., mica) is not options of formulating stable W/O preparations for cosmetic included. In some preferred embodiments, particular prefer 60 and/or dermatological uses, including Such formulations as ence is given to pearlescent pigments prepared using SiO. creams and ointments, which are spreadable in the range from Such pigments, which may also additionally have goniochro room temperature to skin temperature, as well as lotions and matic effects, are available, for example, under the trade name milks, which are more flowable in this temperature range. Sicopearl Fantastico, available from BASF. The stability of emulsions is dependent on their viscosity, In additional embodiments, pigments obtained from 65 in particular on the Viscosity of the external phase. An emul Engelhard/Mearl based on calcium sodium borosilicate sion becomes unstable when the finely dispersed particles which have been coated with titanium dioxide also find use. collect together to form relatively large aggregates, and the US 9,084,734 B2 37 38 droplets which are in contact coalesce. This process is UV-A radiation is much more harmful than UV-B radiation referred to as “coalescence.” The more viscous the external with regard to the triggering of photodynamic, specifically phase of the emulsion, the slower the process of coalescence. phototoxic, reactions and chronic changes in the skin. In Emulsions of “liquid' (flowable) consistency are used in addition, the harmful effects of UV-B radiation can be further various cosmetics (e.g., skin care lotions, cleansing lotions, intensified by exposure to UV-A radiation. face lotions, hand lotions, etc.). These compositions gener It has been shown that UV-A radiation by itself and under ally have a viscosity of from about 2000 mPa's to about very normal everyday conditions, is sufficient to damage 10,000 mPas. The stability of flowable emulsions is deserv collagen and elastin fibers, which are of essential importance ing of particular attention since the considerably greater for the structure and strength of the skin, within a short period. mobility of the particles promotes more rapid coalescence. 10 This leads to chronic light-induced changes in the skin, Such It is known that liquid emulsions typically presently in use that the skin prematurely "ages. The clinical appearance of generally comprise thickeners and are not stable toward rela skin aged by light typically includes increased wrinkles and tively high electrolyte concentrations. This is manifested in lines, and an irregular, furrowed relief. In addition, the skin phase separation of the compositions. However, in some areas affected by light-induced skin aging often show irregu embodiments, it is desirable to use certain electrolytes (e.g., 15 lar pigmentation. In some cases, brown patches, keratoses, water-soluble UV filters), in order to be able to utilize the carcinomas, or malignant melanomas arise. Skin prematurely other physical, chemical or physiological properties thereof. aged as a result of everyday UV exposure is also characterized Although in many cases appropriate choice of the emulsifier has having lowered activity of the Langerhans cells and system can provide remedies to a certain extent, other disad slight, chronic inflammation. Vantages then arise just as often. Approximately 90% of the ultraviolet radiation which For example, some disadvantages result due to the fact that reaches the Earth consists of UV-A rays. While amount of emulsifiers, like ultimately any chemical Substance, may trig UV-B radiation reaching Earth varies widely depending on ger allergic reactions or reactions based on oversensitivity numerous factors (e.g., time of year and day and/or degree of (i.e., hyperSensitivity) of the user. The use of customary cos latitude), the UV-A radiation levels that reach Earth remain metic emulsifiers is generally entirely without risk, although 25 relatively constant on a daily basis, irrespective of the time of for Some individuals, “hypoallergenic’ compositions are nec year and day or geographical factors. Additionally, the major essary and/or preferred. Indeed, in Some particularly sensitive ity of UV-A radiation penetrates the living epidermis, while individuals, certain dermatoses are triggered by exposure to about 70% of the UV-B rays are retained by the horny layer. certain emulsifiers and simultaneous exposure to Sunlight. Preventive protection against UV-A rays, for example by Thus, as known to those in the art, in some compositions, 30 applying light protection filter Substances in the form of a particular emulsifiers are less preferred and/or are avoided. cosmetic or dermatological formulation to the skin, is there It is possible to prepare emulsifier-free preparations. For fore of fundamental importance. example, some preparations have an oily phase which con In general, the light absorption behavior of light protection tains dispersed water droplets (i.e., it is similar to a W/O filter Substances is very well known and documented, largely emulsion). Such systems are sometimes called "hydrodisper 35 due to the fact that most industrialized countries have positive sions' or "oleodispersions.” depending upon which is the lists for the use of such substances, which impose very strict disperse phase and which is the continuous phase. standards on the documentation that accompanies each prod For cosmetic technology, it is, however, neither necessary uct which incorporates these Substances. For the concentra nor possible to dispense with emulsifiers altogether, espe tion of the substances in the finished formulations, the absor cially since there is a certain choice of particularly mild 40 bance values provide a guide, since interaction with emulsifiers. However, the emulsifiers in current use lack a substances within the skin or the surface of the skin itself satisfactorily broad range of choices. Thus, the application often presents variables that may impact how well the com spectrum of correspondingly mild cosmetic preparations positions perform on each individual. However, it is usually which are tolerated by the skin is limited. difficult to estimate beforehand, how uniformly and thickly In addition to the deleterious effects of some emulsifiers, 45 the filter substance is distributed in and on the horny layer of exposure to other factors is known to harm skin and hair. For the skin. example, the harmful effect of the ultraviolet portion of solar To test UV-A protection performance, use is usually made radiation on the skin is generally known. While rays having a of the IPD method (IPD 5 immediate pigment darkening) wavelength of less than 290 nm (i.e., the UVC region) are known to those in the art. This method is similar to the absorbed by the ozone layer in the earth's atmosphere, rays in 50 determination of the Sun protection factor, and provides a the range between 290 nm and 320 nm (i.e., the UVB region), method which indicates how much longer skin protected with cause erythema, simple Sunburn or even burns of varying the light protection composition can be irradiated with UV-A severity. The erythema activity maximum of sunlight is given radiation before the pigmentation which occurs is the same as as the relatively narrow region around 308 nm that produced for unprotected skin. Numerous compounds are known to provide protection 55 Another test method which has become established against harmful UVB radiation. Most commonly, these com throughout Europe is the Australian standard AS/NZS 2604: pounds are derivatives of 3-benzylidenecamphor, of 4-ami 1997. In this method, the absorption of the preparation in the nobenzoic acid, of cinnamic acid, of Salicylic acid, of ben UV-A region is measured. In order to satisfy the standard, the Zophenone, and of 2-phenyl-benzimidazole. preparation must absorb at least 90% of the UV-A radiation in It is also important to have available filter substances for 60 the region 320-360 nm. the range between about 320 nm and about 400 nm, the UVA Of concern in the formulation of sunscreen compositions is region, since its rays can also cause damage. For a long time that the use concentration of known light protection filter it was incorrectly assumed that the long-wave UV-A radiation substances which also exhibit high filter action in the UV-A having a wavelength of between 320 nm and 400 nm had only region are often limited by the very fact that they are com a negligible biological action and that, accordingly, the UV-B 65 bined with other substances which are in the form of solids. rays were responsible for most photodamage to the human Thus, there are certain formulation difficulties associate with skin. However, numerous recent studies have shown that achieving relatively high Sun protection factors and UV-A US 9,084,734 B2 39 40 protection performance. However, those of skill in the art are glycerol, ethylene glycol, ethylene glycol monoethyl or generally aware of means to overcome and/or compensate for monobutyl ether, propylene glycol monomethyl, monoethyl these difficulties. or monobutyl ether, diethylene glycol monomethyl or mono AS light protection filter Substances are generally expen ethyl ether and analogous products. In alternatively preferred sive and Some light protection filter Substances are addition- 5 embodiments, mixtures of two or more of these constituents ally difficult to incorporate into cosmetic and/or dermatologi find use in the present invention. cal preparations in relatively high concentrations, some The term “lipid' is often used as a generic term to refer to embodiments of the present invention were designed to pro fats, oils, waxes and the like. In addition, the terms “oil phase' vide simple and cost-effective preparations which, despite and “lipid phase' are also used synonymously. However, oils having unusually low concentrations of conventional UV-A 10 and fats differ from one another in their polarity, which is light protection filter substances, nevertheless achieve difficult to define. It has been proposed to adopt the interfacial acceptable or even high UV-A protection performance. tension toward water as a measure of the polarity index of an However, as known in the art, UV radiation can also lead to oil or of an oily phase. Thus, it is contemplated that the photochemical reactions which produce products that inter interfacial tension be regarded as a suitable measure of the fere with the skins metabolism. These photochemical reac- 15 polarity of a given oil component. The “interfacial tension” is tion products are predominantly free-radical compounds the force which acts on an imaginary line one meter in length (e.g., hydroxyl radicals). Undefined free-radical photoprod in the interface between two phases. In this measurement, the ucts which form in the skin itself can also exhibit uncontrolled lower the interfacial tension between the oily phase and secondary reactions as a result of their high reactivity. How water, the greater the polarity of the oily phase being ana ever, singlet oxygen, a non-free-radical excited State of the 20 lyzed. The physical unit for this interfacial tension is conven oxygen molecule, can also arise during UV irradiation, as can tionally calculated from the force/length relationship and is short-lived epoxides and many others. Singlet oxygen, for usually expressed in mN/m (millinewtons divided by meters). example, differs from normal triplet oxygen (free-radical It has a positive sign if it endeavours to reduce the interface. ground state) by virtue of its increased reactivity. However, In the converse case, it has a negative sign. As used herein, excited, reactive “free-radical triplet states of the oxygen 25 lipids are regarded as “polar, if their interfacial tension molecule also exist. Thus, in order to prevent these reactions, toward water is less than 30 mN/m. antioxidants and/or free-radical scavengers find use in cos “Polar oils” include those from the group of lecithins and of metic and/or dermatological formulations. fatty acid triglycerides, namely the triglycerol esters of Satu The compounds which are commonly used as light protec rated and/or unsaturated, branched and/or unbranched alkane tion agents in cosmetic and/or dermatological light protection 30 carboxylic acids having a chain length of from 8 to 24, in formulations are generally characterized as providing good particular 12 to 18, carbon atoms. In some embodiments, the light protection. However, they have the disadvantage that it is fatty acid triglycerides are chosen from the group consisting sometimes difficult to incorporate them into the desired for of synthetic, semi-synthetic and natural oils (e.g., olive oil, mulations in a satisfactory manner. Sunflower oil, soya oil, groundnut oil, rapeseed oil, almond As indicated above, the sun protection factor (SPF) indi- 35 oil, palm oil, coconut oil, castor oil, wheatgerm oil, grapeseed cates how much longer the skin protected with the light pro oil, thistle oil, evening primrose oil, macadamia nut oil and tection composition can be irradiated before the erythema the like). However, is it not intended that the present invention reaction which occurs is the same as for unprotected skin (i.e., be limited to compositions that contain particular polar oils. ten times as long compared with unprotected skin for an Additional examples of polar oils that find use in the present SPF=10). Consumers are very aware of the meaning of"SPF" 40 invention include the group of esters of Saturated and/or and choose skin and/or hair care products based on the SPF unsaturated, branched and/or unbranched alkane carboxylic values indicated on products. Consumers expect to receive acids having a chain length of from 3 to 30 carbon atoms and reliable information from manufacturers regarding the Sun saturated and/or unsaturated, branched and/or unbranched protection factor, largely due to increased public awareness of alcohols having a chain length of from 3 to 30 carbon atoms, the association between excess Sun exposure and skin cancer, 45 and from the group of esters of aromatic carboxylic acids and as well as premature aging. In addition, in Some parts of the saturated and/or unsaturated, branched and/or unbranched world, the degradation of the oZone layer is a major concern. alcohols having a chain length of from 3 to 30 carbon atoms. Depending upon the skin type and the Sun exposure expected, In some embodiments, such ester oils are chosen from the consumers choose products with a lower or a higher SPF. group consisting ofisopropyl myristate, isopropyl palmitate, However, there appears to be a tendency for consumers to 50 isopropyl Stearate, isopropyl oleate, n-butyl Stearate, n-hexyl select relatively high SPF factors, particularly for products to laurate, n-decyl oleate, isooctyl Stearate, isononyl Stearate, be applied to children and those with fair skin. In some isononyl isononanoate, 2-ethylhexyl palmitate, 2-ethylhexyl embodiments, the present invention provides compositions laurate, 2-hexyldecyl Stearate, 2-octyldodecyl palmitate, with relatively low concentrations of conventional light pro oleyl oleate, oleylerucate, erucyl oleate, erucyl erucate and tection filter substances, yet with SPF values that are accept- 55 synthetic, semi-synthetic and natural mixtures of Such esters able to consumers. (e.g., jojoba oil). In some preferred embodiments, the basic constituents of In addition, in Some embodiments, the oily phase is chosen the Sunscreen preparations provided by the present invention from the group consisting of dialkyl ethers, as well as Satu include: water or aqueous solutions; aqueous ethanolic solu rated or unsaturated, and branched or unbranched alcohols. In tions; natural oils and/or chemically modified natural oils 60 some particularly preferred embodiments, the oily phase of and/or synthetic oils; fats, waxes and other natural and Syn the compositions of the preferred embodiments also contains thetic fatty substances, preferably esters of fatty acids with Cis-alkylbenzoate, while in alternative embodiments, the alcohols of low carbon number (e.g., with isopropanol, pro preferred embodiments contains only the latter. In yet addi pylene glycol or glycerol), or esters of fatty alcohols with tional embodiments, the oil phase is chosen from the group of alkanoic acids of low carbon number or with fatty acids: 65 Guerbet alcohols (i.e., the group of alcohols named after alcohols, diols or polyols of low carbon number, and ethers Marcel Guerbet who first described their preparation). These thereof, preferably ethanol, isopropanol, propylene glycol, alcohols are formed according to the equation: US 9,084,734 B2 42 additional embodiments, nonpolar oils (e.g., those which are R chosen from the group of branched and unbranched hydro carbons and hydrocarbon waxes, in particular VASELINE(R) R-CH-CH-OH -catalyst A - R-CH-CH-OH i.e., petrolatum, paraffin oil, squalane and squalene, poly olefins and hydrogenated polyisobutenes find use in the by oxidation of an alcohol to an aldehyde, by aldol conden present invention. In some embodiments containing polyole sation of the aldehyde, elimination of water from the aldol fins, polydecenes are the preferred Substances. and hydrogenation of the allylaldehyde. Guerbetalcohols are Fatty and/or wax components which find use in embodi liquid even at low temperatures and result in virtually no skin ments of the present invention include but are not limited to 10 Vegetable waxes, animal waxes, mineral waxes and petro irritations. Thus, they find use as fatting, Superfatting and also chemical waxes. Examples which particularly preferred refatting constituents in skincare and hair care compositions. waxes include candelilla wax, carnauba wax, japan wax, Indeed, the use of Guerbet alcohols is known in the cosmetic esparto grass wax, cork wax, guaruma wax, rice germoil wax, art. In these applications, the species are generally character Sugarcane wax, berry wax, ouricury wax, montan wax, jojoba ized as having the following structure: 15 wax, shea butter, beeswax, shellac wax, spermaceti, lanolin (wool wax), uropygial grease, ceresin, oZokerite (earth wax), paraffin waxes and microcrystalline waxes. H Additional fatty and/or wax components that find use in the R-C-CH-OH present invention include chemically modified waxes and/or synthetic waxes (e.g., those commercially available under the R trade names SYNCROWAX(R) HRC glyceryl tribehenate and SYNSCROWAX(R AW 10 ICs-C fatty acid), which In this structure, R and R2 are usually unbranched alkyl are available from CRODA GmbH), and montan ester waxes, Sasol waxes, hydrogenatedjojoba waxes, synthetic or modi radicals. In some preferred embodiments of the present inven 25 tion the following Guerbet alcohols in which fied beeswaxes (e.g., dimethicone copolyol beeswax and/or R is propyl, butyl, pentyl, hexyl, heptyl or octyl and/or R is Co-so alkylbeeswax), polyalkylene waxes, polyethylene gly hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl col waxes, as well as chemically modified fats (e.g., hydro genated vegetable oils, such as hydrogenated castor oil and/or or tetradecyl find use in the present invention. In additional hydrogenated coconut fatty glycerides), triglycerides (e.g., embodiments, preferred Guerbet alcohols include 2-butyloc 30 trihydroxy Stearin, fatty acids, fatty acid esters, and glycol tanol with the following chemical structure: esters, such as, Co-Cao-alkyl Stearate, Co-Cao-alkylhydrox yStearoyl Stearate and/or glycol montanate). In further H embodiments, the present invention comprises certain orga nosilicone compounds, which have similar physical proper HC-C-CH-OH 35 ties to the specified fatty and/or wax components (e.g., Stear oxytrimethylsilane). In additional embodiments, the fatty and/or wax components are provided individually, while in still further embodiments, they are provided as a mixture. which is commercially available, for example, under the trade Indeed, it is intended that any desired mixture of such oil name ISOFOL(R) 12 (Condea Chemie GmbH), and 2-hexyl 40 and/or wax components will find use in various embodiments decanol with the following chemical structure: of the present invention. In some embodiments, the oily phase is selected from the group consisting of 2-ethylhexyl isostearate, octyldodecanol, H isotridecyl isononanoate, isoeicosane, 2-ethylhexyl cocoate, 45 C-C-alkyl benzoate, caprylic/capric triglyceride, and HC-C-CH-OH dicaprylyl ether. In alternative embodiments, mixtures of various oily phases are provided, including but not limited to mixtures comprising one or more of octyldodecanol, caprylic/capric triglyceride, dicaprylyl ether, C-C-alkyl which is commercially available, for example, under the trade 50 benzoate, 2-ethylhexyl isostearate, isotridecyl isononanoate. name ISOFOL(R) 16 (Condea Chemie GmbH). The following table provides a list of lipids which find use In additional embodiments, mixtures of Guerbet alcohols alone or in combination with other lipids in various embodi find use in compositions of the present invention. For ments of the present invention. The corresponding interfacial example, mixtures of 2-butyloctanol and 2-hexyldecanol find tensions toward water are given in the last column. However, use in some embodiments. The total amount of Guerbet alco 55 it is not intended that the present invention be limited to these hols in the finished cosmetic or dermatological preparations specific components, as other components find use in various is selected from the of range up to about 25.0% by weight, embodiments of the present invention, including mixtures of preferably about 0.5 to about 15.0% by weight, based on the greater or lesser polar components and the like. total weight of the preparations. However, it is not intended that the present invention be limited to any particular concen 60 tration nor range of concentrations, as those of skill in the art know how to prepare compositions having Suitable concen LIPIDS trations for the desired compositions and their use(s). In addi Trade name INCI name (m/Nm) tion, it is contemplated that any mixtures of oil and/or wax ISOFOL (R) 14T Butyl Decanol + Hexyl Decanol + 27.6 components will find use in the present invention. For 65 Hexyl Octanol + Butyl Octanol example, in some embodiments, waxes (e.g., cetyl palmitate) ISOFOL OR 16 Hexyl Decanol 24.3 find use as the sole lipid component of the oil phase. In US 9,084,734 B2 43 44 -continued sented here in general terms by the radicals R to R, where the number of different radicals is not necessarily limited to 4. LIPIDS n can assume values of 3/2 to 20. Fractional values for “n” take into consideration that uneven numbers of siloxyl groups Trade name INCI name (m/Nm) may be present in the cycle. EUTANOL (RG Octyldodecanol 24.8 In some embodiments, phenyltrimethicone is selected as CETIOL (ROE Dicaprylyl Ether 22.1 silicone oil. Other silicone oils suitable for use in various MIGLYOL (R 812 Caprylic Capric Triglyceride 21.3 CEGESOFT (R C24 Octyl Palmitate 23.1 embodiments of the present invention include, but are not Isopropyl Stearate Isopropyl Stearate 21.9 limited to dimethicone, phenyldimethicone, cyclomethicone ESTOL (R) 1540 EEHC Octyl Octanoate 3O.O 10 (octamethylcyclotetrasiloxane), hexamethylcyclotrisilox FINSOLV (RTN C2Cs Alkyl Benzoate 21.8 ane, polydimethylsiloxane, poly(methylphenylsiloxane), CETIOL (RSN Cetearyl Isonoanoate 28.6 DERMOFEEL (R) BGC Butylene Glycol Dicaprylate/Dicapate 21.5 cetyldimethicone, and behenoxydimethicone. In alternative TRIVENT (ROCG Tricaprylin 2O2 embodiments, mixtures of these compounds find use in the MOD Octyldodeceyl Myristate 22.1 present invention, including but not limited to mixtures of COSMACOL (RETI Di-C12-C Alkyl Tartrate 29.4 15 cyclomethicone and isotridecyl isononanoate, and mixtures MIGLYCOLOR. 829 Caprylic Capric Diglyceryl Succinate 29.5 PRISORINE OR 2036 Octyl Isostearate 29.7 of cyclomethicone and 2-ethylhexyl isostearate. It yet addi TEGOSOFT (RSH Stearyl Heptanoate 28.7 tional embodiments, silicone oils of similar constitution, Such ABIL (R) Wax 984O Cetyl Dimethicone 25.1 as the compounds referred to above whose organic side CETIOL (RLC Coco-Caprylate? Caprate 24.8 chains have been derivatized (e.g., polyethoxylated and/or IPP Isopropyl Palmitate 22.5 polypropoxylated) find use in the present invention. These LUVITOL (REHO Cetearyl Octanoate 28.6 include, but are not limited to Such compounds as polysilox CETIOL (R 868 Octyl Stearate 28.4 ane-polyalkyl-polyether copolymers such as cetyldimethi cone copolyol (i.e., cetyldimethicone copolyol (and) polyg In some embodiments, some or all of the oil phase of the lyceryl-4 isostearate (and) hexyl laurate). Indeed, it is not preparations are selected from the group consisting of cyclic 25 intended that the present invention be limited to any specific and/or linear silicones which are also often referred to as silicone oil nor mixture of silicone oils, as various oils find use “silicone oils.” In some embodiments, these silicones or sili in various embodiments of the present invention. cone oils are present as monomers which are generally char In additional embodiments, water in oil (W/O) emulsions acterized by structural elements as follows: find use in the present invention. In some embodiments, W/O 30 emulsifiers are used with or without additional co-emulsifi ers. In still further embodiments, W/O emulsions of the R present further comprise one or more emulsifiers, including, but not limited to one or more of the following compounds: R-O-Si-O-R lecithin, lanolin, microcrystalline wax (Cera microcris R4 35 tallina) in a mixture with paraffin oil (Paraffinum liquidum), oZokerite, hydrogenated castor oil, polyglyceryl-3 oleate, wool wax acid mixtures, wool wax alcohol mixtures, pen Silicones having two or more siloxyl units which find use taerythrithyl isostearate, polyglyceryl-3 diisoStearate, bees in some embodiments of the present invention are generally wax (Cera alba) and Stearic acid, sodium dihydrox characterized by structural elements as follows: 40 ycetylphosphate in a mixture with isopropyl hydroxycetyl ether, methylglucose dioleate, methylglucose dioleate in a mixture with hydroxy Stearate and beeswax, mineral oil in a R R2 mixture with petrolatum and oZokerite and glyceryl oleate and lanolin alcohol, petrolatum in a mixture with oZokerite o-i-o- s 45 and hydrogenated castor oil and glyceryl isostearate and R3 R4 polyglyceyl-3 oleate, PEG-7 hydrogenated castor oil, ozok iii. erite and hydrogenated castor oil, polyglyceryl-4 isostearate, polyglyceryl-4 isostearate in a mixture with cetyldimethicone where the silicon atoms may be substituted by identical or copolyol and hexyl laurate, laurylmethicone copolyol. different alkyl radicals and/or aryl radicals, which are repre 50 cetyldimethicone copolyol, acrylate/Co-Co-alkyl acrylate sented in general terms by the radicals R to Ra, where the crosspolymer, Poloxamer 101, polyglyceryl-2 dipolyhydrox number of different radicals is not necessarily limited to 4 and yStearate, polyglyceryl-3 diisoStearate, polyglyceryl-4 may assume values from 2 to 200,000. dipolyhydroxystearate, PEG-30 dipolyhydroxystearate, dii Cyclic silicones to be used advantageously according to the SoStearoyl polyglyceryl-3 diisostearate, polyglyceryl-2 invention are generally characterized by the structural ele 55 dipolyhydroxy Stearate, polyglyceryl-3 dipolyhydroxy Stear ments as follows ate, polyglyceryl-4 dipolyhydroxyStearate, polyglyceryl-3 dioleate. In yet additional embodiments of the present invention, W/O emulsions of the present invention comprise one or 60 O-Si-O-S more coemulsifiers, including, but not limited to the follow 1ng: glyceryl Stearate in a mixture with ceteareth-20, ceteareth-25, ceteareth-6 in a mixture with stearyl alcohol, cetylstearyl alcohol in a mixture with PEG-40 castor oil and sodium 65 cetylstearyl Sulfate, triceteareth-4 phosphate, sodium where the silicon atoms may be substituted by identical or cetylstearyl sulfate, lecithin trilaureth-4 phosphate, lau different alkyl radicals and/or aryl radicals, which are repre reth-4 phosphate, Stearic acid, propylene glycol Stearate US 9,084,734 B2 45 46 SE, PEG-25 hydrogenated castor oil, PEG-54 hydroge (e.g., cosmetic or skin care preparations) are present in the nated castor oil, PEG-6 caprylic/capric glycerides, glyc range from about 0.1 to about 10.0% by weight, preferably eryl oleate in a mixture with propylene glycol, ceteth-2, about 0.5 to about 5.0% by weight, based on the total weight ceteth-20, polysorbate 60, glyceryl Stearate in a mixture of the preparations. However, it is not intended that the with PEG-100 stearate, laureth-4, ceteareth-3, isostearyl 5 present invention be limited to any specific concentration of glyceryl ether, cetylstearyl alcohol in a mixture with emulsifier and/or co-emulsifier, as various embodiments of Sodium cetylstearyl Sulfate, laureth-23, Steareth-2, glyc the present invention have different preferred concentrations eryl Stearate in a mixture with PEG-30 stearate, PEG-40 and/or concentration ranges. Stearate, glycol distearate, PEG-22 dodecylglycol copoly In some embodiments, the present invention provides mer, polyglyceryl-2 PEG-4 stearate, ceteareth-20, methyl 10 emulsions in various forms, including skin protection creams, glucose sesquistearate, Steareth-10, PEG-20 Stearate, Ste skin lotions, cosmetic milks, Sunscreen creams, and Sun pro areth-2 in a mixture with PEG-8 distearate, steareth-21, tection milks. In some preferred embodiments, these compo steareth-20, isosteareth-20, PEG-45/dodecyl glycol sitions comprise fats, oils, waxes, and/or other fatty Sub copolymer, methoxy-PEG-22/dodecyl glycol copolymer, stances, as well as water, and one or more emulsifiers as are PEG-20 glyceryl Stearate, PEG-8 beeswax, polyglyceryl-2 15 customarily used for Such a type of formulation. laurate, isostearyl diglyceryl Succinate, Stearamidopropyl In addition to the liquid and somewhat more Solid emul PG dimonium chloride phosphate, glyceryl Stearate SE, sions of the cosmetic cleansing lotions and/or cleansing ceteth-20, triethyl citrate, PEG-20 methylglucose ses creams of the present invention, the present invention also quistearate, ceteareth-12, glyceryl Stearate citrate, cetyl provides sprayable cleansing preparations ("cleansing phosphate, triceteareth-4 phosphate, trilaureth-4 phos sprays”), which are used, for example, for removing make-up phate, polyglyceryl methylglucose distearate, potassium or as mild washing lotion. In addition, these cleansing sprays cetylphosphate, isosteareth-10, polyglyceryl-2 Sesquiisos find use in applications for treatment of blemished skin. tearate, ceteth-10, oleth-20, isoceteth-20, glyceryl Stearate These cleansing preparations also find use as "rinse-off in a mixture with ceteareth-20, ceteareth-12, cetylstearyl preparations’ (i.e., products which are rinsed off the skin alcohol and cetyl palmitate, cetylstearyl alcohol in a mix 25 following application). ture with PEG-20 stearate, PEG-30 stearate, PEG-40 stear In addition to the above constituents, various embodiments ate, and PEG-100 stearate. of the present invention include additional components, such In yet additional embodiments in which the oil phase of the as auxiliaries and additives, including but not limited to body preparations consists at least partially of silicone oils, silicone ing agents, fillers, perfume, dyes, emulsifiers, additional emulsifiers find use. In some embodiments, the silicone emul 30 active ingredients (e.g., vitamins and proteins), light protec sifiers are selected from the group of interface-active sub tion agents, stabilizers, insect repellents, alcohol, self-tanning stances, alkylmethicone copolyols, and/or alkyl dimethicone substances, water, salts, antimicrobials, proteases, and/or copolyols, particularly from the group of compounds charac keratinase, etc. Indeed, it is not intended that the present terized by the following chemical structure: invention be limited to any particular components, as long as
it is t t t ic- o- o- o––ct CH3 CH3 p (CH.)3 Y * CH3 O CH-O-CHO-X
