U.S. patent application number 15/404849 was filed with the patent office on 2017-05-04 for compositions for deposition on biological surfaces.
The applicant listed for this patent is The Procter & Gamble Company. Invention is credited to John Christian HAUGHT, Steven Hamilton HOKE, Qingxin LEI, Yakang LIN, Michael REILLY, Lowell Alan SANKER, Koti Tatachar SREEKRISHNA.
Application Number | 20170119639 15/404849 |
Document ID | / |
Family ID | 53055119 |
Filed Date | 2017-05-04 |
United States Patent
Application |
20170119639 |
Kind Code |
A1 |
HAUGHT; John Christian ; et
al. |
May 4, 2017 |
COMPOSITIONS FOR DEPOSITION ON BIOLOGICAL SURFACES
Abstract
Personal care compositions, such as oral care and skin care
compositions containing a flavor/perfume system comprising one or
more coolants. The pleasant cool sensation provided by a coolant is
enhanced in terms of quicker onset, greater intensity, impact or
longer duration, which improves appeal and acceptability of the
compositions to consumers.
Inventors: |
HAUGHT; John Christian;
(West Chester, OH) ; REILLY; Michael; (Lebanon,
OH) ; HOKE; Steven Hamilton; (West Chester, OH)
; LEI; Qingxin; (Liberty Township, OH) ; LIN;
Yakang; (Liberty Towhship, OH) ; SANKER; Lowell
Alan; (Montgomery, OH) ; SREEKRISHNA; Koti
Tatachar; (Mason, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Procter & Gamble Company |
Cincinnati |
OH |
US |
|
|
Family ID: |
53055119 |
Appl. No.: |
15/404849 |
Filed: |
January 12, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14694616 |
Apr 23, 2015 |
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15404849 |
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61982970 |
Apr 23, 2014 |
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61982968 |
Apr 23, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23G 4/06 20130101; A61K
31/4402 20130101; A61P 1/02 20180101; C07C 237/10 20130101; A61K
8/34 20130101; A61K 31/165 20130101; A61Q 19/00 20130101; A61K 8/27
20130101; C07C 233/75 20130101; A61K 31/485 20130101; A23V 2002/00
20130101; A61K 9/28 20130101; A61K 8/46 20130101; A61Q 11/00
20130101; C07C 233/73 20130101; A61K 31/137 20130101; A61K 31/167
20130101; A61Q 5/02 20130101; A61K 9/0043 20130101; A61K 9/0078
20130101; C07C 255/44 20130101; A61K 9/0014 20130101; A61K 2800/244
20130101; A61K 31/09 20130101; A61K 31/192 20130101; A61K 8/42
20130101; A61K 9/06 20130101; A61Q 5/12 20130101; A61Q 9/02
20130101; A61P 43/00 20180101; C07C 323/33 20130101 |
International
Class: |
A61K 8/42 20060101
A61K008/42; A61Q 5/02 20060101 A61Q005/02; A61Q 19/00 20060101
A61Q019/00; A23G 4/06 20060101 A23G004/06; A61Q 5/12 20060101
A61Q005/12; A61K 8/46 20060101 A61K008/46; A61K 8/34 20060101
A61K008/34; A61K 8/27 20060101 A61K008/27; A61Q 11/00 20060101
A61Q011/00; A61Q 9/02 20060101 A61Q009/02 |
Claims
1. A personal care composition providing a cool sensation
comprising a compound having the following structure: ##STR00014##
R.sub.1 is selected from H, alkyl, amino alkyl, alkoxy; Q=H.sub.2,
O, --OR.sub.1, --N(R.sub.1).sub.2, --OPO(OR.sub.1).sub.x,
--PO(OR.sub.1).sub.x, --P(OR.sub.1).sub.x where x=1-2; V=NR.sub.1,
O, --OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x, --P(OR.sub.1).sub.x
where x=1-2; W=H.sub.2, O; X, Y=independently selected from H,
aryl, naphthyl for n=0; X, Y=aliphatic CH.sub.2 or aromatic CH for
n.gtoreq.1 and Z is selected from aliphatic CH.sub.2, aromatic CH,
or heteroatom; A=lower alkoxy, lower alkylthio, aryl, subsitituted
aryl or fused aryl; and stereochemistry is variable at the
positions marked*.
2. The personal care composition of claim 1, wherein the compound
activates at least one of TRPA1, TRPV1, or TRPM8.
3. The personal care composition of claim 2, wherein the compound
at a concentration of about 5.2E-5% provides: a) greater activation
of TRPM8 than WS5 at a concentration of about 30 mM; b) greater
activation of TRPA1 than allyl isothiocyanate at a concentration of
about 50 mM; and c) greater activation of TRPV1 than capsaicin at a
concentration of about 350 nM.
4. The personal care composition of claim 2, wherein the compound
at a concentration of about 5.2E-5% provides: a) at least about
110% activation of TRPM8 when compared to WS5 at a concentration of
about 30 mM; b) at least about 180% activation of TRPA1 when
compared to allyl isothiocyanate at a concentration of about 50 mM;
and c) at least about 100% activation of TRPV1 when compared to
capsaicin at a concentration of about 350 nM.
5. The personal care composition of claim 2, wherein the activation
is determined by measuring intracellular calcium levels.
6. The personal care composition of claim 5, wherein intracellular
calcium level measuring is done using fluorescent dye.
7. The personal care composition of claim 2, wherein the compound
at a concentration of about 5.2E-5% provides greater activation of
TRPM8 than WS5 at a concentration of about 30mM.
8. An isomeric HPLC fraction of the compound of claim 1, wherein
the isomeric HPLC fraction at a concentration of about 12.2 nM had
a higher TRPM8 activation at 10 minutes as compared to WS 5 at a
concentration of about 10 .mu.m.
9. The personal care composition of claim 1 comprising a TRPM8
agonist.
10. The personal care composition of claim 9, wherein the TRPM8
agonists comprises at least one of Menthol; Menthyl Lactate;
N-ethyl-.rho.-menthan-3-carboxamide;
N-ethoxycarbonylmethyl-.rho.-menthan-3-carboxamide;
N-(4-methoxyphenyl)-.rho.-menthan-3-carboxamide;
N-tert-butyl-.rho.-menthan-3-carboxamide;
N,2,3-trimethyl-2-isopropylbutanamide;
N-(4-cyanomethylphenyl)-.rho.-menthanecarboxamide;
N-(4-sulfamoylphenyl)-.rho.-menthanecarboxamide;
N-(4-cyanophenyl).rho.-menthanecarboxamide;
N-(4-acetylphenyl)-.rho.-menthanecarboxamide;
N-(4-hydroxymethylphenyl)-.rho.-menthanecarboxamide;
N-(3-hydroxy-4-methoxyphenyl)-.rho.-menthanecarboxamide;
Isopulegol; and/or (-)-Menthoxypropane-1,2-diol.
11. The personal care composition of claim 1 comprising at least
one of a TRPA1 agonist or TRPV1 agonist.
12. The personal care composition of claim 11, wherein the TRPA1
agonist is at least one of allyl isothiocyanate; menthol; peroxide;
methyl salicylate; cinnamic aldehyde; benzyl alcohol; zinc salts;
and/or vanillin isobutyrate.
13. The personal care composition of claim 11, wherein the TRPV1
agonist is at least one capsaicin; piperine; vanillyl butyl ether;
vanillyl ethyl ether; menthol; peroxide; zinc salts; or an
anti-histamine.
14. The personal care composition of claim 1, wherein the compound
structure comprises: ##STR00015## R.sub.1 is selected from H,
alkyl, amino alkyl, alkoxy; Q=H.sub.2, O, --OR.sub.1,
--N(R.sub.1).sub.2, --OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x,
--P(OR.sub.1).sub.x where x=1-2; V=NR.sub.1, O,
--OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x, --P(OR.sub.1).sub.x
where x=1-2; W=H.sub.2, O; X, Y=independently selected from H,
aryl, naphthyl for n=0; X, Y=aliphatic CH.sub.2 or aromatic CH for
n.gtoreq.1 and Z is selected from aliphatic CH.sub.2, aromatic CH,
or heteroatom; A=lower alkoxy, lower alkylthio, aryl, subsitituted
aryl or fused aryl; and stereochemistry is variable at the
positions marked*.
15. The personal care composition of claim 14, wherein the compound
structure comprises: ##STR00016##
16. The personal care composition of claim 1, where the compound
structure comprises: ##STR00017## R.sub.1 is selected from H,
alkyl, amino alkyl, alkoxy; Q=H.sub.2, O, --OR.sub.1,
--N(R.sub.1).sub.2, --OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x,
--P(OR.sub.1).sub.x where x=1-2; V=NR.sub.1, O,
--OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x, --P(OR.sub.1).sub.x
where x=1-2; W=H.sub.2, O; X, Y=independently selected from H,
aryl, naphthyl for n=0; X, Y=aliphatic CH.sub.2 or aromatic CH for
n.gtoreq.1 and Z is selected from aliphatic CH.sub.2, aromatic CH,
or heteroatom; A=lower alkoxy, lower alkylthio, aryl, subsitituted
aryl or fused aryl; and stereochemistry is variable at the
positions marked*.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to personal care compositions,
such as oral care and skin care compositions, containing a
flavor/perfume system comprising one or more coolants, wherein the
cool sensation provided by the coolant is enhanced in terms of
quicker onset, greater intensity, and/or longer duration, thereby
improving appeal and acceptability of the compositions to
consumers.
BACKGROUND OF THE INVENTION
[0002] Oral care products, such as dentifrice and mouthwash, are
routinely used by consumers as part of their oral care hygiene
regimens. It is well known that oral care products can provide both
therapeutic and cosmetic hygiene benefits to consumers. Therapeutic
benefits include caries prevention which is typically delivered
through the use of various fluoride salts; gingivitis prevention,
by the use of an antimicrobial agent such as stannous fluoride,
triclosan, essential oils; or hypersensitivity control through the
use of ingredients such as strontium chloride or potassium nitrate.
Cosmetic benefits provided by oral care products include the
control of plaque and calculus formation, removal and prevention of
tooth stain, tooth whitening, breath freshening, and overall
improvements in mouth feel impression, which can be broadly
characterized as mouth feel aesthetics. Calculus and plaque along
with behavioral and environmental factors lead to formation of
dental stains, significantly affecting the aesthetic appearance of
teeth. Behavioral and environmental factors that contribute to
teeth staining propensity include regular use of coffee, tea, cola
or tobacco products, and also the use of certain oral products
containing ingredients that promote staining, such as cationic
antimicrobials and metal salts.
[0003] Thus daily oral care at home requires products with multiple
ingredients working by different mechanisms to provide the complete
range of therapeutic and aesthetic benefits, including anticaries,
antimicrobial, antigingivitis, antiplaque, anticalculus and
anti-erosion, as well as antiodor, mouth refreshment, stain
removal, stain control and tooth whitening. In order for daily use
oral care products, such as dentifrice and rinses to provide
complete oral care it is often necessary to combine actives and
additives, many of which have the disadvantage of causing negative
aesthetics during use, in particular unpleasant taste and
sensations and stain promotion. The unpleasant taste and mouth
sensations have been described as having one or more of bitter,
metallic, astringent, salty, numbing, stinging, burning, or
prickling, and even irritating aspects. Typical ingredients for
oral care use that are associated with these aesthetic negatives
include antimicrobial agents such as cetyl pyridinium chloride,
chlorhexidine, stannous and zinc salts; tooth bleaching agents such
as peroxides; antitartar agents such as pyrophosphate,
tripolyphosphate and hexametaphosphate; and excipients such as
baking soda and surfactants. To mitigate the aesthetic negatives
from these ingredients, oral care products are typically formulated
with flavoring agents, sweeteners and coolants to taste as good as
possible and provide a pleasant experience. In particular, it is
desirable for oral care products to provide a refreshing cooling
sensation during and after use. In addition to mitigation of
negative sensations, sensate molecules are formulated into oral
care compositions to convey a signal of efficacy. Such signals of
efficacy include cooling, tingling, numbing, warming, sweetness,
and rheological sensations such as phase change and fizzing or
bubbling.
[0004] A large number of coolant compounds of natural or synthetic
origin have been described. The most well-known compound is
menthol, particularly 1-menthol, which is found naturally in
peppermint oil, notably of Mentha arvensis L and Mentha viridis L.
Of the menthol isomers, the 1-isomer occurs most widely in nature
and is typically what is referred by the name menthol having
coolant properties. L-menthol has the characteristic peppermint
odor, has a clean fresh taste and exerts a cooling sensation when
applied to the skin and mucosal surfaces. Other isomers of menthol
(neomenthol, isomenthol and neoisomenthol) have somewhat similar,
but not identical odor and taste, i.e., some having disagreeable
notes described as earthy, camphor, musty. The principal difference
among the isomers is in their cooling potency. L-menthol provides
the most potent cooling, i.e., having the lowest cooling threshold
of about 800 ppb, i.e., the concentration where the cooling effect
could be clearly recognized. At this level, there is no cooling
effect for the other isomers. For example, d-neomenthol is reported
to have a cooling threshold of about 25,000 ppb and 1-neomenthol
about 3,000 ppb. (R. Emberger and R. Hopp, "Synthesis and Sensory
Characterization of Menthol Enantiomers and Their Derivatives for
the Use in Nature Identical Peppermint Oils," Specialty Chemicals
(1987), 7(3), 193-201). This study demonstrated the outstanding
sensory properties of 1-menthol in terms of cooling and freshness
and the influence of stereochemistry on the activity of these
molecules.
[0005] Among synthetic coolants, many are derivatives of or are
structurally related to menthol, i.e., containing the cyclohexane
moiety, and derivatized with functional groups including
carboxamide, ketal, ester, ether and alcohol. Examples include the
.rho.-menthanecarboxamide compounds, such as
N-ethyl-.rho.-menthan-3-carboxamide, known commercially as "WS-3",
and others in the series, such as WS-5
(N-ethoxycarbonylmethyl-.rho.-menthan-3-carboxamide), WS-12
[N-(4-methoxyphenyl)-.rho.-menthan-3-carboxamide] and WS-14
(N-tert-butyl-.rho.-menthan-3-carboxamide). Examples of menthane
carboxy esters include WS-4 and WS-30. An example of a synthetic
carboxamide coolant that is structurally unrelated to menthol is
N,2,3-trimethyl-2-isopropylbutanamide, known as "WS-23". Additional
examples of synthetic coolants include alcohol derivatives such as
3-(1-menthoxy)-propane-1,2-diol known as TK-10, isopulegol (under
the tradename Coolact P) and .rho.-menthane-3,8-diol (under the
tradename Coolact 38D); menthone glycerol acetal known as MGA;
menthyl esters such as menthyl acetate, menthyl acetoacetate,
menthyl lactate known as Frescolat.RTM. supplied by Haarmann and
Reimer, and monomenthyl succinate under the tradename Physcool from
V. Mane. TK-10 is described in U.S. Pat. No. 4,459,425 to Amano et
al. Other alcohol and ether derivatives of menthol are described
e.g., in GB 1,315,626 and in U.S. Pat. Nos. 4,029,759; 5,608,119;
and 6,956,139. WS-3 and other carboxamide cooling agents are
described for example in U.S. Pat. Nos. 4,136,163; 4,150,052;
4,153,679; 4,157,384; 4,178,459 and 4,230,688. Additional
N-substituted .rho.-menthane carboxamides are described in WO
2005/049553A1 including
N-(4-cyanomethylphenyl)-.rho.-menthanecarboxamide,
N-(4-sulfamoylphenyl)-.rho.-menthanecarboxamide,
N-(4-cyanophenyl).rho.-menthanecarboxamide,
N-(4-acetylphenyl)-.rho.-menthanecarboxamide,
N-(4-hydroxymethylphenyl)-.rho.-menthanecarboxamide and
N-(3-hydroxy-4-methoxyphenyl)-.rho.-menthanecarboxamide. Other
N-substituted .rho.-menthane carboxamides include amino acid
derivatives such as those disclosed in WO 2006/103401 and in US
Pat. Nos. 4,136,163; 4,178,459 and 7,189,760 such as
N-((5-methyl-2-(1-methylethyl)cyclohexyl)carbonyl)glycine ethyl
ester and N-((5-methyl-2-(1-methylethyl)cyclohexyl)carbonyl)alanine
ethyl ester. Menthyl esters including those of amino acids such as
glycine and alanine are disclosed e.g., in EP 310 299 and in U.S.