in which X and Y, independently of one another, are chosen the active component comprising a scaffold and a peptide is from the group H and the branched and unbranched alkyl included. It is further contemplated that the present invention groups, acyl groups and alkoxy groups having 1 to 24 carbon will find use in numerous and various medicinal preparations. atoms, p is a number from 0 to 200, q is a number from 1 to 40, 50 In some preferred embodiments, the present invention pro and r is a number from 1 to 100. Some examples of silicone vides cosmetic and/or topical dermatological preparations emulsifiers which find use in the present invention include, Suitable for use as skin protection creams, cleansing milks, but are not limited to dimethicone copolyols (e.g., ABILR) B 8842, ABIL(R) B 8843, ABILR) B 8847, ABIL(R) B 8851, Sun screen lotions, nourishing creams, day creams, night ABILR) B 8852, ABIL(R) B 8863, ABILR) B 8873, and ABIL(R) creams etc. In some embodiments, the present invention finds B 88183, all of which are commercially available from Th. 55 use a components of drug (i.e., pharmaceutical) composi Goldschmidt AG). An additional example of an interface tions. In additional embodiments, the present invention finds active Substances which finds use in the present invention use in decorative cosmetics (e.g., make-up formulations). includes cetyldimethicone copolyol (ABIL(R) EM90), as well In some particularly preferred embodiments, the present as cyclomethiconedimethicone copolyol (ABTLR EM 97), invention provides Sunscreens useful in cosmetic and/or skin both of which are commercially available from Th. Gold 60 care preparations. In addition to the active ingredient used schmidt AG. An additional emulsifier which has proven use according to the embodiments of the present invention, in ful in various compositions that finds use in embodiments of Some embodiments, these preparations preferably addition the present invention is laurylmethicone copolyol (Dow ally comprise at least one broadband filter and/or at least one Corning R 5200 Formulation Aid), which is commercially UVA filter substance and/or at least one UVB filter substance available from Dow Corning Ltd. 65 and/or at least one inorganic pigment. In preferred embodiments of the present invention, the total In yet further embodiments, the present invention provides amount of emulsifiers used in the personal care compositions personal care compositions which have UV protection com US 9,084,734 B2 47 48 ponents, but which are not primarily Sunscreens. For dazolidinylurea, 5-chloro-2-methyl-4-isothiazolin-3-one, example, in some embodiments, UV-A and/or UV-B filter 2-chloroacetamide, benzalkonium chloride, benzyl alcohol, Substances are incorporated into day creams and/or hair care and formaldehyde donors. Further preservatives that find use compositions. in various embodiments of the present invention include phe In additional embodiments, the personal care compositions nyl hydroxyalkyl ethers, in particular the compounds known of the present invention comprise cosmetically active ingre as “phenoxyethanol.” due to their bactericidal and fungicidal dients, auxiliaries and/or additives, as are customarily used in effects on a number of microorganisms. Such preparations (e.g., antioxidants, preservatives, bacterio Yet otherantimicrobial agents are likewise suitable for use cides, perfumes, antifoams, dyes, pigments which have a in various embodiments of the present invention, including coloring action, thickeners, Surface-active Substances, emul 10 but not limited to 2,4,4-trichloro-2'-hydroxydiphenyl ether sifiers, emollients, moisturizers and/or humectants, fats, oils, (i.e., IRGASANR), 1,6-di(4-chlorophenylbiguanido)hexane waxes or other customary constituents of a cosmetic or der (i.e., CHLORHEXIDIN), 34,4'-trichlorocarbanilide, quater matological formulation, such as alcohols, polyols, poly nary ammonium compounds, oil of cloves, mint oil, thyme mers, foam stabilizers, electrolytes, organic solvents or sili oil, triethylcitrate, FARNESOL.R (3,7,11-trimethyl-2,6,10 cone derivatives). Indeed it is contemplated that various 15 dodecatrien-1-ol) and the active ingredients and/or active compounds will find use in the various embodiments of the ingredient combinations described in DE-3740 186, DE-39 present invention, as appropriate for the product and the user. 38 140, DE-42 04321, DE-4229707, DE-43 09372, DE-44 In still further embodiments, preservatives, such as those 11664, DE-195 41967, DE-195 43 695, DE-195 43 696, used in food and feed applications find use in various com DE-19547 160, DE-19602108, DE-196 02110, DE-19602 positions of the present invention. The following table pro 111, DE-19631 003, DE-19631 004, DE-19634 019, DE-42 vides a list of such compounds, as well as the E number for 29737, DE-4237 081, DE-4324219, DE-4429 467, DE-44 each compound. However, it is not intended that the present 23 410, and DE-19516705, all of which are hereby incorpo invention be limited to these specific preservatives, as it is rated by reference. In still further embodiments, sodium contemplated that additional preservatives will find use in hydrogencarbonate is also included in Some compositions of various embodiments of the present invention. 25 the present invention. However, it is not intended that the present invention be limited to any particular antimicrobial nor combination of anti-microbial, as various compounds Examples of Food Grade Preservatives That Find Use in having such effects will find use in various embodiments of Embodiments of the Present Invention the present invention. 30 2OO Sorbic acid In additional embodiments of the personal care composi 2O1 Sodium Sorbate tions of the present invention, compounds such as anti-irri 2O2 Potassium sorbate tants and/or anti-inflammatory actives are included. In some 2O3 Calcium sorbate embodiments, batyl alcohol (a-octadecyl glyceryl ether), 210 Benzoic acid 211 Sodium benzoate selachyl alcohol (a-9-octadecenyl glyceryl ether), chimyl 212 Potassium benzoate 35 alcohol (a-hexadecyl glyceryl ether), bisabolol, and/or pan 213 Calcium benzoate thenol are included. However, it is not intended that the 214 Ethyl p-hydroxybenzoate 215 p-hydroxybenzoic ethyl ester Nasalt present invention be limited to the incorporation of any spe 216 n-propyl p-hydroxybenzoate cific anti-irritant(s) and/or anti-inflammatory(ies), as various 217 p-hydroxybenzoic-n-propyl ester Nasalt compounds suitable for Such applications find use in the 218 methyl p-hydroxybenzoate 40 present invention. 219 p-hydroxybenzoic methyl ester Nasalt 220 Sulfur dioxide In still further embodiments of the present invention, anti 221 Sodium sulfite oxidants are incorporated in the personal care compositions. 222 Sodium hydrogensulfite It is contemplated that any suitable antioxidants will find use 223 Sodium disulfite in the personal care compositions of the present invention. 224 Potassium disulfite 45 226 Calcium sulfite Suitable antioxidants include, but are not limited to amino 227 Calcium hydrogen sulfite acids (e.g., glycine, histidine, tyrosine, and tryptophan) and 228 Potassium hydrogen sulfite derivatives thereof, imidazoles (e.g. urocanic acid) and 230 Biphenyl (Diphenyl) derivatives thereof, peptides (e.g., D.L-carnosine, D-carnos 231 Orthophenylphenol 232 Sodium orthophenylphenoxide ine, and L-carnosine) and derivatives thereof (e.g., anserine), 50 233 Thiabendazole carotenoids, carotenes (e.g., C.-carotene, 3-carotene, and Y-ly 235 Natamycin copene) and derivatives thereof, chlorogenic acid and deriva 236 Formic acid tives thereof, aurothioglucose, propylthiouracil and other thi 237 Sodium formate ols (e.g., thioredoxin, glutathione, cysteine, cystine, 238 Calcium formate 239 Hexamethylenetetramine cystamine and the glycosyl, N-acetyl, methyl, ethyl, propyl. 249 Potassium nitrite 55 amyl, butyl and lauryl, palmitoyl, oleyl, g-linoleyl, choles 250 Sodium nitrite teryl and glyceryl esters thereof) and salts thereof, dilauryl 251 Sodium nitrate thiodipropionate, distearyl thiodipropionate, thiodipropionic 252 Potassium nitrate 28O Propionic acid acid and derivatives thereof (e.g., esters, ethers, peptides, 281 Sodium propionate lipids, nucleotides, nucleosides and salts), and Sulfoximine 282 Calcium propionate 60 compounds (e.g. buthionine Sulfoximines, homocysteine Sul 283 Potassium propionate foXimine, buthionine Sulfones, penta-, hexa- and heptathion 290 Carbon dioxide ine Sulfoximine) in very small tolerated doses (e.g., typically pmol to mmol/kg), chelating agents (e.g., C.-hydroxy fatty Additional preservatives that find use in various embodi acids, palmitic acid, phytic acid, and lactoferrin), C-hydroxy ments include but are not limited to dibromodicyanobutane 65 acids (e.g. citric acid, lactic acid, and malic acid), humic acid, (2-bromo-2-bromomethylglutarodinitrile), 3-iodo-2-propi bile acid, bile extracts, bilirubin, biliverdin, EDTA, EGTA nylbutylcarbamate, 2-bromo-2-nitropropane-1,3-diol, imi and derivatives thereof, unsaturated fatty acids and deriva US 9,084,734 B2 49 50 tives thereof (e.g., linolenic acids, linoleic acid, oleic acid), between the individual layers. The layer structure is largely folic acid and derivatives thereof, furfurylidenesorbitol and defined by strong, covalent bonds. The stochiometry of the derivatives thereof, ubiquinone and ubiquinol and derivatives sheet silicates is (SiO5) for pure silicate structures and thereof, vitamin Cand derivatives thereof (e.g., Sodium ascor (Al,SiO.'”) for alumosilicates, wherein “m” is a byl phosphate, ascorbyl palmitate, Mg ascorbyl phosphate, number greater than Zero and less than 2. In some embodi and ascorbyl acetate), tocopherols and derivatives (e.g., vita ments in which alumosilicates are present in the absence of min E acetate), coniferyl benzoate of benzoin resin, ferulic pure silicates, each Si" group replaced by Al" requires acid, furfurylideneglucitol, carnosine; butylhydroxytoluene, another singly charged cation to neutralize the charge. The butylhydroxyanisole, nordihydroguaiacic acid, nordihy charge balance is preferably evened out by H', alkali metal droguaiaretic acid, trihydroxybutyrophenone, uric acid and 10 ions or alkaline earth metalions. In alternative embodiments, derivatives thereof, mannose and derivatives thereof, zinc and aluminum is used as a counterion. In contrast to the alumo derivatives thereof (e.g., ZnO, ZnSO4), selenium and deriva silicates, these compounds are referred to as "aluminum sili tives thereof (e.g., selenomethionine), Stilbenes and deriva cates.” “Aluminum alumosilicates.” in which aluminum is tives thereof (e.g., Stilbene oxide, trans-Stilbene oxide) and present both in the silicate network, and also as counterion, the derivatives thereof (e.g., salts, esters, ethers, Sugars, 15 also find use in Some embodiments of the present invention. nucleotides, nucleosides, peptides and lipids) of said active Phyllosilicates are well known in the art (See e.g., Holle ingredients which are suitable for the intended use of the mann et al., Lehrbuch der Anorganischen Chemie Textbook particular embodiment(s) of the present invention. of Inorganic Chemistry, 91st-100th Ed., Walter deGruyter— In some embodiments, the concentration of one or more Verlag 1985: Remy, Lehrbuch der Anorganischen Chemie, antioxidant in the compositions of the present invention is 12" Ed., Akademische Verlagsgesellschaft, Leipzig (1965). preferably from about 0.001 to about 30% by weight, particu The layer structure of montmorillonite is also known (See, larly preferably from about 0.05 to about 20% by weight, and Römpps Chemie-Lexikon, Franckhsche Verlagshandlung, more preferably from about 1 to about 10% by weight, based W. Keller & Co., Stuttgart, 8' Ed., 1985, p. 2668 f). on the total weight of the preparation. In additional embodi Examples of phyllosilicates include the following (montmo ments, in which vitamin E and/or its derivatives are utilized as 25 rillonite is the main mineral comprising the naturally-occur anti-oxidant(s), the preferred range is from about 0.001 to ring bentonites); about 10% by weight, based on the total weight of the formu Montmorillonite Nao ((Ali,Mgos)(OH)2(SOo)) lation. However, it is not intended that the present invention often simplified: AlO4SiOHO*nHO or be limited to any specific antioxidant concentration(s), as Al(OH)/SiO on HO various concentrations will find use in the various embodi 30 Kaolinite Al(OH) (SiOs) ments of the present invention. Illite (KHO),(Mg(OH)2(Si,Al, Olo) or In yet additional embodiments, the present invention pro (K.H.O),(Al(OH)2(Si,Al, Oo)) vides preparations suitable for use as deodorants and/or anti where y=0.7-0.9 perspirants. It is contemplated that any of the active ingredi Beidellite (Ca,Na)(Al(OH)(Alos SiOo)) ents which commonly find use in Such preparations will also 35 Nontronite Nao (Fe(OH)2(Alo S., Oo)) find use in various embodiments of the present invention. Saponite (Ca,Na)oss ((Mg,Fe)(OH)2(Aloss Sisa, Oo)) Additional components that are commonly used in Such Hectorite Nao ((Mg.Li)(OH,F)(SiOo)) preparations also find use in various embodiments of the In some preferred embodiments, inorganic gel formers present invention. Examples of Such actives and inactive including but not limited to aluminum silicates, such as the compounds include, but are not limited to odor maskers (e.g., 40 montmorillonites (bentonites, hectorites and derivatives perfumes), odor absorber (e.g., phyllosilicates described in thereof. Such as quaternium-18 bentonite, quaternium-18 DE-P 40 09347); as well as montmorillonite, kaolinite, illite, hectorites, Stearalkonium bentonites and Stearalkonium hec beidellite, nontronite, Saponite, hectorite, bentonite, Smectite, torites), and also magnesium-aluminum silicates (VEE and Zinc salts of ricinoleic acid. In some embodiments of the GUM(R) grades), and sodium-magnesium silicates (LAPO present invention, the range of active ingredients (i.e., one or 45 NITE(R) grades) find use in the present invention. more compounds) in Such preparations is preferably from Montmorillonites represent clay minerals which are a type about 0.001 to about 30% by weight; more preferably from of dioctahedral Smectites, and are masses which Swell in about 0.05 to about 20% by weight; and most particularly in water, but do not become plastic. The layer packets in the the range of from about 1 to about 10% by weight, based on three-layer structure of the montmorillonites can swell as a the total weight of the preparation. 50 result of reversible incorporation of water (in a 2- to 7-fold In some embodiments of the present invention, the water amount) and other Substances, such as, for example, alcohols, phase has a gel character which, in addition to an effective glycols, pyridine, picoline, ammonium compounds, content of compounds and solvents (as appropriate) prefer hydroxy-aluminosilicate ions etc. The chemical formula ably comprises water, further organic and/or inorganic thick given above provides just an approximation of the formula, as eners, and/or hydrocolloids. 55 montmorillonites have a large capacity for ion exchange. For In some embodiments, inorganic thickeners are selected example, Al can be replaced by Mg, Fe", Fe", Zn, Pb (e.g., from the group consisting of modified, unmodified, naturally from harmful substances in waste waters), Cr, Cu and other occurring, and synthetic phyllosilicates. Although it is gen elements. The resulting negative charge of the octahedral erally preferable to use pure components, in some embodi layers is compensated for by the presence of cations, in par ments, mixtures of different modified and/or unmodified 60 ticular Na" (i.e., sodium montmorillonite) and Ca" (i.e., phyllosilicates find use in various compositions of the present calcium montmorillonite, a compound that is only Swellable invention. As generally known in the art, phyllosilicates are to a very Small extent) in interlayer positions. silicates and alumosilicates in which the silicate or aluminate In alternative embodiments, synthetic magnesium silicates units are linked together via three Si-O or Al-O bonds and/or bentonites find use in the present invention, including and form a wavy sheet or layer structure. The fourth Si-O- 65 but not limited to Such commercially available compounds as or Al-O valence is saturated by cations. Relatively weak OPTIGEL(R) (Siid-Chemie). As indicated above, in some electrostatic interactions (e.g. hydrogen bridge bonds), exist embodiments, aluminum silicates such as the commercially US 9,084,734 B2 51 52 available VEEGUM(R) (R.T. Vanderbilt Comp. Inc), find use OMS, a bentonite paste in Shellsol T.; BENTONER) Gel in the present invention. Various VEEGUM(R) grades which OMS 25, a bentonite paste in isoparaffinic hydrocarbons find use in various embodiments of the present invention are (IDOPARR) H); and BENTONER) Gel IPP, a bentonite paste provided below. in isopropyl palmitate. “Hydrocolloid” is the technological abbreviation for the VEEGUM(R) Grades more correct name “hydrophilic colloid'Hydrocolloids are Regular macromolecules which have a largely linear structure and Grade HV K HS S-728 intermolecular forces of interaction which permit secondary 10 and primary valence bonds between the individual molecules SiO, 55.5 56.9 64.7 69.0 65.3 MgO 13.0 13.0 5.4 2.9 3.3 to form a recticular structure. Some hydrocolloids are water Al2O3 8.9 10.3 14.8 14.7 17.0 soluble natural or synthetic polymers which, in aqueous sys Fe2O3 1.O O.8 1.5 1.8 0.7 tems, form gels or viscous Solutions. These compounds CaO 2.0 2.O 1.1 1.3 1.3 increase the viscosity of water by either binding water mol Na2O 2.1 2.8 2.2 2.2 3.8 15 KO 1.3 1.3 1.9 0.4 O.2 ecules (hydration), or by absorbing and encapsulating the Ashing loss 11.1 12.6 7.6 5.5 7.5 water into their interwoven macromolecules, while restrict ing the mobility of water. These water-soluble polymers rep The above products swell in water to form viscous gels, resent a large group of natural and synthetic polymers that are which have an alkaline reaction. The organophilization of chemically very different, but which share a common feature montmorillonite or bentonites (exchange of the interlayer in their solubility in water or aqueous media. A prerequisite cations for quaternary alkylammonium ions) produces prod for this is that these polymers have a number of hydrophilic ucts (bentones) which are preferably used for dispersion in groups sufficient for solubility in water and are not too greatly organic solvents and oils, fats, ointments, inks, Surface coat crosslinked. These hydrophilic groups can be nonionic, ings and in detergents. 25 anionic or cationic in nature, for example as follows: BENTONER) is a trade name for various neutral and chemically inert gelling agents which are constructed from long-chain, organic ammonium salts and specific types of -NH, -NH-R -OH -SH -O- montmorillonite. BENTONER) gelling agents swell in 30 " organic media, which cause the media to also Swell. The gels -N- -COOH -NH-C-NH2 -NH-C-NH2 are resistant to diluted acids and alkalis, although they par -HN N NH2 tially lose their gelling properties upon prolonged contact with strong acids and alkalis. Because of their organophilic s character, BENTONE(R) gelling agents are only wettable by 35 N- -COO M -SO3 M" water with difficulty. There are various BENTONE(R) gelling agent grades commercially available, including those sold by NH2 Kronos Titan: BENTONER 27, an organically modified -PO2- MP -NH-- X - NRH-- X -NR-- X montmorillonite; BENTONE.R. 34 (dimethyldioctylammo nium bentonite; prepared in accordance with U.S. Pat. No. 40
2.531,427, incorporated herein by reference, which because -- -- of its lipophilic groups, Swells more readily in lipophilic O -NR -NR -- +/ medium than in water); BENTONER) 38, an organically - PR X -CH=y (CH2)n (CH2)n modified montmorillonite, available as a cream-colored to 45 white powder; BENTONER) LT, a purified clay mineral; O SO COO BENTONE(R) Gel MIO, an organically modified montmoril lonite which is Supplied as a very fine Suspension in mineral In some preferred embodiments, the group of the cosmeti oil (SUS-71) (10% bentonite, 86.7% mineral oil and 3.3% 50 cally and dermatologically relevant hydrocolloids are divided wetting agent); BENTONER) Gel IPM, an organically modi into the following groups: organic, natural compounds (e.g., fied bentonite which is suspended in isopropyl myristate agaragar, carrageen, tragacanth, gum arabic, alginates, pec (10% bentonite, 86.7% isopropylmyristate, 3.3% wetting tins, polyoses, guar flour, carobbean flour, starch, dextrins, agent); BENTONE(R) Gel CAO, an organically modified gelatins, and casein); organic, modified natural Substances montmorillonite which is taken up in castor oil (10% bento 55 (e.g., carboxymethylcellulose and other cellulose ethers, nite, 86.7% castor oil and 3.3% wetting agent); BENTONEe hydroxyethylcellulose and hydroxypropylcellulose and Gel Lantrol, an organically modified montmorillonite which, microcristalline cellulose); organic, completely synthetic in paste form, is intended for the further processing, in par compounds (e.g., polyacrylic and polymethacrylic com ticular for the preparation, of cosmetic compositions; 10% pounds, vinyl polymers, polycarboxylic acids, polyethers, bentonite, 64.9 LANTROL(R) (wool wax oil), 22.0 isopropyl 60 polyimines, polyamides, and polyurethanes); and inorganic myristate, 3.0 wetting agent and 0.1 propyl p-hydroxyben compounds (e.g., polysilicic acids, clay minerals, such as zoate: BENTONER) Gel Lan I, a 10% strength BENTONER montmorillonites, Zeolites, and silicas). 27 paste in a mixture of wool wax USP and isopropyl palmi In alternative embodiments, ethylcelluloses find use in tate; BENTONER) Gel Lan II, a bentonite paste in pure liquid 65 compositions of the present invention as stabilizers. Ethylcel wool wax: BENTONE(R) Gel NV, a 15% strength BEN luloses are characterized by the following structure. In this TONER 27 paste in dibutyl phthalate: BENTONER) Gel structure, the RS are either ethyl groups or hydrogen atoms. US 9,084,734 B2 53 54
R R O1 R R R R O HC O r O O HC o O O O R1 O O O O J. O HC y O HC y n R Y. h R R O NR R1 R
In some preferred embodiments, the degree of ethylation in lose, also sometimes referred to as “cellulose gum” (e.g., the ethylcellulose is from about 2.0 to about 3.0, correspond 15 NATROSOL(R) Plus 330 CS: Aqualon) finds use in the present ing to about 40 to about 55%, and more preferably about 48.0 invention. to about 49.5% ethylation. The average molecular mass is In additional embodiments, xanthan (CAS No. 11138-66 preferably chosen Such that the Viscosity of an approximately 2), (i.e., Xanthan gum), an anionic heteropolysaccharide gen 5% strength solution in a mixture of 80 parts of toluene and 20 erally formed by fermentation from maize Sugar and isolated parts of ethanol at 25°C. is 3 to 110 mPas, and more prefer as potassium salt finds use in the present invention. It is ably 9 to 11 mPas. In some particularly preferred embodi produced by Xanthomonas campestris and some other spe ments, the average molar mass is from about 100,000 to about cies under aerobic conditions and has a molecular weight of 400,000 g/mol. In some preferred embodiments, the ethylcel from 2x10 to 24x10. Xanthan is formed from a chain hav lulose concentration in compositions of the present invention ing cellulose with side chains. The structure of the Subgroups ranges from about 0.1 to about 10% by weight, based on the 25 consists of glucose, mannose, glucuronic acid, acetate and total weight of the preparations. Various ethylcelluloses find pyruvate. The number of pyruvate units determines the vis use in the present invention, including but not limited to those cosity of the Xanthan. that are commercially available (e.g., ETHOCEL(R) Standard In still further embodiments, carrageen is used as a gel 10 Premium; Dow Chemicals). former in compositions of the present invention. This com In yet additional embodiments, microcristalline cellulose 30 pound is an extract from North Atlantic red algae (Florideae: finds use as hydrocolloid in compositions of the present Chondrus crispus and Gigartina Stellata) that has a structure invention. Various microcrystalline cellulose preparations similar to that of agar. The term "carrageen” is frequently find use in the present invention, including but not limited to used in reference to a dried algae product and “carrageenan' those that are commercially available (e.g., AVICEL(R), such is used in reference to the extract thereof. The carrageen as AVICEL(R) RC-591, as well as AVICEL(R) RC/CL: 35 precipitated from the hot-water extract of the algae is a col AVICEL(R) CE-15; and AVICEL(R) 500; FMC Corporation orless to sand-colored powder with a molecular weight range Food and Pharmaceutical Products). In some particularly pre from about 100,000 to about 800,000 and a sulfate content of ferred embodiments, AVICEL(R) RC-591 (a modified micro about 25%. Carrageen, which is very readily soluble in warm cristalline cellulose which is made up of 89% microcrystal water, forms a thixotropic gel upon cooling, even if the water line cellulose and 11% sodium carboxymethylcellulose) finds 40 content is 95-98%. The rigidity of the gel is effected by the use in the present invention. double helix structure of the carrageen. Additional hydrocolloids that find use in the present inven In the case of carrageenan, three principle constituents are tion include methylcelluloses methylesters of cellulose). differentiated. The gel-forming “K fraction' consists of D-ga These compounds are characterized by the following struc lactose 4-sulfate and 3,6-anhydro-O-D-galactose, which has tural formula 45 alternate glycoside bonds in the 1.3- and 1.4 position (in contrast, agar contains 3,6-anhydro-O-L-galactose). The non gelling “w fraction' is composed of 1.3-glycosidically linked D-galactose 2-sulfate and 1,4-bonded D-galactose-2,6-disul ROCH2 OR a O fate radicals, and is readily soluble in cold water. Finally, 50 OfRO ORO O s “L-carrageenan, composed of D-galactose 4-Sulfate in 1.3 O bond and 3,6-anhydro-O-D-galactose 2-sulfate in 14 bond, is OR ROCH2 both water-soluble and also gel-forming. The nature of any pi cations which are present (K", NH.", Na", Mg", Ca") also influences the solubility of the carrageens. in which R can be a hydrogen or a methyl group. 55 In yet additional embodiments, chitosan (i.e., partially Cellulose mixed ethers (generally referred to as methylcel deacylated chitin) finds use in various compositions of the luloses, which contain, in addition to a predominating content present invention. Chitosan has film-forming properties and of methyl groups, also 2-hydroxyethyl 2-hydroxypropyl or is characterized as having a silky feel on the skin. One disad 2-hydroxybutyl groups) also find use in some embodiments Vantage for some uses, is its severe stickiness on the skin of the present invention. In some preferred embodiments, 60 which occurs in temporarily (usually) during application. hydroxypropyl)methyl-celluloses (e.g., METHOCEL(R) Due to this stickiness, some preparations are not acceptable to E4M: Dow Chemical Co.) find use in the present invention. consumers. However, chitosan finds use in some prepara In yet further embodiments sodium carboxymethylcellu tions, including hair care compositions, as it is better than lose (i.e., the sodium salt of the glycolic ether of cellulose, for chitin in thickening and/or stabilizing, as well as improving which R in the above structural formula may be hydrogen 65 the adhesion and water resistance of polymeric films. The use and/or CH COONa) finds use in the present invention. In of chitosan is well-known to those of skill in the personal care Some preferred embodiments, sodium carboxymethylcellu art (See e.g., Fiedler, Lexikon der Hilfsstoffe fir Pharmazie, US 9,084,734 B2 55 56 Kosmetik and angrenzende Gebiete, Lexikon of auxiliaries where R is a long-chain alkyl radical, and X and y represent for pharmacy, cosmetics and related fields).3" edition, Editio numbers which symbolize the respective stoichiometric pro Cantor, Aulendorf, 1989, p. 293). Chitosan is characterized portion of each of the comonomers. by the following structural formula: In some embodiments, acrylate copolymers and/or acry late-alkyl acrylate copolymers, include but are not limited to those that are commercially available (e.g., CARBOPOL(R) CHOH CH2OH CHOH 1382, CARBOPOLR 981, and CARBOPOLR 5984; B.F. Goodrich Co., and in particular, polyacrylates from the group OH of CARBOPOL grades 980,981, 1382, 2984, 5984 and Car bomer 2001). In additional embodiments, copolymers of Co HO 3O-alkyl acrylates and one or more monomers of acrylic acid, NH-X NH-X NH-X of methacrylic acid or esters thereof which are crosslinked pi with anallyl ether of sucrose or anallyl ether of pentaerythrito where nassumes values up to about 10 000, and X is either 15 find use in some embodiments of the present invention. the acetyl radical or hydrogen. Chitosan forms by deacetyla Compounds which carry the INCI name “Acrylates/Co-so tion and partial depolymerization (hydrolysis) of chitin, Alkyl Acrylate Crosspolymer also find use in some embodi which is characterized by the structural formula ments of the present invention. In some embodiments, com mercially available polymers (e.g., PEMULENR TR1 and PEMULENR TR2; B.F. Goodrich Co.) find use in some embodiments of the present invention, although it is not CHOH CH2OH CH2OH intended that the present invention be limited to any specific OH acrylate-containing composition. In yet additional embodiments, compounds which carry & NK")K") 25 the INCI name “ammonium acryloyldimethyltaurates/vi HO nylpyrrolidone copolymers' find use in the present invention. NH-CO NH-CO NH-CO These ammonium acryloyldimethyl taurate/vinylpyrrolidone hi, hi, hi, copolymers have the empirical formula C7HNSO, pi CHNO, which corresponds to the following structure: 30 Chitin is an essential constituent of the arthropod (e.g. insects, crabs, and spiders) ectoskeleton, and is also found in the connective and/or supporting tissues of other organisms pi iii. (e.g. mollusks, algae, and fungi). In the region of about pH-6. N O chitosan is positively charged and in that range is also soluble 35 in aqueous systems. It is incompatible with anionic raw mate rials. For this reason, in order to prepare chitosan-containing ic CH2 oil-in-water emulsions, the use of nonionic emulsifiers is H3C e appropriate (See e.g., EP 776 657). In some preferred so NH embodiments, the compositions of the present invention con 40 tain at least one chitosans with a degree of deacetylation of at least about >25%, and more preferably, a range of more than Preferred species of this compound are listed in Chemical about 55 to about 99% (as determined by means of H-NMR). Abstracts under the Registry numbers 58374-69-9, 13162 In some embodiments, chitosans of molecular weights 05-5 and 88-12-0, and are commercially available (e.g., between about 10,000 and about 1,000,000, in particular 45 ARISTOFLEX(R); Clariant GmbH). However, it is not those with molecular weights between 100,000 and 1,000, intended that the present invention be limited to any particular 000 (determined by means of gel permeation chromatogra species. In yet additional embodiments of the present inven phy) find use in the present invention. tion, copolymers/crosspolymers comprising acryloyldim In yet further embodiments, polyacrylates find use as gel ethyl taurate (e.g., SIMUGEL(R) EG and SIMUGEL(R) EG: ling agents in Some compositions of the present invention. 50 Seppic S.A.) find use in Some compositions of the present Suitable polyacrylates include but are not limited to acrylate invention. alkyl acrylate copolymers, in particular those chosen from the Additional completely synthetic hydrocolloids that find group of carbomers or CARBOPOLR) copolymers (B.F. Goo use in the present invention include, but are not limited to drich Co.). In particular, the acrylate-alkyl acrylate copoly anionic polyurethanes which are soluble or dispersible in mers that find use in Some embodiments of the present inven 55 water and which are advantageously obtainable from: tion have the following structure: Aa) at least one compound which contains two or more active hydrogen atoms per molecule, Ab) at least one diol containing acid or salt groups, and Ac) at least one diisocyanate. 60 In some preferred embodiments, the component Aa) is, in particular, a diol, aminoalcohol, diamine, polyesterol, poly etherol with a number-average molecular weight of in each case up to 3000, or mixtures thereof, where up to 3 mol% of said compounds may be replaced by triols or triamines. Pref 65 erence is given to diols and polyesterdiols. In particular, the component Aa) comprises at least 50% by weight, based on the total weight of the component Aa), of a polyesterdiol. US 9,084,734 B2 57 58 Suitable polyesterdiols are all those which are customarily weight solutions in N-methylpyrrolidone at 25°C. and pH 7) used for the preparation of polyurethanes, in particular the of from about 15 to about 100, and preferably about 25 to reaction products of phthalic acid and diethylene glycol, about 50. The K value (i.e., “intrinsic viscosity'), is a param isophthalic acid and 1,4-butanediol, isophthalic acid/adipic eter which is easy to determine by means of Viscosity mea acid and 1.6-hexanediol, and adipic acid and ethylene glycol Surements of polymer Solutions and is therefore frequently or 5-NaSO-isophthalic acid, phthalic acid, adipic acid and used in the industrial sector for characterizing polymers. 1,6-hexanediol. Polyurethanes containing acid groups that find use in some Examples of diols which find use in some embodiments of embodiments of the present invention include, but are not the present invention include, but are not limited to ethylene limited to polyurethanes that are water-soluble or dispersible glycol, propylene glycol, butylene glycol, neopentylglycol, 10 without the aid of emulsifiers after partial or complete neu polyetherols (e.g., polyethylene glycols having molecular tralization. The salts of the polyurethanes generally have better solubility or dispersibility in water than the unneutral weights up to 3000), block copolymers of ethylene oxide and ized polyurethanes. Bases which find use for the neutraliza propylene oxide with number-average molecular weights of tion of the polyurethanes include alkali metal bases (e.g., up to 3000, and block copolymers of ethylene oxide, propy 15 Sodium hydroxide solution, potassium hydroxide solution, lene oxide and butylene oxide which contain the copolymer Soda, Sodium hydrogencarbonate, potassium carbonate or ized alkylene oxide units in randomly distributed manner or potassium hydrogen carbonate) and alkaline earth metal in the form of blocks. Preference is given to ethylene glycol, bases (e.g., calcium hydroxide, calcium oxide, magnesium neopentylglycol, di-, tri-, tetra-, penta- or hexaethylene gly hydroxide or magnesium carbonate, and ammonia and col. Other diols which find use include poly(C.-hydroxycar amines). In some embodiments, 2-amino-2-methylpropanol, boxylic acid)diols. diethylaminopropylamine and triisoproanolamine find par Suitable aminoalcohols that find use in some embodiments ticular use in the neutralization of the polyurethanes contain of the present invention include but are not limited to 2-ami noethanol. 2-(N-methylamino)ethanol, 3-aminopropanol, ing acid groups. In yet additional embodiments, the neutral and 4-aminobutanol. ization of the polyurethanes containing acid groups is carried 25 out using mixtures of two or more bases (e.g. mixtures of In some embodiments, diamines such as ethylenediamine, Sodium hydroxide solution and triisopropanolamine). propylenediamine, 1,4-diaminobutan, 1,6-diaminohexane, Depending on the intended use, neutralization is partial (e.g. and C,c)-diamines which can be prepared by amination of about 20 to about 40%) or complete (i.e., 100%). These polyalkylene oxides with ammonia find use in some compo polymers and their preparation are described in more detail in sitions of the present invention. 30 DE-A-42 25 045, incorporated herein by reference. Component Ab) is, in particular, dimethylolpropanoic acid or B. Water-soluble or -dispersible cationic polyurethanes and a compound with the formula: polyureas of: Ba) at least one diisocyanate, which may have already been reacted beforehand with one or more compounds which 35 contain two or more active hydrogen atoms per mol HO-RR RR-OH and ecule, and No O1 Bb) at least one diol, primary or secondary amino alcohol, primary or secondary diamine or primary or secondary HOOC COOH triamine with one or more tertiary, quaternary or proto O O 40 nated tertiary amino nitrogen atoms. Preferred diisocyanates are as given above under A). Com HO-RR No O1 RR-OH pounds with two or more active hydrogen atoms are diols, aminoalcohols, diamines, polyesterols, polyamidediamines and polyetherols. Suitable compounds of this type are as 45 given above under A). The polyurethanes are prepared as described above under SOMe A). Charged cationic groups can be produced in the polyureas from the tertiary amino nitrogen atoms present either by where RR is in each case a C-C-alkylene group and Me protonation, (e.g., with carboxylic acids, Such as lactic acid), is Na or K. 50 or by quaternization (e.g. with alkylating agents, such as C to Component Ac) is, in particular, hexamethylene diisocy C-alkyl halides) or Sulfates. Examples of Such alkylating anate, isophorone diisocyanate, methyldiphenyl isocyanate agents include, but are not limited to ethyl chloride, ethyl (MDI), and/or tolylene diisocyanate. bromide, methyl chloride, methyl bromide, dimethyl sulfate In some embodiments, the polyurethanes are obtained by and diethyl sulfate. These polymers and their preparation are reacting the compounds of groups Aa) and Ab) under an 55 described in more detail in DE-A-4241 118, which is incor inert-gas atmosphere in an inert Solvent at temperatures of porated herein by reference. from 70 to 130° C. with the compounds of group Ac). This C. Linear polyurethanes with carboxylate groups of: reaction can be carried out, where appropriate, in the presence Ca) a 2.2-hydroxymethyl-substituted carboxylic acid of of chain extenders in order to prepare polyurethanes with the formula relatively high molecular weights. As is customary in the 60 preparation of polyurethanes, the components (Aa)+(Ab): Acare advantageously used in the molar ratio of from 0.8 to HC-OH 1.1:1. The acid number of the polyurethanes is determined by the composition and the concentration of the compounds of RR-C-COOH component (Ab) in the mixture of components (Aa) and (Ab). 65 HC-OH In some embodiments, the polyurethanes have K values according to H. Fikentscher (determined in 0.1% strength by US 9,084,734 B2 59 60 in which RR' is a hydrogen atom or a C-Co-alkyl nium chloride), modified magnesium aluminum silicates group, which is used in an amount which suffices for (e.g., quaternium-18-hectorite, which is commercially avail about 0.35 to about 2.25 milliequivalents of carboxyl able (e.g., BENTONE.R. 38; Rheox), and/or stearalkonium groups to be present in the polyurethane per g of poly hectorite, which is commercially available (e.g., SOFTI urethane, SANR) gel; Hills AG) find use in some personal care compo Cb) about 10 to about 90% by weight, based on the weight sitions of the present invention. However, it is not intended of the polyurethane, of one or more organic compounds that the present invention be limited to any particular cationic with not more than two active hydrogen atoms and polymer. Cc) one or more organic diisocyanates. In some yet further embodiments, some compositions of In some preferred embodiments, the carboxyl groups 10 present in the polyurethane are, finally, at least partially neu the present invention comprise oil thickeners in order to tralized with a suitable base. These polymers and their prepa improve the tactile properties of emulsions. Preferred oil ration are described in EP-A-619 111, incorporated herein by thickeners include, but are not limited to other Solids (e.g., reference. hydrophobic silicon oxides of the AEROSILR type, which D. Carboxyl-containing polycondensation products of anhy 15 are available from Degussa AG). Examples of advantageous drides of tri- or tetracarboxylic acids and diols, diamines or AEROSILR) oxide grades include AEROSIL(R) OX50, aminoalcohols (polyesters, polyamides or polyester AEROSIL(R) 130, AEROSIL(R) 150, AEROSILOR) 200, AERO amides). These polymers and their preparation are SILR 300, AEROSILR 380, AEROSIL(R) MOX 80, AERO described in more detail in DE-A-42 24761, incorporated SIL(R) MOX 170, AEROSIL(R) COK 84, AEROSIL(R) R 202, herein by reference. AEROSIL(R) R 805, AEROSIL(R) R 812, AEROSIL(R) R972, E. Polyacrylates and polymethacrylates, as are described in AEROSILOR) R974 and AEROSILOR) R976. more detail in DE-A-43 14305, 36.27970 and 29 17504, In some additional embodiments, some personal care com all of which are incorporated herein by reference. positions of the present invention comprise at least one “metal The polymers used in some embodiments of the present Soap' (i.e., a salt of a higher fatty acid, with the exception of invention have a K value of from about 15 to about 100, and 25 alkali metal salt), which are function as oil thickeners. more preferably from about 25 to about 50. The polymers are Examples of Such metal Soaps include, but are not limited to generally present in the composition in an amount in the range aluminum Stearate, Zinc Stearate and/or magnesium Stearate. from about 0.2 to about 20% by weight, based on the total In some further embodiments, some personal care compo weight of the compositions. The salt is used in an amount sitions comprise at least one amphoteric and/or Zwitterionic effective for improving the exchangeability of the polymers. 30 Surfactant (e.g., cocamidopropylbetain) and/or moisturizer The salt is generally used in an amount of from about 0.02 to (e.g. betain). Examples of amphoteric Surfactants that find use about 10% by weight, and more preferably from about 0.05 to in such embodiments of the present invention include but are about 5% by weight, and in particular, from about 0.1 to about not limited to acyl/dialkylethylenediamine (e.g., sodium acy 3% by weight, based on the total weight of the composition. lamphoacetate), disodium acylamphodipropionate, disodium The total amount of one or more hydrocolloids in some 35 alkylamphodiacetate, sodium acylamphohydroxypropylsul embodiments of the personal care compositions of the present fonate, disodium acylamphodiacetate, Sodium acylamphop invention is less than about 5% by weight, preferably between ropionate, N-alkylamino acids, for example aminopropyla about 0.05 and about 3.0% by weight, and more preferably lkylglutamide, alkylaminopropionic acid, Sodium between about 0.1 and about 1.0% by weight, based on the alkylimidodipropionate, and lauroamphocarboxyglycinate. total weight of the preparations. 40 In some embodiments, the amount of Surface- or interface In some additional embodiments, interface- and/or Sur active Substances (one or more compounds) in the prepara face-active agents are included in some personal care com tions is preferably between about 0.001 and about 30% by positions of the present invention, including but not limited to weight, and more preferably between about 0.05 and about cationic emulsifiers (e.g., quaternary Surfactants). 20% by weight, in most preferably between about 1 and about Quaternary Surfactants that contain at least one Natom 45 10% by weight, based on the total weight of the preparation. which is covalently bonded to 4 alkyl or aryl groups. This In Some yet additional embodiments, the active ingredients leads, irrespective of the pH, to a positive charge. Alkylbetain, (one or more compounds) comprise at least one lipophilic alkylamidopropylbetain and alkylamidopropylhydroxysul active ingredient. In some embodiments, these lipophilic taine are examples of quaternary Surfactants that find use in active ingredients are selected from the group consisting of Some embodiments of the present invention. 50 acetylsalicylic acid, atropine, azulene, hydrocortisone and The cationic Surfactants provided in Some embodiments of derivatives thereof (e.g., hydrocortisone-17-valerate), B vita the present invention also include, but are not limited to mins, D Vitamins, vitamin B, Vitamin B, Vitamin D, ret quaternary ammonium compounds, in particular benzyltri inoid, bisabolol, unsaturated fatty acids (e.g., the essential alkylammonium chlorides or bromides (e.g., benzyldimeth fatty acids often also referred to as “vitamin F), Y-linolenic ylstearylammonium chloride), alkyltrialkylammonium salts 55 acid, oleic acid, eicosapentenoic acid, docosahexenoic acid (e.g., cetyltrimethylammonium chloride or bromide), alky and derivatives thereof, chloramphenicol, caffeine, prostag ldimethylhydroxyethylammonium chlorides or bromides, landins, thymol, camphor, extracts or other products of a dialkyldimethylammonium chlorides or bromides, alkylami Vegetable and animal origin (e.g. evening primrose oil, bor doethyltrimethylammonium ether sulfates, alkylpyridinium rage oil or currant seed oil, fish oils, cod-liver oil), and cera salts (e.g., lauryl- or cetylpyrimidinium chloride), imidazo 60 mides and ceramide-like compounds, etc. In some embodi line derivatives, and compounds with a cationic character, ments, the active ingredient(s) are refatting Substances (e.g., Such as amine oxides (e.g., alkyldimethylamine oxides or purcellin oil, EUCERITR) and/or NEROCERITR). alkylaminoethyldimethylamine oxides). In some preferred In some yet further embodiments, the active ingredient(s) embodiments, cetyltrimethylammonium salts find use in comprise NO synthesase inhibitors. These embodiments find Some personal care compositions of the present invention. 65 particular use intreatment and/or prophylaxis of the signs and In yet additional embodiments, cationic polymers (e.g., symptoms associated intrinsic and/or extrinsic skin aging, as JAGUAR(R) C 162 hydroxypropyl guar hydroxypropyltrimo well as for the treatment and/or prophylaxis associated with US 9,084,734 B2 61 62 the harmful effects of ultraviolet radiation on the skin. In -continued some preferred embodiments, the NO synthase inhibitor is nitroarginine. FLAVONES In yet some additional embodiments, the active ingredient(s) is/are catechins, bile esters of catechins, and/or 5 aqueous or organic extracts from plants or sections of plants OH Substitution Positions which have a content of catechins or bile esters of catechins (e.g., the leaves of the Theaceae plant family, in particular of the species Camelia sinensis green tea). Their typical 10 ingredients (e.g., polyphenols or catechins, caffeine, Vita Kaempferol ------mins, Sugars, minerals, aminoacids, lipids) find particular use in Some embodiments of the present invention. Quercetin ------In some embodiments, catechins find use in the present Morin ------invention. Catechins are a group of compounds which are Robinetin ------regarded as hydrogenated flavones or anthocyanidines, and 15 Gossypetin ------are derivatives of “catechin' (catechol. 3,3',4',5,7-flavanpen tol, 2-(3,4-dihydroxyphenyl)chroman-3,5,7-triol). Epicat Myricetin ------echin (2R,3R)-3.3',4',5,7-flavanpentol) is also an active ingredient that finds use in some embodiments of the present invention. In nature, flavones are usually present in glycosylated In yet additional embodiments, plant extracts with a con form. tent of catechin, in particular extracts of green tea (e.g., In some further embodiments, the personal care composi extracts from leaves of the plants of the genus Camelia, in tions of the present invention comprise at least one flavonoids particular those used for tea, Such as C. Sinenis, C. assamica, having generic structural formula: C. taliensis. and C. irrawadiensis and hybrids of these species 25 with other species, such as C. japonica) find use in some personal care compositions of the present invention. In some further embodiments, preferred active ingredients include polyphenols and catechins from the group (-)-cat echin, (+)-catechin, (-)-catechin gallate, (-)-gallocatechin 30 gallate, (+)-epicatethin, (-)-epicatechin, (-)-epicatechingal late, (-)-epigallocatechin, and (-)-epigallocatechin gallate. In some additional embodiments of the compositions of the present invention flavone and its derivatives (also often col lectively called “flavones') find used. These compounds have 35 the following basic structure (Substitution positions are Z6 O shown): where Z to Z7, independently of one another, are chosen from the group consisting of H, OH, alkoxy and hydroxy 40 alkoxy, where the alkoxy and hydroxyalkoxy groups can be branched or unbranched and have 1 to 18 carbon atoms, and where Gly is chosen from the group of mono- and oligogly coside radicals. 45 In some alternative embodiments, the personal care com positions of the present invention comprise at least one fla vonoids having the generic structural formula:
Some of the more important flavones which find use in 50 Some personal care compositions of the present invention are provided below. However, it is not intended that the present invention be limited to any particular flavone. 55
FLAVONES
OH Substitution Positions Z6 O 3 5 7 8 2 3' 4' 5" 60 Flavone Flavonol where Z to Z independently of one another, are chosen Chrysin from the group consisting of H, OH, alkoxy and hydroxy Galangin Apigenin alkoxy, where the alkoxy and hydroxyalkoxy groups may be Fisetin - - 65 branched or unbranched and have 1 to 18 carbon atoms, Luteolin where Gly is chosen from the group mono and oligoglycoside radicals. US 9,084,734 B2 63 64 In some preferred embodiments, the composition has the one another, are selected from the group of hexosyl radicals, generic structural formula in particular of rhamnosyl radicals and glucosyl radicals. However, other hexosyl radicals, for example allosyl, altrosyl, galactosyl, gulosyl, idosyl, mannosyl and talosyl, find use in Some embodiments of the present invention. In addition, in Some embodiments, pentosyl radicals find use in the present invention. In some preferred embodiments, the personal care compositions of the present invention comprise Gly-Gly-O O one or more flavone glucoside selected from the group con Gly 10 sisting of a-glucosylrutin, a-glucosylmyricetin, a-glucosyl isoquercitrin, a-glucosylisoquercetin and a-glucosylquercit rin. In some particularly preferred embodiments, the flavone Z6 O glucoside is a-glucosylrutin. In yet some additional embodiments, the personal care 15 compositions of the present invention comprise at least one where Gly, Gly, and Gly, independently of one another, are naringin (e.g., aurantin, naringenin-7-rhamno-glucoside), monoglycoside radicals. Gly, and Gly may also, individu hesperidin 3',5,7-trihydroxy-4-methoxyflavanone-7-rutino ally or together, represent saturations by hydrogen atoms. In side, hesperidoside, hesperetin-7-O-rutinoside), rutin (3,3',4', Some preferred embodiments, Gly, Gly, and Gly, indepen 5,7-pentahydroxyflavone-3-rutinoside, quercetin-3-rutino dently of one another, are selected from the group of hexosyl side, Sophorin, birutan, rutabion, taurutin, phytomelin, radicals, in particular the rhamnosyl radicals and glucosyl melin), troxerutin (3,5-dihydroxy-3',4',7-tris(2-hydroxy radicals. However, hexosyl radicals, for example allosyl, ethoxy)flavone-3-(6-O-(6-deoxy-a-L-mannopyranosyl)-b- altrosyl, galactosyl, gulosyl, idosyl, mannosyl and talosyl, D-glucopyranoside)), monoxerutin (3.3',4',5-tetrahydroxy also find use in some embodiments of the present invention. 7-(2-hydroxyethoxy)flavone-3-(6-O-(6-deoxy-a-L- In yet additional embodiments, pentosyl radicals find use in 25 mannopyranosyl)-b-D-glucopyranoside)), dihydrorobinetin Some personal care compositions of the present invention. (3.3',4',5'.7-pentahydroxyflavanone), taxifolin (3.3',4',5,7- In some embodiments, Z to Zs are, independently of one pentahydroxyflavanone), eriodictyol-7-glucoside (3',4',5,7- another, advantageously chosen from the group consisting of tetrahydroxyflavanone-7 glucoside), flavanomarein (3',4',7, H, OH, methoxy, ethoxy and 2-hydroxyethoxy, and the fla 8-tetrahydroxyflavanone-7 glucoside), and/or isoquercetin Vone glycosides have the structure: 30 (3.3',4',5,7-pentahydroxyflavanone-3-(b-D-glucopyrano side). In some yet further embodiments, the active ingredient is selected from the group consisting of ubiquinones and plastoquinones. Ubiquinones are characterized by the struc tural formula: Z1 Z3 35
Z7 O
40
Z6 O forcis Gly3 45 In some embodiments, the flavone glycosides provided in Ubiquinones are the most widespread and the most investi Some of the personalcare compositions of the present inven gated bioquinones. Ubiquinones are referred to, depending tion have the following structure: on the number of isoprene units linked in the side chain, as Q-1, Q-2, Q-3 etc., or according to the number of carbon 50 atoms, as U-5, U-10, U-15 etc. They preferably arise with Z2 certain chain lengths (e.g. in Some microorganisms and yeasts Z3 where n=6). In most mammals, including humans, Q10 pre dominates. Coenzyme Q10 finds particular use in some HO O 55 embodiments of the present invention. Its structural formula 1S
OH O fy 1-Gly2 60 Gly where Gly, Gly, and Gly, independently of one another, are monoglycoside radicals. Gly, and Gly can also, individually 65 or together, represent Saturations by hydrogenatoms. In alter native embodiments, Gly, Gly, and Gly, independently of US 9,084,734 B2 65 66 Plastoquinones have the general structural formula: the total weight of the preparation. It is further contemplated that those of skill in the art will formulate personal care
compositions of the present invention with active(s) concen trations that are suitable for the intended use of the composi tions. DETAILED DESCRIPTION OF THE INVENTION The present invention provides peptides and Supported peptides for treating various diseases and conditions. In par 10 ticularly preferred embodiments, the present invention pro vides compositions and methods for personal care. In some embodiments, the present invention provides compositions Plastoquinones differ in the number n of isoprene radicals for use in skin and/or hair care, as well as cosmetic compo and are referred to accordingly (e.g. PO-9 n=9). In addition, sitions. In alternative particularly preferred embodiments, the other plastoquinones with varying Substituents on the 15 present invention provides peptides and Supported peptides quinone ring exist in Some embodiments. for treating diseases of the skin, Such as rosacea. In some In some still further embodiments, the present invention particularly preferred embodiments, the Supported peptides comprises at least one creatine and/or creatine derivative. of the present invention are anti-VEGF peptides. In alterna tive particularly preferred embodiments, the anti-VEGF pep Creatine has the following structure: tides are expressed on a scaffold protein. In some most pre ferred embodiments, the scaffold protein comprises BBI. In some preferred embodiments, the present invention pro HN*V CH, OH vides cosmetic and/or pharmaceutical compounds Suitable C-N 12/C for improving the appearance of skin. The present invention in V further provides peptides that block binding of a protein. In CH3 O 25 some preferred embodiments, the protein is VEGF. In some particularly preferred embodiments, the peptide is expressed In some preferred embodiments of the personal care com in a protease-resistant scaffold. In some especially preferred embodiments, the scaffold is a protease inhibitor (e.g., BBI, positions of the present invention creatine phosphate, creatine STI, or Eglin chymotrypsin inhibitor). In some most pre Sulfate, creatine acetate, creatine ascorbate, and/or deriva 30 ferred embodiments, the protease inhibitor is a BBI that has tives esterified at the carboxyl group with mono- or polyfunc been functionally and/or structurally modified. tional alcohols find use. As indicated above, two protein protease inhibitors have In some additional embodiments, the personal care com been isolated from soybeans, the Kunitz-type trypsin inhibi positions of the present invention contain L-carnitine 3-hy tor (soybean trypsin inhibitor, STI) and the Bowman-Birk droxy-4-(trimethylammonio)butyrobetaine. Acylcarnitines 35 protease inhibitor (BBI) (See e.g., Birk, Int. J. Pept. Protein have the following general structure: Res., 25:113-131 (1985; and Kennedy, Am. J. Clin. Neutr., 68:1406 S-1412S 1998). These inhibitors serve as a begin ning scaffold for the variant sequences provided herein to O V produce the protease inhibitor in combination with at least C-R 40 one peptide sequence that has been modified and/or Substi / tuted in the sequence (e.g., BBI-AV or STI-AV). In addition to O alterations in the scaffold comprising the variant sequences, (H3C)N-CH-C-CH-COO other desired proteins used herein include the addition of six H histidine residues at the C-terminus (See, FIGS. 1 and 2). 45 Soybean Trypsin Inhibitor (STI) STI inhibits the proteolytic activity of trypsin by the for where R is chosen from the group of branched and mation of a stable stoichiometric complex (See e.g., Liu, unbranched alkyl radicals having up to 10 carbon atoms, and Chemistry and Nutritional Value of Soybean Components, In: find use in Some embodiments of the present invention. In Soybeans, Chemistry, Technology and Utilization, pp. 32-35. Some preferred embodiments, propionylcarnitine and/orace 50 Aspen Publishers, Inc., Gaithersburg, Md., 1999). STI con tylcarnitine find use. Both enantiomers (D and L form), as sists of 181 amino acid residues with two disulfide bridges well as mixtures and racemates of the D- and L-forms find use and is roughly spherically shaped (See e.g., Song et al., J. in Some personal care compositions of the present invention. Mol. Biol., 275:347-63 (1998). The trypsin inhibitory loop In some further embodiments, the active ingredients of the lies within the first disulfide bridge. The Kunitz-type soybean present invention include, but are not limited to sericoside, 55 trypsin inhibitor (STI) has played a key role in the early study pyridoxol, vitamin K, biotin, and aroma Substances. In addi of proteinases, having been used as the main Substrate in the tion, it is not intended that the active ingredients present in the biochemical and kinetic work that led to the definition of the personal care compositions of the present invention be lim standard mechanism of action of proteinase inhibitors. ited to any particular constituent and/or mixture(s) of actives. Bowman-Birk Inhibitor (BBI) Indeed, it is intended that various actives and mixtures of 60 Bowman-Birk inhibitor proteins are a kinetically and actives will find use in various embodiments of the present structurally well-characterized family of small proteins (60 invention. It is also not intended that the concentration(s) of 90 residues) isolated from leguminous seeds, as well as other Such actives be limited to any particular level. In some plants, including various grasses. They typically have a sym embodiments, the concentration is from about 0.001 to about metrical structure of two tricyclic domains each containing an 30% by weight, while in other embodiments it is from about 65 independent binding loop, although some have one domain 0.05 to about 20% by weight, and in still further embodi and some have more than two domains. The major ~8 kDa ments, it is from about 0.1 to about 10% by weight, based on Bowman-Birk inhibitor isolated from soybeans (BBI) has US 9,084,734 B2 67 68 two separate reactive site loops, loop I inhibits proteases and 9 amino acids in length. These replacement sequences having trypsin-like specificity and loop II inhibits proteases also provide protease inhibition or binding to the targeted with chymotrypsin-like specificity (See e.g., Chen et al., J. proteins. In some embodiments, it is advantages to alter a Biol. Chem., 267: 1990-1994 (1992; Werner and Wemmer, single amino acid. Biochem., 31:999-1010 1992; Lin et al., Eur. J. Biochem. Fusion Proteins 212:549-555 1993; Voss et al., Eur. J. Biochem., 242: 122 In preferred embodiments, each protease inhibitor or vari 131 1996; and Billings et al., Pro. Natl. Acad. Sci.,89:3120 ant thereof is expressed as a fusion protein by the host bacte 31241992). These binding regions each contain a “canoni rial cell. Although cleavage of the fusion polypeptide to cal loop' structure, which is a motif found in a variety of release the desired protein will often be useful, it is not nec serine proteinase inhibitors (Bode and Huber, Eur. J. Bio 10 essary. Protease inhibitors and variants thereof expressed and chem. 204:433-451 1992). STI and BBI are found only in secreted as fusion proteins Surprisingly retain their function. the Soybean seed, and not in any other part of the plant (See The above-defined DNA sequences encoding the corre e.g., Birk, Int. J. Pept. Protein Res., 25:113-131 (1985). sponding amino acid sequences are combined to form a Although numerous isoforms of BBI have been character “fusion DNA sequence.” Such fusion DNA sequences are ized, SEQ ID NO.47 shows the amino acid sequence of the 15 assembled in proper reading frame from the 5' terminus to 3' BBI backbone used in some experiments described herein terminus in the order of first, second, third and fourth DNA comprising approximately 71 amino acid residues (See sequences. As so assembled, the DNA sequence encodes a Example 16). “fusion polypeptide' encoding from its amino-terminus a In soybeans, BBI is produced as a pro-protein with an signal peptide functional as a secretory sequence in a bacte N-terminal pro-peptide that is 19 amino acids in length. Thus, rial species, a secreted polypeptide or portion thereof nor in some embodiments, BBI is produced with all or at least a mally secreted from a bacterial species, a cleavable linker portion of the propeptide. In some embodiments, BBI is trun peptide and a desired polypeptide (e.g., a protease inhibitor cated, with as many as 10 amino acid residues being removed and variants thereof). Various methods are known to those in from either the N- or C-terminal. For example, upon seed the art for the production offusion proteins (See e.g., U.S. Pat. desiccation, some BBI molecules have the C-terminal 9 or 10 25 Nos. 5,411,873, 5,429,950, and 5,679,543, all of which are amino acid residues removed. Thus, proteolysis is generally incorporated herein by reference). Thus, it is intended that highly tolerated prior to the initial disulfide and just after the any suitable method will find use in the present invention. terminal disulfide bond, the consequences of which are usu Expression of Recombinant Protease Inhibitors ally not detrimental to the binding to target protein. However, To the extent that the present invention depends on the it will be appreciated that any one of the isoforms or truncated 30 production of fusion proteins, it relies on routine techniques forms find use in various embodiments of the present inven in the field of recombinant genetics. Basic texts disclosing the tion. general methods of use in this invention include Sambrook et Protease Inhibitor Variants al., Molecular Cloning, A Laboratory Manual ((2nd ed.) As indicated above, the STI and BBI protease inhibitors 1989); Kriegler, Gene Transfer and Expression: A Labora have binding loops that inhibit proteases. The present inven 35 tory Manual (1990); and Ausubel et al., (eds.), Current Pro tion provides protease inhibitor variants with alterations in tocols in Molecular Biology (1994). one or more reactive sites (e.g., Loop I and/or Loop II of BBI). The present invention provides bacterial host cells which In some preferred embodiments, the loops are replaced with have been transduced, transformed or transfected with an sequences that interact with a target protein. expression vector comprising a protease inhibitor-encoding For example, in some embodiments, the loops are replaced 40 nucleic acid sequence. The culture conditions, such as tem with sequences derived from VEGF binding proteins, inhibi perature, pH and the like, are those previously used for the tors of the complement pathway such as C2, C3, C4 or C5 parental host cell prior to transduction, transformation or inhibitors, Compstatin, cytokines, other proteins of interest, transfection are apparent to those skilled in the art. etc. Indeed, it is not intended that the present invention be Basically, a nucleotide sequence encoding a fusion protein limited to any particular sequence Substituted into either of 45 is operably linked to a promoter sequence functional in the these loops, as any Suitable sequence finds use in the present host cell. This promoter-gene unit is then typically cloned into invention. intermediate vectors before transformation into the host cells In some embodiments, variant sequences are selected by for replication and/or expression. These intermediate vectors various methods known in the art, including but not limited to are typically prokaryotic vectors (e.g., plasmids, or shuttle phage display and other Suitable screening methods. For 50 vectors). However, it is not intended that the present invention example, a random peptide gene library is fused with phage be limited to the use of intermediate vectors, as this step is PIII gene so the peptide library will be displayed on the omitted in some preferred embodiments. Surface of the phage. Subsequently, the phage display library In one approach, a bacterial culture is transformed with an is exposed to the target protein and washed with buffer to expression vector having a promoter or biologically active remove non-specific binding (this process is sometimes 55 promoterfragment or one or more (e.g., a series) of enhancers referred to as panning). Finally, the binding phage and PCR which functions in the host cell, operably linked to a nucleic the DNA sequence for the peptide encoded are isolated. acid sequence encoding a protease inhibitor, Such that the a In most embodiments, one of the loops is replaced with a protease is expressed in the cell. In some preferred embodi variant sequence (i.e., peptides; often 3 to 14 amino acids in ments, the DNA sequences encode a protease inhibitor or length, with 5 to 10 amino acids being preferred). Longer 60 variant thereof. In another preferred embodiment, the pro sequences find use in the present invention, as long as they moter is a regulatable one. provide the binding and/or inhibition desired. In addition, Nucleic Acid Constructs/Expression Vectors. peptides Suitable for use as replacements of the binding Natural or synthetic polynucleotide fragments encoding a loop(s) preferably adopt a functional conformation when protease inhibitor (i.e., “PI-encoding nucleic acid contained within a constrained loop (i.e., a loop formed by the 65 sequences') may be incorporated into heterologous nucleic presence of a disulfide bond between two cysteine residues). acid constructs or vectors, capable of introduction into, and In some specific embodiments, the peptides are between 7 replication in, a bacterial cell. The vectors and methods dis US 9,084,734 B2 69 70 closed herein are suitable for use in various host cells for the In some preferred embodiments, heterologous nucleic acid expression of protease inhibitors and variants thereof. Any constructs include the coding sequence for at least one pro vector may be used as long as it is replicable and viable in the tease inhibitor, or variant(s), fragment(s) or splice variant(s) cells into which it is introduced. Large numbers of suitable thereof: (i) in isolation; (ii) in combination with additional vectors and promoters are known to those of skill in the art, coding sequences; Such as fusion protein or signal peptide and are commercially available. Appropriate cloning and coding sequences, where the PI coding sequence is the domi expression vectors are also described in various references nant coding sequence; (iii) in combination with non-coding known to those in the art (See e.g., Sambrook et al., Supra and sequences, such as control elements, such as promoter and Ausubel et al., Supra, expressly incorporated by reference terminator elements or 5' and/or 3' untranslated regions, herein). The appropriate DNA sequence is inserted into a 10 effective for expression of the coding sequence in a Suitable plasmid or vector (collectively referred to herein as “vec host; and/or (iv) in a vector or host environment in which the tors') by any suitable method. In general, the DNA sequence PI coding sequence is a heterologous gene. is inserted into an appropriate restriction endonuclease site(s) In some embodiments, heterologous nucleic acid contain by Standard procedures known to those in the art. ing the appropriate nucleic acid coding sequence, together Appropriate vectors are typically equipped with a select 15 with appropriate promoter and control sequences, is able marker-encoding nucleic acid sequence, insertion sites, employed to introduced into bacterial host cells to permit the and Suitable control elements, such as termination sequences. cells to express at least one protease inhibitor or variant In some embodiments, the vectors comprise regulatory thereof. sequences, including, for example, control elements (i.e., In some embodiments of the present invention, a heterolo promoter and terminator elements or 5' and/or 3' untranslated gous nucleic acid construct is employed to transfer a PI regions), effective for expression of the coding sequence in encoding nucleic acid sequence into a cell in vitro. In some host cells (and/or in a vector or host cell environment in which preferred embodiments, the host cells stably integrate the a modified soluble protein coding sequence is not normally nucleic acid sequences of the present invention. Thus, any expressed), operably linked to the coding sequence. Large suitable method for effectively generating stable transfor numbers of suitable vectors and promoters are knownto those 25 mants finds use in the present invention. of skill in the art, many of which are commercially available In additional embodiments of the present invention, the and known to those in the art. first and second expression cassettes are present on a single Exemplary promoters include both constitutive promoters vector, while in other embodiments these cassettes are present and inducible promoters. Such promoters are well known to on separate vectors. those of skill in the art. Those skilled in the art are also aware 30 In some preferred embodiments, in addition to a promoter that a natural promoter can be modified by replacement, sequence, the expression cassette also contains a transcription substitution, addition or elimination of one or more nucle termination region downstream of the structural gene to pro otides without changing its function. The practice of the vide for efficient termination. In some embodiments, the ter present invention encompasses and is not constrained by Such mination region is obtained from the same gene as the pro alterations to the promoter. The choice of promoter used in the 35 moter sequence, while in other embodiments it is obtained genetic construct is within the knowledge of one skilled in the from another gene. The selection of Suitable transcription art. termination signals is well-known to those of skill in the art. The choice of the proper selectable marker will depend on In addition, it is contemplated that any suitable expression the host cell. Appropriate markers for different bacterial hosts vector will find use in the present invention. Indeed, it is are well known in the art. Typical selectable marker genes 40 contemplated that various conventional vectors used for encode proteins that (a) confer resistance to antibiotics or expression in eukaryotic or prokaryotic cells will be suitable other toxins (e.g., amplicillin, methotrexate, tetracycline, neo and find use with the present invention. Standard bacterial mycin mycophenolic acid, puromycin, Zeomycin, or hygro expression vectors include bacteriophages wand M13, as well mycin; or (b) complement an auxotrophic mutation or a natu as plasmids such as pBR322 based plasmids, pSKF, pET23D, rally occurring nutritional deficiency in the host strain. 