Pat. Nos. 3,111,127; 3,917,613; 3,991,178; 5,703,123; 5,725,865;
5,843,466; 6,365,215; 6,451,844; and 6,884,903. Ketal derivatives
are described, e.g., in U.S. Pat. Nos. 5,266,592; 5,977,166 and
5,451,404. Additional agents that are structurally unrelated to
menthol but have been reported to have a similar physiological
cooling effect include alpha-keto enamine derivatives described in
U.S. Pat. No. 6,592,884 including
3-methyl-2-(1-pyrrolidinyl)-2-cyclopenten-1-one (3-MPC),
5-methyl-2-(1-pyrrolidinyl)-2-cyclopenten-1-one (5-MPC), and
2,5-dimethyl-4-(1-pyrrolidinyl)-3(2H)-furanone (DMPF); icilin (also
known as AG-3-5, chemical name
1-[2-hydroxyphenyl]-4-[2-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one)
described in Wei et al., J. Pharm. Pharmacol. (1983), 35:110-112.
Reviews on the coolant activity of menthol and synthetic coolants
include H. R. Watson, et al. J. Soc. Cosmet. Chem. (1978), 29,
185-200 and R. Eccles, J. Pharm. Pharmacol., (1994), 46,
618-630.
[0006] The present invention provides compositions comprising one
or more coolants, wherein the cooling and refreshing sensation
provided by the coolant(s) is potentiated in terms of onset,
intensity, and/or duration.
SUMMARY OF THE INVENTION
[0007] A compound is provided that comprises the following
structure:
##STR00001##
[0008] R.sub.1 is selected from H, alkyl, amino alkyl, alkoxy;
[0009] Q=H.sub.2, O, --OR.sub.1, --N(R.sub.1).sub.2,
--OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x, --P(OR.sub.1).sub.x
where x=1-2;
[0010] V=NR.sub.1, O, --OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x,
--P(OR.sub.1).sub.x where x=1-2;
[0011] W=H.sub.2, O;
[0012] X, Y=independently selected from H, aryl, naphthyl for
n=0;
[0013] X, Y=aliphatic CH.sub.2 or aromatic CH for n.gtoreq.1 and Z
is selected from aliphatic CH.sub.2, aromatic CH, or
heteroatom;
[0014] A=lower alkoxy, lower alkylthio, aryl, subsitituted aryl or
fused aryl; and
[0015] stereochemistry is variable at the positions marked*.
[0016] A compound is provided having the structure shown above,
wherein the compound at a concentration of about 5.2E-5% provides a
greater activation of TRPM8 than WS5 at a concentration of about 30
mM; a greater activation of TRPA1 than allyl isothiocyanate at a
concentration of about 50 mM; and a greater activation of TRPV1
than capsaicin at a concentration of about 350 nM.
[0017] A compound having the structure shown above is provided,
wherein the compound at a concentration of about 5.2E-5% provides
at least about 100%, 105%, 110%, 115%, 120% 125% or 130% activation
of TRPM8 when compared to WS5 at a concentration of about 30mM; at
least about 100%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%,
210%, 220%, 230% or 240% activation of TRPA1 when compared to allyl
isothiocyanate at a concentration of about 50 mM; and at least
about 95%, 100%, 105%, 110%, or 115% activation of TRPV1 when
compared to capsaicin at a concentration of about 350 nM.
[0018] A compound is provided that comprises the following
structure:
##STR00002##
[0019] R.sub.1 is selected from H, alkyl, amino alkyl, alkoxy;
[0020] Q=H.sub.2, O, --OR.sub.1, --N(R.sub.1).sub.2,
--OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x, --P(OR.sub.1).sub.x
where x=1-2;
[0021] V=NR.sub.1, O, --OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x,
--P(OR.sub.1).sub.x where x=1-2;
[0022] W=H.sub.2, O;
[0023] X, Y=independently selected from H, aryl, naphthyl for
n=0;
[0024] X, Y=aliphatic CH.sub.2 or aromatic CH for n.gtoreq.1 and Z
is selected from aliphatic CH.sub.2, aromatic
[0025] CH, or heteroatom;
[0026] A=lower alkoxy, lower alkylthio, aryl, subsitituted aryl or
fused aryl; and
[0027] stereochemistry is variable at the positions marked*.
[0028] A compound is provided that comprises the following
structure:
##STR00003##
[0029] R.sub.1 is selected from H, alkyl, amino alkyl, alkoxy;
[0030] Q=H.sub.2, O --OR.sub.1i, --N(R.sub.1).sub.2,
--OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x, --P(OR.sub.1).sub.x
where x=1-2;
[0031] V=NR.sub.1, O, --OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x,
--P(OR.sub.1).sub.x where x=1-2;
[0032] W=H.sub.2, O;
[0033] X, Y=independently selected from H, aryl, naphthyl for
n=0;
[0034] X, Y=aliphatic CH.sub.2 or aromatic CH for n.gtoreq.1 and Z
is selected from aliphatic CH.sub.2, aromatic
[0035] CH, or heteroatom;
[0036] A=lower alkoxy, lower alkylthio, aryl, subsitituted aryl or
fused aryl; and
[0037] stereochemistry is variable at the positions marked*.
[0038] A compound is provided that comprises the following
structure:
##STR00004##
[0039] A personal care composition is provided that comprises a
compound having the following structure:
##STR00005##
[0040] R.sub.1 is selected from H, alkyl, amino alkyl, alkoxy;
[0041] Q=H.sub.2, O, --OR.sub.1, --N(R.sub.1).sub.2,
--OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x, --P(OR.sub.1).sub.x
where x=1-2;
[0042] V=NR.sub.1, O, --OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x,
--P(OR.sub.1).sub.x where x=1-2;
[0043] W=H.sub.2, O;
[0044] X, Y=independently selected from H, aryl, naphthyl for
n=0;
[0045] X, Y=aliphatic CH.sub.2 or aromatic CH for n.gtoreq.1 and Z
is selected from aliphatic CH.sub.2, aromatic
[0046] CH, or heteroatom;
[0047] A=lower alkoxy, lower alkylthio, aryl, subsitituted aryl or
fused aryl; and
[0048] stereochemistry is variable at the positions marked*;
and
wherein the compound activates at least one of TRPV1, TRPV1, or
TRPM8.
[0049] These and other features, aspects, and advantages of the
present invention will become evident to those skilled in the art
from the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1 UV chromatogram overlays of three replicate
injections of compound 28, fraction 1. The percentages of relative
peak area are shown above each isomeric compound observed within
this mixture. All peaks appear at nominal m/z 374 in the QDa mass
spectra, indicating that these are isomeric species.
[0051] FIG. 2 UV chromatogram overlays of three replicate
injections of compound 28, fraction 2. The percentages of relative
peak areas are shown above for each isomeric compound observed
within this mixture. All peaks appear at nominal m/z 374 in the QDa
mass spectra, indicating that these are isomeric species. Fraction
2 has higher isomeric purity than fraction 1.
[0052] FIG. 3 UV trace overlays of chromatograms generated during
separate analysis of compound 28, fraction 1 (dashed line) and
fraction 2 (solid line). These overlays demonstrate that some
components are contained within both fractions, while some
components are essentially unique, and the ratio of isomers within
these two fractions differ. All peaks appear at nominal m/z 374 in
the QDa mass spectra, indicating that these are isomeric
species.
DETAILED DESCRIPTION OF THE INVENTION
[0053] The present invention is directed to the discovery that
certain cyclohexanecarboxamide structures deliver the means to
drive a cooling response at low concentrations. It has been
discovered that cyclohexanecarboxamide,
5-methyl-2-(1-methylethyl)-N-(2-phenylethyl)-, (1R,2S,5R) (CAS#
824947-52-6) and cyclohexanecarboxamide,
5-methyl-2-(1-methylethyl)-N-(2-phenylethyl)-, (1R,2S,5R) (CAS#
847564-71-0) structures with 2-amino-propanamide (CAS# 4726-84-5)
have enhanced long lasting cooling properties and
cyclohexanecarboxamide, 5-methyl-2-(1-methylethyl)-N-phenyl-,
(1R,2S,5R) and cyclohexanecarboxamide,
5-methyl-2-(1-methylethyl)-N-1-naphthalenyl-(1R,2S,5R) (CAS#
863091-95-6) structures with an aminoethane (CAS# 75-04-7) moiety
deliver a warming sensation. Both types of cyclohexanecarboxamide
(cooling and warming) are efficacious at low use levels (1-10 ppm).
The stereochemistry assigned to the compounds above is based on the
dominant isomer (1R, 2S, 5R) derived from the menthol starting
material. One or more additional isomers and/or enantiomers may
occur due to the additional chiral sites built out from the amide
linkage.
[0054] Structures built off of the cyclohexanecarboxamide backbone
have been applied as anti-cancer agents as disclosed in WO
2009/067410. As shown in U.S. Pat. No. 4,150,052, only a select few
of the cyclohexanecarboxamide derivatives had noticeable cooling.
The molecules disclosed in WO 2009/067410 were evaluated for their
TRPM8 activity in relation to the destruction of prostate cancer
cells. The data shown herein illustrates that activating TRPM8 does
not necessarily mean that a cooling sensation will be observed.
Thus cooling would have been an undesirable effect and something
they would have avoided.
[0055] The present invention is thus based on the discovery that
select molecules can be used to drive a cooling response when
formulated into consumer products. A second object of this
invention shows the discovery that select cyclohexanecarboxamide
derivatives can provide long lasting cooling at very low levels,
allowing for formulation efficiencies, in particular coolant
compounds (coolants), such as described below.
[0056] All percentages and ratios used hereinafter are by weight of
total composition, unless otherwise indicated. All percentages,
ratios, and levels of ingredients referred to herein are based on
the actual amount of the ingredient, and do not include solvents,
fillers, or other materials with which the ingredient may be
combined as a commercially available product, unless otherwise
indicated.
[0057] All measurements referred to herein are made at 25.degree.
C. unless otherwise specified.
[0058] By "personal care composition" is meant a product, which in
the ordinary course of usage is applied to or contacted with a body
surface to provide a beneficial effect. Body surface includes skin,
for example dermal or mucosal; body surface also includes
structures associated with the body surface for example hair,
teeth, or nails. Examples of personal care compositions include a
product applied to a human body for improving appearance,
cleansing, and odor control or general aesthetics. Non-limiting
examples of personal care compositions include oral care
compositions, such as, dentifrice, mouth rinse, mousse, foam, mouth
spray, lozenge, chewable tablet, chewing gum, tooth whitening
strips, floss and floss coatings, breath freshening dissolvable
strips, denture care product, denture adhesive product; after shave
gels and creams, pre-shave preparations, shaving gels, creams, or
foams, moisturizers and lotions; cough and cold compositions, gels,
gel caps, and throat sprays; leave-on skin lotions and creams,
shampoos, body washes, body rubs, such as Vicks Vaporub; hair
conditioners, hair dyeing and bleaching compositions, mousses,
shower gels, bar soaps, antiperspirants, deodorants, depilatories,
lipsticks, foundations, mascara, sunless tanners and sunscreen
lotions; feminine care compositions, such as lotions and lotion
compositions directed towards absorbent articles; baby care
compositions directed towards absorbent or disposable articles; and
oral cleaning compositions for animals, such as dogs and cats.
[0059] The present invention is also directed towards "oral health
compositions" as used herein refers to compositions in a form that
is deliverable to a mammal in need via the oral cavity, mouth,
throat, nasal passage or combinations thereof. Nonlimiting examples
include liquid compositions, cough syrups, respiratory
preparations, beverage, supplemental water, pills, soft gels,
tablets, capsules, gel compositions, foam compositions, saline wash
and combinations thereof. Liquid compositions, gel compositions can
be in a form that is directly deliverable to the mouth and throat.
These compositions and/or preparations can be delivered by a
delivery device selected from droppers, pump, sprayers, liquid
dropper, saline wash delivered via nasal passageway, cup, bottle,
liquid filled gel, liquid filled gummy, center filled gum, chews,
films, center filled lozenge, gum filled lozenge, pressurized
sprayers, atomizers, air inhalation devices, liquid filled
compressed tablet, liquid filled gelatin capsule, liquid filled
capsule, squeezable sachets, power shots, and other packaging and
equipment, and combinations thereof. The sprayer, atomizer, and air
inhalation devices can be associated with a battery or electric
power source.
[0060] The present invention is also directed towards a respiratory
preparation. In one embodiment the respiratory preparation
comprises a film forming agent; and a thickening agent. The
preparation provides on demand relief. The preparation can work to
physically coat the mouth and throat creating a soothing barrier
over the epithelial cells that line the throat layer. The
preparation can additionally, reduce inflammation and relieve minor
pain associated with a cough and/or sore throat. Preferably the
respiratory preparation would not contain a pharmaceutical
active.
[0061] The present invention is also directed to lotion
compositions and to absorbent articles, particularly disposable
absorbent articles, having a lotion treatment composition applied
thereon. Disposable absorbent articles can be baby diapers or
feminine hygiene articles, including incontinence devices and
catamenial products, such as tampons, sanitary napkins,
pantiliners, interlabial products, and the like. For convenience,
the invention is disclosed below with respect to the embodiment of
a catamenial device, such as a sanitary napkin or pantiliner.
[0062] The absorbent article can comprise any known or otherwise
effective topsheet, such as one which is compliant, soft feeling,
and non-irritating to the body of the wearer. Suitable topsheet
materials include a liquid pervious material that is oriented
towards and contacts the body of the wearer, thereby permitting
body discharges to rapidly penetrate through the topsheet without
allowing fluid to flow back through the topsheet to the skin of the
wearer. The topsheet, while capable of allowing rapid transfer of
fluid through it, also provides for the transfer or migration of
the lotion composition onto an external or internal portion of a
body of the wearer. A suitable topsheet can be made of various
materials, such as woven and nonwoven materials; apertured film
materials including apertured formed thermoplastic films, apertured
plastic films, and fiber-entangled apertured films; hydro-formed
thermoplastic films; porous foams; reticulated foams; reticulated
thermoplastic films; thermoplastic scrims; or combinations thereof,
as is well known in the art of making catamenial products such as
sanitary napkins, pantiliners, incontinence pads, and the like.
[0063] A lotion composition of the present invention comprises at
least one rheology structurant, w typically is a solid. The lotion
composition can further comprise other optional ingredients, like
surface energy modifiers. In one embodiment, a lotion composition
consists essentially of, or consists of, a rheology structurant,
such as a microcrystalline wax, alkyl dimethicone, ethylene glycol
dibehenate, ethylene glycol distearate, glycerol tribehenate,
glycerol tristearate, and ethylene bisoleamide. A present lotion
composition can contain a single rheology structurant or a mixture
of two or more rheology structurants.
[0064] In preparing a lotioned catamenial device according to the
present invention, the lotion composition can be applied to the
outer surface of the absorbent article, such as, for example, the
outer surface of the topsheet. Any of a variety of application
methods that distribute lubricious materials having a molten or
liquid consistency can be used, such as, for example, as set forth
in U.S. Pat. No. 5,968,025 and U.S. Pub. App. No. 2005/0208113.
Suitable methods include but are not limited to spraying, printing
(e.g., flexographic printing), coating (e.g., gravure coating),
extrusion, dipping, or combinations of these application
techniques, e.g., spraying the lotion composition on a rotating
surface, such as a calender roll, that then transfers the
composition to the outer surface of the sanitary napkin topsheet.