45 and fusion expression systems such as MBP, GST, and Lacz. In some embodiments, a selected PI coding sequence is In further embodiments, epitope tags are added to recombi inserted into a Suitable vector according to well-known nant proteins, in order to provide convenient methods of recombinant techniques and used to transform a cell line isolation (e.g., c-myc). capable of PI expression. Due to the inherent degeneracy of Additional elements typically included in expression vec the genetic code, other nucleic acid sequences which encode 50 tors are replicons, a gene encoding antibiotic resistance to Substantially the same or a functionally equivalentamino acid permit selection of bacteria that harbor recombinant plas sequence may be used to clone and express a specific protease mids, and unique restriction sites in nonessential regions of inhibitor, as further detailed above. Therefore it is appreciated the plasmid to allow insertion of heterologous sequences. The that Such Substitutions in the coding region fall within the particular antibiotic resistance gene chosen is not critical, any sequence variants covered by the present invention. Any and 55 of the many resistance genes known in the art are Suitable. all of these sequence variants can be utilized in the same way Introduction of a Protease Inhibitor-Encoding Nucleic Acid as described herein for a parent PI-encoding nucleic acid Sequence into Host Cells. sequence. Those skilled in the art recognize that differing PIs In some preferred embodiments, the methods of the present will be encoded by differing nucleic acid sequences. invention provide host cells that contain a stably integrated In some embodiments, once the desired form of a protease 60 sequence of interest (i.e., PI-encoding nucleic acid). How inhibitor nucleic acid sequence, homologue, variant or frag ever, in alternative embodiments, the methods of the present ment thereof, is obtained, it is modified by any number of invention provide for maintenance of a self-replicating extra ways. Where the sequence involves non-coding flanking chromosomal transformation vector. regions, the flanking regions may be subjected to resection, The invention further provides cells and cell compositions mutagenesis, etc. Thus, transitions, transversions, deletions, 65 which have been genetically modified to comprise an exog and insertions may be performed on the naturally occurring enously provided PI-encoding nucleic acid sequence. In some Sequence. embodiments, a parental host cell is genetically modified by US 9,084,734 B2 71 72 an expression vector. In some embodiments, the vector is a In some embodiments of the present invention, the fraction plasmid, while in other embodiments the vector is a viral of properly folded secretory protein is increased by the addi particle, phage, naked DNA, etc. Thus, it is not intended that tion of chemicals to the growth medium that reduce/oxidize the form of the vector be limited to any particular type of disulfide bonds, and/or alter the general redox potential, and/ vector, as various vectors will find use in the present inven 5 or chemicals that alter solvent properties thus affecting pro tion. tein conformation and aggregation. In particularly preferred Various methods may be employed for delivering an embodiments, a reagent that reduces disulfide bonds, such as expression vector into cells in vitro. Methods of introducing 2-mercaptoethanol, is preferred to increase the fraction of nucleic acids into cells for expression of heterologous nucleic correctly folded protein. However, in other embodiments and acid sequences are also known to the ordinarily skilled arti 10 depending on the medium used, other disulfide reducing or san, including, but not limited to electroporation; protoplast oxidizing agents (e.g., DTT, TCEP, reduced and oxidized fusion with intact cells; transduction; high velocity bombard glutathione, cysteine, cystine, cysteamine, thioglycolate, ment with DNA-coated microprojectiles; infection with S.O., S.O., SOs, SO, S.O., Cu+, etc.), either modified viral (e.g., phage) nucleic acids; chemically-medi used alone or in combination, find use in the present inven ated transformation, competence, etc. In addition, in some 15 tion. It is contemplated that other adjuvants that alter solvent embodiments, heterologous nucleic acid constructs compris properties, (e.g., urea, DMSO, TWEENR-80, etc.), either ing a PI-encoding nucleic acid sequence are transcribed in added to the growth medium alone or preferably in combina vitro, and the resulting RNA introduced into the host cell by tion with disulfide reducing/oxidizing agents, such as BME, any of the suitable methods known in the art. will also increase the fraction of correctly folded secretory Following introduction of a heterologous nucleic acid con protein and find use in various embodiments of the present struct comprising the coding sequence for a protease inhibi invention. In some preferred embodiments, the BME is used tor, the genetically modified cells are cultured in conventional at concentrations ranging from 0.5 to 4 mM, while in other nutrient media modified as appropriate for activating promot embodiments, the concentrations range from 0.1 mM to 10 ers, selecting transformants, and/or amplifying expression of mM. Indeed, those of skill in the art know how to select the a PI-encoding nucleic acid sequence. The culture conditions, 25 best growth medium and growth conditions to optimize the Such as temperature, pH and the like, are those previously effects of the added thiol reducing/oxidizing agents and/or used for the host cell selected for expression, and are apparent other adjuvants, as well as the concentration of thio reducing/ to those skilled in the art. oxidizing agents and/or other adjuvants to use. It is not The progeny of cells into which Such heterologous nucleic intended that the present invention be limited to any particular acid constructs have been introduced are generally consid 30 disulfide reducing/oxidizing agent or adjuvant, as any Suit ered to comprise the PI-encoding nucleic acid sequence able reagents known to those skilled in the art find use in the found in the heterologous nucleic acid construct. present invention. Bacterial Hosts and Expression Fermentation Parameters Appropriate host cells include any Suitable bacterial spe The present invention relies on fermentation procedures cies. In some embodiments, the bacterial hosts serve both as 35 for culturing bacterial species. Fermentation procedures for the expression hosts and the Source of the first and second production of heterologous proteins by bacterial species are nucleic acids. Using the present inventive methods and host well known in the art. Culturing is accomplished in a growth cells, surprising levels of expression have been obtained. The medium comprising an aqueous mineral salts medium, system utilized herein has achieved levels of expression and organic growth factors, the carbon and energy source mate secretion of greater than 0.5 g/l of protease inhibitor. 40 rial, molecular oxygen (for aerobic and facultative bacteria), After the expression vector is introduced into the host cells, and, of course, a starting inoculum of one or more particular the transfected host cells are cultured under conditions favor microorganism species to be employed. ing expression of gene encoding the desired protein. Large In addition to the carbon and energy source, oxygen, batches of transformed cells can be cultured as described assimilable nitrogen, and an inoculum of the microorganism, above. Finally, product is recovered from the culture using 45 it is necessary to Supply Suitable amounts in proper propor techniques known in the art. tions of mineral nutrients to assure proper microorganism Accessory proteins such as thiol-disulfide oxidoreductases growth, maximize the assimilation of the carbon and energy or chaperones find use in some embodiments, as they may be Source by the cells in the microbial conversion process, and beneficial to help fold the secretory protein into its active achieve maximum cellular yields with maximum cell density conformation. Thiol-disulfide oxidoreductases and protein 50 in the fermentation medium. disulfide isomerases catalyze the formation of the correct Various culture media find use in the present invention, as disulfide bonds in the protein. Overexpression of the bdbDC known to those of skill in the art. However, standard bacterial operon in B. subtilis has been shown to be beneficial for the culture media find use in the present invention. In some pre production of a protein with disulfide bonds (See e.g., Meima ferred media formulations, the media include, in addition to et al., J. Biol. Chem., 277:6994-7001, 2002). Chaperones 55 nitrogen, Suitable amounts of phosphorus, magnesium, cal help the secretory protein to fold by binding to exposed cium, potassium, Sulfur, and sodium, in Suitable soluble hydrophobic regions in the unfolded States and preventing assimilable ionic and combined forms, and also present pref unfavorable interactions and prolyl-peptidyl cis-trans erably should be certain trace elements such as copper, man isomerases assist in formation of the proper conformation of ganese, molybdenum, zinc, iron, boron, and iodine, and oth the peptide chain adjacent to proline residues. 60 ers, again in Suitable soluble assimilable form, all as known in In some embodiments of the present invention, the host the art. cells are transformed with an expression vector encoding at In some embodiments, the fermentation reaction involves least one thiol-disulfide oxidoreductase or chaperone. It is not an aerobic process in which the molecular oxygen needed is intended that the present invention be limited to any particular Supplied by a molecular oxygen-containing gas such as air, thiol-disulfide oxidoreductase or chaperone, as any Suitable 65 oxygen-enriched air, or even Substantially pure molecular thiol-disulfide oxidoreductase or chaperone known to those oxygen, provided to maintain the contents of the fermentation skilled in the art will find use in the present invention. vessel with a Suitable oxygen partial pressure effective in US 9,084,734 B2 73 74 assisting the microorganism species to grow in a thriving treatment steps such as Suitable washing steps. The time fashion. In effect, by using an oxygenated hydrocarbon Sub needed to reach this limiting substrate level is not critical and strate, the oxygen requirement for growth of the microorgan may vary with the particular microorganism and fermentation ism is reduced. Nevertheless, molecular oxygen must be Sup process being conducted. However, it is well known in the art plied for growth of aerobic and to a lesser extent, facultative how to determine the carbon source concentration in the organisms. fermentation medium and whether or not the desired level of Although the aeration rate can vary over a considerable carbon Source has been achieved. range, aeration generally is conducted at a rate which is in the Although in some embodiments, the fermentation is con range of about 0.5 to 10, preferably about 0.5 to 7, volumes (at ducted as a batch or continuous operation, fed batch operation the pressure employed and at 25°C.) of oxygen-containing 10 is generally preferred for ease of control, production of uni gas per liquid Volume in the fermentor per minute. This form quantities of products, and most economical uses of all amount is based on air of normal oxygen content being Sup equipment. plied to the reactor, and in terms of pure oxygen the respective If desired, part or all of the carbon and energy source ranges would be about 0.1 to 1.7, or preferably about 0.1 to material and/or part of the assimilable nitrogen Source such as 1.3, Volumes (at the pressure employed and at 25°C.) of 15 ammonia can be added to the aqueous mineral medium prior oxygen per liquid volume in the fermentor per minute. to feeding the aqueous mineral medium into the fermentor. The pressure employed for the microbial conversion pro Indeed, each of the streams introduced into the reactor pref cess can range widely. Pressures generally are within the erably is controlled at a predetermined rate, or in response to range of about 0 to 50 psig, presently preferably about 0 to 30 a need determinable by monitoring Such as concentration of psig, more preferably at least slightly over atmospheric pres the carbon and energy Substrate, pH, dissolved oxygen, oxy Sure, as a balance of equipment and operating cost versus gen or carbon dioxide in the off-gases from the fermentor, cell oxygen solubility achieved. Greater than atmospheric pres density measurable by light transmittancy, or the like. The Sures are advantageous in that such pressures do tend to feed rates of the various materials can be varied so as to obtain increase a dissolved oxygen concentration in the aqueous as rapid a cell growth rate as possible, consistent with efficient ferment, which in turn can help increase cellular growth rates. 25 utilization of the carbon and energy source, to obtain as high At the same time, this is balanced by the fact that high atmo a yield of microorganism cells relative to Substrate charge as spheric pressures do increase equipment and operating costs. possible, but more importantly to obtain the highest produc The fermentation temperature can vary somewhat, but for tion of the desired protein per unit volume. most bacterial species used in the present invention, the tem In either a batch, or the preferred fed batch operation, all perature generally will be within the range of about 20°C. to 30 equipment, reactor, or fermentation means, vessel or con 40°C., generally preferably in the range of about 28°C. to 37 tainer, piping, attendant circulating or cooling devices, and C., depending on the strain of microorganism chosen, as the like, are initially sterilized, usually by employing steam known to those skilled in the art. such as at about 121° C. for at least about 15 minutes. The The microorganisms also require a source of assimilable sterilized reactor then is inoculated with a culture of the nitrogen. The source of assimilable nitrogen can be any nitro 35 selected microorganism in the presence of all the required gen-containing compound or compounds capable of releas nutrients, including oxygen, and the carbon-containing Sub ing nitrogen in a form suitable for metabolic utilization by the strate. The type of fermentor employed is not critical, though microorganism. While a variety of organic nitrogen Source in some embodiments, the 15 L Biolafitte (Saint-Germain compounds. Such as protein hydrolysates, can be employed, en-Laye, France) is preferred. usually cheap nitrogen-containing compounds such as 40 Protein Separations ammonia, ammonium hydroxide, urea, and various ammo In some particularly preferred embodiments, once the nium salts such as ammonium phosphate, ammonium Sulfate, desired protein is expressed, the secreted protein is recovered. ammonium pyrophosphate, ammonium chloride, or various The present invention provides methods of separating a other ammonium compounds can be utilized. Ammonia gas desired protein from its fusion analog. It is specifically con itself is convenient for large scale operations, and can be 45 templated that the methods described herein will find use in employed by bubbling through the aqueous ferment (fermen the separation of proteinase inhibitor and variants from the tation medium) in Suitable amounts. At the same time. Such fusion analog. ammonia can also be employed to assist in pH control. The collection and purification of the desired protein from The pH range in the aqueous microbial ferment (fermen the fermentation broth can also be achieved using procedures tation admixture) should be in the exemplary range of about 50 known to those of skill in the art. The fermentation broth will 2.0 to 8.0. However, pH range optima for certain microorgan generally contain cellular debris, including cells, various Sus isms are dependent on the media employed to some extent, as pended Solids and other biomass contaminants, as well as the well as the particular microorganism, and thus change some desired protein product, which are preferably removed from what with change in media as knownto those skilled in the art. the fermentation broth by means known in the art. Suitable While the average retention time of the fermentation 55 processes for Such removal include conventional Solid-liquid admixture in the fermentor can vary considerably, depending separation techniques (e.g., centrifugation, filtration, dialy in part on the fermentation temperature and culture sis, microfiltration, rotary vacuum filtration, or other known employed, as known in the art. processes), to produce a cell-free filtrate. In some embodi In some embodiments, the fermentation is preferably con ments, it is preferable to further concentrate the fermentation ducted in Such a manner that the carbon-containing Substrate 60 broth or the cell-free filtrate prior to the purification and/or can be controlled as a limiting factor, thereby providing good crystallization process using techniques such as ultrafiltra conversion of the carbon-containing Substrate to cells and tion, evaporation and/or precipitation. avoiding contamination of the cells with a substantial amount Precipitating the proteinaceous components of the Super of unconverted substrate. The latter is not a problem with natant or filtrate may be accomplished by means of a salt (e.g., water-soluble Substrates, since any remaining traces are 65 ammonium sulfate) or low pH (typically less than 3), fol readily removed. It may be a problem, however, in the case of lowed by purification by a variety of chromatographic proce non-water-soluble Substrates, and require added product dures (e.g., ion exchange chromatography, affinity chroma US 9,084,734 B2 75 76 tography, hydrophobic interaction chromatography, protein is diluted in buffer (in some particularly preferred hydrophobic charge induction chromatography etc.) or simi embodiments, a Zwitterionic buffer with TWEENR)-80 at lar art recognized procedures. It is not intended that the basic pH) and activated with bME and a disulfide oxidizing present invention be limited to any particular separation agent (in alternative preferred embodiments, oxidized glu method, as it is contemplated that any method will find use in tathione or sodium sulfite). the present invention. In addition, it is contemplated that conditions will be In certain preferred embodiments, when the expressed screened in order to determine the optimal activation of the desired polypeptide is secreted from the bacterial cells, the protein, if desired. For example, various BME concentrations polypeptide is purified from the growth media. In preferred (0.1-10 mM), oxidizing agent concentrations (0 to 1/20 to 20 embodiments, the expression host cells are removed from the 10 times the BME concentration) pH (7.5-9.5), temperatures media before purification of the polypeptide (e.g. by centrifu (15-40°C.), dilutions (1-20 fold), incubation times (12-72 h), gation). aeration (incubations under inert gas to vigorous mixing When the expressed recombinant desired polypeptide is under oxygen containing gases), buffer types (Tris, CHES, not secreted from the host cell, the host cell is preferably CAPS, Tricine, TAPS, other Zwitterionic buffers, etc.), buffer disrupted and the polypeptide released into an aqueous 15 concentrations (0.1-1 M), and the addition of various adju “extract” which is the first stage of purification. Preferably, vants known to alter solvent properties thereby affecting pro the expression host cells are collected from the media before tein conformation and aggregation (e.g., ethanolamine, the cell disruption (e.g. by centrifugation). The cell disruption DMSO, TWEENR-80, arginine, urea, etc.) are tested in order may be performed by using any suitable means known in the to determine the optimum conditions for the expression sys art, such as by lysozyme or beta-glucanase digestion or by tem used. It is not intended that the present invention be forcing the cells through high pressure (See e.g., Scobes, limited to any particular disulfide reducing/oxidizing agent, Protein Purification, Second edition, Springer-Verlag) dilution, temperature, pH, buffer type or composition, or In some embodiments, the addition of six histidine residues adjuvant, as any suitable reagents known to those skilled in (i.e., a “His Tag”) to the C-terminus is used as an aid in the the art find use in the present invention. purification of the desired protein and its fusion analog. Use 25 Personal Care Compositions of the His tags as a purification aid is well known in the art In particularly preferred embodiments, the present inven (See e.g., Hengen, TIBS 20:285-286 (1995). The 6xhis tion provides cosmetic and/or pharmaceutical compounds for tagged proteins are easily purified using Immobilized Metal improving the appearance of skin comprising at least one ion Affinity Chromatography (IMAC), as known to those polypeptide or a peptide. In some preferred embodiments, the skilled in the art. 30 polypeptide or peptide binds to VEGF. In alternative embodi Purity ments, the binding of the polypeptide or peptide to VEGF For some applications, it is of great importance that the blocks the downstream activity of VEGF. In some embodi protease inhibitors produced using the present invention be ments, the compounds comprise at least one peptide, while in very highly pure (e.g., having a purity of more than 99%). other embodiments, the compounds comprise at least one This is particularly true whenever the desired protein is to be 35 polypeptide. In some preferred embodiments, the peptide used as a therapeutic, but is also necessary for other applica comprises an amino acid sequence selected from the group tions. The methods described herein provide a way of pro consisting of SEQ ID NOS:1-7, 16, and 17. In additional ducing Substantially pure desired proteins. The desired pro preferred embodiments, the peptide has a conserved binding teins described herein are useful in pharmaceutical and sequence, the sequence being XXLWPXWC (SEQ ID personal care compositions. However, it is contemplated that 40 NO:15). In some preferred embodiments, the peptide com proteins of varying purity levels will be produced using the prises an amino acid sequence selected from the group con methods of the present invention and it is not intended that the sisting of SEQ ID NOS:22-24. In alternative preferred proteins produced using the present invention be limited to embodiments, the compounds have a sequence, the sequence any particular level of purity. being at least 70%, preferably 80%, more preferably 90%, Activation of BBI During Purification 45 and most preferably 95% homologous to the sequences set In some embodiments of the present invention, after forth herein. In some preferred embodiments, the polypeptide growth during the purification process, the activity of the has a molecular weight that is preferably between 500 Dal protein is increased by the addition of chemicals that reduce/ tons and 30,000 Daltons, more preferably between 1000 Dal oxidize disulfide bonds and/or alter the general redox poten tons and 10,000 Daltons, and most preferably from 1500 tial, and/or chemicals that alter solvent properties thus affect 50 Daltons to 8,000 Daltons. ing protein conformation and aggregation. In some In some preferred embodiments, the compounds find use in particularly preferred embodiments, addition of a reagent that the improvement of skin in an organism (i.e., Subject) having reduces disulfide bonds, such as 2-mercaptoethanol, is used a skin disorder. In some preferred embodiments, the skin to increase activity of the protein. However, as those skilled in disorder is an angiogenic skin disorder. In additional pre the art appreciate, depending purity and buffer composition, 55 ferred embodiments, the skin disorder is at least one selected other disulfide reducing or oxidizing agents (e.g., DTT, from the group consisting of psoriasis, venous ulcers, acne, TCEP reduced and oxidized glutathione, cysteine, cystine, rosacea, warts, eczema, hemangiomas and lymphangiogen cysteamine, thioglycolate, S.O., S.O., S.O., SOs, esis, etc. In some particularly preferred embodiments, the SO, , Cu+, protein disulfide isomerases, protein thiol-dis skin disorder is rosacea. ulfide oxidoreductases, etc.), either used alone or in combi 60 It is not intended that the present invention be limited to any nation, find use in the present invention. Other adjuvants that particular skin treatment or condition. For example, skin col alter solvent properties, (e.g. ethanolamine, DMSO, oring has been of concern to human beings for many years. In TWEENR-80, arginine, urea, etc.), either added alone or particular, there is a desire to remove the hyperpigmentation preferably in combination with disulfide reducing/oxidizing associated with age spots, freckles, and other areas of dark agents, such as BME, during the purification process also find 65 ening skin due to age or other factors. A uniformly colored use in the present invention by increasing the activity of the complexion (i.e., without areas of redness, darkness or white) protein. In certain preferred embodiments, partially purified is preferred by many individuals. In addition, in Some areas, US 9,084,734 B2 77 78 whitening of the skin is a desired effect, while in others tanned ments, the loop is closed by a disulfide bond. In some pre skin is preferred. Various compounds have been used to ferred embodiments, the polypeptide or peptide binds to achieve depigmentation, including kojic acid, hydrox VEGF. In alternative embodiments, the binding of the yduinone, and retinoids. Much research has been devoted to polypeptide or peptide to VEGF blocks the downstream activ the production of tanned skin without the use of radiation in ity of VEGF. In some particularly preferred embodiments, the order to avoid photodamage. Compounds that have found use peptide is expressed in a protease-resistant scaffold. In some in tanning skin without Sun exposure include dihydroxyac especially preferred embodiments, the scaffold comprises a etone and similar chemicals. However, the results obtained protease inhibitor (e.g., BBI, STI, or Eglin chymotrypsin with these products are usually not optimal, as even and inhibitor). In some most preferred embodiments, the protease precise application is required in order to achieve the desired 10 inhibitor is BBI. result. In addition, many of these compounds are skin irri In some preferred embodiments, the compounds further tantS. comprise at least one peptide. Preferably, the peptide com The chemical and enzymatic basis of melanogenesis is prises an amino acid sequence selected from the group con well known. Melanocytes migrate from the embryonal neural sisting of SEQID NOS: 1-7, 16, and 17. In some alternative crest into the skin to produce the secretory granules known as 15 the peptide comprises an amino acid sequence selected from melanosomes, which produce melanin via melanogenesis. the group consisting of SEQID NOS: 14). In some embodi The melanin produced by these cells is then distributed to ments, the compounds comprise an amino acid sequence keratinocytes via melanocyte dendrites. The key enzyme in selected from the group consisting of SEQ ID NOS:22-24. melanogenesis is tyrosinase, which initiates a cascade of Most preferably, the compounds comprise SEQID NO:22. In reactions which convert tyrosine to melanin, which is a poly Some preferred embodiments, the peptide has a conserved mer. Two tyrosinase-related proteins (TRPs) are known, binding sequence, the sequence being XXLWPXWC (SEQ which share about 40% homology with tyrosinase. These ID NO:15). In some preferred embodiments, the compounds proteins (TRP-1 and TRP-2) have catalytic activities as well have a sequence, the sequence being at least 70%, preferably as regulatory roles in melanogenesis. TRP-1 is reported to be 80%, more preferably 90%, and most preferably 95% identi the most abundant glycoprotein in melanocytes. 25 cal to the sequences set forth herein. The peptide molecular Although the chemical and enzymatic basis of melanogen weight is preferably between 500 Daltons and 45,000 Dal esis is well-documented, its regulation at the cellular level is tons, more preferably between 1000 Daltons and 12,000 Dal only partially understood. Tyrosinase and the TRP's share tons, and most preferably from 1500 Daltons to 10,000 Dal structural and biological properties with the lysosomal-asso tons. In some preferred embodiments, the compounds ciated membrane protein (LAMP) gene family. Thus, it has 30 comprise at least one polypeptide. been contemplated that their targeting to the melanosomal In some preferred embodiments, the compounds are used membrane might induce their activation. It is also contem for the improvement of skin in an organism (i.e., a subject) plated that phosphorylation/dephosphorylation reaction having a skin disorder. In additional preferred embodiments, occurring at the cytoplasmic tails of these proteins could be the skin disorder is at least one selected from the group involved in the regulation of melanogenesis. The beta isoform 35 consisting of psoriasis, Venous ulcers, acne, rosacea, warts, of the Protein Kinase C (PKC) family has been shown to eczema, hemangiomas and lymphangiogenesis, etc. In some regulate human melanogenesis through tyrosinase activation. particularly preferred embodiments, the skin disorder is rosa Gene expression of tyrosinase, TRP-1 and TRP-2 is coordi CCa. nated. In addition, all three enzymes are expressed in human In yet further embodiments, the present invention provides epidermis. 40 cosmetic and/or pharmaceutical compositions comprising at The Protease-activated receptor-2 (PAR-2) is a seventrans least one polypeptide or peptide, as set forth herein, and a membrane G-protein-coupled receptor, that is related to, but physiologically acceptable carrier or excipient. Preferably, distinct from the thrombin receptors (TR also named PAR-1, the compound is present in an amount of about 0.0001% to and PAR-3) in its sequence. Both receptors are activated about 5% by weight based on the total weight of the compo proteolytically by an arginine-serine cleavage at the extracel 45 sition. Also preferably, the compound is present in an amount lular domain. The newly created N-termini then activate these of about 0.001% to about 0.5% by weight based on the total receptors as tethered ligands. Both receptors may be activated weight of the composition. The composition may be in the by trypsin, but only the TRs are activated by thrombin. In form of an emulsified vehicle, such as a nutrient cream or addition, only PAR-2 is activated by mast cell tryptase. Both lotion, a stabilized gel or dispersion system, a treatment receptors may also be activated by the peptides that corre 50 serum, a liposomal delivery system, a topical pack or mask, a spond to their new N-termini, independent of receptor cleav Surfactant-based cleansing system such as a shampoo or body age. In addition, a peptide with the sequence SLIGRL (SEQ wash, an aerosolized or sprayed dispersion or emulsion; a hair ID NO:32), known to be a mouse PAR-2 activating peptide, is or skin conditioner, styling aid, or a pigmented product Such equipotent in the activation of the human receptor. Although as makeup, as well as other Suitable make-up and cosmetic the TR functions are well documented, the biology of the 55 preparations. In some embodiments, the carrier is preferably PAR-2 system has not yet been fully identified. However, a at least one selected from the group consisting of water, role for PAR-2 activation in the inhibition of keratinocyte propylene glycol, ethanol, propanol, glycerol, butylene gly growth and differentiation has been recently described (De col and polyethylene glycol. rian et al., Cell Growth Different., 8:743-749 1997). In additional embodiments, the present invention provides In other preferred embodiments, the present invention pro 60 cosmetic and/or pharmaceutical compounds comprising at vides cosmetic and/or pharmaceutical compounds for least one polypeptide or a peptide Suitable for modulating hair improving the appearance of skin. In these preferred embodi growth. In some preferred embodiments, the compounds ments, the compounds comprise at least one peptide or comprise at least one polypeptide. polypeptide within at least one scaffold, the peptide or In some preferred embodiments, modulation comprises polypeptide being expressed in the scaffold. In some particu 65 treatment of at least one disease or condition that involves loss larly preferred embodiments, the at least one peptide or of hair. In some preferred embodiments, the disease or con polypeptide is a loop. In other particularly preferred embodi dition is at least one selected from the group consisting of US 9,084,734 B2 79 80 inflammatory alopecias, pseudopelade, Scleroderma, tick of FGF-5, TGFB-1 and TGFB-2, as well as inhibitors of the bites, lichen planus, psoriasis, lupus, seborrheic dermatitis, complement pathway such as C2, C3, C4 or C5 inhibitors and loose hair syndrome, hemochromatosis, androgenicalopecia, Compstatin, etc. In additional embodiments, STI and BBI alopecia areata, cancer, conditions that affect defective hair native loops are replaced with sequences that have been iso fiber production, and environmental factors that affect hair lated using various techniques known in the art (e.g., phage production. In some preferred embodiments, the disease is display), such as the VEGF binding proteins described herein. androgenic alopecia or alopecia areata. In some embodiments, a native loop is replaced with a loop In some preferred embodiments, modulation comprises which is 3 to 20 amino acids in length, preferably 5 to 15 hair growth inhibition and/or hair removal for treatment of at amino acids in length, and more preferably 5 to 10 amino least one disease or condition for which decreased hair 10 growth is desirable. In some preferred embodiments, inhibi acids in length. In alternative embodiments, longer sequences tion and/or removal comprises depilation. find use, as long as they provide binding and/or inhibition. In In some preferred embodiments, the invention provides addition, peptides Suitable for use as replacements of the cosmetic and/or pharmaceutical compounds for modulating native loop(s) can form constrained loops (i.e., a loop formed hair growth comprising at least one peptide or polypeptide 15 by the presence to a disulfide bond between two cysteine and at least one scaffold, the peptide or polypeptide being residues). In some particularly preferred embodiments, the contained within the scaffold, preferably the peptide or peptides are between 7 and 9 amino acids in length. polypeptide being a loop, and most preferably, the loop being There are several advantages to using scaffolds to stabilize closed by a disulfide bond. In some preferred embodiments, peptide sequences. In some preferred embodiments, the bio the scaffold comprises STI, Eglin or BBI. In particularly logical activity of the peptide is higher and/or effectively preferred embodiments, the scaffold comprises BBI. In fur modulates biological function as a result of limiting peptide ther preferred embodiments, the peptide or polypeptide com flexibility and reducing the entropic cost of fixing the prises a polypeptide. polypeptide sequence in the bioactive conformation. In addi In yet further embodiments, the present invention provides tion, structural information obtained by X-ray crystallography means for decreasing VEGF activity and/or levels. In some 25 finds use in guiding affinity maturation. Furthermore, in some preferred embodiments, the VEGF activity and/or levels are embodiments, the sequence presented on a structural scaffold decreased in the epidermis. In some embodiments, the is more resistant to proteolytic degradation in different bio method comprising applying an effective amount of at least logical applications. In still further embodiments, the chi one of the compounds described herein to an organism in meric construction is obtained in large amount in low cost need thereof. 