Additionally, the manner of applying the lotion composition to a
portion of a catamenial device can be such that the substrate or
component does not become saturated with the lotion composition.
The lotion composition can be applied to the catamenial device at
any point during assembly. For example, the lotion composition can
also be applied to the outer surface of the topsheet before it is
combined with the other raw materials to form a finished catamenial
device.
[0065] The term "dentifrice", as used herein, includes tooth or
subgingival -paste, gel, or liquid formulations unless otherwise
specified. The dentifrice composition may be a single phase
composition or may be a combination of two or more separate
dentifrice compositions. The dentifrice composition may be in any
desired form, such as deep striped, surface striped, multilayered,
having a gel surrounding a paste, or any combination thereof. Each
dentifrice composition in a dentifrice comprising two or more
separate dentifrice compositions may be contained in a physically
separated compartment of a dispenser and dispensed
side-by-side.
[0066] The term "dispenser", as used herein, means any pump, tube,
or container suitable for dispensing compositions such as
dentifrices.
[0067] The term "teeth", as used herein, refers to natural teeth as
well as artificial teeth or dental prosthesis.
[0068] The term "orally acceptable carrier or excipients" includes
safe and effective materials and conventional additives used in
oral care compositions including but not limited to fluoride ion
sources, anti-calculus or anti-tartar agents, buffers, abrasives
such as silica, alkali metal bicarbonate salts, thickening
materials, humectants, water, surfactants, titanium dioxide,
flavorants, sweetening agents, xylitol, coloring agents, and
mixtures thereof.
[0069] Herein, the terms "tartar" and "calculus" are used
interchangeably and refer to mineralized dental plaque
biofilms.
[0070] The components of the present compositions are described in
the following paragraphs.
TABLE-US-00001 SEQ ID NO Sequence 1 Human TRPV1 DNA sequence 2
Human TRPA1 DNA sequence 3 Human TRPM8 DNA sequence
[0071] The term "TRPV1" or "TRPV1 receptor", as used herein, refers
to the transient receptor potential vanilloid receptor 1, which is
a ligand-gated, non-selective cation channel preferentially
expressed on small-diameter sensory neurons and detects noxious as
well as other substances. The TRPV1 receptor is provided as SEQ ID
NO: 1. The TRPV1 receptor responds to, for example, both noxious
and painful stimuli. A noxious stimulus would include those that
give a burning (i.e. hot) sensation.
[0072] The term "TRPV1 agonist", as used herein, refers to any
compound, which at a concentration of 1 mM gives a calcium flux
count of at least 1000 counts or 20% above the background level of
calcium present in the cell according to the FLIPR method, as
discussed herein. The term "count" is defined as the change in
fluorescence of the cell lines due to the influx of calcium across
the cell membrane, which reacts with the calcium sensitive dye
present within the cells.
[0073] The term "TRPV1 antagonist", as used herein, refers to any
compound which at a concentration of 1 mM gives a reduction in
calcium flux count of at least 1000 counts or 20% below the
activation of TRPV1 receptor by 350 .mu.M capsaicin.
[0074] The term "TRPV1 desensitizer", as used herein, refers to any
compound, which shows agonist activity and causes a decrease in
activation by a known TRPV1 agonist.
[0075] The term "TRPA1" or "TRPA1 receptor", as used herein, refers
to the transient receptor potential cation channel, subfamily A,
member 1, having a large cysteine-rich N-terminus that contains 18
predicted ankyrin repeats. The TRPA1 receptor is provided as SEQ ID
NO: 2. TRPA1 is a ligand-gated, non-selective cation channel
preferentially expressed on small diameter sensory neurons.
[0076] The term "TRPA1 agonist", as used herein, refers to any
compound, which at a concentration of 1 mM gives a calcium flux
count of at least 1000 counts or 20% above the background level of
calcium present in the cell according to the FLIPR method, as
discussed herein. The term "count" is defined as the change in
fluorescence of the cell lines due to the influx of calcium across
the cell membrane, which reacts with the calcium sensitive dye
present within the cells.
[0077] The term "TRPA1 antagonist", as used herein, refers to any
compound, which at a concentration of 1 mM gives a reduction in
calcium flux count of at least 1000 counts or 20% below the
activation of TRPA1 receptor by 50 mM allyl isothiocyanate.
[0078] The term "TRPA1 desensitizer", as used herein, refers to any
compound, which shows agonist activity and causes a decrease in
activation by a known TRPA1 agonist.
[0079] The term "TRPM8" or "TRPM8 receptor", as used herein, refers
to cold- and menthol-sensitive receptor (CMR1) or TRPM8. The TRPM8
nomenclature for the receptor comes from its characterization as a
non-selective cation channel of the transient receptor potential
(TRP) family that is activated by stimuli including low
temperatures, menthol and other chemical coolants. The TRPM8
receptor is provided as SEQ ID NO: 3.
[0080] The cooling receptor conventionally known as TRPM8 or the
menthol receptor has been demonstrated as a means to differentiate
intensity and duration of organic molecules that initiate and
propagate the non-thermal cooling perception (D. D. Mckemy, The
Open Drug Discovery Journal 2:81-88 2010). McKemy reported the EC50
values of many agonists to TRPM8 which span the range of 100 nM to
19 mM, thus showing the channel can be activated across a wide
range of structures at varying concentrations. This channel also
has the nomenclature of CRM1 and TRPP8. The later was designated as
such due to its identification with prostate cells, where it was
employed as a means to identify molecules targeted towards prostate
cancer.
[0081] The term "TRPM8 agonist", as used herein, refers to any
compound, which when added to a TRPM8 receptor, according to the
FLIPR method, as discussed herein, produces any increase in
fluorescence over background.
[0082] The term "TRPM8 antagonist", as used herein, refers to any
compound, which does not show any agonistic activity when directly
added and inhibits activation of the TRPM8 receptor by a known
TRPM8 agonist. Using the FLIPR method, as discussed herein a
molecule that has >20% reduction in calcium flux compared to the
WS5 activated TRPM8 receptor is considered a TRPM8 antagonist.
[0083] The term potency, as defined by the Merck Manual, refers to
the concentration (EC50) or dose (ED50) of a chemistry required to
produce 50% of the chemistry's maximal effect as depicted by a
graded dose-response curve. EC50 equals Kd (Dissociation constant,
which is a measure of 50% of the substance in question bound to the
receptor) when there is a linear relationship between occupancy and
response. Often, signal amplification occurs between receptor
occupancy and response, which results in the EC50 for response
being much less (ie, positioned to the left on the abscissa of the
log dose-response curve) than KD for receptor occupancy. Potency
depends on both the affinity of chemistry for its receptor, and the
efficiency with which chemistry-receptor interaction is coupled to
response. The dose of chemistry required to produce an effect is
inversely related to potency. In general, low potency is important
only if it results in a need to administer the chemistry in large
doses that are impractical. Quantal dose-response curves provide
information on the potency of chemistry that is different from the
information derived from graded dose-response curves. In a quantal
dose-response relationship, the ED50 is the dose at which 50% of
individuals exhibit the specified quantal effect.
[0084] Coolants or compounds that have a physiological cooling
effect particularly on oral and other mucosal surfaces and skin are
common ingredients in a wide variety of products, including edible
compositions, personal care compositions, and in flavor or perfume
compositions. Examples of edible compositions include
confectionery, candies, chocolate, chewing gum, beverages and oral
medicines. Personal care compositions, including oral care
compositions, have been described previously. The pleasant cooling
sensation provided by coolants contributes to the appeal and
acceptability of the products. In particular, oral care products,
such as dentifrices and mouthwashes are formulated with coolants
because they provide breath freshening effects and a clean, cool,
fresh feeling in the mouth.
[0085] It is now well established that sensations such as cool or
cold can be attributed to activation of receptors at peripheral
nerve fibers by a stimulus such as low temperature or a chemical
coolant, which produces electrochemical signals that travel to the
brain, which then interprets, organizes and integrates the incoming
signals into a perception or sensation. Different classes of
receptors have been implicated in sensing cold temperatures or
chemical coolant stimuli at mammalian sensory nerve fibers. Among
these receptors, a major candidate involved in sensing cold has
been identified and designated as cold- and menthol-sensitive
receptor (CMR1) or TRPM8. The TRPM8 nomenclature for the receptor
comes from its characterization as a non-selective cation channel
of the transient receptor potential (TRP) family, which is
activated by stimuli including low temperatures, menthol and other
chemical coolants. However, the precise mechanisms underlying the
perception of a pleasant cooling sensation on skin or oral surfaces
are presently not clearly understood. While it has been
demonstrated that the TRPM8 receptor is activated by menthol and
other coolants, it is not fully understood what other receptors may
be involved, and to what extent these receptors need to be
stimulated or perhaps suppressed in order for the overall perceived
sensation to be pleasant, cooling and refreshing. For example,
menthol is widely used as a cooling agent, but menthol can also
produce other sensations including tingling, burning, prickling and
stinging as well as a minty smell and bitter taste. Thus, it can be
inferred that menthol acts on many different receptors, including
cold, warm, pain and taste receptors.
[0086] Examples of solvents that can be used to solubilize
compounds of the present invention, such as compound 28--as
discussed below, are based upon solubility parameters and cohesion
properties explained by Charles Hansen in "Hansen Solubility
Parameters: A User's Handbook" by Charles M. Hansen, CRC Press
(2007) and in "The CRC Handbook and Solubility Parameters and
Cohesion Parameters," Edited by Allan F. M. Barton (1999). Each
material is defined by three points in 3D space and these three
points are known as the Hansen Solubility Parameters (HSP) which
may be defined as follows.
[0087] Solubility parameters are theoretically calculated numerical
constants, which are a useful tool in predicting the ability of a
solvent material to dissolve a particular solute. When the
solubility parameters of a solvent falls within the solubility
parameter range of a solute, i.e., the material to be dissolved,
solubilization of the solute is likely to occur. There are three
Hansen empirically and theoretically derived solubility parameters,
a dispersion-force component (SD), a polar or dipole interaction
component (.delta..sub.P) and a hydrogen-bonding component (SH).
Each of the three parameters (i.e., dispersion, polar and hydrogen
bonding) represents a different characteristic of solvency, or
solvent capability. In combination, the three parameters are a
measure of the overall strength and selectivity of a solvent. The
Total Hansen solubility parameter, which is the square root of the
sum of the squares of the three parameters mentioned previously,
provides a more general description of the solvency of the
solvents. Individual and total Solubility Parameter units are given
in MPa.sup.0.5. Solubility parameters for a material may then be
plotted in a normal three-dimensional graph. From the location
(.delta..sub.D, .delta..sub.P, .delta..sub.H), a radius is
projected to form a sphere, which encompasses a region of
solubility such that any solvent whose parameters reside within
this space should dissolve the solute in question. The distance
between the HSP coordinate of material (i.e., the solute) to the
HSP coordinates of material (solvent) is designated herein as Ra.
The 3D distance, Ra, is defined by the equation:
Ra.sup.2=4(.delta..sub.D1-.delta..sub.D2).sup.2+(.delta..sub.P1-.delta..s-
ub.P2).sup.2+(.delta..sub.H1-.delta..sub.H2).sup.2 The sphere
equation of Hansen was calculated to center the target molecules of
choice, in this case, compound 28 and the various isomers (L, D,
and neo) and enantiomers of each. The target Polar, Dispersive, and
Hydrogen Bonding HSP are the Hansen solubility parameters of the
target molecule as calculated by "Molecular Modeling Pro" software,
version 5.1.9 (ChemSW, Fairfield Calif., www.chemsw.com) or Hansen
Solubility from Dynacomp Software. The solubility parameters of
every solvent in this analysis were also calculated via this
software. Within the sphere having a radius R.sub.a=14 are solvents
into which compound 28 and isomer materials will be soluble. For
solubility >5% in the selected solvents, the preferred range of
.delta..sub.dispersion is .+-.3 units, from about 15.2 to 21.2
(MPa).sup.0.5. The preferred range of .delta..sub.polarity is .+-.6
units, from about 0 to 10.8 (MPa).sup.0.5. The preferred range of
.delta..sub.Hydrogen bonding is .+-.13 units, from about 0 to 25
(MPa).sup.0.5. The HSP of compound 28 were calculated as
dispersion=17.8, polarity=5.6, and hydrogen bonding=9.0.
Non-limiting examples of flavor and fragrance raw materials having
suitable Hansen Solubility Parameters used to solubilize the
carboxamide derivative include menthone, carvone, pine oil,
cinnamic aldehyde, ethanol, benzyl alcohol, eucalyptol, 1,2-propane
diol, 1,3-propane diol, hexane, ethanolamine, cyclodextrins, and
triacetin.
[0088] Ideally, a coolant can produce a cooling or freshness
sensation similar to that produced by menthol, but without certain
of the disadvantages associated with menthol, such as flavor
modification, bitter aftertaste, off-flavor, strong odor and
burning or irritating sensation, particularly at high
concentrations. It is desirable that the coolant compounds barely
possess a distinctive odor or flavor while providing a pleasant
fresh cool sensation of prolonged duration, in order that the
effect can still be perceived for a considerable time after use,
for example, longer than 15 minutes. Menthol generally provides an
initial high cooling impact, but its effect is somewhat transient
in that the cool sensation drops sharply within a few minutes after
use. By contrast, a number of longer lasting coolant compounds may
fail to provide an immediate cooling perception, i.e., within a few
seconds of application, particularly when used at low levels. Thus
there is a continuing need for means to potentiate the activity of
coolant chemicals, in terms of quickening the onset of the cooling
sensation, intensifying the cooling sensation, especially at lower
concentrations, and producing a longer lasting sensation of cooling
and freshness than what menthol provides.
[0089] As stated previously, the present invention is directed to
the discovery that specific
5-methyl-2-(1-methylethyl)-N-(2-phenylethyl)-, (1R, 2S, 5R)
cyclohexanecarboxamide structures, as shown below, deliver the
means to drive a cooling response at low concentrations.
[0090] Structure I, which includes compounds of the present
invention, as shown below, and which includes compound 28,
represents a genus that has been surprisingly found to be useful as
modulators of TRPM8 activation. Structure I represents a
heteroalkyl substituted aryl or heteroalkyl-aryl substituted alkyl
carboxamide of methanol having the shown below structure and
including any acceptable salts or solvates thereof; wherein:
##STR00006##
[0091] R.sub.1 is selected from H, alkyl, amino alkyl, alkoxy;
[0092] Q=H.sub.2, O, --OR.sub.1, --N(R.sub.1).sub.2,
--OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x, --P(OR.sub.1).sub.x
where x=1-2;
[0093] V=NR.sub.1, O, --OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x,
--P(OR.sub.1).sub.x where x=1-2;
[0094] W=H.sub.2, O;
[0095] X, Y=independently selected from H, aryl, naphthyl for
n=0;
[0096] X, Y=aliphatic CH.sub.2 or aromatic CH for n.gtoreq.1 and Z
is selected from aliphatic CH.sub.2, aromatic
[0097] CH, or heteroatom;
[0098] A=lower alkoxy, lower alkylthio, aryl, subsitituted aryl or
fused aryl; and
[0099] stereochemistry is variable at the positions marked*.
[0100] A number of stereoisomers are contemplated in the above
Structure I, where substitution is allowed and the relative
configuration of each stereo center will dictate the activity
towards the receptor. While it is known that the stereochemistry of
side chain groups may be important to the activity of the molecule,
the activity of these compounds in vivo is highly unpredictable. In
some cases, isomers of the same molecule may have comparable
activity. In other cases, stereoisomers of the same molecule could
have enhanced or diminished activity towards the receptor. In some
cases, individual stereoisomers may have no activity.