30 biological expression systems for industrial applications. In additional embodiments, the present invention provides BBI represents a class of protein scaffolds whose binding applications for hair and/or skin treatment, as well as appli to proteases is mediated by an exposed native loop that is cations wound healing, treatment of proliferative diseases, fixed in a characteristic canonical conformation and which etc. Thus, the present invention provides compositions and fits into the active site in a manner thought to be similar to that methods Suitable for application in?on humans and otherani 35 of a Substrate (Laskowski and Kato, Ann. Rev. Biochem. mals. 49:593-626 1980; and Bode & Huber, supra). The native In additional preferred embodiments, the present invention loop is frequently constrained by the presence of disulfide is directed to at least one protease-resistant scaffold compris bridges and/or extensive hydrogen-bonding networks that act ing at least one peptide or polypeptide and at least one loop. to lock the structure into the correct canonical structure. The Flexible native loops are found on the surface of most protein 40 sequence of this loop determines the specificity of the inhi modules and exist as short stretches of amino acids that con bition, which mirrors the specificity of proteases for their nect regions of defined secondary structure. Although crys Substrates. For example, most trypsin inhibitors have Arg or tallographic and NMR (nuclear magnetic resonance) studies Lys as their P1 residue. Inhibitors of the BBI family have a show that native loops are usually less well defined than network of conserved disulfide bridges that help form a sym alpha-helices and beta-sheets, their conformational freedom 45 metrical structure of two tricyclic domains (Chenet al., Supra; is normally restricted substantially compared with free pep Werner and Wemmer, Supra; and Liu et al., Supra), each tides. Consequently, the binding activities of native loops in containing an independent serine protease binding site. The proteins usually differ significantly from those of the corre native binding loop is contained within a region joined by sponding linear amino acid sequence. However, it is not disulfide bridges formed between cysteine residues. The intended that the present invention be limited to any specific 50 identity of the amino acid residue at the P1 site on each mechanism. domain is the main determinant of the serine protease inhib The loops provided by the present invention bind proteins ited. Native domains possess lysine or arginine for trypsin, such as VEGF (e.g., VEGF-A). Binding the loop to the pro leucine or tyrosine for chymotrypsin and alanine for elastase tein prevents the protein from binding to its target. Thus, (Tsunogae et al., J. Biochem. (Tokyo) 100:243-246 (1986). binding interactions are thought to be disrupted by binding 55 In addition, serine is highly conserved at the P1 position and the loop to the protein. As a result, bioactivity can be altered proline at the P3 position. The individual native loop regions as desired. However, it is not intended that the present inven of BBI are well suited for protein loop grafting of previously tion be limited to any particular mechanism. identified cysteine constrained peptides that bind to targets The present invention further provides protease inhibitors selectively, as described herein. to stabilize the loops. STI, BBI and Eglin Chave native loops 60 Numerous isoforms of BBI have been characterized. For that bind to and inhibit proteases. In some embodiments, STI example, the sequence DDESSKPCCDQCACTKSNP and BBI native loops are replaced with the polypeptides and/ PQCRCSDMRLNSCHSACKSCICALSY or peptides of the invention. In some embodiments, these PAQCFCVDITDFCYE PCKPSEDDKEN (SEQ ID NO:25) sequences are replaced with inhibitors or enhancers of any provides the amino acid sequence of a BBI backbone VEFG, while in other embodiments, the sequences are 65 described herein. In addition, in some embodiments BBI is replaced with inhibitors or enhancers of VEGF-A. In alterna truncated with as many as 10 amino acid residues being tive embodiments, the sequences are replaced with inhibitors removed from either the N- or C-terminal. Any of the iso US 9,084,734 B2 81 82 forms described herein, as well as those additional ones invention. Homology comparisons can be conducted by eye, known in the art, find use as scaffolds in the present invention. or more usually, with the aid of readily available sequence The present invention provides several advantages over comparison programs. Available computer programs can cal creation of for example, chimeric proteins. Transfer of an culate the percent homology between two or more sequences. entire protein can be difficult when, for example, a protein 5 Additionally, percent homology may be calculated over con domain of interest carries more than one important biological tiguous sequences (i.e., one sequence is aligned with the other activity. Maintaining one activity (e.g. functionally signifi sequence and each amino acid in one sequence is directly cant domain-domain interactions) while altering another (e.g. compared with the corresponding amino acid in the other high affinity binding to a co-factor or receptor) can be prob sequence one residue at a time). This is called an “ungapped' 10 alignment. Typically, such ungapped alignments are per lematic. The present invention, as indicated herein, over formed only over a relatively short number of residues. comes that limitation, as in preferred embodiments the loops Although this is a very simple and consistent method, it are transferred, instead of entire domains. fails to take into consideration that, for example, in an other In addition, in Some embodiments, the compounds of the wise identical pair of sequences, one insertion ordeletion will present invention comprise at least one mutation in addition 15 cause following amino acid residues to be put out of align to those set out above. Other mutations, such as deletions, ment, thus potentially resulting in a large reduction in % insertions, Substitutions, transversions, transitions and inver homology when a global alignment is performed. Conse sions, at one or more other locations, also find use in the quently, most sequence comparison methods are designed to present invention. produce optimal alignments that take into consideration pos In some embodiments, the compounds of the present sible insertions and deletions without penalizing unduly the invention also comprise a conservative Substitution that may overall homology score. This is achieved by inserting 'gaps' occur as a like-for-like Substitution (e.g., basic for basic, in the sequence alignment to try to maximize local homology. acidic for acidic, polar for polar etc.). In additional embodi However, these more complex methods assign 'gap pen ments, non-conservative Substitutions are provided (i.e., from alties' to each gap that occurs in the alignment, so that for the one class of residue to another or alternatively involving the 25 same number of identical amino acids, a sequence alignment inclusion of unnatural amino acids Such as ornithine, diami with as few gaps as possible (i.e., reflecting higher relatedness nobutyric acid ornithine, norleucine ornithine, pyriylalanine, between the two compared sequences) will achieve a higher thienylalanine, naphthylalanine and phenylglycine). score than one with many gaps. 'Affine gap costs are typi In some embodiments, the sequences also have deletions, insertions and/or substitutions of amino acid residues that cally used that charge a relatively high cost for the existence 30 of a gap and a smaller penalty for each Subsequent residue in produce a silent change and result in a functionally equivalent the gap. This is one of the most commonly used gap scoring Substance. system. High gap penalties will of course produce optimized In some embodiments, deliberate amino acid substitutions alignments with fewer gaps. Most alignment programs allow are made on the basis of similarity in amino acid properties (e.g., polarity, charge, solubility, hydrophobicity, hydrophi the gap penalties to be modified. However, it is preferred to 35 use the default values when using Such Software for sequence licity, and/or the amphipathic nature of the residues) and it is comparisons. For example when using the GCG Wisconsin therefore useful to group amino acids together in functional Bestfit package the default gap penalty for amino acid groups Amino acids can be grouped together based on the sequences is -12 for a gap and -4 for each extension. properties of their side chain alone. However it is more useful to include mutation data as well. The sets of amino acids thus Calculation of maximum 96 homology therefore firstly 40 requires the production of an optimal alignment, taking into derived are likely to be conserved for structural reasons. consideration gap penalties. A Suitable computer program for These sets can be described in the form of a Venn diagram carrying out such an alignment is the GCG Wisconsin Bestfit (See e.g., Livingstone and Barton, Comput. Appl. BioSci., package (See e.g., Devereux et al., Nuc. Acids Res., 12:387 9:745-756 (1993; and (Taylor, J. Theor. Biol., 119:205-218 1984). Examples of other Software packages than can per 1986). In some embodiments, conservative substitutions are 45 form sequence comparisons include, but are not limited to, made, for example according to the table below that describes the BLAST package FASTA, and the GENEWORKS suite of a generally accepted Venn diagram grouping of amino acids. comparison tools, all of which are well-known to those in the art. Both BLAST and FASTA are available for offline and Set Sub-set online searching. However, for Some applications, it is pre 50 ferred to use the GCG Bestfit program. The BLAST 2 Hydrophobic FWY H K M I L V A G C Aromatic FWY H Sequence package is also available for comparing protein and Aliphatic ILV nucleotide sequences. Polar WY H K R E D C S T N Q Charged HK RED Positively HKR Although the final percent homology can be measured in charged terms of identity, the alignment process itself is typically not Negatively ED 55 based on an all-or-nothing pair comparison. Instead, a scaled charged similarity score matrix is generally used that assigns scores to Small WCA G SPTND Tiny A G S each pair-wise comparison based on chemical similarity or evolutionary distance. An example of Such a matrix com In some embodiments, variantamino acid sequences of the monly used is the BLOSUM62 matrix the default matrix present invention also include Suitable spacer groups inserted 60 for the BLAST suite of programs. GCG Wisconsin programs between any two amino acid residues of the sequence includ generally use either the public default values or a custom ing alkyl groups such as methyl, ethyl or propyl groups in symbol comparison table if supplied. For some applications, addition to amino acid spacers such as glycine or B-alanine it is preferred to use the public default values for the GCG residues. A further form of variation involves the presence of package, or in the case of other Software, the default matrix, one or more amino acid residues in peptoid form. 65 Such as BLOSUM62. In some embodiments, homology comparisons find use in Alternatively, percentage homologies may be calculated identifying homologous sequences that find use in the present using the multiple alignment feature in DNASISTM (Hitachi US 9,084,734 B2 83 84 Software), based on an algorithm, analogous to CLUSTAL ments, the DNA sequence is prepared by polymerase chain (See e.g., Higgins and Sharp, Gene 73:237-244 1988). reaction (PCR) using specific primers, as known in the art. Once the Software has produced an optimal alignment, it is In some embodiments, the nucleotide sequences described possible to calculate the percent of homology, and more pref here and Suitable for use in the methods and compositions erably, the percent of sequence identity. The software typi described here include within them synthetic or modified cally does this as part of the sequence comparison and gen nucleotides. A number of different types of modification to erates a numerical result. oligonucleotides are known in the art. These include, but are In some embodiments, the present invention provides not limited to methylphosphonate and phosphorothioate nucleic acids encoding any of the compounds described backbones and/or the addition of acridine or polylysine herein, as well as complements thereof. In additional pre 10 chains at the 3' and/or 5' ends of the molecule. However, it is ferred embodiments, the invention provides vectors compris not intended that the present invention be limited to any ing a compound, as disclosed herein, cells comprising the particular method, as any Suitable method known to those in compound and methods of expressing the compound. the art for modifying nucleotide sequences find use in the Those of skill in the art appreciate the relationship between present invention. In some embodiments, these modifications nucleic acid sequences and polypeptide sequences, in par 15 are performed in order to enhance the in vivo activity and/or ticular as relate to the genetic code and the degeneracy of this life span of nucleotide sequences. code, and will be able to construct such nucleic acids without In some preferred embodiments, the present invention pro difficulty. For example, one skilled in the art is aware that for vides nucleotide sequences and methods for using nucleotide each amino acid Substitution in a sequence there may be one sequences that are complementary to the sequences presented or more codons that encode the Substitute amino acid. herein, as well as derivatives and/or fragments of these Accordingly, it is evident that, depending on the degeneracy Sequences. of the genetic code with respect to that particular amino acid In some embodiments, the polynucleotides of the present residue, one or more nucleic acid sequences may be generated invention find use in the production of primers and/or probes. corresponding to that polypeptide sequence. Thus, in some embodiments, the polynucleotide sequences Mutations in amino acid sequence and nucleic acid 25 are used to produce PCR primers, primers for other amplifi sequence may be made by any of a number of techniques, as cation methods as known in the art, labeled probes, and/or for known in the art. In particularly preferred embodiments, the cloning methods. In preferred embodiments, these primers, mutations are introduced into parent sequences by means of probes and other fragments are at least 15, preferably at least PCR (polymerase chain reaction) using appropriate primers. 20, and in some more preferable embodiments, at least 25, 30 In some embodiments, the parent enzymes are modified at the 30 or 40 nucleotides. In addition, these primers, probes and amino acid level, while in other embodiments, the enzymes fragments are encompassed by the term “polynucleotide. are modified at the nucleic acid level, in order to generate the In some embodiments, polynucleotides such as DNA poly sequences described herein. In some preferred embodiments, nucleotides and probes are produced recombinantly, while in the present invention provides for the generation of com other embodiments they are produced synthetically. In addi pounds by introducing one or more corresponding codon 35 tional embodiments, these sequences are cloned using stan changes in the nucleotide sequence encoding a compound. It dard methods. In general, primers are produced by Synthetic will be appreciated that the above codon changes will find use means, involving a stepwise manufacture of the desired in various nucleic acid sequences of the present invention. For nucleic acid sequence one nucleotide at a time. Techniques example, in Some embodiments, sequence changes are made for accomplishing this using automated techniques are to any of the homologous sequences described herein. 40 readily available in the art. However, it is not intended that the As indicated above, in some embodiments, the "com present invention be limited to production of polynucleotides pound” comprises the "complete' protein, (i.e., in its entire using any particular method, as any suitable method known to length as it occurs in nature (or as mutated)), while in other those in the art finds use in the present invention. embodiments it comprises a truncated form of a protein. In some embodiments, longer polynucleotides are gener Thus, the compounds of the present invention are either trun 45 ally be produced using recombinant means, for example cated or be “full-length. In addition, in some embodiments, using PCR cloning techniques, as known in the art. In Such the truncation is located at the N-terminal end, while in other embodiments, the primers are typically designed to contain embodiments the truncation is located at the C-terminal end Suitable restriction enzyme recognition sites so that the of the protein. In further embodiments, the compound lacks amplified DNA can be readily cloned into a suitable cloning one or more portions (e.g., Sub-sequences, signal sequences, 50 vector. Preferably, the variant sequences are at least as bio domains or moieties), whether active or not. logically active as the sequences presented herein. In yet further alternative embodiments, the nucleotide In some preferred embodiments, sequences that are pro sequences encoding the compounds are prepared syntheti vided that are complementary to the compound or sequences cally by established Standard methods (e.g. the phosphoroa that are capable of hybridizing to the nucleotide sequences of midite method described by Beucage et al., Tetrahedr. Lett. 55 the compounds (including complementary sequences of 22:1859-1869 1981; or the method described by Mattheset those presented herein), as well as nucleotide sequences that al., EMBO J., 3:801-805 (1984). In the phosphoroamidite are complementary to sequences that can hybridize to the method, oligonucleotides are synthesized (e.g., in an auto nucleotide sequences of the compounds (including comple matic DNA synthesizer), purified, annealed, ligated and mentary sequences of those presented herein). In some pre cloned in appropriate vectors. 60 ferred embodiments, polynucleotide sequences that are In some embodiments of the present invention, the nucle capable of hybridizing to the nucleotide sequences presented otide sequences are either of mixed genomic and synthetic herein under conditions of intermediate to maximal strin origin, mixed synthetic and cDNA origin or mixed genomic gency are provided. and cDNA origin, prepared by ligating fragments of Syn In some preferred embodiments, nucleotide sequences that thetic, genomic or cDNA origin, in accordance with standard 65 can hybridize to the nucleotide sequence of the compound techniques. Each ligated fragment corresponds to various nucleic acid, or the complement thereof, under Stringent con parts of the entire nucleotide sequence. In some embodi ditions (e.g., 50° C. and 0.2xSSC) are provided. More pref US 9,084,734 B2 85 86 erably, the nucleotide sequences can hybridize to the nucle expression vector. It is contemplated that any expression vec otide sequence of the compound, or the complement thereof, tor comprising a compound nucleic acid that is capable of under more highly stringent conditions (e.g. 65° C. and 0.1X expressing the gene encoding the compound nucleic acid in SSC). the selected host organism will find use in the present inven In some embodiments, it is desirable to mutate the tion. The choice of vector depends upon the host cell into sequence in order to prepare a compound. Accordingly, in which it is to be introduced. Thus, in some embodiments, the Some embodiments, mutants are prepared from the com vector is an autonomously replicating vector (i.e., a vector pounds provided herein. In some embodiments, mutations are that exists as an episomal entity, the replication of which is introduced using synthetic oligonucleotides. These oligo independent of chromosomal replication, Such as, for nucleotides contain nucleotide sequences flanking the 10 example, a plasmid, a bacteriophage or an episomal element, desired mutation sites. Various methods known in the art find a minichromosome or an artificial chromosome). Alterna use in this embodiment (See e.g., Morinaga et al., Biotech tively, in some embodiments, the vector integrates into the nol. 2:646-649 (1984; Nelson and Long, Anal. Biochem. host cell genome and replicates together with the chromo 180: 147-151 (1989; and Sarkar and Sommer, Biotechn. SO. 8:404–407 1990). However, additional methods find use in 15 In some preferred embodiments, the expression vector the present invention and it is not intended that the present includes the components of a cloning vector, including but invention be limited to any particular method. not limited to Such components as an element that permits In some preferred embodiments, the sequences used in the autonomous replication of the vector in the selected host methods and compositions described herein is a recombinant organism and one or more phenotypically detectable markers sequence (i.e., a sequence that has been prepared using for selection purposes. In preferred embodiments, the expres recombinant DNA techniques produced using any Suitable sion vector further comprises control nucleotide sequences method known in the art. encoding a promoter, operator, ribosome binding site, trans In additional embodiments, the present invention provides lation initiation signal, and optionally, a repressor gene or one vectors comprising the compound, cells comprising the com or more activator genes. Additionally, in Some embodiments, pound, and methods of expressing the compound. In some 25 the expression vector comprises a sequence coding for an embodiments, the nucleotide sequences used in the methods amino acid sequence capable of targeting the compound to a and compositions described herein are incorporated into a host cell organelle Such as a peroxisome or to a particular host recombinant replicable vector. In some embodiments, the cell compartment. Such a targeting sequence includes but is vector is used to replicate and express the nucleotide not limited to the sequence SKL. For expression under the sequence, in enzyme form, in and/or from a compatible host 30 direction of control sequences, the nucleic acid sequence cell. In some embodiments, expression is controlled using encoding the compound is operably linked to the control control sequences (e.g., regulatory sequences). In some sequences in proper manner with respect to expression. embodiments, proteins produced by a host cell by expression In some preferred embodiments, the polynucleotide in a of the nucleotide sequence are secreted (i.e., into the growth vector is operably linked to a control sequence that is capable medium), while in other embodiments, the proteins are con 35 of providing for the expression of the coding sequence by the tained intracellularly within the host cell. In some embodi host cell (i.e., the vector is an expression vector). In some ments, the coding sequences are designed to include signal embodiments, the control sequences are modified (e.g., by the sequences which direct secretion of the Substance coding addition of further transcriptional regulatory elements) in sequences through a particular prokaryotic or eukaryotic cell order to make the level of transcription directed by the control membrane. In further embodiments, polynucleotides are 40 sequences more responsive to transcriptional modulators. In incorporated into a recombinant replicable vector. In addi Some preferred embodiments, the control sequences com tional embodiments, the vector is used to replicate the nucleic prise promoters. acid in a compatible host cell. In preferred embodiments, the In some preferred embodiments of the vectors, the nucleic vector comprising the polynucleotide sequence is trans acid sequence encoding for the compound is operably com formed into a suitable host cell. While any suitable host finds 45 bined with a suitable promoter sequence. The promoter can use in the present invention, in some preferred embodiments, be any DNA sequence having transcription activity in the host the hosts are selected from the group consisting of bacterial, organism of choice and can be derived from genes that are yeast, insect, fungal, and mammalian cells. homologous or heterologous to the host organism. Examples In some embodiments, compounds and their polynucle of suitable promoters for directing the transcription of the otides are expressed by introducing a polynucleotide into a 50 modified nucleotide sequence. Such as compound nucleic replicable vector, introducing the vector into a compatible acids, in a bacterial host include, but are not limited to the host cell, and growing the host cell under conditions which promoter of the lac operon of E. coli, the Streptomyces coeli bring about replication of the vector. In some embodiments, color agarase gene dagA promoters, the promoters of the the vector is recovered from the host cell. Bacillus licheniformis C.-amylase gene (amyl), the aprE pro In additional embodiments, the compound nucleic acid is 55 moter of Bacillus subtilis, the promoters of the Bacillus operatively linked to transcriptional and translational regula Stearothermophilus maltogenic amylase gene (amyM), the tory elements active in the host cell. In some embodiments, promoters of the Bacillus amyloliquefaciens C.-amylase gene the compound nucleic acid also encodes a fusion protein (amyO), the promoters of the Bacillus subtilis xylA and xylB comprising at least one signal sequence (e.g., those derived genes and a promoter derived from a Lactococcus sp.-derived from the glucoamylase gene from Schwanniomyces occiden 60 promoter including the P170 promoter. When the gene encod talis, C.-factor mating type gene from Saccharomyces cerevi ing the compound is expressed in a bacterial species such as siae and the TAKA-amylase from Aspergillus Oryzae). In E. coli, a Suitable promoter can be selected, for example, from further alternative embodiments, the compound nucleic acid a bacteriophage promoter including a T7 promoter and a encodes a fusion protein comprising a membrane binding phage lambda promoter. For transcription in a fungal species, domain. 65 examples of useful promoters are those derived from the In some preferred embodiments, the compound is genes encoding the Aspergillus Oryzae TAIGA amylase, Rhi expressed at the desired levels in a host organism using an zomucor mieheiaspartic proteinase, A. niger neutral C.-amy US 9,084,734 B2 87 88 lase, A. niger acid stable C-amylase, A. niger glucoamylase, wash, an aerosolized or sprayed dispersion or emulsion, a hair Rhizomucor miehei lipase, A. Oryzae alkaline protease, A. or skin conditioner, styling aid, or a pigmented product Such Oryzae triose phosphate isomerase, and A. nidulans acetami as makeup. Preferably, the carrier is at least compound dase. Examples of Suitable promoters for the expression in a selected from the group consisting of water, propylene glycol, yeast species include but are not limited to the Gal 1 and Gal ethanol, propanol, glycerol, butylene glycol and polyethylene 10 promoters of Saccharomyces cerevisiae and the Pichia glycol. pastoris AOX1 or AOX2 promoters. In some liposomal embodiments, the liposomes comprise Examples of Suitable bacterial host organisms are Gram water and one or more ingredients capable of forming lipid positive species, including, but not limited to members of the bilayer vesicles that can hold one or more functional or active Bacillaceae, (e.g., B. subtilis, B. licheniformis, B. lentus, B. 10 ingredient(s). Non-limiting examples of ingredients capable brevis, B. Stearothermophilus, B. alkalophilus, B. amy of forming lipid bilayer vesicles include: phospholipids, loliquefaciens, B. coagulans, B. lautus, B. megaterium and B. hydrogenated phosphatidylcholine, lecithin, cholesterol and thuringiensis), Streptomyces species (e.g., S. murinus and S. sphingolipids. Preferred liposomes include, without limita lividans) lactic acid bacteria (e.g., Lactococcus spp. Such as tion: a) lipoid liposome 0003 (composed of water and lecithin Lactococcus lactis, Lactobacillus spp. including Lactobacil 15 and glycerin); b) lipoid liposome 0300 (composed of water lus reuteri; Leuconostoc spp.; Pediococcus spp.; and Strep and phosphatidylcholine); c) lipoid liposome 01 11 (com tococcus spp. Alternatively, strains of Gram-negative species posed of water, Ginkgo biloba leaf extract, denatured alcohol, belonging to Enterobacteriaceae (e.g., E. coli) or members of hydrogenated lecithin and cholesterol); d) anti-irritant lipo the Pseudomonadaceae find use in the present invention. Somes (composed of water, cola acuminata seed extract, bis In some embodiments, a suitable yeast host organism is abolol and phospholipids); e) vitamin C and E liposomes selected from various biotechnologically useful yeasts spe (composed of water, phospholipids, tocopheryl acetate and cies, including but not limited to Pichia sp., Hansenula spor ascorbyl palmitate); f) firming liposomes (composed of Kluyveromyces, Yarrowinia, Saccharomyces (e.g., Saccharo water, butylene glycol, pyrus malus (Apple) fruit extract, myces cerevisiae), Schizosaccharomyce (e.g., S. pombe). In phospholipids, tocopheryl acetate and carbomer); and g) Some embodiments, strains of the methylotrophic yeast spe 25 moisturizing liposomes (composed of water, Sodium PCA, cies Pichia pastoris are used as the host organism, while in tocopheryl acetate, Xanthan gum, arginine, lysine, glycine other embodiments, the host organism is a Hansenula spe and proline). cies. Suitable host organisms among filamentous fungi In other embodiments, the personal care composition of the include species of Aspergillus (e.g., A. niger; A. Oryzae, A. present invention further comprise at least one active ingre tubigensis, A. awamori and Aspergillus nidulans). Alterna 30 dient in addition to the scaffolds provide herein. There are tively, strains of Fusarium species (e.g. F. Oxysporum) and numerous active ingredients known to those of skill in the art Rhizomucor (e.g., Rhizomucor miehei) find used as the host that find use in the personal care compositions of the present organism. Additional Suitable strains include, but are not lim invention. Indeed, it is contemplated that any suitable active ited to Thermomyces and Mucor species. ingredient or combination of Suitable active ingredients will In some preferred embodiments, host cells comprising 35 find use in the present invention (See e.g., McCutcheon's polynucleotides are used to express polypeptides, such as the Functional Materials, North American and International Edi compounds disclosed herein, fragments, homologues, vari tions, published by MC Publishing Co. 2003). For example, ants or derivatives thereof. Host cells are cultured under suit in some embodiments, the personal care compositions herein able conditions which allow expression of the proteins. In comprise a skin care active ingredient at a level from about Some embodiments, expression of the polypeptides is consti 40 0.0001% to about 20%, by weight of the composition. In tutive (i.e., the peptides are continually produced), while in another embodiment, the personal care compositions com other embodiments, expression is inducible. In the case of prise a skin care active ingredient from about 0.001% to about inducible expression, protein production is initiated when 5%, by weight of the composition. In yet another embodi required by addition of an inducer substance to the culture ment, the personal care compositions comprise a skin care medium (e.g., dexamethasone or IPTG). Polypeptides can be 45 active ingredient from about 0.01% to about 2%, by weight of extracted from host cells by a variety of techniques known in the composition. the art, including enzymatic, chemical, and/or osmotic lysis Non-limiting examples of functional or active ingredients and physical disruption. Indeed, it is not intended that the that can be delivered via liposomes include: vitamins and present invention be limited to any particular means of har their derivatives, antioxidants, proteins and peptides, kera vesting expressed polypeptides. 50 tolytic agents, bioflavinoids, terpenoids, phytochemicals, and In alternative embodiments, polypeptides are produced extracts of plant, marine or fermented origin. In a preferred recombinantly in any Suitable (including commercially avail embodiment, the composition further comprises askin care or able) in vitro cell-free system, such as the TnTTM (Promega) hair care active. Active ingredients can include any of a wide rabbit reticulocyte system. variety of ingredients such as are known in the art. (See e.g., In additional preferred embodiments, the present invention 55 McCutcheon's Functional Materials, North American and provides cosmetic and/or pharmaceutical compositions com International Editions, (2003), published by MC Publishing prising at least one polypeptide or peptide, as set forth herein, Co.). Preferably, such actives include but are not limited to and a physiologically acceptable carrier or excipient. Prefer antioxidants, such as tocopheryl and ascorbyl derivatives, ably, the compound is present in an amount of about 0.0001% bioflavinoids, terpenoids, synthetics and the like, Vitamins to about 5% by weight, based on the total weight of the 60 and vitamin derivatives, hydroxyl- and polyhydroxy acids composition. Also preferably, the compound is present in an and their derivatives, such as AHAs and BHAs and their amount of about 0.001% to about 0.5% by weight based on reaction products, peptides and polypeptides and their deriva the total weight of the composition. The composition may be tives, such as glycopeptides and lipophilized peptides, heat in the form of an emulsified vehicle. Such as a nutrient cream shock proteins and cytokines, enzymes and enzymes inhibi or lotion, a stabilized gel or dispersion system, a treatment 65 tors and their derivatives, such as proteases, MMP inhibitors, serum, a liposomal delivery system, a topical pack or mask, a catalases, glucose oxydase and Superoxide dismutase, amino Surfactant-based cleansing system such as a shampoo or body acids and their derivatives, bacterial, fungal and yeastfermen US 9,084,734 B2 89 90 tation products and their derivatives, including mushrooms, In further embodiments, an effective amount of one or algae and seaweed and their derivatives, phytosterols and more compounds described herein is also be included in plant and plant part extracts and their derivatives and phos compositions to be applied to keratinous materials such as pholipids and their derivatives, anti-dandruff agents such as nails and hair, including but not limited to those useful as hair Zinc pyrithione and delivery systems containing them, as spray compositions, hair styling compositions, hair sham provided herein and/or known in the art. pooing and/or conditioning compositions, compositions In some preferred embodiments, the skin care active is applied for the purpose of hair growth regulation and compo selected from the group consisting of a Vitamin B3 compo sitions applied to the hair and Scalp for the purpose of treating nent, panthenol, Vitamin E, Vitamin Eacetate, retinol, retinyl seborrhoea, dermatitis and/or dandruff. propionate, retinyl palmitate, retinoic acid, Vitamin C, theo 10 In yet additional embodiments, an effective amount of one bromine, alpha-hydroxyacid, farnesol, phytrantriol, salicylic or more compounds described herein is included in compo acid, palmityl peptapeptide-3 and mixtures thereof. In some sitions suitable for topical application to the skin or hair. preferred embodiments, the Vitamin B3 component is niaci These compositions are provided in any suitable form, namide. In some embodiments, the compositions provided including but not limited to creams, lotions, gels, Suspensions herein comprise a skin care active at a level from about 15 dispersions, microemulsions, nanodispersions, micro 0.0001% to about 20%, preferably from about 0.001% to spheres, hydrogels, emulsions (e.g., oil-in-water and water about 5%, more preferably from about 0.01% to about 2%, by, in-oil, as well as multiple emulsions), and multilaminar gels weight. and the like (See e.g., Schlossman et al., The Chemistry and Exemplary derivatives of the foregoing vitamin B com Manufacture of Cosmetics, (1998, incorporated by refer pounds include nicotinic acid esters, including non-vasodi ence, herein). In some embodiments, the compositions are latingesters of nicotinic acid, nicotinyl amino acids, nicotinyl formulated as aqueous or silicone compositions, while in alcohol esters of carboxylic acids, nicotinic acid N-oxide and other embodiments they are formulated as emulsions of one niacinamide N-oxide. Suitable esters of nicotinic acid include or more oil phases in an aqueous continuous phase (or an nicotinic acid esters of C-C, preferably C-C more pref aqueous phase in an oil phase). erably C-C alcohols. In these embodiments, the alcohols are 25 The type of carrier utilized in the present invention depends Suitably straight-chain or branched chain, cyclic or acyclic, on the type of product form desired for the composition. The saturated or unsaturated (including aromatic), and Substituted carrier can be solid, semi-solid or liquid. Suitable carriers or unsubstituted. The esters are preferably non-vasodilating. include liquids, semi-solids (e.g., creams, lotions, gels, Sticks, Non-vasodilating esters of nicotinic acid include toco ointments, and pastes), sprays and mousses. Preferably the pherol nicotinate and inositol hexanicotinate; tocopherol 30 carrier is in the form of a lotion, cream or a gel, more prefer nicotinate are preferred. A more complete description of vita ably one which has a sufficient thickness or yield point to min B compounds is provided in WO 98/22085. Preferred prevent the particles from sedimenting. In some embodi vitamin B compounds include niacinamide and tocopherol ments, the carrier is inert, while in other embodiments it nicotinate. provides dermatological benefits. In some embodiments, the In additional embodiments, the skin care active comprises 35 carrier is applied directly to the skin and/or hair, while in other at least one retinoid. The retinoid is preferably retinol, retinol embodiments, it is applied via a woven or non-woven wipe or esters (e.g., C-C alkyl esters of retinol, including retinyl cloth. In yet other embodiments, it is in the form of a patch, palmitate, retinyl acetate, retinyl proprionate), retinal, and/or mask or wrap. Instill further embodiments, it is aerosolized or retinoic acid (including all-trans retinoic acid and/or 13-cis otherwise sprayed or pumped onto the skin and/or hair. The retinoic acid), more preferably retinoids other than retinoic 40 carrier chosen is physically and chemically compatible with acid. These compounds are well known in the art and are the essential components described herein, and should not commercially available from a number of Sources (e.g., unduly impair stability, efficacy or other use benefits associ Sigma and Boehringer Mannheim). Preferred retinoids ated with the compositions of the present invention. include retinol, retinyl palmitate, retinyl acetate, retinyl prop Preferred carriers contain a dermatologically acceptable, rionate, retinal, retinoic acid and combinations thereof. More 45 hydrophilic diluent. Suitable hydrophilic diluents include preferred are retinol, retinoic propionate, retinoic acid and water, organic hydrophilic diluents such as C-Co, prefer retinyl palmitate. In some embodiments, the retinoid is ably C-C, preferably, C-C monohydric alcohols and low included as a Substantially pure material, while in other molecular weight glycols and polyols, including propylene embodiments, it is provided as an extract obtained by suitable glycol, polyethylene glycol polypropylene glycol, glycerol, physical and/or chemical isolation from natural (e.g., plant) 50 butylene glycol. 1,2,4-butanetriol, Sorbitol esters, 1.2.6-hex sources. When a retinoid is included in the compositions ametriol, pentylene glycol, hexylene glycol, Sorbitol esters, herein, it preferably comprises from about 0.005% to about ethoxylated ethers, propoxylated ethers, and combinations 2%, preferably from about 0.01% to about 1% retinoid. Ret thereof. The diluent is preferably liquid. Water is a preferred inol is preferably used in an amount of from about 0.01% to diluent. The composition preferably comprises at least about about 0.15%; retinol esters are preferably used in an amount 55 20% of the hydrophilic diluent. of from about 0.01% to about 2% (e.g., about 1%). In some embodiments, Suitable carriers also comprise an In some embodiments, the compositions of the present emulsion comprising a hydrophilic phase, especially an aque invention comprise safe and effective amounts of a dermato ous phase, and a hydrophobic phase (e.g., a lipid, oil or oily logically acceptable carrier that is Suitable for topical appli material). As well known to those skilled in the art, the hydro cation to the skin or hair within which the essential materials 60 philic phase is dispersed in the hydrophobic phase, or vice and optional other materials are incorporated to enable the versa, to form respectively hydrophilic or hydrophobic dis essential materials and optional components to be delivered persed and continuous phases, depending on the composition to the skin or hair at an appropriate concentration. Thus, in of ingredients. The term "dispersed phase' is a term well Some embodiments, the carrier acts as a diluent, dispersant, known to one skilled in the art of emulsion technology, used Solvent or the like for the essential components, ensuring that 65 in reference to the phase which exists as Small particles or these components can be applied and distributed evenly over droplets that are suspended in and Surrounded by a continu the selected target at an appropriate concentration. ous phase. The dispersed phase is also known as the internal US 9,084,734 B2 91 92 or discontinuous phase. The emulsion may be or comprise while helping to provide effective moisturization benefits and (e.g., in a triple or other multi-phase emulsion) an oil-in-water is described in further detail in WO96/03964, incorporated emulsion or a water-in-oil emulsion Such as a water-in-sili herein by reference. cone emulsion. Oil-in-water emulsions typically comprise In some embodiments, the oil-in-water and water-in-oil from about 1% to about 60% (preferably about 1% to about compositions of the present invention comprise from about 30%) of the dispersed hydrophobic phase and from about 1% 0.05% to about 20%, preferably from about 1% to about 15%, to about 99% (preferably from about 10% to about 90%) of preferably from about 2% to about 10%, preferably from the continuous hydrophilic phase, while water-in-oil emul about 2% to about 5% of a dermatologically acceptable emol sions typically comprise from about 1% to about 98% (pref lient. Emollients tend to lubricate the skin, increase the erably from about 40% to about 90%) of the dispersed hydro 10 Smoothness and Suppleness of the skin, prevent or relieve philic phase and from about 1% to about 50% (preferably dryness of the skin and/or protect the skin. Emollients are about 1% to about 30%) of the continuous hydrophobic typically water-immiscible, oily or waxy materials and emol phase. lients can conferaesthetic properties to atopical composition. In further embodiments, the carrier also includes one or A wide variety of suitable emollients are known (See e.g., more components that facilitate penetration through the 15 Sagarin, Cosmetics, Science and Technology, 2nd Edition, upper stratum corneum barrier to the lower levels of the skin. Vol. 1, pp. 32-43 (1972; and WO 00/24372), and find use Examples of penetration enhancers include, but are not lim herein, contains numerous examples of materials suitable as ited to, propylene glycol, aZone, ethoxydiglycol, dimethyl emollients. Additional emollients include, but are not limited isosorbide, urea, ethanol and dimethyl Sulfoxide, as well as to the following: microemulsions, liposomes and nanoemulsions. i) Straight and branched chain hydrocarbons having from In some additional embodiments, the compositions of the about 7 to about 40 carbonatoms, such as mineral oils, dode present invention comprise humectants which are preferably cane, squalane, cholesterol, hydrogenated polyisobutylene, present at a level of from about 0.01% to about 20%, prefer isohexadecane, isoeicosane, isooctahexacontane, isohexap ably from about 0.1% to about 15% and preferably from entacontahectane, and the C7-Co isoparaffins, which are about 0.5% to about 10%. Preferred humectants include, but 25 C7-Cao branched hydrocarbons. Suitable branched chain are not limited to, compounds selected from polyhydric alco hydrocarbons for use herein are selected from isopentacon hols, Sorbitol, glycerol, urea, betaine, D-panthenol, DL-pan taoctactane, petrolatum and mixtures thereof; thenol, calcium pantothenate, royal jelly, panthetine, pan ii) C-C fatty acid esters of C-C carboxylic acids, to theine, panthenyl ethyl ether, pangamic acid, pyridoxin, Cs alkylbenzoates and of C-Co dicarboxylic acids, e.g. pantoyl lactose Vitamin B complex, Sodium pyrrolidone car 30 isononyl isononanoate, isostearyl neopentanoate, isodecyl boxylic acid, hexane-1,2,6,-triol, guanidine or its derivatives, octanoate, isodecyl isononanoate, tridecyl isononanoate, and mixtures thereof. myristyl octanoate, octyl pelargonate, octyl isononanoate, Suitable polyhydric alcohols for use herein include, but are myristyl myristate, myristyl neopentanoate, myristyl not limited to polyalkylene glycols and preferably alkylene octanoate, isopropyl myristate, myristyl propionate, isopro polyols and their derivatives, including propylene glycol, 35 pyl Stearate, isopropyl isostearate, methyl isostearate, behe dipropylene glycol, polypropylene glycol, polyethylene gly nyl behenate, dioctyl maleate, diisopropyl adipate, and diiso col and derivatives thereof, sorbitol, hydroxypropyl sorbitol, propyl dilinoleate and mixtures thereof also find use in the erythritol, threitol, pentaerythritol. Xylitol, glucitol, mannitol, present invention; pentylene glycol, hexylene glycol, butylene glycol (e.g., 1.3- iii) C-C mono- and poly-esters of Sugars and related butylene glycol), hexane triol (e.g., 1.2.6-hexanetriol), trim 40 materials. These esters are derived from a Sugar or polyol ethylol propane, neopentyl glycol, glycerine, ethoxylated moiety and one or more carboxylic acid moieties. Depending glycerine, propane-1,3 diol, propoxylated glycerine and mix on the constituent acid and Sugar, these esters can be in either tures thereof. The alkoxylated derivatives of any of the above liquid or solid form at room temperature. Examples include: polyhydric alcohols are also suitable for use herein. Preferred glucose tetraoleate, the galactose tetraesters of oleic acid, the polyhydric alcohols of the present invention are selected from 45 Sorbitol tetraoleate, Sucrose tetraoleate, Sucrose pentaoleate, glycerine, butylene glycol, propylene glycol, pentylene gly Sucrose hexaoleate, Sucrose heptaoleate. Sucrose octaoleate, col, hexylene glycol, dipropylene glycol, polyethylene gly sorbitol hexaester. Other materials include cottonseed oil or col, hexane triol, ethoxylated glycerine and propoxylated soybean oil fatty acid esters of sucrose. Other examples of glycerine and mixtures thereof. such materials are described in WO 96/16636, incorporated Suitable humectants useful herein are sodium 2-pyrroli 50 by reference herein; done-5-carboxylate (NaPCA), guanidine; glycolic acid and iv) Vegetable oils and hydrogenated vegetable oils. glycolate salts (e.g., ammonium and quaternary alkyl ammo Examples of vegetable oils and hydro-genated vegetable oils nium); lactic acid and lactate salts (e.g., ammonium and qua include safflower oil, grapeseed oil, coconut oil, cottonseed ternary alkyl ammonium); aloe Vera in any of its variety of oil, menhaden oil, palm kernel oil, palm oil, peanut oil, Soy forms (e.g., aloe Vera gel); hyaluronic acid and derivatives 55 bean oil, rapeseed oil, linseed oil, rice bran oil, pine oil, nut thereof (e.g., Salt derivatives Such as sodium hyaluronate); oil, Sesame oil, Sunflower seed oil, partially and fully hydro lactamide monoethanolamine; acetamide monoethanola genated oils from the foregoing sources and mixtures thereof. mine; urea; betaine, panthenol and derivatives thereof, and v) Soluble or colloidally-soluble moisturizing agents. mixtures thereof. Examples include hyaluronic acid and chondroitin Sulfate. In some embodiments, at least part (up to about 5% by 60 In some embodiments, the compositions of the present weight of composition) of a humectant is incorporated into invention contain an emulsifier and/or Surfactant, generally to the compositions of the present invention in the form of an help disperse and Suspend the disperse phase within the con admixture with a particulate cross-linked hydrophobic acry tinuous aqueous phase. A Surfactant may also be useful if the late or methacrylate copolymer, itself preferably present in an product is intended for skin or hair cleansing. For conve amount of from about 0.1% to about 10%, which can be added 65 nience hereinafter, "emulsifiers' are encompassed by the either to the aqueous or disperse phase. This copolymer is term "surfactants.” Thus, as used herein, the term "sur particularly valuable for reducing shine and controlling oil factant(s) refers to Surface active agents, whether used as US 9,084,734 B2 93 94 emulsifiers or for other Surfactant purposes such as skin and aminoalkanoates, imidazolinium and ammonium deriva cleansing. Known, including conventional Surfactants find tives. Other suitable amphoteric and Zwitterionic surfactants use in the present invention, provided that the selected agent are those selected from the group consisting of betaines, is chemically and physically compatible with essential com Sultaines, hydroxysultaines, and branched and unbranched ponents of the composition and provides the desired charac alkanoyl sarcosinates, and mixtures thereof. teristics (See e.g., WO 00/24372). Suitable surfactants In further embodiments, some emulsions of the present include non-silicone derived materials, silicone-derived invention include a silicone containing emulsifier or Surfac materials, and mixtures thereof. tant. A wide variety of silicone emulsifiers find use herein. In further embodiments, the compositions of the present These silicone emulsifiers are typically organically modified invention comprise preferably from about 0.05% to about 10 organopolysiloxanes, also known to those skilled in the art as 30%, more preferably from about 0.5% to 15%, and most silicone surfactants. Useful silicone emulsifiers include, but preferably from about 1% to 10% of a surfactant or mixture of are not limited to dimethicone copolyols. These materials are Surfactants. The exact Surfactant or Surfactant mixture chosen polydimethyl siloxanes which have been modified to include depends upon the pH of the composition, the other compo polyether side chains such as polyethylene oxide chains, nents present and the desired final product aesthetics. 15 polypropylene oxide chains, mixtures of these chains and Among the nonionic Surfactants that are useful herein are polyether chains containing moieties derived from both eth those that can be broadly defined as condensation products of ylene oxide and propylene oxide. Other examples include long chain alcohols (e.g., Cso alcohols), with Sugar or starch alkyl-modified dimethicone copolyols (i.e., compounds polymers (e.g., glycosides). Other useful nonionic Surfactants which contain C-Clso pendant side chains). Still other useful include the condensation products of alkylene oxides with dimethicone copolyols include materials having various cat fatty acids (i.e., alkylene oxide esters of fatty acids). These ionic, anionic, amphoteric, and Zwitterionic pendant moi materials have the general formula RCO(X), OH wherein Ris eties. a Coso alkyl group, X is —OCHCH - (i.e., derived from In some embodiments, the compositions of the present ethylene glycolor oxide) or —OCH2CHCH (i.e., derived invention comprise at least one polymeric thickening agent. from propylene glycolor oxide) and n is an integer from about 25 The polymeric thickening agents useful herein preferably 6 to about 200. Other nonionic surfactants are the condensa have a number average molecular weight of greater than tion products of alkylene oxides with 2 moles of fatty acids about 20,000, more preferably greater than about 50,000, and (i.e., alkylene oxide diesters of fatty acids). These materials most preferably greater than about 100,000. In some embodi have the general formula RCO(X), OOCR wherein R is a ments, the compositions of the present invention comprise Coso alkyl group, X is —OCHCH- (i.e., derived from 30 from about 0.01% to about 10%, preferably from about 0.1% ethylene glycolor oxide) or —OCH2CHCH (i.e., derived to about 8% and more preferably from about 0.2% to about from propylene glycolor oxide) and n is an integer from about 5% by weight of the composition of the polymeric thickening 6 to about 100. In some embodiments, an emulsifier for use agent or mixtures thereof. herein is preferably a fatty acid ester blend based on a mixture Preferred polymer thickening agents for use herein of Sorbitan fatty acid ester and Sucrose fatty acid ester, espe 35 include, but are not limited to non-ionic thickening agents and cially a blend of Sorbitan Stearate and Sucrose cocoate. Fur anionic thickening agents or mixtures thereof. Suitable non ther suitable examples include a mixture of cetearyl alcohols ionic thickening agents include, but are not limited to poly and cetearyl glucosides. However, it is not intended that the acrylamide polymers, crosslinked poly(N-vinylpyrroli present invention be limited to any particular emulsifier, as dones), polysaccharides, natural or synthetic gums, various suitable emulsifiers are known in the art. 40 polyvinylpyrrolidone and polyvinylalcohol. Suitable anionic In additional embodiments, the hydrophilic surfactants thickening agents include, but are not limited to acrylic acid/ useful herein alternatively or additionally include any of a ethyl acrylate copolymers, carboxyvinyl polymers and wide variety of cationic, anionic, Zwitterionic, and amphot crosslinked copolymers of alkyl vinyl ethers and maleic eric Surfactants such as are known in the art (See, e.g., anhydride. Commercially available thickeners (e.g., Car McCutcheon's, Emulsifiers and Detergents, North American 45 bopol; Noveon) find use in some embodiments of the present and International Editions, MC Publishing Co. 2003: U.S. invention. Suitable Carbopol resins may be hydrophobically Pat. No. 5,011,681 U.S. Pat. No. 4,421,769; and U.S. Pat. No. modified, and other suitable resins are described in WO987 3,755,560). 22085, or mixtures thereof. A variety of anionic surfactants are also useful herein (See In some embodiments, the present compositions comprise e.g., U.S. Pat. No. 3,929,678). Exemplary anionic surfactants 50 at least one silicone oil phase. Silicone oil phase(s) generally include, but are not limited to alkoylisethionates (e.g., C2 comprises from about 0.1% to about 20%, preferably from Co), alkyl and alkyl ether Sulfates and salts thereof, alkyl and about 0.5% to about 10%, and more preferably from about alkyl ether phosphates and salts thereof, alkyl methyl taurates 0.5% to about 5%, of the composition. The silicone oil phase (e.g., C.-Co), and Soaps (e.g., Substituted alkylamine and preferably comprises one or more silicone components. alkali metal salts, e.g., Sodium or potassium salts) of fatty 55 In some embodiments, silicone components are fluids, acids. including straight chain, branched and cyclic silicones. Suit Amphoteric and Zwitterionic Surfactants are also useful able siliconefluids useful herein include silicones inclusive of herein. Examples of preferred amphoteric and Zwitterionic polyalkyl siloxane fluids, polyaryl siloxane fluids, cyclic and Surfactants which find use in the compositions of the present linear polyalkylsiloxanes, polyalkoxylated silicones, amino invention are those which are broadly described as derivatives 60 and quaternary ammonium modified silicones, polyalkylaryl of aliphatic secondary and tertiary amines in which the ali siloxanes or a polyether siloxane copolymer and mixtures phatic radical can be straight or branched chain and wherein thereof. Volatile, as well as non-volatile silicone fluids find one of the aliphatic substituents contains from about 8 to use herein. Silicone fluids generally have an average molecu about 22 carbon atoms (preferably Cs-Cs) and one contains lar weight of less than about 200,000. In preferred embodi an anionic water solubilizing group (e.g., carboxy, Sulfonate, 65 ments, suitable silicone fluids have a molecular weight of Sulfate, phosphate, or phosphonate). Examples, include but about 100,000 or less, preferably about 50,000 or less, and are not limited to alkylimino acetates and iminodialkanoates more preferably about 10,000 or less. Preferably the silicone US 9,084,734 B2 95 96 fluid is selected from silicone fluids having a weight average Another class of silicone components suitable for use in a molecular weight in the range from about 100 to about 50,000 silicone oil phase herein includes polydiorganosiloxane and preferably from about 200 to about 40,000. Typically, polyoxyalkylene copolymers containing at least one poly silicone fluids have a viscosity ranging from about 0.65 to diorganosiloxane segment and at least one polyoxyalkylene about 600,000 mm's', preferably from about 0.65 to about segment (See e.g., WO98/22085). Suitable polydiorganosi 10,000 mm.s' at 25°C. The viscosity can be measured by loxane-polyalkylene copolymers are available commercially means of a glass capillary viscometer as set forth in Dow under the tradenames BELSILR) from Wacker-Chemie Corning Corporate Test Method CTM0004, Jul. 29, 1970. GmbH. Aparticularly preferred copolymer fluid blend for use Suitable polydimethyl siloxanes that can be used herein herein includes Dow Corning DC3225C which has the CTFA include commercially available compounds (e.g., from the 10 designation Dimethicone/Dimethicone copolyol. General Electric Company and Dow Corning). Also useful In further embodiments, compositions of the present are essentially non-volatile polyalkylarylsiloxanes, for invention comprise an organic Sunscreen. In some embodi example, polymethylphenylsiloxanes, having viscosities of ments, Suitable Sunscreens have UVA absorbing properties, about 0.65 to 30,000 mm s' at 25° C. (General Electric while others have UVB absorbing properties, and still others Company or from Dow Corning). Cyclic polydimethylsilox 15 comprise a mixture thereof. The exact amount of the Sun anes Suitable for use herein are those having a ring structure screen active varies, depending upon the desired Sun Protec incorporating from about 3 to about 7 (CH)SiO moieties, tion Factor (i.e., the "SPF) of the composition, as well as the preferably about 5 or more. desired level of UV protection. SPF is a commonly used In additional embodiments, silicone gums find use herein. measure of photoprotection of a Sunscreen against erythema. In some preferred embodiments, a silicone oil phase com The SPF is defined as a ratio of the ultraviolet energy required prises a silicone gum or a mixture of silicones including the to produce minimal erythema on protected skin to that silicone gum. Typically, silicone gums have a viscosity at 25° required to produce the same minimal erythema on unpro C. in excess of about 1,000,000 mm s. The silicone gums tected skin in the same individual. Amounts of the Sunscreen include dimethicones as known in the art (See e.g., U.S. Pat. used are preferably from about 2% to about 20%, and more No. 4,152,416; and Noll, Chemistry and Technology of Sili 25 preferably from about 4% to about 14%. Suitable sunscreens cones, Academic Press, New York 1968). Silicone gums include, but are not limited to those approved for use in the such as those described in General Electric Silicone Rubber United States, Japan, Europe and Australia. The compositions Product Data Sheets SE 30, SE 33, SE54 and SE 76, also find of the present invention preferably comprise an SPF of about use in the present invention. Specific examples of silicone 2 to about 30, preferably about 4 about 30, and more prefer gums include polydimethylsiloxane, (polydimethylsiloxane) 30 ably about 4 to about 15. (methylvinylsiloxane) copolymer, poly(dimethylsiloxane) In some embodiments, the compositions of the present (diphenyl)(methylvinylsiloxane) copolymer and mixtures invention comprise one or more UVA absorbing sunscreen thereof. Preferred silicone gums for use herein are silicone actives that absorb UV radiation having a wavelength of from gums having a molecular weight of from about 200,000 to about 320 nm to about 400 nm. Suitable UVA absorbing about 4,000,000 selected from dimethiconol, dimethicone 35 Sunscreen actives include, but are not limited to dibenzoyl copolyol, dimethicone and mixtures thereof. methane (See e.g., Lowe and Shaath (eds.), Sunscreens. In some embodiments, a silicone phase herein preferably Development, Evaluation, and Regulatory Aspects, Marcel comprises a silicone gum incorporated into the composition Dekker, Inc.) derivatives, anthranilate derivatives such as as part of a silicone gum-fluid blend. When the silicone gum methylanthranilate and homomethyl, 1-N-acetylanthranilate, is incorporated as part of a silicone gum-fluid blend, the 40 and mixtures thereof. The UVA absorbing sunscreen active is silicone gum preferably constitutes from about 5% to about preferably present in an amount sufficient to provide broad 40%, especially from about 10% to 20% by weight of the spectrum UVA protection either independently, or in combi silicone gum-fluid blend. Suitable silicone gum-fluid blends nation with, other UV protective actives which may be herein are mixtures consisting essentially of present in the composition. (i) a silicone having a molecular weight of from about 45 Suitable UVA sunscreenactives include dibenzoylmethane 200,000 to about 4,000,000 selected from dimethiconol, fluo sunscreen actives and their derivatives. They include, but are rosilicone and dimethicone and mixtures thereof, and not limited to, those selected from 2-methyldibenzoyl (ii) a carrier which is a silicone fluid, the carrier having a methane, 4-methyldibenzoylmethane, 4-isopropyldibenzoyl viscosity from about 0.65mms' to about 100 mms', methane, 4-tert-butyldibenzoylmethane, 2,4-dimethyldiben wherein the ratio of i) to ii) is from about 10:90 to about 50 Zoylmethane, 2,5-dimethyldibenzoylmethane, 4,4'- 20:80 and wherein said silicone gum-based component has a diisopropylbenzoylmethane, 4-(1,1-dimethylethyl)-4- final viscosity of from about 100 mm s to about 100,000 methoxydibenzoylmethane, 2-methyl-5-isopropyl-4- mms', preferably from 500 mms' to about 10,000 mm methoxydibenzoylmethane, 2-methyl-5-tert-butyl-4- s methoxy-dibenzoylmethane, 2,4-dimethyl-4- Further silicone components suitable for use in a silicone 55 methoxydibenzoylmethane, 2,6-dimethyl-4'-tert-butyl-4- oil phase herein include crosslinked polyorganosiloxane methoxydibenzoylmethane, and mixtures thereof. Preferred polymers, optionally dispersed in a fluid carrier. In general, dibenzoyl sunscreen actives include those selected from 4-(1, when present the crosslinked polyorganosiloxane polymers, 1-dimethylethyl)-4'-methoxydibenzoylmethane, 4-isopropy together with its carrier (if present) comprises from about ldibenzoylmethane, and mixtures thereof. A preferred sun 0.1% to about 20%, preferably from about 0.5% to about 60 SCC active is 4-(1,1-dimethylethyl)-4- 10%, and more preferably from about 0.5% to about 5% of the methoxydibenzoylmethane. composition. Such polymers comprise polyorganosiloxane The sunscreen active 4-(1,1-dimethylethyl)-4'-methoxy polymers crosslinked by a crosslinking agent (See e.g., dibenzoylmethane, which is also known as butyl methoxy WO98/22085). Examples of suitable polyorganosiloxane dibenzoylmethane or "avobenzone is commercially avail polymers for use herein include, but are not limited to methyl 65 able under the names of ParsolR 1789 from Givaudan Roure vinyl dimethicone, methyl vinyl diphenyl dimethicone and (International) S.A., and Eusolex R, 9020 from Merck & Co., methyl vinyl phenyl methyl diphenyl dimethicone. Inc. The Sunscreen 4-isoproplydibenzoylmethane, which is US 9,084,734 B2 97 98 also known as isopropyldibenzoylmethane, is commercially When used in preferred embodiments, the physical sun available from Merck under the name of Eusolex(R) 8020. blocks are present in an amount Such that the present compo In some embodiments, the compositions of the present sitions are transparent on the skin (i.e., non-whitening), pref invention further include one or more UVB sunscreen actives erably from about 0.5% to about 20%, preferably from about that absorb(s) UV radiation having a wavelength of about 290 5 0.5% to about 10%, and more preferably from about 0.5% to nm to about 320 nm. The compositions comprise an amount 5% by weight. When titanium dioxide is used, it can have an of the UVB sunscreen active that is safe and effective in anatase, rutile or amorphous structure. Manufacturers of providing UVB protection either independently, or in combi micronized grade titanium dioxide and Zinc oxide for Sun nation with, other UV protective actives which may be screen use include, but are not limited to Tayca Corporation, present in the compositions. The compositions comprise from 10 Uniqema, Shinetsu Chemical Corporation, Kerr-McGee, Nanophase, Nanosource, Sachtleben, Elementis, and BASF about 0.1% to about 20%, preferably from about 0.1% to Corporation, as well as their distribution agents and those about 12%, and more preferably from about 0.5% to about 8% companies that further process the material for Sunscreen use. by weight, of each UVB absorbing organic Sunscreen, or as Physical Sunblock particles (e.g., titanium dioxide and Zinc mandated by the relevant regulatory authority(s). 15 oxide) can be uncoated or coated with a variety of materials A variety of UVB sunscreen actives are suitable for use including but not limited to amino acids, aluminum com herein (See e.g., U.S. Pat. Nos. 5,087,372; 5,073,371; 5,073, pounds such as alumina, aluminum Stearate, aluminum lau 372; 4.937,370; and 4,999,186). Preferred UVB sunscreen rate, and the like; carboxylic acids and their salts (e.g., Stearic actives are selected from 2-ethylhexyl-2-cyano-3-2-ethyl acid and its salts); phospholipids, such as lecithin; organic hexyl N,N-dimethyl-p-aminobenzoate, p-aminobenzoic 20 silicone compounds; inorganic silicone compounds Such as acid, oxybenzone, homomethyl salicylate, octyl salicylate, silica and silicates and mixtures thereof. In some preferred 4,4'-methoxy-t-butyldibenzoylmethane, 4-isopropyl diben embodiments, the compositions of the present invention com Zoylmethane, 3-benzylidene camphor, 3-(4-methylben prise from about 0.1% to about 15%, preferably from about Zylidene) camphor,3-diphenylacrylate, 2-phenyl-benzimida 0.1% to about 7%, and more preferably from about 0.5% to Zole-5-sulphonic acid (PBSA), cinnamate esters and their 25 about 5%, by weight, of inorganic Sunscreen. derivatives Such as 2-ethylhexyl-p-methoxycinnamate, Sali In some preferred embodiments, the composition of the cylate esters and their derivatives such as triethanolamine present invention also includes preservatives. Such preserva salicylate, ethylhexyl salicylate, octyldimethyl para-ami tives include, but are not limited to pentylene glycol, ethylene nobenzoic acid, camphor derivatives and their derivatives, diamine tetra acetate (EDTA) and their salts, chlorhexidine and mixtures thereof. Preferred organic Sunscreen actives 30 (and its diacetate, dihydrochloride, digluconate derivatives), include 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, 2-phe 1,1,1-trichloro-2-methyl-2-propanol, parachloro metaxyle nyl-benzimidazole-5-Sulphonic acid (PBSA), octyl-p-meth nol, polyhexamethylenebiguanide hydrochloride, dehy oxycinnamate, and mixtures thereof. Salt and acid neutral droacetic acid, diazolidinyl urea, 2,4-dichlorobenzyl alcohol, ized forms of the acidic Sunscreens are also useful herein. 