[0101] Specific compounds of interest may derive from the 1R, 2S,
5R configuration found in natural (-)-menthol. In these cases, the
stereoisomeric derivatives of 1R, 2S, 5R-menthyl carboxamide will
be found in the substituted alkyl side chain fragment of the
molecule. While the 1R, 2S, 5R configuration is known to be
important to activity, the 1S,2S,5R neo-isomer of N-substituted
menthyl carboxamide derivatives has also shown promise.
##STR00007##
[0102] R.sub.1 is selected from H, alkyl, amino alkyl, alkoxy;
[0103] Q=H.sub.2, O, --OR.sub.1, --N(R.sub.1).sub.2,
--OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x, --P(OR.sub.1).sub.x
where x=1-2;
[0104] V=NR.sub.1, O, --OPO(OR.sub.1).sub.x, --PO(OR.sub.1).sub.x,
--P(OR.sub.1).sub.x where x=1-2;
[0105] W=H.sub.2, O;
[0106] X, Y=independently selected from H, aryl, naphthyl for
n=0;
[0107] X, Y=aliphatic CH.sub.2 or aromatic CH for n.gtoreq.1 and Z
is selected from aliphatic CH.sub.2, aromatic
[0108] CH, or heteroatom;
[0109] A=lower alkoxy, lower alkylthio, aryl, subsitituted aryl or
fused aryl; and
[0110] stereochemistry is variable at the positions marked*.
[0111] In the case of compounds 28 and 776 (which would fall under
Structure IA; and are discussed in more detail below), excellent
activity is seen where the amino acid derived side chain (alanine)
contains both the R (28) and S (776) configurations. In these
cases, while not being limited to theory, specific activity among
isomers is determined by the unique structural elements within the
molecule in addition to the exact stereochemistry. While it is
known that molecules having the right balance of hydrogen bonding
groups (i.e. --NHR, --OH, --CONHR, etc.), Log P value, and
molecular weight range are preferred, unique structural elements
can contribute to activity within these preferred ranges. The
current compounds of interest contain polar groups in the
side-chain which are capable of both hydrogen bonding and balancing
the lipophilicity of the overall structure. The stereochemical
features within these molecules also impart a 3D dimensionality to
the structure which can enhance interaction with specific
receptors. It is believed that these unique structural features
lead to enhanced affinity for the receptor which translates into
the prolonged cooling effects which have been observed.
[0112] It has been discovered that cyclohexanecarboxamide,
5-methyl-2-(1-methylethyl)-N-(2-phenylethyl)-, (1R,2S,5R) (CAS#
824947-52-6) and cyclohexanecarboxamide,
5-methyl-2-(1-methylethyl)-N-(2-phenylethyl)-, (1R,2S,5R) (CAS#
847564-71-0) structures (shown above) with 2-amino-propanamide
(CAS# 4726-84-5) have enhanced long lasting cooling properties and
cyclohexanecarboxamide, 5-methyl-2-(1-methylethyl)-N-phenyl-,
(1R,2S,5R) and cyclohexanecarboxamide,
5-methyl-2-(1-methylethyl)-N-1-naphthalenyl-(1R,2S,5R) (CAS#
863091-95-6) structures with an aminoethane (CAS# 75-04-7) moiety
deliver a warming sensation. Both types of cyclohexanecarboxamide
(cooling and warming) are efficacious at low use levels (1-10 ppm).
The advantage of using such low levels of these materials allows
for their formulation into higher water compositions, such as
mouthrinses, without the need for additional processing aids, such
as co-surfactants, oils, or other suspension agents. These
materials may also provide mitigation of off tasting sensations,
such as that derived from metal salts, peroxide, and CPC.
[0113] Other suitable uses for long lasting TRPM8 activity as
exemplified from compound 28, would be for food applications; skin
conditions, such as treatments for non-keratinzed stratified
epithelium; analgesic applications as pain mitigation agents;
reductions in inflammation; additives to cigarettes; topical salves
for muscle pain, for chronic pain from osteoarthritis, and for
chemotherapy induced neuropathy; skin barrier recovery accelerants;
and antipruritic or antiseptic medications; and for
vasoconstriction in relaxed vessels.
[0114] The levels of use for compounds of the present invention,
such as compound 28, depend upon the targeted TRPM8 area of the
body. For example in an oral application of a compound of the
present invention, such as dentifrice, floss, chewing gum, or white
strip, the levels of use may be from about 0.00001% to about 0.1%;
from about 0.00005% to about 0.1%; from about 0.0001% to about
0.05%; or from about 0.001% to about 0.01% by weight of the
composition. When a compound of the present invention is used in a
mouthwash, the level of use may be from about 0.000001% to about
0.01% or from about 0.0001% to about 0.001% by weight of the
composition. When a compound of the present invention, such as
compound 28, is delivered topically, for example in shampoos and
lotions the levels may be from about 0.001% to about 0.5% by weight
of the composition or from about 0.01% to about 0.4% by weight of
the composition.
EXAMPLES
Example 1
[0115] To determine what effect, if any, test compounds (shown in
TABLE 1) had on TRPM8 (SEQ ID NO: 3), TRPA1 (SEQ ID NO: 2), and
TRPV1 (SEQ ID NO: 1) activation the protocols listed below were
used.
TRPM8 Protocol-FLIPR Assay
[0116] To determine whether TRPM8 is activated, the intracellular
calcium ion (Ca.sup.2+) level was measured from transfected cells
with the TRPM8 receptor sequence (SEQ ID NO: 3). HEK-293 (human
embryonic kidney) cells stably transfected with human TRPM8 were
grown in 15 ml growth medium (high glucose DMEM (Dulbecco's
Modification of Eagle's Medium) supplemented with 10% FBS (fetal
bovine serum), 100 ug/ml penicillin/streptomycin, 5 .mu.g/ml
blasticindin, and 100 .mu.g/ml zeocin) in a 75 cm.sup.2 flask for 3
days at 37.degree. C. in a mammalian cell culture incubator set at
5% CO.sub.2. Cells were detached with addition of 2 ml of
trypsin-EDTA buffer (GIBCO.RTM. 25200, Invitrogen, Grand Island,
N.Y.) for about 2-3 min. Trypsin was inactivated by addition of 8
ml growth medium. Cells were transferred to a 50 ml tube and
centrifuged at 850 rpm for 3 minutes to remove medium. After
centrifugation, a pellet of cells was formed in the bottom of the
tube separating them from the supernatant solution. The supernatant
was discarded and the cell pellet was suspended in 1 ml of fresh
growth medium to which 5 .mu.l (12.5 .mu.g) of Fluo-4 AM (Molecular
Probes, Inc., Eugene, Oreg.) calcium indicator was added and
incubated for 30 min with gentle shaking. Fluo-4 AM is a
fluorescent dye used for quantifying cellular Ca.sup.2+
concentrations in the 100 nM to 1 microM range. At the end of 30
minutes, 45 ml of assay buffer (1.times.HBSS (Hank's Balanced Salt
Solution), 20 mM HEPES
(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)) was added to
wash cells and the resulting mixture was then centrifuged at 850
rpm for 3 minutes to remove excess buffer and Fluo-4 AM calcium
indicator.
[0117] The pelleted cells were re-suspended in 10 ml assay buffer
and 90 .mu.l aliquots (50,000 cells) per well delivered to a
96-well assay plate containing 10 .mu.l of test compounds (1 mM in
assay buffer, final concentration 100 .mu.M) or buffer control and
incubated at room temperature for 30 minutes. After 30 minutes, a
plate was placed into a fluorometric imaging plate reader (FLIPR384
from Molecular Devices, Sunnyvale, Calif.) and basal fluorescence
recorded (excitation wave length 488 nm and emission wave length
510 nm). Then 20 .mu.l of 100 mM of TRPM8 agonist WS5 coolant in
the assay buffer was added and fluorescence recorded. For
determining the direct effect of test compounds on TRPM8,
fluorescence was measured immediately after addition of each
compound (TABLES 2 and 3). Additional discussion of the FLIPR
method can be found in Smart et al., Characterization using FLIPR
of human vanilloid VR1 receptor pharmacology, European Journal of
Pharmacology 417, 51-58 (2001) and Liu et al., Development and
validation of a platelet calcium flux assay using a fluorescent
imaging plate reader, Analytical Biochemistry 357, 216-224
(2006).
TRPA1 Protocol--FLIPR Assay
[0118] To determine whether TRPA1 is activated, the intracellular
calcium ion (Ca.sup.2+) level from transfected cells with the TRPA1
receptor sequence (SEQ ID NO: 2) was measured. HEK-293 cells stably
transfected with human TRPA1 were grown in 15 ml growth medium
(high glucose DMEM (Dulbecco's Modification of Eagle's Medium)
supplemented with 10% FBS (fetal bovine serum), 100 .mu.g/ml
penicillin/streptomycin, 100 .mu.g/ml G418) in a 75 cm.sup.2 flask
for 3 days at 37.degree. C. in a mammalian cell culture incubator
set at 5% CO.sub.2. Cells were detached with addition of 10 ml of
PBS (phosphate buffered saline) by hand shaking gently and
transferred to a 50 ml tube and centrifuged at 850 rpm for 3
minutes to remove PBS. After centrifugation, a pellet of cells was
formed in the bottom of the tube separating them from the
supernatant solution. The supernatant was discarded and the cell
pellet suspended in 1 ml of fresh growth medium to which 5 .mu.l
(12.5 .mu.g) of Fluo-4 AM (Molecular Probes, Inc.) calcium
indicator was added and incubated for 30 minutes with gentle
shaking. At the end of the 30 minutes, 45 ml of assay buffer
(1.times.HBSS (Hank's Balanced Salt Solution), 20 mM HEPES
(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)) was added to
wash the cells and the resulting combination was then centrifuged
at 850 rpm for 3 minutes to remove excess buffer and Fluo-4 AM
calcium indicator.
[0119] The pelleted cells were re-suspended in 10 ml assay buffer
and 90 .mu.l aliquots (50,000 cells) per well delivered to a
96-well assay was placed into a fluorometric imaging plate reader
(FLIPR TETRA from Molecular Devices) and basal fluorescence
recorded (excitation wave length 488 nm and emission wave length
510 nm). Then 20 .mu.l of the test compound being tested was added
and fluorescence recorded (TABLES 2 and 3).
[0120] To determine if a compound was an agonist the direct effect
of a test compound was determined. If any increase in fluorescence
over background was noted, then the compound was considered an
agonist. The agonist activity was expressed relative to that
observed with a benchmark agonist such as 50 gm allyl
isothiocyanate for TRPA1.
TRPV1 Protocol--FLIPR Assay
[0121] To determine whether TRPV1 was activated, the intracellular
calcium ion (Ca.sup.+2) levels from cells transfected with the
TRPV1 receptor sequence (SEQ ID NO: 1) were measured. HEK-239 cells
stably transfected with human TRPV1 were grown in 15 ml growth
medium (high glucose DMEM (Dulbecco's Modification of Eagle's
Medium) supplemented with 10% FBS (fetal bovine serum), 100
.mu.g/m1 Penicillin/streptomycin, 100 .mu.g/ml G418) in a 75
cm.sup.2 flask for 3 days at 33.degree. C. in a mammalian cell
culture incubator set at 5% CO.sub.2. Cells were detached with
addition of 10 ml of PBS (phosphate buffered saline) by gentle hand
shaking. Cells were transferred to a 50 ml tube and centrifuged at
850 rpm for 3 minutes to remove PBS. After centrifugation, a pellet
of cells formed in the bottom of the tube separating them from the
supernatant solution. The supernatant was discarded and the cell
pellet suspended in 1 ml of fresh growth medium to which 5 .mu.l
(12.5 .mu.g) of Fluo-4 AM (Molecular Probes, Inc., Eugene, Oreg.)
calcium indicator was added and incubated for 30 minutes with
gentle shaking. At the end of the 30 minutes, 45 ml of assay buffer
(1.times.HBSS (Hank's Balanced Salt Solution), 20 mM HEPES
(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)) was added to
wash the cells and the resulting combination was then centrifuged
at 850 rpm for 3 minutes to remove excess buffer and Fluo-4 AM
calcium indicator.
[0122] The pelleted cells were re-suspended in 10 ml assay buffer
and 90 .mu.l aliquots (50,000 cells) per well delivered to a
96-well assay plate was placed into a fluorometric imaging plate
reader (FLIPR TETRA from Molecular Devices) and basal fluorescence
recorded (excitation wave length 488 nm and emission wave length
510 nm). Then 20 .mu.l of a test compound-being tested as a TRPV1
receptor agonist was added and fluorescence recorded. The observed
value with compound pretreated cells was compared with buffer
control; the difference between the two indicating a measure of
effect of the test compound on the agonist (TABLES 2 and 3).
[0123] If any increase in fluorescence over background was noted,
then the compound was considered an agonist. The agonist activity
was expressed relative to that observed with a benchmark agonist
such as 350 nM Capsaicin for TRPV1.
TABLE-US-00002 TABLE 1 Test Compounds ##STR00008## ##STR00009##
##STR00010## Test Compounds ##STR00011## ##STR00012##
##STR00013##
TABLE-US-00003 TABLE 2 Concentration Tested on HEK 293 receptor
expressing cells Concentration of each test compound TRPM8 TRPA1
TRPV1 180 0.001% 0.001% 0.001% 773 0.004% 0.02% 0.02% 776 0.004%
0.02% 0.02% 777 0.004% 0.02% 0.02% 28 5.2E-6% 5.2E-5% 5.2E-5% 30
5.2E-5% 5.2E-5% 5.2E-5% WS5 30 -- -- microMolar Allyl
Isothiocyanate -- 50 -- (AITC) microMolar Capsaicin -- -- 350
nanoMolar
[0124] TABLE 2 illustrates the concentration of each test compound
when it was tested across the HEK 293 receptor containing
cells.
TABLE-US-00004 TABLE 3 Receptor Activity Tested Compounds TRPM8
TRPA1 TRPV1 180 108.80% 68.02% 0.01% 773 141.07% 198.79% 92.07% 776
139.41% 112.99% 94.61% 777 139.37% 173.88% 60.5% 28 130.42% 242.50%
104.50% 30 109.70% 38.18% 16.24% WS5 100% -- -- Allyl
Isothiocyanate -- 100% -- (AITC) Capsaicin -- -- .sup. 100%
[0125] TABLE 3 showed the cell based receptor activity across the
three receptors (TRPM8, TRPA1, and TRPV1). Compound 28 had
surprisingly high activity across all three receptors, indicating
it could deliver a variety of sensations depending on the
concentration, when tested in vivo.
[0126] TABLE 3 shows the impact of each structure on the following
receptors: TRPM8 (cooling); TRPA1 (burning, numbing, tingling,
irritation); and TRPV1 (warming). The intensity of the described
test compound across three receptors (TRPM8, TRPA1, and TRPV1) was
compared to that of the control test compounds (WS5 for TRPM8,
Allyl Isothiocyanate for TRPA1, and Capsaicin for TRPV1). The
aminoethane moiety on compounds 773 and 777 directed the underlying
phenyl cyclohexanecarboxamide to shift from perceived cooling to
burning (high TRPA1 activity from 777) and warming (high TRPV1
activity from 773). Further, the potency of compound 28 relative to
other carboxamide structures is illustrated from TABLES 2 and 3.
For instance, at a screening level of 0.0000052% compound 28
delivered 130% of WS5 activity and WS5 was tested at 0.003%.
N-(4-cyanomethylphenyl)-p-menthanecarboxamide was tested at 0.001%
to get to 109% of WS5 activity. Therefore, at a single
concentration, compound 28 is 100X more mass efficient than the
next best coolant in class,
N-(4-cyanomethylphenyl)-p-menthanecarboxamide. The EC50 values, as
discussed later, show it is even more mass efficient as it is
diluted, since it retains high activity at very low use levels.