4,4-dimethyl-1,3-oxazolidine, formaldehyde (e.g., 37% In some embodiments, at least one agent is added to any of 35 aqueous solution, with 10-15% methanol to avoid polymer the compositions useful in the present invention to stabilize ization), glutaraldehyde, dimethylidantoin, imidazolidinyl the UVA Sunscreen to prevent it from photo-degrading on urea, 5-Chloro-2-methyl-4-isothiazolin-3-one, ortho-phe exposure to UV radiation and thereby maintaining its UVA nylphenol, 4-hydroxybenzoic acid esters (e.g., “paraben) protection efficacy. A wide range of compounds are reported and its methyl-, ethyl-, propyl-, isopropyl-, butyl-, and isobu to have these stabilizing properties and should be chosen to 40 tyl-esters, trichlosan, 2-phenoxyethanol, phenyl mercuric complement both the UVA Sunscreen and the composition as acetate, borate, nitrate, quaternium-15, salicylate, salicylic a whole (See e.g., U.S. Pat. Nos. 5,972,316; 5,968,485; 5,935, acid and its salts, calcium, calcium Sorbate, Sorbic acid and its 556; 5,827,508; and WO 00/06110). Preferred examples of salts, iodopropanyl butylcarbamate Zinc pyrithione, benzyl stabilizing agents for use in the present invention include alcohol, 5-bromo-5-nitro-1,3-dioxane, 2-bromo-2-nitropro 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, ethyl-2-cyano- 45 pane-1,3-diol, benzoic acid and its salts, sulfites, bisulfites, 3.3-diphenylacrylate, 2-ethylhexyl-3,3-diphenylacrylate, phenyoxyethanol, chloroxylenol, diazolidinyl urea, meth ethyl-3,3-bis(4-methoxyphenyl)acrylate, diethylhexyl 2.6 ylparabens, propylparabens, isoproplyparabens, isobutylpa napthalate and mixtures thereof (Symrise Chemical Com rabens, butylparabens, ethylparaben, phenoxyethanol PG, pany). and benzalkonium chloride. In some embodiments, at least one agent is added to any of 50 A variety of optional ingredients such as neutralizing the compositions useful in the present invention to improve agents, perfumes and perfume solubilizing agents, and color the skin Substantivity of those compositions, particularly to ing agents, also find use in Some of the compositions herein. enhance their resistance to being washed off by water or It is preferred that any additional ingredients enhance the skin rubbed off. Examples include, but are not limited to, acry softness/smoothness benefits of the product. In addition it is lates/C2-alkylmethacrylate copolymer, acrylate/acrylate 55 preferred that any such ingredients do not negatively impact copolymer, dimethicone, dimethiconol, graft-copoly (dim the aesthetic properties of the product. ethylsiloxane/i-butyl methacrylate), lauryl dimethicone, Other optional materials include keratolytic agents, as well PVP/Hexadecane copolymer, PVP/Eicosene copolymer, tri as water-soluble and/or solubilizable preservatives preferably contanyl PVP and trimethoxysiloxysilicate. at a level of from about 0.1% to about 5% (e.g., Germall 115, In addition to organic Sunscreens, in some embodiments, 60 methyl, ethyl, propyl and butyl esters of hydroxybenzoic the compositions of the present invention additionally com acid, benzyl alcohol, DMDM hydantoin iodopropanylbutyl prise inorganic physical Sunblocks (See e.g., TFA Interna carbanate available under the trade name Glydant Plus from tional Cosmetic Ingredient Dictionary, 6' Edition, pp. 1026 Lonza; EDTA, EUXYL(R) K400, Bromopol (2-bromo-2-ni 28 and 1103 (1995: Sayre et al., J. Soc. Cosmet. Chem. tropropane-1,3-diol) and phenoxypropanol); anti-bacterials 41:103-109 1990; and Lowe et al., supra). Preferred inor- 65 (e.g., IRGASANR) and phenoxyethanol (preferably at levels ganic physical Sunblocks include Zinc oxide and titanium of from about 0.1% to about 5%); as well as soluble or dioxide and mixtures thereof. colloidally-soluble moisturizing agents such as hyaluronic US 9,084,734 B2 99 100 acid, chondroitin Sulfate, and starch-grafted sodium poly Optional materials include pigments that, where water acrylates (e.g., SANWETR IM-1000, IM-1500 and insoluble, contribute to and are included in the total level of IM-2500, available from Celanese Superabsorbent Materials, oil phase ingredients. Pigments Suitable for use in the com Portsmith, Va., See e.g., U.S. Pat. No. 4,076,663; vitamins positions of the present invention can be organic and/or inor Such as vitamin A, vitamin C. vitamin E and derivatives ganic. Also included within the term "pigment are materials thereof and building blocks thereof such as phytantriol, and having a low color or luster, such as matte finishing agents, Vitamin K and components thereof Such as the fatty alcohol light scattering agents, and formulation aids such as micas, dodecatrienol; alpha and beta hydroxyacids; aloe Vera; sph seracites, and carbonate salts. Further examples of Suitable ingosines and phytosphingosines, cholesterol; skin whitening pigments include titanium dioxide, iron oxides, glutamate agents: N-acetyl cysteine; colouring agents; antibacterial 10 iron oxides, Zinc oxide, bismuth oxychloride, ultramarine agents such as TCC/TCS, also known as triclosan and trichlo blue (all of which may be either pre-dispersed and/or pre rocarbon; perfumes and perfume solubilizers. Examples of coated or not) D&C dyes and lakes, FD&C colors, natural alpha hydroxy acids include glycolic acid, lactic acid, malic color additives such as carmine, and mixtures thereof. acid, citric acid, glycolic acid in conjunction with ammonium Depending upon the type of composition, a mixture of pig glycolate, alpha-hydroxy ethanoic acid, alpha-hydroxyoc 15 ments is usually used in preferred embodiments of the present tanoic acid, alpha-hydroxycaprylic acid, hydroxycaprylic invention. Preferred pigments for use herein from the view acid, mixed fruit acid, tri-alpha hydroxy fruit acids, triple fruit point of moisturization, skin feel, skin appearance and emul acid, Sugarcane extract, alpha hydroxy and botanicals, 1-al sion compatibility are treated pigments. In some embodi pha hydroxy acid and glycomer in crosslinked fatty acids ments, the pigments are treated with compounds, including (e.g., alpha nutrium). Preferred examples of alpha hydroxy but not limited to amino acids, silicones, lecithin and ester acids are glycolic acid and lactic acid. It is preferred that alpha oils. hydroxy acids are used in levels of up to about 10%. It is not In preferred embodiments, the pH of the compositions intended that the present invention be limited to any particular herein is in the range from about 3.5 to about 10, preferably compound derived from any particular source, as any Suitable from about 4 to about 8, and more preferably from about 5 to additive compound, whether obtained from natural Sources or 25 about 7, wherein the pH of the final composition is adjusted through synthesis in the laboratory find use in the present by addition of acidic, basic or buffer salts as necessary, invention. depending upon the composition of the forms and the pH Other optional materials include water-soluble or solubi requirements of the compounds. lizable preservatives preferably at a level of from about 0.1% The compositions of the present invention are prepared by to about 5% each, such as Germall 115, methyl, ethyl, propyl 30 standard techniques well known to those skilled in the art. In and butyl esters of hydroxybenzoic acid, benzyl alcohol, general the aqueous phase and/or the oil phase are prepared DMDM hydantoin iodopropanyl butylcarbanate available separately, with materials of similar phase partitioning being under the trade name Glydant Plus from Lonza, EDTA, added in any order. If the final product is an emulsion, the two Euxyl R. K400, Bromopol (2-bromo-2-nitropropane-1,3- phases are then combined with vigorous stirring and/or diol), pentylene glycol and phenoxypropanol; anti-bacterials 35 homogenization as necessary, to reduce the size of the inter Such as Irgasan Rand phenoxyethanol (preferably at levels of nal phase droplets. Any ingredients in the formulation with from 0.1% to about 5%). Antibacterial agents such as TCC/ high volatility, or which are susceptible to hydrolysis or TCS, also known as triclosan and trichlorocarbon are also decomposition at high temperatures, are added with gentle useful in compositions of the present invention. stirring towards the end of the process, post emulsification if Neutralizing agents suitable for use in neutralizing acidic 40 applicable. Dosage frequency and amount will depend upon group containing hydrophilic gelling agents herein include the desired performance criteria. Sodium hydroxide, potassium hydroxide, ammonium In some embodiments of the present invention, method of hydroxide, monoethanolamine, diethanolamine, amino decreasing VEGF activity are provided. In these embodi methyl propanol, tris-buffer and triethanolamine. ments, the methods comprise applying to an organism in need Other optional materials that find use in the present inven 45 thereof an effective amount of any one of the compounds set tion include any of the numerous functional and/or active forth herein. In additional preferred embodiments, the present ingredients known to those skilled in the art (See e.g., invention provides compounds for treatment of an organism McCutcheon's Functional Materials, North American and in need thereof, including humans and other animals. International Editions, MC Publishing Co. 2003) As indi cated above, non-limiting examples include keratolytic 50 EXPERIMENTAL agents; Soluble or colloidally-soluble moisturizing agents Such as hyaluronic acid and chondroitin Sulfate; Vitamins The following Examples serve to illustrate certain pre Such as vitamin A, vitamin C, Vitamin E. Vitamin K and ferred embodiments and aspects of the present invention and derivatives thereof and building blocks thereof; phytantriol: are not to be construed as limiting the Scope thereof. fatty alcohols such as dodecatrienol; alpha and beta hydroxy 55 In the experimental disclosure which follows, the follow acids; aloe Vera; sphingosines and phytosphingosines, cho ing abbreviations apply: PI (proteinase inhibitor), BBI (Bow lesterol; skin whitening agents; N-acetyl cysteine; coloring man-Birk inhibitor), STI (Soybean Trypsin inhibitor); ppm agents. Examples of alpha hydroxy acids include glycolic (parts per million); VEGF and VegF (vascular endothelial acid, lactic acid, malic acid, and citric acid (whether derived growth factor); M (molar); mM (millimolar); uM (micromo synthetically or from natural Sources and whether used alone 60 lar); nM (nanomolar); mol (moles); mmol (millimoles); umol or in combination) and their esters or relevant buffered com (micromoles); nmol (nanomoles); gm (grams); mg (milli binations. Other examples of alpha-hydroxy acids include: grams); mg (micrograms); pg (picograms); L (liters); ml and alpha-hydroxy ethanoic acid, alpha-hydroxyoctanoic acid, mL (milliliters); ul and LL (microliters); cm (centimeters); alpha-hydroxycaprylic acid, and hydroxycaprylic acid. Pre mm (millimeters); um (micrometers); nm (nanometers); U ferred examples of alpha hydroxy acids include glycolic acid 65 (units); V (volts); MW (molecular weight); sec (seconds); and lactic acid. It is preferred that alpha hydroxy acids are min(s) (minute/minutes); hr(s) (hour/hours); C. (degrees used in levels of up to about 10%. Centigrade); QS (quantity sufficient); ND (not done); SA (see US 9,084,734 B2 101 102 able): NA (not applicable); rpm (revolutions per minute); tional Center for Biotechnology Information); Applied Bio HO (water); dHO (deionized water); (HCl (hydrochloric systems (Applied Biosystems, Foster City, Calif.); Clontech acid); aa (amino acid); by (base pair); kb (kilobase pair); kD (CLONTECH Laboratories, Palo Alto, Calif); Difco (Difco (kilodaltons); cDNA (copy or complimentary DNA); DNA Laboratories, Detroit, Mich.); Oxoid (Oxoid Inc., Ogdens (deoxyribonucleic acid); ssDNA (single stranded DNA); burg, N.Y.); GIBCO BRL or Gibco BRL (Life Technologies, dsDNA (double stranded DNA); dNTP (deoxyribonucleotide Inc., Gaithersburg, Md.); Millipore (Millipore, Billerica, triphosphate); RNA (ribonucleic acid), MgCl2 (magnesium Mass.); Bio-Rad (Bio-Rad, Hercules, Calif.); Invitrogen (In chloride); NaCl (sodium chloride); w/v (weight to volume); vitrogen Corp., San. Diego, Calif.).; NEB (New England V/v (Volume to Volume); g (gravity); OD (optical density); Biolabs, Beverly, Mass.); Cambrex (Cambrex Bioproducts, Aaos (absorbance at 405 nm), Vmax (the maximum initial 10 velocity of an enzyme catalyzed reaction); FGFrI(IIIc) East Rutherford, N.J.); Sigma (Sigma Chemical Co., St. (FGF-5 receptor); Dulbecco's phosphate buffered solution Louis, Mo.); Pierce (Pierce Biotechnology, Rockford, Takara (DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast (Takara Bio Inc. Otsu, Japan); Roche (Hoffmann-La Roche, Extract, 10 mM. NaCl, 2.5 mM KCl); Terrific Broth (TB; 12 Basel, Switzerland); EM Science (EM Science, Gibbstown, g/l Bacto Tryptone, 24 g/l glycerol. 2.31 g/l KHPO, and 15 N.J.); Qiagen (Qiagen, Inc., Valencia, Calif.); Biodesign 12.54 g/l KHPO); ODs (optical density at 280 nm); (Biodesign Intl. Saco, Maine); Biosource (Biosource, Intl. ODoo (optical density at 600 nm); PAGE (polyacrylamide Camarillo, Calif.); Aptagen (Aptagen, Inc., Herndon, Va.); gel electrophoresis); PBS (phosphate buffered saline 150 Molecular Devices (Molecular Devices, Corp., Sunnyvale, mMNaCl, 10 mM sodium phosphate buffer, pH 7.2); PBST Calif.); R&D Systems (R&D Systems, Minneapolis, Minn.); (PBS+0.25% Tween R. 20); PEG (polyethylene glycol): PCR Stratagene (Stratagene Cloning Systems, La Jolla, Calif); (polymerase chain reaction); RT-PCR (reverse transcription Marsh (Marsh Biosciences, Rochester, N.Y.); Bio-Tek (Bio PCR); SDS (sodium dodecyl sulfate); bME, BME and BME Tek Instruments, Winooski, Vt.): (Biacore (Biacore, Inc., Pis (beta-mercaptoethanol or 2-mercaptoethanol); Tris-HCl (tris cataway, N.J.); PeproTech (PeproTech, Rocky Hill, N.J.); Hydroxymethylaminomethane-hydrochloride); Tricine (N- SynPep (SynPep, Dublin, Calif.); Chemicon (CHEMICON, tris-(hydroxymethyl)-methyl-glycine); CHES (2-(N-cyclo 25 Temecula, Calif.); Clinical Research Laboratories, (Clinical hexylamino) ethane-sulfonic acid); TAPS (3-tris (hydroxymethyl)-methyl-amino-propanesulfonic acid); Research Laboratories, Inc., Piscataway, N.J.); and Microsoft CAPS (3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (Microsoft, Inc., Redmond, Wash.). (dimethylsulfoxide); DTT (1,4-dithio-DL-threitol); Glut and Example 1 GSH (reduced glutathione); GSSG (oxidized glutathione); 30 TCEP (Tris(2-carboxyethyl phosphine); Tris (tris(hy Personal Care Compositions droxymethyl)aminomethane): HEPES (N-2-Hydroxyethyl piperazine-N-2-ethanesulfonic acid); HBS (HEPES buff In this Example, various personal care compositions com ered saline); SDS (sodium dodecylsulfate); Tris-HCl (tris prising any of the compounds of the present invention are Hydroxymethylaminomethane-hydrochloride); Ci (Curies) 35 provided as follows. In these formulations, the amounts are mCi (milliCuries); uCi (microCuries); TLC (thin layer ach given as percentages of the total composition, unless other romatography); O/W (oil in water emulsion); W/O (water in wise indicated. Also, unless otherwise indicated in the fol oil emulsion); W/S (water in silicon emulsion); pickering lowing formulations, the concentration of BBI-AV (referred emulsion (emulsion stabilized with a solid compound); to as “Compound' below) ranges from about 0.01% to about hydrodispersion (emulsifier-free formulations); PIT (phase 40 1.0%. In some formulations, the preferred concentration is in inversion temperature technology used to manufacture spe the range of about 0.1% to about 0.2%, while in other formu cial emulsions); Sticks (any product that is provided in a stick lations, the preferred concentration is in the range of about format, including but not limited to lipsticks, anti-perspirants, 0.05% to about 0.1% (e.g., for some hair growth inhibition deodorants); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms (me embodiments); from about 0.02 to 0.1% (e.g., for some skin syl): Et (ethyl), Me (methyl); Klenow (DNA polymerase I 45 lightening embodiments); from about 0.5% to about 1.0% large (Klenow) fragment); rpm (revolutions per minute); (e.g., for Some skin lightening embodiments); or at concen EGTA (ethylene glycol-bis(B-aminoethyl ether) N.N.N',N'- trations greater than about 0.1% (e.g., for some rosacea treat tetraacetic acid); EDTA (ethylenediaminetetracetic acid); bla ing embodiments). Those of skill in the art know how to (B-lactamase or amplicillin-resistance gene); PDS (plasma determine the suitable (i.e., optimum) concentration of BBI derived bovine serum that has been dialyzed to remove 50 growth factors; dialysis of defibrinated bovine plasma is per AV for each product. In some formed against DMEM for about 6 hours at 4°C., with stir ring, the media is changed and dialysis is continued over night; the dialyzed PDS is collected after 24 hours, and sterile MOISTURIZING BODY WASH (pH 7. filtered twice through a 0.2 um filter); FCS and FBS (fetal calf 55 RAW MATERIAL serum); GE Healthcare (GE Healthcare, Chalfont St. Giles, (INCI Designation) Amount United Kingdom); DNA2.0 (DNA2.0, Menlo Park, Calif); Deionized Water QS OXOID (Oxoid, Basingstoke, Hampshire, UK); MegaZyme Glycerin 4.0 (MegaZyme International Ireland Ltd., Bray Business Park, PEG-6 Caprylic Capric Glycerides 4.0 Palm Kernel Fatty acids 3.0 Bray, Co., Wicklow, Ireland); Corning (Corning Life Sci 60 Sodium Laureth-3 Sulphate 45.0 ences, Corning, N.Y.): (NEN (NEN Life Science Products, Cocamide MEA 3.0 Boston, Mass.); Pharma AS (Pharma AS, Oslo, Norway); Sodium Lauroamphoacetate 2S.O Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis, Soybean Oil 1O.O Polyguaternium-10 O.70 Lewisville, Tex.): ATCC (AmericanType Culture Collection, Preservative, fragrance, color QS Rockville, Md.); Gibco/BRL (Gibco/BRL, Grand Island, 65 Compound 1000 ppm N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Phar macia (Pharmacia Biotech, Pisacataway, N.J.): NCBI (Na US 9,084,734 B2
BODY WASH MOISTURIZING CREAM
RAW MATERIAL pH 8 pH 6.5 pH 7 RAW MATERIAL pH 7 pH 7 pH 7.5 5 (INCI Designation) Amount Amount Amount (INCI Designation) Amount Amount Amount
Deionized water QS QS QS Deionized water QS QS QS Sodium Laureth Sulphate 12 15 8 Glycerine 3 5 10 Petrolatum 3 3 Cocamidopropyl Betaine 8 10 15 10 Cetyl Alcohol 95% 1.5 1.5 Decyl Glucoside O 2 1 Dimethicone Copolyol 2 2 Polyguaternium-10 O.25 O O sopropyl Palmitate 1 1 Polyguaternium-7 O O 0.7 Carbopol 954 (Noveon) 0.7 0.7 Preservative, fragrance, color QS QS QS 15 Dimethicone (350cs) 1 1 Compound 250 ppm 500 ppm 1000 ppm Stearyl Alcohol 97% O.S O.S Stearic acid O.1 O.1 O.1 Peg-100-stearate O.1 O.1 O.1 Titanium Dioxide O.3 O.3 O.3 BODY LOTION Preservative, color, fragrance QS QS QS Compound 50 ppm 250 ppm 1000 ppm RAW MATERIAL pH 7 pH 7 pH 7.5 pH 7 (INCI Designation) Amount Amount Amount Amount Deionized Water QS QS QS QS Glycerine 8 8 O 12 25 Sohexadecane 3 3 3 6 FACIAL CLEANSINGEMULSION Niacinamide O 3 5 6 Sopropyl IsoStearate 3 3 3 3 RAW MATERIAL Polyacrylamide (and) 3 3 3 3 (INCI Designation) Amount soparaffin (and) Laureth-7 Petrolatum 4 4 4 2 30 Water 69.05 Nylon 12 2 2 2.5 2.5 Disodium EDTA O.1 Dimethicone 2 2 2.5 2.5 Glyceryl polymethacrylate (and) 1.O Sucrose Polycottonseed Oil 1.5 1.5 1.5 1.5 Propylene glycol Stearyl Alcohol 97% 1 1 1 1 Glycerin 2.O D Panthenol 1 1 1 1 Xanthan gum O.S DL-alphaTocopherol Acetate 1 1 1 1 O.S 35 Hydroxyethyl cellulose Cetyl Alcohol 95% O.S O.S O.S 1 Tridecyl neopentanoate 4.0 Behenyl Alcohol 1 1 1 O.S Socetyl Stearate 6.0 Cetearyl Alcohol (and) 0.4 0.4 O.S O.S Octyl palmitate 8.0 Cetearyl Glucoside Glyceryl dilaurate 4.0 Stearic Acid O.15 O.15 O.15 O.15 PEG-2O Stearate 2.O PEG-100-Stearate O.15 O.15 O.15 O.15 Glyceryl Stearate (and) Laureth-23 2.O Preservative, fragrance, color QS QS QS QS 40 Laurylpyrrollidone O.S Compounds 250 ppm 500 ppm 750 ppm 1000 ppm Chamomile extract O.2 Aloe vera (200x) O.OS Fragrance, preservative QS Compound SA
ULTRA-HIGHMOISTURIZING EMULSION 45 RAW MATERIAL pH 7 pH 7 (INCI Designation) Amount Amount SURFACTANT-BASED FACIAL CLEANSER
Deionized water QS QS RAW MATERIAL Glycerin 12 5 50 (INCI Designation) Amount PEG 400 O 10 Niacinamide 5 7 Water 62.55 Sohexadecane 5 5 Acrylates/Steareth-20 methacrylate copolymer 3.3 Dimethicone 3 2 Disodium EDTA O.OS Polyacrylamide (and) Isoparaffin (and) 3 3 Glycerin 2.0 Laureth-7 55 Glyceryl polymethacrylate (and) Propylene O.S Sopropyl IsoStearate 2 2 glycol (and) PVMMA copolymer Polymethylsilsesquioxane 2 2 Sodium laureth Sulfate (30%) 17.5 Cetyl Alcohol 95% 1 1 Cetearyl alcohol 1.O Sucrose polycottonseed oil 1 1 Shea butter 1.O D-Panthenol 1 1 Disodium oleamido PEG-2 sulfosuccinate S.O Tocopherol Acetate 1 1 Cocoamidopropyl Betaine 3.0 Stearyl Alcohol 95% O.S O.S 60 Sodium lauroyl sarcosinate 1.O Cetearyl Glucoside O.S O.S PEG-7 glyceryl cocoate 1.O Titanium dioxide O.3 O.3 Isodecyl oleate 1.5 Stearic Acid O.15 O.15 Peppermint extract O.25 PEG-100-Stearate O.15 O.15 Eucalyptus extract O.25 Preservative, fragrance, color QS QS Fragrance, preservative, color, pH adjust QS Compound 250 ppm 100 ppm 65 Compound SA US 9,084,734 B2 105 -continued FACLAL, EXFOLIATING GEL
RAW MATERIAL FACIAL MASK (INCI Designation) Amount RAW MATERIAL Water 64.39 Disodium EDTA O.OS (INCI Designation) Amount Aloe vera (200x) O.O1 Benzophenone-4 O.25 Propylene glycol 4.0 Propylene glycol 1.O Sodium Coco PG-Dimonium Chloride Phosphate 1.O Acrylates/C10-30 alkyl acrylate crosspolymer (2%) 2O.O 10 Glyceryl polymethacrylate (and) Propylene glycol 1O.O Fragrance, preservative, color, pH adjust QS Glyceryl polymethacrylate (and) Propylene 1.O Compound SA glycol (and) PVMMA copolymer Hydrogenatedjojoba oil 1.5 Fragrance, preservative, color, pH adjust QS Compound SA 15 AFTER-SHAVE BALM
RAW MATERIAL (INCI Designation) Amount FACIALTONER Water 82.12 RAW MATERIAL Disodium EDTA O.1 (INCI Designation) Amount Acrylate copolymer 2.O Acrylate/Stareth-20 methacrylate copolymer 1.O Water 93.99 Propylene glycol 3.0 Disodium EDTA O.1 Sodium hydroxide (10%) 1.28 Butylene glycol 2.0 25 Glyceryl Stearate (and) Cetyl alcohol (and) 3.5 Aloe vera (200x) O.1 Stearyl alcohol (and) Behenyl alcohol (and) Allantoin O.1 Palmitic acid (and) Stearic acid (and) Hydroxyethyl Benzophenone-4 O.S cetearamidopropyldimonium chloride Witch hazel extract O.3 Isocetyl Stearate 1.O Propylene glycol (and) Euphrasia extract (and) O.O1 C12-15 alkyl lactate 1.5 Golden seal root extract (and) Green tea extract Octyldodecyl stearate 3.0 PEG-40 hydrogenated castor oil O.S 30 Glyceryl polymethacrylate (and) Propylene 1.O Quaternium-22 O.S glycol (and) PVMMA copolymer Sandlewood oil O.O2 Poly quaternium-11 0.5 Fragrance, preservative, color, pH adjust QS Fragrance, preservative, color, pH adjust QS Compound SA Compound SA 35
EXFOLIATING CREAM EYE GEL
RAW MATERIAL RAW MATERIAL (INCI Designation) Amount 40 (INCI Designation) Amount Water 68.8O Water 89.14 Disodium EDTA O.1 VP/Acrylates/Lauryl methacrylate copolymer O.S PVM/MA decadiene crosspolymer 1.O Glycerin S.O Butylene glycol 3.0 Aminomethylpropanol O.3 PEG-2O Stearate 1.O 45 Aloe vera (200x) O.OS Glyceryl Stearate (and) Laureth-23 2.0 Benzophenone-4 O.1 Diisopropyl adipate 2.0 Glyceryl polymethacrylate (and) Propylene glycol (and) O.2 Isodecyl oleate 2.0 PVM/MA copolymer Isocetyl Stearoyl Stearate S.O Butylene glycol (and) Water (and) Witch hazel extract O.S Myristyl myristate 1.O Butylene glycol (and) Water (and) Cucumber extract O.3 Glyceryl dilaurate 2.0 50 PEG-40 hydrogenated castor oil O.O1 Sodium hydroxide, 10% 2.6 Acrylates/Beheneth-25 methacrylate copolymer 2.4 Glyceryl polymethacrylate (and) Propylene glycol S.O Fragrance, preservative, color, pH adjust QS Glyceryl polymethacrylate (and) Propylene O.S Compound SA glycol (and) PVMMA copolymer Hydrogenatedjojoba oil 3.0 Fragrance, preservative, color, pH adjust QS Compound SA 55 HIGH MELTING POINT LIPSTICK
RAW MATERIAL (INCI Designation) Amount FACIAL MASK 60 Ozokerite wax S.O RAW MATERLAL Candelilla wax 11.0 (INCI Designation) Amount Octyl dodecanol 26.0 C30-45 alkyl methicone S.O Water 76.4 Cyclomethicone 4.8 Disodium EDTA O.1 Petrolatum 3.0 Bentonite 12.5 65 Lanolin oil 9.O Potassium C12-13 Alkyl Phosphate S.O Avocado oil 2.O US 9,084,734 B2 107 108 -continued LIPBALM
HIGH MELTING POINT LIPSTICK RAW MATERLAL (INCI Designation) Amount RAW MATERLAL Petrolatum 47.3 (INCI Designation) Amount Isopropyllanolate 6.O Ozokerite wax 16.5 Candelilla wax 4.5 Oleyl alcohol 8.O Diisopropyl dilinoleate 2SO 10 Retinyl palmitate O.S Pigment cyclomethicone 2S.O Tocopherol acetate O.2 Fragrance, preservative QS Fragrance, preservative QS Compound SA Compound SA
15
WATERPROOF MASCARA LIPSTICK RAW MATERIAL (INCI Designation) Amount RAWMATERIAL (INCI Designation) Amount Water 49.45 Propylene glycol 3.0 Candelilla wax 9.1 Triethanolamine (99%) 3.1 Acrylates. Octylacrylamine Copolymer S.O Isopropyl myristate 9.6 DiisoStearoyl trimethylolpropane siloxy silicate S.O Lanolin S.O 25 Candelilla wax 4.5 Beeswax 4.0 Beeswax 5.5 Ozokerite wax 2.O Paraffin (130/135) 2.O Carnauba wax 1.O Ozokerite wax 2.5 Cetyl alcohol 3.0 Castor oil 53.7 Stearic acid S.O Iron oxides 11.0 Carnauba wax 1.5 30 Fragrance, preservative QS Pigments 7.5 Compound SA Mineral oil 4.0 Fragrance, preservative QS Compound SA 35 ANHYDROUSWATERPROOF MASCARA
RAW MATERIAL (INCI Designation) Amount LIP GLOSS C9-11 Isoparaffin 30.95 RAWMATERIAL 40 Polyethylene 11.O (INCI Designation) Amount Candelilla wax 4.5 Hydroxylated lanolin O.25 Bis-diglyceryl polyacyladipate-1 43.5 Pentaerythrityl rosinate 2.0 Bis-diglyceryl polyacyladipate-2 10 Zinc stearate 1.O Glycerol ricinoleate 10 Silica silylate 1.O Polyisobutene 1000 13 45 Petroleum distillates (and) 3S.O Lanolin wax 10 Quaternium-18 hectorite (and) Candelilla wax 2.5 Propylene Carbonate Mica (and) titanium dioxide 3 Iron oxides 12.O d-Panthenol 5 Fragrance, preservative QS Fragrance, preservative, color QS Compound SA Compound SA 50
WATER-BASED MASCARA LIP GLOSS WITHSUNSCREEN 55 RAW MATERLAL RAW MATERLAL (INCI Designation) Amount (INCI Designation) Amount Water 43.32 Triisostearyl Citrate 58.4 Polyvinyl pyrrollidone (K30) 2.0 Candelilla wax 8.O Hydroxyethyl cellulose 1.O Myristyl lactate 7.5 Triethanolamine (99%) 2.0 Microcrystalline wax S.O 60 Disodium EDTA O.1 Carnauba wax 2.0 Iron Oxides 1O.O Diisopropyl dimmer dilinoleate 1O.O Stearic acid 4.5 Mica (and) Bismuth oxychloride (and) Carmine 6.O Glyceryl monostearate 2.0 Zinc oxide (microfine) 2.0 Beeswax 7.0 Fragrance, preservative QS Carnauba wax 4.5 Compound SA 65 Hydroxylated lanolin 1.O Acrylates copolymer 2O.O US 9,084,734 B2 109 110 -continued -continued
WATER-BASED MASCARA PRESSED POWDER FORMULATIONS
RAWMATERIAL Loose Pressed Eye (INCI Designation) Amount Powder Powder Foundation Blush Shadow Fragrance, preservative QS Compression aids O-25 3-5 2-5 2-7 2-10 Compound SA (e.g., metallic Soaps, 10 waxes) Texture enhancers 1O-40 5-40 1O-40 1O-40 O-30 Colorants 2-10 2-10 S-20 2-10 1-40 (e.g., iron oxides, LIQUID EYELINER organic colors) Pearls O-2O O-10 O-5 O-20 0-60 RAWMATERIAL 15 (e.g. titanated mica, (INCI Designation) Amount bismuth oxychloride) Water 50-70 Wet binder O-3 2-5 2-5 3-10 3-15 Gellant O.S.-1.5 (e.g., Octyldodecyl Wetting agent(s) 1-3 stearoylstearate, di-PPG3 Polyol 4-8 myristyl ether adipate, Colorants 10-20 Alcohol S-10 isocetyl stearate, cetyl Film former 3-8 dimethicone) Fragrance, preservative QS Dry binder O-2 2-5 2-5 3-8 3-8 Compound SA (e.g., calcium silicate, 25 kaolin) Fragrance, preservative QS QS QS QS QS Compound SA SA SA SA SA NAILENAMEL
RAWMATERIAL 30 (INCI Designation) Amount WATER-IN-OIL FOUNDATION Solvent(s) 40-70 Resin(s) 10-20 RAW MATERIAL Plasticizer 3-12 (INCI Designation) Amount Gellant Colorants 35 Cyclomethicone 12.O Fragrance, preservative QS Dimethicone S.O Compound SA Cyclomethicone (and) Dimethicone copolyol 2O.O Laureth-7 O.S Colorants (hydrophobically treated) 2.2 Titanium dioxide (and) methicone 8.5 40 Talc (and) methicone 3.3 CUTICLE TREATMENT Water 37.2 Sodium chloride 2.0 RAWMATERIAL Propylene glycol 8.O (INCI Designation) Amount Fragrance, preservative QS Compound SA Petrolatum 34.8 45 Beeswax 7.2 Ozokerite wax 4.3 Candelilla wax 4.0 Cocoa butter 1.O ANHYDROUSMAKEUPSTICK Shea butter 1.O Glyceryl dilaurate 8.O 50 RAW MATERIAL Ethylhexyl palmitate 2O.O (INCI Designation) Amount C12-15 alkyl lactate 6.O PVP. Eicosene copolymer 3.5 Ozokerite wax S.6 Diisopropyl adipate 2.0 Polyethylene 5.3 Octinoxate 7.5 Glyceryl dilaurate 5.5 Retinyl palmitate O.1 55 Isosteary neopentanoate 13.0 Tocopherol acetate O.1 Octyldodecyl stearoylstearate 12.0 Fragrance, preservative, color, pH adjust QS Myristyl myristate 11.0 Compound SA Ethylhexyl methoxycinnamate 7.5 PVP/Eicosene copolymer O.S Tocopherol acetate O.1 Dimethicone (and) Trimethylsiloxysilicate 8.0 60 Cyclopentasiloxane 9.O PRESSED POWDER FORMULATIONS Mica 1O.O Talc 1.7 Loose Pressed Eye Titanium dioxide (and) Isopropyl titanium triisoStearate 8.86 Powder Powder Foundation Blush Shadow Iron oxides (and) Isopropyl titanium trilisostearate 1.94 Fragrance, preservative QS Fillers 70-95 40-90 40-80 40-80 40-80 65 Compound SA (e.g., talc, mica, Seracite) US 9,084,734 B2 111 112 -continued WATER-IN-SILICONE FOUNDATION SUNSCREEN FORMULAE RAW MATERIAL (INCI Designation) Amount 5 RAW MATERIAL Amount
Cetyl dimethicone copolyol O.45 -- -- Polyglycerol-4 isostearate (and) Cetyl dimethicone copolyol (and) 1.75 (INCI Designation) SPF-2S SPF-15 Hexyl laurate (and) PVMMA copolymer Polyalkylene polysiloxane copolymer O.9 Styrene? Acrylates copolymer (27% solids) 1845 Cetyl dimethicone O.9 Fragrance, preservative QS QS Beeswax 0.7 10 Compound SA SA Castor wax (and) hydrogenated castor oil O.35 Octyl palmitate 7.0 Cyclomethicone 7.95 Phenyl trimethicone 2.2 Titanium dioxide (and) Caprylyl silane 7.5 ron oxides (and) Caprylyl silane 1.1 15 VERY WATER-RESISTANTSUNSCREEN FORMULAE Talc (and) Caprylyl silane 3.8 Cyclomethicone 7.95 RAW MATERIAL - Amount pricone s 5 (INCI Designation) SPF-12 SPF-22 Sodium chloride O.S Water 65.16 46.53 Propylene glycol 5.3 20 Acrylates copolymer 3.0 3.0 Fragrance, preservative QS Disodium EDTA O.1 O.1 Compound SA Butylene glycol 2.0 2.0 Gylceryl polymethacrylate (and) Propylene glycol 1.O 1.O (and) PVMMA copolymer Butylated PVP O.OS O.OS 2s Glyceryl Stearate (and) Behenyl alcohol (and) 4.5 4.5 OIL-IN-WATER FOUNDATION Palmitic acid (and) Stearic acid (and) Lecithin (and) Lauryl alcohol RAW MATERIAL Tricontanyl PVP 1.O 1.O (INCI Designation) Amount Octyl palmitate 2.0 2.0 Octinoxate 7.5 7.5 Water 59.85 30 Oxybenzone 2.0 2.0 Polyvinylpyrrollidone S.O Ethylhexyl salicylate 3.0 3.0 Magnesium aluminum silicate 2.0 Tridecyl neopentanoate 3.0 3.0 Xanthan gum 0.4 Glyceryl dilaurate 0.5 0.5 Trisodium EDTA O.OS Sodium hydroxide (10%) 1.89 1.89 Glyceryl polymethacrylate (and) Propylene glycol (and) PVM/MA 1.0 Cyclopentasiloxane 2.0 2.0 copolymer Butylene glycol 1.O 1.O Polysorbate 20 1.O 35 Styrene? Acrylates copolymer (27% solids) 1845 Kaolin O.8 Fragrance, preservative QS QS Butylene glycol 4.0 Compound SA SA Titanium dioxide 6.OS Iron oxides 1.15 Dimethicone 6.O Ethylhexyl palmitate 2.0 40 PEGPPG-2525Dimethicone 1.O WATER-IN-SILICONE SUNSCREEN Tocopherol acetate O.1 Retinyl palmitate O.1 RAW MATERIAL Silica 3.0 (INCI Designation) Amount Cyclopentasiloxane S.O Fragrance, preservative QS 45 Cetyl PEG/PPG-15/15 butyl ether dimethicone 2.0 Compound SA Mineral oil 3.0 Ethylhexyl palmitate 1.O Ethylhexyl salicylate S.O Hydrogenated castor oil O.S Beeswax O.S SUNSCREEN FORMUL.AE 50 Qingxie 7.5 olyethylene 1.O RAW MATERIAL Amount PEG-30 dipolyhydroxystearate 2.0 o Cyclopentasiloxane S.O Dimethicone S.O (INCI Designation) SPF-2S SPF-15 Sodium chloride O.6 Water 52.65 71.10 55 Acrylates/C12-22 alkylmethacrylate copolymer O.S PVM/MA decadiene crosspolymer O.S O.S Water 66.4 Butylene glycol 3.0 3.0 Fragrance, preservative QS Disodium EDTA O.1 O.1 Compound SA PEG-2O Stearate 1.5 1.5 Glyceryl Stearate (and) Laureth-23 2.0 2.0 Isosteary neopentanoate 1.O 1.O 60 Ethylhexyl palmitate 2.0 2.0 Glyceryl dilaurate O.S O.S LEAVE-ONEHAIR CONDITIONER Octinoxate 7.5 7.5 Oxybenzone 2.0 2.0 RAW MATERIAL Ethylhexyl salicylate 3.0 3.0 (INCI Designation) Amount Sodium hydroxide (10%) 1.3 1.3 Glyceryl polymethacrylate (and) Propylene glycol 3.0 3.0 65 Deionized Water QS Glyceryl polymethacrylate (and) Propylene glycol O.S O.S Isostearamidopropyl Morpholine Lactate 6.O US 9,084,734 B2 113 114 -continued -continued
LEAVE-ONEHAIR CONDITIONER ANTI-DANDRUFF SHAMPOO
RAW MATERLAL 5 (INCI Designation) Amount RAW MATERLAL Hydroxyethylcellulose 1.O (INCI Designation) Amount Preservative, fragrance, color QS Compound 1000 ppm Zinc Pyrithione 40% 4.0 10 Fragrance, preservative, color QS Compound 1000 ppm
CREAM RINSE (pH4 RAWMATERIAL 15 (INCI Designation) Amount CLEAR SHAMPOO Formulations RAW MATERIAL Amounts Deionized Water QS Behentrimonium Chloride 2.0 Trilaureth-4 Phosphate 1.5 (INCI Designation) 1 2 3 4 5 Cetyl alcohol 2.0 20 Texapon N 70 13.OO 15.OO 10.SO 12.SO 10.00 Citric acid QS Dehyton PK 45 7.50 7.00 S.OO S.SO 10.00 Preservative, fragrance, color QS Cetiol HE 2.00 2.50 3.SO SOO 2.30 Compound 1000 ppm Fragrance O.10 O.10 O.10 O.10 O.10 Compound SA SA SA SA SA D-Panthenol USP 1.00 1...SO 18O 1.70 140 25 Preservative O.10 O.10 O.10 O.10 O.10 Citric Acid O.10 O.10 O.10 O.10 O.10 NOURISHING HAIR CONDITIONER/TREATMENT (pH 6) Luviguat Ultra Care 1...SO 1.00 1...SO 1.2O 1.10 Sodium Chloride 1...SO 140 140 1.30 1...SO RAW MATERIAL Water QS QS QS QS QS (INCI Designation) Amount (100) (100) (100) (100) (100) Deionized Water QS 30 Behentrimonium Methosulfate (and) Cetyl Alcohol 4.0 Wheat germ oil 1.0 scol g SHAMPOO Formulations PEG-60 Lanolin 1.O WM - (Amounts)- Panthenol 2.0 (INCI Designation) 1 2 3 4 5 Lupin amino acids 1.O Cocodimonium Hydroxypropyl Hydrolyzed Wheat Protein 1.O Texapon NSO 3S.OO 40.00 30.00 4S.OO 27.00 Fragrance, preservative, color QS Plantacare 2000 S.OO 5.50 4.90 3.50 7.OO Compound 1000 ppm Tego Betain L7 1O.OO S.OO 12.SO 7.50 15.00 Fragrance O.10 O.10 O.10 O.10 O.10 40 Compound SA SA SA SA SA D-Panthenol USP OSO 1.00 O.80 1...SO O.SO Preservative O.10 O.10 O.10 O.10 O.10 CONDITIONING SEHAMPOO Citric Acid O.10 O.10 O.10 O.10 O.10 Rewopal LA 3 OSO 2.00 O.SO OSO 2.00 RAW MATERIAL Sodium Chloride 1...SO 1...SO 1...SO 1...SO 1...SO (INCI Designation) Amount 45 Water QS QS QS QS QS (100) (100) (100) (100) (100) Deionized Water QS Sodium Laureth Sulfate 30% 27.0 Cocamidopropyl Betaine 3.7 Coco-Glucoside (and) Glyceryl Oleate S.O Coco-Glucoside (and) Glycol Distearate (and) Glycerine 3.0 50 CLEAR Guar Hydroxypropyl Trimonium Chloride O.1 CONDITIONING Laureth-2 1.55 SHAMPOO Formulations Fragrance, preservative, color QS RAW MATERIAL (Amounts) Compound 1000 ppm (INCI Designation) 1 2 3 4 5 55 Amphotensid GB 2009 1O.OO 1S.OO 2O.OO 12.00 17.00 Plantacare 2000 S.OO 6.OO 7.00 8.OO 4.OO ANTI-DANDRUFF SHAMPOO Tego Betain L7 1S.OO 12.00 10.00 18.00 2O.OO Luviguat FC 550 O.30 O.2O O.20 O.2O O.30 RAWMATERIAL Fragrance O.10 O.10 O.10 O.10 O.10 (INCI Designation) Amount 60 Compound SA SA SA SA SA Cremophor PS 20 S.OO 1.OO 1.00 7.OO S.OO Deionized Water QS Preservative O.10 O.10 O.10 O.10 O.10 Magnesium Aluminum Silicate 1.O Rewopal LA 3 2.00 1.OO O.SO 2.OO 2.00 Hydroxypropyl Methylcellulose O.8 Citric Acid O.20 O.2O O.20 O.2O O.20 Sodium Olefin Sulfate 40% 3S.O Stepan PEG 600 DS 3.00 2.OO 2.00 3.00 2.50 Lauramide DEA 4.0 Water QS QS QS QS QS Soyamide DEA 1.O 65 (100) (100) (100) (100) (100) Quaternium-70 Hydrolyzed Collagen 2.0 US 9,084,734 B2 116