[0127] Compounds 28 and 30 show additional structures with the
aforementioned moieties 2-amino-propanamide adjacent to a phenyl
ring for cooling; and ethanolamine adjacent to a phenyl ring for
warming and/or burning sensations. When the aminoethane is off a
phenyl ring, the sensorial component of the molecule appeared to
diminish, as shown by the lack of sensation, cooling or warming,
from test compound 30. The aminoethane (test compound 777) on a
naphthalene moiety delivered a burning sensation. Compound 28 was
highly potent on activation of the TRPM8 receptor as shown in TABLE
3 and in the reported cooling sensations by the panelists.
Example 2
[0128] Sensory evaluation studies of coolant activity were
conducted using a methodology patterned after the techniques
described in M. C. Meilgaard, et al., Sensory Evaluation
Techniques, 4th Ed. (2007). Five panelists brushed with a
dentifrice for two minutes from TABLE 4 (SAMPLES A to G), SAMPLES C
to G containing the test compounds in TABLE 1 in a flavor
(peppermint) at 10 parts per million (ppm) and SAMPLE B containing
100 ppm of compound 180 (Cyclohexanecarboxamide,
N-[4-(cyanomethyl)phenyl]-5-methyl-2-(1-methylethyl)), as the
control coolant. After brush expectoration, panelists then rinsed
their mouth with 15 ml of an aqueous rinse and expectorated. As
shown in TABLE 5, panelists then evaluated cooling intensity,
assigning a number between 0 (no cooling) to 90.
[0129] The test compounds from TABLE 1 were placed into dentifrice
4C-4G, shown in TABLE 4 and rated for their intensity and duration
of cooling, as shown in TABLES 5 and 6. The scale was 0, which is
no cooling sensation, to 90, which is a sensation as cold as
ice.
TABLE-US-00005 TABLE 4 Dentifrice formulations containing the
compounds from TABLE 1 SAMPLES A Ingredient (Control) B C D E F G
FD&C Blue #1 0.045% 0.045% 0.045% 0.045% 0.045% 0.045% 0.045%
Color Solution Sodium Fluoride 0.243% 0.243% 0.243% 0.243% 0.243%
0.243% 0.243% CARBOMER 956 0.300% 0.300% 0.300% 0.300% 0.300%
0.300% 0.300% Sodium Saccharin 0.300% 0.300% 0.300% 0.300% 0.300%
0.300% 0.300% Sodium 0.419% 0.419% 0.419% 0.419% 0.419% 0.419%
0.419% Phosphate, Monobasic, Monohydrate Titanium Dioxide 0.525%
0.525% 0.525% 0.525% 0.525% 0.525% 0.525% Carboxymethycel- 0.800%
0.800% 0.800% 0.800% 0.800% 0.800% 0.800% lulose Sodium Peppermint
Flavor 1.000% 1.000% 1.000% 1.000% 1.000% 1.000% 1.000% Coolant 0%
-- -- -- -- -- -- 180 -- 0.01% -- -- -- -- -- Compound 773 -- --
0.001% -- -- -- -- Compound 776 -- -- -- 0.001% -- -- -- Compound
777 -- -- -- -- 0.001% -- -- Compound 28 -- -- -- -- -- 0.001% --
Compound 30 -- -- -- -- -- -- 0.001% Tribasic Sodium 1.100% 1.100%
1.100% 1.100% 1.100% 1.100% 1.100% Phosphate Dodecahydrate Sodium
Lauryl 4.000% 4.000% 4.000% 4.000% 4.000% 4.000% 4.000% Sulfate 28%
Solution Silica, Dental 15.000% 15.000% 15.000% 15.000% 15.000%
15.000% 15.000% Type, NF (Zeodent 119) SORBITOL 54.673% 54.673%
54.673% 54.673% 54.673% 54.673% 54.673% SOLUTION LRS USP Water
Purified, QS* QS* QS* QS* QS* QS* QS* USP, PhEur, JP, JSCI *QS
refers to the term quantum sufficit, meaning as much as suffices,
where the remainder of the formula hole is filled with this
substance.
TABLE-US-00006 TABLE 5 Panelists evaluated cooling properties
Initial 0 15 30 45 60 SAMPLE Time minutes min. min min. minutes C
(773) Sensory 15.0 12.5 9.2 9.2 10.8 D (776) measures 24.0 30.0
24.0 20.0 19 E (777) (0 = none, 20.0 23.8 13.8 8.8 7.5 B (180) 90 =
maximum) 31.0 31.0 25.0 13.0 5.0 F (28) 37.0 44.0 44.0 51.0 61.0 G
(30) 35.0 28.8 20.0 13.8 12.5 A (No Coolant) 27.5 22.5 11.3 6.3
1.3
TABLE-US-00007 TABLE 6 Panelists Sensory Observations SAMPLE Panel
(n = 5) B (180) 5 min delay in sensation Cool initially, then moves
into tingle/burn Sensation lasts for 3 h (concentration dependent)
C (773) 5 min delay in sensation Persistent warming sensation Lasts
for 1 h D (776) 5 min delay in sensation Initial tingle/warm, which
turned into a cooling sensation that moved to the back of the
throat Lasts for 2-3 h E (777) 5 min delay in sensation
Burning/tingle, almost hot sensation Lasts for 30 min. F (28).sup.
Cooling kicks in after 15 min. and lasts over 3 hours. The
sensation starts on the lips and front of the mouth and progresses
to the back of the throat. G (30) No cooling or other sensations
observed
[0130] Results shown in TABLES 5 and 6 showed that compound 28,
shown in TABLE 1, delivered intense long-lasting cooling even at a
low concentration of 10 parts per million, where compound 180 at
100 parts per million was less intense over 60 minutes. Compound 28
provided a cooling sensation from 15 minutes through the duration
of the test. Most panelists commented on the sensation lasting for
more than 3 hours. For compound 28, the intensity of the cooling
was amplified with the drinking of water, as panelists noticed that
it provided a burst of cooling sensation after the water had flowed
over the soft tissues of the gum and throat. Compound 776 also
displayed intense cooling that lasted for two to three hours, even
though it was not as intense as compound 28. Since both compounds
(776 and 28) were tested at 10 parts per million, their perceived
intensity would be expected to be higher and last longer as the
concentration is increased. Surprisingly, compounds 773 and 777
displayed a high degree of burning and/or warming, even though
their TRPM8 activity was greater than the comparative control
coolant molecule WS5. Compound 30 had no sensations, even though it
had TRPM8 activity greater than WS5.
[0131] An observation of the panelists was that certain flavor
types would bring out a burning/tingle sensation prior to the
cooling sensation. For instance, compound 28 was combined with a
Wintergreen flavor, it would display a throat burn/tingle for the
first half hour of use, in addition to the cool sensation. When
combined with a Peppermint flavor, it would be noted as
predominately cooling. These observations were consistent with an
amplifying effect of specific TRP receptors. The Wintergreen flavor
inherently contains two strong TRPA1 activators in Cinnamic
Aldehyde and Methyl Salicylate. Since compound 28 also has a potent
TRPA1 activation in addition to TRPM8, the additional A1 signals
would likely be synergistic in building a stronger A1 signal to the
brain, than without the flavor A1 components. Whereas, Peppermint
contains predominantly TRPM8 agonists, though with much lower TRPM8
EC50's than compound 28, and thus would be synergistic with a
cooling signal. Further, addition of calcium channel enhancers
(calcium soluble chelants), as exemplified in US Pub. No.
2010/0086498, may provide an additional boost to the perceived
cooling by amplifying the TRPM8 sensation. Such non-limiting
examples of coolant enhancers would be phytic acid, polyphosphates
with a chain length of greater than or equal to 3, carboxylate
polymers, such as Gantrez S-97, and polyols.
[0132] Cooling can be further enhanced by combining with select
TRPV1 warming agents. Non-limiting examples of TRPV1 warming agents
would be capsaicin, vanillyl butyl ether, vanillyl ethyl ether,
zingerone, and piperine. Other warming agents have previously been
described in U.S. Pat. No. 6,673,844.
[0133] Combinations of compound 28 with other TRPM8 coolants may
provide a quicker onset of cooling with a higher intensity than
either used alone. Combining compound 28 with another coolant would
allow for even less of compound 28 to be used while still providing
considerable (>3 hours) freshness longevity, which may be
perceived as a cooling sensation. Examples of coolant combinations
that could be used include WS23, menthane diols, menthyl
carboxamide derivatives, such as WS3, WS5,
N-(4-cyanomethylphenyl)-p-menthanecarboxamide, and WS 12.
Example 3
Isomer Characterization of Compound 28
[0134] Two fractions of compound 28, as discussed below, were
collected in gram quantities. The isomeric content of these two
compound 28 fractions was characterized by LC-UV-MS using a Waters
Acuity H Class, Ultra Performance Liquid Chromatograph (UPLC),
equipped with the Sample Manager, Quaternary Solvent Manager,
Tunable Ultraviolet (TUV) detector, and a QDa mass selective,
single-quadrupole mass analyzer (Waters Corporation, Milford, MA).
To prepare for analysis and characterization, a solid sample of
each fraction was weighed and dissolved at approximately 100 .mu.mL
in a solution consisting of 50% deionized water/50% methanol (MeOH,
HPLC grade from EMD Millipore Corporation, Billerica, Mass.) and
also containing 0.1% trifluoroacetic acid (TFA, Sigma Aldrich
Corporation, St. Louis, Mo.).
[0135] The separation of isomers contained within each fraction of
compound 28 was achieved with a 2.1 x 100 mm Acuity UPLC BEH Shield
RP18 column with 1.7 .mu.m particles (Waters Corporation, Milford,
Mass.). A mobile phase gradient was utilized with mobile phase (A)
consisting of water plus 0.1% TFA from Sigma Aldrich, and mobile
phase (B) consisting of MeOH from EMD. The mobile phase composition
was equilibrated prior to injection at 75% (A)/25% (B) and,
following a 5 .mu.l sample injection, the mobile phase composition
was ramped linearly to 100% (B) at 10 minutes. 100% of mobile phase
(B) was held for 3 minutes before ramping back to the original
conditions in 2 minutes. A mobile phase flow rate of 0.4 mL/minute
was maintained throughout. UV traces were obtained by monitoring
detector absorbance at 215 nm. QDa positive ion mass spectra of the
peaks in the UV traces shown within FIGS. 1 to 3 displayed intense
protonated molecular ions at m/z 374, as expected, given the
structure of compound 28, and indicating the components highlighted
within FIGS. 1 to 3 are isomeric species of compound 28.
[0136] UV analysis of compound 28 -fractions 1 and 2 are shown in
FIG's 1 and 2, respectively, indicating excellent retention time
repeatability and a very good separation of the isomers found
within these mixtures. FIG. 3 provides a UV overlay of a
representative analysis from fraction 1 and fraction 2,
highlighting the differences in isomeric composition for these two
fractions of compound 28.
[0137] FIG. 3 shows the HPLC of the isomers of compound 28. The
fraction labeled fraction 1, collected at 7.4 to 7.5 minutes
corresponds to the main isomer and lesser isomers that deliver the
intense cooling and a low EC50 as determined from the TRPM8
activity as shown in TABLES 7, 8 and 9 below. The fraction labeled
fraction 2, collected from 7.20 to 7.38 minutes corresponded to the
isomers of compound 28 with much lower TRPM8 values, which did not
provide a cooling response at the dose tested as shown in TABLES 7,
8 and 9.
[0138] TRPM8 activation was determined by measuring intracellular
calcium ion (Ca.sup.2+) level from transfected cells with the TRPM8
receptor gene, as described in EXAMPLE 1, the results of which are
shown in TABLES 7 and 8.
TABLE-US-00008 TABLE 7 TRPM8 Time Course Activity of compound 28 50
sec % 3 min % 5 min % 10 min % Sample Dose 50 sec of WS5 3 min of
WS5 5 min of WS5 10 min of WS5 Assay Buffer na 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 WS-5 30 uM 10059.7 100.0 9449.3 100.0 9468.0 100.0
9576.0 100.0 Compound 28 fraction 1 100 uM 12646.0 125.7 12520.0
132.5 12844.0 135.7 13187.0 137.7 50 uM 12419.0 123.5 12295.0 130.1
12654.0 133.7 13169.0 137.5 25 uM 13046.0 129.7 13020.0 137.8
13354.0 141.0 14341.0 149.8 12.5 uM 12430.0 123.6 12591.0 133.3
12997.0 137.3 13947.0 145.6 6.25 uM 12229.0 121.6 12775.0 135.2
13098.0 138.3 14102.0 147.3 3.125 uM 11637.0 115.7 12602.0 133.4
12939.0 136.7 13850.0 144.6 1.563 uM 11114.0 110.5 12135.0 128.4
12499.0 132.0 13440.0 140.4 781 nM 9786.0 97.3 12182.0 128.9
12618.0 133.3 13661.0 142.7 390 nM 7592.0 75.5 11373.0 120.4
11968.0 126.4 13121.0 137.0 195 nM 5418.0 53.9 11037.0 116.8
11824.0 124.9 13046.0 136.2 97.6 nM 3963.0 39.4 9744.0 103.1
10711.0 113.1 12011.0 125.4 48.8 nM 2916.0 29.0 8017.0 84.8 8983.0
94.9 10224.0 106.8 24.4 nM 1936.0 19.2 6405.0 67.8 7593.0 80.2
8879.0 92.7 12.2 nM 3018.0 30.0 8783.0 93.0 9830.0 103.8 11065.0
115.5 6.1 nM 884.0 8.8 3452.0 36.5 4426.0 46.7 5375.0 56.1
TABLE-US-00009 TABLE 8 TRPM8 Time Course Activity of Isomer of
compound 28 50 sec % 3 min % 5 min % 10 min % Sample Dose 50 sec of
WS5 3 min of WS5 5 min of WS5 10 min of WS5 Assay Buffer na 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 WS-5 30 uM 10059.7 100.0 9449.3 100.0
9468.0 100.0 9576.0 100.0 Compound 28 fraction 2 100 uM 12246.0
121.7 11826.0 125.2 12074.0 127.5 12287.0 128.3 50 uM 12648.0 125.7
12352.0 130.7 12644.0 133.5 13179.0 137.6 25 uM 12110.0 120.4
11993.0 126.9 12284.0 129.7 13140.0 137.2 12.5 uM 12415.0 123.4
12501.0 132.3 12801.0 135.2 13755.0 143.6 6.25 uM 12236.0 121.6
12644.0 133.8 12891.0 136.2 13793.0 144.0 3.125 uM 11757.0 116.9
12699.0 134.4 12937.0 136.6 13890.0 145.1 1.563 uM 11331.0 112.6
12663.0 134.0 12954.0 136.8 13892.0 145.1 781 nM 10428.0 103.7
12887.0 136.4 13091.0 138.3 14116.0 147.4 390 nM 8766.0 87.1
11955.0 126.5 12301.0 129.9 13026.0 136.0 195 nM 7287.0 72.4
11477.0 121.5 12007.0 126.8 12427.0 129.8 97.6 nM 5007.0 49.8
10101.0 106.9 10747.0 113.5 11375.0 118.8 48.8 nM 2502.0 24.9
7721.0 81.7 8488.0 89.6 9289.0 97.0 24.4 nM 2311.0 23.0 6441.0 68.2
7226.0 76.3 7848.0 82.0 12.2 nM 1814.0 18.0 5446.0 57.6 6224.0 65.7
6809.0 71.1 6.1 nM 1944.0 19.3 4350.0 46.0 4763.0 50.3 4844.0
50.6
[0139] The TRPM8 data shown in TABLES 7 and 8, where TABLE 7
corresponds to fraction 1 and TABLE 8 corresponds to fraction 2,
compares the dose response of the two HPLC separations, of the
isomers (fraction 1, fraction 2) of compound 28. As shown in TABLES
7 and 8, both fractions activate TRPM8 rapidly at 781 nM of each.
However, fraction 1 continued to activate at lower and lower doses
compared to fraction 2. Fraction 1 was 103.8% of the control at 5
minutes of activation from a 12.2 nM dose; whereas, fraction 2 at
the same time point and dose was 65.7% of the control. At 10
minutes of activation, the 12.2 nM dose was 115.5% of the control
for fraction 1 and 71.1% of the control for fraction 2. These
differences in isomers were further illustrated in the EC50 values
as shown in TABLE 9 below.
TABLE-US-00010 TABLE 9 EC50 Calculation of Isomer fractions EC50 in
TRPM8 (.mu.M) 50 sec 3 min 5 min 10 min fraction 2 0.1972 0.05548
0.04567 0.0374 fraction 1 0.3306 0.001476 ~5.235E-008
~1.148E-007
[0140] In an oral application of a compound of the present
invention, such as from a dentifrice, lozenge, floss, chewing gum,
or white strip, when compound 28 is split into isomers or combined,
the levels of use may be from about 10% to about 70% of fraction 1
and about 10% to about 70% of fraction 2 or from about 30% to about
60% of fraction 1 and about 30% to about 60% of fraction 2. When
compound 28, either isomer or combined isomers, is combined with a
TRPA1 agonist, TRPV1 agonist, or both, the level of use of a TRPA1
or TRPV1 agonist would be in the range of about 0.001% to about
0.5% or from about 0.01% to about 0.2% by weight of the composition
of either the TRPA1 or TRPV1 agonists, where both TRPA1 agonists
and/or TRPV1 agonists may be added separately or simultaneously to
the composition containing compound 28. When another TRPM8 agonist,
in addition to compound 28, is used, the level of use of the
additional TRPM8 agonist may be from about 0.001% to about 0.5% or
from about 0.005% to about 0.3% by weight of the composition. If a
TRPM8 enhancer is used, in addition to compound 28, it may be added
in a range of from about 0.001% to about 0.2% or from about 0.005%
to about 0.1% by weight of the composition. Compositions of the
present invention may contain multiple TRPA1 and TRPV1 agonists in
the ranges disclosed above to deliver the enhanced sensorial signal
from compound 28.
[0141] In a topical application of a compound of the present
invention, for example in shampoos and lotions, when compound 28 is
split into isomers or combined, the levels of use may be from about
10% to about 70% of fraction 1 and about 10% to about 70% of
fraction 2 or from about 30% to about 60% of fraction 1 and about
30% to about 60% of fraction 2. When compound 28, either isomer or
combined isomers, is combined with a TRPA1 and/or a TRPV1 agonist,
the level of use of a TRPA1 or TRPV1 agonist may be in the range of
from about 0.001% to about 0.5% or from about 0.01% to about 0.2%
by weight of the composition of either of the TRPA1 or TRPV1
agonists, where both TRPA1 agonists and TRPV1 agonists may be added
separately or simultaneously to the composition containing compound
28. When another TRPM8 agonist is used, in addition to compound 28,
the level of use of the additional TRPM8 agonist may be from about
0.001% to about 0.5% or from about 0.005% to about 0.3% by weight
of the composition. If a TRPM8 enhancer is used, in addition to
compound 28, it may be used in levels of from about 0.001% to about
0.2% or from about 0.005% to about 0.1% by weight of the
composition. The compositions may contain multiple TRPA1 and TRPV1
agonists in the ranges stated to deliver the enhanced sensorial
signal from compound 28.
Example 4
Compound 28 Solubility
TABLE-US-00011 [0142] TABLE 10 Solubility Parameter Calculation
Hydrogen Total Solubility Dispersion Polarity bonding Parameter
Compound (MPa){circumflex over ( )}0.5 (MPa){circumflex over (
)}0.5 (MPa){circumflex over ( )}0.5 (MPa){circumflex over ( )}0.5
Compound 28 17.8 5.6 9.0 20.7
[0143] TABLE 10 outlines the Hansen solubility parameters for
compound 28 and its isomers (fraction 1, fraction 2), as outlined
previously. These parameters help to identify which solvents would
be a good candidate for making stock solutions of >5%. Due to
the low level of use in oral care products, a typical stock
solution of .about.1% would be sufficient to deliver 1 to 10 ppm.
Solvents for the higher (>5%) would utilize Hansen's sphere
calculations and a sphere radius in the range of 5-6 would
sufficiently identify solvents for the higher stock solutions. In
the range of oral care use of stock solutions in the 1-5% range,
solvents such as ethanol, menthol, carvone, anethol, benzyl
alcohol, and the polyols commonly used in oral care products could
be used to make a stock solution.
Example 5
Mouthwash Isomer Cooling
[0144] Mouthwashes were prepared, using conventional methods, which
contained either fraction 1 or fraction 2 of compound 28 and
provided to panelists to assess their cooling properties. The
panelists swished the mouthwash in their mouths for 1 minute prior
to expectoration. After expectoration, the time point of zero was
started and time after rinse was counted from there until 12 hours.
The panelists then rated the perceived refreshing experience as
experienced by breathing in and noting the cool/refreshing
sensation on the interior of the mouth and across the lips. The
self-rated scale was from zero (no cooling) to 100 (maximum
cooling).
TABLE-US-00012 TABLE 11 Mouthwash Formulation Ingredients Control
Sample A Sample B Sample C Cetylpyridinium 0.074% 0.074% 0.074%
0.074% Chloride USP Compound 28 0 0.00005% 0 0.0001% fraction 1
Compound 28 0 0.00005% 0.0001% 0 fraction 2 Superol Vegetable 5% 5%
5% 5% 99.7% Glycerine USP/FCC Poloxamer 407 0.06% 0.06% 0.06% 0.06%
Sucralose NF 0.015% 0.015% 0.015% 0.015% Saccharin Sodium 0.01%
0.01% 0.01% 0.01% USP Granular, High Moist Methyl Paraben 0.02%
0.02% 0.02% 0.02% Propyl Paraben 0.005% 0.005% 0.005% 0.005%
Peppermint Flavor 0.1% 0.1% 0.1% 0.1% Purified Water USP QS QS QS
QS (Bottled) *QS refers to the term quantum sufficit, meaning as
much as suffices, where the remainder of the formula hole is filled
with this substance.
TABLE-US-00013 TABLE 12 Results from panel testing of mouthwash
with coolant fractions Cool burn/thermal diffusion Hours after use
attributes Rinse 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h 9 h Sample A 53 63
60 47 43 40 40 37 27 Sample B 27 10 3 0 0 0 0 0 0 Sample C 15 30 50
60 60 50 45 45 40 Control 5 0 0 0 0 0 0 0 0
[0145] The results in Table 12 showed that the mixture of fraction
1 and 2 of compound 28 (Sample A) delivered higher intensity of
cooling during the first two hours after use compared with either
fraction 1 (Sample C) or fraction 2 (Sample B) alone. The
differences in cooling between fraction 1 (Sample C) and fraction 2
(Sample B) showed that the isomer (fraction 1) of compound 28 as
depicted in FIG. 1 by the peak at 7.4 minutes was the isomer
(fraction 1) that provided long lasting cooling properties. The
isomer (fraction 2) corresponding to the peak from 7.20 to 7.38
minutes in FIG. 2 did not provide a strong nor long lasting cooling
sensation. It was described as more of a burn/tingle than cool and
the sensation trailed off in intensity between 1 and 2 hours. It
does appear that there was synergy between fraction 1 and 2 where
the non-cooling fraction 2 lifted the intensity of fraction 1. The
intensity and duration of cooling for fraction 1 was unlike any
previously tested coolant in that only 1 ppm of total coolant
(compound 28 fraction 1 and 2) was able to deliver a cooling
effect, either singly (fraction 1 or 2) or as a mixture of isomers
(fraction 1 and 2) where even the most powerful commercially
available coolant would take at least 15 ppm to deliver a similar
initial intensity, but would not be able to match the duration of
compound 28.
Example 6
Chewing Gum Cooling
Chewing Gum Preparation
[0146] The chewing gum formulations shown in TABLE 13 were prepared
by melting chewing gum base in a microwave in 20 second increments
until softened. Magnesium stearate was spread on a piece of wax
paper and the softened chewing gum base was placed on the magnesium
stearate coated portion of the wax paper. The gum base was coated
with the magnesium stearate and kneaded with gloved hands until
soft. The gum base was pressed to 1/4 inch thick slabs; powdered
sweetener and spray dried powdered flavor were added to the center
of the surface of the gum. Added liquid flavor and two drops of
colorant on top of the powdered sweetener and spray dried powdered
flavor, letting the liquid flavor and colorant absorb into the
spray dried powdered flavor and powdered sweetener, allowing mixing
it into the gum base without losing any materials. Microwaved as
needed to keep the gum base pliable. Sprinkled magnesium stearate
and powdered sweetener (1.2 grams) onto the wax paper to be able to
roll out without the gum base sticking and also to coat the outside
with a small amount of sweetener. The gum base was set onto the
magnesium stearate and powdered sweetener. Coated a stainless steel
beaker in magnesium stearate and used it to press/roll out the
outside of the gum base for initial sweetness. Used a molded press
to cut the gum base into long strips and cut again in the
perpendicular direction to get squares of .about.1.0 grams each.
For the batch that contains the compound 28 fraction 1, the
compound 28 fraction 1 was added to the liquid flavor and prepared
as described above to give a finished concentration of 1 ppm.
TABLE-US-00014 TABLE 13 Gum Formulation Ingredients Control (%)
Coolant Formulation Compound 28 fraction 1 0.0 0.0001 Spearmint
Flavor Liquid 3.992% .sup. 3.992% .sup. Spearmint spray dried
flavor 8% 8% Sucralose 1% 1% Chewing gum base QS to 25 grams QS to
25 grams Coating of finished gum 1.245 g Xylitol/ 1.245 g Xylitol/
stearate per 1 stearate per 1 gram cube of gum gram cube of gum
[0147] TABLE 14 shows the results of panelists sampling chewing gum
controls and chewing gum containing compound 28 fraction 1.
Panelists chewed the gum for 30 minutes and rated the in use
chewing attributes of the gum. Those attributes were sweetness,
flavor intensity, cooling, bitterness, and perception of freshness.
The panelists rated these attributes on a 0 (no sensation of the
attribute) to 100 (maximum sensation of the attribute). The data
was reported on a gum without the coolant (control) compared to a
gum containing compound 28 fraction 1.
TABLE-US-00015 TABLE 14 Chewing gum panel in use attributes During
Chewing Attributes Sweet- Flavor Bitter- Perception ness Intensity
Cooling ness of Freshness Control Gum 43.3 33.3 30.0 6.7 30.0 Gum
with 56.7 36.7 36.7 6.7 33.3 compound 28 fraction 1
[0148] The gum containing compound 28 fraction 1 showed
improvements in the in use sweetness profile, as shown in TABLE
14.
[0149] After the panelists stopped chewing the gum as described
above, they continued to rate the perception of freshness of their
breath and mouth, as shown in TABLE 15. The panelists monitored the
perception of freshness delivered from the gum after use by
breathing in and noting the cool sensation, along with the
perception of taste, and the overall perceived feeling in their
mouth and on their lips. They rated from 0 (no perception of
freshness) to 100 (maximum perception of freshness) over the course
of 4 hours, rating at each hour. The data is reported in Table 15
below.
TABLE-US-00016 TABLE 15 After Chewing Attributes After Use
Perception of Freshness 1 h 2 h 3 h 4 h Control Gum 6.7 0.0 0.0 0.0
Gum with compound 28 fraction 1 33.3 33.3 30.0 26.7
[0150] The data reported in TABLE 15 showed that the gum containing
compound 28 fraction 1 delivered a long lasting perception of
freshness, as compared to the control gum.
Example 7
[0151] TABLES 16 and 17 show shave prep compositions. The water
soluble polymers (poly-ethylene oxide, hydroxyethylcellulose) were
added to water and mixed by stirring until the polymers were
completely dissolved (about 30 min.). The aqueous mixture was then
heated and the glyceryl oleate, sorbitol and fatty acids added at
about 60.degree. C. and mixed by stirring well while the heating
continues. At 80-85.degree. C. the triethanolamine was added and
mixed for about 20 minutes to form the aqueous soap phase. After
cooling the aqueous soap phase to room temperature
(.about.25.degree. C.), the remaining components (i.e., Lubrajel,
glycerin, fragrance, colorant, botanicals) were added to the
aqueous soap phase and mixed by stirring well to form the gel
concentrate. Water was added if required to bring the batch weight
to 100%, thereby compensating for any water loss due to
evaporation. The concentrate was then combined with the volatile
post-foaming agent under pressure within the filling line and
filled into bottom-gassed aerosol cans with shearing through the
valve under nitrogen pressure.
TABLE-US-00017 TABLE 16 Shave Prep Compositions Samples Ingredients
1 2 3 4 Sorbitol 70% 0.97% 0.97% 0.97% 0.97% Solution Glycerin
0.49% 0.49% 0.49% 0.49% Water QS QS QS QS Hydroxyethyl 0.49% 0.49%
0.49% 0.49% cellulose.sup.1 PEG-90M.sup.2 0.06% 0.06% 0.06% 0.06%
PEG-23M.sup.3 0.05% 0.05% 0.05% 0.05% PTFE 0.15% 0.15% 0.15% 0.15%
Palmitic acid 7.53% 7.53% 7.53% 7.53% Stearic acid 2.53% 2.53%
2.53% 2.53% Glyceryl Oleate 1.94% 1.94% 1.94% 1.94% Triethanolamine
5.88% 5.88% 5.88% 5.88% (99%) Lubrajel Oil.sup.4 0.4% 0.4% 0.4%
0.4% Menthol 0.15% 0.15% 0.15% 0.15% Fragrance 0.87% 0.87% 0.87%
0.87% Other (e.g. Vit E, 0.10% 0.10% 0.10% 0.10% Aloe, etc.)
Compound 776 -- 0.0001% -- -- Compound 28 -- -- 0.0001% 0.1% Dye
0.10% 0.10% 0.10% 0.10% Isopentane (and) 2.85% 2.85% 2.85% 2.85%
Isobutane .sup.1Available as Natrosol 250 HHR from Hercules Inc.,
Wilmington, DE .sup.2Available as Polyox WSR-301 from Amerchol
Corp., Piscataway, NJ .sup.3Available as Polyox WSR N-1 2K from
Amerchol Corp., Piscataway, NJ .sup.4Available as Microslip 519
from Micro Powders Inc., Tarrytown, NY .sup.4Available from
Guardian Laboratories, Hauppauge, NY *QS refers to the term quantum
sufficit, meaning as much as suffices, where the remainder of the
formula hole is filled with this substance
[0152] The pre-shave prep samples shown in TABLE 17 were made by
weighing out the water in a vessel sufficient to hold the entire
batch. Inserted an overhead mixer with impeller into the vessel and
increase agitation to create a vortex. Pre-blended the thickener
and polymer powders (Polyox and HEC). Sprinkled the polymer blend
into the vortex until incorporated. Heated batch to 70.degree. C.
to hydrate the polymers. Added the liquid dispersion polymer (e.g.,
Sepigel) to the batch and mixed until uniform and hydrated,
increasing rpms to maintain good mixing. Added the surfactant
(e.g., Brig 35) and mixed until uniform and dispersed. Cooled batch
to below 45.degree. C. Once below 45.degree. C., added the perfume,
preservatives and other temperature-sensitive additives. Cooled to
below 35.degree. C. and QS with water.
TABLE-US-00018 TABLE 17 Pre-Shave Prep Samples Ingredients 1 2 3 3
Water QS QS QS QS Sepigel 305 (Polyacrylamide & 0.50% 0.50%
0.50% 0.50% C13-C14 Isoparaffin & Laureth-7) Polyox N13K
(PEG-23M) 0.50% 0.50% 0.50% 0.50% Natrosol 250 HHR (HEC) 0.80%
0.80% 0.80% 0.80% Glycerin 99.7% USP/Fcc 5.0% 5.0% 5.0% 5.0% Brij
35 (Laureth 23) 2.0% 2.0% 2.0% 2.0% Disodium EDTA 0.10% 0.10% 0.10%
0.10% Perfume 0.15% 0.15% 0.15% 0.15% Glydant Plus 0.20% 0.20%
0.20% 0.20% Menthol 0.04% 0.04% 0.04% 0.04% Compound 776 0.000001%
0.000001% 0.000001% 0.1% Compound 28 0.000001% 0.000001% 0.000001%
0.1%
Example 8
[0153] Shampoo compositions, as shown below in TABLE 18, were
prepared using conventional methods.