FOAM OFW-EMULSION Formulations CLEAR CONDITIONING SEHAMPOO WITHVOLUMEEFFECT RAW MATERIAL (Amounts) Formulations (INCI Designation) 1 2 RAW MATERIAL (Amounts) Stearic acid S.OO 1.OO Cetyl alcohol 5.50 (INCI Designation) 1 2 3 Cetylstearyl alcohol 2.OO PEG-40 Stearate 8.50 Natriumlaurethsulfat 1O.OO 1O.SO 11.00 10 PEG-2O Stearate 1.OO Ethylhexyl Methoxycinnamat 2.00 1...SO 2.30 Caprylsäure Caprinsäure triglyceride 4.OO 2.OO Compound SA SA SA C12-15 Alkylbenzoate 1O.OO 1S.OO Cocoamidopropylbetain 2.50 2.60 2.2O Cyclomethicone 4.OO Disodium EDTA O.O1 O.10 O.O1 Dimethicone O.SO Compound SA SA 15 Preservative, Perfume, thickener QS QS QS Octylisostearate S.OO Water dem. QS (100) QS (100) QS (100) Myristyl Myristate 2.OO Ceresin 1...SO pH adjusted to 6.0 Glycerine 3.00 Filter Hydroxypropyl distärke phosphate 1.OO 3.SO CONDITIONING SEHAMPOO WITH PEARLESCENT BHT O.O2 Disodium EDTA OSO O.10 Formulations Parfim, Konservierungsmittel QS QS RAW MATERIAL Amounts 25 Colorant QS QS (INCI Designation) 1 2 3 Potassium hydroxide QS QS Water dem. QS (100) QS (100) Polyguarternium-10 O.SO O.SO O.40 Sodiumlaurethsulfat 9.00 8. SO 8.90 pH adjusted to pH adjusted to Cocoamidopropylbetain 2.50 2.60 3.00 65-7.5 S.O-6.O Benzophenon-4 1...SO O.SO 1.00 Emulsion 1 30 Compound SA SA SA Emulsion 2 Pearlescent compound 2.00 2.50 Disodium EDTA O.10 O.15 0.05 Gas (Stickstoff) Preservative, Perfume, thickener QS QS QS Gas (Helium) Water dem. QS (100) QS (100) QS (100) 35 pH adjusted to 6.0
CONDITIONER SHAMPOO WITH PEARLESCENT Formulations CLEAR CONDITIONING SEHAMPOO RAW MATERIAL - (Amounts)- 40 Formulations (INCI Designation) 1 2 3 RAW MATERIAL Amounts Polyguartemium-10 OSO O.SO O40 (INCI Designation) 1 2 3 Sodiumlaurethsulfat 9.00 8.50 8.90 Cocoamidopropylbetain 2.50 2.60 3.00 Polyguarternium-10 O.SO O.SO O.SO Benzophenon-4 1...SO O.SO 1.00 45 Sodiumlaurethsulfat 9.00 8. SO 9.SO Compound SA SA SA Compound SA SA SA Pearlescent compound 2.00 2.50 Benzophenon-3 1.00 1...SO O.SO Disodium EDTA O.10 O.15 O.OS Imidosuccinicacid, Na O.20 O.20 O.80 Preservative, Perfume, thickener QS QS QS Preservative, Perfume, thickener QS QS QS Water dem. QS (100) QS (100) QS (100) Water dem. QS (100) QS (100) QS (100) 50 pH adjusted to 6.0 pH adjusted to 6.0
CLEAR CONDITIONING SEHAMPOO WITHVOLUMEEFFECT CLEAR CONDITIONING SEHAMPOO 55 Formulations Formulations RAW MATERIAL Amounts RAW MATERIAL - (Amounts)- (INCI Designation) 1 2 3 (INCI Designation) 1 2 3 Natriumlaurethsulfate 1O.OO 1O.SO 11.00 Polyguarternium-10 O.SO OSO OSO 60 Ethylhexyl Methoxycinnamat 2.00 1...SO 2.30 Sodiumlaurethsulfat 9.00 8. SO 9.SO Compound SA SA SA Compound SA SA SA Benzophenon-3 1.00 1...SO OSO Cocoamidopropylbetain 2.50 2.60 2.2O Disodium EDTA O.O1 O.10 O.O1 Imidosuccinicacid, Na O.20 O.20 O.80 Preservative, Perfume, thickener QS QS QS Preservative, Perfume, thickener QS QS QS Water dem. QS (100) QS (100) QS (100) Water dem. QS (100) QS (100) QS (100) 65 pH adjusted to 6.0 pH adjusted to 6.0 US 9,084,734 B2 117 118
GEL CREME
Formulations RAW MATERIAL Amounts
(INCI Designation) 1 2 3 4 Acrylat C10-30 Alkylacrylat Crosspolymer O40 O.35 O.40 O.35 Polyacrylicacid O.20 O.22 O.2O O.22 Xanthan Gummi O.10 O.13 O.10 O.13 Cetearylalkohol 3.00 2.50 3.00 2.50 C12-15 Alkylbenzoat 4.OO 4...SO 4.OO 4...SO Caprylic Capric Triglycerid 3.00 3.SO 3.00 3.SO Aminobenzophenon (e.g., UVINULA PLUSTM) 2.OO 1...SO 0.75 1.00 UWASorb K2A 3.00 Ethylhexyl Methoxycinnamat 3.00 1.OO Bis-Ethylhexyloxyphenol methoxyphenyl Triazin 1...SO 2.00 Butyl Methoxydibenzoylmethan 2.OO Disodium Phenyl Dibenzimidazol Tetrasulfonat 2.50 O.SO 2.00 Ethylhexyl Triazon 4.OO 3.00 4.OO Octocrylen 4.OO Diethylhexyl Butamido Triazon 1.OO 2.00 Phenylbenzimidazol Sulfonsäure O.SO 3.00 Methylen Bis-Benzotriazolyl 2.OO O.SO 1...SO Tetramethylbutylphenol Ethylhexysalicylate 3.00 Drometrizol Trisiloxan O.SO Terephthaliden Dicamphor Sulfons?ure 1...SO 1.00 Diethylhexyl-2,6-naphthalate 3.SO 4.OO 7.00 9.00 Titanium dioxide- microfine 1.OO 3.00 Zincoxide- microfine O.25 Compound SA SA SA SA Cyclisches Dimethylpolysiloxane S.OO 5.50 S.OO 5.50 Dimethicon Polydimethylsiloxane 1.OO O60 1.OO O60 Glycerine 1.OO 1.20 1.OO 1.20 Sodium hydoxide QS. QS QS QS Preservative O.30 O.23 O.30 O.23 Perfume 0.20 0.2O Water QS (100) QS (100) QS (100) QS (100) pH adjusted to 6.0
GELCREME
Formulations RAW MATERIAL Amounts
(INCI Designation) 1 2 3 4 Acrylat C10-30 Alkylacrylat Crosspolymer O40 O.35 O.40 O.35 Polyacrylicacid O.20 O.22 O.2O O.22 Xanthan Gummi O.10 O.13 O.10 O.13 Cetearylalkohol 3.00 2.50 3.00 2.50 C12-15 Alkylbenzoat 4.OO 4...SO 4.OO 4...SO Caprylic Capric Triglycerid 3.00 3.SO 3.00 3.SO Aminobenzophenon (e.g., UVINULA PLUSTM) 2.OO 1...SO 0.75 1.00 UWASorb K2A 3.00 Ethylhexyl Methoxycinnamat 3.00 1.OO Bis-Ethylhexyloxyphenol methoxyphenyl Triazin 1...SO 2.00 Butyl Methoxydibenzoylmethan 2.OO Disodium Phenyl Dibenzimidazol Tetrasulfonat 2.50 O.SO 2.00 Ethylhexyl Triazon 4.OO 3.00 4.OO Octocrylen 4.OO Diethylhexyl Butamido Triazon 1.OO 2.00 Phenylbenzimidazol Sulfonsäure O.SO 3.00 Methylen Bis-Benzotriazolyl 2.OO O.SO 1...SO Tetramethylbutylphenol Ethylhexysalicylat 3.00 Drometrizol Trisiloxan O.SO Terephthaliden Dicamphor Sulfons?ure 1...SO 1.00 Diethylhexyl-2,6-naphthalat 3.SO 4.OO 7.00 9.00 Titaniumdioxide- microfine 1.OO 3.00 Zincoxide- microfine O.25 Compound SA SA SA SA Cyclisches Dimethylpolysiloxan S.OO 5.50 S.OO 5.50 Dimethicon Polydimethylsiloxan 1.OO O60 1.OO O60 Glycerin 1.OO 1.20 1.OO 1.20 Sodiumhydoxid QS QS QS QS US 9,084,734 B2
-continued
GELCREME
Formulations RAW MATERIAL Amounts
(INCI Designation) 1 2 3 4 Preservative O.30 O.23 O.30 O.23 Perfume O.20 O.2O Water QS (100) QS (100) QS (100) QS (100) pH adjusted to 6.0
-continued OW SUNSCREEN FORMULATION 15 OW SUNSCREEN FORMULATION Formulation RAW MATERIAL - Anouis- Formulation RAW MATERIAL Amounts (INCI Designations) 1 2 3 4 5 6 7 20 (INCI Designations) 1 2 3 4 5 6 7 Glycerin OSO 100 3.00 1...SO monostearate SE Sodium Carbomer O.2O O.15 O.25 Glyceri 2.OO 1.OO 2.OO 4.OO Vitamin E. Acetat O.6O O.23 O.70 100 Stearate Citrate Fucogel 1000 3.OO 10.00 ES OSO 3.00 2.OO 2.00 Glycin Soja OSO 1...SO 1.OO Cet Ph SE 1.00 25 Ethylhexyloxyglycin O.30 etyl Fnosphate DMDM Hydantoin O.6O O.40 O2O Cetearyl Sulfate 0.75 Glvacil-L O.18 O2O Stearyl Alcohol 3.00 2.00 O60 yac11 Cetyl Alcohol 2.SO 1.10 1...SO 0.60 2.OO Methylparaben O.15 O.25 OSO Compound SA SA SA SA SA SA SA Phenoxyethanol 1.OO O.40 O4O OSO 0.40 Aminobenzophenon 2.00 1.50 0.75 1.00 2.10 4.50 5.00 Trinatrium EDTA O.O2 O.OS (e.g., UVINULA 30 Iminosuccinicacid O2S 1.OO PLUS TM) Ethanol 2.OO 150 3.00 1.20 S.OO UWASOrb K2A Perfume O.1O O.25 O.30 O4O O.2O Ethylhexyl S.OO 6.OO 8.00 Water QS QS QS QS QS QS QS Methoxycinnamate (100) (100) (100) (100) (100) (100) (100) Bis-Ethylhexyl- 1...SO 2.OO 2.50 2.50 oxyphenol 35 methoxyphenyl Triazin Butyl Methoxy- 2.00 2.OO 150 dibenzoylmethane Dinatrium Phenyl 2.50 O.SO 2.OO O.30 HYDRODISPERSION Dibenzimidazol Formulations Tetrasulfonate 40 Ethylhexyl Triazone 4.OO 3.00 4.OO 2.00 RAW MATERIAL - Amounts)- Octocrylen 4.OO 7.50 Diethylhexyl 1.OO 2.OO 1.OO 1.OO (INCI Designation) 1 2 3 4 5 Butamido Triazon Ceteaereth-20 1.OO OSO Phenylbenzimidazol OSO 3.00 Cetyl Alkohol 1.OO Sulfonsaure 45 Sodium Carbomer O.2O O.30 Methylen 2.OO O.SO 150 2.50 Acrylat C10-30 Alkyl Acrylat O.SO O4O 0.10 OSO Bis-Benzotriazolyl Crosspolymer Tetramethylbutyl- Xanthan Gummi O.30 O.15 phenol Compound SA SA SA SA SA Ethylhexysalicylat 3.00 S.OO Aminobenzophenon (e.g., 2.OO 1.SO 0.7S 1.OO 2.10 Drometrizo Trisiloxan O.SO 1.00 UVINULA Terephthaliden 1...SO 1.OO 1.OO OSO 50 Dicamphor Sulfonic Acid Diethylhexyl-2,6- 3.SO 7.00 6.OO 900 naphthalat Titandioxid-microfine 1.00 3.00 3.SO 1...SO HYDRODISPERSION Zinkoxid-microfine O.25 2.00 55 C12-15 Alkyl O.25 4.OO 7.00 Formulations Benzoate RAW MATERIAL - (Amounts)- Dicapryl Ether 3.SO 2.OO Butylenglycol S.OO 6.OO (INCI Designation) 1 2 3 4 5 Dicaprylat Dicaprat PLUSTM Cocoglyceride 6.OO 2.OO 60 E. K2A 3.SO Dimethicon OSO 1.00 2.OO Ethylhexyl Methoxycinnamat S.OO Cyclomethicone 2.OO O.SO O.SO Bis-Ethylhexyloxyphenol 1...SO 2.OO 2SO Shea Butter 2.OO methoxyphenyl Triazin PVP Hexadecen O.2O OSO 1.00 Butyl Methoxydibenzoylmethan 2.OO 2.00 Copolymer Dinatrium Phenyl Dibenzimidazol 2.50 OSO 2.00 Glycerin 3.00 7. SO 7.50 5.00 2.50 65 Tetrasulfonat Xanthan Gum O.15 O.OS O.30 Ethylhexyl Triazon 4.OO 3.OO 4.OO US 9,084,734 B2 121 122 -continued -continued
HYDRODISPERSION HYDRODISPERSION
Formulations 5 Formulations RAW MATERIAL Amounts RAW MATERIAL Amounts
(INCI Designation) 1 2 3 4 5 (INCI Designation) 1 2 3 4 5 Octocrylen 4.OO Shea Butter 2.00 S.OO Diethylhexyl Butamido Triazon 1.00 2.00 1.OO PVP Hexadecen Copolymer O.SO O.SO 1.OO Phenylbenzimidazol Sulfonsãure O.SO 3.00 10 Tricontanyl PVP O.SO 1.OO Methylen Bis-Benzotriazolyl 2.00 O.SO 150 2.50 Ethylhexylglycerin 1.OO O.80 Tetramethylbutylphenol Glycerin 3.00 7. SO 7.50 8.50 Ethylhexysalicylat 3.00 Gvicin Soia 1...SO 1.00 Drometrizo Trisiloxan O.SO Vitamin E. Acetat O.SO O.25 1.00 Terephthaliden Dicamphor 1...SO 1.OO 1.OO Alpha-Glucosilrutin O.60 O.25 Sulfonsiure 15 Fucogel 1000 2.50 OSO 2.00 Diethylhexyl-2,6-naphthalat 7.OO 9.OO DMDM Hydantoin O.60 0.45 0.25 Titaniumdioxide- microfine 1.00 3.00 3.SO Glyacil-S O.20 Zincoxide- microfine O.25 Methylparaben O.SO O.25 0.15 C12-15 Alkyl Benzoat 2.OO 2SO Phenoxyethanol O.SO 0.40 1.00 Dicapryl Ether 4.OO Trinatrium EDTA O.O1 O.OS O.10 Butylenglycol Dicaprylatf 4.OO 2.OO 6.00 2O Ethanol 3.00 2.OO 150 7.00 Dicaprat Perfume O.20 O.OS 0.40 Dicapryl Carbonat 2.OO 6.OO Water QS QS QS QS QS Dimethicon OSO 1.OO (100) (100) (100) (100) (100) Phenyltrimethicon 2.00 O.SO
WO SUNSCREEN EMULSION
Formulations RAWMATERIAL Amounts
(INCI Designation) 1 2 3 4 5 Cetyldimethicon Copolyol 2.50 4.00 Polyglyceryl-2-dipolyhydroxystearat S.OO 4SO PEG-30-dipolyhydroxystearat S.OO Compound SA SA SA SA SA Aminobenzophenon (e.g., UVINULA PLUSTM) 2.OO 1...SO 0.75 1.OO 2.10 UWASorb K2A 2.00 Ethylhexyl Methoxycinnamat S.OO Bis-Ethylhexyloxyphenol methoxyphenyl Triazin 1...SO 2.OO 2.50 Butyl Methoxydibenzoylmethan 2.OO 2.00 Dinatrium Phenyl Dibenzimidazol Tetrasulfonat 2.50 OSO 2.OO Ethylhexyl Triazon 4.OO 3.00 4.OO Octocrylen 4.OO Diethylhexyl Butamido Triazon 1.OO 2.OO 100 Phenylbenzimidazol Sulfonsäure OSO 3.00 Methylen Bis-Benzotriazolyl 2.OO OSO 1...SO 2.50 Tetramethylbutylphenol Ethylhexysalicylat 3.00 Drometrizol Trisiloxan OSO Terephthaliden Dicamphor Sulfonsäure 1...SO 1.OO 100 Diethylhexyl-2,6-naphthalat 7.OO 4.OO Titaniumdioxide- microfine 1.OO 3.00 3.SO Zincoxide- microfine O.25 Mineraloil 12.00 1O.OO 8.00 C12-15 Alkyl Benzoat 9.OO Dicaprylyl Ether 1O.OO 7.00 Butylenglycol Dicaprylat Dicaprat 2.OO 8.OO 4.OO Dicaprylyl Carbonat S.OO 6.OO Dimethicon 4.OO 1.OO S.OO Cyclomethicon 2.OO 2.50 2.00 Shea Butter 3.00 Vaseline 4SO PVP Hexadecen Copolymer OSO O.SO 1.00 Ethylhexylglycerin O.30 1.OO O.SO Glycerin 3.00 7.50 7.50 8.50 Glycin Soia 1.00 1...SO 1.00 MgSO4 1.OO O.SO O.SO MgCl2 1.OO O.70 Vitamin E. Acetat OSO O.25 1.00 Ascorbyl Palmitat OSO 2.OO Fucogel 1000 3.SO 1.00 DMDM Hydantoin O.60 O4O O.20 Methylparaben OSO O.25 O.15 Phenoxyethanol OSO O.40 1.OO US 9,084,734 B2 123 124 -continued
WO SUNSCREEN EMULSION
Formulations RAW MATERIAL Amounts
(INCI Designation) 1 2 3 4 5
Trisodium EDTA O.12 O.OS O.30 Ethanol 3.00 1...SO S.OO Perfume O.20 O.40 O.35 Water QS QS QS QS QS (100) (100) (100) (100) (100)
STICKS
Formulations RAW MATERIAL Amounts
(INCI Designation) 1 2 3 4 Caprylic Capric Triglycerid 12.00 1O.OO 6.OO Octyldodecanol 7.00 14.00 8.00 3.00 Butylene Glycol Dicaprylat Dicaprat 12.00 Pentaerythrityl Tetraisostearat 10.00 6.OO 8.00 7.OO Polyglyceryl-3 Diisostearat 2.50 Bis-Diglyceryl Polyacyladipate-2 9.00 8.OO 10.00 8.OO Cetearyl Alcohol 8.00 11.00 9.00 7.OO Myristyl Myristate 3.SO 3.00 4.OO 3.00 Beeswax S.OO S.OO 6.OO 6.OO Cera Carnauba 1...SO 2.OO 2.00 1...SO Cera Alba O.SO OSO O.SO O4O C16-40 Alkyl Stearat 1...SO 1...SO 1...SO Compound SA SA SA SA Aminobenzophenon (e.g., UVINULA PLUSTM) 2.00 1...SO 0.75 9.OO UWASorb K2A 2.OO 4.OO Ethylhexyl Methoxycinnamat 3.00 Bis-Ethylhexyloxyphenol methoxyphenyl Triazin 1...SO 2.OO Butyl Methoxydibenzoylmethan 2.00 Dinatrium Phenyl Dibenzimidazol Tetrasulfonat 2.50 O.SO 2.OO Ethylhexyl Triazon 4.OO 3.00 4.OO Octocrylen 4.OO Diethylhexyl Butamido Triazon 1.00 2.OO Phenylbenzimidazol Sulfonsãure O.SO 3.00 Methylen Bis-Benzotriazolyl 2.00 O.SO 1...SO Tetramethylbutylphenol Ethylhexysalicylat 3.00 Drometrizol Trisiloxan O.SO Terephthaliden Dicamphor Sulfonsäure 1...SO 1.OO Diethylhexyl-2,6-naphthalat 7.00 Titaniumdioxide- microfine 1.00 3.00 Zincoxide- microfine O.25 Vitamin E. Acetat O.SO 1.OO Ascorbyl Palmitat O.OS O.OS Buxtix Chinensis 2.00 1.OO 1.OO Perfume, BHT O.10 O.25 O3S Ricinus Communis QS QS QS QS (100) (100) (100) (100)
PIT-EMULSION
Formulations RAW MATERIAL Amounts
(INCI Designation) 1 2 3 4 5 6 7 8 Glycerinmonostearat SE OSO 2.OO 3.00 S.OO OSO 4.OO Glyceryl Isostearat 3.SO 4.OO 2.00 Isoceteth-20 OSO 2.00 Ceteareth-12 S.OO 1.00 3.SO SOO Ceteareth-20 S.OO 1.00 3.SO PEG-1OO Stearat 2.80 2.30 3.30 Cetyl Alkohol S.20 120 100 1.30 OSO O.30 Cetyl Palmitat 2.SO 1.20 1...SO OSO 1...SO US 9,084,734 B2 125 126 -continued
PIT-EMULSION
Formulations RAW MATERIAL Amounts
(INCI Designation) 2 3 4 5 6 7 8 Cetyl Dimethicon Copolyol O.SO 1.OO Polyglyceryl-2 0.75 O.30 Compound SA SA SA SA SA SA SA SA Aminobenzophenon (e.g., UVINULA 2.00 1-SO O.7S 1.00 2.10 4...SO S.OO 2.10 PLUS TM) UWASorb K2A 4.OO 1...SO Ethylhexyl Methoxycinnamat S.OO 6.OO 8.00 S.OO Bis-Ethylhexyloxyphenol 1...SO 2.00 2.50 2.SO 2.50 methoxyphenyl Triazin Butyl Methoxydibenzoylmethan 2.OO 2.00 1...SO 2.OO Dinatrium Phenyl Dibenzimidazol 2.50 O.SO 2.00 O.30 Tetrasulfonat Ethylhexyl Triazon 4.OO 3.OO 4.00 2.OO Octocrylen 4.OO 7.50 Diethylhexyl Butamido Triazon 1.00 2.00 1.00 1.OO 1.OO Phenylbenzimidazol Sulfonsãure OSO 3.00 Methylen Bis-Benzotriazolyl 2.00 O.SO 150 2.50 2.50 Tetramethylbutylphenol Ethylhexysalicylat 3.00 S.OO Drometrizo Trisiloxan O.SO 1.OO Terephthaliden Dicamphor 1...SO 1.00 1.00 OSO 1.OO Sulfonsäure Diethylhexyl-2,6-naphthalat 7.OO 10.00 7.50 8.OO Titandioxid-microfine 1.00 3.00 3.SO 1...SO 3.50 Zinkoxid-microfine O.25 2.OO C12-15 Alkyl Benzoat 3.SO 6.35 O.10 Cocoglyceride 3.00 3.00 1.OO Dicapryl Ether 4...SO Dicaprylyl Carbonat 4.30 3.00 7.OO Dibutyl Adipate 0.50 O.30 Phenyltrimethicone 2.00 3.SO 2.OO Cyclomethicon 3.00 Ethyl Galaktomannan OSO 2.00 Hydrierte Coco-Glyceride 3.00 4.OO Abil Wax 2440 1...SO 2.00 PVP Hexadecen Copolymer 1.00 120 Glycerin 4.OO 6.OO S.OO 8.00 1O.OO Vitamin E. Acetat O.20 O3O O.40 O.30 Shea Butter 2.OO 3.60 2.OO odopropyl Butylcarbamat O.12 O.20 Fucogel 1000 O.10 DMDM Hydantoin O.10 O.12 O.13 Methylparaben OSO 0.30 O.35 Phenoxyethanol OSO 0.40 1.00 Octoxyglycerin O.30 1.00 O.35 Ethanol 2.00 2.OO S.OO Trinatrium EDTA O40 O.15 O.2O Perfume O.20 O.20 O.24 O.16 0.1O O.10 Water QS QS QS QS QS QS QS QS (100) (100) (100) (100) (100) (100) (100) (100)
50 -continued GEL CREME GEL CREME Formulations RAW MATERIAL Amounts Formulations 55 RAW MATERIAL Amounts (INCI Designation) 1 2 3 4 Acrylat C10-30 alkylacrylat crosspolymer O4O O.35 0.40 O.3S (INCI Designation) 1 2 3 4 Polyacrylic acid O.2O 0.22 O.2O O.22 Cyclisches Dimethylpolysiloxan 5.00 5.50 S.OO 5.50 Luvigel EM 1...SO 2.SO 2.80 3.50 Dimethicon Polydimethylsiloxan 1.OO O.6O 100 0.60 Xanthan gum 0.10 0.13 0.10 0.13 60 Glycerine 1.OO 1.2O 100 120 Cetearylalkohol 3.OO 2.SO 3.00 2.50 Natrium hydroxide QS QS QS QS C12-15 Alkylbenzoate 4.OO 450 4.OO 450 Preservatives O.30 O.23 O.30 O.23 Caprylic Capric Triglyceride 3.00 3.SO 3.00 3.50 Perfume O.20 O.20 Titan dioxide- microfine 1.OO 1...SO Water QS QS QS QS Zinc oxide- microfine 2.OO O.25 (100) (100) (100) (100) Compound SA SA SA SA 65 Dihydroxyacetone 3.00 S.OO pH adjusted to 6.0 US 9,084,734 B2 127 -continued OW SELFTANNERFORMULATIONS OW MAKE UP Formulations RAW MATERIAL Amounts 5 Formulations (INCI Designation) 1 2 3 4 5 6 7 RAW MATERIAL (Amounts) Glycerin monostearate 0.50 1.00 3.00 1...SO SE (INCI Desigation) 1 2 3 4 5 6 7 Glycerlstearate citrate 2.OO 1.OO 2.OO 4.OO Stearic acid 3.00 2.OO 10 PVP Hexadecen O.2O OSO 1.00 PEG-40 Stearate OSO 2.00 Cetyl phosphate 1.00 Copolymer Cetearylsulfate 0.75 Glycerin 3.OO 7SO 7.50 5.00 2.50 Stearyl alcohol 3.00 2.00 O60 Xanthan Gummi O.15 O.OS O.30 Cetyl alcohol 2.SO 1.10 1...SO 0.60 2.OO Sodium Carbomer O.2O O.15 O.25 Compound SA SA SA SA SA SA SA 15 Vitamin E. Acetat O.6O O.23 O.70 100 Dihydroxyacetone 3.00 S.OO 4 Titanium dioxide- 1.OO 1...SO 1...SO Glycin Soja OSO 1...SO 1.OO microfine Ethylhexyloxyglycin O.30 Zinc oxide-microfine O.25 2.00 DMDM Hydantoin O.6O 0.40 O2O C12-15 Alkylbenzoate O.25 4.OO 7.00 Glyacil-L O.18 O2O Dicapryl ether 3.SO 2.OO Butylenglycol S.OO 6.OO Methylparaben O.15 O.25 OSO Dicaprylaet/Dicaprat Phenoxyethanol 1.OO O.40 O4O OSO 0.40 Cocoglyceride 6.OO 2.OO Trinatrium EDTA O.O2 O.OS Dimethicon OSO 1.OO 2.OO Cyclomethicon 2.OO OSO O.SO Iminosuccinicacid O2S 1.OO Shea butter 2.OO Ethanol 2.OO 150 3.00 1.20 S.OO PVP hexadecen O.2O OSO 1.00 25 Perfume O.10 O2S O.30 O40 O2O copolymer Water QS QS QS QS QS QS QS Glycerin 3.00 7. SO 7.50 5.00 2.50 Xanthan gum O.15 O.OS O.30 (100) (100) (100) (100) (100) (100) (100) Sodium carbomer O.20 O.15 0.25 Vitamin Eacetate O.6O O.23 O.70 100 Fucogel 1000 3.OO 10.00 30 Glycin Soja OSO 1...SO 1.OO Ethylhexyloxy glycin O3O SELFTANNERHYDRODISPERSION DMDM hydantoin O.60 0.40 O2O Glyacil-L O.18 0.2O Formulations Methylparaben O.15 O.25 O.SO RAW MATERIAL Amounts Phenoxyethanol 1.OO 0.40 O.40 OSO 0.40 35 Trinatrium EDTA O.O2 O.OS (INCI Designation) 1 2 3 4 5 Iminobernsteins?ure O2S 1.OO Ceteaereth-20 1.OO OSO Ethanol 2.OO 150 3.00 1.20 S.OO Cetyl Alkohol 1.OO Perfume O.10 O.25 O.30 O.40 O20 Luvigel EM 2.OO 2.SO 2.OO Water QS QS QS QS QS QS QS Acrylat C10-30 Alkyl Acrylat O.SO O4O 0.10 OSO (100) (100) (100) (100) (100) (100) (100) 40 Crosspolymer Xanthan Gummi O.30 O.15 Compound SA SA SA SA SA Dihydroxyaceton 3.00 S.OO Aminobenzophenon (e.g., 2.OO 1...SO 0.75 1.OO 2.10 OW MAKE UP UVINULA PLUSTM) 45 Titandioxid- microfine 1.OO 1.OO 1.00 Formulations Zinkoxid- microfine 1.90 O.25 RAW MATERIAL Amounts C12-15 Alkyl Benzoat 2.OO 2.50 Dicapryl Ether 4.OO (INCI Desigation) 1 2 3 4 5 6 7 Butylenglycol Dicaprylatf 4.OO 2.OO 6.OO Dicaprat Glycerinmonostearat O.SO 100 3.00 1...SO 50 Dicapryl Carbonat 2.OO 6.OO SE Dimethicon OSO 1.OO Glyceri Stearat Citrat 2.OO 1.OO 2.OO 4.00 Phenyltrimethicon 2.OO OSO Stearicacid 3.00 2.OO Shea Butter 2.OO S.OO PEG-40 Stearat O.SO 2.00 PVP Hexadecen Copolymer O.SO OSO 1.00 Cetyl Phosphat 1.00 Tricontanyl PVP O.SO 1.OO Cetearyl Sulfat 0.75 55 Ethylhexylglycerin 1.OO O.80 Stearyl Alkohol 3.00 2.00 0.60 Glycerin 3.OO 7.50 7.50 8.50 Cetyl Alkohol 2.SO 1.10 1...SO 0.60 2.OO Gvicin Soia 1...SO 1.00 Compound SA SA SA SA SA SA SA Vitamin E. Acetat O.SO O.25 1.00 Titaniumoxide 1.O.OO 12.00 9.00 8.SO 11.OO 9.SO 10.00 Alpha-Glucosilrutin O.60 O.25 Ironoxide 2.OO 400 3.00 S.OO 3.40 6.00 440 DMDM Hydantoin O.60 0.45 0.25 Zincoxide 4.OO 2.OO 3.00 Glyacil-S O.20 C12-15 Alkyl Benzoat O.25 4.OO 7.00 60 Methylparaben O.SO O.25 O.15 Dicapryl Ether 3.SO 2.00 Phenoxyethanol O.SO O4O 1.OO Butylenglycol S.OO 6.OO Trinatrium EDTA O.O1 O.OS O.10 Dicaprylat Dicaprat Ethanol 3.OO 2.OO 1...SO 7.00 Cocoglyceride 6.OO 2.00 Parfim O.20 O.OS 0.40 Dimethicon O.SO 1.00 2.00 Water QS QS QS QS QS Cyclomethicon 2.OO O.SO O.SO 65 (100) (100) (100) (100) (100) Shea Butter 2.00 US 9,084,734 B2 129 130 -continued AFTERSUNHYDRODISPERSION AFTER SUN HYDRODISPERSION Formulations - Anouis- 5 Formulations Amounts 1 2 3 4 5 1 2 3 4 5 Ceteaereth-20 1.OO O.SO Cetyl Alkohol 1.OO Tricontanyl PVP OSO 1.OO Luvigel EM 2.OO 2.50 2.OO Ethylhexylglycerin 1.OO O.80 Acrylat C10-30 Alkyl OSO O.30 O4O O.10 0.50 10 Glycerin 3.00 7.50 7.50 8. SO Acrylat Crosspolymer Gvicin Soi 1...SO 1.00 Xanthan Gummi O.30 O.15 ycin Soya Compound SA SA SA SA SA Vitamin E Acetat OSO O.25 1.00 C12-15 Alkyl Benzoat 2.OO 2.50 Alpha-Glucosilrutin O.6O O.25 Dicapryl Ether 4.OO Trinatrium EDTA O.O1 O.OS O.10 Butylenglycol 4.OO 2.OO 6.OO 15 Ethanol 1.OO 1O.OO 8.OO 12.00 9.00 Dicaprylat Dicaprat Perfume O.2O O.OS O.40 Dicapryl Carbonat 2.OO 6.OO Water QS QS QS QS QS Dimethicon OSO 1.OO (100) (100) (100) (100) (100) Phenyltrimethicon 2.OO OSO
WO-EMULSIONS
Formulations RAWMATERIAL Amounts
(INCI Designation) 1 2 3 4 5 Cetyldimethicon Copolyol 2.50 4.OO Polyglyceryl-2-dipolyhydroxystearat S.OO 4SO PEG-30-dipolyhydroxystearat S.OO Compound SA SA SA SA SA Aminobenzophenon (e.g., UVINULA PLUSTM) 2.OO 1...SO 0.75 1.OO 2.10 Titaniumdioxide- microfine 1.OO 3.00 3.SO Zincoxide- microfine O.90 0.25 Mineralöl 12.00 1O.OO 8.00 C12-15 Alkyl Benzoat 9.OO Dicaprylyl Ether 1O.OO 7.00 Butylenglycol Dicaprylat Dicaprat 2.OO 8.OO 4.OO Dicaprylyl Carbonat S.OO 6.OO Dimethicon 4.OO 1.OO S.OO Cyclomethicon 2.OO 25.00 2.00 Shea Butter 3.00 Vaseline 4SO PVP Hexadecen Copolymer OSO O.SO 1.00 Ethylhexylglycerin O.30 1.OO O.SO Glycerin 3.00 7.50 7.50 8.50 Glycin Soia 1.00 1...SO 1.00 MgSO4 1.OO O.SO O.SO MgCl2 1.OO O.70 Vitamin E. Acetat OSO O.25 1.00 Ascorbyl Palmitat OSO 2.OO Fucogel 1000 3.SO 7.OO DMDM Hydantoin O.60 O4O O.20 Methylparaben OSO O.25 O.15 Phenoxyethanol OSO O.40 1.OO Trinatrium EDTA O.12 O.OS O.30 Ethanol 3.00 1...SO S.OO Perfume O.2O O4O O.35 Water QS QS QS QS QS (100) (100) (100) (100) (100)
55
SOLID STABILIZED EMULSIONS (PICKERING EMULSIONS)
Formulations RAW MATERIAL 60 (Amounts)
(INCI Designation) 1 2 3 4 5
Mineral oil 16.00 16.00 Octyldodecanol 65 9.00 9.OO S.OO Caprylic Capric Triglycerid 9.00 9.OO 6.OO US 9,084,734 B2 131 132 -continued
SOLID STABILIZED EMULSIONS (PICKERING EMULSIONS)
Formulations RAW MATERIAL (Amounts)
(INCI Designation) 1 2 3 4
C12-15 Alkyl Benzoat S.OO 8.00 Butylen Glycol Dicaprylat Dicaprat 8.00 Dicaprylyl Ether 9.OO 4.OO Dicaprylyl Carbonat 9.OO Hydroxyoctacosanyl HydroxyStearat 2.OO 2.OO 2.2O 2.50 1...SO Disteardimonium Hectorit 1.OO 0.75 OSO O.25 Cera Microcristallina + Paraffinum Liquidum O3S S.OO Hydroxypropyl Methylcellulose O.10 O.OS Dimethicon 3.00 Compound SA SA SA SA SA Titaniumdioxide + Alumina + Simethicon + Aqua 3.00 Titaniumdioxide + Trimethoxycaprylylsilan 2.OO 4.OO 2.00 4.OO Silica Dimethyl Sillylat 2.50 6.OO 2.50 Bornitrid 1.OO Stärket-Natriummetaphosphat-Polymer 2.OO Tapioca Starke OSO Sodium Chlorid S.OO 7.OO 8.50 3.00 Glycerin 1.00 Trinatrium EDTA 1.OO 1.OO 1.OO 1.00 1.00 Vitamin E. Acetat S.OO 1O.OO 3.00 6.OO 10.00 Ascorbyl Palmitat 1.OO 1.OO 1.00 Methylparaben O.6O O.20 Propylparaben O.20 Phenoxyethanol O.2O Hexamidin Diisethionat O.40 OSO O.40 Diazolidinyl Harnstoff O.08 Ethanol O.23 O.20 Perfume S.OO 3.00 4.OO Water O.20 O.30 O.10 QS (100) QS (100) QS (100) QS (100) QS (100)
STICKS
Formulations RAW MATERIAL Amounts
(INCI Designation) 1 2 3 4 Caprylic Capric Triglycerid 12.00 1O.OO 6.OO Octyldodecanol 7.00 14.00 8.00 3.00 Butylene Glycol Dicaprylat Dicaprat 12.00 Pentaerythrityl Tetraisostearat 10.00 6.OO 8.00 7.OO Polyglyceryl-3 Diisostearat 2.50 Bis-Diglyceryl Polyacyladipate-2 9.00 8.OO 10.00 8.OO Cetearyl Alcohol 8.00 11.00 9.00 7.OO Myristyl Myristate 3.SO 3.00 4.OO 3.00 Beeswax S.OO S.OO 6.OO 6.OO Cera Carnauba 1...SO 2.OO 2.00 1...SO Cera Alba O.SO OSO O.SO O4O C16-40 Alkyl Stearat 1...SO 1...SO 1...SO Compound SA SA SA SA Aminobenzophenon (e.g., UVINULA PLUSTM) 2.00 1...SO 0.75 9.OO Titaniumdioxide- microfine 1.00 3.00 Zincoxide- microfine 1.OO O.25 Vitamin E. Acetat O.SO 1.OO Ascorbyl Palmitat O.OS O.OS Buxtix Chinensis 2.00 1.OO 1.OO Perfume, BHT O.10 O.25 O3S Ricinus Communis QS (100) QS (100) QS (100) QS (100) US 9,084,734 B2 133 134
SELFTANNERPITEMULSION
Formulations Amounts
1 2 3 4 5 6 7 8 Glycerinmonostearat SE OSO 2.OO 3.00 S.OO O.SO 400 Glyceryl Isostearat 3.SO 400 2.00 Isoceteth-20 OSO 2.OO Ceteareth-12 S.OO 1.00 3.SO SOO Ceteareth-20 S.OO 1.00 3.SO PEG-1OO Stearat 2.80 2.30 3.30 Cetyl Alkohol S.20 120 100 130 O.SO O.30 Cetyl Palmitat 2.SO 1.20 1...SO O.SO 1...SO Cetyl Dimethicon Copolyol O.SO 1.OO Polyglyceryl-2 O.75 O.30 Compound SA SA SA SA SA SA SA SA Dihydroxyaceton 3.00 S.OO 4.OO Aminobenzophenon (e.g., UVINULA 2.00 1-SO O.7S 1.OO 2.10 4...SO S.OO 2.10 PLUSTM) Titandioxide-microfine 1.00 1...SO 3.SO 1...SO 1.OO Zinkoxide-microfine 1.OO O.25 2.OO 1...SO C12-15 Alkyl Benzoat 3.SO 6.35 O.10
SELFTANNERPIT-EMULSION
Formulations Amounts
1 2 3 4 5 6 7 8 Cocoglyceride 3.00 3.00 1.00 Dicapryl Ether 4...SO Dicaprylyl Carbonat 4.30 3.OO 7.00 Dibutyl Adipate O.SO O.30 Phenyltrimethicone 2.OO 3.SO 2.OO Cyclomethicon 3.00 Ethyl Galaktomannan O.SO 2.00 Hydrogenated Coco-Glyceride 3.00 4.OO Abil Wax 2440 1...SO 2.OO PVP Hexadecen Copolymer 1.OO 120 Glycerin 4.OO 6.OO SOO 8.OO 10.OO Vitamin E. Acetat O.2O O.30 O40 O.30 Shea Butter 2.00 3.60 2.OO odopropyl Butylcarbamat O.12 O.20 DMDM Hydantoin O.10 O.12 O.13 Methylparaben O.SO O.30 O.35 Phenoxyethanol O.SO 0.40 1.OO Octoxyglycerin O.30 1.00 O.35 Ethanol 2.OO 2.OO S.OO Trinatrium EDTA O40 O.15 O.2O Perfume O.20 O.2O O.24 O.16 O. 10 0.10 (100) (100) (100) (100) (100) (100) (100) (100)
50 -continued OILGELS OILGELS Formulations RAW MATERIAL (Amounts) Formulations 55 RAW MATERIAL Amounts (INCI Designation) 1 2 3 4 (INCI Designation) 1 2 3 4 Caprylic Capric 12.00 10.00 6.OO Triglycerid Myristyl Myristate 3.SO 3.00 4.OO 3.00 Octyldodecanol 7.00 14.00 8.00 3.00 Bentone-34 S.OO S.OO 6.OO 6.OO Butylene Glycol 12.00 60 Propylencarbonat 1S.OO 2O.OO 18.00 19.50 Dicaprylat Dicaprat Compound SA SA SA SA Pentaerythrityl 10.00 6.OO 8.00 7.OO Vitamin E. Acetat O.SO 1.OO Tetraisostearat Ascorbyl Palmitat O.OS O.OS Polyglyceryl-3 2.50 Buxtix Chinensis 2.OO 1.OO 1.OO Diisostearat Perfume, BHT O.10 O.25 O3S Bis-Diglyceryl 9.00 8.00 10.00 8.OO 65 Ricinus Communis QS (100) QS (100) OS (100) QS (100) Polyacyladipate-2 US 9,084,734 B2 135 136 In still further embodiments, the present invention com ferred embodiments are known to those in the art and pro prises at least one inorganic pigment. In some preferred vided previously (See e.g., Colour Index Nummern (CIN), embodiments, these inorganic pigments are based on meta Rowe Colour Index, 3" ed., Society of Dyers and Colourists, loxides and/or other water slightly soluble or insoluble metal Bradford, England 1971). compounds, including but not limited to compounds such as 5 In additional embodiments, pearlescent pigments based on Zinc oxides (ZnO), titanium (TiO), iron (e.g., FeO), Zirco- mica/metaloxide find use, as described above. However, it is nium (ZrO2), Silica (SiO), manganese (e.g., MnO), alu- not intended that the present invention be limited to these minium (Al2O), cer (e.g., CeO), and mixed oxides of these particular pigments, as additional pearlescent pigments find oxides, as well as blends thereof. In some embodiments, the US 1 various embodiments of the present invention. metaloxides are microfine grade, while in other embodi- 10 The following formulations provide additional examples ments, the metaloxides are pigment grade. In further embodi- of the use of the present invention. ments, the metaloxides are a mixture of microfine and pig ment grades. F lati In additional embodiments, the inorganic pigments are is RAW MATERIAL ANY coated (i.e., they are treated on the Surface). In some particu larly preferred embodiments, the surface is coated with a thin, (INCI Designation 1 2 3 4 5 hydrophobic film. In some other particularly preferred Sodium Carbomer O.2 embodiments, the surface is coated with a thin, hydrophilic Acrylates Co-Co Alkyl Acrylate O3 O2 O.6 film. In yet additional embodiments, the present invention Crosspolymer provides compositions comprising various make ups and ' Hydroxypropyl Cellulose 1.O 150 Xanthan Gummi O6 O2 1.O 1.O make up constituents. For example, in some embodiments, Compound O.S. O.1 OO1 O.O1 1.0 the present invention provides various dyes and/or pigments. Dioctyl Butamidotriazon 20 2.0 ico In some embodiments, useful pigments include, but are not Ethylhexyl Triazon 4.O 4.0 S.O limited to titanium dioxide, mica, iron oxides (e.g. Fe2O, Aniso Triazin 1.O O.S 2.O 2.5 Fe,O, Fe0(OH), etc.) and/or stannous oxide. The present Sision 6.O invention further provides colorants, including but not limited PhenylbenzmidazSulfonicacid 2.0 1.O to carmine, blue, chromooxide, ultramarine and/or purple manganese. The colorants and pigments of Some most pre
Formulation RAW MATERIAL (Amounts)
(INCI Designation 1 2 3 4 5 Bisimidazylate 1.O Terephthalylidene Dicamphor O.2 Sulfonic Acid Ethylhexyl Methoxycinnamat 7.5 1O.O S.O Octocrylen S.O Dimethicone-diethylbenzalmalonate 4.0 Ethylhexyl Salicylate Homosalate Butyl Methoxydibenzoylmethan 1.O 1.O 4.0 Titan dioxide 1.O 4.0 Zinc oxide 4.0 Caprylic Capric Triglycerid 2.0 Hydrogenated Coco- 3.0 Glyceride C12-15 Alkyl Benzoat 2.0 2.5 3.0 Dicaprylyl Ether 4.0 Butylenglycol Di- 4.0 2.0 6.O caprylat Dicaprat Dicaprylyl Carbonat 2.0 Cetyl Dimethicon 2.0 O.S 1.O Shea Butter 2.0 PVP Hexadecen Copolymer O.S O.OS O.S Glycerin 3.0 7.5 7.5 2.5 Tocopherol O.S 0.75 O.2 Trisodium EDTA 1.O O.S O.S 1.O 1.5 Natriumcitrat O.2 Zitronensiure O.1 O.1 O.1 DMDM Hydantoin O.6 O.2 Methylparaben O.S O.3 O.15 Phenoxyethanol O.S 0.4 0.4 1.O O.60 Ethanol 3.0 2.0 3.0 1.O Perfume O.2 O.2 O.2 Water QS (100) QS (100) QS (100) QS (100) QS (100) US 9,084,734 B2 137 138 -continued Formulations RAW MATERIAL - (Amounts)- Formulations RAW MATERIAL Amounts (INCI Designation) 1 2 3 4 5 5 Sodium Carbomer O.S 1.5 (INCI Designation) 1 2 3 4 5 6 (C. Co Alkyl Acrylate O4 O1 0.75 Phenylbenzinidazole 2.0 1.O Hydroxypropyl Cellulose O.S O.25 Sulfonicacid Xanthan Gummi O2 O.4 Bisimidazylate 1.5 3.5 Compound 0.5 0.1 0.01 0.01 1.0 10 Terephthalylidene O.2 1.O Dioctyl Butamidotriazon 1.O 2.0 Dicamphor Sulfonic Acid Ethylhexyl 1O.O S.O
Formulations RAW MATERIAL Amounts
(INCI Designation) 1 2 3 4 5 Ethylhexyl Triazon 2.0 2O Aniso Triazin 1.O O.2 3.0 1.O Bisoctyltriazol 8.0 Drometrizole Trisiloxane 4.0 Phenylbenzmidazole Sulfonicacid 1.5 Bisimidazylate 1.5 Terephthalylidene Dicamphor O.S Sulfonic Acid Ethylhexyl Methoxycinnamat 7.5 S.O 1OO Octocrylen 1O.O S.O S.O Dimethicone- 2.5 diethylbenzalmalonate Ethylhexyl Salicylate 3.5 SO Homosalate 4.0 Butyl Methoxydibenzoylmethan O.S Titandioxide 1.5 2.0 1.O 2.5 Zincoxide 1.0 0.5 Caprylic Capric Triglycerid Hydrogenierte Coco-Glyceride C12-15 Alkyl Benzoat S.O Dicaprylyl Ether 7.5 Butylenglycol Dicaprylat Dicaprat Dicaprylyl Carbonat 7.5 Cetyl Dimethicon Shea Butter 3.0 PVP Hexadecen Copolymer O.S 0.75 1.O Glycerin S.O 1O.O Tocopherol O.3 1.5 1.O Trisodium EDTA O.S O.1 OS Natriumcitrat O.3 Zitronensiure O.15 DMDM Hydantoin O3 O.15 Methylparaben 0.4 Phenoxyethanol 1.O Ethanol 7.5 S.O 7.0 Perfume O.25 O2 Water QS (100) QS (100) QS (100) QS (100) QS (100)
-continued Formulations RAW MATERIAL Amounts Formulations 55 RAW MATERIAL Amounts (INCI Designation) 1 2 3 4 5 6 (INCI Designation) 1 2 3 4 5 6 Sodium Carbomer O.S 1.5 1.O O.S Acrylates/Co-Co Alkyl 1.O O.7S 1.0 Methoxycinnamat Acrylate Crosspolymer SNR,len 1O.O 4.0 S.O Hydroxypropyl Cellulose 0.4 1.O 1.O 60 ity, Nimatonate Xanthan Gummi O6 O.2 1.O 1.O Ethylhexyl S.O BBI 4.0 O.S 3.0 2.O 4.0 1.5 Salicylate Dioctyl Butamidotriazon 2.0 2.0 2.O 1.O Homosalate S.O Ethylhexyl Triazon 4.0 S.O 4.O Butyl Methoxy- 1.O 1.O 4.0 O.S Aniso Triazin 1.O 1.O 2.5 1.O dibenzoylmethan Bisoctyltriazol 4.0 65 Titandioxide 1.O 4.0 1.5 Drometrizole Trisiloxane 3.0 Zincoxide 4.0 US 9,084,734 B2 139 140 -continued PRESEHAVE Formulations RAW MATERIAL Amounts % Ingredient INCI
(INCI Designation) 1 2 3 4 5 6 A. 8O Ethanol Alcohol 3.0 Vitamin E Acetate Tocopheryl Acetate Caprylic Capric 2.0 1.0 Bisabolol rac. Bisabolol Triglycerid 0.2 Perfume Paraffinól 1.O 0.1 Menthol Menthol C12-C1s Alkyl 2.0 2.5 3.0 4.0 Luvitol EHO Cetearyl Ethylhexanoate 10 2.0 Eutanol G Octyldodecanol Benzoat 2.0 Miglyol 812 Caprylic Capric Triglyceride Dicaprylyl Ether 4.0 2.0 D-Panthenol USP Panthenol Isohexadecen 4.0 2.0 6.O 2.0 Whitch Hazel Hamamelis Virginiana (Whitch Dicaprylyl 2.0 Distillate Hazel) Distillate Carbonat 2.0 Jojoba Oil Simmondsia Chinensis (Jojoba) Dibutyl Adipat 2.0 O.S 1.O Seed Oil Cylomethicon 3.0 15 SA Compound Jojobadl 2.0 PVP Hexadecen O.S O.OS O.S O.S Copolymer Production: Weigh out the components of Phase A and dis Butylen Glycol 3.0 7.5 7.5 2.5 S.O solve them clearly. Ascorbyl-Palmitate O.S 0.75 O.2 O.3 The after-shave and pre-shave formula provided above contain sufficient BBI-AV (Compound) to provide the desired effect(s). In some embodiments, the concentration of BBI-AV is in the range of about 1,000 ppm to about 10,000 Formulations ppm. In the following formulations, typical concentrations of RAW MATERIAL Amounts 25 BBI-AV used range from about 100 ppm to about 1,000 ppm (INCI Designation) 1 2 3 4 5 6 or from about 1,000 ppm to about 10,000 ppm. However, it is Octoxyglycerin 1.O O.S 1.O not intended that the present invention be limited to this Glycin Soja 2.0 1.5 specific concentration range, as other concentrations find use Trisodium EDTA 1.O O.S O.S 1.S. O.S in other embodiments of the present invention. Caustic acid 1.O O.2 O.25 30 Iodopropyl Butylcarbamat O6 O.2 The following formula provides an example of an after-Sun Phenoxyethanol 0.4 1.0 product comprising the BBI-AV of the present invention. Ethanol S.O 2.O 7.0 Perfume O.2 O.2 O.2 Water QS QS QS QS QS QS AFTERSUNLOTION (100) (100) (100) (100) (100) (100) 35 % Ingredient INCI The following formula provides an example of an after A. 0.4 Carbopol 1342 Acrylates/C10-30 Alkyl Acrylate Crosspolymer shave product comprising the BBI-AV of the present inven 15.0 Luvitol EHO Cetearyl Ethylhexanoate tion. 40 0.2 Bisabolol rac. Bisabolol 1.0 Vitamin E Acetate Tocopheryl Acetate q.s. Perfume B 1.0 D-Panthenol USP Panthenol AFTER SHAVE LOTION 15.0 Ethanol.96% Alcohol % Ingredient INCI 3.0 Glycerin 87% Glycerin 45 SA Compound A. 10.0 Luvitol EHO Cetearyl Ethylhexanoate 64.2 Water dem. Aqua dem. 5.0 Vitamin E Acetate Tocopheryl Acetate C 0.2 Triethanolamine Care Triethanolamine 1.0 Bisabolol rac. Bisabolol 0.1 Perfume 0.3 Carbopol Ultrez 21 Acrylates/C10-30 Alkyl Acrylate Production: Mix the components of Phase A. Dissolve Phase Crosspolymer 50 B and stir it into Phase A whilst homogenizing. Neutralize B 15.0 Ethanol Alcohol with Phase C and homogenize again. 1.0 D-Panthenol USP Panthenol 3.0 Glyerin 87% Glycerin Measure Values: 0.1 Triethanolamine Care Triethanolamine Viscosity: 7500 mPa is Haake Viscotester VT-02 SA Compound pH value: 6.0 QS Water dem. Aqua dem. 55 The following formula provides an example of a facial cleanserproduct comprising the BBI-AV of the present inven tion. Production: Weigh out the components of Phase A and mix them. Dissolve Phase B, stir it into Phase A and homogenize well. 60 FACIAL CLEANSER Measure Values: % Ingredient INCI Viscosity: 18500 mPa is Brookfield RVD II+ A. 10.0 Luvitol EHO Cetearyl Ethylhexanoate pH value: 5.8 10.0 Miglyol 812 Caprylic Capric Triglyceride 1.5 Dow Corning 345 Fluid Cyclopentasiloxane, The following formula provides an example of an after 65 Cyclohexasilosane shave product comprising the BBI-AV of the present inven 2.0 Cremophor CO 40 PEG-40 Hydrogenated Castor Oil tion.