TABLE-US-00019 TABLE 18 Samples Ingredients A B C D E F G H Sodium
Laureth Sulfate (SLE.sub.3S) 6 6 6 Sodium Laureth Sulfate
(SLE.sub.1S) 10.5 10.5 12 12 12 Sodium Lauryl Sulfate (SLS) 1.5 1.5
7 7 7 Cocamidopropyl Betaine 1 1.25 1.5 1.5 1.5 1 1 1 Cocamide MEA
1 1.5 1.5 1.5 1.5 Glycol Distearate 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5
Zinc Pyrithione 1 1 1 1 1 1 1 1 Zinc Carbonate 1.61 1.61 1.61 1.61
1.61 1.61 1.61 1.61 Menthol 0.45 0.45 0.45 0.45 0.45 0.45 0.45
Compound 28 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 Fragrance 0.7
0.7 0.7 0.7 0.7 0.7 0.7 0.7 Guar Hyrdroxypropyltrimonium 0.3 0.3
0.3 0.3 0.3 0.23 0.23 0.23 Chloride (LMW) Polyquaternium-10 0.1 0.1
0.1 (HMW) Polyquaternium 76 (AM:Triquat) 0.01 0.01 0.01 0.01
Stearyl Alcohol 1.29 Cetyl Alcohol 0.71 Dimethicone 1.7 0.8 0.8 0.8
1.7 0.8 0.8 0.8 Hydrochloric acid QS QS QS QS QS QS QS QS
Preservative 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Sodium
Chloride QS QS QS QS QS QS QS QS Sodium Xylene Sulfonate QS QS QS
QS QS QS QS QS Sodium Benzoate (22) 0.27 0.27 0.27 0.27 0.27 0.27
0.27 0.27 Water and Minors (QS to 100%) (23) QS QS QS QS QS QS QS
QS
[0154] Conditioner compositions, as shown below in TABLES 19-23,
were prepared using conventional methods.
Definitions of Components used in TABLES 19-22 [0155] 1.
Polyquaternium-6:Poly(diallyldimethylammonium chloride) supplied
with a tradename Merquat 100 from Lubrizol, having a charge density
of about 6.2 meq/g, and molecular weight of about 150,000 g/mol
[0156] 2. Polyquaternium-6:Poly(diallyldimethylammonium chloride)
supplied with a tradename Merquat 106 from Lubrizol having a charge
density of about 6.2 meq/g, and molecular weight of about 15,000
g/mol [0157] 3. Zinc pyrithione: having a particle size of from
about 1 to about 10 microns [0158] 4. Zinc carbonate: having a
particle size of from about 1 to about 10 microns [0159] 5.
Aminosilicone: Terminal aminosilicone which is available from
Momentive Performance Materials having a viscosity of about 10,000
mPas, and having following formula:
[0159]
(R.sub.1).sub.aG.sub.3-a--Si--(--OSiG.sub.2).sub.n--O--SiG.sub.3--
a(R.sub.1).sub.a
wherein G is methyl; a is an integer of 1; n is a number from 400
to about 600; R.sub.1 is a monovalent radical conforming to the
general formula C.sub.qH.sub.2qL, wherein q is an integer of 3 and
L is --NH.sub.2.
TABLE-US-00020 TABLE 19 Samples Ingredients 1 2 3 4 5
Polyquaternium-6 .sup.1 0.075 -- -- -- 0.075 Polyquaternium-6
.sup.2 0.075 0.075 0.075 Zinc pyrithione .sup.3 0.75 0.75 0.75 0.75
0.75 Zinc carbonate .sup.4 1.6 1.6 1.6 1.6 1.6 Behenyl trimethyl --
-- -- 2.5 ammonium chloride Behenyl trimethyl 2.6 2.6 1.2 -- 2
ammonium methosulfate Dicetyl dimethyl -- -- 0.35 -- -- ammonium
chloride Cetyl alcohol 1 1 1 1 1.4 Stearyl alcohol 2.4 2.4 2.3 2.3
3.4 Aminosilicone .sup.5 0.5 0.5 0.5 0.5 2 Preservatives 0.9 0.9
0.9 0.9 0.9 Compound 28 0 0 0 0 0 Perfume 0.5 0.5 0.5 0.5 0.5
Deionized Water q.s. to q.s. to q.s. to q.s. to q.s. to 100% 100%
100% 100% 100%
TABLE-US-00021 TABLE 20 Samples Ingredients 6 7 8 9 10
Polyquaternium-6 .sup.1 0.075 -- -- -- 0.075 Polyquaternium-6
.sup.2 0.075 0.075 0.075 Zinc pyrithione .sup.3 0.75 0.75 0.75 0.75
0.75 Zinc carbonate .sup.4 1.6 1.6 1.6 1.6 1.6 Behenyl trimethyl --
-- -- 2.5 ammonium chloride Behenyl trimethyl 2.6 2.6 1.2 -- 2
ammonium methosulfate Dicetyl dimethyl -- -- 0.35 -- -- ammonium
chloride Cetyl alcohol 1 1 1 1 1.4 Stearyl alcohol 2.4 2.4 2.3 2.3
3.4 Aminosilicone .sup.5 0.5 0.5 0.5 0.5 2 Preservatives 0.9 0.9
0.9 0.9 0.9 Compound 28 0.09 0.09 0.09 0.09 0.09 Perfume 0.5 0.5
0.5 0.5 0.5 Deionized Water q.s. to q.s. to q.s. to q.s. to q.s. to
100% 100% 100% 100% 100%
TABLE-US-00022 TABLE 21 Samples Ingredients 11 12 13 14 15
Polyquaternium-6 .sup.1 0.075 -- -- -- 0.075 Polyquaternium-6
.sup.2 0.075 0.075 0.075 Zinc pyrithione .sup.3 0.75 0.75 0.75 0.75
0.75 Zinc carbonate .sup.4 -- -- -- -- -- Behenyl trimethyl -- --
-- 2.5 ammonium chloride Behenyl trimethyl 2.6 2.6 1.2 -- 2
ammonium methosulfate Dicetyl dimethyl -- -- 0.35 -- -- ammonium
chloride Cetyl alcohol 1 1 1 1 1.4 Stearyl alcohol 2.4 2.4 2.3 2.3
3.4 Aminosilicone .sup.5 0.5 0.5 0.5 0.5 2 Preservatives 0.9 0.9
0.9 0.9 0.9 Compound 28 0 0 0 0 0 Perfume 0.5 0.5 0.5 0.5 0.5
Deionized Water q.s. to q.s. to q.s. to q.s. to q.s. to 100% 100%
100% 100% 100%
TABLE-US-00023 TABLE 22 Samples Ingredients 16 17 18 19 20
Polyquaternium-6 .sup.1 0.075 -- -- -- 0.075 Polyquaternium-6
.sup.2 0.075 0.075 0.075 Zinc pyrithione .sup.3 0.75 0.75 0.75 0.75
0.75 Zinc carbonate .sup.4 -- -- -- -- -- Behenyl trimethyl -- --
-- 2.5 ammonium chloride Behenyl trimethyl 2.6 2.6 1.2 -- 2
ammonium methosulfate Dicetyl dimethyl -- -- 0.35 -- -- ammonium
chloride Cetyl alcohol 1 1 1 1 1.4 Stearyl alcohol 2.4 2.4 2.3 2.3
3.4 Aminosilicone .sup.5 0.5 0.5 0.5 0.5 2 Preservatives 0.9 0.9
0.9 0.9 0.9 Compound 28 0.09 0.09 0.09 0.09 0.09 Perfume 0.5 0.5
0.5 0.5 0.5 Deionized Water q.s. to q.s. to q.s. to q.s. to q.s. to
100% 100% 100% 100% 100%
[0160] Samples 6-10, in TABLE 20, provided a cooling sensation to
consumers in comparison to Samples 1-5, in TABLE 19. In Samples
16-20, TABLE 22, there was a cooling sensation to consumers in
comparison to Samples 11-15, in TABLE 21.
TABLE-US-00024 TABLE 23 Samples Ingredients 21 22 23 24 25 26
Behenyl trimethyl ammonium chloride 2.25 2.25 Isopropyl alcohol 0.6
0.5 0.5 0.6 0.5 0.5 Behentrimonium methosulfate 1.8 1.8 1.8 1.8
Cetyl alcohol 1.9 1.1 1.1 1.9 1.1 1.1 Stearyl alcohol 4.6 2.8 2.8
4.6 2.8 2.8 Preservatives 0.9 0.9 0.9 0.9 0.9 0.9
Aminosilicone.sup.1 2.8 1.3 0.35 2.8 1.3 0.35 Compound 28 0.09 0.09
0.09 Perfume 0.5 0.5 0.5 0.5 0.5 0.5 Deionized Water q.s. to q.s.
to q.s. to q.s. to q.s. to q.s. to 100% 100% 100% 100% 100%
100%
[0161] Leave on hair compositions, as shown below in TABLE 24, were
prepared using conventional methods.
TABLE-US-00025 TABLE 24 Samples 1 2 3 4 5 6 7 Active Active Active
Active Active Active Active Ingredients wt %) wt % wt % wt % wt %
wt % wt % Water Q.S. QS QS QS QS QS QS Alcohol 100% (Ethanol) 50 50
0 50 60 25 0 Isoproryl Alcohol 0 0 0 0 0 0 0 Acrylates/C10-30 alkyl
acrylate 0.35 0.5 0.2 0 0 0 0 crosspolymer *1 Carbomer *2 0 0 0 0 0
0 0 Polyacrylamide *3 0 0 0 0.5 0 0 0 C13-14 Isoparaffin *3 0 0 0
0.5 0 0 0 Laureth 7 *3 0 0 0 0.1 0 0 0 Polyacrylate crosspolymer-6
*4 0 0 0 0 0.5 0 0 Dehydroxanthan Gum *5 0 0 0 0.25 0 0 0 Cetyl
Alcohol, Sodium 0 0 0 0 0 2.5 0 Polyacrylate, Glyceryl Stearate,
Polysorbate 80, and Caprylic/Capric Triglycerinde *6
Acrylates/Aminoacrylates/C10- 0 0 0 0 0 0 1.5 30 Alkyl PEG-20
Itaconate Copolymer *7 Zinc pyrithione *8a 0.1 0.2 0.07 0.1 0.1 0.1
0.1 Zinc Carbonate *8b 0 0.2 0 0 0 0 0 PEG/PPG 20/23 Dimethicone 0
1 0 0 0 0 0 430 *9 Bis-PEG/PPG-16/16 PEG/PPG 0.7 0 0 1 0 0 0 16/16
Dimethicone *10 Polyquaternium-4 *111 0 0 0 0 0 0 1 Panthenol 0.15
0.5 0 0.15 0 0 0 Niacinamide 2.5 0 0 3 0 0 0 Caffeine 0.75 0 0 1.25
0 0 0 Glycerin 0.5 5 0 5 0 0 0 Argania Spinosa Kernel Oil 0 0 0 0 0
0 0 *12 Propylene Glycol 0 0 1 0 0 0 0 Menthol 0 0 0.5 0 0 0 0
Polyvinylpyrrolidone *13 0 1 0 0 0 0 0 Polyethylene Low Density 0
0.5 0 0 0 0 0 Powder *14 Tapicoa Starch 0 0 1 0 0 0 0
Polymethylsilsesuioxane *15 Benzyl Alcohol 0 0 0.5 0 0 0 0
Methylisothiazolinone *16 0 0 0.05 0 0 0 0 PEG-40 Hydrogenated
Castor 0 0 0.5 0 0 0 0 Oil *17 Tetrahydroxypropyl 0.12 0 0.14 0 0 0
0 Ethylenediamine *18 Triethanolamine *19 0 0.1 0 0 0 0 0 Glycolic
Acid *20 0 0 0 0 0 0 0.25 Citric Acid 0 0 0 0.008 0.005 0.005 0.005
Compound 28 0.09 0.09 0.09 0.09 0.09 0.09 0.09 *1 as in Carbopol
Ultrez 21 available from Lubrizol (Wickliffe, OH) *2 as in Carbopol
Ultrez 30 available from Lubrizol *3 as in Sepigel 305 from Seppic
(Puteaux, France) *4 as in SepiMax Zen from Seppic *5 as in Amaze
XT from AkzoNovel (Amsterdam, Netherlands) *6 as in Jeesperse
CPW-CG-02 from Jeen (Fairfield, New Jersey) *7 as in Structure Plus
from Akzo Nobel *8a as in ZPT from Lonza Personal Care (Basel,
Switzerland) *8b as in Zinc Carbonate from Brueggemann Chemical
(Newtown, PA) *9 as in Silsoft 430 Dimethicone Copolyol from
Momentive (Waterford, NY) *10 as in Abil Care 85 from Evonik
(Zurich, Switzerland) *11 as in Celquat H-100 from Akzo Nobel *12
as in Lipofructyl Argan LS9779 from BASF (Ludwigshafen, Germany)
*13 as in PVP K-30 from ISP Technologies (Wayne, New Jersey) *14 as
in Microthene FN 510-00 from Equistar Chemicals (Houston, TX) *15
as in Dry Flo TS from Akzo Nobel *16 as in Neolone 950 from Rohm
and Haas (Philadelphia, PA) *17 as in Cremophor RH-40 Surfactant
from BASF *18 as in Neutrol Te from BASF *19 as in Trolamine from
Dow Chemical (Houston, TX) *20 as in Glypure from DuPont
(Wilmington, DE)
[0162] The dimensions and values disclosed herein are not to be
understood as being strictly limited to the exact numerical values
recited. Instead, unless otherwise specified, each such dimension
is intended to mean both the recited value and a functionally
equivalent range surrounding that value. For example, a dimension
disclosed as "40 mm" is intended to mean "about 40 mm."
[0163] Every document cited herein, including any cross referenced
or related patent or application and any patent application or
patent to which this application claims priority or benefit
thereof, is hereby incorporated herein by reference in its entirety
unless expressly excluded or otherwise limited. The citation of any
document is not an admission that it is prior art with respect to
any invention disclosed or claimed herein or that it alone, or in
any combination with any other reference or references, teaches,
suggests or discloses any such invention. Further, to the extent
that any meaning or definition of a term in this document conflicts
with any meaning or definition of the same term in a document
incorporated by reference, the meaning or definition assigned to
that term in this document shall govern.
[0164] While particular embodiments of the present invention have
been illustrated and described, it would be obvious to those
skilled in the art that various other changes and modifications can
be made without departing from the spirit and scope of the
invention. It is therefore intended to cover in the appended claims
all such changes and modifications that are within the scope of
this invention.
Sequence CWU 1
1
312520DNAHomo sapiens 1atgaagaaat ggagcagcac agacttgggg gcagctgcgg
acccactcca aaaggacacc 60tgcccagacc ccctggatgg agaccctaac tccaggccac
ctccagccaa gccccagctc 120tccacggcca agagccgcac ccggctcttt
gggaagggtg actcggagga ggctttcccg 180gtggattgcc ctcacgagga
aggtgagctg gactcctgcc cgaccatcac agtcagccct 240gttatcacca
tccagaggcc aggagacggc cccaccggtg ccaggctgct gtcccaggac
300tctgtcgccg ccagcaccga gaagaccctc aggctctatg atcgcaggag
tatctttgaa 360gccgttgctc agaataactg ccaggatctg gagagcctgc
tgctcttcct gcagaagagc 420aagaagcacc tcacagacaa cgagttcaaa
gaccctgaga cagggaagac ctgtctgctg 480aaagccatgc tcaacctgca
cgacggacag aacaccacca tccccctgct cctggagatc 540gcgcggcaaa
cggacagcct gaaggagctt gtcaacgcca gctacacgga cagctactac
600aagggccaga cagcactgca catcgccatc gagagacgca acatggccct
ggtgaccctc 660ctggtggaga acggagcaga cgtccaggct gcggcccatg
gggacttctt taagaaaacc 720aaagggcggc ctggattcta cttcggtgaa
ctgcccctgt ccctggccgc gtgcaccaac 780cagctgggca tcgtgaagtt
cctgctgcag aactcctggc agacggccga catcagcgcc 840agggactcgg
tgggcaacac ggtgctgcac gccctggtgg aggtggccga caacacggcc
900gacaacacga agtttgtgac gagcatgtac aatgagattc tgatcctggg
ggccaaactg 960cacccgacgc tgaagctgga ggagctcacc aacaagaagg
gaatgatgcc gctggctctg 1020gcagctggga ccgggaagat cggggtcttg
gcctatattc tccagcggga gatccaggag 1080cccgagtgca ggcacctgtc
caggaagttc accgagtggg cctacgggcc cgtgcactcc 1140tcgctgtacg
acctgtcctg catcgacacc tgcgagaaga actcggtgct ggaggtgatc
1200gcctacagca gcagcgagac ccctaatcgc cacgacatgc tcttggtgga
gccgctgaac 1260cgactcctgc aggacaagtg ggacagattc gtcaagcgca
tcttctactt caacttcctg 1320gtctactgcc tgtacatgat catcttcacc
atggctgcct actacaggcc cgtggatggc 1380ttgcctccct ttaagatgga
aaaaactgga gactatttcc gagttactgg agagatcctg 1440tctgtgttag
gaggagtcta cttctttttc cgagggattc agtatttcct gcagaggcgg
1500ccgtcgatga agaccctgtt tgtggacagc tacagtgaga tgcttttctt
tctgcagtca 1560ctgttcatgc tggccaccgt ggtgctgtac ttcagccacc
tcaaggagta tgtggcttcc 1620atggtattct ccctggcctt gggctggacc
aacatgctct actacacccg cggtttccag 1680cagatgggca tctatgccgt
catgatagag aagatgatcc tgagagacct gtgccgtttc 1740atgtttgtct
acatcgtctt cttgttcggg ttttccacag cggtggtgac gctgattgaa
1800gacgggaaga atgactccct gccgtctgag tccacgtcgc acaggtggcg
ggggcctgcc 1860tgcaggcccc ccgatagctc ctacaacagc ctgtactcca
cctgcctgga gctgttcaag 1920ttcaccatcg gcatgggcga cctggagttc
actgagaact atgacttcaa ggctgtcttc 1980atcatcctgc tgctggccta
tgtaattctc acctacatcc tcctgctcaa catgctcatc 2040gccctcatgg
gtgagactgt caacaagatc gcacaggaga gcaagaacat ctggaagctg
2100cagagagcca tcaccatcct ggacacggag aagagcttcc ttaagtgcat
gaggaaggcc 2160ttccgctcag gcaagctgct gcaggtgggg tacacacctg
atggcaagga cgactaccgg 2220tggtgcttca gggtggacga ggtgaactgg
accacctgga acaccaacgt gggcatcatc 2280aacgaagacc cgggcaactg
tgagggcgtc aagcgcaccc tgagcttctc cctgcggtca 2340agcagagttt
caggcagaca ctggaagaac tttgccctgg tccccctttt aagagaggca
2400agtgctcgag ataggcagtc tgctcagccc gaggaagttt atctgcgaca
gttttcaggg 2460tctctgaagc cagaggacgc tgaggtcttc aagagtcctg
ccgcttccgg ggagaagtga 252023360DNAHomo sapiens 2atgaagtgca
gcctgaggaa gatgtggcgc cctggagaaa agaaggagcc ccagggcgtt 60gtctatgagg
atgtgccgga cgacacggag gatttcaagg aatcgcttaa ggtggttttt
120gaaggaagtg catatggatt acaaaacttt aataagcaaa agaaattaaa
aacatgtgac 180gatatggaca ccttcttctt gcattatgct gcagcagaag
gccaaattga gctaatggag 240aagatcacca gagattcctc tttggaagtg
ctgcatgaaa tggatgatta tggaaatacc 300cctctgcatt gtgctgtaga
aaaaaaccaa attgaaagcg ttaagtttct tctcagcaga 360ggagcaaacc
caaacctccg aaacttcaac atgatggctc ctctccacat agctgtgcag
420ggcatgaata atgaggtgat gaaggtcttg cttgagcata gaactattga
tgttaatttg 480gaaggagaaa atggaaacac agctgtgatc attgcgtgca
ccacaaataa tagcgaagca 540ttgcagattt tgcttaacaa aggagctaag
ccatgtaaat caaataaatg gggatgtttc 600cctattcacc aagctgcatt
ttcaggttcc aaagaatgca tggaaataat actaaggttt 660ggtgaagagc
atgggtacag tagacagttg cacattaact ttatgaataa tgggaaagcc
720acccctctcc acctggctgt gcaaaatggt gacttggaaa tgatcaaaat
gtgcctggac 780aatggtgcac aaatagaccc agtggagaag ggaaggtgca
cagccattca ttttgctgcc 840acccagggag ccactgagat tgttaaactg
atgatatcgt cctattctgg tagcgtggat 900attgttaaca caaccgatgg
atgtcatgag accatgcttc acagagcttc attgtttgat 960caccatgagc
tagcagacta tttaatttca gtgggagcag atattaataa gatcgattct
1020gaaggacgct ctccacttat attagcaact gcttctgcat cttggaatat
tgtaaatttg 1080ctactctcta aaggtgccca agtagacata aaagataatt
ttggacgtaa ttttctgcat 1140ttaactgtac agcaacctta tggattaaaa
aatctgcgac ctgaatttat gcagatgcaa 1200cagatcaaag agctggtaat
ggatgaagac aacgatgggt gtactcctct acattatgca 1260tgtagacagg
ggggccctgg ttctgtaaat aacctacttg gctttaatgt gtccattcat
1320tccaaaagca aagataagaa atcacctctg cattttgcag ccagttatgg
gcgtatcaat 1380acctgtcaga ggctcctaca agacataagt gatacgaggc
ttctgaatga aggtgacctt 1440catggaatga ctcctctcca tctggcagca
aagaatggac atgataaagt agttcagctt 1500cttctgaaaa aaggtgcatt
gtttctcagt gaccacaatg gctggacagc tttgcatcat 1560gcgtccatgg
gcgggtacac tcagaccatg aaggtcattc ttgatactaa tttgaagtgc
1620acagatcgct tggatgaaga cgggaacact gcacttcact ttgctgcaag
ggaaggccac 1680gccaaagccg ttgcgcttct tctgagccac aatgctgaca
tagtcctgaa caagcagcag 1740gcctcctttt tgcaccttgc acttcacaat
aagaggaagg aggttgttct tacgatcatc 1800aggagcaaaa gatgggatga
atgtcttaag attttcagtc ataattctcc aggcaataaa 1860tgtccaatta
cagaaatgat agaatacctc cctgaatgca tgaaggtact tttagatttc
1920tgcatgttgc attccacaga agacaagtcc tgccgagact attatatcga
gtataatttc 1980aaatatcttc aatgtccatt agaattcacc aaaaaaacac
ctacacagga tgttatatat 2040gaaccgctta cagccctcaa cgcaatggta
caaaataacc gcatagagct tctcaatcat 2100cctgtgtgta aagaatattt
actcatgaaa tggttggctt atggatttag agctcatatg 2160atgaatttag
gatcttactg tcttggtctc atacctatga ccattctcgt tgtcaatata
2220aaaccaggaa tggctttcaa ctcaactggc atcatcaatg aaactagtga
tcattcagaa 2280atactagata ccacgaattc atatctaata aaaacttgta
tgattttagt gtttttatca 2340agtatatttg ggtattgcaa agaagcgggg
caaattttcc aacagaaaag gaattatttt 2400atggatataa gcaatgttct
tgaatggatt atctacacga cgggcatcat ttttgtgctg 2460cccttgtttg
ttgaaatacc agctcatctg cagtggcaat gtggagcaat tgctgtttac
2520ttctattgga tgaatttctt attgtatctt caaagatttg aaaattgtgg
aatttttatt 2580gttatgttgg aggtaatttt gaaaactttg ttgaggtcta
cagttgtatt tatcttcctt 2640cttctggctt ttggactcag cttttacatc
ctcctgaatt tacaggatcc cttcagctct 2700ccattgcttt ctataatcca
gaccttcagc atgatgctag gagatatcaa ttatcgagag 2760tccttcctag
aaccatatct gagaaatgaa ttggcacatc cagttctgtc ctttgcacaa
2820cttgtttcct tcacaatatt tgtcccaatt gtcctcatga atttacttat
tggtttggca 2880gttggcgaca ttgctgaggt ccagaaacat gcatcattga
agaggatagc tatgcaggtg 2940gaacttcata ccagcttaga gaagaagctg
ccactttggt ttctacgcaa agtggatcag 3000aaatccacca tcgtgtatcc
caacaaaccc agatctggtg ggatgttatt ccatatattc 3060tgttttttat
tttgcactgg ggaaataaga caagaaatac caaatgctga taaatcttta
3120gaaatggaaa tattaaagca gaaataccgg ctgaaggatc ttacttttct
cctggaaaaa 3180cagcatgagc tcattaaact gatcattcag aagatggaga
tcatctctga gacagaggat 3240gatgatagcc attgttcttt tcaagacagg
tttaagaaag agcagatgga acaaaggaat 3300agcagatgga atactgtgtt
gagagcagtc aaggcaaaaa cacaccatct tgagccttag 336033315DNAHomo
sapiens 3atgtccttcg agggagccag gctcagcatg aggagccgca gaaatggtac
tatgggcagc 60acccggaccc tgtactccag tgtatctcgg agcacagacg tgtcctacag
tgacagtgat 120ttggtgaatt ttattcaggc aaattttaaa aaacgagaat
gtgtcttctt taccagagac 180tccaaggcca tggagaacat atgcaagtgt
ggttatgccc agagccagca catcgaaggc 240acccagatca accaaaatga
gaagtggaac tacaaaaaac ataccaagga gtttccaaca 300gacgccttcg
gggacattca gtttgagact ctggggaaga aaggcaagta cttacgcttg
360tcctgtgaca ccgactctga aactctctac gaactgctga cccagcactg
gcacctcaaa 420acacccaacc tggtcatttc agtgacgggt ggagccaaaa
actttgcttt gaagccacgc 480atgcgcaaga tcttcagcag gctgatttac
atcgcacagt ctaaaggtgc gtggattctc 540actggaggca ctcactacgg
cctgatgaag tacataggcg aggtggtgag agacaacacc 600atcagcagga
actcagaaga gaacatcgtg gccattggca tcgcagcatg gggcatggtc
660tccaacaggg acaccctcat caggagctgt gatgatgagg gacatttttc
agctcaatac 720atcatggatg actttaccag agaccctcta tacatcctgg
acaacaacca tacccacctg 780ctgcttgtgg acaacggttg tcatggacac
cccacagtgg aagccaagct ccggaatcag 840ctggaaaagt acatctctga
gcgcaccagt caagattcca actatggtgg taagatcccc 900atcgtgtgtt
ttgcccaagg aggtggaaga gagactctaa aagccatcaa cacctctgtc
960aaaagcaaga tcccttgtgt ggtggtggaa ggctcggggc agattgctga
tgtgatcgcc 1020agcctggtgg aggtggagga tgttttaacc tcttccatgg
tcaaagagaa gctggtacgc 1080tttttaccac gcactgtgtc ccggctgcct
gaagaggaaa ttgagagctg gatcaaatgg 1140ctcaaagaaa ttcttgagag
ttctcaccta ctcacagtaa ttaagatgga agaggctgga 1200gatgagattg
tgagcaacgc catttcctat gcgctgtaca aagccttcag cactaatgag
1260caagacaagg acaactggaa tggacagctg aagcttctgc tggagtggaa
ccagttggac 1320cttgccagtg atgagatctt caccaatgat cgccgctggg
agtctgccga ccttcaggag 1380gtcatgttca cggctctcat aaaggacaga
cccaagtttg tccgcctctt tctggagaat 1440ggcctgaatc tgcagaagtt
tctcaccaat gaagtcctca cagagctctt ctccacccac 1500ttcagcaccc
tagtgtaccg gaatctgcag atcgccaaga actcctacaa tgacgcactc
1560ctcacctttg tctggaagtt ggtggcaaac ttccgtcgaa gcttctggaa
agaggacaga 1620agcagcaggg aggacttgga tgtggaactc catgatgcat
ctctcaccac ccggcacccg 1680ctgcaagctc tcttcatctg ggccattctt
cagaacaaga aggaactctc caaggtcatt 1740tgggagcaga ccaaaggctg
tactctggca gccttggggg ccagcaagct tctgaagacc 1800ctggccaaag
ttaagaatga tatcaacgct gctggggaat cggaggaact ggccaatgaa
1860tatgagaccc gagcagtgga gttgttcacc gagtgttaca gcaatgatga
agacttggca 1920gaacagctac tggtctactc ctgcgaagcc tggggtggga
gcaactgtct ggagctggca 1980gtggaggcta cagatcagca tttcatcgct
cagcctgggg tccagaattt cctttctaag 2040caatggtatg gagagatttc
ccgagacacg aagaactgga agattatcct gtgtctattc 2100atcatcccct
tagtgggctg tggcctcgta tcatttagga agaaacccat tgacaagcac
2160aagaagctgc tgtggtacta tgtggccttc ttcacgtcgc ccttcgtggt
cttctcctgg 2220aacgtggtct tctacatcgc cttcctcctg ctgtttgcct
atgtgctgct catggacttc 2280cactcagtgc cacacacccc cgagctgatc
ctctacgccc tggtcttcgt cctcttctgt 2340gatgaagtga ggcagtggta
catgaacgga gtgaattatt tcaccgacct atggaacgtt 2400atggacaccc
tgggactctt ctacttcata gcgggtattg tattccggct ccactcttct
2460aataaaagct cgttgtactc tgggcgcgtc attttctgtc tggattacat
tatattcacg 2520ctaaggctca tccacatttt caccgtcagc aggaacttgg
gacccaagat tataatgctg 2580cagcggatgc tgatcgacgt tttcttcttc
ctgttcctct ttgctgtgtg gatggtggcc 2640tttggcgtgg ccagacaggg
gatcctaagg caaaatgaac agcgctggag atggatcttc 2700cgctctgtca
tctatgagcc ctacctggcc atgtttggcc aggttcccag tgacgtggat
2760agtaccacat atgacttctc ccactgtacc ttctcgggaa atgagtccaa
gccactgtgt 2820gtggagctgg atgagcacaa cctgccccgc ttccctgagt
ggatcaccat tccgctggtg 2880tgcatctaca tgctctccac caatatcctt
ctggtcaacc tcctggtcgc catgtttggc 2940tacacggtag gcattgtaca
ggagaacaac gaccaggtct ggaaattcca gcggtacttc 3000ctggtgcagg
agtactgcaa ccgcctaaac atccccttcc ccttcgttgt cttcgcttat
3060ttctacatgg tggtgaagaa gtgtttcaaa tgctgctgta aagagaagaa
tatggagtct 3120aatgcctgct gtttcagaaa tgaggacaat gagactttgg
cgtgggaggg tgtcatgaag 3180gagaattacc ttgtcaagat caacacgaaa
gccaacgaca actcagagga gatgaggcat 3240cggtttagac aactggactc
aaagcttaac gacctcaaaa gtcttctgaa agagattgct 3300aataacatca agtaa
3315
* * * * *
References